松本 謙一郎 (マツモト ケンイチロウ)

工学研究院 応用化学部門 生物工学分野教授
Last Updated :2025/02/13

■研究者基本情報

学位

  • 博士(工学)

Researchmap個人ページ

研究キーワード

  • ポリヒドロキシアルカン酸
  • 遺伝子組換え植物
  • 進化工学
  • ブロック共重合体
  • ポリマー配列制御
  • 物質生産
  • バイオポリマー
  • PHA
  • バイオプラスチック
  • 乳酸
  • 乳酸・グリコール酸ポリマー
  • バイオプロセス
  • モノマー組成
  • 生分解性プラスチック
  • 重合酵素
  • 形質転換体
  • バイオベースポリマー
  • 進化分子工学
  • 飜訳調節

研究分野

  • ナノテク・材料, 生物分子化学
  • ナノテク・材料, グリーンサステイナブルケミストリー、環境化学
  • 環境・農学, 環境材料、リサイクル技術
  • 環境・農学, 環境負荷低減技術、保全修復技術

担当教育組織

■経歴

経歴

  • 2018年07月 - 現在
    北海道大学, 工学研究院, 教授
  • 2012年03月 - 2018年06月
    北海道大学大学院工学研究院, Applied Chemistry, 准教授
  • 2007年06月 - 2012年02月
    Hokkaido University, Applied Chemistry, Assistant Professor
  • 2007年06月 - 2012年
    北海道大学 工学(系)研究科(研究院), 助教
  • 2005年04月 - 2007年05月
    Tokyo University of Science, Biological Science & Technology, Assistant Professor
  • 2005年 - 2007年
    東京理科大学基礎工学部生物工学科, Faculty of Industrial Science and Technology, Biological Science and Technology, 助教
  • 2003年04月 - 2005年03月
    RIKEN Institute, Special Postdoctoral Researcher
  • 2003年04月 - 2005年03月
    理化学研究所, 基礎科学特別研究員
  • 2002年04月 - 2003年03月
    RIKEN Institute, Postdoctoral Researcher

学歴

  • 1997年04月 - 2002年03月, 東京大学, 工学系研究科, 化学生命工学専攻, 博士(工学), 日本国
  • 1995年04月 - 1997年03月, The University of Tokyo, Engineering, Chemistry and Biotechnology
  • 1993年04月 - 1995年03月, The University of Tokyo, Komaba

委員歴

  • 2023年06月 - 2025年05月
    生物工学会, 英文誌編集委員
  • 2021年06月 - 2023年05月
    日本生物工学会, 理事, 学協会
  • 2022年04月 - 2023年03月
    北海道大学工学研究院応用化学部門, 部門長・コース長
  • 2021年04月 - 2023年03月
    生物工学会北日本支部, 支部長, 学協会
  • 2019年04月 - 2021年03月
    日本生物工学会北日本支部, 副支部長, 学協会

■研究活動情報

受賞

  • 2018年10月, 一般財団法人バイオインダストリー協会, バイオインダストリー奨励賞               
    配列制御型ポリエステル生合成系の開発
    松本謙一郎
  • 2016年, the Minister of Education, Culture, Sports, Science and Technology, Prizes for Science and Technology               
    The Commendation for Science and Technology by the Minister of Education, Culture, Sports, Science and Technology
    松本謙一郎
  • 2008年, The Society of Polymer Science, Japan, Award for Encouragement of Research in Polymer Science               
    松本謙一郎

論文

  • Utilization of duckweed-derived biomass for polyhydroxybutyrate production in Escherichia coli via dual enzymatic saccharification using amylase and cellulase complex               
    Toshihiko Ooi, Rikako Sato, Ken'ichiro Matsumoto
    Bioscience, Biotechnology, and Biochemistry, in press, 2024年09月, [査読有り], [最終著者, 責任著者]
    英語, 研究論文(学術雑誌)
  • Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaCAR for the Biosynthesis of Poly[ (R)-3-hydroxybutyrate-co-6-hydroxyhexanoate]
    Yuka Hozumi, Shin-ichi Hachisuka, Hiroya Tomita, Hiroshi Kikukawa, Ken’ichiro Matsumoto
    Biomacromolecules, 25, 2973, 2979, 2024年05月13日, [査読有り], [最終著者, 責任著者]
    英語, 研究論文(学術雑誌)
  • Insights into widespread disturbance in gene expression and severe growth inhibition observed in transgenic rice producing polyhydroxybutyrate               
    Hiroaki Shimada, Astuo Kawamura, Miki Ogasawara, Aya Tamaki, Tetsuya Yamazaki, Yohei Igarashi, Sota Hara, Chiaki Yamagiwa, Hiroshi Teramura, Hiroaki Kusano, Ken'ichiro Matsumoto
    Plant Biotechnology, in press, 2024年, [査読有り]
    英語, 研究論文(学術雑誌)
  • Toward the production of block copolymers in microbial cells: achievements and perspectives
    Ken’ichiro Matsumoto
    Applied Microbiology and Biotechnology, 108, 164, 2024年01月, [査読有り], [招待有り], [筆頭著者, 最終著者, 責任著者]
    研究論文(学術雑誌)
  • (R/S)-lactate/2-hydroxybutyrate dehydrogenases in and biosynthesis of block copolyesters by Ralstonia eutropha
    Shizuru Ishihara, Izumi Orita, Ken’ichiro Matsumoto, Toshiaki Fukui
    Applied Microbiology and Biotechnology, Springer Science and Business Media LLC, 2023年09月29日, [査読有り]
    英語, 研究論文(学術雑誌), Abstract

    Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaCAR, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaCAR was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent l-valine depletion caused by dominant l-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent d-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent l-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaCAR+, ldhACd-hadACd+ encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaCAR. The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaCAR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell.

    Key points

    (R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha(R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identifiedThe engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB Graphical Abstract
  • Biosynthesis of High-Molecular-Weight Poly(d-lactate)-Containing Block Copolyesters Using Evolved Sequence-Regulating Polyhydroxyalkanoate Synthase PhaCAR
    Hien Thi Phan, Shoko Furukawa, Koto Imai, Hiroya Tomita, Takuya Isono, Toshifumi Satoh, Ken’ichiro Matsumoto
    ACS Sustainable Chemistry and Engineering, 11, 30, 11123, 11129, American Chemical Society (ACS), 2023年07月17日, [査読有り], [最終著者, 責任著者]
    英語, 研究論文(学術雑誌)
  • Real-time NMR analysis of polyhydroxyalkanoate synthase reaction that synthesizes block copolymer comprising glycolate and 3-hydroxybutyrate
    Kengo Yanagawa, Ayaka Kajikawa, Sayaka Sakakibara, Hiroyuki Kumeta, Hiroya Tomita, Ken'ichiro Matsumoto
    Biophysical Chemistry, 296, 107001, 107001, Elsevier BV, 2023年05月, [査読有り], [最終著者, 責任著者]
    研究論文(学術雑誌)
  • Heat-Press Technique for Coating Ammonium Nitrate Granules with Biodegradable Hydrophobic Polymer Blend of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and Poly(ε-caprolactone)
    Chinonso Anthony Ezema, Toshihiko Ooi, Ken’ichiro Matsumoto
    ACS Agricultural Science and Technology, 2, 6, 1179, 1186, American Chemical Society (ACS), 2022年11月23日, [査読有り]
    研究論文(学術雑誌)
  • Versatile aliphatic polyester biosynthesis system for producing random and block copolymers composed of 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates using the sequence-regulating polyhydroxyalkanoate synthase PhaCAR
    Keigo Satoh, Tomoya Kawakami, Nagi Isobe, Loïc Pasquier, Hiroya Tomita, Manfred Zinn, Ken’ichiro Matsumoto
    Microbial Cell Factories, 21, 1, 84, Springer Science and Business Media LLC, 2022年05月, [査読有り], [最終著者, 責任著者]
    英語, 研究論文(学術雑誌), Abstract

    Background

    Polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized by PHA synthases. Naturally occurring PHA copolymers possess a random monomer sequence. The development of PhaCAR, a unique sequence-regulating PHA synthase, has enabled the spontaneous biosynthesis of PHA block copolymers. PhaCAR synthesizes both a block copolymer poly(2-hydroxybutyrate)-b-poly(3-hydroxybutyrate) [P(2HB)-b-P(3HB)], and a random copolymer, poly(3HB-co-3-hydroxyhexanoate), indicating that the combination of monomers determines the monomer sequence. Therefore, in this study, we explored the substrate scope of PhaCAR and the monomer sequences of the resulting copolymers to identify the determinants of the monomer sequence. PhaCAR is a class I PHA synthase that is thought to incorporate long-main-chain hydroxyalkanoates (LMC HAs, > C3 in the main [backbone] chain). Thus, the LMC monomers, 4-hydroxy-2-methylbutyrate (4H2MB), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HHx), as well as 2HB, 3HB, and 3-hydroxypropionate (3HP) were tested.

    Results

    Recombinant Escherichia coli harboring PhaCAR, CoA transferase and CoA ligase genes was used for PHA production. The medium contained the monomer precursors, 2HB, 3HB, 3HP, 4H2MB, 5HV, and 6HHx, either individually or in combination. As a result, homopolymers were obtained only for 3HB and 3HP. Moreover, 3HB and 3HP were randomly copolymerized by PhaCAR. 3HB-based binary copolymers P(3HB-co-LMC HA)s containing up to 2.9 mol% 4H2MB, 4.8 mol% 5HV, or 1.8 mol% 6HHx were produced. Differential scanning calorimetry analysis of the copolymers indicated that P(3HB-co-LMC HA)s had a random sequence. In contrast, combining 3HP and 2HB induced the synthesis of P(3HP)-b-P(2HB). Similarly, P(2HB) segment-containing block copolymers P(3HB-co-LMC HA)-b-P(2HB)s were synthesized. Binary copolymers of LMC HAs and 2HB were not obtained, indicating that the 3HB or 3HP unit is essential to the polymer synthesis.

    Conclusion

    PhaCAR possesses a wide substrate scope towards 2-, 3-, 4-, 5-, and 6-hydroxyalkanoates. 3HB or 3HP units are essential for polymer synthesis using PhaCAR. The presence of a 2HB monomer is key to synthesizing block copolymers, such as P(3HP)-b-P(2HB) and P(3HB-co-LMC HA)-b-P(2HB)s. The copolymers that did not contain 2HB units had a random sequence. This study’s results provide insights into the mechanism of sequence regulation by PhaCAR and pave the way for designing PHA block copolymers., 32094019
  • Directed Evolution of Sequence-Regulating Polyhydroxyalkanoate Synthase to Synthesize a Medium-Chain-Length–Short-Chain-Length (MCL–SCL) Block Copolymer
    Hien Thi Phan, Yumi Hosoe, Maureen Guex, Masayoshi Tomoi, Hiroya Tomita, Manfred Zinn, Ken’ichiro Matsumoto
    Biomacromolecules, 23, 3, 1221, 1231, American Chemical Society (ACS), 2022年03月14日, [査読有り], [最終著者, 責任著者]
    研究論文(学術雑誌), 32094019
  • Biosynthesis of poly(glycolate-co-3-hydroxybutyrate-co-3-hydroxyhexanoate) in Escherichia coli expressing sequence-regulating polyhydroxyalkanoate synthase and medium-chain-length 3-hydroxyalkanoic acid coenzyme A ligase
    Hiroya Tomita, Keigo Satoh, Christopher T Nomura, Ken'ichiro Matsumoto
    Bioscience, Biotechnology, and Biochemistry, 86, 2, 217, 223, Oxford University Press (OUP), 2022年01月24日, [査読有り], [最終著者, 責任著者], [国際誌]
    英語, 研究論文(学術雑誌), ABSTRACT

