研究者紹介システム
| 石井 純 |  |
| イシイ ジュン | |
| 先端バイオ工学研究センター | |
| 准教授 | |
| 応用化学関係 |
研究者情報
所属
【主配置】
先端バイオ工学研究センター【配置】
大学院科学技術イノベーション研究科 科学技術イノベーション専攻
学位
授業科目
- 先端バイオ技術概論, 大学院科学技術イノベーション研究科, 2022
- 先端研究開発プロジェクト研究, 大学院科学技術イノベーション研究科, 2022
- プレゼンテーション演習, 大学院科学技術イノベーション研究科, 2022
- 研究指導, 大学院科学技術イノベーション研究科, 2022
- 先端科学技術特定研究, 大学院科学技術イノベーション研究科, 2022
- 科学技術イノベーション研究1, 大学院科学技術イノベーション研究科, 2022
- 科学技術イノベーション研究2, 大学院科学技術イノベーション研究科, 2022
- 科学技術イノベーション戦略プロジェクト研究, 大学院科学技術イノベーション研究科, 2022
ジャンル
コメントテーマ
研究ニュース
- 2022年06月09日, 有用タンパク質の高生産性化に向けた新たなピキア酵母株の開発方針を提案
- 2022年04月04日, 光で有用物質を高生産する微生物の開発 ―常温・常圧で生産可能な低炭素バイオプロセスへの利用を目指して―
- 2021年09月27日, JST戦略的創造研究推進事業CRESTに石井純准教授、さきがけに末次健司准教授がそれぞれ研究代表者として採択されました
- 2021年03月24日, 酵母の遺伝子スイッチを人工的に作りだす新たな手法を開発
- 2020年12月01日, ピキア酵母においてターミネーター配列がRNA安定性と遺伝子発現量を制御していることを発見 – タンパク質の発現を最適化する技術として期待 –
- 2015年11月20日, 創薬標的により強く結合するタンパク質の選抜方法を開発
研究活動
研究キーワード
研究分野
委員歴
- 2021年06月 - 現在, 日本生物工学会 関西支部, 支部幹事（企画）
- 2021年 - 現在, Frontiers in Chemical Engineering, Associate Editor
- 2017年06月 - 2021年05月, 日本生物工学会, 英文誌編集委員
- 2017年04月 - 2020年03月, 文部科学省 科学技術・学術政策研究所 科学技術予測センター, 専門調査員
受賞
2021年10月 日本生物工学会, 第73回日本生物工学会大会トピックス賞, 光エネルギーを利用した大腸菌におけるメバロン酸のイソプレノールへの変換
日本国国内学会・会議・シンポジウム等の賞
2020年11月 日本農芸化学会関西支部, 日本農芸化学会関西支部第513回講演会 優秀発表賞, 酵母におけるスクアレン生合成経路の改変および下流モノオキシゲナーゼの発現調節
日本国国内学会・会議・シンポジウム等の賞
2020年11月 日本農芸化学会関西支部, 日本農芸化学会関西支部第513回講演会 優秀発表賞, ドーパミン発酵生産性を簡易的に評価するGPCRメタボライトセンサの開発
日本国国内学会・会議・シンポジウム等の賞
2019年12月 日本農芸化学会 関西支部, 日本農芸化学会関西支部 支部例会（第511回講演会） 優秀発表賞, 新規な３機能性融合マーカーを用いた酵母遺伝子スイッチの組織的開発
日本国国内学会・会議・シンポジウム等の賞
2018年11月 The Symposium on Biorefinery and Biprocess Topics, 2018 (iBio-N 2018), Best Poster Award, Construction of a stable, autonomously replicating plasmid vector containing Pichia pastoris centromeric DNA
中華人民共和国国際学会・会議・シンポジウム等の賞
2017年09月 化学工学会 バイオ部会, 化学工学会第49回秋季大会 バイオ部会 優秀ポスター賞, メラトニン濃度をモニタリングするための酵母in vivoメタボライトセンサ
日本国国内学会・会議・シンポジウム等の賞
2014年07月 日本生物工学会 生物工学若手研究者の集い（若手会）, 生物工学若手研究者の集い 夏のセミナー2014 飛翔奨励賞, 上皮成長因子受容体を特異的に認識するAffibody提示バイオナノカプセルの開発
日本国国内学会・会議・シンポジウム等の賞
2013年12月 神戸大学研究基盤センター「若手フロンティア研究会２０１３」, 神戸大学研究基盤センター「若手フロンティア研究会２０１３」 ポスター賞, ヒト受容体のリガンド探索のための酵母バイオセンサーの開発
国内学会・会議・シンポジウム等の賞
2013年09月 化学工学会, 化学工学会第45回秋季大会バイオ部会ポスター賞, ヒト受容体リガンド探索のための酵母蛍光レポーター高感度アッセイシステムの開発とその応用
国内学会・会議・シンポジウム等の賞
2012年09月 International Biotechnology Symposium and Exhibition, IBS2012 Organizing Committee Poster Awards, Complex carriers of affibody-displaying bio-nanocapsule and composition-varied liposomes for HER2-expressing breast cancer cell-specific protein delivery
国際学会・会議・シンポジウム等の賞
論文
Abstract Background The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. Results We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. Conclusions In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.
Springer Science and Business Media LLC, 2023年05月19日, Microbial Cell Factories, 22 (1), 英語[査読有り]
研究論文（学術雑誌）
The discovery of novel antibacterial drugs against infectious diseases has decreased over the past few decades because of their poor cost performance. In this study, we report that the nanoassembly of a short-peptide hydrogelator (P1) endowed novel antifungal selectivity to a conventional antifungal drug, amphotericin B (AmB), which expands its application spectrum. Clinical use of AmB is limited because of poor water solubility and poor selectivity in its toxicity, which often causes harmful effects on tissues. P1 was the low-molecular-weight hydrogelator (LMWHg) that showed low cytotoxicity and was enzymatically degraded. In general, an LMWHg entraps foreign hydrophobic molecules inside the hydrophobic space in the self-assembled body. P1 successfully solubilized AmB in water as a form of a nanocomplex (NC) that had a chain-like structure. The NCs showed remarkably low toxicity toward Saccharomyces cerevisiae as a model fungus when compared with free AmB, meaning that P1 suppressed the antifungal activity of AmB via coassembly. The suppressed antifungal activity of AmB recovered when P1 in the NCs was degraded by a protease to liberate AmB from the coassembly with P1. P1 at a high concentration formed a hydrogel incorporating AmB (AmB-P1 gel), in which the antifungal activity of AmB was suppressed as well as that in the NC. The coassembly with AmB affected the morphology of the P1 self-assembly. While S. cerevisiae that did not secrete proteases formed a colony on the AmB-P1 gel, Aspergillus oryzae that secreted proteases did not grow on the AmB-P1 gel at all, resulting in the selective killing of the fungus. Because some of malignant, infectious fungi secrete proteases, the coassembly strategy of conventional antifungal drugs with self-assembling molecules should lead to "drug repositioning" of approved drugs in the health and medical fields.
American Chemical Society (ACS), 2023年01月27日, ACS Applied Nano Materials, 6 (2), 1432 - 1440, 英語[査読有り]
研究論文（学術雑誌）
- Elsevier BV, 2023年01月, Journal of Chromatography B, 1215, 123588 - 123588, 英語
[査読有り]
研究論文（学術雑誌）
Bioconversion of key intermediate metabolites such as mevalonate into various useful chemicals is a promising strategy for microbial production. However, the conversion of mevalonate into isoprenoids requires a supply of adenosine triphosphate (ATP). Light-driven ATP regeneration using microbial rhodopsin is an attractive module for improving the intracellular ATP supply. In the present study, we demonstrated the ATP-consuming conversion of mevalonate to isoprenol using rhodopsin-expressing Escherichia coli cells as a whole-cell catalyst in a medium that does not contain energy cosubstrate, such as glucose. Heterologous genes for the synthesis of isoprenol from mevalonate, which requires three ATP molecules for the series of reactions, and a delta-rhodopsin gene derived from Haloterrigena turkmenica were cointroduced into E. coli. To evaluate the conversion efficiency of mevalonate to isoprenol, the cells were suspended in a synthetic medium containing mevalonate as the sole carbon source and incubated under dark or light illumination (100 μmol m-2 s-1). The specific isoprenol production rates were 10.0 ± 0.9 and 20.4 ± 0.7 μmol gDCW-1 h-1 for dark and light conditions, respectively. The conversion was successfully enhanced under the light condition. Furthermore, the conversion efficiency increased with increasing illumination intensity, suggesting that ATP regenerated by the proton motive force generated by rhodopsin using light energy can drive ATP-consuming reactions in the whole-cell catalyst.
American Chemical Society (ACS), 2022年12月16日, ACS Synthetic Biology, 11 (12), 3966 - 3972, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established. In this study, we attempted to develop an evolutionary engineering method to improve the proton-pumping activity of rhodopsins. We first introduced random mutations into delta-rhodopsin (dR) from Haloterrigena turkmenica using error-prone PCR to generate approximately 7000 Escherichia coli strains carrying the mutant dR genes. Rhodopsin-expressing E. coli with enhanced proton pumping activity have significantly increased survival rates in prolonged saline water. Considering this, we enriched the mutant E. coli cells with higher proton pumping rates by selecting populations able to survive starvation under 50 μmol m-2 s-1 at 37 °C. As a result, we successfully identified two strains, in which proton pumping activity was enhanced two-fold by heterologous expression in E. coli in comparison to wild-type strains. The combined approach of survival testing using saline water and evolutionary engineering methods used in this study will contribute greatly to the discovery of a novel rhodopsin with improved proton pumping activity. This will facilitate the utilization of rhodopsin in industrial applications.
Elsevier BV, 2022年09月25日, Journal of bioscience and bioengineering, 134 (6), 484 - 490, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
- Wiley, 2022年09月, Microbial Biotechnology, 15 (9), 2364 - 2378, 英語
[査読有り]
研究論文（学術雑誌）
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, 2022年07月08日, Frontiers in Chemical Engineering, 4, 880694, 英語研究論文（学術雑誌）
- Elsevier BV, 2022年07月, Journal of Bioscience and Bioengineering, 134 (1), 1 - 6, 英語
[査読有り]
研究論文（学術雑誌）
In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.
Elsevier, 2022年07月, Metabolic Engineering, 72, 227 - 236, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
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, 2022年06月, Communications Biology, 5 (1), 561, 英語[査読有り]
研究論文（学術雑誌）
- {MDPI} {AG}, 2022年04月08日, Life, 12 (4), 557 - 557, 日本語, 国際誌, 国際共著していない
[査読有り][招待有り]
研究論文（学術雑誌）
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 Nature, 2022年03月16日, Nature Communications, 13 (1), 1405, 英語, 国際誌, 国際共著している[査読有り]
研究論文（学術雑誌）
- Elsevier, 2022年03月, Journal of Bioscience and Bioengineering, 133 (3), 208 - 212, 英語, 国際誌, 国際共著していない
[査読有り]
研究論文（学術雑誌）
The methylotrophic yeast species Komagataella phaffii (synonym: Pichia pastoris) is widely used as a host for recombinant protein production. Although several genetic engineering techniques are being employed on K. phaffii, advanced methods such as in vivo DNA assembly in this yeast species are required for synthetic biology applications. In this study, we established a technique for accomplishing one-step in vivo assembly of multiple DNA fragments and genomic integration in K. phaffii. To concurrently achieve an accurate multiple DNA assembly and a high-efficient integration into the target genomic locus in vivo, a K. phaffii strain, lacking a non-homologous end joining-related protein, DNA ligase IV (Dnl4p), that has been reported to improve gene targeting efficiency by homologous recombination, was used. Using green fluorescent protein along with the lycopene biosynthesis, we showed that our method that included a Dnl4p-defective strain permits direct and easy engineering of K. phaffii strains.
American Chemical Society ({ACS}), 2022年01月30日, ACS Synthetic Biology, 11 (2), 644 - 654, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
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年12月, The Journal of General and Applied Microbiology, 67 (6), 265 - 268, 英語, 国際誌研究論文（学術雑誌）
(-)-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.
Spring Nature, 2021年11月11日, Scientific reports, 11 (1), 22126 - 22126, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
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, 2021年06月, Metabolic Engineering Communications, 12, e00169 - e00169, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
β-Nicotinamide mononucleotide (NMN) is, one of the nucleotide compounds, a precursor of NAD+ and has recently attracted attention as a nutraceutical. Here, we develop a whole-cell biocatalyst using Escherichia coli, which enabled selective and effective high production of NMN from the inexpensive feedstock substrates glucose and nicotinamide (Nam). Notably, we identify two actively functional transporters (NiaP and PnuC) and a high-activity key enzyme (Nampt), permitting intracellular Nam uptake, efficient conversion of phosphoribosyl pyrophosphate (PRPP; supplied from glucose) and Nam to NMN, and NMN excretion extracellularly. Further, enhancement of the PRPP biosynthetic pathway and optimization of individual gene expression enable drastically higher NMN production than reported thus far. The strain extracellularly produces 6.79 g l−1 of NMN from glucose and Nam, and the reaction selectivity from Nam to NMN is 86%. Our approach will be promising for low-cost, high-quality industrial production of NMN and other nucleotide compounds using microorganisms.
Elsevier, 2021年05月, Metabolic Engineering, 65, 167 - 177, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism
Springer Nature, 2021年03月23日, Nature Communications, 12 (1), 1846, 英語, 国際誌, 国際共著していない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.[査読有り]
研究論文（学術雑誌）
Melatonin is an indoleamine neurohormone made by the pineal gland. Its receptors, MTNR1A and MTNR1B, are members of the G-protein-coupled receptor (GPCR) family and are involved in sleep, circadian rhythm, and mood disorders, and in the inhibition of cancer growth. These receptors, therefore, represent significant molecular targets for insomnia, circadian sleep disorders, and cancer. The yeast Saccharomyces cerevisiae is an attractive host for assaying agonistic activity for human GPCR. We previously constructed a GPCR-based biosensor employing a high-sensitivity yeast strain that incorporated both a chimeric yeast-human G alpha protein and a bright fluorescent reporter gene (ZsGreen). Similar approaches have been used for simple and convenient measurements of various GPCR activities. In the current study, we constructed a fluorescence-based yeast biosensor for monitoring the signaling activation of human melatonin receptors. We used this system to analyze point mutations, including previously unreported mutations of the consensus sequences of MTNR1A and MTNR1B melatonin receptors and compared their effects. Most mutations in the consensus sequences significantly affected the signaling capacities of both receptors, but several mutations showed differences between these subtype receptors. Thus, this yeast biosensor holds promise for revealing the functions of melatonin receptors.
Wiley, 2021年02月06日, Biotechnology and Bioengineering, 118 (2), 863 - 876, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
Abstract In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a ‘terminator catalog’ by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3′-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.Oxford University Press ({OUP}), 2020年12月16日, Nucleic Acids Research, 48 (22), 13000 - 13012, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Radiosensitizing therapy for cancer treatment that enhances the effect of existing radiation therapy and enables noninvasive therapy has attracted attention. In this study, to achieve target cell-specific noninvasive cancer treatment using a Z(HER2)-bionanocapsule/liposome (BNC/LP), a carrier that binds to human epidermal growth factor receptor 2 (HER2), we evaluated the delivery of anticancer drugs and radiosensitizers and treatment effects in vitro and in vivo in mice. Target cell-specific cytotoxic activity and antitumor effects were confirmed following delivery of doxorubicin-encapsulated particles. In addition, cell damage due to radiosensitizing effects was confirmed in combination with X-ray irradiation following delivery of particles containing polyacrylic acid-modified titanium peroxide nanoparticles as a radiosensitizer. Furthermore, even when the particles were injected via the tail vein of mice, they accumulated in the tumor and exhibited an antitumor effect because of radiosensitization. Therefore, Z(HER2)-BNC/ LP is expected to be a carrier that releases small-molecule drugs into the target cell cytoplasm and delivers a radiosensitizer such as inorganic nanoparticles, enabling combination therapy with X-rays to the target tumor.
American Chemical Society ({ACS}), 2020年11月16日, ACS Applied Bio Materials, 3 (11), 7743 - 7751, 英語, 国際誌, 国際共著していない研究論文（学術雑誌）
BACKGROUND: Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae. RESULTS: The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production. CONCLUSION: These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.
2019年10月15日, Microbial cell factories, 18 (1), 177 - 177, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
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.
Springer Science and Business Media {LLC}, 2019年05月22日, Nature Communications, 10 (1), 2336 - 2336, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
© 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.
Springer Science and Business Media {LLC}, 2019年05月01日, Nature Communications, 10 (1), 国際共著していない[査読有り]
研究論文（学術雑誌）
- Elsevier {BV}, 2019年04月, Journal of Bioscience and Bioengineering, 127 (4), 451 - 457, 英語
[査読有り]
研究論文（学術雑誌）
- American Chemical Society ({ACS}), 2018年12月21日, ACS Synthetic Biology, 7 (12), 2783 - 2789, 英語, 国際誌, 国際共著していない
[査読有り]
研究論文（学術雑誌）
The non-conventional yeast Komagataella phaffii, formerly Pichia pastoris, is a popular host for recombinant protein production. The relatively lower gene targeting efficiency observed in this species occurs due to high levels of non-homologous recombination activity. In the current study, we explored the function of the K. phaffii homolog of DNA ligase IV (Dnl4p) by creating a DNL4-disrupted strain. To assess the roles of non-homologous end joining (NHEJ)-related proteins in this species, strains deleted for either or both genes encoding Dnl4p or the telomeric Ku complex subunit (Ku70p) were generated. These deletions were constructed by either of two distinct marker-recycling methods (yielding either a seamless gene deletion or a Cre-loxP-mediated gene deletion). The resulting dnl4- and/or ku70-deleted K. phaffii strains were used to evaluate gene targeting efficiency in gene knock-out and gene knock-in experiments. The Dnl4p-defective strain showed improved gene targeting efficiency for homologous recombination compared to the wild-type and Ku70p-deffective strains. The dnl4 ku70 double knock-out strain exhibited a further improvement in gene targeting efficiency. Thus, the K. phaffii dnl4 and dnl4 ku70 deletion strains are expected to serve as useful platforms for functional analysis and strain development in this species.
Oxford University Press ({OUP}), 2018年11月01日, FEMS Yeast Research, 18 (7), foy074, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
The methylotrophic yeast
American Society for Microbiology, 2018年08月25日, Applied and Environmental Microbiology, 84 (15), e02882 - 17, 英語, 国際誌, 国際共著していない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 theP. pastoris -specific autonomously replicating sequence (PARS1 ) permit transformation ofP. 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-lengthCen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids inP. pastoris . Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entireCen2 sequence; this episome facilitates genetic manipulation inP. pastoris , providing high transformation efficiency and plasmid stability.IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeastPichia pastoris , a species that permits mass production of heterologous proteins. To date, the genetic engineering ofP. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicatingPichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as theP. pastoris -specific ARS (PARS1 ), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences ofP. 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 ofP. pastoris is ready to be developed.[査読有り]
研究論文（学術雑誌）
- BioMed Central, 2018年06月, Biotechnology for Biofuels, 11 (1), 180, 英語, 国際誌, 国際共著していない
[査読有り]
研究論文（学術雑誌）
- 2018年06月, Langmuir : the ACS journal of surfaces and colloids, 34 (27), 8065 - 8074, 英語
[査読有り]
研究論文（学術雑誌）
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-Blackwell, 2018年04月30日, Biotechnology Journal, 13 (4), 1700522 - 1700522, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
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., 2018年03月09日, Microbial Cell Factories, 17 (1), 38, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
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., 2018年01月01日, Journal of Bioscience and Bioengineering, 125 (1), 76 - 86, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
The yeast Saccharomyces cerevisiae is a useful eukaryotic host organism for studying GPCRs as monomolecular models. Fluorescent reporter gene assays for GPCRs provide a convenient assay for measuring receptor activity using fluorometric instruments. Generally, these assays detect receptor activation by agonistic ligands as the induction of fluorescent reporter expression, whereas antagonistic activities are detected by competition with agonistic ligands, resulting in decreases in fluorescence intensity. In the current study, we established a system for inverted expression of a fluorescent reporter by incorporating a PEST-tag and finding out a promoter inhibited by activation of the GPCR signaling pathway from yeast endogenous promoters. Because agonists prevent fluorescent reporter expression in this system, antagonists compete with agonists and yield increased fluorescence intensity. We used the yeast endogenous pheromone receptor as a model GPCR to demonstrate the feasibility of our system for positive detection targeted at antagonists. Compared to results when only agonists were added to yeast cells, more than 10-fold higher fluorescence intensity was observed when antagonists were added in combination with agonists. The approach described here has the potential to markedly accelerate the identification of GPCR antagonists by providing rapid and straightforward responses.
AMER CHEMICAL SOC, 2017年08月, ACS SYNTHETIC BIOLOGY, 6 (8), 1554 - 1562, 英語[査読有り]
研究論文（学術雑誌）
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.
American Chemical Society ({ACS}), 2017年06月16日, ACS Synthetic Biology, 6 (6), 928 - 935, 英語[査読有り]
研究論文（学術雑誌）
Volume 3, no. 2, e00389-15, 2015. Page 1, column 1, lines 26 and 27: "silencing mediator for retinoic acid and thyroid hormone receptor" should read "singlemolecule real-time."
