Stepwise optimization of pathway design. A) The proposed pathway operates in two steps, with electrons flowing first from HydA to FD, then from FD to SIR. Substituting PFOR for SIR allows an independent test of the HydA-FD interaction. Substituting FNR for HydA allows an independent test of the FD-SIR interaction. B) PFOR-driven H2 production in vivo with a panel of ferredoxins. H2 production in the presence of PFOR confirms the ability of a ferredoxin to couple with hydrogenase. H2 production in the absence of PFOR suggests that a ferredoxin is reduced by a nonspecific endogenous source, which would tend to confound our genetic selection. C) FNR-rescued growth with a panel of ferredoxins under anaerobic conditions. Growth with FNR confirms the ability of a ferredoxin to couple with SIR. FNR-independent growth suggests that a ferredoxin is not well insulated from endogenous redox sources. All experiments were conducted under anaerobic conditions with the EW11 strain, as described in the methods. Error bars are 95% confidence intervals.