Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase

Sanjeev K Chandrayan,Patrick M Mcternan,R Christopher Hopkins,Junsong Sun,Francis E Jenney,Michael W.W Adams

doi:10.1074/jbc.m111.290916

Sanjeev K Chandrayan, Patrick M Mcternan + Show 4 more

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https://doi.org/10.1074/jbc.m111.290916

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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.

Highlights

Hydrogenases are complex metalloenzymes catalyzing the evolution of hydrogen gas but lacking an efficient system to produce recombinant forms
The Streptag II was chosen for affinity purification as its function is not affected by the chemical reductant, sodium dithionite, which is used to maintain anaerobic conditions during hydrogenase purification
We focused on the hydrogenase protease frxA (PF0975), which cleaves the C terminus of the catalytic subunit (PF0894), and hypF (PF0559), which is involved is the assembly of the diatomic cyanide and carbon monoxide ligands on the iron atom of the [NiFe] catalytic site [17, 19, 23]

Summary

Introduction

Hydrogenases are complex metalloenzymes catalyzing the evolution of hydrogen gas but lacking an efficient system to produce recombinant forms. 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. 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

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