Method for the production of apocarotenoids and uses thereof in the food, cosmetic, nutraceutical and pharmaceutical sector

Abstract

The invention provides methods and materials for recombinantly producing apocarotenoids in bacteria and transgenic plants, wherein said transgenic organisms expressing at least one heterologous gene from a plant species selected from Handroanthus impetiginosus, Paulownia tomentosa, and / or Physalis peruviana. Moreover, the present invention also relates to the genetic constructs comprising the at least one heterologous gene, at least a plasmid and / or at least a vector comprising the same, their use for the expression in bacteria and plants, and methods for obtaining such genetically modified bacteria and plants, which have a high concentration of apocarotenoids compounds, in particular crocins and picrocrocins. Furthermore, the present disclosure also refers to the compositions comprising such apocarotenoids products obtained and uses thereof.

Description

[0001] DESCRIPTION

[0002] METHOD FOR THE PRODUCTION OF APOCAROTENOIDS AND USES THEREOF IN THE FOOD, COSMETIC, NUTRACEUTICAL AND PHARMACEUTICAL SECTOR

[0003] TECHNICAL FIELD

[0004] The invention disclosed herein relates generally to the field of genetic engineering. Particularly, the invention disclosed herein provides methods and materials for recombinantly producing apocarotenoids in bacteria, fungi, and transgenic plants, expressing at least one heterologous gene encoding for carotenoid cleavage dioxygenase 4 (CCD4) enzyme from plant species selected from Handroanthus impetiginosus, Paulownia tomentosa and Physalis peruviana. Moreover, the present disclosure also relates to the genetic constructs comprising at least one heterologous genes, plasmid and / or vectors comprising the same, their use for the expression in bacteria, fungi, and plants, and a method for obtaining such genetically modified bacteria, fungi, and plants, which express the genetic constructs mentioned above, and which have a high concentration of apocarotenoids products, in particular crocins and picrocrocins. Furthermore, the present disclosure also refers to the uses of such apocarotenoid products obtained as food, nutraceutical, cosmetic and pharmaceutical compositions.

[0005] STATE OF THE ART

[0006] Crocins and picrocrocin are glucosylated and water-soluble apocarotenoids produced in nature by a few plant species, being saffron the plant that accumulates these apocarotenoids, in the stigma of saffron, at higher levels. In addition to being ingredients with high added value in foods and nutraceuticals, crocins and their precursor crocetin have cosmetic and medical applications. The biosynthesis of crocins in saffron (Crocus sativus) has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems such as bacteria, tobacco, tomato and potato. In addition to crocins, the apocarotenoid picrocrocin, which also displays many interesting medicinal properties, is also present in saffron. Although gardenia fruits have also been utilized in China as a source of crocins and as pigments, picrocrocin is not present in the fruits of this plant.

[0007] Apocarotenoid compounds are derived from the oxidative cleavage of carotenoids in a reaction catalyzed by a family of enzymes named carotenoid cleavage dioxygenases (CCDs), which cleave carotenoids at their double bonds. Crocetin is generated in Crocus species by the action of CCD2 using zeaxanthin as substrate, and in B. davidii and Verbascum by two CCD4 enzymes, which catalyzed the same reaction. Patent ES2334423B1 discloses sequences of the enzymes CsCCD4a and CsCCD4b, dioxygenase enzymes isolated from Crocus Sativus, which catalyze the cleavage of the bond 9,10; 9", 10" of the carotenoid p-carotene, releasing volatile substances that contribute to the aroma and flavor of said species. On the other hand, the international patent application PCT / EP2022 / 075670 discloses methods and materials for the generation of apocarotenoids: crocetins, crocins and picrocrocins, in plants of the Solanaceae family. International Patent application WO2016012968A1 describes a CCD, specifically CCD2, for the biotechnological production in microorganisms and plants of saffron-derived compounds, specifically dialdehyde crocetin and crocetin from cleavage of the double bonds at position 7,8 and 7', 8'. CCD2 cleaves these bonds from zeaxanthin, but also other carotenoid substrates containing a 3-OH-p ring at the proximal end of the molecule. However, no enzymes catalyzing the transformation of 2,6,6-trimethyl-4- hydroxy-1- carboxaldehyde-1 -cyclohexene (4-Hydroxy-2,6,6-trimethyl-1-cyclohexene-1- carbaldehyde, HTCC) to picrocrocin were detected. Picrocrocin is responsible for the slightly bitter taste of saffron. From picrocrocin, formed during the drying of the spice, safranal, one of the fundamental components of saffron aroma, is produced.

[0008] Given the high cost of apocarotenoids production, mainly due to the expensive extraction processes and the non-availability of a method to produce them through chemical synthesis, there is a constant need to have improved processes for the production of apocarotenoids with a high yield and low costs.

[0009] In 2022, the global carotenoids market value was estimated at $1.8 million and is expected to reach $2.09 million by 2028 at a compound annual growth rate of 5.5% (https: / / www.fortunebusinessinsights.com). The carotenoid market is driven by (i) growing concern over the presence of synthetic carotenoids versus those from natural sources; (ii) increased focus on consumption of natural products; and (iii) increased demand for pharmaceutical, food, cosmetic and nutraceutical products.

[0010] In the last few years, particular attention has been paid to the development of biotechnological methods for their production in high-productivity microbial, fungi or plant systems. Such methods require the availability of genes that are able to mediate the production of these apocarotenoids and their accumulation at high levels. The present invention fits into this field of biotechnological production of apocarotenoids in bacteria, fungi and transgenic plants. Thus, there is a need to provide naturally produced compounds, preferably apocarotenoids, with improved yields.

[0011] DESCRIPTION OF THE INVENTION

[0012] The present disclosure relates to the identification of three enzymes belonging to CCD4 family named as PpCCD4, 35707 and 26288, involved in the cleavage of carotenoid in three different plant species, Handroanthus impetiginosus, Paulownia tomentosa, and Physalis peruviana, respectively. These new three enzymes have been analyzed for apocarotenogenic activity in bacteria, fungi, and plants from the Solanaceae family using a virus-driven system. These three novel enzymes, 26288, 35707, and PpCCD4, identified in H. impetiginosus, P. tomentosa and P. peruviana, respectively, cleave lycopene, p-carotene and zeaxanthin at 7,8,7'8'double bonds to efficiently produce crocetin dialdehyde, although neither crocetin nor crocin has been identified in these three plant species.

[0013] Metabolic analysis of bacterial extracts and dry leaves from plants of the Solanaceae family has shown the efficient activity of these enzymes to produce crocins using different substrates such as lycopene, p-carotene, neurosporene, b-carotene, - carotene, and / or zeaxanthin. Thus, the inventors have identified that the CDD4 enzymes of the invention, PpCCD4, 35707 and 26288 have different substrate specificities (Table 1).

[0014] Table 1. Substrate specificities of each CDD4 enzyme of the present invention. In addition, the experimental data show that accumulations of 0.34%, 0.38% and

[0015] 0.59% of crocins in dry leaves from plants of the Solanaceae family were reached in only two weeks using a recombinant virus expressing the enzymes 26288, 35707 and PpCCD4, independently. Moreover, a similar approach was previously used in the prior art to overexpress the saffron enzyme CsCCD2L transiently, and the enzyme BdCCD4.1 , from B. davidii, which resulted in the synthesis of crocins at 0.21% and 0.076% of leaf dry weight, respectively. These two enzymes, CsCCD2L and BdCCD4.1 , only recognized zeaxanthin as substrate, which can limit their use to tissues where this carotenoid is present. Consequently, in view of the higher substrate specificity for the CCD4 enzymes of the present invention (see Table 1), higher levels of apocarotenoids were obtained compared to those obtained by the prior art enzymes, CsCCD2L and BdCCD4.1, demonstrating how these enzymes, 26288, 35707 and PpCCD4, independently or in any combination thereof, which display particularly a broad substrate spectrum, open new avenues for apocarotenoid biotechnological production in different heterologous system, from microorganisms to plants. In fact, as it is shown in Figure 8, the crocin concentrations in the transgenic tomato fruits obtained from the tomato plants transformed with the CCD4s of the invention (SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 3), were remarkably increased compared to that in the transgenic tomato fruits obtained from the tomato plants transformed with the CsCCD2 (GenBank accession number KP887110.1 - SEQ ID NO: 22).

[0016] In summary, the data provided herein demonstrates the strong potential and feasibility of the CCD4s of the invention as powerful biotechnological tools for significantly enhancing the production of apocarotenoids with high value to both industry and human health. Notably, the results show the highest crocin content ever reported through metabolic engineering in heterologous systems. These levels significantly exceed those achieved using CCD2s known in the art, obtained under the same conditions, highlighting the superior efficiency of CCD4s of the invention over previously known systems. Altogether, this data establishes a novel and viable platform for the large-scale production of apocarotenoids.

[0017] Thus, in a first aspect, the present disclosure provides an isolated and / or exogenous polynucleotide, hereinafter, the polynucleotide of the invention, comprising: i) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%; 99% or 100% identical to any one of the nucleotides sequences selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:5, SEQ ID NO:3, or any combinations thereof; ii) a biologically active fragment thereof; or iii) a nucleotide sequence complementary thereof; wherein the nucleotide sequence of SEQ ID NO: 1, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Physalis, preferably from P. peruviana-, SEQ ID NO: 5, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Handroanthus, preferably from H. impetiginosus; and SEQ ID NO: 3, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Paulownia, preferably from P. tomentosa; a biologically active fragment thereof, or a nucleotide sequence complementary thereof, and preferably, wherein said isolated and / or exogenous polynucleotide encoding a polypeptide having dioxygenase activity able to cleave a broad substrate spectrum such as lycopene, p-carotene and zeaxanthin, among other, at 7,8;7'8' double bonds to efficiently produce apocarotenoids.

[0018] In a second aspect, the present disclosure provides an isolated polypeptide, hereinafter, the polypeptide of the invention, encoded by the polynucleotide of the present invention.

