Rate equations for measured extracellular rates and macromolecular composition data were combined with a stoichiometric model to describe riboflavin production with an industrial Bacillus subtilis strain using errors in variables regression analysis. On the basis of this combined stoichiometric growth model, we explored the topological features of the B. subtilis metabolic reaction network that was assembled from a large amount of literature. More specifically, we simulated maximum theoretical yields of biomass and riboflavin, including the associated flux regimes. Based on the developed model, the importance of experimental data on building block requirements for maximum yield and flux calculations were investigated. These analyses clearly show that verification of macromolecular composition data is important for optimum flux calculations.