It is widely believed that we might be able produce life out of nonliving substances if we possessed the relevant knowledge. Thus synthetic biology is said to be on the way towards artificial life. But this is nonsense: “artificial life” cannot be thought. The idea that biological organisms could be produced reflects a misunderstandig of the concept “life”. Life is formally characterized by the fact that that which in the case of artifacts is three distinct activities – being something, producing it, and using it – is one. For a living being, to be is to be the source of its own activity through its own activity. Hence, if there is an activity of producing distinct from being what is produced – as would have to be the case in artificial life – what is thus produced is not life.

No

Fungi, diverse and impactful organisms, exert both beneficial and harmful effects on plants, animals, and humans. Certain fungi produce auxin or indole-3-acetic acid (IAA), a crucial plant growth hormone that influences various aspects of plant growth and defense mechanisms. Conversely, pathogenic fungi can produce auxin and manipulate auxin signaling in their host plant to promote fungal virulence and infection progression. Targeting the auxin signaling pathway in pathogenic fungi offers a novel strategy for combating fungal infections in both plants and humans. Nevertheless, the auxin biosynthesis pathway and the role of auxin in fungal symbioses is not fully understood, in part, due to the lack of a tool for measuring intracellular auxin with high spatial and temporal resolution. This dissertation presents the first genetically encoded biosensor engineered from the E3 ubiquitin ligase to detect and quantify intracellular auxin in a Saccharomyces cerevisiae model. The biosensor has been applied to begin studying auxin metabolism and biosynthesis in yeast as well as better understand the plant auxin co-receptor proteins from which it is built. Additionally, the biosensor is re-engineered for application in inducible protein degradation, controlled by auxin. This tool could be applied to identify novel protein targets for disrupting pathogenic fungal species. Overall, this research offers valuable tool and platform for studying auxin biosynthesis pathway, plant protein and auxin signaling as well as intracellular proteins in fungi. / Doctor of Philosophy / Fungi affect plants, animals, and humans, in both beneficial and harmful ways. Some fungi aid other organisms, while others cause illness. Certain fungi produce a hormone called auxin, or indole-3-acetic acid (IAA), which is essential for plant growth and many environmental responses. Auxin can also assist plants in defending against harmful fungi. Conversely, fungi that infect plants can utilize auxin to promote their own growth and spread. Some fungi even produce auxin, possibly aiding in their colonization of plants. In human fungal infection, it is suggested that auxin may be involved in virulent traits and disease progression. Targeting the auxin signaling pathway in harmful fungi presents an innovative approach to combat fungal infections in both plants and humans. However, our understanding of fungal auxin biosynthesis pathways and their role in fungal infections are not fully understood due to the lack of tools to measure auxin in cells efficiently and accurately. This study introduces the first biological tool, called a biosensor, engineered from auxin responsive proteins from plants, to detect and measure intracellular auxin in Baker's yeast. The biosensor has been used to investigate auxin production by yeast. Additionally, the biosensor has been re-engineered for application in inducible protein degradation, controlled by auxin. This tool could be applied to identify novel protein targets for disrupting pathogenic fungal species. Overall, this research provides useful tool and platform to study auxin production, plant protein function and particular proteins in fungi.

