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Characterization and overexpression of a [FeFe]-hydrogenasegene of a novel hydrogen-producing bacterium Ethanoligenensharbinense

Xin Zhao, Defeng Xing*, Lu Zhang, Nanqi Ren*

State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of

Technology, P.O. Box 2614, 73 Huanghe Road, Nangang District, Heilongjiang Province, Harbin 150090, PR China

a r t i c l e i n f o

Article history:

Received 1 April 2010

Received in revised form

26 June 2010

Accepted 26 June 2010

Available online 3 August 2010

Keywords:

Biohydrogen

Hydrogen-producing bacterium

Ethanoligenens harbinense

[FeFe]-hydrogenase

Overexpression

* Corresponding authors. Tel./fax: þ86 451 86E-mail addresses: ixdf@yahoo.com.cn (D.

0360-3199/$ e see front matter ª 2010 Profedoi:10.1016/j.ijhydene.2010.06.098

a b s t r a c t

Ethanoligenens, a novel ethanologenic and hydrogen-producing genus, has capability of

hydrogen production at low pH. A [FeFe]-hydrogenase gene with [4Fe-4S] and [2Fe-2S]

clusters from Ethanoligenens harbinense YUAN-3 was cloned and overexpressed in a non-

hydrogen-producing Escherichia coli BL-21. This hydA gene consisted of an open reading

frame of 1743 bp encoding 580 amino acids with an estimated molecular weight of

63 188.1 Da. Six characteristic sequence signatures were present within the H-cluster

domain of [FeFe]-H2ases, and three of them were described previously. The overexpressed

and purified hydrogenases from recombinant cells showed catalytic activity in vitro and

in vivo.

ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1. Introduction understanding physiology and ecology of hydrogen-

Renewable hydrogen is considered a promising energy

carrier of the future since it is clean, carbon neutral and

efficient [1,2]. Biological hydrogen production processes offer

a technique through which the cleanest energy carrier can be

generated from renewable energy sources like biomass or

organic wastewater [3]. In order to improve these processes,

modification of reactor configuration, optimization of reactor

operations, development of two-stage processes, immobili-

zation of whole cells, isolation of hydrogen-producing

bacteria, selection of cheaper raw materials and metabolic

engineering have already been carried out [3,4]. However,

282008.Xing), rnq@hit.edu.cn (Nssor T. Nejat Veziroglu. P

producing bacteria is still important for enhancing H2 yield

and rate of H2 production.

The conventional wisdom is that fermentative hydrogen-

producing bacteria (HPB) are restricted to a few genera, such

as Clostridium, Enterobacter [5,6], which lose the ability to

produce H2 at pH below 5 [7]. However, our previous studies

found significant H2 production by anaerobic sludges via

ethanol-type fermentation in continuous stirred tank reactors

(CSTRs) at pH 4.0e4.5 [8,9]. It was fund that a novel genus

Ethanoligenens as dominant functional population was the

reason of ethanol-type fermentation based on isolation,

alcohol dehydrogenase and genomic evidences [9,10]. Its end

. Ren).ublished by Elsevier Ltd. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 5 ( 2 0 1 0 ) 9 5 9 8e9 6 0 2 9599

products from glucose fermentation were mainly composing

of acetate, ethanol, H2 and CO2, which were different from

clostridial butyrate fermentation [11]. Type strain YUAN-3 of

Ethanoligenens harbinense grow at pH 3.5e9.0, and form

autoaggregating granules at 0.5e5.0 mm (sometimes up to

1.5 cm) in 3- to 4-day-old shake cultures [11,12].

Biological formation and consumption of molecular

hydrogen (H2) are catalyzed by hydrogenases (H2ases), of

which three types are known: [NiFe]-H2ases, [FeFe]-H2ases

and [Fe]-H2ases [13]. [FeFe]-hydrogenases are generally

associated with the formation of H2, which are found in

anaerobic bacteria such as Clostridia, Thermotoga, and Desul-

fovibrio [14]. Hydrogenase gene from different microorgan-

isms was characterized in the last two decades [4,15e20], and

genetic modifications of hydrogenases were performed for

enhancing H2 production [20e23]. Characterizing [FeFe]-

hydrogenase gene of E. harbinense YUAN-3 will provide

insight into H2 metabolism at low pH of ethanol-H2 cop-

roducing-bacterium.

In this study, [FeFe]-hydrogenase gene from E. harbinense

YUAN-3 was isolated and characterized. The open reading

frame (ORF, DNAsequence encoding a protein) of hydA was

overexpressed in non-hydrogen-producing bacterium strain

Escherichia coli BL-21. The overexpressed and purified hydrog-

enase was assayed in vitro and in vivo.

2. Materials and methods

2.1. Bacterial strains and plasmids

E. harbinense YUAN-3, which maintained in our laboratory

[11], was precultured in an anaerobic culture tube

(18 mm � 150 mm) at 35 �C in modified EH medium [12].

E. coli DH5a was used as host for the propagation of

recombinant plasmid and E. coli BL-21 for recombinant

protein production. Plasmid pET28a (Novagen, Madison, WI)

was used to overexpress the hydA gene. pMD 19-T (TaKaRa,

Dalian China) was used as the cloning and sequencing

vector.

2.2. Cloning of hydA gene from E. harbinense YUAN-3and sequence analysis

A 1269-bp fragment of hydrogenase partial gene from E. harbi-

nense YUAN-3 was cloned by Xing [12]. On the base

of this fragment, 2 pairs of cassette primers (CFPS1: 50-TCCATTTCCGTCATGCCGTGCCTGGCTAAA-30 and CFPS2: 50-AAAGCTATTATGCCAAGCTGTTGGATGTGGA-30 for upstream;

CRPS1: 50-GCTTCCTTCCAGCCGTCCATTCCCCGCAC-30 and

CRPS2: 50-GATCTCCACGAAATCGTACTGCACATCGCCTTT-30 fordownstream) were designed and TaKaRa LA PCR� in vitro

Cloning Kit (TaKaRa, Dalian, China) was used for amplifying the

whole hydA ORF.

Computer-assisted sequence analysis was carried out

using expert protein analysis system (ExPASy) proteomics

server (http://expasy.org) [24]. Multiple alignments of amino

acid sequences were performed by ClustalW (http://www.

clustal.org). Secondary structure was predicted from the

amino acid sequences by the PSIPRED protein structure

prediction server (http://www.psipred.net/psiform.html) [25].

3D structure was predicted using amino acid sequence by

ESyPred3D server (http://www.fundp.ac.be/sciences/biologie/

urbm/bioinfo/esypred).

2.3. Sub-cloning, expression and hydrogenase assay

For expressing the hydA, a pair of primers were design-

ed, hydA-HEF (50-CGGGAGGTCTGACATATGGTAAACGTGA-30

forward primer) and hydA-HER (50-GGTGTGTCCGTTTTGGAATTCTTTTTTCGCGG-30 reverse primer) containing Nde

I (in the forward primer) and EcoR I (in the reverse

primer) restriction enzyme cutting sites (underlined). PCR

amplification was performed as the following program: initial

denaturation at 95 �C for 5 min, 35 cycles of denaturation at

95 �C for 35 s, annealing at 68 �C for 35 s, decreasing 0.1 �C per

cycle, extension at 72 �C for 90 s, and the final extension for

7 min. The products and pET28a were digested with Nde I and

EcoR I restriction enzyme, purified and ligated. The ligated

product transformed into E. coli BL-21 and induced to express

at 1 mM IPTG. Induced cells lysed by an ultrasonicator (VCX-

130, SNOICS, USA) and the protein collected. Sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE),

western blotting and two-dimensional gel electrophoresis

(2-DE) were used for confirming the recombinant plasmid can

translate right targeted-protein (Materials and Methods in

Supplementary materials).

Hydrogenase activity of recombinant hydA protein (crude

enzyme overexpressed in E. coli BL-21, hydA-HIS and purified

HIS cleaved) was assayed using reduced methyl viologen as

the substrate [18,26]. E. coli BL-21 containing the recombinant

plasmid was grown in batch system anaerobic adaptation,

and the gas phase was analyzed after 24 h cultured (Materials

and Methods in Supplementary materials).

3. Results and discussion

The ORF consists of 1743 bp encoding 580 amino acids, starts

with ATG codon and ends with TAA. Computer-assisted

sequence analysis indicated that the ORF encodes a protein

with molecular weight of 63 188 Da, and theoretical pI is

5.3. Secondary structure prediction showed the FeFe-

hydrogenase has 256 H-bonds, 21 helices, 14 strands and 41

turns (Fig. S1 in Supplementary materials). The sequence has

been deposited in the GenBank database under the accession

number DQ177326. The alignment of the predicted amino

acid sequence of hydA between E. harbinense YUAN-3 and

relative genera shows the presence of highly conserved

regions (Fig. 1). Six characteristic sequence signatures

within the H-cluster domain of [FeFe]-H2ases were found (in

the frames, Fig. 1), and three of them were described previ-

ously [16].

The results of SDS-PAGE, western blotting (Fig. 2) and 2-DE

indicated the recombinant plasmid can translate right tar-

geted-protein (Fig. S2 in Supplementary materials). The in

vitro assay result showed that the activity of HIS tagged hydA

encoding overexpressed protein is about 13 times more than

the crude enzyme activity, but 1.4 times less active compared

with hydA encoding protein purified HIS cleaved (Fig. 3). One

Fig. 1 e Multiple alignments of amino acid sequences of hydA from Ethanoligenens harbinense YUAN-3 and its related

species. Amino acids in dashed frame are conserved domains.

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enzyme unit of hydrogenase was defined as the amount

of enzyme required for the reduction of micromole MV

per minute [17]. The specific hydrogenase activity was

defined as micromole of H2 production per minute per

microgram of protein. The overexpressed hydrogenase of

E. harbinense YUAN-3 has H2 evolution ability about 7.9 ml

H2/mg protein in 5 min. In vivo hydrogenase assay result

showed recombinant hydA gene is necessary and sufficient

for molecular hydrogen production in non-hydrogen-

producing E. coli BL-21.

It was found for the first time that E. harbinense is

a mesophilic, non-spore-forming, ethanologenic and

hydrogen-producing bacterium, which substantially differs

from Clostridium with butyrate fermentation [11]. There are

Fig. 2 e SDS-PAGE and Western blot analyses of

overexpressed hydrogenase from the E. harbinense

YUAN-3 in E. coli BL-21. Lane 1, purified hydA-HIS from

inclusion-body protein extracted; lane 2 and 4, blank;

lane 3, Precision Protein Standards; lane 5, protein

extracted from induced cells harboring pET28a/hydA;

lane 6, protein extracted from uninduced cells; lane 7,

Western blot analysis with anti-HIS monoclonal

antibody.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 5 ( 2 0 1 0 ) 9 5 9 8e9 6 0 2 9601

obvious differences in ecological niche and physiology

between Ethanoligenens and Clostridium, and Ethanoligenens has

advantage in hydrogen production at low pH over Clostridium

[9e11,27]. Transcriptome and proteome analyses are needed

for further understanding themetabolic pathway related with

hydrogen production of this bacterium. Ethanoligenens, a novel

genus, has potential application in the practice as it’s acido-

philic and autoaggregating growth in the continuous-flow

reactors at low pH [9,10], andwill become anothermode strain

for energy production in the future.

Fig. 3 e In vitro hydrogenase assay of crude enzyme, hydA-

HIS and purified hydA.

4. Conclusions

A [FeFe]-hydrogenase gene (hydA) was cloned from a novel

hydrogen-producing bacterium E. harbinense YUAN-3. The

multiple alignments of predicted amino acid sequence

with other [FeFe]-hydrogenase shows the presence of

highly conserved regions. Six characteristic sequence

signatures were present within the H-cluster domain of

[FeFe]-H2ases. In vitro enzyme assay with the overexpressed

hydrogenase showed that it is catalytically active upon

anaerobic adaptation. In vivo hydrogenase assay confirmed

the presence of H2 gas in the gas mixture obtained from the

batch culture of recombinant E. coli BL-21 under anaerobic

condition.

Acknowledgements

This research was supported by National Nature Science

Foundation of China (Nos. 30870037, 30900046 and 50638020),

the Program for New Century Excellent Talents in University

(No. NCET-10-0066), and the Foundation for Innovative

Research Groups of the National Natural Science Foundation

of China (No. 50821002).

Appendix. Supplementary material

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.ijhydene.2010.06.098.

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