Open Access
Transcriptional and enzymatic profiling of Pleurotus ostreatus laccase genes in submerged and solid-state fermentation cultures
(American Society for Microbiology, 2012) Castanera Andrés, Raúl; Pérez Garrido, María Gumersinda; Omarini, Alejandra; Alfaro Sánchez, Manuel; Pisabarro de Lucas, Gerardo; Faraco, Vicenza; Amore, Antonella; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription- quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.
Open Access
Strain degeneration in pleurotus ostreatus: a genotype dependent oxidative stress process which triggers oxidative stress, cellular detoxifying and cell wall reshaping genes
(MDPI, 2021) Pérez Garrido, María Gumersinda; Lopez-Moya, Federico; Chuina Tomazeli, Emilia; Ibañez Vea, María; Garde Sagardoy, Edurne; López Llorca, Luis V.; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Strain degeneration has been defined as a decrease or loss in the yield of important commercial traits resulting from subsequent culture, which ultimately leads to Reactive Oxygen Species (ROS) production. Pleurotus ostreatus is a lignin-producing nematophagous edible mushroom. Mycelia for mushroom production are usually maintained in subsequent culture in solid media and frequently show symptoms of strain degeneration. The dikaryotic strain P. ostreatus (DkN001) has been used in our lab as a model organism for different purposes. Hence, different tools have been developed to uncover genetic and molecular aspects of this fungus. In this work, strain degeneration was studied in a full-sib monokaryotic progeny of the DkN001 strain with fast (F) and slow (S) growth rates by using different experimental approaches (light microscopy, malondialdehyde levels, whole-genome transcriptome analysis, and chitosan effect on monokaryotic mycelia). The results obtained showed that: (i) strain degeneration in P. ostreatus is linked to oxidative stress, (ii) the oxidative stress response in monokaryons is genotype dependent, (iii) stress and detoxifying genes are highly expressed in S monokaryons with symptoms of strain degeneration, (iv) chitosan addition to F and S monokaryons uncovered the constitutive expression of both oxidative stress and cellular detoxifying genes in S monokaryon strains which suggest their adaptation to oxidative stress, and (v) the overexpression of the cell wall genes, Uap1 and Cda1, in S monokaryons with strain degeneration phenotype indicates cell wall reshaping and the activation of High Osmolarity Glycerol (HOG) and Cell Wall Integrity (CWI) pathways. These results could constitute a hallmark for mushroom producers to distinguish strain degeneration in commercial mushrooms.
Open Access
Antifungal activity of chitosan/poly (ethylene oxide) blend electrospun polymeric fiber mat doped with metallic silver nanoparticles
(MDPI, 2023) Murillo Larrey, Leire; Rivero Fuente, Pedro J.; Sandúa Fernández, Xabier; Rodríguez Trías, Rafael; Pérez Garrido, María Gumersinda; Ingeniería; Ingeniaritza; Institute for Multidisciplinary Research in Applied Biology - IMAB; Institute for Advanced Materials and Mathematics - INAMAT2
In this work, the implementation of advanced functional coatings based on the combination of two compatible nanofabrication techniques such as electrospinning and dip-coating technology have been successfully obtained for the design of antifungal surfaces. In a first step, uniform and beadless electrospun nanofibers of both polyethylene oxide (PEO) and polyethylene (PEO)/chitosan (CS) blend samples have been obtained. In a second step, the dip-coating process has been gradually performed in order to ensure an adequate distribution of silver nanoparticles (AgNPs) within the electrospun polymeric matrix (PEO/CS/AgNPs) by using a chemical reduction synthetic process, denoted as in situ synthesis (ISS). Scanning electron microscopy (SEM) has been used to evaluate the surface morphology of the samples, showing an evolution in average fiber diameter from 157 ± 43 nm (PEO), 124 ± 36 nm (PEO/CS) and 330 ± 106 nm (PEO/CS/AgNPs). Atomic force microscopy (AFM) has been used to evaluate the roughness profile of the samples, indicating that the ISS process induced a smooth roughness surface because a change in the average roughness Ra from 84.5 nm (PEO/CS) up to 38.9 nm (PEO/CS/AgNPs) was observed. The presence of AgNPs within the electrospun fiber mat has been corroborated by UV-Vis spectroscopy thanks to their characteristic optical properties (orange film coloration) associated to the Localized Surface Plasmon Resonance (LSPR) phenomenon by showing an intense absorption band in the visible region at 436 nm. Energy dispersive X-ray (EDX) profile also indicates the existence of a peak located at 3 keV associated to silver. In addition, after doping the electrospun nanofibers with AgNPs, an important change in the wettability with an intrinsic hydrophobic behavior was observed by showing an evolution in the water contact angle value from 23.4° ± 1.3 (PEO/CS) up to 97.7° ± 5.3 (PEO/CS/AgNPs). The evaluation of the antifungal activity of the nanofibrous mats against Pleurotus ostreatus clearly indicates that the presence of AgNPs in the outer surface of the nanofibers produced an important enhancement in the inhibition zone during mycelium growth as well as a better antifungal efficacy after a longer exposure time. Finally, these fabricated electrospun nanofibrous membranes can offer a wide range of potential uses in fields as diverse as biomedicine (antimicrobial against human or plant pathogen fungi) or even in the design of innovative packaging materials for food preservation.
