研究者紹介システム

近藤 昭彦
コンドウ アキヒコ
大学院科学技術イノベーション研究科 科学技術イノベーション専攻
教授
副学長
応用化学関係
Last Updated :2022/05/11

研究者情報

所属

  • 【主配置】

    大学院科学技術イノベーション研究科 科学技術イノベーション専攻
  • 【配置】

    工学部 応用化学科, 大学院工学研究科 応用化学専攻, 先端融合研究環, 先端バイオ工学研究センター, 戦略企画室

学位

  • 工学博士, 京都大学

授業科目

ジャンル

  • 科学・技術 / 工学
  • 科学・技術 / バイオテクノロジー

コメントテーマ

  • バイオテクノロジー
  • バイオマス
  • バイオ燃料
  • バイオリファイナリー
  • 合成生物工学
  • らん藻

研究ニュース

研究活動

研究キーワード

  • バイオマス
  • 応用微生物
  • 酵素
  • バイオリアクター
  • バイオテクノロジー

研究分野

  • ものづくり技術(機械・電気電子・化学工学) / バイオ機能応用、バイオプロセス工学

受賞

  • 2020年10月 神戸大学, 令和2年度 学長表彰(財務貢献者)

    近藤 昭彦

  • 2017年 International Bioprocessing Association, Outstanding Scientist Award-2016

    近藤 昭彦

  • 2016年 American Institute for Medical and Biological Engineering (AIMBE), Fellow

    近藤 昭彦

  • 2016年 膜シンポジウム2016, 膜シンポジウム 学生賞, バイオエタノール生産プロセスにおける浸透圧駆動型膜分離法を利用した糖液濃縮の検討

    渋谷 真史, 安川 政宏, 佐々木 建吾, 田中 裕大, 高橋 智輝, 近藤 昭彦, 松山 秀人

    国内学会・会議・シンポジウム等の賞

  • 2016年 IMSTEC2016, IMSTEC2016 Travel award, Up-concentration of sugar solution by using forward osmosis for bioethanol production process

    渋谷 真史, 安川 政宏, 佐々木 建吾, 田中 裕大, 高橋 智輝, 近藤 昭彦, 松山 秀人

    国際学会・会議・シンポジウム等の賞

  • 2015年 米国出版社協会, 学術出版賞「環境科学」部門賞, Bioprocessing of Renewable Resources to Commodity Bioproducts

    近藤 昭彦

  • 2014年01月 日本農芸化学会, B.B.B.論文賞, Utilization of Lactic Acid Bacterial Genes in Synechocystis sp. PCC 6803 in the Production of Lactic Acid

    Ancy Joseph, Simpei Aikawa, Kengo Sasaki, Yota Tsuge, Fumio Matsuda, Tsutomu Tanaka, Akihiko Kondo

    日本国

    国内学会・会議・シンポジウム等の賞

  • 2013年09月 経済産業省, 「ものづくり日本大賞」経済産業大臣賞(製品・技術開発部門), 世界初マイクロチップでの単一細胞全自動解析・回収装置の商品化開発

    徐 杰, 木村 健一, 金野 徹, 松下 政宏, 黒田 俊一, 近藤 昭彦, 藤井 郁雄

  • 2013年09月 日本生物工学会, 生物工学論文賞, 合成代謝経路と細胞表層工学を利用した組換え大腸菌によるセロビオースからのイソプロパノール生産

    相馬 悠希, 猪熊 健太郎, 田中 勉, 荻野 千秋, 近藤 昭彦, 岡本 正宏, 花井 泰三

    日本国

    国内学会・会議・シンポジウム等の賞

  • 2011年 井植記念会, 第35回井植文化賞(科学技術部門)

    近藤 昭彦

  • 2010年10月 日本生物工学会, 生物工学功績賞, 細胞表層工学技術の広範な展開と合成生物工学の開拓によるバイオ燃料・グリーン化学品生産のための細胞工場の創製―バイオリファイナリーの構築を目指して―

    近藤 昭彦

  • 2010年07月 Renewable Energy 2010, Best Paper Award, Construction of consolidated biopreocesses for production of bio-fuels and chemicals by using cell surface engineered microbial cells

    近藤 昭彦

  • 2009年10月 神戸大学, 第1回学長表彰

    近藤 昭彦

  • 2008年11月 日刊工業新聞社, モノづくり連携大賞 特別賞

    近藤 昭彦

  • 2008年09月 竹田理化工業株式会社, 竹田国際貢献賞

    近藤 昭彦

  • 2007年 フジサンケイビジネスアイ, 第21回 独創性を拓く 先端技術大賞 特別賞, 熱応答性磁性ナノ粒子(Therma-Max)の開発とその実用化~産学連携がもたらした若手研究者たちの挑戦~

    大西 徳幸, 畑 英之, 松井 景明, 近藤 昭彦

  • 2005年 日本イージェイケー株式会社, オルガテクノ大賞 材料・素材部門賞, 熱応答性磁性ナノ粒子 商標名:Therma-Max

    大西 徳幸, 近藤 昭彦

  • 2004年 バイオテクノロジー・ジャパン, 第4回バイオビジネスコンペJapan 最優秀賞, 熱応答性磁性ナノ粒子(商標名:Therma-Max)の開発とその実用化

    大西 徳幸, 近藤 昭彦

  • 2001年 日本生物工学会, 第9回 生物工学論文賞, Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins

    Akihiko Kondo, Jiro Kohda, Yasunori Endo, Tokuhisa Shiromizu, Yoichi Kurokawa, Kazuyo Nishihara, Hideki Yanagi, Takashi Yura, Hideki Fukuda

  • 2000年 日本生物工学会, 第8回 生物工学論文賞, Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent

    Masaru Kaieda, Taichi Samukawa, Takeshi Matsumoto, Kazuhiro Ban, Akihiko Kondo, Yuji Shimada, Hideo Noda, Fumiki Nomoto, Koutaro Ohtsuka, Eiji Izumoto, Hideki Fukuda

  • 1999年 日本生物工学会, 第7回 生物工学論文賞, Protein refolding system using holo-chaperonin from the thermophilic bacterium Thermus thermophilus

    Tadanaru Teshima, Jiro Kohda, Akihiko Kondo, Hideki Taguchi, Masafumi Yohda, Isao Endo, Hideki Fukuda

  • 1993年 化学工学会, 研究奨励賞(内藤雅喜記念賞), 微粒子材料の生物化学工学への応用に関する研究

    近藤 昭彦

論文

  • Ryosuke Fujiwara, Mariko Nakano, Yuuki Hirata, Chisako Otomo, Daisuke Nonaka, Sakiya Kawada, Hikaru Nakazawa, Mitsuo Umetsu, Tomokazu Shirai, Shuhei Noda, Tsutomu Tanaka, Akihiko Kondo

    Elsevier BV, 2022年07月, Metabolic Engineering, 72, 68 - 81, 英語

    研究論文(学術雑誌)

  • Ku Syahidah Ku Ismail, Yuki Matano, Yuri Sakihama, Kentaro Inokuma, Yumiko Nambu, Tomohisa Hasunuma, Akihiko Kondo

    Elsevier BV, 2022年01月, Bioresource Technology, 343, 126071 - 126071, 英語

    研究論文(学術雑誌)

  • Hideo Kawaguchi, Kenji Takada, Taghreed Elkasaby, Radityo Pangestu, Masakazu Toyoshima, Prihardi Kahar, Chiaki Ogino, Tatsuo Kaneko, Akihiko Kondo

    Elsevier BV, 2022年01月, Bioresource Technology, 344, 126165 - 126165, 英語

    研究論文(学術雑誌)

  • Yuichi Kato, Kosuke Inabe, Ryota Hidese, Akihiko Kondo, Tomohisa Hasunuma

    Elsevier BV, 2022年01月, Bioresource Technology, 344, 126196 - 126196, 英語

    研究論文(学術雑誌)

  • Yuma Kobayashi, Kentaro Inokuma, Mami Matsuda, Akihiko Kondo, Tomohisa Hasunuma

    Elsevier BV, 2022年01月, Biotechnology Notes, 3, 1 - 7, 英語

    研究論文(学術雑誌)

  • Hong-Qi Chen, Ming-Ming Zhang, Qi Xing, Pei-Liang Ye, Tomohisa Hasunuma, Akihiko Kondo, Xin-Qing Zhao

    Elsevier BV, 2022年01月, Biochemical Engineering Journal, 178, 108274 - 108274

    研究論文(学術雑誌)

  • Yonghao Li, Peng Zhang, Deying Zhu, Bo Yao, Tomohisa Hasunuma, Akihiko Kondo, Xinqing Zhao

    Elsevier BV, 2022年01月, Biochemical Engineering Journal, 178, 108296 - 108296

    研究論文(学術雑誌)

  • Kosuke Inabe, Ayaka Miichi, Mami Matsuda, Takanobu Yoshida, Yuichi Kato, Ryota Hidese, Akihiko Kondo, Tomohisa Hasunuma

    Nitrogen is essential for the biosynthesis of various molecules in cells, such as amino acids and nucleotides, as well as several types of lipids and sugars. Cyanobacteria can assimilate several forms of nitrogen, including nitrate, ammonium, and urea, and the physiological and genetic responses to these nitrogen sources have been studied previously. However, the metabolic changes in cyanobacteria caused by different nitrogen sources have not yet been characterized. This study aimed to elucidate the influence of nitrate and ammonium on the metabolic profiles of the cyanobacterium Synechocystis sp. strain PCC 6803. When supplemented with NaNO3 or NH4Cl as the nitrogen source, Synechocystis sp. PCC 6803 grew faster in NH4Cl medium than in NaNO3 medium. Metabolome analysis indicated that some metabolites in the CBB cycle, glycolysis, and TCA cycle, and amino acids were more abundant when grown in NH4Cl medium than NaNO3 medium. 15N turnover rate analysis revealed that the nitrogen assimilation rate in NH4Cl medium was higher than in NaNO3 medium. These results indicate that the mechanism of nitrogen assimilation in the GS-GOGAT cycle differs between NaNO3 and NH4Cl. We conclude that the amounts and biosynthetic rate of cyanobacterial metabolites varies depending on the type of nitrogen.

    MDPI AG, 2021年12月14日, Metabolites, 11 (12), 867 - 867

    研究論文(学術雑誌)

  • Nozomu Shibata, Hiroshi Kakeshita, Kazuaki Igarashi, Yasushi Takimura, Yosuke Shida, Wataru Ogasawara, Tohru Koda, Tomohisa Hasunuma, Akihiko Kondo

    Abstract Background Trichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose. Results We found that deletion of an α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (ΔtubB) strain had high enzyme production and the same enzyme profile even if the strain was cultured in a glucose-containing medium. From transcriptome analysis, the ΔtubB strain exhibited upregulation of both cellulase and hemicellulase genes including some that were not originally induced by cellulose. Moreover, cellobiose transporter genes and the other sugar transporter genes were highly upregulated, and simultaneous uptake of glucose and cellobiose was also observed in the ΔtubB strain. These results suggested that the ΔtubB strain was released from CCR. Conclusion Trichoderma reesei α-tubulin is involved in the transcription of cellulase and hemicellulase genes, as well as in CCR. This is the first report of overcoming CCR by disrupting α-tubulin gene in T. reesei. The disruption of α-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.

    Springer Science and Business Media LLC, 2021年12月, Biotechnology for Biofuels, 14 (1), 39 - 39, 英語, 国際誌

    研究論文(学術雑誌)

  • Yuichi Kato, Tomoki Oyama, Kentaro Inokuma, Christopher J. Vavricka, Mami Matsuda, Ryota Hidese, Katsuya Satoh, Yutaka Oono, Jo-Shu Chang, Tomohisa Hasunuma, Akihiko Kondo

    AbstractLight/dark cycling is an inherent condition of outdoor microalgae cultivation, but is often unfavorable for lipid accumulation. This study aims to identify promising targets for metabolic engineering of improved lipid accumulation under outdoor conditions. Consequently, the lipid-rich mutant Chlamydomonas sp. KOR1 was developed through light/dark-conditioned screening. During dark periods with depressed CO2 fixation, KOR1 shows rapid carbohydrate degradation together with increased lipid and carotenoid contents. KOR1 was subsequently characterized with extensive mutation of the ISA1 gene encoding a starch debranching enzyme (DBE). Dynamic time-course profiling and metabolomics reveal dramatic changes in KOR1 metabolism throughout light/dark cycles. During light periods, increased flux from CO2 through glycolytic intermediates is directly observed to accompany enhanced formation of small starch-like particles, which are then efficiently repartitioned in the next dark cycle. This study demonstrates that disruption of DBE can improve biofuel production under light/dark conditions, through accelerated carbohydrate repartitioning into lipid and carotenoid.

    Springer Science and Business Media LLC, 2021年12月, Communications Biology, 4 (1)

    研究論文(学術雑誌)

  • Kengo Sasaki, Daisuke Sasaki, Katsunori Sasaki, Yuto Nishidono, Akihiro Yamamori, Ken Tanaka, Akihiko Kondo

    AbstractDaikenchuto (DKT) is a Japanese traditional herbal (Kampo) medicine containing ginseng, processed ginger, and Japanese or Chinese pepper. We aimed to determine how DKT affects human colonic microbiota. An in vitro microbiota model was established using fecal inocula collected from nine healthy volunteers, and each model was found to retain operational taxonomic units similar to the ones in the original human fecal samples. DKT was added to the in vitro microbiota model culture at a concentration of 0.5% by weight. Next-generation sequencing of bacterial 16S rRNA gene revealed a significant increase in the relative abundance of bacteria related to the Bifidobacterium genus in the model after incubation with DKT. In pure cultures, DKT significantly promoted the growth of Bifidobacterium adolescentis, but not that of Fusobacterium nucleatum or Escherichia coli. Additionally, in pure cultures, B. adolescentis transformed ginsenoside Rc to Rd, which was then probably utilized for its growth. Our study reveals the in vitro bifidogenic effect of DKT that likely contributes to its beneficial effects on the human colon.

    Springer Science and Business Media LLC, 2021年12月, Scientific Reports, 11 (1), 英語

    研究論文(学術雑誌)

  • Kengo Sasaki, Daisuke Sasaki, Yota Tsuge, Masahiko Morita, Akihiko Kondo

    Abstract Background It is desirable to improve the anaerobic digestion processes of recalcitrant materials, such as cellulose. Enhancement of methane (CH4) production from organic molecules was previously accomplished through coupling a bioelectrochemical system (BES); however, scaling-up BES-based production is difficult. Here, we developed a two-stage process consisting of a BES using low-cost and low-reactive carbon sheets as the cathode and anode, and a fixed film reactor (FFR) containing conductive material, i.e., carbon fiber textiles (CFTs) (:BES → FFR). By controlling the cathodic current at 2.7 μA/cm2 without abiotic H2 production, the three-electrode BES system was operated to mimic a microbial electrolysis cell. Results The thermophilic BES (inlet pH: 6.1) and FFR (inlet pH: 7.5) were operated using hydraulic retention times (HRTs) of 2.5 and 4.2 days, respectively, corresponding to a cellulose load of 3555.6 mg-carbon (C)/(L day). The BES → FFR process achieved a higher CH4 yield (37.5%) with 52.8 vol% CH4 in the product gas compared to the non-bioelectrochemical system (NBES) → FFR process, which showed a CH4 yield of 22.1% with 46.8 vol% CH4. The CH4 production rate (67.5 mM/day) obtained with the BER → FFR process was much higher than that obtained using electrochemical methanogenesis (0.27 mM/day). Application of the electrochemical system or CFTs improved the yields of CH4 with the NBES → FFR or BES → non-fixed film reactor process, respectively. Meta 16S rRNA sequencing revealed that putative cellulolytic bacteria (identified as Clostridium species) were present in the BES and NBES, and followed (BES→ and NBES→) FFR. Notably, H2-consuming methanogens, Methanobacterium sp. and Methanosarcina sp., showed increased relative abundances in the suspended fraction and attached fraction of (BES→) FFR, respectively, compared to that of (NBES→) FFR, although these methanogens were observed at trace levels in the BES and NBES. Conclusions These results indicate that bioelectrochemical preprocessing at a low current effectively induces interspecies H2 transfer in the FFR with conductive material. Sufficient electrochemical preprocessing was observed using a relatively short HRT. This type of two-stage process, BES → FFR, is useful for stabilization and improvement of the biogas (CH4) production from cellulosic material, and our results imply that the two-stage system developed here may be useful with other recalcitrant materials.

    Springer Science and Business Media LLC, 2021年12月, Biotechnology for Biofuels, 14 (1)

    研究論文(学術雑誌)

  • Yuma Kobayashi, Kentaro Inokuma, Mami Matsuda, Akihiko Kondo, Tomohisa Hasunuma

    Elsevier BV, 2021年12月, Metabolic Engineering Communications, 13, e00188 - e00188, 英語

    研究論文(学術雑誌)

  • Nunthaphan Vikromvarasiri, Tomokazu Shirai, Akihiko Kondo

    Abstract Background Glycerol is a desirable alternative substrate for 2,3-butanediol (2,3-BD) production for sustainable development in biotechnological industries and non-food competitive feedstock. B. subtilis, a “generally recognized as safe” organism that is highly tolerant to fermentation products, is an ideal platform microorganism to engineer the pathways for the production of valuable bio-based chemicals, but it has never been engineered to improve 2,3-BD production from glycerol. In this study, we aimed to enhance 2,3-BD production from glycerol in B. subtilis through in silico analysis. Genome-scale metabolic model (GSM) simulations was used to design and develop the metabolic pathways of B. subtilis. Flux balance analysis (FBA) simulation was used to evaluate the effects of step-by-step gene knockouts to improve 2,3-BD production from glycerol in B. subtilis. Results B. subtilis was bioengineered to enhance 2,3-BD production from glycerol using FBA in a published GSM model of B. subtilis, iYO844. Four genes, ackA, pta, lctE, and mmgA, were knocked out step by step, and the effects thereof on 2,3-BD production were evaluated. While knockout of ackA and pta had no effect on 2,3-BD production, lctE knockout led to a substantial increase in 2,3-BD production. Moreover, 2,3-BD production was improved by mmgA knockout, which had never been investigated. In addition, comparisons between in silico simulations and fermentation profiles of all B. subtilis strains are presented in this study. Conclusions The strategy developed in this study, using in silico FBA combined with experimental validation, can be used to optimize metabolic pathways for enhanced 2,3-BD production from glycerol. It is expected to provide a novel platform for the bioengineering of strains to enhance the bioconversion of glycerol into other highly valuable chemical products.

    Springer Science and Business Media LLC, 2021年12月, Microbial Cell Factories, 20 (1), 英語

    研究論文(学術雑誌)

  • Tomoki Oyama, Yuichi Kato, Katsuya Satoh, Yutaka Oono, Mami Matsuda, Tomohisa Hasunuma, Akihiko Kondo

    Elsevier BV, 2021年12月, Algal Research, 60, 102544 - 102544

    研究論文(学術雑誌)

  • Hideo Kawaguchi, Tomohisa Hasunuma, Yasuo Ohnishi, Takashi Sazuka, Akihiko Kondo, Chiaki Ogino

    Abstract Background Bio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics. Results Under aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of < 33% saturated oxygen. Based on this metabolome analysis, metabolic pathways were rationally designed for oxygen limitation. An ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture. Conclusions Our results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives. Graphical Abstract

    Springer Science and Business Media LLC, 2021年12月, Microbial Cell Factories, 20 (1), 228, 英語

    研究論文(学術雑誌)

  • Chaofan Zhang, Tomohisa Hasunuma, Su Shiung Lam, Akihiko Kondo, Shih-Hsin Ho

    Elsevier BV, 2021年11月, Bioresource Technology, 340, 125638 - 125638, 英語

    研究論文(学術雑誌)

  • Naofumi Yoshida, Tomoya Yamashita, Tatsunori Osone, Tetsuya Hosooka, Masakazu Shinohara, Seiichi Kitahama, Kengo Sasaki, Daisuke Sasaki, Takeshi Yoneshiro, Tomohiro Suzuki, Takuo Emoto, Yoshihiro Saito, Genki Ozawa, Yushi Hirota, Yasuyuki Kitaura, Yoshiharu Shimomura, Yuko Okamatsu-Ogura, Masayuki Saito, Akihiko Kondo, Shingo Kajimura, Takeshi Inagaki, Wataru Ogawa, Takuji Yamada, Ken-ichi Hirata

    Elsevier BV, 2021年11月, iScience, 24 (11), 103342 - 103342, 英語

    研究論文(学術雑誌)

  • Taiji Yuzawa, Tomokazu Shirai, Ryoko Orishimo, Kazuki Kawai, Akihiko Kondo, Takashi Hirasawa

    Microbiology Research Foundation, 2021年10月, The Journal of General and Applied Microbiology, 67 (4), 142 - 149, 英語

    研究論文(学術雑誌)

  • Joana T. Cunha, Daniel G. Gomes, Aloia Romaní, Kentaro Inokuma, Tomohisa Hasunuma, Akihiko Kondo, Lucília Domingues

    Elsevier BV, 2021年09月, Energy Conversion and Management, 243, 114359 - 114359, 英語

    研究論文(学術雑誌)

  • Shuhei Noda, Yutaro Mori, Ryosuke Fujiwara, Tomokazu Shirai, Tsutomu Tanaka, Akihiko Kondo

    Elsevier BV, 2021年09月, Metabolic Engineering, 67, 1 - 10, 英語

    研究論文(学術雑誌)

  • Phei Er Kee, Hip Seng Yim, Akihiko Kondo, John Chi-Wei Lan, Hui Suan Ng

    Aqueous biphasic electrophoresis system (ABES) incorporates electric fields into the biphasic system to separate the target biomolecules from crude feedstock. Ionic liquid (IL) is regarded as an excellent candidate as the phase-forming components for ABES because of the great electrical conductivity, which can promote the electromigration of biomolecules in ABES, and thereby enhances the separation efficiency of the target biomolecules from crude feedstock. The application of electric fields to the conventional biphasic system expedites the phase settling time of the biphasic system, which eases the subsequent scaling-up steps and reduces the overall processing time of the recovery process. Alkyl sulphate-based IL is a green and economical halide-free surfactant when compared to the other halide-containing IL. The feasibility of halide-free IL-based ABES to recover Kytococcus sedentarius TWHK01 keratinase was studied. Optimum partition coefficient (Ke = 7.53 ± 0.35) and yield (YT = 80.36% ± 0.71) were recorded with IL-ABES comprised of 15.0% (w/w) [EMIM][ESO4], 20.0% (w/w) sodium carbonate and 15% (w/w) crude feedstock. Selectivity (S) of 5.75 ± 0.27 was obtained with the IL-ABES operated at operation time of 5 min with 10 V voltage supplied. Halide-free IL is proven to be a potential phase-forming component of IL-ABES for large-scale recovery of keratinase.

    MDPI AG, 2021年08月17日, Marine Drugs, 19 (8), 463 - 463, 英語

    研究論文(学術雑誌)

  • Jih-Heng Chen, Yuichi Kato, Mami Matsuda, Chun-Yen Chen, Dillirani Nagarajan, Tomohisa Hasunuma, Akihiko Kondo, Jo-Shu Chang

    Elsevier BV, 2021年08月, Bioresource Technology, 334, 125200 - 125200, 英語

    研究論文(学術雑誌)

  • Taku Sakuragawa, Satoshi Wakai, Silai Zhang, Hideo Kawaguchi, Chiaki Ogino, Akihiko Kondo

    Recently, a hyphae-dispersed type of filamentous fungus Aspergillus oryzae was constructed via genetic engineering, and industrial applications are expected due to the ease of handling and to the level of protein production properties. In this study, we constructed cellulase-expressing strains using wild-type and hyphae-dispersed strains to investigate the correlation between protein productivity and metabolism. Compared with the original strain, the hyphae-dispersed cellulase-expressing strain showed elevated cellulase activity, rapid glucose consumption, increased mycelial dry weight, an increased expression of cellulase genes, and activated respiration activity. Comparative metabolomic analysis showed fewer metabolites in the glycolysis and TCA cycles in the dispersed strains than in the original strains. These results indicate that the flux of carbohydrate metabolism in the hyphae-dispersed strains is smoother than that in the original strains. Such efficient metabolic flux would contribute to efficient energy conversion and to sufficient energy supply to anabolisms, such as mycelial growth and protein production. Our findings suggest that the hyphae-dispersed strains could be a useful host not only for protein production but also for the biological production of various chemicals such as organic acids.

    Elsevier BV, 2021年08月, Journal of Bioscience and Bioengineering, 132 (2), 140 - 147, 英語, 国内誌

    研究論文(学術雑誌)

  • Kentaro Inokuma, Yuki Kitada, Takahiro Bamba, Yuma Kobayashi, Takahiro Yukawa, Riaan den Haan, Willem Heber van Zyl, Akihiko Kondo, Tomohisa Hasunuma

    Springer Science and Business Media LLC, 2021年08月, Applied Microbiology and Biotechnology, 105 (14-15), 5895 - 5904, 英語

    研究論文(学術雑誌)

  • Sabrina Wolf, Judith Becker, Yota Tsuge, Hideo Kawaguchi, Akihiko Kondo, Jan Marienhagen, Michael Bott, Volker F. Wendisch, Christoph Wittmann

    Abstract The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity.

    Portland Press Ltd., 2021年06月07日, Essays in Biochemistry, 65 (2), 197 - 212, 英語

    研究論文(学術雑誌)

  • Shota Isogai, Nobuyuki Okahashi, Ririka Asama, Tomomi Nakamura, Tomohisa Hasunuma, Fumio Matsuda, Jun Ishii, Akihiko Kondo

    Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l-phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. In vitro screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3' prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. In vivo, the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3'-prenylnaringenin (3'-PN), in contrast to the previously reported in vivo production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.

    Elsevier BV, 2021年06月, Metabolic Engineering Communications, 12, e00169 - e00169, 英語, 国際誌

    研究論文(学術雑誌)

  • Shinichiro Shoji, Taiki Yamaji, Harumi Makino, Jun Ishii, Akihiko Kondo

    Elsevier BV, 2021年05月, Metabolic Engineering, 65, 167 - 177

    研究論文(学術雑誌)

  • Hiroko Iijima, Atsuko Watanabe, Haruna Sukigara, Kaori Iwazumi, Tomokazu Shirai, Akihiko Kondo, Takashi Osanai

    Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO2 using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO2 to carboxylic acids.

    Elsevier BV, 2021年05月, Metabolic Engineering, 65, 88 - 98, 英語, 国際誌

    研究論文(学術雑誌)

  • Nova Rachmadona, Emmanuel Quayson, Jerome Amoah, Daniel A Alfaro‐Sayes, Shinji Hama, Martha Aznury, Akihiko Kondo, Chiaki Ogino

    Wiley, 2021年05月, Biofuels, Bioproducts and Biorefining, 15 (3), 804 - 814, 英語

    研究論文(学術雑誌)

  • Yutaro Mori, Shuhei Noda, Tomokazu Shirai, Akihiko Kondo

    Abstract The C4 unsaturated compound 1,3-butadiene is an important monomer in synthetic rubber and engineering plastic production. However, microorganisms cannot directly produce 1,3-butadiene using glucose as a renewable carbon source via biological processes. This study constructed a novel artificial metabolic pathway for 1,3-butadiene production from glucose in Escherichia coli by combining the cis,cis-muconic acid (ccMA)-producing pathway and tailored ferulic acid decarboxylase mutants. The rational enzyme design of the substrate-binding site by computational simulation improved 1,3-butadiene-producing ccMA decarboxylation with a small mutant library. We found that changing dissolved oxygen and controlling pH were important factors for 1,3-butadiene production. Using dissolved oxygen–stat fed-batch fermentation in a 1-L jar fermenter, we could produce 2.1 g L− 1 of 1,3-butadiene. These results indicated that using a rational enzyme design, we can produce unnatural/nonbiological compounds (those that are made from petroleum and cannot be produced by microorganisms) using glucose as a renewable carbon source.

    Research Square, 2021年04月, 12, 2195, 英語

  • Shaoze Yuan, Shunsuke Kawasaki, Islam M. Y. Abdellatif, Keiji Nishida, Akihiko Kondo, Tohru Ariizumi, Hiroshi Ezura, Kenji Miura

    Springer Science and Business Media LLC, 2021年04月, Plant Cell Reports, 40 (4), 667 - 676, 英語

    研究論文(学術雑誌)

  • Kengo Sasaki, Tomoyuki Mori, Namiko Hoshi, Daisuke Sasaki, Jun Inoue, Reiko Shinkura, Akihiko Kondo

    Abstract W27 monoclonal immunoglobulin A (IgA) suppresses pathogenic Escherichia coli cell growth; however its effect on the human intestine remains unclear. We thus aimed to determine how W27 IgA affects the human colonic microbiota using the in vitro microbiota model. This model was established using fecal samples collected from 12 healthy volunteers; after anaerobic cultivation, each model was found to retain the genera found in the original human fecal samples. After pre-incubating W27 IgA with the respective fecal sample in aerobic condition, the mixture of W27 IgA (final concentration, 0.5 µg/mL) and each fecal sample was added to the in vitro microbiota model and cultured under anaerobic condition,. Next-generation sequencing of the bacterial 16S rRNA gene revealed that W27 IgA addition significantly decreased the relative abundance of bacteria related to the genus Escherichia in the model. Additionally, at a final concentration of 5 µg/mL, W27 IgA delayed growth in pure culture of Escherichia coli isolated from human fecal samples. Our study thus revealed the suppressive effect of W27 IgA on the genus Escherichia at relatively low-concentrations and the usefulness of an in vitro microbiota model to evaluate the effect of IgA as a gut microbiota regulator.

    Research Square, 2021年03月03日, 英語

  • Masahiro Tominaga, Kenta Nozaki, Daisuke Umeno, Jun Ishii, Akihiko Kondo

    AbstractA wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system’s utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.

    Springer Science and Business Media LLC, 2021年03月, Nature Communications, 12 (1), 英語

    研究論文(学術雑誌)

  • Ning Mao, Nikhil Aggarwal, Chueh Loo Poh, Byung Kwan Cho, Akihiko Kondo, Chenli Liu, Wen Shan Yew, Matthew Wook Chang

    Wiley, 2021年03月, Advanced Genetics, 2 (1), 英語

    研究論文(学術雑誌)

  • Kenta Morita, Yuya Nishimura, Satoko Nakamura, Yuki Arai, Chiya Numako, Kazuyoshi Sato, Masao Nakayama, Hiroaki Akasaka, Ryohei Sasaki, Chiaki Ogino, Akihiko Kondo

    Elsevier BV, 2021年02月, Colloids and Surfaces B: Biointerfaces, 198, 111451 - 111451

    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Ririka Asama, Takuya Tabata, Kenta Morita, Tatsuo Maruyama, Akihiko Kondo, Jun Ishii

    Melatonin is an indoleamine neurohormone made by the pineal gland. Its receptors, MTNR1A and MTNR1B, are members of the G-protein-coupled receptor (GPCR) family and are involved in sleep, circadian rhythm, and mood disorders, and in the inhibition of cancer growth. These receptors, therefore, represent significant molecular targets for insomnia, circadian sleep disorders, and cancer. The yeast Saccharomyces cerevisiae is an attractive host for assaying agonistic activity for human GPCR. We previously constructed a GPCR-based biosensor employing a high-sensitivity yeast strain that incorporated both a chimeric yeast-human G alpha protein and a bright fluorescent reporter gene (ZsGreen). Similar approaches have been used for simple and convenient measurements of various GPCR activities. In the current study, we constructed a fluorescence-based yeast biosensor for monitoring the signaling activation of human melatonin receptors. We used this system to analyze point mutations, including previously unreported mutations of the consensus sequences of MTNR1A and MTNR1B melatonin receptors and compared their effects. Most mutations in the consensus sequences significantly affected the signaling capacities of both receptors, but several mutations showed differences between these subtype receptors. Thus, this yeast biosensor holds promise for revealing the functions of melatonin receptors.

    Wiley, 2021年02月, Biotechnology and Bioengineering, 118 (2), 863 - 876, 英語

    研究論文(学術雑誌)

  • Keiji Nishida, Akihiko Kondo

    Elsevier BV, 2021年01月, Metabolic Engineering, 63, 141 - 147

    研究論文(学術雑誌)

  • Aniruddha Nag, Mohammad Asif Ali, Hideo Kawaguchi, Shun Saito, Yukie Kawasaki, Shoko Miyazaki, Hirotoshi Kawamoto, Deddy Triyono Nugroho Adi, Kumiko Yoshihara, Shunsuke Masuo, Yohei Katsuyama, Akihiko Kondo, Chiaki Ogino, Naoki Takaya, Tatsuo Kaneko, Yasuo Ohnishi

    Wiley, 2021年01月, Advanced Sustainable Systems, 5 (1), 2000193 - 2000193, 英語

    研究論文(学術雑誌)

  • Ayaka Nakashima, Kengo Sasaki, Daisuke Sasaki, Kosuke Yasuda, Kengo Suzuki, Akihiko Kondo

    AbstractThe alga Euglena gracilis (E. gracilis) has recently gained attention as a health food, but its effects on human gut microbiota remain unknown. This study aimed to determine the effect of E. gracilis on gut microbiota and defecation due to modulation of microbiota composition in vitro and in vivo. The in vitro model simulating human colonic microbiota revealed that E. gracilis addition stimulated the growth of commensal Faecalibacterium. Further, E. gracilis addition enhanced butyrate production by Faecalibacterium prausnitzii. Paramylon, an insoluble dietary fibre that accumulates in E. gracilis and is the main component of E. gracilis, did not stimulate Faecalibacterium growth in vitro. Daily ingestion of 2 g of E. gracilis for 30 days increased bowel movement frequency as well as stool volume in 28 human participants. Collectively, these findings indicate that E. gracilis components other than paramylon, stimulate the growth of Faecalibacterium to improve digestive health as well as promote defecation by increasing butyrate production.

    Springer Science and Business Media LLC, 2021年01月, Scientific Reports, 11 (1), 英語

    研究論文(学術雑誌)

  • Takahiro Yukawa, Takahiro Bamba, Gregory Guirimand, Mami Matsuda, Tomohisa Hasunuma, Akihiko Kondo

    Wiley, 2021年01月, Biotechnology and Bioengineering, 118 (1), 175 - 185, 英語

    研究論文(学術雑誌)

  • Musashi Takenaka, Takanobu Yoshida, Yoshimi Hori, Takahiro Bamba, Masao Mochizuki, Christopher J. Vavricka, Takanari Hattori, Yoshihiro Hayakawa, Tomohisa Hasunuma, Akihiko Kondo

    Elsevier BV, 2021年01月, Talanta, 222, 121625 - 121625, 英語

    研究論文(学術雑誌)

  • Kohei Katsurada, Masahiro Tominaga, Misato Kaishima, Hiroko Kato, Toshihide Matsuno, Chiaki Ogino, Akihiko Kondo, Jun Ishii, Katsumi Takayama

    Microbiology Research Foundation, 2021年, The Journal of General and Applied Microbiology, 67 (6), 265 - 268, 英語

    研究論文(学術雑誌)

  • Yoichiro Ito, Goro Terai, Misa Ishigami, Noriko Hashiba, Yasuyuki Nakamura, Takahiro Bamba, Ryota Kumokita, Tomohisa Hasunuma, Kiyoshi Asai, Jun Ishii, Akihiko Kondo

    Abstract In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a ‘terminator catalog’ by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3′-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

    Oxford University Press (OUP), 2020年12月16日, Nucleic Acids Research, 48 (22), 13000 - 13012, 英語

    研究論文(学術雑誌)

  • Johan Hunziker, Keiji Nishida, Akihiko Kondo, Sanae Kishimoto, Tohru Ariizumi, Hiroshi Ezura

    AbstractThe use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes, SlDDB1, SlDET1 and SlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T0 lines, we obtained 10 independent T0 lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.

    Springer Science and Business Media LLC, 2020年12月, Scientific Reports, 10 (1), 20471, 英語

    研究論文(学術雑誌)

  • Emmanuel Quayson, Jerome Amoah, Shinji Hama, Akihiko Kondo, Chiaki Ogino

    Elsevier BV, 2020年12月, Renewable and Sustainable Energy Reviews, 134, 110355 - 110355, 英語

    研究論文(学術雑誌)

  • Naofumi Yoshida, Tomoya Yamashita, Shigenobu Kishino, Hikaru Watanabe, Kengo Sasaki, Daisuke Sasaki, Tokiko Tabata, Yuta Sugiyama, Nahoko Kitamura, Yoshihiro Saito, Takuo Emoto, Tomohiro Hayashi, Tomoya Takahashi, Masakazu Shinohara, Ro Osawa, Akihiko Kondo, Takuji Yamada, Jun Ogawa, Ken-ichi Hirata

    Springer Science and Business Media LLC, 2020年12月, Scientific Reports, 10 (1), 13009, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuya Nishimura, Ryosuke Ezawa, Kenta Morita, Masao Nakayama, Jun Ishii, Ryohei Sasaki, Chiaki Ogino, Akihiko Kondo

    Radiosensitizing therapy for cancer treatment that enhances the effect of existing radiation therapy and enables noninvasive therapy has attracted attention. In this study, to achieve target cell-specific noninvasive cancer treatment using a Z(HER2)-bionanocapsule/liposome (BNC/LP), a carrier that binds to human epidermal growth factor receptor 2 (HER2), we evaluated the delivery of anticancer drugs and radiosensitizers and treatment effects in vitro and in vivo in mice. Target cell-specific cytotoxic activity and antitumor effects were confirmed following delivery of doxorubicin-encapsulated particles. In addition, cell damage due to radiosensitizing effects was confirmed in combination with X-ray irradiation following delivery of particles containing polyacrylic acid-modified titanium peroxide nanoparticles as a radiosensitizer. Furthermore, even when the particles were injected via the tail vein of mice, they accumulated in the tumor and exhibited an antitumor effect because of radiosensitization. Therefore, Z(HER2)-BNC/ LP is expected to be a carrier that releases small-molecule drugs into the target cell cytoplasm and delivers a radiosensitizer such as inorganic nanoparticles, enabling combination therapy with X-rays to the target tumor.

    American Chemical Society (ACS), 2020年11月16日, ACS Applied Bio Materials, 3 (11), 7743 - 7751, 英語

    研究論文(学術雑誌)

  • Emmanuel Quayson, Jerome Amoah, Nova Rachmadona, Kenta Morita, Lawrence Darkwah, Shinji Hama, Ayumi Yoshida, Akihiko Kondo, Chiaki Ogino

    Continuous expansion of agriculture for the production of biofuels may be considered a potential source of greenhouse gas (GHG) emissions due to the ever-increasing amount of waste and fossil fuel-dependent materials involved. Agricultural waste utilization through the circular bioeconomy concept offers a pathway to reduce GHG emissions. Palm oil production, for instance, produces palm kernel shells (PKS) and palm oil mill effluents (POME) as wastes in enormous amounts. PKS and POME account for >60% of solid and liquid waste generated from the mill. In this work, the feasibility of a circular palm bioeconomy is explored where waste PKS is directly converted to activated carbons (AC) in a cost-effective one-step technique (550 degrees C, 10 mL min(-1) N-2) that departs from the conventional two-step (carbonization and activation) technique. Characterization of the synthesized carbons, PKAC, using Fourier-transform infrared spectroscopy and scanning electron microscopy with energy-dispersive X-ray spectroscopy showed oxygen-rich morphological features that were 2-fold higher than in bituminous coal-derived AC. The <10 mu m pore diameters of PKAC were relevant in the immobilization of Aspergillus oryzae whole-cells expressing Fusarium heterosporum lipase (PKAC-FHL). For biodiesel production, the 81.6 IU mg(-1) specific activity of PKAC-FHL yielded >= 97.5 wt% fatty acid methyl ester (FAME) from POME. While offering the benefits of environmental remediation through effluent utilization, the produced FAME showed 48.8 Cetane Number and cold-flow properties that conform to ASTM and EN biodiesel requirements. This waste valorization approach, thus, proposes a green framework for the use of POME and PKS in immobilized lipase-catalyzed methanolysis.

    Elsevier BV, 2020年11月, Biomass and Bioenergy, 142, 105768 - 105768, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shunsuke Oba, Tadahiro Sunagawa, Reiko Tanihiro, Kyoko Awashima, Hiroshi Sugiyama, Tetsuji Odani, Yasunori Nakamura, Akihiko Kondo, Daisuke Sasaki, Kengo Sasaki

    Abstract Yeast mannan (YM) is an indigestible water-soluble polysaccharide of the yeast cell wall, with a notable prebiotic effect on the intestinal microbiota. We previously reported that YM increased Bacteroides thetaiotaomicron abundance in in vitro rat faeces fermentation, concluding that its effects on human colonic microbiota should be investigated. In this study, we show the effects of YM on human colonic microbiota and its metabolites using an in vitro human faeces fermentation system. Bacterial 16S rRNA gene sequence analysis showed that YM administration did not change the microbial diversity or composition. Quantitative real-time PCR analysis revealed that YM administration significantly increased the relative abundance of Bacteroides ovatus and B. thetaiotaomicron. Moreover, a positive correlation was observed between the relative ratio (with or without YM administration) of B. thetaiotaomicron and B. ovatus (r = 0.92), suggesting that these bacteria utilise YM in a coordinated manner. In addition, YM administration increased the production of acetate, propionate, and total short-chain fatty acids. These results demonstrate the potential of YM as a novel prebiotic that selectively increases B. thetaiotaomicron and B. ovatus and improves the intestinal environment. The findings also provide insights that might be useful for the development of novel functional foods.

    Springer Science and Business Media LLC, 2020年10月, Scientific Reports, 10 (1), 17351, 英語

    研究論文(学術雑誌)

  • Complete and draft genome sequences of amino acid-producing Corynebacterium glutamicum strains ATCC 21799 and ATCC 31831 and their genomic islands

    Hideo Kawaguchi, Takashi Sazuka, Akihiko Kondo

    2020年08月, Microbiology Resource Announcements, 9 (32), e00430-20, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Katsuki Murai, Daisuke Sasaki, Shunsuke Kobayashi, Akira Yamaguchi, Hiroto Uchikura, Tomokazu Shirai, Kengo Sasaki, Akihiko Kondo, Yota Tsuge

    Glucose is metabolized through central metabolic pathways such as glycolysis and the pentose phosphate pathway (PPP) to synthesize downstream metabolites including amino acids. However, how the split ratio of carbon flux between glycolysis and PPP specifically affects the formation of downstream metabolites remains largely unclear. Here, we conducted a comprehensive metabolomic analysis to investigate the effect of the split ratio between glycolysis and the PPP on the intracellular concentration of amino acids and their derivatives in Corynebacterium glutamicum. The split ratio was varied by exchanging the promoter of a gene encoding glucose 6-phosphate isomerase (PGI). The ratio was correlated with the pgi transcription level and the enzyme activity. Concentrations of threonine and lysine-derivative 1,5-diaminopentane increased with an increase of the split ratio into the PPP. In contrast, concentrations of alanine, leucine, and valine were increased with an increase of the split ratio into glycolysis. These results could provide a new engineering target for improving the production of the amino acids and the derivatives.

    American Chemical Society (ACS), 2020年07月17日, ACS synthetic biology, 9 (7), 1615 - 1622, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Silai Zhang, Satoshi Wakai, Naoya Sasakura, Hiroko Tsutsumi, Yoji Hata, Chiaki Ogino, Akihiko Kondo

    Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals. We successfully improved productivity by redirecting pyruvate metabolism in the aerobic filamentous fungus Aspergillus oryzae via the deletion of two genes that encode pyruvate decarboxylase and mitochondrial pyruvate carriers. Production of ethanol as a major byproduct was completely inhibited, and the limited translocation of pyruvate into the mitochondria shifted the metabolism from respiration for energy conversion to the effective production of lactate or 2,3-butandiole, even under aerobic conditions. Metabolomic and transcriptomic analyses showed an emphasis on glycolysis and a repressed TCA cycle. Although the dry mycelial weights of the deletion mutants were reduced compared with those of wild type, the titer and yields of the target products were drastically increased. In particular, the redirection of pyruvate metabolism shifted from anabolism for biomass production to catabolism for the production of target chemicals. Conclusively, our results indicate that the redirection of pyruvate metabolism is a useful strategy in the metabolic engineering of aerobic microorganisms.

    Elsevier BV, 2020年07月02日, Metabolic engineering, 61, 225 - 237, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Shoji Narutaki, Ineui Lee, Yasuhito Sugawara, Kahei Akada, Akihiko Kondo

    Elsevier BV, 2020年07月, Biologicals, 66, 1 - 8, 英語

    研究論文(学術雑誌)

  • Rina C. Sakata, Soh Ishiguro, Hideto Mori, Mamoru Tanaka, Kenji Tatsuno, Hiroki Ueda, Shogo Yamamoto, Motoaki Seki, Nanami Masuyama, Keiji Nishida, Hiroshi Nishimasu, Kazuharu Arakawa, Akihiko Kondo, Osamu Nureki, Masaru Tomita, Hiroyuki Aburatani, Nozomu Yachie

    We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.

    Springer Science and Business Media LLC, 2020年07月, Nature Biotechnology, 38 (7), 865 - 869, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Rina C Sakata, Soh Ishiguro, Hideto Mori, Mamoru Tanaka, Kenji Tatsuno, Hiroki Ueda, Shogo Yamamoto, Motoaki Seki, Nanami Masuyama, Keiji Nishida, Hiroshi Nishimasu, Kazuharu Arakawa, Akihiko Kondo, Osamu Nureki, Masaru Tomita, Hiroyuki Aburatani, Nozomu Yachie

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    2020年07月, Nature biotechnology, 38 (7), 901 - 901, 英語, 国際誌

    [査読有り]

  • Joana T. Cunha, Aloia Romaní, Kentaro Inokuma, Björn Johansson, Tomohisa Hasunuma, Akihiko Kondo, Lucília Domingues

    AbstractConsolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which is mainly composed of xylose and may comprise up to 40 % of the total biomass. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiae – the most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose. These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 gram per gram of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.

    Cold Spring Harbor Laboratory, 2020年07月, Biotechnology for Biofuels, 13 (1), 1 - 15, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ryosuke Fujiwara, Shogo Uchio, Mariko Nakano, Chisako Otomo, Yuuki Hirata, Takuya Matsumoto, Shuhei Noda, Tsutomu Tanaka, Akihiko Kondo

    Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value-added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate-producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl-CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP+ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA-MV, increased levels of intracellular acetyl-CoA up to sevenfold higher than the wild-type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying β-glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.

    Wiley, 2020年07月, Biotechnology and Bioengineering, 117 (7), 2153 - 2164, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Hiroko Iijima, Atsuko Watanabe, Haruna Sukigara, Tomokazu Shirai, Akihiko Kondo, Takashi Osanai

    Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L-1 ·day-1 , exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.

    Wiley, 2020年06月, Biotechnology and Bioengineering, 117 (6), 1649 - 1660, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Daisuke Sasaki, Asuka Hannya, Jun Tsubota, Akihiko Kondo

    Springer Science and Business Media LLC, 2020年05月, Scientific Reports, 10 (1), 8516, 英語

    [査読有り]

    研究論文(学術雑誌)

  • High enzymatic recovery and purification of xylooligosaccharides from empty fruit bunch via nanofiltration

    Hans Wijaya, Kengo Sasaki, Prihardi Kahar, Nanik Rahmani, Euis Hermiati, Yopi, Chiaki Ogino, Bambang Prasetya, Akihiko Kondo

    2020年05月, Processes, 8, 619, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Effective bifidogenic growth factors cyclo-Val-Leu and cyclo-Val-Ile produced by Bacillus subtilis C-3102 in the human colonic microbiota model

    Misaki Hatanaka, Hiroto Morita, Yumi Aoyagi, Kengo Sasaki, Daisuke Sasaki, Akihiko Kondo, Teppei Nakamura

    2020年05月, Scientific Reports, 10, 7591, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Daisuke Sasaki, Jun Inoue, Namiko Hoshi, Takayuki Maeda, Tyouichi Yamada, Akihiko Kondo

    The aim of this study was to clarify the effect of the spore-forming and lactic acid-producing probiotic strain, Bacillus coagulans SANK 70258, on human colonic microbiota of healthy subjects and ulcerative colitis patients. A model culture system was employed to construct the in vitro human colonic microbiota, to retain the bacterial species richness and simulate the patient's disordered composition, from the fecal inoculum. Bacterial 16S rRNA gene sequencing confirmed that administration of B. coagulans SANK 70258 (at an initial concentration of 4 × 107-total cells/mL) suppressed bacteria related to the family Enterobacteriaceae in the microbiota models for both healthy subjects (P = 0.016) and ulcerative colitis patients (P = 0.023). In addition, administration of B. coagulans SANK 70258 increased bacteria related to the family Lachnospiraceae (P = 0.031), thereby enhancing butyrate production (P = 0.031) in the microbiota models of healthy subjects. However, these changes were not observed in the microbiota models of ulcerative colitis patients, likely owing to the low abundance of Lachnospiraceae species. This study demonstrates the potential of B. coagulans SANK 70258 to exhibit antimicrobial activity against harmful organisms in patients with ulcerative colitis, while improving the intestinal microenvironment by increasing butyrogenesis in healthy persons. KEY POINTS: • B. coagulans SANK 70258 treatment reduced colonic Enterobacteriaceae species. • B. coagulans SANK 70258 treatment enhanced butyrogenesis in healthy individuals. • B. coagulans SANK 70258 treatment increased Lachnospiraceae in healthy persons. • B. coagulans SANK 70258 improves the colonic microenvironment in ulcerative colitis.

    2020年05月, Applied Microbiology and Biotechnology, 104 (9), 3859 - 3867, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Concentration of Lipase from Aspergillus oryzae Expressing Fusarium heterosporum by Nanofiltration to Enhance Transesterification

    Hans Wijaya, Kengo Sasaki, Prihardi Kahar, Emmanuel Quayson, Nova Rachmadona, Jerome Amoah, Shinji Hama, Chiaki Ogino, Akihiko Kondo

    2020年04月, Processes, 8 (4), 450, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Automatic Redirection of Carbon Flux between Glycolysis and Pentose Phosphate Pathway Using an Oxygen-Responsive Metabolic Switch in Corynebacterium glutamicum

    Shunsuke Kobayashi, Hideo Kawaguchi, Tomokazu Shirai, Kazuaki Ninomiya, Kenji Takahashi, Akihiko Kondo, Yota Tsuge

    2020年03月, ACS Synthetic Biology, 9, 814 - 826, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Biodiesel-mediated biodiesel production: A recombinant Fusarium heterosporum lipase-catalyzed transesterification of crude plant oils

    Emmanuel Quayson, Jerome Amoah, Nova Rachmadona, Shinji Hama, Ayumi Yoshida, Akihiko Kondo, Chiaki Ogino

    2020年03月, Fuel Processing Technology, 199, 106278, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ryota Hidese, Mami Matsuda, Takashi Osanai, Tomohisa Hasunuma, Akihiko Kondo

    d-Lactate is one of the most valuable compounds for manufacturing biobased polymers. Here, we have investigated the significance of endogenous malate dehydrogenase (decarboxylating) (malic enzyme, ME), which catalyzes the oxidative decarboxylation of malate to pyruvate, in d-lactate biosynthesis in the cyanobacterium Synechocystis sp. PCC6803. d-Lactate levels were increased by 2-fold in ME-overexpressing strains, while levels in ME-deficient strains were almost equivalent to those in the host strain. Dynamic metabolomics revealed that overexpression of ME led to increased turnover rates in malate and pyruvate metabolism; in contrast, deletion of ME resulted in increased pool sizes of glycolytic intermediates, probably due to sequential feedback inhibition, initially triggered by malate accumulation. Finally, both the loss of the acetate kinase gene and overexpression of endogenous d-lactate dehydrogenase, concurrent with ME overexpression, resulted in the highest production of d-lactate (26.6 g/L) with an initial cell concentration of 75 g-DCW/L after 72 h fermentation.

    2020年02月21日, ACS synthetic biology, 9 (2), 260 - 268, 英語, 国際誌

    [査読有り]

  • Exploration and Evaluation of Machine Learning-Based Models for Predicting Enzymatic Reactions

    Naoki Watanabe, Masahiro Murata, Teppei Ogawa, Christopher J Vavricka, Akihiko Kondo, Chiaki Ogino, Michihiro Araki

    2020年02月, Journal of Chemical Information and Modeling, 60, 1833 - 1843, 英語

    [査読有り]

  • Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains

    Kentaro Inokuma, Hiroki Kurono, Riaan den, Haan, Willem Heber van Zyl, Tomohisa Hasunuma, Akihiko Kondo

    2020年01月, Metabolic Engineering, 57, 110 - 117, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Metabolic engineering of Escherichia coli for shikimate pathway derivative production from glucose–xylose co-substrate

    Ryosuke Fujiwara, Shuhei Noda, Tsutomu Tanaka, Akihiko Kondo

    2020年01月, Nature Communications, 11, 279, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Dynamic metabolomics for engineering biology: Accelerating learning cycles for bioproduction

    Christopher J. Vavricka, Tomohisa Hasunuma, Akihiko Kondo

    2020年01月, Trends in Biotechnology, 38 (1), 68 - 82, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Matsumoto, Yuki Mori, Tsutomu Tanaka, Akihiko Kondo

    Bioamination methods using microorganisms have attracted much attention because of the increasing demand for environmentally friendly bioprocesses. n-Butylamine production from glucose in Escherichia coli was demonstrated in this study, which has never been reported because of the absence of n-butylamine-producing pathway in nature. We focused on a transaminase-mediated cascade for bioamination from an alcohol or aldehyde. The cascade can convert an alcohol or an aldehyde to the corresponding amine with l-alanine as an amine donor. Here, n-butyraldehyde, which is a metabolic intermediate in the n-butanol producing pathway, is a potential intermediate for producing n-butylamine using this cascade. Hence, the n-butanol-producing pathway and the transaminase-mediated cascade were combined into a synthetic metabolic pathway for producing n-butylamine from glucose. Firstly, we demonstrated the conversion of n-butanol to n-butylamine using a three enzyme-mediated cascade. n-Butanol was successfully converted to n-butylamine in 92% yield in the presence of l-alanine and ammonium chloride. Then, the n-butanol-producing pathway and transaminase-mediated cascade were introduced into E. coli. Using this system, n-butylamine was successfully produced from glucose as a carbon source at a concentration of 53.2 mg L-1 after 96 h cultivation using a ppc (phosphoenolpyruvate carboxylase)-deficient strain. To the best of our knowledge, this is the first report of the direct production of n-butylamine from glucose, and may provide a starting point for the development of microbial methods to produce other bioamines.

    2020年01月, Journal of bioscience and bioengineering, 129 (1), 99 - 103, 英語, 国内誌

    [査読有り]

    研究論文(学術雑誌)

  • Christopher J. Vavricka, Takanobu Yoshida, Yuki Kuriya, Shunsuke Takahashi, Teppei Ogawa, Fumie Ono, Kazuko Agari, Hiromasa Kiyota, Jianyong Li, Jun Ishii, Kenji Tsuge, Hiromichi Minami, Michihiro Araki, Tomohisa Hasunuma, Akihiko Kondo

    © 2019, The Author(s). Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.

    2019年12月01日, Nature Communications, 10 (1)

    [査読有り]

    研究論文(学術雑誌)

  • Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering

    Takahiro Bamba, Takahiro Yukawa, T, Gregory Guirimand, Kentaro Inokuma, Kengo Sasaki, Tomohisa Hasunuma, Akihiko Kondo

    2019年12月, Metabolic Engineering, 56, 17 - 27, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Short-term temporal metabolic behavior in halophilic cyanobacterium Synechococcus sp. strain PCC 7002 after salt shock

    Shimpei Aikawa, Atsumi Nishida, Tomohisa Hasunuma, Jo-Shu Chang, Akihiko Kondo

    2019年12月, Metabolites, 9 (12), 297, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Bifidogenic and butyrogenic effects of young barely leaf extract in an in vitro human colonic microbiota model

    Daisuke Sasaki, Kengo Sasaki, Yasushi Kadowaki, Yasuyuki Aotsuka, Akihiko Kondo

    2019年11月, AMB Express, 9, 182, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Fermentation of pigment-extracted microalgal residue using yeast cell-surface display: direct high-density ethanol production with competitive life cycle impacts

    Xiaochen Huang, Shunwen Bai, Zhuo Liu, Tomohisa Hasunuma, Akihiko Kondo, Shih-Hsin Ho

    2019年11月, Green Chemistry, 22 (1), 153 - 162, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Keisuke Morita, Fumio Matsuda, Koji Okamoto, Jun Ishii, Akihiko Kondo, Hiroshi Shimizu

    BACKGROUND: Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae. RESULTS: The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production. CONCLUSION: These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.

    2019年10月15日, Microbial cell factories, 18 (1), 177 - 177, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Butyryl-CoA:acetate CoA-transferase gene associated with the genus Roseburia is decreased in the gut microbiota of Japanese patients with ulcerative colitis

    Ryohei Shinohara, Kengo Sasaki, Jun Inoue, Namiko Hoshi, Itsuko Fukuda, Daisuke Sasaki, Akihiko Kondo, Ro Osawa

    2019年10月, Bioscience of Microbiota, Food and Health, 38 (4), 159 - 163, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Metabolic engineering to improve 1,5-diaminopentane production from cellobiose using β-glucosidase-secreting Corynebacterium glutamicum

    Rena Matsuura, Mayumi Kishida, Rie Konishi, Yuuki Hirata, Noriko Adachi, Shota Segawa, Kenta Imao, Tsutomu Tanaka, Akihiko Kondo

    2019年10月, Biotechnology Bioengineering, 116 (10), 2640 - 2651, 英語

    [査読有り]

    研究論文(学術雑誌)

  • A novel process for the mixotrophic production of lutein with Chlorella sorokiniana MB-1-M12 using aquaculture wastewater

    Jih-Heng Chen, Yuichi Kato, Mami Matsuda, Chun-Yen Chen, Dillirani Nagarajan, Tomohisa Hasunuma, Akihiko Kondo, Cheng-Di Dong, Duu-Jong Lee, Jo-Shu Chang

    2019年10月, Bioresource Technology, 290, 121786, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Single-Stage Astaxanthin Production Enhances the Nonmevalonate Pathway and Photosynthetic Central Metabolism in Synechococcus sp. PCC 7002

    Tomohisa Hasunuma, Ayako Takaki, Mami Matsuda, Yuichi Kato, Christopher J. Vavricka, Akihiko Kondo

    2019年10月, ACS Synthetic Biology, 8, 2701 - 2709, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Complete genome sequence of Rhodobacter sphaeroides HJ strain, a purple nonsulfur bacterium with high hydrogen production ability from acetate, Microbiology Resource Announcements

    Jyumpei Kobayashi, Akihiko Kondo

    2019年09月, Microbiology Resource Abbiybcements, 8 (37), e00652-19, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Co-fermentation of xylose and glucose from ionic liquid pretreated sugar cane bagasse for bioethanol production using engineered xylose assimilating yeast

    Jerome Amoah, Kazuma Ogura, Quentin Schmetz, Akihiko Kondo, Chiaki Ogino

    2019年09月, Biomass & Bioenergy, 128, 英語

    [査読有り]

    研究論文(学術雑誌)

  • High cell density cultivation of Lipomyces starkeyi for achieving highly efficient lipid production from sugar under low C/N ratio

    Rezky Lastinov Amza, Prihardi Kahar, Ario Betha Juanssilfero, Nao Miyamoto, Hiromi Otsuka, Chie Kihira, Chiaki Ogino, Akihiko Kondo

    2019年09月, Biochemical Engineering Journal, 149 (15), 107236, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Combined cell surface display of β-D-glucosidase (BGL), maltose transporter (MAL11) and overexpression of cytosolic xylose reductase (XR) in Saccharomyces cerevisiae enhance cellobiose/xylose coutilization for xylitol bio-production from lignocellulosic b

    Gregory G, Y. Guirimand, Takahiro Bamba, Mami Matsuda, Kentaro Inokuma, Kenta Morita, Yuki Kitada, Yuma Kobayashi, Takahiro Yukawa, Kengo Sasaki, Chiaki Ogino, Tomohisa Hasunuma, Akihiko Kondo

    2019年09月, Biotechnology Journal, 14 (9), 1800704, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Naofumi Yoshida, Kengo Sasaki, Daisuke Sasaki, Tomoya Yamashita, Hajime Fukuda, Tomohiro Hayashi, Tokiko Tabata, Ro Osawa, Ken-Ichi Hirata, Akihiko Kondo

    AIM: Bacteroides vulgatus and B. dorei have a protective effect against atherosclerosis, suggesting that expansion of these species in the gut microbiota could help patients with coronary artery disease (CAD). This study aimed to investigate the effect of resistant starch (RS) on the gut microbiota and its metabolites in fecal sample cultures from patients with CAD and individuals without CAD, using a single-batch fermentation system. METHODS: Fecal samples from 11 patients with CAD and 10 individuals without CAD were fermented for 30 h with or without RS in the Kobe University Human Intestinal Microbiota Model (KUHIMM). Gut microbiota and the abundance of B. vulgatus and B. dorei were analyzed using 16S ribosomal ribonucleic acid (rRNA) gene sequencing and the quantitative polymerase chain reaction. Short-chain fatty acids were analyzed using high-performance liquid chromatography. RESULTS: Gut microbial analysis showed significantly lower levels of B. vulgatus and B. dorei in the original fecal samples from patients with CAD, which was simulated after 30 h of fermentation in the KUHIMM. Although RS significantly increased the absolute numbers of B. vulgatus and B. dorei, and butyrate levels in CAD fecal sample cultures, the numbers varied among each patient. CONCLUSIONS: The effect of RS on gut microbiota and its metabolites in the KUHIMM varied between CAD and non-CAD fecal sample cultures. The KUHIMM may be useful for preclinical evaluations of the effects of RS on the gut microbiota and its metabolites.

    2019年08月01日, Journal of atherosclerosis and thrombosis, 26 (8), 705 - 719, 英語, 国内誌

    [査読有り]

    研究論文(学術雑誌)

  • Day/night separation of oxygenic energy metabolism and nuclear DNA replication in the unicellular red alga Cyanidioschyzon merolae

    Shin-ya Miyagishima, Atsuko Era, Tomohisa Hasunuma, Mami Matsuda, Shunsuke Hirooka, Nobuko Sumiya, Akihiko Kondo, Takayuki Fujiwara

    2019年07月, mBio, 10 (4), e00833-19, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Reconstruction of metabolic pathway for isobutanol production in Escherichia coli

    Shuhei Noda, Yutaro Mori, Sachiko Oyama, Akihiko Kondo, Michihiro Araki, Tomokazu Shirai

    2019年07月, Microbial Cell Factories, 18, 124, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Lipid production by Lipomyces starkeyi using sap squeezed from felled old oil palm trunks

    Ario Betha Juanssilfero, Prihardi Kahar, Lastinov Amza Rezky, Yopi, Kumar Sudesh, Chiaki Ogino, Bambang Prasetya, Akihiko Kondo

    2019年06月, Journal of Bioscience and Bioengineering, 127 (6), 726 - 731, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Versatility of a dilute acid/butanol pretreatment investigated on various lignocellulosic biomasses to produce lignin, monosaccharides and cellulose in distinct phases

    Quentin Schmetz, Hiroshi Teramura, Kenta Morita, Tomoko Oshima, Aurore Richel, Chiaki Ogino, Akihiko Kondo

    2019年06月, ACS Sustainable Chemistry & Engineering, 7 (13), 11069 - 11079, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Bioenergy and Biorefinery: feedstock, biotechnological conversion and products

    Jerome Amoah, Prihardi Kahar, Chiaki Ogino, Akihiko Kondo

    2019年06月, Biotechnology Journal, 14 (6), 1800494, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Enhanced phenyllactic acid production in Escherichia coli via oxygen limitation and shikimate pathway gene expression

    Hideo Kawaguchi, Hiroki Miyagawa, Sachiko Nakamura‐Tsuruta, Naoki Takaya, Chiaki Ogino, Akihiko Kondo

    2019年06月, Biotechnology Journal, 14 (6), 1800478, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Christopher J Vavricka, Takanobu Yoshida, Yuki Kuriya, Shunsuke Takahashi, Teppei Ogawa, Fumie Ono, Kazuko Agari, Hiromasa Kiyota, Jianyong Li, Jun Ishii, Kenji Tsuge, Hiromichi Minami, Michihiro Araki, Tomohisa Hasunuma, Akihiko Kondo

    In the original version of this Article, the abbreviation of 3,4-dihydroxyphenylacetaldehyde synthase presented in the first paragraph of the Discussion section was given incorrectly as DYPAA. The correct abbreviation for this enzyme is DHPAAS. This error has been corrected in both the PDF and HTML versions of the Article.

    2019年05月22日, Nature communications, 10 (1), 2336 - 2336, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Cell-surface display technology and metabolic engineering of Saccharomyces cerevisiae for enhancing xylitol production from woody biomass

    Gregory Guirimand, Kentaro Inokuma, Takahiro Bamba, Mami Matsuda, Kenta Morita, Kengo Sasaki, Chiaki Ogino, Jean-Guy Berrin, Tomohisa Hasunuma, Akihiko Kondo

    2019年05月, Green Chemistry, 21, 1795 - 1808, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Jun Inoue, Daisuke Sasaki, Namiko Hoshi, Tomokazu Shirai, Itsuko Fukuda, Takeshi Azuma, Akihiko Kondo, Ro Osawa

    2019年05月, Biotechnology Journal, 14, 1800555, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Building a global alliance of biofoundries

    HILLSON Nathan, CADDICK Mark, CAI Yizhi, CARRASCO Jose A, CHANG Matthew Wook, CURACH Natalie C, BELL David J, Le FEUVRE Rosalind, FRIEDMAN Douglas C, FU Xiongfei, GOLD Nicholas D, HERRGARD Markus J, HOLOWKO Maciej B, JOHNSON James R, JOHNSON Richard A, KEASLING Jay D, KITNEY Richard I, KONDO Akihiko, LIU Chenli, MARTIN Vincent JJ, MENOLASCINA Filippo, OGINO Chiaki, PATRON Nicola J, PAVAN Marilene, POH Chueh Loo, PRETORIUS Lsak S, ROSSER Susan J, SCRUTTON Nigel S, STORCH Marko, TEKOTTE Hille, TRAVNIK Evelyn, VIV\CKERS Claudia E, YEW Wen Shan, YUAN Yingjin, ZHAO Huimin, FREEMONT Paul S

    2019年05月, Nature Communications, 10, 2040, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Enhancing lutein production with mixotrophic cultivation of Chlorella sorokiniana MB-1-M12 using different bioprocess operation strategies

    Jih-Heng Chen, Chun-Yen Chen, Tomohisa Hasunuma, Akihiko Kondo, Chien-Hsiang Chang, I-Son Ng, Jo-Shu Chang

    2019年04月, Bioresource Technology, 278, 17 - 25, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shimatani, Zenpei, Ariizumi, Tohru, Fujikura, Ushio, Kondo, Akihiko, Ezura, Hiroshi, Nishida, Keiji

    The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.

    2019年04月, Methods in molecular biology (Clifton, N.J.), 1917, 297 - 307, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Less biomass and intracellular glutamate in anodic biofilms lead to efficient electricity generation by microbial fuel cells

    SASAKI Daisuke, SASAKI Kengo, TSUGE Yota, KONDO Akihiko

    2019年04月, Biotechnology for Biofuels, 12, 72, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation

    Koichi Tamano, Robert Sidney Cox, Kenji Tsuge, Ai Miura, Ayano Itoh, Jun Ishii, Tomohiro Tamura, Akihiko Kondo, Masayuki Machida

    2019年04月, Journal of Bioscience and Bioengineering, 127 (4), 451 - 457, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Modified expression of multi-cellulases in a filamentous fungus Aspergillus oryzae

    Satoshi Wakai, Nanami Nakashima, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, Akihiko Kondo

    2019年03月, Bioresource Technology, 276, 146 - 153, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Valorization of activated carbon as a reusable matrix for the immobilization of Aspergillus oryzae whole-cells expressing Fusarium heterosporum lipase toward biodiesel synthesis

    Emmanuel Quayson, Jerome Amoah, Shinji Hama, Ayumi Yoshida, Kenta Morita, Akihiko Kondo, Chiaki Ogino

    2019年03月, ACS Sustainable Chemistry & Engineering, 7 (5), 5010 - 5017, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Increased flux in acetyl-CoA synthetic pathway and TCA cycle of Kluyveromyces marxianus under respiratory conditions

    SAKIHAMA Yuri, HIDESE Ryota, HASUNUMA Tomohisa, KONDO Akihiko

    2019年03月, Scientific Reports, 9 (1), 5319, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Efficient and supplementary enzyme cocktail from Actinobacteria and plant biomass induction

    竹中 武藏, Lee Jae Min, Prihardi Kahar, 荻野 千秋, 近藤 昭彦

    2019年03月, Biotechnology Journal, 1700744, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yoshifumi Ueno, Shimpei Aikawa, Akihiko Kondo, Seiji Akimoto

    Oxygenic photosynthetic organisms perform photosynthesis efficiently by distributing captured light energy to photosystems (PSs) at an appropriate balance. Maintaining photosynthetic efficiency under changing light conditions requires modification of light-harvesting and energy-transfer processes. In the current study, we examined how green algae regulate their light-harvesting functions in response to different light qualities. We measured low-temperature time-resolved fluorescence spectra of unicellular green algae Chlamydomonas reinhardtii and Chlorella variabilis cells grown under different light qualities. By observing the delayed fluorescence spectra, we demonstrated that both types of green algae primarily modified the associations between light-harvesting chlorophyll protein complexes (LHCs) and PSs (PSII and PSI). Under blue light, Chlamydomonas transferred more energy from LHC to chlorophyll (Chl) located far from the PSII reaction center, while energy was transferred from LHC to PSI via different energy-transfer pathways in Chlorella. Under green light, both green algae exhibited enhanced energy transfer from LHCs to both PSs. Red light induced fluorescence quenching within PSs in Chlamydomonas and LHCs in Chlorella. In Chlorella, energy transfer from PSII to PSI appears to play an important role in balancing excitation between PSII and PSI.

