Introduction

White-rot fungus is the only organism that can effectively break down lignin, which is very resistant to general microbial attack. It is well known that extracellular peroxidases, such as LiP and MnP play major roles in the lignin biodegradation process of white-rot fungi [1–4]. The catalytic mechanisms and molecular genetics of these ligninolytic peroxidases have been studied and have revealed their structural and functional properties [5–10]. It has also been reported that plant and fungal peroxidases may be arranged in a superfamily of three distinct classes; namely class I of bacterial and intracellular peroxidases, class II of fungal secretory peroxidases, and class III of secretory plant peroxidases, respectively [11]. The lignin-degrading peroxidases in class II of fungal secretory peroxidases have been further subdivided into three groups, LiP, MnP, and VP, based on the genetic and protein structural evidence [12]. Recently, phylogenetic analysis of fungal ligninolytic peroxidases was reported, showing the ubiquity and diversity of these enzymes among a wide range of ectomycorrhizal fungi, basidiomycetes and agaricomycetes [13–15].

We have studied the distribution of the extracellular peroxidase reaction of a white-rot fungus P. crassa WD1694 in detail, and showed how the MnP reaction occurred at the hyphal tips [16, 17]. Previously, we reported on the purification and characterization of MnP from P. crassa WD1694 [18]. In this report, we studied the cloning and sequence of genomic mnp genes of P. crassa WD1694 as an initial step toward understanding the molecular genetics of P. crassa WD1694.

Materials and methods

Strains

The white-rot fungus Phanerochaete crassa WD1694 [MAFF420737, Phanerochaete crassa (Lev.) Burdsall] was obtained from the culture collection of the Forestry and Forest Products Research Institute. Escherichia coli strain DH5α was used for transfections with pTA2 vector (Toyobo, Osaka, Japan).

Cultivation conditions

Cultivation was conducted as described previously [16–18].

Purification step

After cultivation for 4 days, culture filtrate was recovered and adsorbed on DEAE-Sepharose CL-6B and further extracted with 0.5 M NaCl in 10 mM acetate buffer (pH 5.5). Eluate was desalted and loaded on a DEAE-Toyopearl column (1 × 5 cm), equilibrated with 10 mM phosphate buffer (pH 6.0) and eluted with a linear 0–0.5 M NaCl gradient in 20 mM acetate buffer, pH 5.5, and fractions with MnP activity were collected, desalted, and concentrated.

Electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were conducted using a Multiphor II electrophoresis system (GE Healthcare UK Ltd., Buckinghamshire, England).

MnP activity staining

A staining solution containing 2 mM β-naphtol, 2 mM 3-amino-9-ethylcarbazole, 100 μM MnSO4, 200 μM H2O2 and 20 % acetone in 80 mM acetate buffer (pH 4.5) was prepared to visualize MnP activity. The IEF gel was placed in the staining solution until the MnP bands were stained and washed with the solution containing 25 % ethanol and 8 % acetic acid. The IEF gel was then further rinsed with distilled water.

Determination of the N-terminal sequence

The purified MnP gave a single band on SDS-PAGE and further separated as 4 bands on IEF. The 4 MnP bands on IEF gel were brown and visible without staining. Each of the MnP bands on the IEF gel was cut out without staining before blotting to prevent cross-contamination. The cut-out gel slice was blotted to the PVDF protein sequencing membrane (Bio-Rad Laboratories, Inc. CA USA) and the N-terminal sequence was determined using the Edman method.

Isolation of genomic DNA

P. crassa WD1694 was grown in nitrogen-limited shake cultures as described previously [16–18]. The mycelia were collected by filtration, freeze-dried, and ground to powder by a Multi-beads shocker (Yasui Kikai, Osaka, Japan). The mycelia powder (100 mg) was completely dissolved in N-cetyl-N,N,N-trimethyl-ammonium bromide (CTAB, 700 μl, 65 °C), and extracted with chloroform:isoamylalcohol (24:1). After centrifuging for 14,000 rpm and 10 min, the supernatant was recovered, washed with chloroform:isoamylalcohol (24:1) and centrifuged. The supernatant had isopropanol added and the precipitate DNA was recovered and washed with ethanol.

Cloning of MnP genes

The first strands of genomic P. crassa mnp genes were obtained using the DNA sequences of Phanerochaete chrysosporium mnp genes as primers; FPA2, FPA2′, FPA3, FPA3′, FPB2, FPB2′, FPB3, and FPB3′ in Table 1 (GenBank accession Nos. M60672.1, S69963.1, and U70998). Four different sequences of the center part of P. crassa mnp genes (P. crassa mnpA2, A3, B2, and B3) were determined as the first strands. Subsequently, the 3′- and 5′-ends of these 4 sequences were determined respectively, by inverse PCR.

