CAJ | 학술논문

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200 s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。
이용과제조전기통과불동물이화학유변화분자생물학방법획득적8주다살균소산생균위출발균주，채용96공판발효배양결합생물검측적고통량사선방법，탐색료원생질체제비、재생、쌍친멸활화원생질체융합등조건，병통과다륜기인조중배획득료다살균소고산균주。결과표명：당균령위65 h、용균매농도위4 mg·ml-1，39℃처리시간20 min시，원생질체적제비솔급재생솔분별위92.30%화7.66%；60℃항온수욕90 min이상화자외조사200 s이상위쌍친멸활조건；PEG농도위50%，재32℃하처리15 min시，원생질체융합솔약위1.18%。최종획득1주산량교출발균주제고료36.07%차유전은정적융합주。
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-1 lysozyme at 39℃for 20 min, 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 min 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.