C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum

• Published in: Applied and environmental microbiology Vol. 72; no. 7; pp. 4743 - 4754
• Main Authors: Kleijn, Roelco J, Winden, Wouter A. van, Ras, Cor, Gulik, Walter M. van, Schipper, Dick, Heijnen, Joseph J
• Format: Journal Article
• Language: English
• Published: American Society for Microbiology 01-07-2006
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.02955-05

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-¹³C]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 ¹³C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of ¹³C-labeled primary metabolites are reported for P. chrysogenum and used for a ¹³C-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 ¹³C-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 ¹³C-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 ¹³C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.