Open Access
Effect of nutritional factors and copper on the regulation of laccase enzyme production in Pleurotus ostreatus
(MDPI, 2022) Durán Sequeda, Dinary Eloísa; Suspes, Daniela; Maestre, Estibenson; Alfaro Sánchez, Manuel; Pérez Garrido, María Gumersinda; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Sierra Ramírez, Rocío; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
This research aimed to establish the relationship between carbon–nitrogen nutritional factors and copper sulfate on laccase activity (LA) by Pleurotus ostreatus. Culture media composition was tested to choose the nitrogen source. Yeast extract (YE) was selected as a better nitrogen source than ammonium sulfate. Then, the effect of glucose and YE concentrations on biomass production and LA as response variables was evaluated using central composite experimental designs with and without copper. The results showed that the best culture medium composition was glucose 45 gL−1 and YE 15 gL−1, simultaneously optimizing these two response variables. The fungal transcriptome was obtained in this medium with or without copper, and the differentially expressed genes were found. The main upregulated transcripts included three laccase genes (lacc2, lacc6, and lacc10) regulated by copper, whereas the principal downregulated transcripts included a copper transporter (ctr1) and a regulator of nitrogen metabolism (nmr1). These results suggest that Ctr1, which facilitates the entry of copper into the cell, is regulated by nutrient-sufficiency conditions. Once inside, copper induces transcription of laccase genes. This finding could explain why a 10–20-fold increase in LA occurs with copper compared to cultures without copper when using the optimal concentration of YE as nitrogen sources.
Open Access
Transposable elements versus the fungal genome: impact on whole-genome architecture and transcriptional profiles
(Public Library of Science, 2016) Castanera Andrés, Raúl; López Varas, Leticia; Borgognone, Alessandra; LaButti, Kurt; Lapidus, Alla; Schmutz, Jeremy; Grimwood, Jane; Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Grigoriev, Igor V.; Stajich, Jason E.; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
Transposable elements (TEs) are exceptional contributors to eukaryotic genome diversity. Their ubiquitous presence impacts the genomes of nearly all species and mediates genome evolution by causing mutations and chromosomal rearrangements and by modulating gene expression. We performed an exhaustive analysis of the TE content in 18 fungal genomes, including strains of the same species and species of the same genera. Our results depicted a scenario of exceptional variability, with species having 0.02 to 29.8% of their genome consisting of transposable elements. A detailed analysis performed on two strains of Pleurotus ostreatus uncovered a genome that is populated mainly by Class I elements, especially LTR-retrotransposons amplified in recent bursts from 0 to 2 million years (My) ago. The preferential accumulation of TEs in clusters led to the presence of genomic regions that lacked intra- and inter-specific conservation. In addition, we investigated the effect of TE insertions on the expression of their nearby upstream and downstream genes. Our results showed that an important number of genes under TE influence are significantly repressed, with stronger repression when genes are localized within transposon clusters. Our transcriptional analysis performed in four additional fungal models revealed that this TE-mediated silencing was present only in species with active cytosine methylation machinery. We hypothesize that this phenomenon is related to epigenetic defense mechanisms that are aimed to suppress TE expression and control their proliferation.
Open Access
Transcriptome metabolic characterization of tuber borchii SP1-A new spanish strain for in vitro studies of the bianchetto truffle
(MDPI, 2023) Chuina Tomazeli, Emilia; Alfaro Sánchez, Manuel; Zambonelli, Alessandra; Garde Sagardoy, Edurne; Pérez Garrido, María Gumersinda; Jiménez Miguel, Idoia; Ramírez Nasto, Lucía; Salman, Hesham; Pisabarro de Lucas, Gerardo; Institute for Multidisciplinary Research in Applied Biology - IMAB
Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.
