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    • 专利摘要:
    Method of obtaining compounds derived from apocarotenoids in microorganisms and plants for use in agricultural and pharmaceutical applications. The present invention relates to an enzymatic method for obtaining apocarotenoid derivatives, specifically crocetin and picrocrocin, which comprises a series of enzymatic reactions carried out by plant enzymes in a host system and other important crops. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2701354A1
    申请号:ES201731043
    申请日:2017-08-21
    公开日:2019-02-21
    发明作者:Gómez Lourdes Gómez；El Kadiri Oussama Ahrazem；Gianfranco Diretto；Moraga Angela Rubio
    申请人:Universidad de Castilla La Mancha；
    IPC主号:
    专利说明:

    [0001]
    [0002] METHOD OF OBTAINING COMPOUNDS DERIVED FROM APOCAROTENOIDS IN MICROORGANISMS AND PLANTS FOR USE IN AGRICULTURAL APPLICATIONS
    [0003]
    [0004] FIELD OF THE INVENTION
    [0005] The present invention falls within the field of biotechnology. More specifically, it relates to the production of apocarotenoids, namely crocetin and picrocrocin, by biotechnological methods for use in agricultural and pharmaceutical applications.
    [0006]
    [0007] BACKGROUND OF THE INVENTION
    [0008] Carotenoids for use in industry are usually obtained by chemical synthesis. However, high production costs, dependence on fossil fuels to obtain energy and for the supply of adequate reactive organic molecular species, and the harmful impacts perceived by the public on the environment, have resulted in intensified efforts to find methods of alternative production, particularly from biological sources.
    [0009]
    [0010] Organisms capable of synthesizing carotenoids include all photosynthetic plants, protists and bacteria, as well as some heterotrophic bacteria and some fungi. In plants, carotenoids are essential for the development and growth of the plant; Mutations that block the accumulation of carotenoids generate pleiotropic effects on the development of chloroplast biogenesis and the development of seeds. Carotenoids are accessory pigments in chloroplast antenna complexes, where they act by increasing the capacity of light capture, adsorbing in the blue-green region of the visible spectrum (450-550 nm) and transferring energy to chlorophyll.
    [0011]
    [0012] Carotenoids also participate in photoprotection processes in plants, and have functions as antioxidants. Other functions of the carotenoids are related to their intense color, being responsible for the intense colorations and pigmentations of flowers and fruits, acting as attractors of pollinators and animals that help the dispersion of the seeds. Carotenoids are also responsible for the pigmentation of invertebrates, birds and fish. In the case of birds, there is a direct relationship between pigmentation due to carotenoids and sexual attractiveness.
    [0013] In plants, the biosynthesis of carotenoids occurs inside chloroplasts, chromoplasts, and amyloplasts, genetically identical plastids, but with a very different architecture of internal membranes, by enzymes encoded by nuclear genes. The major carotenoids in chloroplasts are p-carotene and xanthophylls. In non-photosynthetic chromoplasts the composition and distribution of carotenoid pigments varies considerably from one plant species to another. But, in addition, the composition of the carotenoid compounds present in each plant species is altered in turn, by changes in environmental conditions.
    [0014]
    [0015] Most carotenoids have skeletons of 40 carbon atoms, which consist of eight isoprene units. However, some carotenoids have skeletons with a lower number of 40 carbons. Most of them are the result of the degradation of one or both ends of the 40-carbon molecule, and are called apocarotenoids or diapocarotenoids.
    [0016]
    [0017] The variety of apocarotenoids is determined by the high amount of carotenoid precursors (more than 600 have been identified), the variations in the cutting site within the skeleton, and the subsequent modifications that it undergoes after cutting. Despite its importance, little is known about the factors that regulate its production in plants.
    [0018]
    [0019] One of the most valuable pigments commercially, crocetin, is obtained from stigmas of saffron. Both the saffron pigments and the main components of aroma and taste are formed by the biological oxidation of zeaxanthin. The crocetin is formed from the central zone with conjugated double bonds. First, oxidation occurs to dialdehyde, and then to diacid. Once produced, crocetin binds successively to glucose units, two at each end (the gentobiosa disaccharide) forming the crocin, which is the main pigment of saffron. Due to the presence of sugar groups at the ends of the chain, crocin is soluble in water.
    [0020]
    [0021] Enzymatic transformations of carotenoids in these products are catalyzed by carotenoid cutting oxygenases (CCO). CCOs are present in all living organisms and differ in the specificity of the substrate and at the cutting site. The CCOs of the plants are divided into two subfamilies. Dioxigenases 9-cis-epoxycarotenoids (9-cis-epoxycarotenoiddioxygenases, NCEDs) involved in abscisic acid biosynthesis (ABA) and carotenoid-cutting dioxygenases (CCDs) that include all other enzymes in the CCO family they do not participate in the formation of ABA.
