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KONDO Akihiko
Graduate School of Science, Technology and Innovation / Department of Science, Technology and Innovation
Professor

Researcher basic information

■ Research news
  • 13 Apr. 2021, New method of artificially creating genetic switches for yeast
  • 17 Jan. 2020, Novel protein positioning technique improves functionality of yeast cells -Expected improvements in cell surface engineering across a variety of fields-
  • 06 Sep. 2019, Fe metabolic engineering method succeeds in producing 1,2,4-butanetriol sustainably from biomass
  • 26 Aug. 2016, Researchers succeed in developing a genome editing technique that does not cleave DNA
  • 01 Dec. 2015, New technology selects high-affinity proteins
  • 18 Jun. 2015, Elucidation of chemical ingredients in rice straw
■ Research Keyword
  • 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
■ Research Areas
  • Manufacturing technology (mechanical, electrical/electronic, chemical engineering) / Applied biofunctional and bioprocess engineering

Research activity information

■ Award
  • 2022 The Society for Biotechnology, Japan, Society Award, Innovative approaches to creating smart cells for bioproduction
    Akihiko Kondo

  • Oct. 2020 神戸大学, 令和2年度 学長表彰(財務貢献者)
    近藤 昭彦

  • 2017 International Bioprocessing Association, Outstanding Scientist Award-2016
    KONDO Akihiko

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

  • 2016 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, YASUKAWA MASAHIRO, 佐々木 建吾, 田中 裕大, TAKAHASHI TOMOKI, KONDO AKIHIKO, MATSUYAMA HIDETO
    Japan society

  • 2016 IMSTEC2016, IMSTEC2016 Travel award, Up-concentration of sugar solution by using forward osmosis for bioethanol production process
    渋谷 真史, YASUKAWA MASAHIRO, 佐々木 建吾, 田中 裕大, TAKAHASHI TOMOKI, KONDO AKIHIKO, MATSUYAMA HIDETO
    International society

  • 2015 The Association of American Publishers, PROSE Awards, Winner in Environmental Science, Bioprocessing of Renewable Resources to Commodity Bioproducts
    KONDO Akihiko

  • Jan. 2014 The Japan Society for Bioscience, Biotechnology, and Agrochemistry (JSBBA), Award for Excellence to Authors Publishing in BBB, 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
    Japan society

  • Sep. 2013 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

  • Sep. 2013 The Society for Biotechnology, Japan, Excellent Paper Award, Direct isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display system
    SOMA YUKI, INOKUMA KENTARO, TANAKA TSUTOMU, OGINO CHIAKI, KONDO AKIHIKO, OKAMOTO MASAHIRO, HANAI TAIZO
    Japan society

  • 2011 Iue Memorial Foundation, Iue Memorial Foundation Award
    KONDO Akihiko

  • Oct. 2010 The Society for Biotechnology, Japan, Achievement Award, 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―
    KONDO Akihiko

  • Jul. 2010 Renewable Energy 2010, Best Paper Award, Construction of consolidated biopreocesses for production of bio-fuels and chemicals by using cell surface engineered microbial cells
    KONDO Akihiko

  • Oct. 2009 神戸大学, 第1回学長表彰
    近藤 昭彦

  • Nov. 2008 日刊工業新聞社, モノづくり連携大賞 特別賞
    近藤 昭彦

  • Sep. 2008 竹田理化工業株式会社, 竹田国際貢献賞
    近藤 昭彦

  • 2007 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 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 Biotechnology Japan, Bio Business Competition Japan, Best award, 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 The Society for Biotechnology, Japan, Excellent Paper Award, 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 The Society for Biotechnology, Japan, Excellent Paper Award, 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 The Society for Biotechnology, Japan, Excellent Paper Award, 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 The Society of Chemical Engineers, Excellent young scientist award, 微粒子材料の生物化学工学への応用に関する研究
    KONDO Akihiko

■ Paper
  • Varada Jagadeesh, Nobuyuki Okahashi, Fumio Matsuda, Kenji Tsuge, Akihiko Kondo
    Feb. 2025, ACS Synthetic Biology
    Scientific journal

  • Takahiro Bamba, Ryosuke Munakata, Yuya Ushiro, Ryota Kumokita, Sayaka Tanaka, Yoshimi Hori, Akihiko Kondo, Kazufumi Yazaki, Tomohisa Hasunuma
    Dec. 2024, ACS Synthetic Biology
    Scientific journal

  • Daisuke Nonaka, Yuuki Hirata, Mayumi Kishida, Ayana Mori, Ryosuke Fujiwara, Akihiko Kondo, Yutaro Mori, Shuhei Noda, Tsutomu Tanaka
    Aug. 2024, Biotechnology Journal
    Scientific journal

  • Filemon Jalu Nusantara Putra, Prihardi Kahar, Akihiko Kondo, Chiaki Ogino
    Aug. 2024, Biochemical Engineering Journal
    [Refereed]
    Scientific journal

  • Pamella Apriliana, Prihardi Kahar, Norimasa Kashiwagi, Akihiko Kondo, Chiaki Ogino
    Jun. 2024, Engineering in Life Sciences
    [Refereed]
    Scientific journal

  • Radityo Pangestu, Prihardi Kahar, Chiaki Ogino, Akihiko Kondo
    While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study examined the differences between flocculating and nonflocculating Saccharomyces cerevisiae strains in terms of their metabolic dynamics when incorporating an exogenous lactic acid pathway, across varying cell densities and in the presence of lignocellulose-derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to variations in the nucleotide sequences of the flocculin genes and their regulators. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.
    Apr. 2024, Yeast (Chichester, England), 41(4) (4), 192 - 206, English, International magazine
    Scientific journal

  • Kengo Sasaki, Yasunobu Takeshima, Ayami Fujino, Junya Yamashita, Akira Kimoto, Daisuke Sasaki, Akihiko Kondo, Masaya Akashi, Ryo Okumura
    We developed a simulation model of human oral microbiota using Bio Palette oral medium (BPOM) containing 0.02% glucose and lower bacterial nitrogen sources, derived from saliva and dental plaque. By decreasing the concentration of Gifu anaerobic medium (GAM) from 30 to 10 g L-1 , we observed increased ratios of target pathogenic genera, Porphyromonas and Fusobacterium from 0.5% and 1.7% to 1.2% and 3.5%, respectively, in the biofilm on hydroxyapatite (HA) discs. BPOM exhibited the higher ratios of Porphyromonas and Fusobacterium, and amplicon sequence variant number on HA, compared with GAM, modified GAM and basal medium mucin. Mixing glycerol stocks of BPOM culture solutions from four human subjects resulted in comparable ratios of these bacteria to the original saliva. In this simulation model, sitafloxacin showed higher inhibitory effects on P. gingivalis than minocycline hydrochloride at a low dosage of 0.1 μg mL-1 . Probiotics such as Streptococcus salivarius and Limosilactobacillus fermentum also showed significant decreases in Porphyromonas and Fusobacterium ratios on HA, respectively. Overall, the study suggests that BPOM with low carbon and nutrients could be a versatile platform for assessing the efficacy of antibiotics and live biotherapeutics in treating oral diseases caused by Porphyromonas and Fusobacterium.
    Apr. 2024, Environmental microbiology reports, 16(2) (2), e13243, English, International magazine
    Scientific journal

  • Yuichi Kato, Ryota Hidese, Mami Matsuda, Ryudo Ohbayashi, Hiroki Ashida, Akihiko Kondo, Tomohisa Hasunuma
    Glycogen serves as a metabolic sink in cyanobacteria. Glycogen deficiency causes the extracellular release of distinctive metabolites such as pyruvate and 2-oxoglutarate upon nitrogen depletion; however, the mechanism has not been fully elucidated. This study aimed to elucidate the mechanism of carbon partitioning in glycogen-deficient cyanobacteria. Extracellular and intracellular metabolites in a glycogen-deficient ΔglgC mutant of Synechococcus elongatus PCC 7942 were comprehensively analyzed. In the presence of a nitrogen source, the ΔglgC mutant released extracellular glutamate rather than pyruvate and 2-oxoglutarate, whereas its intracellular glutamate level was lower than that in the wild-type strain. The de novo synthesis of glutamate increased in the ΔglgC mutant, suggesting that glycogen deficiency enhanced carbon partitioning into glutamate and extracellular excretion through an unidentified transport system. This study proposes a model in which glutamate serves as the prime extracellular metabolic sink alternative to glycogen when nitrogen is available.
    Feb. 2024, Communications biology, 7(1) (1), 233 - 233, English, International magazine
    Scientific journal

  • Ryota Kumokita, Takahiro Bamba, Hisashi Yasueda, Ayato Tsukida, Keizo Nakagawa, Tooru Kitagawa, Tomohisa Yoshioka, Hideto Matsuyama, Yasuhito Yamamoto, Satoshi Maruyama, Takahiro Hayashi, Akihiko Kondo, Tomohisa Hasunuma
    Elsevier BV, Feb. 2024, Bioresource Technology, 393, 130144 - 130144
    Scientific journal

  • Radityo Pangestu, Prihardi Kahar, Lutfi Kholida, Urip Perwitasari, Ahmad Thontowi, Fahrurrozi Fahrurrozi, Puspita Lisdiyanti, Yopi Yopi, Chiaki Ogino, Bambang Prasetya, Akihiko Kondo
    Jan. 2024
    [Refereed]

  • Kenya Tanaka, Takahiro Bamba, Akihiko Kondo, Tomohisa Hasunuma
    Microbial biomanufacturing offers a promising, environment-friendly platform for next-generation chemical production. However, its limited industrial implementation is attributed to the slow production rates of target compounds and the time-intensive engineering of high-yield strains. This review highlights how metabolomics expedites bioproduction development, as demonstrated through case studies of its integration into microbial strain engineering, culture optimization, and model construction. The Design-Build-Test-Learn (DBTL) cycle serves as a standard workflow for strain engineering. Process development, including the optimization of culture conditions and scale-up, is crucial for industrial production. In silico models facilitate the development of strains and processes. Metabolomics is a powerful driver of the DBTL framework, process development, and model construction.
    Dec. 2023, Current opinion in biotechnology, 85, 103057 - 103057, English, International magazine
    Scientific journal

  • Ryota Hidese, Ryudo Ohbayashi, Yuichi Kato, Mami Matsuda, Kan Tanaka, Sousuke Imamura, Hiroki Ashida, Akihiko Kondo, Tomohisa Hasunuma
    Abstract The cyanobacterium Synechococcus elongatus PCC 7942 accumulates alarmone guanosine tetraphosphate (ppGpp) under stress conditions, such as darkness. A previous study observed that artificial ppGpp accumulation under photosynthetic conditions led to the downregulation of genes involved in the nitrogen assimilation system, which is activated by the global nitrogen regulator NtcA, suggesting that ppGpp regulates NtcA activity. However, the details of this mechanism have not been elucidated. Here, we investigate the metabolic responses associated with ppGpp accumulation by heterologous expression of the ppGpp synthetase RelQ. The pool size of 2-oxoglutarate (2-OG), which activates NtcA, is significantly decreased upon ppGpp accumulation. De novo 13C-labeled CO2 assimilation into the Calvin-Benson-Bassham cycle and glycolytic intermediates continues irrespective of ppGpp accumulation, whereas the labeling of 2-OG is significantly decreased under ppGpp accumulation. The low 2-OG levels in the RelQ overexpression cells could be because of the inhibition of metabolic enzymes, including aconitase, which are responsible for 2-OG biosynthesis. We propose a metabolic rearrangement by ppGpp accumulation, which negatively regulates 2-OG levels to maintain carbon and nitrogen balance.
    Springer Science and Business Media LLC, Dec. 2023, Communications Biology, 6(1) (1)
    Scientific journal

  • Filemon Jalu Nusantara Putra, Prihardi Kahar, Akihiko Kondo, Chiaki Ogino
    Nov. 2023, Biochemical Engineering Journal
    [Refereed]
    Scientific journal


  • Akihiko Kondo
    Synthetic methylotrophic Yarrowia lipolytica was constructed to convert methanol into biomass components and succinic acid.
    Royal Society of Chemistry (RSC), Oct. 2023, Green Chemistry, 25(1) (1), 183 - 195, English
    [Refereed]
    Scientific journal

  • Kentaro Inokuma, Daisuke Sasaki, Kaoru Kurata, Megumi Ichikawa, Yuya Otsuka, Akihiko Kondo
    Abstract Chondroitin sulfate (CS) is a family of glycosaminoglycans and have a wide range of applications in dietary supplements and pharmaceutical drugs. In this study, we evaluated the effects of several types of CS, differing in their sulfated positions, on the human colonic microbiota and their metabolites. CS (CSA, CSC, and CSE) and non-sulfated chondroitin (CH) were added into an in vitro human colonic microbiota model with fecal samples from 10 healthy individuals. CS addition showed a tendency to increase the relative abundance of Bacteroides, Eubacterium, and Faecalibacterium, and CSC and CSE addition significantly increased the total number of eubacteria in the culture of the Kobe University Human Intestinal Microbiota Model. CSE addition also resulted in a significant increase in short-chain fatty acid (SCFA) levels. Furthermore, addition with CSC and CSE increased the levels of a wide range of metabolites including lysine, ornithine, and Ile-Pro-Pro, which could have beneficial effects on the host. However, significant increases in the total number of eubacteria, relative abundance of Bacteroides, and SCFA levels were also observed after addition with CH, and the trends in the effects of CH addition on metabolite concentrations were identical to those of CSC and CSE addition. These results provide novel insight into the contribution of the colonic microbiota to the beneficial effects of dietary CS.
    Springer Science and Business Media LLC, Jul. 2023, Scientific Reports, 13(1) (1), 12313, English
    [Refereed]
    Scientific journal


  • Daisuke Sasaki, Kengo Sasaki, Aya Abe, Makoto Ozeki, Akihiko Kondo
    Partially hydrolyzed guar gums (PHGGs) are prebiotic soluble dietary fibers. High molecular-weight PHGGs have rapid fermentation and high short-chain fatty acid (SCFA) productivity rates, compared to low molecular-weight PHGGs. Therefore, low molecular-weight PHGGs may have less pronounced prebiotic effects than high molecular-weight PHGGs. However, the effects of PHGGs of different molecular weights on the human intestinal microbiota, as well as their fermentation ability and prebiotic effects, have not been investigated. The aim of this study was to evaluate the effects of two PHGGs of different molecular weights, Sunfiber-R (SF-R; 20 kDa) and Sunfiber-V (SF-V; 5 kDa), on human colonic microbiota and SCFA production. A human intestinal in vitro fermentation model was operated by fecal samples with and without the PHGGs. The addition of 0.2% SF-R or SF-V increased the relative abundance of Bacteroides spp., especially that of Bacteroides uniformis. This increase corresponded to a significant (p = 0.030) and non-significant (p = 0.073) increase in propionate production in response to SF-R and SF-V addition, respectively. Both fibers increased the relative abundance of Faecalibacterium and stimulated an increase in the abundance of unclassified Lachnospiraceae and Bifidobacterium. In conclusion, the low molecular-weight PHGG exerted prebiotic effects on human colonic microbiota to increase SCFA production and bacteria that are beneficial to human health in a manner similar to that of the high molecular-weight forms of PHGG.
    Jul. 2023, Journal of bioscience and bioengineering, 136(1) (1), 67 - 73, English, Domestic magazine
    Scientific journal

  • Misa Doke, Mayumi Kishida, Yuuki Hirata, Mariko Nakano, Mayo Horita, Daisuke Nonaka, Yutaro Mori, Ryosuke Fujiwara, Akihiko Kondo, Shuhei Noda, Tsutomu Tanaka
    Sciscan Publishing Limited, Jul. 2023, Synthetic Biology and Engineering, 1(2) (2), 10012 - 9
    [Refereed]
    Scientific journal

  • Dao Duy Hanh, Taghreed Elkasaby, Hideo Kawaguchi, Yota Tsuge, Chiaki Ogino, Akihiko Kondo
    Itaconic acid (IA) is a value-added chemical currently produced by Aspergillus terreus from edible glucose and starch but not from inedible lignocellulosic biomass owing to the high sensitivity to fermentation inhibitors present in the hydrolysate of lignocellulosic biomass. To produce IA from lignocellulosic biomass, a gram-positive bacterium, Corynebacterium glutamicum, with a high tolerance to fermentation inhibitors was metabolically engineered to express a fusion protein composed of cis-aconitate decarboxylase from A. terreus responsible for IA formation from cis-aconitate and a maltose-binding protein (malE) from Escherichia coli. The codon-optimized cadA_malE gene was expressed in C. glutamicum ATCC 13032, and the resulting recombinant strain produced IA from glucose. IA concentration increased 4.7-fold by the deletion of the ldh gene encoding lactate dehydrogenase. With the Δldh strain HKC2029, an 18-fold higher IA production was observed from enzymatic hydrolysate of kraft pulp as a model lignocellulosic biomass than from glucose (6.15 and 0.34 g/L, respectively). The enzymatic hydrolysate of kraft pulp contained various potential fermentation inhibitors involved in furan aldehydes, benzaldehydes, benzoic acids, cinnamic acid derivatives, and aliphatic acid. Whereas cinnamic acid derivatives severely inhibited IA production, furan aldehydes, benzoic acids, and aliphatic acid improved IA production at low concentrations. The present study suggests that lignocellulosic hydrolysate contains various potential fermentation inhibitors; however, some of them can serve as enhancers for microbial fermentation likely due to the changing of redox balance in the cell.
    Jul. 2023, Journal of bioscience and bioengineering, 136(1) (1), 7 - 12, English, Domestic magazine
    Scientific journal

  • Yuji Haraguchi, Yuichi Kato, Kosuke Inabe, Akihiko Kondo, Tomohisa Hasunuma, Tatsuya Shimizu
    Springer Science and Business Media LLC, Jun. 2023, Archives of Microbiology, 205(7) (7), English
    [Refereed]
    Scientific journal

  • Taghreed Elkasaby, Dao Duy Hanh, Hideo Kawaguchi, Akihiko Kondo, Chiaki Ogino
    Itaconic acid (IA), a C5-dicarboxylic acid, is a potential bio-based building block for the polymer industry. There are three pathways for IA production from natural IA producers; however, most of the engineered strains were used for IA production by heterologous expression of cis-aconitate decarboxylase gene (cadA) from Aspergillus terreus. In this study, IA was produced by an engineered Corynebacterium glutamicum ATCC 13032 expressing two different types of genes from two distinct pathways. The first involves the mammalian immunoresponsive gene1 (Irg1) derived from Mus musculus. The second (termed here the trans-pathway) involves two genes from the natural IA producer Ustilago maydis which are aconitate-delta-isomerase (Adi1) and trans-aconitate decarboxylase (Tad1) genes. The constructed strains developing the two distinct IA production pathways: C. glutamicum ATCC 13032 pCH-Irg1opt and C. glutamicum ATCC 13032 pCH-Tad1optadi1opt were used for production of IA from different carbon sources. The results reflect the possibility for IA production from C. glutamicum expressing the trans-pathway (Adi1/Tad1 genes) and cis-pathway (Irg1 gene) other than the well-known cis-pathway that depends mainly on cadA gene from A. terreus. The developed strain expressing trans-pathway from U. maydis; however, proved to be better at IA production with high titers of 12.25, 11.34, and 11.02 g/L, and a molar yield of 0.22, 0.42, and 0.43 mol/mol from glucose, maltose, and sucrose, respectively, via fed-batch fermentation. The present study suggests that trans-pathway is better than cis-pathway for IA production in engineered C. glutamicum.
    Jun. 2023, Journal of bioscience and bioengineering, English, Domestic magazine
    Scientific journal

  • Yuichi Kato, Kosuke Inabe, Yuji Haraguchi, Tatsuya Shimizu, Akihiko Kondo, Tomohisa Hasunuma
    Abstract l-Lactate is a major waste compound in cultured animal cells. To develop a sustainable animal cell culture system, we aimed to study the consumption of l-lactate using a photosynthetic microorganism. As genes involved in l-lactate utilization were not found in most cyanobacteria and microalgae, we introduced the NAD-independent l-lactate dehydrogenase gene from Escherichia coli (lldD) into Synechococcus sp. PCC 7002. The lldD-expressing strain consumed l-lactate added to basal medium. This consumption was accelerated by expression of a lactate permease gene from E. coli (lldP) and an increase in culture temperature. Intracellular levels of acetyl-CoA, citrate, 2-oxoglutarate, succinate, and malate, and extracellular levels of 2-oxoglutarate, succinate, and malate, increased during l-lactate utilization, suggesting that the metabolic flux from l-lactate was distributed toward the tricarboxylic acid cycle. This study provides a perspective on l-lactate treatment by photosynthetic microorganisms, which would increase the feasibility of animal cell culture industries.
    Springer Science and Business Media LLC, May 2023, Scientific Reports, 13(1) (1), English
    [Refereed]
    Scientific journal

  • Erina Yoshida, Yuichi Kato, Akihiko Kanamoto, Akihiko Kondo, Tomohisa Hasunuma
    Elsevier BV, May 2023, Algal Research, 72, 103144 - 103144, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Mar. 2023, Enzyme and Microbial Technology, 164, 110193 - 110193, English
    Scientific journal

  • Takahiro Yukawa, Takahiro Bamba, Mami Matsuda, Takanobu Yoshida, Kentaro Inokuma, Jungyeon Kim, Jae Won Lee, Yong‐Su Jin, Akihiko Kondo, Tomohisa Hasunuma
    Wiley, Feb. 2023, Biotechnology and Bioengineering, 120(2) (2), 511 - 523, English
    [Refereed]
    Scientific journal

  • Lucky Risanto, Deddy Triyono Nugroho Adi, Triyani Fajriutami, Hiroshi Teramura, Widya Fatriasari, Euis Hermiati, Prihardi Kahar, Akihiko Kondo, Chiaki Ogino
    Lignocellulose is resistant to degradation and requires pretreatment before hydrolytic enzymes can release fermentable sugars. Sulfuric acid has been widely used for biomass pretreatment, but high amount of degradation products usually occurred when using this method. To enhance accessibility to cellulose, we studied the performances of several dilute organic acid pretreatments of sugarcane bagasse and oil palm empty fruit bunch fiber. The results revealed that pretreatment with maleic acid yields the highest xylose and glucose release among other organic acids. The effects of concentration, duration of heating and heating temperature were further studied. Dilute maleic acid 1% (w/w) pretreatment at 180 °C was the key to its viability as a substitute for sulfuric acid. Moreover, maleic acid did not seem to highly promote the formation of either furfural or 5-HMF in the liquid hydrolysate after pretreatment.
    Elsevier BV, Feb. 2023, Bioresource Technology, 369, 128382 - 128382, English, International magazine
    [Refereed]
    Scientific journal

  • Soh Ishiguro, Kana Ishida, Rina C. Sakata, Hideto Mori, Mamoru Takana, Samuel King, Omar Bashth, Minori Ichiraku, Nanami Masuyama, Ren Takimoto, Yusuke Kijima, Arman Adel, Hiromi Toyoshima, Motoaki Seki, Ju Hee Oh, Anne-Sophie Archambault, Keiji Nishida, Akihiko Kondo, Satoru Kuhara, Hiroyuki Aburatani, Ramon I. Klein Geltink, Yasuhiro Takashima, Nika Shakiba, Nozomu Yachie
    Clonal heterogeneity underlies diverse biological processes, including cancer progression, cell differentiation, and microbial evolution. Cell tagging strategies with DNA barcodes have recently enabled analysis of clone size dynamics and clone-restricted transcriptomic landscapes of heterogeneous populations. However, isolating a target clone that displays a specific phenotype from a complex population remains challenging. Here, we present a new multi-kingdom genetic barcoding system, CloneSelect, in which a target cell clone can be triggered to express a reporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells are first barcoded and propagated so their subpopulation can be subjected to a given experiment. A clone that shows a phenotype or genotype of interest at a given time can then be isolated from the initial or subsequent cell pools stored throughout the experimental timecourse. This novel CRISPR-barcode genetics platform provides many new ways of analyzing and manipulating mammalian, yeast, and bacterial systems. Teaser A multi-kingdom CRISPR-activatable barcoding system enables the precise isolation of target barcode-labeled clones from a complex cell population.
    Cold Spring Harbor Laboratory, Jan. 2023

  • Nunthaphan Vikromvarasiri, Shuhei Noda, Tomokazu Shirai, Akihiko Kondo
    Abstract Background Flux Balance Analysis (FBA) is a well-known bioinformatics tool for metabolic engineering design. Previously, we have successfully used single-level FBA to design metabolic fluxes in Bacillus subtilis to enhance (R,R)-2,3-butanediol (2,3-BD) production from glycerol. OptKnock is another powerful technique for devising gene deletion strategies to maximize microbial growth coupling with improved biochemical production. It has never been used in B. subtilis. In this study, we aimed to compare the use of single-level FBA and OptKnock for designing enhanced 2,3-BD production from glycerol in B. subtilis. Results Single-level FBA and OptKnock were used to design metabolic engineering approaches for B. subtilis to enhance 2,3-BD production from glycerol. Single-level FBA indicated that deletion of ackA, pta, lctE, and mmgA would improve the production of 2,3-BD from glycerol, while OptKnock simulation suggested the deletion of ackA, pta, mmgA, and zwf. Consequently, strains LM01 (single-level FBA-based) and MZ02 (OptKnock-based) were constructed, and their capacity to produce 2,3-BD from glycerol was investigated. The deletion of multiple genes did not negatively affect strain growth and glycerol utilization. The highest 2,3-BD production was detected in strain LM01. Strain MZ02 produced 2,3-BD at a similar level as the wild type, indicating that the OptKnock prediction was erroneous. Two-step FBA was performed to examine the reason for the erroneous OptKnock prediction. Interestingly, we newly found that zwf gene deletion in strain MZ02 improved lactate production, which has never been reported to date. The predictions of single-level FBA for strain MZ02 were in line with experimental findings. Conclusions We showed that single-level FBA is an effective approach for metabolic design and manipulation to enhance 2,3-BD production from glycerol in B. subtilis. Further, while this approach predicted the phenotypes of generated strains with high precision, OptKnock prediction was not accurate. We suggest that OptKnock modelling predictions be evaluated by using single-level FBA to ensure the accuracy of metabolic pathway design. Furthermore, the zwf gene knockout resulted in the change of metabolic fluxes to enhance the lactate productivity.
    Springer Science and Business Media LLC, Jan. 2023, Journal of Biological Engineering, 17(1) (1)
    Scientific journal

  • Kentaro Inokuma, Shunya Miyamoto, Kohei Morinaga, Yuma Kobayashi, Ryota Kumokita, Takahiro Bamba, Yoichiro Ito, Akihiko Kondo, Tomohisa Hasunuma
    Wiley, Jan. 2023, Biotechnology and Bioengineering, 120(4) (4), 1097 - 1107, English
    [Refereed]
    Scientific journal



  • Akihiko Kondo
    2023, Internal Medicine
    Scientific journal


  • Hideo Kawaguchi, Shunsuke Masuo, Keiko Wakai, Naoki Takaya, Tomohisa Hasunma, Tatsuo Kaneko, Satoshi Okada, Takashi Sazuka, Chiaki Ogino, Akihiko Kondo
    4-Aminophenylalanine as a diamine monomer to synthesize unique biopolyimides was produced from lignocellulosic biomass by microbial fermentation. Fermentation inhibitors present in lignocellulosic hydrolysate of sorghum bagasse were identified.
    Royal Society of Chemistry (RSC), 2023, RSC Sustainability
    Scientific journal

  • Akihiko Kondo
    Abstract The bottleneck for the production of biofuels from microalgae consists on costly harvesting processes and low lipid production, immobilization technology could play a part on making the production of biofuels more feasible. The aim of this study was to evaluate the effect of alginate immobilization on the growth and lipid productivity of the microalgae Chlorella sorokiniana, so far, the main focus of immobilization technology has been its use for wastewater treatment and nutrient removal from effluents. The microalgae Chlorella sorokiniana was cultured in both free and immobilized forms under optimal autotrophic growth conditions. Microalgae were immobilized in calcium alginate beads generated by mixing algal cells with a sodium alginate solution, followed by extrusion into a CaCl2 solution. The results obtained in this study showed that the growth of the microalgae immobilized in alginate beads, was enhanced and achieved a dry cell weight 1.4-fold higher than that of a free cell culture, a higher light transmittance was also achieved in the alginate immobilized culture, and the lipid productivity was increased from 54.21 ± 2.48 mg l−1 d in the free cell culture to 82.22 ± 8.48 mg l−1 d in the immobilized culture. These results demonstrate the effectiveness of immobilization technology for promoting growth and lipid productivity in the microalgae Chlorella sorokiniana.
    IOP Publishing, Jan. 2023, Journal of Physics: Energy, 5(1) (1), 014019 - 014019, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Jan. 2023, Journal of Chromatography B, 1215, 123588 - 123588, English
    [Refereed]
    Scientific journal

  • Kenya Tanaka, Tomokazu Shirai, Christopher J Vavricka, Mami Matsuda, Akihiko Kondo, Tomohisa Hasunuma
    Abstract Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin–Benson–Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies have shown that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid dynamic changes (on a time scale of seconds) in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enabled estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicated that downstream glycolytic intermediates, including phosphoglycerate and phosphoenolpyruvate, accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant decreases in the initial oxygen evolution rate were observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathways. Accordingly, the degree of decrease in the initial oxygen evolution rate was proportional to the accumulated pool size of glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.
    Oxford University Press (OUP), Dec. 2022, Plant Physiology, English
    [Refereed]
    Scientific journal

  • Varada Jagadeesh, Takanobu Yoshida, Misugi Uraji, Nobuyuki Okahashi, Fumio Matsuda, Christopher J. Vavricka, Kenji Tsuge, Akihiko Kondo
    American Chemical Society (ACS), Dec. 2022, ACS Synthetic Biology, 12(1) (1), 305 - 318, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Dec. 2022, Biochemical Engineering Journal, 188, 108681 - 108681, English
    [Refereed]
    Scientific journal

  • Christopher J. Vavricka, Shunsuke Takahashi, Naoki Watanabe, Musashi Takenaka, Mami Matsuda, Takanobu Yoshida, Ryo Suzuki, Hiromasa Kiyota, Jianyong Li, Hiromichi Minami, Jun Ishii, Kenji Tsuge, Michihiro Araki, Akihiko Kondo, Tomohisa Hasunuma
    Abstract Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes. Therefore, the machine learning discovery of specialized enzymes offers great potential to expand the range of biosynthesis pathways. Benzylisoquinoline alkaloid production is a model example of metabolic engineering with potential to revolutionize the paradigm of sustainable biomanufacturing. Existing bacterial studies utilize a norlaudanosoline pathway, whereas plants contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are still produced using microbial enzymes that remain elusive in plants, and additional downstream missing links remain hidden within highly duplicated plant gene families. In the current study, machine learning is applied to predict and select plant missing link enzymes from homologous candidate sequences. Metabolomics-based characterization of the selected sequences reveals potential aromatic acetaldehyde synthases and phenylpyruvate decarboxylases in reconstructed plant gene-only benzylisoquinoline alkaloid pathways from tyrosine. Synergistic application of the aryl acetaldehyde producing enzymes results in enhanced benzylisoquinoline alkaloid production through hybrid norcoclaurine and norlaudanosoline pathways.
    Springer Science and Business Media LLC, Dec. 2022, Nature Communications, 13(1) (1), 1405 - 1405, English, International magazine
    Scientific journal

  • Ryota Hidese, Mami Matsuda, Mamiko Kajikawa, Takashi Osanai, Akihiko Kondo, Tomohisa Hasunuma
    The four-carbon (C4) dicarboxylic acids, fumarate, malate, and succinate, are the most valuable targets that must be exploited for CO2-based chemical production in the move to a sustainable low-carbon future. Cyanobacteria excrete high amounts of C4 dicarboxylic acids through glycogen fermentation in a dark anoxic environment. The enhancement of metabolic flux in the reductive TCA branch in the Cyanobacterium Synechocystis sp. PCC6803 is a key issue in the C4 dicarboxylic acid production. To improve metabolic flux through the anaplerotic pathway, we have created the recombinant strain PCCK, which expresses foreign ATP-forming phosphoenolpyruvate carboxykinase (PEPck) concurrent with intrinsic phosphoenolpyruvate carboxylase (Ppc) overexpression. Expression of PEPck concurrent with Ppc led to an increase in C4 dicarboxylic acids by autofermentation. Metabolome analysis revealed that PEPck contributed to an increase in carbon flux from hexose and pentose phosphates into the TCA reductive branch. To enhance the metabolic flux in the reductive TCA branch, we examined the effect of corn-steep liquor (CSL) as a nutritional supplement on C4 dicarboxylic acid production. Surprisingly, the addition of sterilized CSL enhanced the malate production in the PCCK strain. Thereafter, the malate and fumarate excreted by the PCCK strain are converted into succinate by the CSL-settling microorganisms. Finally, high-density cultivation of cells lacking the acetate kinase gene showed the highest production of malate and fumarate (3.2 and 2.4 g/L with sterilized CSL) and succinate (5.7 g/L with non-sterile CSL) after 72 h cultivation. The present microbial community engineering is useful for succinate production by one-pot fermentation under dark anoxic conditions.
    American Chemical Society (ACS), Nov. 2022, ACS synthetic biology, 11(12) (12), 4054 - 4064, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Nov. 2022, Journal of Biotechnology, 358, 111 - 117, English
    [Refereed]
    Scientific journal

  • Tomoki Oyama, Yuichi Kato, Ryota Hidese, Mami Matsuda, Minenosuke Matsutani, Satoru Watanabe, Akihiko Kondo, Tomohisa Hasunuma
    Abstract Background Microalgal lipid production has attracted global attention in next-generation biofuel research. Nitrogen starvation, which drastically suppresses cell growth, is a common and strong trigger for lipid accumulation in microalgae. We previously developed a mutant Chlamydomonas sp. KAC1801, which can accumulate lipids irrespective of the presence or absence of nitrates. This study aimed to develop a feasible strategy for stable and continuous lipid production through semi-continuous culture of KAC1801. Results KAC1801 continuously accumulated > 20% lipid throughout the subculture (five generations) when inoculated with a dry cell weight of 0.8–0.9 g L−1 and cultured in a medium containing 18.7 mM nitrate, whereas the parent strain KOR1 accumulated only 9% lipid. Under these conditions, KAC1801 continuously produced biomass and consumed nitrates. Lipid productivity of 116.9 mg L−1 day−1 was achieved by semi-continuous cultivation of KAC1801, which was 2.3-fold higher than that of KOR1 (50.5 mg L−1 day−1). Metabolome and transcriptome analyses revealed a depression in photosynthesis and activation of nitrogen assimilation in KAC1801, which are the typical phenotypes of microalgae under nitrogen starvation. Conclusions By optimizing nitrate supply and cell density, a one-step cultivation system for Chlamydomonas sp. KAC1801 under nitrate-replete conditions was successfully developed. KAC1801 achieved a lipid productivity comparable to previously reported levels under nitrogen-limiting conditions. In the culture system of this study, metabolome and transcriptome analyses revealed a nitrogen starvation-like response in KAC1801.
    Springer Science and Business Media LLC, Sep. 2022, Biotechnology for Biofuels and Bioproducts, 15(1) (1), English
    [Refereed]
    Scientific journal

  • Naoki Watanabe, Masaki Yamamoto, Masahiro Murata, Christopher J. Vavricka, Chiaki Ogino, Akihiko Kondo, Michihiro Araki
    New enzyme functions exist within the increasing number of unannotated protein sequences. Novel enzyme discovery is necessary to expand the pathways that can be accessed by metabolic engineering for the biosynthesis of functional compounds. Accordingly, various machine learning models have been developed to predict enzymatic reactions. However, the ability to predict unknown reactions that are not included in the training data has not been clarified. In order to cover uncertain and unknown reactions, a wider range of reaction types must be demonstrated by the models. Here, we establish 16 expanded enzymatic reaction prediction models developed using various machine learning algorithms, including deep neural network. Improvements in prediction performances over that of our previous study indicate that the updated methods are more effective for the prediction of enzymatic reactions. Overall, the deep neural network model trained with combined substrate-enzyme-product information exhibits the highest prediction accuracy with Macro F1 scores up to 0.966 and with robust prediction of unknown enzymatic reactions that are not included in the training data. This model can predict more extensive enzymatic reactions in comparison to previously reported models. This study will facilitate the discovery of new enzymes for the production of useful substances.
    Sep. 2022, Journal of Physical Chemistry B, 126(36) (36), 6762 - 6770
    Scientific journal

  • Naoki Watanabe, Masaki Yamamoto, Masahiro Murata, Christopher J Vavricka, Chiaki Ogino, Akihiko Kondo, Michihiro Araki
    New enzyme functions exist within the increasing number of unannotated protein sequences. Novel enzyme discovery is necessary to expand the pathways that can be accessed by metabolic engineering for the biosynthesis of functional compounds. Accordingly, various machine learning models have been developed to predict enzymatic reactions. However, the ability to predict unknown reactions that are not included in the training data has not been clarified. In order to cover uncertain and unknown reactions, a wider range of reaction types must be demonstrated by the models. Here, we establish 16 expanded enzymatic reaction prediction models developed using various machine learning algorithms, including deep neural network. Improvements in prediction performances over that of our previous study indicate that the updated methods are more effective for the prediction of enzymatic reactions. Overall, the deep neural network model trained with combined substrate-enzyme-product information exhibits the highest prediction accuracy with Macro F1 scores up to 0.966 and with robust prediction of unknown enzymatic reactions that are not included in the training data. This model can predict more extensive enzymatic reactions in comparison to previously reported models. This study will facilitate the discovery of new enzymes for the production of useful substances.
    Sep. 2022, The journal of physical chemistry. B, 126(36) (36), 6762 - 6770, English, International magazine
    Scientific journal

  • Yoichiro Ito, Misa Ishigami, Noriko Hashiba, Yasuyuki Nakamura, Goro Terai, Tomohisa Hasunuma, Jun Ishii, Akihiko Kondo
    Wiley, Sep. 2022, Microbial Biotechnology, 15(9) (9), 2364 - 2378, English
    [Refereed]
    Scientific journal

  • Radityo Pangestu, Prihardi Kahar, Lutfi Nia Kholida, Urip Perwitasari, Ahmad Thontowi, Fahrurrozi, Puspita Lisdiyanti, Yopi, Chiaki Ogino, Bambang Prasetya, Akihiko Kondo
    Acidic and chemical inhibitor stresses undermine efficient lactic acid bioproduction from lignocellulosic feedstock. Requisite coping treatments, such as detoxification and neutralizing agent supplementation, can be eliminated if a strong microbial host is employed in the process. Here, we exploited an originally robust yeast, Saccharomyces cerevisiae BTCC3, as a production platform for lactic acid. This wild-type strain exhibited a rapid cell growth in the presence of various chemical inhibitors compared to laboratory and industrial strains, namely BY4741 and Ethanol-red. Pathway engineering was performed on the strain by introducing an exogenous LDH gene after disrupting the PDC1 and PDC5 genes. Facilitated by this engineered strain, high cell density cultivation could generate lactic acid with productivity at 4.80 and 3.68 g L-1 h-1 under semi-neutralized and non-neutralized conditions, respectively. Those values were relatively higher compared to other studies. Cultivation using real lignocellulosic hydrolysate was conducted to assess the performance of this engineered strain. Non-neutralized fermentation using non-detoxified hydrolysate from sugarcane bagasse as a medium could produce lactic acid at 1.69 g L-1 h-1, which was competitive to the results from other reports that still included detoxification and neutralization steps in their experiments. This strategy could make the overall lactic acid bioproduction process simpler, greener, and more cost-efficient.
    Springer Science and Business Media LLC, Aug. 2022, Scientific reports, 12(1) (1), 13645 - 13645, English, International magazine
    Scientific journal

  • Jyumpei Kobayashi, Daisuke Sasaki, Kiyotaka Y Hara, Tomohisa Hasunuma, Akihiko Kondo
    BACKGROUND: Glutathione is a valuable tri-peptide that is industrially produced by fermentation using the yeast Saccharomyces cerevisiae, and is widely used in the pharmaceutical, food, and cosmetic industries. It has been reported that addition of L-serine (L-Ser) is effective at increasing the intracellular glutathione content because L-Ser is the common precursor of L-cysteine (L-Cys) and glycine (Gly) which are substrates for glutathione biosynthesis. Therefore, we tried to enhance the L-Ser biosynthetic pathway in S. cerevisiae for improved glutathione production. RESULTS: The volumetric glutathione production of recombinant strains individually overexpressing SER2, SER1, SER3, and SER33 involved in L-Ser biosynthesis at 48 h cultivation was increased 1.3, 1.4, 1.9, and 1.9-fold, respectively, compared with that of the host GCI strain, which overexpresses genes involved in glutathione biosynthesis. We further examined simultaneous overexpression of SHM2 and/or CYS4 genes involved in Gly and L-Cys biosynthesis, respectively, using recombinant GCI strain overexpressing SER3 and SER33 as hosts. As a result, GCI overexpressing SER3, SHM2, and CYS4 showed the highest volumetric glutathione production (64.0 ± 4.9 mg/L) at 48 h cultivation, and this value is about 2.5-fold higher than that of the control strain. CONCLUSIONS: This study first revealed that engineering of L-Ser and Gly biosynthetic pathway are useful strategies for fermentative glutathione production by S. cerevisiase.
    Springer Science and Business Media LLC, Aug. 2022, Microbial cell factories, 21(1) (1), 153 - 153, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Aug. 2022, Journal of the Taiwan Institute of Chemical Engineers, 137, 104198 - 104198, English
    Scientific journal

  • Shota Isogai, Masahiro Tominaga, Akihiko Kondo, Jun Ishii
    Flavonoids, a major group of secondary metabolites in plants, are promising for use as pharmaceuticals and food supplements due to their health-promoting biological activities. Industrial flavonoid production primarily depends on isolation from plants or organic synthesis, but neither is a cost-effective or sustainable process. In contrast, recombinant microorganisms have significant potential for the cost-effective, sustainable, environmentally friendly, and selective industrial production of flavonoids, making this an attractive alternative to plant-based production or chemical synthesis. Structurally and functionally diverse flavonoids are derived from flavanones such as naringenin, pinocembrin and eriodictyol, the major basic skeletons for flavonoids, by various modifications. The establishment of flavanone-producing microorganisms can therefore be used as a platform for producing various flavonoids. This review summarizes metabolic engineering and synthetic biology strategies for the microbial production of flavanones. In addition, we describe directed evolution strategies based on recently-developed high-throughput screening technologies for the further improvement of flavanone production. We also describe recent progress in the microbial production of structurally and functionally complicated flavonoids via the flavanone modifications. Strategies based on synthetic biology will aid more sophisticated and controlled microbial production of various flavonoids.
    Frontiers Media SA, Jul. 2022, Frontiers in Chemical Engineering, 4, 880694, English
    Scientific journal

  • Satoko Niimi-Nakamura, Hideo Kawaguchi, Kouji Uematsu, Hiroshi Teramura, Sachiko Nakamura-Tsuruta, Norimasa Kashiwagi, Yoshinori Sugai, Yohei Katsuyama, Yasuo Ohnishi, Chiaki Ogino, Akihiko Kondo
    The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.
    Microbiology Research Foundation, Jul. 2022, The Journal of General and Applied Microbiology, 68(2) (2), 109 - 116, English, Domestic magazine
    Scientific journal

  • Masahiro Tominaga, Keita Miyazaki, Shoko Hataya, Yasumasa Mitsui, Shuji Kuroda, Akihiko Kondo, Jun Ishii
    Fermentative production of squalene in yeast as an alternative approach to extracting squalene from sharks or plants has attracted significant interest. However, squalene accumulation is limited due to its inevitable high-flux allocation toward ergosterol synthesis. In this study, we described expression control of squalene monooxygenase (Erg1p), the first-step enzyme of ergosterol synthesis from squalene, to significantly reduce squalene loss. We replaced the ERG1 promoter (PERG1) with three natural yeast promoters with different activities (PPCL2, PHCM1, and PTHI2). ERG1 controlled by PTHI2 showed 20 times higher squalene production compared with the wild-type strain, whereas the other two strains exhibited no significant difference. By combining the overexpression of rate-limiting enzyme and the deletion of non-essential competing pathway gene, the yeast Saccharomyces cerevisiae produced up to 379 mg/L of squalene.
    Elsevier BV, Jul. 2022, Journal of Bioscience and Bioengineering, 134(1) (1), 1 - 6, English, Domestic magazine
    [Refereed]
    Scientific journal

