The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown
- Anthony Levasseur1, 2Email author,
- Anne Lomascolo1, 2,
- Olivier Chabrol3,
- Francisco J Ruiz-Dueñas4,
- Eva Boukhris-Uzan5,
- François Piumi1, 2,
- Ursula Kües6,
- Arthur F J Ram7,
- Claude Murat8,
- Mireille Haon1, 2,
- Isabelle Benoit9,
- Yonathan Arfi10,
- Didier Chevret11,
- Elodie Drula12, 13,
- Min Jin Kwon7,
- Philippe Gouret3,
- Laurence Lesage-Meessen1, 2,
- Vincent Lombard12, 13,
- Jérôme Mariette14,
- Céline Noirot14,
- Joohae Park7,
- Aleksandrina Patyshakuliyeva9,
- Jean Claude Sigoillot1, 2,
- Ad Wiebenga9,
- Han A B Wösten15,
- Francis Martin8,
- Pedro M Coutinho12, 13,
- Ronald P de Vries9,
- Angel T Martínez4,
- Christophe Klopp14,
- Pierre Pontarotti3,
- Bernard Henrissat12, 13 and
- Eric Record1, 2
https://doi.org/10.1186/1471-2164-15-486
© Levasseur et al.; licensee BioMed Central Ltd. 2014
Received: 17 January 2014
Accepted: 19 May 2014
Published: 18 June 2014
Abstract
Background
Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology.
Results
The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases.
Conclusions
With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.
Keywords
Background
Filamentous fungi are a source of powerful enzymes for plant biomass breakdown and/or hydrolysis in green and white biotechnology, especially biorefining [1]. The enzymatic modification of lignin-derived aromatic compounds is of strategic importance both for biomass valorization of the other plant-cell-wall compounds in the green chemistry sector and for the biotransformation of these aromatic compounds into high-value products (foods, cosmetics and pharmaceuticals) or industrial compounds (surfactants, adhesives and biomaterials).
The proportions of the constituent polymers of plant cell walls, i.e. cellulose, hemicelluloses, pectin and lignins, fluctuates with botanical origin, tissue, and age of the plant. In response to the structural complexity and heterogeneity of the different plant cell wall polymers, saprophytic fungi produce a complex arsenal of enzymes to gain access to the carbon source. Lignocellulolytic fungi have traditionally been classified into three main fungal groups according to the appearance of the plant material remaining after decomposition [2]. Soft-rot fungi partially degrade plant polysaccharides by mobilizing cellulases and hemicellulases, and cause wood softening [3]. In contrast, brown-rot fungi such as Postia placenta produce enzymes involved in extracellular generation of Fenton’s reagent, where hydroxyl radicals resulting from the reaction between Fe(II) and hydrogen peroxide may ultimately cause cellulose depolymerization [4]. Lignin is apparently only slightly modified in this process, and remains as a crumbly, brownish material. Unlike the above two groups, white-rot fungi are the only organisms able to effectively degrade lignin, in a process called enzymatic combustion [5] where peroxidases cooperate with other oxidoreductases [6]. The decayed wood resulting from attack by white-rot fungi becomes white and stringy. For selective white-rot fungi, the white color is caused by rapid hemicellulose and lignin breakdown of the cell-wall constituents, followed later by cellulose degradation [7].
The white-rot fungus Pycnoporus is very efficient at completely degrading lignin [8]. The Pycnoporus genus belongs to the phylum Basidiomycota, class Agaricomycetes, order Polyporales, family Polyporaceae. The genus Pycnoporus is divided into four species with different geographic origins: P. cinnabarinus is widely distributed especially in the Northern hemisphere, P. coccineus in countries bordering the Indian and Pacific Oceans, P. sanguineus in the tropics and subtropics of both hemispheres, and P. puniceus, a rare species found in Africa, India, Malaysia and New Caledonia. Pycnoporus mycelia and fruiting bodies are characterized by red-to-orange pigmentation due to phenoxazinone pigments including cinnabarin, tramesanguin and cinnabarinic acid [9]. P. cinnabarinus is a heterothallic homobasidiomycete with a tetrapolar mating system. Its life cycle includes a short monokaryotic stage after spore germination, followed after mating by an indefinite dikaryotic stage where karyogamy and meiosis can take place. The fungus is able to produce fruiting body structures and to generate stable monokaryotic cell-lines amenable to genetic improvement by formal genetics and genetic engineering, e.g. development of expression systems for high- level ligninase production [10].
P. cinnabarinus has a large array of copper- and iron-containing metalloenzymes involved in transforming plant-cell-wall aromatics [11, 12] and harbors original metabolic pathways involved in functionalizing these cell-wall aromatics to yield high-added-value compounds such as aromas and antioxidants [13, 14]. P. cinnabarinus is listed as a food- and cosmetic-grade microorganism [15]. Among enzymes involved in lignin degradation, P. cinnabarinus is known to produce high-redox-potential laccase as the predominant enzyme at very high levels of up to 1 g per liter [16, 17]. The potential of Pycnoporus fungi lies in their laccases which find a variety of applications, such as bioconversion of agricultural by-products and raw plant materials into valuable products, biopulping and biobleaching paper pulp [18–22], dye bleaching in the textile and dye industries [23–25], wastewater treatment [26–28], removal of phenolic compounds in beverages [29], biosensor and biofuel cell construction [30], and producing substances of pharmaceutical importance [31].
All studies performed over the last decade support the Pycnoporus genus as a strong contender for green and white biotechnology applications. Here, we describe the sequencing and annotation of the P. cinnabarinus monokaryotic cell-line BRFM137 genome, its growth profiling and its secretome analyses under different culture conditions. Lignocellulolytic repertoires of P. cinnabarinus are highlighted and compared with other fungal counterparts. P. cinnabarinus emerges as a versatile white-rot fungus for biotechnological applications.
Methods
Strain, DNA preparation and culture conditions
Monokaryotic strain P. cinnabarinus BRFM137 [9] was obtained from the International Centre of Microbial Resources dedicated to Filamentous Fungi (CIRM-CF, Marseille, France, http://cirm.esil.univ-mrs.fr/crbmarseille/pages/index_mizenpage.php). Genomic DNA was isolated from ground mycelia powder as described in Lomascolo et al.[32], and a roughly 180 μg sample was sent to GATC Biotech AG (Constanz, Germany) for genome sequencing.
For construction of the cDNA library, P. cinnabarinus BRFM137 was grown as described in Lomascolo et al.[17] with five types of substrate: (i) 20 g/L maltose, (ii) 20 g/l maltose and 0.1 g/l ferulic acid, (iii) 5 g/l maltose and 15 g/l oat spelt xylan (Sigma), (iv) 5 g/l maltose and 15 g/l autoclaved maize bran (ARD, Pomacle, France) and (v) 5 g/l maltose and 15 g/l Avicel cellulose (Sigma). The other constituents of the medium were: diammonium tartrate (1.84 g/l); disodium tartrate (2.3 g/l); KH2PO4 (1.33 g/l); CaCl2 · 2H2O (0.1 g/l); MgSO4 · 7H2O (0.5 g/l); FeSO4 · 7H2O (0.07 g/l); ZnSO4 · 7H2O (0.046 g/l); MnSO4 · H2O (0.035 g/l); CuSO4 · 5H2O (0.007 g/l); yeast extract (1 g/l). After 4–5 days of cultivation, the fungal mycelia from each of the five culture conditions were homogenized in liquid nitrogen, and total RNA was extracted following a standard phenol/chloroform method [33]. RNA from all the culture conditions was pooled and sent to GATC Biotech AG for reverse transcription and sequencing via Illumina technology.