    Chimeric polyhydroxyalkanoate synthase PhaCAR is characterized by the capacity to incorporate unusual glycolate (GL) units and spontaneously synthesize block copolymers. The GL and 3-hydroxybutyrate (3HB) copolymer synthesized by PhaCAR is a random-homo block copolymer, poly(GL-ran-3HB)-b-poly(3HB). In the present study, medium-chain-length 3-hydroxyhexanoate (3HHx) units were incorporated into this copolymer using PhaCAR for the first time. The coenzyme A (CoA) ligase from Pseudomonas oleovorans (AlkK) serves as a simple 3HHx-CoA supplying route in Escherichia coli from exogenously supplemented 3HHx. NMR analyses of the obtained polymers revealed that 3HHx units were randomly connected to 3HB units, whereas GL units were heterogeneously distributed. Therefore, the polymer is composed of 2 segments: P(3HB-co-3HHx) and P(GL-co-3HB-co-3HHx). The thermal and mechanical properties of the terpolymer indicate no contiguous P(3HB) segments in the material, consistent with the NMR results. Therefore, PhaCAR synthesized the novel block copolymer P(3HB-co-3HHx)-b-P(GL-co-3HB-co-3HHx), which is the first block polyhydroxyalkanoate copolymer comprising 2 copolymer segments., 32094019
  • Artificial polyhydroxyalkanoate poly[2-hydroxybutyrate-block-3-hydroxybutyrate] elastomer-like material
    Yuki Kageyama, Hiroya Tomita, Takuya Isono, Toshifumi Satoh, Ken’ichiro Matsumoto
    Scientific Reports, 11, 1, 22446, 22446, Springer Science and Business Media LLC, 2021年12月, [査読有り], [最終著者, 責任著者], [国際誌]
    英語, 研究論文(学術雑誌), AbstractThe first polyhydroxyalkanoate (PHA) block copolymer poly(2-hydroxybutyrate-b-3-hydroxybutyrate) [P(2HB-b-3HB)] was previously synthesized using engineered Escherichia coli expressing a chimeric PHA synthase PhaCAR with monomer sequence-regulating capacity. In the present study, the physical properties of the block copolymer and its relevant random copolymer P(2HB-ran-3HB) were evaluated. Stress–strain tests on the P(88 mol% 2HB-b-3HB) film showed an increasing stress value during elongation up to 393%. In addition, the block copolymer film exhibited slow contraction behavior after elongation, indicating that P(2HB-b-3HB) is an elastomer-like material. In contrast, the P(92 mol% 2HB-ran-3HB) film, which was stretched up to 692% with nearly constant stress, was stretchable but not elastic. The differential scanning calorimetry and wide-angle X-ray diffraction analyses indicated that the P(2HB-b-3HB) contained the amorphous P(2HB) phase and the crystalline P(3HB) phase, whereas P(2HB-ran-3HB) was wholly amorphous. Therefore, the elasticity of P(2HB-b-3HB) can be attributed to the presence of the crystalline P(3HB) phase and a noncovalent crosslinked structure by the crystals. These results show the potential of block PHAs as elastic materials.
  • Evolution of polyhydroxyalkanoate synthesizing systems toward a sustainable plastic industry
    Seiichi Taguchi, Ken'ichiro Matsumoto
    POLYMER JOURNAL, 53, 1, 67, 79, SPRINGERNATURE, 2021年01月, [査読有り]
    英語, 研究論文(学術雑誌), Designing sustainable biobased and/or biodegradable plastics opens up opportunities to achieve a low-carbon society and overcome plastic pollution. Bioplastics manufactured from renewable resources are being designed to feature a minimal carbon footprint and complete biodegradability/compostability. Among them, naturally occurring polyhydroxyalkanoates (PHAs) have currently received increasing attention from academia and industry. A symbolic state-of-the-art PHA industry is a rapidly growing market of PHBH(TM)Kaneka polymers that display excellent marine biodegradability. From an academic perspective, there have been several major breakthroughs in the PHA research field starting with the pioneering works of genetically engineered platforms for the production of artificial PHAs. The discovery of a lactate-polymerizing enzyme enabled us to produce lactate-based PHAs in one-pot microbial systems, whereas polylactide and other relevant copolymers are currently synthesized via biological and chemical processes. This proof-of-concept has been implemented in practical integrated bioprocesses for carbon-neutral polymer production starting from renewable raw bioresources. Challengingly, the photosynthetic machinery RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), has also been applied to synthesize glycolate-based copolymers as a CO(2)fixation model in the current project. Such game-changing technologies contribute to realizing a circular bioeconomy through the utilization of CO2. This review presents the current progress in evolving microbial polymerization systems, including the direct secretion of polymerized products and the creation of sequence-regulated polyesters, which have been considered nearly impossible biological events to date.Polyhydroxyalkanoates (PHAs) are biobased and biodegradable materials. The artificial PHAs, such as lactate-based polymers, synthesized by engineered platforms expand the range of physical properties. The artificial polymers with superior properties are produced mainly from CO2-derived biomass using microbial platform with engineered enzymes. The oligomers can be secreted from cells and derivatized into high-molecular-weight polymers through assembling with other segments. The review summaries recent advances in the biosynthesis and biodegradation of artificial PHAs and oligomers.
  • Biosynthesis of Random-Homo Block Copolymer Poly[Glycolate-ran-3-Hydroxybutyrate (3HB)]-b-Poly(3HB) Using Sequence-Regulating Chimeric Polyhydroxyalkanoate Synthase in Escherichia coli
    Shuzo Arai, Sayaka Sakakibara, Robin Mareschal, Toshihiko Ooi, Manfred Zinn, Ken'ichiro Matsumoto
    FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, 8, 612991, FRONTIERS MEDIA SA, 2020年12月, [査読有り], [責任著者]
    英語, 研究論文(学術雑誌), Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in Escherichia coli expressing the engineered chimeric PHA synthase PhaC(AR) and coenzyme A transferase. The cells produced poly[GL-co-3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaC(AR) is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using H-1 nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL-ran-3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1(P)(s)STQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaC(AR) synthesized a novel random-homo block copolymer, P(GL-ran-3HB)-b-P(3HB)., 32094019
  • Isolation of poly[d-lactate (LA)-co-3-hydroxybutyrate)]-degrading bacteria from soil and characterization of d-LA homo-oligomer degradation by the isolated strains
    Chiaki Hori, Tomohiro Sugiyama, Kodai Watanabe, Jian Sun, Yuu Kamada, Toshihiko Ooi, Takuya Isono, Toshifumi Satoh, Shin-ichiro Sato, Seiichi Taguchi, Ken'ichiro Matsumoto
    Polymer Degradation and Stability, 179, 109231, 109231, Elsevier BV, 2020年09月, [査読有り], [責任著者]
    英語, 研究論文(学術雑誌)
  • Synergy of valine and threonine supplementation on poly(2-hydroxybutyrate-block-3-hydroxybutyrate) synthesis in engineered Escherichia coli expressing chimeric polyhydroxyalkanoate synthase
    Maho Sudo, Chiaki Hori, Toshihiko Ooi, Shoji Mizuno, Takeharu Tsuge, Ken'ichiro Matsumoto
    Journal of Bioscience and Bioengineering, 129, 3, 302, 306, Elsevier BV, 2020年03月, [査読有り], [責任著者], [国内誌]
    英語, 研究論文(学術雑誌), The engineered chimeric polyhydroxyalkanoate (PHA) synthase PhaCAR is composed of N-terminal portion of Aeromonas caviae PHA synthase and C-terminal portion of Ralstonia eutropha (Cupriavidus necator) PHA synthase. PhaCAR has a unique and useful capacity to synthesize the block PHA copolymer poly(2-hydroxybutyrate-block-3-hydroxybutyrate) [P(2HB-b-3HB)] in engineered Escherichia coli from exogenous 2HB and 3HB. In the present study, we initially attempted to incorporate the amino acid-derived 2-hydroxyalkanoate (2HA) units using PhaCAR and the 2HA-CoA-supplying enzymes lactate dehydrogenase (LdhA) and CoA transferase (HadA). Cells harboring the genes for PhaCAR, LdhA, and HadA, as well as for the 3HB-CoA-supplying enzymes β-ketothiolase and acetoacetyl-CoA reductase, were cultivated with supplementation of four hydrophobic amino acids, i.e., leucine, valine (Val), isoleucine (Ile), and phenylalanine, in the medium. No hydrophobic amino acid-derived monomers were incorporated into the polymer, which was most likely because of the strict substrate specificity of PhaCAR; however, P(2HB-co-3HB) was unexpectedly produced with Val supplementation. The copolymer was likely P(2HB-b-3HB) based on proton nuclear magnetic resonance analysis. Based on the endogenous pathways in E. coli, 2HB units are likely derived from threonine (Thr) through deamination and dihydroxylation. In fact, dual supplementation with Thr and Val showed synergy on the 2HB fraction of the polymer. Val supplementation promoted the 2HB synthesis likely by inhibiting the metabolism of 2-ketobutyrate into Ile and/or activating Thr dehydratase. In conclusion, the LdhA/HadA/PhaCAR pathway served as the system for the synthesis of P(2HB-b-3HB) from biomass-derived carbon sources.
  • Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-mediated de novo synthesis of glycolate-based polyhydroxyalkanoate in Escherichia coli.
    Matsumoto K, Saito J, Yokoo T, Hori C, Nagata A, Kudoh Y, Ooi T, Taguchi S
    Journal of bioscience and bioengineering, 128, 3, 302, 306, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2019年09月, [査読有り], [筆頭著者, 責任著者]
    英語, 研究論文(学術雑誌), Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1(Ps)STQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (Delta glcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E. coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1(Ps)STQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1 mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R. rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E. coli but also in photosynthetic organisms. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.
  • Increased Production and Molecular Weight of Artificial Polyhydroxyalkanoate Poly(2-hydroxybutyrate) Above the Glass Transition Temperature Threshold.
    Matsumoto K, Kageyama Y
    Frontiers in bioengineering and biotechnology, 7, 177, 2019年07月, [査読有り], [筆頭著者, 責任著者]
    英語, 研究論文(学術雑誌)
  • High-cell density culture of poly(lactate-co-3-hydroxybutyrate)-producing Escherichia coli by using glucose/xylose-switching fed-batch jar fermentation.
    Chiaki Hori, Takashi Yamazaki, Greg Ribordy, Kenji Takisawa, Ken'ichiro Matsumoto, Toshihiko Ooi, Manfred Zinn, Seiichi Taguchi
    Journal of bioscience and bioengineering, 127, 6, 721, 725, 2019年06月, [査読有り], [国内誌]
    英語, 研究論文(学術雑誌), Poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] is produced in engineered Escherichia coli harboring the genes encoding an LA-polymerizing enzyme (LPE) and monomer-supplying enzymes. In this study, high cell-density fed-batch jar fermentation was developed using xylose and/or glucose as the carbon source. Fed-batch fermentation was initially performed with 20 g/L sugar during the batch phase for 24 h, and subsequent sugar feeding from 24 to 86 h. The feeding rate was increased in a stepwise manner. When xylose alone was used for cultivation, the cells produced the polymer at 11.6 g/L, which was higher than the 4.3 g/L obtained using glucose as the sole carbon source. However, in the first 24 h the growth in the glucose culture was greater than in the xylose culture. Based on these results, glucose was used for cell growth (at the initial stage) and xylose was used for polymer production (at the feeding stage). As expected, in the glucose/xylose switching fermentation method, polymer production was significantly enhanced, eventually reaching 26.7 g/L. The enhanced polymer production obtained by using xylose was presumably due to overflow metabolism. In fact, during xylose feeding, acetic acid excretion was greater than that in case of the glucose grown culture, suggesting the channeling of the metabolic flux from acetyl-CoA towards polymer production over into the tricarboxylic acid cycle in the xylose-fed cultures. Therefore, this sequential glucose/xylose feed strategy is potentially useful for production of acetyl-CoA derived compounds in E. coli.
  • Biosynthesis of novel lactate-based polymers containing medium-chain-length 3-hydroxyalkanoates by recombinant Escherichia coli strains from glucose.
    Goto S, Hokamura A, Shiratsuchi H, Taguchi S, Matsumoto K, Abe H, Tanaka K, Matsusaki H
    Journal of bioscience and bioengineering, 127, 7, 7, 11, 2019年02月, [査読有り], [国内誌]
    英語, 研究論文(学術雑誌), Novel lactate (LA)-based polymers containing medium-chain-length 3-hydroxyalkanoates (MCL-3HA) were produced in fadR-deficient Escherichia coli strains from glucose as the sole carbon source. The genes encoding LA and 3-hydroxybutyrate (3HB) monomers supplying enzymes [propionyl-CoA transferase (PCT), d-lactate dehydrogenase (D-LDH), β-ketothiolase (PhaA), and NADPH-dependent acetoacetyl-CoA reductase (PhaB)], MCL-3HA monomers supplying enzymes [ (R)-3-hydroxyacyl-ACP thioesterase (PhaG) and (R)-3-hydroxyacyl (3HA)-CoA ligase] via fatty acid biosynthesis pathway, and modified polyhydroxyalkanoate (PHA) synthase [PhaC1(STQK)] of Pseudomonas sp. 61-3 were introduced into E. coli LS5218. This resulted in the synthesis of a novel LA-based copolymer, P(LA-co-3HB-co-3HA). 1H-nuclear magnetic resonance (NMR) analysis revealed the composition of P(LA-co-3HB-co-3HA) to be 19.7 mol% LA (C3), 74.9 mol% 3HB (C4), and 5.4 mol% MCL-3HA units of C8 and C10. Furthermore, the recombinant E. coli CAG18497 strain carrying these genes, excluding the phaAB genes, accumulated P(92.0% LA-co-3HA) with a novel monomer composition containing C3, C8, C10, and C12. 13C-NMR analysis showed the existence of LA-3HA sequence in the polymer. The solvent cast film of P(92.0% LA-co-3HA) exhibited transparency similar to poly(lactic acid).
  • Influence of Unusual Co-substrates on the Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates Produced in Multistage Chemostat.
    Nils Hanik, Camila Utsunomia, Shuzo Arai, Ken'ichiro Matsumoto, Manfred Zinn
    Frontiers in bioengineering and biotechnology, 7, 301, 301, 2019年, [国際誌]
    英語, 研究論文(学術雑誌), A two-stage chemostat cultivation was used to investigate the biosynthesis of functionalized medium-chain-length polyhydroxyalkanoate (mcl-PHA) in the β-oxidation weakened strain of Pseudomonas putida KTQQ20. Chemostats were linked in sequence and allowed separation of biomass production in the first stage from the PHA synthesis in the second stage. Four parallel reactors in the second stage provided identical growth conditions and ensured that the only variable was the ratio of decanoic acid (C10) to an unusual PHA monomer precursor, such as 10-undecenoic acid (C11:1) or phenylvaleric acid (PhVA). Obtained PHA content was in the range of 10 to 25 wt%. When different ratios of C10 and C11:1 were fed to P. putida, the produced PHA had a slightly higher molar ratio in favor of C11:1-based 3-hydroxy-10-undecenoate. However, in case of PhVA a significantly lower incorporation of 3-hydroxy-5-phenylvalerate over 3-hydroxydecanoate took place when compared to the ratio of their precursors in the feed medium. A result that is explained by a less efficient uptake of PhVA compared to C10 and a 24% lower yield of polymer from the aromatic fatty acid ( y P H A - M P h V A = 0.25). In addition, PHA isolated from cultivations with PhVA resulted in the number average molecular weight M n ¯ two times lower than the PHA produced from C10 alone. Detection of products from PhVA metabolism in the culture supernatant showed that uptaken PhVA was not entirely converted into PHA, thus explaining the difference in the yield polymer from substrate. It was concluded that PhVA or its related metabolites increased the chain transfer rate during PHA biosynthesis in P. putida KTQQ20, resulting in a reduction of the polymer molecular weight.
  • Enhancement of lactate fraction in poly(lactate-co-3-hydroxybutyrate) synthesized by Escherichia coli harboring the D-lactate dehydrogenase gene from Lactobacillus acetotolerans HT
    Saki Goto, Naoyuki Suzuki, Ken'ichiro Matsumoto, Seiichi Taguchi, Kenji Tanaka, Hiromi Matsusaki
    The Journal of General and Applied Microbiology, 65, 4, 204, 208, Microbiology Research Foundation, 2019年, [査読有り]
    研究論文(学術雑誌)
  • In Vitro Analysis of d-Lactyl-CoA-Polymerizing Polyhydroxyalkanoate Synthase in Polylactate and Poly(lactate- co-3-hydroxybutyrate) Syntheses.
    Matsumoto K, Iijima M, Hori C, Utsunomia C, Ooi T, Taguchi S
    Biomacromolecules, 19, 7, 2889, 2895, AMER CHEMICAL SOC, 2018年07月, [査読有り], [筆頭著者, 責任著者]
    英語, 研究論文(学術雑誌), Engineered D-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaCl(ps)STQK) efficiently produces poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, in vitro analysis of PhaCl(ps)STQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaCl(ps)STQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of 3000, presumably due to the low mobility of the generated polymer. The copolymerization of these substrates only proceeded with a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration in P(LA-co-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that the mobility of polymerized products and LA-CoA concentration are dominant factors characterizing PLA and P(LA-co-3HB) biosynthetic systems.
  • Site-directed saturation mutagenesis of polyhydroxylalkanoate synthase for efficient microbial production of poly[ (R)-2-hydroxybutyrate]
    Chiaki Hori, Kenta Oishi, Ken'ichiro Matsumoto, Seiichi Taguchi, Toshihiko Ooi
    Journal of Bioscience and Bioengineering, 125, 6, 632, 636, Elsevier B.V., 2018年06月01日, [査読有り]
    英語, 研究論文(学術雑誌), In our previous study, artificial polyhydroxyalkanoate (PHA) poly[ (R)-2-hydroxybutyrate] [P(2HB)] was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1Ps(S325T/Q481K). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaC1Ps towards enhanced P(2HB) accumulation in E. coli by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation. By using a screening on agar plates with Nile red, eight colonies were isolated which produced a greater amount of P(2HB) compared to a colony expressing the parent enzyme PhaC1Ps(S325T/Q481K). Among them, the cells expressing PhaC1Ps(S325T/S477R/Q481G) [ST/SR/QG] accumulated polymer at the highest level (up to 2.9-fold). As seen in PhaC1Ps(ST/SR/QG), glycine and basic amino acid residues (K or R) were frequently found at the two positions of the select mutated enzymes. The enzymatic activity of PhaC1Ps(ST/SR/QG) toward 2HB-CoA was approximately 3-fold higher than that of the parent enzyme. Additionally, expression levels of the select mutated enzymes were lower than the parent. These results indicated that PhaC1Ps mutagenesis at the positions 477 and 481 increased specific activity toward 2HB-CoA and it could result in the enhanced production of P(2HB).
  • Enhanced production of lactate-based polyesters in Escherichia coli from a mixture of glucose and xylose by Mlc-mediated catabolite derepression.
    Ryosuke Kadoya, Ken'ichiro Matsumoto, Kenji Takisawa, Toshihiko Ooi, Seiichi Taguchi
    Journal of bioscience and bioengineering, 125, 4, 365, 370, 2018年04月, [査読有り], [国内誌]
    英語, 研究論文(学術雑誌), Lignocellulose-utilizing biorefinery is a promising strategy for the sustainable production of value-added products such as bio-based polymers. Simultaneous consumption of glucose and xylose in Escherichia coli was achieved by overexpression of the gene encoding Mlc, a multiple regulator of glucose and xylose uptake. This catabolite derepression gave the enhancement in the production of poly (15 mol% lactate-co-3-hydroxybutyrate), up to 65% from 50% (wild-type strain) in the cellular contents, of the Mlc-overexpressing strain of E. coli on a mixture of glucose and xylose as carbon sources. Microscopic analysis indicated that the Mlc-overexpressing strain showed the enlargement of cell volume in the presence and absence of polymer production, consequently making an expanded volumetric space available for enhanced polymer accumulation. The enhanced polymer production by the catabolite derepression was also reproducible using the biomass, Miscanthus×giganteus (hybrid Miscanthus), which was cultivated in the farm of Hokkaido University.
  • Dynamic Changes of Intracellular Monomer Levels Regulate Block Sequence of Polyhydroxyalkanoates in Engineered Escherichia coli
    Ken'Ichiro Matsumoto, Chiaki Hori, Ryunosuke Fujii, Masahiro Takaya, Takashi Ooba, Toshihiko Ooi, Takuya Isono, Toshifumi Satoh, Seiichi Taguchi
    Biomacromolecules, 19, 2, 662, 671, American Chemical Society, 2018年02月12日, [査読有り], [筆頭著者]
    英語, 研究論文(学術雑誌), Biological polymer synthetic systems, which utilize no template molecules, normally synthesize random copolymers. We report an exception, a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered Escherichia coli. Using an engineered PHA synthase, block copolymers poly[ (R)-2-hydroxybutyrate(2HB)-b-(R)-3-hydroxybutyrate(3HB)] were produced in E. coli. The covalent linkage between P(2HB) and P(3HB) segments was verified with solvent fractionation and microphase separation. Notably, the block sequence was generated under the simultaneous consumption of two monomer precursors, indicating the existence of a rapid monomer switching mechanism during polymerization. Based on in vivo metabolic intermediate analysis and the relevant in vitro enzymatic activities, we propose a model in which the rapid intracellular 3HB-CoA fluctuation during polymer synthesis is a major factor in generating block sequences. The dynamic change of intracellular monomer levels is a novel regulatory principle of monomer sequences of biopolymers.
  • Incorporation of Glycolate Units Promotes Hydrolytic Degradation in Flexible Poly(glycolate-co-3-hydroxybutyrate) Synthesized by Engineered Escherichia coli
    Ken'ichiro Matsumoto, Tetsufumi Shiba, Yukikazu Hiraide, Seiichi Taguchi
    ACS BIOMATERIALS SCIENCE & ENGINEERING, 3, 12, 3058, 3063, AMER CHEMICAL SOC, 2017年12月, [査読有り]
    英語, 研究論文(学術雑誌), Glycolate (GL)-based polyhydroxyalkanoate (PHA), P[GL-co-3-hydroxybutyrate (3HB)], was characterized with respect to its physical properties and hydrolytic degradability. The copolymers were produced from GL and xylose in recombinant Escherichia coli JW1375 (Delta ldhA) expressing an engineered PHA synthase and monomer supplying enzymes. The GL molar ratio in the copolymer was regulated in the range of 0 to 16 mol % dependent on the concentration of GL supplemented in the medium. Unlike P(3HB) homopolymers which are rigid and opaque, the transparency and elasticity of P(GL-co-3HB) films could be tuned dependent on the GL molar ratio. For example, Young's modulus of the films varied in the range of 1620 to 54 MPa. The hydrothermal treatment of P(GL-co-3HB)s resulted in the generation of water-soluble oligomers, and their concentration was positively correlated with the GL molar ratio in the polymer, indicating that the GL units in the polymer chain promoted the hydrolytic degradation of the polymer. The results of this study demonstrate that the GL molar ratio is a potent determinant for regulating the elasticity and hydrolytic degradability of P(GL-co-3HB).
  • Investigation of the Escherichia coli membrane transporters involved in the secretion of D-lactate-based oligomers by loss-of-function screening
    Camila Utsunomia, Chiaki Hori, Ken'ichiro Matsumoto, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 124, 6, 635, 640, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2017年12月, [査読有り]