  • Prihardi Kahar, Akiho Itomi, Hikari Tsuboi, Miki Ishizaki, Misa Yasuda, Chie Kihira, Hiromi Otsuka, Nurlina binti Azmi, Hana Matsumoto, Chiaki Ogino, Akihiko Kondo
    When lignocellulosic biomass is utilized as a fermentative substrate to produce biochemicals, the existence of a yeast strain resistant to inhibitory chemical compounds (ICCs) released from the biomass becomes critical. To achieve the purpose, in this study, Saccharomyces yeast strains from a NBRC yeast culture collection were used for exploration and evaluated in two different media containing ICCs that mimic one another but resemble the hydrolysate of real biomass. Among them, S. cerevisiae F118 strain shows robustness upon the fermentation with unique flocculation trait that was strongly responsive to ICC stress. When this strain was cultured in the presence of ICCs, its cell wall hydrophobicity increased dramatically, and reduced significantly when the ICCs were depleted, demonstrating that cell-surface hydrophobicity can also act as an adaptive response to the ICCs. Cells from the strain with the highest cell-wall hydrophobicity displayed progressively stronger flocculation, indicating that the F118 strain is having unique robustness under ICC stress. Gene expression perturbation analysis revealed that mot3 gene encoding regulatory Mot3p from the F118 strain was expressed in response to the concentration of ICCs. This gene was found to control expression of ygp1 gene that encoding Ygp1p, one of cell wall proteins. Deep sequencing analysis revealed that the Mot3p of the F118 strain features a unique insertion and deletion of nucleotides that encode glutamine or asparagine residues, particularly in N-terminal domain, as determined by comparison to the Mot3p sequence from the S288c strain, which was employed as a control strain. Furthermore, the cell wall hydrophobicity of the S288c strain was greatly enhanced and became ICC-responsive after gene swapping with the mot3 gene from the F118 strain. The gene-swapped S288c strain fermented 6-fold faster than the wild-type strain, producing 14.5 g/L of ethanol from 30 g/L of glucose consumed within 24 h in a medium containing the ICCs. These such modifications to Mot3p in unique locations in its sequence have a potential to change the expression of a gene involved in cell wall hydrophobicity and boosted the flocculation response to ICC stress, allowing for the acquisition of extraordinary robustness.
    Elsevier BV, Jul. 2022, Metabolic Engineering, 72, 82 - 96, English, International magazine
    Scientific journal

  • Ryosuke Fujiwara, Mariko Nakano, Yuuki Hirata, Chisako Otomo, Daisuke Nonaka, Sakiya Kawada, Hikaru Nakazawa, Mitsuo Umetsu, Tomokazu Shirai, Shuhei Noda, Tsutomu Tanaka, Akihiko Kondo
    Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of β-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICBGlc, and then captured by β-glucosidase.
    Elsevier BV, Jul. 2022, Metabolic Engineering, 72, 68 - 81, English, International magazine
    Scientific journal

  • Ryota Kumokita, Takahiro Bamba, Kentaro Inokuma, Takanobu Yoshida, Yoichiro Ito, Akihiko Kondo, Tomohisa Hasunuma
    American Chemical Society (ACS), Jun. 2022, ACS Synthetic Biology, 11(6) (6), 2098 - 2107, English
    Scientific journal

  • Akihiko Kondo
    Abstract Expression of secreted recombinant proteins burdens the protein secretion machinery, limiting production. Here, we describe an approach to improving protein production by the non-conventional yeast Komagataella phaffii comprised of genome-wide screening for effective gene disruptions, combining them in a single strain, and recovering growth reduction by adaptive evolution. For the screen, we designed a multiwell-formatted, streamlined workflow to high-throughput assay of secretion of a single-chain small antibody, which is cumbersome to detect but serves as a good model of proteins that are difficult to secrete. Using the consolidated screening system, we evaluated >19,000 mutant strains from a mutant library prepared by a modified random gene-disruption method, and identified six factors for which disruption led to increased antibody production. We then combined the disruptions, up to quadruple gene knockouts, which appeared to contribute independently, in a single strain and observed an additive effect. Target protein and promoter were basically interchangeable for the effects of knockout genes screened. We finally used adaptive evolution to recover reduced cell growth by multiple gene knockouts and examine the possibility for further enhancing protein secretion. Our successful, three-part approach holds promise as a method for improving protein production by non-conventional microorganisms.
    Springer Science and Business Media LLC, Jun. 2022, Communications Biology, 5(1) (1), 561, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Jun. 2022, Journal of Environmental Chemical Engineering, 10(3) (3), 107550 - 107550, English
    [Refereed]
    Scientific journal

  • Tomokazu Shirai, Akihiko Kondo
    The optimization of metabolic reaction modifications for the production of target compounds is a complex computational problem whose execution time increases exponentially with the number of metabolic reactions. Therefore, practical technologies are needed to identify reaction deletion combinations to minimize computing times and promote the production of target compounds by modifying intracellular metabolism. In this paper, a practical metabolic design technology named AERITH is proposed for high-throughput target compound production. This method can optimize the production of compounds of interest while maximizing cell growth. With this approach, an appropriate combination of metabolic reaction deletions can be identified by solving a simple linear programming problem. Using a standard CPU, the computation time could be as low as 1 min per compound, and the system can even handle large metabolic models. AERITH was implemented in MATLAB and is freely available for non-profit use.
    Apr. 2022, Biomolecules, 12(5) (5), English, International magazine
    Scientific journal

  • Masahiro Tominaga, Akihiko Kondo, Jun Ishii
    Transcriptional switches can be utilized for many purposes in synthetic biology, including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although to date such switches have mainly been developed in prokaryotes, those for eukaryotes are increasingly being reported as both rational and random engineering technologies mature. In this review, we describe yeast transcriptional switches with different modes of action and how to alter their properties. We also discuss directed evolution technologies for the rapid and robust construction of yeast transcriptional switches.
    MDPI AG, Apr. 2022, Life, 12(4) (4), 557 - 557, English
    [Refereed]
    Scientific journal


  • Kenji Okano, Yu Sato, Shnji Hama, Tsutomu Tanaka, Hideo Noda, Akihiko Kondo, Kohsuke Honda
    BACKGROUND: There has been an increasing demand for optically pure d-lactic and l-lactic acid for the production of stereocomplex-type polylactic acid. The d-lactic acid production from lignocellulosic biomass is important owing to its great abundance in nature. Corn steep liquor (CSL) is a cheap nitrogen source used for industrial fermentation, though it contains a significant amount of l-lactic acid, which decreases the optical purity of d-lactic acid produced. METHOD AND RESULTS: To remove l-lactic acid derived from the CSL-based medium, l-lactate oxidase (LoxL) from Enterococcus sp. NBRC 3427 was expressed in an engineered Lactiplantibacillus plantarum (formally called Lactobacillus plantarum) strain KOLP7, which exclusively produces d-lactic acid from both hexose and pentose sugars. When the resulting strain was applied for d-lactic acid fermentation from the mixed sugars consisting of the major constituent sugars of lignocellulose (35 g L-1 glucose, 10 g L-1 xylose, and 5 g L-1 arabinose) using the medium containing 10 g L-1 CSL, it completely removed l-lactic acid derived from CSL (0.52 g L-1 ) and produced 41.7 g L-1 of d-lactic acid. The l-lactic acid concentration was below the detection limit, and improvement in the optical purity of d-lactic acid was observed (from 98.2% to > 99.99%) by the overexpression of LoxL. CONCLUSION AND IMPLICATIONS: The LoxL-mediated consumption of l-lactic acid would enable the production of optically pure d-lactic acid in any medium contaminated by l-lactic acid.
    Wiley, Apr. 2022, Biotechnology Journal, 17(4) (4), 2100331 - 2100331, English, International magazine
    Scientific journal

  • Teruyuki Nishi, Yoichiro Ito, Yasuyuki Nakamura, Taiki Yamaji, Noriko Hashiba, Masaya Tamai, Yoshihiko Yasohara, Jun Ishii, Akihiko Kondo
    American Chemical Society (ACS), Feb. 2022, ACS Synthetic Biology, 11(2) (2), 644 - 654, English
    Scientific journal

  • Shanti Ratnakomala, Prihardi Kahar, Norimasa Kashiwagi, JaeMin Lee, Motonori Kudou, Hana Matsumoto, Pamella Apriliana, Yopi Yopi, Bambang Prasetya, Chiaki Ogino, Akihiko Kondo
    A novel endo-beta-1,4-mannanase gene was cloned from a novel actinomycetes, Nonomuraea jabiensis ID06-379, isolated from soil, overexpressed as an extracellular protein (47.8 kDa) in Streptomyces lividans 1326. This new endo-1,4-beta-mannanase gene (manNj6-379) is encoded by 445-amino acids. The ManNj6-379 consists of a 28-residue signal peptide and a carbohydrate-binding module of family 2 belonging to the glycoside hydrolase (GH) family 5, with 59-77% identity to GH5 mannan endo-1,4-beta-mannanase. The recombinant ManNj6-379 displayed an optimal pH of 6.5 with pH stability ranging between 5.5 and 7.0 and was stable for 120 min at 50 & DEG;C and lower temperatures. The optimal temperature for activity was 70 & DEG;C. An enzymatic hydrolysis assay revealed that ManNj6-379 could hydrolyze commercial beta-mannan and biomass containing mannan.
    MDPI, Feb. 2022, PROCESSES, 10(2) (2), 269 - 269, English
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Feb. 2022, Current Opinion in Green and Sustainable Chemistry, 33, 100584 - 100584, English
    Scientific journal

  • Sachiko Kashojiya, Yu Lu, Mariko Takayama, Hiroki Komatsu, Luyen Hieu Thi Minh, Keiji Nishida, Kenta Shirasawa, Kenji Miura, Satoko Nonaka, Jun-ichiro Masuda, Akihiko Kondo, Hiroshi Ezura, Tohru Ariizumi
    Abstract Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar “Micro-Tom” and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both “Micro-Tom” and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.
    Oxford University Press (OUP), Jan. 2022, Horticulture Research, 9, uhab004, English, International magazine
    Scientific journal



  • Akihiko Kondo
    2022, Journal of the Taiwan Institute of Chemical Engineers
    Scientific journal


  • Akihiko Kondo
    2022, Frontiers in Bioengineering and Biotechnology
    Scientific journal

  • Akihiko Kondo
    2022, World Journal of Clinical Cases
    Scientific journal

  • Johan Hunziker, Keiji Nishida, Akihiko Kondo, Tohru Ariizumi, Hiroshi Ezura
    Our previous study demonstrated that Target-AID which is the modified CRISPR/Cas9 system enabling base-editing is an efficient tool for targeting multiple genes. Three genes, SlDDB1, SlDET1, and SlCYC-B, responsible for carotenoid accumulation were targeted, and allelic variations were previously obtained by Target-AID. In this research, we characterized the effect of new alleles on plant growth and fruit development, as well as carotenoid accumulation, individually in segregating backcross populations or combined in null self-segregant lines. Only lines carrying homozygous substitutions in the three targeted genes and the segregating backcross population of individual mutations were characterized, resulting in the isolation of two allelic versions for SlDDB1, one associated with SlDET1 and the last one with SlCYC-B. All edited lines showed variations in carotenoid accumulation, with an additive effect for each single mutation. These results suggest that Target-AID base-editing technology is an effective tool for creating new allelic variations in target genes to improve carotenoid accumulation in tomato.
    2022, Frontiers in plant science, 13, 848560 - 848560, English, International magazine
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Jan. 2022, Biochemical Engineering Journal, 178, 108274 - 108274
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Jan. 2022, Biochemical Engineering Journal, 178, 108296 - 108296
    Scientific journal

  • Yuichi Kato, Kosuke Inabe, Ryota Hidese, Akihiko Kondo, Tomohisa Hasunuma
    Metabolomics, an essential tool in modern synthetic biology based on the design-build-test-learn platform, is useful for obtaining a detailed understanding of cellular metabolic mechanisms through comprehensive analyses of the metabolite pool size and its dynamic changes. Metabolomics is critical to the design of a rational metabolic engineering strategy by determining the rate-limiting reaction and assimilated carbon distribution in a biosynthetic pathway of interest. Microalgae and cyanobacteria are promising photosynthetic producers of biofuels and bio-based chemicals, with high potential for developing a bioeconomic society through bio-based carbon neutral manufacturing. Metabolomics technologies optimized for photosynthetic organisms have been developed and utilized in various microalgal and cyanobacterial species. This review provides a concise overview of recent achievements in photosynthetic metabolomics, emphasizing the importance of microalgal and cyanobacterial cell factories that satisfy industrial requirements.
    Elsevier BV, Jan. 2022, Bioresource Technology, 344(Pt A) (Pt A), 126196 - 126196, English, International magazine
    Scientific journal

  • Yuma Kobayashi, Kentaro Inokuma, Mami Matsuda, Akihiko Kondo, Tomohisa Hasunuma
    Elsevier BV, Jan. 2022, Biotechnology Notes, 3, 1 - 7, English
    Scientific journal

  • Hideo Kawaguchi, Kenji Takada, Taghreed Elkasaby, Radityo Pangestu, Masakazu Toyoshima, Prihardi Kahar, Chiaki Ogino, Tatsuo Kaneko, Akihiko Kondo
    Lignocellulosic biomass has great potential as an inedible feedstock for bioplastic synthesis, although its use is still limited compared to current edible feedstocks of glucose and starch. This review focuses on recent advances in the production of biopolymers and biomonomers from lignocellulosic feedstocks with downstream processing and chemical polymer syntheses. In microbial production, four routes composed of existing poly (lactic acid) and polyhydroxyalkanoates (PHAs) and the emerging biomonomers of itaconic acid and aromatic compounds were presented to review present challenges and future perspectives, focusing on the use of lignocellulosic feedstocks. Recently, advances in purification technologies decreased the number of processes and their environmental burden. Additionally, the unique structures and high-performance of emerging lignocellulose-based bioplastics have expanded the possibilities for the use of bioplastics. The sequence of processes provides insight into the emerging technologies that are needed for the practical use of bioplastics made from lignocellulosic biomass.
    Elsevier BV, Jan. 2022, Bioresource Technology, 344, 126165 - 126165, English, International magazine
    Scientific journal

  • Ku Syahidah Ku Ismail, Yuki Matano, Yuri Sakihama, Kentaro Inokuma, Yumiko Nambu, Tomohisa Hasunuma, Akihiko Kondo
    One of the potential bioresources for bioethanol production is Napier grass, considering its high cellulose and hemicellulose content. However, the cost of pretreatment hinders the bioethanol produced from being economical. This study examines the effect of hydrothermal process with dilute acid on extruded Napier grass, followed by enzymatic saccharification prior to simultaneous saccharification and co-fermentation (SScF). Extrusion facilitated lignin removal by 30.2 % prior to dilute acid steam explosion. Optimum pretreatment condition was obtained by using 3% sulfuric acid, and 30-min retention time of steam explosion at 190 °C. Ethanol yield of 0.26 g ethanol/g biomass (60.5% fermentation efficiency) was attained by short-term liquefaction and fermentation using a cellulose-hydrolyzing and xylose-assimilating Saccharomyces cerevisiae NBRC1440/B-EC3-X ΔPHO13, despite the presence of inhibitors. This proposed method not only reduced over-degradation of cellulose and hemicellulose, but also eliminated detoxification process and reduced cellulase loading.
    Elsevier BV, Jan. 2022, Bioresource Technology, 343, 126071 - 126071, English, International magazine
    Scientific journal

  • 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, Dec. 2021, Metabolites, 11(12) (12), 867 - 867, English, International magazine
    Scientific journal

  • Akihiko Kondo
    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, Dec. 2021, Microbial Cell Factories, 20(1) (1), 228, English
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Dec. 2021, Algal Research, 60, 102544 - 102544
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Dec. 2021, Metabolic Engineering Communications, 13, e00188 - e00188, English
    Scientific journal

  • 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, Dec. 2021, Microbial Cell Factories, 20(1) (1), 196 - 196, English, International magazine
    Scientific journal

  • 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, Dec. 2021, Biotechnology for Biofuels, 14(1) (1)
    Scientific journal

  • 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, Dec. 2021, Biotechnology for Biofuels, 14(1) (1), 39 - 39, English, International magazine
    Scientific journal

  • 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, Dec. 2021, Communications Biology, 4(1) (1), 450 - 450, English, International magazine
    Scientific journal

  • 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, Dec. 2021, Scientific Reports, 11(1) (1), English
    Scientific journal

  • Erika Yoshida, Motoki Kojima, Munenori Suzuki, Fumio Matsuda, Kazutaka Shimbo, Akiko Onuki, Yousuke Nishio, Yoshihiro Usuda, Akihiko Kondo, Jun Ishii
    (-)-Carvone is a monoterpenoid with a spearmint flavor. A sustainable biotechnological production process for (-)-carvone is desirable. Although all enzymes in (-)-carvone biosynthesis have been functionally expressed in Escherichia coli independently, the yield was low in previous studies. When cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in a single strain, by-product formation (dihydrocarveol and dihydrocarvone) was detected. We hypothesized that P450 and CDH expression levels differ in E. coli. Thus, two strains independently expressing P450/CPR and CDH were mixed with different ratios, confirming increased carvone production and decreased by-product formation when CDH input was reduced. The optimum ratio of enzyme expression to maximize (-)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method. Thereafter, a single strain expressing both P450/CPR and CDH was constructed to imitate the optimum expression ratio. The upgraded strain showed a 15-fold improvement compared to the initial strain, showing a 44 ± 6.3 mg/L (-)-carvone production from 100 mg/L (-)-limonene. Our study showed the usefulness of the QconCAT proteome analysis method for strain development in the industrial biotechnology field.
    Nov. 2021, Scientific reports, 11(1) (1), 22126 - 22126, English, International magazine
    Scientific journal

  • 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
    The gut microbiome has emerged as a key regulator of obesity; however, its role in brown adipose tissue (BAT) metabolism and association with obesity remain to be elucidated. We found that the levels of circulating branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) were significantly correlated with the body weight in humans and mice and that BCAA catabolic defects in BAT were associated with obesity in diet-induced obesity (DIO) mice. Pharmacological systemic enhancement of BCAA catabolic activity reduced plasma BCAA and BCKA levels and protected against obesity; these effects were reduced in BATectomized mice. DIO mice gavaged with Bacteroides dorei and Bacteroides vulgatus exhibited improved BAT BCAA catabolism and attenuated body weight gain, which were not observed in BATectomized DIO mice. Our data have highlighted a possible link between the gut microbiota and BAT BCAA catabolism and suggest that Bacteroides probiotics could be used for treating obesity.
    Elsevier BV, Nov. 2021, iScience, 24(11) (11), 103342 - 103342, English, International magazine
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Nov. 2021, Bioresource Technology, 340, 125638 - 125638, English
    Scientific journal

  • Taiji Yuzawa, Tomokazu Shirai, Ryoko Orishimo, Kazuki Kawai, Akihiko Kondo, Takashi Hirasawa
    Glycerol is an attractive raw material for the production of useful chemicals using microbial cells. We previously identified metabolic engineering targets for the improvement of glycerol assimilation ability in Saccharomyces cerevisiae based on adaptive laboratory evolution (ALE) and transcriptome analysis of the evolved cells. We also successfully improved glycerol assimilation ability by the disruption of the RIM15 gene encoding a Greatwall protein kinase together with overexpression of the STL1 gene encoding the glycerol/H+ symporter. To understand glycerol assimilation metabolism in the evolved glycerol-assimilating strains and STL1-overexpressing RIM15 disruptant, we performed metabolic flux analysis using 13C-labeled glycerol. Significant differences in metabolic flux distributions between the strains obtained from the culture after 35 and 85 generations in ALE were not found, indicating that metabolic flux changes might occur in the early phase of ALE (i.e., before 35 generations at least). Similarly, metabolic flux distribution was not significantly changed by RIM15 gene disruption. However, fluxes for the lower part of glycolysis and the TCA cycle were larger and, as a result, flux for the pentose phosphate pathway was smaller in the STL1-overexpressing RIM15 disruptant than in the strain obtained from the culture after 85 generations in ALE. It could be effective to increase flux for the pentose phosphate pathway to improve the glycerol assimilation ability in S. cerevisiae.
    Microbiology Research Foundation, Oct. 2021, The Journal of General and Applied Microbiology, 67(4) (4), 142 - 149, English, Domestic magazine
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Sep. 2021, Energy Conversion and Management, 243, 114359 - 114359, English
    Scientific journal

  • Shuhei Noda, Yutaro Mori, Ryosuke Fujiwara, Tomokazu Shirai, Tsutomu Tanaka, Akihiko Kondo
    Microbial metabolic pathway engineering is a potent strategy used worldwide to produce aromatic compounds. We drastically rewired the primary metabolic pathway of Escherichia coli to produce aromatics and their derivatives. The metabolic pathway of E. coli was compartmentalized into the production and energy modules. We focused on the pyruvate-forming reaction in the biosynthesis pathway of some compounds as the reaction connecting those modules. E. coli strains were engineered to show no growth unless pyruvate was synthesized along with the compounds of interest production. Production of salicylate and maleate was demonstrated to confirm our strategy's versatility. In maleate production, the production, yield against the theoretical yield, and production rate reached 12.0 g L-1, 67%, and up to fourfold compared to that in previous reports, respectively; these are the highest values of maleate production in microbes to our knowledge. The results reveal that our strategy strongly promotes the production of aromatics and their derivatives.
    Elsevier BV, Sep. 2021, Metabolic Engineering, 67, 1 - 10, English, International magazine
    Scientific journal

  • Akihiko Kondo
    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, Aug. 2021, Marine Drugs, 19(8) (8), 463 - 463, English
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Aug. 2021, Journal of the Taiwan Institute of Chemical Engineers, 125, 35 - 40, English
    Scientific journal

  • Keiji Nishida, Akihiko Kondo
    Aug. 2021, The CRISPR journal, 4(4) (4), 462 - 463, English, International magazine
    Scientific journal

  • Akihiko Kondo
    Springer Science and Business Media LLC, Aug. 2021, Applied Microbiology and Biotechnology, 105(14-15) (14-15), 5895 - 5904, English
    Scientific journal

  • 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, Aug. 2021, Journal of Bioscience and Bioengineering, 132(2) (2), 140 - 147, English, Domestic magazine
    Scientific journal

  • Jih-Heng Chen, Yuichi Kato, Mami Matsuda, Chun-Yen Chen, Dillirani Nagarajan, Tomohisa Hasunuma, Akihiko Kondo, Jo-Shu Chang
    Microalgae-derived carotenoids have increasingly been considered as feasible green alternatives for synthetic antioxidants. In this study, the lutein high-yielding strain (Chlorella sorokiniana MB-1; henceforth MB-1) and its mutant derivative (C. sorokiniana MB-1-M12; henceforth M12) were evaluated for their growth, biomass production, and lutein accumulation in three different cultivation modes - photoautotrophy, mixotrophy, and heterotrophy. M12 could grow effectively under heterotrophic conditions, but the lutein content was lower, indicating the necessity of photo-induction for lutein accumulation. Metabolic analysis of MB-1 and M12 in autotrophic growth in the presence of carbon dioxide indicated that carbon assimilation and channeling of the fixed metabolites towards carotenoid accumulation was elevated in M12 compared to MB-1. Novel two-stage alternative cultivation strategies (Autotrophic/Heterotrophic and Mixotrophic/Heterotrophic cultures) were applied for enhancing lutein production in M12. Maximum lutein quantity (6.17 mg/g) and production (33.64 mg/L) were obtained with the TSHM strategy that is considered the best two-stage operation.
    Elsevier BV, Aug. 2021, Bioresource Technology, 334, 125200 - 125200, English, International magazine
    Scientific journal

  • 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., Jun. 2021, Essays in Biochemistry, 65(2) (2), 197 - 212, English, International magazine
    Scientific journal

  • 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, Jun. 2021, Metabolic Engineering Communications, 12, e00169 - e00169, English, International magazine
    Scientific journal

  • 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, May 2021, Metabolic Engineering, 65, 88 - 98, English, International magazine
    Scientific journal

  • Akihiko Kondo
    Wiley, May 2021, Biofuels, Bioproducts and Biorefining, 15(3) (3), 804 - 814, English
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, May 2021, Metabolic Engineering, 65, 167 - 177
    Scientific journal

  • Ang Li, Hitoshi Mitsunobu, Shin Yoshioka, Suzuki Takahisa, Akihiko Kondo, Keiji Nishida
    Abstract Structure-based rational engineering of the cytosine base editing system Target-AID was performed to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 was eliminated and additional mutations were introduced to restore enzyme function. The resulting tCDA1EQ was effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized gRNA-independent off-targets, as assessed in yeast and human cells. Combining with the smaller Cas9 ortholog system, the smallest cytosine base editing system was created that is within the size limit of AAV vector.
    Research Square Platform LLC, Apr. 2021

  • Akihiko Kanamoto, Yuichi Kato, Erina Yoshida, Tomohisa Hasunuma, Akihiko Kondo
    The natural pigment fucoxanthin has attracted global attention because of its superior antioxidant properties. The haptophyte marine microalgae Pavlova spp. are assumed to be promising industrial fucoxanthin producers as their lack of a cell wall could facilitate the commercialization of cultured cells as a whole food. This study screened promising Pavlova strains with high fucoxanthin content to develop an outdoor cultivation method for fucoxanthin production. Initial laboratory investigations of P. pinguis NBRC 102807, P. lutheri NBRC 102808, and Pavlova sp. OPMS 30543 identified OPMS 30543 as having the highest fucoxanthin content. The culture conditions were optimized for OPMS 30543. Compared with f/2 and Walne's media, the use of Daigo's IMK medium led to the highest biomass production and highest fucoxanthin accumulation. The presence of seawater elements in Daigo's IMK medium was necessary for the growth of OPMS 30543. OPMS 30543 was then cultured outdoors using acrylic pipe photobioreactors, a plastic bag, an open tank, and a raceway pond. Acrylic pipe photobioreactors with small diameters enabled the highest biomass production. Using an acrylic pipe photobioreactor with 60-mm diameter, a fucoxanthin productivity of 4.88 mg/L/day was achieved in outdoor cultivation. Thus, this study demonstrated the usefulness of Pavlova sp. OPMS 30543 for fucoxanthin production in outdoor cultivation.
    Apr. 2021, Marine biotechnology (New York, N.Y.), 23(2) (2), 331 - 341, English, International magazine
    Scientific journal

  • Shaoze Yuan, Shunsuke Kawasaki, Islam M. Y. Abdellatif, Keiji Nishida, Akihiko Kondo, Tohru Ariizumi, Hiroshi Ezura, Kenji Miura
    KEY MESSAGE: Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.
    Springer Science and Business Media LLC, Apr. 2021, Plant Cell Reports, 40(4) (4), 667 - 676, English, International magazine
    Scientific journal

  • 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, Apr. 2021, NATURE COMMUNICATIONS, 12(1) (1), 2195, English
    Scientific journal

  • 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, Mar. 2021, Scientific Reports, English
    Scientific journal

  • Ning Mao, Nikhil Aggarwal, Chueh Loo Poh, Byung Kwan Cho, Akihiko Kondo, Chenli Liu, Wen Shan Yew, Matthew Wook Chang
    Wiley, Mar. 2021, Advanced Genetics, 2(1) (1), English
    Scientific journal

  • 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, Mar. 2021, Nature Communications, 12(1) (1), English
    Scientific journal

  • Kenta Morita, Yuya Nishimura, Satoko Nakamura, Yuki Arai, Chiya Numako, Kazuyoshi Sato, Masao Nakayama, Hiroaki Akasaka, Ryohei Sasaki, Chiaki Ogino, Akihiko Kondo
    Polyacrylic acid-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers that enhance the therapeutic effect of X-ray irradiation after local injection into tumors. However, the mechanism for this reaction has remained unclear with the exception of the involvement of hydrogen peroxide (H2O2), which is released by PAA-TiOx NPs to a liquid phase during dispersion. In the present study, a clonogenic assay was used to compare PAA-TiOx NPs with free H2O2 molecules to investigate the effect exerted on the radiosensitivity of cancer cells in vitro. A cell-free dialysis method revealed that a portion of the H2O2 adsorbed onto the PAA-TiOx NPs during synthesis could be released during a treatment regimen. The H2O2 release lasted for 7 h, which was sufficient for one radiation treatment procedure. For in vitro experiments, cultured human pancreatic cancer cells took up PAA-TiOx NPs in 10 min after administration. Interestingly, when the cells were washed with a buffer after treatment with either a PAA-TiOx NP or H2O2 solution, the intracellular H2O2 levels remained higher with PAA-TiOx NP treatment compared with the H2O2 solution treatment. Furthermore, the effects of subsequent X-ray irradiation corresponded to the intracellular H2O2 levels. These results indicate that PAA-TiOx NPs are efficient carriers of H2O2 into cancer cells and thus enhance the radiosensitivity.
    Elsevier BV, Feb. 2021, Colloids and Surfaces B: Biointerfaces, 198, 111451 - 111451, English
    Scientific journal

  • 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, Feb. 2021, Biotechnology and Bioengineering, 118(2) (2), 863 - 876, English
    Scientific journal



  • Akihiko Kondo
    2021, Epilepsy & Behavior
    Scientific journal

  • Akihiko Kondo
    2021, World Journal of Clinical Cases
    Scientific journal



  • Akihiko Kondo
    2021, World Journal of Cardiology
    Scientific journal

  • Kohei Katsurada, Masahiro Tominaga, Misato Kaishima, Hiroko Kato, Toshihide Matsuno, Chiaki Ogino, Akihiko Kondo, Jun Ishii, Katsumi Takayama
    We describe a novel expression cassette that enables efficient and constitutive expression of the ZZ domain derived from Staphylococcus aureus protein A on the yeast cell surface to easily prepare yeast-based immunosorbents. Using this expression cassette containing the PGK1 promoter, a secretion signal derived from α-factor, and a Flo1-derived anchor protein, we successfully created a yeast-based immunosorbent for human serum albumin.
    Microbiology Research Foundation, 2021, The Journal of General and Applied Microbiology, 67(6) (6), 265 - 268, English, Domestic magazine
    Scientific journal

  • Keiji Nishida, Akihiko Kondo
    In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.
    Elsevier BV, Jan. 2021, Metabolic Engineering, 63, 141 - 147, English, International magazine
    Scientific journal

  • Akihiko Kondo
    Wiley, Jan. 2021, Advanced Sustainable Systems, 5(1) (1), 2000193 - 2000193, English
    Scientific journal

  • 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, Jan. 2021, Scientific Reports, 11(1) (1), English
    Scientific journal

  • Takahiro Yukawa, Takahiro Bamba, Gregory Guirimand, Mami Matsuda, Tomohisa Hasunuma, Akihiko Kondo
    Wiley, Jan. 2021, Biotechnology and Bioengineering, 118(1) (1), 175 - 185, English
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Jan. 2021, Talanta, 222, 121625 - 121625, English
    Scientific journal

  • 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), Dec. 2020, Nucleic Acids Research, 48(22) (22), 13000 - 13012, English
    Scientific journal

  • 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, Dec. 2020, Scientific Reports, 10(1) (1), 20471 - 20471, English, International magazine
    Scientific journal

  • Akihiko Kondo
    Elsevier BV, Dec. 2020, Renewable and Sustainable Energy Reviews, 134, 110355 - 110355, English
    Scientific journal

  • 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, Dec. 2020, Scientific Reports, 10(1) (1), 13009, English
    [Refereed]
    Scientific journal

  • Kengo Sasaki, Daisuke Sasaki, Asuka Hannya, Jun Tsubota, Akihiko Kondo
    Springer Science and Business Media LLC, Dec. 2020, Scientific Reports, 10(1) (1), 8516, English
    [Refereed]
    Scientific journal

  • 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), Nov. 2020, ACS Applied Bio Materials, 3(11) (11), 7743 - 7751, English
    Scientific journal

  • 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, Nov. 2020, Biomass and Bioenergy, 142, 105768 - 105768, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    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, Oct. 2020, Scientific Reports, 10(1) (1), 17351, English
    Scientific journal

  • 食物中の毒性物質検出のためのパトロール酵母の開発(Development of patrol yeasts for the detection of toxic substances in foods)
    蘇 九龍, 中村 泰之, 北口 哲也, 石井 純, 近藤 昭彦, 小林 典裕, 三宅 司郎, 上田 宏
    (公社)日本生化学会, Sep. 2020, 日本生化学会大会プログラム・講演要旨集, 93回, [3Z09 - 509)], English

  • Hideo Kawaguchi, Takashi Sazuka, Akihiko Kondo
    ABSTRACT We determined the complete and draft genome sequences of two strains of Corynebacterium glutamicum and revealed their genomic islands (GEIs). The two strains, ATCC 21799 and ATCC 31831, were found to have 3,079 and 3,109 coding sequences, respectively, with 13 GEIs each not present in the reference strain, ATCC 13032.
    American Society for Microbiology, Aug. 2020, Microbiology Resource Announcements, 9(32) (32), e00430-20, English
    [Refereed]
    Scientific journal

  • 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), Jul. 2020, ACS synthetic biology, 9(7) (7), 1615 - 1622, English, International magazine
    [Refereed]
    Scientific journal

  • 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, Jul. 2020, Metabolic engineering, 61, 225 - 237, English, International magazine
    [Refereed]
    Scientific journal

  • 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, Jul. 2020, Biotechnology for Biofuels, 13(1) (1), 1 - 15, English
    [Refereed]
    Scientific journal


  • 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, Jul. 2020, Nature biotechnology, 38(7) (7), 865 - 869, English, International magazine
    [Refereed]
    Scientific journal

  • 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.
    Jul. 2020, Nature biotechnology, 38(7) (7), 901 - 901, English, International magazine
    [Refereed]

  • Daichi Satowa, 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, Jul. 2020, Biotechnology and Bioengineering, 117(7) (7), 2153 - 2164, English, International magazine
    [Refereed]
    Scientific journal

  • 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, Jun. 2020, Biotechnology and bioengineering, 117(6) (6), 1649 - 1660, English, International magazine
    [Refereed]
    Scientific journal

  • Misaki Hatanaka, Hiroto Morita, Yumi Aoyagi, Kengo Sasaki, Daisuke Sasaki, Akihiko Kondo, Teppei Nakamura
    May 2020, Scientific Reports, 10, 7591, English
    [Refereed]
    Scientific journal

  • 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
    May 2020, Processes, 8(5) (5), 619, English
    [Refereed]
    Scientific journal

  • 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.
    May 2020, Applied Microbiology and Biotechnology, 104(9) (9), 3859 - 3867, English, International magazine
    [Refereed]
    Scientific journal

  • Shunsuke Kobayashi, Hideo Kawaguchi, Tomokazu Shirai, Kazuaki Ninomiya, Kenji Takahashi, Akihiko Kondo, Yota Tsuge
    Controlling the carbon flux into a desired pathway is important for improving product yield in metabolic engineering. After entering a cell, glucose is channeled into glycolysis and the pentose phosphate pathway (PPP), which decreases the yield of target products whose synthesis relies on NADPH as a cofactor. Here, we demonstrate redirection of carbon flux into PPP under aerobic conditions in Corynebacterium glutamicum, achieved by replacing the promoter of glucose 6-phosphate isomerase gene (pgi) with an anaerobic-specific promoter of the lactate dehydrogenase gene (ldhA). The promoter replacement increased the split ratio of carbon flux into PPP from 39 to 83% under aerobic conditions. The titer, yield, and production rate of 1,5-diaminopentane, whose synthesis requires NADPH as a cofactor, were increased by 4.6-, 4.4-, and 2.6-fold, respectively. This is the largest improvement in the production of 1,5-diaminopentane or its precursor, lysine, reported to date. After aerobic cell growth, pgi expression was automatically induced under anaerobic conditions, altering the carbon flux from PPP to glycolysis, to produce succinate in a single metabolically engineered strain. Such an automatic redirection of metabolic pathway using an oxygen-responsive switch enables two-stage fermentation for efficient production of two different compounds by a single strain, potentially reducing the production costs and time for practical applications.
    Apr. 2020, ACS synthetic biology, 9(4) (4), 814 - 826, English, International magazine
    [Refereed]
    Scientific journal

  • 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
    Apr. 2020, Processes, 8(4) (4), 450, English
    [Refereed]
    Scientific journal

  • Nova Rachmadona, Jerome Amoah, Emmanuel Quayson, Shinji Hama, Ayumi Yoshida, Akihiko Kondo, Chiaki Ogino
    Palm oil mill effluent (POME), a liquid waste from the palm oil industry, presents an alternative source for biodiesel production without interfering with food supply. This study attempted to produce biodiesel from untreated POME with aqueous ethanol using Thermomyces lanuginosus lipase as a biocatalyst. The effects of enzyme concentration, alcohol to oil ratio, and ethanol concentration were considered in the transesterification reaction. The optimum conditions were 2100 U lipase loading, 4 : 1 ethanol to oil molar ratio, and 45% (v/v) ethanol concentration at 40 degrees C reaction, and under 24 hours. The maximum fatty acid ethyl ester (FAEE) yield reached 97.43% (w/w) under these conditions. Integration of dilute ethanol for the conversion of POME to biodiesel could be promising as both feedstocks could be obtained from the same location, and thus reduce the logistical burden on biodiesel production.
    ROYAL SOC CHEMISTRY, Mar. 2020, SUSTAINABLE ENERGY & FUELS, 4(3) (3), 1105 - 1111, English
    Scientific journal

  • Akihiko Kondo
    Mar. 2020, Fuel Processing Technology, 199, 106278, English
    [Refereed]
    Scientific journal

  • 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.
    Feb. 2020, ACS synthetic biology, 9(2) (2), 260 - 268, English, International magazine
    [Refereed]

  • Naoki Watanabe, Masahiro Murata, Teppei Ogawa, Christopher J Vavricka, Akihiko Kondo, Chiaki Ogino, Michihiro Araki
    Feb. 2020, Journal of Chemical Information and Modeling, 60(3) (3), 1833 - 1843, English
    [Refereed]
    Scientific journal



  • Akihiko Kondo
    2020, Pediatrics International
    Scientific journal

  • Akihiko Kondo
    2020, Epilepsy & Behavior Reports
    Scientific journal


  • Christopher J. Vavricka, Tomohisa Hasunuma, Akihiko Kondo
    Jan. 2020, Trends in Biotechnology, 38(1) (1), 68 - 82, English
    [Refereed]
    Scientific journal

  • 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.
    Jan. 2020, Journal of bioscience and bioengineering, 129(1) (1), 99 - 103, English, Domestic magazine
    [Refereed]
    Scientific journal


  • Tomohisa Hasunuma, Ayako Takaki, Mami Matsuda, Yuichi Kato, Christopher J. Vavricka, Akihiko Kondo
    Dec. 2019, ACS Synthetic Biology, 8, 2701 - 2709, English
    [Refereed]
    Scientific journal

  • Kiyotaka Y. Hara, Masaru Saito, Hiroko Kato, Kana Morikawa, Hiroshi Kikukawa, Hironari Nomura, Takanori Fujimoto, Yoko Hirono-Hara, Shigeyuki Watanabe, Kengo Kanamaru, Akihiko Kondo
    Springer Science and Business Media LLC, Dec. 2019, Microbial Cell Factories, 18(1) (1)
    Scientific journal

  • Akihiko Kondo
    Dec. 2019, Metabolic Engineering, 56, 17 - 27, English
    [Refereed]
    Scientific journal

  • 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.
    Dec. 2019, Nature Communications, 10(1) (1)
    [Refereed]
    Scientific journal



  • 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.
    Oct. 2019, Microbial cell factories, 18(1) (1), 177 - 177, English, International magazine
    [Refereed]
    Scientific journal

  • Ryohei Shinohara, Kengo Sasaki, Jun Inoue, Namiko Hoshi, Itsuko Fukuda, Daisuke Sasaki, Akihiko Kondo, Ro Osawa
    Microbial production of butyrate is impaired in patients with ulcerative colitis (UC); however, this inhibition is not well understood in Japanese UC patients. Therefore, we quantitatively analyzed genes encoding butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk) in the gut microbiota of Japanese patients with UC and healthy volunteers (HVs). But showed higher levels than buk. Moreover, patients with UC showed significantly decreased levels of but associated with Roseburia sp./Eubacterium rectale compared with HVs. But, which is associated with Faecalibacterium sp., was maintained in patients with UC, with an unchanged relative abundance of Faecalibacterium sp. microorganisms in patients with UC compared with HVs.
    Oct. 2019, Bioscience of Microbiota, Food and Health, 38(4) (4), 159 - 163, English, Domestic magazine
    [Refereed]
    Scientific journal

  • Rena Matsuura, Mayumi Kishida, Rie Konishi, Yuuki Hirata, Noriko Adachi, Shota Segawa, Kenta Imao, Tsutomu Tanaka, Akihiko Kondo
    Oct. 2019, Biotechnology and Bioengineering, 116(10) (10), 2640 - 2651, English
    [Refereed]
    Scientific journal

  • 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
    In this study, microalgal cultivation was applied as a feasible strategy for treating shrimp culture wastewater (SCW) from a shrimp farm in southern Tainan. Chlorella sorokiniana MB-1-M12 was first grown on BG-11 medium with 0.5% salinity, obtaining a biomass concentration and productivity of 4.35 g/L and 1.56 g/L/d, respectively. When 80% of BG-11 nutrients were added to 75% strength SCW, lutein content and productivity increased to 5.19 mg/g and 5.55 mg/L/d, respectively. A novel operation strategy involving periodic exchange of freshwater and SCW was designed for semi-continuous cultivation of MB-1-M12 strain for optimal biomass and lutein production. The average biomass concentration, productivity, lutein content, and productivity were 3.5 g/L, 1.3 g/L/d, 3.89 mg/g and 5.0 mg/L/d, respectively. Although microalgae have been considered as an alternative natural source of lutein, this work is among the earliest reports describing lutein production from microalgae cultivated with wastewater via a circular economy concept.
    Oct. 2019, Bioresource Technology, 290, 121786 - 121786, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Sep. 2019, Microbiology Resource Announcements, 8(37) (37), e00652-19, English
    [Refereed]
    Scientific journal


  • 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
    Sep. 2019, Biochemical Engineering Journal, 149(15) (15), 107236, English
    [Refereed]
    Scientific journal

  • 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
    Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d-xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro-organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499-SsXR-AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a β-d-glucosidase (Aspergillus aculeatus β-glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5-fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499-XR-ScMAL11-BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.
    Sep. 2019, Biotechnology Journal, 14(9) (9), 1800704, English, International magazine
    [Refereed]
    Scientific journal

  • 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.
    Aug. 2019, Journal of atherosclerosis and thrombosis, 26(8) (8), 705 - 719, English, Domestic magazine
    [Refereed]
    Scientific journal


  • Shuhei Noda, Yutaro Mori, Sachiko Oyama, Akihiko Kondo, Michihiro Araki, Tomokazu Shirai
    BACKGROUND: The microbial production of useful fuels and chemicals has been widely studied. In several cases, glucose is used as the raw material, and almost all microbes adopt the Embden-Meyerhof (EM) pathway to degrade glucose into compounds of interest. Recently, the Entner-Doudoroff (ED) pathway has been gaining attention as an alternative strategy for microbial production. RESULTS: In the present study, we attempted to apply the ED pathway for isobutanol production in Escherichia coli because of the complete redox balance involved. First, we generated ED pathway-dependent isobutanol-producing E. coli. Thereafter, the inactivation of the genes concerning organic acids as the byproducts was performed to improve the carbon flux to isobutanol from glucose. Finally, the expression of the genes concerning the ED pathway was modified. CONCLUSIONS: The optimized isobutanol-producing E. coli produced 15.0 g/L of isobutanol as the final titer, and the yield from glucose was 0.37 g/g (g-glucose/g-isobutanol).
    Jul. 2019, Microbial Cell Factories, 18(1) (1), 124 - 124, English, International magazine
    [Refereed]
    Scientific journal

  • Ario Betha Juanssilfero, Prihardi Kahar, Lastinov Amza Rezky, Yopi, Kumar Sudesh, Chiaki Ogino, Bambang Prasetya, Akihiko Kondo
    The ability of oleaginous yeast Lipomyces starkeyi to efficiently produce lipids when cultivated on sap extracted from felled oil palm trunk (OPT) as a novel inexpensive renewable carbon source was evaluated. OPT sap was found to contain approximately 98 g/L glucose and 32 g/L fructose. Batch fermentations were performed using three different OPT sap medium conditions: regular sap, enriched sap, and enriched sap at pH 5.0. Under all sap medium conditions, the cell biomass and lipid production achieved were approximately 30 g/L and 60% (w/w), respectively. L. starkeyi tolerated acidified medium (initial pH ≈ 3) and produced considerable amounts of ethanol as well as xylitol as by-products. The fatty acid profile of L. starkeyi was remarkably similar to that of palm oil, one of the most common vegetable oil feedstock used in biodiesel production with oleic acid as the major fatty acid followed by palmitic, stearic and linoleic acids.
    Jun. 2019, Journal of Bioscience and Bioengineering, 127(6) (6), 726 - 731, English, Domestic magazine
    [Refereed]
    Scientific journal

  • Quentin Schmetz, Hiroshi Teramura, Kenta Morita, Tomoko Oshima, Aurore Richel, Chiaki Ogino, Akihiko Kondo
    An organosolv pretreatment consisting of an H2SO4/n-butanol biphasic system was designed to separate lignocellulosic biomass in three distinct phases: a cellulose-rich solid residue, hydrolyzed hemicelluloses in an aqueous phase, and lignin dissolved in a hydrophobic butanol phase. In the present study, the versatility of the process was investigated on materials of various compositions and origins: sugar cane bagasse, tall fescue, sugar beet pulp, eucalyptus, beech, and Japanese cedar. The efficiency was assessed in terms of lignin removal from the raw biomass and purity of the recovered cellulosic residue using the Klason method as well as improvement on enzymatic saccharification (increased from 18.7% to 96%). Results were correlated to biomass types and composition, and in comparison to an organic solvent-free method (dilute acid). Up to 81% cellulose purity corresponding to 87% lignin removal was achieved. Results were corroborated by scanning electron microscopy showing an absence of lignin deposition. Lignin molecular weight (GPC), structure (2D-HSQC NMR), recovery, and purity (up to 96%) have been investigated. Moreover, organic compounds responsible for fermentation inhibition were partially solubilized in the butanol, decreasing the concentration in the aqueous phase. Efficient butanol pretreatment applied on hardwood, bagasse, and herbaceous matter is promising. However, Japanese cedar (softwood) was for this process,
    AMER CHEMICAL SOC, Jun. 2019, ACS Sustainable Chemistry & Engineering, 7(13) (13), 11069 - 11079, English
    [Refereed]
    Scientific journal

