Production of frambinone by a recombinant fungal microorganism

• US 10,793,880 B2
• Filed: 06/02/2017
• Issued: 10/06/2020
• Est. Priority Date: 06/03/2016
• Status: Active Grant

• Current Assignee: Lesaffre Et Compagnie
• Original Assignee: Lesaffre Et Compagnie
• Inventors: Farwick, Alexander Matthias, Desfougeres, Thomas, Pignede, Georges, Roussel, Anais, Mouly, Isabelle, Morvan, Brieuc
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US16/302,527
• Publication Number:
  US 20190309330A1
• Time in Patent Office: 1,222 Days
• Field of Search: None
• CPC Class Codes: C07C 49/245 containing six-membered aromatic rings
• CPC Class Codes: C12N 1/16 Yeasts; Culture media therefor
• CPC Class Codes: C12N 1/185 Saccharomyces isolates
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12P 7/22 aromatic
• CPC Class Codes: C12P 7/26 Ketones
• CPC Class Codes: C12R 2001/865 Saccharomyces cerevisiae
• CPC Class Codes: C12Y 401/0108 4-Hydroxyphenylpyruvate decarboxylase (4.1.1.80)
• CPC Class Codes: C12Y 403/01023 Tyrosine ammonia-lyase (4.3.1.23)
• CPC Class Codes: C12Y 403/01024 Phenylalanine ammonia-lyase (4.3.1.24)
• CPC Class Codes: C12Y 602/01012 4-Coumarate-CoA ligase (6.2.1.12)

Abstract

The invention relates to a genetically modified fungal microorganism for the production of frambinone, the microorganism having the following characteristics: —the capacity to produce frambinone from tyrosine; and —a limited capacity or no capacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate; and to the use of same for producing frambinone.

Claims

1. A genetically modified fungal microorganism for the production of frambinone, said microorganism having the following characteristics:

a capacity to produce frambinone from tyrosine; and

a limited capacity or no capacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate,

wherein said genetically modified fungal microorganism has hydroxyphenyl pyruvate decarboxylase (HPPDC) activity less than or equal to 2×

10<sup>−

6</sup> kat per g of protein, and comprises at least one mutation or deletion in at least one of the genes encoding the following enzymes;

deaminase Aro8, deaminase Aro9, decarboxylase Aro10, decarboxylase Pdc5, decarboxylase Pdc6, or alcohol dehydrogenase (ADH).

2. The microorganism according to claim 1 characterized in that the microorganism belongs to the phyla ascomycetes or basidiomycetes.

3. The microorganism of claim 1 characterized in that the microorganism includes at least one heterologous sequence encoding the enzyme 4-coumarate:

CoA ligase (4CL) or benzalacetone synthase (BAS).

4. The microorganism according to claim 3 characterized in that the microorganism additionally comprises at least one heterologous or supernumerary sequence encoding the enzyme tyrosine ammonia lyase (TAL) or benzalacetone reductase (BAR).

5. The microorganism according to claim 3 characterized in that the microorganism additionally comprises at least one heterologous or supernumerary sequence encoding the enzyme phenylalanine ammonia lyase (PAL) or cinnamate 4 hydroxylase (C4H).

6. The microorganism according to claim 1 which is the strain Saccharomyces cerevisiae RK8, registered with the CNCM (National Collection of Microorganism Cultures, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on Apr. 26, 2017 under number I-5200.

7. The microorganism according to claim 1 characterized in that the microorganism belongs to the genus Yarrowia, Debaryomyces, Arxula, Scheffersomyces, Geotrichum, Pichia, or Saccharomyces.

8. The microorganism according to claim 1 characterized in that the microorganism is belongs to the species Yarrowia lipolytica, Debaryomyces hansenii, or Saccharomyces cerevisiae.

9. The microorganism of claim 3 characterized in that the microorganism includes at least one heterologous sequence encoding 4CL and BAS, at least one heterologous or supernumerary sequence encoding TAL and BAR, and/or at least one heterologous or supernumerary sequence encoding PAL or C4H.

10. The microorganism according to claim 1 characterized in that the microorganism comprises at least one mutation or deletion in at least one of the genes encoding Aro10, Pdc5, and/or Pdc6.

11. A process for the production of frambinone comprising culturing the fungal microorganism according to claim 1 in a medium comprising tyrosine.

12. A process for the production of frambinone comprising culturing the fungal microorganism according to claim 1 with a capacity to produce frambinone from tyrosine in a medium comprising tyrosine and a repressor of the path for breaking tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate.

13. The process for the production of frambinone according to claim 12 characterized in that the repressor is glutamate.

14. The process for the production of frambinone according to claim 12 characterized in that the microorganism

belongs to the phyla chosen from among the ascomycetes or basidiomycetes,

includes at least one heterologous sequence encoding the enzyme 4-coumarate:

CoA ligase (4CL) or benzalacetone synthase (BAS),

comprises at least one heterologous or supernumerary sequence encoding the enzyme tyrosine ammonia lyase (TAL) or benzalacetone reductase (BAR), and

comprises at least one heterologous or supernumerary sequence encoding the enzyme phenylalanine ammonia lyase (PAL) or cinnamate 4 hydroxylase (C4H).

15. The process for the production of frambinone according to claim 12 characterized in that the microorganism is the industrial strain Saccharomyces cerevisiae RK4 registered with the CNCM on Jun. 1, 2016 under number I-5101.

16. The process for the production of frambinone according to claim 12 characterized in that the microorganism is the industrial strain Saccharomyces cerevisiae RK5 registered with the CNCM on Apr. 26, 2017 under number I-5199.