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

Abstract

Summary Hydrogen (H2) production by Thermococcus kodakarensis compares very favorably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H2 and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H2 production for reductant disposal. To explore the reductant flux and possible competition for H2 production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H2 production and consumption. The results obtained establish that H2 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 H2 consumption results in a substantial net increase in H2 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 H2 production, revealing a second mechanism to increase H2 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 H2 production. With S° present, they grew slower but synthesized more H2 per unit biomass than T. kodakarensis TS517.

Deletion Strains Reveal Metabolic Roles for Key Elemental Sulfur-Responsive Proteins in Pyrococcus furiosus

“…While this paper was in revision, strains of T. kodakarensis with deletions of homologs of nsr and mbx, and also supposedly of sipA, were reported (19). The phenotypes of the nsr (TS1109) and mbx (TS1105) mutants are similar to those of their P. furiosus counterparts, although we did not observe the higher concentrations of H 2 that were produced by the nsr mutant.…”

Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis

“…The genes of POR, VOR, or PFL were individually deleted in T. kodakarensis by homologous recombination to evaluate the contribution of the enzymes to pyruvate/amino acid oxidation in this hyperthermophile. We used an Hyh-deficient strain, the KUW1⌬hyh mutant, which was constructed by deletion of hyhBGSL (tk2072-tk2069) in T. kodakarensis KUW1 (⌬pyrF ⌬trpE) as a host strain in order to simplify the H 2 -evolving metabolisms by blocking the uptake of the evolved H 2 (26,27). Considering the presence of a common ␥-subunit for POR and VOR, the knockout mutants of POR and VOR were constructed by deletion of the genes encoding ␦, ␣, and ␤ subunits of POR (porDAB) and those of VOR (vorDAB), respectively.…”

“…It also exhibited proton-pumping activity during H 2 evolution, and the resulting proton-motive force was coupled with ATP synthesis driven by the membrane-bound ATP synthase directly or by being converted to sodium-motive force (21)(22)(23)(24). It has been demonstrated genetically that MBH-lacking strains of T. kodakarensis and P. furiosus showed severe growth impairment under the H 2 -evolving growth conditions (26)(27)(28). HYH in these hyperthermophiles acted in H 2 uptake rather than H 2 evolution for the recovery of reducing equivalents from H 2 in the form of NADPH (25)(26)(27)(28).…”

“…It has been demonstrated genetically that MBH-lacking strains of T. kodakarensis and P. furiosus showed severe growth impairment under the H 2 -evolving growth conditions (26)(27)(28). HYH in these hyperthermophiles acted in H 2 uptake rather than H 2 evolution for the recovery of reducing equivalents from H 2 in the form of NADPH (25)(26)(27)(28).…”

“…In Thermococcales hyperthermophiles, two kinds of [NiFe] hydrogenase, membrane-bound hydrogenase (MBH) and cytosolic hydrogenase (HYH), are considered to participate in H 2 metabolism (21)(22)(23)(24)(25)(26)(27)(28). MBH reduces protons to evolve H 2 using reduced ferredoxin (Fd red ), generated via catabolism of growth substrates, as an electron donor.…”