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La infección producida por P. digitatum y P. italicum es una de las principales causas de pérdidas durante la postcosecha de frutos cítricos. Los problemas derivados de la aplicación de fungicidas químicos utilizados en su control justifican la búsqueda de alternativas eficaces. El conocimiento de las bases de la interacción planta-patógeno y los mecanismos de defensa de las plantas son fundamentales en el desarrollo de alternativas para el control de patologías vegetales. Hasta la fecha, hay pocos estudios sobre los procesos implicados en la respuesta de defensa de los frutos frente a la infección por hongos fitopatógenos. Durante el desarrollo de este trabajo hemos empleado diferentes aproximaciones de genómica funcional para profundizar en la respuesta de los frutos cítricos a la infección por hongos del género Penicillium. Hemos elaborado dos bibliotecas de cDNA: RindPdig24 se obtuvo de frutos de mandarina 'Clemenules' a las 24 horas después de ser heridos e infectados por P. digitatum, mientras que PostharvP1 se obtuvo de frutos de mandarina 'Clemenules' heridos y frutos heridos e infectados por P. digitatum a diferentes tiempos, y está enriquecida en cDNAs de longitud completa. Junto a ellas, se analizó una tercera biblioteca substractiva de cDNA elaborada previamente, RindPdigS, enriquecida en genes específicos de infección de naranja 'Navelina'. La secuenciación masiva de clones de estas tres bibliotecas, su anotación y categorización funcional ha permitido obtener una representación global de los genes expresados en el fruto de los cítricos durante el proceso de infección. Así, se han secuenciado un total de 2.505 clones distintos e identificado 1.941 unigenes. Los datos se han integrado en el Proyecto Español de Genómica Funcional de Cítricos (CFGP). El 25% del total de clones no tienen homología en las bases de datos públicas y el 26% de los unigenes no están presentes en ninguna otra biblioteca de cDNA del CFGP. Estos datos indican que la respuesta del f / Alamar Cort, S. (2009). Cambios de expresión génica asociados a la respuesta de los frutos cítricos frente a la infección por hongos del género Penicillium [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/4340

Santé Canada encourage les transformateurs à réduire le contenu en NaCl dans plusieurs aliments, dont les fromages, puisque sa surconsommation est associée à plusieurs problèmes de santé. L'effet causé par la réduction du NaCl dans le Camembert, ou de sa substitution partielle par du KCl, sur la qualité globale de ce fromage n'est pas connu. Le but du projet visait à étudier la cinétique d'incorporation du sodium et du potassium lors du saumurage d'un Camembert commercial afin de produire des fromages réduits en NaCl et substitués par du KCl. Ces nouvelles conditions ont ensuite permis d'étudier le comportement des ferments fongiques d'affinage Penicillium camemberti et Geotrichum candidum. Les résultats ont démontré une modification du profil de croissance des deux espèces, plus accentuée chez G. candidum, en fonction des modifications salines apportées. L'impact causé par ces changements de comportement doit maintenant être évalué au niveau de l'activité spécifique des deux espèces.

Le Rovabio Excel ™ est un cocktail enzymatique complexe sécrété par le champignon filamenteux Penicillium funiculosum. La société ADISSEO commercialise cet additif alimentaire destiné à la nutrition animale car les principales enzymes qui le constituent dégradent les polymères contenus dans les céréales, tels que les polysaccharides non amylacés. Ainsi, le Rovabio Excel™ permet d’améliorer la digestibilité et d’augmenter la valeur nutritionnelle des matières premières agricoles en réduisant la viscosité du bol alimentaire des animaux. Dans le but d’augmenter sa compétitivité, ADISSEO a fait conduire des études sur cette solution pour la caractériser biochimiquement et optimiser son potentiel xylanolytique.Ces travaux de thèse s’inscrivent dans ces projets industriels et ont poursuivi deux objectifs distincts. Le premier correspondait à l’augmentation de la thermostabilité de la protéine XynB du Rovabio Excel™, pour lui permettre de résister à la granulation. Le second concernait XynA, la protéine majoritaire de la solution multienzymatique, qui a été caractérisée biochimiquement. Les premiers résultats de caractérisation biochimique de XynA ont montré que la protéine était 100 fois plus active sur β-1,4-glucane que sur xylane. Des tests complémentaires sur pNP-cellobiose et pNP-β-D-Lactopyranose ont révélé que XynA était 5,2 fois plus active sur pNP-cellobiose et possédait une activité « exo ». Enfin, l’analyse des produits d’hydrolyse d’oligosaccharides composés de 2 à 5 unités de glucose a confirmé que la protéine XynA était une cellobiohydrolase de type I, très sensible à l’inhibition par le cellobiose (IC50 - C2 = 17,7 µM). L’étude la thermostabilité de XynB a confirmé que cette protéine n’était pas naturellement thermostable. Les résultats des travaux d’ingénierie avec l’ajout d’un pont disulfure pour rigidifier la structure 3D de la protéine n’ont pas été probants. En revanche, la création de protéines chimères à partir de protéines plus thermostables (TfxA de Thermomonospora fusca et XynII de Trichoderma reesei) a permis d’améliorer la stabilité thermodynamique de XynB avec des Tm augmentés de plus de 10°C / The Rovabio Excel™ is a complex enzymatic cocktail secreted by the filamentous fungus Penicillium