生物农药多杀菌素的发酵水平限制了其产业化发展.为了提高多杀菌素产量,以刺糖多孢菌 T1(Saccharopolyspora spinose T1)为出发菌,通过常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变和硫酸二乙酯(diethyl sulfate,DES)诱变,并以链霉素和鼠李糖作为筛选因子结合微孔板培养进行筛选,获得高产菌株S.spinosa F5;发酵后其多杀菌素产量为572.3 mg/L,比出发菌提高35.9％;通过单因素实验和响应面实验确定柠檬酸钠、异亮氨酸的最适添加量分别为0.46、0.10 g/L,豆油的最适添加体积分数为1.77％,此时多杀菌素产量为707.2 mg/L.在5 L发酵罐中进行分批发酵,发酵240 h产量接近800 mg/L;进一步分批补料发酵264 h,多杀菌素产量达1 175.5 mg/L.结果表明,ARTP和DES复合诱变育种及添加外源诱导物可以显著提高多杀菌素产量.
Breeding of Spinosad Producing Strains by Combined Mutation and Optimization of Fermentation
The fermentation yield of spinosad,a biological pesticide,limits its industrialization development.To enhance spinosad production,Saccharopolyspora spinose T1 was mutagenized using atmospheric and room temperature plasma(ARTP)and diethyl sulfate(DES).The high-yield strain,S.spinosa F5,was obtained by streptomycin and rhamnose as screening factors combined with microplate culture.After fermentation,the yield of spinosad was 572.3 mg/L,an increase of 35.9％compared to that of the parent strain.Through single factor experiment and response surface test,the optimum addition amounts of sodium citrate and isoleucine were determined to be 0.46 g/L and 0.10 g/L,while the optimum addition volume fraction of soybean oil was 1.77％.Under this condition,the spinosad production reached 707.2 mg/L,and batch fermentation in a 5 L fermenter showed a yield close to 800 mg/L after 240 h fermentation.Further batch-fed fermentation resulted in a production of 1 175.5 mg/L after 264 h fermentation.The results showed that the compound mutation combining ARTP and DES breeding,along with the addition of exogenous inducers,could significantly increase the yield of spinosad.
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