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Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties
Journal of Biotechnology 65 (1998) 163 – 171
Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties Shou Takashima 1, Hiroshi Iikura, Akira Nakamura, Makoto Hidaka, Haruhiko Masaki, Takeshi Uozumi * Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The Uni6ersity of Tokyo, Yayoi 1 -1 -1, Bunkyo-ku, Tokyo 113, Japan Received 10 November 1997; received in revised form 4 February 1998; accepted 8 June 1998
Abstract We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host. In this system, the expression of T. reesei cellulase genes were regulated under the control of A. oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer. The production of recombinant cellulases by A. oryzae transformants reached a maximum after 3 – 4 days of cultivation. In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation. The recombinant cellulases were purified and characterized. The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes. The optimal temperatures and pHs of recombinant cellulases were 50–70°C and 4–5, respectively. Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Aspergillus oryzae; Cellulase; Trichoderma reesei
1. Introduction
* Corresponding author. Tel.: +81 3 56840387; fax: + 81 3 56840387; e-mail: [email protected] 1 Present address: Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan.
Endoglucanases (EG, EC 3.2.1.4), exo-cellobiohydrolases (CBH, EC 3.2.1.91) and b-glucosidases (BGL, EC 3.2.1.21) are three major types of cellulolytic enzymes. Among the cellulolytic fungi, Trichoderma reesei has very strong cellulose-degrading activity. Its cellulase system has been
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widely studied and is considered to be a rational choice for industrial use. The most highly produced cellulase component of T. reesei is CBHI, which accounts for about 60% of the total secreted protein (Uusitalo et al., 1991). CBHII and EGs (most of which is EG1) account for about 20% and 10% of the total secreted protein, respectively, and BGLs account for only 1% of the total secreted protein (Uusitalo et al., 1991). The high production of CBHs should be related to the strong cellulose-degrading activity. T. reesei cellulase genes have been cloned and sequenced, and it has been shown that CBHs and EGs have characteristic domain structures, consisting of the catalytic domain, the cellulosebinding domain and the hinge region between these two domains, which is rich in serine, threonine and proline residues (Shoemaker et al., 1983, Arsdell et al., 1987, Chen et al., 1987, Saloheimo et al., 1988). For expression of T. reesei cellulase genes, yeast has been widely used as a heterogeneous host, although its expression constructs need cDNA forms of cellulase genes. On the other hand, a filamentous fungus Aspergillus oryzae has been used as a host for high expression of both genomic and cDNA forms of fungal cellulase genes from Humicola species (Dalbøge and Heldt-Hansen, 1994, Takashima et al., 1996). In this study, we have constructed an expression system of genomic forms of T. reesei cellulase genes using A. oryzae as a host, and characterized the enzymatic properties of recombinant cellulases.
2. Materials and methods
2.1. Strains, plasmid and media T. reesei QM9414 was used for DNA isolation. The stock culture was stored on agar (1.5%) slants of MY medium (2% malt extract, 0.2% yeast extract). For chromosomal DNA preparation, the spores were inoculated in the liquid MY medium and grown for 2 days at 30°C with shaking, and mycelia were harvested by filtration. A. oryzae M-2-3 (argB − ) was used
as a host for expression of T. reesei cellulase genes, and the stock culture was stored on DPY medium (Tsuchiya et al., 1994). For construction of expression plasmids of T. reesei cellulase genes, fungal expression vector pAMYB118 was used (Takashima et al., 1997), and argB containing plasmid pSal23 (Gomi et al., 1987) and expression vectors were used for transformation of A. oryzae. Czapek–Dox medium was used for fungal transformation (Tsuchiya et al., 1994). For the expression of T. reesei cellulase genes, A. oryzae transformants were cultivated in CD-P medium (Tsuchiya et al., 1994). For general DNA manipulation, Escherichia coli JM109 and E. coli MV1184 were used as hosts for cloning vector pUC118 (Sambrook et al., 1989). E. coli CJ236 was used for site-directed mutagenesis studies (Sambrook et al., 1989).
2.2. Genomic DNA cloning Fungal chromosomal DNA was prepared as described by Tonouchi et al. (1986). Amplification of the DNA fragments encoding portions of T. reesei cellulases was performed using polymerase chain reaction (PCR) with specific primers for T. reesei cellulase genes: for the cbh1 gene (Shoemaker et al., 1983), 5%-TCAACCGCGGACTGGCATC-3% and 5%-CGACGTCTCGAACTGGGTG-3% were used; for the cbh2 gene (Chen et al., 1987), 5%-CAGTTTGTGGTGTATGACTT-3 and 5%-CTGGCTTGACCCAGACAAA-3% were used; for the egl1 gene (Arsdell et al., 1987), 5%-TGAACCAGTACATGCCCAG-3% and 5%-TGGCCAGGATGTTGGATGG-3% were used; for the egl3 gene (Saloheimo et al., 1988), 5%-ACTTGCCTTACCTCGAAGGT-3% and 5%TCTGCATTCAGCTCAGAGTG-3% were used; for the egl5 gene (Saloheimo et al., 1994), 5%CATCGTGACCATGAAGGCAA-3% and 5%-GCCGGAAGAATTCTAGAGAG-3% were used; for the bgl1 gene (Barnett et al., 1991), 5%-ACAGGCAGCACAGCCTTTAC-3% and 5%-GTGCCAGGCATTGACATGTC-3% were used. The amplified DNA fragments were radiolabeled with [a-32P]dCTP using a random primed DNA labeling kit (Boehringer Mannheim) and used as probes for cloning the corresponding T. reesei
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cellulase gene. Southern hybridization and colony hybridization were done as described previously (Takashima et al., 1997). DNA sequencing was done by the dideoxy chain termination method (Sanger et al., 1977) using a BcaBEST sequencing kit (TaKaRa).
