Formate-strengthened NADH supply in Salinivibrio sp. TGB4 improves polyhydroxyalkanoates production.

Polyhydroxyalkanoates (PHAs) are environmentally friendly biopolymers with the potential to replace petroleum-based plastics. However, their large-scale production remains constrained by high costs. Salinivibrio sp. TGB4, which efficiently synthesizes PHA using acetate as a carbon source, represents a promising industrial chassis. In this study, we identified that acetoacetyl-CoA reductase (PhaB), the key enzyme in PHA biosynthesis in Salinivibrio sp. TGB4, exhibits a strong preference for NADH over NADPH. In silico simulations combined with site-directed mutagenesis elucidated the structural basis for this cofactor specificity. To enhance intracellular NADH availability, formate was introduced as an auxiliary source of reducing power via the overexpression of NAD⁺-dependent formate dehydrogenase (FDH). This strategy significantly increased the intracellular NADH/NAD⁺ ratio, boosting poly-3-hydroxybutyrate (PHB) production from 2.29 g/L to 3.94 g/L when acetate was used as the carbon source. Moreover, the elevated NADH/NAD⁺ ratio further facilitated the synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from acetate and valerate. This study provides the first evidence that PHA synthesis in Salinivibrio sp. TGB4 is primarily NADH-dependent and demonstrates that formate oxidation is an effective approach to enhance NADH supply and improve PHA biosynthesis efficiency.