For proteomic analysis, liquid cultures (LC) with non-immobilized or immobilized mycelia on 2 × 2 × 1 cm polyurethane cubes (10 per vial) were run in 250 ml baffled flasks containing 100 ml medium according to Lomascolo et al.[17]. Three LC conditions were used: (i) 20 g/l maltose (LC-M), (ii) 5 g/l maltose, 15 g/l Avicel cellulose (Sigma) and 15 g/l autoclaved maize bran (ARD) (LC-M-MB-A), and (iii) 5 g/l maltose and 15 g/l micronized birchwood (LC-B). Solid-state fermentation (SSF) cultures were also performed with five different substrates: sugarcane bagasse (Orizaba, Mexico), banana skins, wood shavings (Farmer Litter, Weldom, France), hemp (Zolux Litter, Weldom, France) and micronized birchwood. Each substrate was homogenized in water to obtain a moisture content of 70% (w/w). Five grams of substrate (wet weight) was placed in a 250 ml flask and inoculated with 1.2 ml of mycelial suspension (50 ml of nutrient medium and two mycelial mats from precultures) according to a protocol adapted from Meza et al.[34, 35]. For each growth condition, culture supernatants were harvested after 3, 7 and 10 days of cultivation and then pooled.
For growth profiling on 35 carbon sources, P. cinnabarinus BRFM137 was grown on agar plates according to Espagne et al.[36], using either 10 g/l simple carbohydrates or 30 g/l complex carbohydrates. The kraft-lignin was purchased from Sigma (reference: 370959).
Genome sequencing and data assembly
The P. cinnabarinus BRFM137 genome was sequenced by a combination of methods: (i) sequencing of genomic DNA and two normalized cDNA libraries obtained from cultures grown on different substrates (maltose, oat spelt xylan, cellulose and autoclaved maize bran) using 454/GS Roche FLX Titanium technology, (ii) sequencing of genomic DNA with Illumina/Solexa Genome Analyzer II technology, and (iii) sequencing of a 3 kbp paired-end genomic library using Illumina/Solexa Genome Analyzer II technology. The genomic Roche 454 read sets were uploaded to the ng6 storage environment [37]. Reads were cleaned using pyrocleaner [38], which applied a low-complexity filter followed by a read-size filter (over 100 bp) and a duplication-removal filter. The 454 reads were then assembled using wgs-assembler version 6.0. The Illumina mate pair reads were filtered out using the contig alignment information. All short aligned read pairs and long reads were then reassembled to produce contigs and scaffolds using the same assembly software versions. The 454 transcriptome reads of P. cinnabarinus were also cleaned using pyrocleaner, but this time the duplicated sequences were not filtered out. The reads were de novo assembled using tgicl (TIGR Gene Indices clustering tools) and annotated using various databases. The reads and contigs were aligned on the genome using Exonerate to produce gff files. These gff files were uploaded to gbrowse (http://genome-browser.toulouse.inra.fr:9090/cgi-bin/gb2/gbrowse/). Gene prediction was performed using Augustus [39] with the fungal gene model: Phanerochaete chrysosporium. The corresponding gff outputs were also uploaded to the gbrowse environment. Ensembl fungal transcripts of Aspergillus fumigatus, Aspergillus terreus, Aspergillus nidulans, Schizosaccharomyces pombe, Aspergillus clavatus, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae were also aligned on the genome and the results uploaded to gbrowse. For P. cinnabarinus, a biomart environment was set up to link de novo contigs to their genomic alignment location (http://genomebrowser.toulouse.inra.fr:9090/biomart/martview).
All the data are available at the European Nucleotide Archive (ENA), EMBL-EBI, Accession number: [EMBL: PRJEB5237].
Gene prediction and functional annotation
Orthologous groups construction
Orthologous groups (OGs) were built by running OrthoMCL [40] software on the best protein models from: 1) P. cinnabarinus BRFM137, 2) Trametes versicolor, (TaxID: 717944) 3) Postia placenta (TaxID: 561896), 4) Phanerochaete chrysosporium RP-78 (TaxID: 273507), 5) Schizophyllum commune H4-8 (TaxID: 578458), 6) Coprinopsis cinerea okayama7#130 (TaxID: 240176), 7) Laccaria bicolor S238N-H82 (TaxID: 486041), 8) Agaricus bisporus (TaxID: 936046), 9) Gloeophyllum trabeum (TaxID: 670483), 10) Ustilago maydis (TaxID: 5270), 11) Saccharomyces cerevisiae RM11-1a (TaxID: 285006), 12) Schizosaccharomyces pombe (TaxID: 4896), 13) Aspergillus niger (TaxID: 380704), 225 14) Trichoderma reesei QM6a (TaxId: 431241), 15) Nectria haematococca (TaxID: 140110), 16) Neurospora crassa (TaxID: 367110), 17) Myceliophthora thermophila (TaxID: 573729), 18) Chaetomium globosum (TaxID: 306901), 19) Mucor circinelloides (TaxID: 747725), 20) Homo sapiens (TaxID: 9606) and 21) Arabidopsis thaliana (TaxID: 3702). Each OG is a set of proteins across one or more species in the 21 listed genomes that represents putative orthologs and in-paralogs. All-versus-all BLASTP was set a 10−8 cutoff.
Global functional annotation
Global functional annotation was based on the analysis of each OG. All 15788 OGs were used as a seed for the functional annotation process based on the bioinformatics initiative Gene Ontology [41]. OGs containing at least one sequence from P. cinnabarinus were selected (7002 OGs). All sequences included in OG were ordered following the species list above. Sequences from each OG were queried using BLAST against the NCBI non-redundant (NR) protein database. A strict E-value threshold of 10−120 was applied to select homologous sequences retrieved by BlastP. These homologs were mapped to the global Gene Ontology annotation files (ftp://ftp.pir.georgetown.edu/databases/idmapping/idmapping.tb.gz).
If GO information was retrieved for the first sequence, the process was ended; if no information was retrieved for the first sequence in the OG list, the second sequence was used for mapping. In the particular case where several sequences were present in the same species, sequences were ordered by length. All the coding sequences (CDS) not included in OGs were directly BLASTed as described above.
Identification of repeated sequences
RepeatScout [42] was used to identify de novo DNA repeats in the P. cinnarinus genome. Default parameters (with l = 15) were used. The RepeatScout library was then filtered as follows: i) all the sequences less than 100 bp in size were discarded; ii) repeats counting less than ten copies in the genome were removed (as they may correspond to protein-coding gene families) and iii) repeats having significant hits to known proteins in UNIPROT (The UNIPROT Consortium, 2008) other than proteins known to belong to transposable elements (TEs) were removed. The remaining consensus sequences were annotated manually by a TBLASTX search [43] against RepBase [44] to classify them into known TE families. To identify full-length long terminal repeat (LTR) retrotransposons, a second de novo search was performed with LTR_STRUC [45]. The TBLASTX algorithm was to check the full-length candidate LTR retrotransposon sequences for homology against the sequences from the RepBase database. The number of repeat element occurrences and the percent of genome coverage were assessed using RepeatMasker [46] by masking the genome assembly with the consensus sequences coming from the RepeatScout and LTR_STRUC pipelines. MISA (http://pgrc.ipk-gatersleben.de/misa/download/misa.pl) was used with default parameters to identify mono- to hexanucleotide simple sequence repeat (SSR) motifs. Mini-satellites (motif of 7 to 100 bp) and satellites (motif >100 bp) were searched for in the P. cinnabarinus genome using Tandem Repeats Finder software [47] with the following parameters: 2; 7; 7; 80; 10; 50; 500.
