Co-expression Of Cephalosporin C Acylase And Vitreoscilla Hemoglobin In PtsG Gene-defeicient E.coli

Cephalosporin is a semi-synthetic antibiotic that is widely used in the world.In clinical,the intermediate of most cephalosporin antibiotics is 7-aminocephalosporanic acid.7-aminocephalosporanic acid mainly comes from the deacylation of cephalosporin C.The main deacylation methods include chemical method,two-step enzymatic method and one-step enzymatic method.The chemical deacylation method has the disadvantages of unfriendly to environment and harsh reaction conditions,which have been eliminated by the market.The two-step enzyme method has been basically applied to industrial production,but the conversion rate of the two-step enzyme method is low,and the reaction is not easy to control.One-step enzymatic method can directly deacylate cephalosporin C into 7-amino cephalosporanic acid,which has been widely studied for its advantages such as high conversion rate,high economy and friendliness to environment.However,the activity of cephalosporin C acylating enzymes existing in nature is low and unstable,which is difficult for industrial application.Therefore,obtaining a cephalosporin C acylating enzyme with high conversion efficiency becomes the key to solving the problem.Vitreoscilla hemoglobin can assist oxygen to enter the cell,so that the cell can continue to grow under the condition of limited oxygen,which is beneficial to the accumulation of metabolites.The use of vitreoscilla hemoglobin increased the expression of foreign proteins of recombinant strains.The ptsG gene is an important gene in the phosphoenolpyruvate-sugar phosphotransferase system.PTS-deficient strains can significantly reduce the glucose transport rate and balance the carbon flow metabolism in the cell,thereby reducing acetic acid synthesis and conducive to high-density fermentation..Therefore,the knockout of the ptsG gene can facilitate the growth of recombinant strains and increase the expression of foreign proteins.In the preliminary work of the laboratory,the content of base GC was reduced and some the cephalosporin C acylase gene bases were mutated.After the optimization of the medium,the enzyme activity was found to reach 3.68U/mL,which was higher than that of Tsinghua University.After optimizing the fermentation medium,the enzyme activity of the enzyme was increased by 24%.Therefore,experiments based on this gene have certain experimental significance.The experiment successfully constructed recombinant strains pETDuet-CA/pRSFDuet-vgb（ptsG（+）BL21（DE3））that co-expressed vitreoscilla hemoglobin and cephalosporin C acylase;pETDuet-CA/pRSFDuet-vgb（ptsG（-）BL21（DE3））;pRSFDuet-CA/pACYCDuet-1-vgb（ptsG（+）BL21（DE3））;pRSFDuet-CA/pACYCDuet-vgb(ptsG（-）BL21（DE3）,the enzyme activities are 0.67 U/mL,1.40U/mL,1.1 U/mL,2.1 U/mL.In E.coli without ptsG deletion,In recombinant strains,with vitreoscilla hemoglobin,enzyme ctivity of cephalosporin C acylase increased approximately 30% compared to recombinant strains without the hyaglobin hyaline protein.The absence of ptsG allowed Hyaglobin hyaline and cephalosporin C acylation in E.coli The co-expression in is about doubled.After 10 generations pass on of recombinant strains,there is a reasonable decrease in the enzyme activity of the recombinant strains.The optimal IPTG induction concentration is 0.05M.The enzyme activity is the highest when the volume ratio of the medium is 10%.The optimal medium p H is 7.5.The single-factor experiment determined that the optimum external carbon source in the medium is0.5%glycerol,and the highest enzyme activity recombinant strain pRSFDuet-CA/pACYCDuet-vgb(ptsG^（-）BL21（DE3）is 3.5U/mL;the optimal external nitrogen source is 6%beef extract,the highest enzyme activity recombinant strain pRSFDuet-CA/pACYCDuet-vgb(ptsG^（-）BL21（DE3）is 6.5 U/mL.Comparing the recombinant strains constructed with no vgb gene expression and no ptsG gene deletion constructed according gene source,the enzyme activity increased by 77% after media optimization both.Experiments have proved the feasibility of simultaneously expressing cephalosporin C acylase and vitreoscilla hemoglobin in the absence of the ptsG gene,which provides a new path for industrial production applications.