BACKGROUND
filamentous fungi are a source of powerful enzymes for plant biomass breakdown and/or hydrolysis in green and white biotechnology, especially biorefining  <cit> .
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  or industrial compounds .
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  <cit> .
soft-rot fungi partially degrade plant polysaccharides by mobilizing cellulases and hemicellulases, and cause wood softening  <cit> .
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 and hydrogen peroxide may ultimately cause cellulose depolymerization  <cit> .
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  <cit>  where peroxidases cooperate with other oxidoreductases  <cit> .
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  <cit> .
the white-rot fungus pycnoporus is very efficient at completely degrading lignin  <cit> .
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  <cit> .
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  <cit> .
p. cinnabarinus has a large array of copper- and iron-containing metalloenzymes involved in transforming plant-cell-wall aromatics  <cit>  and harbors original metabolic pathways involved in functionalizing these cell-wall aromatics to yield high-added-value compounds such as aromas and antioxidants  <cit> .
p. cinnabarinus is listed as a food- and cosmetic-grade microorganism  <cit> .
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  <cit> .
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 , dye bleaching in the textile and dye industries , wastewater treatment , removal of phenolic compounds in beverages  <cit> , biosensor and biofuel cell construction  <cit> , and producing substances of pharmaceutical importance  <cit> .
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 brfm <dig> 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 brfm <dig>  <cit>  was obtained from the international centre of microbial resources dedicated to filamentous fungi .
genomic dna was isolated from ground mycelia powder as described in lomascolo et al.
<cit> , and a roughly 180 μg sample was sent to gatc biotech ag  for genome sequencing.
for construction of the cdna library, p. cinnabarinus brfm <dig> was grown as described in lomascolo et al.
<cit>  with five types of substrate:  20 g/l maltose,  20 g/l maltose and  <dig>  g/l ferulic acid,  5 g/l maltose and 15 g/l oat spelt xylan ,  5 g/l maltose and 15 g/l autoclaved maize bran  and  5 g/l maltose and 15 g/l avicel cellulose .
the other constituents of the medium were: diammonium tartrate ; disodium tartrate ; kh2po <dig> ; cacl2 · 2h2o ; mgso4 · 7h2o ; feso4 · 7h2o ; znso4 · 7h2o ; mnso4 · h2o ; cuso4 · 5h2o ; yeast extract .
after 4– <dig> 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  <cit> .
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  with non-immobilized or immobilized mycelia on 2 × 2 × 1 cm polyurethane cubes  were run in 250 ml baffled flasks containing 100 ml medium according to lomascolo et al.
<cit> .
three lc conditions were used:  20 g/l maltose ,  5 g/l maltose, 15 g/l avicel cellulose  and 15 g/l autoclaved maize bran  , and  5 g/l maltose and 15 g/l micronized birchwood .
solid-state fermentation  cultures were also performed with five different substrates: sugarcane bagasse , banana skins, wood shavings , hemp  and micronized birchwood.
each substrate was homogenized in water to obtain a moisture content of 70% .
five grams of substrate  was placed in a 250 ml flask and inoculated with  <dig>  ml of mycelial suspension  according to a protocol adapted from meza et al.
<cit> .
for each growth condition, culture supernatants were harvested after  <dig>   <dig> and 10 days of cultivation and then pooled.
for growth profiling on  <dig> carbon sources, p. cinnabarinus brfm <dig> was grown on agar plates according to espagne et al.
<cit> , using either 10 g/l simple carbohydrates or 30 g/l complex carbohydrates.
the kraft-lignin was purchased from sigma .
genome sequencing and data assembly
the p. cinnabarinus brfm <dig> genome was sequenced by a combination of methods:  sequencing of genomic dna and two normalized cdna libraries obtained from cultures grown on different substrates  using 454/gs roche flx titanium technology,  sequencing of genomic dna with illumina/solexa genome analyzer ii technology, and  sequencing of a  <dig> kbp paired-end genomic library using illumina/solexa genome analyzer ii technology.
the genomic roche  <dig> read sets were uploaded to the ng <dig> storage environment  <cit> .
reads were cleaned using pyrocleaner  <cit> , which applied a low-complexity filter followed by a read-size filter  and a duplication-removal filter.
the  <dig> reads were then assembled using wgs-assembler version  <dig> .
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  <dig> 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  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 .
gene prediction was performed using augustus  <cit>  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 .
all the data are available at the european nucleotide archive , embl-ebi, accession number: .
gene prediction and functional annotation
orthologous groups construction
orthologous groups  were built by running orthomcl  <cit>  software on the best protein models from: 1) p. cinnabarinus brfm <dig>  2) trametes versicolor,  3) postia placenta , 4) phanerochaete chrysosporium rp- <dig> , 5) schizophyllum commune h4- <dig> , 6) coprinopsis cinerea okayama7# <dig> , 7) laccaria bicolor s238n-h <dig> , 8) agaricus bisporus , 9) gloeophyllum trabeum , 10) ustilago maydis , 11) saccharomyces cerevisiae rm11-1a , 12) schizosaccharomyces pombe , 13) aspergillus niger ,  <dig> 14) trichoderma reesei qm6a , 15) nectria haematococca , 16) neurospora crassa , 17) myceliophthora thermophila , 18) chaetomium globosum , 19) mucor circinelloides , 20) homo sapiens  and 21) arabidopsis thaliana .
each og is a set of proteins across one or more species in the  <dig> listed genomes that represents putative orthologs and in-paralogs.
all-versus-all blastp was set a 10− <dig> cutoff.
global functional annotation
global functional annotation was based on the analysis of each og.
all  <dig> ogs were used as a seed for the functional annotation process based on the bioinformatics initiative gene ontology  <cit> .
ogs containing at least one sequence from p. cinnabarinus were selected .
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  protein database.
a strict e-value threshold of 10− <dig> was applied to select homologous sequences retrieved by blastp.
these homologs were mapped to the global gene ontology annotation files .
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  not included in ogs were directly blasted as described above.
identification of repeated sequences
repeatscout  <cit>  was used to identify de novo dna repeats in the p. cinnarinus genome.
default parameters  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  and iii) repeats having significant hits to known proteins in uniprot  other than proteins known to belong to transposable elements  were removed.
the remaining consensus sequences were annotated manually by a tblastx search  <cit>  against repbase  <cit>  to classify them into known te families.
to identify full-length long terminal repeat  retrotransposons, a second de novo search was performed with ltr_struc  <cit> .
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  <cit>  by masking the genome assembly with the consensus sequences coming from the repeatscout and ltr_struc pipelines.
misa  was used with default parameters to identify mono- to hexanucleotide simple sequence repeat  motifs.
mini-satellites  and satellites  were searched for in the p. cinnabarinus genome using tandem repeats finder software  <cit>  with the following parameters: 2; 7; 7; 80; 10; 50;  <dig>
carbohydrate-active enzyme and lignin degradation enzyme annotation
all putative proteins were compared to the entries in the cazy database  <cit>  using blastp.
the proteins with e-values smaller than  <dig>  were further screened by a combination of blast searches against individual protein modules belonging to the aa , gh , gt , pl , ce  and cbm  classes .
hmmer  <dig>  <cit>  was used to query against a collection of custom-made hidden markov model  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  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 ; second, the target nucleic acid sequence was translated in three reading frames .
gene intron splice sites were determined based on consensus sequences fitting the gt-ag rule as described in breathnach et al.
<cit> .
identification of proteins in secretomes by lc-ms/ms analysis
proteins from the diafiltered supernatants of p. cinnabarinus brfm <dig> cultures were separated by 1d sds-page electrophoresis according to the protocol of couturier et al.
<cit> .
after protein trypsinolysis, peptide analysis was performed by lc-ms/ms as described in arfi et al.
<cit>  using the pappso platform facilities .
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.
<cit>  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− <dig> was used.
the description of the gene products was taken from the saccharomyces genome database  after identifying the s. cerevisiae orthologs.
RESULTS
characteristics of the p. cinnabarinusgenome
the genome of the monokaryotic strain p. cinnabarinus brfm <dig> was sequenced by  <dig> pyrosequencing and illumina sequencing runs to reach a final 31-fold coverage.
the genome was ultimately assembled into  <dig> scaffolds with n <dig> of 165118 bp.
table  <dig> reports the features of the assembled genome sequences.
the g + c content of the p. cinnabarinus genome was  <dig> %.
genome size was  <dig>  mb and a total of  <dig>  orfs were identified in the structural annotation procedure.
the number of orfs in p. cinnabarinus is close to the average number among the order polyporales.
for instance, phanerochaete chrysosporium, postia placenta, wolfiporia cocos and ceriporiopsis subvermispora count  <dig>   <dig>   <dig> and  <dig> detected orfs in their genomes, respectively  <cit> .
p. cinnabarinus genome size is slightly lower than in p. placenta , c. subvermispora  and w. cocos .table  <dig>
statistical assembly of the
p. cinnabarinus
genome
in general, functional annotation hinges on the propagation of existing functional information via single homology searches.
the resolution of functional inference could be improved by differentiating homologs into orthologs  and paralogs   <cit> .
orthologs are assumed to have more chance of sharing the same function than paralogs.
gene duplication is an essential contributing factor for evolving novel functions, and one of the duplicates could undergo evolutionary events such as sub-functionalization, neofunctionalization, etc.
