U.S. patent application number 11/809646 was filed with the patent office on 2007-10-04 for vanillin production.
This patent application is currently assigned to David Michael & Co., Inc.. Invention is credited to Daphna Havkin-Frenkel, Andrzej Podstolski.
Application Number | 20070231864 11/809646 |
Document ID | / |
Family ID | 21978699 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070231864 |
Kind Code |
A1 |
Havkin-Frenkel; Daphna ; et
al. |
October 4, 2007 |
Vanillin production
Abstract
Novel compositions and methods for improving vanillin production
in cultured Vanilla planifolia and in intact plants are provided.
Transgenic cells and plants having improved vanillin production are
also provided.
Inventors: |
Havkin-Frenkel; Daphna;
(North Brunswick, NJ) ; Podstolski; Andrzej;
(Warsaw, PL) |
Correspondence
Address: |
DRINKER BIDDLE & REATH;ATTN: INTELLECTUAL PROPERTY GROUP
ONE LOGAN SQUARE
18TH AND CHERRY STREETS
PHILADELPHIA
PA
19103-6996
US
|
Assignee: |
David Michael & Co.,
Inc.
|
Family ID: |
21978699 |
Appl. No.: |
11/809646 |
Filed: |
June 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09462576 |
May 25, 2000 |
7226783 |
|
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PCT/US98/14895 |
Jul 15, 1998 |
|
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11809646 |
Jun 1, 2007 |
|
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60052606 |
Jul 15, 1997 |
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Current U.S.
Class: |
435/69.1 ;
435/410; 435/70.1; 800/295 |
Current CPC
Class: |
C12N 9/0077 20130101;
C12P 7/24 20130101; C12N 5/04 20130101; A01H 4/001 20130101; C12N
9/0006 20130101; C12N 9/00 20130101; C12N 15/8243 20130101; C12N
15/8207 20130101 |
Class at
Publication: |
435/069.1 ;
435/410; 435/070.1; 800/295 |
International
Class: |
C12N 5/14 20060101
C12N005/14; A01H 5/00 20060101 A01H005/00; C12P 1/04 20060101
C12P001/04 |
Claims
1. A method for improving production of vanillin in cultured
Vanillin planifolia, which comprises: a) providing a tissue culture
of said Vanilla planifolia; and b) supplementing the culture with a
compound selected from the group consisting of malic acid,
3,4-dihydroxybenzaldehyde, citric acid, pyruvic acid, oxaloacetic
acid, succinic acid, glycosylated lysozyme, and any combination
thereof, in an amount effective to improve the vanillin production
as compared with cultures not supplemented with the compound.
2. The method of claim 1, wherein the tissue culture is an embryo
culture.
3. The method of claim 1, wherein the culture is supplemented with
malic acid at a concentration of between about 0.01% and 5% by
weight of the culture medium.
4. The method of claim 3, wherein the culture is subjected to
mechanical shear stress for 21 days, followed by addition of the
malic acid at a concentration of between about 1% and 3% by weight
of the culture medium.
5. The method of claim 1, wherein the culture is supplemented with
3,4-dihyrdoxybenzaldehyde at a concentration of between about 0.1
and 5 mM.
6. The method of claim 1, wherein the culture is supplemented with
about 0.01 to about 5% by weight of a compound selected from the
group consisting of succinic acid, oxaloacetic acid, citric acid
and pyruvic acid.
7. The method of claim 1, wherein the culture is supplemented with
about 1 to about 100 .mu.g/ml of a glycosylated lysozyme
elicitor.
9. Cultured Vanilla planifolia cells, produced by the method of
claim 1.
10. The cultured Vanilla planifolia cells of claim 9, which produce
at least twice as much vanillin as equivalent cultured cells not
supplemented with the compounds.
11. The cultured Vanilla planifolia cells of claim 9, which produce
at least ten times as much vanillin as equivalent cultured cells
not supplemented with the compounds.
12. A method for improving production of vanillin in cultured
Vanilla planifolia, which comprises: a) providing an embryo culture
of said Vanilla planifolia; and b) subjecting the culture to a
stress condition selected from the group consisting of heat stress
and mechanical shear stress, in an amount and for a time effective
to improve the vanillin production as compared with cultures not
subjected to the stress condition.
13. The method of claim 12, wherein the heat stress comprises
maintaining the cultures between about 33 and 37.degree. C. for
between three and seven days.
14. The method of claim 12, wherein the mechanical shear stress is
imposed by placing the cultures in an impeller-driven incubator,
under conditions whereby the shear stress is caused.
15. Cultured Vanilla planifolia cells, produced by the method of
claim 12.
16. The cultured Vanilla planifolia cells of claim 15, which
produce at least twice as much vanillin as equivalent cultured
cells not subjected to the stress.
