Computer Simulation Of SsbR Protein In Saccharopolyspora Spinosa And Its Functional Verification

Saccharopolyspora spinosa is an important industrial microorganism known for spinosad production,which is a kind of biological insecticides with high efficiency,broad spectrum and low toxicity.In order to improve the yield of spinosad,it is necessary to gain insight into the regulatory mechanisms and networks of spinosad biosynthesis in S.spinosa.Cascade regulation is an important way for actinomycetes to regulate secondary metabolism.This study mainly performed computer simulation and functional verification of cascade regulation key homologous proteins SsbR(Saccharopolyspora spinosa ?-butyrolactone Receptor)in S.spinosa:1?The cascade regulation key homologous proteins SsbR,and its target homologous proteins SstA1,SstA2,and SstA3,eight downstream effector homologous proteins were found in S.spinosa.And computer simulation analysis was taken on SsbR protein.2?QRT-PCR analysis of the expression levels of ssbR gene,sst As genes and effector homologues genes in the fermentation process revealed that the expression levels of ssbR gene and sstAs genes were in opposite trends,which was consistent with the prediction of SsbR protein repress the expression of sstAs genes as a TetR family transcriptional regulator.3?EMSA experiments showed that SsbR protein specifically binded to the sstA2 gene promoter region in vitro,which verified that SsbR protein regulated the expression of sstA2 gene.4?The ssbR gene deletion mutant was constructed by homologous double exchange.The spinosad production of ssbR gene deletion mutant was 26.9% higher than that of the original strain,indicating that SsbR protein has negative regulation of spinosad synthesis in S.spinosa.5?The spinosad produced by ssbR gene deletion mutant was used to develop a spinosad 45% water dispersible granule and greenhouse experiment showed its good insecticidal efficacy.