Original Paper
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file on Synergy |
Acta Biochim Biophys
Sin 2008, 40: 158-165
doi:10.1111/j.1745-7270.2008.00388.x
Improved
heterologous gene expression in Trichoderma reesei by cellobiohydrolase
I gene (cbh1) promoter optimization
Ti Liu, Tianhong Wang*, Xian
Li, and Xuan Liu
State Key
Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
Received: October
25, 2007
Accepted: December
13, 2007
This work was
supported by the grants from the National Natural Science Foundation of China (No.
30470052), the National Basic Research Program (973) of China (No. 2003CB716006
and 2004CB719702) and the Natural Science Research Foundation for the Doctoral
Program of the Higher Education Ministry of China (No. 20040422042)
*Corresponding
author: Tel, 86-531-88366118; Fax, 86-531-88565610; E-mail,
To improve
heterologous gene expression in Trichoderma reesei, a set of optimal
artificial cellobiohydrolase I gene (cbh1) promoters was
obtained. The region from –677 to –724 with three potential
glucose repressor binding sites was deleted. Then the region from –620 to –820 of the
modified cbh1 promoter, including the CCAAT box and the Ace2 binding
site, was repeatedly inserted into the modified cbh1 promoter, obtaining
promoters with copy numbers 2, 4, and 6. The results showed that the glucose
repression effects were abolished and the expression level of the glucuronidase
(gus) reporter gene regulated by these multi-copy promoters was markedly
enhanced as the copy number increased simultaneously. The data showed the great
promise of using the promoter artificial modification strategy to increase
heterologous gene expression in filamentous fungi and provided a set of
optional high-expression vectors for gene function investigation and strain
modification.
Keywords Trichoderma reesei; cbh1 promoter; carbon
catabolite derepression; targeted deletion; multiple-copy strategy
Filamentous fungus Trichoderma reesei is one of the most
efficient cellulase producers and has a long history in producing hydrolytic
enzymes. Several mutant strains can produce cellulases (40 g/L) and the major
cellulase, cellobiohydrolase I (CBH I), accounts for approximately 50%
of all secreted proteins [1]. Thus, cbh1 promoter has been considered
the strongest promoter in T. reesei, and is generally used to construct
high-efficient expression vectors to yield homologous and heterologous
proteins [2,3]. Compared with the high production of homologous proteins in T.
reesei, the yield of the heterologous proteins is rather low. Thus, how to
improve the expression of heterologous proteins has become a significant issue
of fungal molecular biology. Furthermore, the T. reesei genome has been
recently sequenced [4]. The functions of a large number of genes in the
sequenced genome are unknown and need to be elucidated. Therefore, the
construction of high-expression vectors has become an increasingly important
requirement for the study of molecular genetics, as well as for strain
improvement.
In fungi, the production of cellulolytic enzymes is finely regulated
at the level of transcription. Usually, both the pathway-specific regulation,
such as induction and repression, and the wide-domain regulation controls are
operating at the level of transcription, including transcriptional regulation
by the available carbon source, the carbon catabolite repressors (CREI/CreA)
from T. reesei and Aspergillus, the cellulase activator (Ace2)
from T. reesei, and a CCAAT box-binding protein in filamentous fungi
[5]. In T. reesei, the cellulase genes are repressed in the
presence of glucose by the wide-domain carbon catabolite repressors CREI and
CreA of Aspergillus; these genes are induced in the presence of
cellulose or its derivatives. Three putative CREI binding sites present
in the region from –674 to –724 of the cbh1 promoter are considered to be involved in
glucose repression dependent on the binding affinities of the CREI protein in
the glucose medium in T. reesei [6,7]. Ace2 binds to the 5‘-GGCTAATAA-3‘
sequence in the cbh1 promoter (at approximately –783), leading to
positive regulation of primary cellulase genes (cbh1, cbh2, egl1,
and egl2) and xyn2 in cellulose-induced cultures [8]. The CCAAT sequence is one of the most ubiquitous elements in 30% of
eukaryotic promoters. The CCAAT sequence exists at approximately –700 of the cbh1
promoter in T. reesei and is recognized by the Hap protein complex.
The complex consists of three subunits, Hap2, Hap3, and Hap5, which enhance the
overall strength of the promoter activity and increase the expression level of
many genes, such as acetamidase-, amylase-, cellulase-, and xylanase-encoding
genes [9,10]. It is regarded as an essential and functional element for
high-level expression of genes in filamentous fungi [11] and higher eukaryotes.