17. A method for improving vanillin production in Vanilla
planifolia, which comprises genetically engineering the Vanilla
planifolia to overproduce one or more enzymes associated with one
or more steps of vanillin biosynthesis in the Vanilla planifolia,
the steps selected from the group consisting of: chain shortening
of p-coumaric acid to p-hydroxybenzaldehyde; chain shortening of
ferulic acid to vanillin; hydroxylation of p-hydroxybenzyl alcohol
to 3,4-dihydroxybenzyl alcohol or aldehyde; and methylation of
3,4-dihydroxybenzaldehyde to vanillin.
18. The method of claim 17, wherein the enzymes are selected from
the group consisting of: at least one p-hydroxybenzaldehyde
synthase; at least one cytochrome p450 monooxygenase; and at least
one methyl transferase.
19. The method of claim 17, wherein the genetically engineered
Vanilla planifolia is a cell or tissue culture.
20. The method of claim 17, wherein the genetically engineered
Vanilla planifolia is a whole plant.
21. A genetically engineered Vanilla planifolia cell produced by
the method of claim 17.
22. The cell of claim 21, which produces at least twice as much
vanillin as does an equivalent cell which is not comparably
genetically engineered.
23. A genetically engineered Vanilla planifolia plant, regenerated
from the cell of claim 21.
24. The plant of claim 23, which produces at least twice as much
vanillin as does an equivalent plant which is not comparably
genetically engineered.
25. A method for improving vanillin accumulation in cell or tissue
culture of Vanilla planifolia, which comprises inhibiting
production or activity of vanillyl alcohol dehydrogenase in cells
comprising the cell or tissue culture, the inhibition resulting in
the improved vanillin accumulation.
26. The method of claim 25, wherein the inhibiting comprises
genetically engineering the cells to inhibit expression of a gene
encoding the vanillyl alcohol dehydrogenase.
27. A genetically engineered Vanilla planifolia cell or tissue
culture produced by the method of claim 26.
28. The method of claim 25, wherein the inhibiting comprises
treating the culture with an inhibitor of vanillyl dehydrogenase
activity.
29. A method for improving vanillin production and accumulation in
a Vanilla planifolia cell or tissue culture, which comprises: a)
genetically engineering the Vanilla planifolia to overproduce one
or more enzymes associated with one or more steps of vanillin
biosynthesis in the Vanilla planifolia, the steps selected from the
group consisting of: chain shortening of p-coumaric acid to
p-hydroxybenzaldehyde; chain shortening of ferulic acid to
vanillin; hydroxylation of p-hydroxybenzyl alcohol to
3,4-dihydroxybenzyl alcohol or aldehyde; and methylation of
3,4-dihydroxybenzaldehyde to vanillin, thereby resulting in the
improved vanillin production; and b) inhibiting production or
activity of vanillyl alcohol dehydrogenase in cells of the culture,
thereby resulting in the improved vanillin accumulation.
30. A Vanilla planifolia cell or tissue culture produced by the
method of claim 29.
Description
[0001] This is a continuation of U.S. application Ser. No.
09/462,576, filed May 25, 2000, which is a national stage of
PCT/U.S.98/14895 filed Jul. 15, 1998 which claims benefit of U.S.
Provisional Application No. 60/052,606, filed Jul. 15, 1997, which
is herein incorporated in its entirety by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the field of plant tissue culture
and plant genetic engineering to improve agronomic or commercial
properties of plants. In particular, this invention provides novel
compositions, methods and transgenic cells and plants of Vanilla
planifolia, for the improved production of vanillin.
BACKGROUND OF THE INVENTION
[0003] Vanillin is the major principle flavor ingredient in vanilla
extract and is also noted as a nutraceutical because of its
anti-oxidant and antimicrobial properties. Vanillin can be used as
a masking agent for undesirable flavors of other nutraceuticals.
Vanilla extract is obtained from cured vanilla beans, the bean-like
pod produced by Vanilla planifolia, a tropical climbing orchid.
[0004] Vanilla extract is widely used as a flavor by the food and
beverage industry, and is used increasingly in perfumes. The U.S.
annual consumption of vanilla beans, all of which are imported from
foreign countries, is 1,200-1,400 tons, with a market value of
about $100 million. By FDA definition, vanillin can be labeled as
natural only when it is derived from vanilla beans. Currently,
natural vanilla obtained through extraction of vanilla beans as
described below, costs between $1,500 and $3,000 per kilogram.
Vanillin is also produced by molecular breakage of curcumin,
eugenol or piperin at a cost of $1,000/kg or less. However,
vanillin produced by this method can be labeled as a natural flavor
only in non-vanilla flavors. Vanillin chemically synthesized from
guaiacol is consumed at a rate of about 800 tons per year in the
United States for the food and beverage industry, at a cost of
production of about $15/kg.
