Dissertations / Theses on the topic 'Terpene biosynthesis'

Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.

Stinkbugs (Pentatomidae) and other agricultural pests such as bark beetles and flea beetles are known to synthesize terpenoids as aggregation pheromones. Knowledge of the genes and enzymes involved in pheromone biosynthesis may allow engineering of pheromone biosynthetic pathways in plants to develop new forms of trap crops and agricultural practices for pest management. The harlequin bug, Murgantia histrionica, a specialist pest on crucifer crops, produces the sesquiterpene, murgantiol, as a male-specific aggregation pheromone. Similarly, the southern green stink bug, Nezara viridula, a generalist pest worldwide on soybean and other crops, releases sesquiterpene cis-/trans-(Z)-α-bisabolene epoxides as male-specific aggregation pheromone. In both species, enzymes called terpene synthases (TPSs) synthesize precursors of the aggregation pheromones, which are sesquipiperitol and (Z)-α-bisabolene as the precursor of murgantiol and cis-/trans-(Z)-α-bisabolene epoxide, respectively. We hypothesized that enzymes in the family of cytochrome P450 monooxygenases are involved in the conversion of these precursors to the final epoxide products. This study investigated the tissue specificity and sequence of these conversions by performing crude enzyme assays with protein extracts from male tissues. Furthermore, candidate P450 genes were selected by RNA-sequencing and co-expression analysis and the corresponding recombinant proteins tested for enzyme activity. To engineer the pheromone biosynthetic enzymes in plants, transient expression of the TPSs of both stink bugs was performed in Nicotiana benthamiana leaves. Both sesquipiperitol and (Z)-α-bisabolene were found to be produced and emitted from inoculated N. benthamiana leaves. Future work will implement stable transformation to engineer murgantiol biosynthesis in crucifer trap crops and develop similar approaches for pheromone engineering of other agricultural pests.
Master of Science

BACKGROUND:Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.RESULTS:A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.CONCLUSIONS:The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

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.

