Construction Of Spinosad High-yield Strains Saccharopolyspora Spinosa And Function Analysis Of Regulatory Genes Related Biosynthesis Of Spinosad

Spinosad,generated by Saccharopolyspora spinosa,is a new macrolide natural product with high biological insecticidal activity and ideal green insecticide.Which had very good control effect on Lepidoptera?Diptera?Coleoptera and other crop pests.The main active ingredient of spinosad is spinosadA and spinosadD.However,the spinosad biosynthetic capacity of wild type strain was weak,and fermentation cycle was comparatively long,which limited the industrial production of spinosad products.So far,little is known about regulatory genes related to primary metabolism and secondary metabolism.We can make directional genetic modification on Saccharopolyspora spinosa by genetic engineering,increasing or blocking with regulatory genes expression which related to synthesis of spinosad expected to improve spinosad yields.Ribosome recycling factor(RRF)was encoding by Frr gene,playing a key role in the protein synthesis process of ribosomes recycling.Which collapse the complex after the termination of translation,supplying plentiful ribosomes for a new round of protein synthesis.Here we studied the effects of overexpression Frr gene on spinosad biosynthesis and spore formation in Saccharopolyspora spinosa.We had generated the overexpression vector pUC-spn-PermE-Frr.Then,The vector pUC-spn-PermE-Frr was introduced into S.spinosa generating the recombination strain S.spinosa-Frr,The strain S.spinosa-1 whose chromosome was integrated by original plasmid pUC-spn and the Wild type strain S.spinosa was used as control strain in this study.HPLC results revealed that spinosad yield of the recombination strain increased 300%than S.spinosa,and increased 132%than S.spinosa-1,Under the scanning electron microscopy,it was showed that the recombination strain had longer mycelial and more branches,which was similar with S.spinosa,while the strain S.spinosa-1 had shorter mycelial and less branches;These findings revealed that overexpression of Frr in S.spinosa had a effect on sporulation and spinosad biosynthesis in some cases,which has a important significance on strain improvement of S.spinosa.Phosphomannomutase widely exists in eukaryotes,encoding by manB gene.Phosphate-6-mannose can converted into phosphate-1-mannose under the action of phosphomannomutase,which have closely related to primary metabolism and secondary metabolites synthesis process.We can make genetic modification on genes related to primary metabolism in Saccharopolyspora spinosa to improve spinosad yields.Here,the 1.0 kb manB gene segment was amplified by PCR,then cloned intothe vector pOJ260,generating manB disrupted vector pOJ260-manB.This vector was introduced into S.spinosa by conjugation,and transconjugant S.spinosa-?manB was obtained successfully.HPLC results revealed that spinosad yield of the mutant strain increased 180%than S.spinosa.On BHI and TSB agar,the spore formation rate of mutant strain S.spinosa-?manB were advanced,Besides,the spore production was more than S.spinosa.Our results revealed that disruption of manB affected the development of S.spinosa and the biosynthesis of spinosad.In conclusion,this study for the first time achieved function analysis of Frr gene and manB gene in S.spinosa,We also constructed the spinosad high-yield strains successful by the approach of genetical engineering.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.