BUSCADOR RECOLECTA

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., This research was funded by Research Projects RTI2018-099371-B-I00 (MCIU, AEI, FEDER/UE) and AGL2015-66833-R (MINECO) of the Spanish National Research Programme, H2020 MUSA 727624 (EU), and by funds of the Public University of Navarre (UPNA).

Esta tesis doctoral describe el aislamiento, caracterización transcriptómica y compuestos volátiles de una nueva cepa de T. borchii obtenida a partir de un aislado natural. Establecer cultivos de trufas axénicas es una tarea desafiante y rara vez se logra. Sin embargo, tener acceso a diversas cepas es fundamental para avanzar en los estudios de biología molecular y establecer simbiosis micorrízica en estas especies. En este estudio, se realizó un análisis de transcriptoma del micelio vegetativo de T. borchii cultivado en diferentes sustratos en cultivos sumergidos. Nuestro objetivo no era comparar comportamientos en varios medios, sino establecer una comprensión fundamental del comportamiento de T. borchii en cultivos axénicos. En este contexto, la investigación se centró en genes que codifican enzimas integrales de las vías metabólicas centrales (que comprenden la glucólisis, el ciclo del TCA, el ciclo del glioxilato y la cadena respiratoria), genes afiliados a categorías de metabolitos secundarios y genes que gobiernan la generación de compuestos aromáticos volátiles. Además, realizamos un time course del crecimiento del micelio de T. borchii, identificando compuestos volátiles en diferentes tiempos y condiciones de cultivo. También se realizó una evaluación sensorial del micelio. La tesis también abarcó un segmento industrial, donde se probaron tecnologías para la conservación prolongada del micelio.
Más allá de la delimitación de nuevas cepas, los análisis transcriptómicos insinúan que, incluso en condiciones aeróbicas con una expresión genética mínima vinculada a la cadena de transporte de electrones, T. borchii exhibe una mayor síntesis de alcohol deshidrogenasas y quinasas a nivel de sustrato. En particular, también se observó la expresión de genes de desintoxicación contra especies reactivas de oxígeno (ROS)., This doctoral thesis describes the isolation, transcriptomic and volatile compound characterization of a new strain of T. borchii obtained from a natural isolate. Establishing axenic truffles cultures is a challenging task and is rarely accomplished. However, having access to various strains is essential for advancing molecular biology studies and establishing mycorrhizal symbiosis in these species. In this study, transcriptomic analyses of the vegetative mycelium of T. borchii cultivated in different substrates in submerged cultures were conducted. Our objective was not to compare behaviors in several media, but to establish a foundational understanding of T. borchii behavior in axenic cultures. Within this context, the investigation focused on genes encoding enzymes integral to central metabolic pathways (comprising glycolysis, TCA cycle, glyoxylate cycle, and the respiratory chain), genes associated with categories of secondary metabolites, and genes governing the generation of aromatic volatile compounds. Furthermore, a time course of growth of T. borchii mycelium was performed, identifying volatile compounds at different times and culture conditions. Additionally, a sensory assessment of mycelium was conducted. The thesis also encompassed an industrial segment, wherein technologies for the prolonged preservation of the mycelium were tested. Beyond the delineation of new strains, transcriptomic analyses imply that even in aerobic conditions with minimal gene expression linked to the electron transport chain, T. borchii exhibits heightened synthesis of alcohol dehydrogenases and kinases at the substrate level. Notably, the expression of detoxification genes against reactive oxygen species (ROS) was also observed., Este trabajo ha sido financiado por una Beca de Industria del Gobierno de Navarra para la realización de doctorados industriales (Ref. 0011-1408-2020-00000019), ayudas de movilidad Erasmus+ del programa de Campus Iberus (CONS_2017-1-ES01-KA108-036811) y del Gobierno de Navarra, y por fondos de los proyectos RTI2018-099371-B-I00 (MCIN/AEI/10.13039/5011000110333/ y FEDER Una manera de hacer Europa) and AGL2015-66833-R (MCIN/AEI/10.13039/5011000110333/ y FEDER Una manera de hacer Europa) del Plan Estatal de Investigación., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Esta tesis se ha realizado en el marco de un convenio de cotutela entre la Universidad de los Andes y la Universidad Pública de Navarra, El objetivo principal de la investigación fue determinar la influencia de la composición del medio de cultivo y los compuestos lignocelulósicos en la secreción de enzimas lacasas por P. ostreatus en cultivos sumergidos. Utilizando un enfoque estadístico y sistemático que permitió el control de la composición de los medios de cultivo, se encontraron las condiciones nutricionales óptimas que simultáneamente incrementaban el crecimiento fúngico y la actividad lacasa en ausencia y presencia de sulfato de cobre, un inductor reconocido de esta actividad enzimática. En dichas condiciones se indagó sobre los aspectos bioquímicos transcripciones en P. ostreatus relacionados con la secreción de lacasas, lo cual reveló la participación de transportadores de membrana de alta afinidad por el cobre (CTRs) como candidatos intermediarios de la regulación de tres genes lacasas lacc2, lacc6 and lacc10. Los resultados de la evaluación de la composición de los medios de cultivos sugieren que la regulación de estos trasportadores se encuentra estrechamente ligada a condiciones nutricionales suficientes en carbono y nitrógeno, con una participación central del metabolismo del nitrógeno orgánico en dicho proceso.
Este conocimiento fue orientado a determinar el papel de la actividad lacasa en el pretratamiento biológico de la cáscara de arroz con el fin de contribuir a obtener una comprensión más profunda del pretratamiento de la biomasa de lignocelulosa por P. ostreatus en cultivo sumergido., The main objective of this study was to determine the influence of the composition of the culture medium and lignocellulosic compounds on the secretion of laccase enzymes by P. ostreatus in submerged cultures. These studies were done using a statistical and systematic approach that allowed the control of the culture media composition. The optimal nutritional conditions were found that simultaneously increased fungal growth and laccase activity in the absence and presence of copper sulfate, a recognized inducer of laccase. Under these conditions, the biochemical aspects of transcripts in P. ostreatus related to laccase secretion were evaluated , which revealed the participation of membrane transporters with high affinity for copper (CTRs) as intermediate candidates for the regulation of three laccase genes, lacc2, lacc6, and lacc10. Moreover, the evaluation of the results of the culture media composition suggests that the regulation of these transporters is closely linked to sufficient nutritional conditions in carbon and nitrogen, with central participation of the metabolism of organic nitrogen in this process.
With these findings, it was possible to obtain more profound knowledge of the pretreatment of lignocellulosic biomass by P. ostreatus in a submerged culture that was oriented to determine the role of laccase activity in the biological pretreatment of rice husks., The doctoral student was supported by funds of the Gobernación del Departamento del Cesar by the Ministerio de Ciencia y Tecnología en Innovación de Colombia through 681 of 2014 resolution, Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

