We have cloned the gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase (GADH) from Erwinia cypripedii ATCC 29267 in Escherichia coli by performing a direct-expression assay. The positive clone converted D-gluconate to 2-keto-D-gluconate (2KDG) in the culture medium. Nucleotide sequence analysis of the GADH clone revealed that the cloned fragment contained the complete structural genes for a 68-kDa dehydrogenase subunit, a 47-kDa cytochrome c subunit, and a 24-kDa subunit of unknown function and that the genes were clustered with the same transcriptional polarity. Comparison of the deduced amino acid sequences and the NH2-terminal sequences determined for the purified protein indicated that the dehydrogenase, cytochrome c, and 24-kDa subunits contained typical signal peptides of 22, 19, and 42 amino acids, respectively. The molecular masses of the processed subunits deduced from the nucleotide sequences (65, 45, and 20 kDa) coincided well with the molecular masses of subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In E. cypripedii and recombinant E. coli, the GADH was constitutively formed and the activity of GADH was enhanced more than twofold by addition of D-gluconate to the medium. The holoenzyme glucose dehydrogenase of E. coli was reconstituted by addition of pyrroloquinoline quinone to the culture medium, and the conversion of D-glucose or D-gluconate to 2KDG by recombinant E. coli harboring the cloned GADH gene was attempted in batch culture. The conversion yields for D-glucose were 0.95 mol of 2KDG/mol of D-glucose after 16 h of cultivation, and those for D-gluconate were 0.95 mol of 2KDG/mol of D-gluconate after 12 h of cultivation.