Carbohydrate-active enzyme and lignin degradation enzyme annotation
All putative proteins were compared to the entries in the CAZy database [48, 49] using BLASTP. The proteins with E-values smaller than 0.1 were further screened by a combination of BLAST searches against individual protein modules belonging to the AA (Auxiliary Activities), GH (Glycosyl Hydrolases), GT (GlycosylTransferases), PL (Polysaccharide Lyases), CE (Carbohydrate Esterases) and CBM (Carbohydrate-Binding Modules) classes (http://www.cazy.org/). HMMer 3 [50] was used to query against a collection of custom-made hidden Markov model (HMM) profiles constructed for each CAZy family. All identified proteins were then manually curated. Within families, subfamilies were manually defined according to their homology relationships between members of the focal family. Protein sequences obtained from automatic prediction by Augustus software were annotated via this procedure, and all identified proteins were then manually curated.
Structural annotation of the corresponding oxidative encoding genes (number, size and position of introns) was checked manually. To do this, each AA sequence detected was BLASTP-searched against the NCBI non-redundant database. The results with the most satisfactory E-values and coverage were retained.
Then, first the target protein sequence was aligned with the sequence previously selected by BLASTP using ClustalW (http://www.genome.jp/tools/clustalw/); second, the target nucleic acid sequence was translated in three reading frames (http://www.ebi.ac.uk/Tools/st/emboss_sixpack/). Gene intron splice sites were determined based on consensus sequences fitting the GT-AG rule as described in Breathnach et al.[51].
Identification of proteins in secretomes by LC-MS/MS analysis
Proteins from the diafiltered supernatants of P. cinnabarinus BRFM137 cultures were separated by 1D SDS-PAGE electrophoresis according to the protocol of Couturier et al.[52]. After protein trypsinolysis, peptide analysis was performed by LC-MS/MS as described in Arfi et al.[53] using the PAPPSO platform facilities (Jouy-en-Josas, France; http://pappso.inra.fr). Based on the list of peptides, proteins were identified by querying the MS/MS data against the predicted proteins obtained from the P. cinnabarinus genome de novo sequencing data.
Annotation of protein secretion and glycosylation pathways
A. niger proteins related to protein secretion and glycosylation according to Pel et al.[54] and extended with additional proteins were used in a BLASTP search towards the P. cinnabarinus fasta file. The first hits were compared to the A. niger proteins to identify bi-directional BLAST best hits. An E-value cut-off of 10−10 was used. The description of the gene products was taken from the Saccharomyces Genome Database (SGD) after identifying the S. cerevisiae orthologs.
Results and discussion
Characteristics of the P. cinnabarinusgenome
Statistical assembly of the P. cinnabarinus genome
Total scaffolds | 784 |
|---|---|
Total bases in scaffolds | 33 133 717 |
Total span of scaffolds | 33 638 736 |
Coverage | 31.0916 |
Length of genome assembly (Mb) | 33.67 |
ORFs number | 10442 |
GC content (%) | 52.55 |
Average number of exons per gene | 6.7 |
Average exon size (bp) | 257.42 |
Average coding sequence size (bp) | 1774.36 |
N50 scaffold bases | 165118 |
Annotation strategy for P. cinnabarinus based on a phylogenomic approach. Orthologous groups (OGs) were formed from 21 genomes by running the OrthoMCL software using a BLASTP cutoff E- value of 1e−8. OGs containing at least one sequence from P. cinnabarinus were selected (7002 OGs) and used as a seed for the functional annotation process based on the bioinformatics initiative Gene Ontology. Sequences from each OG were BLAST-queried against a NCBI non-redundant (NR) protein database using a cutoff E-value of 10−120. The mapping procedure was carried out with the global Gene Ontology annotation files. The process was ended once GO information was retrieved. For orphan genes, the coding sequences were directly annotated using B2Go procedures.
Annotation of the P. cinnabarinus genome. Classification scheme is summarized in three main GO categories, i.e. biological process, cellular component, molecular function. Some genes have more than one GO annotation.
GO annotation depth. A bottom-up, recursive depth search is carried out to determine the level of GO terms.
Number of repeated sequences in the P. cinnabarinus genome
Number of families | Number of copies | Genome assembly coverage | |
|---|---|---|---|
Class-1 LTR Gypsy-like | 16 (13* + 3**) | 642 | 2.5 |
Class-1 LTR Copia-like | 11 (9* + 2**) | 306 | 0.95 |
Class-1 DIRS | 5 | 62 | 0.2 |
Class-1 LINE | 5 | 163 | 0.7 |
Class-2 DNA Transposons | 8 | 251 | 0.95 |
Uncategorized | 150 | 2831 | 2.91 |
All | 168 | 4255 | 8.21 |
The number of full-length LTR was lower in P. cinnabarinus than in other white-rots [6], although the TE genome coverage was in the range of other white-rot fungi. A total of 1,707 SSRs were identified in the P. cinnabarinus genome corresponding to 350 mono-, 380 di-, 820 tri-, 91 tetra-, 25 penta- and 41 hexanucleotide motifs. A total of 2368 mini-satellites and 10 satellites were identified for a genome coverage of 0.42% and 0.01%, respectively. The number of microsatellites was in the range of those found in other white-rot and Polyporaceae genomes, although the genome of P. cinnabarinus was less rich in mini-satellite and satellite sequences [60].
Carbohydrate metabolism, lignin-degrading oxidoreductases and wood decay
Global composition of AA encoding genes found in P. cinnabarinus BRFM137
Familya | Known activities | Total number |
|---|---|---|
AA1_1 | Laccase | 5 |
AA1_2 | Ferroxidase | 1 |
AA1 | Multicopper oxidase | 1 |
AA2 | Class II peroxidase | 9 (+1 partial) |
AA3_1 | Cellobiose dehydrogenase | 1 |
AA3_2b | Aryl-alcohol oxidase/ | 19 |
Glucose oxidase | ||
AA3_3 | Alcohol oxidase | 2 |
AA3_4 | Pyranose oxidase | 2 |
AA5_1 | Glyoxal oxidase | 7 |
AA6 | 1,4-benzoquinone reductase | 1 |
AA8 | Iron reductase domain | 2 |
AA9 | Lytic polysaccharide monooxygenase | 15 |
Comparison of the CAZy repertoire identified in the selected white-rot and brown-rot fungal genomes
CAZy familiesa | Aude | Cesu | Disq | Fome | Galu | Gano | Hean | Phch | Pust | Pyci | Scco | Sthi | Trve | Copu | Dac | Fopi | Gltr | Popl | Sela | Woco |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
GH1 | 1 | 3 | 4 | 5 | 3 | 3 | 2 | 2 | 1 | 1 | 3 | 3 | 2 | 3 | 1 | 2 | 5 | 2 | 3 | 1 |
