20470: Engineering of Escherichia coli hosts for endotoxin-free production of recombinant proteins and plasmid DNA

Recombinant proteins and DNA manufactured in Escherichia coli are inherently contaminated with cell constituents, especially the abundant lipopolysaccharide (LPS). The lipid A moiety, which anchors the LPS molecule to the outer membrane, is a potent agonist for human TLR4/MD-2-mediated pro-inflammatory activity and, thus, represents the endotoxic principle of LPS. To prevent toxicity, material prepared from E. coli must be free of endotoxin. However, efficient and cost-effective elimination of endotoxin is challenging, and current methods fail to remove endotoxin entirely.

Presented here is an alternative strategy for producing endotoxin-free biomolecules: E. coli host strains that lack all outer membrane agonists for hTLR4/MD-2. These mutants entirely lack LPS, yet remain viable despite exclusively elaborating the tetra-acylated, endotoxic- inactive, lipid A precursor lipid IV_A. Viability requires a suppressor mutation that enables the strain to translocate lipid IV_A across the inner membrane to support outer membrane biogenesis. The initial mutant strains displayed temperature-sensitive growth above 40°C. More robust, temperature-resistant derivatives have been isolated capable of growing exponentially at up to 42°C, suggesting the presence of further suppressor mutation(s). Activation of hTLR4/MD-2 signalling by the mutant cells was many orders of magnitude lower compared with cells of the E. coli wild-type strain, and heterologous proteins and plasmid DNAs prepared from the mutants were free of endotoxic activity. These engineered E. coli strains lacking LPS hold promise for manufacturing of endotoxin-free products, and have been termed “FreE coli^TM” strains.