The acetylation mechanism and engineering strategy of isocitrate lyase for optimizing biosynthesis in Escherichia coli

Lysine acetylation is a widespread post-translational modification that fine-tunes cell physiology and metabolism. Yet its application in metabolic engineering remains underexplored. Here, we report that the key enzyme of the glyoxylate shunt, isocitrate lyase (AceA) in Escherichia coli , undergoes reversible lysine acetylation mediated by both enzymatic and non-enzymatic mechanisms. We demonstrated that acetylation significantly inhibits AceA activity. Through structural analysis and site-directed mutagenesis, we identified key lysine acetylation sites that critically influence enzyme activity. Furthermore, we developed an acetylation-mediated rational design strategy, constructing the AceA(S335Q/S398Q) double mutant, which exhibited a 2.12-fold increase in enzymatic activity and enhanced protein stability compared to the wild-type. Application of this optimized AceA variant in engineered E. coli strains significantly improved the production of some valuable chemicals by reinforcing the glyoxylate shunt and enhancing carbon conversion efficiency. Our work elucidates the regulatory mechanism of AceA acetylation and establishes protein acetylation as a promising and versatile tool for optimizing metabolic pathways for bioproduction.