À l'heure actuelle, il y a un regain d'intérêt pour Clostridium acetobutylicum, le biocatalyseur du procédé Weizmann historique, pour produire le n-butanol un produit chimique de commodité et un bio-carburant alternatif et renouvelable . Ce mémoire de thèse décrit un procédé de recombinaison homologue, utilisant plasmide réplicatif, pour la délétion ou l'introdu ction de gènes chez C. acetobutylicum avec une élimination facile des marqueurs utilisés. La souche de C. acetobutylicum cacl502upp et ce système de recombinaison homologue ont été utilisés dans d'autres expériences d'ingénierie pour obtenir une souche produisant du n-butanol avec une sélectivité élevée et en éliminant la plupart des co-produits. Le mutant final, C. acetobutylicum (C. acetobutylicum CAB1060) a été généré avec succès. Cette souche CAB1060 a été utilisée dans un nouveau procédé de fermentation continu qui utilise i) l'extraction in situ des alcools par distillation sous pression réduite et ii) des cultures à haute densité cellulaire (et ne faisant pas intervenir de procédé membranaire) pour atteindre des titre, rendement et productivité en n-butanol qui n'ont jam ais été obtenus chez aucun micro-organisme.Un second procédé de recombinaison homologue utilisant un plasmide non réplicatif pour la modification de gène sans marqueur est également décrit dans le présent mémoire. Cette méthode permet d'inactiver simultaném ent deux gènes. Il a été utilisé avec succès pour la construction d'un mutant incapable de produire de l'hydrogène et utile, comme souche plate-forme, pour l'ingénierie de C. acetobutylicum pour produire en continu des produits chimiques de commodité et des bio­ carburants. / Current ly, there is a resurgence of interest in Clostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to produce n-butanol for use both as a bulk chemical and as a renewablc alternative transportation fuel. This thesis describes a method of homologous recombination by replicative plasmid to delete or introduce genes in C. acetobutylicum . This method was successfull y used to delete genes, includin g CACJ502, CAC3535, CAC2879 (upp), to generate C. acetobutylicum. These strains are readily transformable without any previous plasmid methylation and can serve as hosts for a "marker-less" genetic exchange system. A mutant C. acetobutylicum (C. acetobuty licum CAB 1060) was successfully genera ted. This final mutant produces mainly bu tanol, with ethanol and traces of acetate at a molar rati o of 7:1 :1 . This CAB 1060 strain was subjected to a new continuous fermentation process using i) in situ extraction of alcohols by distillation under low pressure and ii) high cell density cultures to increase the titer, yield and productivity of n-butanol production to levels that have never been previously açhieved in any organism . A second homologous recombination method using non-replicative plasmid for marker less gene modification is also described in this thesis. This method allows the simultaneou s inactivation of two genes. lt has been successfully used to construct a mutant unable to produce hydrogen and useful, as a platform strain, for further engineering of C. acetobutylicum to continuously produce bulk chemicals and fuels.

Squalene synthase (SS) is an essential enzyme in eukaryotic systems responsible for an important branch point in isoprenoid metabolism that leads to sterol formation. The mechanistic complexity of SS has made it a difficult enzyme to study. The green alga Botryococcus braunii race B possesses several squalene synthase-like (SSL) enzymes that afford a unique opportunity to study the complex mechanism of triterpene biosynthesis. SSL-1 catalyzes presqualene diphosphate (PSPP) formation, which can either be converted to squalene by SSL-2 or botryococcene by SSL-3. A rationally designed mutant study of B. braunii squalene synthase (BbSS) and SSL-3 was conducted to understand structure-function relations among these enzymes. These studies revealed two amino acid positions in SSL-3 (N171, G207) that appeared to control 1’-3 versus 1’-1 linkages. The reciprocal mutations in the corresponding positions of BbSS did not convert this enzyme into a botryococcene synthase. Next, a genetic selection was developed to evolve SSL enzymes towards a fully functional SS. Previous studies have shown that Saccharomyces cerevisiae squalene synthase (ScSS) can be knocked out and although lethal, growth can be restored by providing an exogenous source of ergosterol. Additional studies have shown that successful complementation of the ScSS knockout with a non-fungal SS is possible but requires a fungal SS carboxy- terminus region. Given these observations, proof-of-principle experiments were conducted to demonstrate that SSL-SSL fusion enzymes could complement the ScSS knockout followed by construction of a mutant SSL-SSL fusion enzyme library that was screened in the ScSS knockout yeast line. From this library, mutant SSL-SSL fusion enzymes were identified that were able to complement, which demonstrated the feasibility of this approach as a genetic selection for mutant SSL enzymes. Squalene and botryococcene have valuable industrial applications in vaccine adjuvant formations, cosmetic products, and renewable energy feedstock material. Limitations in natural sources of these molecules have made heterologous production of them an important research target. Algae represent a desirable group of organisms that could be engineered to produce these metabolites because they are photosynthetic and capable of using non-arable farmland. The feasibility, approach, and progress for engineering green algae to produce squalene and botryococcene are discussed.

