Molybdenum or tungsten-dependent formate dehydrogenases (FDH) can reduce carbon dioxide (CO₂) to formate under ordinary conditions, and therefore, are considered promising catalysts for CO₂ fixation. However, to our knowledge, no study on the modification of metal-dependent FDHs has been published, likely because of a lack of convenient expression systems for the recombinant enzymes. We attempted to establish an methodology for the preparation and modification of soluble oxygen-tolerant metal-dependent FDHs, for the following three strategies: (1) Escherichia coli FDH is converted from a membrane bound protein into a soluble protein by deleting the C-terminal membrane-anchor of small subunit (FdoH) and the whole membrane subunit (FdoI) and expressed homologously in E. coli; (2) originally soluble FDHs from Desulfovibrio are expressed heterologously in E. coli; and (3) Desulfovibrio FDHs are genetically engineered by the homologous gene recombination method and expressed homologously in Desulfovibrio. We successfully established the expression systems for (1) and (3), and succeeded in purification of the soluble FDHs with a tag by using affinity columns.