Diurnal Rhythms Result in Significant Changes in the Cellular Protein Complement in the Cyanobacterium Cyanothece 51142

Cyanothece sp. ATCC 51142 is a diazotrophic cyanobacterium notable for its ability to perform oxygenic photosynthesis and dinitrogen fixation in the same single cell. Previous transcriptional analysis revealed that the existence of these incompatible cellular processes largely depends on tightly synchronized expression programs involving ∼30% of genes in the genome. To expand upon current knowledge, we have utilized sensitive proteomic approaches to examine the impact of diurnal rhythms on the protein complement in Cyanothece 51142. We found that 250 proteins accounting for ∼5% of the predicted ORFs from the Cyanothece 51142 genome and 20% of proteins detected under alternating light/dark conditions exhibited periodic oscillations in their abundances. Our results suggest that altered enzyme activities at different phases during the diurnal cycle can be attributed to changes in the abundance of related proteins and key compounds. The integration of global proteomics and transcriptomic data further revealed that post-transcriptional events are important for temporal regulation of processes such as photosynthesis in Cyanothece 51142. This analysis is the first comprehensive report on global quantitative proteomics in a unicellular diazotrophic cyanobacterium and uncovers novel findings about diurnal rhythms

Impact of daytime nitrogen limitation on structure and function of mixed microalgal communities derived from three geographically distinct sources

Increased nitrogen (N) and phosphorus (P) loadings to surface waters lead to algal blooms which cause eutrophication and hypoxia, threatening aquatic ecosystems, human health, and local economies. The adoption of increasingly stringent regulations for effluent N and P loadings, including from wastewater treatment plants (WWTPs), is a successful strategy for addressing this challenge. However, existing technologies for N and P removal are inconsistent with sustainability goals for nutrient recovery and reduced energy consumption. Eukaryotic microalgae are uniquely equipped to address these challenges, with the capacity to remove inorganic and organic N and P from growth media to the point of non-detection and to accumulate carbohydrate and lipid stores which may be further converted to biofuels. While algal wastewater treatment and biofuels are not new lines of research, much previous work has focused on either pure cultures or minimally engineered open systems. Algal biotechnology, using highly engineered open systems, seeks a middle road between these two approaches, offering greater control than open ponds but requiring fewer inputs than axenic systems. The work presented in this thesis explores nutrient dynamics, carbon storage, and community dynamics of mixed algal cultures within the context of algal biotechnology with an aim toward filling key knowledge gaps. Three parallel photobioreactors, each inoculated with mixed microbial communities sourced from WWTP clarifiers and/or surface water in one of three different geographic locations of the United States (Florida, North Carolina, Illinois) were operated in sequencing batch mode with an 8-day solids residence time (SRT) and 14hr:10hr light:dark cycle with nighttime N-feeding and daytime N-limitation. Metrics of community function included nutrient dynamics and biomass composition which were monitored over the course of 82 days and during a subsequent 10-day batch experiment to determine kinetic parameters. Community structure over 82 days was monitored via sequencing of the V4 and V8-V9 regions of the 18S rRNA gene. Structural and functional metrics showed complementary patterns. Functional metrics transitioned from dynamic to stable performance through time while community structure showed a departure from, followed by a return to, a community resembling the initial community. Although the communities in the three reactors remained distinct from each other through time, the most dominant OTUs were shared between all three reactors. The data presented in this experiment demonstrate how system design can induce communities which differ in structure to follow similar extant performance patterns. However, intrinsic storage parameters for carbohydrates and lipids differed widely between reactors, suggesting that more research is needed to elucidate structure-function relationships in microalgal communities to maximize carbon storage potential

Polyhydroxyalkanoate (PHA) biosynthesis from fruit waste at pilot scale: productivity maximisation and polymer tailoring

