ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei

Artigo Acesso aberto Revisado por pares

ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei

2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês

10.1074/jbc.m003624200

ISSN

1083-351X

Autores

Nina Aro, Anu Saloheimo, Marja Ilmén, Merja Penttilä,

Tópico(s)

Plant-Microbe Interactions and Immunity

Resumo

A novel yeast-based method to isolate transcriptional activators was applied to clone regulators binding to the cellulase promoter cbh1 of the filamentous fungusTrichoderma reesei (Hypocrea jecorina). This led to the isolation of the cellulase activator ace2encoding for a protein belonging to the class of zinc binuclear cluster proteins found exclusively in fungi. The DNA-binding domain of ACEII was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and ACEII was shown to bindin vitro to the 5′-GGCTAATAA site present in thecbh1 promoter. This site also contains the proposed binding sequence of the xylanase activator XlnR of Aspergillus niger. Mutation of the GGC triplet abolished ACEII binding. The function of ACEII was studied by analyzing the effects oface2 deletion in the hypercellulolytic T. reesei strain ALKO2221. Deletion of the ace2 gene led to lowered induction kinetics of mRNAs encoding the major cellulases cellobiohydrolases I and II and endoglucanases I and II and to 30–70% reduced cellulase activity when the fungus was grown on medium containing Solka floc cellulose. The expression level of the gene encoding xylanase was also affected. ace2 deletion led to lowered xyn2 expression in cellulose-induced cultivation. Cellulase induction by sophorose was not affected byace2 deletion. A novel yeast-based method to isolate transcriptional activators was applied to clone regulators binding to the cellulase promoter cbh1 of the filamentous fungusTrichoderma reesei (Hypocrea jecorina). This led to the isolation of the cellulase activator ace2encoding for a protein belonging to the class of zinc binuclear cluster proteins found exclusively in fungi. The DNA-binding domain of ACEII was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and ACEII was shown to bindin vitro to the 5′-GGCTAATAA site present in thecbh1 promoter. This site also contains the proposed binding sequence of the xylanase activator XlnR of Aspergillus niger. Mutation of the GGC triplet abolished ACEII binding. The function of ACEII was studied by analyzing the effects oface2 deletion in the hypercellulolytic T. reesei strain ALKO2221. Deletion of the ace2 gene led to lowered induction kinetics of mRNAs encoding the major cellulases cellobiohydrolases I and II and endoglucanases I and II and to 30–70% reduced cellulase activity when the fungus was grown on medium containing Solka floc cellulose. The expression level of the gene encoding xylanase was also affected. ace2 deletion led to lowered xyn2 expression in cellulose-induced cultivation. Cellulase induction by sophorose was not affected byace2 deletion. cellobiohydrolase endoglucanase glutathioneS-transferase kilobase pair(s) base pair(s) 4-methylumbelliferyl-β-d-lactoside hydroxyethyl cellulose The most abundant plant materials produced by photosynthesis are cellulose and hemicelluloses. They can be degraded and used as an energy source by numerous microorganisms that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars, such as to glucose in the case of cellulose. Yet filamentous fungi play a special role because many yeasts, such asSaccharomyces cerevisiae, lack the ability to hydrolyze cellulose and hemicellulose. The cellulolytic system of the soft rot fungus Trichoderma reesei is one of the best characterized among microorganisms. Mutant strains have been reported to produce over 35 g/liter extracellular protein (1Durand H. Clanet H. Tiraby G. Enzyme Microb. Technol. 