Formate-driven efficient pyruvate biosynthesis in Vibrio natriegens

Formic acid (FA), a key one-carbon liquid compound derived directly from CO₂, can serve as a dual-purpose substrate in microbial metabolism, supplying both carbon and energy. Its potential for green biomanufacturing is immense, yet its inherent toxicity and poor metabolizability to most microbes pose a major hurdle in developing efficient microbial cell factories for value-added chemical production. Building on our prior discovery of Vibrio natriegens as a naturally proficient formic acid utilizer, we demonstrate here that formate supplementation as an auxiliary substrate can dramatically boost pyruvate production of V. natriegens from sodium gluconate, achieving a 1.9-fold increase in titer. Transcriptomic analysis revealed that formate presence induces global changes in gene expression. By subsequently downregulating the pyruvate consumption pathway, we engineered a strain that, when co-fed with formate and sodium gluconate, achieved a 49.0% improvement in pyruvate synthesis. Isotopic tracer analysis confirmed substantial formate assimilation, with approximately 9.43% incorporated into biomass. In a fed-batch fermentation, the engineered V. natriegens strain consumed 82.8 g/L sodium gluconate and 37.4 g/L formate (HCOONa·2H₂O) within 51 h, producing 56.4 g/L pyruvate at a rate of 1.1 g/L/h. This work elucidates the stimulatory role of formate in the pyruvate biosynthesis of V. natriegens and establishes a novel strategy for leveraging this feedstock in microbial production.