Plasmid 71 (2014) 16–22

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Short Communication

Enhanced production of heterologous proteins by the filamentous fungus Trichoderma reesei via disruption of the alkaline serine protease SPW combined with a pH control strategy Guoxiu Zhang, Yao Zhu, Dongzhi Wei, Wei Wang ⇑ State Key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, China

a r t i c l e

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Article history: Received 19 November 2013 Accepted 2 January 2014 Available online 11 January 2014 Communicated by Ananda Chakrabarty Keywords: Trichoderma reesei Alkaline serine protease Alkaline endoglucanase

a b s t r a c t The filamentous fungus Trichoderma reesei has received attention as a host for heterologous protein production because of its high secretion capacity and eukaryotic post-translational modifications. However, the heterologous production of proteins in T. reesei is limited by its high expression of proteases. The pH control strategies have been proposed for eliminating acidic, but not alkaline, protease activity. In this study, we verified the expression of a relatively major extracellular alkaline protease (GenBank accession number: EGR49466.1, named spw in this study) from 20 candidates through real-time polymerase chain reaction. The transcriptional level of spw increased about 136 times in response to bovine serum albumin as the sole nitrogen source. Additionally, extracellular protease activity was reduced by deleting the spw gene. Therefore, using this gene expression system, we observed enhanced production and stability of the heterologous alkaline endoglucanase EGV from Humicola insolens using the Dspw strain as compared to the parental strain RUT-C30. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The filamentous fungus Trichoderma reesei is widely investigated for its effective production of cellulolytic enzymes (Zhong et al., 2012). This fungus produces a variety of enzymes, including cellobiohydrolases (CBH, EC 3.2.1.91), endo-b-1,4-glucanases (EG, EC 3.2.1.4), and bglucosidases (BGL, EC 3.2.1.21), which are required for complete biomass hydrolysis (Singhania et al., 2013). Moreover, T. reesei has an excellent extracellular secretion capacity and can secrete large amounts of protein (20–100 g/l) compared to other microbial production/secretion systems, such as Escherichia coli and Saccharomyces cerevisiae ⇑ Corresponding author. Address: East China University of Science and Technology, P.O.B. 311, 130 Meilong Road, Shanghai 200237, China. Fax: +86 21 64250068. E-mail address: [email protected] (W. Wang). 0147-619X/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.plasmid.2014.01.001

(Martinez et al., 2008). Therefore, T. reesei is thought to be one of the most effective hosts for protein production. However, the production of heterologous proteins in T. reesei has only proven to be moderately successful (Peterson and Nevalainen, 2012), and compared with high yields of native proteins, yields of heterologous proteins are very low (Cherry and Fidantsef, 2003; Nevalainen et al., 2005). This may be because of the low secretion rate of incorrectly folded proteins and degradation by proteases (Collén et al., 2005; Arvas et al., 2006). Indeed, in one study, truncation of recombinant proteins produced in T. reesei QM9414 was found to be due to protease activity (Dienes et al., 2007). Several strategies have been developed to increase heterologous proteins yields in filamentous fungi, including modification of codon usage in heterologous genes (Te’o et al., 2000), introduction of a large number of gene copies

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(Withers et al., 1998), the use of strong promoters and efficient fungal secretion signals (Lv et al., 2012), and the development of vacuolar protein sorting receptor-deficient host strains (Yoon et al., 2010). One of the most successful strategies involves the construction of gene fusions of the target gene to genomic sequences encompassing the complete or partial coding region of a highly expressed fungal gene, such as T. reesei cellobiohydrolase 1 (CBH1) (Zou et al., 2012). However, even with successful secretion of higher levels of heterologous proteins, the problem of extracellular proteases remains. At pH 5 and lower, the major protease of T. reesei has been shown to be acid aspartic protease (Haab et al., 1990). Eneyskaya et al. (1999) purified an acid aspartic protease that was responsible for limited proteolysis of fungal carbohydrases. Between pH 2.7 and 3.5, the proteolytic reaction was limited, while proteins were completely digested at lower pH (Eneyskaya et al., 1999). The reduction of aspartic protease secretion through bioprocessing strategies, such as nitrogen source and pH control, has been investigated systematically (Haab et al., 1990; Eneyskaya et al., 1999). When pH was controlled at 6.0, Dienes et al. (2007) purified a trypsin-like alkaline serine protease instead of an acid aspartic protease, as reported by Eneyskaya et al. (1999). All of these studies show that transcription of the extracellular acid protease is inhibited in medium having a neutral pH. The pH control strategy seems to be a simple approach for eliminating acidic protease activity (Haab et al., 1990; Eneyskaya et al., 1999). However, the protein products may be exposed to neutral and alkaline proteases using this strategy (Dienes et al., 2007). Therefore, we hypothesized that knockout of the major alkaline protease gene coupled with a pH control strategy may improve the production of heterologous proteins. In this study, we verified the expression of the gene encoding a relatively major alkaline protease, spw (accession: EGR49466.1), from 20 candidates. SPW is a major extracellular protease of T. reesei RUT-C30. Additionally, we constructed an spw mutant strain by insertional inactivation. As a result, the extracellular protease activity of Dspw strains was reduced, and the production and stability of the alkaline endoglucanase EGV from Humicola insolens were improved. Therefore, T. reesei Dspw strains will be useful for future scientific research or industrial heterologous protein production.