1988; 10: 341-346Crossref Scopus (211) Google Scholar), and nearly all of the secreted protein consists of cellulases and hemicellulases. Of these cellobiohydrolase I encoded by a single gene forms the major part (2Gritzali M. Brown R.D.J. Adv. Chem. Ser. 1979; 181: 237-260Crossref Google Scholar). The synergistic activities of cellobiohydrolases (CBHs),1 endoglucanases (EGs), and β-glucosidases are necessary for the efficient hydrolysis of cellulose. The hemicellulolytic system of Trichoderma consists of a more complex set of enzymes among which are the two endo-β-xylanases, the β-mannanase, and the side chain cleaving enzymes. The production of the main cellulases in Trichoderma is regulated at the transcriptional level depending on the carbon source available (3Kubicek C.P. Penttilä M. Harman G.E. Kubicek C.P. Regulation of Production of Plant Polysaccharide Degrading Enzymes by Trichoderma.in: Taylor & Francis Ltd., London1998Google Scholar), the genes being repressed tightly by glucose and induced up to several thousand fold by cellulose or the disaccharide sophorose (4Ilmén M. Saloheimo A. Onnela M.L. Penttilä M.E. Appl. Environ. Microbiol. 1997; 63: 1298-1306Crossref PubMed Google Scholar, 5Mandels M. Parrish F.W. Reese E.T. J. Bacteriol. 1962; 83: 400-408Crossref PubMed Google Scholar, 6Nisizawa T. Suzuki H. Nakayama M. Nisizawa K. J. Biochem. ( Tokyo ). 1971; 70: 375-385Crossref PubMed Scopus (52) Google Scholar). Carbon catabolite repression of cellulase genes has been extensively studied, and the repressor gene cre1 ofTrichoderma has been shown to mediate glucose repression of cellulase expression (7Ilmén M. Thrane C. Penttilä M. Mol. Gen. Genet. 1996; 251: 451-460Crossref PubMed Google Scholar). In the various conditions studied expression of the main cellulase genes, cbh1, cbh2,egl1, and egl2, has been shown to be coordinate, and expression of the cbh1 gene encoding cellobiohydrolase I has been shown to be always the strongest (4Ilmén M. Saloheimo A. Onnela M.L. Penttilä M.E. Appl. Environ. Microbiol. 1997; 63: 1298-1306Crossref PubMed Google Scholar). Analysis of relative expression levels of various hemicellulase genes on different carbon sources and inducing compounds indicate that several regulatory mechanisms operate, some of which may be shared by genes encoding cellulases and hemicellulases (8Margolles-Clark M. Ilmén M. Penttilä M. J. Biotechnol. 1997; 57: 167-179Crossref Scopus (141) Google Scholar). However, little information is available on the molecular mechanism involved in the strong activation of the cellulase genes or various hemicellulase genes. To isolate cellulase regulators we have developed a novel yeast-based method to isolate transcriptional activators by selecting simultaneously for their promoter binding and activation properties. This led to the discovery of the transcriptional regulator,ace1 (9Saloheimo A. Aro N. Ilmén M. Penttilä M. J. Biol. Chem. 2000; 275: 5817-5825Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In this article we describe the cloning and functional studies of another cellulase regulator, ACEII, obtained in a similar screening. We show that this novel transcription factor binds to the cbh1 promoter and provide evidence thatace2 has a role in the induction of the major cellulase and xylanase genes of Trichoderma. Escherichia colistrain DH5α was used for plasmid construction, and strain BL21(DE3)LysS was used as a host for production of GST fusion proteins.S. cerevisiae strain DBY746 (ATCC44773, α,his3-1, leu2-3, leu2-112,ura3-52, trp1-289, cyh r,cir +) (D. Bothstein, Massachusetts Institute of Technology, Cambridge, MA) was used as a host for the screening of activators. The deletion of ace2 was made in a low protease mutant strain ALKO2221 2A. Mäntylä, unpublished data. originating from the hypercellulolytic strain VTT-D-79125 (10Bailey M.J. Nevalainen K.M.H. Enzyme Microb. Technol. 1981; 3: 153-157Crossref Scopus (254) Google Scholar). Synthetic selection media lacking the appropriate nutrients were used for the plasmid-carrying yeast strains (11Sherman F. Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology.in: Academic Press, London1991Google Scholar). TheTrichoderma strains were grown in minimal medium described by Ilmén et al. (4Ilmén M. Saloheimo A. Onnela M.L. Penttilä M.E. Appl. Environ. Microbiol. 1997; 63: 1298-1306Crossref PubMed Google Scholar) supplemented with either 2% glycerol or 1% Solka floc cellulose (James River Corp.) as the carbon source. In the 6-day-long Solka floc cellulose cultivation, 0.2% proteose peptone was also added. 