Selectivity of the production of vanilloids in a recombinant unicellular host

1. A method for producing a substantially pure vanilloid of formula (I):

R 1 being selected from the group consisting of —CHO; —COOH; —COOR 3 ; —CH 2 OH; —CH 2 COOH; —C(═O)CH 3 ; —CR 3 (OH)COOH; —CHR 3 COOH; —CH 2 NHC(═O)R 3 ; wherein R 3 is a lower alkyl,

R 2 being different from a methyl (—CH 3 ),

comprising the steps of:

a) providing a recombinant unicellular host capable of producing said vanilloid, wherein said recombinant host expresses at least a nucleic acid coding for a 3-dehydroshikimate dehydratase (3DSD) and at least a nucleic acid coding for an aromatic carboxylic acid reductase (ACAR);

b) cultivating said host in a suitable medium; and

c) recovering the produced vanilloid from said host or from the culture supernatant thereof,

wherein said recombinant host expresses at least a nucleic acid coding for a caffeic acid 3-O-methyltransferase polypeptide that is suitable for methylating selectively the 3-OH of a protocatechuic aldehyde.

2. The method according to claim 1 , wherein said recombinant host expresses at least a nucleic acid coding for a caffeic acid 3-O-methyltransferase polypeptide from Medicago sativa, Rosa chinensis , and/or Vanilla planifolia.

3. The method according to claim 1 , wherein in said vanilloid of formula (I), R 2 is H.

4. The method according to claim 1 , wherein in said vanilloid of formula (I), R 1 is —CHO.

5. The method according to claim 1 , wherein the caffeic acid 3-O-methyltransferase polypeptide comprises a sequence selected from SEQ ID NO: 1, 2 or 3.

6. The method according to claim 1 , wherein said recombinant host expresses at least a nucleic acid coding for a phosphopantetheinyl transferase (PPTase).

7. The method according to claim 1 , wherein said recombinant host is a recombinant unicellular microorganism selected from a bacterium, an archaeon, a yeast, a protozoon, an alga, and a fungus.

8. The method according to claim 7 , wherein said recombinant host is selected from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

9. The method according to claim 8 , wherein said recombinant host is Saccharomyces cerevisiae and expresses at least a nucleic acid encoding a 3-dehydroshikimate dehydratase (3DSD), at least a nucleic acid encoding an aromatic carboxylic acid reductase (ACAR), and at least a nucleic acid encoding a phosphopantetheinyl transferase (PPTase).

10. The method according to claim 1 , wherein said recombinant host does not express a functional alcohol dehydrogenase ADH6.

11. The method according to claim 1 , wherein said suitable medium comprises at least one compound selected from glucose, galactose, fructose, arabinose, lactose, mannose, erythrose-4-phosphate, dehydroshikimic acid, catechol, protocatechuic acid, protocatechuic aldehyde, ethanol, glycerol and derivatives thereof.

12. The method according to claim 1 , wherein said suitable medium does not comprise aromatic amino acids.

13. A yeast suitable for producing a substantially pure vanilloid of formula (I):

R 1 being selected from the group consisting of —CHO; —COOH; —COOR 3 ; —CH 2 OH; —CH 2 COOH; —C(═O)CH 3 ; —CR 3 (OH)COOH; —CHR 3 COOH; —CH 2 NHC(═O)R 3 ; wherein R 3 is a lower alkyl,

R 2 being different from a methyl (—CH 3 ),

expressing at least a nucleic acid coding for an aromatic carboxylic acid reductase (ACAR) and a 3-dehydroshikimate dehydratase (3DSD), wherein said yeast expresses at least a nucleic acid coding for a caffeic acid 3-O-methyltransferase polypeptide that is suitable for methylating selectively the 3-OH of a protocatechuic aldehyde.

