The Construction And Fermentation Optimization Of High Copy Gene Engineering Strain Of Glycosyltransferase

Validamycin is a kind of amino alcohols ring antibiotics with the advantages of efficiency,low toxicity,environmentally friendly and less prone to fungal resistance.It is one of the main biological pesticides used by the major rice producing areas of Southeast Asia for controlling of rice sheath blight and Blast.Meanwhile,it is the materials of diabetes medication.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 high copy Streptomyces of glycosyltransferase ValG,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.Then the PCR products and pMD-19 vectors were used for TA cloning.The recombinant plasmids were then transformed to the E.coli DH5a.The recombinant plasmids were extracted and double digested,and then they were connected with shuttle plasmid pIJ8630.The recombinant shuttle plasmid was named pIJ8630-valG.This research tried to insert valG into Streptomyces hygroscopicus by conduction of E.coli and Streptomyces hygroscopicus,transformation of Streptomyces hygroscopicus competent cells from plasmid DNA,electro transformation.After screened by the plates which contend resistance and identified by colony PCR,the target gene fragments were successfully inserted into the Streptomyces hygroscopicus.Fermentation medium and culture conditions were also investigated through PB experiment,responding surface method and so on.The optimal culture conditions were as follows:;6.43%for inoculation amount;The optimal constitution of culture medium was maltose,glucose,soybean meal and com flour with the concentration of 28.5 g/L,10 g/L,30.8 g/L and 10 g/L;KH2PO4 and NaCl with percentage of 1%,and 1%respectively.The results above built a good foundation for further research of improving Validamycin A production by over expression of other gene.