Table 1 Primers used in this study
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To determine the P. crassa mnpA2 gene, primers FPA2 and FPA2′ were used to get the first strand of P. crassa mnpA2. Subsequently, inverse PCR was conducted to get the 3′-end of P. crassa mnpA2 with primers a2p1 and a2p2, and the 5′-end of P. crassa mnpA2 with primers PcrassaF2 and PcrassaR2. The remaining 3 genes, P. crassa mnpA3, B2, and B3 were determined correspondingly. The primers used in these experiments are shown in Table 1.

The genome DNA from P. crassa WD1694 was digested with restriction enzymes and used for self-ligation with a DNA ligation kit (Toyobo) prior to inverse PCR. All PCR procedures were carried out with TaKaRa LA Taq (Takara Bio, Shiga, Japan) with a thermal cycler (Applied Biosystems, CA, USA). The thermal cycle parameters were as follows: a 1-min initial denaturation at 94 °C, 40 cycles of 30 s denaturation at 94 °C, 30 s of annealing at 55 °C, a 3 min extension at 68 °C, and a 10 min final extension at 72 °C. Specific PCR products were purified on 1 % agarose gel and extracted with a QIAquick Gel Extraction Kit (Quiagen, MD, USA). The amplified DNA samples were inserted into the pTA2 vector (Toyobo). Transformation was conducted with COMPENTENT high DH5α (Toyobo).

Results

We cloned and sequenced the genomic genes encoding MnP isozymes from the white-rot fungus P. crassa WD1694. The first fragments of the mnp genes of P. crassa WD1694 were obtained using P. chrysosporium mnp sequences as primers. Subsequently, inverse PCR was conducted repeatedly to get complete mnp genes from P. crassa WD1694.

Genomic clones encoding alleles of 4 MnP isozymes from P. crassa WD1694 were determined. The intron location and alignment for P. crassa mnp genes were determined by comparing each genomic sequence with the P. chrysosporium mnp genes [19–21]. All the intron splice junction sequences in the 4 P. crassa mnp genes conform to the GT-AG rule.

Figure 1 shows the intron alignments for the 4 P. crassa mnp genes sequenced in this study. The intron positions of P. crassa mnpB2, B3, and P. crassa mnp A3 genes were the same as those reported for P. chrysosporium mnp2 and P. chrysosporium mnp3, respectively [20, 21]. However, P. crassa mnpA2 lacked two introns corresponding to the second and third introns of P. chrysosporium mnp2. The pattern of the intron number and position was used to classify the large family of LiP and MnP genes from P. chrysosporium [5]. Our result showed that P. crassa mnpA2 represents another gene subfamily additional to the mnp subfamilies of P. chrysosporium previously reported.

Fig. 1
figure 1

The intron–exon structure of the genomic mnp genes from P. crassa WD1694 in comparison with P. chrysosporium mnp genes Vertical bars indicate intron positions. Identical intron positions among several genes are joined with a dashed line. The signal sequence is shaded

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In order to determine the MnP isozyme secreted in the P. crassa WD1694 culture, we produced and purified MnP from P. crassa WD1694. The purified MnP gave a single band at a molecular weight of 48,300 on SDS-PAGE (Fig. 2). However, it separated on IEF gel for 4 bands around pI 4.55, with very close pI (MnP 1-4: 4.61, 4.59, 4.52, and 4.50) (Fig. 3).

Fig. 2
figure 2

SDS-PAGE of the purified MnP from P. crassa WD1694. Lane1 molecular mass protein markers, lane2 MnP from P. crassa WD1694

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Fig. 3
figure 3

Isoelectric focusing of purified MnP from P. crassa WD1694. Purified MnP was stained with MnP active staining. The bar indicates pI standard

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The N-terminal sequences of the 4 MnP bands were determined experimentally, and it was revealed that the first 13 amino acids of the N-terminal sequence of the 4 MnP bands were identical. The N-terminal sequences of the predicted translation product of P. crassa mnp genes and the experimentally determined first 13 amino acids of MnP proteins were compared in Table 2. The N-terminal sequences of mnpA2, mnpA3, and mnpB2 differed from those of MnP proteins. However, mnpB3 had the same N-terminal sequence as the MnP proteins. These results suggested that only the mnpB3 gene among the 4 P. crassa mnp genes was the origin of P. crassa MnP shown as 4 bands on IEF.

Table 2 The nucleotide sequence and amino acid sequence of N-terminal of mnp genes and MnP protein
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The deduced amino acid sequence and the nucleotide sequence of mnpB3 were shown in Fig. 4. The mnpB3 gene encodes a mature protein of 384 amino acids preceded by a 24 amino acid signal peptide. The predicted molecular weight was 40,000, and the pI value was 4.35. The predicted pI value matched well with those of MnP proteins determined experimentally, but the predicted molecular weight was smaller than the experimentally determined MnP proteins.

