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Tannic acid induces transcription of laccase gene cglcc1 in the white-rot fungus Coriolopsis gallica José M. Carbajo, Howard Junca, María C. Terrón, Tania González, Susana Yagüe, Ernesto Zapico, and Aldo E. González

Abstract: Laccase, a phenoloxidase enzyme secreted by white-rot fungi, has a significant role in the degradation of lignin and environmental pollutants. Coriolopsis gallica is a ligninolytic basidiomycete that produces high levels of this extracellular enzyme. A laccase gene cglcc1 from this fungus has been cloned and sequenced. The capacity of C. gallica to efficiently degrade polyphenols has been successfully applied in our laboratory to the biotreatment and decolorization of several industrial wastewaters. This study focused on the effect of tannic acid, a natural compound widely distributed in plants, on the production of laccase activity by C. gallica. Our results showed an evident increase of extracellular laccase levels when C. gallica was grown in the presence of tannic acid. Concentrations of 50 and 100 µM of this compound increased laccase activity when compared with control samples grown without tannic acid. In addition, we found an increase in laccase transcript levels in C. gallica grown in culture media supplemented with tannic acid. The role of tannic acid was shown to be an inductor of laccase activity in this fungus, due to the enhancement of expression of the laccase gene at the transcriptional level. Key words: laccase, tannic acid, Coriolopsis gallica, induction, gene transcription. Résumé : La laccase est une enzyme phénoloxydase secrétée par les champignons responsables Carbajo et al. de la pourriture blanche et qui joue un rôle significatif dans la dégradation de la lignine et de divers polluants environnementaux. Le Coriolopsis gallica est un basidiomycète qui produit des quantités élevées de cette enzyme extracellulaire. Un gène cglcc1 de la laccase de ce champignon a été cloné et séquencé. La capacité de C. gallica à dégrader efficacement les polyphénols a été appliquée avec succès dans notre laboratoire pour le biotraitement et la décoloration de quelques eaux usées industrielles. La présente étude a vérifié l’effet de l’acide tannique, un produit naturel largement répandu chez les plantes, sur la production de l’activité laccase par C. gallica. Les résultats obtenus ont démontré une nette augmentation des niveaux de laccase extracellulaire lorsque C. gallica était cultivé en présence d’acide tannique. Des concentrations de 50 et 100 µM de ce produit ont fortement augmenté l’activité laccase comparativement à des échantillons de contrôle cultivés en absence d’acide tannique. Nous avons de plus constaté une augmentation des niveaux de transcription de la laccase chez C. gallica cultivé dans des milieux enrichis d’acide tannique. Nous démontrons ainsi le rôle de l’acide tannique comme inducteur de la laccase chez ce champignon à cause d’une augmentation de l’expression du gène et de la transcription de la laccase. Mots clés : laccase, acide tannique, Coriolopsis gallica, induction, transcription d’un gène. [Traduit par la Rédaction] 1047

Introduction White-rot basidiomycetous fungi are gaining interest because of their capability to degrade a wide variety of natural and synthetic materials and environmentally persistent organopollutants, such as chlorophenols, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and industrial dyes

(Pointing 2001). A highly nonspecific ligninolytic enzymatic system secreted by white-rot fungi is known to be involved in this biodegradation (Thurston 1994; Leonowicz et al. 1999). Research on this topic has been undertaken recently in many laboratories because of its great potential in several biotechnological applications, such as bioremediation (Roy-Arcand and Archibald 1991; Pointing 2001), animal feed improve-

Received 8 July 2002. Revision received 20 November 2002. Accepted 28 November 2002. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 7 January 2003. J.M. Carbajo, H. Junca,1 M.C. Terrón, T. González,2 S. Yagüe, E. Zapico,3 and A.E. González.4 Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Velázquez 144, E-28006, Madrid, Spain. 1

Present address: Department of Environmental Microbiology, Gesellschaft für Biotechnologische Forchung (GBF)-National Research Centre for Biotechnology, D-38124 Braunschweig, Germany. 2 Present address: Instituto Cubano de Derivados de la Caña de Azúcar, Havana, C.P. 11000 Cuba. 3 Present address: Biotechnology Department, University of Hamburg, Hamburg, D-21073 Germany. 4 Corresponding author (e-mail: aldo@cib.csic.es). Can. J. Microbiol. 48: 1041–1047 (2002)

