Efficient production of ethyl (R)-4-chloro-3-hydroxybutanoate by a novel alcohol dehydrogenase from Lactobacillus curieae S1L19

Abstract

Ethyl (R)-4-chloro-3-hydroxybutanoate ester [(R)-CHBE] is an important chiral intermediate for the synthesis of chiral drugs. In this study, a novel short-chain, NADH-dependent dehydrogenase (LCRIII) from Lactobacillus curieae S1L19 was discovered to exhibit high activity and enantioselectivity in the production of (R)-CHBE by reduction of ethyl 4-chloroacetoacetate (COBE). LCRIII was heterologously overexpressed in Escherichia coli and the protein was purified to homogeneity. Characterization of LCRIII showed broad substrate specificity towards a variety of ketones. In addition, an efficient cofactor regeneration system was constructed by co-expressing LCRIII and glucose dehydrogenase (GDH) in E. coli cells. Up to 1.5 M (246.8 g/L) COBE could be completely reduced to (R)-CHBE with excellent enantiomeric excess ( > 99% ee) in a monophasic aqueous system. Moreover, the process could be performed even without external addition of cofactors. These results demonstrate the great potential of this process in industrial applications.