.
we therefore based our annotation strategy on the searches for ogs within  <dig> selected genomes followed by similarity searches from each og.
an outline flow of the functional annotation procedure based on this phylogenomic approach is shown in figure  <dig>   <dig>  ogs were retrieved using a best reciprocal hit approach.
the ogs included  <dig>  putative cds from p. cinnabarinus, totaling ~83% of total cds.
based on a sequence homology searches within each og against the nr database using a strict e-value cutoff of 10− <dig>   <dig>  genes were annotated across the go categories.
in addition,  <dig> orphan genes were annotated using the standard blast2go procedure.
the annotation procedure enabled us to annotate  <dig>  cds corresponding to ~52% of total cds .
to compare with the classical method, fewer than 30% of total cds were annotated using the blast2go procedure.
our approach based on ortholog clustering enables us to infer functional information directly from ogs using a subsequent drastic threshold for similarity searches and offers a conceptual framework for inferring information from various genomes.
the  <dig>  annotated genes were grouped into functional groups .
finally, a go tree depth was calculated to assess amount and quality of go annotations .figure  <dig>
annotation strategy for
p. cinnabarinus
based on a phylogenomic approach.
orthologous groups  were formed from  <dig> genomes by running the orthomcl software using a blastp cutoff e- value of 1e− <dig>  ogs containing at least one sequence from p. cinnabarinus were selected  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  protein database using a cutoff e-value of 10− <dig>  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.
repeated sequences were identified in the genome of p. cinnabarinus and a library of  <dig>  consensus sequences was generated using repeatscout  <cit> .
after the different filtering steps, we were left with  <dig> consensus sequences:  <dig>   <dig>   <dig> and  <dig> consensus sequences showed homologies with class  <dig> gypsy, copia, dirs and long interspersed element  retrotransposons, respectively, and  <dig> with class  <dig> transposons .
the remaining  <dig> consensus sequences were uncategorized.
of the  <dig> putative full-length ltrs identified using ltr_struc, three were attributed to gypsy/ty3-like elements and two to copia/ty1-like elements.
the remaining four sequences are excluded from further analyses.
repeatmasker masked  <dig> % of the genome assembly:  <dig> % by repeated elements belonging to unknown/uncategorized families,  <dig> % by class  <dig> gypsy retrotransposons,  <dig>  by class  <dig> copia retrotransposons,  <dig>  by class  <dig> dirs retrotransposons,  <dig>  by class  <dig> line retrotransposons and  <dig>  by class  <dig> dna transposons .table  <dig>
number of repeated sequences in the
p. cinnabarinus
genome
class- <dig> ltr gypsy-like
class- <dig> ltr copia-like
class- <dig> dirs
class- <dig> line
class- <dig> dna transposons
uncategorized
all
*number of elements identified by repeatscout pipeline.
**number of elements identified by ltr_struc pipeline.
the number of full-length ltr was lower in p. cinnabarinus than in other white-rots  <cit> , although the te genome coverage was in the range of other white-rot fungi.
a total of  <dig>  ssrs were identified in the p. cinnabarinus genome corresponding to  <dig> mono-,  <dig> di-,  <dig> tri-,  <dig> tetra-,  <dig> penta- and  <dig> hexanucleotide motifs.
a total of  <dig> mini-satellites and  <dig> satellites were identified for a genome coverage of  <dig> % and  <dig> %, 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  <cit> .
carbohydrate metabolism, lignin-degrading oxidoreductases and wood decay
carbohydrates and lignin are intimately interconnected in all land-plant cell-walls.
the accessibility of all cell-wall components i.e.
cellulose, hemicellulose, pectin and lignin, is strongly limited by the covalent cross-linkages of the constituents which create an intricate network and a physical barrier that resists microbial breakdown.
among the predicted lignin-degrading activities, a total of five laccases , one ferroxidase , one multicopper oxidase , nine ligninolytic peroxidases  including lignin peroxidases , manganese peroxidases  and versatile peroxidases , one cellobiose dehydrogenase containing an iron reductase domain , three aryl-alcohol oxidases and one glucose oxidase , two alcohol oxidases , two pyranose oxidases , seven copper radical oxidases , one benzoquinone reductase , and one iron reductase domain  linked to a cbm <dig> were identified .
p. cinnabarinus was initially considered to lack class-ii peroxidases based on extracellular activities in the culture medium  <cit> .
remarkably, nine class ii peroxidases were annotated and divided into at least four lip, three mnp, one vp and one atypical vp.
on average, white-rot fungi have  <dig> members of the aa <dig> family .
the only exception is s. commune in which the aa <dig> family is absent  <cit> , although it is considered as a white-rot fungus despite limited lignin-degrading ability.
members of family aa <dig> can be considered as one of the most important family markers to differentiate white-rot and brown-rot fungi, since brown-rot  fungi contain no aa <dig> members  <cit> .
in addition to class ii peroxidases, p. cinnabarinus contains several laccases  and one cellobiose dehydrogenase , meaning that this fungus contains a complete, versatile ligninolytic enzymatic spectrum.
a number of enzymes are proposed to supply the hydrogen peroxide required for oxidase activity.
among these, the best established candidate is glyoxal oxidase of family aa5_ <dig>  and p. cinnabarinus has seven candidate gene models in this family.
interestingly, p. cinnabarinus also possesses several other hydrogen peroxide providers, such as gmc oxidoreductases from family aa3_ <dig> which includes at least three aryl-alcohol oxidases.
in summary, the white-rot fungus p. cinnabarinus possesses a complete enzymatic arsenal for lignin breakdown.
the full set of ligninolytic enzymes identified suggests that this fungus may exploit different strategies for ligninolysis, including oxidation mediated by class ii peroxidases requiring hydrogen peroxide or by laccases in the presence of redox mediators, or via fenton chemistry  <cit> .table  <dig>
global composition of aa encoding genes found in
p. cinnabarinus
brfm137
family
a
aa3_2b
aknown family activities are as follows: aa1_1: laccase, aa1_2: ferroxidase, aa1: multicopper oxidase, aa2: class ii peroxidase; aa3_1: cellobiose dehydrogenase; aa3_2: aryl alcohol oxidase, glucose oxidase; aa3_3: alcohol oxidase; aa3_4: pyranose oxidase; aa5_1: glyoxal oxidase, copper radical oxidase; aa6: benzoquinone reductase; aa8: iron reductase domain; aa9: lpmo.
b including  <dig> ao and  <dig> gox.
according to  <cit> .
comparison of the cazy repertoire identified in the selected white-rot and brown-rot fungal genomes
cazy families
a
total gh
186
89
110
106
133
127
101
100
129
71
116
151
114
118
90
100
100
67
80
81
total pl
4
0
1
2
0
0
3
0
7
1
13
7
1
0
0
0
2
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
total cbm
96
38
46
26
53
57
36
47
57
33
43
47
49
31
14
41
19
35
29
18
aa3_2b
total aa
73
48
71
55
61
73
61
53
63
49
46
76
76
35
12
25
27
15
26
16
aselection of the cazy families involved in plant cell wall degradation.
full list of cazy families are provided in additional file 1: table s <dig>
baa3_ <dig> includes only models with similarity to aryl-alcohol oxidase and glucose oxidase
species list
: auricularia delicata ; ceriporiopsis subvermispora ; dichomitus squalens ; fomitiporia mediterranea ; ganoderma lucidum ; ganoderma sp.
; heterobasidion annosum ; phanerochaete chrysosporium ; punctularia strigosozonata ; pycnoporus cinnabarinus ; schizophyllum commune ; stereum hirsutum ; trametes versicolor ; coniophora puteana ; dacryopinax sp.
; fomitopsis pinicola ; gloeophyllum trabeum ; postia placenta ; serpula lacrymans ; wolfiporia cocos .
p. cinnabarinus is fully equipped with putative enzymes from families classically involved in cellulose degradation  and can grow on pure cellulose.
however, p. cinnabarinus possesses the smallest number of gh members among the white-rot fungi.
the p. cinnabarinus genome encodes  <dig> lytic polysaccharide monooxygenases  of family aa <dig>  a number similar to that encoded by other white-rot fungal genomes .
the p. cinnabarinus brfm <dig> genome contains a gene encoding a cdh .
this gene codes for the cdh already described by moukha et al.
<cit> , sigoillot et al.
<cit>  and bey et al.
<cit> .
concerning xylan degradation, only two gh <dig> and two gh <dig> enzymes were identified in p. cinnabarinus, which is less than the average number of representatives in the white-rot group .
no members of family gh <dig> could be found in the p. cinnabarinus genome.
the gh <dig> family includes α-l-arabinofuranosidases acting on terminal non-reducing α-l-arabinofuranose residues in arabinose-containing compounds  <cit> .
terminal arabinose residues are found in the rhamnogalacturonan i from dicot primary cell walls, and glucuronoarabinoxylan from grass primary cell walls, so the absence of gh <dig> could partly constrain the complete degradation of hemicelluloses and pectic polysaccharides in p. cinnabarinus and is consistent with the lack of such cell wall components in wood.
the number of other p. cinnabarinus genes encoding pectinolytic enzymes also seems to be limited.
the members of family gh <dig> are fewer than the average number found in other fungi, and no representative of family gh <dig> including α-l-arabinofuranosidase was found.
also, p. cinnabarinus contains no candidate gene of the pectinolytic families pl <dig> , pl <dig> , pl <dig> , ce <dig>  or gh <dig> .
p. cinnabarinus is the only fungus lacking a family gh <dig> member among the selected white- and brown-rots.
family gh <dig> enzymes degrade galactans and arabinogalactans in the pectic component of plant cell walls.
this genomic repertoire is consistent with the very poor growth of p. cinnabarinus observed on apple pectin and citrus pectin as substrates .figure  <dig>
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 aa <dig>  aa3_ <dig> , aa5_ <dig> , aa <dig>  and cbm <dig>  .
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.
to study the growth ability of p. cinnabarinus, growth profiling was performed on  <dig> carbon sources, including mono-, oligo- or polysaccharides, crude plant biomass, casein and lignin, and the profiles were compared with the cazy gene content of the p. cinnabarinus genome.
on average, growth was better on crude plant biomass substrates than on pure mono- oligo- or polysaccharides .
interestingly, growth on cotton seed hulls was poor, not only compared with the other plant biomass substrates but also compared with several pure substrates.
previous studies suggest that this is probably due to the high lignin content of cotton seed hulls .
the other plant biomass substrates are poorer in lignin , suggesting that high lignin content inhibits growth of p. cinnabarinus in the culture conditions tested.
this correlates with very poor growth on kraft lignin as sole substrate.
as lignin may not be sole carbon source, the poor growth could be related to the possible impurities from lignocellulose introduced during lignin preparation.
growth is better on galactomannan  than on xylan, suggesting a better mannan degrading system.
endomannanases are found in gh <dig> and gh <dig>  but there are no family gh <dig> members in the p. cinnabarinus genome, suggesting that the good growth is mainly due to the gh5_ <dig> endomannanase, together with the three gh <dig> β-mannosidases and one gh <dig> α-galactosidase.
inulin- and sucrose-degrading enzymes are found in gh <dig>  but only one member of family gh <dig> is found in the p. cinnabarinus genome.
considering that growth on sucrose is significantly better than growth on inulin, this gene probably encodes an invertase rather than an inulinase.
these growth profiling studies estimated growth speed by measuring the diameter of the on-plate fungal mycelium on plates.
however, growth is also related to density of mycelium with dense, medium and thin mycelia on the plates.
the fast growth on poor media, especially when no carbon source is added, could also be due to thin mycelial expansion to avoid starvation.figure  <dig>
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
five laccases stricto sensu , one multicopper oxidase  and one ferroxidase  sequence were identified in the genome and in the cdna library, even partially .
structural annotation of the genes  was performed, and the gene lcc3- <dig>  <dig>  coding for laci protein was identified  <cit> .
in  <dig>  otterbein et al.
