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

Abstract

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.Quantification of primary metabolic fluxes in microorganisms provides researchers with an important tool for a more rational approach to metabolic engineering. A part of the cellular metabolism that has received special attention when possible strain improvement strategies have been examined is the flux distribution around branch points (9,30,31). At these metabolic branch points an entering flux diverges in two or more different directions, thus forming a potential target for rerouting fluxes. Under glucose-feeding conditions the first metabolic node encountered by the glucose entering a cell is the node around glucose-6-phosphate (g6p), which results in carbon flux partitioning toward glycolysis, the pentose phosphate pathway (PPP), storage carbohydrates, and in some prokaryotes the Entner-Doudoroff pathway. Each of these pathways has its own unique function in the cell. Glycolysis (combined with the tricarboxylic acid cycle) is the general route for glucose catabolism and energy formation in the cell, while the PPP plays a crucial role in the redox metabolism of the cell. In addition to their catabolic functions these pathways also have anabolic functions as they provide the precursors for the monomers (amino acids, fatty acids, nucleotides, sugar phosphates, etc.) required for growth and product formation. Therefore, accurate determination of the flux distribution around the g6p node provides valuable insight into the functioning of a cell.An importan...

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