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Patent Analysis of

Recombinant host cell for the biosynthesis of vanillin or vanillin beta-D-glucoside

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10000782

Application Number

US14/905228

Application Date

11 July 2014

Publication Date

19 June 2018

Current Assignee

INTERNATIONAL FLAVORS & FRAGRANCES INC.

Original Assignee (Applicant)

INTERNATIONAL FLAVORS & FRAGRANCES INC.

International Classification

C12N9/04,C07H15/203,C12N15/82,C12P19/46,C12N15/70

Cooperative Classification

C12P19/46,C07C47/58,C07H15/203,C12N9/0006,C12N15/70

Inventor

HANSEN, ESBEN HALKJAER,HALLWYL, SWEE CHUANG LIM,HANSEN, KLAVS RIISHEDE

Patent Images

This patent contains figures and images illustrating the invention and its embodiment.

US10000782 Recombinant host cell 1 US10000782 Recombinant host cell 2
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Abstract

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to have reduced levels or activity of one or more alcohol dehydrogenases or aldehyde reductase thereby increasing the production of vanillin or vanillin beta-D-glucoside.

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Claims

1. A recombinant host cell having the following characteristics:(a) the recombinant host cell produces vanillin and/or vanillin glycoside; and(b) the recombinant host cell has a gene disruption or replacement of a first alcohol dehydrogenase gene and a gene disruption or replacement of:(i) two or more second alcohol dehydrogenase genes,(ii) one or more aldehyde reductase genes, or(iii) a combination of the genes of (i) and (ii).

2. The recombinant host cell of claim 1, wherein the first alcohol dehydrogenase gene is Alcohol Dehydrogenase 6 (ADH6).

3. The recombinant host cell of claim 1, wherein the two or more second alcohol dehydrogenase genes are Alcohol Dehydrogenase 7 (ADH7) and Genes de Respuesta a Estres 2 (GRE2).

4. The recombinant host cell of claim 1, wherein the one or more aldehyde reductase genes are Aldehyde Reductase Intermediate 1 (ARI1) or Saccharomyces Aldehyde Reductase YGL039W.

5. The recombinant host cell of claim 1, wherein the recombinant host cell further comprises a nucleic acid encoding an AROM polypeptide, a nucleic acid encoding a catechol-O-methyltransferase (COMT) polypeptide, a nucleic acid encoding a 3-dehydroshikimate dehydratase (3DSD) polypeptide, a nucleic acid encoding an aromatic carboxylic acid reductase (ACAR) polypeptide, a nucleic acid encoding a phosphopantetheine transferase (PPTase) polypeptide, a nucleic acid encoding an uridine 5′-diphosphoglucosyl transferase (UGT) polypeptide and/or a nucleic acid encoding a vanillyl alcohol oxidase (VAO).

6. The recombinant host cell of claim 1, wherein the recombinant host cell is a microorganism.

7. The recombinant host cell of claim 5, wherein the microorganism is Saccharomyces cerevisiae, Schizosaccharomyces pombe or Escherichia coli.

8. The recombinant host cell of claim 1, wherein the recombinant host cell is a plant cell.

9. The recombinant host cell of claim 8, wherein the plant cell is a Physcomitrella cell or tobacco plant cell.

10. A recombinant yeast cell having the following characteristics:(a) the recombinant yeast cell produces vanillin and/or vanillin glycoside; and(b) the recombinant yeast cell has a gene disruption or replacement of:(i) at least three alcohol dehydrogenase genes,(ii) at least one aldehyde reductase gene, or(iii) at least one alcohol dehydrogenase gene and at least one aldehyde reductase gene.

11. The recombinant yeast cell of claim 10, wherein the alcohol dehydrogenase genes of (i) are Alcohol Dehydrogenase 6 (ADH6), Alcohol Dehydrogenase 7 (ADH7), and Genes de Respuesta a Estres 2 (GRE2); and the alcohol dehydrogenase gene of (iii) is ADH6, ADH7, or GRE2.

12. The recombinant yeast cell of claim 10, wherein the aldehyde reductase gene is Aldehyde Reductase Intermediate 1 (ARI1), or Saccharomyces Aldehyde Reductase YGL039W.

13. The recombinant yeast cell of claim 10, wherein the recombinant yeast cell further comprises a nucleic acid encoding an AROM polypeptide, a nucleic acid encoding a catechol-O-methyltransferase (COMT) polypeptide, a nucleic acid encoding a 3-dehydroshikimate dehydratase (3DSD) polypeptide, a nucleic acid encoding an aromatic carboxylic acid reductase (ACAR) polypeptide, a nucleic acid encoding a phosphopantetheine transferase (PPTase) polypeptide, a nucleic acid encoding an uridine 5′-diphosphoglucosyl transferase (UGT) polypeptide and/or a nucleic acid encoding a vanillyl alcohol oxidase (VAO).

14. The recombinant yeast cell of claim 10, wherein the recombinant yeast cell is a member of the genus Saccharomyces.

15. The recombinant yeast cell of claim 14, wherein the recombinant yeast cell is Saccharomyces cerevisiae.

16. A method for producing vanillin and/or vanillin glycoside comprising:(a) providing a recombinant host that produces vanillin and/or vanillin glycoside and has a gene disruption or replacement of:(i) at least three a alcohol dehydrogenase genes,(ii) at least one aldehyde reductase gene, or(iii) at least one alcohol dehydrogenase gene and at least one aldehyde reductase gene;(b) cultivating said recombinant host for a time sufficient for said recombinant host to produce vanillin and/or vanillin glycoside; and(c) isolating vanillin and/or vanillin glycoside from said recombinant host or from the cultivation supernatant, thereby producing vanillin and/or vanillin glycoside.

17. The method of claim 16, wherein the alcohol dehydrogenase genes of (i) are Alcohol Dehydrogenase 6 (ADH6), Alcohol Dehydrogenase 7 (ADH7), and Genes de Respuesta a Estres 2 (CRE2); and the alcohol dehydrogenase gene of (iii) is ADH6, ADH7 or GRE2.

18. The method of claim 16, wherein the aldehyde reductase gene is Aldehyde Reductase Intermediate 1 (ARI1), or Saccharomyces Aldehyde Reductase YGL039W.

19. The method of claim 16, wherein the recombinant host further comprises a nucleic acid encoding an AROM polypeptide, a nucleic acid encoding a catechol-O-methyltransferase (COMT) polypeptide, a nucleic acid encoding a 3-dehydroshikimate dehydratase (3DSD) polypeptide, a nucleic acid encoding an aromatic carboxylic acid reductase (ACAR) polypeptide, a nucleic acid encoding a phosphopantetheine transferase (PPTase) polypeptide, a nucleic acid encoding an uridine 5′-diphosphoglucosyl transferase (UGT) polypeptide and/or a nucleic acid encoding a vanillyl alcohol oxidase (VAO).

20. The method of claim 16, wherein the recombinant host is a microorganism.

21. The method of claim 20, wherein the microorganism is Saccharomyces cerevisiae, Schizosaccharomyces pombe or Escherichia coli.

22. The method of claim 16, wherein the recombinant host is a plant or plant cell.

23. The method of claim 22, wherein the recombinant host is a Physcomitrella plant, a tobacco plant, a Physcomitrella plant cell or a tobacco plant cell.

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Claim Tree

  • 1
    1. A recombinant host cell having the following characteristics:
    • (a) the recombinant host cell produces vanillin and/or vanillin glycoside; and
    • (b) the recombinant host cell has a gene disruption or replacement of a first alcohol dehydrogenase gene and a gene disruption or replacement of:(i) two or more second alcohol dehydrogenase genes,(ii) one or more aldehyde reductase genes, or(iii) a combination of the genes of (i) and (ii).
    • 2. The recombinant host cell of claim 1, wherein
      • the first alcohol dehydrogenase gene is Alcohol Dehydrogenase 6 (ADH6).
    • 3. The recombinant host cell of claim 1, wherein
      • the two or more second alcohol dehydrogenase genes are Alcohol Dehydrogenase 7 (ADH7) and Genes de Respuesta a Estres 2 (GRE2).
    • 4. The recombinant host cell of claim 1, wherein
      • the one or more aldehyde reductase genes are Aldehyde Reductase Intermediate 1 (ARI1) or Saccharomyces Aldehyde Reductase YGL039W.
    • 5. The recombinant host cell of claim 1, wherein
      • the recombinant host cell further comprises
    • 6. The recombinant host cell of claim 1, wherein
      • the recombinant host cell is a microorganism.
    • 8. The recombinant host cell of claim 1, wherein
      • the recombinant host cell is a plant cell.
  • 10
    10. A recombinant yeast cell having the following characteristics:
    • (a) the recombinant yeast cell produces vanillin and/or vanillin glycoside; and
    • (b) the recombinant yeast cell has a gene disruption or replacement of:(i) at least three alcohol dehydrogenase genes,(ii) at least one aldehyde reductase gene, or(iii) at least one alcohol dehydrogenase gene and at least one aldehyde reductase gene.
    • 11. The recombinant yeast cell of claim 10, wherein
      • the alcohol dehydrogenase genes of (i) are Alcohol Dehydrogenase 6 (ADH6), Alcohol Dehydrogenase 7 (ADH7), and Genes de Respuesta a Estres 2 (GRE2); and the alcohol dehydrogenase gene of (iii) is ADH6, ADH7, or GRE2.
    • 12. The recombinant yeast cell of claim 10, wherein
      • the aldehyde reductase gene is Aldehyde Reductase Intermediate 1 (ARI1), or Saccharomyces Aldehyde Reductase YGL039W.
    • 13. The recombinant yeast cell of claim 10, wherein
      • the recombinant yeast cell further comprises
    • 14. The recombinant yeast cell of claim 10, wherein
      • the recombinant yeast cell is a member of the genus Saccharomyces.
  • 16
    16. A method for producing vanillin and/or vanillin glycoside comprising:
    • (a) providing a recombinant host that produces vanillin and/or vanillin glycoside and has a gene disruption or replacement of:(i) at least three a alcohol dehydrogenase genes,(ii) at least one aldehyde reductase gene, or(iii) at least one alcohol dehydrogenase gene and at least one aldehyde reductase gene;
    • (b) cultivating said recombinant host for a time sufficient for said recombinant host to produce vanillin and/or vanillin glycoside; and
    • (c) isolating vanillin and/or vanillin glycoside from said recombinant host or from the cultivation supernatant, thereby producing vanillin and/or vanillin glycoside.
    • 17. The method of claim 16, wherein
      • the alcohol dehydrogenase genes of (i) are Alcohol Dehydrogenase 6 (ADH6), Alcohol Dehydrogenase 7 (ADH7), and Genes de Respuesta a Estres 2 (CRE2); and the alcohol dehydrogenase gene of (iii) is ADH6, ADH7 or GRE2.
    • 18. The method of claim 16, wherein
      • the aldehyde reductase gene is Aldehyde Reductase Intermediate 1 (ARI1), or Saccharomyces Aldehyde Reductase YGL039W.
    • 19. The method of claim 16, wherein
      • the recombinant host further comprises
    • 20. The method of claim 16, wherein
      • the recombinant host is a microorganism.
    • 22. The method of claim 16, wherein
      • the recombinant host is a plant or plant cell.
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Description

BACKGROUND

Vanillin is one of the world's most important flavor compounds with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. Production of natural vanillin from the vanilla pod is a laborious and slow process, which requires hand pollination of the flowers and a 1-6 month curing process of the harvested green vanilla pods (Ramachandra & Ravishankar (2000) J. Sci. Food Agric. 80:289-304). Production of 1 kilogram (kg) of vanillin requires approximately 500 kg of vanilla pods, corresponding to pollination of approximately 40,000 flowers. Today only about 0.25% (40 tons out of 16,000) of vanillin sold annually originates from vanilla pods, while most of the remainder is synthesized chemically from lignin or fossil hydrocarbons, in particular guaiacol. Synthetically produced vanillin is sold for approximately $15 per kg, compared to prices of $1200-4000 per kg for natural vanillin (Walton, et al. (2003) Phytochemistry 63:505-515).

SUMMARY OF THE INVENTION

This invention provides a recombinant host cell having the following characteristics: the recombinant host cell produces vanillin and/or vanillin beta-D-glucoside; and the recombinant host cell has reduced production or activity of a first alcohol dehydrogenase and reduced production of one or more second alcohol dehydrogenases, one or more aldehyde reductases, or a combination thereof. In some embodiments, the first alcohol dehydrogenase is Alcohol Dehydrogenase 6 (ADH6). In another embodiment, the one or more second alcohol dehydrogenases include Alcohol Dehydrogenase 7 (ADH7), Genes de Respuesta a Estres 2 (GRE2), or an ortholog thereof. In a further embodiment, the aldehyde reductase includes Aldehyde Reductase Intermediate 1 (ARI1), Aldehyde Reductase YGL039W, or an ortholog thereof. In certain embodiments, the recombinant host cell further includes a nucleic acid encoding an AROM polypeptide, a nucleic acid encoding a catechol-O-methyltransferase (COMT) polypeptide, a nucleic acid encoding a 3-dehydroshikimate dehydratase (3DSD) polypeptide, a nucleic acid encoding an aromatic carboxylic acid reductase (ACAR) polypeptide, a nucleic acid encoding a phosphopantetheine transferase (PPTase) polypeptide, a nucleic acid encoding an uridine 5′-diphosphoglucosyl transferase (UGT) polypeptide and/or a nucleic acid encoding a vanillyl alcohol oxidase (VAO). Microorganism such as Saccharomyces cerevisiae, Schizosaccharomyces pombe or Escherichia coli are provided as are plant or plant cells such as Physcomitrella or tobacco.

A recombinant yeast cell; a vanillin and/or vanillin glucoside extract isolated from the recombinant host cell or recombinant yeast cell; a consumable, e.g., a food product, pharmaceutical composition, a dietary supplement, a nutraceutical, a dental hygienic composition, a tabletop sweetener, or a cosmetic product containing the extract; and a method for producing vanillin and/or vanillin beta-D-glucoside are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of de novo biosynthesis of vanillin (4) and outline of the different vanillin catabolites and metabolic side products, i.e., dehydroshikimic acid (1), protocatechuic acid (2), protocatechuic aldehyde (3), vanillic acid (5), protocatechuic alcohol (6), vanillyl alcohol (7), and vanillin β-D-glucoside (8), found in an organism expressing 3DSD, ACAR, OMT, and UGT and a phophopantheteine transferase (PPTase). Open arrows show primary metabolic reactions in yeast; black arrows show enzyme reactions introduced by metabolic engineering; diagonally striped arrows show undesired inherent yeast metabolic reactions.

FIG. 2 shows the production of vanillyl alcohol glucoside, vanillyl alcohol, vanillin β-D-glucoside and vanillin in a strain lacking one or more functional alcohol dehydrogenases.

FIG. 3 shows the production of vanillyl alcohol glucoside, vanillyl alcohol, vanillin ρ-D-glucoside and vanillin in a strain lacking gre2 (EFSC2055 gre2) or gre2 and aril (EFSC2055 gre2 aril) as compared to the parental strain EFSC2055. *, p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the discovery that knocking out certain alcohol dehydrogenases and/or aldehyde reductases, or similar enzymes, lowers the amount of vanillic alcohol that is formed as a byproduct. See FIGS. 1 and 2. This is of commercial importance because the presence of alcohol creates inefficiencies in certain steps of the downstream purification of vanillin or vanillin glucoside if the alcohol is allowed to accumulate.