American Society for Microbiology, 2017年, Genome Announcements, 5 (5), 英語[査読有り]
研究論文（学術雑誌）
The expression of epidermal growth factor receptor (EGFR) across a wide range of tumor cells has attracted attention for use as a tumor marker in drug delivery systems. Therefore, binding molecules with the ability to target EGFR have been developed. Among them, we focused on affibodies that are binding proteins derived from staphylococcal protein A. By displaying affibody (Z(EGFR)) binding to EGFR on the surface of a bio-nanocapsule (BNC) derived from a hepatitis B virus (HBV), we developed an altered BNC (Z(EGFR)-BNC) with a high specificity to EGFR-expressing cells. We considered two different types of Z(EGFR) (Z955 and Z1907), and found that the Z1907 dimer-displaying BNC ([Z1907[(2)-BNC) could effectively bind to EGFR-expressing cells and deliver drugs to the cytosol. Since this study showed that [Z1907](2)-BNC could target EGFR-expressing cells, we would use this particle as a drug delivery carrier for various cancer cells expressing EGFR. (C) 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license.
PERGAMON-ELSEVIER SCIENCE LTD, 2017年01月, Bioorganic and Medicinal Chemistry Letters, 27 (2), 336 - 341, 英語[査読有り]
研究論文（学術雑誌）
The budding yeast Saccharomyces cerevisiae is equipped with G protein-coupled receptors (GPCR). Because the yeast GPCR signaling mechanism is partly similar to that of the mammalian system, S. cerevisiae can be used for a host of mammalian GPCR expression and ligand-mediated activation assays. However, currently available yeast systems require several hours to observe the responses because they depend on the expression of reporter genes. In this study, we attempted to develop a simple GPCR assay system using split luciferase and -arrestin, which are independent of the endogenous S. cerevisiae GPCR signaling pathways. We applied the split luciferase complementation assay method to S. cerevisiae and found that it can be used to analyze the ligand response of the human somatostatin receptor in S. cerevisiae. On the contrary, the response of the pheromone receptor Ste2 was not observed by the assay. Thus, the split luciferase complementation should be free from the effect of the endogenous GPCR signaling. Biotechnol. Bioeng. 2017;114: 1354-1361. (c) 2017 Wiley Periodicals, Inc.
WILEY, 2017年06月, Biotechnology and Bioengineering, 114 (6), 1354 - 1361, 英語[査読有り]
研究論文（学術雑誌）
Bacterial phosphoenol pyruvate carboxylase (PPC) and enzymes in the Entner-Doudoroff (ED) pathway were heter-ologously expressed in Saccharomyces cerevisiae to improve the NADPH supply required for the bio-production of chemicals such as isobutanol. The heterologous expression of PPC from Synechocystis sp. PCC6803 increased in the isobutabol titer 1.45-fold (93.2 +/- 1.6 mg/L) in metabolically engineered S. cerevisiae strains producing isobutanol. This result suggested that the pyruvate and NADPH supply for isobutanol biosynthesis was activated by PPC overexpression. On the other hand, the expression of two enzymes organizing the ED pathway (6-phosphogluconate dehydratase [6PGD] and 2-dehydro-3-deoxy-phosphogluconate aldolase [ICDPGA]) had no effect to isobutabol bio-production. Further analysis, however, revealed that additional expression of 6PGD and KDPGA improved the growth rate of S. cerevisiae strain BY4742 gnd1 Delta. A C-13-labeling experiment using [1 - C-13] glucose also suggested that metabolic flow levels in the ED pathway increased slightly with the additional expression. These results showed that the ED pathway was successfully constructed in S. cerevisiae, even though activity of the pathway was too weak to improve isobutanol biosynthesis. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
SOC BIOSCIENCE BIOENGINEERING JAPAN, 2017年09月, Journal of Bioscience and Bioengineering, 124 (3), 263 - 270, 英語[査読有り]
研究論文（学術雑誌）
Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources.
OXFORD UNIV PRESS, 2017年11月, FEMS Yeast Research, 17 (7), fox061, 英語[査読有り]
研究論文（学術雑誌）
This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/ mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, beta-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources. (C) 2017 Elsevier Ltd. All rights reserved.
ELSEVIER SCI LTD, 2017年12月, Bioresource Technology, 245, 1400 - 1406, 英語[査読有り]
研究論文（学術雑誌）
Green fluorescent protein (GFP), which was originally isolated from jellyfish, is a widely used tool in biological research, and homologs from other organisms are available. However, researchers must determine which GFP is the most suitable for a specific host. Here, we expressed GFPs from several sources in codon-optimized and non-codon-optimized forms in the yeast Saccharomyces cerevisiae, which represents an ideal eukaryotic model. Surprisingly, codon-optimized mWasabi and mNeonGreen, which are typically the brightest GFPs, emitted less green fluorescence than did the other five codon-optimized GFPs tested in S. cerevisiae. Further, commercially available GFPs that have been optimized for mammalian codon usage (e.g., EGFP, AcGFP1 and TagGFP2) unexpectedly exhibited extremely low expression levels in S. cerevisiae. In contrast, codon-optimization of the GFPs for S. cerevisiae markedly increased their expression levels, and the fluorescence intensity of the cells increased by a maximum of 101-fold. Among the tested GFPs, the codon-optimized monomeric mUkG1 from soft coral showed the highest levels of both expression and fluorescence. Finally, the expression of this protein as a fusion-tagged protein successfully improved the reporting system's ability to sense signal transduction and protein-protein interactions in S. cerevisiae and increased the detection rates of target cells using flow cytometry.
NATURE PUBLISHING GROUP, 2016年10月, Scientific Reports, 6, 35932, 英語, 国際誌, 国際共著していない[査読有り]
研究論文（学術雑誌）
G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that represent major molecular targets in pharmaceutical and medicinal fields. Many GPCRs have been shown to form not only homodimers, but also heterodimers that can confer large functional and physiological diversity and are therefore expected to offer new opportunities for the discovery of new drugs. Yeast is a useful host organism that can be used to investigate the interactions between eukaryotic protein pairs, as demonstrated by the yeast two-hybrid (Y2H) system. Previously, we established reporter gene assay systems to screen for GPCR dimer pairs based on the splitubiquitin membrane Y2H (mY2H) system. However, conventional systems only induce reporter gene expressions from the OFF to ON states. In this study, we therefore designed a reporter switching system that can switch the expressions between two reporter genes (one from ON to OFF and the other from OFF to ON) in response to the Y2H readout. To invoke reporter switching, we took advantage of Cre/loxP site-specific recombination. Through optimization of Cre-mediated reporter gene recombination using the split-ubiquitin mY2H system, we built a dual-color reporter switching system to discern the formations of GPCR dimers. This system enabled monitoring of the formations of homodimers and heterodimers of human serotonin 1A receptor or beta(2)-adrenergic receptor as well as homodimers of the yeast endogenous pheromone receptor (Ste2p) in yeast cells. Our reporter switching system may be a useful tool for identifying potential molecular targets among GPCR dimers, and is also applicable to other reporter gene assay systems. (C) 2016 Wiley Periodicals, Inc.
WILEY-BLACKWELL, 2016年10月, Biotechnology and Bioengineering, 113 (10), 2178 - 2190, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
We determined the genome sequence of the thermotolerant yeast Kluyveromyces marxianus strain NBRC1777. The genome of strain NBRC1777 is composed of 4,912 open reading frames (ORFs) on 8 chromosomes, with a total size of 10,895,581 bp, including mitochondrial DNA.
American Society for Microbiology, 2016年, Genome Announcements, 3 (2), e00389 - 15, 英語[査読有り]
研究論文（学術雑誌）
Lignocellulosic hydrolysates contain compounds that inhibit microbial growth and fermentation, thereby decreasing the productivity of biofuel and biochemical production. In particular, the heterocyclic aldehyde furfural is one of the most toxic compounds found in these hydrolysates. We previously demonstrated that Corynebacterium glutamicum converts furfural into the less toxic compounds furfuryl alcohol and 2-furoic acid. To date, however, the genes involved in these oxidation and reduction reactions have not been identified in the C. glutamicum genome. Here, we show that Cgl0331 (designated FudC) is mainly responsible for the reduction of furfural into furfuryl alcohol in C. glutamicum. Deletion of the gene encoding FudC markedly diminished the in vivo reduction of furfural to furfuryl alcohol. Purified His-tagged FudC protein from Escherichia coli was also shown to convert furfural into furfuryl alcohol in an in vitro reaction utilizing NADPH, but not NADH, as a cofactor. Kinetic measurements demonstrated that FudC has a high affinity for furfural but has a narrow substrate range for other aldehydes compared to the protein responsible for furfural reduction in E. coli.
SPRINGER, 2016年03月, Applied Microbiology and Biotechnology, 100 (6), 2685 - 2692, 英語[査読有り]
研究論文（学術雑誌）
A novel terpene synthase ( Tps ) gene isolated from Camellia brevistyla was identified as hedycaryol synthase, which was shown to be expressed specifically in flowers. Camellia plants are very popular because they bloom in winter when other plants seldom flower. Many ornamental cultivars of Camellia have been bred mainly in Japan, although the fragrance of their flowers has not been studied extensively. We analyzed floral scents of several Camellia cultivars by gas chromatography-mass spectrometry (GC-MS) and found that Camellia brevistyla produced various sesquiterpenes in addition to monoterpenes, whereas Camellia japonica and its cross-lines produced only monoterpenes, including linalool as the main product. From a flower of C. brevistyla, we isolated one cDNA encoding a terpene synthase (TPS) comprised of 554 amino acids, which was phylogenetically positioned to a sole gene clade. The cDNA, designated CbTps1, was expressed in mevalonate-pathway-engineered Escherichia coli, which carried the Streptomyces mevalonate-pathway gene cluster in addition to the acetoacetate-CoA ligase gene. A terpene product was purified from recombinant E. coli cultured with lithium acetoacetate, and analyzed by H-1-nulcear magnetic resonance spectroscopy (H-1-NMR) and GC-MS. It was shown that a sesquiterpene hedycaryol was produced, because H-1-NMR signals of the purified product were very broad, and elemol, a thermal rearrangement product from hedycaryol, was identified by GC-MS analysis. Spectroscopic data of elemol were also determined. These results indicated that the CbTps1 gene encodes hedycaryol synthase. Expression analysis of CbTps1 showed that it was expressed specifically in flowers, and hedycaryol is likely to be one of the terpenes that attract insects for pollination of C. brevistyla. A linalool synthase gene, which was isolated from a flower of Camellia saluenensis, is also described.
SPRINGER, 2016年04月, Planta, 243 (4), 959 - 972, 英語[査読有り]
研究論文（学術雑誌）
G-protein-coupled receptors (GPCRs) are physiologically important transmembrane proteins that sense signaling molecules such as hormones, neurotransmitters, and various sensory stimuli; GPCRs represent major molecular targets for drug discovery. Although GPCRs traditionally have been thought to function as monomers or homomers, in the recent years these proteins have also been shown to function as heteromers. Heteromerization among GPCRs is expected to generate potentially large functional and physiological diversity and to provide new opportunities for drug discovery. However, due to the existence of numerous combinations, the larger universe of possible GPCR heteromers is unknown, and thus its functional significance is still poorly understood. The oligomerization of GPCRs in living cells now has been demonstrated in mammalian cells and in native tissues by using genetic, biochemical, and physiological approaches, as well as various resonance energy transfer (RET) technologies. In addition, the yeast Saccharomyces cerevisiae, which can serve as a biosensor for monitoring eukaryotic biological processes, can also be used for the identification of functionally significant heteromer pairs of GPCRs. In this review, we focus on studies of GPCR oligomers, and summarize the technologies used to evaluate GPCR oligomerization. We additionally consider the potential limitations of these methods at present, and envision the possible future applications of these techniques.
BENTHAM SCIENCE PUBL LTD, 2016年, Current Medicinal Chemistry, 23 (16), 1638 - 1656, 英語[査読有り]
研究論文（学術雑誌）
2-Phenylethanol (2-PE) is a higher aromatic alcohol that is used in the cosmetics and food industries. The budding yeast Saccharomyces cerevisiae is considered to be a suitable host for the industrial production of higher alcohols, including 2-PE. To produce 2-PE from glucose in S. cerevisiae, we searched for suitable 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes of the Ehrlich pathway for overexpression in strain YPH499, and found that overexpression of the ARO10 and/or ADH1 genes increased 2-PE production from glucose. Further, we screened ten BY4741 single-deletion mutants of genes involved in the competing pathways for 2-PE production, and found that strains aro8 Delta and aat2 Delta displayed increased 2-PE production. Based on these results, we engineered a BY4741 strain that overexpressed ARO10 and contained an aro8 Delta deletion, and demonstrated that the strain produced 96 mg/L 2-PE from glucose as the sole carbon source. As this engineered S. cerevisiae strain showed a significant increase in 2-PE production from glucose without the addition of an intermediate carbon substrate, it is a promising candidate for the large-scale production of 2-PE. (C) 2015 The Society for Biotechnology, Japan. All rights reserved.
SOC BIOSCIENCE BIOENGINEERING JAPAN, 2016年07月, Journal of Bioscience and Bioengineering, 122 (1), 34 - 39, 英語[査読有り]
研究論文（学術雑誌）
Background: Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays beta-mannanase and beta-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. Results: Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered beta-mannanase and beta-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-beta-D-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-beta-D-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. Conclusions: We successfully displayed beta-mannanase and beta-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying beta-mannanase and beta-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering beta-mannanase and beta-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.
BIOMED CENTRAL LTD, 2016年09月, Biotechnology for Biofuels, 9 (1), 188, 英語[査読有り]
研究論文（学術雑誌）
Background: Red yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an antioxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed. Results: Xanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain. Conclusions: In this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in X. dendrorhous.
BIOMED CENTRAL LTD, 2016年09月, Microbial Cell Factories, 15 (1), 155, 英語[査読有り]
研究論文（学術雑誌）
Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived fromS. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae alpha-mating pheromone (MF alpha 1SP) were constructed for cell-surface display of Aspergillus aculeatus beta-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MF alpha 1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MF alpha 1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. (C) 2016 Wiley Periodicals, Inc.
WILEY-BLACKWELL, 2016年11月, Biotechnology and Bioengineering, 113 (11), 2358 - 2366, 英語[査読有り]
研究論文（学術雑誌）
Sake yeasts belong to the budding yeast species Saccharomyces cerevisiae and have high fermentation activity and ethanol production. Although the traditional crossbreeding of sake yeasts is a time-consuming and inefficient process due to the low sporulation rates and spore viability of these strains, considerable effort has been devoted to the development of hybrid strains with superior brewing characteristics. In the present work, we describe a growth selection system for a- and α-type cells aimed at the crossbreeding of industrial yeasts, and performed hybridizations with sake yeast strains Kyokai No. 6, No. 7 and No. 9 to examine the feasibility of this approach. We successfully generated both a- and α-type strains from all parental strains, and acquired six types of hybrids by outcrossing. One of these hybrid strains was subjected to continuous crossbreeding, yielding the multi-hybrid strain, which inherited the genetic characteristics of Kyokai No. 6, No. 7 and No. 9. Notably, because all of the genetic modifications of the yeast cells were introduced using plasmids, these traits can be easily removed. The approach described here has the potential to markedly accelerate the crossbreeding of industrial yeast strains with desirable properties.
Springer Verlag, 2016年12月01日, AMB Express, 6 (1), 45, 英語[査読有り]
研究論文（学術雑誌）
- (公社)日本生物工学会, 2015年09月, 日本生物工学会大会講演要旨集, 平成27年度, 337 - 337, 日本語
Microbes employ cell membranes for reducing exogenous stresses. Polyamino acid display on microbial cell surfaces and their effects on microbial chemical stress tolerance were examined. Growth analysis revealed that displays of polyarginine, polyaspartate and polytryptophan substantially enhanced tolerance of Escherichia coli to NaCl. A titration assay indicated that polyarginine and polyaspartate altered cell surface charges, implying tolerance enhancement via ion atmosphere and/or ionic bond network formations for electrostatic ion repulsion. The enhancement by polytryptophan may have arisen from surface hydrophobicity increase for hydrophobic ion exclusion, because of a strong correlation between hydrophobic characters of amino acids and their effects on tolerance enhancement. The display also enhanced tolerance to other salts and/or alcohols in E. coli and to NaCl in Saccharomyces cerevisiae. Thus polyamino acid display has the potential as an approach for conferring chemical stress tolerance on various microbes.
SPRINGER, 2015年02月, Biotechnology letters., 37 (2), 429 - 435, 英語[査読有り]
研究論文（学術雑誌）
Conferring biomass hydrolysis activity on yeast through genetic engineering has paved the way for the development of groundbreaking processes for producing liquid fuels and commodity chemicals from lignocellulosic biomass. However, the overproduction and misfolding of heterologous and endogenous proteins can trigger cellular stress, increasing the metabolic burden and retarding growth. Improving the efficiency of lignocellulosic breakdown requires engineering of yeast secretory pathway based on system-wide metabolic analysis as well as DNA constructs for enhanced cellulase gene expression with advanced molecular biology tools. Also, yeast is subjected to severe stress due to toxic compounds generated during lignocellulose pretreatment in consolidated saccharification and fermentation processes. The prospect for development of robust yeast strains makes combining evolutionary and rational engineering strategies.
ELSEVIER SCI LTD, 2015年12月, Current Opinion in Chemical Biology, 29, 1 - 9, 英語[査読有り]
研究論文（学術雑誌）
Protein-protein interactions (PPIs) are crucial for the vast majority of biological processes. We previously constructed a G gamma recruitment system to screen PPI candidate proteins and desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. The methods utilized a target protein fused to a mutated G-protein gamma subunit (G gamma(cyto)) lacking the ability to localize to the inner leaflet of the plasma membrane. However, the previous systems were adapted to use only soluble cytosolic proteins as targets. Recently, membrane proteins have been found to form the principal nodes of signaling involved in diseases and have attracted a great deal of interest as primary drug targets. Here, we describe new protocols for the G gamma recruitment systems that are specifically designed to use membrane proteins as targets to overcome previous limitations. These systems represent an attractive approach to exploring novel interacting candidates and affinity-altered protein variants and their interactions with proteins on the inner side of the plasma membrane, with high specificity and selectivity.
NATURE PUBLISHING GROUP, 2015年11月, Scientific Reports, 5, 16723, 英語[査読有り]
研究論文（学術雑誌）
Background: Isobutanol is an important biorefinery target alcohol that can be used as a fuel, fuel additive, or commodity chemical. Baker's yeast, Saccharomyces cerevisiae, is a promising organism for the industrial manufacture of isobutanol because of its tolerance for low pH and resistance to autolysis. It has been reported that gene deletion of the pyruvate dehydrogenase complex, which is directly involved in pyruvate metabolism, improved isobutanol production by S. cerevisiae. However, the engineering strategies available for S. cerevisiae are immature compared to those available for bacterial hosts such as Escherichia coli, and several pathways in addition to pyruvate metabolism compete with isobutanol production. Results: The isobutyrate, pantothenate or isoleucine biosynthetic pathways were deleted to reduce the outflow of carbon competing with isobutanol biosynthesis in S. cerevisiae. The judicious elimination of these competing pathways increased isobutanol production. ILV1 encodes threonine ammonia-lyase, the enzyme that converts threonine to 2-ketobutanoate, a precursor for isoleucine biosynthesis. S. cerevisiae mutants in which ILV1 had been deleted displayed 3.5-fold increased isobutanol productivity. The Delta ILV1 strategy was further combined with two previously established engineering strategies (activation of two steps of the Ehrlich pathway and the transhydrogenase-like shunt), providing 11-fold higher isobutanol productivity as compared to the parent strain. The titer and yield of this engineered strain was 224 +/- 5 mg/L and 12.04 +/- 0.23 mg/g glucose, respectively. Conclusions: The deletion of competitive pathways to reduce the outflow of carbon, including ILV1 deletion, is an important strategy for increasing isobutanol production by S. cerevisiae.
BIOMED CENTRAL LTD, 2015年04月, Microbial Cell Factories, 14, 62, 英語[査読有り]
研究論文（学術雑誌）
Background: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues. Results: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells. Conclusions: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.
BIOMED CENTRAL LTD, 2015年02月, Journal of Nanobiotechnology, 13 (1), 15, 英語[査読有り]
研究論文（学術雑誌）
Olfaction depends on the selectivity and sensitivity of olfactory receptors. Previous attempts at constructing a mammalian olfactory receptor-based artificial odorant sensing system in the budding yeast Saccharomyces cerevisiae suffered from low sensitivity and activity. This result may be at least in part due to poor functional expression of olfactory receptors and/or limited solubility of some odorants in the medium. In this study, we examined the effects of two types of accessory proteins, receptor transporting protein 1 short and odorant binding proteins, in improving odor-mediated activation of olfactory receptors expressed in yeast. We found that receptor transporting protein 1 short enhanced the membrane expression and ligand-induced responses of some olfactory receptors. Coexpression of odorant binding proteins of the silkworm moth Bombyx mori enhanced the sensitivity of a mouse olfactory receptor. Our results suggest that different classes of accessory proteins can confer sensitive and robust responses of olfactory receptors expressed in yeast. Inclusion of accessory proteins may be essential in the future development of practical olfactory receptor-based odorant sensors. (C) 2014 Elsevier Inc. All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2015年02月, Analytical Biochemistry, 471, 1 - 8, 英語[査読有り]
研究論文（学術雑誌）
Recombinant yeast strains that display heterologous amylolytic enzymes on their cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system are considered as promising biocatalysts for direct ethanol production from starchy materials. For the effective hydrolysis of these materials, the ratio optimization of multienzyme activity displayed on the cell surface is important. In this study, we have presented a ratio control system of multienzymes displayed on the yeast cell surface by using different GPI-anchoring domains. The novel gene cassettes for the cell-surface display of Streptococcus bovis alpha-amylase and Rhizopus oryzae glucoamylase were constructed using the Saccharomyces cerevisiae SED1 promoter and two different GPI-anchoring regions derived from Saccharomyces cerevisiae SED1 or SAG1. These gene cassettes were integrated into the Saccharomyces cerevisiae genome in different combinations. Then, the cell-surface alpha-amylase and glucoamylase activities and ethanol productivity of these recombinant strains were evaluated. The combinations of the gene cassettes of these enzymes affected the ratio of cell-surface alpha-amylase and glucoamylase activities and ethanol productivity of the recombinant strains. The highest ethanol productivity from raw starch was achieved by the strain harboring one alpha-amylase gene cassette carrying the SED1-anchoring region and two glucoamylase gene cassettes carrying the SED1-anchoring region (BY-AASS/GASS/GASS). This strain yielded 22.5 +/- 0.6 g/L of ethanol from 100 g/L of raw starch in 120 h of fermentation.
