The Construction And Application Of Gene Engineering Strain Of Glycosyltransferase

Glycosyltransferase is the key enzyme in the process of glycosylation, with the function of conducting glycosylation of antibiotics in its later stage of biological synthesis. The glycosylation products have many biological functions. Validamycin A is produced from the catalysis of validoxylamine A by glycosyltransferase ValG. The aim of this research is to construct engineering Escherichia coli strain with high level expression of glycosyltransferase, which will then be applied to biological synthesis of Validamycin A.Primers were designed according to the gene sequence of glycosyltransferase ValG and the gene sequence was then amplified through PCR employing genomic DNA of streptomyces hygroscopicus as template. After double digestion and connecting operation towards the PCR products and pET-28b(+)vectors, the recombinant plasmids were then transformed to the E. coli DH5a. Transformants with recombinant plasmids were screened and were identified by colony PCR and double digestion of restriction enzyme, with the result that the target gene fragments were successfully inserted into the recombinant plasmids.In this work lactose was taken as the substitution of IPTG for the inducing expression of glycosyltransferase. When IPTG was taken as the inducer, the producing glycosyltransferase took up36.0%of the whole cell protein. The optimal inducing condition of lactose was that100mM lactose was added for induction at30℃when recombinant bacteria grew to a degree when OD600was0.6-0.8. In this condition, glycosyltransferase took up38.5%of the whole cell protein. Result showed that lactose was a better inducer in the research than IPTG, at the same time, fermentation cost had also been reduced.Fermentation medium and culture conditions were also investigated through single factor experiment, responding surface method and so on employing shake culture method. The optimal culture conditions were as follows:10h for seed liquid culture time;6.5for initial pH;2%for inoculation amount;30mL for outfit fluid amount in250mL shake flask; The optimal constitution of culture medium was lactose with a concentration of4.7g/L; yeast extract, ammonium chloride and potassium phosphate with percentage of4.95%,0.27%and11%, respectively; Ca2+ with a concentration of0.88mM.