阿维链霉菌孢子色素生物合成对阿维菌素产量的影响.pdf

生 物 工 程 学 报 Chin J Biotech 2008, October 25; 24(10): 1702-1713 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 阿维链霉菌孢子色素生物合成对阿维菌素产量的影响 朱娟娟, 陶美凤 华中农业大学农业微生物国家重点实验室, 武汉 430070 摘 要: 以野生型阿维链霉菌NRRL8165 为出发菌株, 用PCR 方法克隆孢子色素基因簇直系同源基因(whiE )侧翼片段, a 并构建基因置换载体pHL643 。将pHL643 跨属接合转移进入阿维链霉菌 NRRL8165, 通过置换载体和染色体之间的同 源双交换, 对染色体上的 whiEa 基因簇进行置换, 得到 3 株阿泊拉霉素抗性、硫链丝菌素敏感的重组菌株, 均表现为孢 子色素合成缺陷。通过Southern 杂交分析, 证明whiEa 基因簇被置换。通过摇瓶发酵和HPLC 检测, 发现whiEa 基因簇 置换菌株所产阿维菌素产量明显提高, 表明孢子色素与阿维菌素生物合成之间可能有竞争底物的现象。 关键词: 孢子色素, whiE , 阿维链霉菌, 接合转移, 基因置换, 阿维菌素 a Effect on Production of Avermectins of Spore Pigment Biosynthesis in Streptomyces avermitilis NRRL8165 Juanjuan Zhu, and Meifeng Tao State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University,Wuhan 430070, China Abstract: The flanking fragments of the whiE gene cluster was PCR amplified, cloned and used to construct the gene replacement a plasmid pHL643. pHL643 was conjugated into Streptomyces avermitilis NRRL8165 followed by screening for double crossover event, yielding three apramycin resistance and thiostrepton sensitive isolates named ZJ1, ZJ2 and ZJ3, which were deficient in biosynthesis of the grey spore pigment. The whiEa gene replacement of these isolates was confirmed by Southern hybridization. Fermentation of the mutant strains in shaking flasks and HPLC analyses showed