  • Jerome Amoah, Prihardi Kahar, Chiaki Ogino, Akihiko Kondo
    Biorefinery has been suggested to provide relevant substitutes to a number of fossil products. Feedstocks and conversion technologies have, however, been the bottleneck to the realization of this concept. Herein, feedstocks and bioconversion technologies under biorefinery have been reviewed. Over the last decade, research has shown possibilities of generating tens of new products but only few industrial implementations. This is partly associated with low production yields and poor cost-competitiveness. This review addresses the technical barriers associated with the conversion of emerging feedstocks into chemicals and bioenergy platforms and summarizes the developed biotechnological approaches including advances in metabolic engineering. This summary further suggests possible future advances that would expand the portfolio of biorefinery and speed up the realization of biofuels and biochemicals.
    Jun. 2019, Biotechnology Journal, 14(6) (6), 1800494, English, International magazine
    [Refereed]
    Scientific journal

  • Hideo Kawaguchi, Hiroki Miyagawa, Sachiko Nakamura‐Tsuruta, Naoki Takaya, Chiaki Ogino, Akihiko Kondo
    3-Phenyllactic acid (PhLA) is useful as a start-up material in the pharmaceutical and biorefinery industries. To enhance the production of PhLA from glucose using recombinant Escherichia coli, the effects of glucose concentration and oxygen limitation on PhLA production are assessed in a fed-batch system using dissolved oxygen (DO)-stat method. The highest titer of PhLA (7.3 g L-1 ) is observed with a high concentration of glucose and under oxygen-limited conditions (DO = 0 ppm). Under oxygen limitation, cell growth and the formation of acetate and l-phenylalanine (Phe) by-products after 72 h of cultivation are reduced by 30%, 70%, and 81%, respectively, as compared to that under high DO conditions (DO = 5 ppm). Gene expression levels are compared between low and high DO conditions by quantitative polymerase chain reaction (qPCR) analysis. Several genes in the glycolysis (gapA and pykA), pentose phosphate (tktA), and early shikimate pathways for PhLA biosynthesis (aroF, aroG, and aroH) are upregulated under oxygen limitation. The results suggest that oxygen limitation affects metabolism in the shikimate pathway at both metabolic and transcriptional levels and that controlling the DO level is critical for enhanced production of a variety of aromatic compounds through the shikimate pathway.
    Jun. 2019, Biotechnology Journal, 14(6) (6), 1800478, English, International magazine
    [Refereed]
    Scientific journal

  • 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.
    May 2019, Nature communications, 10(1) (1), 2336 - 2336, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    May 2019, Green Chemistry, 21, 1795 - 1808, English
    [Refereed]
    Scientific journal

  • Kengo Sasaki, Jun Inoue, Daisuke Sasaki, Namiko Hoshi, Tomokazu Shirai, Itsuko Fukuda, Takeshi Azuma, Akihiko Kondo, Ro Osawa
    Compositional alteration of the gut microbiota is associated with ulcerative colitis (UC). Here, a model culture system is established for the in vitro human colonic microbiota of UC, which will be helpful for determining medical interventions. 16S ribosomal RNA sequencing confirms that UC models are successfully developed from fecal inoculum and retain the bacterial species biodiversity of UC feces. The UC models closely reproduce the microbial components and successfully preserve distinct clusters from the healthy subjects (HS), as observed in the feces. The relative abundance of bacteria belonging to the family Lachnospiraceae significantly decreases in the UC models compared to that in HS, as observed in the feces. The system detects significantly lower butyrogenesis in the UC models than that in HS, correlating with the decreased abundance of Lachnospiraceae. Interestingly, the relative abundance of Lachnospiraceae does not correlate with disease activity (defined as partial Mayo score), suggesting that Lachnospiraceae persists in UC patients at a decreased level, irrespective of the alteration in disease activity. Moreover, the system shows that administration of Clostridium butyricum MIYAIRI restores butyrogenesis in the UC model. Hence, the model detects deregulation in the intestinal environment in UC patients and may be useful for simulating the effect of probiotics.
    May 2019, Biotechnology Journal, 14(5) (5), 1800555, English, International magazine
    [Refereed]
    Scientific journal

  • 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
    May 2019, Nature Communications, 10(1) (1), 2040 - 2040, English, International magazine
    [Refereed]
    Scientific journal

  • Jih-Heng Chen, Chun-Yen Chen, Tomohisa Hasunuma, Akihiko Kondo, Chien-Hsiang Chang, I-Son Ng, Jo-Shu Chang
    Apr. 2019, Bioresource Technology, 278, 17 - 25, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    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.
    Apr. 2019, Methods in Molecular Biology, 1917, 297 - 307, English
    [Refereed]
    Scientific journal

  • Koichi Tamano, Robert Sidney Cox, Kenji Tsuge, Ai Miura, Ayano Itoh, Jun Ishii, Tomohiro Tamura, Akihiko Kondo, Masayuki Machida
    Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.
    Apr. 2019, Journal of Bioscience and Bioengineering, 127(4) (4), 451 - 457, English, Domestic magazine
    [Refereed]
    Scientific journal

  • Satoshi Wakai, Nanami Nakashima, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, Akihiko Kondo
    Aspergillus oryzae, a filamentous fungus, can secrete large amounts of enzymes extracellularly. We constructed a genetically engineered A. oryzae that simultaneously produced cellobiohydrolase, endoglucanase, and β-glucosidase by integrating multiple copies of the genes encoding these cellulases into fungal chromosomes. The resulting strain possessed 5-16 copies of each cellulase gene within the chromosome and showed approximately 10-fold higher activity versus single integration strains. Copy number polymorphisms were attributed to differences in flanking region sequence for the integrated gene fragments. Furthermore, we found that the P-sodM/T-glaB set demonstrated the strongest transcription levels per gene copy number. We therefore modified promoter/terminator set and cellulase gene combinations based on this polymorphism and transcription level data, with the resulting transformant showing 40-fold higher cellulolytic activity versus the single integration strain. This designed expression method could be useful for the overexpression of multiple enzymes and pathway flux control-mediated metabolic engineering in A. oryzae.
    Mar. 2019, Bioresource Technology, 276, 146 - 153, English, International magazine
    [Refereed]
    Scientific journal

  • Emmanuel Quayson, Jerome Amoah, Shinji Hama, Ayumi Yoshida, Kenta Morita, Akihiko Kondo, Chiaki Ogino
    The biomass-derived source, low-cost and hydrophobicity/oleophilic advantages of activated carbon (AC) were explored for the immobilization of Aspergillus oryzae whole-cells expressing Fusarium heterosporum lipase. The adsorptive influence of AC favored growth of the cells into its porous interfaces with paralleled exterior dense film formation. Increasing AC weights hindered extracellular lipase activity. Cell aggregation of 0.34 +/- 0.02 mg/BSP was found to be effective in catalyzing an industrially challenging feedstock (68.77% w/w free fatty acids, 20.48% w/w triglycerides) to 98% fatty acid methyl esters (FAME). In a comparative investigation with polyurethane as matrix, higher trans/esterification facilitation was observed with AC. Benefiting from the oleophilicity of AC; denaturation effect from methanol on the lipases was reduced. Surface characterization with FE-SEM, XPS and FT-IR evidenced effective cell-matrix adhesion and a retention of the AC's intrinsic properties. The advantageous tribology of AC ensured recyclability of the matrix for fresh cells immobilization. Comparable FAME (98.08% w/w) was achieved with the recycled matrix in successive batches. The spent-matrix valorization approach, thus, proposes sustainable biorefineries with immobilized lipase catalyzed biodiesel production.
    AMER CHEMICAL SOC, Mar. 2019, ACS Sustainable Chemistry & Engineering, 7(5) (5), 5010 - 5017, English
    [Refereed]
    Scientific journal

  • SAKIHAMA Yuri, HIDESE Ryota, HASUNUMA Tomohisa, KONDO Akihiko
    Yeasts are extremely useful, not only for fermentation but also for a wide spectrum of fuel and chemical productions. We analyzed the overall metabolic turnover and transcript dynamics in glycolysis and the TCA cycle, revealing the difference in adaptive pyruvate metabolic response between a Crabtree-negative species, Kluyveromyces marxianus, and a Crabtree-positive species, Saccharomyces cerevisiae, during aerobic growth. Pyruvate metabolism was inclined toward ethanol production under aerobic conditions in S. cerevisiae, while increased transcript abundances of the genes involved in ethanol metabolism and those encoding pyruvate dehydrogenase were seen in K. marxianus, indicating the augmentation of acetyl-CoA synthesis. Furthermore, different metabolic turnover in the TCA cycle was observed in the two species: malate and fumarate production in S. cerevisiae was higher than in K. marxianus, irrespective of aeration; however, fluxes of both the reductive and oxidative TCA cycles were enhanced in K. marxianus by aeration, implying both the cycles contribute to efficient electron flux without producing ethanol. Additionally, decreased hexokinase activity under aerobic conditions is expected to be important for maintenance of suitable carbon flux. These findings demonstrate differences in the key metabolic trait of yeasts employing respiration or fermentation, and provide important insight into the metabolic engineering of yeasts.
    Mar. 2019, Scientific Reports, 9(1) (1), 5319 - 5319, English, International magazine
    [Refereed]
    Scientific journal

  • Musashi Takenaka, Lee Jae Min, PRIHARDI KAHAR, Chiaki Ogino, Akihiko Kondo
    Actinobacteria plays a key role in the cycling of organic matter in soils. They secret biomass-degrading enzymes that allow it to produce the unique metabolites that originate in plant biomass. Although past studies have focused on these unique metabolites, a large-scale screening of Actinobacteria is yet to be reported to focus on their biomass-degrading ability. In the present study, a rapid and simple method is constructed for a large-scale screening, and the novel resources that form the plant biomass-degrading enzyme cocktail are identified from 850 isolates of Actinobacteria. As a result, Nonomuraea fastidiosa secretes a biomass degrading enzyme cocktail with the highest enzyme titer, although cellulase activities are lower than a commercially available enzyme. So the rich accessory enzymes are suggested to contribute to the high enzyme titer for a pretreated bagasse with a synergistic effect. Additionally, an optimized cultivation method of biomass induction caused to produce the improved enzyme cocktail indicated strong enzyme titers and a strong synergistic effect. Therefore, the novel enzyme cocktails are selected via the optimized method for large-scale screening, and then the enzyme cocktail can be improved via the optimized production with biomass-induction.
    Mar. 2019, Biotechnology Journal, 14(3) (3), 1700744, English, International magazine
    [Refereed]
    Scientific journal

  • 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.
    Mar. 2019, Photosynthesis research, 139(1-3) (1-3), 145 - 154, English, International magazine
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Feb. 2019, Applied Microbiology and Biotechnology, 103(3) (3), 1243 - 1254, English
    [Refereed]
    Scientific journal

  • Yuichi KATO, Yusuke FUJIHARA, Christopher J. VAVRICKA, Jo-Shu CHANG, Tomohisa HASUMUMA, Akihiko KONDO
    BACKGROUND: Light/dark cycling is an inevitable outdoor culture condition for microalgal biofuel production; however, the influence of this cycling on cellular lipid production has not been clearly established. The general aim of this study was to determine the influence of light/dark cycling on microalgal biomass production and lipid accumulation. To achieve this goal, specific causative mechanisms were investigated using a metabolomics approach. Laboratory scale photoautotrophic cultivations of the oleaginous green microalga Chlamydomonas sp. JSC4 were performed under continuous light (LL) and light/dark (LD) conditions. RESULTS: Lipid accumulation and carbohydrate degradation were delayed under the LD condition compared with that under the LL condition. Metabolomic analysis revealed accumulation of phosphoenolpyruvate and decrease of glycerol 3-phosphate under the LD condition, suggesting that the imbalance of these metabolites is a source of delayed lipid accumulation. When accounting for light dosage, biomass yield under the LD condition was significantly higher than that under the LL condition. Dynamic metabolic profiling showed higher levels of lipid/carbohydrate anabolism (including production of 3-phosphoglycerate, fructose 6-phosphate, glucose 6-phosphate, phosphoenolpyruvate and acetyl-CoA) from CO2 under the LD condition, indicating higher CO2 fixation than that of the LL condition. CONCLUSIONS: Photoperiods define lipid accumulation and biomass production, and light/dark cycling was determined as a critical obstacle for lipid production in JSC4. Conversions of phosphoenolpyruvate to pyruvate and 3-phosphoglycerate to glycerol 3-phosphate are the candidate rate-limiting steps responsible for delayed lipid accumulation. The accumulation of substrates including ribulose 5-phosphate could be explained by the close relationship of increased biomass yield with enhanced CO2 fixation. The present study investigated the influence of light/dark cycling on lipid production by direct comparison with continuous illumination for the first time, and revealed underlying metabolic mechanisms and candidate metabolic rate-limiting steps during light/dark cycling. These findings suggest promising targets to metabolically engineer improved lipid production.
    Feb. 2019, Biotechnology for Biofuels, 12, 39 - 39, English, International magazine
    [Refereed]
    Scientific journal


  • Jerome Amoah, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo
    Feb. 2019, Biochemical Engineering Journal, 142, 117 - 123, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    2019, Epileptic Disorders
    Scientific journal


  • Nanik Rahmani, Prihardi Kahar, Puspita Lisdiyanti, Jaemin Lee, Yopi, Bambang Prasetya, Chiaki Ogino, Akihiko Kondo
    A novel strategy for the low-cost, high-yield co-production of xylose and xylooligosaccharides together with no xylose inhibition was developed using a novel heterologous expression of XYN10Ks_480 endo-1,4-β-xylanase with a ricin-type β-trefoil type of domain and XYN11Ks_480 endo-1,4-β-xylanase with a CBM 2 superfamily from the Kitasatospora sp in an actinomycetes expression system. Xylose is the main building block for hemicellulose xylan. Our findings demonstrated high levels of expression and catalytic activity for XYN10Ks_480 during hydrolysis of the extracted xylan of bagasse, and three types of xylan-based substrates were used to produce xylose and xylooligosaccharides. However, hydrolysis by XYN11Ks_480 produced xylooligosaccharides without xylose formation. This study demonstrated how integrating sodium hypochlorite-extracted xylan and enzymatic hydrolysis could provide an alternative strategy for the generation of XOS from lignocellulosic material.
    Jan. 2019, Bioresource Technology, 272, 315 - 325, English, International magazine
    [Refereed]
    Scientific journal

  • Daisuke Sasaki, Kengo Sasaki, Yota Tsuge, Akihiko Kondo
    Background: Using a microbial fuel cell (MFC), we observed that a complex microbial community decomposed starch and transferred electrons to a graphite felt anode to generate current. In spite of the same reactor configuration, inoculum, substrate, temperature, and pH, MFCs produced different current and power density. To understand which factor(s) affected electricity generation, here, we analyzed a complex microbial community in an anodic biofilm and fermentation broth using Illumina MiSeq sequencing and metabolomics. Results: Microbial biomass on the anode was lower in MFCs generating more electricity (0.09-0.16 mg cm-2-anode) than in those generating less electricity (0.60-2.80 mg cm-2-anode), while being equal (3890-4196 mg L-1-broth) in the fermentation broth over the same operational period. Chemical oxygen demand removal and acetate concentration were also similar in fermentation broths. MFCs generating more electricity had relatively more exoelectrogenic bacteria, such as Geobacter sp., but fewer acetate-utilizing Methanosarcina sp. and/or Lactococcus sp. in anodic biofilms. Accordingly, anodic biofilms generating more electricity presented higher levels of most intracellular metabolites related to the tricarboxylic acid cycle and a higher intracellular ATP/ADP ratio, but a lower intracellular NADH/NAD+ ratio. Moreover, the level of intracellular glutamate, an essential metabolite for microbial anabolic reactions, correlated negatively with current density. Conclusion: Microbial growth on the anode and intracellular glutamate levels negatively affect electricity generation by MFCs. Reduced formation of anodic biofilm, in which intracellular glutamate concentration is 33.9 μmol g-cell-1 or less, favors the growth of acetate-utilizing Geobacter sp. on the anode and improves current generation.
    2019, Biotechnology for biofuels, 12, 72 - 72, English, International magazine
    [Refereed]
    Scientific journal

  • Daisuke Sasaki, Kengo Sasaki, Naoko Ikuta, Takahiro Yasuda, Itsuko Fukuda, Akihiko Kondo, Ro Osawa
    This study investigated the effect of various prebiotics (indigestible dextrin, α-cyclodextrin, and dextran) on human colonic microbiota at a dosage corresponding to a daily intake of 6 g of prebiotics per person (0.2% of dietary intake). We used an in vitro human colonic microbiota model based on batch fermentation starting from a faecal inoculum. Bacterial 16S rRNA gene sequence analysis showed that addition of 0.2% prebiotics did not change the diversity and composition of colonic microbiota. This finding coincided with results from a clinical study showing that the microbiota composition of human faecal samples remained unchanged following administration of 6 g of prebiotics over seven days. However, compared to absence of prebiotics, their addition reduced the pH and increased the generation of acetate and propionate in the in vitro system. Thus, even at such relatively low amounts, prebiotics appear capable of activating the metabolism of colonic microbiota.
    Nature Publishing Group, Dec. 2018, Scientific Reports, 8(1) (1), Article number: 435, English
    [Refereed]
    Scientific journal

  • Ryosuke Fujiwara, Shuhei Noda, Tsutomu Tanaka, Akihiko Kondo
    Nov. 2018, ACS Synthetic Biology, 7(11) (11), 2698 - 2705, English
    [Refereed]
    Scientific journal

  • 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, Nov. 2018, JOURNAL OF MEMBRANE SCIENCE, 566, 15 - 24, English
    [Refereed]
    Scientific journal

  • TAKAYAMA Seiya, OZAKI Aiko, KONISHI Rie, OTOMO Chisako, KISHIDA Mayumi, HIRATA Yuuki, MATSUMOTO Takuya, TANAKA Tsutomu, KONDO Akihiko
    BACKGROUND: Economical production of value-added chemicals from renewable biomass is a promising path to sustainability. 3-Hydroxypropionic acid (3-HP) is an important chemical for building a bio-sustainable society. Establishment of 3-HP production from renewable resources such as glucose would provide a bio-sustainable alternative to the production of acrylic acid from fossil resources. RESULTS: Here, we describe metabolic engineering of the fission yeast Schizosaccharomyces pombe to enhance 3-HP production from glucose and cellobiose via the malonyl-CoA pathway. The mcr gene, encoding the malonyl-CoA reductase of Chloroflexus aurantiacus, was dissected into two functionally distinct fragments, and the activities of the encoded protein were balanced. To increase the cellular supply of malonyl-CoA and acetyl-CoA, we introduced genes encoding endogenous aldehyde dehydrogenase, acetyl-CoA synthase from Salmonella enterica, and endogenous pantothenate kinase. The resulting strain produced 3-HP at 1.0 g/L from a culture starting at a glucose concentration of 50 g/L. We also engineered the sugar supply by displaying beta-glucosidase (BGL) on the yeast cell surface. When grown on 50 g/L cellobiose, the beta-glucosidase-displaying strain consumed cellobiose efficiently and produced 3-HP at 3.5 g/L. Under fed-batch conditions starting from cellobiose, this strain produced 3-HP at up to 11.4 g/L, corresponding to a yield of 11.2% (g-3-HP/g-glucose; given that 1 g cellobiose corresponds to 1.1 g glucose upon digestion). CONCLUSIONS: In this study, we constructed a series of S. pombe strains that produced 3-HP via the malonyl-CoA pathway. Our study also demonstrated that BGL display using cellobiose and/or cello-oligosaccharides as a carbon source has the potential to improve the titer and yield of malonyl-CoA- and acetyl-CoA-derived compounds.
    Nov. 2018, Microbial Cell Factories, 17(1) (1), 176 - 176, English, International magazine
    [Refereed]
    Scientific journal

  • Yoichiro Ito, Toru Watanabe, Shimpei Aikawa, Teruyuki Nishi, Tozo Nishiyama, Yasuyuki Nakamura, Tomohisa Hasunuma, Yuji Okubo, Jun Ishii, Akihiko Kondo
    Oxford University Press (OUP), Nov. 2018, FEMS Yeast Research, 18(7) (7), foy074, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Oct. 2018, FEMS Microbiology Letters, 365(20) (20), fny215, English
    [Refereed]
    Scientific journal

  • WIJAYA Hans, SASAKI Kengo, KAHAR Prihardi, YOPI, KAWAGUCHI Hideo, SAZUKA Takashi, OGINO Chiaki, PRASETYA Bambang, KONDO Akihiko
    The aim of this study was to construct a cost-effective method for repeated bioethanol production using membrane (ultrafiltration permeation and nanofiltration concentration)-concentrated sweet sorghum juice by using flocculent Saccharomyces cerevisiae F118 strain. With low initial dry cell concentrations at around 0.28-0.35 g L-1, the S. cerevisiae F118 strain provided an ethanol titer of 86.19 ± 1.15 g L-1 (theoretical ethanol yield of 70.77%), which was higher than the non-flocculent S. cerevisiae BY4741 strain at 33.92 ± 0.99 g L-1 after 24 h fermentation. This result was correlated with higher gene expressions of the sucrose-hydrolysing enzyme invertase, sugar phosphorylation, and pyruvate-to-ethanol pathways in the F118 strain compared with the BY4741 strain. Sequential fed-batch fermentation was conducted, and the F118 strain was easily separated from the fermentation broth via the formation of flocs and sediment. After the 5th cycle of fermentation with the F118 strain, the ethanol concentration reached 100.37 g L-1.
    Oct. 2018, Bioresource Technology, 265, 542 - 547, English, International magazine
    [Refereed]
    Scientific journal

  • Shinji Hama, Maki Kihara, Hideo Noda, Akihiko Kondo
    For the efficient production of lignocellulosic ethanol, a technology of recycling yeast cells at a high-solid load was developed. The yeast Saccharomyces cerevisiae TJ14 grew up to 5.0 × 107 cells/mL at 39 °C during the simultaneous saccharification and fermentation of hydrothermally pretreated rice straw. By exploiting the viability of the cells, a portion of the fermentation residue was recycled, yielding 52.3 g/L ethanol (90.3% yield) at 1.12 g/(L·h) in six batches. This result is attributable to the maintenance of a viable cell count (cells/mL) on the order of 107. In accordance with this principle, the effects of low-cost nutrients on yeast recycling were elucidated, where 0.125% corn steep liquor was deemed sufficient for fermenting rice straw and empty fruit bunch hydrolysates. The supplementation of sugarcane molasses into bagasse was found to be a practical approach, resulting in 63.5–67.7 g/L ethanol and 5.3 × 106–4.4 × 107 cells/mL in six batches. Here, we describe that the high cell-growth potential in industrially relevant media minimizes the retention of lignocellulosic residues in subsequent batches, thus eliminating the problematic operations in yeast recycling.
    Elsevier B.V., Sep. 2018, Biochemical Engineering Journal, 137, 23 - 29, English
    [Refereed]
    Scientific journal

  • Ario Betha Juanssilfero, Prihardi Kahar, Rezky Lastinov Amza, Nao Miyamoto, Hiromi Otsuka, Hana Matsumoto, Chie Kihira, Ahmad Thontowi, Yopi, Chiaki Ogino, Bambang Prasetya, Akihiko Kondo
    A two-stage selection process was applied to eight oleaginous yeast strains from the Lipomyces genera. In the primary selection stage, a nitrogen-limited mineral medium (-NMM) that contained a mixture of glucose and xylose as a carbon source was used to evaluate the lipid-accumulating abilities of the yeast strains. The strains L. doorenjongii, L. orientalis, and L. starkeyi were selected as the potential strains in the primary selection. These three strains exhibited a remarkable ability to simultaneously assimilate glucose and xylose and achieved a cell biomass of more than 30 g/L. The values for lipid content in the selected strains were 57.89 ± 1.92, 56.38 ± 1.93, and 77.14 ± 1.55% for L. doorenjongii, L. orientalis, and L. starkeyi, respectively. In the secondary selection, when the -NMM medium contained an inhibitory chemical compound (ICC), the selected strains showed a different tolerance level against each of the typical inhibitor compounds. However, L. starkeyi accumulated the highest lipid content and yield at 68.24 ± 2.48% and 0.19 ± 0.00 (w/w), respectively. L. starkeyi accumulated high levels of intracellular lipid and tolerated the ICC. The composition of fatty acid methyl esters (FAMEs) was unaltered by the presence of ICC and the major FAMEs consisted of oleic, palmitic, stearic, palmitoleic and linoleic acids.
    Elsevier B.V., Sep. 2018, Biochemical Engineering Journal, 137, 182 - 191, English
    [Refereed]
    Scientific journal


  • Akihiko Kondo
    Sep. 2018, Biotechnology Journal, 13(9) (9), 1700596, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Sep. 2018, Microbial Cell Factories, 17, 151, English
    [Refereed]
    Scientific journal

  • 油脂酵母Lipomyces starkeyiD35株の繰り返し発酵による脂肪酸組成変化解析
    宮本 捺央, Kahar Prihardi, 荻野 千秋, 近藤 昭彦
    (公社)日本生物工学会, Aug. 2018, 日本生物工学会大会講演要旨集, 平成30年度, 113 - 113, Japanese

  • Akihiko Kondo
    Aug. 2018, Data in Brief, 20, 1325 - 1331, English
    [Refereed]
    Scientific journal

  • Izzat F. M. Suffian, Julie T.-W. Wang, Farid N. Faruqu, Julio Benitez, Yuya Nishimura, Chiaki Ogino, Akihiko Kondo, Khuloud T. Al-Jamal
    Jul. 2018, ACS Applied Nano Materials, 1(7) (7), 3269 - 3282, English
    Scientific journal

  • 分裂酵母を用いた有機酸生産技術の開発
    TANAKA TSUTOMU, KONDO AKIHIKO
    化学工業社, Jul. 2018, 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), 7 - 10, Japanese
    Scientific journal

  • Tomohisa Hasunuma, Mami Matsuda, Yuichi Kato, Christopher John Vavricka, Akihiko Kondo
    Succinate is a versatile petrochemical compound that can be produced by microorganisms, often from carbohydrate based carbon sources. Phototrophic cyanobacteria including Synechocystis sp. PCC 6803 can more efficiently produce organic acids such as succinate without sugar supplementation, via photosynthetic production of glycogen followed by glycogen utilization, typically under dark conditions. In this study, Synechocystis 6803 bioproduction of organic acids under dark anoxic conditions was found to increase with elevation of temperature from 30 °C to 37 °C. The further enhancement of succinate bioproduction by overexpression of the rate limiting enzyme phosphoenolpyruvate carboxylase resulted in improved glycogen utilization. To gain more insight into the mechanisms underlying the increased organic acid output, a novel temperature dependent metabolomics analysis was performed. Adenylate energy charge was found to decrease along with elevating temperature, while central metabolites glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glycerol 3-phosphate, malate, fumarate and succinate increased. Temperature dependent 13C-labeling metabolomics analysis further revealed a glycolysis to TCA bottleneck, which could be overcome by addition of CO2, leading to even higher organic acid production. Optimization of initial cell concentration to 25 g-dry cell weight/L, in combination with 100 mM NaHCO3 supplementation, afforded a succinate titer of over 1.8 g/L, the highest reported autotrophic succinate titer. Succinate titers remained high after additional knockout of ackA, resulting in the highest reported autotrophic D-lactate titer as well. The optimization of Synechocystis 6803 organic acid production therefore holds significant promise for CO2 capture and utilization.
    Academic Press Inc., Jul. 2018, Metabolic Engineering, 48, 109 - 120, English
    [Refereed]
    Scientific journal

  • Yota Tsuge, Hideo Kawaguchi, Shogo Yamamoto, Yoshiko Nishigami, Masahiro Sota, Chiaki Ogino, Akihiko Kondo
    Ultraviolet-absorbing chemicals are useful in cosmetics and skin care to prevent UV-induced skin damage. We demonstrate here that heterologous production of shinorine, which shows broad absorption maxima in the UV-A and UV-B region. A shinorine producing Corynebacterium glutamicum strain was constructed by expressing four genes from Actinosynnema mirum DSM 43827, which are responsible for the biosynthesis of shinorine from sedoheptulose-7-phosphate in the pentose phosphate pathway. Deletion of transaldolase encoding gene improved shinorine production by 5.2-fold. Among the other genes in pentose phosphate pathway, overexpression of 6-phosphogluconate dehydrogenase encoding gene further increased shinorine production by 60% (19.1 mg/L). The genetic engineering of the pentose phosphate pathway in C. glutamicum improved shinorine production by 8.3-fold in total, and could be applied to produce the other chemicals derived from sedoheptulose-7-phosphate.
    Jul. 2018, Bioscience, biotechnology, and biochemistry, 82(7) (7), 1252 - 1259, English, International magazine
    [Refereed]
    Scientific journal

  • 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.
    Jul. 2018, Journal of bioscience and bioengineering, 126(1) (1), 119 - 125, English, Domestic magazine
    [Refereed]
    Scientific journal

  • Yasuyuki Nakamura, Teruyuki Nishi, Risa Noguchi, Yoichiro Ito, Toru Watanabe, Tozo Nishiyama, Sinpei Aikawa, Tomohisa Hasunuma, Jun Ishii, Yuji Okubo, Akihiko Kondo
    ABSTRACT The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris. Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability. IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.
    American Society for Microbiology, Jul. 2018, Applied and Environmental Microbiology, 84(15) (15), e02882 - 17, English
    [Refereed]
    Scientific journal

  • Ario B. Juanssilfero, Prihardi Kahar, Rezky L. Amza, Nao Miyamoto, Hiromi Otsuka, Hana Matsumoto, Chie Kihira, Ahmad Thontowi, Yopi, Chiaki Ogino, Bambang Prasetya, Akihiko Kondo
    Oleaginous microbes can convert substrates such as carbon dioxide, sugars, and organic acids to single-cell oils (SCOs). Among the oleaginous microorganisms, Lipomyces starkeyi is a particularly well-suited host given its impressive native abilities, including the capability to utilize a wide variety of carbon sources. In this work, the potential of L. starkeyi NBRC10381 to produce SCOs in a synthetically nitrogen-limited mineral medium (-NMM) was investigated by differing the inoculum size using glucose and/or xylose as a carbon source. Fermentation using glucose and xylose as mixed carbon sources generated the highest production of biomass at 40.8 g/L, and achieved a lipid content of 84.9% (w/w). When either glucose or xylose was used separately, the totals for achieved lipid content were 79.6% (w/w) and 85.1% (w/w), respectively. However, biomass production was higher for glucose than for xylose (30.3 vs. 28.7 g/L, respectively). This study describes the first simultaneous achievement of higher levels of cell mass and lipid production using glucose and/or xylose as the carbon sources in different inoculum sizes.
    Elsevier B.V., Jun. 2018, Journal of Bioscience and Bioengineering, 125(6) (6), 695 - 702, English
    [Refereed]
    Scientific journal


  • Akihiko Kondo
    Jun. 2018, Bio-Protocol, 8(12) (12), e2887, English
    [Refereed]
    Scientific journal

  • Hideo Kawaguchi, Kumiko Yoshihara, Kiyotaka Y. Hara, Tomohisa Hasunuma, Chiaki Ogino, Akihiko Kondo
    Background: l-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to d-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as d-glucose, leading to a preferential utilization of d-glucose over pentoses. In order to simultaneously utilize both d-glucose and l-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis. Results: Corynebacterium glutamicum ATCC 31831 utilized d-glucose and l-arabinose simultaneously at a low concentration (3.6g/L each) but preferentially utilized d-glucose over l-arabinose at a high concentration (15g/L each), although l-arabinose and d-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for d-glucose and l-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of l-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of d-glucose and l-arabinose (15g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized d-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of l-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of d-glucose and l-arabinose. Conclusions: Metabolome analysis identified potential bottlenecks in d-glucose and l-arabinose metabolism and was then applied to the following rational metabolic engineering. Manipulation of only two genes enabled simultaneous utilization of d-glucose and l-arabinose at the same rate in metabolically engineered C. glutamicum. This is the first report of rational metabolic design and engineering for simultaneous hexose and pentose utilization without inactivating the phosphotransferase system.
    BioMed Central Ltd., May 2018, Microbial Cell Factories, 17(1) (1), 76, English
    [Refereed]
    Scientific journal

  • Nicholas M. Thomson, Tomokazu Shirai, Marco Chiapello, Akihiko Kondo, Krishna J. Mukherjee, Easan Sivaniah, Keiji Numata, David K. Summers
    The authors show that quiescent (Q-Cell) Escherichia coli cultures can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater specific productivity of a model metabolite, 3-hydroxybutyrate (3HB), than a control. Q-cells can be created by using the proton ionophore indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks because less metabolic effort is diverted to surplus biomass production. However, the reason for the increased productivity of cells in the quiescent state was previously unknown. In this study, proteome expression patterns between wild-type and Q-cell cultures show that Q-cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Metabolomic data reveal the accumulation of acetyl-coenzyme A and phosphoenolpyruvate: excellent starting points for high-value chemical production. We demonstrate the exploitation of these accumulated metabolites by engineering a simple pathway for 3HB production from acetyl-coenzyme A. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g L−1 of 3HB compared to 18.6 g L−1 in the control. Q-cells therefore have great potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals.
    Wiley-VCH Verlag, May 2018, Biotechnology Journal, 13(5) (5), 1700571, English
    [Refereed]
    Scientific journal

  • Yohei Tashiro, Shinichi Hirano, Morgan M. Matson, Shota Atsumi, Akihiko Kondo
    Here we have developed an electrochemical-biological hybrid system to fix CO2. Natural biological CO2 fixation processes are relatively slow. To increase the speed of fixation we applied electrocatalysts to reduce CO2 to formate. We chose a user-friendly organism, Escherichia coli, as host. Overall, the newly constructed CO2 and formate fixation pathway converts two formate and one CO2 to one pyruvate via glycine and L-serine in E. coli. First, one formate and one CO2 are converted to one glycine. Second, L-serine is produced from one glycine and one formate. Lastly, L-serine is converted to pyruvate. E. coli's genetic tractability allowed us to balance various parameters of the pathway. The carbon flux of the pathway was sufficient to compensate L-serine auxotrophy in the strain. In total, we integrated both electrocatalysis and biological systems into a single pot to support E. coli growth with CO2 and electricity. Results show promise for using this hybrid system for chemical production from CO2 and electricity.
    Academic Press Inc., May 2018, Metabolic Engineering, 47, 211 - 218, English
    [Refereed]
    Scientific journal

  • Kenji Okano, Gentaro Uematsu, Shinji Hama, Tsutomu Tanaka, Hideo Noda, Akihiko Kondo, Kohsuke Honda
    Fermentative production of optically pure lactic acid (LA) has attracted great interest because of the increased demand for plant-based plastics. For cost-effective LA production, an engineered Lactobacillus plantarum NCIMB 8826 strain, which enables the production of optically pure l-LA from raw starch, is constructed. The wild-type strain produces a racemic mixture of d- and l-LA from pyruvate by the action of the respective lactate dehydrogenases (LDHs). Therefore, the gene encoding D-LDH (ldhD) is deleted. Although no decrease in d-LA formation is observed in the ΔldhD mutant, additional disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between d- and l-LA, completely abolished d-LA production. From 100 g L−1 glucose, the ΔldhD ΔlarA-E mutant produces 87.0 g L−1 of l-LA with an optical purity of 99.4%. Subsequently, a plasmid is introduced into the ΔldhD ΔlarA-E mutant for the secretion of α-amylase from Streptococcus bovis 148. The resulting strain could produce 50.3 g L−1 of l-LA from raw corn starch with a yield of 0.91 (g per g of consumed sugar) and an optical purity of 98.6%. The engineered L. plantarum strain would be useful in the production of l-LA from starchy materials.
    Wiley-VCH Verlag, May 2018, Biotechnology Journal, 13(5) (5), 1700517, English
    [Refereed]
    Scientific journal

  • Shinji Hama, Hideo Noda, Akihiko Kondo
    Given the increasing interest in alternative processes for producing biodiesel, we focused on the latest screening of lipases and bioprocess design using multiple feedstocks. The implementation of lipase technology to the biodiesel industry is in the early stages. However, current research has made phenomenal advances in generating lipase variants and in engineering biodiesel production. The structural insights into lipase stability, together with primary screening, have opened up opportunities for acquiring lipase variants that are highly tolerant under industrially relevant conditions. The versatility of lipases is promising for process intensification, where time-consuming and costly steps can possibly be avoided. To judiciously overcome uncertainties in the biodiesel industry, further research on technology development integrated with supply chain models is necessary.
    Elsevier Ltd, Apr. 2018, Current Opinion in Biotechnology, 50, 57 - 64, English
    [Refereed]
    Scientific journal

  • Hal S Alper, Akihiko Kondo
    Elsevier Ltd, Apr. 2018, Current Opinion in Biotechnology, 50, v - vi, English
    [Refereed]
    Scientific journal

  • Hiroki Hashi, Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo
    Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene. However, the fluorescence intensity (sensitivity) of NTSR1-expressing yeast cells is low compared to that of yeast cells expressing other human GPCRs (e.g., human somatostatin receptors). The present study sought to increase the sensitivity of the NTSR1-expressing yeast for use as a fluorescent biosensor, including modification of the expression of human NTSR1 in yeast. Changes in the transcription, translation, and transport of the receptor are attempted by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene. The resulting yeast cells exhibited increased sensitivity to exogenously added peptide. The cells are further engineered by using cell-surface display technology to ensure that the agonistic peptides are secreted and tethered to the yeast cell wall, yielding cells with enhanced NTSR1 activation. This yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases.
    Wiley-VCH Verlag, Apr. 2018, Biotechnology Journal, 13(4) (4), e1700522, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Apr. 2018, AMB Express, 8, 56, English
    [Refereed]
    Scientific journal

  • 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.
    Apr. 2018, Current opinion in biotechnology, 50, 182 - 188, English, International magazine
    [Refereed]
    Scientific journal

  • Satomi Banno, Keiji Nishida, Takayuki Arazoe, Hitoshi Mitsunobu, Akihiko Kondo
    In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool1, 2. However, in prokaryotes, the use of nuclease-mediated genome editing tools has been limited to negative selection for the already modified cells because of its lethality 3, 4. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID)5 adopted in Escherichia coli. Cytidine deaminase PmCDA1 fused to the nuclease-deficient CRISPR-Cas9 system achieved specific point mutagenesis at the target sites in E. coli by introducing cytosine mutations without compromising cell growth. The cytosine-to-thymine substitutions were induced mainly within an approximately five-base window of target sequences on the protospacer adjacent motif-distal side, which can be shifted depending on the length of the single guide RNA sequence. Use of a uracil DNA glycosylase inhibitor6 in combination with a degradation tag (LVA tag)7 resulted in a robustly high mutation efficiency, which allowed simultaneous multiplex editing of six different genes. The major multi-copy transposase genes that consist of at least 41 loci were also simultaneously edited by using four target sequences. As this system does not rely on any additional or host-dependent factors, it may be readily applicable to a wide range of bacteria.
    Nature Publishing Group, Apr. 2018, Nature Microbiology, 3(4) (4), 423 - 429, English
    [Refereed]
    Scientific journal

  • Yuya Nishimura, Terumi Matsui, Jun Ishii, Akihiko Kondo
    Background: To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels. Results: We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA) (ii) overexpression of threonine dehydratase (tdcB) (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC) and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose. Conclusion: These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.
    BioMed Central Ltd., Mar. 2018, Microbial Cell Factories, 17(1) (1), 38, English
    [Refereed]
    Scientific journal

  • Takahiro Bamba, Kentaro Inokuma, Tomohisa Hasunuma, Akihiko Kondo
    Yeast displaying enzymes on the cell surface are used for developing whole-cell biocatalysts. High enzyme activity on the cell surface is required in certain applications such as direct ethanol production from lignocellulosic materials. However, the cell surface enzyme activity is limited by several factors, one of which is the protein amount of the yeast cell wall. In this study, we attempted to improve the incorporation capacity of a displayed heterologous enzyme by disrupting a native cell-wall protein. β-Glucosidase (BGL1) from Aspergillus aculeatus was fused with Saccharomyces cerevisiae Sed1 and displayed on the cell surface of S. cerevisiae BY4741 strain and its SED1 disruptant. Sed1 is one of the most abundant stationary phase yeast cell wall protein. A time course analysis revealed that BGL1 activity of the control strain reached saturation after 48 h of cultivation. In contrast, the BGL1 activity of the SED1 disruptant increased until 72 h of cultivation and was 22% higher than that of the control strain. We also performed relative quantification of cell wall proteins of these strains by nanoscale ultra pressure liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nano-UPLC-MSE). The amount of the cell wall-associated BGL1 per unit dry cell-weight of the SED1 disruptant was 19% higher than that of the control strain. These results suggested that the incorporation capacity of the cell wall for BGL1 was increased by disruption of SED1. Disruption of SED1 would be a promising approach for improving display efficiency of heterologous protein fused with Sed1.
    Elsevier B.V., Mar. 2018, Journal of Bioscience and Bioengineering, 125(3) (3), 346 - 352, English
    [Refereed]
    Scientific journal

  • Hiroshi Teramura, Kengo Sasaki, Tomoko Oshima, Hideo Kawaguchi, Chiaki Ogino, Takashi Sazuka, Akihiko Kondo
    © 2017 We investigated the use of low concentrations of butanol (<40%, all v/v) as an organosolv pretreatment to fractionate lignocellulosic biomass into cellulose, hemicellulose, and lignin. The pretreatment conditions were optimized for sorghum bagasse by focusing on four parameters: butanol concentration, sulfuric acid concentration, pretreatment temperature, and pretreatment time. A butanol concentration of 25% or higher together with 0.5% or higher acid was effective for removing lignin while retaining most of the cellulose in the solid fraction. The highest cellulose (84.9%) and low lignin (15.3%) content were obtained after pretreatment at 200 °C for 60 min. Thus, pretreatment comprising 25% butanol, 0.5% acid, 200 °C, and 60 min process time was considered optimal. Enzymatic saccharification and fermentation by Saccharomyces cerevisiae produced 61.9 g/L ethanol from 200 g/L solid fraction obtained following pretreatment, and 10.2 g/L ethanol was obtained from the liquid fraction by xylose-utilizing S. cerevisiae following membrane nanofiltration to remove butanol.
    Mar. 2018, Bioresource Technology, 252, 157 - 164, English
    [Refereed]
    Scientific journal

  • Shimpei Aikawa, Kentaro Inokuma, Satoshi Wakai, Kengo Sasaki, Chiaki Ogino, Jo-Shu Chang, Tomohisa Hasunuma, Akihiko Kondo
    Background: The cyanobacterium Arthrospira platensis shows promise as a carbohydrate feedstock for biofuel production. The glycogen accumulated in A. platensis can be extracted by lysozyme-degrading the peptidoglycan layer of the bacterial cell walls. The extracted glycogen can be converted to ethanol through hydrolysis by amylolytic enzymes and fermentation by the yeast Saccharomyces cerevisiae. Thus, in the presence of lysozyme, a recombinant yeast expressing α-amylase and glucoamylase can convert A. platensis directly to ethanol, which would simplify the procedure for ethanol production. However, the ethanol titer and productivity in this process are lower than in ethanol production from cyanobacteria and green algae in previous reports. Results: To increase the ethanol titer, a high concentration of A. platensis biomass was employed as the carbon source for the ethanol production using a recombinant amylase-expressing yeast. The addition of lysozyme to the fermentation medium increased the ethanol titer, but not the ethanol productivity. The addition of CaCl2 increased both the ethanol titer and productivity by causing the delamination of polysaccharide layer on the cell surface of A. platensis. In the presence of lysozyme and CaCl2, ethanol titer, yield, and productivity improved to 48 g L-1, 93% of theoretical yield, and 1.0 g L-1 h-1 from A. platensis, corresponding to 90 g L-1 of glycogen. Conclusions: We developed an ethanol conversion process using a recombinant amylase-expressing yeast from A. platensis with a high titer, yield, and productivity by adding both lysozyme and CaCl2. The direct and highly productive conversion process from A. platensis via yeast fermentation could be applied to multiple industrial bulk chemicals.
    BioMed Central Ltd., Feb. 2018, Biotechnology for Biofuels, 11(1) (1), 50, English
    [Refereed]
    Scientific journal

  • Yong-Hao Li, Xiao-Yue Zhang, Fei Zhang, Liang-Cai Peng, Da-Bing Zhang, Akihiko Kondo, Feng-Wu Bai, Xin-Qing Zhao
    Background: Cellulolytic enzymes produced by Trichoderma reesei are widely studied for biomass bioconversion, and enzymatic components vary depending on different inducers. In our previous studies, a mixture of glucose and disaccharide (MGD) was developed and used to induce cellulase production. However, the enzymatic profile induced by MGD is still not defined, and further optimization of the enzyme cocktail is also required for efficient ethanol production from lignocellulosic biomass. Results: In this study, cellulolytic enzymes produced by T. reesei Rut C30 using MGD and alkali-pretreated corn stover (APCS) as inducer were compared. Cellular secretome in response to each inducer was analyzed, which revealed a similar enzyme profile. However, significant difference in the content of cellulases and xylanase was detected. Although MGD induction enhanced β-glucosidase production, its activity was still not sufficient for biomass hydrolysis. To overcome such a disadvantage, aabgl1 encoding β-glucosidase in Aspergillus aculeatus was heterologously expressed in T. reesei Rut C30 under the control of the pdc1 promoter. The recombinant T. reesei PB-3 strain showed an improved β-glucosidase activity of 310 CBU/mL in the fed-batch fermentation, 71-folds higher than that produced by the parent strain. Meanwhile, cellulase activity of 50 FPU/mL was detected. Subsequently, the crude enzyme was applied for hydrolyzing corn stover with a solid loading of 20% through separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation, respectively, for ethanol production. Better performance was observed in the SHF process, through which a total of 119.9 g/L glucose was released within 12 h for concomitant ethanol production of 54.2 g/L. Conclusions: The similar profile of cellulolytic enzymes was detected under the induction of MGD and APCS, but higher amount of cellulases was present in the crude enzyme induced by MGD. However, β-glucosidase activity induced by MGD was not sufficient for hydrolyzing lignocellulosic biomass. High titers of cellulases and β-glucosidase were achieved simultaneously by heterologous expression of aabgl1 in T. reesei and fed-batch fermentation through feeding MGD. We demonstrated that on-site cellulase production by T. reesei PB-3 has a potential for efficient biomass saccharification and ethanol production from lignocellulosic biomass.
    BioMed Central Ltd., Feb. 2018, Biotechnology for Biofuels, 11(1) (1), 49, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    2018, Neurologia Medico-Chirurgica
    Scientific journal

  • Akihiko Kondo
    2018, Epilepsy & Behavior Case Reports
    Scientific journal


  • Microbial platform to synthesize aromatic chemicals and the derivatives
    Akihiko Kondo
    2018, FEBS Open Bio
    Scientific journal

  • Akihiko Kondo
    2018, Internal Medicine
    Scientific journal

  • Metabolic design of Escherichia coli for production of shikimate pathway derivatives
    Akihiko Kondo
    2018, Basic & Clinical Pharmacology & Toxicology
    Scientific journal


  • Metabolic engineering of S. pombe via CRISPR-CAS9 genome editing for 3-hydroxypropionic acid production from glucose and cellobiose
    Akihiko Kondo
    2018, Basic & Clinical Pharmacology & Toxicology
    Scientific journal

  • Akihiko Kondo
    2018, International Journal of Surgery Case Reports
    Scientific journal

  • Metabolic design of Escherichia coli for muconic acid production
    Akihiko Kondo
    2018, FEBS Open Bio
    Scientific journal



  • Akihiko Kondo
    2018, Wilderness and Environmental Medicine
    Scientific journal


  • Akihiko Kondo
    2018, Journal of Visualized Experiments
    Scientific journal

  • Kawaguchi Hideo, Katsuyama Yohei, Du Danyao, Nakamura-Tsuruta Sachiko, Minami Hiromichi, Ogino Chiaki, Ohnishi Yasuo, Kondo Akihiko

    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.

