This work may be considered as a model study for therapeutic protein production, and a theoretical approach to hypothesise new medical strategies to further applied medical questions. A comprehensive generalised metabolic reaction network of Bacillus licheniformis that considers 149 reaction fluxes and 106 metabolites was used in the mass flux balance-based stoichiometric model for the analysis of human leukocyte interferon (IFN-alpha(1)) and erythropoietin (EPO) production capacities of recombinant Bacillus species. The importance of cellular energetics on optimum performance was quantitatively assessed. The metabolic pathways leading to optimised IFN-alpha(1) and EPO overproduction were determined for the two carbon sources that have different reduction degrees (gamma), i.e. glucose (gamma=4.0) and citrate (gamma=3.0), and the variation of the fluxes were obtained. Metabolic capacity analyses showed that maximum IFN-alpha(1) and EPO synthesis rates were, respectively, 0.062 and 0.055 mmol g(-1) DWh(-1) at mu=0h(-1) when glucose uptake rate was 10 mmol g(-1) DWh(-1); and IFN-alpha(1) and EPO synthesis rates decreased, respectively, 1.70- and 1.75-fold when citrate was used as the carbon source. The flux distributions showed that the amino acid composition of the proteins influence the production. Leucine appears to be the most important amino acid for both IFN-alpha(1) and EPO production. Consequently, pyruvate seems to be the critical main branch point and B. pasteurii seems to be the favourable host for therapeutical protein production due to the high leucine uptake capacity. The results encourage the discussion on the potential strategies for improving production of IFN-alpha(1) and EPO, and further enable us to assert medical hypothesis in order to support the immune system of the human body against the deficiencies of the synthesis of IFN-alpha(1) and EPO in the human cells. (C) 2002 Elsevier Science B.V. All rights reserved.