    [0022] These enzymes seem to have a high specificity in terms of the cut point, catalyzing the break in different double bonds, grouping into different subfamilies: for example the CCD1, isolated in Arabidopsis, saffron, tomato, grape and melon, among others (Schwartz et al. 2001. Characterization of a novel carotenoid cleavage dioxygenase from plants J Biol Chem 276, 25208-25211, Simkin et al 2004a The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the volatiles flavor beta-ionone, pseudoionone, and geranylacetone Plant J. 40, 882-892; Mathieu et al., 2005 A Carotenoid Cleavage Dioxygenase from Vitis vinifera L .: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation J. Exp. Bot.
    [0023] 56, 2721-2731; Ibdah et al. 2006 Functional characterization of CmCCD1, a carotenoid cleavage dioxygenase from melon. Phytochem. 67, 1579-1589).
    [0024]
    [0025] One example is patent ES2334423B1, in which sequences of the enzymes CsCCD4a and CsCCD4b, dioxygenase enzymes isolated from Crocus Sativus (saffron), which catalyze the breakdown of the 9-10 bond in apocarotenoids, are disclosed, releasing volatile substances that contribute to the aroma and flavor of said species.
    [0026]
    [0027] In Crocus Sativus a CCD has been characterized, CCD2, with cut point in the links 7-8 and 7'-8 'that uses zeaxanthin as a substrate (Frusciante S, Diretto G, Bruno M, Ferrante P, Pietrella M, Prado- Cabrero A, Rubio-Moraga A, Beyer P, Gomez-Gomez L, Al-Babili S, Giuliano G. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis Proc Natl Acad Sci US A. 2014 Aug 19; 111 (33): 12246-51, doi: 10.1073 / pnas.1404629111).
    [0028]
    [0029] The patent WO2016012968A1 describes a CCD, specifically CCD2, for the biotechnological production in microorganisms and plants of compounds derived from saffron, specifically crocetin dialdehyde and crocetin coming from the breaking of the double bonds in position 7,8 and 7 ', 8' . CCD2 cleaves these zeaxanthin bonds, but also other carotenoid substrates that contain a 3-OH-p ring at the proximal end of the molecule. However, no enzymes were detected that catalyze the transformation of 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene (4-Hydroxy-2,6,6-trimethyl-1-cyclohexene-1- carbaldehyde, HTCC) in picrocrocin. Picrocrocin is responsible for the slightly bitter taste of saffron. From the picrocrocina forms, during the drying of the spice, the safranal, one of the fundamental components of the aroma of saffron.
    [0030] DESCRIPTION OF THE INVENTION
    [0031] The present invention relates to a method of obtaining apocarotenoids, specifically crocetin and picrocrocin, in microorganisms and plants, which comprises a series of enzymatic reactions of zeaxanthin and intermediates. Plant enzymes were identified that catalyze the production of crocetin and picrocrocin that can be exploited for the production of these two apocarotenoids in another host system and other important crops, satisfying the growing demand for natural pigments with added health properties.
    [0032]
    [0033] In a first aspect of the invention, there is provided an isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 1.
    [0034]
    [0035] In another aspect of the invention, there is provided an isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 2.
    [0036]
    [0037] In another aspect of the invention, an encoded enzyme is provided from the nucleotide sequence, which has an amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.
    [0038]
    [0039] The amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2 correspond to the sequences of enzymes BdCCD4.1 and BdCCD4.3, respectively, isolated dioxygenase enzymes in B. davidii which are involved in the production of crocetin dialdehyde in flowers of B. davidii. Both enzymes specifically catalyze the cleavage of the double bonds in position 7,8,7 ', 8' of zeaxanthin producing crocetin dialdehyde. Oxidative cleavage of carotenoids is a generalized process, which is expected to occur not only in all organisms that are able to synthesize carotenoids, but also in animals that obtain carotenoids from their diet. This reaction is mediated by CCDs that initiate the synthesis of biologically important compounds in animals, fungi and plants, such as retinoic acid, trisporic acids, abscisic acid and strigolactones.
    [0040]
    [0041] In another aspect of the invention, there is provided an isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 3.
    [0042]
    [0043] In another aspect of the invention, there is provided an isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 4.
    [0044] In another aspect of the invention, there is provided an isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 5.
    [0045]
    [0046] In another aspect of the invention, there is provided an isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 6.
    [0047]
    [0048] In another aspect of the invention, an encoded enzyme is provided from the nucleotide sequence, which has an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6
    [0049]
    [0050] The amino acid sequences SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, correspond to the enzyme sequences CsALDHI, CsALDH2 and CsALDH3, respectively, aldehyde dehydrogenase enzymes isolated from C. sativus (saffron), whereas the sequence SEQ ID NO: 6 corresponds to the enzyme sequence BdALDHI, enzyme aldehyde dehydrogenase isolated from B. davidii. These four enzymes are capable of converting crocetin dialdehyde into crocetin.
    [0051]
    [0052] In another aspect of the invention, there is provided an isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 7.
