Dissertations / Theses on the topic 'Specialized metabolite'

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
Fireflies (Lampyridae) and certain other families of beetles including the American railroad worms (Phengodidae), Asian starworms (Rhagophthalmidae), and American click-beetles (Elateridae), produce light in a process known as bioluminescence. The bioluminescent systems of beetles, natively used for the purposes of mating communication and/or an aposematic warning signal, are now well understood and have been widely applied in biotechnology and biomedical research. There have been considerable advancements in the engineering of the luciferin substrate, and the luciferase enzyme, for beneficial characteristics such as altered emission wavelength, improved thermostability, and improved catalytic parameters, but despite this substantial effort focused on the biotechnological applications of beetle bioluminescence, major questions remain regarding its natural biochemistry and evolutionary origins.
Four major questions that were unanswered at the beginning of this PhD study were: (1) Do fireflies possess a storage form of their luciferin? (2) What is the evolutionary relationship of bioluminescence amongst the bioluminescent beetles families, and has this trait independently evolved multiple times? (3) How is firefly luciferin biosynthesized? And (4) Are there accessory genes from the bioluminescent beetles which act in bioluminescent metabolism, and might these genes be useful for biotechnological applications? Here I describe the discovery and characterization of the presumed storage form of luciferin in fireflies, sulfoluciferin, and the enzyme which produces it, luciferin-sulfotransferase.
Furthermore, I describe the sequencing, assembly, and characterization of the genome of the North American "Big Dipper" firefly Photinus pyralis, along with the Japanese "heike" firefly Aquatica lateralis genome, and the genome of the Puerto Rican bioluminescent click beetle or "cucubano" Ignelater luminosus. Genomic comparisons amongst these three species support the hypothesis that firefly and click beetle luciferase evolved independently, suggesting an independent evolutionary origin of the bioluminescent systems between these fireflies and click beetles. I also describe stable isotope tracing experiments in live fireflies, establishing that adult and larval fireflies likely do not de novo biosynthesize firefly luciferin, and may instead rely on a "recycling" pathway to re-synthesize luciferin from the luminescence product oxyluciferin. Lastly, I discuss the future directions resulting from this thesis, and the yet unanswered questions.
by Timothy Robert Fallon.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Specialized metabolism (secondary metabolism) in glandular trichomes of sweet basil (Ocimum basilicum L.) and accumulation of specialized metabolites (secondary metabolites) in rhizomes of turmeric (Curcuma longa L.) was investigated using proteomic and metabolomic approaches, respectively. In an effort to further clarify the regulation of metabolism in the glandular trichomes of sweet basil, we utilized a proteomics-based approach that applied MudPIT (multidimensional protein identification technology) and GeLC-MS/MS (gel enhanced LC-MS/MS) to protein samples from isolated trichomes of four different basil lines: MC, SW, SD, and EMX-1. Phosphorylation, ubiquitination and methylation of proteins in these samples were detected using X!tandem. Significant differences in distribution of the 755 non-redundant protein entries demonstrated that the proteomes of the glandular trichomes of the four basil lines were quite distinct. Correspondence between proteomic, EST, and metabolic profiling data demonstrated that both transcriptional regulation and post-transcriptional regulation contribute to the chemical diversity. One very interesting finding was that precursors for different classes of terpenoids, including mono- and sesquiterpenoids, appear to be almost exclusively supplied by the MEP (2-C-methyl-D-erythritol 4- phosphate) pathway, but not the mevolonate pathway, in basil glandular trichomes. Our results suggest that carbon flow can be readily redirected between the phenylpropanoid and terpenoid pathways in this specific cell type. To investigate the impact of genetic, developmental and environmental factors on the accumulation of phytochemicals in rhizomes of turmeric, we performed metabolomic analysis in a 2x2x4 full factorial design experiment using GC-MS, LC-MS, and LC-PDA. Our results showed that growth stage had the largest effect on levels of the three major curcuminoids. Co-regulated metabolite modules were detected, which provided valuable information for identification of phytochemicals and investigation of their biosynthesis. Based on LC-MS/MS data, 4 new diarylheptanoids were tentatively identified in turmeric rhizomes using Tandem-MSASC, a home-made software tool that automatically recognizes spectra of unknown compounds using three approaches. Based on our metabolomic results, we proposed two new strategies, “metabolomics-guided discovery” and “correlation bioassay”, to identify bioactive constituents from plant extracts based on information provided by metabolomic investigation.

Doctor en Ciencias de la Ingeniería, Mención Ingeniería Química y Biotecnología
Streptomyces leeuwenhoekii C34 es una nueva cepa que fue aislada desde la laguna Chaxa ubicada en el Desierto de Atacama, Chile. Esta cepa produce metabolitos especializados con actividad contra Staph. aureus resistente a meticilina (MRSA): chaxamicinas y chaxalactinas. La secuencia genómica de S. leeuwenhoekii C34 se obtuvo mediante las tecnologías de Illumina Miseq y PACbio RS II SMRT. El genoma se utilizó para identificar clústers de genes biosintéticos (BGCs) que codifican para metabolitos especializados a través de minería de genomas, y para desarrollar un modelo a escala genómica (GSM) para estudiar las rutas de biosíntesis de producción de metabolitos especializados. Se encontraron 34 BGCs en el genoma de S. leeuwenhoekii C34, más un BGC ubicado en el plásmido pSLE2. Se encontró tres BGCs para lazo-péptidos. Específicamente, se identificó el producto del BGC del lazo-péptido 3 en el sobrenadante de S. leeuwenhoekii C34 cultivado en medio TSB/YEME y se expresó exitosamente en el huésped heterólogo S. coelicolor M1152. Se confirmó que este lazo-péptido era el mismo que la chaxapeptina, recientemente descrita para S. leeuwenhoekii C58. Por otra parte, se identificó un BGC de 64 kb (locus 1083651 a 1147687) que codifica para un híbrido trans-AT PKS/NRPS. Es probable que el producto de este BGC sea un compuesto halogenado debido a la presencia de un gen, sle09470, que codifica para una enzima cloradora. Para estudiar este clúster de genes, se desarrollaron diferentes cepas derivadas de S. leeuwenhoekii. También, el BGC se clonó en huéspedes heterólogos: S. coelicolor M1152, M1154 and S. albus. A través de análisis de HPLC MS/MS y comparación de perfiles de metabolitos, se identificó un grupo de compuestos con patrón clorado, sin embargo se descartaron como posibles productos del BGC ya que además de encontrarse en las cepas de S. leeuwenhoekii también se encontraron en muestras de S. coelicolor M1152. Por otra parte, se detecto un metabolito con una señal de m/z 611.53 [M + H]+ solamente en las muestras de S. leeuwenhoekii M1614 ( chaxamycin BGC) y M1619 ( chaxamycin BGC; sle09560). Se requieren msá estudios para confirmar si los metabolitos expresados diferencialmente corresponden a un producto del híbrido transAT-PKS/NRPS BGC. Para construir el GSM de S. leeuwenhoekii C34 se desarrolló una interfaz basada en python, que permite: buscar genes de Streptomyces asociados a reacciones en la base de datos KEGG, realizar BLAST local contra S. leeuwenhoekii C34, comparar los dominios de proteínas, descargar información de los metabolitos, construir el GSM y realizar simulaciones usando COBRApy. Las rutas biosintéticas de chaxamicinas, chaxalactinas, desferrioxaminas, ectoina y el producto del híbrido transAT-PKS/NRPS BGC (híbrido PK-NP) se incluyeron en el modelo. El modelo, iVR1007, consiste de 1722 reacciones, 1463 metabolitos y 1007 genes, y se validó usando información experimental de crecimiento en diferentes fuentes de carbono, nitrógeno y fósforo, mostrando un 83.7 % de precisión. El modelo se usó para encontrar deleción y sobre-expresión de genes no intuitivas que predicen un aumento en la producción de precursores de chaxamicinas, chaxalactinas e híbrido PK-NP. Las modificaciones predichas podrán ser usadas para realizar ingeniería metabólica de S. leeuwenhoekii C34 para incrementar la producción de metabolitos especializados.