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DOI: 10.1139/W02-107

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ment (Akin et al. 1993), pulp and paper production (Messner and Srebotnik 1994), and wastewater treatment (Terrón et al. 1993; Garg and Modi 1999). Studies on lignin-degrading enzymes have been mainly focused on peroxidases (lignin and manganese peroxidases) and laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2). Although biochemical, genetic, and regulatory aspects of peroxidases have been relatively well studied (Cullen 1997), information on laccases is still scarce. Nevertheless, the important role of laccases in degradation of lignin and other phenolic molecules has been confirmed (Eggert et al. 1997). To date, there are few reports focused on the regulatory mechanisms involved in laccase production (Collins and Dobson 1997; Mansur et al. 1998; Palmieri et al. 2000). Still, these studies are of interest given that large quantities of the enzyme will be required for further practical applications in bioremediation and biotechnology (Cullen 1997). There are several studies demonstrating increased laccase activity levels in basidiomycetes caused by natural and synthetic aromatic substances (Pickard and Westlake 1970; Arora and Sandhu 1984). In contrast, there are few reports on the effect of phenolics and other substances on the enhancement of laccase gene transcription (Linden et al. 1991; Collins and Dobson 1997; Mansur et al. 1998). Some of these inducers are synthetic molecules, such as 1hydroxybenzotriazole, cycloheximide, and 2,5-xylidine, which show a significant degree of toxicity to animal and human health. Given the toxicity associated with these substances, the search for less harmful compounds that are able to increase laccase levels is of environmental importance. The ligninolytic basidiomycete Coriolopsis gallica has been selected in our laboratory, based on the results of a previous screening performed among more than 90 fungal species to select the most efficient decolorizer of a lignincontaining paper-industry effluent; a laccase enzyme is suggested to be involved in this process (Calvo et al. 1998). Moreover, another strain of C. gallica has recently been reported to be an effective pollutant degrader (Pickard et al. 1999). To date, we have evidence of only one genomic sequence for a laccase gene in C. gallica, even though there are reports of gene families for laccase in some white-rot fungi (Yaver and Golightly 1996; Mansur et al. 1997; Palmieri et al. 2000), and in spite of our numerous attempts to find more laccase sequences in this fungus (Zapico 1999). This laccase gene (cglcc1) has been cloned and sequenced (GenBank accession No.: AY017340). Different types of industrial effluents (paper industry, distillery, beer factory, and oil mill wastewaters) have been shown to increase laccase activity in various white-rot fungi (Mansur et al. 1997; Calvo et al. 1998; Pérez et al. 1998; González et al. 2000; Tsioulpas et al. 2002). All of these effluents derive from industries that use plants as a raw material, and the presence of tannic compounds has been reported in some of them (Maestro-Durán et al. 1993). After lignin, tannins are the most abundant group of plant polyphenols and share with lignin a series of common structural polyphenolic features (William et al. 1986). Tannic acid (TA) is the tannin most widely distributed in nature. The possible role of TA as a laccase inductor in C. gallica was studied from a physiological and molecular point of view, and the results are presented in this work.