La partie oligosaccharidique des glycoconjugués, présents à la surface des cellules eucaryotes, intervient dans de nombreux processus biologiques de reconnaissance et d'adhésion cellulaire. L'essor de la glycobiologie au cours des vingt dernières années a permis de définir, à partir de l'implication de ces structures glycaniques, de nombreuses applications thérapeutiques potentielles. Cependant la fabrication de nouveaux médicaments à partir d'oligosaccharidiques requiert leur disponibilité en grande quantité mais leurs obtentions par purification ou par méthode de synthèse chimique et enzymatique restent difficiles et couteuses et donnent un rendement faible. Le laboratoire CERMAV a récemment développé une technologie cellulaire, non polluante, capable de produire rapidement et en grande quantité un certain nombre d'oligosaccharides d'intérêt biologique. Le procédé baptisé « usine cellulaire » repose sur la co-expression de glycosyltransférases recombinantes chez la bactérie Escherichia coli. La sulfatation des glycanes est un élément important de leurs propriétés biologiques. Celle-ci dépend de l'activité de sulfotransférases, dont l'activité chez Escherichia coli a été peu étudiée. Par contre, ces enzymes utilisent des accepteurs oligosaccharidiques dont la synthèse est maîtrisée par le procédé d'usine cellulaire. Nous avons exprimé des gènes de sulfotransférases afin de permettre in vivo la sulfatation d'accepteurs oligosaccharidiques endogènes produits dans la bactérie. Les familles de molécules synthétisées dériveront des motifs GlcAlac, Lacto-N-néotétraose, et LewisX dont la synthèse in vivo est maîtrisée. Nous avons également réalisé une synthèse combinée chimio-enzymatique d'une néoglycoprotéine porteuse d'un analogue de l'épitope HNK-1, déterminant sulfaté impliqué notamment dans la régénération des motoneurones. Finalement nous avons entrepris une étude préliminaire d'adaptation du procédé d'« usine cellulaire » à la synthèse de glycanes sulfatés chez Saccharomyces cerevisiae. / The oligosaccharide moety of glycoconjugates, present on the eukaryotic cell surface, is involved in many biological processes of recognition and cell adhesion. The rise of glycobiology over the last twenty years has helped to define, from the involvement of these glycan structures, many potential therapeutic applications. However, the manufacture of new drugs from oligosaccharide requires their availability in large quantities but their varieties by purification or by method of chemical and enzymatic synthesis remain difficult and expensive and lead to low yield. CERMAV has recently developed a clean and fast cell technology, which is able of producing large quantities of several oligosaccharides of biological interest. The process called "cell factory" is based on the co-expression of recombinant glycosyltransferases in Escherichia coli. Sulfation of glycans is an important part of their biological properties and depends on the activity of sulfotransferase, whose activity in Escherichia coli has not been well studied. But these enzymes use oligosaccharide acceptor whose synthesis is controlled by the process of cell factory. We expressed genes of sulfotransferase to allow the in vivo sulfation of endogenous oligosaccharide acceptor produced in the bacterium. Families of molecules synthesized drift patterns of GlcAlac, Lacto-N-neotetraose, and Lewisx whose synthesis in vivo is controlled. We also performed a combined chemo-enzymatic synthesis of a neoglycoprotein bearing an analogue of the epitope HNK-1, involved particularly in the regeneration of motoneurons. Finally we tried to adapt the method of "cell factory” for the synthesis of sulfated glycans in a eukaryotic organism such as yeast Saccharomyces cerevisiae.