GH2 | 7 | 4 | 4 | 2 | 3 | 3 | 3 | 2 | 4 | 3 | 4 | 3 | 5 | 5 | 3 | 4 | 4 | 3 | 3 | 3 |
GH3 | 14 | 6 | 8 | 8 | 12 | 13 | 12 | 11 | 14 | 7 | 12 | 17 | 13 | 13 | 9 | 12 | 11 | 6 | 11 | 8 |
GH5 | 43 | 18 | 19 | 20 | 19 | 18 | 16 | 19 | 18 | 17 | 16 | 20 | 22 | 21 | 24 | 19 | 19 | 17 | 20 | 18 |
GH6 | 2 | 1 | 1 | 2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 2 | 0 | 0 | 0 | 0 | 1 | 0 |
GH7 | 8 | 3 | 4 | 2 | 3 | 3 | 1 | 9 | 5 | 3 | 2 | 3 | 4 | 2 | 0 | 0 | 0 | 0 | 0 | 0 |
GH10 | 4 | 6 | 5 | 4 | 7 | 10 | 2 | 6 | 5 | 2 | 5 | 6 | 6 | 3 | 3 | 2 | 3 | 3 | 1 | 4 |
GH11 | 3 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
GH12 | 1 | 2 | 3 | 3 | 3 | 3 | 4 | 2 | 2 | 3 | 1 | 5 | 5 | 4 | 1 | 2 | 2 | 2 | 1 | 2 |
GH13 | 10 | 7 | 10 | 6 | 9 | 8 | 8 | 9 | 10 | 7 | 13 | 14 | 7 | 6 | 11 | 7 | 9 | 7 | 7 | 11 |
GH15 | 2 | 3 | 2 | 1 | 3 | 3 | 5 | 2 | 4 | 1 | 3 | 3 | 4 | 2 | 2 | 4 | 2 | 2 | 2 | 2 |
GH26 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 0 | 0 |
GH27 | 5 | 4 | 6 | 4 | 6 | 3 | 4 | 3 | 5 | 1 | 1 | 5 | 4 | 4 | 2 | 4 | 3 | 3 | 3 | 3 |
GH28 | 14 | 6 | 7 | 17 | 13 | 10 | 8 | 4 | 13 | 4 | 3 | 17 | 11 | 13 | 6 | 12 | 10 | 8 | 7 | 9 |
GH29 | 3 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 1 | 0 | 2 | 4 | 0 | 4 | 2 | 0 | 1 | 0 | 1 | 0 |
GH31 | 11 | 5 | 6 | 5 | 6 | 7 | 10 | 6 | 8 | 5 | 4 | 8 | 5 | 12 | 6 | 5 | 5 | 4 | 5 | 5 |
GH32 | 2 | 0 | 2 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 3 | 2 | 1 | 3 | 1 | 0 | 0 | 0 |
GH35 | 6 | 1 | 3 | 2 | 10 | 7 | 4 | 3 | 4 | 2 | 4 | 7 | 2 | 2 | 1 | 2 | 2 | 1 | 3 | 2 |
GH36 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
GH43 | 28 | 2 | 7 | 7 | 11 | 12 | 4 | 4 | 7 | 2 | 19 | 12 | 3 | 6 | 5 | 7 | 6 | 1 | 2 | 1 |
GH45 | 2 | 2 | 1 | 0 | 2 | 2 | 2 | 2 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 1 | 0 | 0 | 0 |
GH51 | 3 | 2 | 2 | 1 | 2 | 2 | 1 | 2 | 3 | 0 | 2 | 3 | 2 | 3 | 2 | 4 | 4 | 1 | 1 | 4 |
GH53 | 1 | 6 | 1 | 1 | 1 | 1 | 1 | 1 | 2 | 0 | 1 | 2 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 |
GH54 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
GH62 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
GH74 | 1 | 1 | 1 | 4 | 1 | 1 | 1 | 4 | 2 | 1 | 1 | 2 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 |
GH78 | 4 | 1 | 5 | 2 | 5 | 4 | 2 | 1 | 7 | 1 | 3 | 3 | 3 | 2 | 0 | 3 | 2 | 3 | 2 | 3 |
GH88 | 2 | 1 | 1 | 2 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
GH93 | 1 | 0 | 1 | 0 | 2 | 2 | 0 | 0 | 1 | 0 | 2 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
GH95 | 1 | 1 | 1 | 2 | 1 | 2 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 |
GH105 | 3 | 0 | 1 | 1 | 1 | 1 | 2 | 0 | 2 | 0 | 2 | 2 | 1 | 0 | 0 | 2 | 2 | 0 | 0 | 0 |
GH115 | 2 | 2 | 2 | 3 | 4 | 3 | 1 | 1 | 1 | 2 | 2 | 2 | 2 | 2 | 2 | 1 | 2 | 1 | 1 | 2 |
GH131 | 2 | 1 | 3 | 2 | 3 | 3 | 2 | 3 | 2 | 3 | 2 | 3 | 3 | 2 | 1 | 1 | 1 | 0 | 2 | 0 |
Total GH | 186 | 89 | 110 | 106 | 133 | 127 | 101 | 100 | 129 | 71 | 116 | 151 | 114 | 118 | 90 | 100 | 100 | 67 | 80 | 81 |
PL1 | 2 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 4 | 0 | 5 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
PL3 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
PL4 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 3 | 1 | 3 | 3 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 0 |
PL9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Total PL | 4 | 0 | 1 | 2 | 0 | 0 | 3 | 0 | 7 | 1 | 13 | 7 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 0 |
CE1 | 4 | 2 | 0 | 0 | 2 | 2 | 1 | 4 | 2 | 3 | 11 | 1 | 3 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
CE8 | 2 | 2 | 3 | 3 | 3 | 3 | 3 | 2 | 6 | 1 | 2 | 5 | 2 | 2 | 3 | 2 | 2 | 2 | 2 | 1 |
CE12 | 2 | 0 | 2 | 2 | 1 | 1 | 2 | 0 | 1 | 0 | 2 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Total CE | 8 | 4 | 5 | 5 | 6 | 6 | 6 | 6 | 9 | 4 | 15 | 9 | 5 | 2 | 3 | 2 | 3 | 2 | 2 | 1 |
CBM1 | 43 | 17 | 17 | 6 | 14 | 18 | 17 | 30 | 21 | 17 | 5 | 17 | 23 | 2 | 1 | 0 | 1 | 0 | 8 | 0 |
CBM5 | 8 | 3 | 5 | 5 | 10 | 9 | 5 | 3 | 4 | 3 | 3 | 10 | 6 | 11 | 1 | 4 | 4 | 5 | 5 | 4 |
CBM12 | 2 | 1 | 1 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 |
CBM13 | 4 | 6 | 11 | 4 | 9 | 8 | 1 | 5 | 7 | 4 | 17 | 4 | 6 | 6 | 2 | 11 | 1 | 15 | 5 | 5 |
CBM18 | 1 | 1 | 1 | 0 | 2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 |
CBM20 | 4 | 4 | 2 | 2 | 3 | 3 | 3 | 2 | 4 | 1 | 1 | 5 | 4 | 2 | 1 | 2 | 2 | 1 | 2 | 1 |
CBM21 | 3 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 3 | 2 | 3 | 1 | 2 | 2 | 3 | 2 | 2 | 2 | 1 | 1 |
CBM35 | 2 | 0 | 1 | 1 | 0 | 1 | 2 | 1 | 1 | 0 | 1 | 2 | 1 | 0 | 1 | 1 | 2 | 1 | 2 | 1 |
CBM38 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
CBM42 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
CBM43 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 |
CBM48 | 3 | 2 | 3 | 3 | 3 | 3 | 2 | 1 | 3 | 2 | 4 | 3 | 3 | 3 | 2 | 3 | 3 | 3 | 2 | 3 |
CBM50 | 21 | 1 | 2 | 2 | 8 | 10 | 1 | 1 | 10 | 1 | 5 | 1 | 1 | 1 | 1 | 15 | 2 | 4 | 1 | 0 |
CBM63 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Total CBM | 96 | 38 | 46 | 26 | 53 | 57 | 36 | 47 | 57 | 33 | 43 | 47 | 49 | 31 | 14 | 41 | 19 | 35 | 29 | 18 |
AA1_1 | 0 | 7 | 11 | 10 | 13 | 16 | 14 | 0 | 12 | 5 | 2 | 15 | 7 | 6 | 0 | 5 | 4 | 2 | 4 | 3 |
AA1_2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 2 | 2 | 1 | 2 | 1 | 1 | 1 | 1 | 1 |
AA1_3 | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
AA2 | 18 | 16 | 12 | 17 | 8 | 9 | 7 | 16 | 11 | 9 | 0 | 6 | 26 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
AA3_1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 2 | 0 |
AA3_2b | 3 | 6 | 11 | 2 | 4 | 8 | 13 | 3 | 6 | 4 | 2 | 15 | 5 | 0 | 0 | 1 | 6 | 3 | 0 | 0 |
AA3_3 | 6 | 3 | 4 | 3 | 3 | 5 | 3 | 3 | 4 | 2 | 4 | 7 | 4 | 5 | 1 | 4 | 2 | 4 | 5 | 5 |
AA3_4 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 2 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
AA4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 3 | 0 | 0 | 0 |
AA5_1 | 8 | 3 | 9 | 4 | 9 | 9 | 5 | 7 | 9 | 7 | 2 | 8 | 9 | 6 | 3 | 4 | 2 | 2 | 3 | 4 |
AA5_2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
AA6 | 4 | 0 | 1 | 3 | 1 | 2 | 2 | 4 | 2 | 1 | 4 | 1 | 1 | 2 | 1 | 1 | 3 | 1 | 2 | 1 |
AA7 | 2 | 0 | 4 | 0 | 4 | 4 | 3 | 0 | 1 | 0 | 4 | 3 | 0 | 0 | 3 | 5 | 0 | 0 | 0 | 0 |
AA8 | 2 | 2 | 2 | 1 | 2 | 2 | 2 | 2 | 1 | 2 | 3 | 2 | 2 | 4 | 0 | 0 | 0 | 0 | 4 | 0 |
AA9 | 20 | 9 | 15 | 13 | 15 | 16 | 10 | 15 | 14 | 15 | 22 | 16 | 18 | 10 | 0 | 4 | 4 | 2 | 5 | 2 |
AA11 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Total AA | 73 | 48 | 71 | 55 | 61 | 73 | 61 | 53 | 63 | 49 | 46 | 76 | 76 | 35 | 12 | 25 | 27 | 15 | 26 | 16 |
Growth speed of P. cinnabarinus on different substrates.