    [0053]
    [0054] In another aspect of the invention, an encoded enzyme is provided from the nucleotide sequence, which has an amino acid sequence of SEQ ID NO: 7.
    [0055]
    [0056] The amino acid sequence SEQ ID NO: 7 correspond to the sequence of the enzyme UGT709G1, enzyme glucosyltransferase (Uridinadiphosphate glucuronyltransferase, UGT) which recognizes and glycosylates HTCC to produce picrocrocin. The isolated enzyme UGT709G1 allows the biotechnological production of picrocrocin, the precursor of safranal flavor, present in a stable non-volatile form for several applications.
    [0057]
    [0058] In another aspect of the invention, there is provided a method of obtaining apocarotenoid derivatives in microorganisms and plants comprising the expression in said microorganisms and / or plants of the enzyme having the amino acid sequence SEQ ID NO: 1 or SEQ ID NO. : 2 to obtain crocetin dialdehyde.
    [0059]
    [0060] In another aspect of the invention, there is provided a method of obtaining apocarotenoid derivatives in microorganisms and plants comprising the expression in said microorganisms and / or plants of the enzyme having the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 for obtaining crocetin.
    [0061]
    [0062] In another aspect of the invention, there is provided a method for obtaining apocarotenoid derivatives in microorganisms and plants comprising the expression in said microorganisms and / or plants of the enzyme having the amino acid sequence SEQ ID NO: 7 for obtaining picrocrocin.
    [0063]
    [0064] FREE TEXT OF THE LIST OF SEQUENCES
    [0065] Below is a translation of the free text in English that appears in the sequence list.
    [0066] SEQ ID NO: 1. Protein BdCCD4.1
    [0067] SEQ ID NO: 2. Protein BdCCD4.3
    [0068] SEQ ID NO: 3. CsALDHI protein
    [0069] SEQ ID NO: 4. Protein CsALDH2
    [0070] SEQ ID NO: 5. Protein CsALDH3
    [0071] SEQ ID NO: 6. BdALDHI Protein
    [0072] SEQ ID NO: 7. Protein UGT709G1
    [0073] SEQ ID NO: 8. Primer for RACE-PCR 3 'and 5'- Direct-BdCCD4.1
    [0074] SEQ ID NO: 9. Primer for RACE-PCR 3 'and 5'- Reverse-BdCCD4.1
    [0075] SEQ ID NO: 10. Primer for RACE-PCR 3 'and 5'- Direct-BdCCD4.3
    [0076] SEQ ID NO: 11. Primer for RACE-PCR 3 'and 5'- Reverse-BdCCD4.3
    [0077] SEQ ID NO: 12. Primer for RACE-PCR 3 'and 5'- Direct-BdALDH1
    [0078] SEQ ID NO: 13. Primer for RACE-PCR 3 'and 5'- Reverse-BdALDH1
    [0079] SEQ ID NO: 14. Primer for RACE-PCR 3 'and 5'- Direct-CsALDH1
    [0080] SEQ ID NO: 15. Primer for RACE-PCR 3 'and 5'- Reverse-CsALDH1
    [0081] SEQ ID NO: 16. Primer for RACE-PCR 3 'and 5'- Direct-CsALDH2
    [0082] SEQ ID NO: 17. Primer for RACE-PCR 3 'and 5'- Reverse-CsALDH2
    [0083] SEQ ID NO: 18. Primer for RACE-PCR 3 'and 5'- Direct-CsALDH3
    [0084] SEQ ID NO: 19. Primer for RACE-PCR 3 'and 5'- Reverse-CsALDH3
    [0085] SEQ ID NO: 20. Primer for amplification of full-length cDNAs-Direct-BdCCD4.1
    [0086] SEQ ID NO: 21. Primer for amplification of full-length cDNAs-Inverse-BdCCD4.1
    [0087] SEQ ID NO: 22. Primer for amplification of full-length cDNAs-Direct-BdCCD4.3
    [0088] SEQ ID NO: 23. Primer for amplification of full-length cDNAs-Inverse-BdCCD4.3
    [0089] SEQ ID NO: 24. Primer for amplification of full-length cDNAs-Direct-BdALDH1
    [0090] SEQ ID NO: 25. Primer for amplification of full-length cDNAs-Reverse-BdALDH1
    [0091] SEQ ID NO: 26. Primer for amplification of full-length cDNAs-Direct-CsALDH1
    [0092] SEQ ID NO: 27. Primer for amplification of full-length cDNAs-Inverse-CsALDH1
    [0093] SEQ ID NO: 28. Primer for amplification of full-length cDNAs-Direct-CsALDH2
    [0094] SEQ ID NO: 29. Primer for amplification of full-length cDNAs-Inverse-CsALDH2
    [0095] SEQ ID NO: 30. Primer for amplification of full-length cDNAs-Direct-CsALDH3