2.3. Site-directed mutagenesis For construction of expression plasmids of T. reesei cellulase genes, appropriate restriction sites were introduced just before the translational initiation codon of each cellulase gene by site-directed mutagenesis (Kunkel, 1985). To introduce EcoRI site just before the start codon of the cbh2 gene, the mutagenic primer 5%-TGATCTTACAAGCGAATTCAGGTGAGCTG-3% was used. To introduce EcoRI site just before the start codon of the egl1 gene, the mutagenic primer 5%-GGGACAACAAGAATTCCTAAGATAGGGGG-3% was used. To introduce EcoRI site just before the start codon of the egl5 gene, the mutagenic primer 5%-CTTCCATCTCGTGAATTCCTTGTAACCAT-3% was used. To introduce SmaI site just before the start codon of the bgl1 gene, the mutagenic primer 5%-ATTCTGTTGAGCCCGGGCAGAAATGCGTTAC-3% was used.
2.4. Fungal transformation Transformation of A. oryzae was done according to the method of Gomi et al. (1987).
2.5. Enzyme assay The enzyme activities towards carboxymethylcellulose, Avicel, xylan, p-nitrophenyl-b-D-glucoside and p-nitrophenyl-b-D-cellobioside were measured as described previously with minor modification (Takashima et al., 1996, 1997). For the optimal temperature, the enzyme activities were measured as described above at various temperatures for the appropriate time. For the thermal stability, the enzyme solutions were treated at various temperatures for 10 min without substrate, then the remaining activities were measured. For the optimal pH, the enzyme activi-
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ties were measured as described above at various pHs. For pH stability, the enzyme solutions were treated at various pHs at 4°C for 20 h, then the enzyme solutions were adjusted to pH 4 or 5 and the remaining activities were measured. One unit of the enzyme was defined as the activity producing 1 mmol min − 1 of reducing sugars in glucose or xylose equivalents or p-nitrophenol under these assay conditions.
2.6. Protein purification To purify the recombinant cellulases produced by A. oryzae transformants, they were cultivated for 4 days in CD-P medium and the culture supernatants were obtained by filtration. Except the filtrate containing recombinant CBHI, these filtrates were buffered by adding 1/10 vol. of 100 mM Tris–HCl buffer, pH 7.5, and gently mixed for 1 h at room temperature with SuperQ Toyopearl 650M (Tosoh), previously equilibrated with 10 mM Tris–HCl buffer, pH 7.5. After centrifugation, solid ammonium sulfate was added to the supernatants to 40% saturation and the precipitates were removed by filtration. The samples were loaded on a column of Phenyl Toyopearl 650M (2.5× 5.0 cm, Tosoh), previously equilibrated with 50 mM Tris–HCl buffer, pH 7.5, containing 40% saturated ammonium sulfate. The enzymes were eluted with a linear gradient of 40–0% saturation of ammonium sulfate in the same buffer. Fractions showing cellulase activity were collected and ultrafiltrated with Centriprep 30 (Millipore), and the purified cellulases were obtained. For purification of CBHI, the culture filtrate was processed by Phenyl Toyopearl column chromatography as described above, and fractions showing cellulase activity were collected and ultrafiltrated with Centriprep 30. Then the desalted sample was buffered by adding 1/10 vol. of 100 mM Tris–HCl buffer, pH 7.5, and loaded on a column of MonoQ 10/10 (Pharmacia), previously equilibrated with 10 mM Tris–HCl buffer, pH 7.5. The enzyme was eluted with a linear gradient of 0–0.5 M NaCl in the same buffer, and the purified CBHI was obtained. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) was done as described by Laemmli (1970).
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Fig. 1. T. reesei cellulase genes used for construction of expression vectors. The coding regions are shown by rectangles. Introns are shown by black rectangles. Untranslated regions are shown by bars.
Protein content was measured by a dye-binding assay kit (Protein Assay Kit, Bio-Rad) using gglobulin as the standard.
3. Results
3.1. Construction of expression plasmids PCRs were performed using the specific primers for each of T. reesei cellulase gene and the amplified fragments encoding a portion of each of T. reesei cellulase gene were radiolabeled and used as probes for screening of the corresponding genomic clones of T. reesei cellulase genes. Thus the 6.8-kb PstI fragment containing the cbh1 gene, the 4.1-kb EcoRI fragment containing the cbh2 gene, the 4.2-kb EcoRI fragment containing the egl1 gene, the 7.4-kb EcoRI fragment containing the egl3 gene, the 6.3-kb HindIII fragment containing the egl5 gene and the 6.0-kb HindIII fragment containing the bgl1 gene were cloned using the colony hybridization technique. For construction of the expression system of T. reesei cellulase genes in A. oryzae, we used the expression vector pAMYB118, in which the expression of the cellulase genes should be regu-
lated under the amyB (encoding Taka-amylase) promoter control that is inducible by maltose. From the cloned DNA fragment containing the cbh1 gene, the 4.3-kb SacII fragment was excised and blunt ended, and subcloned into the SmaI site of pAMYB118, giving rise to the expression vector pAMYB-TrCBHI. From the cloned DNA fragment containing the egl3 gene, the 2.0-kb EcoRV fragment was excised and subcloned into the SmaI site of pAMYB118, giving rise to the expression vector pAMYBTrEGIII. To construct the expression vectors of other cellulase genes, site-directed mutageneses were performed to introduce the appropriate restriction sites just before the translational initiation codon of these genes, and, using the introduced restriction sites, appropriate DNA fragments containing each cellulase gene (the 3.6-kb EcoRI fragment for the cbh2 gene, the 3.2-kb EcoRI-HindIII fragment for the egl1 gene, the 3.3-kb EcoRI-HindIII fragment for the egl5 gene and the 2.7-kb SmaI fragment for the bgl1 gene; Fig. 1) were excised and subcloned into pAMYB118. Thus the expression vectors of the cbh2, egl1, egl5 and bgl1 genes were constructed, giving rise to pAMYB-TrCBHII, pAMYB-TrEGI, pAMYB-TrEGV and pAMYBTrBGL1, respectively.