    2019年03月, Photosynthesis research, 139 (1-3), 145 - 154, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Sustainable production of glutathione from lignocellulose-derived sugars using engineered Saccharomyces cerevisiae

    KOBAYASHI Jyumpei, SASAKI Daisuke, BAMBA Takahiro, HASUNUMA Tomohisa, KONDO Akihiko

    2019年02月, Applied Microbiology and Biotechnology, 103 (3), 1243 - 1254, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Light/dark cycling causes delayed lipid accumulation and increased photoperiod-based biomass yield by altering metabolic flux in oleaginous Chlamydomonas sp.

    Yuichi KATO, Yusuke FUJIHARA, Christopher J. VAVRICKA, Jo-Shu CHANG, Tomohisa HASUMUMA, Akihiko KONDO

    2019年02月, Biotechnology for Biofuels, 12, 39, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Disruption of poly (3-hydroxyalkanoate) depolymerase gene and overexpression of three poly (3-hydroxybutyrate) biosynthetic genes improve poly (3-hydroxybutyrate) production from nitrogen rich medium by Rhodobacter sphaeroides.

    KOBAYASHI Jyumpei, KONDO Akihiko

    2019年02月, Microbial Cell Factories, 18, 40, 英語

    [査読有り]

    研究論文(学術雑誌)

  • 5-Hydroxymethylfurfural production from salt-induced photoautotrophically cultivated Chlorella sorokiniana

    AMOAH Jerome, HASUNUMA Tomohisa, OGINO Chiaki, KONDO Akihiko

    2019年02月, Biochemical Engineering Journal, 142, 117 - 123, 英語

    [査読有り]

    研究論文(学術雑誌)

  • GH-10 and GH-11 Endo-1,4-β-xylanase enzymes from Kitasatospora sp. produce xylose and xylooligosaccharides from sugarcane bagasse with no xylose inhibition

    Nanik Rahmani, Prihardi Kahar, Puspita Lisdiyanti, Jaemin Lee, Yopi, Bambang Prasetya, Chiaki Ogino, Akihiko Kondo

    2019年01月, Bioresource Technology, 272, 315 - 325, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yihan Zhang, Keizo Nakagawa, Masafumi Shibuya, Kengo Sasaki, Tomoki Takahashi, Takuji Shintani, Tomohisa Yoshioka, Eiji Kamio, Akihiko Kondo, Hideto Matsuyama

    The hydrothermal pretreatment of rice straw generates a liquid fraction that contains low concentrations of sugars, as well as fermentation inhibitors which are not favourable for efficient bioethanol production. For the development of a forward osmosis (FO) process to concentrate sugars and remove fermentation inhibitors, we fabricated polyamide-polysulfone (PA-PSf) membranes treated with NaClO solution. The NaClO treatment improved the water permeability (A value) and the permselectivity of fermentation inhibitors against sugars. Maximum separation of xylose from furfural and acetic acid was achieved when the treated membranes had A values of 5.5-7 L m(-2) h(-1) bar(-1). Using an optimized membrane with A value of approximately 6 L m(-2) h(-1) bar(-1), a liquid fraction from hydrothermally pretreated rice straw was concentrated in a one-step FO process to increase sugar concentrations and decrease fermentation inhibitor concentrations. The ethanol concentration and yield (ethanol/total sugar) by xylose-utilizing Saccharomyces cerevisiae reached 18.0 g/L and 0.40, respectively, which were much higher than those obtained with commercial FO membranes. These results clearly indicate the possibility of introducing a simple and low-cost FO process into bioethanol production from lignocellulosic biomass by incorporating NaClO treatment to FO membrane.

    ELSEVIER SCIENCE BV, 2018年11月, JOURNAL OF MEMBRANE SCIENCE, 566, 15 - 24, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Enhancing 3-hydroxypropionic acid production in combination with sugar supply engineering by cell surface-display and metabolic engineering of Schizosaccharomyces pombe

    TAKAYAMA Seiya, OZAKI Aiko, KONISHI Rie, OTOMO Chisako, KISHIDA Mayumi, HIRATA Yuuki, MATSUMOTO Takuya, TANAKA Tsutomu, KONDO Akihiko

    2018年11月, Microbial Cell Factories, 17, 176, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Deletion of DNA ligase IV homolog confers higher gene targeting efficiency on homologous recombination in Komagataella phaffii

    Yoichiro Ito, Toru Watanabe, Shimpei Aikawa, Teruyuki Nishi, Tozo Nishiyama, Yasuyuki Nakamura, Tomohisa Hasunuma, Yuji Okubo, Jun Ishii, Akihiko Kondo

    2018年11月, FEMS Yeast Research, 18 (7), foy074, 英語

    [査読有り]

    研究論文(学術雑誌)

  • De novo design of biosynthetic pathways for bacterial production of bulk chemicals and biofuels

    Kenji Okano, Kohsuke Honda, Hironori Taniguchi, Akihiko Kondo

    2018年10月, FEMS Micobiology Letters, 365 (20), fny215, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Repeated ethanol fermentation from membrane-concentrated sweet sorghum juice using the flocculating yeast Saccharomyces cerevisiae F118 strain

    WIJAYA Hans, SASAKI Kengo, KAHAR Prihardi, YOPI, KAWAGUCHI Hideo, SAZUKA Takashi, OGINO Chiaki, PRASETYA Bambang, KONDO Akihiko

    2018年10月, Bioresource Technology, 265, 542 - 547, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Muconic acid production using gene-level fusion proteins in Escherichia coli

    FUJIWARA Ryosuke, NODA Shuhei, TANAKA Tsutomu, KONDO Akihiko

    2018年10月, ACS Synthetic Biology, 7 (11), 2698 - 2705, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Inheritance of co-edited genes by CRISPR-based targeted nucleotide substitutions in rice

    SHIMATANI Zenpei, FUJIKURA Ushio, ISHII Hisaki, MATSUI Yusuke, SUZUKI Minoru, UEKE Yuki, TAOKA Ken-ichiro, TERADA Rie, NISHIDA Keiji, KONDO Akihiko

    2018年10月, Plant Physiology and Biochemistry, 131, 78 - 83, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Development of cell recycle technology incorporating nutrient supplementation for lignocellulosic ethanol fermentation using industrial yeast Saccharomyces cerevisiae

    Shinji Hama, Maki Kihara, Hideo Noda, Akihiko Kondo

    2018年09月, Biochemical Engineering Journal, 137, 23 - 29, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Widespread effect of N-acetyl-D-glucosamine assimilation on the metabolisms of amino acids, purines, and pyrimidines in Scheffersomyces stipitis

    INOKUMA Kentaro, MATSUDA Mami, SASAKI Daisuke, HASUNUMA Tomohisa, KONDO Akihiko

    2018年09月, Microbial Cell Factories, 17, 153, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Targeted nucleotide editing technologies for microbial metabolic engineering

    ARAZOE Takayuki, KONDO Akihiko, NISHIDA Keiji

    2018年09月, Biotechnology Journal, 13 (9), 1700596, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Selection of oleaginous yeasts capable of high lipid accumulation during challenges from inhibitory chemical compounds

    Juanssilfero BA, Prihardi Kahar, Rezky Lastinov Amza, 宮本 捺央, 大塚 裕美, Hana Matsumoto, 紀平 知枝, Ahmad Thontowi, Yopi, 荻野 千秋, Prasetya B, 近藤 昭彦

    2018年09月, Biochemical Engineering Journal, 137, 182 - 191, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Development of a strictly regulated xylose-induced expression system in Streptomyces,

    Noguchi Y, Kashiwagi N, Uzura A, 荻野 千秋, 近藤 昭彦, Ikeda H, Sota M

    2018年09月, Microbial Cell Factories, 17, 151, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Herbicide tolerance-assisted multiplex targeted nucleotide substitution in rice

    SHIMATANI Zenpei, FUJIKURA Ushio, ISHII Hisaki, TERADA Rie, NISHIDA Keiji, KONDO Akihiko

    2018年08月, Data in Brief, 20, 1325 - 1331, 英語

    [査読有り]

    研究論文(学術雑誌)

  • 分裂酵母を用いた有機酸生産技術の開発

    田中 勉, 近藤 昭彦

    化学工業社, 2018年07月, ケミカルエンジニヤリング, 特集=革新技術が拓く新しい可能性, 63 (7), 7 - 10, 日本語

    研究論文(学術雑誌)

  • Temperature enhanced succinate production concurrent with increased central metabolism turnover in the cyanobacterium Synechocystis sp. PCC 6803

    HASUNUMA Tomohisa, MATSUDA Mami, KATO Yuichi, VAVRICKA JR John Christopher, KONDO Akihiko

    2018年07月, Metabolic Engineering, 48, 109 - 120, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Metabolic engineering of Corynebacterium glutamicum for production of sunscreen shinorine

    Yota Tsuge, Hideo Kawaguchi, Shogo Yamamoto, Yoshiko Nishigami, Masahiro Sota, Chiaki Ogino, Akihiko Kondo

    2018年07月, Bioscience, Biotechnology, and Biochemistry, 82 (7), 1252 - 1259, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kenta Morita, Takahiro Suzuki, Yuya Nishimura, Kazuhisa Matsumoto, Chiya numako, Kazuyoshi Sato, Masao Nakayama, Ryohei Sasaki, Chiaki Ogino, Akihiko Kondo

    Polyacrylic acid (PAA)-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers. PAA-TiOx NPs were synthesized from commercial TiO2 nanoparticles that were modified with PAA and functionalized by H2O2 treatment. To realize practical clinical uses for PAA-TiOx NPs, their tissue distribution and acute toxicity were evaluated using healthy mice and mice bearing tumors derived from xenografted MIAPaCa-2 human pancreatic cancer cells. Healthy mice were injected with PAA-TiOx NPs at 25 mg/kg body weight via the tail vein, and tumor-bearing mice were injected either into the tumor locally or via the tail vein. The concentration of PAA-TiOx NPs in major organs was determined over time using inductively coupled-plasma atomic emission spectrometry. After 1 h, 12% of the PAA-TiOx NP dose had accumulated in the tumor, and 2.8% of the dose remained after 1 week. Such high accumulation could be associated with enhanced permeability and retention effects of the tumor, as PAA-TiOx NPs are composed of inorganic particles and polymers, without tumor-targeting molecules. The liver accumulated the largest proportion of the injected nanoparticles, up to 42% in tumor-bearing mice. Blood biochemical parameters were also investigated after intravenous injection of PAA-TiOx NPs in healthy mice. PAA-TiOx NPs invoked a slight change in various liver-related biochemical parameters, but no liver injury was observed over the practical dose range. In the future, PAA-TiOx NPs should be modified to prevent accumulation in the liver and minimize risk to patients.

    2018年07月, Journal of Bioscience and Bioengineering, 126 (1), 119 - 125, 英語, 国内誌

    [査読有り]

    研究論文(学術雑誌)

  • Genotypic effects on sugar and by-products of liquid hydrolysates and on saccharification of acid-insoluble residues of from wheat straw

    Ohno R, Teramura H, 荻野 千秋, 近藤 昭彦, Takumi S

    2018年07月, Genes & Genetic Systems, 93, 1 - 7, 英語

    [査読有り]

    研究論文(学術雑誌)

  • A stable, autonomously replicating plasmid vector containing Pichia pastoris centromeric DNA

    Yasuyuki Nakamura, Teruyuki Nishi, Risa Noguchi, Yoichiro Ito, Toru Watanabe, Tozo Nishiyama, Sinpei Aikawa, Tomohisa Hasunuma, Jun Ishii, Yuji Okubo, Akihiko Kondo

    2018年07月, Applied and Environmental Microbiology, 84 (15), e02882 - 17, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Engineering human epidermal growth receptor 2-targeting hepatitis B virus core nanoparticles for siRNA delivery in vitro and in vivo

    SUFFIAN Izzat F. M, WANG Julie T.-W, FARUQU Farid N, BENITEZ Julio, NISHIMURA Yuya, OGINO Chiaki, KONDO Akihiko, AL-JAMAL Khuloud T

    2018年06月, ACS Applied Nano Materials, 1 (7), 3269 - 3282, 英語

    研究論文(学術雑誌)

  • Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi

    Juanssilfero AB, Prihardi Kahar, Rezky Lastinov Amza, 宮本 捺央, 大塚 裕美, Hana Matsumoto, 紀平 知枝, Ahmad Thontowi, Yopi, 荻野 千秋, Prasetya B, 近藤 昭彦

    2018年06月, Journal of Bioscience and Bioengineering, 126 (6), 695 - 702, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Changes in the microbial consortium during dark hydrogen fermentation in a bioelectrochemical system increases methane production during a two-stage process

    SASAKI Kengo, SASAKI Daisuke, TSUGE Yota, MORITA Masahiko, KONDO Akihiko

    2018年06月, Biotechnology for Biofuels, 11, 173, 英語

    [査読有り]

    研究論文(学術雑誌)

  • A pyruvate carbon flux tugging strategy for increasing 2,3-butanediol production and reducing ethanol subgeneration in the yeast Saccharomyces cerevisiae

    Jun Ishii, Keisuke Morita, Kengo Ida, Hiroko Kato, Shohei Kinoshita, Shoko Hataya, Hiroshi Shimizu, Akihiko Kondo, Fumio Matsuda

    BioMed Central, 2018年06月, Biotechnology for Biofuels, (11), 180, 英語

    [査読有り]

    研究論文(学術雑誌)

  • A procedure for precise determination of glutathione produced by Saccharomyces cerevisiae

    KOBAYASHI Jyumpei, SASAKI Daisuke, KONDO Akihiko

    2018年06月, Bio-protocol, 8 (12), e2887, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Efficient 3-hydroxybutyrate production by quiescent Escherichia coli microbial cell factories is facilitated by indole-induced proteomic and metabolomic changes

    Nicholas M. Thomson, Tomokazu Shirai, Marco Chiapello, Akihiko Kondo, Krishna J. Mukherjee, Easan Sivaniah, Keiji Numata, David K. Summers

    2018年05月, Biotechnology Journal, 13 (5), 1700571, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Electrical-biological hybrid system for CO2 reduction

    Yohei Tashiro, Shinichi Hirano, Morgan M. Matson, Shota Atsumi, Akihiko Kondo

    2018年05月, Metabolic Engineering, 47, 211 - 218, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of D-glucose and L-arabinose

    KAWAGUCHI Hideo, YOSHIHARA K, HARA KY, HASUNUMA Tomohisa, OGINO Chiaki, KONDO Akihiko

    2018年05月, Microbial Cell Factories, 17, 76, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Metabolic engineering of Lactobacillus plantarum for direct L-lactic acid production from raw corn starch

    OKANO Kenji, UEMATSU Gentaro, HAMA Shinji, TANAKA Tsutomu, NODA Hideo, KONDO Akihiko, HONDA Kohsuke

    2018年05月, Biotechnology Journal, 13 (5), 1700517, 英語

    [査読有り]

    研究論文(学術雑誌)

  • How lipase technology contributes to evolution of biodiesel production using multiple feedstocks

    Shinji Hama, Hideo Noda, Akihiko Kondo

    2018年04月, Current Opinion in Biotechnology, 50, 57 - 64, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Modifying expression modes of human neurotensin receptor type 1 alters sensing capabilities for agonists in yeast signaling biosensor

    Hiroki Hashi, Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo

    WILEY, 2018年04月, Biotechnology Journal, 13 (4), e1700522, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Genetic and physiological basis for antibody production by Kluyveromyces marxianus

    NAMBU-NISHIDA Yumiko, NISHIDA Keiji, HASUNUMA Tomohisa, KONDO Akihiko

    2018年04月, AMB Express, 8, 56, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Daisuke Sasaki, Kazuhide Kamiya, Shuji Nakanishi, Akihiko Kondo, Souichiro Kato

    Microbial electrochemical systems (MESs) are expected to be put into practical use as an environmental technology that can support a future environmentally friendly society. However, conventional MESs present a challenge of inevitably increasing initial investment, mainly due to requirements for a large numbers of electrode assemblies. In this review, we introduce electrochemical biotechnologies that are under development and can minimize the required electrode assemblies. The novel biotechnologies, called electro-fermentation and indirect electro-stimulation, can drive specific microbial metabolism by electrochemically controlling intercellular and extracellular redox states, respectively. Other technologies, namely electric syntrophy and microbial photo-electrosynthesis, obviate the need for electrode assemblies, instead stimulating targeted reactions by using conductive particles to create new metabolic electron flows.

    2018年04月, Current opinion in biotechnology, 50, 182 - 188, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Deaminase-mediated multiplex genome editing in Escherichia coli

    BANNO Satomi, NISHIDA Keiji, ARAZOE Takayuki, MITSUNOBU Hitoshi, KONDO Akihiko

    2018年04月, Nature Microbiology, 3, 423 - 429, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae

    Yuya Nishimura, Terumi Matsui, Jun Ishii, Akihiko Kondo

    BioMed Central, 2018年03月, Microbial Cell Factories, 17 (1), 38, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Enhanced cell-surface display of a heterologous protein using SED1 anchoring system in SED1-disrupted Saccharomyces cerevisiae strain

    Takahiro Bamba, Kentaro Inokuma, Tomohisa Hasunuma, Akihiko Kondo

    2018年03月, Journal of Bioscience and Bioengineering, 125 (3), 306 - 310, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Effective usage of sorghum bagasse: optimization of organosolv pretreatment using 25% 1-butanol and subsequent nanofiltration membrane separation

    Hiroshi Teramura, Kengo Sasaki, Tomoko Oshima, Hideo Kawaguchi, Chiaki Ogino, Takashi Sazuka, Akihiko Kondo

    2018年03月, Bioresource Technology, 252, 157 - 164, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Optimization of cellulolytic enzyme components through engineering Trichoderma reesei and on-site fermentation using the soluble inducer for cellulosic ethanol production from corn stover

    Yong-Hao Li, Xiao-Yue Zhang, Fei Zhang, Liang-Cai Peng, Da-Bing Zhang, Akihiko Kondo, Feng-Wu Bai, Xin-Qing Zhao

    2018年02月, Biotechnology for Biofuels, 11, 49, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars

    Nanik Rahmani, Prihardi Kahar, Puspita Lisdiyanti, Euis Heermiati, Jaemin. Lee, Yopi, Bambang Prasetya, Chiaki Ogino, Akihiko Kondo

    2018年02月, Bioscience, Biotechnology, and Biochemistry, 82 (5), 904 - 915, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Direct and highly productive conversion of cyanobacteria Arthrospira platensis to ethanol with CaCl2 addition

    AIKAWA S, INOKUMA Kentaro, WAKAI Satoshi, SASAKI Kengo, OGINO Chiaki, J. S. Chang, HASUNUMA Tomohisa, KONDO Akihiko

    2018年02月, Biotechnology for Biofuels, 11, 50, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Matsumoto, Yuki Isogawa, Tsutomu Tanaka, Akihiko Kondo

    A tetrameric streptavidin (SA)-appended LPETG tag was site-specifically linked to azido-containing tri-glycine via sortase A catalysis and the resulting azido-modified SA (SA-N3) was retained in the biotin-binding pocket. SA-N3 was polymerized with dibenzylcyclooctyne-modified branched poly(ethyleneglycol) (DBCO-PEG) using azido-modified branched PEG (N3-PEG) as a spacer via copper-free click chemistry. The resulting SA-based hydrogel exhibited gel-like mechanical properties and could immobilize biotin-modified molecules through biotin-SA affinity. Glucose dehydrogenase (GDH) was immobilized in the SA-based hydrogel, and the hydrogel was then coated on a glassy carbon electrode (GCE) and used for the biocatalytic oxidation of glucose. The designed GCE exhibited better performance and stability compared with GDH chemically adsorbed onto a GCE. In addition, the designed GCE anode and a Pt-carbon cathode were assembled into a glucose/O-2 fuel cell that provided a maximum power density and open circuit voltage of 11.8 +/- 0.56 W cm(-2) and 0.17 V, respectively.

    ELSEVIER ADVANCED TECHNOLOGY, 2018年01月, BIOSENSORS & BIOELECTRONICS, 99, 56 - 61, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation

    Yumiko Nambu-Nishida, Yuri Sakihama, Jun Ishii, Tomohisa Hasunuma, Akihiko Kondo

    Elsevier, 2018年01月, Journal of Bioscience and Bioengineering, 125 (1), 76 - 86, 英語

    [査読有り]

    研究論文(学術雑誌)

  • 川口 秀夫, 勝山 陽平, ト タンヤオ, 鶴田 祥子, 南 博道, 荻野 千秋, 大西 康夫, 近藤 昭彦

    Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by expressing the genes encoding a tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344 and 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

    一般社団法人 日本エネルギー学会, 2018年, バイオマス科学会議発表論文集, 13 (0), 135 - 136, 日本語

    [査読有り]

  • Matsumoto T, Tanaka T, Kondo A

    Metabolic engineering has been an important approach for microbial bio-production. To produce bio-chemicals with engineered microorganisms, metabolic pathways have been edited using several common strategies, including gene disruption, gene overexpression, and gene attenuation. Here, we demonstrated metabolic channeling based on enzymatic metabolic enzyme ligation as a noteworthy approach for enhancing a desired metabolic flux. To achieve metabolic channeling , the metabolic enzymes should be in close proximity in cells. In the literature, several methodologies have been recently applied to achieve metabolic channeling . Meanwhile, we have proposed a strategy for possessing metabolic enzymes in close proximity, by utilizing sortase A as a stapler to tether such enzymes in Escherichia coli. By tethering metabolic enzymes that catalyze the reactions before and after a target metabolite, the metabolic flux may be enhanced. This chapter describes the approach for enhancing acetate-producing flux by sortase-A-assisted metabolic ligation in E. coli.

    2018年, Methods in molecular biology (Clifton, N.J.), 1772, 125 - 136, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Low amounts of dietary fibre increase in vitro production of short-chain fatty acids without changing human colonic microbiota structure

    Daisuke Sasaki, Kengo Sasaki, Naoko Ikuta, Takahiro Yasuda, Itsuko Fukuda, Akihiko Kondo, Ro Osawa

    2018年, Scientific Reports, 8, Article number: 435, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Naoko Okai, Takaya Masuda, Yasunobu Takeshima, Kosei Tanaka, Ken-Ichi Yoshida, Masanori Miyamoto, Chiaki Ogino, Akihiko Kondo

    Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a lignin-derived phenolic compound abundant in plant biomass. The utilization of FA and its conversion to valuable compounds is desired. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a precursor of polymers and plastics and a constituent of food. A microbial conversion system to produce PCA from FA was developed in this study using a PCA-producing strain of Corynebacterium glutamicum F (ATCC 21420). C. glutamicum strain F grown at 30 °C for 48 h utilized 2 mM each of FA and vanillic acid (4-hydroxy-3-methoxybenzoic acid, VA) to produce PCA, which was secreted into the medium. FA may be catabolized by C. glutamicum through proposed (I) non-β-oxidative, CoA-dependent or (II) β-oxidative, CoA-dependent phenylpropanoid pathways. The conversion of VA to PCA is the last step in each pathway. Therefore, the vanillate O-demethylase gene (vanAB) from Corynebacterium efficiens NBRC 100395 was expressed in C. glutamicum F (designated strain FVan) cultured at 30 °C in AF medium containing FA. Strain C. glutamicum FVan converted 4.57 ± 0.07 mM of FA into 2.87 ± 0.01 mM PCA after 48 h with yields of 62.8% (mol/mol), and 6.91 mM (1064 mg/L) of PCA was produced from 16.0 mM of FA after 12 h of fed-batch biotransformation. Genomic analysis of C. glutamicum ATCC 21420 revealed that the PCA-utilization genes (pca cluster) were conserved in strain ATCC 21420 and that mutations were present in the PCA importer gene pcaK.

    2017年12月, AMB Express, 7 (1), 130 - 130, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • M. Mahfuza Khatun, Xinshui Yu, Akihiko Kondo, Fengwu Bai, Xinqing Zhao

    In this work, the consolidated bioprocessing (CBP) yeast Saccharomyces cerevisiae MNII/cocdBEC3 was transformed by an artificial zinc finger protein (AZFP) library to improve its thermal tolerance, and the strain MNII-AZFP with superior growth at 42 degrees C was selected. Improved degradation of acid swollen cellulose by 45.9% led to an increase in ethanol production, when compared to the control strain. Moreover, the fermentation of Jerusalem artichoke stalk (JAS) by MNII-AZFP was shortened by 12 h at 42 degrees C with a concomitant improvement in ethanol production. Comparative transcriptomics analysis suggested that the AZFP in the mutant exerted beneficial effect by modulating the expression of multiple functional genes. These results provide a feasible strategy for efficient ethanol production from JAS and other cellulosic biomass through CBP based-fermentation at elevated temperatures. (C) 2017 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245 (B), 1447 - 1454, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Current advances on fermentative biobutanol production using third generation feedstock

    Yue Wang, Shih-Hsin Ho, Hong-Wei Yen, Dillirani Nagarajan, Nan-Qi Ren, Shuangfei Li, Zhangli Hu, Duu-Jong Lee, Akihiko Kondo, Jo-Shu Chang

    2017年12月, Biotechnology Advances, 35 (8), 1049 - 1059, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kaoru Onodera, Shinji Hama, Ayumi Yoshida, Hideo Noda, Akihiko Kondo

    Here, we developed a fed-batch process for the cultivation of multistress tolerant Saccharomyces cerevisiae engineered to display p-glucosidase (BGL) on the cell surface. Although cell growth was promoted by increasing the amount of oxygen supplied, increasing the overall volumetric oxygen transfer coefficient rather decreased the specific BGL activity of yeast cells probably because of the high agitation speed used. To suppress the formation of ethanol, the method of feeding glucose was investigated. Namely, the timely addition of glucose and glucose feed rate were found crucial for increasing both cell concentration and specific BGL activity. When the lowest concentration of dissolved oxygen at 9 h was used as an indicator, the lowest glucose feed rate of 1 g/(L.h) provided the maximum BGL activity of 673.6 U/g-dry cell, which is 1.6-fold higher than that in batch cultivation. By employing the fed-batch technique together with the improved gene cassette containing the SEEM promoter and anchoring domain, we found that the cell concentration and specific BGL activity continued to increase, reaching 15.4 g-dry cell/L and 1866.6 U/g-dry cell after 48 h, respectively. The results show the advantage of the fed-batch process for realizing the potential of cell-surface display systems in S.cerevisiae. (C) 2017 Published by Elsevier B.V.

    ELSEVIER SCIENCE BV, 2017年12月, Biochemical Engineering Journal, 128, 195 - 200, 英語

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    研究論文(学術雑誌)

  • Norimasa Kashiwagi, Chiaki Ogino, Akihiko Kondo

    Bioproduction using microbes from biomass feedstocks is of interest in regards to environmental problems and cost reduction. Streptomyces as an industrial microorganism plays an important role in the production of useful secondary metabolites for various applications. This strain also secretes a wide range of extracellular enzymes which degrade various biopolymers in nature, and it consumes these degrading substrates as nutrients. Hence, Streptomyces can be employed as a cell factory for the conversion of biomass-derived substrates into various products. This review focuses on the following two points: (1) Streptomyces as a producer of enzymes for degrading biomass-derived polysaccharides and polymers; and, (2) wild-type and engineered strains of Streptomyces as a host for chemical production from biomass-derived substrates. (C) 2017 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245, 1655 - 1663, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hideo Kawaguchi, Chiaki Ogino, Akihiko Kondo

    The worldwide market for plastics is rapidly growing, and plastics polymers are typically produced from petroleum-based chemicals. The overdependence on petroleum-based chemicals for polymer production raises economic and environmental sustainability concerns. Recent progress in metabolic engineering has expanded fermentation products from existing aliphatic acids or alcohols to include aromatic compounds. This diversity provides an opportunity to expand the development and industrial uses of high-performance bio-based polymers. However, most of the biomonomers are produced from edible sugars or starches that compete directly with food and feed uses. The present review focuses on recent progress in the microbial conversion of biomass into bio-based polymers, in which fermentative products from renewable feedstocks serve as biomonomers for the synthesis of bio-based polymers. In particular, the production of biomonomers from inedible lignocellulosic feedstocks by metabolically engineered microorganisms and the synthesis of bio-based engineered plastics from the biological resources are discussed. (C) 2017 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245 (B), 1664 - 1673, 英語

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    研究論文(学術雑誌)

  • Mannan endo-1,4-β-mannosidase from Kitasatospora sp. isolated in Indonesia and its potential for production of mannooligosaccharides from mannan polymers

    Nanik Rahmani, Norimasa Kashiwagi, JaeMin Lee, Satoko Niimi-Nakamura, Hana Matsumoto, Prihardi Kahar, Puspita Lisdiyanti, Yopi, Bambang Prasetya, Chiaki Ogino, Akihiko Kondo

    2017年12月, AMB Express, 7, 100, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Satoshi Wakai, Takayoshi Arazoe, Chiaki Ogino, Akihiko Kondo

    Filamentous fungi exhibit versatile abilities, including organic acid fermentation, protein production, and secondary metabolism, amongst others, and thus have applications in the medical and food industries. Previous genomic analyses of several filamentous fungi revealed their further potential as host microorganisms for bioproduction. Recent advancements in molecular genetics, marker recycling, and genome editing could be used to alter transformation and metabolism, based on optimized design carbolated with computer science. In this review, we detail the current applications of filamentous fungi and describe modern molecular genetic tools that could be used to expand the role of these microorganisms in bioproduction. The present review shed light on the possibility of filamentous fungi as host microorganisms in the field of bioproduction in the future. (C) 2017 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245 (B), 1314 - 1326, 英語

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    研究論文(学術雑誌)

  • Jerome Amoah, Naoya Ishizue, Miki Ishizaki, Misa Yasuda, Kenji Takahashi, Kazuaki Ninomiya, Ryosuke Yamada, Akihiko Kondo, Chiaki Ogino

    This work aimed to study the use of consolidated bioprocess (CBP) yeast expressing five cellulase genes (BGL, XYNII, EGII, CBHI and CBHII) for ethanol production from ionic liquid-pretreated bagasse and Laubholz unbleached Kraft pulp (LUKP). A proposed screening method shows that the optimal cellulase ratio varies for each biomass substrate, and thus it is essential to breed CBP yeast having optimal cellulase-displaying ratio for the target biomass. CBP yeast specialized towards bagasse produced 0.93 g/l ethanol whiles that for LUKP produced 0.71 g/l ethanol, which is approximately 4 and 2-fold, respectively, higher than that of the wild type. The cell-surface displayed enzymes synergistically contributed to the degradation of the biomass. The developed CBP yeast is a potential cheap source for consolidated bioprocessing of ethanol and the proposed screening method can be used for matching CBP yeast to a target biomass. (C) 2017 Published by Elsevier Ltd.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245 (B), 1413 - 1420, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Prihardi Kahar, Eny Ida Riyanti, Hiromi Otsuka, Hana Matsumoto, Chie Kihira, Chiaki Ogino, Akihiko Kondo

    This study provides insight observation based on the gene expression and the metabolomic analysis of the natural robust yeast Saccharomyces cerevisiae NBRC849 during the fermentation in the medium containing inhibitory chemical complexes (ICC) at different concentrations. The tolerance mechanisms involved in the strain might have existed through the upregulation of genes involved in NAD(H)/NADP (H) cofactors generations (ALD6, ZWF1, GND1), membrane robustness for efflux pump (YOR1, PDR5, TPO3) and cation/polyamine transport (TPO3). The alteration of metabolic flux to the shikimic pathway was also found in this strain, resulted in the enhanced formation of aromatic amino acid required for cell survival. Enhanced expression of these genes as well as the increase of metabolic flux to shikimic pathway were suggested to result in the robustness of non-flocculating S. cerevisiae haploid strain. (C) 2017 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245 (B), 1436 - 1446, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Improvement of Xylose Fermentation Ability under Heat and Acid Co-Stress in Saccharomyces cerevisiae Using Genome Shuffling Technique

    Kentaro Inokuma, Ryo Iwamoto, Takahiro Bamba, Tomohisa Hasunuma, Akihiko Kondo

    2017年12月, Frontiers in Bioengineering and Biotechnology, 5, 81, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Alex Prima, Kiyotaka Y. Hara, Apridah Cameliawati Djohan, Norimasa Kashiwagi, Prihardi Kahar, Jun Ishii, Hideki Nakayama, Fumiyoshi Okazaki, Bambang Prasetya, Akihiko Kondo, Yopi, Chiaki Ogino

    This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/ mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, beta-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources. (C) 2017 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245 (B), 1400 - 1406, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuichi Kato, Shih-Hsin Ho, Christopher J. Vavricka, Jo-Shu Chang, Tomohisa Hasunuma, Akihiko Kondo

    The aim of this study was to improve biomass production of the green microalga Chlamydomonas sp. JSC4 under high salinity conditions. For this purpose, heavy ion beam-coupled mutagenesis and evolutionary engineering were performed using JSC4 as the parent strain. After long-term and continuous cultivation with high salinity, salt-resistant strains that grow well even in the presence of 7% sea salt were successfully obtained. Transcriptional analysis revealed inactivation of starch-to-lipid biosynthesis switching, which resulted in delayed starch degradation and decreased lipid content in the salt-resistant strains. Cellular aggregation and hypertrophy during high salinity were relieved in these strains, indicating strong resistance to salt stress. These results suggest that high salinity stress, not the salinity condition itself, is important for activating lipid accumulation mechanisms in microalgae. (C) 2017 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245 (part B), 1484 - 1490, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Engineering metabolic pathways in Escherichia coli for constructing a “microbial chassis” for biochemical production

    MATSUMOTO Takuya, TANAKA Tsutomu, KONDO Akihiko

    2017年12月, Bioresource Technology, 245 (B), 1362 - 1368, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Jerome Amoah, Shih-Hsin Ho, Shinji Hama, Ayumi Yoshida, Akihito Nakanishi, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo

    Lipid from Chlamydomonas sp. JSC4 was used as a feedstock for biodiesel production. The lipid was found to contain high amounts of phospholipids and free fatty acid in addition to the triglycerides. Two enzymatic methods for the efficient conversion of the heterogenous lipid to fatty acid methyl esters (FAME) were carried out. The method using either a lipase cocktail containing Candida cylindracea lipase and Thermomyces lanuginosus lipase combination (m I) or immobilized Fusarium heterosporum lipase-expressing Aspergillus oryzae whole-cells (m II) were both successful. However, the method using lipase cocktail showed 30.8% relative stability after the fourth batch, whereas the whole-cell biocatalyst showed 98.1%. Although the whole-cell biocatalyst tolerated a wide range of water content, an exploration of the effect of water-methanol interaction on the biocatalytic process showed that 24% water and 7: 1 methanol to oil ratio is more favorable for FAME production. A higher initial methanol consumption rate facilitated a more stable system with the whole-cell biocatalyst, producing over 97% FAME in 32 h. The efficient conversion of a highly heterogenous substrate in the presence of high amounts of water could be an effective technique for the enzymatic conversion of microalgal lipids.

    ELSEVIER SCIENCE BV, 2017年12月, ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS, 28, 16 - 23, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kenta Imao, Rie Konishi, Mayumi Kishida, Yuuki Hirata, Shota Segawa, Noriko Adachi, Rena Matsuura, Yota Tsuge, Takuya Matsumoto, Tsutomu Tanaka, Akihiko Kondo

    Xylooligosaccharide-assimilating Corynebacterium glutamicum strains were constructed using metabolic engineering and cell surface display techniques. First, C. glutamicum was metabolically engineered to create lysine-producing strains. Beta-xylosidase BSU17580 derived from Bacillus subtilis was then expressed on the C. glutamicum cell surface using PorH anchor protein, and enzymes involved in the xylose assimilation pathway were also expressed. Metabolic engineering had no effect on the activity of beta-xylosidase. The engineered strains efficiently consumed xylooligosaccharides and produced 12.4 mM of lysine from 11.9 g/L of xylooligosaccharides as the carbon source. Finally, co-expression of lysine decarboxylase enabled production of 11.6 mM of 1,5-diaminopentane (cadaverine) from 13 g/L of consumed xylooligosaccharides. (C) 2017 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年12月, BIORESOURCE TECHNOLOGY, 245 (B), 1684 - 1691, 英語

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    研究論文(学術雑誌)

  • Musashi Takenaka, Tomokazu Amino, Yusuke Miyachi, Chiaki Ogino, Akihiko Kondo

    A sensing system was constructed to monitor the target peptide via two aptamer-based sensors pinching. First, aptamers against somatostatin (SST) were selected via the systematic evolution of ligands by exponential enrichment, and four aptamers were selected from a single-stranded DNA library. Their specificities to SST were evaluated via surface plasmon resonance and atomic force microscopy (AFM). Next, two aptamers with higher specificities to SST were used as aptamer-based sensors; one aptamer was modified with a chip, and the other was modified with a probe. Based on AFM system, the probe was surveyed on the chip in SST solutions, simultaneously measuring an interactive force. The label-free SST could be detected, and then the change in its concentration could be monitored at levels that ranged from 2 to 2000 nM. The interactive force of a single pair was approximately 45 pN, and the molecule number was associated with the interactive force. Therefore, we could firstly select the aptamers against somatostatin, and the sandwich-like monitoring system can be used to promote peptide sensor or monitoring system using an aptamer-based sensor. (C) 2017 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2017年11月, SENSORS AND ACTUATORS B-CHEMICAL, 252, 813 - 821, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Musashi Takenaka, Tomokazu Amino, Yusuke Miyachi, Chiaki Ogino, Akihiko Kondo

    A split aptamers-based aptasensor is applied to peptide sensing using atomic force microscopy (AFM). Somatostatin (SST) is targeted as a model peptide. The aptamer against SST is split, and each part is chemically modified with a probe or a chip. Hybridized aptamers can detect label-free SSTs and monitor changes in their concentration from 2 nM to 2000 nM as an index of interactive force. The change in an interactive force could be firstly evaluated via Hill model. Additionally, the analysis based on the fitting enables clarification of the differences in hybridization and in interaction, as well as an estimation of the number of SSTs. The proposed sensing system can promote peptide sensing based on the use of an aptasensor. (C) 2017 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2017年11月, SENSORS AND ACTUATORS B-CHEMICAL, 252, 600 - 605, 英語

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    研究論文(学術雑誌)

  • Kiyotaka Y. Hara, Jyumpei Kobayashi, Ryosuke Yamada, Daisuke Sasaki, Yuki Kuriya, Yoko Hirono-Hara, Jun Ishii, Michihiro Araki, Akihiko Kondo

    Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources.

    OXFORD UNIV PRESS, 2017年11月, FEMS YEAST RESEARCH, 17 (7), fox061, 英語

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    研究論文(学術雑誌)

  • Shuhei Noda, Tomokazu Shirai, Yutaro Mori, Sachiko Oyama, Akihiko Kondo

    Maleate is one of the most important dicarboxylic acids and is used to produce various polymer compounds and pharmaceuticals. Herein, microbial production of maleate is successfully achieved, to our knowledge for the first time, using genetically modified Escherichia coli. A synthetic pathway of maleate is constructed in E. coli by combining the polyketide biosynthesis pathway and benzene ring cleavage pathway. The metabolic engineering approach used to fine-tune the synthetic pathway drastically improves maleate production and demonstrates that one of the rate limiting steps exists in the conversion of chorismate to gentisate. In a batch culture of the optimised transformant, grown in a 1-L jar fermentor, the amount of produced maleate reaches 7.1 g L-1, and the yield is 0.221 mol mol(-1). Our results suggest that the construction of synthetic pathways by combining a secondary metabolite pathway and the benzene ring cleavage pathway is a powerful tool for producing various valuable chemicals.

    NATURE PUBLISHING GROUP, 2017年10月, NATURE COMMUNICATIONS, 8 (1), 1153, 英語

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    研究論文(学術雑誌)

  • MERT MJ, ROSE SH, La GRANGE DC, BAMBA Takahiro, HASUNUMA Tomohisa, KONDO Akihiko, van ZYL WH

    The yeast Saccharomyces cerevisiae cannot utilize xylose, but the introduction of a xylose isomerase that functions well in yeast will help overcome the limitations of the fungal oxido-reductive pathway. In this study, a diploid S. cerevisiae S288c[2n YMX12] strain was constructed expressing the Bacteroides thetaiotaomicron xylA (XI) and the Scheffersomyces stipitis xyl3 (XK) and the changes in the metabolite pools monitored over time. Cultivation on xylose generally resulted in gradual changes in metabolite pool size over time, whereas more dramatic fluctuations were observed with cultivation on glucose due to the diauxic growth pattern. The low G6P and F1,6P levels observed with cultivation on xylose resulted in the incomplete activation of the Crabtree effect, whereas the high PEP levels is indicative of carbon starvation. The high UDP-d-glucose levels with cultivation on xylose indicated that the carbon was channeled toward biomass production. The adenylate and guanylate energy charges were tightly regulated by the cultures, while the catabolic and anabolic reduction charges fluctuated between metabolic states. This study helped elucidate the metabolite distribution that takes place under Crabtree-positive and Crabtree-negative conditions when cultivating S. cerevisiae on glucose and xylose, respectively.

    SPRINGER HEIDELBERG, 2017年10月, Journal of Industrial Microbiology and Biotechnology, 44 (10), 1459 - 1470, 英語

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    研究論文(学術雑誌)

  • Hitoshi Mitsunobu, Jun Teramoto, Keiji Nishida, Akihiko Kondo

    Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated manipulation of genomic information is becoming more versatile by combining nuclease-deficient CRISPR systems with a wide variety of effectors including base-editing deaminases, transcriptional regulators, and epigenetic modifiers. The programmable binding ability of CRISPR systems is essential when the systems are employed as targeting domains to recruit the effectors to specific genomic loci. The discovery of a variety of Cas9 orthologs and engineered variants enables high-fidelity genome editing and a wider selection of genomic targets, and CRISPR-mediated deaminases enable more precise and predictable genome editing compared with CRISPR nuclease-based editing. Finally, combining transcriptional regulators with CRISPR systems can control expression of specific genes in a genome. Some applications and future challenges of CRISPR-derived tools are also discussed.

    ELSEVIER SCIENCE LONDON, 2017年10月, TRENDS IN BIOTECHNOLOGY, 35 (10), 983 - 996, 英語

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    研究論文(学術雑誌)

  • Yoshifumi Ueno, Shimpei Aikawa, Kyosuke Niwa, Tomoko Abe, Akio Murakami, Akihiko Kondo, Seiji Akimoto

    The light-harvesting antennas of oxygenic photosynthetic organisms capture light energy and transfer it to the reaction centers of their photosystems. The light-harvesting antennas of cyanobacteria and red algae, called phycobilisomes (PBSs), supply light energy to both photosystem I (PSI) and photosystem II (PSII). However, the excitation energy transfer processes from PBS to PSI and PSII are not understood in detail. In the present study, the energy transfer processes from PBS to PSs in various cyanobacteria and red algae were examined in vivo by selectively exciting their PSs or PBSs, and measuring the resulting picosecond to nanosecond time-resolved fluorescences. By observing the delayed fluorescence spectrum of PBS-selective excitation in Arthrospira platensis, we demonstrated that energy transfer from PBS to PSI via PSII (PBS -> PSII -> PSI transfer) occurs even for PSI trimers. The contribution of PBS -> PSII -> PSI transfer was species dependent, being largest in the wild-type of red alga Pyropia yezoensis (formerly Porphyra yezoensis) and smallest in Synechococcus sp. PCC 7002. Comparing the time-resolved fluorescence after PSs- and PBS-selective excitation, we revealed that light energy flows from CP43 to CP47 by energy transfer between the neighboring PSII monomers in PBS-PSII supercomplexes. We also suggest two pathways of energy transfer: direct energy transfer from PBS to PSI (PBS -> PSI transfer) and indirect transfer through PSII (PBS -> PSII -> PSI transfer). We also infer that PBS -> PSI transfer conveys light energy to a lower-energy red chlorophyll than PBS -> PSII -> PSI transfer.

    SPRINGER, 2017年09月, Photosynthesis Research, 133 (1-3), 235 - 243, 英語

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    研究論文(学術雑誌)

  • Aya Onishi, Shimpei Aikawa, Akihiko Kondo, Seiji Akimoto

    Nitrogen is among the most important nutritious elements for photosynthetic organisms such as plants, algae, and cyanobacteria. Therefore, nitrogen depletion severely compromises the growth, development, and photosynthesis of these organisms. To preserve their integrity under nitrogen-depleted conditions, filamentous nitrogen-fixing cyanobacteria reduce atmospheric nitrogen to ammonia, and self-adapt by regulating their light-harvesting and excitation energy-transfer processes. To investigate the changes in the primary processes of photosynthesis, we measured the steady-state absorption and fluorescence spectra and time-resolved fluorescence spectra (TRFS) of whole filaments of the nitrogen-fixing cyanobacterium Anabaena variabilis at 77 K. The filaments were grown in standard and nitrogen-free media for 6 months. The TRFS were measured with a picosecond time-correlated single photon counting system. Despite the phycobilisome degradation, the energy-transfer paths within phycobilisome and from phycobilisome to both photosystems were maintained. However, the energy transfer from photosystem II to photosystem I was suppressed and a specific red chlorophyll band appeared under the nitrogen-depleted condition.

    SPRINGER, 2017年09月, PHOTOSYNTHESIS RESEARCH, 133 (1-3), 317 - 326, 英語

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    研究論文(学術雑誌)

  • Keisuke Morita, Yuta Nomura, Jun Ishii, Fumio Matsuda, Akihiko Kondo, Hiroshi Shimizu

    Bacterial phosphoenol pyruvate carboxylase (PPC) and enzymes in the Entner-Doudoroff (ED) pathway were heter-ologously expressed in Saccharomyces cerevisiae to improve the NADPH supply required for the bio-production of chemicals such as isobutanol. The heterologous expression of PPC from Synechocystis sp. PCC6803 increased in the isobutabol titer 1.45-fold (93.2 +/- 1.6 mg/L) in metabolically engineered S. cerevisiae strains producing isobutanol. This result suggested that the pyruvate and NADPH supply for isobutanol biosynthesis was activated by PPC overexpression. On the other hand, the expression of two enzymes organizing the ED pathway (6-phosphogluconate dehydratase [6PGD] and 2-dehydro-3-deoxy-phosphogluconate aldolase [ICDPGA]) had no effect to isobutabol bio-production. Further analysis, however, revealed that additional expression of 6PGD and KDPGA improved the growth rate of S. cerevisiae strain BY4742 gnd1 Delta. A C-13-labeling experiment using [1 - C-13] glucose also suggested that metabolic flow levels in the ED pathway increased slightly with the additional expression. These results showed that the ED pathway was successfully constructed in S. cerevisiae, even though activity of the pathway was too weak to improve isobutanol biosynthesis. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2017年09月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 124 (3), 263 - 270, 英語

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    研究論文(学術雑誌)

  • Shuhei Noda, Akihiko Kondo

    Along with the development of metabolic engineering and synthetic biology tools, various microbes are being used to produce aromatic chemicals. In microbes, aromatics are mainly produced via a common important precursor, chorismate, in the shikimate pathway. Natural or non-natural aromatics have been produced by engineering metabolic pathways involving chorismate. In the past decade, novel approaches have appeared to produce various aromatics or to increase their productivity, whereas previously, the targets were mainly aromatic amino acids and the strategy was deregulating feedback inhibition. In this review, we summarize recent studies of microbial production of aromatics based on metabolic engineering approaches. In addition, future perspectives and challenges in this research area are discussed.

    ELSEVIER SCIENCE LONDON, 2017年08月, TRENDS IN BIOTECHNOLOGY, 35 (8), 785 - 796, 英語

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    研究論文(学術雑誌)

  • Kengo Sasaki, Yota Tsuge, Hideo Kawaguchi, Masahiro Yasukawa, Daisuke Sasaki, Takashi Sazuka, Eiji Kamio, Chiaki Ogino, Hideto Matsuyama, Akihiko Kondo

    The juice from sweet sorghum cultivar SIL-05 (harvested at physiological maturity) was extracted, and the component sucrose and reducing sugars (such as glucose and fructose) were subjected to a membrane separation process to purify the sucrose for subsequent sugar refining and to obtain a feedstock for repeated bioethanol production. Nanofiltration (NF) of an ultrafiltration (UF) permeate using an NTR-7450 membrane (Nitto Denko Corporation, Osaka, Japan) concentrated the juice and produced a sucrose-rich fraction (143.2 g L-1 sucrose, 8.5 g L-1 glucose, and 4.5 g L-1 fructose). In addition, the above NF permeate was concentrated using an ESNA3 NF membrane to provide concentrated permeated sugars (227.9 g L-1) and capture various amino acids in the juice, enabling subsequent ethanol fermentation without the addition of an exogenous nitrogen source. Sequential batch fermentation using the ESNA3 membrane concentrate provided an ethanol titer and theoretical ethanol yield of 102.5-109.5 g L-1 and 84.4-89.6%, respectively, throughout the five-cycle batch fermentation by Saccharomyces cerevisiae BY4741. Our results demonstrate that a membrane process using UF and two types of NF membranes has the potential to allow sucrose purification and repeated bioethanol production.

    2017年08月, Applied microbiology and biotechnology, 101 (15), 6007 - 6014, 英語, 国際誌

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    研究論文(学術雑誌)

  • Metabolic engineering of Schizosaccharomyces pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose

    OZAKI Aiko, KONISHI Rie, OTOMO Chisako, KISHIDA Mayumi, TAKAYAMA Seiya, MATSUMOTO Takuya, TANAKA Tsutomu, KONDO Akihiko

    2017年08月, Metabolic Engineering Communications, 5, 60 - 67, 英語

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    研究論文(学術雑誌)

  • Yumiko Nambu-Nishida, Keiji Nishida, Tomohisa Hasunuma, Akihiko Kondo

    Kluyveromyces marxianus, a non-conventional thermotolerant yeast, is potentially useful for production of ethanol and other products. This species has a strong tendency to randomly integrate transforming DNA fragments, making necessary the development of more precise methods for gene targeting. In this study, we first demonstrated that K. marxianus NBRC1777 is cold-tolerant, and then established a highly efficient and precise technique for gene editing by introducing genes encoding deaminase-mediated targeted point mutagenesis (Target-AID) and clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas9). We used Target-AID to introduce targeted point mutations that disrupted Nej1 or Dnl4, genes that are involved in non-homologous end-joining (NHEJ). Both of the resulting mutant strains showed enhanced proportions of homology-mediated integration compared to the wild-type parent. In combination with target cleavage by CRISPR-Cas9, markerless integration was performed using short (-50 bp) flanking homologous sequences. Together, these tools render this species fully tractable for gene manipulation, permitting targeted genetic changes in the cold- and thermo-tolerant yeast K. marxianus.

    NATURE PUBLISHING GROUP, 2017年08月, SCIENTIFIC REPORTS, 7 (1), 8993, 英語

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    研究論文(学術雑誌)

  • Hideo Kawaguchi, Yohei Katsuyama, Du Danyao, Prihardi Kahar, Sachiko Nakamura-Tsuruta, Hiroshi Teramura, Keiko Wakai, Kumiko Yoshihara, Hiromichi Minami, Chiaki Ogino, Yasuo Ohnishi, Akihiko Kondo

    Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (ae<currency>2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

    SPRINGER, 2017年07月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 101 (13), 5279 - 5290, 英語

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    研究論文(学術雑誌)

  • Targeted nucleotide substitution in mammalian cell by Target-AID

    ARAZOE Takahiro, NISHIDA Keiji, KONDO Akihiko

    2017年06月, Bio-protocol, 7, 11, 英語

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    研究論文(学術雑誌)

  • Yosuke Fukutani, Jun Ishii, Akihiko Kondo, Takeaki Ozawa, Hiroaki Matsunami, Masafumi Yohda

    The budding yeast Saccharomyces cerevisiae is equipped with G protein-coupled receptors (GPCR). Because the yeast GPCR signaling mechanism is partly similar to that of the mammalian system, S. cerevisiae can be used for a host of mammalian GPCR expression and ligand-mediated activation assays. However, currently available yeast systems require several hours to observe the responses because they depend on the expression of reporter genes. In this study, we attempted to develop a simple GPCR assay system using split luciferase and -arrestin, which are independent of the endogenous S. cerevisiae GPCR signaling pathways. We applied the split luciferase complementation assay method to S. cerevisiae and found that it can be used to analyze the ligand response of the human somatostatin receptor in S. cerevisiae. On the contrary, the response of the pheromone receptor Ste2 was not observed by the assay. Thus, the split luciferase complementation should be free from the effect of the endogenous GPCR signaling. Biotechnol. Bioeng. 2017;114: 1354-1361. (c) 2017 Wiley Periodicals, Inc.

    WILEY, 2017年06月, BIOTECHNOLOGY AND BIOENGINEERING, 114 (6), 1354 - 1361, 英語

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    研究論文(学術雑誌)

  • Shintaro Ryo, Jun Ishii, Toshihide Matsimo, Yasuyuki Nakamura, Daiki Matsubara, Masahiro Tominaga, Akihiko Kondo

    The GAL expression system is the most frequently used induction technique in the yeast Saccharomyces cerevisiae. Here we report a simple but powerful genetic circuit for use with the GAL induction system. Briefly, an artificial positive feedback circuit was incorporated into the GAL regulatory network. We selected green fluorescent protein (GFP) as a reporter of GAL] induction, and designed a strain that expressed a constitutively active Gal3 mutant protein (Gal3(c)) under control of the GAL10 promoter. In the resulting strain, GALL and GAL10 promoters regulate the expression of GFP and GALS(c), respectively. Because Gal3(c) sequesters the Gal80 repressor away from the Gal4 transcriptional activator in the same manner as the galactose-bound Gal3, the expressed Gal3(c) protein provokes further expression of GFP and Gal3(c), yielding further enhancement of GAL induction. Thus, this GAL3(c)-mediated positive feedback circuit permits substantially enriched induction of a target gene at extremely low concentrations, or even in the absence, of galactose, while maintaining the strict glucose-mediated repression of the target.

    AMER CHEMICAL SOC, 2017年06月, ACS SYNTHETIC BIOLOGY, 6 (6), 928 - 935, 英語

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    研究論文(学術雑誌)

  • Zhuo Liu, Kentaro Inokuma, Shih-Hsin Ho, Riaan den Haan, Willem H. van Zyl, Tomohisa Hasunuma, Akihiko Kondo

    Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;114: 1201-1207. (c) 2017 Wiley Periodicals, Inc.

    WILEY, 2017年06月, BIOTECHNOLOGY AND BIOENGINEERING, 114 (6), 1201 - 1207, 英語

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    研究論文(学術雑誌)

  • Ryosuke Yamada, Kazunori Nakashima, Nanami Asai-Nakashima, Wataru Tokuhara, Nobuhiro Ishida, Satoshi Katahira, Noriho Kamiya, Chiaki Ogino, Akihiko Kondo

    Among the many types of lignocellulosic biomass pretreatment methods, the use of ionic liquids (ILs) is regarded as one of the most promising strategies. In this study, the effects of four kinds of ILs for pretreatment of lignocellulosic biomass such as bagasse, eucalyptus, and cedar were evaluated. In direct ethanol fermentation from biomass incorporated with ILs by cellulase-displaying yeast, 1-butyl-3-methylimidazolium acetate ([Bmim][OAc]) was the most effective IL. The ethanol production and yield from [Bmim][OAc]-pretreated bagasse reached 0.81 g/L and 73.4% of the theoretical yield after fermentation for 96 h. The results prove the initial concept, in which the direct fermentation from lignocellulosic biomass effectively promoted by the pretreatment with IL.

    HUMANA PRESS INC, 2017年05月, APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 182 (1), 229 - 237, 英語

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    研究論文(学術雑誌)

  • Masafumi Shibuya, Masahiro Yasukawa, Kengo Sasaki, Yasuhiro Tanaka, Tomoki Takahashi, Akihiko Kondo, Hideto Matsuyama

    Forward osmosis (FO), driven by high osmotic pressure, was used for the first time for bioethanol production from lignocellulosic biomass. A commercial membrane, TFC-ES (Hydration Technology Innovation), was used and 3.6 M triethylamine (TEA) was chosen as the draw solution because the concentration ratio against a model xylose solution was similar to that achieved with 2.5 M NaCI. The liquid fraction of hot-water-pretreated rice straw was concentrated by using the FO membrane. The initial sugar concentration of 199 mM increased to 825 and 1612 mM after 48 and 72 h of FO concentration, respectively, and these values were much higher than those obtained from nanofiltration. Simultaneous saccharification and fermentation of the liquid fraction after 48 h of FO concentration by xylose-fermenting recombinant Saccharornyces cerevisiae produced 17.7 g/L of ethanol after 24 h fermentation. Thus, the FO process has tremendous potential to up-concentrate sugars obtained from lignocellulosic biomass. (C) 2017 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2017年05月, BIOCHEMICAL ENGINEERING JOURNAL, 121, 13 - 16, 英語

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    研究論文(学術雑誌)

  • Zenpei Shimatani, Sachiko Kashojiya, Mariko Takayama, Rie Terada, Takayuki Arazoe, Hisaki Ishii, Hiroshi Teramura, Tsuyoshi Yamamoto, Hiroki Komatsu, Kenji Miura, Hiroshi Ezura, Keiji Nishida, Tohru Ariizumi, Akihiko Kondo

    We applied a fusion of CRISPR-Cas9 and activation-induced cytidine deaminase (Target-AID) for point mutagenesis at genomic regions specified by single guide RNAs (sgRNAs) in two crop plants. In rice, we induced multiple herbicide-resistance point mutations by multiplexed editing using herbicide selection, while in tomato we generated marker-free plants with homozygous heritable DNA substitutions, demonstrating the feasibility of base editing for crop improvement.

    NATURE PUBLISHING GROUP, 2017年05月, NATURE BIOTECHNOLOGY, 35 (5), 441 - +, 英語

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    研究論文(学術雑誌)

  • Shih-Hsin Ho, Akihito Nakanishi, Yuichi Kato, Hiroaki Yamasaki, Jo-Shu Chang, Naomi Misawa, Yuu Hirose, Jun Minagawa, Tomohisa Hasunuma, Akihiko Kondo

    Biodiesel production using microalgae would play a pivotal role in satisfying future global energy demands. Understanding of lipid metabolism in microalgae is important to isolate oleaginous strain capable of overproducing lipids. It has been reported that reducing starch biosynthesis can enhance lipid accumulation. However, the metabolic mechanism controlling carbon partitioning from starch to lipids in microalgae remains unclear, thus complicating the genetic engineering of algal strains. We here used "dynamic" metabolic profiling and essential transcription analysis of the oleaginous green alga Chlamydomonas sp. JSC4 for the first time to demonstrate the switching mechanisms from starch to lipid synthesis using salinity as a regulator, and identified the metabolic rate-limiting step for enhancing lipid accumulation (e. g., pyruvate-to-acetyl-CoA). These results, showing salinity-induced starch-tolipid biosynthesis, will help increase our understanding of dynamic carbon partitioning in oleaginous microalgae. Moreover, we successfully determined the changes of several key lipid-synthesis- related genes (e. g., acetyl-CoA carboxylase, pyruvate decarboxylase, acetaldehyde dehydrogenase, acetylCoA synthetase and pyruvate ferredoxin oxidoreductase) and starch-degradation related genes (e. g., starch phosphorylases), which could provide a breakthrough in the marine microalgal production of biodiesel.

    NATURE PUBLISHING GROUP, 2017年04月, SCIENTIFIC REPORTS, 7 (7), 45471, 英語

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    研究論文(学術雑誌)

  • Dillirani Nagarajan, Duu-Jong Lee, Akihiko Kondo, Jo-Shu Chang

    One of the best options to alleviate the problems associated with global warming and climate change is to reduce burning of fossil fuels and search for new alternative energy resources. In case of biodiesel and bioethanol production, the choice of feedstock and the process design influences the GHG emissions and appropriate methods need to be adapted. Hydrogen is a zero-carbon and energy dense alternative energy carrier with clean burning properties and biohydrogen production by microalgae can reduce production associated GHG emissions to a great extent. Biohydrogen can be produced through dark fermentation using sugars, starch, or cellulosic materials. Microalgae-based biohydrogen production is recently regarded as a promising pathway for biohydrogen production via photolysis or being a substrate for anaerobic fermentation. This review lists the methods of hydrogen production by microalgae. The enzymes involved and the factors affecting the biohydrogen production process are discussed. The bottlenecks in microalgae-based biohydrogen production are critically reviewed and future research areas in hydrogen production are presented. (C) 2016 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2017年03月, BIORESOURCE TECHNOLOGY, 227, 373 - 387, 英語

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    研究論文(学術雑誌)

  • Ishigaki M, Nakanishi A, HASUNUMA Tomohisa, KONDO Akihiko, MORISHIMA T, OKUNO T, OZAKI Y

    In the present study, the high-speed quantitative evaluation of glycogen concentration accumulated in bioethanol feedstock Synechocystis sp. PCC6803 was performed using a near-infrared (NIR) imaging system with a hyperspectral NIR spectral camera named Compovision. The NIR imaging system has a feature for high-speed and wide area monitoring and the two-dimensional scanning speed is almost 100 times faster than the general NIR imaging systems for the same pixel size. For the quantitative analysis of glycogen concentration, partial least squares regression (PLSR) and moving window PLSR (MWPLSR) were performed with the information of glycogen concentration measured by high performance liquid chromatography (HPLC) and the calibration curves for the concentration within the Synechocystis sp. PCC6803 cell were constructed. The results had high accuracy for the quantitative estimation of glycogen concentration as the best squared correlation coefficient R-2 was bigger than 0.99 and a root mean square error (RMSE) was less than 2.9%. The present results proved not only the potential for the applicability of NIR spectroscopy to the high-speed quantitative evaluation of glycogen concentration in the bioethanol feedstock but also the expansivity of the NIR imaging instrument to in-line or on-line product evaluation on a factory production line of bioethanol in the future.

    SAGE PUBLICATIONS INC, 2017年03月, Applied Spectroscopy, 71 (3), 463 - 471, 英語

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    研究論文(学術雑誌)

  • Izzat Fahimuddin Bin Mohamed Suffian, Mitla Garcia-Maya, Paul Brown, Tam Bui, Yuya Nishimura, Amir Rafiq Bin Mohammad Johari Palermo, Chiaki Ogino, Akihiko Kondo, Khuloud T. Al-Jamal

    An E. coli expression system offers a mean for rapid, high yield and economical production of Hepatitis B Virus core (HBc) particles. However, high-level production of HBc particles in bacteria is demanding and optimisation of HBc particle yield from E. coli is required to improve laboratory-scale productivity for further drug delivery applications. Production steps involve bacterial culture, protein isolation, denaturation, purification and finally protein assembly. In this study, we describe a modified E. coli based method for purifying HBc particles and compare the results with those obtained using a conventional purification method. HBc particle morphology was confirmed by Atomic Force Microscopy (AFM). Protein specificity and secondary structure were confirmed by Western Blot and Circular Dichroism (CD), respectively. The modified method produced similar to 3-fold higher yield and greater purity of wild type HBc particles than the conventional method. Our results demonstrated that the modified method produce a better yield and purity of HBc particles in an E. coli-expression system, which are fully characterised and suitable to be used for drug delivery applications.

    NATURE PUBLISHING GROUP, 2017年03月, SCIENTIFIC REPORTS, 7, 43160, 英語

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    研究論文(学術雑誌)

  • Jerome Amoah, Emmanuel Quayson, Shinji Hama, Ayumi Yoshida, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo

    The presence of high levels of free fatty acids (FFA) in oil is a barrier to one-step biodiesel production. Undesirable soaps are formed during conventional chemical methods, and enzyme deactivation occurs when enzymatic methods are used. This work investigates an efficient technique to simultaneously convert a mixture of free fatty acids and triglycerides (TAG). A partial soybean hydrolysate containing 73.04% free fatty acids and 24.81% triglycerides was used as a substrate for the enzymatic production of fatty acid methyl ester (FAME). Whole-cell Candida antarctica lipase B-expressing Aspergillus oryzae, and Novozym 435 produced only 75.2 and 73.5% FAME, respectively. Fusarium heterosporum lipase-expressing A. oryzae produced more than 93% FAME in 72 h using three molar equivalents of methanol. FFA and TAG were converted simultaneously in the presence of increasing water content that resulted from esterification. Therefore, F. heterosporum lipase with a noted high level of tolerance of water could be useful in the industrial production of biodiesel from feedstock that has high proportion of free fatty acids.

    WILEY-V C H VERLAG GMBH, 2017年03月, BIOTECHNOLOGY JOURNAL, 12 (3), 1600400, 英語

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    研究論文(学術雑誌)

  • Kenji Okano, Shinji Hama, Maki Kihara, Hideo Noda, Tsutomu Tanaka, Akihiko Kondo

    Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the EY-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8-17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4-129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.

    SPRINGER, 2017年03月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 101 (5), 1869 - 1875, 英語

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    研究論文(学術雑誌)

  • Musashi Takenaka, Takuya Kobayashi, Kentaro Inokuma, Tomohisa Hasunuma, Tatsuo Maruyama, Chiaki Ogino, Akihiko Kondo

    The surface of yeast cells has been an attractive interface for the effective use of cellulose. Surface enzymes, however, are difficult to visualize and evaluate. In this study, two kinds of unique anchoring regions were used to display the cellulase, endoglucanase (EG), on a yeast cell surface. Differences in the display level and the localization of EG were observed by atomic force microscopy. By surveying the yeast cell surface with a chemically modified cantilever, the interactive force between the cellulose and EG was measured. Force curve mapping revealed differences in the display levels and the localization of EG according to anchoring regions. The proposed methodology enables visualization of displayed enzymes such as EG on the yeast cell surface. (C) 2016 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2017年03月, COLLOIDS AND SURFACES B-BIOINTERFACES, 151, 134 - 142, 英語

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    研究論文(学術雑誌)

  • Jyumpei Kobayashi, Daisuke Sasaki, Kiyotaka Y. Hara, Tomohisa Hasunuma, Akihiko Kondo

    Background: Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes. Results: We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for gamma-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1(S32A) gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene. Conclusions: This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.

    BIOMED CENTRAL LTD, 2017年03月, MICROBIAL CELL FACTORIES, 16, 44, 英語

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    研究論文(学術雑誌)

  • Izzat Fahimuddin Bin Mohamed Suffian, Julie Tzu-Wen Wang, Naomi O. Hodgins, Rebecca Klippstein, Mitla Garcia-Maya, Paul Brown, Yuya Nishimura, Hamed Heidari, Sara Bals, Jane K. Sosabowski, Chiaki Ogino, Akihiko Kondo, Khuloud T. Al-Jamal

    Hepatitis B Virus core (HBc) particles have been studied for their potential as drug delivery vehicles for cancer therapy. HBc particles are hollow nano-particles of 30-34 nm diameter and 7 nm thick envelopes, consisting of 180-240 units of 21 kDa core monomers. They have the capacity to assemble/dis-assemble in a controlled manner allowing encapsulation of various drugs and other biomolecules. Moreover, other functional motifs, i.e. receptors, receptor binding sequences, peptides and proteins can be expressed. This study focuses on the development of genetically modified HBc particles to specifically recognise and target human epidermal growth factor receptor-2 (HER2)-expressing cancer cells, in vitro and in vivo, for future cancer therapy. The non-specific binding capacity of wild type HBc particles was reduced by genetic deletion of the sequence encoding arginine-rich domains. A specific HER2-targeting was achieved by expressing the ZHER2 affibodies on the HBc particles surface. In vitro studies showed specific uptake of ZHER2-AHBc particles in HER2 expressing cancer cells. In vivo studies confirmed positive uptake of ZHER2-ABBc particles in HER2-expressing tumours, compared to non-targeted AHBc particles in intraperitoneal tumour-bearing mice models. The present results highlight the potential of these nanocarriers in targeting HER2-positive metastatic abdominal cancer following intra-peritoneal administration. (C) 2016 The Authors. Published by Elsevier Ltd.

    ELSEVIER SCI LTD, 2017年03月, BIOMATERIALS, 120, 126 - 138, 英語

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    研究論文(学術雑誌)

  • Han-Hsiu Hsu, Michihiro Araki, Masao Mochizuki, Yoshimi Hori, Masahiro Murata, Prihardi Kahar, Takanobu Yoshida, Tomohisa Hasunuma, Akihiko Kondo

    Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput "omics" methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture.

    NATURE PUBLISHING GROUP, 2017年03月, SCIENTIFIC REPORTS, 7, 43518, 英語

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    研究論文(学術雑誌)

  • Musashi Takenaka, Yuzo Okumura, Tomokazu Amino, Yusuke Miyachi, Chiaki Ogino, Akihiko Kondo

    DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350 pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFMSELEX. (C) 2016 Elsevier Ltd. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2017年02月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 27 (4), 954 - 957, 英語

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    研究論文(学術雑誌)

  • Feng Zhang, Houng-Wei Wang, Keisuke Tominaga, Michitoshi Hayashi, Tomohisa Hasunuma, Akihiko Kondo

    This work illustrates several theoretical fundamentals for the application of THz vibrational spectroscopy to molecular characterization in the solid state using two different types of saccharide systems as examples. Four subjects have been specifically addressed: (1) the qualitative differences in the molecular vibrational signatures monitored by THz and mid-IR vibrational spectroscopy; (2) the selection rules for THz vibrational spectroscopy as applied to crystalline and amorphous systems; (3) a normal mode simulation, using a-l-xylose as an example; and (4) a rigorous mode analysis to quantify the percentage contributions of the intermolecular and intramolecular vibrations to the normal mode of interest.

