Formate-driven in situ CO2 recycling for efficient succinic acid bioproduction in engineered Actinobacillus succinogenes

Succinic acid (SA) biosynthesis presents a promising sustainable alternative to petrochemical routes, yet industrial potential is often hampered by inefficient CO 2 fixation and redox imbalances. To systematically address these bottlenecks, a synergistic metabolic engineering strategy was developed in Actinobacillus succinogenes by co-expressing the endogenous gene of formate dehydrogenase (FdoG) and the heterologous gene of phosphoenolpyruvate carboxylase (PPC). This design established a self-reinforcing metabolic loop where FdoG-mediated formate oxidation generated in situ CO 2 and NADH, which were immediately channeled into the PPC-driven carboxylation pathway. Physiological characterizations, utilizing real-time off-gas analysis, demonstrated that this pathway effectively functions as an endogenous CO 2 generator, decoupling production from external mass transfer limitations. Furthermore, carbon flux distribution analysis confirmed the synergistic efficacy, revealing a 12.11% increase in succinate-directed flux and a significant reduction in byproduct accumulation compared to the wild type. To fully exploit this formate-utilization potential, an optimized fed-batch fermentation with pulsed formate feeding was implemented. Consequently, the engineered strain AS-PF achieved a remarkable succinate titer of 96.64 g/L, with a productivity of 1.38 g/ (L·h), corresponding to an overall product yield of 0.78 g/g based on the total consumed mixed carbon source (glucose and formate). This study establishes a sustainable and efficient platform for SA bioproduction by synergistically integrating formate metabolism-driven CO 2 recycling with enhanced biosynthetic pathways, thereby advancing the valorization of C1 compounds.