2026-10-12T05:11:49Z
https://api.figshare.com/v2/oai
oai:figshare.com:article/33719440
2026-09-14T06:08:36Z
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month_year_09_2026
Engineered Orthogonal
Translation Systems from Metagenomic
Libraries Expand the Genetic Code
Kosuke Seki (5991629)
Michael T. A. Nguyen (18098869)
Petar I. Penev (8124389)
Jillian F. Banfield (161078)
Farren J. Isaacs (1323855)
Michael C. Jewett (1323852)
Biochemistry
Space Science
Genetics
Molecular Biology
Biotechnology
Chemical Sciences not elsewhere classified
Cancer
opens new opportunities
noncanonical amino acids
natively encodes tryptophan
genetic code expansion
uca </ sub
metagenomic libraries expand
integrating metagenomic bioprospecting
experimental pipeline based
orthogonal translation systems
free gene expression
open uga codon
uga codon
ochre </
metagenomic data
uga codons
orthogonal pair
orthogonal aminoacyl
throughput methods
small collection
ncaa incorporation
lacks uag
integrated computational
hydroxytryptophan incorporation
highly active
free screening
chemical repertoire
cellular contexts
ap1 trprs
250 combinations
Genetic code expansion with noncanonical amino acids
(ncAAs) opens
new opportunities for the design and engineering of proteins by broadening
their chemical repertoire. 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. 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.
2026-09-14T00:00:00Z
Dataset
Dataset
10.1021/acssynbio.6c00373.s003
https://figshare.com/articles/dataset/Engineered_Orthogonal_Translation_Systems_from_Metagenomic_Libraries_Expand_the_Genetic_Code/33719440
CC BY-NC 4.0