    英語, 研究論文(学術雑誌), D-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of D-LA and D-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LA-polymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. con, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds. Among the deletants of the outer membrane-associated proteins, Delta ompF and Delta ompG exhibited diminished D-LAOs secretion and elevated intracellular D-LAO accumulation. When the ompF and ompG expression levels were down- and up-regulated with plasmids harboring these genes, the secreted amounts of the D-LAOs were changed in correspondence with their expression levels. These results suggest that porins mediate D-LAOs transport through the outer membrane. In particular, OmpF is likely to be the major porin involved in the spontaneous secretion of D-LAOS due to the high basal expression of ompF in the parental strain. Among the deletants of the inner membrane-associated proteins, the Delta mngA, Delta argT, Delta macA, Delta citA and Delta cpxA strains were selected by the screening. These genes are also candidate transporters related to D-LAO secretion, suggesting the presence of multiple secretion routes across the inner membrane. To the best of our knowledge, this is the first report on the mechanism of the microbial secretion of oligoesters. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Synthesis of lactate (LA)-based poly(ester-urethane) using hydroxyl-terminated LA-based oligomers from a microbial secretion system
    Camila Utsunomia, Tatsuya Saito, Ken'ichiro Matsumoto, Chiaki Hori, Takuya Isono, Toshifumi Satoh, Seiichi Taguchi
    JOURNAL OF POLYMER RESEARCH, 24, 10, 167, 167, SPRINGER, 2017年09月, [査読有り]
    英語, 研究論文(学術雑誌), D-Lactate (LA)-based oligomers (D-LAOs), consisting of D-LA and D-3-hydroxybutyrate (D-3HB), are biobased compounds which are produced and spontaneously secreted by recombinant Escherichia coli. By supplementing the bacterial cultivation with diethylene glycol (DEG), the oligomers featuring hydroxyl groups at both ends of their structures, the D-LAOs-DEG, can be efficiently biosynthesized. In the present work, we attempted to verify the feasibility of D-LAOs-DEG as building blocks to be assembled into LA-based poly(ester-urethane) via polyaddition reactionwith diisocyanate. The polymeric products were demonstrated by SEC and the urethane bound formation in the polymer was determined by FT-IR analysis, indicating that the polymerization was successfully performed. These results suggested that the one-step biosynthesized D-LAOs-DEG are potential substrates for the synthesis of LA-based poly(ester-urethane) and can be further applied to the synthesis of other LA copolymers.
  • In vivo target exploration of apidaecin based on Acquired Resistance induced by Gene Overexpression (ARGO assay)
    Ken'ichiro Matsumoto, Kurato Yamazaki, Shun Kawakami, Daichi Miyoshi, Toshihiko Ooi, Shigeki Hashimoto, Seiichi Taguchi
    SCIENTIFIC REPORTS, 7, 12136, 12136, NATURE PUBLISHING GROUP, 2017年09月, [査読有り]
    英語, 研究論文(学術雑誌), Identifying the target molecules of antimicrobial agents is essential for assessing their mode of action. Here, we propose Acquired Resistance induced by Gene Overexpression (ARGO) as a novel in vivo approach for exploring target proteins of antimicrobial agents. The principle of the method is based on the fact that overexpression of the expected target protein leads to reduced sensitivity to the antimicrobial agent. We applied this approach to identify target proteins of the antimicrobial peptide apidaecin, which is specifically effective against Gram-negative bacteria. To this end, a set of overexpression Escherichia coli clones was tested, and peptide chain release factor 1, which directs the termination of translation, was found as a candidate, suggesting that apidaecin inhibits the termination step of translation. This finding was confirmed in vivo and in vitro by evaluating the inhibitory activity of apidaecin towards lacZ reporter gene expression, which is tightly dependent on its stop codon. The results of this study demonstrate that apidaecin exerts its antimicrobial effects partly by inhibiting release factors.
  • Microbial secretion of lactate-enriched oligomers for efficient conversion into lactide: A biological shortcut to polylactide
    Camila Utsunomia, Ken'ichiro Matsumoto, Sakiko Date, Chiaki Hori, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 124, 2, 204, 208, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2017年08月, [査読有り]
    英語, 研究論文(学術雑誌), Recently, we have succeeded in establishing the microbial platform for the secretion of lactate (LA)-based oligomers (D-LAOs), which consist of D-LA and D-3-hydroxybutyrate (D-3HB). The secretory production of D-LAOs was substantially enhanced by the supplementation of diethylene glycol (DEG), which resulted in the generation of DEG-capped oligomers at the carboxyl terminal (referred as D-LAOS-DEG). The microbial D-LAOs should be key compounds for the synthesis of lactide, an important intermediate for polylactides (PLAs) production, eliminating the costly chemo-oligomerization step in the PLA production process. Therefore, in order to demonstrate a proof-of-concept, here, we attempted to convert the D-LAOS-DEG into lactide via metal-catalyzed thermal depolymerization. As a result, D-LAOS-DEG containing 68 mol% LA were successfully converted into lactide, revealing that the DEG bound to D-LAOs-DEG does not inhibit the conversion into lactide. However, the lactide yield (4%) was considerably lower than that of synthetic LA homooligomers (33%). We presumed that 3HB units in the polymer chain blocked the lactide formation, and therefore, we investigated the LA enrichment in the oligomers. As the results, the combination of an LA-overproducing Escherichia coli mutant (Mid and ApflA) with the use of xylose as a carbon source exhibited synergistic effect to increase LA fraction in the oligomers up to 89 mol%. The LA-enriched D-LAOS-DEG were converted into lactide with greater yield (18%). These results demonstrated that a greener shortcut route for PLA production can be created by using the microbial D-LAOS secretion system. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Genome-wide screening of transcription factor deletion targets in Escherichia coli for enhanced production of lactate-based polyesters
    Ryosuke Kadoya, Yu Kodama, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 123, 5, 535, 539, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2017年05月, [査読有り]
    英語, 研究論文(学術雑誌), Engineered Escherichia coli is a useful platform for production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] from renewable sugars. Here we screened all non-lethal transcription factor deletions of E. coli for efficient production of the polymer. This approach aimed at drawing out the latent potential of the host for efficient polymer production via indirect positive effects. Among 252 mutants from Keio Collection tested, eight mutants (Delta pdhR, Delta cspG, Delta yneJ, Delta chbR, Delta yiaU, Delta creB, Delta ygfI and Delta nanK) accumulated greater amount of polymer (6.2-10.1 g/L) compared to the parent strain E. coli BW25113 (5.1 g/L). The mutants increased polymer production per cell (1.1-1.5-fold) without significant change in cell density. The yield of the polymer from glucose was also higher for the selected mutants (0.34-0.38 g/g) than the parent strain (0.27 g/g). Therefore, the deletions of transcription factors should channel the carbon flux towards polymer production. It should be noted that the screening employed in this study identified beneficial mutants without analyzing causal relationship between the mutation and the enhanced polymer production. This approach, therefore, should be applicable to broad range of fermentation productions. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Effect of acetate as a co-feedstock on the production of poly(lactate-co-3-hydroxyalkanoate) by pflA-deficient Escherichia coli RSC10
    Lucia Salamanca-Cardona, Ryan A. Scheel, Kouhei Mizuno, N. Scott Bergey, Arthur J. Stipanovic, Ken'ichiro Matsumoto, Seiichi Taguchi, Christopher T. Nomura
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 123, 5, 547, 554, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2017年05月, [査読有り]
    英語, 研究論文(学術雑誌), Developing Escherichia coli strains that are tolerant to acetate toxicity is important in light of an increased interest in the efficient utilization of lignocellulosic biomass feedstocks for the biosynthesis of value-added products. In this study, four strains known to produce polyhydroxyalkanoates (PHAs) from the typical hemicellulosic sugar xylose were tested for their tolerance to acetate. E. coli RSC10 was found to be tolerant of acetate, both in growth and fermentation studies. In the presence of acetate the strain showed a >2-fold increase in overall yields compared to using xylose alone as the feedstock. More importantly, the strain was found to be able to utilize acetate as a feedstock for biosynthesis of PHAs, with complete depletion of acetate (25 mM) at 9 h when acetate was the sole feedstock. Higher concentrations of acetate showed greater inhibition of fermentation than growth with a reduction of 90% in PHA yields at 100 mM. Additionally, the present work provides data to support the potential of acetate as a modulator for the control of composition of PHAs that incorporate lactate (LA) monomers into the copolymer from hemicellulose derived sugars. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Xylose-based hydrolysate from eucalyptus extract as feedstock for poly(lactate-co-3-hydroxybutyrate) production in engineered Escherichia coli
    Kenji Takisawa, Toshihiko Ooi, Ken'ichiro Matsumoto, Ryosuke Kadoya, Seiichi Taguchi
    PROCESS BIOCHEMISTRY, 54, 102, 105, ELSEVIER SCI LTD, 2017年03月, [査読有り]
    英語, 研究論文(学術雑誌), Woody extract-derived hemicellulosic hydrolysate, which was obtained from dissolving pulp manufacturing, was utilized as feedstock for the production of poly(lactate-co-3-hydroxybutyrate) P(LA-co-3HB)] in engineered Escherichia coil. The hydrolysate was composed of mainly xylose and galactose, and contained impurities mainly acetate, which was found to inhibit the polymer synthesis rather than the cell growth. Thus, acetate and other impurities were removed through active charcoal and ion-exchange columns. Using the purified hydrolysate, P(LA-co-3HB) was successfully produced (cell dry weight 8.6 g/L, polymer concentration 5.4 g/L, LA fraction 5.5 mol%, polymer content 62.4%), the amount of which was comparable to that obtained using reagent grade xylose and galactose. Therefore, the hydrolysate from woody extract is considered as an abundant, inexpensive and efficient feedstock applicable to consolidated process for P(LA-co-3HB) production, when the removal of acetic acid was satisfactorily accomplished. (C)2016 Elsevier Ltd. All rights reserved.
  • Sucrose supplementation suppressed the growth inhibition in polyhydroxyalkanoate-producing plants
    Takeshi Yoshizumi, Miwa Yamada, Mieko Higuchi-Takeuchi, Ken'ichiro Matsumoto, Seiichi Taguchi, Minami Matsui, Keiji Numata
    PLANT BIOTECHNOLOGY, 34, 1, 39, 43, JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, 2017年03月, [査読有り]
    英語, 研究論文(学術雑誌), Polyhydroxyalkanoate (PHA) is a thermoplastic polymer with several advantageous properties, including biomass origin, biocompatibility, and biodegradability. PHA is synthesized in transgenic plants harboring 3 enzymatic genes: phaA, phaB, and phaC (collectively referred to as phaABC). PHA-producing plants exhibit severe growth inhibition that leads to extremely low PHA accumulation when these enzymes are localized in the cytosol. This growth inhibition could be attributed to the deleterious effects of the PHA biosynthetic pathway on endogenous essential metabolites or to PHA cytotoxicity itself. We performed precise morphological observations of phaABC-overexpressing Arabidopsis (ABC-ox), which displayed typical growth inhibition. On growth medium without sucrose, ABC-ox exhibited a pale green phenotype, dwarfism, including small cotyledons and true leaves, and short roots. ABC-ox partially recovered from this growth inhibition when the growth medium was supplemented with 1% sucrose. This recovery was reversed after ABC-ox grown on 1% sucrose medium was transferred to soil. ABC-ox grown on 1% sucrose medium not only demonstrated recovery from growth inhibition but were also the only examined plants with PHA accumulation, suggesting that growth inhibition was not caused by PHA cytotoxicity but rather by a lack of essential metabolites.
  • Microbial Secretion of D-Lactate-Based Oligomers
    Camila Utsunomia, Ken'ichiro Matsumoto, Seiichi Taguchi
    ACS SUSTAINABLE CHEMISTRY & ENGINEERING, 5, 3, 2360, 2367, AMER CHEMICAL SOC, 2017年03月, [査読有り]
    英語, 研究論文(学術雑誌), Here, we report the one-step secretory production of D-lactate (LA)-based oligomers (D-LAOS) by engineered Escherichia coli from glucose. Notably, D-LAOs are spontaneously secreted into the medium without the introduction of exogenous exporters. This study was originated from the finding that a small amount of oligo[LA-co-3hydroxybutyrate(3HB)] was secreted by the cells expressing the D-specific LA-polymerizing enzyme (LPE). To increase DLAOs production, we attempted to increase the frequency of chain transfer (CT) reaction of LPE using CT agents, which bear alcoholic moiety. As the result, addition of diethylene glycol into the culture medium was found to be particularly effective. The highest yield of D-LAOs production of 8.3 +/- 1.5 g L-1 from 20 g L-1 glucose was 57% of the theoretical maximum. Furthermore, we demonstrated the covalent conjugation of diethylene glycol at the carboxyl terminal of D-LAOs by NMR analyses. The secreted D-LAOs have the potential to be used as precursors of lactide, enabling the establishment of a greener shortcut route in the process of polylactides (PLAs) production.
  • Co-crystallization phenomena in biosynthesized isotactic poly [ (R)-lactate-co-(R)-2-hydroxybutyratels with various lactate unit ratios
    Taizo Kabe, Ken'ichiro Matsumoto, Satsuki Terai, Takaaki Hikima, Masaki Takata, Masahiro Miyake, Seiichi Taguchi, Tadahisa Iwata
    POLYMER DEGRADATION AND STABILITY, 132, 137, 144, ELSEVIER SCI LTD, 2016年10月, [査読有り]
    英語, 研究論文(学術雑誌), Poly[ (R)-lactate-co-(R)-2-hydroxybutyrate], which is biosynthesized as a random co-polyester by microbial organisms, with various ratios of lactic acid (LA) to hydroxybutyrate monomers has been isothermally crystallized to investigate its thermal properties and crystal structure changes. Differential scanning calorimetry on the isothermally crystallized samples detected endothermic peaks due to the melting of the crystals at all LA unit ratios. Melting temperatures of the copolymers increased from ca. 107 degrees C-146 degrees C as the LA unit ratio increased. Glass transition temperatures also increased from 24 degrees C to 44 degrees C with increasing LA unit ratio. A melting temperature was observed even at LA unit ratios around 50%. Wide-angle X-ray measurements also revealed that co-crystallization occurred at all samples, including at LA unit ratios of ca. 50%. Despite of "jumping" phenomena many co-crystallizable random copolymers having, the lattice constants changed "linearly" from resembling poly[ (R)-lactate] to poly [ (R)-2-hydroxybutyrate] depending on the LA unit ratio. This unique co-crystallization behavior is discussed in detail. (C) 2016 Elsevier Ltd. All rights reserved.
  • Consolidated bioprocessing of poly(lactate-co-3-hydroxybutyrate) from xylan as a sole feedstock by genetically-engineered Escherichia coli
    Lucia Salamanca-Cardona, Ryan A. Scheel, Norman Scott Bergey, Arthur J. Stipanovic, Ken'ichiro Matsumoto, Seiichi Taguchi, Christopher T. Nomura
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 122, 4, 406, 414, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2016年10月, [査読有り]
    英語, 研究論文(学術雑誌), Consolidated bioprocessing of lignocellulose is an attractive strategy for the sustainable production of petroleum based alternatives. One of the underutilized sources of carbon in lignocellulose is the hemicellulosic fraction which largely consists of the polysaccharide xylan. In this study, Escherichia coli JW0885 (pyruvate formate lyase activator protein mutant, pflA(-)) was engineered to express recombinant xylanases and polyhydroxyalkanoate (PHA)-producing enzymes for the biosynthesis of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] from xylan as a consolidated bioprocess. The results show that E. coli JW0885 was capable of producing P(LA-co-3HB) when xylan was the only feedstock and different feeding and growth parameters were examined in order to improve upon initial yields. The highest yields of P(LA-co-3HB) copolymer obtained in this study occurred when xylan was added during mid-exponential growth after cells had been grown at high shaking speeds (290 rpm). The results showed an inverse relationship between total PHA production and LA-monomer incorporation into the copolymer. Proton nuclear magnetic resonance (H-1 NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analyses corroborate that the polymers produced maintain physical properties characteristic of LA-incorporating PHB-based copolymers. The present study achieves the first ever engineering of a consolidated bioprocessing bacterial system for the production of a bioplastic from a hemicelluosic feedstock. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.
  • Microbial production of poly(lactate-co-3-hydroxybutyrate) from hybrid Miscanthus-derived sugars
    Jian Sun, Camila Utsunomia, Shohei Sasaki, Ken'ichiro Matsumoto, Toshihiko Yamada, Toshihiko Ooi, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 80, 4, 818, 820, TAYLOR & FRANCIS LTD, 2016年04月, [査読有り]
    英語, 研究論文(学術雑誌), P[ (R)-lactate-co-(R)-3-hydroxybutyrate] [P(LA-co-3HB)] was produced in engineered Escherichia coli using lignocellulose-derived hydrolysates from Miscanthusxgiganteus (hybrid Miscanthus) and rice straw. Hybrid Miscanthus-derived hydrolysate exhibited no negative effect on polymer production, LA fraction, and molecular weight of the polymer, whereas rice straw-derived hydrolysate reduced LA fraction. These results revealed that P(LA-co-3HB) was successfully produced from hybrid Miscanthus-derived sugars.
  • Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp C34 and Alcaligenes faecalis T1
    Jian Sun, Ken'ichiro Matsumoto, Yuta Tabata, Ryosuke Kadoya, Toshihiko Ooi, Hideki Abe, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 99, 22, 9555, 9563, SPRINGER, 2015年11月, [査読有り]
    英語, 研究論文(学術雑誌), Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZ(Vs)) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(d-LA-co-d-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZ(Vs) for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZ(Af)) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZ(Vs) generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZ(Vs) cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZ(Vs) but also PhaZ(Af) hydrolyzed all of these substrates, namely PhaZ(Af) also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[ (R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZ(Vs) exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZ(Af). Therefore, the cleaving capability of PhaZs used here toward the d-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.
  • Indirect positive effects of a sigma factor RpoN deletion on the lactate-based polymer production in Escherichia coli
    Ryosuke Kadoya, Yu Kodama, Ken'ichiro Matsumoto, Seiichi Taguchi
    BIOENGINEERED, 6, 5, 307, 311, TAYLOR & FRANCIS INC, 2015年09月, [査読有り]
    英語, 研究論文(学術雑誌), The production of bacterial polyesters, polyhydroxyalkanoates (PHAs), has been improved by several rational approaches such as overexpression and/or engineering of the enzymes directly related to PHA biosynthetic pathways. In this study, a new approach at transcription level has been applied to a new category of the copolymer of lactate (LA) and 3-hydroxybutyrate (3HB), P(LA-co-3HB). When the 4 disrupting mutants of sigma factors in Escherichia coli, rpoN, rpoS, fliA, fecI, were used as platforms for production of P(LA-co-3HB), increases in the production level and LA fraction of the copolymer were observed for the mutant strain with rpoN disruption. These positive impacts on the polymer production were caused in an indirect manner via changes in the multiple genes governed by RpoN. A genome-wide engineering by sigma factors would be a versatile approach for the production of value-added products of interest and available for combination with the other beneficial tools.
  • Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase
    Toshinori Yokoo, Ken'ichiro Matsumoto, Takashi Ooba, Kenjiro Morimoto, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 79, 6, 986, 988, TAYLOR & FRANCIS LTD, 2015年06月, [査読有り]
    英語, 研究論文(学術雑誌), Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cultured cells to produce poly(3-hydroxybutyrate) [P(3HB)]. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.
  • MtgA Deletion-Triggered Cell Enlargement of Escherichia coli for Enhanced Intracellular Polyester Accumulation
    Ryosuke Kadoya, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    PLOS ONE, 10, 6, e0125163, e0125163, PUBLIC LIBRARY SCIENCE, 2015年06月, [査読有り]
    英語, 研究論文(学術雑誌), Bacterial polyester polyhydroxyalkanoates (PHAs) have been produced in engineered Escherichia coli, which turned into an efficient and versatile platform by applying metabolic and enzyme engineering approaches. The present study aimed at drawing out the latent potential of this organism using genome-wide mutagenesis. To meet this goal, a transposon-based mutagenesis was carried out on E. coli, which was transformed to produce poly (lactate-co-3-hydroxybutyrate) from glucose. A high-throughput screening of polymer-accumulating cells on Nile red-containing plates isolated one mutant that produced 1.8-fold higher quantity of polymer without severe disadvantages in the cell growth and monomer composition of the polymer. The transposon was inserted into the locus within the gene encoding MtgA that takes part, as a non-lethal component, in the formation of the peptidoglycan backbone. Accordingly, the mtgA-deleted strain E. coli JW3175, which was a derivate of superior PHA-producing strain BW25113, was examined for polymer production, and exhibited an enhanced accumulation of the polymer (7.0 g/l) compared to the control (5.2 g/l). Interestingly, an enlargement in cell width associated with polymer accumulation was observed in this strain, resulting in a 1.6-fold greater polymer accumulation per cell compared to the control. This result suggests that the increase in volumetric capacity for accumulating intracellular material contributed to the enhanced polymer production. The mtgA deletion should be combined with conventional engineering approaches, and thus, is a promising strategy for improved production of intracellularly accumulated biopolymers.
  • Enhanced cellular content and lactate fraction of the poly(lactate-co-3-hydroxybutyrate) polyester produced in recombinant Escherichia coli by the deletion of σ factor RpoN.
    Kadoya R, Kodama Y, Matsumoto K, Taguchi S
    Journal of bioscience and bioengineering, 119, 4, 427, 429, 4, 2015年04月, [査読有り]
