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High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.

Kuchenreuther JM, Grady-Smith CS, Bingham AS, George SJ, Cramer SP, Swartz JR - PLoS ONE (2010)

Bottom Line: Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation.Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified.These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, USA.

ABSTRACT

Background: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.

Principal findings: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms.

Significance: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

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E. coli growth and anaerobic expression of heterologous active [FeFe] hydrogenases.All data are for cultures of E. coli strain BL21(DE3) ΔiscR, and both iron and cysteine were included in the growth medium. (Fig. 1A) Optical density at 600 nm (shown on a logarithmic scale) of cultures during aerobic (orange circles) and anaerobic (blue circles) growth phases for cells containing the pACYCDuet-1–hydGX–hydEF and pET-21(b) shydA1*–Strep-tag II plasmids. The pH of culture media (×) was also measured. Data for cultures with cells containing the pET-21(b) shydA–Strep-tag II plasmid instead of pET-21(b) shydA1*–Strep-tag II were similar and are not shown. (Fig. 1B) Cell lysate-based hydrogenase activities (µmol MV reduced·min−1·mg−1 total protein) for active CpI (red squares) and HydA1 hydrogenase (green triangles) were determined using the methyl viologen reduction assay. Data are the average for n = 3 cultures examined ± standard deviations. (Fig. 1C) SDS-PAGE analysis for the soluble fractions of final cell lysates after the anoxic co-expression of HydA1 or CpI and the HydE, HydF, and HydG maturases: (Lane 1) the molecular weight markers are from the Mark12TM protein ladder (Invitrogen); (Lane 2) soluble cell lysate protein content for E. coli strain BL21(DE3) ΔiscR following expression of no heterologous proteins from recombinant DNA plasmids; (Lane 3) co-expression of only the HydE, HydF, and HydG maturases; (Lane 4) co-expression of HydE, HydF, HydG, and HydA1–Strep-tag II; and (Lane 5) co-expression of HydE, HydF, HydG, and CpI–Strep-tag II.
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pone-0015491-g001: E. coli growth and anaerobic expression of heterologous active [FeFe] hydrogenases.All data are for cultures of E. coli strain BL21(DE3) ΔiscR, and both iron and cysteine were included in the growth medium. (Fig. 1A) Optical density at 600 nm (shown on a logarithmic scale) of cultures during aerobic (orange circles) and anaerobic (blue circles) growth phases for cells containing the pACYCDuet-1–hydGX–hydEF and pET-21(b) shydA1*–Strep-tag II plasmids. The pH of culture media (×) was also measured. Data for cultures with cells containing the pET-21(b) shydA–Strep-tag II plasmid instead of pET-21(b) shydA1*–Strep-tag II were similar and are not shown. (Fig. 1B) Cell lysate-based hydrogenase activities (µmol MV reduced·min−1·mg−1 total protein) for active CpI (red squares) and HydA1 hydrogenase (green triangles) were determined using the methyl viologen reduction assay. Data are the average for n = 3 cultures examined ± standard deviations. (Fig. 1C) SDS-PAGE analysis for the soluble fractions of final cell lysates after the anoxic co-expression of HydA1 or CpI and the HydE, HydF, and HydG maturases: (Lane 1) the molecular weight markers are from the Mark12TM protein ladder (Invitrogen); (Lane 2) soluble cell lysate protein content for E. coli strain BL21(DE3) ΔiscR following expression of no heterologous proteins from recombinant DNA plasmids; (Lane 3) co-expression of only the HydE, HydF, and HydG maturases; (Lane 4) co-expression of HydE, HydF, HydG, and HydA1–Strep-tag II; and (Lane 5) co-expression of HydE, HydF, HydG, and CpI–Strep-tag II.

Mentions: Co-expression of the [FeFe] hydrogenases and the maturases was induced under strictly anoxic conditions at an optical density (OD600) of ∼0.4. To facilitate anaerobic metabolism, glucose (0.5% w/v) and the electron acceptor fumarate (25 mM) were added to the complex growth medium. Aerobic growth rates were exponential (0.45 hr−1), while anaerobic growth rates were linear and eventually ceased after 24 hr at final OD600 measurements ranging from 1.5 to 3.0 (Fig. 1A). Substrate limitations and acetate accumulation may have contributed to the slowed anoxic growth. Without glucose addition, the culture density did not increase during the anaerobic incubation period. This lack of growth resulted in a lower cell concentration, which thus decreased the total amount of hydrogenase produced per culture volume.


High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.

