Genome Shuffling beween Streptomyces and Saccharopolyspora Spinosa to Select the High Efficiency and Low Toxicity Strain
|School||Zhejiang University of Technology|
|Keywords||Streptomyces avermitilis Saccharopolyspora spinosa HPLC preparation of protoplast genomeshuffling|
Avermectins was a novel microbial broad-spectrum insecticide and one of the most effective insecticide,acaricide,parasiticide. Spinosad, which was a novel microbial broad-spectrum insecticide, was secondary metaboli -tes from the aerobic fermentation of a naturally occurring actinomycetes bacterium, Saccharopolyspora spinosa. In this dissertation, the determinati- on of spinosad and avermectin by HPLC, the optimum conditions for preparing and regenerating avermectin producer’-s protoplast,the genome shuffling between Streptomyces avermitilis and Saccharopolyspora spinosa to select the high efficiency and low toxicity strains were studied.Analyzing method of spinosad by HPLC was introduced. The chromatographic conditions were as follows: Agilent Zorbax XDB-C18 column(5μm,4.6×250mm),mobile phase is Methanol/ acetonitrile/ water= 45/45/10 (v/v).The results also show that flow rate ,detective wavelength, v -olume,the temperature of column were 1.0mL/min,245nm,20μL, 30℃respectively. The condition for preparation and regenaration of protoplast was optimized .The effects of seed age,the glycin concentration,the lysozyme concentration,the lysozyme treating time and temperature on preparation of protoplast were studied,and the regeneration medium was optimized.A set of media and conditions for formation and regeneration of protolasm was obtained. The result showed that using 2 mg/mL lysozyme at 32℃for 20 mintutes protoplasts were obtained from mycelia of Streptomyces avermitilis which had cultured in a medium cotaining 0.5% glycin.The research is aim to improve the Streptomyces avermitilis,Sacch- aropolyspora spinosa by genome shuffling and select the high efficiency and low toxicity strains. ZGD-2 was mutanted by Co60 and protoplast’s UV in order to obtain a population called pop-YUV. Then pop-YDS and pop-YUV protoplasts were fused recursively there rounds. The results show that the population of F3, which was obtained after three rounds of protoplast fusion,remarkably improved the uotput. Finally five shuffled strains GS-4,GS-1, GSD-1, JSS-1 and JSS-2.The output of GS-4, GS-1 on avermectin are 107.8% than ZGD-2, and the GSD-1 on spinosad is 24.4% than parent strain .