Open Access
Nutritional value of protein from vegetative mycelia of edible mushroom Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Parada Albarracín, Julián Andrés; Urdaneta, Elena; Marzo Pérez, Florencio; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
The present work was designed to study the effects of supplementation a control diet with P. ostreatus mycelium for evaluation a nutritional value of mycoprotein and possible cholesterol lowering.
Open Access
Mapping the Pleurotus ostreatus genome
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Castellón Gadea, Jordi; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus is a commercially important edible mushroom commonly known as oyster mushroom which has also important biotechnical applications. Industrial production of P.ostreatus is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. In a previous study a linkage map of P. ostreatus strain N001 was constructed, which provided a basis for performing an efficient QTL (Quantitative trait loci) analysis based in a population of 80 sibling monokaryons. The map is based on the segregation of RAPD markers, RFLP markers, phenotypic characters and cloned genes. Nevertheless the linkage map is just a first step towards the selection of the appropiate parentals for new breeds. In order to organize and improve the access to the data and information accumulated in the previous works mentioned above, a Microsoft® Excel Linkage Map Matrix (MELMM) was designed and created. On this linkage map matrix we could have an easy and functional view of the P. ostreatus linkage map data, such as, recombination frequencies, genotypes information and degree of similarity between monokaryons that will help us in the design of breeding crosses aimed at improving QTLs of agronomic interest of new commercial strains.
Open Access
Ligninolytic peroxidase gene expression by Pleurotus ostreatus: differential regulation in lignocellulose medium and effect of temperature and pH
(Elsevier, 2014) Fernández Fueyo, Elena; Castanera Andrés, Raúl; Ruiz Dueñas, Francisco J.; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25 C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25 C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn2+ (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3 > vp2/vp3/mnp1/mnp2/mnp6 > mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10 C or 37 C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10 C treatment, while many of them were alternatively upregulated (often 6 h after the thermal shock) and downregulated (12 h) at 37 C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion.
Open Access
Transposon-associated epigenetic silencing during Pleurotus ostreatus life cycle
(Oxford University Press, 2018) Borgognone, Alessandra; Castanera Andrés, Raúl; Morselli, Marco; López Varas, Leticia; Rubbi, Liudmilla; Pisabarro de Lucas, Gerardo; Pellegrini, Matteo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Transposable elements constitute an important fraction of eukaryotic genomes. Given their mutagenic potential, host-genomes have evolved epigenetic defense mechanisms to limit their expansion. In fungi, epigenetic modifications have been widely studied in ascomycetes, although we lack a global picture of the epigenetic landscape in basidiomycetes. In this study, we analysed the genome-wide epigenetic and transcriptional patterns of the white-rot basidiomycete Pleurotus ostreatus throughout its life cycle. Our results performed by using high-throughput sequencing analyses revealed that strain-specific DNA methylation profiles are primarily involved in the repression of transposon activity and suggest that 21 nt small RNAs play a key role in transposon silencing. Furthermore, we provide evidence that transposon-associated DNA methylation, but not sRNA production, is directly involved in the silencing of genes surrounded by transposons. Remarkably, we found that nucleus-specific methylation levels varied in dikaryotic strains sharing identical genetic complement but different subculture conditions. Finally, we identified key genes activated in the fruiting process through the comparative analysis of transcriptomes. This study provides an integrated picture of epigenetic defense mechanisms leading to the transcriptional silencing of transposons and surrounding genes in basidiomycetes. Moreover, our findings suggest that transcriptional but not methylation reprogramming triggers fruitbody development in P. ostreatus.
