Engineered formate dehydrogenase from Chaetomium thermophilum, a promising enzymatic solution for biotechnical CO2 fixation.

Objectives: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO₂. 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 CO₂ 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 HCO₃⁻. 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 HCO₃⁻ 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 CO₂ utilization and will serve as scaffolds to be further developed in order to meet industrial requirements. [ABSTRACT FROM AUTHOR]