In Streptomyces bacteria, the expression of many antibiotic biosynthetic clusters is controlled by both cluster-specific regulators and more globally-acting regulators; however, much remains unknown about the factors that govern antibiotic production. In Streptomyces venezuelae, we have discovered that the broadly-conserved nucleoid-associated protein Lsr2, plays a major role in repressing specialized metabolic cluster gene expression. To understand how Lsr2 exerts its gene silencing effects, we focused our attention on the well-studied, but transcriptionally silent, chloramphenicol cluster in S. venezuelae. We established that Lsr2 represses transcription of the chloramphenicol cluster by binding DNA both within the cluster and at distal positions. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not under Lsr2 control. We discovered that CmlR functions to ‘counter-silence’ Lsr2 activity, alleviating Lsr2 repression and permitting chloramphenicol production, by recruiting RNA polymerase. Lsr2 plays a central role in controlling antibiotic production in Streptomyces; however, beyond this counter-silencing activity, little is known about how Lsr2 is regulated. We identified regulators that could control the expression of lsr2, and found that Lsr2 and LsrL, an Lsr2 homologue that is encoded by all streptomycetes, interact directly with each other, and that their respective DNA-binding activities are altered by the presence of the other protein. These data suggest that LsrL may impact Lsr2 activity in regulating antibiotic production in Streptomyces. Beyond Lsr2, we wanted to develop a comprehensive understanding of the regulatory proteins that impact biosynthetic gene cluster expression. To define the regulatory protein occupancy of antibiotic clusters, we developed ‘in vivo protein occupancy display-high resolution’ (IPOD-HR) technology for use in Streptomyces. This work will lay the foundation for establishing a comprehensive regulatory network map for biosynthetic clusters in Streptomyces, and guide future work aimed at stimulating the expression of metabolic clusters in any Streptomyces species. / Thesis / Doctor of Philosophy (PhD) / Streptomyces bacteria produce the majority of naturally-derived antibiotics, and they have the genetic potential to produce many more antibiotics and antibiotic-like compounds (‘specialized metabolites’). Specialized metabolism is controlled by multiple regulatory systems. In Streptomyces venezuelae, we have discovered that the nucleoid-associated protein, Lsr2, represses the expression of most specialized metabolic clusters, and manipulating Lsr2 activity can stimulate antibiotic production. To better understand how Lsr2 exerts its repressive effect, we explored how Lsr2 controlled the production of a known antibiotic. We ultimately identified multiple regulators that could impact the expression and/or activity of Lsr2. Building on the regulatory foundation provided by Lsr2, we then set out to establish a comprehensive regulatory network that governs biosynthetic gene cluster expression. Collectively, this work improves our understanding of antibiotic gene regulation in Streptomyces bacteria, and has the potential to guide novel strategies aimed at stimulating the production of new antibiotics in Streptomyces.

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.

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.