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Fig. 2. Recombinant cellulase production by A. oryzae transformants. Culture supernatants (15 ml, each lane) of (a) A. oryzae M-2-3, (b) transformant producing EGI, (c) transformant producing CBHI and (d) transformant producing BGLI were analysed by SDS-PAGE. The 50-kDa band seen on all panels corresponds to the endogeneous A. oryzae Taka-amylase (Dalbøge and Heldt-Hansen, 1994).
3.2. Expression of T. reesei cellulase genes in A. oryzae The expression vectors (20 mg each) were introduced into A. oryzae M-2-3, an arginine auxotroph, by co-transformation with 20 mg of an argB-containing plasmid, pSal23. The arg + transformants were isolated and grown in 10 ml of CD-P medium (containing maltose as a carbon source) for 4 days at 30°C and assayed for enzyme activities. The clones showing the highest activity of each enzyme were selected from the transformants and further analysed. The cellulase activities of these transformants reached a maximum after 3 or 4 days of cultivation. These transformants showed several to hundreds times cellulase activities compared to the control transformant transformed with pAMYB118 and pSal23. Culture supernatants of transformants were analysed by SDS-PAGE (Fig. 2). A significant band that was not observed in the culture supernatant of A.
oryzae was observed in those of transformants. These bands were identified as recombinant cellulases by purification and characterization, including their molecular weight (Fig. 4). The cellulase activities of some transformants were greatly reduced in the late stage of cultivation, probably because of the proteolysis of T. reesei cellulases by the endogenous proteases of A. oryzae (Fig. 2). To investigate whether T. reesei cellulase genes had integrated into the genome of the transformants, we performed Southern hybridization of chromosomal DNA of transformants after complete digestion with excess amount of restriction enzymes for 20 h. The results showed that a few to multiple copies of T. reesei cellulase genes had integrated into the genome of the transformants (Fig. 3), although some weakly hybridizing bands seen in Fig. 3 may be the result of the disruption of the introduced genes. However, no relation was observed between the copy numbers of the integrated genes and intensity of cellulase activity.
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3.3. Purification of recombinant cellulases produced by A. oryzae From the culture supernatants of transformants producing recombinant cellulases after 4 days of cultivation in CD-P medium, we purified each
Fig. 4. SDS-PAGE of purified recombinant T. reesei cellulases. Lane 1, BGLI; lane 2, EGI; lane 3, EGIII; lane 4, EGV; lane 5, CBHI; lane 6, CBHII. M, molecular mass standard.
Fig. 3. Southern blot analyses of A. oryzae transformants. Chromosomal DNAs of A. oryzae M-2-3 (lane 1), control transformant transformed with pAMYB118 and pSal23 (lane 2) and transformant producing recombinant cellulase (lanes 3, 4) were digested with restriction enzymes and analyzed by Southern blot. Southern blot analyses of the transformants producing: (a) CBHI, digested with PstI (P) (lanes 1–3) and with SphI (S) (lane 4); (b) CBHII, digested with EcoRI (E) (lanes 1 – 3) and with SphI (S) (lane 4); (c) BGLI, digested with SmaI (Sm) and HindIII (H) (lanes 1–3) and with HindIII (lane 4); (d) EGI, digested with EcoRI (E) and HindIII (H) (lanes 1 – 3) and with HindIII (lane 4); (e) EGIII, digested with EcoRI (E) and HindIII (H) (lanes 1–3) and with EcoRI (lane 4); (f) EGV, digested with EcoRI (E) (lanes 1–3) and with HindIII (H) (lane 4).
enzyme by combination of anion-exchange chromatography and hydrophobic chromatography (Fig. 4). Although the amount of recombinant cellulases produced by each transformant differed, some transformants produced several hundred mg ml − 1 of recombinant cellulases in the culture supernatant. The molecular masses of recombinant cellulases produced by A. oryzae were estimated by SDS-PAGE, which were more or less larger than those of native enzymes produced by T. reesei but smaller than those of recombinant cellulases produced by yeast (Table 1). These molecular masses of native and recombinant enzymes were also more or less larger than the corresponding calculated molecular masses of each enzyme (Table 1), suggesting the glycosylated forms of these enzymes. Periodic acid–Schiff staining of recombinant cellulases produced by A. oryzae showed glycosylation of these enzymes (data not shown), suggesting that the discrepancies between the molecular masses of native cellulases and recombinant cellulases were caused by the difference of the mode of glycosylation. Comparison of the molecular masses of recombinant cellulases produced by A. oryzae with the corresponding calculated molecular masses of each enzyme shows that the levels of glycosylation of CBHI, CBHII, EGI and EGV are substantially higher than those of EGIII and BGLI. The optimal temperatures of these recombinant cellulases were between 50 and 70°C and these
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Table 1 Comparison of the molecular masses of native and recombinant T. reesei cellulases Enzyme
Amino acid residues of mature protein
Molecular mass (kDa) Calculated
CBHI CBHII EGI EGIII EGV BGLI a
From From c From d From b
496 447 437 397 225 713
52.2 47.2 46 42.2 22.8 72.3
Producer T. reesei
Yeast
A. oryzae
60 a 55 b 50 a 48 c – 75 d
100–200 a – 60–200 a – – 90 d
75 67–75 67 55 35–40 80
Arsdell et al. (1987). Henrissat et al. (1985). Macarro´n et al. (1993). Cummings and Fowler (1996).
enzymes were stable up to 45°C for at least 10 min (Table 2). The optimal pHs of these enzymes were between 4 and 5. These enzymes except BGL1 were stable at least between pH 3 and 6 at 4°C for 20 h, but BGLI was rather unstable at a pH of less than 5 (Table 2).
