Multilevel Systematic\nOptimization To Achieve Efficient\nIntegrated Expression of <i>Escherichia coli</i>

Genomic integration of heterologous genes is the preferred\napproach\nin industrial fermentation-related strains due to the drawbacks associated\nwith plasmid-mediated microbial fermentation, including additional\ngrowth burden, genetic instability, and antibiotic contamination.\nSynthetic biology and genome editing advancements have made gene integration\nconvenient. Integrated expression is extensively used in the field\nof biomanufacturing and is anticipated to become the prevailing method\nfor expressing recombinant proteins. Therefore, it is pivotal to strengthen\nthe expression of exogenous genes at the genome level. Here, we systematically\noptimized the integrated expression system of Escherichia\ncoli from 3 aspects. First, the integration site <i>slmA</i> with the highest expression activity was screened out\nof 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters\nin E. coli and combined them with the\nT7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly\nhigher expression strength than the T7 promoter, achieving a 3.3-fold\nincrease in expression levels. Finally, to further enhance the T7\nexpression system, we proceeded with overexpression of T7 RNA polymerase\nat the chassis cell level. The resulting constitutive efficient integrated\nexpression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression\ncompared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli\nwas applied to the integrated expression of nitrilase and hyaluronidase,\nachieving stable and efficient enzyme expression, with enzyme activities\nof 22.87 and 12,195 U·mL<sup>–1</sup>, respectively, comparable\nto plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient\nmethod of gene expression without the need for antibiotics or inducers,\nmaking it a robust tool for synthetic biology, enzyme engineering,\nand related applications.