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bioRxiv · 10.1101/2024.11.27.625662

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

Ficaretta, E. D.·Yared, T. J.·Bhattacharjee, S.·Voss, L. A.·Huang, R. L.·Chatterjee, A.
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Abstract

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 N{varepsilon}-acetyl-methyllysine, a recently discovered post-translational modification in mammalian cells.

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Ficaretta, E. D., Yared, T. J., Bhattacharjee, S., Voss, L. A., Huang, R. L., Chatterjee, A.. 2024-11-27. Optimized directed evolution of E. coli leucyl-tRNA synthetase adds many noncanonical amino acids into the eukaryotic genetic code including ornithine and Nepsilon-acetyl-methyllysine. https://doi.org/10.1101/2024.11.27.625662

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