13c-Metabolic Flux Analysis of the Pentose Phosphate Pathway Using GC-MS Analysis of RNA and Glycogen

The pentose phosphate pathway (PPP) plays a key role in cellular metabolism, generating precursors for amino acid and nucleotide biosynthesis, as well as cofactors in the form of NADPH. Additionally, the PPP has been the target of metabolic engineering strategies for the production of various industrially relevant compounds. ¹³C- Metabolic flux analysis (¹³C-MFA) has proven to be a powerful tool in gaining a quantitative understanding of metabolic pathways. However, there are inherent difficulties in measuring fluxes in the PPP due to the reversibility of the sugar phosphate interconversions and the relative paucity of measured fragments derived from upper central carbon metabolism in typical GC-MS analysis of proteinogenic amino acids.

In this work, we present a method to incorporate glycogen and RNA ¹³C-labeling to gain significant new observability in the PPP for flux analysis. These measurements provide direct information on glucose 6-phosphate (G6P) and ribose 5-phosphate (R5P), respectively. A significant advantage over existing methods is that relatively little biomass is required for these measurements. Two fragments are obtained for each sugar, providing additional positional labeling information. These methods were applied for the analysis of both E. coli and CHO cells, using parallel labeling experiments that have been specifically optimized for this purpose. In each system, ¹³C-MFA was able to resolve net and exchange PPP fluxes using only glycogen and RNA data. This approach reduces the amount of biomass and sample analysis required by providing direct labeling information of PPP intermediates. Overall, this method provides a rigorous technique for measuring PPP fluxes. More generally, these measurements can also be added to compliment commonly used amino acid labeling information for precise ¹³C-MFA.