Study On The Breeding And Fermentation Process Of Spinosad-Producing Bacterium Saccharopolyspora Spinosa

Spinosad is a kind of biopesticide isolated from the fermentation broth of Saccharopolyspora spinosa.Its main components are Spinosad A and Spinosad D.Compared with traditional chemical pesticides,it has advantages of rapid degradation,strong selectivity,and environmental safety,and is widely used in field to control pests such as diptera,isoptera,hymenoptera,lepidoptera and other pests.Spinosad is mainly produced through the fermentation of Saccharopolyspora spinosa,but the domestic Spinosad production capacity is low and cannot meet the market demand.This study mainly improves the yield of Spinosad through strain mutation breeding,genome shuffling breeding and subsequent fermentation process optimization,so as to lay a foundation for the industrialization of Spinosad.Firstly,the original strain was mutated by UV irradiation and nitrosoguanidine After several rounds of mutation breeding,the yield of 8U-19(110.77 mg/L)and 6N-1(95.76 mg/L)were obtained with increased and stable yield,which were 3.26 times and 2.69 times higher than that of the original strain(25.98 mg/L)respectively.Before genome shuffling breeding,Response Surface Methodology(RSM)was used to optimize the conditions of protoplasm preparation,regeneration,and fusion.The optimum conditions were determined as follows:treatment temperature was 28℃,treatment time was 60.23min,lysozyme concentration was 11.8mg/mL,preparation number was 0.92 × 108 and regeneration rate was about 3.67%.In the condition of 50%PEG2000,the protoplast was fused for 5 min and the fusion rate was 0.58%.A strain S.17-1 with yield of 122.23 mg/L was obtained by two rounds of genome shuffling breeding.In order to further improve the yield and explore the fermentation process and suitable conditions for the recombinant strain,the fermentation process and fermentation medium were optimized to maximize the production performance of S.17-1 under suitable conditions.The optimized fermentation process was as follows:the inclined surface culture time was 8d,the seed age was 48h,the inoculation amount was 20%,the fermentation temperature was 28℃ the initial pH of the fermentation medium was 7.4.The fermentation time was 8d.Then the suitable seed culture(g/L)was selected according to the literature:glucose 15,starch 10,soybean cake powder 3,cottonseed protein 3,soybean peptone 25,MgSO4·7H2O 2,pH7.2;The optimized fermentation medium was(g/L):glucose 50;Dextrin 20;Fish meal peptone 30;NaCl 20;Na2HPO4·3 H2O 1;FeSO4·7H2O 0.06;CaCO35;Arg 1.5;pH 7.2;The yield of Spinosad after medium optimization was 728.72 mg/L,which was 4.96 times higher than that before optimization.