1 mm α-sophorose (Serva) was added to the glycerol medium to induce cellulase expression. ace2 was isolated from a T. reeseicDNA library expressed in S. cerevisiae as described by Saloheimo et al. (9Saloheimo A. Aro N. Ilmén M. Penttilä M. J. Biol. Chem. 2000; 275: 5817-5825Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). To isolate the chromosomalace2 gene the genomic λ library of T. reeseiQM9414 (12Vanhanen S. Penttilä M. Lehtovaara P. Knowles J. Curr. Genet. 1989; 15: 181-186Crossref PubMed Scopus (38) Google Scholar) was screened with the ace2 cDNA from pAS26. The chromosomal ace2 gene was subcloned as a 6.5-kbHindIII-EcoRI fragment into pZErO-1 vector (Invitrogen). This plasmid, pAS33, contained 0.6 and 4.9 kb, respectively, of the ace2 5′- and 3′-flanking sequences. To construct the GST-ACEII fusion expression vector, a DNA fragment encompassing the ACEII DNA-binding domain (amino acids 1–58) was amplified by polymerase chain reaction and cloned in frame with the GST gene in the pGEX-2T plasmid (Amersham Pharmacia Biotech) to obtain pARO17. Production of GST-ACEII1–58 inE. coli BL21(DE3) cells transformed with pARO17 and purification by glutathione Sepharose 4B (Amersham Pharmacia Biotech) were performed according to the supplier's manual. Short double-stranded DNA for binding assays was made by annealing complementary oligonucleotides designed to produce a 3′ recessed end that was then filled in with [α-32P]dCTP using the Klenow fragment of DNA polymerase I. Fragments longer than 100 bp were amplified by polymerase chain reaction using sequence specific primers, gel-purified, and labeled with [γ-32P]ATP by using T4 polynucleotide kinase (New England Biolabs). Binding assays were performed at room temperature in a 20-μl reaction mixture containing 50 mm Tris (pH 8.0), 50 mmKCl, 10% glycerol, 4 mm spermidine, 2 μg of poly(dI·dC), 2.5 μm ZnCl2 with 0.5–1 μg of the GST-ACEII1–58 fusion protein and 1 ng (about 20,000 cpm) of the labeled double-stranded DNA. In competition experiments unlabeled DNA was added to the reaction in 20–200-fold excess over the labeled DNA fragment. Electrophoresis was performed as described by Saloheimo et al. (9Saloheimo A. Aro N. Ilmén M. Penttilä M. J. Biol. Chem. 2000; 275: 5817-5825Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The plasmid containing the ace2 deletion cassette was constructed as follows. The hygromycin resistance cassette was cloned from pRLMEX30 (13Mach R.L. Schindler M. Kubicek C.P. Curr. Genet. 1994; 25: 567-570Crossref PubMed Scopus (145) Google Scholar) as a XhoI-HindIII fragment into the XhoI-HindIII cut pBluescript KS+ (Stratagene) to create pARO21. An Asp718-SalI fragment from pAS33 containing 2.2 kb of the 3′-flanking sequence oface2 was cloned into the Asp718-XhoI sites of pARO21. Then the NruI fragment containing 1.6 kb of the 5′-flanking sequence of ace2 gene was cloned directly from a λ clone containing chromosomal ace2 into theSmaI site of pARO21, which already contained the 3′-flanking sequences of ace2, to obtain pAS40. The deletion cassette was released from pAS40 by an Asp718-BamHI digestion, and 5 μg was used for transformation of the ALKO2221 strain according to Penttilä et al. (14Penttilä M. Nevalainen H. Rättö M. Salminen E. Knowles J. Gene ( Amst. ). 1987; 61: 155-164Crossref PubMed Scopus (539) Google Scholar) except that the transformants were selected on plates containing 100 μg/ml hygromycin. Fungal DNA was isolated from transformants by the Easy-DNA kit according to the manufacturer's protocol number 3 (Invitrogen) and subjected to Southern analysis to verify the replacement of theace2 gene in the genome and the presence of no other copies of the deletion cassette. For the 6-day cultivation on Solka floc cellulose 50 ml of growth medium in two parallel shake flasks of ace2 deletants and three of the host strain ALKO2221 were inoculated with 107 spores. The cultures were carried out in 250-ml flasks with shaking at 200 rpm at 28 °C. Culture medium samples were collected for enzyme activity measurements from each flask. For RNA isolation mycelia were combined from the two parallel shake flasks of the ace2 deletants. Mycelia from the host cultures were treated separately. In the experiment designed to study induction kinetics of cellulases,ace2 deletants and ALKO2221 were each cultivated in three parallel 2-liter shake flasks on 400 ml of glycerol medium for 72 h, after which mycelia of each strain were