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

Le manuscrit de thèse traite de la synthèse totale de molécules naturelles de structures particulièrement intrigantes. Deux familles de molécules naturelles issues du monde marin ont été ciblées dans ce travail : les halichonadines et les araiosamines.- La famille des halichonadines nous plongent dans le domaine des terpènes d’origine marine. Isolées d’éponges du genre Halichondria, deux structures ont particulièrement retenues notre attention : les halichonadines K et L. En effet, non seulement, ces deux molécules complexes contiennent une partie terpénique de type eudesmane (halichonadine C, un isonitrile naturel) mais aussi un cœur central de nature peptidique constitué, notamment, d’une pipéridine disubstituées par des fonctions acide carboxylique. Une partie est dédiée à comprendre comment dans la nature, des molécules de type isonitrile sont produites et peuvent réagir dans des voies de biosynthèse. Par ailleurs, le travail expérimental s’est organisé de la façon suivante :1- Concevoir une synthèse efficace de l’halichonadine C. Une stratégie au départ de la santonine est développée. La présence d’un groupement isopropyle sur la structure finale s’est avérée poser un nombre important de problèmes. Cependant, un composé très avancé a été obtenu figurant tout le squelette et l’atome d’azote nécessaire à la finalisation de la synthèse de l’halichonadine C.2- Concevoir une synthèse du cœur central permettant de contrôler la stéréochimie relative des deux fonctions carboxyliques en alpha de l’atome d’azote. Plusieurs stratégies ont été étudiées faisant appel notamment à une double addition de Michael ou à des réactions inspirées de la synthèse de la tropinone. Le cœur central est ainsi accessible en un nombre très limité d’étapes.Les résultats sont très encourageants et la quasi-totalité des pièces du puzzle sont là pour entrevoir rapidement la synthèse totale des halichonadines K et L.- La partie consacrée aux araisoamines (A-D, extraites d’éponges du genre Clathria) est exploratoire et permet de proposer des pistes synthétiques prometteuses pour l’accès bio-inspiré à ces molécules naturelles constituants un défi pour le chimiste. Un des défis relevés dans le travail est de concevoir des analogues synthétiques d’intermédiaires biosynthétiques très réactifs tels que des indoles aldéhydiques très instables. Une méthode pour générer in situ de telles entités a été étudiée. Les premières expériences ont été appliquées à la synthèse des « pyridiniums de Discodermia » et apparaissent prometteurs.Les travaux menés s’inscrivent dans « l’art de la synthèse totale » mais sont aussi toujours en lien avec le souci de mieux comprendre « chimiquement » comment des architectures moléculaires complexes s’assemblent au cours des voies de biosynthèse des substances naturelles
The work described in this PhD dissertation is dedicated to the total synthesis of intriguing natural product structures. Two distinct families of natural substances of marine origin have been targeted in this work: the halichonadins and the araiosamines.- With the halichonadins, we plunge into the marine terpene chemistry. Isolated from sponges of the genus Halichondria, two structures have particularly drawn our attention: halichonadins K and L. Indeed, besides two subunits of terpene origin (namely halichonadin C, a natural isonitrile) with an eudesmane skeleton, a central core of peptidic origin is also original (especially a carboxylic acid disubstituted piperidine ring). A part of the work is dedicated to understanding how, in nature, isonitrile natural products may be formed and may react. The experimental part is organized according to the two following topics:1- Devise an efficient and straightforward total synthesis of halichonadin C. A strategy starting from santonin has been studied and developed. The presence of an isopropyl pending group has attracted many synthetic problems. Anyhow, an advanced intermediate comprising the whole skeleton and the crucial nitrogen atom of the target has been reached and provides good hopes for the access to halichonadin C.2- Conceiving a strategy of the stereocontroled access to the central piperidine ring of halichonadins K and L. Several strategies have been evaluated including the recourse to double Michael additions and reactions inspired by Robinson’s tropinone synthesis. The peptidic central core is now accessible in a limited number of steps.Most of the pieces of the puzzle are in our hands at the end of this PhD to secure a rapid access to the complex targets that constitutes halichonadins K and L.- The chapter dedicated to araiosamines (A-D, isolated from sponges of the genus Clathria) is exploratory and allows to propose promising strategies for a bio-inspired synthesis that constitutes true challenges for the organic chemists. One of the challenges to take up is to prepare highly reactive indole aldehyde units that could be foreseen as chemical equivalents of postulated biosynthetic intermediates. A method to generate in situ such units is studied. The first applications have been directed to the synthesis of “Discodermia pyridiniums” and appear to be promising towards the total synthesis of these molecules.The work conducted during this PhD take place in the framework of the “art of total synthesis”. But, in our strategies, the chemical understanding of biosynthetic pathways is never far away

Isoprenoids are a class of secondary metabolites that are widely distributed in Nature. This thesis describes the synthesis and feeding of isotopically labelled enriched substrates to elucidate features of a new mevalonate independent pathway to isoprenoids. Chapter 2 describes studies with a whole plant system, Mentha citrata, which produces the monoterpene linalyl acetate, and a bacterium Escherichia coli which produces ubiquinone-8. Feeding experiments with stable isotopically enriched compounds demonstrate that the terpene unit of linalyl acetate is biosynthesised via the mevalonate independent pathway, hicorporation of deuterium from [6,6-(^2)H(_2)]-glucose, [(^2)H(_3)]-alanine and [(^13)C(^2)H(_3)]-alanine into luialyl acetate show that the conversion to isopentenyl pyrophosphate does not proceed via a series of dehydrations. Feeding experiments with putative intermediates bearing deuterium into E. coli show that none of the intermediates are incorporated. This suggests that E. coli lacks a kinase to activate exogenously administered substrates fed as the free alcohols. Chapter 3 outlines biosynthesis studies on a meroterpene rosnecatone produced by the fungus R. necatrix. Intact incorporation of (^13)C from the feeding of [l,2-(^13)C(_2)]-acetate shows that the terpenoid moiety is produced via the mevalonic acid pathway and the non-terpenoid unit is polyketide derived. Incorporation of deuterium from [6,6-(^2)H(_2)]-glucose fully describes the pentaketide that delivers the non-terpenoid fragment. The effect on the metabolite production of changing the growth conditions of R. necatrix is investigated. Changing from a static culture to a submerged cultures causes an increased rate of growth, an upregulation of the production of cytochalasan E and a cessation of rosnecatone production. Screening of rosnecatone against two Human cancer cell lines shows IC(_50) values of 4.48µM and 5.78µM.

Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.

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

The stink bugs, (Pentatomidae) harlequin bug (Murgantia histrionica), brown marmorated stink bug (Halyomorpha halys), and southern green stink bug (Nezara viridula) are significant agricultural pests both in the United States and globally. The aggregation or sex pheromones produced by these insects are known to be bisabolene-type sesquiterpenoids; however, the biosynthetic pathways in the formation of these pheromones are unknown. Here we provide evidence that Pentatomidae produce sesquiterpene aggregation pheromones de novo and discuss the evolution of terpene biosynthesis in stink bugs. According to transcriptome analyses, the investigated stink bug species express at least two isoprenyl diphosphate synthases (IDSs), one of which makes (E,E)-farnesyl diphosphate (FPP) as the general precursor in sesquiterpene synthesis, whereas other IDS-type proteins function as terpene synthases (TPSs) generating intermediates in sesquiterpene pheromone formation. The TPS genes are expressed in a sex- and tissue-specific manner. Based on phylogenetic analysis, these IDS-type TPSs arose from trans-IDS progenitors in divergence from bona fide IDS proteins. Compared to microbes and plants, the evolution of TPS function from IDS progenitors in insects appears to have occurred more recently. The discovery of TPS genes in stink bugs provides valuable insight into pentatomid and insect terpene biosynthesis. Moreover, the identified genes may be used in developing alternative management strategies for stink bug pests.
PHD

Cette thèse a pour objectif de développer une nouvelle approche bio-inspirée des processus de polymérisation conduisant à la formation de polyterpènes et ultérieurement du caoutchouc naturel, un polyisoprène de structure purement 1,4-cis de forte masse molaire dont les propriétés sont encore inégalées par ses homologues synthétiques. Pour cette raison la production mondiale de caoutchouc naturel demeure stratégique et est toujours proche de 10 millions de tonnes, soit environ 45% de la demande mondiale annuelle en élastomères. Alors que les voies de synthèse développées jusqu’à présent pour accéder à des homologues du caoutchouc naturel sont basées sur la polymérisation par voie anionique ou par coordination de l’isoprène,l’approche suivie par la nature pour synthétiser les polyterpènes est basée sur la polymérisation à caractère cationique d’un autre monomère, le pyrophosphate d’isopentényle (IPP) qui est la brique de construction universelle de la famille des terpènes. Le projet a pour objet de tenter de reproduire au plus près le procédé naturel et d’utiliser, d’une part, la voie cationique pour polymériser des modèles de l’IPP et l’isoprène en utilisant des homologues du pyrophosphate dediméthylallyle (DMAPP, amorceur employé par la nature) comme amorceurs et des acides de Lewis pour mimer les cations divalents (Zn2+, Mg2+ or Mn2+) qui assistent les enzymes au cours des étapes d’ionisation et d’activation mises en jeu au cours de la biosynthèse, afin d’accéder à des polyterpènes approchant la structure des produits naturels. Contrairement à la nature, nous avons obtenus des oligomères de structure 1,4-trans avec de plus, de nombreuses structures cyclisées. D’autre part, nous avons également cherché à améliorer les propriétés d’un polyisoprène 1,4-cis obtenu par polymérisation anionique afin d’approcher celles du caoutchouc naturel. Nous avons développé une fonctionnalisation des chaines de polyisoprène 1,4-cis permettant d’ancrer des groupements urées susceptibles d’établir un réseau physique de liaisons hydrogènes. Nous avons montré que les polyisoprènes modifiés possèdent des propriétés viscoélastiques comparables à celles du caoutchouc naturel non vulcanisé obtenu par coagulation du latex
Although synthetic rubbers, including high cis-content polyisoprene (PIP), are used in a broad range ofapplications, they are far from achieving the performances of natural rubber (NR), a 100% 1,4-cis polyisoprenewith very high molar mass. Therefore, NR produced exclusively by hevea (whereas more than 2,500 plant species are known to produce polyterpene-based polymers) is still dominant in many engineering applications since its exceptional properties grants this polymer a strategic resource material which holds a significant marketshare (about 45%).. The only alternative plant species under cultivation, Parthenium argentatum, also calledguayule, produces a latex yielding rubber with properties close to those of hevea rubber, and marketed as “non allergenic natural rubber” but with a higher cost complex extraction processes The NR biosynthesis process isdescribed as a polymerization process involving a series of enzymatic reactions using isopentenyl pyrophosphate(IPP) as elementary building brick. Besides, synthetic PIPs with high cis-content were already produced fromisoprene monomer by Ziegler-Natta, lanthanide-based or anionic-type polymerizations. Currently, no syntheticPIPs mimics the performance of NR, maybe because 100% 1,4-cis PIP could so far not been produced synthetically. A close inspection of the NR biosynthesis process led us to postulate that this latter is consistent with a transferless, stereospecific carbocationic-type polymerization mechanism. We then propose to develop this new bio-inspired cationic-like polymerization approach with the aim to produce polyterpenes and then NR homologues of tailored molar mass and microstructure that could exhibit properties close to natural polyterpenes by using IPP homologues and isoprene as monomers, DMAPP homologues asinitiators and Lewis acids to mimick the divalent cations (Zn2+, Mg2+ or Mn2+) that assist the enzymes during the initiation end activation steps. For the cationic polymerization of isoprene, oligomers with 1,4-trans and cyclized structures were obtained. We also develop new routes to modify polyisoprenes obtained by anionic polymerization in order to establish hydrogen interaction. To this end, different urea groups were grafted and the modified polyisoprene exhibite delastomeric properties close to the one of a non-vulcanized NR obtained by latex coagulation

