Vanillin synthase

Patent metadata

Field	Value
Publication number	US-10689672-B2
Application number	US-201314438200-A
Country	US
Kind code	B2
Filing date	Nov 5, 2013
Priority date	Nov 5, 2012
Publication date	Jun 23, 2020
Grant date	Jun 23, 2020

Abstract

The invention relates to methods for producing vanillin and related compounds. The methods involve use of a vanillin synthase capable of catalyzing side chain cleavage of ferulic acid to form vanillin. The invention also relates to host organisms expressing such vanillin synthases useful in the methods.

First claim

The invention claimed is:

1. A method of producing vanillin, vanillyl alcohol, vanillin glucoside, and/or vanillyl alcohol glucoside, the method comprising the steps of:
(a) providing a microbial organism that comprises a heterologous nucleic acid encoding a vanillin synthase, wherein the vanillin synthase is capable of catalyzing conversion of ferulic acid or a ferulic acid derivative to form vanillin, vanillyl alcohol, vanillin glucoside, and/or vanillyl alcohol glucoside;
(b) cultivating the microbial organism in the presence of ferulic acid and/or a ferulic acid derivative in culture medium supporting growth of the microbial organism; and
(c) isolating vanillin, vanillyl alcohol, vanillyl alcohol glucoside, and/or vanillin glucoside from the microbial organism and/or from the culture medium;
wherein the vanillin synthase capable of catalyzing the conversion of ferulic acid or a ferulic acid derivative to form vanillin comprises the amino acid sequence of SEQ ID NO: 1 or a functional homolog thereof,
wherein the functional homolog thereof comprises:
(i) an amino acid sequence with at least 99% identity to SEQ ID NO: 1;
(ii) an amino acid sequence with at least 99% identity to SEQ ID NO: 17;
(iii) an amino acid sequence with at least 99% identity to amino acids 62 to 356 of SEQ ID NO: 1;
(iv) an amino acid sequence with at least 98% identity to amino acids 138 to 356 of SEQ ID NO: 1;
(v) an amino acid sequence with at least 98% identity to SEQ ID NO: 21; or
(vi) an amino acid sequence with at least 93% identity to the sequence encoded by SEQ ID NO: 24.

2. The method of claim 1 , wherein step (b) comprises cultivating the microbial organism in the presence of ferulic acid.

3. The method of claim 2 , wherein the culture medium comprises at least 1 mM ferulic acid.

4. The method of claim 1 , wherein step (b) comprises cultivating the microbial organism in the presence of a ferulic acid derivative.

5. The method of claim 4 , wherein the ferulic acid derivative is ferulic acid glucose ester, ferulic acid glucoside, feruloyl-coA, ferulic acid shikimate, or feruloyl-quinate.

6. The method of claim 4 , wherein the ferulic acid derivative is ferulic acid glucoside.

7. The method of claim 6 , wherein the culture medium comprises at least 1 mM ferulic acid glucoside.

8. The method of claim 6 , wherein step (c) comprises isolating vanillin glucoside.

9. The method of claim 1 , further comprising deglucosylating the vanillin glucoside.

10. The method of claim 9 , wherein deglucosylating vanillin glucoside comprises contacting the vanillin glucoside with a glucosidase.

11. The method of claim 10 , wherein the glucosidase is a beta-glucosidase.

12. The method of claim 1 , wherein the medium comprises molasses.

13. The method of claim 1 , wherein the medium comprises eugenol.

14. The method of claim 1 , wherein the microbial organism comprises reduced, disrupted, or eliminated ferulic acid decarboxylase (FADase) activity.

15. The method of claim 14 , wherein an endogenous phenylacrylic acid decarboxylase (PAD1) or ferulic acid decarboxylase (FDC1) gene in the microbial organism is reduced, inactivated, disrupted, or deleted, in full or in part.

16. The method of claim 14 , wherein the expression of an endogenous PAD1 or FDC1 gene, or the expression of a protein encoded by an endogenous PAD1 or FDC1 gene in the microbial organism is reduced, disrupted, or eliminated, in full or in part.

17. The method of claim 14 , wherein the activity of a protein encoded by an endogenous PAD1 or FDC1 gene in the microbial organism is reduced, disrupted, or eliminated, in full or in part.

18. The method of claim 1 , wherein the vanillin synthase capable of catalyzing conversion of ferulic acid or a ferulic acid derivative to form vanillin comprises the amino acid sequence of SEQ ID NO: 21.

19. The method of claim 1 , wherein the vanillin synthase capable of catalyzing the conversion of ferulic acid or a ferulic acid derivative to form vanillin comprises-amino acids 138 to 356 of SEQ ID NO: 1.

20. The method of claim 19 , wherein the vanillin synthase capable of catalyzing the conversion of ferulic acid or a ferulic acid derivative to form vanillin further comprises amino acids 62 to 137 of SEQ ID NO: 1.

21. The method of claim 20 , wherein the vanillin synthase capable of catalyzing the conversion of ferulic acid or a ferulic acid derivative to form vanillin further comprises amino acids 22 to 61 of SEQ ID NO: 1.

22. The method of claim 1 , wherein the vanillin synthase capable of catalyzing the conversion of ferulic acid or a ferulic acid derivative to form vanillin comprises the amino acid sequence of SEQ ID NO: 1.

23. The method of claim 1 , wherein the vanillin synthase capable of catalyzing the conversion of ferulic acid or a ferulic acid derivative to form vanillin comprises the amino acid sequence encoded by SEQ ID NO: 24.

Assignees

• Evolva Sa
• Univ Copenhagen

Inventors

• Lindberg Møller Birger
• Hansen Esben Halkjaer
• Hansen Jørgen
• Janeshawari Gallage Nethaji

Classifications

• A23L27/24

  prepared by fermentation · CPC title

• C12N15/8243

  involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12P7/24 Primary

  containing a carbonyl group · CPC title