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.

Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

The leucyl-tRNA synthetase (EcLeuRS)/tRNA pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNA. Here, we overcome this limitation by evolving tRNA directly in mammalian cells by using a virus-assisted selection scheme.

sp. KT-1 PahZ2 Structure Reveals a Role for Conformational Dynamics in Peptide Bond Hydrolysis.

Poly(aspartic acid) (PAA) is a common water-soluble polycarboxylate used in a broad range of applications. PAA biodegradation and environmental assimilation were first identified in river water bacterial strains, sp. KT-1 and sp.