Engineering a genomically recoded organism with one stop codon

Michael W. Grome, Michael T. A. Nguyen, Daniel W. Moonan, Kyle Mohler, Kebron Gurara, Shenqi Wang, Colin Hemez, Benjamin J. Stenton, Yunteng Cao, Felix Radford, Maya Kornaj, Jaymin Patel, Maisha Prome, Svetlana Rogulina, David Sozanski, Jesse Tordoff, Jesse Rinehart () and Farren J. Isaacs ()
Additional contact information
Michael W. Grome: Yale University
Michael T. A. Nguyen: Yale University
Daniel W. Moonan: Yale University
Kyle Mohler: Yale University
Kebron Gurara: Yale University
Shenqi Wang: Yale University
Colin Hemez: Yale University
Benjamin J. Stenton: Yale University
Yunteng Cao: Yale University
Felix Radford: Yale University
Maya Kornaj: Yale University
Jaymin Patel: Yale University
Maisha Prome: Yale University
Svetlana Rogulina: Yale University
David Sozanski: Yale University
Jesse Tordoff: Yale University
Jesse Rinehart: Yale University
Farren J. Isaacs: Yale University

Nature, 2025, vol. 639, issue 8054, 512-521

Abstract: Abstract The genetic code is conserved across all domains of life, yet exceptions have revealed variations in codon assignments and associated translation factors1–3. Inspired by this natural malleability, synthetic approaches have demonstrated whole-genome replacement of synonymous codons to construct genomically recoded organisms (GROs)4,5 with alternative genetic codes. However, no efforts have fully leveraged translation factor plasticity and codon degeneracy to compress translation function to a single codon and assess the possibility of a non-degenerate code. Here we describe construction and characterization of Ochre, a GRO that fully compresses a translational function into a single codon. We replaced 1,195 TGA stop codons with the synonymous TAA in ∆TAG Escherichia coli C321.∆A4. We then engineered release factor 2 (RF2) and tRNATrp to mitigate native UGA recognition, translationally isolating four codons for non-degenerate functions. Ochre thus utilizes UAA as the sole stop codon, with UGG encoding tryptophan and UAG and UGA reassigned for multi-site incorporation of two distinct non-standard amino acids into single proteins with more than 99% accuracy. Ochre fully compresses degenerate stop codons into a single codon and represents an important step toward a 64-codon non-degenerate code that will enable precise production of multi-functional synthetic proteins with unnatural encoded chemistries and broad utility in biotechnology and biotherapeutics.

Date: 2025
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DOI: 10.1038/s41586-024-08501-x

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