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Materials and methods Chemicals TA (tannic acid powder pure, United States Pharmacopoeia (USP) empirical formula C 76H 52O 46), was obtained from Merck (Darmstadt, Germany). 2,2′-Azino-bis(3ethylbenzthiazoline-6-sulfonate) (ABTS) was purchased from Boehringer Mannheim (Mannheim, Germany) and 2,6-dimethoxyphenol from Fluka (Buch, Switzerland). All other chemicals were reagent grade obtained from Merck, Boehringer Mannheim, or Sigma–Aldrich Corp., St. Louis, Mo. Organism and maintenance Coriolopsis gallica A-241 was obtained from the IJFM (Instituto Jaime Ferrán de Microbiología) collection. The fungal culture was maintained on malt agar slants (2% malt extract, 2% Bacto agar, Difco, Detroit, Mich.), grown for 10 days at 28°C and stored at 4°C. Culture conditions The fungus was grown on agar plates with modified Czapek’s medium (Guillén et al. 1990) for 7 days at 28°C. Ten plugs (1 cm2) were cut and inoculated under sterile conditions into 500-mL culture flasks containing 300 mL of Kirk growth medium (nitrogen-limited defined medium) (Kirk et al. 1986). After incubation for 48 h at 28°C in an orbital shaker (200 rpm), 10 mL of the culture medium containing little fragments of fungal mycelia was used to inoculate 250-mL Erlenmeyer flasks containing 90 mL of Kirk medium. Samples were incubated at 28°C and 125 rpm for 7 days. After this time, a filter-sterilized (0.22 µm) solution of 50 mM TA in distilled water was added to the culture medium to reach a final concentration of 50, 100, or 200 µM. Controls without TA were also grown. In all cases, the final pH of the culture media was 4.6 ± 0.1, which is optimum for C. gallica growth (Calvo et al. 1998). Laccase activity was measured in the extracellular fluid throughout the following 9 days of incubation. Enzymatic activities assays Laccase activity was determined in the extracellular fluid of fungal cultures by the method of Wolfenden and Willson (1982), using ABTS as the substrate. Lignin and manganese peroxidases were measured as described by Tien and Kirk (1984) and Paszczy½ski et al. (1988), respectively. One unit of enzymatic activity is defined as the formation of 1 µmol of product per min. Zymograms of fungal laccase activity Polyacrylamide gel electrophoresis was performed at alkaline pH under nondenaturating conditions using a MiniProtean (Bio-Rad Laboratories, Hercules, Calif.) electrophoresis cell. The separating gel contained 12% acrylamide, and the buffer solution was 375 mM Tris–HCl (pH 8.8). The stacking gel contained 5% acrylamide, and the buffer solution was 125 mM Tris–HCl (pH 6.8). The electrode buffer solution contained 25 mM Tris–HCl and 122 mM glycine (pH 8.8). Prestained molecular weight standards (Bio-Rad Laboratories) were used. All attempts to determine total proteins in the extracellular medium were inaccurate because of © 2002 NRC Canada

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the interference caused by TA. This problem was overcome by loading the same amount (25 µL) of extracellular fluid from induced and control cultures. To detect laccase activity, the gels were previously equilibrated in 100 mM acetate buffer at pH 5.0 and stained using 10 mM 2,6-dimethoxyphenol as the substrate.

1043 Fig. 1. Time course for laccase activity monitored in the extracellular medium of Coriolopsis gallica grown in the absence () and in the presence of 50 µM (䉱) and 100 µM (Ž) tannic acid. Time is expressed in days after the addition of tannic acid. Experimental data are the means of three experiments, and the experimental error was never greater than 5%.

RNA preparations Total RNA was prepared using the Fast RNA Kit following the manufacturer’s instructions (BIO 101 Inc., San Diego, Calif., U.S.A.) from fresh C. gallica mycelium collected at day 5 of incubation with different concentrations of TA and from controls without TA. PCR PCR was performed to obtain the hybridization probes for cglcc1 and gpd1 (glyceraldehyde-3-phosphate-dehydrogenase gene from C gallica). Taq DNA polymerase (Perkin Elmer Corp., Norwalk, Conn.) was used as recommended by the manufacturer, using a Rapidcycler (Idaho Technology, Idaho Falls, Idaho, U.S.A.) thermocycler with a PCR temperature program of 95°C for 1 min, followed by 30 cycles of 95°C for 40 s, 55°C for 40 s, 72°C for 1 min, and a final extension at 72°C for 8 min. All the primers were used at a final concentration of 0.4 µM. The hybridization probe for cglcc1 was obtained using as a template the plasmid pYES1, which contains the full length cDNA of the C. gallica laccase gene (GenBank acc. No. AF263467). The primers 5RT (5′-GCGATTGGCCCCAAGACTG-3′) and 3RT (5′-CAGTGGCTGCGTGTTCACAC-3′) were designed to anneal at specific sites of this gene, amplifying an intragenic region of 690 bp. The probe to detect gpd1 transcripts, used as a signal of constitutive expression, was obtained using primers DIM (5′TCAACGGTTTCGGTCGTATT-3′) and RM2 (5′-GTGGACGGTGGTCATGAGAC-3′) that amplify a highly conserved fragment of 515 bp of the gpd1 gene from C. gallica (GenBank acc. No. AF297874). The template DNA was the plasmid pHJC5 that included a partial gpd1 cDNA, synthesized previously by RT-PCR and cloned on pGEM-T (Promega, Madison, Wisc.). Labeling of probes was performed using the PCRamplified fragments of cglcc1 and gpd1 that were run on a 1% agarose gel and eluted with the UltraClean-15 DNA purification kit (MO BIO, Solana Beach, California). One microgram of each purified fragment was diluted in H2O to a total volume of 16 µL, then the DNA samples were denatured in a boiling water bath for 10 min and chilled on ice. After that, 4 mL of digoxigenin (DIG) High Prime (Boehringer Mannheim) were added, mixed, and spinned down briefly. The reaction was then incubated at 37°C overnight and stopped with 2 µL of 0.2 M EDTA. Northern analysis The expression of cglcc1 and gpd1 genes was estimated using the hybridization probes described above. Approximately 10 µg of total RNA were electrophoresed overnight at 1.2 V/cm in a 1.2% agarose–formaldehyde gel with 40 mM MOPS (morpholinepropanesulfonic acid) – 10 mM sodium acetate (pH 7.0) – 1 mM EDTA. RNA band intensities were densitometrically measured after ethidium bromide

