title: Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.
abstract: The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Chandrayan
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McTernan
authors.forename: R Christopher
authors.initials: RC
authors.lastname: Hopkins
authors.forename: Junsong
authors.initials: J
authors.lastname: Sun
authors.forename: Francis E
authors.initials: FE
authors.lastname: Jenney
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
mesh_terms: D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000235:genetics; D056804:Biofuels; D020134:Catalytic Domain / Q000502:physiology; D003593:Cytoplasm / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000096:biosynthesis* / Q000235:genetics; D008320:Maltose / Q000378:metabolism / Q000494:pharmacology; D060847:Metabolic Engineering; D009876:Operon / Q000502:physiology; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013549:Sweetening Agents / Q000378:metabolism / Q000494:pharmacology
chemical_list: D019843:Archaeal Proteins; D056804:Biofuels; D013549:Sweetening Agents; D008320:Maltose; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase