High glutamate demand enables simultaneous consumption of glycerol and citrate despite carbon catabolite repression in engineered Bacillus subtilis strains

The increasing demand for biopolymers has positioned poly-γ-glutamic acid (γ-PGA) as a promising alternative to fossil-based polymers due to its biodegradability and biocompatibility. γ-PGA biosynthesis in Bacillus subtilis is closely linked to intracellular glutamate availability, which is typically maintained via the supply of an exogenous glutamate source, a cost-intensive factor for industrial production. This study investigates the metabolic interplay between glycerol, citrate, and glutamate during γ-PGA synthesis, focusing on how cellular glutamate demand influences carbon source utilization. We demonstrate that reducing exogenous glutamate supply induces demand-driven co-consumption of glycerol and citrate, which is usually inhibited by carbon catabolite repression. In the absence of exogenous glutamate, the B. subtilis strain PG10 produced 8.4 g L^-1 γ-PGA, indicating significant de novo glutamate synthesis. A deletion analysis of known citrate transporters identified CimH as the key translocation system enabling citrate uptake under glutamate-limiting conditions. Further isotope labeling confirmed that citrate serves as a glutamate precursor during glutamate demand and is not used as a gluconeogenic substrate. Proteome analysis revealed a regulatory shift towards enhanced glutamate biosynthesis in the absence of exogenous glutamate, accompanied by reduced overflow metabolism and adaptive changes in central carbon and nitrogen metabolism. To our knowledge, carbon source co-utilization is a so far unknown response of B. subtilis 168 to glutamate scarcity. Uncovering the regulatory network involved offers a powerful tool by enabling biotechnological exploitation of this drastic change in carbon flux to boost the production of various products dependent on tricarboxylic acid cycle intermediates.