Catharanthus roseus est une plante médicinale produisant divers types d’alcaloïdes indoliques monoterpéniques (AIM) d’intérêt en santé humaine. Ainsi, les AIM dimères comme la vinblastine et la vincristine sont utilisés en chimiothérapie anticancéreuse et les alcaloïdes monomères de type hétéroyohimbine présentent diverses activités pharmacologiques. La fabrication de ces molécules dans la plante est fort complexe. Elle requiert un haut niveau de compartimentation tissulaire et subcellulaire et met en jeu plus d’une trentaine d’étapes enzymatiques, dont certaines sont encore très mal connues. Dans ce contexte, l’objectif de la thèse a consisté à élucider plusieurs étapes enzymatiques de la voie de biosynthèse des AIM. Nos travaux ont permis de caractériser de nouvelles isoformes enzymatiques de la famille des cytochromes P450 ainsi que les réductases qui leur sont associées. Ils ont abouti à l’identification de nouvelles déshydrogénases et mis en évidence, in planta, leurs interactions avec la strictosidine synthase suggérant une biosynthèse orientée vers les divers alcaloïdes de type hétéroyohimbine. Enfin, en ayant recours à l’ingénierie métabolique, un segment de la voie de biosynthèse a été transféré dans la levure Saccharomyces cerevisiae, lui conférant la capacité de bio-transformer la tabersonine en vindoline, l’un des deux précurseurs finaux des alcaloïdes dimères. / Catharanthus roseus is a medicinal plant producing various types of monoterpene indole alkaloids (MIA) with a great interest in human health. Dimeric alkaloids such as vinblastine and vincristine are used in cancer chemotherapy and monomeric heteroyohimbine alkaloids exhibit various pharmacological activities. The production of these molecules in the plant is very complex. It requires a high level of tissular and subcellular compartmentalization and involves more than thirty enzymatic steps, some of which are largely unknown. In this context, the aim of this thesis was to elucidate several enzymatic steps of the MIA biosynthetic pathway. Our work allowed us to characterize new enzyme isoforms of cytochrome P450 and their associated reductases. They also resulted in the identification of new dehydrogenases and highlighted their interactions with the strictosidine synthase suggesting a directed biosynthesis towards various heteroyohimbine type of alkaloids. Finally, engineered yeast containing a segment of the MIA biosynthetic pathway was able to convert tabersonine into vindoline, one of the two final precursors of the dimeric alkaloids.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / The direct use of vegetable oils as a biofuel suffers from problems such as high viscosity, low volatility and poor cold temperature properties. 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) have lower viscosity and freezing temperature than regular vegetable oils. However, by modifying their fatty acid composition, further improvement in their fuel properties is possible. Our goal was to develop plants that synthesize seed oils with further improved fuel properties. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes acetyl-TAGs by the acetyl-CoA dependent acylation of diacylglycerol (DAG). Knowledge of the substrate specificity of EaDAcT for its acetyl-CoA donor and DAG acceptor substrates is important to generate the required acetyl-TAG composition in seed oil. A rapid method to quantify acetyl-TAGs was developed based on electrospray ionization mass spectrometry to gain information about the substrate specificity of EaDAcT. This method is as accurate and more rapid than the traditional radiolabeled substrate based assay and additionally provides information on acetyl-TAG molecular species present. Using this assay, EaDAcT specificity for different chain length acyl-CoA and DAGs was tested. It was found that although EaDAcT can use other short chain length acyl-CoAs as acyl donors, it has high preference for acetyl-CoA. Further, EaDAcT can acetylate a variety of DAGs with short, medium and long chain length fatty acids with high preference for DAGs containing unsaturated fatty acids. To generate acetyl-TAGs with lower molecular mass, EaDAcT was transformed into transgenic Camelina sativa lines producing high amounts of medium chain fatty acids (MCFAs). EaDAcT expression was also combined with the knockdown of DGAT1 and PDAT enzymes, which compete with EaDAcT for their common DAG substrate. High acetyl-TAG yielding homozygous T3 transgenic lines were generated but the incorporation of MCFAs into acetyl-TAGs was inefficient. A small increase in the viscosity of acetyl-TAGs from these lines was observed compared to acetyl-TAGs produced in wild type Camelina plant. The combined effect of insufficient lowering of molecular mass and increased fatty acid saturation levels of acetyl-TAGs might be responsible for this increased viscosity. Overall, it was concluded that the molecular mass and the saturation levels of fatty acids of acetyl-TAGs need to be considered at the same time in future attempts to further decrease their viscosity.