La rose est l'une des plantes ornementales les plus populaires, dont les composés volatils sont non seulement impliqués dans les interactions des fleurs avec l’environnement au sens large, mais aussi largement utilisés dans l’industrie des arômes et parfums. Le chapitre 1 décrit l'histoire de la culture de la rose, les usages de son parfum, les connaissances actuelles sur la biosynthèse des composés de ce parfum, ainsi que les voies de biosynthèse des composés volatils qui ont été récemment élucidées chez différentes plantes. Les chapitres expérimentaux 2 et 3 analysent les fonctions de deux gènes exprimés dans les pétales de rose. Ils codent pour des protéines Nudix hydrolase 1 (NUDX1). Le gène NUDX1-1 (nommé RhNUDX1 dans la publication) a été découvert en comparant les transcriptomes de deux cultivars de rose, Rosa x hybrida cv. 'Papa Meilland' (PM) très parfumé et R. x hybrida cv. 'Rouge Meilland' (RM), dépourvu de parfum. Le gène RhNUDX1-1 n'est exprimé que chez PM et son expression est corrélée avec la production de monoterpènes dans les pétales, en particulier de géraniol. Lors de l'étude d'une descendance issue du croisement de R. chinensis cv. ‘Old Blush’ (OB) et de R. x wichurana (Rw), le gène orthologue RcNUDX1-1a, présentant la même fonction, a été caractérisé chez OB. Un gène paralogue, RwNUDX1-2, a été découvert chez Rw et il a été démontré que son expression présentait une corrélation avec la production sesquiterpènes, en particulier de E,E-farnesol. Une série d'analyses in vitro et in vivo ainsi qu'une analyse de corrélation ont permis de vérifier la fonction de RhNUDX1-1, qui hydrolyse le géranyl diphosphate (GPP) en géranyl monophosphate (GP). Une phosphatase non identifiée pourrait catalyser la transformation du GP en géraniol. Des expériences de fusion avec la Green Fluorescent Protein (GFP), suivies de transformation transitoire de feuilles de tabac, ont révélé que RhNUDX1-1 était localisée dans le cytoplasme. Les mêmes approches (analyses QTL, essais enzymatiques et expression transitoire) ont également été appliquées à RwNUDX1-2, démontrant sa fonction dans la production de E,E-farnesol. La cartographie de RwNUDX1-2 et la localisation subcellulaire de la protéine sont encore à l'étude. De plus, la cristallographie des protéines et la modélisation ont été employées pour étudier le mécanisme de l'interaction NUDX1-substrat et les acides aminés potentiellement importants pour la reconnaissance du substrat. Collectivement, ces données révèlent une voie alternative pour la biosynthèse des terpènes, en particulier le géraniol et E,E-farnesol, via l'hydrolyse des prényl diphosphates par les enzymes NUDX1. Nos résultats montrent que la production de composés volatils dans les pétales est fortement corrélée avec l’expression des gènes des voies de biosynthèse. Par conséquent, la régulation transcriptionnelle de RcNUDX1-1a et RwNUDX1-2 joue probablement un rôle important dans la production de parfum. Les promoteurs de RcNUDX1-1a, RcNUDX1-1b, et RwNUDX1-2 et deux facteurs de transcription (FT), RcbHLH79 (OB TF) et RwbHLH79 (Rw TF) ont ainsi été isolés et testés (Chapitre 4). Les FT candidats ont été choisis lors d’une analyse RNA-Seq (Chapitre 5). En utilisant des tests d'expression transitoire avec le gène rapporteur GUS (β-glucuronidase) dans les pétales de rose, il a été montré que les trois promoteurs pouvaient entraîner l'expression de GUS. Les deux FT ont ensuite été introduits dans des feuilles de tabac avec les promoteurs testés, pour voir s'ils étaient capables d'activer ces promoteurs. Aucune transactivation significative n'a été détectée, même si Rw TF semblait pouvoir activer une construction témoin (promoteur du gène de la tomate TPS5. Les transcriptomes de quatre cultivars de rose, dont deux produisent du géraniol mais pas de E,E-farnesol et deux autres produisent du E,E-farnesol mais pas de géraniol, ont été analysés (Chapitre 5) et ont abouti à une liste de FT putatifs pour une étude plus approfondie