Fig. 4
figure 4

The nucleotide and predicted amino acid sequence of P. crassa WD1694 mnpB3 gene. The underlined amino acid sequence is the predicted signal peptide. Introns are indicated by small letters

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Discussion

We cloned and sequenced the mnp gene from the P. crassa WD1694 genome comprehensively. Four mnp genes, mnpA2, mnpA3, mnpB2, and mnpB3 were determined. The exon–intron structure of P. crassa mnpA3, B2, and B3 was similar to those of P. chrysosporium mnp2 and mnp3, respectively [20, 21]. P. crassa mnpA2 had only 5 introns, which lacked two introns corresponding to the second and third introns of P. chrysosporium mnp2. The exon–intron structure was used to classify the large family of LiP and MnP genes from P. chrysosporium [5]. Recently, the exon–intron structure of mnp genes from Formitiporia mediterranea, Physisporinus rivulosus, and Phlebia radiata has been reported [22–24]. The exon–intron structure of P. crassa mnpA2 differed from these reports, and represents another gene subfamily additional to the mnp subfamilies previously reported.

It has been reported that fungal class II ligninolytic peroxidases were divided into three families, LiP, MnP and VP [12]. Several groups reported the phylogenetic analysis of fungal ligninolytic peroxidases that also showed similar three groupings, mainly constructed with LiP, MnP and VP [13–15]. Our results showed that MnP of P. crassa WD1694 belongs to the classical MnP group, which oxidizes manganese (II) but not veratryl alcohol. The results correlate to the catalytic property of P. crassa WD1694 MnP in our previous reports [16–18].

Although P. crassa mnpA2 had a different structure that lacked 2 introns, the intron position and length of exon from other regions were very similar to P. crassa mnpA3 and P. crassa mnpB2. In general, MnP isozymes from same origin belong to the same group of ligninolytic peroxidases [12–15]. P. crassa mnpA2 should belong to the classic MnP group, like other mnp genes obtained from P. crassa WD1694.

The purified P. crassa WD1694 MnP gave a single band on SDS-PAGE, and was separated into 4 bands by isoelectric focusing. Generally, MnP isozymes of a white-rot fungus have the same molecular weight with different pI [25–29]. In these cases, each isozyme is encoded by a different gene [5]. In this study, we determined 4 different mnp gene sequences and anticipated that these genes should correspond to the 4 MnP bands. However, only P. crassa mnpB3 had the same N-terminal sequence as the experimentally determined N-terminal sequence of MnP, while the other 3 genes, mnpA2, A3, and B2 had different N-terminal sequences. These results suggested that the 4 MnP bands arose from a single gene.

P. crassa mnpB3 existed as an allele with different nucleotides at eight positions of coding region sequences, two of which were translated regions and the remaining six untranslated regions. However, both allele of mnpB3 had the same predicted pI value, meaning the allele was not the reason for separation of the 4 MnP bands.

One possibility of multiple products from single gene is splicing variation. Recently, incomplete splicing of mco transcripts and incomplete processing of peroxidase transcripts from P. chrysosporium have been reported [30, 31]. Alternative splicing of introns in exocellobiohydrolase and in cytochrome P450 monooxygenase genes has also been reported for P. chrysosporium [32, 33]. Although there have been no reports of alternative splicing or incomplete processing of MnP, splicing variation should produce multiple products of different molecular weight. In our results, since the 4 MnP bands had the same molecular weight, it is unlikely that the 4 MnP bands arose from splicing variation.

It has been indicated that MnP was a glycoprotein modified with glycosylation or phosphorylation and that these posttranslational modifications could differ between MnP isozymes encoded by a single gene [5, 27, 34, 35]. The encoded mature protein of mnpB3 had a molecular weight of 40,000, although the molecular weight determined for the purified MnP protein was 48,300. The gap between the encoded mature protein of mnpB3 and the purified MnP protein of P. crassa WD1694 could be the difference between MnP proteins generated from the mnpB3 gene. Additional analysis is required to determine whether the multiplicity of P. crassa WD1694 MnP arose due to posttranslational modification or for other reasons.

In this study, we did not detect MnP from other P. crassa mnp genes, mnpA2, mnpA3, and mnpB2. However, the expression of P. crassa MnP isozymes could be affected by culture conditions such as the concentration of manganese or nutrient nitrogen [29, 36, 37].

In conclusion, analysis of 4 genomic mnp genes from P. crassa WD1694 revealed that P. crassa WD1694 MnP belongs to the classical MnP type among the fungal class II peroxidases. The purified P. crassa WD1694 MnP consisted of 4 isozymes of equivalent molecular weight and N-terminal sequence, and different pI. Comparison of the N-terminal sequences between 4 MnP isozymes and deduced sequences from P. crassa mnp genes suggested that 4 MnP isozymes arose from a single gene. These results showed that the hyphal tip MnP reaction of P. crassa WD1694 was caused by the classical type MnP.