combined, and an equal amount was transferred into two shake flasks containing Solka floc cellulose medium and one shake flask containing glycerol medium (control). Samples of mycelium were collected for RNA isolation 9, 12, 15, 18, and 32 h after transfer. For sophorose induction studies strains were grown on glycerol medium for 72 h after which 1 mm sophorose was added, and mycelial samples were collected 1, 2, 3, and 6 h after the sophorose addition. Total RNA was isolated with the Trizol reagent kit (Life Technologies, Inc.). The probes for Northern analyses were the entire cDNAs of cbh1, cbh2 (15Penttilä M.E. Andre L. Lehtovaara P. Bailey M. Teeri T.T. Knowles J.K. Gene ( Amst. ). 1988; 63: 103-112Crossref PubMed Scopus (151) Google Scholar),egl1 (16Penttilä M.E. Andre L. Saloheimo M. Lehtovaara P. Knowles J.K. Yeast. 1987; 3: 175-185Crossref PubMed Scopus (112) Google Scholar), and egl2 (previously calledegl3) (17Saloheimo M. Lehtovaara P. Penttilä M. Teeri T.T. Ståhlberg J. Johansson G. Pettersson G. Claeyssens M. Tomme P. Knowles J.K. Gene ( Amst. ). 1988; 63: 11-22Crossref PubMed Scopus (269) Google Scholar) released from vector sequences. The probes for the β-xylanases xyn1 and xyn2 (18Törrönen A. Mach R.L. Messner R. Gonzalez R. Kalkkinen N. Harkki A. Kubicek C.P. BioTechnology. 1992; 10: 1461-1465Crossref PubMed Scopus (195) Google Scholar, 19Saarelainen R. Paloheimo M. Fagerstrom R. Suominen P.L. Nevalainen K.M. Mol. Gen. Genet. 1993; 241: 497-503Crossref PubMed Scopus (61) Google Scholar) were 350-bp fragments prepared by polymerase chain reaction (8Margolles-Clark M. Ilmén M. Penttilä M. J. Biotechnol. 1997; 57: 167-179Crossref Scopus (141) Google Scholar). The membranes were hybridized with the actin fragment and a glyceraldehyde-3-phosphate dehydrogenase encoding (gpd1) cDNA fragment as internal RNA loading controls. The probes were labeled using the random primed DNA labeling kit (Roche Molecular Biochemicals) and [α-32P]dCTP (Amersham Pharmacia Biotech). The amounts of the hybridized mRNAs were quantified by densitometric scanning using ImageQuant software of Phosphoimager SI (Molecular Dynamics) and normalized for the total amount of mRNA by using the amount of gpd1 and actin mRNA as a loading control for each filter. CBHI and EGI activity in the culture supernatants were measured using 4-methylumbelliferyl-β-d-lactoside (MUL) (Sigma) as a substrate according to van Tilbeurgh et al. (20van Tilbeurgh H. Claeyssens M. de Bruyne C.K. FEBS Lett. 1982; 149: 152-156Crossref Scopus (149) Google Scholar) using 0.17 mm MUL and 10 min of incubation time at pH 5.0 and 50 °C. The endoglucanase activity in the culture supernatant was measured as liberation of reducing sugars using 1% hydroxyethyl cellulose (HEC) (Fluka) as a substrate in 10 min of incubation time at 50 °C and pH 4.8. Total protein amount was measured according to Lowry et al. (21Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) from proteins precipitated from the culture medium with 20% trichloroacetic acid. The amino acid similarities of ACEII with other proteins were calculated, and the multiple alignment of Fig.2 was generated with the Clustal W program (22Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56001) Google Scholar) with a gap opening penalty of 10.0 and a gap extension penalty of 0.2. The similarity searches of data bases were done using the BLAST program with an existence gap penalty of 11 and an extension gap penalty of 1. All procedures not described above were carried out using standard methods. To isolate the transcriptional activators of cellulase genes, the 1.15-kb-long promoter of the T. reesei cbh1 gene was fused to theS. cerevisiae HIS3 gene to provide a reporter construct, pAS3, in which the HIS3 gene is not expressed without activation from the cbh1 promoter (9Saloheimo A. Aro N. Ilmén M. Penttilä M. J. Biol. Chem. 2000; 275: 5817-5825Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). ATrichoderma cDNA expression library present on a multicopy vector was transformed to a his3 mutant yeast containing the reporter construct pAS3, and the transformants were selected on plates containing no histidine. One of the growing colonies contained a cDNA library plasmid named pAS26. pAS26 supported growth only when the reporter