Enzymes and genes used for producing vanillin

• US 20030092143A1
• Filed: 09/03/2002
• Published: 05/15/2003
• Est. Priority Date: 12/14/1999
• Status: Abandoned Application

• Current Assignee: Symrise Gmbh & Co. Kg (Symrise AG)
• Original Assignee: Symrise Gmbh & Co. Kg (Symrise AG)
• Inventors: Priefert, Horst, Achterholt, Sandra, Steinbuchel, Alexander, Rabenhorst, Jurgen
• Application Number:
  US10/149,485
• Publication Number:
  US 20030092143A1
• Time in Patent Office: Days
• US Class Current: 435/147
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 7/24 containing a carbonyl group

Abstract

Enzymes obtained from Amycolatopsis sp. HR167 (DSMZ 9992) can be used for synthesizing vanillin from ferulic acid. DNA which codes for these enzymes and host cells which are transformed using this DNA can be used for producing vanillin.

Claims

1. An enzyme from Amycolatopsis sp. for the synthesis of vanillin from ferulic acid.

2. The enzyme as claimed in claim 1, selected from the group of feruloyl-CoA synthetases or enoyl-CoA hydratase/aldolases.

3. The enzyme as claimed in claims 1 and 2, which exerts feruloyl-CoA synthetase activity and comprises an amino acid sequence which is at least 70% identical to a sequence according to SEQ ID NO:

2 over a distance of at least 20 consecutive amino acids.

4. The enzyme as claimed in claims 1 and 2, which exerts enoyl-CoA hydratase/aldolase activity and comprises an amino acid sequence which is at least 70% identical to a sequence according to SEQ ID NO:

3 over a distance of at least 20 consecutive amino acids.

5. A nucleic acid comprising a nucleotide sequence which codes for an enzyme as claimed in claims 1 to 4 and functional equivalents thereof.

6. The nucleic acid as claimed in claim 5, characterized in that it is single-stranded or double-stranded DNA or RNA.

7. The nucleic acid as claimed in claims 5 and 6, characterized in that it is fragments of genomic DNA or cDNA.

8. The nucleic acid as claimed in claims 5 to 7, characterized in that the nucleotide sequence corresponds to a sequence according to SEQ ID NO:

1 over a distance of at least 20 nucleotides of at least 70% identity.

9. A DNA construct comprising a nucleic acid as claimed in any of claims 5 to 8 and a heterologous promoter.

10. A vector comprising a nucleic acid as claimed in any of claims 5 to 8 or a DNA construct as claimed in claim 9.

11. A cosmid clone, comprising a nucleic acid as claimed in any of claims 5 to 9.

12. A host cell, comprising a nucleic acid as claimed in any of claims 5 to 8 or a DNA construct as claimed in claim 9 or 10.

13. The host cell as claimed in claim 12, characterized in that it is a prokaryotic cell.

14. The host cell as claimed in claim 13, characterized in that it is Escherichia coli.

15. The host cell as claimed in claim 12, characterized in that it is a eukaryotic cell.

16. The host cell as claimed in claim 15, characterized in that it is a unicellularly or filamentously growing fungus.

17. The host cell as claimed in claim 15, characterized in that it is a plant cell.

18. A method for preparing an enzyme as claimed in claims 1 to 4, characterized in, comprising

a) culturing a host cell as claimed in any of claims 12 to 17 under conditions which ensure expression of the nucleic acid as claimed in any of claims 5 to 7, or

b) expressing a nucleic acid as claimed in any of claims 5 to 11 in an in-vitro system, and

c) obtaining the enzyme from the cell, the culture medium or the in-vitro system.

19. A method for preparing feruloyl-coenzymeA from ferulic acid, characterized in that the reaction takes place in the presence of feruloyl-CoA synthetase.

20. A method for preparing 4-hydroxy-3-methoxyphenyl-β

-hydroxypropionyl-coenzymeA, characterized in that the reaction takes place in the presence of enoyl-CoA hydratase/aldolase.

21. A method for preparing vanillin from 4-hydroxy-3-methoxyphenyl-β

-hydroxypropionyl-coenzymeA, characterized in that the reaction takes place in the presence of enoyl-CoA hydratase/aldolase.