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Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-01-04T16:33:42Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) jorge_andres_rueda_ardila.pdf: 2023531 bytes, checksum: f404efb27a2e6ec9624c8477f8ce4680 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-01-17T18:47:58Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) jorge_andres_rueda_ardila.pdf: 2023531 bytes, checksum: f404efb27a2e6ec9624c8477f8ce4680 (MD5) / Made available in DSpace on 2018-01-17T18:47:58Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) jorge_andres_rueda_ardila.pdf: 2023531 bytes, checksum: f404efb27a2e6ec9624c8477f8ce4680 (MD5) Previous issue date: 2017 / A enzima L-asparaginase ? atualmente utilizada na ind?stria de alimentos e na ind?stria farmac?utica devido ? facilidade de catalisar a rea??o de hidr?lise da L-asparagina em aspartato e am?nia. Esta propriedade tem aplica??o na ind?stria dos alimentos, pois evita a produ??o de compostos carcinog?nicos como as acrilamidas. Por outro lado, na ind?stria farmac?utica, esta rea??o enzim?tica det?m o crescimento de c?lulas leuc?micas devido ? falta de L-asparagina que estas c?lulas devem afrontar. Conforme as c?lulas leuc?micas t?m pouca ou nenhuma asparagina sintetase, as rotas metab?licas dependem exclusivamente da absor??o desse amino?cido do meio fisiol?gico. V?rias pesquisas foram feitas desde que a L-asparaginase mostrou a habilidade de reduzir alguns c?nceres na d?cada de 50. Estas pesquisas incluem a triagem de micro-organismos produtores, a otimiza??o de meios de cultura para melhorar a produ??o e a procura de uma metodologia que consiga purificar a enzima a partir do estrato bruto. Tais pesquisas se intensificaram recentemente no Brasil devido a uma crise de abastecimento gerada pela interrup??o da importa??o pelo fornecedor. A L-asparaginase ? um princ?pio ativo de alta demanda para tratar a leucemia linfobl?stica aguda e por isso deve ser produzida no Brasil. Este estudo foi realizado utilizando a linhagem Penicillium sp. T8.3 e teve como objetivo aprimorar a produ??o da enzima ajustando as condi??es de cultivo, usando glicerol e L-asparagina (como fontes de carbono e nitrog?nio, respectivamente) e pH como as tr?s vari?veis de entrada. Os experimentos foram desenvolvidos aplicando ferramentas da estat?stica multivari?vel como planejamento fatorial, desenho composto central para gerar um modelo matem?tico emp?rico. Foram analisadas a concentra??o de am?nio e a atividade enzim?tica produzida nos bioprocessos. A atividade enzim?tica foi determinada em rea??o conduzida a 37 ?C e pH 7,0, condi??es semelhantes ? do meio fisiol?gico. Todos os dados estat?sticos foram gerados com o programa Statistica 7.0 ?. Foi produzida uma atividade m?xima de 12,7 U por bioprocesso estacion?rio conduzido em meio ajustado com 15,5 g.L-1 de glicerol, 5,6 g.L-1 de L-asparagina e pH 4,8. Desse modo, o ajuste das condi??es de cultivo permitiu elevar a produ??o em mais de 30 vezes e alcan?ar uma atividade enzim?tica superior ? maioria dos relatos da literatura que tratam da produ??o da enzima f?ngica. O modelo estat?stico previu a produ??o enzim?tica com 77% de acerto, mostrando sua validade experimental e o potencial da linhagem T8.3 para a produ??o de L-asparaginase eucarionte. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The enzyme L-asparaginase is nowadays used in both pharmaceutics and food industry because of its ability to catalyze the reaction of hydrolysis of L-asparaginase into ammonia and aspartate. This feature is useful in the food industry because it hinders the formation of carcinogenic compounds such as acrylamide. On the other hand, in the pharmaceutical industry, this enzymatic reaction prevents some leukemic cells from growing due to asparagine depletion. Since these cells have low levels or no asparagine synthase, their metabolic routes depend on amino acid absorption from physiological medium. Several researches have been carried out since L-asparaginase showed to reduce some cancers in the 50?s. These researches include the screening of producing-microorganisms, optimization of the culture media to increase enzyme production, and development of methodology to purify the enzyme from crude