大肠杆菌-链霉菌穿梭表达载体pMF的构建及其应用

Construction of Escherichia coli-Streptomyces shuttle expression plasmid pMF

摘要:

摘要：【目的】构建能定点整合到链霉菌（Streptomyces）染色体上的高效表达载体。【方法】以链霉菌自杀型表达载体pLSB2为基础，通过插入链霉菌噬菌体ΦC31整合酶基因int和attP位点(Phage attachment site)，构建了能在大肠杆菌和链霉菌之间进行接合转移并定点整合到链霉菌染色体上的表达载体pMF。将pMF转化大肠杆菌ET12567(pUZ8002)，并分别接合转移天蓝色链霉菌（Streptomyces coelicolor M145）、变铅青链霉菌（Streptomyces

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.