[0019] In a third aspect, the present disclosure provides a nucleic acid construct or expression vector, hereinafter, the nucleic acid construct of the invention or expression vector of the invention, comprising at least one of the polynucleotide of the present invention or any combinations thereof, wherein preferably the at least one polynucleotide or any combination thereof is operably linked to one or more control sequences that direct the production of at least one polypeptide of the present invention, or any combinations thereof, in a cell, preferably in an expression host cell.

[0020] In a fourth aspect, the present disclosure provides a recombinant expression host cell, hereinafter, the host cell of the invention, comprising at least one of the polynucleotide of the invention or any combinations thereof, encoding the at least one of the polypeptides of the invention or any combinations thereof, wherein the at least one polynucleotide or any combination thereof is operably linked to one or more control sequences that direct the production of the at least one polypeptide or any combinations thereof in a cell, and wherein preferably the cell is a host cell, preferably selected from the list consisting of a bacterial cell, a fungal cell, or a plant cell.

[0021] In a fifth aspect, the present disclosure provides a transgenic non-human organism, hereinafter the transgenic non-human organism of the invention, comprising at least a polynucleotide of the invention or any combinations thereof, at least a polypeptide of the invention or any combinations thereof, a nucleic acid construct or expression vector of the invention, or a recombinant host cell of the invention, wherein the transgenic non-human organism is preferably a bacterium, a fungus, or a plant.

[0022] In a sixth aspect, the present disclosure provides the use of at least one isolated polynucleotide of the invention or any combination thereof, the at least one isolated polypeptide of the invention or any combinations thereof, the nucleic acid construct or expression vector of the invention, the recombinant host cell of the invention, or the transgenic non-human organism of the invention, for the production of apocarotenoids, preferably wherein the apocarotenoids are selected from the list consisting of B-cyclocitral, 6-methyl-5-hepten-2-one, crocetin, crocin, safranal, picrocrocin, geranyl acetone, or any combinations thereof.

[0023] In a seventh aspect, the present disclosure provides a method of producing apocarotenoids, hereinafter the method of producing apocarotenoids of the present invention, wherein the method comprising introducing the at least one polynucleotide of the invention or any combination thereof, the at least one isolated polypeptide of the invention or any combination thereof; or the nucleic acid construct or the expression vector of the invention, into a host cell, or into a transgenic non-human organism, preferably a bacterium, a fungus or a plant, wherein the method optionally further comprises extracting, isolating and purifying at least one apocarotenoid from the culture of the host cell or the tissues of the transgenic non-human organism.

[0024] In an eighth aspect, the present disclosure provides an extract and / or harvestable parts of the transgenic non-human organisms of the invention, hereinafter the extract and / or the harvestable parts of the transgenic non-human organisms of the invention, comprising at least one apocarotenoid compound according to the present disclosure.

[0025] In a ninth aspect, the present disclosure provides a composition comprising the extract and / or the harvestable parts of the transgenic non-human organisms of the invention, comprising the at least one apocarotenoid according to the invention, and optionally, excipients and / or carriers. In a particular embodiment, the composition of the invention is a cosmetic, nutritional, nutraceutical or pharmaceutical composition.

[0026] In a tenth aspect, the present disclosure provides an extract and / or harvestable parts of the transgenic non-human organism of the invention, or the pharmaceutical composition of the invention, for use as medicine.

[0027] In the eleventh aspect, the present disclosure provides an extract and / or harvestable parts of the transgenic non-human organisms according to the present invention, or the pharmaceutical composition of the invention, for use in the prevention and / or treatment of diseases of the central nervous system diseases such as age-related neurodegenerating diseases, preferably Alzheimer's and Parkinson's diseases; epilepsy, convulsion, and insomnia; cardiovascular diseases such as hypertension, hyperlipidemia, and atherosclerosis; cancer; inflammation, hepatitis, cirrhosis, hypercholesterolemia, depression and diabetes.

[0028] In a twelfth aspect, the present disclosure provides a system or kit for producing at least one apocarotenoid compound, hereinafter the system or kit of the invention, comprising at least a polynucleotide of the invention or any combination thereof, at least a polypeptide of the invention or any combination thereof, at least a nucleic acid construct or expression vector of the invention, or at least a recombinant host cell of the invention, or a transgenic non-human organism of the invention.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To complete the description and in order to provide for a better understanding of the invention, a set of drawings is provided. Said drawings form an integral part of the description and illustrate aspects and embodiments disclosed herein, which should not be interpreted as restricting the scope of the invention, but just as an example of how the invention can be carried out. The drawings comprise the following figures:

[0031] Figure 1. In the panel A and D correspond with the two stages of fruits development used for RNAseq transcriptome analyses.

[0032] Figure 2. A) Amino acid sequence alignment of BoCCD4.3 (CCd4.3 protein belonging to Bixa orellana and having the GenBank accession number: QTZ19663), GjCCD4a (CCd4a protein belonging to Gardenia jasminoides and having the GenBank accession number: ARU08109), BdCCD4.1 (CCd4.1 protein belonging to Buddleja davidii and having the GenBank accession number: APU54674), BdCCD4.3 (CCd4.3 protein belonging to Buddleja davidii and having the GenBank accession number: APU54676), VgCCD4.1 (CCd4.1 protein belonging to Verbascum giganteum and having the GenBank accession number: WQF66837) and VsCCD4.1 (CCd4.1 protein belonging to Verbascum sinuatum and having the GenBank accession number: WQF66840), and PpCCD4 (SEQ ID NO: 2). Conserved amino acid residues are depicted with asterisk. B) Percent Identity Matrix created by Clustal2.1.

[0033] Figure 3. A) Comparative analyses among plant CCDs involved in the biosynthesis of crocetin in different plant species. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 2.78440340 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2500 replicates) are shown next to the branches. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. This analysis involved 11 amino acid sequences: BoCCd4.3 (CCd4.3 protein belonging to Bixa orellana and having the GenBank accession number: QTZ19663), GjCCD4a (CCd4a protein belonging to Gardenia jasminoides and having the GenBank accession number: ARU08109), BdCCD4.1 (CCd4.1 protein belonging to Buddleja davidii and having the GenBank accession number: APU54674), BdCCD4.3 (CCd4.3 protein belonging to Buddleja davidii and having the GenBank accession number: APU54676), VgCCD4.1 (CCd4.1 protein belonging to Verbascum giganteum and having the GenBank accession number: WQF66837), VsCCD4.1 (CCd4.1 protein belonging to Verbascum sinuatum and having the GenBank accession number: WQF66840), CsCCD2L (CCD2L protein belonging to Crocus sativus and having the GenBank accession number: ALM23547.1), CancCCD2 (CCD2 protein belonging to Crocus angustifolius and having the GenBank accession number: ALM23546.1), CaCCD2 (CCD2 protein belonging to Crocus ancyrensis and having the GenBank accession number: AKN09908.), CchryCCD2 (CCD2 protein belonging to Crocus chrysanthus and having the GenBank accession number: ALM23545.1), FhCCD2 (CCD2 protein belonging to Freesia hybrida and having the GenBank accession number: BDB95543.1), 26288 (SEQ ID NO: 6), 35707 (SEQ ID NO: 4), and PpCCD4 (SEQ ID NO: 2). All positions containing gaps and missing data were eliminated (complete deletion option). There was a total of 491 positions in the final dataset. Evolutionary analyses were conducted in MEGA X. B) Percent Identity Matrix created by Clustal2.1 Figure 4. Chromatograms obtained from E.coli pThio-26288, pThio-35707, and pThio-PpCCD4 cultures producing p-carotene.

[0034] Figure 5. Inoculation of N. benthamiana plants that stably express Nib with TEV recombinant clones that express the different CCDs. Pictures of representative plants mock-inoculated and agroinoculated with the different CCD4 of the invention: TEV- PpCCD4, TEV-26288, and TEV-35707, as well as with the TEV-CCD2 as positive control, taken at 14 days post infection (dpi).

[0035] Figure 6. Crocins production in infected N. benthamiana plants. A) representative chromatograms at 440 nm of polar extracts from leaves of TEV-PpCCD4, TEV- 26288, TEV-35707, and TEV-control. Inset is shown the absorbance spectra of crocin. B) Quantification of crocins levels in polar extracts of leaves of TEV-PpCCD4, TEV-26288, TEV-35707, and TEV-control, and comparison with levels in TEV- CCD2L, used as positive control for crocins production.

[0036] Figure 7. Analyses of apolar extracts from leaves of infected plants with the TEV constructs. A) Relative levels of carotenoids in TEV-PpCCD4, TEV-26288, TEV- 35707, and TEV-control. B) Relative levels of apolar extracts from infected leaves with the different TEV constructs.

[0037] Figure 8. Crocins production in tomato fruits. Area quantification of crocins levels in polar extracts of tomato Fruits transformed with the different CCDs of the invention, and comparison with levels in wild type (WT) tomato plants and with tomato plants previously transformed with CCD2L, used as positive control for crocins production.

[0038] DETAILED DESCRIPTION OF THE INVENTION.

[0039] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature.

[0040] As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogues of the DNA or RNA generated using nucleotide analogues. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences. In one embodiment, cDNA is preferred. Thus, in all of the aspects described herein that use nucleic acids, cDNA can be used unless otherwise specified. The terms “nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein also include variants, mutants, biologically active fragments, and / or modifications of the polynucleotides described herein.

[0041] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds. The terms "proteins" and "polypeptides" as used herein also include variants, mutants, biologically active fragments, and / or modifications of the polypeptides described herein.

[0042] A nucleic acid or polynucleotide is “isolated” when it is at least partially or completely separated from other components, including but not limited to for example, other proteins, nucleic acids, cells, etc. Similarly, a polypeptide, protein or peptide is “isolated” when it is at least partially or completely separated from other components, including but not limited to for example, other proteins, nucleic acids, cells, etc. Preferably, the isolated polynucleotide or the isolated polypeptide is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, and most preferably at least 100% free from other components with which they are naturally associated.

