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EP-4739098-A1 - METHODS OF PRODUCING NATURAL SWEETENERS

EP4739098A1EP 4739098 A1EP4739098 A1EP 4739098A1EP-4739098-A1

Abstract

Methods for modification of the cucurbitacin biosynthetic pathway by inhibition of cucurbitacin biosynthetic pathway enzymes in plants and plant cells, production of cucurbit plants and plant cells having modified cucurbitacin and cucurbitacin derivatives, plants and plant cells modified thereby and their propagation, and compositions comprising the modified plants and plant cells are provided.

Inventors

  • SCHAFFER, Arthur Aaron
  • SCHAFFER, Amos Avraham
  • COHEN, SHAHAR
  • KLUTZKY, Galina
  • DORON-FAIGENBOIM, Adi
  • ESELSON, Elena
  • DAVIDOVICH-RIKANATI, Rachel

Assignees

  • The State of Israel, Ministry of Agriculture & Rural Development, Agricultural Research Organization (ARO) (Volcani Center)
  • Nature Sweet Ltd.

Dates

Publication Date
20260513
Application Date
20240704

Claims (20)

  1. 1. A method of producing a plant or plant cell with a modified cucurbitacin content, the method comprising down-regulating expression of at least one cucurbitacin biosynthetic pathway gene in the plant or plant cell, thereby modifying cucurbitacin expression in said plant or plant cell.
  2. 2. A method of producing a plant or plant cell with a modified cucurbitacin content, the method comprising growing the plant or plant cell of claim 1 with modified expression of the at least one cucurbitacin biosynthetic pathway gene.
  3. 3. The method of claim 1 or 2, wherein said down-regulation is by genome editing.
  4. 4. A plant or plant cell modified to have reduced expression of at least one gene of the cucurbitacin biosynthetic pathway, wherein said cucurbit plant or plant cell is obtainable according to the method of claim 1.
  5. 5. The plant or plant cell of claim 4, wherein said plant or plant cell is an elite plant or plant cell.
  6. 6. The plant or plant cell of claim 4, wherein said plant or plant cell is a hybrid plant or plant cell.
  7. 7. The plant or plant cell of claim 4, wherein said plant or plant cell is an inbred plant or plant cell.
  8. 8. An inbred plant or plant cell having a nucleic acid sequence alteration of at least one gene of the cucurbitacin biosynthetic pathway.
  9. 9. An elite plant or plant cell having a nucleic acid sequence alteration of at least one gene of the cucurbitacin biosynthetic pathway.
  10. 10. A hybrid plant or plant cell having a nucleic acid sequence alteration of at least one gene of the cucurbitacin biosynthetic pathway.
  11. 11. The plant or plant cell of any one of claims 4-10, wherein said plant or plant cell comprises at least one tetracyclic triterpene capable of glucosylation by a UDP- glucoronosyltransferase (UGT).
  12. 12. The plant or plant cell of claim 11, wherein said UGT is a plant UGT.
  13. 13. The plant or plant cell of claim 11, wherein said UGT is selected from the group consisting of S. grosvenorii UGTs selected from the group consisting of UGT74-345-2, UGT73- 348-2, UGT94-289-1, UGT73-327-2, UGT73-251-5, UGT73-251-6, UGT75-281-2, UGT85-269- 4, UGT85-269-1, UGT94-289-2 and UGT94-289-3.
  14. 14. The plant or plant cell of claim 11, wherein said UGT is a UGT having an amino acid sequence selected from the group consisting of SEQ ID NOs. 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 and 149.
  15. 15. The plant or plant cell of claim 11, wherein said UGT is a UGT encoded by a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs. 128, 129, 130, 131, 132, 133, 135 and 137.
  16. 16. An extract of the plant or plant cell of any one of claims 4-11, comprising at least one tetracyclic triterpene capable of glucosylation by a UGT.
  17. 17. The method of any one of claims 1-3, or plant or plant cell of any one of any one of claims 4-15, or the extract of claim 16, wherein said plant or plant cell is of a bitter cucurbit species.
  18. 18. The method of any one of claims 1-3, or plant or plant cell of any one of any one of claims 4-15, or the extract of claim 16, wherein said plant or plant cell is of a species naturally expressing said at least one cucurbitacin biosynthetic pathway gene.
  19. 19. The method of any one of claims 1-3, or plant or plant cell of any one of any one of claims 4-15, or the extract of claim 16, wherein said plant or plant cell is of a species genetically modified to express said at least one cucurbitacin biosynthetic pathway gene.
  20. 20. The method of any one of claims 1-3, or plant or plant cell of any one of any one of claims 4-15, or the extract of claim 16, wherein said plant or plant cell is an Iberis amara plant or plant cell.