Therefore, the present invention is a recombinant host that is capable of producing vanillin or vanillin glucoside, but fails to produce, or has reduced production of, one or more alcohol dehydrogenases and/or one or more aldehyde reductases. A recombinant host that produces or is capable of producing vanillin or vanillin glucoside is a host cell that expresses the necessary biosynthetic enzymes to produce vanillin or vanillin glucoside from a primary substrate, e.g., glucose, or from an intermediate molecule, e.g., dehydroshikimic acid, protocatechuic acid, protocatechuic aldehyde, or vanillic acid. See FIG. 1.

A recombinant host that fails to produce an enzyme, has reduced production of an enzyme, or lacks a functional enzyme, includes an organism that have been recombinantly modified such that the gene encoding the enzyme is knocked out, an organism with one or more point mutations in the enzyme which reduces or diminishes enzyme activity, or an organism wherein the promoter of the gene encoding the enzyme has been modified or removed so that the enzyme is not expressed or expressed at a reduced level compared to a wild-type organism.

Many methods for genetic modification of target genes are known to one skilled in the art and may be used to create the recombinant host of this invention. Modifications that may be used to reduce or eliminate expression of a target enzyme are disruptions that include, but are not limited to, deletion of the entire gene or a portion of the gene encoding an enzyme; inserting a DNA fragment into a gene encoding the enzyme (in either the promoter or coding region) so that the enzyme is not expressed or expressed at lower levels; introducing a mutation into the coding region for the enzyme, which adds a stop codon or frame shift such that a functional enzyme is not expressed; and introducing one or more mutations into the coding region of an enzyme to alter amino acids so that a non-functional or a less enzymatically active enzyme is expressed. In addition, expression of an enzyme may be blocked by expression of an antisense RNA or an interfering RNA, and constructs may be introduced that result in cosuppression. In addition, the synthesis or stability of the transcript may be lessened by mutation. Similarly, the efficiency by which an enzyme is translated from mRNA may be modulated by mutation. All of these methods may be readily practiced by one skilled in the art making use of the known sequences encoding the alcohol dehydrogenases and/or aldehyde reductases of this invention.

Alcohol dehydrogenase and aldehyde reductase sequences from a variety of organisms are well-known in the art and the selection of the target gene(s) will be dependent upon the host selected. Representative alcohol dehydrogenase (ADH) and aldehyde reductase sequences, which may be targeted in accordance with the present invention are listed in Table 1. One skilled in the art may choose specific modification strategies to eliminate or lower the expression of an alcohol dehydrogenase and/or aldehyde reductase as desired to facilitate vanillin and/or vanillin glucoside production.


TABLE 1
Amino Acid
Nucleotide
Sequence
Sequence
SEQ ID
SEQ ID
Source
Target
Accession No.
NO:
Accession No.
NO:
S. cerevisiae
Adh6
NP_014051
1
NM_001182831
2
S. cerevisiae
Adh7
NP_010030
3
NM_001178812
4
S. cerevisiae
GRE2
NP_014490
5
NM_001183405
6
S. cerevisiae
YDR541C
NP_010830
7
NM_001180849
8
S. cerevisiae
ARI1
NP_011358
9
NM_001181022
10
S. cerevisiae
YGL039W
NP_011476
11
NM_001180904
12
S. pombe
SPAC513.07
NP_593981
13
NM_001019407
14
Arabidopsis thaliana
CAD3
NP_179780
15
NM_127758
16
A. thaliana
CAD9
NP_195643
17
NM_120093
18
A. thaliana
CAD2
NP_179765
19
NM_127743
20
A. thaliana
AT1G51410
NP_175552
21
NM_104019
22
A. thaliana
AT5G19440
NP_197445
23
NM_121949
24

In some embodiments, the recombinant host cells has reduced production or activity of a first alcohol dehydrogenase and reduced production of one or more second alcohol dehydrogenases, one or more aldehyde reductases, or a combination thereof. In particular embodiments, the first alcohol dehydrogenase is ADH6 or an ortholog thereof, e.g., CAD9, CAD3 or CAD2 from A. thaliana. In another embodiment, the one or more second alcohol dehydrogenases are selected from the group of ADH7, GRE2 (Genes de Respuesta a Estres 2), or an ortholog thereof, e.g., AT1G51410 or AT5G19440; and the aldehyde reductase is selected from the group of ARI1 (Aldehyde Reductase Intermediate 1), Aldehyde Reductase YGL039W, or an ortholog thereof, e.g., SPAC513.07 or YDR541C).

DNA sequences surrounding one or more of the above-referenced sequence are also useful in some modification procedures and are available for yeasts such as for Saccharomyces cerevisiae in the complete genome sequence coordinated by NCBI (National Center for Biotechnology Information) with identifying BioProject Nos. PRJNA128, PRJNA13838, PRJNA43747, PRJNA48559, PRJNA52955, PRJNA48569, PRJNA39317. Additional examples of yeast genomic sequences include that of Schizosaccharomyces pombe, which is included in BioProject Nos. PRJNA127, PRJNA13836, and PRJNA20755. Genomic sequences of plants are also known in the art and the genomic sequence of Arabidopsis thaliana is included in BioProject Nos. PRJNA116, PRJNA10719, PRJNA13190, and PRJNA30811. Other genomic sequences can be readily found by one of skill in the art in publicly available databases.

In particular, DNA sequences surrounding an alcohol dehydrogenase or aldehyde reductase coding sequence are useful for modification methods using homologous recombination. For example, sequences flanking the gene of interest are placed on either side of a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the gene of interest. Also partial gene sequences and flanking sequences bounding a selectable marker gene may be used to mediate homologous recombination whereby the marker gene replaces a portion of the target gene. In addition, the selectable marker may be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the gene of interest without reactivating the latter. The site-specific recombination leaves behind a recombination site which disrupts expression of the alcohol dehydrogenase or aldehyde reductase. The homologous recombination vector may be constructed to also leave a deletion in the gene of interest following excision of the selectable marker, as is well known to one skilled in the art.

Deletions may be made using mitotic recombination as described in Wach, et al. ((1994) Yeast 10:1793-1808). This method involves preparing a DNA fragment that contains a selectable marker between genomic regions that may be as short as 20 bp, and which bound a target DNA sequence. This DNA fragment can be prepared by PCR amplification of the selectable marker gene using as primers oligonucleotides that hybridize to the ends of the marker gene and that include the genomic regions that can recombine with the yeast genome. The linear DNA fragment can be efficiently transformed into yeast and recombined into the genome resulting in gene replacement including with deletion of the target DNA sequence.

Moreover, promoter replacement methods may be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression such as described in Mnaimneh, et al. ((2004) Cell 118(1):31-44).

Hosts cells of use in this invention include any organism capable of producing vanillin and/or vanillin glucoside either naturally or synthetically, e.g., by recombinant expression of one or more genes of the vanillin and/or vanillin glucoside biosynthetic pathway (FIG. 1). A number of prokaryotes and eukaryotes are suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, gram-positive bacteria, yeast or other fungi. A species and strain selected for use as a vanillin and/or vanillin glucoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).

Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species may be suitable. For example, suitable species may be in a genus Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces Yarrowia and Lactobacillus. Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Physcomitrella patens, Rhodoturula glutinis 32, Rhodoturula mucilaginosa, Phaffia rhodozyma UBV-AX, Xanthophyllomyces dendrorhous, Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis and Yarrowia lipolytica. In some embodiments, a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, or Saccharomyces cerevisiae. In some embodiments, a microorganism can be a prokaryote such as Escherichia coli, Rhodobacter sphaeroides, or Rhodobacter capsulatus. It will be appreciated that certain microorganisms can be used to screen and test genes of interest in a high throughput manner, while other microorganisms with desired productivity or growth characteristics can be used for large-scale production of vanillin beta-D-glucoside.

Specific non-limiting examples of useful recombinant hosts are described in WO 01/40491, as well as in Hansen et al. (2009) Appl. Environ. Microbiol. 75:2765-2774 and Brochado, et al. (2010) Microbial Cell Factories 9:84, wherein the recombinant host according to this invention contains a heterologous nucleic acid encoding a mutant COMT polypeptide and/or mutant AROM polypeptide instead of the OMT genes described in WO 01/40491.

One preferred recombinant host to use with the present invention is S. cerevisiae, which may be recombinantly engineered as described herein. S. cerevisiae is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. There are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms. The VG4 strain of S. cerevisiae (Brochado, et al. (2010) Microb. Cell Fact. 9:84) is particularly useful. VG4 has the genotype of pdc1Δgdh1Δ↑GDH2.

Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production, and can also be used as the recombinant microorganism platform. Thus, the recombinant host may be Aspergillus spp. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for the production of food ingredients such as vanillin and vanillin glucoside.

E. coli, another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Thus, the recombinant host may be E. coli. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.

Rhodobacter can be used as the recombinant microorganism platform. Thus, the recombinant host may be Rhodobacter spp. Similar to E. coli, there are libraries of mutants available as well as suitable plasmid vectors, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for E. coli can be used to make recombinant Rhodobacter microorganisms.

Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera is becoming an important type of cell for production of plant secondary metabolites, which can be difficult to produce in other types of cells. Thus, the recombinant host may be a Physcomitrella spp.

In some embodiments, the recombinant host is a plant or plant cells that includes the one or more genes of the vanillin and/or vanillin glucoside biosynthetic pathway. A plant or plant cell can be modified to express the vanillin and/or vanillin glucoside biosynthetic pathway with a concurrent knockout of one or more alcohol dehydrogenases and/or aldehyde reductases. The plant or plant cells can be stably transformed to retain the introduced nucleic acid with each cell division. A plant or plant cell can also be transiently transformed such that the heterologous nucleic acid is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced nucleic acid with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.

Transgenic plant cells used in methods described herein can constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a heterologous nucleic acid, for example a recombinant nucleic acid construct into other lines, to transfer a heterologous nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. As used herein, a transgenic plant also refers to progeny of an initial transgenic plant provided the progeny inherits the transgene. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct.

Transgenic plants can be grown in suspension culture, or tissue or organ culture. For the purposes of this invention, solid and/or liquid tissue culture techniques can be used. When using solid medium, transgenic plant cells can be placed directly onto the medium or can be placed onto a filter that is then placed in contact with the medium. When using liquid medium, transgenic plant cells can be placed onto a flotation device, e.g., a porous membrane that contacts the liquid medium.

When transiently transformed plant cells are used, a reporter sequence encoding a reporter polypeptide having a reporter activity can be included in the transformation procedure and an assay for reporter activity or expression can be performed at a suitable time after transformation. A suitable time for conducting the assay typically is about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days. The use of transient assays is particularly convenient for rapid analysis in different species, or to confirm expression of a heterologous polypeptide whose expression has not previously been confirmed in particular recipient cells.

Techniques for introducing nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrobacterium-mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation; see U.S. Pat. Nos. 5,538,880; 5,204,253; 6,329,571; and 6,013,863. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.

A population of transgenic plants can be screened and/or selected for those members of the population that have a trait or phenotype conferred by expression of the transgene. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of a polypeptide or nucleic acid described herein. Physical and biochemical methods can be used to identify expression levels. These include Southern analysis or PCR amplification for detection of a polynucleotide; northern blots, S1 RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or nucleic acids. Methods for performing all of the referenced techniques are known.

As an alternative, a population of plants with independent transformation events can be screened for those plants having a desired trait, such as production of vanillin glucoside and lack of vanillic alcohol production. Selection and/or screening can be carried out over one or more generations, and/or in more than one geographic location. In some cases, transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant. In addition, selection and/or screening can be applied during a particular developmental stage in which the phenotype is expected to be exhibited by the plant.

Depending on the particular organism used in this invention, the recombinant host cell can naturally or recombinantly express genes encoding an AROM (A=Multifunctional Enzyme), OMT (O-methyltransferase), COMT (Catechol-O-Methyl Transferase), 3DSD (3-dehydroshikimate dehydratase), ACAR (aromatic carboxylic acid reductase), UGT (uridine 5′-diphosphoglucosyl transferase), or PPTase (phosphopantetheine transferase) (FIG. 1).

Recombinant expression means that the genome of a host cell has been augmented through the introduction of one or more recombinant genes, which include regulatory sequences that facilitate the transcription and translation of a protein of interest. While embodiments include stable introduction of recombinant genes into the host genome, autonomous or replicative plasmids or vectors can also be used within the scope of this invention. Moreover, the present invention can be practiced using a low copy number, e.g., a single copy, or high copy number (as exemplified herein) plasmid or vector.

Generally, the introduced recombinant gene is not originally resident in the host that is the recipient of the recombinant gene, but it is within the scope of the invention to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms, plant cells, and plants.

The term “recombinant gene” refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. “Introduced,” or “augmented” in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene may be a DNA sequence from another species, or may be a DNA sequence that originated from or is present in the same species, but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA.

A recombinant gene encoding a polypeptide described herein includes the coding sequence for that polypeptide, operably linked, in sense orientation, to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.

In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. The term “heterologous nucleic acid” as used herein, refers to a nucleic acid introduced into a recombinant host, wherein said nucleic acid is not naturally present in said host. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism. A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.

“Regulatory region” refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically includes at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.

The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.

One or more genes, for example one or more heterologous nucleic acids, can be combined in a recombinant nucleic acid construct in “modules” useful for a discrete aspect of vanillin and/or vanillin glucoside production. Combining a plurality of genes or heterologous nucleic acids in a module, facilitates the use of the module in a variety of species. For example, a vanillin gene cluster can be combined such that each coding sequence is operably linked to a separate regulatory region, to form a vanillin module for production in eukaryotic organisms. Alternatively, the module can express a polycistronic message for production of vanillin and/or vanillin glucoside in prokaryotic hosts such as species of Rodobacter, E. coli, Bacillus or Lactobacillus. In addition to genes useful for vanillin or vanillin glucoside production, a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.

It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism). As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.

As indicated, recombinant hosts can express one or more enzymes involved in the biosynthesis of the vanillin or vanillin glucoside, as well as additional genes or biosynthetic modules that improve efficiency with which energy and carbon sources are converted to vanillin and its glucoside, and/or to enhance productivity from the cell culture or plant. In certain embodiments, the recombinant host endogenously ore recombinantly expresses genes encoding AROM, COMT, 3DSD, ACAR, UGT, and/or PPTase.

AROM is a penta-functional enzyme complex encoded in yeast by the ARO1 gene. The gene is 4764 bp long and encodes a corresponding polypeptide 1588 amino acids in length. AROM performs five consecutive enzymatic conversions, i.e., converting DAHP (3-deoxy-D-arabino-heptulosonic acid-7-phosphate) into 3-DHQ (3-dehydroquinate), which is converted to 3-DHS (3-dehydroshikimic acid), which is converted to shikimate, which is converted to shikimate-3-P (shikimate 3-phosphate), which is converted into EPSP (5-enolpyruvylskimate 3-phosphate), all en route to cellular biosynthesis of the aromatic amino acids tyrosine, tryptophan and phenylalanine. According to some embodiments of this invention, the AROM enzyme possesses at least four of the five enzymatic activities of the S. cerevisiae AROM polypeptide, i.e., 3-dehydroquinate dehydratase activity, 3-dehydroquinate synthase activity, 3-phosphoshikimate 1-carboxyvinyltransferase activity, shikimate 3-dehydrogenase (NADP+) activity, and shikimate kinase activity.

