Discovery and characterization of Novel NAD+-dependent formate dehydrogenases (FDHs) for enzymatic Carbon Dioxide-Formate Interconversion

Formate dehydrogenases (FDHs) have become important biocatalysts for the reduction of CO₂ and the oxidation of formate with potential uses in the manufacture of biofuel and industrial carbon capture. However, these enzymes’ cofactor selectivity, stability, and efficiency remain significant obstacles to their broad application. In order to identify the key sequence determinants of FDH’s thermostability and catalytic capabilities, we applied a directed evolution approach as well as phylogenetic-driven selection of novel enzymes. A comprehensive screening and mutagenesis approach, including site-directed, site-saturation, and random mutagenesis, was employed to identify and optimize novel FDH variants. The resulting enzymes were expressed, purified, and characterized through kinetic assays. In addition, we identified new FDHs using phylogenetic analysis, such as MkaFDH, which demonstrated robust thermostability by continuing to function at 60°C as confirmed by far-UV circular dichroism (CD) and its high catalytic efficiency (kcat/Km= 0.44 s-1mM-1) suggested its potential industrial application. According to kinetic experiments, PboFDH showed the capacity to use NADP⁺ as a substitute cofactor with 0.045 U/mg, whereas TarFDH effectively catalyzed the reduction of CO₂ to formate with promising efficiency arriving at 0.15 s-1mM-1. Additionally, activity tests suggested metal-independent catalysis by showing that the presence of divalent metal ions, such as Mn2+, did not increase FDH activity and further research into pH, buffer conditions, and PEG effects yielded important information for enzyme optimization. This work expands our understanding of FDH diversity and function, offering a foundation for future applications in sustainable CO₂ conversion and bio-based energy systems. The engineered FDHs presented here hold promise for industrial-scale CO₂ reduction and formate-driven bio-catalysis.