    [0096] SEQ ID NO: 31. Primer for amplification of full-length cDNAs-Inverse-CsALDH3
    [0097] SEQ ID NO: 32. Primer for cloning in expression vectors-Direct-BdCCD4.1 SEQ ID NO: 33. Primer for cloning in expression vectors-Inverse-BdCCD4.1 SEQ ID NO: 34. Primer for cloning in expression vectors-Direct-BdCCD4.3 SEQ ID NO: 35. Primer for cloning in expression vectors-Inverse-BdCCD4.3 SEQ ID NO: 34. Primer for cloning in expression vectors-Direct-UGT709G1 SEQ ID NO: 35. Primer for cloning in expression vectors-Inverse-UGT709G1 SEQ ID NO: 38. Primer for cloning in expression vectors-Direct-BdALDH1 SEQ ID NO: 39. Primer for cloning in expression vectors-Inverse -BdALDH1 SEQ ID NO: 40. Primer for cloning in expression vectors-Direct-CsALDH1 SEQ ID NO: 41. Primer for cloning in expression vectors-Inverse-CsALDH1 SEQ ID NO: 42. Primer for cloning in vectors of expression-Direct-CsALDH2 SEQ ID NO: 43. Primer for cloning in In-Expression vectors verso- CsALDH2 SEQ ID NO: 44. Primer for cloning in expression vectors-Direct-CsALDH3 SEQ ID NO: 45. Primer for cloning in Expression-Inverse vectors- CsALDH3 SEQ ID NO: 46. Primer for qRT-PCR -Direct-BdCCD4.1
    [0098] SEQ ID NO: 47. Primer for qRT-PCR-Reverse-BdCCD4.1
    [0099] SEQ ID NO: 48. Primer for qRT-PCR-Direct-BdCCD4.3
    [0100] SEQ ID NO: 49. Primer for qRT-PCR-Reverse-BdCCD4.3
    [0101] SEQ ID NO: 50. Primer for qRT-PCR-Direct-UGT709G1
    [0102] SEQ ID NO: 51. Primer for qRT-PCR-Reverse-UGT709G1
    [0103] SEQ ID NO: 52. Primer for qRT-PCR-Direct-BdALDH1
    [0104] SEQ ID NO: 53. Primer for qRT-PCR-Reverse-BdALDH1
    [0105] SEQ ID NO: 54. Primer for qRT-PCR-Direct-CsALDH1
    [0106] SEQ ID NO: 55. Primer for qRT-PCR-Reverse-CsALDH1
    [0107] SEQ ID NO: 56. Primer for qRT-PCR-Direct-CsALDH2
    [0108] SEQ ID NO: 57. Primer for qRT-PCR-Reverse- CsALDH2
    [0109] SEQ ID NO: 58. Primer for qRT-PCR-Direct-CsALDH3
    [0110] SEQ ID NO: 59. Primer for qRT-PCR-Reverse- CsALDH3
    [0111]
    [0112] BRIEF DESCRIPTION OF THE FIGURES
    [0113] Figure 1 Biosynthetic route for obtaining crocetin and picrocrocin, where the enzymes of the invention that are involved in each stage of the route to obtain the products derived from apocarotenoids are highlighted.
    [0114] Figure 2. Crocin and zeaxanthin levels in different stages of development of white flowers of B. davidii.
    [0115] Figure 3. Unbranched phylogenetic tree of CCD proteins based on sequence similarity.
    [0116] Figure 4. Expression levels of BdCCD4.1 and BdCCD4.3 in the different stages of floral development in B.davidii.
    [0117] Figure 5. Chromatogram obtained from the HPLC-DAD analysis of the E. coli plasmids pThio-BdCCD4.1, pThio-BdCCD4.3, pThio-control (1 and 2) containing Zeaxanthin and a sample of crocetin dialdehyde (C). The presence of a peak (P1) is highlighted in the chromatogram of pThio-BdCCD4.1 and pThio-BdCCD4.3 which indicates the presence of crocetin dialdehyde (C). Figure 6. Unbranched phylogenetic tree of ALDH proteins based on sequence similarity.
    [0118] Figure 7A and B. Expression levels of CsALDHs and levels of crocin and crocetin in the different stages of stigma development.
    [0119] Figure 8. Unbranched phylogenetic tree of the UGT protein based on sequence similarity.
    [0120] Figure 9. UV spectra and retention times to evaluate the in vitro enzymatic activity of UGT709G1 on HTCC.
    [0121] Figure 10A and B. Expression levels of UGT and levels of HTCC and picrocrocin in the different stages of stigma development.
    [0122] DESCRIPTION OF MODES OF REALIZATION
    [0123] Having described the present invention, it is further illustrated by the following examples.
    [0124]
    [0125] Example 1. Biosynthesis and accumulation of crocetin and crocin during the development of white flowers of B. davidii.