Bio-based and biodegradable plastics are an ecological alternative to conventional petroleum-derived polyolefins. Polyhydroxyalkanoates (PHA) have drawn significant attention as one of the most promising biopolymers due to its biocompatibility and biodegradable character. In particular, the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) – P(3HB-co-3HV) – has exceptional thermal and mechanical properties, which can be manipulated by varying its monomeric composition. PHA production using non-aseptic mixed microbial cultures (MMCs) enables the use of waste/surplus feedstocks, contributing simultaneously to the implementation of a circular economy approach and to reduce the process operational costs associated to the traditional pure cultures’ PHA production. The MMC process usually comprises three steps: the acidogenic fermentation, the enrichment of a MMC in PHA-storers and finally the PHA production. The monomeric composition of the P(3HB-co-3HV) produced by MMCs largely depends on the fraction of each soluble fermentation product (SFP) produced in the first step of the process, which can be precursors of either 3-hydroxybutyrate (3-HB) or 3-hydroxyvalerate (3-HV). This Thesis project was focused on solving, at pilot scale, the main difficulties (at the production level) that currently prevent industrial implementation of the PHA production process by MMC: the high manufacturing costs associated to the low process performance and the ability to consistently manipulate and tailor the polymer composition (and thus its properties). A three-stage MMC process was implemented at pilot scale. Fruit waste (FW) was selected as feedstock, since it contains high concentration of carbon and is globally generated in large volumes, causing several environmental and economic problems. FW is a nutrient-deficient feedstock, thus enabling the implementation of an uncoupled carbon and nitrogen feeding strategy in the selection reactor. The first study conducted in this PhD assesses, for the first time, the impact of the sludge retention time (SRT) (2 and 4 days) and the organic loading rate (OLR) (from 2.6 to 14.5 gCOD.L-1.d-1) on the growth versus polymer storage dynamics of PHA-storers selected under the uncoupled feeding strategy. The results showed that, similar biomass volumetric productivities were achieved for each OLR tested, regardless the SRT. However, the culture selected at 4 d SRT showed superior specific storage rates and accumulation capacity resulting in a global PHA productivity (4.6 ± 0.3 g.L-1.d-1) that was 80% above that of 2 d SRT (2.6 ± 0.2 g.L-1.d-1 ). This study underlined the importance of achieving a good balance between culture growth and accumulation capacity to increase the viability of the PHA-producing process from wastes. The same pilot plant set-up and feedstock (FW) were used to incorporate different effective operating conditions in the three stages of the process with the objective of boosting the overall PHA production performance (namely, PHA content on biomass, global productivity and overall yield). The OLR and pH of the acidogenic reactor were successfully adjusted targeting a high fermentation yield (0.74 gCOD.gCOD-1) and the production of a fermentate highly enriched in butyrate (56.8%, gCOD-basis), resulting in enhanced PHA production steps. A well selected MMC was obtained as a result of uncoupling the carbon to the nitrogen feeding, and the biomass volumetric productivity attained the unprecedented value of 1.96 g.L-1.d-1 as a response to the high OLR (8.7 gCOD.L-1.d-1) imposed. The culture selected at the optimal OLR achieved a high storage yield (0.98 gCOD.gCOD-1), and the continuous feeding strategy led to a maximum PHA content of 80.5% (w/w) at the end of accumulation assays. The obtained global PHA productivity (8.1 g-PHA.L-1.d-1) and overall process yield (0.45 gCOD.gCOD-1) are the highest values reported for MMC using a real waste feedstock at pilot scale. Moreover, a P(3HB-co-3HV) copolymer with a 3-HV content of 0.24 (g-basis) and a molecular weight of 311 KDa was produced, making this material an ideal candidate for packaging applications, the largest market of plastic usage. Lastly, the possibility of tailoring the precursors that influence the P(3HB-co-3HV) composition was investigated, by controlling the operating pH (between 4.69 and 6.34) of a continuous upflow anaerobic sludge blanket (UASB) reactor fed with FW. The impact of the operating conditions imposed was assessed by evaluating the microbial community profile, the corresponding performance and the impact on polymer composition. The fermentation yield of