Post-translational modifications (PTMs) are pivotal regulators of protein function, influencing processes such as enzymatic activity, stability and molecular interactions, yet their precise study has been hampered by the lack of tools to produce proteins with site-specific and homogeneous modifications. Traditional approaches rely on enzyme-based modulation, chemical synthesis or site-directed mutagenesis to mimic modifications. However, these methods are often indirect and lack precision, as the enzymes used in these approaches exhibit broad substrate specificity and mutagenesis cannot fully replicate the functional properties of the native modification.

By constructing and optimizing orthogonal aaRS–tRNA pairs for efficient amber codon suppression in bacterial and mammalian cells, this approach enabled the incorporation of lysine lactylation at defined sites in target proteins. Using this method, we successfully introduced lactylation into p53 at two lysine residues in its DNA-binding domain and systematically investigated the functional impact on p53 activity both in vitro and in vivo. By circumventing the ambiguity associated with mutation-based or enzyme-based approaches, this system provides a direct and reliable tool for studying lysine lactylation and related acylations. Additionally, the orthogonal nature of the system ensures minimal interference with native cellular machinery, allowing robust and physiologically relevant experiments.