Efficient Genetic Code Expansion Without Host Genome Modifications

Supplementing translation with non-canonical 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 E. coli strains with native ribosomes, we develop a novel 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 allows us to identify 12 mutually orthogonal tRNA–synthetase pairs. Enabled by these findings, we evolve and optimize five tRNA–synthetase pairs to incorporate a broad repertoire of ncAAs at orthogonal quadruplet codons. Finally, 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. Given the generality of our approach and streamlined resources, our findings will accelerate innovations in multiplexed genetic code expansion and enable the discovery of chemically diverse biomolecules for researcher-defined applications. ### Competing Interest Statement AHB and AC have filed a Provisional Patent Application through The Scripps Research Institute on sequences and activities of tRNAs, proteins, enzymes, and bacterial strains described in this manuscript. The authors otherwise declare no competing interests.