Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes.

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes.

Laccases (EC 1.10.3.2; benzenediol: oxygen oxidoreductases) are multicopper enzymes belonging to the "blue" oxidase group that catalyze the oxidation of a wide variety of organic and inorganic compounds, including diphenols, polyphenols, diamines, and aromatic amines [1]. Laccases are prevalent enzymes, especially among plants and fungi [2, 3], and fungal laccases are the most frequently studied. The potential for biodegradation of various pollutants by laccase-producing microorganisms or purified laccases is one of the most exciting subjects in environmental biotechnology research [4]. There has been growing interest in the use of fungal laccases for applications such as bio-bleaching, catalysis of complex chemical conversions in the paper industry, textile dye decolorization, and detoxification of environmental pollutants [5, 6, 7, 8]. Numerous studies have focused on the molecular characterization of fungal laccases, as well as on methods for improving laccase production levels.

F. velutipes is one of the major actively cultivated mushroom species in the world; over 300,000 tons of this mushroom are produced per year [9, 10]. In a recent study, we determined the whole genome sequence of F. velutipes and identified 12 putative laccase genes [11]. In the whole genome sequence, it was revealed F. velutipes retains many genes encoding laccase compared with either Postia placenta, Laccaria bicolor, Schizophyllum commune, or Phanerochaete chrysosporium. Thus, it is reasonably assumed that F. velutipes has potential ability for lignin degradation. Laccase genes have been isolated from different mushroom species and their copper-ligand domain that includes one cysteine and ten histidine residues were characterized [12, 13].

The aim of this study was to identify and characterize laccase genes in the F. velutipes genome in order to increase the availability of these industrially useful enzymes. Using genome information from F. velutipes, we cloned and sequenced the cDNAs of laccase genes and defined the organization of their exon-introns, copper-binding sites, and signal peptides. In addition, we examed the expressional induction of individual laccase genes by copper.

MATERIALS AND METHODS

Strains and growth conditions

Flammulina velutipes monokaryotic strain KACC42780 was obtained from the Korean Agricultural Culture Collection (KACC; Rural Development Administration, Korea; http://www.genebank.go.kr/) and was grown at 26℃ on mushroom complete medium (MCM) agar (0.2% peptone, 2% glucose, 0.2% yeast extract, 0.05% MgSO4, 0.046% KH2PO4, 0.1% K2HPO4, and 1.5% agar) for 14 days. To induce laccase expression, mycelia were grown in MCM medium supplemented at the time of inoculation with various concentrations of copper sulfate (CuSO4). For genomic DNA and total RNA isolation from mycelia, a 300-mL Erlenmeyer flask containing 50 mL MCM medium was inoculated with fresh plugs from a plate (five mycelial plugs per flask) and incubated at 26℃ for 2 wk without agitation.

Laccase gene identification

The genome-wide gene identification of laccases was conducted by applying a combination of several methods, including ab initio gene structure prediction (Fgenesh; http://www.softberry.com), a homology-based approach (Fgenesh+; http://www.softberry.com), and transcriptome-based gene identification (Cufflinks; http://cufflinks.cbcb.umd.edu/manual.html) [11] to the F. velutipes whole genome sequence (AQHU00000000). Gene prediction using the AUGUSTUS tool [14] with default parameters based on Coprinopsis cinerea was also performed. Functional annotation of the predicted genes was conducted using BLAST ver. 2.2.17 software with a series of protein databases, including the NCBI nucleotide (nt; http://blast.ncbi.nlm.nih.gov/Blast.cgi), and nonredundant set (nr; http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Total RNA preparation, cDNA synthesis, and reverse transcriptase (RT)-PCR

Mycelia samples were ground to a fine powder under liquid nitrogen using a mortar and pestle and stored at -80℃. Total RNA was prepared from tissue samples (100 mg) using TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY, USA) according to manufacturer's instructions. Total RNA (10 µg) was treated for 30 min at 37℃ with 1 U of RQ1 RNase-free DNase (Promega, Madison, WI, USA). cDNA synthesis and RT-PCR analysis were performed using 1 µg RNA in a 20-µL reaction volume with oligo-dT18 and ImProm-II reverse transcriptase (Promega). Reactions were first incubated at 25℃ for 5 min, next at 42℃ for 60 min, and finally at 70℃ for 10 min to inactivate the reverse transcriptase. PCRs were conducted in a 50-µL reaction mixture containing 10 mM dNTP mixture, 10 pmol of each specific primer (Table 1), one unit Taq-polymerase (TaKaRa Korea Biomedical Inc., Seoul, Korea), 10× PCR buffer (100 mM Tris-Cl, pH 8.3, 500 mM KCl, and 25 mM MgCl2), and 1 µL cDNA product.