extracts. Such researches have recently been intensified in Brazil due to a supply crisis resulting from interruption of the importer activities. L-asparaginase is a pharmaceutical of high demand to treat acute lymphoblastic leukemia, and therefore, it must be produced by Brazil. This study was carried out with the strain Penicillium sp. T8.3, and aimed to improve enzyme production by adjusting culture conditions, and by evaluating glycerol and L-asparagine ? as carbon and nitrogen sources, respectively ? and pH as input variables. The experiments were developed by using multivariable statistic tools such as factorial planning and central composite design to generate an empirical mathematical model. It was analyzed the ammonium concentration and the enzymatic activity produced in the bioprocesses. L-asparaginase activity was determined in reactions conduced at 37 ?C and at pH 7.0, similar to the physiological conditions. All statistical data were obtained with the software Statistica 7.0 ?. A maximum activity of 12.7 U was produced by stationary bioprocess in media adjusted with 15.5 g.L-1 glycerol, 5.6 g.L-1 L-asparagine, and pH 4,8. Thus, adjustment of culture conditions allowed to increase production by 30 times, and to reach an enzyme activity higher than those reported by most of the literature that deals with production of the fungal enzyme. The statistical model predicted enzyme production with 77% of accuracy, showing its experimental validity and the potential of strain T8.3 to produce the eukaryote L-asparaginase. / La enzima L-asparaginasa es usada actualmente en la industria de alimentos y en la industria farmac?utica debido a su facilidad para catalizar la reacci?n de degradaci?n de L-asparagina en amoniaco y aspartato. Esta caracter?stica es ?til en la industria de alimentos puesto a que evita la producci?n de compuestos cancer?genos como la acrilamida. Por otro lado, en la industria farmac?utica, esta reacci?n detiene el crecimiento de c?lulas leuc?micas debido al desabastecimiento de L-asparagina al que las c?lulas se enfrentan. Ya que estas c?lulas tienen poca o ninguna asparagina sintetasa, sus rutas metab?licas dependen exclusivamente de la absorci?n de amino?cidos desde el medio fisiol?gico. Varias investigaciones se han llevado a cabo desde que la L-asparaginasa mostr? su capacidad para reducir algunos c?nceres en los a?os 50. Estas investigaciones incluyen la clasificaci?n de microorganismos como productores de esta enzima, la mejora de los medios de cultivo para optimizar la producci?n, y la b?squeda de una metodolog?a para purificarla desde el extracto celular. Estos temas de investigaci?n han ganado inter?s en Brasil debido a que el proveedor de este f?rmaco detuvo sus servicios. La L-asparaginasa presenta una alta demanda para tratar la leucemia linfobl?stica aguda y por lo tanto debe ahora producirse en Brasil. Este estudio se llev? a cabo utilizando una cepa Penicillium sp. T8.3 cuyo objetivo fue optimizar la producci?n de la enzima mediante ajustes en las condiciones de cultivo usando glicerol y L-asparagina (como fuentes de carbono y de nitr?geno, respectivamente) y pH como las tres variables de entrada. Los experimentos fueron desarrollados utilizando herramientas de estad?stica multivariable como un planeamiento factorial y un dise?o de compuesto central para obtener un modelo matem?tico emp?rico. Se analizaron la concentraci?n de amoniaco y la actividad enzim?tica en los procesos biotecnol?gicos. La actividad enzim?tica fue determinada en una reacci?n a 37 ?C y pH 7.0, condiciones similares al medio fisiol?gico. Todos los datos estad?sticos fueron obtenidos del programa Statistica 7.0 ?. Se obtuvo una actividad m?xima de 12.7 U en proceso biotecnol?gico estacionario en un medio ajustado con 15.5 g.L-1 de glicerol, 5.6 g.L-1 de L-asparagina y pH 4.8. Este ajuste de las condiciones de cultivo logr? aumentar la producci?n en m?s de 30 veces, bien como alcanzar una actividad enzim?tica superior a la mayor?a de los relatos de literatura que tratan de la producci?n de la enzima de hongos. El modelo estad?stico predijo la producci?n enzim?tica en 77% de acierto, mostrando su validez experimental y el potencial de la cepa T8.3 para la producci?n de la L-asparaginasa eucariota.