    [0126]
    [0127] The process of biosynthesis and accumulation of crocetin and crocin during the development of white flowers of B. davidii was analyzed . A total of five stages of development were selected and defined:
    [0128] ■ Stage I corresponds to the cocoon closed and completely white.
    [0129] ■ Stage II corresponds to the cocoon closed with a yellowish corolla tube.
    [0130] ■ Stage III corresponds to the anthesis stage with flowers showing an orange corolla tube.
    [0131] ■ Stage IV corresponds to the post-anthesis phase with flowers showing a dark orange corolla tube.
    [0132] ■ Stage V corresponds to the senescence stage.
    [0133]
    [0134] The flowers were analyzed at the selected stages of development for crocetin and crocin accumulation. Crocids were already detected in stage I, but at very low levels compared to stage V, which showed a 30-fold increase in crocin content and allowed the characterization of crocin. By contrast, the levels of zeaxanthin, the precursor of crocetin, were higher in stage I, and their levels decreased as the flower develops, with the lowest levels in stage V (Figure 2).
    [0135]
    [0136] Example 2. Identification of a candidate gene for crocetin formation in B. davidii flowers .
    [0137]
    [0138] The partial cDNAs were obtained by RT-PCR using degenerate oligonucleotides for the CCD enzymes. The obtained sequences were used for the design of oligonucleotides specific for RACE-PCR. Two full length cDNAs were obtained from flowers in the anthesis and named as BdCCD4.1 and BdCCD4.3 according to their identity with other plants with CCD enzymes.
    [0139]
    [0140] BdCCD4.1 encodes a protein of 580 amino acids with a molecular mass of 64.56 kD. The BdCCD4.1 protein (SEQ ID NO: 1) showed the highest identity, 66%, to an enzyme Putative CCD4 from Sesamum indicum (XP_011082281.1) and 55% identity with VvCCD4a (AGT63321.1), the carotenoid excision dioxygenase 4a from Vitis vinifera.
    [0141]
    [0142] BdCCD4.3 encodes a protein of 579 amino acids with a molecular mass of 64.39 kD. The BdCCD4.3 protein (SEQ ID NO: 2) showed the highest identity, 68%, to a putative CCD4 enzyme from Scutellaria baicalensis (AGN03860.1). The two sequences were used to construct a phylogenetic tree in order to determine its position in the different sub families of CCDs in plants (Figure 3). BdCCD4.1 and BdCCD4.3 appear in a group outside the main group of CCD4 of dicotyledonous species.
    [0143]
    [0144] The sequences of CCDs were analyzed for the presence of N-terminal and C-terminal signals and for membrane anchoring signals using web programs based on ChloroP 1.1 and TargetP v1.1. It was predicted that BdCCD4.1 and BdCCD4.3 had an N-terminal signal peptide to target plastids.
    [0145]
    [0146] Example 3. Analysis of BdCCD expression during floral development in B.davidii
    [0147]
    [0148] The expression patterns of the BdCCD genes were analyzed during the development of B. davidii flores. For this purpose, the five stages of development characterized for crocin accumulation (described in Example 1) were selected for real-time quantitative PCR. From this analysis, BdCCD4.1 and BdCCD4.3 showed relatively high expression levels and their expression levels follow the accumulation of crocin in the flowers. The maximum expression levels for BdCCD4.1 and BdCCD4.3 were reached in the anthesis (IV) stage, whereas no transcripts were detected in senescent flowers (Figure 4).
    [0149]
    [0150] Example 4. Activity of BdCCDs in E. coli cells
    [0151]
    [0152] Total RNA was isolated from corollas and stigmas of B. davidii and C. sativus, respectively, at different stages of development using Ambion PolyAtrack, following the protocols of the manufacturer (Ambion Inc., Austin, TX, USA). First strand cDNAs were synthesized by reverse transcription (RT) from 2 pg of total RNA using an oligo dT primer of 18 base pairs and a first strand cDNA synthesis kit (GE Healthcare Life Sciences, Buckinghamshire, UK ) according to the manufacturer's instructions.
    [0153]
    [0154] The B. davidii cDNAs were used for the PCR reaction for the isolation of CCD. The corresponding primers were used (see Table 1), under thermal cycle parameters were 2 min at 95 ° C, 10x (30s at 95 ° C, 30s at 55 ° C-0.3 ° C / cycles and 1min 30s to 72 ° C), 35x (30s at 95 ° C, 30s at 50 ° C and 1min 30s at 72 ° C) and finally 5 min at 72 ° C, for the two isolates.
    [0155]
    [0156] The PCR products obtained in the reactions were separated on a 1% agarose gel, purified, ligated into pSpark-TA (Canvax, Córdoba, Spain) and then introduced into E. coli.
