糖多孢红霉菌酮还原酶域在羰基还原中的应用

Application of ketoreductase domain in polyketide synthase from Saccharopolyspora erythraea to carbonyl reduction

为研究糖多孢红霉菌聚酮合成酶模块1的酮还原酶域,催化羰基还原的底物特异性和立体选择性,PCR扩增了该酶域的编码基因(eryKR1),并将其克隆到表达载体pET-28a,得到重组质粒pET-eryKR1,转化到Escherichia coli BL21(DE3)后,获得了重组菌株E.coli BL21(pET-eryKR1).将E.coli BL21(pET-eryKR1)和异源表达枯草芽孢杆菌葡萄糖脱氢酶基因的重组大肠杆菌E.coli BL21(pET-gdh1)进行双重组菌耦合,对4-氯乙酰乙酸乙酯、苯乙酮、2-辛酮和环己酮4种底物进行转化还原,利用气相色谱分析转化液,结果显示E.coli BL21(pET-eryKR1)对环己酮的还原效果较好,产物环己醇的得率最高可达93.24%,而对其他3种底物几乎没有还原能力.利用E.coli BL21(pET-eryKR1)2催化2-甲基环己酮不对称还原,产物主要为顺式-2-甲基环己醇,产率可达41.87%,产物的对映体过量值为74.81%.

In order to define substrate specificity and enantioselectivity of ketoreductase domain in the first module of polyketide synthase from Saccharopolyspora erythraea, the eryKR1 gene coding this domain was amplified by PCR （polymerase chain reaction） and cloned into vector pET-28a to construct recombinant plasmid pET-eryKR1. The plasmid was then transformed into Esckerichia coli BL21 （DE3） to obtain recombinant E. coli BL21 （pET eryKR1）. The recombinant and E. coli BL21 （pET- gdhl） harboring Bacillus subtilis glucose dehydrogenase gene fermented together with ethyl 4-chloro- 3-oxobutanoate, acetophenone, 2-octanone or cyclohexanone as reduction substrate, respectively. Gas chromatography analysis of these ferments showed that among the four substrates recombinants could not reduce other than cyclohexanone, and the best yield of cyclohexanol could reach 93.24% Then E. coli BL21 （pET-eryKR1）2 was chosen to ferment together with 2-methylcyclohexanone. The result showed that this strain could mainly reduce 2-methylcyclohexanone to produce cis-2-methylcyclohexanol, the yield was 41.87%, and enantiomeric excess was 74.81%.