Engineered Escherichia coli with Superior Recombinant Protein Production and Reduced Acetate Overflow Compared with Industrial Strains

Background: Escherichia coli strains are widely used as cell factories for recombinant protein production. Among these, E. coli BL21 is often preferred over K‑12 strains due to its lower acetate yield and higher biomass production. However, acetate overflow remains a significant challenge, negatively affecting both biomass yield and protein expression. Previous studies have shown that coordinated activation of the TCA cycle and the PTA‑ACS node via deletion of pka and arcA in E. coli K‑12 BW25113 can reduce acetate excretion fourfold. Results: Here, we evaluated an E. coli K‑12 BW25113 strain with pka and arcA deletions for the expression of single-chain variable fragments (scFvs) derived from 4D5MOC-B, a monoclonal antibody binding to epithelial cell adhesion molecule (EpCAM), a key target in tumor immunotherapy. The engineered strain, RV04 (BW25113 ΔpkaΔarcA), demonstrated superior growth compared to BW25113 and BL21. In the minimal M9 medium, RV04 reached a maximum cell density 44% higher than the wild type and 11% higher than BL21. In enriched M9 medium, it achieved a μ_max of 0.775 ± 0.003 h⁻¹ and a maximum cell density of 2.1095 ± 0.0205, even under metabolic burden. While the wild type accumulated up to 3.99 g/L of acetate without reuptake, RV04 completely eliminated acetate in M9 medium and resisted its accumulation even when the glucose concentration was tripled. Under these conditions, RV04 maintained acetate levels comparable to BW25113 in standard M9 while supporting higher cell density and protein production. These optimizations resulted in significantly enhanced recombinant protein production. In LB medium, RV04 produced 5% more protein than BL21 and 44.8% more than the wild type, while in enriched M9 medium, it outperformed BL21 and BW25113 by 7.1% and 59.5%, respectively. Furthermore, RV04 presented markedly greater protein expression than did the other commercial strains; RV04 produced 33.8% more protein than Shuffle, 145.7% more than Origami, and over tenfold more than Rosetta. Conclusions: Our findings demonstrate that the BW25113 ΔpkaΔarcA effectively mitigates acetate overflow, enhances growth, and substantially increases recombinant protein yield, making RV04 a strong candidate for large-scale industrial bioprocesses.