[Seminar] Engineering Formate Dehydrogenase for Applications in Biocatalysis

Title: Engineering Formate Dehydrogenase for Applications in Biocatalysis

Speaker: Carlos G. Acevedo-Rocha

From: DTU Biosustain, Technical University of Denmark

Abstract: Formate dehydrogenase (FDH) catalyzes the reversible oxidation of formate to carbon dioxide (CO₂). FDHs can be classified in metal-dependent and metal-free. The former ones are usually oxygen-sensitive and contain molybdenum (Mo) or tungsten (W) at their active sites as well as diverse cofactors (molybdopterin, iron-sulfur clusters, or FAD) and electron acceptors (cytochrome, NAD⁺, or ferredoxin), which require the concerted action of several multi-component systems and are thus difficult to scale up for industrial applications. On the other hand, metal-free FDHs are simpler systems that use NAD(P)⁺ or NAD(P)H for hydride and electron transfer. Most metal-free FDHs predominantly catalyse formate oxidation, but it has been shown that protein engineering can improve the reverse reaction; namely, reduction of CO₂ to formate, opening potential applications for carbon capture. Most metal-free FDHs use the cofactor NAD⁺, but there are also FDHs that can use the cofactor NADP⁺, thus opening the potential for cofactor regeneration in oxidoreductase-based biocatalysis. In this talk, our ongoing efforts towards engineering metal-free FDH in both the forward and reverse reactions will be presented. Our strategy combines high-throughput selection assays based on auxotrophy (for either NADPH or formate) and state-of-the-are experimental (e.g., error-prone PCR and deep mutational scanning) and computational methods based on machine learning (e.g., structure-based design and protein language models). To investigate the effect of the mutations from a mechanistic perspective, we are performing Molecular Dynamics simulations.