FEMS Microbiology Letters 246 (2005) 229234

www.fems-microbiology.org

Overexpression of a hydrogenase gene in Clostridium

paraputricum to enhance hydrogen gas production
Kenji Morimoto a, Tetsuya Kimura b, Kazuo Sakka b,*
, Kunio Ohmiya c

a
Rare Sugar Research Center, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
b
Faculty of Bioresources, Mie University, Tsu 514-8507, Japan
c
Agricultural High-Tech Research Center, Meijo University, Tenpaku, Nagoya 468-8502, Japan

Received 28 January 2005; received in revised form 11 April 2005; accepted 12 April 2005

First published online 29 April 2005

Edited by R.P. Gunsalus

Abstract

A [Fe]-hydrogenase gene (hydA) was cloned from Clostridium paraputricum M-21 in Escherichia coli using a conserved DNA
sequence of clostridial hydrogenase genes amplied by PCR as the probe. The hydA gene consisted of an open reading frame of
1749 bp encoding 582 amino acids with an estimated molecular mass of 64,560 Da. It was ligated into a shuttle vector, pJIR751,
originally constructed for Clostridium perfringens and E. coli, and expressed in C. paraputricum. Hydrogen gas productivity of
the recombinant increased up to 1.7-fold compared with the wild-type. In the recombinant, overexpression of hydA abolished lactic
acid production and increased acetic acid production by over-oxidation of NADH, which is required for reduction of pyruvic acid to
lactic acid in the wild-type.
2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

Keywords: Clostridium paraputricum; Hydrogenase; Hydrogen gas production

1. Introduction tive and obligatory anaerobic bacteria have been re-

Since hydrogen gas is an idealistic clean energy mate-
rial that does not generate carbon dioxide gas after com-
bustion, its sustainable production from biomass is in
demand worldwide [1]. To compensate for environmen-
tal problems by reducing carbon dioxide gas generation
and overcome shortages of fossil energy in the future,
utilization of abundant biomasses such as chitin, a ma-
jor marine waste, is expected. Gaseous hydrogen is
widely produced by many microorganisms, but is virtu-
ally absent from higher organisms. Anaerobic microor-
ganisms are involved in hydrogen production,
especially photosynthetic microorganisms, and faculta-
*
Corresponding author. Tel.: +81 59 231 9621; fax: +81 59 231 9684.
E-mail address: sakka@bio.mie-u.ac.jp (K. Sakka).
ported to produce hydrogen gas from soluble and
insoluble biomass such as agricultural by-products and
marine wastes [111]. Some chitin-degrading bacteria
such as Aeromonas [12], Serratia [13], and Bacillus [14]
have been reported, although hydrogen gas productivity
has not been characterized in detail in these microorgan-
isms. Anaerobic bacteria generally have the ability to
produce hydrogen gas during catabolism of carbohy-
drates and [Fe]-hydrogenases (EC 1.12.7.2) are known
to release hydrogen gas from the reduced form of ferre-
doxin in Clostridium and Desulfovibrio species (Fig. 1)
[1518].
It is well known that many clostridial species evolve
hydrogen gas as a fermentation product during growth.
Information about hydrogenase genes and their
products has been reported from a few clostridia, for

