Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

Abstract

Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

Natural Competence in the Hyperthermophilic ArchaeonPyrococcus furiosusFacilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases

“…Studying the pathways of hydrogen production in P. furiosus is of great interest because of the increasing concern for production of alternative energy sources, such as hydrogen, to replace fossil fuels. Hyperthermophiles such as P. furiosus could potentially have an important role in biological hydrogen production systems (15,42), and although the cytoplasmic hydrogenases have been characterized in vitro (21,22), as demonstrated herein, their in vivo functions have yet to be established. We anticipate that the ability to investigate their functions in vivo through genetic manipulation, together with the roles of related enzymes, will provide insight into the mechanisms of hydrogen uptake and production in P. furiosus as well as in related hyperthermophiles.…”

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli

“…It is not clear whether the hypCABDFE genes are essential. Recently, an active [NiFe] hydrogenase from P. furiosus was expressed in E. coli when only the structural genes and the protease were expressed (Sun et al, 2010). Because these experiments were performed under anaerobic conditions, it is likely that accessory proteins from E. coli assisted the maturation of the heterologous hydrogenase.…”

“…For example, expression of the Thiocapsa roseopersicina hynSL genes in E. coli failed to yield an active hydrogenase (Shirshikova et al, 2009). Recently, however, an active NADP-dependent [NiFe] hydrogenase from Pyrococcus furiosus was expressed in E. coli and required expression of only an additional protease for activity (Sun et al, 2010).…”

Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

“…4-Hydroxybutyrate-CoA ligase (Msed_0406) was cloned into pET46-Ek/LIC with an N-terminal His 6 tag as described previously (9). 4-Hydroxybutyryl-CoA dehydratase (Msed_1321) was cloned with an N-terminal His 6 tag into a modified pETA vector into which the anaerobic hya promoter from Escherichia coli had been inserted to allow for anaerobically regulated expression (10). Crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399) was cloned into pET21b without a His tag, and ␤-ketothiolase (Msed_0656) was cloned into pCDF-Ek/LIC with an N-terminal His 6 tag.…”