Microorganisms And Methods For Producing Substituted Phenols

• US 20140087428A1
• Filed: 06/18/2012
• Published: 03/27/2014
• Est. Priority Date: 06/17/2011
• Status: Active Grant

• Current Assignee: Symrise AG
• Original Assignee: Alexander Steinbuchel
• Inventors: Lambrecht, Stefan, Pesaro, Manuel, Hilmer, Jens-Michael, Kroll, Jens, Hansen, Gunda, Steinbchel, Alexander, Fleige, Christian
• Granted Patent: US 9,567,618 B2
• Time in Patent Office: Days
• US Class Current: 435/92
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12P 19/32 having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
• 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
• CPC Class Codes: C12Y 102/01067 Vanillin dehydrogenase (1.2.1.67)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention is concerned with methods for producing vanillin, feroyl-CoA, ferulic acid, coniferyl aldehyde and/or coniferyl alcohol. Also, the invention relates to microorganisms useful in such production method, and to the construction of such microorganisms.

Claims

1. Method for producing a product selected from the group consisting of vanillin, feruloyl-CoA, ferulic acid, coniferyl aldehyde and coniferyl alcohol, comprising adding an educt selected from the group consisting of eugenol, coniferyl alcohol, coniferyl aldehyde and ferulic acid to a microorganism of order Actinomycetales, wherein the microorganism does not comprise a gene coding for a vanillin dehydrogenase.

2. The method according to claim 1, wherein the vanillin dehydrogenase has an amino acid sequence according to any of SEQ ID 1, 2 or 3.

3. The method according to claim 1, wherein the microorganism comprises a vanillin dehydrogenase gene inactivated by an insert or a deletion.

4. The method according to claim 1, wherein the microorganism comprises a gene selected from the group consisting of:

an eugenol hydroxylase gene of Pseudomonas sp. DSMZ 7063, a vanillyl alcohol oxidase gene of Penicillium simplicissimium, and an eugenol oxidase gene of Rhodococcus jostii RHA1,

a coniferyl alcohol dehydrogenase gene of Pseudomonas sp. DSMZ 7063, an aromatic alcohol dehydrogenase gene (adhA;

accession number AB213394) of Rhodococcus opacus TKN14, an aryl alcohol dehydrogenase gene of Acinetobacter sp. ADP1, an aryl alcohol dehydrogenase gene of Rhodococcus erythropolis PR4, Lactobacillus plantarum WCFS 1 (lp_—

3054;

accession number CAB69495) and Gordonia polyisoprenivorans Kd2 (adhA, accession number ZP_—

09272609.1),

a coniferyl aldehyde dehydrogenase of Pseudomonas sp. DSMZ 7063, a benzaldehyd dehydrogenase of Acinetobacter sp. ADP1, an aldehyd-dehydrogenase of Arabidopsis thaliana (ALDH2C4, accession number NM_—

113359.3), a benzaldehyd dehydrogenase of Pseudomonas putida mt-2 (XylC, accession number U15151.1), and a dodecanal dehydrogenase Gluconobacter oxydans 521H (accession number YP_—

190934),

a feruloyl-CoA-synthetase of Pseudomonas sp. DSMZ 7063,

an enoyl-CoA-hydratase/aldolase of Pseudomonas sp. DSMZ 7063,

and a gene coding for an enzyme having a sequence similarity of at least 95% to the sequence of any of the aforementioned genes.

11. Method according to claim 1 for producing a substance selected from the group consisting of:

vanillin from any educt elected from eugenol, coniferyl alcohol, coniferyl aldehyde, ferulic acid and feruloyl-CoA,

feruloyl-CoA from any educt elected from eugenol, coniferyl alcohol, coniferyl aldehyde and ferulic acid,

ferulic acid from any educt elected from eugenol, coniferyl alcohol and coniferyl aldehyde,

coniferyl aldehyde from any educt elected from eugenol and coniferyl alcohol, and

coniferyl alcohol from eugenol as educt,

comprising the steps of;

providing the microorganism of claim 5, and

exposing the microorganism to an educt.

14. The method according to claim 1, wherein the microorganism comprises a microorganism of genus Amycolatopsis.

15. The method according to claim 1, wherein the microorganism comprises a microorganism of genus Streptomyces.

16. The method according to claim 1, wherein the vanillin dehydrogenase has an amino acid sequence having a sequence similarity of at least 90% to an amino acid sequence according to any of SEQ ID 1, 2 or 3.

17. The method according to claim 1, wherein the microorganism comprises a left and a right flanking region, each having a length of 50-1500 nucleotides and a sequence similarity of at least 95% to a sequence of identical length within 2000 nucleotides to the left and right, respectively, of an Amycolatopsis or Streptomyces wild type vanillin dehydrogenase gene, wherein the nucleic acid sequence between the left and right flanking region does not comprise a gene coding for a vanillin dehydrogenase.

5. Microorganism of order Actinomycetales, wherein the microorganism does not comprise a gene coding for a vanillin dehydrogenase.

6. The microorganism of claim 5, wherein the vanillin dehydrogenase has an amino acid sequence having a sequence similarity of at least 90% to an amino acid sequence according to any of SEQ ID 1, 2 or 3.

7. The microorganism of claim 5, wherein the microorganism comprises a vanillin dehydrogenase gene inactivated by an insert or a deletion.

