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

Chandrayan, Sanjeev K.; McTernan, Patrick M.; Hopkins, Robin; Sun, Junsong; Jenney, Francis E.; Adams, Michael W. W.

doi:10.1074/jbc.m111.290916

Cited by 50 publications

(41 citation statements)

References 40 publications

(56 reference statements)

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“…Hydrogen is used in P. furiosus by the native soluble hydrogenase I (SHI) that reduces NADP to NADPH (21). SHI is extremely active, even at 70°C, and a P. furiosus strain engineered to overexpress the enzyme has been previously developed (14).…”

Section: Resultsmentioning

confidence: 99%

“…The five genes encoding the three enzymes (E1αβγ, E2, E3) of M. sedula SP1 were combined into a single synthetic operon with transcription driven by a P. furiosus S-layer promoter (P slp ), a native, constitutive promoter of the highly expressed S-layer protein (encoded by locus PF1399) of P. furiosus (14). The M. sedula ribosomal binding sites (RBS) for E1(γ), E2, and E3 were replaced with RBSs for known highly expressed P. furiosus proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It cannot use carbon dioxide as its sole carbon source. A genetic system is available for P. furiosus based on a competent strain with a known sequence (13) that has allowed both homologous (14,15) and heterologous (9) overexpression of genes. A novel means of metabolic control was recently reported in P. furiosus that exploited the difference in the temperature dependence of the host's metabolism and the inserted foreign synthetic pathway (9).…”

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confidence: 99%

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Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

Keller

Schut

Lipscomb

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

116

103

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Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

Keller

Schut

Lipscomb

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

116

103

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“…A stable replicating shuttle vector is also available (18). In addition, recombinant forms that contain inserted DNA have been generated, such as highly active promoters or forms encoding affinity tags for rapid protein purification (7,24). For example, an affinity-tagged, catalytically active, H 2 gas-producing subcomplex of P. furiosus hydrogenase was produced at approximately 100 times the level of the native hydrogenase (24).…”

mentioning

confidence: 99%

“…A more robust genetic method that allows for selection on complex growth media for agmatine auxotrophy has also been developed, involving a thermostable compound required for polyamine biosynthesis (50). This was used to produce an affinity-tagged (Strep) version of the native form of the cytoplasmic hydrogenase of P. furiosus (7).…”

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Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome

et al. 2012

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The model archaeon Pyrococcus furiosus grows optimally near 100°C on carbohydrates and peptides. Its genome sequence (NCBI) was determined 12 years ago. A genetically tractable strain, COM1, was very recently reported, and here we describe its genome sequence. Of 1,909,827 bp in size, it is 1,571 bp longer (0.1%) than the reference NCBI sequence. The COM1 genome contains numerous chromosomal rearrangements, deletions, and single base changes. COM1 also has 45 full or partial insertion sequences (ISs) compared to 35 in the reference NCBI strain, and these have resulted in the direct deletion or insertional inactivation of 13 genes. Another seven genes were affected by chromosomal deletions and are predicted to be nonfunctional. In addition, the amino acid sequences of another 102 of the 2,134 predicted gene products are different in COM1. These changes potentially impact various cellular functions, including carbohydrate, peptide, and nucleotide metabolism; DNA repair; CRISPRassociated defense; transcriptional regulation; membrane transport; and growth at 72°C. For example, the IS-mediated inactivation of riboflavin synthase in COM1 resulted in a riboflavin requirement for growth. Nevertheless, COM1 grew on cellobiose, malto-oligosaccharides, and peptides in complex and minimal media at 98 and 72°C to the same extent as did both its parent strain and a new culture collection strain (DSMZ 3638). This was in spite of COM1 lacking several metabolic enzymes, including nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase and beta-glucosidase. The P. furiosus genome is therefore of high plasticity, and the availability of the COM1 sequence will be critical for the future studies of this model hyperthermophile.

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Extreme Thermophiles as Metabolic Engineering Platforms: Strategies and Current Perspective

Loder

Zeldes

Conway

et al. 2016

Industrial Biotechnology

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

Cited by 50 publications

References 40 publications

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome

Extreme Thermophiles as Metabolic Engineering Platforms: Strategies and Current Perspective

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