Mapping Photoautotrophic Carbon Metabolism Using the INCA 13C Flux Analysis Platform

Although steady-state ¹³C -labeling experiments are widely used to quantify fluxes in heterotrophic organisms, autotrophs assimilate carbon solely from CO₂ and therefore produce a uniform steady-state ¹³C-labeling pattern when grown on ¹³CO₂. This makes steady-state ¹³C metabolic flux analysis (MFA) ineffective for studying autotrophic metabolism. However, transient measurements of isotope incorporation following a step change from unlabeled to labeled CO₂ can be used to estimate photoautotrophic fluxes by applying isotopically nonstationary MFA (INST-MFA). We have recently developed a package of MATLAB routines called INCA that automates the computational workflow of INST-MFA. INCA is the first publically available software package that can perform INST-MFA on networks of arbitrary size and complexity. To establish proof of concept, we first applied INCA to map fluxes in the model cyanobacterium Synechocystis sp. PCC 6803 growing under photoautotrophic conditions. Our study relied on both GC-MS and LC-MS/MS to quantify labeling trajectories of 15 intracellular metabolites following administration of ¹³C-labeled bicarbonate to a photobioreactor culture. Overall, we were able to precisely quantify the rates of all Calvin cycle and TCA pathway reactions, as well as several “wasteful” side reactions that contribute to suboptimal photoautotrophic growth of Synechocystis.

We next adapted our INST-MFA model to a terrestrial plant system. We performed in vivo isotopic labeling of Arabidopsis thaliana leaves with ¹³CO₂, measured the transient labeling of 30 metabolite fragment ions using mass spectrometry, and estimated fluxes throughout leaf photosynthetic metabolism using INCA. Leaves were exposed to either 200 or 500 µmol m^-2s^-1 light, with or without prior acclimation. Approximately 1,200 independent mass isotopomer measurements were regressed to estimate 110 fluxes under each condition. Photorespiration flux was significantly increased under high light conditions, despite concomitant increases in carboxylation flux that led to enhanced sucrose production. Interestingly, we observed an inverse relationship between intermediate pool sizes and Calvin cycle fluxes as light intensity increased. Additionally, we identified enhanced hexose exchange between the chloroplast and cytosol as a potential short-term adaptation to high light that was suppressed by acclimation. Taken together, these studies have established ¹³C INST-MFA and the INCA software package as a comprehensive platform to map carbon fluxes in cyanobacteria, plants, and other photoautotrophic organisms.