Engineered orthogonal translation systems from metagenomic libraries expand the genetic code

Seki, K.; Nguyen, M. T. A.; Penev, P. I.; Isaacs, F. J.; Banfield, J. F.; Jewett, M. C.

doi:10.1101/2025.10.30.685624

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bioRxiv · 10.1101/2025.10.30.685624

Engineered orthogonal translation systems from metagenomic libraries expand the genetic code

Seki, K.·Nguyen, M. T. A.·Penev, P. I.·Isaacs, F. J.·Banfield, J. F.·Jewett, M. C.

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Abstract

Genetic code expansion with non-canonical amino acids (ncAAs) opens new opportunities for the function and design of proteins by broadening their chemical repertoire. Unfortunately, ncAA incorporation 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 developed an integrated computational and experimental pipeline to profile the orthogonality of >200 tRNAs, test >1,250 combinations of aaRS:tRNA pairs, and identify the AP1 TrpRS:tRNATrpUCA as an orthogonal pair that natively encodes tryptophan at the UGA codon. We demonstrate that the AP1 TrpRS:tRNATrpUCA is highly active in cell-free and cellular contexts. We then use Ochre, 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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Seki, K., Nguyen, M. T. A., Penev, P. I., Isaacs, F. J., Banfield, J. F., Jewett, M. C.. 2025-10-30. Engineered orthogonal translation systems from metagenomic libraries expand the genetic code. https://doi.org/10.1101/2025.10.30.685624

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