Abstract
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 CO2(g) and HCO3−. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) 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 kcat 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.
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Acknowledgements
We acknowledge Dr. Matthew P. Thompson for providing ScADH. This work was fully supported by a grant from the TUBITAK (Grant Number: 214Z292).
Supporting information
Supplementary Figure 1—Michaelis–Menten plots of CtFDH mutants-catalyzed HCO3− and CO2 reduction.
Supplementary Figure 2—Synthesis of 4-phenyl-2-butanone by ScADH and CtFDH coupled system.
Supplementary Table 1—The primers used in this study.
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Çakar, M.M., Ruupunen, J., Mangas-Sanchez, J. et al. Engineered formate dehydrogenase from Chaetomium thermophilum, a promising enzymatic solution for biotechnical CO2 fixation. Biotechnol Lett 42, 2251–2262 (2020). https://doi.org/10.1007/s10529-020-02937-7
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DOI: https://doi.org/10.1007/s10529-020-02937-7