Dans cette étude, le palmier Astrocaryum sciophilum a été choisi comme modèle pour l'étude de ses endophytes foliaires. Du fait de sa longévité, nous avons cherché à mettre en évidence une communauté compétitive d’endophytes en fonction de l’âge de ses feuilles. Afin d’évaluer si les métabolites produits par ces endophytes pourraient être utilisés en santé humaine, les extraits de chaque endophyte ont été testés contre Staphylococcus aureus résistant à la méticilline (SARM) ainsi que pour leur activité quorum quenching (QQ). En parallèle, afin d’identifier un rôle écologique de protection de la plante par ces endophytes, des co-cultures ont été réalisées avec le phytopathogène Fusarium oxysporum. Plusieurs extraits de souches ont été sélectionnés afin d’isoler et d’identifier le ou les métabolites responsables des activités biologiques observées. Différents outils analytiques ont permis de guider le processus d’isolement (LC-MS/MS, réseaux moléculaires et imagerie par spectrométrie de masse). L’étude de la communauté d’endophytes isolée des feuilles âgées n’a pas mis en évidence un arsenal chimique plus compétitif. Toutefois, deux souches bactériennes du genre Luteibacter sp. ont montré un extrait actif sur SARM et de nombreux extraits de bactéries présentent une activité QQ. Par la suite, le métabolome secondaire du genre Colletotrichum a été étudié à l’aide des réseaux moléculaires et un champignon de la famille des Xylariaceae a été étudié pour son activité contre F. oxysporum. Dans le cadre de cette thèse, sept souches endophytes ont été étudiées chimiquement permettant l’isolement et l’identification de 42 molécules dont dix sont nouvelles
The palm Astrocaryum sciophilum is the host plant model chosen in this work. Indeed, due to the longevity of its leaves, we expected to highlight a competitive community of endophytes within the oldest leaves. Thus, 197 endophytes have been isolated and identified from different leaves of six palm specimens. In order to evaluate whether the compounds produced by these microorganisms could be used for the treatment of human disease, the ethyl acetate extracts of each endophyte were tested against methicillin-resistant Staphylococcus aureus (MRSA) as well as for a quorum quenching (QQ) activity. Simultaneously, co-culture were carried with the fungi Fusarium oxysporum in order to highlight endophytes providing plant protection against phytopathogens. We selected extracts in order to isolate and identify the bioactive metabolites. Various analytical tools have been used to improve the isolation process (LC-MS/MS, molecular networking or MS imaging).The study of the endophytic community isolated from older leaves did not show a more competitive chemical arsenal. However, two Luteibacter strains exhibited an ethyl acetate extract active against MRSA and several bacteria provide quorum quenching extracts. The metabolome of Colletotrichum genus was studied using molecular networking and a fungus from the Xylariaceae family was studied for its capacity to inhibit F. oxysporum’s growth. In our study, seven endophyte strains were chemically investigated leading to the isolation and identification of 42 molecules whose ten are new

Background: Specialized adult care of phenylketonuria (PKU) patients is of increasing importance. Adult outpatient clinics for inherited errors of metabolism can help to achieve this task, but experience is limited. Ten years after establishment of a coordinated transition process and specialised adult care for inherited metabolic diseases, adult PKU care was evaluated with respect to metabolic control, therapy satisfaction, life satisfaction, sociodemographic data, economical welfare as well as pregnancy outcome. Methods: All PKU patients transferred from paediatric to adult care between 2005 and 2015 were identified. A retrospective data analysis and a cross-sectional survey in a sub-cohort of 30 patients including a questionnaire for assessing quality of life (FLZm) were performed as a single-centre investigation at the metabolic department of the University Hospital Leipzig, Germany. For statistical analysis, Mann-Whitney-U-test, t-test for independent samples, ANOVA and chi square test were used as appropriate. Results: 96 PKU patients (56 females/40 males; median age 32 years, range 18–62) were included. In the last 3-year period, 81 % of the transferred patients still kept contact to the adult care centre. Metabolic control was stable over the evaluation period and dried blood phenylalanine concentrations mostly remained within the therapeutic range (median 673.0 μmol/l, range 213.0–1381.1). Sociodemographic data, economical welfare and life satisfaction data were comparable to data from the general population. However, differences could be revealed when splitting the cohort according to time of diagnosis and to management during childhood. 83 % of the PKU adults were satisfied with the transition process and current adult care. 25 completed pregnancies were supervised; three newborns, born after unplanned pregnancy, showed characteristic symptoms of maternal PKU syndrome. Conclusions: Continuous care for adult PKU patients in a specialized outpatient clinic is successful, leading to good to satisfactory metabolic control and social outcomes. Uninterrupted good metabolic treatment throughout childhood and adolescence positively influences educational, professional and economic success in later life. Further effort in specialized paediatric and adult metabolic care is needed to prevent loss of follow-up and to support the recommended life-long treatment and/or care.

Depuis plus de 80 ans, le métabolisme spécialisé nous fournit de nombreuses molécules utilisées en médecine, en particulier comme anti-infectieux. Aujourd’hui, avec l’augmentation mondiale de la résistance aux antimicrobiens, de nouveaux antibiotiques sont indispensables. Une des réponses à cette pénurie grave pourrait provenir de la biologie synthétique. Dans le domaine du métabolisme spécialisé, la biologie synthétique est utilisée en particulier pour la biosynthèse de métabolites non naturels. Parmi les métabolites spécialisés, les peptides non ribosomiques constituent une cible attrayante, car ils nous ont déjà fourni des molécules à haute valeur clinique (ex. les antibiotiques vancomycine et daptomycine). De plus, la plupart sont synthétisés par des enzymes multimodulaires appelées synthétases de peptides non ribosomiques (NRPS), et sont diversifiés davantage par des enzymes de décoration. Ainsi, ces voies de biosynthèse se prêtent particulièrement à la biosynthèse combinatoire, consistant à combiner des gènes de biosynthèse provenant de divers groupes de gènes ou, dans le cas des NRPS, à combiner des modules ou domaines pour créer de nouvelles enzymes. Cependant, si plusieurs études ont établi la faisabilité de telles approches, de nombreux obstacles subsistent avant que les approches combinatoires de biosynthèse soient totalement efficaces pour la synthèse de nouveaux métabolites. Les travaux présentés ici s’inscrivent dans le cadre d’un projet visant à comprendre les facteurs limitant les approches de biosynthèse combinatoire basées sur les NRPS, en utilisant une approche de biologie synthétique. Nous avons choisi de travailler avec les NRPS responsables de la biosynthèse des pyrrolamides. En effet, ces NRPS sont constitués uniquement de modules et de domaines autonomes, et donc particulièrement adaptés aux manipulations génétiques et biochimiques. La caractérisation du groupe de gènes de biosynthèse du pyrrolamide anthelvencine constitue la première partie de cette thèse et nous a fourni de nouveaux gènes pour notre étude. La deuxième partie a consisté à construire de vecteurs intégratifs modulaires, outils essentiels pour la construction et l’assemblage de cassettes génétiques. La dernière partie présente la reconstruction du groupe de gènes du pyrrolamide congocidine, basée sur la construction et l’assemblage de cassettes de gènes synthétiques. Dans l’ensemble, ces travaux ouvrent la voie à de futures expériences de biosynthèse combinatoire, expériences qui devraient contribuer à une meilleure compréhension du fonctionnement précis des NRPS
For more than 80 years, specialized metabolism has provided us with many molecules used in medicine, especially as anti-infectives. Yet today, with the rise of antimicrobial resistance worldwide, new antibiotics are crucially needed. One of the answers to this serious shortage could arise from synthetic biology. In the field of specialized metabolism, synthetic biology is used in particular to biosynthesize unnatural metabolites. Among specialized metabolites, non-ribosomal peptides constitute an attractive target as they have already provided us with clinically valuable molecules (e.g. the vancomycin and daptomycin antibiotics). In addition, most are synthesized by multimodular enzymes called non-ribosomal peptide synthetases (NRPS) and further diversified by tailoring enzymes. Thus, such biosynthetic pathways are particularly amenable to combinatorial biosynthesis, which consists in combining biosynthetic genes coming from various gene clusters or, in the case of NRPSs, combining modules or domains to create a new enzyme. Yet, if several studies have established the feasibility of such approaches, many obstacles remain before combinatorial biosynthesis approaches are fully effective for the synthesis of new metabolites. The work presented here is part of a project aiming at understanding the limiting factors impeding NRPS-based combinatorial biosynthesis approaches, using a synthetic biology approach. We chose to work with the NRPSs involved in the biosynthesis of pyrrolamides. Indeed, these NRPS are solely constituted of stand-alone modules and domains, and thus, particularly amenable to genetic and biochemical manipulations. The characterization of the biosynthetic gene cluster of the pyrrolamide anthelvencin constitutes the first part of this thesis, and provided us with new genes for our study. The second part involved the construction of modular integrative vectors, essential tools for the construction and assembly of gene cassettes. The final part presents the successful refactoring of the congocidine pyrrolamide gene cluster, based on the construction and assembly of synthetic gene cassettes. Altogether, this work paves the way for future combinatorial biosynthesis experiments that should help deciphering the detailed functioning of NRPSs

