Figure 1. Overview of the experimental and model-based analyses to determine the metabolite levels, physiological parameters and intracellular metabolic fluxes of replicatively aging budding yeast.
Samples were harvested at various time points (corresponding to different cell ages) from a column-based cultivation system (Janssens et al., 2015), designed to enrich aged mother cells. The fractional abundance of mother, daughter and dead cells in each sample was determined by flow cytometry and a combined dye-staining with propidium iodide and avidin–FITC. Aliquots were used to determine the intracellular metabolite concentrations, c̄i, by LC-MS/MS and the cell count, ncell(t), by flow cytometry, extracellular metabolites (i.e. substrates and products), cS(t) and cP(t), by HPLC and the integral of oxygen and carbon transfer rates, OTR and CTR (i.e. total consumed oxygen and produced carbon dioxide) by a Respiration Activity Monitoring System (RAMOS), in the mixed population samples. Next, the age-dependent intracellular metabolite concentrations (ci) were inferred from the acquired population-average data using non-negative least square regression (NNLS) and the physiological parameters (growth (µ) and metabolite exchange rates (q)) of mother (mo) and daughter (da) cells) from an ordinary differential equation (ODE) model. The inferred physiological parameters and intracellular metabolite levels of aged mother cells were then analyzed using a combined stoichiometric-thermodynamic metabolic model and regression analysis to obtain the intracellular metabolic flux distribution.