Methods and materials for Biosynthesis of Mogroside Compounds

A method for transferring a sugar moiety to a mogrol, a glycosylated mogroside compound, or both the mogrol and the glycosylated mogroside compound, comprising contacting the mogrol, the glycosylated mogroside compound, or both the mogrol and the glycosylated mogroside compound with one or more recombinant polypeptides capable of glycosylation at C3′-OH, C24′-OH, C3′-OH and C24′-OH of the mogrol, at C3′-OH or C24′-OH of the glycosylated mogroside compound, or both C3′-OH, C24′-OH, C3′-OH and C24′-OH of the mogrol and C3′-OH or C24′-OH of the glycosylated mogroside compound or beta-1,2-glycosylation of the C6′ position, and/or beta-1,6-glycosylation of the C2′ position of the 3-O-glucose and/or the 24-O-glucose of the glycosylated mogroside compound and one or more UDP-sugars, under suitable reaction conditions for the transfer of one or more sugar moieties from the one or more UDP-sugars to the mogrol, the glycosylated mogroside compound, or both the mogrol and the glycosylated mogroside compound;

• and
• wherein a mogroside compound glycosylated at C3′-OH, a mogroside compound glycosylated at C24′-OH, a mogroside compound glycosylated at C3′-OH and C24′-OH, a mogroside compound di-glycosylated at C24′ position, a mogroside compound tri-glycosylated at C24′ position, a mogroside compound glycosylated at C3′-OH and tri-glycosylated at C24′ position, a mogroside compound di-glycosylated at C3′-OH and tri-glycosylated at C24′ position, a mogroside compound di-glycosylated at C3′-OH and tri-glycosylated at C24′ position and oxidized at C11-OH, a mogroside compound di-glycosylated at C3′-OH and di-glycosylated at C24′ position, an isomer thereof, and/or a mogroside composition thereof is produced upon transfer of the sugar moiety.

A method of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound, comprising whole cell bioconversion of a plant-derived or a synthetic mogrol precursor or a mogroside precursor in a cell culture medium of a recombinant host cell using:

• (a) a polypeptide capable of catalyzing conversion of oxido-squalene to produce cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:1 or 43;
• (b) a polypeptide capable of catalyzing conversion of dioxido-squalene to produce 24,25 epoxy cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:1 or 43;
• (c) a polypeptide capable of catalyzing hydroxylation of cucurbitadienol to produce 11-hydroxy-cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO:44;
• (d) a polypeptide capable of catalyzing conversion of 11-hydroxy-cucurbitadienol to produce mogrol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NOs:38 or 40;
• (e) a polypeptide capable of catalyzing epoxidation of 11-hydroxy-cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO:44;
• (f) a polypeptide capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NOs:38 or 40;
• (g) a polypeptide capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO:44;
• (h) a polypeptide capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO:44;
• and/or
• (i) a polypeptide capable of glycosylation at C3′-OH, C24′-OH, C3′-OH and C24′-OH of the mogrol, at C3′-OH or C24′-OH of the glycosylated mogroside compound, or both C3′-OH, C24′-OH, C3′-OH and C24′-OH of the mogrol and C3′-OH or C24′-OH of the glycosylated mogroside compound or beta-1,6-glycosylation of the C2′ position of the 24-O-glucose, and/or beta-1,2-glycosylation of the C6′ position of the 3-O-glucose and/or the 24-O-glucose of the glycosylated mogroside compound;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:
• SEQ ID NOs:21-25, 48, 50, or 53 and/or a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by the nucleotide sequence set forth in any one of SEQ ID NOs:26-36;
• wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell;
• wherein the recombinant host cell is a microorganism that is a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell;
• and producing the mogrol precursor, the mogroside precursor, and/or the mogroside compound thereby.

An in vitro method of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound, comprising adding:

• (a) a polypeptide capable of catalyzing conversion of oxido-squalene to produce cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:1 or 43;
• (b) a polypeptide capable of catalyzing conversion of dioxido-squalene to produce 24,25 epoxy cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:1 or 43;
• (c) a polypeptide capable of catalyzing hydroxylation of cucurbitadienol to produce 11-hydroxy-cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO:44;
• (d) a polypeptide capable of catalyzing conversion of 11-hydroxy-cucurbitadienol to produce mogrol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NOs:38 or 40;
• (e) a polypeptide capable of catalyzing epoxidation of 11-hydroxy-cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO:44;
• (f) a polypeptide capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NOs:38 or 40;
• (g) a polypeptide capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO:44;
• (h) a polypeptide capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO:44;
• and/or
• (i) a polypeptide capable of glycosylation at C3′-OH, C24′-OH, C3′-OH and C24′-OH of the mogrol, at C3′-OH or C24′-OH of the glycosylated mogroside compound, or both C3′-OH, C24′-OH, C3′-OH and C24′-OH of the mogrol and C3′-OH or C24′-OH of the glycosylated mogroside compound or beta-1,6-glycosylation of the C2′ position of the 24-O-glucose, and/or beta-1,2-glycosylation of the C6′ position of the 3-O-glucose and/or the 24-O-glucose of the glycosylated mogroside compound;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:
• SEQ ID NOs:21-25, 48, 50, or 53 and/or a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by the nucleotide sequence set forth in any one of SEQ ID NOs:26-36;
• and a plant-derived or a synthetic mogrol precursor or a mogroside precursor to a reaction mixture;
• wherein at least one of the polypeptides is a recombinant polypeptide;
• and producing the mogrol precursor, the mogroside precursor, and/or the mogroside compound thereby.