[0005] Natural vanilla extract currently produced from vanilla
beans is presently the most desirable form of vanilla, due to the
recent demand for natural food ingredients. The areas of the world
capable of supporting vanilla cultivation are limited, due to its
requirement for a warm, moist and tropical climate with frequent,
but not excessive rain, and moderate sunlight. The primary growing
region for vanilla is around the Indian Ocean, in Madagascar,
Comoros, Reunion and Indonesia.
[0006] The production of vanilla beans is a lengthy process that is
highly dependent on suitable soil and weather conditions. Beans
(pod-like fruit) are produced after 4-5 years of cultivation.
Flowers must be hand-pollinated, and fruit production takes about
8-10 months. The characteristic flavor and aroma develops in the
fruit after a process called "curing," lasting an additional 3-6
months. For a complete review of the vanilla growing and curing
process, see D. Havkin Frenkel & R. Dorn, "Vanilla," Chapter 4
in Spices: Flavor Chemistry and Antioxidant Properties, (Eds. Risch
& Ho), American Chemical Society, Washington, 1997.
[0007] Interest has focused recently on plant cell and tissue
culture as an approach to control quality and yield of vanilla
production and to solve some of the agronomic problems associated
with growing vanilla. Plant tissue culture should be useful for
three objectives: (1) micropropagation of vanilla plants; (2)
production of vanillin and other secondary products associated with
vanilla flavor; and (3) improving production of vanillin in culture
or in intact plants by elucidating and manipulating the
biosynthetic pathways of vanillin and other flavor compounds. In
connection with this last objective, efforts have been made to
commercialize production of vanillin, the most valuable component
of vanilla, by using plant cell culture. However, these efforts
have not resulted in economically significant amounts of vanillin
production, perhaps due in part to the heretofore incomplete
understanding of the vanillin biosynthetic pathway.
[0008] From the foregoing, it can be seen that improvement of
vanillin production, either in tissue culture or in intact plants,
would be of significant agronomic and economic advantage.
Accordingly, it is an object of this invention to provide means for
obtaining high yields of vanillin from cultured cells and tissues.
It is another object of this invention to improve vanillin
production in intact vanilla plants.
SUMMARY OF THE INVENTION
[0009] Novel compositions and methods for improving vanillin
production in cultured Vanilla planifolia and in intact plants are
provided. These cultures and plants are expected to be of
significant agronomic and economic value.
[0010] According to one aspect of the invention, a method for
improving production of vanillin in cultured Vanilla planifolia is
provided. The method comprises supplementing the culture with a
compound selected from the group consisting of malic acid,
3,4-dihydroxybenzaldehyde, citric acid, pyruvic acid, oxaloacetic
acid, succinic acid, glycosylated lysozyme, and any combination
thereof, in an amount effective to improve the vanillin production
as compared with cultures not supplemented with the compound.
[0011] In preferred embodiments of the invention, the tissue
culture is an embryo culture. In another preferred embodiment, the
culture is supplemented with malic acid at a concentration of
between about 0.01% and 5% by weight of the culture medium. In
another preferred embodiment, the culture is supplemented with
3,4-dihyrdoxybenzaldehyde at a concentration of between about 0.1
and 5 mM. In another embodiment, the culture is supplemented with
about 0.01 to about 5% by weight of a compound selected from the
group consisting of succinic acid, oxaloacetic acid, citric acid
and pyruvic acid. In yet another embodiment, the culture is
supplemented with about 1 to about 100 .mu.g/ml of a glycosylated
lysozyme elicitor.
[0012] According to another aspect of the invention, cultured
Vanilla planifolia cells, produced by the aforementioned method,
are provided. These cells preferably produce at least twice as much
vanillin as equivalent cultured cells not supplemented with the
listed compounds.
[0013] In an particularly preferred embodiment the cells produce at
least ten times, and most preferably 50 to 100 times, as much
vanillin as equivalent cultured cells not supplemented with the
compounds.
[0014] According to another aspect of the invention, a second
method for improving production of vanillin in cultured Vanilla
planifolia is provided. This method comprises subjecting the
culture to a stress condition selected from the group consisting of
heat stress and mechanical shear stress, in an amount and for a
time effective to improve the vanillin production as compared with
cultures not subjected to the stress condition. In a preferred
embodiment, the heat stress comprises maintaining the cultures
between about 33 and 37.degree. C. for between three and seven
days. In another embodiment, the mechanical shear stress is imposed
by placing the cultures in an impeller-driven incubator, under
conditions whereby the shear stress is caused.
[0015] Cultured Vanilla planifolia cells produced by the
aforementioned method are also provided. In a preferred embodiment,
these cells produce at least twice as much vanillin as equivalent
cultured cells not subjected to the stress.
