Abstract
AbstractA genomically recoded Escherichia coli strain that lacks all amber codons and release factor 1 (C321.ΔA) enables efficient genetic encoding of chemically diverse, non-canonical amino acids (ncAAs) into proteins. While C321.ΔA 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.
Publisher
Cold Spring Harbor Laboratory
Reference77 articles.
1. Lodish, H. , Berk, A. , Zipursky, S. L. , Matsudaira, P. , Baltimore, D. , & Darnell, J. (2000). Molecular Cell Biology (4th ed.). W. H. Freeman .
2. Use of the UGA terminator as a tryptophan codon in yeast mitochondria.
3. Natural expansion of the genetic code;Nature Chemical Biology,2007
4. Next-generation genetic code expansion
5. Cell-Free Exploration of the Natural Product Chemical Space;Chembiochem: A European Journal of Chemical Biology,2021