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.

Thesis advisor: Abhishek Chatterjee / Genetic code expansion (GCE) is an enabling technology whereby noncanonical amino acids (ncAAs) can be site-specifically incorporated into proteins of interest, allowing for vast applications and an improved understanding of structure-function relationships in biology. GCE stands out as a versatile platform due to the use of a variety of engineered aminoacyl-tRNA synthetase (aaRS)/transfer RNA (tRNA) pairs, and it has endowed proteins with over 200 distinct ncAAs in both prokaryotic and eukaryotic systems. My dissertation outlines endeavors aimed at broadening the chemical diversity of α-amino side chains and substrates beyond α-amino acids in both prokaryotic and eukaryotic organisms through the utilization of GCE technology. This was achieved by creating universal GCE platforms called altered translational machinery (ATM) strains, which eliminate the limitations of orthogonality for the evolution of aaRS/tRNA pairs. This expansion enables the use of the same aaRS/tRNA pair for ncAA incorporation functionalities into multiple domains of life. Moreover, the diversity of ncAAs that can be genetically encoded in eukaryotic cells was enhanced by evolving the E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNA pair using a yeast-based selection system. This advancement facilitated the incorporation of novel ncAAs into proteins within mammalian cells. Additionally, I worked toward developing a platform for introducing monomers into the genetic code beyond α-amino acids. This involved developing an aaRS evolution platform that doesn't rely on translation as a selectable readout. Finally, I worked towards the creation of polyester-polyamide oligomers with sequence control as a step towards the goal of generating sequence-defined biopolymers with new-to-nature backbone chemistries. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Yes / We present the Infobiotics Workbench (IBW), a user-friendly, scalable, and integrated computational environment for the computer-aided design of synthetic biological systems. It supports an iterative workflow that begins with specification of the desired synthetic system, followed by simulation and verification of the system in high- performance environments and ending with the eventual compilation of the system specification into suitable genetic constructs. IBW integrates modelling, simulation, verification and bicompilation features into a single software suite. This integration is achieved through a new domain-specific biological programming language, the Infobiotics Language (IBL), which tightly combines these different aspects of in silico synthetic biology into a full-stack integrated development environment. Unlike existing synthetic biology modelling or specification languages, IBL uniquely blends modelling, verification and biocompilation statements into a single file. This allows biologists to incorporate design constraints within the specification file rather than using decoupled and independent formalisms for different in silico analyses. This novel approach offers seamless interoperability across different tools as well as compatibility with SBOL and SBML frameworks and removes the burden of doing manual translations for standalone applications. We demonstrate the features, usability, and effectiveness of IBW and IBL using well-established synthetic biological circuits. / The work of S.K. is supported by EPSRC (EP/R043787/1). N.K., A.W., and B.B. acknowledge a Royal Academy of Engineering Chair in Emerging Technologies award and an EPSRC programme grant (EP/N031962/1).

Tumors employ a variety of genetic resistance mechanisms to evade immune responses and immunotherapies such as PD-1 blockade. The pleiotropic cytokine interferon-gamma (IFNγ) is a potent immune effector and critical for patient response to PD-1 blockade, yet conventional systemic delivery is hindered by severe dose limiting toxicities. As such, the effects of exogenously introduced IFNγ either as monotherapy or in combination with PD-1 blockade in the context of different tumor genetic background remain poorly understood. Synthetic biology allows programming of microbes for tumor-specific delivery of therapeutic candidates that are otherwise not possible using conventional administration strategies. Herein, we engineered a strain of probiotic bacteria that home to tumors and locally release IFNγ. We validated the efficacy of our therapeutic strain, either as monotherapy or in combination with PD-1 blockade, in multiple murine tumor models. Within this dissertation, we demonstrate that a single intratumoral injection of these IFNγ-producing bacteria is sufficient to drive systemic tumor antigen–specific antitumor immunity, without observable toxicity. Although cancer cells employ various resistance mechanisms to evade immune responses, bacteria-derived IFNγ additionally overcomes primary resistance to PD-1 blockade via activation of cytotoxic CD4⁺Foxp3⁻ and CD8⁺ T cells. Moreover, by activating NK cells, bacteria-derived IFNγ also overcome acquired resistance mechanisms to PD-1 blockade, specifically loss of function mutations in IFNγ signaling and antigen presentation pathways. Collectively, this dissertation highlights the promise of combining IFNγ-producing bacteria with PD-1 blockade as a therapeutic strategy for overcoming immunotherapy-resistant, locally advanced, and metastatic disease.