[0016] According to another aspect of the invention, a method for
improving vanillin production in Vanilla planifolia, is provided,
which comprises genetically engineering the Vanilla planifolia to
overproduce one or more enzymes associated with one or more steps
of vanillin biosynthesis in the Vanilla planifolia. The steps are
selected from the group consisting of: chain shortening of
p-coumaric acid to p-hydroxybenzaldehyde; chain shortening of
ferulic acid to vanillin; hydroxylation of p-hydroxybenzyl alcohol
to 3,4-dihydroxybenzyl alcohol or aldehyde; and methylation of
3,4-dihydroxybenzaldehyde to vanillin. The enzymes preferably are
selected from the group consisting of: at least one
p-hydroxybenzaldehyde synthase; at least one cytochrome p450
monooxygenase; and at least one methyl transferase.
[0017] In one embodiment of the aforementioned method, the
genetically engineered Vanilla planifolia is a cell or tissue
culture. In another embodiment, it is a whole plant. Genetically
engineered Vanilla planifolia cells or plants produced by the
aforementioned method are also provided. These cells or plants
preferably produce at least twice as much vanillin as does an
equivalent cell which is not comparably genetically engineered.
[0018] According to yet another aspect of the invention, a method
for improving vanillin accumulation in cell or tissue culture of
Vanilla planifolia is provided, which comprises inhibiting
production or activity of vanillyl alcohol dehydrogenase in cells
comprising the cell or tissue culture, the inhibition resulting in
the improved vanillin accumulation. In one embodiment, the
inhibiting comprises genetically engineering the cells to inhibit
expression of a gene encoding the vanillyl alcohol dehydrogenase.
In another embodiment, the inhibiting comprises treating the
culture with an inhibitor of vanillyl dehydrogenase activity.
Cultures produced by the aforementioned method are also
provided.
[0019] According to still another aspect of the present invention,
a method for improving vanillin production and accumulation in a
Vanilla planifolia cell or tissue culture is provided, which
comprises: (a) genetically engineering the Vanilla planifolia to
overproduce one or more enzymes associated with one or more steps
of vanillin biosynthesis in the Vanilla planifolia, the steps
selected from the group consisting of: chain shortening of
p-coumaric acid to p-hydroxybenzaldehyde; chain shortening of
ferulic acid to vanillin; hydroxylation of p-hydroxybenzyl alcohol
to 3,4-dihydroxybenzyl alcohol or aldehyde; and methylation of
3,4-dihydroxybenzaldehyde to vanillin, thereby resulting in the
improved vanillin production; and (b) inhibiting production or
activity of vanillyl alcohol dehydrogenase in cells of the culture,
thereby resulting in the improved vanillin accumulation. A Vanilla
planifolia cell or tissue culture produced by the aforementioned
method is also provided.
[0020] Additional features and advantages of the present invention
will be understood by reference to the drawings, detailed
description and examples that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1. Schematic diagram showing the biosynthetic pathway
of vanillin in Vanilla planifolia.
[0022] FIG. 2. Graph showing the conversion of p-coumaric acid to
p-hydroxybenzaldehyde as catalyzed by p-hydroxybenzaldehyde
synthase in V. planifolia embryo culture.
[0023] FIG. 3. Graph showing uptake of exogenously added vanillin
and its transformation to vanillyl alcohol by vanillyl alcohol
dehydrogenase in V. planifolia embryo culture. V(T)=vanillin in
tissue; V(M)=vanillin in medium; VAL(T)=vanillyl alcohol in tissue;
VAL(M)=vanillyl alcohol in medium.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0024] Various terms relating to the biological molecules of the
present invention are used hereinabove and also throughout the
specification and claims. The terms "substantially the same,"
"percent similarity" and "percent identity" are defined in detail
below.
[0025] With reference to nucleic acids of the invention, the term
"isolated nucleic acid" is sometimes used. This term, when applied
to DNA, refers to a DNA molecule that is separated from sequences
with which it is immediately contiguous (in the 5' and 3'
directions) in the naturally occurring genome of the organism from
which it was derived. For example, the "isolated nucleic acid" may
comprise a DNA molecule inserted into a vector, such as a plasmid
or virus vector, or integrated into the genomic DNA of a procaryote
or eucaryote. An "isolated nucleic acid molecule" may also comprise
a cDNA molecule.
[0026] With respect to RNA molecules of the invention the term
"isolated nucleic acid" primarily refers to an RNA molecule encoded
by an isolated DNA molecule as defined above. Alternatively, the
term may refer to an RNA molecule that has been sufficiently
separated from RNA molecules with which it would be associated in
its natural state (i.e., in cells or tissues), such that it exists
in a "substantially pure" form (the term "substantially pure" is
defined below).
[0027] With respect to protein, the term "isolated protein" or
"isolated and purified protein" is sometimes used herein. This term
refers primarily to a protein produced by expression of an isolated
nucleic acid molecule of the invention. Alternatively, this term
may refer to a protein which has been sufficiently separated from
other proteins with which it would naturally be associated, so as
to exist in "substantially pure" form.