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
Highly expressed captured genes and cross-kingdom domains present in Helitrons create novel diversity in Pleurotus ostreatus and other fungi
(BioMed Central, 2014) Castanera Andrés, Raúl; Pérez Garrido, María Gumersinda; López Varas, Leticia; Sancho, Rubén; Santoyo Santos, Francisco; Alfaro Sánchez, Manuel; Gabaldón Estevan, Juan Antonio; Pisabarro de Lucas, Gerardo; Oguiza Tomé, José Antonio; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Background: Helitrons are class-II eukaryotic transposons that transpose via a rolling circle mechanism. Due to their ability to capture and mobilize gene fragments, they play an important role in the evolution of their host genomes. We have used a bioinformatics approach for the identification of helitrons in two Pleurotus ostreatus genomes using de novo detection and homology-based searching. We have analyzed the presence of helitron-captured genes as well as the expansion of helitron-specific helicases in fungi and performed a phylogenetic analysis of their conserved domains with other representative eukaryotic species. Results: Our results show the presence of two helitron families in P. ostreatus that disrupt gene colinearity and cause a lack of synteny between their genomes. Both putative autonomous and non-autonomous helitrons were transcriptionally active, and some of them carried highly expressed captured genes of unknown origin and function. In addition, both families contained eukaryotic, bacterial and viral domains within the helitron’s boundaries. A phylogenetic reconstruction of RepHel helicases using the Helitron-like and PIF1-like helicase conserved domains revealed a polyphyletic origin for eukaryotic helitrons. Conclusion: P. ostreatus helitrons display features similar to other eukaryotic helitrons and do not tend to capture host genes or gene fragments. The occurrence of genes probably captured from other hosts inside the helitrons boundaries pose the hypothesis that an ancient horizontal transfer mechanism could have taken place. The viral domains found in some of these genes and the polyphyletic origin of RepHel helicases in the eukaryotic kingdom suggests that virus could have played a role in a putative lateral transfer of helitrons within the eukaryotic kingdom. The high similarity of some helitrons, along with the transcriptional activity of its RepHel helicases indicates that these elements are still active in the genome of P. ostreatus.
Open Access
Genetic breeding of edible mushrooms: from the genome to the production of new varieties of Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pisabarro de Lucas, Gerardo; Peñas Parrila, María Manuela; Pérez Garrido, María Gumersinda; Park, Sang-Kyu; Eizmendi Goicoechea, Arantza; Parada Albarracín, Julián Andrés; Palma Dovis, Leopoldo; Idareta Olagüe, Eneko; Jurado Cabanillas, Javier; Castellón Gadea, Jordi; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The breeding of new varieties of industrially cultivated edible mushrooms must proceed in the framework defined by the breeding objectives, the biological characteristics of the material and the legal and cultural constraints imposed to the breeding technology to be used. This last aspect is of the greatest importance in the case of a food that is considered in European countries as high quality and closer to nature than other industrially produced foods. This fact prevents the use of genetic-engineering based technologies for breeding, as the consumers would hardly accept genetically modified mushrooms. Consequently, mushroom breeding should be based on time-consuming processes of classic breeding. Molecular biology, however, can offer to the breeders useful tools for speeding up the selection process, for evaluating the new bred lines and, last but not least, to identify and eventually protect legally the outcome of their breeding programs.
Open Access
Non-additive transcriptional profiles underlie dikaryotic superiority in Pleurotus ostreatus laccase activity
(Public Library of Science, 2013) Castanera Andrés, Raúl; Omarini, Alejandra; Santoyo Santos, Francisco; Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Background: The basidiomycete Pleurotus ostreatus is an efficient producer of laccases, a group of enzymes appreciated for their use in multiple industrial processes. The aim of this study was to reveal the molecular basis of the superiority of laccase production by dikaryotic strains compared to their parental monokaryons. Methodology/Principal Findings: We bred and studied a set of dikaryotic strains starting from a meiotic population of monokaryons. We then completely characterised the laccase allelic composition, the laccase gene expression and activity profiles in the dikaryotic strain N001, in two of its meiotic full-sib monokaryons and in the dikaryon formed from their mating. Conclusions/Significance: Our results suggested that the dikaryotic superiority observed in laccase activity was due to nonadditive transcriptional increases in lacc6 and lacc10 genes. Furthermore, the expression of these genes was divergent in glucose- vs. lignocellulose-supplemented media and was highly correlated to the detected extracellular laccase activity. Moreover, the expression profile of lacc2 in the dikaryotic strains was affected by its allelic composition, indicating a putative single locus heterozygous advantage.