Genetic regulatory circuits control metabolism, development, and environmental response across all kingdoms of life. Genetic circuit engineering facilitates sustainable and efficient production of biopharmaceutical, chemical, fiber, and food products that keep humans healthy, nourished, and clothed. However, the complexity of most genetic regulatory circuits, particularly in the context of multicellular eukaryotes, often prevents them from being leveraged as tools or applied technologies with bioeconomic relevance. However, synthetic biology enables the transfer of genes, circuits, networks, and even whole chromosomes between organisms. This approach can be leveraged to port genetic circuits into simple model organisms to control existing and engineer new cellular functions. Still, porting genes to non-native contexts can affect circuit function due to unknown factors. For this reason, iterative design-build-test-learn (DBTL) cycles are necessary for optimizing circuits in new contexts. To facilitate the DBTL cycle, automation approaches can be deployed for streamlining synthetic genetic circuits optimization. Here, I provide a case study for how using synthetic biology and automation – a biofoundry approach – has facilitated engineering of the auxin signaling pathway in a synthetic yeast system. Auxin is a phytohormone involved in nearly every aspect of plant growth and development, and this striking versatility designates it as a target for biotechnology development and a candidate for engineering. First, I provide a literature review of the history of synthetic auxin engineering in yeast, a survey of tools available for expanding yeast synthetic biology, and a summary of applicable automation tools and platforms. Next, I describe and validate a platform called AssemblyTron, which deploys liquid handling robotics for DNA assembly and can serve as the foundation of a biofoundry platform. I then introduce TidyTron, which is a protocol library for automated wash and reuse of single use lab plastics to promote biofoundry sustainability. Next, I expand the AssemblyTron package by providing protocols for mutant and modular indexed plasmid library assembly. Finally, I describe a modular indexed plasmid library (toolkit) for rapid assembly of auxin circuit variants and validate it by building and optimizing an auxin circuit. / Doctor of Philosophy / Genetic mechanisms allow humans, plants, and microbes to grow, breathe, speak, and survive. The DNA that encodes these genetic mechanisms produces protein machines that make chemicals, transfer them, and respond to them in other cells. This process is called signaling, and the protein machines involved make a circuit. In biotechnology, we harness natural genetic circuits to create important products like biopharmaceuticals, food, and clothes. However, the genetic circuits that make valuable proteins/chemicals are usually located on chromosomes along with every other gene involved in building an advanced, multicellular organism (called the genome). Synthetic biology allows us to choose just the DNA that encodes a genetic circuit of interest and put it into the chromosome of a simpler organism with faster growth, smaller genome, etc., which allow us to engineer it more easily. However, transferring a gene circuit to a new organism can cause problems, and it is usually necessary to try many versions of gene circuits to find one that works. Using robots to do synthetic biology can make it faster and less error-prone, which enables more versions of the genetic circuit to be tested. Here, I describe a biofoundry approach where I combined synthetic biology and robotics to speed up the process of building and optimizing the auxin plant hormone signaling pathway. Auxin is a small molecule that plants produce and transfer throughout their leaves, stems, and roots to turn growth on or off (e.g., auxin causes plants to do things like bend towards the sun). I focus on auxin because my goal is to manipulate the auxin pathway to rationally control plant growth. First, I provide a recap of existing work in the field of auxin synthetic biology, tools for transferring auxin circuits into simpler organisms, and available robotics that can speed up auxin synthetic biology. Next, I introduce a software called AssemblyTron, which I developed for building and modifying genes (a process called DNA assembly) with a robot. Next, I discuss how I used the same robot to wash and reuse plastic pipette tips and plates to improve lab sustainability. I then discuss an extended version of AssemblyTron that can be used for more advanced DNA assembly applications like making 10s – 100000s of versions of gene circuits at the same time. Finally, I introduce a collection of auxin circuit DNA parts that can be assembled interchangeably for rapid synthetic auxin engineering.