Table 1

Primers used for RT-PCR

RT-PCR, reverse transcription polymerase chain reaction.

Sequence analysis

DNA was sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer instructions. Sequences were analyzed on an ABI Prism 3730 genetic analyzer (Applied Biosystems), after which the sequence data were further analyzed using the Lasergene software (DNASTAR Inc., Madison, WI, USA). The nucleotide and amino acid (aa) sequences of the laccases were aligned using the BioEdit program (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Prediction of signal peptides for the F. velutipes laccases was conducted using the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/). N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified using the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/). The GenBank accession numbers of the sequences reported in this paper are KM276550 (fvLac-1), KM276551 (fvLac-2), KM276552 (fvLac-3), and KM276553 (fvLac-4).

Laccase activity and zymogram assays

Laccase activity was determined using a modified 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS; Sigma, St. Louis, MO, USA) oxidation assay as previously reported [15]. The assay mixture contained 9 µL ABTS (1.8 mM, Sigma) and 10 µL culture supernatant in 181 µL of sodium acetate buffer (50mM, pH 4.5). Oxidation of ABTS was monitored by determining the increase of absorbance at 420 nm (ε420, 36,000/M/cm). One unit of laccase activity was defined as the amount of substrate in micromoles transformed per minute, reported in units per volume. All experiments were performed in triplicate by using three replicates of each set of conditions at each time point. Laccase activity was determined using zymograms with a modified sodium dodecyl sulfate polyacrylamide gel electrophoresis technique [15, 16]. The separating and stacking gels were 12% and 5% acrylamide, respectively, and the electrode reservoir solution contained 25 mM Tris and 192 mM glycine, pH 8.4. Gels were stained for laccase activity using 5 mM ABTS as the substrate. The total extracellular protein concentration in the culture supernatants was measured using the Bradford assay with bovine serum albumin as the standard.

RESULTS AND DISCUSSION

Identification of laccase genes in the F. velutipes genome

The predicted amino acid sequences of F. velutipes genes, determined using an approach combining several techniques (Fgenesh, Fgenesh+, and cufflinks) [11], were compared against the NCBI-nr database using BLASTP. This examination showed that nine of the predicted proteins shared sequence similarity with fungal laccases (Table 2). Gene prediction using the AUGUSTUS tool uncovered 14 laccase genes in F. velutipes, a higher number than revealed using the combination approach (Table 2). Eight of the laccase genes were identified by both prediction approaches (Table 2, Fig. 1). Fungal laccases are secreted, glycosylated proteins with two disulfide bonds and four copper atoms distributed between a mononuclear site (T1, where the substrate is reduced) and a trinuclear cluster (T2/T3, where oxygen is bound and reduced to H2O) [6]. Thus, electrons are transferred from substrate molecules to the trinuclear T2/T3 center via the T1-bound copper; subsequent to the electron transfer, the dioxygen in the trinuclear center is reduced to two molecules of H2O [17, 18]. The protein sequences of previously predicted laccase proteins indicate that all these enzymes contain four conserved copperbinding regions, as well as highly conserved copper-binding ligands consisting of ten histidine residues and one cysteine residue [13]. Of the 15 putative laccase genes identified in F. velutipes, only four genes (fvLac-1, fvLac-2, fvLac-3, and fvLac-4) conformed to the fungal laccase characteristics described above (Fig. 1). The amino acid sequence of the other 11 genes did not contain either the four complete copper-binding regions (ten histidine residues and one cysteine residue) or the four cysteine residues involved in the formation of the disulfide bridges (Fig. 1). Using cDNA sequence analysis, the open reading frame size of laccase genes fvLac-1, fvLac-2, fvLac3, and fvLac-4 were estimated to be 1,551 bp (516 aa), 1,557 bp (518 aa), 1,548 bp (515 aa), and 1,602 bp (533 aa), respectively. The intron positions of fvLac-1, fvLac-2, fvLac3, and fvLac-4 were determined analyzed by aligning between their genomic DNA and cDNA sequences. Obtained by cDNA sequencing. These comparisons revealed that the genomic DNA of fvLac-1, fvLac-2, fvLac3, and fvLac-4 contain 17, 17, 13, and 16 introns, respectively, with an average intron size of 52.5 bp, and that all the splicing sites follow the GT-AG rule (Supplementary Figs. 1, 2, 3, 4). Figs. 2 and 3 show the alignment of the predicted amino acid sequences of genes fvLac-1, fvLac-2, fvLac3, and fvLac-4 with those of previously reported fungal laccases. The F. velutipes laccases share 45.5~63.9% homology with the laccases of other fungi, including Pleurotus ostreatus (poxc [GenBank accession No. Z34848], pox1 [GenBank accession No. Z34847], poxa1b [GenBank accession No. AJ005018], pox3 [EMBL accession No. FM202671], pox4 [EMBL accession No. FM202672], and poxa3 [EMBL accession No. AJ344434]), Ganoderma lucidum (GenBank accession No. ACR24357), Lentinula edodes (GenBank accession No. AAF13037), Pycnoporus cinnabarinus (GenBank accession No. O59896), and Trametes versicolor (GenBank accession No. BAA23284). The predicted amino acid sequences of F. velutipes fvLac-1 and fvLac-2 showed the highest level of homology (75.3%) (Fig. 3). In addition, the amino acid residues required for copper-binding disulfide bridge formation were completely conserved in the four F. velutipes laccases (Fig. 2). One putative N-glycosylation site (Asn-Xaa-Ser/Thr) was identified in fvLac-1 (Asn-454) and two putative sites were identified in fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac-4 (Asn-402 and Asn-457). The initial 19~20 residues of the four laccases conformed to the structure of a signal peptide typical of extracellular enzymes, i.e., a positively charged amino terminus, a hydrophobic stretch, and small amino acid residues [19]. These characteristic structures showed that the fvLac-1, fvLac-2, fvLac3, and fvLac-4 genes encode mature laccases consisting of 496, 498, 496, and 497 amino acid residues, respectively (Fig. 2).

