Protoplast Formation and Regeneration Conditions of Streptomyces gilvosporeus

Natamycin is a very attractive antifungal agent with wide applications in medical and food industries, whose production can be increased by breeding high natamycin-producing strain using protoplast fusion. In this paper, Streptomyces gilvosporeus SG-2002 was used as a starting strain and conditions for protoplast formation and regeneration were investigated, including mycelia medium, glycine concentration, seed age, lysozyme concentration, enzymatic temperature and treatment time. Moreover, the addition of nutrient substances (potassium dihydrogen phosphate, L-proline, BSA and PVP) to regeneration medium were discussed and optimized to improve regeneration efficiency. Results showed that an efficient yield of protoplasts (4.0times10⁶ cells/mL) with a relatively high regeneration rate was produced when mycelia were precultured in S medium containing 1% glycine for 30 h and then incubated in the buffer P with the lysozyme concentration 40 mg/g(DCW) for 60 min at 30degC with shaking at 100 rpm. A medium containing PVP was more effective on increasing regeneration efficiency than BSA. The components of optimized regeneration medium were as follows: potassium dihydrogen phosphate 0.13 g/L, L-proline 5.00 g/L, PVP 0.20 g/L, other components were the same as R5´ regeneration medium. Under above conditions, protoplast regeneration rate was increased to 16.5%, which was 284% higher than that of initial conditions.