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Engineered orthogonal translation systems from metagenomic libraries expand the genetic code.

Kosuke Seki1, Michael T A Nguyen2,3, Petar I Penev4,5

  • 1Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.

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|November 24, 2025
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Scientists discovered a new orthogonal translation system from metagenomic data to incorporate non-canonical amino acids (ncAAs) into proteins. This system enables genetic code expansion, advancing protein engineering and design.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Genetic code expansion using non-canonical amino acids (ncAAs) enhances protein functionality and design possibilities.
  • Current methods for discovering orthogonal aminoacyl-tRNA synthetase (aaRS) and tRNA pairs are limited by low throughput and a small collection of available systems.

Purpose of the Study:

  • To discover, characterize, and engineer novel orthogonal translation systems for genetic code expansion.
  • To develop an integrated computational and experimental pipeline for efficient discovery of aaRS:tRNA pairs.
  • To enable the incorporation of specific ncAAs, such as 5-hydroxytryptophan, into proteins at the UGA codon.

Main Methods:

  • Metagenomic data mining to identify potential orthogonal tRNA and aaRS candidates.
  • High-throughput screening of over 1,250 aaRS:tRNA combinations for orthogonality.
  • Characterization of the identified orthogonal system in cell-free and cellular environments.
  • Engineering of an aaRS variant for specific ncAA incorporation in a genomically recoded bacterial strain (Ochre E. coli).

Main Results:

  • Discovery and characterization of the AP1 TrpRS:tRNATrpUCA orthogonal pair, which decodes the UGA codon with tryptophan.
  • Demonstration of high activity of the AP1 system in both cell-free and cellular contexts.
  • Successful engineering of an AP1 TrpRS variant for 5-hydroxytryptophan incorporation at the UGA stop codon in Ochre E. coli.

Conclusions:

  • The integrated pipeline of metagenomic bioprospecting, cell-free screening, and cell-based engineering significantly accelerates the discovery and optimization of orthogonal translation systems.
  • This work expands the toolkit for genetic code expansion, paving the way for novel protein designs and functions.
  • The developed strategy provides a robust platform for future advancements in synthetic biology and protein engineering.