[0028] The term "substantially pure" refers to a preparation
comprising at least 50-60% by weight the compound of interest
(e.g., nucleic acid, oligonucleotide, protein, etc.). More
preferably, the preparation comprises at least 75% by weight, and
most preferably 90-99% by weight, the compound of interest. Purity
is measured by methods appropriate for the compound of interest
(e.g. chromatographic methods, agarose or polyacrylamide gel
electrophoresis, HPLC analysis, and the like).
[0029] With respect to oligonucleotides or hybridization generally,
the term "specifically hybridizing" refers to the association
between two single-stranded nucleotide molecules of sufficiently
complementary sequence to permit such hybridization under
pre-determined conditions generally used in the art (sometimes
termed "substantially complementary"). In particular, the term
refers to hybridization of an oligonucleotide with a substantially
complementary sequence contained within a single-stranded DNA or
RNA molecule of the invention, to the substantial exclusion of
hybridization of the oligonucleotide with single-stranded nucleic
acids of non-complementary sequence.
[0030] The term "promoter region" refers to the 5' regulatory
regions of a gene.
[0031] The term "reporter gene" refers to genetic sequences which
may be operably linked to a promoter region forming a transgene,
such that expression of the reporter gene coding region is
regulated by the promoter and expression of the transgene is
readily assayed.
[0032] The term "selectable marker gene" refers to a gene product
that when expressed confers a selectable phenotype, such as
antibiotic resistance, on a transformed cell or plant.
[0033] The term "operably linked" means that the regulatory
sequences necessary for expression of the coding sequence are
placed in the DNA molecule in the appropriate positions relative to
the coding sequence so as to effect expression of the coding
sequence. This same definition is sometimes applied to the
arrangement of coding sequences and transcription control elements
(e.g. promoters, enhancers, and termination elements) in an
expression vector.
[0034] The term "DNA construct" refers to genetic sequence used to
transform plants and generate progeny transgenic plants. These
constructs may be administered to plants in a viral or plasmid
vector. Other methods of delivery such as Agrobacterium T-DNA
mediated transformation and transformation using the biolistic
process are also contemplated to be within the scope of the present
invention. The transforming DNA may be prepared according to
standard protocols such as those set forth in "Current Protocols in
Molecular Biology", eds. Frederick M. Ausubel et al., John Wiley
& Sons, 1995.
II. Description
[0035] In an effort to obtain commercially feasible tissue culture
yields of vanillin and related compounds, the inventors have now
elucidated the biosynthetic pathway by which these compounds are
produced out of several possible pathways which have been proposed,
and have determined the rate-limiting step in the biosynthesis. The
important discovery of the correct pathway, and the rate limiting
step in particular, has contributed to the development of
high-yield tissue culture for vanillin production.
[0036] Another important feature of the present invention is the
use of embryo cultures of vanilla plants for the purpose of
producing vanillin at an economically feasible level. Embryo
culture of Vanilla planifolia is described in detail in Example
1.
[0037] The vanillin biosynthetic pathway is shown in FIG. 1 and
described in detail in Example 2. As can be seen from FIG. 1,
p-coumaric acid is produced from L-phenylalanine via the shikimic
acid pathway. The first key step in the pathway is the chain
shortening of p-coumaric acid to form p-hydroxybenzyaldehyde, then
p-hydroxybenzyl alcohol. The next key step is the hydroxylation of
p-hydroxybenzyl alcohol to 3,4-dihydroxybenzyl alcohol, then
3,4-dihydroxybenzaldehyde (sometimes referred to herein as
"proaldehyde"). This is believed to be the rate limiting step in
the pathway. Proaldehyde is next methylated to form vanillin
(3-methoxy-4-hydroxybenzaldehyde). In cultured cells, much of the
vanillin produced is reduced to vanillyl alcohol, which is a
detrimental occurrence inasmuch as it depletes the culture of
accumulated vanillin.
[0038] The enzymes involved in the vanillin biosynthetic pathway
are believed to be the following. The chain shortening of
p-coumaric acid to form p-hydroxybenzaldahyde is catalyzed by at
least one chain-shortening enzyme, sometimes referred to herein as
p-hydroxybenzaldahyde synthase. The partial purification and
characterization of a p-hydroxybenzaldahyde synthase from V.
planifolia is described in Example 6.
[0039] The enzyme catalyzing the rate-limiting hydroxylation of
p-hydroxybenzyl alcohol to 3,4-dihydroxybenzyl alcohol is believed
to be a cytochrome P450 monooxygenase. Strategies for cloning the
gene(s) encoding the enzyme(s) are described in greater detail
below and in Example 7.
