Expression systems for soluble metal-dependent formate dehydrogenase

Abstract

Molybdenum or tungsten-dependent formate dehydrogenases (FDH) can reduce carbon dioxide (CO 2 ) to formate under ordinary conditions, and therefore, are considered promising catalysts for CO 2 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 metaldependent 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.

Protein Engineering of the Soluble Metal-dependent Formate Dehydrogenase from Escherichia coli

“…Recombinant FDH proteins were expressed and partially purified from the supernatant of the cell lysate, as described previously. 8 In-gel visualization of enzyme activity and western blotting analyses were performed as described previously. 8…”

“…8 In-gel visualization of enzyme activity and western blotting analyses were performed as described previously. 8…”

“…For fraction determination of that containing the partially purified FDH, we performed western blotting, as described previously. 8…”

“…To construct plasmids for fusion protein expression of soluble EcFDH-O (EcFDHs-O*) and PsaE, we inserted the linker sequence and psaE gene to the C-terminus of the truncated fdoH gene in pUC-fdoGHtst′. 8 The amino acid sequences of the linkers that we examined are shown in Table S2. We transformed BWΔΔ (Table S1) with the resultant plasmids, cultivated transformants micro-aerobically, and analyzed their FDH activities by the in-gel enzyme assay.…”

“…Previously, we reported an expression system for the metaldependent FDH from E. coli (EcFDH-O encoded by fdoGHI operon), in which the active recombinant metal-dependent FDH-O was successfully produced by optimizing the transcription level. 8 EcFDH-O is a membrane-bound enzyme complex composed of three subunits: α (FdoG), β (FdoH), and γ (FdoI) (Fig. 1A).…”