[0043] The term "exogenous" in the context of a polynucleotide refers to the polynucleotide when present in a cell, or in a cell-free expression system, in an altered amount compared to its native state. In one embodiment, the cell is a cell that does not naturally comprise the polynucleotide. However, the cell may be a cell that comprises a non-endogenous polynucleotide resulting in an altered, preferably increased, amount of production of the encoded polypeptide. An exogenous polynucleotide of the invention includes polynucleotides that have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems in which are subsequently purified away from at least some other components.

[0044] The terms “derived from” and “obtained from” refer to not only a protein or compounds, such as apocarotenoids in the context of the present invention, produced or producible by a recombinant host cell or a transgenic non-human organism in question, but also a protein encoded by a DNA sequence isolated from such host cell or a transgenic non-human organism containing such DNA sequence. Additionally, the term refers to a protein which is encoded by a DNA sequence of synthetic and / or cDNA origin and which has the identifying characteristics of the protein in question.

[0045] The term “identical” in the context of two polynucleotide or polypeptide sequences refers to the nucleic acids or amino acids in the two sequences that are the same when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms described below and known in the art. “% identity” or “percent identity” refers to protein sequence identity. Percent identity may be determined using standard techniques known in the art. The percent amino acid identity shared by sequences of interest can be determined by aligning the sequences to directly compare the sequence information, e.g., by using a program such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described, for example, in Altschul et al., J Mol Biol, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993). A percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “reference” sequence including any gaps created by the program for optimal / maximum alignment. BLAST algorithms refer to the “reference” sequence as the “query” sequence. The CLUSTAL W algorithm is another example of a sequence alignment algorithm (See, Thompson et al., Nucleic Acids Res, 22:4673- 4680, 1994).

[0046] Understanding the homology between molecules can reveal the evolutionary history of the molecules as well as information about their function; if a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the new protein's biochemical function. The most fundamental relationship between two entities is homology; two molecules are said to be homologous if they have been derived from a common ancestor. Homologous molecules, or homologs, can be divided into two classes, paralogs and orthologs. Paralogs are homologs that are present within one species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs that are present within different species and have very similar or identical functions. A protein superfamily is the largest grouping (clade) of proteins for which common ancestry can be inferred. Usually, this common ancestry is based on sequence alignment and mechanistic similarity. Superfamilies typically contain several protein families which show sequence similarity within the family.

[0047] The inventors have surprisingly identified three genes encoding for three enzymes named PpCCD4 (SEQ ID NO: 2), 35707 (SEQ ID NO: 4) and 26288 (SEQ ID NO: 6), respectively that are involved in the cleavage of lycopene, p-carotene and / or zeaxanthin at 7,8,7'8'double bonds to efficiently produce apocarotenoids, wherein such enzymes belong to three different plants species, P. peruviana, P. tomentosa and H. impetiginosus, respectively, although neither crocetin nor crocin has been identified in these three plant species.

[0048] Experimental data show that higher levels of apocarotenoids using the CCD4 enzymes of the present invention, independently, or in any combination thereof, are obtained compared to those levels previously reported for CsCCD2L (Figures 6 to 8) and BdCCd4.1, demonstrating how these CCD4 enzymes of the present invention, PpCCD4, 35707 and 26288, independently or in any combination thereof, display particularly a broad substrate spectrum (Table 1), and open new avenues for apocarotenoid biotechnological production in different heterologous systems, from microorganisms to plants. Thus, in a first aspect, the present disclosure provides an isolated and / or exogenous polynucleotide, hereinafter the polynucleotide of the invention, comprising: i) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%; 99% or 100% identity to any one of the nucleotide sequences selected from the group consisting of SEQ ID NO:1 (PpCCD4), SEQ ID NO:5 (26288), SEQ ID NO:3 (35707), or any combinations thereof; ii) a biologically active fragment thereof; or iii) a nucleotide sequence complementary thereof; wherein the nucleotide sequence of SEQ ID NO: 1, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Physalis, preferably from P. peruviana, SEQ ID NO: 5, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Handroanthus, preferably from H. impetiginosus; and SEQ ID NO: 3, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Paulownia, preferably from P. tomentosa; and the nucleotide sequence of i), ii), iii) or iv) being SEQ ID NO: 1.

[0049] In a preferred embodiment of the isolated and / or exogenous polynucleotide of the invention, they are further characterized by encoding a polypeptide having dioxygenase activity able to cleave lycopene, p-carotene and zeaxanthin at 7,8;7'8' double bonds to efficiently produce at least one apocarotenoid, according to the present invention.

[0050] In a preferred embodiment of the isolated and / or exogenous polynucleotide of the invention, said nucleotide sequences of the invention encoding for an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%; 99% or 100% identity to any one of the sequences selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 4, SEQ ID NO: 2, belong to H. impetiginosus, P. tomentosa, and P. peruviana, respectively; as well as encoding for an biologically active fragment thereof, or encoding for an amino acid sequence complementary thereof.

[0051] In a more preferred embodiment, the nucleotide sequences of the invention, can be used independently but also in any combination thereof to increase the synthesis of apocarotenoids in the host cell or organism according to the present invention. Thus, any combination of nucleotides sequences disclosed in the first aspect of the present invention is useful for the purposes of the present invention. Therefore, as an example, useful combinations to increase the apocarotenoid synthesis could be selected from SEQ ID NO:5 + SEQ ID NO: 3; SEQ ID NO: 5 + SEQ ID NO: 1 ; SEQ ID NO: 3 + SEQ ID NO: 1 and SEQ ID NO: 5+SEQ ID NO: 3 + SEQ ID NO: 1, in any order thereof.

[0052] In another aspect, the present disclosure provides an isolated polypeptide, hereinafter the polypeptide of the invention, encoded by the polynucleotide of the present invention.

[0053] In a more preferred embodiment, the polypeptides sequences of the invention as disclosed above, can be used independently but also, in any combination thereof to increase the synthesis of apocarotenoids in the host cell or host or transgenic organism according to the present invention. Thus, any combination of polypeptides of the present invention is useful for the purposes of the present invention. Therefore, as an example, useful combinations to increase the apocarotenoid synthesis is selected from SEQ ID NO:6 + SEQ ID NO: 4; SEQ ID NO: 6 + SEQ ID NO: 2; SEQ ID NO: 4 + SEQ ID NO: 2 and SEQ ID NO: 6 + SEQ ID NO: 4 + SEQ ID NO: 2, in any order thereof.

[0054] A “PpCCD4 polypeptide” or “PpCCD4 protein” has the amino acid sequence of SEQ ID NO: 2, encoded by the nucleotide sequence of SEQ ID NO: 1 and belongs to P. peruviana. In a preferred embodiment the PpCCD4 polypeptide has an amino acid sequence with an identity of at least 50%, more preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even more preferably 99% with the sequence SEQ ID NO: 2. In the same sense, the PpCCD4 nucleotide sequence has a nucleotide sequence with an of at least 50%, more preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even more preferably 99% with the sequence SEQ ID NO: 1.

[0055] A “35707 polypeptide” or “35707 protein” has the amino acid sequence of SEQ ID NO: 4, encoded by the nucleotide sequence of SEQ ID NO: 3 and belongs to P. tomentosa. In a preferred embodiment the 35707 polypeptide has an amino acid sequence with an identity of at least 50%, more preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even more preferably 99% with the sequence SEQ ID NO: 4. In the same sense, the 35707 nucleotide sequence has a nucleotide sequence with an of at least 50%, more preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even more preferably 99% with the sequence SEQ ID NO: 3.

[0056] A “26288 polypeptide” or “26288 protein” has the amino acid sequence of SEQ ID NO: 6, encoded by the nucleotide sequence of SEQ ID NO: 5 and belongs to H. impetiginosus. In a preferred embodiment the 26288 polypeptide has an amino acid sequence with an identity of at least 50%, more preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even more preferably 99% with the sequence SEQ ID NO: 6. In the same sense, the 26288 nucleotide sequence has a nucleotide sequence with an of at least 50%, more preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even more preferably 99% with the sequence SEQ ID NO: 5.

[0057] The term "biologically active fragment" as used herein, for example with reference to SEQ ID NO: 1 to 6, or any sequence mentioned herein, refers to a variant gene or peptide / protein sequence or part of the gene or peptide / protein sequence which retains the biological function of the full non-variant sequence. Biologically active fragments can be any size as long as they maintain the defined activity. A functional equivalent sequence also comprises a variant of the gene of interest encoding a peptide which has sequence alterations that do not affect the function of the resulting protein, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in nonconserved residues, to the wild type sequences as shown herein and is biologically active.

[0058] Thus, it is understood, as those skilled in the art will appreciate, that the aspects of the invention, including the methods and uses, encompass not only a nucleic acid or peptide as described herein but also functional variants or homologs thereof that do not affect the biological activity and function of the resulting protein. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do however not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also produce a functionally equivalent product. Each of the proposed modifications is well within the routine skill in the art, as it determines of retention of biological activity of the encoded products.

[0059] In a preferred embodiment of the isolated and / or exogenous polynucleotide of the invention, said polynucleotide(s) is(are) operably linked to one or more control sequences that direct the production of the polypeptide(s) of the invention in a cell, preferably wherein the cell is an expression host cell.

[0060] In another aspect, the present disclosure provides a nucleic acid construct or expression vector, hereinafter the nucleic acid construct of the invention or the expression vector of the invention, comprising the at least one of the polynucleotide of the present invention as previously disclosed, or any combination thereof, in any order, such as a combination of SEQ ID NO: 5 + SEQ ID NO: 3; SEQ ID NO: 5 + SEQ ID NO: 1; SEQ ID NO: 3 + SEQ ID NO: 1, or SEQ ID NO: 5 + SEQ ID NO: 3 + SEQ ID NO: 1, wherein preferably the at least one polynucleotide or any combination thereof as previously mentioned, is operably linked to one or more control sequences that direct the production of a polypeptide of the present invention, as previously disclosed, in a cell, preferably in an expression host cell.

