Multiplexed automated genome engineering of a recoded E. coli strain to enable the production of low-endotoxin recombinant therapeutic proteins with an expanded genetic code

• Justin Swing, Chemical Engineering, University of Delaware

• Aditya Kunjapur, Chemical & Biomolecular Engineering, University of Delaware

Site-specific incorporation of nonstandard amino acids (nsAAs) is a powerful tool to modify recombinant proteins. nsAAs possess diverse chemical functionalities not seen in the 20 proteinogenic amino acids, and they may alter the physical and chemical properties of the proteins in which they are incorporated. This technology is a powerful tool for the development of next-generation biopharmaceuticals and can be used in numerous ways, such as facilitating bioconjugation reactions, increasing the stability for therapeutic peptides, breaking immune self-tolerance, and enhancing the immunogenicity of foreign antigens for vaccine design. Escherichia coli is generally used for the production of recombinant proteins containing nsAAs; however, E. coli and essentially all Gram-negative bacteria contain lipopolysaccharide (LPS), also known as endotoxin, which induces pro-inflammatory and pyrogenic activity in mammals. As a result, therapeutic proteins expressed in E. coli require extensive downstream purification to remove LPS. Instead of this, E. coli can be genetically engineered to reduce the immunogenicity of its LPS by modifying the structure of lipid A to its non-toxic precursor, lipid IVA. We have applied this approach using multiplex automated genome engineering (MAGE) to develop an engineered E. coli strain capable of: (1) high fidelity nsAA incorporation and (2) endotoxin-free therapeutic protein production. Using this strain, we will begin to create various therapeutic proteins with nsAAs incorporated to enable increased stability, immunogenicity, and enable bioconjugation.