Description

METHODS OF PRODUCING NATURAL SWEETENERS RELATED APPLICATION/S This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/525,322 filed on July 6, 2023, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING STATEMENT The XML file, entitled 100786 Sequence Listing. XML, created on July 4, 2024, comprising 256,208 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to methods of producing modified cucurbitane-type triterpenoids that can be utilized in the production of sweet glycosylated triterpenoids known as mogrosides and compositions comprising same and uses thereof. Mogrosides are triterpene-derived specialized secondary metabolites found in the fruit of the Cucurbitaceaea family plant Siraitia grosvenorii (Luo Han Guo). Their biosynthesis in fruit involves the synthesis of mogrol, a tetra-hydroxy cucurbitane triterpenoid, followed by a number of consecutive glucosylations of the aglycone mogrol to the final sweet products mogroside IV and mogroside V (Figure 6). The parent aglycone compound mogrol is derived by successive hydroxylations of cucurbitadienol, the initial product of the stereospecific triterpene synthase, cucurbitadienol synthase. Cucurbitadienol subsequently undergoes hydroxylations, by a combination of epoxidation and action of epoxide hydrolase, at the C24 and C25 positions, and an additional hydroxylation at Cl 1 by a cytochrome P450 enzyme, leading to mogrol, as described in Itkin et al, 2016 (Figure 1). The mogrol is subsequently glucosylated at the C3 and C24 positions to varying degrees, from 1 to 6 glucosyl groups, in a temporally successive pattern during fruit development and the glucosylated mogrol compounds are termed mogrosides. The sweetness strength of the mogrosides increases with the additional glucose moieties such that M6 (with 6 glucosyl groups) is sweeter than M5, followed by M4, respectively (Kasai R., et al., Sweet cucurbitane glycosides from fruits of Siraitia siamensis (chi-zi luo-han-guo), a Chinese folk medicine. Agric Biol Chem 1989, 53(12):3347-3349). The purified mogroside V, has been approved as a high-intensity sweetening agent in Japan (Jakinovich, W ., Jr., Moon, C., Choi, Y. H., & Kinghorn, A. D. 1990. Evaluation of plant extracts for sweetness using the Mongolian gerbil. Journal of Natural Products, 53, 190-195) and the extract has gained generally recognized as safe (GRAS) status in the USA as a non-nutritive sweetener and flavor enhancer. Mogroside V has been known in the food industry as a natural non-sugar food sweetener, with a sweetening capacity of -250 times that of sucrose (Kasai R., et al., Sweet cucurbitane glycosides from fruits of Siraitia siamensis (chi-zi luo-han-guo), a Chinese folk medicine. Agric Biol Chem 1989, 53(12):3347-3349.). Moreover, additional health benefits of mogrosides have been revealed in recent studies (Li et al., Chemistry and pharmacology of Siraitia grosvenoriv. a review. Chin J Nat Med. 2014 12(2):89-102.). Extraction of mogrosides from the Siraitia fruit can yield a product of varying degrees of purity, often accompanied by undesirable aftertaste. In addition, yields of mogroside from cultivated Siraitia fruit are limited due to low plant yields and particular cultivation requirements of the plant. It is therefore advantageous to be able to produce sweet mogroside compounds in an alternative plant species, amenable to high yield cultivation. Fruit of other species in the Cucurbitaceae family accumulate members of the cucurbitane- type triterpenoid family, such as cucurbitacins. The cucurbitacins, however, are extremely bitter. Non-glycosylated mogrol and non-glycosylated cucurbitacin differ from each other in the number and positions of oxygenations (either as hydroxyl groups, or as carbonyl groups), by dehydrogenations and by acetylations of hydroxyl groups. Mogrol synthesis from squalene precursors proceeds from squalene to diepoxysqualene via successive epoxidations by squalene epoxidase. The diepoxysqualene (two epoxy groups, one each at each of the penultimate terminal positions of the 30-carbon squalene molecule) is further transformed to mogrol as described above. Cucurbitacins are a family of triterpenoid compounds comprising over 20 members, differing in the number of hydroxyl, carbonyl, and acetyl groups on the cucurbitadienol skeleton. Squalene epoxide undergoes cyclization to the cucurbitadienol skeleton via cucurbitadienol synthase. The cucurbitadienol skeleton may undergo further chemical modifications, including numerous hydroxylation s, acetylations, dehydrogenations and reductions, the combination of which determines the final cucurbitacin compound. For example, Cucurbitacin E, which is the major cucurbitacin in wild bitter watermelon is shown in Fig. 2 and contains various oxy