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Original Paper
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Acta Biochim Biophys
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doi:10.1111/j.1745-7270.2007.00260.x |
Cloning, sequencing and expression analysis of the first cellulase gene encoding� cellobiohydrolase 1 from a cold-adaptive Penicillium chrysogenum FS010
Yunhua HOU1,2, Tianhong WANG1*,
Hao LONG1, and Huiyuan ZHU1,2
1 State Key Laboratory of Microbial Technology,
Shandong University, Jinan 250100, China;
2 Department of Food and Biologic Engineering,
Shandong Institute of Light Industry, Jinan 250323, China
Received: November
1, 2006�������
Accepted: December
16, 2006
This
work was supported by the grants from the Major State Basic Research
Development Program (973) of China (N0. 2003CB716006 and 2004CB719702), the
Natural Science Foundation of Shandong Province (No. L2003D01) and the Open
Foundation of State Key laboratory of Microbial Technology, Shandong University
*Corresponding
author: 86-0531-88566118; Fax, 86-531-88565610; E-mail, [email protected]
Abstract������� A cellobiohydrolase 1 gene (cbh1) was cloned from Penicillium chrysogenum FS010 by a modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). DNA sequencing shows that cbh1 has an open reading frame of 1590 bp, encoding a putative protein of 529 amino acid residues. The deduced amino acid sequence revealed that CBHI has a modular structure with a predicted molecular mass of 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region. The putative gene product is homologous to fungal cellobiohydrolases in Family 7 of the glycosyl hydrolases. A novel cbh1 promoter (1.3 kb) was also cloned and sequenced, which contains seven putative binding sites (5'-SYGGRG-3') for the carbon catabolite repressor CRE1. Effect of various carbon sources to the cbh1 transcription of P. chrysogenum was examined by Northern analysis, suggesting that the expression of cbh1 is regulated at transcriptional level. The cbh1 gene in cold-adaptive fungus P. chysogenum was expressed as an active enzyme in Saccharomyces cerevisiae H158. The recombinant CBHI accumulated intracellularly and could not be secreted into the medium.
Key words������� Penicillium chrysogenum; cellobiohydrolase; TAIL-PCR; promoter of cbh1
Cellulose is the world�s most abundant
biopolymer, and as such, its degradation is of considerable ecological,
agricultural� and commercial importance. Cellobiohydrolase 1 (CBHI EC 3.2.1.91)
is a retaining exo-cellulase that hydrolyzes� the b-1,4-linkages of a cellulose chain from its reducing
end liberating b-cellobiose as the
main product. It belongs to Family 7 of the glycosyl hydrolases [1]. The GH
Family 7 comprises enzymes responsible for hydrolysis� of b-1,4-D-glucosidic linkages in
cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of
the chains [2]. Fungal cellobiohydrolase 1 enzymes share a modular structure
consisting of a fungal type cellulose�-binding module and a catalytic binding
domain separated by a proline/serine/threonine rich linker peptide [3]. CBHs
play a key role in degradation of crystalline cellulose. Cellobiohydrolases
genes were cloned and characterized� from a number of fungal sources including Penicillium
janthinellum [4], Trichoderma reesei [3,5], Phanerochaete
chrysosporium [6,7], Aspergillus aculeatus [8], Aspergillus niger
[9], Fusarium oxysporum [10], Irpex lacteus [11-13], and the thermophilic fungus Talaromyces
emersonii [14,15]. Although cellulose utilizations by terricolous fungi
have been widely investigated [16], studies on CBHs from marine fungi have
rarely been reported [17]. Identification and characterization of these genes
from marine fungi are of great importance in the ocean carbon cycle.
