Study On Saccharopolyspora Erythraea Expression Vector

Erythromycin is the secondary metabolite of Saccharopolyspora erythraea. It is a broad spectrum macrolide antibiotic and clinically used widely. Erythromycin has already developed three generations. Ketolide, the third-generation erythromycin can not only strengthen the acidity stability, but also kill or restrain many drug-resistant bacteria. At present, the third generation is also completed by modifying the structures of erythromycin by chemical means. In order to avoid complicated chemical steps, now genetic engineering methods are used to synthesize the third-generation erythromycin.The synthesis process includes two parts: the synthesis and post-modification of 6-deoxy-erythronolide B, 6-dEB. Based on changing erythromycin biosynthesis genes in S. erythraea chromosome, many erythromycin analogs had been synthesized. To modify the structures of erythromycin analogs in S. erythraea, stable expression vectors need to be constructed.Existing plasmids and inherited materials are used to construct expression vector pZMW which could be integrated into S. erythraea chromosome and multi-copy expression vector pZM. As most Streptomyces promoters are invalid in S. erythraea, the promoter of erythromycin-resistant gene, ermE, and Streptomyces chromosomic integration site, attP, and apramycin-resistant gene, apr from plasmid pSET152 are utilized to construct the expression vector pZMW. The vector is 10260bp containing PermE promoter, fd terminator, multiple clone site, aminobenzylpenicillin and apramycin resistance genes, Streptomyces chromosomic integration site, attP.The multi-copy expression vector pZM is a shuttle plasmid which can replicate in Escherichia coli, Streptomyces and S. erythraea. It contains PermE promoter, fd terminator, multiple clone site, thiostrepton and ampicillin resistance genes, and ColE1 ori and pJV1 ori replicons which make the vector replicate in E. coli and S. erythraea. This vector is 9880bp.The most important function of expression vectors pZMW and pZM is to express exogenous genes. As their stabilities stand more important positions, stability research on pZMW and pZM would be done. The plasmid pZMW was isolated from E. coli. S. erythraea A226 protoplast was prepared and transformed with plasmid pZMW by PEG-mediation. The A226 transformant culture was transferred 5 to 10 generations in TSB media without antibiotics. Total DNA of pZMW transformants was extracted on a small scale in order to PCR identification. And S. erythraea A226 protoplast was prepared and transformed with plasmid pZM. The A226 transformant culture was transferred 5 to 10 generations in TSB media without antibiotics and plasmid DNA of pZM transformants was prepared. Stability research on expression vector indicates that pZMW and pZM are stable expression vectors in S. erythraea.But pZMW, the chromosome integrated expression vector, has two E. coli replicons. The result is that the plasmid is unstable in E. coli. Because of two resistance genes, it increases the length of expression vector and it is not suitable for the expression of exogenous genes. In order to solve the problems mentioned above, the promoter of erythromycin-resistant gene where the site BamH I was inactivated was amplified by PCR from genomic DNA of S. erythraea, and ligated to plasmid pET-22b(+). Fd terminator from plasmid pKA was utilized, then multiple clone site was inserted. Finally, plasmid pSET152 where the sites EcoR I and EcoR V were inactivated was utilized to construct the chromosome integrated expression vector pZW. The pZW contains one E. coli replicon and one resistance gene. The length is reduced by 2530bp. In S. erythraea, pZW could express reporter genes thiostrepton resistance gene and enhanced green fluorescin gene. And it is more suitable to express exogenous genes in S. erythraea stably.