construct was also present in the cell, indicating activation of HIS3 expression through binding to the cbh1 promoter (Fig.1). The cDNA in plasmid pAS26 contains an open reading frame coding for a protein of 341 amino acids with a calculated molecular mass of 38 kDa. This new gene was named ace2 for activator of cellulase expression (GenBankTMaccession number AF220671). The N-terminal part of the deduced ACEII protein has a typical zinc binuclear cluster DNA-binding domain of the fungal type (Zn(II)2Cys6), first characterized in S. cerevisiae (23Pan T. Coleman J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2077-2081Crossref PubMed Scopus (164) Google Scholar). Fig. 2 shows an alignment of the ACEII zinc binuclear cluster domain with the corresponding domains of 12 other proteins showing the highest similarity with the ACEII DNA-binding domain and that of XlnR, a factor regulating xylanase expression in Aspergillus nidulans. The DNA-binding domain of ACEII is most similar (70%) to the DNA-binding domains found in ACU-15 (SWISS-PROT entry P87000), a positive regulator of acetate induction of Neurospora crassa, and in theA. nidulans FacB (70%), the major regulatory protein involved in acetamide and acetate utilization (24Todd R.B. Murphy R.L. Martin H.M. Sharp J.A. Davis M.A. Katz M.E. Hynes M.J. Mol. Gen. Genet. 1997; 254: 495-504Crossref PubMed Scopus (60) Google Scholar). Aside from the zinc binuclear cluster domain, no significant similarities with known proteins or sequences contained in the expressed sequence tag data bases were found by the BLAST program. The overall similarity of ACU-15 and FacB with ACEII is low (25 and 23%, respectively). However, the DNA-binding domain of ACEII is followed by a histidine-rich area (amino acids 53–66) in which a His-Xaa sequence is repeated seven times. A similar histidine repeat of unknown function is found in the Zn(II)2Cys6-type aflatoxin regulatory protein AflR of two Aspergillus species (25Woloshuk C.P. Foutz K.R. Brewer J.F. Bhatnagar D. Cleveland T.E. Payne G.A. Appl. Environ. Microbiol. 1994; 60: 2408-2414Crossref PubMed Google Scholar, 26Chang P.K. Ehrlich K.C., Yu, J. Bhatnagar D. Cleveland T.E. Appl. Environ. Microbiol. 1995; 61: 2372-2377Crossref PubMed Google Scholar). Furthermore, a glutamine-rich region (82QQQEQQQGQPQHPPPPVQ99) resembling the glutamine-rich activation domains of other transcription factors,e.g. the human transcription factor Sp1 (27Gill G. Pascal E. Tseng Z.H. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 192-196Crossref PubMed Scopus (473) Google Scholar), is present in ACEII. Because thecbh1 promoter used in the initial screening was about 1.15 kb, further experiments were needed to characterize the ACEII-binding site more precisely. Therefore, the ACEII segment coding for the zinc binuclear cluster domain (amino acids 1–58) was fused to GST, and the ability of the resulting of GST-ACEII1–58 hybrid to bind to DNA was studied by electrophoretic mobility shift assays with four polymerase chain reaction amplified fragments: fragment 1 (from −133 to −392 upstream from ATG), fragment 2 (−621 to −941), fragment 3 (−886 to −1177), and fragment 4 (−1116 to −1421). DNA-protein complexes were detected with fragments 2 and 3 (data not shown). Because binding to fragment 2 produced the strongest specific shift, it was further characterized by amplifying shorter DNA fragments and designing specific oligonucleotides to further delimit the ACEII-binding region. Of the four partially overlapping oligonucleotides named 2B (−843 to −809), 2C (−818 to −780), 2D (−789 to −764), and 2G (−772 to −736), GST-ACEII1–58binding was observed only to oligonucleotide 2D. No competition was observed by the addition of nonspecific DNA to the binding reaction, whereas addition of excess of oligonucleotide 2D clearly competed out the binding (data not shown). Interestingly, 2D (Fig.3) contains a sequence 5′-GGCTAA that is similar to the one (5′-GGCTAAA) recently shown to bind the xylanase activator XlnR of Aspergillus niger (28van Peij N.N. Visser J. de Graaff L.H. Mol. Microbiol. 