Penicillium chrysogenum is an important industrial organism� due to
its capacity to produce penicillin, which is still one of the main
commercial antibiotics, and the saprobic ascomycete fungus is also known for
its ability of secreting a variety of cellulolytic enzymes [18]. In addition, a
wide spectrum of lytic enzymes is secreted by P. chrysogenum, including
hemicellulases, xylanases, and amylolytic enzymes [18-20]. P. chrysogenum FS010 isolated� from
Huanghai Sea was identified as P. chrysogenum by the analysis of
18s rDNA sequence (AY593254) and small
subunit ribosomal RNA sequence (AY553613) reported previously [21]. The optimal
temperature� of the crude CBHI from P. chrysogenum FS010 was 35 �C,
whereas the optimal temperatures of other CBHs from moderate thermophilic fungi
were usually� 50-60 �C, suggesting�
that the CBHI from marine P. chrysogenum FS010 had great advantages in
hydrolyzing the crystaline cellulase at room temperature. However, cellulase
genes have not been isolated and characterized from this fungus.
In this paper, we cloned the cbh1
from the cold-adaptive� P. chrysogenum FS010 by a modified TAIL-PCR
approach and examined the transcription of this cbh1 gene. The hetero�logous
expression of CBHI in yeast was also studied.
Materials and Methods
Strains, plasmids and media
P. chrysogenum strain FS010 [21], was used as a DNA donor in this
study. Stock cultures were kept on potato glucose agar and subcultured monthly.
P. chrysogenum FS010 conidia were inoculated in minimal medium as
described� by Mandels and Andreotti [22] at a final concentration� of 108 conidia/L. Flasks were incubated in an
orbital shaker (220 rpm) at 15 �C for 48 h. The mycelium� was recovered by
filtration on a nylon filter (30 mm spore)
washed with 0.9% (w/v) NaCl and dried by pressing
between� two filter papers. To examine the effects of various� carbon sources
(1%, w/v) on cellulase expression, the
replacement technique described by Sternberg and Mandels [23] was used. The
induction time of various carbon sources was 18 h. Avicel cellulose, sophorose,
gentibiose, cellobiose and sorbitol were purchased from Sigma-Aldrich (St.
Louis, USA).
Escherichia coli DH5a and the plasmid pGEM-T (Promega, Madison, USA) were
used for general DNA manipulations and for DNA sequencing. Saccharomyces
cerevisiae H158 (his- leu- ura-) and the expression vector pAJ401 (ura3, 2 m plasmid replicate origin, PGK promoter,
and PGK terminator) derived from plasmid pFL60 [24] were used for the
heterologous expression of CBHI in S. cerevisiae H158.
General recombinant DNA techniques
The genomic DNA was isolated from P.
chrysogenum FS010 using the method developed by Raeder and Broda [25]. DNA
fragments were recovered from agarose gels by using the E.Z.N.A gel extraction
kit (Omega Bio-Tek, Jinan, China) and PCR clean-up system (Promega). The
purification of plasmid and other general DNA manipulation procedures were
carried out as described by Sambrook and Russell [26].
Cloning and sequencing of the full-length cbh1 gene by TAIL-polymerase chain reaction
The multiple alignment (DNAMAN) using the
primary structure of known fungal CBHIs including P. janthinellum cbh1,
T. reesei cbh1, P. chrysosporium cbh1, and A. aculeatus cbh1
shows high conservation of the protein sequences V-L-D-A-N-W-R-X-V-H and N-M-L-W-L-D-S-D-Y-P
(data not shown). Based on the conserved sequences, two degenerate
oligonucleotide primers were designed and synthesized to amplify a fragment of
the cbh1 gene: forward, 5'-NTCATTMACGCCAYCTGG-3'; reverse,
5'-MCTMTCGAGCCACAACAT-3' (N, M, and Y represent A/G/C/T, C/G, and A/T,
respectively). Genomic DNA of P. chrysogenum FS010 was used as the
template. Polymerase chain reaction (PCR) was performed under the following
conditions: an initial denaturation at 94 �C for 5 min followed by 35 cycles of
amplification (94 �C for 30 s, 54 �C for 30 s, and 72 �C for 1 min), and an
additional extension step at 72 �C for 10 min. The amplified� fragment (1062-bp
PCR product) was analyzed by gel electro�phoresis and purified, then cloned
into the pGEM-T vector (Promega), and its nucleotide sequence was determined.
