Study On Screening And Protoplast Preparation Of Spinosad-produced Strains

Spinosad represents a new class of fermentation-derived tetracyclic macrolides with unique activity against a variety of lepidopterous pests and possesses very favorable mammalian toxicity and environmental profiles. Additionally, spinosad exhibits a great deal of selectivity towards beneficial insects, especially predators enabling extensive utility in DBM and other IPM programs. Because of its various beneficial features, spinosad looks forward to great practical merits and thus broad marketing prospects.After S. spinosa 5-11 was mutagenized with UV, the spinosad resistant mutants were screened directly on the gradient plants containing different concentrations of spinosad. Overproduction of spinosad by the mutant S. spinosa 107-2 was obtained and its fermentation output was increased by 59.1% compared with that of the starting strain.The effects of glucose and inorganic phosphate on mycelium growth and spinosad production with Saccharopolyspora spinosa were studied. The Results showed that the increase of glucose concentration from 18.6g/L to 58.8g/L could promote the mycelium growth and spinosad production. And when the glucose concentration increased from 58.8g/L to 79.6g/L, no obvious change in mycelium growth but slight drop in spinosad production was observed, while increased from 79.6g/L to 115.3g/L, substantial decrease in both mycelium growth and spinosad production occured. The increase of phosphate concentration from 3.68 mmol/L to 29.41mmol/L rendered corresponding increase in mycelium growth and spinosad production. When phosphate concentration increased from 29.41 mmol/L to 44.12mmol/L, mycelium growth slightly increased and dropped in spinosad production, while increased from 44.12mmol/L to 57.62mmol/L, both mycelium growth and spinosad production decreased sharply. Conclusively, the optimal initial concentration of glucose and phosphate is 58.8g/L and 29.41 mmol/L, respectively. The spinosad fermentation in the production medium containing 58.8g/L glucose and 29.41 mmol/L phosphate was scaled up in 5L fermentation and the spinosad production reached 507mg/L, which was 28% higher than that in the flask fermentation.The preliminary conditions for protoplast preparation of S. spinosa were studied, and the protoplast formation rate reached 16.1%.