Mucosal passive immunization is the transfer of active antibodies from one organism to the mucosal surfaces of another organism for preventing or treating infectious diseases. Mucosal passive immunization has a great potential for the prevention and treatment of enteric infections like Rotavirus, which causes more than 114 million episodes of diarrhoea annually with a death toll of more than 450.000 per year. However, the high cost of recombinant antibodies with the current manufacturing systems based on mammalian cells hampers the production of the high antibody quantities required for passive immunization strategies. Alternative expression platforms such as plants could provide higher scalability and reduced costs. Moreover, the use of edible plant organs, which are Generally¿Regarded¿As¿ Safe (GRAS), could reduce manufacturing costs even further by easing the requirements for antibody purification. We analyze here the feasibility of utilizing fruits as inexpensive biofactories of human antibodies that can be orally delivered as crude extracts or partially purified formulations in mucosal passive immunization strategies. In the first section of this thesis, the construction of tomato plants producing a model human Immunoglobulin A (IgA) against rotavirus in their fruits is described. As a result, an elite homozygous line was obtained whose fruits produced on average 41 ¿g of IgA per gram of fresh weigh, equivalent to 0.69 mg IgA per gram of dry tomato powder. Minimally processed products derived from IgA¿expressing tomatoes were shown to strongly inhibit virus infection in an in vitro neutralization assay. Moreover, in order to make IgA¿expressing tomatoes easily distinguishable from wild¿type tomatoes, they were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. The resulting transgenically¿labelled purple tomatoes contained not only high levels of recombinant neutralizing human IgA but also increased amounts of anthocyanins. In the second section of the thesis the composition of IgA¿expressing tomatoes was analyzed in search of possible unintended effects that could compromise the GRAS status of the final product. To this end, transgenic IgA¿tomatoes were compared with wild type tomatoes and also commercial tomato varieties using proteomic and metabolomic approaches. 2D¿DIGE gels coupled with LC¿MSMS for protein identification showed that all the uptrend differential proteins detected corresponded only to immunoglobulin chains or antibody fragments. On the other hand, non¿targeted metabolite data obtained by UPLC¿MS / Juárez Ortega, P. (2014). Production of recombinant Immunoglobulin A in plants for passive immunotherapy [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37015

In order to decrease our dependence on non-renewable petrochemical resources, it is urgently required to establish sustainable biomass-based biorefineries. Replacing fossil fuels with renewable biomass as a raw feedstock for the production of chemicals and biofuels is a main driving force of biorefinering. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents cellular constraints so that it can implement some biotransformations better than whole-cell fermentation, which spends a fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries. Hydrogen gas is a promising future energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen from renewable biomass will be vital to the hydrogen economy. Substrate costs contribute to a major portion of the production cost for low-value bulk biocommodities, such as hydrogen. The reconstitution of 17 thermophilic enzymes enabled to construct an artificial enzymatic pathway converting all glucose units of starch, regardless of the branched and linear contents, to hydrogen gas at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. Using a biomimetic electron transport chain, a maximum volumetric productivity was increased by more than 200-fold to 90.2 mmol of H2/L/h at a high starch concentration from the original study in 2007. In order to promote economics of biorefineries, the production of a sugar phosphate and a fourth-generation sweetener is under development. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. Under the optimized condition, 32 mM Xu5P was produced from 50 mM xylose and polyphosphate, achieving a 64% conversion yield, after 36 h at 45 °C. L-arabinose, a FDA-approved zero-calorie sweetener, was produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and xylulose 4-epimerase (Xu4E). Promiscuous activity of Xu4E, a monosaccharide C4-epimerase, was discovered for the first time. Directed evolution of Xu4E enabled to increase the catalytic function of C4-epimerization on D-xylulose as a substrate by more than 29-fold from the wild-type enzyme. Together, these results demonstrate that the in vitro synthetic biosystem as a feasible biomanufacturing platform has great engineering, and can be used to convert renewable biomass resources to a spectrum of marketable products and renewable energy. As