天蓝淡红链霉菌SIPI-1482中酮还原酶基因dnrU的阻断突变研究

Research on Disruption of a Ketoreductase Gene dnrU in Streptomyces coeruleorubidus SIPI-1482

目的:研究柔红霉素产生菌天蓝淡红链霉菌SIPI-1482中酮还原酶基因dnrU阻断后的产物(13s)-13-二氢柔红霉素及其他发酵产物的变化。方法:利用同源重组的原理,以大肠杆菌质粒pUC18为基础构建了dnrU基因交换质粒,通过在SIPI-1482染色体上的dnrU基因中插入安普霉素抗性基因来筛选dnrU的阻断突变株。结果和结论:PCR验证表明成功地阻断了dnrU基因。dnrU基因敲除后,重组菌发酵产物中(13s)-13-二氢柔红霉素消失,而其他发酵中间产物也有一定变化。

Objective： To investigate the effect of disruption of a ketoreductase gene dnrU on metabolites of daunorubicin producer Streptomyces coeruleorubidus SIPI-1482. Methods： The metabolites of SIPI-1482 includes daunorubicin, （13s）- 13-dihydrodaunorubicin, ε-rhodomycinone, etc, among which （13s）-13-dihydrodaunorubicin was transformed from daunorubicin by a ketoreductase encoded by dnrU gene. A recombinant plasmid was constructed based on E.coli vector pUC18, and the aparamycin resistant gene was inserted into dnrU gene in SIPI-1482 chromosome to screen the dnrU block mutant. Results ＆ Conclusion： dnrU was successfully disrupted via homologous recombination which was confirmed by PCR. （13s）-13-dihydrodaunorubicin was absent from the mutant metabolites and other fermentation products were also changed after dnrU was disrupted.