柔红霉素生物合成基因簇的克隆以及基因打靶研究

【作者】 袁天杰； 胡又佳；

【机构】 中国医药工业研究总院,上海医药工业研究院；

【摘要】 本研究通过构建柔红霉素产生菌天蓝淡红链霉菌DM的基因组文库,获得2230个重组克隆。利用菌落原位杂交和PCR两种筛选方法获得黏粒Cosmid 8-22,其插入片段的大小为41431bp,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,构建PCR-Targeting系统,同时敲除dauW,dnrX基因。HPLC分析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(DNR),was used to construct the gene library,A total of 2230 recombinants were obtained.The recombinant cosmid 8-22 was obtained by colony in situ hybridization and PCR amplification,the length of insert fragment of Cosmid 8-22 was 41431bp 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 can disrupt the gene dauW,dnrX at the same time.The HPLC result was 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