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(13)C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H.

Ostermann S, Richhardt J, Bringer S, Bott M, Wiechert W, Oldiges M - Metabolites (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bio- and Geosciences-IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany. s.ostermann@fz-juelich.de.

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.

No MeSH data available.


Related in: MedlinePlus

Growth (full lines) of Δupp (Δ, black) and Δgnd (o, red) strains. Glucose (dashed lines) and acetate (dotted lines) concentration in supernatant at a sequentially sampled cultivation.
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metabolites-05-00455-f003: Growth (full lines) of Δupp (Δ, black) and Δgnd (o, red) strains. Glucose (dashed lines) and acetate (dotted lines) concentration in supernatant at a sequentially sampled cultivation.

Mentions: The offline measured values for OD, glucose consumption, and acetate secretion are displayed in Figure 3. In the first 6 h, glucose consumption and acetate formation were identical for both strains and likewise the growth rate. After 6 h of cultivation, the glucose consumption was dramatically reduced, matching a reported threshold of 6–8 mM glucose concentration to induce a glucose transport system in other organisms [20,21]. It took another 4 h to completely consume the glucose present in the culture medium.


(13)C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H.

Ostermann S, Richhardt J, Bringer S, Bott M, Wiechert W, Oldiges M - Metabolites (2015)

Growth (full lines) of Δupp (Δ, black) and Δgnd (o, red) strains. Glucose (dashed lines) and acetate (dotted lines) concentration in supernatant at a sequentially sampled cultivation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588806&req=5

metabolites-05-00455-f003: Growth (full lines) of Δupp (Δ, black) and Δgnd (o, red) strains. Glucose (dashed lines) and acetate (dotted lines) concentration in supernatant at a sequentially sampled cultivation.
Mentions: The offline measured values for OD, glucose consumption, and acetate secretion are displayed in Figure 3. In the first 6 h, glucose consumption and acetate formation were identical for both strains and likewise the growth rate. After 6 h of cultivation, the glucose consumption was dramatically reduced, matching a reported threshold of 6–8 mM glucose concentration to induce a glucose transport system in other organisms [20,21]. It took another 4 h to completely consume the glucose present in the culture medium.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bio- and Geosciences-IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany. s.ostermann@fz-juelich.de.

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.

No MeSH data available.


Related in: MedlinePlus