d-lactate drives lysine d-lactylation to regulate metabolism in Escherichia coli.

l-lactate-derived lysine l-lactylation (KL-la) has emerged as a key regulator in diverse cellular processes and disease pathogenesis. While lactate predominantly exists as the l-isomer in eukaryotes, both l- and d-lactate are present in some bacteria. However, it remains unclear whether d-lactate can drive post-translational modification to exert biological functions. Here, we reported that d-lactate-derived lysine d-lactylation (KD-la) serves as a post-translational modification in Escherichia coli. We demonstrated that acetate CoA-transferase (YdiF) catalyzes the formation of d-lactyl-CoA, the key d-lactyl donor, connecting d-lactate to KD-la. Notably, we identified 86 KD-la sites on 71 proteins in E. coli. In addition, our data demonstrated that anerobic conditions enhance glycolysis, increasing d-lactate production vial-lactate dehydrogenase A (LdhA) and further elevating KD-la levels. We also found that CobB functions as an endogenous de-d-lactylase. Our experiment further showed that K257 of GapA can regulate bacterial growth, whereas CobB can remove KD-la at this site, suggesting a potential CobB-mediated KD-la role. Briefly, this study shows that KD-la directly driven by d-lactate exists as a regulatory mechanism and provides insights into the functional roles of d-lactate in prokaryotes.