Optimized directed evolution of leucyl-tRNA synthetase adds many noncanonical amino acids into the eukaryotic genetic code including ornithine and N -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 -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 N -acetyl-methyllysine, a recently discovered post-translational modification in mammalian cells.