Dynamic Lysine Acetylation Disrupts Isocitrate Lyase Function and Enables Metabolic Optimisation

Area of Science:

• Biochemistry
• Microbial Metabolism
• Enzyme Regulation

Background:

• Proteomic studies indicate Escherichia coli isocitrate lyase (ICL) is acetylated, impacting its activity.
• The precise molecular mechanisms and specific lysine residues involved in ICL acetylation were previously undefined.

Purpose of the Study:

• To elucidate the molecular basis of ICL acetylation in E. coli.
• To define the role of specific lysine residues in ICL regulation.
• To explore the application of ICL regulation for metabolic engineering.

Main Methods:

• Investigated acetyl-phosphate-dependent acetylation and CobB deacetylase reversibility.
• Performed site-specific mutagenesis (K13R, K308R) to assess regulatory impact.
• Engineered lysine-to-arginine substitutions directly into the chromosomal aceA gene.
• Evaluated metabolic flux, biomass yield, and bioproduction (lycopene).

Main Results:

• Acetylation at K13 and K308 inhibits ICL activity by destabilizing its tetrameric structure and promoting degradation.
• Lysine-to-arginine substitutions at K13 and K308 enhance carbon flux, metabolic flexibility, and biomass yield.
• A chromosomal KR mutant (aceA K13R/K308R) maintained growth rates, reduced acetate overflow, and improved metabolic balance.
• This engineered strain achieved a 61% increase in lycopene production.

Conclusions:

• ICL acetylation is a reversible post-translational regulatory mechanism within the glyoxylate shunt.
• Targeted modification of ICL acetylation sites offers a powerful strategy for metabolic engineering.
• This regulatory approach can optimize microbial cell factories for sustainable bioproduction of valuable compounds.