[0061] As used in the present disclosure, the term “nucleic acid construct” refers to a DNA molecule / s not normally associated in nature, capable of genomic integration in a host, comprising one or more transgene DNA sequences that have been linked in a functionally operative manner using well-known recombinant DNA techniques. A “plurality” of nucleic acid constructs refers to two or more.

[0062] As used herein, the term “vector” refers to a nucleic acid construct used to introduce or transfer nucleic acid(s) into a target cell or tissue. A vector is typically used to introduce foreign DNA into a cell, tissue or organism. Vectors include plasmids, cloning vectors, bacteriophages, viruses (e.g., viral vector), cosmids, expression vectors, shuttle vectors, and the like. A vector typically includes an origin of replication, a multicloning site, and a selectable marker. The process of inserting a vector into a target cell is typically referred to as transformation. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11 , Charon4A, Charon21A, etc. may be used as a phage vector or cosmid vector, and pBR-based, pUC-based. pBluescriptll-based, pGEM-based, pTZ-based, pCL-based, pET-based, etc. may be used as a plasmid vector. The vectors usable in the present disclosure is not particularly limited and known expression vectors may be used.

[0063] As used herein in the context of introducing a nucleic acid sequence into a cell, tissue, or organism, the term “introduced” refers to any method suitable for transferring the nucleic acid sequence into the cell, tissue, or organism. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic alteration of a cell which results from the uptake, optional genomic incorporation, and expression of genetic material (e.g., DNA).

[0064] “Expression cassette” or “expression vector” refers to a nucleic acid construct or vector generated recombinantly or synthetically for the expression of at least a nucleic acid, or various nucleic acids of interest (e.g., a foreign nucleic acid or transgene) in a target cell. The nucleic acid / s of interest typically expresses protein(s) of interest. An expression vector or expression cassette typically comprises a promoter nucleotide sequence that drives or promotes the expression of the foreign nucleic acid(s). The expression vector or cassette also typically includes other specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Some expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell or genome of the host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors for expression of a protein from a nucleic acid sequence incorporated into the expression vector is within the knowledge of those of skill in the art.

[0065] “Promoter” or “control sequences” refers to a nucleic acid sequence located upstream or 5' to a translational start codon of an open reading frame (or protein-coding region) of a gene(s) and that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. “Constitutive promoters” are functional in most or all tissues of a plant throughout plant development. Tissue-, organ- or cell-specific promoters are expressed only or predominantly in a particular tissue, organ, or cell type, respectively. Rather than being expressed “specifically” in a given tissue, organ, or cell type, a promoter may display “enhanced” expression, i.e. , a higher level of expression, in one part (e.g., cell type, tissue, or organ) of the host compared to other parts of the host.

[0066] As used herein, the term "operably linked to one or more control sequences" or "operably linked to one or more promoters" means the gene, or DNA sequence, is positioned or connected to the promoter in such a way as to ensure its functioning. The promoter is any sequence sufficient to allow the DNA to be transcribed. After the gene and promoter sequences are joined, upon activation of the promoter, the gene will be expressed.

[0067] As used herein, the term “heterologous” sequence refers to a sequence that originates from a foreign source or species or, if from the same source, is modified from its original form.

[0068] “Recombinant” nucleic acid is made by an artificial combination of two or more otherwise separated segments of sequences, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. Techniques for nucleic-acid manipulation are well-known in the art. This term also applies to a host cell, which comprises a foreign nucleic acid.

[0069] The terms “transformed,” “recombinant”, “transfected,” or “transgenic” refer to a cell, tissue, organ, or organism into which has been introduced a foreign nucleic acid(s), such as a recombinant construct. Preferably, the introduced nucleic acid is integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced nucleic acid is inherited by subsequent progeny. A “transgenic” “recombinant”, “transfected,” or “transgenic” cell or organism also includes progeny of the cell or organism and progeny produced.

[0070] In another aspect, the present disclosure provides a recombinant expression host cell, hereinafter the host cell of the invention, comprising the at least one polynucleotide of the invention or any combination thereof as previously mentioned, encoding the at least one polypeptide of the invention or any combination thereof as previously mentioned, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the at least one polypeptide or any combinations of polypeptides as previously mentioned, in a cell. In a preferred embodiment, the recombinant expression host cell is a host cell selected from the list consisting of a bacterial cell, a fungal cell, or a plant cell.

[0071] In a preferred embodiment, the recombinant expression host cell of the invention is a bacterial cell selected from the list consisting of: E. coli.

[0072] In another preferred embodiment, the recombinant expression host cell of the invention is a fungal cell selected from the list consisting of: Fusarium spp and Aspergillus spp.

[0073] In another preferred embodiment, the recombinant expression host cell of the invention is a plant cell selected from the Solanaceae family, such as tobacco, tomato, potato; more preferably tomato; or belonging to the Apiaceae family, more preferably wild carrot, carrot; or belonging to the Amaranthaceous family, more preferably selected from the group consisting of: spinach; or belonging to the Asteraceae family, more preferably selected from the group consisting of: lettuce; or belonging to the Cucurbitaceae family, more preferably selected from the group consisting of: zucchini, cushaw squash, squash, pumpkin; or belonging to the Poaceae family, more preferably selected from the group consisting of: rice, corn.

[0074] In another aspect, the present disclosure provides a transgenic non-human organism, hereinafter, the transgenic non-human organism of the invention, comprising at least a polynucleotide of the invention or any combination thereof as previously mentioned, at least a polypeptide of the invention or any combination thereof as previously mentioned, a nucleic acid construct or expression vector of the invention, or a recombinant host cell of the invention.

[0075] In a preferred embodiment, the transgenic non-human organism is preferably a bacterium, a fungus, or a plant, as disclosed previously. In a more preferred embodiment, the transgenic non-human organism is a plant and, in a more preferred embodiment, the transgenic plant is selected from the Solanaceae family, such as tobacco, tomato, potato; more preferably tomato; or belonging to the Apiaceae family, more preferably wild carrot, carrot; or belonging to the Amaranthaceous family, more preferably selected from the group consisting of: spinach; or belonging to the Asteraceae family, more preferably selected from the group consisting of: lettuce; or belonging to the Cucurbitaceae family, more preferably selected from the group consisting of: zucchini, cushaw squash, squash, pumpkin; or belonging to the Poaceae family, more preferably selected from the group consisting of: rice, corn.

[0076] The term "plant" as used herein as a noun refers to whole plants such as, for example, a plant growing in a field for commercial plant or grain production. A "plant part" refers to vegetative structures (for example, leaves, stems), roots, floral organs / structures, seed (including embryo, endosperm, and seed coat), plant tissue (for example, vascular tissue, ground tissue, and the like), fruits, cells and progeny of the same.

[0077] A "transgenic plant" refers to a plant that contains a gene construct ("transgene") not found in a wild-type plant of the same species, variety or cultivar. A "transgene" as referred to herein has the normal meaning in the art of biotechnology and includes a genetic sequence which has been produced or altered by recombinant DNA or RNA technology and which has been introduced into the plant cell. The transgene may include genetic sequences derived from a plant cell. Typically, the transgene has been introduced into the plant by human manipulation such as, for example, by transformation but any method can be used as one of the skilled in the art recognizes.

[0078] The at least one polynucleotide or any combination of the polynucleotides of the present invention may be expressed constitutively in the transgenic plants during all stages of development. Depending on the use of the plant or plant organs, the polypeptide(s) may be expressed in a stage-specific manner. Furthermore, the polynucleotides may be expressed tissue-specifically, preferably, in fruits.

[0079] The various aspects of the invention described herein clearly extend to any plant / bacterial / fungal cell or any plant / bacteria / fungi produced, obtained or obtainable by any of the methods known by the skilled person to obtain transformed / transfected non-human organisms, and to all plant / bacteria / fungi parts and propagules thereof unless otherwise specified. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the methods known by the skilled person, the only requirement being that progeny exhibit the same genotypic and / or phenotypic characteristic(s) as those produced by the transgenic parent according to the invention. In another aspect, the present disclosure provides the use of at least one isolated polynucleotide of the invention or any combination thereof as previously mentioned herein, the at least one isolated polypeptide of the invention or any combination thereof as previously mentioned herein, the nucleic acid construct or expression vector of the invention, the recombinant host cell of the invention, or the transgenic non-human organism of the invention, for the production of at least one apocarotenoid compound.

[0080] In a preferred embodiment, the apocarotenoid compounds are selected from the list consisting of B-cyclocitral, 6-methyl-5-hepten-2-one, crocetin, crocin, safranal, picrocrocin, geranyl acetone, or any combinations thereof.

[0081] In a more preferred embodiment, the apocarotenoids generated by the CCD activities of the proteins of the present invention are shown in Table 2.

[0082] Table 2. Apocarotenoids generated by the CCD activities of the PpCCD, 35707 and 26288 of the present invention.

[0083] In another aspect, the present disclosure provides a method of producing at least one apocarotenoid compound, hereinafter the method of producing apocarotenoids of the present invention, wherein the method comprising introducing at least one polynucleotide or any combination of polynucleotides of the invention, the at least one isolated polypeptide or any combination of polypeptides of the invention; or the nucleic acid construct or the expression vector of the invention, into a host cell, or into a transgenic non-human organism as previously disclosed, wherein the method optionally further comprises extracting, isolating and purifying apocarotenoids from the culture of the host cell or from the tissues of the transgenic non-human organism.

[0084] The apocarotenoid compounds recovery step may be performed by a suitable method known in the pertinent field. Specifically, known methods of recovering apocarotenoid compounds are not particularly limited thereto, but methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., HPLC, ion exchange, affinity, hydrophobicity and size exclusion) may be used, but are not limited thereto.

[0085] In a preferred embodiment of the method of the present invention for the production of apocarotenoids, the at least one apocarotenoid compounds are selected from the list consisting of B-cyclocitral, 6-methyl-5-hepten-2-one, crocetin, crocin, safranal, picrocrocin, geranyl acetone, or any combinations thereof.