funiculosum. The ADISSEO company sells it as food additive for animal feed because the main enzymes degrade polymers contained in grains, such as non-starch polysaccharides. Thus, the Rovabio Excel™ improves the digestibility and increases the nutritional value of agricultural raw materials by reducing the viscosity of the diet of animals. In order to increase its competitiveness, ADISSEO did conduct studies on this solution to characterize it biochemically and maximize its xylanolytic potential.This thesis takes part of this industrial project and have pursued two distinct objectives. The first corresponds to the increase in the thermostability of the protein XynB from the Rovabio Excel™, to enable it to resist at the granulation process. The second was XynA, the major protein of the multienzyme solution, which was characterized biochemically.Initial results of biochemical characterization of XynA showed that the protein was 100 times more active on β-1,4-glucan on xylan. Additional tests on pNP-cellobiose and pNP-β-D-Lactopyranose revealed that XynA was 5.2 times more active on pNP-cellobiose and possess an "exo-acting" activity. Finally, the analysis of products from oligosaccharides hydrolysis, composed of 2 to 5 units of glucose, confirmed that the protein XynA was a type I cellobiohydrolase, very sensitive to inhibition by cellobiose (IC50-C2 = 17.7 µM).The thermostability of XynB study has confirmed that this protein was not thermostable naturally. The results of the engineering work with the addition of a disulfide bridge to rigidify the 3D structure of the protein were not conclusive. However, the creation of chimeric proteins with more thermostable proteins (TfxA from Thermomonospora fusca and XynII from Trichoderma reesei) has improved the thermodynamic stability of XynB with Tm increased by more than 10°C

A diversidade de organismos oriundos do ambiente marinho constitui uma fonte significativa de substâncias estruturalmente inéditas e biologicamente ativas, dentre as quais, diversas inspiraram o desenvolvimento de novas classes de agentes terapêuticos. Neste contexto, macroalgas vermelhas do gênero Bostrychia (Rhodomelaceae) foram coletadas em praias do litoral norte do estado de São Paulo e têm sido objeto de estudos químicos e biológicos, no Laboratório de Química Orgânica do Ambiente Marinho (LQOAM - NPPNS) da FCFRPUSP, sob a supervisão da Profa. Dra. Hosana M. Debonsi. As algas da espécie Bostrychia radicans demonstraram potencial quando avaliadas as atividade citotóxica, tripanocida, leishmanicida e antimicrobiana; além de um perfil químico interessante, evidenciado pelo isolamento de substâncias inéditas na literatura. Neste contexto, o presente trabalho descreve a continuidade do estudo químico da espécie B. radicans, coletada no Manguezal do Rio Escuro, em Ubatuba-SP; bem como o potencial biológico desta espécie, além da avaliação da atividade de enzimas fenolsulfatases na espécie. Ainda, no sentido de explorar novas fontes promissoras para o isolamento de substâncias bioativas, este trabalho descreve o isolamento de micro-organismos endofíticos associados à espécie B. radicans. Foram isoladas 45 linhagens de micro-organismos; dentre as quais foram selecionadas nove linhagens para obtenção de extratos e realização de triagens química e biológica. A partir desta triagem inicial, foi realizado o estudo químico dos fungos Xylaria sp., Penicillium brevicompactum e Phomopsis longicolla. A partir da Xylaria sp. foram isoladas as seguintes substâncias: ácido 2,5-diidroxibenzóico, 8-diidroxinaftol 1-O-a-glucopiranosídeo, 8-metóxi-3-metil-1- isocromanona e ácido pilifórmico. O estudo químico de Penicillium brevicompactum resultou no isolamento das substâncias: ácido micofenólico, asperfenamato, brevianamida A, brevianamida C e brevianamida oxindol, substância inédita como produto natural. A partir de Phomopsis longicolla foi isolado o dicerandrol C. Este trabalho descreve ainda o potencial biológico de algumas destas substâncias isoladas. O estudo químico e biológico de microorganismos realizado no LQOAM estimulou a consolidação de uma colaboração com o Prof. Dr. Isidro C. Gonzalez, através da realização de um estágio de 12 meses no Laboratório Botrytis (Departamento de Química Orgânica, Universidade de Cádiz). Durante este período foi realizado o estudo químico do fitopatógeno Botrytis cinerea, visando o isolamento de novos metabólitos ou toxinas; além do estudo da biogênese destas substâncias, através de ensaios utilizando precursores isotopicamente marcados. / The diversity of organisms from the marine environment is a significant source of structurally novel and biologically active substances, several of which have inspired the development of new classes of therapeutic agents. In this context, red macroalgae belonging to Bostrychia genus (Rhodomelaceae) were collected on beaches of the north coast of São Paulo State and have been studied chemically and biologically in the Laboratory of Organic Chemistry of the Marine Environment - (LQOAM - NPPNS) at FCFRP-USP, under Prof. Hosana M. Debonsi supervision. Algae Bostrychia radicans species showed cytotoxic, trypanocidal, antileishmanial and antimicrobial potential, besides a interesting chemical profile, evidenced by the isolation of new compounds in the literature. In this context, this work describes the sequential chemical study of B. radicans species, collected at the Rio Escuro Mangrove, Ubatuba-SP, as well as the biological potential of this