Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production

Summary Hydrogen (H 2 ) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H 2 and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H 2 production for reductant disposal. To explore the reductant flux and possible competition for H 2 production in vivo , T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H 2 production and consumption. The results obtained establish that H 2 is generated predominantly by the membrane‐bound hydrogenase complex (Mbh), confirm the essential role of the SurR (TK1086p) regulator in vivo , delineate the roles of sulfur (S°) regulon proteins and demonstrate that preventing H 2 consumption results in a substantial net increase in H 2 production. Constitutive expression of TK1086 ( surR ) from a replicative plasmid restored the ability of T. kodakarensis TS1101 (ΔTK1086) to grow in the absence of S° and stimulated H 2 production, revealing a second mechanism to increase H 2 production. Transformation of T. kodakarensis TS1101 with plasmids that express SurR variants constructed to direct the constitutive synthesis of the Mbh complex and prevent expression of the S° regulon was only possible in the absence of S° and, under these conditions, the transformants exhibited wild‐type growth and H 2 production. With S° present, they grew slower but synthesized more H 2 per unit biomass than T. kodakarensis TS517.