Roses are one of the most popular ornamental plants, whose volatiles are not only involved in environmental interactions but also widely used for industries. Chapter 1 describes the cultivation history of roses, usages of rose fragrance, knowledge on the biosynthesis of rose scent compounds, as well as non-canonical biosynthesis pathways of other plant volatiles. Experimental chapters (Chapter 2 and 3) analyse the functions of two genes expressed in rose petals, both encoding Nudix hydrolase 1 (NUDX1) protein. NUDX1-1 gene (named RhNUDX1) was first discovered by comparing the transcriptomes of two rose cultivars, the scented Rosa x hybrida cv. ‘Papa Meilland’ (PM) and the unscented R. x hybrida cv. ‘Rouge Meilland’ (RM). RhNUDX1-1 was only expressed in scented PM and its expression exhibited a positive correlation with the monoterpenoid production in petals, especially geraniol. When studying a rose progeny of R. chinensis cv. ‘Old Blush’ (OB) and R. x wichurana (Rw), an orthologous gene RcNUDX1-1a was found in OB, whose expression also had positive correlation with geraniol emission. A paralogous gene in Rw, RwNUDX1-2, was discovered and it was shown that its expression displayed a correlation with the sesquiterpenoid production, especially E,E-farnesol. A series of in vitro and in vivo assays as well as correlation analyses verified the function of RhNUDX1-1, which hydrolysed geranyl diphosphate (GPP) to geranyl monophosphate (GP). The transformation of GP into geraniol is supposed to be processed by an, as yet, unidentified phosphatase. The prediction of the localisation together with green fluorescent protein (GFP) fusion experiments revealed that RhNUDX1-1 was located in the cytosol. A series of approaches (QTL analyses, enzymatic assays and transient expression studies) were also applied to RwNUDX1-2, demonstrating its function in the production of E,E-farnesol. Mapping of RwNUDX1-2 and subcellular localization of the protein are still under investigation. Furthermore, protein crystallography and protein modelling illustrated the NUDX1-substrate interaction and proposed several residues that may be important for substrate recognition, although further experimental and computational data are required to gain more insight into the enzymatic mechanism. Collectively, these data revealed an alternative pathway for the biosynthesis of terpenoids, especially geraniol and E,E-farnesol, in rose, via the hydrolysis of prenyl diphosphates by NUDX1 enzymes. Transcriptional regulation of RcNUDX1-1a or RwNUDX1-2 probably plays an important role in the scent production by rose petals. Therefore, three promoters, pOB1a (promoter of RcNUDX1-1a), pOB1b (promoter of RcNUDX1-1b, not expressed in rose petals), pRw (promoter of RwNUDX1-2) were cloned and tested (Chapter 4). In addition, two transcription factors (TFs), RcbHLH79 (OB TF) and RwbHLH79 (Rw TF) candidates were chosen via RNA-Seq analysis as their expression correlated with expression of RcNUDX1-1a or RwNUDX1-2, respectively (Chapter 5). Using transient expression assays with a reporter gene, β-glucuronidase (GUS) in rose petals, it was shown that all three promoters could drive the expression of GUS, suggesting that all of them are active. However, quantification of promoter activities is still needed. OB TF and Rw TF were introduced into Nicotiana benthamiana leaves together with the promoters driving GUS , to determine if they were able to activate these promoters. However, no significant transactivation was detected in any promoter-TF combination. The expression of the TF in the progeny was also analysed but, due to the similarity of the sequences of family members, no conclusive data were obtained. Transcriptomes of the petals four roses, two of which produce geraniol but not E,E-farnesol and two that produce E,E-farnesol but not geraniol, were analysed (Chapter 5) and this resulted in a list of putative scent related genes and transcription factors for further study

