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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

Mass isotopomer proportions of the 13C labeling states of alanine for (A) Δupp and (B) Δgnd mutant strains. The process time indicates the sampling points; the red line indicates the respective glucose concentration. The isotopic state of alanine can be unlabeled (m.0, black), single (m.1.0 blue dotted, m.1.1 blue dashed), double (m.2, grey), or triple (m.3, dark grey) 13C labeled. Mean values and standard deviations of at least two replicates are shown.
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metabolites-05-00455-f004: Mass isotopomer proportions of the 13C labeling states of alanine for (A) Δupp and (B) Δgnd mutant strains. The process time indicates the sampling points; the red line indicates the respective glucose concentration. The isotopic state of alanine can be unlabeled (m.0, black), single (m.1.0 blue dotted, m.1.1 blue dashed), double (m.2, grey), or triple (m.3, dark grey) 13C labeled. Mean values and standard deviations of at least two replicates are shown.

Mentions: The specific labeling distribution in alanine, formed after degradation of [1,2-13C] glucose, is shown in Figure 4. For the PPP-negative Δgnd strain, with EDP as the exclusive catabolic pathway, a 50/50 mixture of m.0 and m.2 signal was expected due to the formation of unlabeled GAP, double-labeled pyruvate, and, subsequently, alanine. Two hours after inoculation, 66% of alanine m.2 was found (Figure 4B). After an additional two hours, this was reduced to the expected theoretical value of 51%, then staying constant during the remaining cultivation. The signal for m.0 is a mixed signal from GAP and degraded unlabeled amino acids from the cultivation medium. It cannot be determined from the data whether the conversion rate of GAP to pyruvate or the uptake rate of precursors from the cultivation medium was changing. The observed reduction of growth rate after 6 h cannot be detected by the labeling state of alanine. Hence, a potential change of intracellular metabolism did not affect the alanine labeling state, i.e., the fueling and composition of the pyruvate pool. Identical growth rates for the PPP-negative strain Δgnd and the strain Δupp in the first 6 h show without a doubt that EDP pathway activity is sufficient for G. oxydans and PPP is not essential for growth on glucose in the first phase, as was also shown in bioreactor cultivations with higher substrate concentrations [10].


(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)

Mass isotopomer proportions of the 13C labeling states of alanine for (A) Δupp and (B) Δgnd mutant strains. The process time indicates the sampling points; the red line indicates the respective glucose concentration. The isotopic state of alanine can be unlabeled (m.0, black), single (m.1.0 blue dotted, m.1.1 blue dashed), double (m.2, grey), or triple (m.3, dark grey) 13C labeled. Mean values and standard deviations of at least two replicates are shown.
© Copyright Policy
Related In: Results  -  Collection

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

metabolites-05-00455-f004: Mass isotopomer proportions of the 13C labeling states of alanine for (A) Δupp and (B) Δgnd mutant strains. The process time indicates the sampling points; the red line indicates the respective glucose concentration. The isotopic state of alanine can be unlabeled (m.0, black), single (m.1.0 blue dotted, m.1.1 blue dashed), double (m.2, grey), or triple (m.3, dark grey) 13C labeled. Mean values and standard deviations of at least two replicates are shown.
Mentions: The specific labeling distribution in alanine, formed after degradation of [1,2-13C] glucose, is shown in Figure 4. For the PPP-negative Δgnd strain, with EDP as the exclusive catabolic pathway, a 50/50 mixture of m.0 and m.2 signal was expected due to the formation of unlabeled GAP, double-labeled pyruvate, and, subsequently, alanine. Two hours after inoculation, 66% of alanine m.2 was found (Figure 4B). After an additional two hours, this was reduced to the expected theoretical value of 51%, then staying constant during the remaining cultivation. The signal for m.0 is a mixed signal from GAP and degraded unlabeled amino acids from the cultivation medium. It cannot be determined from the data whether the conversion rate of GAP to pyruvate or the uptake rate of precursors from the cultivation medium was changing. The observed reduction of growth rate after 6 h cannot be detected by the labeling state of alanine. Hence, a potential change of intracellular metabolism did not affect the alanine labeling state, i.e., the fueling and composition of the pyruvate pool. Identical growth rates for the PPP-negative strain Δgnd and the strain Δupp in the first 6 h show without a doubt that EDP pathway activity is sufficient for G. oxydans and PPP is not essential for growth on glucose in the first phase, as was also shown in bioreactor cultivations with higher substrate concentrations [10].

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