Enhanced The Production Of Spinosad By Drug Resistance Screening And Genome Shuffling In Saccharopolyspora Spinosa

Spinosyns are natural metabolites produced by fermentation of the aetinomyceteSaccharopolyspora spinosa. This kind of antibiotics has the safety of biologicalpesticide. It can fast kill insects and fast decompose, however it is safe to mammals.So it has very broad market prospects. In our country, more research of spinosad stillstay in the experimental stage. It is meaningful to improve the strain to produce morespinosad. In this research, we combined the genome shuffling and drug resistancescreening to improve the product of spinosad in S.spinosa.Firstly, this research examined the best conditions of protoplast preparation,regeneration and fusion. The best conditions of protoplast preparation were that sporesuspension was inoculated into50ml of CSM media and added into9sterile glasses toprovide the appropriate mechanical shearing force at30℃，200r/min, for72h.Mycelium was collected at100000r/min, used PB solution washing twice,0.2%lysozyme enzymolysis for60min at34℃,filtered with400mesh of gauze, keptat-70℃. The best condition of regeneration were that the protoplast was spread ontothe NO.3regeneration medium and cultured at30℃.The best conditions of fusionwere that mixed the protoplast with1:1and processed with40%PEG4000for5min.Secondly, we examined the minimum inhibitory concentration of ATCC49460toerythromycin and neomycin and constructed the mutant library with drug resistancescreening. The minimum inhibitory concentration of S.spinosa to erythromycin andneomycin are40μg/ml and30μg/ml. The mutant library was obtained by UVtreatment of protoplast for70s. Determination of production was used HPLC. Finally,we obtained strain Ery-13(erythromycin resistance） and Neo-127(neomycin resistance), which production were180μg/ml and158μg/ml respectively. Crossresistance test of the two strains shown that Ery-13was sensitive to neomycin andNeo-127was sensitive to erythromycin.Lastly, they were used as starting strain for four rounds of genome shuffling.With the deepening of genome shuffling, the production of spinosad was increased.Finally, we obtained a strain that had erythromycin and neomycin resistance at thesame time. Its production was331μg/mL, which was6.6fold than that of parentalstrain. After the fermentation characteristics measurement, we concluded that thegrowth rate and consumption of glucose and amino nitrogen were much higher thanthat of parental strain.The results show that it was feasible to enhance the production by combing thegenome shuffling and drug resistance screening. It provide a new method for otherstrains breeding.