Submitted by Regina Correa (rehecorrea@gmail.com) on 2016-09-21T13:34:29Z No. of bitstreams: 1 DissLM.pdf: 2911132 bytes, checksum: 362b734e96e18d3619903ec85dba4bb5 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-23T18:32:59Z (GMT) No. of bitstreams: 1 DissLM.pdf: 2911132 bytes, checksum: 362b734e96e18d3619903ec85dba4bb5 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-23T18:33:06Z (GMT) No. of bitstreams: 1 DissLM.pdf: 2911132 bytes, checksum: 362b734e96e18d3619903ec85dba4bb5 (MD5) / Made available in DSpace on 2016-09-23T18:33:14Z (GMT). No. of bitstreams: 1 DissLM.pdf: 2911132 bytes, checksum: 362b734e96e18d3619903ec85dba4bb5 (MD5) Previous issue date: 2016-03-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The use of a simplified consolidated bioprocess for the conversion of biomass using enzymes produced in-house (integrated into the process of converting lignocellulosic biomass) may enable significant increases in the production of bioethanol, without the need for expansion of cultivated area. With this motivation, the present work had as objective the study of the steps of production of (hemi)cellulases by solid-state fermentation (SSF) and the use of whole fermentation medium (WFM) for saccharification of biomass and subsequent alcoholic fermentation within the context of a consolidated bioprocess for the 2G ethanol production. To this end, firstly, the selection of the cultivation conditions for the production of enzymes directly using the sugarcane bagasse pretreated by steam explosion (SEB) as SSF substrate was studied. Thus, different fungi were cultivated in isolation and in co-cultivation, including the study of the use of wheat bran and lactose as inducers in the production of enzymes. In order to optimize the enzymatic hydrolysis (EH) step with the WFM from SSF, it was evaluated the influence of the operating parameters and the use of soy protein, Tween 80, PEG 1500 and bovine serum albumin (BSA) as additives. Finally, the alcoholic fermentation step was performed for the selected condition for the overall validation of this bioprocess. The results obtained in the conditions which maximize the production of enzymes, using SEB as substrate, were: addition of lactose at 0.075 g/g of substrate and wheat bran in the mass ratio 1:1 relative to the substrate, which led to an increase of activity of 73% for beta-glucosidase, 67% for endoglucanase, and 72% for xylanase, compared to the control condition using SEB as the substrate, without the presence of lactose or wheat bran. Cultivations under SSF followed by the EH step with the WFM showed that the co-cultivation were, in most cases, better in the conversion of SEB into fermentable sugars. The co-cultivation that presented the best result when compared to the best-isolated cultivation of Aspergillus niger was the Trichoderma reesei with Aspergillus oryzae, which has promoted an increase of 47% in the glucose production. The evaluation of operating parameters (pH, temperature and stirring) during the enzymatic hydrolysis step with the WFM from SSF showed that the saccharification process performed under the condition of pH 4.8, 200 RPM and 50 °C presented the best conversion of lignocellulosic biomass. This operational condition is the same already used in the conventional process of enzymatic hydrolysis with commercial extract enzymes. The addition of additives (soy protein and Tween 80) improved the saccharification process, whereas the addition of soy protein (0.5% w/v) lead to a 50% increase in glucose release. The alcoholic fermentation carried out for validating the overall integration process at the best condition of cultivation /hydrolysis gave a yield of 83.5% of the theoretical yield and volumetric productivity of ethanol (QP) 4.77 g/L.h. These results show that all process steps can be performed sequentially in the same reactor, thus denoting the proposed consolidated bioprocess. / A utilização de um bioprocesso consolidado simplificado para a conversão de biomassa usando enzimas produzidas in-house (integrada ao processo de conversão da biomassa lignocelulósica) pode possibilitar incrementos significativos na produção de bioetanol, sem a necessidade de expansão da área cultivada. Com esta motivação, o presente trabalho teve como objetivo o estudo das etapas de produção de (hemi)celulases por fermentação em estado sólido (FES) e a utilização do meio fermentado integral (MFI) para sacarificação da biomassa e posterior fermentação alcoólica, dentro do contexto de um bioprocesso consolidado para a produção de etanol 2G. Para este fim, primeiramente, foi estudada a seleção das condições de cultivo para a produção de enzimas usando diretamente o bagaço de cana-de-açúcar pré-tratado por explosão a vapor (BEX) como substrato da FES. Assim, diferentes fungos foram cultivados de forma isolada e em cocultivo, incluindo o estudo de farelo de trigo e lactose como indutores na produção de enzimas. Para otimizar a etapa de hidrólise enzimática (HE) com o MFI da FES, avaliou-se a influência dos parâmetros operacionais e o uso de proteína de soja, Tween 80, PEG 1500 e albumina de soro bovino (BSA) como aditivos. Por fim, para a melhor condição foi realizada a etapa de fermentação alcoólica para a validação global