Plant-Based Production of Metabolites and Nanoparticles Using Potyvirus Vectors

Tesis por compendio[ES] La biotecnología de plantas actual, y la llamada agricultura molecular aspiran a convertir las plantas en "biofábricas" sostenibles para producir compuestos de valor como proteínas, metabolitos o nanopartículas de interés farmacéutico o industrial. Los virus de plantas constituyen una de las principales causas de enfermedades vegetales. Son capaces de secuestrar la maquinaria celular del huésped, y de ahí surgió la idea de reconvertir los virus de plantas en herramientas para la biotecnología de plantas como vectores de expresión transitoria y andamios para nanomateriales. Los carotenoides son metabolitos relevantes debido a sus propiedades nutricionales y beneficiosas para la salud. El primer objetivo fue manipular la ruta de biosíntesis de carotenoides para producir los muy apreciados apocarotenoides de azafrán, siendo estos los productos de la escisión de carotenoides. Para ello, se diseñó un vector derivado del virus del grabado del tabaco (TEV; género Potyvirus) manipulado para expresar unas enzimas específicas, dioxigenasas de escisión de carotenoides (CCD) de Crocus sativus y Buddleja davidii. Los análisis metabólicos de los tejidos infectados demostraron que, tras sólo dos semanas, se alcanzaron cantidades notables de crocinas y picrocrocina en plantas adultas de Nicotiana benthamiana. Sólo la expresión de CsCCD2L de C. sativus dio una acumulación en hoja de 0.2% de crocinas y 0.8% de picrocrocina en peso seco. La coexpresión de CsCCD2L con otra enzima carotenogénica, como la fitoeno sintasa de Pantoea ananatis (PaCrtB), usando el mismo vector aumentó la acumulación de crocinas al 0.35%. Pese a ser cantidades inferiores a las encontradas en fuentes naturales, este sistema mediado por virus representa el primer sistema heterólogo capaz de producir crocinas. Los compuestos fenólicos son otro amplio grupo de metabolitos secundarios en plantas muy apreciados también. Los curcuminoides son polifenoles con alta actividad antioxidante que se encuentran naturalmente en el rizoma de la cúrcuma (Curcuma longa). El segundo objetivo fue establecer un sistema para la producción heteróloga de curcuminoides utilizando vectores virales. Para ello, se desarrolló un sistema viral doble, basado en TEV y en el virus X de la patata (PVX; género Potexvirus), capaz de coexpresar diferentes enzimas biosintéticas en las mismas células. Este sistema se usó para expresar la dicétido-CoA sintasa 1 (DCS1) y la curcumina sintasa 3 (CURS3) de C. longa en plantas de N. benthamiana. El análisis metabólico confirmó la producción exitosa de curcuminoides usando dos vectores virales. Posteriormente se analizó la coexpresión de DCS1 y CURS3 usando un solo vector viral derivado de TEV, obteniendo una producción más eficiente, aumentando al doble la acumulación de curcumina. Tras un análisis temporal usando el vector TEVΔN-DCS1-CURS3, se vio que a los 11 días se lograba una acumulación máxima de 22 ± 4 µg/g peso seco. Las nanopartículas virales (VNP) también han atraído la atención en biotecnología por su uso potencial como componentes básicos para nuevos materiales en nanotecnología y medicina. Los nanoanticuerpos son los dominios variables de los anticuerpos de camélidos de sólo cadena pesada (VHH) que han ganado interés como moléculas terapéuticas por su estructura simple, tamaño pequeño y alta especificidad. El último objetivo de este trabajo fue producir VNPs decoradas con un nanoanticuerpo codificadas genéticamente. El virus del mosaico amarillo del calabacín (ZYMV; género Potyvirus) y TEV se utilizaron como andamios para producir VNPs decoradas con un nanoanticuerpo contra la proteína verde fluorescente en plantas de calabacín y N. benthamiana, respectivamente. Confirmándose el ensamblaje y unión de ambas VNPs contra GFP. En conjunto, el trabajo presentado en esta tesis contribuye al concepto de que los virus de plantas, convenientemente manipulados, pueden convertirse en poderosas herramientas en biotecnología vegetal y agricultura molecular.