To isolate the 5'-end of the cbh1
gene fragment, TAIL-PCR was performed according to the protocol developed by
Liu et al. [27] with a modification. The modification� is shown on the use
of an asymmetric thermocycling pattern� of the tertiary PCR. The PCR pattern
was: 94 �C for 4 min (1 cycle); 94 �C for 30 s, 61 �C for 1 min, and 72 �C for
2 min, 94 �C for 30 s, 61 �C for 1 min, and 72 �C for 2 min, 94 �C for 30 s, 40
�C for 1 min, and 72 �C for 2 min (12 cycles); and 72 �C for 10 min (1 cycle).
Five arbitrary degenerate primes (AD) such as AD1 (5'-NTGCANTNT�GCNGTT-3'),
AD2 (5'-NGT�CA�GN��NNGANANGAA-3'), AD3 (5'-NGT�GNG�AN�AN�CA�N�CAG-3'),
AD4 (5'-TGN�GNGANANCANAG-3') and AD5 (5'-AGNGNAGNA�NCANAGC-3'),
in which N represents A/G/C/T, were designed. Three interlaced specific reverse
primers complementary to the known nucleotide sequence (1062-bp PCR product)
were synthesized [Fig. 1(A), sp1, sp2 and sp3]. The tertiary PCR
products were separated by electrophoresis on 1.0% agarose� gels. The correct
PCR product was purified, and then cloned into the pGEM-T vector. Its
nucleotide sequence� was determined.
According to the method described above,
three interlaced� specific sense primers according to the known nucleotide
sequence were designed [Fig. 1(B), sp1.1, sp2.1 and sp3.1] to isolate
the 3'-end of cbh1 fragment. Five arbitrary degenerate primers
were also used (AD1, AD2, AD3, AD4 and AD5).
DNA manipulations and sequence analysis
DNA was sequenced by an ABI 377 automated
DNA sequencer (ABI, Foster City, USA). Database similarity searches were
performed using the National Centre for Biotechnological Information (NCBI)
online program BLAST [28] against protein (BlastX) and nucleotide (BlastN)
sequences stored in GenBank. Multiple sequence alignments were done by DNAMAN
program. The protein sequence was analyzed by CBS Prediction Server [29,30] and
ExPASy server [31].
Southern and Northern blot analyses
Chromosomal DNA (5.0 mg) from P. chrysogenum FS010 was
digested to completion overnight with BamHI, EcoRV, PstI
and XhoI (TaKaRa, Dalian, China), separated on a 0.8% agarose gel, and
transferred to Hybond-N+ filter
(Amersham, Piscataway, USA). The full-length cbh1 gene was fluorescein-labeled
using an ECL random prime labeling and detection system (Amersham), and used as
a probe to determine the copy numbers of the cbh1 gene of P.
chrysogenum FS010. Total RNA was isolated from powdered mycelia with Trizol
reagent (Sangon, Shanghai, China) according to the supplier�s manual. For
Northern blot analysis, 10 mg of
total RNA was separated on a 1.2% agarose/formaldehyde gel. After capillary
blotting to Hybond-N+ membrane, the filter
was probed with a fluorescein-labeled full-length cbh1 cDNA probe. 18S
rRNA was used as a loading control. Southern hybridization and Northern
hybridization were performed according to the supplier�s instructions. The
signal intensity was determined by the Genetool
software (Cambridge, UK).
Construction of a shuttle expression vector and transformation� of S.
cerevisiae
Total RNA induced by filter paper was
isolated using SV Total RNA Isolation System (Promega). The cbh1 cDNA
gene was amplified from P. chrysogenum first-strand cDNA, using primers,
corresponding to the putative� amino-terminal and carboxyl-terminal sequences
from the 5' and 3' TAIL-PCR products, cbh1 sense primer 5'-GCGCGAATTCATGGCTTCCACTTCTCCTTCA�AGA-3'
and the cbh1 anti-sense primer 5'-GCGC�CTCGACTACAGGCACTGCGAGTAGTAATCA-3'.