deste bioprocesso. Os resultados obtidos para as condições que maximizam a produção de enzimas, utilizando o BEX como substrato, foram: inserção de lactose na concentração de 0,075 g/g de substrato e farelo de trigo na proporção 1:1 em massa em relação ao substrato, o que levou a um aumento de atividade de 73% para betaglicosidase, 67% para endoglucanase e 72% para xilanase, em relação à condição controle utilizando apenas BEX como substrato, sem a presença de lactose ou farelo. Os cultivos em FES seguidos pela etapa de HE com o MFI mostrou que os cocultivos foram, na maioria dos casos, melhores na conversão do BEX a açúcares fermentescíveis. O cocultivo que apresentou o melhor resultado quando comparado ao melhor cultivo isolado de Aspergillus niger foi o de Trichoderma reesei com Aspergillus oryzae, o qual promoveu um aumento de 47% na produção de glicose. A avaliação dos parâmetros operacionais (pH, temperatura e agitação) durante a etapa de hidrólise enzimática com o MFI da FES mostrou que o processo de sacarificação realizado sob a condição de pH 4,8, 200 rpm e 50 ºC apresentou a melhor conversão da biomassa lignocelulósica. Essa condição operacional é a mesma já empregada no processo convencional de HE com extrato comercial de enzimas. A utilização de aditivos (proteína de soja e Tween 80) melhorou o processo de sacarificação, sendo que a adição da proteína de soja (0,5%, m/v) promoveu um aumento de 50% na liberação de glicose. Assim, realizou-se a fermentação alcoólica para a validação do processo global de integração para a melhor condição do conjunto cultivo/hidrólise, obtendo-se um rendimento de etanol de 83,5% do rendimento teórico e uma produtividade volumétrica de etanol (QP) de 4,77 g/L.h. Esses resultados permitiram a validação do bioprocesso consolidado proposto, indicando que todas as etapas podem ser realizadas sequencialmente no mesmo reator.

La maîtrise du développement des moisissures retrouvées dans le contexte agro-alimentaire parmi les flores microbiennes d'altération ou technologiques répond à des enjeux économiques et sanitaires importants. Le développement des moisissures peut être affecté par des facteurs abiotiques comme la température, l'activité de l'eau (aw) ou la composition gazeuse. L'évaluation de l'effet de ces facteurs via des outils de mycologie prévisionnelle vise à prévoir l'altération fongique des aliments. Ce travail a pour objectif d'explorer l'impact des conditions environnementales sur la physiologie de moisissures d'intérêt pour l'industrie agro-alimentaire.L'effet de la température, de l'aw ajustée par du glycérol ou du chlorure de sodium (NaCl) du pH et de la composition gazeuse a été évalué sur la germination des spores et/ou la croissance radiale de cinq moisissures: Paecilomyces niveus, Mucor lanceolatus, Penicillium brevicompactum, Penicillium expansum et Penicillium roqueforti. Au niveau appliqué, ces travaux ont montré que l'effet du NaCl ou de la composition gazeuse peuvent être inclus dans une approche de mycologie prévisionnelle. Le choix des souches représentatives d'une espèce fongique et l'état physiologique des spores utilisées comme inoculum ont un impact significatif sur les modèles prédictifs.Au niveau fondamental, des marqueurs ont été recherchés pour évaluer l'effet des facteurs abiotiques sur la physiologie des spores. La température et l'aw ont un effet significatif sur l'état physiologique des spores et leur germination. La recherche de marqueurs moléculaire, contribuera aux connaissances de l'effet des facteurs abiotiques sur la physiologie des moisissures. / In the food processing industry, controlling the development of filamentous fungi encountered as spoilers or technological cultures address significant economic and sanitary issues. Fungal development in foods is mainly determined by abiotic factors including temperature, water activity (aw) or the headspace gas composition. The quantification of these respective effects through a predictive mycology approach aims at preventing fungal food spoilage. The present work aims at exploring the effect of environmental conditions on the physiology of filamentous fungi of interest in the food processing industry.The effect of temperature, aw (adjusted with glycerol of sodium chloride), pH and headspace gas composition was evaluated on conidial germination and/or radial growth of five fungi isolated from dairy products: Paecilomyces niveus, Mucor lanceolatus, Penicillium brevicompactum, Penicillium expansum and Penicillium roqueforti. The present work suggests that the specific effects of sodium chloride or gas composition could be included in predictive mycology approaches. It was also demonstrated that the selection of strains representative of a fungal species and the physiological state of conidia utilized as inoculum have a significant effect on the final predictive models.At the fundamental level, markers were investigated to study the effect of abitoic factors on the physiological state of spores. The temperature and aw significantly affected the physiological state of spores and their germination kinetics. The investigation of markers at the molecular level could provide better knowledges on the effect of abiotic factors on the physiology of filamentous fungi.