future efforts are addressed to overcome remaining challenges, for example, decreasing enzyme production costs, prolonging enzyme lifetime, engineering biomimetic coenzymes to replace natural coenzymes, and so on. This in vitro synthetic biology platform would become a cornerstone technology for biorefinery industries and advanced biomanufacturing (Biomanufacturing 4.0). / Ph. D. / The carbon cycle is the circulation and transformation of carbon back and forth between living things and the environment. With the fixed amount of carbon dioxide in the atmosphere, the carbon cycle has been in the balance of exchanges between living things and the environment. As we evolve with increasing demand on crude oil, however, significant amounts of carbon are being released into the atmosphere much faster than they would have been released naturally. This rapid release is the primary cause of currently observed global warming. In order to decrease our dependence on petrochemical products, the biorefinery was introduced as the sustainable processing of biomass into a spectrum of alternatives to products from petrochemical refineries. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents whole cell’s constraints, so that it can implement some biotransformations better than whole-cell fermentation spending a significant fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen gas, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries. Hydrogen gas is a promising energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen will be vital to the hydrogen economy. We demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch to hydrogen a theoretic yield, which is three times of the theoretical yield from dark microbial fermentation. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. This minimal in vitro enzymatic pathway was optimized for improved conversion yield and productivity. L-arabinose, a FDA-approved zero-calorie sweetener, was also produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and hypothetical enzyme xylulose 4-epimerase (Xu4E), a monosaccharide 4-epimerase that can convert D-xylulose to L-ribulose. Xu4E activities due to substrate promiscuity of some natural 4-epimerases were discovered for the first time. Three rounds of directed evolution have been conducted to increase the catalytic function of carbon 4-epimerization on D-xylulose. As the result, the catalytic activity of Xu4E was improved by more than 29-fold from the wild-type enzyme.

Tesis por compendio / [ES] La mejora genética vegetal tiene como objetivo la obtención de plantas con rasgos mejorados o características novedosas que podrían ayudar a superar los objetivos de sostenibilidad. Para este fin, la biotecnología vegetal necesita incorporar nuevas herramientas de ingeniería genética que combinen una mayor precisión con una mayor capacidad de mejora. Las herramientas de edición genética recientemente descubiertas basadas en la tecnología CRISPR/Cas9 han abierto el camino para modificar los genomas de las plantas con una precisión sin precedentes. Por otro lado, los nuevos enfoques de biología sintética basados en la modularidad y la estandarización de los elementos genéticos han permitido la construcción de dispositivos genéticos cada vez más complejos y refinados aplicados a la mejora genética vegetal. Con el objetivo final de expandir la caja de herramientas biotecnológicas para la mejora vegetal, esta tesis describe el desarrollo y la adaptación de dos nuevas herramientas: una nueva endonucleasa específica de sitio (SSN) y un interruptor genético modular para la regulación de la expresión transgénica. En una primera parte, esta tesis describe la adaptación de CRISPR/Cas12a para la expresión en plantas y compara la eficiencia de las variantes de Acidaminococcus (As) y Lachnospiraceae (Lb) Cas12a con Streptococcus pyogens Cas9 (SpCas9) descritos anteriormente en ocho loci de Nicotiana benthamiana usando expresión transitoria. LbCas12a mostró la actividad de mutagénesis promedio más alta en los loci analizados. Esta actividad también se confirmó en experimentos de transformación estable realizados en tres plantas modelo diferentes, a saber, N. benthamiana, Solanum lycopersicum y Arabidopsis thaliana. Para este último, los efectos mutagénicos colaterales fueron analizados en líneas segregantes sin la endonucleasa Cas12a, mediante secuenciación del genoma descartándose efectos indiscriminados. En conjunto, los resultados muestran que LbCas12a es una alternativa viable a SpCas9 para la edición genética en plantas. En una segunda parte, este trabajo describe un interruptor genético reversible destinado a controlar la expresión génica en plantas con mayor precisión que los sistemas inducibles tradicionales. Este interruptor, basado en el sistema de recombinación del fago PhiC31, fue construido como un dispositivo modular hecho de partes de ADN estándar y diseñado para controlar el estado transcripcional (encendido o apagado) de dos genes de interés mediante