SPRINGER, 2015年02月, Applied Microbiology and Biotechnology, 99 (4), 1655 - 1663, 英語[査読有り]
研究論文（学術雑誌）
An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, a-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.
ROYAL SOC CHEMISTRY, 2015年, Nanoscale, 7 (11), 4956 - 4963, 英語[査読有り]
研究論文（学術雑誌）
The monoamine neurotransmitter serotonin (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. One such receptor, the 5-HT1A receptor (HTR1A), is the most widely studied subtype and represents a significant molecular target in medicinal and therapeutic fields. Yeast-based fluorescent reporter systems have proven to be especially useful for GPCR assays, since detection using a fluorescent reporter considerably simplifies measurement procedures. However, previously reported systems using enhanced green fluorescent protein (EGFP) as the reporter in yeast still showed low signal-to-noise (S/N) ratios, making EGFP difficult to apply as an easily accessible tool. Therefore, we constructed a refined yeast-based GPCR biosensor employing a high-sensitivity strain that incorporated both a G-engineered receptor and a fluorescent reporter (ZsGreen). As we report here, the refined yeast-based fluorescent biosensor was applied successfully to antagonist characterization and analysis of site-directed mutants of the HTR1A receptor. Pindolol, a known antagonist of HTR1A, specifically inhibited agonist-induced signaling, demonstrating the ease of evaluating inhibition effects using our reporter strain. Characterization of site-specific receptor mutants confirmed the role of specific targeted residues, including the highly conserved DRY motif, in the activation of HTR1A. Thus, our refined yeast biosensor strain, which incorporates a ZsGreen reporter and an engineered G receptor, is expected to serve as a simple and practical sensing tool for evaluating the ligand candidates and defining residues important to the function of human GPCRs. Biotechnol. Bioeng. 2015;112: 1906-1915. (c) 2015 Wiley Periodicals, Inc.
WILEY-BLACKWELL, 2015年09月, Biotechnology and Bioengineering, 112 (9), 1906 - 1915, 英語[査読有り]
研究論文（学術雑誌）
A novel degradable polyanion, poly(phthalic ethylene glycol ester), was synthesized in one pot in a single step. The degradable polyanion assembles with various polycations to form layer-by-layer films that can encapsulate physiologically active biomolecules. Polyanion degradation can induce film disassembly and release of the encapsulated functional protein.
ROYAL SOC CHEMISTRY, 2015年, Chemical Communications, 51 (98), 17447 - 17450, 英語[査読有り]
研究論文（学術雑誌）
- (公社)日本生物工学会, 2014年08月, 日本生物工学会大会講演要旨集, 平成26年度, 161 - 161, 日本語
- 2014年08月, Bio-protocol, 4 (16), e1206, 英語Signaling assays for detection of human G-protein-coupled receptors in yeast
[査読有り]
研究論文（学術雑誌）
Neurotensin receptor type-I (NTSR1) is a member of the G-protein-coupled receptor (GPCR) family. The natural ligand of NTSR1 is neurotensin (NT), a neuromodulator of the central nervous system. Because NT is also involved in many oncogenic actions, the signaling mediator NTSR1 is a significant molecular target in medicinal and therapeutic fields. In the current study, we constructed a fluorescence-based microbial yeast biosensor that can monitor the activation of human NTSR1 signaling responding to its agonist. To increase the sensitivity of the biosensor, a yeast strain with the green fluorescent protein (GFP) reporter gene was genetically engineered to enhance binding with human NTSRI expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein a-subunit (G alpha) in yeast Gpalp were substituted with the equivalent human G alpha, sequences (Gpal/G alpha(q) transplant). After optimizing the assay conditions, the G alpha-engineered yeast demonstrated significantly improved sensing for NTSR1 signaling. Because detection using a GFP fluorescence reporter considerably simplifies the measurement procedure, this microbial fluorescence sensor holds promise for use in the diagnosis of NTSR1-associated diseases and the development of agonists. (C) 2013 Elsevier Inc. All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2014年02月, Analytical Biochemistry, 446 (1), 37 - 43, 英語[査読有り]
研究論文（学術雑誌）
Understanding the role of G-protein-coupled receptor (GPCR) dimerization in cellular function has now become a major research focus. The potentially large functional and physiological diversity of dimerization among GPCRs is expected to provide opportunities for novel drug discovery. However, there is currently a lack of cell-based assays capable of specific profiling for the functional consequences of dimerization linked to ligand-mediated signaling. Here, we present an advanced method to simultaneously analyze the dimerization and ligand response of GPCRs using two yeast-based systems for split-ubiquitin two-hybrid assay and G-protein signaling assay. To permit simultaneous detection, we established a two-color (dual-color) fluorescence reporter gene assay using enhanced green fluorescent protein (EGFP) and a far-red derivative of the tetrameric fluorescent protein DsRed-Express2 (E2-Crimson). In the present study, we tested our method first by analyzing dimerization and ligand-mediated signaling by the yeast endogenous pheromone receptor (Ste2p). Second, we showed that the system facilitated mutational analysis of domains involved in dimerization and signaling by Ste2p. Third, we successfully demonstrated that the system could simultaneously monitor homo- and hetero-dimerization and somatostatin-induced signaling in the test case of the human SSTR5 somatostatin receptor. Our strategy is expected to provide a useful tool for the elucidation of molecular biological functions of GPCR dimers and for the screening of GPCR dimer-specific agonistic ligands. Biotechnol. Bioeng. 2014;111: 586-596. (c) 2013 Wiley Periodicals, Inc.
WILEY, 2014年03月, Biotechnology and Bioengineering, 111 (3), 586 - 596, 英語[査読有り]
研究論文（学術雑誌）
Background: An affibody-displaying bio-nanocapsule (Z(HER2)-BNC) with a hepatocyte specificity derived from hepatitis B virus (HBV) was converted into an affibody, Z(HER2), that recognizes HER2 receptors. This affibody was previously reported to be the result of the endocytosis-dependent specific uptake of proteins and siRNA into target cancer cells. To assist the endosomal escape of inclusions, a helper lipid with pH-sensitive fusogenic ability (1,2-dioleoyl-sn-glycero-3-phos phoethanolamine; DOPE) was conjugated with a Z(HER2)-BNC. Findings: In this study, we displayed a pH-sensitive fusogenic GALA peptide on the surface of a particle in order to confer the ability of endosomal escape to a Z(HER2)-BNC. A GALA-displaying Z(HER2)-BNC purified from yeast uneventfully formed a particle structure. Furthermore, endosomal escape of the particle was facilitated after endocytic uptake and release of the inclusions to the cytoplasm without the cell toxicity. Conclusion: The genetic fusion of a GALA peptide to the virus-like particle confers the ability of endosomal escape.
BIOMED CENTRAL LTD, 2014年04月, Journal of Nanobiotechnology, 12 (1), 11, 英語[査読有り]
研究論文（学術雑誌）
Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S.cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering.
WILEY-BLACKWELL, 2014年05月, FEMS Yeast Research, 14 (3), 399 - 411, 英語[査読有り]
研究論文（学術雑誌）
Techniques using nanotechnology in the detection and treatment of cancers have made great progress in multidisciplinary fields. The advances in drug delivery systems (DDSs) have been supported mainly by the development of varied nanoparticles (NPs). Although the NPs based on organic and inorganic materials are integral parts in DDSs, bio-nanoparticles containing biopolymer and virus-like particles (VLPs) are attractive biomaterials for DDSs because of their unique features originating in bio-based materials, such as biocompatibility, biodegradability and low immunogenicity. It is notable that these NPs additionally have a great advantage to enable the easy and flexible alteration of their features by genetic engineering approaches. Controlling the sequence and oligomeric process of polypeptide genes permits a variety of choices in type or size of biopolymeric NPs (e.g., elastin-like polypeptide NPs). In contrast, the functional genes are often inserted into the coding sequences for self-assembled proteins to give the VLPs (e.g., hemagglutinating virus of Japan, adeno-associated virus, human immunodeficiency virus-1, simian virus 40 and hepatitis B virus) additional functions. Thus, genetic engineering readily allow alterations of the properties of NPs (e.g., particle shape, size and stability) and grant of new abilities (e.g., cell-specificity and drug loading and release). In this review, we introduce recent advances in bio-nanoparticles from the standpoint of engineering.
AMER SCIENTIFIC PUBLISHERS, 2014年09月, Journal of Biomedical Nanotechnology, 10 (9), 2063 - 2085, 英語[査読有り]
研究論文（学術雑誌）
Molecules that can control protein-protein interactions (PPIs) have recently drawn attention as new drug pipeline compounds. Here, we report a technique to screen desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. We previously constructed a screening system based on a target protein fused to a mutated G-protein gamma subunit (G gamma(cyto)) lacking membrane localization ability. This ability, required for signal transmission, is restored by recruiting G gamma(cyto) into the membrane only when the target protein interacts with an artificially membrane-anchored candidate protein, thereby allowing interacting partners (G gamma recruitment system) to be searched and identified. In the present study, the G gamma recruitment system was altered by integrating the cytosolic expression of a third protein as a competitor to set a desirable affinity threshold. This enabled the reliable selection of both affinity-enhanced and affinity-attenuated protein variants. The presented approach may facilitate the development of therapeutic proteins that allow the control of PPIs.
PUBLIC LIBRARY SCIENCE, 2014年09月, Plos One, 9 (9), e108229, 英語[査読有り]
研究論文（学術雑誌）
The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD(+) and NADPH/NADP(+) ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.
SPRINGER, 2014年10月, Applied Microbiology and Biotechnology, 98 (20), 8675 - 8683, 英語[査読有り]
研究論文（学術雑誌）
Angiotensin II (Ang II) type 1 receptor (AGTR1) is a G-protein-coupled receptor (GPCR). Its natural ligand, Ang II, is an important effector molecule controlling blood pressure and volume in the cardiovascular system, and is consequently involved in various diseases such as hypertension and heart failure. Thus, the signaling mediator, AGTR1, is a significant molecular target in medicinal and therapeutic fields. Yeast is a useful organism for sensing GPCR signaling because it provides a simplified version of the complicated machinery used by mammalian cells for signal transduction. Although yeast cells can successfully transmit a signal through a variety of human GPCRs expressed in the cell membrane, there have been no reports of the functional activation of AGTR1-mediated signaling in yeast cells. In the present study, we introduced a single mutation into human AGTR1 and used yeast-human chimeric G alpha to exert the functional activation of AGTR1 in yeast cells. The engineered yeast cells expressing AGTR1 mutated at Asn295 and the chimeric G alpha successfully transmitted the signal inside the yeast cells in response to Ang II peptide and its analogs (Ang III and Ang IV peptides) added to the assay medium. Further, we demonstrated that the autocrine Ang II peptide and its analog, produced and secreted by the engineered yeast cells, could by themselves promote AGTR1-mediated signaling. This means that screening for agonistic peptides with various sequences from a self-produced genetic library would be a viable strategy. Thus, the constructed yeast biosensor, integrating an Asn295-mutated AGTR1 receptor, will be valuable in the design of drugs to treat AGTR1-related diseases. (C) 2014 Wiley Periodicals, Inc.
WILEY-BLACKWELL, 2014年11月, Biotechnology and Bioengineering, 111 (11), 2220 - 2228, 英語[査読有り]
研究論文（学術雑誌）
Synthetic bioengineering is a strategy for developing useful microbial strains with innovative biological functions. Novel functions are designed and synthesized in host microbes with the aid of advanced technologies for computer simulations of cellular processes and the system-wide manipulation of host genomes. Here, we review the current status and future prospects of synthetic bioengineering in the yeast Saccharomyces cerevisiae for bio-refinery processes to produce various commodity chemicals from lignocellulosic biomass. Previous studies to improve assimilation of xylose and production of glutathione and butanol suggest a fixed pattern of problems that need to be solved, and as a crucial step, we now need to identify promising targets for further engineering of yeast metabolism. Metabolic simulation, transcriptomics, and metabolomics are useful emerging technologies for achieving this goal, making it possible to optimize metabolic pathways. Furthermore, novel genes responsible for target production can be found by analyzing large-scale data. Fine-tuning of enzyme activities is essential in the latter stage of strain development, but it requires detailed modeling of yeast metabolic functions. Recombinant technologies and genetic engineering are crucial for implementing metabolic designs into microbes. In addition to conventional gene manipulation techniques, advanced methods, such as multicistronic expression systems, marker-recycle gene deletion, protein engineering, cell surface display, genome editing, and synthesis of very long DNA fragments, will facilitate advances in synthetic bioengineering. (C) 2012 Elsevier B.V. All rights reserved.
ELSEVIER SCIENCE BV, 2013年01月, Journal of Biotechnology, 163 (2), 204 - 216, 英語[査読有り]
研究論文（学術雑誌）
Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase (MDH2), and pyruvate carboxylase (PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38 +/- 0.01 g g(-1) xylose consumed) was improved from that of the control strain (0.31 +/- 0.01 g g(-1) xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1-MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1-MDH2-PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells.
SPRINGER, 2013年02月, Applied Microbiology and Biotechnology, 97 (4), 1669 - 1678, 英語[査読有り]
研究論文（学術雑誌）
Several alcohol dehydrogenase (ADH)-related genes have been identified as enzymes for reducing levels of toxic compounds, such as, furfural and/or 5-hydroxymethylfurfural (5-HMF), in hydrolysates of pretreated lignocelluloses. To date, overexpression of these ADH genes in yeast cells have aided ethanol production from glucose or glucose/xylose mixture in the presence of furfural or 5-HMF. However, the effects of these ADH isozymes on ethanol production from xylose as a sole carbon source remain uncertain. We showed that overexpression of mutant NADH-dependent ADH1 derived from TMB3000 strain in the recombinant Saccharomyces cerevisiae, into which xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway of Pichia stipitis has been introduced, improved ethanol production from xylose as a sole carbon source in the presence of 5-HMF. Enhanced furan-reducing activity is able to regenerate NAD+ to relieve redox imbalance, resulting in increased ethanol yield arising from decreased xylitol accumulation. In addition, we found that overexpression of wild-type ADH1 prevented the more severe inhibitory effects of furfural in xylose fermentation as well as overexpression of TMB3000-derived mutant. After 120 h of fermentation, the recombinant strains overexpressing wild-type and mutant ADH1 completely consumed 50 g/L xylose in the presence of 40 mM furfural and most efficiently produced ethanol (15.70 g/L and 15.24 g/L) when compared with any other test conditions. This is the first report describing the improvement of ethanol production from xylose as the sole carbon source in the presence of furan derivatives with xylose-utilizing recombinant yeast strains via the overexpression of ADH-related genes. © 2012 Springer-Verlag.
2013年03月, Applied Microbiology and Biotechnology, 97 (6), 2597 - 2607, 英語[査読有り]
研究論文（学術雑誌）
Capsid-like particles consisting of a hepatitis B core (HBc) protein have been studied for their potential as carriers for drug delivery systems (DDS). The hollow HBc particle, which is formed by the self-assembly of core proteins comprising 183 aa residues, has the ability to bind to various cells non-specifically via the action of an arginine-rich domain. In this study, we developed an engineered HBc particle that specifically recognizes and targets human epidermal growth factor receptor-related 2 (HER2)-expressing breast cancer cells. To despoil the non-specific binding property of an HBc particle, we genetically deleted the C-terminal 150-183 aa part of the core protein that encodes the arginine-rich domain (delta HBc). Then, we genetically inserted a Z(HER2) affibody molecule into the 78-81 aa position of the core protein to confer the ability of target-cell-specific recognition. The constructed Z(HER2)-displaying HBc (Z(HER2)-delta HBc) particle specifically recognized HER2-expressing SKBR3 and MCF-7 breast cancer cells. In addition, the Z(HER2)-delta HBc particle exhibited different binding amounts in accordance with the HER2 expression levels of cancer cells. These results show that the display of other types of affibody molecules on HBc particles would be an expandable strategy for targeting several kinds of cancer cells that would help enable a pinpoint DDS.
OXFORD UNIV PRESS, 2013年03月, Journal of Biochemistry, 153 (3), 251 - 256, 英語[査読有り]
研究論文（学術雑誌）
A bio-nanocapsule derived from the hepatitis B virus (HBV) is expected to be useful as a drug delivery system carrier. Because various types of bio-nanocapsules have been developed, a simple and versatile purification method for bio-nanocapsules would be useful. Therefore, this study was focused on establishing a simple purification method using affinity chromatography by inserting a histidine tag (His-tag) into a bio-nanocapsule. The method achieved a simple, one-step purification with a yield that was 2.5-fold higher than conventional ultracentrifugation, and thus would be a desirable alternative method for recombinant virus-like particle purification. Crown Copyright (c) 2013 Published by Elsevier B.V. All rights reserved.
ELSEVIER SCIENCE BV, 2013年05月, Journal of Virological Methods, 189 (2), 393 - 396, 英語[査読有り]
研究論文（学術雑誌）
The biorefinery manufacturing process for producing chemicals and liquid fuels from biomass is a promising approach for securing energy and resources. To establish cost-effective fermentation of lignocellulosic biomass, the consolidation of sacccharification and fermentation processes is a desirable strategy, but requires the development of microorganisms capable of cellulose/hemicellulose hydrolysis and target chemical production. Such an endeavor requires a large number of prerequisites to be realized, including engineering microbial strains with high cellulolytic activity, high product yield, productivities, and titers, ability to use many carbon sources, and resistance to toxic compounds released during the pretreatment of lignocellulosic biomass. Researchers have focused on either engineering naturally cellulolytic microorganisms to improve product-related properties or modifying non-cellulolytic organisms with high product yields to become cellulolytic. This article reviews recent advances in the development of microorganisms for the production of renewable chemicals and advanced biofuels, as well as ethanol, from lignocellulosic materials through consolidated bioprocessing. (C) 2012 Elsevier Ltd. All rights reserved.
ELSEVIER SCI LTD, 2013年05月, Bioresource Technology, 135, 513 - 522, 英語[査読有り]
研究論文（学術雑誌）
Background: Small interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction. However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required. Results: In this study, we used a carrier that combined Z(HER2)-displaying bio-nanocapsule (derived from hepatitis B virus surface antigen) and liposomes in a complex in order to investigate the feasibility of effective and target-cell-specific RNAi applications. As a result, by observing RNAi only in HER2-expressing breast cancer cells, using our proposed methodology, we successfully demonstrated target-cell-specific delivery and effective function expression of siRNA. Conclusions: These findings show that, in the field of nucleic acid medicine, Z(HER2)-BNC/ LP can be a useful carrier for siRNA delivery, and could also become a useful tool for gene silencing and to accomplish protein knock-down.
BIOMED CENTRAL LTD, 2013年06月, Journal of Nanobiotechnology, 11 (1), 19, 英語[査読有り]
研究論文（学術雑誌）
Potentially immeasurable heterodimer combinations of human G-protein-coupled receptors (GPCRs) result in a great deal of physiological diversity and provide a new opportunity for drug discovery. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood. Thus, the identification of GPCR dimer pairs has been a major challenge. Here, we established a specialized method to screen potential heterodimer partners of human GPCRs based on the split-ubiquitin membrane yeast two-hybrid system. We demonstrate that the mitogen-activated protein kinase (MAPK) signal-independent method can detect ligand-induced conformational changes and rapidly identify heterodimer partners for target GPCRs. Our data present the abilities to apply for the intermolecular mapping of interactions among GPCRs and to uncover potential GPCR targets for the development of new therapeutic agents.
PUBLIC LIBRARY SCIENCE, 2013年06月, PLoS ONE, 8 (6), e66793, 英語[査読有り]
研究論文（学術雑誌）
Metabolic inhibitors were applied for chemical regulation of central carbon metabolism in Saccharomyces cerevisiae. S. cerevisiae was treated with 10 metabolic inhibitors with various modes of action, and their activities were evaluated using a growth inhibition assay. Among the 6 active inhibitors, the effects of pyrazole (alcohol dehydrogenase inhibitor) and TTA (2-thenoyltrifluoloacetone, succinate dehydrogenase inhibitor) were analyzed in detail. The flask-scale batch-fermentation test showed that ethanol yield was reduced to 0.10 +/- 0.01 g g(-1) and glycerol yield increased to 0.26 +/- 0.01 g g(-1) on treatment with pyrazole at 5.0 g L-1, indicating that multiple isozymes of alcohol dehydrogenase were simultaneously inhibited. The multi-targeted metabolic profiling analysis revealed that, although the TTA and pyrazole treatments affected the profiles of all central carbon metabolites in distinct manners, the level of fructose-1,6-bisphosphate commonly increased in the TTA- and pyrazole-treated S. cerevisiae by an unknown mechanism. These results demonstrate that chemical regulation of the central carbon metabolism could be used as an alternative tool to control microbial cell factories for bioproduction, or as a chemical probe to investigate the metabolic systems of useful microorganisms. (C) 2013, The Society for Biotechnology, japan. All rights reserved.