L’objectif de cette thèse est de développer une nouvelle souche de champignon filamenteux pour la production d’acide lactique. Pour répondre à cet objectif, nous avons tout d’abord d’exploré la biodiversité fongique à la recherche de champignons filamenteux capables de produire de l’acide lactique ou présentant de bonnes prédispositions pour la production d’acides organiques sans neutralisation du pH. Grâce à ce criblage, une souche sauvage d’Aspergillus brasiliensis a été sélectionnée et utilisée pour construire, grâce à l’ingénieure métabolique, les premières souches d’Aspergillus capables de produire de l’acide lactique à pH acide. Ces souches ont ensuite été caractérisées et les conditions de culture en fioles et fermenteurs volumes ont été étudiées. Cette étude des conditions de culture donne des résultats prometteurs et dévoile de nombreuses voies possibles pour continuer à améliorer la production d’acide lactique par ces organismes. / The objective of this thesis is to develop a new filamentous fungal strain for the production of lactic acid. To meet this goal, we first explored the fungal biodiversity in order to find filamentous fungi able to produce lactic acid or having good predispositions for the production of organic acids without pH neutralization. Through this screening, a wild type strain of Aspergillus brasiliensis was selected and used to construct, using the metabolic engineer, new strains capable of producing lactic acid at an acidic pH. These strains were then characterized and culture conditions in flasks and bioreactors were studied. The study of culture conditions shows promising results and reveals many possible ways to further improve the production of lactic acid by these organisms.

Pour se défendre des effets délétères des éléments transposables, les pezizomycotina ont développé un système de défense génétique et épigénétique appelé « Repeat Induced Point Mutation » (RIP). Chez N. crassa, le RIP survient dans la cellule dicaryotique avant la caryogamie et conduit à la méthylation de novo des cytosines (5mC) inclues dans les séquences répétées de chacun des noyaux parentaux haploïdes. De plus, certaines de ces cytosines sont la cible d’un processus de mutation qui les transforme en thymines. Cette étape est suivie par la mise en place locale de l’hétérochromatine constitutive permettant une répression transcriptionnelle durable des séquences cibles du RIP au cours des divisions nucléaires. L’acteur majeur du RIP correspond à une cytosine méthyltransférase putative appelée RID (RIP Defective). Bien que son génome ne montre pas une quantité significative de 5mC, l’inactivation de PaRid chez Podospora anserina aboutit à un blocage du développement sexué survenant après la fécondation. Dans ce contexte, nous avons voulu déterminer si la fonction de PaRid dans le développement sexué consiste à éteindre l’expression de gènes cibles via l’installation de foyers d’hétérochromatine constitutive aux loci concernés. Pour ce faire, nous avons identifié les gènes PaKmt1 et PaHP1, codant respectivement l’histone méthyltransférase PaKmt1 (l’homologue de SU(VAR)39 qui catalyse la tri-méthylation du résidu H3K9 (H3K9me3) et PaHP1 (l’homologue de Heterochromatin Protein 1 qui se lie à H3K9me3). Les deux protéines interviennent dans une même voie de régulation qui aboutit à la mise en place de l’hétérochromatine constitutive. Par opposition, PaKmt6, homologue de l’histone méthyltransférase E(Z), correspond à la sous-unité catalytique du complexe PRC2 qui catalyse la marque H3K27me3 pour permettre l’établissement de l’hétérochromatine facultative. Nos