| Project/Area Number |
10356004
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| Research Category |
Grant-in-Aid for Scientific Research (A).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SAKAYU Shimizu Kyoto University, Agric., Professor, 農学研究科, 教授 (70093250)
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| Co-Investigator(Kenkyū-buntansha) |
KITA Keiko Tottori University, Engineering, Associate Professor, 工学部, 助教授 (70234226)
OGAWA Jun Kyoto University, Agric., Assistant Professor, 農学研究科, 助手 (70281102)
KATAOKA Michihiko Kyoto University, Agric., Associate Professor, 農学研究科, 助教授 (90252494)
HASEGAWA Junzo Kaneka Co., Takasago Institute, Researcher, 高砂研究所, 基幹研究員
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥30,300,000 (Direct Cost: ¥30,300,000)
Fiscal Year 2000: ¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1999: ¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1998: ¥17,200,000 (Direct Cost: ¥17,200,000)
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| Keywords | Chiral building block / Carbonyl reductase / Asymmetric reduction / Aldehyde reductase / Ethyl 4-chloro-3-hydroxybutanoate / 4-700-3-ヒドロキシ酪酸エチル / 4-クロロ-3ヒドロキシ酪酸エチル / 4-クロロアセト酢酸エチル |
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| Research Abstract |
Chiral alcohols with additional functional groups are useful building blocks for the synthesis of enantiomeric pure pharmaceuticals and other chemicals. We constructed a novel bioreduction system for the production of chiral alcohols (ethyl 4-chloro-3-hydroxybutanoate, CHBE) using microbial carbonyl reductases as catalysts. In this system, E.coli transformant cells co-expressing a carbonyl reductase (from Sporobolomyces salmonicolor or Candida magnoliae) and glucose dehydrogenase (GDH) genes were used, because GDH co-produced with carbonyl reductase in E.coli cells regenerated NADPH required for the reduction reaction through oxidation of glucose to gluconolactone.
Aldehyde reductase (ART) of S.salmonicolor or carbonyl reductase (S1) of C.magnoliae were applied to the co-expression system for (R)- or (S)-CHBE production, respectively. These enzymes catalyze NADPH-dependent stereospecific reduction of ethyl 4-chloroacetoacetate (CAAE) to (R)- or (S)-CHBE.The reduction reaction is carried out in a mixture containing CAAE, glucose, NADP^+ and E.coli cells co-expressing AR1 (or S1) and GDH genes. Using E.coli cells expressing AR1 and GDH genes, 300 g/l of CAAE was stoichiometrically converted to (R)-CHBE (92% e.e.). E.coli cells expressing S1 and GDH genes produced about 350 g/l of (S)-CHBE with optical purity of 96% e.e.
This bioreduction system using E.coli cells co-expressing carbonyl reductase and cofactor-regeneration enzyme (GDH) genes is thought to be applicable to the production of various kinds of other chiral alcohols, by replacing the carbonyl reductase gene for other appropriate reductase genes.
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