02.Gonzalezetal2003

Mycol. Res. 107 (6): 727–735 (June 2003). f The British Mycological Society

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DOI: 10.1017/S0953756203007937 Printed in the United Kingdom.

Identification of a new laccase gene and confirmation of genomic predictions by cDNA sequences of Trametes sp. I-62 laccase family

Tania GONZA´LEZ#, Marı´ a del Carmen TERRO´N, Ernesto ZAPICO$, Susana YAGU¨E, Alejandro TE´LLEZ·, Howard JUNCA## and Aldo GONZA´LEZ* Centro de Investigaciones Biolo´gicas, Vela´zquez 144, E-28006 Madrid, Spain.

E-mail : aldo@cib.csic.es Received 21 October 2002; accepted 17 April 2003.

The strain Trametes sp. I-62 (CECT 20197) is a white-rot fungus with great potential for biotechnological applications in the fields of industrial waste water decolorization and clean up. Three laccase genes: lcc1, lcc2 and lcc3 have been cloned and sequenced from this basidiomycete. In this work, the coding regions of the corresponding cDNAs have been synthesized, cloned, and sequenced. They are 1563, 1563 and 1575 bp in length, respectively. Former putative intron/ exon structures from genomic DNA are fully confirmed by match analysis with our cDNA sequences. Using Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR–RFLP) analysis, an additional laccase cDNA was also identified, corresponding to a new gene, lcc1A, which displayed 99.6 % identity with lcc1 at protein level. Such high similarity between lcc1 and lcc1A sequences, and the comparison with reports from other basidiomycete laccases, suggest that in this strain these two genes are allelic variants.

INTRODUCTION Laccases (benzenediol : oxygen oxidoreductases, EC1.10.3.2) are a heterogeneous group of multi-copper oxidative enzymes widespread in plants and fungi (Thurston 1994). Although in bacteria laccase activity has been rarely described, it has been detected in Azospirillum lipoferum (Givaudan et al. 1993) and more recently in two marine bacteria and in Bacillus subtilis endospore coat (Solano & Sanchez-Amat 1999, Martins et al. 2002). Recently, a revision on the presence of laccase in bacteria has been published (Alexandre & Zhulin 2000). A variety of different, and sometimes contradictory, physiological functions has been proposed for this type of oxidative enzyme. In fungi, laccases have been involved in pigmentation (Leatham 1985, Coll et al. 1993a, b), fruiting-body formation (Wood 1980), pathogenicity (Larson, Choi & Nuss 1992, Williamson 1997) and lignin degradation (Ander * Corresponding author.

Present addresses: # Instituto Cubano de Derivados de la Can˜a de Azu´car, Havana, Cuba; $ Biotechnology Department, University of Hamburg, D-21073, Hamburg, Germany; · Departmento de Biotecnologı´ a, Universidad Auto´noma Metropolitana Iztapalapa, S. Rafael Atlixco nx 186, Col. Vicentina, C.O. 09340 Me´xico D.F., Me´xico; ## Department of Environmental Microbiology, GBFNational Research Centre for Biotechnology, D-38124 Braunschweig, Germany.

& Eriksson 1976, Hatakka 1994, Bourbonnais et al.

1995, Call & Mu¨cke 1997). Although the physiological role of this enzyme has not as yet been definitively clarified, it is known that laccases catalyse the oxidation of different aromatic compounds (mono-, di-, and polyphenols, aminophenols, and diamines) by reducing molecular oxygen to water (Reinhammar 1984).

Laccases have great potential as industrial enzymes due to their capacity to degrade a broad diversity of natural and synthetic materials such as lignin (Kirk & Farrell 1987, Hatakka, Mohammandi & Lundell 1989), chlorophenols (Roy-Arcand & Archibald 1991) polycyclic aromatic hydrocarbons (Field et al. 1993), tannins (Yagu¨e et al. 2000), melanoidins (Gonza´lez et al.

2000) and azo dyes (reviewed by Husain & Jan 2000).

Different applications for these enzymes would include the upgrading of animal feed (Hatakka et al. 1989, Akin et al. 1993), pulp and paper production, textile dye bleaching, bioremediation and effluent detoxification, washing powders components, removal of phenols from wines (Servili et al. 2000), and transformation of antibiotics and steroids (Breen & Singleton 1999).

