Co-expression Of Formate Dehydrogenase And (R)-hydroxyacetophenone Reductase In Escherichia Coli

Cofactor is non-recyclable in the asymmetric reduction of biocatalysis, which is the bottleneck of industrialization. Although whole-cell can provide a certain amount of cofactors by its own metabolism, but the requirement of cofactor will increase with substrate concentration, according to stoichiometric balance,the supply of cofactor becomes the main limiting factor. In this paper, the codons of R-hydroxyacetophenone reductase gene (rcr) from Candida parapsilosis CCTCC M203011 were optimized on the basis of codon usage bias in Escherichia coli. The formate dehydrogenase gene (fdh) was amplified from Candida boidinii genomes by PCR technique. Then the purified PCR products of fdh and codon optimized rcr were inserted into a co-expression vector pETDuetTM-1. The positive plasmid was transformed into codon optimized E. coli Rosetta strain and the efficient co-expression system of R-hydroxyacetophenone reductase (rCR) and formate dehydrogenase (FDH) was constructed. The biotransformation experiments were carried out using 2-hydroxyacetophenone and suitable sodium formate as substrates with 0.1 g recombinant E. coli cells as catalysts. The results showed that the product (R)-1-phenyl-1,2-ethanediol was produced with high optical purity and yield after optimization of the reaction conditions, . This work supplied a foundation for biosynthesis of (R)-1-phenyl-1,2-ethanediol coupled with the cofactor regeneration by the method of genetic engineering. The main results were described as follows:(1)Based on the rcr gene sequence and codon usage bias in E. coli, three pairs of primers Rcr-F1 and Rcr-R1, Rcr-F2 and Rcr-R2, Rcr-F3 and Rcr-R2 were designed, two segments of rcr were cloned respectively by PCR amplification using genomic DNA of C. parapsilosis as the template. The two fragments were combined by splicing overlapping extension-PCR (SOE-PCR) method and the whole codon optimized gene rcr was obtained. The purified PCR product was inserted into pMD19-T vector and the recombinant plasmid T-rcr was constructed and sequenced. The results showed that the full length of rcr was 1011 bp and encoding 336 amino acids.(2)Based on the reported fdh gene sequence, primers Fdh-F and Fdh-R were designed, the gene fdh was cloned by PCR amplification using the primers and genomic DNA of C. boidinii as the template. The purified PCR product was inserted into pMD19-T vector and the recombinant plasmid T- fdh was obtained and sequenced. The results showed that the full length of fdh was 1095 bp and encoding 364 amino acids.(3)The rcr and fdh genes were cloned into the co-expression vector pETDuetTM-1 and the recombinant co-expression plasmid pETDuet-rcr-fdh was constructed, The recombinant plasmid pETDuet-rcr-fdh was transformed into codon optimized E. coli Rosetta strain and the positive clone E. coli Rosetta/pETDuet-rcr-fdh was obtained. SDS-PAGE analysis showed that 1 mmol/L IPTG would induce the expressions of rCR and FDH with the molecular weights of 37 kDa and 40 kDa simultaneously. Through the shaking-flask condition research, the optimum induction conditions had been determined as follow: 20% medium volume, [OD600]=0.8 point of induction, induction temperature 30℃, IPTG concentration 0.8 mmol/L, induction duration 16 h. Because two DNA fragments were designed so as to encode 6×His-tag domains followed by the open read frames of rcr and fdh, the two expressed fusion proteins were purified by Ni2+-affinity chromatography and the preliminary purification of two recombinant proteins were obtained.(4) The biotransformation experiments were carried out using recombinant E. coli cells as catalyst and using both 2-hydroxyacetophenone and sodium formate as substrates. Effects of different conditions on asymmetric reduction were inverstigated. When substrate and washed cell concentration were 5 g/L and 10% (w/v), the optimum reaction conditions were: pH7.0, temperature 30oC, reaction time 48 h, the optical purity and yield of (R)-1-phenyl-1,2-ethanediol reached 100% enantiomeric excess and 64.9%, respectively. When concentration optimized sodium formate and suitable ZnSO4 were added into the reation mixture, the product was produced with high optical purity of 100% e.e. and yield of 85.9%.