In addition, the activity of the cbh2 promoter in T. reesei has
been shown to depend on the Hap protein complex binding to the CCAAT box [10]. The Escherichia coli -glucuronidase gene (UidA or gus)
has been used as a successful marker gene in several transgenic organisms such
as plants [12,13], yeasts [14–16], and filamentous fungi [17] because of the unparalleled
sensitivity of the encoded enzyme, the ease with which it can be quantified in
cell-free extracts and visualized histochemically in cells and tissues, and
its stability and activity in a wide pH range.In this study, a strategy was designed to improve heterologous gene
expression in filamentous fungi with artificial modification of the promoter A
set of optimal and effective vectors can evidently improve the expression
level of heterologous proteins in Trichoderma and other filamentous fungi,
as well as provide a useful and efficient tool for academic research and
industrial applications.
Materials and Methods
Strains, plasmids, primers, and culture conditions The protease-deficient strain T. reesei RutC-30 M3 [18], a
derivative of cre1 mutant strain RutC-30, was used as the
recipient strain. Plasmids were propagated in E. coli DH5a. The vector
backbone used in constructing the plasmid was pUC19 (L09137). The E. coli
cultivations were carried out overnight at 37 ?C in Luria-Bertani medium with
50–100
mg/ml
ampicillin.Primers used in this study were listed in Table 1.Media of T. reesei were prepared as described previously
[19]. The fungal mycelia for DNA isolations were obtained after the strains
were cultivated at 28 ?C on a rotary shaker (250 rpm) for 2 d in minimal medium
(MM) containing 2% proteose peptone. MM for T. reesei contained (g/L,
final concentrations): MgSO4, 0.6; (NH4)2SO4, 5; KH2PO4, 15; CaCl2, 0.6; Na citrate2H2O, 3;
and microelements FeSO4?7H2O, 0.005; MnSO4?H2O, 0.0016; ZnSO47H2O,
0.0014; and CoCl2, 0.002. The pH of the medium was 5.5. Growth
medium (MM containing 20 g/L glucose) was used for initial flask cultivation.
Induction media prepared by replacing glucose with 2% lactose were used for RNA
extraction and detection of enzyme activity. In transformation experiments,
solid MM containing 2% glucose, 1 M sorbitol, and 100 mg/ml hygromycin was used to
screen the positive transformants.
DNA and RNA manipulation
Total RNA was isolated from the mycelia cultivated in the induction
medium according to the method of Verwoerd et al [20]. RNase-free DNase
I was used to remove DNA contamination. RNA concentration and quantity were
spectrophotometrically assessed. Genomic DNA was extracted from all available
mycelia according to the method of Penttil et al [21]. DNA and RNA
manipulations were carried out using standard procedures [22].
Construction of expression
vectors
Construction of expression
vectors
The PstI/EcoRI fragment containing the cbh1
terminator of T. reesei and multiple restriction sites from vector pTRIL
[23] was inserted into pUC19 digested with the same endonucleases [24] to
generate the resulting vector pT.
The gus gene was amplified from vector pNOM102 [25] using primers A and B (Table 1). The polymerase chain
reaction (PCR) was carried out as follows: 97 ?C for 5 min, then 30 cycles of
amplification (94 ?C for 30 s, 57.5 ?C for 30 s, 72 ?C for 1 min), then 72 ?C
for 10 min. The amplified fragment was digested with XhoI and XbaI,
cloned into pT, and generated vector pTG.The two regions, –16 to –1301 and –16 to –868, of cbh1 promoter were obtained by PCR, using primer
pairs C, D and E, D (Table 1), respectively. The two amplified fragments
were digested with PstI and KpnI, and inserted into pTG to
construct expression vectors pL and pC, respectively. The fragments, –16 to –676 and –725 to –1301, of promoter were amplified using primer pairs E, F and G, D (Table
1), respectively. The region from –677 to –724 of the cbh1 promoter
was deleted by overlap PCR with primers E and D (Table 1) using the two
amplified fragments mixture as the template. The PCR was carried out as
follows: 97 ?C for 5 min, then 30 cycles of amplification (94 ?C for 30 s, 50
?C for 30 s, 72 ?C for 1 min), then 72 ?C for 10 min. The amplified fragment
was digested with PstI/KpnI and inserted into pTG to obtain
vector DpC.A 200 bp DNA fragment (–620 to –820) containing the CCAAT
box and Ace2 binding sites located in the modified cbh1 promoter of DpC was amplified
using primers E and H (Table 1). After treatment with T4 DNA polymerase
and digestion with PstI, the DNA fragment was inserted back between the
PstI and StuI sites of DpC, constructing vector Dp2C. Similarly,
vectors Dp4C and Dp6C containing 4 and 6 copies of the 200 bp fragment, respectively,
were obtained with primers M13R and I (Table 1), using Dp2C as the
template.