[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/10251/185046

Tesis por compendio / [EN] Synthetic Biology is an emerging interdisciplinary field that aims to apply the engineering principles of modularity, abstraction and standardization to genetic engineering. The nascent branch of Synthetic Biology devoted to plants, Plant Synthetic Biology (PSB), offers new breeding possibilities for crops, potentially leading to enhanced resistance, higher yield, or increased nutritional quality. To this end, the molecular tools in the PSB toolbox need to be adapted accordingly, to become modular, standardized and more precise. Thus, the overall objective of this Thesis was to adapt, expand and refine DNA assembly tools for PSB to enable the incorporation of functional specifications to the description of standard genetic elements (phytobricks) and to facilitate the construction of increasingly complex and precise multigenic devices, including genome editing tools. The starting point of this Thesis was the modular DNA assembly method known as GoldenBraid (GB), based on type IIS restriction enzymes. To further optimize the GB construct-making process and to better catalog the phytobricks collection, a database and a set of software-tools were developed as described in Chapter 1. The final webbased software package, released as GB2.0, was made publicly available at www.gbcloning.upv.es. A detailed description of the functioning of GB2.0, exemplified with the building of a multigene construct for anthocyanin overproduction was also provided in Chapter 1. As the number and complexity of GB constructs increased, the next step forward consisted in the refinement of the standards with the incorporation of experimental information associated to each genetic element (described in Chapter 2). To this end, the GB package was reshaped into an improved version (GB3.0), which is a self-contained, fully traceable assembly system where the experimental data describing the functionality of each DNA element is displayed in the form of a standard datasheet. The utility of the technical specifications to anticipate the behavior of composite devices was exemplified with the combination of a chemical switch with a prototype of an anthocyanin overproduction module equivalent to the one described in Chapter 1, resulting in a dexamethasone-responsive anthocyanin device. Furthermore, Chapter 3 describes the adaptation and functional characterization of CRISPR/Cas9 genome engineering tools to the GB technology. The performance of the adapted tools for gene editing, transcriptional activation and repression was successfully validated by transient expression in N. benthamiana. Finally, Chapter 4 presents a practical implementation of GB technology for precision plant breeding. An intragenic construct comprising an intragenic selectable marker and a master regulator of the flavonoid biosynthesis was stably transformed in tomato resulting in fruits enhanced in flavonol content. All together, this Thesis shows the implementation of increasingly complex and precise genetic designs in plants using standard elements and modular tools following the principles of Synthetic Biology. / [ES] La Biología Sintética es un campo emergente de carácter interdisciplinar que se fundamenta en la aplicación de los principios ingenieriles de modularidad, abstracción y estandarización a la ingeniería genética. Una nueva vertiente de la Biología Sintética aplicada a las plantas, la Biología Sintética Vegetal (BSV), ofrece nuevas posibilidades de mejora de cultivos que podrían llevar a una mejora de la resistencia, a una mayor productividad, o a un aumento de la calidad nutricional. Sin embargo, para alcanzar este fin las herramientas moleculares disponibles en estos momentos para BSV deben ser adaptadas para convertirse en modulares, estándares y más precisas. Por ello se planteó como objetivo general de esta Tesis adaptar, expandir y refinar las herramientas de ensamblaje de DNA de la BSV para permitir la incorporación de especificaciones funcionales en la descripción de elementos genéticos estándar (fitobricks) y facilitar la construcción de estructuras multigénicas cada vez más complejas y precisas, incluyendo herramientas de editado genético. El punto de partida de esta Tesis fue el método de ensamblaje modular de ADN GoldenBraid (GB) basado en enzimas de restricción tipo IIS. Para optimizar el proceso de ensamblaje y catalogar la colección de fitobricks generados se desarrollaron una base de datos y un conjunto de herramientas software, tal y como se describe en el Capítulo 1. El paquete final de software se presentó en formato web como GB2.0, haciéndolo accesible al público a través de www.gbcloning.upv.es. El Capítulo 1 también proporciona una descripción detallada del funcionamiento de GB2.0 ejemplificando su uso con el ensamblaje de una construcción multigénica para la producción de antocianinas. Con el aumento en número y complejidad de las construcciones GB, el siguiente paso necesario fue el refinamiento de los estándar con la incorporación de la información experimental asociada a cada elemento genético (se describe