Non-limiting examples of AROM polypeptides include the Saccharomyces cerevisiae polypeptide available under GENBANK Accession No. X06077; the Schizosaccharomyces pombe polypeptide available under GENBANK Accession No. NP_594681.1; Schizosaccharomyces japonicas polypeptide available under GENBANK Accession No. XP_002171624; Neurospora crassa polypeptide available under GENBANK Accession No. XP_956000; and the Yarrowia lipolytica polypeptide available under GENBANK Accession No. XP_505337.

According to one embodiment of this invention, the AROM polypeptide is a mutant AROM polypeptide with decreased shikimate dehydrogenase activity. When expressed in a recombinant host, the mutant AROM polypeptide redirects metabolic flux from aromatic amino acid production to vanillin precursor production, i.e., 3-DHS. See WO 2013/022881. In certain embodiments, the mutant AROM polypeptide described herein can have one or more modifications in domain 5 (e.g., a substitution of one or more amino acids, a deletion of one or more amino acids, insertions of one or more amino acids, or combinations of substitutions, deletions, and insertions).

In some embodiments, a modified AROM polypeptide is fused to a polypeptide catalyzing the first committed step of vanillin biosynthesis, 3DSD. A polypeptide having 3DSD activity and that is suitable for use in a fusion polypeptide includes the 3DSD polypeptide from Podospora pauciseta, Ustilago maydis, Rhodoicoccus jostii, Acinetobacter sp., Aspergillus niger or Neurospora crassa. See, GENBANK Accession Nos. CAD60599), XP_001905369.1, XP_761560.1, ABG93191.1, AAC37159.1, and XM_001392464.

Alternatively, or in addition to, the recombinant host can express a COMT polypeptide. Non-limiting examples of COMT polypeptides of use in this invention include COMT polypeptides in the family classified under EC number 2.1.1.6, such as the Homo sapiens (Hs) polypeptide available under GENBANK Accession No. NM_000754; an Arabidopsis thaliana polypeptide available under GENBANK Accession No. AY062837; or a Fragaria×ananassa (strawberry) polypeptide available under GENBANK Accession No. AF220491. Human COMT polypeptide exists as several variants and the COMT polypeptide may be any of these variants. Other suitable mammalian COMT polypeptides of use in this invention include, but are not limited to, those isolated from Pan troglodytes (GENBANK Accession No. XP_514984), Macaca mulatta (GENBANK Accession No. AFJ70145), Equus caballus (GENBANK Accession No. NP_001075303), Canis lupus familiaris (GENBANK Accession No. AAR20324), Cricetulus griseus (GENBANK Accession No. EGV97595), Sus scrofa (GENBANK Accession No. NP_001182259), and Bos taurus (GENBANK Accession No. NP_001095787). Other exemplary COMT polypeptides from plant and microorganism sources include, but are not limited to, those isolated from Rosa chinensis (GENBANK Accession No. CAD29457), Prunus dulcis (GENBANK Accession No. CAA58218), Gossypium hirsutum (GENBANK Accession No. ACT32028), Jatropha curcas (GENBANK Accession No. ACT87981), Eucalyptus camaldulensis (ADB82906), Candida orthopsilosis (GENBANK Accession No. CCG25047), Pichia stipitis (GENBANK Accession No. ABN67921), and Spathaspora passalidarum (GENBANK Accession No. EGW29958). In certain embodiments, the COMT polypeptide of the invention is obtained from Phytophthera infestans (GENBANK Accession No. XP_002899214), Catharanthus roseus (GENBANK Accession No. EGS21863), Yarrowia lipolytica (GENBANK Accession No. XP 500451), Ciona intestinalis (GENBANK Accession No. XP_002121420 or XP_002131313), Capsasproa owczarzaki (GENBANK Accession No. EFW46044), Chaetomium therophilum (GENBANK Accession No. EGS21863), Clavispora lusitaniae (GENBANK Accession No. XP_002899214), Paracoccidioides sp. ‘lutzii’ Pb01 (GENBANK Accession No. XP_002793380), Vanilla planifolia (see SEQ ID NO:56 of PCT/US2012/049842), Coffea Arabica (GENBANK Accession No. AAN03726), Rattus norvegicus (GENBANK Accession No. NP_036663), Mus musculus (GENBANK Accession No. NP_031770), Crenarchaeote (GENBANK Accession No. ABZ07345), Mycobacterium vanbaleeni (GENBANK Accession No. ABM14078), or Schizosaccharomyces pombe (GENBANK Accession No. NP_001018770.

In some embodiments, a mutant COMT polypeptide is used to improve biosynthesis of vanillin beta-D-glucoside. For example, mutant COMT polypeptides can have one or more of the following properties: increased turnover; preferential methylation at the meta (3′) position, rather than at the para (4′) position such that production of vanillin is favored over isovanillin; or better specificity for the vanillin pathway substrates, protocatechuic acid and protocatechuic aldehyde. See WO 2013/022881. A mutant COMT polypeptide can have one or more mutations (e.g., a substitution of one or more amino acids, a deletion of one or more amino acids, insertions of one or more amino acids, or combinations of substitutions, deletions, and insertions) in, for example, the substrate binding site. For example, a mutant COMT polypeptide can have one or more amino acid substitutions in the substrate binding site of human COMT.

In one embodiment, a mutant COMT polypeptide is provided, which is capable of catalyzing methylation of an —OH group of protocatechuic acid, wherein said methylation results in generation of at least 4 times more vanillic acid compared to iso-vanillic acid. In another embodiment, the mutant COMT polypeptide is capable of catalyzing methylation of an —OH group of protocatechuic aldehyde, wherein said methylation results in generation of at least 4 times more vanillin compared to iso-vanillin.

In some embodiments, the host harbors a nucleic acid encoding mutant AROM polypeptide and optionally a wild-type COMT polypeptide. In another embodiment, the host of this invention harbors a nucleic acid encoding mutant COMT polypeptide and optionally a wild-type AROM polypeptide. In yet another embodiment, the host of this invention harbors a nucleic acid encoding mutant AROM polypeptide and optionally a mutant COMT polypeptide.

Suitable 3DSD polypeptides are known. A 3DSD polypeptide according to the present invention may be any enzyme with 3-dehydroshikimate dehydratase activity. Preferably, the 3DSD polypeptide is an enzyme capable of catalyzing conversion of 3-dehydro-shikimate to protocatechuate and H2O. A 3DSD polypeptide according to the present invention is preferably an enzyme classified under EC 4.2.1.118. For example, a suitable polypeptide having 3DSD activity includes the 3DSD polypeptide made by Podospora pauciseta, Ustilago maydis, Rhodoicoccus jostii, Acinetobacter sp., Aspergillus niger or Neurospora crassa. See, GENBANK Accession Nos. CAD60599, XP_001905369.1, XP_761560.1, ABG93191.1, AAC37159.1, and XM_001392464. Thus, the recombinant host may include a heterologous nucleic acid encoding the 3DSD polypeptide of Podospora anserina, Ustilago maydis, Rhodoicoccus jostii, Acinetobacter sp., Aspergillus niger or Neurospora crassa or a functional homologue of any of the aforementioned sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.

Suitable ACAR polypeptides are also known in the art. An ACAR polypeptide according to the present invention may be any enzyme having aromatic carboxylic acid reductase activity. Preferably, the ACAR polypeptide is an enzyme capable of catalyzing conversion protocatechuic acid to protocatechuic aldehyde and/or conversion of vanillic acid to vanillin. An ACAR polypeptide according to the present invention is preferably an enzyme classified under EC 1.2.1.30. For example a suitable ACAR polypeptide is made by Nocardia sp. See, e.g., GENBANK Accession No. AY495697. Thus, the recombinant host may include a heterologous nucleic acid encoding the ACAR polypeptide of Nocardia sp. or a functional homologue thereof sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.

Suitable PPTase polypeptides are known. A PPTase polypeptide according to the present invention may be any enzyme capable of catalyzing phosphopantetheinylation. Preferably, the PPTase polypeptide is an enzyme capable of catalyzing phosphopantetheinylation of ACAR. For example, a suitable PPTase polypeptide is made by E. coli, Corynebacterium glutamicum, or Nocardia farcinica. See GENBANK Accession Nos. NP_601186, BAA35224, and YP_120266. Thus, the recombinant host may include a heterologous nucleic acid encoding the PPTase polypeptide of E. coli, C. glutamicum, or N. farcinica or a functional homologue of any of the aforementioned sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.

Glucosylation of vanillin is particularly useful. Vanillin-β-D-glucoside is the storage form of vanillin found in the vanilla pod. It is non-toxic to most organisms, including yeast, and has a higher solubility in water, as compared to vanillin. In addition, the formation of vanillin-β-D-glucoside most likely directs the biosynthesis toward vanillin production. UGT72E2 (Hansen, et al. (2009) Appl. Environ. Microbiol. 75:2765-27740) exhibited high substrate specificity toward vanillin. In concordance with this observation, its expression in the vanillin producing S. cerevisiae strain resulted in almost all vanillin being converted into vanillin-β-D-glucoside. The ability to turn vanillin into vanillin-3-D-glucoside in vivo is important, because microbial production of non-glucosylated vanillin beyond the 0.5-1 g/liter scale would be hampered by the toxicity of free vanillin. Glucosylation serves to circumvent the inhibitory effect.

Accordingly, the recombinant host of this invention can also express a UGT polypeptide. A UGT polypeptide may be any UDP-Glucose:Aglycon-Glucosyltransferase. Preferably the UGT polypeptides can catalyze the glucosylation of vanillin (i.e., to produce vanillin beta-D-glucoside). Thus, the UGT polypeptide may be a Family 1 glycosyltransferease. Preferred UGT polypeptides according to the invention are classified under EC 2.4.1. Suitable UGT polypeptides include the UGT71C2, UGT72B1, UGT72E2, UGT84A2, UGT89B1, UGT85B1, and arbutin synthase polypeptides. See, e.g., GENBANK Accession Nos. AC0005496, NM_116337, and NM_126067. The A. thaliana UGT72E2 is particularly useful (see, e.g., Hansen, et al. (2009) supra). Thus, the recombinant host may include a heterologous nucleic acid encoding the UGT71C2, UGT72B1, UGT72E2, UGT84A2, UGT89B1, UGT85B1, or arbutin synthase or a functional homologue of any of the aforementioned sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith. Other useful UGTs are described in WO 01/40491.

As a further embodiment of this invention, a VAO enzyme (EC 1.1.3.38) can also be expressed by host cells to oxidize any residual vanillyl alcohol into vanillin. VAO enzymes are known in the art and include, but are not limited to enzymes from filamentous fungi such as Fusarium monilifomis (GENBANK Accession No. AFJ11909) and Penicillium simplicissium (GENBANK Accession No. P56216; Benen, et al. (1998) J. Biol. Chem. 273:7865-72) and bacteria such as Modestobacter marinus (GENBANK Accession No. YP_006366868), Rhodococcus jostii (GENBANK Accession No. YP_703243.1) and R. opacus (GENBANK Accession No. EHI39392).

In some cases, it is desirable to inhibit one or more functions of an endogenous polypeptide in order to divert metabolic intermediates toward vanillin or vanillin glucoside biosynthesis. For example, pyruvate decarboxylase (PDC1) and/or glutamate dehydrogenase activity can be reduced. In such cases, a nucleic acid that inhibits expression of the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain. Alternatively, mutagenesis can be used to generate mutants in genes for which it is desired to inhibit function.

To demonstrate expression and activity of one or more of the above-referenced enzymes expressed by the recombinant host, levels of products, substrates and intermediates, e.g., dehydroshikimic acid, protocatechuic acid, protocatechuic aldehyde, vanillin, and vanillin beta-D-glucoside produced by the recombinant host can be determined by extracting samples from culture media for analysis according to published methods.

Recombinant hosts described herein can be used in methods to produce vanillin or vanillin glucoside. For example, if the recombinant host is a microorganism, the method can include growing the recombinant microorganism in a culture medium under conditions in which vanillin and/or vanillin glucoside biosynthesis genes are expressed. The recombinant microorganism may be grown in a batch, fed batch or continuous process or combinations thereof. Typically, the recombinant microorganism is grown in a fermentor at a defined temperature(s) in the presence of a suitable nutrient source, e.g., a carbon source, for a desired period of time to produce a desired amount of vanillin and/or vanillin glucoside.

Therefore, this invention also provides a method for producing vanillin and/or vanillin beta-D-glucoside by providing a recombinant host that produces vanillin and/or vanillin beta-D-glucoside and has reduced production or activity of at least one (or two, three, four, five, six, seven, eight, nine or ten) alcohol dehydrogenase, at least one aldehyde reductase, or at least one alcohol dehydrogenase and at least one aldehyde reductase; cultivating said recombinant host, e.g., in the presence of a suitable carbon source, for a time sufficient for said recombinant host to produce vanillin and/or vanillin glucoside; and isolating vanillin and/or vanillin glucoside from said recombinant host or from the cultivation supernatant. In one embodiment, the recombinant host produces a reduced amount of vanillic alcohol in comparison to a host that expresses the one or more functional alcohol dehydrogenases or one or more aldehyde reductases.

In certain embodiments, it is preferred that the recombinant host expresses at least one 3DSD and at least one ACAR, which preferably may be one of the 3DSD's and ACAR's described herein. In embodiments where the recombinant host expresses an ACAR capable of catalyzing conversion of vanillic acid to vanillin, then the method may also include determining the level of generated vanillin and iso-vanillin. The recombinant host may also express at least one UGT capable of catalyzing glucosylation of vanillin and isovanillin, in which case the levels of vanillin-glucoside and iso-vanillin-glucoside may be determined instead of the levels of vanillin and iso-vanillin, respectively. Alternatively, this may be determined by generating a recombinant host harboring a heterologous nucleic acid encoding the mutant COMT polypeptide to be tested, and feeding protocatechuic acid to said recombinant host, followed by determining the level of generated iso-vanillic acid and vanillic acid.

Similarly, an in vitro assay or a recombinant host cell can be used to determine whether a mutant COMT polypeptide is capable of catalyzing methylation of an —OH group of protocatechuic aldehyde, wherein said methylation results in generation of at least X times more vanillin compared to iso-vanillin. However, in this assay, protecatechuic aldehyde is used as starting material and the level of vanillin and iso-vanillin is determined.

Likewise, an in vitro assay or a recombinant host cell can be used to determine whether a given mutant COMT polypeptide is capable of catalyzing methylation of an —OH group of protocatechuic alcohol, wherein said methylation results in generation of at least X times more vanillyl alcohol compared to iso-vanillyl alcohol. However, in this assay, protecatechuic alcohol is used as starting material and the level of vanillyl alcohol and iso-vanillyl alcohol is determined.