3.4. Catalytic properties The purified cellulases were tested for substrate specificities (Table 3). Recombinant EGI showed broad substrate specificities and high activity when compared with other cellulases investigated here. EGI produced by yeast also showed broad substrate specificities and high activity toward several substrates such as b-glucan, hydroxyethyl cellulose, RBB-xylan, methylumbelliferyl cellobioside and methylumbelliferyl lactoside (Saloheimo et al., 1994). EGV showed narrow substrate specificities and rather weak activity toward carboxymethylcellulose compared with EGI and EGIII. This tendency was also observed in the case of recombinant EGV produced by yeast (Saloheimo et al., 1994). Recombinant CBHI and CBHII showed relatively low activity toward Avicel when compared with native enzymes (Tomme et al., 1988). Probably the modes of glycosylation of recombinant CBHI and CBHII differ from native enzymes so that recombinant CBHI and CBHII may not attack insoluble substrate effectively.
4. Discussion In this study, we have established a high expression system of T. reesei cellulase genes using A. oryzae as a host and analysed enzymatic properties of recombinant cellulases. The T. reesei cellulase system has a very strong cellulolytic activity so that its application to industrial use seems to be attractive. So far, overexpression of T. reesei cellulase genes has been performed by using yeast as a main host (Zurbriggen et al., 1990), but cDNA clones are needed when using yeast expression system in general. In this study, we have shown that genomic clones of T. reesei cellulase genes work well in the A. oryzae expression system. Probably, the splicing mechanism of A. oryzae is similar to that of T. reesei. A. oryzae has been used in the industrial process for a long time and considered to be a safe host in the recombinant studies like yeast. So, application of the A. oryzae expression system of T. reesei cellulase genes to industrial use may be realized. In the case of yeast, highly glycosylated forms of recombinant cellulases have been produced (Arsdell et al., 1987, Cummings and Fowler, 1996). In some cases, high glycosylation may cause the decrease of the enzymatic activity. On the other hand, A. oryzae produces recombinant cellulases whose glycosylation levels are similar to those of native enzymes, although some glycosyla-
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Table 2 Enzymatic properties of recombinant cellulases Enzyme
Optimal temperature (°C)
Thermal stability (°C)
Optimal pH
pH stability
Substrate
CBHI CBHII EGI EGIII EGV BGLI
60 55 60 70 50–65 70
50 50 45 55 80 55
4 5 4 4 4 4
2–10 3–8 2–8 2–6 2–6 5–9
PNPC Avicel CMC CMC CMC PNPG
a
The enzyme retains more than 75% of the relative activity under the indicated temperature. The enzyme retains more than 75% of the relative activity between the indicated pH range. CMC, carboxymethylcellulose; PNPC, p-nitrophenyl-b-D-cellobioside; PNPG, p-nitrophenyl-b-D-glucoside.
b
tion patterns seem to differ from those of native enzymes. Among the recombinant cellulases produced by A. oryzae, EGI showed broad substrate specificities and high activity toward several substrates. EGI is a major endoglucanase component of T. reesei cellulase system (Uusitalo et al., 1991) and its enzymatic properties seem to reflect strong cellulolytic activity of T. reesei cellulase system. Although CBHI and CBHII are the main cellulase components of T. reesei cellulase system, these enzymes showed rather narrow substrate specificities and weak activity toward several substrates. But these exoglucanases show strong cellulolytic activity when combined with other cellulase components such as endoglucanases (Henrissat et al., 1985). This exo–endo mode of synergism is necessary for efficient hydrolysis of insoluble cellulosic substrates (Henrissat et al., 1985). Addition of these recombinant cellulase components to other cellulase systems may bring about such synergistic actions and improve the overall cellulolytic activity. Table 3 Substrate specificity of recombinant cellulases (U mg−1) Enzyme
Avicel
CMC
Xylan
PNPG
PNPC
CBHI CBHII EGI EGIII EGV BGLI
0.0105 0.0205 0.0140 0.0116 ND ND
ND ND 59.8 30.7 9.22 ND
ND Trace 46.2 ND ND ND
Trace ND ND ND ND 767.7
0.355 ND 9.19 ND ND 0.894
ND, not detected.
The activity of T. reesei EGV is rather weak, so its activity was detected by expression cloning technique for the first time (Saloheimo et al., 1994). So EGV is considered to be a minor cellulase component in T. reesei cellulase system and its purification from T. reesei culture supernatant may be difficult. The expression system of A. oryzae would be useful for analyses of such minor enzymes as well as cellulase components. Acknowledgements We wish to thank Dr K. Kitamoto for generously providing the expression system of A. oryzae. This work was performed using the facilities of the Biotechnology Reseach Center, The University of Tokyo. References Arsdell, J.N.V., Kwok, S., Schweickart, V.L., Ladner, M.B., Gelfand, D.H., Innis, M.A., 1987. Cloning, characterization, and expression in Saccharomyces cere6isiae of endoglucanase I from Trichoderma reesei. Biotechnology 5, 60 – 64. Barnett, C.C., Berka, R.M., Fowler, T., 1991. Cloning and amplification of the gene encoding an extracellular b-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates. Biotechnology 9, 562 – 567. Chen, C.M., Gritzali, M., Stafford, D.W., 1987. Nucleotide sequence and deduced primary structure of cellobiohydrolase II from Trichoderma reesei. Biotechnology 5, 274 – 278. Cummings, C., Fowler, T., 1996. Secretion of Trichoderma reesei b-glucosidase by Saccharomyces cere6isiae. Curr. Genet. 29, 227 – 233.