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

Breakthroughs in molecular and synthetic biology through de novo gene synthesis are stimulating new vaccines, pharmaceutical applications, and functionalized biomaterials, and advancing the knowledge of the function of cells. This evolution in biological processing motivates the study of a class of manufacturing systems, defined here as genetic manufacturing systems, which produce a final product with a genetic construct. Genetic manufacturing systems rely on rare molecular events for success, resulting in waste and repeated work during the deoxyribonucleic acid (DNA) fabrication process. Inspection and real time monitoring strategies are possible as mitigation tools, but it is unclear if these techniques are cost efficient and value added for the successful creation of custom genetic constructs. This work investigates resource allocation strategies for DNA fabrication environments, with an emphasis on inspection allocation. The primary similarities and differences between traditional manufacturing systems and genetic manufacturing systems are described. A serial, multi-stage inspection allocation mathematical model is formulated for a genetic manufacturing system utilizing gene synthesis. Additionally, discrete event simulation is used to evaluate inspection strategies for a fragment synthesis process and multiple fragment assembly operation. Results from the mathematical model and discrete event simulation provide two approaches to determine the appropriate inspection strategies with respect to total cost or total flow time of the genetic manufacturing system. / Ph. D. / Breakthroughs in molecular and synthetic biology through <i>de novo</i> gene synthesis are stimulating new vaccines, pharmaceutical applications, and functionalized biomaterials, and advancing the knowledge of the function of cells. This evolution in biological processing motivates the study of a class of manufacturing systems, defined here as genetic manufacturing systems, which produce a final product with a genetic construct. Genetic manufacturing systems rely on rare molecular events for success, resulting in waste and repeated work during the deoxyribonucleic acid (DNA) fabrication process. Inspection and real time monitoring strategies are possible as mitigation tools, but it is unclear if these techniques are cost efficient and value added for the successful creation of custom genetic constructs. This work investigates resource allocation strategies for DNA fabrication environments, with an emphasis on inspection allocation. The primary similarities and differences between traditional manufacturing systems and genetic manufacturing systems are described. A serial, multi-stage inspection allocation mathematical model is formulated for a genetic manufacturing system utilizing gene synthesis. Additionally, discrete event simulation is used to evaluate inspection strategies for a fragment synthesis process and multiple fragment assembly operation. Results from the mathematical model and discrete event simulation provide two approaches to determine the appropriate inspection strategies with respect to total cost or total flow time of the genetic manufacturing system.

The advent of synthetic transcriptional regulators built mainly on human-derived proteins, namely synthetic Zinc Finger Transcription Regulators (synZiFTRs), has enabled fine-tuned control of therapeutically significant genes in primary T cells. However, their clinical relevance could be enhanced by amplifying synthetic gene circuit activation and expanding the synZiFTR toolkit with standardized compo-nents for the construction of more complex circuits. This study describes the de-velopment of the next iteration of synZiFTR, the synZiFTR2.0, incorporating the human-derived transcription elongation domain, IWS1. We present an engi-neered version 2.0 of GZV- and 4OHT/TMX-regulated gene switches, exhibiting a robust increase in transcriptional output upon drug induction. Furthermore, the synZiFTR toolkit was expanded and utilized to examine the feasibility of con-structing a two-input AND logic gate. Interestingly, the integration of IWS1 un-veiled a potential role of PP1-NUTS phosphatase in enhancing synthetic circuit output, though the precise mechanism warrants further investigation. The intro-duction of synZiFTR2.0 is projected to boost its clinical applicability, particularly in settings where circuit output strength is contingent on disease context that is often uncertain. / 2025-10-03T00:00:00Z

Plant form is a product of emergent processes of cell division, patterning and morphogenesis. These fundamental processes remain poorly characterised in plants. However, engineering approaches can provide new tools and frameworks for the study and manipulation of plant development. This dissertation describes the development of engineering frameworks for reprogramming of the early diverging land plant Marchantia polymorpha (Marchantia). I describe the generation of genomic and transcriptomic datasets for Marchantia, which has provided the basis for the compilation of a gene-centric registry of DNA parts for engineering (MarpoDB). I describe the development of Loop assembly, an efficient and standardised DNA assembly system based on Type IIS restriction enzymes for recursive fabrication of DNA circuits with high efficiency. MarpoDB was used to mine new DNA parts compatible with Loop assembly which were used to generate plant transformation vectors for labelling of cellular features to study aspects of growth and development. I performed image analysis of genetic markers for segmentation and quantification of cellular properties in germinating gemmae. I implemented high-efficiency Cas9-mediated mutagenesis in Marchantia for use in functional molecular genetics studies. Furthermore, I produced inducible systems for expression of heterologous elements by transactivation which showed negligible levels of basal activity. It was possible to use this system for induction of gene expression in single cells. Finally, these new frameworks were applied to study the gametophytic meristem in Marchantia gemmae. I mapped the expression of several putative candidate homologues for higher plant meristem regulators, performed overexpression and loss-of-function studies for homologues of WUSCHEL, CLAVATA3 and SHOOT MERISTEMLESS. A strategy for misregulation of endogenous genes was developed using inducible transactivation, and was used with cellular markers for WUSCHEL and CLAVATA3 homologues in Marchantia.

Enhancing productivity in bioprocesses, especially for biofuel production, is crucial for achieving an environmentally and economically sustainable biotechnology industry.Metabolic channelling occurs in nature when the intermediate between two consecutive enzymes in a pathway is directed from the first enzyme to the second avoiding diffusion in the cytosol. This would be very advantageous in bioprocesses as it would increase efficiency of a particular pathway, reducing side products and protecting the cells from potential toxic intermediates. In recent years different strategies for emulating channelling effect wereproposed and used with very promising results. Clustering of enzymes seems to be the simplest way to create metabolic channelling. In this master thesis, four different strategies to co-localize enzymes in clusters are compared. The metabolic pathway chosen as a model was ethanol production by pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh). Chimeric proteins were genetically engineered and transformed in E. coli creating different strains. Ethanol production by the different strains was measured to compare production efficiency. Cell growth and protein expression were used for further understanding of the results. Strengths and weaknesses of each strategy and proposals for further improvement were discussed.