This study aimed to improve essential oil composition by modifying terpene production in Lavandula x intermedia cv Grosso via mutagenesis to more closely resemble the oil of the commercially valuable essential oil from L. angustifolia or the medicinally active essential oil from L. latifolia. Additionally this study aimed to identify genes that control essential oil production in lavenders, and to determine the effect of essential oil composition on biological activity, specifically insecticidal and insect repellent properties. This study resulted in an improved method for the efficient regeneration of Grosso lavender, and applied this method to generate ten unique mutants. The transcriptomes of some mutants were sequenced, and thirty seven differentially expressed transcripts were identified as being involved in the biosynthesis and production of essential oil terpenes. The transcript expression results were confirmed by real-time quantitative polymerase chain reaction analysis. The lavender essential oil showing greatest biological activity against an invasive pest, spotted wing drosophila, was identified as Lavandula latifolia cv Medikus and the active constituents were identified through fumigation and spray toxicity assays as the monoterpenes 1,8-cineole and linalool. These oils showed strong fumigation and contact toxicity. In all, this thesis presents the generation, screening and analysis of unique L. x intermedia essential oil mutants, which represent both potential new commercial cultivars and model organisms for the investigation of the regulation and biosynthesis of essential oil terpenes.
Irving K. Barber School of Arts and Sciences (Okanagan)
Biology, Department of (Okanagan)
Graduate

Plants produce a large number of specialized or secondary compounds that aid in their reproduction and protection against biotic and abiotic stress. In this work I investigated the metabolism and function of terpenes, the largest class of specialized metabolites, in switchgrass and carrot. Switchgrass (Panicum virgatum L.), a perennial C4 grass of the Tallgrass Prairie, represents an important species in natural and anthropogenic grasslands of North America. Its natural resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. I have investigated the metabolism of terpenes in switchgrass leaves and roots in response to herbivory or defense hormone treatments and the application of drought. With a focus on volatile terpene metabolites, I functionally characterized over thirty genes (terpene synthases, TPSs), of which one third could be correlated with the production and release of volatile monoterpenes and sesquiterpenes that likely function in direct chemical defense or in the attraction of insect predators or parasitoids. Drought stress application caused switchgrass roots to accumulate a larger amount of oxygenated terpenes and presumably non-volatile terpenes, the function of which in direct or indirect drought stress protection requires further investigation. I also examined the metabolic dynamics and role of the monoterpene borneol, which accumulates at high concentrations in the roots of switchgrass and to a lower extent in the roots of the close relative Setaria viridis, in root microbe interactions. Although we demonstrated a successful RNAi based knock down of the borneol terpene synthase TPS04, we found no immediate evidence that borneol significantly modifies bacterial communities in the root. Further studies on Setaria and equivalent RNAi lines in switchgrass will provide more detailed and needed insight to decipher the role of monoterpene accumulation in grasses interactions with mutualists, pathogens, and pests. In an applied project, I investigated terpene specialized metabolism in carrot (Daucus carota L.) to identify genetic determinants of carrot aroma and flavor. To determine central enzymes which contribute to the terpene component of carrot volatile blends, we first analyzed tissue specific expression patterns of carrot terpene synthase genes (TPS) in the genomic model carrot (cv. DH1) and in roots of four aromatically unique colored carrot genotypes (orange-4943B, red-R6637, yellow-Y9244A and purple-P7262). We selected nineteen key biosynthetic enzymes involved in terpene formation and compared in vitro products from recombinant proteins with native volatile profiles obtained from DH1 and colored carrot genotypes. We biochemically characterized several highly expressed TPSs with direct correlations to major compounds of carrot flavor and aroma including germacrene-D (DcTPS11), (DcTPS30) and -terpinolene (DcTPS03). Random forest analysis of colored carrot volatiles revealed that nine terpene compounds are sufficient for distinguishing the flavor and aroma of raw colored carrots. Interestingly, accumulation of specific terpene compounds rather than chemical diversity is responsible for differences in sensory quality traits in colored genotypes. As accumulations of specific terpene compounds can contribute to the undesired flavor in carrot, our report provides a detailed roadmap for future breeding efforts to enhance carrot flavor and aroma.
Doctor of Philosophy

Orientador: Carmen Silvia Fernandes Boaro
Resumo: Espécies vegetais são capazes de produzir diversidade de substâncias, que desempenham funções importantes para sua sobrevivência e adaptação ao ecossistema. O metabolismo primário, é essencial para o crescimento, desenvolvimento, maturação e reprodução de qualquer espécie. O metabolismo especializado, dependente do primário, é responsável por originar o óleo essencial, que são misturas de metabólitos especializados voláteis, representados principalmente por monoterpenos e sesquiterpenos. Cada espécie vegetal produz um óleo essencial de composição característica específica, podendo ser influenciado por fatores bióticos e abióticos. A fenologia pode influenciar processos bioquímicos e rotas metabólicas capazes de modificar a formação de substâncias biologicamente ativas, alterando diretamente o conteúdo e a qualidade dos óleos essenciais. Sendo assim, o objetivo deste trabalho foi avaliar se as fases fenológicas, vegetativa e reprodutiva modificam o desempenho fotossintético e o perfil do óleo essencial de Xylopia aromatica (Lam.) Mart., influenciando sua atividade biológica na defesa antioxidante e ação antifúngica. As variáveis, fluorescência da clorofila a, trocas gasosas, carboidratos, atividade enzimática e peroxidação lipídica, potencial água, conteúdo relativo de água das folhas, extração, rendimento, caracterização química e atividade antifúngica do óleo essencial de Xylopia aromatica foram avaliadas em 24 plantas, 12 no estádio vegetativo e 12 no reprodutivo, coletadas... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Research aimed at the knowledge of plant species allows the elaboration of projects that aim at the understanding of development, conservation of biodiversity and sustainable exploitation of natural resources. The primary metabolism, represented by photosynthesis and the specialized one, that synthesizes the essential oil, can be influenced by the environmental and phenological conditions, which can influence the chemical profile of the essential oil and the biological activity in the vegetal defense, including against fungi, bacteria and virus. Compounds from the specialized metabolism present biological activity and potential for the production of bactericides and fungicides. Therefore, it is necessary to know the stage of development of plant species in which the substances of interest, with economic potential, are more concentrated, thus orienting, if appropriate, the collection period, aiming at the conservation and sustainable use. There are scientific studies that reveal biological activity of essential oils, as observed for the genus Xylopia, but none of them relates the primary and specialized metabolism to the stage of development in which the species is found. In this way, the objective of this research was to evaluate if the phenological, vegetative and reproductive phases of Xylopia aromatica (Lam.) Mart. modify the photosynthetic performance and the profile of the essential oil, which may influence its biological activity in the antioxidant defense and antifunga... (Complete abstract click electronic access below)
Mestre

