Genetic Incorporation of Ε‐n‐benzoyllysine by Engineering Methanomethylophilus Alvus Pyrrolysyl‐tRNA Synthetase

Post-translational modifications regulate protein structure and function. Lysine benzoylation is a newly discovered histone modification with unique physiological relevance. To construct proteins with this modification site-specifically, we generated orthogonal tRNAPyl -MaBzKRS pairs by engineering Methanomethylophilus alvus pyrrolysyl-tRNA synthetase, allowing the genetic incorporation of ϵ-N-benzoyllysine (BzK) into proteins with high efficiency in E. coli and mammalian cells. Two types of MaBzKRS were identified to incorporate BzK using mutations located at different positions of the amino acid binding pocket. These MaBzKRS are small in size and highly expressed, which will afford broad utilities in studying the biological effects of lysine benzoylation.