    The Japan Institute of Energy, 2018, Proceedings of the Conference on Biomass Science, 13(0) (0), 135 - 136, Japanese
    [Refereed]

  • 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, Methods in molecular biology (Clifton, N.J.), 1772, 125 - 136, English, International magazine
    [Refereed]
    Scientific journal

  • 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, Jan. 2018, BIOSENSORS & BIOELECTRONICS, 99, 56 - 61, English
    [Refereed]
    Scientific journal

  • Yumiko Nambu-Nishida, Yuri Sakihama, Jun Ishii, Tomohisa Hasunuma, Akihiko Kondo
    To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, PTDH3, PFBA1, and PTDH1 were favorable for high expression, and PSED1, PHXT7, PPDC1, PTEF1, PTPI1, and PPGK1 were acceptable for medium–high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. PTEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium–high expression in glucose media. PZWF1 and PSOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. PALD3 and PTKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation.
    Elsevier B.V., Jan. 2018, Journal of Bioscience and Bioengineering, 125(1) (1), 76 - 86, English
    [Refereed]
    Scientific journal

  • Nanik Rahmani, Prihardi Kahar, Puspita Lisdiyanti, Euis Hermiati, Jaemin Lee, Yopi, Bambang Prasetya, Chiaki Ogino, Akihiko Kondo
    The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification.
    Japan Society for Bioscience Biotechnology and Agrochemistry, 2018, Bioscience, Biotechnology and Biochemistry, 82(5) (5), 904 - 915, English
    [Refereed]
    Scientific journal

  • Kengo Sasaki, Daisuke Sasaki, Yota Tsuge, Masahiko Morita, Akihiko Kondo
    Background: Bioelectrochemical systems (BESs) are an innovative technology developed to influence conventional anaerobic digestion. We examined the feasibility of applying a BES to dark hydrogen fermentation and its effects on a two-stage fermentation process comprising hydrogen and methane production. The BES used low-cost, low-reactivity carbon sheets as the cathode and anode, and the cathodic potential was controlled at - 1.0 V (vs. Ag/AgCl) with a potentiostat. The operation used 10 g/L glucose as the major carbon source. Results: The electric current density was low throughout (0.30-0.88 A/m2 per electrode corresponding to 0.5-1.5 mM/day of hydrogen production) and water electrolysis was prevented. At a hydraulic retention time of 2 days with a substrate pH of 6.5, the BES decreased gas production (hydrogen and carbon dioxide contents: 52.1 and 47.1%, respectively), compared to the non-bioelectrochemical system (NBES), although they had similar gas compositions. In addition, a methane fermenter (MF) was applied after the BES, which increased gas production (methane and carbon dioxide contents: 85.1 and 14.9%, respectively) compared to the case when the MF was applied after the NBES. Meta 16S rRNA sequencing revealed that the BES accelerated the growth of Ruminococcus sp. and Veillonellaceae sp. and decreased Clostridium sp. and Thermoanaerobacterium sp., resulting in increased propionate and ethanol generation and decreased butyrate generation; however, unknowingly, acetate generation was increased in the BES. Conclusions: The altered redox potential in the BES likely transformed the structure of the microbial consortium and metabolic pattern to increase methane production and decrease carbon dioxide production in the two-stage process. This study showed the utility of the BES to act on the microbial consortium, resulting in improved gas production from carbohydrate compounds.
    2018, Biotechnology for biofuels, 11, 173 - 173, English, International magazine
    [Refereed]
    Scientific journal

  • Ryoko Ohno, Hiroshi Teramura, Chiaki Ogino, Akihiko Kondo, Shigeo Takumi
    © 2018, Genetics Society of Japan. All rights reserved. Wheat straw is one of the major attractive resources for low-cost raw materials for renewable energy, biofuels and biochemicals. However, like other sources of lignocellulosic biomass, straw is a heterogeneous material due to its mixed origin from different tissue and cell types. Here, to examine the genotypic effects on biorefinery usage of wheat straw, straw obtained from different wheat cultivars and experimental lines was pretreated with dilute acid. Significant differences between cultivars were observed in the concentrations of glucose and toxic byproducts of the liquid hydrolysates. A higher content of xylose than glucose was found in liquid hydrolysates from wheat straw, and the xylose content appeared to be affected by both environmental and genetic factors. Analysis using chromosome substitution lines of the common wheat cultivar Chinese Spring showed that chromosomes 2A and 3A from other wheat cultivars, Hope and Timstein, significantly increased the xylose content. However, no significant relationship was observed between the liquid hydrolysate xylose content and the glucose content obtained from enzymatic saccharification of the acid-insoluble residue. These results highlight the potential of wheat breeding to improve biomass-related traits in wheat straw.
    2018, Genes and Genetic Systems, 93(1) (1), 1 - 7, English
    [Refereed]
    Scientific journal

  • Zenpei Shimatani, Ushio Fujikura, Hisaki Ishii, Yusuke Matsui, Minoru Suzuki, Yuki Ueke, Ken-ichiro Taoka, Rie Terada, Keiji Nishida, Akihiko Kondo
    The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus. Inheritance of the introduced mutations, as well as the removal of transgenes, are key issues that must be addressed in order to fully develop Target-AID as a plant breeding technique. Here we report the transmission of such mutations from the callus to regenerants and their progenies, leading to a generation of selectable marker-free (SMF) herbicide tolerant rice plants with simultaneous multiplex nucleotide substitutions. These findings demonstrate that Target-AID can be developed into novel plant breeding technology which enables improvement of multiplex traits at one time in combination with sophisticated targeted base editing with the simplicity and versatility of CRISPR/Cas9 system.
    Elsevier Masson SAS, 2018, Plant Physiology and Biochemistry, 131, 78 - 83, English
    [Refereed]
    Scientific journal

  • 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.
    Dec. 2017, AMB Express, 7(1) (1), 130 - 130, English, International magazine
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(B) (B), 1447 - 1454, English
    [Refereed]
    Scientific journal

  • 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
    Biobutanol is gaining more attention as a potential alternative to ethanol, and the demand for fermentative biobutanol production has renewed interest. The main challenge faced in biobutanol production is the availability of feedstock. Using conventional agricultural biomass as feedstock is controversial and less efficient, while microalgae, the third generation feedstock, are considered promising feedstock for biobutanol production due to their high growth rate and high carbohydrates content. This review is primarily focused on biobutanol production by using carbohydrate-rich microalgal feedstock. Key technologies and challenges involved in producing butanol from microalgae are discussed in detail and future directions are also presented.
    Elsevier Inc., Dec. 2017, Biotechnology Advances, 35(8) (8), 1049 - 1059, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIOCHEMICAL ENGINEERING JOURNAL, 128, 195 - 200, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245, 1655 - 1663, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(B) (B), 1664 - 1673, English
    [Refereed]
    Scientific journal

  • Nanik Rahmani, Norimasa Kashiwagi, Jae Min Lee, Satoko Niimi-Nakamura, Hana Matsumoto, Prihardi Kahar, Puspita Lisdiyanti, Yopi, Bambang Prasetya, Chiaki Ogino, Akihiko Kondo
    Mannan endo-1,4-β-mannosidase (commonly known as β-mannanase) catalyzes a random cleavage of the β-d-1,4-mannopyranosyl linkage in mannan polymers. The enzyme has been utilized in biofuel production from lignocellulose biomass, as well as in production of mannooligosaccharides (MOS) for applications in feed and food industries. We aimed to obtain a β-mannanase, for such mannan polymer utilization, from actinomycetes strains isolated in Indonesia. Strains exhibiting high mannanase activity were screened, and one strain belonging to the genus Kitasatospora was selected. We obtained a β-mannanase from this strain, and an amino acid sequence of this Kitasatospora β-mannanase showed a 58–71% similarity with the amino acid sequences of Streptomyces β-mannanases. The Kitasatospora β-mannanase showed a significant level of activity (944 U/mg) against locust bean gum (0.5% w/v) and a potential for oligosaccharide production from various mannan polymers. The β-mannanase might be beneficial particularly in the enzymatic production of MOS for applications of mannan utilization.
    Springer Verlag, Dec. 2017, AMB Express, 7(1) (1), 100, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(B) (B), 1314 - 1326, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(B) (B), 1413 - 1420, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(B) (B), 1436 - 1446, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Dec. 2017, Frontiers in Bioengineering and Biotechnology, 5, 81, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(B) (B), 1400 - 1406, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(part B) (part B), 1484 - 1490, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Dec. 2017, Bioresource Technology, 245(B) (B), 1362 - 1368, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS, 28, 16 - 23, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2017, BIORESOURCE TECHNOLOGY, 245(B) (B), 1684 - 1691, English
    [Refereed]
    Scientific journal

  • 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, Nov. 2017, SENSORS AND ACTUATORS B-CHEMICAL, 252, 813 - 821, English
    [Refereed]
    Scientific journal

  • 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, Nov. 2017, SENSORS AND ACTUATORS B-CHEMICAL, 252, 600 - 605, English
    [Refereed]
    Scientific journal

  • 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, Nov. 2017, FEMS YEAST RESEARCH, 17(7) (7), fox061, English
    [Refereed]
    Scientific journal

  • 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, Oct. 2017, NATURE COMMUNICATIONS, 8(1) (1), 1153, English
    [Refereed]
    Scientific journal

  • M. J. Mert, S. H. Rose, D. C. la Grange, T. Bamba, T. Hasunuma, A. Kondo, W. H. van Zyl
    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, Oct. 2017, JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, 44(10) (10), 1459 - 1470, English
    [Refereed]
    Scientific journal

  • 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, Oct. 2017, TRENDS IN BIOTECHNOLOGY, 35(10) (10), 983 - 996, English
    [Refereed]
    Scientific journal

  • Paul Marques Alves, Anke Becker, Helmut Blöcker, Raquel Lia Chan, George Guo-Qiang Chen, Martin Fussenegger, Akihiko Kondo, Robert Kourist, Helmut Schwab, Jin-Ho Seo, Christoph W. Sensen, Maria Sensen, Friedrich Scrienc
    Elsevier B.V., Sep. 2017, Journal of Biotechnology, 257, 1, English
    [Refereed]
    Scientific journal

  • 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, Sep. 2017, PHOTOSYNTHESIS RESEARCH, 133(1-3) (1-3), 235 - 243, English
    [Refereed]
    Scientific journal

  • 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, Sep. 2017, PHOTOSYNTHESIS RESEARCH, 133(1-3) (1-3), 317 - 326, English
    [Refereed]
    Scientific journal

  • 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, Sep. 2017, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 124(3) (3), 263 - 270, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2017, TRENDS IN BIOTECHNOLOGY, 35(8) (8), 785 - 796, English
    [Refereed]
    Scientific journal

  • 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.
    Aug. 2017, Applied microbiology and biotechnology, 101(15) (15), 6007 - 6014, English, International magazine
    [Refereed]
    Scientific journal

  • 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
    Aug. 2017, Metabolic Engineering Communications, 5, 60 - 67, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2017, SCIENTIFIC REPORTS, 7(1) (1), 8993, English
    [Refereed]
    Scientific journal

  • 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, Jul. 2017, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 101(13) (13), 5279 - 5290, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Jun. 2017, Bio-Protocol, 7, 11, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2017, BIOTECHNOLOGY AND BIOENGINEERING, 114(6) (6), 1354 - 1361, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2017, ACS SYNTHETIC BIOLOGY, 6(6) (6), 928 - 935, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2017, BIOTECHNOLOGY AND BIOENGINEERING, 114(6) (6), 1201 - 1207, English
    [Refereed]
    Scientific journal

  • 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, May 2017, APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 182(1) (1), 229 - 237, English
    [Refereed]
    Scientific journal

  • 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, May 2017, BIOCHEMICAL ENGINEERING JOURNAL, 121, 13 - 16, English
    [Refereed]
    Scientific journal

  • 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, May 2017, NATURE BIOTECHNOLOGY, 35(5) (5), 441 - +, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2017, SCIENTIFIC REPORTS, 7(7) (7), 45471, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2017, BIORESOURCE TECHNOLOGY, 227, 373 - 387, English
    [Refereed]
    Scientific journal

  • Mika Ishigaki, Akihito Nakanishi, Tomohisa Hasunuma, Akihiko Kondo, Tetsu Morishima, Toshiaki Okuno, Yukihiro Ozaki
    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, Mar. 2017, APPLIED SPECTROSCOPY, 71(3) (3), 463 - 471, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2017, SCIENTIFIC REPORTS, 7, 43160, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2017, BIOTECHNOLOGY JOURNAL, 12(3) (3), 1600400, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2017, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 101(5) (5), 1869 - 1875, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2017, COLLOIDS AND SURFACES B-BIOINTERFACES, 151, 134 - 142, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2017, MICROBIAL CELL FACTORIES, 16, 44, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2017, BIOMATERIALS, 120, 126 - 138, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2017, SCIENTIFIC REPORTS, 7, 43518, English
    [Refereed]
    Scientific journal

  • 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, Feb. 2017, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 27(4) (4), 954 - 957, English
    [Refereed]
    Scientific journal

  • 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, Feb. 2017, CHEMISTRY-AN ASIAN JOURNAL, 12(3) (3), 324 - 331, English
    [Refereed]
    Scientific journal

  • Application of LC-MS/MS analysis for time-lapse amino acid metabolomics in CHO cell culture.
    Hsu H, Hasunuma T, Araki M, Yoshida T, Hori Y, Murata M, Kondo A
    Feb. 2017, Shimadzu Journal, 5(1) (1), 17 - 21, English
    [Refereed]
    Scientific journal




  • Akihiko Kondo
    2017, Annals of Allergy, Asthma & Immunology
    Scientific journal


  • Akihiko Kondo
    2017, Resuscitation
    Scientific journal


  • INOKUMA Kentaro, HASUNUMA Tomohisa, KONDO Akihiko

    The recombinant yeast strains displaying the heterologous cellulolytic enzymes on the cell surface using the glycosylphosphatidylinositol (GPI) anchoring system are considered promising biocatalysts for the consolidated bioethanol production from lignocellulosic biomass. In this study, we constructed novel gene cassettes for the efficient cellulase display on yeast cell surface. We revealed that simultaneous utilization of the GPI anchoring region derived from Saccharomyces cerevisiae SED1 and its original promoter in a gene cassette enabled highly-efficient enzyme integration into the cell wall. The β-glucosidase and endoglucanase activities of recombinant yeast cells transduced with the novel gene cassette were 8.4- and 106-fold higher than those of conventional strains. The novel gene cassette also improved cell-surface hemicellulase activity. These results suggest that the novel gene cassette has the wide applicability for efficient cell-surface display of heterologous enzymes and that recombinant yeast cells displaying enzymes using these cassettes are promising biocatalysts for the efficient ethanol production from biomass resources.

    The Japan Institute of Energy, 2017, Proceedings of the Conference on Biomass Science, 12, 37 - 38, Japanese

  • 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, Jan. 2017, BIOTECHNOLOGY FOR BIOFUELS, 10, 27, English
    [Refereed]
    Scientific journal

  • Shibuya, M., Sasaki, K., Tanaka, Y., Yasukawa, M., Takahashi, T., Kondo, A., Matsuyama, H.
    2017, Bioresource Technology, 235, 405 - 410, English
    [Refereed]
    Scientific journal

  • 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, Genome Announcements, 5(5) (5), English
    [Refereed]
    Scientific journal

  • 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, PloS one, 12(7) (7), e0180991, English, International magazine
    [Refereed]
    Scientific journal

  • 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 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, Jan. 2010, ENZYME AND MICROBIAL TECHNOLOGY, 46(1) (1), 51 - 55, English
    [Refereed]
    Scientific journal

  • Kenji Okano, Qiao Zhang, Shogo Yoshida, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo
    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, Jan. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(3) (3), 643 - 650, English
    [Refereed]
    Scientific journal

  • 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, Jan. 2010, JOURNAL OF BIOTECHNOLOGY, 145(1) (1), 79 - 83, English
    [Refereed]
    Scientific journal

  • 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, Jan. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85(3) (3), 413 - 423, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(24) (24), 7858 - 7861, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2009, BIOCONTROL SCIENCE, 14(4) (4), 171 - 175, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2009, JOURNAL OF BIOCHEMISTRY, 146(6) (6), 867 - 874, English
    [Refereed]
    Scientific journal

  • Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo
    SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108(1) (1), S36 - S36, English
    [Refereed]
    Scientific journal

  • 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, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108(5) (5), 359 - 364, English
    [Refereed]
    Scientific journal

  • 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, Nov. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108(5) (5), 369 - 371, English
    [Refereed]
    Scientific journal

  • Hiroyuki Yamamoto, Hideki Yamaji, Yuichiro Abe, Kazuo Harada, Danang Waluyo, Eiichiro Fukusaki, Akihiko Kondo, Hiromu Ohno, Hideki Fukuda
    Dimensionality reduction is an important technique for preprocessing of high-dimensional data. Because only one side of the original data is represented in a low-dimensional subspace, useful information may be lost. In the present study, novel dimensionality reduction methods were developed that are suitable for metabolome data, where observation varies with time. Metabolomics deal with this type of data, which are often obtained in microorganism fermentation processes. However, no dimensionality reduction method that utilizes information from the original data in a positive manner has been reported to date. The ordinary dimensionality reduction methods of principal component analysis (PCA), partial least squares (PLS), orthonormalized PLS (OPLS), and regularized Fisher discriminant analysis (RFDA) were extended by introducing differential penalties to the latent variables in each class. A nonlinear extension of this approach, using kernel methods, was also proposed in the form of kernel-smoothed PCA, PLS, OPLS, and FDA. Since all of these methods are formulated as generalized eigenvalue problems, the solutions can be computed easily. These methods were then applied to intracellular metabolite data of a xylose-fermenting yeast in ethanol fermentation. Visualization in the low-dimensional subspace suggests that smoothed PCA successfully preserves the information about the time course of observations during fermentation, and that RFDA can produce high separation among different strains. © 2009 Elsevier B.V. All rights reserved.
    Oct. 2009, Chemometrics and Intelligent Laboratory Systems, 98(2) (2), 136 - 142, English
    [Refereed]
    Scientific journal

  • 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, Sep. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(17) (17), 5536 - 5543, English
    [Refereed]
    Scientific journal

  • 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, Sep. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 84(4) (4), 733 - 739, English
    [Refereed]
    Scientific journal

  • Ken Horii, Takashi Adachi, Tetsuya Matsuda, Tsutomu Tanaka, Hiroshi Sahara, Seiji Shibasaki, Chiaki Ogino, Yoji Hata, Mitsuyoshi Ueda, Akihiko Kondo
    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, Aug. 2009, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 59(4) (4), 297 - 301, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(15) (15), 5175 - 5178, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2009, BIOTECHNOLOGY LETTERS, 31(8) (8), 1259 - 1264, English
    [Refereed]
    Scientific journal

  • Yan Zhou, Shin'ichiro Kajiyama, Kouichi Itoh, Takanori Tanino, Nobuo Fukuda, Tsutomu Tanaka, Akihiko Kondo, Kiichi Fukui
    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, Aug. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 84(2) (2), 375 - 382, English
    [Refereed]
    Scientific journal

  • 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, Jul. 2009, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 19(13) (13), 3619 - 3622, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2009, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 58(1-4) (1-4), 93 - 97, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(12) (12), 4149 - 4154, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 83(4) (4), 783 - 789, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2009, JOURNAL OF BIOCHEMISTRY, 145(6) (6), 701 - 708, English
    [Refereed]
    Scientific journal

  • 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, May 2009, RENEWABLE ENERGY, 34(5) (5), 1354 - 1358, English
    [Refereed]
    Scientific journal

  • Ryosuke Yamada, Yohei Bito, Takashi Adachi, Tsutomu Tanaka, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo
    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, May 2009, ENZYME AND MICROBIAL TECHNOLOGY, 44(5) (5), 344 - 349, English
    [Refereed]
    Scientific journal

  • Nobuo Fukuda, Jun Ishii, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo
    In the current study, we report the construction of a novel system for the detection of protein-protein interactions using yeast G-protein signaling. It is well established that the G-protein γ subunit (Gγ) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-terminus, and that this localization of Gγ is required for signal transduction. In our system, mutated Gγ (Gγcyto) lacking membrane localization ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G-protein signal was observed when Gγcyto was expressed in the cytoplasm of yeast cells from which the endogenous Gγ gene had been deleted. In order to demonstrate the potential use of our system, we utilized the Staphylococcus aureus ZZ domain and the Fc portion of human immunoglobulin G (IgG) as a model interaction pair. To design our detection system for protein-protein interaction, the ZZ domain was altered so that it associates with the inner leaflet of the plasma membrane, and the Fc part was then fused to Gγcyto. The Fc- Gγcyto fusion protein migrated towards the membrane via the ZZ-Fc interaction, and signal transduction was therefore restored. This signal was successfully detected by assessing growth inhibition and transcription in response to G-protein signaling. Finally, several Z variants displaying affinity constants ranging from 8.0 × 103 to 6.8 × 108 m-1 were prepared, and it was demonstrated that our system was able to discriminate subtle differences in affinity. In conclusion, our system appears to be a reliable and versatile technique for detection of protein-protein interactions, and may prove useful in future protein interaction studies. © 2009 FEBS.
    May 2009, FEBS Journal, 276(9) (9), 2636 - 2644, English
    [Refereed]
    Scientific journal

  • 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, May 2009, JOURNAL OF MAGNETISM AND MAGNETIC MATERIALS, 321(10) (10), 1686 - 1688, English
    [Refereed]
    Scientific journal

  • 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, May 2009, BIOTECHNOLOGY LETTERS, 31(5) (5), 623 - 627, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82(6) (6), 1067 - 1078, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82(5) (5), 883 - 890, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2009, BIOCHEMICAL ENGINEERING JOURNAL, 44(1) (1), 2 - 12, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82(6) (6), 1037 - 1047, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2009, PLANT BIOTECHNOLOGY, 26(1) (1), 39 - 46, English
    [Refereed]
    Scientific journal

  • 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
    Mar. 2009, Renewable Energy, Vol. 34, No. 5, pp. 1354-1358, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2009, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 19(5) (5), 1473 - 1476, English
    [Refereed]
    Scientific journal

  • 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, Feb. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82(1) (1), 59 - 66, English
    [Refereed]
    Scientific journal

  • 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, Feb. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82(2) (2), 387 - 395, English
    [Refereed]
    Scientific journal

  • Toshihiro Tateno, Yusuke Okada, Takeyuki Tsuchidate, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo
    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, Feb. 2009, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 82(1) (1), 115 - 121, English
    [Refereed]
    Scientific journal


  • Akihiko Kondo
    2009, IFPT'6: PROGRESS ON POST-GENOME TECHNOLOGIES, PROCEEDINGS
    Scientific journal

  • Akihiko Kondo
    2009, Journal of Bioscience and Bioengineering
    Scientific journal





  • 加藤 寛子, 山田 亮祐, 蓮沼 誠久, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2009, 化学工学会 研究発表講演要旨集, 2009, 4 - 4, Japanese

  • 浜 真司, 倉谷 伸行, Tamalampudi Sriappareddy, 近藤 昭彦, 野田 秀夫, 福田 秀樹
    公益社団法人 化学工学会, 2009, 化学工学会 研究発表講演要旨集, 2009, 769 - 769, Japanese

  • 松井 かずさ, 荻野 千秋, 田中 勉, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2009, 化学工学会 研究発表講演要旨集, 2009, 727 - 727, Japanese

  • ?? 真司, 玉谷 直紀, 吉田 あゆみ, 倉谷 伸行, 近藤 昭彦, 福田 秀樹, 野田 秀夫
    公益社団法人 化学工学会, 2009, 化学工学会 研究発表講演要旨集, 2009, 675 - 675, Japanese

  • 澤本 詩織, 坂本 祟幸, 田中 勉, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2009, 化学工学会 研究発表講演要旨集, 2009, 631 - 631, Japanese

  • Ogino Chiaki, Kondo Akihiko
    平成14年度に日本政府の総合戦略「バイオマス・ニッポン」が策定され、バイオマスの有効利用による持続的に発展可能な社会の実現が提言されている。この戦略は、(1)バイオマスの有効利用に基づく地球温暖化防止や循環型社会形成の達成、(2)日本独自のバイオマス利用法の開発による戦略的産業の育成を目指すものである。また、地球規模での環境保護の観点から、バイオマス原料は日本のみならず、世界中から安価かつ豊富な資源の積極的な利用が求められている。さらに石油資源枯渇や価格高騰の影響より、これまでの石油資源依存型の「オイルリファイナリー」から、バイオマスベースの「バイオリファイナリー」社会への転換が急務とされている。米国においても、1999年にクリントン大統領が「バイオ製品およびバイオマスエネルギーの開発・普及に関する大統領令」を発令し、バイオマスエネルギーの利用率を2010年までに10%までに拡大する方針を指示し、ヨーロッパにおいても同様の方針が打ち出されている状況である。このような社会的背景も重なり、現在、グルコースから生産されるバイオエタノールはブラジル、米国をはじめとして多くの地域でガソリンと混合され、自動車用燃料として利用されている。しかしながら、食料価格高騰の問題原因ともなっており、代替原料が模索されている。木質系バイオマスはデンプンよりもはるかに豊富に存在し、また古紙や廃材、農産廃棄物など処理が問題化しているものを利用するため原料が安価であることなどから、将来の石油代替エネルギー資源として特に有望視されている。
    Japanese Society of Grassland Science, 2009, Japanese Journal of Grassland Science, 55(3) (3), 270 - 273, Japanese

  • 中島 一紀, 荒井 章吾, 谷野 孝徳, 荻野 千秋, 近藤 昭彦, 福田 秀樹
    公益社団法人 化学工学会, 2009, 化学工学会 研究発表講演要旨集, 2009, 912 - 912, Japanese

  • Akiyoshi Hoshino, Noriyoshi Manabe, Sanshiro Hanada, Kouki Fujioka, Masato Yasuhara, Kondo Akihiko, 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. Fluorescent nanoparticles, such as nanocrystal quantum dots (QDs), have potential to be applied to molecular biology and bioimaging, since some nanocrystals emit higher and longer lasting fluorescence than conventional organic probes do. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced the 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/geneconstruct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as 1) the reading direction of gene fragments, 2) the quantity of gene fragments attached on the surface of QD-constructs, 3) the surface electronic charges varied according to the structure of 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/geneconstruct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs was disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes. These results suggest that inappropriate treatment and disposal of QDs may still have risks to the environmental pollution including human health under cert in conditions. Here we propose the further research for the immune and physiological responses in not only immune cells but also other cells, in order to clear the effect of all other nanoscale products as well as nanocrystal QDs.© 2009 SPIE.
    2009, Progress in Biomedical Optics and Imaging - Proceedings of SPIE, 7189, English
    [Refereed]
    International conference proceedings

  • 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, IFPT'6: PROGRESS ON POST-GENOME TECHNOLOGIES, PROCEEDINGS, 238 - 238, English
    [Refereed]
    International conference proceedings

  • 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, Biocontrol Science, 14(4) (4), 171 - 175, English
    [Refereed]
    Scientific journal

  • Kenji Okano, Qiao Zhang, Satoru Shinkawa, Shogo Yoshida, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo
    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, Jan. 2009, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 75(2) (2), 462 - 467, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2008, JOURNAL OF BIOCHEMISTRY, 144(6) (6), 701 - 707, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2008, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 81(4) (4), 637 - 645, English
    [Refereed]
    Scientific journal

  • 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, Dec. 2008, NANOTECHNOLOGY, 19(49) (49), English
    [Refereed]
    Scientific journal

  • 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, Dec. 2008, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 81(4) (4), 711 - 719, English
    [Refereed]
    Scientific journal

  • Satoshi Saitoh, Tsutomu Tanaka, Akihiko Kondo
    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, Dec. 2008, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 106(6) (6), 594 - 597, English
    [Refereed]
    Scientific journal

  • Nobuhiro Nakamura, Ryosuke Yamada, Satoshi Katahira, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo
    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, Sep. 2008, ENZYME AND MICROBIAL TECHNOLOGY, 43(3) (3), 233 - 236, English
    [Refereed]
    Scientific journal

  • 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, Sep. 2008, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 80(4) (4), 735 - 743, English
    [Refereed]
    Scientific journal

  • Junji Ito, Hiroshi Sahara, Masahiko Kaya, Yoji Hata, Seiji Shibasaki, Koji Kawata, Saori Ishida, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo
    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, Sep. 2008, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 55(1-2) (1-2), 69 - 75, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2008, ENZYME AND MICROBIAL TECHNOLOGY, 43(2) (2), 115 - 119, English
    [Refereed]
    Scientific journal

  • 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, Jul. 2008, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 79(6) (6), 1009 - 1018, English
    [Refereed]
    Scientific journal

  • Kenro Tokuhiro, Nobuhiro Ishida, Akihiko Kondo, Haruo Takahashi
    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, Jun. 2008, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 79(3) (3), 481 - 488, English
    [Refereed]
    Scientific journal

  • Atsushi Kotaka, Hiroki Bando, Masahiko Kaya, Michiko Kato-Murai, Kouichi Kuroda, Hiroshi Sahara, Yoji Hata, Akihiko Kondo, Mitsuyoshi Ueda
    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, Jun. 2008, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 105(6) (6), 622 - 627, English
    [Refereed]
    Scientific journal

  • Jun Ishii, Tsutomu Tanaka, Shizuka Matsumura, Kenji Tatematsu, Shunichi Kuroda, Chiaki Ogino, Hideki Fukuda, Akihiko Kondo
    Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.
    May 2008, Journal of Biochemistry, Vol 143, pp. 667-674(5) (5), 667 - 74, English, International magazine
    [Refereed]
    Scientific journal

  • Masahiko Kaya, Junji Ito, Atsushi Kotaka, Kengo Matsumura, Hiroki Bando, Hiroshi Sahara, Chiaki Ogino, Seiji Shibasaki, Kouichi Kuroda, Mitsuyoshi Ueda, Akihiko Kondo, Yoji Hata
    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, May 2008, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 79(1) (1), 51 - 60, English
    [Refereed]
    Scientific journal

  • 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, May 2008, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 72(5) (5), 1376 - 1379, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    Apr. 2008, Biotechnology Progress, Vol. 24, No. 2, pp. 352-357, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2008, BIOCHEMICAL ENGINEERING JOURNAL, 39(1) (1), 185 - 189, English
    [Refereed]
    Scientific journal

  • 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, Feb. 2008, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 74(4) (4), 1117 - 1123, English
    [Refereed]
    Scientific journal


  • 若村 香菜子, 田中 勉, 石井 純, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 944 - 944, Japanese

  • 戸川 翔太, 石井 純, 田中 勉, 立松 健司, 黒田 俊一, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 971 - 971, Japanese

  • 井口 裕介, 石井 純, 田中 勉, 立松 健司, 黒田 俊一, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 970 - 970, Japanese

  • 柳瀬 修平, 山田 亮祐, 田中 勉, 荻野 千秋, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 990 - 990, Japanese

  • 新川 智, 岡野 憲司, 張 イ肖, 田中 勉, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 988 - 988, Japanese

  • 甲田 梨沙, 田中 勉, 近藤 昭彦, 福田 秀樹
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 997 - 997, Japanese

  • 甲田 梨沙, 沼田 崇男, 田中 勉, 近藤 昭彦, 福田 秀樹
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 399 - 399, Japanese

  • 阿部 祐一郎, 山田 亮介, 中村 延博, 田中 勉, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 394 - 394, Japanese

  • 石井 純, 田中 勉, 松村 静香, 立松 健司, 黒田 俊一, 荻野 千秋, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 348 - 348, Japanese

  • 安住 友希, 倉田 直弥, 宍戸 卓矢, 上田 政和, 妹尾 昌治, 多田 宏子, 谷澤 克行, 黒田 俊一, 田中 勉, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2008, 化学工学会 研究発表講演要旨集, 2008, 320 - 320, Japanese

  • 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, Dec. 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 77(3) (3), 533 - 541, English
    [Refereed]
    Scientific journal

  • Akiyoshi Hoshino, Noriyuki Ohnishi, Masato Yasuhara, Kenji Yamamoto, Akihiko Kondo
    Magnetic particles have been used widely in both biotechnological and medical fields, including for immunoassay, enzyme immobilization, drug transport, and immunological diagnosis. Especially particles with bioactive molecules such as antibodies and streptavidin are very useful tools for cell separation. Here we report affinity selection of neutrophils and macrophages from peritoneal inflammatory cells performed by thermoresponsive magnetic nanoparticles conjugated with macrophage-specific anti-F4/80 antibody. The magnetic nanoparticles, which are capped with thermoresponsive polymers, are aggregated by heating the particles over 30 degrees C and show their intrinsic magnetism. The neutrophils are concentrated approximately 90% by these magnetic nanoparticles without any activation, indicating that this novel cell separation method could fulfill a wide range of applications in analysis of the isolation of fragile cells such as neutrophils.
    AMER CHEMICAL SOC, Nov. 2007, BIOTECHNOLOGY PROGRESS, 23(6) (6), 1513 - 1516, English
    [Refereed]
    Scientific journal

  • 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, Nov. 2007, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 49(1-4) (1-4), 92 - 97, English
    [Refereed]
    Scientific journal

  • MATSUI Yoshimitsu, OKADA Sanae, UCHIMURA Tai, KONDO Akihiko, SATOH Eiichi
    We analyzed the functional structure of Streptococcus bovis 148 α-amylase (AmyA) to determine the starch binding domain (SBD) of this enzyme. Several derivative AmyAs and putative SBD segments were constructed and assayed their adsorption ability onto raw starch. On the basis of the results, a putative linker domain region and two SBDs (SBDα and SBDβ) were found at the C-terminal region of AmyA. SBDα and SBDβ showed 56% amino acid sequence similarity; these SBDs were considered to be tandem repeat motif. In addition, we constructed a chimeric enzyme CsaB, which consisted of S. bovis 148 irresolvable intracellular α-amylase (AmyB) as for insoluble starch and a putative linker plus two SBDs. This enzyme succeeded in conferring starch adsorption and hydrolysis abilities, which suggests that SBD reacts with the surface of raw starch and breaks down its structure.
    The Japanese Society of Applied Glycoscience, Oct. 2007, Journal of Applied Glycoscience, 54(4) (4), 217 - 222, English

  • Sriappareddy Tamalampudi, Shinji Hama, Takanori Tanino, Mahabubur Rahman Talukder, Akihiko Kondo, Hideki Fukuda
    Organic esters are used in various industries such as perfumery, flavour and pharmaceutical intermediates. The use of biocatalysts for esterification and transesterification reaction under ambient reaction conditions gives better products for use in flavour and fragrance industries. In the current study, enantioselective transesterification reaction was developed by using recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica. The recombinant fungal cells were immobilized on Biomass Support Particles (BSPs) to facilitate the reusability of whole-cell biocatalyst. The immobilized CALB expressing whole-cell biocatalyst was used for the optical resolution of (RS)-1-phenylethanol by enantioselective transesterification with vinyl acetate as acyl donor. The activity of the whole-cell biocatalyst was optimized and compared with the other whole-biocatalysts E. coli displaying CALB, S. cerevisiae displaying ROL and A. oryzae whole-cell biocatalyst expressing tglA lipase. The initial activity of immobilized CALB expressing A. oryzae was at least 15-folds higher than that of A. oryzae expressing tglA lipase. The maximum yield of (R)-1-phenylethyl acetate reached 88.1% with an enantiomeric excess (ee) of > 99% after 3.5 h reaction, while tglA lipase expressing A. oryzae showed 90% yield and 95% ee after 48 h. The recombinant A. oryzae retained its activity in hexane, heptane, toluene, cyclohexane and octane. Moreover, whole-cell biocatalyst maintained its activity for more than 15 batch reaction cycles. Current study demonstrated the applicability of recombinant whole-cell biocatalyst to bioconversion process in non-aqueous medium. © 2007 Elsevier B.V. All rights reserved.
    Sep. 2007, Journal of Molecular Catalysis B: Enzymatic, 48(1-2) (1-2), 33 - 37, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 76(1) (1), 151 - 158, English
    [Refereed]
    Scientific journal

  • 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, Jul. 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75(6) (6), 1319 - 1325, English
    [Refereed]
    Scientific journal

  • Kenji Okano, Sakurako Kimura, Junya Narita, Hideki Fukuda, Akihiko Kondo
    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, Jul. 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75(5) (5), 1007 - 1013, English
    [Refereed]
    Scientific journal

  • 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, Jul. 2007, ENZYME AND MICROBIAL TECHNOLOGY, 41(1-2) (1-2), 156 - 161, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2007, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 103(6) (6), 572 - 574, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2007, BIOCHEMICAL ENGINEERING JOURNAL, 34(3) (3), 273 - 278, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75(3) (3), 549 - 555, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2007, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1774(6) (6), 671 - 678, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75(4) (4), 821 - 828, English
    [Refereed]
    Scientific journal

  • 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, May 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 75(2) (2), 387 - 395, English
    [Refereed]
    Scientific journal

  • 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.
    Apr. 2007, Journal of Controlled Release, 118(3) (3), 348 - 356, English
    [Refereed]
    Scientific journal

  • M. Ueda, Y. Iwasaki, T. Yamada, A. Kondo, M. Seno, K. Tanizawa, S. Kuroda, M. Sakamoto, M. Kitajima
    NATURE PUBLISHING GROUP, Apr. 2007, CANCER GENE THERAPY, 14(4) (4), 440 - 440, English

  • 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, Apr. 2007, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 74(6) (6), 1213 - 1220, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2007, BIOCHEMICAL ENGINEERING JOURNAL, 33(3) (3), 232 - 237, English
    [Refereed]
    Scientific journal


  • 森口 みゆき, 石井 純, 立松 健司, 黒田 俊一, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2007, 化学工学会 研究発表講演要旨集, 2007, 844 - 844, Japanese

  • 岡田 勇介, 舘野 俊博, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2007, 化学工学会 研究発表講演要旨集, 2007, 818 - 818, Japanese

  • 張 イ肖, 岡野 憲司, 木村 桜子, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2007, 化学工学会 研究発表講演要旨集, 2007, 817 - 817, Japanese

  • 福田 展雄, 石井 純, 芝崎 誠司, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2007, 化学工学会 研究発表講演要旨集, 2007, 325 - 325, Japanese

  • 谷野 孝徳, 大野 卓見, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2007, 化学工学会 研究発表講演要旨集, 2007, 413 - 413, Japanese

  • 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, Jan. 2007, BIOCHEMICAL ENGINEERING JOURNAL, 33(1) (1), 60 - 65, English
    [Refereed]
    Scientific journal

  • Fluorescence detection system for human G protein - Coupled receptor signaring in yeast
    Jun Ishii, Akihiko Kondo
    PHOENIX PUBL & MEDIA NETWORK, 2007, PROGRESS ON POST-GENOME TECHNOLOGIES, 57 - 57, English
    [Refereed]
    International conference proceedings

  • 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.
    Jan. 2007, Journal of Bioscience and Bioengineering, 103(1) (1), 95 - 97, English
    [Refereed]
    Scientific journal

  • 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, Nov. 2006, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 73(2) (2), 366 - 373, English
    [Refereed]
    Scientific journal

  • 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.
    Nov. 2006, Applied Microbiology and Biotechnology, 73(1) (1), 60 - 66, English
    [Refereed]
    Scientific journal

  • 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, Oct. 2006, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 72(6) (6), 1136 - 1143, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2006, BIOTECHNOLOGY PROGRESS, 22(4) (4), 989 - 993, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2006, BIOTECHNOLOGY PROGRESS, 22(4) (4), 994 - 997, English
    [Refereed]
    Scientific journal

  • 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.
    Jul. 2006, Biotechnology Progress, 22(4) (4), 998 - 1002, English
    [Refereed]
    International conference proceedings

  • Jun Ishii, Shizuka Matsumura, Sakurako Kimura, Kenji Tatematsu, Shun'ichi Kuroda, Hideki Fukuda, Akihiko Kondo
    The mechanism of G protein-coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone-responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into the FUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high-throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods. © 2006 American Chemical Society and American Institute of Chemical Engineers.
    Jul. 2006, Biotechnology Progress, 22(4) (4), 954 - 960, English
    [Refereed]
    International conference proceedings

  • 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, May 2006, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 70(5) (5), 564 - 572, English
    [Refereed]
    Scientific journal

  • 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.
    May 2006, Applied Microbiology and Biotechnology, 70(5) (5), 573 - 579, English
    [Refereed]
    Scientific journal

  • Rinji Akada, Takao Kitagawa, Shohei Kaneko, Daiso Toyonaga, Sachiko Ito, Yoshito Kakihara, Hisashi Hoshida, Shigeru Morimura, Akihiko Kondo, Kenji Kida
    Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra- or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat-generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5-fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome. Copyright © 2006 John Wiley & Sons, Ltd.
    Apr. 2006, Yeast, 23(5) (5), 399 - 405, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2006, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 101(4) (4), 328 - 333, English
    [Refereed]
    Scientific journal

  • Noriyuki Ohnishi, Hirotaka Furukawa, Hata Hideyuki, Jing-Ming Wang, Chung-Il An, Eiichiro Fukusaki, Kazunori Kataoka, Katsuhiko Ueno, Akihiko Kondo
    Thermoresponsive magnetic nanoparticles with an upper critical solution temperature (UCST) in aqueous solution were synthesized for the first time. Named Therma-Max, the material was synthesized by redox copolymerization of N-acryloyl glycinamide with a monomer form of biotin using methacrylated dextran-magnetite. While the resulting Therma-Max was completely dispersed at temperatures above the UCST (18°C) and could not be separated by a permanent magnet, it was rapidly flocculated when the temperature fell below the UCST and was easily separated by a permanent magnet. The flocculated particles dispersed completely when the temperature was raised to above the UCST. Because biotin was immobilized on the Therma-Max, avidin and antibodies were subsequently immobilized with good efficiency. Furthermore, transiently transfected Arabidopsis protoplasts, which have surface display of CD4 antigen, were efficiently captured and enriched by using a biotinylated anti-CD4 antibody in combination with avidin-conjugated Therma-Max. Also, the silkworm storage protein (SP2) was efficiently separated from the silkworm hemolymph by using biotinylated anti-IgG antibody and anti-SP2 antibody in combination with avidin-conjugated Therma-Max. In both cases, it was confirmed that specificity and adsorption capacity were markedly improved by converting the conventional micro-size fine magnetic particles to nano-size particles. These results show the potential of Therma-Max with a UCST in bioaffinity separation of cells and biomolecules. Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved.
    Mar. 2006, Nanobiotechnology, 2(1-2) (1-2), 43 - 49, English
    [Refereed]
    Scientific journal

