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US 10,612,064 B2Granted
Production of steviol glycosides in recombinant hosts
AssigneeEvolva SA
InventorsHelene Lunde Robertson, Iben Nordmark Andersen, Adam Matthew Takos, Swee Chuang Lim Hallwyl, Francesca Ambri, Manuel Quiros Asensio, Michael Dalgaard Mikkelsen, Jens Houghton-Larsen, Veronique Douchin, Jane Dannow Dyekjaer, Simon Carlsen, Nina Nicoline Rasmussen, Esben Halkjaer Hansen
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Abstract
The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.
Claims19
A recombinant host that produces a steviol glycoside precursor and/or one or more steviol glycosides in a cell culture, comprising:
- (a) a gene encoding a polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene;
- wherein the polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:54;
- (b) a gene encoding a polypeptide that reduces cytochrome P450 complex;
- wherein the polypeptide that reduces cytochrome P450 complex comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:22;
- and
- (c) a gene encoding a polypeptide that synthesizes steviol from ent-kaurenoic acid;
- wherein the polypeptide that synthesizes steviol from ent-kaurenoic acid comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:82;
- and further comprising one or more of:
- (d) a gene encoding a polypeptide that synthesizes geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);
- wherein the polypeptide that synthesizes geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) comprises a polypeptide having at least 95 sequence identity to the amino acid sequence set forth in SEQ ID NO:49;
- (e) a gene encoding a polypeptide that synthesizes ent-copalyl diphosphate from GGPP;
- wherein the polypeptide that synthesizes ent-copalyl diphosphate from GGPP comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:37;
- or
- (f) a gene encoding a polypeptide that synthesizes ent-kaurene from ent-copalyl diphosphate;
- wherein the polypeptide that synthesizes ent-kaurene from ent-copalyl diphosphate comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:6;
- wherein at least one of the genes is a recombinant gene;
- and
- wherein the host is a fungal cell.
15 more elements
A recombinant host that produces a steviol glycoside precursor in a cell culture, comprising:
- (a) a gene encoding a polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene comprising a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:54;
- (b) a gene encoding a polypeptide that reduces cytochrome P450 complex comprising a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:22;
- and
- (c) a gene encoding a polypeptide that synthesizes steviol from ent-kaurenoic acid comprising a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:82;
- wherein at least one of the genes is a recombinant gene.
2 more elements
Dependencies are inferred from the claim text.