Asymmetry Reduction By Coupled Two Genetic Engineering Strain Systems For The Production Of (S)-4-chloro-3-hydroxybutanoate

Enantiometrieally pure hydroxy compounds are important intermediates in synthesisof chiral drug. Using microorganism or enzymes to asymmetrical synthesis of chiralcompounds has become an active field in research. Because of mild reactionconditions,high conversion ratio and outstanding stereochemical specificity, biocatalysishas become the most promising method in the asymmetric synthesis.Ethyl S-4-chloro-3-hydorxybuytrate(S-CHBE) is a key intermediate to synthetizehydroxy methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. It is an economicway to asymmetic reduction ethyl4-chloroacetoacetate (COBE) to synthesis ethylS-4-chloro-3-hydorxybuytrate.In this study, the NAD(P)H-dependent Ketoreductase gene（KRED）was synthesisedaccording to the sequence of amino acids derived from Candida magnoliae (Genbank Ace.No. JC7338; GI:11360538), and expressed by the recombinant E. coli BL21/pET28a-kred.Effective expression of ketoreductase in recombination strain E. coli BL21/pET28a-kred bythe optimization medium and IPTG induction conditions. The specific activity of KREDwas11.49U/mg proteins from the initial enzyme activity (9.28U/mg protein). E.coliBL21/pET28a-kred was applied to catalyze the asymmetric reduction of COBE toS-CHBE.Asymmetric reduction of COBE to synthesis S-CHBE by the recombination strain E.coli BL21/pET28a-kred often need to add NAD(P)H as co-enzyme, due to the E. coli itselfhas no perfect co-enzyme regeneration system. In order to solve co-enzyme NAD(P)Hregeneration during reaction, glucose dehydrogenase (GDH, E.C.I.1.1.47), was clonedfrom Bacillus subtilis and a recombinant strain E. coli BL21/pET28a-gdh was constructedto expressing glucose dehydrogenase, which is able to regenerate NAD(P)+to NAD(P)Hcoupled with the oxidation of glucose.The two-strain coupled system of E. coli BL21/pET28a-kred and BL21/pET28a-gdhwas studied in the biotransformation of COBS to S-CHBE in aqueous phase. The systemcan be operated without any addition of NADP+and glucose dehydrogenase. The yield andthe enantiomeric excess（e.e）of S-CHBE reached92.6%and100%, respectively, wheninitial COBE concentration was24.2g/L,the wet cell concentration was20g/L and initial glucose concentration was50g/L. The optimized bioreduction conditions for two-straincoupled system are:25C,150r/min of rotate speed and the1:1in weight ratio of wet cellsfor BL21/pET28a-kred to BL21/pET28a-gdh.