Discovery and characterization of formate dehydrogenases for enzymatic conversion of CO2

Abstract

Among the developments aimed at addressing the climate crisis, the field of carbon capture and utilization (CCU) contains promising technologies. Enzymatic solutions for CO2 conversion are of interest given their high selectivity and mild reaction conditions. Metal-dependent formate dehydrogenases (FDHs) are particularly interesting since it is the only class of enzymes capable of producing a liquid in-demand product using CO2 and electricity as the only substrates. However, due to the incredible FDH diversity, selection of promising candidates for CCU proves nontrivial. Additionally, their inherent complexity and preference for anaerobic conditions have been shown to confer challenges for expression and characterization. This is reflected in literature, where a majority of studies are based on natively expressed FDHs and no study characterizes more than a single metal-dependent FDH variant at a time. This thesis aims to develop and present a novel classification scheme for metal-dependent FDHs that allows discussion and selection of FDH candidates relevant for CCU. Additionally, through characterization of relevant FDHs, this thesis aims at expanding and consolidating the current understanding of FDHs in a CCU context. By comparing structural and compositional qualities of all metal-dependent FDHs from the RefSeq sequence database, a classification scheme consisting of six FDH ‘types’ was developed. Here, the only two previously described oxygen tolerant FDH variants, RcFDH and CnFDH, were both classified as ‘type 5 FDHs’. These variants and a third novel type 5 FDH, RsFDH, were subsequently expressed recombinantly, purified, and then characterized in terms of co-factor saturation, pH and temperature optima, catalytic parameters, and oxygen tolerance. Interestingly, it was observed that hypothesized best practices for expression of metal-dependent FDHs were inferior in terms of yield and specific activity to a previously demonstrated simpler protocol. In terms of catalytic rate, CnFDH clearly outperformed the other type 5 FDHs, although RsFDH showed a higher relative preference for CO2 reduction. Remarkably, oxygen tolerance turned out to involve complex dynamics between the enzyme, substrates, oxygen, and nitrate ions. Adding to this complexity, all three FDHs displayed previously unreported interaction with phosphate. In conclusion, this thesis expands the previous knowledge on metal-dependent FDHs by demonstrating that type 5 FDHs represent a distinct sub-group, which is able to convert CO2 under aerobic conditions with complex catalytic dynamics. Combined with a broad investigation of expression and purification methods, this thesis provides valuable insights for future research on these fascinating enzymes.