0378-1097/$22.00 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
doi:10.1016/j.femsle.2005.04.014
230 K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229234
glucose Fd
2
H2
1 C. paraputricum was grown anaerobically at 45 C in
ATP NADH
pyruvate lactate
CO2
H2 Fd
acetyl-CoA
ATP
acetate butyrate
Fig. 1. Outline of biological hydrogen gas production from glucose by
fermentative microorganisms: 1, ferredoxinNAD+ reductase (EC
1.18.1.3); 2, ferredoxin hydrogenase (EC 1.12.7.2).
example, [Fe]-hydrogenase I of Clostridium pasteuria-
num [15,16], hydrogenase A of Clostridium perfringens
[17], and hydrogenase A of Clostridium acetobutylicum
P262 [18]. A number of clostridial species are also
known to degrade and ferment various biomass poly-
mers such as polysaccharides and proteins to obtain en-
ergy and reducing powers such as the proton/electron
and reduced compounds in cells. Since anaerobic bacte-
ria possess a mechanism to remove excessive reducing
powers as hydrogen gas using hydrogenase, it is possible
that they produce huge amounts of hydrogen gas during
growth on biomass materials.
Clostridium paraputricum M-21 was isolated and
characterized as a chitin-degrading hydrogen-producing
anaerobe in our laboratory [8,9]. Two chitinase and two
b-N-acetylglucosaminidase genes of this bacterium were
characterized along with their translated products [19
22]. In our preceding paper [23], we reported the con-
struction of the hostvector system of C. paraputricum
M-21, allowing us to improve its biomass-degrading and
hydrogen gas-producing abilities.
In the present study, we isolated the hydA gene
encoding a [Fe]-hydrogenase from C. paraputricum
M-21, which was isolated from soil as a chitin-degrading
hydrogen-producing anaerobe, and studied the eect of
hydA overexpression on the production of hydrogen gas.
As a result, we found that the recombinant clone over-
expressing the hydA gene produced 1.7 times as much
hydrogen gas as the parental clone, along with a drastic
reduction in lactic acid production.
2. Materials and methods
2.1. Bacterial strains, plasmids, and growth conditions
C. paraputricum M-21 was isolated and character-
ized as a chitin-degrading hydrogen-producing bacte-
rium, was described previously [8]. Plasmid pJIR751
was obtained from Dr. J.I. Rood (Monash University,
Australia) [24] and used to overexpress the hydA gene.
modied GS medium (pH 6.5) supplemented with 1%
N-acetylglucosamine (GlcNAc). Cultivations were con-
ducted in test tubes under static condition or in a 1-l
jar fermenter (B.E. Marubishi Lab., Tokyo) containing
500 ml of the medium with agitation at 250 rpm. Re-
combinant C. paraputricum was cultivated under the
same conditions but with erythromycin (10 lg/ml).
The following plasmids were used as the cloning and
sequencing vectors for Escherichia coli: pT7Blue (Nova-
gen, Madison, WI), pBluescript II KS (
) and pBlue-
script II KS (+) (Stratagene, La Jolla, CA), and
Charomid 9-28 (Nippon Gene, Tokyo). E. coli XL1-
Blue and DH5a were grown aerobically at 37 C in Lur-
iaBerrani (LB) medium supplemented with ampicillin
(100 lg/ml) and IPTG (50 lg/ml) when necessary.
2.2. Cloning of the C. paraputricum hydA gene
Chromosomal DNA of C. paraputricum M-21 was
isolated according to the procedure of Silhavy et al.
[25], partially digested with EcoRI, and separated on
0.4% Agarose H gel (Nippon Gene). DNA fragments
with appropriate sizes were recovered from the agarose
gel using the GeneClean (Bio101, La Jolla, CA) proce-
dure, and ligated into the EcoRI site of Charomid 9-
28. Ligation and transformation of E. coli DH5a were
carried out according to the protocol of the Charomid
cloning kit. A pair of PCR primers were designed
according to the sequences highly conserved in the
[Fe]-hydrogenase genes from Clostridium and Desulf-
ovibrio species to obtain a partial region of a hydroge-
nase gene from C. paraputricum M-21. The following
forward and reverse primers were used: 5 0 -
TTYGGNGCNGAYATGACNATHATGGARGA-3 0
and 5 0 -CANCCNCCNKGRCANGCCATNACYTC-
3 0 , respectively. A 700-bp PCR fragment amplied
from C. paraputricum chromosomal DNA was li-
gated into pT7Blue and introduced into E. coli XL1-
Blue. The cloned DNA fragment was then amplied
and labeled with digoxigenin-11 dUTP (Roche Diag-
nostics GmbH, Penzberg) by PCR with the primers de-
scribed above. The labeled DNA fragment was used as
a probe for colony hybridization to clone the full-
length [Fe]-hydrogenase gene (hydA) from the C.
paraputricum genome library.
2.3. DNA sequencing
Nucleotide sequencing was carried out on a LICOR
model 4000L automated DNA sequencer (Lincoln,
Neb.), with appropriate dye primers and a series of sub-
clones. Nucleotide sequence data was analyzed with
GENETYX computer software (Software Development
 K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229234
Co. Ltd., Tokyo, Japan). Homology searches in DDBJ
were carried out with the BLAST program.
2.4. Construction of pJIR751hyd
For overexpression of hydA in C. paraputricum M-
21, the hydA gene was subcloned into an E. coliC. per-
fringens shuttle vector, pJIR751, as follows: plasmid
pHYD101 containing the full-length hydA gene was di-
gested with XbaI and SpeI then a 2.3-kbp DNA frag-
ment containing hydA was ligated into pJIR751, which
had been digested with XbaI in advance, yielding
pJIR751hyd.
2.5. Electroporation of C. paraputrofocum M-21
C. paraputricum M-21 was transformed with
pJIR751hyd according to the electroporation proce-
dure described previously [23].
2.6. Analysis of hydrogen gas production
The total amount of gas produced by the wild-type
and recombinant strains from a 500-ml culture in a 1-l
jar fermenter was measured with a wet gas meter (W-
NK Da-0.5A, Shinagawa, Co., Tokyo) connected to
the jar fermenter by rubber tubing during cultivation.
The absorbance was measured at 600 nm for evaluating
bacterial cell growth with a double-beam spectropho-
tometer (UV-150-02, Shimadzu Co., Kyoto). The com-
position of the fermentation gas was analyzed using a
gas chromatography system (model GC-323 equipped
with Molecular Sieve 5A and Porapak Q columns and
a TCD detector; GL Sciences Inc., Tokyo). Separation
was carried out at 50 C using argon gas as the carrier
gas. Organic acids produced were analyzed using an
HPLC system, GL Sciences EZ Chrom Elite analyzer
equipped with a Shodex Rspak KC-811 column and a
GL Sciences UV 620 detector. Separation was con-
ducted at 40 C using 1 mM HClO4 as an eluent and
ST3-R as a regent at a ow rate of 1.0 ml/min.
2.7. Nucleotide sequence accession number
The nucleotide sequence reported in this paper is
available in the DDBJ, EMBL, and GenBank nucleotide
sequence databases under accession number AB159510.
3. Results
3.1. Cloning and sequencing of the hydA gene from
C. paraputricum M-21
Employing PCR and colony hybridization methods,
a 9.0-kbp DNA fragment expected to contain the full-
231
length hydA gene and its anking region was cloned
from C. paraputricum M-21 using Charomid 9-28.
Sequencing of the inserted DNA fragment identied
an open reading frame of 1749 bp, which encoded a no-
vel hydrogenase (HydA) of 582 amino acids with a pre-
dicted molecular mass of 64,560 Da. The predicted
molecular mass was in good agreement with that of
many clostridial [Fe]-hydrogenases [15,17,18]. A puta-
tive ribosomal-binding site (GGAGG) and
35 and