S. Takashima et al. / Journal of Biotechnology 65 (1998) 163–171 Dalbøge, H., Heldt-Hansen, H.P., 1994. A novel method for efficient expression cloning of fungal enzyme genes. Mol. Gen. Genet. 243, 253–260. Gomi, K., Iimura, Y., Hara, S., 1987. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem. 51, 2549 – 2555. Henrissat, B., Driguez, H., Viet, C., Schu¨lein, M., 1985. Synergism of cellulases from Trichoderma reesei in the degradation of cellulose. Biotechnology 3, 722–726. Kunkel, T.A., 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488 – 492. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Macarro´n, R., Acebal, C., Castillo´n, M.P., Manuel Domı´nguez, J., Mata, I.D.L., Pettersson, G., Tomme, P., Claeyssens, M., 1993. Mode of action of endoglucanase III from Trichoderma reesei. Biochem. J. 289, 867–873. Saloheimo, M., Lehtovaara, P., Penttila¨, M., Teeri, T.T., Stu¨hlberg, J., Johansson, G., Pettersson, G., Claeyssens, M., Tomme, P., Knowles, J.K.C., 1988. EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. Gene 63, 11–21. Saloheimo, A., Henrissat, B., Hoffre´n, A.-M., Teleman, O., Penttila¨, M., 1994. A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast. Mol. Microbiol. 13, 219–228. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 – 5467.
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Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties Shou Takashima 1, Hiroshi Iikura, Akira Nakamura, Makoto Hidaka, Haruhiko Masaki, Takeshi Uozumi * Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The Uni6ersity of Tokyo, Yayoi 1 -1 -1, Bunkyo-ku, Tokyo 113, Japan Received 10 November 1997; received in revised form 4 February 1998; accepted 8 June 1998
Abstract We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host. In this system, the expression of T. reesei cellulase genes were regulated under the control of A. oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer. The production of recombinant cellulases by A. oryzae transformants reached a maximum after 3 – 4 days of cultivation. In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation. The recombinant cellulases were purified and characterized. The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes. The optimal temperatures and pHs of recombinant cellulases were 50–70°C and 4–5, respectively. Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Aspergillus oryzae; Cellulase; Trichoderma reesei
1. Introduction
* Corresponding author. Tel.: +81 3 56840387; fax: + 81 3 56840387; e-mail: [email protected] 1 Present address: Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan.
Endoglucanases (EG, EC 3.2.1.4), exo-cellobiohydrolases (CBH, EC 3.2.1.91) and b-glucosidases (BGL, EC 3.2.1.21) are three major types of cellulolytic enzymes. Among the cellulolytic fungi, Trichoderma reesei has very strong cellulose-degrading activity. Its cellulase system has been
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widely studied and is considered to be a rational choice for industrial use. The most highly produced cellulase component of T. reesei is CBHI, which accounts for about 60% of the total secreted protein (Uusitalo et al., 1991). CBHII and EGs (most of which is EG1) account for about 20% and 10% of the total secreted protein, respectively, and BGLs account for only 1% of the total secreted protein (Uusitalo et al., 1991). The high production of CBHs should be related to the strong cellulose-degrading activity. T. reesei cellulase genes have been cloned and sequenced, and it has been shown that CBHs and EGs have characteristic domain structures, consisting of the catalytic domain, the cellulosebinding domain and the hinge region between these two domains, which is rich in serine, threonine and proline residues (Shoemaker et al., 1983, Arsdell et al., 1987, Chen et al., 1987, Saloheimo et al., 1988). For expression of T. reesei cellulase genes, yeast has been widely used as a heterogeneous host, although its expression constructs need cDNA forms of cellulase genes. On the other hand, a filamentous fungus Aspergillus oryzae has been used as a host for high expression of both genomic and cDNA forms of fungal cellulase genes from Humicola species (Dalbøge and Heldt-Hansen, 1994, Takashima et al., 1996). In this study, we have constructed an expression system of genomic forms of T. reesei cellulase genes using A. oryzae as a host, and characterized the enzymatic properties of recombinant cellulases.
2. Materials and methods
2.1. Strains, plasmid and media T. reesei QM9414 was used for DNA isolation. The stock culture was stored on agar (1.5%) slants of MY medium (2% malt extract, 0.2% yeast extract). For chromosomal DNA preparation, the spores were inoculated in the liquid MY medium and grown for 2 days at 30°C with shaking, and mycelia were harvested by filtration. A. oryzae M-2-3 (argB − ) was used
as a host for expression of T. reesei cellulase genes, and the stock culture was stored on DPY medium (Tsuchiya et al., 1994). For construction of expression plasmids of T. reesei cellulase genes, fungal expression vector pAMYB118 was used (Takashima et al., 1997), and argB containing plasmid pSal23 (Gomi et al., 1987) and expression vectors were used for transformation of A. oryzae. Czapek–Dox medium was used for fungal transformation (Tsuchiya et al., 1994). For the expression of T. reesei cellulase genes, A. oryzae transformants were cultivated in CD-P medium (Tsuchiya et al., 1994). For general DNA manipulation, Escherichia coli JM109 and E. coli MV1184 were used as hosts for cloning vector pUC118 (Sambrook et al., 1989). E. coli CJ236 was used for site-directed mutagenesis studies (Sambrook et al., 1989).
2.2. Genomic DNA cloning Fungal chromosomal DNA was prepared as described by Tonouchi et al. (1986). Amplification of the DNA fragments encoding portions of T. reesei cellulases was performed using polymerase chain reaction (PCR) with specific primers for T. reesei cellulase genes: for the cbh1 gene (Shoemaker et al., 1983), 5%-TCAACCGCGGACTGGCATC-3% and 5%-CGACGTCTCGAACTGGGTG-3% were used; for the cbh2 gene (Chen et al., 1987), 5%-CAGTTTGTGGTGTATGACTT-3 and 5%-CTGGCTTGACCCAGACAAA-3% were used; for the egl1 gene (Arsdell et al., 1987), 5%-TGAACCAGTACATGCCCAG-3% and 5%-TGGCCAGGATGTTGGATGG-3% were used; for the egl3 gene (Saloheimo et al., 1988), 5%-ACTTGCCTTACCTCGAAGGT-3% and 5%TCTGCATTCAGCTCAGAGTG-3% were used; for the egl5 gene (Saloheimo et al., 1994), 5%CATCGTGACCATGAAGGCAA-3% and 5%-GCCGGAAGAATTCTAGAGAG-3% were used; for the bgl1 gene (Barnett et al., 1991), 5%-ACAGGCAGCACAGCCTTTAC-3% and 5%-GTGCCAGGCATTGACATGTC-3% were used. The amplified DNA fragments were radiolabeled with [a-32P]dCTP using a random primed DNA labeling kit (Boehringer Mannheim) and used as probes for cloning the corresponding T. reesei
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cellulase gene. Southern hybridization and colony hybridization were done as described previously (Takashima et al., 1997). DNA sequencing was done by the dideoxy chain termination method (Sanger et al., 1977) using a BcaBEST sequencing kit (TaKaRa).
