构建有序排列的除虫链霉菌基因组的柯斯文库用于工业生产菌株的遗传改造  

Construction of an ordered cosmid library of S.avermitilis for genetic modification of the industrial strains

作　　者：夏海洋[1] 黄隽 胡敏杰[1] 沈美娟[1] 谢鹏飞[1] 张亮[1] 王海彬 覃重军[1] 

机构地区：[1]中国科学院上海生命科学研究院植物生理生态研究所合成生物学重点实验室,上海200032 [2]浙江海正药业股份有限公司生物技术中心,台州318000

出　　处：《中国抗生素杂志》2009年第7期403-405,444,共4页Chinese Journal of Antibiotics

基　　金：科技部863项目(2007AA021503);中科院知识创新工程重要方向性项目(KSCX2-YW-G-014);国家自然科学基金(30325003;30770045;30870067)

摘　　要：以容纳DNA大片段的柯斯质粒Supercos-1为载体,构建了除虫链霉菌的基因组文库。对约1000个柯斯质粒上插入片段的两端进行测序,并利用全基因组序列的信息,构建了覆盖约91%基因组的有序排列的柯斯文库。利用λ-Red高效DNA重组和大肠埃希菌-链霉菌接合转移技术,建立了除虫链霉菌的基因组遗传操作系统。运用该系统可以在除虫链霉菌工业菌株中进行高效、精确的基因敲除工作和连续的基因缺失研究,获得可以用来生产多拉菌素的工程菌株。A cosmid library of Streptomyces avermitilis genome was constructed by using vector Supercos-1. Sequencing of the insertion ends of nearly 1000 cosmids showed that they covered 91% genomic sequences of the strain. A genetic manipulation system was established by the k-Red mediated recombination and conjugation from E. coli to Streptomyces. Genes of the strain were knocked out with high efficiency and site-preciseness, and even sequential gene knocking out was also practical. Several industrial strains with modifications of antibiotic biosynthetic pathways,producing doramectin for example, were obtained.

关 键 词：除虫链霉菌 有序基因文库 基因敲除 工业菌株 

分 类 号：Q784[生物学—分子生物学]