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., This research was funded by Research Projects RTI2018-099371-B-I00 (MCIN/AEI/10.13039/5011000110333/ and FEDER Una manera de hacer Europa) and AGL2015-66833-R (MCIN/AEI/10.13039/5011000110333/ and FEDER Una manera de hacer Europa) of the Spanish National Research Programme, H2020 MUSA 727624 (EU), and by funds of the Public University of Navarre (UPNA). E.C.T. was supported by a grant of the Gobierno de Navarra (Ref. 0011-1408-2020-00000019) for Industrial Ph.D. students and by mobility grants from Erasmus+ program of Campus Iberus (CONS_2017-1-ES01-KA108-036811) and by a mobility grant of the Gobierno de Navarra, Spain.

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., This research was funded by project number RTI2018-099371-B-I00 (MCIU, AEI, FEDER/ UE) of the Spanish National Research Plan and the Public University of Navarre (UPNA) funds. D.D.-S. was supported by funds from the Gobernación del Departamento del Cesar by the Ministerio de Ciencia y Tecnología e Innovación de Colombia through 681 of the 2014 resolution.

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., This research was funded by Research Projects RTI2018-099371-B-I00 (MCIU, AEI, FEDER/UE) and AGL2015-66833-R (MINECO) of the Spanish National Research Programme, H2020 MUSA 727624 (EU), and by funds of the Public University of Navarre (UPNA).

The LysM domain is a highly conserved carbohydrate-binding module that recognizes polysaccharides containing N-acetylglucosamine residues. LysM domains are found in a wide variety of extracellular proteins and receptors from viruses, bacteria, fungi, plants and animals. LysM proteins are also present in many species of mammalian fungal pathogens, although a limited number of studies have focused on the expression and determination of their putative roles in the infection process. This review summarizes the current knowledge and recent studies on LysM proteins in the main morphological groups of fungal pathogens that cause infections in humans and other mammals. Recent advances towards understanding the biological functions of LysM proteins in infections of mammalian hosts and their use as potential targets in antifungal strategies are also discussed., This work was supported by research project RTI2018-099371-B-I00 of the Spanish National Research Plan and by additional institutional support from the Public University of Navarre.