Methods and materials for biosynthesis of mogroside compounds

A recombinant host cell capable of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound in a cell culture, the recombinant host cell comprising:

• (a) a gene encoding 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 gene encoding 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 gene encoding 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 gene encoding 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 set forth in SEQ ID NOs:
• 38 or 40;
• (e) a gene encoding 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 gene encoding 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 set forth in SEQ ID NOs:38 or 40;
• (g) a gene encoding 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 gene encoding 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 gene encoding a polypeptide capable of catalyzing glycosylation of mogrol and/or glycosylated mogrol to produce a 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:
• 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 genes is a recombinant gene.