This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kleijn, R. J. Articles by Heijnen, J. J. Search for Related Content PubMed PubMed Citation Articles by Kleijn, R. J. Articles by Heijnen, J. J. Agricola Articles by Kleijn, R. J. Articles by Heijnen, J. J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, July 2006, p. 4743-4754, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.02955-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

¹³C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands,¹ Beijerinck Laboratory, DSM Research, A. Fleminglaan 1, 2611 XT Delft, The Netherlands²

Received 14 December 2005/ Accepted 26 April 2006

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.