la inversión alternativa de un elemento regulador central de ADN. El estado del interruptor puede ser operado externa y reversiblemente por la acción de los actuadores de recombinación y su cinética, memoria y reversibilidad fueron ampliamente caracterizados en experimentos de transformación transitoria y estable en N. benthamiana. En conjunto, esta tesis muestra el diseño y la caracterización funcional de herramientas para la ingeniería del genómica y biología sintética de plantas que ahora ha sido completada con el sistema de edición genética CRISPR/Cas12a y un interruptor genético reversible y biestable basado en el sistema de recombinación del fago PhiC31. / [CA] La millora genètica vegetal té com a objectiu l'obtenció de plantes amb trets millorats o característiques noves que podrien ajudar a superar els objectius de sostenibilitat. Amb aquesta finalitat, la biotecnologia vegetal necessita incorporar noves eines d'enginyeria genètica que combinen una major precisió amb una major capacitat de millora. Les eines d'edició genètica recentment descobertes basades en la tecnologia CRISPR/Cas9 han obert el camí per modificar els genomes de les plantes amb una precisió sense precedents. D'altra banda, els nous enfocaments de biologia sintètica basats en la modularitat i l'estandardització dels elements genètics han permès la construcció de dispositius genètics cada vegada més complexos i sofisticats aplicats a la millora genètica vegetal. Amb l'objectiu final d'expandir la caixa d'eines biotecnològiques per a la millora vegetal, aquesta tesi descriu el desenvolupament i l'adaptació de dues noves eines: una nova endonucleasa específica de lloc (SSN) i un interruptor genètic modular per a la regulació de l'expressió transgènica . En una primera part, aquesta tesi descriu l'adaptació de CRISPR/Cas12a per a l'expressió en plantes i compara l'eficiència de les variants de Acidaminococcus (As) i Lachnospiraceae (Lb) Cas12a amb la ben establida Streptococcus pyogens Cas9 (SpCas9), en vuit loci de Nicotiana benthamiana usant expressió transitòria. LbCas12a va mostrar l'activitat de mutagènesi mitjana més alta en els loci analitzats. Aquesta activitat també es va confirmar en experiments de transformació estable realitzats en tres plantes model diferents, a saber, N. benthamiana, Solanum lycopersicum i Arabidopsis thaliana. Per a aquest últim, els efectes mutagènics col·laterals van ser analitzats en línies segregants sense l'endonucleasa Cas12a, mitjançant seqüenciació completa del genoma i descartant efectes indiscriminats. En conjunt, els resultats mostren que LbCas12a és una alternativa viable a SpCas9 per a l'edició genètica en plantes. En una segona part, aquest treball descriu un interruptor genètic reversible destinat a controlar l'expressió gènica en plantes amb major precisió que els sistemes induïbles tradicionals. Aquest interruptor, basat en el sistema de recombinació del bacteriòfag PhiC31, va ser construït com un dispositiu modular fet de parts d'ADN estàndard i dissenyat per controlar l'estat transcripcional (encès o apagat) de dos gens d'interès mitjançant la inversió alternativa d'un element regulador central d'ADN. L'estat de l'interruptor pot ser operat externa i reversiblement per acció dels actuadors de recombinació i la seva cinètica, memòria i reversibilitat van ser àmpliament caracteritzats en experiments de transformació transitòria i estable en N. benthamiana. En conjunt, aquesta tesi mostra el disseny i la caracterització funcional d'eines per a l'enginyeria del genòmica i biologia sintètica de plantes que ara ha sigut completat amb el sistema d'edició genètica CRISPR/Cas12a i un interruptor genètic biestable i reversible basat en el sistema de recombinació del bacteriòfag PhiC31. / [EN] Plant breeding aims to provide plants with improved traits or novel features that could help to overcome sustainability goals. To this end, plant biotechnology needs to incorporate new genetic engineering tools that combine increased precision with higher breeding power. The recently discovered genome editing tools based on CRISPR/Cas9 technology have opened the way to modify plant¿s genomes with unprecedented precision. On the other hand, new synthetic biology approaches based on modularity and standardization of genetic elements have enabled the construction of increasingly complex and refined genetic devices applied to plant breeding. With the ultimate goal of expanding the toolbox of plant breeding techniques, this thesis describes the development and adaptation to plant systems of two new breeding tools: a site-specific nuclease (SSNs), and a modular gene switch for the regulation of transgene expression. In a first part, this thesis describes the adoption of the SSN CRISPR/Cas12a for plant expression and compares the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described Streptococcus pyogens Cas9 (SpCas9) in eight Nicotiana benthamiana loci using transient expression experiments. LbCas12a showed highest average