[EN] Until recently, the genetic improvement of tomato (Solanum lycopersicum) was focused in agronomic traits, such as yield and biotic or abiotic stresses; therefore the interest in tomato fruit quality is relatively new. The tomato fruit surface can be considered both an agronomic trait as well as a quality trait, because it has an effect on consumer impression in terms of color and glossiness but also it underlies the resistance/sensitivity to cracking or water loss with consequences on fruit manipulation (e.g. transport and processing). The cuticle is deposited over the cell wall surrounding the epidermal cells and it is the first barrier in the plant-environment interface. The cuticle composition includes two main groups of metabolites: cuticular waxes and cutin. Other metabolites can be founded into the cuticle matrix, as triterpenoids and flavonoids. Those minor cuticular components are involved in the correct functionality of the cuticle. Understanding cuticle biosynthesis and genetic regulation requires the development of fast and simple analytical methodologies to study those specialized metabolites using large populations (e.g. mutant collections or introgression lines), together with the identification of genes and genomic regions responsible of their production. This thesis aims to contribute to our understanding of the molecular programs underlying tomato fruit quality by providing: i) a general protocol to profile cuticular waxes in different species, including tomato; ii) a QTL map for cuticular composition (i.e. cuticular waxes and cutin monomers) using the Solanum pennellii introgression line population; iii) a detailed protocol of the reverse genetic tool so-called Fruit-VIGS to assist in the study of gene function in tomato fruit; and iv) a thorough characterization of the first null allele for the transcription factor SlMYB12 (i.e. Slmyb12-pf) in tomato fruit which provides new insights into the regulation of the flavonoid biosynthetic pathway in the fruit peel by high resolution mass spectrometry and RNA-Seq approaches.
[ES] Hasta hace poco, la mejora genética del cultivo del tomate (Solanum lycopersicum) había estado centrada principalmente en caracteres agronómicos, como la productividad y la resistencia a estreses, tanto bióticos como abióticos. Así, el interés en la calidad del fruto de tomate es relativamente reciente. La superficie del fruto del tomate puede considerarse tanto un carácter agronómico como de calidad, pues influye en la primera impresión de los consumidores en términos de color y brillo, así como también en los procesos de resistencia o sensibilidad a la rotura ('cracking') o a la pérdida de agua. Estos factores determinan el aspecto del fruto y condicionan atributos relacionados con su manipulación (transporte y procesado). La cutícula se deposita sobre la pared celular de las células epidérmicas y es la primera barrera que interacciona con el ambiente. Está constituida por dos grandes tipos de metabolitos: las ceras cuticulares y la cutina. Otros metabolitos pueden aparecer embebidos en la matriz cuticular, como es el caso de los triterpenoides y los flavonoides. Estos metabolitos contribuyen a la correcta funcionalidad de la cutícula. La compresión de la biosíntesis y regulación génica de la cutícula requiere del desarrollo de metodologías de análisis sencillas y rápidas para el estudio de estos metabolitos especializados en grandes poblaciones (colecciones de mutantes o líneas de introgresión), así como para la identificación de genes y regiones génicas responsables de la producción y acumulación de dichos compuestos, pudiendo ser muy útiles para implementar programas de mejora de la calidad del tomate. El objetivo de esta tesis es contribuir a la comprensión sobre los programas moleculares subyacentes a la calidad del fruto de tomate, proporcionando: i) un protocolo general de análisis del contenido de ceras cuticulares en diferentes especies, incluyendo el tomate; ii) un mapa de QTL de la composición cuticular (incluyendo ceras y monómeros de cutina) obtenido con la población de líneas de introgresión de Solanum pennellii; iii) un protocolo detallado de uso de la herramienta de genética reversa Fruit-VIGS con el que realizar estudios de funciones génicas en fruto de tomate; y iv) una minuciosa caracterización de un nuevo alelo nulo del factor de transcripción SlMYB12 (Slmyb12-pf) en fruto de tomate, proporcionando nueva información sobre la regulación de la ruta biosintética de los flavonoides en la piel del fruto, utilizando espectrometría de masas de alta resolución y de nuevas tecnologías de secuenciación.
[CAT] Fins fa poc de temps, la millora genètica de la tomata (Solanum lycopersicum) anava dirigida fonamentalment als caràcters de tipus agronòmic, com la productivitat i la tolerància a estressos biòtics o abiòtics, resultant que l'interés per la qualitat dels fruits és relativament nou. La superfície de la tomata pot ser considerada tant com un caràcter agronòmic com un de qualitat, ja que és l'aspecte de la superfície del fruit el que confereix al consumidor la primera impressió de color, brillantor, però és també la pell del fruit la responsable de la diferent susceptibilitat del fruit a desenvolupar clevills o que el fruit sofrisca més o menys pèrdues d'aigua, tot tenint importants conseqüències en la manipulació (i.e. transport i processament del fruit). La cutícula és dipositada per sobre de la paret cel·lular que envolta la capa de cèl·lules epidèrmiques i constitueix la primera barrera en la interfase planta-medi ambient. La composició de la cutícula presenta dos grups principals de metabòlits: les ceres i la cutina. També es poden trobar altres metabòlits els triterpenoids i el flavonoids. Aquests darrers components cuticulars menors són implicats en el correcte funcionament de la cutícula. Per tal de comprendre la biosíntesi i la regulació genètica de la cutícula cal desenvolupar tecnologies analítiques senzilles i rapides que permeten estudiar aquests metabòlits especialitzats en poblacions grans de plantes (i.e. Col·leccions de mutants o de línies d'introgressió), a més de la identificació de gens i regions genòmiques que són responsables de la seua producció. Aquesta tesi té com a objectiu contribuir a millorar la nostra comprensió dels programes moleculars que afecten determinats aspectes de la qualitat de la tomata mitjançant els següents objectius: i) proporcionar un protocol general per obtenir perfils de ceres cuticulars en diferents espècies, inclosa la tomata; ii) obtenir un mapa de QTL per a la composició cuticular (i.e. ceres cuticulars i monòmers de cutina) mitjançant la utilització de la població de línies d'introgressió de Solanum pennelli; iii) descriure amb detall el protocol d'una eina de revers genètica denominada Fruit-VIGS que resulta molt adequada per estudiar funció gènica a la tomata; y iv) fer una caracterització exhaustiva del primer al·lel nul del factor de transcripció SlMYB12 (ie. Slmyb12-pf) en tomata la qual proporciona informació nova sobre la regulació de la ruta de biosíntesi de flavonoides en la pell de la tomata mitjançant espectrometria de masses d'alta resolució i RNAseq.
Fernández Moreno, JP. (2015). TOMATO FLESHY FRUIT QUALITY IMPROVEMENT: CHARACTERIZATION OF GENES AND GENOMIC REGIONS ASSOCIATED TO SPECIALIZED METABOLISM IN TOMATO FLESHY FRUIT [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/55505
TESIS
Premiado

Plants have evolved a variety of constitutive and induced chemical defense mechanisms against biotic stress. Emission of volatile compounds from plants facilitates interactions with both beneficial and pathogenic organisms. However, knowledge of the chemical defense in roots is still limited. In this study, we have examined the root-specific biosynthesis and function of volatile terpenes in the model plant Arabidopsis. When infected with the root rot pathogen Pythium irregulare, Arabidopsis roots release the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT), which is a common constituent of volatile blends emitted from insect-damaged foliage. We have identified a single cytochrome P450 monooxygenase of the CYP705 family that catalyzes a root-specific oxidative degradation of the C30-triterpene precursor arabidiol thereby causing the release of DMNT and a C19-degradation product named arabidonol. We found that DMNT shows inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro, and that DMNT biosynthetic mutant plants were more susceptible to P. irregulare infection. We provide evidence based on genome synteny and phylogenetic analysis that the arabidiol biosynthetic gene cluster containing the arabidiol synthase (ABDS) and CYP705A1 genes possibly emerged via local gene duplication followed by de novo neofunctionalization. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. Additionally, we demonstrated that the arabidiol cleavage product, arabidonol, is further modified by yet unknown enzymatic reactions into three products, which are found in root exudates. We suggested a pathway for their biosynthesis based on precursor feeding experiments and NMR analysis. Although DMNT biosynthetic genes are clustered on chromosome 4 along with several potential modification genes, we did not find a possible role of these genes in the derivatization of arabidonol. Preliminary experimental results using genetic and biochemical approaches for identifying genes involved in the modification steps are also presented.
In summary, this study demonstrates an alternative route for volatile terpene formation belowground different from aboveground plant tissues via triterpene degradation and provides evidence for an unexplored triterpene catabolism pathway in Arabidopsis.
Ph. D.

