ISSN 1672-9145 Acta Biochimica et Biophysica Sinica 2004, 36(10): 667–672 CN 31-1940/Q

Novel Cellulase Profile of Trichoderma reesei Strains Constructed by cbh1 Gene

Replacement with eg3 Gene Expression Cassette

Tian-Hong WANG*, Ti LIU, Zhi-Hong WU, Shi-Li LIU, Yi LU, and Yin-Bo QU
The State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

Abstract To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases
while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of
the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous
recombination. Disruption of the cbh1 gene was confirmed by PCR, Southern dot blot and Western hybri-
dization analysis in two transformants denoted as L13 and L29. The filter paper-hydrolyzing activity of strain
L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively
modest increase suggested that the eg3 cDNA under the control of the cbh1 promoter was not efficiently
transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could
be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such
strains are of interest from the basic science perspective and also have potential industrial applications.

Key words Trichoderma reesei; gene replacement; gene disruption; cbh1; eg3

The filamentous fungus Trichoderma reesei is consi-
dered to be the most efficient cellulase producer, and has
a long history in the production of hydrolytic enzymes,
which was widely used in the food and feed industries
and recently also used in the textile, pulp and paper indus-
tries [1,2]. With the development of molecular manipu-
lation technology and filamentous fungi transformation
systems, it is possible to genetically modify the filamen-
tous fungi strains with recombinant DNA technology.
The cellulolytic system of T. reesei is composed of two
cellobiohydrolases (CBHI and CBHII) and at least five
endoglucanases (EGI, EGII, EGIII, EGIV and EGV)
[3,4]. The best strain can secrete up to 40 g protein per
liter of culture, most of which is cellubiohydrolase I [5].
Thus, the promoter of cbh1 gene is considered to be one
of the strongest promoters in T. reesei. Endoglucanase III
is classified as a family A cellulase and considered to be
the endoglucanase with the highest catalytic efficiency
Received: June 14, 2004 Accepted: August 14, 2004
This work was supported by the grants from the National Natural
Science Foundation of China (No. 30070014), the Natural Science
Foundation of Shandong Province (No. Y2000D12) and the Major
State Basic Research Development Program of China (No.
2003CB716006)
*Corresponding author: Tel, 86-531-8364384-8109; Fax, 86-531-
8565234; E-mail, wangtianhong@sdu.edu.cn
among all the endoglucanases in T. reesei [6,7]. The tex-
tile and paper industries need some of the cellulase com-
ponents to modify the properties of the fabric surface with-
out reducing the integrity of fibers. Unfortunately, CBHI
does affect fibers’ integrity [8]. Thus industry requires
novel strains with altered cellulase profile more appropri-
ate for fabric surface modification. The development of
genetic engineering in filamentous fungi provides us with
the possibility of reconstructing the ratio of cellulase com-
ponents produced by the organism.
In our previous work, we cloned the strong promoter
and terminator of cellobiohydrolase I gene cbh1 from
cellulose-producing filamentous fungus T. reesei strain
QM9414. Using these sequences we constructed a pUC19
based expression vector, pTRIL, with multiple cloning
sites PstI, ApaLI, KpnI, SmaI, SacI and XhoI between
the promoter and terminator of cbh1 [9]. Here we report
the use of the pTRIL plasmid to construct the Pcbh1-eg3-
Tcbh1 expression cassette and the use of this cassette to
generate T. reesei strains with decreased cellobiohydro-
lases activity but elevated endoglucanase activity via
homologous recombination. Our results will further
help the understanding of the gene regulation of different
cellulase components of T. reesei and potentially lead
to the development of cellulose-producing strains for
668 Acta Biochimica et Biophysica Sinica Vol. 36, No. 10

