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

Objectives: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO 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 and HCO. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (k/K) compared to that of the wild-type for HCO 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 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH.

Conclusions: Two variants presenting up to five-fold increase in catalytic efficiency and k were obtained and characterised. They constitute a promising enzymatic alternative for CO utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.