[0040] The enzyme catalyzing the methylation of
3,4-dihydroxybenzaldehyde to vanillin has been determined to be an
O-methyltransferase. This methyl transferase may be purified from
cultured vanilla cells or from intact plants, according to one of
several methods available in the art for purifying O-methyl
transferase. In a preferred embodiment, it is purified according to
the method of Edwards & Dixon, Arch. Biochem. Biophys. 287:
372-379, 1991.
[0041] The enzyme catalyzing the conversion of vanillin to vanillyl
alcohol has been determined to be an alcohol dehydrogenase, which
the inventors have named vanillyl alcohol dehyrdogenase (VAD). The
purification of VAD from cultured cells of V. planifolia and its
characterization are described in Example 8.
[0042] In the present invention, two general approaches are used to
improve vanillin production in cultured cells and, in some
instances, in intact vanilla plants. The first approach employs
manipulation of tissue culture conditions to increase vanillin
accumulation in cultured cells. The second approach employs genetic
manipulation of the vanillin biosynthetic pathway by up-regulating
or down-regulating, as appropriate, enzymes involved in the
vanillin biosynthetic pathway or in the conversion of vanillin to
vanillyl alcohol. These approaches are described below.
[0043] A. Improving Vanillin Production in Tissue Culture by
Manipulation of Culture Conditions
[0044] It has been discovered in accordance with the invention that
addition of certain "elicitor" compounds provides a surprisingly
high yield of vanillin and related compounds in plant tissue
culture, particularly embryo culture. These elicitor compounds
include malic acid, citric acid, succinic acid, pyruvic acid and
oxaloacetic acid. No method heretofore described has employed these
compounds in plant tissue culture to stimulate production of
vanillin and similar compounds.
[0045] Malic acid is especially successful in this respect. The use
of malic acid in an amount effective to increase production of
vanillin and related compounds in plant tissue is an important part
of the present invention. Malic acid may be used with any type of
plant tissue, under any form of cultivation or in any conditions
known for plant tissue culture. For instance, Table 6 in Example 5
shows that 3% malic acid used in elicitation of embryo cultures
increases vanillin yield from 5 to 72 mg/100 g tissue. Citric acid,
succinic acid, pyruvic acid and oxaloacetic acid may also be used
in amounts effective to increase yields of vanillin and related
compounds at least two- three-fold in plant tissue culture, such as
at about 0.1% to about 5.0%, preferably from about 0.5 to 3.0%, and
otherwise as discussed for malic acid here and in the Examples.
[0046] Another useful elicitor of vanillin production in cultured
vanilla is the glycosylated lysozayme protein elicitor described in
U.S. Pat. No. 5,552,307 to Kessler et al. As shown in Example 5,
treatment with this elicitor more than doubles the amount of
vanillin produced in cultured vanilla cells.
[0047] Another elicitor of vanillin production in cultured cells is
heat stress, i.e. placing the cultures at 33-37.degree. C. for an
extended period of time. Heat stress of this nature has been found
to increase production of vanillin and related compounds in
cultured cells by at least 2-3-fold. Similarly, shear stress, as
described in greater detail in the examples, increases production
of vanillin and related compounds in cultured cells by at least 2-3
fold.
[0048] Also in accordance with the present invention, vanillin
production in cultured cells may be improved by feeding the
cultures with an excess of any of the precursors or intermediates
in the vanillin biosynthetic pathway. For instance, proaldehyde
(3,4-dihydroxybenzaldahyde, the immediate precursor of vanillin)
can be used with any type of plant tissue under any form of
cultivation or any conditions known for plant tissue culture to
stimulate vanillin synthesis. A proaldehyde concentration of 0.1 to
5.0 mM is especially useful. Examples of specific conditions for
addition of proaldehyde are set forth in Example 3. Furthermore,
Example 3 also describes that precursors can be fed to intact green
vanilla beans to improve vanillin production in the beans.
[0049] B. Improving Vanillin Production in Tissue Culture and
Intact Plants by Manipulation of Enzymes of the Vanillin
Biosynthetic Pathway
[0050] Manipulation of the enzymes involved in the vanillin
biosynthetic pathway is another approach used in accordance with
this invention to improve vanillin production in vanilla tissue
culture and in intact plants. As discussed below and in the
examples, the inventors have either isolated these enzymes or
devised means for their isolation using standard methodologies
known in the art, as described in greater detail in the Examples.
These enzymes may be added to or inhibited in plant cultures
directly, or plant tissues may be engineered for altered expression
of the genes encoding the enzymes, by one of several methods as
described below.
[0051] The first key enzyme in the vanillin biosynthetic pathway is
the enzyme referred to herein as the "chain shortening enzyme" or
p-hydroxybenzaldehyde synthase, which catalyzes the conversion of
p-coumaric acid to p-hydroxybenzaldehyde. Though the chain
shortening enzyme may be referred to in the singular, it is
possible that this activity is performed by more than one enzyme.
