基于基因组重排技术的多杀菌素高产菌株选育

Enhanced production of spinosad with strains of Saccharopolyspora spinosa by genome shuffling

利用课题组前期通过不同物理化学诱变和分子生物学方法获得的8株多杀菌素产生菌为出发菌株,采用96孔板发酵培养结合生物检测的高通量筛选方法,探索了原生质体制备、再生、双亲灭活和原生质体融合等条件,并通过多轮基因组重排获得了多杀菌素高产菌株。结果表明:当菌龄为65 h、溶菌酶浓度为4 mg·ml-1,39℃处理时间20 min时,原生质体的制备率及再生率分别为92.30%和7.66%;60℃恒温水浴90 min以上和紫外照射200s以上为双亲灭活条件;PEG浓度为50%,在32℃下处理15 min时,原生质体融合率约为1.18%。最终获得1株产量较出发菌株提高了36.07%且遗传稳定的融合株。

In this study, genome shuffling of eight original strains obtained by various methods of chemical and physical mutations was used to achieve rapid improvement of spinosad production. The conditions of preparation, regeneration, parents inactivation and fusion of protoplast of the original strain were investigated. The strain grown for 65 h was treated by 4 mg .ml-l lysozyme at 39℃ for 20 rain, and preparation rate and regeneration rate were 92.30% and 7.66%, respectively. The protoplasts were inactivated by heating at 60℃ for more than 90 rain and UV treatment more than 200 s, respectively. The inactivated protoplasts were fused and regenerated in polyethylene glycol （PEG 6000, 50%） for 15 min, and fusion rate was about 1.18%. After three rounds of genome shuffling, a high yielding strain, designated as S.spinosa 3-652, was isolated. The increased yield was about 36.07% higher than that of the original strain. Subculture experiments indicated that the high producers were stable.