<cit>  demonstrated the presence of a second laccase isoenzyme, called lac <dig>  in the culture medium of p. cinnabarinus brfm <dig>
lac <dig> was purified and its n-terminal sequence was determined  <cit> .
however, the corresponding gene has never been identified and cloned, and the biochemical properties of the lac <dig> protein have never been determined.
based on the n-terminal sequence, we were able to determine the corresponding gene sequence, named lac <dig>  from the strain brfm <dig> genome sequencing data .
the five laccase-encoding genes have a size of about  <dig> – <dig>  kb interrupted by  <dig> to  <dig> introns.
based on the intron and exon positions of each gene, we were able to classify the various laccase genes into three groups .
lac2/lac <dig>  and lac1/lac <dig>  pairs have a similar structural organization with homologous intron positions, whereas the lac <dig> gene is organized slightly differently .
the lac <dig> gene comprised  <dig> exons but showed a slightly different structure from lac <dig> and lac <dig>  and an experimentally-found stop codon was confirmed in exon  <dig> .
in contrast to other laccase-encoding genes, the full-length lac <dig> 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 s <dig> .
in the p. cinnabarinus brfm <dig> genome, several laccase-encoding genes were identified on the same scaffold.
for instance, the lac <dig> and lac <dig> genes were separated by approximately 23 kb in the same reading frame on scaffold  <dig>
descriptions of the p. cinnabarinus ligninolytic peroxidases
we have shown that the p. cinnabarinus genome encodes a large set of ligninolytic peroxidases of family aa <dig>  nine full-length aa <dig> sequences were detected from the genomic dna of p. cinnabarinus brfm <dig> .
after an initial automatic classification as lips and mnps, they were manually reclassified following the strategy described by ruiz-dueñas et al.
<cit>  for manual annotation of the complete inventory of heme peroxidases of pleurotus ostreatus.
this protocol was based on a combined analysis of the deduced amino acid sequences and structural homology models obtained using the crystal structures of related enzymes as templates.
the identified members of family aa <dig> share common structural features, including four disulfide bridges and residues coordinating two calcium ions, a proximal histidine , and distal histidine and arginine residues , as shown in figure  <dig>  the presence of specific catalytic residues  <cit>  allowed us to classify the nine members of family aa <dig>  firstly, three short mnps  characterized both by the presence of a manganese oxidation site formed by two glutamates and one aspartate at the internal heme propionate region, and by a shorter c-terminal tail than that of long and extralong mnps  <cit> .
secondly, four lips  containing a 174-trp residue exposed to the solvent responsible for oxidation of high-redox-potential aromatic compounds.
thirdly, one vp  including both a catalytic trp residue exposed to the solvent and a manganese oxidation site; fourth, one atypical vp  differing from vps in one of the three acidic residues of the manganese oxidation site.
a partial sequence for the first  <dig> amino acids of the n-terminal end of an additional putative class ii peroxidase was also identified and could be hypothetically annotated as a lip <dig>  the above set of aa <dig> peroxidases identified in p. cinnabarinus is close to that identified in trametes versicolor   <cit> , although the total number of sequences is lower in pycnoporus.
two genes encoding heme peroxidases of a recently discovered superfamily of heme-thiolate peroxidases   <cit>  were also identified in p. cinnabarinus.figure  <dig>
molecular models for the nine class-ii heme peroxidases  found in the
p. cinnabarinus
genome.
mnp models  present a mn2+ oxidation site characteristic of typical mnps, formed by two glutamates and one aspartate at the internal heme propionate region; lip models  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  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  contains an aspartate residue  in a position occupied by a glutamate in vps and mnps.
two axial histidines, one acting as heme iron ligand  and the second  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  <cit>  using p. eryngii vpl  and p. chrysosporium liph <dig> and liph <dig>  crystal structures as templates.
these peroxidases are widely distributed in fungal genomes, including those from soft-rot, brown-rot and white-rot fungi  <cit> .
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  <cit> .
consequently, similar reactions are expected to be catalyzed by the htps identified in the p. cinnabarinus genome sequence.
all lip and mnp genes except mnp <dig> and lip <dig> were also found in the cdna library .
the mnp genes present lengths of  <dig> – <dig>  kb,  and and count 4– <dig> introns according to gene.
the two genes encoding vp and the four lips showed relatively similar sizes  and were interrupted by six introns, for coding sequences of similar length .
considering the analysis of the intron/exon structure, a division of family aa <dig> into several subgroups could be proposed.
vp and lip genes share a similar structural organization and form one group , whereas mnp genes are a more heterogeneous group in terms of gene structure, i.e.
exons  <dig> and  <dig> of the mnp2/mnp <dig> pair merge into a single exon in mnp <dig> while exons  <dig>   <dig> and  <dig> of the mnp1/mnp <dig> pair correspond to a single exon in mnp <dig>  finally, atypical-vp gene was totally different in length , number and structure of exons/introns compared with the other class ii peroxidase genes analyzed .
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 mnp <dig>  lip <dig>  lip <dig> and lip <dig> genes, each separated by about 2 kb and oriented in the same transcriptional direction on the  <dig> scaffold.
johansson and nyman  <cit>  had already described in t. versicolor, a similar cluster of three genes encoding two lips  and one mnp  in a genomic region of 10 kb, oriented in the same transcriptional direction and separated by approximately  <dig>  kb.
in addition, the intron/exon organization of these t. versicolor genes pointed to a similar structure for the two lpgiii and lpgiv , whereas the mpg <dig> gene was slightly different .
after analyzing the recently-sequenced t. versicolor genome sequence  <cit> , we identified an additional lip gene   <dig>  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 , these sequences appear closely related to those located at the same positions in the cluster identified in p. cinnabarinus .
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  <cit> .
htp <dig>  htp <dig> and lip <dig> genes also clustered on scaffold  <dig> at  <dig>  kb  and  <dig>  kb  apart .
similarly, two of the three htp genes identified in t. versicolor, only  <dig>  kb apart, form a cluster on scaffold  <dig> 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 mnp <dig>  lip <dig>  lip <dig> and lip <dig> 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 brfm <dig> cdna library .
the cloning of partial lip-like genes is described in additional file 9: data s <dig> .
figure  <dig> provides a dendrogram showing sequence relationships between  <dig> protein sequences of basidiomycete class ii peroxidases  <cit> , including those identified in the genome of p. cinnabarinus.
five peroxidase groups can be distinguished.
cluster a consists of  <dig> short mnps where the three p. cinnabarinus mnps appear closely related to seven of the  <dig> short mnps identified in the t. versicolor genome sequence  <cit> , and relatively distant from the  <dig> vps from p. eryngii, p. ostreatus, p. pulmonarius, p. sapidus, b. adusta and spongipellis sp.
also included in this cluster.
a well-defined cluster b contains all the lip  sequences, including the  <dig> lips from p. cinnabarinus closely related to the  <dig> lips identified in t. versicolor, as well as the only p. cinnabarinus vp grouped together with the two other vps  contained in this cluster.
cluster c consists of  <dig> short mnps, four vps and seven atypical vps, plus the only atypical vp identified in p. cinnabarinus which is grouped together with vps and atypical vps from other species , all of them clustered together with p. cinnabarinus within the core polyporoid clade.
the clearly-differentiated cluster d is composed of intermixed long and extralong mnps absent in p. cinnabarinus and characterized by the presence of 10– <dig> and 20– <dig> extra amino acid residues at the c-terminal end, respectively , and by containing one more disulfide bridge than lips, short mnps and vps .
different groups of generic peroxidases  and atypical mnps  are located next to the root of the dendrogram in the cluster d.figure  <dig>
dendrogram of  <dig> sequences of class-ii basidiomycete heme peroxidases  showing the position of nine sequences from the
p. cinnabarinus
genome .
evolutionary analysis was performed with mega <dig> using poisson distances and an unweighted pair group method with arithmetic mean clustering.
the cytochrome c peroxidase from p. ostreatus, monokaryon pc <dig>  was used to root the tree .
the dendogram was used to illustrate the clustering of sequences .
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 , with the rest collected from genbank  <cit> .
fungal abbreviations are as follows: bjead, bjerkandera adusta ; dicsq, dichomitus squalens ; fomme, fomitiporia mediterranea ; gansp, ganoderma sp.
; hetan, heterobasidion annosum ; phlbr, phlebia brevispora ; pycci, pycnoporus cinnabarinus; stehi, stereum hirsutum ; trace, trametopsis cervina; and trave, trametes versicolor .
other fungal species with peroxidase sequences included in the collapsed clusters are: agaricus bisporus , auricularia delicata , bjerkandera sp , ceriporiopsis rivulosa, coprinellus disseminatus, coprinopsis cinerea , fomitopsis pinicola , ganoderma applanatum, ganoderma australe, ganoderma formosanum, ganoderma lucidum, gelatoporia subvermispora , basidiomycete izu- <dig>  laccaria bicolor , lentinula edodes, phanerochaete chrysosporium , phanerochaete sordida, phlebia radiata, pleurotus eryngii, pleurotus ostreatus , pleurotus pulmonarius, pleurotus sapidus, punctularia strigosozonata , rhodonia placenta , spongipellis sp., taiwanofungus camphoratus, wolfiporia cocos .