The conversion of p-coumaric acid to p-hydroxybenzaldehyde is not
considered to be the rate-limiting step in vanillin biosynthesis in
cultured cells. However, it may play a more important
rate-controlling function in intact vanilla beans. In either case,
it is believed that up-regulation or some other form of
supplementation of this enzyme will enhance vanillin production in
cultured cells and in intact plants.
[0052] Another chain shortening enzyme that is expected to be
useful for practice of the invention is the chain-shortening
enzyme(s) that catalyze the conversion of ferulic acid to vanillin.
These enzymes may be used in conjunction with a growth medium
containing ferulic acid, to stimulate production of vanillin.
[0053] The next key enzyme in the vanillin biosynthetic pathway is
the oxygenase that catalyzes hydroxylation of p-hydroxybenzyl
alcohol to 3,4-dihydroxybenzyl alcohol. This enzyme is believed to
be a cytochrome P450 monooxygenase, and this step is believed to be
the rate-limiting step in the vanillin biosynthetic pathway in
cultured cells. For these reasons, up-regulation or some other form
of supplentation of this enzyme in cultured cells and in intact
plants.
[0054] The next key enzyme in the vanillin biosynthetic pathway is
the methyl transferase that catalyzes the conversion of
3,4-dihydroxybenzaldehyde (proaldehyde) to vanillin. Though this
enzyme is not believed to be rate-controlling either in cultured
vanilla or in intact plants, up-regulation or some other form of
supplementation of this enzyme should augment vanillin
accumulation.
[0055] The next key enzyme, vanillyl alcohol dehydrogenase (VAD)
actually catalyzes the destruction of vanillin rather than its
synthesis. VAD is a novel enzyme found in vanilla tissue culture,
but not in any significant amount in vanilla beans. The inventors
have found that vanilla beans produce and accumulate vanillin as a
final product, whereas in tissue culture, vanillin is produced but
is converted by VAD and stored as vanillyl alcohol. Accordingly,
down-regulation of VAD in cultured cells is a key feature of
improved vanillin production from cultured vanilla.
[0056] In some instances it may be possible to add one or more of
the above-listed enzymes directly to a vanilla cell or tissue
culture, to enhance the biosynthetic activity of endogenous enzymes
and increase vanillin production. However, it is preferred for
practice of the present invention to augment or reduce activity of
one or more of these enzymes by internal manipulation; e.g.
up-regulation by genetic engineering to increase transcription or
translation of endogenous genes or transgenes, or down-regulation
by expression of antisense molecules or antibodies that
specifically bind to genes encoding the enzymes or the enzymes
themselves, respectively, or by expression of non-functional
mutants, or by an overexpression/co-suppression effect.
[0057] In order to genetically manipulate the vanillin biosynthetic
pathway, it is necessary to have in hand nucleic acid molecules
that encode selected key enzymes of that pathway. The availability
of purified or semi-purified biosynthetic pathway enzymes, as
described in greater detail in the Examples, enables obtaining
their encoding nucleic acid sequences by a variety of methods known
in the art. Such methods can be found in general references such as
Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory
(1989) (hereinafter "Sambrook et al.") or Ausubel et al. (eds)
Current Protocols in Molecular Biology, John Wiley & Sons
(1997) (hereinafter "Ausubel et al.").
[0058] In a preferred embodiment, antibodies immunologically
specific for a selected key enzyme in the vanillin biosynthetic
pathway are produced, then used to screen a cDNA library made
either from cultured vanilla cells or from intact plants. In an
alternative embodiment, purified enzymes are partially or fully
sequenced, and a set of degenerate oligonucleotide probes is
produced, which encodes part or all of the sequence. These probes
may be used to screen either a genomic or cDNA library by standard
means or via PCR amplification.
[0059] One common formula for calculating the stringency conditions
required to achieve hybridization between nucleic acid molecules of
a specified sequence homology (Sambrook et al., 1989):
T.sub.m=81.5.degree. C.+16.6Log [Na+]+0.41(% G+C)-0.63 (%
formamide)-600/#bp in duplex
[0060] As an illustration of the above formula, using [N+]=[0.368]
and 50% formamide, with GC content of 42% and an average probe size
of 200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA
duplex decreases by 1-1.5.degree. C. with every 1% decrease in
homology. Thus, targets with greater than about 75% sequence
identity would be observed using a hybridization temperature of
42.degree. C.
[0061] Nucleic acids encoding vanillin biosynthetic pathway
enzymes, obtained in accordance with the present invention, may be
maintained as DNA in any convenient cloning vector. In a preferred
embodiment, clones are maintained in plasmid cloning/expression
vector, such as pGEM-T (Promega Biotech, Madison, Wis.) or
pBluescript (Stratagene, La Jolla, Calif.), either of which is
propagated in a suitable E. coli host cell.
[0062] Specific cloning strategies for the various key enzymes of
the vanillin biosynthetic pathway are set forth in the Examples.