specific industrial applications.
Materials and Methods
Strains, vectors and media
Plasmids, E. coli strains and filamentous fungi strains
were listed in Table 1. Growth conditions, media, and
genomic DNA isolation for filamentous fungi were as
described in the literature [10].
General procedures
Recombinant DNA technology and double-stranded DNA
sequencing were carried out by standard procedures [11].
Construction of recombinant plasmid pTRIL-eg3
The eg3 coding sequence was amplified by PCR from
the constructed pAJ401-eg3 using primer A and B. Primer
A is identical to nucleotide sequence at position 4–23 of
the T. reesei eg3 cDNA, and primer B is complementary
to the nucleotide sequence downstream of the eg3 gene
terminator in the plasmid pAJ401-eg3.
Primer A: 5'-ACTACTGCAGAACAAGTCCGTGGCT-
CCATT-3' (PstI site underlined)
Primer B: 5'-GCGCTCTAGAATGACCATGATTACG-
CCAAG-3' (XbaI site underlined)
The amplified fragment was digested with PstI and
XbaI, and cloned into PstI-XbaI sites of pTRIL to created
plasmid pTRIL-eg3.
DNA sequencing
A 1.7 kb T. reesei eg3 cDNA fragment were sequenced
using the dideoxy sequencing technique [15] with primer
5'-GCTCTCCCCATCTACTCATC-3' which is identical
to the nucleotides from position –96 to –77 of the cbh1
promoter, and primer G (Table 2) which is identical to
the nucleotides from position –301 to –281 of the cbh1
promoter, respectively. At the same time, a 416 bp frag-
ment obtained by PCR with primer G and H (Table 2) was
sequenced using the same method.
Transformation of Trichoderma reesei and isolation
of the positive transformants
The Pcbh1-eg3-Tcbh1 cassette was amplified by PCR from
the plasmid pTRIL-eg3 using oligonucleotide C and D as
primers (Table 2), and co-transformed with plasmid
pAN7-1 carrying a hygromycin resistance cassette into
the recipient T. reesei strain Rut C30. The plates and
media for T. reesei transformation and hygromycin B
selection were the same as described by Penttilä et al.
[10]. The hygromycin-resistant transformants were
selected on minimal medium containing 100 µg/ml
hygromycin B. Then, the positive transformants were

Table 1 The strains and plasmids used in this study

Strains and plasmids Genotype and characters Origin and reference

E. coli DH5α F–, SupE44, ∆(argF lacZya)U169, φ80lacZ ∆M15, hsdR17(rk–, mk+), Hanahan D [12]
recA1, endA1, gyrA96, thi-1, relA1, λ–
T. reesei Rut C30 Mutant strain in which glucose repressor gene Cre1 was destroyed Presented by Dr. Penttilä M,
(Ilmen 1996) Biotechnology Institute,
VTT, Finland
T. reesei Rut C30L13 and The EGIII-overproducing and CBHI-negative strain This study
C30L29
pAJ401-eg3 The eg3 gene cDNA from T. reesei was inserted between the EcoRI and Xiao ZZ et al. [13]
XhoI sites of plasmid pAJ401
pAN7-1 The fungal plasmid with the hph gene under the control of A. nidulans Punt PJ et al. [14]
gpd promoter and the trpA terminator
pTRIL 4.7 kb plasmid in which the MCS with PstI, ApaLI, KpnI, SmaI, SacI Wang TH et al. [9]
and XhoI sites was inserted between the 1355 bp T. reesei cbh1
promoter and the 595 bp terminator fragments
pTRIL-eg3 T. reesei eg3 was inserted into the PstI and XbaI sites of pTRIL under This study
the control of the cbh1 promoter. MW 6.4 kb
Oct., 2004 Tian-Hong WANG et al.: Novel Cellulase Profile of T. reesei Strains Constructed with eg3 Expression Cassette 669

Table 2 PCR primers

Primer Location (bp) Sequence Amplified segment

Size (bp) Name

C P cbh1 –1355 to –133 5'-AATTCTGGAGACGGCTTGTT-3' 3761 Pcbh1-eg3-Tcbh1 cassette

D T cbh1 +2377 to +2396 5'-CACGAAGAGCGGCGATTCTA-3'
E cbh1 –10 to +11 5'-GACTGGCATCATGTATCGGAA-3' 631 cbh1 gene fragment
F cbh1 +600 to +621 5'-TACTTGCTCACGCCACCATCC-3'
G cbh1 –301 to –281 5'-AGGATCGAACACACTGCTGC-3' 416 Partial sequence of cbh1 promoter
H eg3 +96 to +115 5'-GTCCGCTCCAACCCATACCT-3' and eg3 structure gene