    [0157]
    [0158] The full-length cDNAs were cloned into the EcoRI site of the pBAD-Thio vector (Invitrogen) by recombination using the In-Fusion® HD Cloning Plus CE kit (Clontech) and specific primers (see Table 1). The resulting expression plasmids: pThio- BdCCD4.1 and pThio-BdCCD4.3 were sequenced to confirm correct assembly and lack of sequence errors. Then, the vectors were transformed into Escherichia coli BL21 manipulated with plasmids for the production of zeaxanthin, lycopene, 5-carotene and pcarotene. The E.coli transformants were grown overnight at 30 ° C in 4 ml of LB medium supplemented with ampicillin (100pg / ml) and chloramphenicol (60pg / ml). The cultured cells were transferred to 100 ml of 2xYT medium supplemented with ampicillin (50 pg / ml) and chloramphenicol (30pg / ml) and further cultured at 30 ° C until an OD600 of 0.8 was reached. The cells were then induced with 0.2% arabinose and grown overnight at 20 ° C. The cells were cultured by centrifugation (6,000 rpm for 10 minutes) and the pigments were extracted repeatedly with a total volume of 10 ml of acetone until all the pigments were visibly removed. The solvent was evaporated under N2 gas and the pigments were resuspended with 0.3 ml of MeOH: tert-methyl-butyl-ether (50:50). After centrifugation (13,000 rpm for 10 min), the extracts were analyzed by HPLC.
    [0159]
    [0160] The results obtained are shown in Figure 5. The presence of a peak (P1) is highlighted in the chromatogram of pThio-BdCCD4.1 and pThio-BdCCD4.3 which indicates the presence of crocetin dialdehyde (C). Confirmed by HPLC-MS.
    [0161]
    [0162] Example 5. Identification of ALDH genes of C. sativus and B. davidii
    [0163] ALDHs constitute a superfamily of NAD (P) + - dependent oxidoreductases, widely distributed throughout the body, which catalyze the conversion of aldehydes into the corresponding acids. Eukaryotic ALDH are divided into several families, based on specific homology criteria established by the ALDH Genes Nomenclature Committee (AGNC). ALDHs share a conserved ALDH domain and differ in the presence of variable amino acids and carboxylic extensions. To identify the ALDH involved in the crocetin biosynthesis, a strategy was used based on homology, taking advantage of the specific motifs conserved among the ALDH involved in the oxidation of apocarotenoids.
    [0164]
    [0165] A population of cDNA was prepared by reverse transcription of poly (A) + from total RNA isolated from stigmas of saffron and Buddleja flowers , and used as a template for the amplification reaction. The amplified products obtained with the degenerate primers were cloned and analyzed. Sequence analyzes of several PCR products revealed homologies to aldehyde dehydrogenases and a RACE-PCR approach was used to isolate complete clones. Four cDNAs were obtained: CsALDH1, CsALDH2, CsALDH3 and BdALDH1.
    [0166]
    [0167] CsALDH1 codes for a protein of 537 amino acids. The CsALDH1 protein (SEQ ID NO: 3) showed 83% identity with an Elaeis guineensis protein (XP_010914166.1). CsALDH2 codes for a protein of 535 amino acids and showed an identity of 81% with Elaeis guineensis (XP_010914166.1). CsALDH3 codes for a protein of 482 amino acids, and showed 79% identity with a protein for Phoenix dactylifera (XP_008781593). BdALDH1 codes for a protein of 476 amino acid residues, and showed 82% identity with a protein from Sesamum indicum (XP_011069490.1).
    [0168]
    [0169] The isolated sequences were used to construct a phylogenetic tree in order to determine the ALDHs involved in the modification of apocarotenoids (Figure 6).
    [0170]
    [0171] Example 6. Activity analysis of ALDHs in E. coli cells
    [0172]
    [0173] Total RNA was isolated from corollas and stigmas of B. davidii and C. sativus, respectively, at different stages of development using Ambion PolyAtrack, following the protocols of the manufacturer (Ambion Inc., Austin, TX, USA). First strand cDNAs were synthesized by reverse transcription (RT) from 2 pg of total RNA using an oligo dT primer of 18 base pairs and a first strand cDNA synthesis kit (GE Healthcare Life Sciences, Buckinghamshire, UK ) according to the manufacturer's instructions.
    [0174]
    [0175] The B. davidii cDNAs were used for the PCR reaction for the isolation of ALDH. The corresponding primers were used (see Table 1), under thermal cycle parameters were 2 min at 95 ° C, 10x (30s at 95 ° C, 30s at 55 ° C-0.3 ° C / cycles and 1min 30s at 72 ° C), 35x (30s at 95 ° C, 30s at 50 ° C and 1min 30s at 72 ° C) and finally 5 min at 72 ° C, for the two isolates. For the identification of C. sativus ALDH the primers used were the same as those used for the identification of ALDH from B. davidii, as well as PCR conditions.
