糖多孢红霉菌A226的原生质体转化和染色体同源整合

Protoplast transformation and homologous chromosomal integration of Saccharopolyspora erythraea A226

糖多孢红霉菌的原生质体转化和染色体同源整合,是红霉素生物合成基因改造的重要途径。本研究对糖多孢红霉菌A226原生质体制备和转化条件进行了优化,结果表明以对数生长后期和稳定期菌丝体制备的原生质体转化效率较高;质粒、原生质体和PEG-T缓冲液体积比例为15:40:200(μl)时转化效果较好;比重小原生质体的转化效率虽高,但在转化子中有效整合的比例较低;PEG1000和PEG3350对转化效率没有显著差异;而Yamamoto转化系统优于Weber转化系统。PCR鉴定、抑菌活性鉴定和质谱分析均表明,转化质粒已整合到染色体红霉素合成基因位点。

Protoplast transformation and homologous chromosomal integration of S erythraea was an important path in changing gene involved in erythromycin biosynthesis.This study optimized the conditions used in protoplast prepara-tion,transformation and homologous chromosomal integration of S.erythraea A226strain.The results showed that protoplasts made from mycelia in late lag-phase and stationary phase had higher efficiency in transformation;When the volume proportion of plasmid,protoplast and PEG-T buffer was15:40:200(μl),the transformation condition was optimal;the lighter protoplasts had more transformants than the heavier ones,but they had lower proportion of valid integratants;PEG1000and PEG3350made no difference in transformation;Yamamoto transformation system had higher efficiency in protoplast transformation of S erythraea A226than Weber transformation system.PCR identifica-tion,test of repression on B subtilis PUB110and MS analysis all gave the conclusion that the pWHM2201plasmid had integrated in the chromosomal site involved in erythromycin biosynthesis.