Several reviews on laccase function and applications has been recently published (Leonowicz et al. 2001, Mayer & Staples 2002).

The biotechnological importance of laccases has encouraged the search for genes coding for them in

New fungal lcc gene, confirmation of genomic DNAs different organisms. A laccase gene sequence was first described in the ascomycetous fungus Neurospora crassa (Germann & Lerch 1986). Two other gene sequences were reported, one of them in the hyphomycete: Aspergillus nidulans (Aramayo & Timberlake 1990), and the other, in the basidiomycete Coriolus hirsutus (Kojima et al. 1990). Perry et al. (1993) described the presence of two laccase genes in the same chromosome of the basidiomycete Agaricus bisporus, thus reporting the first laccase gene family in fungi.

Subsequently, four gene families have been described in Rhizoctonia solani (Wahleithner et al. 1996) and Pleurotus sajor-caju (Soden & Dobson 2001) ; three in Pleurotus ostreatus (Giardina et al. 1995, 1996, 1999), a family of five genes in different chromosomes of Trametes villosa (Yaver et al. 1996, Yaver & Golightly 1996) and two genes in Trametes versicolor (Jo¨nsson et al. 1995, Mikuni & Morohoshi 1997, Ong, Pollock & Smith 1997).

At first, the diversity of fungal laccase isozymes was thought to be the result of post-translational modifications of the same gene product. The characterization of the several laccase gene families mentioned above suggested that at least in part, this biochemical diversity would be the result of the multiplicity of laccase genes in fungal genomes. However, in most fungi, laccase activity is produced at levels which are too low for industrial purposes, and it is clear that any realistic application of these enzymes requires their production from an inexpensive source. For this reason concerted efforts are being made dedicated to express cloned laccase genes in heterologous hosts (Kojima et al. 1990, Saloheimo & Niku-Paavola 1991, Gouka et al. 2001).

A key step in the production of recombinant enzymes from eukaryotes is the isolation, characterization and molecular cloning of their cDNAs. The white-rot fungus Trametes sp. I-62 (CECT 20197) is a strain with a wide biotechnological potential (Pointing 2001). The high detoxification capacity of distillery effluents shown by this fungus, and the possible role of laccases in this process have been studied in our laboratory (Gonza´lez et al. 2000). Moreover, Mansur and coworkers had already described the biochemical diversity of laccases in this strain, and they cloned three laccase genes (lcc1, lcc2 and lcc3) of a probably larger family (Mansur et al. 1997).

In the present work, cDNA sequences of the lcc1, lcc2 and lcc3 laccase genes from Trametes sp. I-62 were cloned, sequenced and characterized. A new laccase cDNA sequence was also identified, and evidence is shown that it corresponds to another laccase gene from this strain.

MATERIALS AND METHODS Organism and culture conditions The basidiomycete used in this study, Trametes sp. I-62 (CECT 20197), was isolated from decayed wood of

728 Pinar del Rı´ o, Cuba (Mansur et al. 1997). The voucher specimen from which this isolate was made is preserved in the National Research Institute on Tropical Agriculture (INIFAT), Habana ; it was determined as Trametes sp. by Joost A. Stalpers (CBS) but could not be named to species rank. The fungal culture was grown on agar plates with a modified Czapeck medium (Guille´n, Martı´ nez & Martı´ nez 1990) for 7 d at 28 xC. Submerged cultures of the fungus were prepared by the inoculation of eight plugs (1 cm2) from these plates, under sterile conditions, into 500 ml culture flasks containing 300 ml of the same growth medium and four glass beads (1.5 cm diam). After incubation for 24 h at 28 x in an orbital shaker (100 rpm), a 7.5 ml inoculum was transferred into 250 ml Erlenmeyer flasks containing 75 ml (total volume) of Kirk medium (Kirk et al.

1986). The culture was incubated for 7 d under the same conditions of temperature and agitation.

Laccase activity Laccase activity was determined in the extracellular fluid of fungal cultures (Wolfenden & Willson 1982), using ABTS (2,2k-azinobis-3-ethylbenzthiazoline6-sulphonate) as the substrate. One unit of laccase activity was defined as that catalyzing formation of 1 mmol of oxidized ABTS minx1.