In conclusion, white-rot fungi possess more representatives of lignocellulolytic enzymes than the brown-rot group, especially in the families AA2 (12 vs. 0) AA3_2 (6.3 vs. 1.4), AA5_1 (6.8 vs. 3.4), AA9 (15.2 vs. 3.8) and CBM1 (18.8 vs. 1.7) (Table 4). Based on these results, P. cinnabarinus clearly belongs to the classical white-rot fungi, with a distribution typical of all the specific families found for this nutritional strategy.
Growth profiling of P. cinnabarinus on different substrates.
Gene structure and localization of the ligninolytic repertoire in P. cinnabarinus
Descriptions of the P. cinnabarinus laccases (AA1_1)
Five laccases stricto sensu (AA1_1), one multicopper oxidase (Mco, AA1) and one ferroxidase (AA1_2) sequence were identified in the genome and in the cDNA library, even partially (Additional file 3: Table S3). Structural annotation of the genes (designated lac1 to lac5) was performed, and the gene lcc3-1 15 (or lac1) coding for LacI protein was identified [17, 67]. In 2000, Otterbein et al.[68] demonstrated the presence of a second laccase isoenzyme, called Lac2, in the culture medium of P. cinnabarinus BRFM137.
Lac2 was purified and its N-terminal sequence was determined [68]. However, the corresponding gene has never been identified and cloned, and the biochemical properties of the Lac2 protein have never been determined. Based on the N-terminal sequence, we were able to determine the corresponding gene sequence, named lac2, from the strain BRFM137 genome sequencing data (Additional file 4: Table S4). The five laccase-encoding genes have a size of about 2.1–2.3 kb interrupted by 10 to 12 introns. Based on the intron and exon positions of each gene, we were able to classify the various laccase genes into three groups (Additional file 5: Figure S1). Lac2/lac5 (12 introns) and lac1/lac3 (10 introns) pairs have a similar structural organization with homologous intron positions, whereas the lac4 gene is organized slightly differently (length of exons and introns). The lac4 gene comprised 11 exons but showed a slightly different structure from lac1 and lac3, and an experimentally-found stop codon was confirmed in exon 6 (Additional file 5: Figure S1). In contrast to other laccase-encoding genes, the full-length lac4 mRNA could not be found. The multiplicity of laccase genes and their groupings are common features in fungi and are discussed in Additional file 6: Data S1 [69–80]. In the P. cinnabarinus BRFM137 genome, several laccase-encoding genes were identified on the same scaffold. For instance, the lac1 and lac3 genes were separated by approximately 23 kb in the same reading frame on scaffold 185007.
Descriptions of the P. cinnabarinus ligninolytic peroxidases (AA2)
Molecular models for the nine class-II heme peroxidases (AA2) found in the P. cinnabarinus genome. MnP models (A-C) present a Mn2+ oxidation site characteristic of typical MnPs, formed by two glutamates and one aspartate at the internal heme propionate region; LiP models (D-G) exhibit a Trp residue exposed to the solvent, which has been involved in high-redox-potential aromatic compound oxidation by typical LiPs; the VP model (H) obtained for the only peroxidase of this family identified in the genome analysis evidences both the Mn2+ oxidation site and the Trp residue exposed to the solvent, characteristic of members of this class-II family; the atypical VP (I) contains an aspartate residue (Asp36) in a position occupied by a glutamate in VPs and MnPs. Two axial histidines, one acting as heme iron ligand (proximal histidine) and the second (distal histidine) contributing to the heme reaction with peroxide, together with an arginine residue characterizing class-II peroxidases are also shown in the nine molecular homology models. Four disulfide bridges are depicted as green sticks. These homology models were obtained at the Swiss-Model protein-homology server [83] using P. eryngii VPL (PDB entries 4FCS, 2VKA and 3FJW) and P. chrysosporium LiPH2 and LiPH8 (PDB entries 1LLP, 1B80 and 1B82) crystal structures as templates.
These peroxidases are widely distributed in fungal genomes, including those from soft-rot, brown-rot and white-rot fungi [6, 84, 86, 87]. However, only a few of them have so far been studied, with those from Leptoxyphium fumago and Agrocybe aegerita being the best characterized. They are known to catalyze halogenation reactions and to possess catalase, peroxidase and peroxygenase activities [88]. Consequently, similar reactions are expected to be catalyzed by the HTPs identified in the P. cinnabarinus genome sequence.
All lip and mnp genes except MnP2 and LiP6 were also found in the cDNA library (Additional file 3: Table S3). The mnp genes present lengths of 1.4–1.5 kb, (Additional file 7: Table S5) and and count 4–6 introns according to gene. The two genes encoding VP and the four LiPs showed relatively similar sizes (about 1.45 kb) and were interrupted by six introns, for coding sequences of similar length (about 1.1 kb).
Considering the analysis of the intron/exon structure, a division of family AA2 into several subgroups could be proposed. vp and lip genes share a similar structural organization and form one group (Additional file 8: Figure S2 A), whereas mnp genes are a more heterogeneous group in terms of gene structure, i.e. exons 2 and 3 of the mnp2/mnp3 pair merge into a single exon in mnp1 while exons 3, 4 and 5 of the mnp1/mnp2 pair correspond to a single exon in mnp3. Finally, atypical-vp gene was totally different in length (1728 bp), number and structure of exons/introns compared with the other class II peroxidase genes analyzed (Additional file 8: Figure S2 A).
