Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor.

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and ß-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates.

Production of Potyvirus-Derived Nanoparticles Decorated with a Nanobody in Biofactory Plants.

Viral nanoparticles (VNPs) have recently attracted attention for their use as building blocks for novel materials to support a range of functions of potential interest in nanotechnology and medicine. Viral capsids are ideal for presenting small epitopes by inserting them at an appropriate site on the selected coat protein (CP). VNPs presenting antibodies on their surfaces are considered highly promising tools for therapeutic and diagnostic purposes. Due to their size, nanobodies are an interesting alternative to classic antibodies for surface presentation. Nanobodies are the variable domains of heavy-chain (VHH) antibodies from animals belonging to the family Camelidae, which have several properties that make them attractive therapeutic molecules, such as their small size, simple structure, and high affinity and specificity. In this work, we have produced genetically encoded VNPs derived from two different potyviruses-the largest group of RNA viruses that infect plants-decorated with nanobodies. We have created a VNP derived from zucchini yellow mosaic virus (ZYMV) decorated with a nanobody against the green fluorescent protein (GFP) in zucchini (Cucurbita pepo) plants. As reported for other viruses, the expression of ZYMV-derived VNPs decorated with this nanobody was only made possible by including a picornavirus 2A splicing peptide between the fused proteins, which resulted in a mixed population of unmodified and decorated CPs. We have also produced tobacco etch virus (TEV)-derived VNPs in Nicotiana benthamiana plants decorated with the same nanobody against GFP. Strikingly, in this case, VNPs could be assembled by direct fusion of the nanobody to the viral CP with no 2A splicing involved, likely resulting in fully decorated VNPs. For both expression systems, correct assembly and purification of the recombinant VNPs was confirmed by transmission electron microscope; the functionality of the CP-fused nanobody was assessed by western blot and binding assays. In sum, here we report the production of genetically encoded plant-derived VNPs decorated with a nanobody. This system may be an attractive alternative for the sustainable production in plants of nanobody-containing nanomaterials for diagnostic and therapeutic purposes.

Carotenoid fortification of zucchini fruits using a viral RNA vector.

BACKGROUND: Carotenoids are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content. Although the usefulness of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial. RESULTS: Here, we demonstrate an alternative biotechnological strategy for carotenoid fortification of edible fruits in which no transgenic DNA is involved. A viral RNA vector derived from zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated to zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers. After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color. Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as α- and ß-carotene (provitamin A), lutein and phytoene, in both rind and flesh. CONCLUSION: Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved.

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system.

Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, for the color and the unique taste of the saffron spice, known as red gold due to its high price. Several studies have also shown the health-promoting properties of these compounds. However, their high costs hamper the wide use of these metabolites in the pharmaceutical sector. We have developed a virus-driven system to produce remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants in only two weeks. The system consists of viral clones derived from tobacco etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. In an attempt to improve apocarotenoid content in N. benthamiana, co-expression of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene synthase (PaCrtB) and saffron ß-carotene hydroxylase 2 (BCH2), was performed using the same viral system. This combinatorial approach led to an additional crocin increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. Considering that saffron apocarotenoids are costly harvested from flower stigma once a year, and that Buddleja spp. flowers accumulate lower amounts, this system may be an attractive alternative for the sustainable production of these appreciated metabolites.