CRISPR/Cas9-Mediated Deletion of lpxM and lpxL Reduces Endotoxin Activity in Escherichia coli W3110 Without Compromising Recombinant Protein Production

Background/Objectives: Escherichia coli (E. coli) W3110 is a common host for recombinant protein production. However, lipopolysaccharide (LPS) endotoxin pollution brings extra challenges for downstream purification and safety testing of biotherapeutic proteins. This work set out to build low-endotoxin E. coli mutants by editing lipid A synthesis genes. We further tested their growth profiles, endotoxin levels, and ability to produce recombinant anti-vWF-A1 protein. Methods: The CRISPR/Cas9 genome-editing system was applied to individually and combinatorially delete lpxM and lpxL, two key genes in lipid A synthesis in E. coli W3110. We characterized bacterial growth kinetics, measured LPS endotoxin activity, expressed anti-vWF-A1 protein in wild-type and mutant strains, and quantified protein yield and residual endotoxin in purified anti-vWF-A1 preparations. Results: Three mutants, W3110ΔlpxM, W3110ΔlpxL and W3110ΔlpxMΔlpxL, were generated. Deletion of lpxL (single or combined with lpxM) extended the lag phase and decreased the maximum specific growth rate, while ΔlpxM showed only minor growth defects; all strains achieved similar final biomass. LPS endotoxin was markedly reduced in mutants, with the ΔlpxMΔlpxL double mutant showing a 92.1% reduction. When expressing anti-vWF-A1, purified protein from mutants contained significantly less endotoxin, and protein yields were comparable across strains (202–210 mg/L, 96.2–99.5% of wild-type yield). Conclusions: Deletion of lpxM and lpxL effectively lowers endotoxin loads without substantially compromising recombinant anti-vWF-A1 productivity. This gene-editing strategy provides a feasible route to engineer low-endotoxin E. coli chassis for the manufacture of recombinant therapeutic proteins.