Gluconobacter is a genus of acetic acid bacteria which are able to oxidize a broad range of sugars, sugar alcohols and sugar acids, and which accumulate large amounts of the corresponding oxidized products in their culture medium. The production of ketogluconates by Gluconobacter strains has been reported to be catalyzed by two types of membrane-bound gluconate dehydrogenases. One is FAD-gluconate dehydrogenase (FAD-GADH), an FAD-containing, 2-keto-D-gluconate (2KGA)-yielding enzyme, and the other is PQQ-glycerol dehydrogenase (PQQ-GLDH), a PQQ-containing, 5-keto-D-gluconate (5KGA)-yielding enzyme. In this study, in order to have selective 5KGA production system able to be done without serious temperature control, high 5KGA-producing strain under higher temperature was developed.