SOC BIOSCIENCE BIOENGINEERING JAPAN, 2013年07月, Journal of Bioscience and Bioengineering, 116 (1), 59 - 64, 英語[査読有り]
研究論文（学術雑誌）
Background: Isobutanol is an important target for biorefinery research as a next-generation biofuel and a building block for commodity chemical production. Metabolically engineered microbial strains to produce isobutanol have been successfully developed by introducing the Ehrlich pathway into bacterial hosts. Isobutanol-producing baker's yeast (Saccharomyces cerevisiae) strains have been developed following the strategy with respect to its advantageous characteristics for cost-effective isobutanol production. However, the isobutanol yields and titers attained by the developed strains need to be further improved through engineering of S. cerevisiae metabolism. Results: Two strategies including eliminating competing pathways and resolving the cofactor imbalance were applied to improve isobutanol production in S. cerevisiae. Isobutanol production levels were increased in strains lacking genes encoding members of the pyruvate dehydrogenase complex such as LPD1, indicating that the pyruvate supply for isobutanol biosynthesis is competing with acetyl-CoA biosynthesis in mitochondria. Isobutanol production was increased by overexpression of enzymes responsible for transhydrogenase-like shunts such as pyruvate carboxylase, malate dehydrogenase, and malic enzyme. The integration of a single gene deletion lpd1 Delta and the activation of the transhydrogenase-like shunt further increased isobutanol levels. In a batch fermentation test at the 50-mL scale from 100 g/L glucose using the two integrated strains, the isobutanol titer reached 1.62 +/- 0.11 g/L and 1.61 +/- 0.03 g/L at 24 h after the start of fermentation, which corresponds to the yield at 0.016 +/- 0.001 g/g glucose consumed and 0.016 +/- 0.0003 g/g glucose consumed, respectively. Conclusions: These results demonstrate that downregulation of competing pathways and metabolic functions for resolving the cofactor imbalance are promising strategies to construct S. cerevisiae strains that effectively produce isobutanol.
BIOMED CENTRAL LTD, 2013年12月, Microbial Cell Factories, 12 (1), 119, 英語[査読有り]
研究論文（学術雑誌）
G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer's disease and Parkinson's disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).
PUBLIC LIBRARY SCIENCE, 2013年12月, PLoS ONE, 8 (12), e82237, 英語[査読有り]
研究論文（学術雑誌）
For the development of a biomimetic odor-sensing system, we investigated the effects of replacing the N-terminus of an olfactory receptor (OR) on its functional expression in the budding yeast, Saccharomyces cerevisiae. Using the mouse olfactory receptor OR226 (mOR226), three types of chimeric ORs were constructed by replacing N-terminal regions of mOR226 with the corresponding regions of the rat I7 receptor, which is known to be functionally expressed in yeast. The replacement of the N-terminal region of mOR226 dramatically affected the expression and localization of the receptor and improved the sensing ability of the yeast cells for the odorant. Furthermore, the replacement of the endogenous yeast G-protein a subunit (Gpa1) by the OR-specific Golf drastically elevated the odorant-sensing ability of the yeast cells and caused the cells to display a dose-dependent responsiveness to the odorant. Because of the suitability of yeast cells for screening large-scale libraries, the strategy presented here would be useful for the establishment of advanced biomimetic odor-sensing systems. Biotechnol. Bioeng. 2012;109: 205212. (c) 2011 Wiley Periodicals, Inc.
WILEY-BLACKWELL, 2012年01月, Biotechnology and Bioengineering, 109 (1), 205 - 212, 英語[査読有り]
研究論文（学術雑誌）
Bio-nanocapsules (BNCs) are hollow nanoparticles composed of the L protein of hepatitis B virus (HBV) surface antigen (HBsAg), which can specifically introduce genes and drugs into various kinds of target cells. Although the classic electroporation method has typically been used to introduce highly charged molecules such as DNA, it is rarely adopted for proteins due to its very low efficiency. In this study, a novel approach to the preparation of BNC was established whereby a target protein was pre-encapsulated during the course of nanoparticle formation. Briefly, because of the process of BNC formation in a budding manner on the endoplasmic reticulum (ER) membrane, the association of target proteins to the ER membrane with lipidation sequences (ER membrane localization sequences) could directly generate protein-encapsulating BNC in collaboration with co-expression of the L proteins. Since the membrane-localized proteins are automatically enveloped into BNCs during the budding event, this method can be protect the proteins and BNCs from damage caused by electroporation and obviate the need for laborious consideration to study the optimal conditions for protein encapsulation. This approach would be a useful method for encapsulating therapeutic candidate proteins into BNCs. (C) 2011 Elsevier B.V. All rights reserved.
ELSEVIER SCIENCE BV, 2012年01月, Journal of Biotechnology, 157 (1), 124 - 129, 英語[査読有り]
研究論文（学術雑誌）
Here we present a successful transplantation of the GAL genetic regulatory circuitry into the G-protein signaling pathway in yeast. The GAL regulon represents a strictly regulated transcriptional mechanism that we have transplanted into yeast to create a highly robust induction system to assist the detection of on-off switching in G-protein signaling. In our system, we engineered yeast to drive the positive GAL regulatory gene in response to agonist-promoted G-protein signaling and to induce transcription of a green fluorescent protein (GFP) reporter gene under the control of the GAL structural gene promoter. Consequently, in response to agonist stimulation of G-protein-coupled receptors (GPCRs), the engineered yeast achieved more than a 150-fold increase in reporter intensity in up to 98% of cells, as determined by flow cytometric sorting. Surprisingly, agonist-stimulated induction of the GFP reporter gene was higher than that by galactose. Our approach to boost reporter gene induction could be applicable in establishing more efficient yeast-based flow cytometric screening systems for agonistic ligands for heterogeneous GPCRs. (c) 2012 Elsevier Inc. All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年05月, Analytical Biochemistry, 424 (1), 27 - 31, 英語[査読有り]
研究論文（学術雑誌）
The production of higher alcohols by engineered bacteria has received significant attention. The budding yeast, Saccharomyces cerevisiae, has considerable potential as a producer of higher alcohols because of its capacity to naturally fabricate fusel alcohols, in addition to its robustness and tolerance to low pH. However, because its natural productivity is not significant, we considered a strategy of genetic engineering to increase production of the branched-chain higher alcohol isobutanol, which is involved in valine biosynthesis. Initially, we overexpressed 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) in S. cerevisiae to enhance the endogenous activity of the Ehrlich pathway. We then overexpressed Ilv2, which catalyzes the first step in the valine synthetic pathway, and deleted the PDC1 gene encoding a major pyruvate decarboxylase with the intent of altering the abundant ethanol flux via pyruvate. Through these engineering steps, along with modification of culture conditions, the isobutanol titer of S. cerevisiae was elevated 13-fold, from 11 mg/l to 143 mg/l, and the yield was 6.6 mg/g glucose, which is higher than any previously reported value for S. cerevisiae. (C) 2012 Elsevier B.V. All rights reserved.
ELSEVIER SCIENCE BV, 2012年05月, Journal of Biotechnology, 159 (1-2), 32 - 37, 英語[査読有り]
研究論文（学術雑誌）
G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.
PUBLIC LIBRARY SCIENCE, 2012年05月, PLoS ONE, 7 (5), e37136, 英語[査読有り]
研究論文（学術雑誌）
Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is industrially produced by fermentation using Saccharomyces cerevisiae. Before the glutathione fermentation process with S. cerevisiae, a glucose extraction process from starchy materials is required. This glucose extraction is usually carried out by converting starchy materials to starch using high-temperature cooking and subsequent hydrolysis by amylases to convert starch to glucose. In this study, to develop an energy-saving glutathione production process by reducing energy consumption during the cooking step, we efficiently produced glutathione from low-temperature cooked rice using amylase-expressing S. cerevisiae. The combination of the amylase-expressing yeast with low-temperature cooking is potentially applicable to a variety of energy-saving bio-production methods of chemicals from starchy bio-resources.
WILEY-BLACKWELL, 2012年05月, Biotechnology Journal, 7 (5), 686 - 689, 英語[査読有り]
研究論文（学術雑誌）
Flow cytometry enables comparative quantification, population analysis, and high-throughput screening of agonist-mediated G-protein-coupled receptor (GPCR) signaling in genetically engineered yeasts. By using flow cytometry, we found that transformation of yeast cells with a low plasmid number is critical both for the construction of large screening libraries and for stable signal transmission in cell ensembles. Based on these findings, we constructed an engineered yeast strain for the improved identification of signal promotion by C alpha(i)-specific human GPCRs using flow cytometry. (C) 2012 Elsevier Inc. All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2012年07月, Analytical Biochemistry, 426 (2), 129 - 133, 英語[査読有り]
研究論文（学術雑誌）
To increase isobutanol production in Saccharomyces cerevisiae, the valine biosynthetic pathway was activated by overexpression of the relevant enzymes in the mitochondria and the cytosol. Native mitochondrial enzymes were overepxressed in the cytosol by deleting the mitochondrial transit peptides. The metabolically engineered S. cerevisiae possessing the cytosolic pathway showed increased isobutanol production (63 +/- 4 mg/L).
TAYLOR & FRANCIS LTD, 2012年11月, Bioscience, Biotechnology and Biochemistry, 76 (11), 2139 - 2141, 英語[査読有り]
研究論文（学術雑誌）
A bio-nanocapsule (BNC), a hollow particle composed of hepatitis B virus (HBV) surface antigen (HBsAg), and liposome (LP) conjugation method (BNC/LP) has been recently developed by Jung et al. (2008). The BNC/LP complex carrier could successfully deliver fluorescence-labeled beads (100 nm) into liver cells. In this study, we report the promising delivery of proteins incorporated in the complex carriers, which were prepared by the BNC/LP conjugation method with specificity-altered BNC and composition-varied LPs. The specificity-altered BNC, Z(HER2)-BNC was developed by replacing the hepatocyte recognition site of BNC with Z(HER2) binding to HER2 receptor specifically. Using green fluorescent protein (GFP; 27 kDa) and cellular cytotoxic protein (exotoxin A; 66 kDa) for the delivery, we herein present the impact of different charges attributed to the composition of the LP on specific cell targeting and cellular uptake of the complex carriers. In addition, we demonstrate that the mixture prepared by mixing LPs with helper lipid possessing endosomal escaping ability boosts the functional expression of the cellular cytotoxic exotoxin A activity specifically. Finally, we further show the blending ratio of the LP mixture and Z(HER2)-BNC is a critical factor in determining the highly-efficient expression of the cytotoxic activity of exotoxin A.
INFORMA HEALTHCARE, 2012年12月, Journal of Drug Targeting, 20 (10), 897 - 905, 英語[査読有り]
研究論文（学術雑誌）
The goal of this work was to improve the bioluminescence-based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast-expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G-protein a-subunit (Gpa1p) with the olfactory-specific Gaolf, the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence-based odor-sensing system using OR-expressing yeast. Biotechnol. Bioeng. 2012; 109: 31433151. (C) 2012 Wiley Periodicals, Inc.
WILEY, 2012年12月, Biotechnology and Bioengineering, 109 (12), 3143 - 3151, 英語[査読有り]
研究論文（学術雑誌）
Background: The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results: In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions: Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering.
BIOMED CENTRAL LTD, 2011年01月, Microbial Cell Factories, 10, 英語[査読有り]
研究論文（学術雑誌）
Weak and transient protein-protein interactions are associated with biological processes, but many are still undefined because of the difficulties in their identification. Here, we describe a redesigned method to screen transient protein-protein interactions by using a novel signal amplification circuit, which is incorporated into yeast to artificially magnify the signal responding to the interactions. This refined method is based on the previously established G gamma recruitment system, which utilizes yeast G-protein signaling and mating growth selection to screen interacting protein pairs. In the current study, to test the capability of our method, we chose mutants of the Z-domain derived from Staphylococcus aureus protein A as candidate proteins, and the Fc region of human IgG as the counterpart. By introduction of an artificial signal amplifier into the previous G gamma recruitment system, the signal transduction responding to transient interactions between Z-domain mutants and the Fc region with significantly low affinity (8.0 x 10(3) M(-1)) was successfully amplified in recombinant haploid yeast cells. As a result of zygosis with the opposite mating type of wildtype haploid cells, diploid colonies were vigorously and selectively generated on the screening plates, whereas our previous system rarely produced positive colonies. This new approach will be useful for exploring the numerous transient interactions that remain undefined because of the lack of powerful screening tools for their identification.
WILEY-BLACKWELL, 2011年09月, FEBS Journal, 278 (17), 3086 - 3094, 英語[査読有り]
研究論文（学術雑誌）
Background: While Saccharomyces cerevisiae is a promising host for cost-effective biorefinary processes due to its tolerance to various stresses during fermentation, the metabolically engineered S. cerevisiae strains exhibited rather limited production of higher alcohols than that of Escherichia coli. Since the structure of the central metabolism of S. cerevisiae is distinct from that of E. coli, there might be a problem in the structure of the central metabolism of S. cerevisiae. In this study, the potential production of higher alcohols by S. cerevisiae is compared to that of E. coli by employing metabolic simulation techniques. Based on the simulation results, novel metabolic engineering strategies for improving higher alcohol production by S. cerevisiae were investigated by in silico modifications of the metabolic models of S. cerevisiae. Results: The metabolic simulations confirmed that the high production of butanols and propanols by the metabolically engineered E. coli strains is derived from the flexible behavior of their central metabolism. Reducing this flexibility by gene deletion is an effective strategy to restrict the metabolic states for producing target alcohols. In contrast, the lower yield using S. cerevisiae originates from the structurally limited flexibility of its central metabolism in which gene deletions severely reduced cell growth. Conclusions: The metabolic simulation demonstrated that the poor productivity of S. cerevisiae was improved by the introduction of E. coli genes to compensate the structural difference. This suggested that gene supplementation is a promising strategy for the metabolic engineering of S. cerevisiae to produce higher alcohols which should be the next challenge for the synthetic bioengineering of S. cerevisiae for the efficient production of higher alcohols.
BIOMED CENTRAL LTD, 2011年09月, Microbial Cell Factories, 10, 英語[査読有り]
研究論文（学術雑誌）
G-protein-coupled receptors (GPCRs) are considered as important targets for drug discovery. The yeast Saccharomyces cerevisiae is an attractive host for high-throughput screening of agonistic ligands for human GPCRs because it can simplify the complicated signaling pathways that are present in mammalian cell lines. Unfortunately, many human GPCRs induce only partial signal activation in yeast cells depending on their coupling efficiency with yeast G-proteins. This problem often results in unsatisfactory detection sensitivity, thereby resulting in a limitation to yeast-based detection systems. Here we introduce a new highly sensitive detection method that provides robust agonist detection of human GPCRs. Our strategy is designed to invoke feedback activation of signals within yeast G-protein signaling pathways. Briefly, agonist stimulation of human GPCRs triggers expression of an artificial signal activator that amplifies signaling. We chose human somatostatin receptor subtype 5 (hSSTR5) as a model of a human GPCR. Investigation of the response of hSSTR5-expressing yeast to various concentrations of somatostatin demonstrated that feedback activation of the signal can successfully improve the detection limit and the maximum level of signaling. This novel approach will enhance the usefulness of yeast-based screening of agonistic ligands for a variety of human GPCRs. (C) 2011 Elsevier Inc. All rights reserved.
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2011年10月, Analytical Biochemistry, 417 (2), 182 - 187, 英語[査読有り]
研究論文（学術雑誌）
Here we expand the yeast cell surface display system to display non-natural, functional molecules. The short biotin acceptor peptide (BAP) sequence of biotin ligase from E. coli(BirA) was genetically introduced to the N-terminus of the anchor protein, Flo428. Through co-expression of BAP-fused Flo428 with BirA, biotinylated BAP could be displayed on the yeast cell surface. Subsequent addition of streptavidin-FITC resulted in the display of streptavidin-FITC, and, the display of biotin-FITC was successful using streptavidin as a linker. Our strategy provides a powerful tool for displaying functional molecules on yeast cell surfaces. (c) 2009 Elsevier B.V. All rights reserved.
ELSEVIER SCIENCE BV, 2010年01月, Journal of Biotechnology, 145 (1), 79 - 83, 英語[査読有り]
研究論文（学術雑誌）
We have developed a new approach based on the G gamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established G gamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein c subunit (G gamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered G gamma that lacks membrane localization upon deletion of the lipid modi. cation site (G gamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with G gamma(cyto) are expressed. As protein-protein interactions bring G gamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-G gamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced G gamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.
WILEY-BLACKWELL PUBLISHING, INC, 2010年04月, FEBS Journal, 277 (7), 1704 - 1712, 英語[査読有り]
研究論文（学術雑誌）
N-linked oligosaccharides or asparagine residues are often involved in G protein-coupled receptor functions. Focusing on Asn13 and Asn26 positioned on N-linked glycosylation motifs in the amino-terminal domain of human somatostatin receptor subtype-5 (hSSTR5), we performed site-directed mutagenesis and evaluated the mutants by using yeast cells as the host strain. This is because analysing the complicated signalling in mammalian cell lines is simplified by the utilization of the monopolistic pheromone signalling pathway in yeast. Western blot analysis and confocal laser scanning microscope observation showed that Asn13 and/or Asn26 mutations had no effects on cell-surface expression of hSSTR5 in yeast. By using an engineered yeast strain of Saccharomyces cerevisiae, which induces the expression of the green fluorescent protein (GFP) reporter gene in response to the agonist-specific signal transduction, it was demonstrated that a single mutation of two asparagine residues attenuated the somatostatin-specific signalling levels, and the double mutant significantly lost the signalling ability. These results clearly show the importance of these asparagine residues in the agonist-specific signalling of hSSTR5, although it was not enough to identify the consequence of oligosaccharides.
OXFORD UNIV PRESS, 2010年06月, Journal of Biochemistry, 147 (6), 867 - 873, 英語[査読有り]
研究論文（学術雑誌）
The yeast Saccharomyces cerevisiae is known as an available host for human G-protein-coupled receptor (GPCR) ligand screening. Although several types of yeast signal sequences (SS) attached with the GPCRs could improve their productivities and facilitate transportation of the GPCRs to the yeast plasma membrane, the effects of additional SS on ligand-specific signalling functions of GPCRs are not reported. Here, we demonstrated the controlling signalling properties by addition of SS using engineered yeast as a host. Prepro and pre regions of alpha-factor and amino-terminal sequence of Ste2 (Ste2N) were used as SS, and somatostatin (SST) receptor subtype-5 (SSTR5) was used as a model GPCR. We also constructed a yeast-based fluorescent assay system for monitoring the activation levels of SSTR5 signalling by a green fluorescent protein (GFP) reporter gene. The production levels and localisation patterns of the SS-attached SSTR5 were more significantly improved than those of wild-type SSTR5. In addition, we successfully controlled the pharmacological efficacy and potency by introducing SS. Among four types of SSTR5 receptors, Ste2N-SSTR5 responded at the lowest ligand concentration. This finding will be informative for constructing optimal yeast-based ligand screening systems to discriminate the cells on the basis of signalling levels.
OXFORD UNIV PRESS, 2010年06月, Journal of Biochemistry, 147 (6), 875 - 884, 英語[査読有り]
研究論文（学術雑誌）
A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.
SPRINGER, 2010年11月, Applied Microbiology and Biotechnology, 88 (5), 1215 - 1221, 英語[査読有り]
研究論文（学術雑誌）
For elucidating protein-protein interactions, many methodologies have been developed during the past two decades. For investigation of interactions inside cells under physiological conditions, yeast is an attractive organism with which to quickly screen for hopeful candidates using versatile genetic technologies, and various types of approaches are now available. Among them, a variety of unique systems using the guanine nucleotide-binding protein (G-protein) signaling pathway in yeast have been established to investigate the interactions of proteins for biological study and pharmaceutical research. G-proteins involved in various cellular processes are mainly divided into two groups: small monomeric G-proteins, and heterotrimeric G-proteins. In this minireview, we summarize the basic principles and applications of yeast-based screening systems, using these two types of G-protein, which are typically used for elucidating biological protein interactions but are differentiated from traditional yeast two-hybrid systems.
WILEY-BLACKWELL, 2010年05月, FEBS Journal, 277 (9), 1982 - 1995, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
- SOUTHEAST UNIV PRESS, 2009年, IFPT'6: PROGRESS ON POST-GENOME TECHNOLOGIES, PROCEEDINGS, 238 - 238, 英語CONSTRUCTION OF A NOVEL DETECTION SYSTEM FOR PROTEIN-PROTEIN INTERACTIONS USING YEAST G-PROTEIN SIGNALING
[査読有り]
研究論文（国際会議プロシーディングス）
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 gamma subunit (G gamma) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-terminus, and that this localization of G gamma is required for signal transduction. In our system, mutated G gamma (G gamma(cyto)) lacking membrane localization ability was genetically prepared by deletion of the lipid modification site. Complete disappearance of G-protein signal was observed when G gamma(cyto) was expressed in the cytoplasm of yeast cells from which the endogenous G gamma 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 G gamma(cyto). The Fc-G gamma(cyto) 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 x 10(3) to 6.8 x 10(8) 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.
