Co-expression of formate dehydrogenase from Candida boidinii and (R)- specific carbonyl reductase from Candida parapsilosis CCTCCM203011 in Escherichia coli

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