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.