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13 C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum
In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U- 13 C]gluconate, followe...
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Published in: | Applied and environmental microbiology 2006-07, Vol.72 (7), p.4743-4754 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-
13
C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus
Penicillium chrysogenum
. For comparison, a
13
C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of
P. chrysogenum
. For the first time mass isotopomer measurements of
13
C-labeled primary metabolites are reported for
P. chrysogenum
and used for a
13
C-based MFA. Estimation of the PPP split ratio of
P. chrysogenum
at a growth rate of 0.02 h
−1
yielded comparable values for the gluconate tracer method and the
13
C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the
13
C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the
13
C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio. |
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ISSN: | 0099-2240 1098-5336 |
DOI: | 10.1128/AEM.02955-05 |