Improvement Of Spinosyn-producing Strain By Genome Shuffling

Spinosyns is a secondary metabolite produced by the soil actinomycetes Saccharopolyspora spinosa.For its security of biological pesticide and high-efficiency of chemical pesticide,spinosyns became a potent and broad-spectrum insecticidal macrolide with unique toxicity mechanism.Spinosyn A and spinosyn D are two major effective factors in the products of S.spinosa fermentation.Spinosyns has been commercially produced in overseas and have not been accomplished in China currently.Therefore,it is very valuable to Investigate and develop spinosyns further as a new generation of antibiotic.The aim of this work is to increase the productivity of spinosyns fermentation.After particularity study in strain improvement by genome shuffling and culture condition optimization,spinosyns productivity is increased greatly.The main research contents and results are as follows:The experimental study found that the strain tolerance and the level of spinosad titer is positively related,while erythromycin concentration less than or equal to 20U/mL and streptomycin concentrations less than or equal to 216?g/mL.Established a method for S.spinosa protoplast formation and regeneration.The results showed that the S.spinosa protoplast yield was the highest under these conditions:the collected mycelium grown in S medium with 0.75%glycin and 20 glass beads of 2-3mm diameter,sucrose concentration of the two seed medium select 10%,the buffer of P-buffer select the TES,the 4 medium was suitable for regeneration of S.spinosa protoplast.Also found that the regenerative ability,spore full and metabolic vigorous growth strains,whose production is relatively high.Through three rounds of genome rearrangement,Spinosad production of the strains has been greatly improved.The spinosad production of HL204 recombinant strain has improved than the original strain,which were increased by 1.21 times.The paper identified the screening methods of the recombinant strain of S.spinosa and Streptomyces avermitilis,and successfully screened 13 recombinants.The spinosad production of UV163,E523 and Z276 recombinants strains have improved than the original strain,which were increased by 1.76 times,1.45 times and 1.91 times.Protoplast mortality will increase after electric shock,camphor fumigation and ultraviolet light to promote financial reduce the rate of protoplast regeneration,at the same time improve the fusion rate.Lastly,plotted the Z276 strains shaking flasks fermentation time course curve.Meanwhile,the effects of precursors on the biosynthesis of spinosad were also studied.The fermentation potency of spinosad had increased by 11.8%,when flask fermentation with shaking in the optimized medium feeding 0.3%isobutyric acid at 60h.Glucose is the optimum carbon in fed-batch fermentation synthetic spinosad.While Single and three times filled with glucose,the fermentation potency of spinosad had increased by 21.1%and 25.4%.