Methods of producing mogrosides and compositions comprising the same and uses thereof

Abstract

Provided are isolated mogroside and mogrol biosynthetic pathway enzyme polypeptides useful for mogroside biosynthesis. The mogroside biosynthetic pathway enzymes of the invention include squalene epoxidase (SE), epoxy hydratase (EH), cytochrome p450 (Cyp), cucurbitadienol synthase (CDS), and udp-glucosyltransferase (UGT). Also provided are methods of producing mogroside using the isolated mogroside and mogrol biosynthetic enzyme polypeptides, the methods comprising contacting a mogrol and / or glycosylated mogrol (mogroside) with at least one UDP glucose glucosyltransferase (UGT) enzyme polypeptide of the invention that catalyzes glucosylation of the mogrol and / or glycosylated mogrol to produce a mogroside with an additional glucosyl component, thereby producing a mogroside. Alternatively or additionally provided are methods of synthesizing mogrol, the methods comprising contacting a mogrol precursor substrate with one or more mogrol biosynthetic pathway enzyme polypeptides as described herein that catalyze synthesis of mogrol from the mogrol precursor substrate, thereby synthesizing mogrol.

Claims

1. An isolated uridine diphosphate-glucosyltransferase polypeptide, comprising the same amino acid sequence as the polypeptide encoded by SEQ ID NO:33, wherein the polypeptide catalyzes the main-chain glucosylation of mogroside at C24 and the main-chain glucosylation of mogroside at C3.

2. An isolated uridine diphosphate-glucosyltransferase polypeptide consisting of an amino acid sequence as shown in SEQ ID NO:38, wherein the polypeptide catalyzes the branching glucosylation of mogroside at (1-2) and (1-6) positions of C3 and at (1-2) and (1-6) positions of C24.

3. An isolated uridine diphosphate-glucosyltransferase polypeptide consisting of an amino acid sequence as shown in SEQ ID NO:38, wherein the polypeptide catalyzes the branch glucosylation of mogroside M5 to mogroside M6.

4. The isolated uridine diphosphate-glucosyltransferase polypeptide of claim 2 or 3, wherein the polypeptide catalyzes: (a) Branch glucosylation of mogroside at positions (1-2) and (1-6) of C3; (b) Branch glucosylation of mogrosides at positions (1-2) and (1-6) of C24, and (c) Branch glucosylation of mogroside M5 to mogroside M6.

5. An isolated uridine diphosphate-glucosyltransferase polypeptide selected from the same amino acid sequence as the polypeptide encoded by SEQ ID NO:33, the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence shown in SEQ ID NO:38, wherein the polypeptide catalyzes the branch glucosylation of mogroside IV (M4) to mogroside V (M5).

6. The isolated uridine diphosphate-glucosyltransferase polypeptide of claim 5, wherein the amino acid sequence is as shown in SEQ ID NO:

6.

7. The isolated uridine diphosphate-glucosyltransferase polypeptide of any one of claims 1-6, wherein the uridine diphosphate-glucosyltransferase is a plant uridine diphosphate-glucosyltransferase.

8. The isolated uridine diphosphate-glucosyltransferase polypeptide of claim 7, wherein the plant is a plant of the Cucurbitaceae family.

9. The isolated uridine diphosphate-glucosyltransferase polypeptide of claim 7, wherein the plant is monk fruit.

10. A method for synthesizing mogroside, the method comprising contacting at least one uridine diphosphate-glucosyltransferase polypeptide or combination thereof as described in any one of claims 1-9 with at least one uridine diphosphate-glucosyltransferase substrate mogroside precursor.

11. The method of claim 10, wherein the amino acid sequence of the at least one uridine diphosphate-glucosyltransferase polypeptide is selected from the polypeptide encoded by SEQ ID NO:33, SEQ ID NO:38 and SEQ ID NO:

6.

12. The method of claim 10, wherein the at least one uridine diphosphate-glucosyltransferase polypeptide comprises the uridine diphosphate-glucosyltransferase polypeptide of claim 1 and the uridine diphosphate-glucosyltransferase polypeptide of claim 2.

13. The method of claim 10, wherein the uridine diphosphate-glucosyltransferase substrate mogroside precursor substrate is mogroside, the method comprising: (a) Production of mogroside by contacting at least one mogroside precursor substrate with a mogroside pathway enzyme, wherein: (i) When the mogroside precursor product contains diepoxysqualene and the mogroside precursor substrate contains squalene or squalene oxide, the mogroside pathway enzyme contains a squalene epoxysqualene polypeptide as shown in SEQ ID NO:14 or SEQ ID NO:16, wherein the polypeptide catalyzes the synthesis of diepoxysqualene from squalene or squalene oxide, thereby producing diepoxysqualene. (ii) When the mogroside precursor product comprises 3-hydroxy, 24-25-epoxycucurbitadienol and the mogroside precursor substrate comprises diepoxysqualene, the mogroside pathway enzyme comprises a cucurbitadienol synthase polypeptide as shown in SEQ ID NO:12, thereby producing 3-hydroxy, 24-25-epoxycucurbitadienol. (iii) When the product comprises 3,24,25-trihydroxycucurbita diol and the substrate comprises 3-hydroxy,24-25-epoxycucurbita diol, the mogroside pathway enzyme comprises an epoxidase (EH) polypeptide comprising an amino acid sequence as shown in SEQ ID NO:18, SEQ ID NO:22, or SEQ ID NO:24, wherein the polypeptide catalyzes the synthesis of 3,24,25-trihydroxycucurbita diol from 3-hydroxy,24-25-epoxycucurbita diol, thereby producing 3,24,25-trihydroxycucurbita diol. (iv) When the product comprises mogroside and the mogroside precursor substrate comprises 3,24,25-trihydroxycucurbitadienol, the mogroside pathway enzyme is a cytochrome P450 enzyme as shown in SEQ ID NO:10, thereby producing 3,11,24,25-tetrahydroxycucurbitadienol, and (b) The method of any one of claims 10-12 to synthesize the mogroside from the mogroside.

