title: (13)C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H.
abstract: Gluconobacter oxydans 621H is used as an industrial production organism due to its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. With glucose as the carbon source, up to 90% of the initial concentration is oxidized periplasmatically to gluconate and ketogluconates. Growth on glucose is biphasic and intracellular sugar catabolism proceeds via the Entner-Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP). Here we studied the in vivo contributions of the two pathways to glucose catabolism on a microtiter scale. In our approach we applied specifically (13)C labeled glucose, whereby a labeling pattern in alanine was generated intracellularly. This method revealed a dynamic growth phase-dependent pathway activity with increased activity of EDP in the first and PPP in the second growth phase, respectively. Evidence for a growth phase-independent decarboxylation-carboxylation cycle around the pyruvate node was obtained from (13)C fragmentation patterns of alanine. For the first time, down-scaled microtiter plate cultivation together with (13)C-labeled substrate was applied for G. oxydans to elucidate pathway operation, exhibiting reasonable labeling costs and allowing for sufficient replicate experiments.
authors.affiliation: Institute of Bio- and Geosciences-IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany. s.ostermann@fz-juelich.de.
authors.forename: Steffen
authors.initials: S
authors.lastname: Ostermann
authors.affiliation: Institute of Bio- and Geosciences-IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany. j.richhardt@fz-juelich.de.
authors.forename: Janine
authors.initials: J
authors.lastname: Richhardt
authors.affiliation: Institute of Bio- and Geosciences-IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany. st.bringer-meyer@fz-juelich.de.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bringer
authors.affiliation: Institute of Bio- and Geosciences-IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany. m.bott@fz-juelich.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
authors.affiliation: Institute of Bio- and Geosciences-IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany. w.wiechert@fz-juelich.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wiechert
authors.affiliation: Institute of Bio- and Geosciences-IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany. m.oldiges@fz-juelich.de.
authors.forename: Marco
authors.initials: M
authors.lastname: Oldiges
keywords: 13C tracer; Entner–Doudoroff pathway; glucose; microtiter cultivation; pentose phosphate pathway