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Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon—out of up to 6 synonyms—to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coliwith a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme—with simple corrections at just seven positions—to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA.

We recently characterized a new class of pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs from Methanomassiliicocalesthat are active and orthogonal in Escherichia coli. The aminoacyl-tRNA synthetases (aaRSs) of these pairs lack the N-terminal domain that is essential for tRNA recognition and in vivoactivity in the Methanosarcina mazei(Mm) PylRS but share a homologous active site with MmPylRS; this facilitates the transplantation of mutations discovered with existing PylRS systems into the new PylRS systems to reprogram their substrate specificity for the incorporation of noncanonical amino acids (ncAAs). Several of the new PylRS/PyltRNA pairs, or their evolved variants [including Methanomethylophilus alvus(Ma) PylRS/MaPyltRNA(6)CUA], are mutually orthogonal to the MmPylRS/MmPyltRNA pair, and the active sites of the Mmpair and Mapair can be diverged to enable the incorporation of distinct ncAAs in response to distinct codons via orthogonal translation in E. coli. Here we demonstrate that MaPylRS/MaPyltRNA(6)CUAis orthogonal to the aaRSs and tRNAs in mammalian cells and directs efficient incorporation of ncAAs into proteins. Moreover, we confirm that the MaPylRS/MaPyltRNA(6) and MmPylRS/MmPyltRNA pairs are mutually orthogonal in mammalian cells and demonstrates that these pairs can be used to encode distinct ncAAs into a protein in mammalian cells. Thus, the MaPylRS/MaPyltRNA(6)CUApair provides an additional pair that is orthogonal in both E. coliand mammalian systems and is mutually orthogonal to the most widely used system for genetic code expansion. Our results provide a foundation for expanding the scope of genetic code expansion and may also facilitate strategies for proteome-wide ncAA tagging with mutually orthogonal systems.