The following PCR cycling parameters were used: 94 �C for 5 min (1 cycle), 94
�C for 1 min, 59 �C 30 s, 72 �C for 1.5 min (35 cycles), and 72 �C for 10 min.
The amplified� PCR product was digested with these two enzymes, and then
purified by PCR Clean-Up system. The DNA fragment of approximately 1.6-kb
containing the cbh1 cDNA gene was cloned downstream of the PGK promoter
of EcoRI/XhoI treated pAJ401. The recombinant plasmid was
designated as pAJ401-cbh1. pAJ401-cbh1 was transformed into CaCl2 competent E. coli DH5a cells. After propagation in E. coli,
the transformant plasmid (5 mg) was
purified and transformed into the yeast S. cerevisiae strain H158 by
electroporation (Bio-Rad, Hercules, USA) according to the manufacturer�s
instructions. The yeast transformants were selected on synthetic complete
medium lacking uracil (SC-URA) medium plates. The pAJ401-cbh1 transformant
identified by yeast colony PCR was grown in liquid SC-URA for 3 d at 30 �C.
After the incubation, cell-free extracts from cell pellets were prepared and
analyzed with 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The preparation of cell-free extracts was carried out as described
by Kushnirov [32].
SDS-PAGE analysis
12% SDS-PAGE was performed as described by Laemmli
[33]. Protein concentrations were determined by Bradford method [34] with
bovine serum albumin as the standard.
Activity assay
Hydrolytic activities produced by
recombinant yeast cells were assayed based on the method of Takashima et al.
[35]. p-nitrophenyl-b-D-cellobioside
(pNPC) (Sigma-Aldrich) was used to as a substrate. One unit of CBHI was defined
as the amount of protein that produces 1.0 mmol of pNP per minute under the standard assay
conditions.
Nucleotide sequence accession number
The genomic and cDNA sequence of cbh1
gene have been deposited in GenBank under the accession number AY790330 and
AY973993, respectively.
Results and Discussion
Cloning and analysis of the primary structure of the P. chrysogenum cbh1 gene
Under experimental conditions described
using degenerate oligonucleotide primers with homology to other cbh1
genes, a specific fragment of 1062-bp was amplified from P. chrysogenum
chromosomal DNA. Sequence analysis confirmed that the PCR product was homologous
to the cbh1 gene family (data not shown). Based on the sequence of the
gene fragment, a modified TAIL-PCR was performed to clone the entire cbh1 gene
(Fig. 1). The full-length cbh1 was successfully and rapidly
isolated. To examine whether the sequence obtained from TAIL-PCR reactions and
sequencing was correctly deduced, a 2940-bp DNA fragment, including 1316-bp of
the region upstream of the putative initiator ATG and 34-bp of the region
downstream of the stop codon, was amplified by PCR as a continuous fragment
using genomic DNA as a template and sequenced.
The nucleotide sequence of the 2940-bp DNA
fragment was determined for both strands. One open reading frame (ORF) was
located between nucleotides 1 and 1590, and its molar G+C content was 56.04%. The
sequence of the 5' flanking region of the cbh1 gene was
determined to nt -1316. The putative
translation start site was designated as +1. A putative TATA box was found at
nt -41, and three putative CAAT box was found
at nt -207, -412 and -589 respectively.
Two putative binding sites were found at nt -418 and -809 for
the transcriptional activator ACEII [36]. In the 5' upstream region of
the cbh1 gene, seven carbon catabolite-repressor binding consensus
sequences (5'-SYGGRG-3') [37] that possibly mediate carbon
catabolite repression by a CREA-homologue were found at nt -230, -232, -252, -259, -296, -572 and -776 respectively. Sequence analysis showed
that the cbh1 promoter region from P. chrysogenum FS010 has no
homology with those cbh1 promoter from T. reesei [37], Thermoascus
aurantiacus [38] and Trichoderma koningii [39], suggesting� that the
cloned cbh1 promoter is a novel promoter. The cbh1 promoter�
would be useful for development of a high efficient regulated expression system
for P. chrysogenum.