[0086] In a preferred embodiment of the method of producing at least one apocarotenoid of the present invention, the host cell is preferably a bacterium cell, a fungal cell, or a plant host cell, more preferably a plant host cell, according to those disclosed in the present invention.

[0087] In another aspect, the present disclosure provides an extract and / or harvestable parts and / or recombinant biomass of the transgenic non-human organisms of the invention, hereinafter the extract and / or the harvestable parts and / or recombinant microbial biomass of the transgenic non-human organisms of the invention, comprising at least one apocarotenoid compound or any combination thereof according to the present invention.

[0088] As used herein, the term "extract" refers to any portion of a host cell or non-human transgenic organism of the invention comprising at least one polypeptide or any combination of polypeptides of the invention, preferably also comprising at least one polynucleotide or any combination of polynucleotides of the invention, or at least one vector of the invention or having increased levels of at least one apocarotenoid compound or any combination thereof as disclosed in the present invention. This term includes apocarotenoids secreted from the host cell, and hence encompasses culture supernatants, or included in the harvestable parts of the transgenic non-human organisms, preferably transgenic plants.

[0089] In a preferred embodiment, the harvestable parts of the transgenic non-human organisms, preferably transgenic plants are selected from seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. More preferably, the harvestable parts are selected from seeds, tubers and fruits, and they are characterized by having increased levels of apocarotenoids as disclosed herein as compared to the harvestable parts of wild-type plants.

[0090] The terms “increase”, “improve” or “enhance” are interchangeably used herein and they refer to an increase, for example, by at least 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, for example at least 15% or 20%, or 25%, 30%, 35%, 40% or 50% in comparison to a control or wild-type plant.

[0091] The present disclosure also refers to products derived from harvestable parts of the transgenic plants of the invention, preferably directly derived, from a harvestable part of such plant, such as dry pellets or powders, oil, fat and fatty acids, fiber or proteins. In another aspect, the present disclosure provides a composition comprising the extract and / or the harvestable parts of the transgenic non-human organisms of the present invention, as previously disclosed, and optionally at least one excipient and / or carrier.

[0092] The term "excipient" refers to a substance that helps the absorption of any of the components of the product of the invention, stabilizes these components or helps the preparation of composition in the sense of giving it consistency or providing flavors that make it more pleasant. Thus, the excipients could have the function of keeping the components together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

[0093] The term “vehicle” or “carrier” is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the composition. Therefore, the carrier is a substance that is used to dilute any of the components of the composition of the present invention to a given volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the composition.

[0094] In a preferred embodiment, the composition of the invention is selected from a cosmetic, nutritional, nutraceutical or pharmaceutical composition.

[0095] In a particular embodiment, the composition of the invention comprises at least one apocarotenoid according to the present invention together with a vehicle suitable for food, being a nutritional composition; or with a vehicle suitable for medicine, being a pharmaceutical composition; or with a vehicle suitable for cosmetic use, being a cosmetic composition.

[0096] Therefore, according to this particular embodiment the composition of the invention is a food product. In the sense used in this description, the term "food product" includes any substance or product of any type that is solid or liquid, natural or transformed, which due to its features, uses, components, preparation and state of preservation, is susceptible to being commonly or suitably used for any of the following purposes: a) for normal human or animal nutrition or as components having no nutritional value; or b) as dietary products or additives, in special cases of human or animal food. Likewise, said term also includes natural materials or feeds and prepared products of any origin which, separately or duly mixed with one another, are suitable for animal food. As it is used herein, the term "food product" also includes nutraceutical compositions, i.e., compositions suitable for use in humans or animals comprising one or more natural products with therapeutic action or providing a benefit for health or which have been associated with the prevention or reduction of diseases, and includes dietary supplements presented in a non-food matrix (e.g., capsules, powder, etc.) of a concentrated bioactive natural product normally present (or lacking) in foods and which, when taken at a dose exceeding that existing in those foods has a favorable effect on health that is greater than normal food could have. A food ready for consumption is a food that does not need to be diluted by means of, for example, an aqueous solution suitable for consumption. In theory, the ingredients present in a food ready for consumption are balanced and additional ingredients do not need to be added to the food to make them ready for consumption, as is considered by one skilled in the art. A concentrated food is a food in which one or more ingredients are present at a higher concentration than it is in a food ready for consumption, so for use thereof it must be diluted by means of, for example, an aqueous solution suitable for consumption. Illustrative, non-limiting examples, of foods provided by this invention include both food products intended for human consumption, and feeds and concentrated products for animal food, such as feeds and food concentrates intended for use in aquaculture or in any animal, whether said animal is a domestic or wild production animal, such as dogs, cats, bovines, ovines, suidae, equines, etc.

[0097] The use of the at least one isolated polynucleotide or any combination of the polynucleotides of the invention, the vector of the invention, the host cell of the invention, the transgenic non-human organism of the invention wherein said transgenic non-human organism is a plant, or the at least one isolated peptide or any combination of peptides of the invention, in the preparation of a food product, constitutes an additional aspect of this invention. In a particular embodiment, said food product is a substance or product susceptible to being used in foods, feeds, food additives, dietary supplements, nutraceutical compositions, etc., for both human and animal food. Thus, the present disclosure also relates to food products and food supplements comprising preferably the harvestable parts of the transgenic plant of the invention, more preferably, tubers, fruits and / or seeds, or the apocarotenoid obtained by the culture of any of the transgenic non-human organism according to the present invention. The food products and food supplements have increased apocarotenoid compounds as disclosed herein, compared to food products or food supplements comprising harvestable parts of control or wild-type plants, which do not comprise the polynucleotides / polypeptides of the invention.

[0098] In a preferred embodiment the food product or food supplement is preferably a tomato, a potato, a tomato-based product and a potato-based product. In a more preferred embodiment, the tomato-based product is selected from the list consisting of ketchup, a tomato-based sauce, a pizza sauce, tomato soup or tomato juice, among others, and the potato-based product is selected from the list consisting of potato chips, potato flakes, or potato flour, among others.

[0099] In another particular embodiment, the composition of the invention is a pharmaceutical composition comprising, in addition to at least one apocarotenoid compound or any combination thereof according to the present invention and optionally, a pharmaceutically acceptable vehicle. Compositions of this type are suitable for administration of at least one apocarotenoid compound or any combination thereof present in the composition of the invention to a subject. Generally, said pharmaceutically acceptable vehicle will be chosen depending on the nature of the active component (e.g., at least one apocarotenoid compound of the invention), of the chosen presentation form, for example, solid (e.g., tablets, capsules, coated tablets, granules, suppositories, etc.) or liquid (e.g., solutions, suspensions, emulsions, etc.) and on the chosen administration route, for example, oral, parenteral (e.g., intramuscular, subcutaneous, intravenous, etc.), rectal, etc. In each case, the pharmaceutically acceptable excipients suitable for the chosen pharmaceutical dosage form and administration route will be chosen. Information about excipients suitable for the formulation of pharmaceutical compositions intended for administration by oral, parenteral or rectal route, and about the production of said pharmaceutical compositions are known for those skilled in the art.

[0100] The pharmaceutical composition provided by this invention can be applied or administered to a subject in need of prevention and / or treatment. As it is used herein, the term "subject" refers to a member of an animal species, preferably a mammal, and includes, but is not limited to domestic animals, primates and humans; preferably, the subject is a male or female human being, of any age or race.

[0101] The pharmaceutical composition of the invention may comprise another active substance. In addition to the requirement of therapeutic efficacy, where said pharmaceutical composition may require the use of other therapeutic agents, there may be additional fundamental reasons that compel or strongly recommend the use of a combination of at least one apocarotenoid compound or any combination thereof, of the invention and another therapeutic agent. The term "active principle" is any matter, whatever its origin, human, animal, plant, chemical or, other, to which an appropriate activity is attributed to constitute a medicine.

[0102] In each case the form of presentation of the medicament or the pharmaceutical composition will be adapted to the type of administration used, therefore, the composition of the present invention can be presented in the form of solutions or any other form of clinically permitted administration and in a therapeutically effective amount. The pharmaceutical composition of the invention can be formulated in solid, semi-solid, liquid or, gaseous forms, such as tablet, capsule, powder, granule, ointment, solution, suppository, injectable, inhalant, gel, syrup, nebulizer, microsphere or aerosol, preferably in the form of a tablet, capsule, powder, granule, solution, suppository or syrup.

[0103] The above-mentioned compositions may be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.

[0104] The apocarotenoids and compositions of the present invention can be used together with other medicaments in combination therapies. The other drugs may be part of the same composition or of a different composition, for administration at the same time or at different.

[0105] In another particular embodiment, the composition of the invention is a cosmetic composition comprising, in addition to at least one apocarotenoid compound according to the present invention optionally, a cosmetically acceptable vehicle. In the present invention, the cosmetic composition may be formulated in any form. For example, cosmetics prepared using the cosmetic composition are creams, packs, lotions, essences, face lotions, foundations, makeup bases, and the like, and in order to achieve the object of the present invention, it may be manufactured and commercialized in any form of these formulations but is not limited to the above examples.

[0106] In the present invention, the cosmetic composition may further include one or more vehicles suitable for cosmetic use, which can be selected from the group consisting of purified water, polyhydric alcohol, surfactant, viscosity modifier, chelating agent, emulsifier, pH adjuster, acid-alkali agent, antioxidant, humectant, brightener, preservative, flavoring agent, fragrance, gelling agent, stabilizer, colorant, and pigment.

[0107] In addition, the present invention provides a cosmetic composition for preventing skin aging.

[0108] In another aspect, the present disclosure also relates to the extract, harvestable parts of the transgenic non-human organisms, or the pharmaceutical composition of the invention for use as medicine or medicament.

[0109] In another aspect, the present disclosure also relates to the extract, harvestable parts of the transgenic non-human organisms or the pharmaceutical composition of the invention for use in the prevention and / or treatment of diseases of neurological diseases, cardiovascular diseases, sleep disorders, metabolic diseases, cancer, mental health diseases, inflammatory condition, or liver disease, preferably epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, dementia, Bell's palsy, Huntington's disease, cerebral palsy, meningitis, heart failure, arrhythmia, stroke, atherosclerosis, cardiomyopathy, peripheral artery disease, coronary artery disease, angina, aortic disease, aneurysm, high blood pressure, Insomnia, Circadian rhythm sleep disorder, narcolepsy, idiopathic hypersomnia, parasomnia, REM sleep behavior disorder, Delayed sleep phase disorder, Excessive daytime sleepiness, Type 1 diabetes, type 2 diabetes, hemochromatosis, metabolic syndrome, Phenylketonuria, Gaucher disease, Wilson's disease, breast cancer, lung cancer, prostate cancer, colorectal cancer, melanoma, bladder cancer, kidney cancer, lymphoma, leukemia, myeloma, sarcoma, lymphoma, endometrial cancer, brain tumor, Head and neck cancer, Pancreatic cancer, depression, bipolar disorder, schizophrenia, anxiety disorder, Post-Traumatic Stress Disorder, Neurodevelopmental disorder, Eating Disorder, Rheumatoid arthritis, Psoriatic arthritis, Hepatitis, Acute inflammation, Inflammatory bowel disease, Asthma, Chronic Obstructive Pulmonary Disease, hepatitis A, hepatitis B, hepatitis C, fibrosis, cirrhosis, fatty liver disease, Alpha- 1 antitrypsin deficiency, Hemochromatosis, or Primary sclerosing cholangitis.

[0110] In another aspect, the present disclosure provides a system or kit for producing apocarotenoid compounds, hereinafter, the system or kit of the invention, comprising at least a polynucleotide of the invention or any combination thereof, at least a polypeptide of the invention or any combination thereof, at least a nucleic acid construct or expression vector of the invention, or at least a recombinant host cell of the invention, or a transgenic non-human organism of the invention.

[0111] EXAMPLES

[0112] Following are examples of the invention by means of assays carried out by the inventors, which evidence the effectiveness of the product of the invention. The following examples serve to illustrate the invention and must not be considered to limit the scope thereof.

[0113] MATERIALS AND METHODS

[0114] Extraction and analysis of carotenoids and apocarotenoids by high-performance liquid chromatography with diode array detector (HPLC-DAD)

[0115] Leaf tissues were ground in liquid nitrogen with a mixer mill MM400 (Retsch GmbH, Haan, Germany) in a 2 mL Eppendorf tube. Extraction was performed as previously described (Marti et al., 2020). In brief, 50% methanol was added to the homogenate, and the mix vortexed, sonicated and centrifuged to recover polar metabolites. The pellet was extracted with 2:1 methanokchloroform, mixed for an additional 10 min followed by centrifugation. The apolar phase was evaporated under N2 gas and the dried residues were stored with the polar extracts at -80 °C until analysis by HPLC. All assays were performed in triplicate.

[0116] The HPLC-DAD and the high-performance liquid chromatography with diode array detector and High-Resolution-Mass-Spectrometry (HPLC-DAD-HRMS) methods used for the analysis and detection of crocins, and carotenoids have been previously described (Diretto et al., 2019, Marti et al., 2020). In brief, chromatographic separation was carried out on a C30 reverse-phase column (100 x 3.0 mm; YMC Europe) with a mobile phase composed of methanol (A), water / methanol 20 / 80 (v / v) containing 0.2% ammonium acetate (B), and tert-methyl butyl ether (C) at a total flow rate of 800 l min-1. The separation program used 95% A / 5% B for 1.3 min, followed by 80% A / 5% B / 15% C for 2.0 min and a subsequent 9.2 min linear gradient to 30% A / 5% B / 65% C. Mass ionization was performed with an APCI probe, operating in both + and - voltage conditions. Nitrogen was used at 20 and 10 units as a sheath and auxiliary gas, respectively. The vaporizer and capillary temperature were set at 300 °C and 250 °C, respectively. The discharge current was 5.5 pA, while the S-lens RF level was set at 50. A mass range of 110 / 1600 m / z was used both, in positive and negative voltage with the following parameters: resolution set at 70000, microscan 1, AGC target 1e6, and maximum injection time equal to 50. Metabolite identification was done by comparison of retention times, UV-visible spectra, and MS data of authentic standards.

[0117] Phylogenetic analysis

[0118] The amino acid sequences of BoCCd4.3 (CCd4.3 protein belonging to Bixa orellana and having the GenBank accession number: QTZ19663), GjCCD4a (CCd4a protein belonging to Gardenia jasminoides and having the GenBank accession number: ARU08109), BdCCD4.1 (CCd4.1 protein belonging to Buddleja davidii and having the GenBank accession number: APU54674), BdCCD4.3 (CCd4.3 protein belonging to Buddleja davidii and having the GenBank accession number: APU54676), VgCCD4.1 (CCd4.1 protein belonging to Verbascum giganteum and having the GenBank accession number: WQF66837), VsCCD4.1 (CCd4.1 protein belonging to Verbascum sinuatum and having the GenBank accession number: WQF66840), CsCCD2L (CCD2L protein belonging to Crocus sativus and having the GenBank accession number: ALM23547.1), CancCCD2 (CCD2 protein belonging to Crocus angustifolius and having the GenBank accession number: ALM23546.1), CaCCD2 (CCD2 protein belonging to Crocus ancyrensis and having the GenBank accession number: AKN09908.), CchryCCD2 (CCD2 protein belonging to Crocus chrysanthus and having the GenBank accession number: ALM23545.1), FhCCD2 (CCD2 protein belonging to Freesia hybrida and having the GenBank accession number: BDB95543.1), 26288 (SEQ ID NO: 6), 35707 (SEQ ID NO: 4), and PpCCD4 (SEQ ID NO: 2), were aligned using the BLOSUM62 matrix with the ClustalW (http: / / www.clustal.org) algorithm-based AlignX module from MEGA Version 11.0 (http: / / www.megasoftware.net / mega.htmL). The alignments were saved and executed by MEGA Version 11.0 to generate a Neighbour Joining Tree with bootstrapping (2,500 replicates) analysis and handling gaps with pairwise deletion.

[0119] Activity assays in E. coli cells

[0120] The genes corresponding to PpCCD4 (SEQ ID NO: 1), 35707 (SEQ ID NO: 3) and 26288 (SEQ ID NO 5) were cloned into the EcoRI site of the pThio-Dan1 (Invitrogen) vector by recombination using the In-Fusion® HD Cloning Plus CE kit (Clontech) and specific primers (Table 3).

[0121] Table 3. oligonucleotide sequences used for the activity assays.

[0122] The resulting expression plasmids, named pThio-PpCCD4 (SEQ ID NO: 19), pThio- 26288 (SEQ ID NO: 20), and pThio-35707 (SEQ ID NO: 21) were sequenced to confirm the correct assembly and sequence. Each construct was used to transform E. coli BL21 containing the plasmids and PAC-BETA (https: / / www.addgene.org) to produce p-carotene. The transformants were cultured overnight at 30 °C in 4 mL 2YT medium supplemented with ampicillin (100 pg mL-1) and chloramphenicol (60 pg mL'1). The cultured cells were transferred to 50 mL 2xYT medium supplemented with ampicillin (50 pg mL'1) and chloramphenicol (30 pg mL'1) and further cultured at 30 °C until an QD600 of 0.8 was reached. Cells were then induced with 2% arabinose and grew overnight at 20 °C.

[0123] For HPLC analysis of the obtained products, the cells were harvested by centrifugation (8,000 rpm for 10 min) and pigments consisting of carotenoids such as lycopene, p-carotene, and zeaxanthin; and apocarotenoids such as crocetin, were extracted repeatedly in a total volume of 10 mL of acetone until all pigments were visibly removed. The solvent was evaporated under N2 and the pigments resuspended with 0.5 mL of tert-methyl-butyl-ether. The extracts were analyzed as previously described (Ahrazem et al., 2022). All solvents used were HPLC-MS grade (Merck Millipore). For GC-MS analyses, once the cells were induced, a total of 20 mL were transferred to special vials for volatile collection as previously described (Morote et al., 2023). In brief, the cells were induced with arabinose, and 20 mL of the culture were transferred to special vials for volatile collection by using the magnetic stir bar extraction method applied to headspace (HS-SBSE). In HS-SBSE, the magnetic stir bar is placed in an open glass adapter inside a closed headspace vial, so the analytes are extracted from the vapor phase above the cell culture. The culture was grown during 24 h at 30 °C with shaking, after this time the magnetic stir bars were collected and analyzed by GC-MS. Compounds were identified by comparing the Rl and mass spectra with those of synthetic reference compounds (p-ionone, geranylacetone, and p-cyclocitral were purchased from Sigma) and with library entries of the National Institute of Standards and Technology NIST 2014. Activity assays in N. benthamiana

[0124] Plasmids were constructed by PCR amplification of cDNA of PpCCD4 (SEQ ID NO: 1), 26288 (SEQ ID NO: 5), 35707 (SEQ ID NO: 3), and CsCCD2 (GenBank accession number KP887110.1 - SEQ ID NO: 22) with the oligonucleotides disclosed in Table 3, and Gibson DNA assembly. The resulting plasmids contain a wild-type Tobacco etch virus (TEV) cDNA (GenBank DQ986288, with two silent and neutral mutations G273A and A1119G) flanked by the CaMV 35S promoter and terminator in a binary vector derived from pCLEAN-G181 (Marti et al., 2020). The plasmid to express the recombinant TEVANIb-aGFP clone was previously described (Marti et al., 2020). The plasmids were named as: TEV-PpCCD4.1, TEV- 26288, TEV-35707 and pGTEV-CCD2L.

