Use of octaketide synthases to produce kermesic acid and flavokermesic acid

Patent metadata

Field	Value
Publication number	US-10415066-B2
Application number	US-201615735051-A
Country	US
Kind code	B2
Filing date	Jun 10, 2016
Priority date	Jun 10, 2015
Publication date	Sep 17, 2019
Grant date	Sep 17, 2019

Abstract

A method for producing an octaketide derived aromatic compound of interest (e.g. carminic acid), wherein the method comprises (I): heterologous expression of a recombinantly introduced Type III polyketide synthase (PKS) gene encoding an octaketide synthase (OKS) to obtain non-reduced octaketide in vivo within the recombinant host cell and (II): converting in vivo the non-reduced octaketide of step (I) into a C14-C34 aromatic compound of interest (e.g. carminic acid).

First claim

The invention claimed is:

1. A method for producing an octaketide derived aromatic compound, wherein the method comprises:
(I): contacting in vivo in a recombinant host cell comprising a recombinantly introduced Type III polyketide synthase (PKS) gene encoding an octaketide synthase (OKS), wherein the OKS is of a different genus than the host cell:
(i): a starter unit and an extender unit with said OKS such that the starter and extender units convert into a non-reduced octaketide;

(II): converting in vivo within the recombinant host cell the non-reduced octaketide of step (I) into a C 14 -C 34 aromatic aglycon compound via at least one in trans acting aromatase/cyclase, wherein the aromatic aglycon compound is not SEK4 and/or SEK4B;
(III): the recombinant host cell further comprises a glycosyltransferase gene encoding a glycosyltransferase having at least 90% sequence identity to SEQ ID NO: 2 or amino acids 1 to 468 of SEQ ID NO:2, which glycosylates the aromatic aglycon compound produced in step (II) into a C 14 -C 34 aromatic glycoside compound; and
(IV): isolating the aromatic glycoside compound of step (III) so as to get a composition, wherein the composition comprises less than 1% w/w dry matter of recombinant host cell material, and wherein the recombinant host cell is a plant cell or a filamentous fungal cell.

2. The method of claim 1 , wherein the recombinant host cell is a growing recombinant host cell and step (I) and step (II) comprise:
(I): contacting in vivo in a growing recombinant host cell comprising a recombinantly introduced Type III polyketide synthase (PKS) gene encoding an octaketide synthase (OKS), wherein the OKS is of a different genus than the host cell:
(i): a starter unit and an extender unit with said OKS such that the starter and extender units convert into a non-reduced octaketide; and

(II): converting in vivo within the growing recombinant host cell the non-reduced octaketide of step (I) into a C 14 -C 34 aromatic aglycon compound, wherein the aromatic aglycon compound is not SEK4 and/or SEK4B.

3. The method according to claim 1 , wherein the plant cell is Nicotiana sp.

4. The method according to claim 1 , wherein the plant cell is an Aspergillus cell.

5. The method according to claim 1 , wherein the Type III polyketide synthase (PKS) gene of step (I) is a PKS gene from a plant and wherein the plant is a plant selected from the group consisting of: Aloe spp. and Hypericum spp.

6. The method of claim 5 , wherein the octaketide synthase (OKS) in step (I) is a OKS comprising an amino acid sequence, which has at least 70% sequence identity with amino acids 1 to 403 of SEQ ID NO:7 (AaOKS).

7. The method of claim 5 , wherein the octaketide synthase (OKS) in step (I) is a OKS comprising an amino acid sequence, which has at least 70% identity with amino acids 1 to 393 of SEQ ID NO:15 (HpPKS2).

8. The method according to claim 1 , wherein the starter unit is acetyl-CoA and/or malonyl-CoA.

9. The method according to claim 1 , wherein the:
recombinant host cell is a host cell selected from the group consisting of: Aspergillus and Nicotiana sp.; and
the OKS of a different genus than the host cell is an OKS selected from the group consisting of: OKS from Aloe spp.) and Hypericum spp.

10. The method according to claim 1 , wherein the aromatic aglycon compound is a C 16 aromatic aglycon compound.

11. The method according to claim 10 , wherein the C 16 aromatic aglycon compound is flavokermesic acid (FK) or kermesic acid (KA).

12. The method of claim 1 , wherein the aromatase/cyclase is a aromatase/cyclase comprising an amino acid sequence, which has at least 70% sequence identity with amino acids 1 to 169 of SEQ ID NO:17 ( Streptomyces ZhuI); and/or wherein the aromatase/cyclase is a aromatase/cyclase comprising an amino acid sequence, which has at least 70% sequence identity with amino acids 1 to 256 of SEQ ID NO:19 ( Streptomyces ZhuJ).

13. The method of claim 1 , wherein the aromatic glycoside compound of interest is a C 16 aromatic glycoside compound and wherein the C 16 aromatic glycoside compound is a flavokermesic acid glycoside or a kermesic acid glycoside.

14. The method of claim 13 , wherein the C 16 aromatic glycoside compound is a flavokermesic acid glycoside and the flavokermesic acid glycoside is Dell.

15. The method of claim 13 , wherein the C 16 aromatic glycoside compound is a kermesic acid glycoside and the kermesic acid glycoside is carminic acid (CA).

Assignees

• Univ Danmarks Tekniske
• Univ Copenhagen

Inventors

• Møller Birger Lindberg
• Madsen Bjørn
• Stærk Dan
• Okkels Finn Thyge
• Andersen-Ranberg Johan
• Kongstad Kenneth Thermann
• Binderup Kim
• Bennedsen Mads
• Nafisi Majse
• Khorsand-Jamal Paiman
• Kannangara Rubini Maya
• Mortensen Uffe Hasbro
• Thrane Ulf
• Frandsen Rasmus John Normand

Classifications

• C12P7/26

  Ketones · CPC title

• C12P17/06

  containing a six-membered hetero ring, e.g. fluorescein · CPC title

• C12P7/66 Primary

  containing the quinoid structure · CPC title

• C12N9/1048

  Glycosyltransferases (2.4) · CPC title

• C12N9/93

  Ligases (6) · CPC title