Structure and expression properties of the endo-β-1,4-glucanase A gene from the filamentous fungus Aspergillus nidulans

FEMS Microbiology Letters 175 (1999) 239^245

Structure and expression properties of the endo-L-1,4-glucanase A gene from the ¢lamentous fungus Aspergillus nidulans Go Chikamatsu, Kengo Shirai, Masashi Kato, Tetsuo Kobayashi *, Norihiro Tsukagoshi Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya-shi, Aichi 464-8601, Japan Received 10 October 1998; received in revised form 12 April 1999; accepted 16 April 1999

Abstract Endo-L-1,4-glucanase A (EG A) of Aspergillus nidulans was purified to homogeneity, and its genomic gene (eglA) was cloned based on partial amino acid sequences of the purified enzyme and sequenced. The eglA gene comprised 1228 bp with four putative introns and encoded a polypeptide of 326 amino acids bearing high homology to the family A cellulases. The eglA promoter activity in A. nidulans was examined using the A. oryzae Taka-amylase A gene as a reporter. Expression of the reporter gene was induced by carboxymethylcellulose and cellobiose, and repressed by glucose, galactose, mannose, xylose, sorbitol, glycerol and succinate. Lactose neither induced nor repressed the expression. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Aspergillus nidulans; Endo-L-1,4-glucanase; Cellulase; Gene regulation

1. Introduction Cellulose is the most abundant biomass on earth and utilized as an energy source by cellulolytic organisms. Filamentous fungi produce a complex spectrum of cellulases, consisting of endo-L-1,4-glucanases (EGs), cellobiohydrolases (CBHs) and Lglucosidases (BGLs), of which the production is regulated by availability of the carbon source as well described in an industrial fungus, Trichoderma

* Corresponding author. Tel.: +81 (52) 7894086; Fax: +81 (52) 7894087; E-mail: [email protected]

reesei. In this organism, expression of the cbh1 and cbh2 genes encoding CBHs and the egl1, egl2 and egl5 genes encoding EGs are coordinately induced by cellulose, sophorose, cellobiose and lactose, while their expression is tightly repressed by glucose [1]. Functional analyses of the cbh1 promoter have revealed that the sequence mediating the induction lies within a 161-bp region upstream of the translation initiator ATG [2]. Factors binding to the cbh2 promoter have been detected by protein-DNA binding assays [3]. However, no major areas of homology have been observed between the cbh1 and cbh2 promoters, and functions of the DNA binding factors are still unknown. Aspergillus nidulans is a genetic model fungus. It

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 0 1 - 3

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can grow on a completely de¢ned medium, and its life cycle is easy to manipulate and allows ¢ne structure genetic analysis. Thus, regulation of A. nidulans cellulases could be studied in detail at the genetic level and further at the molecular level. In this paper, endo-L-1,4-glucanase A (EG A), one of the EGs produced in A. nidulans, was puri¢ed to homogeneity and its enzymatic properties were determined. Based on the amino acid sequences determined chemically, a genomic gene (eglA) encoding EG A was cloned and sequenced. This is the ¢rst cellulase gene cloned from A. nidulans. Furthermore, the expression properties of the eglA gene was examined by using the A. oryzae Taka-amylase A gene (taaG2) [4] as a reporter.

2. Materials and methods 2.1. Puri¢cation of EG A A. nidulans G191 (pyrG89, pabaA1, fwA1, uaY9) was grown aerobically at 37³C in a medium consisting of 1% carboxymethylcellulose (CMC), 1% polypeptone, 0.5% K2 HPO4 , 0.1% NaNO3 , 0.05% MgSO4 W7H2 O, 0.1% uridine, 2 mg l31 p-aminobenzoic acid (pH 6.5). Proteins in the culture ¢ltrate (1.2 l) were precipitated by the addition of ammonium sulfate at 70% saturation, and dissolved in and dialyzed against 50 mM sodium phosphate bu¡er (pH 6.5) containing 87 mg l31 phenylmethylsulfonyl £uoride. The resultant crude enzyme solution was puri¢ed by DEAE-Toyopearl 650M (Tosoh, Japan) column chromatography with a linear gradient of 0^ 1.0 M NaCl in 50 mM sodium phosphate bu¡er (pH 6.5). The EG active fractions were concentrated by Centricon 10 (Amicon Co.), and further puri¢ed by a gel ¢ltration column of Sephacryl G200 SH (Pharmacia). 2.2. Enzyme assays EG reaction was carried out at 37³C in 50 mM sodium phosphate bu¡er (pH 6.5) containing 0.1% CMC as a substrate. Amylase reaction was carried out at 37³C in 20 mM acetate bu¡er (pH 5.9) containing 10 mM CaCl2 and 1% soluble starch. The amount of reducing sugar liberated was determined

