Photoautotrophic metabolism involves the utilization of light energy to fix freely available CO₂ into complex organic molecules. ¹³C-MFA is widely used for quantification of flux-phenotypes under varying environmental and genetic conditions. However, inherent limitations have prevented the application of steady state ¹³C-MFA to purely autotrophic metabolism, wherein CO₂ is the sole carbon source. This is due to the fact that, in autotrophic systems under conditions of isotopic steady state, every single carbon atom in every downstream molecule has the same relative labeling as the single input carbon of CO₂, irrespective of flux distribution. However, the trajectory of label incorporation during the period preceding isotopic steady state is sensitive to fluxes. This transient labeling information is utilized in recently developed techniques of nonstationary ¹³C-MFA, which enable estimation of metabolic fluxes from measurements of dynamic changes in ¹³C labeling patterns of intracellular metabolite pools in response to a step change in CO₂ labeling.

In this work, we utilize the nonstationary ¹³C- MFA technique with the elementary metabolite unit (EMU) formulation to estimate central carbon fluxes under photoautotrophic conditions for the first time. The technique is applied to a prokaryotic cyanobacterium, Synechocystis sp. PCC 6803. Reactions of the central carbon network consisting of glycolysis/gluconeogenesis, pentose phosphate/Calvin cycle, and the TCA/glyoxylate shunt, are modeled. Tandem mass spectrometry based techniques are used to measure mass isotopomer distributions as well as concentrations (pool sizes) of intracellular metabolites. Metabolite pool size measurements are subject to several inaccuracies during the quenching and extraction procedure. Therefore, the effect of number and accuracy of pool size measurements on flux identifiability will also be discussed.