1998; 27: 131-142Crossref PubMed Scopus (262) Google Scholar). To study whether this sequence in 2D was the recognition site for GST-ACEII1–58, four mutations of nucleotide triplets were made to the 2D oligonucleotide. Fig. 3 shows that the mutation of the three nucleotides upstream from the 5′-GGCTAA sequence in thecbh1 promoter did not affect the binding of GST-ACEII1–58; instead the following three nucleotides (GGC) were essential for binding. The mutations of the following two TAA triplets in the promoter each reduced ACEII binding. Thecbh1 promoter also contains regions identical to the proposed XlnR-binding site (5′-GGCTAAA). One is found at position −825, present in the oligo 2B, but GST-ACEII1–58 did not bind to it (data not shown). To investigate the role the of ace2 gene in carbon utilization, the protein coding region of the ace2 chromosomal fragment was replaced with the hygromycin selectable marker hph in the T. reesei strain ALKO2221. The ace2 deletants VTT-D-99729, VTT-D-99757, and VTT-D-99758 were selected for further experiments. Because the induction and high level expression of cellulase genes occurs in the presence of cellulose as the sole carbon source, the possible negative effect of ace2 deletion on cellulase expression in cellulose-based cultivations was analyzed. In the first approach several parallel cultures of the three deletants and the host strain were cultivated on medium containing Solka floc cellulose as the sole carbon source. Cellobiohydrolase and endoglucanase activities were measured from culture supernatants with MUL and HEC as substrates, respectively (Table I). The values for MUL mainly reflect the amount of CBHI and EGI produced by the fungus, and the values for HEC reflect the amount of endoglucanases (e.g. EGI and EGII). The ace2 deletants clearly produced less cellobiohydrolase and endoglucanase activities than the host strain. At day 3 the ace2 deletants only had on the average 20% of the cellobiohydrolase activity when compared with the host strain ALKO2221, and after 6 days the cellobiohydrolase and endoglucanase activities produced by the ace2 deletants were on the average 55% of the activities of ALKO2221. Biomass determination cannot be made from the cultures containing cellulose particles, but the pH level of the medium indicated thatace2 deletant may have grown slower than the host. This would be expected if the lower cellulase levels would lead to reduced amount of sugar available for the fungus. No difference in biomass accumulation was observed on glucose- or glycerol-based cultures.Table ICellulase activitiesTimeStrainsVTT-D-99729VTT-D-99757VTT-D-99758ALKO22213 daysMUL (nkat/ml)0.320.240.191.24HEC (nkat/ml)5.85.14.218.3Total protein (mg/ml)0.070.060.050.136 daysMUL (nkat/ml)25201834HEC (nkat/ml)251202177382Total protein (mg/ml)1.61.41.22.4Cellulase activities (1 nkat = 1 nmol of methylumbelliferyl or reducing sugar released from the substrate(s) for MUL and HEC, respectively) were measured using MUL and HEC as substrates and total protein amounts in Solka floc cellulose cultivation of the three ace2deletant strains VTT-D-99729, VTT-D-99757, and VTT-D-99758 and their host strain ALKO2221 at two different time points of the culture. The values for ace2 deletants are the mean values from two parallel shake flasks, and the values for the ALKO2221 strain are the mean values from three parallel flasks. Open table in a new tab Cellulase activities (1 nkat = 1 nmol of methylumbelliferyl or reducing sugar released from the substrate(s) for MUL and HEC, respectively) were measured using MUL and HEC as substrates and total protein amounts in Solka floc cellulose cultivation of the three ace2deletant strains VTT-D-99729, VTT-D-99757, and VTT-D-99758 and their host strain ALKO2221 at two different time points of the culture. The values for ace2 deletants are the mean values from two parallel shake flasks, and the values for the ALKO2221 strain are the mean values from three parallel flasks. To verify that the effect of ace2 deletion is seen also at transcriptional levels at later time points of the culture and not only at the enzyme level, the expression of three of the main cellulase genes, cbh1, cbh2, and egl2, was analyzed by Northern analysis from the mycelia collected after 6 days of cultivation. Transcript levels of all of the cellulases analyzed were lower in the three ace2 deletants than in their host strain ALKO2221; the mean values of the transformants were at