(228k) Production of Ralstonia Eutropha Formate Dehydrogenases for CO2 Conversion

Xuejun Yu^a, Dimitri Niks^c, Russ Hille^c, Xin Ge^b, Ashok Mulchandani^b*

^aDepartment of Bioengineering, ^bDepartment of Chemical Engineering, ^cDepartment of Biochemistry, University of California Riverside, 900 University Ave, Riverside, CA 92521, USA

* adani@engr.ucr.edu

Formate dehydrogenases (FDHs) are a group of redox enzymes that catalyze the oxidation reaction of formic acid to carbon dioxide by generating two protons and two electrons. Recently, several FDHs able to catalyze the reverse reaction, the reduction of carbon dioxide to formic acid under appropriate conditions, have been reported. Two main challenges are associated with CO2 conversion at industrial scales: oxygen sensitivity of most CO2-reducing FDHs, and difficulty of production and purification from their homologous hosts. In this study, we cloned and overexpressed an oxygen tolerant Ralstonia eutropha FDH (ReFDH) in Escherichia coli for CO2 conversion to formic acid. This soluble ReFDH was found to be tetrameric (α�γδ) containing one molybdenum active center, one FMN and seven Fe/S centers. To facilitate heterologous production of fully active ReFDH in E. coli, we constructed R. eutropha molybdopterin cofactor synthetic pathway in E. coli and fused ReFDH gene with a His6 tag. Produced ReFDH was purified with Ni-NTA columns and size-exclusion chromatography, and the amount of protein-bound molybdenum and iron-sulfur clusters were quantified by electron paramagnetic resonance (EPR) spectroscopy. The cofactor analysis showed that the bis-MGD was bound to the FdsA subunit. The results from kinetic study showed that soluble ReFDH catalyzed the reduction of CO2 to formic acid. This ReFDH overexpression system allows us to study the electron transfer mechanisms inside of this protein, as well as to develop a highly efficient biocatalytic process for transformation of the abundant but waste greenhouse gas carbon dioxide to formic acid that has applications as fuel for formic acid fuel-cells and chemical feedstock for synthesis of other value-added chemicals.