Cloning, Expression And Functional Research Of SpnG Gene And SpnK Gene From Saccharopolyspora Spinosa

Spinosad is widely used in agriculture as a potent insect control agent and animal health product because of its high efficiency against target insects and environment-friendly characteristics. The biosynthesis of spinosad in wild-type Saccharopolyspora spinosa is very low, engineering Saccharopolyspora spinosa is widely used. Enhancing spinosad biosynthesis in Saccharopolyspora spinosa through increasing the gene dose of gene in spinosyn cluster was studied.In this paper, the 1206 bp spnG gene and 1249 bp spnK gene were amplified by PCR from Saccharopolyspora spinosa and cloned into a pMD18-T for sequencing spnG gene and spnK, respectively. The spnG gene and spnK gene PCR product were then subcloned to the pET-28a. And 43 kDa protein and 45 kDa were over-expressed when E. coli BL21 (DE3)(pET-28a-spnG) and E. coli BL21(DE3)(pET-28a-spnK) were induced with IPTG. MS spectrum identification of SpnG and SpnK showed a good result respectively. The results provide material guarantee for purification and study the dynamic characteristics of the enzymetic reaction of the two proteins.The E. coli-Streptomyces shuttle expression vector pMF was chosen and then cloned the spnG gene and spnK from S. spinosa SP06081 into it respectively and pMF-spnG and pMF-spnK were engineered. Then the vector of pMF-spnG was conjugated into Streptomyces coelicolor M145 and Streptomyces lividans TK24. The original strain and conjugants were cultured. The color caused by the secondary metabolite Red of transformant are much dark than the two control strains, respectively. The spore formation of TK24/pMF-spnG was apparently earlier than the two control strains. Fermentation results showed that:the Act production in conjugant M145/pMF-spnG was increased by 210% and 190% than the control. The Act production of conjugant TK24/pMF-spnG was was increased by 2200% and 1800% than the control. SpnG of 43 kDa was detected by SDS-PAGE in the whole-cell protein of M145/pMF-spnG and TK24/pMF-spnG, and further demonstrated by MS. It was the spnG gene expressed in two Streptomyces that enhance the production of secondary metabolites.spnG gene and spnK gene were first cloned from Saccharopolyspora spinosa, subcloned to the vector of pMF and the function of spnG gene was studied in the two streptomyces strain. Our results set the stage for future duplicating spnG gene and spnK gene in Saccharopolyspora spinosa.