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The bacterium <i>Escherichia coli</i> 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 <i>cas9</i> and λ_Red recombineering genes. Then, we constructed plasmids for expressing gRNA, DNA cargo, and the <i>Vibrio cholerae</i> 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 λ_Red recombineering. With this toolkit, we can exploit the synergistic functions of CRISPR-Cas, λ_Red 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.