Characterization of a multicopper oxidase gene cluster in Phanerochaete chrysosporium and evidence of altered splicing of the mco transcripts

Abstract

A cluster of multicopper oxidase genes (mco1, mco2, mco3, mco4) from the lignin-degrading basidiomycete Phanerochaete chrysosporium is described. The four genes share the same transcriptional orientation within a 25 kb region. mco1, mco2 and mco3 are tightly grouped, with intergenic regions of 2?3 and 0?8 kb, respectively, whereas mco4 is located 11 kb upstream of mco1. All are transcriptionally active, as shown by RT-PCR. Comparison of cDNAs and the corresponding genomic sequences identified 14-19 introns within each gene. Based on homology and intron composition, two subfamilies of mco sequences could be identified. The sequences have copper-binding motifs similar to ferroxidase proteins, but different from fungal laccases. Thus, these sequences constitute a novel branch of the multicopper oxidase family. Analysis of several cDNA clones obtained from poly(A) RNA revealed the presence of transcripts of various lengths. Splice variants from mco2, mco3 and mco4 were characterized. They generally exhibited the presence of one to five introns, whereas other transcripts lacked some exons. In all cases, the presence of introns leads to frame shifts that give rise to premature stop codons. In aggregate, these investigations show that P. chrysosporium possesses a novel family of multicopper oxidases which also feature clustering and incomplete processing of some of their transcripts, a phenomenon referred to in this paper as 'altered splicing'. INTRODUCTIONWhite rot fungi are the only organisms capable of efficiently degrading lignin to CO 2 and H 2 O. Among these, the basidiomycete Phanerochaete chrysosporium has been the most extensively characterized (Gold & Alic, 1993;Kirk & Cullen, 1998). The oxidative enzymes secreted by this fungus are also capable of degrading a wide variety of recalcitrant compounds, both natural and anthropogenic (Kirk & Farrell, 1987). When growing on wood, P. chrysosporium is highly aggressive towards lignin. This property, together with its ability to grow at rather elevated temperatures (about 40 u C) in compost, makes it a suitable micro-organism for biotechnological applications, such as biopulping and soil bioremediation. Its genome has been sequenced (Martinez et al., 2004) and is available at an interactive website (http://genome.jgi-psf.org/whiterot1/ whiterot1.home.html).Three different types of extracellular peroxidases have been described as being actively involved in lignin degradation by white rot fungi. These are manganese peroxidases (MnPs), lignin peroxidases (LiPs) and hybrid peroxidases which share properties of both MnPs and LiPs. For a recent review, see Martinez (2002). The genome of P. chrysosporium contains five and ten genes encoding MnP and LiP, respectively (Cullen, 1997;Martinez et al., 2004).Laccase is another type of extracellular enzyme that has been implicated in lignin degradation. Laccases catalyse the one-electron oxidation of phenols, aromatic amines and other electron-rich substrates, with the concomitant fourelectron reduction of O 2 to 2H 2 O. This vers...

Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

“…However, C. subvermispora is dikaryotic, allowing the possibility that the isoforms are allelic. The 79% nucleotide identity between the two oxoX genes is lower than that of Ͼ95% which is usually associated with alleles in basidiomycetes (22,38). Nevertheless, using homokaryotic strains, it was possible to demonstrate that the C and G isoforms do indeed represent two alleles of the oxalate oxidase rather than different genes.…”

Telomere Organization in the Ligninolytic Basidiomycete Pleurotus ostreatus

“…Several authors have reported that the genes involved in lignin degradation appeared to form clusters resulting from genome duplications (42). A cluster of laccase genes was mapped to the LG6 subtelomeric region at 250 kb, TRF XhoI 2100 (Fig.…”

The copper-dependent ACE1 transcription factor activates the transcription of the mco1 gene from the basidiomycete Phanerochaete chrysosporium

“…home.html) lacks laccase-encoding sequences. Instead, we identified four clustered MCO genes (designated mco1 to mco4) distantly related to laccases (Larrondo et al, 2003(Larrondo et al, , 2004. Heterologous expression of mco1-cDNA in Aspergillus nidulans showed that the substrate specificity of the recombinant MCO1 differs from that of laccases.…”

“…The four clustered mco genes (mco1 to mco4) are located on Scaffold 9 of the assembly version 2.0 of the P. chrysosporium genome database (Larrondo et al, 2004). Their respective promoter regions were defined and searched for the presence of regulatory elements as described in Methods.…”