Methods and Materials for Biosynthesis of Mogroside Compounds
AssigneeEvolva SA
InventorsJens Houghton-Larsen, Katarzyna Krzystanek, Angelika Semmler, Iver Klavs Riishede Hansen, Soren Damkiaer, Gary Liu, Yaoquan Liu, Jorgen Hansen, Sathish Kumar, Muthuswamy Panchapagesa Murali, Nina Nicoline Rasmussen
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Abstract
Methods for recombinant and enzymatic production of mogroside compounds and compositions containing mogroside compounds are provided by this invention.
Claims32
A recombinant host comprising one or more of:
- (a) a gene encoding a squalene epoxidase polypeptide;
- (b) a gene encoding a cucurbitadienol synthase polypeptide;
- (c) a gene encoding a cytochrome P450 polypeptide;
- (d) a gene encoding a cytochrome P450 reductase polypeptide;
- (e) a gene encoding an epoxide hydrolase polypeptide;
- (f) a gene encoding a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48;
- (g) a gene encoding a UGT430 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62;
- (h) a gene encoding a UGT1697 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68;
- (i) a gene encoding a UGT11789 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72;
- (j) a gene encoding a UGT98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53;
- (k) a gene encoding a UGTSK98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50;
- wherein at least one of the genes is a recombinant gene;
- wherein the host is capable of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound.
A recombinant host comprising one or more of:
- (a) one or more genes encoding one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienol;
- (b) one or more genes encoding one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadienol;
- (c) one or more genes encoding one or more enzymes capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
- (d) one or more genes encoding one or more enzymes capable of catalyzing hydroxylation of cucurbitadienol to produce 11-hydroxy-cucurbitadienol;
- (e) one or more genes encoding one or more enzymes capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadienol;
- or
- (f) one or more genes encoding one or more enzymes capable of catalyzing epoxidation of 11-hydroxy-cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
- (g) one or more genes encoding one or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol;
- or
- (h) one or more genes encoding one or more enzymes capable of catalyzing glycosylation of a mogroside precursor to produce a mogroside compound;
- wherein at least one of the genes is a recombinant gene.
A method of producing a mogrol precursor in vitro, comprising:
- (a) contacting dioxidosqualene with one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienol;
- or
- (b) contacting oxidosqualene with one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadienol;
- or
- (c) contacting 24,25 epoxy cucurbitadienol with one or more enzymes capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
- or
- (d) contacting cucurbitadienol with one or more enzymes capable of catalyzing hydroxylation of cucurbitadienol to produce 11-hydroxy-cucurbitadienol;
- or
- (e) contacting cucurbitadienol with one or more enzymes capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadienol;
- or
- (f) contacting 11-hydroxy-cucurbitadienol with one or more enzymes capable of catalyzing epoxidation of 11-hydroxy-cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol.
A method of producing a mogrol in vitro, comprising contacting 11-hydroxy-24,25 epoxy cucurbitadienol with one or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol.
A method of producing a mogroside compound in vitro, comprising contacting a mogroside precursor with one or more enzymes capable of catalyzing glycosylation of the mogroside precursor to produce a mogroside compound.
A method of producing a mogroside compound, comprising contacting a recombinant host expressing one or more of:
- (a) a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48;
- (b) a UGT430 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62;
- (c) a UGT1697 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68;
- (d) a UGT11789 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72;
- (e) a UGT98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53;
- or
- (f) a UGTSK98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50 with a mogroside precursor.
The bacterial cell according to 22, wherein the bacterial cell comprises Escherichia bacteria cells, Lactobacillus bacteria cells, Lactococcus bacteria cells, Cornebacterium bacteria cells, Acetobacter bacteria cells, Acinetobacter bacteria cells, or Pseudomonas bacterial cells.
Dependencies are inferred from the claim text.