Etude de 2 reactions enzymatiques impliquees dans la biosynthese des terpenes: l'isomerisation du pyrophosphate d'isopentyle en pyrophosphate de dimethylallyle, par la ipp-dmapp isomerase, et la synthese de pyrophosphate de geranyle et du pyrophosphate de farnesyle, par la prenyltransferase. Modelisation de ces 2 reactions enzymatiques par l'utilisation d'analogues de structure des intermediaires biosynthetiques supposes. Application a la synthese de divers ethers et esters terbutyliques dans des conditions douces

Molecular biological techniques represent powerful tools with applications to a wide variety of biomedical problems. Described herein is the use of metabolic engineering to manipulate sterol biosynthesis for medicinal purposes. Also presented is the cloning of terpene biosynthetic genes in plants of agricultural and pharmacological importance. Two examples of metabolic engineering of the Saccharomyces cerevisiae sterol biosynthetic pathway are illustrated in Part I. We first demonstrated the development of genetically engineered S. cerevisiae strains that efficiently produce meiosis activating sterols (MAS), which are difficult to obtain by chemical synthesis or isolation from natural sources. Homologous recombination was used to construct an erg24Deltaerg25DeltahemlDelta mutant, which accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25DeltahemlDelta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). This in vivo MAS production needs no added substrate, is technically simpler than chemical synthesis, and provides an inexhaustible source of MAS in relatively high purity and yield. In a different approach to metabolic engineering, we generated recombinant S. cerevisiae strains to screen drugs against the pathogenic parasite Trypanosoma cruzi. The native sterol Delta8-Delta7 isomerase in S. cerevisiae was replaced by the heterologous T. cruzi sterol isomerase and the corresponding human enzyme respectively. The relative effectiveness of the tested compounds on these two strains was compared using simple plate assay and validated by chromatographic methods. This differential screening method identifies inhibitors that specifically target the T. cruzi sterol isomerase with minimal side-effect on the parallel human enzyme and avoids the direct handling of deadly T. cruzi cells. Part II of this thesis describes the characterizations of two plant terpene biosynthetic genes. The saponins in model legume Medicago truncatula may negatively affect the forage digestibility of ruminants, and the aglycones of these saponins are most likely derived from triterpene beta-amyrin. A putative beta-amyrin synthase clone uncovered from M. truncatula Expressed Sequence Tag libraries was expressed in S. cerevisiae , and the recombinant enzyme cyclized 2,3-oxidosqualene cleanly into beta-amyrin. Also, a monoterpene synthase gene from Artemisia annua L., a medicinal plant with anti-malaria activity, was expressed in Escherichia coli, and the in vitro enzymatic reaction with geranyl pyrophosphate afforded (-)-beta-pinene and (-)-alpha-pinene (94:6) as the cyclization products.