the UASB was maintained quite stable (between 0.72 and 0.79 gCOD.gCOD-1) during the entire operational period. On the other hand, the 3-HV bioprecursors fraction in effluent was highly affected by pH, resulting in the production of P(3HB-co-3HV) copolymers with quite different monomeric compositions. Overall, the 3-HV content of the produced polymers varied from 0.16 to a maximum of 0.44 (gCOD-basis) when the pH of the acidogenic reactor increased from 4.69 to 5.92. Moreover, the end-stream SFP composition were similar for identical operational pH values tested in different periods, showing that despite the changes occurring in the system, the selected cultures were resilient and able to produce a consistent profile of fermentation products (and thus a constant PHA monomeric composition). Additionally, the IWA Anaerobic Digestion Model No. 1 (ADM1) was expanded to include the pathway of valerate production from lactate, aiming to dynamically predict the profile of the PHA bioprecursors produced. Calibration and validation procedures were done against data from two distinct pilot-scale UASB reactors fed with FW and describing different pH and OLR dynamics. The model was capable to predict the different PHA bioprecursors production in the UASB reactors with overall strong correlations with the experimental data for different OLRs over the pH range between 4.20 and 5.16, providing a useful tool for process optimisation and tailoring of the PHA monomer composition. This thesis highlights the importance of understanding in-depth the impact of key operating conditions on the PHA production process with MMC to increase its overall viability. Moreover, it shows that predicting and regulating the acidogenic process is essential to promote an adequate PHA bioprecursors composition production, which are both promising results towards the full-scale implementation of the PHA production from MMC.Plásticos biodegradáveis de origem biológica são uma alternativa ecológica às poliolefinas convencionais derivadas do petróleo. Os polihidroxialcanoatos (PHAs) têm vido a ganhar prestígio como um dos bioplásticos mais promissores devido ao seu carater biocompatível e biodegradável. Em particular, o copolímero poli(3-hidroxibutirato-co-3-hidroxivalerato) – P(3HB-co-3HV) – tem excelentes propriedades mecânicas e térmicas, que podem ser manipuladas variando a sua composição monomérica. A produção de PHA usando culturas microbianas mistas (MMCs) não assépticas permite o uso de resíduos/sub-produtos como substratos, contribuindo simultaneamente para a implementação de uma economia circular e para reduzir os custos operacionais associados ao tradicional processo de produção de PHA com culturas puras. O processo de produção com MMC envolve normalmente 3 passos: uma fermentação acidogénica, o enriquecimento de uma MMC em bactérias acumuladoras de PHA e, por último, a produção de PHA. A composição monomérica do P(3HB-co-3HV) produzido por MMCs depende fortemente da fração de cada produto de fermentação solúvel (SFP) produzido no primeiro passo do processo, que podem ser precursores de 3-hidroxibutirato (3-HB) ou de 3-hidroxivalerato (3-HV) Este projeto de Tese teve como foco principal resolver, à escala piloto, as principais dificuldades (ao nível da produção) que impendem a implementação do processo de produção de PHAs a partir de MMCs à escala industrial: os custos de produção altos associados à baixa performance do processo e a habilidade de manipular consistentemente a composição do polímero (e portanto as suas propriedades). O processo de três passos de produção de PHAs foi implementado à escala piloto. Resíduos de fruta estragada (FW) foram selecionados como substrato, visto que contêm altas concentrações de carbono e são globalmente gerados em grandes volumes, causando diversos problemas ambientais e económicos. FW são um substrato deficiente em nutrientes, o que permite implementar uma estratégia de alimentação em que o fornecimento de carbono é desacoplado do azoto no reator de seleção. O primeiro estudo realizado durante este PhD teve como objetivo estudar, pela primeira vez, o impacto do tempo de retenção de lamas (SRT) (2 e 4 dias) e da taxa de carga orgânica (OLR) (de 2.6 a 14.5 gCOD.L-1.d-1) nas dinâmicas de crescimento versus armazenamento de polímero de culturas produtoras de PHA sujeitas ao regime de alimentação desacoplado. Os resultados mostraram que, semelhantes produtividades volumétricas de biomassa são observadas para cada OLR imposta, independentemente do SRT. No entanto, a cultura selecionada a um SRT de 4 d mostrou uma taxa especifica de produção de polímero e uma