by Nelson's photometric adaptation of the method of Somogyi [5]. The amount of each enzyme which released 1 Wmol of reducing sugar equivalent to glucose per min was de¢ned to represent one unit of activity. 2.3. Molecular cloning of the EG A gene (eglA) A pair of degenerated primers, 5P-TGGYTNGGNACNAAYGARGC and 5P-GTRTTNGTNCCNGANCCRTC, were designed based on the partial amino acid sequences of EG A. A part of the eglA gene was ampli¢ed by PCR using the above primers with the A. nidulans total DNA as a template, and cloned into the SmaI site of pUC119 (pCF15). The EcoRIBamHI fragment of pCF15 containing the insert was labeled with digoxigenin dUTP using a DNA Labeling kit (Boehringer Mannheim Biochemicals), and used as the probe for cloning of the entire eglA genomic gene. Southern hybridization analysis and colony hybridization were performed as described previously [6]. 2.4. Construction of the amylase reporter plasmid pTaaG2 carries a 3.2-kb EcoRI fragment containing the taaG2 gene (taaG2) [4] on pUC119. The 5Ppart of the taaG2 gene (311 to +416, referring to the translational start site as +1) was ampli¢ed by PCR with pTaaG2 as a template using a pair of primers, 5P-GAAGGCATTCATGATGGTCG and 5P-CTGGGGCAGCTGGGCTGTAA. The former primer contained a single base change, T to C, at 31 to generate a BspHI site. The latter primer contained the intrinsic PvuII site in the taaG2 coding region. The entire 5P-noncoding region of the eglA gene (SacI-BspHI fragment, 954 bp), the PCR ampli¢ed 5P-region of taaG2 (BspHI-PvuII fragment, 408 bp), and the rest of taaG2 (PvuII-EcoRI fragment from pTaaG2, 2137 bp) were joined and inserted between SacI and EcoRI sites on pUC119 (pCMta10). The XbaI-EcoRI fragment on pCMta10 containing the eglA promoter fused to the taaG2 coding region was then inserted into pTG1, which carries the pyr4 gene of Neurospora crassa on pUC18 [7]. The resultant plasmid, pGCMta6, was introduced into A. nidulans G191 by the protoplast transformation procedure [8].

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2.5. Other methods Protein was determined by a Bio-Rad protein assay kit with bovine IgG as the standard. SDS-polyacrylamide gel electrophoresis (PAGE) was carried out as described previously [9]. The NH2 -terminal amino acid sequences of the puri¢ed enzyme and a peptide generated by V8 protease digestion were determined with an ABI protein sequencer model 476A. Nucleotide sequences were determined with an ABI DNA sequencer Model 373A using a ABI PRISM1 Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer).

3. Results and discussion 3.1. Puri¢cation and enzymatic properties of EG A A. nidulans extracellularly produced three EGs designated EG A^C when grown on the inducing carbon source CMC, and the production of all the EGs was repressed by addition of glucose (Fig. 1A). Lactose, which is known to be one of the cellulase inducers in T. reesei, neither induced nor repressed the production of EGs in A. nidulans. EG A was puri¢ed to homogeneity as described in Section 2 (Fig. 1B). The molecular mass of the enzyme was determined to be 35 kDa by SDS-PAGE as well as by the gel ¢ltration. EG A exhibited a pH optimum at 6.5 and was stable over a wide pH range; over 80% of the activity was retained in the range of 4.5^7.5 after incubation for 2 h at 37³C. The enzyme displayed optimal activity at around 50³C. Over 50% of the activity remained after incubation for 1 h at temperatures between 30 and 70³C. The pure EG A showed a speci¢c activity of 21 U mg31 protein toward CMC. EG A also utilized Avicel as a substrate with a reduced activity of 0.32 U mg31 protein. However, no activity was detected toward xylan, p-nitrophenyl-L-glucopyranoside and p-nitrophenyl-L-cellobioside. 3.2. Isolation and structural features of the eglA gene The NH2 -terminal amino acid sequence of EG A was chemically determined to be AFTWLGTNEA-

