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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

Selection and fragmentation of an ionized alanine molecule in the used triple quadrupole MS. The circled numbers indicate the carbon atoms of the alanine molecule derived from the carbon position of the substrate glucose; the red dots symbolize the inert gas particles (N2) for collision with the mother ion.
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metabolites-05-00455-f006: Selection and fragmentation of an ionized alanine molecule in the used triple quadrupole MS. The circled numbers indicate the carbon atoms of the alanine molecule derived from the carbon position of the substrate glucose; the red dots symbolize the inert gas particles (N2) for collision with the mother ion.

Mentions: The index number at the carbon atom indicates its position in the carbon backbone based on IUPAC nomenclature i.e., naturally labeled, uniformly [13C], [1-13C]-, and [1,2-13C]-labeled. The selection and fragmentation of alanine in a triple quadrupole MS are shown in Figure 6.


(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)

Selection and fragmentation of an ionized alanine molecule in the used triple quadrupole MS. The circled numbers indicate the carbon atoms of the alanine molecule derived from the carbon position of the substrate glucose; the red dots symbolize the inert gas particles (N2) for collision with the mother ion.
© Copyright Policy
Related In: Results  -  Collection

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

metabolites-05-00455-f006: Selection and fragmentation of an ionized alanine molecule in the used triple quadrupole MS. The circled numbers indicate the carbon atoms of the alanine molecule derived from the carbon position of the substrate glucose; the red dots symbolize the inert gas particles (N2) for collision with the mother ion.
Mentions: The index number at the carbon atom indicates its position in the carbon backbone based on IUPAC nomenclature i.e., naturally labeled, uniformly [13C], [1-13C]-, and [1,2-13C]-labeled. The selection and fragmentation of alanine in a triple quadrupole MS are shown in Figure 6.

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