sup 13^C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicilliurn chrysogen urn

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-^sup 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 ^sup 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 ^sup 13^C-labeled primary metabolites are reported for P. chrysogenum and used for a ^sup 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 ^sup 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 ^sup 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 ^sup 13^C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio. [PUBLICATION ABSTRACT]