capacidade de acumulação superiores, apresentando uma produtividade global de PHA (4.6 ± 0.3 g.L-1.d-1) 80% superior ao atingido pela cultura selecionada a um SRT de 2 d (2.6 ± 0.2 g.L-1.d-1 ). Este estudo realçou a importância de conseguir um bom equilíbrio entre o crescimento da cultura e a sua capacidade de acumulação, de forma a aumentar a viabilidade do processo de produção de PHA a partir de resíduos. A mesma instalação piloto e substrato (FW) foram usados no seguinte estudo, onde várias condições de operação eficazes foram incorporadas de uma só vez nos três passos do processo com o objetivo de impulsionar a performance global da produção de PHA (nomeadamente, o conteúdo de PHA na biomassa, a produtividade global e o rendimento global). A OLR e o pH do reator acidogénico foram ajustados com sucesso de forma a atingir um elevado rendimento de fermentação (0.74 gCOD.gCOD-1) e produzir um fermentado enriquecido em butirato (56.8%, base gCOD), que irá beneficiar a performance da cultura nos próximos passos do processo. Uma MMC bem selecionada foi obtida como resultado da aplicação da estratégia de desacoplamento do carbono do azoto, e a produtividade volumétrica de biomassa atingiu o valor sem precedentes de 1.96 g.L-1.d-1, como resposta à alta OLR (8.7 gCOD.L-1.d-1) imposta. A cultura selecionada à OLR ótima mostrou um elevado rendimento de produção de PHA (0.98 gCOD.gCOD-1), e a estratégia de alimentação continua levou à obtenção de um conteúdo máximo de PHA na biomassa de 80.5% (w/w) no final dos ensaios de acumulação. A produtividade global de PHA (8.1 g-PHA.L-1.d-1) e o rendimento global do processo (0.45 gCOD.gCOD-1) obtidos foram os valores mais altos reportados para MMC usando um resíduo real como substrato à escala piloto. Alem disso, foi produzido um copolímero P(3HB-co-3HV) com 0.24 (base g) de monómeros 3-HV e um peso molecular de 311 KDa, o que faz deste material um excelente candidato para ser usado em embalagens, o sector de mercado que apresenta o consumo de plástico mais elevado. Por último, a possibilidade de controlar a produção dos percursores que influenciam a composição do P(3HB-co-3HV) foi investigada através da manipulação do pH (entre 4.69 e 6.34) de um reator anaeróbio de fluxo ascendente (UASB) alimentado com FW. O impacto das diferentes condições de operação impostas foi avaliado através da observação do perfil da comunidade microbiana selecionada, da sua performance e do impacto na composição dos diferentes polímeros produzidos. O rendimento de fermentação do UASB manteve-se relativamente estável (entre 0.72 e 0.79 gCOD.gCOD-1) durante todo o período de operação. Por outro lado, a fração de precursores de 3-HV no efluente foi bastante afetada pelo pH, o que resultou na produção de copolímeros P(3HB-co-3HV) com diferentes composições monoméricas. De forma global, o conteúdo de 3-HV nos polímeros produzidos variou de 0.16 até um máximo de 0.44 (base gCOD) quando o pH do reator acidogénico aumentou de 4.69 para 5.92. Além disso, a composição das correntes de SFP produzidas a valores de pH semelhantes, mas testados em diferentes períodos da operação, foram idênticas, mostrando que, apesar das mudanças que ocorrem no sistema, as culturas selecionadas foram resilientes a capazes de produzir um perfil de produtos de fermentação consistente (resultando numa composição monomérica de polímero constante). Além do trabalho experimental relatado, o modelo de digestão anaeróbia No. 1 (ADM1) foi expandido de forma a incluir a via de produção de valerato a partir de lactato, com o objetivo de se prever dinamicamente o perfil de precursores de PHA produzido no reator UASB descrito. Os procedimentos de calibração e validação foram realizados usando dados de dois reatores UASB piloto distintos, ambos alimentados com FW e sujeitos a diferentes dinâmicas de pH e OLR. O modelo foi capaz de prever a produção dos diferentes precursores de PHA nos reatores UASB e atingir fortes correlações com os dados experimentais obtidos para várias OLRs dentro do intervalo de pH de 4.20 a 5.16, providenciando uma ferramenta útil para otimização do processo e para manipular a composição monomérica de PHA. Esta tese realça a importância de efetivamente perceber o impacto de condições de operação chave no processo de produção de PHA com MMC de forma a aumentar a sua viabilidade global. Alem disso, mostrou que prever e regular o processo acidogénico é essencial para promover a produção de precursores de PHA com uma composição adequada, o que são ambos resultados promissores no sentido da implementação à escala industrial do processo de produção de PHA a partir de MMC