    WILEY-V C H VERLAG GMBH, 2017年02月, CHEMISTRY-AN ASIAN JOURNAL, 12 (3), 324 - 331, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Application of LC-MS/MS analysis for time-lapse amino acid metabolomics in CHO cell culture.

    Hsu H, HASUNUMA Tomohisa, Araki Michihiro, Yoshida Takanobu, Hori Yoshimi, Murata Masahiro, KONDO Akihiko

    2017年02月, Shimadzu Journal, 5 (1), 17 - 21, 英語

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    研究論文(学術雑誌)

  • Shih-Hsin Ho, Yi-Di Chen, Ching-Yu Chang, Yen-Ying Lai, Chun-Yen Chen, Akihiko Kondo, Nan-Qi Ren, Jo-Shu Chang

    Background: Although outdoor cultivation systems have been widely used for mass production of microalgae at a relatively low cost, there are still limited efforts on outdoor cultivation of carbohydrate-rich microalgae that were further used as feedstock for fermentative bioethanol production. In particular, the effects of seasonal changes on cell growth, CO2 fixation, and carbohydrate production of the microalgae have not been well investigated. Results: This work demonstrates the feasibility of using outdoor tubular photobioreactors (PBR) for whole-year-round cultivation of a carbohydrate-rich microalga Scenedesmus obliquus CNW-N in southern Taiwan. Time-course profile of the carbohydrate content under nitrogen-deficient conditions was monitored to assess the seasonal changes. The optimal CO2 fixation rate and carbohydrate productivity were 430.2 mg L-1 d(-1)and 111.8 mg L(-1)d(-1), respectively, which were obtained during the summer time. Under nitrogen starvation, the microalgal biomass can accumulate nearly 45-50% of carbohydrates, mainly composed of glucose that accounted for 70-80% of the total carbohydrates in the microalgal cells. This glucose-rich microalgal biomass is apparently a very suitable carbon source for bioethanol fermentation. Conclusion: This work shows the feasibility of combining CO2 fixation and bioethanol production using microalgae grown in outdoor photobioreactors as feedstock. The understanding of the seasonal changes in the carbohydrate productivity makes this approach more practically viable. The novel strategy proposed in this study could be a promising alternative to the existing technology dealing with CO2 mitigation and biofuels production.

    BIOMED CENTRAL LTD, 2017年01月, BIOTECHNOLOGY FOR BIOFUELS, 10, 27, 英語

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    研究論文(学術雑誌)

  • Yuya Nishimura, Ryosuke Ezawa, Jun Ishii, Chiaki Ogino, Akihiko Kondo

    The expression of epidermal growth factor receptor (EGFR) across a wide range of tumor cells has attracted attention for use as a tumor marker in drug delivery systems. Therefore, binding molecules with the ability to target EGFR have been developed. Among them, we focused on affibodies that are binding proteins derived from staphylococcal protein A. By displaying affibody (Z(EGFR)) binding to EGFR on the surface of a bio-nanocapsule (BNC) derived from a hepatitis B virus (HBV), we developed an altered BNC (Z(EGFR)-BNC) with a high specificity to EGFR-expressing cells. We considered two different types of Z(EGFR) (Z955 and Z1907), and found that the Z1907 dimer-displaying BNC ([Z1907[(2)-BNC) could effectively bind to EGFR-expressing cells and deliver drugs to the cytosol. Since this study showed that [Z1907](2)-BNC could target EGFR-expressing cells, we would use this particle as a drug delivery carrier for various cancer cells expressing EGFR. (C) 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license.

    PERGAMON-ELSEVIER SCIENCE LTD, 2017年01月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 27 (2), 336 - 341, 英語

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    研究論文(学術雑誌)

  • Shibuya, M., Sasaki, K., Tanaka, Y., Yasukawa, M., Takahashi, T., Kondo, A., Matsuyama, H.

    2017年, Bioresource Technology, 235, 405 - 410, 英語

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    研究論文(学術雑誌)

  • Kentaro Inokuma, Jun Ishii, Kiyotaka Y. Hara, Masao Mochizuki, Tomohisa Hasunuma, Akihiko Kondo

    Volume 3, no. 2, e00389-15, 2015. Page 1, column 1, lines 26 and 27: "silencing mediator for retinoic acid and thyroid hormone receptor" should read "singlemolecule real-time."

    American Society for Microbiology, 2017年, Genome Announcements, 5 (5), 英語

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    研究論文(学術雑誌)

  • Kengo Sasaki, Daisuke Sasaki, Naoko Okai, Kosei Tanaka, Ryohei Nomoto, Itsuko Fukuda, Ken-Ichi Yoshida, Akihiko Kondo, Ro Osawa

    Accumulating evidence suggests that dietary taurine (2-aminoethanesulfonic acid) exerts beneficial anti-inflammatory effects in the large intestine. In this study, we investigated the possible impact of taurine on human colonic microbiota using our single-batch fermentation system (Kobe University Human Intestinal Microbiota Model; KUHIMM). Fecal samples from eight humans were individually cultivated with and without taurine in the KUHIMM. The results showed that taurine remained largely undegraded after 30 h of culturing in the absence of oxygen, although some 83% of the taurine was degraded after 30 h of culturing under aerobic conditions. Diversity in bacterial species in the cultures was analyzed by 16S rRNA gene sequencing, revealing that taurine caused no significant change in the diversity of the microbiota; both operational taxonomic unit and Shannon-Wiener index of the cultures were comparable to those of the respective source fecal samples. In addition, principal coordinate analysis indicated that taurine did not alter the composition of bacterial species, since the 16S rRNA gene profile of bacterial species in the original fecal sample was maintained in each of the cultures with and without taurine. Furthermore, metabolomic analysis revealed that taurine did not affect the composition of short-chain fatty acids produced in the cultures. These results, under these controlled but artificial conditions, suggested that the beneficial anti-inflammatory effects of dietary taurine in the large intestine are independent of the intestinal microbiota. We infer that dietary taurine may act directly in the large intestine to exert anti-inflammatory effects.

    2017年, PloS one, 12 (7), e0180991, 英語, 国際誌

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    研究論文(学術雑誌)

  • Hiroshi Teramura, Kengo Sasaki, Hideo Kawaguchi, Fumio Matsuda, Jun Kikuchi, Tomokazu Shirai, Takashi Sazuka, Masanori Yamasaki, Shigeo Takumi, Chiaki Ogino, Akihiko Kondo

    Bio-refinery processes require use of the most suitable lignocellulosic biomass for enzymatic saccharification and microbial fermentation. Glucose yield from biomass solid fractions obtained after dilute sulfuric acid (1%) pretreatment (at 180 degrees C) was investigated using 14, 8, and 16 varieties of rice, wheat, and sorghum, respectively. Biomass solid fractions of each crop showed similar cellulose content. However, glucose yield after enzymatic hydrolysis (cellulase loading at 6.6 filter paper unit/g-biomass) was different among the varieties of each crop, indicating genotypic differences for rice, wheat, and sorghum. Nuclear magnetic resonance method revealed that the high residual level of lignin aromatic regions decreased glucose yield from solid fraction of sorghum.

    TAYLOR & FRANCIS LTD, 2017年, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 81 (8), 1650 - 1656, 英語

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    研究論文(学術雑誌)

  • Ryutaro Morita, Hiroshi Teramura, Chiaki Ogino, Akihiko Kondo, Hiroshi Fukayama

    Production of bioethanol from rice straw has attracted attention from the point of effective use of agricultural residue. Starch content is an important determinant for bioethanol production from rice straw. The overexpression of CO2 -responsive CCT protein (CRCT), which is the positive regulator of starch synthesis in vegetative organs, notably increased the starch content in rice straw. To produce the bioethanol from rice straw, the dilute acid pretreatment is a general pretreatment method. Importantly, the glucose yields in liquid hydrolyzate after dilute acid pretreatment was markedly increased in the CRCT overexpression lines compared with non-transgenic rice. In addition, the overexpression of CRCT enhanced the biomass production. In contrast, CRCT did not affect on the glucose yields from cellulose in acid-insoluble residue obtained after dilute acid pretreatment. With respect to byproduct in liquid hydrolyzate which inhibits the fermentation, the formic acid content was increased, whereas the furfural, 5-hydroxymethylfurfural and acetic acid contents were unchanged by the overexpression of CRCT. These results demonstrate that genetic engineering of CRCT is an effective method to increase the bioethanol production from rice straw.

    TAYLOR & FRANCIS LTD, 2017年, PLANT PRODUCTION SCIENCE, 20 (4), 441 - 447, 英語

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    研究論文(学術雑誌)

  • Ryosuke Fujiwara, Shuhei Noda, Tsutomu Tanaka, Akihiko Kondo

    To produce styrene from a biomass-derived carbon source, Streptomyces lividans was adopted as a host strain. The gene encoding ferulic acid decarboxylase from Saccharomyces cerevisiae (FDC1) was introduced into S. lividans, and the resulting S. lividans transformant successfully expressed FDC1 and converted trans-cinnamic acid (CA) to styrene. A key factor in styrene production using microbes is the recovery of volatile styrene. In the present study, we selected polystyrene resin beads XRD-4 as the absorbent agent to recover styrene produced using S. lividans transformants, which enabled recovery of styrene from the culture broth. For styrene production from biomass-derived carbon sources, S. lividans/FDC1 was cultured together with S. lividans/p-encP, which we previously reported as a CA-producing S. lividans strain. This coculture system combined with the recovery of styrene using XAD-4 allowed the production of styrene from glucose, cellobiose, or xylo-oligosaccharide, respectively. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2016年12月, Journal of Bioscience and Bioengineering, 122 (6), 730 - 735, 英語

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    研究論文(学術雑誌)

  • Quentin Schmetz, Guillaume Maniet, Nicolas Jacquet, Hiroshi Teramura, Chiaki Ogino, Akihiko Kondo, Aurore Richel

    It is commonly accepted that the current society needs to partially substitute fossil resources by renewable ones. Among many solutions, one approach consists in the development of biorefinery involving lignocellulosic biomass to produce bio-based materials and fuels. This study focuses on the comprehension of an organosolv treatment designed to break the complex lignocellulosic structure for high purity lignin extraction from tall fescue (Festuca arundinacea Schreb.). This grass benefits from an increasing interest in Western Europe and has been suggested as feedstock for biorefinery. However, its use as material for high purity lignin production has not been determined yet. Ethanol/water, 92/8% [v/v] with H2SO4 0.32 M was investigated at pilot scale under conventional heating (5 degrees C min(-1) during 30 min and stabilized at 148 degrees C for 5 min). Precipitated lignin were analyzed as well as the composition of side stream products (recovered cellulosic pulp and the aqueous hydrolysate). Lignin has been recovered at a purity level of 90% with a yield of 60%. The main contaminants were nitrogen containing compounds and degraded hemicelluloses. 2D-HSQC NMR (Two Dimension-Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance) revealed a co-extraction of ferulates and coumarates function as well as arabinoxylan. Cellulose was recovered at 53% purity with 60% yield. The conditions appear to be too harsh for tall fescue and led to significant amount of cellulose degradation. A process using a lower alcohol concentration will be developed to provide better yields of both cellulose and lignin. (C) 2016 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2016年12月, INDUSTRIAL CROPS AND PRODUCTS, 94, 308 - 317, 英語

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    研究論文(学術雑誌)

  • Tomita Y, Yoshioka K, Iijima H, Nakashima A, Iwata O, Suzuki K, HASUNUMA Tomohisa, KONDO Akihiko, HIRAI MY, OSANAI T

    Euglena gracilis is a eukaryotic, unicellular phytoflagellate that has been widely studied in basic science and applied science. Under dark, anaerobic conditions, the cells of E. gracilis produce a wax ester that can be converted into biofuel. Here, we demonstrate that under dark, anaerobic conditions, E. gracilis excretes organic acids, such as succinate and lactate, which are bulk chemicals used in the production of bioplastics. The levels of succinate were altered by changes in the medium and temperature during dark, anaerobic incubation. Succinate production was enhanced when cells were incubated in CM medium in the presence of NaHCO3. Excretion of lactate was minimal in the absence of external carbon sources, but lactate was produced in the presence of glucose during dark, anaerobic incubation. E gracilis predominantly produced L lactate; however, the percentage of D-lactate increased to 28.4% in CM medium at 30 degrees C. Finally, we used a commercial strain of E. gracilis for succinate production and found that nitrogen-starved cells, incubated under dark, anaerobic conditions, produced 869.6 mg/L succinate over a 3-day incubation period, which was 70-fold higher than the amount produced by nitrogen-replete cells. This is the first study to demonstrate organic acid excretion by E. gracilis cells and to reveal novel aspects of primary carbon metabolism in this organism.

    FRONTIERS MEDIA SA, 2016年12月, Frontiers in Microbiology, 7, 2050, 英語

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    研究論文(学術雑誌)

  • Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells

    Nobuo Fukuda, Misato Kaishima, Jun Ishii, Akihiko Kondo, Shinya Honda

    2016年12月, AMB Express, 6, 45, 英語

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    研究論文(学術雑誌)

  • Daisuke Sasaki, Kengo Sasaki, Yota Tsuge, Akihiko Kondo

    The metabolic state of microflora (mixed microbial cultures) in microbial fuel cells (MFCs) is currently unclear. Metabolomic analyses were conducted of microflora growing on the anodic electrodes of MFCs operated at pH 7.0, 5.5, or 4.0 and utilizing starch as the major carbon substrate. A much higher current was produced at pH 7.0 than at pH 5.5 and 4.0, correlating with an increased population ratio of Geobacter species to the total bacteria growing on the electrode. Most intracellular metabolites related to the tricarboxylic acid (TCA) cycle were present at a higher level at pH 7.0 than at pH 5.5 and 4.0, and the levels of metabolites correlated well with the obtained current densities. A high intracellular adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio at pH 7.0, compared to at pH 5.5 and 4.0, likewise supported current production. Overall, the metabolomic analyses demonstrated that activation of the TCA cycle and increased ATP generation are critical parameters for electricity generation by microflora.

    BIOMED CENTRAL LTD, 2016年12月, AMB EXPRESS, 6, 125, 英語

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    研究論文(学術雑誌)

  • Kenta Morita, Serika Miyazaki, Chiya Numako, Shinya Ikeno, Ryohei Sasaki, Yuya Nishimura, Chiaki Ogino, Akihiko Kondo

    An induction of polyacrylic acid-modified titanium dioxide with hydrogen peroxide nanoparticles (PAA-TiO2/H2O2 NPs) to a tumor exerted a therapeutic enhancement of X-ray irradiation in our previous study. To understand the mechanism of the radiosensitizing effect of PAA-TiO2/H2O2 NPs, analytical observations that included DLS, FE-SEM, FT-IR, XAFS, and Raman spectrometry were performed. In addition, highly reactive oxygen species (hROS) which PAA-TiO2/H2O2 NPs produced with X-ray irradiation were quantified by using a chemiluminescence method and a EPR spin-trapping method. We found that PAA-TiO2/H2O2 NPs have almost the same characteristics as PAA-TiO2. Surprisingly, there were no significant differences in hROS generation. However, the existence of H2O2 was confirmed in PAA-TiO2/H2O2 NPs, because spontaneous hROS production was observed w/o X-ray irradiation. In addition, PAA-TiO2/H2O2 NPs had a curious characteristic whereby they absorbed H2O2 molecules and released them gradually into a liquid phase. Based on these results, the H2O2 was continuously released from PAA-TiO2/H2O2 NPs, and then released H2O2 assumed to be functioned indirectly as a radiosensitizing factor.

    TAYLOR & FRANCIS LTD, 2016年12月, FREE RADICAL RESEARCH, 50 (12), 1319 - 1328, 英語

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    研究論文(学術雑誌)

  • Hideo Kawaguchi, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo

    The feedstocks used for the production of bio-based chemicals have recently expanded from edible sugars to inedible and more recalcitrant forms of lignocellulosic biomass. To produce biobased chemicals from renewable polysaccharides, several bioprocessing approaches have been developed and include separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP). In the last decade, SHF, SSF, and CBP have been used to generate macromolecules and aliphatic and aromatic compounds that are capable of serving as sustainable, drop-in substitutes for petroleum-based chemicals. The present review focuses on recent progress in the bioprocessing of microbially produced chemicals from renewable feedstocks, including starch and lignocellulosic biomass. In particular, the technological feasibility of bio-based chemical production is discussed in terms of the feedstocks and different bioprocessing approaches, including the consolidation of enzyme production, enzymatic hydrolysis of biomass, and fermentation.

    CURRENT BIOLOGY LTD, 2016年12月, CURRENT OPINION IN BIOTECHNOLOGY, 42, 30 - 39, 英語

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    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Akihiko Kondo

    Wiley-VCH Verlag, 2016年11月26日, Industrial Biotechnology: Products and Processes, 161 - 185, 英語

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    論文集(書籍)内論文

  • Takuya Matsumoto, Kou Furuta, Tsutomu Tanaka, Akihiko Kondo

    We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling. Here, sortase A expression was controlled by the Inc promoter to trigger metabolic channeling by the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG). We tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A. These results show that sortase A-mediated enzyme ligation enhances an acetate producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation offers a metabolic channeling approach to redirect a central flux to a desired flux.

    AMER CHEMICAL SOC, 2016年11月, ACS SYNTHETIC BIOLOGY, 5 (11), 1284 - 1289, 英語

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    研究論文(学術雑誌)

  • Kentaro Inokuma, Takahiro Bamba, Jun Ishii, Yoichiro Ito, Tomohisa Hasunuma, Akihiko Kondo

    Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived fromS. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae alpha-mating pheromone (MF alpha 1SP) were constructed for cell-surface display of Aspergillus aculeatus beta-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MF alpha 1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MF alpha 1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. (C) 2016 Wiley Periodicals, Inc.

    WILEY-BLACKWELL, 2016年11月, BIOTECHNOLOGY AND BIOENGINEERING, 113 (11), 2358 - 2366, 英語

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    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Takamichi Hashimoto, Jun Ishii, Akihiko Kondo

    G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that represent major molecular targets in pharmaceutical and medicinal fields. Many GPCRs have been shown to form not only homodimers, but also heterodimers that can confer large functional and physiological diversity and are therefore expected to offer new opportunities for the discovery of new drugs. Yeast is a useful host organism that can be used to investigate the interactions between eukaryotic protein pairs, as demonstrated by the yeast two-hybrid (Y2H) system. Previously, we established reporter gene assay systems to screen for GPCR dimer pairs based on the splitubiquitin membrane Y2H (mY2H) system. However, conventional systems only induce reporter gene expressions from the OFF to ON states. In this study, we therefore designed a reporter switching system that can switch the expressions between two reporter genes (one from ON to OFF and the other from OFF to ON) in response to the Y2H readout. To invoke reporter switching, we took advantage of Cre/loxP site-specific recombination. Through optimization of Cre-mediated reporter gene recombination using the split-ubiquitin mY2H system, we built a dual-color reporter switching system to discern the formations of GPCR dimers. This system enabled monitoring of the formations of homodimers and heterodimers of human serotonin 1A receptor or beta(2)-adrenergic receptor as well as homodimers of the yeast endogenous pheromone receptor (Ste2p) in yeast cells. Our reporter switching system may be a useful tool for identifying potential molecular targets among GPCR dimers, and is also applicable to other reporter gene assay systems. (C) 2016 Wiley Periodicals, Inc.

    WILEY-BLACKWELL, 2016年10月, BIOTECHNOLOGY AND BIOENGINEERING, 113 (10), 2178 - 2190, 英語

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    研究論文(学術雑誌)

  • Kentaro Inokuma, Tomohisa Hasunuma, Akihiko Kondo

    N-acetyl-D-glucosamine (GlcNAc) is the building block of chitin, which is one of the most abundant renewable resources in nature after cellulose. Therefore, a microorganism that can utilize GlcNAc is necessary for chitin-based biorefinery. In this study, we report on the screening and characterization of yeast strains for bioethanol production from GlcNAc. We demonstrate that Scheffersomyces (Pichia) stipitis strains can use GlcNAc as the sole carbon source and produce ethanol. S. stipitis NBRC1687, 10007, and 10063 strains consumed most of the 50 g/L GlcNAc provided, and produced 14.5 +/- 0.6, 15.0 +/- 0.3, and 16.4 +/- 0.3 g/L of ethanol after anaerobic fermentation at 30 degrees C for 96 h. The ethanol yields of these strains were approximately 81, 75, and 82 % (mol ethanol/mol GlcNAc consumed), respectively. Moreover, S. stipitis NBRC10063 maintained high GlcNAc-utilizing capacity at 35 degrees C, and produced 12.6 +/- 0.7 g/L of ethanol after 96 h. This strain also achieved the highest ethanol titer (23.3 +/- 1.0 g/L) from 100 g/L GlcNAc. To our knowledge, this is the first report on ethanol production via fermentation of GlcNAc by naturally occurring yeast strains.

    BIOMED CENTRAL LTD, 2016年10月, AMB EXPRESS, 6, 83, 英語

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    研究論文(学術雑誌)

  • Yoshifumi Ueno, Shimpei Aikawa, Akihiko Kondo, Seiji Akimoto

    Cyanobacteria and red algae control the energy distributions of two photosystems (PSI and PSII) by changing the energy transfer among phycobilisome (PBS), PSI, and PSII. However, whether PSII -> PSI energy transfer (spillover) occurs in the intact megacomplexes composed of PBS, PSI, and PSII (PBS-PSII-PSI megacomplexes) in vivo remains controversial. In this study, we measured the delayed fluorescence spectra of PBS-selective excitation in cyanobacterial and red algal cells. In the absence of spillover, 7% of the PBS (at most) would combine with PSII, inconsistent with the PBSs' function as the antenna pigment-protein complexes of PSII. Therefore, we conclude that spillover occurs in vivo in PBS-PSII-PSI megacomplexes of both cyanobacteria and red algae.

    AMER CHEMICAL SOC, 2016年09月, JOURNAL OF PHYSICAL CHEMISTRY LETTERS, 7 (18), 3567 - 3571, 英語

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    研究論文(学術雑誌)

  • Ryosuke Fujiwara, Shuhei Noda, Yoshifumi Kawai, Tsutomu Tanaka, Akihiko Kondo

    To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species. The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme's k(cat)/K-m value was 0.54 mM (-1) s(-1). Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l(-1) from 10 g glucose l(-1). A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

    SPRINGER, 2016年09月, BIOTECHNOLOGY LETTERS, 38 (9), 1543 - 1549, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Mami Okamoto, Tomokazu Shirai, Yota Tsuge, Ayami Fujino, Daisuke Sasaki, Masahiko Morita, Fumio Matsuda, Jun Kikuchi, Akihiko Kondo

    Rice straw was mechanically milled using a process consuming 1.9 MJ/kg-biomass, and 10 g/L of unmilled or milled rice straw was used as the carbon source for methane fermentation in a digester containing carbon fiber textile as the supporting material. Milling increased methane production from 226 to 419 mL/L/day at an organic loading rate of 2180 mg-dichromate chemical oxygen demand/L/day, corresponding to 260 mL(CH4/gVS). Storage of the fermentation effluent at room temperature decreased the weight of the milled rice straw residue from 3.81 to 1.00 g/L. The supernatant of the effluent was subjected to nanofiltration. The black concentrates deposited on the nanofiltration membranes contained 53.0-57.9% lignin. Solution nuclear magnetic resonance showed that lignin aromatic components such as p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) were retained primarily, and major lignin interunit structures such as the beta-O-4-H/G unit were absent. This combinational process will aid the complete utilization of rice straw. (C) 2016 Published by Elsevier Ltd.

    ELSEVIER SCI LTD, 2016年09月, BIORESOURCE TECHNOLOGY, 216, 830 - 837, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Keiji Nishida, Takayuki Arazoe, Nozomu Yachie, Satomi Banno, Mika Kakimoto, Mayura Tabata, Masao Mochizuki, Aya Miyabe, Michihiro Araki, Kiyotaka Y. Hara, Zenpei Shimatani, Akihiko Kondo

    The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.

    AMER ASSOC ADVANCEMENT SCIENCE, 2016年09月, SCIENCE, 353 (6305), aaf8729, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Zhuo Liu, Shih-Hsin Ho, Tomohisa Hasunuma, Jo-Shu Chang, Nan-Qi Ren, Akihiko Kondo

    Waste biorefinery aims to maximize the output of value-added products from various artificial/agricultural wastes by using integrated bioprocesses. To make waste biorefinery economically feasible, it is thus necessary to develop a low-cost, environment-friendly technique to perform simultaneous biodegradation and bioconversion of waste materials. Cell-surface display engineering is a novel, cost-effective technique that can auto-immobilize proteins on the cell exterior of microorganisms, and has been applied for use with waste biofinery. Through tethering different enzymes (e.g., cellulase, lipase, and protease) or metal-binding peptides on cell surfaces, various yeast strains can effectively produce biofuels and biochemicals from sugar/protein-rich waste materials, catalyze waste oils into biodiesels, or retrieve heavy metals from wastewater. This review critically summarizes recent applications of yeast cell-surface display on various types of waste biorefineries, highlighting its potential and future challenges with regard to commercializing this technology. (C) 2016 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2016年09月, BIORESOURCE TECHNOLOGY, 215, 324 - 333, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Iijima H, Shirai T, Okamoto M, Pinto F, Tamagnini P, Hasunuma Tomohisa, KONDO Akihiko, HIRAI Y, OSANAI T

    Cyanobacterial hydrogenases are important owing to the association between hydrogen metabolism and cell physiology and the production of future renewable energy. Many studies have examined hydrogen productivity, transcriptional regulation of hydrogenases, and the biochemistry of hydrogenases; however the relationship between hydrogen and primary carbon metabolism using metabolomic techniques has not been elucidated. Here, we studied the effect of the genetic manipulation of a hydrogenase on primary carbon metabolism in the model unicellular cyanobacterium Synechocystis sp. PCC 6803. Metabolomic analysis revealed that the hoxH mutant with reduced hoxH transcripts exhibited increased sugar phosphates in dark, anaerobic conditions. Organic acids, lactate, succinate, fumarate, and malate increased substantially by the hoxH mutation both inside and outside of cells in dark, anaerobic conditions. Transcriptome analysis revealed higher expression of genes encoding the RNA polymerase sigma factor SigE, which is a positive regulator of sugar catabolism, and 6-phosphogluconate dehydrogenase in the hoxH mutant than in the wild-type strain. Immunoblotting results showed that sugar catabolic enzymes and SigE proteins increased in the hoxH mutant. These results demonstrate the wide alterations of primary metabolism by the genetic manipulation of a hydrogenase subunit in this cyanobacterium. (C) 2016 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2016年09月, Algal Research, 18, 305 - 313, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Jun Ishii, Fumiyoshi Okazaki, Apridah Cameliawati Djohan, Kiyotaka Y. Hara, Nanami Asai-Nakashima, Hiroshi Teramura, Ade Andriani, Masahiro Tominaga, Satoshi Wakai, Prihardi Kahar, Yopi, Bambang Prasetya, Chiaki Ogino, Akihiko Kondo

    Background: Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays beta-mannanase and beta-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. Results: Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered beta-mannanase and beta-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-beta-D-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-beta-D-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. Conclusions: We successfully displayed beta-mannanase and beta-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying beta-mannanase and beta-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering beta-mannanase and beta-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.

    BIOMED CENTRAL LTD, 2016年09月, Biotechnology for Biofuels, 9 (1), 188, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Keisuke Yamamoto, Kiyotaka Y. Hara, Toshihiko Morita, Akira Nishimura, Daisuke Sasaki, Jun Ishii, Chiaki Ogino, Noriyuki Kizaki, Akihiko Kondo

    Background: Red yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an antioxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed. Results: Xanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain. Conclusions: In this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in X. dendrorhous.

    BIOMED CENTRAL LTD, 2016年09月, MICROBIAL CELL FACTORIES, 15 (1), 155, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Wang Y, Chiu S. Y, Ho H. S, Liu Z, Liu Z. HASUNUMA Tomohi, Hasunuma T, Chang T. T, Chang F. K, Ren Q. N, KONDO Akihiko

    Biofuels from microalgae is now a hot issue of great potential. However, achieving high starch productivity with photoautotrophic microalgae is still challenging. A feasible approach to enhance the growth and target product of microalgae is to conduct mixotrophic cultivation. The appropriate acetate addition combined with CO2 supply as dual carbon sources (i.e., mixotrophic cultivation) could enhance the cell growth of some microalgae species, but the effect of acetate-mediated mixotrophic culture mode on carbohydrate accumulation in microalgae remains unclear. Moreover, there is still lack of the information concerning how to increase the productivity of carbohydrates from microalgae under acetate-amended mixotrophic cultivation and how to optimize the engineering strategies to achieve the goal. This study was undertaken to develop an optimal acetate-contained mixotrophic cultivation system coupled with effective operation strategies to markedly improve the carbohydrate productivity of Chlorella sorokiniana NIES-2168. The optimal carbohydrate productivity of 695 mg/L/d was obtained, which is the highest value ever reported. The monosaccharide in the accumulated carbohydrates is mainly glucose (i.e., 85-90%), which is very suitable for bio-alcohols fermentation. Hence, by applying the optimal process developed in this study, C. sorokiniana NIES-2168 has a high potential to serve as a feedstock for subsequent biofuels conversion.

    WILEY-V C H VERLAG GMBH, 2016年08月, Biotechnology Journal, 11 (8), 1072 - 1081, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Sakiko Ueda, Yuhki Kawamura, Hiroko Iijima, Mitsuharu Nakajima, Tomokazu Shirai, Mami Okamoto, Akihiko Kondo, Masami Yokota Hirai, Takashi Osanai

    Potassium (K+) is an essential macronutrient for all living organisms including cyanobacteria. Cyanobacteria are a group of bacteria performing oxygenic photosynthesis, widely studied in basic and applied sciences. The primary metabolism of the unicellular cyanobacterium Synechocystis sp. PCC 6803 is altered by environmental conditions, and it excretes organic acids and hydrogen under dark, anaerobic conditions. Here we demonstrated that K+ widely changes the primary carbon metabolism of this cyanobacterium. Succinate and lactate excretion from the cells incubated under dark, anaerobic conditions was enhanced in the presence of K+, while hydrogen production was repressed. The addition of K+ and the genetic manipulation of acetate kinase AckA and an RNA polymerase sigma factor SigE additively increased succinate and lactate production to 141.0 and 217.6 mg/L, which are 11 and 46 times, compared to the wild-type strain without K+, respectively. Intracellular levels of 2-oxoglutarate, succinate, fumarate, and malate increased by K+ under dark, anaerobic conditions. This study provides the evidence of the considerable effect of K+ on the biosynthesis of anionic metabolites in a unicellular cyanobacterium.

    NATURE PUBLISHING GROUP, 2016年08月, SCIENTIFIC REPORTS, 6, 32354, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yue Wang, Sheng-Yi Chiu, Shih-Hsin Ho, Zhuo Liu, Tomohisa Hasunuma, Ting-Ting Chang, Kuan-Fu Chang, Jo-Shu Chang, Nan-Qi Ren, Akihiko Kondo

    Biofuels from microalgae is now a hot issue of great potential. However, achieving high starch productivity with photoautotrophic microalgae is still challenging. A feasible approach to enhance the growth and target product of microalgae is to conduct mixotrophic cultivation. The appropriate acetate addition combined with CO2 supply as dual carbon sources (i.e., mixotrophic cultivation) could enhance the cell growth of some microalgae species, but the effect of acetate-mediated mixotrophic culture mode on carbohydrate accumulation in microalgae remains unclear. Moreover, there is still lack of the information concerning how to increase the productivity of carbohydrates from microalgae under acetate-amended mixotrophic cultivation and how to optimize the engineering strategies to achieve the goal. This study was undertaken to develop an optimal acetate-contained mixotrophic cultivation system coupled with effective operation strategies to markedly improve the carbohydrate productivity of Chlorella sorokiniana NIES-2168. The optimal carbohydrate productivity of 695 mg/L/d was obtained, which is the highest value ever reported. The monosaccharide in the accumulated carbohydrates is mainly glucose (i.e., 85-90%), which is very suitable for bio-alcohols fermentation. Hence, by applying the optimal process developed in this study, C. sorokiniana NIES-2168 has a high potential to serve as a feedstock for subsequent biofuels conversion.

    WILEY-V C H VERLAG GMBH, 2016年08月, BIOTECHNOLOGY JOURNAL, 11 (8), 1072 - 1081, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Risa Takagi, Kengo Sasaki, Daisuke Sasaki, Itsuko Fukuda, Kosei Tanaka, Ken-ichi Yoshida, Akihiko Kondo, Ro Osawa

    We devised a single-batch fermentation system to simulate human colonic microbiota from fecal samples, enabling the complex mixture of microorganisms to achieve densities of up to 1011 cells/mL in 24 h. 16S rRNA gene sequence analysis of bacteria grown in the system revealed that representatives of the major phyla, including Bacteroidetes, Firmicutes, and Actinobacteria, as well as overall species diversity, were consistent with those of the original feces. On the earlier stages of fermentation (up to 9 h), trace mixtures of acetate, lactate, and succinate were detectable; on the later stages (after 24 h), larger amounts of acetate accumulated along with some of propionate and butyrate. These patterns were similar to those observed in the original feces. Thus, this system could serve as a simple model to simulate the diversity as well as the metabolism of human colonic microbiota. Supplementation of the system with several prebiotic oligosaccharides (including fructo-, galacto-, isomalto-, and xylo-oligosaccharides; lactulose; and lactosucrose) resulted in an increased population in genus Bifidobacterium, concomitant with significant increases in acetate production. The results suggested that this fermentation system may be useful for in vitro, preclinical evaluation of the effects of prebiotics prior to testing in humans.

    PUBLIC LIBRARY SCIENCE, 2016年08月, PLOS ONE, 11 (8), e0160533, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tanimura K, Matsumot, T., T, Nakayama H, 田中 勉, 近藤 昭彦

    We successfully enhanced the productivity of ectoine with Halomonas elongata by improvement of the transport of sugar. First, we carried out screening for sugar transporters capable of improving glucose and xylose consumption. We found two transporters: b3657 from Escherichia coli, which is capable of improving glucose consumption, and HEO_0208 from H. elongata, which is capable of improving xylose consumption. Using transporter-overexpressing strains, the productivity of ectoine was improved. These results indicate that sugar consumption is important for efficient ectoine production. As result of phenotypic analysis of a HEO_0208 deletion strain, we discovered that HEO_0208 is the major xylose transporter in H. elongata. This is the first report demonstrating improvement of ectoine productivity by enhancing the transport of sugar. (C) 2016 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2016年07月, Enzyme and Microbial Technology, 89, 63 - 68, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tanimura K, Takashima S, Matsumoto T, 田中 勉, 近藤 昭彦

    We engineered efficient 2,3-butanediol (23BD) production from cellobiose using Bacillus subtilis. First, we found that B. subtilis harboring an empty vector could produce 23BD from cellobiose. However, productivity using cellobiose as a carbon source was lower than that when using glucose. This lower productivity was improved by adding purified beta-glucosidase from Thermobifida fusca YX (Tfu_0937) in the fermentation. Encouraged by these findings, we found that hydrolysis of cellobiose to glucose was an important reaction of 23BD biosynthesis in B. subtilis using cellobiose. Hence, we created efficient 23BD production from cellobiose using exogenous Tfu_0937-expressing B. subtilis. Using the engineered strain, 21.2 g L-1 of 23BD was produced after 72 h of cultivation. The productivity and yield were 0.294 g L-1 h(-1) and 0.35 g 23BD/g cellobiose, respectively. We successfully demonstrated efficient 23BD production from cellobiose by using BGL-expressing B. subtilis.

    SPRINGER, 2016年07月, Applied Microbiology and Biotechnology, 100 (13), 5781 - 5789, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Masao Nakayama, Ryohei Sasaki, Chiaki Ogino, Tsutomu Tanaka, Kenta Morita, Mitsuo Umetsu, Satoshi Ohara, Zhenquan Tan, Yuya Nishimura, Hiroaki Akasaka, Kazuyoshi Sato, Chiya Numako, Seiichi Takami, Akihiko Kondo

    Background: Biological applications of nanoparticles are rapidly increasing, which introduces new possibilities to improve the efficacy of radiotherapy. Here, we synthesized titanium peroxide nanoparticles (TiOxNPs) and investigated their efficacy as novel agents that can potently enhance the effects of radiation in the treatment of pancreatic cancer. Methods: TiOxNPs and polyacrylic acid-modified TiOxNPs (PAA-TiOxNPs) were synthesized from anatase-type titanium dioxide nanoparticles (TiO(2)NPs). The size and morphology of the PAA-TiOxNPs was evaluated using transmission electron microscopy and dynamic light scattering. The crystalline structures of the TiO(2)NPs and PAA-TiOxNPs with and without X-ray irradiation were analyzed using X-ray absorption. The ability of TiOxNPs and PAA-TiOxNPs to produce reactive oxygen species in response to X-ray irradiation was evaluated in a cell-free system and confirmed by flow cytometric analysis in vitro. DNA damage after X-ray exposure with or without PAA-TiOxNPs was assessed by immunohistochemical analysis of gamma-H2AX foci formation in vitro and in vivo. Cytotoxicity was evaluated by a colony forming assay in vitro. Xenografts were prepared using human pancreatic cancer MIAPaCa-2 cells and used to evaluate the inhibition of tumor growth caused by X-ray exposure, PAA-TiOxNPs, and the combination of the two. Results: The core structures of the PAA-TiOxNPs were found to be of the anatase type. The TiOxNPs and PAA-TiOxNPs showed a distinct ability to produce hydroxyl radicals in response to X-ray irradiation in a dose-and concentration-dependent manner, whereas the TiO(2)NPs did not. At the highest concentration of TiOxNPs, the amount of hydroxyl radicals increased by >8.5-fold following treatment with 30 Gy of radiation. The absorption of PAA-TiOxNPs enhanced DNA damage and resulted in higher cytotoxicity in response to X-ray irradiation in vitro. The combination of the PAA-TiOxNPs and X-ray irradiation induced significantly stronger tumor growth inhibition compared to treatment with either PAA-TiOxNPs or X-ray alone (p < 0.05). No apparent toxicity or weight loss was observed for 43 days after irradiation. Conclusions: TiOxNPs are potential agents for enhancing the effects of radiation on pancreatic cancer and act via hydroxyl radical production; owing to this ability, they can be used for pancreatic cancer therapy in the future.

    BIOMED CENTRAL LTD, 2016年07月, RADIATION ONCOLOGY, 11 (1), 91, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Li Shen, Yuya Nishimura, Fumio Matsuda, Jun Ishii, Akihiko Kondo

    2-Phenylethanol (2-PE) is a higher aromatic alcohol that is used in the cosmetics and food industries. The budding yeast Saccharomyces cerevisiae is considered to be a suitable host for the industrial production of higher alcohols, including 2-PE. To produce 2-PE from glucose in S. cerevisiae, we searched for suitable 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes of the Ehrlich pathway for overexpression in strain YPH499, and found that overexpression of the ARO10 and/or ADH1 genes increased 2-PE production from glucose. Further, we screened ten BY4741 single-deletion mutants of genes involved in the competing pathways for 2-PE production, and found that strains aro8 Delta and aat2 Delta displayed increased 2-PE production. Based on these results, we engineered a BY4741 strain that overexpressed ARO10 and contained an aro8 Delta deletion, and demonstrated that the strain produced 96 mg/L 2-PE from glucose as the sole carbon source. As this engineered S. cerevisiae strain showed a significant increase in 2-PE production from glucose without the addition of an intermediate carbon substrate, it is a promising candidate for the large-scale production of 2-PE. (C) 2015 The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2016年07月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 122 (1), 34 - 39, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Jerome Amoah, Shih-Hsin Ho, Shinji Hama, Ayumi Yoshida, Akihito Nakanishi, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo

    The presence of phospholipid has been a challenge in liquid enzymatic biodiesel production. Among six lipases that were screened, lipase AY had the highest hydrolysis activity and a competitive transesterification activity. However, it yielded only 21.1% FAME from oil containing phospholipids. By replacing portions of these lipases with a more robust bioFAME lipase, CalT, the combination of lipase AY-CalT gave the highest FAME yield with the least amounts of free fatty acids and partial glycerides. A higher methanol addition rate reduced FAME yields for lipase DF-CalT and A10D-CalT combinations while that of lipase AY-CalT combination improved. Optimizing the methanol addition rate for lipase AY-CalT resulted in a FAME yield of 88.1% at 2 h and more than 95% at 6 h. This effective use of lipases could be applied for the rapid and economic conversion of unrefined oils to biodiesel. (C) 2016 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2016年07月, BIORESOURCE TECHNOLOGY, 211, 224 - 230, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

    Misato Kaishima, Jun Ishii, Toshihide Matsuno, Nobuo Fukuda, Akihiko Kondo

    2016年06月, Scientific Reports, 6, 35932, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takashi Goda, Hiroshi Teramura, Miki Suehiro, Kengo Kanamaru, Hideo Kawaguchi, Chiaki Ogino, Akihiko Kondo, Masanori Yamasaki

    Rice straw is a promising resource for bioethanol production. Because the glucose content of pretreatment liquid hydrolysates is highly correlated with ethanol yield, the selection of appropriate rice cultivars is essential. The glucose content in liquid hydrolysates of pretreated rice straws of 208 diverse cultivars was evaluated in natural field in 2013 and 2014 using a novel high-throughput system. The glucose content of the rice straw samples varied across cultivars and was affected by environmental factors such as temperature and solar radiation. Several high-quality cultivars exhibiting high glucose content in both years were identified. The results of this study can aid in development of novel rice cultivars suitable as both feedstocks for bioethanol production and cooking.

    TAYLOR & FRANCIS LTD, 2016年05月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 80 (5), 863 - 869, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Improved sugar-free succinate production by Synechocystis sp. PCC 6803 following identification of the limiting steps in glycogen catabolism.

    HASUNUMA Tomohisa, MATSUDA Mami, KONDO Akihiko

    2016年05月, Metabolic Engineering Communications, 3, 130 - 141, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Jun-ichiro Hattan, Kazutoshi Shindo, Tomoko Ito, Yurica Shibuya, Arisa Watanabe, Chie Tagaki, Fumina Ohno, Tetsuya Sasaki, Jun Ishii, Akihiko Kondo, Norihiko Misawa

    A novel terpene synthase ( Tps ) gene isolated from Camellia brevistyla was identified as hedycaryol synthase, which was shown to be expressed specifically in flowers. Camellia plants are very popular because they bloom in winter when other plants seldom flower. Many ornamental cultivars of Camellia have been bred mainly in Japan, although the fragrance of their flowers has not been studied extensively. We analyzed floral scents of several Camellia cultivars by gas chromatography-mass spectrometry (GC-MS) and found that Camellia brevistyla produced various sesquiterpenes in addition to monoterpenes, whereas Camellia japonica and its cross-lines produced only monoterpenes, including linalool as the main product. From a flower of C. brevistyla, we isolated one cDNA encoding a terpene synthase (TPS) comprised of 554 amino acids, which was phylogenetically positioned to a sole gene clade. The cDNA, designated CbTps1, was expressed in mevalonate-pathway-engineered Escherichia coli, which carried the Streptomyces mevalonate-pathway gene cluster in addition to the acetoacetate-CoA ligase gene. A terpene product was purified from recombinant E. coli cultured with lithium acetoacetate, and analyzed by H-1-nulcear magnetic resonance spectroscopy (H-1-NMR) and GC-MS. It was shown that a sesquiterpene hedycaryol was produced, because H-1-NMR signals of the purified product were very broad, and elemol, a thermal rearrangement product from hedycaryol, was identified by GC-MS analysis. Spectroscopic data of elemol were also determined. These results indicated that the CbTps1 gene encodes hedycaryol synthase. Expression analysis of CbTps1 showed that it was expressed specifically in flowers, and hedycaryol is likely to be one of the terpenes that attract insects for pollination of C. brevistyla. A linalool synthase gene, which was isolated from a flower of Camellia saluenensis, is also described.

    SPRINGER, 2016年04月, PLANTA, 243 (4), 959 - 972, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Liu Z, Ho S.H, Sasaki Kengo, Haan R, Inokuma Kentaro, Ogino Chiaki, van Zyl W.H, Hasunuma Tomohisa, Kondo Akihiko

    Cellulosic biofuel is the subject of increasing attention. The main obstacle toward its economic feasibility is the recalcitrance of lignocellulose requiring large amount of enzyme to break. Several engineered yeast strains have been developed with cellulolytic activities to reduce the need for enzyme addition, but exhibiting limited effect. Here, we report the successful engineering of a cellulose-adherent Saccharomyces cerevisiae displaying four different synergistic cellulases on the cell surface. The cellulase-displaying yeast strain exhibited clear cell-to-cellulose adhesion and a "tearing" cellulose degradation pattern; the adhesion ability correlated with enhanced surface area and roughness of the target cellulose fibers, resulting in higher hydrolysis efficiency. The engineered yeast directly produced ethanol from rice straw despite a more than 40% decrease in the required enzyme dosage for high-density fermentation. Thus, improved cell-to-cellulose interactions provided a novel strategy for increasing cellulose hydrolysis, suggesting a mechanism for promoting the feasibility of cellulosic biofuel production.

    NATURE PUBLISHING GROUP, 2016年04月, Scientific Reports, 6, 24550, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Gregory Guirimand, Kengo Sasaki, Kentaro Inokuma, Takahiro Bamba, Tomohisa Hasunuma, Akihiko Kondo

    Xylitol, a value-added polyol deriving from D-xylose, is widely used in both the food and pharmaceutical industries. Despite extensive studies aiming to streamline the production of xylitol, the manufacturing cost of this product remains high while demand is constantly growing worldwide. Biotechnological production of xylitol from lignocellulosic waste may constitute an advantageous and sustainable option to address this issue. However, to date, there have been few reports of biomass conversion to xylitol. In the present study, xylitol was directly produced from rice straw hydrolysate using a recombinant Saccharomyces cerevisiae YPH499 strain expressing cytosolic xylose reductase (XR), along with beta-glucosidase (BGL), xylosidase (XYL), and xylanase (XYN) enzymes (co-) displayed on the cell surface; xylitol production by this strain did not require addition of any commercial enzymes. All of these enzymes contributed to the consolidated bioprocessing (CBP) of the lignocellulosic hydrolysate to xylitol to produce 5.8 g/L xylitol with 79.5 % of theoretical yield from xylose contained in the biomass. Furthermore, nanofiltration of the rice straw hydrolysate provided removal of fermentation inhibitors while simultaneously increasing sugar concentrations, facilitating high concentration xylitol production (37.9 g/L) in the CBP. This study is the first report (to our knowledge) of the combination of cell surface engineering approach and membrane separation technology for xylitol production, which could be extended to further industrial applications.

    SPRINGER, 2016年04月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100 (8), 3477 - 3487, 英語

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    研究論文(学術雑誌)

  • Twigged streptavidin polymer as a scaffold for protein assembly.

    Matsumoto T, Isogawa Y, Minamihata K, Tanaka Tsutomu, Kondo Akihiko

    2016年03月, Journal of Biotechnology, 255, 61 - 66, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yota Tsuge, Motonori Kudou, Hideo Kawaguchi, Jun Ishii, Tomohisa Hasunuma, Akihiko Kondo

    Lignocellulosic hydrolysates contain compounds that inhibit microbial growth and fermentation, thereby decreasing the productivity of biofuel and biochemical production. In particular, the heterocyclic aldehyde furfural is one of the most toxic compounds found in these hydrolysates. We previously demonstrated that Corynebacterium glutamicum converts furfural into the less toxic compounds furfuryl alcohol and 2-furoic acid. To date, however, the genes involved in these oxidation and reduction reactions have not been identified in the C. glutamicum genome. Here, we show that Cgl0331 (designated FudC) is mainly responsible for the reduction of furfural into furfuryl alcohol in C. glutamicum. Deletion of the gene encoding FudC markedly diminished the in vivo reduction of furfural to furfuryl alcohol. Purified His-tagged FudC protein from Escherichia coli was also shown to convert furfural into furfuryl alcohol in an in vitro reaction utilizing NADPH, but not NADH, as a cofactor. Kinetic measurements demonstrated that FudC has a high affinity for furfural but has a narrow substrate range for other aldehydes compared to the protein responsible for furfural reduction in E. coli.

    SPRINGER, 2016年03月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100 (6), 2685 - 2692, 英語

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    研究論文(学術雑誌)

  • Yixing Zhang, Amit Kumar, Philip R. Hardwidge, Tsutomu Tanaka, Akihiko Kondo, Praveen V. Vadlani

    d-lactic acid is of great interest because of increasing demand for biobased poly-lactic acid (PLA). Blending poly-l-lactic acid with poly-d-lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d-lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l-lactate-deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ldhL1-pCU-PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d-lactic acid yield and productivity. d-lactic acid (27.3 g L-1) and productivity (0.75 g L-1 h(-1)) was obtained from corn stover and d-lactic acid (22.0 g L-1) and productivity (0.65 g L-1 h(-1)) was obtained from sorghum stalks using ldhL1-pCU-PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d-lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d-lactic acid. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271-278, 2016

    WILEY, 2016年03月, Biotechnology Progress, 32 (2), 271 - 278, 英語

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    研究論文(学術雑誌)

  • Disruption of PHO13 improves ethanol production via the xylose isomerase pathway

    BAMBA Takahiro, HASUNUMA Tomohisa, KONDO Akihiko

    2016年03月, AMB Express, 6 (1), 4, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroshi Teramura, Kengo Sasaki, Tomoko Oshima, Fumio Matsuda, Mami Okamoto, Tomokazu Shirai, Hideo Kawaguchi, Chiaki Ogino, Ko Hirano, Takashi Sazuka, Hidemi Kitano, Jun Kikuchi, Akihiko Kondo

    Background: The primary components of lignocellulosic biomass such as sorghum bagasse are cellulose, hemicellulose, and lignin. Each component can be utilized as a sustainable resource for producing biofuels and bio-based products. However, due to their complicated structures, fractionation of lignocellulosic biomass components is required. Organosolv pretreatment is an attractive method for this purpose. However, as organosolv pretreatment uses high concentrations of organic solvents (>50 %), decreasing the concentration necessary for fractionation would help reduce processing costs. In this study, we sought to identify organic solvents capable of efficiently fractionating sorghum bagasse components at low concentrations. Results: Five alcohols (ethanol, 1-propanol, 2-propanol, 1-butanol, and 1-pentanol) were used for organosolv pretreatment of sorghum bagasse at a concentration of 12.5 %. Sulfuric acid (1 %) was used as a catalyst. With 1-butanol and 1-pentanol, three fractions (black liquor, liquid fraction containing xylose, and cellulose-enriched solid fraction) were obtained after pretreatment. Two-dimensional nuclear magnetic resonance analysis revealed that the lignin aromatic components of raw sorghum bagasse were concentrated in the black liquor fraction, although the major lignin side-chain (beta-O-4 linkage) was lost. Pretreatment with 1-butanol or 1-pentanol effectively removed p-coumarate, some guaiacyl, and syringyl. Compared with using no solvent, pretreatment with 1-butanol or 1-pentanol resulted in two-fold greater ethanol production from the solid fraction by Saccharomyces cerevisiae. Conclusions: Our results revealed that a low concentration (12.5 %) of a highly hydrophobic solvent such as 1-butanol or 1-pentanol can be used to separate the black liquor from the solid and liquid fractions. The efficient delignification and visible separation of the lignin-rich fraction possible with this method simplify the fractionation of sorghum bagasse.

    BIOMED CENTRAL LTD, 2016年02月, BIOTECHNOLOGY FOR BIOFUELS, 9, 27, 英語

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    研究論文(学術雑誌)

  • Kohji Nobuta, Hiroshi Teramura, Hiroaki Ito, Chizuru Hongo, Hideo Kawaguchi, Chiaki Ogino, Akihiko Kondo, Takashi Nishino

    Four types of kenaf bast fibers were prepared via a combination of Wise treatments, for delignification, and alkaline treatments, for the removal of hemicellulose. Each type of kenaf bast fiber with different refining processes were nano fibrillated by grinding. Resulting, cellulose nanofiber (CNF) sheet was obtained from CNF by vacuum filtration (Scheme 1). The structures and properties of these CNF sheets then were investigated to determine how the CNF components had affected these properties. All of the CNFs from different refining processes were classified as a cellulose I-beta type by X-ray diffraction. However, the mechanical properties (Young's modulus, tensile strength and toughness) of the CNF sheet with Wise treatment were higher than the properties of the other three CNF sheets. These results strongly suggested that alkaline treatment was unnecessary for the removal of hemicellulose, and that the application of the Wise treatment effectively imparted high mechanical properties to the cellulose microfiber.

    SPRINGER, 2016年02月, CELLULOSE, 23 (1), 403 - 414, 英語

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    研究論文(学術雑誌)

  • Shuhei Noda, Tomokazu Shirai, Sachiko Oyama, Akihiko Kondo

    A synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coil. An L-phenylalanine-overproducing E. coil strain was engineered to enhance the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds in microbes. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E.coli as a base strain, we tested our system by carrying out the production of salicylate, a highdemand aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this test case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. In order to demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1-3 g/L in test tube-scale culture. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2016年01月, METABOLIC ENGINEERING, 33, 119 - 129, 英語

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    研究論文(学術雑誌)

  • Naoko Okai, Takanori Miyoshi, Yasunobu Takeshima, Hiroaki Kuwahara, Chiaki Ogino, Akihiko Kondo

    Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) serves as a building block for polymers and pharmaceuticals. In this study, the biosynthetic pathway for PCA from glucose was engineered in Corynebacterium glutamicum. The pathway to PCA-employed elements of the chorismate pathway by using chorismate-pyruvate lyase (CPL) and 4-hydroxybenzoate hydroxylase (4-HBA hydroxylase). As C. glutamicum has the potential to synthesize the aromatic amino acid intermediate chorismate and possesses 4-HBA hydroxylase, we focused on expressing Escherichia coli CPL in a phenylalanine-producing strain of C. glutamicum ATCC21420. To secrete PCA, the gene (ubiC) encoding CPL from E. coli was expressed in C. glutamicum ATCC 21420 (strain F(UbiC)). The formation of 28.8 mg/L of extracellular 4-HBA (36 h) and 213 ± 29 mg/L of extracellular PCA (80 h) was obtained by the C. glutamicum strain F(UbiC) from glucose. The strain ATCC21420 was also found to produce extracellular PCA. PCA fermentation was performed using C. glutamicum strain F(UbiC) in a bioreactor at the optimized pH of 7.5. C. glutamicum F(UbiC) produced 615 ± 2.1 mg/L of PCA from 50 g/L of glucose after 72 h. Further, fed-batch fermentation of PCA by C. glutamicum F(UbiC) was performed with feedings of glucose every 24 h. The maximum production of PCA (1140.0 ± 11.6 mg/L) was achieved when 117.0 g/L of glucose was added over 96 h of fed-batch fermentation.

    2016年01月, Applied microbiology and biotechnology, 100 (1), 135 - 45, 英語, 国際誌

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    研究論文(学術雑誌)

  • Tomokazu Shirai, Takashi Osanai, Akihiko Kondo

    Background: Designing optimal intracellular metabolism is essential for using microorganisms to produce useful compounds. Computerized calculations for flux balance analysis utilizing a genome-scale model have been performed for such designs. Many genome-scale models have been developed for different microorganisms. However, optimal designs of intracellular metabolism aimed at producing a useful compound often utilize metabolic reactions of only the host microbial cells. In the present study, we added reactions other than the metabolic reactions with Synechosystis sp. 6803 as a host to its genome-scale model, and constructed a metabolic model of hybrid cells (SyHyMeP) using computerized analysis. Using this model provided a metabolic design that improves the theoretical yield of succinic acid, which is a useful compound. Results: Constructing the SyHyMeP model enabled new metabolic designs for producing useful compounds. In the present study, we developed a metabolic design that allowed for improved theoretical yield in the production of succinic acid during glycogen metabolism by Synechosystis sp. 6803. The theoretical yield of succinic acid production using a genome-scale model of these cells was 1.00 mol/mol-glucose, but use of the SyHyMeP model enabled a metabolic design with which a 33 % increase in theoretical yield is expected due to the introduction of isocitrate lyase, adding activations of endogenous tree reactions via D-glycerate in Synechosystis sp. 6803. Conclusions: The SyHyMeP model developed in this study has provided a new metabolic design that is not restricted only to the metabolic reactions of individual microbial cells. The concept of construction of this model requires only replacement of the genome-scale model of the host microbial cells and can thus be applied to various useful microorganisms for metabolic design to produce compounds.

    BIOMED CENTRAL LTD, 2016年01月, MICROBIAL CELL FACTORIES, 15 (1), 13, 英語

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    研究論文(学術雑誌)

  • 川口,秀夫, 寺村,浩, 中村,聡子, 荻野,千秋, 原,清敬, 蓮沼,誠久, 老沼,研一, 高谷,直樹, 平野,恒, 佐塚,隆志, 北野,英己, 近藤,昭彦

    Sorghum bagasse pretreated with diluted acid, which was predominantly composed of glucan (59%) and xylan (7.2%), was used as a lignocellulosic feedstock to produce D-phenyllactic acid (PhLA) by a recombinant Escherichia coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens. Compared to filter paper hydrolysate, the PhLA yield was reduced by 35% during fermentation with enzymatic hydrolysate of sorghum bagasse as a carbon source, and metabolomics analysis revealed that intracellular levels of erythrose-4-phosphate and phosphoenolpyruvate and NAD(P)H regeneration for PhLA production from glucose markedly reduced. Compared to the separate hydrolysis and fermentation (SHF) with sorghum bagasse hydrolysate, simultaneous saccharification and fermentation (SSF) of sorghum bagasse under glucose limitation conditions yielded 4.8-fold more PhLA with less accumulation of eluted components of p-coumaric acid and aldehydes, which inhibited PhLA fermentation. These results suggest that gradual hydrolysis of sorghum bagasse during SSF reduces the accumulation of both glucose and fermentation inhibitors, collectively leading to increased PhLA yield.

    一般社団法人 日本エネルギー学会, 2016年01月, バイオマス科学会議発表論文集, 11, 35 - 36, 日本語

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    研究論文(国際会議プロシーディングス)

  • Kengo Sasaki, Kiyotaka Y. Hara, Hideo Kawaguchi, Takashi Sazuka, Chiaki Ogino, Akihiko Kondo

    This study aimed to optimize extracellular glutathione production by a Saccharomyces cerevisiae engineered strain and to concentrate the extracellular glutathione by membrane separation processes, including ultrafiltration (UF) and nanofiltration (NF). Synthetic defined (SD) medium containing 20 g L-1 glucose was fermented for 48 h; the fermentation liquid was passed through an UF membrane to remove macromolecules. Glutathione in this permeate was concentrated for 48 h to 545.1 +/- 33.6 mg L-1 using the NF membrane; this was a significantly higher concentration than that obtained with yeast extract peptone dextrose (YPD) medium following 96 h NF concentration (217.9 +/- 57.4 mg L-1). This higher glutathione concentration results from lower cellular growth in SD medium (final OD600 = 6.9 +/- 0.1) than in YPD medium (final OD600 = 11.0 +/- 0.6) and thus higher production of extracellular glutathione (16.0 +/- 1.3 compared to 9.2 +/- 2.1 mg L-1 in YPD medium, respectively). Similar fermentation and membrane processing of sweet sorghum juice containing 20 g L-1 total sugars provided 240.3 +/- 60.6 mg L-1 glutathione. Increased extracellular production of glutathione by this engineered strain in SD medium and subsequent UF permeation and NF concentration in shortend time may help realize industrial recovery of extracellular glutathione. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2016年01月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 121 (1), 96 - 100, 英語

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    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Takatoshi Sakamoto, Akihiko Kondo

    Improving the production of ethanol from xylose is an important goal in metabolic engineering of Saccharomyces cerevisiae. Furthermore, S. cerevisiae must produce ethanol in the presence of weak acids (formate and acetate) generated during pre-treatment of lignocellulosic biomass. In this study, weak acid-containing xylose fermentation was significantly improved using cells that were acclimated to the weak acids during pre-cultivation. Transcriptome analyses showed that levels of transcripts for transcriptional/translational machinery-related genes (RTC3 and ANB1) were enhanced by formate and acetate acclimation. Recombinant yeast strains overexpressing RTC3 and ANB1 demonstrated improved ethanol production from xylose in the presence of the weak acids, along with improved tolerance to the acids. Novel metabolic engineering strategy based on the combination of short-term acclimation and system-wide analysis was developed, which can develop stress-tolerant strains in a short period of time, although conventional evolutionary engineering approach has required long periods of time to isolate inhibitor-adapted strains.

    SPRINGER, 2016年01月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100 (2), 1027 - 1038, 英語

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    研究論文(学術雑誌)

  • Yixing Zhang, Praveen V. Vadlani, Amit Kumar, Philip R. Hardwidge, Revathi Govind, Tsutomu Tanaka, Akihiko Kondo

    D-lactic acid is used as a monomer in the production of poly-D-lactic acid (PDLA), which is used to form heat-resistant stereocomplex poly-lactic acid. To produce cost-effective D-lactic acid by using all sugars derived from biomass efficiently, xylose-assimilating genes encoding xylose isomerase and xylulokinase were cloned into an L-lactate-deficient strain, Lactobacillus plantarum. The resulting recombinant strain, namely L. plantarum NCIMB 8826 a triangle ldhL1-pLEM-xylAB, was able to produce D-lactic acid (at optical purity > 99 %) from xylose at a yield of 0.53 g g(-1). Simultaneous utilization of glucose and xylose to produce D-lactic acid was also achieved by this strain, and 47.2 g L-1 of D-lactic acid was produced from 37.5 g L-1 glucose and 19.7 g L-1 xylose. Corn stover and soybean meal extract (SBME) were evaluated as cost-effective medium components for D-lactic acid production. Optimization of medium composition using response surface methodology resulted in 30 % reduction in enzyme loading and 70 % reduction in peptone concentration. In addition, we successfully demonstrated D-lactic acid fermentation from corn stover and SBME in a fed-batch fermentation, which yielded 61.4 g L-1 D-lactic acid with an overall yield of 0.77 g g(-1). All these approaches are geared to attaining high D-lactic acid production from biomass sugars to produce low-cost, highly thermostable biodegradable plastics.

    SPRINGER, 2016年01月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100 (1), 279 - 288, 英語

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    研究論文(学術雑誌)

  • Yota Tsuge, Hideo Kawaguchi, Kengo Sasaki, Akihiko Kondo

    Synthetic polymers are widely used in daily life. Due to increasing environmental concerns related to global warming and the depletion of oil reserves, the development of microbial-based fermentation processes for the production of polymer building block chemicals from renewable resources is desirable to replace current petroleum-based methods. To this end, strains that efficiently produce the target chemicals at high yields and productivity are needed. Recent advances in metabolic engineering have enabled the biosynthesis of polymer compounds at high yield and productivities by governing the carbon flux towards the target chemicals. Using these methods, microbial strains have been engineered to produce monomer chemicals for replacing traditional petroleum-derived aliphatic polymers. These developments also raise the possibility of microbial production of aromatic chemicals for synthesizing high-performance polymers with desirable properties, such as ultraviolet absorbance, high thermal resistance, and mechanical strength. In the present review, we summarize recent progress in metabolic engineering approaches to optimize microbial strains for producing building blocks to synthesize aliphatic and high-performance aromatic polymers.

    BIOMED CENTRAL LTD, 2016年01月, MICROBIAL CELL FACTORIES, 15, 19, 英語

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    研究論文(学術雑誌)

  • Jerome Amoah, Shih-Hsin Ho, Shinji Hama, Ayumi Yoshida, Akihito Nakanishi, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo

    The presence of phospholipids in oil has been a major hurdle in the production of biodiesel using immobilized Aspergillus oryzae whole-cell biocatalysts. A density of phospholipids within the range of 10-30% could reduce both the rate of production and the final yield of biodiesel. Phospholipids in the oil leads to the formation of water-in-oil phospholipid-based reverse micelles. The water that activates the enzymatic process is observed to be trapped inside these reverse micelles. This has resulted in the inactivation of the reaction systems and has subsequently led to the deactivation of the immobilized lipase by the extended residence time of the added methanol. A reaction system involving gentle agitation and higher amount of water was found to reduce the reverse micelles formation. This simple technique improved the conversion efficiency by approximately 3-folds, producing a final biodiesel of more than 90%, using immobilized A. oryzae whole cells expressing Fusarium heterosporum lipase. This demonstrates that, the above technique could be successfully applied to the enzymatic biodiesel conversion of oils containing high amounts of phospholipids such as that from microalgae. (C) 2015 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2016年01月, BIOCHEMICAL ENGINEERING JOURNAL, 105, 10 - 15, 英語

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    研究論文(学術雑誌)

  • Jieun Song, Daisuke Sasaki, Kengo Sasaki, Souichiro Kato, Akihiko Kondo, Kazuhito Hashimoto, Shuji Nakanishi

    Comprehensive metabolomic analysis of anode-respiring Geobacter sulfurreducens cells revealed that intracellular levels of metabolites related to the tricarboxylic acid (TCA) cycle, gluconeogenesis, consumptions of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), and adenosine triphosphate (ATP) generation correlated well with the activity of microbial anode-respiration detected as microbial electric current. Use of a glassy carbon electrode as the anode material in a three-electrode system resulted in a higher microbial current at +0.2 V (vs. Ag/AgCl) than at -0.2 V. A larger current flow resulted in higher concentrations of TCA cycle metabolites and lower concentrations of gluconeogenesis metabolites. Metabolomic analysis also revealed that higher anode respiration activity resulted in a higher ATP/adenosine diphosphate (ADP) ratio and lower ratios of NADH/NAD(+) and NADPH/NADP(+). These findings provide direct experimental evidence that microbial anode-respiration activity controlled by the anode potential influences both the flux of central metabolic pathways and the redox balance in microbial fuel cells. (C) 2015 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2016年01月, PROCESS BIOCHEMISTRY, 51 (1), 34 - 38, 英語

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    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo

    G-protein-coupled receptors (GPCRs) are physiologically important transmembrane proteins that sense signaling molecules such as hormones, neurotransmitters, and various sensory stimuli; GPCRs represent major molecular targets for drug discovery. Although GPCRs traditionally have been thought to function as monomers or homomers, in the recent years these proteins have also been shown to function as heteromers. Heteromerization among GPCRs is expected to generate potentially large functional and physiological diversity and to provide new opportunities for drug discovery. However, due to the existence of numerous combinations, the larger universe of possible GPCR heteromers is unknown, and thus its functional significance is still poorly understood. The oligomerization of GPCRs in living cells now has been demonstrated in mammalian cells and in native tissues by using genetic, biochemical, and physiological approaches, as well as various resonance energy transfer (RET) technologies. In addition, the yeast Saccharomyces cerevisiae, which can serve as a biosensor for monitoring eukaryotic biological processes, can also be used for the identification of functionally significant heteromer pairs of GPCRs. In this review, we focus on studies of GPCR oligomers, and summarize the technologies used to evaluate GPCR oligomerization. We additionally consider the potential limitations of these methods at present, and envision the possible future applications of these techniques.

    BENTHAM SCIENCE PUBL LTD, 2016年, CURRENT MEDICINAL CHEMISTRY, 23 (16), 1638 - 1656, 英語

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    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Jun Ishii, Akihiko Kondo

    Conferring biomass hydrolysis activity on yeast through genetic engineering has paved the way for the development of groundbreaking processes for producing liquid fuels and commodity chemicals from lignocellulosic biomass. However, the overproduction and misfolding of heterologous and endogenous proteins can trigger cellular stress, increasing the metabolic burden and retarding growth. Improving the efficiency of lignocellulosic breakdown requires engineering of yeast secretory pathway based on system-wide metabolic analysis as well as DNA constructs for enhanced cellulase gene expression with advanced molecular biology tools. Also, yeast is subjected to severe stress due to toxic compounds generated during lignocellulose pretreatment in consolidated saccharification and fermentation processes. The prospect for development of robust yeast strains makes combining evolutionary and rational engineering strategies.

    ELSEVIER SCI LTD, 2015年12月, CURRENT OPINION IN CHEMICAL BIOLOGY, 29, 1 - 9, 英語

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    研究論文(学術雑誌)

  • Katsunori Yoshikawa, Shimpei Aikawa, Yuta Kojima, Yoshihiro Toya, Chikara Furusawa, Akihiko Kondo, Hiroshi Shimizu

    Arthrospira (Spirulina) platensis is a promising feedstock and host strain for bioproduction because of its high accumulation of glycogen and superior characteristics for industrial production. Metabolic simulation using a genome-scale metabolic model and flux balance analysis is a powerful method that can be used to design metabolic engineering strategies for the improvement of target molecule production. In this study, we constructed a genome-scale metabolic model of A. platensis NIES-39 including 746 metabolic reactions and 673 metabolites, and developed novel strategies to improve the production of valuable metabolites, such as glycogen and ethanol. The simulation results obtained using the metabolic model showed high consistency with experimental results for growth rates under several trophic conditions and growth capabilities on various organic substrates. The metabolic model was further applied to design a metabolic network to improve the autotrophic production of glycogen and ethanol. Decreased flux of reactions related to the TCA cycle and phosphoenolpyruvate reaction were found to improve glycogen production. Furthermore, in silico knockout simulation indicated that deletion of genes related to the respiratory chain, such as NAD(P)H dehydrogenase and cytochrome-c oxidase, could enhance ethanol production by using ammonium as a nitrogen source.