staining, and the RNA was then blotted by capillary transfer to a Hybond-N membrane (Amersham Biosciences Corp., Piscataway, N.J.) in 20× SSPE (0.36 M NaCl, 20 mM NaH2PO4, 2 mM EDTA, pH 7.7). The membrane was then hybridized with the homologous DIG-labeled probes under high stringency conditions, following the manufacturer’s instructions (Boehringer Mannheim). Hybridization signals and RNA bands were quantified by densitometry using Imagequant Software (Molecular Dynamics, Sunnyvale, Calif.). The transcript levels of the cglcc1 gene were calculated by dividing their hybridization signal percentages by the corresponding gpd1 constitutive signals. Experimental reproducibility All the experiments were performed three times. The standard deviation in all the analytical assays was always less than 5%.

Results Influence of different concentrations of TA on laccase production: spectrophotometric time course A time course experiment for laccase activity in the extracellular fluid of C. gallica showed that it was clearly influenced by the addition of different concentrations of TA (Fig. 1). At day 2 of fungal growth in the presence of 50 µM TA, laccase values were higher than those of the controls and reached a maximum after 4 days. From day 4 to 9 of incubation with 50 µM TA, the activity decreased, although at day 9, it was still higher than controls. At a final concentration of 100 µM TA, the increase of laccase activity was much higher than that detected at 50 µM but occurred later, reaching a maximum after 7 days following TA addition. The highest TA concentration assayed (200 µM) caused a partial inhibition of C. gallica growth. The same observation regarding the negative effect of tannic compounds on the growth of several filamentous fungi has been reported (Davidson et al. 1938; Nobles 1948; Scalbert 1991) and for © 2002 NRC Canada

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1044 Fig. 2. Zymograms of laccase activity monitored in the extracellular fluid of Coriolopsis gallica grown in the absence (lanes b) and in the presence of 50 µM (lanes c) and 100 µM (lanes d) tannic acid at different days of incubation after the addition of tannic acid. Molecular weight standards are represented by lanes a.

this reason, the results of laccase activity at this TA concentration were considered not to be comparable. In all cases, except in controls without TA, an increase of brown color in the extracellular fluid was observed during the first days after the addition of TA (data not shown), at which time laccase activity also started to be detected (Fig. 1). Moreover, the color intensity was directly correlated with TA concentration in the cultures containing 50 and 100 µM of TA. This color became less intense as the length of incubation increased, along with increasing values of laccase activity in the extracellular fluid. Lignin peroxidase and manganese peroxidase were not detected in the extracellular fluid of C. gallica grown with different concentrations of TA nor in the controls without TA. Influence of different concentrations of TA on laccase production: zymograms The results of zymograms of laccase activity from day 1 to 9 after the addition of various concentrations of TA are shown in Fig. 2. The increase in the laccase activity detected in the fungal culture containing 50 and 100 µM TA (Fig. 1) was also observed in the zymograms (Fig. 2). As in the spectrophotometric assays, the effect of TA on increased laccase activity was observed earlier in the medium containing 50 µM TA than in the culture with 100 µM TA (Fig. 2). In the last 5 days of incubation, the induction of laccase production became stronger in the cultures containing 100 µM TA, whereas a decrease in laccase signals were observed in the presence of 50 µM TA, although levels were still higher than controls at day 9. In addition, the density of the induced enzymes bands was greater than those of controls without TA. Moreover, the bands of laccase activity in the controls showed a sharp pattern during the first 4 days assayed, becoming more diffuse towards the latter part of the experiment (Fig. 2, lanes b).