The TAIL-PCR approach developed by Liu and
Whittier [40] is a simple and efficient technique for genomic walking in plant
molecular biology [27,41], which does not require any restriction or ligation
steps. But to the best of our knowledge, it has never been employed for the
isolation of full-length genes from fungi. In this paper, a modified TAIL-PCR
method in combination with degenerate PCR was recruited to clone the CBHI
encoding gene and the cbh1 promoter from a cold-adaptive P.
chrysogenum FS010. Our results indicated that bioinformatics analysis in
combination with TAIL-PCR protocol would facilitate the fungal full-length gene
cloning and the development of filamentous fungi molecular biology.
Structure of the CBHI protein
The ORF encodes a protein of 529 amino
acid residues, with a deduced molecular mass of about 56-kDa. At the N terminus
of the deduced sequence, a putative signal sequence was identified by the
SignalP 3.0 server system (http://www.cbs.dtu.dk/services/),
with cleavage predicted� to occur after amino acid 26 of the pre-protein. Three
potential N-glycosylation sites were found at Asn-295, Asn-442, and Asn-505.
Comparison of the deduced CBHI amino acid sequence from P. chrysogenum
with those available on databases reveals identity values of 70.37%, 66.91%,
62.08%, 62.00% and 56.69% respectively with the CBHI from P. janthinellum
(GenBank accession� No. CAA41780), A. aculeatus (GenBank accession� No.
BAA25183), Penicillium occitanis (GenBank accession No. AAT99321), T.
emersonii (GenBank accession No. AAL89553) and Trichoderma viride
(GenBank accession No. AAQ76092). All of the homologous sequences� belong to Family
7 of the glycosyl hydrolase, which suggests that CBHI of P. chrysogenum is also
a member. Among the conserved residues, the amino acid equivalent to Glu-237
was identified as a potential nucleophile� in the displacement reaction and
that equivalent� to Glu-242 was identified as a potential proton donor [35]. An
alignment of the deduced polypeptide sequence shows that the modular structure
is conserved, with an N-terminal� catalytic domain (aa 27-460) linked via a threonine rich linker
(aa 461-493) region to the carboxyl terminal
carbohydrate�-binding module (aa 501-528).
Prosite pattern� search performed on the deduced FS010 protein sequence�
suggests a fungal cellulose-binding domain [42] signature pattern
C-G-G-x(4,7)-G-x(3)-C-x(4,5)-C-x(3,5)-[NHGS]-x-[FYWM]-x(2)-Q-C (the four
cysteine residues� are involved in disulfide bonds) between amino acid 501 and
528.
Restriction analysis of the P. chrysogenum cbh1 gene
In order to examine whether the cbh1
gene is present in only one or multiple copies in the P. chrysogenum
genome, Southern blotting was performed using total chromosomal DNA digested
with different restriction enzymes (Fig. 2). A single hybridizing band
is present in all the digestions. The hybridization result shows that P.
chrysogenum has a single copy of the cbh1 gene in its chromosomal
DNA, which is the same as the reported findings in T. reesei, T.
viride, and T. aurantiacus [37]. In contrast, P.
janthinellum [4] has multiple copies of cbh1 gene.
Northern blotting of P. chrysogenum cbh1 transcription�
To gain insight into the regulation of the cbh1
gene in P. chrysogenum FS010, Northern blot was carried out by using the
full-length cbh1 gene probe, under high stringency. To ensure equal
loading of each RNA sample, the membrane was rehybridized with 18S rRNA
(approx. 1.5 kb) probe. The data obtained from the Northern blot analysis shown
in Fig. 3 indicated that Avicel strongly induces cbh1
transcription. The signal intensity of Avicel induction was defined as 100%.