2. Materials and methods 2.1. Strains and media E. coli DH5a was used as the host strain for the recombinant DNA manipulations. T. reesei RUT-C30 (ATCC 56765), a hypersecreting and catabolite derepressed mutant (Peterson and Nevalainen, 2012), was used as a host for heterologous protein expression. H. insolens (Schülein, 1997) was used as the source of the alkaline endoglucanase EGV. Luria–Bertani (LB) medium was used to culture E. coli and Agrobacterium tumefaciens. Basal fermentation medium (BFM) (2050 g/l a carbon source, 0.6 g/l urea,

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10 g/l (NH4)2SO4, 5 g/l KH2PO4, 0.5 g/l CaCl22H2O, 0.6 g/l MgSO47H2O, 3 g/l peptone, 1 g/l yeast extract, 6 g/l corn paddle, 1 g/l Tween 80, 10 mg/l FeSO47H2O, 3.2 mg/l MnSO44H2O, 2.8 mg/l ZnSO47H2O and 20 mg/l CoCl26H2O) was used for fungal cultures and alkaline endoglucanases fermentations. Cultures were grown at 28 °C in shaking flasks with protease-inducible medium (PIM) (20 g/l a nitrogen source, 20 g/l glucose, 4.0 g/l KH2PO4, 0.5 g/l CaCl22H2O, 0.6 g/l MgSO44H2O, 10 mg/l FeSO47H2O, 3.2 mg/l MnSO44H2O, 2.8 mg/l ZnSO47H2O and 20 mg/l CoCl26H2O) for RNA extraction and protease production. 2.2. Identification and sequence analysis of protease genes from T. reesei Sixty predicted protease genes from T. reesei were identified by searching the NCBI protein database (see Supplementary material S1). After SignalP v4.1 prediction analysis (http://www.cbs.dtu.dk/services/SignalP/), 20 genes were selected for testing as the secreted protease (see Supplementary material S2). 2.3. RNA preparation T. reesei strains were ground in PIM containing 20 g/l (NH4)2SO4 at 28 °C and pH 6.0. After 2 days of cultivation, mycelia were centrifuged and supplied with fresh PIM containing 20 g/l bovine serum albumin (BSA) protein for 4 h. Then, about 400 mg of mycelia was used for extraction of RNA. Total RNA was extracted using the TRI Reagent Solution (Applied Biosystems) and purified with an additional on-column DNase digestion using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Reverse transcription was performed with 500 ng of total RNA using PrimeScript 1st strand cDNA Synthesis Kit (TianGen) according to the manufacturer’s instructions. Integrity of RNA preparations was checked with an Agilent 2100 bioanalyzer and quantification was done on a ND1000 spectrophotometer (Thermo). 2.4. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) For qRT-PCR, the UltraSYBR Mixture (CWBIO) was used with 320 nM of forward and reverse primers (see Supplementary material S2) and 5 ll of 50-fold diluted cDNA in a final volume of 25 ll. Thermocycling was carried out in a ABI StepOne thermocycler. Every qRT-PCR was done in triplicates on 96 well microplates including negative (water) and positive controls (genomic DNA) and analyzed with ABI software. In addition, for all samples negative controls using an RT mix without reverse transcriptase were performed in order to exclude contamination of samples with genomic DNA. Primers were validated by creating standard curves with tenfold serial dilutions of genomic DNA, a primer pair being considered as valid if amplification efficiency ranged between 85% and 115%. Melting curves were realised after each qRT-PCR run, to confirm the specificity of amplification and the absence of primer dimers. The qRT-PCR program consisted of an

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initial denaturation step at 95 °C and 40 amplification cycles of 10 s at 95 °C and 30 s at 60 °C.

