Construction of expression vectors for protein production in Gluconobacter oxydans

The characteristic ability of Gluconobacter oxydans to incompletely oxidize numerous sugars, sugar acids, polyols, and alcohols has been exploited in several biotechnological processes, for example vitamin C production. The genome sequence of G. oxydans 621H is known but molecular tools are needed for the characterization of putative proteins and for the improvement of industrial strains by heterologous and homologous gene expression. To this end, promoter regions for the genes encoding G. oxydans ribosomal proteins L35 and L13 were introduced into the broad-host-range plasmid pBBR1MCS-2 to construct two new expression vectors for gene expression in Gluconobacter spp. These vectors were named pBBR1p264 and pBBR1p452, respectively, and have many advantages over current vectors for Gluconobacter spp. The uidA gene encoding β-D-glucuronidase was inserted downstream of the promoter regions and these promoter-reporter fusions were used to assess relative promoter strength. The constructs displayed distinct promoter strengths and strong (pBBR1p264), moderate (pBBR1p452) and weak (pBBR1MCS-2 carrying the intrinsic lac promoter) promoters were identified.