Genome-scale mapping models and algorithms for stationary and instationary MFA-based metabolic flux elucidation

Conference Dates

July 16-20, 2017


Metabolic models used in 13C metabolic flux analysis (13C-MFA) generally include a limited number of reactions primarily from central metabolism, neglecting degradation pathways and atom transition contributions for reactions outside central metabolism. This study addresses the impact on prediction fidelity of scaling-up core bacterial and cyanobacterial mapping models to a genome-scale carbon mapping (GSCM) models, imEco726 (668 reaction and 566 metabolites) and imSyn711 (731 reactions, 679 metabolites) for E. coli and Synechocystis PCC 6803, respectively, representing a ten-fold increase in model size. The GSCM models are constructed using the CLCA algorithm following reduction of the corresponding metabolic models, iAF1260 and iSyn731, using experimentally measured biomass and product yield during growth on glucose and CO2, respectively. The mapping models are then deployed for flux elucidation using isotopic steady-state MFA for E. coli to recapitulate experimentally observed labeling distributions of 12 measured amino acids, and isotopic instationary MFA for Synechocystis, to recapitulate labeling dynamics of 15 central metabolites. In both models, 80% of all fluxes varies less than onetenth of the basis carbon substrate uptake rate primarily due to the flux coupling with biomass production. Overall, we find that both the topology and estimated values of the metabolic fluxes remain largely consistent between the core and GSMM models for E. coli. Stepping up to a genome-scale mapping model leads to wider flux inference ranges for 20 key reactions present in the core model. The glycolysis flux range doubles due to the possibility of active gluconeogenesis, the TCA flux range expanded by 80% due to the availability of a bypass through arginine consistent with labeling data, and the transhydrogenase reaction flux was essentially unresolved due to the presence of as many as five routes for the inter-conversion of NADPH to NADH afforded by the genome-scale model. By globally accounting for ATP demands in the GSMM model the unused ATP decreased drastically with the lower bound matching the maintenance ATP requirement. A non-zero flux for the arginine degradation pathway was identified to meet biomass precursor demands as detailed in the iAF1260 model. Significant flux range shifts were observed using a GSCM model compared to a core model in Synechocystis arising from the inclusion of 18 novel carbon paths in the GSCM model. In particular, no flux is channeled through the oxidative pentose phosphate pathway, resulting in a reduced carbon fixation flux. In addition, a higher flux is seen through the Transaldolase reaction, serving as a bypass route to Fructose bisphosphatase. Serine and glycine are found to be synthesized from 3-phosphoglycerate and the photorespiratory pathway, respectively. Pyruvate is synthesized exclusively via the malate bypass with trace contributions from pyruvate kinase. Furthermore, trace flux is predicted through the lower TCA cycle indicating either pathway incompleteness or dispensability during photoautotrophic growth. Despite these differences, 80% of all reactions in both genome-scale models are resolved to within 10% of the respective substrate uptake rate due to the presence of 411 and 407 growth-coupled reactions in E. coli and Synechocystis, respectively. Flux ranges obtained with GSCM models are compared with those obtained upon projecting core model ranges on to a genome-scale metabolic model to elucidate the loss of information and erroneous biological inferences about pathway usage arising from assumptions contained within core models, reaffirming the importance of using mapping models with global carbon path coverage in 13C metabolic flux analysis.

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