  • 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, Feb. 2006, BIOCHEMICAL ENGINEERING JOURNAL, 28(1) (1), 73 - 78, English
    [Refereed]
    Scientific journal

  • BIOT 247-Preparation of lipases derivatives with high activity and stability by isopropanol treatment and immobilization on macroporous resin
    Akihiko Kondo
    2006, Abstracts of Papers of the American Chemical Society
    Scientific journal

  • 森口 みゆき, 石井 純, 立松 健司, 黒田 俊一, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2006, 化学工学会 研究発表講演要旨集, 2006, 986 - 986, Japanese

  • 沼田 崇男, 福水 崇裕, 濱 真司, 山地 秀樹, 近藤 昭彦, 福田 秀樹
    公益社団法人 化学工学会, 2006, 化学工学会 研究発表講演要旨集, 2006, 950 - 950, Japanese

  • 谷野 孝徳, 大野 卓見, 鄭 源英, 渡辺 有香, 加藤 倫子, 植田 充美, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2006, 化学工学会 研究発表講演要旨集, 2006, 947 - 947, Japanese

  • 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, Drug Delivery System, 21(4) (4), 435 - 443, English
    [Refereed]
    Scientific journal

  • Development of a single cell analysis system of agonist for drug discovery
    Akihiko Kondo, Jun Ishii, Shizuka Matsumura, Hideki Fukuda
    SOUTHEAST UNIV PRESS, 2006, Progress on Post-Genome Technologies, 30 - 30, English
    [Refereed]
    International conference proceedings

  • 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, Jan. 2006, IUBMB LIFE, 58(1) (1), 1 - 6, English
    [Refereed]
    Scientific journal

  • 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, Jan. 2006, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 72(1) (1), 269 - 275, English
    [Refereed]
    Scientific journal

  • 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, Jul. 2005, FEBS JOURNAL, 272(14) (14), 3651 - 3660, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2005, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 99(4) (4), 354 - 360, English
    [Refereed]
    Scientific journal

  • 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, Apr. 2005, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 99(4) (4), 354 - 360, English
    [Refereed]
    Scientific journal

  • 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, Mar. 2005, BIOCHEMICAL ENGINEERING JOURNAL, 23(1) (1), 45 - 51, English
    [Refereed]
    Scientific journal

  • 宍戸 卓矢, 村岡 優, 上田 政和, 妹尾 昌治, 多田 宏子, 谷澤 克行, 黒田 俊一, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2005, 化学工学会 研究発表講演要旨集, 2005, 327 - 327, Japanese

  • 岡野 憲司, 成田 純也, 瀬脇 智満, 成 文喜, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2005, 化学工学会 研究発表講演要旨集, 2005, 255 - 255, Japanese

  • 谷野 孝徳, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2005, 化学工学会 研究発表講演要旨集, 2005, 256 - 256, Japanese

  • ? 真司, 近藤 昭彦, 福田 秀樹
    公益社団法人 化学工学会, 2005, 化学工学会 研究発表講演要旨集, 2005, 288 - 288, Japanese

  • 伊藤 環, 片平 悟史, 近藤 昭彦, 福田 秀樹
    公益社団法人 化学工学会, 2005, 化学工学会 研究発表講演要旨集, 2005, 240 - 240, Japanese

  • 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, 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, Japanese
    Scientific journal

  • K Machi, M Azuma, K Igarashi, T Matsumoto, H Fukuda, A Kondo, H Oshima
    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, Oct. 2004, MICROBIOLOGY-SGM, 150, 3163 - 3173, English
    [Refereed]
    Scientific journal

  • 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, Oct. 2004, BIOCHEMICAL ENGINEERING JOURNAL, 21(2) (2), 155 - 160, English
    [Refereed]
    Scientific journal

  • 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, Sep. 2004, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 70(9) (9), 5407 - 5414, English
    [Refereed]
    Scientific journal

  • 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, Aug. 2004, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 65(3) (3), 301 - 305, English
    [Refereed]
    Scientific journal

  • H Shigechi, J Koh, Y Fujita, T Matsumoto, Y Bito, M Ueda, E Satoh, H Fukuda, A Kondo
    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, Aug. 2004, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 70(8) (8), 5037 - 5040, English
    [Refereed]
    Scientific journal

  • M Ueda, A Kondo
    ELSEVIER SCIENCE BV, Jun. 2004, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28(4-6) (4-6), 137 - 137, English
    [Refereed]

  • 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, Jun. 2004, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 97(6) (6), 423 - 425, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2004, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 64(6) (6), 823 - 828, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2004, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28(4-6) (4-6), 235 - 239, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2004, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28(4-6) (4-6), 247 - 251, English
    [Refereed]
    Scientific journal

  • 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, Jun. 2004, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28(4-6) (4-6), 259 - 264, English
    [Refereed]
    Scientific journal

  • 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 binding affinity, stability, and catalytic activity. Conformation of the surface-displayed hetero-dimeric Fab fragment mainly depended on the intermolecular disulfide bond between the Lc and Fd fragments. The conformation of 6139 Fab1 was more stable than that of Fab2. In the reducing environment of solution containing 25 nM DTT, the function of 6D9 Fab2 was almost completely lost. The successful display of 6D9 Fab1 on yeast-cell surface provides a novel approach to the engineering of catalytic antibodies. (C) 2004 Elsevier B.V All rights reserved.
    ELSEVIER SCIENCE BV, Jun. 2004, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 28(4-6) (4-6), 241 - 246, English
    [Refereed]
    Scientific journal

  • Over-expression system of phospholipase D from actinomycete by Streptomyces lividans
    OGINO Chiaki, KANEMASU M, HAYASHI Y, KURODA S, KONDO Akihiko, SHMIZU N, TANIZAWA K, FUKUDA Hideki
    Jun. 2004, Applied Microbiology and Biotechnology, Vol 64, pp. 823-828, English
    [Refereed]
    Scientific journal

  • J Ito, Y Fujita, M Ueda, H Fukuda, A Kondo
    To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414, the genes encoding the cellulose-binding domain (CBD) of EGII, cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T. reesei QM9414, were fused with the catalytic domain of EGII and expressed in Saccharomyces cerevisiae. Display of each of the recombinant EGIIs was confirmed using immunofluorescence microscopy. In the case of EGII-displaying yeast strains in which the CBD of EGII was replaced with the CBD of CBHI or CBHII, the binding affinity to Avicel and hydrolytic activity toward phosphoric acid swollen Avicel were similar to that of a yeast strain displaying wild-type EGII. On the other hand, the three yeast strains displaying EGII with two or three tandemly aligned CBDs showed binding affinity and hydrolytic activity higher than that of the yeast strain displaying wild-type EGII. This result indicates that the hydrolytic activity of yeast strains displaying recombinant EGII increases with increased binding ability to cellulose.
    AMER CHEMICAL SOC, May 2004, BIOTECHNOLOGY PROGRESS, 20(3) (3), 688 - 691, English
    [Refereed]
    Scientific journal

  • J Ito, Y Fujita, M Ueda, H Fukuda, A Kondo
    To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414, the genes encoding the cellulose-binding domain (CBD) of EGII, cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T. reesei QM9414, were fused with the catalytic domain of EGII and expressed in Saccharomyces cerevisiae. Display of each of the recombinant EGIIs was confirmed using immunofluorescence microscopy. In the case of EGII-displaying yeast strains in which the CBD of EGII was replaced with the CBD of CBHI or CBHII, the binding affinity to Avicel and hydrolytic activity toward phosphoric acid swollen Avicel were similar to that of a yeast strain displaying wild-type EGII. On the other hand, the three yeast strains displaying EGII with two or three tandemly aligned CBDs showed binding affinity and hydrolytic activity higher than that of the yeast strain displaying wild-type EGII. This result indicates that the hydrolytic activity of yeast strains displaying recombinant EGII increases with increased binding ability to cellulose.
    AMER CHEMICAL SOC, May 2004, BIOTECHNOLOGY PROGRESS, 20(3) (3), 688 - 691, English
    [Refereed]
    Scientific journal

  • H Shigechi, Y Fujita, J Koh, M Ueda, H Fukuda, A Kondo
    Direct ethanol fermentation from corn starch cooked at low temperature (80degreesC) was carried out using flocculent Saccharomyces cerevisiae strains co-expressing Rhizopus oryzae glucoamylase and Bacillus stearothermophilus alpha-amylase. The yeast strain YF207/pGA11/pAA12 co-displaying the two enzymes was able to produce 18 g/l of ethanol from low-temperature-cooked corn starch (50 g/l) after 36 h of fermentation, an ethanol concentration almost equal to that produced from corn starch cooked at conventional high temperature (120 degreesC). The yeast strain YF207/pGA11/pSAA11, displaying glucoamylase on the cell surface and secreting alpha-amylase, was similarly able to produce ethanol from low-temperature-cooked corn starch, but with a time lag in the initial phase of fermentation. With an initial corn-starch concentration of 90 g/l, which is easily prepared at low temperature and avoids gel formation by the starch, the former strain was able to produce 30 g/l of ethanol after 36 h of fermentation. The yeast strain co-displaying glucoamylase and alpha-amylase is thus very effective in ethanol production from corn starch cooked under energy-saving low-temperature conditions. (C) 2003 Elsevier B.V. All rights reserved.
    ELSEVIER SCIENCE SA, May 2004, BIOCHEMICAL ENGINEERING JOURNAL, 18(2) (2), 149 - 153, English
    [Refereed]
    Scientific journal

  • H Shigechi, Y Fujita, J Koh, M Ueda, H Fukuda, A Kondo
    Direct ethanol fermentation from corn starch cooked at low temperature (80degreesC) was carried out using flocculent Saccharomyces cerevisiae strains co-expressing Rhizopus oryzae glucoamylase and Bacillus stearothermophilus alpha-amylase. The yeast strain YF207/pGA11/pAA12 co-displaying the two enzymes was able to produce 18 g/l of ethanol from low-temperature-cooked corn starch (50 g/l) after 36 h of fermentation, an ethanol concentration almost equal to that produced from corn starch cooked at conventional high temperature (120 degreesC). The yeast strain YF207/pGA11/pSAA11, displaying glucoamylase on the cell surface and secreting alpha-amylase, was similarly able to produce ethanol from low-temperature-cooked corn starch, but with a time lag in the initial phase of fermentation. With an initial corn-starch concentration of 90 g/l, which is easily prepared at low temperature and avoids gel formation by the starch, the former strain was able to produce 30 g/l of ethanol after 36 h of fermentation. The yeast strain co-displaying glucoamylase and alpha-amylase is thus very effective in ethanol production from corn starch cooked under energy-saving low-temperature conditions. (C) 2003 Elsevier B.V. All rights reserved.
    ELSEVIER SCIENCE SA, May 2004, BIOCHEMICAL ENGINEERING JOURNAL, 18(2) (2), 149 - 153, English
    [Refereed]
    Scientific journal

  • J Kohda, T Yamada, T Yoshida, T Maruyama, M Yohda, H Fukuda, A Kondo
    The alpha and beta subunits of group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1, were produced in Escherichia coli. Thermococcus KS-1 alpha and beta chaperonins were purified from a crude cell extract by heat treatment and subsequent chromatographic purification in the presence and absence of Mg2+ to produce hexadecameric and monomeric form, respectively. The monomeric alpha and beta subunits were able to form homo-hexadecarners in the presence of Mg2+. In the absence of ATP, the alpha and beta homo-hexadecamers arrested the refolding of guanidine hydrochloride-denatured Bacillus stearothermophilus leucine dehydrogenase (LeuDH) and Thermus flavus malate dehydrogenase (MDH), which were released by the addition of ATP at 50-65degreesC. In the presence of ATP, the alpha and beta homo-hexadecamers facilitated the refolding of LeuDH and MDH. The alpha homo-hexadecamer showed greater complex stability and greater ability to facilitate the refolding of enzymes than the beta homo-hexadecamer. On the other hand, both the alpha and beta monomers facilitated the refolding of the proteins in the absence of ATR Thermococcus KS-1 chaperonin homo-hexadecamers and monomers could both therefore be used as molecular tools in biotechnology. (C) 2003 Elsevier B.V. All rights reserved.
    ELSEVIER SCIENCE SA, Apr. 2004, BIOCHEMICAL ENGINEERING JOURNAL, 18(1) (1), 73 - 79, English
    [Refereed]
    Scientific journal

  • A Kondo, M Ueda
    In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.
    SPRINGER-VERLAG, Mar. 2004, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 64(1) (1), 28 - 40, English
    [Refereed]
    Scientific journal

  • Y Fujita, J Ito, M Ueda, H Fukuda, A Kondo
    A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase 11 and cellobiohydrolase 11 and Aspergillus aculeatus beta-glucosidase I were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase H showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase 11, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase 11, and cellobiohydrolase 11 enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase I and endoglucanase 11 could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.
    AMER SOC MICROBIOLOGY, Feb. 2004, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 70(2) (2), 1207 - 1212, English
    [Refereed]
    Scientific journal

  • A. Kondo, T. Oogami, K. Sato, Y. Tanaka
    Cathodic arc ion plated chromium nitride (CrN) coatings were deposited on WC-Co substrates at negative substrate bias voltages in a range from 0 to 200 V. The properties of CrN coatings were investigated, including the structure, hardness, adhesion and residual stress. The performance of cutting tests of CrN-coated endmills in copper was also investigated correlating with the properties. At a negative substrate bias voltage of 30 V, the adhesion strength of the coating was maximized, and the grain size became relatively small. The endmill deposited at the same bias voltage exhibited the best cutting performance of all the other samples. The cutting performance of CrN-coated endmills were superior to that of (Ti,Al)N coated endmills or uncoated endmills. The relationships between the cutting performance and film properties were discussed in terms of the wear system of the cutting tools. © 2003 Elsevier B.V. All rights reserved.
    Elsevier, Jan. 2004, Surface and Coatings Technology, 177-178, 238 - 244, English
    [Refereed]
    Scientific journal

  • Khaw Teik-Seong, Katakura Yoshio, Koh Jun, Kondo Akihiko, Shioya Suteaki
    Single step bioconversion of raw starch to ethanol has raised considerable interest in recent years. We have developed yeast strains displaying α-amylase and glucoamylase on yeast cell surface for this purpose. The advantage of this system is that the displayed enzymes can be recovered easily with the yeast after fermentation compared to the case that these amylolytic enzymes are secreted into medium. However, display of α-amylase on cell surface may reduce the apparent association rate of the enzyme to the substrate because both of them are huge molecules, resulting a lower apparent conversion rate. The recovery of the yeast cells together with the amylolytic enzymes becomes easier when a flocculating yeast strain is used as the host strain. However, flocculation of the arming yeast may again reduce the apparent conversion rate. To establish an industrial ethanol production process with a good cost performance ratio, the advantages and disadvantages of each strategy should be evaluated quantitatively. Therefore, from a viewpoint of kinetics, we compared the performance of the fermentation systems in the case that α-amylase is displayed on the cell surface or secreted in the medium, and in the cases that a flocculating or non-flocculating strain is used as the host strain. A kinetic model has been developed to simulate the direct ethanol fermentation from raw corn starch by arming yeast. The simulation results were found agree well with experimental data obtained from direct ethanol fermentation.
    The Society of Chemical Engineers, Japan, 2004, Asian Pacific Confederation of Chemical Engineering congress program and abstracts, 2004, 789 - 789, English

  • Fukuda Hideki, Kondo Akihiko, Noda Hideo
    With a view to utilizing Rhizopus oryzae cells immobilized within biomass support particles (BSPs) as a whole-cell biocatalyst for biodiesel fuel production, an investigation was made of the effect of cross-linking treatment with glutaraldehyde on the stability of lipase activity. Although the lipase activity of the BSP-immobilized cells decreased considerably in the presence of the methyl esters produced by methanolysis, the activity of cells treated with 0.1% glutaraldehyde solution showed no significant decrease during twenty batch cycles, with the methyl ester content of the reaction mixture reaching 60-83% in each cycle. We also constructed a novel cell-surface display system using as a new type of cell-wall anchor the 3297-bp of the 3' region of the FLO1 gene, and in this system the N-terminus of the Rhizopus oryzae lipase (ROL) was fused to the FLO1 protein. In the use of Saccharomyces cerevisiae cells displaying FLO1 gene as a whole-cell biocatalyst methyl ester content synthesized reached 78.3% after 72 h. These findings indicate that, given the simplicity of the lipase production process, the use of whole cell biocatalysts using either R. oryzae cells immobilized within BSPs or S. cerevisiae cells displaying ROL offers a promising means of biodiesel fuel production for industrial application.
    The Society of Chemical Engineers, Japan, 2004, Asian Pacific Confederation of Chemical Engineering congress program and abstracts, 2004, 455 - 455, English

  • Kondo Akihiko, Fujita Yasuya, Fukuda Hideki
    Display of novel enzymes on the yeast cell surface is very powerful method to develop the efficient whole cell biocatalysts, because the diffusion problem of substrate and product is circumvented. In addition, the displayed enzymes are regarded as a kind of self-immobilized enzyme on the cell surfaces. In this study, we have developed the novel methods to display enzymes using cell wall anchoring proteins -agglutinin. To show their applicability to the productions of biofuels and chemicals from biomass, the ethanol production from cellulosic materials was investigated by using yeast cells displaying cellulolytic enzymes. Due to the display of these enzymes, cellulosic materials were sequentially hydrolyzed to glucose on the yeast cell surface, immediately utilized and converted to ethanol by intracellular enzymes. The yield in terms of grams of ethanol produced per grams of carbohydrate utilized was over 0.45, which corresponds to over 89% of theoretical yield. These results demonstrate that a combination of cell surface displayed enzymes and intracellular metabolic system is a very effective approach to develop cells with novel catalytic and metabolic abilities for industrial applications.
    The Society of Chemical Engineers, Japan, 2004, Asian Pacific Confederation of Chemical Engineering congress program and abstracts, 2004, 427 - 427, English

  • Pinpoint drug delivery system using hollow bio-nanoparticles
    T Yamada, M Seno, A Kondo, M Ueda, K Tanizawa, S Kuroda
    Hepatitis B virus envelope L proteins produced in yeast cells form hollow nanoparticles (L particles, average diameter 220 nm) displaying human liver-specific receptor. Recently, the L particles were found to incorporate genes, proteins, and drugs, and act as an efficient pinpoint delivery system to human liver-derived tissues in xenograft models. By substituting the epidermal growth factor (EGF) for human liver-specific receptor, the mutated L particles showed the affinity to the EGF receptor, not to human liver. Other similar HBV envelope proteins, e.g., M and S particles, have already been commercialized for hepatitis B vaccine, strongly suggesting the safety of L particles in human. These results indicate that the hollow bio-nanoparticles are a promising candidate for the next-generation platform of DDS, especially that related to gene therapy.
    SOC POLYMER SCIENCE JAPAN, 2004, KOBUNSHI RONBUNSHU, 61(12) (12), 606 - 612, Japanese
    [Refereed]
    Scientific journal

  • 熱応答性磁性ナノ粒子の開発とポストゲノムシーケンスへの応用
    大西 徳幸, 近藤 昭彦
    シーエムシー出版, 2004, バイオインダストリー, 21(8),31-38(8) (8), 31 - 38, Japanese
    Scientific journal

  • 熱応答性磁性ナノ粒子(Therma-Max)の開発とそのバイオ領域への応用
    大西 徳幸, 近藤 昭彦
    日刊工業新聞社, 2004, 工業材料, 52,57-61(10) (10), 57 - 61, Japanese
    Scientific journal

  • 熱応答性磁性ナノ粒子(Therma-Max)の開発とそのバイオ領域への応用
    大西 徳幸, 近藤 昭彦
    日刊工業新聞社, 2004, MATERIAL ATAGE, 4(5),54-57(10) (10), 57 - 61, Japanese
    Scientific journal

  • 熱応答性高分子を利用した磁性ナノ粒子の新しい分離法
    近藤 昭彦, 大西 徳幸
    日本農芸化学会, 2004, 化学と生物, 485,212-216(4) (4), 213 - 216, Japanese
    Scientific journal

  • 中空バイオナノ粒子を用いたピポイントドラッグデリバリーシステム
    YAMADA Tadanori, SENOO Masaharu, KONDO Akihiko, UEDA Masakazu, TANIZAWA Katsuyuki, KURODA Syunnichi
    2004, 高分子論文集, 61,606-612, Japanese
    [Refereed]
    Scientific journal

  • 中空バイオナノ粒子によるピンポイントDDS
    近藤 昭彦
    2004, フォルマシア, 40,1013-1017, Japanese
    Scientific journal

  • 中空バイオナノ粒子が拓く新しい医療技術
    黒田 俊一, 山田 忠徳, 妹尾 昌治, 近藤 昭彦, 上田 政和, 谷澤 克行
    化学工業社, 2004, 化学工業, 40,936-942(12) (12), 936 - 942, Japanese
    Scientific journal

  • 多彩な細胞表層ディスプレイシステム
    近藤 昭彦
    日本生物工学会, 2004, 生物工学会誌, 82,454(9) (9), 454 - 454, Japanese
    Scientific journal

  • アーミング技術を用いた微生物によるバイオマス変換の高効率化
    近藤 昭彦
    2004, グリーンプラジャーナル, 12,10-13, Japanese
    Scientific journal

  • FUJITA Yasuya, ITO Junji, UEDA Mitsuyoshi, FUKUDA Hideki, KONDO Akihiko
    American Society for Microbiology, 2004, Applied and Environmental Microbiology, 70(2),1207-1212(2) (2), 1207 - 1212, English
    [Refereed]
    Scientific journal

  • Over-expression system for secretory phospholipase D by Streptomyces livedanse
    OGINO Chiaki, KANEMASU Masayuki, HAYASI Yasuhiro, KONDO AKIHIKO, SHIMIZU Noriaki, TOKUYAMA Shinji, TAHARA Y, KURODA Syunicni, TANIZAWA Katsyuyuki, FUKUDA Hideki
    2004, Applied and Microbiogy and Biotechnology, 64,823-828, English
    [Refereed]
    Scientific journal

  • Tadanori Yamada, Masakazu Ueda, Masaharu Seno, Akihiko Kondo, Katsuyuki Tanizawa, Shun'ichi Kuroda
    The hepatitis B virus (HBV) surface antigen (HBsAg) L particle is a hollow nano-scale particle. HBsAg L particles have many properties that make them useful for in vivo gene transfer vectors and drug delivery systems. Gene therapy so far has required the in vivo pinpoint delivery of genetic materials into the target organs and cells. Gene transfer by HBsAg L particles might be an attractive method, since their tropism is the same as that of HBV. The HBsAg L particles are able to deliver therapeutic payloads with high specificity to human hepatocytes. In addition, the specificity of L particle can be altered by displaying various cell-binding molecules on the surface. Our results indicate that the L particle is suitable for a cell- and tissue-specific gene/drug transfer vector. In this review, we discuss HBsAg L particles as a gene/drug transfer vector and its potential for the treatment of infectious diseases. © 2004 Bentham Science Publishers Ltd.
    2004, Current Drug Targets - Infectious Disorders, 4(2) (2), 163 - 167, English
    [Refereed]
    Scientific journal

  • Enantioselective Transesterification Using Lipase-Displaying Yeast Whole-Cell Biocatalyst
    MATSUMOTO Takeshi, ITO Meguru, FUKUDA Hideki, KONDO Akihiko
    日本生物工学会, 2004, Applied Microbiology and Biotechnology, 64 481-485, 88 - 88, English
    [Refereed]
    Scientific journal

  • Enantioselective Transesterification Using Lipase-Displaying Yeast Whole-Cell Biocatalyst
    MATSUMOTO Takeshi, ITO M, FUKUDA Hideki, KONDO Akihiko
    日本生物工学会, 2004, Applied Microbiology and Biatechnology, 64,481-485, 88 - 88, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    2004, Journal of Molecular Catalysis B: Enzymatic, 28(4-6) 253-257, English
    [Refereed]
    Scientific journal

  • Construction of Ethanol-Telerant yeast Strains with Combinatorial Library-Selected Peptides
    MATSUMOTO Takeshi, ISHIKAWA Shinya, FUKUDA Hideki, KONDO Akihiko
    2004, Journal of Molecular Catalysis B, 28(4-6),253-257, English
    [Refereed]
    Scientific journal

  • Preparation of yeast strains displaying IgG binding domain ZZ and enhanced green fluorescent protein for novel antigen detection systems
    R Shimojyo, H Furukawa, H Fukuda, A Kondo
    To develop novel immunofluorescence-labeling and antigen-detection systems, the ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G, and enhanced green fluorescent protein (EGFP) were displayed on the cell surface of Saccharomyces cerevisiae by cell-surface engineering using the C-terminus 318 amino acids of Flo1 protein. Two systems were constructed, one for co-display of ZZ and EGFP, and one for display of a fusion protein of the two. In both cases, two proteins on the cell surface successfully retain their activities.
    SOC BIOSCIENCE BIOENGINEERING JAPAN, Nov. 2003, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 96(5) (5), 493 - 495, English
    [Refereed]
    Scientific journal

  • H Furukawa, R Shimojyo, N Ohnishi, H Fukuda, A Kondo
    Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30degreesC, above which the flocculated particles-which absorbed a large amount of avidin due to their large surface area-were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.
    SPRINGER-VERLAG, Oct. 2003, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 62(5-6) (5-6), 478 - 483, English
    [Refereed]
    Scientific journal

  • C Ogino, S Kuroda, S Tokuyama, A Kondo, N Shimizu, K Tanizawa, H Fukuda
    This review is focusing on an industrially important enzyme, phospholipase D (PLD), exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. The transphosphatidylation activity of PLD is particularly useful for converting phosphatidylcholine (PC) into other phospholipids. During the last decade, the genes coding for PLD have been identified from various species including mammals, plants, yeast, and bacteria. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far. We recently isolated and characterized the PLD gene from Streptoverticillium cinnamoneum, producing a secretory PLD. Furthermore, we constructed an overexpression system for the secretory enzyme in an active and soluble form using Streptomyces lividans as a host for transformation of the PLD gene. The Stv. cinnamoneum PLD was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food and pharmaceutical industries. (C) 2003 Elsevier B.V. All rights reserved.
    ELSEVIER SCIENCE BV, Sep. 2003, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 23(2-6) (2-6), 107 - 115, English
    [Refereed]
    Scientific journal

  • Y Lin, T Tsumuraya, T Wakabayashi, S Shiraga, Fujii, I, A Kondo, M Ueda
    A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.
    SPRINGER-VERLAG, Aug. 2003, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 62(2-3) (2-3), 226 - 232, English
    [Refereed]
    Scientific journal

  • K. Sato, N. Ichimiya, A. Kondo, Y. Tanaka
    Cathodic arc ion-plated (Al,Ti)N coatings were deposited on WC-Co substrates by several bias voltages. The residual stress of the films, measured by the X-ray diffraction (XRD) 2θ-sin2 ψ method, was found to vary significantly with the negative bias 2 voltages. The composition and microstructure of the films were investigated by electron probe microanalysis and transmission electron microscopy, respectively, and these were also changed by the bias voltages. The changes in composition and microstructure were related to the film strains induced by ion bombardment, and were evaluated by the peak shifts and half-value widths of XRD profiles. At higher bias voltages, the films were mainly composed of granular structures with a size of 100 nm. With the decrease of negative substrate bias voltage, the microstructure changed to a columnar structure with a column size of 120 nm. Carbide drills deposited with (Al,Ti)N coating with lower bias voltages showed better cutting performance in the machining of carbon steel compared with those deposited with coating under high bias voltages. The wear resistance of drill margins is critical for the drilling performance in this test, and the dense columnar (Al,Ti)N films with low strains showed greatly improved wearresistant characteristics of the drills. © 2002 Elsevier Science B.V. All rights reserved.
    Jan. 2003, Surface and Coatings Technology, 163-164, 135 - 143, English
    [Refereed]
    Scientific journal

  • 片平 悟史, 藤田 靖也, 植田 充美, 田中 渥夫, 福田 秀樹, 近藤 昭彦
    公益社団法人 化学工学会, 2003, 化学工学会 研究発表講演要旨集, 2003, 139 - 139, Japanese

  • 小田 充宏, 海江田 優, 浜 真司, 近藤 昭彦, 福田 秀樹
    公益社団法人 化学工学会, 2003, 化学工学会 研究発表講演要旨集, 2003, 130 - 130, Japanese

  • N. Sato, T. Matsumoto, M. Ueda, A. Tanaka, H. Fukuda, A. Kondo
    We investigated the influence of anchor length on the reactivity to polymer substrate of enzyme displayed on yeast cell surfaces. Using various lengths [42, 102, 146, 318, 428, and 1,326 amino acids (aa)] of the C-terminal region of the Saccharomyces cerevisiae Flo1 protein (Flo1p), which plays a major role in yeast flocculation, six display systems with various anchor lengths were constructed. In these systems, the target protein was displayed on the yeast cell surface under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL). Cell-surface display of Rhizopus oryzae glucoamylase by these systems was induced and confirmed in all systems by immunofluorescence microscopy and immunoblotting. Flow-cytometer measurement of the fluorescence intensity of immunofluorescence-labeled yeast cells displaying glucoamylase indicated that glucoamylase displayed with longer anchors, especially those of 428 and 1,326 aa in length, had higher reactivity to antibodies. The reactivity of starch to displayed glucoamylase, which was evaluated by plate assay, increased with anchor length, as did the cell growth-rate in starch-containing medium. These results indicate that cell-surface display systems using 428- and 1,326-aa length anchors of Flo1p are effective for the display of enzymes on the outer surface of yeast cells.
    2003, Applied Microbiology and Biotechnology, 60(4) (4), 469 - 474, English
    [Refereed]
    Scientific journal

  • Efficient secretory overexpression of Bacillus subtilis pectate lyase in Escherichia coli and single-step purification
    T Matsumoto, D Katsura, A Kondo, H Fukuda
    We successfully constructed an efficient system for secretory production of the pectate lyase gene from Bacillus subtilis IFO3134 in Escherichia coli. Pectate lyase possesses pectin-releasing activity and is expected to find applications in enzymatic cotton scouring. The gene encoding the mature region of pectate lyase was cloned and fused to the pelB gene encoding the periplasmic secretion signal and the fusion gene was expressed under the control of the T7 promoter in a culture of E coli BL21DE3 grown in a jar fermentor using an 1713 synthetic medium containing 1% casamino acids. Cultivation was controlled using a DO-stat to keep DO below 2.5% by feeding glucose and controlling agitation. When OD600 reached 7.0, 50 muM of IPTG was added to induce expression of the pectate lyase gene. After 8 h induction, pectin-releasing activity in the culture broth reached 2200 U/ml. This is about three times higher than in native B. subtilis expression systems. Recombinant pectate lyase secreted in the culture broth was purified easily in a single step using hydrophobic chromatograph. The purified recombinant pectate lyase showed similar polygalacturonate-hydrolysis and pectin-releasing activities to that purified from B. subtilis. (C) 2002 Elsevier Science B.V. All rights reserved.
    ELSEVIER SCIENCE BV, Dec. 2002, BIOCHEMICAL ENGINEERING JOURNAL, 12(3) (3), 175 - 179, English
    [Refereed]
    Scientific journal

  • Yasuya Fujita, Shouji Takahashi, Mitsuyoshi Ueda, Atsuo Tanaka, Hirofumi Okada, Yasushi Morikawa, Takashi Kawaguchi, Motoo Arai, Hideki Fukuda, Akihiko Kondo
    For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on α-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His6) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.
    Oct. 2002, Applied and Environmental Microbiology, 68(10) (10), 5136 - 5141, English
    [Refereed]
    Scientific journal

  • Takeshi Matsumoto, Hideki Fukuda, Mitsuyoshi Ueda, Atsuo Tanaka, Akihiko Kondo
    We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.
    Sep. 2002, Applied and Environmental Microbiology, 68(9) (9), 4517 - 4522, English
    [Refereed]
    Scientific journal

  • Effect of oxidized and reduced forms of Escherichia coli DsbC on protein refolding
    J Kohda, N Kawahara, H Fukuda, A Kondo
    DsbC, which catalyzes disulfide isomerization, was overproduced in the periplasm of Escherichia coli and purified from the periplasmic fraction by osmotic shock and anion-exchange chromatography. The active site of the purified DsbC was found to be an oxidized form (ox-DsbC) which could be converted to the reduced form (red-DsbC) by the addition of dithiothreitol. The effect of ox- and red-DsbC on the refolding of chemically denatured and reduced proteins with different numbers of disulfide bonds and free cysteine-thiol groups was investigated. Ox-DsbC facilitated the refolding of proteins with multiple disulfide bonds in both oxidative and reductive environments, while red-DsbC facilitated refolding only in the former. On the other hand, only red-DsbC facilitated the refolding of proteins with multiple free cysteine-thiol groups but either form of DsbC did not facilitate the refolding of proteins with only one cysteine-thiol group. It is therefore important to choose the form which suits the properties of the protein. Holo-chaperonin from Thermus thermophilus and DsbC demonstrated a synergistic effect on protein refolding.
    SOC BIOSCIENCE BIOENGINEERING JAPAN, Aug. 2002, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 94(2) (2), 130 - 134, English
    [Refereed]
    Scientific journal

  • Hisayori Shigechi, Ken Uyama, Yasuya Fujita, Takeshi Matsumoto, Mitsuyoshi Ueda, Atsuo Tanaka, Hideki Fukuda, Akihiko Kondo
    To develop novel yeasts with high starch-to-ethanol productivity, we constructed two cell-surface-engineered flocculent Saccharomyces cerevisiae strains one co-displaying glucoamylase and α-amylase on the cell surface and the other displaying glucoamylase and secreting α-amylase into the culture medium. With starch as the carbon source, both yeast strains grew faster under aerobic conditions than strains displaying only glucoamylase. In fed-batch fermentation of ethanol, these recombinant yeasts co-expressing sequential amylolytic enzymes also showed higher starch decomposition and ethanol production abilities than yeast cells displaying only glucoamylase, with the concentration of ethanol produced reaching 60 g/l after approximately 100 h fermentation under anaerobic conditions. Both co-display and secretion of α-amylase are thus effective in improving ethanol production from starchy materials in glucoamylase-displaying yeast cells. © 2002 Elsevier Science B.V. All rights reserved.
    Jun. 2002, Journal of Molecular Catalysis - B Enzymatic, 17(3-5) (3-5), 179 - 187, English
    [Refereed]
    Scientific journal

  • Yasuya Fujita, Satoshi Katahira, Mitsuyoshi Ueda, Atsuo Tanaka, Hirofumi Okada, Yasushi Morikawa, Hideki Fukuda, Akihiko Kondo
    We constructed a yeast-based whole-cell biocatalyst displaying Trichoderma reesei xylanase II (XYNII) on the cell-surface and endowed the yeast-cells with the ability to degrade xylan. The fusion gene encoding the mature region of XYNII and the C-terminal half (320 amino acid residues from the C-terminal end) of yeast α-agglutinin (XYNII-α-agglutinin) was constructed and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The expression system of fusion gene encoding XYNII-α-agglutinin tagged with RGSHis6 consisting of arginine, glycine, serine, and histidine hexamer (RGSHis6-XYNII-α-agglutinin) was also constructed. Immunofluorescence labeling to confirm cell-surface display of the RGSHis6-XYNII-α-agglutinin fusion protein, and confirmation of similar xylanase activity in yeast-cells expressing XYNII-α-agglutinin and RGSHis6-XYNII-α-agglutinin but not in the culture medium, indicated that XYNII was displayed on the cell-surface in the active form. The XYNII-displaying yeast-based whole-cell biocatalyst showed highest XYNII activity at pH 5.0 and 40°C, respectively. This whole-cell biocatalyst is expected to find application not only in the first step of fermentation of xylan to ethanol but also in xylooligosaccharide production. © 2002 Elsevier Science B.V. All rights reserved.
    Jun. 2002, Journal of Molecular Catalysis - B Enzymatic, 17(3-5) (3-5), 189 - 195, English
    [Refereed]
    Scientific journal

  • Jin-Min Nam, Yasuya Fujita, Terukazu Arai, Akihiko Kondo, Yasushi Morikawa, Hirofumi Okada, Mitsuyoshi Ueda, Atsuo Tanaka
    The genes encoding cellulose binding domain (CBD) from cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) of the filamentous fungus Trichoderma reesei were expressed on the cell surface of the yeast Saccharomyces cerevisiae by cell surface engineering. The CBD genes were fused to the gene encoding the Rhizopus oryzae glucoamylase secretion signal sequence, and expressed under the control of the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) promoter. Each of CBDs was successfully displayed on the yeast cell surface by fusing their genes to the gene encoding the 3′-half of α-agglutinin of S. cerevisiae having a glycosylphosphatidylinositol anchor attachment signal. Tandemly aligned CBHI (CBD1) and CBHII (CBD2) fusion gene was also constructed to display simultaneously both CBDs on the cell surface of S. cerevisiae. Binding affinity of the CBD-displaying yeast cells to a cellulose substrate was similar between the CBD1- and CBD2-displaying yeast cells. However, the cells displaying the fusion protein of CBD1 and CBD2 showed much higher binding affinity to cellulose than either of the single CBD-displaying yeast cells. The binding affinity of the cells was increased by treating the cellulose with phosphoric acid. © 2002 Elsevier Science B.V. All rights reserved.
    Jun. 2002, Journal of Molecular Catalysis - B Enzymatic, 17(3-5) (3-5), 197 - 202, English
    [Refereed]
    Scientific journal

  • Takeshi Matsumoto, Shouji Takahashi, Mitsuyoshi Ueda, Atsuo Tanaka, Hideki Fukuda, Akihiko Kondo
    Yeast whole cell biocatalysts, which intracellularly overproduced a recombinant lipase with a pro-sequence from Rhizopus oryzae IFO4697 (rProROL) were constructed, and the content of active lipase in Saccharomyces cerevisiae cells was maximized by optimizing the cultivation procedure. rProROL was overproduced intracellularly under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) as the inducible system and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter as the constitutive expression system. Enhancement of expression level of ProROL gene at the initial cultivation phase inhibited rProROL accumulation in yeast cells both in GAPDH promoter system by using high glucose concentration at 30°C and in UPR-ICL system by using non-fermentable carbon sources. The highest intracellular lipase activity of 350.6 IU/l was obtained in the inducible UPR-ICL system with an initial glucose concentration of 0.5% at 30°C. To prepare the efficient whole cell biocatalyst by intracellular overproduction of lipase, utilization of inducible UPR-ICL and the optimization of cultivation conditions such as temperature, carbon source and its initial concentration are important. © 2002 Elsevier Science B.V. All rights reserved.
    Jun. 2002, Journal of Molecular Catalysis - B Enzymatic, 17(3-5) (3-5), 143 - 149, English
    [Refereed]
    Scientific journal

  • Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production
    K Ban, S Hama, K Nishizuka, M Kaieda, T Matsumoto, A Kondo, H Noda, H Fukuda
    With a view to utilizing Rhizopus onzae cells immobilized within biomass support particles (BSPs) as a whole-cell biocatalyst for biodiesel fuel production, an investigation was made of the effect of cross-linking treatment with glutaraldehyde (GA) on the stability of lipase activity. Although the lipase activity of the BSP-immobilized cells decreased considerably in the presence of the methyl esters produced by methanolysis, the activity of cells treated with 0.1% GA solution showed no significant decrease during six batch cycles, with the methyl ester content of the reaction mixture reaching 70-83% in each cycle. In contrast, without GA treatment, activity decreased gradually with each cycle to give a methyl ester content of only 50% at the sixth batch cycle. These findings indicate that, given the simplicity of the lipase production process and the long-term stability of lipase activity, the use of whole-cell biocatalysts immobilized within BSPs and treated with GA solution offers a promising means of biodiesel fuel production for industrial application. (C) 2002 Elsevier Science B.V. All rights reserved.
    ELSEVIER SCIENCE BV, Jun. 2002, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 17(3-5) (3-5), 157 - 165, English
    [Refereed]
    Scientific journal

  • Improvement of transphosphatidylation reaction model of phospholipase D from Streptoverticillium cinnamoneum
    C Ogino, Y Yasuda, A Kondo, N Shimizu, H Fukuda
    A new reaction model for transphosphatidylation and hydrolysis reactions by PLD has been constructed and simulated under various experimental conditions. The reaction model is based on the Ping-Pong mechanism and composed of PLD-phosphatidyl complex intermediates. In order to accommodate the inhibition of the initial reaction rate at high nucleophile concentration, a phosphatidyl-enzyme complex associating with two nucleophile molecules [P-PLD-X-2] is introduced into the transphosphatidylation scheme of the proposed reaction model. In both cases (phosphatidylethanolamine (PE) production, and phosphatidylglycerol (PG) production from phosphatidylcholine (PC)), the simulation results agreed well with the experimental data. While the PE production rate at the initial state was inhibited at high concentrations of the nucleophile (ethanolamine), the PG production rate did not show inhibition. Combination of the PE and PG production schemes is expected to make multi-phospholipid production from PC possible, thus, making this model of potential use for phospholipid synthesis in engineering applications. (C) 2002 Elsevier Science B.V All rights reserved.
    ELSEVIER SCIENCE SA, Mar. 2002, BIOCHEMICAL ENGINEERING JOURNAL, 10(2) (2), 115 - 121, English
    [Refereed]
    Scientific journal

  • Improvement of productivity of active form of glutamate racemase in Escherichia coli by coexpression of folding accessory proteins
    J Kohda, Y Endo, N Okumura, Y Kurokawa, K Nishihara, H Yanagi, T Yura, H Fukuda, A Kondo
    Since two classes of folding accessory proteins, molecular chaperones and foldases, prevent the misfolding of newly synthesized polypeptides in the cell, their coexpression could be expected to improve the productivity of soluble and active recombinant proteins. Escherichia coli cytoplasmic glutamate racemase (GluR), which has five cysteine thiol groups and no disulfide bond, was selected as a model enzyme and overexpressed in E. coli. The effects of coexpressing a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active GluR in E. coli were examined. A relatively large amount of active GluR produced by mild induction with 10 muM isopropyl-beta-D-thiogalactopyranoside (IPTG). Active GluR productivity was further increased 2.2-2.3-fold by coexpression of GroEL/ES, Trx, or DsbB-DsbD (DsbBD), while it was decreased by coexpression of DnaK-DnaJ-GrpE and TF. These results demonstrate that coexpression of appropriate folding accessory proteins could significantly improve the productivity of active form of proteins in E. coli. (C) 2002 Elsevier Science B.V. All rights reserved.
    ELSEVIER SCIENCE SA, Feb. 2002, BIOCHEMICAL ENGINEERING JOURNAL, 10(1) (1), 39 - 45, English
    [Refereed]
    Scientific journal

  • Development of new surface display systems for combinatorial bioengineering
    A Kondo, T Matsumoto, H Fukuda
    The novel cell surface display system using flocculation functional domain of Flo1p, namely 1-1099 amino acids (FS protein) was developed. In this system, the N-terminus of the target protein was fused to the FS protein. Recombinant lipase with pro-sequence from Rhizopus oryzae (rProROL) was displayed on the cell surface by this new system. Lipase activity of yeast cells reached 61.3 IU/g-dry cell after 194-h cultivation. This cell surface lipase activity was much higher than that reported previously. On the other hand, detectable lipase activity was not observed in the culture media. The variants, which show the different ratio of methanolysis and hydrolysis activities from that of the wild type lipase, were obtained from combinatorial cell surface display library using this new system, indicating the effectiveness of the new cell surface display system.
    INT INST INFORMATICS & SYSTEMICS, 2002, 6TH WORLD MULTICONFERENCE ON SYSTEMICS, CYBERNETICS AND INFORMATICS, VOL VI, PROCEEDINGS, 431 - 435, English
    [Refereed]
    International conference proceedings

  • A. Kondo, H. Shigechi, M. Abe, K. Uyama, T. Matsumoto, S. Takahashi, M. Ueda, A. Tanaka, M. Kishimoto, H. Fukuda
    A Strain of host yeast YF207, which is a tryptophan auxotroph and shows strong flocculation ability, was obtained from Saccharomyces diastaticus ATCC60712 and S. cerevisiae W303-1B by tetrad analysis. The plasmid pGA11, which is a multicopy plasmid for cell-surface expression of the Rhyzopus oryzae glucoamylase/α-agglutinin fusion protein, was then introduced into this flocculent yeast strain (YF207/pGA11). Yeast YF207/pGA11 grew rapidly under aerobic condition (dissolved oxygen 2.0 ppm), using soluble starch. The harvested cells were used for batch fermentation of soluble starch to ethanol under anaerobic condition and showed high ethanol production rates (0.71 g h-1 l-1) without a time lag, because glucoamylase was immobilized on the yeast cell surface. During repeated utilization of cells for fermentation, YF207/pGA11 maintained high ethanol production rates over 300 h. Moreover, in fed-batch fermentation with YF207/pGA11 for approximately 120 h, the ethanol concentration reached up to 50 g l-1. In conclusion, flocculent yeast cells displaying cell-surface glucoamylase are considered to be very effective for the direct fermentation of soluble starch to ethanol.
    2002, Applied Microbiology and Biotechnology, 58(3) (3), 291 - 296, English
    [Refereed]
    Scientific journal