en el Capítulo 2). Para este fin, el paquete de software de GB se reformuló en una nueva versión (GB3.0), un sistema de ensamblaje auto-contenido y completamente trazable en el que los datos experimentales que describen la funcionalidad de cada elemento genético se muestran en forma de una hoja de datos estándar. La utilidad de las especificaciones técnicas para anticipar el comportamiento de dispositivos biológicos compuestos se ejemplificó con la combinación de un interruptor químico y un prototipo de un módulo de sobreproducción de antocianinas equivalente al descrito en el Capítulo 1, resultando en un dispositivo de producción de antocianinas con respuesta a dexametasona. Además, en el Capítulo 3 se describe la adaptación a la tecnología GB de las herramientas de ingeniería genética CRISPR/Cas9, así como su caracterización funcional. La funcionalidad de estas herramientas para editado génico y activación y represión transcripcional se validó con el sistema de expresión transitoria en N.benthamiana. Finalmente, el Capítulo 4 presenta una implementación práctica del uso de la tecnología GB para hacer mejora vegetal de manera precisa. La transformación estable en tomate de una construcción intragénica que comprendía un marcador de selección intragénico y un regulador de la biosíntesis de flavonoides resultó en frutos con un mayor contenido de flavonoles. En conjunto, esta Tesis muestra la implementación de diseños genéticos cada vez más complejos y precisos en plantas utilizando elementos estándar y herramientas modulares siguiendo los principios de la Biología Sintética. / [CA] La Biologia Sintètica és un camp emergent de caràcter interdisciplinar que es fonamenta amb l'aplicació a la enginyeria genètica dels principis de modularitat, abstracció i estandarització. Una nova vessant de la Biologia Sintètica aplicada a les plantes, la Biologia Sintètica Vegetal (BSV), ofereix noves possibilitats de millora de cultius que podrien portar a una millora de la resistència, a una major productivitat, o a un augment de la qualitat nutricional. Tanmateix, per poder arribar a este fi les eines moleculars disponibles en estos moments per a la BSV han d'adaptar-se per convertir-se en modulars, estàndards i més precises. Per això es plantejà com objectiu general d'aquesta Tesi adaptar, expandir i refinar les eines d'ensamblatge d'ADN de la BSV per permetre la incorporació d'especificacions funcionals en la descripció d'elements genètics estàndards (fitobricks) i facilitar la construcció d'estructures multigèniques cada vegada més complexes i precises, incloent eines d'edidat genètic. El punt de partida d'aquesta Tesi fou el mètode d'ensamblatge d'ADN modular GoldenBraid (GB) basat en enzims de restricció tipo IIS. Per optimitzar el proces d'ensamblatge i catalogar la col.lecció de fitobricks generats es desenvolupà una base de dades i un conjunt d'eines software, tal i com es descriu al Capítol 1. El paquet final de software es presentà en format web com GB2.0, fent-se accessible al públic mitjançant la pàgina web www.gbcloning.upv.es. El Capítol 1 també proporciona una descripció detallada del funcionament de GB2.0, exemplificant el seu ús amb l'ensamblatge d'una construcció multigènica per a la producció d'antocians. Amb l'augment en nombre i complexitat de les construccions GB, el següent pas fou el refinament dels estàndards amb la incorporació de la informació experimental associada a cada element genètic (es descriu en el Capítol 2). Per a aquest fi, el paquet de software de GB es reformulà amb una nova versió anomenada GB3.0. Aquesta versió consisteix en un sistema d'ensamblatge auto-contingut i complemtament traçable on les dades experimentals que descriuen la funcionalitat de cada element genètic es mostren en forma de fulla de dades estàndard. La utilitat de les especificacions tècniques per anticipar el comportament de dispositius biològics compostos s'exemplificà amb la combinació de un interruptor químic i un prototip d'un mòdul de sobreproducció d'antocians equivalent al descrit al Capítol 1. Aquesta combinació va tindre com a resultat un dispositiu de producció d'antocians que respón a dexametasona. A més a més, al Capítol 3 es descriu l'adaptació a la tecnologia GB de les eines d'enginyeria genètica CRISPR/Cas9, així com la seua caracterització funcional. La funcionalitat d'aquestes eines per a l'editat gènic i activació i repressió transcripcional es validà amb el sistema d'expressió transitòria en N. benthamiana. Finalment, al Capítol 4 es presenta una implementació pràctica de l'ús de la tecnologia GB per fer millora vegetal de mode precís. La transformació estable en tomaca d'una construcció intragènica que comprén un marcador de selecció intragènic i un regulador de la biosíntesi de flavonoïdes resultà en plantes de tomaca amb un major contingut de flavonols en llur fruits. En conjunt, esta Tesi mostra la implementació de dissenys genètics cada vegada més complexos i precisos en plantes utilitzant elements estàndards i eines modulars seguint els principis de la Biologia Sintètica. / Vázquez Vilar, M. (2016). DESIGN OF GENETIC ELEMENTS AND SOFTWARE TOOLS FOR PLANT SYNTHETIC BIOLOGY [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68483 / Premios Extraordinarios de tesis doctorales / Compendio

