东方拟无枝酸菌HCCB10007中vcm8基因的敲除及无氯万古霉素产生菌的构建

Knockout of the vcm8 gene from Amycolatopsis orientalis HCCB 10007 and construction of a didechlorovancomycin-producing strain

目的:利用基因敲除的方法构建无氯万古霉素产生菌。方法:利用PCR-Targeting的方法获得破坏粘粒cLYLH15(Δvcm8::apr),通过接合转移敲除东方拟无枝酸菌HCCB10007中万古霉素生物合成基因簇中的卤化酶基因vcm8。结果:筛选得到1株突变子A.orientalisdvcm8(Δvcm8::apr)。结论:该菌不再代谢产生万古霉素,转而合成无氯万古霉素。经传代试验证明该菌株遗传稳定,可作为无氯万古霉素产生菌。

Objective：To construct a didechlorovancomycin-producing strain by the gene-knockout method. Methods： PCR-targeting method was applied to construct the blocking vector cLYLH15 （△vcm8： ：apr）. The cosmid eLYLHI5 was introduced into E. coli ET12567 （pUZ8002） and transferred into A. orientalis HCCB10007 by conjugation. Results： A representative scm8 mutant was obtained and named as A. orientalis dvcm8 （ △vcm8 ： ： apr）. Conclusion： Vancomycin is absent from metabolites of the resulting transformant while another compound was found there, which is then confirmed as didechlorovancomycin （DDCV） based on its MS analysis and genetic background. Strain A. orientalis dvcm8 has been proved to be stable on genetic as well as production level, and it can be used as a DDCV-producing strain.