Citrate Supplementation Modulates Medium Viscosity and Poly‐γ‐Glutamic Acid Synthesis by Engineered <i>B. subtilis</i> 168

The industrially attractive biopolymer poly-γ-glutamic acid (γ-PGA) is commonly produced by species of the genus <i>Bacillus</i> by co-feeding different carbon- and nitrogen-sources. Recent studies have highlighted the pivotal role of co-metabolization of a rapidly degradable carbon source such as glycerol together with citrate for γ-PGA production, independently fueling biomass generation as well as tricarboxylic acid (TCA) cycle precursor supply. With this study, we report that the sole presence of citrate in the production medium greatly influences growth behavior, γ-PGA production, and the viscosity of microbial cultures during biopolymer synthesis. Independent of the citrate concentration in the medium, only minor amounts of citrate were imported by <i>B. subtilis</i> 168 in the presence of glycerol due to carbon catabolite repression. However, a high citrate concentration resulted in a 6-fold increase in γ-PGA titer compared to low exogenous citrate levels. Data suggests that citrate was not used as a precursor in γ-PGA synthesis but rather influenced the fate of imported glutamate. The citrate concentration also affected medium viscosity as depletion resulted in a remarkable spike in culture broth viscosity. Additionally, cellular proteome analysis at different levels of citrate availability revealed significant changes in protein abundance involved in motility and fatty acid degradation. <i>Practical Application:</i> This research provides critical insights into optimizing γ-PGA production in <i>Bacillus subtilis</i>, particularly by using citrate supplementation to control medium viscosity and improve production yields. The study reveals that citrate not only plays a role in controlling viscosity but also influences intracellular glutamate metabolism, a key factor for γ-PGA synthesis. Citrate interacts with divalent cations such as Mg²⁺ and Ca²⁺, reducing electrostatic interactions and thus decreasing viscosity in the medium. Additionally, while citrate uptake is limited due to carbon catabolite repression (CCR), even the minimal presence of citrate impacts growth and production. The findings suggest that citrate may trigger unexplored regulatory mechanisms affecting glutamate utilization. Their understanding opens new avenues for industrial optimization, which focus on enhancing glutamate synthesis pathways and exploring novel citrate-sensing mechanisms. Overall, this research lays the groundwork for improving the efficiency and consistency of γ-PGA production by fine-tuning media components and understanding their metabolic effects.