Development of an efficient bio-production system using thermostable enzyme modules

• Project/Area Number: 25820402
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biofunction/Bioprocess
• Research Institution: Osaka University
• Principal Investigator: Okano Kenji 大阪大学, 工学(系)研究科(研究院), 助教 (40623335)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2015: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2013: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: In vitro代謝工学 / 耐熱性酵素 / 非リン酸化型Entner-Doudoroff経路 / 乳酸 / 人工オペロン / in vitro代謝工学 / 非リン酸化Entner-Doudoroff経路

Outline of Final Research Achievements

Novel and efficient bio-production system using thermostable enzyme modules was developed. In this system, recombinant Escherichia coli strains having a heterologous thermophilic enzyme were directly used as catalysts at high temperatures. The denaturation of indigenous enzymes at high temperatures resulted in a one-step preparation of highly selective thermotolerant biocatalytic modules. The combination of these modules enabled to construct in vitro synthetic pathways specialized for chemical manufacture.
By using this technique, in this study, the non-phosphorylated Entner-Doudoroff glycolysis pathway, which was found in some hyperthermophile, was constructed. By coupling this pathway with lactate dehydrogenase, lactate was produced from glucose with 100% molar yield. Moreover, an artificial operon including the genes encoding eight thermophilic enzymes was successfully constructed and co-expression of eight enzymes in a single recombinant E. coli strains was achieved.

Research Products

• [Journal Article] In vitro代謝工学による1-ブタノール生産 (2015)
  • Author(s): 岡野憲司、本田孝祐
  • Journal Title: バイオサイエンスとインダストリー Volume: 73 Pages: 481-482
• [Journal Article] Direct conversion of glucose to malate by synthetic metabolic engineering (2013)
  • Author(s): Ye, X., Honda, K., Morimoto, Y., Okano, K. and Ohtake, H.
  • Journal Title: J. Biotechnol. Volume: 164 Issue: 1 Pages: 34-40
  • DOI: 10.1016/j.jbiotec.2012.11.011
  • Peer Reviewed
• [Journal Article] Development of continuous bioconversion system using thermophilic whole-cell biocatalyst (2013)
  • Author(s): Ninh, P. H., Honda, K. , Yokohigashi, Y. , Okano, K., Omasa, T. and Ohtake, H.
  • Journal Title: Appl. Environ. Microbiol. Volume: 79 Issue: 6 Pages: 1996-2001
  • DOI: 10.1128/aem.03752-12
  • Peer Reviewed
• [Journal Article] Directed evolution of thermotolerant malic enzyme for improved malate production (2013)
  • Author(s): Morimoto, Y., Honda, K., Ye X., Okano, K. and Ohtake, H.
  • Journal Title: J. Biosc. Bioeng. Volume: 117 Issue: 2 Pages: 147-152
  • DOI: 10.1016/j.jbiosc.2013.07.005
  • NAID: 110009804064
  • Peer Reviewed
• [Journal Article] In vitro production of n-butanol from glucose (2013)
  • Author(s): Krutsakorn, B., Honda, K., Ye, X., Imagawa, T., Bei, X., Okano, K. and Ohtake, H.
  • Journal Title: Metabolic Eng. Volume: 20 Pages: 84-91
  • DOI: 10.1016/j.ymben.2013.09.006
  • Peer Reviewed
• [Journal Article] Construction of an in vitro bypassed pyruvate decarboxylation pathway using thermostable enzyme modules and its application to N-acetylglutamate production (2013)
  • Author(s): Borimas Krutsakorn, Takashi Imagawa, Kohsuke Honda, Kenji Okano, Hisao Ohtake
  • Journal Title: Microbial Cell Factory Volume: 12 Issue: 1 Pages: 91-91
  • DOI: 10.1186/1475-2859-12-91
  • Peer Reviewed
• [Presentation] Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering (2015)
  • Author(s): Kenji Okano, Kohsuke Honda, Hisao Ohtake
  • Organizer: 21th Young Asian Biochemical Engineers' Community Symposium (YABEC2015)
  • Place of Presentation: Elysian Changchon Resort, Chuncheon, Korea
  • Year and Date: 2015-10-14
  • Int'l Joint Research / Invited
• [Presentation] Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering (2015)
  • Author(s): Kenji Okano, Kohsuke Honda, Hisao Ohtake
  • Organizer: Japanese-German Graduate Externship Program, Biotechnology and Chemistry for Green Growth
  • Place of Presentation: 大阪大学銀杏会館
  • Year and Date: 2015-03-09 – 2015-03-11
  • Invited
• [Presentation] In vitro metabolic engineering employing thermophilic enzymes -a novel, simple technology for designing a chimeric metabolic pathway- (2014)
  • Author(s): Kenji Okano, Kohsuke Honda, Hisao Ohtake
  • Organizer: 20th Young Asian Biochemical Engineers’ Community Symposium (YABEC2014)
  • Place of Presentation: Nathonal Chung Cheng University
  • Year and Date: 2014-11-05 – 2014-11-08
• [Presentation] In vitro代謝工学による非リン酸化型Entner-Doudoroff経路の構築および乳酸生産への応用 (2014)
  • Author(s): 岡野憲司、朱倩沁、本田孝祐、大竹久夫
  • Organizer: 第66回日本生物工学会大会
  • Place of Presentation: 札幌コンベンションセンター
  • Year and Date: 2014-09-09 – 2014-09-11
• [Presentation] In vitro代謝工学-バイオプロダクションのための新たなパラダイム構築- (2014)
  • Author(s): 岡野憲司、本田孝祐、大竹久夫
  • Organizer: 2014年度生物工学若手研究者の集い 夏のセミナー
  • Place of Presentation: 神戸セミナーハウス
  • Year and Date: 2014-07-12 – 2014-07-13
• [Presentation] 合成代謝工学による非リン酸化Entner-Doudoroff経路の構築および乳酸生産への応用 (2014)
  • Author(s): 岡野憲司、朱倩沁、本田孝祐、大竹久夫
  • Organizer: 化学工学会第79年会
  • Place of Presentation: 岐阜大学