Fig 1. Metabolic changes required for the evolution of the acetate and glycerol cross-feeding strains.

(A) Minimal number of reactions requiring a change in flux in the ancestor for acetate and glycerol cross-feeding to evolve. Most reactions requiring a flux change belong to glycolysis and gluconeogenesis, the pentose phosphate pathway, the citric acid cycle or from transport processes. The remaining reactions (‘others’) come from multiple pathways that comprise oxidative phosphorylation, alternate carbon metabolism, pyruvate metabolism, glycine and serine metabolism, alanine and aspartate metabolism, folate metabolism, anaplerotic reactions, or that are unassigned to a pathway. In the table we also include the number of genes associated with the reactions requiring a flux change, the number of operons into which these genes fall, as well as the number of regulons. (B) Central carbon metabolism of E. coli. Every orange circle represents a metabolite and every line a reaction. Thick grey lines indicate a non-zero flux in the ancestral strain (|a_i|>0.001 mmolgDW^-1 h^-1). (C) to (F) As in (B), but each panel shows reactions with non-zero flux in cross-feeding strains (blue for producers, red for consumers) in addition to non-zero fluxes in the ancestor (grey). (S1 Fig allows ‘zooming in’ to see metabolite names, reaction names, and flux values.) Panels (G) to (J) show, on the left of each panel, the number of reactions that are activated in a producer (blue) or consumer (red) relative to the ancestor (‘on’), or the reactions that are inactivated relative to the ancestor (‘off’, grey). On the right of each panel, the amount of flux change is shown for reactions that change their flux relative to the ancestor.