    [0176]
    [0177] The full-length cDNAs for the 4 isolated ALDHs ( CsALDHI, CsALDH2, CsALDH3 and BdALDHI) were cloned into the EcoRI site of the pET28 vector by recombination using the In-Fusion® HD Cloning Plus CE kit (Clontech) and specific primers (Table 1 ). The resulting expression plasmids: pET-CsALDH1 , pET-CsALDH2, pET-CsALDH3 and pET- BdALDHI were sequenced to confirm correct assembly and lack of sequence errors. Next, the vectors were transformed into Escherichia coli BL21 manipulated with plasmids for the production of zeaxanthin and host pThio-BdCCD4.3 or pBAD / Thio. Colonies were inoculated overnight in a 2xYT medium supplemented with ampicillin (50 pg / ml), kanamycin (25 pg / ml) and chloramphenicol (30pg / ml) and further cultured at 30 ° C until an OD600 of 0.8 was reached. . Expression of the recombinant proteins was induced by the addition of 0.1 mm of isopropyl-thio-pD-galactoside and 0.2% (v / v) of arabinose and grown overnight at 20 ° C. The cells were cultured by centrifugation at 12000g for 1 minute and the pigments were extracted by dissolving "pellets" in 5 ml of acetone, sonicated and centrifuged at 12000g for 10 minutes. The extraction was repeated, and the extracts were combined, dried and analyzed by HPLC for the detection of crocetin and crocetin dialdehyde.
    [0178]
    [0179] Example 7. Expression of CsALDHs in the stigma tissue
    [0180] The expression profiles of isolated CsADLH1-3 were analyzed during stigma development and compared with crocetin, picrocrocin and crocin levels during the different stages of development. CsALDH2 was expressed at relatively low levels, and reached a peak expression in the red stage, and decreased thereafter.
    [0181]
    [0182] CsALDH1 was highly expressed in all the development stages analyzed and reached the highest levels in pre-anthesis and anthesis (Figure 7). The transcript levels of CsALDH3 increased from yellow to the anthesis stage (Figure 7). Comparison of the expression levels of each CsALDH with the levels of picrocrocin, crocetin and crocin, showed a better correlation of CsALDH1 with crocetin and CsALDH2 and CsALDH3 showed a better correlation with crocine levels (Figure 8).
    [0183] In summary, four ALDH enzymes were isolated and analyzed, three of them from C. sativus and one fourth from B. davidii and it was found that all of them are capable of converting crocetin dialdehyde into crocetin (Figure 1).
    [0184]
    [0185] Example 8. Identification and cloning of UGT709G1 genes
    [0186] To identify UGTs from saffron stigmas, a strategy based on homology was used, taking advantage of the specific UGT motifs located in the C-terminal region. A population of cDNA was prepared by reverse transcription of poly (A) + from total RNA isolated from saffron stigmas and used as a template for the amplification reaction. The amplified products obtained with the degenerate primers were cloned and analyzed. Sequence analyzes of several PCR products revealed homologies to glycosyltransferases, including those previously characterized from saffron. A new clone obtained for full-length isolation was selected. The clone obtained called UGT709G1 (according to the UGT Nomenclature Committee) contains a putative open reading frame of 1548 bp encoding 515 amino acid residues with a calculated molecular mass of 57.5 kDa. UGT709G1 had a typical PSPG-box sequence that included highly conserved key residues for substrate recognition and catalysis of UGTs: a histidine residue (His-19) highly conserved between PSPG acts as a key catalytic residue to activate the hydroxy group of the glucosyl acceptor molecule to facilitate the formation of a glycosidic bond.
    [0187]
    [0188] Phylogenetic analysis based on the deduced amino acid sequences of UGT709G1 (SEQ ID NO: 7) and other plant UGTs suggested that UGT709G1 belonged to group G of the PSPGs family (Figure 8). Functionally, members of group G were implicated in the biosynthesis of the genidoid iridoids (GjUGT6) in Jaminoides gardenia, 7-deoxyloganic acid (CrUGT8) in Catharanthus roseus, cyanogenic glycosides (UGT85B1) in Sorghum (Sorghum bicolor), and cytokinins (UGT85A1 ) in Arabidopsis thaliana. The amino acid sequence comparisons showed 43 and 40% sequence identity with CrUGT8 and GjUGT6 respectively, which participate in the glycosylation of secondary metabolites related to monoterpenoids. While the highest identity, 54%, was found with an uncharacterized protein of Elaeis guineensis (XP_010923185.1).
    [0189]
    [0190] To examine the catalytic function of UGT709G1, its open reading frame was expressed in E. coli as a GST fusion protein. After the purification of the UGT recombinant by affinity chromatography, was assayed for its O-glucosyltransferase activity using various secondary metabolites and HTCC as acceptor substrates in the presence of the UDP-Glc donor. UGT709G1 rapidly and efficiently converted HTCC into a product with a retention time and UV spectrum identical to that of picrocrocin, while no such reaction product was detected with the other substrates tested (Figure 9). The E. coli harboring the control vector did not produce HTCC glycosylation products.