  • Y Nakamura, S Shibasaki, M Ueda, A Tanaka, H Fukuda, A Kondo
    The ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G (IgG), was displayed on the cell surfaces of yeast Saccharomyces cerevisiae by cell-surface engineering using the C-terminal half of a-agglutinin under control of the 5'-upstream region of the isocitrate lyase gene from Candida tropicalis (UPR-ICL). Display of ZZ on the cell surface was confirmed by immunofluorescence microscopy. Enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA using the S. cerevisiae cells displaying ZZ detected IgG and antigen (human serum albumin) down to a concentration of 1-10 ng/ml in both cases. The detection range covered by these assay systems was wide and could be varied by adjusting the amount of cells and reaction times with horseradish peroxidase (HRP) substrate. Moreover, yeast cells displaying ZZ were successfully used for repeated affinity purification of IgG from serum. These results indicate that S. cerevisiae displaying ZZ may constitute novel and genetically renewable whole-cell immunoadsorbents widely applicable to immunoassays and affinity purification.
    SPRINGER-VERLAG, Nov. 2001, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 57(4) (4), 500 - 505, English
    [Refereed]
    Scientific journal

  • Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles
    K Ban, M Kaieda, T Matsumoto, A Kondo, H Fukuda
    As part of a research program aimed at producing biodiesel fuel from plant oils enzymatically cells of Rhizopus oryzae (R. oryzae) IGO4697 (with a 1,3-positional specificity lipase) immobilized within biomass support particles (BSPs) were investigated for the methanolysis of soybean oil. The R. oryzae cells easily became immobilized within the BSPs during batch operation. To enhance the methanolysis activity of the immobilized cells under the culture conditions used, various substrate-related compounds were added to the culture medium. Among the compounds tested, olive oil or oleic acid was significantly effective. In contrast, no glucose was necessary. Immobilized cells were treated with several organic solvents, but none gave higher activity than untreated cells. When methanolysis was carried out with stepwise additions of methanol using BSP-immobilized cells, in the presence of 15% water the methyl esters (MEs) content in the reaction mixture reached 90% - the same level as that using the extracellular lipase. The process presented here, using a whole cell biocatalyst, is considered to be promising for biodiesel fuel production in industrial applications. (C) 2001 Elsevier Science B.V. Ah rights reserved.
    ELSEVIER SCIENCE SA, Jul. 2001, BIOCHEMICAL ENGINEERING JOURNAL, 8(1) (1), 39 - 43, English
    [Refereed]
    Scientific journal

  • コンビナトリアル・バイオエンジニアリング 細胞表層ディスプレイ法による新機能細胞の創製
    藤田靖也, 松本健史, 近藤昭彦, 植田充美
    シ-エムシ-, Jun. 2001, Bio Ind, 18(6) (6), 56 - 63, Japanese
    [Refereed]

  • Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system
    M Kaieda, T Samukawa, A Kondo, H Fukuda
    Methyl esters synthesized from plant oil and methanol by the methanolysis reaction are potentially important as a biodiesel fuel. The methanolysis of soybean oil by lipases from various microorganisms was investigated. Several of the lipases were found to catalyze methanolysis in a water-containing system without an organic solvent. The lipases from Candida rugosa, Pseudomonas cepacia, and Pseudomonas fluorescens displayed particularly high catalytic ability. The reaction rates of methanolysis catalyzed by the C. rugosa and P, fluorescens lipases decreased significantly when the water content was low, showing that water prevents the inactivation of these lipases by methanol, On the other hand, the methanolysis reaction rate catalyzed by the P, cepacia lipase remained high even under a low water content. In addition, the P, cepacia lipase gave high methyl ester contents in the reaction mixture up to 2 or 3 molar equivalents of methanol to oil, which is attributed to the P. cepacia lipase having substantial methanol resistance. For the same methanol content, the reaction rates of methanolysis catalyzed by the P. cepacia lipase increased with decreasing water content, and hence lipases strongly resistant to high methanol, such as that from P, cepacia, are desirable for use in methanolysis reaction processes.
    SOC BIOSCIENCE BIOENGINEERING JAPAN, Jan. 2001, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 91(1) (1), 12 - 15, English
    [Refereed]
    Scientific journal

  • Effective biofuel production by an intelligent bioreactor
    H Fukuda, A Kondo, H Noda
    With the aim of contributing to efforts to solve global energy and environmental problems, a joint research project - Effective Biofuel Production by an Intelligent Bioreactor - has been set up with participants representing several universities, research institute, and industrial companies. Biofuel obtained from biomass resources is seen as an important source of 'clean energy' by virtue of features such as its biodegradability and low carbon and sulphur dioxide contents. By utilising an 'intelligent' bioreactor containing immobilised 'arming cells', it is expected that practical biofuel production can be achieved at a considerably lower cost than with conventional processes.
    SPRINGER, 2001, ENGINEERING AND MANUFACTURING FOR BIOTECHNOLOGY, VOL 4, 4, 449 - 455, English
    [Refereed]
    International conference proceedings

  • Technology 細胞表層ディスプレイ系の開発と応用
    近藤 昭彦, 植田 充美
    メディカルドゥ, 2001, 遺伝子医学, 5(2) (2), 299 - 304, Japanese
    [Refereed]

  • T. Matsumoto, S. Takahashi, M. Kaieda, M. Ueda, A. Tanaka, H. Fukuda, A. Kondo
    Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomyces cerevisiae MT8-1. The gene encoding lipase from R. oryzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rProROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5′-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 °C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 °C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization.
    2001, Applied Microbiology and Biotechnology, 57(4) (4), 515 - 520, English
    [Refereed]
    Scientific journal

  • Identification of novel membrane-bound phospholipase D from Streptoverticillium cinnamoneum, possessing only hydrolytic activity
    C Ogino, Y Negi, H Daido, M Kanemasu, A Kondo, S Kuroda, K Tanizawa, N Shimizu, H Fukuda
    A membrane-bound phospholipase D (PLD) has been identified and isolated in a soluble form from an actinomycete, Streptoverticillium cinnamoneum. The enzyme has a monomeric structure with a molecular size of about 37 kDa, bring the smallest among the enzymes so far reported. The enzyme catalyzes the hydrolysis of phosphatidylethanolamine and phosphatidylserine as preferred substrates, but not the transphosphatidylation reaction of their phospholipid groups to ethanol. Together with the absence of immunochemical cross-reactivity, these enzymatic properties demonstrate that the membrane-bound enzyme is distinct from the extracellular enzyme recently characterized and cloned from the same bacterial strain [C. Ogino ct al., J. Biochem. 125 (1999) 263-269] and is therefore regarded as a novel prokaryotic PLD. (C) 2001 Elsevier Science B.V. All rights reserved.
    ELSEVIER SCIENCE BV, Jan. 2001, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS, 1530(1) (1), 23 - 31, English
    [Refereed]
    Scientific journal

  • Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins
    A Kondo, J Kohda, Y Endo, T Shiromizu, Y Kurokawa, K Nishihara, H Yanagi, T Yura, H Fukuda
    Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E, coli were examined. Active HRP was produced by very mild induction with 1 muM isopropyl-beta -D-thiogalactopyranoside (IPTG) at 37 degreesC, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.
    SOC BIOSCIENCE BIOENGINEERING JAPAN, Dec. 2000, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 90(6) (6), 600 - 606, English
    [Refereed]
    Scientific journal

  • Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol
    A Kondo, Y Liu, M Furuta, Y Fujita, T Matsumoto, H Fukuda
    Flocculent yeast Saccharomyces cerevisiae YF234 (MATa ura3-52 trp1 Delta2 his ade 2-1 can1-100 sta1 FLO8) cells overexpressing glyoxalase I and having strong flocculation ability were permeabilized with isopropyl alcohol and ethanol under various conditions. The treatment with 40% isopropyl alcohol significantly improves the initial reaction rates of recombinant flocculent yeast cells. Moreover, the reactivity of permeabilized flocculent yeast cells was similar to that of dispersed cells with EDTA. On the other hand, the flocculation ability of yeast cells was not affected by the treatment with alcohol solutions of various concentrations and treatment time length. Therefore, the recombinant flocculent yeast cells permeabilized with alcohol are very effective whole cell biocatalysts. (C) 2000 Elsevier Science Inc. All rights reserved.
    ELSEVIER SCIENCE INC, Dec. 2000, ENZYME AND MICROBIAL TECHNOLOGY, 27(10) (10), 806 - 811, English
    [Refereed]
    Scientific journal

  • Efficient production of protopectinases by Bacillus subtilis using medium based on soybean flour
    T Matsumoto, Y Sugiura, A Kondo, H Fukuda
    We have developed a culture system for efficient production of protopectinases (PPases) by Bacillus subtilis. PPase shows the pectin-releasing activity and is expected to be utilized in the enzymatic cotton scouring. B. subtilis IFO3134 was cultivated using defatted soybean flour as a main component of culture media. This strain produced three different types of PPases, namely PPase-C, -N and -R performing endo-arabinase activity, pectate-lyase activity and pectin-lyase activity, respectively. The effects of alkaline solubilization and autoclave treatments to extract nutrients from soybean flour and initial soybean flour concentration (20-80g/l) on production bf PPases in batch fermentation were investigated. Alkaline solubilization of soybean flour with NaOH remarkably reduced enzyme productivity. In addition, a higher initial concentration of soybean flour reduced the enzyme productivity of cells. The pectin-releasing activity was the largest and reached up to 2200-2400 U/ml, when the culture medium containing an initial soybean flour concentration of 40 g/l was autoclaved far 45-60 min without alkaline solubilization treatment. (C) 2000 Elsevier Science S.A. All rights reserved.
    ELSEVIER SCIENCE BV, Oct. 2000, BIOCHEMICAL ENGINEERING JOURNAL, 6(2) (2), 81 - 86, English
    [Refereed]
    Scientific journal

  • Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil
    T Samukawa, M Kaieda, T Matsumoto, K Ban, A Kondo, Y Shimada, H Noda, H Fukuda
    The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated. Methanolysis progressed much faster when Novozym 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. The initial reaction rate of methanolysis catalyzed by both the non-treated and preincubated enzyme decreased significantly with increasing water content. The initial reaction rate increased with increasing methanol content, showed a maximum, and thereafter decreased when the methanol content was increased further. The variation of the initial reaction rate with the methanol content was therefore analyzed using a Michaelis-Menten-type equation with substrate inhibition. Based on this equation, a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired methanol content was determined. When preincubated Novozym 435 was used, the ME content reached over 97% within 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25-0.4 h intervals.
    SOC BIOSCIENCE BIOENGINEERING JAPAN, Aug. 2000, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 90(2) (2), 180 - 183, English
    [Refereed]
    Scientific journal

  • Preparation of high-activity whole cell biocatalysts by permeabilization of recombinant yeasts with alcohol
    Y Liu, Y Fujita, A Kondo, H Fukuda
    Recombinant yeast cells intracellularly overexpressing three different enzymes were permeabilized with alcohol under various conditions. The effects of enzyme stability in alcohol and enzyme molecular weight on the activities of permeabilized cells and enzyme leakage during incubation were examined. Saccharomyces cerevisiae YPH250 overexpressing glyoxalase I (GloI), S. cerevisiae MT8-1 overexpressing isocitrate lyase (ICL), and Pichia pastoris GS115 overexpressing beta-galactosidase (beta-gal) were used as model recombinant yeast systems. In all cases, the percentage of alcohol used for the treatment significantly affected the activity of permeabilized whole cell biocatalysts; cells showed high activity when treated with 40% Isopropyl alcohol. The activity of whole cell biocatalysts was also significantly affected by the stability of the enzyme In alcohol solution; permeabilized yeast cells overexpressing ICL, which had low stability, showed rather ion; activity. Although the enzyme leakage from permeabilized cells was rather low in all cases, the molecular weight of the enzyme appeared to affect the extent of enzyme leakage during incubation. Permeabilized cells of P, pastoris overexpressing beta-gal (540 kDa) retained particularly high activity during incubation and could be used as an immobilized whole cell biocatalysts.
    SOC BIOSCIENCE BIOENGINEERING JAPAN, Jun. 2000, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 89(6) (6), 554 - 558, English
    [Refereed]
    Scientific journal

  • Shuji Sonezaki, Shinichi Yagi, Emika Ogawa, Akihiko Kondo
    To develop a stable immuno-assay system for quantification of human hemoglobin (Hb), the interaction between various antibodies and Hb was studied using a surface plasmon resonance (SPR) biosensor in the BIAcore equipment (Amersham Pharmacia Biotech) with an immobilized anti-Hb antibody sensor chip. When polyclonal antibodies were used, the immuno-reactivity of purified and commercially available Hb decreased drastically with incubation times up to 14 h. This instability of immuno-reactivity of Hb is attributable to the conformational changes in Hb induced by oxidation. On the other hand, of the sixteen monoclonal antibodies tested, four antibodies (MSU-102, -103, -106 and -115) were found to maintain their immuno-reactivities at least up to 24 h. During long-term storage, however, the immuno-reactivity of Hb with these monoclonal antibodies decreased significantly. The chemical ββ-cross-linking of Hb was effectively able to stabilize the structure of Hb and immuno-reactivity with monoclonal antibodies such as MSU-103 for periods at least up to 70 days. Therefore, the combination of specific monoclonal antibodies such as MSU-103 and a ββ-cross-linked Hb standard could be used for the quantification of Hb. Copyright (C) 2000 Elsevier Science B.V.
    Apr. 2000, Journal of Immunological Methods, 238(1-2) (1-2), 99 - 106, English
    [Refereed]
    Scientific journal

  • Development of efficient protein refolding systems using chaperonins
    J Kohda, A Kondo, T Teshima, H Fukuda
    Application of chaperonins to in vitro protein refolding was investigated. E. coli chaperonin GroEL/ES and Thermus thermophilus holo-chaperonin (T. thermophilus holo-cpn) were used for construction of protein refolding systems. The systems based on immobilized chaperonin, fusion chaperonin and chaperonin in combination with ultrafiltration were investigated. Fusion chaperonin with the hexa-histidine affinity tag (GroEL-(His)(6)) was efficiently purified by affinity column. The refolding activity of GroEL-(His)(6) was proved to be similar to that of native one. GroEL/ES was immobilized by covalent bond. In addition, GroEL-(His)(6) was immobilized on metal chelate resin by affinity interaction. These immobilized chaperonins retained sufficiently high refolding activities and were reused efficiently. Especially, immobilized GroEL-(His)(6) showed a high refolding activity. On the other hand, in ultrafiltration system, I: thermophilus holo-cpn, which forms a stable complex, was efficiently separated from refolded proteins and repeatedly used for protein refolding. The selection of the system is mainly dependent on the type of chaperonin most effective for the refolding of target proteins.
    ELSEVIER SCIENCE BV, 2000, BIOSEPARATION ENGINEERING, 16, 119 - 124, English
    [Refereed]
    International conference proceedings

  • Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent
    M Kaieda, T Samukawa, T Matsumoto, K Ban, A Kondo, Y Shimada, H Noda, F Nomoto, K Ohtsuka, E Izumoto, H Fukuda
    A new enzymatic method of synthesizing methyl esters from plant oil and methanol in a solvent-free reaction system was developed. It is anticipated that such plant oil methyl esters can be used as a biodiesel fuel in the future. Lipase from Rhizopus oryzae efficiently catalyzed the methanolysis of soybean oil in the presence of 4-30 wt% water in the starting materials; however the lipase was nearly inactive in the absence of water. The methyl ester (ME) content in the reaction mixture reached 80-90 wt% by stepwise additions of methanol to the reaction mixture. The kinetics of the reaction appears to be in accordance with the successive reaction mechanism. That is, the oil is first hydrolyzed to free fatty acids and partial glycerides, and the fatty acids produced are then esterified with methanol. Although R. oryzae lipase is considered to exhibit 1(3)regiospecificity, a certain amount of 1,3-diglyceride was obtained during the methanolysis and hydrolysis of soybean oil by R. oryzae lipase solution. Therefore, the high ME content in the reaction mixture is probably attributable to the acyl migration from the sn-2 position to the sn-l or sn-3 position in partial glycerides.
    SOC BIOSCIENCE BIOENGINEERING JAPAN, Dec. 1999, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 88(6) (6), 627 - 631, English
    [Refereed]
    Scientific journal

  • Preparation of thermo-sensitive magnetic microspheres and their application to bioprocesses
    A Kondo, H Fukuda
    Ultrafine magnetite particles were prepared by the co-precipitation method. Poly(styrene/N-isopropylacrylamide/methacrylic acid) latex particles containing ultrafine magnetite [magnetic P(St/NIPAM/MAA)] were prepared by two-step emulsifier-free emulsion polymerization. Most of the synthesized magnetic latex particles showed the reversible transition between flocculation and dispersion by controlling temperature, and the thermoflocculated magnetic latex particles were separated quickly in a magnetic field. Copolymerization of divinylbenzene with St at the first step enhanced the particle growth and was effective to improve magnetic separation of thermo-flocculated latex particles. Porcine trypsin was covalently immobilized onto the thermo-sensitive magnetic latex particles with high efficiency by the carbodiimide method. The immobilized trypsin showed high activity, and hence the thermo-sensitive magnetic latex particles are effective for enzyme reaction processes. (C) 1999 Elsevier Science B.V. All rights reserved.
    ELSEVIER SCIENCE BV, Aug. 1999, COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS, 153(1-3) (1-3), 435 - 438, English
    [Refereed]
    Scientific journal

  • Production of S-lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols
    Y Liu, H Hama, Y Fujita, A Kondo, Y Inoue, A Kimura, H Fukuda
    The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts. Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S-lactoylglutathione in the presence of glutathione, was used as the model system. The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells. Especially, the initial S-lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4 degrees C were high and were 364 and 582 times larger than those of untreated cells, respectively. These permeabilized yeast cells retained high activities during repeated batch reactions. Even in third batch reaction, they showed approximately 70-80% of the activity in the first batch. The plasma membrane of S. cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability. The initial S-lactoylglutathione production rates of these permeabilized cells were 1.5-2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells. These results demonstrate that the recombinant S. cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts. (C) 1999 John Wiley & Sons, Inc.
    JOHN WILEY & SONS INC, Jul. 1999, BIOTECHNOLOGY AND BIOENGINEERING, 64(1) (1), 54 - 60, English
    [Refereed]
    Scientific journal

  • Purification, characterization, and sequence determination of phospholipase D secreted by Streptoverticillium cinnamoneum
    C Ogino, Y Negi, T Matsumiya, K Nakaoka, A Kondo, S Kuroda, S Tokuyama, U Kikkawa, T Yamane, H Fukuda
    Phospholipase D (PLD), secreted into the culture medium of an actinomycete, Streptoverticillium cinnamoneum, has been purified to homogeneity and characterized. The Stv. cinnamoneum PLD efficiently catalyzes both the hydrolysis and transphosphatidylation of various phospholipids, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS), However, the substrate specificity differs between the two reactions; PE serves as the most preferred substrate for the hydrolysis, but PC and PS are better substrates than PE for the transphosphatidylation. In addition, the transphosphatidylation but not the hydrolysis of PE and PC is markedly activated on the addition of metal ions, especially Al3+. Nucleotide and amino acid sequence determination of the Sty. cinnamoneum PLD revealed the presence of common structural motifs identified in all PLD sequences from various species.
    OXFORD UNIV PRESS, Feb. 1999, JOURNAL OF BIOCHEMISTRY, 125(2) (2), 263 - 269, English
    [Refereed]
    Scientific journal

  • Bioconversion using immobilized recombinant flocculent yeast cells carrying a fused enzyme gene in an 'intelligent' bioreactor
    Y Liu, A Kondo, H Ohkawa, N Shiota, H Fukuda
    Immobilized recombinant cells of the flocculent yeast Saccharomyces diastaticus carrying an expression plasmid for a fused enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 reductase were used in the bioconversion reaction from acetanilide (AA) to p-acetaminophene (p-AAP). Immobilization of the strain within reticulated polyurethane foam biomass support particles (BSPs) was effected passively in situ in a fluidized-bed bioreactor using 'draw and fill' operation. In repeated batch reactions both the final product concentration and the production rate were notably improved compared with the results obtained using freely suspended cells without BSPs. Cells immobilized within BSPs exhibited a significantly high level of expression of the fused enzyme. In addition, a high proportion of plasmid-carrying cells was maintained among the immobilized cells, in contrast to a much lower proportion among freely suspended cells released from the BSPs. Since the bioreactor became packed with highly expressing cells immobilized within BSPs as a consequence of spontaneous screening, it was termed an 'intelligent' bioreactor, and is believed to offer significant potential for the further development of efficient production processes. (C) 1998 Elsevier Science S.A. All rights reserved.
    ELSEVIER SCIENCE SA, Dec. 1998, BIOCHEMICAL ENGINEERING JOURNAL, 2(3) (3), 229 - 235, English
    [Refereed]
    Scientific journal

  • Effects of adsorption conditions on kinetics of protein adsorption and conformational changes at ultrafine silica particles
    A Kondo, H Fukuda
    Human hemoglobin (HHb, a very soft protein) was adsorbed on ultrafine silica particles. Effects of various adsorption conditions on the kinetics of adsorption and conformational changes in HHb were studied. The conformational changes in HHb upon adsorption were monitored by both circular dichroism and absorbance spectra. The adsorption rates were measured using an aqueous two-phase system to very quickly separate the ultrafine silica particles. The rate of conformational changes upon adsorption was significantly affected by pH, ionic strength, and temperature. At high temperature, large conformational changes in HHb were accomplished soon after attachment to the surfaces. On the other hand, at low temperature and/or high ionic strength, the extent of conformational changes accompanying attachment to the surfaces was small, and slow conformational changes occurred after adsorption. Moreover, the rate of conformational changes upon adsorption decreased with increasing adsorption. Renaturation of HHb during desorption was also measured and found to be accomplished within a short time. The percentage of desorbed HHb decreased with increasing adsorption time, because the extent of conformational changes in adsorbed HHb increased, Based on these results, the general tendency of the kinetics of conformational changes in proteins upon adsorption on the solid surfaces are discussed. (C) 1998 Academic Press.
    ACADEMIC PRESS INC, Feb. 1998, JOURNAL OF COLLOID AND INTERFACE SCIENCE, 198(1) (1), 34 - 41, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    1998, Journal of the Electrochemical Society
    Scientific journal

  • Yoshiyuki Ishii, Kaoru Murakami, Hiroaki I-Ogawa, Akihiko Kondo, Yasuhiko Kato
    GroES, the co-chaperonin of Escherichia coli, was overproduced and purified using the maltose binding protein (MBP) fusion vector. The MBP-GroES fusion protein was expressed in a soluble form in E. coli and purified to homogeneity using amylose resin in a single step. The MBP-GroES fusion protein was found to form a heptamer, reduce the ATPase activity of chaperonin GroEL and stimulate GroEL-mediated protein refolding, to a similar extent as the wild-type GroES. Furthermore, the MBP-GroES fusion protein bound GroEL, and this complex was recovered easily from the refolding mixture by amylose resin. These results suggest that the combination of the MBP-GroES fusion protein and GroEL is effective for the construction of a repeated refolding system of proteins.
    Elsevier Sci B.V., 1998, Journal of Fermentation and Bioengineering, 85(1) (1), 69 - 73, English
    [Refereed]
    Scientific journal

  • Tadanaru Teshima, Shinobu Mashimo, Akihiko Kondo, Hideki Fukuda
    Escherichia coli chaperonin GroEL with a hexa-histidine affinity tag (His)6 fused to its C-terminal (GroEL(His)6) was overproduced in E. coli. GroEL-(His)6 was expressed in a soluble form in E. coli and purified to homogeneity in a single step by nickel chelate resin. GroEL-(His)6 was found to form a tetradecamer and showed a similar ATPase activity to native GroEL. GroEL-(His)6 mediated refolding of guanidine hydrochloride-unfolded yeast enolase and reactivation of thermally inactivated yeast enolase to similar extents as native GroEL. The structure and function of GroEL are thus little affected by the fusion of the (His)6 tag to the C-terminal of GroEL. GroEL- (His)6 was efficiently immobilized on nickel chelate Cellulofine, and immobilized GroEL-(His)6 retained a high ability to mediate protein refolding. Therefore, GroEL-(His)6 is advantageous in the application of chaperonin to protein refolding systems.
    Elsevier Sci B.V., 1998, Journal of Fermentation and Bioengineering, 86(4) (4), 357 - 362, English
    [Refereed]
    Scientific journal

  • Yoshiyuki Ishii, Syuji Sonezaki, Yasushi Iwasaki, Eiichi Tauchi, Yoshikazu Shingu, Koichi Okita, Hiroaki I.-Ogawa, Yasuhiko Kato, Akihiko Kondo
    DnaJ is a molecular chaperone, which contains a zinc finger-like motif and cooperates with DnaK to mediate the folding of newly synthesized and denatured proteins. DnaJ was overproduced and purified using the maltose binding protein (MBP) fusion vector. The fusion protein (MBP-DnaJ) was expressed in a soluble form in Escherichia coli and purified to homogeneity using amylose resin in a single step. The UV-visible absorption spectrum of MBP-DnaJ showed peaks at 355 and 475 nm. Moreover, these absorption peaks disappeared upon treatment with ethylenediaminetetraacetic acid (EDTA) or p-hydroxy-mercuriphenylsulfonic acid (PMPS). Inductively coupled plasma (ICP) spectrometry demonstrated that MBP-DnaJ contains Fe ions as well as Zn ions, MBP-DnaJ mediated the replication of the λ phage in vivo, stimulated the ATPase activity of DnaK and prevented the aggregation of denatured rhodanase, indicating that fusion of MBP to the N-terminal of DnaJ does not affect the functions of DnaJ. To study the roles of bound metal ions, metal-free MBP-DnaJ, and MBP-DnaJ containing 2 Zn ions were prepared. MBP-DnaJ containing Fe and Zn ions, and MBP-DnaJ containing 2 Zn ions stimulated the ATPase activity of DnaK, prevented the aggregation of denatured rhodanase and bound to DNA to similar extents. On the other hand, metal-free MBP-DnaJ showed much lower DNA-binding ability and lower ability to prevent rhodanese aggregation. Therefore, the bound metal species do not affect the function of the zinc finger-like motif of DnaJ, whereas removal of the metal ions from DnaJ diminishes its binding ability as to DNA and denatured proteins.
    Japanese Biochemical Society, 1998, Journal of Biochemistry, 124(4) (4), 842 - 847, English
    [Refereed]
    Scientific journal

  • Reactivation of thermally inactivated enzymes by free and immobilized chaperonin GroEL/ES
    T Teshima, A Kondo, H Fukuda
    Thermally inactivated bovine deoxyribonuclease I (DNase I) and yeast enolase were reactivated by GroEL/ES from Escherichia coli. In both cases, GroEL/ ES was found to have the ability to reactivate inactivated enzymes in an ATP-dependent manner. GroEL/ ES can interact with the enzymes that were denatured at high temperature and convert them to the active conformations. To test the applicability of GroEL/ES to the reactivation processes of thermally inactivated enzymes, GroEL/ES was immobilized using formyl-Cellulofine (GroEL/ES-Cellulofine) and its performance was studied. GroEL/ES-Cellulofine retained a sufficiently high ability to reactivate enzymes. Moreover, GroEL/ES-Cellulofine could be used repeatedly, indicating high durability. These results indicate that immobilized chaperonin is effective for reactivation of enzymes that are thermally inactivated in various bioprocesses.
    SPRINGER VERLAG, Jul. 1997, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 48(1) (1), 41 - 46, English
    [Refereed]
    Scientific journal

  • Yoshiyuki Ishii, Tadanaru Teshima, Akihiko Kondo, Kaoru Murakami, Shuji Sonezaki, I. -Ogawa Hiroaki, Yasuhiko Kato, Hideki Fukuda
    To clarify the efficient refolding conditions of enzymes using chaperonin GroE from Escherichia coli, the effects of various factors on the chaperonin-mediated refolding of enzymes from 4M guanidine hydrochloride (GdnHCl) were investigated. Three enzymes, Bacillus subtilis α-amylase, bovine deoxyribonuclease I (DNase I) and yeast enolase were used as the model systems. In all enzymes, the maximum recovery of activities (90-150% with respect to the enzyme activities before denaturation) was attained in the presence of 2mM ATP and 2-5-fold molar excess of GroE 21-mer or GroEL 14-mer over enzyme molecules. Since the recovery of enzyme activity by GroEL is close to that by GroE, GroEL as well as GroE are applicable for the refolding systems of these enzymes. GroE significantly enhanced the recovery of enzyme activities at around 25-40 °C, pH 6-9 and up to rather high final GdnHCl and enzyme concentrations. Therefore, the chaperonin efficiently mediates the enzyme refolding under wide operation conditions. To test the reusability of chaperonins in enzyme refolding, GroEL was separated from refolded enzymes by ultrafiltration and recycled. GroEL repeatedly mediated the refolding of enzymes by choosing appropriate membranes. Therefore, protein refolding processes based on chaperonins are promising.
    Elsevier Science S.A., May 1997, Chemical Engineering Journal, 65(2) (2), 151 - 157, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo
    1997, Colloid and Polymer Science
    Scientific journal

  • Hiroyuki Furuta, Terukazu Arai, Hideki Hama, Naofumi Shiomi, Akihiko Kondo, Hideki Fukuda
    Production of glucoamylase by cells of a flocculent yeast, Saccharomyces diastaticus, immobilized within porous biomass support particles (BSPs) was studied in repeated batch fermentation. Since many of the immobilized cells could be utilized as seeds for each subsequent batch cycle, the glucoamylase productivity in repeated batch fermentation was 70% higher than that obtained in batch fermentation without BSPs.
    Elsevier Sci B.V., 1997, Journal of Fermentation and Bioengineering, 84(2) (2), 169 - 171, English
    [Refereed]
    Scientific journal

  • Hideki Yamaji, Masato Nakagawa, Kanji Tomioka, Akihiko Kondo, Hideki Fukuda
    For spectrophotometric liposome immunoassay of a specific antibody, antigen (cytochrome c)-coupled liposomes containing the substrate glucose-6-phosphate (G6P) as a marker were prepared from 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPhyPC). DPhyPC liposomes showed good stability against G6P leakage, which was below 8% after incubation for 70 d at 4°C. When cytochrome c-coupled liposomes were incubated with anti-cytochrome c antibody and complement, after which glucose-6-phosphate dehydrogenase and NAD+ were added, the absorbance at 340 nm increased with the amount of anti-cytochrome c antibody added.
    Elsevier Sci B.V., 1997, Journal of Fermentation and Bioengineering, 83(6) (6), 596 - 598, English
    [Refereed]
    Scientific journal

  • Preparation of thermo-sensitive magnetic hydrogel microspheres and application to enzyme immobilization
    A Kondo, H Fukuda
    Magnetic hydrogel microspheres mere prepared by co-polymerizing N-isopropylacrylamide (NIPAM), methacrylic acid (MAA) and N,N'-methylene-bis-acrylamide (MBA) in the presence of ultrafine magnetite particles (average particle diameter of 10-20 nm) prepared by co-precipitation. The synthesized magnetic hydrogel microspheres with varying MAA content were monodispersed, and their average diameters ranged from 150-250 nm. All the magnetic hydrogel microspheres showed a reversible transition between flocculation and dispersion as a function of temperature (4-40 degrees C), and the thermo-flocculated microspheres were separated quickly in the magnetic field. Enzymes were immobilized onto these thermo-sensitive magnetic hydrogel microspheres by two different methods. Trypsin was covalently immobilized onto the magnetic hydrogel microspheres in high yield by the carbodiimide method. In the case of microspheres with higher MAA content, immobilized trypsin demonstrated higher activity. On the other hand, a fusion enzyme consisting of affinity tag AG (immunoglobulin G binding domains) and beta-galactosidase (beta gal) was immobilized onto the magnetic hydrogel microspheres with covalently immobilized human gamma-globulin by affinity interaction. The immobilized AG beta gal retained high activity even in the case of microspheres with a low MAA content. Therefore, the thermosensitive magnetic hydrogel microspheres are useful carriers for enzyme immobilization.
    SOC FERMENTATION BIOENGINEERING, JAPAN, 1997, JOURNAL OF FERMENTATION AND BIOENGINEERING, 84(4) (4), 337 - 341, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Takafumi Urabe, Kohji Yoshinaga
    Ultrafine silica particles were modified with various polymer-silane coupling agents. Since the light scattering intensity of these particles was low, the conformation of adsorbed enzyme can be evaluated by spectrophotometric methods, such as circular dichroism (CD). Adsorption isotherms of Bacillus subtilis α-amylase and the activities of adsorbed α-amylase were measured under various adsorption conditions. Moreover, to discuss the extent of conformational changes upon adsorption, the α-helix content of adsorbed α-amylase was elucidated from the CD spectrum measurements. The amount of adsorption was strongly affected by the type of particles and increased with increasing surface hydrophobicity. A stronger interaction at more-hydrophobic surfaces was found to cause larger conformational changes and to reduce the activity of adsorbed enzyme. Therefore, the surface properties have a large influence on the molecular states of enzymes on solid surfaces. However, in all the particles, the amount of adsorption decreased with increasing pH and decreasing temperature. Lower affinity between particles and α-amylase at higher pH and/or lower temperature reduces the extent of conformational changes and increases the relative activity.
    Elsevier Science B.V., Apr. 1996, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 109, 129 - 136, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Jun Mihara
    Human hemoglobin and horse myoglobin were adsorbed on the two types of ultrafine silica (silica 1 and 2), titania, and zirconia particles under various conditions. The order of the affinity of the ultrafine particles to these proteins was silica 1 < silica 2 < zirconia = titania under the same adsorption condition. The conformational changes in these proteins upon adsorption and desorption were monitored by both circular dichroism and absorbance spectra. In both spectrum measurements, a similar tendency was observed, indicating that the adsorption of proteins causes the changes not only in the secondary structure but also in the tertiary structure. In all the particles, the extent of conformational changes upon adsorption increased with decreasing pH and adsorption amount. On the other hand, the extent of conformational changes was significantly affected by the particle type (its order was silica 1 < silica 2 < zirconia = titania) under the same adsorption condition. The percentage of desorption and the extent of refolding of desorbed proteins were lower when the extent of conformational changes in the adsorbed states was higher. These results clearly demonstrate that higher affinity between particle surfaces and proteins causes larger conformational changes and consequently leads to higher irreversibility in adsorption and conformational states.
    Academic Press Inc., Jan. 1996, Journal of Colloid and Interface Science, 177(1) (1), 214 - 221, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Hideki Fukuda, Yoshiharu Iwabuchi, Ikuo Fujii
    To develop a bioreactor system using catalytic antibodies, the reaction properties of immobilized catalytic antibodies in aqueous dimethyl sulfoxide (DMSO) were studied. As a model system, the catalytic antibody 17E11, which selectively hydrolyzes the ester group at C-4 of the carbohydrates with two 4-(N-acetyl-amino)phenylacetyl groups at C-3 and C-4 to produce a monoester 4-oh exclusively, was used. 17E11 was immobilized onto CNBr-activated Sepharose 4B (17E11-sepharose 4B) and Formyl-cellulofine (17E11-cellulofine). 17E11-sepharose 4B and 17E11-cellulofine retained a high catalytic activity in 10% DMSO/50 mm Tris-HCl buffer, pH 8.2, and showed the activity up to 40% DMSO. Moreover, both the immobilized catalytic antibodies were stable and repeatedly used for the hydrolysis of the substrate in 10% DMSO/50 mM Tris- HCl, pH 8.2, without detectable activity reduction, indicating a high durability. The immobilized catalytic antibodies are advantageous for quick production and separation of products which are unstable under the reaction conditions. Model calculations based on the kinetic parameters obtained experimentally showed that unstable 4-OH can be produced with a high yield using an appropriate amount of immobilized 17E11. These results clearly show the effectiveness of immobilized catalytic antibodies and their applicability to bioreactor systems.
    Society of Fermentation and Bioengineering, Japan, 1996, Journal of Fermentation and Bioengineering, 82(5) (5), 452 - 457, English
    [Refereed]
    Scientific journal

  • Hideki Fukuda, Yuji Turugida, Takahiro Nakajima, Eiji Nomura, Akihiko Kondo
    Production of phospholipase D (PLD) by Streptoverticillium cinnamoneum immobilized within porous particles was investigated in repeated batch fermentation. The enzyme productivity in repeated batch fermentation was 2.2-fold that obtained in batch fermentation without immobilization, since many of the immobilized cells could be utilized as seed cells for each subsequent batch cycle.
    Kluwer Academic Publishers, 1996, Biotechnology Letters, 18(8) (8), 951 - 956, English
    [Refereed]
    Scientific journal

  • S. Sonezaki, K. Okita, T. Oba, Y. Ishii, A. Kondo, Y. Kato
    Interaction between the fusion protein MBP-Lon, formed by maltose-binding protein and Lon protease, and the plasmid pBR322 was studied to clarify the DNA-binding behavior of the Lon protease. Since the MBP-Lon fusion protein that was bound to the plasmid was strongly adsorbed by amylose resin, complex formation and dissociation were determined by quantifying the unadsorbed plasmid using agarose gel electrophoresis. The autolysis of MBP-Lon fusion protein was suppressed when the protein was bound to the plasmid. The plasmid was completely dissociated from MBP-Lon fusion protein by the addition of the protein substrates of Lon protease (i.e. α-casein and denatured bovine serum albumin). In addition, at high temperatures, MBP-Lon fusion protein lost its plasmid-binding ability, although it fully retained ATP-dependent protease activity. These results suggest that Lon protease loses DNA-binding ability when cells are exposed to abnormal conditions and the amount of damaged proteins increases. On the other hand, DNA probably plays an important role in controlling the Lon protease activity in cells under normal conditions by entrapping the enzyme. © 1995 Springer-Verlag.
    Springer-Verlag, Dec. 1995, Applied Microbiology and Biotechnology, 44(3-4) (3-4), 484 - 488, English
    [Refereed]
    Scientific journal

  • A. Kondo, T. Urabe
    Bacillus subtilis α-amylase, which contains a relatively large amount of α-helix, was adsorbed on two types of ultrafine silica particles (silica-1 and-2, average diameter 15 nm) under various conditions. The changes in circular dichroism (CD) spectra of α-amylase upon adsorption were measured, and the extent of conformational changes was estimated from the reduction in α-helix content. In additions the activities of adsorbed α-amylase were measured at pH 5.2 using corn starch and p-nitrophenylbenzyl α-maltopentaoside (BG5P). In the ultrafine silica-2 particles, the extent of both activity reductions and conformational changes upon adsorption was much larger than that in the ultrafine silica-1 particles and increased with decreasing pH and amount of adsorption. The extent of activity reductions correlated closely with the conformational changes. On the other hand, the effect of reduction in α-amylase activity upon adsorption measured by BG5P was smaller than that measured by starch, indicating that the lack of accessibility of the active site to a large substrate also reduces the activity of adsorbed α-amylase. However, the effects of particle type and adsorption conditions on the extent of activity reductions by the accessibility resistance were small. Therefore, variation of the activity of adsorbed α-amylase is mainly attributable to the extent of conformational changes upon adsorption. Based on these results, a procedure to prepare adsorbed α-amylase with high activity was investigated. © 1995 Springer-Verlag.
    Springer-Verlag, Oct. 1995, Applied Microbiology and Biotechnology, 43(5) (5), 801 - 807, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Takafumi Urabe
    α-Amylases from Bacillus subtilis and Bacillus licheniformis, which has high thermostability, were adsorbed on ultrafine silica particles at various temperatures. The activities of adsorbed αamylases were measured using corn starch as a substrate. In addition, the circular dichroism spectra of adsorbed α-amylases were measured, and the α-helix contents were calculated to discuss the extent of conformational changes quantitatively. The extent of activity reductions upon adsorption of B. subtilis α-amylase on the ultrafine silica particles was correlated closely with that of conformational changes, and both of them were significantly increased by raising temperature from 4 to 40°C. Moreover, they showed stronger temperature dependences at lower adsorption amount and pH. On the other hand, the extent of activity reductions and conformational changes upon adsorption of B. licheniformis α-amylase on the ultrafine silica particles was much smaller and was not affected by temperature up to 40°C, indicating the strong influence of stability of enzymes on their molecular states on the solid surfaces. The mechanism of thermal deactivation of α-amylases upon adsorption was clearly explained by the direct observation of their conformational states on the solid surfaces. It should be noted that the thermal unfolding of adsorbed enzymes takes place in a much lower temperature range than that of free ones and is strongly affected by the adsorption conditions. © 1995 by Academic Press, Inc.
    Sep. 1995, Journal of Colloid And Interface Science, 174(1) (1), 191 - 198, English
    [Refereed]
    Scientific journal

  • Kohji Yoshinaga, Kimihiko Kondo, Akihiko Kondo
    1995, Polymer Journal, 27(1) (1), 98 - 100, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Tadanaru Teshima
    Affinity tag AG consisting of immunoglobulin G (lgG)‐binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of β‐galactosidase (βgal) from Escherichia coli. Poly(methylmethacrylate/N‐isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N‐isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human γ‐globulin (HγGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGβgal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGβgal per unit amount of immobilized HγGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HγGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HγGb–P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HγGb molecules. Immobilized AGβgal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. © 1995 John Wiley & Sons, Inc. Copyright © 1995 John Wiley & Sons, Inc.
    1995, Biotechnology and Bioengineering, 46(5) (5), 421 - 428, English
    [Refereed]
    Scientific journal

  • S. Sonezaki, Y. Ishii, K. Okita, T. Sugino, A. Kondo, Y. Kato
    To overproduce extremely unstable SulA protein, which is the cell-division inhibitor of Escherichia coli, we fused the sulA gene to the maltose-binding protein (MBP) fusion vectors with or without the signal sequence (plasmids pMAL-p-SulA and pMAL-c-SulA respectively). The amount of the full-length fusion protein expressed from the plasmid pMAL-p-SulA (pre-MBP-SulA) in E. coli was much larger than that expressed from the plasmid pMAL-c-SulA (MBP-SulA). A major amount of the pre-MBP-SulA fusion protein was expressed in a soluble form and affinity-purified by amylose resin. Since site-specific cleavage of the fusion protein with factor Xa resulted in the precipitation of SulA protein, the pre-MBP-SulA fusion protein was used to study the degradation of SulA protein by E. coli Lon protease in vitro. It was found that only the SulA portion of the fusion protein was degraded by Lon protease in an ATP-dependent manner. This result provides direct evidence that Lon protease plays an important role in the rapid degradation of SulA protein in cells. © 1995, Springer-Verlag. All rights reserved.
    1995, Applied Microbiology and Biotechnology, 43(2) (2), 304 - 309, English
    [Refereed]
    Scientific journal

  • S. Sonezaki, A. Kondo, T. Oba, Y. Ishii, Y. Kato, H. Nakayama
    Lon protease, which plays a major role in degradation of abnormal proteins in Escherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector. The MBP-Lon fusion protein was expressed in a soluble form in E. coli and purified to homogeneity by amylose resin in a single step. Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration. In this simple method, Lon protease was purified to homogeneity. Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-l-phenylalanyl-l-leucyl-phenylalanyl-β-d-methoxynaphthylamide) and protein (α-casein) in the presence of ATP. Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease. MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes. © 1994 Springer-Verlag.
    Springer-Verlag, Nov. 1994, Applied Microbiology and Biotechnology, 42(2-3) (2-3), 313 - 318, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Hiroko Kamura, Ko Higashitani
    Ultrafine magnetite particles were prepared by a co-precipitation method. The poly-(styrene/N-isopropylacrylamide/methacrylic acid) latex particles containing ultrafine magnetite [magnetic P(St/NIPAM/MAA)] were prepared by two-step emulsifier-free emulsion polymerization. The minimum NaCl concentration for flocculation of these magnetic latex particles (critical flocculation concentration, CFC) decreased with increasing temperature. These temperature dependence of CFC, namely its thermo-sensitivity, originated from NIPAM. At a certain NaCl concentration, some of the magnetic latex particles showed reversible transition between flocculation and dispersion by controlling the temperature, and the thermo-flocculated magnetic latex particles were separated quickly in a magnetic field. Bovine serum albumin (BSA) was covalently immobilized onto the magnetic P(St/NIPAM/MAA) latex particles with high efficiency by the carbodiimide method. These thermo-sensitive magnetic immunomicrospheres were effective for the immunoaffinity purification of anti-BSA antibodies from antiserum. © 1994 Springer-Verlag.
    Springer-Verlag, Mar. 1994, Applied Microbiology and Biotechnology, 41(1) (1), 99 - 105, English
    [Refereed]
    Scientific journal

