Enhancing the utility of unnatural amino acid synthetases by manipulating broad substrate specificity

Many unnatural amino acid synthetases have been evolved to enable the site-specific in vivo incorporation of many useful functionalities into proteins. While these unnatural amino acid-tRNA synthetase-tRNA(CUA) pairs do not incorporate endogenous amino acids, their substrate specificity has not been assessed for other unnatural amino acids. Here we demonstrate that the unnatural synthetases can be permissive to many unnatural amino acid substrates. The utility of unnatural synthetases can be further expanded by manipulating the synthetase active sites by mutagenesis. Here we have also shown that an L-2-naphthylalanine synthetase can be converted into a permissive L-4-benzoylphenylalanine synthetase with a single mutation without compromising fidelity. Permissive unnatural amino acid synthetases should significantly expand the tool set available for manipulation of proteins.