Construction of Probiotic Escherichia coli Nissle 1917 as a Next- Generation Expression Platform and Its Application in Therapeutic Protein Production

Biologics produced in Escherichia coli BL21(DE3) require rigorous removal of lipopolysaccharide (LPS), also termed endotoxin, as its presence can elicit severe immune responses and life threatening complications in humans. Escherichia coli Nissle 1917 (EcN) is a clinically approved probiotic with unique biosafety characteristics due to its LPS-deficient phenotype, and has only 0.86% of the LPS activity observed in BL21(DE3), so its markedly lower immunostimulatory potential makes it an attractive chassis for low-LPS protein production. Using GFP as a model protein for comparison, its yield in EcN is only ~ 30% of that observed in BL21(DE3) owing to lower cell density (OD₆₀₀) and per-cell protein yield. Transcriptomic profiling revealed that heterologous protein induction in EcN suppresses key biosynthetic and energy-generating pathways—including ribosome biogenesis, translation, and the tricarboxylic acid (TCA) cycle—thereby constraining heterologous protein synthesis. To boost intracellular protein synthesis in EcN, we first inserted the T7 RNA polymerase gene into the chromosome, creating EcN::T7. We then used CRISPR/Cas9-mediated genome editing technology to delete ompT, iclR, and arcA, yielding the high-yield mutant EcN::T7Δ ompT Δ iclR Δ arcA . This engineered strain produced 3.2-fold more reporter protein than its parental strain EcN::T7, reaching ~ 70% of the yield observed in BL21(DE3). When applied to interferon α-2b (IFNα-2b) production, mutant EcN::T7Δ ompT Δ iclR Δ arcA achieved 89.3% of BL21(DE3)’s titer while exhibiting post-purification LPS levels comparable to BL21(DE3). Notably, the the IFNα-2b retained full biological activity. By eliminating the need of extensive LPS removal procedures, this strategy positions EcN as a cost-effective and clinically compliant platform for biomanufacturing.