Antimicrobial resistant microorganisms are predicted to pose an existential threat to humanity inside of the next 3 decades. Characterisation of novel acting antimicrobial small molecules from microorganisms has historically counteracted this evolutionary arms race, however the bountiful source of pharmaceutically relevant bioactive specialised metabolites discovered in the Golden era of drug discovery has long since dried up. The clinicians' arsenal of useful antimicrobials is diminishing, and a fresh perspective on specialised metabolite discovery is necessary. This call to action is being answered, in part, through advances in genome sequencing, bioinformatics predictions and the development of next generation synthetic biology tools aiming to translate the biological sciences into an engineering discipline. To expedite our route to new pharmaceutically relevant specialised metabolites using the synthetic biology toolbox several bottlenecks need to be addressed, and are tackled here in. Biosynthetic gene clusters (BGCs) represent blueprints to pharmaceuticals, however to date the vast wealth of knowledge about biosynthetic gene clusters is inconsistently reported and sporadically disseminated throughout the literature and databases. To bring the reporting of BGCs in line with engineering principles we designed and built a community supported standard, the Minimum Information about a Biosynthetic Gene cluster (MIBiG), for reporting BGCs in a consistent manner, and centralised this information in an easy to operate and open access repository for rapid retrieval of information, an essential resource for the bioengineer. Prioritisation represents the next bottleneck in specialised metabolite discovery. Bioinformatics tools have predicted a cache of thousands of BGCs within publicly available genome sequences, however high experimental attrition rates drastically slows characterisation of the corresponding specialised metabolite. We designed and built an Output Ordering and Prioritisation System (OOPS), to rank thousands of BGCs in parallel against molecular biology relevant parameters, pairing BGCs with appropriate heterologous expression hosts and facilitating a judicious choice of BGCs for characterisation to reduce experimental attrition. To fully realise the potential of synthetic biology in specialised metabolite discovery a genetically amenable heterologous host, capable of completing rapid design-build-test-learn cycles, is necessary. This cannot be achieved for the pharmaceutically important type II polyketides, as their biosynthetic machinery is largely restricted to Actinobacteria. Using MIBiG datasets, antiSMASH and BLASTP we identify 5 sets of soluble type II polyketide synthases (PKS) in Escherichia coli for the first time. We construct and test the robustness of a plug-and-play scaffold for bioproduction of aromatic polyketides using one PKS in E. coli, yielding anthraquinones, dianthrones and benzoisochromanequinones intermediates. Through bioprospecting for biological 'parts' to expand the chemical diversity of our plug-and-play scaffold we describe a new lineage of type II PKSs predominantly from non-Actinobacteria. The standards, softwares, and plug-and-play scaffold and biosynthetic 'parts' described here-in will act as an engine for rapid and automated bioproduction of existing, and novel, pharmaceutically relevant aromatic polyketides in E. coli using the synthetic biology toolbox.

Le sclaréol est un diterpène produit par les organes floraux de la sauge sclarée (Salvia sclarea, Lamiaceae). Il est utilisé en parfumerie pour l’hémisynthèse de l’ambroxide, une substance caractérisée par une odeur ambrée et une grande capacité de fixation des parfums. L’augmentation de la demande mondiale en sclaréol stimule actuellement les tentatives d’accroître le rendement de la production de sclaréol à partir de la sauge sclarée. L’objectif du travail présenté dans ce manuscrit était d’améliorer notre compréhension de la biosynthèse du sclaréol et de sa régulation chez la sauge sclarée, afin de mettre en évidence des stratégies d’augmentation du contenu en sclaréol de la sauge sclarée. L'analyse de la surface des calices de sauge sclarée par imagerie par spectrométrie de masse suggère que le sclaréol est principalement sécrété par des structures épidermiques spécialisées appelées trichomes glandulaires. De plus, nous avons mis en évidence les contributions respectives des deux voies de biosynthèse des terpènes présentes chez les plantes, les voies MVA et MEP, à la biosynthèse de trois terpènes de la sauge sclarée. Des expériences de marquage au ¹³C indiquent que le sclaréol et l’acétate de linalyle sont tous deux issus de la voie MEP, alors que le β-caryophyllène semble être d’origine mixte. Nous avons également étudié le rôle potentiel d’une phytohormone, le méthyljasmonate, dans la régulation de la production de sclaréol chez la sauge sclarée. Enfin, nous avons exploré la diversité génétique et phénotypique de populations croates de sauge sclarée sauvage, et montrons que ces populations représentent une ressource génétique distincte par rapport aux populations de référence. L’ensemble de ces résultats met en évidence des pistes prometteuses pour l'amélioration génétique ciblée des performances de la sauge sclarée
Sclareol is a diterpene produced by floral organs of clary sage (Salvia sclarea, Lamiaceae). It is used in perfume industry for the hemisynthesis of ambroxide, a high-valued perfume component characterized by an amber scent and a high perfume fixation capacity. The global demand for sclareol currently rises, prompting attempts at increasing the yield of sclareol production from clary sage. The purpose of the work presented in this manuscript was to improve knowledge on sclareol biosynthesis and its regulation in clary sage, in order to highlight strategies aiming at enhancing clary sage sclareol content. The analysis of the surface of clary sage calyces by mass spectrometry imaging suggests that sclareol is mainly secreted by specialized epidermal structures called glandular trichomes. Moreover, we have highlighted the respective contributions of the two terpenoid biosynthesis pathways present in plants, MVA and MEP pathways, to the biosynthesis of three terpenoids of clary sage. ¹³C-labeling experiments indicate that sclareol and linalyl acetate both originate from the MEP pathway, whereas β-caryophyllene seems to be of mixed origin. We have also investigated the potential role of a phytohormone, methyljasmonate, in the regulation of sclareol production in clary sage. Finally, we have explored the genetic and phenotypic diversity of Croatian wild clary sage populations and show that these populations represent a distinct genetic resource compared to reference populations. Taken together, these results highlight promising avenues for targeted genetic enhancement of clary sage performances

Le nombre des génomes bactériens séquencés disponibles dans les bases des données ne cesse d’augmenter. Grâce au développement d’outils bio informatiques, l’exploration de ces données génomiques est devenue beaucoup plus aisée. Ces analyses génomiques révèlent qu’un important réservoir de gènes du métabolisme spécialisé, et potentiellement de métabolites bioactifs, reste encore à explorer. J’ai étudié le métabolisme spécialisé d’une souche de Streptomyces appelée Streptomyces sp.TN58, isolée à partir d’un échantillon de sol tunisien et retenue pour son spectre d’activité biologique assez large. Son génome a été séquencé dans le cadre de ce travail. Je me suis particulièrement intéressée à la biosynthèse de deux familles de métabolites spécialisés, les acyl alpha-L-rhamnopyranosides et les dicétopipérazines.Les acyl alpha-L-rhamnopyranosides sont des composés qui possèdent un groupement rhamnose lié à un groupement acyle. Ils présentent plusieurs activités d’intérêt médical (antitumorale, antifongique, antibactérienne…). Leur biosynthèse par des Streptomyces a déjà été décrite, mais aucune étude de leur voie de biosynthèse n’est disponible dans la littérature. La souche Streptomyces sp.TN58 était connue pour produire deux molécules de cette famille. J’ai montré qu’elle en produisait une troisième et j’ai recherché quels gènes dirigeaient leur biosynthèse. J’ai pu identifier les gènes impliqués dans la biosynthèse du précurseur rhamnose et montrer qu’ils sont impliqués dans la biosynthèse des acyl alpha-L-rhamnopyranosides. Les gènes localisés au voisinage de ceux qui dirigent la biosynthèse du rhamnose ne sont pas impliqués dans la biosynthèse des acyl alpha-L-rhamnopyranoides. Cette organisation est originale, car tous les gènes impliqués dans la biosynthèse d’un métabolite spécialisé ne sont pas groupés, contrairement à ce qui est classiquement trouvé chez les Streptomyces et plus généralement chez les microorganismes. L’analyse du génome de Streptomyces sp. TN58 a permis l’identification d’autres gènes candidats, mais l’inactivation de certains de ces gènes n’abolit pas la biosynthèse des trois molécules d’acyl alpha-L-rhamnopyranoside. Ceci peut suggérer plusieurs enzymes promiscuitaires pourraient être impliquées dans la biosynthèse des acyl alpha-L-rhamnopyranosides.Les dicétopipérazines sont des dérivés de dipeptides cycliques et constituent une classe de produits naturels possédant des activités biologiques diverses, mais leur rôle physiologique chez l’organisme producteur reste peu connu. Elles peuvent être synthétisées par des mégacomplexes enzymatiques, les synthétases de peptides non ribosomiques (NRPS), ou par des cyclodipeptides synthases (CDPS). Ces dernières sont des enzymes de petite taille utilisant des ARN de transfert amino-acylés comme substrat. Les cyclodipeptides synthétisés peuvent subir différentes modifications, ce qui explique la diversité de leur structure chimique. L’analyse du génome de Streptomyces sp.TN58 a permis d’identifier un cluster de deux gènes (codant une CDPS et un cytochrome P450) homologues à des gènes impliqués dans la biosynthèse d’une dicétopipérazine (la mycocyclosine) chez Mycobacterium tuberculosis. J’ai identifié les produits dont la biosynthèse est dirigée par ces gènes. J’ai construit des souches mutées pour tester l’hypothèse d’un rôle de ces produits dans la signalisation pour la différenciation morphologique et la production d’antibiotiques chez Streptomyces sp.TN58. Les premiers résultats obtenus semblent en accord avec cette hypothèse
The number of sequenced bacterial genomes available in databases is steadily increasing. With the development of bioinformatics tools, the exploration of these genomic data has become much easier. These genomic analyzes reveal that an important reservoir of genes for specialized metabolism, and potentially bioactive metabolites, remains to be explored. The vast majority of bacterial specialized metabolism was therefore ignored. I studied the specialized metabolism of a Streptomyces strain called Streptomyces sp.TN58, isolated from a Tunisian soil sample and retained for its broad spectrum of biological activity. Its genome has been sequenced in the frame of this work. I was particularly interested in the biosynthesis of two families of specialized metabolites, acyl alpha-L-rhamnopyranosides and diketopiperazines (DKPs).Acyl alpha-L-rhamnopyranosides are compounds having a rhamnose group linked to an acyl group. They possess a variety of biological activities of medical interest (anti-tumor, antifungal, antibacterial…). Their production by Streptomyces sp. has been described previously but no study of their biosynthetic pathway is available in literature. Streptomyces sp.TN58 strain was known to produce two molecules of this family. I showed that it produced a third one and I looked for the genes directing their biosynthesis. I have identified the genes involved in the biosynthesis of the rhamnose precursor and shown that their inactivation abolished the biosynthesis of acyl alpha-L-rhamnopyranosides. However, the genes located in the vicinity of the rhamnose biosynthetic genes are not involved in acyl alpha-L-rhamnopyranoside biosynthesis. This organization is unusual because all the genes directing the biosynthesis of a specialized metabolite are not clustered, contrarily to what is usually found in Streptomyces and more generally in microorganisms. A genome-mining approach allowed the identification of candidate genes, but the inactivation of some of these genes did not abolish the biosynthesis of the three acyl alpha-L-rhamnopyranoside molecules. This suggests that several rather promiscuous enzymes might be involved in the biosynthesis of acyl alpha-L-rhamnopyranosides.DKPs are cyclic dipeptide derivatives. This class of natural products possesses a wide variety of biological activities, but their physiological role in the producing organism remains often unknown. DKPs can be synthesized by non-ribosomal peptide synthases (NRPSs) or by cyclodipetide synthases (CDPSs). Contrarily to NRPSs which are enzymatic megacomplexes using amino acids as substrate, CDPSs are small enzymes using amino-acylated tRNAs as a substrate. The synthesized cyclodipeptides can undergo various modifications, which explains the diversity of DKP chemical structures. Mining the genome of Streptomyces sp. TN58 allowed the identification of a cluster of two genes (encoding a CDPS and a cytochrome P450) homologous to genes involved in the biosynthesis of a DKP (mycocyclosin) in Mycobacterium tuberculosis. I managed to identify the DKP synthesized. I constructed mutant strains to test the hypothesis that these DKPs could play a role as signaling molecules for morphological differentiation and antibiotic production in Streptomyces sp.TN58. Preliminary results seem to support this hypothesis

