Catalysis-dependent condensation of citrate synthase involved in glutamate overproduction in Corynebacterium glutamicum

Studies suggest that protein condensation regulates cellular metabolism. The overproduction of extracellular glutamate in Corynebacterium glutamicum causes a global increase in metabolic flux toward glutamate synthesis without increasing metabolic enzyme levels. This suggests a post-translational mechanism of metabolic regulation. In this study, we determined that citrate synthase (CgCS) formed droplet-like condensates in C. glutamicum cells. CgCS condensation was observed in growing cells containing catalytically active CgCS. In vivo and in vitro studies revealed that oxaloacetate, a substrate of CS, regulated condensate formation and suggested that substrate-assisted conformational change in CgCS was important for condensation, i.e. catalysis-dependent condensation. Notably, the impairment of CgCS condensation was correlated with defective extracellular glutamate production. In glutamate production-deficient strains, intracellular amino acid profiles fluctuated, although the metabolism to supply 2-oxoglutarate was active. Our results suggest that CgCS condensation plays a distinct role in glutamate overproduction-related metabolism. These findings support the development of engineered condensate-based regulatory modules.