Fig. 1. EG production in A. nidulans (A) and SDS-PAGE analysis of the puri¢ed EG A (B). A: A. nidulans was grown aerobically at 37³C for 24 h in the medium described in Section 2 except that CMC was replaced with various carbon sources. Culture ¢ltrates (20 Wl) of A. nidulans grown on CMC (lane 1), CMC+lactose (lane 2), lactose (lane 3), CMC+glucose (lane 4) and glucose (lane 5) were subjected to native PAGE analysis (12%). After electrophoresis, the gel was soaked in 1% CMC for 30 min with gentle shaking at room temperature, washed several times with distilled water, and incubated in 50 mM sodium phosphate bu¡er (pH 6.5) at 37³C for 30 min. EG activity bands were visualized by staining the gel in 1% Congo red at room temperature for 30 min followed by destaining in 1 M NaCl. Then the gel was soaked in 10% (v/v) acetic acid for better observation of the activity bands. A, B and C represent EG A, B, and C, respectively. B: The puri¢ed EG A was subjected to SDS-PAGE (15%). EG A was visualized by Coomassie brilliant blue staining (a) and by activity staining (b). Prestained SDSPAGE standards (Bio-Rad) containing phosphorylase B (105 kDa), bovine serum albumin (82 kDa), ovalbumin (49 kDa), carbonic anhydrase (33.3 kDa), soybean trypsin inhibitor (28.6 kDa), and lysozyme (19.4 kDa) were used for molecular mass estimation. Activity staining was carried out as described above except that the gel was soaked in 1% Triton X-100 and then rinsed with distilled water prior to incubation with CMC.

GAEFG. V8 protease digestion of the enzyme generated two peptides of 22 kDa and 13 kDa, and the NH2 -terminal amino acid sequence for the smaller peptide was determined to be MHQYLDSDGSGTNTA. A part of the eglA gene was ampli¢ed by PCR with a pair of degenerated primers corresponding to the determined amino acid sequences and the genomic DNA as a template. The ampli¢ed DNA fragment of approximately 0.6 kb was used as a probe in Southern hybridization analysis of the genomic DNA digested by various restriction endonucleases. A 3.2-kb SacI fragment which hybridized to the probe was cloned onto pUC119 (pNCM27) and sequenced (Fig. 2). The coding region of eglA comprised 1228 bp. Comparisons with other endo-L-1,4-glucanases re-

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Fig. 2. Nucleotide and derived amino acid sequences of the eglA gene. The amino acid sequences determined chemically are underlined. Putative introns are shown by lower-case letters. Possible TATA box, CCAAT sequences, CreA binding sites, and XlnR site are boxed. The nucleotide sequence of eglA has been assigned DDBJ accession number AB009402.

6

vealed four short putative introns of 52^71 bp, all of which followed the GT-AG rule at the intron/exon junctions [10]. The derived amino acid sequence comprised a putative signal peptide of 19 amino acid residues and the mature EG A of 307 amino acid residues with a calculated molecular mass of 33 888 Da. EG A showed a high degree of sequence identity to the family 5 cellulases [11,12]: 62.5% identity to A. niger endoglucanase B [13], 61.9% to Humicola insolens endo-L-1,4-glucanase [14], 43.3% to Pseudomonas solanacearum endoglucanase [15], 35.3% to Cryptococcus £avus CMCase 1 [16], 34.1% to T. reesei endoglucanase II [17], and 30.8% to Macrophomia phaseolina L-1,4-endoglucanase [18]. Although some family 5 cellulases have been shown to possess a cellulose binding domain, EG A did not possess one. The 5P-noncoding region of the sequence was screened for various consensus sequences. A TATA box-like sequence, TATTTAA, was found at 375 to 369 referring to the translation start site as +1, which was followed by a CT-rich sequence at 348 to 335. The CCAAT sequence, which is known as an upstream activating sequence in higher eukaryotes [19] as well as in ¢lamentous fungi [20,21],

Fig. 3. Structure of the reporter plasmid pGCMta6. Nucleotide and amino acid sequences at the eglA promoter/taaG2 junction are shown at the top. Thick arrows represent the pyr4 gene of N. crassa and the ampicillin resistance gene (Ampr ) derived from pUC18.