14. The method of claim 13, wherein the squalene epoxygenase is a plant squalene epoxygenase.

15. The method of claim 13, wherein the epoxide hydratase polypeptide is a plant epoxide hydratase polypeptide.

16. The method of any one of claims 10-15, wherein the mogroside is selected from mogroside I-A1, mogroside I-E1, mogroside IIE, mogroside III, symmonoside, mogroside V and mogroside VI.

17. The method of any one of claims 10-16, further comprising isolating the mogroside.

18. The method of any one of claims 10-16, wherein the method is carried out in recombinant cells expressing at least one polypeptide of any one of claims 1-9 or any combination thereof.

19. The method of claim 18, wherein the at least one polypeptide is selected from the polypeptide encoded by SEQ ID NO:33, the polypeptide shown in SEQ ID NO:38, and the polypeptide shown in SEQ ID NO:

6.

20. An isolated polynucleotide comprising a nucleic acid sequence encoding an isolated polypeptide of any one of claims 1-9.

21. The isolated polynucleotide of claim 20, wherein the nucleic acid sequence is selected from SEQ ID NO. 5, 33 and 37.

22. A nucleic acid construct comprising the isolated polynucleotide of claim 20 or 21 and a cis-regulatory element for directing the expression of said isolated polynucleotide.

23. The nucleic acid construct of claim 22, wherein the cis-regulatory element comprises a promoter.

24. A lysate of a host cell comprising the polynucleotide of claim 20 or 21 or the nucleic acid construct of claim 22 or 23, wherein the host cell is selected from yeast cells, bacteria, and plant cells, and wherein the nucleic acid sequence is not naturally expressed in the host cell.

25. The lysate of claim 24, wherein the plant cells are selected from the list comprising: *Acacia* spp., *Acer* spp., *Actinidia* spp., *Aesculus* spp., *Agathis australis*, *Albizia amara*, *Alsophila tricolor*, *Andropogon* spp., *Arachis* spp., *Areca catechu*, *Cinnamomum camphora*, *Astragalus cicer*, *Baikiaea plurijuga*, *Betula* spp., *Brassica* spp., *Bruguiera gymnorrhiza*, *Burkea africana*, *Butea* frondosa, Cadaba farinosa, Calliandra spp., Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Papaya, Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronavirus variant, Cotoneaster serotina, Crataegus spp., Cucumber spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp.), Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dioclea spp., Dolichos spp.Dorycnium rectum, Pyramidal Barnyardgrass (Echinochloa pyramidalis), Eleusine coracana, Erythrina spp., Eucalyptus, False Ebony, Velvety Golden Imperata, Buckwheat, Feijoa sellowlana, Fragaria spp., Flemingia spp., Riverbank Vine, Geranium thunbergii, Ginkgo (GinAgo biloba), Glycine javanica, Mexican Syringa, Gossypium hirsutum, Silver Jasmine, Guibourtia coleosperma, Hedysarum spp., Glutinous Grass, Yellow Imperata, Barley (Hordeum vulgare), Red Imperata, Hypericum erectum, indigofera, iris, leptarrhena pyrolifolia, lespedeza, capernia, leucaena leucocephala, loudetia simplex, lotus, macrotyloma axillare, magnolia, cassava, alfalfa, metasequoia glyptostroboides, banana, tobacco, onobrychis, ornithopus, oryza, peltophorum africanum, pennisetum, avocado, phaseolus spp.), Canary Island date palm (Phoenix canariensis), New Zealand sisal (Phormium cookianum), Photinia spp., White spruce (Picea glauca), Pinus spp., Pea, New Zealand Podocarpus totara, Pogonarthria fleckii, Populus spp.), Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Raspberry, Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Red lemma, Golden pine, Sequoia sempervirens, Sequoia dendron giganteum, Sorghum bicolor, Spinacia spp.), Spirobolus fimbriatus, dwarf styrax, Tadehagi spp., Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Wattonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, Brussels sprouts Sprouts), cabbage, rapeseed, carrots, cauliflower, celery, green kale, flax, loose-leaf kale, lentils, oilseed rape, okra, onions, potatoes, rice, soybeans, rice straw, beets, sugarcane, sunflowers, tomatoes, pumpkin tea, and trees.

26. A lysate of a host cell comprising the nucleic acid construct of claim 22 or 23, wherein the host cell is selected from yeast cells, bacteria, and plant cells, and wherein the nucleic acid sequence of the nucleic acid construct is not naturally expressed in the host cell.

27. The lysate of claim 25, wherein the host cell is a plant cell of the Cucurbitaceae family.

28. The lysate of any one of claims 24-27, wherein the host cell produces at least one mogroside glucosylated product or mogroside precursor glucosylated product of at least one uridine diphosphate-glucosyltransferase polypeptide of any one of claims 1-9 in the host cell.

29. The lysate of any one of claims 24-28, said lysate comprising at least one mogroside glucosylated product or mogroside precursor glucosylated product of at least one uridine diphosphate-glucosyltransferase polypeptide of any one of claims 1-9.

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