Plant-based production of metabolites and nanoparticles using potyvirus vectors
- Autores: Mari Carmen Martí Botella
- Directores de la Tesis: José Antonio Daròs Arnau (dir. tes.), Carmelo López del Rincón (tut. tes.)
- Lectura: En la Universitat Politècnica de València ( España ) en 2022
- Idioma: español
- Tribunal Calificador de la Tesis: Fernando Ponz Ascaso (presid.), Carmen Hernández Fort (secret.), Hadrien Peyret (voc.)
- Programa de doctorado: Programa de Doctorado en Biotecnología por la Universitat Politècnica de València
- Materias:
- Enlaces
- Tesis en acceso abierto en: RiuNet
- Resumen
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 (TEV¿N-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 TEV¿N-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.