Catharanthus roseus est une plante tropicale de la famille des Apocynacées d’intérêt thérapeutique en raison de sa capacité à synthétiser des alcaloïdes indoliques monoterpéniques (AIM) utilisés en chimiothérapie anticancéreuse. La teneur en AIM in planta est très faible notamment en raison d’une haute compartimentalisation cellulaire et subcellulaire de la voie de biosynthèse. Si la compartimentalisation cellulaire était bien caractérisée, très peu de données de localisation subcellulaire in situ étaient disponibles au début de cette thèse. Une connaissance fine de cette compartimentalisation est cependant nécessaire pour identifier les transports inter-compartiment de métabolites intermédiaires, limitant potentiellement le flux métabolique, afin d’améliorer ensuite le rendement de biosynthèse des AIM par ingénierie métabolique. Dans ce contexte nous avons réalisé une étude exhaustive de la localisation subcellulaire des enzymes de cette voie par imagerie GFP dans des cellules de C. roseus transformées par biolistique permettant d’établir un nouveau modèle intégré d’organisation cellulaire et subcellulaire de la biosynthèse des AIM. / Catharanthus roseus is a tropical plant from the Apocynaceae family with a great therapeutic value due to its ability to synthesize monoterpene indole alkaloids (MIA) used in cancer treatment. The yields of these molecules in planta are very low due to a very high level of compartmentation of the biosynthetic pathway at both cellular and subcellular levels. While the cellular compartmentation was widely characterized, very few in situ subcellular localization data were available at the beginning of this PhD. An accurate knowledge of this compartmentation is necessary to identify intermediate metabolites transport events from one compartment to another one, in order to increase the MIA biosynthesis yield by metabolic engineering approaches. In this context we have proceed to the exhaustive study of the subcellular localization of these enzymes by in vivo GFP imaging in C. roseus cells transformed by biolistic. Potential interprotein interactions of these enzymes have also been studied by BiFC. Altogether, our results enabled us to draw an integrated model of the cellular and subcellular organization of MIA biosynthesis in situ.

