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
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
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.

Pérez Garrido, María Gumersinda

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