Plants produce secondary (or specialized) metabolites to respond to a variety of environmental changes and threats. Especially, volatile compounds released by plants facilitate short and long distance interaction with both beneficial and harmful organisms. Comparatively little is known about the organization and role of specialized metabolism in root tissues. In this study, we have investigated the root-specific formation and function of volatile terpenes in the model plant Arabidopsis. As one objective, we have characterized the two root-specific terpene synthases, TPS22 and TPS25. Both enzymes catalyze the formation of several volatile sesquiterpenes with (E)-β-farnesene as the major product. TPS22 and TPS25 are expressed in the root in distinct different cell type-specific patterns and both genes are induced by jasmonic acid. Unexpectedly, both TPS proteins are localized to mitochondria, demonstrating a subcellular localization of terpene specialized metabolism in compartments other than the cytosol and plastids. (E)-β-Farnesene is produced at low concentrations suggesting posttranslational modifications of the TPS proteins and/or limited substrate availability in mitochondria. We hypothesize that the mitochondrial localization of TPS22 and TPS25 reflects evolutionary plasticity in subcellular compartmentation of TPS proteins with emerging or declining activity. Since (E)-β-farnesene inhibits Arabidopsis root growth in vitro, mitochondrial targeting of both proteins may fine tune (E)-β-farnesene concentrations to prevent possible autotoxic or inhibitory effects of this terpene in vivo. We further investigated the role of volatile terpenes in Arabidopsis roots in interaction with the soil-borne oomycete, Pythium irregulare. Infection of roots with P. irregulare causes emission of the C11-homoterpene (or better called C4-norterpene) 4,8-dimethylnona-1,3,7-triene (DMNT), which is a common volatile induced by biotic stress in aerial parts of plants but was not previously known to be produced in plant roots. We demonstrate that DMNT is synthesized by a novel, root-specific pathway via oxidative degradation of the C30-triterpene, arabidiol. DMNT exhibits inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro. Moreover, arabidiol and DMNT biosynthetic mutants were found to be more susceptible to P. irregulare infection and showed higher rates of Pythium colonization in comparison to wild type plants. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues.
Ph. D.

Plants produce a vast number of low-molecular-weight chemicals (so called secondary or specialized metabolites) that confer a selective advantage to the plant, such as defense against herbivory or protection from changing environmental conditions. Many of these specialized metabolites are used for their medicinal properties, as lead compounds in drug discovery, or to impart our food with different tastes and scents. These chemicals are produced by various pathways of enzyme-mediated reactions in plant cells. It is suspected that enzymes in plant specialized metabolism evolved from those in primary metabolism. Understanding how plants evolved to produce these diverse metabolites is of primary interest, as it can lead to the engineering of plants to be more resistant to both biotic and abiotic stress, or to produce more complex small molecule compounds that are difficult to derive. To that end, the first objective was to develop a schema for rational protein engineering using meta-analyses of a well-characterized sesquiterpene synthase family encoding two closely-related but different types of enzymes, using quantitative measures of natural selection on amino-acid positions previously demonstrated as important for neofunctionalization between two terpene synthase gene families. The change in the nonsynonymous to synonymous mutation rate ratio (dN/dS) between these two gene families was large at the sites known to be responsible for interconversion. This led to a metric (delta dN/dS) that might have some predictive power. This natural selection-oriented approach was tested on two related enzyme families involved in either nicotine/tropane alkaloid biosynthesis (putrescine N-methyltransferase) or primary metabolism (spermidine synthase) by attempting to interconvert a spermidine synthase to encode putrescine N-methyltransferase activity based upon past patterns of natural selection. In contrast to the HPS/TEAS system, using delta dN/dS metrics between SPDS and PMT and site directed mutagenesis of SPDS did not result in the desired neofunctionalization to PMT activity. Phylogenetic analyses were performed to investigate the molecular evolution of plant N-methyltransferases involved in three alkaloid biosynthetic pathways. The results from these studies indicated that unlike O-MTs that show monophyletic origins, plant N-MTs showed patterns indicating polyphyletic origins. To provide the foundation for future molecular-oriented studies of urushiol production in poison ivy, the complete poison ivy root and leaf transcriptomes were sequenced, assembled, and analyzed.
Ph. D.