    英語, 研究論文(学術雑誌), A new approach at the transcriptional level was applied to lactate-based polyester production. Four sigma factor disruptants, Delta rpoN, Delta rpoS, Delta fliA and Delta fecl, of Escherichia con were used as hosts for poly(lactate-co-3-hydroxybutyrate) production from glucose. Among them, Delta rpoN caused dual positive effects of polymer production, enhanced cellular content and lactate fraction. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.
  • Improved production of poly(lactic acid)-like polyester based on metabolite analysis to address the rate-limiting step
    Ken'ichiro Matsumoto, Kota Tobitani, Shunsuke Aoki, Yuyang Song, Toshihiko Ooi, Seiichi Taguchi
    AMB EXPRESS, 4, 1, 1, 5, BIOMED CENTRAL LTD, 2014年11月, [査読有り]
    英語, 研究論文(学術雑誌), The biosynthesis of poly(lactic acid) (PLA)-like polymers, composed of >99 mol% lactate and a trace amount of 3-hydroxybutyrate, in engineered Corynebacterium glutamicum consists of two steps; the generation of the monomer substrate lactyl-coenzyme A (CoA) and the polyhydroxyalkanoate (PHA) synthase-catalyzed polymerization of lactyl-CoA. In order to increase polymer productivity, we explored the rate-limiting step in PLA-like polymer synthesis based on quantitative metabolite analysis using liquid chromatography mass spectroscopy (LC-MS). A significant pool of lactyl-CoA was found during polymer synthesis. This result suggested that the rate-limitation occurred at the polymerization step. Accordingly, the expression level of PHA synthase was increased by means of codon-optimization of the corresponding gene that consequently led to an increase in polymer content by 4.4-fold compared to the control. Notably, the codon-optimization did not significantly affect the concentration of lactyl-CoA, suggesting that the polymerization reaction was still the rate-limiting step upon the overexpression of PHA synthase. Another important finding was that the generation of lactyl-CoA was concomitant with a decrease in the acetyl-CoA level, indicating that acetyl-CoA served as a CoA donor for lactyl-CoA synthesis. These results show that obtaining information on the metabolite concentrations is highly useful for improving PLA-like polymer production. This strategy should be applicable to a wide range of PHA-producing systems.
  • Deletion of the pflA gene in Escherichia coli LS5218 and its effects on the production of polyhydroxyalkanoates using beechwood xylan as a feedstock
    Lucia Salamanca-Cardona, Ryan A. Scheel, Benjamin R. Lundgren, Arthur J. Stipanovic, Ken'ichiro Matsumoto, Seiichi Taguchi, Christopher T. Nomura
    BIOENGINEERED, 5, 5, 284, 287, LANDES BIOSCIENCE, 2014年09月, [査読有り]
    英語, 研究論文(学術雑誌), Engineering of microorganisms to directly utilize plant biomass as a feedstock for the biosynthesis of value-added products such as bioplastics is the aim of consolidated bioprocessing. In previous research we successfully engineered E. coli LS5218 to produce polyhydroxyalkanoates (PHAs) from xylan. In this study we report further genetic modifications to Escherichia coli LS5218 in order to increase the lactic acid (LA) fraction in poly(lactic acid-co-3-hydroxyalkanoate) P(LA-co-HA) copolymers. Deletion of the pflA gene resulted in increased content of LA repeating units in the copolymers by over 3-fold compared with the wild type; however, this increase was offset by reduced yields in cell mass. Additionally, when acetate was used as a feedstock LA monomer incorporation reached 18.5 (mol%), which suggests that acetate can be used as a feedstock for the production of P(LA-co-HA) copolymers by E. coli.
  • Engineering Escherichia coli for Improved Production of Short-Chain-Length-co-Medium-Chain-Length Poly[ (R)-3-hydroxyalkanoate] (SCL-co-MCL PHA) Copolymers from Renewable Nonfatty Acid Feedstocks
    Ryan C. Tappel, Wenyang Pan, N. Scott Bergey, Qin Wang, Ivory L. Patterson, Obiajulu A. Ozumba, Ken'ichiro Matsumoto, Seiichi Taguchi, Christopher T. Nomura
    ACS SUSTAINABLE CHEMISTRY & ENGINEERING, 2, 7, 1879, 1887, AMER CHEMICAL SOC, 2014年07月, [査読有り]
    英語, 研究論文(学術雑誌), Polyhydroxyalkanoates (PHAs) are biorenewable and biodegradable polyesters that have garnered attention as alternatives to more common petroleum-based polymers. One of the current limitations for the widespread use of PHAs is the inability to produce PHA polymers with desired material properties. Previous studies have shown that PHA copolymers consisting primarily of one short-chain-length (SCL) repeating unit and a small concentration of medium-chain-length (MCL) repeating units have physical properties resembling the petroleum-based plastic polyethylene. In addition, these SCL-co-MCL PHA copolymers have been investigated for biomedical applications such as tissue engineering. However, bacterial production of these SCL-co-MCL PHA copolymers is often at a much lower yield compared to SCL PHA biosynthesis produced from simple sugars such as glucose. Here, we report the highest yield to date of SCL-co-MCL PHA copolymers produced from glucose. Two separate biosynthetic pathways for SCL and MCL PHAs were introduced into Escherichia coli LS5218, and copolymer production experiments were carried out in batch fermentations. The PHA copolymers produced consisted of repeating units with 4, 6, 8, 10, and 12 carbons at mol % concentrations similar to that of other SCL-co-MCL PHA copolymers reported to have desirable physical properties. The PHA repeating unit compositions, structures, and linkages between individual repeating unit types were analyzed by GC and NMR. The thermal properties of purified PHA copolymers were also examined. The engineered strain developed in this study (E. coli LS5218-STQKABGK) provides a platform to further increase PHA copolymer yields from unrelated carbon sources in a non-native PHA producing bacterial strain.
  • Deletion of the pflA gene in Escherichia coli LS5218 and its effects on the production of polyhydroxyalkanoates using beechwood xylan as a feedstock
    Lucia Salamanca-Cardona, Ryan A. Scheel, Benjamin R. Lundgren, Arthur J. Stipanovic, Ken’ichiro Matsumoto, Seiichi Taguchi, Christopher T. Nomura
    Bioengineered Bugs, 5, 5, 284, 287, Landes Bioscience, 2014年06月20日, [査読有り]
    英語, 研究論文(学術雑誌)
  • Enhanced production of poly(lactate-co-3-hydroxybutyrate) from xylose in engineered Escherichia coli overexpressing a galactitol transporter
    John Masani Nduko, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 98, 6, 2453, 2460, SPRINGER, 2014年03月, [査読有り]
    英語, 研究論文(学術雑誌), Poly(lactate-co-3-hydroxybutyrate) (P(LA-co-3HB)) was previously produced from xylose in engineered Escherichia coli. The aim of this study was to increase the polymer productivity and LA fraction in P(LA-co-3HB) using two metabolic engineering approaches: (1) deletions of competing pathways to lactate production and (2) overexpression of a galactitol transporter (GatC), which contributes to the ATP-independent xylose uptake. Engineered E. coli mutants (Delta pflA, Delta pta, Delta ackA, Delta poxB, Delta dld, and a dual mutant; Delta pflA + Delta dld) and their parent strain, BW25113, were grown on 20 g l(-1) xylose for P(LA-co-3HB) production. The single deletions of Delta pflA, Delta pta, and Delta dld increased the LA fraction (58-66 mol%) compared to BW25113 (56 mol%). In particular, the Delta pflA + Delta dld strain produced P(LA-co-3HB) containing 73 mol% LA. Furthermore, GatC overexpression increased both polymer yields and LA fractions in Delta pflA, Delta pta, and Delta dld mutants, and BW25113. The Delta pflA + gatC strain achieved a productivity of 8.3 g l(-1), which was 72 % of the theoretical maximum yield. Thus, to eliminate limitation of the carbon source, higher concentration of xylose was fed. As a result, BW25113 harboring gatC grown on 40 g l(-1) xylose reached the highest P(LA-co-3HB) productivity of 14.4 g l(-1). On the other hand, the Delta pflA + Delta dld strain grown on 30 g l(-1) xylose synthesized 6.4 g l(-1) P(LA-co-3HB) while maintaining the highest LA fraction (73 mol%). The results indicated the usefulness of GatC for enhanced production of P(LA-co-3HB) from xylose, and the gene deletions to upregulate the LA fraction in P(LA-co-3HB). The polymers obtained had weight-averaged molecular weights in the range of 34,000-114,000.
  • Enzyme and metabolic engineering for the production of novel biopolymers: crossover of biological and chemical processes
    Ken'ichiro Matsumoto, Seiichi Taguchi
    CURRENT OPINION IN BIOTECHNOLOGY, 24, 6, 1054, 1060, CURRENT BIOLOGY LTD, 2013年12月, [査読有り]
    英語, 研究論文(学術雑誌), The development of synthetic biology has transformed microbes into useful factories for producing valuable polymers and/or their precursors from renewable biomass. Recent progress at the interface of chemistry and biology has enabled the production of a variety of new biopolymers with properties that substantially differ from their petroleum-derived counterparts. This review touches on recent trials and achievements in the field of biopolymer synthesis, including chemo-enzymatically synthesized aliphatic polyesters, wholly biosynthesized lactate-based polyesters, polyhydroxyalkanoates and other unusual bacterially synthesized polyesters. The expanding diversities in structure and the material properties of biopolymers are key for exploring practical applications. The enzyme and metabolic engineering approaches toward this goal are discussed by shedding light on the successful case studies.
  • Biosynthetic polyesters consisting of 2-hydroxyalkanoic acids: current challenges and unresolved questions
    Ken'ichiro Matsumoto, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97, 18, 8011, 8021, SPRINGER, 2013年09月, [査読有り]
    英語, 研究論文(学術雑誌), 2-Hydroxyalkanoates (2HAs) have become the new monomeric constituents of bacterial polyhydroxyalkanoates (PHAs). PHAs containing 2HA monomers, lactate (LA), glycolate (GL), and 2-hydroxybutyrate (2HB) can be synthesized by engineered microbes in which the broad substrate specificities of PHA synthase and propionyl-CoA transferase are critical factors for the incorporation of the monomers into the polymer chain. LA-based polymers, such as P[LA-co-3-hydroxybutyrate (3HB)], have the properties of pliability and stretchiness which are distinctly different from those of the rigid poly(lactic acid) (PLA) and P(3HB) homopolymers. This versatile platform is also applicable to the biosynthesis of GL- and 2HB-based polymers. In the case of the synthesis of 2HB-based polymers, the enantiospecificity of PHA synthase enabled the production of isotactic (R)-2HB-based polymers, including P[ (R)-2HB], from racemic precursors of 2HB. P(2HB) is a pliable material, in contrast to PLA. Furthermore, to obtain a new 2HA-polymerizing PHA synthase, the class I PHA synthase from Ralstonia eutropha was engineered so as to achieve the first incorporation of LA units. The analysis of the polymer synthesized using this new LA-polymerizing PHA synthase unexpectedly focused a spotlight on the studies on block copolymer biosynthesis.
  • One-Pot Microbial Production, Mechanical Properties, and Enzymatic Degradation of Isotactic P[ (R)-2-hydroxybutyrate] and Its Copolymer with (R)-Lactate
    Ken'ichiro Matsumoto, Satsuki Terai, Ayako Ishiyama, Jian Sun, Taizo Kabe, Yuyang Song, John Masani Nduko, Tadahisa Iwata, Seiichi Taguchi
    BIOMACROMOLECULES, 14, 6, 1913, 1918, AMER CHEMICAL SOC, 2013年06月, [査読有り]
    英語, 研究論文(学術雑誌), P[ (R)-2-hydroxybutyrate] [P((R)-2HB)] is an aliphatic polyester analogous to poly(lactic acid) (PLA). However, little has been known for its properties because of a high cost of commercially available chiral 2HB as a starting substance for chemical polymer synthesis. In this study, P[ (R)-2HB] and P[ (R)-2HB-co-(R)-lactate] [P((R)-2HB-co-(R)-LA)] with a new monomer combination were successfully synthesized in recombinant Escherichia coli LS5218 from less-expensive racemic 2HB using an R-specific polyester synthase. The cells expressing an engineered polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 and propionyl-CoA transferase from Megasphaera elsdenii were grown on LB medium containing 2HB and glucose in a shake flask and accumulated up to 17 wt% of P[ (R)-2HB] with optical purity of >99.1%. In addition, the same cells cultured in a jar-fermentor produced P(86 mol% 2HB-co-LA) copolymer. Notably, the molecular weights (M-w) of P(2HB) (27000) and P(2HB-co-LA) (39000) were 2- and 3-fold higher than that of P(2HB) previously synthesized by chemical polycondensation. P(2HB) was processed into a transparent film by solvent-casting and it had flexible properties with elongation at break of 173%, which was contrast to the rigid PLA. Regarding mechanical properties, P(2HB-co-LA) was tougher but less stretchy than P(2HB). These results demonstrated that P(2HB) has useful properties and LA units in 2HB-based polymers can act as a controllable modulator of the material properties. In addition, P[ (R)-2HB] was efficiently degraded by treatment of Novozym 42044 (lipase) but not Savinase 16L (protease), indicating that the degrading behavior of the polymer was similar to that of P[ (R)-LA].
  • Engineering of class i lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature
    Anna Ochi, Ken'Ichiro Matsumoto, Takashi Ooba, Kohei Sakai, Takeharu Tsuge, Seiichi Taguchi
    Applied Microbiology and Biotechnology, 97, 8, 3441, 3447, 2013年04月, [査読有り]
    英語, 研究論文(学術雑誌), Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaCRe) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaCRe was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1PsSTQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323-17327, 2008). The LA-polymerizing activity of the PhaCReA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaCRe(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaCReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The 13C and 1H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 105 Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA×-LA triad sequence, suggesting that the polymer synthesized by PhaCRe mutant may not be a random copolymer, but presumably had a block sequence. © 2012 Springer-Verlag.
  • Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase
    Ken'ichiro Matsumoto, Takehiro Okei, Inori Honma, Toshihiko Ooi, Hirobumi Aoki, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 97, 1, 205, 210, SPRINGER, 2013年01月, [査読有り]
    英語, 研究論文(学術雑誌), (R)-3-hydroxybutyrate [ (R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [ (R)-3HB-CoA] by beta-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L-1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L-1, with a productivity of 0.22 g L-1 h(-1). The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of C-13 carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.
  • Single-step production of polyhydroxybutyrate from starch by using α-amylase cell-surface displaying system of Corynebacterium glutamicum.
    Song Y, Matsumoto K, Tanaka T, Kondo A, Taguchi S
    Journal of bioscience and bioengineering, 115, 1, 12, 14, 1, 2013年01月, [査読有り]
    英語, 研究論文(学術雑誌), Direct polyhydroxybutyrate (PHB) production from starch was for the first time achieved using engineered Corynebacterium glutamicum expressing PHB biosynthetic genes and displaying a-amylase on its cell surface. The engineered strain accumulated 6.4 wt% PHB from starch which was higher than that obtained from glucose (4.9 wt%). (C) 2012, The Society for Biotechnology, Japan. All rights reserved.
  • Effectiveness of xylose utilization for high yield production of lactate-enriched P(lactate-co-3-hydroxybutyrate) using a lactate-overproducing strain of Escherichia coli and an evolved lactate-polymerizing enzyme
    John Masani Nduko, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    METABOLIC ENGINEERING, 15, 159, 166, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2013年01月, [査読有り]
    英語, 研究論文(学術雑誌), Xylose, which is a major constituent of lignocellulosic biomass, was utilized for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)], having transparent and flexible properties. The recombinant Escherichia coli JW0885 (pflA(-)) expressing LA-polymerizing enzyme (LPE) and monomer supplying enzymes grown on xylose produced a copolymer having a higher LA fraction (34 mol%) than that grown on glucose (26 mol%). This benefit of xylose was further enhanced by combining it with an evolved LPE (ST/FS/QK), achieving a copolymer production having 60 mol% LA from xylose, while glucose gave a 47 mol% LA under the same condition. The overall carbon yields from the sugars to the polymer were similar for xylose and glucose, but the ratio of the LA and 3HB units in the copolymer was different. Notably, the P(LA-co-3HB) yield from xylose (7.3 g l(-1)) was remarkably higher than that of P(3HB) (4.1 g l(-1)), indicating P(LA-co-3HB) as a potent target for xylose utilization. (C) 2012 Elsevier Inc. All rights reserved.
  • Directed evolution and structural analysis of nadph-dependent acetoacetyl coenzyme A(acetoacetyl-CoA) reductase from ralstonia eutropha reveals two mutations responsible for enhanced kinetics
    Ken'ichiro Matsumoto, Yoshikazu Tanaka, Tsuyoshi Watanabe, Ren Motohashi, Koji Ikeda, Kota Tobitani, Min Yao, Isao Tanaka, Seiichi Taguchi
    Applied and Environmental Microbiology, 79, 19, 6134, 6139, 19, 2013年, [査読有り]
    英語, 研究論文(学術雑誌), NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along withβ-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutrophawas engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu(Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, thePhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an importantrole in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex withNADPHand acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity. © 2013, American Society for Microbiology. All Rights Reserved.
  • Engineered Corynebacterium glutamicum as an endotoxin-free platform strain for lactate-based polyester production
    Yuyang Song, Ken'ichiro Matsumoto, Miwa Yamada, Aoi Gohda, Christopher J. Brigham, Anthony J. Sinskey, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 93, 5, 1917, 1925, SPRINGER, 2012年03月, [査読有り]
    英語, 研究論文(学術雑誌), The first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D-lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by beta-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.
  • Polyhydroxyalkanoates production from cellulose hydrolysate in Escherichia coli LS5218 with superior resistance to 5-hydroxymethylfurfural
    John Masani Nduko, Wakako Suzuki, Ken'ichiro Matsumoto, Hirokazu Kobayashi, Toshihiko Ooi, Atsushi Fukuoka, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 113, 1, 70, 72, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2012年01月, [査読有り]
    英語, 研究論文(学術雑誌), Poly[3-hydroxybutyrate-co-3-hydroxyvalerate(3HV)] was produced in recombinant Escherichia coli LS5218 from ruthenium-catalyzed cellulose hydrolysate and propionate. The strain was found to be resistant to 5-hydroxymethylfurfural (5-HMF), which is a major inhibitory byproduct generated in the cellulose hydrolysis reaction. The 3HV fraction was successfully regulated in the range of 5.6-40 mol%. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.
  • Biological Lactate-Polymers Synthesized by One-Pot Microbial Factory: Enzyme and Metabolic Engineering
    John Masani Nduko, Ken'ichiro Matsumoto, Seiichi Taguchi
    BIOBASED MONOMERS, POLYMERS, AND MATERIALS, 1105, 213, 235, AMER CHEMICAL SOC, 2012年, [査読有り]
    英語, 研究論文(国際会議プロシーディングス), Poly lactic acid (PLA) is a representative commodity polyester synthesized from renewable resources. The benefits of PLA over the conventional petrochemical-based plastics are; it is biodegradable, it requires less energy to produce and reduces greenhouse gas production. PLA is mainly produced via a two step bio-chemo process where lactate produced by microorganisms is converted into lactides which are then polymerized by metal catalysts to form high molecular weight PLA. In 2008, microbial LA-based polyester production systems were for the first time established as microbial cell factories (MCFs). This paradigm shift is based on a conversion of a two-step chemical process into a single-step MCF. This breakthrough was triggered by the discovery of an engineered LA-polymerizing enzyme (LPE). LPE was discovered among a collection of polyhydroxyalkanoate (PHA) synthase mutants from Pseudomonas sp. 61-3 through a long-term evolutionary engineering study. The MCF has been advanced by combination of further evolution of LPE with metabolic engineering for improved monomer precursor production. This chapter reviews the backgrounds on the establishment of the MCFs for the synthesis of LA-based polyesters to near PLA homopolymer, structures and properties of LPE-catalyzed polymers and challenges of LA-based polymer production system as well as future perspectives on the microbial LA-based polyesters.
  • Biosynthesis of glycolate-based polyesters containing medium-chain-length 3-hydroxyalkanoates in recombinant Escherichia coli expressing engineered polyhydroxyalkanoate synthase
    Ken'ichiro Matsumoto, Ayako Ishiyama, Kohei Sakai, Tetsufumi Shiba, Seiichi Taguchi
    JOURNAL OF BIOTECHNOLOGY, 156, 3, 214, 217, ELSEVIER SCIENCE BV, 2011年12月, [査読有り]
    英語, 研究論文(学術雑誌), Glycolate(GL)-based polyesters were for the first time produced in the recombinant Escherichia coli fatty acid beta-oxidation pathway reinforcing mutant LS5218, using extracellularly added GL as a monomer precursor. Cells expressing a Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase (PhaC1STQK) from Pseudomonas sp. 61-3, propionyl-CoA transferase from Megasphaera elsdenii and enoyl-CoA hydratase from Pseudomonas aeruginosa grown on GL and dodecanoate were found to produce novel copolymers of GL with 3-hydroxyalkanoates (3HAs) (C(4)-C(12)), P(GL-co-3HA), with a weight-average molecular weight of 34,000. The (1)H and (13)C NMR analyses of the copolymer revealed the incorporation of GL units into the polymer chain. This result demonstrates that PhaC1STQK polymerized glycolyl-CoA as a monomer substrate. Additionally, the novel lactate(LA)-based polyester P(LA-co-3HA) was produced by substituting GL with LA, indicating that the method is versatile and allows the production of a variety of biopolymers. (C) 2011 Elsevier B.V. All rights reserved.
  • Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha
    Xuerong Han, Yasuharu Satoh, Toshifumi Satoh, Ken'ichiro Matsumoto, Toyoji Kakuchi, Seiichi Taguchi, Tohru Dairi, Masanobu Munekata, Kenji Tajima
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 92, 3, 509, 517, SPRINGER, 2011年11月, [査読有り]
    英語, 研究論文(学術雑誌), A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 x 10(5), and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T (g), T (m), and T (d) (10%) were observed at -1.1A degrees C, 158.8A degrees C, and 252.7A degrees C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.
  • Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli
    Miwa Yamada, Ken'ichiro Matsumoto, Shu Uramoto, Ren Motohashi, Hideki Abe, Seiichi Taguchi
    JOURNAL OF BIOTECHNOLOGY, 154, 4, 255, 260, ELSEVIER SCIENCE BV, 2011年07月, [査読有り]
    英語, 研究論文(学術雑誌), In order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Young's modulus values of the P(LA-co-3HB) s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB) s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers. (C) 2011 Elsevier B. V. All rights reserved.
  • Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Corynebacterium glutamicum using propionate as a precursor
    Ken'ichiro Matsumoto, Kei Kitagawa, Sung-Jin Jo, Yuyang Song, Seiichi Taguchi
    JOURNAL OF BIOTECHNOLOGY, 152, 4, 144, 146, ELSEVIER SCIENCE BV, 2011年04月, [査読有り]
    英語, 研究論文(学術雑誌), Lipopolysaccharides free P[3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)] production was achieved using recombinant Corynebacterium glutamicum harboring polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha. Cells grown on glucose with feeding of propionate as a precursor of 3HV unit accumulated 8-47 wt% of P(3HB-co-3HV). The 3HV fraction in the copolymer was varied from 0 to 28 mol% depending on the propionate concentrations. (C) 2010 Elsevier B.V. All rights reserved.
  • Improved polyhydroxybutyrate (PHB) production in transgenic tobacco by enhancing translation efficiency of bacterial PHB biosynthetic genes
    Ken'ichiro Matsumoto, Kenjiro Morimoto, Aoi Gohda, Hiroald Shimada, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 111, 4, 485, 488, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2011年04月, [査読有り]
    英語, 研究論文(学術雑誌), Polyhydroxybutyrate [P(3HB)] was produced in the transgenic tobacco harboring the genes encoding acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha (Cupriavidus necator) with optimized codon usage for expression in tobacco. P(3HB) contents in the transformants (0.2 mg/g dry cell weight in average) harboring the codon-optimized phaB gene was twofold higher than the control transformants harboring the wild-type phaB gene. The immunodetection revealed an increased production of PhaB in leaves, indicating that the enhanced expression of PhaB was effective to increase P(3HB) production in tobacco. In contrast, codon-optimization of the phaC gene exhibited no apparent effect on P(3HB) production. This result suggests that the efficiency of PhaB-catalyzed reaction contributed to the flux toward P(3HB) biosynthesis in tobacco leaves. 2010, The Society for Biotechnology, Japan. All rights reserved.
  • Biosynthesis of a lactate (LA)-based polyester with a 96 mol% la fraction and its application to stereocomplex formation
    Fumi Shozui, Ken'Ichiro Matsumoto, Ren Motohashi, Jian Sun, Toshifumi Satoh, Toyoji Kakuchi, Seiichi Taguchi
    Polymer Degradation and Stability, 96, 4, 499, 504, 2011年04月, [査読有り]
    英語, 研究論文(学術雑誌), A poly(lactic acid) (PLA)-like terpolyester consisting of 96 mol% lactate (LA), 1 mol% 3-hydroxybutyrate and 3 mol% 3-hydroxyvalerate was produced in recombinant Escherichia coli LS5218 expressing LA-polymerizing enzyme (LPE). The strain was grown on glucose with a feeding of valerate as the monomer precursor. The glass transition and melting temperatures of the terpolyester were close to those of chemically synthesized poly(L-LA)s (PLLAs) having similar molecular weights. Additionally, a blend of the terpolyester, which was composed entirely of (R)-LA (D-LA) due to the strict enantiospecificity of LPE, with PLLA formed a stereocomplex with higher melting temperature (201.9 °C). These results indicate that the biological PLA-like polyester produced via this one-step microbial process has comparable thermal properties to chemically synthesized PLAs. © 2011 Elsevier Ltd. All rights reserved.
  • Chemo-microbial conversion of cellulose into polyhydroxybutyrate through ruthenium-catalyzed hydrolysis of cellulose into glucose
    Ken'ichiro Matsumoto, Hirokazu Kobayashi, Koji Ikeda, Tasuku Komanoya, Atsushi Fukuoka, Seiichi Taguchi
    BIORESOURCE TECHNOLOGY, 102, 3, 3564, 3567, ELSEVIER SCI LTD, 2011年02月, [査読有り]
    英語, 研究論文(学術雑誌), Cellulose-derived glucose generated using the supported ruthenium catalyst was applied to poly(3-hydroxybutyrate) [P(3HB)] production in recombinant Escherichia coli. By the reaction with the catalyst at 220 degrees C. 15-20 carbon mol% of cellulose was converted into glucose. The hydrolysate also contained byproducts such as fructose, mannose, levoglucosan, oligomeric cellulose, 5-hydroxymethylfurfural (5-HMF), and furfural together with unidentified compounds. Setting the reaction temperature lower (215 degrees C) improved the ratio of glucose to 5-HMF, which was a main inhibiting factor for the cell growth. Indeed, the recombinant E. coli exhibited better performance on the hydrolysate generated at 215 degrees C and accumulated P(3HB) up to 42 wt%, which was the same as the case of the same concentration of analytical grade glucose. The result indicated that the ruthenium-mediated cellulose hydrolysis has a potency as a useful biorefinery process for production of bio-based plastic from cellulosic biomass. (C) 2010 Elsevier Ltd. All rights reserved.
  • A New Pathway for Poly(3-hydroxybutyrate) Production in Escherichia coli and Corynebacterium glutamicum by Functional Expression of a New Acetoacetyl-coenzyme A Synthase
    Ken'ichiro Matsumoto, Miwa Yamada, Chean Ring Leong, Sung-Jin Jo, Tomohisa Kuzuyama, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 75, 2, 364, 366, TAYLOR & FRANCIS LTD, 2011年02月, [査読有り]
    英語, 研究論文(学術雑誌), A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] was developed in Escherichia coli and Corynebacterium glutamicum by an acetoacetyl-coenzyme A (CoA) synthase (AACS) recently isolated from terpenoid-producing Streptomyces sp. strain CL190. Expression of AACS led to significant productions of P(3HB) in E. coli (10.5 wt %) and C. glutamicum (19.7 wt %).
  • A Novel Factor FLOURY ENDOSPERM2 Is Involved in Regulation of Rice Grain Size and Starch Quality
    Kao-Chih She, Hiroaki Kusano, Kazuyoshi Koizumi, Hiromoto Yamakawa, Makoto Hakata, Tomohiro Imamura, Masato Fukuda, Natsuka Naito, Yumi Tsurumaki, Mitsuhiro Yaeshima, Tomohiko Tsuge, Ken'ichiro Matsumoto, Mari Kudoh, Eiko Itoh, Shoshi Kikuchi, Naoki Kishimoto, Junshi Yazaki, Tsuyu Ando, Masahiro Yano, Takashi Aoyama, Tadamasa Sasaki, Hikaru Satoh, Hiroaki Shimada
    PLANT CELL, 22, 10, 3280, 3294, AMER SOC PLANT BIOLOGISTS, 2010年10月, [査読有り]
    英語, 研究論文(学術雑誌), Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.
  • Production of P(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) Terpolymers Using a Chimeric PHA Synthase in Recombinant Ralstonia eutropha and Pseudomonas putida
    Jian Sun, Fumi Shozui, Miwa Yamada, Ken'ichiro Matsumoto, Kazuma Takase, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74, 8, 1716, 1718, TAYLOR & FRANCIS LTD, 2010年08月, [査読有り]
    英語, 研究論文(学術雑誌), Recombinant strains of Ralstonia eutropha and Pseudomonas putida harboring a chimeric polyhydroxyalkanoate (PHA) synthase, which consisted of PHA synthases of Aeromonas caviae and R. eutropha, produced 3-hydroxybutyrate (3HB)-based PHA copolymers comprised of 3-hydroxyhexanoate and 3-hydroxyoctanoate units from dodecanoate (87-97 mol % 3HB), indicating that the chimeric PHA synthase possesses desirable substrate specificity leading to the production of 3HB-rich copolymers.
  • A New Beneficial Mutation in Pseudomonas sp 61-3 Polyhydroxyalkanoate (PHA) Synthase for Enhanced Cellular Content of 3-Hydroxybutyrate-Based PHA Explored Using Its Enzyme Homolog as a Mutation Template
    Fumi Shozui, Jian Sun, Yuyang Song, Miwa Yamada, Kohei Sakai, Ken'ichiro Matsumoto, Kazuma Takase, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74, 8, 1710, 1712, TAYLOR & FRANCIS LTD, 2010年08月, [査読有り]
    英語, 研究論文(学術雑誌), A newly isolated mutation (Gl508Leu) and a combination of it with previously discovered beneficial mutations in polyhydroxyalkanoate synthase 1 from Pseudomonas sp. 61-3 were found to enhance the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoate)s in recombinant Escherichia coli.
  • Establishment of a metabolic pathway to introduce the 3-hydroxyhexanoate unit into LA-based polyesters via a reverse reaction of beta-oxidation in Escherichia coli LS5218
    Fumi Shozui, Ken'ichiro Matsumoto, Ren Motohashi, Miwa Yamada, Seiichi Taguchi
    POLYMER DEGRADATION AND STABILITY, 95, 8, 1340, 1344, ELSEVIER SCI LTD, 2010年08月, [査読有り]
    英語, 研究論文(学術雑誌), New lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate (3HB)-co-3-hydroxyhexananoate (3HHx)]s, were produced in recombinant Escherichia coli LS5218 harboring three genes encoding LA-polymerizing enzyme (LPE), propionyl-coenzyme A (CoA) transferase (PCT) and (R)-specific enoyl-CoA hydratase (PhaJ4). When the recombinant LS5218 was grown on glucose with the feeding of butyrate, 3HB-CoA and 3HHx-CoA were supplied, probably via reverse reactions of the beta-oxidation pathway and PhaJ4. LPE copolymerized the two monomers 3HB-CoA and 3HHx-CoA with LA-CoA, which was generated by PCT, to yield the terpolymers. Gas chromatography analysis revealed that the terpolymers consisted of 2.7-34 mol% LA, 38-81 mol% 3HB and 17-33 mol% 3HHx units, which can be varied depending on the butyrate concentration fed in the medium. In addition, H-1-C-13 COSY NMR analysis provided evidence for a linkage between LA and 3HHx units in the polymer. (C) 2010 Elsevier Ltd. All rights reserved.
  • Quick and efficient method for genetic transformation of biopolymer-producing bacteria
    Qin Wang, Alexander P. Mueller, Chean Ring Leong, Ken'ichiro Matsumoto, Seiichi Taguchi, Christopher T. Nomura
    Journal of Chemical Technology and Biotechnology, 85, 6, 775, 778, 2010年06月, [査読有り]
    英語, 研究論文(学術雑誌), In order to genetically modify microorganisms capable of producing polyhydroxyalkanoate (PHA) biopolymers, a simple and rapidmethod to prepare freshly plated Pseudomonas cells for transformation via electroporation was developed. This method can be used to transfer both replicative plasmids and linear DNA to knock out genes into the cells. The transformation efficiencies were in the range of ≥107 transformants μg-1 DNA for replicative plasmids and ≥106 transformants μg-1 DNA for linear DNA, which are comparable with commercially available competent cells. Furthermore, this transformation procedure can be performed in less than 10 min, saving a great deal of time compared with traditional methods. Knockout mutants of several Pseudomonas species were generated by transformation of linear DNA and these mutations were verified by PCR and analysis of PHA content. © 2009 Society of Chemical Industry.
  • Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus
    Ken'ichiro Matsumoto, Yuichi Mukai, Daiki Ogata, Fumi Shozui, John Masani Nduko, Seiichi Taguchi, Toshihiko Ooi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 86, 5, 1431, 1438, SPRINGER, 2010年05月, [査読有り]
    英語, 研究論文(学術雑誌), The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 A degrees C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 A degrees C and for 1 month at 30 A degrees C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 A degrees C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.
  • Flow cytometric analysis of the contributing factors for antimicrobial activity enhancement of cell-penetrating type peptides: Case study on engineered apidaecins
    Ken'ichiro Matsumoto, Yoshitake Orikasa, Kenta Ichinohe, Shigeki Hashimoto, Toshihiko Ooi, Seiichi Taguchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 395, 1, 7, 10, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2010年04月, [査読有り]
    英語, 研究論文(学術雑誌), Contributing factors for the antimicrobial activity enhancement of N-terminally engineered mutants of cell-penetrating apidaecins were analyzed based on their cell-penetration efficiency. The flow cytometric analysis of the engineered apidaecins labeled with carboxyfluorescein (FAM) revealed their enhanced cell-penetrating efficiencies into Escherichia coli that should be one of key factors causing the enhanced antimicrobial activity. It is noteworthy that, for one mutant, the enhancement in antimicrobial activity (18-fold higher than wild type) was greater than that of cell penetration (5.9-fold), suggesting that the N-terminal mutation may reinforce both interaction with unidentified intracellular target(s) and cell-penetration efficiency. (C) 2010 Elsevier Inc. All rights reserved.
  • Adjustable Mutations in Lactate (LA)-Polymerizing Enzyme for the Microbial Production of LA-Based Polyesters with Tailor-Made Monomer Composition
    Miwa Yamada, Ken'ichiro Matsumoto, Kotaro Shimizu, Shu Uramoto, Takanori Nakai, Fumi Shozui, Seiichi Taguchi
    BIOMACROMOLECULES, 11, 3, 815, 819, AMER CHEMICAL SOC, 2010年03月, [査読有り]
    英語, 研究論文(学術雑誌), Lactate (LA)-polymerizing enzyme (LPE) is a newly established class of polyhydroxyalkanoate (PHA) synthase, which can incorporate LA units into a polymer chain. We previously synthesized P(LA-co-3-hydroxybutyrate)s [P(LA-co-3HB)s] in recombination Escherichia coli using the first LPE, which is the Ser325Thr/Glu481Lys mutant of PHA synthase from Pseudomonas sp. 61-3 [PhaCl(Ps)ST/QK]. In this study, we finely regulated LA fraction in the copolymer by saturated mutations at position 392 (F392X), which corresponds to the activity-enhancing mutations at position 420 of PHA synthase from Ralstonia eutropha. Among the 19 saturated mutants of LPe at position 392, 17 mutants produced P(La-co-3HB)s with various LA fractions (16-45 mol %), whereas PhaCl(Ps)ST/QK produced P(LA-co-3HB) with 26 mol % LA under the same culture condition. In particular, the F392S mutation exhibited the highest LA fraction of 45 mol %, and also increased polymer content (62 wt %) compared with PhaCl(Ps)ST/QK (44 wt %). Combination of the F392S mutant and anaerobic culture conditions, which promote LA production, led to a further increase in LA fraction up to 62 mol%. The P(LA-co-3HB)s with various LA fractions exhibited altered melting temperatures and melting enthalpy depending on their monomer composition. Accordingly, the mutations at position 392 in LPE greatly contributed to fine-tuning of the LA fraction in the copolymers that is useful for regulating LA fraction-dependent thermal properties.
  • Biosynthesis of novel terpolymers poly(lactate-co-3-hydroxybutyrate-co-3-hydroxyvalerate)s in lactate-overproducing mutant Escherichia coli JW0885 by feeding propionate as a precursor of 3-hydroxyvalerate
    Fumi Shozui, Ken'ichiro Matsumoto, Takanori Nakai, Miwa Yamada, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85, 4, 949, 954, SPRINGER, 2010年01月, [査読有り]
    英語, 研究論文(学術雑誌), Novel lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate(3HB)-co-3-hydroxyvalerate(3HV)]s (PLBVs), were produced in LA-overproducing mutant, Escherichia coli JW0885, which was found to be a superior host for the efficient production of LA-based polyesters. Recombinant E. coli JW0885 harboring the genes encoding LA-polymerizing enzyme (Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3) and three monomer supplying enzymes [propionyl-CoA transferase, beta-ketothiolase, and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-dependent acetoacetyl-CoA reductase] was aerobically grown on glucose with feeding of propionate as a precursor of 3-hydroxyvaleryl-CoA (3HV-CoA). Gas chromatography and nuclear magnetic resonance (NMR) analyses revealed that polymers accumulated in the cells were composed of LA, 3HB, and 3HV units, thus being identified as terpolymers, PLBVs. In addition, H-1-NMR analysis suggested the existence of LA-3HV sequence in the terpolymer. When 100 mg/l of sodium propionate was added into the medium, 3HV fraction in the terpolymer linearly reached up to 7.2 mol%, while LA fraction was inversely decreased. This phenomenon could be due to the change in metabolic fluxes of lactyl-CoA (LA-CoA) and 3HV-CoA depending on the concentration of propionate fed into the medium.
  • Enzymatic and whole-cell synthesis of lactate-containing polyesters: toward the complete biological production of polylactate
    Ken'ichiro Matsumoto, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 85, 4, 921, 932, SPRINGER, 2010年01月, [査読有り]
    英語, 研究論文(学術雑誌), The importance of polylactic acid, a representative bio-based polyester, has been established on a worldwide scale in response to emerging global environmental problems such as green house gas emission and limited petroleum consumption. The current methods for generating this bio-based polymer involve biological synthesis and lactic acid (LA) fermentation, followed by chemical ring-opening polymerization. Among the research community working on polyhydroxyalkanoate polyesters, the prospect of direct biological synthesis of LA into a polymeric form is very attractive from the academic and industrial perspectives. In 2008, this challenge was met for the first time by the discovery of an "LA-polymerizing enzyme". Using this novel enzyme, the metabolic engineering approach outlined here provided an entirely new, single organism generation of the polymer. This is a major breakthrough in the field. In this review, we provide an overview of the whole-cell synthesis of LA-containing polyesters in comparison with conventional lipase-catalyzed polymer synthesis in terms of both the concepts and strategies of their synthetic processes.
  • A unique post-translational processing of an exo-beta-1,3-glucanase of Penicillium sp KH10 expressed in Aspergillus oryzae
    Toshihiko Ooi, Hirokazu Sato, Ken'ichiro Matsumoto, Seiichi Taguchi
    PROTEIN EXPRESSION AND PURIFICATION, 67, 2, 126, 131, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009年10月, [査読有り]
    英語, 研究論文(学術雑誌), To characterize an exo-beta-1,3-glucanase (ExgP) of an isolated fungal strain with high laminarin degradation activity, identified as Penicillium sp. KH10, heterologous secretory expression of the ExgP was performed in Aspergillus oryzae. Deduced amino acid sequence of the exgP gene possibly consisted of 989 amino acids which showed high sequence similarity to those of fungal exo-beta-1,3-glucanases belonging to the glycoside hydrolase (GH) family 55. Notably, the purified recombinant ExgP showed a single protein peak in the native state (by gel-permeation chromatographic analysis), but showed two protein bands in the denatured state (by SDS-polyacrylamide gel electrophoresis). These two polypeptides exhibited activity in a coexisting state even under reducing conditions, suggesting that non-covalent association of both polypeptides took place. Taken together with the nucleotide sequence information, the ExgP precursor (104 kDa) would be proteolytically processed (cleaved) to generate two protein fragments (42 and 47 kDa) and the processed products (polypeptide fragments) would be assembled each other by a non-covalent interaction. Moreover, one of the matured ExgP polypeptides was N-glycosylated by the post-translational modi. cation. (C) 2009 Elsevier Inc. All rights reserved.
  • Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters
    Fumi Shozui, Ken'ichiro Matsumoto, Takahiro Sasaki, Seiichi Taguchi
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 84, 6, 1117, 1124, SPRINGER, 2009年10月, [査読有り]
    英語, 研究論文(学術雑誌), Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1(Ps)) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1(Ps) that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436-2442). In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1(Ps) was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1(Ps) mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired mutants.
  • The Hydrophobicity in a Chemically Modified Side-Chain of Cysteine Residues of Thanatin Is Related to Antimicrobial Activity against Micrococcus luteus
    Yoshitake Orikasa, Kenta Ichinohe, Junki Saito, Shigeki Hashimoto, Ken'ichiro Matsumoto, Toshihiko Ooi, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73, 7, 1683, 1684, TAYLOR & FRANCIS LTD, 2009年07月, [査読有り]
    英語, 研究論文(学術雑誌), The chemically modified thanatins with the methyll group (CH3), ethyl group (C2H5), and normal-octyl group (C8H17) at the side-chain of cysteine residues were synthesized. The octyl group modified form exhibited 8-fold higher antimicrobial activity against Micrococcus luteus than wild type thanatin. It was found that there was an equilateral correlation between antimicrobial activity and side-chain hydrophobicity at the cysteine residues in thanatin.
  • A novel superior factor widely controlling the rice grain quality
    Kao-Chih She, Hiroaki Kusano, Kazuyoshi Koizumi, Hiromoto Yamakawa, Makoto Hakata, Tomohiro Imamura, Masato Fukuda, Natsuka Naito, Yumi Tsurumaki, Ken'ichiro Matsumoto, Mari Kudoh, Eiko Itoh, Shoshi Kikuchi, Naoki Kishimoto, Junshi Yazaki, Tsuyu Ando, Masahiro Yano, Takashi Aoyama, Tadamasa Sasaki, Hikaru Satoh, Hiroaki Shimada
    Nature Precedings, Springer Science and Business Media LLC, 2009年05月26日
    研究論文(学術雑誌), AbstractSynthesis of storage starch and protein accumulation is the main action of endosperm organogenesis in term of the economic importance of rice. This event is strongly disturbed by abiotic stresses such as high temperature; thus, the upcoming global warming will cause a crisis with a great impact on food production^1,2^. The enzymes for the protein storage and starch synthesis pathway should work in concert to carry out the organogenesis of rice endosperm^3-5^, but the regulatory mechanism is largely unknown. Here we show that a novel regulatory factor, named OsCEO1, acts as the conductor of endosperm organogenesis during the rice grain filling stage. The physiological properties of floury-endosperm-2 (flo2) mutants showed many similarities to symptoms of grains developed under high-temperature conditions, suggesting important roles of the responsible gene in sensitivity to high-temperature stress. Our map-based cloning identified the responsible gene for the flo2 mutant, OsCEO1, which has no homology to any genes of known function. The OsCEO1 belongs to a novel conserved gene family and encodes a protein composed of 1,720 amino acid residues containing a TPR (tetratricopeptide repeat) motif, which is considered to mediate a protein-protein interaction. The yeast two-hybrid analysis raised an unknown protein showing homology to a late embryogenesis abundant protein and a putative basic helix-loop-helix protein as candidates for the direct interactor for OsCEO1, whereas no enzyme genes for the synthesis of storage substances were detected. The flo2 mutant exhibited reduced expression of several genes for putative regulatory proteins as well as many enzymes involved in storage starch and proteins. These results suggest that OsCEO1 is a superior conductor of the novel regulatory cascade of endosperm organogenesis and may have important roles in the response to high-temperature stress.
  • Comparative Enzymatic Analysis of Azoreductases from Bacillus sp B29
    Toshihiko Ooi, Takeshi Shibata, Ken'ichiro Matsumoto, Shinichi Kinoshita, Seiichi Taguchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73, 5, 1209, 1211, TAYLOR & FRANCIS LTD, 2009年05月, [査読有り]
    英語, 研究論文(学術雑誌), We cloned and expressed two genes encoding azoreductase homologes, AzrB and AzrC, from Bacillus sp. B29. Purified recombinant AzrB and AzrC were homodimers with 23kDa identical subunits, and were flavoproteins. NADH was preferred as electron donors for both azoreductases. The azoreductases showed optimal activities at 70 degrees C (AzrB) and 55 degrees C (AzrC), and retained activities up to 55 degrees C (AzrB) and 50 degrees C (AzrC) after incubation for 1 h. Other enzymatic properties, including the substrate specificities of both azoreductases, were also investigated.
  • Chimeric Enzyme Composed of Polyhydroxyalkanoate (PHA) Synthases from Ralstonia eutropha and Aeromonas caviae Enhances Production of PHAs in Recombinant Escherichia coli