14. A method for converting a protocatechuic aldehyde into a substantially pure vanilloid of formula (I):

R 1 being selected from the group consisting of —CHO; —COOH; —COOR 3 ; —CH 2 OH; —CH 2 COOH; —C(═O)CH 3 ; —CR 3 (OH)COOH; —CHR 3 COOH; —CH 2 NHC(═O)R 3 ; wherein R 3 is a lower alkyl,

R 2 being different from a methyl (—CH 3 ),

and comprising the steps of:

a) providing a recombinant unicellular host capable of producing said vanilloid, wherein said recombinant host expresses at least a nucleic acid coding for an aromatic carboxylic acid reductase (ACAR) and does not express nucleic acids coding for the following enzymes: a phenylalanine ammonia lyase (PAL) or a tyrosine ammonia lyase (TAL) or a phenylalanine/tyrosine ammonia lyase (PAL/TAL), a coA ligase, and a crotonase;

b) cultivating said host in a suitable medium; and

c) recovering the produced vanilloid from said host or from the culture supernatant thereof,

wherein said recombinant host expresses at least a nucleic acid coding for a caffeic acid 3-O-methyltransferase polypeptide that is suitable for methylating selectively the 3-OH of a protocatechuic aldehyde.

15. A method for converting a protocatechuic aldehyde into a substantially pure vanilloid of formula (I):

R 1 being selected from the group consisting of —CHO; —COOH; —COOR 3 ; —CH 2 OH; —CH 2 COOH; —C(═O)CH 3 ; —CR 3 (OH)COOH; —CHR 3 COOH; —CH 2 NHC(═O)R 3 ; wherein R 3 is a lower alkyl,

R 2 being different from a methyl (—CH 3 ),

and comprising the steps of:

a) providing a recombinant unicellular host capable of producing said vanilloid, wherein said recombinant host expresses at least a nucleic acid coding for an aromatic carboxylic acid reductase (ACAR) and at least one nucleic acid coding for a 3-dehydroshikimate dehydratase (3DSD);

b) cultivating said host in a suitable medium; and

c) recovering the produced vanilloid from said host or from the culture supernatant thereof,

wherein said recombinant host expresses at least a nucleic acid coding for a caffeic acid 3-O-methyltransferase polypeptide that is suitable for methylating selectively the 3-OH of a protocatechuic aldehyde.

16. A yeast suitable for converting a protocatechuic aldehyde into a substantially pure vanilloid of formula (I):

R 1 being selected from the group consisting of —CHO; —COOH; —COOR 3 ; —CH 2 OH; —CH 2 COOH; —C(═O)CH 3 ; —CR 3 (OH)COOH; —CHR 3 COOH; —CH 2 NHC(═O)R 3 ; wherein R 3 is a lower alkyl,

R 2 being different from a methyl (—CH 3 ),

expressing at least a nucleic acid coding for an aromatic carboxylic acid reductase (ACAR), wherein said yeast expresses at least a nucleic acid coding for a caffeic acid 3-O-methyltransferase polypeptide that is suitable for methylating selectively the 3-OH of a protocatechuic aldehyde, and does not express nucleic acids coding for the following enzymes: a phenylalanine ammonia lyase (PAL) or a tyrosine ammonia lyase (TAL) or a phenylalanine/tyrosine ammonia lyase (PAL/TAL), a coA ligase, and a crotonase.

17. A yeast suitable for converting a protocatechuic aldehyde into a substantially pure vanilloid of formula (I):

R 1 being selected from the group consisting of —CHO; —COOH; —COOR 3 ; —CH 2 OH; —CH 2 COOH; —C(═O)CH 3 ; —CR 3 (OH)COOH; —CHR 3 COOH; —CH 2 NHC(═O)R 3 ; wherein R 3 is a lower alkyl,

R 2 being different from a methyl (—CH 3 ),

expressing at least a nucleic acid coding for an aromatic carboxylic acid reductase (ACAR) and at least a nucleic acid coding for a 3-dehydroshikimate dehydratase (3DSD), wherein said yeast expresses at least a nucleic acid coding for a caffeic acid 3-O-methyltransferase polypeptide that is suitable for methylating selectively the 3-OH of a protocatechuic aldehyde.

18. A composition comprising a vanilloid obtained by the method according to claim 1 .

19. A method for providing a flavor or a fragrance to a composition, comprising a step of producing a substantially pure vanilloid in said composition by the method according to claim 1 .

20. A composition comprising a vanilloid obtained by the method according to claim 14 .

21. A composition comprising a vanilloid obtained by the method according to claim 15 .

22. A method for providing a flavor or a fragrance, comprising a step of producing a composition comprising a vanilloid obtained by the method according to claim 14 .

23. A method for providing a flavor or a fragrance, comprising a step of producing a composition comprising a vanilloid obtained by the method according to claim 15 .