Optimization of transformation procedures in avermectin high-producing Streptomyces avermitilis

The optimal pH conditions for efficient transformation of protoplasts and intact cells were established in avermectin high-producing mutants, ATCC31780 and L-9. Among all factors tested, protoplast buffer pH was elucidated as the most important factor influencing transformation efficiency. The optimal pH of the protoplast buffer for the regeneration of ATCC31780 was 6.5, and using this condition, 4.5 × 10⁶ transformants per g of pIJ702 were produced. At pH 6.3, the maximal number of L-9 transformants was 1.6 × 10⁵ per g of the same plasmid. However, the protoplasting process decreased avermectin productivity to half or one-sixth of ATCC31780 or L-9, respectively. To avoid the productivity loss, electroporation of intact cells without lysozyme treatment was developed for these mutant strains even though this method was approximately 100-fold less efficient. In this method, the initial pH of culture medium was elucidated as a critical factor and optimized for both transformation efficiency and avermectin productivity.