analyzed by PCR with primer E and F (Table 2) specifi- lyzed in one minute during the initial period of hydrolysis.
cally amplified cbh1 to select the strains in which the cbh1 All measurements were performed with a spectrophoto-
gene was disrupted. Further PCR was carried out by with meter (Thermo Spectronic HeλioS β).
primer G and H (Table 2) to specifically amplify Pcbh1-eg3-
Tcbh1 cassette to select the strains in which the cassette
was inserted into the correct chromosomal position. Results and Discussion
Southern dot blot hybridization
Isolation of eg3 gene and construction of plasmid
Genomic DNA was extracted as described by Raeder
pTRIL-eg3
and Brode [16]. Southern dot blot analysis was performed
with an ECL direct nucleic acid labeling and detection kit A 1.7 kb fragment that included the 1.5 kb coding
(Amersham Pharmacia Biotech, Buckinghamshire, UK) region of eg3 gene and 0.2 kb of the 3' flanking vector
according to the manufacturer’s instructions. sequence was amplified as expected. This fragment was
inserted into the PstI-XbaI sites of the pTRIL, producing
Western hybridization analysis
the recombinant plasmid pTRIL-eg3 (Fig. 1). Plasmid
The T. reesei culture was grown in minimal medium pTRIL-eg3 contained the 1.5 kb eg3 gene cDNA under
for 3 d. The mycelia were harvested and then inoculated the control of the 1350 bp cbh1 promoter from T. reesei
into filter paper liquid medium and cultivated for 3 d. The (Fig. 2). The eg3 fragment encoding 397 amino acids was
mycelia were removed by centrifugation at 10,000 g at correctly fused into pTRIL after the ATG start codon as
4 °C for 10 min. The supernatants were ultrafiltered, and confirmed by sequencing.
then the concentrated liquid was analyzed by the Western
Trichoderma reesei co-transformation and isolation of
hybridization analysis using CBHI-antibodies. The remaining
the transformants lacking the cbhl gene
samples were stored at –20 °C for further analysis.
Pcbh1-eg3-Tcbh1 cassette amplified from the pTRIL-eg3
Enzymatic assays
was co-transformed with plasmid pAN7-1 into T. reesei
The enzymatic activity of exocellulase in the culture
supernatants was measured as the release of reducing su-
gars from filter paper according to the method reported by
Mandels et al. [17]. The endoglucanase activity was mea-
sured using sodium carboxymethyl cellulose as a substrate
(CMCase activity) as described by Wood et al. [18]. Pro-
tein concentrations were determined from the trichloro-
acetic acid-precipitated T. reesei culture media using the
method of Lowry et al. [19], with bovine serum albumin
as the standard. One unit of enzyme activity corresponds Fig. 1 Gel electrophoresis of PCR to amplify eg3 gene
to 1 µmol of β-1,4-glycosidic bonds of substrate hydro- 1, DL2000 marker; 2–4, amplified products of PCR.
670 Acta Biochimica et Biophysica Sinica
Fig. 2 The profile of pTRIL-eg3
strain Rut C30 as described above. 112 hygromycin-
resistant transformants were obtained and cultivated in
shaker flasks on cellulase-inducing medium. Chromosomal
DNAs were extracted from all these transformants. PCR
amplification identified two that were lack of cbh1 among
these transformants, and designated as T. reesei Rut
C30L13 and L29 respectively (Fig. 3).
Fig. 3 Gel electrophoresis of PCR products showing loss of
amplification of the chbl locus in genome in two hygromycin B-
resistant transformants L29 and L13
1, DL2000 marker; 2, T. reesei Rut C30; 3, Rut C30L29; 4, Rut C30L23; 5, T.
reesei Rut C30 (pAN7-1); 6, Rut C30L13.
PCR result indicated that the Pcbh1-eg3-Tcbh1 cassette had
been inserted into the chromosomal DNA of both L13 strain