2.3. Site-directed mutagenesis For construction of expression plasmids of T. reesei cellulase genes, appropriate restriction sites were introduced just before the translational initiation codon of each cellulase gene by site-directed mutagenesis (Kunkel, 1985). To introduce EcoRI site just before the start codon of the cbh2 gene, the mutagenic primer 5%-TGATCTTACAAGCGAATTCAGGTGAGCTG-3% was used. To introduce EcoRI site just before the start codon of the egl1 gene, the mutagenic primer 5%-GGGACAACAAGAATTCCTAAGATAGGGGG-3% was used. To introduce EcoRI site just before the start codon of the egl5 gene, the mutagenic primer 5%-CTTCCATCTCGTGAATTCCTTGTAACCAT-3% was used. To introduce SmaI site just before the start codon of the bgl1 gene, the mutagenic primer 5%-ATTCTGTTGAGCCCGGGCAGAAATGCGTTAC-3% was used.
2.4. Fungal transformation Transformation of A. oryzae was done according to the method of Gomi et al. (1987).
2.5. Enzyme assay The enzyme activities towards carboxymethylcellulose, Avicel, xylan, p-nitrophenyl-b-D-glucoside and p-nitrophenyl-b-D-cellobioside were measured as described previously with minor modification (Takashima et al., 1996, 1997). For the optimal temperature, the enzyme activities were measured as described above at various temperatures for the appropriate time. For the thermal stability, the enzyme solutions were treated at various temperatures for 10 min without substrate, then the remaining activities were measured. For the optimal pH, the enzyme activi-
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ties were measured as described above at various pHs. For pH stability, the enzyme solutions were treated at various pHs at 4°C for 20 h, then the enzyme solutions were adjusted to pH 4 or 5 and the remaining activities were measured. One unit of the enzyme was defined as the activity producing 1 mmol min − 1 of reducing sugars in glucose or xylose equivalents or p-nitrophenol under these assay conditions.
2.6. Protein purification To purify the recombinant cellulases produced by A. oryzae transformants, they were cultivated for 4 days in CD-P medium and the culture supernatants were obtained by filtration. Except the filtrate containing recombinant CBHI, these filtrates were buffered by adding 1/10 vol. of 100 mM Tris–HCl buffer, pH 7.5, and gently mixed for 1 h at room temperature with SuperQ Toyopearl 650M (Tosoh), previously equilibrated with 10 mM Tris–HCl buffer, pH 7.5. After centrifugation, solid ammonium sulfate was added to the supernatants to 40% saturation and the precipitates were removed by filtration. The samples were loaded on a column of Phenyl Toyopearl 650M (2.5× 5.0 cm, Tosoh), previously equilibrated with 50 mM Tris–HCl buffer, pH 7.5, containing 40% saturated ammonium sulfate. The enzymes were eluted with a linear gradient of 40–0% saturation of ammonium sulfate in the same buffer. Fractions showing cellulase activity were collected and ultrafiltrated with Centriprep 30 (Millipore), and the purified cellulases were obtained. For purification of CBHI, the culture filtrate was processed by Phenyl Toyopearl column chromatography as described above, and fractions showing cellulase activity were collected and ultrafiltrated with Centriprep 30. Then the desalted sample was buffered by adding 1/10 vol. of 100 mM Tris–HCl buffer, pH 7.5, and loaded on a column of MonoQ 10/10 (Pharmacia), previously equilibrated with 10 mM Tris–HCl buffer, pH 7.5. The enzyme was eluted with a linear gradient of 0–0.5 M NaCl in the same buffer, and the purified CBHI was obtained. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) was done as described by Laemmli (1970).
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Fig. 1. T. reesei cellulase genes used for construction of expression vectors. The coding regions are shown by rectangles. Introns are shown by black rectangles. Untranslated regions are shown by bars.
Protein content was measured by a dye-binding assay kit (Protein Assay Kit, Bio-Rad) using gglobulin as the standard.
3. Results
3.1. Construction of expression plasmids PCRs were performed using the specific primers for each of T. reesei cellulase gene and the amplified fragments encoding a portion of each of T. reesei cellulase gene were radiolabeled and used as probes for screening of the corresponding genomic clones of T. reesei cellulase genes. Thus the 6.8-kb PstI fragment containing the cbh1 gene, the 4.1-kb EcoRI fragment containing the cbh2 gene, the 4.2-kb EcoRI fragment containing the egl1 gene, the 7.4-kb EcoRI fragment containing the egl3 gene, the 6.3-kb HindIII fragment containing the egl5 gene and the 6.0-kb HindIII fragment containing the bgl1 gene were cloned using the colony hybridization technique. For construction of the expression system of T. reesei cellulase genes in A. oryzae, we used the expression vector pAMYB118, in which the expression of the cellulase genes should be regu-
lated under the amyB (encoding Taka-amylase) promoter control that is inducible by maltose. From the cloned DNA fragment containing the cbh1 gene, the 4.3-kb SacII fragment was excised and blunt ended, and subcloned into the SmaI site of pAMYB118, giving rise to the expression vector pAMYB-TrCBHI. From the cloned DNA fragment containing the egl3 gene, the 2.0-kb EcoRV fragment was excised and subcloned into the SmaI site of pAMYB118, giving rise to the expression vector pAMYBTrEGIII. To construct the expression vectors of other cellulase genes, site-directed mutageneses were performed to introduce the appropriate restriction sites just before the translational initiation codon of these genes, and, using the introduced restriction sites, appropriate DNA fragments containing each cellulase gene (the 3.6-kb EcoRI fragment for the cbh2 gene, the 3.2-kb EcoRI-HindIII fragment for the egl1 gene, the 3.3-kb EcoRI-HindIII fragment for the egl5 gene and the 2.7-kb SmaI fragment for the bgl1 gene; Fig. 1) were excised and subcloned into pAMYB118. Thus the expression vectors of the cbh2, egl1, egl5 and bgl1 genes were constructed, giving rise to pAMYB-TrCBHII, pAMYB-TrEGI, pAMYB-TrEGV and pAMYBTrBGL1, respectively.