The level of isovanillin and vanillin may be determined by any suitable method useful for detecting these compounds, wherein said method can distinguish between isovanillin and vanillin. Such methods include for example HPLC. Similarly, the level of iso-vanillic acid, vanillic acid, iso-vanillyl alcohol and vanillyl alcohol may be determined using any suitable method useful for detecting these compounds, wherein said method can distinguish between isovanillin and vanillin. Such methods include for example HPLC.

Carbon sources of use in the method of this invention include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the vanillin and/or vanillin glucoside. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose containing polymer. In embodiments employing yeast as a host, for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.

After the recombinant host has been grown in culture for the desired period of time, vanillin and/or vanillin beta-D-glucoside can then be recovered from the culture using various techniques known in the art, e.g., isolation and purification by extraction, vacuum distillation and multi-stage re-crystallization from aqueous solutions and ultrafiltration (Böddeker, et al. (1997) J. Membrane Sci. 137:155-158; Borges da Silva, et al. (2009) Chem. Eng. Des. 87:1276-1292). Two-phase extraction processes, employing either sulphydryl compounds, such as dithiothreitol, dithioerythritol, glutathione, or L-cysteine (U.S. Pat. No. 5,128,253), or alkaline KOH solutions (WO 94/13614), have been used in the recovery of vanillin as well as for its separation from other aromatic substances. Vanillin adsorption and pervaporation from bioconverted media using polyether-polyamide copolymer membranes has also been described (Böddeker, et al. (1997) supra; Zucchi, et al. (1998) J. Microbiol. Biotechnol. 8:719-722). Macroporous adsorption resins with crosslinked-polystyrene framework have also been used to recover dissolved vanillin from aqueous solutions (Zhang, et al. (2008) Eur. Food Res. Technol. 226:377-383). Ultrafiltration and membrane contactor (MC) techniques have also been evaluated to recover vanillin (Zabkova, et al. (2007) J. Membr. Sci. 301:221-237; Scuibba, et al. (2009) Desalination 241:357-364). Alternatively, conventional techniques such as percolation or supercritical carbon dioxide extraction and reverse osmosis for concentration could be used. If the recombinant host is a plant or plant cells, vanillin or vanillin glucoside can be extracted from the plant tissue using various techniques known in the art.

In some embodiments, vanillin or vanillin beta-D-glucoside can be produced using whole cells that are fed raw materials that contain precursor molecules. The raw materials may be fed during cell growth or after cell growth. The whole cells may be in suspension or immobilized. The whole cells may be in fermentation broth or in a reaction buffer. In some embodiments a permeabilizing agent may be required for efficient transfer of substrate into the cells.

In some embodiments, the vanillin or vanillin beta-D-glucoside is isolated and purified to homogeneity (e.g., at least 90%, 92%, 94%, 96%, or 98% pure). In other embodiments, the vanillin or vanillin beta-D-glucoside is isolated as an extract from a recombinant host. In this respect, vanillin or vanillin beta-D-glucoside may be isolated, but not necessarily purified to homogeneity. Desirably, the amount of vanillin or vanillin beta-D-glucoside produced can be from about 1 mg/l to about 20,000 mg/L or higher. For example about 1 to about 100 mg/L, about 30 to about 100 mg/L, about 50 to about 200 mg/L, about 100 to about 500 mg/L, about 100 to about 1,000 mg/L, about 250 to about 5,000 mg/L, about 1,000 to about 15,000 mg/L, or about 2,000 to about 10,000 mg/L of vanillin or vanillin beta-D-glucoside can be produced. In general, longer culture times will lead to greater amounts of product. Thus, the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days.

It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant microorganisms rather than a single microorganism. When a plurality of recombinant microorganisms is used, they can be grown in a mixed culture to produce vanillin and/or vanillin glucoside.

Extracts of isolated, and optionally purified, vanillin or vanillin beta-D-glucoside find use in flavoring consumables such as food products, dietary supplements, nutraceuticals, pharmaceutical compositions, dental hygienic compositions, and cosmetic products.

The phrase “food product,” as used herein, includes, but is not limited to, fruits, vegetables, juices, meat products such as ham, bacon and sausage; egg products, fruit concentrates, gelatins and gelatin-like products such as jams, jellies, preserves, and the like; milk products such as ice cream, sour cream and sherbet; icings, syrups including molasses; corn, wheat, rye, soybean, oat, rice and barley products, nut meats and nut products, cakes, cookies, confectionaries such as candies, gums, fruit flavored drops, and chocolates, chewing gum, mints, creams, icing, ice cream, pies and breads, beverages such as coffee, tea, carbonated soft drinks, such as COKE and PEPSI, non-carbonated soft drinks, juices and other fruit drinks, sports drinks such as GATORADE, coffee, teas, iced teas, cola, alcoholic beverages, such as beers, wines and liquors, and KOOL-AID.

Food products also include condiments such as herbs, spices and seasonings, flavor enhancers. A food product also includes prepared packaged products, such as dietetic sweeteners, liquid sweeteners, granulated flavor mixes which upon reconstitution with water provide non-carbonated drinks, instant pudding mixes, instant coffee and tea, coffee whiteners, malted milk mixes, pet foods, livestock feed, tobacco, and materials for baking applications, such as powdered baking mixes for the preparation of breads, cookies, cakes, pancakes, donuts and the like. Food products also include diet or low-calorie food and beverages containing little or no sucrose. Other examples of food products envisioned in accordance with the present invention are described below and throughout the specification.

In another embodiment, the food products are fruits, vegetables, juices, meat products such as ham, bacon and sausage; egg products, fruit concentrates, gelatins and gelatin-like products such as jams, jellies, preserves, and the like; milk products such as ice cream, sour cream and sherbet; icings, syrups including molasses; corn, wheat, rye, soybean, oat, rice and barley products, nut meats and nut products, cakes, cookies, confectionaries such as candies, gums, fruit flavored drops, and chocolates, creams, icing, ice cream, pies and breads.

In another embodiment, the consumable is a pharmaceutical composition. Preferred compositions are pharmaceutical compositions containing vanillin and/or vanillin beta-D-glucoside and one or more pharmaceutically acceptable excipients. These pharmaceutical compositions can be used to formulate pharmaceutical drugs containing one or more active agents that exert a biological effect. As such, the pharmaceutical composition preferably further include one or more active agents that exert a biological effect. Such active agents include pharmaceutical and biological agents that have an activity. Such active agents are well known in the art. See, e.g., The Physician's Desk Reference. Such compositions can be prepared according to procedures known in the art, for example, as described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., USA. In one embodiment, such an active agent includes bronchodilators, anorexiants, antihistamines, nutritional supplements, laxatives, analgesics, anesthetics, antacids, H2-receptor antagonists, anticholinergics, antidiarrheals, demulcents, antitussives, antinauseants, antimicrobials, antibacterials, antifungals, antivirals, expectorants, anti-inflammatory agents, antipyretics, and mixtures thereof. In one embodiment, the active agent is an antipyretics or analgesics, e.g., ibuprofen, acetaminophen, or aspirin; laxatives, e.g., phenolphthalein dioctyl sodium sulfosuccinate; appetite depressants, e.g., amphetamines, phenylpropanolamine, phenylpropanolamine hydrochloride, or caffeine; antacidics, e.g., calcium carbonate; antiasthmatics, e.g., theophylline; antidiuretics, e.g., diphenoxylate hydrochloride; agents active against flatulence, e.g., simethecon; migraine agents, e.g., ergotaminetartrate; psychopharmacological agents, e.g., haloperidol; spasmolytics or sedatives, e.g., phenobarbitol; antihyperkinetics, e.g., methyldopa or methylphenidate; tranquilizers, e.g., benzodiazepines, hydroxinmeprobramates or phenothiazines; antihistaminics, e.g., astemizol, chloropheniramine maleate, pyridamine maleate, doxlamine succinate, bromopheniramine maleate, phenyltoloxamine citrate, chlorocyclizine hydrochloride, pheniramine maleate, and phenindamine tartrate; decongestants, e.g., phenylpropanolamine hydrochloride, phenylephrine hydrochloride, pseudoephedrine hydrochloride, pseudoephedrine sulfate, phenylpropanolamine bitartrate, and ephedrine; beta-receptor blockers, e.g., propanolol; agents for alcohol withdrawal, e.g., disulfiram; antitussives, e.g., benzocaine, dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, and chlophedianol hydrochloride; fluorine supplements, e.g., sodium fluoride; local antibiotics, e.g., tetracycline or cleocine; corticosteroid supplements, e.g., prednisone or prednisolone; agents against goiter formation, e.g., colchicine or allopurinol; antiepileptics, e.g., phenytoine sodium; agents against dehydration, e.g., electrolyte supplements; antiseptics, e.g., cetylpyridinium chloride; NSAIDs, e.g., acetaminophen, ibuprofen, naproxen, or salts thereof; gastrointestinal active agents, e.g., loperamide and famotidine; various alkaloids, e.g., codeine phosphate, codeine sulfate, or morphine; supplements for trace elements, e.g., sodium chloride, zinc chloride, calcium carbonate, magnesium oxide, and other alkali metal salts and alkali earth metal salts; vitamins; ion-exchange resins, e.g., cholestyramine; cholesterol-depressant and lipid-lowering substances; antiarrhythmics, e.g., N-acetylprocainamide; and expectorants, e.g., guaifenesin.

Active substances which have a particularly unpleasant taste include antibacterial agents such as ciprofloxacin, ofloxacin, and pefloxacin; antiepileptics such as zonisamide; macrolide antibiotics such as erythromycin; beta-lactam antibiotics such as penicillins and cephalosporins; psychotropic active substances such as chlorpromazine; active substances such as sulpyrine; and agents active against ulcers, such as cimetidine.

The pharmaceutical compositions of this invention are administered to a subject in any form suitable to achieve their intended purpose. Preferably, however, the composition is one which can be administered buccally or orally. Alternatively, the pharmaceutical composition can be an oral or nasal spray. The subject is any animal, such as a human, although the invention is not intended to be so limited. Other suitable animals include canines, felines, dogs, cats, livestock, horses, cattle, sheep, and the like. A veterinary composition, as used herein, refers to a pharmaceutical composition that suitable for non-human animals. Such veterinary compositions are known in the art.

In another embodiment, the pharmaceutical composition is a liquid dosage form for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

The pharmaceutical composition of the present invention can be in the form of a chewable tablet. Chewable tablets are known in the art. See, e.g., U.S. Pat. Nos. 4,684,534 and 6,060,078, each of which is incorporated by reference in its entirety. Any kind of medicament can be contained in the chewable tablet, preferably a medicament of bitter taste, natural plant extracts or other organic compounds. More preferably, vitamins such as vitamin A, vitamin B, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin E and vitamin K; natural plant extracts such as Sohgunjung-tang extracts, Sipchundaebo-tang extracts and Eleutherococcus senticosus extracts; organic compounds such as dimenhydrinate, meclazine, acetaminophen, aspirin, phenylpropanolamine, and cetylpyridinium chloride; or gastrointestinal agents such as dried aluminum hydroxide gel, domperidone, soluble azulene, L-glutamine and hydrotalcite can be contained in the core.

The pharmaceutical composition of the present invention can be an orally disintegrating composition. Orally disintegrating tablets are known in the art. See, e.g., U.S. Pat. Nos. 6,368,625 and 6,316,029, each of which is hereby incorporated by reference in its entirety.

The pharmaceutical composition of the present invention can be a solid dosage form, including vanillin or vanillin beta-D-glucoside and a water and/or saliva activated effervescent granule, such as one having a controllable rate of effervescence. The effervescent composition can further comprise a pharmaceutically active compound. Effervescent pharmaceutical compositions are known in the art. See, e.g., U.S. Pat. No. 6,649,186, which is incorporated by reference in its entirety. The effervescent composition can be used in pharmaceutical, veterinary, horticultural, household, food, culinary, pesticidal, agricultural, cosmetic, herbicidal, industrial, cleansing, confectionery and flavoring applications. Formulations incorporating the effervescent composition containing vanillin or vanillin beta-D-glucoside can further include one or more additional adjuvants and/or active ingredients which can be chosen from those known in the art, including flavors, diluents, colors, binders, filler, surfactant, disintegrant, stabilizer, compaction vehicles, and non-effervescent disintegrants.

The pharmaceutical composition can be a film-shaped or wafer-shaped pharmaceutical composition. Such a film-shaped or wafer-shaped pharmaceutical composition can be configured, for example, as quickly disintegrating administration forms, e.g., administration forms disintegrating within a period of 1 second up to 3 minutes, or as slowly disintegrating administration forms, e.g., administration forms disintegrating within a period of 3 to 15 minutes. The indicated disintegration times can be set to the above-mentioned ranges by using, for example, matrix-forming polymers which have different disintegrating, or solubility, characteristics. Thus, by mixing the corresponding polymer components, the disintegration time can be adjusted. In addition, disintegrants are known which “draw” water into the matrix and cause the matrix to burst open from within. As a consequence, certain embodiments of the invention include such disintegrants for the purpose of adjusting the disintegration time.

Suitable are polymers for use in the film-shaped or wafer-shaped pharmaceutical composition include cellulose derivatives, polyvinyl alcohol (e.g. MOWIOL), polyacrylates, polyvinyl pyrrolidone, cellulose ethers, such as ethyl cellulose, as well as polyvinyl alcohol, polyurethane, polymethacrylates, polymethyl methacrylates and derivatives and copolymerizates of the aforementioned polymers.

In certain embodiments, the total thickness of the film-shaped or wafer-shaped pharmaceutical composition according to the invention is preferably 5 μm up to 10 mm, preferably 30 μm to 2 mm, and with particular preference 0.1 mm to 1 mm. The pharmaceutical preparations can be round, oval, elliptic, triangular, quadrangular or polygonal shape, but they can also have any rounded shape.

The pharmaceutical composition of the present invention can be in the form of an aerosol. The aerosol composition can further include a pharmaceutically active agent. Aerosol compositions are known in the art. See, e.g., U.S. Pat. No. 5,011,678, which is hereby incorporated by reference in its entirety. As a nonlimiting example, an aerosol composition according to the present invention can include a medically effective amount of a pharmaceutically active substance, vanillin or vanillin beta-D-glucoside and a biocompatible propellant, such as a (hydro/fluoro)carbon propellant.

In one embodiment of the present invention, the pharmaceutical composition is a nutritional composition. Examples of nutritional compositions having an undesirable taste include, but are not necessarily limited to, enteral nutrition products for treatment of nutritional deficit, trauma, surgery, Crohn's disease, renal disease, hypertension, obesity and the like, to promote athletic performance, muscle enhancement or general well being or inborn errors of metabolism such as phenylketonuria. In particular, such nutritional formulations can contain one or more amino acids which have a bitter or metallic taste or aftertaste. Such amino acids include, but are not limited to, an essential amino acids such as an L isomer of leucine, isoleucine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine or valine.

In one embodiment, the consumable of the present invention is a dental hygienic composition containing vanillin and/or vanillin beta-D-glucoside. Dental hygienic compositions are known in the art and include, but are not necessarily limited to, toothpaste, mouthwash, plaque rinse, dental floss, dental pain relievers (such as ANBESOL), and the like.