The sophorose, cellobiose, gentiobiose, lactose and xylose induced 64%, 41%,
30%, 26% and 19% of cbh1 expression. D-glucose, fructose and sorbitol
could not induce any detectable levels of FS010 cbh1 expression. Effects
of various carbon sources to the cbh1 transcription showed that CBHI of P.
chrysogenum are inducible. Although sophorose, cellobiose, gentiobiose,
lactose and xylose could induce the cbh1 transcription, the natural
inducer of P. chrysogenum CBHI awaits further study. The addition
of 1% glucose for 2 h to P. chrysogenum mycelia, previously� cultured on
Avicel (48 h), resulted in abolition of the cbh1 signal (data not
shown), indicating that the P. chrysogenum cbh1 expression was
subject to carbon catabolite repression.
Glucose repression in Trichoderma
and Aspergillus species� are mediated by the catabolite repressor Cre1
and CreA, respectively. These repressive proteins bound to specific target
sequences in the promoters of cellulase genes and downregulated their
transcription. Analysis of the novel cbh1 promoter from P. chrysogenum
FS010 showed that six SYGGRG motifs are present. It is therefore likely that,
as in other fungi, glucose repression in P. chrysogenum is also mediated
through a CreA homologue.
Heterologous expression in S. cerevisiae H158
Although the amino acid sequence deduced
from the nucleotide sequence of cbh1 is homologous with other CBHs,
whether it codes a CBH remained to be identified. Using RT-PCR, the cbh1
cDNA was amplified and sequenced. The comparison of the cbh1 cDNA
sequence to the cbh1 genomic sequence shows that P. chrysogenum
FS010 cbh1 gene is not interrupted by introns. The same result was
obtained for the A. aculeatus cbh1 gene [8], whereas all the other
fungal cbh1 genes sequenced, including� from P. janthinellum, T.
reesei, A. nidulans and N. crassa had their structural genes
interrupted by introns at various positions. The expression plasmid was
constructed as described above and designated as pAJ401-cbh1, which was
introduced into S. cerevisiae H158.
A 62-kDa protein band from the cell-free
extracts of H158-cbh1 was shown on SDS-PAGE, whereas no 62-kDa protein band
from cell-free extracts of H158 harboring� the plasmid pAJ401 was detected on
SDS-PAGE (Fig. 4). Due to the hyperglycosylation in yeast, the molecular
weight of recombinant CBHI (approx. 62 kDa) is different� with the deduced size
of the cbh1 cDNA translate (53.5 kDa). The recombinant CBHI activity of
cell extracts was measured against the pNPC under standard conditions. The
specific activity of recombinant CBHI was 64.3 U/mg (total protein), suggesting that the cbh1 cDNA
from strain FS010 was successfully expressed in the S.
cerevisiae H158. The comparison of the recombinant CBHI activity� (64.3 U/mg) to the CBHI activity of P.
chrysogenum FS010 (594.2 U/mg, total
protein, unpublished data) showed that the CBHI activity in yeast transformant
was low. No CBHI activity was detected in the supernatant of H158-cbh1cultures,
suggesting that the recombinant CBHI could not be secreted into the medium.
To further confirm the 62-kDa protein was
the product� of the cDNA, the protein band was cut and analyzed by Edman
degradation. The N-terminus amino acids analyzed were Q-V-G-T-S, identical to
the deduced amino acid sequence from the 27th to 31st, indicating that the signal
peptide of recombinant CBHI was recognized and cut in S. cerevisiae
H158. Considering this 62-kDa recombinant protein had cellobiohydrolase
activity and the N-terminal amino acid sequence, we concluded that P.
chrysogenum FS010 cbh1 cDNA encoded a cellobiohydrolase.
Cellulase had been applied in a wide array of biotechnology ranging from biofuel production, paper making, food processing, biostoning, environmental bioremediation to stereoselective tools for separation of drug enantiomers [3,6,16,18]. Our research provides a new member for cellulase family and a novel experimental material for detailed� research of the cellulase action mechanism. The investigation of P. chrysogenum cellulase gene and upstream� regulatory sequence would be beneficial to the improvement of utilization of cellulosic substrate and research� on the mechanism of cellulose degradation in P. chrysogenum. Further work will be needed to characterize� the high-efficient expression of cbh1 and the transcriptional factors in the P. chrysogenum.
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