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Fig. 2. Recombinant cellulase production by A. oryzae transformants. Culture supernatants (15 ml, each lane) of (a) A. oryzae M-2-3, (b) transformant producing EGI, (c) transformant producing CBHI and (d) transformant producing BGLI were analysed by SDS-PAGE. The 50-kDa band seen on all panels corresponds to the endogeneous A. oryzae Taka-amylase (Dalbøge and Heldt-Hansen, 1994).
3.2. Expression of T. reesei cellulase genes in A. oryzae The expression vectors (20 mg each) were introduced into A. oryzae M-2-3, an arginine auxotroph, by co-transformation with 20 mg of an argB-containing plasmid, pSal23. The arg + transformants were isolated and grown in 10 ml of CD-P medium (containing maltose as a carbon source) for 4 days at 30°C and assayed for enzyme activities. The clones showing the highest activity of each enzyme were selected from the transformants and further analysed. The cellulase activities of these transformants reached a maximum after 3 or 4 days of cultivation. These transformants showed several to hundreds times cellulase activities compared to the control transformant transformed with pAMYB118 and pSal23. Culture supernatants of transformants were analysed by SDS-PAGE (Fig. 2). A significant band that was not observed in the culture supernatant of A.
oryzae was observed in those of transformants. These bands were identified as recombinant cellulases by purification and characterization, including their molecular weight (Fig. 4). The cellulase activities of some transformants were greatly reduced in the late stage of cultivation, probably because of the proteolysis of T. reesei cellulases by the endogenous proteases of A. oryzae (Fig. 2). To investigate whether T. reesei cellulase genes had integrated into the genome of the transformants, we performed Southern hybridization of chromosomal DNA of transformants after complete digestion with excess amount of restriction enzymes for 20 h. The results showed that a few to multiple copies of T. reesei cellulase genes had integrated into the genome of the transformants (Fig. 3), although some weakly hybridizing bands seen in Fig. 3 may be the result of the disruption of the introduced genes. However, no relation was observed between the copy numbers of the integrated genes and intensity of cellulase activity.
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3.3. Purification of recombinant cellulases produced by A. oryzae From the culture supernatants of transformants producing recombinant cellulases after 4 days of cultivation in CD-P medium, we purified each
Fig. 4. SDS-PAGE of purified recombinant T. reesei cellulases. Lane 1, BGLI; lane 2, EGI; lane 3, EGIII; lane 4, EGV; lane 5, CBHI; lane 6, CBHII. M, molecular mass standard.
Fig. 3. Southern blot analyses of A. oryzae transformants. Chromosomal DNAs of A. oryzae M-2-3 (lane 1), control transformant transformed with pAMYB118 and pSal23 (lane 2) and transformant producing recombinant cellulase (lanes 3, 4) were digested with restriction enzymes and analyzed by Southern blot. Southern blot analyses of the transformants producing: (a) CBHI, digested with PstI (P) (lanes 1–3) and with SphI (S) (lane 4); (b) CBHII, digested with EcoRI (E) (lanes 1 – 3) and with SphI (S) (lane 4); (c) BGLI, digested with SmaI (Sm) and HindIII (H) (lanes 1–3) and with HindIII (lane 4); (d) EGI, digested with EcoRI (E) and HindIII (H) (lanes 1 – 3) and with HindIII (lane 4); (e) EGIII, digested with EcoRI (E) and HindIII (H) (lanes 1–3) and with EcoRI (lane 4); (f) EGV, digested with EcoRI (E) (lanes 1–3) and with HindIII (H) (lane 4).
enzyme by combination of anion-exchange chromatography and hydrophobic chromatography (Fig. 4). Although the amount of recombinant cellulases produced by each transformant differed, some transformants produced several hundred mg ml − 1 of recombinant cellulases in the culture supernatant. The molecular masses of recombinant cellulases produced by A. oryzae were estimated by SDS-PAGE, which were more or less larger than those of native enzymes produced by T. reesei but smaller than those of recombinant cellulases produced by yeast (Table 1). These molecular masses of native and recombinant enzymes were also more or less larger than the corresponding calculated molecular masses of each enzyme (Table 1), suggesting the glycosylated forms of these enzymes. Periodic acid–Schiff staining of recombinant cellulases produced by A. oryzae showed glycosylation of these enzymes (data not shown), suggesting that the discrepancies between the molecular masses of native cellulases and recombinant cellulases were caused by the difference of the mode of glycosylation. Comparison of the molecular masses of recombinant cellulases produced by A. oryzae with the corresponding calculated molecular masses of each enzyme shows that the levels of glycosylation of CBHI, CBHII, EGI and EGV are substantially higher than those of EGIII and BGLI. The optimal temperatures of these recombinant cellulases were between 50 and 70°C and these
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Table 1 Comparison of the molecular masses of native and recombinant T. reesei cellulases Enzyme
Amino acid residues of mature protein
Molecular mass (kDa) Calculated
CBHI CBHII EGI EGIII EGV BGLI a
From From c From d From b
496 447 437 397 225 713
52.2 47.2 46 42.2 22.8 72.3
Producer T. reesei
Yeast
A. oryzae
60 a 55 b 50 a 48 c – 75 d
100–200 a – 60–200 a – – 90 d
75 67–75 67 55 35–40 80
Arsdell et al. (1987). Henrissat et al. (1985). Macarro´n et al. (1993). Cummings and Fowler (1996).
enzymes were stable up to 45°C for at least 10 min (Table 2). The optimal pHs of these enzymes were between 4 and 5. These enzymes except BGL1 were stable at least between pH 3 and 6 at 4°C for 20 h, but BGLI was rather unstable at a pH of less than 5 (Table 2).
