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<p><i>Escherichia coli</i> strains are widely utilized as cell factories for recombinant protein production. However, acetate overflow remains a significant challenge that negatively impacts both biomass yield and protein expression. Here, we evaluated a previously engineered <i>E. coli</i> K 12 BW25113 strain with <i>pka</i> and <i>arcA</i> deletions (RV04) for the expression of a single-chain variable fragments (scFv) derived from 4D5MOC-B, a monoclonal antibody that binds to epithelial cell adhesion molecule (EpCAM) as a biologically important marker for tumor immunotherapy. According to our results, RV04 strain demonstrated a significant growth advantage over both BW25113 and BL21 strains. In minimal M9 medium, RV04 exhibited a maximum cell density that was 44% higher than wild‑type and 11% higher than BL21. In enriched M9 medium, RV04 achieved a remarkable maximum specific growth rate (µ_max) of 0.775 ± 0.003&#xa0;h⁻¹ and a maximum cell density of 2.1095 ± 0.0205, even under metabolic load. Regarding acetate accumulation, RV04 fully eliminated acetate accumulation within 24&#xa0;h, whereas BW25113 accumulated acetate up to 0.521&#xa0;g L<sup>− 1</sup> under the same minimal medium conditions. Similarly, in enriched M9 medium, RV04 maintained significantly lower acetate levels (1.65&#xa0;g L<sup>− 1</sup> at 24&#xa0;h) compared to BW25113 (3.99&#xa0;g L<sup>− 1</sup>), despite increased biomass and protein production. These results confirm that RV04 can control acetate overflow more efficiently than the wild type under both minimal and enriched conditions. The combination of using the genetically modified strain and medium enrichment strategy resulted in significantly increased recombinant protein production. In LB medium, RV04 produced 5% more protein than BL21 and 44.8% more than the wild‑type, while in enriched synthetic M9 medium, it outperformed BL21 and BW25113 by 7.1% and 59.5%, respectively. Furthermore, RV04 demonstrated markedly enhanced protein expression compared to other commercial strains; it produced approximately 33.8% more protein than SHuffle, 145.7% more than Rosetta, and over sevenfold more than Origami B. Our findings demonstrate that RV04 effectively mitigates acetate overflow, enhances growth, and substantially increases the recombinant protein titer under both minimal and enriched culture conditions. These features make RV04 a strong candidate for large-scale industrial bioprocessing operations.</p> Graphical Abstract <p></p>

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Enhanced anti-EpCAM ScFv production and reduced acetate overflow in a BW25113-derived E. coli strain with ArcA and Pka deletions

  • 期刊论文

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  • 发表日期：

    2025年12月26日

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  • Majid Basafa,
  • Aidin Behravan,
  • Hossein Vahidi,
  • Atieh Hashemi

摘要

Escherichia coli strains are widely utilized as cell factories for recombinant protein production. However, acetate overflow remains a significant challenge that negatively impacts both biomass yield and protein expression. Here, we evaluated a previously engineered E. coli K 12 BW25113 strain with pka and arcA deletions (RV04) for the expression of a single-chain variable fragments (scFv) derived from 4D5MOC-B, a monoclonal antibody that binds to epithelial cell adhesion molecule (EpCAM) as a biologically important marker for tumor immunotherapy. According to our results, RV04 strain demonstrated a significant growth advantage over both BW25113 and BL21 strains. In minimal M9 medium, RV04 exhibited a maximum cell density that was 44% higher than wild‑type and 11% higher than BL21. In enriched M9 medium, RV04 achieved a remarkable maximum specific growth rate (µ_max) of 0.775 ± 0.003 h⁻¹ and a maximum cell density of 2.1095 ± 0.0205, even under metabolic load. Regarding acetate accumulation, RV04 fully eliminated acetate accumulation within 24 h, whereas BW25113 accumulated acetate up to 0.521 g L− 1 under the same minimal medium conditions. Similarly, in enriched M9 medium, RV04 maintained significantly lower acetate levels (1.65 g L− 1 at 24 h) compared to BW25113 (3.99 g L− 1), despite increased biomass and protein production. These results confirm that RV04 can control acetate overflow more efficiently than the wild type under both minimal and enriched conditions. The combination of using the genetically modified strain and medium enrichment strategy resulted in significantly increased recombinant protein production. In LB medium, RV04 produced 5% more protein than BL21 and 44.8% more than the wild‑type, while in enriched synthetic M9 medium, it outperformed BL21 and BW25113 by 7.1% and 59.5%, respectively. Furthermore, RV04 demonstrated markedly enhanced protein expression compared to other commercial strains; it produced approximately 33.8% more protein than SHuffle, 145.7% more than Rosetta, and over sevenfold more than Origami B. Our findings demonstrate that RV04 effectively mitigates acetate overflow, enhances growth, and substantially increases the recombinant protein titer under both minimal and enriched culture conditions. These features make RV04 a strong candidate for large-scale industrial bioprocessing operations.

Graphical Abstract