庆大霉素生物合成基因genD1的研究

Research of genD1 in gentamicin biosynthesis gene cluster

为研究genD1基因功能,在绛红色小单孢菌GK1101(Δgen K)上构建genD1基因缺失工程菌并分析其代谢产物组分变化。首先构建用于genD1基因框内敲除的同源重组质粒pGD14,经接合转移导入绛红色小单孢菌GK1101,经影印筛选获得genD1缺失工程菌GD1989。再发酵并提取其代谢产物,采用质谱法等检测代谢产物。结果表明,工程菌GD1989不再合成庆大霉素C1a和C2b,主要积累庆大霉素A。对工程菌GD1989喂养庆大霉素X2的发酵产物经检测含有庆大霉素C1a、C2b和JI-20A。genD1基因失活导致庆大霉素生物合成代谢流中断,说明genD1基因负责加洛糖胺C-4″位的甲基化。

In order to verify the function of genD1,a recombinant plasmid pGD14 was constructed for blockingup genD1. pGD14 was introduced into Micromonospora purpurea GK1101（ Δgen K）） by conjugation. A desired mutant strain GD1989 was obtained by PCR analysis. Flask shaking experiments and MS analysis showed that the mutant strain GD1989 only produced gentamicin A instead of gentamicin C complex,and the product of GD1989 feeding on gentamicin X2 contained gentamicin C complex. It was speculated that genD1 gene might be responsible for methylation at C-4″ of garosamine.