VALIDATION OF A METABOLIC NETWORK FOR SACCHAROMYCES-CEREVISIAE USING MIXED SUBSTRATE STUDIES

Setting up a metabolic network model for respiratory growth of Sacchar omyces cerevisiae requires the estimation of only two (energetic) stoi chiometric parameters: (1) the operational P0 ratio and (2) a growth-r elated maintenance factor h. It is shown, both theoretically and pract ically, how chemostat cultivations with different mixtures of two subs trates allow unique values to be given to these unknowns of the propos ed metabolic model. For the yeast and model considered, an effective P 0 ratio of 1.09 mol of ATP/mol of 0 (95% confidence interval 1.07-1.11 ) and a k factor of 0.415 mol of ATP/C-mol of biomass (0.385-0.445) we re obtained from biomass substrate yield data on glucose/ethanol mixtu res. Symbolic manipulation software proved very valuable in this study as it supported the proof of theoretical identifiability and signific antly reduced the necessary computations for parameter estimation. In the transition from 100% glucose to 100% ethanol in the feed, four met abolic regimes occur. Switching between these regimes is determined by cessation of an irreversible reaction and initiation of an alternativ e reaction. Metabolic network predictions of these metabolic switches compared well with activity measurements of key enzymes. As a second v alidation of the network, the biomass yield of S. cerevisiae on acetat e was also compared to the network prediction. An excellent agreement was found for a network in which acetate transport was modeled with a proton symport, while passive diffusion of acetate gave significantly higher yield predictions.