UDP-glycosyltransferases

• US 10,947,515 B2
• Filed: 01/30/2020
• Issued: 03/16/2021
• Est. Priority Date: 03/16/2015
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Inventors: Boer, Viktor Marius, Van Leeuwen, Johannes Gustaaf Emst, Zwartjens, Priscilla, Kolen, Catharina Petronella Antonia Maria
• Primary Examiner(s): Ekstrom, Richard C
• Application Number:
  US16/777,427
• Publication Number:
  US 20200224180A1
• Time in Patent Office: 411 Days
• Field of Search: None
• CPC Class Codes: A23L 2/60 Sweeteners
• CPC Class Codes: A23L 27/36 Terpene glycosides
• CPC Class Codes: C12N 1/14 Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• CPC Class Codes: C12N 1/16 Yeasts; Culture media therefor
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/56 having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12Y 204/01017 Glucuronosyltransferase (2.4.1.17)

Abstract

The present invention relates to a recombinant host comprising a recombinant nucleic acid sequence encoding a polypeptide having at least about: a. 85% identity to the amino acid sequence set forth in SEQ ID NO: 1; b. 85% identity to the amino acid sequence set forth in SEQ ID NO: 3; c. 85% identity to the amino acid sequence set forth in SEQ ID NO: 6; d. 85% identity to the amino acid sequence set forth in SEQ ID NO: 9; e. 85% identity to the amino acid sequence set forth in SEQ ID NO: 11; f. 85% identity to the amino acid sequence set forth in SEQ ID NO: 14; g. 85% identity to the amino acid sequence set forth in SEQ ID NO: 17; h. 85% identity to the amino acid sequence set forth in SEQ ID NO: 20; i. 85% identity to the amino acid sequence set forth in SEQ ID NO: 22; or j. 85% identity to the amino acid sequence set forth in SEQ ID NO: 25.

Claims

1. A recombinant host comprising a recombinant nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

25.

2. The recombinant host of claim 1 which is capable of producing a glycosylated diterpene.

3. The recombinant host of claim 1, further 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.

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

5. The recombinant host of claim 1 which comprises a recombinant nucleic acid sequence encoding one or more of:

(i) a polypeptide having UGT74G1 (UGT3) activity;

(ii) a polypeptide having UGT85C2 (UGT1) activity; and

(iii) a polypeptide having UGT76G1 (UGT4) activity.

6. The recombinant host of claim 1 which comprises a recombinant nucleic acid sequence encoding an additional polypeptide having UGT2 activity.

7. 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.

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

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

10. The recombinant host of claim 1, comprising one or more recombinant nucleic acid sequence(s) encoding hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase and geranylgeranyl diphosphate synthase.

11. The recombinant host of claim 1 which comprises a nucleic acid sequence encoding one or more of:

a polypeptide having hydroxymethylglutaryl-CoA reductase activity;

a polypeptide having farnesyl-pyrophosphate synthetase activity; and

a polypeptide having geranylgeranyl diphosphate synthase activity.

12. A process for the preparation of a glycosylated diterpene which comprises fermenting the recombinant host of claim 2 in a suitable fermentation medium, and optionally recovering the glycosylated diterpene.

13. The process of claim 12 for the preparation of a glycosylated diterpene, wherein the process is carried out on an industrial scale.

14. A method for converting a first glycosylated diterpene into a second glycosylated diterpene, which method comprises:

contacting said first glycosylated diterpene with the recombinant host of claim 1, a cell free extract derived from such a recombinant host or an enzyme preparation derived from either thereof;

thereby to convert the first glycosylated diterpene into the second glycosylated diterpene.

15. The method of claim 14, wherein the second glycosylated diterpene is steviol-19-diside, steviolbioside, stevioside, RebE, RebD or 13-[(β

-D-glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β

-D-glucopyranosyl-β

-D-glucopyranosyl ester.

16. The method of claim 15, wherein the first glycosylated diterpene is steviol-13-monoside, steviol-19-monoside, rubusoside, stevioside, rebaudioside A or 13-[(β

-D-glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β

-D-glucopyranosyl-β

-D-glucopyranosyl ester and the second glycosylated diterpene is steviol-19-diside, steviolbioside, stevioside, RebE, RebD or 13-[(β

-D-glucopyranosyl)oxy)kaur-16-en-18-oic acid 2-O-β

-D-glucopyranosyl-β

-D-glucopyranosyl ester.

17. A nucleic acid construct comprising a polynucleotide sequence encoding a polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

25.

18. The nucleic acid construct of claim 17 which is an expression vector, wherein the polynucleotide sequence is operably linked to at least one control sequence for the expression of the polynucleotide sequence in a host cell.

19. A method of producing the nucleic acid construct of claim 17, comprising:

(a) cultivating a recombinant host cell comprising a recombinant nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO;

25 under conditions conducive to the production of the nucleic acid construct by the host cell, and optionally,

(b) recovering the nucleic acid construct.