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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 chlorine-free vancomycin producing bacterium by gene knockout method. Methods: The cosmid cLYLH15 (Δvcm8 :: apr) was cloned by PCR-Targeting, and the halohydrin gene vcm8 in the vancomycin biosynthetic gene cluster was knocked down by ligase transfer. Results: A mutant A.orientalisdvcm8 (Δvcm8 :: apr) was screened. Conclusion: The bacteria no longer produce vancomycin metabolism, in turn, the synthesis of chlorine-free vancomycin. The passage test proved that the strain is genetically stable, can be used as chlorine-free vancomycin-producing bacteria.