Production of steviol glycosides in microorganisms
AssigneeEvolva SA
InventorsGanesh M. Kishore, Michael Motion, Paula M. Hicks, Jorgen Hansen, Jens Houghton-Larsen, Esben Halkjær Hansen, Michael Dalgaard Mikkelsen, Sabina Tavares, Charlotte Blom
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Abstract
Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements.
Claims40
A recombinant host comprising:
- a) a recombinant gene encoding a UGT91D2 polypeptide:
- b) a recombinant gene encoding a UGT85C polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:3 such as a UGT85C polypeptide comprising one or more amino acid substitutions at residues 9, 10, 13, 15, 21, 27, 60, 65, 71, 87, 91, 220, 243, 270, 289, 298, 334, 336, 350, 368, 389, 394, 397, 418, 420, 440, 441, 444, and 471 of SEQ ID NO:3;
- and/or
- c) a recombinant gene encoding a UGT76G1 polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:7, and having one or more amino acid substitutions at residues 29, 74, 87, 91, 116, 123, 125, 126, 130, 145, 192, 193, 194, 196, 198, 199, 200, 203, 204, 205, 206, 207, 208, 266, 273, 274, 284, 285, 291, 330, 331, and 346.
-12. (canceled)
-24. (canceled)
(canceled)
-34. (canceled)
-38. (canceled)
(canceled)
An isolated nucleic acid:
- a) encoding a polypeptide having at least 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:5, such as i) a polypeptide comprising at least one amino acid substitution at residues 1-19, 27-38, 44-87, 96-120, 125-141, 159-184, 199-202, 215-380, or 387-473 of SEQ ID NO:5;
- ii) a polypeptide comprising an amino acid substitution at one or more residues selected from the group consisting of residues 30, 93, 99, 122, 140, 142, 144, 148, 152, 153, 156, 195, 196, 199, 206, 207, 211, 213, 221, 286, 343, 364, 427, and 438 of SEQ ID NO:5;
- iii) a polypeptide comprising an arginine at residue 206, a cysteine at residue 207, and an arginine at residue 343 of SEQ ID NO:5;
- or iv) a polypeptide comprising a phenylalanine at residue 30, a glutamine at residue 93, a valine at residue 99, a phenylalanine at residue 122, a tyrosine at residue 140, a cysteine at residue 142, an isoleucine at residue 144, a threonine at residue 148, a leucine at residue 152, an alanine at residue 153, a serine at residue 156, a methionine at residue 195, a glutamic acid at residue 196, a glutamic acid at residue 199, a methionine at residue 211, a phenylalanine at residue 213, a phenylalanine at residue 221, an alanine at residue 286, a proline at residue 364, an asparagine at residue 427, or an alanine at residue 438 of SEQ ID NO:5;
- or
- b) encoding a polypeptide having greater than 90% sequence identity to the amino acid sequence of UGT91D2e or UGT91D2m, excluding the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14;
- or
- c) having greater than 90% sequence identity to one of the nucleotide sequences set forth in SEQ ID NOs:
- 18-25, 34-36, 4-43, 48, 49, 52-55, 60-64, 70-72, 77, or 79.
-47. (canceled)
An isolated polypeptide having an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:5 or an isolated polypeptide having greater than 90% sequence identity to the amino acid sequence of UGT91D2e or UGT91D2m, excluding the amino acid sequence of SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14.
A recombinant host, said host comprising:
- (i) a gene encoding a geranylgeranyl diphosphate synthase such as a geranylgeranyl diphosphate synthase having greater than 90% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs:
- 121-128;
- (ii) a gene encoding a bifunctional copalyl diphosphate synthase and kaurene synthase, or a gene encoding a copalyl diphosphate synthase such as a copalyl diphosphate synthase having greater than 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:
- 129-131 and a gene encoding a kaurene synthase such as a kaurene synthase having greater than 90% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs:
- 132-135;
- (iii) a gene encoding a kaurene oxidase such as a kaurene oxidase having greater than 90% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs:
- 138-141;
- and
- (iv) a gene encoding a steviol synthetase such as a steviol synthetase having greater than 90% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs:
- 142-146;
- wherein at least one of said genes is a recombinant gene.
-56. (canceled)
-62. (canceled)
-70. (canceled)
(canceled)
-75. (canceled)
A recombinant Saccharomyces strain comprising:
- (i) a gene encoding a bifunctional copalyl diphosphate synthase and kaurene synthase, or
- (ii) a gene encoding a copalyl diphosphate synthase and a gene encoding a kaurene synthase;
- wherein said strain produces ent-kaurene upon expression of said copalyl diphosphate synthase and said kaurene synthase.
-90. (canceled)
A recombinant host, said host comprising:
- (i) a recombinant gene encoding a UGT91D2;
- (ii) a recombinant gene encoding a UGT74G1;
- iii) a recombinant gene encoding a UGT85C2;
- (iv) a recombinant gene encoding a UGT76G1;
- and
- (v) optionally
- (a) a gene encoding a bifunctional copalyl diphosphate synthase and kaurene synthase, or a gene encoding a copalyl diphosphate synthase and a gene encoding a kaurene synthase;
- (b) a gene encoding a kaurene oxidase;
- (c) a gene encoding a steviol synthetase;
- and
- (d) a gene encoding a geranylgeranyl diphosphate synthase,
- wherein said host produces at least one steviol glycoside such as rebaudioside A, rebaudioside D or rebaudioside E when cultured under conditions in which each of said genes is expressed,
- wherein said host further can include a gene encoding a rhamnose synthetase, and produce at least one steviol glycoside such as rebaudioside C or dulcoside A when cultured under conditions in which each of said genes is expressed;
- or
- wherein said host further can include a gene encoding a UDP-glucose dehydrogenase;
- and a gene encoding a UDP-glucuronic acid decarboxylase, and produce at least one steviol glycoside such as rebaudioside F when cultured under conditions in which each of said genes is expressed.
-98. (canceled)
-104. (canceled)
A method for transferring a second sugar moiety to the C-2′ of a glucose in a steviol glycoside, said method comprising contacting said steviol glycoside with a 13-monoglucoside beta 1,2 glycosyltransferase or a 19 monoglucoside beta 1,2 glucosyltransferase and a UDP-sugar under suitable reaction conditions for the transfer of said second sugar moiety to said steviol glycoside, wherein the steviol glycoside optionally can be selected from the group consisting of steviol-13-O-glucoside, rubusoside, stevioside, and Rebaudioside A.
-111. (canceled)
-120. (canceled)
Dependencies are inferred from the claim text.