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

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.利用入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 λ-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.