(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 CCTCCM203011 in Escherichia coli

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pETDuetTM-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。

Abstract:

[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 pETDuetTM-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 and fdh 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 Rosetta/pETDuet-rcr without fdh gene for cofactor regeneration, the optical purity and yield of product from the asymmetric reduction of 2-hydroxyacetophenone by E. coli Rosetta/ pETDuet-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