RNA preparation Fresh mycelium samples (approx. 1 g) were harvested daily and those corresponding to the maximal laccase activity were used for total RNA extraction, that was performed by using the Fast RNA kit-Red, following the indications of the manufacturer (BIO 101, Montreal). Total RNA concentration was determined spectrophotometrically.

RT–PCR First-strand cDNA synthesis was carried out from 2 mg of total RNA by using a cDNA synthesis kit (Roche) according to the manufacturer’s instructions.

For PCR amplification, a 5 ml volume from each RT reaction mixture was mixed with primers, Taq polymerase (PerkinElmer) and the rest of components used in standard PCR reactions (Sambrook, Fritsch & Maniatis 1989). cDNA amplification was performed in a Rapidcycler (Idaho Technology, Idaho Falls) thermocycler with a PCR temperature program of 95 x for 5 min, followed by 35 cycles of 95 x for 45 s, 61 x for 30 s, 72 x for 2 min, and a final extension of 72 x for 7 min. MgCl2 final concentration in all PCR reactions was 2.5 mM. Specific primers to amplify the complete codifying regions of cDNAs, and used in the sequencing reactions were designed from the Trametes sp. I-62 lcc1, lcc2 and lcc3 laccase gDNA sequences previously reported (Mansur, Sua´rez & Gonza´lez 1998). Fig. 1 shows primers sequences and annealing

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Fig. 1. Binding sites and sequences of primers used in the PCR reactions to amplify the coding sequence of lcc1, lcc2 and lcc3 laccase genes from Trametes sp. I-62. Primer annealing sites are indicated by arrows. Coding and intravening sequences are represented by clear and dark regions respectively. ‘cd ’ primers were used in the amplification of lcc gene cDNAs and ‘ icd ’ primers were used in the sequencing reactions.

sites for each gene. DNA samples were electrophoresed on 1% agarose gels, and visualized under UV light after staining with ethidium bromide. cDNA cloning and restriction analysis PCR products were purified from agarose gels using the ‘Ultra Clean DNA purification Kit ’ from MOBIO, and cloned in the pGEM-T vector (‘pGEM-T Vector System ’, Promega). Prior to sequencing, the clones corresponding to lcc1, lcc2 and lcc3 cDNAs, were checked by digestion with endonuclease enzymes that were expected to produce characteristic restriction patterns according to the restriction maps of corresponding gDNA sequences.

DNA sequencing and analysis Nucleotide sequences of inserts from selected clones were determined in the Sequencing Facility at Centro de Investigaciones Biolo´gicas (Madrid) by using an automated 3700 DNA sequencer (PerkinElmer, Applied Biosystems, Hitachi) and the sequencing kit Big Dye based on fluorescence labelling reaction from the same manufacturer. All DNAs were sequenced on both strands. 5k and 3k ends of the cDNA cloned in the pGEM-T vector were sequenced using the T7 and M13 reverse ‘universal ’ primers. Sequencing of internal regions was done using specific primers for each cDNA (Fig. 1). cDNA and putative protein sequences were analysed with ALIGN, BESTFIT and BLAST programs integrated in the macroprogram ‘GCG Wisconsin ’ developed by Devereux, Haeberli & Smithies (1984). The sequences of Trametes sp. I-62 lcc1, lcc1A, lcc2 and lcc3 cDNAs, reported in this paper, have been assigned the GenBank accession nos. AF548032, AF548033, AF548034, AF548035, respectively.

RESULTS Synthesis, cloning and sequencing of lcc gene cDNAs In order to isolate RNA for cDNA synthesis, Trametes sp. I-62 was grown in conditions that result in high levels of laccase, with the highest enzymatic activity being reached at the fifth day of culture. The mycelium harvested at this day was used to purify total RNA. This was reverse transcribed into single-stranded cDNA, that was finally amplified by PCR using specific primers, as described above.