In another embodiment, the consumable of the present invention is a cosmetic product containing vanillin and/or vanillin beta-D-glucoside. For example, but not by way of limitation, the cosmetic product can be a face cream, lipstick, lip gloss, and the like. Other suitable compositions of the invention include lip balm, such as CHAPSTICK or BURT'S BEESWAX Lip Balm, further containing vanillin and/or vanillin beta-D-glucoside.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: Yeast Strains with Reduced Alcohol Dehydrogenase/Aldehyde Reductase Activity

Yeast strains lacking alcohol dehydrogenase and/or aldehyde reductase were produced in a parent strain of yeast that was capable of producing vanillin/vanillin beta-D-glucoside. The parent strain, EFSC2932, was created by integrating exogenous OMT, UGT and 3DSD genes into the genome of strain EFSC2055 (Genotype: Mata his3D1 leu2D0 met15D0 ura3D0 adh6::LEU2 bgl1::KanMX4 PTPI1::3DSD[AurC]::(HsOMT::MET15[NatMX])::ACAR[HphMX]::UGT7 2E2[HIS3] ECM3::(CorPPTase-ScHAP4). Genes encoding OMT, UGT and 3DSD were amplified by polymerase chain reaction (PCR) with gene-specific primers using X7 DNA polymerase. Vectors for chromosomal integration of gene expression were constructed using the uracil-specific excision reagent (USER) cloning method and a vector system adapted from Mikkelsen, et al. ((2012) Metab. Eng. 14:104-111). The genes were fused to Ashbya gossypii TEF1 promoter and S. cerevisiae PGK1 promoter by USER cloning. The resulting constructs were integrated into the following chromosome positions: XII-1 (pTEF1::HsOMT L198Y::pPGK::HsOMTDNA20), XII-2 (pTEF1::HsOMT L198Y+pPGK::UGT72E2) and X-2 (pTEF1::HsOMT L198Y+pPGK::Pa3DSD).

Candidate genes encoding alcohol dehydrogenases or aldehyde reductases were selected and knocked out in various combinations. Cultures of each knockout were analyzed using HPLC-UV to quantify vanillin glucoside/isovanillin glucoside and related products. HPLC analysis was carried out with an AGILENT 1100 series system with binary pump and a Phenomenex Synergi Polar-RP 2.5 u 100 Å 100×2.00 mm column, which separates precursors and Isovanillin and vanillin. A flat gradient was run with water/acetonitrile+0.1% trifluoroacetic acid. A 8.9 minute program+1.1 minute postrun was carried out as presented in Table 2.


TABLE 2
Time
% Acetonitrile
Flow ml/min.
0
5
0.5
0.7
5
0.5
5.7
27
0.5
6.2
100
0.5
6.6
100
0.7
7.8
100
1.0
8.1
100
1.0
8.6
5
0.8
8.9
5
0.6

Vanillin glucoside and isovanillin glucoside were quantified by integrating the area of the HPLC peaks and comparing the same with a standard curve. The combinations of genes disrupted and the resulting effect on vanillyl alcohol production are provided in Table 3.


TABLE 3
Strain
Gene Disruption
Designation
1
2
3
Result
EFSC2906
adh6
adh7
adh5
No effect
EFSC2907
adh6
adh7
gre3
No effect
EFSC2908
adh6
adh7
ypr127w
No effect
EFSC2909
adh6
adh7
ycr102c
No effect
EFSC2911
adh6
adh7
ari1
No effect
EFSC2912
adh6
adh7
zta1
No effect
EFSC2913
adh6
adh7
ycr102c
No effect
EFSC2929
adh6
ari1
ygl039w
Some
effect
EFSC2930
adh6
adh7
ylr460c
No effect
EFSC2931
adh6
adh7
ygl039w
No effect
EFSC2932
adh6
adh7
gre2
Vanillyl
alcohol
reduced by
77%

This analysis indicated that, when knocked out, the combination of adh6, adh7 and gre2 provided a 77% decrease in vanillyl alcohol and vanillyl alcohol glucoside production (see FIG. 2).

To further analyze the effect of aril, the aril and gre2 genes were deleted in strain EFSC2055 containing adh6 and adh7 gene knockouts. Strain EFSC2055 and the derived single (EFSC2055 gre2) and double mutant (EFSC 2055 gre2 aril) strains were cultivated for three days in Delft-molasses media (Delft+2% glucose+4% Cane molasses) containing 3 mM of vanillin. EFSC2055 and derived strains encompass the UDP-glycosyltransferase UGT72E2 which is capable of glycosylating vanillin and vanillyl alcohol. Reduction of vanillin to vanillyl alcohol and vanillyl alcohol glycoside was quantified by HPLC-UV. As shown in FIG. 3, the gre2 deletion alone significantly reduced the formation of vanillyl alcohol glycoside from vanillin (t-test, P<0.01). However, the double deletion of both gre2 and aril in strain EFSC2055 completely abolished formation of vanillyl alcohol glycoside and significantly reduced formation of vanillyl alcohol (t-test, P<0.01).

Given the observed effect of aril and YGL039W deletions, it is contemplated that knocking out one or both of these genes in combination with adh6, adh7 or gre2 (e.g., adh6/adh7/gre2/YGL039W; adh6/adh7/gre2/aril/YGL039W; adh6/gre2/aril; adh6/gre2/YGL039W; adh6/gre2/aril/YGL039W) will also yield a decrease in vanillyl alcohol and vanillyl alcohol glucoside production.

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Met Leu Tyr Pro Glu Lys Phe Gln Gly Ile Gly Ile Ser Asn Ala Lys

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Asp Trp Lys His Pro Lys Leu Val Ser Phe Asp Pro Lys Pro Phe Gly

20 25 30

Asp His Asp Val Asp Val Glu Ile Glu Ala Cys Gly Ile Cys Gly Ser

35 40 45

Asp Phe His Ile Ala Val Gly Asn Trp Gly Pro Val Pro Glu Asn Gln

50 55 60

Ile Leu Gly His Glu Ile Ile Gly Arg Val Val Lys Val Gly Ser Lys

65 70 75 80

Cys His Thr Gly Val Lys Ile Gly Asp Arg Val Gly Val Gly Ala Gln

85 90 95

Ala Leu Ala Cys Phe Glu Cys Glu Arg Cys Lys Ser Asp Asn Glu Gln

100 105 110

Tyr Cys Thr Asn Asp His Val Leu Thr Met Trp Thr Pro Tyr Lys Asp

115 120 125

Gly Tyr Ile Ser Gln Gly Gly Phe Ala Ser His Val Arg Leu His Glu

130 135 140

His Phe Ala Ile Gln Ile Pro Glu Asn Ile Pro Ser Pro Leu Ala Ala

145 150 155 160

Pro Leu Leu Cys Gly Gly Ile Thr Val Phe Ser Pro Leu Leu Arg Asn

165 170 175

Gly Cys Gly Pro Gly Lys Arg Val Gly Ile Val Gly Ile Gly Gly Ile

180 185 190

Gly His Met Gly Ile Leu Leu Ala Lys Ala Met Gly Ala Glu Val Tyr

195 200 205

Ala Phe Ser Arg Gly His Ser Lys Arg Glu Asp Ser Met Lys Leu Gly

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Ala Asp His Tyr Ile Ala Met Leu Glu Asp Lys Gly Trp Thr Glu Gln

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Tyr Ser Asn Ala Leu Asp Leu Leu Val Val Cys Ser Ser Ser Leu Ser

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305 310 315 320

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325 330 335

Ala Phe Thr Arg Met Glu Ser Gly Asp Val Lys Tyr Arg Phe Thr Leu

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Val Asp Tyr Asp Lys Lys Phe His Lys

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tgccacactg gggtaaaaat cggtgaccgt gttggtgttg gtgcccaagc cttggcgtgt 300

tttgagtgtg aacgttgcaa aagtgacaac gagcaatact gtaccaatga ccacgttttg 360

actatgtgga ctccttacaa ggacggctac atttcacaag gaggctttgc ctcccacgtg 420

aggcttcatg aacactttgc tattcaaata ccagaaaata ttccaagtcc gctagccgct 480

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115 120 125

Ala Ala Val Phe Asp Met Ala Lys Glu Asn Asp Lys Ser Leu Thr Phe

130 135 140

Asn Glu Glu Ser Trp Asn Pro Ala Thr Trp Glu Ser Cys Gln Ser Asp

145 150 155 160

Pro Val Asn Ala Tyr Cys Gly Ser Lys Lys Phe Ala Glu Lys Ala Ala

165 170 175

Trp Glu Phe Leu Glu Glu Asn Arg Asp Ser Val Lys Phe Glu Leu Thr

180 185 190

Ala Val Asn Pro Val Tyr Val Phe Gly Pro Gln Met Phe Asp Lys Asp

195 200 205

Val Lys Lys His Leu Asn Thr Ser Cys Glu Leu Val Asn Ser Leu Met

210 215 220

His Leu Ser Pro Glu Asp Lys Ile Pro Glu Leu Phe Gly Gly Tyr Ile

225 230 235 240

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245 250 255

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260 265 270

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275 280 285

Asn Ile Pro Val Gly Lys Pro Gly Ser Gly Ala Thr His Asn Thr Leu

290 295 300

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305 310 315 320

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325 330 335

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340

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acggaggcct ttggtaacaa cccaaaattc tccatggaag ttgtcccaga catatctaag 180

