Construction Of Spinosyn High-yield Strains Via Genetically Modifying Saccharopolyspora Spinosa

Spinosyns,generated by Saccharopolyspora spinosa,is a novel macrolide natural product with high biological insecticidal activity and an ideal green insecticides.Howerer,the spinosyns biosynthetic capacity of wild-type S.spinosa strain is weak and the spinosyns yield is very low,therefore,oriented engineering genetically of S.spinosa is expected to be an effective way to improve spinosyns yields.In this study,the cyclic AMP receptor protein gene(crp)and cell division activator gene(ssgA)were amplified by PCR and placed in the control of enhanced promoter PermE by restriction digestion and ligation,respectively.Fragments PermE-crp and PermE-ssgA were subcloned into E.coli-Streptomyces shuttle vector pUC-spn stored in our lab by method of Red/ET homologous recombinational technology,and the recombinant vectors pUC-spn-PermE-crp and pUC-spn-PermE-ssgA were constructed.These recombinant vectors were transfered into S.spinosa through conjugation separately and were integrated into the chromosome of S.spinosa by single-cross homologous recombination,and recombinational engineering strains S.spinosa-Crp and S.spinosa-SsgA with genetic stability were obtained successfully.Scanning electron microscopy results revealed that,mycelium of engineering strain S.spinosa-SsgA exhibited higher degree of fragmentation and fewer branches in comparision with the control strain,and formed a certain number of spore septum;We analysed spinosad production by high performance liquid chromatography,Shake flask fermentation results indicated that compared with the control strain,spinosad production of engineering strain S.spinosa-Crp increased by 1.28 times,increased by 1.55 times in the engineering strain S.spinosa-SsgA;RT-qPCR results displayed the transcriptional levels of corresponding genes in engineering strains upregulated.These results indicate that overexpression of crp and ssgA had certain impacts on mycelial morphology and growth of S.spinosa,and promoted the biosynthesis of spinosad effectively.Butenyl-spinosyns,produced by Saccharopolyspora pogona,which shows high similarity to spinosyns in molecular structure,and its insecticidal activity is higher compared with spinosyns,however,the butenyl-spinosyns yield of wild-type S.pogona is very low.3'-O-methyl rhamnose is not only an important component for activity of butenyl-spinosad,but also composition of cell wall.To improve synthesis of the key substrate 3'-O-methyl-rhamnose,we amplified rhamnose biosynthetic genes gtt,epi and gdh+kre which arranged in genome of S.spinosa scatterly by three sections and cloned them into E.coli-Streptomyces shuttle vector pJN100,thus rhamnose expression vector pJNRM was obtained;This was transfered into S.pogona through conjugation and engineering strain SPOG-RM was constructed in which rha genes were double.PCR results showed that the vector pJNRM had been integrated into the genome of S.pogona successfully;Real-time PCR results revealed that transcriptional levels of gtt,epi,kre and gdh in SPOG-RM were improved by 54.1-fold,26.8-fold,14.0-fold and 11.9-fold respectivly;HPLC results represented that butenyl-spinosyn yield in SPOG-RM improved by 214%compared with the original strain.In conclusion,this study for the first time achieved overexpression of positive regulatory genes ssgA and crp in S.spinosa,as well as overexpression of rhamnose biosynthesis gene in S.pogona through the approach of genetical engineering.The biosynthesis of spinosyns and butenyl-spinosyns were effectively promoted,which proved the efficient feasibility of this method.These results lay an important foundation for improvming production of spinosyns compound by overexpression of other positive regulatory genes and rate-limiting enzymes.