Coenzyme nicotinamide adenine dinucleotide (NAD, including the oxidized form-- NAD+ and reduced form--NADH) and the phosphorylated form--nicotinamide adenine dinucleotide phosphate (NADP, including NADP+ and NADPH) are two of the most important biological electron carriers. Most NAD(P) dependent redox enzymes show a preference of either NADP or NAD as an electron acceptor or donor depending on their unique metabolic roles. In biocatalysis, the low enzymatic activities with unnatural coenzymes have made it difficult to replace costly NADP with economically advantageous NAD or other biomimetic coenzyme for catalysis. This is a significant challenge that must be addressed should in vitro biocatalysis be a viable option for the practical production of low-value biocommodities (i.e., biohydrogen). There is a significant need to first address the coenzyme selectivity of the NADP-dependent dehydrogenases and evolve mutated enzymes that accept biomimetic coenzymes. This is a major focus of this dissertation. Establishment of efficient screening methods to identify beneficial mutants from an enzymatic library is the most challenging task of coenzyme engineering of dehydrogenases. To fine tune the coenzyme preference of dehydrogenases to allow economical hydrogen production, we developed a double-layer Petri-dish based screening method to identify positive mutant of the Moorella thermoacetica 6PGDH (Moth6PGDH) with a more than 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This method was also used to screen the thermostable mutant of a highly active glucose 6-phosphate dehydrogenase from the mesophilic host Zymomonas mobilis. The resulting best mutant Mut 4-1 showed a more than 124-fold improvement of half-life times at 60oC without compromising the specific activity. The screening method was further upgraded for the coenzyme engineering of Thermotaga maritima 6PGDH (Tm6PGDH) on the biomimetic coenzyme NMN+. Through six-rounds of directed evolution and screening, the best mutant showed a more than 50-fold improvement in catalytic efficiency on NMN+ and a more than 6-fold increased hydrogen productivity rate from 6-phosphogluconate and NMN+ compared to those of wild-type enzyme. Together, these results demonstrated the effectiveness of screening methods developed in this research for coenzyme engineering of NAD(P) dependent dehydrogenase and efficient use of the less costly coenzyme in ivSB based hydrogen production. / Ph. D. / NADP and NAD are two of the most important electron carriers in cellular metabolism, and they play distinctive roles in anabolism and catabolism, respectively. Most NAD(P)-dependent dehydrogenases exhibit a strong preference for either NADP or NAD. This coenzyme preference, however, make it nearly impossible to replace the costly NADP with less costly NAD or biomimetic coenzymes in the biocatalysis application. How to engineer dehydrogenases through directed evolution and effective screening method to accept NAD or biomimetic coenzymes, is critical and the focus of this dissertation. The use of in vitro synthetic biosystem (ivSB) to produce hydrogen form starch, is one of the most important in vitro synthetic biology projects, and it depends on NADP coenzyme. With other issues in this system solved, the efficient use of dehydrogenases along with low cost and stable coenzyme is the last obstacle to hydrogen production through industrial biomanufacturing. However, the 6-phosphogluconate dehydrogenase (6PGDH), one of the rate-limiting enzymes in this biosystem, exhibits a strong coenzyme preference for NADP⁺ . For producing low-cost hydrogen, the coenzyme engineering of this dehydrogenase is urgently required. Its activity with less costly NAD or biomimetic coenzymes must be improved. The establishment of an effective screening method is the most challenging task for coenzyme engineering of dehydrogenases. In this research, we developed a Petri-dish double-layer based screening method for coenzyme engineering of thermophilic 6PGDH for activity for NAD⁺ . This screening method was also used to improve the thermostability of a highly active glucose 6-phosphate dehydrogenase from a mesophilic host, where the evolved mutant had a greatly improved thermostability without losing activity. The screening method was further upgraded to develop for coenzyme engineering on biomimetic coenzyme NMN⁺ . The engineered mutant showing a more than 50-fold increase in catalytic efficiency on NMN⁺ was used to develop the first biomimetic coenzyme dependent electron transfer chain for hydrogen production. This screening method is suitable to change the coenzyme selectivity of series of NAD(P)-dependent redox enzymes and show great potential in improving other properties, such as thermostability, substrate scope and optimal pH, of different dehydrogenases. With this method developed, we can efficiently use the low cost stable coenzyme in the biocatalysis, and break the last obstacle to industrial biomanufacturing of hydrogen production.