10 regions (TTGAAC and AAAAAT with a 18-bp
spacing) were located upstream of the hydA ATG start
codon. Transcription of the hydA gene was expected to
end in rho-factor dependence, because there was no
clear stem-loop structure downstream of the stop codon.
Comparisons of the amino acid sequence of HydA
with entries in the DDBJ database indicated that this en-
zyme is highly homologous with some clostridial [Fe]-
hydrogenases (EC 1.12.7.2) as expected; for example,
HydA of C. acetobutylicum P262 (sequence identity
75.1%) [18], HydI of C. pasteurianum W5 (69.4%) [15],
HydA of C. perfringens NTCT8237 (71.3%) [17], and
HydA of Clostridium thermocellum (47.6%, DDBJ acces-
sion no. AAD33071). Fig. 2 shows alignment of amino
acid sequences of clostridial hydrogenases. In addition,
C. paraputricum HydA showed a certain similarity to
some large subunits of [Fe]-hydrogenases in Desulfovib-
rio species such as Desulfovibrio vulgaris subsp. oxami-
cus Monticell (sequence identity 45.4%) [26] and D.
vulgaris subsp. vulgaris str. Hildenborough (45.1%) [27].
3.2. Hydrogen gas production by C. paraputricum M-21
carrying multiple copies of hydA
When C. paraputricum M-21 was cultivated in the
fermenter containing 500 ml of GS medium (pH 6.5)
with 1% GlcNAc as the carbon source at 45 C, the total
volume of fermentation gas evolved was about 2 l per li-
tre of medium. The ratio of H2 to CO2 was 2:1 and the
hydrogen gas yield was 1.4 mol/mol GlcNAc (Fig. 3(a)).
Plasmid pJIR751hyd contained the hydA structural
gene along with its anking region containing the possi-
ble promoter region. This plasmid was introduced and
expressed in C. paraputricum M-21. Although the ratio
of H2 to CO2 in the fermentation gas of the wild-type
and recombinant strains was constantly 2:1, the total
volume of fermentation gas produced by the recombi-
nant was about 3.5 l per litre of the medium; hydrogen
gas yield was 2.4 mol/mol GlcNAc, 1.7-fold higher than
that of the wild type (Fig. 3(b)). These results suggested
that enforced hydrogenase activity accelerated oxidation
of ferredoxin to release hydrogen gas. The composition
of organic acids produced by the recombinant and host
was determined and compared (Table 1). The amount of
acetic acid remarkably increased in parallel with an in-
crease in hydrogen gas evolution, while on the contrary,
the amount of lactic acid drastically decreased (Table 1).
232 K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229234