WILEY-BLACKWELL PUBLISHING, INC, 2009年05月, FEBS Journal, 276 (9), 2636 - 2644, 英語[査読有り]
研究論文（学術雑誌）
To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.
OXFORD UNIV PRESS, 2009年06月, Journal of Biochemistry, 145 (6), 701 - 708, 英語[査読有り]
研究論文（学術雑誌）
- 公益社団法人 化学工学会, 2008年, 化学工学会 研究発表講演要旨集, 2008, 348 - 348, 日本語
- 2008年04月, Biotechnology Progress, 24 (2), 英語
[査読有り]
研究論文（学術雑誌）
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.
OXFORD UNIV PRESS, 2008年05月, Journal of Biochemistry, 143 (5), 667 - 674, 英語[査読有り]
研究論文（学術雑誌）
A yeast protein fragment complementation assay (PCA) system based on dihydrofolate reductase (DHFR) is difficult to be operated because it is not as sensitive to trimethoprim (TMP) as the system using a prokaryotic microorganism. Here, the PCA system using DHFR, specific inhibitors, and a substrate in the yeast Saccharomyces cerevisiae was newly developed. As a model, the human oncoprotein Ras and the Ras-binding domain (RBD) of Raf-1 were individually and genetically fused to DHFR fragment, and each genetic construct was coexpressed under the control of the GAL1 promoter. An interaction between Ras and RBD could be evaluated on the basis of cell proliferation. To establish the experimental conditions for the yeast PCA system based on the DHFR reconstitution, we examined yeast host strains and the concentration of inhibitory additives to prevent endogenous DHFR activity, namely, TMP and sulfanilamide, and the substrate of DHFR, namely, folic acid. The transformant harboring wild-type Ras or its variants showed positive interaction signals, and the order of interactions for combination corresponded to the results of other in vitro assays. Moreover, combinatorial mutated Ras-binding domains were constructed, and the interaction of RBDs with Ras using this yeast PCA system was examined. As a result, various types of mutated clone for RBD were obtained. These demonstrations suggest that the yeast PCA system based on DHFR can be one of good, convenient, and inexpensive tools for investigating eukaryotic protein-protein interactions in vivo.
SPRINGER, 2008年09月, Applied Microbiology and Biotechnology, 80 (4), 735 - 743, 英語[査読有り]
研究論文（学術雑誌）
- 公益社団法人 化学工学会, 2007年, 化学工学会 研究発表講演要旨集, 2007, 325 - 325, 日本語
- PHOENIX PUBL & MEDIA NETWORK, 2007年, PROGRESS ON POST-GENOME TECHNOLOGIES, 57 - 57, 英語Fluorescence detection system for human G protein - Coupled receptor signaring in yeast
[査読有り]
研究論文（国際会議プロシーディングス）
We determined whether the cocultivation of yeast cells displaying a ZZ-domain and secreting an Fc fusion protein can be a novel tool for the recovery of secreted recombinant proteins. The ZZ-domain from Staphylococcus aureus protein A was displayed on the cell surface of Saccharomyces cerevisiae under the control of the GAL1 promoter. Strain S. cerevisiae BY4742 cells displaying the ZZ-domain on their surface were used for cocultivation with cells that produce a target protein fused to the Fc fragment as an affinity tag. The enhanced green fluorescent protein or Rhizopus oryzae lipase was genetically fused to the N and C termini of the Fc fragment of human immunoglobulin G, respectively. Through analysis by fluorescence-activated cell sorting and enzymatic assay, it was demonstrated that these fusion proteins are successfully produced in the medium and recovered by affinity binding with the cell surface displaying the ZZ-domain. These results suggest that the ZZ-domain-displaying cell and Fc fusion protein-secreting cell can be applied to use in synergistic process of production and recovery of secreted recombinant proteins.
SPRINGER, 2007年06月, Applied Microbiology and Biotechnology, 75 (4), 821 - 828, 英語[査読有り]
研究論文（学術雑誌）
We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein-protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fe part of human immumoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.
SPRINGER, 2007年08月, Applied Microbiology and Biotechnology, 76 (1), 151 - 158, 英語[査読有り]
研究論文（学術雑誌）
- 公益社団法人 化学工学会, 2006年, 化学工学会 研究発表講演要旨集, 2006, 986 - 986, 日本語
- SOUTHEAST UNIV PRESS, 2006年, Progress on Post-Genome Technologies, 30 - 30, 英語Development of a single cell analysis system of agonist for drug discovery
[査読有り]
研究論文（国際会議プロシーディングス）
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.
2006年07月, Biotechnology Progress, 22 (4), 954 - 960, 英語[査読有り]
研究論文（学術雑誌）
MISC
- 2023年05月, Bioscience & Industry, 81 (3), 234 - 236, 日本語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
記事・総説・解説・論説等（学術雑誌）
- 2021年12月, 島津製作所 Application Notes, 72, 日本語出芽酵母代謝工学株が生産するファイトケミカルの高分解能質量分析による構造確認 –プレニルナリンゲニンの分析–
記事・総説・解説・論説等（学術雑誌）
- 2020年, 日本農芸化学会大会講演要旨集(Web), 2020デルタロドプシンの変異体解析
- 2020年, 日本農芸化学会大会講演要旨集(Web), 2020デルタロドプシンによるATP再生を伴う物質生産性の向上
- 2019年, 日本農芸化学会大会講演要旨集(Web), 2019光駆動ATP再生系を導入したVmax細胞の開発
- 2019年, 日本生物工学会大会講演要旨集, 71stデルタロドプシン発現が出芽酵母の代謝に及ぼす影響
- 2019年, 日本生物工学会大会講演要旨集, 71stロドプシンを利用した大腸菌における光駆動ATP再生の物質生産への応用
- 2019年, 化学工学会年会研究発表講演要旨集(CD-ROM), 84th大腸菌による物質生産への光駆動ATP再生の利用に向けて
- 2018年03月05日, 日本農芸化学会大会講演要旨集(Web), 2018, ROMBUNNO.3A29p01 (WEB ONLY), 日本語Combi‐OGAB法を利用した最適コドンの探索
- 2018年, 日本生物工学会大会講演要旨集, 70th光駆動ATP再生系によるVmax細胞の創製
- 2018年, 日本農芸化学会大会講演要旨集(Web), 2018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
- (公社)日本生物工学会, 2017年08月, 日本生物工学会大会講演要旨集, 平成29年度, 200 - 200, 日本語GPCR拮抗薬のポジティブ検出を可能とする反転型レポーター発現系
- 2016年, 日本農芸化学会大会講演要旨集(Web), 2016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
- 日本生物工学会, 2015年, 日本生物工学会大会講演要旨集, 67, 108 - 108, 日本語1P-080 ヒトニューロテンシン受容体におけるリガンド探索のための酵母バイオセンサー(タンパク質工学,一般講演)
- 日本生物工学会, 2014年, 日本生物工学会大会講演要旨集, 66, 18 - 18, 日本語1P-006 Gタンパク質共役型受容体の二量体形成検出のためのゲノム編集技術の開発(遺伝子工学,一般講演)
- 日本生物工学会, 2014年, 日本生物工学会大会講演要旨集, 66, 251 - 251, 日本語3P-226 pH 応答性ペプチドGALAを表層提示したバイオナノカプセルのエンドソーム脱出(生物化学工学,一般講演)
- 日本生物工学会, 2014年, 日本生物工学会大会講演要旨集, 66, 258 - 258, 日本語3P-253 出芽酵母Saccharomyces cerevisiaeでの嗅覚受容体匂い分子応答における匂い結合タンパク質の効果(バイオセンシング,分析化学,一般講演)
- 日本生物工学会, 2014年, 日本生物工学会大会講演要旨集, 66, 258 - 258, 日本語3P-254 出芽酵母における分割ルシフェラーゼを利用したGPCR リガンド応答検出法の応用(バイオセンシング,分析化学,一般講演)
- 日本生物工学会, 2014年, 日本生物工学会大会講演要旨集, 66, 133 - 133, 日本語2P-108 芳香植物のテルペン合成酵素遺伝子のカタログ化 : ツバキの花を例に(代謝工学,一般講演)
- 日本生物工学会, 2014年, 日本生物工学会大会講演要旨集, 66, 161 - 161, 日本語2P-220 二次代謝産物を指標とした有用油性酵母のクラスター解析(生合成,天然物化学,一般講演)
- WILEY-BLACKWELL, 2013年09月, YEAST, 30, 210 - 210, 英語Metabolic pathway engineering of yeast Saccharomyces cerevisiae for isobutanol production
研究発表ペーパー・要旨（国際会議）
- WILEY-BLACKWELL, 2013年09月, YEAST, 30, 182 - 182, 英語A selection system exploiting yeast signal transduction machinery to create desirably affinity-altered protein variants
研究発表ペーパー・要旨（国際会議）
- 日本生物工学会, 2013年, 日本生物工学会大会講演要旨集, 65, 38 - 38, 日本語1P-083 G蛋白質共役型受容体の二量体形成およびシグナル伝達の同時解析システム(タンパク質工学,一般講演)
- 日本生物工学会, 2013年, 日本生物工学会大会講演要旨集, 65, 54 - 54, 日本語1P-145 イソブタノール生産酵母におけるトランスヒドロゲナーゼ様シャントの活性化(一般講演(代謝工学))
- 日本生物工学会, 2013年, 日本生物工学会大会講演要旨集, 65, 154 - 154, 日本語2P-200 出芽酵母Saccharomyces cerevisiaeでの嗅覚受容体の機能的発現におけるアクセサリータンパク質の効果(バイオセンシング,分析化学,一般講演)
- 日本生物工学会, 2013年, 日本生物工学会大会講演要旨集, 65, 231 - 231, 日本語3P-174 上皮成長因子受容体を特異的に認識するAffibody提示バイオナノカプセルの開発(生物化学工学,一般講演)
- 2012年11月05日, 化学工学 = Chemical engineering, 76 (11), 722 - 722, 日本語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
- 日本生物工学会, 2012年, 日本生物工学会大会講演要旨集, 64, 194 - 194, 日本語4Ca11 出芽酵母を用いた嗅覚受容体リガンドアッセイシステムの最適化(酵素学,酵素工学/タンパク質工学,一般講演)
- 2011年, 日本生物工学会大会講演要旨集, 63rdAffibody提示Bio-nanocapsuleを用いたHER2発現癌細胞へのタンパク質送達システム
- 日本生物工学会, 2011年, 日本生物工学会大会講演要旨集, 63, 180 - 180, 日本語2Ip13 Affibody提示Bio-nanocapsuleを用いたHER2発現癌細胞へのタンパク質送達システム(生物化学工学,一般講演)
- 日本生物工学会, 2010年, 日本生物工学会大会講演要旨集, 22, 197 - 197, 日本語2S-Ba05 1細胞アッセイ技術に基づくGPCR解析、アゴニスト探索法の開発(1分子1細胞アッセイ技術に基づく生物工学の最近の潮流,シンポジウム,伝統の技と先端科学技術の融合)
- SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S61 - S62, 英語
研究発表ペーパー・要旨（国際会議）
- SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S164 - S164, 英語
研究発表ペーパー・要旨（国際会議）
- SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S159 - S160, 英語
研究発表ペーパー・要旨（国際会議）
- SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S53 - S53, 英語
研究発表ペーパー・要旨（国際会議）
- SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S108 - S108, 英語
研究発表ペーパー・要旨（国際会議）
- SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年11月, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108, S36 - S36, 英語
研究発表ペーパー・要旨（国際会議）
- 2009年, 日本薬学会年会要旨集, 129th (1)分子ディスプレイによる創薬基盤技術の展開
- 日本生物工学会, 2008年, 日本生物工学会大会講演要旨集, 20, 63 - 63, 日本語2Ap01 ビオチン提示酵母を用いた新規細胞表層提示システムの開発(生物化学工学,一般講演)
- 日本生物工学会, 2005年, 日本生物工学会大会講演要旨集, 17, 62 - 62, 日本語2A12-3 酵母シグナル伝達経路を利用したリガンドスクリーニングシステムの開発(遺伝子工学・核酸工学,一般講演)
- 日本生物工学会, 2013年, 日本生物工学会大会講演要旨集, 65, 38 - 38, 日本語1P-082 酵母蛍光レポーターの改変によるヒト由来7回膜受容体の高感度リガンド検出システム(タンパク質工学,一般講演)
- 2018年, 日本生物工学会大会講演要旨集, 70th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
書籍等出版物
- 共著, Precision Medicine, 2021年12月, 第4巻,第13号,60-65, 日本語酵母細胞を用いた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
学術書
- 共著, アグリバイオ, 2021年07月, 第5巻,第7号,58-63, 日本語酵母細胞を用いた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
学術書
- 共著, 月刊細胞, 2021年05月, 第53巻,第5号,37-40, 日本語酵母シグナル伝達経路を利用した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
学術書
- 共編者(共編著者), ゲノム編集技術を応用した製品開発とその実用化 ～研究開発動向・課題解決策・技術予測と市場展望～（第4章 ゲノム編集によるスマートセルインダストリーの技術開発とその課題解決，第7節, 技術情報協会, 2021年02月26日, 日本語長鎖DNA合成技術による有用物質生産微生物の構築とその課題解決
学術書
- 単著, 羊土社, 2021年01月, 2021年1月号,第39巻,第1号,8-12, 日本語新型コロナで変わる時代の実験自動化・遠隔化「微生物での発酵生産と実験の自動化」
学術書
- 共編者(共編著者), スマートセルインダストリー –微生物細胞を用いた物質生産の展望–（第1編 ハイスループット合成・分析・評価技術，第2章 ハイスループット微生物構築・評価技術，第1節）, シーエムシー出版, 2018年06月, 日本語微生物を用いた物質生産とハイスループット微生物構築技術
学術書
- 共編者(共編著者), Peripheral Membrane Proteins (Chapter 3 "Biosensing techniques in yeast"), InTechOpen, 2018年06月, 英語G-protein signaling and protein-protein interaction assays for monitoring ligand stimulation and oligomer formation of heterologous GPCRs
学術書
- 共著, 現代化学, 2018年01月, 2018年1月号,第562号,36-41, 日本語ゲノム合成の潮流のインパクト 〜微生物による物質生産〜
学術書
- 共著, 月刊BIO INDUSTRY, 2017年06月, 2017年6月号,第34巻,第6号,54-62, 日本語低分子抗体医薬の開発展望
学術書
- 共著, 化学と生物, 2015年10月, 第53巻,第10号,689-695, 日本語バイオリファイナリーの現状と展望
学術書
- 共著, 生物工学会誌, 2015年09月, 第93巻, 第9号, 523-526, 日本語合成生物工学によるモノづくり微生物のデザインに向けて
学術書
- 共編者(共編著者), 化学便覧 応用化学編 第7版 （第Ⅶ編 バイオ科学技術，第26章 バイオマテリアル，第3節）, 丸善出版, 2014年01月, 日本語ヒドロキシ化合物
学術書
- 共著, 安全工学, 2013年08月, 第52巻,第4号,249-255, 日本語バイオリファイナリー社会に向けた燃料・化学品生産
学術書
- 共著, 生物工学会誌, 2013年06月, 第91巻,第6号,314-318, 日本語革新的なものづくり実現のための「合成生物工学」
学術書
- 共編者(共編著者), 次世代医薬開発に向けた抗体工学の最前線 （第Ⅱ編 抗体の改変技術，第4章 親和性の向上，第4節）, シーエムシー出版, 2012年12月, 日本語酵母による抗体フラグメントおよび抗体様結合性タンパク質の改変技術
学術書
- 単著, 生物工学会誌, 2011年12月, 第89巻,第12号,759, 日本語ノミニケーションノススメ
- 単著, 内藤財団時報, 2011年09月, 第88号,47, 日本語GPCRと酵母との出会い
- 共編者(共編著者), シングルセル解析の最前線 （第３章 細胞内生体分子群の実測定量解析，第4節）, シーエムシー出版, 2010年03月, 日本語フローサイトメトリーとGFPレポーターによるG蛋白質シグナルのシングルセル解析
学術書
- 共著, 未来材料, 2007年06月, 2007年6月号,40-45, 日本語タンパク質間相互作用とシグナル伝達解析¬ −酵母細胞を用いたハイスループット技術の開発-−
学術書
- 共著, 一細胞定量解析の最前線 −ライフサーベイヤ構築に向けて− （第３章 細胞内生体分子群の動態シグナルの解析，第６節）, シーエムシー出版, 2006年12月, 日本語酵母細胞内シグナル定量解析の創薬への応用
学術書
- 共著, 生物工学会誌, 2006年08月, 第84巻,第8号,319-321, 日本語コンビナトリアル・バイオエンジニアリングによる新しい創薬システム
学術書
講演・口頭発表等
- 2021年度膜工学春季講演会・膜工学サロン, 2022年03月29日, 日本語, 神戸大学先端膜工学研究センター／一般社団法人先端膜工学研究機構, オンライン, 日本国, 国内会議, 国際共著していない代謝工学と合成生物学によるモノづくり微生物の開発
[招待有り]
口頭発表（招待・特別）
- SONY “Life Science Spring Webinar 2022”, 2022年03月23日, 日本語, SONY, オンライン, 日本国, 国内会議, 国際共著していないバイオエコノミー社会実現に向けた合成生物学の基盤開発と微生物での物質生産への展開
[招待有り]
口頭発表（招待・特別）
- 化学工学会第87年会, 2022年03月17日, 日本語, 化学工学会, 日本, 日本国, 国内会議, 国際共著していないロドプシンを利用した光駆動ATP再生によるイソプレノールおよび3ヒドロキシプロピオン酸生産の向上
口頭発表（一般）
- 化学工学会第87年会, 2022年03月16日, 日本語, 化学工学会, オンライン, 日本国, 国内会議, 国際共著していない酵母シグナル伝達機構を利用したタンパク質間相互作用分子探索および改変体創出
ポスター発表
- 化学工学会第87年会, 2022年03月16日, 日本語, 化学工学会, オンライン, 日本国, 国内会議, 国際共著していないロドプシンによる光駆動プロトンポンプが大腸菌の弱酸耐性に及ぼす影響
ポスター発表
- 日本農芸化学会2022年度大会, 2022年03月18日, 日本語, 日本農芸化学会, オンライン, 日本国, 国内会議, 国際共著していない微生物生産能向上を目的とした微生物ロドプシンの改変
ポスター発表
- 日本農芸化学会関西支部第519回講演会, 2022年02月05日, 日本語, 日本農芸化学会関西支部, オンライン, 日本国, 国内会議, 国際共著していない酵母シグナル伝達機構を利用したタンパク質間相互作用分子探索および改変体創出
口頭発表（一般）
- 第12回スクリーニング学研究会（企業セミナー：テカンジャパン）, 2021年11月26日, 日本語, スクリーニング学研究会, オンライン, 日本国, 国内会議, 国際共著していない微生物モノづくりにおける合成生物学と実験自動化:バイオエコノミー社会の実現に向けて
[招待有り]
公開講演，セミナー，チュートリアル，講習，講義等
- バイオDXの最前線 CREST 「データ駆動型・AI駆動を中心としたデジタルトランスフォーメーションによる生命科学研究の革新」キックオフシンポジウム, 2021年11月21日, 日本語, 科学技術振興機構（JST）／理化学研究所 生命機能科学研究センター, オンラインn, 日本国, 国内会議, 国際共著していないデータ駆動型の次世代微生物進化育種に向けて
[招待有り]
口頭発表（招待・特別）
- Laboratory Automation月例勉強会 / 2021.10，, 2021年10月30日, 日本語, ラボラトリーオートメーション研究会, オンライン, 日本国, 国内会議, 国際共著していないいっぱいデータを出したいがため微生物の組換え実験を自動化する
[招待有り]
公開講演，セミナー，チュートリアル，講習，講義等
- 第73回日本生物工学会大会, 2021年10月29日, 日本語, 日本生物工学会, オンライン, 日本国, 国内会議, 国際共著していない光駆動プロトンポンプを発現させたメバロン酸生産大腸菌の代謝解析
ポスター発表
- 第73回日本生物工学会大会, 2021年10月29日, 日本語, 日本生物工学会, オンライン, 日本国, 国内会議, 国際共著していないH＋ポンプ能向上を目的とした微生物ロドプシンの改変
ポスター発表
- 第73回日本生物工学会大会, 2021年10月28日, 日本語, 日本生物工学会, オンライン, 日本国, 国内会議, 国際共著していない光エネルギーを利用した大腸菌におけるメバロン酸のイソプレノールへの変換
