Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression

The production of exogenous recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we developed a rational and highly efficient approach to improve bacterial engineering, using HPV16-L1 as a protein candidate. Comparing the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress, we found that leaky expression accelerated host energy metabolism and altered ribosome synthesis assembly, and inducing overexpression of the heterologous protein under low-temperature conditions led to an upregulation in polysaccharide transport, and cell motility, and a downregulation in oxidation-reduction, and anaerobic respiration. Subsequently, we selected and knocked out 36 hub genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL, ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24°C or 37°C induction. This study highlights a rational and efficient strategy to increase the yield of recombinant proteins in model or non-model organisms, and lays a solid foundation for the engineering of bacterial strains for recombinant technological advances.