Shikimate pathway disruption in yeast induces metabolite self‐assembly into toxic aggregates

The shikimate pathway in Saccharomyces cerevisiae is pivotal for the biosynthesis of aromatic amino acids, which are essential for protein synthesis and cellular homeostasis. Here, we investigated the regulatory mechanisms within this pathway, focusing on the two key enzymes: phospho‐2‐dehydro‐3‐deoxyheptonate aldolase, tyrosine‐inhibited (Aro4) and phospho‐2‐dehydro‐3‐deoxyheptonate aldolase, phenylalanine‐inhibited (Aro3). Deletion of either the ARO4 or ARO3 genes led to significant intracellular accumulation of phenylalanine or tyrosine, respectively, upon feeding with the corresponding metabolite. Remarkably, we discovered that this metabolite accumulation promotes the formation of amyloid‐like assemblies within cells, as evidenced by amyloid‐specific staining and antibody recognition towards phenylalanine and tyrosine assemblies. These assemblies strongly correlated with reduced cell viability. Treatment with common amyloid inhibitors significantly mitigated the formation of these toxic assemblies, improving cell growth, and viability in the mutant strains. Additionally, although overexpression of ARO3 in the aro4 Δ background partially rescued the growth phenotype, combining ARO3 overexpression with trimethylamine N ‐oxide (TMAO) treatment provided an additive growth rescue effect. Our findings highlight the role of cellular self‐assembly in regulating the shikimate pathway and reveal a previously unknown link between metabolic dysregulation and the formation of toxic amyloid‐like structures. This discovery offers new insights into inherited metabolic disorders and potential therapeutic approaches.