Transformation of T. reesei
and isolation of positive transformants
The GUS expression vectors with different modified promoters were
co-transformed into the recipient T. reesei RutC-30 M3 protoplasts [18] with pAN7-1 vector containing a hygromycin resistance cassette [26]. The total amount of transforming DNA was 8 mg (4 mg expression
vector and 4 mg pAN7-1 vector). After cultivating at 28 ?C for 3 d, the
hygromycin-resistant transformants were selected in solid MM containing 100 mg/ml hygromycin
B and 1 M sorbitol. Transformants were regenerated on potato dextrose agar with
100 mg/ml hygromycin B. Mitotically stable transformants were obtained by
at least three sequential transfers of conidia from non-selective to selective
media. The potential gus-positive transformants were analyzed by PCR
with primer pairs J, K and L, M (Table 1). The PCR reaction conditions were
as follows: 97 ?C for 5 min, then 30 cycles of amplification (94 ?C for 30 s,
51.4 ?C for 30 s or 52.6 ?C for 30 s, 72 ?C for 1 min), then 72 ?C for 10 min.
The two amplified fragments were sequenced using the dideoxy sequencing
technique. At the same time, the potential gus-positive transformants
were further analyzed by PCR using primers 5‘-CTATACGCCATTTGAAGCC-3‘
(gus gene) and M13/pUC reverse primer to confirm the modifications of
the promoter directing the GUS expression.
Dot blot hybridization analysis
Genomic DNA was extracted after cultivation in the glucose medium
according to the method of Penttil et al [21]. The total DNA of
the transformants was extracted and the dot blot hybridization analysis was
carried out with an Enhanced chemiluminescence direct nucleic acid labeling and
detecting kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to
the manufacturer? instructions. DNA from a non-transformed T. reesei was used
as a control. After hybridization and washing, the membrane was exposed to
X-ray film for 15 min. The amplified gus gene fragment using primers J
and K was used as the probe.
Reverse transcription (RT)-PCR
analysis
Total RNA was extracted from freeze-dried mycelia cultivated in lactose
medium. RT-PCR was carried out with primers J and K (Table 1) according
to the protocol described by the RT-PCR kit (Promega, Madison, USA).
Approximately 2 mg total RNA was used to synthesize the first-strand cDNA with the
reverse transcriptase (Promega). The PCR reaction was carried out as follows:
94 ?C for 5 min, then 30 cycles of amplification (94 ?C for 30 s, 53 ?C for 1
min, 72 ?C for 1 min), then 72 ?C for 10 min.
Semi-quantitative PCR analysis
Total DNA was extracted from the mycelia cultivated in 2% glucose
medium. The PCR of the gus gene was carried out with primers J and K (Table
1) with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
as the inner control with primers 5‘-TCCGCAACGCTGTTGACAC-3‘
and 5‘-TGGGACGGTTGTAGTTCACC-3‘. The PCR reactions were
carried out as follows: 97 ?C for 5 min, then 30 cycles of amplification (94 ?C
for 30 s, 52 ?C for 30 s, 72 ?C for 1 min), then 72 ?C for 10 min. The reaction
products were analyzed by electrophoresis with 1% agarose gel containing
ethidium bromide, and the intensity of PCR products was semi-quantified by the
GeneTools from Syngene to detect the integrated optical density.
Enzyme
activity assay
The transformants were cultivated in 30 ml of 2% (W/V)
lactose medium for 48 h at 28 ?C, shaking at 200 rpm. Then 2% (W/V)
lactose was added once more, and the transformants were cultivated for another
24 h to detect GUS activity [15]. One unit of activity was defined as the
amount of enzyme required to produce 1 nmol of nitrophenyl per minute at 37 ?C.
To study the glucose repression/derepression effect, the transformants pC and DpC were first
cultivated in 2% (W/V) lactose medium for 48 h, then 2% (W/V)
lactose and 2% (W/V) lactose-glucose were added into the medium
and cultivated for another 24 h to obtain the culture for the determination of
GUS activity.
Results
Construction of expression
vectors
The expression vectors pL and pC, with the region –869 to –1301 deleted [Fig.
1(A)], were constructed according to the method mentioned above. On the
basis of the vector pC, the motif containing three CREI binding sites was
deleted and the vector DpC was constructed [Fig. 1(A)].After PCR amplification and treatment with polymerase, the 200 bp
DNA fragment (–620 to –820 region) containing the CCAAT box and Ace2 binding sites was
inserted into the vector DpC, generating Dp2C [Fig. 1(B)]. Similarly, vectors Dp4C and Dp6C, containing
4 and 6 copies of the 200 bp fragment, respectively, were also obtained [Fig.
1(B)].