mutagenesis activity in the loci assayed. This activity was also confirmed in stable genome editing experiments performed in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, off-target effects in Cas12a-free segregating lines were discarded at genomic level by deep sequencing. Collectively, the results show that LbCas12a is a viable alternative to SpCas9 for plant genome engineering. In a second part, this work describes the engineering of a new reversible genetic switch aimed at controlling gene expression in plants with higher precision than traditional inducible systems. This switch, based on the bacteriophage PhiC31 recombination system, was built as a modular device made of standard DNA parts and designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally and reversibly operated by the action of the recombination actuators and its kinetics, memory, and reversibility were extensively characterized in N. benthamiana using both transient expression and stable transgenics. Altogether, this thesis shows the design and functional characterization of refined tools for genome engineering and synthetic biology in plants that now has been expanded with the CRISPR/Cas12a gene editing system and the phage PhiC31-based toggle switch. / Bernabé Orts, JM. (2019). Development and characterization of two new tools for plant genetic engineering: A CRISPR/Cas12a-based mutagenesis system and a PhiC31-based gene switch [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133055 / Compendio

<p dir="ltr">More than 60% of all antifungal, antimicrobial, and anti-cancer drugs come from natural sources, such as plants, fungi, and bacteria; we call these nature-derived drugs Natural Products (NPs). One ‘super producer’ of NPs is the bacterial genus <i>Streptomyces</i>, with over 70% of therapeutic NP sourced from <i>Streptomyces</i>. These chemical weapons are regulated by sensitive signaling systems that respond to environmental conditions by activating ‘NP gene cluster’ expression, enzymes that biosynthesize NPs. Essential components of these signaling systems are Cluster Situated Regulators (CSRs); specific regulatory proteins of this class are transcription factors which can repress NP gene cluster expression until they sense a chemical signal. Previous research identified gamma-butyrolactones (GBLs), butenolides (BNs), and methylenomycin furans (MMFs) -small, low-concentration signaling molecules that affect morphological development- as ligands to a multitude of CSRs. These hormones are capable of inducing NP expression, thereby triggering production of therapeutically relevant NPs such as avermectin or streptomycin. This regulatory strategy is bioinformatically predicted to regulate potentially hundreds of novel NPs. My research objective is to elucidate signaling systems of <i>Streptomyces</i> which induce or enhance NP expression. To execute this, I first collaborated with organic chemists to derive a hybrid synthetic/biocatalytic approach to synthesizing these GBL-type hormones. I have developed a highly modular plasmid-based fluorescence reporter assay to identify CSR/ligand pairs. While these assays can aid in Natural Product discovery, they also have broad applicability for building novel genetic circuitry for use in synthetic biology.</p>

The interconnection of molecular machines with different functionalities to form molecular communication systems can increase the number of design possibilities and overcome the limited reliability of the individual molecular machines. Artificial information exchange using molecular signals would also expand the capabilities of single engineered cell populations by providing them a way to cooperate across heterogeneous cell populations for the applications of synthetic biology and lab-on-a-chip systems. The realization of molecular communication systems necessitates analysis and design of the communication channel, where the information carrying molecular signal is transported from the transmitter to the receiver. In this thesis, significant progress towards the use of microfluidic channels to interconnect molecular transmitter and receiver pairs is presented. System-theoretic analysis of the microfluidic channels are performed, and a finite-impulse response filter is designed using microfluidic channels. The spectral density of the propagation noise is studied and the additive white Gaussian noise channel model is developed. Memory due to inter-diffusion of the transmitted molecular signals is also modeled. Furthermore, the interference modeling is performed for multiple transmitters and its impact on the communication capacity is shown. Finally, the efficient sampling of the signal transduction by engineered bacterial receivers connected to a microfluidic channel is investigated for the detection of the pulse-amplitude modulated molecular signals. This work lays the foundation for molecular communication over microfluidic channels that will enable interconnection of engineered molecular machines.

