Breeding Strains Of Agricultural Antibiotic 9512-3 With High-Yield

Agricultural antibiotic 9512-3 is a new macrolide compounds, which is produced a high-performance, low toxicity, broad-spectrum antibiotics insecticidal by fermentation. They are the most effective agricultural pesticides and antiparasitic agents, and widely used in veterinary and agricultural fields. In this paper, optimization of Streptomyces avermitilis carried out through analysis of resistant mutation and genome shuffling after established the protoplast preparation conditions.A stable and high-yielding strain Dor-90 was obtained from Primitive strain by stable experimentation. Obtains strain UL-33 and D7 after a series of mutagenesis, and celebrates magnamycin resistant plate screening through the sulfuric acid chain mildew element, obtains strain Y34 and strain Z18.Results showed that conditions for protoplast preparation for Streptomyces avermtitlis were following. The 30mL YEME media( plus 0.5% glycine) in 250mL flask inoculated with spore suspension and incubated at 28℃for 42h on a rotary shaker at 200rpm. Mycelia were pelletted through centrifugation at 3000g and washed with 10.3% sucrose. Mycelia were resuspended with P buffer (contain 4mg/mL lysozyme) and incubated at 32℃for 60min to release protoplast. 40%PEG1000 was suit for protoplast fusion.As the parent carries on the genome team take Y34, Z18, D7 and AA732 to rearrange the breeding research, fermentation analysis showed that the ratios of strains with high avermectin production increase with the further shuffling. After five round recursive fusion, the fusion rate reached 42.1% and the system were stable. The strain F5-41 was obtained, which can produce 478.5μg/mL. It was increased 52.8% more than the original strain UL-33. Subculture experiment indicated that the F5-41 was genetically stable.