ctggacgcat ttgaccatgt tttccaaaag cacggcaagg atatcaagat agttctacat 240

acggcctctc cattctgctt tgatatcact gacagtgaac gcgatttatt aattcctgct 300

gtgaacggtg ttaagggaat tctccactca attaaaaaat acgccgctga ttctgtagaa 360

cgtgtagttc tcacctcttc ttatgcagct gtgttcgata tggcaaaaga aaacgataag 420

tctttaacat ttaacgaaga atcctggaac ccagctacct gggagagttg ccaaagtgac 480

ccagttaacg cctactgtgg ttctaagaag tttgctgaaa aagcagcttg ggaatttcta 540

gaggagaata gagactctgt aaaattcgaa ttaactgccg ttaacccagt ttacgttttt 600

ggtccgcaaa tgtttgacaa agatgtgaaa aaacacttga acacatcttg cgaactcgtc 660

aacagcttga tgcatttatc accagaggac aagataccgg aactatttgg tggatacatt 720

gatgttcgtg atgttgcaaa ggctcattta gttgccttcc aaaagaggga aacaattggt 780

caaagactaa tcgtatcgga ggccagattt actatgcagg atgttctcga tatccttaac 840

gaagacttcc ctgttctaaa aggcaatatt ccagtgggga aaccaggttc tggtgctacc 900

cataacaccc ttggtgctac tcttgataat aaaaagagta agaaattgtt aggtttcaag 960

ttcaggaact tgaaagagac cattgacgac actgcctccc aaattttaaa atttgagggc 1020

agaatataa 1029

<210> SEQ ID NO: 7

<211> LENGTH: 344

<212> TYPE: PRT

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQENCE: 7

Met Ser Asn Thr Val Leu Val Ser Gly Ala Ser Gly Phe Ile Ala Leu

1 5 10 15

His Ile Leu Ser Gln Leu Leu Lys Gln Asp Tyr Lys Val Ile Gly Thr

20 25 30

Val Arg Ser His Glu Lys Glu Ala Lys Leu Leu Arg Gln Phe Gln His

35 40 45

Asn Pro Asn Leu Thr Leu Glu Ile Val Pro Asp Ile Ser His Pro Asn

50 55 60

Ala Phe Asp Lys Val Leu Gln Lys Arg Gly Arg Glu Ile Arg Tyr Val

65 70 75 80

Leu His Thr Ala Ser Pro Phe His Tyr Asp Thr Thr Glu Tyr Glu Lys

85 90 95

Asp Leu Leu Ile Pro Ala Leu Glu Gly Thr Lys Asn Ile Leu Asn Ser

100 105 110

Ile Lys Lys Tyr Ala Ala Asp Thr Val Glu Arg Val Val Val Thr Ser

115 120 125

Ser Cys Thr Ala Ile Ile Thr Leu Ala Lys Met Asp Asp Pro Ser Val

130 135 140

Val Phe Thr Glu Glu Ser Trp Asn Glu Ala Thr Trp Glu Ser Cys Gln

145 150 155 160

Ile Asp Gly Ile Asn Ala Tyr Phe Ala Ser Lys Lys Phe Ala Glu Lys

165 170 175

Ala Ala Trp Glu Phe Thr Lys Glu Asn Glu Asp His Ile Lys Phe Lys

180 185 190

Leu Thr Thr Val Asn Pro Ser Leu Leu Phe Gly Pro Gln Leu Phe Asp

195 200 205

Glu Asp Val His Gly His Leu Asn Thr Ser Cys Glu Met Ile Asn Gly

210 215 220

Leu Ile His Thr Pro Val Asn Ala Ser Val Pro Asp Phe His Ser Ile

225 230 235 240

Phe Ile Asp Val Arg Asp Val Ala Leu Ala His Leu Tyr Ala Phe Gln

245 250 255

Lys Glu Asn Thr Ala Gly Lys Arg Leu Val Val Thr Asn Gly Lys Phe

260 265 270

Gly Asn Gln Asp Ile Leu Asp Ile Leu Asn Glu Asp Phe Pro Gln Leu

275 280 285

Arg Gly Leu Ile Pro Leu Gly Lys Pro Gly Thr Gly Asp Gln Val Ile

290 295 300

Asp Arg Gly Ser Thr Thr Asp Asn Ser Ala Thr Arg Lys Ile Leu Gly

305 310 315 320

Phe Glu Phe Arg Ser Leu His Glu Ser Val His Asp Thr Ala Ala Gln

325 330 335

Ile Leu Lys Lys Gln Asn Arg Leu

340

<210> SEQ ID NO: 8

<211> LENGTH: 1035

<212> TYPE: DNA

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQENCE: 8

atgtctaata cagttctagt ttctggcgct tcaggtttta ttgccttgca tatcctgtca 60

caattgttaa aacaagatta taaggttatt ggaactgtga gatcccatga aaaagaagca 120

aaattgctaa gacaatttca acataaccct aatttaactt tagaaattgt tccggacatt 180

tctcatccaa atgctttcga taaggttctg cagaaacgtg gacgtgagat taggtatgtt 240

ctacacacgg cctctccttt tcattatgat actaccgaat atgaaaaaga cttattgatt 300

cccgcgttag aaggtacaaa aaacatccta aattctatca agaaatatgc agcagacact 360

gtagagcgtg ttgttgtgac ttcttcttgt actgctatta taacccttgc aaagatggac 420

gatcccagtg tggtttttac agaagagagt tggaacgaag caacctggga aagctgtcaa 480

attgatggga taaatgctta ctttgcatcc aagaagtttg ctgaaaaggc tgcctgggag 540

ttcacaaaag agaatgaaga tcacatcaaa ttcaaactaa caacagtcaa cccttctctt 600

ctttttggtc ctcaactttt cgatgaagat gtgcatggcc atttgaatac ttcttgcgaa 660

atgatcaatg gcctaattca taccccagta aatgccagtg ttcctgattt tcattccatt 720

tttattgatg taagggatgt ggccctagct catctgtatg ctttccagaa ggaaaatacc 780

gcgggtaaaa gattagtggt aactaacggt aaatttggaa accaagatat cctggatatt 840

ttgaacgaag attttccaca attaagaggt ctcattcctt tgggtaagcc tggcacaggt 900

gatcaagtca ttgaccgcgg ttcaactaca gataatagtg caacgaggaa aatacttggc 960

tttgagttca gaagtttaca cgaaagtgtc catgatactg ctgcccaaat tttgaagaag 1020

cagaacagat tatga 1035

<210> SEQ ID NO: 9

<211> LENGTH: 347

<212> TYPE: PRT

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQENCE: 9

Met Thr Thr Asp Thr Thr Val Phe Val Ser Gly Ala Thr Gly Phe Ile

1 5 10 15

Ala Leu His Ile Met Asn Asp Leu Leu Lys Ala Gly Tyr Thr Val Ile

20 25 30

Gly Ser Gly Arg Ser Gln Glu Lys Asn Asp Gly Leu Leu Lys Lys Phe

35 40 45

Asn Asn Asn Pro Lys Leu Ser Met Glu Ile Val Glu Asp Ile Ala Ala

50 55 60

Pro Asn Ala Phe Asp Glu Val Phe Lys Lys His Gly Lys Glu Ile Lys

65 70 75 80

Ile Val Leu His Thr Ala Ser Pro Phe His Phe Glu Thr Thr Asn Phe

85 90 95

Glu Lys Asp Leu Leu Thr Pro Ala Val Asn Gly Thr Lys Ser Ile Leu

100 105 110

Glu Ala Ile Lys Lys Tyr Ala Ala Asp Thr Val Glu Lys Val Ile Val

115 120 125

Thr Ser Ser Thr Ala Ala Leu Val Thr Pro Thr Asp Met Asn Lys Gly

130 135 140

Asp Leu Val Ile Thr Glu Glu Ser Trp Asn Lys Asp Thr Trp Asp Ser

145 150 155 160

Cys Gln Ala Asn Ala Val Ala Ala Tyr Cys Gly Ser Lys Lys Phe Ala

165 170 175

Glu Lys Thr Ala Trp Glu Phe Leu Lys Glu Asn Lys Ser Ser Val Lys

180 185 190

Phe Thr Leu Ser Thr Ile Asn Pro Gly Phe Val Phe Gly Pro Gln Met

195 200 205

Phe Ala Asp Ser Leu Lys His Gly Ile Asn Thr Ser Ser Gly Ile Val

210 215 220

Ser Glu Leu Ile His Ser Lys Val Gly Gly Glu Phe Tyr Asn Tyr Cys

225 230 235 240

Gly Pro Phe Ile Asp Val Arg Asp Val Ser Lys Ala His Leu Val Ala

245 250 255

Ile Glu Lys Pro Glu Cys Thr Gly Gln Arg Leu Val Leu Ser Glu Gly

260 265 270

Leu Phe Cys Cys Gln Glu Ile Val Asp Ile Leu Asn Glu Glu Phe Pro

275 280 285

Gln Leu Lys Gly Lys Ile Ala Thr Gly Glu Pro Ala Thr Gly Pro Ser

290 295 300

Phe Leu Glu Lys Asn Ser Cys Lys Phe Asp Asn Ser Lys Thr Lys Lys

305 310 315 320

Leu Leu Gly Phe Gln Phe Tyr Asn Leu Lys Asp Cys Ile Val Asp Thr

325 330 335

Ala Ala Gln Met Leu Glu Val Gln Asn Glu Ala

340 345

<210> SEQ ID NO: 10

<211> LENGTH: 1044

<212> TYPE: DNA

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQENCE: 10

atgactactg ataccactgt tttcgtttct ggcgcaaccg gtttcattgc tctacacatt 60

atgaacgatc tgttgaaagc tggctataca gtcatcggct caggtagatc tcaagaaaaa 120

aatgatggct tgctcaaaaa atttaataac aatcccaaac tatcgatgga aattgtggaa 180

gatattgctg ctccaaacgc ctttgatgaa gttttcaaaa aacatggtaa ggaaattaag 240

attgtgctac acactgcctc cccattccat tttgaaacta ccaattttga aaaggattta 300

ctaacccctg cagtgaacgg tacaaaatct atcttggaag cgattaaaaa atatgctgca 360

gacactgttg aaaaagttat tgttacttcg tctactgctg ctctggtgac acctacagac 420

atgaacaaag gagatttggt gatcacggag gagagttgga ataaggatac atgggacagt 480

tgtcaagcca acgccgttgc cgcatattgt ggctcgaaaa agtttgctga aaaaactgct 540

tgggaatttc ttaaagaaaa caagtctagt gtcaaattca cactatccac tatcaatccg 600

ggattcgttt ttggtcctca aatgtttgca gattcgctaa aacatggcat aaatacctcc 660

tcagggatcg tatctgagtt aattcattcc aaggtaggtg gagaatttta taattactgt 720

ggcccattta ttgacgtgcg tgacgtttct aaagcccacc tagttgcaat tgaaaaacca 780

gaatgtaccg gccaaagatt agtattgagt gaaggtttat tctgctgtca agaaatcgtt 840

gacatcttga acgaggaatt ccctcaatta aagggcaaga tagctacagg tgaacctgcg 900

accggtccaa gctttttaga aaaaaactct tgcaagtttg acaattctaa gacaaaaaaa 960

ctactgggat tccagtttta caatttaaag gattgcatag ttgacaccgc ggcgcaaatg 1020

ttagaagttc aaaatgaagc ctaa 1044

<210> SEQ ID NO: 11

<211> LENGTH: 348

<212> TYPE: PRT

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQENCE: 11

Met Thr Thr Glu Lys Thr Val Val Phe Val Ser Gly Ala Thr Gly Phe

1 5 10 15

Ile Ala Leu His Val Val Asp Asp Leu Leu Lys Thr Gly Tyr Lys Val

20 25 30

Ile Gly Ser Gly Arg Ser Gln Glu Lys Asn Asp Gly Leu Leu Lys Lys

35 40 45

Phe Lys Ser Asn Pro Asn Leu Ser Met Glu Ile Val Glu Asp Ile Ala

50 55 60

Ala Pro Asn Ala Phe Asp Lys Val Phe Gln Lys His Gly Lys Glu Ile

65 70 75 80

Lys Val Val Leu His Ile Ala Ser Pro Val His Phe Asn Thr Thr Asp

85 90 95

Phe Glu Lys Asp Leu Leu Ile Pro Ala Val Asn Gly Thr Lys Ser Ile

100 105 110

Leu Glu Ala Ile Lys Asn Tyr Ala Ala Asp Thr Val Glu Lys Val Val

115 120 125

Ile Thr Ser Ser Val Ala Ala Leu Ala Ser Pro Gly Asp Met Lys Asp

130 135 140

Thr Ser Phe Val Val Asn Glu Glu Ser Trp Asn Lys Asp Thr Trp Glu

145 150 155 160

Ser Cys Gln Ala Asn Ala Val Ser Ala Tyr Cys Gly Ser Lys Lys Phe

165 170 175

Ala Glu Lys Thr Ala Trp Asp Phe Leu Glu Glu Asn Gln Ser Ser Ile

180 185 190

Lys Phe Thr Leu Ser Thr Ile Asn Pro Gly Phe Val Phe Gly Pro Gln

195 200 205

Leu Phe Ala Asp Ser Leu Arg Asn Gly Ile Asn Ser Ser Ser Ala Ile

210 215 220

Ile Ala Asn Leu Val Ser Tyr Lys Leu Gly Asp Asn Phe Tyr Asn Tyr

225 230 235 240

Ser Gly Pro Phe Ile Asp Val Arg Asp Val Ser Lys Ala His Leu Leu

245 250 255

Ala Phe Glu Lys Pro Glu Cys Ala Gly Gln Arg Leu Phe Leu Cys Glu

260 265 270

Asp Met Phe Cys Ser Gln Glu Ala Leu Asp Ile Leu Asn Glu Glu Phe

275 280 285

Pro Gln Leu Lys Gly Lys Ile Ala Thr Gly Glu Pro Gly Ser Gly Ser

290 295 300

Thr Phe Leu Thr Lys Asn Cys Cys Lys Cys Asp Asn Arg Lys Thr Lys

305 310 315 320

Asn Leu Leu Gly Phe Gln Phe Asn Lys Phe Arg Asp Cys Ile Val Asp

325 330 335

Thr Ala Ser Gln Leu Leu Glu Val Gln Ser Lys Ser

340 345

<210> SEQ ID NO: 12

<211> LENGTH: 1047

<212> TYPE: DNA

<213> ORGANISM: Saccharomyces cerevisiae

<400> SEQENCE: 12

atgactactg aaaaaaccgt tgtttttgtt tctggtgcta ctggtttcat tgctctacac 60

gtagtggacg atttattaaa aactggttac aaggtcatcg gttcgggtag gtcccaagaa 120

aagaatgatg gattgctgaa aaaatttaag agcaatccca acctttcaat ggagattgtc 180

gaagacattg ctgctccaaa cgcttttgac aaagtttttc aaaagcacgg caaagagatc 240

aaggttgtct tgcacatagc ttctccggtt cacttcaaca ccactgattt cgaaaaggat 300

ctgctaattc ctgctgtgaa tggtaccaag tccattctag aagcaatcaa aaattatgcc 360

gcagacacag tcgaaaaagt cgttattact tcttctgttg ctgcccttgc atctcccgga 420

gatatgaagg acactagttt cgttgtcaat gaggaaagtt ggaacaaaga tacttgggaa 480

agttgtcaag ctaacgcggt ttccgcatac tgtggttcca agaaatttgc tgaaaaaact 540

gcttgggatt ttctcgagga aaaccaatca agcatcaaat ttacgctatc aaccatcaac 600

ccaggatttg tttttggccc tcagctattt gccgactctc ttagaaatgg aataaatagc 660

tcttcagcca ttattgccaa tttggttagt tataaattag gcgacaattt ttataattac 720

agtggtcctt ttattgacgt tcgcgatgtt tcaaaagctc atttacttgc atttgagaaa 780

cccgaatgcg ctggccaaag actattctta tgtgaagata tgttttgctc tcaagaagcg 840

ctggatatct tgaatgagga atttccacag ttaaaaggca agatagcaac tggcgaacct 900

ggtagcggct caaccttttt gacaaaaaac tgctgcaagt gcgacaaccg caaaaccaaa 960

aatttattag gattccaatt taataagttc agagattgca ttgtcgatac tgcctcgcaa 1020

ttactagaag ttcaaagtaa aagctaa 1047

<210> SEQ ID NO: 13

<211> LENGTH: 336

<212> TYPE: PRT

<213> ORGANISM: Schizosaccharomyces pombe

<400> SEQENCE: 13

Met Ser Gly Lys Leu Val Leu Val Thr Gly Val Thr Gly Phe Ile Gly

1 5 10 15

Ala His Val Ala Glu Gln Leu Leu Gln Ala Gly Tyr Arg Val Arg Gly

20 25 30

Thr Val Arg Ser Met Glu Lys Ala Asp Glu Leu Ile Arg Leu Asn Pro

35 40 45

Gly Leu Lys Asp Lys Ile Glu Phe Val Ile Val Lys Asp Val Ser Ala

50 55 60

Ser Asn Ala Phe Asp Gly Val Leu Lys Asp Val Glu Leu Ile Cys His

65 70 75 80

Ile Ala Ser Pro Phe Phe Val Glu Asn Val Thr Asp Asn Lys Ser Gln

85 90 95

Leu Leu Asp Pro Ala Val Lys Gly Thr Leu Gly Ile Leu Glu Ala Ala

100 105 110

Gln Gly Val Lys Ser Ile Lys Arg Ile Val Ile Thr Ser Ser Phe Ala

115 120 125

Ala Val Gly Asn Phe Gln Ile Asp Pro His Asn Asn Lys Val Tyr Thr

130 135 140

Glu Lys Asp Trp Asn Pro Ile Thr Tyr Glu Glu Ala Leu Thr Thr Asp