species. Also, the phenolsulphatases enzyme activity was evaluated in this species. Still, in order to explore new promising sources for the isolation of bioactive substances, this study describes the isolation of endophytic microorganisms associated to B. radicans. In this way, 45 strains of microorganisms were isolated and nine strains were selected for extracts preparation; and subsequently chemical and biological screenings. Based on the biological screening and chemical profile analyses, the large-scale fermentation of the endophytic fungi Xylaria sp., Penicillium brevicompactum and Phomopsis longicolla was carried out. The chromatographic purification of the bioactive acethyl acetate extract from Xylaria sp. allowed the isolation of 2,5-dihydroxybenzoic acid, 8- dihydroxynaphtol 1-O-a-glucopyranoside, 8-methoxy-3-methyl-1-isochromanone and piliformic acid. Chemical studies of Penicillium brevicompactum resulted in the isolation of: mycophenolic acid, asperphenamate, brevianamide A, brevianamide C and brevianamide oxindole, isolated for the first time as a natural product. From Phomopsis longicolla was isolated dicerandrol C. This thesis also describes the potential biological of some of these isolated compounds. The chemical and biological studies of microorganisms achieved in LQOAM encouraged the consolidation of a collaboration work with Prof. Dr. Isidro C. Gonzalez, through the completion of a 12-month internship at the Laboratory Botrytis (Department of Organic Chemistry, University of Cádiz). During this period, was conducted the chemical study of plant pathogen Botrytis cinerea, aiming the isolation of new metabolites or toxins, in addition to studying the biogenesis of these substances, through experiments using isotopically labeled precursors.

P. roqueforti est une moisissure connue comme contaminant des ensilages et des aliments, mais aussi utilisée comme ferment pour la production de fromages à pâte persillée. Cette espèce est également connue pour son potentiel de production de métabolites secondaires à impact positif, comme les arômes, mais aussi à impact négatif, tels que des mycotoxines (dont la roquefortine C -ROQC- et l’acide mycophénolique -MPA-). Dans la première partie de ce travail, l’occurrence de la ROQC, du MPA et de l’aflatoxine M1 (seule mycotoxine réglementée dans les produits laitiers), dans une collection de 86 fromages à pâte persillée de différentes variétés et origines géographiques (15 pays), a été réalisée. Il apparaît que, si l’aflatoxine est inférieure au seuil de détection (LOD) de la méthode, les concentrations en ROQC et MPA sont quant à elles très variables et qu’une co-occurrence de ces mycotoxines existe dans 51% des échantillons testés. Dans la seconde partie, la toxicité de ces mycotoxines sur modèles cellulaires intestinal ou monocytaires a été évaluée. L’étude de cytotoxicité en mono-exposition a permis d’établir des valeurs de référence (CI50); d’autre part, un effet synergique des deux mycotoxines aux plus fortes concentrations testées a été révélé sur la lignée cellulaire intestinale (Caco-2). L’implication du mécanisme apoptotique après une exposition précoce (3 et 6 heures) à la ROQC et/ou au MPA a aussi été mise en évidence sur le modèle THP-1 ainsi qu’une absence d’implication de l’apoptose après 24 heures de mono ou de co-exposition sur modèle intestinal Caco-2. Enfin, dans la troisième partie, une étude de facteurs biotique ou abiotiques pouvant potentiellement moduler la production de ROQC et de MPA a été réalisée. L’évaluation du potentiel mycotoxigénique de 96 souches de P. roqueforti a mis en évidence que le milieu cheese-agar (mimant la composition physico-chimique du fromage) était beaucoup moins favorable que le milieu YES pour la production des deux mycotoxines et une grande variabilité dans la capacité de production des souches a été observée. L’étude de l’influence des facteurs abiotiques a montré qu’une température de 12°C ou une concentration en NaCl de 8% ou encore une atmosphère contenant 5% d’O2 diminuait significativement la production de ROQC et de MPA indépendamment de la croissance, alors que les autres facteurs (pH et présence de précurseurs de la ROQC) n’avaient pas d’effet dans les conditions testées. / P. roqueforti is a mould associated with silage and food contamination but also used as a ripening culture for the production of blue-veined cheese. Moreover, this species is also known for its potential to produce secondary metabolites. These metabolites can have a positive impact (e.g. aromas) or a negative impact (e.g. mycotoxins, including roquefortine C -ROQC- and mycophenolic acid -MPA-). In the framework of this work, we first studied the occurrence of MPA, ROQC and aflatoxin M1 (the only regulated mycotoxin in dairy products) within an 86 blue-veined cheese collection (representing 15 countries). Aflatoxin contents were always below the method detection limit. For MPA and ROQC, concentrations were highly variable and a co-occurrence of both mycotoxins was observed in 51% of the tested samples. Then, the toxicity of the 2 mycotoxins was established on various cell models (intestinal and monocytic). Mono-exposure studies allowed IC50 determination. Moreover, a synergistic effect was observed on Caco-2 cells when MPA and ROQC were used for co-exposure at the highest tested concentrations. While an apoptotic mechanism was observed at early THP-1 exposure stages (3 and 6h), no apoptose occurred for Caco-2 cells after 24 h of mono or co-exposure. Finally, a study of the biotic and abiotic factors potentially modulating P. roqueforti MPA and ROQC production was performed. Mycotoxigenic potential evaluation of 96 de P. roqueforti strains highlighted that a cheese-agar medium (mimicking cheese physico-chemical composition) was less favourable than the synthetic YES medium for mycotoxin production. A large variability in terms of mycotoxin production was also observed among the tested strains. Besides, while a significant effect of the temperature (12°C) and NaCl (8%) and O2 (5%) concentrations was observed on ROQC and MPA production, no significant effect of pH and ROQC amino-acid precursor addition could be detected on the production of both mycotoxins.