[0125] A. tumefaciens C58C1 competent cells, previously transformed with the helper plasmid pCLEAN-S48 (Thole et al., 2007), were electroporated separately with the plasmids TEV-PpCCD4.1, TEV- 26288, TEV-35707, and pGTEV-CCD2L, and selected in plates with 50 pg / ml kanamycin, 50 pg / ml rifampicin, and 7.5 pg / ml tetracycline. Liquid cultures of selected colonies were prepared to infiltrate one leaf of one-month-old transgenic N. benthamiana that stably expresses the RNA-dependent RNA polymerase (Nib) of Tobacco etch virus (TEV Nib). After infiltration, plants were kept under controlled conditions in a growth chamber at 25°C under a 12 h day-night photoperiod. Leaf tissue was collected after 14 days after infection (dpi) and analyzed for apocarotenoids and carotenoids content.

[0126] Plant material and growth conditions

[0127] Tomato (S. lycopersicum cv. Moneymaker (MM)) was used as the wild-type (WT) and the genetic background for all tomato plant transformations. Tomato fruits were collected at the ripe stage and pericarp tissues, once separated from the seeds and placenta, were cut into pieces, and immediately frozen in liquid nitrogen until further analyses.

[0128] Tomato transformations

[0129] Binary plasmids comprising the genes corresponding to PpCCD4 (SEQ ID NO: 1), 35707 (SEQ ID NO: 3), 26288 (SEQ ID NO 5) and CsCCD2 (GenBank accession number KP887110.1 - SEQ ID NO: 22) were transferred to Agrobacterium tumefaciens LBA4404 strain by electroporation and then used for tomato stable transformation into S. lycopersicum (var. Moneymaker), as described previously (Ellul, P. et al. Theor Appl Genet. 2003, 106, 231-238). Transgenic plants were selected for their ability to develop roots on kanomycin selection media and the genomic DNA was further verified using PCR to check for the presence of the corresponding genes (see below). All in vitro steps were carried out in a long-day growth chamber (16 h light / 8 h dark, 24°C, 60-70% humidity, 250 pmol / m2 / s). Positive transgenic tomato plants were transferred to soil and grown in a greenhouse with a 14 / 10 h day / night photoperiod at 22 °C.

[0130] Polar metabolites, i.e. crocins and picrocrocins, were extracted from 50 mg and 5 mg of lyophilized fruit tissues, respectively. The tissue was extracted in cold 75% methanol. The soluble fractions were analyzed using high performance liquid chromatography-diode array detector-high resolution mass spectrometry (HPLC- DAD-HRMS) and HPLC-DAD as previously described (Diretto, G. et al. New Phytol. 2019; 16079).

[0131] Metabolites were identified using co-migration with standards, by matching the UV spectrum of each peak against that of a standard when available, on the basis of literature data, and m / z accurate masses, as reported in the Pubchem database (http: / / pubchem.ncbi.nlm.nih.gov / ) for monoisotopic mass identification or using the Metabolomics Fiehn Lab Mass Spectrometry Adduct Calculator (http: / / fiehnlab.ucdavis.edu / staff / kind / Metabolomics / MS-Adduct-Calculator / ) in the case of adduct detection. Pigments were quantified by integrating the peak areas that were converted to concentrations by comparison with the standards and as reported previously (Marti, M. et al. Metabolic Engineering. 2020, 61, 238-250).

[0132] Example 1. Identification of carotenoids cleavage dioxygenases in Physalis peruviana.

[0133] Developed transcriptomes from two developmental stages of fruits of P. peruviana (Fig. 1, see A and D) were compared to Arabidopsis CCD enzymes using BLAST analyses. The search resulted in the identification of different contigs homologs to CCDs and among them two contigs showed homology to CCD4 genes. Only the full- length clone which showed less identity with the Arabidopsis CCD4 enzyme was selected for further analyses by comparing it with those CCD4 enzymes from different plants that showed a 7, 8:7’, 8’ cleavage activity (Fig. 2). The sequence was also analyzed for N-terminal targeting signals. The CCD4 enzymes, named as PpCCD4 (SEQ ID NO: 2) has an N-terminal transit peptide for plastids targeting.

[0134] Example 2. Identification of carotenoids cleavage dioxygenases in Paulownia tomentosa.

[0135] Developed transcriptomes from two developmental stages of flowers of P. tomentosa were compared to B. davidii CCD4.1 and 4.3 enzymes using BLAST analyses. The search resulted in the identification of four different contigs homologs to these CCDs. Among these CCDs, we selected the one showing the highest identity and named as 35707 (SEQ ID NO: 3). The sequence was also analyzed for N-terminal targeting signals. The CCD4 enzyme, named as 35707 (SEQ ID NO: 4). has an N-terminal transit peptide for plastids targeting.

[0136] Example 3. Identification of carotenoids cleavage dioxygenases in Handroanthus impetiginosus.

[0137] Developed transcriptomes from two developmental stages of fruits of P. peruviana were compared to Arabidopsis CCD enzymes using BLAST analyses. The search resulted in the identification of twelve different contigs homologs to CCD4. Among them, we selected the contig 26288 for further analyses. The sequence was also analyzed for N-terminal targeting signals. The CCD4 enzyme, named as 26288 (SEQ ID NO: 6) has an N-terminal transit peptide for plastids targeting.

[0138] Further, the amino acid 26288 sequence (SEQ ID NO: 6) was compared with the sequences of PpCCD4 (SEQ ID NO: 2), 35707 (SEQ ID NO: 4), and with all characterized CCD enzymes in plants that have been reported to catalyze a 7, 8; 7, 8 cleavage (Fig. 3). 26288 (SEQ ID NO: 6) showed the highest identity with 35707 (SEQ ID NO: 4) (60.09%); 35707 (SEQ ID NO: 4) showed the highest identity with BdCCD4.1 (CCd4.1 protein belonging to Buddleja davidii and having the GenBank accession number: APU54674), and finally, PpCCD4 (SEQ ID NO: 2) showed the highest identity with GjCCD4a (CCd4a protein belonging to Gardenia jasminoides and having the GenBank accession number: ARU08109) (59.16%).

[0139] Example 4. Functional analyses of recombinant CCD4 proteins. The in vivo activity of PpCCD4 (SEQ ID NO: 2), 26288 (SEQ ID NO: 6) and 35707 (SEQ ID NO: 4) in E. coli as well as in N. benthamiana plants was examined. Carotenoids such as lycopene, p-carotene, and zeaxanthin were used as substrates to assay the enzymatic activity in vivo in E. coli. While for the assay of TEV- PpCCD4.1, TEV- 26288, TEV-35707 and pGTEV-CCD2L in N. benthamiana, were tested as follows: in brief, plasmids were constructed by PCR amplification of cDNA of PpCCD4 (SEQ ID NO:1), 26288 (SEQ ID NO: 5), 35707 (SEQ ID NO: 3) and CsCCD2 (GenBank accession number KP887110.1 - SEQ ID NO: 22) and Gibson DNA assembly. The resulting plasmid contains a wild-type TEV cDNA (GenBank DQ986288, with two silent and neutral mutations G273A and A1119G) flanked by the CaMV 35S promoter and terminator in a binary vector derived from pCLEAN-G181. A. tumefaciens C58C1 competent cells, previously transformed with the helper plasmid pCLEAN-S48, were electroporated with the plasmids and selected in plates with 50 pg / ml kanamycin, 50 pg / ml rifampicin, and 7.5 pg / ml tetracycline. Liquid cultures of selected colonies were prepared to infiltrate one leaf of one-month-old transgenic N. benthamiana that stably expresses TEV Nib. After infiltration, plants were kept under controlled conditions in a growth chamber at 25°C under a 12 h day- night photoperiod. Leaf tissue was collected 14 days after infection (dpi) and analyzed for apocarotenoids and carotenoids content.

[0140] For the assays in E. coli, the full-length cDNAs of PpCCD4 (SEQ ID NO:1), 35707 (SEQ ID NO: 3) and 26288 (SEQ ID NO: 5), excluding the first amino acids predicted to be the transit peptide for plastid localization, were independently cloned into the expression vector pThio-Dan1 (Invitrogen) and the recombinant plasmids containing PpCCD4, 35707 and 26288 were each introduced in E. coli cells that produced and accumulated different carotenoid substrates. The reaction products were characterized by GC-MS and HPLC-DAD-HRMS. The volatile p-cyclocitral was detected in the headspace of all p-carotene-producing E. coli cells expressing the three recombinant proteins, independently, p-cyclocitral is generated as a result of a 7,8;7'8' cleavage activity on p-carotene. Therefore, the obtained results suggested that all the tested enzymes catalyzed the cleavage at the 7,8;7'8' double bonds.

[0141] Furthermore, to identify the non-volatile apocarotenoid produced by the activity of the three recombinant enzymes, the pigments produced by carotenoid-accumulating E. coli expressing PpCCD4, 26288 and 35707, independently were analyzed by HPLC- DAD-HRMS. Compared with the empty vector, the UVA / is absorbance spectrum obtained from the p-carotene background from bacterial cells expressing PpCCD4, 26288, and 35707, independently showed a peak that matched crocetin dialdehyde based on UV / Vis spectrum and retention time (Fig. 4). Crocetin dialdehyde was confirmed by MS analyses (Fig. 4).

[0142] Subsequently, a recombinant virus to express PpCCD4.1 (TEV-VgCCD4.1), 26288 (TEV-26288), 35707 (TEV-35707) and CsCCD2 (GenBank accession number KP887110.1 - SEQ ID NO: 22) in N. benthamiana was constructed. As shown before, all the analyzed CCDs are predicted to be plastidic enzymes that must contain the native amino-terminal transit peptide. Thus, to target the enzymes to the plastids, we inserted the cDNAs in a position in the virus genome that corresponds to the amino terminus of the viral polyprotein. In addition, all the constructs also contained, at the 3' end, a sequence for an artificial NlaPro cleavage site to allow the release of the heterologous protein from the viral polyprotein, we used the -8 / +3 site, which splits Nib and CP in TEV. The obtained recombinant clones were agroinoculated in N. benthamiana plants. As a control, some plants were agroinoculated with TEV-aGFP that expresses GFP located at the most amino-terminal position in the viral polyprotein and TEV-CCD2L were used as a positive control. Symptoms of infection were observed approximately 8 dpi in plants agroinoculated with TEV-PpCCD4.1 , TEV-26288, TEV-35707, TEV-CCD2L, and TEV-aGFP. A distinctive yellow pigmentation was observed in tissues of plants agroinoculated with TEV-PpCCD4.1, TEV-26288, TEV-35707, and TEV-CCD2L, at approximately 14 dpi (Fig. 5). Symptomatic leaves from plants infected with TEV-aGFP, and TEV-PpCCD4.1 , TEV- 26288, TEV-35707 and TEV-CCD2L were subjected to extraction and analysis to determine their crocins and carotenoid profiles.

