金色链霉菌J13基因敲除体系的构建

Constuction of the Gene Knockout System of Streptomyces aureofaciens J13

目的:建立金色链霉菌基因敲除体系,敲除金色链霉菌J13中的ctcF基因,研究工程菌的代谢变化。方法:采用基因置换和框内缺失技术,对ctcF基因进行敲除。结果:利用接合转移的方法,将质粒pFD109导入到金色链霉菌J13中,经抗性筛选及PCR验证获得ctcF基因置换菌株金色链霉菌A1-20;将质粒pFD111经接合转移导入到A1-20中,获得ctcF基因框内缺失菌株金色链霉菌K2-46;以上工程菌的发酵组分经HPLC分析,发现金霉素发酵单位显著降低。结论:获得的接合子发生双交换的概率可达18%以上,建立及验证了金色链霉菌基因敲除体系的实用性和可操作性,并初步推测ctcF为金霉素生物合成中的调控基因。

Objective:To establish the gene knockout system of Streptomyces aureofaciens,disrupt the gene ctcF in S.aureofaciens J13 and research the change in metabolism of engineering strains.Method:Disrupting ctcF by gene replaced and in-frame deleted.Result:The recombinant plasmid pFD109 was transtformed into S.aureofaciens J13 by conjugation.After antibiotics resistance screening and PCR identification,it was confirmed that ctcF was replaced in S.aureofaciens A1-20;Then the plasmid pFD111 was transtformed into S.aureofaciens A1-20 with the same method,and ctcF in-frame deletion strain S.aureofaciens K2-46 was obtained;After HPLC analysis to the fermentation components of engineer strains,it was found that the fermentation unit of chlortetracycline reduced significantly.Conclusion:The chance of double crossing strain in the conjugons was more than 18%.It was proved that the gene knockout system of S.aureofaciens was exercisable and practical,and it was supposed ctcF was regulator gene in biosynthesis of chlortetracycline.