Engineered Aminoacyl-tRNA Synthetases with Improved Selectivity toward Noncanonical Amino Acids

A wide range of noncanonical amino acids (ncAAs) can be incorporated into proteins in living cells by using engineered aminoacyl-tRNA synthetase/tRNA pairs. However, most engineered tRNA synthetases are polyspecific; that is, they can recognize multiple rather than one ncAA. Polyspecificity of engineered tRNA synthetases imposes a limit to the use of genetic code expansion because it prevents specific incorporation of a desired ncAA when multiple ncAAs are present in the growth media. In this study, we employed directed evolution to improve substrate selectivity of polyspecific tRNA synthetases by developing substrate-selective readouts for flow-cytometry-based screening with the simultaneous presence of multiple ncAAs. We applied this method to improve the selectivity of two commonly used tRNA synthetases, p-cyano-L-phenylalanyl aminoacyl-tRNA synthetase (pCNFRS) and N-epsilon-acetyl-lysyl aminoacyl-tRNA synthetase (AcKRS), with broad specificity. Evolved pCNFRS and AcKRS variants exhibit significantly improved selectivity for ncAAs p-azido-L-phenylalanine (pAzF) and m-iodo-L-phenylalanine (mlF), respectively. To demonstrate the utility of our approach, we used the newly evolved tRNA synthetase variant to produce highly pure proteins containing the ncAA mlF, in the presence of multiple ncAAs present in the growth media. In summary, our new approach opens up a new avenue for engineering the next generation of tRNA synthetases with improved selectivity toward a desired ncAA.