Paving the crossroad of biorefinery

This thesis focuses on anaerobic digestion and, more specifically, on its role in biorefinery and on the dynamic behaviour of the underlying microbiomes. In recent decades, significant progress has been made in the field of anaerobic digestion. Innovative methods, especially highthroughput sequencing approaches, have allowed for a deeper understanding of biotechnologically relevant biocenosis. However, the exact behaviour of the relevant microbiomes under different conditions has not been thoroughly researched. In order to shed light on the diversity of the underlying biocenosis, this thesis compares multiple biogas production facilities in Germany. It also provides the first multi-OMICs characterization of separated acidification stages at mesophilic and thermophilic conditions. At the phylum level, three key microbiomes are identified, which are specific for sewage sludge, highly viscous codigester sludge, and leachate from leach-bed systems. All three microbiomes are strongly related to their underlying environmental parameters (Chemical oxygen demand, total organic carbon, total nitrogen contents, conductivity, total volatile fatty acids, total solids, volatile solids, pH, and volume of biogas). Through various experiments, new methods for acidifying biomass in pretreatment stages were investigated. One of the main contributions of this thesis is to highlight the importance of separated acidification stages as crossroad for multiple industries. Separated acidification potentially allows for the production of multiple organic acids, the usage of many varieties of waste, and the production of hydrogen simultaneously. Moreover, separated acidification might facilitate the usage of substrates that are difficult to digest, such as lignocellulose grass biomass or nitrogen-rich chicken dung. Indeed, this thesis demonstrates that both substrates can contribute to successful liquefaction. In searching for further possible applications based on acidification stages, we developed the first Microbial Thermoelectric Cell (MTC), which is compatible with anaerobic digestion and suitable for use in the pre-treatment stage. The MTC allows for the simultaneous production of ethanol and electric energy. Remnants might be used in a subsequent methane-producing stage. In addition, in seeking further new pretreatment methods, we investigated the possibility of combining thermal pre-treatment with microbe-driven acidification. Surprisingly, we observed only minimal impacts of heat-shocks in the microbial composition. Therefore, it might be possible in the future to combine heatshocks with acidification processes to improve biomass pre-treatment. Furthermore, this possibility highlights the robustness of microbiomes from anaerobic digestion processes. Finally, we isolated news strains from the acidification of grass biomass, with foreseeable roles in anaerobic digestion.This thesis focuses on anaerobic digestion and, more specifically, on its role in biorefinery and on the dynamic behaviour of the underlying microbiomes. In recent decades, significant progress has been made in the field of anaerobic digestion. Innovative methods, especially highthroughput sequencing approaches, have allowed for a deeper understanding of biotechnologically relevant biocenosis. However, the exact behaviour of the relevant microbiomes under different conditions has not been thoroughly researched. In order to shed light on the diversity of the underlying biocenosis, this thesis compares multiple biogas production facilities in Germany. It also provides the first multi-OMICs characterization of separated acidification stages at mesophilic and thermophilic conditions. At the phylum level, three key microbiomes are identified, which are specific for sewage sludge, highly viscous codigester sludge, and leachate from leach-bed systems. All three microbiomes are strongly related to their underlying environmental parameters (Chemical oxygen demand, total organic carbon, total nitrogen contents, conductivity, total volatile fatty acids, total solids, volatile solids, pH, and volume of biogas). Through various experiments, new methods for acidifying biomass in pretreatment stages were investigated. One of the main contributions of this thesis is to highlight the importance of separated acidification stages as crossroad for multiple industries. Separated acidification potentially allows for the production of multiple organic acids, the usage of many varieties of waste, and the production of hydrogen simultaneously. Moreover, separated acidification might facilitate the usage of substrates that are difficult to digest, such as lignocellulose grass biomass or nitrogen-rich chicken dung. Indeed, this thesis demonstrates that both substrates can contribute to successful liquefaction. In searching for further possible applications based on acidification stages, we developed the first Microbial Thermoelectric Cell (MTC), which is compatible with anaerobic digestion and suitable for use in the pre-treatment stage. The MTC allows for the simultaneous production of ethanol and electric energy. Remnants might be used in a subsequent methane-producing stage. In addition, in seeking further new pretreatment methods, we investigated the possibility of combining thermal pre-treatment with microbe-driven acidification. Surprisingly, we observed only minimal impacts of heat-shocks in the microbial composition. Therefore, it might be possible in the future to combine heatshocks with acidification processes to improve biomass pre-treatment. Furthermore, this possibility highlights the robustness of microbiomes from anaerobic digestion processes. Finally, we isolated news strains from the acidification of grass biomass, with foreseeable roles in anaerobic digestion

Introducing deep learning -based methods into the variant calling analysis pipeline

