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Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

Sun J, Hopkins RC, Jenney FE, McTernan PM, Adams MW - PLoS ONE (2010)

Bottom Line: Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2).The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus.The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

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Related in: MedlinePlus

Analysis of E. coli promoters with a lacZ gene fragment β-galactosidase reporter assay.Plasmids were transformed into DH10B. The specific activity of lacZ was measured by a modified Miller [30] assay calculated as 200(OD420-t1-OD420-t2) min−1mg−1, where t1 and t2 are the start and end time points, respectively.
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pone-0010526-g002: Analysis of E. coli promoters with a lacZ gene fragment β-galactosidase reporter assay.Plasmids were transformed into DH10B. The specific activity of lacZ was measured by a modified Miller [30] assay calculated as 200(OD420-t1-OD420-t2) min−1mg−1, where t1 and t2 are the start and end time points, respectively.

Mentions: A series of four compatible vectors based on the DUET vector system (Novagen) had been modified previously to contain GATEWAY™ recombination sites (Invitrogen) [25], which allows coexpression of as many as eight separate genes or operons under the control of T7 promoters. This cloning approach also inserts 19 amino acids (GSITSLYKKAGSENYFQGG, ∼2.0 kDa) at the N-terminus of the first gene of the operon in each plasmid. Since the standard T7 promoter does not function in E. coli under anaerobic conditions, four different promoters were investigated for anaerobic heterologous expression of the Pf genes in E. coli. Three were native E. coli hydrogenase promoters that are anaerobically-induced (hya, hyb, and hyc, [26]-[28]), while the fourth (hyp, [29]) induces expression of E. coli hydrogenase-processing genes. Expression of the lacZ gene by these four promoters (Fig. 2) and several of the Pf hyd genes, including hypCDAB and hycI, were examined using RT-PCR (data not shown). The hya promoter (Phya) was induced by anaerobiosis and gave the highest level of expression. The T7 promoters on all expression vectors were therefore replaced with Phya (Fig. 3). One vector (pDEST-C3A) was further modified to include the tRNA genes from plasmid pRIL (Stratagene), which is typically required for efficient expression of Pf genes in E. coli due to differing codon usage, creating the plasmid pC3A-RIL. The complete list of expression vectors used in this study is given in Table 1.


Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

Sun J, Hopkins RC, Jenney FE, McTernan PM, Adams MW - PLoS ONE (2010)

Analysis of E. coli promoters with a lacZ gene fragment β-galactosidase reporter assay.Plasmids were transformed into DH10B. The specific activity of lacZ was measured by a modified Miller [30] assay calculated as 200(OD420-t1-OD420-t2) min−1mg−1, where t1 and t2 are the start and end time points, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865534&req=5

pone-0010526-g002: Analysis of E. coli promoters with a lacZ gene fragment β-galactosidase reporter assay.Plasmids were transformed into DH10B. The specific activity of lacZ was measured by a modified Miller [30] assay calculated as 200(OD420-t1-OD420-t2) min−1mg−1, where t1 and t2 are the start and end time points, respectively.
Mentions: A series of four compatible vectors based on the DUET vector system (Novagen) had been modified previously to contain GATEWAY™ recombination sites (Invitrogen) [25], which allows coexpression of as many as eight separate genes or operons under the control of T7 promoters. This cloning approach also inserts 19 amino acids (GSITSLYKKAGSENYFQGG, ∼2.0 kDa) at the N-terminus of the first gene of the operon in each plasmid. Since the standard T7 promoter does not function in E. coli under anaerobic conditions, four different promoters were investigated for anaerobic heterologous expression of the Pf genes in E. coli. Three were native E. coli hydrogenase promoters that are anaerobically-induced (hya, hyb, and hyc, [26]-[28]), while the fourth (hyp, [29]) induces expression of E. coli hydrogenase-processing genes. Expression of the lacZ gene by these four promoters (Fig. 2) and several of the Pf hyd genes, including hypCDAB and hycI, were examined using RT-PCR (data not shown). The hya promoter (Phya) was induced by anaerobiosis and gave the highest level of expression. The T7 promoters on all expression vectors were therefore replaced with Phya (Fig. 3). One vector (pDEST-C3A) was further modified to include the tRNA genes from plasmid pRIL (Stratagene), which is typically required for efficient expression of Pf genes in E. coli due to differing codon usage, creating the plasmid pC3A-RIL. The complete list of expression vectors used in this study is given in Table 1.

Bottom Line: Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2).The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus.The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

Show MeSH
Related in: MedlinePlus