Les chercheurs en biologie de synthèse programment l’ADN pour construire des systèmes biologiques capables de répondre à certaines conditions de manière prédéfinie. Cette capacité pourrait avoir un impact sur plusieurs domaines, de la médecine à la fermentation industrielle. Le traitement de signal par des circuits biologiques synthétiques est en train d’être démontré à large échelle, mais hélas la variété des signaux d’entrée capables de contrôler ces circuits est pour l’instant limitée. Ce manque de diversité est un obstacle majeur au développement de nouvelles applications car en général chaque application requiert une réponse à des signaux de nature particulière qui lui sont spécifiques. Cette thèse cherche à apporter des solutions au manque de signaux d’entrée appropriés contrôlant les circuits biologiques en développant deux nouvelles stratégies d’induction. La première stratégie vise à étendre la diversité chimique des signaux d’entrée. A l’inverse des approches existantes, qui reposent sur la modification des systèmes de détections naturels tels que les riboswitchs ou les facteurs de transcription allostériques, j’ai cherché ici à modifier directement des molécules préalablement non-détectables afin de les rendre détectables par les systèmes de détection actuels. Pour ce faire, la transformation chimique des molécules cibles est réalisée in situ grâce à l’expression de voies métaboliques synthétiques dans la cellule. Afin de pouvoir utiliser cette stratégie de manière systématique, j’ai employé la conception assistée par ordinateur et puisé dans l’ensemble des réactions biochimiques connues afin de prédire des voies de détections pour de nouvelles molécules. J’ai ensuite implémenté in vivo plusieurs prédictions qui ont permis à E. coli de détecter de nouveaux composés. Au-delà de l’intérêt de cette méthode en biotechnologie, cela montre que le métabolisme peut jouer un rôle dans le transfert d’information, en plus de son rôle dans le transfert de matière et d’énergie, ce qui soulève la question de l’utilisation potentielle de cette stratégie de détection par la nature. Un second axe présente une façon d’épargner l’utilisation d’inducteurs chimiques pour les programmes biologiques simples, et propose d’utiliser des inducteurs biologiques à la place. Lorsqu’une seule étape d’induction ou de répression de gènes est nécessaire, comme c’est le cas en fermentation industrielle, je propose de remplacer la coûteuse étape d’induction chimique par l’infection simultanée de toutes les cellules d’une population par des particules virales capables d’injecter en temps réel l’ensemble des informations nécessaires pour déclencher l’activité biologique recherchée. A des fins de fermentation, j’ai développé des particules virales modifiées qui reprogramment dynamiquement le métabolisme d’une large population de bactérie au moment opportun et les forcent à produire des molécules à haute valeur ajoutée. / Synthetic biologists program DNA with the aim of building biological systems that react under certain conditions in a predefined way. This ability could have impact in several fields, from medicine to industrial fermentation. While the scalability of synthetic biological circuits in terms of signal processing in now almost demonstrated, the variety of input signals for these circuits is limited. Because each application typically requires a circuit to react to case-specific molecules, the lack of input diversity is a major obstacle to the development of new applications. Two axis are developed over the course of this thesis to try to address input-related problems. The main axis consists in a new strategy aiming at systematically and immediately increasing the chemical diversity of inputs for synthetic circuits. Current approaches to expand the number of potential inputs focus on re-engineering sensing systems such as riboswitches or allosteric transcription factors to make them react to previously non-detectable molecules. On the contrary, here we developed a method to transform the non-detectable molecules themselves into molecules for which sensing systems already exist. These chemical transformations are realized in situ by expressing synthetic metabolic pathways in the cell. In order to systematize this strategy, we leveraged computer-aided design to predict ways of detecting new molecules by digging into all known biochemical reactions. We then implemented several predictions in vivo that successfully enabled E. coli to detect new chemicals. Aside from the interest of the method for biotechnological applications, this shows that in addition to transferring matter and energy, metabolism can also play a role in transferring information, raising the question of potential occurrences of this sensing strategy in nature. A second axis introduce a way to exempt simple programs from the need for a chemical input, and explore the use of a biological input instead. In situations where a single timely induction or repression of multiple genes is required, such as in industrial fermentation processes, we propose to replace expensive chemical induction by simultaneous infection of all the members of a growing population of cells with viral particles inputting in real-time all the necessary information for the task at hand. In the context of fermentation, we developed engineered viral particles that can dynamically reprogram the metabolism of a large population of bacteria at the optimal stage of growth and force them to produce value-added chemicals.