ポスター発表
- 第73回日本生物工学会大会, 2021年10月28日, 日本語, 日本生物工学会, オンライン, 日本国, 国内会議, 国際共著していないXanthophyllomyces dendrorhousによるレチナールの生産
ポスター発表
- 第1回神戸大学先端バイオ工学センター成果発表会, 2021年10月11日, 日本語, 先端バイオ工学センター, オンライン, 日本国, 国内会議, 国際共著していない進化工学による酵母遺伝子スイッチの開発とテルペノイドセンサへの応用
公開講演，セミナー，チュートリアル，講習，講義等
- 第1回神戸大学先端バイオ工学センター成果発表会, 2021年10月11日, 日本語, 先端バイオ工学センター, オンライン, 日本国, 国内会議, 国際共著していないピキア酵母による低分子抗体高生産化
公開講演，セミナー，チュートリアル，講習，講義等
- 第1回神戸大学先端バイオ工学センター成果発表会, 2021年10月11日, 日本語, 先端バイオ工学センター, オンライン, 日本国, 国内会議, 国際共著していない分泌タンパク質精製のハイスループット自動化
ポスター発表
- 第1回神戸大学先端バイオ工学センター成果発表会, 2021年10月11日, 日本語, 神戸大学先端バイオ工学センター, オンライン, 日本国, 国内会議, 国際共著していないメタノール資化性酵母Pichia pastorisによる有用化合物生産プロセス開発
ポスター発表
- 第1回神戸大学先端バイオ工学センター成果発表会, 2021年10月11日, 日本語, 神戸大学先端バイオ工学センター, オンライン, 日本国, 国内会議, 国際共著していないメタノール資化性酵母Pichia pastorisにおけるゲノム編集技術の開発
ポスター発表
- 第1回神戸大学先端バイオ工学センター成果発表会, 2021年10月11日, 日本語, 神戸大学先端バイオ工学センター, オンライン, 日本国, 国内会議, 国際共著していない大腸菌における有用物質生産に適した鉄硫黄タンパク質発現系の構築
ポスター発表
- The 15th International Congress on Yeasts (ICY15), 英語, online, オーストリア共和国, 国際会議, 国際共著していないDevelopment of engineering tools for methylotrophic yeast Pichia pastoris and their applications to small antibody secretory production
口頭発表（招待・特別）
- 酵母研究会第89回講演会, 2021年08月06日, 日本語, 酵母研究会, オンライン, 日本国, 国内会議, 国際共著していない酵母における合成生物学のための基盤ツール開発と有用物質生産細胞育種への応用
[招待有り]
口頭発表（招待・特別）
- 日本化学会第101年会春季年会 イノベーション共創プログラム（CIP）（スマートセルインダストリーという未来）, 2021年03月22日, 日本語, 日本化学会, オンライン, 日本国, 国内会議, 国際共著していない酵母を用いたセルファクトリーとスマートセル創出に向けた基盤技術開発
[招待有り]
口頭発表（招待・特別）
- 日本農芸化学会関西支部第513回講演会, 2020年11月28日, 日本語, 日本農芸化学会関西支部, オンライン, 日本国, 国内会議, 国際共著していない酵母におけるスクアレン生合成経路の改変および下流モノオキシゲナーゼの発現調節
口頭発表（一般）
- 日本農芸化学会関西支部第513回講演会, 2020年11月28日, 日本語, 日本農芸化学会関西支部, オンライン, 日本国, 国内会議, 国際共著していないドーパミン発酵生産性を簡易的に評価するGPCRメタボライトセンサの開発
口頭発表（一般）
- International Union of Microbiological Societies 2020 Congresses (IUMS 2020), 英語, online, 大韓民国, 国際会議, 国際共著していないDevelopment of engineering tools for methylotrophic yeast Pichia pastoris and their applications to small antibody secretory production
口頭発表（招待・特別）
- Laboratory Automation Developers Conference 2020（LADEC2020）, 2020年10月02日, 日本語, Laboratory Automation Supplier's Association, オンライン, 日本国, 国内会議, 国際共著していない自動分注装置を用いた微生物形質転換システムの構築
[招待有り]
口頭発表（招待・特別）
- 化学工学会第51回秋季大会, 2020年09月25日, 化学工学会, オンライン, 日本国, 国内会議, 国際共著していないGタンパク質共役型受容体を利用したドーパミンセンサの開発とスクリーニングへの応用
ポスター発表
- 第93回日本生化学会大会, 2020年09月食品中の有毒物質を検出するためのパトロール酵母の開発
- 化学工学会第85年会, 2020年03月, 化学工学会, 国内会議低毒性とゲル化能を両立した短鎖ペプチドゲル化剤
口頭発表（一般）
- 日本農芸化学会2020年度大会, 2020年03月, 日本語, 日本農芸化学会, 日本国, 国内会議デルタロドプシンの変異体解析
口頭発表（一般）
- 日本農芸化学会2020年度大会, 2020年03月, 日本語, 日本農芸化学会, 日本国, 国内会議デルタロドプシンによるATP再生を伴う物質生産性の向上
口頭発表（一般）
- 日本農芸化学会関西支部 支部例会（第511回講演会）, 2019年12月07日, 日本語, 国内会議進化デザインによる新規テルペノイドセンサの開発
口頭発表（一般）
- 日本農芸化学会関西支部 支部例会（第511回講演会）, 2019年12月07日, 日本語, 国内会議メラトニン生産性を簡便かつハイスループットに評価するメタボライトセンサの開発
口頭発表（一般）
- 日本農芸化学会関西支部 支部例会（第511回講演会）, 2019年12月07日, 日本語, 国内会議新規な３機能性融合マーカーを用いた酵母遺伝子スイッチの組織的開発
口頭発表（一般）
- The 10th Symposium on Innovative Bioproduction Taichung (iBioT2019), 2019年11月08日, 英語, Tunghai University, Taichung, 台湾, 国際会議Construction of metabolite sensor using yeast signal transduction machinery for monitoring melatonin production
[招待有り]
口頭発表（招待・特別）
- 2019 Asian Synthetic Biology Association (ASBA) Meeting (ASBA2019), 2019年10月28日, 英語, JW Marriott Hotel Chengdu, Chengdu, 中華人民共和国, 国際会議Construction of melatonin metabolite sensor using yeast signal transduction machinery
[招待有り]
口頭発表（招待・特別）
- 第5回Laboratory Automation勉強会, 2019年10月19日, 日本語, ラボラトリーオートメーション研究会, 神戸大学 百年記念館 六甲ホール, 日本国, 国内会議合成生物学とバイオエコノミー
[招待有り]
公開講演，セミナー，チュートリアル，講習，講義等
- 第57回日本生物物理学会年会, 2019年09月25日, 日本生物物理学会, 宮崎県・シーガイアコンベンションセンター, 国内会議マイクロドロップレットを用いた G タンパク質共役型受容体ペプチドアゴニスト探索法のフィジ ビリティスタディ
- 第92回日本生化学会大会（シンポジウム「実験自動化の今」）, 2019年09月20日, 日本語, 日本生化学会, パシフィコ横浜, 国内会議大腸菌・酵母の半自動形質転換システムとハイスループット生産性評価
[招待有り]
口頭発表（招待・特別）
- 第71回日本生物工学会大会, 2019年09月18日, 日本語, 日本生物工学会, 岡山大学 津島キャンパス, 国内会議陽性/陰性選択マーカーと蛍光タンパク質から成る3機能性新規融合タンパク質を用いた酵母遺伝子スイッチの開発
口頭発表（一般）
- 第71回日本生物工学会大会, 2019年09月18日, 日本語, 日本生物工学会, 岡山大学 津島キャンパス, 国内会議デルタロドプシン発現が出芽酵母の代謝に及ぼす影響
口頭発表（一般）
- 第71回日本生物工学会大会, 2019年09月18日, 日本語, 日本生物工学会, 岡山大学 津島キャンパス, 国内会議Pichia pastorisにおいて分泌シグナル配列の１アミノ酸置換はタンパク質分泌生産量を劇的に増大させる
口頭発表（一般）
- 第71回日本生物工学会大会, 2019年09月17日, 日本語, 日本生物工学会, 岡山大学 津島キャンパス, 国内会議ロドプシンを利用した大腸菌における光駆動ATP再生の物質生産への応用
口頭発表（一般）
- 酵母遺伝学フォーラム第52回研究報告会, 2019年09月05日, 日本語, 酵母遺伝学フォーラム, 静岡市清水文化会館マリナート, 国内会議光駆動ATP再生系による出芽酵母型Vmax細胞の創製
口頭発表（一般）
- 生物工学若手研究者の集い 夏のセミナー2019, 2019年07月20日, 日本語, 日本生物工学会 生物工学若手研究者の集い（若手会）, 琵琶湖国定公園 近江白浜 政府登録旅館 白浜荘, 国内会議分泌タンパク質精製のハイスループット自動化
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2019, 2019年07月20日, 日本語, 日本生物工学会 生物工学若手研究者の集い（若手会）, 琵琶湖国定公園 近江白浜 政府登録旅館 白浜荘, 国内会議酵母を用いた癌標的膜タンパク質に対する相互作用阻害分子のスクリーニング系構築
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2019, 2019年07月20日, 日本語, 日本生物工学会 生物工学若手研究者の集い（若手会）, 琵琶湖国定公園 近江白浜 政府登録旅館 白浜荘, 国内会議抗体生産性に関わる遺伝子探索のためのPichia pastorisゲノム編集技術の構築
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2019, 2019年07月20日, 日本語, 日本生物工学会 生物工学若手研究者の集い（若手会）, 琵琶湖国定公園 近江白浜 政府登録旅館 白浜荘, 国内会議メラトニン発酵生産性を簡便に評価する酵母メタボライトセンサの開発
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2019, 2019年07月20日, 日本語, 日本生物工学会 生物工学若手研究者の集い（若手会）, 琵琶湖国定公園 近江白浜 政府登録旅館 白浜荘, 国内会議ノカルジチオシン二次代謝生合成遺伝子クラスターの大腸菌発現系構築
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2019, 2019年07月20日, 日本語, 日本生物工学会 生物工学若手研究者の集い（若手会）, 琵琶湖国定公園 近江白浜 政府登録旅館 白浜荘, 国内会議デルタロドプシンを用いた光エネルギーによるATP再生能力強化酵母株の構築
ポスター発表
- 日本農芸化学会2019年度大会, 2019年03月27日, 日本語, 日本農芸化学会, 東京農業大学 世田谷キャンパス, 国内会議光駆動ATP再生系によるVmax細胞の創製
口頭発表（一般）
- 化学工学会第84年会, 2019年03月14日, 日本語, 化学工学会, 芝浦工業大学 豊洲キャンパス, 国内会議大腸菌による物質生産への光駆動ATP再生の利用に向けて
口頭発表（一般）
- 日本薬学会第139年会, 2019年03月, 日本語, 日本薬学会, 幕張メッセ, 国内会議Gタンパク質共役型受容体ペプチドアゴニスト創出法のハイスループット化
口頭発表（一般）
- 第41回日本分子生物学会年会, 2018年11月29日, 日本語, 日本分子生物学会, パシフィコ横浜, 国内会議DNAシャッフリングを利用した出芽酵母におけるコドン最適化ルールの抽出
ポスター発表
- 2018 Asian Synthetic Biology Association (ASBA) Meeting (ASBA2018), 2018年11月, 英語, Hyatt Regency Jeju, 国際会議Yeast-based in vivo metabolite sensor using signal transduction machinery
口頭発表（一般）
- The Symposium on Biorefinery and Biprocess Topics, 2018 (iBio-N 2018), 2018年11月, 英語, Nanjing, China, 国際会議Construction of a stable, autonomously replicating plasmid vector containing Pichia pastoris centromeric DNA
ポスター発表
- 第70回日本生物工学会大会（シンポジウム「新時代の物質生産宿主開発の方法論： ゲノムを大規模に編集する。代謝計測から設計図を書く。」）, 2018年09月07日, 日本語, 日本生物工学会, 関西大学 千里山キャンパス, 日本国, 国内会議物質生産宿主としての酵母の代謝経路改変とゲノム改変に向けた合成生物工学的手法開発
[招待有り]
口頭発表（招待・特別）
- 第70回日本生物工学会大会, 2018年09月, 日本語, 関西大学 千里山キャンパス, 国内会議出芽酵母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
ポスター発表
- 第70回日本生物工学会大会, 2018年09月, 日本語, 日本生物工学会, 関西大学 千里山キャンパス, 国内会議酵母細胞における生理活性物質メラトニンをモニタリングするためのGタンパク質共役型受容体(GPCR)を用いたメタボライトセンサの開発
口頭発表（一般）
- 第70回日本生物工学会大会, 2018年09月, 日本語, 日本生物工学会, 関西大学 千里山キャンパス, 国内会議光駆動ATP再生系によるVmax細胞の創製
口頭発表（一般）
- 第70回日本生物工学会大会, 2018年09月, 日本語, 日本生物工学会, 関西大学 千里山キャンパス, 国内会議遺伝子組換え効率向上に向けたDNAリガーゼIV欠損Pichia pastoris株の開発
口頭発表（一般）
- 第70回日本生物工学会大会, 2018年09月, 日本語, 日本生物工学会, 関西大学 千里山キャンパス, 国内会議Pichia pastorisにおける自律複製型ベクターを用いた効率的なDNAマルチアセンブル法
口頭発表（一般）
- 第70回日本生物工学会大会, 2018年09月, 日本語, 日本生物工学会, 関西大学 千里山キャンパス, 国内会議Pichia pastorisにおけるセントロメアDNA配列を用いた自律複製型プラスミドベクターの開発
口頭発表（一般）
- 第20回 新産業技術促進検討会, 2018年07月, 日本語, モノづくり日本会議/日刊工業新聞社, 国内会議ハイスループット微生物構築・評価技術の開発
ポスター発表
- 第3回デザイン生命工学研究会, 2018年03月, 日本語, デザイン生命工学研究, 今帰仁村コミュニティセンター, 国内会議発酵制御に向けたグルコースを感知するin vivoバイオセンサーの構築
ポスター発表
- 第3回デザイン生命工学研究会, 2018年03月, 日本語, デザイン生命工学研究, 今帰仁村コミュニティセンター, 国内会議出芽酵母における代謝経路デザインと高級アルコール生産
ポスター発表
- 日本農芸化学会2018年度大会, 2018年03月, 日本語, 日本農芸化学会, 名城大学 天白キャンパス, 国内会議麹菌を用いた遊離型高度不飽和脂肪酸の生産化
口頭発表（一般）
- 第3回デザイン生命工学研究会, 2018年03月, 日本語, デザイン生命工学研究, 今帰仁村コミュニティセンター, 国内会議酵母シグナル伝達機構を利用した膜タンパク質に対する相互作用を阻害する分子スクリーニングシステムの構築
ポスター発表
- 日本農芸化学会2018年度大会, 2018年03月, 日本語, 日本農芸化学会, 名城大学 天白キャンパス, 国内会議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
口頭発表（一般）
- 第12回日本ゲノム微生物学会年会, 2018年03月, 日本語, 日本ゲノム微生物学会, 京都大学 桂キャンパス, 国内会議P. pastorisにおける自律複製型ベクターを用いた効率的なDNAマルチアセンブル法
口頭発表（一般）
- 日本農芸化学会2018年度大会, 2018年03月, 日本語, 名城大学 天白キャンパス, 国内会議Combi-OGAB法を用いた最適コドンの探索
口頭発表（一般）
- The 9th International Symposium of Innovative BioProduction Kobe (iBioK), 2018年02月, 英語, Kobe, Japan, 国際会議Enhanced protein secretion by accumulation of novel effective factors in Pichia pastoris
ポスター発表
- The 9th International Symposium of Innovative BioProduction Kobe (iBioK), 2018年02月, 英語, Kobe, Japan, 国際会議Development of G-protein-coupled receptor (GPCR)-based metabolite sensor for monitoring physiological chemical melatonin in eukaryotic yeast cells
ポスター発表
- The 9th International Symposium of Innovative BioProduction Kobe (iBioK), 2018年02月, 英語, Kobe, Japan, 国際会議Development of combinatorial gene assemble system and application to improvement of organic solvent tolerance in Escherichia coli
ポスター発表
- The 9th International Symposium of Innovative BioProduction Kobe (iBioK), 2018年02月, 英語, Kobe, Japan, 国際会議A new autonomous replicating plasmid vector containing Pichia pastoris centromeric DNA
ポスター発表
- 2017 Asian Symposium on innovative Biorefinery in Singapore (i-BioS 2017), 2017年12月, 英語, Nanjing, China, 国際会議Dynamic regulation of ethanol production in yeast by inactive Cas9 system
ポスター発表
- 第17回糸状菌分子生物学研究会, 2017年11月, 日本語, 糸状菌分子生物学研究会, 佐賀市立東与賀文化ホール, 国内会議麹菌を用いた遊離型高度不飽和脂肪酸の生産化
ポスター発表
- 第68回日本生物工学会大会, 2017年09月, 日本語, 富山国際会議場, 国内会議胞子形成過程を回避した酵母の人為的有性生殖法
ポスター発表
- 第69回日本生物工学会大会（ランチョンセミナー）, 2017年09月, 日本語, 早稲田大学 西早稲田キャンパス, 国内会議長鎖DNA合成技術による高機能遺伝子デザインの可能性
[招待有り]
公開講演，セミナー，チュートリアル，講習，講義等
- 化学工学会第49回秋季大会, 2017年09月, 日本語, 化学工学会, 名古屋市, 国内会議メラトニン濃度をモニタリングするための酵母in vivoメタボライトセンサ
ポスター発表
- 第69回日本生物工学会大会, 2017年09月, 日本語, 日本生物工学会, 早稲田大学 西早稲田キャンパス, 国内会議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
ポスター発表
- 第49回化学工学会秋季大会, 2017年09月, 英語, 化学工学会, 名古屋市, 国内会議Self-assembly of pentapeptide-based hydrogelators for encapsulation and release of functional compounds
口頭発表（一般）
- 第69回日本生物工学会大会, 2017年09月, 日本語, 日本生物工学会, 早稲田大学 西早稲田キャンパス, 国内会議Pichia pastorisのタンパク質分泌生産における新規有用因子の獲得とその蓄積による効果の検証
ポスター発表
- 第69回日本生物工学会大会, 2017年09月, 日本語, 日本生物工学会, 早稲田大学 西早稲田キャンパス, 国内会議Pichia pastorisにおけるセントロメア配列を含む自律複製型プラスミドの開発
ポスター発表
- 第69回日本生物工学会大会, 2017年09月, 日本語, 早稲田大学 西早稲田キャンパス, 国内会議GPCR拮抗薬のポジティブ検出を可能とする反転型レポーター発現系
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2017, 2017年07月, 日本語, 日本生物工学会 若手会, ツネイシしまなみビレッジ, 国内会議特定の生理活性物質を濃度依存的に感知する酵母メタボライトセンサの開発
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2017, 2017年07月, 日本語, 日本生物工学会 若手会, ツネイシしまなみビレッジ, 国内会議酵母代謝改変のための遺伝子のノックダウン/ノックアウト法の開発
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2017, 2017年07月, 日本語, 日本生物工学会 若手会, ツネイシしまなみビレッジ, 国内会議酵母遺伝子スイッチの進化工学のためのON/OFF選抜法の開発
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2017, 2017年07月, 日本語, 日本生物工学会 若手会, ツネイシしまなみビレッジ, 国内会議酵母シグナル伝達を利用したGγ recruitment systemによるタンパク質-ペプチド間相互作用の検出と変異体スクリーニング
ポスター発表
- 第11回日本ゲノム微生物学会年会, 2017年03月, 日本語, 日本ゲノム微生物学会, 慶應義塾大学湘南藤沢キャンパス, 国内会議油性酵母Lipomyces starkeyiを利用したテルペン生産系の開発
口頭発表（一般）
- 日本農芸化学会2017年度大会, 2017年03月, 日本語, 日本農芸化学会, 京都女子大学, 国内会議油性酵母 (Lipomyces starkeyi) によるアルテミシニン前駆体の生産
口頭発表（一般）
- 第11回日本ゲノム微生物学会年会, 2017年03月, 日本語, 日本ゲノム微生物学会, 慶應義塾大学湘南藤沢キャンパス, 国内会議酵母を用いたHMG-CoA reductase阻害剤FR901512生合成遺伝子クラスター解析と新規物質生産の可能性
口頭発表（一般）
- 第11回日本ゲノム微生物学会年会, 2017年03月, 日本語, 日本ゲノム微生物学会, 慶應義塾大学湘南藤沢キャンパス, 国内会議酵母でのモノづくり細胞のエンジニアリング
[招待有り]
口頭発表（招待・特別）
- 第19回化学工学会学生発表会, 2017年03月, 日本語, 化学工学会関西支部, 豊中市, 国内会議メラトニン濃度をモニタリングするための酵母in vivoバイオセンサー
口頭発表（一般）
- 日本農芸化学会2017年度大会, 2017年03月, 日本語, 日本農芸化学会, 京都女子大学, 国内会議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
口頭発表（一般）
- The 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2016), 2016年10月, 英語, Dublin, Ireland, 国際会議In vitro selection of novel peptide agonists for human somatostatin receptor subtype-2 using a water-in-oil microdroplet platform
口頭発表（一般）
- 第68回日本生物工学会大会, 2016年09月, 日本語, 日本生物工学会, 富山国際会議場, 国内会議イソブタノール生産酵母の構築：オルガネラ局在の検討
口頭発表（一般）
- The 14th International Congress on Yeasts (ICY2016), 2016年09月, 英語, ICY14組織委員会, Awaji, Japan, 国際会議Metabolic and cell surface engineering to produce isobutanol from lignocellulosic baiomass in yeast Saccharomyces cerevisiae
ポスター発表
- The 14th International Congress on Yeasts (ICY2016), 2016年09月, 英語, ICY14組織委員会, 淡路市, 国際会議Enhanced protein secretion by accumulation of novel effective factors in Pichia pastoris
ポスター発表
- The 14th International Congress on Yeasts (ICY2016), 2016年09月, 英語, Awaji, Japan, 国際会議Engineering of mitochondrial isobutanol production in Saccharomyces cerevisiae
ポスター発表
- The 14th International Congress on Yeasts (ICY2016), 2016年09月, 英語, Awaji, Japan, 国際会議A new autonomous replicating plasmid vector containing centromere DNA sequence of Pichia pastoris
ポスター発表
- 第6回植物生理化学会シンポジウム, 2016年07月, 日本語, 大阪府立大学中百舌鳥キャンパス, 国内会議微生物バイオによるテルペン系化合物の生産法開発
口頭発表（一般）
- 生物工学若手研究者の集い 夏のセミナー2016, 2016年07月, 日本語, 生物工学会, 府中市, 国内会議培地中のグルコース濃度をモニタリングできる酵母in vivo バイオセンサの開発
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2016, 2016年07月, 日本語, 生物工学会, 府中市, 国内会議出芽酵母における酵素連結法を用いた代謝フラックス改変