Made available in DSpace on 2016-08-17T18:39:33Z (GMT). No. of bitstreams: 1 3024.pdf: 4531108 bytes, checksum: 1e95a8ef5ff0d05c05375d174d890b9e (MD5) Previous issue date: 2010-04-22 / Universidade Federal de Minas Gerais / Fungi are known as one of the most promising sources of unprecedent bioactive compounds, however, marine derived fungi are still very poorly studied. These microorganisms present high percentages of active metabolites and they are commonly isolated from marine invertebrates. At this study, the associated microbiota of the marine sponge Dragmacidon reticulatum was investigated in order to know the microbial diversity and explore the biotechnological potential of those strains for the production of bioactive secondary metabolites. Fragments from D. reticulatum were incubated on Petri dishes with different growth media. Forty five strains were isolated, although only 40 presented satisfactory growth for the preservation and production of extracts. From these, 32 strains were analysed for their genetic polymorphism through the ARDRA method that showed 24 different ribotypes. Extracts produced from the growing media of the 40 strains were evaluated for their cytotoxic and/or antimicrobial activity. From those, 22 presented biological activities in at least one of the bioassays, and had their chemical profiles evaluated in HPLC-UV-MS. Considering only the different ribotypes, 20 strains were selected by the biological activity of their extracts and were submitted to molecular identification. These isolates were attributed to the Aspergillus, Penicillium, Cladosporium, Myrmecridium, Microphaeropsis, Sporidesmium and Westerdykella genera. One strain was identified as actinobacteria from the Streptomyces sp. genus, and had its extract investigated, because the marine derived actinobacteria are also potential sources of bioactive compounds. The isolation of the unprecedented compounds _,_-dehydrocurvularin, the I-6, O-2 and JKL-4, produced by the fungus Penicillium decaturense, demonstrated the effectiveness of the screening procedures used in strain selection as producers of metabolites of biotechnical interest, and the need of more studies with this strain and the other strains selected in this study (Streptomyces sp., Westerdykella sp. and Sporidesmium sp.). The results confirmed the biotechnological potential presented by marine derived fungi. / Os fungos são conhecidos como uma das fontes mais promissoras de compostos bioativos inéditos, no entanto, fungos derivados do ambiente marinho ainda são pouco estudados. Estes microrganismos apresentam altas porcentagens de metabólitos ativos e são comumente isolados de invertebrados marinhos. No presente estudo, a esponja marinha Dragmacidon reticulatum foi selecionada para ter sua microbiota associada investigada, de maneira a conhecer a diversidade de microrganismos associados a esta esponja e explorar o potencial biotecnológico destas linhagens para a produção de metabólitos secundários bioativos. Fragmentos da esponja D. reticulatum foram incubados em placas de Petri contendo diferentes meios de crescimento. Foram isoladas 45 linhagens, das quais 40 apresentaram crescimento satisfatório para preservação e produção de extratos. Destas, 32 foram avaliadas quanto ao seu polimorfismo genético por meio do método ARDRA. Os resultados da avaliação da diversidade genética permitiram identificar 24 ribotipos distintos das linhagens microbianas isoladas. Extratos produzidos a partir do cultivo das 40 linhagens isoladas foram avaliados quanto à sua atividade citotóxica e/ou antimicrobiana. Destes, 22 apresentaram atividade biológica em pelo menos um dos bioensaios, e tiveram seu perfil químico avaliados em CLAE-UV-EM. Considerando apenas os ribotipos distintos, 20 linhagens foram selecionadas pela atividade biológica de seus extratos e foram submetidas à identificação molecular. Estes isolados foram atribuídos aos gêneros Aspergillus, Penicillium, Cladosporium, Myrmecridium, Microphaeropsis, Sporidesmium e Westerdykella. Uma das linhagens foi identificada como uma actinobactéria do gênero Streptomyces sp., e teve seu extrato investigado, pois actinobactérias derivadas do ambiente marinho também são fontes potenciais de compostos bioativos. O isolamento da _,_- dehidrocurvularina e dos compostos I-6, O-2 e JKL-4 inéditos na literatura, a partir do fungo Penicillium decaturense, evidenciou a efetividade da seleção das linhagens como produtoras de metabólitos de interesse biotecnológico e a necessidade de continuidade dos estudos deste isolado e das outras linhagens selecionadas neste estudo (Streptomyces sp., Westerdykella sp. e Sporidesmium sp.). Os resultados confirmam o potencial biotecnológico apresentado por fungos derivados do ambiente marinho.