    PUBLIC LIBRARY SCIENCE, 2015年12月, PLOS ONE, 10 (12), e0144430, 英語

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    研究論文(学術雑誌)

  • Kiyotaka Y. Hara, Akihiko Kondo

    Adenosine-5'-triphosphate (ATP) is consumed as a biological energy source by many intracellular reactions. Thus, the intracellular ATP supply is required to maintain cellular homeostasis. The dependence on the intracellular ATP supply is a critical factor in bioproduction by cell factories. Recent studies have shown that changing the ATP supply is critical for improving product yields. In this review, we summarize the recent challenges faced by researchers engaged in the development of engineered cell factories, including the maintenance of a large ATP supply and the production of cell factories. The strategies used to enhance ATP supply are categorized as follows: addition of energy substrates, controlling pH, metabolic engineering of ATP-generating or ATP-consuming pathways, and controlling reactions of the respiratory chain. An enhanced ATP supply generated using these strategies improves target production through increases in resource uptake, cell growth, biosynthesis, export of products, and tolerance to toxic compounds.

    BIOMED CENTRAL LTD, 2015年12月, MICROBIAL CELL FACTORIES, 14, 198, 英語

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    研究論文(学術雑誌)

  • Toyoshima M, Aikawa S, Yamagishi T, Kondo Akihiko, Kawai H

    Microalgae are considered to be efficient bio-resources for biofuels and bio-based chemicals because they generally have high productivity. The filamentous cyanobacterium Arthrospira (Spirulina) platensis has been widely used for food, feed, and nutrient supplements and is usually cultivated in open ponds. In order to extend the surface area for growing this alga, we designed a pilot-scale floating closed culture system for cultivating A. platensis on open water and compared the growth and quality of the alga harvested at both subtropical and temperate regions. The biomass productivity of A. platensis NIES-39 was ca. 9 g dry biomass m(-2) day(-1) in summer at Awaji Island (warm temperature region) and ca. 10 and 6 g dry biomass m(-2) day(-1) in autumn and winter, respectively, at Ishigaki Island, (subtropical region) in Japan. If seawater can be used for culture media, culture cost can be reduced; therefore, we examined the influence of seawater salt concentrations on the growth of A. platensis NIES-39. Growth rates of A. platensis NIES-39 in diluted seawater with enrichment of 2.5 g L-1 NaNO3, 0.01 g L-1 FeSO4 center dot 7H(2)O, and 0.08 g L-1 Na(2)EDTA were considerably lower than SOT medium, but the biomass productivity (dry weight) was comparable to SOT medium. This is explained by the heavier cell weight of the alga grown in modified seawater media compared to the alga grown in SOT medium. Furthermore, A. platensis grown in modified seawater-based medium exhibited self-flocculation and had more loosely coiled trichomes.

    SPRINGER, 2015年12月, Journal of Applied Phycology, 27 (6), 2191 - 2202, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hideo Kawaguchi, Kengo Sasaki, Kouji Uematsu, Yota Tsuge, Hiroshi Teramura, Naoko Okai, Sachiko Nakamura-Tsuruta, Yohei Katsuyama, Yoshinori Sugai, Yasuo Ohnishi, Ko Hirano, Takashi Sazuka, Chiaki Ogino, Akihiko Kondo

    The production of the bioplastic precursor 3-amino-4-hydroxybenzoic acid (3,4-AHBA) from sweet sorghum juice, which contains amino acids and the fermentable sugars sucrose, glucose and fructose, was assessed to address the limitations of producing bio-based chemicals from renewable feedstocks. Recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI derived from Streptomyces griseus produced 3,4-AHBA from the sweet sorghum juice of cultivar SIL-05 at a final concentration (1.0 g l(-1)) that was 5-fold higher than that from pure sucrose. Fractionation of sweet sorghum juice by nanofiltration (NF) membrane separation (molecular weight cut-off 150) revealed that the NF-concentrated fraction, which contained the highest concentrations of amino acids, increased 3,4-AHBA production, whereas the NF-filtrated fraction inhibited 3,4-AHBA biosynthesis. Amino acid supplementation experiments revealed that leucine specifically enhanced 3,4-AHBA production by strain KT01. Taken together, these results suggest that sweet sorghum juice is a potentially suitable feedstock for 3,4-AHBA production by recombinant C. glutamicum. (C) 2015 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2015年12月, BIORESOURCE TECHNOLOGY, 198, 410 - 417, 英語

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    研究論文(学術雑誌)

  • Misato Kaishima, Jun Ishii, Nobuo Fukuda, Akihiko Kondo

    Protein-protein interactions (PPIs) are crucial for the vast majority of biological processes. We previously constructed a G gamma recruitment system to screen PPI candidate proteins and desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. The methods utilized a target protein fused to a mutated G-protein gamma subunit (G gamma(cyto)) lacking the ability to localize to the inner leaflet of the plasma membrane. However, the previous systems were adapted to use only soluble cytosolic proteins as targets. Recently, membrane proteins have been found to form the principal nodes of signaling involved in diseases and have attracted a great deal of interest as primary drug targets. Here, we describe new protocols for the G gamma recruitment systems that are specifically designed to use membrane proteins as targets to overcome previous limitations. These systems represent an attractive approach to exploring novel interacting candidates and affinity-altered protein variants and their interactions with proteins on the inner side of the plasma membrane, with high specificity and selectivity.

    NATURE PUBLISHING GROUP, 2015年11月, Scientific Reports, 5, 16723, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Cell surface engineering of industrial microorganisms for biorefining applications.

    Tanaka Tsutomu, Kondo Akihiko

    2015年11月, Biotechnology Advances, 33, 1403 - 1411, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Jyumpei Kobayashi, Misaki Tanabiki, Shohei Doi, Akihiko Kondo, Takashi Ohshiro, Hirokazu Suzuki

    The plasmid pGKE75-cat(A138T), which comprises pUC18 and the cat(A138T) gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CAT(A138T)), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-cat(A138T) in an error-prone thermophile, generating the mutant plasmid pGKE75(alpha beta)-cat(A138T) responsible for substantial chloramphenicol resistance at 65 degrees C. pGKE75(alpha beta)-cat(A138T) contained no mutation in the cat(A138T) gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75(alpha beta)-cat(A138T) is attributable to increases in intracellular CAT(A138T) and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75(alpha beta)-cat(A138)T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75(alpha beta)-cat(A138T) using chloramphenicol selection at 60 degrees C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli.

    AMER SOC MICROBIOLOGY, 2015年11月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 81 (21), 7625 - 7632, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hara Kiyotaka, Aoki N, KOBAYASHI Jyumpei, Kiriyama K, Nishida Keiji, Araki Michihiro, Kondo Akihiko

    Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

    SPRINGER, 2015年11月, Applied Microbiology and Biotechnology, 99 (22), 9771 - 9778, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takashi Osanai, Tomokazu Shirai, Hiroko Iijima, Yuka Nakaya, Mami Okamoto, Akihiko Kondo, Masami Y. Hirai

    Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.

    FRONTIERS MEDIA SA, 2015年10月, FRONTIERS IN MICROBIOLOGY, 6, 1064, 英語

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    研究論文(学術雑誌)

  • Nobuko Sumiya, Yasuko Kawase, Jumpei Hayakawa, Mami Matsuda, Mami Nakamura, Atsuko Era, Kan Tanaka, Akihiko Kondo, Tomohisa Hasunuma, Sousuke Imamura, Shin-ya Miyagishima

    Nitrogen starvation is known to induce the accumulation of triacylglycerol (TAG) in many microalgae, and potential use of microalgae as a source of biofuel has been explored. However, nitrogen starvation also stops cellular growth. The expression of cyanobacterial acyl-acyl carrier protein (ACP) reductase in the unicellular red alga Cyanidioschyzon merolae chloroplasts resulted in an accumulation of TAG, which led to an increase in the number and size of lipid droplets while maintaining cellular growth. Transcriptome and metabolome analyses showed that the expression of acyl-ACP reductase altered the activities of several metabolic pathways. The activities of enzymes involved in fatty acid synthesis in chloroplasts, such as acetyl-CoA carboxylase and pyruvate dehydrogenase, were up-regulated, while pyruvate decarboxylation in mitochondria and the subsequent consumption of acetyl-CoA by the tricarboxylic acid (TCA) cycle were down-regulated. Aldehyde dehydrogenase, which oxidizes fatty aldehydes to fatty acids, was also up-regulated in the acyl-ACP reductase expresser. This activation was required for the lipid droplet accumulation and metabolic changes observed in the acyl-ACP reductase expresser. Nitrogen starvation also resulted in lipid droplet accumulation in C. merolae, while cell growth ceased as in the case of other algal species. The metabolic changes that occur upon the expression of acyl-ACP reductase are quite different from those caused by nitrogen starvation. Therefore, there should be a method for further increasing the storage lipid level while still maintaining cell growth that is different from the metabolic response to nitrogen starvation.

    OXFORD UNIV PRESS, 2015年10月, PLANT AND CELL PHYSIOLOGY, 56 (10), 1962 - 1980, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Chiaki Ogino, Akihiko Kondo

    Phospholipids (PLs) containing specific polar head groups and fatty acids, artificially synthesized from a complex mixture of natural PLs, have considerable industrial applications. The biocatalytic approaches to synthesizing structured PLs are of great interest because the enzymes used show high selectivity and performance under mild conditions, leading to the generation of products that cannot easily be obtained by chemical catalysis. Although the limited supply of phospholipases (e.g., phospholipase D) has thus far been an obstacle to the widespread use of enzymatic processing, recent advances in enzyme preparation have opened up various applications for PL modification. In this review, attempts to increase the productivity and utility of microbial phospholipases and lipases are presented. We also summarize recent developments in enzyme-catalyzed modification of PLs, focusing particularly on the relevant reactions, bioreactor design, and novel proof-of-concept experiments.

    SPRINGER, 2015年10月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 99 (19), 7879 - 7891, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuto Hata, Takuya Matsumoto, Tsutomu Tanaka, Akihiko Kondo

    In the present study, sortase A-mediated immobilization of enzymes was used for the preparation of immobilized enzymes. Thermobifida fusca YX -glucosidase (BGL) or Streptococcus bovis 148 -amylase (AmyA) were produced with C-terminal sortase A recognition sequences. The resulting fusion proteins were successfully immobilized on nanoparticle surfaces using sortase A. Some properties (activity, stability, and reusability) of the immobilized fusion proteins were evaluated. Both immobilized BGL and immobilized AmyA prepared by the sortase A-mediated technique retained their catalytic activity, exhibiting activities 3.0- or 1.5-fold (respectively) of those seen with the same enzymes immobilized by chemical crosslinking. Immobilized enzymes prepared by the sortase A-mediated technique did not undergo dramatic changes in stability compared with the respective free enzymes. Thus, the sortase A-mediated technique provides a promising method for immobilization of active, stable enzymes.

    WILEY-V C H VERLAG GMBH, 2015年10月, MACROMOLECULAR BIOSCIENCE, 15 (10), 1375 - 1380, 英語

    [査読有り]

    研究論文(学術雑誌)

  • バイオリファイナリーの現状と展望

    蓮沼 誠久, 石井 純, 荻野 千秋, 近藤 昭彦

    2015年10月, 化学と生物, 53 (10), 689 - 695, 日本語

    研究論文(学術雑誌)

  • 合成生物工学によるモノづくり微生物のデザインに向けて

    石井純, 荒木 通啓, 中津井 雅彦, 崎濱 由梨, 柘植 陽太, 蓮沼 誠久, 近藤 昭彦

    2015年09月, 生物工学会誌, 93 (9), 523 - 526, 日本語

    研究論文(学術雑誌)

  • Zhuo Liu, Kentaro Inokuma, Shih-Hsin Ho, Riaan den Haan, Tomohisa Hasunuma, Willem H. van Zyl, Akihiko Kondo

    Background: Engineering Saccharomyces cerevisiae to produce heterologous cellulases is considered as a promising strategy for production of bioethanol from lignocellulose. The production of cellulase is usually pursued by one of the two strategies: displaying enzyme on the cell surface or secreting enzyme into the medium. However, to our knowledge, the combination of the two strategies in a yeast strain has not been employed. Results: In this study, heterologous endoglucanase (EG) and cellobiohydrolase I (CBHI) were produced in a beta-glucosidase displaying S. cerevisiae strain using cell-surface display, secretion, or a combined strategy. Strains EG-D-CBHI-D and EG-S-CBHI-S (with both enzymes displayed on the cell surface or with both enzymes secreted to the surrounding medium) showed higher ethanol production (2.9 and 2.6 g/L from 10 g/L phosphoric acid swollen cellulose, respectively), than strains EG-D-CBHI-S and EG-S-CBHI-D (with EG displayed on cell surface and CBHI secreted, or vice versa). After 3-cycle repeated-batch fermentation, the cellulose degradation ability of strain EG-D-CBHI-D remained 60 % of the 1st batch, at a level that was 1.7-fold higher than that of strain EG-S-CBHI-S. Conclusions: This work demonstrated that placing EG and CBHI in the same space (on the cell surface or in the medium) was favorable for amorphous cellulose-based ethanol fermentation. In addition, the cellulolytic yeast strain that produced enzymes by the cell-surface display strategy performed better in cell-recycle batch fermentation compared to strains producing enzymes via the secretion strategy.

    BIOMED CENTRAL LTD, 2015年09月, BIOTECHNOLOGY FOR BIOFUELS, 8, 162, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo

    The monoamine neurotransmitter serotonin (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. One such receptor, the 5-HT1A receptor (HTR1A), is the most widely studied subtype and represents a significant molecular target in medicinal and therapeutic fields. Yeast-based fluorescent reporter systems have proven to be especially useful for GPCR assays, since detection using a fluorescent reporter considerably simplifies measurement procedures. However, previously reported systems using enhanced green fluorescent protein (EGFP) as the reporter in yeast still showed low signal-to-noise (S/N) ratios, making EGFP difficult to apply as an easily accessible tool. Therefore, we constructed a refined yeast-based GPCR biosensor employing a high-sensitivity strain that incorporated both a G-engineered receptor and a fluorescent reporter (ZsGreen). As we report here, the refined yeast-based fluorescent biosensor was applied successfully to antagonist characterization and analysis of site-directed mutants of the HTR1A receptor. Pindolol, a known antagonist of HTR1A, specifically inhibited agonist-induced signaling, demonstrating the ease of evaluating inhibition effects using our reporter strain. Characterization of site-specific receptor mutants confirmed the role of specific targeted residues, including the highly conserved DRY motif, in the activation of HTR1A. Thus, our refined yeast biosensor strain, which incorporates a ZsGreen reporter and an engineered G receptor, is expected to serve as a simple and practical sensing tool for evaluating the ligand candidates and defining residues important to the function of human GPCRs. Biotechnol. Bioeng. 2015;112: 1906-1915. (c) 2015 Wiley Periodicals, Inc.

    WILEY-BLACKWELL, 2015年09月, BIOTECHNOLOGY AND BIOENGINEERING, 112 (9), 1906 - 1915, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yoshifumi Ueno, Shimpei Aikawa, Akihiko Kondo, Seiji Akimoto

    Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed.

    SPRINGER, 2015年08月, PHOTOSYNTHESIS RESEARCH, 125 (1-2), 211 - 218, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Aya Onishi, Shimpei Aikawa, Akihiko Kondo, Seiji Akimoto

    Some filamentous cyanobacteria (including Anabaena) differentiate into heterocysts under nitrogen-depleted conditions. During differentiation, the phycobiliproteins and photosystem II in the heterocysts are gradually degraded. Nitrogen depletion induces changes in the pigment composition of both vegetative cells and heterocysts, which affect the excitation energy transfer processes. To investigate the changes in excitation energy transfer processes of Anabaena variabilis filaments grown in standard medium (BG11) and a nitrogen-free medium (BG11(0)), we measured their steady-state absorption spectra, steady-state fluorescence spectra, and time-resolved fluorescence spectra (TRFS) at 77 K. TRFS were measured with a picosecond time-correlated single photon counting system. The pigment compositions of the filaments grown in BG11(0) changed throughout the growth period; the relative phycocyanin levels monotonically decreased, whereas the relative carotenoid (Car) levels decreased and then recovered to their initial value (at day 0), with formation of lower-energy Cars. Nitrogen starvation also altered the fluorescence kinetics of PSI; the fluorescence maximum of TRFS immediately after excitation occurred at 735, 740, and 730 nm after 4, 8, and 15 days growth in BG11(0), respectively. Based on these results, we discuss the excitation energy transfer dynamics of A. variabilis filaments under the nitrogen-depleted condition throughout the growth period.

    SPRINGER, 2015年08月, PHOTOSYNTHESIS RESEARCH, 125 (1-2), 191 - 199, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kenta Niki, Shimpei Aikawa, Makio Yokono, Akihiko Kondo, Seiji Akimoto

    Currently, cyanobacteria are regarded as potential biofuel sources. Large-scale cultivation of cyanobacteria in seawater is of particular interest because seawater is a low-cost medium. In the present study, we examined differences in light-harvesting and energy transfer processes in the cyanobacterium Synechococcus sp. PCC 7002 grown in different cultivation media, namely modified A medium (the optimal growth medium for Synechococcus sp. PCC 7002) and f/2 (a seawater medium). The concentrations of nitrate and phosphate ions were varied in both media. Higher nitrate ion and/or phosphate ion concentrations yielded high relative content of phycobilisome. The cultivation medium influenced the energy transfers within phycobilisome, from phycobilisome to photosystems, within photosystem II, and from photosystem II to photosystem I. We suggest that the medium also affects charge recombination at the photosystem II reaction center and formation of a chlorophyll-containing complex.

    SPRINGER, 2015年08月, PHOTOSYNTHESIS RESEARCH, 125 (1-2), 201 - 210, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Iijima H, Shirai T, Okamoto M, Kondo Akihiko, Yokota Hirai M, Osanai T

    The study of the primary metabolism of cyanobacteria in response to light conditions is important for environmental biology because cyanobacteria are widely distributed among various ecological niches. Cyanobacteria uniquely possess circadian rhythms, with central oscillators consisting from three proteins, KaiA, KaiB, and KaiC. The two-component histidine kinase SasA/Hik8 and response regulator RpaA transduce the circadian signal from KaiABC to control gene expression. Here, we generated a strain overexpressing rpaA in a unicellular cyanobacterium Synechocystis sp. PCC 6803. The rpaA-overexpressing strain showed pleiotropic phenotypes, including slower growth, aberrant degradation of an RNA polymerase sigma factor SigE after the light-to-dark transition, and higher accumulation of sugar catabolic enzyme transcripts under dark conditions. Metabolome analysis revealed delayed glycogen degradation, decreased sugar phosphates and organic acids in the tricarboxylic acid cycle, and increased amino acids under dark conditions. The current results demonstrate that in this cyanobacterium, RpaA is a regulator of primary metabolism and involved in adaptation to changes in light conditions.

    FRONTIERS MEDIA SA, 2015年08月, Frontiers in Microbiology, 6, 888, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Shino Mizuno, Maki Kihara, Tsutomu Tanaka, Chiaki Ogino, Hideo Noda, Akihiko Kondo

    This study focused on the process development for the D-lactic acid production from cellulosic feedstocks using the Lactobacillus plantarum mutant, genetically modified to produce optically pure D-lactic acid from both glucose and xylose. The simultaneous saccharification and fermentation (SSF) using delignified hardwood pulp (5-15% loads) resulted in the lactic acid titers of 55.2-84.6 g/L after 72 h and increased productivities of 1.77-2.61 g/L/h. To facilitate the enzymatic saccharification of high-load pulp at a fermentation temperature, short-term (610 min) pulverization of pulp was conducted, leading to a significantly improved saccharification with the suppressed formation of formic acid by-product. The short-term milling followed by SSF resulted in a lactic acid titer of 102.3 g/L, an optical purity of 99.2%, and a yield of 0.879 g/g-sugars without fed-batch process control. Therefore, the process presented here shows promise for the production of high-titer D-lactic acid using the L. plantarum mutant. (C) 2015 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2015年07月, BIORESOURCE TECHNOLOGY, 187, 167 - 172, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takashi Osanai, Tomokazu Shirai, Hiroko Iijima, Ayuko Kuwahara, Iwane Suzuki, Akihiko Kondo, Masami Yokota Hirai

    Cyanobacteria possess circadian clocks consisting of KaiABC proteins, and circadian rhythm must closely relate to the primary metabolism. A histidine kinase, SasA, interacts with KaiC to transduce circadian signals and widely regulates transcription in Synechococcus sp. PCC 7942, although the involvement of SasA in primary metabolism has not been demonstrated at metabolite levels. Here, we generated a strain overexpressing hik8 (HOX80), an orthologue of SasA in Synechocystis sp. PCC 6803. HOX80 grew slowly under light conditions and lost viability under continuous dark conditions. Transcript levels of genes related to sugar catabolism remained higher in HOX80 under dark conditions. Metabolomic analysis revealed that under light conditions, glycogen was undetectable in HOX80, and there were decreased levels of metabolites of sugar catabolism and increased levels of amino acids, compared with those in the wild-type strain. HOX80 exhibited aberrant degradation of SigE proteins after a light-to-dark transition and immunoprecipitation analysis revealed that Hik8 directly interacts with KaiC1. The results of this study demonstrate that overexpression of hik8 widely alters sugar and amino acid metabolism, revealing the involvement of Hik8 in primary metabolism under both light and dark conditions in this cyanobacterium.

    WILEY-BLACKWELL, 2015年07月, ENVIRONMENTAL MICROBIOLOGY, 17 (7), 2430 - 2440, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroshi Teramura, Kengo Sasaki, Tomoko Oshima, Shimpei Aikawa, Fumio Matsuda, Mami Okamoto, Tomokazu Shirai, Hideo Kawaguchi, Chiaki Ogino, Masanori Yamasaki, Jun Kikuchi, Akihiko Kondo

    A renewable raw material, rice straw is pretreated for biorefinery usage. Solution-state two-dimensional (2D) H-1-(13) C hetero-nuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, was used to analyze 13 cultivars of rice straw before and after dilute acid pretreatment, to characterize general changes in the lignin and polysaccharide components. Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate, guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin aliphatic linkages or ferulate decreased. Decreased heterogeneity in the intensities of three peaks related to cellulose components in acid-insoluble residues resulted in similar glucose yield (0.45-0.59 g/g-dry biomass). Starch-derived components showed positive correlations (r = 0.71 to 0.96) with glucose, 5-hydroxymethyl-furfural (5-HMF), and formate concentrations in the liquid hydrolysates, and negative correlations (r = -0.95 to -0.97) with xylose concentration and acid-insoluble residue yield. These results showed the fate of lignin and polysaccharide components by pretreatment, suggesting that lignin aromatic regions and cellulose components were retained in the acid insoluble residues and starch-derived components were transformed into glucose, 5-HMF, and formate in the liquid hydrolysate.

    PUBLIC LIBRARY SCIENCE, 2015年06月, PLOS ONE, 10 (6), e0128417, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Yota Tsuge, Daisuke Sasaki, Hideo Kawaguchi, Takashi Sazuka, Chiaki Ogino, Akihiko Kondo

    Sequential batch fermentation from sweet sorghum juice concentrated by membrane separation (ultrafiltration permeation and nanofiltration concentration) to increase sugar contents, was investigated. Ethanol production at 5th batch fermentation by Saccharomyces cerevisiae BY4741 attained 113.7 +/- 3.1 g L-1 (89.1 +/- 2.2% of the theoretical ethanol yield) from 270.0 +/- 22.6 g L-1 sugars, corresponding to 98.7% of ethanol titer attained at the 1st batch fermentation. This titer was comparable to ethanol production of 115.8 +/- 0.6 g L-1 (87.1 +/- 2.7% of the theoretical ethanol yield) obtained at 5th batch fermentation with 3 g L-1 yeast extract and 6 g L-1 polypeptone. Increase of cell density in the concentrated sweet sorghum juice was observed during sequential batch fermentation, as indicated by increased OD600. Utilization of sweet sorghum juice as the sole source, membrane separation, and S. cerevisiae was a cost-effective process for high ethanol production. (C) 2015 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2015年06月, BIORESOURCE TECHNOLOGY, 186, 351 - 355, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Mami Okamoto, Tomokazu Shirai, Yota Tsuge, Hiroshi Teramura, Daisuke Sasaki, Hideo Kawaguchi, Tomohisa Hasunuma, Chiaki Ogino, Fumio Matsuda, Jun Kikuchi, Akihiko Kondo

    Background: Hydrothermal pretreatment of lignocellulosic biomass such as rice straw can dissolve part of the lignin and hemicellulose into a liquid fraction, thus facilitating enzyme accessibility to cellulose in bioethanol production process. Lignin is awaited to be recovered after hydrothermal pretreatment for utilization as value-added chemical, and lignin recovery also means removal of fermentation inhibitors. To recover lignin with high content from the liquid fraction, it is necessary to separate lignin and hemicellulose-derived polysaccharide. Therefore, the following processes were applied: membrane separation with nanofiltration (NF) and enzymatic hydrolysis by hemicellulase. To clarify lignin-concentrated fraction obtained during these processes, the fates of lignin and polysaccharide components were pursued by a solution NMR method and confirmed by compositional analysis of each fraction. Results: After hydrothermal pretreatment of rice straw, the NF concentrate of the supernatant of liquid fraction was hydrolyzed by hemicellulase and the resulting black precipitate was recovered. In this black precipitate, the intensity of NMR spectra related to lignin aromatic regions increased and those related to polysaccharides decreased, compared to rice straw, the solid fraction after hydrothermal pretreatment, and the NF concentrate. The lignin content of the black precipitate was 65.8 %. Lignin in the black precipitate included 52.9 % of the acid-insoluble lignin and 19.4 % of the soluble lignin in the NF concentrate of supernatant of liquid fraction. Conclusion: A precipitate with high lignin content was obtained from supernatants of the liquid fraction. These results suggested that precipitation of lignin was enhanced from concentrated mixtures of lignin and hemicellulosic polysaccharides by hydrolyzing the polysaccharides. Precipitation of lignin can contribute to lignin recovery from lignocellulosic biomass and, at the same time, allow more efficient ethanol production in the subsequent fermentation process.

    BIOMED CENTRAL LTD, 2015年06月, BIOTECHNOLOGY FOR BIOFUELS, 8, 88, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Yota Tsuge, Daisuke Sasaki, Hiroshi Teramura, Kentaro Inokuma, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo

    Mechanical milling and membrane separation were applied to simultaneous saccharification and co-fermentation from hydrothermally pretreated rice straw. Mechanical milling with minimized 4 cycles enabled 37.5 +/- 3.4 g L-1 and 45.3 +/- 4.4 g L-1 of ethanol production after 48 h by xylose-fermenting Saccharomyces cerevisiae from solid fractions (200 and 250 g L-1) of pretreated rice straw with 5 filter paper unit g-biomass(-1) cellulase (respectively, 77.3 +/- 7.1% and 74.7 +/- 7.3% of theoretical ethanol yield). Use of a membrane-based process including nanofiltration and ultrafiltration increased the sugar concentrations in the liquid fraction of pretreated rice straw and addition of this liquid fraction to 250 g L-1 solid fraction increased ethanol production to 52.0 +/- 0.4 g L-1 (73.8 +/- 0.6% of theoretical ethanol yield). Mechanical milling was effective in increasing enzymatic hydrolysis of the solid fraction and membrane separation steps increased the ethanol titer during co-fermentation, leading to a proposal for combining these processes for ethanol production from whole rice straw. (C) 2015 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2015年06月, BIORESOURCE TECHNOLOGY, 185, 263 - 268, 英語

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    研究論文(学術雑誌)

  • Noda S, Shirai T, Mochida K, Matsuda F, Oyama S, Okamoto M, Kondo Akihiko

    To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC), which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L) than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

    PUBLIC LIBRARY SCIENCE, 2015年05月, PLOS ONE, 10 (5), e0125488, 英語

    [査読有り]

    研究論文(学術雑誌)

  • CBPによるバイオエタノール生産技術の開発

    猪熊 健太郎, 蓮沼 誠久, 近藤 昭彦

    2015年05月, バイオインダストリー, 32 (5), 26 - 31, 日本語

    研究論文(学術雑誌)

  • Hideo Kawaguchi, Hiroshi Teramura, Kouji Uematsu, Kiyotaka Y. Hara, Tomohisa Hasunuma, Ko Hirano, Takashi Sazuka, Hidemi Kitano, Yota Tsuge, Prihardi Kahar, Satoko Niimi-Nakamura, Ken-Ichi Oinuma, Naoki Takaya, Shigemitsu Kasuga, Chiaki Ogino, Akihiko Kondo

    Dilute acid-pretreated sorghum bagasse, which was predominantly composed of glucan (59%) and xylose (7.2%), was used as a lignocellulosic feedstock for D-phenyllactic acid (PhLA) production by a recombinant Escherichia coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens. During fermentation with enzymatic hydrolysate of sorghum bagasse as a carbon source, the PhLA yield was reduced by 35% compared to filter paper hydrolysate, and metabolomics analysis revealed that NAD(P)H regeneration and intracellular levels of erythrose-4-phosphate and phosphoenolpyruvate for PhLA biosynthesis markedly reduced. Compared to separate hydrolysis and fermentation (SHF) with sorghum bagasse hydrolysate, simultaneous saccharification and fermentation (SSF) of sorghum bagasse under glucose limitation conditions yielded 4.8-fold more PhLA with less accumulation of eluted components, including p-coumaric acid and aldehydes, which inhibited PhLA fermentation. These results suggest that gradual enzymatic hydrolysis during SSF enhances PhLA production under glucose limitation and reduces the accumulation of fermentation inhibitors, collectively leading to increased PhLA yield. (c) 2015 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2015年04月, BIORESOURCE TECHNOLOGY, 182, 169 - 178, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Improving polyglucan production in cyanobacteria and microalgae via cultivation design and metabolic engineering.

    Aikawa S, Ho S.H, Nakanishi A, Chang J.S, Hasunuma Tomohisa, Kondo Akihiko

    2015年04月, Biotechnology Journal, 10 (6), 886 - 898, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Kaoru Onodera, Ayumi Yoshida, Hideo Noda, Akihiko Kondo

    Given the complex fungal morphology, we provide an alternative to enhancing secretory protein production by Aspergillus oryzae. Immobilized A. oryzae, constructed to overexpress phospholipase A(1) (PLA1) and cultivated in the presence of reticulated polyurethane foams, attained the extracellular PLA1 activities of 51.2-62.0 U/ml after 96-120h, higher than those of suspension cells (36.9-53.5 U/ml). Moreover, the extracellular PLA1 activity of the immobilized cells at 0.5% polypeptone concentration was 34.8 U/ml, which is more than one-half of the maximum activity attained using 2% polypeptone concentration. Further investigations suggested the contribution of high growth rates of immobilized cells toward the enhanced PLA1 production. The macroscopic morphology, which affects the supply of oxygen and nutrients to the interior of cell pellets, is likely the reason for the high growth rates. This is based on the findings that, at 0.5% polypeptone concentration, the suspension cells formed mycelial clumps growing to a diameter of 10 mm, whereas the immobilized cells maintained a dense film with a thickness of 0.4 mm at the surface of the reticulated matrix. Together with the potential utility of repeated-batch cultivation, immobilized cell culture can be a powerful tool for targeted control of fungal morphology in bioprocesses using A. oryzae. (C) 2014 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2015年04月, BIOCHEMICAL ENGINEERING JOURNAL, 96, 1 - 6, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Ida, Jun Ishii, Fumio Matsuda, Takashi Kondo, Akihiko Kondo

    Background: Isobutanol is an important biorefinery target alcohol that can be used as a fuel, fuel additive, or commodity chemical. Baker's yeast, Saccharomyces cerevisiae, is a promising organism for the industrial manufacture of isobutanol because of its tolerance for low pH and resistance to autolysis. It has been reported that gene deletion of the pyruvate dehydrogenase complex, which is directly involved in pyruvate metabolism, improved isobutanol production by S. cerevisiae. However, the engineering strategies available for S. cerevisiae are immature compared to those available for bacterial hosts such as Escherichia coli, and several pathways in addition to pyruvate metabolism compete with isobutanol production. Results: The isobutyrate, pantothenate or isoleucine biosynthetic pathways were deleted to reduce the outflow of carbon competing with isobutanol biosynthesis in S. cerevisiae. The judicious elimination of these competing pathways increased isobutanol production. ILV1 encodes threonine ammonia-lyase, the enzyme that converts threonine to 2-ketobutanoate, a precursor for isoleucine biosynthesis. S. cerevisiae mutants in which ILV1 had been deleted displayed 3.5-fold increased isobutanol productivity. The Delta ILV1 strategy was further combined with two previously established engineering strategies (activation of two steps of the Ehrlich pathway and the transhydrogenase-like shunt), providing 11-fold higher isobutanol productivity as compared to the parent strain. The titer and yield of this engineered strain was 224 +/- 5 mg/L and 12.04 +/- 0.23 mg/g glucose, respectively. Conclusions: The deletion of competitive pathways to reduce the outflow of carbon, including ILV1 deletion, is an important strategy for increasing isobutanol production by S. cerevisiae.

    BIOMED CENTRAL LTD, 2015年04月, Microbial Cell Factories, 14, 62, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Xiaoting Ye, Kana Morikawa, Shih-Hsin Ho, Michihiro Araki, Keiji Nishida, Tomohisa Hasunuma, Kiyotaka Y. Hara, Akihiko Kondo

    Background: Measurement of mitochondrial ATP synthesis is a critical way to compare cellular energetic performance. However, fractionation of mitochondria requires large amounts of cells, lengthy purification procedures, and an extreme caution to avoid damaging intact mitochondria, making it the highest barrier to high-throughput studies of mitochondrial function. To evaluate 45 genes involved in oxidative phosphorylation in Saccharomyces cerevisiae, we aimed to develop a simple and rapid method to measure mitochondrial ATP synthesis. Results: To obtain functional mitochondria, S. cerevisiae cells were lysed with zymolyase followed by two-step, low-then high-speed centrifugation. Using a firefly luciferin-luciferase assay, the ATP synthetic activity of the mitochondria was determined. Decreasing the ATP synthesis in the presence of mitochondrial inhibitors confirmed functionality of the isolated crude mitochondria. Deletion of genes encoding mitochondrial ATP synthesis-related protein showed their dependency on the oxidative phosphorylation in S. cerevisiae. Conclusions: Compared with conventional procedures, this measurement method for S. cerevisiae Mitochondrial ATP Synthetic activity in High-throughput (MASH method) is simple and requires a small amount of cells, making it suitable for high-throughput analyses. To our knowledge, this is the first report on a rapid purification process for yeast mitochondria suitable for high-throughput screening.

    BIOMED CENTRAL LTD, 2015年04月, Microbial Cell Factories, 14, 56, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Complete genome sequence of Kluyveromyces marxianus NBRC1777, a nonconventional thermotolerant yeast

    Kentaro Inokuma, Jun Ishii, Kiyotaka Hara, Masao Mochizuki, Tomohisa Hasunuma, Akihiko Kondo

    2015年04月, Genome Announcements, 3 (2), e00389 - 15, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Effective saccharinefication of kraft pulp by using a cecllulase cocktail prepared from genetically engineered Aspergillus oryzae.

    Yamada R, Yoshie T, Sakai S, Wakai S, Asai-Nakashima N, Okazaki Fumiyoshi, Ogino Chiaki, Hisada H, Tsutsumi H, Hata Y, Kondo Akihiko

    2015年03月, Bioscience, Biotechnology, and Biochemistry, 79 (6), 1034 - 1037, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yota Tsuge, Tomohisa Hasunuma, Akihiko Kondo

    Recent increasing attention to environmental issues and the shortage of oil resources have spurred political and industrial interest in the development of environmental friendly and cost-effective processes for the production of bio-based chemicals from renewable resources. Thus, microbial production of commercially important chemicals is viewed as a desirable way to replace current petrochemical production. Corynebacterium glutamicum, a Gram-positive soil bacterium, is one of the most important industrial microorganisms as a platform for the production of various amino acids. Recent research has explored the use of C. glutamicum as a potential cell factory for producing organic acids such as lactate and succinate, both of which are commercially important bulk chemicals. Here, we summarize current understanding in this field and recent metabolic engineering efforts to develop C. glutamicum strains that efficiently produce L- and D-lactate, and succinate from renewable resources.

    SPRINGER HEIDELBERG, 2015年03月, JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, 42 (3), 375 - 389, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Michihiro Araki, Robert Sidney Cox, Hiroki Makiguchi, Teppei Ogawa, Takeshi Taniguchi, Kohei Miyaoku, Masahiko Nakatsui, Kiyotaka Y. Hara, Akihiko Kondo

    Motivation: Construction of synthetic metabolic pathways promises sustainable production of diverse chemicals and materials. To design synthetic metabolic pathways of high value, computational methods are needed to expand present knowledge by mining comprehensive chemical and enzymatic information databases. Several computational methods have been already reported for the metabolic pathway design, but until now computation complexity has limited the diversity of chemical and enzymatic data used. Results: We introduce a computational platform, M-path, to explore synthetic metabolic pathways including putative enzymatic reactions and compounds. M-path is an iterative random algorithm, which makes efficient use of chemical and enzymatic databases to find potential synthetic metabolic pathways. M-path can readily control the search space and perform well compared with exhaustively enumerating possible pathways. A web-based pathway viewer is also developed to check extensive metabolic pathways with evaluation scores on the basis of chemical similarities. We further produce extensive synthetic metabolic pathways for a comprehensive set of alpha amino acids. The scalable nature of M-path enables us to calculate potential metabolic pathways for any given chemicals.

    OXFORD UNIV PRESS, 2015年03月, BIOINFORMATICS, 31 (6), 905 - 911, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuri Sakihama, Tomohisa Hasunuma, Akihiko Kondo

    The hydrolysis of lignocellulosic biomass liberates sugars, primarily glucose and xylose, which are subsequently converted to ethanol by microbial fermentation. The rapid and efficient fermentation of xylose by recombinant Saccharomyces cerevisiae strains is limited by weak acids generated during biomass pretreatment processes. In particular, acetic acid negatively affects cell growth, xylose fermentation rate, and ethanol production. The ability of S. cerevisiae to efficiently utilize xylose in the presence of acetic acid is an essential requirement for the cost-effective production of ethanol from lignocellulosic hydrolysates. Here, an acetic acid-responsive transcriptional activator, HAA1, was overexpressed in a recombinant xylose-fermenting S. cerevisiae strain to yield BY4741X/HAA1. This strain exhibited improved cell growth and ethanol production from xylose under aerobic and oxygen limited conditions, respectively, in the presence of acetic acid. The HAA1p regulon enhanced transcript levels in BY4741X/HAA1. The disruption of PHO13, a p-nitrophenylphosphatase gene, in BY4741X/HAA1 led to further improvement in both yeast growth and the ability to ferment xylose, indicating that HAM overexpression and PHO13 deletion act by different mechanisms to enhance ethanol production. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2015年03月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 119 (3), 297 - 302, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shih-Hsin Ho, Akihito Nakanishi, Xiaoting Ye, Jo-Shu Chang, Chun-Yen Chen, Tomohisa Hasunuma, Akihiko Kondo

    Background: Marine microalgae are among the most promising lipid sources for biodiesel production because they can be grown on nonarable land without the use of potable water. Marine microalgae also harvest solar energy efficiently with a high growth rate, converting CO2 into lipids stored in the cells. Both light intensity and nitrogen availability strongly affect the growth, lipid accumulation, and fatty acid composition of oleaginous microalgae. However, very few studies have systematically examined how to optimize lipid productivity by adjusting irradiance intensity, and the metabolic dynamics that may lead to improved lipid accumulation in microalgae have not been elucidated. Little is known about the mechanism of lipid synthesis regulation in microalgae. Moreover, few studies have assessed the potential of using marine microalgae as oil producers. Results: In this work, a newly isolated marine microalga, Chlamydomonas sp. JSC4, was selected as a potential lipid producer, and the effect of photobioreactor operations on cell growth and lipid production was investigated. The combined effects of light intensity and nitrogen depletion stresses on growth and lipid accumulation were further explored in an effort to markedly improve lipid production and quality. The optimal lipid productivity and content attained were 312 mg L-1 d(-1) and 43.1% per unit dry cell weight, respectively. This lipid productivity is the highest ever reported for marine microalgae. Metabolic intermediates were profiled over time to observe transient changes during lipid accumulation triggered by combined stresses. Finally, metabolite turnover was also assessed using an in vivo C-13-labeling technique to directly measure the flow of carbon during lipid biosynthesis under stress associated with light intensity and nitrogen deficiency. Conclusions: This work demonstrates the synergistic integration of cultivation and dynamic metabolic profiling technologies to develop a simple and effective strategy for enhancing oil production in a marine microalga. The knowledge obtained from this study could be useful in assessing the feasibility of marine microalgae biodiesel production and for understanding the links between dynamic metabolic profiles and lipid biosynthesis during the course of microalgal cultivation.

    BIOMED CENTRAL LTD, 2015年03月, BIOTECHNOLOGY FOR BIOFUELS, 8, 48, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shuhei Noda, Yoshifumi Kawai, Tsutomu Tanaka, Akihiko Kondo

    Streptomyces lividans was adopted as a host strain for 4-vinylphenol (4VPh) production directly from cellulose. In order to obtain novel phenolic acid decarboxylase (PAD) expressed in S. lividans, PADs distributed among Streptomyces species were screened. Three novel PADs, derived from Streptomyces sviceus, Streptomyces hygroscopicus, and Streptomyces cattleya, were successfully obtained and expressed in S. lividans. S. sviceus PAD (SsPAD) could convert p-hydroxycinnamic acid (pHCA) to 4VPh more efficiently than the others both in vitro and in vivo. For 4VPh production directly from cellulose, L-tyrosine ammonia lyase derived from Rhodobacter sphaeroides and SsPAD were introduced into endoglucanase-secreting S. lividans, and the 4VPh biosynthetic pathway was constructed therein. The created transformants successfully produced 4VPh directly from cellulose. (C) 2014 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2015年03月, BIORESOURCE TECHNOLOGY, 180, 59 - 65, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hirokazu Suzuki, Jun Ishii, Akihiko Kondo, Ken-ichi Yoshida

    Microbes employ cell membranes for reducing exogenous stresses. Polyamino acid display on microbial cell surfaces and their effects on microbial chemical stress tolerance were examined. Growth analysis revealed that displays of polyarginine, polyaspartate and polytryptophan substantially enhanced tolerance of Escherichia coli to NaCl. A titration assay indicated that polyarginine and polyaspartate altered cell surface charges, implying tolerance enhancement via ion atmosphere and/or ionic bond network formations for electrostatic ion repulsion. The enhancement by polytryptophan may have arisen from surface hydrophobicity increase for hydrophobic ion exclusion, because of a strong correlation between hydrophobic characters of amino acids and their effects on tolerance enhancement. The display also enhanced tolerance to other salts and/or alcohols in E. coli and to NaCl in Saccharomyces cerevisiae. Thus polyamino acid display has the potential as an approach for conferring chemical stress tolerance on various microbes.

    SPRINGER, 2015年02月, BIOTECHNOLOGY LETTERS, 37 (2), 429 - 435, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Izzat Fahimuddin Bin Mohamed Suffian, Yuya Nishimura, Kenta Morita, Sachiko Nakamura-Tsuruta, Khuloud T. Al-Jamal, Jun Ishii, Chiaki Ogino, Akihiko Kondo

    Background: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues. Results: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells. Conclusions: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

    BIOMED CENTRAL LTD, 2015年02月, JOURNAL OF NANOBIOTECHNOLOGY, 13 (1), 15, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yosuke Fukutani, Ayaka Hori, Satoshi Tsukada, Ryoichi Sato, Jun Ishii, Akihiko Kondo, Hiroaki Matsunami, Masafumi Yohda

    Olfaction depends on the selectivity and sensitivity of olfactory receptors. Previous attempts at constructing a mammalian olfactory receptor-based artificial odorant sensing system in the budding yeast Saccharomyces cerevisiae suffered from low sensitivity and activity. This result may be at least in part due to poor functional expression of olfactory receptors and/or limited solubility of some odorants in the medium. In this study, we examined the effects of two types of accessory proteins, receptor transporting protein 1 short and odorant binding proteins, in improving odor-mediated activation of olfactory receptors expressed in yeast. We found that receptor transporting protein 1 short enhanced the membrane expression and ligand-induced responses of some olfactory receptors. Coexpression of odorant binding proteins of the silkworm moth Bombyx mori enhanced the sensitivity of a mouse olfactory receptor. Our results suggest that different classes of accessory proteins can confer sensitive and robust responses of olfactory receptors expressed in yeast. Inclusion of accessory proteins may be essential in the future development of practical olfactory receptor-based odorant sensors. (C) 2014 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2015年02月, ANALYTICAL BIOCHEMISTRY, 471, 1 - 8, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kentaro Inokuma, Takanobu Yoshida, Jun Ishii, Tomohisa Hasunuma, Akihiko Kondo

    Recombinant yeast strains that display heterologous amylolytic enzymes on their cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system are considered as promising biocatalysts for direct ethanol production from starchy materials. For the effective hydrolysis of these materials, the ratio optimization of multienzyme activity displayed on the cell surface is important. In this study, we have presented a ratio control system of multienzymes displayed on the yeast cell surface by using different GPI-anchoring domains. The novel gene cassettes for the cell-surface display of Streptococcus bovis alpha-amylase and Rhizopus oryzae glucoamylase were constructed using the Saccharomyces cerevisiae SED1 promoter and two different GPI-anchoring regions derived from Saccharomyces cerevisiae SED1 or SAG1. These gene cassettes were integrated into the Saccharomyces cerevisiae genome in different combinations. Then, the cell-surface alpha-amylase and glucoamylase activities and ethanol productivity of these recombinant strains were evaluated. The combinations of the gene cassettes of these enzymes affected the ratio of cell-surface alpha-amylase and glucoamylase activities and ethanol productivity of the recombinant strains. The highest ethanol productivity from raw starch was achieved by the strain harboring one alpha-amylase gene cassette carrying the SED1-anchoring region and two glucoamylase gene cassettes carrying the SED1-anchoring region (BY-AASS/GASS/GASS). This strain yielded 22.5 +/- 0.6 g/L of ethanol from 100 g/L of raw starch in 120 h of fermentation.

    SPRINGER, 2015年02月, Applied Microbiology and Biotechnology, 99 (4), 1655 - 1663, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology

    Tanaka Tsutomu, Kondo Akihiko

    2015年02月, FEMS Yeast Research, 15, 1 - 9, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Sarvesh Kumar Srivastava, Chiaki Ogino, Akihiko Kondo

    Biological synthesis of nanoparticles has been present in living organisms over the course of evolution to serve a variety of purposes. In this chapter, we discuss the latest trends and application for nanoparticle synthesis via plants, algae, yeast, bacteria, fungi, etc. There exists several review articles among others documenting studies about various biogenic sources and associated nanoparticle synthesis we have rather emphasized on recent research works which probed into novel applications of these bio-nanoparticles along with some important historical fi ndings. Also, we have discussed the challenges faced by biogenic methods along with possible areas to tweak in order to standardize this synthesis technique. Biogenic synthesis of nanoparticles has the potential to provide cost-effective, eco-friendly alternative to work as “biological nanofactories”/functionalization method once the attention has been shifted to understand the underlying mechanism, its in vitro replication and obtaining shape/size control over the nanoparticles being synthesized.

    Springer International Publishing, 2015年01月01日, Green Processes for Nanotechnology: From Inorganic to Bioinspired Nanomaterials, 237 - 257, 英語

    [査読有り]

    論文集(書籍)内論文

  • Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces lividans as a host

    NODA S, MATSUMOTO Takuya, TANAKA Tsutomu, KONDO Akihiko

    2015年01月, Microbial Cell Factories, 14 (1), 5, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Matsumoto, Shota Shimada, Yuto Hata, Tsutomu Tanaka, Akihiko Kondo

    We here describe a unique beta-D-glucosidase (BGL; Blon_0625) derived from Bifidobacterium longum subsp. infantis ATCC 15697. The Blon_0625 gene was expressed by recombinant Escherichia coli. Purified recombinant Blon_0625 retains hydrolyzing activity against both p-nitrophenyl-beta-D-glucopyranoside (pNPG; 17.3 +/- 0.24 U mg(-1)) and p-nitrophenyl-beta-D-xylopyranoside (pNPX; 16.7 +/- 0.32 U mg(-1)) at pH 6.0, 30 degrees C. To best of our knowledge, no previously described BGL retains the same level of both pNPGase and pNPXase activity. Furthermore, Blon_0625 also retains the activity against 4-nitrophenyl-alpha-L-arabinofranoside (pNPAf; 5.6 +/- 0.09 U mg(-1)). In addition, the results of the degradation of phosphoric acid swollen cellulose (PASC) or xylan using endoglucanase from Thermobifida fusca YX (Tfu_0901) or xylanase from Kitasatospora setae KM-6054 (KSE_59480) show that Blon_0625 acts as a BGL and as a beta-D-xylosidase (XYL) for hydrolyzing oligosaccharides. These results clearly indicate that Blon_0625 is a multi-functional glycoside hydrolase which retains the activity of BGL, XYL, and also alpha-L-arabinofuranosidase. Therefore, the utilization of multi-functional Blon_0625 may contribute to facilitating the efficient degradation of lignocellulosic materials and help enhance bioconversion processes. Copyright (C) 2014 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2015年01月, ENZYME AND MICROBIAL TECHNOLOGY, 68, 10 - 14, 英語

    [査読有り]

    研究論文(学術雑誌)

  • KUDOU Motonori, OKAZAKI F, ASAI-NAKASHIMA N, OGINO Chiaki, KONDO Akihiko

    Cold-adapted beta-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes.

    SPRINGER, 2015年01月, Biotechnology Letters, 37 (1), 89 - 94, 英語

    [査読有り]

    研究論文(学術雑誌)

  • 代謝工学を利用したCorynebacterium glutamicumによる乳酸およびコハク酸の生産

    近藤 昭彦, 柘植 洋太

    2015年, バイオサイエンスとインダストリー, 73 (5), 362 - 368, 日本語

    研究論文(学術雑誌)

  • 合成生物工学の未来展望

    近藤 昭彦, 植田 充美

    2015年, 生物工学会誌, 93 (9), 522, 日本語

    研究論文(学術雑誌)

  • Green Processes for Nanotechnology, Chapter 8, Nanoparticle Synthesis

    Srivastave S.K, Ogino Chiaki, 近藤 昭彦

    2015年, Biogenic Approach, Springer, 237 - 257, 日本語

    研究論文(学術雑誌)

  • Chun Wan, Mingming Zhang, Qing Fang, Liang Xiong, Xinqing Zhao, Tomohisa Hasunuma, Fengwu Bai, Akihiko Kondo

    The mechanisms of how zinc protects the cells against acetic acid toxicity and acts as an antioxidant are still not clear. Here we present results of the metabolic profiling of the eukaryotic model yeast species Saccharomyces cerevisiae subjected to long term high concentration acetic acid stress treatment in the presence and absence of zinc supplementation. Zinc addition decreased the release of reactive oxygen species (ROS) in the presence of chronic acetic acid stress. The dynamic changes in the accumulation of intermediates in central carbon metabolism were observed, and higher contents of intracellular alanine, valine and serine were observed by zinc supplementation. The most significant change was observed in alanine content, which is 3.51-fold of that of the control culture in cells in the stationary phase. Subsequently, it was found that 0.5 g L-1 alanine addition resulted in faster glucose consumption in the presence of 5 g L-1 acetic acid, and apparently decreased ROS accumulation in zinc-supplemented cells. This indicates that alanine exerted its antioxidant effect at least partially through the detoxification of acetic acid. In addition, intracellular glutathione (GSH) accumulation was enhanced by zinc addition, which is related to the protection of yeast cells from the oxidative injury caused by acetic acid. Our studies revealed for the first time that zinc modulates cellular amino acid metabolism and redox balance, especially biosynthesis of alanine and glutathione to exert its antioxidant effect.

    ROYAL SOC CHEMISTRY, 2015年, METALLOMICS, 7 (2), 322 - 332, 英語

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    研究論文(学術雑誌)

  • Musashi Takenaka, Yusuke Miyachi, Jun Ishii, Chiaki Ogino, Akihiko Kondo

    An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, a-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

    ROYAL SOC CHEMISTRY, 2015年, Nanoscale, 7 (11), 4956 - 4963, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ku Syahidah Ku Ismail, Takatoshi Sakamoto, Tomohisa Hasunuma, Xin-Qing Zhao, Akihiko Kondo

    Lignocellulosic biomass is a potential substrate for ethanol production. However, pretreatment of lignocellulosic materials produces inhibitory compounds such as acetic acid, which negatively affect ethanol production by Saccharomyces cerevisiae. Supplementation of the medium with three metal ions (Zn2+, Mg2+, and Ca2+) increased the tolerance of S. cerevisiae toward acetic acid compared to the absence of the ions. Ethanol production from xylose was most improved (by 34%) when the medium was supplemented with 2 mM Ca2+, followed by supplementation with 3.5 mM Mg2+ (29% improvement), and 180 mu M Zn2+ (26% improvement). Higher ethanol production was linked to high cell viability in the presence of metal ions. Comparative transcriptomics between the supplemented cultures and the control suggested that improved cell viability resulted from the induction of genes controlling the cell wall and membrane. Only one gene, FIT2, was found to be up-regulated in common between the three metal ions. Also up-regulation of HXT1 and TKL1 might enhance xylose consumption in the presence of acetic acid. Thus, the addition of ionic nutrients is a simple and cost-effective method to improve the acetic acid tolerance of S. cerevisiae.

    WILEY-V C H VERLAG GMBH, 2014年12月, BIOTECHNOLOGY JOURNAL, 9 (12), 1519 - 1525, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shih-Hsin Ho, Xiaoting Ye, Tomohisa Hasunuma, Jo-Shu Chang, Akihiko Kondo

    Although the potential for biofuel production from microalgae via photosynthesis has been intensively investigated, information on the selection of a suitable operation strategy for microalgae-based biofuel production is lacking. Many published reports describe competitive strains and optimal culture conditions for use in biofuel production; however, the major impediment to further improvements is the absence of effective engineering strategies for microalgae cultivation and biofuel production. This comprehensive review discusses recent advances in understanding the effects of major environmental stresses and the characteristics of various engineering operation strategies on the production of biofuels (mainly biodiesel and bioethanol) using microalgae. The performances of microalgae-based biofuel-producing systems under various environmental stresses (i.e., irradiance, temperature, pH, nitrogen depletion, and salinity) and cultivation strategies (i.e., fed-batch, semi-continuous, continuous, two-stage, and salinity-gradient) are compared. The reasons for variations in performance and the underlying theories of the various production strategies are also critically discussed. The aim of this review is to provide useful information to facilitate development of innovative and feasible operation technologies for effectively increasing the commercial viability of microalgae-based biofuel production. (c) 2014 Elsevier Inc. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2014年12月, BIOTECHNOLOGY ADVANCES, 32 (8), 1448 - 1459, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Mami Matsuda, Youhei Senga, Shimpei Aikawa, Masakazu Toyoshima, Ginga Shimakawa, Chikahiro Miyake, Akihiko Kondo

    Background: To ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability. Results: In the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O-2 photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O2 evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo C-13-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain. Conclusions: Overexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO2 incorporation is enhanced by the flv3 overexpression. Increase in O-2 evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

    BIOMED CENTRAL LTD, 2014年12月, BIOTECHNOLOGY FOR BIOFUELS, 7, 493, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Satoshi Wakai, Toshihide Yoshie, Nanami Asai-Nakashima, Ryosuke Yamada, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, Akihiko Kondo

    Lactic acid is a commodity chemical that can be produced biologically. Lactic acid-producing Aspergillus oryzae strains were constructed by genetic engineering. The A. oryzae LDH strain with the bovine L-lactate dehydrogenase gene produced 38 g/L of lactate from 100 g/L of glucose. Disruption of the wild-type lactate dehydrogenase gene in A. oryzae LDH improved lactate production. The resulting strain A. oryzae LDHD871 produced 49 g/L of lactate from 100 g/L of glucose. Because A. oryzae strains innately secrete amylases, A. oryzae LDHD871 produced approximately 30 g/L of lactate from various starches, dextrin, or maltose (all at 100 g/L). To our knowledge, this is the first report describing the simultaneous saccharification and fermentation of lactate from starch using a pure culture of transgenic A. oryzae. Our results indicate that A. oryzae could be a promising host for the bioproduction of useful compounds such as lactic acid. (C) 2014 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2014年12月, BIORESOURCE TECHNOLOGY, 173, 376 - 383, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kiyotaka Y. Hara, Michihiro Araki, Naoko Okai, Satoshi Wakai, Tomohisa Hasunuma, Akihiko Kondo

    Fine chemicals that are physiologically active, such as pharmaceuticals, cosmetics, nutritional supplements, flavoring agents as well as additives for foods, feed, and fertilizer are produced by enzymatically or through microbial fermentation. The identification of enzymes that catalyze the target reaction makes possible the enzymatic synthesis of the desired fine chemical. The genes encoding these enzymes are then introduced into suitable microbial hosts that are cultured with inexpensive, naturally abundant carbon sources, and other nutrients. Metabolic engineering create efficient microbial cell factories for producing chemicals at higher yields. Molecular genetic techniques are then used to optimize metabolic pathways of genetically and metabolically well-characterized hosts. Synthetic bioengineering represents a novel approach to employ a combination of computer simulation and metabolic analysis to design artificial metabolic pathways suitable for mass production of target chemicals in host strains. In the present review, we summarize recent studies on bio-based fine chemical production and assess the potential of synthetic bioengineering for further improving their productivity.

    BIOMED CENTRAL LTD, 2014年12月, MICROBIAL CELL FACTORIES, 13 (1), 173, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kiyotaka Y. Hara, Toshihiko Morita, Masao Mochizuki, Keisuke Yamamoto, Chiaki Ogino, Michihiro Araki, Akihiko Kondo

    Background: Red yeast, Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) that is widely used in the aquaculture, food, pharmaceutical and cosmetic industries. Recently, the potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Addition of mevalonate, the common precursor for isoprenoid biosynthesis, has been shown to be critical to enhance the astaxanthin content in X. dendrorhous. However, addition of mevalonate is unrealistic during industrial isoprenoid production because it is an unstable and costly chemical. Therefore, up-regulating the intracellular mevalonate supply by enhancing the mevalonate synthetic pathway though genetic engineering is a promising strategy to improve isoprenoid production in X. dendrorhous. However, a system to strongly express multiple genes has been poorly developed for X. dendrorhous. Results: Here, we developed a multiple gene expression system using plasmids containing three strong promoters in X. dendrorhous (actin, alcohol dehydrogenase and triose-phosphate isomerase) and their terminators. Using this system, three mevalonate synthetic pathway genes encoding acetoacetyl-CoA thiolase, HMG-CoA synthase and HMG CoA reductase were overexpressed at the same time. This triple overexpressing strain showed an increase in astaxanthin production compared with each single overexpressing strain. Additionally, this triple overexpression of mevalonate synthetic pathway genes together with genes involved in beta-carotene and astaxanthin synthesis showed a synergetic effect on increasing astaxanthin production. Finally, astaxanthin production was enhanced by 2.1-fold compared with the parental strain without a reduction of cell growth. Conclusions: We developed a system to strongly overexpress multiple genes in X. dendrorhous. Using this system, the synthetic pathway of mevalonate, a common substrate for isoprenoid biosynthesis, was enhanced, causing an increase in astaxanthin production. Combining this multiple gene overexpression system with a platform strain that overproduces mevalonate has the potential to improve industrial production of various isoprenoids in X. dendrorhous.

    BIOMED CENTRAL LTD, 2014年12月, MICROBIAL CELL FACTORIES, 13 (1), 175, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hitoshi Suzuki, Thu V. Vuong, Yunchen Gong, Kin Chan, Chi-Yip Ho, Emma R. Master, Akihiko Kondo

    Expansin and expansin-related proteins loosen plant cell wall architectures and are widely distributed in several types of organisms, including plants, fungi and bacteria. Here we describe sequence diversity and unique gene expression profiles of multiple expansin-related proteins identified in the basidiomycete, Phanerochaete carnosa. The protein sequences were homologous to loosenin, an expansin-related protein reported in the basidiomycete, Bjerkandera adusta. We identified homologous sequences of each of those P. carnosa proteins in many basidiomycete species. Twelve P. carnosa loosenin-like proteins (LOOLs) were classified into two subgroups according to sequence homology. Conservation of polysaccharide-binding amino acid residues was stricter in subgroup A. Subgroup A sequences included a conserved 8-9 amino acid insertion in a polysaccharide-binding groove whereas subgroup B contained a 12-18 amino acid insertion next to the binding groove. The P. carnosa genome also encodes the expansin-related protein, DREX1, which adopts a loosenin-like structure but has lower sequence homology to other LOOLs. The gene expression analysis of those proteins showed distinct patterns that were not significantly related to subgroupings. The variation in the protein sequences and gene expression patterns, and wide distribution among the basidiomycota, suggest that the diverse cell wall loosening proteins contribute to effective plant cell wall association and utilization by basidiomycetes. (C) 2014 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2014年11月, FUNGAL GENETICS AND BIOLOGY, 72, 115 - 123, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo

    Angiotensin II (Ang II) type 1 receptor (AGTR1) is a G-protein-coupled receptor (GPCR). Its natural ligand, Ang II, is an important effector molecule controlling blood pressure and volume in the cardiovascular system, and is consequently involved in various diseases such as hypertension and heart failure. Thus, the signaling mediator, AGTR1, is a significant molecular target in medicinal and therapeutic fields. Yeast is a useful organism for sensing GPCR signaling because it provides a simplified version of the complicated machinery used by mammalian cells for signal transduction. Although yeast cells can successfully transmit a signal through a variety of human GPCRs expressed in the cell membrane, there have been no reports of the functional activation of AGTR1-mediated signaling in yeast cells. In the present study, we introduced a single mutation into human AGTR1 and used yeast-human chimeric G alpha to exert the functional activation of AGTR1 in yeast cells. The engineered yeast cells expressing AGTR1 mutated at Asn295 and the chimeric G alpha successfully transmitted the signal inside the yeast cells in response to Ang II peptide and its analogs (Ang III and Ang IV peptides) added to the assay medium. Further, we demonstrated that the autocrine Ang II peptide and its analog, produced and secreted by the engineered yeast cells, could by themselves promote AGTR1-mediated signaling. This means that screening for agonistic peptides with various sequences from a self-produced genetic library would be a viable strategy. Thus, the constructed yeast biosensor, integrating an Asn295-mutated AGTR1 receptor, will be valuable in the design of drugs to treat AGTR1-related diseases. (C) 2014 Wiley Periodicals, Inc.

    WILEY-BLACKWELL, 2014年11月, Biotechnology and Bioengineering, 111 (11), 2220 - 2228, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Daisuke Sasaki, Kengo Sasaki, Yota Tsuge, Masahiko Morita, Akihiko Kondo

    Central metabolite profiles from glucose in microbial communities during methanogenic process were compared between a stable methanogenic reactor (MR) and a deteriorated reactor (DR). The concentrations of intracellular metabolites related to the Embden-Meyerhof and pentose phosphate pathways, with the exception of pyruvate, remained high in the MR, showing increased carbon flux in the glycolysis pathway during stable methanogenesis. Extracellular acetate temporarily accumulated in the MR, consistent with higher ATP level in the MR. Intracellular concentrations of the intermediates in the reductive branch of tricarboxylic acid cycle, malate, fumarate, and succinate were higher in the DR. Low NADH/NAD(+) ratio both in the MR and DR would suggest NADH consumption during acetate and lactate/succinate production in the MR and DR, respectively. Intracellular glutamate levels were higher in the MR, correlating with lower NADPH/NADP(+) ratio concentrations in the MR. These findings contribute to a better understanding of the metabolic state during stable methanogenesis. (C) 2014 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2014年11月, BIORESOURCE TECHNOLOGY, 172, 83 - 90, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Norimasa Kashiwagi, Michiru Miyake, Shuichi Hirose, Masahiro Sota, Chiaki Ogino, Akihiko Kondo

    The end products from starch hydrolysis by amylases have important applications in various industries. Here, two amylases derived from two Streptomyces species that hydrolyze soluble starch from potato produced maltotriose as the primary maltooligosaccharide product. The genes, annotated as putative glycoside hydrolases, were cloned and expressed in Streptomyces lividans. These amylases displayed hydrolysis activity from pH 3 to 9.5 and were not affected by Ca2+. Optimal production of maltotriose was between 20 and 30 A degrees C at pH 6.5. At the optimal temperature, both amylases produced maltotriose-rich end products rather than either maltose or maltotetraose.

    SPRINGER, 2014年11月, BIOTECHNOLOGY LETTERS, 36 (11), 2311 - 2317, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Yota Tsuge, Daisuke Sasaki, Tomohisa Hasunuma, Takatoshi Sakamoto, Yuri Sakihama, Chiaki Ogino, Akihiko Kondo

    Oligomeric sugars in the liquid fraction of hot water-pretreated rice straw are more amenable to membrane process than monomeric sugars, as lower pressure is required. Following membrane process was employed: nanofiltration (NF) concentration -> (dilution -> NF concentration) x 2 times -> enzymatic hydrolysis (EH) -> ultrafiltration (UF) permeation [Implication: NF for recovery of oligomeric sugars, dilution and NF for removal of low molecular weight fermentation inhibitors, UF for removal of high molecular weight fermentation inhibitors and recovery of monomeric sugars after EH]. This process provided the liquid fraction containing 111.4 g L-1 of sugars, corresponding to 681.0 mM as monomeric sugars, from the original liquid fraction (181.1 mM monomeric sugars). Concentrations of low molecular weight fermentation inhibitors, acetic and formic acids, were decreased to 24% and 48%, respectively. Xylose-fermenting recombinant Saccharomyces cerevisiae produced 34.5 +/- 2.2 g L-1 ethanol from the 0.8 times liquid fraction (76% of theoretical yield). (C) 2014 Published by Elsevier Ltd.

    ELSEVIER SCI LTD, 2014年10月, BIORESOURCE TECHNOLOGY, 169, 380 - 386, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kengo Sasaki, Yota Tsuge, Daisuke Sasaki, Hiroshi Teramura, Satoshi Wakai, Hideo Kawaguchi, Takashi Sazuka, Chiaki Ogino, Akihiko Kondo

    The aim of this investigation was to attain high ethanol concentration by concentrating sweet sorghum juice using a two-step membrane separation process. Ultrafiltration permeation of the juice was used to remove residues, followed by nanofiltration concentration to increase the sugar concentration. The concentrated juice containing 180.0 g L-1 sucrose, 59.3 g L-1 glucose and 49.3 g L-1 fructose supplemented with nitrogen sources (10 and 20 g L-1 of yeast extract and polypeptone, respectively) was fermented by Saccharomyces cerevisiae BY4741 to produce 133.5 g L-1 of ethanol (87.6% of theoretical yield) after 48 h fermentation. Importantly, the addition of lower concentrations of exogenous nitrogen sources (3 and 6 g L-1 of yeast extract and polypeptone, respectively) or no exogenous nitrogen sources resulted in the production of 131.4 and 132.8 g L-1 of ethanol (84.8% and 86.0% of theoretical yield), respectively, after 48 h fermentation. (C) 2014 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2014年10月, BIORESOURCE TECHNOLOGY, 169, 821 - 825, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Yoshimi Hori, Takatoshi Sakamoto, Misa Ochiai, Haruyo Hatanaka, Akihiko Kondo

    Background: Bioethanol produced by the yeast Saccharomyces cerevisiae is currently one of the most promising alternatives to conventional transport fuels. Lignocellulosic hemicelluloses obtained after hydrothermal pretreatment are important feedstock for bioethanol production. However, hemicellulosic materials cannot be directly fermented by yeast: xylan backbone of hemicelluloses must first be hydrolyzed by heterologous hemicellulases to release xylose, and the yeast must then ferment xylose in the presence of fermentation inhibitors generated during the pretreatment. Results: A GIN11/FRT-based multiple-gene integration system was developed for introducing multiple functions into the recombinant S. cerevisiae strains engineered with the xylose metabolic pathway. Antibiotic markers were efficiently recycled by a novel counter selection strategy using galactose-induced expression of both FLP recombinase gene and GIN11 flanked by FLP recombinase recognition target (FRT) sequences. Nine genes were functionally expressed in an industrial diploid strain of S. cerevisiae: endoxylanase gene from Trichoderma reesei, xylosidase gene from Aspergillus oryzae, beta-glucosidase gene from Aspergillus aculeatus, xylose reductase and xylitol dehydrogenase genes from Scheffersomyces stipitis, and XKS1, TAL1, FDH1 and ADH1 variant from S. cerevisiae. The genes were introduced using the homozygous integration system and afforded hemicellulolytic, xylose-assimilating and inhibitor-tolerant abilities to the strain. The engineered yeast strain demonstrated 2.7-fold higher ethanol titer from hemicellulosic material than a xylose-assimilating yeast strain. Furthermore, hemicellulolytic enzymes displayed on the yeast cell surface hydrolyzed hemicelluloses that were not hydrolyzed by a commercial enzyme, leading to increased sugar utilization for improved ethanol production. Conclusions: The multifunctional yeast strain, developed using a GIN11/FRT-based marker recycling system, achieved direct conversion of hemicellulosic biomass to ethanol without the addition of exogenous hemicellulolytic enzymes. No detoxification processes were required. The multiple-gene integration technique is a powerful approach for introducing and improving the biomass fermentation ability of industrial diploid S. cerevisiae strains.

    BIOMED CENTRAL LTD, 2014年10月, MICROBIAL CELL FACTORIES, 13 (1), 145, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yota Tsuge, Yoshimi Hori, Motonori Kudou, Jun Ishii, Tomohisa Hasunuma, Akihiko Kondo

    The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD(+) and NADPH/NADP(+) ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.

