Hydrogenase-based oxidative biocatalysis without oxygen

Biocatalysis-based synthesis can provide a sustainable and clean platform for producing chemicals. Many oxidative biocatalytic routes require the cofactor NAD⁺ as an electron acceptor. To date, NADH oxidase (NOX) remains the most widely applied system for NAD⁺ regeneration. However, its dependence on O₂ implies various technical challenges in terms of O₂ supply, solubility, and mass transfer. Here, we present the suitability of a NAD⁺ regeneration system in vitro based on H₂ evolution. The efficiency of the hydrogenase-based system is demonstrated by integrating it into a multi-enzymatic cascade to produce ketoacids from sugars. The total NAD⁺ recycled using the hydrogenase system outperforms NOX in all different setups reaching up to 44,000 mol per mol enzyme. This system proves to be scalable and superior to NOX in terms of technical simplicity, flexibility, and total output. Furthermore, the system produces only green H₂ as a by-product even in the presence of O₂.