2.5. Cloning procedures The vectors ppk1s and pamdS were constructed as follows. PCR amplifications of hygromycin and acetamidase gene cassettes were done using primers HF/R and AF/F, respectively, using the pPK2 (Lv et al., 2012) and p3SR2 (Penttilä et al., 1987) plasmids as the templates. The PCR products were digested with PacI/HindIII and inserted at the PacI/HindIII restriction site of plasmid pPK2 to generate plasmids ppk1s and pamdS, respectively (Fig. 1A, B). The SPW disruption vector pSB004 was created as follows. Noncoding regions 1.7 kb upstream and 1.6 kb downstream of the spw gene were amplified using the primer pairs SPW5F/R and SPW3F/R, respectively (Table 1). The two PCR fragments were digested with PacI/SpeI and ligated into the PacI/SpeI sites of pamdS to construct the pSB001 and pSB002 vectors, respectively (see Fig. SM1 in Supplementary material S3). The pSB003 vector (see Fig. SM1 in Supplementary material S3) was created by ligating the purified smaller fragment (from pamdS digested with PacI/SpeI) into the PacI/ XbaI sites of pSB002 (exploiting the compatibility of cohesive ends generated by XbaI and SpeI). Then, the pSB004 plasmid (Fig. 1C) was created by ligating the purified smaller fragment (from pSB001 digested with PacI/SpeI) into the PacI/ XbaI sites of pSB003. For creation of the alkaline endoglucanase expression vector pWEF31-V, PCR amplification of EGV was carried out with primers VF/R (Table 1). The 0.64-kb EGV gene

was subcloned into the StuI/PacI sites of pWEF31 (Lv et al., 2012) to generate the expression vector pWEF31-V (see Fig. SM1 in Supplementary material S3). 2.6. Transformation of T. reesei and characterization of the transformants The spw disruption vector pSB004 was introduced into T. reesei RUT-C30 by Agrobacterium-mediated transformation (Lv et al., 2012). Transformants were created essentially as described by Penttilä et al. (1987). After transformation and purification, 12 transformants were screened for deletion of SPW by PCR (Fig. SM4 in Supplementary material S3). For each expression vector transformation, hygromycin-resistant transformants were collected, and we verified that the expression cassettes were successfully integrated into the genomes by PCR, which showed the translation product of both heterologous endoglucanase and the native cbh1 gene. The confirmed transformants were further subcultured for 2–4 generations and examined for endoglucanase activity. 2.7. Alkaline protease production by T. reesei Protease production from T. reesei strains was carried out in a 7-L jar fermenter (BIOTECH-5BG-7000; Baoxing BIO-ENGINEERING EQUIPMENT Co. Ltd., Shanghai, China) with a final working volume of 2 L. Seed cultivation was performed as follows: conidia of strains (107 conidia) were inoculated into 200 ml of PIM containing 20 g/l polypeptone in a 1000-ml flask and cultivated by rotation

Fig. 1. Schematic illustration of pPK1s, pamdS, and pSB004. (A) ppk1s; (B) pamdS; (C) pSB004.