    Ken'ichiro Matsumoto, Kazuma Takase, Yoko Yamamoto, Yoshiharu Doi, Seiichi Taguchi
    BIOMACROMOLECULES, 10, 4, 682, 685, AMER CHEMICAL SOC, 2009年04月, [査読有り]
    英語, 研究論文(学術雑誌), Chimeric enzymes composed of polyhydroxyalkanoate (PHA) synthases from Ralstonia eutropha (Cupriavidus necator) (PhaC(Re)) and Aeromonas caviae (PhaC(Ac)) were constructed. PhaC(Re) is known for its potent enzymatic activity among the characterized PHA synthases. PhaC(Ac) has broad substrate specificity and synthesizes short-chain-length (SCL)/medium-chain-length (MCL) PHA. We attempted to create chimeric enzymes inheriting both of the advantageous properties. Among eight chimeras, AcRe 12, with 26% of the N-terminal of PhaCA, and 74% of the C-terminal of PhaC(Re), exhibited comparable P(3-hydroxybutyrate) accumulation as parental enzymes in Escherichia coli JM109. Thus, AcRe12 was applied to SCL/MCL PHA production using E. coli LS5218 as the host. AcRe12 accumulated higher amount of PHA (50 wt %) than the parental enzymes. Furthermore, the PHA consisted of 2 mol % 3-hydroxyhexanoate as well as 3-hydroxybutyrate. Therefore, the chimeric PHA synthase, AcRe 12, inherited the character of both of the parental enzymes and thus exhibits improved enzymatic properties.
  • Microbial Production of Lactate-Enriched Poly[ (R)-lactate-co-(R)-3-hydroxybutyrate] with Novel Thermal Properties
    Miwa Yamada, Ken'ichiro Matsumoto, Takanori Nakai, Seiichi Taguchi
    BIOMACROMOLECULES, 10, 4, 677, 681, AMER CHEMICAL SOC, 2009年04月, [査読有り]
    英語, 研究論文(学術雑誌), Considerable enrichment of the lactate (LA) fraction in Poly [ (R)-LA-co-(R)-3-hydroxybutyrate (3HB)] has been achieved, reaching up to 47 mol % from the previous 6 mol % [S. Taguchi et al. Proc. Natl. Acad. Sci. U.S.A. 2008,105 (45), 17323-17327], when recombinant Escherichia coli W3110, harboring the LA-polymerizing enzyme gene together with three genes for supplying monomers, lactyl-coenzyme A (CoA), and 3-hydroxybutyryl-CoA, were grown on glucose under anaerobic conditions. The molecular weights of the copolymer were M(n) = 1.5 x 10(4) g/mol and M(w) = 2.0 x 10(4) g/mol, respectively. Notably, alkali ne-hydrolyzed product analysis revealed that only the (R)-enantiomer of LA was incorporated in the copolymer. Furthermore, the triad sequence of LA-LALA was clearly detected in the polymer chain based on NMR analysis. A remarkably contrasting melting temperature was observed between the copolymer obtained here P[ (R)-LA-co-(R)-3HB] (157 degrees C) and the chemically synthesized P[ (S)-LA-co-(R)-3HB] (105 degrees C) with similar monomer composition.
  • Production of Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate (PHA) Copolymer in the Plastid of Arabidopsis thaliana Using an Engineered 3-Ketoacyl-acyl Carrier Protein Synthase III
    Ken'ichiro Matsumoto, Takaaki Murata, Rina Nagao, Christopher T. Nomura, Satoshi Arai, Yuko Arai, Kazuma Takase, Hideo Nakashita, Seiichi Taguchi, Hiroaki Shimada
    BIOMACROMOLECULES, 10, 4, 686, 690, AMER CHEMICAL SOC, 2009年04月, [査読有り]
    英語, 研究論文(学術雑誌), Short-chain-length/medium-chain-length (SCL/MCL) polyhydroxyalkanoate (PHA) was produced in the plastids of Arabidopsis thaliana. Phe87Thr (F87T) mutated 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) from Escherichia coli, and Ser325Thr/Gln481Lys (ST/QK) mutated polyhydroxyalkanoate (PHA) synthase (PhaC1) from Joseudomonas sp. 61-3, along with the beta-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) from Ralstonia eutropha (Cupriavidus necator) genes were introduced into Arabidopsis. The transgenic Arabidopsis produced PHA copolymers composed of monomers consisting of 4-14 carbons. The introduction of the engineered PHA synthase resulted in a 10-fold increase in PHA content compared to plants expressing the wild-type PHA synthase. In addition, expression of the engineered fabH gene in the plastid led to an increase in the amount of the SCL monomer, 3-hydroxybutyrate, incorporated into PHA, and contributed to supply of MCL monomers for PHA production.
  • Dual production of poly (3-hydroxybutyrate) and glutamate using variable biotin concentrations in Corynebacterium glutamicum
    Sung-Jin Jo, Chean Ring Leong, Ken'ichiro Matsumoto, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 107, 4, 409, 411, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2009年04月, [査読有り]
    英語, 研究論文(学術雑誌), We previously synthesized poly(3-hydroxybutyrate) [P(3HB)] in recombinant Corynebacterium glutamicum, a prominent producer of amino acids. In this study, a two-step cultivation was established for the dual production of glutamate and P(3HB) due to the differences in the optimal concentration of biotin. Glutamate was extracellularly produced first under the biotin-limited condition of 0.3 mu g/L. Production was then shifted to P(3HB) by addition of biotin to a total concentration of 9 mu g/L. The final products obtained were 18 g/L glutamate and 36 wt% of P(3HB). (c) 2008, The Society for Biotechnology, Japan. All rights reserved.
  • Kinetic Analysis of Engineered Polyhydroxyalkanoate Synthases with Broad Substrate Specificity
    Ken'ichiro Matsumoto, Fumi Shozui, Yasuharu Satoh, Kenji Tajima, Masanobu Munekata, Seiichi Taguchi
    POLYMER JOURNAL, 41, 3, 237, 240, SOC POLYMER SCIENCE JAPAN, 2009年
    英語, 研究論文(学術雑誌)
  • A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme
    Seiichi Taguchi, Miwa Yamadaa, Ken'ichiro Matsumoto, Kenji Tajima, Yasuharu Satoh, Masanobu Munekata, Katsuhiro Ohno, Katsunori Kohda, Takashi Shimamura, Hiromi Kambe, Shusei Obata
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105, 45, 17323, 17327, NATL ACAD SCIENCES, 2008年11月, [査読有り]
    英語, 研究論文(学術雑誌), Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 x 10(5), as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.
  • FabG mediates polyhydroxyalkanoate production from both related and nonrelated carbon sources in recombinant Escherichia coli LS5218
    Christopher T. Nomura, Tomoyo Tanaka, Tenai E. Eguen, Alexandria S. Appah, Ken'ichiro Matsumoto, Seiichi Taguchi, C. Leo Ortiz, Yoshiharu Doi
    BIOTECHNOLOGY PROGRESS, 24, 2, 342, 351, WILEY-BLACKWELL, 2008年03月, [査読有り]
    英語, 研究論文(学術雑誌), Polyhydroxyalkanoates (PHAs) composed of a mixture of short-chain-length-medium-chain-length (SCL-MCL) hydroxyacyl monomers are biologically produced polyesters that have properties ranging from thermoplastic to elastomeric, dependent on the molar ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of properties and applications for SCL-MCL PHA copolymers, it is important to develop and characterize novel metabolic pathways for SCL-MCL PHA production. The current study shows that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes enhances the production of SCL-MCL PHA copolymer from both related and nonrelated carbon sources in Escherichia coli LS5218, indicating the flexibility of FabG as a monomer-supplying enzyme for biological PHA production.
  • Improvement of poly(3-hydroxybutyrate) [P(3HB)] production in Corynebacterium glutamicum by codon optimization, point mutation and gene dosage of P(3HB) biosynthetic genes
    Sung-Jin Jo, Ken'ichiro Matsumoto, Chean Ring Leong, Toshihiko Ooi, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 104, 6, 457, 463, SOC BIOSCIENCE BIOENGINEERING JAPAN, 2007年12月, [査読有り]
    英語, 研究論文(学術雑誌), In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon (phaCAB(Re)) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been applied to improve P(3HB) production in recombinant C glutamicum. One is a codon optimization of the N-terminal-coding region of the PHA synthase (PhaC(Re)) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaC,, with a modified gene encoding a mutation of Gly4Asp (G4D), which enhanced the production of PhaCRe and P(3HB) in Escherichia coli. The introduction of these engineered PHA synthase genes into C glutamicum enhanced the production of PhaC(Re) and P(3HB). Interestingly, we found that these gene modifications also caused increases in the concentration of the translation products of the genes encoding monomer-supplying enzymes, beta-ketothiolase (PhaA(Re)) and acetoacetyl-CoA reductase (PhaB(Re)). This finding prompted us to carry out a gene dosage of phaAB(Re) for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaAB(Re) with codon optimization. The molecular weight of P(3HB) was also increased by approximately 2-fold, as was P(3HB) content. Microscopic observation revealed that the volume of the cells accumulating P(31113) was increased by more than 4-fold compared with the non -P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.
  • Synthesis of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymers in peroxisome of the transgenic Arabidopsis thaliana harboring the PHA synthase gene from Pseudomonas sp 61-3
    Ken'ichiro Matsumoto, Yuko Arai, Rina Nagao, Takaaki Murata, Kazuma Takase, Hideo Nakashita, Seiichi Taguchi, Hiroaki Shimada, Yoshiharu Doi
    JOURNAL OF POLYMERS AND THE ENVIRONMENT, 14, 4, 369, 374, SPRINGER/PLENUM PUBLISHERS, 2006年10月, [査読有り]
    英語, 研究論文(学術雑誌), In this paper, the photosynthetic production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymers is reported. The wild-type and highly active doubly mutated PHA synthase 1 (S325T/Q481K, abbreviated ST/QK) genes from Pseudomonas sp. 61-3 were introduced into Arabidopsis thaliana. Peroxisome targeting signal 1 (PTS1) was used to target PHA synthases into the peroxisome to synthesize PHA from the intermediates of the beta-oxidation pathway. The transgenic Arabidopsis produced PHA copolymers consisting of 40-57 mol% 3-hydroxybutyrate, 21-49 mol% 3-hydroxyvalerate, 8-18 mol% 3-hydroxyhexanoate, and 2-8 mol% 3-hydroxyoctanoate. The maximum PHA contents were 220 mu g/g cell dry weight (cdw) in leaves, and 36 mu g/g cdw in stems, respectively. The expression of the ST/QK mutated PHA synthase in leaves gene did not lead to significant difference in PHA content and monomer composition of PHAs, compared to the wild-type PHA synthase gene, suggesting that the supply of monomers may be a rate-determining step of PHA biosynthesis in the peroxisome. However, in stems, there were significant differences dependent on whether the wild-type or ST/QK mutated PHA synthase was expressed. These results suggest that tissue-specific monomer availability is important in determining the final mol% composition of PHA copolymers produced by the peroxisome in plants.
  • In vivo and in vitro characterization of Ser477X mutations in polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp 61-3: Effects of beneficial mutations on enzymatic activity, substrate specificity, and molecular weight of PHA
    Ken'ichiro Matsumoto, Emi Aoki, Kazuma Takase, Yoshiharu Doi, Seiichi Taguchi
    BIOMACROMOLECULES, 7, 8, 2436, 2442, AMER CHEMICAL SOC, 2006年08月, [査読有り]
    英語, 研究論文(学術雑誌), Evolutionary engineered polyhydroxyalkanoate (PHA) synthases from Pseudomonas sp. 61-3 enhance PHA accumulation and enable the monomer composition of PHAs to be regulated. We characterized a newly screened Ser477Arg (S477R) mutant of PHA synthase by in vivo analyses of P(3-hydroxybutyrate) [P(3HB)] homopolymer and P(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] copolymer productions in the recombinants of Escherichia coli. The results indicated that the S477R mutation contributed to a shift in substrate specificity to smaller monomers containing a 3HB unit rather than to an enhancement in catalytic activity. Multiple mutations of S477R with other beneficial mutations, for example, Ser325Cys, exhibited synergistic effects on both an increase in PHA production (from 9 wt % to 21 wt %) and an alteration of substrate specificity. Furthermore, the effects of complete amino acid substitutions at position 477 were characterized in terms of in vivo PHA production and in vitro enzymatic activity. The five mutations, S477Ala(A)/Phe(F)/His(H)/Arg(R)/Tyr(Y), resulted in a shift in substrate specificity to smaller monomer units. The S477Gly(G) mutant greatly enhanced activity toward all different sizes of substrates with carbon numbers ranging from 4 to 12. These results indicated that the residue 477 contributes to both the catalytic activity and substrate specificity of PHA synthase. In recombinant E. coli, the S477A/F/G/H/R/Y mutations consistently led to increases (up to 6 times that of wild-type enzyme) in weight average molecular weights of P(3HB) homopolymers. On the basis of our studies, we created a structural feasibility accounting for the mutational effects on enzymatic activity and substrate specificity of PHA synthase.
  • Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae
    K Matsumoto, R Nagao, T Murata, Y Arai, T Kichise, H Nakashita, S Taguchi, H Shimada, Y Doi
    BIOMACROMOLECULES, 6, 4, 2126, 2130, AMER CHEMICAL SOC, 2005年07月, [査読有り]
    英語, 研究論文(学術雑誌), In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.
  • Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: Enhancement of PHA production and alteration of polymer molecular weight
    K Matsumoto, K Takase, E Aoki, Y Doi, S Taguchi
    BIOMACROMOLECULES, 6, 1, 99, 104, AMER CHEMICAL SOC, 2005年01月, [査読有り]
    英語, 研究論文(学術雑誌), In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1p,) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.
  • Alteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by in vitro evolution: in vivo and in vitro enzyme assays
    K Takase, K Matsumoto, S Taguchi, Y Doi
    BIOMACROMOLECULES, 5, 2, 480, 485, AMER CHEMICAL SOC, 2004年03月, [査読有り]
    英語, 研究論文(学術雑誌), In our previous study, in vitro evolution of type 11 polyhydroxyalkanoate (PHA) synthase (PhaC1(Ps))from Pseudomonas sp. 61-3 yielded eleven mutant enzymes capable of synthesizing homopolymer of (R)-3-hydroxybutyrate [P(3HB)] in recombinant Escherichia coli JM109. These recombinant strains were capable of accumulating up to approximately 400-fold more P(3HB) than strains expressing the wild-type enzyme. These mutations enhanced the ability of the enzyme to specifically incorporate the 3HB-coenzyme A (3HB-CoA) substrate or improved catalytic efficiency toward the various monomer substrates Of C-4 to C-12 (R)-3-hydroxyacyl-CoAs which can intrinsically be channeled by PhaC1(Ps), into P(3HB-co-3HA) copolymerization. In this study, beneficial amino acid substitutions of PhaC1(Ps) were analyzed based on the accumulation level and the monomer composition of P(3HB-co-3HA) copolymers generated by E. coli LS5218 [fadR601 atoC(Con)] harboring the monomer supplying enzyme genes. Substitutions of Set by Thr(Cys) at position 325 were found to lead to an increase in the total amount of P(3HB-co-3HA) accmumulated, whereas 3HB fractions in the P(3HB-co-3HA) copolymer were enriched by substitutions of Gln by Lys(Arg, Met) at position 481. This strongly suggests that amino acid substitutions at positions 325 and 481 are responsible for synthase activity and/or substrate chain-length specificity of PhaC1(Ps),. These in vivo results were supported by the in vitro results obtained from synthase activity assays using representative single and double mutants and synthetic substrates, (R,S)-3HB-CoA and (R,S)-3-hydroxydecanoyl-CoA. Notably, the position 481 was found to be a determinant for substrate chain-length specificity of PhaC1(Ps).
  • Biosynthesis of biodegradable polyester: Current research and future direction
    K Matsumoto, Y Doi
    JOURNAL OF SYNTHETIC ORGANIC CHEMISTRY JAPAN, 61, 5, 489, 495, SOC SYNTHETIC ORGANIC CHEM JPN, 2003年05月, [査読有り]
    日本語, 研究論文(学術雑誌), In last half century, petrochemical polymers have been developed, however the high stability of these polymers has led to an increase in the pollution of plastic waste. Biodegradable polymers are one Solution to this problem. Polyhydroxyalkanoates (PHA) are a family of polyesters synthesized by microbes and can be used as biodegradable thermoplastics which may substitute for petrochemical polymers. At present, there are two main reasons why PHAs are not used commercially. First, the production cost of PHA is too expensive. Second, the physical properties of PHA are relatively poor compared with those of petrochemical polymers. In order to address these problems, the metabolic pathway of PHA biosynthesis was altered by genetic engineering to improve both physical properties of PHA and efficiency of PHA production. Engineering of PHA polymerase encoding genes has generated mutant PHA polymerases which are capable of higher PHA accumulation. To further address the issue of PHA production costs, transgenic plants harboring PHA biosynthesis genes from bacteria were generated. These transgenic plants have the potential to produce PHA at low cost. Further research in these areas will make the goal of having PHA commercially available, achievable.
  • Biosynthesis of biodegradable polyesters from renewable carbon sources by recombinant bacteria
    S Taguchi, H Matsusaki, K Matsumoto, K Takase, K Taguchi, Y Doi
    POLYMER INTERNATIONAL, 51, 10, 899, 906, WILEY-BLACKWELL, 2002年10月
    英語, 研究論文(学術雑誌), Based on the metabolic pathways for polyhydroxyalkanoate (PHA) biosynthesis, we succeeded in establishing the recombinant Pseudomonas sp 61-3 strains that synthesize random copolyesters consisting of (R)-3-hydroxybutyrate (3HB) and (R)-medium-chain-length 3-hydroxyalkanoate (mcl-3HA) units, P(3HB-co-3HA), with very high 3HB compositions (up to 94 mol%) from glucose. The mechanical properties of P(94% 3HB-co-3HA) copolyester were very similar to those of low-density polyethylene. We carried out the molecular cloning and characterization of a PhaG(Ps) encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase of Pseudomonas sp 61-3. It was concluded that the PhaGPs gene product is involved in providing mcl-3HA-CoA from glucose in the original strain. Heterologous expression of the PhaGPs gene with the PhaC1(Ps) gene encoding PHA synthase from Pseudomonas sp 61-3 was performed in the PhbC(Re) negative mutant (PHB(-)4) of Ralstonia eutropha. The recombinant PHB-4 strain successfully produced PHA copolyesters consisting of 3HB and mcl-3HA units of 6-12 carbon atoms from sugars. The 3HB fraction in copolyesters was very high (95-97 mol%). The PHA content in the recombinant strain could further be increased by the additional introduction of the PhbAB(Re) genes from R eutropha encoding beta-ketothiolase and NADPH-dependent acetoacetyl-coenzyme A reductase. Moreover, we have established an in vivo assay system to analyze mutational effects of R eutropha synthase (PhbCRe) on the level of PHB accumulation in recombinant strains of Escherichia coli. The activity of the PhbCRe could be efficiently estimated using the in vivo system constructed here, and would be useful for in vitro evolution of PhbCRe. (C) 2002 Society of Chemical Industry.
  • Isolation and characterization of polyhydroxyalkanoates inclusions and their associated proteins in Pseudomonas sp 61-3
    K Matsumoto, H Matsusaki, K Taguchi, M Seki, Y Doi
    BIOMACROMOLECULES, 3, 4, 787, 792, AMER CHEMICAL SOC, 2002年07月, [査読有り]
    英語, 研究論文(学術雑誌), Two types of polyester inclusions of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] were isolated from crude extract of Pseudomonas sp. 61-3. Proteins associated with each inclusion were separated by SDS-PAGE. PHA synthase 1 (PhaC1(Ps)), PhaF(Ps), and PhaI(Ps) were identified from P(3HB-co-3HA) inclusions by N-terminal amino acid sequences analyses, as well as PHB synthase (PhbC(Ps)) and 24-kDa unknown protein were identified from P(3HB) inclusions. The structural genes of PhaF(Ps) and PhaI(Ps) were located downstream of the pha locus. The relative PHA/PHB synthase activities of each inclusion were measured for various 3-hydroxyacyl-coenzyme As of 4-12 carbon atoms. Direct atomic force microscopy observation of P(3HB) and P(3HB-co-3HA) inclusions demonstrated that the two types of inclusions had different morphologies.
  • Direct observation of polyhydroxyalkanoate chains by atomic force microscopy
    K Sudesh, Z Gan, K Matsumoto, Y Doi
    ULTRAMICROSCOPY, 91, 1-4, 157, 164, ELSEVIER SCIENCE BV, 2002年05月, [査読有り]
    英語, 研究論文(学術雑誌), Atomic force microscopy in the tapping mode was used to investigate aqueous acetone-treated polyhydroxyalkanoate (PHA) inclusions freshly isolated from a recombinant bacterium. The PHA is a copolymer containing about 95mol% 3-hydroxybutyrate units while the rests are units of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate. Polymer chains extending to several micrometers in length were observed on glass cover slips upon the evaporation of the aqueous acetone. The polymer chains seem to exist in the form of fibrillar aggregates. The height of the microfibrils was about 1 nm. Upon prolonged standing at ambient conditions, the microfibrils dissociated into finer strands of about 0.5 mn in height. The results suggest that biosynthesized PHA are stored in the inclusions in an amorphous state but with minimal chain entanglement. This is possible because the PHA chains exist in the form of fibrillar aggregates that may be the product of a special biosynthesis mechanism. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) copolymer from sugars by recombinant Ralstonia eutropha harboring the phaC1Ps and the phaGPs genes of Pseudomonas sp. 61-3
    K. Matsumoto, S. Nakae, K. Taguchi, H. Matsusaki, M. Seki, Y. Doi
    Biomacromolecules, 2, 3, 934, 939, 3, 2001年, [査読有り]
    英語, 研究論文(学術雑誌), Heterologous expression of the phaGPs and the phaCIPs genes encoding 3-hydroxyacyl acyl carrier protein-coenzyme A transacylase and polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp. 61-3, respectively, was performed in the phbCRe negative mutant, Ralstonia eutropha PHB-4. The recombinant strain of the R. eutropha PHB-4 produced PHA copolymers consisting of 3-hydroxybutyrate (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-3HA) units of 6-12 carbon atoms from sugars. The 3HB fraction in copolymers was very high (95-97 mol %). The PHA content in the recombinant strain could further be increased by the additional introduction of the phbABRe genes from R. eutropha encoding β-ketothiolase and NADPH-dependent acetoacetyl-coenzyme A reductase. Differential scanning calorimetry analysis of the PHA copolymers produced by the recombinant R. eutropha PHB-4 have indicated that the PHA is a random copolymer of 3HB and mcl-3HA units.
  • Cloning and characterization of the pseudomonas sp. 61-3 phaG gene involved in polyhydroxyalkanoate biosynthesis
    K. Matsumoto, H. Matsusaki, S. Taguchi, M. Seki, Y. Doi
    Biomacromolecules, 2, 1, 142, 147, 1, 2001年, [査読有り]
    英語, 研究論文(学術雑誌), Pseudomonas sp. 61-3 produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] random copolymer consisting of monomeric units of 4-12 carbon atoms from sugars. The phaGPs gene encoding (R)-3-hydroxyacyl-acyl carrier protein coenzyme A transferase was cloned from this strain, and homologous expression of this gene under the control of the lac or the native promoter was investigated. Additional copies of the phaGPs gene in Pseudomonas sp. 61-3 led to an increase in both the polyhydroxyalkanoate (PHA) content in the cells and the fraction of medium-chain-length 3HA units in PHA. Disruption of the chromosomal phaGPs gene resulted in an increase in the fraction of the 3HB unit in PHA. The site-directed mutagenesis of the phaGPs gene was carried out to investigate the role of a HX4D motif which has been proposed to be related to PhaG activity.