[CA] La biotecnologia de plantes actual i la anomenada agricultura molecular aspiren a convertir les plantes en "biofàbriques" sostenibles per a produir compostos de valor com a proteïnes, metabòlits o nanopartícules d'interès farmacèutic o industrial. Els virus de plantes constitueixen una de les principals causes de malalties vegetals. Son capaços de segrestar la maquinària cel·lular de l'hoste, i d'ací va sorgir la idea de reconvertir els virus en eines per la biotecnologia de plantes com a vectors d'expressió transitòria i bastides per a nanomaterials. Els carotenoides són metabòlits rellevants a causa de les seues propietats nutricionals i beneficioses per a la salut. El primer objectiu va ser manipular la ruta de biosíntesi de carotenoides per a produir els valuosos apocarotenoides de safrà, sent aquests els productes de l'escissió de carotenoides. Per a això, es va dissenyar un vector derivat del virus del gravat del tabac (TEV; gènere Potyvirus) manipulat per a expressar uns enzims específics, dioxigenases d'escissió de carotenoides (CCD) de Crocus sativus i Buddleja davidii. Les anàlisis metabòliques dels teixits infectats van demostrar que, després de només dues setmanes, es van aconseguir quantitats notables de crocines i picrocrocina en plantes adultes de Nicotiana benthamiana. Només l'expressió de CsCCD2L de C. sativus va donar com a resultat una acumulació en fulla de 0.2% de crocines i 0.8% de picrocrocina en pes sec. La coexpressió de CsCCD2L amb un altre enzim carotenogènic, com la fitoé sintasa de Pantoea ananatis (PaCrtB), usant el mateix vector viral va augmentar l'acumulació de crocines al 0.35%. Malgrat ser quantitats inferiors a les trobades en fonts naturals, aquest sistema mediat per virus representa el primer sistema heteròleg capaç de produir crocines. Els compostos fenòlics són un altre ampli grup de metabòlits secundaris en plantes, també molt valuosos. Els curcuminoides són polifenols amb alta activitat antioxidant que es troben naturalment en el rizoma de la cúrcuma (Curcuma longa). El segon objectiu va ser establir un sistema per a la producció heteròloga de curcuminoides utilitzant vectors virals. Per a això, es va desenvolupar un sistema viral doble, basat en TEV i en el virus X de la creïlla (PVX; gènere Potexvirus, família Alphaflexiviridae), capaç de coexpressar diferents enzims biosintètics en les mateixes cèl·lules. Aquest sistema es va usar per a expressar la dicétid-CoA sintasa 1 (DCS1) i la curcumina sintasa 3 (CURS3) de C. longa en plantes de N. benthamiana. L'anàlisi metabòlica va confirmar la producció reeixida de curcuminoides. Posteriorment es va analitzar la coexpresió de DCS1 i CURS3 usant un sol vector viral derivat de TEV, obtenint una producció més eficient, augmentant al doble l'acumulació de curcumina. Una anàlisi temporal usant el vector TEVΔN-DCS1-CURS3 va mostrar que als 11 dies s'aconseguia una acumulació màxima de 22 ± 4 µg/g pes sec. Les nanopartícules virals (VNP) també han atret l'atenció en biotecnologia pel seu ús potencial com a components bàsics per a nous materials en nanotecnologia i medicina. Els nanoanticossos són els dominis variables dels anticossos de camèlids de només cadena pesada (VHH) que han guanyat interès com a molècules terapèutiques per la seua estructura simple, grandària xicoteta i alta especificitat. L'últim objectiu d'aquest treball va ser produir VNPs decorades amb un nanoanticos codificades genèticament. El virus del mosaic groc de la carabasseta (ZYMV; gènere Potyvirus) i TEV es van utilitzar com a bastides per a produir VNPs decorades amb un nanocos contra la proteïna verda fluorescent en plantes de carabasseta i N. benthamiana, respectivamente. Confirmant-se l'assemblatge i unió de les VNPs contra GFP. En conjunt, el treball presentat en aquesta tesi contribueix al concepte que els virus de plantes, convenientment manipulats, poden convertir-se en poderoses eines en biotecnologia vegetal i agricultura molecular.