Table 1 Sequences of primers used for PCR amplification. Primer

Sequence

HF

50 -aggaacTTAATTAA(PacI)GTTAACTCTAGA(XbaI)GAGCTCTGTACAGTGACCGGTGACTCTTTC-30

HR

50 -aggaacAAGCTT(HindIII)ATTTAAAT(SwaI)GAGCTCACTAGT(SpeI)AAGAAGGATTACCTCTAAACAAGTG-30

AF

50 -aggaacTTAATTAA(PacI)GTTAACTCTAGA(XbaI)GATGTGTCTACGCCAGGACCGAGCAAG-30

AR

50 -aggaacAAGCTT(HindIII)ATTTAAAT(SwaI)GAGCTCACTAGT(SpeI)CTGGAAACGCAACCCTGAAGGGA-30

SPW5F

50 -aggaacTTAATTAA(PacI)GTTAACTCTAGA(XbaI)GCATTTGGACTGGCACGAGAAC-30

SPW5R

50 -aggaacACTAGT(SpeI)TCAAGCTCTTGGCATCTAGTGTCAA-30

SPW3F

50 -aggaacTTAATTAA(PacI)GTTAACTCTAGA(XbaI)GTTCAGAAGGTGCGATTGGAGAAGT-30

SPW3R

50 -aggaacACTAGT(SpeI)GTGTATCCGCCATTCAACCAGAGG-30

VF

50 -aggaacAGGCCT(StuI)GCCGACGGCCGCAGCACCCGC-30

VR

50 -aggaacTTAATTAA(PacI)TTAGGGGATCTGGACGGCGGGGAA-30

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(180 rpm) at 28 °C for 2 days. This culture was poured into 1.8 L fresh PIM containing 20 g/l polypeptone. Cultivation was carried out at 28 °C with 25% dissolved oxygen and 2 vvm (volumes of air per volume of liquid per minute) of aeration for 3 days. The pH was adjusted to 6.0 by the addition of 10% NaOH. A 10-ml aliquot of the culture was collected daily, and the supernatant was recovered by centrifugation (10,000g for 10 min at 4 °C) in order to determine the protease activity. 2.8. Endoglucanase production by transformed T. reesei Endoglucanase production of the transformants was carried out in a 7-L jar fermenter with a final working volume of 4 L. Seed cultivation was performed as follows: conidia of strains (107 conidia) were inoculated into 200 ml BFM containing 20 g/l glucose in a 1-L flask and cultivated by rotation (180 rpm) at 28 °C for 2 days. This culture was poured into 3.8 L fresh BFM containing 10 g/l microcrystalline cellulose and 10 g/l fiber material. Cultivation was carried out at 28 °C with 25% dissolved oxygen and 2 vvm of aeration for 5 days. Microcrystalline cellulose (30 g/l) was supplied to the culture after 48 h of cultivation. The pH was adjusted to 6.0 by the addition of 10% NH3H2O. A 10-ml aliquot of the culture was collected daily, and the supernatant was recovered by centrifugation (10,000g for 10 min at 4 °C) in order to determine the cellulolytic activity. 2.9. Enzyme assays Endoglucanase activity was determined by measuring reducing sugars from enzymatic reaction using the Somogyi–Nelson method (Nelson, 1944; Somogyi, 1952). Endogenous endoglucanase activity was measured in 50 mM sodium acetate buffer (pH 5.0) at 50 °C, while alkaline endoglucanase activity was measured in 50 mM sodium acetate buffer (pH 8.0) at 50 °C. General protease activity was determined as described by Lourien et al. (1985) with modifications. An aliquot of 0.225 ml of enzyme solution was mixed with 0.150 ml BSA stock solution (1% w/v in 0.02 M buffer of different pH values, 0.01 M 2-mercaptoethanol) and incubated for 60 min at 30 °C in a microcentrifuge tube. The reaction was then terminated, and the undigested substrate was precipitated by the addition of 0.375 ml of 5% trichloroacetic acid. After centrifugation, 0.400 ml of 1 M Na2CO3 was added to the supernatant, and the protein concentration of the supernatant was measured using a Folin Assay kit (Dingguo, China). For determination of the optimal pH on BSA, T. reesei protease activity was assayed at pH 6.0 and 7.0 (0.02 M citrate and phosphate buffers, respectively). 3. Results 3.1. Analysis of T. reesei RUT-C30 protease Sixty putative proteases from T. reesei that were mined from the NCBI database were screened for the presence of possible signal peptides. Twenty candidates were identified as possible secreted proteins. Gene expression levels