(Fig. 4, lane 1) and L29 strain (Fig. 4, lane 4). The 416 bp
fragment sequencing results indicated that the strains con-
tained partial sequence of cbh1 promoter and eg3 struc-
ture gene.
Fig. 4 Gel electrophoresis of PCR products showing the
amplified Pcbh1-eg3-Tcbh1 cassette 416 bp fragment
1, Rut C30L13; 2, Rut C30; 3, Rut C30 (pAN7-1); 4, Rut C30L29; 5, DL2000
marker.
Vol. 36, No. 10
Dot blot hybridization and Western blot analysis
Southern dot blot hybridization and Western blot analy-
sis demonstrated that in T. reesei strain Rut C30L13 (Fig.
5, dot 6) and L29 (Fig. 5, dot 2) the cbhI gene was absent,
and neither strain secreted CBHI as expected (L13, Fig. 6
lane 1; L29, Fig. 6 lane 2). The results suggested that the
Pcbh1-eg3-Tcbh1 cassette replaced the cbh1 gene by homolo-
gous recombination. While in Rut C30L23, a heterogenous
recombination but not homologous recombination
happened, as the cbh1 gene still existed in this strain (Fig.
5, dot 4).
Fig. 5 Dot blot hybridization of demonstrating deletion of
the cbhl gene in the T. reesei strain L13 and L29
1, T. reesei Rut C30; 2, Rut C30L29; 3, pTRIL; 4, Rut C30L23; 5, Rut C30
(pAN7-1); 6, Rut C30L13.
Fig. 6 SDS-PAGE (A) and Western blot analysis (B) of the
supernatants from different T. reesei strain
1, Rut C30L13; 2, Rut C30L29; 3, protein molecular weight marker; 4, Rut C30;
5, Rut C30 (pAN7-1).
Enzyme production by the transformant strain Rut
C30L29
The filter paper activity and endoglucanase activity
(measured against CMC) of the culture supernatant from
transformant L29 were measured (Fig. 7, Table 3). The
results indicated that the filter paper-hydrolyzing activity
of the strain Rut C30L29 was reduced to 60% of that of
the parent strain Rut C30, while the CMCase activity was
increased by 30% than that of parent strain. These results
were consistent with that of replacement of the CBH1 gene
via homologous recombination with the Pcbh1-eg3-Tcbh1
Oct., 2004 Tian-Hong WANG et al.: Novel Cellulase Profile of T. reesei Strains Constructed with eg3 Expression Cassette
Fig. 7 The filter paper-hydrolyzing activity (A) and CMCase activity (B) of Rut C30L29
The results were average from three parallel flasks of each strain.
Table 3 Cellulase activities produced by T. reesei strains
Strain FPA CMCase
(U/mg protein) (U/mg protein)
Rut C30 0.038 0.162
Rut C30L29 0.022 0.322
expression cassette.
Karhunen [20] reported that expression of one copy
of the eg1 cDNA under the control of the cbh1 promoter
at the cbh1 locus produced less EGI compared with CBHI
produced by the wild-type cbh1 locus. In our experiment,
the expression of one copy of eg3 cDNA under the
control of the cbh1 promoter resulted in a 30% increase
CMCase activity compared with the parent strain. This
result was similar to that reported by Karhunen [20].
In this study the production of endoglucanase enzymes
was improved in the biotechnically important filamentous
fungus T. reesei. Expressing of one copy of eg3 cDNA
under the control of the strong cbh1 promoter in T. reesei,
CBHI-negative strains with high endoglucanase activity
were constructed. The T. reesei strain Rut C30L29 con-
structed in this study is beneficial to molecular biological
research and potentially has industrial applications. The
enzyme with novel cellulase profiles derived from the
transformants L13 and L29 could possibly be used in the
textile industry, such as cotton finishing, improving the
stonewashing effect. The new strains may also provide a
more economical way to produce cellulase for feed
modification.
Acknowledgements