  • Ko Higashitani, Takuya Yamamura, Yumiko Isshiki, Akiko Kage, Akihiko Kondo
    Composite particles are formed using negative and amphoteric monodispersed latices as the core and shell respectively, and the experimental conditions to obtain maximum coverage of the surface of core particles with amphoteric particles and the mechanism of the deposition process are investigated. As results, (1) the experimental conditions for forming the composite particles are clarified (2) the maximum coverage of the core surface with amphoteric particles is found not to be greater than 0.3 and (3) the coverage is mainly determined by the strength of the repulsive force between deposited particles. The relation between the coverage and the zeta potential of composite particles is also clarified. © 1994, Hosokawa Powder Technology Foundation. All rights reserved.
    1994, KONA Powder and Particle Journal, 12, 119 - 124, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Tetsuya Kaneko, Ko Higashitani
    1994, Biotechnology and Bioengineering, 44(3) (3), 395 - 395, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Tetsuya Kaneko, Ko Higashitani
    The latex particles composed of poly(styrene/N‐isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N‐isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier‐free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo‐sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo‐sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo‐sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA‐immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo‐sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti‐BSA antibodies from antiserum. © 1994 John Wiley & Sons, Inc. Copyright © 1994 John Wiley & Sons, Inc.
    1994, Biotechnology and Bioengineering, 44(1) (1), 1 - 6, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Takafumi Urabe, Ko Higashitani
    Bioconversions of corn starch and bovine hemoglobin in an aqueous two-phase system using α-amylase and papain immobilized on ultrafine silica particles (average diameter 15 nm) were studied. Both α-amylase and papain were immobilized onto the ultrafine silica particles with high efficiency by covalent cross-linking with glutaraldehyde, and both enzymes showed high activities. Since these immobilized enzymes were totally partitioned to the polyethylene glycol (PEG)-rich top phase in PEG/dextran aqueous two phase systems, the products were recovered from the bottom phase. These reaction systems were found to be effective for extractive bioconversions. © 1994.
    1994, Journal of Fermentation and Bioengineering, 77(6) (6), 700 - 703, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Kouichi Imura, Kouzo Nakama, Ko Higashitani
    Poly(methyl methacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles were prepared by emulsifier-free emulsion polymerization. These submicrometer-size latex particles showed a reversible transition between dispersion and flocculation (thermoflocculation) by lowering and raising temperature. This thermosensitivity originated from the thermosensitive nature of NIPAM. Papain was covalently immobilized onto the thermosensitive latex particles with high efficiency by the carbodiimide method. Moreover, the latex particles with covalently immobilized papain were separated by thermoflocculation, dispersed by lowering temperature and reused. Papain immobolized on the P(MMA/NIPAM/MAA) latex particles showed an extremely high activity toward casein and retained the activity without any definite loss during repeated thermocytes. © 1994.
    1994, Journal of Fermentation and Bioengineering, 78(3) (3), 241 - 245, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Shuji Uchimura, Ko Higashitani
    Carboxylated poly(styrene/acrylamide) [P(St/AAm)-H] latex particles with relatively high hydrophilicity and colloidal stability were prepared by emulsifier-free emulsion polymerization. Various amounts of antibodies against hen egg-white lysozyme (HEL), bakers yeast invertase, ovalbumin and bovine serum albumin (BSA) were covalently immobilized onto the latex particles by the carbodiimide method. The initial rates of the immunological agglutination of these antibody-immobilized latex particles by the addition of antigens were quantified by the absorbance changes at a wavelength of 680 nm. The immunological agglutination rates showed a similar tendency irrespective of the antigen-antibody system. That is, the initial agglutination rate (i) increased with an increasing amount of immobilized antibody, (ii) was largest at an ionic strength of approximately 0.1 at pH 7.0, and (iii) decreased with increasing pH. These tendencies were similar to those in the immunological agglutination of antigen-immobilized latex particles by the addition of antibodies. Based on these results, the main factors which control immunological agglutination rates are discussed. © 1994.
    1994, Journal of Fermentation and Bioengineering, 78(2) (2), 164 - 169, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Tetsuya Kaneko, Ko Higashitani
    Affinity microspheres were prepared by immobilizing human γ-globulin (HγGb) onto carboxylated poly (styrene/acrylamide) latex particles [P(St/AAm)-H average diameter 0.33 μm], which were prepared by emulsifier-free emulsion polymerization. HγGB was covalently immobilized onto the latex particles with high efficiency by the carbodiimide method. A fusion protein (ZZB1B2) of immunoglobulin G and albumin-binding domains (ZZ and B1B2, respectively) was expressed intracellularly and extracellularly in Escherichia coli and was purified by the affinity microspheres. In poly (ethylene glycol) (PEG)/potassium phosphate aqueous two-phase system, the affinity microspheres were partitioned into the PEG-rich top phase, while cells and cell debris of E. coli were displaced into the salt-rich bottom phase. Therefore, ZZB1B2 was directly purified from cell disintegrate or culture broth by combining the affinity microspheres with the aqueous two-phase partitioning, and its purity was almost the same as that purified by conventional affinity chromatography. Therefore, by this purification method, the primary purification process and the subsequent high resolution purification process are combined, and the number of purification steps can be reduced. © 1993 Springer-Verlag.
    Springer-Verlag, Nov. 1993, Applied Microbiology and Biotechnology, 40(2-3) (2-3), 365 - 369, English
    [Refereed]
    Scientific journal

  • Kohji Yoshinaga, Akihiko Kondo, Ko Higashitani, Taketoshi Kito
    Surface modifications of monodispersed colloidal silica with poly(ethylene glycol) (PEG-COOH) silane coupling agents having a carboxyl end group, the successive immobilization of bovine serum albumin (BSA) and the application of the protein composites as an immunolatex were investigated. The reaction of the colloidal silica suspended in ethanol with the PEG-COOH coupling agents in the presence of small amounts of aqueous ammonia successfully gave PEG-COOH/SiO2 composites, retaining the particle size. Covalent binding of BSA to PEG/SiO2 composites was achieved by an activated ester method in the presence of an excess of the protein in pH 7.0 buffer. The agglutination tests using the BSA-PEG/SiO2 particles with anti-BSA antibody and γ-globulin derived from rabbits suggested applicability of the protein-PEG/SiO2 composites as immunoassay latexes. © 1993.
    Sep. 1993, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 77(2) (2), 101 - 107, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Shinya Oku, Fumiyasu Murakami, Ko Higashitani
    Conformational changes in hemoglobin (a "soft" protein) and catalase (a relatively "soft" protein) upon adsorption on ultrafine silica particles (average diameter 15 nm) have been studied by circular dichroism (CD). Because the light scattering intensity of the ultrafine silica particles is very low, the secondary structure of protein molecules on solid surfaces can be estimated from the CD spectra of the suspension of the ultrafine silica particles on which proteins are adsorbed. The extent of the changes in the secondary structure of both the protein molecules was found to increase with decreasing pH and adsorbed amount. Hemoglobin showed the changes in the secondary structure up to high pH and high adsorbed amount. Therefore, soft proteins are susceptible to conformational changes upon adsorption on solid surfaces. These CD spectrum data give us some quantitative information about the conformational changes in protein molecules upon adsorption on solid surfaces. © 1993.
    Aug. 1993, Colloids and Surfaces B: Biointerfaces, 1(3) (3), 197 - 201, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Fumiyasu Murakami, Masako Kawagoe, Ko Higashitani
    Negatively charged ultrafine silica particles (average diameter 20 nm) were used as support materials for adsorption immobilization of porcine trypsin, horseradish peroxidase, and bovine catalase under various conditions, and the changes in the enzyme activities and the circular dichroism (CD) spectra of these enzymes upon adsorption were measured. Since the light scattering intensity of the ultrafine particles was very low, the activities and the CD spectra of the enzymes adsorbed on the particle surfaces could be measured. The enzymes adsorbed at pH around and above their isoelectric points (pI) showed high activities. On the other hand, the enzymes adsorbed at pHs below their pI had significantly diminished activities and showed large CD spectral changes upon adsorption. The extent of CD spectral changes in the enzymes upon adsorption correlated very closely with that of the activity reduction. Therefore, the conformational changes in enzymes upon adsorption are one of the important factors that reduce the activities of adsorbed enzymes. These results demonstrate that the ultrafine particles are not only a novel support for enzyme immobilization but also are helpful for the molecular understanding of the immobilized enzymes. © 1993 Springer-Verlag.
    Springer-Verlag, Aug. 1993, Applied Microbiology and Biotechnology, 39(6) (6), 726 - 731, English
    [Refereed]
    Scientific journal

  • STABILITY OF ANTIBODY FRAGMENTS SECRETED TO THE CULTURE MEDICUM OF ESCHERICHIA-COLI
    Akihiko Kondo
    1993, Protein engineering
    Scientific journal

  • Hideto Matsuyama, Takashi Yamamoto, Setsuo Furuyoshi, Masaaki Teramoto, Akihiko Kondo
    1993, Bioscience, Biotechnology and Biochemistry, 57(6) (6), 992 - 993, English
    [Refereed]

  • Ko Hierashitani, Takuya Yamamura, Yumiko Isshiki, Akiko Kage, Akihiko Kondo
    Composite particles are formed using negative and amphoteric monodispersed latices as the core and shell respectively, and the experimental conditions to gain maximum coverage of the surface of core particles with amphoteric particles and the mechanism of the deposition process are investigated. As the results, (1) the experimental conditions for forming the composite particles are clarified (2)the maximum coverage of the core surface with amphoteric particles is found not to be greater than 0.3 and (3) the coverage is mainly determined by the strength of the repulsive force between deposited particles. The relation between the coverage and the zeta potential of composite particles is also clarified. © 1992, The Society of Chemical Engineers, Japan. All rights reserved.
    1992, KAGAKU KOGAKU RONBUNSHU, 18(5) (5), 637 - 642, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Katsuhiro Yoshioka, Ko Higashitani
    The effect of copolymer composition on adsorption and covalent immobilization of γ-globulin (γ-Gb), a model protein of antibodies, on microspheres was studied by using styrene/methacrylic acid [P(St/MAA)] and styrene/2-hydroxyethyl methacrylate [P(St/HEMA)] microspheres synthesized by emulsifier free emulsion polymerization. Amounts of γ-Gb adsorbed on microspheres with 11.5 and 13.8mol% of copolymerized hydrophilic MAA and HEMA respectively were low compared with the PSt microspheres. Moreover, adsorption of γ-Gb on these hydrophilic microspheres was highly reversible, while that with the PSt microspheres was highly irreversible. On the other hand, γ-Gb was covalently immobilized on the P(St/MAA) microspheres by the carbodiimide method and on the P(St/HEMA) microspheres by the CNBr method. In particular, the microspheres with 11.5% of MAA immobilized r-Gb with high efficiency by the carbodiimide method and hence is suitable for covalent immobilization of antibodies. © 1992, The Society of Chemical Engineers, Japan. All rights reserved.
    1992, KAGAKU KOGAKU RONBUNSHU, 18(3) (3), 353 - 359, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Ko Higashitani
    To elucidate some general principles of protein adsorption, the adsorption isotherms of ribonuclease A (RNase A), cytochrome c, lysozyme, α-lactalbumin, ovalbumin, and bovine serum albumin (BSA) on colloidal particles of polystyrene, styrene/2-hydroxyethyl methacrylate, and silica were measured as a function of pH and ionic strength. These proteins were different, especially with respect to molecular flexibility. All adsorption isotherms showed plateaus. While affinities of all proteins for hydrophilic particles decreased significantly with increasing pH, those for hydrophobic particles were high irrespective of pH. At an ionic strength of 0.01, the pH dependence of the plateau adsorption of small proteins (RNase A, cytochrome c, lysozyme, and α-lactalbumin) was affected by the surface properties of the particles, while that of large proteins (ovalbumin and BSA) showed the maximum at around their isoelectric points irrespective of the type of particles. On the other hand, the pH dependence of the plateau adsorption was related to the surface properties at an ionic strength of 0.1 irrespective of the type of protein. The above dependence of protein adsorption on properties of solid surfaces and adsorption conditions did not vary with the molecular flexibility of the proteins, although the structural changes in the flexible protein molecules upon adsorption promoted adsorption. Therefore, both the protein-surface interactions, such as hydrophobic and electrostatic ones, and the lateral interaction between adsorbed molecules are dominant factors that control protein adsorption on these particles. © 1992.
    1992, Journal of Colloid And Interface Science, 150(2) (2), 344 - 351, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Ryo Yamasaki, Ko Higashitani
    Antigen-immobilized colloidal particles (immunomicrospheres) of submicrometer size were used for the immunoaffinity separation of antibodies. Carboxylated latex particles (P(St/AAm)-H) were prepared by copolymerization of styrene (St)/acrylamide (AAm) and by subsequent hydrolysis and were coupled covalently with antigen (bovine serum albumin BSA) by the carbodiimide method. The anti-BSA antibody binding capacity of the immunomicrospheres increased with increasing amount of immobilized BSA up to the value which corresponds to the high packing density of the antibody molecules on the surface. The binding ratios of the immunomicrospheres, defined as the ratio of mol antibody adsorbed to mol BSA immobilized, were comparative to those of the conventional immunoadsorbents for affinity chromatography and did not decrease during repeated cycles of adsorption and desorption. By use of the immunomicrospheres, the antibodies were successfully purified from antisera. © 1992.
    1992, Journal of Fermentation and Bioengineering, 74(4) (4), 226 - 229, English
    [Refereed]
    Scientific journal

  • Ko Higashitani, Nobuhisa Tanise, Atsuro Yoshiba, Akihiko Kondo
    The breakup of flocs composed of a small number of constituent particles by orifice contractile flow is investigated extensively by measuring the size of broken flocs of PSL particles on a Coulter counter. It is found that (1) flocs of maximum strength are obtained when they are formed in an electrolyte concentration greater than the critical coagulation concentration, and the strength is independent of the valency of electrolyte (2) the average size of broken flocs and the maximum number of constituent particles in a broken floc are successfully expressed as a function of the energy dissipation at the orifice and (3) the mechanism of floc breakup in the contractile flow is different from that by ultrasonication (flocs are split into smaller agglomerates in the former case, but particles are ripped off one by one from the surface of flocs in the latter case). © 1992, The Society of Chemical Engineers, Japan. All rights reserved.
    1992, Journal of Chemical Engineering of Japan, 25(5) (5), 502 - 507, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Fumiyasu Murakami, Ko Higashitani
    The conformational changes in well‐characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm‐whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the “soft” protein molecules (myoglobin, hemoglobin, and BSA), while they were insingnificant in the “rigid” proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in α‐helix content upon adsorption. Moreover, the native α‐helix content, which is given as the percentage of the α‐helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well‐known hypotheses for the confirmational chages in protein molecules upon adsorption. © 1992 John Wiley & Sons, Inc. Copyright © 1992 John Wiley & Sons, Inc.
    1992, Biotechnology and Bioengineering, 40(8) (8), 889 - 894, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Takeharu Kawano, Ko Higashitani
    Hen egg-white lysozyme (HEL), ovalbumin and bovine serum albumin (BSA) were covalently immobilized onto styrene/methacrylic acid [P(St/MAA)] copolymer latex particles by the carbodiimide method. The initial rates of the immunological agglutination of these particles initiated by the addition of antibodies were quantified by the absorbance changes at a wavelength of 680 nm. The sensitivity of the immunological agglutination of the particles with covalently immobilized antigens was higher than that with physically adsorbed ones. The immunological agglutination kinetics showed a similar tendency irrespective of antigen-antibody systems. That is, the initial agglutination rates (i) increased with increasing immobilized amount of antigens, (ii) were largest in the ionic strength range of 0.02 to 0.05 at pH 7 and (iii) decreased with increasing pH. These results indicate that the electrostatic interactions of particle-particle and particle-antibody are main factors which control the immunological agglutination. On the other hand, the sensitivity of the immunological agglutination increased with increasing molecular size of antigens. © 1992.
    1992, Journal of Fermentation and Bioengineering, 73(6) (6), 435 - 439, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Shinya Oku, Ko Higashitani
    The structural changes in horse cytochrome c, bovine ribonuclease A (RNase A), sperm whale myoglobin, ovalbumin, human hemoglobin, and bovine serum albumin (BSA) during adsorption on ultrafine silica particles have been studied. Since the intensity of light scattered by the ultrafine silica particles (average diameter 15 nm) is negligible, the structure of adsorbed protein molecules on the surface is directly estimated by measuring the circular dichroism (CD) spectra of the suspension of particles on which protein molecules were adsorbed. While the CD spectra of soft proteins such as hemoglobin and BSA were changed extensively by adsorption on ultrafine silica particles, those of rigid proteins such as cytochrome c and RNAse A were changed little. Changes in the CD spectrum of BSA during adsorption increased with decreasing pH. Thus, the magnitude of the structural changes is affected by both the flexibility of the protein molecules and the affinities of the particles for proteins. The BSA molecules desorbed from the ultrafine silica particles by addition of morpholine showed CD spectra similar to those of native BSA and hence were refolded. Thus, the conformational changes in BSA molecules produced by adsorption were highly reversible. The adsorption amounts of all these proteins on ultrafine silica particles were maximum at around their isoelectric points regardless of structural adaptability. © 1991 Academic Press, Inc. All rights reserved.
    1991, Journal of Colloid And Interface Science, 143(1) (1), 214 - 221, English
    [Refereed]
    Scientific journal

  • Kristina Köhler, Charlotte Ljungquist, Akihiko Kondo, Andres Veide, Björn Nilsson
    We describe a novel method to partition recombinant proteins into the polymer-rich top phase in poly(ethylene glycol) (PEG)4000/potassium phosphate aqueous two-phase systems. The concept is based on fusion of a gene fragment encoding a short peptide sequence to the product gene of interest thereby changing the partitioning properties of the expressed product protein as a fusion to the peptide. The model protein in this study, ZZ, is a two domain molecule based on staphylococcal protein A (SPA) which distributes evenly in PEG/salt systems. A tetrapeptide sequence, AlaTrpTrpPro (designated the partitioning peptide), was designed by molecular modeling techniques to include exposed tryptophan residues and to have a coding DNA sequence which is possible to polymerize in an obligate head-to-tail fashion at the DNA level. Gene fragments encoding one and three partitioning peptides, respectively, were fused to the 3′ end of the ZZ gene and the fusion proteins were produced intracellularly in Escherichia coli. The partition coefficients of ZZ proteins containing zero, one and three fused partitioning peptides were determined in three PEG 4000/potassium phosphate aqueous two-phase systems of different compositions. In all three phase systems, there were dramatic effects on the partition coefficient by the fused partitioning peptides. In the phase system with the largest effects, the partition coefficient was enhanced from 1.6 to 11.6 by fusing one tetrapeptide sequence to the 147 amino acid model ZZ protein. By the fusion of three partitioning peptides, the coefficient was increased to 96. These results show that fused partitioning peptides have a dramatic effect on the partitioning of a protein in aqueous two-phase systems which may open up new possibilities to utilize aqueous two-phase extraction techniques as a primary recovery step of recombinant proteins.
    Presse Dienstleistungsgesellschaft mbH und Co. KG, 1991, Nature Biotechnology, 9(7) (7), 642 - 646, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Shinichi Furukawa, Masato Taira, Ko Higashitani
    The antibodies to hydrophilic and hydrophobic peptides representing residues 85-94 of cytochrome c (P1) and 148-158 of tobacco mosaic virus protein (P2), respectively, were produced in rabbits. Peptide-keyhole limpets hemocyanin (KLH) conjugates and peptide-polyvinylpyrrolidone (PVP) mixtures were used for immunization. From the binding experiments of analogous synthetic peptides in immunoadsorbents, the major fractions of all the polyclonal anti-peptide antibodies were found to bind to the antigenic determinants localized in the four to six C-terminal amino acids of the peptides. In both the anti-P1 and anti-P2 antibodies produced by immunizing peptide-KLH conjugates, the C-terminal carboxylic acid group was essential for antibody binding. On the other hand, the effect of pH and ionic strength on the adsorbed amount of the antibodies depended on the peptide. That is, the adsorbed amount of P1 to both the anti-P1 antibodies produced by the different immunization methods decreased significantly with increasing ionic strength, while the adsorbed amount of P2 to the antibodies exhibited a minimum at around ionic strength 1.0. These results indicate that the dependence of antibody affinities on pH and ionic strength is related to the properties of antigenic determinants. © 1991.
    1991, Journal of Fermentation and Bioengineering, 72(6) (6), 409 - 412, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Shinya Oku, Ko Higashitani
    The effect of surface properties on the adsorption of bovine γ‐globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2‐hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2‐hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of γ‐globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for γ‐globulin at alkaline pH, while PS showed high affinities for γ‐globulin over the whole range of pH and ionic strength. The γ‐globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of γ‐globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength. Copyright © 1991 John Wiley & Sons, Inc.
    1991, Biotechnology and Bioengineering, 37(6) (6), 537 - 543, English
    [Refereed]
    Scientific journal

  • Kristina Köhler, Charlotta Ljungquist, Akihiko Kondo, Andres Veide, Björn Nilsson
    We describe a novel method to partition recombinant proteins into the polymerrich top phase in poly(ethylene glycol) (PEG)4000/potassium phosphate aqueous two-phase systems. The concept is based on fusion of a gene fragment encoding a short peptide sequence to the product gene of interest thereby changing the partitioning properties of the expressed product protein as a fusion to the peptide. The model protein in this study, ZZ, is a two domain molecule based on staphylococcal protein A (SPA) which distributes evenly in PEG/salt systems. A tetrapeptide sequence, AlaTrpTrpPro (designated the partitioning peptide), was designed by molecular modeling techniques to include exposed tryptophan residues and to have a coding DNA sequence which is possible to polymerize in an obligate head-to-tail fashion at the DNA level. Gene fragments encoding one and three partitioning peptides, respectively, were fused to the 3′ end of the ZZ gene and the fusion proteins were produced intracellularly in Escherichia coli. The partition coefficients of ZZ proteins containing zero, one and three fused partitioning peptides were determined in three PEG 4000/potassium phosphate aqueous two-phase systems of different compositions. In all three phase systems, there were dramatic effects on the partition coefficient by the fused partitioning peptides. In the phase system with the largest effects, the partition coefficient was enhanced from 1.6 to 11.6 by fusing one tetrapeptide sequence to the 147 amino acid model ZZ protein. By the fusion of three partitioning peptides, the coefficient was increased to 96. These results show that fused partitioning peptides have a dramatic effect on the partitioning of a protein in aqueous two-phase systems which may open up new possibilities to utilize aqueous two-phase extraction techniques as a primary recovery step of recombinant proteins. © 1991 Nature Publishing Group.
    1991, Bio/Technology, 9(7) (7), 642 - 646, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Takeharu Kawano, Fumio Itoh, Ko Higashitani
    The influence of the properties of antigens and particles on the immunological agglutination kinetics of the antigen-coated latex particles was studied. Horse cytochrome c, hen egg-white lysozyme (HEL), bovine serum albumin (BSA), and Aspergillus sp. glucose oxidase were physically adsorbed onto the surfactant free latices of styrene-methacrylic acid (MAA) copolymer (p(S/MAA)) and polystyrene (PS). The initial rates of the immunological agglutination of these protein-coated particles initiated by the addition of antibodies were quantified by the absorbance change at a wavelength of 680 nm. The initial agglutination rates of the particles covered with smaller antigens were lower. This effect of the molecular size of antigens was larger in P(S/MAA), because small antigens are probably buried in the hydrous polymethacrylic acid layer on the surface of particles. Thus, both the molecular size of antigens and the surface properties of particles affect the sensitivity of the immunological agglutination. On the other hand, the dependence of the initial rate of the immunological agglutination on the ionic strength and pH was similar irrespective of antigen-particle systems. The initial agglutination rates were largest at an ionic strength of approximately 0.05 at pH 7.0 and decreased with increasing pH. This dependence of the sensitivity on the pH and ionic strength is attributed to the electrostatic interactions of particle-particle and antibody-particle. © 1990.
    Dec. 1990, Journal of Immunological Methods, 135(1-2) (1-2), 111 - 119, English
    [Refereed]
    Scientific journal

  • EIZO SADA, SHIGEO KATOH, AKIHIKO KONDO, YOSHINORI SOHMA
    1990, Annals of the New York Academy of Sciences, 613(1) (1), 874 - 877, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Hiroyuki Takamatsu, Shigeo Katoh, Eizo Sada
    The effects of charged residues in peptide antigens on the binding characteristics of polyclonal antipeptide antibodies were studied using immunoadsorbents prepared by coupling the antibodies to CNBr‐activated Sepharose 4B. Among the antipeptide antibodies, an antibody to the peptide without charged residues showed the most stable interaction with the peptide to the changes in pH. Conversely, the binding affinity of antibodies to the pep‐tides with histidine residues having a unique pKa value of 6.0 decreased steeply with pH at around 6.0. The binding affinity of an antibody to the peptide with many charged residues decreased steeply with an increase in the ionic strength (adjusted by NaCl). Since circular dichroism (CD) spectrum measurements indicate that these peptides show disordered structures in the pH range of adsorption measurement, the dependence of peptide‐antibody interaction on environmental conditions is attributed to the characteristics of side chains of the peptides. These results indicate that the dependence of the binding affinity of antipeptide antibodies on pH and the ionic strength is dominantly affected by the number and the pKa values of charged residues in the peptides. Copyright © 1990 John Wiley & Sons, Inc.
    1990, Biotechnology and Bioengineering, 35(2) (2), 146 - 151, English
    [Refereed]
    Scientific journal

  • Akihiko Kondo, Masaaki Kishimura, Shigeo Katoh, Eizo Sada
    Immunoadsorbents were modified with monomethoxy‐polyethylene glycol (PEG average molecular weights of 5000 (PEG‐5000) and 1900 (PEG‐1900)) activated with cyanuric acid (activated PEG) by four different methods. In the two methods, anti‐BSA antibodies were modified with activated PEG with and without protection of antigen binding sites with BSA and then were coupled to CNBr‐activated Sepharose 4B. In the other two methods, Immunoadsorbents, which were prepared by coupling anti‐BSA antibodies to CNBr‐activated Sepharose 4B, were modified with activated PEG with and without the protection. The effects of PEG modification by these four methods on the binding ratio (the ratio of the numbers of moles of antigen adsorbed to the numbers of moles of binding sites of antibody coupled), the antigen binding property and the resistance to proteolytic digestion of immunoadsorbents were studied. The decrease in the binding ratio by the modification with activated PEG was small enough to use modified immunoadsorbents for industrial purification processes. The resistance to proteolytic digestion of immunoadsorbents was improved by modification with activated PEG. The modification without protection of antigen binding sites gave higher resistance to proteolytic digestion than that with protection, while the former caused larger decrease in the binding ratio of modification. The immunoadsorbents modified with activated PEG‐5000 showed higher resistance to proteolytic digestion than those modified with activated PEG‐1900. Copyright © 1989 John Wiley & Sons, Inc.
    1989, Biotechnology and Bioengineering, 34(4) (4), 532 - 540, English
    [Refereed]
    Scientific journal

  • Eizo Sada, Shigeo Katoh, Atsuo Kiyokawa, Akihiko Kondo
    Polyclonal anti-myoglobin antibodies were fractionated into five subpopulations directed against five specific antigenic sites, respectively. The equilibrium characteristics of each subpopulation and orginal anti-myoglobin immobilized to CNBr-activated Sepharose 4B were compared. The four subpopulations of antibodies lost their binding abilities at around pH 4.5 because of the conformational changes of myoglobin. However, the subpopulation directed against the region containing three histidine residues dissociated with the antigenic site at higher pH, and such equilibrium characteristics were considered to be caused by the dissociation characteristics of histidine residues. Therefore, the effects of histidine modification in BSA on the adsorption capacities of original anti-BSA antibody and a pH sensitive fraction of it were compared. The adsorption capacity of the pH sensitive fraction showed greater decrease than that of original antibody by histidine modification in BSA. These results imply that the antigenic sites in which histidine residues play an important role for the binding to antibodies show equilibrium characteristics sensitive to pH. © 1988 Springer-Verlag.
    Springer-Verlag, Feb. 1988, Applied Microbiology and Biotechnology, 27(5-6) (5-6), 528 - 532, English
    [Refereed]
    Scientific journal

  • Eizo Sada, Shigeo Katoh, Hirotoshi Miyoshi, Kuniko Yamanaka, Akihiko Kondo
    Antisera against charged (p‐azobenzoate and p‐azoben zenesulfonate) and uncharged (dinitrophenyl) haptenic groups were produced in rabbits, and the equilibrium characteristics of hapten‐antibody were measured by use of immunoadsorbents. The antibody to the uncharged hapten formed a stable binding with the hapten to the changes in ionic strength and pH. On the other hand, the antibodies to the charged haptens showed affinities sensitive to the changes in pH and ionic strength. Therefore, the effect of the pKa of ionizable haptens on the pH dependence of the hapten‐antibody binding was studied by comparing the interactions between a series of para‐substituted benzoic acids and the anti‐p‐azobenzoate antibody. The pH dependence of the interactions was strongly affected by the pKa of ionizable groups in haptens. Furthermore, the equilibrium characteristics of anti‐p‐aminobenzoyl dipeptides were compared. The characteristics of interactions were affected by the features of amino acid residues. Copyright © 1988 John Wiley & Sons, Inc.
    1988, Biotechnology and Bioengineering, 32(4) (4), 467 - 474, English
    [Refereed]
    Scientific journal

  • Eizo Sada, Shigeo Katoh, Atsuo Kiyokawa, Akihiko Kondo
    Polyclonal anti‐BSA antibodies were ractionated by stepwise elution from an immobilized BSA column by decreasing pH or increasing the concentration of NaSCN. The binding affinities of each fraction and original globulin under physiological conditions and their dependence on pH and ionic environments were compared. Fractions with high association constant under physiological conditions did not necessarily show antigen binding affinity over a wide pH range, but they retained a high affinity at higher ionic strength of NaSCN. Consequently, by combining these two fractionation procedures, a fraction with high affinity and which dissociated at moderate pH was obtained. It is clear that high affinity is not always incompatible with ease of dissociation accompanying a change in conditions. Copyright © 1988 John Wiley & Sons, Inc.
    1988, Biotechnology and Bioengineering, 31(7) (7), 635 - 642, English
    [Refereed]
    Scientific journal

  • Eizo Sada, Shigeo Katoh, Akihiko Kondo, Seiji Ishida
    Two columns of 100 mm and 200 mm in diameter for bench-scale liquid chromatography were designed to obtain a small dead volume and an appropriately uniform distribution of liquid, and the values of the height equivalent to a theoretical plate were measured for three kinds of packing beads, Sepharose 4B, Cellulofine GC-700 m and Silica. A suitable pressure drop through a distribution plate and/or a packed bed helps to distribute liquid flow uniformly over the column diameter, and reasonable HETP values were obtained. © 1987.
    1987, Journal of Fermentation Technology, 65(1) (1), 111 - 113, English
    [Refereed]
    Scientific journal

  • Eizo Sada, Shigeo Katoh, Akihiko Kondo, Atsuo Kiyokawa
    The effects of ligand concentration and coupling method on the adsorption equilibrium in immuno-affinity chromatography were studied by use of polyclonal anti-BSA antibody and Fab fragments. Immobilized IgG and Fab fragments showed similar adsorption behavior, and the adsorption equilibrium was not affected by the coupling method or the concentration of immobilized ligands. However, the binding ratio, defined as the ratio of mole antigen adsorbed to mole binding site, depended on the coupling method and decreased with increasing ligand concentration. © 1986, The Society of Chemical Engineers, Japan. All rights reserved.
    1986, JOURNAL OF CHEMICAL ENGINEERING OF JAPAN, 19(6) (6), 502 - 506, English
    [Refereed]
    Scientific journal

  • Eizo Sada, Shigeo Katoh, Kiyoshi Sukai, Masaaki Tohma, Akihiko Kondo
    The effects of pH, ionic strength, anion species, and antibody concentration on the adsorption equilibrium between immobilized antibodies and antigens were studied by use of anti‐BSA, anti‐HSA, anti‐BlgG, and monoclonal anti‐HSA coupled to Sepharose 4B. The polyclonal antibodies possessed average binding affinities of the order of 108M−1, and the heterogeneity was accounted for by assuming a normal distribution of the free energy of antibody–antigen combination. The monoclonal antibody, on the other hand, showed a homogeneous affinity of the Langmuir type. Bound antigens could be eluted by lowering pH or adding a chaotropic anion, and their purity was very high. The antibody ligand was sufficiently stable for repeated use. Copyright © 1986 John Wiley & Sons, Inc.
    1986, Biotechnology and Bioengineering, 28(10) (10), 1497 - 1502, English
    [Refereed]
    Scientific journal

■ MISC
  • Development of a highly accurate and high-throughput in vitro human colonic microbiota culture model
    佐々木大介, 佐々木大介, 新谷知也, 松木泰, 近藤昭彦
    2024, 腸内細菌学雑誌, 38(2) (2)

  • ピキア酵母の多重遺伝子欠損による難分泌性抗体タンパク質の生産性向上
    伊藤洋一郎, 石井純, 近藤昭彦
    May 2023, Bioscience & Industry, 81(3) (3), 234 - 236, Japanese
    Introduction scientific journal

  • Maintaining downstream glycolytic intermediates ensures rapid start of photosynthesis in cyanobacteria
    田中謙也, 田中謙也, 田中謙也, 松田真実, 白井智量, 白井智量, 近藤昭彦, 近藤昭彦, 近藤昭彦, 蓮沼誠久, 蓮沼誠久, 蓮沼誠久
    2023, 日本植物生理学会年会(Web), 64th

  • バイオ物質生産に資するスマートセル創出に向けた革新的アプローチ
    近藤昭彦
    Jan. 2023, 生物工学会誌, 101(1) (1), 2 - 14, Japanese
    Introduction scientific journal

  • 特集 CO2から有用物質を生産し低炭素社会実現へ:「バイオものづくり」が導く低炭素社会
    近藤昭彦
    Jan. 2023, JST news, 1, 4 - 5, Japanese
    Introduction scientific journal

  • Filemon Jalu Nusantara Putra, Prihardi Kahar, Akihiko Kondo, Chiaki Ogino
    As the third most plentiful biopolymer after other lignocellulosic derivates such as cellulose and hemicellulose, lignin carries abundant potential as a substitute for petroleum-based products. However, the efficient, practical, value-added product valorization of lignin remains quite challenging. Although several studies have reviewed the valorization of lignin by microorganisms, this present review covers recent studies on the valorization of lignin by employing yeast to obtain products such as single-cell oils (SCOs), enzymes, and other chemical compounds. The use of yeasts has been found to be suitable for the biological conversion of lignin and might provide new insights for future research to develop a yeast strain for lignin to produce other valuable chemical compounds.
    MDPI AG, 04 Oct. 2022, Processes, 10(10) (10), 2004 - 2004, English
    [Refereed]

  • 第3章 生物工学の研究100年 育種技術 -突然変異から代謝工学へ-
    近藤昭彦, 蓮沼誠久, 秀瀬涼太
    Oct. 2022, 日本生物工学会100年史, 29 - 31, Japanese
    Introduction scientific journal

  • バイオエコノミーの展望
    近藤昭彦, 荻野千秋
    Apr. 2022, 化学工学 [特集]「生物機能を利用したモノづくり」に貢献するプロセス強化, 86(4) (4), 151 - 152, Japanese
    Introduction scientific journal

  • Development of high fucoxanthin production technology using Haptophyta Pavlova sp. based on metabolome knowledges
    吉田江里菜, 加藤悠一, 金本昭彦, 近藤昭彦, 近藤昭彦, 近藤昭彦, 蓮沼誠久, 蓮沼誠久
    2022, 日本生物工学会大会講演要旨集, 74th

  • Development of novel cyanobacteria with L-lactate utilization ability
    加藤悠一, 稲辺宏輔, 辻彩花, 原口裕次, 清水達也, 近藤昭彦, 近藤昭彦, 蓮沼誠久, 蓮沼誠久
    2022, 日本生物工学会大会講演要旨集, 74th

  • A quantitative characterization of rapid metabolic dynamics during activation of Calvin cycle in cyanobacteria
    田中謙也, 白井智量, 白井智量, 松田真実, 近藤昭彦, 近藤昭彦, 近藤昭彦, 蓮沼誠久, 蓮沼誠久, 蓮沼誠久
    2022, 日本生物工学会大会講演要旨集, 74th

  • Development of aldehyde-deformylating oxygenase with substrate specificificity toward various carbon chain length aldehydes
    工藤恒, VAVRICKA Christopher J., 伏見圭司, 蓮沼誠久, 近藤昭彦
    2022, 日本生物工学会大会講演要旨集, 74th

  • Development of alpha-keto acid decarboxylase for tailor-made bio-production
    伏見圭司, 秀瀬涼太, 秀瀬涼太, VAVRICKA Christopher J., 工藤恒, 蓮沼誠久, 蓮沼誠久, 近藤昭彦, 近藤昭彦
    2022, 日本生物工学会大会講演要旨集, 74th

  • Cost-effective production of D-lactic acid from corncobs by simultaneous saccharification and fermentation using genetically engineered lactic acid bacteria
    久保早友理, 熊田陽一, 堀内淳一, 岡野憲司, 近藤昭彦, 田中勉
    2022, 日本生物工学会大会講演要旨集, 74th

  • 酵母細胞を用いたG蛋白質共役型受容体リガンドスクリーニングシステムの開発とメタボライトセンサへの応用
    中村泰之, 石井純, 近藤昭彦
    Dec. 2021, Precision Medicine, 4(13) (13), 60 - 65, Japanese
    Introduction scientific journal

  • 酵母細胞を用いたG蛋白質共役型受容体リガンド検出システムの開発とメタボライトセンサへの応用
    中村泰之, 近藤昭彦, 石井純
    Jul. 2021, アグリバイオ, 5(7) (7), 58 - 63, Japanese
    Introduction scientific journal

  • 電気化学的前処理/通電式担体充填による2段発酵を廃水・廃棄物処理へ適用
    佐々木建吾, 佐々木大介, 近藤昭彦
    May 2021, 化学と生物, 59(12) (12), 247 - 253, Japanese
    Introduction scientific journal

  • Development of a novel fucoxanthin production technology using Haptophyta Pavlova sp. based on the comprehensive metabolic profiling
    吉田江里菜, 加藤悠一, 金本昭彦, 蓮沼誠久, 近藤昭彦
    2021, 日本農芸化学会大会講演要旨集(Web), 2021

  • パーム油産業廃棄物POMEを原料とする微生物による酵素・化学品への変換
    荻野千秋, RACHMADORA Nova, KAHAR Prihardi, 近藤昭彦
    2021, 化学工学会年会研究発表講演要旨集(CD-ROM), 86th

  • Breeding of organic acid assimilation yeast for bio-refinery
    壷井ひかり, KAHAR Prihardi, 近藤昭彦, 荻野千秋
    2021, 日本生物工学会大会講演要旨集, 73rd

  • Polyhydroxyalkanoic acid production by an oleaginous yeast strain Lipomyces starkeyi
    中村優里, 壷井ひかり, KAHAR Prihardi, 近藤昭彦, 荻野千秋
    2021, 日本生物工学会大会講演要旨集, 73rd

  • 好冷性海洋細菌が産生するβ-1,3-キシラナーゼの機能解析
    谷口瑞季, 工藤基徳, 荻野千秋, 近藤昭彦, 田丸浩, 青木恭彦, 岡崎文美
    2020, 日本農芸化学会中部支部例会講演要旨集(Web), 187th

  • A COMPUTATIONAL TOOL TO DESIGN UNIQUE GUIDE RNAS FOR TARGET-AID MEDIATED GENOME-WIDE BASE EDITING
    Kenta Katayama, Jun Teramoto, Akihiko Kondo, Keiji Nishida
    Nov. 2019, Frontiers in Genome Engineering 2019
    [Refereed]
    Summary international conference

  • Current research topics for sustainable society by biomass and assimilanting of CO2
    Ogino Chiaki, Kondo Akihiko
    Nov. 2019, Journal of Society of Automotive Engineers of Japan, 73(11) (11), 107 - 114, Japanese
    Introduction scientific journal

  • 独自の創業支援組織をつくりバイオベンチャーを連続起業
    近藤 昭彦
    Jun. 2019, SMBSマネジメントプラス, 10 - 11, Japanese
    Introduction commerce magazine

  • 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
    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.
    01 May 2019, Nature communications, 10(1) (1), 2015 - 2015, English, International magazine

  • 日本人潰瘍性大腸炎患者の腸内細菌叢における2種の酪酸産生遺伝子の定量
    篠原 涼平, 佐々木 建吾, 井上 潤, 星 奈美子, 福田 伊津子, 佐々木 大介, 近藤 昭彦, 大澤 朗
    (公財)日本ビフィズス菌センター, Apr. 2019, 腸内細菌学雑誌, 33(2) (2), 105 - 105, Japanese

  • スマートセル開発のためのバイオ技術とデジタル技術の革新と融合 バイオ生産に資するDBTLサイクルの構築に向けて
    向山 正治, 近藤 昭彦
    Mar. 2019, バイオサイエンスとインダストリー, 77(2) (2), 199 - 201, Japanese
    Introduction scientific journal

  • 廃棄果実を用いたバイオエタノールの生産技術
    都築正男, 石井純, 近藤昭彦
    2019, 奈良県産業振興総合センター研究報告, (45) (45)

  • 油脂酵母Lipomyces starkeyiの遺伝子的改変による長鎖長脂肪酸の生産
    宮本捺央, LASTINOV Amza Rezky, KAHAR Prihardi, 荻野千秋, 近藤昭彦
    2019, 日本生物工学会大会講演要旨集, 71st

  • 好冷性海洋細菌Psychroflexus torquis由来β-1,3-キシラナーゼの酵素化学的諸性質
    谷口瑞季, 工藤基徳, 荻野千秋, 近藤昭彦, 田丸浩, 青木恭彦, 岡崎文美
    2019, 日本生物工学会大会講演要旨集, 71st

  • セルロース系バイオマスを原料とするカフェ酸発酵のためのゲノム編集による大腸菌代謝改変の作製と培養法の検討
    川口秀夫, 宮崎翔子, 寺本潤, 西田敬二, 南博道, 佐塚隆志, 近藤昭彦, 近藤昭彦
    2019, 日本生物工学会大会講演要旨集, 71st

  • in viro培養システムによる食物繊維のヒト腸管細菌叢への影響評価
    SASAKI DAISUKE, SASAKI KENGO, KONDO AKIHIKO, OSAWA RO
    Dec. 2018, ルミナコイド研究(日本食物繊維学会誌), 22(2) (2), 63 - 74, Japanese
    Introduction scientific journal

  • 多様な糖を用いた高収率物質生産技術の開発, 特集/バイオテクノロジーが拓く新世代
    猪熊 健太郎, 若井 暁, 近藤 昭彦
    Nov. 2018, ケミカルエンジニヤリング, 63(11) (11), 43 - 51, Japanese
    Introduction scientific journal

  • 機械学習を用いた酵素のEC number予測
    渡邉直暉, 村田昌浩, 荻野千秋, 近藤昭彦, 荒木通啓, 荒木通啓
    07 Aug. 2018, 日本生物工学会大会講演要旨集, 70th, 191, Japanese

  • 出芽酵母2,3‐ブタンジオール生産に向けた不活性型Cas9によるエタノール生合成経路の抑制
    森田啓介, 松田史生, 石井純, 近藤昭彦, 清水浩
    07 Aug. 2018, 日本生物工学会大会講演要旨集, 70th, 278, Japanese

  • 過酸化チタンナノ粒子の併用による放射線増感治療法の開発
    西村 勇哉, 森田 健太, 荻野 千秋, 犬伏 祥子, 佐々木 良平, 近藤 昭彦
    日本DDS学会, May 2018, 日本DDS学会学術集会プログラム予稿集, 34回, 204 - 204, Japanese

  • ミトコンドリア代謝の抑制による出芽酵母2,3‐ブタンジオール生産能力の向上
    森田啓介, 松田史生, 岡本浩二, 石井純, 近藤昭彦, 清水浩
    05 Mar. 2018, 日本農芸化学会大会講演要旨集(Web), 2018, ROMBUNNO.2A10p06 (WEB ONLY), Japanese

  • ゲノム合成の潮流のインパクト 〜微生物による物質生産〜
    TSUGE KENJI, ISHII JUN, ARAKI MICHIHIRO, KONDO AKIHIKO
    東京化学同人, Jan. 2018, 現代化学, (562) (562), 36 - 41, Japanese
    [Invited]
    Introduction commerce magazine

  • Improvement in the Bio–ethanol Production by FO Process
    SHIBUYA MASAFUMI, NAKAGAWA KEIZO, SASAKI KENGO, KONDO AKIHIKO, MATSUYAMA HIDETO
    The Membrane Society of Japan, Jan. 2018, MEMBRANE, 43(1) (1), 21 - 26, Japanese
    [Refereed][Invited]
    Introduction scientific journal

  • Ryoko Ohno, Hiroshi Teramura, Chiaki Ogino, Akihiko Kondo, Shigeo Takumi
    Jan. 2018, Wheat Information Service, 125, eWIS125.3, English
    [Refereed]
    Report scientific journal

