Steviol glycoside transport

Patent metadata

Field	Value
Publication number	US-12285033-B2
Application number	US-202418428486-A
Country	US
Kind code	B2
Filing date	Jan 31, 2024
Priority date	Aug 13, 2015
Publication date	Apr 29, 2025
Grant date	Apr 29, 2025

Abstract

A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least about 50% sequence identity thereto. A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least about 50% sequence identity thereto.

First claim

What is claimed is:

1. A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29 or an amino acid sequence having at least about 95% sequence identity thereto, wherein the recombinant host comprises one or more recombinant nucleic acid sequence encoding:
(i) a polypeptide having UDP-glycosyltransferase 74G1 (UGT74G1) activity;
(ii) a polypeptide having UDP-glycosyltransferase 2 (UGT2) activity;
(iii) a polypeptide having UDP-glycosyltransferase 85C2 (UGT85C2) activity; and
(iv) a polypeptide having UDP-glycosyltransferase 76G1 (UGT76G1) activity.

2. A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29 or an amino acid sequence having at least about 95% sequence identity thereto, wherein the recombinant host comprises one or more recombinant nucleic acid sequence encoding:
(i) a polypeptide having UDP-glycosyltransferase 74G1 (UGT74G1) activity;
(ii) a polypeptide having UDP-glycosyltransferase 2 (UGT2) activity;
(iii) a polypeptide having UDP-glycosyltransferase 85C2 (UGT85C2) activity;
(iv) a polypeptide having UDP-glycosyltransferase 76G1 (UGT76G1) activity;
(v) a polypeptide having ent-copalyl pyrophosphate synthase activity;
(vi) a polypeptide having ent-Kaurene synthase activity;
(vii) a polypeptide having ent-Kaurene oxidase activity;
(viii) a polypeptide having kaurenoic acid 13-hydroxylase activity.

3. The recombinant host of claim 1 , comprising a recombinant nucleic acid which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:29 or an amino acid sequence having at least about 95% sequence identity thereto.

4. The recombinant host of claim 1 , comprising one or more recombinant nucleotide sequence(s) encoding:
a polypeptide having ent-copalyl pyrophosphate synthase activity;
a polypeptide having ent-Kaurene synthase activity;
a polypeptide having ent-Kaurene oxidase activity; and
a polypeptide having kaurenoic acid 13 -hydroxylase activity.

5. The recombinant host of claim 1 , comprising a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity.

6. The recombinant host of claim 1 , wherein the host belongs to one of the genera Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces, Pachysolen, Yarrowia, Yamadazyma or Escherichia.

7. The recombinant host of claim 5 , wherein the recombinant host is a Saccharomyces cerevisiae cell, a Yarrowia lipolytica cell, a Candida krusei cell, or an Escherichia coli cell.

8. The recombinant host of claim 1 , wherein the ability of the host to produce geranylgeranyl diphosphate (GGPP) is upregulated.

9. The recombinant host of claim 1 , comprising a nucleic acid sequence encoding one or more of:
a polypeptide having hydroxymethylglutaryl-CoA reductase activity; or
a polypeptide having farnesyl-pyrophosphate synthetase activity.

10. A process for the preparation of a steviol glycoside which comprises fermenting the recombinant host of claim 1 in a suitable fermentation medium and, optionally, recovering the steviol glycoside.

11. The process of claim 10 , wherein the process is carried out on an industrial scale.

12. A fermentation broth comprising the recombinant host of claim 1 .

13. The fermentation broth of claim 12 , further comprising a steviol glycoside obtainable by fermenting, in a suitable fermentation medium, a recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29 or an amino acid sequence having at least about 95% sequence identity thereto, wherein the recombinant host comprises one or more recombinant nucleic acid sequence encoding:
(i) a polypeptide having UGT74G1 activity;
(ii) a polypeptide having UGT2 activity;
(iii) a polypeptide having UGT85C2 activity; and
(iv) a polypeptide having UGT76G1 activity.

Assignees

• Dsm Ip Assets Bv

Inventors

• Boer Viktor Marius
• Zwartjens Priscilla
• Van Den Berg Eric

Classifications

• C12N9/2402

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C12N1/16

  Yeasts; Culture media therefor · CPC title

• C07K14/40

  from Candida · CPC title

• C07K14/395

  from Saccharomyces · CPC title

• C07K14/39 Primary

  from yeasts · CPC title