Tungsten-dependent formate dehydrogenase of Cupriavidus necator as an O2-tolerant biocatalyst for cofactor regeneration and CO2 fixation

Cupriavidus necator’s tungsten-dependent NAD + -reducing formate dehydrogenase ( Cn FDW) has strong potential as a cofactor-recycling biocatalyst, as it remains active under ambient air (∼21% O 2 ) and elevated O 2 levels (up to 50%). Using formate as a clean reductant, Cn FDW enables efficient NADH regeneration for diverse oxidoreductases and oxygenases under aerobic conditions, achieving complete conversion of representative substrates within 8 h and retaining high product yields (92–87%) upon a 10-fold increase in reaction volume and higher substrate concentrations. In reverse reaction, coupling of Cn FDW to the O 2 -tolerant NAD + -reducing hydrogenase of C. necator ( Cn SH) enables H 2 -driven CO 2 reduction to formate. In anaerobic conditions, concentrations of up to 64 mM formate were achieved, when using methyl viologen instead of NAD + /H as electron mediator. Overall, these results establish Cn FDW as an O 2 tolerant biocatalyst for both, aerobic cofactor recycling and sustainable H 2 -driven CO 2 valorisation to formate.