Methods of producing mogrosides and compositions comprising same and uses thereof

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.

1 - 23 . (canceled) 
     
     
         24 . A method of synthesizing a mogrol or mogrol precursor product from a mogrol precursor substrate, the method comprising contacting at least one mogrol precursor substrate with a mogroside pathway enzyme, wherein:
 (a) when said mogrol precursor product comprises diepoxy squalene and said mogrol precursor substrate comprises squalene or oxidosqualene, said mogroside pathway enzyme comprises a squalene epoxidase polypeptide at least 94% identical to SEQ ID NO: 14 or 89% identical to SEQ ID NO: 16, wherein said polypeptide catalyzes diepoxysqualene synthesis from squalene or oxidosqualene, thereby producing diepoxy squalene,   (b) when said mogrol precursor product comprises 3 hydroxy, 24-25 epoxy cucurbitadienol and said mogrol precursor substrate comprises diepoxy squalene, said mogrol pathway enzyme comprises a cucurbitadienol synthetase polypeptide at least 60% homologous or identical to SEQ ID NO: 12, thereby producing a 3 hydroxy, 24-25 epoxy cucurbitadienol,   (c) when said product comprises 3, 24, 25 trihydroxy cucurbitadienol and said substrate comprises 3-hydroxy, 24-25 epoxy cucurbitadienol, the mogrol pathway enzyme comprises an epoxy hydratase polypeptide at least 75% identical to SEQ ID NO: 18, SEQ ID NO: 22 or SEQ ID NO: 24, said polypeptide catalyzing 3, 24, 25 trihydroxy cucurbitadienol synthesis from 3-hydroxy, 24-25 epoxy cucurbitadienol, thereby producing a 3, 24, 25 trihydroxy cucurbitadienol,   (d) when said product comprises mogrol and said mogrol precursor substrate comprises 3, 24, 25 trihydroxy cucurbitadienol, said mogrol pathway enzyme is Cytochrome P 450 enzyme at least 60% homologous or identical to SEQ ID NO: 10, thereby producing 3, 11, 24, 25 tetrahydroxy cucurbitadienol (mogrol).   
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 24 , wherein producing said mogrol product comprises at least one of:
 (i) contacting said squalene or oxido squalene with said squalene epoxidase enzyme polypeptide, thereby producing diepoxy squalene;   (ii) contacting said diepoxy squalene with a cucurbitadienol synthase, thereby producing 3 hydroxy, 24-25 epoxy cucurbitadienol;   (iii) contacting said 3 hydroxy, 24-25 epoxy cucurbitadienol with said epoxy hydratase enzyme, thereby producing 3, 24, 25 trihydroxy cucurbitadienol;   (iv) contacting said 3, 24-25 trihydroxy cucurbitadienol with said Cytochrome P 450 enzyme, thereby producing the mogrol product (3, 11, 24, 25 tetrahydroxy cucurbitadienol),   (i) and (iv),   (ii) and (iv),   (iii) and (iv),   (i), (ii) and (iii),   (i), (ii) and (iv),   (i), (iii) and (iv),   (ii), (iii) and (iv) and   all of (i), (ii), (iii) and (iv).   
     
     
         27 - 34 . (canceled) 
     
     
         35 . A method of synthesizing a mogroside, the method comprising contacting at least one UGT polypeptide selected from the group consisting of a UGT polypeptide at least 34% identical to SEQ ID NO: 34, which catalyzes (a) primary glucosylation of mogrol at C24; (b) primary glucosylation of mogroside at C3; and (c) branching glucosylation of mogroside at C3, a UGT polypeptide at least 89% identical to SEQ ID NO: 38 which catalyzes branching glucosylation of mogroside at the (1-2) and (1-6) positions of C3 and branching glucosylation of mogroside at the (1-2) and (1-6) positions of C24, and a UTG polypeptide at least 84% identical to SEQ ID NO: 6 which catalyzes branching glucosylation of mogroside IV (M4) to mogroside V (M5) or a combination thereof with at least one UGT substrate mogroside precursor. 
     
