System for the production of aromatic molecules in Streptomyces

• US 8,344,119 B2
• Filed: 02/20/2008
• Issued: 01/01/2013
• Est. Priority Date: 02/21/2007
• Status: Active Grant

• Current Assignee: V. MANE FILS
• Original Assignee: V. MANE FILS
• Inventors: Lambert, Fanny, Zucca, Joseph, Mane, Jean
• Primary Examiner(s): Kosson, Rosanne
• Application Number:
  US12/528,000
• Publication Number:
  US 20100184172A1
• Time in Patent Office: 1,777 Days
• Field of Search: 536/23.2, 435/253.5, 435/147, 435/146, 435/156, 435/190
• US Class Current: 536/23.2
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/22 aromatic
• CPC Class Codes: C12P 7/24 containing a carbonyl group
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids

Abstract

A process for the production of natural ferulic acid, coniferyl alcohol and/or vanillin, includes the bio-conversion of eugenol by a bacteria belonging to the Streptomyces genes including at least one nucleotide sequence SEQ ID NO:1 or SEQ ID NO:8 or any nucleotide sequence having at least 70%, preferably 80% and very preferably 90%, identity with the sequence SEQ ID NO:1 or SEQ ID NO:8.

Claims

1. A method for producing natural ferulic acid, coniferyl alcohol, vanillin or mixtures thereof, comprising:

a) culturing a bacterium belonging to the genus Streptomyces in a fermentation medium comprising eugenol, said bacterium comprising the nucleotide sequence of SEQ ID NO;

1 or SEQ ID NO;

8, or a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO;

1 or SEQ ID NO;

8, wherein the polypeptide encoded by the polynucleotide has vanillyl alcohol oxidase activity; and

b) isolating the ferulic acid, coniferyl alcohol, vanillin or mixtures thereof produced in step (a).

2. The method according to claim 1, comprising:

adding eugenol, continuously or in a single dose, as a bioconversion substrate to the fermentation medium in a quantity between 5 g/l and 50 g/l; and

extracting vanillin from the fermentation medium.

3. The method according to claim 1, comprising:

adding eugenol, in a single dose, as a bioconversion substrate to the fermentation medium in a quantity between 5 g/l and 40 g/l; and

extracting coniferyl alcohol from the fermentation medium.

4. The method according to claim 1, comprising:

adding eugenol, continuously, as a bioconversion substrate to the fermentation medium in a quantity between 5 g/l and 30 g/l; and

extracting ferulic acid from the fermentation medium.

5. The method according to claim 1, wherein said bacterium is cultured in a fermentation medium having a pH less than 10.

6. The method according to claim 1, wherein said bacterium is cultured at a temperature between 27°

C. and 40°

C.

7. The method according to claim 1, wherein said bacterium is obtained by:

a) cloning the nucleotide sequence of SEQ ID NO;

1 or SEQ ID NO;

8 into an expression vector;

b) transforming the expression vector obtained in step (a) into a competent E. coli bacterium;

c) conjugating a bacterium belonging to the genus Streptomyces with the E. coli bacterium obtained in step (b); and

d) selecting Streptomyces cells comprising the vector of step (a) obtained in step (c) in the presence of eugenol.

8. The method according to claim 1, wherein the vanillin, ferulic acid and/or coniferyl alcohol is produced in the fermentation medium at a level of 10 to 25 g/l.

9. The method according to claim 1, wherein the ferulic acid is converted to vanillin in said bacterium belonging to genus Streptomyces by a biochemical or enzymatic route.

10. The method according to claim 1, wherein the bacterium comprises the nucleotide sequence of SEQ ID NO:

1 or SEQ ID NO;

8.

11. The method according to claim 7, wherein the expression vector is a bifunctional vector.

12. The method according to claim 1, wherein said bacterium is cultured at a temperature between 36°

C. and 37°

C.