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Transcription analysis of cglcc1 The possible correlation between the increase in laccase activity observed in culture media supplemented with TA and the levels of cglcc1 transcripts was analyzed by Northern blotting. RNA was prepared from C. gallica mycelium harvested on day 5 after the addition of 50, 100, and 200 µM of TA and from controls. Good quality RNA was obtained from controls and from samples with 50 and 100 µM, but all the attempts to extract RNA from mycelium incubated in the presence of 200 µM TA were unsuccessful. The interference of tannic compounds in the extraction of RNA has previously been reported in the case of polyphenolic-rich materials (John 1992). In the same way, TA also causes an inaccurate spectrophotometric quantification of RNA; this problem was overcome by using the hybridization signal of gpd1 from C. gallica as a loading control. The effect of TA on cglcc1 expression is shown in Fig. 3, which also shows the ratio between the hybridization signals of cglcc1 and gpd1 (Fig. 3D). In the range of TA concentrations tested, the results showed the existence of a clear correlation between TA concentration and induction of cglcc1 expression. Our results showed that relative levels of cglcc1 transcripts in cultures containing 50 and 100 µM TA were higher than controls without TA, in agreement with the laccase activity determined spectrophotometrically and the zymogram data obtained on day 5. This observation indicates that TA can have an important effect on the induction of cglcc1 gene transcription, suggesting that these transcripts are translated to an active laccase protein.

Discussion Until now, little work has been done regarding the regulation of laccase gene expression in white-rot fungi (Eggert et al. 1996; Collins and Dobson 1997; Mansur et al. 1998; Palmieri et al. 2000). The addition of inducers is one of the simplest methods to increase the yield of enzyme production. It has been shown that tannic compounds are able to increase laccase activity in several basidiomycetes (Pickard and Westlake 1970; Arora and Sandhu 1984) and ascomycetes (Kim et al. 1995), although, as far as we know, the molecular basis of such induction has not been reported. The results presented here demonstrate that the expression of the laccase gene cglcc1 of C. gallica is transcriptionally inducible by TA, giving rise to an enhancement of laccase activity. The induction of laccase in several fungi by a number of low molecular weight phenolic compounds (KoroljovaSkorobogat’ko et al. 1998), aromatic acids (Farnet et al. 1999), a variety of flavonoids (Pickard and Westlake 1970), and different lignin preparations (Arora and Sandhu 1984) has been demonstrated at the physiological level. Our results showed that TA can be considered as an efficient inducer of laccase production by C. gallica, as revealed by the increase of enzymatic activity detected and, as will be discussed later, by the enhancement of the laccase gene at the transcription level. An increased level of laccase activity in C. gallica and other white-rot fungi growing in the presence of diverse types of industrial wastewaters has been frequently detected (Ardon et al. 1998; Pérez et al. 1998; Calvo et al. 1998; © 2002 NRC Canada

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Fig. 3. (A) Total RNA samples from mycelium of Coriolopsis gallica harvested on day 5 after the addition of 0, 50, and 100 mM of TA (lanes 1, 2, and 3, respectively). Northern blot analysis of cglcc1 gene expression (B) and gpd1 gene transcripts (C). (D) cglcc1 transcript levels represented as the ratio between the hybridization signals of cglcc1 and gpd1.

González et al. 2000; Yagüe et al. 2000; Tsioulpas et al. 2002), and the presence of tannins has been described in some of them (Maestro-Durán et al. 1993; Yagüe et al. 2000). Our results suggest that laccase induction produced by this type of industrial effluents could be ascribed, in part, to TA or to structurally related molecules. A detoxification of toxic phenols by means of polymerization reactions has been suggested as one of the physiological roles of fungal laccases (Bollag et al. 1988; Thurston 1994). Our observation that an increase of color in extracellular fluid of cultures containing TA coincided with detection of laccase supports the proposed role of the enzyme in the detoxification of phenolic compounds by polymerization reactions. In addition, the fact that this color became less intense with time, along with increasing values of laccase activity, suggests the involvement of laccase in the further degradation of these compounds (Bollag et al. 1988) without disregarding the possible contribution of other enzymes. Studies using different basidiomycetous strains are being carried out in our laboratory to obtain further information on this subject. Laccase induction by TA could be indicative of an environmental switching response developed by fungi to oxidize and, therefore, to decrease the potentially toxic effect of TA and related compounds. Recently, some of our results regarding the decolorization of tannin-rich wastewaters also suggested a toxic effect of polyphenols on the growth of C. gallica and a further adaptation of the fungus along with a polymerization of phenol compounds (Yagüe et al. 2000). Similarly, an induction of laccase by other potential toxic compounds in connection with an adaptative fungal response to diminish their toxic effect has also been suggested for 2,5-xylidine in Pycnoporus cinnabarinus (Eggert et al. 1996), 2,5-xylidine and 1-hydroxybenzotriazole in Trametes versicolor (Collins and Dobson 1997), and for undefined