Fig. 2. Alignment of [Fe]-hydrogenases of C. paraputricum (C. par), C. acetobutylicum P262 (C. ace), C. pasteurianum W5 (C. pas), C. perfringens
NTCT8237 (C. per), and C. thermocellum (C. the). Amino acids which are conserved in all sequences are highlighted. A His residue and 19 Cys
residues responsible for holding FeS clusters are shown with sharp signs (#). , gap left to improve alignment. Numbers refer to amino acid residues
at the start of the respective lines; all sequences are numbered from Met-1 of the peptide.

An increase in acetic acid with an increase in hydrogen
gas evolution seems to be reasonable since their produc-
tions result from the same metabolic pathway (Fig. 1).
The production of lactic acid was negligible in the
hydrogenase-fortied recombinant (Table 1), indicating
that the conversion of pyruvic acid to lactic acid was al-
most shut down. The growth rate of the recombinant
clone was identical to that of the host and the trans-
formant containing pJIR751hyd as judged by measure-
ment of absorbance at 600 nm (Fig. 3).
4. Discussion
In our previous papers, we reported the hydrogen gas
productivity of C. paraputricum M-21 from chitinous
materials [8,9] and suggested that the enhancement of
hydrogenase activity using genetic engineering would
improve hydrogen gas production. Kaji et al. [17] re-
ported that disruption of the hydA gene encoding a
[Fe]-hydrogenase by homologous recombination in C.
perfringens strain 13 abolished its hydrogen gas produc-
tion from glucose, suggesting that this gene was respon-
sible for hydrogen gas production. Therefore, we cloned
a C. paraputricum M-21 [Fe]-hydrogenase gene that
was a homologue of the C. perfringens hydA gene.
[Fe]-hydrogenases belong to a category of metal-con-
taining hydrogenases including [NiFe] and [NiFeSe]
hydrogenases [28]. The amino acid sequence of
C. paraputricum M-21 HydA showed strong similarity
to that of clostridial [Fe] hydrogenases, particularly that
from C. acetobutylicum P262, and moderate similarity to
 K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229234 233

[29] reported that a signicant decrease in the rate of

hydrogen gas production correlated with the shift of
metabolic phase from acidogenesis to solventogenesis.
The metabolic pathway in acidogenic Clostridium has
several possible end products, including butyrate, ace-
tate, lactate, CO2, and H2 (Fig. 1). Acetate and butyrate
fermentation on glucose by typical acidogenic Clostrid-
ium species ideally proceeds according to the following
equation [30]:

Glucose ! 0.8 butyrate 0.4 acetate 2 CO2 2.4 H2

Although C. paraputricum M-21 also produced an

enormous volume of hydrogen gas during the exponen-
tial growth phase [8,9], the yield of hydrogen gas from 1
mol of glucose did not reach 2.4 mol (1.4 mol). When C.
paraputricum M-21 was cultured with 1% GlcNAc, not
only acetic and butyric acids but also lactic acid was
produced from GlcNAc (Table 1). Since hydrogen gas
is not coproduced with lactic acid (Glucose ! 2 lactic
acid), the production of lactic acid would reduce the
yield of hydrogen gas production. On the other hand,
when the C. paraputricum M-21 recombinant over-
Fig. 3. Enhanced hydrogen gas production by the overexpression of expressing hydA was cultured with 1% GlcNAc, the re-
the hydA gene in C. paraputricum. Non-transformant cells (a) and combinant produced higher amounts of acetic acid
recombinant cells harboring pJIR751hyd (b) were cultivated in 500 and negligible amounts of lactic acid in the culture uid
ml of GS medium containing 1% GlcNAc.
compared with the host organism: the yield of acetic
acid, lactic acid and butyric acid from 1 mol of glucose
Table 1 was calculated as 0.93, 0.01, and 0.24 mol, respectively.
Composition of organic acids produced from GlcNAc by C. parapu- Improvement of hydrogen gas production in the recom-
tricum M-21
binant was caused by reduction in the amount of lactic
Plasmid Organic acid (mM) acid and enhancement in the amount of acetic acid. It
Lactic Acetic Butyric Formic Propionic seems apparent that the enhanced hydrogenase activity
acid acid acid acid acid caused over-oxidation of NADH to NAD+, and conse-
None 29.3 38.1 12.7 4.87 0.03 quently the depletion of NADH to reduce pyruvic acid
pJIR751 28.6 33.9 14.4 5.13 0.04 to lactic acid (Fig. 1). However, the production of buty-
pJIR751hyd 0.42 51.8 13.1 5.90 0.02 ric acid was not aected by the overexpression of hydA.
Further improvement of hydrogen gas production might
those of a large subunit of hydrogenases from Desulf- be achieved by the inhibition of electron ow to butyric
ovibrio species; no sequence similarity with those of acid by the disruption of the gene responsible for butyric
[NiFe] or [NiFeSe] hydrogenases was seen. The acid production.
three-dimensional structure of a [Fe]-hydrogenase In conclusion, the hydA-expressing recombinant of C.
(HydI) of C. pasteurianum was previously reported paraputricum M-21 produced an increased amount of
[16]. In HydI and some other clostridial hydrogenases, hydrogen gas from GlcNAc, along with increased acetic
19 cysteine residues and a histidine residue are conserved acid production and reduced production of lactic acid.
as shown in Fig. 2, and these residues are known to fas- Further studies are necessary to further improve the
ten one [2Fe2S] cluster, three [4Fe4S] clusters, and one hydrogen gas productivity of C. paraputricum M-21
H cluster that functions as an active center [16]. by gene disruption leading to the inhibition of butyric
Hydrogen gas production has been discussed in a acid production.
number of studies using various clostridia, especially in
C. acetobutylicum. Sugar metabolism of this bacterium
is composed of two phases: an acidogenesis phase and Acknowledgements
solventogenesis phase [18]. During acidogenesis in clos-
tridia, a large amount of electron ow is directed to This work was supported in part by a Grant-in-Aid
hydrogen gas production while sugars are converted to for University and Society Collaboration (Grant No.
organic acids such as acetic acid (Fig. 1). Kim et al. 12794004), the Ministry of Education, Culture, Sports,
234 K. Morimoto et al. / FEMS Microbiology Letters 246 (2005) 229234
Science, and Technology of Japan, and by the NEDO
project High eciency bioenergy conversion prohect
development of high eciency hydrogenmethane fer-
mentation process using organic wastes.
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