东方拟无枝酸菌中vcm14基因的敲除对万古霉素合成的影响

Effect of gene disruption of vcm14 on vancomycin production in Amycolatopsis orientalis

PCR-targeting是一种用于敲除或阻断放线菌染色体上某一基因的快速高效的方法。本研究利用该方法在大肠埃希菌中构建破坏粘粒cLYLH17(Δvcm14::apr),通过结合转移首次敲除东方拟无枝酸菌HCCB10007中万古霉素生物合成基因簇中推测的haloperoxidase基因——vcm14,得到一株双交换阻断突变株A.orientalis dvcm14(Δvcm14::apr)。该菌不产生万古霉素及其类似物,证实vcm14基因的编码蛋白不是haloperoxidase,且该蛋白不参与万古霉素的卤化反应,可能是万古霉素生物合成途径中的一个关键酶。本研究为阐明万古霉素生物合成途径中的卤化过程提供了有益线索。

vcm14 is a putative haloperoxidase gene in the vancomycin biosynthesis gene cluster in Amycolatopsis orientalis HCCB10007. PCR-targeting method was applied to construct the gene disruption vector cLYLH17（△vcm14：：apr）. The cosmid cLYLH17 was then introduced into E. coli ET12567 （pUZ8002） and transferred into A. orientalis HCCB10007 by conjugation. Apramycin-resistant exconjugants were identified and purified, while the disruption event was confirmed by PCR. A representative vcm14 mutant was designated A. orientalis dvcm14（△vcm14：： apr）. Absence of vancomycin and its analogs production from the resulting exconjugants suggests that the protein coded by vcm14 is not a haloperoxidase and this protein may be a key enzyme in the vancomycin biosynthesis pathway.