N-Acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo

Posttranslational modifications play a crucial role in modulating protein structure and function. Genetic incorporation of unnatural amino acids into a specific site of a protein facilitates the systematic study of protein modifications including acetylation. We here report the directed evolution of pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei to create N-acetyl lysyl-tRNA synthetases (AcKRSs) using a new selection system based on the killing activity of the toxic ccdB gene product. The amino acid specificity of these and of published [1] and [2] AckRSs was tested in vitro and in vivo, and the enzyme-kinetic properties of the AckRSs were evaluated for the first time.

► N-acetyl lysyl-tRNA synthetases were evolved by a CcdB-based selection. ► N-acetyl lysine specificity was validated by both in vitro and in vivo approaches. ► Kinetic properties of the evolved synthetases were evaluated for the first time. ► The evolved synthetases will facilitate the systematic study of protein acetylation.