compounds present in distillery vinasses in Trametes sp. I62 (Mansur et al. 1997). At the transcriptional level, induction of laccase in the ascomycete Neurospora crassa has been well characterized (Linden et al. 1991; Tamaru et al. 1994). Moreover, Schouten and coworkers (2002) have recently described the induction of transcripts for the laccase gene bclcc2 from the ascomycete Botrytis cinerea when a solution of tannic acid was added to the culture medium. In contrast, in basidiomycetous fungi, studies regarding induction of laccase transcription are still scarce (Eggert et al. 1996; Collins and Dobson 1997; Mansur et al. 1998; Palmieri et al. 2000). Our results showed a direct relation between an increase in cglcc1 transcript levels and the enhancement of laccase activity values produced by C. gallica in the presence of TA. The presence of various laccase isozymes in the extracellular fluid of C. gallica growing in TA is suggested by the thick shape of the bands attained in the zymograms presented in this study. Laccase isozymes can be ascribed to a laccase gene family or to postranslational modifications of a single protein. The latter could be the case for cglcc1, given that several potential places of glycosylation can be found in the putative amino acidic sequence deduced from this gene. Glycosylation is a common feature that has been demonstrated in many other fungal laccase proteins from basidiomycetes (Perry et al. 1993; Giardina et al. 1996). The increase of laccase activity could be, among others, the result of several aspects: (i) an increased production of laccase mRNA, (ii) an increased stability of laccase mRNA transcripts, (iii) increased production of the active protein, and (iv) an increased half-life of laccase protein. Work is currently underway to examine some of these possibilities. The induction of laccase production in different filamentous fungi seems to be specific for certain aromatic © 2002 NRC Canada

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compounds; therefore, some precise mechanisms of transcriptional activation are likely to be involved. We have found a putative xenobiotic response element (XRE) in the promoter of cglcc1 of C. gallica (GenBank acc. No. AY017340). The sequence GTGCCAT, reported by Fujisawa-Sehara et al. (1988) (coincidences with the XRE consensus sequence are underlined), is present 115 bp upstream of the TATA box. The presence of these putative XREs has been reported in several white-rot fungal lcc promoters (Coll et al. 1993; Giardina et al. 1995; Collins and Dobson 1997; Mansur et al. 1997), suggesting that transcription of laccase genes may be activated by stressing compounds, such as TA. The high degree of toxicity for human and animal health shown by some synthetic laccase inducers makes their use rather limited as potential enhancers of laccase production on an industrial scale in the future. TA is one of the first reported molecules of natural origin that is able to increase laccase levels, making its use, in principle, more suitable from an industrial standpoint. In addition, the synthetic origin of some of the inducers studied to date make it difficult to elucidate their role in nature. In contrast, tannic compounds are natural molecules widely distributed in the environment, mainly as components of the external surfaces of plants, which represent the first defense mechanism against external aggressions. The results presented here permit us to propose an essential role of tannins as inducers of fungal laccases in the initial steps of wood degradation in natural systems.

Acknowledgements We are grateful to G. del Solar, M. Espinosa, and A.D.W. Dobson for their critical reading of the manuscript. Research was financed by Comisión Interministerial de Ciencia y Tecnología (CICYT, Madrid, Spain) BIO 97-0655-E. H. Junca acknowledges financial support from G. Díaz de Junca. T. González and E. Zapico acknowledge support from a Mutis Programme doctoral grant from Agencia Española de Cooperación Internacional (AECI) (Spain). S. Yagüe acknowledges a grant from Ministerio de Ciencia y Tecnologia. M.C. Terrón acknowledges a postdoctoral grant from Conserjería de Educación y Cultura de la Comunidad Autónoma de Madrid (Spain).

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01.Carbajoetal2002  
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1 Present address: Department of Environmental Microbiology, Gesellschaft für Biotechnologische Forchung (GBF)-National Research Centre for...

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