    SPRINGER, 2014年10月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 98 (20), 8675 - 8683, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroyuki Morita, Tomohisa Hasunuma, Maria Vassileva, Akihiko Kondo, Roumiana Tsenkova

    Fuel ethanol produced from lignocettulose by the yeast Saccharomyces cerevisiae becomes an increasingly important alternative to fossil fuels. Selection of S. cerevisiae strains, which can effectively produce ethanol from xylose is crucial to improve the fuel yield from lignocellulose. In the present study, a universal calibration system was developed by the combination of time series, fermentation near infrared (NIR) spectral data analysis and reference high-performance analysis of a single yeast strain which enabled the evaluation of the ethanol production ability of a wide variety of xylose-fermenting yeast strains. Subtraction of xylose and ethanol concentrations at 0 h for each clone, as well as the respective spectra, reduced subtle errors of the fermentation components naturally occurring in multiple experiments to clearly visualize the difference of fermentation ability between strains. Also, NI R spectra showed specific peaks in difference spectra calculated from the subtraction treatment. A robust univariate linear regression model led to high prediction accuracy of xylose consumption (R-2 > 0.99) and ethanol production (R-2 > 0.98) for a wide variety of yeast strains when using only the distinct spectral pattern of a single-strain. Here, a novel method for screening of high-performing yeast strains has been developed requiring only a simple single-strain calibration model. The advantages of NIR spectroscopy such as rapid and convenient experimental preparation using electromagnetic waves in the region, which provide deep penetration into an aqueous sample, are successfully exploited in the proposed screening method.

    ROYAL SOC CHEMISTRY, 2014年09月, ANALYTICAL METHODS, 6 (17), 6628 - 6634, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuya Nishimura, Jun Ishii, Chiaki Ogino, Akihiko Kondo

    Techniques using nanotechnology in the detection and treatment of cancers have made great progress in multidisciplinary fields. The advances in drug delivery systems (DDSs) have been supported mainly by the development of varied nanoparticles (NPs). Although the NPs based on organic and inorganic materials are integral parts in DDSs, bio-nanoparticles containing biopolymer and virus-like particles (VLPs) are attractive biomaterials for DDSs because of their unique features originating in bio-based materials, such as biocompatibility, biodegradability and low immunogenicity. It is notable that these NPs additionally have a great advantage to enable the easy and flexible alteration of their features by genetic engineering approaches. Controlling the sequence and oligomeric process of polypeptide genes permits a variety of choices in type or size of biopolymeric NPs (e.g., elastin-like polypeptide NPs). In contrast, the functional genes are often inserted into the coding sequences for self-assembled proteins to give the VLPs (e.g., hemagglutinating virus of Japan, adeno-associated virus, human immunodeficiency virus-1, simian virus 40 and hepatitis B virus) additional functions. Thus, genetic engineering readily allow alterations of the properties of NPs (e.g., particle shape, size and stability) and grant of new abilities (e.g., cell-specificity and drug loading and release). In this review, we introduce recent advances in bio-nanoparticles from the standpoint of engineering.

    AMER SCIENTIFIC PUBLISHERS, 2014年09月, JOURNAL OF BIOMEDICAL NANOTECHNOLOGY, 10 (9), 2063 - 2085, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Misato Kaishima, Nobuo Fukuda, Jun Ishii, Akihiko Kondo

    Molecules that can control protein-protein interactions (PPIs) have recently drawn attention as new drug pipeline compounds. Here, we report a technique to screen desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. We previously constructed a screening system based on a target protein fused to a mutated G-protein gamma subunit (G gamma(cyto)) lacking membrane localization ability. This ability, required for signal transmission, is restored by recruiting G gamma(cyto) into the membrane only when the target protein interacts with an artificially membrane-anchored candidate protein, thereby allowing interacting partners (G gamma recruitment system) to be searched and identified. In the present study, the G gamma recruitment system was altered by integrating the cytosolic expression of a third protein as a competitor to set a desirable affinity threshold. This enabled the reliable selection of both affinity-enhanced and affinity-attenuated protein variants. The presented approach may facilitate the development of therapeutic proteins that allow the control of PPIs.

    PUBLIC LIBRARY SCIENCE, 2014年09月, PLOS ONE, 9 (9), e108229, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Signaling assays for detection of human G-protein-coupled receptors in yeast

    Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo

    2014年08月, Bio-protocol, 4 (16), e1206, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Seiji Akimoto, Makio Yokono, Erina Yokono, Shimpei Aikawa, Akihiko Kondo

    In photosynthetic organisms, the interactions among pigment protein complexes change in response to light conditions. In the present study, we analyzed the transfer of excitation energy from the phycobilisome (PBS) and photosystem (PS) II to PSI in the cyanobacterium Synechocystis sp. PCC 6803. After 20 mm of dark adaptation, Synechocystis cells were illuminated for 5 mm with strong light with different spectral profiles, blue, green, two kinds of red, and white light. After illumination, the energy-transfer characteristics were evaluated using steady-state fluorescence and picosecond time-resolved fluorescence spectroscopy techniques. The fluorescence rise and decay curves were analyzed by global analysis to obtain fluorescence decay-associated spectra, followed by spectral component analysis. Under illumination with strong light, the contribution of the energy transfer from the PSII to PSI (spillover) became greater, and that of the energy transfer from the PBS to PSI decreased; the former change was larger than the latter. The energy transfer pathway to PSI was sensitive to red light. We discuss the short-term adaptation of energy-transfer processes in Synechocystis under strong-light conditions. (C) 2014 Elsevier Masson SAS. All rights reserved.

    ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, 2014年08月, PLANT PHYSIOLOGY AND BIOCHEMISTRY, 81, 149 - 154, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Respiration accumulates Calvin cycle intermediates for the rapid start of photosynthesis in Synechocystis sp. PCC 6803

    SHIMAKAWA G, HASUNUMA Tomohisa, KONDO Akihiko, MATSUDA M, MAKINO A, MIYAKE C

    2014年08月, Bioscience, Biotechnology, and Biochemistry, 78 (12), 1997 - 2007, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kazuma Ogura, Kazuaki Ninomiya, Kenji Takahashi, Chiaki Ogino, Akihiko Kondo

    Background: Lignocellulosic biomass from plant biomass, especially softwoods, are well-known to present difficulties during attempts at hydrolysis due to their rigid structure. Pretreatment of lignocellulosic biomass with ionic liquids (ILs) is attractive as this requires to a low input of energy. However, IL pretreatment has the disadvantage of the presence of large amounts of water. Recently, it was reported that a small amount of acid has a positive effect on the degradation of biomass in IL with water. In this study the pretreatment of Japanese cedar, the most abundant softwood in Japan, was investigated using a combination of IL, acid and metal ions. Results: First, the novel ionic liquid pretreatment was investigated by changing the pretreatment solvent and the anti-solvent. A mixture of IL, acid and ferric oxide (Fe3+) ion was most effective for pretreatment, and an acetone-water mixture was also most effective on the precipitation of biomass. These optimized pretreatment combinations attained a higher degree of glucose release from the pretreated biomass. The amount of cellulose was concentrated from to a level of 36 to 84% of the insoluble fraction by the optimized pretreatment. Based on this result, it was assumed that the extraction of the lignin fraction from the biomass into an anti-solvent solution was attained. Finally, this optimized pretreatment was applied to the enzymatic hydrolysis of Japanese cedar at high-solid biomass loading, and 110 g/L of glucose production was attained. In addition, the ethanol fermentation with this hydrolyzed solution by Saccharomyces cerevisiae achieved 50 g/L ethanol production, and this yield reached 90% of the theoretical yield. Conclusions: We developed an effective pretreatment protocol by changing to a pretreatment solvent containing IL, acid, metal ion and anti-solvent. The optimized pretreatment has an effect on softwood and separately retrieved lignin as a by-product. The saccharified solution at high-solid biomass loading was converted to ethanol in a high yield. This proposed methodology would boost the performance of the bioconversion of low-cost materials to other chemicals, and would not be limited to only ethanol but also would include other target chemicals.

    BIOMED CENTRAL LTD, 2014年08月, BIOTECHNOLOGY FOR BIOFUELS, 7, 120, 英語

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    研究論文(学術雑誌)

  • Kiyotaka Y. Hara, Toshihiko Morita, Yusuke Endo, Masao Mochizuki, Michihiro Araki, Akihiko Kondo

    Astaxanthin is a valuable carotenoid that is widely used in the aquaculture, food, pharmaceutical, and cosmetic industries. Xanthophyllomyces dendrorhous is a carotenoid-synthesizing yeast strain that produces astaxanthin as its main pigment. Although metabolic engineering using gene manipulation is a valuable way to improve astaxanthin production, a gene expression system for X. dendrorhous has been poorly developed. In this study, three known promoters of X. dendrorhous, glycerol-3-phosphate dehydrogenase (gpd) promoter (Pgpd), glucose dehydrogenase (gdh) promoter (Pgdh), and actin (act) promoter (Pact), were evaluated for use in the overexpression of target proteins using green fluorescence protein (GFP) as an expression level indicator protein. The actin promoter, Pact, showed the highest expression level of GFP when compared with Pgpd and Pgdh. Additionally, to obtain new promoters for higher expression of target protein in X. dendrorhous, intracellular GFP intensity was evaluated for 13 candidate promoters. An alcohol dehydrogenase promoter, Padh4, showed more efficient expression of GFP rather than Pact. Overexpression of crtE gene encoding rate-limiting enzyme of carotenoid synthesis under the adh4 promoter yielded an increase in intracellular astaxanthin content of about 1.7-fold compared with the control strain. The promoters identified in this study must be useful for improving carotenoids production in X. dendrorhous.

    SPRINGER, 2014年08月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 98 (15), 6787 - 6793, 英語

    [査読有り]

    研究論文(学術雑誌)

  • アーミング酵母による統合型バイオプロセスの開発

    蓮沼 誠久, 近藤 昭彦

    2014年07月, 日本エネルギー学会誌, 93 (7), 580 - 585, 日本語

    研究論文(学術雑誌)

  • Chiak Ogino, Tetsuya Matsuda, Fumiyoshi Okazaki, Tsutomu Tanaka, Akihiko Kondo

    Effective secretion of green fluorescent protein (GFP) was investigated by the screening signal sequences for GFP secretion in Aspergillus oryzae. GFP production in A. oryzae was evaluated using fusions with signal sequences from Taka-amylase A (TAA), glucoamylase A, glucoamylase S, and triacylglycerol lipase. The TAA signal sequence promoted the highest protein secretion of GFP. Fusing this signal sequence with an N-terminal 28-amino acid region (N28 fragment) from the Rhizopus oryzae lipase signal sequence increased protein secretion. In addition, using multiple copies of this signal sequence, instead of the N28 fragment, also induced protein secretion. These results show that using multiple signal sequences or combining a signal sequence with the N28 fragment can be used to improve heterogeneous protein secretion in A. oryzae. (C) 2014 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2014年07月, PROCESS BIOCHEMISTRY, 49 (7), 1078 - 1083, 英語

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    研究論文(学術雑誌)

  • Hideo Kawaguchi, Kouji Uematsu, Chiaki Ogino, Hiroshi Teramura, Satoko Niimi-Nakamura, Yota Tsuge, Tomohisa Hasunuma, Ken-Ichi Oinuma, Naoki Takaya, Akihiko Kondo

    Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10-50 filter paper unit (FPU) g(-1) kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L-1) in a complex medium (pH 7.0) at 37 degrees C The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing log L-1 glucose compared to 100 g L-1. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions. (C) 2014 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2014年07月, BIOCHEMICAL ENGINEERING JOURNAL, 88, 188 - 194, 英語

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    研究論文(学術雑誌)

  • TANAKA Tsutomu, HIRATA Y, NAKANO M, KAWABATA H, KONDO Akihiko

    We demonstrated direct utilization of xylooligosaccharides using beta-xylosidase-displaying Escherichia coll. After screening active beta-xylosidases, BSU17580 from Bacillus subtilis or Tfu1616 from Thermobifida fusca YX, were successfully displayed on the E. coli cell surface using Blc or HdeD as anchor proteins, and these transformants directly assimilated xylooligosaccharides as a carbon source. The final OD 600 in minimal medium containing 2% xylooligosaccharides was 1.09 (after 12 h of cultivation) and 130 (after 40 h of cultivation). We then constructed an E. coli strain displaying both beta-glucosidase and beta-xylosidase. beta-glucosidase- and beta-xylosidase-displaying E. coli was successfully grown on a 1% cellobiose and 1% xylooligosaccharides mixture, and the OD 600 was 1.76 after 10 h of cultivation, which was higher and reached faster than that grown on a glucose/xylose mixture (1.20 after 30 h of cultivation).

    AMER CHEMICAL SOC, 2014年07月, ACS Synthetic Biology, 3 (7), 446 - 453, 英語

    [査読有り]

    研究論文(学術雑誌)

  • 酵素触媒法によるバイオディーゼル燃料生産システムのスケールアップ

    濱 真司, 吉田 あゆみ, 小野寺 かおる, 蔵田 兼司, 近藤 昭彦, 野田 秀夫

    2014年06月, 生物工学会誌, 92 (6), 262 - 269, 日本語

    研究論文(学術雑誌)

  • 「バイオプロダクション次世代農工連携研究」と拠点形成への展望

    近藤 昭彦, 荻野 千秋

    2014年06月, 紙パルプ技術タイムス, 57 (6), 51 - 56, 日本語

    研究論文(学術雑誌)

  • Two-step production of D-lactate from mixed sugars by growing and resting cells of metabolically engineered Lactobacillus plantarum.

    TSUGE Yota, KAWAGUCHI Hideo, SASAKI Kengo, TANAKA Tsutomu, KONDO Akihiko

    2014年06月, Applied Microbiology and Biotechnology, 98 (11), 4911 - 4918, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shih-Hsin Ho, Akihito Nakanishi, Xiaoting Ye, Jo-Shu Chang, Kiyotaka Hara, Tomohisa Hasunuma, Akihiko Kondo

    Background: Biodiesel production from marine microalgae has received much attention as microalgae can be cultivated on non-arable land without the use of potable water, and with the additional benefits of mitigating CO2 emissions and yielding biomass. However, there is still a lack of effective operational strategies to promote lipid accumulation in marine microalgae, which are suitable for making biodiesel since they are mainly composed of saturated and monounsaturated fatty acids. Moreover, the regulatory mechanisms involved in lipid biosynthesis in microalgae under environmental stress are not well understood. Results: In this work, the combined effects of salinity and nitrogen depletion stresses on lipid accumulation of a newly isolated marine microalga, Chlamydomonas sp. JSC4, were explored. Metabolic intermediates were profiled over time to observe transient changes during the lipid accumulation triggered by the combination of the two stresses. An innovative cultivation strategy (denoted salinity-gradient operation) was also employed to markedly improve the lipid accumulation and lipid quality of the microalga, which attained an optimal lipid productivity of 223.2 mg L-1 d(-1) and a lipid content of 59.4% per dry cell weight. This performance is significantly higher than reported in most related studies. Conclusions: This work demonstrated the synergistic integration of biological and engineering technologies to develop a simple and effective strategy for the enhancement of oil production in marine microalgae.

    BIOMED CENTRAL LTD, 2014年06月, BIOTECHNOLOGY FOR BIOFUELS, 7, 97, 英語

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    研究論文(学術雑誌)

  • Motonori Kudou, Yuka Kubota, Nanami Nakashima, Fumiyoshi Okazaki, Kazunori Nakashima, Chiaki Ogino, Akihiko Kondo

    The uses of ionic liquids (ILs) to alter the performance of enzymes and to pretreat cellulosic biomass have been investigated. In the present study, the activity of beta-glucosidase from Themotoga maritime (TmBgl1A) was studied in a phosphate buffer containing imidazolium-based ILs. Among the imidazolium-based ILs that were tested, 1-butyl-3-methylimidazolium acetate [Bmim][OAc] showed the most improvement for the hydrolysis activity of TmBgl1A at pH 7.0. From the steady-state emission spectra, [Bmim][OAc] quenched the intrinsic fluorescence of TmBgl1A. The improvement in the activity of TmBgl1A induced by [Bmim][OAc] could be linked to the flexibility of the conformation of TmBgl1A. (C) 2014 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2014年06月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 104, 17 - 22, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shimpei Aikawa, Atsumi Nishida, Shih-Hsin Ho, Jo-Shu Chang, Tomohisa Hasunuma, Akihiko Kondo

    Background: Oxygenic photosynthetic microorganisms such as cyanobacteria and microalgae have attracted attention as an alternative carbon source for the next generation of biofuels. Glycogen abundantly accumulated in cyanobacteria is a promising feedstock which can be converted to ethanol through saccharification and fermentation processes. In addition, the utilization of marine cyanobacteria as a glycogen producer can eliminate the need for a freshwater supply. Synechococcus sp. strain PCC 7002 is a fast-growing marine coastal euryhaline cyanobacteria, however, the glycogen yield has not yet been determined. In the present study, the effects of light intensity, CO2 concentration, and salinity on the cell growth and glycogen content were investigated in order to maximize glycogen production in Synechococcus sp. strain PCC 7002. Results: The optimal culture conditions for glycogen production in Synechococcus sp. strain PCC 7002 were investigated. The maximum glycogen production of 3.5 g L-1 for 7 days (a glycogen productivity of 0.5 g L-1 d(-1)) was obtained under a high light intensity, a high CO2 level, and a nitrogen-depleted condition in brackish water. The glycogen production performance in Synechococcus sp. strain PCC 7002 was the best ever reported in the alpha-polyglucan (glycogen or starch) production of cyanobacteria and microalgae. In addition, the robustness of glycogen production in Synechococcus sp. strain PCC 7002 to salinity was evaluated in seawater and freshwater. The peak of glycogen production of Synechococcus sp. strain PCC 7002 in seawater and freshwater were 3.0 and 1.8 g L-1 in 7 days, respectively. Glycogen production in Synechococcus sp. strain PCC 7002 maintained the same level in seawater and half of the level in freshwater compared with the optimal result obtained in brackish water. Conclusions: We conclude that Synechococcus sp. strain PCC 7002 has high glycogen production activity and glycogen can be provided from coastal water accompanied by a fluctuation of salinity. This work supports Synechococcus sp. strain PCC 7002 as a promising carbohydrate source for biofuel production.

    BIOMED CENTRAL LTD, 2014年06月, BIOTECHNOLOGY FOR BIOFUELS, 7, 88, 英語

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    研究論文(学術雑誌)

  • Jun Ishii, Takashi Kondo, Harumi Makino, Akira Ogura, Fumio Matsuda, Akihiko Kondo

    Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S.cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering.

    WILEY-BLACKWELL, 2014年05月, FEMS YEAST RESEARCH, 14 (3), 399 - 411, 英語

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    研究論文(学術雑誌)

  • Kengo Sasaki, Yota Tsuge, Daisuke Sasaki, Akihiko Kondo

    Under conditions conductive to growth, Corynebacterium glutamicum showed higher lactate yield from glucose (1.62 +/- 0.04) in a bioelectrochemical reactor including 0.2 mM of anthraquinone 2,6-disulfonate with the electrode potential regulated at -0.6 V (vs. Ag/AgCl) than in a non-regulated environment (1.10 +/- 0.03), clarifying that low cathodic potential is beneficial for lactate production. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2014年05月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 117 (5), 598 - 601, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ryosuke Yamada, Toshihide Yoshie, Satoshi Wakai, Nanami Asai-Nakashima, Fumiyoshi Okazaki, Chiaki Ogino, Hiromoto Hisada, Hiroko Tsutsumi, Yoji Hata, Akihiko Kondo

    Background: Kojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications. Thus, an efficient and cost-effective kojic acid production process would be valuable. However, little is known about the complete set of genes for kojic acid production. Currently, kojic acid is produced from glucose. The efficient production of kojic acid using cellulose as an inexpensive substrate would help establish cost-effective kojic acid production. Results: A kojic acid transcription factor gene over-expressing the A. oryzae strain was constructed. Three genes related to kojic acid production in this strain were transcribed in higher amounts than those found in the wild-type strain. This strain produced 26.4 g/L kojic acid from 80 g/L glucose. Furthermore, this strain was transformed with plasmid harboring 3 cellulase genes. The resultant A. oryzae strain successfully produced 0.18 g/L of kojic acid in 6 days of fermentation from the phosphoric acid swollen cellulose. Conclusions: Kojic acid was produced directly from cellulose material using genetically engineered A. oryzae. Because A. oryzae has efficient protein secretion ability and secondary metabolite productivity, an A. oryzae-based cell factory could be a platform for the production of various kinds of bio-based chemicals.

    BIOMED CENTRAL LTD, 2014年05月, MICROBIAL CELL FACTORIES, 13, 71, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kenji Okano, Tsutomu Tanaka, Akihiko Kondo

    Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA and LA-based polymers production using several kinds of genetically modified microorganisms, such as lactic acid bacteria, Escherichia coli, Corynebacterium glutamicum, and yeasts. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass have been significantly improved, as well as LA-based polymers. The drawbacks as well as improvements of LA production by fermentation are discussed.

    Wiley Blackwell, 2014年04月28日, Bioprocessing of Renewable Resources to Commodity Bioproducts, 353 - 380, 英語

    [査読有り]

    論文集(書籍)内論文

  • Tomohisa Hasunuma, Ryosuke Yamada, Akihiko Kondo

    Numerous environmental and social benefits could result from the replacement of petroleum-based transport fuels with bioethanol converted from renewable biomass. One of the key elements for the development of environmentally benign ethanol production is the construction of biomass-hydrolyzing yeast strains. The commonly used yeast Saccharomyces cerevisiae is a superior ethanol producer with demonstrated fast sugar consumption, high ethanol yield from glucose, and high resistance to ethanol. Hence, many researchers have targeted the heterologous expression of biomass-degrading enzymes in yeast to utilize glucose released from biomass for the production of ethanol by itself. In particular, cell-surface engineering is a powerful tool for yeast engineering. The display of amylolytic and cellulolytic enzymes on the yeast cell surface has accomplished direct ethanol production from starchy and cellulosic biomass. Moreover, reutilization of the cell surface-engineered yeast has the advantage of reducing enzyme cost, enabling reuse of enzymes on the cell surface by collecting the cells. For the efficient production of ethanol from biomass, improved assimilation of a wide variety of substrates could be achieved by overexpressing or deleting genes encoding traits responsible for yeast fermentability. Establishing economically feasible fermentation processes requires a marked increase in ethanol product titers due to the high energy demands of product recovery steps, as well as the high capital and production costs associated with bioethanol production equipment. A combination of biomass-degrading enzyme capacity and metabolic engineering in yeast strains could be an effective approach to developing cells with novel fermentation ability for industrial applications, and development of functional consolidated bioprocessing.

    Wiley Blackwell, 2014年04月28日, Bioprocessing of Renewable Resources to Commodity Bioproducts, 201 - 226, 英語

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    論文集(書籍)内論文

  • 植物の機能解明と新たな機能材料創出の可能性

    寺村 浩, 延田 紘治, 川口 秀夫, 荻野 千秋, 西野 孝, 近藤 昭彦

    2014年04月, バイオインダストリー, 31 (4), 62 - 68, 日本語

    研究論文(学術雑誌)

  • Shinji Hama, Kohsuke Nakano, Kaoru Onodera, Masashi Nakamura, Hideo Noda, Akihiko Kondo

    This study was conducted to realize the potential application of cellulose acetate to enzymatic processing, followed by microbial ethanol fermentation. To eliminate the effect of steric hindrance of acetyl groups on the action of cellulase, cellulose acetate was subjected to deacetylation in the presence of 1N sodium hydroxide and a mixture of methanol/acetone, yielding 88.8-98.6% at 5-20% substrate loadings during a 48 h saccharification at 50 degrees C. Ethanol fermentation using Saccharomyces cerevisiae attained a high yield of 92.3% from the initial glucose concentration of 44.2 g/L; however, a low saccharification yield was obtained at 35 degrees C, decreasing efficiency during simultaneous saccharification and fermentation (SSF). Pre-saccharification at 50 degrees C prior to SSF without increasing the total process time attained the ethanol titers of 19.8 g/L (5% substrate), 38.0 g/L (10% substrate), 55.9 g/L (15% substrate), and 70.9 g/L (20% substrate), which show a 12.0-16.2% improvement in ethanol yield. (C) 2014 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2014年04月, BIORESOURCE TECHNOLOGY, 157, 1 - 5, 英語

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    研究論文(学術雑誌)

  • Naoko Okai, Chihiro Takahashi, Kazuki Hatada, Chiaki Ogino, Akihiko Kondo

    Gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by Corynebacterium glutamicum that expresses Escherichia coli glutamate decarboxylase (GAD) B encoded by gadB. This strain was engineered to produce GABA more efficiently from biomass-derived sugars. To enhance GABA production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pknG (encoding serine/threonine protein kinase G), which controls the activity of 2-oxoglutarate dehydrogenase (Odh) in the tricarboxylic acid cycle branch point leading to glutamate synthesis. We succeeded in expressing GadB in a C. glutamicum strain harboring a deletion of pknG. C. glutamicum strains GAD and GAD Delta pknG were cultured in GP2 medium containing 100 g L-1 glucose and 0.1 mM pyridoxal 5'-phosphate. Strain GAD Delta pknG produced 31.1 +/- 0.41 g L-1 (0.259 g L-1 h(-1)) of GABA in 120 hours, representing a 2.29-fold higher level compared with GAD. The production yield of GABA from glucose by GAD Delta pknG reached 0.893 mol mol(-1).

    BIOMED CENTRAL LTD, 2014年04月, AMB EXPRESS, 4, 20, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuya Nishimura, Koichi Takeda, Ryosuke Ezawa, Jun Ishii, Chiaki Ogino, Akihiko Kondo

    Background: An affibody-displaying bio-nanocapsule (Z(HER2)-BNC) with a hepatocyte specificity derived from hepatitis B virus (HBV) was converted into an affibody, Z(HER2), that recognizes HER2 receptors. This affibody was previously reported to be the result of the endocytosis-dependent specific uptake of proteins and siRNA into target cancer cells. To assist the endosomal escape of inclusions, a helper lipid with pH-sensitive fusogenic ability (1,2-dioleoyl-sn-glycero-3-phos phoethanolamine; DOPE) was conjugated with a Z(HER2)-BNC. Findings: In this study, we displayed a pH-sensitive fusogenic GALA peptide on the surface of a particle in order to confer the ability of endosomal escape to a Z(HER2)-BNC. A GALA-displaying Z(HER2)-BNC purified from yeast uneventfully formed a particle structure. Furthermore, endosomal escape of the particle was facilitated after endocytic uptake and release of the inclusions to the cytoplasm without the cell toxicity. Conclusion: The genetic fusion of a GALA peptide to the virus-like particle confers the ability of endosomal escape.

    BIOMED CENTRAL LTD, 2014年04月, JOURNAL OF NANOBIOTECHNOLOGY, 12 (1), 11, 英語

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    研究論文(学術雑誌)

  • リグノセルロース系バイオマスからのバイオ燃料・バイオ化学品生産プロセスの開発

    近藤 昭彦

    2014年03月, 繊維と工業, 70 (3), 99 - 102, 日本語

    研究論文(学術雑誌)

  • リグノセルロースからの高効率エタノール生産に向けた新規代謝改変酵母の創製

    蓮沼 誠久, 近藤 昭彦

    2014年03月, オレオサイエンス, 14 (3), 95 - 101, 日本語

    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Norika Takemoto, Jun Ishii, Akihiko Kondo

    Understanding the role of G-protein-coupled receptor (GPCR) dimerization in cellular function has now become a major research focus. The potentially large functional and physiological diversity of dimerization among GPCRs is expected to provide opportunities for novel drug discovery. However, there is currently a lack of cell-based assays capable of specific profiling for the functional consequences of dimerization linked to ligand-mediated signaling. Here, we present an advanced method to simultaneously analyze the dimerization and ligand response of GPCRs using two yeast-based systems for split-ubiquitin two-hybrid assay and G-protein signaling assay. To permit simultaneous detection, we established a two-color (dual-color) fluorescence reporter gene assay using enhanced green fluorescent protein (EGFP) and a far-red derivative of the tetrameric fluorescent protein DsRed-Express2 (E2-Crimson). In the present study, we tested our method first by analyzing dimerization and ligand-mediated signaling by the yeast endogenous pheromone receptor (Ste2p). Second, we showed that the system facilitated mutational analysis of domains involved in dimerization and signaling by Ste2p. Third, we successfully demonstrated that the system could simultaneously monitor homo- and hetero-dimerization and somatostatin-induced signaling in the test case of the human SSTR5 somatostatin receptor. Our strategy is expected to provide a useful tool for the elucidation of molecular biological functions of GPCR dimers and for the screening of GPCR dimer-specific agonistic ligands. Biotechnol. Bioeng. 2014;111: 586-596. (c) 2013 Wiley Periodicals, Inc.

    WILEY, 2014年03月, BIOTECHNOLOGY AND BIOENGINEERING, 111 (3), 586 - 596, 英語

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    研究論文(学術雑誌)

  • Ancy Joseph, Shimpei Aikawa, Kengo Sasaki, Fumio Matsuda, Tomohisa Hasunuma, Akihiko Kondo

    The effect of phycobilisome antenna-truncation in the cyanobacterium Synechocystis sp. PCC 6803 on biomass production and glycogen accumulation have not yet been fully clarified. To investigate these effects here, the apcE gene, which encodes the anchor protein linking the phycobilisome to the thylakoid membrane, was deleted in a glucose tolerant strain of Synechocystis sp. PCC 6803. Biomass production of the apcE-deleted strain under photoautotrophic and atmospheric air conditions was 1.6 times higher than that of strain PCC 6803 (1.32 +/- 0.01 versus 0.84 +/- 0.07 g cell-dry weight L-1, respectively) after 15 days of cultivation. In addition, the glycogen content of the apcE-deleted strain (24.2 +/- 0.7%) was also higher than that of strain PCC 6803 (11.1 +/- 0.3%). Together, these results demonstrate that antenna truncation by deleting the apcE gene was effective for increasing biomass production and glycogen accumulation under photoautotrophic and atmospheric air conditions in Synechocystis sp. PCC 6803.

    BIOMED CENTRAL LTD, 2014年03月, AMB EXPRESS, 4, 17, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Structural evaluation of the DNA aptamer for ATP DH25.42 by AFM., Nucleosides

    MIYACHI Y, OGINO Chiaki, KONDO Akihiko

    2014年02月, Nucleosides, Nucleotides and Nucleic Acids, 33 (1), 31 - 39, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Jun Ishii, Asami Oda, Shota Togawa, Akira Fukao, Toshinobu Fujiwara, Chiaki Ogino, Akihiko Kondo

    Neurotensin receptor type-I (NTSR1) is a member of the G-protein-coupled receptor (GPCR) family. The natural ligand of NTSR1 is neurotensin (NT), a neuromodulator of the central nervous system. Because NT is also involved in many oncogenic actions, the signaling mediator NTSR1 is a significant molecular target in medicinal and therapeutic fields. In the current study, we constructed a fluorescence-based microbial yeast biosensor that can monitor the activation of human NTSR1 signaling responding to its agonist. To increase the sensitivity of the biosensor, a yeast strain with the green fluorescent protein (GFP) reporter gene was genetically engineered to enhance binding with human NTSRI expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein a-subunit (G alpha) in yeast Gpalp were substituted with the equivalent human G alpha, sequences (Gpal/G alpha(q) transplant). After optimizing the assay conditions, the G alpha-engineered yeast demonstrated significantly improved sensing for NTSR1 signaling. Because detection using a GFP fluorescence reporter considerably simplifies the measurement procedure, this microbial fluorescence sensor holds promise for use in the diagnosis of NTSR1-associated diseases and the development of agonists. (C) 2013 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2014年02月, Analytical Biochemistry, 446, 37 - 43, 英語

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    研究論文(学術雑誌)

  • Nobuo Yoshimoto, Kenji Tatematsu, Masumi Iijima, Tomoaki Niimi, Andres D. Maturana, Ikuo Fujii, Akihiko Kondo, Katsuyuki Tanizawa, Shun'ichi Kuroda

    Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In similar to 3.2 x 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening.

    NATURE PUBLISHING GROUP, 2014年02月, SCIENTIFIC REPORTS, 4, 4242, 英語

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    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Ku Syahidah Ku Ismail, Yumiko Nambu, Akihiko Kondo

    Lignocellulosic biomass dedicated to bioethanol production usually contains pentoses and inhibitory compounds such as furfural that are not well tolerated by Saccharomyces cerevisiae. Thus, S. cerevisiae strains with the capability of utilizing both glucose and xylose in the presence of inhibitors such as furfural are very important in industrial ethanol production. Under the synergistic conditions of transaldolase (TAL) and alcohol dehydrogenase (ADH) overexpression, S. cerevisiae MT8-1X/TAL ADH was able to produce 1.3-fold and 2.3-fold more ethanol in the presence of 70 mM furfural than a TAL-expressing strain and a control strain, respectively. We also tested the strains' ability by mimicking industrial ethanol production from hemicellulosic hydrolysate containing fermentation inhibitors, and ethanol production was further improved by 16% when using MT8-1X/TAL-ADH compared to the control strain. Transcript analysis further revealed that besides the pentose phosphate pathway genes TKL1 and TAL1, ADH7 was also upregulated in response to furfural stress, which resulted in higher ethanol production compared to the TAL-expressing strain. The improved capability of our modified strain was based on its capacity to more quickly reduce furfural in situ resulting in higher ethanol production. The co-expression of TAL/ADH genes is one crucial strategy to fully utilize undetoxified lignocellulosic hydrolysate, leading to cost-competitive ethanol production. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2014年02月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 117 (2), 165 - 169, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroyuki Matsuoka, Kazuya Hashimoto, Aki Saijo, Yuki Takada, Akihiko Kondo, Mitsuyoshi Ueda, Hiroshi Ooshima, Taro Tachibana, Masayuki Azuma

    A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. -Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of -glucosidase activity using p-nitrophenyl--glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright (c) 2014 John Wiley & Sons, Ltd.

    WILEY-BLACKWELL, 2014年02月, YEAST, 31 (2), 67 - 76, 英語

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    研究論文(学術雑誌)

  • Takashi Osanai, Akira Oikawa, Tomokazu Shirai, Ayuko Kuwahara, Hiroko Iijima, Kan Tanaka, Masahiko Ikeuchi, Akihiko Kondo, Kazuki Saito, Masami Yokota Hirai

    WILEY-BLACKWELL, 2014年02月, ENVIRONMENTAL MICROBIOLOGY, 16 (2), 512 - 524, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Joseph Ancy, Aikawa Shimpei, Sasaki Kengo, Teramura Hiroshi, Hasunuma Tomohisa, Matsuda Fumio, Osanai Takashi, Hirai Masami Yokota, Kondo Akihiko

    2014年01月31日, FEBS LETTERS, 588 (3), 466 - 471

    [査読有り]

  • Robert Sidney Cox, Masahiko Nakatsuil, Hiroki Makiguchi, Teppei Ogawa, Akihiko Kondo, Michihiro Araki

    2014年01月01日, Synthetic Biology: Engineering, Evolution, and Design Conference 2014, SEED 2014, 54 - 56

  • Ancy Joseph, Shimpei Aikawa, Kengo Sasaki, Hiroshi Teramura, Tomohisa Hasunuma, Fumio Matsuda, Takashi Osanai, Masami Yokota Hirai, Akihiko Kondo

    Rre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Delta rre37 was due to low expression of glycogen anabolic genes. In addition to low 2-oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose-6-phosphate, fructose-1,6-bisphosphate, and glyceraldehyde-3-phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2-oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2014年01月, FEBS LETTERS, 588 (3), 466 - 471, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kentaro Inokuma, Tomohisa Hasunuma, Akihiko Kondo

    Background: The recombinant yeast strains displaying the heterologous cellulolytic enzymes on the cell surface using the glycosylphosphatidylinositol (GPI) anchoring system are considered promising biocatalysts for direct conversion of lignocellulosic materials to ethanol. However, the cellulolytic activities of the conventional cellulase-displaying yeast strains are insufficient for the hydrolysis of cellulose. In this study, we constructed novel gene cassettes for the efficient cellulose utilization by cellulase-displaying yeast strains. Results: The novel gene cassettes for the cell-surface display of Aspergillus aculeatus beta-glucosidase (BGL1) and Trichoderma reeseii endoglucanase II (EGII) were constructed using the promoter and the GPI anchoring region derived from Saccharomyces cerevisiae SED1. The gene cassettes were integrated into the S. cerevisiae genome, then the beta-glucosidase activity of these recombinant strains was evaluated. We revealed that simultaneous utilization of the SED1 promoter and Sed1 anchoring domain in a gene cassette enabled highly-efficient enzyme integration into the cell wall. The beta-glucosidase activity of recombinant yeast cells transduced with the novel gene cassette was 8.4-fold higher than that of a conventional strain. The novel EGII-displaying strain also achieved 106-fold higher hydrolysis activity against the water-insoluble cellulose than a conventional strain. Furthermore, direct ethanol production from hydrothermally processed rice straw was improved by the display of T. reeseii EGII using the novel gene cassette. Conclusions: We have developed novel gene cassettes for the efficient cell-surface display of exo- and endo-type cellulolytic enzymes. The results suggest that this gene cassette has the wide applicability for cell-surface display and that cellulase-displaying yeasts have significant potential for cost-effective bioethanol production from lignocellulosic biomass.

    BIOMED CENTRAL LTD, 2014年01月, BIOTECHNOLOGY FOR BIOFUELS, 7 (1), 8, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Akihito Nakanishi, Shimpei Aikawa, Shih-Hsin Ho, Chun-Yen Chen, Jo-Shu Chang, Tomohisa Hasunuma, Akihiko Kondo

    Biodiesel production from microalgae has become a popular research topic. In this study, Chlamydomonas sp. JSC4 isolated from the southern coast of Taiwan was selected for a detailed study on cell growth and lipid accumulation under marine salinity (3.5% sea salt). Proper CO2 was supplied as the improvement of lipid productivity. Under the optimal condition, the highest lipid productivity was 169.1 mg/L/d, which was significantly higher than those reported in current studies for marine green algae. To date, only very few studies have reported a marine algae strain with both high cell growth and lipid productivity. This study demonstrated that a newly isolated marine green alga Chlamydomonas sp. JSC4 would be a feasible oil producer due to its high biomass production and lipid productivity under marine salinity. (C) 2013 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2014年01月, BIORESOURCE TECHNOLOGY, 152, 247 - 252, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Akihiko Kondo, Virendra S. Bisaria

    Wiley-VCH Verlag, 2014年, Biotechnology Journal, 9 (12), 1459 - 1461, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Sarvesh Kumar Srivastava, Chiaki Ogino, Akihiko Kondo

    A novel green one-pot approach for surface modification of graphene oxide into organosulfur modified graphene nanosheets (OS-GNS) is being reported. Alliin (garlic phytochemical) mediated organothiol linkages over OS-GNS facilitated one-step attachment of pre-synthesized gold nanoparticles. In absence of alliin treatment, no Au NP attachment was observed.

    ROYAL SOC CHEMISTRY, 2014年, RSC ADVANCES, 4 (12), 5986 - 5989, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Sarvesh Kumar Srivastava, Jonathan Sabate del Rio, Ciara K. O'Sullivan, Chiaki Ogino, Akihiko Kondo

    We report an eco-friendly, one-pot, room-temperature method for the rapid synthesis of electrocatalytically active Au@Pt (50 nm) bimetallic nanoparticles via a tryptophan (Trp) mediated supramolecular interface in an aqueous environment. Our results demonstrate a simple universal approach for high shell-metal loading where a pre-stabilized tryptophan polymerized-Au core serves as a template to facilitate subsequent deposition of Pt. We observed that the amine-stabilized poly-Trp bi-layer has an enhancing effect on the electrocatalytic potential of Au@Pt NPs by the virtue of an amine stabilized interface, thereby enhancing the HER activity over glassy carbon electrodes. Several characterization techniques were used to confirm the inherent core-shell morphology of the resulting Au@Pt NPs. This Trp mediated facile green synthesis strategy has the potential to synthesize an array of Au-core containing bimetallic nanoparticles with enhanced catalytic activity and stable structure integration.

    ROYAL SOC CHEMISTRY, 2014年, RSC ADVANCES, 4 (89), 48458 - 48464, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yota Tsuge, Toshihiro Tateno, Kengo Sasaki, Tomohisa Hasunuma, Tsutomu Tanaka, Akihiko Kondo

    We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying α-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40°C, which are higher than the optimal growth temperature of 30°C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated.

    2013年12月17日, AMB Express, 3 (1), 72 - 72, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • T. Matsumoto, S. Shimada, K. Yamamoto, T. Tanaka, A. Kondo

    The present study reports the design of a novel bioanode to deeply oxidize glucose in an enzymatic biofuel cell (EFC). This enzymatic glucose cell utilizes three co-immobilized enzymes: NAD-dependent glucose dehydrogenase (GDH), NAD(P)+-dependent gluconate-5-dehydrogenase (Ga5DH), and diaphorase (DI). Glucose is oxidized to gluconate by NAD-dependent GDH, gaining two electrons per glucose the gluconate obtained as a by-product is oxidized at the C5 carbon to 5-keto-gluconate by Ga5DH. Operation of our bioanode enabled the oxidation of glucose in two stages, resulting in the gain of four electrons. The three-enzyme EFC provides a maximum power density of 10.51 ± 1.72 μW cm-2, which is about 1.6 times higher than the maximum power density of an EFC using a bioanode based on the co-immobilization of two enzymes (GDH and DI). Our results hold promise for increasing the current density of EFCs, and for application in glucose biosensor. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

    2013年12月, Fuel Cells, 13 (6), 960 - 964, 英語

    [査読有り]

    研究論文(学術雑誌)

  • HWANG SY, NAKASHIMA K, OKAI Naoko, OKAZAKI F, MIYAKE M, HANAZONO K, OGINO Chiaki, KONDO Akihiko

    Amylases from Streptontyces are useful in the production of maltooligosaccharides, but they have weak thermal stability at temperatures higher than 40 degrees C. In this study, alpha-amylase (SAV5981 gene of Streptomyces avermitilis) was expressed from Streptomyces lividans 1326 and purified by ammonium sulfate fractionation followed by anionic chromatography (Q-HP sepharose). The properties of the purified SAV5981 amylase were determined by the starch-iodine method. The effect of metal ions on amylase activity was investigated. The optimal temperature shifted from 25 to 50 degrees C with the addition of the Ca2+ ion. The thermal stability of SAV5981 was also dramatically enhanced by the addition of 10 mm CaCl2. Improvement of the thermal stability of SAV5981 was examined by CD spectra in the presence and the absence of the Ca2+ ion. Thin-layer chromatography (TLC) analysis and HPLC analysis of starch degradation revealed that SAV5981 mainly produced maltose and maltotriose, not glucose. The maltoorigosaccharide-producing amylase examined in this study has the potential in the industrial application of oligosaccharide production.

    TAYLOR & FRANCIS LTD, 2013年12月, Bioscience, Biotechnlolgy, and Biochemistry, 77 (12), 2449 - 2453, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Fumio Matsuda, Jun Ishii, Takashi Kondo, Kengo Ida, Hironori Tezuka, Akihiko Kondo

    Background: Isobutanol is an important target for biorefinery research as a next-generation biofuel and a building block for commodity chemical production. Metabolically engineered microbial strains to produce isobutanol have been successfully developed by introducing the Ehrlich pathway into bacterial hosts. Isobutanol-producing baker's yeast (Saccharomyces cerevisiae) strains have been developed following the strategy with respect to its advantageous characteristics for cost-effective isobutanol production. However, the isobutanol yields and titers attained by the developed strains need to be further improved through engineering of S. cerevisiae metabolism. Results: Two strategies including eliminating competing pathways and resolving the cofactor imbalance were applied to improve isobutanol production in S. cerevisiae. Isobutanol production levels were increased in strains lacking genes encoding members of the pyruvate dehydrogenase complex such as LPD1, indicating that the pyruvate supply for isobutanol biosynthesis is competing with acetyl-CoA biosynthesis in mitochondria. Isobutanol production was increased by overexpression of enzymes responsible for transhydrogenase-like shunts such as pyruvate carboxylase, malate dehydrogenase, and malic enzyme. The integration of a single gene deletion lpd1 Delta and the activation of the transhydrogenase-like shunt further increased isobutanol levels. In a batch fermentation test at the 50-mL scale from 100 g/L glucose using the two integrated strains, the isobutanol titer reached 1.62 +/- 0.11 g/L and 1.61 +/- 0.03 g/L at 24 h after the start of fermentation, which corresponds to the yield at 0.016 +/- 0.001 g/g glucose consumed and 0.016 +/- 0.0003 g/g glucose consumed, respectively. Conclusions: These results demonstrate that downregulation of competing pathways and metabolic functions for resolving the cofactor imbalance are promising strategies to construct S. cerevisiae strains that effectively produce isobutanol.

    BIOMED CENTRAL LTD, 2013年12月, MICROBIAL CELL FACTORIES, 12, 119, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroshi Teramura, Tomoko Oshima, Fumio Matsuda, Kengo Sasaki, Chiaki Ogino, Masanori Yamasaki, Akihiko Kondo

    Lignocellulosic biomass, such as rice straw, is often utilized as a bioresource after being hydrolyzed using dilute acid and separated into liquid hydrolysate and acid-insoluble residue. However, the biomass component that determines the distribution between liquid hydrolysate and acid-insoluble residue has not yet been clarified. In this study, the glucose content in the liquid hydrolysate and weight of acid-insoluble residue of 13 rice cultivars were analyzed. Starch content was positively correlated with glucose content in the liquid hydrolysate, and negatively correlated with acid-insoluble residue weight. These results indicate that the glucose in the liquid hydrolysate is mainly liberated from starch rather than cellulose in the rice straw. These observations suggest that starch content is a good indicator of the glucose distribution between the liquid hydrolysate and insoluble residue. (C) 2013 Published by Elsevier Ltd.

    ELSEVIER SCI LTD, 2013年12月, BIORESOURCE TECHNOLOGY, 149, 520 - 524, 英語

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    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo

    G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer's disease and Parkinson's disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).

    PUBLIC LIBRARY SCIENCE, 2013年12月, PLOS ONE, 8 (12), e82237, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Masahiko Nakatsui, Michihiro Araki, Akihiko Kondo

    Background: One of the most important projects in the post-genome-era is the systemic identification of biological network. The almost of studies for network identification focused on the improvement of computational efficiency in large-scale network inference of complex system with cyclic relations and few attempted have been done for answering practical problem occurred in real biological systems. In this study, we focused to evaluate inferring performance of our previously proposed method for inferring biological network on simple network motifs. Results: We evaluated the network inferring accuracy and efficiency of our previously proposed network inferring algorithm, by using 6 kinds of repeated appearance of highly significant network motifs in the regulatory network of E. coli proposed by Shen-Orr et al and Herrgard et al, and 2 kinds of network motif in S. cerevisiae proposed by Lee et. al. As a result, our method could reconstruct about 40% of interactions in network motif from time-series data set. Moreover the introduction of time-series data of one-factor disrupted model could remarkably improved the performance of network inference. Conclusions: The results of network inference examination of E. coli network motif shows that our network inferring algorithm was able to apply to typical topology of biological network. A continuous examination of inferring well established network motif in biology would strengthen the applicability of our algorithm to the realistic biological network.

    BIOMED CENTRAL LTD, 2013年12月, BMC SYSTEMS BIOLOGY, 7 (suppl 6), 54, 英語

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    研究論文(学術雑誌)

  • Seiji Akimoto, Makio Yokono, Shimpei Aikawa, Akihiko Kondo

    In cyanobacteria, the interactions among pigment-protein complexes are modified in response to changes in light conditions. In the present study, we analyzed excitation energy transfer from the phycobilisome and photosystem II to photosystem I in the cyanobacterium Arthrospira (Spirulina) platensis. The cells were grown under lights with different spectral profiles and under different light intensities, and the energy-transfer characteristics were evaluated using steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopy techniques. The fluorescence rise and decay curves were analyzed by global analysis to obtain fluorescence decay-associated spectra. The direct energy transfer from the phycobilisome to photosystem I and energy transfer from photosystem II to photosystem I were modified depending on the light quality, light quantity, and cultivation period. However, the total amount of energy transferred to photosystem I remained constant under the different growth conditions. We discuss the differences in energy-transfer processes under different cultivation and light conditions.

    SPRINGER, 2013年11月, PHOTOSYNTHESIS RESEARCH, 117 (1-3), 235 - 243, 英語

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    研究論文(学術雑誌)

  • Kengo Sasaki, Daisuke Sasaki, Yuri Sakihama, Hiroshi Teramura, Ryosuke Yamada, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo

    Concentrating sugars using membrane separation, followed by ethanol fermentation by recombinant xylose-assimilating Saccharomyces cerevisiae, is an attractive technology. Three nanofiltration membranes (NTR-729HF, NTR-7250, and ESNA3) were effective in concentrating glucose, fructose, and sucrose from dilute molasses solution and no permeation of sucrose. The separation factors of acetate, formate, furfural, and 5-hydroxymethyl furfural, which were produced by dilute acid pretreatment of rice straw, over glucose after passage through these three membranes were 3.37-11.22, 4.71-20.27, 4.32-16.45, and 4.05-16.84, respectively, at pH 5.0, an applied pressure of 1.5 or 2.0 MPa, and 25 degrees C. The separation factors of these fermentation inhibitors over xylose were infinite, as there was no permeation of xylose. Ethanol production from approximately two-times concentrated liquid hydrolysate using recombinant S. cerevisiae was double (5.34-6.44 g L-1) that compared with fermentation of liquid hydrolysate before membrane separation (2.75 g L-1). (C) 2013 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年11月, BIORESOURCE TECHNOLOGY, 147, 84 - 88, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ryosuke Yamada, Tomohisa Hasunuma, Akihiko Kondo

    With the exhaustion of fossil fuels and with the environmental issues they pose, utilization of abundant lignocellulosic biomass as a feedstock for biofuels and bio-based chemicals has recently become an attractive option. Lignocellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin and has a very rigid and complex structure. It is accordingly much more expensive to process than starchy grains because of the need for extensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Efficient and cost-effective methods for the production of biofuels and chemicals from lignocellulose are required. A consolidated bioprocess (CBP), which integrates all biological steps consisting of enzyme production, saccharification, and fermentation, is considered a promising strategy for reducing production costs. Establishing an efficient CBP using lignocellulosic biomass requires both lignocellulose degradation into glucose and efficient production of biofuels or chemicals from glucose. With this aim, many researchers are attempting to endow selected microorganisms with lignocellulose-assimilating ability. In this review, we focus on studies aimed at conferring lignocellulose-assimilating ability not only to yeast strains but also to bacterial strains by recombinant technology. Recent developments in improvement of enzyme productivity by microorganisms and in improvement of the specific activity of cellulase are emphasized. (C) 2013 Elsevier Inc. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2013年11月, BIOTECHNOLOGY ADVANCES, 31 (6), 754 - 763, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Muhammad Arba, Shimpei Aikawa, Kenta Niki, Makio Yokono, Akihiko Kondo, Seiji Akimoto

    Excitation energy transfer of Arthrospira platensis cells grown in f/2 medium (a high salinity medium) and SOT medium (a control) was investigated by steady-state and time-resolved spectroscopies. Growth in f/2 medium induced changes in absorption and fluorescence spectra as well as in the energy transfer pathways. Excitation energy captured by phycobilisome (PBS) was transferred directly to photosystem (PS) I, instead of being first transferred to an intermediate (PBS -> PSII -> PSI), as observed in SOT medium. The respiration rate increased while photosynthetic rate reduced in f/2 medium. Possible causes of the differences in light-harvesting and energy-transfer processes between the two media are discussed. (C) 2013 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2013年11月, CHEMICAL PHYSICS LETTERS, 588, 231 - 236, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takaya Miyazaki, Shuhei Noda, Tsutomu Tanaka, Akihiko Kondo

    Background: Protein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation enzymes derived from Thermobifida fusca YX and S. lividans. Results: Three candidate signal peptides were isolated and evaluated for their protein secretion ability using beta-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the beta-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the beta-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the beta-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the beta-glucosidase fused to S. cinnamoneus phospholipase D signal peptide. Conclusion: In the present study, we focused on signal peptide sequences derived from biomass degradation enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic beta-glucosidase.

    BIOMED CENTRAL LTD, 2013年10月, MICROBIAL CELL FACTORIES, 12, 88, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Feng Zhang, Daisuke Adachi, Sriappareddy Tamalampudi, Akihiko Kondo, Keisuke Tominaga

    Fourier-transform infrared spectroscopy with the attenuated total reflection method provides a fast, reliable technique to monitor the conversion of oil to fatty acid methyl esters (FAME) during biodiesel production. In this work, we employed a linear calibration method to monitor the generation of FAME in the transesterification of soybean oil and methanol, catalyzed by immobilized Candida antarctica lipase B (CALB). The interaction of the immobilized CALB catalyst with the substrates and products improved its catalytic performance. The rate law changed from zero-order in the first run to quasi-first-order in the subsequent three reaction runs. The quasi-first-order rate law was characterized by a phenomenological compressed exponential function.

    AMER CHEMICAL SOC, 2013年10月, ENERGY & FUELS, 27 (10), 5957 - 5961, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ku Syahidah Ku Ismail, Takatoshi Sakamoto, Tomohisa Hasunuma, Akihiko Kondo

    Agricultural residues comprising lignocellulosic materials are excellent sources of pentose sugar, which can be converted to ethanol as fuel. Ethanol production via consolidated bioprocessing requires a suitable microorganism to withstand the harsh fermentation environment of high temperature, high ethanol concentration, and exposure to inhibitors. We genetically enhanced an industrial Saccharomyces cerevisiae strain, sun049, enabling it to uptake xylose as the sole carbon source at high fermentation temperature. This strain was able to produce 13.9 g/l ethanol from 50 g/l xylose at 38 A degrees C. To better understand the xylose consumption ability during long-term, high-temperature conditions, we compared by transcriptomics two fermentation conditions: high temperature (38 A degrees C) and control temperature (30 A degrees C) during the first 12 h of fermentation. This is the first long-term, time-based transcriptomics approach, and it allowed us to discover the role of heat-responsive genes when xylose is the sole carbon source. The results suggest that genes related to amino acid, cell wall, and ribosomal protein synthesis are down-regulated under heat stress. To allow cell stability and continuous xylose uptake in order to produce ethanol, hexose transporter HXT5, heat shock proteins, ubiquitin proteins, and proteolysis were all induced at high temperature. We also speculate that the strong relationship between high temperature and increased xylitol accumulation represents the cell's mechanism to protect itself from heat degradation.

    SPRINGER HEIDELBERG, 2013年09月, JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, 40 (9), 1039 - 1050, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shih-Hsin Ho, Akihiko Kondo, Tomohisa Hasunuma, Jo-Shu Chang

    Engineering strategies were applied to improve the cell growth, CO2 fixation ability, and carbohydrate productivity of a Scenedesmus obliquus CNW-N isolate. The resulting carbohydrate-rich microalgal biomass was subsequently utilized as feedstock for ethanol fermentation. The microalga was cultivated with 2.5% CO2 in a photobioreactor on different operation modes. Semi-batch operations with 50% replacement of culture medium resulted in the highest CO2 fixation rate (1546.7 mg L-1 d(-1)), carbohydrate productivity (467.6 mg L-1 d(-1)), and bioethanol yield (0.202 g/g biomass). This performance is better than most reported values in the literature. The microalgal biomass can accumulate nearly 50% carbohydrates, as glucose accounted for nearly 80% of the total carbohydrate content. This glucose-predominant carbohydrate composition of the microalga is well suited for fermentative bioethanol production. Therefore, using the proposed carbohydrate-rich microalgal biomass both as the carbon sink and as the feedstock provides a feasible alternative to current carbon-reduction and bioethanol-production strategies. (C) 2013 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年09月, BIORESOURCE TECHNOLOGY, 143, 163 - 171, 英語

    [査読有り]

    研究論文(学術雑誌)

  • KATO H, MATSUDA F, YAMADA R, NAGATA K, SHIRAI T, HASUNUMA Tomohisa, KONDO Akihiko

    Cocktail delta-integration was applied to improve ethanol production from xylose in Saccharomyces cerevisiae. Two hundred of recombinant S. cerevisiae strains possessing various copies of XYL1, XYL2, and XKS1 genes were constructed by cocktail delta-integration. Efficient strains with efficient ethanol production from xylose were successfully obtained by the fermentation test. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2013年09月, Journal of Bioscience and Bioengineering, 116 (3), 333 - 336, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Won-Kyung Hong, Sun-Yeon Heo, Baek-Rock Oh, Chul Ho Kim, Jung-Hoon Sohn, Ji-Won Yang, Akihiko Kondo, Jeong-Woo Seo

    In the present study, we established a genetic system for manipulating the oleaginous heterotrophic microalgae Aurantiochytrium sp. KRS101, using cycloheximide resistance as the selectable marker. The gene encoding ribosomal protein L44 (RPL44) of Aurantiochytrium sp. KRS101 was first identified and characterized. Proline 56 was replaced with glutamine, affording cycloheximide resistance to strains encoding the mutant protein. This resistance served as a novel selection marker. The gene encoding the Delta 12-fatty acid desaturase of Mortierella alpina, used as a reporter, was successfully introduced into chromosomal DNA of Aurantiochytrium sp. KRS101 via 18S rDNA-targeted homologous recombination. Enzymatic conversion of oleic acid (C18:1) to linoleic acid (C18:2) was detected in transformants but not in the wild-type strain.

    SPRINGER, 2013年09月, BIOPROCESS AND BIOSYSTEMS ENGINEERING, 36 (9), 1191 - 1197, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Noriko Adachi, Chihiro Takahashi, Naoko Ono-Murota, Rie Yamaguchi, Tsutomu Tanaka, Akihiko Kondo

    We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum, and direct l-lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans, was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of l-lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced l-lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum. © 2013 Springer-Verlag Berlin Heidelberg.

    2013年08月, Applied Microbiology and Biotechnology, 97 (16), 7165 - 7172, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kosuke Tanimura, Hideki Nakayama, Tsutomu Tanaka, Akihiko Kondo

    In this study, the water-retaining cyclic amino acid ectoine was produced from a variety of sugars, including glucose, xylose, cellobiose, and glucose/xylose mixture using engineered Halomonas elongata. When grown on xylose as the sole carbon source, H. elongata produced 333 mmol/kg fresh cell weight (FW) of ectoine, which was 1.4-fold higher than that produced from glucose. To improve ectoine production, an ectD deficient H. elongata mutant was constructed. The engineered H. elongata produced 377 mmol/kg FIN of ectoine from a glucose/xylose mixture. Ectoine was also produced from rice straw hydrolysate. These results show that H. elongata can produce ectoine from a variety of sugars derived from lignocellulosic biomass and thus has tremendous potential as a host for producing useful compounds from biomass resources. (C) 2013 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年08月, BIORESOURCE TECHNOLOGY, 142, 523 - 529, 英語

    [査読有り]

    研究論文(学術雑誌)

  • ナノ粒子とX線照射を併用した深部ガンの非侵襲的治療法の開発

    田野 亜都里, 森田 健太, 荻野 千秋, 佐藤 和好, 沼子 千弥, 中山 雅央, 佐々木 良平, 近藤 昭彦

    2013年08月, 日本生物工学会大会講演要旨集平成25年度, 244, 日本語

    研究論文(その他学術会議資料等)

  • Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

    KIRIYAMA K, HARA Kiyotaka, KONDO Akihiko

    2013年08月, Applied Microbiology and Biotechnology, 97 (16), 7399 - 7404, 英語

    [査読有り]

    研究論文(学術雑誌)

  • OKAZAKI F, NAKASHIMA N, OGINO Chiaki, KONDO Akihiko

    The biochemical properties of a putative beta-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against beta-1,3-xylan and released beta-1,3-xylobiose and beta-1,3-xylotriose as main products. It displayed maximum activity at 85 A degrees C during a 10-min incubation, and its activity half-life was 23.9 h at 85 A degrees C. Enzyme activity was stable in the pH range 3-10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble beta-1,3-xylan K (m) value was 10.35 mg/ml and the k (cat) value was 588.24 s(-1). The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.

    SPRINGER, 2013年08月, Applied Microbiology and Biotechnology, 97 (15), 6749 - 6757, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shuhei Noda, Yoshifumi Kawai, Takaya Miyazaki, Tsutomu Tanaka, Akihiko Kondo

    We screened for high-activity endoglucanase (EG) as a first step toward the creation of cellulose-assimilating Streptomyces lividans transformants. EGs derived from Thermobifida fusca YX, Tfu0901, and S. lividans, cellulase B (CelB), were successfully expressed. Genes encoding Tfu0901 or CelB were introduced into S. lividans using the integrative vector pTYM18 and the high-copy-number vector pUC702, and EG activity was detected in the supernatant of each transformant. To achieve coexpression of EG and transglutaminase, the transglutaminase gene was introduced into EG-secreting S. lividans using pUC702. S. lividans coexpressing EG and transglutaminase effectively assimilated phosphoric acid swollen cellulose. The yield of Streptomyces cinnamoneus transglutaminase in the culture supernatant was 7.2 mg/L, which was 18 times higher than that of the control strain. To demonstrate the versatility of our system, we also created an EG-producing S. lividans transformant capable of coexpressing endoxylanase. The EG-secreting S. lividans transformants constructed here can be used to produce other useful compounds through cellulose fermentation.

    SPRINGER, 2013年07月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97 (13), 5711 - 5720, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Fumio Matsuda, Tomokazu Shirai, Jun Ishii, Akihiko Kondo

    Metabolic inhibitors were applied for chemical regulation of central carbon metabolism in Saccharomyces cerevisiae. S. cerevisiae was treated with 10 metabolic inhibitors with various modes of action, and their activities were evaluated using a growth inhibition assay. Among the 6 active inhibitors, the effects of pyrazole (alcohol dehydrogenase inhibitor) and TTA (2-thenoyltrifluoloacetone, succinate dehydrogenase inhibitor) were analyzed in detail. The flask-scale batch-fermentation test showed that ethanol yield was reduced to 0.10 +/- 0.01 g g(-1) and glycerol yield increased to 0.26 +/- 0.01 g g(-1) on treatment with pyrazole at 5.0 g L-1, indicating that multiple isozymes of alcohol dehydrogenase were simultaneously inhibited. The multi-targeted metabolic profiling analysis revealed that, although the TTA and pyrazole treatments affected the profiles of all central carbon metabolites in distinct manners, the level of fructose-1,6-bisphosphate commonly increased in the TTA- and pyrazole-treated S. cerevisiae by an unknown mechanism. These results demonstrate that chemical regulation of the central carbon metabolism could be used as an alternative tool to control microbial cell factories for bioproduction, or as a chemical probe to investigate the metabolic systems of useful microorganisms. (C) 2013, The Society for Biotechnology, japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2013年07月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 116 (1), 59 - 64, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Production of lipids containing high levels of docosahexaenoic acid from empty palm fruit bunches by Aurantiochytrium sp. KRS101

    HONG WK, YU A, HEO SY, OH BR, KIM CH, SOHN JH, YANG JW, KONDO Akihiko, SEO JW

    2013年07月, Bioprocess and Biosystems Engineering, 36 (7), 959 - 963, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Aqueous size-exclusion chromatographic method for the quantification of cyanobacterial native glycogen

    IZUMI Y, AIKAWA S, MATSUDA F, HASUNUMA Tomohisa, KONDO Akihiko

    2013年07月, Journal of Chromatography B, 930, 90 - 97, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shimpei Aikawa, Ancy Joseph, Ryosuke Yamada, Yoshihiro Izumi, Takahiro Yamagishi, Fumio Matsuda, Hiroshi Kawai, Jo-Shu Chang, Tomohisa Hasunuma, Akihiko Kondo

    Oxygenic photosynthetic microorganisms such as cyanobacteria and microalgae have attracted attention as feedstocks for next-generation biofuels. To date, however, there have been no reports on efficient bioethanol production from cyanobacterial glycogen by yeast fermentation. Additionally, multiple pretreatment and enzymatic hydrolysis steps of polysaccharides are required for conventional ethanol production from agricultural crops and microalgae. Here, we investigate direct ethanol production from Arthrospira (Spirulina) platensis, a fast-growing halophilic cyanobacterium that accumulates large amounts of glycogen, using lysozyme and a recombinant amylase-expressing yeast strain to eliminate the need for biomass pretreatment and amylase hydrolysis. In the direct conversion process from A. platensis to ethanol, 6.5 g L-1 (ethanol productivity of 1.08 g per L per day) of ethanol was produced. The total ethanol yield based on glycogen consumption was 86% of theoretical yield, which to our knowledge, is the highest yield of bioethanol from an oxygenic photosynthetic microorganism. The present findings indicate that A. platensis is a remarkable carbohydrate feedstock in the form of glycogen, which is a promising material for the production of bioethanol and various other commercially valuable chemicals.

    ROYAL SOC CHEMISTRY, 2013年06月, ENERGY & ENVIRONMENTAL SCIENCE, 6 (6), 1844 - 1849, 英語

    [査読有り]

    研究論文(学術雑誌)

  • バイオマスプラスチックとバイオリファイナリー

    岡井 直子, 近藤 昭彦

    2013年06月, 配管技術, 55 (7), 30 - 35, 日本語

    研究論文(学術雑誌)

  • Yuya Nishimura, Hiroaki Mieda, Jun Ishii, Chiaki Ogino, Toshinobu Fujiwara, Akihiko Kondo

    Background: Small interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction. However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required. Results: In this study, we used a carrier that combined Z(HER2)-displaying bio-nanocapsule (derived from hepatitis B virus surface antigen) and liposomes in a complex in order to investigate the feasibility of effective and target-cell-specific RNAi applications. As a result, by observing RNAi only in HER2-expressing breast cancer cells, using our proposed methodology, we successfully demonstrated target-cell-specific delivery and effective function expression of siRNA. Conclusions: These findings show that, in the field of nucleic acid medicine, Z(HER2)-BNC/ LP can be a useful carrier for siRNA delivery, and could also become a useful tool for gene silencing and to accomplish protein knock-down.

    BIOMED CENTRAL LTD, 2013年06月, JOURNAL OF NANOBIOTECHNOLOGY, 11, 19, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose

    NAKATANI Y, YAMADA R, OGINO Chiaki, KONDO Akihiko

    2013年06月, Microbial Cell Factories, 12 (1), 66, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo

    Potentially immeasurable heterodimer combinations of human G-protein-coupled receptors (GPCRs) result in a great deal of physiological diversity and provide a new opportunity for drug discovery. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood. Thus, the identification of GPCR dimer pairs has been a major challenge. Here, we established a specialized method to screen potential heterodimer partners of human GPCRs based on the split-ubiquitin membrane yeast two-hybrid system. We demonstrate that the mitogen-activated protein kinase (MAPK) signal-independent method can detect ligand-induced conformational changes and rapidly identify heterodimer partners for target GPCRs. Our data present the abilities to apply for the intermolecular mapping of interactions among GPCRs and to uncover potential GPCR targets for the development of new therapeutic agents.

    PUBLIC LIBRARY SCIENCE, 2013年06月, PLOS ONE, 8 (6), e66793, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Dynamic metabolic profiling of cyanobacterial glycogen biosynthesis under conditions of nitrate depletion

    HASUNUMA Tomohisa, KIKUYAMA F, MATSUDA M, AIKAWA S, IZUMI Y, KONDO Akihiko

    2013年06月, Journal of Experimental Botany, 64 (10), 2943 - 2954, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Direct ethanol production from cellulose by cellulase and cellodextrin transporter co-expressing Sccharomyces cerevisiae

    NAKATANI Y, YAMADA R, OGINO Chiaki, KONDO Akihiko

    2013年06月, AMB Express, 3 (1), 34, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Won-Kyung Hong, Sun-Yeon Heo, Hye-Mi Park, Chul Ho Kim, Jung-Hoon Sohn, Akihiko Kondo, Jeong-Woo Seo

    The gene encoding squalene synthase (SQS) of the lipid-producing heterotrophic microalga Aurantiochytrium sp. KRS101 was cloned and characterized. The krsSQS gene is 1,551 bp in length and has two exons and one intron. The open reading frame of the gene is 1,164 bp in length, yielding a polypeptide of 387 predicted amino acid residues with a molecular mass of 42.7 kDa. The deduced krsSQS sequence shares at least four conserved regions known to be required for SQS enzymatic activity in other species. The protein, tagged with His(6), was expressed into soluble form in Escherichia coli. The purified protein catalyzed the conversion of farnesyl diphosphate to squalene in the presence of NADPH and Mg2+. This is the first report on the characterization of an SQS from a Thraustochytrid microalga.

    KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY, 2013年06月, JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, 23 (6), 759 - 765, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yoshifumi Kawai, Shuhei Noda, Chiaki Ogino, Yasunobu Takeshima, Naoko Okai, Tsutomu Tanaka, Akihiko Kondo

    Background: p-Hydroxycinnamic acid (pHCA) is an aromatic compound that serves as a starting material for the production of many commercially valuable chemicals, such as fragrances and pharmaceuticals, and is also used in the synthesis of thermostable polymers. However, chemical synthesis of pHCA is both costly and harmful to the environment. Although pHCA production using microbes has been widely studied, there remains a need for more cost-effective methods, such as the use of biomass as a carbon source. In this study, we produced pHCA using tyrosine ammonia lyase-expressing Streptomyces lividans. In order to improve pHCA productivity from cellulose, we constructed a tyrosine ammonia lyase- and endoglucanase (EG)-expressing S. lividans transformant and used it to produce pHCA from cellulose.Results: A Streptomyces lividans transformant was constructed to express tyrosine ammonia lyase derived from Rhodobacter sphaeroides (RsTAL). The transformant produced 786 or 736 mg/L of pHCA after 7 days of cultivation in medium containing 1% glucose or cellobiose as the carbon source, respectively. To enhance pHCA production from phosphoric acid swollen cellulose (PASC), we introduced the gene encoding EG into RsTAL-expressing S. lividans. After 7 days of cultivation, this transformant produced 753, 743, or 500 mg/L of pHCA from 1% glucose, cellobiose, or PASC, respectively.Conclusions: RsTAL-expressing S. lividans can produce pHCA from glucose and cellobiose. Similarly, RsTAL- and EG-expressing S. lividans can produce pHCA from glucose and cellobiose with excess EG activity remaining in the supernatant. This transformant demonstrated improved pHCA production from cellulose. Further enhancements in the cellulose degradation capability of the transformant will be necessary in order to achieve further improvements in pHCA production from cellulose. © 2013 Kawai et al. licensee BioMed Central Ltd.

    2013年05月07日, Microbial Cell Factories, 12 (1), 45, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shuhei Noda, Takaya Miyazaki, Tsutomu Tanaka, Ogino Chiaki, Akihiko Kondo

    Streptomyces mobaraensis transglutaminase (MTG) is one of the most useful transglutaminases due to its rather broad substrate specificity and independence of Ca2+. To achieve efficient production of active-form MTG using Streptomyces lividans as a host, we created three vector constructs consisting of the signal peptide sequence (pld signal) derived from the phospholipase D gene of Streptomyces cinnamoneus, prepro-domain of S. cinnamoneus transglutaminase, and the sequence encoding mature MTG, and then generated three over-expressing S. lividans strains. We successfully demonstrated that S. lividans can be used as a host for the efficient production of mature, active-form MTG. © 2013 Elsevier B.V.

    2013年05月05日, Biochemical Engineering Journal, 74, 76 - 80, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ancy Joseph, Shimpei Aikawa, Kengo Sasaki, Yota Tsuge, Fumio Matsuda, Tsutomu Tanaka, Akihiko Kondo

    Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO2 has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plazztarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Coexpression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 +/- 0.02 or 0.14 +/- 0.02 mm after 18 d of cultivation.

    TAYLOR & FRANCIS LTD, 2013年05月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77 (5), 966 - 970, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Sarvesh Kumar Srivastava, Ryosuke Yamada, Chiaki Ogino, Akihiko Kondo

    Sidewall modification of multiwalled carbon nanotubes (abbreviated as MWCNTs) was achieved using Allivum sativum (garlic) extract by an acid-free green process. These organosulfur modified-MWCNTs were then decorated with gold nanoparticles and examined by transmission electron microscopy. The presence of organosulfurs over the modified nanotube surface was confirmed. Nanotube surface modification and subsequent presence of thiols as an active linker was confirmed by Raman spectroscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray and X-ray photoelectron spectroscopy. In the absence of these organosulfurs (thiols), no gold nanoparticle attachment was observed. Both small (1-8 nm) and large (12-20 nm) gold nanoparticles were found to decorate the modified nanotube surface suggesting coalescence among nanoparticles. (C) 2013 Elsevier Ltd. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2013年05月, CARBON, 56, 309 - 316, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Daisuke Adachi, Shinji Hama, Kazunori Nakashima, Takayuki Bogaki, Chiaki Ogino, Akihiko Kondo

    For enzymatic biodiesel production from plant oil hydrolysates, an Aspergillus oryzae whole-cell biocatalyst that expresses Candida antarctica lipase B (r-CALB) with high esterification activity was developed. Each of soybean and palm oils was hydrolyzed using Candida rugosa lipase, and the resultant hydrolysates were subjected to esterification where immobilized r-CALB was used as a catalyst. In esterification, r-CALB afforded a methyl ester content of more than 90% after 6 h with the addition of 1.5 M equivalents of methanol. Favorably, stepwise additions of methanol and a little water were unnecessary for maintaining the lipase stability of r-CALB during esterification. During long-term esterification in a rotator, r-CALB can be recycled for 20 cycles without a significant loss of lipase activity, resulting in a methyl ester content of more than 90% even after the 20th batch. Therefore, the presented reaction system using r-CALB shows promise for biodiesel production from plant oil hydrolysates. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年05月, BIORESOURCE TECHNOLOGY, 135, 410 - 416, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Ayumi Yoshida, Naoki Tamadani, Hideo Noda, Akihiko Kondo

    An engineering approach was applied to an efficient biodiesel production from waste cooking oil. In this work, an enzymatic packed-bed reactor (PBR) was integrated with a glycerol-separating system and used successfully for methanolysis, yielding a methyl ester content of 94.3% and glycerol removal of 99.7%. In the glycerol-separating system with enhanced retention time, the effluent contained lesser amounts of glycerol and methanol than those in the unmodified system, suggesting its promising ability to remove hydrophilic impurities from the oil layer. The PBR system was also applied to oils with high acid values, in which fatty acids could be esterified and the large amount of water was extracted using the glycerol-separating system. The long-term operation demonstrated the high lipase stability affording less than 0.2% residual triglyceride in 22 batches. Therefore, the PBR system, which facilitates the separation of hydrophilic impurities, is applicable to the enzymatic biodiesel production from waste cooking oil. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年05月, BIORESOURCE TECHNOLOGY, 135, 417 - 421, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Akihiko Kondo

    The increased global demand for biofuels has prompted the search for alternatives to edible oils for biodiesel production. Given the abundance and cost, waste and nonedible oils have been investigated as potential feedstocks. A recent research interest is the conversion of such feedstocks into biodiesel via enzymatic processes, which have considerable advantages over conventional alkali-catalyzed processes. To expand the viability of enzymatic biodiesel production, considerable effort has been directed toward process development in terms of biodiesel productivity, application to wide ranges of contents of water and fatty acids, adding value to glycerol byproducts, and bioreactor design. A cost evaluation suggested that, with the current enzyme prices, the cost of catalysts alone is not competitive against that of alkalis. However, it can also be expected that further process optimization will lead to a reduced cost in enzyme preparation as well as in downstream processes. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年05月, BIORESOURCE TECHNOLOGY, 135, 386 - 395, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tsutomu Tanaka, Sayoko Matsumoto, Mari Yamada, Ryosuke Yamada, Fumio Matsuda, Akihiko Kondo

    Here, we demonstrate display of beta-glucosidase (BGL) on the surface of Schizosaccharomyces pombe cells using novel anchor proteins. A total of four candidate anchor proteins (SPBC21D10.06c, SPBC947.04, SPBC19C7.05, and SPBC359.04c) were selected from among almost all of S. pombe membrane proteins. The C-terminus of each anchor protein was genetically fused to the N-terminus of BGL, and the fusion protein was expressed using S. pombe as a host. The highest cell surface-associated BGL activity (107 U/10(5) cells was achieved with SPBC359.04c serving as the anchor, followed by SPBC947.04 (44 U/10(5) cells) and SPBC21D10.06c (38 U/10(5) cells). S. pombe displaying BGL with SPBC359.04c as an anchor showed the highest growth on 2 % cellobiose (10.7 x 10(7) cells/mL after 41 h of cultivation from an initial density of 0.1 x 10(7) cells/mL). Additionally, culturing BGL-displaying S. pombe in medium containing cellobiose as the sole carbon source did not affect protein expression, and ethanol fermentation from cellobiose was successfully demonstrated using BGL-displaying S. pombe. This is the first report describing a cell surface display system for the functionalization of S. pombe.

    SPRINGER, 2013年05月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97 (10), 4343 - 4352, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Characterization and optimization of carbohydrates production from an indigenous microalga Chlorella vulgaris FSP-E

    HO SH, HUANG SW, CHEN CY, HASUNUMA Tomohisa, KONDO Akihiko

    2013年05月, Bioresource Technology, 135, 157 - 165, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuki Matano, Tomohisa Hasunuma, Akihiko Kondo

    The aim of this study is to develop a scheme of cell recycle batch fermentation (CRBF) of high-solid lignocellulosic materials. Two-phase separation consisting of rough removal of lignocellulosic residues by low-speed centrifugation and solid-liquid separation enabled effective collection of Saccharomyces cerevisiae cells with decreased lignin and ash. Five consecutive batch fermentation of 200 g/L rice straw hydrothermally pretreated led to an average ethanol titer of 34.5 g/L. Moreover, the display of cellulases on the recombinant yeast cell surface increased ethanol titer to 42.2 g/L. After, five-cycle fermentation, only 3.3 g/L sugar was retained in the fermentation medium, because cellulase displayed on the cell surface hydrolyzed cellulose that was not hydrolyzed by commercial cellulases or free secreted cellulases. Fermentation ability of the recombinant strain was successfully kept during a five-cycle repeated batch fermentation with 86.3% of theoretical yield based on starting biomass. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年05月, BIORESOURCE TECHNOLOGY, 135, 403 - 409, 英語

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    研究論文(学術雑誌)

  • Shih-Hsin Ho, Shu-Wen Huang, Chun-Yen Chen, Tomohisa Hasunuma, Akihiko Kondo, Jo-Shu Chang

    This study aimed to evaluate the potential of using a carbohydrate-rich microalga Chlorella vulgaris FSP-E as feedstock for bioethanol production via various hydrolysis strategies and fermentation processes. Enzymatic hydrolysis of C. vulgaris FSP-E biomass (containing 51% carbohydrate per dry weight) gave a glucose yield of 90.4% (or 0.461 g (g biomass)(-1)). The SHF and SSF processes converted the enzymatic microalgae hydrolysate into ethanol with a 79.9% and 92.3% theoretical yield, respectively. Dilute acidic hydrolysis with 1% sulfuric acid was also very effective in saccharifying C vulgaris FSP-E biomass, achieving a glucose yield of nearly 93.6% from the microalgal carbohydrates at a starting biomass concentration of 50 g L-1. Using the acidic hydrolysate of C vulgaris FSP-E biomass as feedstock, the SHF process produced ethanol at a concentration of 11.7 g L-1 and an 87.6% theoretical yield. These findings indicate the feasibility of using carbohydrate-producing microalgae as feedstock for fermentative bioethanol production. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年05月, BIORESOURCE TECHNOLOGY, 135, 191 - 198, 英語

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    研究論文(学術雑誌)

  • Daisuke Adachi, FookHee Koh, Shinji Hama, Chiaki Ogino, Akihiko Kondo

    To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50 degrees C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50 degrees C and was maintained even after an incubation of 24-h at 60 degrees C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50 degrees C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production. (C) 2013 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2013年05月, ENZYME AND MICROBIAL TECHNOLOGY, 52 (6-7), 331 - 335, 英語

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    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Fumiyoshi Okazaki, Naoko Okai, Kiyotaka Y. Hara, Jun Ishii, Akihiko Kondo

    The biorefinery manufacturing process for producing chemicals and liquid fuels from biomass is a promising approach for securing energy and resources. To establish cost-effective fermentation of lignocellulosic biomass, the consolidation of sacccharification and fermentation processes is a desirable strategy, but requires the development of microorganisms capable of cellulose/hemicellulose hydrolysis and target chemical production. Such an endeavor requires a large number of prerequisites to be realized, including engineering microbial strains with high cellulolytic activity, high product yield, productivities, and titers, ability to use many carbon sources, and resistance to toxic compounds released during the pretreatment of lignocellulosic biomass. Researchers have focused on either engineering naturally cellulolytic microorganisms to improve product-related properties or modifying non-cellulolytic organisms with high product yields to become cellulolytic. This article reviews recent advances in the development of microorganisms for the production of renewable chemicals and advanced biofuels, as well as ethanol, from lignocellulosic materials through consolidated bioprocessing. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年05月, BIORESOURCE TECHNOLOGY, 135, 513 - 522, 英語

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    研究論文(学術雑誌)

  • SUZUKI Hirokazu, OKAZAKI Fumiyoshi, KONDO Akihiko, YOSHIDA Ken-ichi

    Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-β-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a β-glucosidase resulted in the unexpected generation of a thermostable, highly specific β-fucosidase, concomitant with large increases in β-glucosidase, β-cellobiosidase, α-arabinofuranosidase, β-mannosidase, β-glucuronidase, β-xylopyranosidase, and β-fucosidase activities and a dramatic decline in 6-phospho-β- glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-β-glycosidase and to generate a thermostable β-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts. © 2012 Springer-Verlag.

    2013年04月, Applied Microbiology and Biotechnology, 97 (7), 2929 - 2938, 英語

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    研究論文(学術雑誌)

  • Tomokazu Shirai, Fumio Matsuda, Mami Okamoto, Akihiko Kondo

    We performed metabolome and metabolite-metabolite correlation analyses for eight single-gene deletion mutants of Saccharomyces cerevisiae to evaluate the physiology of glucose metabolism. The irreversible enzyme reactions can become bottlenecks when intracellular metabolism is perturbed by direct interference from the central metabolic pathway by gene deletions or by a deletion of transcriptional regulator. Metabolome data reveal that transcriptional factor, gcr2, regulates the reaction that converts 3-phosphoglycerate into phosphoenolpyruvate. Metabolome data also suggest that the reaction catalyzed by pyruvate kinase makes one of the rate-limiting reactions throughout the glycolytic pathway.

    SPRINGER, 2013年04月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97 (8), 3569 - 3577, 英語

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    研究論文(学術雑誌)

  • 電気化学システムを用いた模擬生ごみからの水素発酵の制御

    佐々木 建吾, 佐々木 大介, 森田 仁彦, 五十嵐 泰夫, 近藤 昭彦

    2013年03月, 水素エネルギーシステム, 38 (1), 33 - 37, 日本語

    研究論文(学術雑誌)

  • Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with cell-surface engineered yeast strains

    MATANO Yuki, HASUNUMA Tomohisa, KONDO Akihiko

    2013年03月, Applied Microbiology and Biotechnology, 97 (5), 2231 - 2237, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR-XDH pathway

    Jun Ishii, Yoshimura Kazuya, Tomohisa Hasunuma, Akihiko Kondo

    2013年03月, Applied Microbiology and Biotechnology, 97 (6), 2597 - 2607, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuya Nishimura, Wakiko Mimura, Izzat Fahimuddin Mohamed Suffian, Tomokazu Amino, Jun Ishii, Chiaki Ogino, Akihiko Kondo

    Capsid-like particles consisting of a hepatitis B core (HBc) protein have been studied for their potential as carriers for drug delivery systems (DDS). The hollow HBc particle, which is formed by the self-assembly of core proteins comprising 183 aa residues, has the ability to bind to various cells non-specifically via the action of an arginine-rich domain. In this study, we developed an engineered HBc particle that specifically recognizes and targets human epidermal growth factor receptor-related 2 (HER2)-expressing breast cancer cells. To despoil the non-specific binding property of an HBc particle, we genetically deleted the C-terminal 150-183 aa part of the core protein that encodes the arginine-rich domain (delta HBc). Then, we genetically inserted a Z(HER2) affibody molecule into the 78-81 aa position of the core protein to confer the ability of target-cell-specific recognition. The constructed Z(HER2)-displaying HBc (Z(HER2)-delta HBc) particle specifically recognized HER2-expressing SKBR3 and MCF-7 breast cancer cells. In addition, the Z(HER2)-delta HBc particle exhibited different binding amounts in accordance with the HER2 expression levels of cancer cells. These results show that the display of other types of affibody molecules on HBc particles would be an expandable strategy for targeting several kinds of cancer cells that would help enable a pinpoint DDS.

    OXFORD UNIV PRESS, 2013年03月, JOURNAL OF BIOCHEMISTRY, 153 (3), 251 - 256, 英語

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    研究論文(学術雑誌)

  • An affinity chromatography method used to purify His-tag-displaying bio-nanocapsules

    Yuya Nishimura, Koichi Takeda, Jun Ishii, Chiaki Ogino, Akihiko Kondo

    2013年03月, Journal of Virological Methods, 189 (2), 393 - 396, 英語

    [査読有り]

    研究論文(学術雑誌)

  • イオン液体前処理バイオマスを用いたエタノール同時糖化発酵

    荻野 千秋, 山田 亮祐, 近藤 昭彦

    2013年02月, 化学工業, 64 (2), 52 - 55, 日本語

    研究論文(学術雑誌)

  • Baek-Rock Oh, Won-Kyung Hong, Sun-Yeon Heo, Lian Hua Luo, Akihiko Kondo, Jeong-Woo Seo, Chul Ho Kim

    In the present study, mutant strain of Klebsiella pneumoniae with deletion of the crr gene encoding EIIA(Glc) (a component of the glucose-specific phosphoenolpyruvate-dependent transferase system [PTS]) was prepared. This eliminated the ability of the strain to mediate carbon catabolite repression (CCR). Production of 1,3-propanediol (1,3-PD) from glycerol by the crr mutant strain was enhanced (compared to that of the parent) in the presence of glucose. Using molasses as a co-substrate of glycerol, the maximum yield of 1,3-PD was 60.4% greater (81.2 g/l) than that obtained when glycerol was used alone, under optimum fermentation conditions. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2013年02月, BIORESOURCE TECHNOLOGY, 130, 719 - 724, 英語

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    研究論文(学術雑誌)

  • Hiroyuki Suga, Fumio Matsuda, Tomohisa Hasunuma, Jun Ishii, Akihiko Kondo

    Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase (MDH2), and pyruvate carboxylase (PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38 +/- 0.01 g g(-1) xylose consumed) was improved from that of the control strain (0.31 +/- 0.01 g g(-1) xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1-MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1-MDH2-PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells.

    SPRINGER, 2013年02月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97 (4), 1669 - 1678, 英語

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    研究論文(学術雑誌)

  • Sarvesh Kumar Srivastava, Ryosuke Yamada, Chiaki Ogino, Akihiko Kondo

    Room-temperature extracellular biosynthesis of gold nanoparticles (Au NPs) was achieved using Escherichia coli K12 cells without the addition of growth media, pH adjustments or inclusion of electron donors/stabilizing agents. The resulting nanoparticles were analysed by ultraviolet-visible (UV-vis) spectrophotometry, atomic force microscopy, transmission electron microscopy and X-ray diffraction. Highly dispersed gold nanoplates were achieved in the order of around 50 nm. Further, the underlying mechanism was found to be controlled by certain extracellular membrane-bound proteins, which was confirmed by Fourier transformation-infrared spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis. We observed that certain membrane-bound peptides are responsible for reduction and subsequent stabilization of Au NPs (confirmed by zeta potential analysis). Upon de-activation of these proteins, no nanoparticle formation was observed. Also, we prepared a novel biocatalyst with Au NPs attached to the membrane-bound fraction of E. coli K12 cells serving as an efficient heterogeneous catalyst in complete reduction of 4-nitrophenol in the presence of NaBH4 which was studied with UV-vis spectroscopy. This is the first report on bacterial membrane-Au NP nanobiocomposite serving as an efficient heterogeneous catalyst in complete reduction of nitroaromatic pollutant in water.

    SPRINGER, 2013年02月, NANOSCALE RESEARCH LETTERS, 8 (1), 70, 英語

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    研究論文(学術雑誌)

  • Daisuke Adachi, Risa Koda, Shinji Hama, Ryosuke Yamada, Kazunori Nakashima, Chiaki Ogino, Akihiko Kondo

    We attempted to integrate lipase-catalyzed ethanolysis into fermentative bioethanol production. To produce bioethanol, ethanol fermentation from brown rice was conducted using a tetraploid Saccharomyces cerevisiae expressing alpha-amylase and glucoamylase. The resultant ethanol was distilled and separated into three fractions with different concentrations of water and fusel alcohols. In ethanolysis using the first fraction with 89.3% ethanol, a recombinant Aspergillus oryzae whole-cell biocatalyst expressing Fusarium heterosporum lipase (r-FHL) afforded the highest ethyl ester content of 94.0% after 96 h. Owing to a high concentration of water in the bioethanol solutions, r-FHL, which works best in the presence of water when processing ethanolysis, was found to be more suitable for the integrative process than a commercial immobilized Candida antarctica lipase. In addition, r-FHL was used for repeated-batch ethanolysis, resulting in an ethyl ester content of more than 80% even after the fifth batch. Fusel alcohols such as 1-butanol and isobutyl alcohol are thought to decrease the lipase activity of r-FHL. Using this process, a high ethyl ester content was obtained by simply mixing bioethanol, plant oil, and lipase with an appropriate adjustment of water concentration. The developed process model, therefore, would contribute to biodiesel production from only biomass-derived feedstocks. (c) 2012 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2013年02月, ENZYME AND MICROBIAL TECHNOLOGY, 52 (2), 118 - 122, 英語

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    研究論文(学術雑誌)

  • Nobuo Yoshimoto, Akiko Kida, Xu Jie, Masaya Kurokawa, Masumi Iijima, Tomoaki Niimi, Andres D. Maturana, Itoshi Nikaido, Hiroki R. Ueda, Kenji Tatematsu, Katsuyuki Tanizawa, Akihiko Kondo, Ikuo Fujii, Shun'ichi Kuroda

    When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e. g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.

    NATURE PUBLISHING GROUP, 2013年02月, SCIENTIFIC REPORTS, 3, 1191, 英語

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    研究論文(学術雑誌)

  • SONG Y, MATSUMOTO K, TANAKA Tsutomu, KONDO Akihiko, TAGUCHI S

    Direct polyhydroxybutyrate (PHB) production from starch was for the first time achieved using engineered Corynebacterium glutamicum expressing PHB biosynthetic genes and displaying a-amylase on its cell surface. The engineered strain accumulated 6.4 wt% PHB from starch which was higher than that obtained from glucose (4.9 wt%). (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2013年01月, Journal of Bioscience and Bioengineering, 115 (1), 12 - 14, 英語

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    研究論文(学術雑誌)

  • Gene expression cross-profiling in genetically modified industrial Saccharomyces cerevisiae strains during high-temperature ethanol production from xylose

    ISMAIL K.S.K, SAKAMOTO Takatoshi, HATANAKA H, HASUNUMA Tomohisa, KONDO Akihiko

    2013年01月, Journal of Biotechnology, 163, 50 - 60, 英語

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    研究論文(学術雑誌)

  • Akihiko Kondo, Jun Ishii, Kiyotaka Y. Hara, Tomohisa Hasunuma, Fumio Matsuda

    Synthetic bioengineering is a strategy for developing useful microbial strains with innovative biological functions. Novel functions are designed and synthesized in host microbes with the aid of advanced technologies for computer simulations of cellular processes and the system-wide manipulation of host genomes. Here, we review the current status and future prospects of synthetic bioengineering in the yeast Saccharomyces cerevisiae for bio-refinery processes to produce various commodity chemicals from lignocellulosic biomass. Previous studies to improve assimilation of xylose and production of glutathione and butanol suggest a fixed pattern of problems that need to be solved, and as a crucial step, we now need to identify promising targets for further engineering of yeast metabolism. Metabolic simulation, transcriptomics, and metabolomics are useful emerging technologies for achieving this goal, making it possible to optimize metabolic pathways. Furthermore, novel genes responsible for target production can be found by analyzing large-scale data. Fine-tuning of enzyme activities is essential in the latter stage of strain development, but it requires detailed modeling of yeast metabolic functions. Recombinant technologies and genetic engineering are crucial for implementing metabolic designs into microbes. In addition to conventional gene manipulation techniques, advanced methods, such as multicistronic expression systems, marker-recycle gene deletion, protein engineering, cell surface display, genome editing, and synthesis of very long DNA fragments, will facilitate advances in synthetic bioengineering. (C) 2012 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2013年01月, JOURNAL OF BIOTECHNOLOGY, 163 (2), 204 - 216, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ability of a perfluoropolymer membrane to tolerate by-products of ethanol fermentation broth from dilute acid-pretreated rice straw

    SASAKI Kengo, MATSUDA Fumio, HASUNUMA Tomohisa, OGINO Chiaki, URAIRI M, YOSHIDA K, KONDO Akihiko

    2013年01月, Biocehmical Engineering Journal, 70, 135 - 139, 英語

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    研究論文(学術雑誌)

  • Naoki Ikeda, Mari Miyamoto, Noriko Adachi, Mariko Nakano, Tsutomu Tanaka, Akihiko Kondo

    In this study, we demonstrate the one-step production of cadaverine (1,5-diaminopentane) from cellobiose using an Escherichia coli strain displaying beta-glucosidase (BGL) on its cell surface. L-lysine decarboxylase (CadA) derived from E. coli and BGL from Thermobifida fusca YX (Tfu0937) fused to the anchor protein Blc from E. coli were co-expressed using E. coli as a host. The expression of CadA was confirmed by Western blotting and BGL activity on the cell surface was evaluated using pNPG as a substrate. Growth on cellobiose as the sole carbon source was also achieved. The OD600 value of the BGL and CadA co-expressing strain was 8.0 after 48 h cultivation, which is higher than that obtained by growth on glucose (5.4 after 48 h cultivation). The engineered strain produced cadaverine from cellobiose more effectively than from glucose: 6.1 mM after 48 h from 28 g/L of consumed cellobiose, vs. 3.3 mM from 20 g/L of consumed glucose.

    BIOMED CENTRAL LTD, 2013年, AMB EXPRESS, 3, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Zhenquan Tan, Kazuyoshi Sato, Seiichi Takami, Chiya Numako, Mitsuo Umetsu, Kohei Soga, Masao Nakayama, Ryohei Sasaki, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo, Kazuhiro Yamamoto, Takeshi Hashishin

    2013年, RSC Adv, 3 (1), 19268 - 19271, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Sarvesh Kumar Srivastava, Takashi Hasegawa, Ryosuke Yamada, Chiaki Ogino, Minoru Mizuhata, Akihiko Kondo

    A facile, eco-friendly, room-temperature method for rapid one-pot synthesis of Au@Pd bimetallic nanoparticles exhibiting a core-shell morphology (similar to 60 nm) has been developed based on the successive reduction of Au(III) and Pd(II) precursors with tryptophan (Trp) in an aqueous environment. The unique supramolecular chemistry arising due to the hydrogen bonded indole group layer over the Au core seemed critical in the formation Pd shell. The core-shell morphology and surface analysis of the resulting Au@Pd nanoparticles were confirmed by aberration corrected scanning transmission electron microscopy followed by X-ray photoelectron spectroscopy. The formation of the core (Au) and shell (Pd) was also confirmed by Energy Dispersive X-Ray elemental scanning analysis. The resulting Au, Pd and Au@Pd nanoparticles were also analysed by UV-Vis spectroscopy, X-ray diffraction and Dynamic Light Scattering. Our results suggest a simple coordination mechanism where the pre-stabilized poly-Trp Au core serves as a template to facilitate the subsequent reduction of Pd(II) via active carboxyl groups. This study effectively demonstrates for the first time that core-shell nanoparticle synthesis (reduction and stabilization) can be effectively achieved by simple amino acids like Trp in an aqueous reaction mixture.

    ROYAL SOC CHEMISTRY, 2013年, RSC ADVANCES, 3 (40), 18367 - 18372, 英語

    [査読有り]

    研究論文(学術雑誌)

  • An approach for dynamical network reconstruction of simple netswork motifs

    NAKATSUI Masahiko, ARAKI Michihiro, KONDO Akihiko

    2013年, BMC System Biology, 7 (suppl 6), 54, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuya Nishimura, Jun Ishii, Fumiyoshi Okazaki, Chiaki Ogino, Akihiko Kondo

    A bio-nanocapsule (BNC), a hollow particle composed of hepatitis B virus (HBV) surface antigen (HBsAg), and liposome (LP) conjugation method (BNC/LP) has been recently developed by Jung et al. (2008). The BNC/LP complex carrier could successfully deliver fluorescence-labeled beads (100 nm) into liver cells. In this study, we report the promising delivery of proteins incorporated in the complex carriers, which were prepared by the BNC/LP conjugation method with specificity-altered BNC and composition-varied LPs. The specificity-altered BNC, Z(HER2)-BNC was developed by replacing the hepatocyte recognition site of BNC with Z(HER2) binding to HER2 receptor specifically. Using green fluorescent protein (GFP; 27 kDa) and cellular cytotoxic protein (exotoxin A; 66 kDa) for the delivery, we herein present the impact of different charges attributed to the composition of the LP on specific cell targeting and cellular uptake of the complex carriers. In addition, we demonstrate that the mixture prepared by mixing LPs with helper lipid possessing endosomal escaping ability boosts the functional expression of the cellular cytotoxic exotoxin A activity specifically. Finally, we further show the blending ratio of the LP mixture and Z(HER2)-BNC is a critical factor in determining the highly-efficient expression of the cytotoxic activity of exotoxin A.

    INFORMA HEALTHCARE, 2012年12月, JOURNAL OF DRUG TARGETING, 20 (10), 897 - 905, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yosuke Fukutani, Jun Ishii, Keiichi Noguchi, Akihiko Kondo, Masafumi Yohda

    The goal of this work was to improve the bioluminescence-based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast-expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G-protein a-subunit (Gpa1p) with the olfactory-specific Gaolf, the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence-based odor-sensing system using OR-expressing yeast. Biotechnol. Bioeng. 2012; 109: 31433151. (C) 2012 Wiley Periodicals, Inc.

    WILEY, 2012年12月, BIOTECHNOLOGY AND BIOENGINEERING, 109 (12), 3143 - 3151, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kentaro Kiriyama, Kiyotaka Y. Hara, Akihiko Kondo

    A novel extracellular glutathione fermentation method using engineered Saccharomyces cerevisiae was developed by following three steps. First, a platform host strain lacking the glutathione degradation protein and glutathione uptake protein was constructed. This strain improved the extracellular glutathione productivity by up to 3.2-fold compared to the parental strain. Second, the ATP-dependent permease Adp1 was identified as a novel glutathione export ABC protein (Gxa1) in S. cerevisiae based on the homology of the protein sequence with that of the known human glutathione export ABC protein (ABCG2). Overexpression of this GXA1 gene improved the extracellular glutathione production by up to 2.3-fold compared to the platform host strain. Finally, combinatorial overexpression of the GXA1 gene and the genes involved in glutathione synthesis in the platform host strain increased the extracellular glutathione production by up to 17.1-fold compared to the parental strain. Overall, the metabolic engineering of the glutathione synthesis, degradation, and transport increased the total (extracellular + intracellular) glutathione production. The extracellular glutathione fermentation method developed in this study has the potential to overcome the limitations of the present intracellular glutathione fermentation process in yeast.

    SPRINGER, 2012年11月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 96 (4), 1021 - 1027, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Akihiko Kondo

    To build an energy and material secure future, a next generation of renewable fuels produced from lignocellulosic biomass is required. Although lignocellulosic biomass, which represents an abundant, inexpensive and renewable source for bioethanol production, is of great interest as a feedstock, the complicated ethanol production processes involved make the cost of producing bioethanol from it higher compared to corn starch and cane juice. Therefore, consolidated bioprocessing (CBP), which combines enzyme production, saccharification and fermentation in a single step, has gained increased recognition as a potential bioethanol production system. CBP requires a highly engineered microorganism developed for several different process-specific characteristics. The dominant strategy for engineering a CBP biocatalyst is to express multiple components of a cellulolytic system from either fungi or bacteria in the yeast Saccharomyces cerevisiae. The development of recombinant yeast strains displaying cellulases and hemicellulases on the cell surface represents significant progress toward realization of CBP. Regardless of the process used for biomass hydrolysis. CBP-enabling microorganisms encounter a variety of toxic compounds produced during biomass pretreatment that inhibit microbial growth and ethanol yield. Systems biology approaches including disruptome screening, transcriptomics, and metabolomics have been recently exploited to gain insight into the molecular and genetic traits involved in tolerance and adaptation to the fermentation inhibitors. In this review, we locus on recent advances in development of yeast strains with both the ability to directly convert lignocellulosic material to ethanol and tolerance in the harsh environments containing toxic compounds in the presence of ethanol. (c) 2011 Elsevier Inc. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2012年11月, BIOTECHNOLOGY ADVANCES, 30 (6), 1207 - 1218, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Fumiyoshi Okazaki, Jun-ichi Aoki, Soichiro Tabuchi, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V-HH) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V-HH with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V-HH reached 73.8 mg/1 in a Sakaguchi flask. The V-HH fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V-HH fragment was specifically recognized and could bind to the EGFR with a high affinity.

    SPRINGER, 2012年10月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 96 (1), 81 - 88, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ayumi Yoshida, Shinji Hama, Naoki Tamadani, Hideo Noda, Hideki Fukuda, Akihiko Kondo

    A continuous production of biodiesel from an aqueous plant oil emulsion was attempted using immobilized fungal whole cells. Six packed-bed reactors were connected in series and operated with stepwise methanol addition. In the first column, more than 3% water was necessary for methanolysis to proceed. Despite the low initial water content of 0.36%, the methyl ester content in the effluent from the second column increased similarly in a wide range of contents of water (0-20%) added, which shows the water-content-independent reaction in this column in contrast to the reaction in the first column. Further investigations using reaction mixture models suggested a contribution of the composition of the reaction mixture to the phenomenon. On the basis of these findings, the sequential methanolysis through six columns was attempted, where the upper layer of the effluent from each column was supplied into the next column without further addition of water. Consequently, an aqueous plant oil emulsion with 3% water was converted into the final product with 96.1% methyl ester and 0.15% water. Therefore, the system developed is useful for producing biodiesel enzymatically from water-containing feedstocks. (C) 2012 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2012年10月, BIOCHEMICAL ENGINEERING JOURNAL, 68, 7 - 11, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Matsumoto, Tsutomu Tanaka, Akihiko Kondo

    Bioconjugates are valuable tools in many fields, including protein engineering and environmental and therapeutic research. Chemical methods are commonly used to synthesize protein-protein and protein-functional molecule bioconjugates because they permit easy tethering through covalent bonds. However, chemical methods often produce heterogeneous products and lead to degradation of protein activity due to random modifications. Recently, a number of techniques for modifying proteins or synthesizing bioconjugates have been reported, including more sophisticated chemical modification methods, utilization of noncovalent affinity, and protein splicing. Enzymatic methods in particular have attracted much attention due to the substrate specificity of enzymes, which enables site-specific tethering of proteins to other proteins or functional molecules. Here, we discuss newly developed methods for protein modification and bioconjugate synthesis that exploit the properties of acyltransferases, ligases, and other enzymes.

    2012年09月, Biotechnology journal, 7 (9), 1137 - 46, 英語, 国際誌

    [査読有り]

    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Akihiko Kondo

    Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a single process, is a promising strategy for effective ethanol production from lignocellulosic materials because of the resulting reduction in utilities, the substrate and other raw materials and simplification of operation. CBP requires a highly engineered microbial strain capable of hydrolyzing biomass with enzymes produced on its own and producing high-titer ethanol. Recently, heterologous production of cellulolytic enzymes has been pursued with yeast hosts, which has realized direct conversion of cellulose to ethanol. Specifically, the development of cell surface engineering, which provides a display of cellulolytic enzymes on the yeast cell surface, facilitates effective biomass hydrolysis concomitantly with ethanol production. On the other hand, the difference in optimum temperature between saccharification and fermentation is a drawback of efficient ethanol production in the simultaneous saccharification and fermentation (SSF). The application of thermotolerant yeast strains engineered to the SSF process would overcome the drawback by performing hydrolysis and fermentation at elevated temperature. In this review, we focus on the recent advances in the application of thermotolerant yeast to CBP and SSF of lignocellulosic material to ethanol. The development of thermotolerant and ethanologenic yeast strains with the ability to hydrolyze lignocellulosic materials is emphasized for high-temperature CBP. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2012年09月, PROCESS BIOCHEMISTRY, 47 (9), 1287 - 1294, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Fumio Matsuda, Akihiko Kondo

    To build an energy and material secure future, we must pioneer the next generation of renewable fuels and chemicals using environmentally benign production processes. Since biomass represents an abundant carbon-neutral renewable resource for the production of biofuels, numerous environmental and social benefits could result from the replacement of petroleum-based transport fuels with bioethanol converted from biomass. One of the key technologies for the development of biorefineries is cell surface engineering, which is a powerful tool for engineering and functionalizing many organisms. Using the technology, various kinds of functional proteins, such as enzymes, can be expressed on the cell surface without loss of cell activity. The display of amylolytic and cellulolytic enzymes on the surface of Saccharomyces cerevisiae has accomplished direct ethanol production from starchy and cellulosic biomass. Moreover, the display of hemicellulase on the surface of S. cerevisiae that has a xylose-assimilating metabolic pathway has enabled production of ethanol from hemicellulosic materials. Furthermore, reutilization of the cell-surface engineered yeast has an advantage in the reduction of enzyme cost, which enables reuse of enzymes on the cell surface by collecting the cells. Thus, cell surface engineering is a promising technology for the development of a consolidated bioprocess by integrating enzyme production, saccharification and fermentation. Regardless of the biomass hydrolysis, metabolic engineering of microorganisms is emphasized for the efficient production of ethanol from biomass. Specifically, lignocellulosic hydrolysates contain high concentrations of inhibitors that negatively affect metabolism and ethanol yields. To circumvent these difficulties, robust S. cerevisiae strains that efficiently ferment mixtures of hexose and pentose sugars in the presence of various chemical contexts for industrial ethanol production should be constructed through metabolic engineering approaches. A combination of a cell-surface displayed enzyme system and an intracellular metabolic engineering system is a very effective approach for developing cells with improved fermentation ability for industrial applications. The technology (synthetic bioengineering) will open up new applications of cell factories to industrially important processes.

    Springer Netherlands, 2012年08月01日, Systems Metabolic Engineering, 9789400745346, 329 - 348, 英語

    [査読有り]

    論文集(書籍)内論文

  • Seiji Akimoto, Makio Yokono, Fumiya Hamada, Ayaka Teshigahara, Shimpei Aikawa, Akihiko Kondo

    Cyanobacteria change the quantity and/or quality of their pigment-protein complexes in response to light conditions. In the present study, we analyzed excitation relaxation dynamics in the cyanobacterium, Arthrospira (Spirulina) platensis, grown under lights exhibiting different spectral profiles, by means of steady-state absorption and picosecond time-resolved fluorescence spectroscopies. It was found that F760, which is the PSI red-chlorophyll characteristic of A. platensis, contributes to slower energy-transfer phase in the PSI of A platens is. Excitation energy transfers in phycobilisome and those from PSII to PSI were modified depending on the light quality. Existence of quencher was suggested in PSI of the blue-light grown cells. Phycobilisomes in the green-light grown cells and the far-red-light grown cells transferred excitation energy from phycobilisome to chlorophyll without loss of energy. In these cells, excitation energy was shared between two photosystems. Fast energy transfer was established in phycobilisome under the yellow-light condition where only the phycobilisome can absorb the cultivation light. Differences in light-harvesting and energy-transfer processes under different cultivation-light conditions are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. (C) 2012 Elsevier BV. All rights reserved.

    ELSEVIER SCIENCE BV, 2012年08月, BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 1817 (8), 1483 - 1489, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Suger consumption and ethanol fermentation by transporter-overexpressed xylose metabolizing Saccharomyces cerevisiae harboring a xyloseisomerase pathway

    TANINO T, ITO T, OGINO Chiaki, OHMURA Naoto, OHSHIMA T, KONDO Akihiko

    2012年08月, Journal of Bioscience and Bioengineering, 114 (2), 209 - 211, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Recent developments in yeast cell surface display toward extended applications in biotechnology

    TANAKA Tsutomu, YAMADA Ryosuke, OGINO Chiaki, KONDO Akihiko

    2012年08月, Applied Microbiology and Biotechnology, 95, 577 - 591, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Chihiro Takahashi, Junki Shirakawa, Takeyuki Tsuchidate, Naoko Okai, Kazuki Hatada, Hideki Nakayama, Toshihiro Tateno, Chiaki Ogino, Akihiko Kondo

    Gamma-amino butyric acid (GABA) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. Here, we report a simple and robust system to produce GABA from glucose using the recombinant Corynebacterium glutamicum strain GAD, which expresses GadB, a glutamate decarboxylase encoded by the gadB gene of Escherichia coli W3110. As confirmed by HPLC analysis. GABA fermentation by C. glutamicum GAD cultured at 30 degrees C in GABA Production 1 (GP1) medium containing 50 g/L glucose without the addition of glutamate yielded 8.07 +/- 1.53 g/L extracellular GABA after 96h. Addition of 0.1 mM pyridoxal 5'-phosphate (PLP) was found to enhance the production of GABA, whereas Tween 40 was unnecessary for GABA fermentation. Using the optimized GABA Production 2 (GP2) medium, which contained 50 g/L glucose and 0.1 mM PLP, fermentation was performed in a flask at 30 degrees C with 10% (v/v) seed culture of C. glutamicum GAD. GABA was produced in the culture supernatant with a yield of 12.37 +/- 0.88 g/L after 72 h with a space-time yield of 0.172 g/L/h, which is the highest yield obtained to date for GABA from fermentation with glucose as a main carbon source. (C) 2012 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2012年08月, ENZYME AND MICROBIAL TECHNOLOGY, 51 (3), 171 - 176, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Seiji Akimoto, Makio Yokono, Fumiya Hamada, Ayaka Teshigahara, Shimpei Aikawa, Akihiko Kondo

    Cyanobacteria change the quantity and/or quality of their pigment-protein complexes in response to light conditions. In the present study, we analyzed excitation relaxation dynamics in the cyanobacterium, Arthrospira (Spirulina) platensis, grown under lights exhibiting different spectral profiles, by means of steady-state absorption and picosecond time-resolved fluorescence spectroscopies. It was found that F760, which is the PSI red-chlorophyll characteristic of A. platensis, contributes to slower energy-transfer phase in the PSI of A platens is. Excitation energy transfers in phycobilisome and those from PSII to PSI were modified depending on the light quality. Existence of quencher was suggested in PSI of the blue-light grown cells. Phycobilisomes in the green-light grown cells and the far-red-light grown cells transferred excitation energy from phycobilisome to chlorophyll without loss of energy. In these cells, excitation energy was shared between two photosystems. Fast energy transfer was established in phycobilisome under the yellow-light condition where only the phycobilisome can absorb the cultivation light. Differences in light-harvesting and energy-transfer processes under different cultivation-light conditions are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. (C) 2012 Elsevier BV. All rights reserved.

    ELSEVIER SCIENCE BV, 2012年08月, BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 1817 (8), 1483 - 1489, 英語

    [査読有り]

    研究論文(学術雑誌)

  • 合成生物工学によるバイオ燃料生産のための微生物細胞工場の創製

    蓮沼 誠久, 近藤 昭彦

    2012年07月, 生物工学会誌, 90 (7), 386 - 391, 日本語

    研究論文(学術雑誌)

  • Jun Ishii, Miyuki Moriguchi, Kiyotaka Y. Hara, Seiji Shibasaki, Hideki Fukuda, Akihiko Kondo

    Flow cytometry enables comparative quantification, population analysis, and high-throughput screening of agonist-mediated G-protein-coupled receptor (GPCR) signaling in genetically engineered yeasts. By using flow cytometry, we found that transformation of yeast cells with a low plasmid number is critical both for the construction of large screening libraries and for stable signal transmission in cell ensembles. Based on these findings, we constructed an engineered yeast strain for the improved identification of signal promotion by C alpha(i)-specific human GPCRs using flow cytometry. (C) 2012 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年07月, Analytical Biochemistry, 426 (2), 129 - 133, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuki Soma, Kentaro Inokuma, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo, Masahiro Okamoto, Taizo Hanai

    Efficient bio-production from lignocellulosic biomass is required for the purpose of developing an inexpensive, practical bio-refinery process. As one approach to address this problem, we genetically engineered Escherichia coli to produce isopropanol directly from cellobiose via the cellobiose degradation by Beta-Glucosidase (BGL) on the cell surface. First, we investigated the cellobiose consumption of two E. coli strains with the BGL protein from Thermobifida filsca YX (Tfu0937) fused to the anchor protein Blc (Tfu0937/Blc) using different fusion sites. Next, we introduced the synthetic pathway for isopropanol production into those strains and compared their isopropanol production in the presence of glucose. Based on the results of these assays, TA212/pTA411, which was introduced Tfu-Blc fused protein expression system and the synthetic pathway for isopropanol production, was selected for the directly isopropanol production from cellobiose. TA212/pTA411 produced 69.0 +/- 11.6 mM isopropanol at 21 h of fermentation, whereas TA212/pTA147, which did not introduced the BGL/anchor fused protein but was introduced the synthetic pathway for isopropanol production, showed no cellobiose consumption and no isopropanol production during fermentation. To our knowledge, this is the first report of the production of a bio-product from cellobiose using E. coli. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2012年07月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 114 (1), 80 - 85, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Construction of an artificial pathway for isobutanol biosynthesis in the cytosol of Saccharomyces cerevisiae

    Fumio Matsuda, Takashi Kondo, Kengo Ida, Hironori Tezuka, Jun Ishii, Akihiko Kondo

    2012年07月, Bioscience, Biotechnlolgy, and Biochemistry, 76 (11), 2139 - 2141, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroko Kato, Hiroaki Suyama, Ryosuke Yamada, Tomohisa Hasunuma, Akihiko Kondo

    To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 A degrees C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.

    SPRINGER, 2012年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 94 (6), 1585 - 1592, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kiyotaka Y. Hara, Kentaro Kiriyama, Akiko Inagaki, Hideki Nakayama, Akihiko Kondo

    Glutathione (GSH) is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using . In this study, we demonstrated that engineering in sulfate assimilation metabolism can significantly improve GSH production. The intracellular GSH content of MET14 and MET16 over-expressing strains increased up to 1.2 and 1.4-fold higher than that of the parental strain, respectively, whereas those of APA1 and MET3 over-expressing strains decreased. Especially, in the MET16 over-expressing strain, the volumetric GSH concentration was up to 1.7-fold higher than that of the parental strain as a result of the synergetic effect of the increases in the cell concentration and the intracellular GSH content. Additionally, combinatorial mutant strains that had been engineered to contain both the sulfur and the GSH synthetic metabolism synergistically increased the GSH production. External addition of cysteine to is well known as a way to increase the intracellular GSH content; however, it results a decrease in cell growth. This study showed that the engineering of sulfur metabolism in proves more valuable than addition of cysteine as a way to boost GSH production due to the increases in both the intracellular GSH content and the cell growth.

    SPRINGER, 2012年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 94 (5), 1313 - 1319, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroko Kato, Yoshihiro Izumi, Tomohisa Hasunuma, Fumio Matsuda, Akihiko Kondo

    A method for a widely targeted analysis was developed for the metabolic profiling of yeast central metabolism. The widely targeted method consists of 2 analyses, namely, gas chromatography-quadrupole-mass spectrometry (GC-Q-MS) operated in selected ion monitoring mode with 25 m/z channels, and liquid chromatography triple-stage quadrupole (LC-QqQ)-MS operated in multiple reaction monitoring mode. This platform was set up to identify and quantify preselected 99 compounds, including sugars, sugar phosphates, organic adds, amino acids, and cofactors. The method showed good sensitivity and a wide dynamic range. For example, limits of detection for lactate and L-phenylalanine were 1.4 fmol and 2.0 fmol, respectively. The dynamic ranges for GC-Q-MS analysis and LC-QqQ-MS analysis were approximately 10(2)-10(5) and 10(3)-10(4), respectively. The metabolite profiles of 2 yeast strains, YPH499 and BY4741, under glucose-fermenting conditions were compared using the developed method. Although YPH499 and BY4741 were derived from an identical experimental strain, the profiling analysis successfully revealed a variation in metabolic phenotypes among experimental yeast strains demonstrating that the widely targeted method could be a robust and useful method for the investigation of metabolic phenotypes of Saccharomyces cerevisiae. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2012年05月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 113 (5), 665 - 673, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shintaro Ryo, Jun Ishii, Yusuke Iguchi, Nobuo Fukuda, Akihiko Kondo

    Here we present a successful transplantation of the GAL genetic regulatory circuitry into the G-protein signaling pathway in yeast. The GAL regulon represents a strictly regulated transcriptional mechanism that we have transplanted into yeast to create a highly robust induction system to assist the detection of on-off switching in G-protein signaling. In our system, we engineered yeast to drive the positive GAL regulatory gene in response to agonist-promoted G-protein signaling and to induce transcription of a green fluorescent protein (GFP) reporter gene under the control of the GAL structural gene promoter. Consequently, in response to agonist stimulation of G-protein-coupled receptors (GPCRs), the engineered yeast achieved more than a 150-fold increase in reporter intensity in up to 98% of cells, as determined by flow cytometric sorting. Surprisingly, agonist-stimulated induction of the GFP reporter gene was higher than that by galactose. Our approach to boost reporter gene induction could be applicable in establishing more efficient yeast-based flow cytometric screening systems for agonistic ligands for heterogeneous GPCRs. (c) 2012 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年05月, ANALYTICAL BIOCHEMISTRY, 424 (1), 27 - 31, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Matsumoto, Ryosuke Takase, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

    Modification of proteins with small molecules is a widely used and powerful tool in biological research. Enzymatic approaches are particularly promising because substrate specificity allows for site-specific modification. Sortase A, a transpeptidase from Staphylococcus aureus, cleaves between the T and G residues in the sequence LPXTG, and subsequently links the carboxyl group of the T residue to an amino group of N-terminal glycine oligomers by a native peptide bond. Although Gram-positive bacteria have several kinds of sortases, there are few reports concerning their expression and substrate specificity. Here, we demonstrate site-specific protein modification with primary amine-containing molecules catalyzed by Lactobacillus plantarum sortase. Enhanced green fluorescent protein (EGFP) was employed as a model protein, and an amine-containing biotin molecule was site-specifically conjugated with LPQTSEQ-tagged EGFP. We developed a novel Lactobacillus plantarum sortase that has different substrate specificity compared to Staphylococcus aureus sortase. Amine-directed protein modification was achieved using the Lactobacillus plantarum sortase ''LPQTSEQ'' sequence original recognition tag. Our results demonstrate a promising method for expanding the capabilities of site-specific protein-small molecule modification.

    WILEY-BLACKWELL, 2012年05月, BIOTECHNOLOGY JOURNAL, 7 (5), 642 - 648, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kunihiko Hiraiwa, Masakazu Ueda, Hiroya Takeuchi, Takashi Oyama, Tomoyuki Irino, Takahisa Yoshikawa, Akihiko Kondo, Yuko Kitagawa

    Background. In this study, we investigated the ability of magnetic resonance imaging (MRI) after interstitial administration of thermoresponsive magnetic nanoparticles to detect the sentinel lymph node (SLN). Materials and Methods. Postcontrast MRI scans were acquired following subcutaneous injection of thermoresponsive magnetic nanoparticles into the thoracic wall of rats. The signal-to-noise ratio of axillary lymph nodes was calculated to assess whether the SLN could be detected by MRI. In a second experiment, after injecting thermoresponsive magnetic nanoparticles, i.e., Therma-Max 36, Therma-Max 42, Therma-Max 55, and Ferridex, into the subserosa of the cecum of rats, the injection sites, the SLNs, and the distant lymph nodes were resected and examined histologically in order to determine which nanoparticles, if any, were specifically retained in the SLN. Results. MRI showed that the signal-to-noise ratio of axillary SLNs was significantly lower 24 h after injection of Therma-Max 42 than on the precontrast images (P < 0.05). Histologic evaluation revealed that Therma-Max 36 aggregated at body temperature and did not migrate to the SLN. Therma-Max 42, on the other hand, aggregated, and the particles became large enough to be retained in the SLNs. Therma-Max 55 and Ferridex did not aggregate, and they both migrated to the SLNs and the distant lymph nodes. Conclusions. The results of this study showed that thermoresponsive magnetic nanoparticles could be targeted to the SLN by adjusting the temperature at which they aggregate, and that they could be used as a contrast agent for SLN mapping by MRI. (C) 2012 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年05月, JOURNAL OF SURGICAL RESEARCH, 174 (1), 48 - 55, 英語

    [査読有り]

    研究論文(学術雑誌)

  • YAMAKAWA S, YAMADA Ryosuke, TANAKA Tsutomu, OGINO Chiaki, KONDO Akihiko

    A diploid yeast strain displaying both alpha-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of alpha-amylase was optimized for cell surface display, as there have been no reports of alpha-amylase-displaying yeast. The modified yeast displaying both glucoamylase and alpha-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native alpha-amylase-displaying yeast. Using the glucoamylase and modified alpha-amylase co-displaying diploid strain, we repeated fermentation from 100 g/l of raw starch for 23 cycles without the loss of alpha-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production. (C) 2012 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2012年05月, Enzyme and Microbial Technology, 50 (6-7), 343 - 347, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takashi Kondo, Hironori Tezuka, Jun Ishii, Fumio Matsuda, Chiaki Ogino, Akihiko Kondo

    The production of higher alcohols by engineered bacteria has received significant attention. The budding yeast, Saccharomyces cerevisiae, has considerable potential as a producer of higher alcohols because of its capacity to naturally fabricate fusel alcohols, in addition to its robustness and tolerance to low pH. However, because its natural productivity is not significant, we considered a strategy of genetic engineering to increase production of the branched-chain higher alcohol isobutanol, which is involved in valine biosynthesis. Initially, we overexpressed 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) in S. cerevisiae to enhance the endogenous activity of the Ehrlich pathway. We then overexpressed Ilv2, which catalyzes the first step in the valine synthetic pathway, and deleted the PDC1 gene encoding a major pyruvate decarboxylase with the intent of altering the abundant ethanol flux via pyruvate. Through these engineering steps, along with modification of culture conditions, the isobutanol titer of S. cerevisiae was elevated 13-fold, from 11 mg/l to 143 mg/l, and the yield was 6.6 mg/g glucose, which is higher than any previously reported value for S. cerevisiae. (C) 2012 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2012年05月, JOURNAL OF BIOTECHNOLOGY, 159 (1-2), 32 - 37, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ranjita Biswas, Masaru Yamaoka, Hideki Nakayama, Takashi Kondo, Ken-ichi Yoshida, Virendra S. Bisaria, Akihiko Kondo

    Production of 2,3-butanediol by Bacillus subtilis takes place in late-log or stationary phase, depending on the expression of bdhA gene encoding acetoin reductase, which converts acetoin to 2,3-butanediol. The present work focuses on the development of a strain of B. subtilis for enhanced production of 2,3-butanediol in early log phase of growth cycle. For this, the bdhA gene was expressed under the control of P (alsSD) promoter of AlsSD operon for acetoin fermentation which served the substrate for 2,3-butanediol production. Addition of acetic acid in the medium induced the production of 2,3-butanediol by 2-fold. Two-step aerobic-anaerobic fermentation further enhanced 2,3-butanediol production by 4-fold in comparison to the control parental strain. Thus, addition of acetic acid and low dissolved oxygen in the medium are involved in activation of bdhA gene expression from P (alsSD) promoter in early log phase. Under the conditions tested in this work, the maximum production of 2,3-butanediol, 2.1 g/l from 10 g/l glucose, was obtained at 24 h. Furthermore, under the optimized microaerophilic condition, the production of 2,3-butanediol improved up to 6.1 g/l and overall productivity increased by 6.7-fold to 0.4 g/l h in the engineered strain compared to that in the parental control.

    SPRINGER, 2012年05月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 94 (3), 651 - 658, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Keisuke Fujitomi, Tomoya Sanda, Tomohisa Hasunuma, Akihiko Kondo

    For efficient bioethanol production from lignocellulosic biomass by Saccharomyces cerevisiae, it is necessary to improve cellular tolerance to toxic compounds released during the pretreatment of biomass. The gene encoding p-nitrophenylphosphatase, PHO13, was disrupted in a recombinant xylose-fermenting S. cerevisiae strain, which improved ethanol production from xylose in the presence of three major inhibitors, acetic and formic acids, and furfural. In medium supplemented with 30 mM acetic acid, the ethanol yield obtained by the Delta PHO13 mutant was 0.45 g-ethanol/g-xylose. Notably, the specific ethanol productivity of the mutant in the presence of 90 mM furfural was fourfold higher than that of the control strain. The PHO13-disrupted strain produced ethanol from rice straw hydrolysate obtained by liquid hot-water pretreatment with a greater than fourfold higher xylose consumption rate than the control. Together, our findings demonstrate that PHO13 deletion is a simple, but effective, approach for improving cellulosic bioethanol production by S. cerevisiae. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2012年05月, BIORESOURCE TECHNOLOGY, 111, 161 - 166, 英語

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    研究論文(学術雑誌)

  • Jun Ishii, Nobuo Yoshimoto, Kenji Tatematsu, Shun'ichi Kuroda, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

    PUBLIC LIBRARY SCIENCE, 2012年05月, PLOS ONE, 7 (5), e37136, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kiyotaka Y. Hara, Songhee Kim, Kentaro Kiriyama, Hideyo Yoshida, Shogo Arai, Jun Ishii, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is industrially produced by fermentation using Saccharomyces cerevisiae. Before the glutathione fermentation process with S. cerevisiae, a glucose extraction process from starchy materials is required. This glucose extraction is usually carried out by converting starchy materials to starch using high-temperature cooking and subsequent hydrolysis by amylases to convert starch to glucose. In this study, to develop an energy-saving glutathione production process by reducing energy consumption during the cooking step, we efficiently produced glutathione from low-temperature cooked rice using amylase-expressing S. cerevisiae. The combination of the amylase-expressing yeast with low-temperature cooking is potentially applicable to a variety of energy-saving bio-production methods of chemicals from starchy bio-resources.

    WILEY-BLACKWELL, 2012年05月, BIOTECHNOLOGY JOURNAL, 7 (5), 686 - 689, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ayumi Yoshida, Shinji Hama, Naoki Tamadani, Hideki Fukuda, Akihiko Kondo

    To improve enzymatic biodiesel production, we developed a packed-bed reactor (PBR) system using recombinant cells, into which a strong enolase promoter and 5' untranslated region were introduced. Aspergillus oryzae expressing Fusarium heterosporum lipase was immobilized within biomass support particles (BSPs) during cultivation and used directly as a whole-cell biocatalyst. BSP-immobilized A. oryzae carrying three copies of the expression cassette showed a higher performance than previously developed cell systems, resulting in 87.5% conversion after 10 passes in PBR. The performance was also affected by operational factors including residence time and methanol feeding. After optimization, the PBR system attained a final methyl ester content of 96.1% with a residence time of 140 min per pass and stepwise addition of 4.25 molar equivalents of methanol to oil for 6 passes. Moreover, lipase activity was maintained for 5 batch cycles. Therefore, the developed PBR employing a high-lipase-expression system is considered useful for improving enzymatic biodiesel production. (C) 2011 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2012年04月, BIOCHEMICAL ENGINEERING JOURNAL, 63, 76 - 80, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takatoshi Sakamoto, Tomohisa Hasunuma, Yoshimi Hori, Ryosuke Yamada, Akihiko Kondo

    The cost of the lignocellulose-hydrolyzing enzymes used in the saccharification process of ethanol production from biomass accounts for a relatively high proportion of total processing costs. Cell surface engineering technology has facilitated a reduction in these costs by integrating saccharification and fermentation processes into a recombinant microbe strain expressing heterologous enzymes on the cell surface. We constructed a recombinant Saccharomyces cerevisiae that not only hydrolyzed hemicelluloses by codisplaying endoxylanase from Trichoderma reesei, beta-xylosidase from Aspergillus oryzae, and beta-glucosidase from Aspergillus aculeatus but that also assimilated xylose through the expression of xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. The recombinant strain successfully produced ethanol from rice straw hydrolysate consisting of hemicellulosic material containing xylan, xylooligosaccharides, and cellooligosaccharides without requiring the addition of sugar-hydrolyzing enzymes or detoxication. The ethanol titer of the strain was 8.2 g/l after 72 h fermentation, which was approximately 2.5-fold higher than that of the control strain. The yield (grams of ethanol per gram of total sugars in rice straw hydrolysate consumed) was 0.41 g/g, which corresponded to 82% of the theoretical yield. The cell surface-engineered strain was thus highly effective for consolidating the process of ethanol production from hemicellulosic materials. (C) 2011 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2012年04月, JOURNAL OF BIOTECHNOLOGY, 158 (4), 203 - 210, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shuhei Noda, Eiichi Kitazono, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    Background: Benzoic acid is one of the most useful aromatic compounds. Despite its versatility and simple structure, benzoic acid production using microbes has not been reported previously. Streptomyces are aerobic, Gram-positive, mycelia-forming soil bacteria, and are known to produce various kinds of antibiotics composed of many aromatic residues. S. maritimus possess a complex amino acid modification pathway and can serve as a new platform microbe to produce aromatic building-block compounds. In this study, we carried out benzoate fermentation using S. maritimus. In order to enhance benzoate productivity using cellulose as the carbon source, we constructed endo-glucanase secreting S. maritimus. Results: After 4 days of cultivation using glucose, cellobiose, or starch as a carbon source, the maximal level of benzoate reached 257, 337, and 460 mg/l, respectively. S. maritimus expressed beta-glucosidase and high amylase-retaining activity compared to those of S. lividans and S. coelicolor. In addition, for effective benzoate production from cellulosic materials, we constructed endo-glucanase-secreting S. maritimus. This transformant efficiently degraded the phosphoric acid swollen cellulose (PASC) and then produced 125 mg/l benzoate. Conclusions: Wild-type S. maritimus produce benzoate via a plant-like beta-oxidation pathway and can assimilate various carbon sources for benzoate production. In order to encourage cellulose degradation and improve benzoate productivity from cellulose, we constructed endo-glucanase-secreting S. maritimus. Using this transformant, we also demonstrated the direct fermentation of benzoate from cellulose. To achieve further benzoate productivity, the L-phenylalanine availability needs to be improved in future.

    BIOMED CENTRAL LTD, 2012年04月, MICROBIAL CELL FACTORIES, 11 (1), 49, 英語

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    研究論文(学術雑誌)

  • Shimpei Aikawa, Yoshihiro Izumi, Fumio Matsuda, Tomohisa Hasunuma, Jo-Shu Chang, Akihiko Kondo

    Arthrospira (Spirulina) platensis, a fast-growing halophilic cyanobacterium able to accumulate glycogen, was investigated for its feasibility to serve as feedstock for fermentative production of biofuels and chemicals. The culture conditions most appropriate for glycogen production were identified. Glycogen production was maximized by the depleting nitrate source under a high light intensity of 700 mu mol photons m(-2) s(-1). With optimal control of both light intensity and nitrate supply, glycogen production of A. platensis reached nearly 1.03 g L-1 (a glycogen productivity of 0.29 g L-1 d(-1)), which is, to the best of our knowledge, the highest alpha-polyglucan (glycogen or starch) production performance ever reported in microalgae. The outcome of this work supports A. platensis as a promising carbohydrate source for biorefinery. (C) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2012年03月, BIORESOURCE TECHNOLOGY, 108, 211 - 215, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuki Matano, Tomohisa Hasunuma, Akihiko Kondo

    Economically feasible processes for industrial cellulosic ethanol production requires increasing the final ethanol titer during fermentation due to the high energy demands of the subsequent ethanol distillation. In the present study, high-yield ethanol production was achieved by short-term liquefaction and fermentation of lignocellulose biomass in a novel drum-type rotary fermentation system using a yeast strain developed for cell-surface display of fungal endoglucanase, cellobiohydrolase, and beta-glucosidase. In the presence of 10 FPU/g-biomass cellulase added, the recombinant cellulolytic strain produced 1.4-fold higher ethanol (89% of theoretical yield) from high-solid (200 g-dry weight/L) rice straw within 72 h of fermentation than wild type strain. Cell-surface engineering successfully reduced the amount of commercial enzyme required for the fermentation of cellulose. This study demonstrates that cellulases displayed on the yeast cell surface are capable of hydrolyzing cellulose that was not hydrolyzed by commercial cellulases, leading to increased sugar utilization for improved ethanol production. (c) 2012 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2012年03月, BIORESOURCE TECHNOLOGY, 108, 128 - 133, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Anjali Madhavan, Aradhana Srivastava, Akihiko Kondo, Virendra S. Bisaria

    Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.

    INFORMA HEALTHCARE, 2012年03月, CRITICAL REVIEWS IN BIOTECHNOLOGY, 32 (1), 22 - 48, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Matsumoto, Tsutomu Tanaka, Akihiko Kondo

    A microparticle surface was designed by the unique method incorporating streptavidin biotin affinity and sortase A (SrtA)-catalyzed transpeptidation. Leucine-proline-glutamate-threonine-glycine-tagged streptavidin (Stav-LPETG) was immobilized on the surface using streptavidin biotin affinity, and GGGGG-tagged red fluorescent protein (Gly5-RFP) was conjugated with SrtA. Biotinylated fluorescein isothiocyanate (biotin FITC) was then bound to residual biotin-binding sites in Stav-LPETG. The resulting particles had RFP and FITC immobilized on the surface via Stav-LPETG, and RFP- and FITC-associated fluorescence was observed using fluorescence microscopy. Finally, GGG-tagged glucose oxidase and biotinylated horseradish peroxidase were immobilized on the microparticle surface, resulting in a functional particle capable of detecting glucose. This particle can be repeatedly used and is more sensitive in detecting glucose than particles prepared using chemical modification. Our method provides a simple strategy for site-specific coimmobilization on molecular surfaces and expands the use of protein hybrid devices.

    AMER CHEMICAL SOC, 2012年02月, LANGMUIR, 28 (7), 3553 - 3557, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kiyotaka Y. Hara, Songhee Kim, Hideyo Yoshida, Kentaro Kiriyama, Takashi Kondo, Naoko Okai, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources.

    SPRINGER, 2012年02月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 93 (4), 1495 - 1502, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yosuke Fukutani, Tomoko Nakamura, Maiko Yorozu, Jun Ishii, Akihiko Kondo, Masafumi Yohda

    For the development of a biomimetic odor-sensing system, we investigated the effects of replacing the N-terminus of an olfactory receptor (OR) on its functional expression in the budding yeast, Saccharomyces cerevisiae. Using the mouse olfactory receptor OR226 (mOR226), three types of chimeric ORs were constructed by replacing N-terminal regions of mOR226 with the corresponding regions of the rat I7 receptor, which is known to be functionally expressed in yeast. The replacement of the N-terminal region of mOR226 dramatically affected the expression and localization of the receptor and improved the sensing ability of the yeast cells for the odorant. Furthermore, the replacement of the endogenous yeast G-protein a subunit (Gpa1) by the OR-specific Golf drastically elevated the odorant-sensing ability of the yeast cells and caused the cells to display a dose-dependent responsiveness to the odorant. Because of the suitability of yeast cells for screening large-scale libraries, the strategy presented here would be useful for the establishment of advanced biomimetic odor-sensing systems. Biotechnol. Bioeng. 2012;109: 205212. (c) 2011 Wiley Periodicals, Inc.

    WILEY-BLACKWELL, 2012年01月, BIOTECHNOLOGY AND BIOENGINEERING, 109 (1), 205 - 212, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yuya Nishimura, Takuya Shishido, Jun Ishii, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    Bio-nanocapsules (BNCs) are hollow nanoparticles composed of the L protein of hepatitis B virus (HBV) surface antigen (HBsAg), which can specifically introduce genes and drugs into various kinds of target cells. Although the classic electroporation method has typically been used to introduce highly charged molecules such as DNA, it is rarely adopted for proteins due to its very low efficiency. In this study, a novel approach to the preparation of BNC was established whereby a target protein was pre-encapsulated during the course of nanoparticle formation. Briefly, because of the process of BNC formation in a budding manner on the endoplasmic reticulum (ER) membrane, the association of target proteins to the ER membrane with lipidation sequences (ER membrane localization sequences) could directly generate protein-encapsulating BNC in collaboration with co-expression of the L proteins. Since the membrane-localized proteins are automatically enveloped into BNCs during the budding event, this method can be protect the proteins and BNCs from damage caused by electroporation and obviate the need for laborious consideration to study the optimal conditions for protein encapsulation. This approach would be a useful method for encapsulating therapeutic candidate proteins into BNCs. (C) 2011 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2012年01月, JOURNAL OF BIOTECHNOLOGY, 157 (1), 124 - 129, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shuhei Noda, Takaya Miyazaki, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    Transglutaminase from Streptoverticillium cinnamoneum (StvcMTG) was produced using recombinant Streptomyces lividans. When grown on glycerol and xylose as sole carbon sources, S. lividans/StvcMTG produced 360 and 530 mg of StvcMTG per liter, respectively. With starch and xylan, the strain produced 230 and 400 mg of StvcMTG per liter, respectively. Recombinant S. lividans/encP, which expresses phenylalanine ammonia lyase from Streptomyces maritimus, produced 160 mg/L of cinnamic acid from cellulose. These results show that S. lividans can assimilate various carbon sources and produce useful compounds in desirable quantities. (C) 2011 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2012年01月, BIORESOURCE TECHNOLOGY, 104, 648 - 651, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Won-Kyung Hong, Chul Ho Kim, Dina Rairakhwada, Seonghun Kim, Byung-Ki Hur, Akihiko Kondo, Jeong-Woo Seo

    We examined the growth of a novel oleaginous microalga, Aurantiochytrium sp. KRS101, using cellulosic materials as nutrients, and the resultant production of lipids containing high levels of docosahexaenoic acid (DHA). The microalgal strain could grow using either carboxymethylcellulose or cellobiose as a carbon source, and produced lipids containing high levels of DHA (49-58% of total fatty acids). In line with this growth behavior, carboxymethylcellulase and cellobiohydrolase activities were evident in both cell-free lysates and culture broths. Additionally, an industrial cellulosic biomass, palm oil empty fruit bunches (POEFB), a by-product of the palm oil industry, were utilized by the microalgal strain for cell growth and lipid production.

