Integration Site Library for Efficient Construction of Plasmid-Free Microbial Cell Factories in <i>Escherichia coli</i>

Enhanced production stability and efficiency along with a decrease in production costs are required to build efficient microbial cell factories. Target genes can be integrated into the genome to enhance genetic stability, reduce reliance on antibiotics, and alleviate the metabolic burden. However, selecting the optimal insertion site for the desired gene expression levels remains challenging. Therefore, 18 commonly used<i>Escherichia coli</i>integration sites were systematically characterized in this study. Promoters of different strengths were combined with integration sites, yielding a differential intensity range of up to 93-fold. This indicated the versatility and precision of this approach for controlling gene expression levels. Referring to the library, pathway genes were strategically integrated into the<i>E. coli</i>genome based on their respective expression levels. Genetically stable and highly efficient engineered strains that could biosynthesize arbutin and <i>p</i>-aminobenzoic acid were constructed.