| Natamycin is a kind of 26-membered ring polyene macrolide antibiotics produced by actinomycetes such as Streptomyces natalensis, Streptomyces chatanoogensis, Streptomyces gilvosporeus. This antibiotic is predominantly a strong, broad-spectrum antifungal agent. Because of its low toxicity to the mammalian cells compared to other antifungal compounds, natamycin is widely used in food industries and medicial treatment. In this dissertation, the condition for preparation, regeneration and fusion of protoplast, and improvement of natamycin producing strain by the genome shuffling were studied in order to improve the production of natamycin. The main research contents and results are as follows.The condition for preparation and regeneration of the protoplast was optimized. The effects of seed age, the glycin concentration, the lysozyme concentration, the lysozyme treating time and temperature on preparation of protoplast were studied, and the regeneration medium was optimized. A set of media and conditions for formation and regeneration of protoplasm was obtained.The condition for protoplast fusion was also studied. The protoplasms were inactivated by UV and heat respectively and then fusion between biparental inactivated protoplasms was carried out. The method of protoplast fusion was set up.After protoplast of S.gilvosporeus SG-1 was mutated with UV, the streptomycin resistance mutatants were screened on the agar medium containing streptomycin, and four mutant strains with high natamycin production, named str~r-69 (2364 mg/L), str~r-89 (2468 mg/L), str~r-l 11 (2433 mg/L), and str~r-129 (2398 mg/L) were obtained. These 4 strains with high ntamycin production were selected as parent strains for genome shuffling. After 2 cycle of recursive protoplast fusion, a shuffled strain S. gilvosporeus GS-74 was obtained, whose natamycin productiion reached 3574 mg/L, which is increased by 1.17 times in comparison with that of the original strain S. gilvosporeus SG-1.
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