résultats ont montré que l’absence de PaKmt1 et PaHP1 ne provoquent que des défauts mineurs. A l’inverse, l’inactivation du gène PaKmt6 conduit à un ensemble de défauts sévères : croissance végétative altérée, surproduction des gamètes mâles, malformations critiques des fructifications, production très réduite d’ascospores dont la germination est pour partie déficiente. Une étude d’épistasie a montré que les protéines PaRid et PaKmt6 interviennent chacune dans deux voies développementales distinctes. Par ailleurs, nous avons établi par immuno-précipitation de la chromatine les profils de distribution à l’échelle du génome entier des modifications H3K9me3, H3K27me3 et H3K4me3. Caractéristique rare, la marque H3K9me3 colocalise avec H3K27me3 sur des gènes transcriptionnellement réprimés et les séquences répétées ripées. Conformément à sa fonction canonique, H3K4me3 est présente en 5’ des gènes transcrits et est exclue des domaines H3K9me3 et H3K27me3. Comme attendue, PaKmt6 est essentielle à la mise en place de la marque H3K27me3, mais, de manière surprenante, elle serait aussi impliquée dans le dépôt et/ou le maintien d’une partie des marques H3K9me3, dévoilant ainsi une voie de méthylation non canonique de ces résidus. / In pezizomycotina, transposable elements are targeted by a genome defense system named Repeat Induced Point Mutation (RIP). First described in Neurospora crassa, RIP occurs before karyogamy in each parental haploid nucleus of the dikaryotic cells and results, within the repeats, in de novo methylation of cytosine (5mC) and mutations, mainly C to T transitions. This initial step triggers local assembly of constitutive heterochromatin, which allows transcriptional gene silencing. RID (RIP Defective) is a putative cytosine methyltransferase essential for RIP. Despite the absence of 5mC in its genome, PaRid inactivation in Podospora anserina results in sexual reproduction arrest right after fertilization. In this context, we asked whether PaRid is required to silence expression of some of sexual development-specific genes by nucleation of constitutive heterochromatin. To this end, we identified PaKmt1 and PaHp1 genes encoding respectively the histone methyltransferase PaKmt1 (SU(VAR)39 homologue protein) and the heterochromatin protein 1 (PaHP1). To assemble constitutive heterochromatin, PaKmt1 catalyses tri-methylation of H3K9 (H3K9me3), latter on bound by PaHP1. By contrast, the E(Z) histone methyltransferase homologue PaKmt6, as part of the PRC2 complex, catalyses tri-methylation of H3K27 (H3K27me3) to form facultative heterochromatin. Our results showed that loss of either PaKmt1 or PaHP1 does not cause major defects. Conversely, PaKmt6 gene inactivation results in severe defects: altered mycelium and vegetative growth rate, overproduction of male gamete, development of crippled fructifications, reduced production ascospores, part of which does not germinate. Furthermore, epistatic study showed that PaRid and PaKmt6 likely act in two different developmental pathways, with respect to sexual reproduction. In addition, using chromatin immuno-precipitation we characterized H3K9me3, H3K27me3 and H3K4me3 genome-wide distribution patterns. We observed an uncommon overlapping distribution between H3K9me3 and H3K27me3 on transcriptionally repressed genes and RIP target repeats. As expected, H3K4me3 localizes in 5’ of the transcribed genes and is excluded from the H3K9me3 and H3K27me3 domains. As expected, PaKmt6 is essential for H3K27me3 modification, but surprisingly, could also be responsible for some of the H3K9me3 setting up or maintenance.

