Dual-Plasmid Mini-Tn5 System to Stably Integrate Multicopy of Target Genes in <i>Escherichia coli</i>

The efficiency of valuable metabolite production by engineered microorganisms underscores the importance of stable and controllable gene expression. While plasmid-based methods offer flexibility, integrating genes into host chromosomes can establish stability without selection pressure. However, achieving site-directed multicopy integration presents challenges, including site selection and stability. We introduced a stable multicopy integration method by using a novel dual-plasmid mini-Tn5 system to insert genes into <i>Escherichia coli</i>'s genome. The gene of interest was combined with a removable antibiotic resistance gene. After the selection of bacteria with inserted genes, the antibiotic resistance gene was removed. Optimizations yielded an integration efficiency of approximately 5.5 × 10^-3 per recipient cell in a single round. Six rounds of integration resulted in 19 and 5 copies of the <i>egfp</i> gene in the RecA⁺ strain MG1655 and the RecA^- strain XL1-Blue MRF', respectively. Additionally, we integrated a polyhydroxybutyrate (PHB) synthesis gene cluster into <i>E. coli</i> MG1655, yielding an 8-copy integration strain producing more PHB than strains with the cluster on a high-copy plasmid. The method was efficient in generating gene insertions in various <i>E. coli</i> strains, and the inserted genes were stable after extended culture. This stable, high-copy integration tool offers potential for diverse applications in synthetic biology.