Breeding Of Spinosad High-Production Strain And Optimization Of Fermentation

Spinosad was a microbial pesticides,which was a secondary metabolite produced by aerobic fermentation of Saccharopolyspora spinosa.Because of its safety in use,environmentally friendly and high-effective insecticidal activity,spinosad has become the most promising bio-pesticides.There was a new screening method about Saccharopolyspora spinosa was used in this research.The main content has the following three parts.The wild type of Saccharopolyspora spinosa has a low production.The traditional method of nitrosoguanidine mutagenesis is aim to get high-yield mutant strains.NTG with the concentration of 1mg/mL was used to mutate S.spinosa ASAG 36 in this study.Primary screened two mutant strains which increased more than 20%of spinosad production and one which increased more than 50%,but all of them are genetic instability.Fermentation medium optimization was used for S.spinosa ASAG 36 to improve the objective spinosad production.The optimum fermentation medium for spinosad production were as follows:Glucose 50 g/L,peptone milk 10 g/L,cotton seed flour 10 g/L,dextrin 15g/L,yeast extract paste 10 g/L,NaCl 0.5 g/L,MgSO₄·7H₂O 0.5 g/L,KH₂PO₄ 0.5 g/L,CaCO₃ 3 g/L.We also explored the seed and fermentation growth curve.Experiment date showed that when the first-class seed cultured 56 to 60 hours and second-class seed cultured42 to 46 hours,they were in late logarithmic phase state.In fermentation cultured,in 96 to136 h and 168 to 192 h,the spinosad synthesized in large amounts.And when fermentation cultured for 192 hours,the maximum production of spinosad reached to 462.21?g/mL.Explore amino acids and organic acids whether effect the production of spinosad.First,we used single-factor experiment to study eighteen animo acids and five organic acids whether effect the production of spinosad.Then orthogonal experiment was used to optimize the five promotive amino acids and the four promotive organic acids.Experiment data showed that the best components of the five amino acids were arginine?0.75 mmol/L?,glycine?0.75 mmol/L?,alanine?4 mmol/L?,glutamine?1.25 mmol/L?,serine?1 mmol/L?,and the yield of spinosad was 388.56?g/mL,which was increased 14.95%than the original medium?340.23?g/mL?.And the best components of the four organic acids were succinic?0.1 g/L?,oxaloacetic acids?0.75 g/L?,malic?0.15 g/L?,citric?0.25 g/L?,and the yield of spinosad was 410.65?g/mL,which was increased 22.72%than the original medium?334.61?g/mL?.