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 .
the ability of hydrogen peroxide to generate hydroxyl radicals  also points to another role of hydrogen peroxide in the biodegradation of wood, where these hydroxyl radicals  could initiate the attack of lignocellulose  <cit> .
for these reasons, research into hydrogen peroxide-producing enzymes – especially aa3_ <dig>  and aa5_ <dig>  – has surged.
the subfamily aa5_ <dig> contains glyoxal oxidases  and copper radical oxidases , which are enzymes related to glyoxal oxidases containing conserved active site residues but that diverge in terms of other structural features  <cit> .
in p. cinnabarinus, seven aa5_ <dig> enzymes have been identified in p. cinnabarinus brfm <dig>  including three glyoxal oxidases stricto sensu and four “radical copper oxidases” .
furthermore, these three glox and four cro were also expressed in the cdna library.
glox and cro encoding genes  have diverse characteristics in p. cinnabarinus.
the gene sizes ranged from  <dig>  to  <dig>  kb and were interrupted by one to  <dig> introns, corresponding to coding sequences ranging from  <dig>  to 3 kb .
the structure of the gene called cro <dig> stands out  <dig> from the others, with a large number  of introns.
in contrast, the sequences identified as glox sensu stricto share comparable size  and structure  and form a homogeneous group.
based on the analysis of the intron/exon structure of each pycnoporus aa5_ <dig> encoding gene , we could propose dividing aa5_ <dig> into three subgroups corresponding to:  the glox sequences, which had strong intron position homology,  the cro <dig>  cro <dig> and cro <dig> sequences, and  the very different cro <dig> sequence.
moreover, the three glox genes formed a cluster oriented in the same transcriptional direction and grouped on the same scaffold, with glox <dig> and glox <dig> separated by only  <dig>  kb .
this type of organization has also been found for the genes named cro <dig>  cro <dig> and cro <dig> in p. chrysosporium .
additional file 14: figure s <dig> and additional file 15: data s <dig> report the structural comparison between the glox <dig> protein sequence from p. cinnabarinus and that of gaox   <cit> .
secretome analyses and lignocellulosic degradation
several recent studies have shown that the diversity  of hemicellulolytic and ligninolytic enzymes or isoenzymes produced by basidiomycetes depends on substrate used and mode of cultivation  or solid-state fermentation ) .
agro-residues such as fruit 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  <cit> .
lignocellulosic residues such as straw, bran and wood chips favor the peroxidase production by most basidiomycetes  <cit> .
lc promotes the production of laccases and hydrolases while ssf promotes the production of peroxidases, including mnps  <cit> .
we thus ran several p. cinnabarinus brfm <dig> cultures via both lc and ssf in presence of simple or complex “natural” substrates to compositionally analyze the corresponding secretomes .
analysis of the p. cinnabarinus secretomes detected  <dig> proteins in lc-m ,  <dig> proteins in lc-b ,  <dig> proteins in lc-m-mb-a , and  <dig> proteins in ssf cultures.
most of the secreted proteins in our culture conditions consisted of carbohydrate-active enzymes , which represented 55% and 52% of the total proteins detected in lc-m-mb-a and ssf, respectively, and 41% and 47% in lc-m and lc-b, respectively .
cazyme distributions were compared according to the different cultures conditions .
interestingly, the lpmos of family aa <dig> were only identified in the conditions including complex substrates, and no aa <dig> protein was found in the control condition with maltose.
moreover, different aa <dig> proteins were produced in response to different growth conditions.
for instance, three aa <dig> proteins were produced only with birchwood, whereas two different aa <dig> proteins were identified in cultures with maize bran and avicel.
this result indicates that there is a differential regulation of the lpmo genes that is dependent on growth substrates and/or on temporal scale.
indeed, the aa9-encoding genes may also be constrained by strict short expression during substrate-supported fungal growth.
in recent studies, a preponderance of aa <dig> was produced exclusively in sugar beet pulp conditions  <cit> .
the detailed distribution of the cellulolytic and ligninolytic proteins detected in secretomes is described in additional file 16: table s <dig>  interestingly, all the representatives of the ligninolytic aa families were identified in these conditions, although with different distribution patterns depending on growth conditions .
three aa1_ <dig> laccases  were identified in all conditions studied here, demonstrating that these enzymes are widely and constitutively produced by the fungus.
contrary to laccases, members belonging to the class ii peroxidases  were only identified in the secretomes from ssf cultures  and in lc-m .
despite the major role of family peroxidases aa <dig> in lignin degradation, no aa <dig> protein was detected in the conditions using the hardwood substrate .
the class ii peroxidases could be constrained by a fine-tuned regulation or, alternatively, be not produced in our growth conditions.
the expression and regulation of class ii peroxidase-encoding genes depend on environmental signals such as concentration of carbon and nitrogen, exposure to metal ions and xenobiotics, temperature shock, and daylight  <cit> .figure  <dig>
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 gh <dig>  gh <dig>  gh <dig> and gh <dig>  which are principally involved in cellulose breakdown.
however, the endo-β- <dig> -glucanases of the subfamily gh5_ <dig> 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 , including members of families ce <dig>  ce <dig>  gh <dig>  gh <dig>  gh <dig>  moreover, family ce <dig> members were only found when maize bran was used in the cultures.
among the known activities in the ce <dig> family, feruloyl esterase activity mobilizes key enzymes acting on ferulic and diferulic acid bridges embedded in the hemicellulose from plant cell walls  <cit> .
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  <cit> .
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 .
transport of secretory proteins is expected to take place both via a pathway dependent on a signal recognition particle  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  complex and the exocyst complex .
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  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  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 .
the machinery to deal with misfolded or unfolded proteins ) is also conserved, although we were unable to identify a clear ortholog of the hac1/xbp <dig> transcription factor in the p. cinnabarinus genome.
hac1/haca  or xbp <dig>  is a bzip transcription factor that is uniquely activated by an unconventional splicing event mediated by ire1p  and trl1p   <cit> .
the presence of proteins involved in hac <dig> activation, such as the sensor  and the trna ligase , 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  system, which targets misfolded proteins for degradation in the proteasome, is conserved, since we identified orthologous proteins to the erad and proteasome .
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 -anchor biosynthesis, .
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 -linked- glycosylation) as well as the transfer of the oligosaccharide to asparagine residues  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 , which are carried out by protein mannosyl transferase , are also conserved.
like in other fungi, p. cinnabarinus has a genome that contains multiple pmt homologs.
glycosylphosphatidylinositol -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  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
<cit> .
galactofuranosylation is a type of modification found on glycoproteins in aspergillus species  <cit> , 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  have a mammalian-like structure and are devoid of the highly antigenic galactofuranose residues found in expression hosts such as a. niger see  <cit>  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  <cit> .
pycnoporus species are heterothallic agaricomycetes with two mating type loci controlling the fungal lifecycle  <cit> .
one mating type locus  in the tetrapolar agaricomycetes encodes two types of homeodomain transcription factors  in divergently-transcribed gene pairs, whereas the other  contains genes for pheromones and pheromone precursors, respectively  <cit> .
thea mating type locus
hd <dig> and hd <dig> mating type proteins from p. chrysosporium  were successfully used to screen the pycnoporus est contigs.
pycnoporus, like other basidiomycetes  <cit> , has at least one hd <dig> and one hd <dig> gene for homeodomain transcription factors.
the hd <dig> protein a1- <dig> deduced from contig > gcto4wp02f0tdf.f.pc.
<dig> dna:contig contig::gcto4wp02f0tdf.f.pc.1:1:2252: <dig> is  <dig> aa long.
its n-terminal domain is related to the n-terminal of mating type proteins from other species  and is expected to act in heterodimerization with compatible hd <dig> proteins while discriminating hd <dig> proteins from the same mating type  <cit> .
the two classes of homeodomain proteins encoded in basidiomycete mating type loci are defined by their distinct homeodomain sequences  <cit> .
hd <dig> 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  in helix iii are tolerated  <cit> .
in the pycnoporus a1- <dig> protein, the position of the hd <dig> homeodomain is only recognized by sequence alignment with related hd <dig> proteins from other species .
the dna-recognition sequence in helix iii is degenerated and helix ii has undergone a deletion.
previous research failed to find the expected conserved hd <dig> motif in respective proteins of postia placenta
<cit> .
we note from other species that a defective hd <dig> homeodomain does not inevitably cause loss-of-function in mating type regulation provided that the hd <dig> homeodomain in a hd1-hd <dig> heterodimer continues to function  <cit> .
a hd <dig> gene for the  <dig> aa-long protein a2- <dig> was found on > gcto4wp02f01pn.f.pc.
<dig> dna:contig contig::gcto4wp02f01pn.f.pc.1:1:2027: <dig>  the protein has a classical  <dig> amino acid-long homeodomain with all invariant residues in the dna-binding motif which is highly sequence-conserved with hd <dig> mating type proteins from other agaricomycetes .
interestingly, contig gcto4wp02f0tdf.f.pc.
<dig> contains not only the full-length coding sequence of protein a1- <dig>  but also, downstream on the opposite strand, the 3-terminal half of gene β-fg for an unknown fungal protein , which in most agaricomycetes flanks one side of the homeodomain transcript factor locus  <cit> .
at the other side of the loci, a mip gene for a mitochondrial intermediate peptidase is usually present  <cit> .
p. chrysosporium and p. placenta differ from other analyzed agaricomycetes in the relative order of their single hd <dig> gene to mip and β-fg.
these are the two species where hd <dig> 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  <cit> .
contig gcto4wp02f0tdf.f.pc.
<dig> indicates that pycnoporus is another species with this same inverted arrangement.
p. chrysosporium  and p. placenta , like pycnoporus , belong to the polyporales, and an inversion event early in evolution is likely  <cit> .
theb 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  <cit> .
the five p. chrysosporium proteins were used to screen the pycnoporus est contigs, and five hits were found.
three models contained full-length  or nearly complete  orfs for g protein-coupled transmembrane receptors .
the other two contained a 5′ half of a gene  and a 3′ half of a gene , respectively, and it is possible that these two est contigs present the same gene .
sequence analysis of the nearly- complete proteins using clustalw for alignment , genedoc  for manual corrections, and the neighbor-joining function in mega <dig> software  <cit>  indicates that pciste <dig>  groups with the two non-mating-type g protein-coupled transmembrane receptors of p. chrysosporium , whereas pciste <dig>  and pciste <dig>  cluster with the b-mating-type-orthologous receptors, respectively .
this finding suggests that p. cinnabarinus, like other tetrapolar agaricales, has b-mating-type-specific and non-mating-type genes for pheromone receptors  <cit> .
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 gcto4wp02fnfo <dig> f.pc.
<dig> and gcto4wp02f7kns.f.pc.
<dig>  respectively.
in both phylogenetic trees, the partial pheromone receptors group with pciste <dig>  and with the b orthologous pchste <dig>  of p. chrysosporium, which is evidence that the sequences may come from the same gene.
as in several other species  <cit> , 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  <dig> aa and the mature pheromones are  <dig> to  <dig> aa-long peptides, which are difficult to find in blast searches even at lowest stringency due to strongly divergent sequences  <cit> .
searches starting with the five identified p. chrysosporium pheromone precursor sequences  <cit>  were unsuccessful, but sequences from serpula lacrymans  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  motif at the c-terminus and a mda/df-motif at the n-terminus .
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  <cit> .