this time point 70, 70, and 80% for cbh1, cbh2, andegl2 mRNA, respectively, of the values of the host (data not shown). The finding that ace2 deletion resulted in reduced expression levels of cellulases after 3 and 6 days of cultivation on Solka floc cellulose prompted us to analyze the role of ace2in induction kinetics of cellulase genes at earlier time points in cellulose-based cultures. For this study we cultivated two parallel shake flasks of both the ace2 deletant VTT-D-99729 and ALKO2221. The strains were first pregrown to accumulate biomass on glycerol, a neutral carbon source with respect to cellulase expression (4Ilmén M. Saloheimo A. Onnela M.L. Penttilä M.E. Appl. Environ. Microbiol. 1997; 63: 1298-1306Crossref PubMed Google Scholar), whereafter the mycelia were transferred to Solka floc cellulose to induce expression and to glycerol medium as a control. After 6, 9, 12, 15, 18, and 32 h of the transfer, mycelium was harvested, and Northern blot analyses were performed. The differences between theace2 deletant and the host were seen in the level of induction of the cbh1, cbh2, and egl1(Fig. 4) mRNAs studied. Quantification of the signals showed that on the average the signals of the three cellulase mRNAs of the ace2 deletant were 30–75% of the amount seen in the ALKO2221 strain at time points 9–18 h after transfer but were closer to the control levels at later time points (Fig. 4 B). No cellulase signals were detected from the mycelia transferred to the glycerol medium (data not shown). Taken together the results obtained on Solka floc cellulose medium show that cellulase transcript levels of the ace2 mutant strain are on the average lower throughout the cultivation until day 6. The difference is greater at the early stages of cultivation, indicating that induction has become slower in the mutant. It was of interest whether ace2 functions also in the induction caused by a known strong and rapid cellulase inducer, the disaccharide sophorose. Each of the strains VTT-D-99729, VTT-D-99757, and ALKO2221 were grown in two parallel shake flasks on glycerol medium for 4 days, after which sophorose was added into the culture. mRNA analyses were performed 1, 2, 3, and 6 h after sophorose addition. The induction of cbh1 (Fig. 5) and of cbh2 and egl1 (data not shown) was followed by Northern analysis. The levels of cellulase mRNA were very similar between the two independent ace2 deletants and the control strain ALKO2221. This result suggests that ace2deletion does not affect sophorose induction of cbh1 and the other cellulase genes. We wanted to study whether ace2 deletion would also affect the transcription of the two main endo-β-xylanase genes, xyn1and xyn2, encoding enzymes that hydrolyze the main chain of xylan. The same filter as probed for cellulase expression was probed with the xyn1- and xyn2-specific probes. After transfer of mycelia from glycerol medium to Solka floc cellulose medium the xyn1 gene was induced weakly at 18 h and more strongly at 32 h after transfer to cellulose medium (Fig.4 A). The expression of xyn2 was induced more strongly already at 18 h. Fig. 4 shows that the expression ofxyn2 is much more weakly induced in the ace2deletant than in the host strain ALKO2221, and the quantification reveals that the xyn2 signal in VTT-D-99729 is only 30–45% of that of the host strain ALKO2221 at 15, 18, and 32 h. No significant difference in the xyn1 expression level between the host and the ace2 deletant strain can be seen in this experiment (Fig. 4 B). Despite the importance of plant material breakdown in basic biology and biotechnology, very little is yet known about the molecular mechanisms that regulate expression of the genes encoding the tens or even hundreds of hydrolytic enzymes needed for the efficient breakdown of the polymeric substrates, cellulose and hemicellulose. In addition to the glucose repressor protein CREI/CREA (7Ilmén M. Thrane C. Penttilä M. Mol. Gen. Genet. 1996; 251: 451-460Crossref PubMed Google Scholar, 29Dowzer C.E. Kelly J.M. Mol. Cell. Biol. 1991; 11: 5701-5709Crossref PubMed Scopus (333) Google Scholar) the only other regulatory protei

Ver no editor

Altmetric

PlumX

Entrar

Lembrar minha senha

Receber meu e-mail de confirmação

ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei