Construction of Escherichia coli-Streptomyces shuttle expression plasmid pMF

Abstract:

Abstract: ［Objective］ To construct an E. coli-streptomyces shuttle vector pMF that can integrate into the genome of streptomyces by site-specific integration.［Methods］We inserted the integrase gene ΦC31 int and attP site into pLSB2, a suicidal streptomyces plasmid. The resulting conjugably transferable vector which contains the activator promoter system actⅡ-ORF4/PactⅠfrom Strepromyces coelicolor A3(2) could be integrated into the genome of streptomyces by site-specific integration.［Results］The 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 confirmed functional protein expression by cloning a putative S-adenosylmethionine synthetase (SAM-s) gene from Sacc. spinosa S08-4 into pMF and conjugated into S. coelicolor M145. Protein expression were confirmed using Western blotting.［Conclution］pMF can be used as an effective tool for site-specific integration expression of foreign gene in streptomyces.