Strengthening The Glycosyl Transferase Engineering Strain UDP-Glucose Synthesis Pathwayand Application

In the process of validamycin fermentation,the precursor Validoxylamine A will accumulate in quantity,which greatly affected the quality of products.In order to solve this problem,the engineering strain of glycosyltransferase that use for catalyzing validoxylame A glycosylation have constructed and preliminary applied in the biotransformation of Validoxylamine A glycosylation to generate validamycin A,however,the result is not ideal.In this research,the engineering bacteria is successfully built,which can coexpress the glycosyltransferase,the UDP-glucose pyrophosphorylase and the phosphoglucomutase.Their expression will be investigated,which will then be applied to biological synthesis of Validamycin A.Based on the research of the condition for catalyzing Validoxylamine A glycosylation by the engineering bacteria,the optimal condition of the catalytic will be found.Primers were designed according to the gene sequence of the glycosyltransferase valG,the UDP-glucose pyrophosphorylase galU and the phosphoglucomutase pgm.The gene sequence for the TA-cloning wasthen amplified through PCR.After double digestion and connecting operation towards the pMD19-T-valG?pMD19-T-galU?pMD19-T-pgm and pETDuet-1vectors,the recombinant plasmids were then successfully constructed.Transformants with recombinant plasmids were screened and were identified by colony PCR and single-double digestion of restriction enzyme,with the result that the target gene fragments were successfully inserted into the recombinant plasmids.Therefore,The engineering bacteria is named E.coli BL21(DE3)-p ETDuet-valG-galU-pgm.In this work the engineering bacteria induced with lactose or IPTG which coexpress the glycosyltransferase,the UDP-glucose pyrophosphorylase and the phosphoglucomutase has the different situation about the protein expression.The optimal inducing condition of lactose was that 80 mM lactose was added to induce 8 h at 25 ? when recombinant bacteria grew to a degree when the time was 3 h.In this condition,the glycosyltransferase,the UDP-glucose pyrophosphorylase and the phosphoglucomutase took up respectively 10.3%,13.6%,10.4%of the whole cell protein and the weight of the wet bacteria is 11.3 mg/mL.The result showed that lactose was a better inducer in the research than IPTG about the optimal protein expression and the intracellular quantity of UDPG synthesized by the engineering bacteria can get 6-7 times than the control.Moreover,the dates show that the optimal condition for the resting cell catalysis Validoxylamine A to glycosylate is the cells used forcatalytic has best capacity after being induced by 80 mM lactose,the bacteria concentration in the catalytic system should be choosed 0.1 g/mL,when the inputing time of the substrate is put off to 22 h the mole yield of Validoxylamine A can improve by 7%,with the 3% of the cellobiose and the 0.25% of the substrate concentration the mole yield of Validoxylamine A can also reach more than 95%.It is necessary to control the catalytic cycle for avoiding the product degradation.