As a result of the PCR reactions, a single product was obtained for each of the three laccase genes of Trametes sp. I-62. The size of the products was approximately 1600 bp, as expected from the putative cDNA deduced from the genomic DNA sequences reported by Mansur et al. (1997). The amplified cDNAs were cloned and the resulting constructions were subjected to restriction analysis as a first step in the sequence analysis of these inserts. All cDNA clones corresponding to lcc2 and lcc3 genes showed the expected restriction pattern. However, lcc1 cDNA clones were segregated into two different groups (both having the same proportion). One of them produced identical restriction patterns as expected from the gDNA sequence, but the other group showed unexpected patterns upon digestion with ApaI and BamHI enzymes.

Inserts from the last group of clones could not be digested with ApaI, as it should be if they had the expected sequence ; and the digestion with BamHI produced only two restriction fragments instead of the three that should be obtained. These results indicate the lost of both the single ApaI site, and of one of the BamHI recognition sites in the cDNAs from this group of clones.

Two clones carrying cDNA of each lcc2 and lcc3 genes, together with two further clones from both

New fungal lcc gene, confirmation of genomic DNAs

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Fig. 2. Alignment of a new laccase cDNA sequence (lcc1A) from Trametes sp. I-62, isolated by RT–PCR (on top), with the cDNA sequence of the lcc1 gene from this fungus. Differences are indicated by *.

groups of lcc1 cDNA clones were sequenced. Clones of the two different groups derived from lcc1 cDNA restriction analysis have a very similar nucleotide sequence (Fig. 2). They differ in nine nucleotides, resulting only in a two amino acid change between the putative proteins. These small differences prompted us to perform an additional analysis to verify that the new cDNA sequence corresponded to another laccase gene, and that nucleotide changes were not due to mistakes introduced in the cDNA synthesis and/or amplification.

Partial cloning of the new laccase gene by PCR–RFLP Fig. 3 shows the strategy designed to isolate a fragment of gDNA corresponding to the possible new laccase gene. The basis of the analysis is the difference in the restriction patterns obtained from the digestion with ApaI and BamHI endonucleases. At first, the 5k half of the lcc1 gene from genomic DNA was PCR-amplified, using the specific primers shown in Fig. 3. This permitted the cloning of a PCR product of approximately 1 kb (Fig. 4A) that was purified and digested with ApaI,

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(A)

(B)

(C)

Fig. 3. Strategy to confirm the presence of the Trametes sp. I-62 lcc1A laccase gene. Coding and intravening sequences are represented by clear and dark regions respectively. The coding region differs from that of lcc1 in only nine base substitutions.

(A) These base changes cause the lost of an ApaI recognition site and of one of the two BamHI sites. (B) The 5k half of the gene is amplified, and the PCR product is digested with ApaI, BamHI and KpnI. (C) Last enzyme must produce the same pattern for both genes. Undigested DNA bands obtained upon treatment with ApaI and BamHI must correspond to the new gene (lcc1A).

In order to verify this assumption, this DNA was cloned and sequenced.

BamHI, and KpnI enzymes. As it was expected, part of the PCR product from the lcc1 gene amplification was digested with ApaI (Fig. 4B) and BamHI (Fig. 4C) and fragments of the expected size were obtained. However, approximately half of the total amount of this product remained undigested, as would occur if another laccase gene which differs from lcc1 in these two restriction sites actually exists. Digestion with KpnI endonuclease, which has two recognition sites in this fragment, was done in order to confirm that the PCR product derived exclusively from both lcc1 and the new laccase gene, and not from other different sequences. In this case the PCR product was completely digested, and the three expected restriction fragments were obtained (Fig. 4D).

Fragments of approximately 1 kb undigested with ApaI and BamHI, corresponding to the putative new laccase gene were subsequently purified and cloned in the pGEM-T vector. Clones containing both inserts were sequenced. The sequences of the new lcc cDNA isolated in this work, and that of the genomic DNA fragment entirely coincide in the exons regions. This confirms that they correspond to the same gene: a laccase gene different from lcc1, which was assigned as lcc1A.

Characterization of the lcc cDNA sequences The DNA sequences of lcc1, lcc2 and lcc3 cDNAs obtained by RT–PCR consisted of 1563, 1563 and 1575 bp, respectively. That is, lcc2 and lcc3 cDNA lengths agree with those predicted from the genomic DNA sequences (Mansur et al. 1997). That from lcc1 differs in the presence of three additional bases that do not cause any reading frame displacement. They only introduce a new amino acid (valine) in the composition of the putative protein. Comparison between cDNA and genomic DNA sequences of the lcc1, lcc2 and lcc3 genes confirmed the structure proposed by these authors in relation to the number and position of intron/exons in each one of those genes (data not shown).