145 150 155 160

Asn Gly Ile Val Ala Tyr Cys Ala Ser Lys Lys Leu Ala Glu Glu Ala

165 170 175

Ala Arg Glu Tyr Val Lys Glu Lys Lys Pro Ser Tyr Asp Ile Cys Thr

180 185 190

Ile Asn Pro Pro Tyr Val Tyr Gly Pro Pro Ile His Pro Met Lys Asn

195 200 205

Met Asp Ser Leu Asn Thr Ser Asn Gln Ile Phe Trp Lys Leu Ile Asp

210 215 220

Gly Ser Lys Glu Ala Thr Pro Phe Tyr Tyr Tyr Tyr Val Asp Val Arg

225 230 235 240

Asp Val Ala Ala Ala His Val Phe Ala Leu Glu Asn Ala Lys Leu Ser

245 250 255

Asn Gly Arg Met Leu Val Ser Lys Gly Val Phe Thr Thr Gly Asp Ile

260 265 270

Cys Lys Val Leu Arg Lys Glu Phe Pro Asn Lys Ser Asp Val Ile Ala

275 280 285

Glu Pro Val Asp Ile Thr Val Asp Pro Ser Phe Phe Lys Leu Asp Asn

290 295 300

Ser Phe Ser Lys Ser Leu Gly Phe Lys Tyr His Ser Asp Glu Glu Cys

305 310 315 320

Tyr Val Asp Thr Ala Lys Lys Leu Trp Glu Arg Ala Glu Glu Phe Lys

325 330 335

<210> SEQ ID NO: 14

<211> LENGTH: 1759

<212> TYPE: DNA

<213> ORGANISM: Schizosaccharomyces pombe

<400> SEQENCE: 14

tccataaatc tgaaaactgc tttttatatc tttttaccat gaatagaata gctgtatgcc 60

aaagcgggat actaaattta cacaaccatt tattttcatt ttctttctaa tttgtattaa 120

caaaactttt attgctcaga agcaatagct gaccattgtt tacatcccta cttctttact 180

aaatttccgt attacgaaac tctcctgtat tcaacaagtt ttgtgagact tttactataa 240

tgcgtaagaa atggcagaac gaaaacttga gaaagtcaag ttatccattt cacgtcatgt 300

tatagcccat taataatatt atatttgatt actaaaaaaa tgggaacaat cggatgcgca 360

acttccgaag aaaaatatgg aacaaatttt tcgttgccta ctcattttct tatatattac 420

aaggaaagtc tacttccatt gtcaacaagt gatatttcag gtataatcac ttatattttt 480

tagaacaaac acaaaacctt caaagatgag tggaaaattg gtccttgtta ctggtgttac 540

aggatttatt ggagcccacg tcgctgagca gctcctgcaa gccggctacc gtgtgcgtgg 600

tacggtgaga agcatggaaa aggcagacga gcttattaga ctcaatcccg gattaaagga 660

taaaattgag tttgtaatcg tcaaggatgt ctctgcttct aacgcttttg acggtgtttt 720

gaaagatgtg gaattgattt gtcatatagc atcaccattc tttgtggaaa acgttactga 780

caacaaatcc caacttttgg accctgccgt gaagggtact ctcggcattt tagaggctgc 840

tcaaggcgta aagagtatta agcgcattgt tattacttca tcttttgccg cagtgggcaa 900

tttccaaatc gaccctcata acaataaggt gtatactgag aaggattgga atcccattac 960

ctatgaggag gcattgacca ctgataatgg cattgtagca tactgtgctt cgaagaaact 1020

cgccgaagag gctgctcgag agtatgtaaa ggaaaagaag ccttcttatg atatttgtac 1080

gatcaatcct ccttatgtgt atggtccacc aattcaccct atgaaaaata tggactcttt 1140

aaatacctcc aatcaaatat tttggaaatt aatcgatgga tcgaaggaag ccactccatt 1200

ttattattac tatgttgatg tccgtgatgt agctgctgct cacgtttttg cccttgagaa 1260

cgccaagttg tctaacggcc gtatgcttgt ctccaagggt gtattcacta ctggtgatat 1320

ttgcaaggtc cttcgcaaag aattccccaa caagtccgat gttattgccg agcccgtcga 1380

tataactgtg gatcccagtt tcttcaagtt agacaactca ttttccaagt ctcttggatt 1440

caaatatcat tccgacgagg aatgctatgt tgacactgct aagaagttat gggaacgtgc 1500

tgaggagttt aaataaaatg tttacaaacg ttgggttgag cattcttttg ttacgaattc 1560

atttttgctc cttatttatt tcacgatttc ttctttcaat ttatatcgct acgacatata 1620

tagactttag acatattaaa ttattagaat ttcattgcat ttagataaaa gacttttttc 1680

gatagtgctt gtcggtacac tttgttgtat acaatttttt caatattttg aaatttttta 1740

ctctatcaat cgatttgcg 1759

<210> SEQ ID NO: 15

<211> LENGTH: 375

<212> TYPE: PRT

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 15

Met Val Asp Gln Asn Arg Ala Phe Gly Trp Ala Ala Asn Asp Glu Ser

1 5 10 15

Gly Val Leu Ser Pro Phe His Phe Ser Arg Arg Glu Asn Gly Glu Asn

20 25 30

Asp Val Thr Val Lys Ile Leu Phe Cys Gly Val Cys His Ser Asp Leu

35 40 45

His Thr Ile Lys Asn His Trp Gly Phe Ser Arg Tyr Pro Ile Ile Pro

50 55 60

Gly His Glu Ile Val Gly Ile Ala Thr Lys Val Gly Lys Asn Val Thr

65 70 75 80

Lys Phe Lys Glu Gly Asp Arg Val Gly Val Gly Val Ile Ile Gly Ser

85 90 95

Cys Gln Ser Cys Glu Ser Cys Asn Gln Asp Leu Glu Asn Tyr Cys Pro

100 105 110

Lys Val Val Phe Thr Tyr Asn Ser Arg Ser Ser Asp Gly Thr Arg Asn

115 120 125

Gln Gly Gly Tyr Ser Asp Val Ile Val Val Asp His Arg Phe Val Leu

130 135 140

Ser Ile Pro Asp Gly Leu Pro Ser Asp Ser Gly Ala Pro Leu Leu Cys

145 150 155 160

Ala Gly Ile Thr Val Tyr Ser Pro Met Lys Tyr Tyr Gly Met Thr Lys

165 170 175

Glu Ser Gly Lys Arg Leu Gly Val Asn Gly Leu Gly Gly Leu Gly His

180 185 190

Ile Ala Val Lys Ile Gly Lys Ala Phe Gly Leu Arg Val Thr Val Ile

195 200 205

Ser Arg Ser Ser Glu Lys Glu Arg Glu Ala Ile Asp Arg Leu Gly Ala

210 215 220

Asp Ser Phe Leu Val Thr Thr Asp Ser Gln Lys Met Lys Glu Ala Val

225 230 235 240

Gly Thr Met Asp Phe Ile Ile Asp Thr Val Ser Ala Glu His Ala Leu

245 250 255

Leu Pro Leu Phe Ser Leu Leu Lys Val Ser Gly Lys Leu Val Ala Leu

260 265 270

Gly Leu Leu Glu Lys Pro Leu Asp Leu Pro Ile Phe Pro Leu Val Leu

275 280 285

Gly Arg Lys Met Val Gly Gly Ser Gln Ile Gly Gly Met Lys Glu Thr

290 295 300

Gln Glu Met Leu Glu Phe Cys Ala Lys His Lys Ile Val Ser Asp Ile

305 310 315 320

Glu Leu Ile Lys Met Ser Asp Ile Asn Ser Ala Met Asp Arg Leu Val

325 330 335

Lys Ser Asp Val Arg Tyr Arg Phe Val Ile Asp Val Ala Asn Ser Leu

340 345 350

Leu Pro Glu Ser Ser Ala Glu Ile Leu Thr Glu His Val Asp His Gly

355 360 365

Val Ser Ile Thr Ser Arg Phe

370 375

<210> SEQ ID NO: 16

<211> LENGTH: 1128

<212> TYPE: DNA

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 16

atggtagatc aaaatagagc atttggttgg gcggccaacg acgaatcggg cgtcctctct 60

ccatttcatt tctctagaag agaaaatggt gagaacgatg taacagtgaa gatcttgttc 120

tgtggtgttt gtcactctga tcttcatacc atcaagaacc attggggatt ctctcgttac 180

cccattattc ccgggcatga aatcgttgga atagcaacaa aagttgggaa gaatgtgaca 240

aagtttaaag aaggagaccg agtaggagta ggcgtaataa tcggttcatg ccaatcatgt 300

gaatcatgta accaagactt agaaaactat tgtcctaaag tcgttttcac atacaactct 360

cgttcctctg acggaaccag aaaccaaggt ggttattccg acgtaattgt tgtcgatcac 420

cgctttgtcc taagcattcc cgacggttta ccaagcgatt caggcgcgcc gctgctctgt 480

gctggaatca ctgtgtacag tcccatgaag tattatggca tgactaaaga atcagggaaa 540

cgtttaggtg tgaatggact tggtggactt ggtcatatcg ctgttaagat tggtaaagcc 600

tttggtttaa gagttactgt gattagtagg tcatcagaga aagagagaga agcaattgat 660

cgactcggtg ctgattcgtt tcttgttaca acggattctc aaaagatgaa ggaagcagtt 720

ggaactatgg atttcattat cgatacggta tcagcagaac atgctctatt acctttgttt 780

agtttgctta aagtgagtgg aaagcttgtg gctttaggct tgctggagaa gccactcgac 840

ctgccaattt tccctctagt tctcggaagg aaaatggtgg gaggaagtca gattggaggg 900

atgaaggaga cacaagagat gcttgagttc tgtgccaagc ataaaatcgt ttcggatatt 960

gagctcataa agatgagtga tatcaactct gcgatggacc gtttggttaa atctgatgtc 1020

aggtaccggt tcgtgatcga tgtggccaac tctttactcc ctgagtcgtc agctgagatt 1080

ttaacggagc atgtcgacca tggagtctcg atcacgtcta gattctga 1128

<210> SEQ ID NO: 17

<211> LENGTH: 360

<212> TYPE: PRT

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 17

Met Ala Lys Ser Pro Glu Thr Glu His Pro Asn Lys Val Phe Gly Trp

1 5 10 15

Gly Ala Arg Asp Lys Ser Gly Val Leu Ser Pro Phe His Phe Ser Arg

20 25 30

Arg Asp Asn Gly Glu Asn Asp Val Thr Val Lys Ile Leu Phe Cys Gly

35 40 45

Val Cys His Thr Asp Leu His Thr Ile Lys Asn Asp Trp Gly Tyr Ser

50 55 60

Tyr Tyr Pro Val Val Pro Gly His Glu Ile Val Gly Ile Ala Thr Lys

65 70 75 80

Val Gly Lys Asn Val Thr Lys Phe Lys Glu Gly Asp Arg Val Gly Val

85 90 95

Gly Val Ile Ser Gly Ser Cys Gln Ser Cys Glu Ser Cys Asp Gln Asp

100 105 110

Leu Glu Asn Tyr Cys Pro Gln Met Ser Phe Thr Tyr Asn Ala Ile Gly

115 120 125

Ser Asp Gly Thr Lys Asn Tyr Gly Gly Tyr Ser Glu Asn Ile Val Val

130 135 140

Asp Gln Arg Phe Val Leu Arg Phe Pro Glu Asn Leu Pro Ser Asp Ser

145 150 155 160

Gly Ala Pro Leu Leu Cys Ala Gly Ile Thr Val Tyr Ser Pro Met Lys

165 170 175

Tyr Tyr Gly Met Thr Glu Ala Gly Lys His Leu Gly Val Ala Gly Leu

180 185 190

Gly Gly Leu Gly His Val Ala Val Lys Ile Gly Lys Ala Phe Gly Leu

195 200 205

Lys Val Thr Val Ile Ser Ser Ser Ser Thr Lys Ala Glu Glu Ala Ile

210 215 220

Asn His Leu Gly Ala Asp Ser Phe Leu Val Thr Thr Asp Pro Gln Lys

225 230 235 240

Met Lys Ala Ala Ile Gly Thr Met Asp Tyr Ile Ile Asp Thr Ile Ser

245 250 255

Ala Val His Ala Leu Tyr Pro Leu Leu Gly Leu Leu Lys Val Asn Gly

260 265 270

Lys Leu Ile Ala Leu Gly Leu Pro Glu Lys Pro Leu Glu Leu Pro Met

275 280 285

Phe Pro Leu Val Leu Gly Arg Lys Met Val Gly Gly Ser Asp Val Gly

290 295 300

Gly Met Lys Glu Thr Gln Glu Met Leu Asp Phe Cys Ala Lys His Asn

305 310 315 320

Ile Thr Ala Asp Ile Glu Leu Ile Lys Met Asp Glu Ile Asn Thr Ala

325 330 335

Met Glu Arg Leu Ala Lys Ser Asp Val Arg Tyr Arg Phe Val Ile Asp

340 345 350

Val Ala Asn Ser Leu Ser Pro Pro

355 360

<210> SEQ ID NO: 18

<211> LENGTH: 1744

<212> TYPE: DNA

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 18

acacaacata aacccaatct ctcactcaga ctaagacaga gtcagaaaca atggcgaaat 60

ctccagaaac agagcatccg aacaaagtct ttggttgggg tgctagagac aaatccggtg 120

ttctctctcc ttttcacttc tctagaagag acaatggtga aaatgatgtg acagtgaaga 180

tcttgttctg tggagtttgc cacactgatt tacacaccat caaaaacgac tggggatact 240

cgtattaccc agtagttcca gggcatgaaa tcgttgggat cgctacaaaa gttggtaaga 300

acgtgactaa attcaaagaa ggagatcgtg tcggagtagg agtgatcagt ggctcgtgcc 360

aatcttgcga atcttgtgac caagatcttg aaaactactg tcctcaaatg tctttcacat 420

acaatgcgat tggatccgat ggaaccaaga attacggtgg ctattcggag aacattgtgg 480

ttgatcaacg gtttgttttg cggtttccgg agaatttacc gagcgattcg ggtgcgccgt 540

tgctgtgtgc tggaatcact gtgtatagtc caatgaagta ttatggtatg actgaggcag 600

ggaagcattt aggggttgct ggacttggtg ggcttggtca tgttgctgtt aagattggta 660

aagcttttgg tttgaaagtt actgtcatta gttcttcttc tacgaaagca gaggaagcca 720

ttaatcatct tggtgctgat tcgtttcttg tcacaactga tcctcagaaa atgaaggctg 780

caattggaac aatggactac attatcgata cgatatcagc agtacatgct ctgtatccgt 840

tgctcggttt actcaaagtc aacggaaagc tcattgcttt aggcttacct gagaagcctc 900

tcgagctacc aatgttccct cttgttctcg gaaggaaaat ggttggagga agtgacgtgg 960

gagggatgaa ggagacacaa gagatgcttg atttctgcgc taagcacaac attacagctg 1020

atattgaatt gattaagatg gatgagatta acactgcgat ggagaggctt gctaagtctg 1080

atgttaggta caggttcgtg atcgacgtgg ctaactcctt gagccctcca tgaatgatcc 1140

ggatctaaga attgagcatt gaggaggctt taaatctatg tcataatctt ggtgtttgtt 1200

tgtgtctctc gagttatctt cgttttctgc tttcggtttg agaatcggtt tcttctcaga 1260

caagttacct tatttcgttg ttttcttctc atgttctgtt tcctgagaga aactctttct 1320

gattccagat aagactttga tccattttca gtttgctaat aatataaaga tgggtctaaa 1380

acctaaacag ctttaaactc tttatgattt taaagaatct caatatgcag aatcattgga 1440

ttgtgatcat ttaaacatga gcgtttatta attgatatga aagttactat caaactattt 1500

tgggagacgg agccggagaa gagtcgccgg agtttggatt ctgacaaaag ccacgaagga 1560

cattagcttc ctcaggcgta aatccgagag aagtgagcat cgccggccag caattggtgg 1620

tgatgatgtc tacggattcg caacaactaa caccaagttt tgtttcaccg ttgaggaaga 1680

agagaacgat ctcgttggtg catgatttca gctcgtataa tgcgttccag cattccatca 1740

gtcc 1744

<210> SEQ ID NO: 19

<211> LENGTH: 376

<212> TYPE: PRT

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 19

Met Val Asp Gln Asn Lys Ala Phe Gly Trp Ala Ala Asn Asp Glu Ser

1 5 10 15

Gly Val Leu Ser Pro Phe His Phe Ser Arg Arg Glu Asn Gly Glu Asn

20 25 30

Asp Val Thr Val Lys Ile Leu Phe Cys Gly Val Cys His Ser Asp Leu

35 40 45

His Thr Ile Lys Asn His Trp Gly Phe Ser Arg Tyr Pro Ile Ile Pro

50 55 60

Gly His Glu Ile Val Gly Ile Ala Thr Lys Val Gly Lys Asn Val Thr

65 70 75 80

Lys Phe Lys Glu Gly Asp Arg Val Gly Val Gly Val Ile Ile Gly Ser

85 90 95

Cys Gln Ser Cys Glu Ser Cys Asn Gln Asp Leu Glu Asn Tyr Cys Pro

100 105 110

Lys Val Val Phe Thr Tyr Asn Ser Arg Ser Ser Asp Gly Thr Ser Arg

115 120 125

Asn Gln Gly Gly Tyr Ser Asp Val Ile Val Val Asp His Arg Phe Val

