Improving genomically recoded Escherichia coli to produce proteins containing non‐canonical amino acids

A genomically recoded Escherichia coli strain that lacks all amber codons and release factor 1 (C321.∆A) enables efficient genetic encoding of chemically diverse non-canonical amino acids (ncAAs) into proteins. While C321.∆A has opened new opportunities in chemical and synthetic biology, this strain has not been optimized for protein production, limiting its utility in widespread industrial and academic applications. To address this limitation, the construction of a series of genomically recoded organisms that are optimized for cellular protein production is described. It is demonstrated that the functional deactivation of nucleases (e.g., rne, endA) and proteases (e.g., lon) increases production of wild-type superfolder green fluorescent protein (sfGFP) and sfGFP containing two ncAAs up to ≈5-fold. Additionally, a genomic IPTG-inducible T7 RNA polymerase (T7RNAP) cassette into these strains is introduced. Using an optimized platform, the ability to introduce two identical N6 -(propargyloxycarbonyl)-L -Lysine residues site specifically into sfGFP with a 17-fold improvement in production relative to the parent strain is demonstrated. The authors envision that their library of organisms will provide the community with multiple options for increased expression of proteins with new and diverse chemistries.