In the genome of P. cinnabarinus, we noted that some class II peroxidase genes were grouped on the same scaffold, forming a cluster of peroxidases. This was the case for mnp3, lip1, lip2 and lip3 genes, each separated by about 2 kb and oriented in the same transcriptional direction on the 184983 scaffold. Johansson and Nyman [89] had already described in T. versicolor, a similar cluster of three genes encoding two LiPs (LPGIII, LPGIV) and one MnP (MPG1) in a genomic region of 10 kb, oriented in the same transcriptional direction and separated by approximately 2.4 kb. In addition, the intron/exon organization of these T. versicolor genes pointed to a similar structure for the two LPGIII and LPGIV (about 1470 bp in length, including six introns), whereas the MPG1 gene was slightly different (1400 bp interrupted by five introns).
After analyzing the recently-sequenced T. versicolor genome sequence [6], we identified an additional lip gene (1441 bp in length, including six introns) 6.8 kb upstream of the above sequences, completing the same cluster of three lip and one mnp genes as that observed in P. cinnabarinus. Compared with other class II peroxidases (see the dendrogram in Figure 7), these sequences appear closely related to those located at the same positions in the cluster identified in P. cinnabarinus (mnp3/mnp2, lip1/lip12, lip2/lip2 and lip3/lip1 in P. cinnabarinus/T. versicolor). The co-localization of these genes in both genomes suggests they may occupy a large orthologous genomic region that has been preserved in these two closely-related species sharing a common ancestor [84]. htp1, htp2 and lip6 genes also clustered on scaffold 184962 at 7.9 kb (htp1 and htp2) and 34.1 kb (htp2 and lip6) apart (Additional file 8: Figure S2 B). Similarly, two of the three htp genes identified in T. versicolor, only 1.2 kb apart, form a cluster on scaffold 12 but are arranged in the same transcriptional direction, whereas those from P. cinnabarinus are found in a transcriptionally convergent orientation, and the nearest class II gene is located 64 kb away. This suggests that unlike what is observed for mnp3, lip1, lip2 and lip3 genes, the organization of htp genes does not appear to be conserved between these two species of the core polyporoid clade. Almost all of these peroxidase genes were transcribed, as they were recovered in the P. cinnabarinus BRFM137 cDNA library (Additional file 3: Table S3). The cloning of partial lip-like genes is described in Additional file 9: Data S2 [90–93].
Dendrogram of 223 sequences of class-II basidiomycete heme peroxidases (AA2) showing the position of nine sequences from the P. cinnabarinus genome (orange background). Evolutionary analysis was performed with MEGA5 using Poisson distances and an unweighted pair group method with arithmetic mean clustering. The cytochrome c peroxidase from P. ostreatus, monokaryon PC9, was used to root the tree (http://phylobench.vital-it.ch/raxml-bb/). The dendogram was used to illustrate the clustering of sequences (clusters A to E). Clusters with no P. cinnabarinus sequences included were collapsed. Most of the sequences were obtained from the analysis of fungal genome sequences deposited at the US Department of Energy Joint Genome Institute (JGI), with the rest collected from GenBank [86]. Fungal abbreviations are as follows: BJEAD, Bjerkandera adusta (JGI); DICSQ, Dichomitus squalens (JGI); FOMME, Fomitiporia mediterranea (JGI); GANSP, Ganoderma sp. (JGI); HETAN, Heterobasidion annosum (JGI); PHLBR, Phlebia brevispora (JGI); PYCCI, Pycnoporus cinnabarinus; STEHI, Stereum hirsutum (JGI); TRACE, Trametopsis cervina; and TRAVE, Trametes versicolor (JGI). Other fungal species with peroxidase sequences included in the collapsed clusters are: Agaricus bisporus (JGI), Auricularia delicata (JGI), Bjerkandera sp (JGI), Ceriporiopsis rivulosa, Coprinellus disseminatus, Coprinopsis cinerea (JGI), Fomitopsis pinicola (JGI), Ganoderma applanatum, Ganoderma australe, Ganoderma formosanum, Ganoderma lucidum, Gelatoporia subvermispora (JGI), basidiomycete IZU-154, Laccaria bicolor (JGI), Lentinula edodes, Phanerochaete chrysosporium (JGI), Phanerochaete sordida, Phlebia radiata, Pleurotus eryngii, Pleurotus ostreatus (JGI), Pleurotus pulmonarius, Pleurotus sapidus, Punctularia strigosozonata (JGI), Rhodonia placenta (JGI), Spongipellis sp., Taiwanofungus camphoratus, Wolfiporia cocos (JGI).
Descriptions of other AA proteins involved in ligninolysis
Other putative AA proteins produce the hydrogen peroxide necessary for the catalytic cycle of hydrogen peroxide-dependent fungal peroxidases (LiP, MnP, VP). The ability of hydrogen peroxide to generate hydroxyl radicals (OH•) also points to another role of hydrogen peroxide in the biodegradation of wood, where these hydroxyl radicals (OH•) could initiate the attack of lignocellulose [94]. For these reasons, research into hydrogen peroxide-producing enzymes – especially AA3_2 (aryl alcohol oxidases) and AA5_1 (glyoxal oxidases) – has surged. The subfamily AA5_1 contains glyoxal oxidases (called Glox) and copper radical oxidases (called Cro), which are enzymes related to glyoxal oxidases containing conserved active site residues but that diverge in terms of other structural features [95]. In P. cinnabarinus, seven AA5_1 enzymes have been identified in P. cinnabarinus BRFM137, including three glyoxal oxidases stricto sensu and four “radical copper oxidases” (Additional file 10: Table S6). Furthermore, these three glox and four cro were also expressed in the cDNA library.
Glox and Cro encoding genes (AA5_1) have diverse characteristics in P. cinnabarinus. The gene sizes ranged from 1.85 to 4.45 kb and were interrupted by one to 22 introns, corresponding to coding sequences ranging from 1.6 to 3 kb (Additional file 11: Table S7). The structure of the gene called cro2 stands out 19 from the others, with a large number (22) of introns. In contrast, the sequences identified as glox sensu stricto share comparable size (1.85 kb) and structure (three introns) and form a homogeneous group. Based on the analysis of the intron/exon structure of each Pycnoporus AA5_1 encoding gene (Additional file 12: Figure S3 A), we could propose dividing AA5_1 into three subgroups corresponding to: (i) the glox sequences, which had strong intron position homology, (ii) the cro1, cro3 and cro4 sequences, and (iii) the very different cro4 sequence. Moreover, the three glox genes formed a cluster oriented in the same transcriptional direction and grouped on the same scaffold, with glox2 and glox3 separated by only 1.1 kb (Additional file 12: Figure S3 B). This type of organization has also been found for the genes named cro3, cro4 and cro5 in P. chrysosporium ([95]; Additional file 13: Data S3 [95–97]). Additional file 14: Figure S4 and Additional file 15: Data S4 report the structural comparison between the Glox1 protein sequence from P. cinnabarinus and that of Gaox (PDB reference 1GOG) [97].
Secretome analyses and lignocellulosic degradation
Several recent studies have shown that the diversity (number and type) of hemicellulolytic and ligninolytic enzymes or isoenzymes produced by basidiomycetes depends on substrate used and mode of cultivation (liquid culture (LC) or solid-state fermentation (SSF)) [98–102]. Agro-residues such as fruit peels (banana, mandarin, melon, peach and apple peels) are rich in cellulose, hemicellulose, lignin, soluble sugars and aromatic compounds, and were found to be substrates favoring the production of glycoside hydrolases and laccases in white-rot basidiomycetes [99]. Lignocellulosic residues such as straw, bran and wood chips favor the peroxidase production by most basidiomycetes [99]. LC promotes the production of laccases and hydrolases while SSF promotes the production of peroxidases, including MnPs [101, 102]. We thus ran several P. cinnabarinus BRFM137 cultures via both LC and SSF in presence of simple or complex “natural” substrates to compositionally analyze the corresponding secretomes (Additional file 16: Table S8).
