Methods of producing mogrosides and compositions comprising same and uses thereof

Inactive Publication Date: 2017-10-05
THE STATE OF ISRAEL MINIST OF AGRI & RURAL DEV AGRI RES ORG ARO VOLCANI CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0084]According to some embodiments of the present invention concentration of t

Problems solved by technology

Extraction of mogrosides from the fruit can yield a product of varying degrees of purity, often accompanied by undesirable aftertaste.
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  • Methods of producing mogrosides and compositions comprising same and uses thereof
  • Methods of producing mogrosides and compositions comprising same and uses thereof
  • Methods of producing mogrosides and compositions comprising same and uses thereof

Examples

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example 1

Temporal Pattern of Mogroside Accumulation

[0336]Mogroside accumulation during development of the Siraitia fruit is shown in FIGS. 4A and 4B. Targeted metabolic profiling of Siraitia mogrosides during fruit ripening was carried out on methanolic extracts of the frozen powders and analyzed by HPLC with photodiode array and mass spec detection. Results reveal their unique temporal distribution. Mogrosides were limited to the developing fruit and were not observed in the root, stem or leaf tissue.

[0337]Already in the youngest stage of immature fruit analyzed, at 15 DAA (Days After Anthesis), the majority of the mogrols were present in the di-glucosylated form in which the C-3 and C-24 mogrol carbons are each mono-glucosylated. Non-glucosylated, mono-glucosylated or alternative M2 compounds, in which the second glucosyl moiety was present as a branched glucose on one of the primary glucose moieties, were not observed, indicating that the initial metabolic steps of mogroside glucosylation...

example 2

Identification of Siraitia Cucurbitadienol Synthase (SgCDS) as the Enzyme which Cyclicizes Both 2,3-Monoepoxysqualene and 2,3;22,23-Diepoxysqualene, Leading to, Respectively, Cucurbitadienol and 24,25-Epoxycucurbitadienol

[0343]The preferred substrate for the synthesis of the novel trans-C24,C25-dihydroxycucurbitadienol is 2,3;22,23-diepoxysqualene which is symmetrically epoxidated at both ends of the squalene molecule at the squalene numbered positions of C2,3 and C22,23 (FIG. 3). 2,3;22,23-diepoxysqualene is synthesized by the enzyme squalene epoxidase (SQE) which is ubiquitous in squalene metabolizing organisms, including the yeast strain GIL77. The yeast strain GIL77 is one of the strains in which the yeast gene erg7 encoding lanosterol synthase is mutated and non-functional, thereby making available the 2,3-epoxysqualene precursor to the cucrbitadienol synthase cyclization reaction and allowing for the synthesis of cucurbitadienol. This has previously been shown for the Cucurbit...

example 3

Identification of S. Grosvenorii Epoxy Hydratase Enzymes Catalyzing the Hydration of 24,25-Epoxycucurbitadienol in Mogrol Biosynthesis

[0347]In order to identify candidate Siraitia epoxy hydratase genes that may be involved in mogrol biosynthesis a detailed transcriptome analysis of 6 stages of developing Siraitia fruit was performed. The fruit stages were 15, 34, 55, 77, 93 and 103 days after fruit set, and used for the productions of transcriptome and mogroside metabolome that are described above. Data mining of Siraitia transcriptome led to the identification and isolation of 4 candidate epoxy hydratase enzymes (contigs 73966, 86123, 102640 and 28382) with high levels of expression early in fruit development (FIGS. 7 and 21-24).

[0348]The epoxy hydratase genes were expressed in GIL77 yeast, and the products assayed for production of 24,25-dihydroxycucurbitadienol from 24,25-epoxycucurbitadienol, the product of the previously described SgCDS reaction. FIGS. 8A and 8B show the effect...

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Abstract

Isolated mogroside and mogrol biosynthetic pathway enzyme polypeptides useful in mogroside biosynthesis are provided. Mogroside biosynthetic pathway enzymes of the invention include squalene epoxidase (SE), expoxy hydratase (EH), cytochrome p450 (Cyp), cucurbitadienol synthase (CDS) and udp-glucosyl-transferase (UGT). Also provided are methods of producing a mogroside using the isolated mogroside and mogrol biosynthetic enzyme polypeptides, the methods comprising contacting a mogrol and/or a glycosylated mogrol (mogroside) with at least one UDP glucose glucosyl transferase (UGT) enzyme polypeptide of the invention catalyzing glucosylation of the mogrol and/or the glucosylated mogrol to produce a mogroside with an additional glucosyl moietie(s), thereby producing the mogroside. Alternatively or additionally provided is a method of synthesizing a mogrol, the method comprising contacting a mogrol precursor substrate with one or more mogrol biosynthetic pathway enzyme polypeptides as described herein catalyzing mogrol synthesis from the mogrol precursor substrate, thereby synthesizing the mogrol.

Description

FIELD AND BACKGROUND OF THE INVENTION[0001]The present invention, in some embodiments thereof, relates to methods of producing mogrosides and compositions comprising same and uses thereof.[0002]Mogrosides are triterpene-derived specialized secondary metabolites found in the fruit of the Cucurbitaceae family plant Siraitia grosvenorii (Luo Han Guo). Their biosynthesis in fruit involves number of consecutive glucosylations of the aglycone mogrol to the final sweet products mogroside V and mogroside VI (FIG. 1).[0003]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 grosvenorii: a review. ...

Claims

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Application Information

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IPC IPC(8): C12P33/12C12N9/02A23L27/30C12N9/88A23L2/60C07J17/00C12N9/90
CPCC12P33/12C07J17/005C12N9/0083C12N9/90C12N9/88A23V2002/00C12Y114/99007C12Y504/99033C12Y402/01A23L2/60A23L27/36C12N9/0071C12P19/56C12N9/14C12P33/20C12Y204/02017
Inventor ITKIN, MAXIMDAVIDOVICH-RIKANATI, RACHELCOHENPORTNOY, VITALYDORON-FAIGENBOIM, ADIPETREIKOV, MARINASHEN, SHMUELTADMOR, YAAKOVBURGER, YOSEFLEWINSOHN, EFRAIMKATZIR, NURITSCHAFFER, ARTHUR A.OREN, ELAD
Owner THE STATE OF ISRAEL MINIST OF AGRI & RURAL DEV AGRI RES ORG ARO VOLCANI CENT
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