Table 2

The predicted laccase genes of Flammulina velutipes identified by BLAST analysis against the NCBI-nr database

CA, combiined approches (Fgenesh, Fgensh+, and cufflinks); AU, AUGUSTUS tool.

Fig. 1

Amino acid sequence alignment of Flammulina velutipes laccase genes identified using either the combination approach or the AUGUSTUS tool. Histidine (His) and Cysteine (Cys) residues predicted to be involved in the binding of copper are highlighted with gray boxes. Arrows indicate Cys residues involved in the formation of disulfide bridges.

Fig. 2

Amino acid sequence alignment of laccase genes from Flammulina velutipes and other Basidiomycetes. His and Cys residues predicted to be involved in the binding of copper are highlighted with gray boxes. Potential N-glycosylation sites (N-XS/T) are highlighted with boxes. Arrows indicate Cys residues involved in the formation of disulfide bridges. Triangles indicate the position of signal peptide cleavage sites predicted by SignalP V4.1. Positions of identical amino acid residues are marked with asterisks below the sequence. Colons and dots indicate the positions of amino acid residues with strong and weak similarity, respectively.

Fig. 3

A, Comparison of amino acids similarity of four laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) from Flammulina velutipes with laccases from Pleurotus ostreatus (pox1, pox3, pox4, poxa1b, poxa3, and poxc), Ganoderma lucidum, Lentinula edodes, P. cinnabarinus, and Trametes versicolor; B, The phylogenetic relationship of the laccases.

The effect of copper on laccase activity and transcription

Copper (CuSO4) has been reported to be a strong inducer of laccases in several species, including P. ostreatus [20], Phanerochaete chrysosporium [16], and T. versicolor [21]. In addition, copper has been shown to induce both laccase transcription and activity [21]. The increase in laccase activity is proportional to the level of copper used. In order to evaluate the effect of CuSO4 on laccase production in F. velutipes, we first tested laccase activity in response to growth with various concentrations of CuSO4. Laccase activity in a medium containing 0.5 mM CuSO4 drastically increased from day 3 and showed a peak activity on day 9 (3.03 U/mL) (Fig. 4A). This level of activity is approximately 450% (0.55 U/mL) higher than that in F. velutipes grown without CuSO4 (Fig. 1B). Laccase activity in cells grown without CuSO4 gradually increased from day 3 to a peak activity of 2.25 U/mL on day 9 (Fig. 1). Laccase activity in cells grown with 0.25 mM CuSO4 was lower on days 6 and 9 than that in F. velutipes cells grown without CuSO4. Interestingly, laccase activity in cells grown with 1 mM CuSO4 had drastically decreased by day 9, and was lower overall than that of the other conditions tested. Several studies have indicated that although copper can induce both laccase transcription and activity, even very low concentrations of laccase are toxic to most fungi [21, 22]. To evaluate the effect of apple pomace on the production of laccase enzyme, we utilized native polyacrylamide gel electrophoresis to examine the level of laccase activity in 0.33-µg protein samples collected on different days (3, 6, and 9) from the culture supernatants of cells supplemented with various concentrations of CuSO4 (0, 0.25, 0.5, 1, and 2 mM). As shown in Fig. 4B, an increased level of laccase activity was apparent in the 0.5 mM CuSO4 samples on both days 6 and 9. Although the highest level of activity was observed for the 0.5mM CuSO4 day 6 sample (Fig, 4B), it was not significantly increased compared to the activity shown in Fig. 4A.