A diversidade de organismos oriundos do ambiente marinho constitui uma fonte significativa de substâncias estruturalmente inéditas e biologicamente ativas, dentre as quais, diversas inspiraram o desenvolvimento de novas classes de agentes terapêuticos. Neste contexto, macroalgas vermelhas do gênero Bostrychia (Rhodomelaceae) foram coletadas em praias do litoral norte do estado de São Paulo e têm sido objeto de estudos químicos e biológicos, no Laboratório de Química Orgânica do Ambiente Marinho (LQOAM - NPPNS) da FCFRPUSP, sob a supervisão da Profa. Dra. Hosana M. Debonsi. As algas da espécie Bostrychia radicans demonstraram potencial quando avaliadas as atividade citotóxica, tripanocida, leishmanicida e antimicrobiana; além de um perfil químico interessante, evidenciado pelo isolamento de substâncias inéditas na literatura. Neste contexto, o presente trabalho descreve a continuidade do estudo químico da espécie B. radicans, coletada no Manguezal do Rio Escuro, em Ubatuba-SP; bem como o potencial biológico desta espécie, além da avaliação da atividade de enzimas fenolsulfatases na espécie. Ainda, no sentido de explorar novas fontes promissoras para o isolamento de substâncias bioativas, este trabalho descreve o isolamento de micro-organismos endofíticos associados à espécie B. radicans. Foram isoladas 45 linhagens de micro-organismos; dentre as quais foram selecionadas nove linhagens para obtenção de extratos e realização de triagens química e biológica. A partir desta triagem inicial, foi realizado o estudo químico dos fungos Xylaria sp., Penicillium brevicompactum e Phomopsis longicolla. A partir da Xylaria sp. foram isoladas as seguintes substâncias: ácido 2,5-diidroxibenzóico, 8-diidroxinaftol 1-O-a-glucopiranosídeo, 8-metóxi-3-metil-1- isocromanona e ácido pilifórmico. O estudo químico de Penicillium brevicompactum resultou no isolamento das substâncias: ácido micofenólico, asperfenamato, brevianamida A, brevianamida C e brevianamida oxindol, substância inédita como produto natural. A partir de Phomopsis longicolla foi isolado o dicerandrol C. Este trabalho descreve ainda o potencial biológico de algumas destas substâncias isoladas. O estudo químico e biológico de microorganismos realizado no LQOAM estimulou a consolidação de uma colaboração com o Prof. Dr. Isidro C. Gonzalez, através da realização de um estágio de 12 meses no Laboratório Botrytis (Departamento de Química Orgânica, Universidade de Cádiz). Durante este período foi realizado o estudo químico do fitopatógeno Botrytis cinerea, visando o isolamento de novos metabólitos ou toxinas; além do estudo da biogênese destas substâncias, através de ensaios utilizando precursores isotopicamente marcados. / The diversity of organisms from the marine environment is a significant source of structurally novel and biologically active substances, several of which have inspired the development of new classes of therapeutic agents. In this context, red macroalgae belonging to Bostrychia genus (Rhodomelaceae) were collected on beaches of the north coast of São Paulo State and have been studied chemically and biologically in the Laboratory of Organic Chemistry of the Marine Environment - (LQOAM - NPPNS) at FCFRP-USP, under Prof. Hosana M. Debonsi supervision. Algae Bostrychia radicans species showed cytotoxic, trypanocidal, antileishmanial and antimicrobial potential, besides a interesting chemical profile, evidenced by the isolation of new compounds in the literature. In this context, this work describes the sequential chemical study of B. radicans species, collected at the Rio Escuro Mangrove, Ubatuba-SP, as well as the biological potential of this species. Also, the phenolsulphatases enzyme activity was evaluated in this species. Still, in order to explore new promising sources for the isolation of bioactive substances, this study describes the isolation of endophytic microorganisms associated to B. radicans. In this way, 45 strains of microorganisms were isolated and nine strains were selected for extracts preparation; and subsequently chemical and biological screenings. Based on the biological screening and chemical profile analyses, the large-scale fermentation of the endophytic fungi Xylaria sp., Penicillium brevicompactum and Phomopsis longicolla was carried out. The chromatographic purification of the bioactive acethyl acetate extract from Xylaria sp. allowed the isolation of 2,5-dihydroxybenzoic acid, 8- dihydroxynaphtol 1-O-a-glucopyranoside, 8-methoxy-3-methyl-1-isochromanone and piliformic acid. Chemical studies of Penicillium brevicompactum resulted in the isolation of: mycophenolic acid, asperphenamate, brevianamide A, brevianamide C and brevianamide oxindole, isolated for the first time as a natural product. From Phomopsis longicolla was isolated dicerandrol C. This thesis also describes the potential biological of some of these isolated compounds. The chemical and biological studies of microorganisms achieved in LQOAM encouraged the consolidation of a collaboration work with Prof. Dr. Isidro C. Gonzalez, through the completion of a 12-month internship at the Laboratory Botrytis (Department of Organic Chemistry, University of Cádiz). During this period, was conducted the chemical study of plant pathogen Botrytis cinerea, aiming the isolation of new metabolites or toxins, in addition to studying the biogenesis of these substances, through experiments using isotopically labeled precursors.

