Table · dataset · 2026
Engineered Orthogonal Translation Systems from Metagenomic Libraries Expand the Genetic Code
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Genetic code expansion with noncanonical amino acids (ncAAs) opens new opportunities for the design and engineering of proteins by broadening their chemical repertoire.
Description
Unfortunately, ncAA incorporation into proteins is limited both by a small collection of orthogonal aminoacyl-tRNA synthetases (aaRSs) and tRNAs and by low-throughput methods to discover them. Here, we report the discovery, characterization, and engineering of a UGA suppressing orthogonal translation system mined from metagenomic data.
We develop an integrated computational and experimental pipeline based on cell-free gene expression to screen the orthogonality of >200 tRNAs, test >1,250 combinations of aaRS/tRNA pairs, and identify the AP1 TrpRS/tRNA<sup>Trp</sup><sub>UCA</sub> as an orthogonal pair that natively encodes tryptophan at the UGA codon. We demonstrate that the AP1 TrpRS/tRNA<sup>Trp</sup><sub>UCA</sub> is highly active in cell-free and cellular contexts.
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We then use <i>Ochre</i>, a genomically recoded Escherichia coli strain that lacks UAG and UGA codons, to engineer an AP1 TrpRS variant capable of 5-hydroxytryptophan incorporation at an open UGA codon. We anticipate that our strategy of integrating metagenomic bioprospecting with cell-free screening and cell-based engineering will accelerate the discovery and optimization of orthogonal translation systems for genetic code expansion.
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Where it is published
- DOI doi.org/10.1021/acssynbio.6c00373.s003 ↗
DOI / persistent id · from figshare com
Catalogue records · 1
- OAI-PMH record api.figshare.com/v2/oai?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Af… ↗
metadata API · from figshare com
Topics
Provenance · 1 source records, 18 field assertions
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|---|---|---|---|
| figshare | oai:figshare.com:article/33719440 | 9 d ago | JSON v1 |
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