13C-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-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.
Kleijn
Roelco J
RJ
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. R.J.Kleijn@tnw.tudelft.nl
van Winden
Wouter A
WA
Ras
Cor
C
van Gulik
Walter M
WM
Schipper
Dick
D
Heijnen
Joseph J
JJ
D002247:Carbon Isotopes / Q000378:metabolism; D003470:Culture Media; D005942:Gluconates / Q000378:metabolism*; D005947:Glucose; D006019:Glycolysis; D009172:Mycology / Q000379:methods*; D010408:Penicillium chrysogenum / Q000254:growth & development / Q000378:metabolism*; D010427:Pentose Phosphate Pathway / Q000502:physiology*; D015203:Reproducibility of Results
D002247:Carbon Isotopes; D003470:Culture Media; D005942:Gluconates; D005947:Glucose; C030691:gluconic acid