The mature proteins encoded by lcc1, lcc2 and lcc3 cDNAs are predicted to be 499 amino acids in length.

The rest of the characteristics of these deduced protein sequences, such as the signal peptide, the potential sites of N-glycosylation and the copper-binding motifs were as proposed by Mansur et al. (1997), and they are indicated in the amino acid sequences shown in Fig. 5.

Furthermore, lcc1 and lcc3 products display 71.9 % identity at the amino acid level, whereas lcc2 product

New fungal lcc gene, confirmation of genomic DNAs

Fig. 4. Confirmation of lcc1A gene by PCR–RFLP. (A) Fragment of lcc1 gene amplified from Trametes sp. I-62 genomic DNA. (B–C) Restriction analysis of the amplified fragment digested with ApaI and BamHI, respectively, which cut lcc1 gene, but not lcc1A. Changes in the sequence of lcc1A cause lost of both restriction sites. (D) Complete digestion of the amplified fragment with KpnI confirms that all the PCR product is derived from lcc1 and lcc1A genes. It has two recognition sites for this enzyme that generate three characteristic bands. M=molecular weight marker.

shares 67.4 and 75.5 % identity with Lcc1 and Lcc3 proteins, respectively. With regard to lcc1A, as mentioned previously, its cDNA sequence differs from that of lcc1 only in nine nucleotide changes, located in the 5k region of the gene (Fig. 2). The two proteins deduced from their cDNA sequences have identical amino acid number. Only two of the nucleotide changes resulted in amino acid changes : a phenylalanine instead of leucine in the signal-peptide region from Lcc1A, and an isoleucine is replaced by valine in the corresponding mature protein. It should be noted that as these four amino acids belong to the group of neutral and hydrophobic amino acids, the influence of these changes in the final protein characteristics should be less than if they were from different groups. The rest of nucleotide changes correspond to silent mutations. As shown in Fig. 5, the most important characteristics of both proteins are almost identical. The two amino acid changes represent only a 0.4 % divergence in the deduced amino acid sequence of these gene products.

DISCUSSION The identification and genomic cloning of three laccase genes from Trametes sp. I-62 was reported by Mansur et al. in 1997. Nevertheless, the cDNA sequences of these genes had not been yet isolated. Here, we report on the cloning of the lcc1, lcc2 and lcc3 together with a novel lcc1A, cDNA permitting a fuller examination

732 of the laccase gene family in this biotechnologically important fungus.

The comparison between the sequence of each cDNA and the corresponding genomic sequence allowed us to: (1) confirm the predicted structures according to the number and position of intron/exons; and (2) deduce the sequences of mature proteins. Analysis of the structural characteristic of lcc1, lcc2 and lcc3 genes from Trametes sp. I-62, and their evolutive relationship with laccase genes from other basidiomycetes were broadly discussed by Mansur et al. (1997). Taking into account the structural analysis of the three lcc genes, and the differences in the nucleotide sequences and in the number and position of introns, these authors concluded that they were not alleles. Moreover, the results obtained by Southern Blot analysis suggested that the laccase family of Trametes sp. I-62 is formed by up to five different genes.

In the present work, a cDNA sequence which only differs from that of lcc1 in nine base changes has been isolated. This represents a 99.6 % identity at the protein level. A fragment of the genomic sequence of this gene, named lcc1A, was isolated by PCR–RFLP analysis.

