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Applied and Environmental Microbiology, February 2003, p. 933-937, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.933-937.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

Masaru Wada,^1* Ayumi Yoshizumi,¹ Yumiko Noda,¹ Michihiko Kataoka,² Sakayu Shimizu,² Hiroshi Takagi,¹ and Shigeru Nakamori¹

Department of Bioscience, Fukui Prefectural University, Matsuoka-cho, Fukui 910-1195 ,¹ Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan²

Received 1 August 2002/ Accepted 21 October 2002

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.

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* Corresponding author. Mailing address: Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka-cho, Fukui 910-1195, Japan. Phone: 81-776-61-6000. Fax: 81-776-61-6015. E-mail: masaru{at}fpu.ac.jp.

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Applied and Environmental Microbiology, February 2003, p. 933-937, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.933-937.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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