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2016, 2016年07月, 日本語, 生物工学会, 府中市, 国内会議メラトニン濃度をモニタリングするための酵母in vivoバイオセンサの開発
ポスター発表
- Metabolic Engineering 11, 2016年06月, 英語, Metabolic Engineering 11, Awaji, Japan, 国際会議Metabolic and cell surface engineering to produce isobutanol from lignocellulosic biomass in yeast Saccharomyces cerevisiae
ポスター発表
- Metabolic Engineering 11, 2016年06月, 英語, Metabolic Engineering 11, 淡路市, 国際会議Heterologous gene expression in Saccharomyces cerevisiae for higher isobutanol production
ポスター発表
- Metabolic Engineering 11, 2016年06月, 英語, Metabolic Engineering 11, 淡路市, 国際会議Expression of varied GFPs in Saccharomyces cerevisiae: comparison of codon-optimized and non-codon-optimized GFPs
ポスター発表
- Metabolic Engineering 11, 2016年06月, 英語, Metabolic Engineering 11, 淡路市, 国際会議Development of selection method for directed evolution of genetic switches in Saccharomyces cerevisiae
ポスター発表
- Metabolic Engineering 11, 2016年06月, 英語, Metabolic Engineering 11, 淡路市, 国際会議Development of Platform Yeast Strain Capable of Direct Fermentation of Raw Biomass to Ethanol
ポスター発表
- Metabolic Engineering 11, 2016年06月, 英語, Metabolic Engineering 11, 淡路市, 国際会議1-Propanol production of S. cerevisiae engineering 2-ketobutyrate biosynthetic pathway
ポスター発表
- 日本農芸化学会2016年度大会, 2016年03月, 日本語, 国内会議大腸菌における1,4-ブタンジオール人工生合成経路構築
口頭発表（一般）
- 日本農芸化学会2016年度大会, 2016年03月, 日本語, 国内会議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
口頭発表（一般）
- 日本農芸化学会2016年度大会, 2016年03月, 日本語, 国内会議マンナンバイオマスからのエタノール生産：βｰマンナナーゼとβｰマンノシダーゼを細胞表層に提示した出芽酵母の開発
口頭発表（一般）
- 日本農芸化学会2016年度大会, 2016年03月, 日本語, 国内会議パスウェイエンジニアリングを用いたフリージア・エアリーパープル及びエアリーピーチの新規セキステルペン合成酵素遺伝子の機能解析
口頭発表（一般）
- 日本農芸化学会2016年度大会, 2016年03月, 日本語, 国内会議タキサジエンの10位を水酸化する新規シトクロムP450 遺伝子の単離と機能解析
口頭発表（一般）
- The 6th iBioK Asian Workshop, 2016年03月, 日本語, Kobe, 国際会議From mannan and lignocellulosic biomass to biochemicals: cell surface display and metabolic engineering in yeast Saccharomyces cerevisiae
口頭発表（一般）
- 日本農芸化学会2016年度大会, 2016年03月, 日本語, 国内会議2,3-ブタンジオール高生産出芽酵母株の構築
口頭発表（一般）
- The 2015 International Chemical Congress of Pacific Basin Societies (Pacifichem 2015), 2015年12月, 英語, 国際会議Specific drug delivery for target cancer tumor using affibody-displaying bionanocapsule/liposome complex
口頭発表（一般）
- The 2015 International Chemical Congress of Pacific Basin Societies (Pacifichem 2015), 2015年12月, 英語, 国際会議Live cell imaging and membrane protein mapping with atomic force microscope
口頭発表（一般）
- The 2015 International Chemical Congress of Pacific Basin Societies (Pacifichem 2015), 2015年12月, 英語, 国際会議High-resolution, quantitative analysis for measuring heterogeneities of G-protein signaling at single-cell levels in yeast
口頭発表（一般）
- The 2015 International Chemical Congress of Pacific Basin Societies (Pacifichem 2015), 2015年12月, 英語, 国際会議Desired alteration of protein affinities using competitive screening system
口頭発表（一般）
- 33th Yeast Workshop, 2015年11月, 日本語, 国内会議酵母シグナル伝達を利用した結合タンパク質の選択的スクリーニング
口頭発表（一般）
- 33th Yeast Workshop, 2015年11月, 日本語, 国内会議酵母シグナル伝達を用いた標的膜タンパク質に対するバイオメディカル分子スクリーニングシステム
口頭発表（一般）
- 33th Yeast Workshop, 2015年11月, 日本語, 国内会議ヒトニューロテンシン受容体のリガンド検出のための酵母バイオセンサーの開発
口頭発表（一般）
- 第67回日本生物工学会大会 国際シンポジウム, 2015年10月, 日本語, 国内会議大腸菌パスウェイエンジニアリングによるパクリタキセル前駆体タキサジエン酸化物の生産
口頭発表（一般）
- 第67回日本生物工学会大会 国際シンポジウム, 2015年10月, 日本語, 国内会議フリージア・エアリーパープルから新規セスキテルペン合成酵素遺伝子の単離と機能解析
口頭発表（一般）
- 第67回日本生物工学会大会 国際シンポジウム, 2015年10月, 日本語, 国内会議ヒトニューロテンシン受容体におけるリガンド探索のための酵母バイオセンサー
口頭発表（一般）
- 第67回日本生物工学会大会 国際シンポジウム, 2015年10月, 日本語, 国内会議カンツバキから新規セスキテルペン合成酵素遺伝子の単離と機能解析
口頭発表（一般）
- 化学工学会第47回秋季大会, 2015年09月, 日本語, 国内会議合成生物学的アプローチを目指した酵母プロティンタグの開発
口頭発表（一般）
- 日本農芸化学会2015年度大会, 2015年03月, 日本語, 日本農芸化学会, 岡山市, 国内会議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
口頭発表（一般）
- 化学工学会第80年会, 2015年03月, 日本語, 化学工学会, 江東区, 国内会議酵母における1-propanol発酵生産
口頭発表（一般）
- 第17回化学工学会学生発表会, 2015年03月, 日本語, 化学工学会, 徳島市, 国内会議ヒトニューロテンシン受容体のリガンド探索のための酵母1細胞分析用バイオセンサー
口頭発表（一般）
- 合成生物工学シンポジウム, 2014年11月, 日本語, 文部科学省先端融合領域イノベーション創出拠点形成プログラム 「バイオプロダクション次世代農工連携拠点 」, 神戸大学 統合研究拠点 コンベンションホール, 国内会議酵母における合成生物工学ツールの開発
[招待有り]
口頭発表（招待・特別）
- 「細胞を創る」研究会7.0, 2014年11月, 日本語, 「細胞を創る」研究会, 東京大学 弥生講堂一条ホール, 国内会議G 蛋白質共役型受容体の二量体形成およびシグナル伝達同時解析のための二色蛍光レポーターによる酵母細胞設計
口頭発表（一般）
- The 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2014), 2014年10月, 英語, San Antonio, TX, USA, 国際会議Peptide-based ligand screening system for G protein-coupled receptors using water-in-oil microdroplets
口頭発表（一般）
- The 31st International Specialised Symposium on Yeast (ISSY 31), 2014年10月, 日本語, Vipava and Nova Gorica, Slovenia, 国際会議Genetic engineering for altering metabolic flow in Saccharomyces cerevisiae to produce higher alcohols
口頭発表（一般）
- 第66回日本生物工学会大会, 2014年09月, 日本語, 日本生物工学会, 札幌市, 国内会議二次代謝産物を指標とした有用油性酵母のクラスター解析
ポスター発表
- 第66回日本生物工学会大会, 2014年09月, 日本語, 日本生物工学会, 札幌市, 国内会議大腸菌のパスウェイエンジニアリングによる安価な基質からのパクリタキセル前駆体タキサジエンの効率的生産
ポスター発表
- 第66回日本生物工学会大会, 2014年09月, 日本語, 日本生物工学会, 札幌市, 国内会議出芽酵母における分割ルシフェラーゼを利用したGPCR リガンド応答検出法の応用
ポスター発表
- 第66回日本生物工学会大会, 2014年09月, 日本語, 日本生物工学会, 札幌市, 国内会議出芽酵母 Saccharomyces cerevisiae での嗅覚受容体匂い分子応答における匂い結合タンパク質の効果
ポスター発表
- 第66回日本生物工学会大会, 2014年09月, 日本語, 日本生物工学会, 札幌市, 国内会議pH 応答性ペプチドGALA を表層提示したバイオナノカプセルのエンドソーム脱出
ポスター発表
- 第66回日本生物工学会大会, 2014年09月, 日本語, 日本生物工学会, 札幌市, 国内会議G タンパク質共役型受容体の二量体形成検出のためのゲノム編集技術の開発
ポスター発表
- 生物工学若手研究者の集い夏のセミナー, 2014年07月, 日本語, 生物工学会, 神戸市, 国内会議ｐH応答性膜融合ペプチドGALAを表層提示したバイオナノカプセルのエンドソーム脱出
ポスター発表
- 生物工学若手研究者の集い夏のセミナー, 2014年07月, 日本語, 生物工学会, 神戸市, 国内会議色素生産を指標としたCoA代謝経路の評価系
ポスター発表
- 生物工学若手研究者の集い夏のセミナー, 2014年07月, 日本語, 生物工学会, 神戸市, 国内会議上皮成長因子受容体を特異的に認識するAffibody提示バイオナノカプセルの開発
ポスター発表
- 生物工学若手研究者の集い夏のセミナー, 2014年07月, 日本語, 生物工学会, 神戸市, 国内会議酵母シグナル伝達機構を基盤としたアフィニティ改変タンパク質スクリーニングシステムの開発
ポスター発表
- 生物工学若手研究者の集い夏のセミナー, 2014年07月, 日本語, 生物工学会, 神戸市, 国内会議アンジオテンシン受容体のリガンド探索のための酵母バイオセンサーの開発
ポスター発表
- 生物工学若手研究者の集い夏のセミナー, 2014年07月, 日本語, 生物工学会, 神戸市, 国内会議G蛋白質共役型受容体の二量体形成に応答して機能するゲノム編集技術の開発
ポスター発表
- Metabolic Engineering X, 2014年06月, 英語, Society for Biological Engineering, Vancouver, Canada, 国際会議Metabolic engineering of yeast central metabolism for higher alcohol production
口頭発表（一般）
- Metabolic Engineering X, 2014年06月, 英語, Society for Biological Engineering, Vancouver, Canada, 国際会議Genetic engineering to produce higher alcohols in yeast Saccharomyces cerevisiae
口頭発表（一般）
- 日本農芸化学会2014年度大会, 2014年03月, 日本語, 日本農芸化学会, 川崎市, 国内会議代謝シミュレーションにもとづく酵母イソブタノール生産能の向上
口頭発表（一般）
- 日本農芸化学会2014年度大会, 2014年03月, 日本語, 日本農芸化学会, 川崎市, 国内会議ED経路導入による酵母中央代謝経路の拡張
口頭発表（一般）
- 「細胞を創る」研究会6.0, 2013年11月, 日本語, 「細胞を創る」研究会, 鶴岡メタボロームキャンパス レクチャーホール, 国内会議酵母の遺伝情報を操作する
[招待有り]
口頭発表（招待・特別）
- 2013 Asian Symposium on innovative Biorefinery in Beijing (i-BioB 2013), 2013年10月, 日本語, Beijing, China, 国際会議Metabolic engineering of yeast Saccharomyces cerevisiae for increased isobutanol production
口頭発表（一般）
- 第65回日本生物工学会大会, 2013年09月, 日本語, 日本生物工学会, 広島市, 国内会議上皮成長因子受容体を特異的に認識するAffibody提示バイオナノカプセルの開発
ポスター発表
- 第65回日本生物工学会大会, 2013年09月, 日本語, 日本生物工学会, 広島市, 国内会議出芽酵母Saccharomyces cerevisiaeでの嗅覚受容体の機能的発現におけるアクセサリータンパク質の効果
ポスター発表
- 第65回日本生物工学会大会, 2013年09月, 日本語, 日本生物工学会, 広島市, 国内会議酵母蛍光レポーターの改変によるヒト由来7回膜受容体の高感度リガンド検出システム
ポスター発表
- 化学工学会第45回秋季大会, 2013年09月, 日本語, 化学工学会, 岡山市, 国内会議ヒト受容体リガンド探索のための酵母蛍光レポーター高感度アッセイシステムの開発とその応用
ポスター発表
- 第65回日本生物工学会大会, 2013年09月, 日本語, 日本生物工学会, 広島市, 国内会議イソブタノール生産酵母におけるトランスヒドロゲナーゼ様シャントの活性化
ポスター発表
- 第65回日本生物工学会大会, 2013年09月, 日本語, 日本生物工学会, 広島市, 国内会議G蛋白質共役型受容体の二量体形成およびシグナル伝達の同時解析システム
ポスター発表
- The 26th International Conference on Yeast Genetics and Molecular Biology (Yeast2013), 2013年08月, 英語, Committee of the 26th International Conference on Yeast Genetics and Molecular Biology, Frankfurt am Main, Germany, 国際会議Metabolic pathway engineering of yeast Saccharomyces cerevisiae for isobutanol production
ポスター発表
- The 19th Symposium of Young Asian Biochemical Engineers' Community (YABEC2013), 2013年08月, 英語, 生物工学会, Xinjiang,China, 国際会議A yeast-based simultaneous method to analyze dimerization and signaling of G-protein-coupled receptor by dual-color reporter
ポスター発表
- The 26th International Conference on Yeast Genetics and Molecular Biology (Yeast2013), 2013年08月, 英語, Committee of the 26th International Conference on Yeast Genetics and Molecular Biology, Frankfurt am Main, Germany, 国際会議A selection system exploiting yeast signal transduction machinery to create desirably affinity-altered protein variants.
ポスター発表
- The 19th Symposium of Young Asian Biochemical Engineers' Community (YABEC2013), 2013年08月, 英語, 生物工学会, Xinjiang,China, 国際会議A selection method of desirably affinity-altered protein mutants using cellular signaling in living yeast cells
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2013, 2013年07月, 日本語, 生物工学会, 宮崎市, 国内会議ヒト由来7回膜受容体の酵母蛍光レポーター改変型高感度リガンド検出システムの開発とその応用
ポスター発表
- 生物工学若手研究者の集い 夏のセミナー2013, 2013年07月, 日本語, 生物工学会, 宮崎市, 国内会議Saccharomyces cerevisiaeにおけるイソブタノール高生産のための代謝改変
ポスター発表
- World Biotechnology Congress 2013 (WBC2013), 2013年06月, 英語, Eureka Conferences, Boston, USA, 国際会議Metabolically engineered yeast Saccharomyces cerevisiae for increased isobutanol production
[招待有り]
口頭発表（招待・特別）
- 第35回日本分子生物学会年会, 2012年12月, 英語, 日本分子生物学会, 福岡市, 国内会議Developing a Screening Method for Desirably Altering Affinities of Protein Mutants Based on Yeast Cellular Signaling
口頭発表（一般）
- International Joint Symposium on Single-Cell Analysis (The 6th International Workshop on Approaches to Single-Cell Analysis & The 8th International Forum on Post-Genome Technologies), 2012年11月, 英語, The Society for Single-Cell Surveyor, 京都市, 国際会議Interaction survey between GPCR and ligand on the surface of yeast by AFM
ポスター発表
- International Joint Symposium on Single-Cell Analysis (The 6th International Workshop on Approaches to Single-Cell Analysis & The 8th International Forum on Post-Genome Technologies), 2012年11月, 英語, The Society for Single-Cell Surveyor, 京都市, 国際会議Improvement of reporter sensitivity for human G-protein-coupled receptor signaling assay in yeast cells by flow cytometry
ポスター発表
- International Joint Symposium on Single-Cell Analysis (The 6th International Workshop on Approaches to Single-Cell Analysis & The 8th International Forum on Post-Genome Technologies), 2012年11月, 英語, The Society for Single-Cell Surveyor, 京都市, 国際会議A technique to screen heterdimerizable partners against target human G-protein-coupled receptors in living yeast cells
ポスター発表
- International Joint Symposium on Single-Cell Analysis (The 6th International Workshop on Approaches to Single-Cell Analysis & The 8th International Forum on Post-Genome Technologies), 2012年11月, 英語, The Society for Single-Cell Surveyor, 京都市, 国際会議A selection method of desirably affinity-altered protein mutants using cellular signaling in living yeast cells
ポスター発表
- 第64回日本生物工学会大会, 2012年10月, 日本語, 日本生物工学会, 神戸市, 国内会議出芽酵母を用いた嗅覚受容体リガンドアッセイシステムの最適化
口頭発表（一般）
- The 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS 2012), 2012年10月, 英語, Okinawa, Japan, 国際会議Yeast-based ligand assay system for detecting G protein-coupled receptor activation in water-in-oil droplets
口頭発表（一般）
- The 18th Symposium of Young Asian Biochemical Engineers' Community (YABEC2012), 2012年10月, 英語, Society for Biological Engineering, Tokushima, Japan, 国際会議Genetic engineering of valine biosynthesis for isobutanol production in Saccharomyces cerevisiae
口頭発表（一般）
- The 18th Symposium of Young Asian Biochemical Engineers' Community (YABEC2012), 2012年10月, 英語, Society for Biological Engineering, Tokushima, Japan, 国際会議A yeast-based screening method to identify hetero-dimerizable partners against target human G-protein-coupled receptor
口頭発表（一般）
- The 18th Symposium of Young Asian Biochemical Engineers' Community (YABEC2012), 2012年10月, 英語, Society for Biological Engineering, Tokushima, Japan, 国際会議A new technology to screen desirably affinity-altered proteins based on yeast signal transduction machinery
口頭発表（一般）
- 日本農芸化学会関西支部大会, 2012年09月, 日本語, 日本農芸化学会, 亀岡市, 国内会議非天然型経路を導入した新規イソブタノール生産酵母の開発
口頭発表（一般）
- The 15th International Biotechnology Symposium (IBS 2012), 2012年09月, 英語, The Korean Society for Biotechnology and Bioengineering, Deague, Korea, 国際会議Complex carriers of affibody-displaying bio-nanocapsule and composition-varied liposomes for HER2-expressing breast cancer cell-specific protein delivery
口頭発表（一般）
- The 13th International Congress on Yeasts (ICY2012), 2012年08月, 英語, ICY 2012 Organizing Committee, Madison, WI, USA, 国際会議Genetically enhanced valine biosynthesis and the Ehrlich pathway for isobutanol production in Saccharomyces cerevisiae
口頭発表（一般）
- Metabolic EngineeringⅨ, 2012年06月, 英語, Engineering Conferences International, Biarritz, France, 国際会議Genetic engineering to enhance the Ehrlich pathway and alter carbon flux for increased isobutanol production by Saccharomyces cerevisiae
口頭発表（一般）