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 aa <dig> peroxidases  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  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.
electronic supplementary material
additional file 1: table s1: list of annotated genes in p. cinnabarinus.
additional file 2: table s2: list of the lignocellulolytic repertoire encoding-genes in p. cinnabarinus brfm <dig>
additional file 3: table s3: annotation of the lignin oxidoreductases predicted from the genome of p. cinnabarinus brfm <dig>
additional file 4: table s4: characteristics of laccase genes from p. cinnabarinus brfm <dig>
additional file 5: figure s1: molecular characterization of p. cinnabarinus brfm <dig> laccase genes.
additional file 6: data s1: multiplicity of fungal laccase encoding-genes and gene organization 69
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additional file 7: table s5: characteristics of peroxidase genes from p. cinnabarinus brfm <dig>
additional file 8: figure s2: molecular characterization of peroxidase genes  and schematic organization of peroxidase gene clusters on genomic dna from p. cinnabarinus brfm <dig> .
additional file 9: data s2: amplification of lip-like genes in white-rot fungi 90
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additional file 10: table s6: annotation of the aa5_ <dig> proteins predicted from the genome of p. cinnabarinus brfm <dig>
additional file 11: table s7: characteristics of aa5_ <dig> genes from p. cinnabarinus brfm <dig>
additional file 12: figure s3: molecular characterization of aa5_ <dig> genes  and schematic organization of glyoxal oxidase gene clusters on genomic dna from p. cinnabarinus brfm <dig> .
additional file 13: data s3: copper radical oxidases of p. chrysosporium
.
additional file 14: figure s4: modeling of the active site of the theoretical glox <dig> predicted in p. cinnabarinus brfm <dig> and comparison with galactose oxidase from dactylium dendroides.
additional file 15: data s4: structural comparison between glox <dig> and gaox proteins  <cit> .
additional file 16: table s8: list of lignocellulolytic enzymes identified in the secretomes of p. cinnabarinus brfm <dig> in different growth conditions.
additional file 17: table s9: list of unique and common genes identified in the different growth conditions.
additional file 18: table s10:
p. cinnabarinus genome annotation related to protein secretion pathways.
additional file 19: table s11:
p. cinnabarinus genome annotation related to protein glycosylation pathways.
additional file 20: figure s5: alignments of the n-terminal regions of hd <dig>  and hd <dig> mating type proteins  of pycnoporus and other agaricomycetes.
additional file 21: figure s6: alignment of sequences of putative g protein-coupled transmembrane pheromone receptors deduced from est contigs of p. cinnabarinus  and sequences of pheromone receptors of p. chrysosporium .
additional file 22: figure s7: neighbour-joining trees  of sequences of a.  complete, b. n-terminal and c. c-terminal halves of pheromone receptors of p. cinnabarinus and p. chrysosporium .
the classification in non-mating type and b orthologs follows the analysis of niculita-hierzel et al.
<cit> .
additional file 23: figure s8: alignment of sequences of putative b mating type pheromone precursors  and of putative precursors for non-mating-type pheromone-like peptides .
competing interests
the authors declare that they have no competing interests.
authors’ contribution
al, alo, er designed the research; al, alo, oc, ebu, fp, mh, ya, dc, ed, mjk, pg, llm, vl, jm, cn, jp, ap, jcs, aw, ck performed the research; al, alo, fjrd, ebu, uk, afjr cm, ib, habw, fm, pmc, rpdv, atm, ck, pp, bh analyzed the data; al, alo, er wrote the paper; al coordinated the study.
all authors have read and approved the final manuscript.

 filamentous fungi are a source of powerful enzymes for plant biomass breakdown and/or hydrolysis in green and white biotechnology, especially biorefining. 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 or industrial compounds. 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. soft-rot fungi partially degrade plant polysaccharides by mobilizing cellulases and hemicellulases, and cause wood softening. 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 and hydrogen peroxide may ultimately cause cellulose depolymerization. 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 where peroxidases cooperate with other oxidoreductases. 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. the white-rot fungus pycnoporus is very efficient at completely degrading lignin. 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. 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. p. cinnabarinus has a large array of copper- and iron-containing metalloenzymes involved in transforming plant-cell-wall aromatics and harbors original metabolic pathways involved in functionalizing these cell-wall aromatics to yield high-added-value compounds such as aromas and antioxidants. p. cinnabarinus is listed as a food- and cosmetic-grade microorganism. 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. 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, dye bleaching in the textile and dye industries, wastewater treatment, removal of phenolic compounds in beverages, biosensor and biofuel cell construction, and producing substances of pharmaceutical importance. 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 brfm 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 brfm was obtained from the international centre of microbial resources dedicated to filamentous fungi. genomic dna was isolated from ground mycelia powder as described in lomascolo et al., and a roughly 180 μg sample was sent to gatc biotech ag for genome sequencing. for construction of the cdna library, p. cinnabarinus brfm was grown as described in lomascolo et al. with five types of substrate: 20 g/l maltose, 20 g/l maltose and  g/l ferulic acid, 5 g/l maltose and 15 g/l oat spelt xylan, 5 g/l maltose and 15 g/l autoclaved maize bran and 5 g/l maltose and 15 g/l avicel cellulose. the other constituents of the medium were: diammonium tartrate; disodium tartrate; kh2po; cacl2 · 2h2o; mgso4 · 7h2o; feso4 · 7h2o; znso4 · 7h2o; mnso4 · h2o; cuso4 · 5h2o; yeast extract. after 4– 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. 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 with non-immobilized or immobilized mycelia on 2 × 2 × 1 cm polyurethane cubes were run in 250 ml baffled flasks containing 100 ml medium according to lomascolo et al.. three lc conditions were used: 20 g/l maltose, 5 g/l maltose, 15 g/l avicel cellulose and 15 g/l autoclaved maize bran, and 5 g/l maltose and 15 g/l micronized birchwood. solid-state fermentation cultures were also performed with five different substrates: sugarcane bagasse, banana skins, wood shavings, hemp and micronized birchwood. each substrate was homogenized in water to obtain a moisture content of 70%. five grams of substrate was placed in a 250 ml flask and inoculated with  ml of mycelial suspension according to a protocol adapted from meza et al.. for each growth condition, culture supernatants were harvested after and 10 days of cultivation and then pooled. for growth profiling on carbon sources, p. cinnabarinus brfm was grown on agar plates according to espagne et al., using either 10 g/l simple carbohydrates or 30 g/l complex carbohydrates. the kraft-lignin was purchased from sigma. genome sequencing and data assembly the p. cinnabarinus brfm genome was sequenced by a combination of methods: sequencing of genomic dna and two normalized cdna libraries obtained from cultures grown on different substrates using 454/gs roche flx titanium technology, sequencing of genomic dna with illumina/solexa genome analyzer ii technology, and sequencing of a kbp paired-end genomic library using illumina/solexa genome analyzer ii technology. the genomic roche read sets were uploaded to the ng storage environment. reads were cleaned using pyrocleaner, which applied a low-complexity filter followed by a read-size filter and a duplication-removal filter. the reads were then assembled using wgs-assembler version. 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 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 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. gene prediction was performed using augustus 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. all the data are available at the european nucleotide archive, embl-ebi, accession number:. gene prediction and functional annotation orthologous groups construction orthologous groups were built by running orthomcl software on the best protein models from: 1) p. cinnabarinus brfm 2) trametes versicolor, 3) postia placenta, 4) phanerochaete chrysosporium rp-, 5) schizophyllum commune h4-, 6) coprinopsis cinerea okayama7#, 7) laccaria bicolor s238n-h, 8) agaricus bisporus, 9) gloeophyllum trabeum, 10) ustilago maydis, 11) saccharomyces cerevisiae rm11-1a, 12) schizosaccharomyces pombe, 13) aspergillus niger, 14) trichoderma reesei qm6a, 15) nectria haematococca, 16) neurospora crassa, 17) myceliophthora thermophila, 18) chaetomium globosum, 19) mucor circinelloides, 20) homo sapiens and 21) arabidopsis thaliana. each og is a set of proteins across one or more species in the listed genomes that represents putative orthologs and in-paralogs. all-versus-all blastp was set a 10− cutoff. global functional annotation global functional annotation was based on the analysis of each og. all ogs were used as a seed for the functional annotation process based on the bioinformatics initiative gene ontology. ogs containing at least one sequence from p. cinnabarinus were selected. 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 protein database. a strict e-value threshold of 10− was applied to select homologous sequences retrieved by blastp. these homologs were mapped to the global gene ontology annotation files. 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 not included in ogs were directly blasted as described above. identification of repeated sequences repeatscout was used to identify de novo dna repeats in the p. cinnarinus genome. default parameters 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 and iii) repeats having significant hits to known proteins in uniprot other than proteins known to belong to transposable elements were removed. the remaining consensus sequences were annotated manually by a tblastx search against repbase to classify them into known te families. to identify full-length long terminal repeat retrotransposons, a second de novo search was performed with ltr_struc. 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 by masking the genome assembly with the consensus sequences coming from the repeatscout and ltr_struc pipelines. misa was used with default parameters to identify mono- to hexanucleotide simple sequence repeat motifs. mini-satellites and satellites were searched for in the p. cinnabarinus genome using tandem repeats finder software with the following parameters: 2; 7; 7; 80; 10; 50; carbohydrate-active enzyme and lignin degradation enzyme annotation all putative proteins were compared to the entries in the cazy database using blastp. the proteins with e-values smaller than were further screened by a combination of blast searches against individual protein modules belonging to the aa, gh, gt, pl, ce and cbm classes. hmmer was used to query against a collection of custom-made hidden markov model 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 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; second, the target nucleic acid sequence was translated in three reading frames. gene intron splice sites were determined based on consensus sequences fitting the gt-ag rule as described in breathnach et al.. identification of proteins in secretomes by lc-ms/ms analysis proteins from the diafiltered supernatants of p. cinnabarinus brfm cultures were separated by 1d sds-page electrophoresis according to the protocol of couturier et al.. after protein trypsinolysis, peptide analysis was performed by lc-ms/ms as described in arfi et al. using the pappso platform facilities. 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. 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− was used. the description of the gene products was taken from the saccharomyces genome database after identifying the s. cerevisiae orthologs.  