Orientador: Marcia Ortiz Mayo Marques
Resumo: Lychnophora pinaster Mart. (Asteraceae) é uma espécie de ocorrência restrita ao Estado de Minas Gerais e utilizada pela população local por suas propriedades anti-inflamatória e analgésica. O uso indiscriminado pela população e destruição de seu habitat contribuíram para que a espécie entrasse em risco de extinção. Estudos de populações de L.pinaster oriundas do sul e do norte do estado de Minas Gerais reportam diferentes composições de seus óleos essenciais e níveis de diversidade genética, sendo necessários estudos de maior abrangência geográfica que permitam sua caracterização em diversas regiões do estado. Diante disso, este estudo objetivou avaliar a composição química dos óleos essenciais e diversidade genética de populações de Lychnophora pinaster Mart. coletadas em diferentes regiões de Minas Gerais. As populações avaliadas nos estudos químicos são oriundas de duas regiões: Diamantina (DIMa), Olhos D’Àgua (OD) e Grão Mogol (GM), da região Norte de Minas Gerais; e três da Região Metropolitana de Belo Horizonte, Caeté/Rio Acima (CTRA), Nova Lima/Serra da Calçada (NLSC) e Serra da Moeda (SM). Os óleos essenciais foram extraídos por hidrodestilação, a composição química analisada por CG-EM e CGxCG-EM e os resultados avaliados por métodos quimiométricos. Análises de solos revelaram diferenças entre os solos das populações. DIMa e SM apresentaram os maiores rendimentos dos óleos essenciais (ambas 0,12%), enquanto o menor foi o de NLSC (0,04%). Não foi detectada diferença de... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Lychnophora pinaster Mart. (Asteraceae) is a species restricted to the State of Minas Gerais and used by the local population for its anti-inflammatory and analgesic properties. The indiscriminate use by the population and the destruction of its habitat contributed to the species becoming at risk of extinction. Studies of L. pinaster populations from the south and north of the state report different compositions of their essential oils and levels of genetic diversity, requiring studies of greater geographic scope that allow their characterization in several regions of the state. Given this scenario, this study aimed to evaluate the chemical composition of essential oils and genetic diversity of populations of Lychnophora pinaster Mart. collected in different regions of Minas Gerais. The populations evaluated in the chemical studies come from two regions: Diamantina (DIMa), Olhos D’Àgua (OD) and Grão Mogol (GM), from the northern region of Minas Gerais; and three from the Metropolitan Region of Belo Horizonte, Caeté/Rio Acima (CTRA), Nova Lima/Serra da Calçada (NLSC) and Serra da Moeda (SM). The essential oils were extracted by hydrodistillation, the chemical composition analyzed by CG-EM and CGxCG-EM and the results evaluated by chemometric methods. Soil analyzes revealed differences between the populations' soils. DIMa and SM showed the highest yields of essential oils (both 0.12%), while the lowest was that of NLSC (0.04%). No difference in yield was detected depending on t... (Complete abstract click electronic access below)
Doutor

Le genre Pelargonium fait partie de la famille des Geraniaceae et réunit plus de 280 espèces, ainsi que de nombreux hybrides et variétés sélectionnés depuis le 18e siècle. Ces accessions regroupent notamment de pélargoniums commercialisés en tant que plantes ornementales (comme les P. x hortorum) mais également des pélargoniums odorants (comme les P. x hybridum cv rosat) qui sont cultivés pour leur huile essentielle (HE). L’HE de P. rosat est stockée dans des structures glandulaires (trichomes glandulaires) présentes sur les feuilles et se compose principalement de mono- et sesqui-terpénoïdes. Ces composés organiques volatils sont à l’origine du parfum « géranium », prisé des parfumeurs pour son profil olfactif complexe et rappelant l’odeur de rose. L’objectif de cette thèse était de mieux comprendre la diversité de terpénoïdes présents dans l’HE de pélargonium, de décrypter les mécanismes sous-jacents à la biosynthèse de ces nombreux composés odorants et plus particulièrement d’analyser les enzymes impliquées dans leur production. Afin de répondre à cet objectif, des études biochimiques et transcriptomiques ont été menées. Celles-ci ont permis de mettre en place une approche multi-omique afin d’étudier le terpénome de dix accessions de pélargoniums odorants, de caractériser structurellement et fonctionnellement des enzymes impliquées dans la biosynthèse de ce terpénome et d’analyser l’effet d’un stress climatique sur celui-ci
The Pelargonium genus belongs to the Geraniaceae family and includes more than 280 species as well as multiple hybrids and varieties, which have been selected by botanists since the 18th century. Among these accessions, several can be found on the market as ornemental plant (e.g. P. x hortorum) whereas some are cultivated for essential oil (EO) production (e.g. P. x hybridum cv rosat). P. rosat EO is stored in glandular trichomes from leaves and is mainly composed of mono- and sesqui-terpenoids. The resulting volatile organic compound mixture offers a characteristic “geranium” scent. Due to its sophisticated odour reminding of the rose scent, this scent is highly pursued by perfumers and fragrance industries. The purpose of this thesis was to improve our understanding of the terpenoid diversity in pelargonium EO and decipher mechanims underlying their biosynthesis, in particular by characterising enzymes responsible for their production. To this aim, biochemical and transcriptomic studies have been performed. Therefore, a multi-omic approach has been implemented to analyse the terpenome from ten scented-pelargoniums. Moreover, structural and functional analysis of several enzymes involved in terpenoid biosynthesis have been performed and the effect of a climatic stress on the EO composition has been studied

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum sensing molecule hormaomycin, and antituberculotic griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L-tyrosine- or L-leucine-derived 4-alkyl-L-proline derivatives (APDs) in their structures. APD biosynthesis involves a set of up to six homologous proteins. According to their proposed order in the biosynthesis of 4-propyl-L-proline, a model APD of lincosamide lincomycin, the homologous proteins were named Apd1 - Apd6. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction and in vitro tests of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1 -pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. The two successive F420H2-dependent reduction...