     
         36 - 42 . (canceled) 
     
     
         43 . The method of  claim 35 , wherein said UGT substrate mogroside precursor substrate is a mogrol, and optionally,
 wherein said mogroside is selected from the group consisting of mogroside I-A1, mogroside I-E1, mogroside IIE, mogroside III, siamenoside, mogroside V and mogroside VI.   
     
     
         44 - 45 . (canceled) 
     
     
         46 . The method of  claim 35 , being performed in a recombinant cell exogenously expressing at least one UGT polypeptide selected from the group consisting of a UGT polypeptide at least 34% identical to SEQ ID NO: 34, which catalyzes (a) primary glucosylation of mogrol at C24; (b) primary glucosylation of mogroside at C3; and (c) branching glucosylation of mogroside at C3, a UGT polypeptide at least 89% identical to SEQ ID NO: 38 which catalyzes branching glucosylation of mogroside at the (1-2) and (1-6) positions of C3 and branching glucosylation of mogroside at the (1-2) and (1-6) positions of C24, and a UTG polypeptide at least 84% identical to SEQ ID NO: 6 which catalyzes branching glucosylation of mogroside IV (M4) to mogroside V (M5) or any combination thereof. 
     
     
         47 . The method of  claim 46 , wherein said at least one polypeptide is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO; 22 and SEQ ID NO: 24. 
     
     
         48 . A composition comprising a mogroside generated according to the method of  claim 46 . 
     
     
         49 - 50 . (canceled) 
     
     
         51 . A nucleic acid construct comprising an isolated polynucleotide comprising a nucleic acid sequence encoding a UGT polypeptide selected from the group consisting of SEQ ID NOs. 5, 9, 11, 13, 15, 17, 21, 23, 33 and 37 and a cis-acting regulatory element for directing expression of the isolated polynucleotide. 
     
     
         52 . The nucleic acid construct of  claim 51 , wherein said cis-acting regulatory element comprises a promoter. 
     
     
         53 . A host cell comprising the nucleic acid construct of  claim 51 , heterologously expressing said isolated polynucleotide. 
     
     
         54 . The host cell of  claim 53 , being of a microorganism. 
     
     
         55 . The host cell of  claim 53 , wherein said host cell is selected from the group consisting of yeast, bacteria, and plant. 
     
     
         56 - 58 . (canceled) 
     
     
         59 . The host cell of  claim 55 , wherein said plant is of the Cucurbitaceae family. 
     
     
         60 . The host cell of  claim 55 , wherein said cell is a plant and said plant cell forms a part of a fruit or root of said plant. 
     
     
         61 . The host cell of  claim 53  producing a mogroside or mogroside precursor in the host cell. 
     
     
         62 . A cell lysate of the host cell of  claim 53 . 
     
     
         63 . A composition enriched in mogroside VI to a total concentration of mogroside VI of at least 10% (wt/wt). 
     
     
         64 . A composition comprising mogroside VI (M6) and at least one of mogro side II (M2) and mogroside V (M5). 
     
     
         65 . (canceled) 
     
     
         66 . The composition of  claim 64 , wherein a concentration of said mogroside VI or mogroside V is sufficient to cause an enhancement in flavor. 
     
     
         67 . The composition of  claim 66 , wherein a concentration of said mogroside VI is at least 0.2 ppm. 
     
     
         68 . The composition of  claim 66 , being a sweetener. 
     
     
         69 . The composition of  claim 68 , further comprising at least one flavor ingredient selected from the group consisting of sucrose, fructose, glucose, high fructose corn syrup, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharine, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), rubusoside, rebaudioside A, stevioside, stevia, trilobtain. 
     
     
         70 . The composition of  claim 66 , being a consumable composition. 
     
     
         71 . The composition of  claim 66 , further comprising one or more additional flavor ingredients. 
     
     
         72 . The composition of  claim 70 , being a beverage. 
     
     
         73 . (canceled) 
     
     
         74 . The composition of  claim 72 , being Coca-Cola® and the like. 
     
     
         75 . The composition of  claim 70 , being a solid consumable. 
     
     
         76 . (canceled) 
     
     
         77 . The composition of  claim 70 , being a foodstuff.