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from fungi grown in PIM containing (NH4)SO4 (20 g/l) or BSA (20 g/l) as the solo nitrogen source, were measured after 4 h by qRT-PCR. Values were normalized to b-actin (see Supplementary material S2). Following exposure to BSA, the transcription of proteases increased. The mRNA levels of EGR52488.1 (named APW1 in this study), EGR52940.1 (named APW2 in this study), EGR52804.1 (CB895877), and EGR49466.1 (named SPW in this study) were increased by 22-, 15-, 48-, and 136-fold, respectively. The aspartic proteases APW1 and APW2 (calculated molecular weight of 34 kDa, with estimated molecular weight of 32 kDa) may be the major protease at pH 5 and lower, purified by Eneyskaya et al. (1999). The proteolysis of aspartic protease was highly affected by the pH of the culture medium. Maintaining conditions for fungal growth, especially pH and concentrations of mono- and oligosaccharides, can minimize the proteolysis of aspartic proteases. The trypsin-like alkaline serine protease CB895877 was investigated by Dienes et al. (2007) in detail. The sequence of SPW (a peptide of 409 amino acids, see Fig. SM2 in Supplementary material S3) gave a calculated nominal molecular weight (MW) of 42 kDa and an isoelectric point (IP) of 7.10. Analysis of the protein sequence with SignalP v4.1 predicted that the coded protein was destined to the secretory pathway, by a signal peptide of 20 amino acids. Two Lys–Arg (KR) sequences, potential sites for KEX2-like proteases (Goller et al., 1998), were found in the sequence at positions 65–66 and 119–120. The protein presented the typical features of the active site of subtilisin-type serine proteinases (catalytic triad formed by the functional residues Asp, His, and Ser at positions 161, 192, and 254, respectively). These characteristics define the spw-encoded protein SPW as a serine-type peptidase included in the subtilase family (catalogued as S8) (Rose et al., 1999). Based on similarities to other fungal serine proteases, we expected the mature protease to contain 289 residues, with a MW of 29 kDa and an IP of 8.94, resulting from cleavage after the second KR sequence (Markaryan et al., 1996). The 50 untranslated sequence of the spw gene was analyzed for the presence of regulatory motifs using MatInspector v2.2 (Quandt et al., 1995). The putative TATA and CAAT boxes were located at 107 and 137 upstream from the ATG, respectively. Five consensus HGATAR motifs, possible binding sites for nitrogen regulators, such as AreA in A. nidulans (Ravagnani et al., 1997), were found at positions 158, 170, 514, 632, and 968. Two copies of the motif GCCARG, the recognition site for the PacC protein mediating pH regulation in Aspergillus (Tilburn et al., 1995), were found at 303 and 560 (see Fig. SM3 in Supplementary material S3). An NCBI BLAST search of the amino acid sequence of SPW showed that the closest match to the deduced amino acid sequence was the sequence of Tvsp1, a serine protease that plays a role in the biocontrol process in the biocontrol agent T. virens (GenBank accession number: AAO63588.1), sharing 91% identity with SPW. 3.2. Knockout of the spw gene High transcription of spw does not necessarily indicate high protein expression. Therefore, we wanted to investigate

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the effects of spw disruption on heterologous protein production. After pSB004 transformation, 12 transformants were screened for deletion of spw by PCR. Three transformants were shown to have deletions in the spw gene (see Fig. SM4 in Supplementary material S3). To ensure that the obtained Dspw strains exhibited fully functional for heterologous proteins expression systems, we tested whether loss of spw would result in decreased extracellular protease activity and increased endogenous endoglucanase production. Compared with T. reesei RUT-C30, the Dspw strain lost approximately 55% of its total extracellular protease activity at pH 7.0 and 44% activity at pH 6.0 (Fig. 2) after 3 days of incubation in PIM containing 20 g/l polypeptone. Moreover, after 5 days of incubation in BFM, there was no obvious improvement in endogenous endoglucanase activity between the Dspw strain and the original strain (data not shown). 3.3. Alkaline endoglucanase production and proteolysis In order to investigate alkaline endoglucanase productivity, the expression vector pWEF31-V was used to transform the Dspw strain and its parent RUT-C30. After fermentation, the endoglucanase activity was analyzed for each transformants (pWEF31-V/Dspw and pWEF31-V/ RUT-C30). The endoglucanase activity of pWEF31-V/Dspw culture (30.25 IU/ml) increased by about 8% compared to that of pWEF31-V/RUT-C30 culture (28.02 IU/ml) after 5 days of incubation in BFM. The dependence of the stability of the alkaline endoglucanase fermentation broth on pH was investigated. After filtration sterilization, the broth was adjusted to various pHs (6.0 and 7.0) and incubated at 4 °C for 30 days. After incubation, the endoglucanase activities (at pH 8.0) were decreased (Fig. 3). As shown in Fig. 3, the alkaline endoglucanase culture of the pWEF31-V/Dspw strain was more stable than that of the pWEF31-V/RUT-C30 strain.

Fig. 2. Protease activity of Dspw against the RUT-C30 strain. Protease activity was investigated at pH 6.0 and 7.0. Data were normalized to the protease activity of RUT-C30 at pH 6.0. Error bars indicate means ± SEMs (n = 3 samples) from the same experiment.

