Rationally Breeding And Expression Of AviD Gene In Streptomyces Viridochromogenes

Avilamycin is a new type of digestive enhancer and metabolic modulater, which is producted by Streptomyces viridochromogenes, belonging to oligosaccharide antibiotic. Avilamycin has high biological safety level and efficiency, and is widely used as feed additive for animals. At present, the low antibiotic productivity of strain and the expensive product price limit the application of avilamycin in China. So rational screening methods and metabolic engineering method were taken in this study to breed avilamycin high-product strain.In order to improve the yield of avilamycin producting strain, S. viridochromogenes NRRL2860preserved in our laboratory being used as original strain, UV-LiCl complex mutagenesis, microwave-acridin orange complex mutagenesis, CaCl2, avilamycin, streptomycin resistance screening method were taken in this study. And a high-product strain MY-20with stable genetic character was obtained. The fermentation result show that MY-20showing high-yielding character, with avilamycin A and B yield at1.Og/L and3.08g/L respectively after168hour fermention, which was up to488%and584%of original strain NRRL2860.Meanwhile, metabolic engineering method was tried to improve avilamycin yield. A conjugal transfer method in S. viridochromogenes and E.coli was established. Using pSET152as transfer plasmid, MS medium and the1:1radio between the donor E.coli ET12567(pUZ8002) and the acceptor S. viridochromogenes, was optimum condition of transfer system between S. viridochromogenes and E.coli. The key gene aviD in avilamycin (?)ynthesis pathway was overexpressed through the conjugal transfer method,and two recombination strain TF-1,TF-2was got, with avilamycin A yield improveing150%, and avilamycin B disappeared in shake culture. The result show that the biosynthesis of avilamycin effective constituent was enhanced effectively through overexpress aviD gene which encoding starting enzyme dNTP-glucose synthase in biosynthesis of D-olivose and,2-deoxy-D-evalose groups in avilamycin molecule.