  • 細胞系譜を一斉にトレーシングするDNAバーコードテクノロジー
    谷内江 望, 増山 七海, 石黒 宗, 森 秀人, 増井 修, 松尾 采佳, 西田 敬二, 栫 健太郎, 近藤 昭彦, 冨田 勝, 油谷 浩幸, 古関 明彦
    生命科学系学会合同年次大会運営事務局, Dec. 2017, 生命科学系学会合同年次大会, 2017年度, [JST - 01], Japanese

  • 哺乳動物の全細胞系譜追跡にむけたDNAバーコード技術の開発
    増山 七海, 石黒 宗, 森 秀人, 増井 修, 関 元昭, 松尾 采佳, 西田 敬二, 栫 健太郎, 近藤 昭彦, 冨田 勝, 油谷 浩幸, 古関 明彦, 谷内江 望
    生命科学系学会合同年次大会運営事務局, Dec. 2017, 生命科学系学会合同年次大会, 2017年度, [2P - 1314], English

  • バイオリファイナリー技術の最近の進歩
    近藤 昭彦
    Nov. 2017, アグリバイオ, 11(1(12)) (1(12)), Japanese
    Introduction scientific journal

  • ピルビン酸代謝フローの転換による出芽酵母2,3‐ブタンジオール生産性の向上
    森田啓介, 松田史生, 岡本浩二, 石井純, 近藤昭彦, 清水浩
    08 Aug. 2017, 日本生物工学会大会講演要旨集, 69th, 287, Japanese

  • Deaminase- mediated genome editing
    Keiji Nishida, Zenpei Shimatani, Akihiko Kondo
    植物化学調節学会, Jun. 2017, Regulation of Plant Growth & Development, 52(1) (1), 15 - 18, Japanese
    Introduction scientific journal

  • 低分子抗体医薬の開発展望
    NAKAMURA YASUYUKI, ITO YOICHIRO, 梅津 光央, ISHII JUN, KONDO AKIHIKO
    シーエムシー出版, Jun. 2017, 月刊BIO INDUSTRY, 34(6) (6), 54 - 62, Japanese
    [Invited]
    Introduction commerce magazine

  • マイトファジー抑制による出芽酵母の細胞質物質生産能力向上
    森田啓介, 松田史生, 岡本浩二, 石井純, 近藤昭彦, 清水浩
    05 Mar. 2017, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.4C26a01 (WEB ONLY), Japanese

  • 微粉砕・メタン発酵・高度膜分離による稲わらの利用
    佐々木建吾, 白井智量, 柘植陽太, 佐々木大介, 菊地淳, 近藤昭彦
    05 Mar. 2017, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.4J28a09 (WEB ONLY), Japanese

  • リグノセルロース系バイオマスからのバイオモノマー発酵生産 (特集 バイオマス利活用のイノベーション)
    川口 秀夫, 近藤 昭彦
    化学工業社, Mar. 2017, ケミカルエンジニヤリング = Chemical engineering, 62(3) (3), 169 - 176, Japanese

  • Kentaro Inokuma, Jun Ishii, Kiyotaka Y. Hara, Masao Mochizuki, Tomohisa Hasunuma, Akihiko Kondo
    AMER SOC MICROBIOLOGY, Feb. 2017, MICROBIOLOGY RESOURCE ANNOUNCEMENTS, 5(5) (5), English
    Others

  • バイオ燃料生産に向けた耐塩性緑藻の進化工学的育種と塩ストレス耐性による油脂生産低下メカニズムの解明
    加藤悠一, HO Shih-Hsin, CHANG Jo-Shu, 蓮沼誠久, 近藤昭彦
    2017, 日本農芸化学会大会講演要旨集(Web), 2017

  • デンプン合成制御因子CRCTが稲わらを用いたバイオエタノール生産に及ぼす効果
    森田隆太郎, 森田隆太郎, 寺村浩, 荻野千秋, 近藤昭彦, 深山浩
    2017, 日本作物学会講演会要旨集, 243rd

  • バイオエタノール生産プロセスにおける糖液濃縮を指向したNF-FOハイブリッドシステムの構築
    渋谷真史, 佐々木建吾, 安川政宏, 田中裕大, 高橋智輝, 近藤昭彦, 松山秀人
    2017, 化学工学会年会研究発表講演要旨集(CD-ROM), 82nd

  • バイオテクノロジー革新によるバイオプラスチック研究開発の加速
    近藤 昭彦
    2017, バイオプラジャーナル, 65, 巻頭, Japanese
    Introduction scientific journal

  • 無機とバイオの融合ナノ粒子によるがん治療戦略
    Nishimura Yuuya, Morita Kenta, Suzuki Takahiro, OGINO Chiaki, KONDO Akihiko
    ホソカワミクロン, 2017, 粉砕, 60(60) (60), 13 - 19, Japanese
    [Refereed]
    Introduction scientific journal

  • 未利用バイオマスからのD-乳酸生産に向けた乳酸菌の代謝工学研究
    岡野憲司, 濵真司, Tsutom Tanaka, 野田秀夫, KONDO Akihiko
    農林水産・食品産業技術振興協会, 2017, JATAFFジャーナル, 5(3) (3), 33 - 37, Japanese
    [Refereed]
    Introduction scientific journal

  • リグノセルロース系バイオマスからのバイオモノマー発酵生産
    Kawaguchi Hideo, KONDO Akihiko
    2017, ケミカルエンジニヤリング, 62(3) (3), 13 - 20, Japanese
    [Refereed]
    Introduction scientific journal

  • Production of platform chemicals from sustainable resources:
    Tsuge, Y, Kondo A
    Springer, 2017, Production of amino acids(L-glutamic acid and L-lysine) from biomass, Feng Z (Ed), 437 - 455, English
    [Refereed]
    Introduction scientific journal

  • 原子間力顕微鏡による酵母細胞表層セルラーゼの局在評価
    InokumaKentarou, TAKENAKA Musashi, OGINO Chiaki, Hasunuma Tomohisa, KONDO Akihiko
    日本生物工学会, Nov. 2016, 生物工学, 94(11) (11), 698 - 700, Japanese
    [Refereed]
    Introduction scientific journal

  • 疎水性アルコールを用いた植物バイオマスの効率的な成分分離法の開発
    寺村浩, 佐々木建吾, 白井智量, 川口秀夫, 荻野千秋, 菊地淳, 佐塚隆志, 近藤昭彦, 近藤昭彦
    14 Oct. 2016, リグニン討論会講演集, 61st, 180‐181, Japanese

  • 次世代バイオエタノール製造技術
    InokumaKentarou, Hasunuma Tomohisa, KONDO Akihiko
    シーエムシー出版, Oct. 2016, バイオマスエネルギーの技術と市場, 第4章, 33 - 45, Japanese
    [Refereed]
    Introduction scientific journal

  • イソブタノール生産酵母の構築:オルガネラ局在の検討
    森田啓介, 松田史生, 伊田賢吾, 石井純, 近藤昭彦, 清水浩
    25 Aug. 2016, 日本生物工学会大会講演要旨集, 68th, 186, Japanese

  • 凝集性酵母における新規な阻害剤耐性機構の解明
    糸見明穂, 紀平和枝, KAHAR Prihardi, 荻野千秋, 近藤昭彦
    25 Aug. 2016, 日本生物工学会大会講演要旨集, 68th, 316, Japanese

  • Tsutomu Tanaka, Hiroki Nishikawa, Naoki Ikeda, Akihiko Kondo
    ELSEVIER SCIENCE BV, Jul. 2016, NEW BIOTECHNOLOGY, 33, S191 - S191, English
    Summary international conference

  • Matsumoto Takuya, Tanaka Tsutomu, Kondo Akihiko
    ELSEVIER SCIENCE BV, Jul. 2016, NEW BIOTECHNOLOGY, 33, S194 - S194, English
    Summary international conference

  • Shuhei Noda, Tomokazu Shirai, Akihiko Kondo
    ELSEVIER SCIENCE BV, Jul. 2016, NEW BIOTECHNOLOGY, 33, S193 - S193, English
    Summary international conference

  • Yeast breeding for fuels and chemicals production from Indonesia Culture Collection
    KAHAR Prihardi, OTSUKA Hiromi, KIHARA Chie, ITOMI Akiho, LEE Jaemin, THONTOWI Ahmad, OCTAVIANA Senlio, JALU Filemon, KANTI Atit, YOPI, OGINO Chiaki, PRASETYA Bambang, KONDO Akihiko
    13 Mar. 2016, 化学工学会年会研究発表講演要旨集(CD-ROM), 81st, ROMBUNNO.N316, English

  • Biorefinery research in Kobe and Indonesia
    OGINO Chiaki, LEE JaeMin, KAHAR Prihardi, KONDO Akihiko
    13 Mar. 2016, 化学工学会年会研究発表講演要旨集(CD-ROM), 81st, ROMBUNNO.N302, English

  • 異種遺伝子発現による出芽酵母イソブタノール合成活性化
    森田啓介, 松田史生, 石井純, 近藤昭彦, 清水浩
    05 Mar. 2016, 日本農芸化学会大会講演要旨集(Web), 2016, 3F211 (WEB ONLY), Japanese

  • マンナンバイオマスからのエタノール生産:β‐マンナナーゼとβ‐マンノシダーゼを細胞表層に提示した出芽酵母の開発
    石井純, 岡崎文美, DJOHAN Apridah Cameliawati, 原清敬, 浅井菜々実, ANDRIANI Ade, 寺村浩, KAHAR Prihardi, YOPI, PRASETYA Bambang, 荻野千秋, 近藤昭彦
    05 Mar. 2016, 日本農芸化学会大会講演要旨集(Web), 2016, 4A022 (WEB ONLY), Japanese

  • 2,3‐ブタンジオール高生産出芽酵母株の構築
    松田史生, 伊田賢吾, 木下翔平, 森田啓介, 石井寛子, 富田淳美, 石井純, 近藤昭彦, 清水浩
    05 Mar. 2016, 日本農芸化学会大会講演要旨集(Web), 2016, 2F159 (WEB ONLY), Japanese

  • アルコールの添加が希硫酸前処理の効率に与える影響の解析
    寺村浩, 佐々木建吾, 白井智量, 川口秀夫, 荻野千秋, 菊地淳, 佐塚隆志, 近藤昭彦
    05 Mar. 2016, 日本農芸化学会大会講演要旨集(Web), 2016, 3A030 (WEB ONLY), Japanese

  • スーパーバイオマス:第6章 バイオリファイナリー
    OGINO CHIAKI, KAWAGUCHI HIDEO, KONDO AKIHIKO
    Feb. 2016, 慶応義塾大学出版会, 87 - 104, Japanese
    Introduction scientific journal

  • バイオエタノール生産プロセス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
    2016, 膜シンポジウム, (28) (28)

  • 正浸透膜法による糖液濃縮がバイオエタノール発酵プロセスに与える効果
    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, 松山秀人
    2016, 化学工学会秋季大会研究発表講演要旨集(CD-ROM), 48th

  • 紙パルプの同時糖化発酵によるカフェ酸の微生物生産
    川口秀夫, 勝山陽平, DU Danyao, 鶴田祥子, 南博道, 荻野千秋, 大西康夫, 近藤昭彦
    2016, 日本農芸化学会大会講演要旨集(Web), 2016

  • 微生物でのゲノム編集の利用と拡大技術
    KONDO Akihiko, 西田敬二, 荒添貴之
    裳華房, 2016, ゲノム編集入門, 第3章, 40 - 55, Japanese
    [Refereed]
    Introduction scientific journal

  • 農産廃棄物循環利用バイオエタノール製造システムに関する研究
    北口敏弘, KONDO Akihiko, 宮下和夫
    日本環境衛生センター, 2016, 生活と環境, 9(9) (9), 67 - 71, Japanese
    [Refereed]
    Introduction scientific journal

  • 2P-111 Isolation and functional analysis of a novel sesquiterpene synthase gene from Freesia X hybrida 'Ishikawa f1'
    Higuchi,Yuki, Hattan,Jun-ichiro, Ohno,Fumina, Ito,Tomoko, Shibuya,Yurika, Watanabe,Arisa, Ishii,Jun, Kondo,Akihiko, Shindo,Kazutoshi, Misawa,Norihiko
    日本生物工学会, 25 Sep. 2015, 日本生物工学会大会講演要旨集, 67, 202, Japanese

  • Masao Nakayama, Ryohei Sasaki, Toru Mukohara, Chiaki Ogino, Kenta Morita, Mitsuo Umetsu, Satoshi Ohara, Kazuyoshi Sato, Chiya Numako, Seiichi Takami, Akihiko Kondo
    AMER ASSOC CANCER RESEARCH, Aug. 2015, CANCER RESEARCH, 75, English
    Summary international conference

  • 2次元NMRを用いた希硫酸前処理後固体画分の組成解析
    TERAMURA HIROSHI, SASAKI KENGO, AIKAWA SHIMPEI, MATSUDA FUMIO, OKAMOTO MAMI, SHIRAI TOMOKAZU, KAWAGUCHI HIDEO, OGINO CHIAKI, YAMASAKI MASANORI, KIKUCHI JUN, KONDO AKIHIKO
    09 Mar. 2015, 日本植物生理学会年会要旨集, 56th, 284, Japanese

  • 長期継代培養によるエタノール非生産酵母の増殖改善株の取得
    森田啓介, 富田淳美, 松田史生, 石井純, 近藤昭彦, 清水浩
    05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 3A21P11 (WEB ONLY), Japanese

  • 実バイオマスを微生物変換するための新しい酵母プラットフォームの探索
    KAHAR Prihardi, LEE Jaemin, 荻野千秋, 近藤昭彦
    05 Mar. 2015, 日本農芸化学会大会講演要旨集(Web), 2015, 3B33A01 (WEB ONLY), Japanese

  • MALDI-TOF/MSを用いた発現タンパク質プロファイル測定方法の開発
    原清敬, 望月正雄, 蓮沼誠久, 中津井雅彦, 荒木通啓, 近藤昭彦
    2015, 日本ゲノム微生物学会年会要旨集, 9th

  • バイオリファイナリー利用に向けた稲わらの多様性
    合田喬, 寺村浩, 末廣美紀, 高山隆一, 最相大輔, 山本洋, 金丸研吾, 川口秀夫, 荻野千秋, 近藤昭彦, 山崎将紀
    2015, 育種学研究, 17

  • 1P-080 Yeast-based biosensor for ligand screening of human nerotensin receptor
    Hashi Hiroki, Nakamura Yasuyuki, Ishii Jun, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 108 - 108, Japanese

  • 1P-079 Development of non-nuclease genome editing tool
    Nishida Keiji, Banno Satomi, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 108 - 108, Japanese

  • 1P-025 Improvement of astaxanthin production using metabolically engineered Xanthophyllomyces dendrorhous
    Yamamoto Keisuke, Hara Kiyotaka, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 95 - 95, Japanese

  • 3P-198 Characterics studies of Streptoverticillium cinnamoneum phospholipase D promoter for gene expression
    Kashiwagi Norimasa, Nishioka Masato, Matsumoto Hana, Hirose Shuichi, Sota Masahiro, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 320 - 320, Japanese

  • 2P-195 Development of metabolic channeling for efficient production of butanol using Escherichia coli.
    Furuta Kou, Matsumoto takuya, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 223 - 223, Japanese

  • 2P-113 Metabolic engineering of Escherichia coli designed in view of the reaction of chorismate pyruvate lyase
    Noda Shuhei, Shirai Tomokazu, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 203 - 203, Japanese

  • 2P-196 C-Terminal-oriented Immobilization of Enzymes Using Sortase A-mediated Technique
    Matsumoto Takuya, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 223 - 223, Japanese

  • 2S-Ca04 Development of microbial cell factories for biorefinery
    Tomohisa Hasunuma, Akihiko Kondo
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 250 - 250, English

  • 1P-179 Development of biofuel production from chitinous substrates
    Inokuma Kentaro, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 133 - 133, Japanese

  • 1P-206 Combined cell-surface display- and secretion-based strategies for production of cellulosic ethanol with Saccharomyces cerevisiae :
    Liu Zhuo, Inokuma Kentaro, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 140 - 140, English

  • 3P-083 Development of 5-aminolevulinic acid production using engineered S. cerevisiae
    Hara Kiyotaka, Saito Masaru, Morikawa Kana, Kato (Ishii) Hiroko, Nomura Hironari, Fujimoto Takanori, Kanamaru Kengo, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 291 - 291, Japanese

  • 2P-097 Metabolic engineering of L-Lactate producing Aspergillus oryzae.
    Sasakura Naoya, Wakai Satoshi, Asai Nanami, Ogino Chiaki, Tsutsumi Hiroko, Hata Youji, Kondo Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 199 - 199, Japanese

  • 2P-014 Cadaverine production from biomss using Corynebacterium glutamicum
    Imao Kenta, Segawa Shota, Tanaka Tsutomu, KONDO Akihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 178 - 178, Japanese

  • 2P-110 Isolation and functional analysis of a novel sesquiterpene synthase gene from "Kantsubaki" Camellia hiemalis
    Hattan Jun-ichiro, Shindo Kazutoshi, Ohno Fumina, Higuchi Yuhki, Ishii Jun, Kondo Akihiko, Misawa Norihiko
    日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 202 - 202, Japanese

  • 好気・低濃度グルコース条件下におけるSaccharomyces cerevisiaeの転写解析
    崎濱由梨, 蓮沼誠久, 蓮沼誠久, 近藤昭彦, 近藤昭彦
    2015, 日本生物工学会大会講演要旨集, 67th

  • Saccharomyces cerevisiaeの糖代謝における転写制御ネットワークの予測
    南部由美子, 南部由美子, 崎濱由梨, 荒木道啓, 蓮沼誠久, 近藤昭彦, 近藤昭彦
    2015, 日本生物工学会大会講演要旨集, 67th

  • Tsutomu Tanaka, Mariko Nakano, Yuuki Hirata, Akihiko Kondo
    ELSEVIER SCIENCE BV, Sep. 2014, JOURNAL OF BIOTECHNOLOGY, 185, S52 - S52, English
    Summary international conference

  • Shuhei Noda, Tomokazu Shirai, Keiichi Mochida, Sachiko Oyama, Mami Okamoto, Fumio Matsuda, Kondo Akihiko
    ELSEVIER SCIENCE BV, Sep. 2014, JOURNAL OF BIOTECHNOLOGY, 185, S102 - S102, English
    Summary international conference

  • Virendra S. Bisaria, Akihiko Kondo
    For the development of a sustainable, industrial society to meet our demands of energy and materials, it is being increasingly realized that we will have to shift from our dependence on petroleum to the use of renewable resources, such as starch- and cellulose-based plant materials. Historically till recently, petroleum-based resources were mainly targeted for research and development, and subsequent commercialization of the products derived therefrom. However, their rising costs and the anticipated threat to the earth's environment are providing the required incentive to find sustainable alternative resources. Biorefineries, based on renewable resources, shall enable the production of biofuels as well as commodity chemicals (those produced in excess of about 1 million tons per year). These processes which are based on carbohydrates (such as starch and cellulose) are also favorable from a chemical point of view because the functional groups that are introduced by costly oxidative process steps into naphta are already present in them. The commodity bioproducts can be produced by microbial processes. Most of them are natural products of microorganisms or can be produced by suitable pathway engineering of industrial organisms. As these bioproducts contain functional groups, they are extremely useful as starting materials for the chemical industry for synthesis of a wide variety of products such as polymers, surfactants, lubricants, and resins.
    Wiley Blackwell, 28 Apr. 2014, Bioprocessing of Renewable Resources to Commodity Bioproducts, 1 - 555, English
    Others

  • モデルベース機械学習による配合注射薬外観変化予測アルゴリズムの開発
    中津井雅彦, 角山香織, 権丈紀子, 荒木通啓, 栄田敏之, 近藤昭彦, 奥野恭史
    (公社)日本薬学会, Mar. 2014, 日本薬学会年会要旨集(CD-ROM), 134th(4) (4), ROMBUNNO.30AMM-197 - 171, Japanese

  • 過酸化チタンナノ粒子による放射線増感の検討
    中山 雅央, 佐々木 良平, 西村 英輝, 吉田 賢史, 宮脇 大輔, 赤坂 浩亮, 椋本 成俊, 村岡 修, 原田 文, ノル・シャズリナ, 上原 和之, 田中 勉, 荻野 千秋, 近藤 昭彦
    (公社)日本医学放射線学会, Feb. 2014, Japanese Journal of Radiology, 32(Suppl.) (Suppl.), 37 - 37, Japanese

  • 遺伝子発現における時系列データ・制御様式の整合性評価
    中津井雅彦, 中津井雅彦, 荒木通啓, 近藤昭彦
    2014, 日本分子生物学会年会プログラム・要旨集(Web), 37th

  • M-path: 拡張代謝パスウェイデータベースの構築
    牧口大旭, 小川哲平, 中津井雅彦, ROBERT Cox Sidney, III, 近藤昭彦, 谷口岳志, 宮奥康平, 荒木通啓
    2014, 日本分子生物学会年会プログラム・要旨集(Web), 37th

  • 3P-045 Effects of hydrolysate of lignocellulosic biomass on phenyllactate fermentation by recombinant Escherichia coli
    Kawaguchi Hideo, Teramura Hiroshi, Niimi-Nakamura Satoko, Ogino Chiaki, Hara Kiyotaka, Hasunuma Tomohisa, Oinuma Ken-Ichi, Takaya Naoki, Hirano Ko, Sazuka Takashi, Kitano Hidemi, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 206 - 206, Japanese

  • 3P-044 Improvement of CHO cells useful in bio-pharmaceutical development, construction of data leading to the culture state elucidation
    Iga Tomoyo, Hasunuma Tomohisa, Araki Michihiro, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 205 - 205, Japanese

  • 1P-134 Development of the novel secretion signal sequence for highly-efficient cell surface display and secretory production of proteins by yeasts
    Inokuma Kentaro, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 51 - 51, Japanese

  • 3P-144 Comparison of microbial metabolites between methane fermentation and acid fermentation
    Sasaki Daisuke, Sasaki Kengo, Tsuge Yota, Nakanishi Shuji, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 230 - 230, Japanese

  • 3P-205 Metabolite profiling of oceanic cyanobacteria under salt stress condition
    Nishida Atsumi, Aikawa Shimpei, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 246 - 246, Japanese

  • 1P-006 Genome editing system for detecting dimerization of G-protein-coupled receptors
    Hashimoto Takamichi, Nakamura Yasuyuki, Ishii Jun, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 18 - 18, Japanese

  • 3P-230 Development of photoenergetic yeast cell factories by redirecting delta-rhodopsin to the mitochondria :
    Xiaoting Ye, Kiyotaka Hara, Akihiko Kondo
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 252 - 252, English

  • 3P-227 Enzymatic degradation of cellulose/hemicellulose with multi-functional beta-glucosidase
    Matsumoto Takuya, Hata Yuto, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 251 - 251, Japanese

  • 3P-225 Highly-oriented immobilized enzymes using sortase A-mediated transpeptidation
    Hata Yuto, Matsumoto Takuya, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 251 - 251, Japanese

  • 3P-229 Effect of PHO13 gene disruption on xylose production in xylose-fermenting yeast harboring xylose isomerase gene
    Bamba Takahiro, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 252 - 252, Japanese

  • 3P-226 A display of pH-sensitive fusogenic GALA peptide facilitates endosomal escape for a Bio-nanocapsule
    Nishimura Yuya, Ezawa Ryosuke, Ishii Jun, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 251 - 251, Japanese

  • 3P-253 Effect of the odorant binding protein on the ligand response of Saccharomyces cerevisiae expressing odorant receptors
    Hori Ayaka, Fukutani Yosuke, Tsukada Satoshi, Sato Ryoichi, Ishii Jun, Kondo Akihiko, Matsunami Hiroaki, Yohda Masafumi
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 258 - 258, Japanese

  • 3P-254 Application of the GPCR ligand response detection using split luciferase in budding yeast
    Asakawa Masashi, Fukutani Yosuke, Ishii Jun, Kondo Akihiko, Ozawa Takeaki, Yohda Masafumi
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 258 - 258, Japanese

  • 2P-132 Development of lipid-producing system depending on estimation of cell composition and metabolic analysis for marine green alga Chlamydomonas sp. JSC4
    Nakanishi Akihito, Ho Shih-Hsin, Chang Jo-Shu, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 139 - 139, Japanese

  • 2P-136 Effect of illumination coupled with nitrogen depletion on biodiesel production of a marine microalga Chlamydomonas sp. JSC4 :
    Ho Shih-Hsin, Nakanishi Akihito, Ye Xiaoting, Chang Jo-Shu, Hara Kiyotaka, Kondo Akihiko, Hasunuma Tomohisa
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 140 - 140, English

  • 2P-209 Development of a design tool for novel metabolic pathways for productions of valuable chemicals
    Shirai Tomokazu, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 159 - 159, Japanese

  • 2P-110 Direct cadaverine production from oligosaccharide using Escherichia coli coexpressing beta-glucosidase and beta-xylosidase
    Ikeda Naoki, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 134 - 134, Japanese

  • 2P-094 Analysis of high-yield, high-efficient homo-butanol-fermentative Clostridia
    Mukoyama Masaharu, Nananosho Masahiro, Ichige Eita, Dobashi Yukio, Nakayama Shunichi, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 130 - 130, Japanese

  • 2P-113 Improvement of glutathione production using engineered yeast
    Endo Yusuke, Hara Kiyotaka, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 135 - 135, Japanese

  • 2P-108 Cataloguing terpene synthase genes of fragrant plants : example of Camellia flowers
    Hattan Jun-ichiro, Tagaki Chie, Ohno Fumina, Ishii Jun, Kondo Akihiko, Harimoto Takashi, Misawa Norihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 133 - 133, Japanese

  • 2P-112 Lactic acid production from cellobiose using Schizosaccharomyces pombe displaying beta-glucosidase on the surface
    Ogawa Kousuke, Matsumoto Sayoko, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 134 - 134, Japanese

  • 2P-095 High-level ethanol production from cyanobacterium Spirulina by amylase displayed yeast
    Aikawa Shimpei, Inokuma Kentaro, Sasaki Kengo, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 130 - 130, Japanese

  • 1P-219 Membrane process increased ethanol production from pretreated rice straw.
    Sasaki Kengo, Tsuge Yota, Sasaki Daisuke, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 72 - 72, Japanese

  • 1P-110 Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions
    Tsuge Yota, Hori Yoshimi, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 44 - 44, Japanese

  • 1P-044 Simultaneous saccharification and fermentation of D-lactic acid from starch by Aspergillus oryzae
    Wakai Satoshi, Asai-Nakashima Nanami, Ogino Chiaki, Tsutsumi Hiroko, Hata Yoji, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 28 - 28, Japanese

  • 1P-112 Production of p-aminobenzoic acid by Streptomyces lividans expressing pabAB
    Okai Naoko, Satou Yoshihiro, Ohno Maya, Takeshima Yasunobu, Masuda Takaya, Miyamoto Masanori, Toida Kouzou, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 45 - 45, Japanese

  • 2P-220 Cluster analysis of oleaginous yeasts based on their secondary metabolites
    Kojima Motoki, Suzuki Munenori, Harada Hisashi, Misawa Norihiko, Ishii Jun, Kondo Akihiko, Harimoto Takashi
    日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 161 - 161, Japanese

  • Direct isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display system
    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
    日本生物工学会, 2014, 生物工学会誌 : seibutsu-kogaku kaishi, 92(2) (2), 71 - 71, Japanese

  • キシロース資化性酵母における遺伝子発現プロファイルの経時変化解析
    南部由美子, 崎濱由梨, 蓮沼誠久, 蓮沼誠久, 近藤昭彦, 近藤昭彦
    2014, 日本生物工学会大会講演要旨集, 66th

  • Kluyveromyces marxianusの好気条件での糖代謝系の解析
    崎濱由梨, 蓮沼誠久, 蓮沼誠久, 近藤昭彦, 近藤昭彦
    2014, 日本生物工学会大会講演要旨集, 66th

  • バイオマスからの微生物によるバイオモノマー生産
    OGINO Chiaki, NAKAMURA-NIIMI Satoko, OKAI Naoko, WAKAI Satoshi, KAWAGUCHI Hideo, KONDO Akihiko
    化学工業社, 2014, ケミカルエンジニヤリング, 59(7) (7), 506 - 515, Japanese
    Introduction scientific journal

  • Metabolic pathway engineering of yeast Saccharomyces cerevisiae for isobutanol production
    Jun Ishii, Fumio Matsuda, Akihiko Kondo
    WILEY-BLACKWELL, Sep. 2013, YEAST, 30, 210 - 210, English
    Summary international conference

  • A selection system exploiting yeast signal transduction machinery to create desirably affinity-altered protein variants
    Misato Kaishima, Nobuo Fukuda, Jun Ishii, Akihiko Kondo
    WILEY-BLACKWELL, Sep. 2013, YEAST, 30, 182 - 182, English
    Summary international conference

  • Evaluation of control mechanisms for Saccharomyces cerevisiae central metabolic pathway using metabolic analysis of single-gene deletion mutants
    Shuhei Noda, Tomokazu Shirai, Fumio Matsuda, Mami Okamoto, Akihiko Kondo
    WILEY-BLACKWELL, Sep. 2013, YEAST, 30, 170 - 170, English
    Summary international conference

  • バイオリファイナリー社会に向けた燃料・化学品生産
    ISHII JUN, HASUNUMA TOMOHISA, MATSUDA FUMIO, KONDO AKIHIKO
    Aug. 2013, 安全工学, 52(4) (4), 249 - 255, Japanese
    Introduction scientific journal

  • Kazuhiro Yamamoto, Takuya Matsumoto, Shota Shimada, Tsutomu Tanaka, Akihiko Kondo
    The present study reports the design of a novel bioanode to directly utilize starch as a fuel in an enzymatic biofuel cell. The enzymatic fuel cell is based on three enzymes (alpha-amylase, glucoamylase and glucose oxidase). The carbon paste electrode containing these three enzymes and tetrathiafulvalene can both saccharize and oxidize starchy biomass. In cyclic voltammetry, catalytic currents were successfully observed with both glucose and starchy white rice used as a substrate. Finally, a membrane-less white rice/O2 biofuel cell was assembled and the electrochemical performance was evaluated. The three enzyme based electrode was used as a bioanode and an immobilized bilirubin oxidase (derived from Myrothecium verrucaria) electrode was used as a biocathode. The biofuel cell delivered an open circuit voltage of 0.522V and power density of up to 99.0 μWcm(-2). Our results show that a readily available fuel can be used for enzymatic fuel cells, and will lead to new designs.
    25 Jun. 2013, New biotechnology, 30(5) (5), 531 - 5, English, International magazine

  • 革新的なものづくり実現のための「合成生物工学」
    ISHII JUN, HASUNUMA TOMOHISA, MATSUDA FUMIO, KONDO AKIHIKO
    日本生物工学会, Jun. 2013, 生物工学会誌, 91(6) (6), 314 - 318, Japanese
    Introduction scientific journal

  • Simultaneous Saccharification and Fermentation of Ethanol from Ionic Liquid Treated Biomass
    荻野 千秋, 山田 亮祐, 近藤 昭彦
    小峰工業出版, Feb. 2013, 化學工業, 64(2) (2), 136 - 139, Japanese

  • コリネ菌の細胞表層提示技術を用いたデンプンからの乳酸様ポリマーの生産
    門屋亨介, 門屋亨介, SONG Yuyang, 飛谷康太, 松本謙一郎, 田中勉, 近藤昭彦, 田口精一, 田口精一
    2013, 日本農芸化学会北海道支部講演会講演要旨, 2013

  • バイオプロセスデザインプラットフォームを用いたアミノ酸のバイオプロセスデータベースの開発
    牧口大旭, 小川哲平, 中津井雅彦, COX Robert Sidney, III, 近藤昭彦, 荒木通啓
    2013, 日本分子生物学会年会プログラム・要旨集(Web), 36th

  • 1P-118 Metabolic engineering for homogentisic acid production in Streptomyces lividans
    Niimi-Nakamura Satoko, Kawaguchi Hideo, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 47 - 47, Japanese

  • 1P-082 High sensitive detection system for ligand screening of human G-protein-coupled receptors by improvement of yeast-based fluorescence reporter assay
    Nakamura Yasuyuki, Ishii Jun, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 38 - 38, Japanese

  • 1P-083 An advanced method to simultaneously analyze the dimerization and signal transduction of G-protein-coupled receptors
    Takemoto Norika, Nakamura Yasuyuki, Ishii Jun, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 38 - 38, Japanese

  • 1P-105 Simultaneous saccharification and fermentation of cellulose for phenyllactic acid production
    Kawaguchi Hideo, Niimi-Nakamura Satoko, Ogino Chiaki, Oinuma Kenichi, Takaya Naoki, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 44 - 44, Japanese

  • 1P-104 Identification of genes related to improved ethanol production from xylose in presence of acetate and formate by regulation of metabolism in Saccharomyces cerevisiae
    Sakamoto Takatoshi, Hori Yoshimi, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 43 - 43, Japanese

  • 1P-117 Production of protocatechuate by Corynebacterium glutamicum
    Okai Naoko, Takeshima Yasunobu, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 47 - 47, Japanese

  • 2P-089 Highly-oriented immobilized cellulase using sortase A-mediated transpeptidation
    Hata Yuto, Matsumoto Takuya, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 126 - 126, Japanese

  • 2P-114 Astaxanthine production using engineered Xanthophyllomyces dendrorhous
    Morita Toshihiko, Hara Kiyotaka, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 133 - 133, Japanese

  • 2S-Ba02 Development of innovative bioprocess based on multiomics data
    Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 81 - 81, Japanese

  • 1P-185 Development of high lipid producing system by green alga Chlamydomonas orbicularis under sea salt condition
    Nakanishi Akihito, Ho Shih-Hsin, Aikawa Shimpei, Chang Jo-Shu, Kondo Akihiko, Hasunuma Tomohisa
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 64 - 64, Japanese

  • 1P-196 Phototrophic cultivation of a marine microalga Chlamydomonas orbicularis for CO_2 fixation and biodiesel production: Effect of medium composition, nitrogen depletion, and sea salt concentration :
    Ho Shih-Hsin, Nakanishi Akihito, Aikawa Shimpei, Chang Jo-Shu, Kondo Akihiko, Hasunuma Tomohisa
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 66 - 66, English

  • 1P-186 Glycogen production for biorefinery using oceanic cyanobacteria
    Nishida Atsumi, Aikawa Shimpei, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 64 - 64, Japanese

  • 1P-146 Improvement of ethanol production from xylose by regulation of the PHO pathway in Saccharomyces cerevisiae
    Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 54 - 54, Japanese

  • 1P-191 The effect of sea-salts on the cell-growth and photosynthesis in a remarkable carbon feedstock Spirulina
    Aikawa Shimpei, Akimoto Seiji, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 65 - 65, Japanese

  • 1P-209 Characterization of anaerobic digestion by metabolomic analysis derived from microbial community
    SASAKI Daisuke, SASAKI Kengo, NAKANISHI Shuji, HASHIMOTO Kazuhito, KONDO Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 70 - 70, Japanese

  • 1P-184 Development of novel yeast cell surface display system for consolidated bioethanol production from lignocellulosic biomass
    Inokuma Kentaro, Hasunuma Tomohisa, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 63 - 63, Japanese

  • 1P-145 Activation of transhydrogenase-like shunt for isobutanol production in Saccharomyces cerevisiae
    Ishii Jun, Matsuda Fumio, Kondo Takashi, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 54 - 54, Japanese

  • 1P-218 Pretreatment of lignocellulosic biomass by acid-catalysed process in aqueous ionic liquid solution
    Ogura Kazuma, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 72 - 72, Japanese

  • 2P-200 Effect of the accessory proteins on expressing functional odorant receptor with yeast
    Hori Ayaka, Fukutani Yosuke, Ishii Jun, Kondo Akihiko, Matsunami Hiroaki, Yohda Masafumi
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 154 - 154, Japanese

  • 2P-148 Sugar concentration in liquid hydrolysate of dilute acid pretreated rice straw by nanofiltration
    Sasaki Kengo, Hasunuma Tomohisa, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 141 - 141, Japanese

  • 3P-201 Production of polyesters from starch in Corynebacterium glutamicum by using α-amylase cell-surface displaying system
    Kadoya Ryosuke, Song Yuyang, Tobitani kouta, Matsumoto Ken'ichiro, Tanaka Tsutomu, Kondo Akihiko, Taguchi Seiichi
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 238 - 238, Japanese

  • 3P-174 Development of affibody-displaying bio-nanocapsule for specific delivery to EGFR-expressing cells
    Ezawa Ryosuke, Nishimura Yuya, Ishii Jun, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 231 - 231, Japanese

  • 3P-182 Glucose dehydrogenase and gluconate dehydrogenase co-immobilized bioanode for biofuel cell
    Shimada Shota, Matsumoto Takuya, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 233 - 233, Japanese

  • 3P-161 Direct putrescine production from cellobiose using Escherichia coli displaying beta-glucosidase
    Ikeda Naoki, Miyamoto Mari, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 228 - 228, Japanese

  • 3P-183 Oriented co-immobilization of enzymes through sortase A mediated-methodology
    Matsumoto Takuya, Hata Yuto, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 234 - 234, Japanese

  • 特集によせて(<特集>新しい資源の創出に向かって-合成生物工学の隆起-)
    植田 充美, 近藤 昭彦
    日本生物工学会, 2013, 生物工学会誌 : seibutsu-kogaku kaishi, 91(6) (6), 308 - 308, Japanese

  • 統合バイオプロセスによる環境調和型セルロース系エタノール生産に資する前処理技術の開発
    崎濱由梨, 俣野結城, 蓮沼誠久, 蓮沼誠久, 近藤昭彦, 近藤昭彦
    2013, 日本生物工学会大会講演要旨集, 65th

  • 発酵阻害物存在下におけるキシロースからのエタノール発酵向上酵母の構築
    崎濱由梨, 蓮沼誠久, 近藤昭彦
    2013, 日本農芸化学会大会講演要旨集(Web), 2013

  • 神戸大学大学院工学研究科応用化学専攻 生物化学工学研究室
    近藤 昭彦, 荻野 千秋, 田中 勉, 蓮沼 誠久, 石井 純, 原 清敬, 山田 亮祐, 岡井 直子, 工藤 基徳, 佐々木 建吾, 川口 秀夫, 柘植 陽太
    05 Nov. 2012, 化学工学 = Chemical engineering, 76(11) (11), 722 - 722, Japanese

  • Novel Radiosensitization Through Reactive Oxygen Species Generation Using Titanium Peroxide Nanoparticle Compounds Against Pancreas Cancer
    M. Nakayama, R. Sasaki, C. Ogino, T. Tanaka, M. Umetsu, S. Ohara, Z. Tan, K. Sato, C. Numako, A. Kondo
    ELSEVIER SCIENCE INC, Nov. 2012, INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS, 84(3) (3), S700 - S700, English
    Summary international conference

  • 糖質バイオマスから多様なポリエステルを生産するコリネ菌微生物工場の開発
    SONG YUYANG, MATSUMOTO KEN'ICHIRO, OI TOSHIHIKO, TANAKA TSUTOMU, KONDO AKIHIKO, TAGUCHI SEIICHI
    日本生物工学会, 25 Sep. 2012, 日本生物工学会大会講演要旨集, 64th, 27 - 27, Japanese

  • Shuhei Noda, Tsutomu Tanaka, Chiaki Ogino, Akihiko Kondo
    ELSEVIER SCIENCE BV, Sep. 2012, NEW BIOTECHNOLOGY, 29, S50 - S50, English
    Summary international conference

  • Takuya Matsumoto, Tsutomu Tanaka, Akihiko Kondo
    ELSEVIER SCIENCE BV, Sep. 2012, NEW BIOTECHNOLOGY, 29, S97 - S98, English
    Summary international conference

  • Tsutomu Tanaka, Yuuki Hirata, Hitomi Kawabata, Akihiko Kondo
    ELSEVIER SCIENCE BV, Sep. 2012, NEW BIOTECHNOLOGY, 29, S51 - S51, English
    Summary international conference

  • Cyanobacterium Synechocystis sp. PCC6803 におけるO2存性オルタナティブ・エレクトロン・フロー (AEF)の生理的役割
    林 良祐, 清水 聡子, 山本 宏, 藍川 晋平, 蓮沼 誠久, 秋本 誠志, 近藤 昭彦, 三宅 親弘
    Mar. 2012, 第53回日本植物生理学会, Japanese

  • 特集によせて(バイオリファイナリーの今,そして未来,<特集>バイオ技術10年の軌跡,創立90周年記念特別企画)
    植田 充美, 近藤 昭彦
    日本生物工学会, 2012, 生物工学会誌 : seibutsu-kogaku kaishi, 90(7) (7), 381 - 381, Japanese

  • 4Ip19 High temperature ethanol fermentation from lignocellulose and cell recycling using thermotolerant yeast
    Nakano Kohsuke, Hama Shinji, Ichiki Kenichiro, Harashima Satoshi, Kondo Akihiko, Noda Hideo
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 247 - 247, Japanese

  • 4Ip22 Saccharification and fermentation of biomass by the ionic liquid pretreatment with the engineered xylose fermentable yeast
    Ogura Kazuma, Nakashima Nanami, Yamada Ryousuke, Hasunuma Tomohisa, Kamiya Noriho, Ishida Nobuhiro, Saito Satosi, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 248 - 248, Japanese

  • 4Ip11 Enhancement of fermentation efficiency on softbiomass with yeast strains displaying hemicellulases
    Murayama Hirokazu, Nakagawa Takuya, Hisada Hiromoto, Tsutsumi Hiroko, Hata Yoji, Kondo Akihiko, Ueda Mitsuyoshi
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 245 - 245, Japanese

  • 4Hp09 Cellulase and glucose dehydrogenase co-immobilized bioanode for biofuel cell
    Shimada Shota, Yamamoto Kazuhiro, Matsumoto Takuya, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 234 - 234, Japanese

  • 4Hp15 Efficient D-lactic acid fermentation from cellulosic biomass using pentose-assimilating lactic acid bacteria
    Mizuno Shino, Kihara Maki, Takenaka Sayaka, Hama Shinji, Kondo Akihiko, Noda Hideo
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 236 - 236, Japanese

  • 4Ha05 Construction of biomass-degradation enzymes library from Streptomyces and exploring novel high-level secretion signal
    Miyazaki Takaya, Noda Shuhei, Tanaka Tsutomu, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 230 - 230, Japanese

  • 3Hp19 Production of ectoine from biomass using Halomonas elongata
    Tanimura Kosuke, Nakayama Hideki, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 156 - 156, Japanese

  • 3Hp07 Production of L-Lysine from cellobiose using Corynebacterium glutamicum expressing beta-glucosidase
    ADACHI Noriko, ONO Naoko, HIRATA Yuuki, TAKAHASHI Chihiro, OKAI Naoko, TANAKA Tsutomu, KONDO Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 153 - 153, Japanese

  • 2Gp16 Metabolome analysis of filamentous fungus Rhizopus oryzae showing different behaviors of lipase secretion
    Yoshida Ayumi, Hama Shinji, Hasunuma Tomohisa, Yamamoto Hiroyuki, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 65 - 65, Japanese

  • 2Gp09 Improvement of astaxanthin production using engineered Xanthophyllomyces dendrorhous
    Morita Toshihiko, Hara Kiyotaka, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 63 - 63, Japanese

  • 2Gp06 Oxidized glutathione production using engineered Saccharomyces cerevisiae
    Hara Kiyotaka, Kiriyama Kentaro, Aoki Naoko, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 63 - 63, Japanese

  • 4Cp07 Highly-oriented co-immobilization of proteins on streptavidin-supported particles
    Matsumoto Takuya, Hata Yuto, Tanaka Tsutomu, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 196 - 196, Japanese

  • 4Dp13 Benzoic acid fermentation from starch and cellulose using the polyketide synthesis pathway of Streptomyces maritimus
    Noda Shuhei, Tanaka Tsutomu, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 208 - 208, Japanese

  • 4Dp02 Production of cadaverine by Corynebacterium glutamicum
    Okai Naoko, Niba Emma, Nakayama Hideki, Matsuda Fumio, Ogino Chiaki, Kondo Akihiko
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 205 - 205, Japanese

  • 4Ca11 An improved bioluminescence-based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor
    Fukutani Yosuke, Ishii Jun, Noguchi Keiichi, Kondo Akihiko, Yohda Masafumi
    日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 194 - 194, Japanese

  • イオン液体耐性セルラーゼの単離と酵素特性解析
    窪田有華, 工藤基徳, 仲島菜々実, 中島一紀, 岡崎文美, 荻野千秋, 近藤昭彦
    2012, 酵素工学研究会講演会講演要旨集, 68th

  • イオン液体耐性セルラーゼの探索と機能解析
    窪田有華, 山口剛示, 仲島菜々実, 工藤基徳, 中島一紀, 岡崎文美, 荻野千秋, 近藤昭彦
    2012, 日本生物工学会大会講演要旨集, 64th

  • 海洋性超好熱菌Thermotoga neapolitana由来β-1,3-キシラナーゼの耐熱性を導く要因の解析
    山崎清志, 岡崎文美, 荻野千秋, 近藤昭彦, 三上文三, 榑林陽一, 鶴田宏樹
    2012, 日本農芸化学会大会講演要旨集(Web), 2012

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