    SPRINGER, 2012年01月, BIOPROCESS AND BIOSYSTEMS ENGINEERING, 35 (1-2), 129 - 133, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Preface to the special edition

    M. Ueda, A. Kondo

    2012年, Seibutsu-kogaku Kaishi, 90 (7), 381-, 英語

    [査読有り]

  • バイオリファイナリーとバイオプラスチック

    岡井 直子, 蓮沼 誠久, 近藤 昭彦

    2012年, ペトロテック, 35 (10), 700 - 706, 日本語

    研究論文(学術雑誌)

  • Hirokazu Nagaoka, Yasunobu Sato, Xiaomao Xie, Hideyuki Hata, Masaru Eguchi, Nobulci Sakurai, Takeshi Watanabe, Hiroshi Saitoh, Akihiko Kondo, Satoru Sugita, Noriyuki Ohnishi

    Because current homogeneous immunoassays show some limitations, particularly low sensitivity, we developed a new immunoassay to overcome these limitations. The approach was based on magnetic nanoparticles with a thermoresponsive polymer layer, a negatively charged polymer, and streptavidin-biotin-based antibody-antigen detection and yielded higher sensitivity than commonly used heterogeneous immunoassays. Because no special equipment is needed, it can be applied to currently available absorbance-based systems for high-throughput assays.

    AMER CHEMICAL SOC, 2011年12月, ANALYTICAL CHEMISTRY, 83 (24), 9197 - 9200, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Sriappareddy Tamalampudi, Ayumi Yoshida, Naoki Tamadani, Nobuyuki Kuratani, Hideo Noda, Hideki Fukuda, Akihiko Kondo

    A process model for efficient glycerol separation during methanolysis in an enzymatic packed-bed reactor (PBR) was developed. A theoretical glycerol removal efficiency from the reaction mixture containing over 30% methyl esters was achieved at a high flow rate of 540 ml/h. To facilitate a stable operation of the PBR system, a batch reaction prior to continuous methanolysis was conducted using oils with different acid values and immobilized lipases pretreated with methyl esters. The reaction system successfully attained the methyl ester content of over 30% along with reduced viscosity and water content. Furthermore, to obtain a high methyl ester content above 96% continuously, long-term lipase stability was confirmed by operating a bench-scale PBR system for 550 h, in which the intermediates containing methyl esters and residual glycerides were fed into the enzyme-packed columns connected in series. Therefore, the developed process model is considered useful for industrial biodiesel production. (C) 2011 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2011年11月, BIORESOURCE TECHNOLOGY, 102 (22), 10419 - 10424, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroki Bando, Hiromoto Hisada, Hiroki Ishida, Yoji Hata, Yoshio Katakura, Akihiko Kondo

    A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-beta-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the alpha-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30A degrees C or 37A degrees C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

    SPRINGER, 2011年11月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 92 (3), 561 - 569, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yusuke Miyachi, Chiaki Ogino, Tomokazu Amino, Akihiko Kondo

    Atomic force microscopy (AFM) can dynamically detect the adhesion or affinity force between a sample surface and a cantilever. This feature is useful as a detection method using aptamers - single-strand DNA that recognizes its target with very high affinity. The present study proposes a novel DNA aptamer-based sensing system using AFM. In this study, thrombin was chosen as the target molecule, and a DNA aptamer-based AFM sensing system based on competition was developed. The affinity force between the gold chip and the cantilever decreased as the concentration of thrombin increased. Moreover, a low detection limit of 0.2 nM was achieved. Therefore, the AFM sensing system used would be appropriate for the measurement of various chemical compounds. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2011年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 112 (5), 511 - 514, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Fumio Matsuda, Masahiro Hayashi, Akihiko Kondo

    Comparative metabolic profiling analysis was performed to investigate light- and aeration-dependent regulation of central metabolism in Englena gracilis. The metabolic profiles of E. gracilis were significantly altered in response to changes in aeration conditions. While many glycolytic intermediates and amino acids accumulated in aerobically grown E. gracilis, a significant reduction in these metabolites was observed for cells under anaerobic conditions, which resulted in elevated production of wax ester.

    TAYLOR & FRANCIS LTD, 2011年11月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 75 (11), 2253 - 2256, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shogo Yoshida, Kenji Okano, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    In order to achieve efficient d-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (Delta ldhL1-xpk1::tkt-Delta xpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of Delta ldhL1-xpk1::tkt-Delta xpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-d-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of d-lactic acid of 99.5%. Finally, we successfully demonstrated homo-d-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-d-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.

    SPRINGER, 2011年10月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 92 (1), 67 - 76, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Nobuo Fukuda, Jun Ishii, Misato Kaishima, Akihiko Kondo

    G-protein-coupled receptors (GPCRs) are considered as important targets for drug discovery. The yeast Saccharomyces cerevisiae is an attractive host for high-throughput screening of agonistic ligands for human GPCRs because it can simplify the complicated signaling pathways that are present in mammalian cell lines. Unfortunately, many human GPCRs induce only partial signal activation in yeast cells depending on their coupling efficiency with yeast G-proteins. This problem often results in unsatisfactory detection sensitivity, thereby resulting in a limitation to yeast-based detection systems. Here we introduce a new highly sensitive detection method that provides robust agonist detection of human GPCRs. Our strategy is designed to invoke feedback activation of signals within yeast G-protein signaling pathways. Briefly, agonist stimulation of human GPCRs triggers expression of an artificial signal activator that amplifies signaling. We chose human somatostatin receptor subtype 5 (hSSTR5) as a model of a human GPCR. Investigation of the response of hSSTR5-expressing yeast to various concentrations of somatostatin demonstrated that feedback activation of the signal can successfully improve the detection limit and the maximum level of signaling. This novel approach will enhance the usefulness of yeast-based screening of agonistic ligands for a variety of human GPCRs. (C) 2011 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年10月, ANALYTICAL BIOCHEMISTRY, 417 (2), 182 - 187, 英語

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    研究論文(学術雑誌)

  • Tomoya Sanda, Tomohisa Hasunuma, Fumio Matsuda, Akihiko Kondo

    A major challenge associated with the fermentation of lignocellulose-derived hydrolysates is improved ethanol production in the presence of fermentation inhibitors, such as acetic and formic acids. Enhancement of transaldolase (TAL) and formate dehydrogenase (FDH) activities through metabolic engineering successfully conferred resistance to weak acids in a recombinant xylose-fermenting Saccharomyces cerevisiae strain. Moreover, hybridization of the metabolically engineered yeast strain improved ethanol production from xylose in the presence of both 30 mM acetate and 20 mM formate. Batch fermentation of lignocellulosic hydrolysate containing a mixture of glucose, fructose and xylose as carbon sources, as well as the fermentation inhibitors, acetate and formate, was performed for five cycles without any loss of fermentation capacity. Long-term stability of ethanol production in the fermentation phase was not only attributed to the coexpression of TAL and FDH genes, but also the hybridization of haploid strains. (C) 2011 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2011年09月, BIORESOURCE TECHNOLOGY, 102 (17), 7917 - 7924, 英語

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    研究論文(学術雑誌)

  • Satoru Shinkawa, Kenji Okano, Shogo Yoshida, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Delta ptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.

    SPRINGER, 2011年09月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 91 (6), 1537 - 1544, 英語

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    研究論文(学術雑誌)

  • Tsutomu Tanaka, Hitomi Kawabata, Chiaki Ogino, Akihiko Kondo

    We demonstrated direct assimilation of cellooligosaccharide using Escherichia coli displaying beta-glucosidase (BGL). BGL from Thermobifida fusca YX (Tfu0937) was displayed on the E. coli cell surface using a novel anchor protein named Blc. This strain was grown successfully on 0.2% cellobiose, and the optical density at 600 nm (OD(600)) was 1.05 after 20 h.

    AMER SOC MICROBIOLOGY, 2011年09月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77 (17), 6265 - 6270, 英語

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    研究論文(学術雑誌)

  • SAITOH S, TANAKA Tsutomu, KONDO Akihiko

    We constructed a recombinant industrial Saccharomyces cerevisiae yeast strain OC2-AXYL2-ABGL2-Xyl2 by inserting two copies of the beta-glucosidase (BGL) and beta-xylosidase (XYL) genes, and a gene cassette for xylose assimilation in the genome of yeast strain OC-2HUT. Both BGL and XYL were expressed on the yeast cell surface with high enzyme activities. Using OC2-AXYL2-ABGL2-Xyl2, we performed ethanol fermentation from a mixture of powdered cellulose (KC-flock) and Birchwood xylan, with the additional supplementation of a 30-g/l Trichoderma reesei cellulase complex mixture. The ethanol yield (gram per gram of added cellulases) of the strain OC2-AXYL2-ABGL2-Xyl2 increased approximately 2.5-fold compared to that of strain OC2-Xyl2, which lacked beta-glucosidase and beta-xylosidase activities. Notably, the concentration of additional T. reesei cellulase was reduced from 30 to 24 g/l without affecting ethanol production. The BGL- and XYL-displaying industrial yeast of the strain OC2-AXYL2-ABGL2-Xyl2 represents a promising yeast for reducing cellulase consumption of ethanol fermentation from lignocellulosic biomass by compensating for the inherent weak BGL and XYL activities of T. reesei cellulase complexes.

    SPRINGER, 2011年09月, Applied Microbiology and Biotechnology, 91 (6), 1553 - 1559, 英語

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    研究論文(学術雑誌)

  • Nobuo Fukuda, Jun Ishii, Akihiko Kondo

    Weak and transient protein-protein interactions are associated with biological processes, but many are still undefined because of the difficulties in their identification. Here, we describe a redesigned method to screen transient protein-protein interactions by using a novel signal amplification circuit, which is incorporated into yeast to artificially magnify the signal responding to the interactions. This refined method is based on the previously established G gamma recruitment system, which utilizes yeast G-protein signaling and mating growth selection to screen interacting protein pairs. In the current study, to test the capability of our method, we chose mutants of the Z-domain derived from Staphylococcus aureus protein A as candidate proteins, and the Fc region of human IgG as the counterpart. By introduction of an artificial signal amplifier into the previous G gamma recruitment system, the signal transduction responding to transient interactions between Z-domain mutants and the Fc region with significantly low affinity (8.0 x 10(3) M(-1)) was successfully amplified in recombinant haploid yeast cells. As a result of zygosis with the opposite mating type of wildtype haploid cells, diploid colonies were vigorously and selectively generated on the screening plates, whereas our previous system rarely produced positive colonies. This new approach will be useful for exploring the numerous transient interactions that remain undefined because of the lack of powerful screening tools for their identification.

    WILEY-BLACKWELL, 2011年09月, FEBS Journal, 278 (17), 3086 - 3094, 英語

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    研究論文(学術雑誌)

  • Fumio Matsuda, Chikara Furusawa, Takashi Kondo, Jun Ishii, Hiroshi Shimizu, Akihiko Kondo

    Background: While Saccharomyces cerevisiae is a promising host for cost-effective biorefinary processes due to its tolerance to various stresses during fermentation, the metabolically engineered S. cerevisiae strains exhibited rather limited production of higher alcohols than that of Escherichia coli. Since the structure of the central metabolism of S. cerevisiae is distinct from that of E. coli, there might be a problem in the structure of the central metabolism of S. cerevisiae. In this study, the potential production of higher alcohols by S. cerevisiae is compared to that of E. coli by employing metabolic simulation techniques. Based on the simulation results, novel metabolic engineering strategies for improving higher alcohol production by S. cerevisiae were investigated by in silico modifications of the metabolic models of S. cerevisiae. Results: The metabolic simulations confirmed that the high production of butanols and propanols by the metabolically engineered E. coli strains is derived from the flexible behavior of their central metabolism. Reducing this flexibility by gene deletion is an effective strategy to restrict the metabolic states for producing target alcohols. In contrast, the lower yield using S. cerevisiae originates from the structurally limited flexibility of its central metabolism in which gene deletions severely reduced cell growth. Conclusions: The metabolic simulation demonstrated that the poor productivity of S. cerevisiae was improved by the introduction of E. coli genes to compensate the structural difference. This suggested that gene supplementation is a promising strategy for the metabolic engineering of S. cerevisiae to produce higher alcohols which should be the next challenge for the synthetic bioengineering of S. cerevisiae for the efficient production of higher alcohols.

    BIOMED CENTRAL LTD, 2011年09月, MICROBIAL CELL FACTORIES, 10, 英語

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    研究論文(学術雑誌)

  • Fumio Matsuda, Masanori Yamasaki, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo

    Biomass properties of rice straws were compared among eight cultivars that formed a mini diverse set. The ethanol productivity from rice straws was evaluated employing a laboratory-scale method based on dilute acid-hydrolysis pretreatment. The results indicated significant variation in biomass properties among the cultivars.

    TAYLOR & FRANCIS LTD, 2011年08月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 75 (8), 1603 - 1605, 英語

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    研究論文(学術雑誌)

  • Hideyo Yoshida, Kiyotaka Y. Hara, Kentaro Kiriyama, Hideki Nakayama, Fumiyoshi Okazaki, Fumio Matsuda, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    We developed a novel enzymatic glutathione (GSH) production system using Saccharomyces cerevisiae as a whole-cell biocatalyst, and improved its GSH productivity by metabolic engineering. We demonstrated that the metabolic engineering of GSH pathway and ATP regeneration can significantly improve GSH productivity by up to 1.7-fold higher compared with the parental strain, respectively. Furthermore, the combination of both improvements in GSH pathway and ATP regeneration is more effective (2.6-fold) than either improvement individually for GSH enzymatic production using yeast. The improved whole-cell biocatalyst indicates its great potential for applications to other kinds of ATP-dependent bioproduction.

    SPRINGER, 2011年08月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 91 (4), 1001 - 1006, 英語

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    研究論文(学術雑誌)

  • OKAZAKI Fumiyoshi, OGINO Chiaki, KONDO Akihiko, MIKAMI B, KUREBAYASHI Yoichi, TSURUTA Hiroki

    Crystals of beta-1,3-xylanase (1,3-beta-D-xylan xylanohydrolase; EC 3.2.1.32) from Thermotoga neapolitana strain DSM 4359 with maximum dimensions of 0.2 x 0.1 x 0.02 mm were grown using the sitting-drop vapour-diffusion method at 293 K over 24 h. The crystals diffracted to a resolution of 1.82 angstrom, allowing structure determination. The crystals belonged to space group P2(1), with unit-cell parameters a = 39.061, b = 75.828, c = 52.140 angstrom; each asymmetric unit cell contained a single molecule.

    WILEY-BLACKWELL, 2011年07月, Acta Crystallographica Section F-Structural Biology and Crystallization Communications, 67, 779 - 781, 英語

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    研究論文(学術雑誌)

  • Shinji Hama, Kazunori Miura, Ayumi Yoshida, Hideo Noda, Hideki Fukuda, Akihiko Kondo

    The enzymatic process presents an advantage of producing specified phospholipids that rarely exist in nature. In this study, we investigated the regiospecific modification of phosphatidylcholine (PC) in the sn-1 position using immobilized Rhizopus oryzae. In a reaction mixture containing egg yolk PC and exogenous lauric acid (LA) in n-hexane, lipase-producing R. oryzae cells immobilized within biomass support particles (BSPs) showed a much higher transesterification activity than lipase powders. To improve the product yield, several parameters including substrate ratio and reaction time were investigated, resulting in the incorporation of 44.2% LA into the product PC after a 48-h reaction. The analysis of the molecular structure showed that a large proportion of exogenous LA (> 90%) was incorporated in the sn-1 position of the enzymatically modified PC. Moreover, the BSP-immobilized R. oryzae maintained its activity for more than 12 batch cycles. The presented results, therefore, suggest the applicability of BSP-immobilized R. oryzae as a whole-cell biocatalyst for the regiospecific modification of phospholipids.

    SPRINGER, 2011年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 90 (5), 1731 - 1738, 英語

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    研究論文(学術雑誌)

  • Hiroyuki Morita, Tomohisa Hasunuma, Maria Vassileva, Roumiana Tsenkova, Akihiko Kondo

    Recently, genetic engineering efforts have been made to develop recombinant Saccharomyces cerevisiae strains able to utilize xylose, an inexpensive and abundant carbon source. However, their construction and selection processes are limited by the speed and expenses of the existing testing methods, thus a rapid and equally precise method will significantly increase the number of tested strains. Here, near infrared (NIR) spectroscopy is proposed as a successful alternative method for screening recombinant xylose-fermenting S. cerevisiae. Supernatant samples of fermentation solutions from one diploid and three haploid recombinant strains were collected along the fermentation process. NIR spectra of the diluted supernatant provided effective differentiation of strains consistent with their phenotypic and genotypic features. This result could be used as a feedback for multicomponent analysis, in order to develop regression model for quantification of consumed glucose and xylose, produced ethanol, glycerol, and xylitol. Robust partial least-squares regression models with high prediction accuracy that are effective with any strain were achieved for all components when the modeling was performed with combined data of all strains, achieving 0.21-1.49 g/L of standard error of prediction with calibration, prediction, limit of detection and limit of quantification in the range of 1.0-4.5 and 3.0-13.4 g/L, respectively.

    AMER CHEMICAL SOC, 2011年06月, ANALYTICAL CHEMISTRY, 83 (11), 4023 - 4029, 英語

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    研究論文(学術雑誌)

  • Shinji Hama, Sriappareddy Tamalampudi, Ayumi Yoshida, Naoki Tamadani, Nobuyuki Kuratani, Hideo Noda, Hideki Fukuda, Akihiko Kondo

    From the viewpoint of process engineering in enzymatic biodiesel production, this paper provides a practical basis for development of the packed-bed reactor (PBR) on a bench scale. A key component of this work is the separation of glycerol byproduct in continuous operation. To facilitate measuring and separating the byproduct, we constructed the PBR integrated with a glycerol-separating system. Methyl ester content in the effluent increased with increasing pass number of the reaction mixture, whereas the efficiency of glycerol removal was affected significantly by the flow rate in PBR. Further optimization revealed that the PBR could be operated over a long period realizing both a high methyl ester content and an efficient glycerol removal, obtaining a product that meets the biodiesel standard for residual glycerides. To remove soluble glycerol present in the biodiesel, adsorptive purification using ion-exchange resin was applied to the PBR system. The initial glycerol content of approximately 0.053 wt% was reduced to less than 0.003 wt%, which was maintained for more than 140 bed volumes and repeated after regeneration of the resin using methanol. Therefore, this developed model incorporating the enzymatic PBR and glycerol-separating system is promising for practical biodiesel production, in which no wastewater treatment is necessary. (C) 2011 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2011年06月, BIOCHEMICAL ENGINEERING JOURNAL, 55 (1), 66 - 71, 英語

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    研究論文(学術雑誌)

  • Daisuke Adachi, Shinji Hama, Takao Numata, Kazunori Nakashima, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    An Aspergillus oryzae whole-cell biocatalyst which coexpresses Fusarium heterosporum lipase (FHL) and mono- and di-acylglycerol lipase B (mdlB) in the same cell has been developed to improve biodiesel production. By screening a number of transformants, the best strain was obtained when FHL gene was integrated into A. oryzae chromosome using (S)C selection marker while mdlB was integrated using niaD selection marker. The reaction system using the lipase-coexpressing whole-cells was found to be superior in biodiesel production to others such as lipase-mixing and two-step reactions, affording the highest reaction rate and the highest ME content (98%). Moreover, an ME content of more than 90% was maintained during 10 repeated batch cycles. The whole-cell biocatalyst developed in this work would be promising biocatalysts for efficient biodiesel production. (C) 2011 Elsevier Ltd. All rights reserved.

    ELSEVIER SCI LTD, 2011年06月, BIORESOURCE TECHNOLOGY, 102 (12), 6723 - 6729, 英語

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    研究論文(学術雑誌)

  • Tomohiro Takaya, Risa Koda, Daisuke Adachi, Kazunori Nakashima, Junpei Wada, Takayuki Bogaki, Chiaki Ogino, Akihiko Kondo

    In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5' , methanolysiuntranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL#1) and double copies (pSENSU-FHL#2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL#1 and #2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL#2s could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time.

    SPRINGER, 2011年05月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 90 (3), 1171 - 1177, 英語

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    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Kyung-mo Sung, Tomoya Sanda, Kazuya Yoshimura, Fumio Matsuda, Akihiko Kondo

    Recombinant yeast strains highly tolerant to formic acid during xylose fermentation were constructed. Microarray analysis of xylose-fermenting Saccharomyces cerevisiae strain overexpressing endogenous xylulokinase in addition to xylose reductase and xylitol dehydrogenase from Pichia stipitis revealed that upregulation of formate dehydrogenase genes (FDH1 and FDH2) was one of the most prominent transcriptional events against excess formic acid. The quantification of formic acid in medium indicated that the innate activity of FDH was too weak to detoxify formic acid. To reinforce the capability for formic acid breakdown, the FDH1 gene was additionally overexpressed in the xylose-metabolizing recombinant yeast. This modification allowed the yeast to rapidly decompose excess formic acid. The yield and final ethanol concentration in the presence of 20 mM formic acid is as essentially same as that of control. The fermentation profile also indicated that the production of xylitol and glycerol, major by-products in xylose fermentation, was not affected by the upregulation of FDH activity.

    SPRINGER, 2011年05月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 90 (3), 997 - 1004, 英語

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    研究論文(学術雑誌)

  • TSUCHIDATE Takeyuki, TATENO Toshihiro, OKAI Naoko, TANAKA Tsutomu, OGINO Chiaki, KONDO Akihiko

    We demonstrate glutamate production from beta-glucan using endoglucanase (EG)-expressing Corynebacterium glutamicum. The signal sequence torA derived from Escherichia coli K12, which belongs to the Tat pathway, was suitable for secreting EG of Clostridium thermocellum using C. glutamicum as a host. Using the torA signal sequence, endoglucanase from Clostridium cellulovorans 743B was successfully expressed, and the secreted EG produced 123 mg of reducing sugar from 5 g of beta-glucan at 30 A degrees C for 72 h, which is the optimal condition for C. glutamicum growth. Subsequently, glutamate fermentation from beta-glucan was carried out with the addition of Aspergillus aculeatus beta-glucosidase produced by recombinant Aspergillus oryzae. Using EG-secreting C. glutamicum, 178 mg/l of glutamate was produced from 15 g of beta-glucan. This is the first report of glutamate fermentation from beta-glucan using endoglucanase-secreting C. glutamicum.

    SPRINGER, 2011年05月, Applied Microbiology and Biotechnology, 90 (3), 895 - 901, 英語

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    研究論文(学術雑誌)

  • Shuhei Noda, Takaya Miyazaki, Takanori Miyoshi, Michiru Miyake, Naoko Okai, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans, and its expression was confirmed by Western blot analysis. After 4 days cultivation using glucose as carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose or xylan as carbon source, the maximal level of cinnamic acid reached 460, 300, and 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds.

    SPRINGER HEIDELBERG, 2011年05月, JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, 38 (5), 643 - 648, 英語

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    研究論文(学術雑誌)

  • Ayumi Yoshida, Shinji Hama, Kazunori Nakashima, Akihiko Kondo

    Water activity (a(w)) is a crucial parameter affecting enzymatic synthetic reactions in organic media. In this paper, we report on the a(w) dependence of surface-displayed lipases, genetically immobilized on yeast cells via fusion with cell wall proteins. When Saccharomyces cerevisiae displaying Rhizopus oryzae lipase was used for esterification in n-hexane, equilibrating the dried cells with water prior to the reaction markedly increased the reaction rate. An equilibration of the cells with various saturated salt solutions showed that the reaction rate increased with increasing a(w) of the salt solution, to give the best performance at a(w) of 1.0. Interestingly, this trend was extremely different from those of lipases in powder or resin-immobilized form. To determine whether the cell surface is responsible for the unique a(w) profiles, an investigation was carried out similarly using other lipase sources and yeast strains, which indicated that, in all the cells examined, a higher a(w) resulted in a higher reaction rate. Moreover, increasing a(w) was found to increase the cell surface hydrophobicity determined by an aqueous-hydrocarbon biphasic partitioning assay. These results indicate that lipases displayed on yeast cells show a unique a(w) dependence probably because of the variation in cell surface characteristics. (C) 2011 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2011年04月, ENZYME AND MICROBIAL TECHNOLOGY, 48 (4-5), 334 - 338, 英語

    [査読有り]

    研究論文(学術雑誌)

  • APIWATANAPIWAT W, MURATA Y, KOSUGI A, YAMADA Ryosuke, KONDO Akihiko, ARAI T, RUGTHAWORN P, MORI Y

    In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying alpha-amylase (alpha-AM), glucoamylase, endoglucanase, cellobiohydrase, and beta-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley beta-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.

    SPRINGER, 2011年04月, Applied Microbiology and Biotechnology, 90 (1), 377 - 384, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ryosuke Yamada, Syun-ichi Yamakawa, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Efficient ethanol producing yeast Saccharomyces cerevisiae cannot produce ethanol from raw starch directly. Thus the conventional ethanol production required expensive and complex process. In this study, we developed a direct and efficient ethanol production process from high-yielding rice harvested in Japan by using amylase expressing yeast without any pretreatment or addition of enzymes or nutrients. Ethanol productivity from high-yielding brown rice (1.1 g/L/h) was about 5-fold higher than that obtained from purified raw corn starch (0.2 g/L/h) when nutrients were added. Using an inoculum volume equivalent to 10% of the fermentation volume without any nutrient supplementation resulted in ethanol productivity and yield reaching 1.2 g/L/h and 101%, respectively, in a 24-h period. High-yielding rice was demonstrated to be a suitable feedstock for bioethanol production. In addition, our polyploid amylase-expressing yeast was sufficiently robust to produce ethanol efficiently from real biomass. This is first report of direct ethanol production on real biomass using an amylase-expressing yeast strain without any pretreatment or commercial enzyme addition. (C) 2011 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2011年04月, ENZYME AND MICROBIAL TECHNOLOGY, 48 (4-5), 393 - 396, 英語

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    研究論文(学術雑誌)

  • Takuya Matsumoto, Shiori Sawamoto, Takayuki Sakamoto, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

    Streptavidin is tetrameric protein which has tight and specific biotin binding affinity, and streptavidin modification of proteins or small molecules is widely used for biotechnology tool. Here, we demonstrate site-specific streptavidin-protein conjugation using enzymes. We focused on sortase A, a transpeptidase from Staphylococcus aureus. A streptavidin-tagged LPETG motif (Stav-LPETG) was expressed in Escherichia coli. We achieved soluble streptavidin expression in E. coli without refolding using a cold shock expression system. Then we successfully conjugated Stav-LPETG with pentaglycine-appended green fluorescence protein (Gly5-GFP) or triglycine-appended glucose oxidase (Gly3-GOD) using sortase A. SDS-PAGE analysis showed site-specific tetrameric streptavidin-protein conjugation with the tagged proteins. In addition, the functions of a Stav-GOD conjugate, i.e., biotin-binding and glucose oxidase activity, were significantly higher compared to those of streptavidin-GOD conjugates prepared by chemical modification. (C) 2011 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2011年03月, JOURNAL OF BIOTECHNOLOGY, 152 (1-2), 37 - 42, 英語

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    研究論文(学術雑誌)

  • Hideyo Yoshida, Shogo Arai, Kiyotaka Y. Hara, Ryosuke Yamada, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. We demonstrated that expression of amylase genes in glutathione-producing S. cerevisiae enables direct use of starch as a carbon source, thus eliminating the Crabtree effect that is caused by excess glucose. Consequently, cell growth and glutathione productivity were significantly improved. This approach is potentially applicable to a variety of fermentative processes for production of value-added chemicals under aerobic conditions.

    SPRINGER, 2011年03月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 89 (5), 1417 - 1422, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Tomoya Sanda, Ryosuke Yamada, Kazuya Yoshimura, Jun Ishii, Akihiko Kondo

    Background: The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results: In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions: Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering.

    BIOMED CENTRAL LTD, 2011年01月, MICROBIAL CELL FACTORIES, 10, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Chiaki Ogino, Shuhei Noda, Akihiko Kondo, Naoko Okai, Tsutomu Tanaka

    Cinnamic acid production was demonstrated using Streptomyces as a host. A gene encoding phenylalanine ammonia lyase (PAL) from Streptomyces maritimus was introduced into Streptomyces lividans and its expression was confirmed by western blot analysis. After 4 days cultivation using glucose as a carbon source, the maximal level of cinnamic acid reached 210 mg/L. When glycerol (30 g/L) was used as the carbon source, the maximal level of produced cinnamic acid reached 450 mg/L. In addition, using raw starch, xylose and xylan as the carbon source, the maximal level of cinnamic acid reached 460, 300, 130 mg/L, respectively. We demonstrated that S. lividans has great potential to produce cinnamic acid as well as other aromatic compounds. © 2011 IEEE.

    2011年, 2011 Defense Science Research Conference and Expo, DSR 2011, 英語

    [査読有り]

    研究論文(国際会議プロシーディングス)

  • Ryosuke Yamada, Naho Taniguchi, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Background: Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and b-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass. Results: The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours. Conclusions: We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzymeexpressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.

    BioMed Central Ltd., 2011年, Biotechnology for Biofuels, 4 (1), 8, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression

    YAMADA Ryosuke, TANIGUCHI N, TANAKA Tsutomu, OGINO Chiaki, FUKUDA Hideki, KONDO Akihiko

    2011年, Biotechnology for Biofuels, 4:8, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kazunori Nakashima, Koji Yamaguchi, Naho Taniguchi, Shogo Arai, Ryosuke Yamada, Satoshi Katahira, Nobuhiro Ishida, Haruo Takahashi, Chiaki Ogino, Akihiko Kondo

    The efficient production of biofuels from cellulosic biomass is a current imperative. The present study demonstrates direct bioethanol production from cellulose by cellulase-displaying yeast (arming yeast) using ionic liquid pretreatment. In this approach, all of the bioethanol production processes (pretreatment, saccharification, and fermentation) were conducted in a single vessel. In a preliminary test, the arming yeast showed resistance to cellulose-dissolving ionic liquids below a concentration of approximately 200 mM. Cellulose degradation and ethanol productivity by the arming yeast was dramatically enhanced by pretreatment with ionic liquids. The addition of free cellulases during fermentation significantly enhanced ethanol productivity in the arming yeast system, producing approximately 90% ethanol yield. Furthermore, in a preliminary examination, 82% of the ionic liquids in the culture medium could be recovered and recycled for the pretreatment of cellulose.

    ROYAL SOC CHEMISTRY, 2011年, GREEN CHEMISTRY, 13 (10), 2948 - 2953, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takayuki Sakamoto, Shiori Sawamoto, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

    A ZZ domain (ZZ) and alkaline phosphatase (AP), luciferase (Luc), or glucose oxidase (GOD) were conjugated using Sortase A (SrtA) from Staphylococcus aureus. The specific peptidyl linker for SrtA was genetically used to the C-terminus of ZZ, and the other linker was fused to the N-terminus of AP, Luc, or GOD, respectively. The resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged ZZ and AP, Luc, or GOD were site-specifically conjugated by SrtA through he extra peptidyl linkers in vitro. The SrtA reaction had little influence on either the antibody-binding activity of the ZZ moiety or the enzymatic activity of AP, Luc, or GOD moieties of the conjugates. Additionally, antibody-ZZ-proteins were yielded easily by mixing antibody with ZZ-AP, ZZ-Luc, or ZZ-GOD, allowing their use in an enzyme-linked immunosorbent assay. These results suggest that the enzymatic approach with SrtA facilitates the construction of ZZ-proteins. Furthermore, mixing antibody and ZZ-proteins produces a wide variety of antibody-ZZ-proteins.

    AMER CHEMICAL SOC, 2010年12月, BIOCONJUGATE CHEMISTRY, 21 (12), 2227 - 2233, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takanori Tanino, Atsushi Hotta, Tomonori Ito, Jun Ishii, Ryosuke Yamada, Tomohisa Hasunuma, Chiaki Ogino, Naoto Ohmura, Takayuki Ohshima, Akihiko Kondo

    A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity. © 2010 Springer-Verlag.

    2010年11月, Applied Microbiology and Biotechnology, 88 (5), 1215 - 1221, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Construction of a xylose-metabolizing Saccharomyces cerevisiae by integration of xylose isomerase gene into the genome and investigation of the effect of xylITOl on fermentation

    Takanori Tanino, Atsushi Hotta, Tomonori Ito, Jun Ishii, Ryosuke Yamada, Tomohisa Hasunuma, Chiaki Ogino, Naoto Ohmura, Takayuki Ohima, Akihiko Kondo

    2010年11月, Applied Microbiology and Biotechnology, 88, 1215-1221, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shuhei Noda, Yuko Ito, Nobuaki Shimizu, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    Streptomyces lividans is known to produce large amounts of proteins in culture supernatants. In this report, to expand the secretory expression system with a strong promoter derived from phospholipase D of Streptoverticillium cinnamoneum, we expressed three kinds of proteins: transglutaminase from Sty. cinnamoneum (StvcMTG) and beta-1,4-endoglucanase and beta-glucosidase from Thermobifida fusca YX. The StvcMTG gene was introduced into S. lividans using the shuttle vector pUC702 for Escherichia coli and S. lividans, and high level secretory production of StvcMTG (230 mu g/ml in the culture supernatant) was achieved. The other prokaryotic proteins, beta-1,4-endoglucanase and beta-glucosidase, were also expressed in (His)(6)-tag fused form into culture supernatants and retained high activity. Furthermore, complete purification was achieved by conventional column or affinity column chromatography for each recombinant protein with 1 mg/ml over protein concentration. Three independent proteins were thus successfully expressed and purified, and we expect to use this system for the expression of other valuable heterologous proteins. (C) 2010 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, 2010年10月, PROTEIN EXPRESSION AND PURIFICATION, 73 (2), 198 - 202, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Shishido, Hiroaki Mieda, Sang Youn Hwang, Yuya Nishimura, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    A novel HER2-targeted carrier was developed using bionanocapsules (BNCs). Bionanocapsules (BNCs) are 100-nm hollow nanoparticles composed of the L-protein of hepatitis B virus surface antigen. An affibody of HER2 was genetically displayed on the BNC surface (Z(HER2)-BNC). For the investigation of binding affinity, Z(HER2)-BNC was incubated with the cancer cell lines SK-BR-3 (HER2 positive), and MDA-MB-231 (HER2 negative). For analysis of HER2 targeting specificity, Z(HER2)-BNC or Z(WT)-BNC (without affibody) was incubated with both SK-BR-3 and MDA-MB-231 cells by time lapse and concentration. For the delivery of encapsulated molecules (calcein), fluorescence of Z(HER2)-BNC mixed with liposomes was also compared with that of Z(WT)-BNC and nude liposomes by incubation with SK-BR-3 cells. As a result, Z(HER2)-BNC-liposome complex demonstrated the delivery to HER2-expressing cells (SK-BR-3) with a high degree of specificity. This indicates that genetically engineered BNCs are promising carrier for cancer treatment. (C) 2010 Elsevier Ltd. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2010年10月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 20 (19), 5726 - 5731, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Risa Koda, Takao Numata, Shinji Hama, Sriappareddy Tamalampudi, Kazunori Nakashima, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    We demonstrated ethanolysis of rapeseed oil to produce biodiesel fuel using lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst. We prepared two types of whole-cell biocatalyst: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL) and recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL). Both w-ROL and r-FHL successfully catalyzed the ethanolysis of rapeseed oil, and the fatty acid ethyl ester yield was as high as 79% (w-ROL) or 94% (r-FHL). In the case of r-FHL, the residual monoglycerides (MGs) and diglycerides (DGs) were no more than 0.73 and 0.18%, respectively. In addition, r-FHL could be recycled for the ethanolysis of rapeseed oil, retaining over 85% fatty acid ethyl ester yield by the fifth cycle. r-FHL was revealed to be a promising catalyst for biodiesel production using rapeseed oil and ethanol. (C) 2010 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2010年09月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 66 (1-2), 101 - 104, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shuhei Yanase, Tomohisa Hasunuma, Ryosuke Yamada, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification-fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 degrees C and 37 degrees C, while the activity of cellulolytic enzymes is highest at around 50 degrees C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus beta-glucosidase on the cell surface, which successfully converts a cellulosic beta-glucan to ethanol directly at 48 degrees C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield ( in grams of ethanol produced per gram of beta-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.

    SPRINGER, 2010年09月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 88 (1), 381 - 388, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kazusa Matsui, Miki Karasaki, Maiko Segawa, Sang Youn Hwang, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    Cancer cell-specific photokilling was successfully demonstrated using antibody-immobilized TiO2 nanoparticles with only 1 J cm(-2) UV irradiation.

    ROYAL SOC CHEMISTRY, 2010年09月, MEDCHEMCOMM, 1 (3), 209 - 211, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Soichiro Tabuchi, Junji Ito, Takashi Adachi, Hiroki Ishida, Yoji Hata, Fumiyoshi Okazaki, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.

    SPRINGER, 2010年08月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87 (5), 1783 - 1789, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Satoshi Saitoh, Tomohisa Hasunuma, Tsutomu Tanaka, Akihiko Kondo

    The co-utilization of sugars, particularly xylose and glucose, during industrial fermentation is essential for economically feasible processes with high ethanol productivity. However, the major problem encountered during xylose/glucose co-fermentation is the lower consumption rate of xylose compared with that of glucose fermentation. Here, we therefore attempted to construct high xylose assimilation yeast by using industrial yeast strain with high beta-glucosidase activity on the cell surface. We first constructed the triple auxotrophic industrial strain OC2-HUT and introduced four copies of the cell-surface-displaying beta-glucosidase (BGL) gene and two copies of a xylose-assimilating gene into its genome to generate strain OC2-ABGL4Xyl2. It was confirmed that the introduction of multiple copies of the BGL gene increased the cell-surface BGL activity, which was also correlated to the observed increase in xylose-assimilating ability. The strain OC2-ABGL4Xyl2 was able to consume xylose during cellobiose/xylose co-fermentation (0.38 g/h/g-DW) more rapidly than during glucose/xylose co-fermentation (0.18 g/h/g-DW). After 48 h, 5.77% of the xylose was consumed despite the co-fermentation conditions, and the observed ethanol yield was 0.39 g-ethanol/g-total sugar. Our results demonstrate that a BGL-displaying and xylose-assimilating industrial yeast strain is capable of efficient xylose consumption during the co-fermentation with cellobiose. Due to its high performance for fermentation of mixtures of cellobiose and xylose, OC2-ABGL4Xyl2 does not require the addition of beta-glucosidase and is therefore a promising yeast strain for cost-effective ethanol production from lignocellulosic biomass.

    SPRINGER, 2010年08月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87 (5), 1975 - 1982, 英語

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    研究論文(学術雑誌)

  • Shinji Hama, Ayumi Yoshida, Kazunori Nakashima, Hideo Noda, Hideki Fukuda, Akihiko Kondo

    The cell surface engineering system, in which functional proteins are genetically displayed on microbial cell surfaces, has recently become a powerful tool for applied biotechnology. Here, we report on the surfactant modification of surface-displayed lipase to improve its performance for enzymatic synthesis reactions. The lipase activities of the surfactant-modified yeast displaying Rhizopus oryzae lipase (ROL) were evaluated in both aqueous and nonaqueous systems. Despite the similar lipase activities of control and surfactant-modified cells in aqueous media, the treatment with nonionic surfactants increased the specific lipase activity of the ROL-displaying yeast in n-hexane. In particular, the Tween 20-modified cells increased the cell surface hydrophobicity significantly among a series of Tween surfactants tested, resulting in 8-30 times higher specific activity in organic solvents with relatively high log P values. The developed cells were successfully used for the enzymatic synthesis of phospholipids and fatty acid methyl esters in n-hexane, whereas the nontreated cells produced a significantly low yield. Our results thus indicate that surfactant modification of the cell surface can enhance the potential of the surface-displayed lipase for bioconversion.

    SPRINGER, 2010年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87 (2), 537 - 543, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Syun-ichi Yamakawa, Ryosuke Yamada, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    We successfully demonstrated batch ethanol fermentation repeated ten times from raw starch with high ethanol productivity. We constructed a yeast diploid strain coexpressing the maltose transporter AGT1, alpha-amylase, and glucoamylase. The introduction of AGT1 allows maltose and maltotriose fermentation as well as the improvement of amylase activities. We also found that alpha-amylase activity during fermentation was retained by the addition of 10 mM calcium ion and that the highest alpha-amylase activity was 9.26 U/ml during repeated fermentation. The highest ethanol productivity was 2.22 g/l/h at the fourth batch, and after ten cycles, ethanol productivity of more than 1.43 g/l/h was retained, as was alpha-amylase activity at 6.43 U/ml.

    SPRINGER, 2010年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87 (1), 109 - 115, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shota Togawa, Jun Ishii, Atsushi Ishikura, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    N-linked oligosaccharides or asparagine residues are often involved in G protein-coupled receptor functions. Focusing on Asn13 and Asn26 positioned on N-linked glycosylation motifs in the amino-terminal domain of human somatostatin receptor subtype-5 (hSSTR5), we performed site-directed mutagenesis and evaluated the mutants by using yeast cells as the host strain. This is because analysing the complicated signalling in mammalian cell lines is simplified by the utilization of the monopolistic pheromone signalling pathway in yeast. Western blot analysis and confocal laser scanning microscope observation showed that Asn13 and/or Asn26 mutations had no effects on cell-surface expression of hSSTR5 in yeast. By using an engineered yeast strain of Saccharomyces cerevisiae, which induces the expression of the green fluorescent protein (GFP) reporter gene in response to the agonist-specific signal transduction, it was demonstrated that a single mutation of two asparagine residues attenuated the somatostatin-specific signalling levels, and the double mutant significantly lost the signalling ability. These results clearly show the importance of these asparagine residues in the agonist-specific signalling of hSSTR5, although it was not enough to identify the consequence of oligosaccharides.

    OXFORD UNIV PRESS, 2010年06月, JOURNAL OF BIOCHEMISTRY, 147 (6), 867 - 873, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yusuke Iguchi, Jun Ishii, Hideki Nakayama, Atsushi Ishikura, Keiko Izawa, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo

    The yeast Saccharomyces cerevisiae is known as an available host for human G-protein-coupled receptor (GPCR) ligand screening. Although several types of yeast signal sequences (SS) attached with the GPCRs could improve their productivities and facilitate transportation of the GPCRs to the yeast plasma membrane, the effects of additional SS on ligand-specific signalling functions of GPCRs are not reported. Here, we demonstrated the controlling signalling properties by addition of SS using engineered yeast as a host. Prepro and pre regions of alpha-factor and amino-terminal sequence of Ste2 (Ste2N) were used as SS, and somatostatin (SST) receptor subtype-5 (SSTR5) was used as a model GPCR. We also constructed a yeast-based fluorescent assay system for monitoring the activation levels of SSTR5 signalling by a green fluorescent protein (GFP) reporter gene. The production levels and localisation patterns of the SS-attached SSTR5 were more significantly improved than those of wild-type SSTR5. In addition, we successfully controlled the pharmacological efficacy and potency by introducing SS. Among four types of SSTR5 receptors, Ste2N-SSTR5 responded at the lowest ligand concentration. This finding will be informative for constructing optimal yeast-based ligand screening systems to discriminate the cells on the basis of signalling levels.

    OXFORD UNIV PRESS, 2010年06月, JOURNAL OF BIOCHEMISTRY, 147 (6), 875 - 884, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shuhei Yanase, Ryosuke Yamada, Shohei Kaneko, Hideo Noda, Tomohisa Hasunuma, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and beta-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.

    WILEY-V C H VERLAG GMBH, 2010年05月, BIOTECHNOLOGY JOURNAL, 5 (5), 449 - 455, 英語

    [査読有り]

    研究論文(学術雑誌)

  • KOTAKA, A, SAHARA, H, KURODA, K, KONDO Akihiko, UEDA, M, HATA, Y

    We determined the genetic background that would result in a more optimal display of heterologously expressed p-glucosidase (BGL) on the cell surface of yeast Saccharomyces cerevisiae. Amongst a collection of 28 strains carrying deletions in genes for glycosylphosphatidyl inositol (GPI)-anchored proteins, the Delta sed1 and Delta tos6 strains had significantly higher BGL-activity whilst maintaining wild type growth. Absence of Sed1p, which might facilitate incorporation of anchored BGL on the cell-surface, could also influence the activity of BGL on the cell surface with the heterologous gene being placed under the control of the SED1 promoter. For the evaluation of its industrial applicability we tested this system in heterologous and homogenous SED1-disruptants of sake yeast, a diploid S. cerevisiae strain, in which either the SED1 ORF or the complete gene including the promoter was deleted by use of the high-efficiency loss of heterozygosity method. Evaluation of disruptants displaying BGL showed that deletion of the SED1 ORF enhanced BGL activity on the cell surface, while additional deletion of the SED1 promoter increased further BGL activity on the cell surface. Compared to heterozygous disruption, homozygous disruption resulted generally in a higher BGL activity. Thus, homozygous deletion of both SED1 gene and promoter resulted in the most efficient display of BGL reaching a 1.6-fold increase of BGL-activity compared to wild type. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2010年05月, Journal of Bioscience and Bioengineering, 109 (5), 442 - 446, 英語

    [査読有り]

    研究論文(学術雑誌)

  • YAMADA Ryosuke, TANIGUCHI, N, TANAKA Tsutomu, OGINO Chiaki, FUKUDA Hideki, KONDO Akihiko

    Background: The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as beta-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail delta-integration. Results: In cocktail delta-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail delta-integrant strain was about half that of a conventional delta-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression. Conclusions: To our knowledge, this is the first report on the expression of cellulase genes by delta-integration and optimization of various foreign genes by delta-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.

    BIOMED CENTRAL LTD, 2010年05月, Microbial Cell Factories, 9, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Nobuo Fukuda, Jun Ishii, Tsutomu Tanaka, Akihiko Kondo

    We have developed a new approach based on the G gamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established G gamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein c subunit (G gamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered G gamma that lacks membrane localization upon deletion of the lipid modi. cation site (G gamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with G gamma(cyto) are expressed. As protein-protein interactions bring G gamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-G gamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced G gamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.

    WILEY-BLACKWELL PUBLISHING, INC, 2010年04月, FEBS Journal, 277 (7), 1704 - 1712, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yusuke Miyachi, Nobuaki Shimizu, Chiaki Ogino, Akihiko Kondo

    Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an 'AFM-SELEX' cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.

    OXFORD UNIV PRESS, 2010年03月, NUCLEIC ACIDS RESEARCH, 38 (4), 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tomohisa Hasunuma, Kazuo Harada, Shin-Ichi Miyazawa, Akihiko Kondo, Eiichiro Fukusaki, Chikahiro Miyake

    Understanding of the control of metabolic pathways in plants requires direct measurement of the metabolic turnover rate. Sugar phosphate metabolism, including the Calvin cycle, is the primary pathway in C-3 photosynthesis, the dynamic status of which has not been assessed quantitatively in the leaves of higher plants. Since the flux of photosynthetic carbon metabolism is affected by the CO2 fixation rate in leaves, a novel in vivo C-13-labelling system was developed with (CO2)-C-13 for the kinetic determination of metabolic turnover that was the time-course of the C-13-labelling ratio in each metabolite. The system is equipped with a gas-exchange chamber that enables real-time monitoring of the CO2 fixation rate and a freeze-clamp that excises a labelled leaf concurrently with quenching the metabolic reactions by liquid nitrogen within the photosynthesis chamber. Kinetic measurements were performed by detecting mass isotopomer abundance with capillary electrophoresis-tandem mass spectrometry. The multiple reaction monitoring method was optimized for the determination of each compound for sensitive detection because the amount of some sugar phosphates in plant cells is extremely small. Our analytical system enabled the in vivo turnover of sugar phosphates to be monitored in fresh tobacco (Nicotiana tabacum) leaves, which revealed that the turnover rate of glucose-1-phosphate (G1P) was significantly lower than that of other sugar phosphates, including glucose-6-phosphate (G6P). The pool size of G1P is 12 times lower than that of G6P. These results indicate that the conversion of G6P to G1P is one of the rate-limiting steps in the sugar phosphate pathway.

    OXFORD UNIV PRESS, 2010年03月, JOURNAL OF EXPERIMENTAL BOTANY, 61 (4), 1041 - 1051, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Ken Horii, Takashi Adachi, Takanori Tanino, Tsutomu Tanaka, Atsushi Kotaka, Hiroshi Sahara, Tsuneharu Hashimoto, Nobuyuki Kuratani, Seiji Shibasaki, Chiaki Ogino, Hideo Noda, Yoji Hata, Mitsuyoshi Ueda, Akihiko Kondo

    We used cutinase from the filamentous fungi Aspergillus oryzae to produce dairy flavors. Secretory and displayed forms of cutinase were investigated using salt-free butter, which is composed mostly of triacylglycerides, as the substrate. The secretory form of cutinase, which was produced in recombinant A. oryzae, was suitable for producing butyric acids (16.8 mol%). Also, cutinase displayed on the cell surface of the yeast Saccharomyces cerevisiae as a fusion protein with alpha-agglutinin released butyric acid at a 2.7-fold rate (45.4 mol%) higher than that of the secreted form. Yeasts carrying two copies of cutinase genes into their chromosomes, which were constructed using the HELOH method, released free fatty acids rapidly and showed 2-fold higher lipase activity compared with yeasts carrying one copy of the cutinase gene. (C) 2010 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2010年03月, ENZYME AND MICROBIAL TECHNOLOGY, 46 (3-4), 194 - 199, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Novel strategy for yeast construction using δ-integration and cell fusion to efficiently produce ethanol from raw starch

    YAMADA, Ryosuke, TANAKA, Tsutomu, OGINO, Chiaki, FUKUDA, Hideki, KONDO, Akihiko

    2010年02月, Applied Microbiology and Biotechnology,, 85, 1491-1498, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shogo Arai, Kazunori Nakashima, Takanori Tanino, Chiaki Ogino, Akihiko Kondo, Hideki Fukuda

    The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wildtype Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF(4)] or [Bmim][BF(4)] biphasic systems following a 24h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted monoglyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF(4)] for 72 h. The stability of w-ROL in [Bmim][BF(4)] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present Study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts. (C) 2009 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2010年01月, ENZYME AND MICROBIAL TECHNOLOGY, 46 (1), 51 - 55, 英語

    [査読有り]

    研究論文(学術雑誌)

  • OKANO, K, ZHANG, Q, YOSHIDA, Shogo, TANAKA, Tsutomu, OGINO, Chiaki, FUKUDA, Hideki, KONDO, Akihiko

    In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (a dagger ldhL1) bacterial strain. CelA expression and its degradation of beta-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting a dagger ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley beta-glucan was carried out with the addition of Aspergillus aculeatus beta-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from beta-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.

    SPRINGER, 2010年01月, Applied Microbiology and Biotechnology, 85 (3), 643 - 650, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tsutomu Tanaka, Shinsuke Masunari, Jun Ishii, Kanako Wakamura, Maiko Segawa, Hideki Fukuda, Akihiko Kondo

    Here we expand the yeast cell surface display system to display non-natural, functional molecules. The short biotin acceptor peptide (BAP) sequence of biotin ligase from E. coli(BirA) was genetically introduced to the N-terminus of the anchor protein, Flo428. Through co-expression of BAP-fused Flo428 with BirA, biotinylated BAP could be displayed on the yeast cell surface. Subsequent addition of streptavidin-FITC resulted in the display of streptavidin-FITC, and, the display of biotin-FITC was successful using streptavidin as a linker. Our strategy provides a powerful tool for displaying functional molecules on yeast cell surfaces. (c) 2009 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2010年01月, JOURNAL OF BIOTECHNOLOGY, 145 (1), 79 - 83, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kenji Okano, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed.

    SPRINGER, 2010年01月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85 (3), 413 - 423, 英語

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    研究論文(学術雑誌)

  • Tomonori Ito, Atsushi Hotta, Atsuko Uchida, Takanori Tanino, Chiaki Ogino, Akihiko Kondo, Naoto Ohmura

    Glucose and xylose are major component of the lignocellulosic biomass, and the efficient utilization of not only glucose but also xylose has been required for large-scale ethanol production. In order to establish a highly efficient bio-ethanol production process, this study tried to enhance xylose asimilation ability by introducing the metabolic pathway. Xylose isomerase (XI) pathway (called Pathway I in this study) was selected as a xylose metabolic pathway, and introduced into Saccharomyces cerevisiae genome by integration of XI expression cassette. The resulting recombinant yeast (MT8-1/XKδXI) attained successfully production of ethanol from not only xylose as the sole carbon source but also mixed sugar of xylose and glucose. The xylose consumption rate of the fermentation from xylose was estimated to be a second-order reaction model, and from mixed sugars was estimated to be a first-order reaction model, respectively. These results were expected to be a glucose effect on fermentation from xylose. Copyright © 2010 AIDIC Servizi S.r.l.

    Italian Association of Chemical Engineering - AIDIC, 2010年, Chemical Engineering Transactions, 20, 103 - 108, 英語

    [査読有り]

    研究論文(国際会議プロシーディングス)

  • Tomonori Ito, Atsushi Hotta, Atsuko Uchida, Takanori Tanino, Chiaki Ogino, Akihiko Kondo, Naoto Ohmura

    Glucose and xylose are major component of the lignocellulosic biomass, and the efficient utilization of not only glucose but also xylose has been required for large-scale ethanol production. In order to establish a highly efficient bio-ethanol production process, this study tried to enhance xylose asimilation ability by introducing the metabolic pathway. Xylose isomerase (XI) pathway (called Pathway I in this study) was selected as a xylose metabolic pathway, and introduced into Saccharomyces cerevisiae genome by integration of XI expression cassette. The resulting recombinant yeast (MT8-1/XK delta XI) attained successfully production of ethanol from not only xylose as the sole carbon source but also mixed sugar of xylose and glucose. The xylose consumption rate of the fermentation from xylose was estimated to be a second-order reaction model, and from mixed sugars was estimated to be a first-order reaction model, respectively. These results were expected to be a glucose effect on fermentation from xylose.

    AIDIC SERVIZI SRL, 2010年, IBIC2010: 2ND INTERNATIONAL CONFERENCE ON INDUSTRIAL BIOTECHNOLOGY, 20, 103 - 108, 英語

    [査読有り]

    研究論文(国際会議プロシーディングス)

  • Kenji Okano, Shogo Yoshida, Ryosuke Yamada, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    The production of optically pure D-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose L-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of D-lactic acid was produced from 50 g/liter of xylose.

    AMER SOC MICROBIOLOGY, 2009年12月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75 (24), 7858 - 7861, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Evaluation of the Biodegradability of Polyurethane and Its Derivatives by Using Lipase-Displaying Arming Yeast

    Seiji Shibasaki, Al Kawabata, Takanori Tanino, Akihiko Kondo, Mitsuyoshi Ueda, Mamoru Tanaka

    Candida antarctica lipase B (CALB) has been used to polymerize and degrade polyesters. We developed a convenient method for Investigating the biodegradability of plastics that involves the use of CALB-displaying "arming yeast." Polyurethane containing dulcitol units was prepared and used as the model material. Additionally, standard polyurethane with no dulcitol units was prepared by reacting 2,4-toluene dilsocyanate with ethylene glycol. These polymers were incubated with CALB-displaying yeast cells. The polyurethane containing dulcitol was degraded, while the standard polyurethane was relatively unaffected. Arming yeast displaying appropriate enzymes can be used to investigate the biodegradability of synthetic plastics. It was also revealed that arming yeasts were applicable to evaluate the degradation of the film state of polyurethane.

    SOC ANTIBACTERIAL & ANTIFUNGAL AGENTS, JAPAN, 2009年12月, BIOCONTROL SCIENCE, 14 (4), 171 - 175, 英語

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    研究論文(学術雑誌)

  • Takuya Shishido, Yuki Azumi, Takeshi Nakanishi, Mitsuo Umetsu, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Bionanocapsule (BNC) is hollow nanoparticle composed of the l-protein of the hepatitis B virus surface antigen. BNC allows targeted delivery of either genes or drugs only to hepatocytes, but not to other cell types. In this study, we attempted to alter the specificity of BNC by insertion of biotin-acceptor peptide (BAP), which is efficiently biotinylated using biotin ligase BirA from Escherichia coli. Using streptavidin as a linker, biotinylated BNC could be display various biotinylated ligands that are otherwise difficult to fuse with BNC, such as antibodies, synthetic peptides and functional molecules. BAP-fused BNC was efficiently biotinylated and effectively displayed streptavidin. Furthermore, we demonstrated that biotinylated BNC was internalized into targeted cells via biotinylated Nanobody displayed on the BNC surface. Biotinylated BNC permit display of diverse ligands, and thus have potential as a versatile carrier for drug delivery to a variety of target cells.

    OXFORD UNIV PRESS, 2009年12月, JOURNAL OF BIOCHEMISTRY, 146 (6), 867 - 874, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108 (1), S36 - S36, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hiroshi Sahara, Atsushi Kotaka, Akihiko Kondo, Mitsuyoshi Ueda, Yoji Hata

    By application of the high-efficiency loss of heterozygosity (HELOH) method for disrupting genes in diploid sake yeast (Kotaka et al., Appl. Microbiol. Biotechnol., 82, 387-395 (2009)), we constructed, from a beterozygous integrant, a homozygous diploid that overexpresses the alcohol acetyltransferase gene ATF2 from the SEDI promoter, without the need for sporulation and mating. Under the conditions of sake brewing, the homozygous integrant produced 1.4 times more isoamyl acetate than the parental, heterozygous strain. Furthermore, the homozygous integrant was more genetically stable than the heterozygous recombinant. Thus, the HELOH method can produce homozygous, recombinant sake yeast that is ready to be grown on an industrial scale using the well-established procedures of sake brewing. The HELOH method, therefore, facilitates genetic modification of this rarely sporulating diploid yeast strain while maintaining those characteristics required for industrial applications. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108 (5), 359 - 364, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takanori Tanino, Takumi Ohm, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    The effect of the reaction conditions on the ester synthesis reaction with CALB displaying yeast whole cells was determined. Utilization of hydrophobic organic solvent improved the efficiency of the ester synthesis reaction. Also the initial water content was important for the expression of the ester synthesis activity of CALB displaying yeast whole cells. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108 (5), 369 - 371, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Dimensionality reduction using PCA, PLS, OPLS, and RFDA with differential penalty to latent variables

    YAMAMOTO Hiroyuki, YAMAJI Hideki, ABE Yuichiro, HARADA Kazuo, WALUYO Danang, FUKUSAKI Eiichiro, KONDO Akihiko, Ohno Hiromu, FUKUDA Hideki

    2009年10月, Chemometrics and Intelligent Laboratory Systems, Vol. 98, No. 2, pp. 136-142, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kenro Tokuhiro, Masayoshi Muramatsu, Chikara Ohto, Toshiya Kawaguchi, Shusei Obata, Nobuhiko Muramoto, Masana Hirai, Haruo Takahashi, Akihiko Kondo, Eiji Sakuradani, Sakayu Shimizu

    (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter(-1)) rather than GGOH (0.2 mg liter(-1)) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter(-1) GGOH and 6.5 mg liter(-1) squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter(-1) GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

    AMER SOC MICROBIOLOGY, 2009年09月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75 (17), 5536 - 5543, 英語

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    研究論文(学術雑誌)

  • Toshihiro Tateno, Kazuki Hatada, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

    We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used alpha-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum.

    SPRINGER, 2009年09月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 84 (4), 733 - 739, 英語

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    研究論文(学術雑誌)

  • HORII, Ken, ADACHI, T, MATSUDA, T, TANAKA, Tsutomu, SAHARA, H, SHIBASAKI, S, OGINO, Chiaki, HATA, Y, UEDA, M, KONDO, Akihiko

    beta-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than P-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides. (C) 2008 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2009年08月, Journal of Molecular Catalysis B: ,, 59 (4), 297 - 301, 英語

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    研究論文(学術雑誌)

  • Kenji Okano, Shogo Yoshida, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Optically pure D-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose L-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of D-lactic acid was produced from 50 g/liter of arabinose.

    AMER SOC MICROBIOLOGY, 2009年08月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75 (15), 5175 - 5178, 英語

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    研究論文(学術雑誌)

  • Ken Horii, Takashi Adachi, Takanori Tanino, Tsutomu Tanaka, Hiroshi Sahara, Seiji Shibasaki, Chiaki Ogino, Yoji Hata, Mitsuyoshi Ueda, Akihiko Kondo

    A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed beta-glucosidase resisted pepsin digestion compared with secreted, free beta-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted beta-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed beta-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted beta-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed beta-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.

    SPRINGER, 2009年08月, BIOTECHNOLOGY LETTERS, 31 (8), 1259 - 1264, 英語

    [査読有り]

    研究論文(学術雑誌)

  • ZHOU, Y, Kajiyama, S, ITOh, K, TANINO, T, FUKUDA, Nobuo, TANAKA, Tsutomu, KONDO, Akihiko

    Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying beta-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.

    SPRINGER, 2009年08月, Applied Microbiology and Biotechnology,, 84 (2), 375 - 382, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yusuke Miyachi, Nobuaki Shimizu, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    DNA aptamers, which bind specific molecule, such as 8-OHdG, with high affinity were investigated using an in vitro selection strategy called systematic evolution of ligands by exponential enrichment (SELEX). However, 8-OHdG was difficult to immobilize on a carrier for SELEX. Therefore, a DNA aptamer binding to 8-OHdG was selected using GMP-agarose as an analogue from a library of about 4(60) random ssDNA sources. As a result, three aptamer candidates were selected. Among the selected DNA aptamers, the No. 22 DNA aptamer exhibited a high affinity for 8-OHdG. The dissociation constant, K(D), of No. 22 DNA aptamer was on the order of 0.1 mu mol/L. This result suggests that using an analogue will be a useful new SELEX method for obtaining various aptamers that are difficult to immobilize on a matrix. (C) 2009 Elsevier Ltd. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2009年07月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 19 (13), 3619 - 3622, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Takao Numata, Sriappareddy Tamalampudi, Ayumi Yoshida, Hideo Noda, Akihiko Kondo, Hideki Fukuda

    The accumulation of partial glycerides such as monoglyceride (MG) and diglyceride (DG) is one of the rate-limiting steps in plant oil methanolysis catalyzed by Rhizopus oryzae producing triacylglycerol lipase. To convert partial glycerides efficiently into their corresponding methyl esters (MEs), we attempted to use a mono- and diacylglycerol lipase (mdIB) derived from Aspergillus oryzae. By considering cost efficiency, R. oryzae and recombinant mdIB-producing A. oryzae were immobilized independently within polyurethane foam biomass support particles and directly utilized as a whole-cell biocatalyst. The mdIB-producing A. oryzae effectively exhibited Substrate specificity toward MG and DG and was then used for the methanolysis of intermediate products (approximately 82% ME), which were produced using R. oryzae. In the presence of 5% water, the use of mdIB-producing A. oryzae resulted in less than 0.1% of MG and DG. whereas a considerable amount of triglyceride was present in the final reaction mixture. On the basis of these results, we developed a packed-bed reactor (PBR) system, which consists of the first column with R. oryzae and the second column containing both R. oryzae and mdIB-producing A. oryzae. Ten repeated-batch methanolysis cycles in the PBR maintained a high ME content of over 90% with MG and DC at 0.08-0.69 and 0.22-1.45%, respectively, indicating that the PBR system can be used for long-term repeated-batch methanolysis with partial glycerides at low levels. The proposed method is therefore effective for improving enzymatic biodiesel production. (C) 2008 Published by Elsevier B.V.

    ELSEVIER SCIENCE BV, 2009年06月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 58 (1-4), 93 - 97, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Junji Ito, Akihiko Kosugi, Tsutomu Tanaka, Kouichi Kuroda, Seiji Shibasaki, Chiaki Ogino, Mitsuyoshi Ueda, Hideki Fukuda, Roy H. Doi, Akihiko Kondo

    We constructed a novel cell surface display system to control the ratio of target proteins on the Saccharomyces cerevisiae cell surface, using two pairs of protein-protein interactions. One protein pair is the Z domain of protein A derived from Staphylococcus aureus and the Fc domain of human immunoglobulin G. The other is the cohesin (Coh) and dockerin (Dock) from the cellulosome of Clostridium cellulovorans. In this proposed displaying system, the scaffolding proteins (fusion proteins of Z and Coh) were displayed on the cell surface by fusing with the 3' half of alpha-agglutinin, and the target proteins fused with Fc or Dock were secreted. As a target protein, a recombinant Trichoderma reesei endoglucanase II (EGII) was secreted into the medium and immediately displayed on the yeast cell surface via the Z and Fc domains. Display of EGII on the cell surface was confirmed by hydrolysis of beta-glucan as a substrate, and EGII activity was detected in the cell pellet fraction. Finally, two enzymes, EGII and Aspergillus aculeatus beta-glucosidase 1, were codisplayed on the cell surface via Z-Fc and Dock-Coh interactions, respectively. As a result, the yeast displaying two enzymes hydrolyzed beta-glucan to glucose very well. These results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.

    AMER SOC MICROBIOLOGY, 2009年06月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75 (12), 4149 - 4154, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shohei Kaneko, Tsutomu Tanaka, Hideo Noda, Hideki Fukuda, Rinji Akada, Akihiko Kondo

    The introduction of several kinds of genes into the yeast chromosome is a powerful tool in many fields from fundamental study to industrial application. Here, we describe a general strategy for one-step gene integration and a marker recycling method. Forty base pairs of a short sequence derived from a region adjacent to the HIS3 locus were placed between cell surface displaying beta-glucosidase (BGL) and URA3 marker genes. HIS3 deletion and BGL-URA3 fragment integration were achieved via a PCR fragment consisting of the BGL-URA3 fragment attached to homology sequences flanked by the HIS3 targeting locus. The obtained his3::URA3 disruptants were plated on a 5-FOA plate to select for the URA3 deletion due to repeated sequences at both sides of URA3 gene. In all selected colonies, BGL genes were integrated at the targeted HIS3 locus and URA3 was completely deleted. In addition, introduced BGL was efficiently expressed, and the transformants fermented cellobiose to ethanol effectively. As our strategy creates next transformation markers continuously together with gene integration, this method can serve as a simple and powerful tool for multiple genetic manipulations in yeast engineering.

    SPRINGER, 2009年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 83 (4), 783 - 789, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Jun Ishii, Keiko Izawa, Shizuka Matsumura, Kanako Wakamura, Takanori Tanino, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.

    OXFORD UNIV PRESS, 2009年06月, JOURNAL OF BIOCHEMISTRY, 145 (6), 701 - 708, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Akihiko Kosugi, Akihiko Kondo, Mitsuyoshi Ueda, Yoshinori Murata, Pilanee Vaithanomsat, Warunee Thanapase, Takamitsu Arai, Yutaka Mori

    Cassava (Manihot esculenta Crantz) pulp, produced in large amounts as a by-product of starch manufacturing, is a major biomass resource in Southeast Asian countries. It contains abundant starch (approximately 60%) and cellulose fiber (approximately 20%). To effectively utilize the cassava pulp, an attempt was made to convert its components to ethanol using a sake-brewing yeast displaying glucoamylase on the cell surface. Saccharomyces cerevisiae Kyokai no. 7 (strain K7) displaying Rhizopus oryzae glucoamylase, designated strain K7G, was constructed using the C-terminal-half region of at-agglutinin. A sample of cassava pulp was pretreated with a hydrothermal reaction (140 degrees C for 1 h), followed by treatment with a Trichoderma reesei cellulase to hydrolyze the cellulose in the sample. The K7G strain fermented starch and glucose in pretreated samples without addition of amylolytic enzymes, and produced ethanol in 91% and 80% of theoretical yield from 5% and 10% cassava pulp, respectively. (c) 2008 Elsevier Ltd. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2009年05月, RENEWABLE ENERGY, 34 (5), 1354 - 1358, 英語

    [査読有り]

    研究論文(学術雑誌)

  • YAMADA, Ryosuke, BITO, Y, ADACHI, T, TANAKA, Tsutomu, OGINO, Chiaki, FUKUDA, Hideki, KONDO, Akihiko

    The goal of this research was to construct a stable and efficient process for the production of ethanol from raw starch, using a recombinant Saccharomyces cerevisiae. which is productive even under conditions such as non-selection or long-term operation. Three recombinant yeast strains were used, two haploid strains (MT8-1SS and NBRC1440SS) and one diploid strain (MN8140SS). The recombinant strains were constructed by integrating the glucoamylase gene from Rhizopus oryzae fused with the 3'-half of the alpha-agglutinin gene as the anchor protein, and the alpha-amylase gene from Streptococcus bovis, respectively, into their chromosomal DNA by homologous recombination. The diploid strain MN8140SS was constructed by mating these opposite types of integrant haploid strains in order to enhance the expression of integrated amylase genes. The diploid strain had the highest ethanol productivity and reusability during fermentation from raw starch. Moreover, the ethanol production rate of the integrant diploid strain was maintained when batch fermentation was repeated three times (0.67. 0.60, and 0.67 g/l/h in each batch). These results clearly show that a diploid strain developed by mating two integrant haploid strains is useful for the establishment of an efficient ethanol production process. (C) 2009 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2009年05月, Enzyme and Microbial Technology, 44 (5), 344 - 349, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Construction of a novel detection system for protein-protein interactions using yeast G-protein signaling

    Nobuo Fukuda, Jun Ishii, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

    2009年05月, FEBS Journal, 276, 2636 - 2644, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Xiaomao Xie, Noriyuki Ohnishi, Yuki Takahashi, Akihiko Kondo

    The magnetic nanoparticles (MNPs) Therma-Max (TM) were used as a carrier to develop an automated sandwich chemiluminescent enzyme immunoassay (CLEIA) to detect thyroid-stimulating hormone (TSH) in a sensitive and specific way. The Therma-Max (TM) particles allow for automation because, unlike magnetic microspheres, they are completely dispersed in aqueous solution and allow for accurate automatic handling. Signal intensities detected with MNPs were 8-fold higher than those found with conventional micron-sized magnetic particles. A reproducibility study suggests that these particles allow for a stable detection method, as the coefficient of variation(CV) is less than 6% (n = 10). (C) 2009 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2009年05月, JOURNAL OF MAGNETISM AND MAGNETIC MATERIALS, 321 (10), 1686 - 1688, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Shishido, Naoya Kurata, Myung Eui Yoon, Tsutomu Tanaka, Hideki Yamaji, Hideki Fukuda, Akihiko Kondo

    Insect cell expression systems are widely used to produce active recombinant proteins. Here, we have developed a high-level expression vector containing a selectable marker for continuous production of recombinant proteins in insect cells. The plasmid, pXIHAbla, developed in this study, established a polyclonal cell line 8 days shorter than pXINSECT-DEST38 and pBmAneo. In addition, pXIHAbla exhibited an approximately fivefold higher average enhanced GFP expression level and approximately a twofold higher bionanocapsule secretion level than pXINSECT-DEST38. Using this plasmid, insect cells that highly express active proteins have been easily established.