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

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

Many economically important crop systems exhibit an Age-Related Resistance (ARR) response whereby mature plants become resistant to pathogens they were susceptible to when younger. The signaling pathways and mechanisms of ARR have not been well studied. Arabidopsis displays ARR in response to P. syringae pv tomato (Pst). Several studies provide evidence that intercellular salicylic acid (SA) accumulation is required for ARR and SA acts as a direct antimicrobial agent to limit bacterial growth and biofilm-like aggregate formation. SA accumulation mutants are ARR defective; however, a modest level of resistance is occasionally observed leading to the hypothesis that other compounds contribute to ARR as antimicrobial agents. Previous studies demonstrated that CYP71A13 (a key enzyme in indolic biosynthesis) is expressed during the ARR response. I demonstrated that CYP71A12 functionally compensated for CYP71A13 during ARR, as cyp71a12/cyp71a13-1 mutants were consistently ARR-defective compared to their respective single mutants. I demonstrated that dihydrocamalexic acid (DHCA) accumulated in intercellular washing fluids (IWFs) collected from plants during the ARR response using high resolution mass spectrometry-based profiling. DHCA was detected in IWFs collected from wild-type ARR-competent plants and, was absent in IWFs from ARR-incompetent cyp71a12/cyp71a13 mutants. In vitro DHCA antimicrobial activity against P. syringae was not observed, but exogenous infiltration of DHCA into the leaf intercellular space restored ARR in cyp71a12/cyp71a13 mutants Unlike SA which exhibits direct antimicrobial activity against P. syringae, DHCA does not and instead may affect pathogen virulence in other ways. My research provides evidence that intercellular DHCA contributes to ARR in response to P. syringae in Arabidopsis. Understanding the genes and metabolites contributing to ARR will provide useful information for future crop breeding and genetic modification that will mitigate agricultural losses due to disease. / Thesis / Master of Science (MSc) / During Age-Related Resistance (ARR), mature plants including some crop plants become resistant to pathogens they were susceptible to when younger. How ARR works is poorly understood. My objective was to identify potential antimicrobial metabolites contributing to ARR in Arabidopsis against the bacterial pathogen Pseudomonas syringae. Genetic analyses combined with mass-spectrometry based metabolite profiling demonstrates that two cytochromes P450, CYP71A12 and CYP71A13 contribute to ARR. My research provides evidence that DHCA accumulates in the leaf intercellular space in ARR-competent plants, where it may act to inhibit the bacterial infection process. DHCA has low antimicrobial activity against P. syringae suggesting its mechanism of action is not directly antimicrobial. Importantly, application of DHCA to the leaf intercellular space of cyp71a12/cyp71a13 restored ARR, confirming that DHCA contributes to ARR in Arabidopsis. Understanding ARR will provide useful information for future crop breeding and genetic modification that will mitigate agricultural losses due to disease.

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.

<p>Specialized metabolites are chemical tools produced by organisms to aid in their interaction with the surrounding environment. These diverse compounds can often function as metabolic weapons (<em>e.g.</em> antibiotics), structural components (<em>e.g.</em> lignins), or even attractants (<em>e.g.</em> flavonoids). Because of their frequent utilization in niche environments, specialized metabolite production is often lineage- or even species-specific. Therefore, knowledge between specialized metabolic systems is often nontransferable, which poses a major obstacle in the characterization of these bioactive and commercially relevant compounds. Beyond resolving the chemical composition of a specialized metabolite, the identification of responsible pathway genes and the evolutionary processes responsible for their formation is an arduous task. These gaps in knowledge are further widened by the lack of genomic resources available for specialized metabolite producing species. In this work, we present the genome assemblies of two organisms, each with unique specialized metabolic pathways: the Chinese medicinal plant <em>Lithospermum erythrorhizon </em>and the toxic golden alga <em>Prymnesium parvum. </em>Leveraging the predicted proteome of <em>L. erythrorhizon</em>, we investigated the evolutionary history of specialized metabolic genes responsible for the production of shikonin, a 1,4-naphthoquinone specialized metabolite. We identified a retrotransposition-mediated duplication event responsible for the creation of the core shikonin biosynthesis gene, <em>PGT</em>. In addition, we performed a global coexpression network analysis to identify regulatory and enzymatic gene candidates involved in the shikonin biosynthesis pathway. We also built phylogenetic trees of known and candidate shikonin genes to reveal patterns of lineage-specific gene duplication and retroduplication. Like plants, unicellular algae are known for their production of diverse, often toxic, specialized metabolites. However, these species are often enigmatic. For example, previous studies have documented large phenotypic variation in both toxin chemotypes and levels among different strains of <em>P. parvum</em>. To investigate the genetic basis of this variation, we generated near chromosome level assemblies of two <em>P. parvum </em>strains and performed a broad genome survey of thirteen additional strains. As a result, we identified a commonly studied reference strain, UTEX 2797, as a hybrid with two distinct subgenomes. We also provide evidence of significant variation in haploid genome size across the species. Collectively, these studies supply genetic resources for the future study of these organisms, as well as provide insight into the evolution of their specialized metabolic pathways.</p>

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.

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...