1. Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.
- Author
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Chandrayan SK, McTernan PM, Hopkins RC, Sun J, Jenney FE Jr, and Adams MW
- Subjects
- Archaeal Proteins genetics, Biofuels, Catalytic Domain physiology, Cytoplasm genetics, Gene Expression, Hydrogenase genetics, Maltose metabolism, Maltose pharmacology, Operon physiology, Pyrococcus furiosus genetics, Pyrococcus furiosus growth & development, Sweetening Agents metabolism, Sweetening Agents pharmacology, Archaeal Proteins biosynthesis, Cytoplasm enzymology, Hydrogen metabolism, Hydrogenase biosynthesis, Metabolic Engineering, Pyrococcus furiosus enzymology
- 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.
- Published
- 2012
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