Engineered formate dehydrogenase from Chaetomium thermophilum , a promising enzymatic solution for biotechnical CO 2 fixation

Objectives Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO 2 . The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum ( Ct FDH) with enhanced reduction activity in the conversion of CO 2 into formic acid. Results Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO 2(g) and HCO 3 − . The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency ( k cat /K M ) compared to that of the wild-type for HCO 3 − reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 ( Sc ADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type Ct FDH. Conclusions Two variants presenting up to five-fold increase in catalytic efficiency and k cat were obtained and characterised. They constitute a promising enzymatic alternative for CO 2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.