[ES] El empleo de organismos vivos como biofactorías ha ganado una atención significativa en la industria debido a la creciente demanda de sistemas de producción sostenibles y la escasez de recursos. Entre sus muchas aplicaciones, las biofactorías pueden ser diseñadas para producir feromonas de insectos, las cuales sirven como alternativa ecológica a los pesticidas para el control de plagas en la agricultura. Como prueba de concepto, en esta tesis doctoral se caracterizaron plantas de Nicotiana benthamiana modificadas genéticamente con una ruta multigénica para producir las feromonas de polillas (Z)-11-hexadecenol (Z11-16OH) y (Z)-11-hexadecenil acetato (Z11-16OAc). Las plantas resultantes produjeron cantidades moderadas de ambas feromonas (111.4 µg g-1 FW y 11.8 µg g-1 FW para Z11-16OH y Z11-16OAc, respectivamente), y tasas de emisión diarias de ~10 ng g-1 FW para cada feromona. La producción de feromonas afectó negativamente al desarrollo de las plantas, probablemente debido a la sustancial carga metabólica y posible toxicidad de estos productos. Una estrategia para superar estas limitaciones es diseñar un sistema de expresión condicional que permita a las plantas crecer con normalidad antes de inducir la producción de feromonas. Para ello desarrollamos un conjunto de promotores sintéticos personalizables, llamados GB_SynP, activables con dCasEV2.1, un activador transcripcional potente y programable desarrollado recientemente para la inducción de genes en plantas. Los promotores GB_SynP permitieron una regulación precisa de los transgenes, con unos niveles de transcripción robustos y modulables en el estado "encendido" (presencia de dCasEV2.1 y la correspondiente guía de ARN), y una expresión mínima en el estado "apagado". Para implementar el sistema de producción condicional de feromonas en plantas se generó una nueva ruta multigénica para la biosíntesis de feromonas de polilla bajo el control de los promotores GB_SynP. Paralelamente, el activador dCasEV2.1 se reguló transcripcionalmente mediante el módulo CUP2:GAL4 sensible a sulfato de cobre, un inductor químico ampliamente utilizado en la agricultura. La funcionalidad del sistema se probó mediante expresión transitoria en N. benthamiana, resultando en unos rendimientos en el estado "encendido" de 32.7 µg g-1 FW y 25 µg g-1 FW para Z11-16OH y Z11-16OAc, respectivamente, y unos niveles insignificantes en ausencia de cobre. Sin embargo, la expresión en estable de esta ruta en N. benthamiana produjo unos niveles de expresión de los transgenes significativamente menores y una marcada disminución en la producción de feromonas. Esto supone que el sistema en su forma actual resulte inviable como biofactoría de feromonas en términos prácticos. La optimización de este sistema debe centrarse en mejorar la cascada de activación, en el uso de especies de plantas alternativas con mayor biomasa, y/o en incrementar las tasas de emisión en planta. Como alternativa a la producción de feromonas en plantas, la intercambiabilidad de piezas génicas entre plantas y hongos filamentosos puede aprovecharse para crear biofactorías fúngicas de feromonas. En este sentido, nuestro grupo adaptó previamente el sistema GoldenBraid a hongos filamentosos, llamado FungalBraid. En esta tesis ampliamos la colección de FungalBraid incorporando 27 piezas nuevas que incluyen diferentes marcadores de selección y promotores constitutivos e inducibles, los cuales se caracterizaron funcionalmente en Penicillium digitatum y P. chrysogenum. Además, se expresaron con éxito los promotores GB_SynP en P. digitatum, en combinación con el sistema de dCas9 activadora contenido en el vector pAMA18. Aunque los niveles de expresión de GB_SynP en hongos filamentosos fueron menores que los observados previamente en plantas, ésta y otras herramientas disponibles en la colección FungalBraid pueden utilizarse en el futuro para el desarrollo de biofactorías fúngicas que produzcan feromonas de insectos y otras biomoléculas de alto valor. / [CA] L'ús d'organismes vius com biofàbriques ha guanyat una atenció significativa a la indústria a causa de la creixent demanda de sistemes de producció sostenible i l'escassetat de