Production of steviol glycosides in recombinant hosts
First Claim
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1. 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;
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(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;
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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.
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Abstract
The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.
94 Citations
19 Claims
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1. A recombinant host that produces a steviol glycoside precursor and/or one or more steviol glycosides in a cell culture, comprising:
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(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. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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2. A recombinant host that produces a steviol glycoside precursor in a cell culture, comprising:
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(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. - View Dependent Claims (3)
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Specification