Metabolic Engineering of <i>Bacillus subtilis</i> for the Production of Poly-γ-Glutamic Acid from Glycerol Feedstock

Poly-γ-glutamic acid (γ-PGA) is an attractive biopolymer for medical, agri-food, and environmental applications. Although microbial synthesis by <i>Bacilli</i> fed on waste streams has been widely adopted, the obtainment of efficient sustainable production processes is still under investigation by bioprocess and metabolic engineering approaches. The abundant glycerol-rich waste generated in the biodiesel industry can be used as a carbon source for γ-PGA production. Here, we studied fermentation performance in different engineered <i>Bacillus subtilis</i> strains in glycerol-based media, considering a <i>swrA⁺ degU32^Hy</i> mutant as the initial producer strain and glucose-based media for comparison. Modifications included engineering the biosynthetic <i>pgs</i> operon regulation (replacing its native promoter with P_hyspank), precursor accumulation (<i>sucCD</i> or <i>odhAB</i> deletion), and enhanced glutamate racemization (<i>racE</i> overexpression), predicted as crucial reactions by genome-scale model simulations. All interventions increased productivity in glucose-based media, with P_hyspank-<i>pgs</i> ∆<i>sucCD</i> showing the highest γ-PGA titer (52 g/L). Weaker effects were observed in glycerol-based media: ∆<i>sucCD</i> and P_hyspank-<i>pgs</i> led to slight improvements under low- and high-glutamate conditions, respectively, reaching ~22 g/L γ-PGA (26% increase). No performance decrease was detected by replacing pure glycerol with crude glycerol waste from a biodiesel plant, and by a 30-fold scale-up. These results may be relevant for improving industrial γ-PGA production efficiency and process sustainability using waste feedstock. The performance differences observed between glucose and glycerol media also motivate additional computational and experimental studies to design metabolically optimized strains.