Expanding the Scope of Genetically Encoded Lysine Post‐Translational Modifications with Lactylation, β‐Hydroxybutyrylation and Lipoylation

Abstract Post‐translational modifications (PTMs) occurring on lysine residues, especially diverse forms of acylations, have seen rapid growth over the past two decades. Among them, lactylation and β‐hydroxybutyrylation of lysine side‐chains are newly identified histone marks and their implications in physiology and diseases have aroused broad research interest. Meanwhile, lysine lipoylation is highly conserved in diverse organisms and well known for its pivotal role in central metabolic pathways. Recent findings in the proteomic profiling of protein lipoylation have nonetheless suggested a pressing need for an extensive investigation. For both basic and applied research, it is necessary to prepare PTM‐bearing proteins particularly in a site‐specific manner. Herein, we use genetic code expansion to site‐specifically generate these lysine PTMs, including lactylation, β‐hydroxybutyrylation and lipoylation in proteins in E . coli and mammalian cells. Notably, using strategies including activity‐based selection, screening and rational design, unique pyrrolysyl‐tRNA synthetase variants were successfully evolved for each of the three non‐canonical amino acids, which enabled efficient production of recombinant proteins. Through encoding these ncAAs, we examined the deacylase activities of mammalian sirtuins to these modifications, and importantly we unfold the lipoamidase activity of several sirtuins.