Photoautotrophic metabolism is the process by which plants and other photosynthetic organisms use light energy to fix freely available CO₂ into complex organic molecules. This represents the primary source of all food on earth as well as raw materials for bio-based production of fuels and chemicals. The ability to perform quantitative studies using isotope tracers and metabolic flux analysis (MFA) is critical for detecting pathway bottlenecks and deciphering flux regulation in these systems. Although ¹³C is the preferred isotope tracer for studying central carbon metabolism in heterotrophic systems, photoautotrophs assimilate carbon solely from CO₂ and therefore produce a uniform steady-state ¹³C-labeling pattern that is insensitive to fluxes. However, transient measurements of isotope incorporation following a step change from unlabeled to labeled CO₂ can be used to estimate fluxes successfully with newly developed techniques of isotopically nonstationary MFA (INST-MFA). We have assembled a package of computational routines that achieves more than 5000-fold speedup relative to previous INST-MFA approaches. These tools now permit comprehensive flux analysis of photoautotrophic metabolism, complementing previous studies that have been limited to heterotrophic or mixotrophic conditions.

We have applied the INST-MFA approach to study the metabolism of Synechocystis sp. PCC 6803, a model photosynthetic organism, under autotrophic conditions using both GC/MS and LC/MS/MS to quantify the trajectories of metabolite labeling that result from introduction of ¹³C-labeled bicarbonate. The INST-MFA flux map was compared to values predicted by a linear programming (LP) method that does not require experimental measurements but instead assumes that carbon and light utilization are regulated to provide optimal growth. Although the LP predicts that there should be no flux through the oxidative pentose phosphate pathway, the experimental results indicate that around 10% of the fixed carbon is lost via this pathway. Due in part to these losses, 142 ± 12 moles of CO₂ must be fixed to yield a net gain of 100 C-moles of biomass. This is significantly more than the 111 moles of CO₂ predicted by the LP model, indicating that growth is suboptimal with respect to carbon utilization. Another notable result of the flux analysis is that Synechocystis appears to use transaldolase rather than aldolase as its primary route to regenerate S7P in the Calvin cycle. This is an interesting result, since plants are known to use aldolase exclusively for this purpose. Lastly, the flux analysis confirms that the oxygenation side reaction of RuBisCO is insignificant, as expected due to the potent CO₂-concentrating effect of carboxysomes in this organism.