DNA assembly and rational design are cornerstones of synthetic biology. While many DNA assembly standards have been published in recent years, only the Modular Cloning standard, or MoClo, has the advantage of publicly available part libraries for use in plant, yeast, and mammalian systems. No multipart modular library has previously been developed for use in prokaryotes. Building upon the existing MoClo assembly framework, we developed a collection of DNA parts and optimized MoClo protocols for use in E. coli. We present this assembly standard and library along with part characterization, design strategies, potential applications, and troubleshooting. Developed as part of the Cross-disciplinary Integration of Design Automation Research (CIDAR) lab collection of tools, the CIDAR MoClo Library is publicly available and contains promoters, ribosomal binding sites, coding sequences, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from previously published protocols. The CIDAR MoClo Library is the first bacterial DNA part library compatible with a multipart assembly standard. To demonstrate the utility of the CIDAR MoClo system in a traditional biology context, we used the library and previous expression data to create a series of dual expression plasmids. In this manner, we produced a dual expression plasmid capable of expressing equimolar amounts of two variants of rabbit aldolase, a His-tagged wildtype protein and a single-amino-acid substitution mutant deficient in binding actin. This expression plasmid will enable the production of dimer-of-dimer heterotetramers needed for structural determination of the actin-aldolase interaction by electron microscopy. To employ CIDAR MoClo in a synthetic biology context, we produced a bioelectronic pH-mediated genetic logic gate with DNA circuits built using MoClo and integrated with Raspberry Pi computers, Twitter, and 3D printed components. Logic gates are an increasingly common biological tool with applications in cellular memory and biological computation. MoClo facilitates rapid iteration of genetic designs, better enabling the development of cellular logic. The CIDAR MoClo Library and assembly standard enable rapid design-build-test cycles in E. coli making this system advantageous for use in many areas of synthetic biology as well as traditional biological research.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25206号 / 医科博第162号 / 新制||医科||11(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 竹内 理, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p dir="ltr">Sterile manufacturing is multifaceted. Each aspect seeks to improve the process of producing drug products absent of impurities. Bacteriophages can benefit greatly from sterile manufacturing which would amplify their already vast range of applications. Novel bacteriophage discovery and annotation, implemented within a classroom setting, can aid in building the foundation of bacteriophages for use in clinical applications. High-Performance Liquid Chromatography (HPLC) can detect impurities and test compatibilities in the final product. Oxytocin and its interaction with tranexamic acid (TXA) provide an excellent example of how HPLC use can be critical in sterile manufacturing as well as build a baseline for which bacteriophages may be utilized in sterile production. A quality scorecard for drug products provides an additional metric that can be used by governing agencies and consumers to analyze drug products of similar bioequivalence and subsequently grade them. The development of a scorecard will provide a guideline to improve the sterile manufacturing of drug products and biologics such as bacteriophages. A literary analysis of lipid nanoparticles presents a future application for synthetically manufactured bacteriophages. The conclusions gathered from this work can be utilized as a case study for working professionals who aim to implement advancements in sterile manufacturing within their industry.</p>