Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis

Abstract

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.

Figure 1. Simplified example of carbon labeling in a photoautotrophic system

Following a switch from natural CO₂ to ¹³CO₂, intracellular metabolites become gradually labeled over time. 3-phosphoglycerate (3PGA) becomes ¹³C-labeled due to carbon fixation by RuBisCO (RBC), resulting in labeling of pentose-5-phosphate (P5P) intermediates of the Calvin-Benson-Bassham (CBB) cycle. Once steady-state labeling is achieved, all metabolites are uniformly ¹³C-labeled irrespective of fluxes and intracellular pool sizes. Labeling patterns observed during the isotopically transient period, however, can be computationally analyzed to determine fluxes.

Figure 2. Overview of INST-MFA procedure

Following introduction of ¹³C-labeled bicarbonate, a series of metabolite samples were obtained by rapid quenching and extraction of cyanobacterial cells. Dynamic changes in isotope labeling patterns were assessed using GC-MS and LC-MS/MS, followed by computational analysis of these trajectories to estimate metabolic pathway fluxes.

Figure 3. ¹³C-Labeling trajectories of selected central carbon metabolites

(A) Experimentally measured mass isotopomer abundances (data points) and INST-MFA model fits (solid lines). Error bars represent standard measurement errors. Raw mass isotopomer data are shown without correction for natural isotope abundance. (B) Average ¹³C-enrichments of selected ion fragments. Mass isotopomer distributions were corrected for natural isotope abundance using the method of Fernandez et al. (1996), and average ¹³C enrichment was calculated using the formula $\frac{1}{N}{\displaystyle {\sum }_{i=1}^{N}Mi\times i}$, where N is the number of carbon atoms in the metabolite and Mi is the fractional abundance of the ith mass isotopomer. The top axis shows the full labeling trajectory and the bottom axis shows an enlarged view of the highlighted region from 0–5 min. Ions shown are for 3-phosphoglycerate (3PGA), fructose-6-phosphate (F6P), ribulose-1,5-bisphosphate (RUBP), malate (MAL), glycerate (GA), succinate (SUC), citrate (CIT) and fumarate (FUM). Nominal masses of M0 mass isotopomers are shown in parentheses.

Figure 3. ¹³C-Labeling trajectories of selected central carbon metabolites

(A) Experimentally measured mass isotopomer abundances (data points) and INST-MFA model fits (solid lines). Error bars represent standard measurement errors. Raw mass isotopomer data are shown without correction for natural isotope abundance. (B) Average ¹³C-enrichments of selected ion fragments. Mass isotopomer distributions were corrected for natural isotope abundance using the method of Fernandez et al. (1996), and average ¹³C enrichment was calculated using the formula $\frac{1}{N}{\displaystyle {\sum }_{i=1}^{N}Mi\times i}$, where N is the number of carbon atoms in the metabolite and Mi is the fractional abundance of the ith mass isotopomer. The top axis shows the full labeling trajectory and the bottom axis shows an enlarged view of the highlighted region from 0–5 min. Ions shown are for 3-phosphoglycerate (3PGA), fructose-6-phosphate (F6P), ribulose-1,5-bisphosphate (RUBP), malate (MAL), glycerate (GA), succinate (SUC), citrate (CIT) and fumarate (FUM). Nominal masses of M0 mass isotopomers are shown in parentheses.

Figure 4. Synechocystis flux map determined under photoautotrophic growth conditions

Net fluxes are shown normalized to a net CO₂ uptake rate of 100. Values are represented as M ± SE, where M is the median of the 95% flux confidence interval and SE is the estimated standard error of M calculated as (UB95 – LB95)/3.92. (UB95 and LB95 are the upper and lower bounds of the 95% confidence interval, respectively.) Arrow thickness is scaled proportional to net flux. Dotted arrows indicate fluxes to biomass formation.

Figure 5. Isotope labeling in photorepiratory pathways

(A) Schematic diagram of the complete 2PG metabolism of Synechocystis showing the fate of the first two carbon atoms of ribulose-1,5-bisphosphate. The three photorespiratory pathways diverge at glyoxylate into the plant-like C2 cycle (via glycine and serine), the bacterial-like glycerate pathway (via tartronic semialdehyde), and the decarboxylation branch (via oxalate). The first two pathways re-converge at glycerate. (B) ¹³C enrichments of photorespiratory pathway intermediates after 4.2 minutes of ¹³CO₂ labeling. Average enrichments were calculated as described in the caption to Figure 3B. The compositions of selected 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), and glycerate (GA) fragment ions are listed in Table I. The carbon atoms of serine (SER) and glycine (GLY) contained in the selected fragment ions were taken from Huege et al. (2007): SER218 (C1-C2), SER204 (C2-C3), GLY276 (C1-C2), GLY174 (C2).

Figure 5. Isotope labeling in photorepiratory pathways

(A) Schematic diagram of the complete 2PG metabolism of Synechocystis showing the fate of the first two carbon atoms of ribulose-1,5-bisphosphate. The three photorespiratory pathways diverge at glyoxylate into the plant-like C2 cycle (via glycine and serine), the bacterial-like glycerate pathway (via tartronic semialdehyde), and the decarboxylation branch (via oxalate). The first two pathways re-converge at glycerate. (B) ¹³C enrichments of photorespiratory pathway intermediates after 4.2 minutes of ¹³CO₂ labeling. Average enrichments were calculated as described in the caption to Figure 3B. The compositions of selected 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), and glycerate (GA) fragment ions are listed in Table I. The carbon atoms of serine (SER) and glycine (GLY) contained in the selected fragment ions were taken from Huege et al. (2007): SER218 (C1-C2), SER204 (C2-C3), GLY276 (C1-C2), GLY174 (C2).

Figure 6. Average steady-state ¹³C enrichment of CBB cycle intermediates

Average enrichments were calculated as described in the caption to Figure 3B. The labeling of all CBB intermediates achieved steady state at times greater than 8 minutes after tracer administration. Averages of at least two steady-state time points are shown. Error bars indicate standard errors of the mean values.