The knowledge on the secondary metabolites (SMs) produced by archaea is a field where few research and experimental testing ha s been done, when comparing to bacteria or fungi.The class Halobacteria, composed by haloarchaea that need high concentrations of salt to survive, has been one of the best well studied in the domain Archae a, in this regard. Some examples include the class model organisms Haloferax mediterranei, Haloferax volcanii and Halobacterium salinarum. Haloarchaea produce halocins that are peptides with anti-archaeal activity, but information regarding their biosynthesis and structure is still limited. Genes involved in the production of other peptides with post-translational modifications (RiPPs) have also been identifie d but, so far, none have been isolated or characterized. It is also known that the main carotenoid produced by haloarch aea is bacterioruberin, which has higher antioxidant capacity than β-carotene. Haloarchaea can also produce β-carotene (mainly as a precursor molecule) and canthaxanthin. The objectives of this study were: i) to characterize the diversity of SMs encoded in haloarchaeal genomes, in particular the diversity of genes involved in the production of carotenoids and ii) to evaluate the antibacterial activity of H. mediterranei ATCC 33500 and the impact of the production of bacterioruberin on its antimicrobial activity. Herein, 67 complete genomes of haloarchaea were analysed and 182 biosynthetic gene clusters (BGC) where 49% of them shared no homology. The BGCs identified encode the production of terpenes (70%), RiPPs (16%) and siderophores (14%). A closer inspection of the terpene BGCs allowed their division into two groups: those encoding a bifunctional protein lycopene cyclase/phytoene synthase gene (crtB, 53%) and those encoding a squalene synthase (sqs, 46.5%). The crtB gene was mostly found associated with the biosynthetic genes of bacterioruberin. However, in 22% of the strains, genes from retinal biosynthesis or genes not related with the production of carotene s were found in the proximity of crtB. The results obtained confirmed that all haloarchaeal strains should be able to produce bacterio ruberin. In addition , about 64% of the species may produce β -carotene as a precursor of retinal biosynthesis and 3% as a final product and/or precursor of canthaxanthin synthesis. A gene that putatively encodes a β - carotene ketolase (CrtO) was identified in Haloterrigena turkmenica. To date, these enzymes have not been characterized in haloarch aea, but may be involved in the biosynthesis of cant haxanthin . The crtB gene was associated to the bacterioruberin synthesis pathway through the generation of a kn ock -out mutant and carotenoid quantification. In addition to its anti -archaeal activity , it was determined that H. mediterranei is also able to inhibit the growth of Bacillus cereus. Finally, the H. mediterranei mutants lacking the crtB or sqs genes revealed the same antimicrobial profile as the wildtype strain. Thus, it was concluded that this bioactivity is not influenced by the production of bacterioruberin nor squalenes
O conhecimento dos metabolitos secundários (MS) produzidos por arqueas é bastante limitado, quando comparado com os de bactérias e fungos. No domínio Arquea, a classe Halobacteria, que é constituída por haloarqueas que necessitam de elevadas concentrações de sal para sobreviverem, tem sido das mais bem estudadas a este nível. Alguns exemplos incluem organismos modelo de classes como Haloferax mediterranei, Haloferax volcanii e Halobacterium salinarum. As haloarqueas produzem halocinas, que são péptidos com atividade antiarquea, mas dos quais existe pouca informação relativamente à sua biossíntese e estrutura. Genes envolvidos na produção de outros péptidos com modificações pós-traducionais (RiPPs) foram também já identificados, mas nenhum destes compostos foi isolado ou caracterizado. O principal carotenóide produzido por haloarqueas é a bacterioruberina, que tem despertado o interesse da comunidade científica devido à sua capacidade antioxidante ser superior à do β-caroteno. As haloarqueas também podem produzir β-caroteno (principalmente como percursor) e cantaxantina. Os objetivos deste estudo foram: i) caracterizar a diversidade de MSs codificados nos genomas de haloarqueas, em particular a diversidade dos genes envolvidos na produção de carotenóides e ii) avaliar a atividade antibacteriana de H. mediterranei ATCC 33500 e o impacto da produção de bacterioruberina na sua actividade antimicrobiana. Analisaram-se 67 genomas completos de haloarqueas nos quais foram identificados 182 clusters biossintéticos (CB) sendo que 49% deles não apresentaram homologia entre si. Os CBs detetados codificam a produção de terpenos (70%), RiPPs (16%) e sideróferos (14%). A análise dos CBs de terpenos permitiu a sua divisão em dois grupos: os que codificam a proteína bifuncional licopeno ciclase/fitoeno sintase (crtB, 53%) e os que codificam esqualeno sintases (sqs, 46,5%). O gene crtB foi encontrado maioritariamente associado a genes que codificam as proteínas necessárias para a produção de bacterioruberina. No entanto, em 22% das estirpes, encontraram-se na sua proximidade genes da síntese de retinal ou genes não relacionados com a produção de carotenos. A análise mais detalhada de todos os genes envolvidos no processo de carotenogénese permitiu confirmar que todas as haloarqueas devem produzir bacterioruberina. Para além deste caroteno, cerca de 64% das espécies deverão produzir β-caroteno como precursor da síntese de retinal e 3% como produto final e/ou precursor da síntese de cantaxantina. Foi identificado um gene em Haloterrigena turkmenica, que poderá codificar uma β-caroteno cetolase (CrtO). Até à data, estas enzimas não foram caracterizadas em haloarquea, mas podem estar envolvidas na biossíntese de cantaxantina. Através de mutantes sem o gene crtB, verificou-se que este gene está associado à produção de bacterioruberina. Foi também determinado que, para além de outras haloarqueas, H. mediterranei é capaz de inibir o crescimento de Bacillus cereus. Finalmente, os mutantes de H. mediterranei sem os genes crtB ou sqs revelaram ter o mesmo perfil antimicrobiano da estirpe original. Concluiu-se, portanto, que esta bioatividade não é influenciada pela produção de bacterioruberina nem de nenhum composto com origem em esqualenos
Mestrado em Microbiologia

