Development of biotransformation process for asymmetric reduction with novel anti-Prelog NADH-dependent alcohol dehydrogenases

Alcohol dehydrogenases or carbonyl reductases have been extensively developed for the asymmetric reduction of ketones to chiral alcohols, which are important pharmaceutical precursors. Ideal biotransformation using alcohol dehydrogenases requires (i) the identification of novel enzymes with broad substrate range, high substrate tolerance, enantioselectivity and preference for cheaper cofactor (NADH) and (ii) the development of an optimized biocatalytic process with a mechanism for efficient cofactor recycling. This report details the mining and identification of a subfamily of novel NADH-dependent alcohol dehydrogenases with anti-Prelog stereo selectivity, that exhibit high specific activity on beta-ketoesters. Further, an efficient biocatalytic process has been developed using ADH from Acetobacter aceti mined in this study for the enantioselective reduction of up to 10 M ethyl 4-chloro-3-oxobutanoate to (S)-Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The process employed lyophilized cell -free extract and reduction was achieved with > 99% yield and high enantiomeric excess ( > 99% ee) in 24 h using a biphasic reaction system. A space-time yield of approximately 650 g/L. d and cofactor TTN of 10(6) has been achieved using the process, with potential application in industrial biocatalysis.