Protoplast transformation and homologous chromosomal integration of Saccharopolyspora erythraea A226

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.