Genome Shuffling Breeding Of Saccharopolyspora Spinosa And Study Of Rapid Screening Model

Saccharopolyspora spinosa could produce one kind of novel microbial broad-spectrum insecticide——Spinosad, which was a broad-spectrum insecticide with contact toxicity and ingestion toxicity. In this dissertation, we improved the spinosyn-producing strain by genome shuffling and established a fast screening model for spinosad screening.In this study, we established a fast screening model for spinosad-producing strains which made use of the relevance between the fatality rate of Drosophila larvae and the concentration of spinosad. This model used 3rd instar Drosophila larvae as indicating animals, Saccharopolyspora spinosa lawn as the object for detecting, and the detection time is 2 hours. Using this fast screening model we could screen out higher yield spinosad-produing strains by comparing the fatality rate of Drosophila larvae. The Rescreening standard was the fatality rate above 50%.In this study, the conditions for protoplastp reparation and regeneration were optimized. Considered the frequency of protoplast preparation and regeneration, the results showed that obtained mycelia of S. spinosa which had cultured in S medium containing 0.75% glycin and using 3 mg/ml lysozyme at 36℃for 50 minutes to obtain protoplasts then cultured protoplasts in number 4 regenerative medium were the must properly conditions.Based on the biosynthetic pathway and metabolic regulation of S. spinosa, we used phosphate UV-induced mutation to obtain the high productive strain. One of them was S.spinosa UV-405 which could grow well on the medium with 25U/mL erythromycin and it's productivity reached 345.62mg/L, which was 83.18% higher than that of the parent strain S.spinosa L2. The other strain was S.spinosa UV-502 which could grow well on the medium with 10mg/mL acetate and it's productivity reached 308.24mg/L, which was 63.37% higher than that of the parent strain S.spinosa L2.Finally, we used UV-405 and UV-502 as parental strains, after 4 turns of protoplast fusion we obtained S.spinosa 4 whose productivity reached 479.16mg/L, which was 38.64% higher than parent strain UV40-6 and 1.54 times higher than parent S.spinosa L2.