Metabolomics Reveals Acetate-Formate compatibility for Dual-Carbon PHB biosynthesis in Cupriavidus necator

The production of polyhydroxybutyrate (PHB) from organic substrates is often limited by energetic imbalance when single-carbon sources are used. Formate, while easily generated via electrochemical CO₂ reduction, fails to sustain robust biomass and polymer yields when utilized as a primary carbon source. Similarly, acetate provides the necessary skeletal structures for PHB biosynthesis, but lacks the intrinsic energy to drive high-yield accumulation. To bridge this gap, we implemented a dual-carbon framework in wild-type Cupriavidus necator H16 that decouples energy supply from carbon assimilation, in which formate oxidation functions as a dedicated source of ATP and NADH, allowing acetate to be preferentially directed toward biosynthesis. Following optimization of physiological parameters, the process was evaluated under batch, fed-batch, and pH-stat cultivation modes, achieving a PHB content of 84% and a titer of 7.3 ± 0.9 g L^-1. Relative quantitative metabolomics further indicated that formate-derived energy alleviated acetate-associated energetic constraints, resulting in a more efficient carbon conversion into biopolymer. Collectively, this study provides a systematic evaluation of acetate-formate substrate compatibility for PHB production in C. necator, offering a design principle for integrating organic acid feedstocks into sustainable bioplastic processes.