characteristics of the p. cinnabarinusgenome the genome of the monokaryotic strain p. cinnabarinus brfm was sequenced by pyrosequencing and illumina sequencing runs to reach a final 31-fold coverage. the genome was ultimately assembled into scaffolds with n of 165118 bp. table  reports the features of the assembled genome sequences. the g + c content of the p. cinnabarinus genome was %. genome size was  mb and a total of orfs were identified in the structural annotation procedure. the number of orfs in p. cinnabarinus is close to the average number among the order polyporales. for instance, phanerochaete chrysosporium, postia placenta, wolfiporia cocos and ceriporiopsis subvermispora count and detected orfs in their genomes, respectively. p. cinnabarinus genome size is slightly lower than in p. placenta, c. subvermispora and w. cocos.table statistical assembly of the p. cinnabarinus genome in general, functional annotation hinges on the propagation of existing functional information via single homology searches. the resolution of functional inference could be improved by differentiating homologs into orthologs and paralogs. orthologs are assumed to have more chance of sharing the same function than paralogs. gene duplication is an essential contributing factor for evolving novel functions, and one of the duplicates could undergo evolutionary events such as sub-functionalization, neofunctionalization, etc.. we therefore based our annotation strategy on the searches for ogs within selected genomes followed by similarity searches from each og. an outline flow of the functional annotation procedure based on this phylogenomic approach is shown in figure  ogs were retrieved using a best reciprocal hit approach. the ogs included putative cds from p. cinnabarinus, totaling ~83% of total cds. based on a sequence homology searches within each og against the nr database using a strict e-value cutoff of 10− genes were annotated across the go categories. in addition, orphan genes were annotated using the standard blast2go procedure. the annotation procedure enabled us to annotate cds corresponding to ~52% of total cds. to compare with the classical method, fewer than 30% of total cds were annotated using the blast2go procedure. our approach based on ortholog clustering enables us to infer functional information directly from ogs using a subsequent drastic threshold for similarity searches and offers a conceptual framework for inferring information from various genomes. the annotated genes were grouped into functional groups. finally, a go tree depth was calculated to assess amount and quality of go annotations.figure annotation strategy for p. cinnabarinus based on a phylogenomic approach. orthologous groups were formed from genomes by running the orthomcl software using a blastp cutoff e- value of 1e− ogs containing at least one sequence from p. cinnabarinus were selected 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 protein database using a cutoff e-value of 10− 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. repeated sequences were identified in the genome of p. cinnabarinus and a library of consensus sequences was generated using repeatscout. after the different filtering steps, we were left with consensus sequences: and consensus sequences showed homologies with class gypsy, copia, dirs and long interspersed element retrotransposons, respectively, and with class transposons. the remaining consensus sequences were uncategorized. of the putative full-length ltrs identified using ltr_struc, three were attributed to gypsy/ty3-like elements and two to copia/ty1-like elements. the remaining four sequences are excluded from further analyses. repeatmasker masked % of the genome assembly: % by repeated elements belonging to unknown/uncategorized families, % by class gypsy retrotransposons, by class copia retrotransposons, by class dirs retrotransposons, by class line retrotransposons and by class dna transposons.table number of repeated sequences in the p. cinnabarinus genome class- ltr gypsy-like class- ltr copia-like class- dirs class- line class- dna transposons uncategorized all *number of elements identified by repeatscout pipeline. **number of elements identified by ltr_struc pipeline. the number of full-length ltr was lower in p. cinnabarinus than in other white-rots, although the te genome coverage was in the range of other white-rot fungi. a total of ssrs were identified in the p. cinnabarinus genome corresponding to mono-, di-, tri-, tetra-, penta- and hexanucleotide motifs. a total of mini-satellites and satellites were identified for a genome coverage of % and %, 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. carbohydrate metabolism, lignin-degrading oxidoreductases and wood decay carbohydrates and lignin are intimately interconnected in all land-plant cell-walls. the accessibility of all cell-wall components i.e. cellulose, hemicellulose, pectin and lignin, is strongly limited by the covalent cross-linkages of the constituents which create an intricate network and a physical barrier that resists microbial breakdown. among the predicted lignin-degrading activities, a total of five laccases, one ferroxidase, one multicopper oxidase, nine ligninolytic peroxidases including lignin peroxidases, manganese peroxidases and versatile peroxidases, one cellobiose dehydrogenase containing an iron reductase domain, three aryl-alcohol oxidases and one glucose oxidase, two alcohol oxidases, two pyranose oxidases, seven copper radical oxidases, one benzoquinone reductase, and one iron reductase domain linked to a cbm were identified. p. cinnabarinus was initially considered to lack class-ii peroxidases based on extracellular activities in the culture medium. remarkably, nine class ii peroxidases were annotated and divided into at least four lip, three mnp, one vp and one atypical vp. on average, white-rot fungi have members of the aa family. the only exception is s. commune in which the aa family is absent, although it is considered as a white-rot fungus despite limited lignin-degrading ability. members of family aa can be considered as one of the most important family markers to differentiate white-rot and brown-rot fungi, since brown-rot fungi contain no aa members. in addition to class ii peroxidases, p. cinnabarinus contains several laccases and one cellobiose dehydrogenase, meaning that this fungus contains a complete, versatile ligninolytic enzymatic spectrum. a number of enzymes are proposed to supply the hydrogen peroxide required for oxidase activity. among these, the best established candidate is glyoxal oxidase of family aa5_ and p. cinnabarinus has seven candidate gene models in this family. interestingly, p. cinnabarinus also possesses several other hydrogen peroxide providers, such as gmc oxidoreductases from family aa3_ which includes at least three aryl-alcohol oxidases. in summary, the white-rot fungus p. cinnabarinus possesses a complete enzymatic arsenal for lignin breakdown. the full set of ligninolytic enzymes identified suggests that this fungus may exploit different strategies for ligninolysis, including oxidation mediated by class ii peroxidases requiring hydrogen peroxide or by laccases in the presence of redox mediators, or via fenton chemistry.table global composition of aa encoding genes found in p. cinnabarinus brfm137 family a aa3_2b aknown family activities are as follows: aa1_1: laccase, aa1_2: ferroxidase, aa1: multicopper oxidase, aa2: class ii peroxidase; aa3_1: cellobiose dehydrogenase; aa3_2: aryl alcohol oxidase, glucose oxidase; aa3_3: alcohol oxidase; aa3_4: pyranose oxidase; aa5_1: glyoxal oxidase, copper radical oxidase; aa6: benzoquinone reductase; aa8: iron reductase domain; aa9: lpmo. b including ao and gox. according to. comparison of the cazy repertoire identified in the selected white-rot and brown-rot fungal genomes cazy families a total gh 186 89 110 106 133 127 101 100 129 71 116 151 114 118 90 100 100 67 80 81 total pl 4 0 1 2 0 0 3 0 7 1 13 7 1 0 0 0 2 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 total cbm 96 38 46 26 53 57 36 47 57 33 43 47 49 31 14 41 19 35 29 18 aa3_2b total aa 73 48 71 55 61 73 61 53 63 49 46 76 76 35 12 25 27 15 26 16 aselection of the cazy families involved in plant cell wall degradation. full list of cazy families are provided in additional file 1: table s baa3_ includes only models with similarity to aryl-alcohol oxidase and glucose oxidase species list: auricularia delicata; ceriporiopsis subvermispora; dichomitus squalens; fomitiporia mediterranea; ganoderma lucidum; ganoderma sp.; heterobasidion annosum; phanerochaete chrysosporium; punctularia strigosozonata; pycnoporus cinnabarinus; schizophyllum commune; stereum hirsutum; trametes versicolor; coniophora puteana; dacryopinax sp.; fomitopsis pinicola; gloeophyllum trabeum; postia placenta; serpula lacrymans; wolfiporia cocos. p. cinnabarinus is fully equipped with putative enzymes from families classically involved in cellulose degradation and can grow on pure cellulose. however, p. cinnabarinus possesses the smallest number of gh members among the white-rot fungi. the p. cinnabarinus genome encodes lytic polysaccharide monooxygenases of family aa a number similar to that encoded by other white-rot fungal genomes. the p. cinnabarinus brfm genome contains a gene encoding a cdh. this gene codes for the cdh already described by moukha et al., sigoillot et al. and bey et al.. concerning xylan degradation, only two gh and two gh enzymes were identified in p. cinnabarinus, which is less than the average number of representatives in the white-rot group. no members of family gh could be found in the p. cinnabarinus genome. the gh family includes α-l-arabinofuranosidases acting on terminal non-reducing α-l-arabinofuranose residues in arabinose-containing compounds. terminal arabinose residues are found in the rhamnogalacturonan i from dicot primary cell walls, and glucuronoarabinoxylan from grass primary cell walls, so the absence of gh could partly constrain the complete degradation of hemicelluloses and pectic polysaccharides in p. cinnabarinus and is consistent with the lack of such cell wall components in wood. the number of other p. cinnabarinus genes encoding pectinolytic enzymes also seems to be limited. the members of family gh are fewer than the average number found in other fungi, and no representative of family gh including α-l-arabinofuranosidase was found. also, p. cinnabarinus contains no candidate gene of the pectinolytic families pl, pl, pl, ce or gh. p. cinnabarinus is the only fungus lacking a family gh member among the selected white- and brown-rots. family gh enzymes degrade galactans and arabinogalactans in the pectic component of plant cell walls. this genomic repertoire is consistent with the very poor growth of p. cinnabarinus observed on apple pectin and citrus pectin as substrates.figure 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 aa aa3_, aa5_, aa and cbm. 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. to study the growth ability of p. cinnabarinus, growth profiling was performed on carbon sources, including mono-, oligo- or polysaccharides, crude plant biomass, casein and lignin, and the profiles were compared with the cazy gene content of the p. cinnabarinus genome. on average, growth was better on crude plant biomass substrates than on pure mono- oligo- or polysaccharides. interestingly, growth on cotton seed hulls was poor, not only compared with the other plant biomass substrates but also compared with several pure substrates. previous studies suggest that this is probably due to the high lignin content of cotton seed hulls. the other plant biomass substrates are poorer in lignin, suggesting that high lignin content inhibits growth of p. cinnabarinus in the culture conditions tested. this correlates with very poor growth on kraft lignin as sole substrate. as lignin may not be sole carbon source, the poor growth could be related to the possible impurities from lignocellulose introduced during lignin preparation. growth is better on galactomannan than on xylan, suggesting a better mannan degrading system. endomannanases are found in gh and gh but there are no family gh members in the p. cinnabarinus genome, suggesting that the good growth is mainly due to the gh5_ endomannanase, together with the three gh β-mannosidases and one gh α-galactosidase. inulin- and sucrose-degrading enzymes are found in gh but only one member of family gh is found in the p. cinnabarinus genome. considering that growth on sucrose is significantly better than growth on inulin, this gene probably encodes an invertase rather than an inulinase. these growth profiling studies estimated growth speed by measuring the diameter of the on-plate fungal mycelium on plates. however, growth is also related to density of mycelium with dense, medium and thin mycelia on the plates. the fast growth on poor media, especially when no carbon source is added, could also be due to thin mycelial expansion to avoid starvation.figure 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 five laccases stricto sensu, one multicopper oxidase and one ferroxidase sequence were identified in the genome and in the cdna library, even partially. structural annotation of the genes was performed, and the gene lcc3- coding for laci protein was identified. in otterbein et al. demonstrated the presence of a second laccase isoenzyme, called lac in the culture medium of p. cinnabarinus brfm lac was purified and its n-terminal sequence was determined. however, the corresponding gene has never been identified and cloned, and the biochemical properties of the lac protein have never been determined. based on the n-terminal sequence, we were able to determine the corresponding gene sequence, named lac from the strain brfm genome sequencing data. the five laccase-encoding