Ce mémoire de maîtrise est une étude d’un phénomène émergent, le biohacking. Depuis 2008 et la création du groupe Boslab à Boston, le biohacking se pratique dans différents groupes autour du monde. Les biohackers se réunissent autour d’un vivant que l’on ne découvre plus mais que l’on fabrique. Ils hackent, bricolent le vivant et son code génétique, comme l’on hackerait un programme informatique. À travers une ethnographie qui suit la création du groupe de biohacking de Montréal, mais aussi à travers une ethnographie en passant dans différents groupes d’Europe et d’Amérique du Nord, je propose de comprendre le phénomène du biohacking à travers une étude médiatique du vivant. Ainsi, je propose de penser le vivant comme un medium, entendu comme un intermédiaire, un moyen, mais surtout un milieu. Un milieu qui permet de placer la notion de relation au centre de la réflexion, plutôt que sur l’objet en lui même. Un milieu dans lequel des groupes se développent, vivent et cohabitent à l’intérieur d’une communauté plus grande. Des groupes qui échangent des matériaux, des connaissances et des pratiques, entres eux, mais aussi avec les grandes institutions. Cette recherche propose de repenser notre rapport au vivant pour comprendre un phénomène à la base d’une révolution scientifique et sociale. / This thesis is a study of an emerging phenomenon, biohacking. Since 2008, and the creation of the Boslab in Boston, biohacking is practiced in different groups around the world. Biohackers meet around the idea that the living is no longer discovered but made. They hack, tinker the living and its genetic code, like one would hack a computer program. Through an ethnography that follows the creation of the biohacking group of Montreal, but also through an ethnography in different groups in Europe and North America, I propose to understand the phenomenon of biohacking through a media study of the living. I propose to think of the living as a medium, understood as an intermediary, a support, but above all an environment. A medium which places the notion of relation at the center of the reflection. An environment in which groups develop, live and cohabit in a larger community. These groups exchange materials, knowledge and practices, among themselves, but also with major institutions. This research proposes to rethink our relationship with the living to understand a phenomenon which could very well be the basis of a scientific and social revolution, biohacking.

An aqueous droplet in a solution of lipids in oil acquires a lipid monolayer coat, and two such droplets adhere to form a bilayer at their interface. Networks of droplets have been constructed in this way that function as light sensors, batteries and electrical circuits by using membrane proteins incorporated into the bilayers. However, the droplets have been confined to a bulk oil phase, which precludes direct communication with physiological environments. Further, the networks typically have been assembled manually, which limits their scale and complexity. This thesis addresses these limitations, and thereby enables prospective medical and technological applications for droplet networks. In the first part of the work, defined droplet networks are encapsulated within mm-scale drops of oil in water to form structures called multisomes. The encapsulated droplets adhere to one another and to the surface of the oil drop to form interface bilayers that allow them to communicate with each other and with the surrounding aqueous environment through membrane pores. The contents of the droplets can be released by changing the pH or temperature of the surrounding solution. Multisomes have potential applications in synthetic biology and medicine. In the second part of the work, a three-dimensional printing technique is developed that allows the construction of complex networks of tens of thousands of heterologous droplets ~50 µm in diameter. The droplets form a self-supporting material in bulk oil or water analogous to biological tissue. The mechanical properties of the material are calculated to be similar to those of soft tissues. Membrane proteins can be printed in specific droplets, for example to establish a conductive pathway through an otherwise insulating network. Further, the networks can be programmed by osmolarity gradients to fold into designed shapes. Printed droplet networks can serve as platforms for soft devices, and might be interfaced with living tissues for medical applications.

This thesis is concerned with large-scale systems of Ordinary Differential Equations that model Biomolecular Reaction Networks (BRNs) in Systems and Synthetic Biology. It addresses the strategies of model reduction and decomposition used to overcome the challenges posed by the high dimension and stiffness typical of these models. A number of developments of these strategies are identified, and their implementation on various BRN models is demonstrated. The goal of model reduction is to construct a simplified ODE system to closely approximate a large-scale system. The error estimation problem seeks to quantify the approximation error; this is an example of the trajectory comparison problem. The first part of this thesis applies semi-definite programming (SDP) and dissipativity theory to this problem, producing a single a priori upper bound on the difference between two models in the presence of parameter uncertainty and for a range of initial conditions, for which exhaustive simulation is impractical. The second part of this thesis is concerned with the BRN decomposition problem of expressing a network as an interconnection of subnetworks. A novel framework, called layered decomposition, is introduced and compared with established modular techniques. Fundamental properties of layered decompositions are investigated, providing basic criteria for choosing an appropriate layered decomposition. Further aspects of the layering framework are considered: we illustrate the relationship between decomposition and scale separation by constructing singularly perturbed BRN models using layered decomposition; and we reveal the inter-layer signal propagation structure by decomposing the steady state response to parametric perturbations. Finally, we consider the large-scale SDP problem, where large scale SDP techniques fail to certify a system’s dissipativity. We describe the framework of Structured Storage Functions (SSF), defined where systems admit a cascaded decomposition, and demonstrate a significant resulting speed-up of large-scale dissipativity problems, with applications to the trajectory comparison technique discussed above.