We thank Dr. Roberta Greenwood (Shandong
671
University, China) and Dr. Peter L. Nagy (The Medical
School of University of Iowa, USA) for preparation of
this manuscript.
References
1 Béguin P. Molecular biology of cellulose degradation. Annu Rev Microbiol,
1990, 44: 219–248
2 Oksanen T, Pere J, Paavilainen L, Buchert J, Viikari L. Treatment of recycled
kraft pulps with Trichoderma reesei hemicellulases and cellulases. J Biotechnol,
2000, 78(1): 39–48
3 Saloheimo M, Nakari-Setälä T, Tenkanen M, Penttilä M. cDNA cloning of a
Trichoderma reesei cellulase and demonstration of endoglucanase activity by
expression in yeast. Eur J Biochem, 1997, 249(2): 584–591
4 Srisodsuk M. Mode of Action of Trichoderma reesei Cellobiohydrolase I on
Crystalline Cellulose. Finland: VTT Publications, Espoo, 1994
5 Durand H, Clanet M, Tiraby G. Genetic improvement of Trichoderma reesei
for large scale cellulase production. Enzyme Microb Technol, 1988, 10: 341–
346
6 Saloheimo M, Lehtovaara P, Penttilä M, Teeri TT, Stahlberg J, Johansson
G, Pettersson G et al. EGIII, a new endoglucanase from Trichoderma reesei:
The characterization of both gene and enzyme. Gene, 1988, 63(1): 11–22
7 Macarron R, Acebal C, Castillon MP, Dominguez JM, de la Mata I, Pettersson
G, Tomme P et al. Mode of action of endoglucanase III from Trichoderma
reesei. Biochem J, 1993, 289 (3): 867–873
8 Nevalainen H, Harkki A, Penttilä M, Saloheimo M, Teeri T, Knowles J.
Trichoderma reesei as a production organism for enzyme for the pulp and
paper Industry. In: Kirk TK, Chang HM eds. Biotechnology in Pulp and
Paper Manufacture. Application and Fundamental Investigations. Boston:
Utterworth-Heinemann, 1990, 593–599
9 Wang TH, Wu ZH, Liu SL, Qu YB. Construction of heterologous genes
expression system of filamentous fungus Trichoderma reesei. Chinese Journal
of Biochemistry and Molecular Biology, 2003, 19(6): 736–742
10 Penttilä M, Nevalainen H, Ratto M, Salminen E, Knowles J. A versatile
transformation system for the cellulolytic filamentous fungus Trichoderma
reesei. Gene, 1987, 61(2): 155–164
11 Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory
Manual. 2nd ed. New York: Cold Spring Harbor, 1989
12 Hanahan D. Studies on transformation of Escherichia coli with plasmids.
J Mol Biol, 1983, 166(4): 557–580
13 Xiao ZZ, Wang T, Wang TH, Qu YB, Gao PJ. Cloning and expression of
672 Acta Biochimica et Biophysica Sinica
Trichoderma reesei endoglucanase III (EG III) gene in Saccharomyces
cerevasiae. Acta Microbiology Sinica, 2001, 41(4): 391–396
14 Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA.
Transformation of Aspergillus based on the hygromycin B resistance marker
from Escherichia coli. Gene, 1987, 56(1): 117–124
15 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating
inhibitors. Proc Natl Acad Sci USA, 1977, 74(12): 5463–5467
16 Raeder M, Brode P. Rapid preparation of DNA from filamentous fungi.
Letters in Applied Microbiology, 1985, 1: 17–20
17 Mandels M, Andreotti R, Roche C. Measurement of saccharifying cellulase.
Vol. 36, No. 10
In: Gaden EL, Mandels MH, Reese ET, Spano LA eds. Bio/Technology and
Bioengineering Symposium No. 6. New York: John Wiley and Sons, 1976,
21–33
18 Wood TM, Bhat KM. Methods for measuring cellulase activities. Methods
Enzymol, 1988, 160: 87–112
19 Lowry OH, Rosebrough NJ, Farr Al, Randall RJ.Protein measurement with
the Folin phenol reagent. J Biol Chem, 1951, 193: 265–275
20 Karhunen T, Mantyla A, Nevalainen KM, Suominen PL. High frequency
one-step gene replacement in Trichoderma reesei. I. Endoglucanase I
overproduction. Mol Gen Genet, 1993, 241(5-6): 515–522
Edited by
Zhen-Zhen GONG