Studying lysine acetylation of citric acid cycle enzymes by genetic code expansion

Lysine acetylation is one of the most abundant post-translational modifications in nature, affecting many key biological pathways in both prokaryotes and eukaryotes. It has not been long since technological advances led to understanding of the roles of acetylation in biological processes. Most of those studies were based on proteomic analyses, which have identified thousands of acetylation sites in a wide range of proteins. However, the specific role of individual acetylation event remains largely unclear, mostly due to the existence of multiple acetylation and dynamic changes of acetylation levels. To solve these problems, the genetic code expansion technique has been applied in protein acetylation studies, facilitating the incorporation of acetyllysine into a specific lysine position to generate a site-specifically acetylated protein. By this method, the effects of acetylation at a specific lysine residue can be characterized with minimal interferences. Here, we summarized the development of the genetic code expansion technique for lysine acetylation and recent studies on lysine acetylation of citrate acid cycle enzymes in bacteria by this approach, providing a practical application of the genetic code expansion technique in protein acetylation studies.