genes have a size of about –  kb interrupted by to introns. based on the intron and exon positions of each gene, we were able to classify the various laccase genes into three groups. lac2/lac and lac1/lac pairs have a similar structural organization with homologous intron positions, whereas the lac gene is organized slightly differently. the lac gene comprised exons but showed a slightly different structure from lac and lac and an experimentally-found stop codon was confirmed in exon. in contrast to other laccase-encoding genes, the full-length lac 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 s. in the p. cinnabarinus brfm genome, several laccase-encoding genes were identified on the same scaffold. for instance, the lac and lac genes were separated by approximately 23 kb in the same reading frame on scaffold descriptions of the p. cinnabarinus ligninolytic peroxidases we have shown that the p. cinnabarinus genome encodes a large set of ligninolytic peroxidases of family aa nine full-length aa sequences were detected from the genomic dna of p. cinnabarinus brfm. after an initial automatic classification as lips and mnps, they were manually reclassified following the strategy described by ruiz-dueñas et al. for manual annotation of the complete inventory of heme peroxidases of pleurotus ostreatus. this protocol was based on a combined analysis of the deduced amino acid sequences and structural homology models obtained using the crystal structures of related enzymes as templates. the identified members of family aa share common structural features, including four disulfide bridges and residues coordinating two calcium ions, a proximal histidine, and distal histidine and arginine residues, as shown in figure  the presence of specific catalytic residues allowed us to classify the nine members of family aa firstly, three short mnps characterized both by the presence of a manganese oxidation site formed by two glutamates and one aspartate at the internal heme propionate region, and by a shorter c-terminal tail than that of long and extralong mnps. secondly, four lips containing a 174-trp residue exposed to the solvent responsible for oxidation of high-redox-potential aromatic compounds. thirdly, one vp including both a catalytic trp residue exposed to the solvent and a manganese oxidation site; fourth, one atypical vp differing from vps in one of the three acidic residues of the manganese oxidation site. a partial sequence for the first amino acids of the n-terminal end of an additional putative class ii peroxidase was also identified and could be hypothetically annotated as a lip the above set of aa peroxidases identified in p. cinnabarinus is close to that identified in trametes versicolor, although the total number of sequences is lower in pycnoporus. two genes encoding heme peroxidases of a recently discovered superfamily of heme-thiolate peroxidases were also identified in p. cinnabarinus.figure molecular models for the nine class-ii heme peroxidases found in the p. cinnabarinus genome. mnp models present a mn2+ oxidation site characteristic of typical mnps, formed by two glutamates and one aspartate at the internal heme propionate region; lip models 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 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 contains an aspartate residue in a position occupied by a glutamate in vps and mnps. two axial histidines, one acting as heme iron ligand and the second 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 using p. eryngii vpl and p. chrysosporium liph and liph crystal structures as templates. these peroxidases are widely distributed in fungal genomes, including those from soft-rot, brown-rot and white-rot fungi. 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. consequently, similar reactions are expected to be catalyzed by the htps identified in the p. cinnabarinus genome sequence. all lip and mnp genes except mnp and lip were also found in the cdna library. the mnp genes present lengths of –  kb, and and count 4– introns according to gene. the two genes encoding vp and the four lips showed relatively similar sizes and were interrupted by six introns, for coding sequences of similar length. considering the analysis of the intron/exon structure, a division of family aa into several subgroups could be proposed. vp and lip genes share a similar structural organization and form one group, whereas mnp genes are a more heterogeneous group in terms of gene structure, i.e. exons and of the mnp2/mnp pair merge into a single exon in mnp while exons and of the mnp1/mnp pair correspond to a single exon in mnp finally, atypical-vp gene was totally different in length, number and structure of exons/introns compared with the other class ii peroxidase genes analyzed. 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 mnp lip lip and lip genes, each separated by about 2 kb and oriented in the same transcriptional direction on the scaffold. johansson and nyman had already described in t. versicolor, a similar cluster of three genes encoding two lips and one mnp in a genomic region of 10 kb, oriented in the same transcriptional direction and separated by approximately  kb. in addition, the intron/exon organization of these t. versicolor genes pointed to a similar structure for the two lpgiii and lpgiv, whereas the mpg gene was slightly different. after analyzing the recently-sequenced t. versicolor genome sequence, we identified an additional lip gene  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, these sequences appear closely related to those located at the same positions in the cluster identified in p. cinnabarinus. 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. htp htp and lip genes also clustered on scaffold at  kb and  kb apart. similarly, two of the three htp genes identified in t. versicolor, only  kb apart, form a cluster on scaffold 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 mnp lip lip and lip 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 brfm cdna library. the cloning of partial lip-like genes is described in additional file 9: data s. figure  provides a dendrogram showing sequence relationships between protein sequences of basidiomycete class ii peroxidases, including those identified in the genome of p. cinnabarinus. five peroxidase groups can be distinguished. cluster a consists of short mnps where the three p. cinnabarinus mnps appear closely related to seven of the short mnps identified in the t. versicolor genome sequence, and relatively distant from the vps from p. eryngii, p. ostreatus, p. pulmonarius, p. sapidus, b. adusta and spongipellis sp. also included in this cluster. a well-defined cluster b contains all the lip sequences, including the lips from p. cinnabarinus closely related to the lips identified in t. versicolor, as well as the only p. cinnabarinus vp grouped together with the two other vps contained in this cluster. cluster c consists of short mnps, four vps and seven atypical vps, plus the only atypical vp identified in p. cinnabarinus which is grouped together with vps and atypical vps from other species, all of them clustered together with p. cinnabarinus within the core polyporoid clade. the clearly-differentiated cluster d is composed of intermixed long and extralong mnps absent in p. cinnabarinus and characterized by the presence of 10– and 20– extra amino acid residues at the c-terminal end, respectively, and by containing one more disulfide bridge than lips, short mnps and vps. different groups of generic peroxidases and atypical mnps are located next to the root of the dendrogram in the cluster d.figure dendrogram of sequences of class-ii basidiomycete heme peroxidases showing the position of nine sequences from the p. cinnabarinus genome. evolutionary analysis was performed with mega using poisson distances and an unweighted pair group method with arithmetic mean clustering. the cytochrome c peroxidase from p. ostreatus, monokaryon pc was used to root the tree. the dendogram was used to illustrate the clustering of sequences. 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, with the rest collected from genbank. fungal abbreviations are as follows: bjead, bjerkandera adusta; dicsq, dichomitus squalens; fomme, fomitiporia mediterranea; gansp, ganoderma sp.; hetan, heterobasidion annosum; phlbr, phlebia brevispora; pycci, pycnoporus cinnabarinus; stehi, stereum hirsutum; trace, trametopsis cervina; and trave, trametes versicolor. other fungal species with peroxidase sequences included in the collapsed clusters are: agaricus bisporus, auricularia delicata, bjerkandera sp, ceriporiopsis rivulosa, coprinellus disseminatus, coprinopsis cinerea, fomitopsis pinicola, ganoderma applanatum, ganoderma australe, ganoderma formosanum, ganoderma lucidum, gelatoporia subvermispora, basidiomycete izu- laccaria bicolor, lentinula edodes, phanerochaete chrysosporium, phanerochaete sordida, phlebia radiata, pleurotus eryngii, pleurotus ostreatus, pleurotus pulmonarius, pleurotus sapidus, punctularia strigosozonata, rhodonia placenta, spongipellis sp., taiwanofungus camphoratus, wolfiporia cocos. 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. the ability of hydrogen peroxide to generate hydroxyl radicals also points to another role of hydrogen peroxide in the biodegradation of wood, where these hydroxyl radicals could initiate the attack of lignocellulose. for these reasons, research into hydrogen peroxide-producing enzymes – especially aa3_ and aa5_ – has surged. the subfamily aa5_ contains glyoxal oxidases and copper radical oxidases, which are enzymes related to glyoxal oxidases containing conserved active site residues but that diverge in terms of other structural features. in p. cinnabarinus, seven aa5_ enzymes have been identified in p. cinnabarinus brfm including three glyoxal oxidases stricto sensu and four “radical copper oxidases”. furthermore, these three glox and four cro were also expressed in the cdna library. glox and cro encoding genes have diverse characteristics in p. cinnabarinus. the gene sizes ranged from to  kb and were interrupted by one to introns, corresponding to coding sequences ranging from to 3 kb. the structure of the gene called cro stands out from the others, with a large number of introns. in contrast, the sequences identified as glox sensu stricto share comparable size and structure and form a homogeneous group. based on the analysis of the intron/exon structure of each pycnoporus aa5_ encoding gene, we could propose dividing aa5_ into three subgroups corresponding to: the glox sequences, which had strong intron position homology, the cro cro and cro sequences, and the very different cro sequence. moreover, the three glox genes formed a cluster oriented in the same transcriptional direction and grouped on the same scaffold, with glox and glox separated by only  kb. this type of organization has also been found for the genes named cro cro and cro in p. chrysosporium. additional file 14: figure s and additional file 15: data s report the structural comparison between the glox protein sequence from p. cinnabarinus and that of gaox. secretome analyses and lignocellulosic degradation several recent studies have shown that the diversity of hemicellulolytic and ligninolytic enzymes or isoenzymes produced by basidiomycetes depends on substrate used and mode of cultivation or solid-state fermentation ). agro-residues such as fruit 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. lignocellulosic residues such as straw, bran and wood chips favor the peroxidase production by most basidiomycetes. lc promotes the production of laccases and hydrolases while ssf promotes the production of peroxidases, including mnps. we thus ran several p. cinnabarinus brfm cultures via both lc and ssf in presence of simple or complex “natural” substrates to compositionally analyze the corresponding secretomes. analysis of the p. cinnabarinus secretomes detected proteins in lc-m, proteins in lc-b, proteins in lc-m-mb-a, and proteins in ssf cultures. most of the secreted proteins in our culture conditions consisted of carbohydrate-active enzymes, which represented 55% and 52% of the total proteins detected in lc-m-mb-a and ssf, respectively, and 41% and 47% in lc-m and lc-b, respectively. cazyme distributions were compared according to the different cultures conditions. interestingly, the lpmos of family aa were only identified in the conditions including complex substrates, and no aa protein was found in the control condition with maltose. moreover, different aa proteins were produced in response to different growth conditions. for instance, three aa proteins were produced only with birchwood, whereas two different aa proteins were identified in cultures with maize bran and avicel. this result indicates that there is a differential regulation of the lpmo genes that is dependent on growth substrates and/or on temporal scale. indeed, the aa9-encoding genes may also be constrained by strict short expression during substrate-supported fungal growth. in recent studies, a preponderance of aa was produced exclusively in sugar beet pulp conditions. the detailed distribution of the cellulolytic and ligninolytic proteins detected in secretomes is described in additional file 16: table s interestingly, all the representatives of the ligninolytic aa families were identified in these conditions, although with different distribution patterns depending on growth conditions. three aa1_ laccases were identified in all conditions studied here, demonstrating that these enzymes are widely and constitutively produced by the fungus. contrary to laccases, members belonging to the class ii peroxidases were only identified in the secretomes from ssf cultures and in lc-m. despite the major role of family peroxidases aa in lignin degradation, no aa protein was detected in the conditions using the hardwood substrate. the class ii peroxidases could be constrained by a fine-tuned regulation or, alternatively, be not produced in our growth conditions. the expression and regulation of class ii peroxidase-encoding genes depend on environmental signals such as concentration of carbon and nitrogen, exposure to metal ions and xenobiotics, temperature shock, and daylight.figure 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 gh gh gh and gh which are principally involved in cellulose breakdown. however, the endo-β- -glucanases of the subfamily gh5_ 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, including members of families ce ce gh gh gh moreover, family ce members were only found when maize bran was used in the cultures. among the known activities in the ce family, feruloyl esterase activity mobilizes key enzymes acting on ferulic and diferulic acid bridges embedded in the hemicellulose from plant cell walls. 