In the first part of this research, we constructed a Genome scale Metabolic Model (GEM) of Taxus cuspidata, a medicinal plant used to produce paclitaxel (Taxol®). The construction of the T. cuspidata GEM was predicated on recent acquisition of a transcriptome of T. cuspidata metabolism under methyl jasmonate (MJ) elicited conditions (when paclitaxel is produced) and unelicited conditions (when paclitaxel is not produced). Construction of the draft model, in which transcriptomic data from elicited and unelicited conditions were included, utilized tools including the ModelSEED developed by Argonne National Laboratory. Although a model was successfully created and gapfilled by ModelSEED using their software, we were not able to reproduce their results using COBRA, a widely accepted FBA software package. Further work needs to be done to figure out how to run ModelSEED models on commonly available software. In the second part of this research, we modeled the MJ elicited/defense response phenotype in Arabidopsis thaliana. Previously published models of A. thaliana were tested for suitability in modeling the MJ elicited phenotype using publicly available computation tools. MJ elicited and unelicited datasets were compared to ascertain differences in metabolism between these two phenotypes. The MJ elicited and unelicited datasets were significantly different in many respects, including the expression levels of many genes associated with secondary metabolism. However, it was found that the expression of genes related to growth and central metabolism were not generally significantly different for the MJ+ and MJ- datasets, the pathways associated with secondary metabolism were incomplete and could not be modeled, and FBA methods did not show the difference in growth that was expected. These results suggest that behavior associated with the MJ+ phenotype such as slow growth and secondary metabolite production may be controlled by factors not easily modeled with transcriptome data alone. Additional research was performed in the area of cryosectioning and immunostaining of fixed Taxus aggregates. Protocols developed for this work can be found in Appendix B.

Growing concerns of the negative effects on the environment and dependency of fossil fuelsare major driving forces for finding novel sustainable production pathways for plastic.Metabolic engineering has emerged as a powerful tool to enable microorganisms to producenon-native metabolites. The aim of this project was recombinant production of 4-hydroxybutyrate (4-HB) by expressing two enzymes in the model organism Escherichia coli.α-ketoglutarate decarboxylase (SucA) from Mycobacterium smegmatis followed by 4-hydroxybutyrate dehydrogenase (4-HBd) from Clostridium kluyveri was expressed inEscherichia coli. Results showed that the genes were successfully transformed and expressedin E. coli and after protein purification a concentration of 0.9 g/L SucA and 9.8 g/L 4-HBdwas achieved. Furthermore, some protein activity was detected by a coupled reaction withSucA and 4-HBd. When the enzymes got coupled together a change in NADH concentrationcould be detected spectrophotometrically. The enzymes were also tested for substratespecificity by using substrates with various carbon chain lengths and a decrease in NADHconcentration was seen. However, a decrease in the negative control for the experiments wasalso seen indicating a breakdown of NADH over time rather than consumption. Therefore, noconclusion could be drawn about the promiscuity of the enzymes. Lastly a single plasmidssystem was tested where both the genes were ligated on the same plasmid (pCDF duet) andexpressed successfully in E. coli Bl21DE3. / Ökad oro för miljön samt behovet av fossila resurser för produktion av plaster har gjort detnödvändigt att skapa nya och mer hållbara produktions vägar. Genetisk modifikation av olikaorganismer har utvecklats som ett starkt redskap för att få mikroorganismer att framställametaboliter som de normalt inte producerar. Målet med detta projekt var rekombinantproduktion av gamma hydroxibutansyra (4-HB) genom att uttrycka två enzym i modellorganismen Escherichia coli. Dessa enzym bestod av α-ketoglutarat dekarboxylas (SucA) frånMycobacterium smegmatis samt 4-hydroxybutyrate dehydrogenas (4-HBd) från Clostridiumkluyveri. Resultaten visade att proteinerna lyckades utryckas i E. coli med en koncentration av0,9 g/L SucA och 9,8 g/L 4-HBd som uppnåddes efter rening. Utöver detta detekterades ävenviss enzymaktivitet genom att kopplad enzymreaktion mellan 4-HBd och SucA och mätakonsumtionen av NADH spektrofotometriskt över tid. Enzymen testades även försubstratspecificitet genom att köra reaktionen med substrat med olika längd på kolkedjan. Dåkunde en minskning i NADH koncentrationen ses men det gjordes det även för de negativakontrollerna vilket indikerar nedbrytning av NADH och inte konsumtion av NADH. Ingaslutsatser angående enzymens substratspecificitet kunde därför dras. Det sista som gjordes varatt sätta in båda generna i ett en plasmidsystem där båda generna sattes in på samma plasmid(pCDF duet) och uttrycktes framgångsrikt i E. coli Bl21DE3.

No description available.