Evolving Bacterial Fitness with an Expanded Genetic Code

Abstract

Since the fixation of the genetic code, evolution has largely been confined to 20 proteinogenic amino acids. The development of orthogonal translation systems that allow for the codon-specific incorporation of noncanonical amino acids may provide a means to expand the code, but these translation systems cannot be simply superimposed on cells that have spent billions of years optimizing their genomes with the canonical code. We have therefore carried out directed evolution experiments with an orthogonal translation system that inserts 3-nitro-L-tyrosine across from amber codons, creating a 21 amino acid genetic code in which the amber stop codon ambiguously encodes either 3-nitro-L-tyrosine or stop. The 21 amino acid code is enforced through the inclusion of an addicted, essential gene, a beta-lactamase dependent upon 3-nitro-L-tyrosine incorporation. After 2000 generations of directed evolution, the fitness deficit of the original strain was largely repaired through mutations that limited the toxicity of the noncanonical. While the evolved lineages had not resolved the ambiguous coding of the amber codon, the improvements in fitness allowed new amber codons to populate protein coding sequences.

Addicting Escherichia coli to New-to-Nature Reactions

“…Notably, in an effort to mimic the localization observed for Ru1 by adding 3nY onto agar plates (10 μL from a 20-mM stock solution), we observed a distinct halo pattern at the location at which 3nY was provided (Figure H and Figure S4). This marked difference with respect to supplying Ru1 is consistent with the previously observed toxicity of this ncAA at high concentrations . Thus, these results highlight the advantage of producing 3nY over time by a biocompatible catalyst, which does not show any apparent toxicity when applied onto agar plates containing live E. coli.…”

“…This marked difference with respect to supplying Ru1 is consistent with the previously observed toxicity of this ncAA at high concentrations. 31 Thus, these results highlight the advantage of producing 3nY over time by a biocompatible catalyst, which does not show any apparent toxicity when applied onto agar plates containing live E. coli.…”

Tumour neoantigen mimicry by microbial species in cancer immunotherapy

“…Many microbes that colonise the human body can synthesise all 20 proteinogenic amino acids of humans, 95 , 96 and viruses can harness the host cell translational machinery to exclusively use the endogenous amino acid repertoire. It is believed that the human gastrointestinal tract harbours approximately 10 14 microbes, which, together, equate to roughly 1000 times the number of cells and 10000 times the DNA content of the human body.…”

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

“…By engineering the suppressor tRNA Tyr with two substitutions in the anticodon loop (G34C/G37A), Rauch et al further improved nTyr-incorporation efficiency [ 131 ]. Recently, Tack et al utilized an engineered β-lactamase that is structurally dependent on nTyr-incorporation to study the evolving fitness of bacteria with an expanded genetic code and they found that after 2000 generations of directed evolution, the fitness deficit of cells related to nTyr toxicity was overcome by adaptive mutations [ 132 ]. The most commonly mutated or deleted genes were amino acid transporters in the hydroxyl and aromatic amino acid permease family such as the tyrosine-specific permease TyrP and the tryptophan permease Mtr.…”