Parameters screened for optimization Selected enzymes Final hydrogen concentration (mM) Initial enzyme concentration (E0) E3, E4, E13 582 Enzyme turn-over (kcat) E3, E4, E13 800 Enzyme affinity for substrate (Km) E4 and E13 236

Table 2: Hydrogen final concentration value reached by the system after single stage optimization runs. Here, E0, kcat and K0 for only E3 (glucose-6-phosphate dehydrogenase), E4 (6-phosphogluconic dehydrogenase) and E13 (NADP+ dependant hydrogen dehydrogenase) were optimized each separately while the parameters for the other enzymes were kept constant at their default values (see Method). The modifications were evaluated in order to obtain the best final hydrogen concentration (mM) after a simulation time of 9000 min with starting concentrations of 70 mM for cellobiose, 70 mM for inorganic phosphate and 1 mM for NADP. Additional results are featured in Figure 3.