Illustration of knockout experiments. Panel A: Illustration of the sub-network that explains why deletion of pta or ackA is wrongly predicted as lethal by OT. According to the model, the alternative route via acs is inhibited by the presence of external pyruvate, which is always excreted when biomass is produced. Thus, there cannot be an uptake of external acetate. In vivo, however, the excreted pyruvate is negligible. Panel B: Illustration of the sub-network that explains why deletion of ppc is wrongly predicted as viable by OT. The glyoxylate shunt is activated if no external glucose but external acetate is present. According to the model, acetate is always excreted when biomass is produced. Therefore, the glyoxylate shunt is always upregulated, if glucose is not contained in the growth medium. Arrows indicate metabolic reactions, squares indicate activation and T-shaped lines inhibition. Dotted arrows indicate schematic reactions, which abstract a set of metabolic reactions.