Venn diagram showing CAZyme distributions among the P. cinnabarinus secretomes from different growth conditions. LC: liquid culture, B: birchwood, M: maltose, M-MB-A: maltose + maize bran + Avicel, SSF: solid-state fermentation.
A number of cellulolytic enzymes were produced in all conditions studied. For instance, secretomes contained members of the families GH3, GH6, GH7 and GH12, which are principally involved in cellulose breakdown. However, the endo-β-1,4-glucanases of the subfamily GH5_5 were only produced when birchwood was used in the culture medium. We also identified a number of xylan-degrading enzymes produced only in the LC-M-MB-A (maltose, maize bran, Avicel), including members of families CE1, CE15, GH3, GH5, GH10. Moreover, family CE1 members were only found when maize bran was used in the cultures. Among the known activities in the CE1 family, feruloyl esterase activity mobilizes key enzymes acting on ferulic and diferulic acid bridges embedded in the hemicellulose from plant cell walls [105]. Maize fiber xylan features among the most complex heteroxylans and is highly substituted by feruloylated branches yielding a large in ferulic acid content of up to 3% of the dry mass [106]. Thus, the breakdown of this substrate required varied enzymes, as suggested by the diversity of xylanolytic enzymes produced by P. cinnabarinus in presence of maize bran.
Protein secretion and glycosylation pathways
The main lignocellulolytic enzymes of P. cinnabarinus are extracellular, and the proteins are secreted and processed during secretion by the secretion and glycosylation systems of the fungus. Analysis of the genes involved in protein secretion and glycosylation shows that P. cinnabarinus contains the entire machinery needed for protein secretion via the classical secretory pathway (Additional file 18: Table S10 and Additional file 19: Table S11, respectively). Transport of secretory proteins is expected to take place both via a pathway dependent on a signal recognition particle (SRP) and via an SRP-independent pathway, as genes for both pathways were identified. Protein transport from one compartment to the next in the secretory pathway is carried out by various protein complexes, such as the COPI/COPII complexes, Transport Protein Particule (TRAPP) complex and the exocyst complex (Additional file 18: Table S10).
The genome contains homologs of subunits in these complexes and indicates that the complexes are highly conserved in P. cinnabarinus. We also screened for V- and T-SNAREs (Soluble NSF Attachment Protein Receptors) in the genome and for secretion-related GTPases. Both the SNARE proteins and secretion-related GTPases are expected to function at discrete steps in the secretory pathway, and for most proteins we were able to identify a bi-directional best hit, indicating conservation of these functions, probably at the same step along the secretory pathway.
The endoplasmic reticulum (ER) is an important organelle that harbors the enzymes required for proper folding of secretory proteins. P. cinnabarinus is fully equipped with the enzymes needed for protein folding and disulfide bridge formation (Additional file 18: Table S10). The machinery to deal with misfolded or unfolded proteins (the Unfolded Response Pathway (UPR)) is also conserved, although we were unable to identify a clear ortholog of the Hac1/XBP1 transcription factor in the P. cinnabarinus genome. Hac1/HacA (in fungi) or Xbp1 (mammalian cells) is a bZIP transcription factor that is uniquely activated by an unconventional splicing event mediated by Ire1p (acting as sensor and endonuclease) and Trl1p (acting as ligase) [107]. The presence of proteins involved in Hac1 activation, such as the sensor (Ire1p) and the tRNA ligase (Trl1p), in the P. cinnabarinus genome suggests that this same UPR mechanism via HacA activation is also present in P. cinnabarinus. The removal of misfolded protein via the ER-associated degradation (ERAD) system, which targets misfolded proteins for degradation in the proteasome, is conserved, since we identified orthologous proteins to the ERAD and proteasome (Additional file 18: Table S10).
We also analyzed the presence of genes related to post-translational modifications in the secretory system including protein N- and O-glycosylation as well as glycosylphosphatidylinositol (GPI)-anchor biosynthesis, (Additional file 19: Table S11). The biosynthetic genes required for the formation of nucleotide sugar GDP-mannose, UDP-glucose, UDP-N-acetylglucosamine and UDP-galactose together with transporter to localize the nucleotide sugars in the ER or Golgi lumen were identified. The genes encoding the proteins for stepwise synthesis of the dolicholphosphate-linked oligosaccharide (ALG genes; asparagine (N)-linked- glycosylation) as well as the transfer of the oligosaccharide to asparagine residues (OST-complex) are conserved and found in the genome of P. cinnabarinus. Similarly, genes homologous to the attachment of the mannose residue to serine or threonine residues (O-linked glycosylation), which are carried out by protein mannosyl transferase (PMT), are also conserved. Like in other fungi, P. cinnabarinus has a genome that contains multiple PMT homologs. Glycosylphosphatidylinositol (GPI)-anchor biosynthesis and transfer of the pre-assembled GPI anchor also takes place in the ER. Most of the genes involved in GPI-anchor biosynthesis were identified. The genes encoding Golgi-localized proteins that are involved in outer chain elongation (Och1p/Mnn9p mannosyltransferase complexes) are not present in the P. cinnabarinus genome.
The genes encoding proteins that are expected to add the second and third mannosyltransferase to O-chains are present, but genes homologous to α-1-3-mannosyltransferase that add the fourth or fifth mannose to O-chains were not identified. Thus the post-transcriptional glycosylation events in the Golgi appear to be severely curtailed in P. cinnabarinus to much the same extent as previously reported for the basidiomycete S. commune[61, 108]. Galactofuranosylation is a type of modification found on glycoproteins in Aspergillus species [109], but the genes involved in this process are absent in P. cinnabarinus. This raises prospects for using P. cinnabarinus to produce pharmaceutical proteins, as the glycostructures (N- and O-chains) have a mammalian-like structure and are devoid of the highly antigenic galactofuranose residues found in expression hosts such as A. niger see [109] for review.
Mating-type loci and their genes in P. cinnabarinus
In the past, the fungal lifecycle of P. cinnabarinnus was studied in order to select monokaryotic lines with characteristics specifically tied to lignocellulose degradation [17]. Pycnoporus species are heterothallic Agaricomycetes with two mating type loci controlling the fungal lifecycle [8, 110]. One mating type locus (A locus) in the tetrapolar Agaricomycetes encodes two types of homeodomain transcription factors (HD1 and HD2) in divergently-transcribed gene pairs, whereas the other (B locus) contains genes for pheromones and pheromone precursors, respectively [111, 112].
The A mating type locus
HD1 and HD2 mating type proteins from P. chrysosporium (ADN97192.1, ADN97171.1) were successfully used to screen the Pycnoporus EST contigs. Pycnoporus, like other basidiomycetes [110], has at least one HD1 and one HD2 gene for homeodomain transcription factors. The HD1 protein a1-1 deduced from contig > GCTO4WP02F0TDF.f.pc.1 dna:contig contig::GCTO4WP02F0TDF.f.pc.1:1:2252:1 is 495 aa long. Its N-terminal domain is related to the N-terminal of mating type proteins from other species (Additional file 20: Figure S5 A) and is expected to act in heterodimerization with compatible HD2 proteins while discriminating HD2 proteins from the same mating type [111]. The two classes of homeodomain proteins encoded in basidiomycete mating type loci are defined by their distinct homeodomain sequences [113]. HD1 proteins have a TALE-class homeodomain with three extra amino acids in-between Helix I and Helix II of the three-helical DNA-binding domain. Some amino acid exchanges in the conserved DNA-recognition motif (WFxNxR) in Helix III are tolerated [112]. In the Pycnoporus a1-1 protein, the position of the HD1 homeodomain is only recognized by sequence alignment with related HD1 proteins from other species (Additional file 20: Figure S5 A). The DNA-recognition sequence in Helix III is degenerated and Helix II has undergone a deletion. Previous research failed to find the expected conserved HD1 motif in respective proteins of Postia placenta[4]. We note from other species that a defective HD1 homeodomain does not inevitably cause loss-of-function in mating type regulation provided that the HD2 homeodomain in a HD1-HD2 heterodimer continues to function [114].