recursos. Entre les seues moltes aplicacions, les biofàbriques poden ser dissenyades per a produir feromones d'insectes, les quals serveixen com a alternativa ecològica als pesticides per al control de plagues a l'agricultura. Com a prova d'aquest concepte, en aquesta tesi doctoral es van caracteritzar plantes de Nicotiana benthamiana modificades genèticament plantes de amb una ruta multigènica per a produir les feromones d'arnes (Z)-11-hexadecenol (Z11-16OH) i (Z)-11-hexadecenil acetat (Z11-16OAc). Les plantes resultants van produir quantitats moderades de totes dues feromones (111.4 µg g-1 FW i 11.8 µg g-1 FW per a Z11-16OH i Z11-16OAc, respectivament), i taxes d'emissió diàries d'aproximadament 10 ng g-1 FW per a cada feromona. La producció de feromones en aquestes plantes va afectar negativament el seu desenvolupament, probablement a causa de la substancial càrrega metabòlica i possible toxicitat d'aquests productes. Una estratègia per superar aquestes limitacions és dissenyar un sistema d'expressió condicional que permeta a les plantes créixer amb normalitat abans d'induir la producció de feromones. Per això hem desenvolupat un conjunt de promotors sintètics personalitzables, anomenats GB_SynP, activables amb dCasEV2.1, un activador transcripcional potent i programable desenvolupat recentment per a la inducció de gens en plantes. Els promotors GB_SynP van permetre una regulació precisa des transgens, amb uns nivells de transcripció robustos i modulables a l'estat "encès" (presència de dCasEV2.1 i la corresponent guia d'ARN), i una expressió mínima a l'estat "apagat". Per implementar el sistema de producció condicional de feromones en plantes es va generar una nova ruta multigènica per a la biosíntesi de feromones d'arna sota el control dels promotors GB_SynP. Paral·lelament, l'activador dCasEV2.1 es va regular transcripcionalment al mòdul CUP2:GAL4 sensible al sulfat de coure, un inductor químic àmpliament utilitzat en l'agricultura. La funcionalitat del sistema es va provar mitjançant expressió transitòria en N. benthamiana, resultant en uns rendiments a l'estat "encès" de 32.7 g-1 FW i 25 µg g-1 FW per a Z11-16OH i Z11-16OAc, respectivament, i uns nivells insignificants en absència de coure. No obstant això, l'expressió estable d'aquesta ruta a N. benthamiana va produir uns nivells d'expressió dels transgens significativament menors i una marcada disminució en la producció de feromones. Això suposa que el sistema en la seua forma actual resulte inviable com a biofàbrica de feromones en termes pràctics. L'optimització d'aquest sistema ha de centrar-se en millorar la cascada d'activació, en l'ús d'espècies de plantes alternatives amb major biomassa, i/o en incrementar les taxes d'emissió a la planta. Com a alternativa a la producció de feromones en plantes, la intercanviabilitat de peces gèniques entre plantes i fongs filamentosos pot aprofitar-se per crear biofàbriques fúngiques de feromones. En aquest sentit, el nostre grup va adaptar prèviament el sistema GoldenBraid a fongs filamentosos, anomenat FungalBraid. En aquesta tesi, vam ampliar la col·lecció de FungalBraid incorporant 27 peces noves que inclouen diferents marcadors de selecció i promotors constitutius i induïbles, els quals es van caracteritzar funcionalment a Penicillium digitatum i P. chrysogenum. A més, es van expressar amb èxit els promotors GB_SynP en P. digitatum, en combinació amb el sistema de dCas9 activadora contingut en el vector pAMA18. Encara que els nivells d'expressió de GB_SynP en fongs filamentosos van ser menors que els observats prèviament en plantes, aquesta i altres eines disponibles a la col·lecció FungalBraid poden utilitzar-se en el futur per al desenvolupament de biofàbriques fúngiques que produeixin feromones d'insectes i altres biomolècules de gran valor. / [EN] The use of living organisms as biofactories have gained significant attention in the industry due to the increasing demand for sustainable production systems and the shortage of resources. Among their many applications, biofactories can be engineered to produce insect pheromones, which serve as eco-friendly alternatives to pesticides for pest management in agriculture. As a proof of concept, in this thesis we characterized Nicotiana benthamiana plants engineered with a multigene pathway to produce the moth pheromones (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc). The resulting transgenic plants produced modest amounts of both pheromones (111.4 µg g-1 FW and 11.8 µg g-1 FW for Z11-16OH and Z11-16OAc, respectively), and daily emission rates of ~10 ng g-1 FW for each pheromone. Pheromone production in these plants significantly affected their fitness, likely due to the substantial metabolic burden and possible toxicity of lipid-derived products. One strategy to address