- Metabolic EngineeringⅨ, 2012年06月, 英語, Engineering Conferences International, Biarritz, France, 国際会議Effect of metabolic inhibitors on yeast central metabolism
口頭発表（一般）
- Japan-Finland Biotechnology Symposium 2012, 2012年06月, 英語, Sendai, Japan, 国際会議Combinatorial screen of human G-protein-coupled receptor heterodimer pairs by using a split-ubiquitin yeast two-hybrid system
口頭発表（一般）
- Japan-Finland Biotechnology Symposium 2012, 2012年06月, 英語, Sendai, Japan, 国際会議A new technology to screen desirably affinity-altered proteins
口頭発表（一般）
- 日本農芸化学会2012年度大会, 2012年03月, 日本語, 日本農芸化学会, 京都市, 国内会議Saccharomyces cerevisiaeにおけるイソブタノール生産性向上のためのEhrlich経路強化と炭素フラックス改変戦略
口頭発表（一般）
- 第34回日本分子生物学会年会, 2011年12月, 英語, 日本分子生物学会, 横浜市, 国内会議Amplification and smoothing of signal activation from human G-protein-coupled receptor for enriching agonist stimulation in yeast
ポスター発表
- The 17th Symposium of Young Asian Biochemical Engineers' Community (YABEC2011), 2011年10月, 英語, Society for Biological Engineering, Incheon, Korea, 国際会議A yeast two-hybrid method to identify heterodimerization pairs of human G protein-coupled receptors
口頭発表（一般）
- 第63回日本生物工学会大会, 2011年09月, 日本語, 日本生物工学会, 小金井市, 国内会議次世代バイオアルコール生産酵母の代謝デザイン
口頭発表（一般）
- 第63回日本生物工学会大会, 2011年09月, 日本語, 日本生物工学会, 小金井市, 国内会議マウス嗅覚受容体の出芽酵母での機能的発現における N 末端およびC 末端配列置換の効果
口頭発表（一般）
- 第63回日本生物工学会大会, 2011年09月, 日本語, 日本生物工学会, 小金井市, 国内会議フラン化合物存在下でのXR-XDH 発現酵母を用いたキシロース発酵におけるNADH 依存性Adh1過剰発現のインパクト
口頭発表（一般）
- 第63回日本生物工学会大会, 2011年09月, 日本語, 日本生物工学会, 小金井市, 国内会議バイオマス資源からの BIO-1,3-Butanediol 発酵生産
口頭発表（一般）
- 第63回日本生物工学会大会, 2011年09月, 日本語, 日本生物工学会, 小金井市, 国内会議タンパク質アファニティを自在に改変するための新規スクリーニングシステムの開発
口頭発表（一般）
- 第63回日本生物工学会大会, 2011年09月, 日本語, 日本生物工学会, 小金井市, 国内会議G 蛋白質共役型受容体のヘテロダイマー形成ペアの探索のための新規検出システムの開発
口頭発表（一般）
- 第63回日本生物工学会大会, 2011年09月, 日本語, 日本生物工学会, 小金井市, 国内会議Affibody 提示Bio-nanocapsule を用いたER2 発現癌細胞へのタンパク質送達システム
口頭発表（一般）
- 化学工学会第76年会, 2011年03月, 日本語, 日本化学会, 東京都 小金井市, 国内会議代謝工学を活用したイソブタノール生産酵母の創製
口頭発表（一般）
- 化学工学会第15回学生発表会, 2011年03月, 日本語, (社)化学工学会, 神戸市, 国内会議酵母GPCRシグナル伝達系を用いたタンパク質アフィニティ改変技術の開発
口頭発表（一般）
- 日本農芸化学会2011年度大会, 2011年03月, 日本語, 日本農芸化学会, 京都市, 国内会議バイオマス資源からの BIO-1,3-Butanediol 発酵生産-2 -グルコースから 1,3-Butanediol の合成-
口頭発表（一般）
- 日本農芸化学会2011年度大会, 2011年03月, 日本語, 日本農芸化学会, 京都市, 国内会議バイオマス資源からの BIO-1,3-Butanediol 発酵生産-1
口頭発表（一般）
- The 5th International Workshop on Approaches to Single-Cell Analysis, 2011年03月, 英語, Tokyo, Japan, 国際会議Single-cell analysis revealed correlation between yeast GPCR signaling and plasmid retention
ポスター発表
- The 5th International Workshop on Approaches to Single-Cell Analysis, 2011年03月, 英語, Tokyo, Japan, 国際会議Expression and functional characterization of olfactory receptors in Saccharomyces cerevisiae
ポスター発表
- 日本化学会第91春季年会, 2011年03月, 日本語, 化学工学会, 横浜市, 国内会議AFMを用いた生細胞表層のレセプター・リガンド間相互作用解析と細胞応答の観察
口頭発表（一般）
- The 2010 International Chemical Congress of Pacific Basin Societies (Pacifichem 2010), 2010年12月, 英語, Honolulu, Hawaii, USA, 国際会議High-resolution, quantitative signalling assay for G-protein-coupled receptors by flow cytometry
口頭発表（一般）
- 第62回日本生物工学会大会, 2010年10月, 日本語, (社)日本生物工学会, 宮崎市, 国内会議ユビキチン分割体を用いたG 蛋白質共役型受容体二量体化検出システムとドメイン解析への応用
ポスター発表
- 第62回日本生物工学会大会, 2010年10月, 日本語, (社)日本生物工学会, 宮崎市, 国内会議キシロース資化性Saccharomyces cerevisiae におけるアルコールデヒドロゲナーゼ過剰発現およびフラン化合物存在下でのキシロース発酵特性
ポスター発表
- 第62回日本生物工学会大会, 2010年10月, 日本語, (社)日本生物工学会, 宮崎市, 国内会議1細胞アッセイ技術に基づくGPCR 解析、アゴニスト探索法の開発
口頭発表（一般）
- 化学工学会第42回秋季大会, 2010年09月, 日本語, (社)化学工学会, 京都市, 国内会議力学的指標による生細胞表面におけるリガンド‐受容体の相互作用解析
ポスター発表
- 化学工学会第42回秋季大会, 2010年09月, 日本語, (社)化学工学会, 京都市, 国内会議ガラクトース転写誘導系を利用した酵母リガンド検出系の開発
ポスター発表
- 日本化学会第90春季年会, 2010年03月, 日本語, 日本化学会, 東大阪市, 国内会議原子間力顕微鏡を用いた細胞表層におけるリガンド・レセプター間相互作用測定系の構築
口頭発表（一般）
- 化学工学会第75年会, 2010年03月, 日本語, (社)化学工学会, 鹿児島市, 国内会議G蛋白質共役型受容体における二量体化解析のための新規検出システム
口頭発表（一般）
- APBioChEC'09, 2009年11月, 英語, APBioChEC'09 Committee, 神戸市, 国際会議Split-ubiquitin system for analyzing oligomerization of G protein-coupled receptor
ポスター発表
- APBioChEC'09, 2009年11月, 英語, APBioChEC'09 Committee, 神戸市, 国際会議Investigation of the interaction between GPCR and ligand by AFM equipped with bio-molecule modified cantilever
ポスター発表
- APBioChEC'09, 2009年11月, 英語, APBioChEC'09 Committee, 神戸市, 国際会議Functional analysis of mutant human somatostatin receptor using a yeast-based fluorescence reporter assay
ポスター発表
- APBioChEC'09, 2009年11月, 英語, APBioChEC'09 Committee, 神戸市, 国際会議Expression and signaling analyses of human G protein-coupled receptor in yeast
ポスター発表
- APBioChEC'09, 2009年11月, 英語, APBioChEC'09 Committee, 神戸市, 国際会議Construction of a novel detection system for protein–protein interactions using yeast G-protein signaling
口頭発表（一般）
- APBioChEC'09, 2009年11月, 英語, APBioChEC'09 Committee, 神戸市, 国際会議Bioethaol fermentation from mixed sugar by the recombinant yeast with xyloseisomerase pathway
ポスター発表
- 第61回日本生物工学会大会, 2009年09月, 日本語, 名古屋市, 国内会議酵母Ｇタンパク質シグナルを用いたタンパク質間相互作用解析法の開発
口頭発表（一般）
- The 6th International Forum on Post-Genome Technologies (IFPT'6), 2009年09月, 英語, Beijing, China, 国際会議Construction of a novel detection system for protein-protein interactions using yeast G-protein signaling
ポスター発表
- International Workshop HITS 2009, 2009年02月, 英語, Tokyo, Japan, 国際会議Construction of novel detection system for protein-protein interaction using yeast
ポスター発表
- 第31回日本分子生物学会年会・第81回日本生化学会大会合同大会, 2008年12月, 日本語, 日本分子生物学会年会日本生化学会, 神戸市, 国内会議酵母G蛋白質共役型受容体蛍光アッセイシステムによるヒトソマトスタチンレセプター細胞外ループドメイン2の変異解析
ポスター発表
- The 14th Symposium of Young Asian Biochemical Engineers' Community (YABEC2008), 2008年11月, 英語, Tokyo, Japan, 国際会議Mutational analysis for extracellular loop-2 of human somatostatin receptor by yeast-based fluorescence signalling assay
ポスター発表
- Bioseparation for Biorecignition and Bionanotechnology Conference, 2008年10月, 英語, Ansan, Korea, 国際会議Rapid and Efficient Selection of Yeast Displaying a Target Protein Using Thermo-responsive Magnetic Nanoparticles
ポスター発表
- 化学工学会 第40回 秋季大会, 2008年09月, 日本語, 化学工学会, 宮城県仙台市, 国内会議酵母蛍光レポーターアッセイによるヒトソマトスタチンレセプター変異体の機能解析
ポスター発表
- 化学工学会 第40回 秋季大会, 2008年09月, 日本語, 化学工学会, 宮城県仙台市, 国内会議酵母でのヒトG蛋白質共役型受容体発現に関する分泌シグナル配列の影響
ポスター発表
- 化学工学会 第40回 秋季大会, 2008年09月, 日本語, 化学工学会, 宮城県仙台市, 国内会議ペプチド転移酵素を用いた細胞内タンパク質連結技術の開発
ポスター発表
- The 3rd International Workshop on Approaches to Single-Cell Analysis, 2008年09月, 英語, Zurich, Switzerland, 国際会議Mutational analysis of human somatostatin receptor by yeast-based signalling assay
ポスター発表
- 第60回 日本生物工学会2008年度大会, 2008年08月, 日本語, 日本生物工学会, 宮城県仙台市, 国内会議ビオチン提示酵母を用いた新規細胞表層提示システムの開発
口頭発表（一般）
- 日本薬学会第128年会, 2008年03月, 日本語, 日本薬学会, 横浜市, 国内会議酵母ディスプレイ法によるタンパク質の分泌生産/同時回収システム
ポスター発表
- 化学工学会第10回化学工学会学生発表会大阪大会(西日本地区), 2008年03月, 日本語, 化学工学会, 吹田市, 国内会議酵母PCAシステムによるタンパク質分子間相互作用の検出
口頭発表（一般）
- 化学工学会第73年会, 2008年03月, 日本語, 化学工学会, 浜松市, 国内会議フローサイトメーター(FCM)を利用した酵母GPCRシグナリング解析技術
口頭発表（一般）
- 第6回最先端バイオテクノロジー若手発表会, 2008年02月, 日本語, 化学工学会関西支部化学工学会バイオ部会, 西宮市, 国内会議酵母GPCRシグナリングにおけるフローサイトメトリー解析技術
ポスター発表
- 第30回日本分子生物学会年会・第80回日本生化学会大会・合同大会, 2007年12月, 日本語, 日本分子生物学会年会日本生化学会, 横浜市, 国内会議分子ディスプレイ法によるタンパク質の同時生産/回収システムの構築
口頭発表（一般）
- 化学工学会第39回秋季大会, 2007年09月, 日本語, 化学工学会, 札幌市, 国内会議酵母を用いたリガンド検出システムの開発
ポスター発表
- 第59回 日本生物工学会大会, 2007年09月, 日本語, 広島大学, 国内会議温度応答性磁性ナノ粒子を用いた迅速かつ高効率なアフィニティ分子選択法の確立
ポスター発表
- The 5th International Forum on Post-Genome Technologies (IFPT'5), 2007年09月, 英語, Suzhou, China, 国際会議Fluorescence detection system for human G protein-coupled receptor signaling in yeast
ポスター発表
- The 2nd International Workshop on Approaches to Single-Cell Analysis, 2007年09月, 英語, Tokyo, Japan, 国際会議Fluorescence detection system for heterologous G protein-coupled receptor in yeast
ポスター発表
- 化学工学会第72年会, 2007年03月, 日本語, 化学工学会, 京都市, 国内会議酵母表層ディスプレイを用いた迅速かつ高効率なタンパク質アフィニティ選択法の確立
口頭発表（一般）
- 日本農芸化学会2007年度大会, 2007年03月, 日本語, 日本農芸化学会, 世田谷区, 国内会議蛍光レポーターを用いたヒトGタンパク質共役型受容体に対する高感度な酵母アッセイシステム
口頭発表（一般）
- The 12th Symposium of Young Asian Biochemical Engineers' Community (YABEC2006), 2006年11月, 英語, Kaohsiung, Taiwan, 国際会議Development of ligand detection system for heterologous G protein-coupled receptor using yeast
ポスター発表
- 日本生物工学会2006年度大会, 2006年09月, 日本語, 日本生物工学会, 豊中市, 国内会議酵母細胞表層ディスプレイを利用した新規タンパク質間相互作用解析システムの開発
口頭発表（一般）
- 日本生物工学会2006年度大会, 2006年09月, 日本語, 日本生物工学会, 豊中市, 国内会議酵母による異種Gタンパク質共役型受容体リガンド検出システムの開発
口頭発表（一般）
- 化学工学会第38回秋季大会, 2006年09月, 日本語, 化学工学会, 福岡市, 国内会議フェロモン応答性タンパク質を利用した新規レポーターによるリガンド検出システムの開発
ポスター発表
- The 4th International Forum on Post-Genome Technologies (4’IFPT), 2006年09月, 英語, Hangzhou, China, 国際会議Development of a single cell analysis system of agonist for drug discovery
ポスター発表
- The 1st International Workshop on Approaches to Single-Cell Analysis, 2006年06月, 英語, Uppsala, Sweden, 国際会議Development of a single cell analysis system of agonist for drug discovery
ポスター発表
- 日本農芸化学会2006年度大会, 2006年03月, 日本語, 日本農芸化学会, 京都市, 国内会議Zドメイン表層提示細胞の機能改変と抗体関連分子の精製への応用
口頭発表（一般）
- 第4回最先端バイオテクノロジー公開セミナー, 2006年02月, 日本語, 化学工学会関西支部化学工学会バイオ部会, 西宮市, 国内会議EGFP-HIS3融合遺伝子をFUS1遺伝子座に組込んだ酵母細胞におけるシグナル伝達の解析
ポスター発表
- 第６回コンビナトリアル・バイオエンジニアリングシンポジウム, 2006年01月, 日本語, コンビナトリアル・バイオエンジニアリング研究会, 理化学研究所, 国内会議リガンド・レセプター共提示酵母によるハイスループット創薬システムの開発
口頭発表（招待・特別）
- 日本生物工学会2005年度大会, 2005年11月, 日本語, 日本生物工学会, つくば市, 国内会議酵母シグナル伝達経路を利用したリガンドスクリーニングシステムの開発
口頭発表（一般）
- 日本生物工学会2004年度大会, 2004年09月, 日本語, 日本生物工学会, 名古屋市, 国内会議酵母シグナル伝達によるリガンド検出システム
口頭発表（一般）
所属学協会
共同研究・競争的資金等の研究課題
- 日本医療研究開発機構（AMED）, 次世代治療・診断実現のための創薬基盤技術開発事業 / バイオ医薬品の高度製造技術の開発 / 高性能な国産細胞株の構築, 高機能遺伝子デザイン技術研究組合（TRAHED），神戸大学，アステラス製薬，味の素，東京大学，東京工業大学，東北大学, 2018年05月 - 2021年03月, 研究分担者高性能な国産細胞株の構築
- 日本医療研究開発機構（AMED）, 次世代治療・診断実現のための創薬基盤技術開発事業 / バイオ医薬品の高度製造技術の開発 / 先端的バイオ製造技術開発, 高機能遺伝子デザイン技術研究組合（TRAHED），神戸大学，Bio-energy, 2018年05月 - 2021年03月, 研究代表者バイオ医薬品の多品種・大量製造に適した微生物による高度生産技術の開発
- 新エネルギー・産業技術総合開発機構（NEDO）, 植物等の生物を用いた高機能品生産技術の開発 / 微生物による高機能品生産技術開発, 2019年 - 2021年02月, 研究分担者希少アミノ酸エルゴチオネイン高生産スマートセルの開発
- 新エネルギー・産業技術総合開発機構（NEDO）, 植物等の生物を用いた高機能品生産技術の開発, 2016年10月 - 2021年02月, 研究分担者高生産性微生物創製に資する情報解析システムの開発
- 科学技術振興機構（JST）, 先端融合領域イノベーション創出拠点形成プログラム, 神戸大学, 2008年 - 2019年03月, 研究分担者バイオプロダクション次世代農工連携拠点
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 大阪大学, 2015年04月01日 - 2018年03月31日
バイオマス原料から、化成品原料のブタノールを生産する出芽酵母の構築を目指した。そのために、細胞内代謝中間体濃度の絶対定量値から各反応のギブス自由エネルギー変化を推定し、代謝律速を同定する熱力学レベルの解析法を確立した。エタノール非生産酵母株の代謝状態を精密に測定することに成功し、代謝フローをエタノール生合成から他経路へと転換するには、細胞内コンパートメントを考慮したNADH過剰の解消が必要なことを明らかにした。これらの知見をもとに、ピルビン酸のミトコンドリア輸送に着目し、新たなイソブタノール高生産株の構築に成功した。
- 経済産業省（METI） / 日本医療研究開発機構（AMED）, 次世代治療・診断実現のための創薬基盤技術開発事業, 高機能遺伝子デザイン技術研究組合（TRAHED），神戸大学，産業技術総合研究所，慶應義塾大学，カネカ，アステラス製薬，プレシジョン・システム・サイエンス，インシリコバイオロジー，東京工業大学，東北大学，東京大学，弘前大学，金澤工業大学，大阪府立大学, 2014年10月 - 2018年03月, 研究分担者国際基準に適合した次世代抗体医薬等の製造技術のうち高生産宿主構築の効率化基盤技術の開発に係るもの
- 科学技術振興機構（JST）, 未来社会創造事業（MIRAI）, 2017年11月, 研究分担者光駆動ATP再生系によるVmax細胞の創製
競争的資金
- 経済産業省（METI）, 経済産業省委託事業, 高機能遺伝子デザイン技術研究組合（TRAHED），神戸大学，産業技術総合研究所，慶應義塾大学，プレシジョン・システム・サイエンス，Spiber，小島プレス工業，味の素，三菱化学，カネカ，神戸天然物化学，アステラス製薬，インシリコバイオロジー，クミアイ化学工業，次世代天然物化学技術研究組合，東北大学，北海道大学，京都大学，国立遺伝学研究所，千葉大学，東京工業大学，石川県立大学，鳥取大学，理化学研究所，バイオインダストリー協会, 2012年10月 - 2017年03月, 研究分担者革新的バイオマテリアル実現のための高機能化ゲノムデザイン技術開発
- 国際協力機構（JICA） / 科学技術振興機構（JST）, 地球規模課題対応国際科学技術協力（SATREPS）, 神戸大学，インドネシア科学院（LIPI）, 2012年04月 - 2017年03月, 研究分担者インドネシアにおける統合バイオリファイナリーシステムの開発
- 三菱ケミカル株式会社, NEDO委託プロジェクト「植物等の生物を用いた高機能品生産技術の開発」, 2017年【NEDO：三菱ケミカル】共同研究員受入
競争的資金
- 日本学術振興会, 学術研究助成基金助成金／若手研究(B), 若手研究(B), 神戸大学, 2014年04月 - 2016年03月, 研究代表者
Gタンパク質シグナルに応答するスイッチングゲノム編集技術として、Cre/loxP組換えシステムを利用した「遺伝子が抜け落ちて代わりに別の遺伝子が発現する」系を確立した。これにより、Gタンパク質シグナルを検知して2つの遺伝子発現の「ON→OFF」と「OFF→ON」を同時に引き起こすことが可能となった。本技術を利用して、Gタンパク質共役型受容体（GPCR）の二量体形成シグナルに応答して2つの遺伝子発現を切替えることのできるシステムを開発し、酵母内在性Ste2受容体とヒト由来セロトニン受容体（HTR1A）のホモ二量体形成や、ヒト由来アドレナリン受容体（ADRB2）のヘテロ二量体形成の検出に成功した。
競争的資金
- 新エネルギー・産業技術総合開発機構（NEDO）, 先導的産業技術創出事業費助成金, 大阪大学，神戸大学, 2011年10月 - 2015年09月, 研究分担者新規代謝デザインにもとづく次世代バイオ燃料（イソブタノール）生産酵母の開発
- 日本学術振興会, 科学研究費助成事業 若手研究(B), 若手研究(B), 大阪大学, 2013年04月01日 - 2015年03月31日
酵母を多様なバイオ化成品原料を大量生産可能な宿主へと改良するために、バクテリアにユニークな中心代謝反応を追加導入し、酵母の中心代謝機能を拡張することを試みた。大腸菌のエントナー-ドウドロフ (ED) および光合成微生物のホスホエノールピルビン酸カルボキシラーゼ (PPC)経路を導入した酵母株を作成し、経路が機能していること、高級アルコール類の一つであるイソブタノール生産能力の向上に寄与することを示した。
- 日本学術振興会（JSPS）, 二国間交流事業共同研究, 神戸大学，インドネシア科学院（LIPI）, 2011年 - 2013年, 研究分担者酵母細胞表層工学によるエタノール・マンノオリゴ糖生産の統合バイオプロセス開発
- 公益財団法人 内藤記念科学振興財団, 第42回内藤記念科学奨励金・研究助成, 神戸大学, 2010年10月 - 2012年09月, 研究代表者7回膜受容体シグナル伝達機構解析のための新規バイオセンサの開発
- 科学技術振興機構（JST）, 研究シーズ探索プログラム（融合分野）, 神戸大学, 2009年 - 2010年, 研究分担者原子間力顕微鏡を利用した力学的生物界面のナノスケール現象解析
産業財産権
二重特異性抗体
特願2017-237252, 2017年12月11日, 国立大学法人神戸大学, 株式会社カネカ, 国立大学法人東北大学, 特開2019-104699, 2019年06月27日特許権
新規宿主細胞及びそれを用いた目的タンパク質の製造方法
特願2016-172841, 2016年09月05日, 国立大学法人神戸大学, 株式会社カネカ, 特開2018-38286, 2018年03月15日特許権
イソブタノール生産酵母
特願2013-94568, 2013年04月26日, 国立大学法人神戸大学, 特開2014-212762, 2014年11月17日特許権
高親和性のタンパク質間相互作用検出・スクリーニング方法
特願2010-017509, 2010年01月28日, 国立大学法人神戸大学, 特許5574479, 2014年07月11日特許権
レセプター結合性物質のスクリーニング方法
特願2003-417482, 2003年12月16日, バイオ･エナジー株式会社, 特許第5224491号, 2013年03月22日特許権
イソプロピルアルコール生産酵母及びイソプロピルアルコール生産方法
特願2010-226668, 2010年10月06日, 三井化学株式会社, 国立大学法人神戸大学, 特開2011-97929, 2011年05月19日特許権