Cloning Of The Aldehyde Reductase Gene From S.erythraea And The Application Of Synthesizing Chiral Alcohol

Chiral alcohols with special functional groups are important building blocks for synthesizing chiral drug, and synthesizing chiral alcohols by the asymmetric reduction of carbonyl compounds has become an important part of chiral synthesis. Biocatalysis plays a very important role in carbonyl asymmetric reduction because of its mild reaction conditions, less stressful environment and efficient stereoselectivity. Screening the most suitable biocatalyst is critical. In 2006, Bali reported that ketoreductase of polyketides synthase in Saccharopolyspora erythraea as one of the members of short-chain dehydrogenase superfamily existed optimistic ability of biological selective reduction, especially to the substrates whose structures contain cyclohexanone.In this work, DNA of S.erythraea was extracted.The primer sequences were designed according to the result Alexandros reported.We cloned the mild eryKR1 gene. Based on the nucleotide sequences of the ketoreductase from the first extension module of the erythromycin polyketide synthase, and the nucleotide sequences of ketoreductase of polyketide synthase in Actinorhodin (ActKR) in the Genbank, gene specific primers were designed. Through the overlapping PCR manner, the gene sites determining its substrate specificity in the ketoreductase (KR1) domain is replaced by that determining its specificity in ActKR, and we get the mutated KR1 domain DNA fragment eryKR1M. We cloned eryKR1 and eryKR1M into vector pET-28a, built the plasmid pET-eryKR1M and introduced the plasmid pET-eryKR1M and pET-eryKR1 into Escherichia coli BL21.Then we built the plasmid pET-GDH with the same method as the coenzyme regeneration. After 6 hours of inducing by IPTG, we detected the expression of the protein eryKR1M ,eryKR1,GDH by SDS-PAGE. At last, we examined its effect on four kinds of different substrates (ethyl 4-chloro-3-oxobutanoate, acetophenone, 2-octanone and cyclohexanone) using the fermentation technology.The results showed that the best annealing temperature was 68℃.The protein sequences of the cloned genes had the high homology compared with the reported protein sequences(reached 98%). The target gene of pET-eryKR1M differented from the gene of the ketoreductase from the first extension module of the erythromycin polyketide synthase only in the sites determing the specificity of the substrates, and was replaced by the sites of ketoreductase of polyketide synthase in Actinorhodin(ActKR), which is accordant with our initial presumption. In addition, through SDS-PAGE, all the target protein eryKR1M,eryKR1,GDH were detected. At last, the results of fermentation showed the wild gene existed optimic effects on Cyclohexanone ,which suggests that the engineering bacteria would be used for the biocatalysis of chiral alcohols the in the future.