Steady-state and dynamic flux balance analysis of ethanol production by Saccharomyces cerevisiae.

Steady-state and dynamic flux balance analysis (DFBA) was used to investigate the effects of metabolic model complexity and parameters on ethanol production predictions for wild-type and engineered Saccharomyces cerevisiae. Three metabolic network models ranging from a single compartment representation of metabolism to a genome-scale reconstruction with seven compartments and detailed charge balancing were studied. Steady-state analysis showed that the models generated similar wild-type predictions for the biomass and ethanol yields, but for ten engineered strains the seven compartment model produced smaller ethanol yield enhancements. Simplification of the seven compartment model to two intracellular compartments produced increased ethanol yields, suggesting that reaction localisation had an impact on mutant phenotype predictions. Further analysis with the seven compartment model demonstrated that steady-state predictions can be sensitive to intracellular model parameters, with the biomass yield exhibiting high sensitivity to ATP utilisation parameters and the biomass composition. The incorporation of gene expression data through the zeroing of metabolic reactions associated with unexpressed genes was shown to produce negligible changes in steady-state predictions when the oxygen uptake rate was suitably constrained. Dynamic extensions of the single and seven compartment models were developed through the addition of glucose and oxygen uptake expressions and transient extracellular balances. While the dynamic models produced similar predictions of the optimal batch ethanol productivity for the wild type, the single compartment model produced significantly different predictions for four implementable gene insertions. A combined deletion/overexpression/insertion mutant with improved ethanol productivity capabilities was computationally identified by dynamically screening multiple combinations of the ten metabolic engineering strategies. The authors concluded that extensive compartmentalisation and detailed charge balancing can be important for reliably screening metabolic engineering strategies that rely on modification of the global redox balance and that DFBA offers the potential to identify novel mutants for enhanced metabolite production in batch and fed-batch cultures.