柔红霉素产生菌SIPI-1482中dnmV基因功能的阻断及恢复

dnmV基因产物为柔红霉素生物合成途径中TDP-6-脱氧己糖C4酮基还原酶，破坏该基因能阻断柔红糖胺的合成，进而阻断柔红霉素的产生。从天蓝淡红链霉菌(S. coeruleorubidus)SIPI-1482基因组DNA中经PCR扩增出dnmV及其上游dnmU基因片段，并由此构建了用于阻断dnmV基因的同源重组质粒pYG817，转化SIPI-1482菌株后成功地破坏了dnmV基因，发酵结果显示阻断突变株不再代谢产生柔红霉素，为引入新的基因来改变代谢产物的糖基结构打下了基础。通过导入dnmV基因表达质粒可重建该突变株的生物合成途径，恢复产生柔红霉素，但产量比出发菌株要低。

Disruption and Compensation of dnmV gene from Daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482

TDP-4-ketohexulose reductase, encoded by dnmV, is important in daunorubicin biosynthesis. To obtain a daunorubicin block mutant, double cross-over plasmid pYG817 was constructed by inserting apramycin resistant gene and amplified dnmV together with upstream dnmU into vector pUC18. dnmV was successfully disrupted after transformation of daunorubicin-producing strain SIPI-1482 by pYG817. Daunorubicin was absent from metabolites of the resulting transformant, and its biosynthesis can be reconstituted by introducing dnmV expression plasmid into the disruptant, although the yield is lower than wild-type SIPI-1482, according to HPLC analysis. This mutant can be a good candidate for production of anthracycline such as epi-daunorubicin by introducing exogenous gene into the host.