Optimized directed evolution of E. coli leucyl‐tRNA synthetase adds many noncanonical amino acids into the eukaryotic genetic code including ornithine and Nε‐acetyl‐methyllysine

Site‐specific incorporation of noncanonical amino acids (ncAAs) into proteins in eukaryotes has predominantly relied on the pyrrolysyl‐tRNA synthetase/tRNA pair. However, access to additional easily engineered pairs is crucial for expanding the structural diversity of the ncAA toolbox in eukaryotes. The Escherichia coli‐derived leucyl‐tRNA synthetase (EcLeuRS)/tRNA pair presents a particularly promising alternative. This pair has been engineered to charge a small yet structurally diverse group of ncAAs in eukaryotic cells. However, expanding the substrate scope of EcLeuRS has been difficult due to the suboptimal yeast‐based directed evolution platform used for its engineering. In this study, we address this limitation by optimizing the yeast‐based directed evolution platform for efficient selection of ncAA‐selective EcLeuRS mutants. Using the optimized selection system, we demonstrate rapid isolation of many novel EcLeuRS mutants capable of incorporating various ncAAs in mammalian cells, including ornithine and Nε‐acetyl‐methyllysine, a recently discovered post‐translational modification in mammalian cells.