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 (CtFDH) 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 (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 _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.