was found at regions 3401 to 3397 and 3339 to 3335. CREA binding sites responsible for carbon catabolite repression in A. nidulans [22] were also located at four positions, three of them were clustered within 3237 and 3201 and the other was located at around 3310. A putative binding site for XlnR, which regulates xylanolytic and endoglucanase gene expression in A. niger, was found at 3411 to 3405 [13,23]. 3.3. Function of the eglA promoter A reporter plasmid pGCMta6 was constructed by fusing the eglA promoter to the taaG2 coding region as described in Section 2 (Fig. 3) and introduced into A. nidulans to determine speci¢cally the eglA promoter activity, since A. nidulans produced three Table 1 Carbon source dependence of the eglA promoter activity Carbon substrates

CMC Lactose (24 h) Lactose+CMC Lactose+Avicel Lactose (56 h) Lactose+sophorose Lactose+cellobiose Lactose+gentibiose CMC+glucose CMC+galactose CMC+mannose CMC+xylose CMC+sorbitol CMC+glycerol CMC+succinate

A. nidulans carrying: pGCMta6

pTG1

1.53 0.20 1.86 0.61 0.55 0.60 2.50 0.54 0.13 0.23 ND ND 0.02 0.03 0.01

0.01 0.03 0.06 NT NT NT NT NT 0.01 NT NT NT NT NT NT

A. nidulans carrying pGCMta6 or pTG1 (vector alone) was grown for 24 h at 37³C on various carbon substrates, and extracellular amylase activity was measured as described in Section 2. The concentration of each carbon substrate was 1% except for sophorose, cellobiose and gentibiose. These disaccharides were added at a ¢nal concentration of 0.1% after 24 h growth on 1% lactose, and amylase activity of the culture ¢ltrates was examined after 32 h of the addition. The activity is shown as U ml31 of culture. ND, not detected ; NT, not tested.

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kinds of EG (Fig. 1). Five randomly selected transformants were examined for amylase production in the presence and absence of CMC. All of them produced approximately 10 times higher amylase activity on CMC than on starch, indicating that amylase production is under control of the eglA promoter in these transformants. For further experiments we chose one of the transformants, which produced 1.5 U ml31 of amylase in the presence of CMC and 0.2 U ml31 in its absence. Carbon source dependence of expression of the fusion gene was analyzed by measuring amylase activity of the transformant after growth on various carbon substrates as shown in Table 1. High level expression of the fusion gene was observed on CMC as a carbon source, where amylase activity reached 1.53 U ml31 culture. However, little amylase activity was detected in A. nidulans carrying the vector pTG1 alone. The fusion gene was slightly expressed on lactose, while high level expression was observed on lactose plus CMC (1.86 U ml31 ). Relatively high level expression was also observed on lactose plus crystalline cellulose, Avicel (0.61 U ml31 ). These results indicate that lactose neither induces nor represses the expression and that the eglA gene is positively regulated in the presence of CMC and Avicel. However, CMC and Avicel should not be the true inducers, since they are high molecular substances and could not permeate cell membranes. In T. reesei and in Penicillium purpurogenum, cellulose is thought to be ¢rst degraded by cellulases produced at the basal level, and then converted to the true inducer, such as sophorose or gentibiose, respectively [24,25]. As shown in Table 1, neither sophorose nor gentibiose induced the fusion gene expression in A. nidulans, while cellobiose showed strong inducing activity. In A. niger, expression of endoglucanase genes is induced by xylose under the control of XlnR [13]. In contrast, the fusion gene expression was repressed by xylose in A. nidulans, although a possible XlnR site was found within the eglA promoter as described above. Thus, at present, it seems that each organism recognizes a di¡erent molecule as a cellulase inducer. However, further work will be necessary to identify the true inducer in A. nidulans. The high level expression on CMC was repressed by addition of other carbon sources such as glucose, galactose, mannose, xylose, sorbitol, glycerol and

succinate. This indicates that the eglA promoter activity is regulated by carbon catabolite repression probably mediated by CREA binding sites described above. However, it is yet to be determined how many and which binding sites actually function, and whether all the repressing carbon substrates reduce the expression through a CREA-dependent mechanism. Comparison of the sequence of the eglA promoter with those of 5P-£anking regions of other fungal cellulase genes revealed no major areas of homology, and therefore it is di¤cult to predict the cis-element responsible for cellulase regulation at this stage. The fusion gene used here will facilitate promoter analysis by in vitro mutagenesis and protein-DNA binding assays for identi¢cation of cis-elements and transfactors involved in regulation of the eglA gene. Moreover, taking advantage of established genetics and DNA manipulation techniques in A. nidulans, regulatory mechanisms of the cellulase gene, including signaling pathways, may be studied in detail at the molecular level.

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