13C-Metabolic Flux Analysis at Its Limits: Identifiability of Anaplerotic Reactions in Corynebacterium Glutamicum

¹³C-Metabolic Flux Analysis has matured into a cornerstone of modern omics technologies [1], providing key insights in metabolic engineering applications as well as in basic pathway regulation [2]. Several studies performing ¹³C-MFA on Corynebacterium glutamicum, an industrial workhorse exhibiting the simultaneous presence of five anaplerotic reactions, indicated non-identifiable fluxes for anaplerotic reactions [3, 4]. In light of these results we thoroughly investigated the question whether stationary isotopic data always allow inferring all fluxes for the best case of full isotopomer information.

Applying a suite of numerical methods and exact integer arithmetic we prove in full generality that for C. glutamicum the anaplerotic reactions of the wildtype are non-identifiable from stationary ¹³C-isotopomer data, even if the labeling enrichment in each metabolite is completely known. We analyzed different scenarios of active anaplerotic reactions to conclude that assuming single reaction steps to be non-active does not necessarily render the remaining reactions identifiable. The omission of one reaction changes the topology of the network, induces a different label flow and thus alters the information content of the respective ¹³C-enrichment. The results for different assumptions of active anaplerotic reactions are directly transferable to other compartmentation-free organisms [5].

On the one hand, our results show that fluxes in complex networks can remain non-observable regardless of the measurement information provided. Even parallel labeling experiments will not yield additional information. On the other hand, the gained structural information from our analysis allowed to deduce identifiable combinations of measurements and network topologies for C. glutamicum and, thus, represents further a priori knowledge for the future design of ¹³C-MFA experiments.

[1] Wiechert W., Nöh K. (2013) Isotopically non-stationary metabolic flux analysis: complex yet highly informative. Curr Opin Biotechnol 24(6):979-986

[2] Niedenführ S., Wiechert W., Nöh K. (2015) How to measure metabolic fluxes: a taxonomic guide for ¹³C fluxomics. Curr Opin Biotechnol 34:82-90

[3] Noack S., Nöh K., Moch M., Oldiges M., Wiechert W.  (2012) Stationary versus non-stationary 13C-MFA: A comparison using a consistent dataset. J Biotech 154(2-3):179-190

[4] van Ooyen J, Noack S, Bott M, Reth A, Eggeling L (2012) Improved L-lysine production with Corynebacterium glutamicum and systemic insight into citrate synthase flux and activity. Biotechnol Bioeng 109(8):2070-81

[5] Kappelmann J., Wiechert W., Noack S. (2015) Cutting the Gordian Knot: Identifiability of Anaplerotic reactions in Corynebacterium glutamicum. Biotechnol Bioeng 113(3):661-674