Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase

Abstract

Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O2-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining ‘minimal’ hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg−1 in H2 production using the artificial electron donor methyl viologen) and thermostable (t1/2 ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H2 in vitro without the need for an intermediate electron carrier.

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

“…The SP1 expression cassette (Fig. 1B) was cloned into plasmid pSPF300 (15), generating the plasmid pALM506-1, to be used for targeted insertion of the synthetic SP1 operon into the P. furiosus ΔpdaD strain (Fig. S1).…”

“…It cannot use carbon dioxide as its sole carbon source. A genetic system is available for P. furiosus based on a competent strain with a known sequence (13) that has allowed both homologous (14,15) and heterologous (9) overexpression of genes. A novel means of metabolic control was recently reported in P. furiosus that exploited the difference in the temperature dependence of the host's metabolism and the inserted foreign synthetic pathway (9).…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…The reduced ferredoxin can then serve as an additional energy source via membrane-bound hydrogenase and ATP synthase (42,456,(469)(470)(471)(472)(473). Acetyl-CoA is converted to acetate in one step involving acetyl-CoA synthetase (ADP forming) coupling the reaction with the formation of ATP from ADP and P i via substrate-level phosphorylation.…”

Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome

“…A stable replicating shuttle vector is also available (18). In addition, recombinant forms that contain inserted DNA have been generated, such as highly active promoters or forms encoding affinity tags for rapid protein purification (7,24). For example, an affinity-tagged, catalytically active, H 2 gas-producing subcomplex of P. furiosus hydrogenase was produced at approximately 100 times the level of the native hydrogenase (24).…”

“…In addition, recombinant forms that contain inserted DNA have been generated, such as highly active promoters or forms encoding affinity tags for rapid protein purification (7,24). For example, an affinity-tagged, catalytically active, H 2 gas-producing subcomplex of P. furiosus hydrogenase was produced at approximately 100 times the level of the native hydrogenase (24). A more robust genetic method that allows for selection on complex growth media for agmatine auxotrophy has also been developed, involving a thermostable compound required for polyamine biosynthesis (50).…”