Open Access
In silico analysis of the expression profile of AA9 Lytic Polysaccharide Monooxygenases (LPMOs) and the CDH Cellobiose Dehydrogenase enzyme in wood-degrader Agaricomycetes. The Pleurotus ostreatus case
(Elsevier, 2024-08-22) Jiménez Miguel, Idoia; Roscales, Gabriel; Garde Sagardoy, Edurne; Chuina Tomazeli, Emilia; Honda, Yoichi; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Barry, Kerrie; Grigoriev, Igor V.; Ramírez L.; Ramírez Nasto, Lucía; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Lignocellulose, the Earth's most abundant biopolymer, is degraded by wood-decaying fungi, specifically white rot fungi (WRF) and brown rot fungi (BRF), which use different strategies. This study examines the expression profiles of the AA9 and CDH enzymes of three WRF species (Heterobasidion annosum, Phanerochaete chrysosporium, and Pleurotus ostreatus) and two BRF species (Fomitopsis pinicola and Rhodonia placenta) from the Agaricomycetes class, grown on poplar wood or glucose as the sole carbon source. Mycelia were collected between days 10 and 12, revealing distinct lignocellulose degradation strategies between WRF and BRF, evidenced by the upregulation of AA9 LPMO (lytic polysaccharide monooxygenases) and AA3_1 (Cellobiose Dehydrogenase) genes, with the co-occurrence of both types of transcripts at the time of mycelial collection. The genome analysis showed variability in the number of AA9LPMO genes between WRF and BRF, which were differentially regulated depending on the carbon source. WRF exhibited a significant upregulation of AA9 LPMO genes,. In Phanerochaete chrysosporium, only one AA9LPMO gene was homologous to Pleurotus ostreatus, which had the highest number of AA9LPMO genes among the WRF studied. Some AA9 LPMO genes in Pleurotus ostreatus were associated to transposable elements (TEs, mainly footprints of LTRs) and grouped in clustered. LTRs were found either in the flanking or within the gene coding regions with no effect on gene transcription. In silico analysis of the AA9LPMO proteins in WRF uncovered distinct features at their C-terminal ends. Most of them lacked an appended module, but those with a CBM1 were highly induced in poplar wood media. The proportion of AA9 proteins with a CBM1 module was similar in Phanerochaete chrysosporium and Heterobasidion irregulare, but lower in Pleurotus ostreatus, which contained more AA9LPMO genes overall. In Pleurotus ostreatus, AA9LPMO proteins were grouped into three clades based on their C oxidizing type, with each clade containing proteins with specific features. The abundance (redundancy) of AA9LPMO genes in WRF especially associated to footprints LTRs in Pleurotus ostreatus suggests these genes may have other roles beyond lignocellulose degradation.
Open Access
Identification and functional characterisation of ctr1, a Pleurotus ostreatus gene coding for a copper transporter
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Peñas Parrila, María Manuela; Azparren Larraya, María Goretti; Domínguez, A.; Sommer, H.; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Copper homeostasis is primordial for life maintenance and especially relevant for ligning-degrading fungi whose phenol-oxidase enzymes depend on this micronutrient for their activity. In this paper we report the identification of a gene (ctr1), coding for a copper transporter in the white rot fungus Pleurotus ostreatus, in a cDNA library constructed from four-days old vegetative mycelium growing in submerged culture. The results presented here indicate that: (1) ctr1 functionally complements the respiratory deficiency of a yeast mutant defective in copper transport supporting the transport activity of the Ctr1 protein; (2) ctr1 transcription is detected in all P. ostreatus developmental stages (with exception of lamellae) and is negatively regulated by the presence of copper in the culture media; (3) ctr1 is a single copy gene that maps to P. ostreatus linkage group III; and (4) the regulatory sequence elements found in the promoter of ctr1 agree with those found in other copper related genes described in other systems. These results provide the first description of a copper transporter in this white rot fungus and open the possibility of further studies on copper metabolism in higher basidiomyetes.
Open Access
Molecular characterization of A cellobiohydrolase gene family in the fungus Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Eizmendi Goikoetxea, María Arantzazu; Sannia, Giovanni; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Cellulose is the most abundant biological polymer on Earth. Its chemical composition consists of D-glucose units linked by β-1,4- glycosidic bonds forming linear polymeric chains with a reducing and a non-reducing end. Cellulose chains may either adhere to each other, via hydrophobic and van der Waals interactions, forming crystalline structures or remain more loosely packaged (amorphous cellulose). Consequently, the physical structure and morphology of native cellulose is complex and not uniform. Biological degradation of cellulose depends on the action of three types of enzymes: endoglucanases (E.C.3.2.1.4), cellobiohydrolases (E.C.3.2.1.91) and β-glucosidases (E.C.3.2.1.21). All them hydrolyse β-1,4-glycosidic bonds but they differ on the substrate specificity. Endoglucanases hydrolyse the amorphous regions of the cellulose fibbers generating new reducing and non-reducing ends, cellobiohydrolases attack the molecule ends yielding cellobiose units, and β-glucosidases hydrolyse cellobiose molecules yielding glucose. Cellobiohydrolases can be classified into two groups: type I (CBHI) and type II (CBHII), each having opposite chain-end specificities. CBHI prefer the reducing ends while CBHII act at non-reducing ends. By the screening of a genomic library from the basidiomycete Pleurotus ostreatus var. florida, we have isolated five cbhI genes, named cbhI1, cbhI2, cbhI3, cbhI4 and cbhI5, proving the occurrence of a multigenic family coding for this enzymatic activity. Using this sequences as probe, it has been possible to know the conditions in which are expressed those genes. This has allowed the synthesis of the each gene cDNA and, by comparison of this sequence with the corresponding genomic sequence, the characterization of their structure. On the other hand, using the RFLP technique and a progeny of 80 monokaryons derived from the dikaryon N001, the five genes have been mapped on the linkage map of P. ostreatus var. florida mapping the cbhI1 to the chromosome IV and the others to the chromosome VI.
