Integration Site\nLibrary for Efficient Construction\nof Plasmid-Free Microbial Cell Factories in Escherichia\ncoli

Enhanced production stability and\nefficiency along with\na decrease\nin production costs are required to build efficient microbial cell\nfactories. Target genes can be integrated into the genome to enhance\ngenetic stability, reduce reliance on antibiotics, and alleviate the\nmetabolic burden. However, selecting the optimal insertion site for\nthe desired gene expression levels remains challenging. Therefore,\n18 commonly usedEscherichia coliintegration\nsites were systematically characterized in this study. Promoters of\ndifferent strengths were combined with integration sites, yielding\na differential intensity range of up to 93-fold. This indicated the\nversatility and precision of this approach for controlling gene expression\nlevels. Referring to the library, pathway genes were strategically\nintegrated into theE. coligenome based\non their respective expression levels. Genetically stable and highly\nefficient engineered strains that could biosynthesize arbutin and <i>p</i>-aminobenzoic acid were constructed.