Chemical Evolution of a Bacterial Proteome

Hoesl, Michael Georg; Oehm, M. Sc. Stefan; Durkin, Patrick; Darmon, Elise; Peil, Lauri; Aerni, Hans‐Rudolf; Rappsilber, Juri; Rinehart, Jesse; Leach, David R. F.; Söll, Dieter; Budiša, Nediljko

doi:10.1002/anie.201502868

Cited by 73 publications

(109 citation statements)

References 41 publications

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“…The most reasonable strategy would be to use properly configured metT / metUdeficient E. coli MG1655 strain which might be slowly adapted to use a different amino acid in place of Met using an evolution strategy recently reported by our group. [33,34] This configuration would also require the availability of mutually orthogonal MetRSs capable to generate the context-dependent AUG-codon reassignments: one ncAA (or Met itself) can be encoded site-specifically at the Nterminus and the other ncAAs (or Met itself) can be inserted globally at the internal AUG positions. …”

Section: Discussionmentioning

confidence: 99%

“…[32] Given its physiological importance, it might be more challenging to completely replace Met with a ncAA, similarly to the recently reported complete trophic reassignment of Trp with L-β-(thieno[3,2-b]pyrrolyl)alanine under experimentally designed evolutionary pressure. [33,34] Nevertheless, like Trp, Met is one of the least common amino acids in proteins: it represents only about 2.5 % of all residues, [35] mostly buried in the hydrophobic core of proteins where it is rarely directly involved in catalytic function. This feature makes it a good candidate for proteome-wide substitution using the SPI method.…”

Section: Global Reassignment Of the Aug Codonmentioning

confidence: 99%

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

Simone¹,

Acevedo‐Rocha²,

Hoesl³

et al. 2016

Croat. Chem. Acta

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Global Reassignment Of the Aug Codonmentioning

confidence: 99%

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

Simone¹,

Acevedo‐Rocha²,

Hoesl³

et al. 2016

Croat. Chem. Acta

Self Cite

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“…In an alternative strategy, Hoesl and colleagues used serial passaging to systematically replace tryptophan with L-beta-(thieno[3,2-b]pyrrolyl)alanine (Tpa) in the E. coli genome [62]. Working on a tryptophan auxotrophic E. coli strain, selection was carried out by lowering indole (tryptophan precursor) while maintaining high beta-thieno[3,2-b]pyrrole concentrations.…”

Section: Engineering Further Containment: Semantic Containmentmentioning

confidence: 99%

Synthetic biology approaches to biological containment: pre-emptively tackling potential risks

Torres

Krüger

Csibra

et al. 2016

Essays in Biochemistry

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Biocontainment comprises any strategy applied to ensure that harmful organisms are confined to controlled laboratory conditions and not allowed to escape into the environment. Genetically engineered microorganisms (GEMs), regardless of the nature of the modification and how it was established, have potential human or ecological impact if accidentally leaked or voluntarily released into a natural setting. Although all evidence to date is that GEMs are unable to compete in the environment, the power of synthetic biology to rewrite life requires a pre-emptive strategy to tackle possible unknown risks. Physical containment barriers have proven effective but a number of strategies have been developed to further strengthen biocontainment. Research on complex genetic circuits, lethal genes, alternative nucleic acids, genome recoding and synthetic auxotrophies aim to design more effective routes towards biocontainment. Here, we describe recent advances in synthetic biology that contribute to the ongoing efforts to develop new and improved genetic, semantic, metabolic and mechanistic plans for the containment of GEMs.

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“…This work suggests a selective advantage of an expanded genetic code in nature. In another study, evolved E. coli cells that completely replaces Trp with an analog (Tpa) in the proteome appear longer and thinner, and need to accummulate beneficial mutations to support robust growth [100]. …”

Section: Physiological Impact Of Rewiring Protein Synthesismentioning

confidence: 99%

Rewiring protein synthesis: From natural to synthetic amino acids

Fan

Evans

Ling

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. Scope of review This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. Major conclusions The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. General significance Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms.

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Chemical Evolution of a Bacterial Proteome

Cited by 73 publications

References 41 publications

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

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

Synthetic biology approaches to biological containment: pre-emptively tackling potential risks

Rewiring protein synthesis: From natural to synthetic amino acids

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