Conforme novas tecnologias e metodologias estão surgindo, e pesquisadores estão sedentos por ferramentas moleculares mas rápidas, mais eficientes e fáceis de usar, dominar os princípios e tecnologias do design de vetores e padronização de partes biológicas tornaram-se desafios fundamentais. Isso está abrindo espaço para o surgimento de uma disciplina inteiramente nova chamada Biologia Sintética. Esta área de estudo inovadora combina partes e módulos biológicos para criar sistemas mais confiáveis e robustos. Linhagens fúngicas são comumente alvo desses estudos, não apenas porque muitos achados fundamentais em relação à clonagem molecular surgiram das lições dadas por elas, mas também devido a um imenso e inexplorado potencial desses organismos em uma ampla gama de aplicações - desde biocombustíveis e produção de químicos finos até terapias biomédicas. Neste contexto, a presente dissertação é dividida em duas partes: a primeira diz respeito ao design e construção de uma ferramenta modular e versátil para ser aplicada em várias linhagens de fungos. Essa ferramenta é um plasmídeo binário para transformação mediada pro Agrobacterium tumefaciens, que foi construído em quatro diferentes versões contendo GFP ou mCherry como proteínas repórter e um gene sintético de resistência à higromicina como marcador de seleção. O vetor foi validado em Paracoccidioides lutzii, um patógeno oportunista humano dimórfico que é muito importante para medicina, mas ainda carecia de ferramentas genéticas eficientes. A segunda parte consiste na criação de uma biblioteca de promotores para a levedura oleaginosa Rhodosporidium toruloides, um promissor hospedeiro para a produção de bioprodutos a partir de biomassa, uma vez que pode eficientemente consumir açúcares C5 e C6 e aromáticos derivados da lignina. Vinte e nove promotores foram testados em um cassete de duplo-repórter - compreendendo ambas as proteínas fluorescentes GFP e mRuby - utilizando citometria de fluxo para análise de células únicas. A coleção de promotores apresentados neste trabalho é a maior disponível para R. toruloides até o momento e foi um avanço indispensável para superar a10 escassez de ferramentas para este organismo. Notavelmente, também apresentamos os primeiros promotores bidirecionais descritos para essa levedura e otimizamos o protocolo de transformação. Portanto, a Biologia Sintética foi eficientemente aplicada para expandir a coleção de partes biológicas padronizadas e otimizar vetores para transformação e manipulação genética de fungos. Estas ferramentas são de valor imediato e são aplicáveis a desafios muito distintos, mas igualmente importantes: a busca de novas soluções para a saúde humana e para uma economia bio-sustentável. / As new technologies and methodologies are surfacing, and researchers are now eager for fast, enhanced and easy-to-use molecular tools, mastering the principles and technologies of vector design and standardization of biological parts have become fundamental challenges. This is making room for the rise of an entirely novel discipline called Synthetic Biology. This innovative field of study combines biological parts and modules to create more reliable and robust systems. Fungal strains are commonly the target of these studies, not only because several fundamental findings regarding molecular cloning arose from lessons given by them, but also due to an immense and much unexplored potential of those organisms in a wide range of applications - ranging from biofuels and fine chemicals production to biomedical therapies. In this context, the present dissertation is divided in two parts: the first one concerns the design and construction of a modular and versatile tool to be applied in several fungal strains. This tool is a plasmid binary vector for Agrobacterium tumefaciens-mediated transformation, which was built in four different versions containing either GFP or mCherry as reporter proteins and a synthetic hygromycin resistance gene as selection marker. The vector was validated in Paracoccidioides lutzii, a dimorphic human opportunist pathogen that is very important for health care but was still lacking efficient genetic tools. The second part consists in the creation of a promoter library for the oleaginous yeast Rhodosporidium toruloides, a promising host for the production of bioproducts from biomass since it can efficiently consume C5 and C6 sugars and lignin-derived aromatics. Twenty-nine promoters were tested with a dual-reporter cassette - comprising both GFP and mRuby fluorescent