these developmental abnormalities consists of engineering conditional transgene expression systems, thus allowing plants to grow normally before inducing the production of pheromones. To achieve this goal, in this thesis we developed a set of customizable synthetic promoters called GB_SynP, which can be activated by dCasEV2.1, a strong programable transcriptional activator recently developed for plant gene regulation. These GB_SynP promoters enabled tight regulation of single and multiple transgenes, with robust and tunable transcription levels in the ON state (presence of dCasEV2.1 loaded with the corresponding gRNA), and minimal or undetectable expression in the OFF state. To implement a conditional expression system for pheromone production in plants, a newly engineered multigene pathway for the biosynthesis of moth pheromones was constructed under the control of GB_SynP promoters. In parallel, the dCasEV2.1 activator was transcriptionally regulated with the CUP2:GAL4 sensor for copper sulphate, an agronomically-compatible chemical trigger. The functionality of this system was tested transiently in N. benthamiana, resulting in estimated yields of 32.7 µg g-1 FW and 25 µg g-1 FW for Z11-16OH and Z11-16OAc respectively in the ON state, and negligible levels in the absence of copper. However, stable transformation of the same copper-regulated pheromone pathway in N. benthamiana plants resulted in significantly lower transgene expression levels, which translated into a great reduction of pheromone yields. This makes the system in its current form a non-viable pheromone biofactory in practical terms. Further optimization should focus on the improvement of the activation cascade, the use of alternative plant hosts with more biomass, and/or the enhancement of emission rates in planta. As an alternative to pheromone production in plants, the interchangeability of DNA parts between plants and filamentous fungi could also be exploited to create fungal biofactories for pheromone production. In this regard, our research group previously adapted the GoldenBraid system for filamentous fungi, which we named FungalBraid. In this thesis, we expanded the FungalBraid collection by incorporating 27 new DNA parts, including different selection markers and several constitutive and inducible promoters, all of which were functionally characterized in Penicillium digitatum and P. chrysogenum. Furthermore, we successfully expressed the GB_SynP promoters developed for plants in P. digitatum, in combination with the non-integrative pAMA18-derived vector for the expression of a dCas9-based activator. Although further optimization of GB_SynP in filamentous fungi is required, as expression levels were lower than those previously observed in plants, this and the other tools available in the FungalBraid collection can be effectively employed in the future for the development of fungal biofactories that produce insect pheromones and other high value biomolecules. / Este trabajo ha sido financiado mediante la Ayuda para la Formación de Profesorado Universitario FPU18/02019 (Ministerio de Educación, Cultura y Deporte), así como por el proyecto europeo SUSPHIRE (PCI2018-092893, Era- CoBiotech), y los proyectos de Plan Nacional I+D PID2019-108203RB-100 y PID2021-125858OB-100 (Ministerio de Ciencia e Innovación). / Moreno Giménez, E. (2023). Synthetic biology tools for production of insect pheromones in plants and filamentous fungi [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/201180

in English β-N-Acetylhexosaminidase (EC 3.2.1.52) is exoglycosidase, which exhibits the unique properties in the filamentous fungi. Enzyme from these organisms are dimeric, inducible and secreted extracelluary. It is expresed as preproprotein, consists of a signal sequence, a large propeptid and a catalytic subunit. Although the enzyme is widely distributed, its structure differs in varies organisms. Bacteria have only monomeric hexosaminidase. Human enzymes are dimeric as well as fungal, but only hexosaminidase from filamentous fungi have the catalytic subunit noncovalently associated with the propeptide. Propeptide is a essential for the enzyme activity. It exists a homologues model of the catalytic subunit of β-N-acetylhexosaminidase from Penicillium oxalicum, but the structure of the propeptide has not yet been solved. The first part of this diploma thesis deals with the optimization of production and purification conditions. The second part deals with structural studies of β-N-acetylhexosaminidases from the filamentous fungi Penicillium oxalicum CCF 3438. These studies were carried out using chemical cross-linking and high resolution mass spectrometry. The combination of these methods revealed region of the noncovalent interaction of the catalytic subunit with the propeptide.