(R)-专一性羰基还原酶与甲酸脱氢酶基因在大肠杆菌中的共表达  

Co-expression of formate dehydrogenase from Candida boidinii and (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011 in Escherichia coli

作　　者：孙莹[1] 张荣珍[1] 徐岩 

机构地区：[1]江南大学生物工程学院教育部工业生物技术重点实验室,无锡214122

出　　处：《微生物学报》2008年第12期1629-1633,共5页Acta Microbiologica Sinica

基　　金：国家"973项目"(2003CB716008);国家"863计划"(2007AA02Z200;2006AA020104);国家自然基金项目(20776060);新世纪优秀人才支持计划(NCET-04-0498)~~

摘　　要：【目的】通过研究(R)-专一性羰基还原酶和甲酸脱氢酶基因在大肠杆菌中的共表达,解决较高底物浓度下不对称转化反应的辅酶限制性问题。【方法】分别以近平滑假丝酵母(Candida parapsilosis CCTCC M203011)和博伊丁假丝酵母(Candida boidinii)基因组为模板,采用PCR方法扩增得到(R)-专一性羰基还原酶基因(rcr)和甲酸脱氢酶基因(fdh),克隆到共表达载体pETDuet^(TM)-1中进行表达。共表达质粒pETDuet-rcr-fdh转化稀有密码子优化型菌株E.coli Rosetta,获得重组菌E.coli Rosetta/pETDuet-rcr-fdh。【结果】在30℃条件下,经1 mmol/L IPTG诱导表达8 h后,SDS-PAGE结果表明(R)-专一性羰基还原酶和甲酸脱氢酶均有明显的表达,其相对分子质量分别为37 kDa和40 kDa。以高浓度(6 g/L)2-羟基苯乙酮为底物时,0.1 g重组菌细胞催化产生(R)-苯基乙二醇,产物光学纯度为100%e.e.,产率为85.9%。与无甲酸脱氢酶参与辅酶再生循环的重组菌E.coli Rosetta/pETDuet-rcr相比,产物光学纯度和产率分别提高了1.3和2.7倍。【讨论】该重组菌的构建为基因工程法生物合成(R)-苯基乙二醇的工业应用奠定了基础。[Objective] To overcome coenzyme restriction in the asymmetric reduction at high substrate concentration, we constructed a recombinant Escherichia coli with （R）-sepcificity carbonyl reductase coupled with formate dehydrogenase for cofactor regeneration. [Methods] The R-carbonyl reductase gene （rcr） and formate dehydrogenase gene （fdh） were amplified from Candida parapsilosis and Candida boidinii genomes by PCR technique respectively. Then the purified PCR products were inserted into a co-expression vector pETDuet^TM-1 to construct plasmid pETDuet-rcr-fdh. The positive plasmid was transformed into codon optimized E. coli Rosetta, and a recombinant strain E. coli Rosetta/pETDuet-rcr-fdh was obtained. [Results] SDS-PAGE analysis showed that two enzymes were expressed simultaneously. Isopropyl-β-D-thiogalactopyranoside （1 mmol/L） induced at 30℃ the expression of both proteins encoded by rcr andfdh genes with the molecular weights of 37 kDa and 40 kDa. The biotransformation experiments were done using 2-hydroxyacetophenone and sodium formate as substrates. When the concentration of 2-hydroxyacetophenone was 6 g/L, the （R）-1-phenyl-1,2-ethanediol was produced with the high optical purity of 100% enantiomeric excess and a yield of 85,9%. Compared with the recombinant strain E. coli RosettalpETDuet-rcr withoutfdh gene for cofactor regeneration, the optical purity and yield of product from the asymmetric reduction of 2-hydroxyacetophenone by E. coli RosettalpETDuet-rcr-fdh were increased by 1.3 and 2.7 times, respectively. [Conclusion] This method supplied a foundation for biosynthesis of （R）-1-phenyl-1,2-ethanediol for the cofactor regeneration by genetic engineering.

关 键 词：羰基还原酶 甲酸脱氢酶 共表达 辅酶再生 不对称还原 

分 类 号：Q78[生物学—分子生物学]