Illustration of the procedure to assign reaction directions by heuristic rules. For the assignment steps 2a–2c (cf. Fig. 1), the applied heuristic rules are displayed. Generally, the rules defined a reaction as irreversible in the direction of consumption of a high-energy co-substrate. The rules, however, were not applied if the respective reaction simultaneously produced CO2. The vertical arrows indicate the consecutive application of the rules: if no assignment was possible with a particular heuristic rule, the next rule along the arrow was employed. The consumption of a co-substrate with a higher energetic content was prefered over the consumption of a co-substrate with a lower energetic content. In case the cycle contains more than one reaction producing the same highly energetic co-substrate, all these reaction steps are defined as irreversible. In pair cycles (2a), the reaction that produces the only generated co-substrate was defined as irreversible as long as it did not consume CO2. In case of CO2 consumption, however, it follows that the other reaction also produces CO2 and we define this reaction direction as irreversible. As only one co-substrate pair is converted in each pair cycle, the assignment was achieved by applying the heuristic rules consecutively while omitting the first rule as indicated in the figure. In the analysis of the remaining energy producing cycles (2b), the cycles can contain reaction steps that produce different kinds of co-substrates. Here, in the first place we restricted CO2 consumption, which is in general indicating a thermodynamically infeasible reaction step. If no CO2 consuming reaction was preset, the production of highly energetic co-substrates were disabled with the indicated priorities. Note that NADPH and NADH producing reactions, here, were assigned with the same priority (not illustrated in the figure). In the bypass analysis (2c), reaction directions were assigned for CO2 consuming or nucleotide triphosphates producing reactions. Preliminary studies showed that only these heuristic rules were fully reliable in this assignment step, and thus, we only applied these two rules.