[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

Die Entwicklung einer mukosal verabreichbaren, effektiven DNA-Vakzine gegen Infektionskrankheiten oder Tumorerkrankungen auf der Basis invasiver attenuierter Bakterien ist eine vielversprechende Alternative zu bisherigen parenteralen Strategien der genetischen Vakzinierung. Innerhalb dieser Arbeit wurden Salmonellen-Impfstämme für die orale Übertragung eines eukaryontischen Expressionsplasmids mit dem kleinen Oberflächenantigen des Hepatitis-B-Virus (HBsAg) als Modellantigen optimiert. Die kontinuierliche Sezernierung von Plasmiden als filamentöse Phagenpartikel wurde als ein erster Ansatz getestet, um mit lebenden Bakterien eine DNA-Vakzine innerhalb infizierter Zellen freizusetzen. Die Salmonellen-vermittelte Phagensekretion in der Wirtszelle ist jedoch nicht effizient genug, die Expression des Transgens zu vermitteln. Alternativ wurde ein Ansatz gewählt, durch eine spontan induzierte Lyse der Impfbakterien, Plasmid-DNA in die Wirtszelle zu übertragen. Dazu wurde ein neuartiges bakterielles Autolysesystem etabliert, basierend auf einem Zwei-Phasen-Expressionssystem und von Bakteriophagen abgeleiteten Lysedeterminanten. Dieses System ermöglicht erstmals die kontinuierliche Freisetzung von Plasmid-DNA und Proteinen aus einzelnen, lysierenden Salmonellen innerhalb einer sonst gesunden bakteriellen Gesamtpopulation. Innerhalb infizierter COS7-Zellen führt die Freisetzung des porenformierenden Proteins Listeriolysin O durch autolytische Salmonellen zur Zerstörung der Vakuole, in der die Impfbakterien replizieren, und erleichtert somit den Transfer der Plasmid-DNA aus den Bakterien in das Zytoplasma der Wirtszelle. Die Lysedeterminante und die eukaryontische Expressionskassette für HBsAg wurden auf einem Plasmid kombiniert, sowie eine Kassette zur konstitutiven Expression des Histon-ähnlichen Proteins aus Thermotoga maritima (TmHU) in ein solches Konstrukt integriert. TmHU stabilisiert die Plasmiderhaltung unter nicht selektiven Bedingungen und besitzt das Potential, die Effizienz der DNA-Translokation innerhalb der Wirtszelle zu erhöhen. Durch die orale Gabe optimierter autolytischer Impfbakterien konnte eine potente HBsAg-spezifische Antikörperantwort sowie eine zytotoxische zelluläre Antwort induziert werden. Bereits die einmalige Gabe der autolytischen Bakterien induzierte eine höhere antigenspezifische Antikörperantwort, als die herkömmliche intramuskuläre DNA-Vakzine. Das im Rahmen dieser Arbeit entwickelte Konzept autolytischer Salmonellen stellt also eine neuartige, effiziente Strategie für den mukosalen DNA-Transfer dar. Die Übertragung des Konzeptes der Autolyse auf andere bakterielle Trägersysteme ist möglich und kann zur Erweiterung des Anwendungspektrums bakterieller Vektoren beitragen. / The development of an effective mucosal DNA vaccine against infectious diseases or tumors based on invasive attenuated bacteria is a very promising alternative to common parenteral routes of genetic vaccination. This work aimed at the optimization of Salmonella vaccine strains for the oral delivery of an eukaryotic expression plasmid encoding the small Hepatitis B Virus surface antigen (HBsAg), here used as model antigen. The continuous secretion of plasmids as filamentous phage particles was first tested as a mean for the delivery of the DNA vaccine by living bacteria inside infected host cells. However, Salmonella-mediated phage secretion inside cells did not suffice for the induction of transgene expression. As alternative approach, inducible spontanous lysis of bacteria was used to mediate the release of plasmid DNA into host cells. For this purpose a novel bacterial autolytic system was established on the basis of a two-phase expression system and lysis determinants derived from bacteriophages. This system allows for the first time the continuous release of plasmid DNA and proteins from only few lysing Salmonella within an otherwise healthy bacterial population. Inside COS7 cells the release of the pore-forming protein listeriolysin O by autolytic Salmonella mediates the destruction of the Salmonella-harbouring vacuole, thereby facilitating the transfer of plasmid DNA from bacteria into the host cell cytoplasm. The lysis determinant was combined with the eukaryotic expression cassette for HBsAg on one plasmid. In addition, a cassette for the constitutive expression of TmHU, a histon-like protein derived from Thermotoga maritima, was integrated in such vector. TmHU stabilizes the plasmid propagation in the absence of selective pressure and has the potential to increase the efficiency of plasmid translocation inside the host cell. The oral administration of the optimized autolytic bacteria stimulated a potent HBsAg-specific antibody response as well as a cytotoxic cellular response. Already a single inoculation of the oral vaccine induced a higher specific antibody response than the conventional intramuscular DNA vaccine. Therefore the concept of autolytic Salmonella carrier strains developed in this work constitutes a novel efficient strategy for mucosal DNA delivery. The transfer of this concept to other bacterial carriers is possible and may widen the application field for bacterial vectors.

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