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<p>Biologics produced in <i>Escherichia coli</i> BL21(DE3) require rigorous removal of lipopolysaccharide (LPS), since even traces can trigger severe immune responses and potentially fatal sequelae in humans. In contrast, the clinically licensed probiotic <i>Escherichia coli</i> Nissle 1917 (EcN) is inherently LPS-deficient, displaying only 0.86% of the LPS activity observed in BL21(DE3). Due to its markedly reduced immunostimulatory activity, EcN is an attractive chassis for manufacturing proteins with minimal LPS burden. On the basis of the constructed strain EcN::T7 by inserting the T7 RNA polymerase gene into chromosome, using GFP as a model protein, this strain produced only 30% of the yield obtained from BL21(DE3) because both cell density (OD₆₀₀) and per-cell productivity were lower. To boost intracellular protein synthesis, we then used CRISPR/Cas9-mediated genome editing technology to knock out <i>ompT</i>, <i>iclR</i>, and <i>arcA</i>, yielding the high-producer EcN::T7Δ<i>ompT</i>Δ<i>iclR</i>Δ<i>arcA</i>. This triple-deletion mutant produced 3.2-fold more reporter protein than its parental strain EcN::T7, reaching 70% of the BL21(DE3) output. When this strain was used to produce recombinant IFNα-2b, the final protein yield reached 89.3% of that achieved in BL21(DE3). Even without extra endotoxin-removal steps, the IFNα-2b purified from EcN::T7Δ<i>ompT</i>Δ<i>iclR</i>Δ<i>arcA</i> contained the same low level as the product from the BL21(DE3) that had been through extensive downstream cleaning. By eliminating the need for costly LPS removal, the engineered EcN::T7Δ<i>ompT</i>Δ<i>iclR</i>Δ<i>arcA</i> becomes an economical, clinical-ready biomanufacturing platform.</p>

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Construction of probiotic Escherichia coli Nissle 1917 as a next-generation expression platform and its application in therapeutic protein production

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月24日

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  • Chenglin Wang,
  • Lun Luo,
  • Ziqiao Dai,
  • Wantong Zhang,
  • Yi Ma,
  • Jufang Wang

摘要

Biologics produced in Escherichia coli BL21(DE3) require rigorous removal of lipopolysaccharide (LPS), since even traces can trigger severe immune responses and potentially fatal sequelae in humans. In contrast, the clinically licensed probiotic Escherichia coli Nissle 1917 (EcN) is inherently LPS-deficient, displaying only 0.86% of the LPS activity observed in BL21(DE3). Due to its markedly reduced immunostimulatory activity, EcN is an attractive chassis for manufacturing proteins with minimal LPS burden. On the basis of the constructed strain EcN::T7 by inserting the T7 RNA polymerase gene into chromosome, using GFP as a model protein, this strain produced only 30% of the yield obtained from BL21(DE3) because both cell density (OD₆₀₀) and per-cell productivity were lower. To boost intracellular protein synthesis, we then used CRISPR/Cas9-mediated genome editing technology to knock out ompT, iclR, and arcA, yielding the high-producer EcN::T7ΔompTΔiclRΔarcA. This triple-deletion mutant produced 3.2-fold more reporter protein than its parental strain EcN::T7, reaching 70% of the BL21(DE3) output. When this strain was used to produce recombinant IFNα-2b, the final protein yield reached 89.3% of that achieved in BL21(DE3). Even without extra endotoxin-removal steps, the IFNα-2b purified from EcN::T7ΔompTΔiclRΔarcA contained the same low level as the product from the BL21(DE3) that had been through extensive downstream cleaning. By eliminating the need for costly LPS removal, the engineered EcN::T7ΔompTΔiclRΔarcA becomes an economical, clinical-ready biomanufacturing platform.