The multiplicity of genes having only slight differences in their nucleotide sequences is a phenomenon frequently found in fungi. Nevertheless, the great similarity between sequences can be the product of the duplication of ancestral genes that constitute families, or simply the result of allelic differences. As mentioned in the introduction of the present work, laccase genes have been described in different species of basidiomycete fungi. Although less frequent, there are some reports on the allelic sequences of these genes. The existence of a laccase family in a single organism could represent an evolutionary mechanism to expand its catabolic capabilities associated with the adaptation to the environment. Similar enzymes can evolve by the introduction of changes in their active or structural sites, and are selected according to their degradation potential against a wide range of toxic compounds to the fungus. This variability can be also enhanced and/ or assessed in allelic copies. Coincidently, the first description of laccase genes from a basidiomycete fungus involved the isolation of cDNA and genomic DNA sequences coding two allelic forms of Coriolus hirsutus laccases (Kojima et al. 1990). The coding regions of these alleles differed in only 18 bases changes representing a single amino acid difference in the corresponding putative proteins. In the basidiomycete Rhizoctonia solanii, from which a family of four laccase genes has been reported, two cDNA sequences that differ in a few nucleotides with respect to the corresponding genomic DNAs have also been described.

This produces changes in only five and four amino acids of the protein sequences deduced from the lcc3 and lcc4 genes, respectively. Therefore, the authors proposed that they represented allelic forms of both genes (Wahleithner et al. 1996). Finally, Zhao & Kwan (1999) have described two genomic sequences that

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Fig. 5. Alignment of the putative Lcc1, Lcc1A, Lcc2 and Lcc3 amino acid sequences deduced from the corresponding cDNA sequences of these Trametes sp. I-62 genes (they are shown from the upper side to the bottom, in this order). Areas of dark background indicate common amino acids. Copper-binding domains are indicated in boxes. The predicted cleavage sites of the signal peptides are indicated by arrows.

represent allelic variants of the lcc1 gene from Lentinula edodes. These alleles differ in 45 nucleotides producing seven amino acid changes between the deduced proteins. Mansur et al. (1997) found that all the three laccase genes they sequenced had the highest similarities with laccases from other basidiomycetes, not between them ; on the contrary, the sequence we are reporting here, lcc1A, has the highest identity with laccase lcc1 from the same organism, Trametes sp. I-62. If the differences between the pairs of alleles reported from the three basidiomycetous species mentioned above are compared with those existing between lcc1 and lcc1A genes, it is possible to suggest that these two genes are alleles. This would be entirely passing given that Trametes sp. I-62 is an heterokaryon strain. So that,

other copies of lcc2 and lcc3 must be also present in Trametes sp. I-62. Nevertheless, looking at the high similarity between lcc1 and lcc1A, it could be not surprising that the allelic copies have not any detectable change in their coding sequences respect to those of lcc2 and lcc3. The real importance of our hypothesis about the existence of allelic copies in this fungus then, is that its laccase family would be less numerous than expected from the first studies by Mansur et al.

(1997). Southern Blot analysis performed by these authors using specific probes to detect lcc1, lcc2 and lcc3 genes under restrictive hybridization conditions, suggested the existence of other laccase genes which differed from these 3 ones. One of the additional hybridization bands was detected in the genomic DNA

New fungal lcc gene, confirmation of genomic DNAs digested with BamHI endonuclease. The lost of one BamHI recognition site was one of the features found to distinguish lcc1A from lcc1. Then, the additional band observed, having a higher size than that corresponding to lcc1 would be the result of the hybridization of lcc1A. In order to confirm this hypothesis, it is necessary to obtain a monokaryotic mycelium from Trametes sp. I-62 and to study the segregation of genes in monokaryons. Physical maps, sequencing of new laccase genes, analysis of laccase activity using specific substrates, and induction studies, among others, will support a theoretical frame to correlate laccase coding sequences with their possible role on biological fitness, biotechnological applications, and rational enzyme design. This will contribute to a better understanding of a gene family that, due to its great potential in biotechnological applications, deserves future investigation.

ACKNOWLEDGEMENTS We are grateful to Gloria del Solar, Manuel Espinosa and Alan D. W. Dobson for their critical reading of the manuscript. We wish also to acknowledge the valuable help of Juan Pascual and Lisandro Rodon in the design of some of the figures. Authors wish to thank the CICYT (Madrid) BIO 95-2065-E and BIO 97-0655 for the financial support. Tania Gonza´lez acknowledges support from a Mutis Programme doctoral grant from AECI (Spain), and Marı´ a C. Terro´n, a postdoctoral grant from Conserjerı´ a de Educacio´n y Cultura de la Comunidad Auto´noma de Madrid.

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Corresponding Editor: S. B. Pointing