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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

Offline measurement of final OD (white, dashed) and final pH value (grey) after 24 h of cultivation for all combinations of mutant strain and 13C-labeled glucose.
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metabolites-05-00455-f001: Offline measurement of final OD (white, dashed) and final pH value (grey) after 24 h of cultivation for all combinations of mutant strain and 13C-labeled glucose.

Mentions: All four strains were cultivated in a microtiter plate (MTP) format in the Biolector® on a defined medium with 13C-labeled glucose as the carbon and energy source. To exclude an influence of the 13C labeling state of the substrate, all four strains were cultivated with differently labeled species of glucose in at least two biological and three technical replicates. The measurement of the growth phenotype (final pH and OD600) did not reveal any influence of the substrate labeling state, as shown in Figure 1.


(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)

Offline measurement of final OD (white, dashed) and final pH value (grey) after 24 h of cultivation for all combinations of mutant strain and 13C-labeled glucose.
© Copyright Policy
Related In: Results  -  Collection

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

metabolites-05-00455-f001: Offline measurement of final OD (white, dashed) and final pH value (grey) after 24 h of cultivation for all combinations of mutant strain and 13C-labeled glucose.
Mentions: All four strains were cultivated in a microtiter plate (MTP) format in the Biolector® on a defined medium with 13C-labeled glucose as the carbon and energy source. To exclude an influence of the 13C labeling state of the substrate, all four strains were cultivated with differently labeled species of glucose in at least two biological and three technical replicates. The measurement of the growth phenotype (final pH and OD600) did not reveal any influence of the substrate labeling state, as shown in Figure 1.

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