Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate

An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S )-4-chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, whic h is a useful chiral building block for the synthesis of pharmaceuticals. T he gene encoding the enzyme was cloned and sequenced. The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da. The deduced amino acid sequ ence showed a high degree of similarity to those of the other members of th e short-chain alcohol dehydrogenase superfamily. The S1 gene was overexpres sed in Escherichia coli under the control of the lac promoter. The enzyme e xpressed in E. coli was purified to homogeneity and had the same catalytic properties as the enzyme from C. magnoliae did. An E. coli transformant red uced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomer ic excess, in an organic solvent two-phase system.