Improving genomically recoded Escherichia coli for the production of proteins containing non-canonical amino acids

• Published in: bioRxiv
• Main Authors: Perez, Jessica G, Carlson, Erik D, Weisser, Oliver, Kofman, Camila, Seki, Kosuke, Des Soye, Benjamin J, Karim, Ashty S, Jewett, Michael C
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 11.12.2021
• Subjects: Amino acids; Codons; Conflicts of interest; Deactivation; DNA-directed RNA polymerase; E coli; Escherichia coli; Genetic diversity; Green fluorescent protein; Lysine; Nuclease; Protein folding; Proteins; RNA polymerase
• DOI: 10.1101/2021.12.10.472167

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, we describe the construction of a series of genomically recoded organisms that are optimized for cellular protein production. We demonstrate 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, we introduce a genomic IPTG-inducible T7 RNA polymerase (T7RNAP) cassette into these strains. Using an optimized platform, we demonstrated the ability to introduce 2 identical N6-(propargyloxycarbonyl)-L-Lysine residues site specifically into sfGFP with a 17-fold improvement in production relative to the parent. We envision that our library of organisms will provide the community with multiple options for increased expression of proteins with new and diverse chemistries. Competing Interest Statement M.C.J. has a financial interest in Pearl Bio. M.C.J.s interests are reviewed and managed by Northwestern University in accordance with their conflict of interest policies. All other authors declare no conflicts of interest.