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

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 degrees) 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 (Delta TK1086) to grow in the absence of S degrees 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 degrees regulon was only possible in the absence of S degrees and, under these conditions, the transformants exhibited wild-type growth and H(2) production. With S degrees present, they grew slower but synthesized more H(2) per unit biomass than T. kodakarensis TS517.