The yeast Saccharomyces cerevisiae possesses the endogenous precursor for diterpene production but does not biosynthesize diterpenes. Part I of this thesis describes metabolic engineering of S. cerevisiae to achieve diterpene biosynthesis at 500-fold increased production levels (5 mg/L) relative to levels observed in wild type yeast. Induced expression of S. cerevisiae genes geranylgeranyl pyrophosphate synthase (BTS1) and a truncated form of 3-hydroxy-3-methylglutaryl CoA reductase (HMG1) in yeast carrying the upc2-1 allele afforded accumulating geranylgeranyl pyrophosphate, the universal precursor for diterpene biosynthesis. The precursor demonstrated efficient cyclization to 7,13-abietadiene upon coexpression of transformed Abies grandis abietadiene synthase in a multiple copy yeast shuttle vector. Similarly, metabolic engineering of S. cerevisiae allowed investigation of achieving attenuated sesquiterpene production in vivo; those results and possible physiological implications to yeast are discussed. The recombinant strains serve as an alternative means of access to natural products via a novel in vivo production system. Part II describes the characterization of three sterol biosynthetic enzymes. Higher plants catalyze the cyclization of (S)-2,3-oxidosqualene to cycloartenol. A single point mutation demonstrated altered sterics contributing to catalytic product specificity; two point mutations are characterized and evaluated. Cycloartenol constitutes a cyclopropyl sterol structure not found in other eukaryotes. In an effort to better understand the roles served by the cyclopropyl sterols, the characterization of the gene responsible for their metabolism is described. Understanding this point of evolutionary divergence can facilitate accurate phylogenetic analysis among eukaryotes.