Biological interpretation of the genetic variation enhances our understanding of normal and pathological phenotypes, and may lead to the development of new therapeutics. However, it is heavily dependent on the genomic data analysis, which might be inaccurate due to the various sequencing errors and inconsistencies caused by these errors. Modern analysis pipelines already utilize heuristic and statistical techniques, but the rate of falsely identified mutations remains high and variable, particular sequencing technology, settings and variant type. Recently, several tools based on deep neural networks have been published. The neural networks are supposed to find motifs in the data that were not previously seen. The performance of these novel tools is assessed in terms of precision and recall, as well as computational efficiency. Following the established best practices in both variant detection and benchmarking, the discussed tools demonstrate accuracy metrics and computational efficiency that spur further discussion

In Situ Resource Utilization (ISRU) Technical Interchange Meeting

This volume contains abstracts that have been accepted for presentation at the In Situ Resource Utilization (ISRU) Technical Interchange Meeting, February 4-5, 1997, at the Lunar and Planetary Institute, Houston, Texas. Abstracts are arranged in order of presentation at the meetings, with corresponding page numbers shown in the enclosed agenda. Logistics, administration, and publication support for this meeting were provided by the staff of the Publications and Program Services Department at the Lunar and Planetary Institute

APPLICATION OF MICROBIAL ELECTROCHEMICAL SYSTEMS FOR VALORISATION OF CHEESE WHEY IN BIOREFINERY FRAMEWORK

In the last decades, the current unsustainable fossil-based economic model has been worldwide disputed by policies and public opinion. As consequence, the exploitation of biomasses has arisen as pivotal towards a green and circular economy. In this context, waste biorefineries would represent the optimal technical solution. Firstly, the integration of feasible bioprocess can generate a mix of biofuels and bioproducts, according to the cascade principle, thus making possible to hit the market with products characterised by either significant market size or high market value, guaranteeing economic sustainability. In addition, the use of organic wastes as alternative to dedicated biomasses would significantly tackle costs of waste management and related environmental impacts. Due to their qualitative homogeneity and volumes of production, agro-industrial residues are currently pointed out as suitable for multi-step valorisation in biorefinery. However, their valorisation is currently aimed to few products, like biogas and compost, characterised by low market value. Therefore, the full achievement of waste biorefineries potential has to be achieved yet, since it would greatly impact the economic and environmental resilience of the whole agro-industrial sector, in particular for smaller supply chains. Sheep milk supply chain is a notable example in this respect: even though it represents a small portion of European milk market, it is a fundamental source of income in few southern regions like Sardinia, and it cyclically experiences economic difficulties. This research aims to evaluate the integration of Microbial Electrochemical Systems (MESs) in a biorefinery framework for the valorisation of cheese whey, as the main by-product of dairy industry. It started by assessing the state of art of available bioprocesses for feedstock valorisation. The literature review highlighted the current weakness of MESs treating this substrate, but also found how their integration as downstream process of Dark Fermentation (DF) can significantly enhance the power output generation in comparison to standalone processes. Consequently, a general overview on DF and MESs was provided, to also stress out how MESs can also expand material outputs generated during DF. The experimental work focused then on the application of Electro fermentation of lactate rich effluents from DF to propionate and acetate, which are seldom reported as main metabolites in DF broths. Then, a novel Microbial Fuel Cell for electricity generation is presented and characterised by mathematical modelling, aiming to a deeper understanding of reactor design to favour future systems scale up. Last experimental work gives a proof of concept of hydrogen production by Microbial Electrochemical Cells, underlining the further energy recovery and carbon removal achievable by their implementation. Finally, two biorefinery schemes are presented and analysed, pointing out their novelty and potential benefits to cheese making plants

Development of CRISPR-based programmable transcriptional regulators and their applications in plants