    SPRINGER, 2009年05月, BIOTECHNOLOGY LETTERS, 31 (5), 623 - 627, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Anjali Madhavan, Sriappareddy Tamalampudi, Kazunari Ushida, Daisuke Kanai, Satoshi Katahira, Aradhana Srivastava, Hideki Fukuda, Virendra S. Bisaria, Akihiko Kondo

    The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37 degrees C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.

    SPRINGER, 2009年04月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82 (6), 1067 - 1078, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Kenro Tokuhiro, Nobuhiro Ishida, Eiji Nagamori, Satoshi Saitoh, Toru Onishi, Akihiko Kondo, Haruo Takahashi

    Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.

    SPRINGER, 2009年04月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82 (5), 883 - 890, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hideki Fukuda, Akihiko Kondo, Sriappareddy Tamalampudi

    The dependency on depleting natural resources is a challenge for energy security that can be potentially answered by bioenergy. Bioenergy is derived from starchy and lignocellulosic biomass in the form of bioethanol or from vegetable oils in the form of biodiesel fuel. The acid and enzymatic methods have been developed for the hydrolysis of biomass and for transesterification of plant oils. However, acid hydrolysis results in the production of unnatural compounds which have adverse effects on yeast fermentation. Recent advancements in the yeast cell surface engineering developed strategies to genetically immobilize amylolytic. cellulolytic and xylanolytic enzymes on yeast cell surface for the production of fuel ethanol from biomass. Whereas in the case of biodiesel fuel production, alkali catalysis gives high levels of conversion in short reaction times. But complexity in the separation of produced biodiesel fuel from glycerol by-product led to intensive research on lipase enzyme and immobilized whole-cell biocatalysts. This system facilitates the easy separation of glycerol with the advantage of cost effectiveness. This review gives an insight in to the recent technological developments in the production of bioenergy, i.e., bioethanol and biodiesel fuel using surface engineered yeast and whole-cell biocatalysts. (C) 2008 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2009年04月, BIOCHEMICAL ENGINEERING JOURNAL, 44 (1), 2 - 12, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Anjali Madhavan, Sriappareddy Tamalampudi, Aradhana Srivastava, Hideki Fukuda, Virendra S. Bisaria, Akihiko Kondo

    Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h(-1), while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h(-1). The adapted strain could ferment 20 g l(-1) of xylose to ethanol with a yield of 0.37 g g(-1) and production rate of 0.026 g l(-1) h(-1). Raising the fermentation temperature from 30 degrees C to 35 degrees C resulted in a substantial increase in the ethanol yield (0.43 g g(-1)) and production rate (0.07 g l(-1) h(-1)) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g(-1).

    SPRINGER, 2009年04月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82 (6), 1037 - 1047, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Metabolic pathway engineering by plastid transformation is a powerful tool for production of compounds in higher plants

    Tomohisa Hasunuma, Akihiko Kondo, Chikahiro Miyake

    Plastid transformation is a powerful tool for the production of useful compounds in higher plants through metabolic engineering, because it has many advantages over conventional nuclear transformation: high-level foreign protein accumulation, no need for a transit peptide, absence of gene silencing, and convenient transgene stacking in an operon. Plastid transformation has recently yielded remarkable results in the production of highly valued biopharmaceutical proteins and in conferring herbicide and insect resistance. Metabolic pathway engineering by plastid transformation has also produced higher levels of useful compounds than nuclear transformation. furthermore, recent reports have shown the functional regulation of transgene expression from the plastid genome. In this review, we have focused on the progress of plastid transformation in material production from the aspect of biosynthetic pathway engineering, discussing the issues for future expansion of plastid transformation.

    JAPANESE SOC PLANT CELL & MOLECULAR BIOL, 2009年03月, PLANT BIOTECHNOLOGY, 26 (1), 39 - 46, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Production of ethanol from cassava pulpvia fermentation with a surface-engineered yeast strain displaying glucoamylase.

    KOSUGI A, KONDO Akihiko, UEDA M, MURATA Y, VAITHANOMSAT P, THANAPASE W. ARAI T, MORI Y

    2009年03月, Renewable Energy, Vol. 34, No. 5, pp. 1354-1358, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Shishido, Daisaku Yonezawa, Kiyokazu Iwata, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Bionanocapsule (BNC) is a hollow nanoparticle composed of L-protein of the hepatitis B virus surface antigen. BNC can deliver genes or drugs into specific human hepatocytes, but delivery is limited to hepatocytes. In this study, we attempted to alter the specificity of BNCs by genetically introducing cell-penetrating peptides (CPPs), such as arginine-rich peptides, into BNCs. The CPP-fused BNC was efficiently internalized into various cell lines in a short period without significant cytotoxicity. These results show that CPP-BNC could be applied as an efficient carrier for gene and drug delivery. (C) 2009 Elsevier Ltd. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, 2009年03月, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 19 (5), 1473 - 1476, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takanori Tanino, Tohru Aoki, Won-young Chung, Yuka Watanabe, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    A Candida antarctica lipase B (CALB)-displaying yeast whole-cell biocatalyst was constructed with the integration of the CALB cell-surface display expression cassette in the yeast genome and cell fusion by mating. Lipase hydrolytic activity of the yeast whole-cell biocatalyst subsequently increased, in both a- and alpha-type yeast cells, with the number of copies of the CALB cell-surface display expression cassette introduced, and reached 43.6 and 32.2 U/g-dry cell at 168 h cultivation, respectively. The lipase hydrolytic activity of whole cells in diploid yeast cells containing eight copies of the CALB cell-surface expression cassette reached 117 U/g-dry cell, and this value is approximately ninefold higher than that of the previously reported haploid CALB cell-surface displaying yeast using a multi-copy plasmid (Tanino et al. Appl. Microbial Biotechnol 75:1319-1325, 2007). This improved novel CALB-displaying yeast whole-cell biocatalyst could repeatedly catalyze the polyester, polybutylene adipate, synthesis reaction, using adipic acid and 1, 4-butandiol as the monomer molecules, four times in succession. This is the first report of the polymer synthesis using enzyme displaying yeast as the catalyst. The ratios of cyclic compounds in the polybutylene adipates synthesized with the CALB-displaying yeast whole-cells were lower than that in the polybutylene adipate synthesized with conventional metal catalysis. From these results, it appears that the use of CALB-displaying yeast cells could be useful for the polyester synthesis reaction, with reduced by-product production.

    SPRINGER, 2009年02月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82 (1), 59 - 66, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Atsushi Kotaka, Hiroshi Sahara, Akihiko Kondo, Mitsuyoshi Ueda, Yoji Hata

    Sake yeast, a diploid Saccharomyces cerevisiae strain, is useful for industry but difficult to genetically engineer because it hardly sporulates. Until now, only a few recessive mutants of sake yeast have been obtained. To solve this problem, we developed the high-efficiency loss of heterozygosity (HELOH) method, which applies a two-step gene disruption. First, a heterozygous disruptant was constructed by gene replacement with URA3, followed by marker recycling on medium containing 5-fluoroorotic acid (5-FOA). Subsequently, spontaneous loss of heterozygosity (LOH) yielding a homozygous disruptant was selected for in a second round of gene integration. During this step, the wild-type allele of the heterozygous disruptant was marked by URA3 integration, and the resulting transformants were cultivated in non-selective medium to induce recombination and then grown on medium with 5-FOA to enrich for mutants that had undergone LOH. Although the frequency with which LOH occurs is extremely low, many homozygous disruptants were obtained with the HELOH method. Thus, we were able to efficiently construct homozygous disruptants of diploid sake yeast without sporulation, and sake yeast strains with multiple auxotrophies and a protease deficiency could be constructed. The HELOH method, therefore, facilitated the utilization of diploid sake yeast for genetic engineering purposes.

    SPRINGER, 2009年02月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82 (2), 387 - 395, 英語

    [査読有り]

    研究論文(学術雑誌)

  • TATENO Toshihiro, OKADA Yusuke, TSUCHIDATE Takeyuki, TANAKA Tsutomu, OGINO Chiaki, FUKUDA Hideki, KONDO Akihiko

    Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 alpha-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli-C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5'-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation ofl-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources.

    SPRINGER, 2009年02月, Applied Microbiology and Biotechnology, 82 (1), 115 - 121, 英語

    [査読有り]

    研究論文(学術雑誌)

  • OKANO Kenji, ZHANG Qiao, SHINKAWA Satoru, YOSHIDA Shogo, TANAKA Tsutomu, FUKUDA Hideki, KONDO Akihiko

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.

    AMER SOC MICROBIOLOGY, 2009年01月, Applied and Environmental Microbiology, 75 (2), 462 - 467, 英語

    [査読有り]

    研究論文(学術雑誌)

  • CONSTRUCTION OF A NOVEL DETECTION SYSTEM FOR PROTEIN-PROTEIN INTERACTIONS USING YEAST G-PROTEIN SIGNALING

    Nobuo Fukuda, Jun Ishii, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

    SOUTHEAST UNIV PRESS, 2009年, IFPT'6: PROGRESS ON POST-GENOME TECHNOLOGIES, PROCEEDINGS, 238 - 238, 英語

    [査読有り]

    研究論文(国際会議プロシーディングス)

  • Seiji Shibasaki, Al Kawabata, Takanori Tanino, Akihiko Kondo, Mitsuyoshi Ueda, Mamoru Tanaka

    Candida antarctica lipase B (CALB) has been used to polymerize and degrade polyesters. We developed a convenient method for investigating the biodegradability of plastics that involves the use of CALB-displaying "arming yeast." Polyurethane containing dulcitol units was prepared and used as the model material. Additionally, standard polyurethane with no dulcitol units was prepared by reacting 2,4-toluene dilsocyanate with ethylene glycol. These polymers were incubated with CALB-displaying yeast cells. The polyurethane containing dulcitol was degraded, while the standard polyurethane was relatively unaffected. Arming yeast displaying appropriate enzymes can be used to investigate the biodegradability of synthetic plastics. It was also revealed that arming yeasts were applicable to evaluate the degradation of the film state of polyurethane.

    2009年, Biocontrol Science, 14 (4), 171 - 175, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Naoya Kurata, Takuya Shishido, Masaru Muraoka, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    Bionanocapsules (BNCs) are nanoparticles with a high biocompatibility composed of the L protein of the hepatitis B virus surface antigen. BNC can deliver bioactive molecules to hepatocytes efficiently and specifically. However, delivery is limited to hepatocytes and incorporation of proteins into BNC is quite troublesome. Here, in order to alter the specificity of BNC and to achieve efficient protein delivery, we developed engineered BNC displaying the ZZ domain of protein A and incorporating enhanced green fluorescent protein (EGFP) inside the particles using an insect cell expression system. The ZZ domain displayed on the surface of BNC binds to anti-epidermal growth factor receptor (EGFR) antibodies, allowing specific delivery of EGFP to HeLa cells. The engineered BNCs are a promising and powerful tool for efficient and cell-specific protein delivery.

    OXFORD UNIV PRESS, 2008年12月, JOURNAL OF BIOCHEMISTRY, 144 (6), 701 - 707, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Sriappareddy Tamalampudi, Yuya Suzuki, Ayumi Yoshida, Hideki Fukuda, Akihiko Kondo

    In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn-2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn-2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn-2 to the sn-1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.

    SPRINGER, 2008年12月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 81 (4), 637 - 645, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Akiyoshi Hoshino, Noriyoshi Manabe, Kouki Fujioka, Sanshiro Hanada, Masato Yasuhara, Akihiko Kondo, Kenji Yamamoto

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

    IOP PUBLISHING LTD, 2008年12月, NANOTECHNOLOGY, 19 (49), 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takashi Adachi, Junji Ito, Kouji Kawata, Masahiro Kaya, Hiroki Ishida, Hiroshi Sahara, Yoji Hata, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of beta-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, beta-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.

    SPRINGER, 2008年12月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 81 (4), 711 - 719, 英語

    [査読有り]

    研究論文(学術雑誌)

  • SAITOH S, TANAKA Tsutomu, KONDO Akihiko

    We constructed a double auxotrophic OC-2 industrial diploid strain of Saccharomyces cerevisiae and introduced 4 copies of cell surface displaying beta-glucosidase (BGL) genes into the chromosome. The engineered OC-2 strain showed 5-fold higher BGL activity compared with the yeast carrying 2 copies of BGL gene and directly produced ethanol from cellobiose.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2008年12月, Journal of Bioscience and Bioengineering, 106 (6), 594 - 597, 英語

    [査読有り]

    研究論文(学術雑誌)

  • NAKAMURA Nobuhiro, YAMADA Ryosuke, KITAHARA S, TANAKA Tsutomu, FUKUDA Hideki, KONDO Akihiko

    Co-utilization of several sugars, especially xylose and glucose, is essential for economically feasible processes with high ethanol productivity. However, the majorproblem during xylose/glucose co-fermentation is that xylose is used very slowly until after glucose is completely consumed. Here, we demonstrated an effective co-fermentation process using xylose- and cellobiose-assimilating recombinant Saccharomyces cerevisiae. The recombinant yeast is able to consume xylose during xylose/cellobiose co-fermentation as rapidly as during glucose fermentation. After 72 h, 95.6% of xylose was consumed, despite the co-fermentation conditions, and the ethanol yield was 0.358 g-ethanol/g-total sugar. (C) 2008 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2008年09月, Enzyme and Microbial Technology, 43 (3), 233 - 236, 英語

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    研究論文(学術雑誌)

  • Seiji Shibasaki, Kozue Sakata, Jun Ishii, Akihiko Kondo, Mitsuyoshi Ueda

    A yeast protein fragment complementation assay (PCA) system based on dihydrofolate reductase (DHFR) is difficult to be operated because it is not as sensitive to trimethoprim (TMP) as the system using a prokaryotic microorganism. Here, the PCA system using DHFR, specific inhibitors, and a substrate in the yeast Saccharomyces cerevisiae was newly developed. As a model, the human oncoprotein Ras and the Ras-binding domain (RBD) of Raf-1 were individually and genetically fused to DHFR fragment, and each genetic construct was coexpressed under the control of the GAL1 promoter. An interaction between Ras and RBD could be evaluated on the basis of cell proliferation. To establish the experimental conditions for the yeast PCA system based on the DHFR reconstitution, we examined yeast host strains and the concentration of inhibitory additives to prevent endogenous DHFR activity, namely, TMP and sulfanilamide, and the substrate of DHFR, namely, folic acid. The transformant harboring wild-type Ras or its variants showed positive interaction signals, and the order of interactions for combination corresponded to the results of other in vitro assays. Moreover, combinatorial mutated Ras-binding domains were constructed, and the interaction of RBDs with Ras using this yeast PCA system was examined. As a result, various types of mutated clone for RBD were obtained. These demonstrations suggest that the yeast PCA system based on DHFR can be one of good, convenient, and inexpensive tools for investigating eukaryotic protein-protein interactions in vivo.

    SPRINGER, 2008年09月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 80 (4), 735 - 743, 英語

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    研究論文(学術雑誌)

  • ITO J, SAHARA H, KAYA M, HATA Y, SHIBASAKI S, KAWATA K, ISHIDA S, OGINO Chiaki, FUKUDA Hideki, KONDO Akihiko

    The yeast Saccharomyces cerevisiae GRI-117-UK was transformed to either display or secrete beta-glucosidase 1 (BGL1) from the koji mold, Aspergillus oryzae. The P-glucosidase activity of BGL1-displaying yeast strains reached 405.9 U/g dry cell mass after 72 h of cultivation in YPD medium. The optimal pH and temperature for BGL1 displayed on the cell surfaces of the yeast were 5.0 and 55 degrees C, while the optima for BCL1 secreted by the yeast were 4.0 and 55 degrees C. The displayed BGL1 was stable at higher pH compared with the secreted BGL1. In addition, the thermostability of BGL1 was improved by displaying the enzyme on the yeast cell surfaces. In addition, the displayed and secreted forms of BGL1 had similar substrate specificity. beta-Glucosidase hydrolyzes daidzin and genistin, which are the glycoside forms of soybean isoflavones, to the aglycones. Isoflavone aglycones were efficiently produced by BGL1-displaying yeast from an isoflavone mixture: at optimal temperature and pH the rate of aglycone production was at least 15.8 g/(1h). After 144 h of reaction, almost isoflavones were converted to its aglycone by BGL1-displaying yeast. The results of the present study demonstrate that BGL1-displaying yeast strains are effective whole cell biocatalysts of isoflavone aglycone production. (c) 2008 Elsevier B. V. All rights reserved.

    ELSEVIER SCIENCE BV, 2008年09月, Journal of Molecular Catalysis B, 55 (1-2), 69 - 75, 英語

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    研究論文(学術雑誌)

  • Satoshi Katahira, Meguru Ito, Hisae Takema, Yasuya Fujita, Takanori Tanino, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

    Enhancing the sugar uptake ability of the yeast Saccharomyces cerevisiae is a potentially important factor for efficient ethanol production during fermentation of lignocellulosic biomass. Here, we attempted to express a Pichia stipitis gene encoding a sugar transporter, SUT1, in a xylose-assimilating S. cerevisiae strain that expresses xylose reductase, xylosedehydrogenase and xylulokinase. We next investigated xylose fermentation, glucose fermentation and glucose and xylose co-fermentation using the Sut1-expressing S. cerevisiae strain. Expression of Sut1 in xylose-assimilating S. cerevisiae increased both xylose uptake ability and ethanol productivity during xylose fermentation. Moreover, glucose uptake ability and ethanol productivity during glucose fermentation also increased by expressing of Sut1. The yield of ethanol during xylose and glucose co-fermentation by the Sut1-expressing yeast strain (0.44 g/g-consumed sugar) was significantly higher than that of the parental strain (0.39 g/g-consumed sugar). (C) 2008 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2008年08月, ENZYME AND MICROBIAL TECHNOLOGY, 43 (2), 115 - 119, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Sriappareddy Tamalampudi, Naoki Shindo, Takao Numata, Hideki Yamaji, Hideki Fukuda, Akihiko Kondo

    To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL-green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL-GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.

    SPRINGER, 2008年07月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 79 (6), 1009 - 1018, 英語

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    研究論文(学術雑誌)

  • TOKUHIRO K, ISHIDA N, KONDO Akihiko, TAKAHASHI H

    The Aspergillus aculeatus beta-glucosidase 1 (bgl1) gene was expressed in a lactic-acid-producing Saccharomyces cerevisiae strain to enable lactic fermentation with cellobiose. The recombinant beta-glucosidase enzyme was expressed on the yeast cell surface by fusing the mature protein to the C-terminal half region of the alpha-agglutinin. The beta-glucosidase expression plasmids were integrated into the genome. Three strong promoters of S. cerevisiae, the TDH3, PGK1, and PDC1 promoters, were used for beta-glucosidase expression. The specific beta-glucosidase activity varied with the promoter used and the copy number of the bgl1 gene. The highest activity was obtained with strain PB2 that possessed two copies of the bgl1 gene driven by the PDC1 promoter. PB2 could grow on cellobiose and glucose minimal medium at the same rate. Fermentation experiments were conducted in non-selective-rich media containing 95 g l(-1) cellobiose or 100 g l(-1) glucose as a carbon source under microaerobic conditions. The maximum rate of L-lactate production by PB2 on cellobiose (2.8 g l(-1) h(-1)) was similar to that on glucose (3.0 g l(-1) h(-1)). This indicates that efficient fermentation of cellobiose to L-lactate can be accomplished using a yeast strain expressing beta-glucosidase from a mitotically stable genomic integration plasmid.

    SPRINGER, 2008年06月, Applied Microbiology and Biotechnology, 79 (3), 481 - 488, 英語

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    研究論文(学術雑誌)

  • KOTAKA A, BANDO H, KAYA M, KATO M, KURODA K, SAHARA H, HATA Y, KONDO Akihiko, UEDA M

    Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucasidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GR1-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results; showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2008年06月, Journal of Bioscience and Bioengineering, 105 (6), 622 - 627, 英語

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    研究論文(学術雑誌)

  • Yeast-based fluorescence reporter assay of G protein-coupled receptor signaling for flow cytometric screening: FAR1-disruption recovers loss of episomal plasmid caused by signaling in yeast

    Jun Ishii, Tsutomu Tanaka, Shizuka Matsumura, Kenji Tatematsu, Shunichi Kuroda, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo

    2008年05月, Journal of Biochemistry, Vol 143, pp. 667-674, 英語

    [査読有り]

    研究論文(学術雑誌)

  • KAYA M, ITO J, KOTAKA A, MATSUMURA K, BANDO H, SAHARA H, OGINO Chiaki, SHIBASAKI S, KURODA K, UEDA M, KONDO Akihiko, HATA Y

    For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of beta-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with alpha-agglutinin. Three beta-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these beta-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit beta-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying beta-glucosidase.

    SPRINGER, 2008年05月, Applied Microbiology and Biotechnology, 79 (1), 51 - 60, 英語

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    研究論文(学術雑誌)

  • Atsushi Kotaka, Hiroshi Sahara, Yoji Hata, Yasuhisa Abe, Akihiko Kondo, Michiko Kato-Murai, Kouichi Kuroda, Mitsuyoshi Ueda

    Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.

    TAYLOR & FRANCIS LTD, 2008年05月, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 72 (5), 1376 - 1379, 英語

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    研究論文(学術雑誌)

  • Rapid and efficient selection of yeast displaying a target protein using thermo-responsive magnetic nanoparticles

    Nobuo Fukuda, Jun Ishii, Tsutomu Tanaka, Hideki Fukuda, Noriyuki Ohnishi, Akihiko Kondo

    2008年04月, Biotechnology Progress, Vol. 24, No. 2, pp. 352-357, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Sriappareddy Tamalampudi, Mahabubur Rahman Talukder, Shinji Hama, Takao Numata, Akihiko Kondo, Hideki Fukuda

    The large percentage of biodiesel fuel (BDF) cost associated with feedstock oil and enzyme. In order to reduce the cost of BDF production, the lipase producing whole cells of Rhizopus oryzae (ROL) immobilized onto biomass support particles (BSPs) was used for the production of BDF from relatively low cost non-edible oil from the seeds of Jatropha curcas. The activity of ROL was compared with that of commercially available most effective lipase (Novozym 435). Different alcohols as a hydroxyl donor are tested, and methanolysis of Jatropha oil progresses faster than other alcoholysis regardless of lipases used. The maximum methyl esters content in the reaction mixture reaches 80 wt.% after 60 h using ROL, whereas it is 76% after 90 It using Novozym 435. Both the lipases can be used for repeated batches and both lipases exhibit more than 90% of their initial activities after five cycles. Our results suggest that whole-cell ROL immobilized on BSP is a promising biocatalyst for producing BDF from oil. (C) 2007 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2008年04月, BIOCHEMICAL ENGINEERING JOURNAL, 39 (1), 185 - 189, 英語

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    研究論文(学術雑誌)

  • Kenji Okano, Qiao Zhang, Sakurako Kimura, Junya Narita, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

    Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA domain) of AcmA (a major autolysin from Lactococcus lactis). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and alpha-amylase from Streptococcus bovis 148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of L. lactis cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the cA domain were used as an anchor protein, 82% of alpha-amylase activity was detected on the cells. The raw starch-degrading activity of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus. In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of Lactobacillus plantarum and Lactobacillus casei.

    AMER SOC MICROBIOLOGY, 2008年02月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 74 (4), 1117 - 1123, 英語

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    研究論文(学術雑誌)

  • Toshihiro Tateno, Hideki Fukuda, Akihiko Kondo

    Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes alpha-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40 degrees C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37 degrees C, respectively. The alpha-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.

    SPRINGER, 2007年12月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 77 (3), 533 - 541, 英語

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    研究論文(学術雑誌)

  • Junji Ito, Takumi Ebe, Seiji Shibasaki, Hideki Fukuda, Akihiko Kondo

    For efficient alkyl glucoside production from cellooligosaccharides, we constructed a yeast strain for alkyl glucoside synthesis by genetically inducing the display of beta-glucosidase 1 (BGL1) from the filamentous fungus Aspergillus aculeatus No. F-50 on the cell surface. The localization of BGL1 on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying BGL1 catalyzed alkyl glucoside synthesis from p-nitrophenyl beta-D-glucoside and primary alcohols. The highest yield of alkyl glucoside was 27.3% of the total sugar. The substrate specificities of the BGL1-displaying yeast strain and almond beta-glucosidase were compared using different-chain-length cellooligosaccharides. The BGL1-displaying yeast showed efficient alkyl glucoside production from not only glucose but also cellohexaose. This yeast is applicable as a whole-cell biocatalyst for alkyl glucoside production from cellulose hydrolysates. (c) 2007 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2007年11月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 49 (1-4), 92 - 97, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tamalampudi S, Hama S, Tanino T, Talukder MMR, Kondo A, Fukuda H

    2007年09月03日, J. Mol. Catal. B: Enzym., 48 (1), 33 - 37

    [査読有り]

  • Nobuo Fukuda, Jun Ishii, Seiji Shibasaki, Mitsuyoshi Ueda, Hideki Fukuda, Akihiko Kondo

    We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein-protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fe part of human immumoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.

    SPRINGER, 2007年08月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 76 (1), 151 - 158, 英語

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    研究論文(学術雑誌)

  • Takanori Tanino, Takumi Ohno, Tohru Aoki, Hideki Fukuda, Akihiko Kondo

    We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60 degrees C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.

    SPRINGER, 2007年07月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75 (6), 1319 - 1325, 英語

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    研究論文(学術雑誌)

  • Okano K, Kimura S, Narita J, Fukuda H, Kondo A

    To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting alpha-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l(-1) h(-1) lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l(-1) h(-1) lactate). Maximum volumetric lactate productivity was further increased (1.57 g l(-1) h(-1) lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of L-lactate) was achieved. In this study, we propose a new approach to lactate production by alpha-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.

    SPRINGER, 2007年07月, Applied Microbiology and Biotechnology, 75 (5), 1007 - 1013, 英語

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    研究論文(学術雑誌)

  • Chiaki Ogino, Masayuki Kanemasu, Masashi Fukumoto, Takafumi Kubo, Takamichi Yoshino, Akihiko Kondo, Hideki Fukuda, Nobuaki Shimizu

    Using biomass support particles (BSPs) as a cell immobilized matrix, immobilized recombinant Streptomyces lividans continuously produced phospholipase D (PLD) in a yield of about 1.5 x 10(4) U/L in each of eight batches. In contrast to the original strain Streptoverticillium cinnamoneum, this heterologous expression system with an immobilization method is capable of producing secretory PLD with an 8-fold greater efficiency. The presence of both glucose and tryptone in the initial culture medium also promoted secretory production, and PLD activity around 3.0 x 10(4) U/L were achieved. In addition, the promoter region of PLD ORF was deduced, and three types of plasmid having different lengths of promoter sequence were constructed. The deduced sequence had same effect on either of PLD production or mycelium immobilization, and the transformants harboring each of three plasmids showed the similar cultivation profiles (3.0 x 10(4) U/L). A combination of the immobilization method with BSPs and S. lividans transformant harboring the deduced plasmid has the potential for producing secretory PLD in the culture supernatant continuously. (c) 2007 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, 2007年07月, ENZYME AND MICROBIAL TECHNOLOGY, 41 (1-2), 156 - 161, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takuya Shishido, Masaru Muraoka, Hideki Yamaji, Akihiko Kondo, Hideki Fukuda

    L particles, composed of the L protein of the hepatitis B virus surface antigen, are candidates for a specific gene and drug delivery system. We previously constructed stably transfected insect cells for L particle production. In this study, the cells were successfully immobilized within porous biomass support particles (BSPs) in shake-flask culture. The immobilized cells showed a high specific productivity, comparable to the maximum productivities in static and shake-flask cultures of nonimmobilized cells.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2007年06月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 103 (6), 572 - 574, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Shinji Hama, Hideki Yamaji, Takahiro Fukumizu, Takao Numata, Sriappareddy Tamalampudi, Akihiko Kondo, Hideo Noda, Hideki Fukuda

    A packed-bed reactor (PBR) system using fungus whole-cell biocatalyst was developed for biodiesel fuel production by plant oil methanolysis. Lipase-producing Rhizopus oryzae cells were immobilized within 6 mm x 6 mm x 3 mm cuboidal polyurethane foam biomass support particles (BSPs) during batch cultivation in a 20-1 air-lift bioreactor. Emulsification of the reaction mixture containing soybean oils and water improved the methanolysis reaction rate. Using a high flow rate for the reaction mixture in the PBR caused exfoliation of the immobilized cells from the BSPs, while the inefficient mixing of the reaction mixture at low flow rates allowed the BSPs to be covered with a hydrophilic layer of high methanol concentration, leading to a significant decrease in lipase activity. A high methyl ester content of over 90% was achieved at a flow rate of 251/h in the first cycle of repeated batch methanolysis and a high value of around 80% was maintained even after the tenth cycle. Comparison with methanolysis reaction in a shaken bottle suggested that the PBR enhances repeated batch methanolysis by protecting immobilized cells from physical damage and excess amounts of methanol. The process presented here is therefore considered to be promising for industrial biodiesel-fuel production. (c) 2007 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2007年06月, BIOCHEMICAL ENGINEERING JOURNAL, 34 (3), 273 - 278, 英語

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    研究論文(学術雑誌)

  • Michiko Kato, Jun Fuchimoto, Takanori Tanino, Akihiko Kondo, Hideki Fukuda, Mitsuyoshi Ueda

    To prepare a whole-cell biocatalyst of a stable lipase at a low price, mutated Candida antarctica lipase B (mCALB) constructed on the basis of the primary sequences of CALBs from C antarctica CBS 6678 strain and from C. antarctica LF 058 strain was displayed on a yeast cell surface by Lx-agglutinin as the anchor protein for easy handling and stability of the enzyme. When mCALB was displayed on the yeast cell surface, it showed a preference for short chain fatty acids, an advantage for producing flavors; although when Rhizopus oryzae lipase (ROL) was displayed, the substrate specificity was for middle chain lengths. When the thermal stability of mCALB on the cell surface was compared with that of ROL on a cell surface, T-1/2, the temperature required to give a residual activity of 50% for heat treatment of 30 min, was 60 degrees C for mCALB and 44 degrees C for ROL indicating that mCALB displayed on cell surface has a higher thermal stability. Furthermore, the activity of the displayed mCALB against p-nitrophenyl butyrate was 25-fold higher than that of soluble CALB, as reported previously. These findings suggest that mCALB-displaying yeast is more practical for industrial use as the whole-cell biocatalyst.

    SPRINGER, 2007年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75 (3), 549 - 555, 英語

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    研究論文(学術雑誌)

  • Chiaki Ogino, Hidenori Daido, Yuka Ohmura, Namiko Takada, Yoshiki Itou, Akihiko Kondo, Hideki Fukuda, Nobuaki Shimizu

    The gene that encodes phospholipase D (PLD) from Streptoverticillium cinnamoneum contains three consensus regions (Region 1, 11 and Was shown in Fig. 1A) that are conserved among the PLD superfamily. The glycine-glycine (GG) motif in Region I and the glycine-serine (GS) motif in Region IV are also conserved in the PLD superfamily. These (GG and GS) motifs are located 7 residues downstream from each HKD motif. In an investigation of fifteen GG/GS motif mutants, generated as fusion proteins with maltose-binding protein (MBP-PLDs), three highly active mutants were identified. Three of the mutants (G215S, G216S, and G216S-S489G) contained a serine residue in the GG motif, and exhibited approximately a 9-27-fold increased transphosphatidylation activity to DPPC compared with recombinant wild type MBP-PLD. When heat stability was compared between three mutants and the recombinant wild type, only G216S-S489G showed heat labile properties. It appears that the 489th serine residue in the GS motif also contributes to the thermal stability of the enzyme. In addition, the GG/GS motif was very close to the active center residue, including two HKD motifs, as shown by computer modeling. The findings suggest that the GG/GS motif of PLD is a key motif that affects catalytic function and enzymatic stability. (c) 2007 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2007年06月, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1774 (6), 671 - 678, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Seiji Shibasaki, Ai Kawabata, Jun Ishii, Shunsuke Yagi, Tetsuya Kadonosono, Michiko Kato, Nobuo Fukuda, Akihiko Kondo, Mitsuyoshi Ueda

    We determined whether the cocultivation of yeast cells displaying a ZZ-domain and secreting an Fc fusion protein can be a novel tool for the recovery of secreted recombinant proteins. The ZZ-domain from Staphylococcus aureus protein A was displayed on the cell surface of Saccharomyces cerevisiae under the control of the GAL1 promoter. Strain S. cerevisiae BY4742 cells displaying the ZZ-domain on their surface were used for cocultivation with cells that produce a target protein fused to the Fc fragment as an affinity tag. The enhanced green fluorescent protein or Rhizopus oryzae lipase was genetically fused to the N and C termini of the Fc fragment of human immunoglobulin G, respectively. Through analysis by fluorescence-activated cell sorting and enzymatic assay, it was demonstrated that these fusion proteins are successfully produced in the medium and recovered by affinity binding with the cell surface displaying the ZZ-domain. These results suggest that the ZZ-domain-displaying cell and Fc fusion protein-secreting cell can be applied to use in synergistic process of production and recovery of secreted recombinant proteins.

    SPRINGER, 2007年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75 (4), 821 - 828, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Sriappareddy Tamalampudi, M. D. Mahabubur Rahman Talukder, Shinji Hama, Takanori Tanino, Yuya Suzuki, Akihiko Kondo, Hideki Fukuda

    To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tg1A) from A.. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of paranitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.

    SPRINGER, 2007年05月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75 (2), 387 - 395, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Tadahiro Nagaoka, Takayuki Fukuda, Shinnosuke Yoshida, Hirohito Nishimura, Dongwei Yu, Shun'ichi Kuroda, Katsuyuki Tanizawa, Akihiko Kondo, Masakazu Ueda, Hidenori Yamada, Hiroko Tada, Masaharu Seno

    The bio-nanocapsules (BNCs) composed of the recombinant envelope L-protein of hepatitis B virus constitute efficient delivery vectors specifically targeting human hepatocytes. Here, we have tried to enhance the stability of the BNCs because the L-proteins in the BNCs were aggregated due to random disulfide bridging when stored for a long period at 4 °C. The envelope protein contains fourteen cysteine residues in the S domain. Aggregation of the envelope proteins might be avoided if unessential cysteine residues are replaced or removed because the irreversible alkylation of the free sulfhydryl group protects against the aggregation and enhances the efficiency of encapsulation. In this study, the possibility of reducing the number of cysteine residues in the S domain to enhance the stability of the BNCs was assessed. The replacement of each cysteine residue by site-directed mutation showed that nine of fourteen cysteine residues were not essential to obtaining BNCs secreted into the culture media. Furthermore, upon evaluating the combination of these mutations, it was found that eight residues of replacement were acceptable. The mutant BNCs with replaced eight cysteine residues were not only more resistant against trypsin, but also more effective in transducing genes into human hepatoma-derived HepG2 cells than the original type BNC. Thus, we demonstrated that the minimized number of cysteine residues in the S domain could enhance the stability of the BNCs. © 2006 Elsevier B.V. All rights reserved.

    2007年04月23日, Journal of Controlled Release, 118 (3), 348 - 356, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Toshihiro Tateno, Hideki Fukuda, Akihiko Kondo

    We engineered a Corynebacterium glutamicum strain displaying a-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of alpha-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was carried out using C glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed L-lysine fermentation at various temperatures (30-40 degrees C) and pHs (6.0-7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest L-lysine yield was recorded at 30 degrees C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l L-lysine was produced in 24 h. The L-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C glutamicum displaying alpha-amylase has a potential to directly convert soluble starch to amino acids.

    SPRINGER, 2007年04月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 74 (6), 1213 - 1220, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takanori Tanino, Erina Noguchi, Sakurako Kimura, Hiroshi Sahara, Youji Hata, Hideki Fukuda, Akihiko Kondo

    The cell-surface display of the Flo1p anchor system with a flocculation functional domain was examined under various cultivation conditions. As a model system, lipase from Rhizopus oryzae with the pro sequence was genetically fused to the Flo1 short (FS) anchor (FSProROL) and displayed on the sake yeast cell-surface under the control of the SED800 promoter (pSED800). The nutrients and carbon source in the culture media affected the display of the fusion protein FSProROL on the sake yeast cell-surface. The lipase activity in whole cells cultivated in poor media, without peptone and/or yeast extracts, were higher than those cultivated in rich media. In addition, glucose and maltose were effective carbon sources for increasing the lipase activity in whole cells, and the addition of di- or tri-saccharide as the carbon source reduced the release of the lipase activity into the culture supernatants. The initial glucose concentration was found to influence the total lipase activity and it mainly affected the lipase activity in whole cells. Under the optimum condition, sake yeast was found to show high cell density and high lipase activity in short time cultivation. (c) 2006 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2007年03月, BIOCHEMICAL ENGINEERING JOURNAL, 33 (3), 232 - 237, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Md. Mahabubur Rahman Talukder, Sriappareddy Tamalampudy, Chong Jia Li, Le Yanglin, Jinchuan Wu, Akihiko Kondo, Hideki Fukuda

    A simple and effective preparation of lipases for use in organic solvents is hereby proposed. Lipases in aqueous solution were treated with isopropanol, immediately followed by immobilization onto a commercially available macroporous resin CRBO2 (crosslinked polystyrene with N-methylglucamine as a functional group). The dual modification of lipases by (1) isopropanol treatment and (2) immobilization improved the activity and stability of lipases more significantly than either of the two treatments alone. The degree of lipase activation was dependent on isopropanol-buffer (v/v) ratio and the source of lipase used. Among the lipases tested, Rhizopus oryzae lipase was more significantly activated. The maximum specific activity of R. oryzae lipase after dual modification was 94.9 mmol h(-1) g(-1), which was, respectively, 3.3-, 2.5- and 1.5-fold of untreated free, untreated immobilized and treated free lipases. The conformations of the treated and untreated free lipases were investigated by circular dichroism (CD) measurement. Changes in the far- and near-UV CD spectra of lipase indicate that lipase activation is accompanied by changes in secondary and tertiary structures of lipases. The increase in negative molar elipticity at 222 nm suggests that the alpha-helical content of lipase increase after pretreatment. (c) 2006 Elsevier B.V.. All rights reserved.

    ELSEVIER SCIENCE SA, 2007年01月, BIOCHEMICAL ENGINEERING JOURNAL, 33 (1), 60 - 65, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Teik Seong Khaw, Yoshio Katakura, Kazuaki Ninomiya, Churairat Moukamnerd, Akihiko Kondo, Mitsuyoshi Ueda, Suteaki Shioya

    The specific ethanol production rate of raw starch by arming yeast cells displaying α-amylase and glucoamylase increased significantly when the cells and starch granules settled together. The specific ethanol production rate also increased when the size distribution of starch granules was almost same as that of the yeast cells. These results indicate that the surface contact between starch granules and yeast cells is important for increasing the apparent specific activity of α-amylase, which was the rate-limiting factor of the direct fermentation. © 2007 The Society for Biotechnology, Japan.

    2007年01月, Journal of Bioscience and Bioengineering, 103 (1), 95 - 97, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Fluorescence detection system for human G protein - Coupled receptor signaring in yeast

    Jun Ishii, Akihiko Kondo

    PHOENIX PUBL & MEDIA NETWORK, 2007年, PROGRESS ON POST-GENOME TECHNOLOGIES, 57 - 57, 英語

    [査読有り]

    研究論文(国際会議プロシーディングス)

  • Junya Narita, Saori Ishida, Kenji Okano, Sakurako Kimura, Hideki Fukuda, Akihiko Kondo

    The 5'-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5' stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses alpha-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the alpha-amylase mRNA was proved by measuring alpha-amylase activity in the culture supernatant and the relative expression of alpha-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving alpha-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.

    SPRINGER, 2006年11月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 73 (2), 366 - 373, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Khaw Teik Seong, Yoshio Katakura, Kazuaki Ninomiya, Yohei Bito, Satoshi Katahira, Akihiko Kondo, Mitsuyoshi Ueda, Suteaki Shioya

    In the direct ethanol fermentation of raw starch by arming yeast with α-amylase and glucoamylase, it is preferable to use a flocculent yeast because it can be recovered without centrifugation. Three types of arming yeast system, I (nonflocculent), II (mildly flocculent), and III (heavily flocculent), were constructed and their fermentation performances were compared. With an increase in the degree of flocculation, specific ethanol production rate for soluble starch decreased (0.19, 0.17, and 0.12 g g-dry-cell-1 h -1 for systems I, II, and III, respectively), but that for raw starch did not decrease as much as expected (0.06, 0.06, and 0.04 g g-dry-cell -1 h-1 for systems I, II and III, respectively). Microscopic observation revealed that many starch granules were captured in the yeast flocs in system III during the direct ethanol fermentation of raw starch. It was suggested that the capture of starch granules increases apparent substrate concentration for amylolytic enzymes in arming yeast cell flocs thus, the specific ethanol production rate of system III was kept at a level comparable to those of the other systems. © 2006 Springer-Verlag.

    2006年11月, Applied Microbiology and Biotechnology, 73 (1), 60 - 66, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Satoshi Katahira, Atsuko Mizuike, Hideki Fukuda, Akihiko Kondo

    The sulfuric acid hydrolysate of lignocellulosic biomass, such as wood chips, from the forest industry is an important material for fuel bioethanol production. In this study, we constructed a recombinant yeast strain that can ferment xylose and cellooligosaccharides by integrating genes for the intercellular expressions of xylose reductase and xylitol dehydrogenase from Pichia stipitis, and xylulokinase from Saccharomyces cerevisiae and a gene for displaying beta-glucosidase from Aspergillus acleatus on the cell surface. In the fermentation of the sulfuric acid hydrolysate of wood chips, xylose and cellooligosaccharides were completely fermented after 36 h by the recombinant strain, and then about 30 g/l ethanol was produced from 73 g/l total sugar added at the beginning. In this case, the ethanol yield of this recombinant yeast was much higher than that of the control yeast. These results demonstrate that the fermentation of the lignocellulose hydrolysate is performed efficiently by the recombinant Saccharomyces strain with abilities for xylose assimilation and cellooligosaccharide degradation.

    SPRINGER, 2006年10月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 72 (6), 1136 - 1143, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Takanori Tanino, Hideki Fukuda, Akihiko Kondo

    A Pichia pastoris cell-surface display system was constructed using a Flo1p anchor system, which was developed in Saccharomyces cerevisiae. The lipase from Rhizopus oryzae with a pro sequence (ProROL) was used as the model protein and was genetically fused to the anchor consisting of amino acids 1-1099 of Flo1p (FS anchor). The resulting fusion protein FSProROL was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). The fluorescence microscopy of immunolabeled P. pastoris cells revealed that ProROL was displayed on the cell surface, and Western blot analysis revealed that the fusion protein FSProROL was noncovalently attached to the cell wall and highly glycosylated. The lipase activity of P. pastoris cells was affected by the methanol concentration for the induction phase. Surprisingly, the activity of lipase displayed on the cells incubated at 60 degrees C was not only stable but also increased to about 6.5 times the initial value after 4 h incubation.

    AMER CHEMICAL SOC, 2006年08月, BIOTECHNOLOGY PROGRESS, 22 (4), 989 - 993, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Hirotaka Furukawa, Takanori Tanino, Hideki Fukuda, Akihiko Kondo

    Streptavidin derived from Streptomyces avidinii was displayed on the cell surface of the yeast Saccharomyces cerevisiae by cell-surface engineering using two types of plasmid for the expression of a native subunit and an anchored subunit fused with the C-terminus of 318 amino acids of Flo1p containing a glycosylphosphatidylinositol anchor attachment signal. The displayed streptavidin had the binding ability for biotinylated compounds. This was confirmed by fluorescence microscopy after the adsorption of yeast cells displaying streptavidin and biotinylated fluorescein isothiocyanate. On the other hand, streptavidin produced by cells harboring only the plasmid for the expression of the anchored subunit showed a very low binding activity for biotinylated compounds. Cells displaying streptavidin may constitute novel whole-cell affinity adsorbents widely used for immunoassay and biosensing. This coexpression method will ensure that proteins, such as homo- and hetero-oligomeric proteins, are displayed on the cell surface in an active form.

    AMER CHEMICAL SOC, 2006年08月, BIOTECHNOLOGY PROGRESS, 22 (4), 994 - 997, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Yurie Nakamura, Takeshi Matsumoto, Fumiki Nomoto, Mitsuyoshi Ueda, Hideld Fukuda, Akihiko Kondo

    Rhizopus oryzae lipase (ROL) was displayed on the cell surface of Saccharomyces cerevisiae via the Flo1 N-terminal region (1100 amino acids), which corresponds to a flocculation functional domain. The activity of lipase-displaying yeast whole-cell biocatalysts was enhanced 7.3-fold by incubation of the yeast cells at 20 °C in distilled water for 8 days after 8 day cultivation. The amount of lipase molecules present in cell wall and intracellular fractions was found to be increased 4.5- and 1.8-fold, respectively, by incubation, which proves that ROL molecules are expressed during incubation. The ROL-displaying yeast whole-cell biocatalyst with enhanced activity was successfully catalyzed by optical resolution of the pharmaceutical precursor (R,S)-1-benzyloxy-3-chloro-2-propyl monosuccinate. Moreover, it showed stable activity through at least eight reaction cycles. These results demonstrate that ROL-displaying yeast cells with enhanced activity by incubation in distilled water are very effective in industrial bioconversion processes. © 2006 American Chemical Society and American Institute of Chemical Engineers.

    2006年07月, Biotechnology Progress, 22 (4), 998 - 1002, 英語

    [査読有り]

    研究論文(国際会議プロシーディングス)

  • Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein

    Jun Ishii, Shizuka Matsumura, Sakurako Kimura, Kenji Tatematsu, Shun'ichi Kuroda, Hideki Fukuda, Akihiko Kondo

    2006年07月, Biotechnology Progress, 22 (4), 954 - 960, 英語

    [査読有り]

    研究論文(学術雑誌)

  • J Narita, K Okano, T Tateno, T Tanino, T Sewaki, MH Sung, H Fukuda, A Kondo

    We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.

    SPRINGER, 2006年05月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 70 (5), 564 - 572, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Teik Seong Khaw, Yoshio Katakura, Jun Koh, Akihiko Kondo, Mitsuyoshi Ueda, Suteaki Shioya

    Four types of cell-surface-engineered yeast Saccharomyces cerevisiae displaying glucoamylase, namely, systems A, B, C, and D, were constructed to evaluate their performance in direct ethanol fermentation from raw corn starch. Systems A and B were glucoamylase-displaying nonflocculent yeast (YF237) types that secrete α-amylase into the culture medium and codisplay α-amylase on the cell surface, respectively. Systems C and D were flocculent yeast counterparts (YF207) for systems A and B, respectively. In batch fermentations, the specific ethanol production rates of systems A, B, C, and D were 0.18, 0.06, 0.06, and 0.04 g (g cell)-1 h-1, respectively. In repeated fermentations, the specific ethanol production rate of system A decreased with the number of repetitions, whereas, that of system B was maintained. In all systems, the rate-limiting step was the conversion of starch to oligosaccharide because oligosaccharide and glucose were not accumulated throughout the fermentations. © Springer-Verlag 2005.

    2006年05月, Applied Microbiology and Biotechnology, 70 (5), 573 - 579, 英語

    [査読有り]

    研究論文(学術雑誌)

  • S Hama, S Tamalampudi, T Fukumizu, K Miura, H Yamaji, A Kondo, H Fukuda

    To identify the lipase responsible for the methanolysis activity of fungus whole-cell biocatalysts, the lipase localization of Rhizopus oryzae cells was determined. Western blot analysis showed that R. oryzae cells produce two types of lipase with different molecular masses of 34 and 31 kDa; the former (ROL34) was bound to the cell wall, whereas the latter (ROL31) was mainly bound to the cell membrane. It was found that cell immobilization within reticulated polyurethane foam biomass support particles strongly inhibits the secretion of membrane-bound lipase into the culture medium. An investigation of the relationship between ROL34 and ROL31 suggested that ROL31 originates from the cleavage of a 28-amino-acid residue at the N-terminus of ROL34. The addition of olive oil to the culture medium led to the retention of increased amounts of lipase within the cell. This phenomenon was further confirmed by an immunofluorescence labeling of hyphal cells. When cells were cultivated with various substrate-related compounds, such as olive oil and oleic acid, the intracellular methanolysis activity strongly correlated with the relative amounts of the membrane-bound lipase, which suggests that ROL31 localized in the membrane plays a crucial role in the methanolysis activity of R. oryzae cells.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2006年04月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 101 (4), 328 - 333, 英語

    [査読有り]

    研究論文(学術雑誌)

  • H Michida, S Tamalampudi, SS Pandiella, C Webb, H Fukuda, A Kondo

    New probiotic foods are expected to develop out of the increasing demand for a health-oriented society. Fermented cereal has recently been looked to as a way of delivering lactic acid bacteria. The present study investigated the effect of cereal extracts and cereal fiber on the viability of the lactic acid bacterium Lactobacillus plantarum under gastrointestinal tract conditions, with simulated gastric juice and bile juice used for the evaluation of gastrointestinal tolerance. The cells were cultured using cereal media that contained cereal extract and cereal fiber to immobilize the cells. Malt and barley were used as cereal. Malt and barley extracts were also used to examine the effect of cereal extracts on gastrointestinal tolerance. Regarding gastric tolerance, the addition of cereal extracts significantly improved the viability of L. plantarum while immobilization within cereal fiber slightly improved its viability. The effect of malt extract was more positive than that of barley extract. Meanwhile, immobilization within cereal fiber played a major role in bile tolerance and the presence of cereal extracts further enhanced the tolerance of L. plantartun to bile juice. In both cereal media, cell immobilization within cereal fiber and the presence of cereal extracts had a synergistic effect on the gastrointestinal tolerance. (c) 2005 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2006年02月, BIOCHEMICAL ENGINEERING JOURNAL, 28 (1), 73 - 78, 英語

    [査読有り]

    研究論文(学術雑誌)

  • DW Yu, T Fukuda, Tuoya, S Kuroda, K Tanizawa, A Kondo, M Ueda, T Yamada, H Tada, M Seno

    The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.

    TAYLOR & FRANCIS INC, 2006年01月, IUBMB LIFE, 58 (1), 1 - 6, 英語

    [査読有り]

    研究論文(学術雑誌)

  • J Narita, K Okano, T Kitao, S Ishida, T Sewaki, MH Sung, H Fukuda, A Kondo

    We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying a-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.

    AMER SOC MICROBIOLOGY, 2006年01月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 72 (1), 269 - 275, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Akihiko Kondo, Shun-ichi Kuroda, Katsuyuki Tanizawa, Masaharu Seno, Masakazu Ueda

    We succeeded overproduction of the HBV envelope L particles with an approximate average particle size of 80 nm in yeast cells. Because the L particle is an empty bionanoparticles containing no viral DNA, it can be used as a safe and efficient carrier for human liver-specific delivery (pinpoint delivery) of drug and gene. In addition, genetically engineered L particles that are able to target to various organs were constructed by deleting the hepatocyte binding domain of L protein (pre-S region) and displaying targeting peptide or protein ligands. Therefore, bionanoparticles are a novel nano-carrier applicable to the broad range of pinpoint DDS. © 2006, THE JAPAN SOCIETY OF DRUG DELIVERY SYSTEM. All rights reserved.

    2006年, Drug Delivery System, 21 (4), 435 - 443, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Development of a single cell analysis system of agonist for drug discovery

    Akihiko Kondo, Jun Ishii, Shizuka Matsumura, Hideki Fukuda

    SOUTHEAST UNIV PRESS, 2006年, Progress on Post-Genome Technologies, 30 - 30, 英語

    [査読有り]

    研究論文(国際会議プロシーディングス)

  • DW Yu, C Amano, T Fukuda, T Yamada, S Kuroda, K Tanizawa, A Kondo, M Ueda, H Yamada, H Tada, M Seno

    A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

    BLACKWELL PUBLISHING, 2005年07月, FEBS JOURNAL, 272 (14), 3651 - 3660, 英語

    [査読有り]

    研究論文(学術雑誌)

  • DW Yu, C Amano, T Fukuda, T Yamada, S Kuroda, K Tanizawa, A Kondo, M Ueda, H Yamada, H Tada, M Seno

    A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

    BLACKWELL PUBLISHING, 2005年07月, FEBS JOURNAL, 272 (14), 3651 - 3660, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Jaruwan Maneesri, AZUMA Masayuki, SASAKI Yumiko, IGARASHI Koichi, MATSUMOTO Takeshi, FUKUDA Hideki, KONDO Akihiko, OOSHIMA Hiroshi

    Most proteins involved in the synthesis of the GPI core structure of Saccharomyces cerevisiae are essential for growth. To explore the relationship between the GPI anchor structure and beta-1,6-glucan synthesis, we screened deletion mutants in genes involved in GPI synthesis for osmotic remedial growth. Heterozygous diploid strains were dissected on medium with osmotic support and slow growth of the mcd4 deletion mutant was observed. The med4 mutant showed abnormal morphology and cell aggregation, and was hypersensitive to SDS, hygromycin B and K1 killer toxin. Incorporation of GPI cell wall proteins was examined using a GPI-Flo1 fusion protein. The result suggested that the mcd4 deletion causes a decrease in GPI cell wall proteins levels. The mutation also caused a decrease in mannan levels and an increase in alkali-insoluble beta-1,6-glucan and chitin levels in the cell wall.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2005年04月, Journal of Bioscience and Bioengineering, 99 (4), 354 - 360, 英語

    [査読有り]

    研究論文(学術雑誌)

  • J Maneesri, M Azuma, Y Sakai, K Igarashi, T Matsumoto, H Fukuda, A Kondo, H Ooshima

    Most proteins involved in the synthesis of the GPI core structure of Saccharomyces cerevisiae are essential for growth. To explore the relationship between the GPI anchor structure and beta-1,6-glucan synthesis, we screened deletion mutants in genes involved in GPI synthesis for osmotic remedial growth. Heterozygous diploid strains were dissected on medium with osmotic support and slow growth of the mcd4 deletion mutant was observed. The med4 mutant showed abnormal morphology and cell aggregation, and was hypersensitive to SDS, hygromycin B and K1 killer toxin. Incorporation of GPI cell wall proteins was examined using a GPI-Flo1 fusion protein. The result suggested that the mcd4 deletion causes a decrease in GPI cell wall proteins levels. The mutation also caused a decrease in mannan levels and an increase in alkali-insoluble beta-1,6-glucan and chitin levels in the cell wall.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2005年04月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 99 (4), 354 - 360, 英語

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    研究論文(学術雑誌)

  • M Oda, M Kaieda, S Hama, H Yamaji, A Kondo, E Izumoto, H Fukuda

    For biodiesel-fuel production by methanolysis of plant oils, Rhizopus oryzae cells producing a 1,3-positional specificity lipase were cultured with polyurethane foam biomass support particles (BSPs) in a 201 air-lift bioreactor, and the cells immobilized within BSPs were used as whole-cell biocatalyst in repeated batch-cycle methanolysis reaction of soybean oil. The whole-cell biocatalyst had a higher durability in the methanolysis reaction when obtained from air-lift bioreactor cultivation than from shake-flask cultivation. Following repeated methanolysis reaction using the whole-cell biocatalyst, analysis of the reaction mixture composition indicated that monoglycerides (MGs) decreased and free fatty acids (FFAs) increased with increasing water content in the reaction mixture, and that MGs, diglycerides (DGs), and triglycerides (TGs) increased with increasing number of reaction cycles. The isomers of MGs and DGs generated during the 20th methanolysis reaction cycle consisted of 2-MGs and 1,2(2,3)-DGs, respectively. The hydrolytic activity of the whole-cell biocatalyst, on the other hand, was stable regardless of the number of reaction cycles. It was demonstrated thus that the whole cell biocatalyst promotes acyl migration of partial glycerides, and that the facilitatory effect is increased by increase in the water content of the reaction mixture but it is lost gradually with increasing number of reaction cycles. (C) 2004 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2005年03月, BIOCHEMICAL ENGINEERING JOURNAL, 23 (1), 45 - 51, 英語

    [査読有り]

    研究論文(学術雑誌)

  • ピンポイントデリバリー用バイオナノキャリアの開発とがん遺伝子治療への応用

    近藤 昭彦

    2005年, 厚生労働科学研究費補助金萌芽的先端医療技術推進研究事業平成16年度研究報告書, 83(2),91, 日本語

    研究論文(学術雑誌)

  • MACHI Kazuki, AZUMA Masayuki, IGARASHI Koichi, MATSUMOTO Takeshi, FUKUDA Hideki, KONDO Akihiko, OOSHIMA Hiroshi

    Although ROT1 is essential for growth of Saccharomyces cerevisiae strain BY4741, the growth of a rot1Delta haploid was partially restored by the addition of 0(.)6 M sorbitol to the growth medium. Rot1p is predicted to contain 256 amino acids, to have a molecular mass of 29 kDa, and to possess a transmembrane domain near its C-terminus. Candida albicans and Schizosaccharomyces pombe have Rot1p homologues with high identity that also have predicted transmembrane domains. To explore the role of Rot1p, the phenotypes of the rot1Delta haploid were analysed. Deletion of ROT1 caused cell aggregation and an abnormal morphology. Analysis of the cell cycle showed that rot1Delta cells are delayed at the G2/M phase. The rot1Delta cells were resistant to K1 killer toxin and hypersensitive to SIDS and hygromycin B, suggesting that they had cell wall defects. Indeed, greatly reduced levels of alkali-soluble and -insoluble 1,6-beta-glucan, and increased levels of chitin and 1,3-beta-glucan, were found in rot1Delta cells. Furthermore, the phenotypes of rot1Delta cells resemble those of disruption mutants of the KRE5 and BIG1 genes, which show greatly reduced levels of cell wall 1,6-beta-glucan. Incorporation of glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in big1Delta and rot1Delta cells was examined using a GFP-Flo1 fusion protein. GFP fluorescence was detected both on the cell surface and in the culture medium, suggesting that, in these mutants, mannoproteins may become only weakly bound to the cell wall and some of these proteins are released into the medium. Electron microscopic analyses of rot1Delta and big1Delta cells showed that the electron-dense mannoprotein rim staining was more diffuse and paler than that in the wild-type, and that the outer boundary of the cell wall was irregular. A big1Deltarot1Delta double mutant had a growth rate similar to the corresponding single mutants, suggesting that Rot1p and Big 1 p have related functions in 1,6-beta-glucan synthesis.

    SOC GENERAL MICROBIOLOGY, 2004年10月, Microbiology(UK), 150, 3163 - 3173, 英語

    [査読有り]

    研究論文(学術雑誌)

  • S Hama, H Yamaji, M Kaieda, M Oda, A Kondo, H Fukuda

    To stabilize the lipase activity of Rhizopus oryzae cells as whole-cell biocatalysts, the effect of cell membrane fatty acid composition on biodiesel-fuel production was investigated. The fatty acid composition of the cell membrane was easily controllable by addition of various fatty acids to the culture medium. Oleic or linoleic acid-enriched cells showed higher initial methanolysis activity than saturated fatty acid-enriched cells, among which palmitic acid-enriched cells exhibited significantly greater enzymatic stability than unsaturated fatty acid-enriched cells. It was assumed that fatty acids significantly affect the permeability and rigidity of the cell membrane, and that higher permeability and rigidity lead to increases in methanolysis activity and enzymatic stability, respectively. When the optimal fatty acid ratio of 0.67, indicated by R-f [=oleic acid/(oleic acid + palmitic acid)], was adopted for repeated methanolysis reactions, both methanolysis activity and enzymatic stability were maintained at significantly elevated levels, with methyl ester content of around 55% even in the 10th batch cycles. (C) 2004 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE SA, 2004年10月, BIOCHEMICAL ENGINEERING JOURNAL, 21 (2), 155 - 160, 英語

    [査読有り]

    研究論文(学術雑誌)

  • S Katahira, Y Fujita, A Mizuike, H Fukuda, A Kondo

    Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan. We used a cell surface engineering system based on alpha-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase 11 (XYNII) from Trichoderma reesei QM9414 and beta-xylosidase (XyIA) from Aspergillus oryzae NiaD300, on the cell surface. In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XyIA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface. These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA. In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S. cerevisiae strain was constructed by codisplaying XYNII and XyIA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g. These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.

    AMER SOC MICROBIOLOGY, 2004年09月, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 70 (9), 5407 - 5414, 英語

    [査読有り]

    研究論文(学術雑誌)

  • M Kaieda, M Nagayoshi, S Hama, A Kondo, H Fukuda

    In the present study, we used gene manipulation to construct a recombinant Aspergillus oryzae strain overexpressing lipase and investigated its application to the optical resolution of chiral compounds. A. oryzae niaD300, which was derived from the wild-type strain RIB40, was used as the host strain. The tglA gene, which encodes a triacylglycerol lipase, was cloned from the A. oryzae niaD300 chromosomal genome, then reintroduced, with and without a secretion-signal sequence, into the genome and expressed under the control of the improved glaA promoter of plasmid pNGA142. The resulting recombinant strain overexpressing A. oryzae lipase was immobilized within biomass-support particles and used as a whole-cell biocatalyst. The immobilized lipase-overexpressing strain with secretion-signal sequence showed high activity and was used to selectively synthesize (R)-1-phenylethyl acetate from (RS)-1-phenylethanol and vinyl acetate. After 48 h reaction at 30degreesC with molecular sieve 4A, the yield and enantiomeric excess (%ee) of (R)-1-phenylethyl acetate reached approximately 90 and 95%ee, respectively. The whole-cell biocatalyst for optical resolution of chiral compounds produced in this study maintained its activity over 25 batch-reaction cycles.

    SPRINGER, 2004年08月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 65 (3), 301 - 305, 英語

    [査読有り]

    研究論文(学術雑誌)

  • SHIGECHI Hisayori, KOH Jun, FUIJITA Yasuya, MATSUMOTO Takeshi, BITO Youhei, UEDA Mitsuyoshi, SATOH Eiichi, FUKUDA Hideki, KONDO AKIHIKO

    Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis a-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.

    AMER SOC MICROBIOLOGY, 2004年08月, Applied and Environmental Microbiology, 70 (8), 5037 - 5040, 英語

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    研究論文(学術雑誌)

  • M Ueda, A Kondo

    ELSEVIER SCIENCE BV, 2004年06月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28 (4-6), 137 - 137, 英語

    [査読有り]

  • Efficient production of L-(+)-lactic acid from raw starch by Streptococcus bovis 148

    J Narita, S Nakahara, H Fukuda, A Kondo

    Streptococcus bovis 148 was found to produce L-(+)-lactic acid directly from soluble and raw starch substrates at pH 6.0. Productivity was highest at 37degreesC, with 14.7 g/l lactic acid produced from 20 g/l raw starch. The yield and optical purity of L-lactic acid were 0.88 and 95.6%, respectively.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, 2004年06月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 97 (6), 423 - 425, 英語

    [査読有り]

    研究論文(学術雑誌)

  • C Ogino, M Kanemasu, Y Hayashi, A Kondo, N Shimizu, S Tokuyama, Y Tahara, S Kuroda, K Tanizawa, H Fukuda

    The structural gene for phospholipase D (PLD) of an actinomycete, Streptoverticillium cinnamoneum, together with its promoter region was introduced into Streptomyces lividans using a shuttle vector-pUC702-for Escherichia coli and S. lividans. The transformant was found to secrete a large amount of PLD (about 2.0x10(4) U/l, 42 mg/l) when cultured in a jar fermentor. Both an initial glucose concentration of 17.5 g/l and the feeding of carbon and nitrogen sources are effective for efficient secretion of PLD; under these culture conditions, the amount of PLD secreted reached a maximum level (about 5.5x10(4) U/l, 118 mg/l) after about 60 h. In contrast to the original producer, Stv. cinnamoneum, which secretes only a small amount of PLD (about 1.1x10(3) U/l, 2 mg/l) along with other extracellular proteins, this heterologous expression system is markedly more efficient in production of secretory PLD.

    SPRINGER-VERLAG, 2004年06月, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 64 (6), 823 - 828, 英語

    [査読有り]

    研究論文(学術雑誌)

  • H Shibamoto, T Matsumoto, H Fukuda, A Kondo

    We constructed a combinatorial yeast library through cell-surface display of the pro- and mature region of lipase from Rhizopus oryzae (ProROL) and obtained clones retaining lipase activity in fluorescent plate assay. The initial reaction rates of hydrolysis and methanolysis could be measured directly as whole-cell biocatalyst without complex treatments such as concentration, purification, and immobilization. The selected clones showed wide-ranging variation of reaction specificity. The K138R mutant showed a 1.3-fold shift of reaction specificity toward methanolysis compared to the wild type, while the V-95D, I53V, P-96S/F196Y, and Q128H/Q197L mutants showed shifts toward hydrolysis of 1.6-5.9-fold. Predictions of the mutants' three-dimensional structure suggested that the hydrogen-bond distance between threonine 83 and aspartic acid 92 may influence reaction specificity, which shifted toward hydrolysis in mutants where this distance was shorter than in the wild type, but toward methanolysis where it was longer. The positions of amino acid residues (aa) 53, 138 and 196 in ProROL are considered the sites that influence hydrogen-bond distance and change reaction specificity. Construction of a surface-displayed combinatorial library in yeast cells is thus a powerful tool in accelerating the combinatorial approach to enzyme engineering and novel whole-cell biocatalyst development. (C) 2004 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2004年06月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28 (4-6), 235 - 239, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Y Lin, S Shiraga, T Tsumuraya, Fujii, I, T Matsumoto, A Kondo, M Ueda

    A combinatorial library of the Fab fragment of a catalytic antibody able to hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol was constructed on yeast-cell surface. Interesting clones were selected using fluorescence-activated cell sorting (FACS). When binding affinity to a transition-state analog was detected, evolution of the catalytic antibody was carried out in vitro on yeast-cell surface. A number of variants with enhanced catalytic activity and binding affinity were obtained. The results showed that the improvement of catalytic antibody, which can be performed easily on yeast-cell surface using the cell-surface engineering system, is a good example of the application of protein library construction. (C) 2004 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2004年06月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28 (4-6), 247 - 251, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Y Lin, S Shiraga, T Tsumuraya, Fujii, I, T Matsumoto, A Kondo, M Ueda

    A combinatorial library of the Fab fragment of a catalytic antibody able to hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol was constructed on yeast-cell surface. Interesting clones were selected using fluorescence-activated cell sorting (FACS). When binding affinity to a transition-state analog was detected, evolution of the catalytic antibody was carried out in vitro on yeast-cell surface. A number of variants with enhanced catalytic activity and binding affinity were obtained. The results showed that the improvement of catalytic antibody, which can be performed easily on yeast-cell surface using the cell-surface engineering system, is a good example of the application of protein library construction. (C) 2004 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2004年06月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28 (4-6), 247 - 251, 英語

    [査読有り]

    研究論文(学術雑誌)

  • T Tanino, T Matsumoto, H Fukuda, A Kondo

    We constructed a novel system for periplasmic localization of target proteins, using yeast external invertase (INV) as anchor protein, in which the C- or N-terminal of the target protein was fused to the invertase and the fusion proteins expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (GAPDH). Unlike in conventional cell-surface display, the system enables the target fusion protein to localize in yeast periplasm in a free state. As a model, enhanced green fluorescence protein (EGFP) was localized in yeast periplasm using the new system. Yeast-periplasm localization of INV-EGFP and EGFP-INV fusion proteins was confirmed by fluorescence microscopy and immunoblotting: green fluorescence was observed at the cell outline and, in western blot analysis, most fusion proteins were detected in the cell-surface fraction, indicating that the fusion proteins had been transported to the cell-surface layer. In addition, in both C- and N-terminal fusion, invertase showed activity, indicating dimer formation. These results demonstrate that invertase is a useful anchor for localizing target protein in the yeast periplasm. (C) 2004 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, 2004年06月, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28 (4-6), 259 - 264, 英語

    [査読有り]

    研究論文(学術雑誌)

  • Y Lin, S Shiraga, T Tsumuraya, T Matsumoto, A Kondo, Fujii, I, M Ueda

    Two forms of the Fab fragment of the catalytic antibody 6D9 were individually displayed on yeast-cell surface in fusion to the C-terminal half of a-agglutinin: one was 6D9 Fab1, in which the light chain of the Fab (Lc) fragment is displayed on cell surface and the heavy chain of the Fab (Fd) fragment is secreted and linked to the Lc fragment with a disulfide bond; the other was 6D9 Fab2, in which the Fd fragment is displayed on cell surface and the Lc fragment is secreted and linked to the Fd fragment with a disulfide bond. Analysis by flow cytometry indicated that some 6139 Fab2 fragments were unable to construct an appropriate conformation, and that most of the 6D9 Fab I fragments displayed on yeast-cell surface exhibited higher