How Many Biochemistries Are Available To Build a Cell?

Acevedo‐Rocha, Carlos G.; Schulze‐Makuch, Dirk

doi:10.1002/cbic.201500379

Cited by 10 publications

(4 citation statements)

References 55 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

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“…Most proteins contain only one or a few Trp residues. As highlighted previously, [4] the evolution of E. coli with a synthetic genetic code endowed with [3,2]Tpa represented one of the most significant variations in the chemical makeup that a living cell encountered thus far in the laboratory. At that time, however, the adaptation mechanism was not elucidated.…”

Section: Figurementioning

confidence: 99%

How many Mutations are needed to Evolve the Chemical Makeup of a Synthetic Cell?

Lefèvre‐Morand,

Nikel,

Acevedo‐Rocha

2024

ChemBioChem

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The chemical evolution of a synthetic cell endowed with a synthetic amino acid as building block, analog to tryptophan, required the emergence of key mutations in genes involved in, inter alia, the general stress response, amino acid metabolism, stringent response, and chemotaxis. Understanding adaptation mechanisms to non‐canonical biomass components will inform strategies for engineering synthetic metabolic pathways and cells.

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“…To achieve a Trp‐independent reassignment (i.e. ‘real’ codon reassignment) at all UGG codons across E. coli 's genome – an experimental strategy for biocontainment still needs to be developed and executed (Acevedo‐Rocha and Schulze‐Makuch, ).…”

Section: Code Engineering Via Experimental Evolutionmentioning

confidence: 99%

Xenomicrobiology: a roadmap for genetic code engineering

Acevedo‐Rocha

Budiša

2016

Microbial Biotechnology

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SummaryBiology is an analytical and informational science that is becoming increasingly dependent on chemical synthesis. One example is the high‐throughput and low‐cost synthesis of DNA, which is a foundation for the research field of synthetic biology (SB). The aim of SB is to provide biotechnological solutions to health, energy and environmental issues as well as unsustainable manufacturing processes in the frame of naturally existing chemical building blocks. Xenobiology (XB) goes a step further by implementing non‐natural building blocks in living cells. In this context, genetic code engineering respectively enables the re‐design of genes/genomes and proteins/proteomes with non‐canonical nucleic (XNAs) and amino (ncAAs) acids. Besides studying information flow and evolutionary innovation in living systems, XB allows the development of new‐to‐nature therapeutic proteins/peptides, new biocatalysts for potential applications in synthetic organic chemistry and biocontainment strategies for enhanced biosafety. In this perspective, we provide a brief history and evolution of the genetic code in the context of XB. We then discuss the latest efforts and challenges ahead for engineering the genetic code with focus on substitutions and additions of ncAAs as well as standard amino acid reductions. Finally, we present a roadmap for the directed evolution of artificial microbes for emancipating rare sense codons that could be used to introduce novel building blocks. The development of such xenomicroorganisms endowed with a ‘genetic firewall’ will also allow to study and understand the relation between code evolution and horizontal gene transfer.

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How Many Biochemistries Are Available To Build a Cell?

Cited by 10 publications

References 55 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

How many Mutations are needed to Evolve the Chemical Makeup of a Synthetic Cell?

Xenomicrobiology: a roadmap for genetic code engineering

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