8. The microorganism of claim 5, wherein the microorganism comprises a gene selected from the group consisting of:

an eugenol hydroxylase gene of Pseudomonas sp. DSMZ 7063, a vanillyl alcohol oxidase gene of Penicillium simplicissimium, and an eugenol oxidase gene of Rhodococcus jostii RHA1,

a coniferyl alcohol dehydrogenase gene of Pseudomonas sp. DSMZ 7063, an aromatic alcohol dehydrogenase gene (adhA;

accession number AB213394) of Rhodococcus opacus TKN14, an aryl alcohol dehydrogenase gene of Acinetobacter sp. ADP1, an aryl alcohol dehydrogenase gene of Rhodococcus erythropolis PR4, Lactobacillus plantarum WCFS 1 (lp_—

3054;

accession number CAB69495) and Gordonia polyisoprenivorans Kd2 (adhA, accession number ZP_—

09272609.1),

a coniferyl aldehyde dehydrogenase of Pseudomonas sp. DSMZ 7063, a benzaldehyd dehydrogenase of Acinetobacter sp. ADP1, an aldehyd-dehydrogenase of Arabidopsis thaliana (ALDH2C4, accession number NM_—

113359.3), a benzaldehyd dehydrogenase of Pseudomonas putida mt-2 (XylC, accession number U15151.1), and a dodecanal dehydrogenase Gluconobacter oxydans 521H (accession number YP_—

190934),

a feruloyl-CoA-synthetase of Pseudomonas sp. DSMZ 7063,

an enoyl-CoA-hydratase/aldolase of Pseudomonas sp. DSMZ 7063,

and a gene coding for an enzyme having a sequence similarity of at least 95% to the sequence of any of the aforementioned genes.

9. The microorganism of claim 5, wherein the microorganism is of genus Amycolatopsis or of Amycolatopsis sp. ATCC 39116, with one or more of the following modifications:

the gene for vanillin dehydrogenase is inactivated by an insertion of an antibiotic resistance gene into the gene coding for vanillin dehydrogenase; and

the microorganism additionally comprises a vanillyl alcohol oxidase gene of Penicillium simplicissimium and/or an eugenol hydroxylase gene of Rhodococcus jostii RHA1; and

optionally the microorganism additionally comprises a coniferyl alcohol dehydrogenase gene of Pseudomonas sp. DSMZ 7063 and a coniferyl aldehyde dehydrogenase of Pseudomonas sp. DSMZ 7063.

10. Use of the microorganism of claim 5 for the production of a substance selected from the group consisting of:

vanillin from any of eugenol, coniferyl alcohol, coniferyl aldehyde, ferulic acid and feruloyl-CoA,

feruloyl-CoA from any of eugenol, coniferyl alcohol, coniferyl aldehyde and ferulic acid,

ferulic acid from any of eugenol, coniferyl alcohol and coniferyl aldehyde,

coniferyl aldehyde from any of eugenol and coniferyl alcohol, and

coniferyl alcohol from eugenol.

12. Method of constructing the microorganism of claim 5 comprising:

providing a microorganism of order Actinomycetales,

transforming the microorganism with an omega element to inactivate a gene coding for a vanillin dehydrogenase, and

selecting a recombinant obtained by the transformation step.

13. The method of claim 12 further comprising:

transforming the microorganism with a vector and;

selecting the microorganism comprising the vector;

wherein the vector is selected from the group consisting of;

an eugenol hydroxylase gene of Pseudomonas sp. DSMZ 7063, a vanillyl alcohol oxidase gene of Penicillium simplicissimium, and an eugenol oxidase gene of Rhodococcus jostii RHA1,

a coniferyl alcohol dehydrogenase gene of Pseudomonas sp. DSMZ 7063, an aromatic alcohol dehydrogenase gene (adhA;

accession number AB213394) of Rhodococcus opacus TKN14, an aryl alcohol dehydrogenase gene of Acinetobacter sp. ADP1, an aryl alcohol dehydrogenase gene of Rhodococcus erythropolis PR4, Lactobacillus plantarum WCFS 1 (lp_—

3054;

accession number CAB69495) and Gordonia polyisoprenivorans Kd2 (adhA, accession number ZP_—

09272609.1),

a coniferyl aldehyde dehydrogenase of Pseudomonas sp. DSMZ 7063, a benzaldehyd dehydrogenase of Acinetobacter sp. ADP1, an aldehyd-dehydrogenase of Arabidopsis thaliana (ALDH2C4, accession number NM_—

113359.3), a benzaldehyd dehydrogenase of Pseudomonas putida mt-2 (XylC, accession number U15151.1), and a dodecanal dehydrogenase Gluconobacter oxydans 521H (accession number YP_—

190934),

a feruloyl-CoA-synthetase of Pseudomonas sp. DSMZ 7063,

an enoyl-CoA-hydratase/aldolase of Pseudomonas sp. DSMZ 7063,

and a gene coding for an enzyme having a sequence similarity of at least 95% to the sequence of any of the aforementioned genes.

18. The microorganism of claim 5, wherein the microorganism comprises a microorganism of genus Amycolatopsis.

19. The microorganism of claim 5, wherein the microorganism comprises a microorganism of genus Streptomyces.

20. The microorganism of claim 5, wherein the microorganism comprises a left and a right flanking region, each having a length of 50-1500 nucleotides and a sequence similarity of at least 95% to a sequence of identical length within 2000 nucleotides to the left and right, respectively, of an Amycolatopsis or Streptomyces wild type vanillin dehydrogenase gene, wherein the nucleic acid sequence between the left and right flanking region does not comprise a gene coding for a vanillin dehydrogenase.