Open Access
Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis
(National Academy of Sciences, 2012) Fernández Fueyo, Elena; Ruiz Dueñas, Francisco J.; Ferreira, Patricia; Floudas, Dimitrios; Lavín Trueba, José Luis; Oguiza Tomé, José Antonio; Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Santoyo Santos, Francisco; Producción Agraria; Nekazaritza Ekoizpena
Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.
Open Access
Isolation, molecular characterization and location of telomeric sequences of the basidiomycete Pleurotus ostreatus var. florida
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The white rot fungus Pleurotus ostreatus is an edible basidiomycete of increasing biotechnological interest due to its ability to degrade both wood and chemicals related to lignin degradation products. Telomeres are specialized structures at the end of all eukaryotic chromosomes. Ensure chromosome stability and protect the ends from degradation and from fusing with other chromosomes. Telomeres sequences are extraordinary highly conserved in evolution. The loss of telomeric repeats triggers replicative senescence in cells. For identification of restriction telomeric fragments in a previously described linkage map of Pleurotus ostreatus var. florida (Larraya et al., 2000), dikaryotic and eighty monokaryotic genomic DNAs were digested with diferents restriction enzymes (BamHI, BglII, HindIII, EcoRI, PstI, SalI, XbaI and XhoI) electrophoresed and transferred to nylon membranes. Numerous polymorphic bands were observed when membranes were hibridized with human telomericd probe (TTAGGG)132 (heterologous probe). Telomeric restriction fragments were genetically mapped to a previously described linkage map of Pleurotus ostreatus var.florida, using RFLPs identified by a human telomeric probe (tandemly repeating TTAGGG hexanucleotide). Segregation of each telomeric restriction fragment was recorded as the presence vs. absence of a hibridizing band. Segregation data for seventy three telomeric restriction fragments was used as an input table to be analysed as described by Ritter et al. (1990) and by Ritter and Salamini (1996) by using the MAPRF program software. Seventeen out of twenty two telomeres were identified. Telomere and telomere-associated (TA) DNA sequences of the basidiomycete Pleurotus ostreatus were isolated by using a modified version of single- specific-primer polymerase chain reaction (SSP-PCR) technique (Sohapal et al., 2000). Telomeres of Pleurotus ostreatus contain at least twenty five copies of non-coding tandemly repeated sequence (TTAGGG).
Open Access
Metatranscriptomics sheds light on the links between the functional traits of fungal guilds and ecological processes in forest soil ecosystems
(Blackwell Scientific Publications Ltd, 2023) Auer, Lucas; Buée, Marc; Fauchery, Laure; Lombard, Vincent; Barry, Kerrie; Clum, Alicia; Copeland, Alex; Daum, Chris; LaButti, Kurt; Singan, Vasanth; Yoshinaga, Yuko; Martineau, Christine; Castillo Martínez, Federico; Alfaro Sánchez, Manuel; Imbert Rodríguez, Bosco; Ramírez Nasto, Lucía; Castanera Andrés, Raúl; Pisabarro de Lucas, Gerardo; Finlay, Roger; Lindahl, Björn D.; Olson, Ake; Séguin, Armand; Kohler, Annegret; Henrissat, Bernard; Grigoriev, Igor V.; Martin, Francis; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB
Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.