Modification of the membrane-bound glucose oxidation system in Gluconobacter oxydans significantly increases gluconate and 5-keto-D-gluconic acid accumulation.

Gluconobacter oxydans DSM 2343 (ATCC 621H)catalyzes the oxidation of glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of the industrially important L-(+)-tartaric acid. To further increase 5-KGA production in G. oxydans, the mutant strain MF1 was used. In this strain the membrane-bound gluconate-2-dehydrogenase activity, responsible for formation of the undesired by-product 2-keto-D-gluconic acid, is disrupted. Therefore, high amounts of 5-KGA accumulate in the culture medium. G. oxydans MF1 was equipped with plasmids allowing the overexpression of the membrane-bound enzymes involved in 5-KGA formation. Overexpression was confirmed on the transcript and enzymatic level. Furthermore, the resulting strains overproducing the membrane-bound glucose dehydrogenase showed an increased gluconic acid formation, whereas the overproduction of gluconate-5-dehydrogenase resulted in an increase in 5-KGA of up to 230 mM. Therefore, these newly developed recombinant strains provide a basis for further improving the biotransformation process for 5-KGA production.

A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-D-gluconic acid.

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-D-gluconic acid (2-KGA) and 5-keto-D-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important L-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO2, 0.05% pCO2) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.

Genetic and biological diversity among populations of Leishmania major from Central Asia, the Middle East and Africa.

Evidence is provided for genetic and biological variation among Leishmania major strains that correlates with their geographical origin. The host-parasite relationship also appears to be specific. Great gerbils, Rhombomys opimus, and fat sand rats, Psammomys obesus, are the main reservoir hosts in Central Asia and the Middle East, respectively. However, the Central Asian parasite failed to infect the Middle Eastern rodent host in the laboratory, and vice versa. A permissively primed intergenic polymorphic (PPIP)-PCR and a single-stranded conformation polymorphism (SSCP)-PCR exposed genetic polymorphism among 30 strains of L. major from different geographical regions. This was verified by subsequent sequencing of DNA from the same strains using four genomic targets: (a) the NADH-dehydrogenase (NADH-DH) gene, (b) the 6-phosphogluconate dehydrogenase (6PGD) gene, (c) the ribosomal internal transcribed spacers, and (d) an anonymous DNA sequence originally amplified with random primers. All the genetic markers indicated that the nine Central Asian strains were a separate homogenous genetic group. The Middle Eastern strains formed another geographical group that displayed heterogeneity corresponding with their different Middle Eastern locations. Molecular markers and host-parasite relationships confirmed that Central Asian and Middle Eastern strains are genetically and biologically distinct sub-populations of L. major. Three African strains of L. major were genetically closer to the Middle Eastern strains, and a representative one did infect fat sand rats, but they had distinct permissively primed inter-genic polymorphic PCR patterns and internal transcribed spacer 2 types.