Fig. 4

Laccase activity of Flammulina velutipes. Time course examination of laccase activity in F. velutipes cells supplemented with different concentrations of CuSO4 (0, 0.25, 0.5, 1, and 2 mM) (A). Zymogram of laccase isoenzymes in culture supernatants of P. ostreatus. Samples contained 0.33 µg protein collected from culture supernatants supplemented with different concentrations of CuSO4 (0, 0.25, 0.5, 1, and 2 mM) on different days (3, 6, and 9) (B). Staining was performed with 5 mM ABTS in 50 mM sodium acetate buffer (pH 5.2).

To confirm and further elucidate the effects of CuSO4 on the mRNA transcription levels of the laccase genes, including fvLac-1, fvLac-2, fvLac3, and fvLac-4, we conducted semiquantitative RT-PCR. This analysis clearly demonstrated the effect of CuSO4 on the induction of transcription of these laccase genes (Fig. 5). Although laccase activity was relatively lower in cells cultured with 0.25 mM CuSO4 than in cells cultured with either 0.5 or 1mM CuSO4 (Fig. 4), the transcripts of all four laccases (fvLac-1, fvLac-2, fvLac3, and fvLac-4) could be detected in day 3 samples by RTPCR (Fig. 5A). This might be due to the rapid effect of copper on induction during the early phase of fungal growth. The fv-Lac-1 gene exhibited the highest transcript levels in all the 0.5mM CuSO4 samples analyzed. Moreover, the transcript level of fv-Lac-1 increased in correlation with CuSO4 concentration, while fv-Lac-3 mRNA was barely detectable even under inducing conditions except in the day 3 sample. Interestingly, the fvLac-2, fvLac3, and fvLac-4 mRNAs were detectable in the 0.5 mM CuSO4 in the sample of 9 days, but not in other days. Similarly, the fv-Lac-1 and fvLac-4 mRNAs were detectable only in the 1mM CuSO4 day 9 sample (Fig. 5). Fernandez-Larrea and Stahl [22] reported that free copper ions, and the production of toxic compounds, could result in oxidative stress at an advanced stage of fungal growth, which could be responsible for late transcriptional induction.

Fig. 5

Reverse trascription-PCR assays (A) and mRNA transcription levels (B) of Flammulina velutipes laccase genes. Total RNA was isolated from mycelia grown with different concentrations of CuSO4 (0, 0.25, 0.5, and 1 mM) and collected at different time points (days 3, 6, and 9).

In order to identify putative response elements in the promoter regions of the laccase genes, we analyzed the nucleotide sequences extending 500 bp upstream from the start codons of the four laccase genes (Supplementary Fig. 5). This analysis allowed us to identify the unique distribution of several putative response elements. The promoter region of fvLac-1 revealed a potential antioxidant responsive element motif known to be involved in the phenol antioxidant response in mammalian cells, and previously detected in the promoters of P. ostreatus laccases (pox3, pox4, and poxa1b) [23], as well as in the P. sajor-caju lac4 promoter [12]. A putative stress responsive element corresponding with the consensus sequence CCCCT [24] was identified in the fvLac-3 promoter (Supplementary Fig. 5).

The four laccase genes were first overexpressed in an Escherichia coli system (data not shown). However, the overexpressed laccases formed insoluble inclusion bodies [25] lacking enzymatic activity, consistent with a previous study describing the difficulty of overexpressing recombinant forms of fungal laccases in E. coli systems [26]. Therefore, the development of an expression system for the production of higher levels of these useful enzymes would be greatly advantageous. The results of this study indicate that further experiments are required to elucidate the enzymatic characteristics of laccases, and to obtain higher production levels of these proteins.