130 135 140

Leu Ser Ile Pro Asp Gly Leu Pro Ser Asp Ser Gly Ala Pro Leu Leu

145 150 155 160

Cys Ala Gly Ile Thr Val Tyr Ser Pro Met Lys Tyr Tyr Gly Met Thr

165 170 175

Lys Glu Ser Gly Lys Arg Leu Gly Val Asn Gly Leu Gly Gly Leu Gly

180 185 190

His Ile Ala Val Lys Ile Gly Lys Ala Phe Gly Leu Arg Val Thr Val

195 200 205

Ile Ser Arg Ser Ser Glu Lys Glu Arg Glu Ala Ile Asp Arg Leu Gly

210 215 220

Ala Asp Ser Phe Leu Val Thr Thr Asp Ser Gln Lys Met Lys Glu Ala

225 230 235 240

Val Gly Thr Met Asp Phe Ile Ile Asp Thr Val Ser Ala Glu His Ala

245 250 255

Leu Leu Pro Leu Phe Ser Leu Leu Lys Val Asn Gly Lys Leu Val Ala

260 265 270

Leu Gly Leu Pro Glu Lys Pro Leu Asp Leu Pro Ile Phe Ser Leu Val

275 280 285

Leu Gly Arg Lys Met Val Gly Gly Ser Gln Ile Gly Gly Met Lys Glu

290 295 300

Thr Gln Glu Met Leu Glu Phe Cys Ala Lys His Lys Ile Val Ser Asp

305 310 315 320

Ile Glu Leu Ile Lys Met Ser Asp Ile Asn Ser Ala Met Asp Arg Leu

325 330 335

Ala Lys Ser Asp Val Arg Tyr Arg Phe Val Ile Asp Val Ala Asn Ser

340 345 350

Leu Leu Pro Glu Ser Ser Ala Glu Ile Leu Thr Glu Gln Val Asp His

355 360 365

Gly Val Ser Ile Thr Ser Arg Phe

370 375

<210> SEQ ID NO: 20

<211> LENGTH: 1141

<212> TYPE: DNA

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 20

agaaacaaat atggtagatc aaaataaagc atttggttgg gcggccaacg acgaatcggg 60

cgtcctctct ccatttcatt tctctagaag agaaaatggt gagaacgatg taacagtgaa 120

gatcttgttc tgtggtgttt gtcactctga tcttcatacc atcaagaacc attggggatt 180

ctctcgttac cccattattc ccgggcatga aatcgttgga atagcaacaa aagttgggaa 240

gaatgtgaca aagtttaaag aaggagaccg agtaggagta ggcgtaataa tcggttcatg 300

ccaatcatgt gaatcatgta accaagactt agaaaactat tgtcctaaag tcgttttcac 360

atacaactct cgttcctctg acggaaccag cagaaaccaa ggtggttatt ccgacgtaat 420

tgttgtcgat caccgctttg tcctaagcat tcccgatggt ttaccaagcg attcaggcgc 480

gccgctgctc tgtgctggaa tcactgtgta cagtcccatg aagtattatg gcatgactaa 540

agaatcaggg aaacgtttag gtgtgaatgg acttggtgga cttggtcata tcgctgttaa 600

gattggtaaa gcctttggtt taagagttac tgtgattagt aggtcatcag aaaaagagag 660

agaagcaatt gatcggcttg gtgctgattc gtttcttgtt acaacggatt ctcaaaagat 720

gaaggaagcg gttggaacta tggatttcat tatcgatacg gtatcagcag aacatgctct 780

attaccgttg tttagtttgc ttaaagtgaa tggaaagctt gtggctttag gcttaccgga 840

gaagccactc gacctgccaa ttttctctct agttctcgga aggaaaatgg tgggaggaag 900

tcagattgga gggatgaagg agacacaaga gatgcttgag ttctgtgcca agcataaaat 960

cgtttcggat attgagctca taaagatgag tgatatcaac tctgcgatgg accgtttggc 1020

taaatctgat gtcaggtacc ggttcgtgat cgatgtggcc aactctttac tccctgagtc 1080

gtcagctgag attttaacgg agcaggtgga ccatggagtc tcgatcacgt ctagattctg 1140

a 1141

<210> SEQ ID NO: 21

<211> LENGTH: 325

<212> TYPE: PRT

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 21

Met Ser Ser Glu Glu Glu Lys Thr Val Cys Val Thr Gly Ala Ser Gly

1 5 10 15

Tyr Ile Ala Ser Trp Ile Val Lys Leu Leu Leu Leu Arg Gly Tyr Thr

20 25 30

Val Lys Ala Ser Val Arg Asp Pro Asn Asp Pro Arg Lys Thr Glu His

35 40 45

Leu Leu Ala Leu Glu Gly Ala Glu Glu Arg Leu Lys Leu Phe Lys Ala

50 55 60

Asn Leu Leu Glu Glu Gly Ser Phe Asp Ser Ala Ile Asp Gly Cys Glu

65 70 75 80

Gly Val Phe His Thr Ala Ser Pro Phe Tyr His Asp Val Lys Asp Pro

85 90 95

Gln Ala Glu Leu Leu Asp Pro Ala Val Lys Gly Thr Ile Asn Val Leu

100 105 110

Ser Ser Cys Leu Lys Thr Ser Ser Val Lys Arg Val Val Leu Thr Ser

115 120 125

Ser Ile Ala Ala Val Ala Phe Asn Gly Met Pro Arg Thr Pro Glu Thr

130 135 140

Ile Val Asp Glu Thr Trp Phe Ala Asp Pro Asp Tyr Cys Arg Ala Ser

145 150 155 160

Lys Leu Trp Tyr Val Leu Ser Lys Thr Leu Ala Glu Asn Ala Ala Trp

165 170 175

Lys Phe Ala Lys Glu Asn Asn Leu Gln Leu Val Ser Ile Asn Pro Ala

180 185 190

Met Val Ile Gly Pro Leu Leu Gln Pro Thr Leu Asn Thr Ser Ala Ala

195 200 205

Ala Val Leu Ser Leu Ile Lys Gly Ala Gln Thr Phe Pro Asn Ala Thr

210 215 220

Phe Gly Trp Val Asn Val Lys Asp Val Ala Asn Ala His Ile Gln Ala

225 230 235 240

Phe Glu Asn Pro Asp Ala Asp Gly Arg Tyr Cys Leu Val Glu Arg Val

245 250 255

Ala His Tyr Ser Glu Val Val Asn Ile Leu His Asp Leu Tyr Pro Asp

260 265 270

Phe Gln Leu Pro Glu Lys Cys Ala Asp Glu Lys Ile Tyr Ile Pro Thr

275 280 285

Tyr Lys Val Ser Lys Glu Lys Ala Glu Ser Leu Gly Val Glu Phe Val

290 295 300

Pro Leu Glu Val Ser Ile Lys Glu Thr Val Glu Ser Leu Arg Asp Lys

305 310 315 320

Gly Phe Ile Arg Phe

325

<210> SEQ ID NO: 22

<211> LENGTH: 978

<212> TYPE: DNA

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 22

atgagcagcg aagaagagaa gacagtgtgt gtgacaggag cttcaggtta cattgcttca 60

tggatcgtta agcttcttct tctccgtggt tacaccgtta aagcctctgt tcgtgatcca 120

aatgatccaa ggaaaacaga gcatttgctt gcattggaag gagcagagga aaggcttaaa 180

ttgttcaaag caaacttgtt agaagaaggc tctttcgatt cagcaatcga tggttgcgaa 240

ggagttttcc acaccgcatc gccattctat catgacgtca aggaccctca ggctgagtta 300

cttgatccgg cagtgaaagg aacaatcaat gttctaagct cttgtttgaa gacttcctcg 360

gttaagagag tcgtcttaac ctcatcgata gcagctgttg ctttcaatgg aatgcctcga 420

acacccgaaa ccatagttga cgaaacttgg ttcgccgatc ctgactattg cagagcttcc 480

aagctatggt atgtactctc gaagacatta gctgaaaacg cagcgtggaa attcgcaaaa 540

gagaacaatt tacagctggt ttcgataaat ccggctatgg tgattggtcc tctcttacag 600

ccaacgctaa acactagtgc tgctgcagta ctaagcttga tcaaaggagc acaaacgttt 660

cctaatgcga cgttcgggtg ggttaacgtt aaagatgtag cgaacgctca tattcaagcg 720

tttgagaatc cagatgctga tgggagatac tgtttagtgg agagagttgc tcattactct 780

gaagttgtta acattttgca tgatctttac cctgattttc aactccctga gaagtgtgca 840

gatgaaaaga tatatattcc aacatataaa gtgtctaaag agaaagcaga gtctcttggg 900

gttgagtttg tgccattgga agttagcatt aaagagactg tagagagctt gagagataaa 960

gggttcatca gattttga 978

<210> SEQ ID NO: 23

<211> LENGTH: 326

<212> TYPE: PRT

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 23

Met Ala Asn Ser Gly Glu Gly Lys Val Val Cys Val Thr Gly Ala Ser

1 5 10 15

Gly Tyr Ile Ala Ser Trp Leu Val Lys Phe Leu Leu Ser Arg Gly Tyr

20 25 30

Thr Val Lys Ala Ser Val Arg Asp Pro Ser Asp Pro Lys Lys Thr Gln

35 40 45

His Leu Val Ser Leu Glu Gly Ala Lys Glu Arg Leu His Leu Phe Lys

50 55 60

Ala Asp Leu Leu Glu Gln Gly Ser Phe Asp Ser Ala Ile Asp Gly Cys

65 70 75 80

His Gly Val Phe His Thr Ala Ser Pro Phe Phe Asn Asp Ala Lys Asp

85 90 95

Pro Gln Ala Glu Leu Ile Asp Pro Ala Val Lys Gly Thr Leu Asn Val

100 105 110

Leu Asn Ser Cys Ala Lys Ala Ser Ser Val Lys Arg Val Val Val Thr

115 120 125

Ser Ser Met Ala Ala Val Gly Tyr Asn Gly Lys Pro Arg Thr Pro Asp

130 135 140

Val Thr Val Asp Glu Thr Trp Phe Ser Asp Pro Glu Leu Cys Glu Ala

145 150 155 160

Ser Lys Met Trp Tyr Val Leu Ser Lys Thr Leu Ala Glu Asp Ala Ala

165 170 175

Trp Lys Leu Ala Lys Glu Lys Gly Leu Asp Ile Val Thr Ile Asn Pro

180 185 190

Ala Met Val Ile Gly Pro Leu Leu Gln Pro Thr Leu Asn Thr Ser Ala

195 200 205

Ala Ala Ile Leu Asn Leu Ile Asn Gly Ala Lys Thr Phe Pro Asn Leu

210 215 220

Ser Phe Gly Trp Val Asn Val Lys Asp Val Ala Asn Ala His Ile Gln

225 230 235 240

Ala Phe Glu Val Pro Ser Ala Asn Gly Arg Tyr Cys Leu Val Glu Arg

245 250 255

Val Val His His Ser Glu Ile Val Asn Ile Leu Arg Glu Leu Tyr Pro

260 265 270

Asn Leu Pro Leu Pro Glu Arg Cys Val Asp Glu Asn Pro Tyr Val Pro

275 280 285

Thr Tyr Gln Val Ser Lys Asp Lys Thr Arg Ser Leu Gly Ile Asp Tyr

290 295 300

Ile Pro Leu Lys Val Ser Ile Lys Glu Thr Val Glu Ser Leu Lys Glu

305 310 315 320

Lys Gly Phe Ala Gln Phe

325

<210> SEQ ID NO: 24

<211> LENGTH: 1273

<212> TYPE: DNA

<213> ORGANISM: Arabidopsis thaliana

<400> SEQENCE: 24

caaatatacg cattcgtcat ttggtccctc cacttagaga gaagcagaca gaacataact 60

aaaatcgaga aaaatggcaa acagtggtga aggtaaagtg gtgtgtgtaa caggagcctc 120

cggttacatc gcctcatggc tcgtcaagtt cctacttagc cgtggctaca ctgttaaggc 180

ctccgtccgt gatcccagtg atccgaaaaa gacacaacac ttagtttcac tagaaggtgc 240

aaaggaaaga cttcacttgt tcaaagcaga ccttttggaa caaggttctt tcgactctgc 300

tattgatggt tgccatggag ttttccacac tgcttctcca ttttttaatg atgccaaaga 360

cccacaggct gaacttattg atcctgcggt caaggggacg cttaacgttt tgaattcgtg 420

cgccaaagcc tcttcggtta agagggttgt tgtaacctcc tccatggctg ccgttggtta 480

caatggaaaa ccacgcacac ctgatgttac cgtcgatgaa acttggttct ctgatcctga 540

gctttgcgag gcctccaaga tgtggtatgt tctatccaag actttggcgg aagatgcagc 600

ttggaaactc gctaaagaga aaggcttaga cattgttact attaacccgg ctatggtgat 660

cggtcctctc ctacagccaa ctctgaacac gagtgctgct gctatattaa acttaatcaa 720

tggtgcaaag actttcccaa acttgagttt cggatgggtt aatgtaaaag acgtagccaa 780

tgcgcacatc caagcatttg aggtcccttc agctaatggg cgttattgtt tggtcgagcg 840

tgtcgttcac cactccgaga ttgttaacat tctacgtgag ctttacccaa atctcccact 900

acctgaaagg tgtgtggacg agaatcccta cgtgccaacg tatcaagtgt ccaaggataa 960

aacgaggagc cttggcatag actacatacc cttgaaggtt agcatcaagg agaccgtcga 1020

gtccttgaag gaaaaaggtt tcgcacagtt ctgagaaagc atttgagcca atggatttaa 1080

tccagattag ataaagtatt tggaagacta tttcagaaat aatatttgga acatgtcaat 1140

gttctcaagg agatattagt atgttcttgt gtactttatt gttgttccat caaatgagtt 1200

acttttcctt ttatttcttg ttagtaagat atcaaataag aattcgagtc aagtgagcat 1260

ctttacactg atg 1273

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Patent Valuation

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Reveal the value <>

13.37/100 Score

Market Attractiveness

It shows from an IP point of view how many competitors are active and innovations are made in the different technical fields of the company. On a company level, the market attractiveness is often also an indicator of how diversified a company is. Here we look into the commercial relevance of the market.

39.0/100 Score

Market Coverage

It shows the sizes of the market that is covered with the IP and in how many countries the IP guarantees protection. It reflects a market size that is potentially addressable with the invented technology/formulation with a legal protection which also includes a freedom to operate. Here we look into the size of the impacted market.

73.99/100 Score

Technology Quality

It shows the degree of innovation that can be derived from a company’s IP. Here we look into ease of detection, ability to design around and significance of the patented feature to the product/service.

60.0/100 Score

Assignee Score

It takes the R&D behavior of the company itself into account that results in IP. During the invention phase, larger companies are considered to assign a higher R&D budget on a certain technology field, these companies have a better influence on their market, on what is marketable and what might lead to a standard.

20.06/100 Score

Legal Score

It shows the legal strength of IP in terms of its degree of protecting effect. Here we look into claim scope, claim breadth, claim quality, stability and priority.

Citation

Title Current Assignee Application Date Publication Date
Synthesis of vanillin from a carbon source BOARD OF TRUSTEES OPERATING MICHIGAN STATE UNIVERSITY 16 September 1999 16 April 2002
Vanillin production DAVID MICHAEL & CO., INC. 01 June 2007 04 October 2007
Compositions and methods for the biosynthesis of vanillan or vanillin beta-d-glucoside INTERNATIONAL FLAVORS & FRAGRANCES INC.,EVOLVA SA 07 August 2012 28 August 2014
Compositions and methods for the biosynthesis of vanillin or vanillin beta-d-glucoside INTERNATIONAL FLAVORS & FRAGRANCES INC.,EVOLVA SA,HANSEN, JOERGEN,HANSEN, ESBEN HALKJAER,SOMPALLI, HONEY POLUR 07 August 2012 14 February 2013
See full citation <>

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