S-4-氯-3-羟基丁酸乙酯(S-CHBE)是多种药物的手性中间体,可由4-氯乙酰乙酸乙酯(COBE)不对称还原合成。该研究对来自Candida magnoliae AKU4643的S1羰基还原酶基因进行E.coli密码子优化,获得的S1'基因序列并进行人工合成,利用该序列构建了基因工程菌BL21(DE3)/pET28a-S1',提高了羰基还原酶表达效率,经诱导条件优化后羰基还原酶活力达11.49 U/mg蛋白;将BL21(DE3)/pET28a-S1'与E.coli BL21(DE3)/pET28a-gdh进行偶联,在单一水相中未添加辅酶条件下转化10 h,产物的摩尔转化率和对映体过量值(e.e.)分别达92.1%和100%。

S-Ethyl-4-chloro-3-hydroxybutanoate( S-CHBE) is used as a chiral building block for the synthesis of pharmaceutical target compounds. S-CHBE can be produced by the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate( COBE). In present study,the S1' gene was delivered from the NADPH-dependent carbonyl reductase gene( S1) of Candida magnoliae AKU4643 followed artificially synthesizing and codon optimizing for E. coli host. The recombinant strain BL21( DE3) / pET28a-S1 ' with S1 ' gene showed more effective expression of carbonyl reductase gene. The highest activity of carbonyl reductase was achieved 11. 49U / mg protein after induction conditions optimization. Furthermore,the asymmetric reduction of COBE to CHBE was performed using the recombinant E. coli BL21( DE3) /pET28a-S1' coupling with E. coli BL21/pET28a-gdh in an aqueous system and the enantiomeric excess( e. e.) of S-CHBE was arrived to 92. 1% and 100% respectively after 10h bioconversion without adding coenzyme NADPH or NADP+.