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Original Paper	Pdf file on Synergy omments
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, [email protected]

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 hetero�logous 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 hetero�logous 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 hetero�logous 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

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'-CTATACGCCA�TTTGAAGCC-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抯 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'-TCCGC�AA�C�GCTGTTGACAC-3' and 5'-TGGGACGG�TTGTA�G�T�TC�ACC-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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