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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

Scheme of a hypothetical reaction sequence from pyruvate via acetoin back to pyruvate, including the carbon transition. Atoms are labeled 13C (black) or 12C (white). The numbers originate from carbon position in the substrate glucose: (A) unlabeled precursors and 13C-labeled CO2 transformed to alanine generating m.1.0 signal; (B) double-labeled pyruvate originated from [1,2-13C]-labeled glucose transformed to alanine, generating an m.1.1 signal.
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metabolites-05-00455-f005: Scheme of a hypothetical reaction sequence from pyruvate via acetoin back to pyruvate, including the carbon transition. Atoms are labeled 13C (black) or 12C (white). The numbers originate from carbon position in the substrate glucose: (A) unlabeled precursors and 13C-labeled CO2 transformed to alanine generating m.1.0 signal; (B) double-labeled pyruvate originated from [1,2-13C]-labeled glucose transformed to alanine, generating an m.1.1 signal.

Mentions: For acetaldehyde, a hypothetical reaction sequence explaining the observed labeling data was identified. The constitutively expressed pyruvate decarboxylase (pdc, GOX1081) converts 1 mol pyruvate to 1 mol acetaldehyde and 1 mol CO2 as main reaction, but is also able to condense 2 mol acetaldehyde to 1 mol acetoin under suitable reaction conditions [27], as shown in Figure 5. Next, a thus-far unspecified enzyme would have to carboxylate acetoin and simultaneously split the carboxylation product into one pyruvate and one acetaldehyde. Such a reaction sequence would allow the cell to get rid of toxic acetaldehyde and recover CO2. The resulting transition of 13C labeling would be a perfect match for the observed m.1.0 (Figure 5A) and m.1.1 (Figure 5B) mass isotopomers.


(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)

Scheme of a hypothetical reaction sequence from pyruvate via acetoin back to pyruvate, including the carbon transition. Atoms are labeled 13C (black) or 12C (white). The numbers originate from carbon position in the substrate glucose: (A) unlabeled precursors and 13C-labeled CO2 transformed to alanine generating m.1.0 signal; (B) double-labeled pyruvate originated from [1,2-13C]-labeled glucose transformed to alanine, generating an m.1.1 signal.
© Copyright Policy
Related In: Results  -  Collection

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

metabolites-05-00455-f005: Scheme of a hypothetical reaction sequence from pyruvate via acetoin back to pyruvate, including the carbon transition. Atoms are labeled 13C (black) or 12C (white). The numbers originate from carbon position in the substrate glucose: (A) unlabeled precursors and 13C-labeled CO2 transformed to alanine generating m.1.0 signal; (B) double-labeled pyruvate originated from [1,2-13C]-labeled glucose transformed to alanine, generating an m.1.1 signal.
Mentions: For acetaldehyde, a hypothetical reaction sequence explaining the observed labeling data was identified. The constitutively expressed pyruvate decarboxylase (pdc, GOX1081) converts 1 mol pyruvate to 1 mol acetaldehyde and 1 mol CO2 as main reaction, but is also able to condense 2 mol acetaldehyde to 1 mol acetoin under suitable reaction conditions [27], as shown in Figure 5. Next, a thus-far unspecified enzyme would have to carboxylate acetoin and simultaneously split the carboxylation product into one pyruvate and one acetaldehyde. Such a reaction sequence would allow the cell to get rid of toxic acetaldehyde and recover CO2. The resulting transition of 13C labeling would be a perfect match for the observed m.1.0 (Figure 5A) and m.1.1 (Figure 5B) mass isotopomers.

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