[ES] La Biología Sintética de Plantas tiene como objetivo rediseñar las plantas para que adquieran características y funcionalidades novedosas a través de circuitos reguladores ortogonales. Para lograr este objetivo, se deben desarrollar nuevas herramientas moleculares con la capacidad de interactuar con factores endógenos de manera potente y específica. CRISPR/Cas9 surgió como una herramienta prometedora que combina la capacidad personalizable de unión al DNA, a través de la versión catalíticamente inactivada de la proteína Cas9 (dCas9), con la posibilidad de anclar dominios autónomos de activación transcripcional (TADs) a su estructura para lograr una regulación específica de la expresión génica. Los activadores transcripcionales programables (PTAs) pueden actuar como procesadores específicos, ortogonales y versátiles para el desarrollo de nuevos circuitos genéticos en las plantas. En busca de dCas9-PTA optimizados, se llevó a cabo una evaluación combinatoria de diferentes arquitecturas dCas9 con un catálogo de varios TAD. La mejor herramienta resultante de esta comparación, denominada dCasEV2.1, se basa en la estrategia scRNA y la combinación de los dominios de activación EDLL y VPR con un bucle multiplexable gRNA2.1, que es una versión mutada del gRNA2.0 descrito previamente. En este trabajo, el activador programable dCasEV2.1 demostró ser una herramienta potente y específica, logrando tasas de activación más altas que otras estrategias dCas9 disponibles en plantas. Se observaron tasas de activación sin precedentes dirigidas a genes endógenos en N. benthamiana, acompañadas de una estricta especificidad en todo el genoma, lo que hace que esta herramienta sea adecuada para la regulación estricta de redes reguladoras complejas. Como prueba de concepto, se diseñaron cuatro programas de activación para distintas ramas de la ruta de los flavonoides, buscando obtener enriquecimientos metabólicos específicos en hojas de N. benthamiana. El análisis metabólico de las hojas metabólicamente reprogramadas mediante dCasEV2.1 reveló un enriquecimiento selectivo de los metabolitos diana y sus derivados glicosilados, que se correlacionaron con el programa de activación empleado. Estos resultados demuestran que dCasEV2.1 es una herramienta eficaz para la ingeniería metabólica y un componente clave en los circuitos genéticos destinados a reprogramar los flujos metabólicos. Finalmente, basándonos en dCasEV2.1, desarrollamos un sistema optimizado de regulación de genes inducidos por virus (VIGR) que utiliza un vector Potato Virus X (PVX) para el suministro de los programas de activación CRISPR codificados con gRNA. Este enfoque permite controlar el transcriptoma de la planta a través de una aplicación sistémica basada en aerosol de componentes CRISPR a plantas adultas. El nuevo sistema PVX-VIGR produjo una fuerte activación transcripcional en varios genes diana endógenos, incluidos tres factores de transcripción MYB-like seleccionados. Las activaciones específicas de MYB condujeron a perfiles metabólicos distintivos, demostrando que las aplicaciones potenciales de la herramienta dCasEV2.1 en plantas incluyen la obtención de perfiles metabólicos personalizados utilizando un suministro basado en aerosol de instrucciones de reprogramación transcripcional codificadas por gRNA. En resumen, esta tesis proporciona herramientas novedosas para la activación transcripcional fuerte, ortogonal y programable en plantas, con una caja de herramientas ampliada para el suministro de los programas de activación.[CA] La Biologia Sintètica de Plantes té com objectiu redissenyar les plantes per que obtinguen característiques i funcionalitats innovadores mitjançant circuits reguladors ortogonals. Per arribar a aquest objectiu, s'han de desenvolupar noves ferramentes moleculars amb la capacitat d'interactuar amb factor endògens d'una manera potent i específica. CRISPR/Cas9 va sorgir com una ferramenta prometedora que combina la capacitat personalitzable d'unió al DNA, mitjançant la versió catalíticament inactivada de la proteïna Cas9 (dCas9), amb la possibilitat de fixar dominis autònoms de activació transcripcional (TADs) a la seua estructura per aconseguir una regulació específica de la expressió gènica. Els activadors transcripcionals programables (PTAs) poden actuar com a processadors específics, ortogonals i versàtils per al desenvolupament de nous circuits genètics a les plantes. Buscant dCas9-PTA optimitzats, es va realitzar una avaluació combinatòria de distintes arquitectures dCas9 amb un catàleg de diversos TAD. La millor ferramenta segons aquesta comparació, anomenada dCasEV2.1, es basa en la estratègia scRNA i la combinació del dominis d'activació EDLL i VPR amb un bucle multiplexable gRNA2.1, que es una versió mutada del gRNA2.0 descrit prèviament. En aquest treball, el activador programable dCasEV2.1 es va mostrar com una ferramenta potent i específica, aconseguint nivells d'activació majors que altes estratègies dCas9 disponibles en plantes. Es van observar taxes d'activació sense precedents dirigides a gens endògens en N. benthamiana, junt a una estricta especificitat en tot el genoma, indicant