    [0191]
    [0192] Example 9. Expression of UGT709G1 in the stigma tissue
    [0193] The transcription levels of UGT709G1 were determined in the organs of the saffron plant by RT-PCR in real time, using Pfu polymerase (Promega, Madison, USA). The oligonucleotide sequences represented by SEQ ID NO: 19 and 20 were used for the cloning of UGT709G1.
    [0194]
    [0195] The resulting product was gel purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA). The gene was cloned into the vector pGEX4T2 by recombination using the In-Fusion® HD Cloning Plus CE kit (Clontech) and specific primers (Table 1). The resulting expression plasmid, ie pGEX-UGT709G1, was sequenced to confirm correct assembly and lack of sequence errors. Next, pGEX-UGT709G1 was transformed into Eschenchia coli BL21 (Novagen) cells. After transformation the colonies were selected in LB containing 100 pg / ml ampicillin plates. The individual colonies were grown overnight in 5 ml of 2YT medium with ampicillin (50 pg / ml), at 25 ° C, and 2.5 ml of the culture was used to inoculate 200 ml of fresh 2YT-AMP medium.
    [0196]
    [0197] The cells were cultured at 25 ° C until an A600 of 0.8 was reached, after which the 1 mM IPTG culture was induced and allowed to grow for 16 h at 25 ° C. The cells were harvested by centrifugation at 5,000 g for 10 minutes and resuspended in 10 ml of PBS. The resuspended cells were sonicated with a microtiter probe on ice until the viscosity disappeared. After sonication, the samples were centrifuged at 10,000 g for 30 min. The supernatant and the pellet were analyzed by PAGE (polyacrylamide gel electrophoresis) / SDS for the solubility of the fusion protein by Coomassie staining . The glutathione S-transferase (GST) -UGT709G1 fusion protein was purified by column according to the manufacturer's instructions (GE Healthcare Life Sciences). The protein concentration was determined by Bradford using serum albumin as standard.
    [0198]
    [0199] The relative abundance was compared with the levels of HTCC and picrocrocin in seven stages of stigma development (Figure 10A). In the stigma tissue the expression of UGT709G1 was regulated in the development, and the transcripts were detected mainly in the early stages of development and between these stages the highest levels of expression were detected in the orange stage (Figure 10B) and gradually decreased. in the following stages.
    [0200] Table 1. Oligonucleotide sequences used for isolation, cloning and analysis of CCDs (BdCCD4.1 and BdCC4.3), ALDH (BdALDH1 and CsALDH1-3) and UGT (UGT709G1)
    [0201]
    [0202]
    权利要求:
    Claims (17)
    [1]
    1. Isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 1.
    [2]
    2. Isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 2.
    [3]
    3. Isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 3.
    [4]
    4. Isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 4.
    [5]
    5. Isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 5.
    [6]
    6. Isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 6.
    [7]
    7. Isolated polynucleotide encoding a peptide comprising the amino acid sequence SEQ ID NO: 7.
    [8]
    8. Enzyme encoded from the nucleotide sequence according to claim 1, having the amino acid sequence SEQ ID NO: 1.
    [9]
    9. Enzyme encoded from the nucleotide sequence according to claim 2, having the amino acid sequence SEQ ID NO: 2.
    [10]
    10. Enzyme encoded from the nucleotide sequence according to claim 3, having the amino acid sequence SEQ ID NO: 3.
    [11]
    11. Enzyme encoded from the nucleotide sequence according to claim 4, having the amino acid sequence SEQ ID NO: 4.
    [12]
    12. Enzyme encoded from the nucleotide sequence according to claim 5, having the amino acid sequence SEQ ID NO: 5.
    [13]
    13. Enzyme encoded from the nucleotide sequence according to claim 6, having the amino acid sequence SEQ ID NO: 6.
    [14]
    14. Enzyme encoded from the nucleotide sequence according to claim 7, having the amino acid sequence SEQ ID NO: 7.
    [15]
    15. Method for obtaining apocarotenoid derivatives in microorganisms and / or plants, characterized in that it comprises the expression in said microorganisms and / or plants of the enzyme having the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2 for obtaining crocetin dialdehyde.
    [16]
    16. Method of production according to claim 15, characterized in that it comprises the expression in said microorganisms and / or plants of the enzyme having the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 for obtaining crocetin.
    [17]
    17. Obtaining method according to claims 15 to 16, characterized in that it comprises the expression in said microorganisms and / or plants of the encoded enzyme having the amino acid sequence SEQ ID NO: 7 for obtaining picrocrocin.
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    WO2019038459A1|2019-02-28|
    ES2701354B2|2019-06-24|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2334423A1|2008-06-27|2010-03-09|Universidad De Castilla La Mancha|Recombinant dioxygenases. |
    WO2016012968A1|2014-07-23|2016-01-28|Ente Per Le Nuove Tecnologie, L'energia E L'ambiente |A carotenoid dioxygenase and methods for the biotechnological production in microorganisms and plants of compounds derived from saffron|
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