[EN] Modern plant biotechnology and molecular farming aim to convert plants into sustainable 'biofactories' to produce valuable compounds as proteins, metabolites or nanoparticles of pharmaceutical or industrial interest. Plant viruses, constitute a major cause of plant diseases inducing devastating crop losses. Based on their ability to hijack the host cell machinery, it arose the idea of repurposing plant viruses from foes to friends into tools for plant biotechnology as transient expression vectors and scaffolds for nanomaterials. Carotenoids are relevant metabolites based on their nutritional and health-promoting properties. The first goal of this work was to manipulate the carotenoid biosynthesis pathway to produce highly appreciated saffron apocarotenoids. For this purpose, a vector derived from Tobacco etch virus (TEV; genus Potyvirus, family Potyviridae) was engineered to express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that, after only two weeks, remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants were reached. The sole virus-driven expression of C. sativus CsCCD2L resulted in an accumulation of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight (DW). Co-expression of CsCCD2L with another carotenogenic enzyme, such as Pantoea ananatis phytoene synthase (PaCrtB), using the same viral vector increased crocin accumulation to 0.35%. Although these amounts are still far from those accumulating in natural sources, such as saffron stigma, this virus-driven system represents the first heterologous system able to produce crocins. Phenolic compounds represent another broad group of plant secondary metabolites highly appreciated for their health promoting properties. Curcuminoids are polyphenols with high antioxidant activity that are naturally found in turmeric (Curcuma longa) rhizome. The second goal of this work was to establish a system for the heterologous production of curcuminoids using viral vectors. To this aim, a double-virus vector system, based on TEV and Potato virus X (PVX; genus Potexvirus, family Alphaflexiviridae), able to co-express different biosynthetic enzymes in the same cells was developed. This system was used to express C. longa diketide-CoA synthase 1 (DCS1) and curcumin synthase 3 (CURS3) in N. benthamiana plants. Metabolic analysis confirmed the successful production of curcuminoids. Curcumin quantification indicated that sequential inoculation of both viral vectors was more efficient than co-inoculation. Co-expression of DCS1 and CURS3 was next analysed using a single viral vector derived from TEV (TEVΔN-DCS1-CURS3). This resulted in a more efficient approach as it led to a 2-fold increase in curcumin accumulation (11.7 ± 1.5 µg/g DW). A time-course analysis using the TEVΔN-DCS1-CURS3 vector showed that a maximum accumulation of 22 ± 4 µg/g DW was achieved at 11 days post-inoculation. Viral nanoparticles (VNPs) have also attracted attention in biotechnology for their potential use as building blocks for novel materials in nanotechnology and medicine. Nanobodies are the variable domains of heavy-chain (VHH) camelid antibodies that have sparked interest as therapeutic molecules due to their simple structure, small size and high specificity. The last goal of this work was to produce genetically encoded VNPs decorated with a nanobody. Zucchini yellow mosaic virus (ZYMV; genus Potyvirus, family Potyviridae) and TEV were used as scaffolds to produce VNPs decorated with a nanobody against the green fluorescent protein in zucchini (Cucurbita pepo) and N. benthamiana plants, respectively. Assembly and binding functionality of both VNPs against GFP was confirmed. Altogether, the work presented in this thesis contribute to the concept that plant viruses, conveniently manipulated, can turn into powerful tools in plant biotechnology and molecular farming.This work was supported by grants BIO2016-77000-R, PID2020-114691RB-I00 and BIO2017-83184-R from the Spanish Ministerio de Ciencia, Innovación y Universidades (co-financed European Union FEDER funds). M.M. was the recipient of a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (FPU16/05294).Martí Botella, MC. (2022). Plant-Based Production of Metabolites and Nanoparticles Using Potyvirus Vectors [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/187155Compendi

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