Kuchenreuther JM, Grady-Smith CS, Bingham AS, George SJ, Cramer SP, Swartz JR - PLoS ONE (2010)

E. coli growth and anaerobic expression of heterologous active [FeFe] hydrogenases.All data are for cultures of E. coli strain BL21(DE3) ΔiscR, and both iron and cysteine were included in the growth medium. (Fig. 1A) Optical density at 600 nm (shown on a logarithmic scale) of cultures during aerobic (orange circles) and anaerobic (blue circles) growth phases for cells containing the pACYCDuet-1–hydGX–hydEF and pET-21(b) shydA1*–Strep-tag II plasmids. The pH of culture media (×) was also measured. Data for cultures with cells containing the pET-21(b) shydA–Strep-tag II plasmid instead of pET-21(b) shydA1*–Strep-tag II were similar and are not shown. (Fig. 1B) Cell lysate-based hydrogenase activities (µmol MV reduced·min−1·mg−1 total protein) for active CpI (red squares) and HydA1 hydrogenase (green triangles) were determined using the methyl viologen reduction assay. Data are the average for n = 3 cultures examined ± standard deviations. (Fig. 1C) SDS-PAGE analysis for the soluble fractions of final cell lysates after the anoxic co-expression of HydA1 or CpI and the HydE, HydF, and HydG maturases: (Lane 1) the molecular weight markers are from the Mark12TM protein ladder (Invitrogen); (Lane 2) soluble cell lysate protein content for E. coli strain BL21(DE3) ΔiscR following expression of no heterologous proteins from recombinant DNA plasmids; (Lane 3) co-expression of only the HydE, HydF, and HydG maturases; (Lane 4) co-expression of HydE, HydF, HydG, and HydA1–Strep-tag II; and (Lane 5) co-expression of HydE, HydF, HydG, and CpI–Strep-tag II.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991362&req=5

pone-0015491-g001: E. coli growth and anaerobic expression of heterologous active [FeFe] hydrogenases.All data are for cultures of E. coli strain BL21(DE3) ΔiscR, and both iron and cysteine were included in the growth medium. (Fig. 1A) Optical density at 600 nm (shown on a logarithmic scale) of cultures during aerobic (orange circles) and anaerobic (blue circles) growth phases for cells containing the pACYCDuet-1–hydGX–hydEF and pET-21(b) shydA1*–Strep-tag II plasmids. The pH of culture media (×) was also measured. Data for cultures with cells containing the pET-21(b) shydA–Strep-tag II plasmid instead of pET-21(b) shydA1*–Strep-tag II were similar and are not shown. (Fig. 1B) Cell lysate-based hydrogenase activities (µmol MV reduced·min−1·mg−1 total protein) for active CpI (red squares) and HydA1 hydrogenase (green triangles) were determined using the methyl viologen reduction assay. Data are the average for n = 3 cultures examined ± standard deviations. (Fig. 1C) SDS-PAGE analysis for the soluble fractions of final cell lysates after the anoxic co-expression of HydA1 or CpI and the HydE, HydF, and HydG maturases: (Lane 1) the molecular weight markers are from the Mark12TM protein ladder (Invitrogen); (Lane 2) soluble cell lysate protein content for E. coli strain BL21(DE3) ΔiscR following expression of no heterologous proteins from recombinant DNA plasmids; (Lane 3) co-expression of only the HydE, HydF, and HydG maturases; (Lane 4) co-expression of HydE, HydF, HydG, and HydA1–Strep-tag II; and (Lane 5) co-expression of HydE, HydF, HydG, and CpI–Strep-tag II.
Mentions: Co-expression of the [FeFe] hydrogenases and the maturases was induced under strictly anoxic conditions at an optical density (OD600) of ∼0.4. To facilitate anaerobic metabolism, glucose (0.5% w/v) and the electron acceptor fumarate (25 mM) were added to the complex growth medium. Aerobic growth rates were exponential (0.45 hr−1), while anaerobic growth rates were linear and eventually ceased after 24 hr at final OD600 measurements ranging from 1.5 to 3.0 (Fig. 1A). Substrate limitations and acetate accumulation may have contributed to the slowed anoxic growth. Without glucose addition, the culture density did not increase during the anaerobic incubation period. This lack of growth resulted in a lower cell concentration, which thus decreased the total amount of hydrogenase produced per culture volume.

Bottom Line: Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation.Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified.These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, USA.

ABSTRACT

Background: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.

Principal findings: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms.

Significance: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

Show MeSH
Related in: MedlinePlus