A HD2 gene for the 569 aa-long protein a2-1 was found on > GCTO4WP02F01PN.f.pc.1 dna:contig contig::GCTO4WP02F01PN.f.pc.1:1:2027:1. The protein has a classical 60 amino acid-long homeodomain with all invariant residues in the DNA-binding motif which is highly sequence-conserved with HD2 mating type proteins from other Agaricomycetes (Additional file 20: Figure S5 B).
Interestingly, contig GCTO4WP02F0TDF.f.pc.1 contains not only the full-length coding sequence of protein a1-1 (>scf185007.g8) but also, downstream on the opposite strand, the 3-terminal half of gene β-fg for an unknown fungal protein (>scf185007.g7), which in most Agaricomycetes flanks one side of the homeodomain transcript factor locus [115]. At the other side of the loci, a mip gene for a mitochondrial intermediate peptidase is usually present [116]. P. chrysosporium and P. placenta differ from other analyzed Agaricomycetes in the relative order of their single HD1 gene to mip and β-fg. These are the two species where HD1 gene neighbors β-fg and not mip and is transcribed in the same direction as mip, suggesting that there has been an inversion of the mating type locus [115, 117]. Contig GCTO4WP02F0TDF.f.pc.1 indicates that Pycnoporus is another species with this same inverted arrangement. P. chrysosporium (Phanerochaetaceae) and P. placenta (Fomitopsidaceae), like Pycnoporus (Coriolaceae), belong to the Polyporales, and an inversion event early in evolution is likely [118].
The B mating type locus
The bipolar P. chrysosporium contains five genes for pheromone receptors, three of which cluster together in a locus orthologous to the B mating type locus of tetrapolar species, whereas two others belong to the still-unexplored non-mating-type G protein-coupled transmembrane receptors of the Agaricales [115, 117]. The five P. chrysosporium proteins were used to screen the Pycnoporus EST contigs, and five hits were found. Three models contained full-length (PciSTE3.2, PciSTE3.3) or nearly complete (PciSTE3.4) ORFs for G protein-coupled transmembrane receptors (Additional file 21: Figure S6). The other two contained a 5′ half of a gene (PciSTE3N) and a 3′ half of a gene (PciSTE3C), respectively, and it is possible that these two EST contigs present the same gene (Additional file 21: Figure S6). Sequence analysis of the nearly- complete proteins using ClustalW for alignment (http://www.clustal.org/clustal2/), GeneDoc (http://www.psc.edu/biomed/genedoc/) for manual corrections, and the neighbor-joining function in MEGA4 software [119] indicates that PciSTE3.4 groups with the two non-mating-type G protein-coupled transmembrane receptors of P. chrysosporium (Additional file 22: Figure S7 A), whereas PciSTE3.2 and PciSTE3.3 cluster with the B-mating-type-orthologous receptors, respectively (Additional file 22: Figure S7 B,C). This finding suggests that P. cinnabarinus, like other tetrapolar Agaricales, has B-mating-type-specific and non-mating-type genes for pheromone receptors [112, 115]. We also analyzed the N-terminal ends and the C-terminal ends of the proteins separately and together with the protein halves deduced from the incomplete EST contigs GCTO4WP02FNFO2.f.pc.1 and GCTO4WP02F7KNS.f.pc.1, respectively. In both phylogenetic trees, the partial pheromone receptors group with PciSTE3.2 and with the B orthologous PchSTE3.2 of P. chrysosporium, which is evidence that the sequences may come from the same gene. As in several other species [112, 115], there are thus at least three expressed paralogous candidate genes for B-mating-type function in P. cinnabarinus.
Pheromone precursors are short peptide chains of up to about 100 aa and the mature pheromones are 9 to 14 aa-long peptides, which are difficult to find in BLAST searches even at lowest stringency due to strongly divergent sequences [112, 120]. Searches starting with the five identified P. chrysosporium pheromone precursor sequences [112, 117] were unsuccessful, but sequences from Serpula lacrymans (http://genome.jgi-psf.org/SerlaS7_3_2/SerlaS7_3_2.home.html) and cross-searches with the detected P. cinnabarinus pheromone precursors identified a total of seven potential 39-to-65-aa-long pheromone precursors. All possess the typical CAAX (cysteine-aliphatic-aliphatic-any amino acid) motif at the C-terminus and a MDA/DF-motif at the N-terminus (Additional file 23: Figure S8). Three are very distinct in sequence, as is typical for B-mating-type pheromone precursors, whereas four others share more similarity, resembling the precursors of presumed non-mating-type pheromone-like peptides [112, 115].
Conclusions
The P. cinnabarinus genome contains the genes encoding the full enzymatic portfolio for lignin degradation, notably peroxidases and numerous auxiliary enzymes for the generation of hydrogen peroxide. Several laccase-encoding gene models and AA2 peroxidases (MnP, LiP or VP) were identified in P. cinnabarinus. A large number of these genes are expressed as they have been detected as transcripts in the cDNA library. Furthermore, secretome analysis showed effective and differential secretion of several peroxidases, copper radical oxidases, and aryl alcohol, glucose and pyranose oxidases under our culture conditions. These genes structurally organize into a set of clusters and intron/exon position homologies. The physical proximity of these genes (lac, lip, mnp, glox) suggests that this organization may result from chromosomal rearrangements such as local duplications. Furthermore, the different isoenzymes annotated in P. cinnabarinus evidenced high diversity in terms of primary sequence and predicted biochemical characteristics. In P. cinnabarinus, post-transcriptional glycosylation capabilities appear reduced to the strict minimum, making it a promising candidate for heterologous protein production in biotechnological applications. In conclusion, P. cinnabarinus is shown to be an outstanding and representative model white-rot fungi for studying the enzyme machinery involved in the degradation and/or transformation of lignocellulosic materials.
Declarations
Acknowledgements
This work was funded by the Commission of the European Communities through the BIORENEW project (NMP2-CT-2006-026456 “White Biotechnology for added-value products from re newable plant polymers: Design of tailor-made biocatalysts and new industrial bioprocesses”) and the PEROXICATS project (KBBE-2010-4-265397 “Novel and more robust fungal peroxidases as industrial biocatalysts.”), and by the Spanish Ministerio de Economía y Competitividad (MINECO) through the HIPOP project (BIO2011-26694, “Screening and engineering of new high-redox-potential peroxidases”). The authors also thank the MICA and CEPIA departments of the INRA [French National Institute for Agricultural Research] for their support. Francisco J. Ruiz-Dueñas received a Ramon y Cajal contract from the Spanish MINECO. We thank Anne-Hélène Jan and Julie Pitrat (School of Biological Engineering and Applied Microbiology Polytech Marseille, France) for their assistance in the in silico annotation of oxidoreductases, and Peter J. Punt and Marie Noëlle Rosso for their insightful comments.
Authors’ Affiliations
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