que aquesta ferramenta és adequada per a la regulació estricta de xarxes reguladores complexes. Como proba de concepte, se van dissenyar quatre programes d'activació per a diferent branques de la ruta dels flavonoides, cercant obtenir enriquiments metabòlics específics en fulles de N. benthamiana. L'anàlisi metabòlic de les fulles metabòlicament reprogramades mitjançant dCasEV2.1 va revelar un enriquiment selectiu del metabòlits diana i els seus derivats glicosilats que es correlacionen amb el programa d'activació emprat. Aquests resultats demostren que dCasEV2.1 és una ferramenta eficaç per a l'enginyeria metabòlica i un component clau als circuits genètics destinats a reprogramar els fluxos metabòlics. Finalment, en base a dCasEV2.1, desenvoluparem un sistema optimitzat de regulació de gens induïts per virus (VIGR) que utilitza un vector Potato Virus X (PVX) per al subministrament dels programes d'activació CRISPR codificats amb gRNA. Aquesta aproximació permet controlar el transcriptoma de la planta mitjançant l'aplicació sistèmica basada en aerosol de components CRISPR a plantes adultes. El nou sistema PVX-VIGR va produir una gran activació transcripcional en diversos gens diana endògens, inclosos tres factors de transcripció MYB-like seleccionats prèviament. Les activacions específiques de MYB conduïren a perfils metabòlics distintius, demostrant que les aplicacions potencials de la ferramenta dCasEV2.1 en plantes inclouen la obtenció de perfils metabòlics personalitzats emprant un subministrament basat en aerosol de instruccions de reprogramació transcripcional codificades per gRNA. En resum, aquesta tesis proporciona noves ferramentes per a l'activació transcripcional forta, ortogonal i programable en plantes, amb una caixa de ferramentes eixamplada per al subministraments dels programes d'activació.[EN] Plant Synthetic Biology aims to redesign plants to acquire novel traits and functionalities through orthogonal regulatory circuits. To achieve this goal, new molecular tools with the capacity of interacting with endogenous factors in a potent and specific manner must be developed. CRISPR/Cas9 emerged as promising tools which combine a customizable DNA-binding activity through the catalytically inactivated version of Cas9 protein (dCas9) with the possibility to anchor autonomous transcriptional activation domains (TADs) to its structure to achieve a specific regulation of the gene expression. The Programmable Transcriptional Activators (PTAs) could act as specific, orthogonal and versatile processor components in the development of new genetic circuits in plants. In search for optimized dCas9-PTAs, a combinatorial evaluation of different dCas9 architectures with a catalogue of various TADs was performed. The best resulting tool of this comparison, named dCasEV2.1, is based on the scRNA strategy and the combination of EDLL and VPR activation domains with a multiplexable gRNA2.1 loop, which is a mutated version of the previously described gRNA2.0. In this work, the dCasEV2.1 programable activator was proved to be a strong and specific tool, achieving higher activation rates than other available dCas9 strategies in plants. Unprecedented activation rates were observed targeting endogenous genes in N. benthamiana, accompanied by strict genome-wide specificity that makes this tool suitable to perform a tight regulation of complex regulatory networks. As a proof of concept, a design of four activation programs to activate different branches of the flavonoid pathway and obtain specific metabolic enrichments in N. benthamiana leaves was performed. The metabolic analysis on the dCasEV2.1 metabolically reprogrammed leaves revealed a selective enrichment of the targeted metabolites and their glycosylated derivatives that correlated with the activation program employed. These results demonstrate that dCasEV2.1 is a powerful tool for metabolic engineering and a key component in genetic circuits aimed at reprogramming metabolic fluxes. Finally, based on dCasEV2.1, we developed an optimized Viral Induced Gene Regulation (VIGR) system that makes use of a Potato Virus X (PVX) vector for the delivery of the gRNA-encoded CRISPR activation programs. This approach offers a way to control the plant transcriptome through a spray-based systemic delivery of CRISPR components to adult plants. The new PVX-VIGR system led to strong transcriptional activation in several endogenous target genes, including three selected MYB-like transcription factors. Specific MYB activations lead to distinctive metabolic profiles, showing that the potential applications of the dCasEV2.1 tool in plants include the obtention of custom metabolic profiles using a spray-based delivery of gRNA-encoded transcriptional reprogramming instructions. In sum, this thesis provides novel tools for strong, orthogonal and programmable transcriptional activation in plants, with an expanded toolbox for the delivery of the activation programs.Selma García, S. (2022). Development of CRISPR-based programmable transcriptional regulators and their applications in plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185046TESI