Biocatalytic Conversion of CO₂ to Formaldehyde and Methanol Using Efficient Dehydrogenases

Efficient enzymatic conversion of carbon dioxide (CO₂) into value-added chemicals offers a sustainable route for carbon utilization; however, the identification of highly active biocatalysts remains a major challenge. In this study, a novel formate dehydrogenase from Xanthobacter sp. 91 (XaFDH) exhibited markedly enhanced CO₂ reduction activity compared with Candida boidinii FDH, showing up to 7-fold higher specific activity and 15-fold greater catalytic efficiency (k_cat/K_m). Kinetic analysis revealed a substantially lower K_m for bicarbonate, while isothermal titration calorimetry demonstrated enhanced NADH affinity, suggesting that substrate binding affinity is key determinant of catalytic performance. Structural and molecular docking analyses further indicated that enlarged substrate entry tunnel and favorable hydride-transfer geometry of carbonate contributed to the improved catalysis. Downstream dehydrogenases were comparatively screened and selected to establish a multienzyme cascade system for the conversion of CO₂ to formaldehyde and methanol. A formaldehyde dehydrogenase from Pseudomonas lalkuanensis (PlFaldDH) exhibited outstanding formate-reduction activity, with 34-fold higher activity than the widely used benchmark PpFaldDH. The established XaFDH-FaldDH cascade produced high formaldehyde up to 306.02 ± 6.30 μM. Furthermore, coupling the system with alcohol dehydrogenase (ADH) enabled the direct conversion of gaseous CO₂ to methanol through the XaFDH-FaldDH-ADH cascade, yielding up to 3.03 mM methanol (3.5 μmol/h) in the presence of an NADH-regeneration system. Collectively, these findings establish XaFDH and the associated dehydrogenase cascade as highly efficient biocatalysts for CO₂ valorization and highlight their potential for sustainable formaldehyde and methanol biosynthesis.