The Cloning And Functional Studing Of Regulatory Genes Related Biosynthesis Of Spinosad

Spinosad,generated by Saccharopolyspora spinosa,are part of a new class of highly efficient and environment-friendly antibiotic insecticides.However,the spinosad biosynthetic capacity of wild-type S.spinosa strain is weak,therefore,oriented engineering genetically of S.spinosa to improve spinosad yields is practical and meaningful.It was reported that polyphosphate kinase gene(ppk)and leucyl aminopeptidase gene(pep A)play negative roles in the control of antibiotic production in Streptomycets.Disruption of these genes can effectively improve production of the secondary metabolites.Parts of these homologous genes of ppk and pep A were amplified and cloned into Escherichia coli-Streptomyces shuttle vector p OJ260 to generate p OJ260-ppk and p OJ260-pep A,which were transformed into S.spinosa by conjugation.Transconjugants in which ppk gene or pep A gene was successfully disrupted were chosen for further study.Mycelia in S.sp-?ppk and S.sp-?pep A displayed a much higher degree of fragmentation and more branches when compared with wild strain.The growth rate of S.sp-?ppk and S.sp-?pep A were delayed and their biomass were reduced.Shake flask fermentation demonstrated that spinosad yield increased by 1.19 and 1.37 times in S.sp-?ppk and S.sp-?pep A strain.3'-O-methyl rhamnose is not only an important component for activity of spinosad,but also composition of cell wall.To improve synthesis of this key substrate,rhamnose expression vector p JNRM containing rhamnose biosynthetic genes was transfered into S.spinosa through conjugation and engineering strain S.sp-RM was constructed in which rha genes were doubled.PCR results showed that the vector p JNRM had been integrated into the genome of S.spinosa successfully;Real-time PCR results revealed that transcript levels of gtt,epi,kre and gdh in S.sp-RM were improved significantly;HPLC results represented that spinosad yields in S.sp-RM increased by 1.72 times compared with the original strain.In conclusion,this study for the first time achieved disruption of ppk and pep A in S.spinosa,as well as over-expression of rhamnose biosynthesis gene in S.spinosa through the approach of genetical engineering.The biosynthesis of spinosad were effectively promoted,which proved the efficient feasibility of this method.These results laid an important foundation for enhancing production of spinosad by disruption of other negative regulators or over-expression of other positive regulatory genes and rate-limiting enzymes.