3.4. Catalytic properties The purified cellulases were tested for substrate specificities (Table 3). Recombinant EGI showed broad substrate specificities and high activity when compared with other cellulases investigated here. EGI produced by yeast also showed broad substrate specificities and high activity toward several substrates such as b-glucan, hydroxyethyl cellulose, RBB-xylan, methylumbelliferyl cellobioside and methylumbelliferyl lactoside (Saloheimo et al., 1994). EGV showed narrow substrate specificities and rather weak activity toward carboxymethylcellulose compared with EGI and EGIII. This tendency was also observed in the case of recombinant EGV produced by yeast (Saloheimo et al., 1994). Recombinant CBHI and CBHII showed relatively low activity toward Avicel when compared with native enzymes (Tomme et al., 1988). Probably the modes of glycosylation of recombinant CBHI and CBHII differ from native enzymes so that recombinant CBHI and CBHII may not attack insoluble substrate effectively.
4. Discussion In this study, we have established a high expression system of T. reesei cellulase genes using A. oryzae as a host and analysed enzymatic properties of recombinant cellulases. The T. reesei cellulase system has a very strong cellulolytic activity so that its application to industrial use seems to be attractive. So far, overexpression of T. reesei cellulase genes has been performed by using yeast as a main host (Zurbriggen et al., 1990), but cDNA clones are needed when using yeast expression system in general. In this study, we have shown that genomic clones of T. reesei cellulase genes work well in the A. oryzae expression system. Probably, the splicing mechanism of A. oryzae is similar to that of T. reesei. A. oryzae has been used in the industrial process for a long time and considered to be a safe host in the recombinant studies like yeast. So, application of the A. oryzae expression system of T. reesei cellulase genes to industrial use may be realized. In the case of yeast, highly glycosylated forms of recombinant cellulases have been produced (Arsdell et al., 1987, Cummings and Fowler, 1996). In some cases, high glycosylation may cause the decrease of the enzymatic activity. On the other hand, A. oryzae produces recombinant cellulases whose glycosylation levels are similar to those of native enzymes, although some glycosyla-
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Table 2 Enzymatic properties of recombinant cellulases Enzyme
Optimal temperature (°C)
Thermal stability (°C)
Optimal pH
pH stability
Substrate
CBHI CBHII EGI EGIII EGV BGLI
60 55 60 70 50–65 70
50 50 45 55 80 55
4 5 4 4 4 4
2–10 3–8 2–8 2–6 2–6 5–9
PNPC Avicel CMC CMC CMC PNPG
a
The enzyme retains more than 75% of the relative activity under the indicated temperature. The enzyme retains more than 75% of the relative activity between the indicated pH range. CMC, carboxymethylcellulose; PNPC, p-nitrophenyl-b-D-cellobioside; PNPG, p-nitrophenyl-b-D-glucoside.
b
tion patterns seem to differ from those of native enzymes. Among the recombinant cellulases produced by A. oryzae, EGI showed broad substrate specificities and high activity toward several substrates. EGI is a major endoglucanase component of T. reesei cellulase system (Uusitalo et al., 1991) and its enzymatic properties seem to reflect strong cellulolytic activity of T. reesei cellulase system. Although CBHI and CBHII are the main cellulase components of T. reesei cellulase system, these enzymes showed rather narrow substrate specificities and weak activity toward several substrates. But these exoglucanases show strong cellulolytic activity when combined with other cellulase components such as endoglucanases (Henrissat et al., 1985). This exo–endo mode of synergism is necessary for efficient hydrolysis of insoluble cellulosic substrates (Henrissat et al., 1985). Addition of these recombinant cellulase components to other cellulase systems may bring about such synergistic actions and improve the overall cellulolytic activity. Table 3 Substrate specificity of recombinant cellulases (U mg−1) Enzyme
Avicel
CMC
Xylan
PNPG
PNPC
CBHI CBHII EGI EGIII EGV BGLI
0.0105 0.0205 0.0140 0.0116 ND ND
ND ND 59.8 30.7 9.22 ND
ND Trace 46.2 ND ND ND
Trace ND ND ND ND 767.7
0.355 ND 9.19 ND ND 0.894
ND, not detected.
The activity of T. reesei EGV is rather weak, so its activity was detected by expression cloning technique for the first time (Saloheimo et al., 1994). So EGV is considered to be a minor cellulase component in T. reesei cellulase system and its purification from T. reesei culture supernatant may be difficult. The expression system of A. oryzae would be useful for analyses of such minor enzymes as well as cellulase components. Acknowledgements We wish to thank Dr K. Kitamoto for generously providing the expression system of A. oryzae. This work was performed using the facilities of the Biotechnology Reseach Center, The University of Tokyo. References Arsdell, J.N.V., Kwok, S., Schweickart, V.L., Ladner, M.B., Gelfand, D.H., Innis, M.A., 1987. Cloning, characterization, and expression in Saccharomyces cere6isiae of endoglucanase I from Trichoderma reesei. Biotechnology 5, 60 – 64. Barnett, C.C., Berka, R.M., Fowler, T., 1991. Cloning and amplification of the gene encoding an extracellular b-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates. Biotechnology 9, 562 – 567. Chen, C.M., Gritzali, M., Stafford, D.W., 1987. Nucleotide sequence and deduced primary structure of cellobiohydrolase II from Trichoderma reesei. Biotechnology 5, 274 – 278. Cummings, C., Fowler, T., 1996. Secretion of Trichoderma reesei b-glucosidase by Saccharomyces cere6isiae. Curr. Genet. 29, 227 – 233.
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