Function Analysis Of Spinosad Biosynthetic Genes Psda And BldD In Saccharopolyspora Spinosa

Spinosad,generated by soil actinomycetes Saccharopolyspora spinosa,is a new macrolide natural product with high biological insecticidal activity and ideal green insecticide.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 the rate limiting steps and metabolic regulatory genes related to spinosad biosynthesis.Exploring the structure and regulation of different regulators in S.spinosa could provide a basis for directed genetic manipulation of spinosad-producing strains by genetic engineering,and expected to be a new breakthrough approach to improve spinosad yields.The polysaccharide deacetylase(PSDA),which was firstly isolated from Bacillus subtilis,had an important role in the cell wall formation and spore germination of B.subtilis.Our previous proteomic analysis of S.spinosa revealed that there was a strong correlation between the trend of polysaccharide deacetylase expression and spinosad biosynthesis during S.spinosa fermentation.Here,the 1.1 kb psda gene segment was amplified by PCR,then cloned into Escherichia coli-Streptomyces shuttle vector p OJ260,generating psda disrupted vector p OJ260-psda.This vector was introduced into S.spinosa by conjugation,and transconjugant S.spinosa-?psda was obtained successfully.On BHI and TSB agar,the spore formation and germination rate of mutant S.spinosa-?psda were delayed.Shaking flask fermentation demonstrated that the biosynthsis of spinosad could not be detected on HPLC analysis level in S.spinosa-?psda.Our results revealed that disruption of psda affected the development of S.spinosa negatively,and repressed the biosynthesis ofspinosad.BldD was a global transcriptional regulator,participating in morphological development and secondary metabolism in Streptomyces.Here,we studied the effects of bldD gene overexpression on spore formation and spinosad biosynthesis in S.spinosa.The ORF of bldD was placed under the control of the promoter for erythromycin resistance gene Perm E by overlap PCR.This was cloned into E.coli-Streptomyces shuttle vector p UC-spn,generating recombinant vector p UC-spn-Perm E-bldD.This vector was introduced into S.spinosa from E.coli S17 through conjugation,and integrated to the chromosome of S.spinosa via homologous recombination.The resultant conjugant,S.spinosa-BldD,was genetically stable.On BHI and TSB agar,the sporulation of recombination strain S.spinosa-BldD was apparently inhibited.Shaking flask fermentation demonstrated that the recombination strain overproduced 1.35-fold spinosad compared with the control strain.These findings revealed that overexpression of bldD from S.spinosa had a negative effect on sporulation,and effectively promoted spinosad biosynthesis in S.spinosa.In conclusion,this study cloned psda and bldD gene from S.spinosa for the first time,and explored their roles in morphological differentiation and spinosad biosynthesis process of S.spinosa.These results laid an important foundation for studying regulatory network of spinosad biosynthesis.