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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

Transition scheme of glucose via the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP). The circles indicate carbon atoms in the isotopic states 12C (white) and 13C (black); depending on the substrate, the products may contain the 13C labeling (grey). The numbers of the carbon position in glucose is based on IUPAC nomenclature. The numbers in the remaining metabolites originate from the glucose carbon position.
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metabolites-05-00455-f002: Transition scheme of glucose via the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP). The circles indicate carbon atoms in the isotopic states 12C (white) and 13C (black); depending on the substrate, the products may contain the 13C labeling (grey). The numbers of the carbon position in glucose is based on IUPAC nomenclature. The numbers in the remaining metabolites originate from the glucose carbon position.

Mentions: For the labeling approach used in the present work, it is irrelevant whether single steps of the conversion from glucose to 6-phosphogluconate occur intra- or extracellularly, since the carbon backbone is not altered by this conversion. Intracellular gluconate can be converted to 5-ketogluconate by a gluconate-5-dehydrogenase (gno, GOX2187), or phosphorylated by a gluconate kinase (gnk, GOX1709) [2,16]. The metabolite 6-phosphogluconate (6-PG) exclusively serves as a precursor molecule for both intracellular glucose degradation pathways, i.e., PPP and EDP, and ends up in the common C3-molecule pyruvate. All carbon transition information was deduced with the Omix [17] software tool from the KEGG database [18]. In Figure 2, the schematic carbon transition is displayed.


(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)

Transition scheme of glucose via the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP). The circles indicate carbon atoms in the isotopic states 12C (white) and 13C (black); depending on the substrate, the products may contain the 13C labeling (grey). The numbers of the carbon position in glucose is based on IUPAC nomenclature. The numbers in the remaining metabolites originate from the glucose carbon position.
© Copyright Policy
Related In: Results  -  Collection

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

metabolites-05-00455-f002: Transition scheme of glucose via the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP). The circles indicate carbon atoms in the isotopic states 12C (white) and 13C (black); depending on the substrate, the products may contain the 13C labeling (grey). The numbers of the carbon position in glucose is based on IUPAC nomenclature. The numbers in the remaining metabolites originate from the glucose carbon position.
Mentions: For the labeling approach used in the present work, it is irrelevant whether single steps of the conversion from glucose to 6-phosphogluconate occur intra- or extracellularly, since the carbon backbone is not altered by this conversion. Intracellular gluconate can be converted to 5-ketogluconate by a gluconate-5-dehydrogenase (gno, GOX2187), or phosphorylated by a gluconate kinase (gnk, GOX1709) [2,16]. The metabolite 6-phosphogluconate (6-PG) exclusively serves as a precursor molecule for both intracellular glucose degradation pathways, i.e., PPP and EDP, and ends up in the common C3-molecule pyruvate. All carbon transition information was deduced with the Omix [17] software tool from the KEGG database [18]. In Figure 2, the schematic carbon transition is displayed.

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