aprK基因敲除的氨甲酰妥布霉素工程菌的构建  

Construction of carbamoyltobramcin-producing engineering S. tenebrarius by disruption of aprK gene

作　　者：李辉[1] 温淑平[1] 洪文荣[1] 

机构地区：[1]福州大学生物科学与工程学院,福州350108

出　　处：《中国药科大学学报》2013年第4期368-373,共6页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目(No.31070093);国家"重大新药创制"科技重大专项资助项目(No.2012ZX09201101-008)~~

摘　　要：以温敏型穿梭质粒pKC1139为基础,克隆安普霉素辛二糖合成酶基因aprK上下游序列作为同源交换臂,构建用于敲除aprK的重组质粒pBK5。pBK5转化E.coli ET12567后经接合转移导入黑暗链霉菌Tt-49,得到单交换菌株ST315。ST315经松弛传代后通过影印培养与PCR筛选得到aprK阻断突变株ST316。ST316发酵效价约1 500 ug/mL,发酵产物经TLC分析,发现安普霉素的生物合成被阻断,得到了一株主要产氨甲酰妥布霉素的工程菌。To construct a genetic engineering S.tenebrarius for production of carbamoyltobramycin.Recombinant plasmid pBK5 derivated from pKC1139 was constructed for disrupting gene aprK.pBK5 was introduced into S.tenebrariusTt-49 by conjugation.The single crossover mutant ST315 was obtained and cultivated for several rounds of sporulation in the absence of erythromycin.A desired double crossover mutant ST316 was achieved by PCR.TLC analysis indicated that ST316 produced high-yield carbamoyltobramycin,in which apramycin biosynthesis was blocked and the yield of carbamoyltobramycin was about 1 500 ug/mL.

关 键 词：黑暗链霉菌 安普霉素 氨甲酰妥布霉素 基因工程 

分 类 号：Q784[生物学—分子生物学]