Abstract
Since the fixation of the genetic code, evolution has largely been confined to 20 proteinogenic amino acids. The development of orthogonal translation systems that allow for the codon-specific incorporation of noncanonical amino acids may provide a means to expand the code, but these translation systems cannot be simply superimposed on cells that have spent billions of years optimizing their genomes with the canonical code. We have therefore carried out directed evolution experiments with an orthogonal translation system that inserts 3-nitro-L-tyrosine across from amber codons, creating a 21 amino acid genetic code in which the amber stop codon ambiguously encodes either 3-nitro-L-tyrosine or stop. The 21 amino acid code is enforced through the inclusion of an addicted, essential gene, a beta-lactamase dependent upon 3-nitro-L-tyrosine incorporation. After 2000 generations of directed evolution, the fitness deficit of the original strain was largely repaired through mutations that limited the toxicity of the noncanonical. While the evolved lineages had not resolved the ambiguous coding of the amber codon, the improvements in fitness allowed new amber codons to populate protein coding sequences.