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. 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. transport of secretory proteins is expected to take place both via a pathway dependent on a signal recognition particle 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 complex and the exocyst complex. 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 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 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. the machinery to deal with misfolded or unfolded proteins ) is also conserved, although we were unable to identify a clear ortholog of the hac1/xbp transcription factor in the p. cinnabarinus genome. hac1/haca or xbp is a bzip transcription factor that is uniquely activated by an unconventional splicing event mediated by ire1p and trl1p. the presence of proteins involved in hac activation, such as the sensor and the trna ligase, 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 system, which targets misfolded proteins for degradation in the proteasome, is conserved, since we identified orthologous proteins to the erad and proteasome. 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 -anchor biosynthesis,. 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 -linked- glycosylation) as well as the transfer of the oligosaccharide to asparagine residues 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, which are carried out by protein mannosyl transferase, are also conserved. like in other fungi, p. cinnabarinus has a genome that contains multiple pmt homologs. glycosylphosphatidylinositol -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 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. galactofuranosylation is a type of modification found on glycoproteins in aspergillus species, 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 have a mammalian-like structure and are devoid of the highly antigenic galactofuranose residues found in expression hosts such as a. niger see 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. pycnoporus species are heterothallic agaricomycetes with two mating type loci controlling the fungal lifecycle. one mating type locus in the tetrapolar agaricomycetes encodes two types of homeodomain transcription factors in divergently-transcribed gene pairs, whereas the other contains genes for pheromones and pheromone precursors, respectively. thea mating type locus hd and hd mating type proteins from p. chrysosporium were successfully used to screen the pycnoporus est contigs. pycnoporus, like other basidiomycetes, has at least one hd and one hd gene for homeodomain transcription factors. the hd protein a1- deduced from contig > gcto4wp02f0tdf.f.pc. dna:contig contig::gcto4wp02f0tdf.f.pc.1:1:2252: is aa long. its n-terminal domain is related to the n-terminal of mating type proteins from other species and is expected to act in heterodimerization with compatible hd proteins while discriminating hd proteins from the same mating type. the two classes of homeodomain proteins encoded in basidiomycete mating type loci are defined by their distinct homeodomain sequences. hd 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 in helix iii are tolerated. in the pycnoporus a1- protein, the position of the hd homeodomain is only recognized by sequence alignment with related hd proteins from other species. the dna-recognition sequence in helix iii is degenerated and helix ii has undergone a deletion. previous research failed to find the expected conserved hd motif in respective proteins of postia placenta. we note from other species that a defective hd homeodomain does not inevitably cause loss-of-function in mating type regulation provided that the hd homeodomain in a hd1-hd heterodimer continues to function. a hd gene for the aa-long protein a2- was found on > gcto4wp02f01pn.f.pc. dna:contig contig::gcto4wp02f01pn.f.pc.1:1:2027: the protein has a classical amino acid-long homeodomain with all invariant residues in the dna-binding motif which is highly sequence-conserved with hd mating type proteins from other agaricomycetes. interestingly, contig gcto4wp02f0tdf.f.pc. contains not only the full-length coding sequence of protein a1- but also, downstream on the opposite strand, the 3-terminal half of gene β-fg for an unknown fungal protein, which in most agaricomycetes flanks one side of the homeodomain transcript factor locus. at the other side of the loci, a mip gene for a mitochondrial intermediate peptidase is usually present. p. chrysosporium and p. placenta differ from other analyzed agaricomycetes in the relative order of their single hd gene to mip and β-fg. these are the two species where hd 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. contig gcto4wp02f0tdf.f.pc. indicates that pycnoporus is another species with this same inverted arrangement. p. chrysosporium and p. placenta, like pycnoporus, belong to the polyporales, and an inversion event early in evolution is likely. theb 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. the five p. chrysosporium proteins were used to screen the pycnoporus est contigs, and five hits were found. three models contained full-length or nearly complete orfs for g protein-coupled transmembrane receptors. the other two contained a 5′ half of a gene and a 3′ half of a gene, respectively, and it is possible that these two est contigs present the same gene. sequence analysis of the nearly- complete proteins using clustalw for alignment, genedoc for manual corrections, and the neighbor-joining function in mega software indicates that pciste groups with the two non-mating-type g protein-coupled transmembrane receptors of p. chrysosporium, whereas pciste and pciste cluster with the b-mating-type-orthologous receptors, respectively. this finding suggests that p. cinnabarinus, like other tetrapolar agaricales, has b-mating-type-specific and non-mating-type genes for pheromone receptors. 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 gcto4wp02fnfo f.pc. and gcto4wp02f7kns.f.pc. respectively. in both phylogenetic trees, the partial pheromone receptors group with pciste and with the b orthologous pchste of p. chrysosporium, which is evidence that the sequences may come from the same gene. as in several other species, 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 aa and the mature pheromones are to aa-long peptides, which are difficult to find in blast searches even at lowest stringency due to strongly divergent sequences. searches starting with the five identified p. chrysosporium pheromone precursor sequences were unsuccessful, but sequences from serpula lacrymans 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 motif at the c-terminus and a mda/df-motif at the n-terminus. 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.  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 aa peroxidases 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 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. electronic supplementary material additional file 1: table s1: list of annotated genes in p. cinnabarinus. additional file 2: table s2: list of the lignocellulolytic repertoire encoding-genes in p. cinnabarinus brfm additional file 3: table s3: annotation of the lignin oxidoreductases predicted from the genome of p. cinnabarinus brfm additional file 4: table s4: characteristics of laccase genes from p. cinnabarinus brfm additional file 5: figure s1: molecular characterization of p. cinnabarinus brfm laccase genes. additional file 6: data s1: multiplicity of fungal laccase encoding-genes and gene organization 69 70 71 72 73 74 75 76 77 78 79 additional file 7: table s5: characteristics of peroxidase genes from p. cinnabarinus brfm additional file 8: figure s2: molecular characterization of peroxidase genes and schematic organization of peroxidase gene clusters on genomic dna from p. cinnabarinus brfm. additional file 9: data s2: amplification of lip-like genes in white-rot fungi 90 91 92 additional file 10: table s6: annotation of the aa5_ proteins predicted from the genome of p. cinnabarinus brfm additional file 11: table s7: characteristics of aa5_ genes from p. cinnabarinus brfm additional file 12: figure s3: molecular characterization of aa5_ genes and schematic organization of glyoxal oxidase gene clusters on genomic dna from p. cinnabarinus brfm. additional file 13: data s3: copper radical oxidases of p. chrysosporium. additional file 14: figure s4: modeling of the active site of the theoretical glox predicted in p. cinnabarinus brfm and comparison with galactose oxidase from dactylium dendroides. additional file 15: data s4: structural comparison between glox and gaox proteins. additional file 16: table s8: list of lignocellulolytic enzymes identified in the secretomes of p. cinnabarinus brfm in different growth conditions. additional file 17: table s9: list of unique and common genes identified in the different growth conditions. additional file 18: table s10: p. cinnabarinus genome annotation related to protein secretion pathways. additional file 19: table s11: p. cinnabarinus genome annotation related to protein glycosylation pathways. additional file 20: figure s5: alignments of the n-terminal regions of hd and hd mating type proteins of pycnoporus and other agaricomycetes. additional file 21: figure s6: alignment of sequences of putative g protein-coupled transmembrane pheromone receptors deduced from est contigs of p. cinnabarinus and sequences of pheromone receptors of p. chrysosporium. additional file 22: figure s7: neighbour-joining trees of sequences of a. complete, b. n-terminal and c. c-terminal halves of pheromone receptors of p. cinnabarinus and p. chrysosporium. the classification in non-mating type and b orthologs follows the analysis of niculita-hierzel et al.. additional file 23: figure s8: alignment of sequences of putative b mating type pheromone precursors and of putative precursors for non-mating-type pheromone-like peptides. competing interests the authors declare that they have no competing interests. authors’ contribution al, alo, er designed the research; al, alo, oc, ebu, fp, mh, ya, dc, ed, mjk, pg, llm, vl, jm, cn, jp, ap, jcs, aw, ck performed the research; al, alo, fjrd, ebu, uk, afjr cm, ib, habw, fm, pmc, rpdv, atm, ck, pp, bh analyzed the data; al, alo, er wrote the paper; al coordinated the study. all authors have read and approved the final manuscript. 
in addition, p. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. the distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. p. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. 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. 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. the megabase genome of p. cinnabarinus was sequenced and assembled, and the predicted genes were functionally annotated using a phylogenomic procedure. 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. growth profiling of p. cinnabarinus was performed on 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 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.
 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.  the megabase genome of p. cinnabarinus was sequenced and assembled, and the 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. 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 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pycnoporus cinnabarinusgenome annotationcazyauxiliary activitiesoxidoreductasewhite-rot fungilignocelluloseissue-copyright-statement© the author 2014 