Biosynthesis Of Ethyl(S)-4-chloro-3-hydroxybutanoate

Ethyl(S)-4-chloro-3-hydroxybutanoate((S)-CHBE)is a key chiral intermediate for synthesis of cholesterol-lowering HMG-CoA reductase inhibitors,which can also applied in the synthesis of other desired chiral drug intermediates by chlorine replacement and reduction.The asymmetric reducion of ethyl 4-chloroacetoacetate(COBE)to(S)-CHBE by biocatalysis method is advantageous due to its low cost,mild reaction conditions,high yield,and high level of enantioselectivity.It has become a promising method for preparation of(S)-CHBE.In this paper,gas chromatography method for the detection of COBE and(S)-CHBE was established.One microbial strain ZJB-12164 was isolated from soil samples which could be used for the production of(S)-CHBE with high activity and enantioselectivity(e.e.>99%).On the basis of the morphology,physiological characteristics and 18S rDNA analysis,ZJB-12164 was identified as Candida sorboxylosa.Secondly,the medium components and cultivation conditions of C.sorboxylosa ZJB-12164 were optimized.The results showed that the optimal medium components were as follows(g/L):glucose 10.0,yeast extract 50.0,KH2P04 2.5 and NaCl 1.0.The optimal cultivation conditions were as follows:medium loading 30 mL/500 mL,inoculum size 1%(V/V),and initial pH 5.0.Under the optimal conditions,the biomass of C.sorboxylosa ZJB-12164 reached 11.2 g dcw/L and the carbonyl reductase activity reached 202.4 U/L.Finally,the asymmetric reduction of COBE by whole-cells of C.sorboxylosa ZJB-12164 was studied.The data showed that the optimal reaction temperature and cosubstrate glucose concentration were 35? and 40 g/L,respectively.The optimal buffer was pH 8.0 phosphate buffer solution.When 50 g/L wet cells were used,the optimal substrate concentration was 60 mM.In the case of fed-batch catalysis with the control of reaction pH,after 8 hours,the concentration and yield of(S)-CHBE reached 207.9 mM and 86.6%,respectively.The results demonstrated that C.sorboxylosa ZJB-12164 may paly an important role in synthesis of ethyl(S)-4-chloro-3-hydroxybutanoate.This study put forward a low-cost,efficient and proprietary bioprocess for the production of(S)-4-chloro-3-hydroxybutanoate.