A Toolkit for Effective and Successive Genome Engineering of Escherichia coli

The bacterium Escherichia coli has been well-justified as an effective workhorse for industrial applications. In this study, we developed a toolkit flexible genome engineering of microorganism, including site-specific insertion heterologous genes and inactivation endogenous genes, such that bacterial hosts can be effectively engineered biomanufacturing. We first constructed base strain by genomic implementation the cas9 ?Red recombineering genes. Then, plasmids expressing gRNA, DNA cargo, Vibrio cholerae Tn6677 transposon type I-F CRISPR-Cas machinery. Genomic cargo up to 5.5 kb was conducted using transposon-associated system, whereas gene mediated classic CRISPR-Cas9 system coupled with recombineering. With toolkit, exploit synergistic functions CRISPR-Cas, recombineering, successive manipulations. As demonstration, used derive plasmid-free production poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) knock-in knockout several key high editing efficiencies.