Engineering a genomically recoded organism with one stop codon

• 成果类型: Article
• 署名作者: Grome, Michael W.; Nguyen, Michael T. A.; Moonan, Daniel W.; Mohler, Kyle; Gurara, Kebron; Wang, Shenqi; Hemez, Colin; Stenton, Benjamin J.; Cao, Yunteng; Radford, Felix; Kornaj, Maya; Patel, Jaymin; Prome, Maisha; Rogulina, Svetlana; Sozanski, David; Tordoff, Jesse; Rinehart, Jesse; Isaacs, Farren J.
• 署名单位: Yale University; Yale University; Yale University; Yale University; Yale University
• 刊物名称: Nature
• ISSN/ISSBN: 0028-3229
• DOI: 10.1038/s41586-024-08501-x
• 发表日期: 2025-03-13
• 关键词: release-factors; amino-acid; genetic-code; in-vitro; translation; genome; rna; readthrough; mutations; EVOLUTION

摘要

The genetic code is conserved across all domains of life, yet exceptions have revealed variations in codon assignments and associated translation factors1, 2-3. 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 triangle TAG Escherichia coli C321.triangle 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.