Dual-Plasmid Mini-Tn5\nSystem to Stably Integrate Multicopy\nof Target Genes in <i>Escherichia coli</i>

The\nefficiency of valuable metabolite production by engineered\nmicroorganisms underscores the importance of stable and controllable\ngene expression. While plasmid-based methods offer flexibility, integrating\ngenes into host chromosomes can establish stability without selection\npressure. However, achieving site-directed multicopy integration presents\nchallenges, including site selection and stability. We introduced\na stable multicopy integration method by using a novel dual-plasmid\nmini-Tn5 system to insert genes into Escherichia coli’s genome. The gene of interest was combined with a removable\nantibiotic resistance gene. After the selection of bacteria with inserted\ngenes, the antibiotic resistance gene was removed. Optimizations yielded\nan integration efficiency of approximately 5.5 × 10<sup>–3</sup> per recipient cell in a single round. Six rounds of integration\nresulted in 19 and 5 copies of the <i>egfp</i> gene in the\nRecA<sup>+</sup> strain MG1655 and the RecA<sup>–</sup> strain\nXL1-Blue MRF′, respectively. Additionally, we integrated a\npolyhydroxybutyrate (PHB) synthesis gene cluster into E. coli MG1655, yielding an 8-copy integration strain\nproducing more PHB than strains with the cluster on a high-copy plasmid.\nThe method was efficient in generating gene insertions in various E. coli strains, and the inserted genes were stable\nafter extended culture. This stable, high-copy integration tool offers\npotential for diverse applications in synthetic biology.