【摘要】 本研究通过构建柔红霉素产生菌天蓝淡红链霉菌Streptomyces coeruleorubidus DM的基因组文库,获得2 230个重组克隆。利用菌落原位杂交和PCR筛选获得黏粒Cosmid 8-22,其插入片段大小为41 431 bp,含36个完整的开放阅读框,基本包含了完整的柔红霉素生物合成基因簇。利用黏粒Cosmid 8-22成功构建了基因打靶系统,同时敲除部分dau W和完整的dnr X基因。HPLC结果显示,敲除突变株的柔红霉素产量较出发菌提高了3~4倍。更多还原

【Abstract】 Genome of Streptomyces coeruleorubidus DM, the producing strain of daunorubicin, was used to construct the gene library, and a total of 2 230 recombinants were obtained. The recombinant Cosmid 8-22 was obtained by colony in situ hybridization and PCR amplification. The insert fragment of Cosmid 8-22 was 41 431 bp in length and harbored 36 intact ORFs which almostly contained the complete DNR biosynthetic gene cluster. The Cosmid 8-22 was used to establish the PCR-targeting system, which could disrupt partial dau W and complete dnr X at the same time. The HPLC results showed that the DNR production of mutant strain was increased significantly, which was nearly about 3- 4 folds higher than the parental strain.更多还原