Engineering a genomically recoded organism with one stop codon

The genetic code is conserved across all domains of life, yet exceptions have revealed variations in codon assignments and associated translation factors^1-3. 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 tRNA^Trp 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.