A Toolkit for Effective and Successive Genome Engineering of Escherichia coli

Fermentation(2022)

引用 1|浏览1
暂无评分
摘要
The bacterium Escherichia coli has been well-justified as an effective workhorse for industrial applications. In this study, we developed a toolkit for flexible genome engineering of this microorganism, including site-specific insertion of heterologous genes and inactivation of endogenous genes, such that bacterial hosts can be effectively engineered for biomanufacturing. We first constructed a base strain by genomic implementation of the cas9 and λRed recombineering genes. Then, we constructed plasmids for expressing gRNA, DNA cargo, and the Vibrio cholerae Tn6677 transposon and type I-F CRISPR-Cas machinery. Genomic insertion of a DNA cargo up to 5.5 kb was conducted using a transposon-associated CRISPR-Cas system, whereas gene inactivation was mediated by a classic CRISPR-Cas9 system coupled with λRed recombineering. With this toolkit, we can exploit the synergistic functions of CRISPR-Cas, λRed recombineering, and Tn6677 transposon for successive genomic manipulations. As a demonstration, we used the developed toolkit to derive a plasmid-free strain for heterologous production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by genomic knock-in and knockout of several key genes with high editing efficiencies.
更多
查看译文
关键词
Escherichia coli,genome engineering,lambda(Red) recombineering,transposon-associated CRISPR-Cas,poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
AI 理解论文
溯源树
样例
生成溯源树,研究论文发展脉络
Chat Paper
正在生成论文摘要