Fig. 3. Stability of the alkaline endoglucanase fermentation broth of T. reesei strains. After filtration sterilization, each broth was incubated at 4 °C for 30 days at pH 6.0 or 7.0. The stability of the alkaline endoglucanase fermentation broth was measured after 30 days of storage. Alkaline endoglucanase activity was determined in 50 mM sodium phosphate buffer, pH 8.0, at 50 °C. Error bars indicate means ± SEMs (n = 3 samples) from the same experiment.

4. Discussion Although T. reesei has attracted increasing attention as a host organism for protein production, extracellular proteolytic degradation of homologous and heterologous proteins is still a major issue (Dienes et al., 2007). Additionally, heterologous proteins are often degraded more robustly than homologous proteins (Dienes et al., 2007). Luderer et al. (1991) reported that the degradation products of Cel7B can be observed at pHs below 5.0, indicating that an acidic protease was responsible for proteolytic activity. Proteolytic degradation of cellulases and other extracellular proteins produced by Trichoderma strains has been attributed to acidic proteases (Delgado-Jarana et al., 2002). Therefore, it may be possible to regulate protease gene expression or proteolytic activity through the control of cultivation pH. The activity of acidic proteases of T. harzianum was found to be regulated primarily by the pH of culture media (Delgado-Jarana et al., 2000). The pH control strategy has been applied to repress/inhibit extracellular proteases and increase heterologous protein production in Aspergillus niger (Jarai and Buxton, 1994; O’Donnell et al., 2001). Thus, the pH control strategy seems to be a simple approach for eliminating acidic protease activity. However, use of this strategy may result in increased exposure of protein products to neutral and alkaline proteases. When exposed to BSA, four protease genes, encoding APW1, APW2, CB895877 and SPW, exhibited the greatest increased in transcription in this study. The acid aspartic proteases APW1 and APW2 may be the major proteases at pH 5 and lower, purified by Eneyskaya et al. (1999). Additionally, the proteolysis of aspartic protease can be minimized at pHs higher than 5.0 (Haab et al., 1990; Eneyskaya et al., 1999). The trypsin-like alkaline serine protease CB895877 was investigated by Dienes et al. (2007) in detail. However, no previous reports described the investigation of SPW.

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The analysis of spw transcription confirmed that serine protease activity was induced by the presence of an organic nitrogen source. This is consistent with the presence of potential sites for nitrogen (AreA) regulation in the promoter region. Moreover, the presence of a PacC binding site suggested possible control by pH, as pH plays an important role in the regulation of many proteases (St Leger et al., 1998). Deletion of the spw gene in T. reesei RUT-C30 by homologous recombination had a dramatic effect on total extracellular protease activity. This result showed that the SPW enzyme was a major extracellular protease in T. reesei that could be activated by maintaining pH at 6.0. Some residual proteolytic activity was detected in the Dspw mutant, indicating the existence of additional alkaline proteases (CB895877, EGR50513.1, EGR45552.1, EGR46243.1 and EGR45779.1). Endoglucanases derived from fungi have their highest activity at acidic pHs. When the pH value increased above 7.0, the enzyme activity almost completely disappeared, thereby limiting the application of fungal endoglucanases under neutral or alkaline conditions (Qin et al., 2008). Alkaline endoglucanases, with activities in the pH range from 6.0 to 8.0, are less aggressive against cotton than acid endoglucanases and do not readily compromise the strength of the fabric (Nakane et al., 2005). In this study, we heterologously expressed EGV, an alkaline endoglucanase from H. insolens, in the Dspw strain and the original strain. As shown in Fig. 3, the alkaline endoglucanase culture of the pWEF31-V/Dspw strain was more stable than that of the pWEF31-V/RUT-C30 strain when they were preserved at 4 °C at neutral pHs between 6.0 and 7.0 for 30 days. In conclusion, we disrupted a major alkaline protease gene in T. reesei RUT-C30. Combined with a pH control strategy, the production and stability of alkaline endoglucanase was improved. Our current knowledge about these proteolytic enzymes from Trichoderma is limited, and the present work provided important insights into the functions of alkaline proteases from T. reesei. Therefore, in future studies, we will disrupt additional protease genes to construct a more robust strain. Acknowledgments This research was supported by National Basic Research Program of China (973, Program No. 2012CB721103), the National High Technology Research and Development Program of China (863, Program No. 2012AA022206) and Fundamental Research Funds of the Central Universities. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.plasmid.2014.01.001. References Arvas, M., Pakula, T., Lanthaler, K., Saloheimo, M., Valkonen, M., Suortti, T., Robson, R., Penttilä, M., 2006. Common features and interesting

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