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.

FIGURE 1.

Model of affinity-tagged SHI showing subunit and cofactor content. The Strep-tag II is located at the N terminus of PF0891. Adapted from Ref. .

FIGURE 2.

Marked knock-in strategy to modify operon (PF0891–0894) encoding SHI. A schematic representation of the knock-in cassette is presented. The abbreviations are: UFR, upstream flanking region; Pgdh-pyrF, marker driven by the promoter for the glutamate dehydrogenase gene; Pslp-strep-tagII, S-Layer promoter with codon-optimized 8-amino acid Strep-tag II sequence; DFR, downstream flanking region.

FIGURE 3.

Increased catalytic activity and amount of catalytic subunit of SHI in OE-SHI strain. The bar graph compares the MV-linked hydrogenase activity in cytoplasmic extracts (S100) of the parent COM1 and OE-SHI strains. The error bars represent standard deviations obtained from three independent experiments. The corresponding immunoanalysis of the extracts is shown below using anti-PF0894 (catalytic subunit, see Fig. 1) with anti-PF1281 (superoxide reductase) as the internal loading control.

FIGURE 4.

Relative mRNA abundance in OE-SHI and COM1 strains. The relative levels were determined by qPCR of the mRNA encoding PF0894 (the catalytic subunit of SHI), PF0975 (frxA), and PF0559 (hypF). The error bars represent standard deviations obtained using triplicate independent samples.

FIGURE 5.

Comparison of growth and H₂ production and maltose consumption by OE-SHI and COM1 strains. A, growth of the two strains using maltose as the carbon source and consumption of maltose during growth in closed bottles at 95 °C. B, corresponding production of H₂ during growth. The error bars represent standard deviation obtained from three independent samples.

FIGURE 6.

Electrophoretic analysis of OE-SHI hydrogenase. The purified enzyme was analyzed by conventional SDS-PAGE except that the protein was incubated with the SDS-loading buffer for 10 min (lane 1) or for 60 min (lane 3) prior to electrophoresis. Native SHI (treated for 10 min) is shown in lane 2. The arrow indicates the high molecular weight catalytically active protein band seen in lanes 1 and 2 (see Ref. 25). The center lane (M) contains the protein molecular weight ladder (Invitrogen) with corresponding masses as indicated in kDa.