[0143] Analysis of the polar metabolic fraction of tissues infected with TEV-PpCCD4.1 , TEV- 26288, TEV-35707 and TEV-CCD2L showed a series of peaks with maximum absorbance around 440 nm (Fig. 6A), corresponding to crocins with different degree of glucosylation, as revealed by HPLC-HRMS (Fig. 6A), and their levels were quantified. The crocins concentration of tissues infected with TEV-PpCCD4.1, TEV- 26288, and TEV-35707 of the present invention were remarkably increased compared to that obtained from tissued infected with TEV-CCD2L (Fig. 6B). The peaks or the crocins concentration were not observed in the counterpart extracts from tissues from mock-inoculated plants or plants infected with the TEV-aGFP control. Subsequently, the levels of carotenoids and chlorophylls in the apolar fractions were also investigated (Fig. 7). The comparison between tissues infected with TEV-aGFP and TEV-PpCCD4.1, TEV-26288, and TEV-35707, showed differences at metabolite level in both carotenoids and chlorophylls. At the carotenoid level, the most striking differences were in the contents of phytoene, violaxanthin, lutein, and a-carotene. Phytoene levels were increased in TEV-PpCCD4.1, TEV-26288, and TEV-35707, and strongly reduced in TEV-aGFP-infected leaves (Fig. 7A). However, the levels of violaxanthin, lutein and a-carotene were reduced in TEV-PpCCD4.1 , TEV-26288, and TEV-35707, compared with TEV-aGFP-infected leaves (Fig. 7B). Chlorophyll and pheophytin were all reduced in TEV-PpCCD4.1 , TEV-26288, and TEV-35707 (Fig. 7B). Overall, these experiments revealed a remarkable accumulation of 3.47±1.19, 3.82±0.97, and 5.92±1.53 mg of crocins per gram of N. benthamiana DW leaf tissue using TEV-PpCCD4.1, TEV-26288, and TEV-35707.

[0144] Example 5. Quantification of crocins production in tomato fruit.

[0145] Transgenic and control (WT) tomato fruits were crushed and the serum juice obtained after centrifugation. Crocins were detected at 440 nm in all the transgenic serum juices, but not in the control tomato serum juice (Figure 8). However, as it is shown in Figure 8, the crocin concentrations in the transgenic tomato fruits obtained from the tomato plants transformed with the CCD4 of the invention (SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 3), were remarkably increased compared to that in the transgenic tomato fruits obtained from the tomato plants transformed with the CCD2 (GenBank accession number KP887110.1 - SEQ ID NO: 22).

[0146] In summary, the data provided herein demonstrates the strong potential and feasibility of the CCD4s of the invention as powerful biotechnological tools for significantly enhancing the production of apocarotenoids with high value to both industry and human health. Notably, the results show the highest crocin content ever reported through metabolic engineering in heterologous systems. These levels significantly exceed those achieved using CCD2s known in the art, highlighting the superior efficiency of CCD4s of the invention over previously known systems. Altogether, this data establishes a novel and viable platform for the large-scale production of apocarotenoids.

Claims

CLAIMS1. An isolated and / or exogenous polynucleotide(s) comprising at least a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%; 99% or 100% identity to any one of the nucleotides sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 3, or any combination thereof; a biologically active fragment thereof; or a nucleotide sequence complementary thereof; wherein the nucleotide sequence of SEQ ID NO: 1, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Physalis, preferably from P. peruviana; SEQ ID NO: 5, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Handroanthus, preferably from H. impetiginosus; and SEQ ID NO: 3, a biologically active fragment thereof, or a nucleotide sequence complementary thereof, belongs to the genus Paulownia, preferably from P. tomentosa; .

2. An isolated polypeptide(s) encoded by the polynucleotide sequence(s) according to claim 1.

3. An isolated and / or exogenous polynucleotide(s) according to claim 1 , wherein the at least one polynucleotide or any combination thereof, is / are operably linked to one or more control sequences that direct the production of at least one polypeptide according to claim 2 in a cell, preferably wherein the cell is an expression host cell.

4. A nucleic acid construct or expression vector comprising at least one of the polynucleotides of any one of claims 1 or 3, wherein preferably the at least one polynucleotide is operably linked to one or more control sequences that direct the production of at least one polypeptide according to claim 2 in a cell, preferably wherein the cell is an expression host cell.

5. A recombinant expression host cell comprising at least one polynucleotide according to claim 1, encoding at least one polypeptide according to claim 2, wherein the at least one polynucleotide is operably linked to one or more control sequences that direct the production of the at least one polypeptide, and whereinpreferably the host cell is a bacterial cell, a fungal cell, or a plant cell.

6. A transgenic non-human organism comprising at least a polynucleotide according to claim 1 or 3, at least a polypeptide according to claim 2, at least one nucleic acid construct or expression vector according to claim 4, or a recombinant host cell according to claim 5, wherein the transgenic non-human organism is preferably a bacterium, a fungus, or a plant.

7. Use of at least one isolated and / or exogenous polynucleotide according to any one of claims 1 or 3, at least one isolated polypeptide according to claim 2; at least one nucleic acid construct or expression vector according to claim 4, a recombinant host cell according to claim 5, or a transgenic non-human organism according to claim 6, for the production of apocarotenoids, preferably wherein the apocarotenoids are selected from the list consisting of B-cyclocitral, 6-methyl-5- hepten-2-one, crocetin, crocin, safranal, picrocrocin, geranyl acetone, or any combinations thereof.

8. A method of producing at least an apocarotenoid comprising introducing at least one polynucleotide according to any one of claims 1 or 3, at least one isolated polypeptide according to claim 2; or at least a nucleic acid construct or expression vector according to claims 4, into a host cell, or into a transgenic non- human organism, preferably a bacterium, a fungus or a plant, wherein the method optionally further comprises extracting, isolating and purifying apocarotenoids from the culture of the host cell or from the tissues of the transgenic non-human organism.

9. The method according to claim 8, wherein the transgenic non-human organism is a plant, preferably belonging to the Solanaceae family, more preferably being selected from the group consisting of: tobacco, tomato, potato; or belonging to the Apiaceae family, more preferably wild carrot, carrot; or belonging to the Amaranthaceous family, more preferably selected from the group consisting of: spinach; or belonging to the Asteraceae family, more preferably selected from the group consisting of: lettuce; or belonging to the Cucurbitaceae family, more preferably selected from the group consisting of: zucchini, cushaw squash, squash, pumpkin; or belonging to the Poaceae family, more preferably selected from the group consisting of: rice, corn.

10. The method according to claim 8 or 9, wherein the apocarotenoids are selected from the list consisting of B-cyclocitral, 6-methyl-5-hepten-2-one, crocetin, crocin, safranal, picrocrocin, geranyl acetone, or any combinations thereof.

11. An extract and / or harvestable parts of the transgenic non-human organism according to claim 6, comprising apocarotenoids.

12. A composition comprising the extract and / or the harvestable parts of the transgenic non-human organisms according to claim 11 , and optionally at least one excipient and / or carrier, preferably, wherein the composition is selected from a cosmetic, nutritional, nutraceutical or pharmaceutical composition.

13. An extract and / or harvestable parts of the transgenic non-human organisms according to claim 11 , or the pharmaceutical composition according to claim 12, for use as medicine.

14. An extract and / or harvestable parts of the transgenic non-human organisms according to claim 11, or the pharmaceutical composition according to claim 12, for use in the prevention and / or treatment of neurological diseases, cardiovascular diseases, sleep disorders, metabolic diseases, cancer, mental health diseases, inflammatory condition, or liver disease, preferably epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, dementia, Bell's palsy, Huntington's disease, cerebral palsy, meningitis, heart failure, arrhythmia, stroke, atherosclerosis, cardiomyopathy, peripheral artery disease, coronary artery disease, angina, aortic disease, aneurysm, high blood pressure, Insomnia, Circadian rhythm sleep disorder, narcolepsy, idiopathic hypersomnia, parasomnia, REM sleep behavior disorder, Delayed sleep phase disorder, Excessive daytime sleepiness, Type 1 diabetes, type 2 diabetes, hemochromatosis, metabolic syndrome, Phenylketonuria, Gaucher disease, Wilson's disease, breast cancer, lung cancer, prostate cancer, colorectal cancer, melanoma, bladder cancer, kidney cancer, lymphoma, leukemia, myeloma, sarcoma, lymphoma, endometrial cancer, brain tumor, Head and neck cancer, Pancreatic cancer, depression, bipolar disorder, schizophrenia, anxiety disorder, Post-Traumatic Stress Disorder, Neurodevelopmental disorder, Eating Disorder, Rheumatoid arthritis, Psoriatic arthritis, Hepatitis, Acute inflammation, Inflammatory bowel disease, Asthma, Chronic Obstructive Pulmonary Disease, hepatitis A, hepatitis B, hepatitis C, fibrosis, cirrhosis, fatty liver disease, Alpha-1antitrypsin deficiency, Hemochromatosis, or Primary sclerosing cholangitis .

15. A system or kit for producing at least an apocarotenoid compound comprising at least a polynucleotide according to claim 1 or 3, at least a polypeptide according to claim 2, at least a nucleic acid construct or expression vector according to claim 4, at least a recombinant host cell according to claim 5, or at least a transgenic non-human organism according to claim 6.

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