(194ai) Elucidation of Carbon Flux Topology Representing Photoautotrophic Growth in Synechocystis PCC 6803 Using Genome-Scale Isotopic Instationary Metabolic Flux Analysis

The reliability of flux estimation in photoautotrophic systems depends on the completeness and accuracy of carbon path coverage of the metabolic model. In this study, metabolic fluxes under photoautotrophic growth conditions in Synechocystis PCC 6803 are quantified by re-analyzing an existing dataset using genome-scale isotopic instationary ¹³C-Metabolic Flux Analysis (INST-MFA) with the carbon mapping model imSyn617. The mapping model traverses 18 novel carbon paths spanning Calvin cycle, photorespiration, an expanded glyoxylate metabolism, and corrinoid biosynthetic pathways and 190 additional metabolites absent in core models currently used for MFA. Flux elucidation reveals that 88% of the assimilated bicarbonate is fixed by RuBisCO and 12% off-gassed as CO₂. This sub-optimal carbon fixation is compensated by conversion of fixed CO₂ to biomass with near 100% efficiency while minimizing the yield of organic acids and glycogen. A newly discovered modality is the bifurcated topology of glycine metabolism using parts of photorespiration and the phosphoserine pathways to avoid carbon losses associated with glycine oxidation. The TCA cycle is shown to be incomplete with a bifurcated topology, fueled by anaplerotic metabolism and pyruvate oxidation. Inactivity of futile cycles and alternate routes results in pathway usage and (in)dispensability predictions consistent with experimental findings. The resolved flux map confirms maximization of biomass yield as the cellular objective with carbon fixation as the major metabolic bottleneck. Flux prediction departures from the ones obtained with the core model demonstrate the importance of constructing mapping models with global coverage to reliably glean new biological insights using labeled substrates.