13 C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum
| Autor: | Joseph J. Heijnen, Cor Ras, Walter M. van Gulik, Roelco J. Kleijn, Wouter A. van Winden, Dick Schipper |
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| Rok vydání: | 2006 |
| Předmět: |
Primary metabolism
Mycology Chemostat Penicillium chrysogenum Pentose phosphate pathway Gluconates Applied Microbiology and Biotechnology Isotopomers Pentose Phosphate Pathway TRACER Metabolic flux analysis Carbon Isotopes Chromatography Cellular metabolism Ecology biology Reproducibility of Results Physiology and Biotechnology biology.organism_classification Culture Media Glucose Biochemistry Glycolysis Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 72:4743-4754 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.02955-05 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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