Efficient genetic code expansion without host genome modifications

Supplementing translation with noncanonical amino acids (ncAAs) can yield protein sequences with new-to-nature functions but existing ncAA incorporation strategies suffer from low efficiency and context dependence. We uncover codon usage as a previously unrecognized contributor to efficient genetic code expansion using non-native codons. Relying only on conventional Escherichia coli strains with native ribosomes, we develop a plasmid-based codon compression strategy that minimizes context dependence and improves ncAA incorporation at quadruplet codons. We confirm that this strategy is compatible with all known genetic code expansion resources, which allowed us to identify 12 mutually orthogonal transfer RNA (tRNA)–synthetase pairs. Enabled by these findings, we evolved and optimized five tRNA–synthetase pairs to incorporate a broad repertoire of ncAAs at orthogonal quadruplet codons. Lastly, we extend these resources to an in vivo biosynthesis platform that can readily create >100 new-to-nature peptide macrocycles bearing up to three unique ncAAs. Our approach will accelerate innovations in multiplexed genetic code expansion and the discovery of chemically diverse biomolecules.