その他活動・業績

書籍等出版物

  • リアルタイムNMR法によるグリコール酸ベースブロック共重合体の酵素合成機構の解析
    柳川 謙吾, 梶川 彩香, 榊原 早也果, 久米田 博之, 冨田 宏矢, 松本 謙一郎
    NPO法人 環境バイオテクノロジー学会, 2023年, 日本語
  • Poly(lactic acid): Synthesis, Structures, Properties, Processing, Applications, and End of Life, 2nd Edition               
    Tadahisa Iwata, Hideki Abe, Yoshihiro Kikkawa, Ken’ichiro Matsumoto, Part of Chapter 22
    Wiley, 2022年06月, 9781119767442, [分担執筆]

講演・口頭発表等

  • 長主鎖モノマーを含む非天然型ポリヒドロキシアルカン酸の律速段階解析に基づく代謝工学的改変               
    田近誠也, 蜂須賀真一, 菊川寛史, 松本謙一郎
    日本栄養・食糧学会北海道支部大会講演要旨集, 2023年08月05日, 日本語, ポスター発表
    2023年08月05日 - 2023年08月05日
  • ブロック配列型微生物ポリエステルの酵素分解系の構築               
    伊関叶互, 蜂須賀真一, 菊川寛史, 松本謙一郎
    日本栄養・食糧学会北海道支部大会講演要旨集, 2023年08月05日, 日本語, ポスター発表
    2023年08月05日 - 2023年08月05日
  • ポリ乳酸セグメントを含む新規ブロック共重合体の微生物合成と物性制御               
    今井昂渡, 蜂須賀真一, 菊川寛史, 冨田宏矢, 松本謙一郎
    日本栄養・食糧学会北海道支部大会講演要旨集, 2023年08月05日, 日本語, ポスター発表
    2023年08月05日 - 2023年08月05日
  • ポリヒドロキシアルカン酸生合成過程におけるエネルギー代謝動態観察               
    富士航至, 梶川彩香, 冨田宏矢, 蜂須賀真一, 菊川寛史, 今村博臣, 松本謙一郎
    日本栄養・食糧学会北海道支部大会講演要旨集, 2023年08月05日, 日本語, ポスター発表
    2023年08月05日 - 2023年08月05日
  • Versatile aliphatic polyester biosynthesis system for producing random and block copolymers using engineered enzymes               
    Ken'ichiro Matsumoto
    ETH-Hokudai Joint symposium, 2023年03月08日, 英語, シンポジウム・ワークショップパネル(指名)
    [招待講演]
  • ポリヒドロキシアルカン酸合成系を利用したスクリーニングによるRuBisCOの高活性多重変異体の獲得               
    芥川柚月, 渡邉秀平, 藤原悠暉, 蜂須賀真一, 菊川寛史, 蘆田弘樹, 松本謙一郎
    日本栄養・食糧学会北海道支部大会講演要旨集, 2023年
    2023年 - 2023年
  • 粘弾性PHAブロック共重合体の 合成と生分解               
    松本謙一郎
    生物工学会2022年年次大会シンポジウム, 2022年10月18日, 日本語, 口頭発表(招待・特別)
    2022年10月17日 - 2022年10月20日, [招待講演]
  • 微生物の中にプラスチック工場を作る               
    北鍾総会, 2022年10月08日, 日本語, 公開講演,セミナー,チュートリアル,講習,講義等
    [招待講演]
  • Biosynthesis and properties of sequence-regulated artificial polyhydroxyalkanoates               
    Ken'ichiro Matsumoto
    International Symposium on Biological Polymers, 2022年09月14日, 英語, 口頭発表(基調)
    [招待講演]
  • 進化分子工学による新規バイオ プラスチックの微生物合成               
    松本謙一郎
    トランスフォーマティブ化学生命融合研究大学院プログラム, 2022年04月27日, 日本語, 公開講演,セミナー,チュートリアル,講習,講義等
    [招待講演]
  • 組換えRalstonia eutrophaによるポリヒドロキシアルカン酸ブロック共重合体の生合成               
    石原静流, 折田和泉, 松本謙一郎, 福居俊昭
    日本農芸化学会大会講演要旨集(Web), 2022年
    2022年 - 2022年
  • 新規生分解性プラスチックの生産に向けた配列制御型共重合が可能なRalsotonia eutropha改変株の開発               
    石原静流, 折田和泉, 松本謙一郎, 福居俊昭
    日本生物工学会大会講演要旨集, 2022年
    2022年 - 2022年
  • 粘弾性PHAブロック共重合体の合成と生分解               
    松本謙一郎
    日本生物工学会大会講演要旨集, 2022年
    2022年 - 2022年
  • 改変型重合酵素を用いたグリコール酸ベースポリマー合成のin vitro解析               
    山本一輝, 柳川謙吾, 冨田宏矢, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • 改変型重合酵素PhaCARによる軟質性グリコール酸ポリマーの生合成               
    田中聖也, 佐藤圭悟, 冨田宏矢, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • 中鎖モノマー認識に向けたポリヒドロキシアルカン酸合成酵素の基質特異性の改変               
    向井太一, HIEN Phan Thi, 冨田宏矢, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • (R)-2-ヒドロキシブタン酸ベースポリマー分解菌の探索               
    井口綾実, 冨田宏矢, 大井俊彦, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • 組み換え大腸菌を利用したポリヒドロキシアルカン酸生合成系スクリーニングによる光合成細菌由来RuBisCO高機能変異体の獲得               
    渡邉秀平, 藤原悠輝, 永田暁洋, 工藤悠希, 松本直己, 齋藤樹里, 喜多幸, 冨田宏矢, 蘆田弘樹, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • 配列制御型PHA酸合成酵素の基質による配列制御性の解析               
    田近誠也, 佐藤圭悟, 冨田宏矢, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • 配列制御型ポリヒドロキシアルカン酸合成酵素による新規ブロック共重合体の生合成および物性解析               
    川上智也, 冨田宏矢, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • 機能改変型ポリヒドロキシアルカン酸合成酵素のモノマー配列制御の特性解析               
    富士航至, HIEN Phan Thi, 細江有美, MUREEN Guex, 友居昌慶, 冨田宏矢, MANFRED Zinn, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • ポリヒドロキシアルカン酸生合成系を利用したスクリーニングによる光合成細菌由来RuBisCO高機能変異体の創出               
    渡邉秀平, 藤原悠暉, 永田暁洋, 工藤悠希, 松本直己, 齋藤樹里, 喜多幸, 冨田宏矢, 蘆田弘樹, 松本謙一郎
    日本生物工学会大会講演要旨集, 2022年
    2022年 - 2022年
  • 配列制御型ポリエステル合成酵素を用いたエラストマー様共重合体の生合成               
    今井昂渡, 影山友基, 冨田宏矢, 磯野拓也, 佐藤敏文, 松本謙一郎
    日本農芸化学会東北支部大会プログラム・講演要旨集, 2022年
    2022年 - 2022年
  • モノマー配列が制御された生分解性ポリエステルの微生物合成               
    松本謙一郎
    日本高分子学会講演会ポリマーフロンティア21, 2021年10月15日, 日本語, 口頭発表(招待・特別)
    2021年10月15日 - 2021年10月15日, [招待講演]
  • 微生物分解性プラスチックの開発と活用法               
    松本謙一郎
    北海道経済連合会 環境・エネルギーセミナー, 2021年01月18日, 日本語, 公開講演,セミナー,チュートリアル,講習,講義等
    [招待講演]
  • 微生物産生ブロック共重合体ポリマーの構造機能相関の解析               
    佐藤 圭悟、影山 友基、藤井 隆之輔、松本 謙一郎
    日本農芸化学会2020年度大会, 2020年03月27日, 日本語, 口頭発表(一般)
  • 新規配列制御型グリコール酸ポリマーの生合成と構造解析               
    梶川彩香、榊原早也果、荒井修造、大井俊彦、堀千明、松本謙一郎
    日本農芸化学会2020年度大会, 2020年03月27日, 日本語, 口頭発表(一般)
  • ブロックポリヒドロキシアルカン酸の微生物合成と構 造・物性解析               
    松本 謙一郎, 影山 友基, 堀 千明, 大井 俊彦, 磯野 拓也, 佐藤 敏文, 田口 精一
    第68回高分子討論会, 2019年09月25日, 日本語, 口頭発表(一般)
  • Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) 反応を経由したグリコール酸ポリマー生合成系とその応用               
    大潟祥梧、永田暁洋、齋藤樹理、横尾俊憲、工藤悠希、堀千明、大井俊彦、田口精一、松本謙一郎
    第71回日本生物工学会大会, 2019年09月16日, 日本語, 口頭発表(一般)
  • Ribulose 1,5-bisphosphate carboxylase/oxygenase(RubisCO)反応を経由したグリコール酸ポリマー生合成系構築とその応用               
    大潟祥梧, 永田暁洋, 齋藤樹理, 横尾俊憲, 工藤悠希, 堀千明, 大井俊彦, 田口精一, 松本謙一郎
    日本生物工学会大会講演要旨集, 2019年
    2019年 - 2019年
  • 非天然ポリエステル生合成系の開発と配列制御型ポリマーへの展開               
    松本謙一郎, 松本謙一郎, 松本謙一郎, 堀千明, 大井俊彦, 大井俊彦, 磯野拓也, 佐藤敏文, 石井大輔, 石井大輔, 岩田忠久, 岩田忠久, 田口精一, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2019年
    2019年 - 2019年
  • ルビスコ経路を利用した組換え大腸菌におけるグリコール酸ポリマー生合成系の構築               
    永田暁洋, 齋藤樹理, 横尾俊憲, 工藤悠希, 堀千明, 大井俊彦, 田口精一, 松本謙一郎, 松本謙一郎
    日本農芸化学会大会講演要旨集(Web), 2019年
    2019年 - 2019年
  • X線繊維回折法によるポリ[ (R)-2-ヒドロキシブタン酸]の結晶構造解析               
    丸林弘典, 金子はるひ, 木村聡, 木村聡, 松本謙一郎, 松本謙一郎, 田口精一, 田口精一, 岩田忠久, 岩田忠久, 岩田忠久
    繊維学会予稿集, 2018年
    2018年 - 2018年
  • オリゴ酪酸およびオリゴ乳酸とポリマー分解酵素間の相互作用の分子動力学シミュレーションを用いた検討               
    織田耕平, 鎌田佑, 松本謙一郎, 大井俊彦, 田口精一, 佐藤信一郎
    化学系学協会北海道支部冬季研究発表会(Web), 2018年
    2018年 - 2018年
  • ポリ[ (R)-2-ヒドロキシブタン酸]の結晶構造解析               
    丸林弘典, 金子はるひ, 木村聡, 木村聡, 松本謙一郎, 松本謙一郎, 田口精一, 田口精一, 岩田忠久, 岩田忠久, 岩田忠久
    繊維学会予稿集, 2018年
    2018年 - 2018年
  • 組換え大腸菌による乳酸ベースオリゴマーの分泌生産と高分子合成への応用               
    松本謙一郎, UTSUNOMIA Camila, 堀千明, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2018年
    2018年 - 2018年
  • 転写因子欠失株を用いた乳酸ベースポリマー生産大腸菌のリモデリング               
    門屋亨介, 門屋亨介, 児玉悠, 松本謙一郎, 大井俊彦, 大井俊彦, 田口精一, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2018年
    2018年 - 2018年
  • 透明性を有する新規乳酸ベースポリマーの生合成               
    後藤早希, 龍野菜々美, 松本謙一郎, 阿部英喜, 田口精一, 田中賢二, 松崎弘美, 松崎弘美
    日本農芸化学会大会講演要旨集(Web), 2018年
    2018年 - 2018年
  • モノマー組成制御された乳酸ベースオリゴマーの分泌生産               
    廣江綾香, 廣江綾香, 増永凛, 佐藤千絵, 松本謙一郎, 田口精一, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2018年
    2018年 - 2018年
  • グルコース/キシロース切替え流加法による乳酸ベースポリマー生産組換え大腸菌の高密度培養               
    山崎貴史, RIBORDY Greg, 堀千明, 堀千明, 松本謙一郎, 松本謙一郎, 大井俊彦, 大井俊彦, ZINN Manfred, 田口精一, 田口精一
    日本生物工学会大会講演要旨集, 2018年
    2018年 - 2018年
  • 植物バイオマス原料から微生物合成する多元ポリ乳酸の部材化               
    田口精一, 田口精一, 田口精一, 廣江綾香, 廣江綾香, 石井大輔, 石井大輔, 大井俊彦, 大井俊彦, 松本謙一郎, 松本謙一郎, 岩田忠久, 岩田忠久
    高分子学会予稿集(CD-ROM), 2018年
    2018年 - 2018年
  • 動的配列制御に基づくブロック共重合ポリエステル生合成系の発見とポリマー構造解析
    松本謙一郎, 藤井隆之輔, 堀千明, 大井俊彦, 磯野拓也, 佐藤敏文, 田口精一
    高分子学会予稿集(CD-ROM), 2018年, 日本語
    2018年 - 2018年
  • ポリ[ (R)‐2‐ヒドロキシブチレート]に対する結晶構造解析の試み               
    牧野恭平, 木村聡, 竹村彰夫, 加部泰三, 岩田忠久, 松本謙一郎, 田口精一, 引間孝明, 高田昌樹
    繊維学会予稿集, 2015年06月08日, 日本語
  • 様々な新規微生物産性ポリエステルの熱物性と光学特性               
    加部泰三, 壇上隆寛, 岩田忠久, 松本謙一郎, 田口精一, 柘植丈晴, 信川省吾, 山口正之
    高分子学会予稿集(CD-ROM), 2015年05月12日, 日本語
  • ポリ[ (R)‐2‐ヒドロキシブチレート]の結晶構造解析               
    牧野恭平, 木村聡, 竹村彰夫, 加部泰三, 岩田忠久, 松本謙一郎, 田口精一, 引間孝明, 高田昌樹
    高分子学会予稿集(CD-ROM), 2015年05月12日, 日本語
  • 組換え大腸菌による糖からの生分解性共重合ポリエステルの生合成               
    外村彩夏, 西村綾乃, 後藤早希, 脇田和, 田口精一, 松本謙一郎, 柘植丈治, 田中賢二, 松崎弘美
    日本農芸化学会大会講演要旨集(Web), 2015年03月05日, 日本語
  • 組換え大腸菌を用いた高光学活性(R)‐3‐ヒドロキシブタン酸の生産におけるCoA転移酵素高活性変異体の効果               
    三吉大地, 瀬戸望, 松本謙一郎, 大井俊彦, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2015年03月05日, 日本語
  • 組換え大腸菌を利用した高光学純度(S)‐3‐ヒドロキシブタン酸の生産               
    瀬戸望, 大井俊彦, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2015年03月05日, 日本語
  • 耐酸性乳酸菌Lactobacillus acetotolerans HTの乳酸脱水素酵素遺伝子のクローニングと生分解性共重合ポリエステルの生合成               
    後藤早希, 外村彩夏, 田口精一, 松本謙一郎, 田中賢二, 松崎弘美
    日本農芸化学会大会講演要旨集(Web), 2015年03月05日, 日本語
  • 大腸菌転写制御因子欠失株を用いたP(LA‐co‐3HB)の高生産化               
    児玉悠, 門屋亨介, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2015年03月05日, 日本語
  • 組換え大腸菌によるグリコール酸ベースポリマー合成とポリマーの物性評価               
    平出幸和, 斯波哲史, SUN Jian, 大井俊彦, 田口精一, 松本謙一郎
    日本農芸化学会大会講演要旨集(Web), 2015年03月05日, 日本語
  • 組み換え体大腸菌で生産する資源循環型微生物ポリマーの高生産化に関与する新規遺伝子の探索               
    門屋亨介, 松本謙一郎, 大井俊彦, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2015年03月05日, 日本語
  • 組換え大腸菌を用いたグリコール酸ベースポリマーの生合成と酵素分解性               
    平出幸和, 斯波哲史, SU Jian, 大井俊彦, 田口精一, 松本謙一郎
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • 生合成乳酸ベースポリマーの高生産化を目指した宿主大腸菌の遺伝子改変               
    児玉悠, 門屋亨介, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • 進化工学的手法によるポリヒドロキシアルカン酸合成酵素の乳酸重合能力の強化               
    青木駿介, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • Lactobacillus acetotolerans HTの乳酸脱水素酵素遺伝子を利用した乳酸ユニットを含む生分解性プラスチックの生合成               
    後藤早希, 佐藤美咲, 田中賢二, 松本謙一郎, 田口精一, 松崎弘美
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • 乳酸ポリマー合成コリネ菌の代謝解析に基づく高生産化               
    飛谷康太, 笹森哲弥, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • グリコール酸ポリマーの生合成経路の構築               
    横尾俊憲, 平出幸和, 田口精一, 松本謙一郎
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • 二酸化炭素からプラスチックを合成する人工生命システムの創製―最短の合成ルートは?―               
    松本謙一郎, 横山俊憲, 大井俊彦, 田口精一
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • ゲノム工学的手法を用いた微生物ポリマー高生産化宿主の創成               
    門屋亨介, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • 高分子量2‐ヒドロキシブタン酸ベースポリマーの生合成および物性解析               
    高谷真宏, 大場貴史, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2014年08月05日, 日本語
  • 微生物産生ポリ[ (R)‐ラクテート‐co‐(R)‐2‐ヒドロキシブチレート]の結晶性および熱的性質               
    加部泰三, 岩田忠久, 松本謙一郎, 田口精一, 丸林弘典, 引間孝明, 高田昌樹
    高分子学会予稿集(CD-ROM), 2014年05月09日, 日本語
  • 植物バイオマス由来の単糖混合物を炭素源としたバイオポリエステルの生産               
    大井俊彦, 佐々木勝平, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2014年03月05日, 日本語
  • 微生物を利用したキラルP(2‐ヒドロキシ酪酸)の合成とポリマー解析               
    三宅政裕, 松本謙一郎, 寺井彩月, 加部泰三, 岩田忠久, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2014年03月05日, 日本語
  • バイオマスから高効率で乳酸プラスチックを生産する微生物工場の開発とポリマー物性評価               
    松本謙一郎, 前田理久, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2014年03月05日, 日本語
  • ポリ[ (R)‐2‐ヒドロキシブチレート]の基礎物性評価と構造解析の試み               
    牧野恭平, 加部泰三, 竹村彰夫, 岩田忠久, 田口精一, 松本謙一郎, 引間孝明, 高田昌樹, 丸林弘典
    繊維学会予稿集, 2014年, 日本語
  • 組換えコリネ型細菌によるポリ乳酸様ポリマー生産の増強               
    飛谷康太, 笹森哲弥, 松本謙一郎, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2013年11月27日, 日本語
  • 抗菌スペクトルを改変した抗菌ペプチド「アピデシン」特殊変異体の創成とその抗菌活性               
    山崎蔵亨, 岩村雄太, 松本謙一郎, 橋本茂樹, 大井俊彦, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2013年11月27日, 日本語
  • コリネ菌の細胞表層提示技術を用いたデンプンからの乳酸様ポリマーの生産               
    門屋亨介, SONG Yuyang, 飛谷康太, 松本謙一郎, 田中勉, 近藤昭彦, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2013年11月27日, 日本語
  • 微生物重合系に基づく乳酸ポリマーからの拡張               
    田口精一, 松本謙一郎
    高分子学会予稿集(CD-ROM), 2013年08月28日, 日本語
  • 不飽和モノマーを導入した2‐ヒドロキシブタン酸ベースポリマーの生合成とその応用               
    CHOI Yoongyoo, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2013年08月25日, 日本語
  • ポリヒドロキシアルカン酸重合酵素の機能改変によるポリマー構造制御               
    松本謙一郎, 越智杏奈, 大場貴史, 高谷真宏, 田口精一
    日本生物工学会大会講演要旨集, 2013年08月25日, 日本語
  • 植物バイオマスを用いた組換え大腸菌による共重合ポリエステルの生産               
    佐々木勝平, 大井俊彦, NDUKO John, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2013年08月25日, 日本語
  • デンプンを炭素源に用いたポリ乳酸様ポリマーの合成               
    門屋亨介, SONG Yuyang, 飛谷康太, 松本謙一郎, 田中勉, 近藤昭彦, 田口精一
    日本生物工学会大会講演要旨集, 2013年08月25日, 日本語
  • 糖を利用した組換え大腸菌内でのグリコール酸ベースポリマーの生産               
    斯波哲史, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2013年08月25日, 日本語
  • 非可食性植物バイオマスからのP(3HB)の微生物生産               
    大井俊彦, 佐々木勝平, NDUKO John, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2013年08月25日, 日本語
  • 2‐ヒドロキシブタン酸ベースポリマーの微生物合成とその物性解析               
    三宅政裕, 寺井彩月, 松本謙一郎, 加部泰三, 岩田忠久, 田口精一
    日本生物工学会大会講演要旨集, 2013年08月25日, 日本語
  • Corynebacterium glutamicumをプラットフォームとしたポリ乳酸様ポリマー合成系の効率化のための代謝工学および培養工学的アプローチ               
    飛谷康太, SONG Yuyang, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2013年03月05日, 日本語
  • 改変型重合酵素による2‐ヒドロキシブタン酸ベースポリマーの生合成と物性解析               
    青木駿介, 大場貴史, 越智杏奈, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2013年03月05日, 日本語
  • 動力学的解析および立体構造に基づいた進化型アセトアセチル‐CoAレダクターゼの高活性化メカニズムの解明               
    渡辺剛志, 松本謙一郎, 本橋廉, 池田弘二, 田中良和, MIN Yao, 田中勲, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2013年03月05日, 日本語
  • 微生物ポリエステル合成系酵素の機能改変と多元ポリ乳酸の創製               
    松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2013年03月05日, 日本語
  • PLAを蓄積した大腸菌の透過型電子顕微鏡観察               
    若林愛子, 佐藤道夫, 松本謙一郎, 田口精一, 前田理久
    日本顕微鏡学会関東支部講演会予稿集, 2013年03月05日, 日本語
  • PhaB高活性体を用いた光学活性(R)‐3‐ヒドロキシブタン酸生産の向上               
    大井俊彦, 本間以祝, 渡辺剛志, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2013年03月05日, 日本語
  • Ralstonia eutropha由来PHA重合酵素の改変による2‐ヒドロキシブタン酸重合能力の強化               
    越智杏奈, 大場貴史, 坂井浩平, 松本謙一郎, 柘植丈治, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2013年03月05日, 日本語
  • Ralstonia eutropha由来ポリヒドロキシアルカン酸重合酵素の改変によるClass I乳酸重合酵素の創出               
    越智杏奈, 大場貴史, 坂井浩平, 松本謙一郎, 柘植丈治, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2012年11月01日, 日本語
  • 進化工学的改変による高活性PhaBの速度論的解析とPHB生産性の向上               
    渡辺剛志, 宋育陽, 松本謙一郎, 大井俊彦, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2012年11月01日, 日本語
  • セルロース系糖質バイオマスを原料とした高乳酸分率ポリマーの効率的生産               
    佐々木勝平, NDUKO John Masani, 松本謙一郎, 大井俊彦, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2012年11月01日, 日本語
  • 可変領域への変異導入とその組み合わせによる抗菌ペプチド「アピデシン」の抗菌スペクトル改変               
    三宅政裕, 岩村雄太, 松本謙一郎, 橋本茂樹, 大井俊彦, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2012年11月01日, 日本語
  • 糖質バイオマスから多様なポリエステルを生産するコリネ菌微生物工場の開発               
    SONG Yuyang, 松本謙一郎, 大井俊彦, 田中勉, 近藤昭彦, 田口精一
    日本生物工学会大会講演要旨集, 2012年09月25日, 日本語
  • 2‐ヒドロキシブタン酸ベース新奇バイオプラスチックの微生物合成と物性解析               
    寺井彩月, 石山絢子, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2012年09月25日, 日本語
  • CoA転移酵素を用いた大腸菌による3‐ヒドロキシブタン酸の生産               
    大井俊彦, 松本謙一郎, 大慶岳洋, 本間以祝, 田口精一
    日本生物工学会大会講演要旨集, 2012年09月25日, 日本語
  • 乳酸重合活性を有するClass Iポリヒドロキシアルカン酸(PHA)重合酵素の創出               
    大場貴史, 越智杏奈, 坂井浩平, 松本謙一郎, 柘植丈治, 田口精一
    日本生物工学会大会講演要旨集, 2012年09月25日, 日本語
  • Microbial Plastic Factory : ポリ乳酸から多元ポリ乳酸の時代へ(バイオリファイナリーの今,そして未来,<特集>バイオ技術10年の軌跡,創立90周年記念特別企画)               
    松本 謙一郎, 田口 精一
    生物工学会誌 : seibutsu-kogaku kaishi, 2012年07月25日, 日本語
  • 抗菌ペプチド「アピデシン」の可変領域への変異導入とその抗菌活性への影響               
    三宅政裕, 岩村雄太, 松本謙一郎, 橋本茂樹, 大井俊彦, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2012年03月05日, 日本語
  • 進化工学による新規乳酸重合酵素の探索               
    越智杏奈, 大場貴史, 坂井浩平, 松本謙一郎, 柘植丈治, 田口精一
    日本農芸化学会大会講演要旨集(Web), 2012年03月05日, 日本語
  • 進化工学的改変により得られたアセトアセチル‐CoAレダクターゼ高活性変異体の解析               
    渡辺剛志, 早坂知士, 池田弘二, 本橋廉, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2011年08月25日, 日本語
  • 進化型PhaBによる組換えタバコにおけるPHB生産性の増強               
    大場貴史, 江口綾, 森本健二郎, 本橋廉, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2011年08月25日, 日本語
  • 組換え大腸菌による乳酸およびグリコール酸ベース新奇バイオプラスチックの生合成               
    石山絢子, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2011年08月25日, 日本語
  • 進化工学的手法による高乳酸分率のポリ(乳酸‐co‐3‐ヒドロキシブタン酸)が合成可能な乳酸重合酵素の探索               
    寺井彩月, 正瑞文, 松本謙一郎, 大井俊彦, 田口精一
    日本生物工学会大会講演要旨集, 2011年08月25日, 日本語
  • 真菌に対する抗菌ペプチドThanatin誘導体の作用評価               
    大島雄, 斉藤洵己, 橋本茂樹, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2011年08月25日, 日本語
  • 生体触媒の連携が実現する"一気通貫型"の乳酸ポリマー生合成プロセス : 乳酸重合酵素の発見から物性解析まで               
    松本 謙一郎, 田口 精一
    高分子論文集, 2011年05月25日, 日本語
  • 高乳酸分率のポリ(乳酸‐co‐3‐ヒドロキシブタン酸)[P(LA‐co‐3HB)]が合成可能な乳酸重合酵素の探索               
    寺井彩月, 正瑞文, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集, 2011年03月05日, 日本語
  • 高活性化PhaBの導入による組換えタバコでのPHB生産性の向上               
    大場貴史, 江口綾, 森本健二郎, 本橋廉, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集, 2011年03月05日, 日本語
  • 抗菌ペプチド「アピデシン」のN末端領域分子デザインによる高活性変異体の作製と抗菌スペクトル評価               
    若林雅明, 岩村雄太, 松本謙一郎, 橋本茂樹, 大井俊彦, 田口精一
    日本農芸化学会大会講演要旨集, 2011年03月05日, 日本語
  • 乳酸ベース共重合体P(lactate‐co‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)の生合成および物性評価               
    孫健, 本橋廉, 正瑞文, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集, 2011年03月05日, 日本語
  • PHA生合成系におけるモノマー供給酵素アセトアセチル‐CoAレダクターゼの高活性変異体の酵素反応解析               
    渡辺剛志, 早坂知士, 池田弘二, 本橋廉, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集, 2011年03月05日, 日本語
  • 抗菌ペプチドThanatin誘導体の酵母様真菌に対する抗菌活性               
    大島雄, 斉藤洵己, 橋本茂樹, 松本謙一郎, 田口精一
    日本農芸化学会大会講演要旨集, 2011年03月05日, 日本語
  • Poly(lactate‐co‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)の生合成および物性評価               
    渡辺剛志, 本橋廉, 松本謙一郎, 田口精一
    高分子学会北海道支部研究発表会講演要旨集, 2011年02月01日, 日本語
  • グルコースを炭素源とした乳酸ベースポリマーの微生物生産               
    合田葵, 宋育陽, 松本謙一郎, 山田美和, 田口精一
    高分子学会北海道支部研究発表会講演要旨集, 2011年02月01日, 日本語
  • アミノ酸残基置換による抗菌ペプチド「アピデシン」の抗菌スペクトルの改変               
    岩村雄太, 山形享子, 若林雅明, 松本謙一郎, 橋本茂樹, 大井俊彦, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2011年, 日本語
  • 高乳酸分率ポリ(乳酸‐co‐3‐ヒドロキシブタン酸)合成のための乳酸重合酵素の進化工学的改変               
    坂井浩平, 寺井彩月, 正瑞文, 越智杏奈, 松本謙一郎, 大井俊彦, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2011年, 日本語
  • 乳酸およびグリコール酸ベース新奇バイオプラスチックの微生物生産               
    斯波哲史, 石山絢子, 松本謙一郎, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2011年, 日本語
  • 組換え大腸菌を利用した(R)‐3‐ヒドロキシ酪酸の生産               
    大慶岳洋, 本間以祝, 松本謙一郎, 渡辺剛志, 大井俊彦, 田口精一
    日本農芸化学会北海道支部講演会講演要旨, 2011年, 日本語
  • Pseudomonas sp.61‐3由来PHA重合酵素の新規高活性変異体の取得と機能解析               
    坂井浩平, 正瑞文, 松本謙一郎, 高瀬和真, 田口精一
    日本生物工学会大会講演要旨集, 2010年09月25日, 日本語
  • 抗菌ペプチドThanatinのシステイン部位に導入されたアルキル基の側鎖長が抗菌活性に及ぼす影響               
    斉藤洵己, 折笠善丈, 橋本茂樹, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2010年09月25日, 日本語
  • N末分子デザインによる抗菌ペプチド「アピデシン」の高活性体創出               
    山形享子, 橋本茂樹, 折笠善丈, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2010年09月25日, 日本語
  • 組換え大腸菌を用いたポリ(乳酸‐co‐3‐ヒドロキシブタン酸‐co‐3‐ヒドロキシヘキサン酸)の生産               
    正瑞文, 本橋廉, 山田美和, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2010年09月25日, 日本語
  • ポリヒドロキシアルカン酸生合成系のモノマー供給酵素acetoacetyl‐CoA reductaseの進化工学的改変               
    早坂知士, 本橋廉, 正瑞文, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2010年09月25日, 日本語
  • 新奇かつデザイナブルなバイオポリエステル合成のための重合酵素の機能改変               
    松本謙一郎, 田口精一
    日本蛋白質科学会年会プログラム・要旨集, 2010年05月15日, 日本語
  • 3‐ヒドロキシブタン酸ベースバイオポリマーの組成制御を目指したモノマー供給酵素の進化工学的改変               
    正瑞文, 本橋廉, 松本謙一郎, 大井俊彦, 田口精一
    日本農芸化学会大会講演要旨集, 2010年03月05日, 日本語
  • ルテニウム触媒によるセルロース糖化物を用いたポリヒドロキシブタン酸(PHB)の微生物生産               
    池田弘二, 松本謙一郎, 駒野谷将, 小林広和, 大井俊彦, 福岡淳, 田口精一
    日本農芸化学会大会講演要旨集, 2010年03月05日, 日本語
  • 乳酸重合酵素への新規変異導入による乳酸コポリマーの分率制御               
    山田美和, 清水康多郎, 浦本周, 中井孝憲, 松本謙一郎, 大井俊彦, 田口精一
    日本農芸化学会大会講演要旨集, 2010年03月05日, 日本語
  • ジャーファーメンターを用いた乳酸ベースポリマーの微生物発酵生産               
    浦本周, 山田美和, 松本謙一郎, 阿部英喜, 大井俊彦, 田口精一
    日本農芸化学会大会講演要旨集, 2010年03月05日, 日本語
  • 3‐ヒドロキシ吉草酸ユニットを含む新規乳酸ベース3元共重合体の微生物合成               
    正瑞文, 中井孝憲, 山田美和, 松本謙一郎, 田口精一
    高分子学会北海道支部研究発表会講演要旨集, 2010年01月26日, 日本語
  • 立体選択的乳酸ベースポリマーのバイオ合成と構造解析               
    山田美和, 中井孝憲, 清水康多郎, 浦本周, 松本謙一郎, 大井俊彦, 田口精一
    高分子学会北海道支部研究発表会講演要旨集, 2010年01月26日, 日本語
  • 担持ルテニウム触媒によるセルロース糖化反応を利用したポリヒドロキシブタン酸(PHB)の微生物生産               
    松本謙一郎, 池田弘二, 駒野谷将, 小林広和, 福岡淳, 田口精一
    日本生物工学会シンポジウム講演要旨集, 2010年, 日本語
  • 乳酸ポリマー生産用微生物工場の開発               
    田口精一, 山田美和, 松本謙一郎, 中井孝憲, 清水康多郎
    高分子学会予稿集(CD-ROM), 2009年09月01日, 日本語
  • 組換え大腸菌を用いた新規乳酸ベース3元共重合体の生産               
    正瑞文, 中井孝憲, 山田美和, 松本謙一郎, 田口精一
    日本生物工学会大会講演要旨集, 2009年08月25日, 日本語
  • 抗菌ペプチドThanatinおよびその高活性化学修飾体の細菌に対する作用               
    折笠善丈, 斉藤洵己, 橋本茂樹, 松本謙一郎, 大井俊彦, 田口精一
    日本生物工学会大会講演要旨集, 2009年08月25日, 日本語
  • Bacillus sp.B29由来アゾ還元酵素基質複合体の結晶構造解析               
    緒方大記, 田口精一, 松本謙一郎, 藤原孝彰, 田中勲, YAO Min, 大井俊彦
    日本生物工学会大会講演要旨集, 2009年08月25日, 日本語
  • 抗菌ペプチドApidaecinの高活性化のための一戦略:細胞内導入量の向上               
    山形享子, 一戸健太, 折笠善丈, 橋本茂樹, 松本謙一郎, 大井俊彦, 田口精一
    日本生物工学会大会講演要旨集, 2009年08月25日, 日本語
  • バイオプラスチック生産のための微生物工場               
    山田 美和, 松本 謙一郎, 田口 精一
    繊維学会誌, 2008年11月10日, 日本語
  • 酵素進化工学に基づくバイオポリマー生物生産システムの開発研究               
    松本 謙一郎, 田口 精一
    バイオサイエンスとインダストリー = Bioscience & industry, 2007年07月01日, 日本語