proteins - using flow cytometer for single-cell analysis. The assortment of promoters presented in this work is the largest set available for R. toruloides until now and was an imperative advancement to overcome the scarcity of tools for this organism. Remarkably, we also presented the first bidirectional promoters described for this yeast and optimized the transformation protocol. Thus, we efficiently applied Synthetic Biology to expand the collection of standard biological parts and to12 optimize vectors for fungal transformation and genetic manipulation. These tools are of immediate value and are applicable for very distinct but equally important challenges: the pursuit of new solutions for human health and for a sustainable biobased economy

En biologie synthétique, il existe plusieurs manières d’adresser les problèmes soulevés dans plusieurs domaines comme la thérapeutique, les biofuels, les biomatériaux ou encore les biocapteurs. Nous avons choisi de nous concentrer sur l’une d’entre elles : les réseaux de régulation génétique (RRG). Un constat peut être fait : la diversité des problèmes résolus grâce aux RRGs est bridée par la complexité de ces RRGs, qui a atteint une limite. Quelles solutions s’offrent aux biologistes, pour repousser cette limite et continuer d’augmenter la complexité de leur système ? Cette thèse a pour but de fournir aux biologistes les outils nécessaires à la conception et à la simulation de RRGs complexes. Un examen de l’état de l’art en la matière nous a mené à adapter les outils de la micro-électronique à la biologie ainsi qu’à créer un algorithme de programmation génétique pour la conception des RRGs. D’une part, nous avons élaboré les modèles Verilog A de différents systèmes biologiques (passe-bande, proie-prédateur, repressilator, XOR) ainsi que de la diffusion spatiotemporelle d’une molécule. Ces modèles fonctionnent très bien avec plusieurs simulateurs électroniques (Spectre et NgSpice). D’autre part, les premières marches vers l’automatisation de la conception de RRGs ont été gravies. En effet, nous avons développé un algorithme capable d’optimiser les paramètres d’un RRG pour remplir un cahier des charges donné. De plus, la programmation génétique a été utilisée pour optimiser non seulement les paramètres d’un RRG mais aussi sa topologie. Ces outils ont su prouver leur utilité en apportant des réponses pertinentes à des problèmes soulevés lors du développement de systèmes biologiques. Ce travail a permis de montrer que notre approche, à savoir adapter les outils de la micro-électronique et utiliser des algorithmes de programmation génétique, est valide dans le contexte de la biologie synthétique. L’assistance que notre environnement de développement fournit au biologiste devrait encourager l’émergence de systèmes plus complexes. / In synthetic biology, Gene Regulatory Networks (GRN) are one of the main ways to create new biological functions to solve problems in various areas (therapeutics, biofuels, biomaterials, biosensing). However, the complexity of the designed networks has reached a limit, thereby restraining the variety of problems they can address. How can biologists overcome this limit and further increase the complexity of their systems? The goal of this thesis is to provide the biologists with tools to assist them in the design and simulation of complex GRNs. To this aim, the current state of the art was examined and it was decided to adapt tools from the micro-electronic field to biology, as well as to create a Genetic Programming algorithm for GRN design. On the one hand, models of diffusion and of other various systems (band-pass, prey-predator, repressilator, XOR) were created and written in Verilog A. They are already implemented and well-functioning on the Spectre solver as well as a free solver, namely NgSpice. On the other hand, the first steps of automatic GRN design were achieved. Indeed, an algorithm able to optimize the parameters of a given GRN according to a specification was developed. Moreover, Genetic Programming was applied to GRN design, allowing the optimization of both the topology and the parameters of a GRN. These tools proved their usefulness for the biologists’ community by efficiently answering relevant biological questions arising in the development of a system. With this work, we were able to show that adapting microelectronics and Genetic Programming tools to biology is doable and useful. By assisting design and simulation, such tools should promote the emergence of more complex systems.