Transformation and isolation
of T. reesei transformants
Six expression vectors were co-transformed with the pAN7-1 vector
according to the method mentioned above. Twenty hygromycin-resistant transformants
of each vector were obtained. These transformants were identified by PCR that
the gus gene was inserted into the chromosomal DNA. The 800 bp PCR
product with primers J and K (Fig. 2) was sequenced further confirming
the existence of the gus gene in the chromosomal DNA. As expected, the
sequencing results of the 654 bp fragment with primers L and M (Fig. 3)
contained a partial sequence of cbh1 promoter and the gus gene,
confirming the integration of the expression vector into the transformant genome.
The complementary PCR results using the M13/pUC primer confirmed the
modifications of the cbh1 promoter integrated into the transformant
chromosomal DNA. Thus, 10 PCR-positive transformants of each vector were obtained.
For the convenience of depiction, only one PCR-positive transformant for each
vector was selected and designated T. reesei pL, pC, DpC, Dp2C, Dp4C, and Dp6C.
Dot blot hybridization and
RT-PCR analysis
Dot blot hybridization and RT-PCR analysis showed that the gus
gene not only existed in the chromosomal DNA of T. reesei pL, pC, DpC, Dp2C, Dp4C, and Dp6C (Fig. 4),
but also was successfully transcribed in these transformants (Fig. 5). At the same time, as Fig. 6 shown, the semi-quantitative PCR
analysis showed these transformants contained the same copy of gus
expression vector using GAPDH as the inner control (Table 2).
Enzyme activity assay
The GUS activity of T. reesei DpC cultivated in 2% lactose
medium was 1.8- and 1.4-fold higher than that of T. reesei pL and pC,
respectively [Fig. 7(A)]. Furthermore, the GUS activity of the T.
reesei DpC in glucose and lactose medium was the same as that in the lactose
medium [Fig. 7(B)], whereas the activity of T. reesei pC in glucose
medium was just 40% of the activity obtained in the lactose medium. This
phenomenon verified that deletion of CREI binding sites in T. reesei DpC could result
in enhancement of heterologous gene expression and alleviation of the carbon
catabolite repression.The GUS activity of the T. reesei Dp4C was 1.4- and 2.4-fold
higher than that of DpC and pL, respectively [Fig. 7(A)]. The result showed that
the GUS activity was enhanced in the T. reesei transformants as the copy
number of the region containing the CCAAT box and the Ace2 binding site
increased from one to four. Interestingly, the GUS activity of T. reesei Dp6C was almost
the same as that of T. reesei Dp4C [Fig. 7(A)]. The data further
verified that the region from –620 to –820 of the cbh1 promoter contains the binding sites of the
transcription activators. Therefore, the introduction of multiple copies of
this region into the promoter could distinctly increase heterologous gene
expression.
Discussion
The deletion of the carbon catabolite repression binding sites
existed in cbh1 promoter not only eliminated the glucose repression
effect, but also increased promoter activity and the expression level of
heterologous protein in T. reesei. The results were similar with that of
CreA binding sites deletion in Aspergillus nidulans [27] and multicopy
inhibitor of growth protein (MIG) binding sites deletion in Saccharomyces
cerevisiae [28]. The results also supported the finding that deletion of
the putative binding site at about approximately –720 or upstream to –750 can relieve
the carbon catabolite repression [6]. The data further elucidated that CREI
binding sites were involved in cbh1 expression, confirming the role of CREI in
regulating cellulase and xylanase expression in T. reesei [29]. The introduction of multiple copies (2, 4 and 6) of the cis-acting
elements in modified promoters significantly improved the transcriptional
activity and expression levels of heterologous genes. This result was
consistent with that obtained from Aspergillus niger [17] and Aspergillus
oryzae [30]. Therefore, the CCAAT sequence and Ace2 binding
sites could be critical for the transcriptional regulation of the hydrolase
genes in filamentous fungus, although its flanking sequence might also have a
role in regulating gene expression. With the copy number varying from 1 to 4,
the expression level of heterologous gene increased, whereas the
activity of the 6-copy promoter was nearly the same as that of the 4-copy
promoter. The result could be mainly ascribed to a titration effect [31], as it is similar to the titration phenomenon observed after
introducing multiple copies of the amdS gene into A. nidulans [32].
We have highly expressed the erythropoietin protein in T. reesei
using the modified 4-copy promoter successfully (data not shown). With the accomplishment of the T. reesei genome project, many
unknown genes will be detected. As a consequence, functions of a large quantity
of genes in the sequenced genome are unknown and need to be elucidated. The
construction of the high-expression vectors is beneficial for elucidation of
gene function. Our results showed the feasibility of artificial modification of
the regulatory region by a deletion and multi-copy strategy to effectively improve
heterologous gene expression. The constructed expression plasmids in our study
should be a useful tool for elevating expression of heterologous proteins,
development of basic research, and genetic modification of T. reesei and
other filamentous fungi.
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