In-Cell Synthesis of N ^ε -acetyl-L-lysine for Facile Protein Acetylation

N ^ε -acetylation of proteins is a crucial post-translational modification (PTM) that occurs across all kingdoms of life and plays key roles in nearly every cellular process. Due to its broad significance, there is a strong demand for chemical biology tools that enable efficient, site-specific acetylation of target proteins in live cells. Genetic code expansion (GCE) has emerged as a powerful tool for introducing N ^ε -acetylation at specific lysine residues in proteins. However, achieving adequate expression levels typically requires adding 2-10 mM of chemically synthesized N ^ε -Acetyl-L-lysine (AcK) to the culture medium, which can be cumbersome and costly. To overcome this limitation, we present the first proof-of-concept for a one-pot acetylation platform that simplifies protein acetylation in both E. coli and mammalian cells. Our approach begins with the discovery of 10 novel lysine acetyltransferases (KATs) capable of biosynthesizing AcK from basic carbon sources. When these enzymes are co-expressed with the genetic incorporation machinery for AcK, they facilitate streamlined, site-specific acetylation of any target protein without compromising E. coli viability. This innovative platform not only broadens the range of unnatural amino acids (UAAs) that can be biosynthesized and incorporated but also provides a powerful tool for probing the histone and non-histone acetylation events in live cells. In addition, this technique offers an eco-friendly and scalable method to produce synthetic acetylated proteins, which will provide practical value in acetylation/deacetylation-related research such as chemical biology, biotechnology, and drug development.