Analysis of protein condensation for enzymes involved in extracellular glutamate overproduction in Corynebacterium...

During extracellular glutamate overproduction in Corynebacterium glutamicum, an increase in metabolic flux toward glutamate synthesis occurs without an increase in the transcriptional or translational levels of relevant enzymes, suggesting the involvement of unprecedented post-translational regulatory mechanisms. Here, we investigated the relationship of enzyme condensation with the metabolic shifts that lead to glutamate overproduction. We systematically analyzed the subcellular localization of enzymes involved in glutamate overproduction using fluorescent protein tagging to identify enzyme condensates in C. glutamicum. Our analyses revealed punctate localization, suggesting condensate formation for glyceraldehyde 3-phosphate dehydrogenase, enolase, pyruvate carboxylase, citrate synthase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (ODH) complexes, and the intrinsic ODH inhibitor OdhI. Notably, enolase frequently formed condensates under glutamate-producing conditions, without changes in protein abundance. Our findings provide key insights into the intracellular dynamics of central carbon metabolic enzymes and offer a novel perspective on the mechanisms underlying glutamate overproduction in C. glutamicum.