Cloning And Function Analysis Of Two Regulation Genes From Saccharopolyspora Spinosa

In this paper, the 700bp SAM-s gene and 400 bp ssgA-sp gene were first amplified by PCR from Saccharopolyspora spinosa. The SAM-s gene PCR product was cloned downstream of promoter PT7 of pET-28a(+), an Escherichia coli expression vector. And a 25kDa protein was over-expressed when E. coli BL21(DE3)(pETsams) was induced with IPTG, but the products existed in the form of inclusion bodies.To further explore the function of these two regulatory genes, we first need to construct a suitable expression vector for Streptomyces. The plasmid pLSB2, a derivative of pCJR24 which was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes, includes the pathway-specific activator gene actⅡ-ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actⅠpromoter and the associated ribosome binding site are located upstream of an Nde I site (5'-CATATG-3') which encompasses the actl start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActⅡ-ORF4 activator. We inserted the integrase geneφC31 int and attP site, which derived from plasmid pSET152, into pLSB2, a suicidal streptomyces plasmid. The resulting conjugably transferable vector can integrate into the genome of streptomyces by site-specific integration. We initially demonstrated that plasmid pMF was conjugably transferred with high frequency into S. coelicolor M145,S. lividans TK24 and Saccharopolyspora erythraea 2338 from E. coli. Southern blotting results showed that pMF was able to integrate into the genome of streptomyces.We also cloned the putative S-adenosylmethionine synthetase (SAM-s) gene and ssgA-sp gene from Sacc. spinosa S08-4 into pMF under the control of PactⅢ/PactⅠand conjugated into S. coelicolor M145. The original strain and conjugants were cultured. Results showed that:(1) The Act production of conjugant M145/pMFsam was 3.2 times of the control strain M145/pMF, while there was not much differences between the two control strains M145 and M145/pMF. We found that the spores formation were apparently inhibited in M145/pMFsam, and Act and Red pigment formation was earlier than the two control strains on GYM and R4C medium. (2) ssgA-sp gene could complemented the defective phenotype of the ssgA disrupted mutant strain GSA3. Overexpression of ssgA-sp resulted in a dramatic morphological change of the vegetative hyphae of S. lividans, without forming lump-like mycelium network.The cloning and function analysis of these two genes in steptomyces indicated that the putative SAM-s and ssgA-sp gene may play an important role in morphological differentiation and secondary metabolism process of actinomyces.