Tese de mestrado. Biologia (Ecologia Marinha). Universidade de Lisboa, Faculdade de Ciências, 2012
Temperature is considered as one of the most important physical variables controlling the life of marine organisms. Thus, understanding how they will respond to future thermal scenarios is of great relevance. The present study aimed to investigate, for the first time, the spawning success, embryonic development and metabolic rates of a specialized soft coral-feeding nudibranch, Armina maculata, under different thermal scenarios, which reflect: i) the present-day average spring (18ºC), early-summer (22ºC) and summer (24ºC) temperatures, and ii) the projected near-future warming (27ºC, +3ºC, IPCC 2007), a temperature that the species already experiences during heat wave events in Sado’s estuary. At 22ºC, A.maculata’s adults spawned more frequently and the embryos showed higher survival rates (~88%), indicating this temperature as the optimal for the reproduction of this species. On the other hand, the project near-future warming (27ºC) promoted metabolic suppression in adults, i.e., it triggered the shut-down of several biochemical functions. Additionally, under such thermal scenario, spawning events were scarce, ceased after three weeks, feeding intake was absent and the individuals perished after seven weeks of incubation. Furthermore, early ontogenetic stages (blastula, gastrula, trocophore, active veliger, newly-hatched, veliger) showed concomitant negative effects with increasing temperature. Embryonic development was dramatically shortened followed by increased energy expenditure rates. Moreover, egg mass analysis showed visible deleterious effects on embryo’s development and survival, with the latter decreasing up to 45%. Hence, the ability of A.maculata to overcome the developmental and physiological challenges faced at the projected near-future warming scenario will dictate its long-term survival success.
As alterações climáticas emergiram no último século como uma preocupação global. O aquecimento da superfície dos oceanos é uma realidade incontestável e as consequências desta tendência podem ter efeitos catastróficos nas comunidades marinhas. Estudos preveem que a superfície dos oceanos sofra um aumento de 3ºC até 2100 ditando grandes alterações nas características das grandes massas de água, como o aumento do seu nível médio, que causará um forte impacte nas comunidades costeiras, ou mudanças nas correntes, com consequências graves a nível de dispersão larvar e disponibilidade alimentar, por exemplo. Compreender como irão as comunidades marinhas responder às rápidas mudanças nos ecossistemas constitui uma prioridade na investigação científica. A temperatura é um dos principais fatores físicos que influencia os processos fisiológicos nos organismos marinhos ectotérmicos, tais como taxas de respiração, crescimento, alimentação e reprodução. No entanto, a resposta de cada organismo às variações de temperatura está dependente dos seus limites de tolerância térmica e estágio de desenvolvimento. Além destes limites, a sobrevivência e fitness de um organismo podem estar comprometidos, principalmente numa perspetiva a longo termo. Assim, um organismo exposto prolongadamente a elevadas temperaturas aumenta o seu metabolismo exponencialmente até um nível em que a capacidade respiratória e cardiovascular atingem o seu máximo, seguindo-se um conjunto de reações fisiológicas, tais como, falhas no sistema enzimático, desnaturalização de proteínas e danos membranares que podem resultar na morte do indivíduo. Embora a ordem Nudibranchia seja a maior dentro dos Heterobranchia (Euthyneura, Nudipleura), poucos estudos reportam o efeito da temperatura na ecologia fisiológica dos nudibrânquios e não existem referências quanto à ecologia de Armina maculata. Como seres invertebrados e bênticos, que habitam principalmente as zonas intertidal e subtidal, o efeito do aumento da temperatura pode representar um obstáculo à persistência de muitas espécies. Os nudibrânquios são uma espécie carnívora, alimentando-se de um espectro de seres bênticos e sésseis, que não sejam largamente explorados por outros taxa. Adicionalmente, muitas espécies de nudibrânquios apresentam associações predador-presa, designando-se por espécies estonofágicas, estando intimamente ligados à distribuição e sobrevivência da sua única fonte de recursos alimentares. Assim sendo, a extinção local de determinada presa pode conduzir paralelamente à extinção da espécie de nudibrânquio que dela depende. Esta condição agrava-se perante um cenário de aquecimento global e, consequentemente, a capacidade de resposta e adaptação dos nudibrânquios deve ser avaliada conjuntamente com a capacidade de resposta de adaptação da sua presa. Num cenário futuro de altas temperaturas, onde é esperado um aumento nas taxas metabólicas dos organismos, uma diminuição nas reservas nutricionais pode resultar em falhas no sucesso reprodutor, já que grande parte da energia de um nudibrânquio adulto é direcionada para a reprodução. Atendendo-se ao fato de que a maioria dos nudibrânquios são espécies semélparas com um ciclo de vida anual, entende-se que o estado nutricional do individuo, bem como, as condições ambientais prevalentes durante o período de copulação, desova e eclosão larvar, estejam intimamente ligados ao sucesso reprodutor da espécie. Estudos sobre o sucesso reprodutor do nudibrânquio Adalaria proxima demonstraram que a temperatura afetava significativamente todas as atividades relacionadas com a reprodução, nomeadamente, periodicidade na colocação de posturas, duração do período de desova e viabilidade das massas de ovos. Assim sendo, compreende-se que, embora os nudibrânquios adultos tenham uma maior plasticidade na resposta às variações anuais de temperatura, o seu investimento reprodutor seja direcionado para um curto e único período de tempo, durante o qual as temperaturas são as ideais para assegurar a sobrevivência da descendência. A maioria das espécies de nudibrânquios possuem uma estratégia reprodutora do tipo “r”, isto é, investem em várias em posturas ao longo do período reprodutor, sendo que cada postura é constituída por milhares de ovos, de forma a superar o efeito da seleção natural sobre a grande variabilidade individual da descendência. Esta estratégia revela-se de especial importância para espécies de nudibrânquios que tenham desenvolvimento larvar planctotrófico. A capacidade de adaptação das espécies de nudibrânquios às adversidades do aquecimento global, pode ficar comprometida logo nos primeiros estágios de vida. Na verdade, é esperado que estes sejam os mais vulneráveis às mudanças de temperatura, por possuírem uma janela de tolerância térmica muito menor que a dos adultos. Estudos realizados em vários moluscos heterobrânquios, demonstraram que o aumento da temperatura diminuía o tempo de desenvolvimento embrionário, com efeitos negativos concomitantes na sobrevivência e morfologia dos embriões. Adicionalmente, a diminuição do período de embriogénese pode levar a um desfasamento temporal entre disponibilidade alimentar e eclosão larvar. No futuro, as larvas recém-eclodidas terão uma maior necessidade de alimento, devido às altas taxas metabólicas esperadas, pelo que a falta de recursos poderá comprometer a sua sobrevivência. A atividade metabólica, por sua vez, pode ser considerada como a função fisiológica de um organismo mais dependente das variações de temperatura. Vários estudos foram realizados na perspetiva de identificar os efeitos das variações térmicas no metabolismo de invertebrados marinhos, comprovando-se que o aumento da temperatura é sempre acompanhado de um aumento nas taxas de consumo de oxigénio. Situações de stress ambiental como extremos de temperatura ou privação de alimento, resultam na ativação de uma resposta de supressão metabólica, em que o organismo diminui os seus gastos energéticos ao máximo, procurando sobreviver até surgirem condições ambientais mais favoráveis. Verifica-se também que a taxa de aumento no consumo de oxigénio é mais significativa nos primeiros estágios de vida de um organismo marinho, bem como em nudibrânquios, corroborando a premissa de que estes estágios são os mais vulneráveis e a sua performance e sobrevivência influenciarão diretamente toda a dinâmica populacional da espécie. Neste sentido, o objetivo do presente estudo foi avaliar, pela primeira vez, o impacte de um cenário futuro de aquecimento na ecologia fisiológica do nudibrânquio A.maculata. Indivíduos adultos da espécie A.maculata foram capturados no estuário do Sado (costa oeste de Portugal) e mantidos sob quatro cenários térmicos diferentes, que refletiam: i) a temperatura média atual da primavera (18ºC), início do verão (22ºC) e verão (24ºC) e, ii) a temperatura prevista para o verão sob um cenário futuro de aquecimento (27ºC, +3ºC, IPCC 2007), experienciada já presentemente pela espécie durante ondas de calor no verão, no estuário do Sado. Para se avaliar os efeitos da temperatura na alimentação e reprodução, os indivíduos adultos foram colocados aos pares em aquários individualizados, permitindo a copulação, visto esta espécie ser hermafrodita mas apenas realizar fecundação cruzada, e alimentados ad libitum com a sua presa, o octocoral Veretillum cynomorium. Após iniciado o período reprodutor, as posturas colocadas foram incubadas nas mesmas condições que os adultos e o seu desenvolvimento foi acompanhado diariamente, verificando-se o efeito do aquecimento na morfologia, sobrevivência e tempo de embriogénese. Por fim, foram medidas as taxas de consumo de oxigénio e determinada a sensibilidade térmica (valores de Q10) desta espécie em adultos e embriões. Embora fosse expetado que as altas taxas metabólicas verificadas nos adultos a temperaturas elevadas (24, 27ºC) fossem acompanhadas por um aumento na ingestão de alimento, tal não se verificou. Na verdade, as maiores taxas foram verificadas a 18 e 22ºC, onde se verificou também a preferência pela presa V.cynomorium. Esta diminuição na procura de alimento pode ser explicada pela ativação de uma resposta de supressão metabólica, que resultou numa diminuição da atividade dos indivíduos e no decréscimo da capacidade respiratória, reprodutora e locomotora, levando à morte dos exemplares, após sete semanas de incubação a 27ºC. Concomitantemente, o cenário de aquecimento (27ºC) teve consequências devastadoras para esta espécie em vários aspetos da sua reprodução. Os adultos mantidos a esta temperatura apenas colocaram posturas durante as primeiras três semanas de teste, as quais demonstraram deformações morfológicas (ex. variações na cor, diminuição no tamanho das capsulas, embriões e larvas recém-eclodidas, nos últimos estágios da embriogénese) e cujo tempo de desenvolvimento até à eclosão foi significativamente encurtado (~5 dias) e acompanhado por altas taxas de mortalidade embrionária (~55%). Como esperado, o consumo de oxigénio aumentou linearmente com o aumento da temperatura, verificando-se a premissa de que este aumento seria mais substancial nos embriões do que nos adultos, visto que os estágios iniciais são os mais vulneráveis a variações térmicas. Por outro lado, os resultados apontam o cenário de 22ºC como a temperatura ótima para a reprodução desta espécie. Elevada frequência de desova associada a altas taxas de sobrevivência embrionária (~88%) e sucesso no desenvolvimento ontogenético, sugerem que esta espécie se reproduza preferencialmente no início do verão. Os resultados deste estudo demonstraram que a temperatura desempenha um papel crucial no ciclo de vida do nudibrânquio A.maculata, determinando vários aspetos das suas respostas fisiológicas a nível alimentar, reprodutivo e metabólico. No entanto, a sobrevivência desta espécie, estará dependente da sua capacidade de adaptação às condições de stress extremo no previsto cenário de aquecimento global.

The clinically used antibiotic lincomycin consists of an amino-sugar and an amino-acid moiety. The incorporated amino-acid 4-propyl-L-prolin (PPL) is very important for the linomycin bioactivity, as evidenced by the lower activity of the related antibiotic celesticetin, which incorporates proteinogenic L-prolin instead. Gene clusters for the biosynthesis of both lincosamides are published and reflect a common basis - biosynthesis of amino-sugar precursor and condensation reactions. Additionally, in the biosynthetic gene cluster for lincomycin there is a sub-cluster of genes encoding the biosynthesis of PPL, the alkylated proline derivative (APD). PPL has a common biosynthetic origin with other APDs that are part of the structures of antitumor pyrrolobenzodiazepines and the signal molecule hormaomycin, which is also reflected in the presence of homologous genes in their gene clusters. The acquired knowledge on PPL biosynthesis thus can be applied to a larger group of natural products. The first overall concept of APD biosynthesis was published forty years ago. The milestone was the year 1995 when the gene cluster for lincomycin biosynthesis was published and specific gene products have been proposed for individual biosynthetic steps. The functional proof of proteins has been performed so far just...