Development of production process for chiral building blocks using microbial carbonyl reductases

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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.