所属学協会

  • 高分子学会               
  • 農芸化学会               
  • 生物工学会               

共同研究・競争的資金等の研究課題

  • 特殊配列を有する新規高分解性プラスチックの生合成と合成機構解明
    科学研究費助成事業 基盤研究(B)
    2020年04月01日 - 2025年03月31日
    松本 謙一郎, 大井 俊彦
    本研究課題は、微生物産生ポリエステルであり生分解性プラスチックとして利用できるポリヒドロキシアルカン酸(PHA)の構造制御に関するものである。PHAの構造制御は、材料物性に直結する重要な技術課題である。PHAは、通常共重合体の合成においてはランダム共重合体しか作ることができないが、申請者はブロック共重合体を合成できる方法を独自に見出している。この合成手法を応用することで、非天然モノマーであるグリコール酸を含むポリマーを得ることができた。得られた共重合体は、同一分子内にランダム共重合体と単独重合体の構造を有する、特殊な配列構造を有していた。ランダム共重合体部分のグリコール酸分率は推定約70モル%に達し。本結果は、これまで全く例のない特殊な構造を持つPHAの合成を可能にするものであり、かつ、グリコール酸分率の高い構造を合成する新規な方法を与えた。グリコール酸ポリマーは高い加水分解性を持つことで知られることから、PHAに加水分解性を付与する効果が期待された。これらの成果はFrontiers in Bioengineering and Biotechnology誌に発表した。さらに、ブロック共重合体の化学構造の拡張に取り組んだ。使用している配列制御型重合酵素は、3-hydroxyhexanoate (3HHx)モノマーに弱い活性を有する。3HHxは材料に柔軟性を付与する性質がある事が知られ、PHAの重要な構成成分の一つである。そこで、進化工学的手法を用いて3HHxモノマーに対する活性が向上した変異体を創出した。こうして得られた新たな重合酵素を用いることで、3HHxモノマーから構成される新規ブロック共重合体を合成することができた。本成果はBiomacromolecules誌に発表した。
    日本学術振興会, 基盤研究(B), 北海道大学, 20H04368
  • 配列制御機構を利用した高粘弾性ポリマーの微生物生産
    科学研究費助成事業 基盤研究(B)
    2017年04月01日 - 2020年03月31日
    松本 謙一郎, 大井 俊彦, 佐藤 敏文
    微生物産生ポリエステルは、石油ではなく再生可能なバイオマスを原料として合成でき、さらに自然環境中で生分解されごみが蓄積しないことから、持続可能性が高く、環境低負荷なプラスチックとして着目されている。2019年からは国内でも実用化され、用途および使用量の拡大が課題である。目的達成のためには、プラスチックの物性をより幅広く制御することが重要な技術課題である。これまで、ランダム共重合化により軟質性を付与するという戦略で材料開発が行われてきた。本研究では、モノマーが重合する順序を制御する全く新しい手法により新規ポリマーを合成し、既存のポリマーとは異なる有用な物性を示すことを明らかにできた。
    日本学術振興会, 基盤研究(B), 北海道大学, 17H01902
  • 分解機構に基づいた新規生分解性制御プラスチックの創製
    科学研究費助成事業 基盤研究(C)
    2017年04月01日 - 2020年03月31日
    大井 俊彦, 田口 精一, 松本 謙一郎, 堀 千明
    石油由来のプラスチックによる海洋汚染が世界的問題となっている。その中で自然環境中で分解し水と二酸化炭素まで分解できる生分解性プラスチックが注目されている。本研究課題では、我々が新規に開発したバイオプラスチックである乳酸コポリマー[P(LA-co-3HB)]の乳酸(LA)と3-ヒドロキシブタン酸(3-HB)のポリマー分子チュノ両モノマー組成と環境微生物による分解性を評価した。
    初めに乳酸コポリマーの微生物生産法を検討し、乳酸分率を制御した培養方法を確立した。得られたコポリマーをフィルム化して環境中での分解を評価するとともに、分解に関与する環境微生物の系統解析を実施した。
    日本学術振興会, 基盤研究(C), 北海道大学, 17K00619
  • 「補酵素A再生系」に基づく高光学純度ヒドロキシ酸の高効率生産
    科学研究費助成事業 基盤研究(C)
    2014年04月01日 - 2017年03月31日
    大井 俊彦, 田口 精一, 松本 謙一郎
    我々が新規に開発したグルコースから高光学純度の(R)-3-ヒドロキシ酪酸((R)-3HBの微生物生合成システムを、高効率で(R)-3HB生産可能な微生物工場を創成する基盤技術を確立した。
    生合成中間体である3種のCoA体の細胞内蓄積量を定量する新規分析法を開発し、アセチル-CoAへのフラックス向上が生産性に影響を与えることを見出し、グルコース消費速度の高い宿主菌株を選択した。培地中に添加するグルコース及び酢酸濃度の至適化を行い15g/Lの(R)-3HB生産性を達成した。一方添加する酢酸濃度が高いと生育が阻害されるので、培地交換培養法により効率的な高光学純度(R)-3HB生産法を達成できた。
    日本学術振興会, 基盤研究(C), 北海道大学, 26420791
  • 非天然モノマー導入による配列制御型ポリマーの合成
    科学研究費助成事業 基盤研究(B)
    2014年04月01日 - 2017年03月31日
    松本 謙一郎, 大井 俊彦, 田口 精一, 佐藤 敏文
    再生可能なバイオマスから合成可能な微生物産生ポリエステルの有効活用のためには、優れた材料物性を発揮させることが重要な課題となる。通常、微生物合成を用いるとランダム共重合体が合成されるが、我々はランダム性が低いポリマーが得られる条件を偶然見出した。本研究課題では、この発見を端緒に、新規なポリマー合成系の確立を目指した。モノマー配列の一次構造を解析するとともに、機械的・熱的物性を測定した。さらに、酵素を細胞から単離精製して生化学的な解析を行い、関連する酵素の活性を評価した。これら一連の実験結果に基づいて、対象としたポリエステル合成系について、合成から物性に至るまで幅広い情報を得ることができた。
    日本学術振興会, 基盤研究(B), 北海道大学, 研究代表者, 競争的資金, 26281043
  • バイオマス高度利用による乳酸ポリマー生産のための次世代微生物工場の創成
    科学研究費助成事業 基盤研究(B)
    2011年04月01日 - 2014年03月31日
    田口 精一, 松本 謙一郎, 佐藤 敏文, 佐藤 敏文
    独自に開発した乳酸重合酵素を駆使して乳酸ベースポリマーの微生物生産系を構築している。大腸菌を使用した系では、乳酸分率を0~50%の範囲で、新たにコリネ型細菌を利用した場合は、逆に100%に迫る高分率組成のコポリマーの合成が可能となった。たとえば、30%乳酸ポリマーの場合は、11g/Lまで増大することがわかった。ポリマー物性に関しては、ポリ乳酸あるいはPHBのホモポリマーと比べて、乳酸の分率に応じて柔軟性が増大した。さらに、脱リグニン処理した植物バイオマス原料をセルラーゼ入り酵素製剤によって得た酵素糖化液を利用して、微生物ポリマーの合成が可能であることを実証することができた。
    日本学術振興会, 基盤研究(B), 北海道大学, 23310059
  • 多様なバイオポリエステルを合成する資源循環型フレックス微生物工場の開発
    科学研究費助成事業 若手研究(A)
    2011年04月01日 - 2014年03月31日
    松本 謙一郎
    ある種の微生物は細胞内にポリエステルを蓄積することが知られ、このポリマーはプラスチックの物性を示すことから、バイオベースプラスチックの候補物質として期待される。しかし、天然で合成される3-ヒドロキシ酪酸から構成されるポリマーは、脆性を示すことから応用例が限定的であった。本研究課題では、人工的に改変したポリエステル生合成系を用いて、種々の非天然ポリエステルを合成し、その物性を明らかにした。とくに、2-ヒドロキシ酪酸のポリマーの微生物合成では、機械物性が評価できる程度の量のポリマーの合成に初めて成功した。本ポリマーは優れた透明性と伸張性を持っており、天然型のポリマーとは全く異なる物性を示した。
    日本学術振興会, 若手研究(A), 北海道大学, 研究代表者, 競争的資金, 23681015
  • 非可食性バイオマス糖化物に適応するバイオプラスチック製造微生物工場の開発
    科学研究費助成事業 基盤研究(C)
    2011年 - 2013年
    大井 俊彦, 田口 精一, 松本 謙一郎
    セルロースの触媒加水分解では、糖の過分解で生じる5HMFが副生され宿主の生育を阻害することから、5HMF耐性を持った大腸菌を検索し、使用する糖濃度の範囲でポリマー生産性に障害とならない宿主大腸菌LS5218株を選抜した。また、改良した微生物工場がキシロースとグルコースの単独使用だけでなく、混合糖液においてもポリマー生産に十分適応可能であることを示した。実際に非可食バイオマスから調製した各種不純物を含む混合糖液においても十分なポリマー生産能力を示した。
    以上の研究結果から、本研究目的である非可食バイオマス糖化物を炭素源としたバイオポリエステルを生産するまでの一貫プロセスの開発することができた
    日本学術振興会, 基盤研究(C), 北海道大学, 23580452
  • 新規乳酸ポリエステルを合成する遺伝子組換え植物の創出
    科学研究費助成事業 若手研究(B)
    2009年 - 2010年
    松本 謙一郎
    ポリ乳酸は、微生物発酵によって合成される乳酸を重合させたバイオマス由来のプラスチックである。申請者らは、乳酸を重合可能な特殊な重合酵素を導入した遺伝子組換え植物を作成し、植物細胞内による新奇ポリエステル合成を検討した。この目的のために、ポリエステルの合成に必要なモノマー供給および重合酵素の遺伝子を導入した組換え植物を作成した。また、導入した遺伝子の発現と、ポリエステル合成を確認できた。
    日本学術振興会, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 21710087
  • 酵素進化工学が先導する新規モノマー導入型バイオポリマーの完全生合成システムの創成
    科学研究費助成事業 基盤研究(B)
    2008年 - 2010年
    田口 精一, 佐藤 敏文, 松本 謙一郎
    再生可能バイオマス原料から高性能バイオポリマーを微生物生産させるシステム(微生物工場)の開発を行った。最も大きなブレイクスルーは、「乳酸重合酵素=LPE」の発見である。従来の微生物ポリマーPHA(ポリヒドロキシアルカン酸)の重合酵素の改変研究を積み重ねてきた過程で見出された。この酵素を大腸菌に遺伝子導入することにより、バイオマス由来グルコースから乳酸を取り込んだポリマーが“一気通貫"に細胞内に合成された。また、代謝と酵素の複合改変を試みたところ、ポリマー中の乳酸分率が6%から47%に上昇した。さらに、合成ポリマーの基礎物性を調べ、従来のポリ乳酸(PLA)と微生物ポリマーPHB(ポリヒドロキシブタン酸)との比較において、ユニーク性を見出した。
    日本学術振興会, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 20310050
  • ポリエステル生産の効率化のための組換え植物の生育阻害機構の解明とその改善
    科学研究費助成事業 若手研究(B)
    2007年 - 2008年
    松本 謙一郎
    バイオプラスチック(PHA)の合成遺伝子を導入した遺伝子組換えイネは、細胞内にPHA を作ることができる。しかしPHA を蓄積することにより、植物の生育が悪くなることが知られていた。
    そこで、その原因を解明するため、組換え株と野生株の遺伝子の働きを網羅的に解析し、組換え株の中で遺伝子の働きにどのような変化が起こったのかを調べた。
    その結果、組換え株で影響を受けていると考えられる遺伝子を数個見つけることができた。
    日本学術振興会, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 19710076
  • trans-スプライシングに連鎖した非コードRNAによる新規な翻訳調節機構の解析
    科学研究費助成事業 基盤研究(B)
    2005年 - 2007年
    島田 浩章, 菊池 尚志, 松本 謙一郎
    プロティンキナーゼSPK遺伝子の第1エキソンは翻訳領域の逆鎖には機能未知な別の遺伝子であるOsMaclが存在する。OsMac1と相同性のある遺伝子を検索したところ、この他に2つの遺伝子が見つかり、それぞれをOsMac2、OsMac3と名付けた。OsMac1の発現は発芽直後に高く、成長するにつれて発現量が低下した。GFPとの融合タンパク質を用いた細胞内局在部位の解析を行ったところ、OsMac1の転写産物は細胞膜に局在することがわかった。このことからOsMac1はストレス応答に関与するものと推測された。ポリヒドロキシアルカン酸を生産させた形質転換体イネではさまざまなストレス応答遺伝子の変動が認められたため、この形質転換体カルスでのOsMac1の発現量の変動を調べた。GC/MSによりポリヒドロキシアルカン酸の生産を確認した後、この個体からRNAの抽出を試み、OsMac1遺伝子の発現量の変動を調べたが、この遺伝子の発現量の変動は認められなかった。ところで、OsMac1の主たるORFの前方に複数の短いORFが存在することがわかった。また複数のスプライシングバリアントが存在した。これらにより主たるORFからの翻訳が制御されている可能性が示唆されたため、この領域の下流にGUS遺伝子を結合し、形質転換カルスでのGUSタンパク質の翻訳を検定した。その結果、上流ORFが存在するにもかかわらず、下流のORFが翻訳されることがわかった。おそらく、上流ORFを読み飛ばす何らかな機構が存在するのであろうと示唆された。
    日本学術振興会, 基盤研究(B), 東京理科大学, 連携研究者, 競争的資金, 17310115
  • 生分解性プラスチックを合成する遺伝子組換え植物の創出
    科学研究費助成事業 若手研究(B)
    2004年 - 2005年
    松本 謙一郎
    ポリヒドロキシアルカン酸(PHA)は微生物が合成する貯蔵物質であり、生分解性を有するプラスチックとして利用できることから、環境低負荷型のエコ材料として期待されている。当研究課題では、微生物のPHA生合成系遺伝子群を植物に導入することにより、太陽光と二酸化炭素からPHAを直接生産するシステムの開発を目指した。これにより、PHAの実用化への最大の障壁である生産コストが削減できる可能性がある。PHAはモノマー組成に依存して物性が変化することが知られており、炭素数が4(短鎖)のモノマーユニットを主成分とし、少量の炭素数が6以上(中鎖)のモノマーユニットを含む共重合体は、柔軟性がよく幅広い応用範囲が期待できる。しかし、植物細胞内には有効な中鎖のモノマー供給系が存在しないため、共重合体の合成は非常に困難であった。そこで当研究室グループで作成された新規な中鎖モノマー供給酵素である大腸菌由来変異型3-ketoacyl-ACP synthase III (FabH)を用いて、植物の葉緑体内での共重合体の合成を試みた。Pseudomonas sp.61-3及びWautersia eutropha由来のPHA生合成系遺伝子群と変異FabH遺伝子を導入したシロイヌナズナの組換え株は、PHA生合成系遺伝子群のみを導入した組換え株と比較して約2倍のPHAを蓄積した。さらにごく微量ではあるが、炭素数が6〜14のモノマーユニットを含む短鎖・中鎖共重合体が合成された。これより、植物の葉緑体内で中鎖のモノマーを供給する代謝経路の構築に成功した。また、変異型FabHは中鎖よりも短鎖モノマーの供給能力のほうが高いことが分かった。
    日本学術振興会, 若手研究(B), 独立行政法人理化学研究所->東京理科大学, 研究代表者, 競争的資金, 16710059