Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

Abstract

Genetic encoding of noncanonical amino acids (ncAAs) through sense codon reassignment is an efficient tool for expanding the chemical functionality of proteins. Incorporation of multiple ncAAs, however, is particularly challenging. This work describes the first attempts to reassign the sense methionine (Met) codon AUG to two different ncAAs in bacterial protein translation. Escherichia coli methionyl-tRNA synthetase (MetRS) charges two tRNAs with Met: tRNA fMet initiates protein synthesis (starting AUG codon), whereas elongator tRNA Met participates in protein elongation (internal AUG codon(s)). Preliminary in vitro experiments show that these tRNAs can be charged with the Met analogues azidohomoalanine (Aha) and ethionine (Eth) by exploiting the different substrate specificities of EcMetRS and the heterologous MetRS / tRNA Met pair from the archaeon Sulfolobus acidocaldarius, respectively. Here, we explored whether this configuration would allow a differential decoding during in vivo protein initiation and elongation. First, we eliminated the elongator tRNA Met from a methionine auxotrophic E. coli strain, which was then equipped with a rescue plasmid harboring the heterologous pair. Although the imported pair was not fully orthogonal, it was possible to incorporate preferentially Eth at internal AUG codons in a model protein, suggesting that in vivo AUG codon reassignment is possible. To achieve full orthogonality during elongation, we imported the known orthogonal pair of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) / tRNA Pyl and devised a genetic selection system based on the suppression of an amber stop codon in an important glycolytic gene, pfkA, which restores enzyme functionality and normal cellular growth. Using an evolved PylRS able to accept Met analogues, it should be possible to reassign the AUG codon to two different ncAAs by using directed evolution.

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

“…In addition to the host metabolic engineering strategies described above, these issues could be further addressed by improving the performance of endogenous MetRS using directed evolution of its substrate specificity. This can also be done with imported heterologous MetRS enzymes from other phylogenetically distant sources as previously described [6]. Next, further optimisation of the fermentation protocol could be accomplished by, for example, by switching to recombinant expression in batch cultures.…”

“…Met contains a unique thioether unit, whose sulphur atom (although often involved in S/π interactions with adjacent aromatic amino acids) can easily be replaced by methylene [3], oxygen [2], selenium [4] and even tellurium [5]. Many non-canonical isosteric analogues and surrogates of Met, which are translationally active, are activated by methionyl-tRNA synthetase (MetRS) with a kinetic turnover similar to those of the native substrate [6]. For this reason, the plasticity of substrate binding in wild-type MetRS can be used to co-translate a relatively large number of analogues and surrogates such as metoxinine [2], ethionine [7], homopropargylglycine [8] and azidohomoalanine [9].…”

Hijacking Translation Initiation for Synthetic Biology

“…Expressing this mutant E. coli MetRS in HEK293T afforded specific incorporation of Anl at the initiating position and Met at internal AUG positions . A similar strategy was used in E. coli employing what was thought to be an orthogonal MetRS from Sulfolobus acidocaldarius ( Sa ‐MetRS) with unique ncAA recognition . It was hypothesized that deleting elongator tRNA Met from the E. coli genome with rescue by Sa ‐MetRS/ Sa ‐tRNA Met would allow for specific incorporation of ethionine ( 43 , Eth) at elongator AUG codons facilitated by Sa ‐MetRS/ Sa ‐tRNA Met while incorporation of azidohomoalanine ( 54 , Aha) could be restricted to the initiator position, incorporated by endogenous MetRS/tRNA fMet .…”

“…However, this strategy was met with limited success as it was found that the E. coli and S. acidocaldarius MetRS enzymes were not completely orthogonal. To overcome this problem, it was proposed that a demonstrated orthogonal aaRS/tRNA pair, such as the pyrrolysyl‐tRNA synthetase (PylRS)/tRNA Pyl could be evolved for Met recognition and used to replace elongator tRNA Met . Regardless of the method used to improve site‐selectivity, RSI will still cause global integration of the amino acid analogue into the host proteome.…”

Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?

“…leads to a genetic firewall . In cases when CMOs are not able to create the essential synthetic nutrient (trophic containment) , combination of the genetic firewall with this “alien ersatz” increases the degree of biocontainment . The likelihood of spreading genetic material from genetically modified organisms (GMOs) into the environment is still underestimated and not well studied .…”