Cloning And Expression Of CgKR2 And The Construction Of The Plasmid Addiction System

(R)-2-hydroxy-4-phenylbutyrate is the key second alcohol,which is used to synthesize angiotensin-converting enzyme inhibitors.Using ketoreductase to produce(R)-2-hydroxy-4-phenylbutyrate has a great of advantages.The process requires NADH or NADPH to transfer hydrogen,so the expensive coenzymes are necessary.In order to make ketoreductase catalyzed successfully and to reduce production cost,the commonly used technology is to build a co-expression system of ketone reductase and dehydrogenase in the industry.After a review of related literatures,we choosed the CgKR2 gene in Candida glabrata and BmGDH gene in Bacillus megaterium as target genes.We cloned the two target genes.Prokaryotic expression vector “pET17b-TC-CgKR2,pET17b-TC-BmGDH” were constructed through gene targeting cloning technology.We built the two cistron expression structure of the two genes“T7 promoter-T7 RBS-CgKR2-T7 RBS-BmGDH-T7 terminator”.The expression vector “pET17b-TC-CgKR2-BmGDH” was constructed by overlap extension and gene targeted cloning technology.After induction,the recombinant strains BL21-pET17b-TCCgKR2-BmGDH and BL21-pET17b-TC-CgKR2 both highly expressed proteins,and the molecular weight of proteins are consistent with expection.This laid a solid foundation for the next step to analysis enzyme activity of CgKR2.To make CgKR2 gene has a efficiently expression and to improve the plasmid stability,this work has built a plasmid addiction system.By knockout of an essential gene in the E.coli genome and transfer it to the expression plasmid,a plasmid addiction systems was constructed.Triosephosphate isomerase(tpi A)and glucosamine synthase(glmS)were selected as target genes.The two genes were cloned and ligaed to pUCm-T vector,and then transformed into E.coli strain DH5?.On the basis of the pUCm-tpiA and pET17b-TC,we built the complementary expression plasmid “pUCm-tpiA-pET17b-TC”.Temperature sensitive plasmid pMAK705 was used to construct the gene knockout system.By cloning of the homologous sequences of target gene and integrated it into the pMAK705,recombinant plasmid pMAK705-tpiA and pMAK705-glmS was constructed.