Once cloned DNA is obtained, it may be used to genetically
manipulate the vanillin biosynthetic pathway by enhancing or
inhibiting, as appropriate, selected enzymes of the pathway.
[0063] Transgenic plants can be generated using standard plant
transformation methods known to those skilled in the art. These
include, but are not limited to, Agrobacterium vectors, PEG
treatment of protoplasts, biolistic DNA delivery, UV laser
microbeam, gemini virus vectors, calcium phosphate treatment of
protoplasts, electroporation of isolated protoplasts, agitation of
cell suspensions with microbeads coated with the transforming DNA,
direct DNA uptake, liposome-mediated DNA uptake, and the like. Such
methods have been published in the art. See, e.g., Methods for
Plant Molecular Biology (Weissbach & Weissbach, eds., 1988);
Methods in Plant Molecular Biology (Schuler & Zielinski, eds.,
1989); Plant Molecular Biology Manual (Gelvin, Schilperoort, Verma,
eds., 1993); and Methods in Plant Molecular Biology--A Laboratory
Manual (Maliga, Klessig, Cashmore, Gruissem & Varner, eds.,
1994).
[0064] The method of transformation depends upon the plant to be
transformed. The biolistic DNA delivery method is useful for
nuclear transformation, and is preferred for practice of the
present invention. Transformation of V. planifolia using the
biolistic method is described in detail in Example 9. In another
embodiment of the invention, Agrobacterium vectors, particularly
binary vectors such as BIN19, are used for transformation of plant
nuclei.
[0065] Nucleic acids encoding vanillin biosynthetic enzymes may be
placed under a powerful constitutive promoter, such as the rice
actin promoter or the maize ubiquitin promoter, both of which are
particularly useful for gene expression in monocots. Other
constitutive promoters that may also prove useful include the
Cauliflower Mosaic Virus (CaMV) 35S promoter or the figwort mosaic
virus 35S promoter. Alternatively, transgenic plants expressing one
or more of the genes under an inducible promoter (either their own
promoter or a heterologous promoter) are also contemplated to be
within the scope of the present invention. Inducible plant
promoters include the tetracycline repressor/operator controlled
promoter.
[0066] Using a biolistic delivery system for transformation, the
coding region of interest, under control of a constitutive or
inducible promoter as described above, is linked to a nuclear drug
resistance marker, such as kanamycin resistance. Biolistic
transformation of plant nuclei is accomplished according to the
following procedure:
[0067] (1) the gene is inserted into a selected vector;
[0068] (2) transformation is accomplished by bombardment with
DNA-coated microparticles, as described in Example 9;
[0069] (3) plant tissue is then transferred onto the selective
medium to identify transformed tissue; and
[0070] (4) identified transformants are regenerated to intact
plants or are maintained as cultured cells.
[0071] It should be recognized that the amount of expression, as
well as the tissue specificity of expression of the transgenes in
transformed plants can vary depending on the position of their
insertion into the nuclear genome. Such position effects are well
known in the art. For this reason, several nuclear transformants
should be regenerated and tested for expression of the
transgene.
[0072] In some instances, it may be desirable to down-regulate or
inhibit expression of endogenous enzymes, such as VAD in cultured
V. planifolia. Accordingly, VAD-encoding nucleic acid molecules, or
fragments thereof, may also be utilized to control the production
of VAD. In one embodiment, full-length VAD gene antisense molecules
or antisense oligonucleotides, targeted to specific regions of
VAD-encoding mRNA that are critical for translation, are used. The
use of antisense molecules to decrease expression levels of a
pre-determined gene is known in the art. In a preferred embodiment,
antisense molecules are provided in situ by transforming plant
cells with a DNA construct which, upon transcription, produces the
antisense sequences. Such constructs can be designed to produce
full-length or partial antisense sequences.
[0073] In another embodiment, overexpression of a VAD-encoding gene
is induced to generate a co-suppression effect. This excess
expression serves to promote down-regulation of both endogenous and
exogenous VAD genes. Alternatively, transgenic plants can be
created containing mutations in the region encoding the active site
of the enzyme, thereby creating a pool of non-functional enzyme in
the plant cells, which competes for substrate (i.e., vanillin), but
is unable to catalyze the conversion to the undesired product
(vanillyl alcohol).
[0074] From the foregoing discussion, it can be seen that genetic
manipulation of the enzymes involved in the vanillin biosynthetic
pathway will produce engineered plant tissue culture and, if
desired, intact plants capable of high yield of vanillin and
related compounds of value. This approach, alone or combined with
the alternative approach of stimulating vanillin production in
cultured cells by supplementation with elicitors or biosynthetic
precursors, result in improved production of vanillin from a
variety of sources, in accordance with the present invention.
[0075] The following specific examples are provided to illustrate
embodiments of the invention. They are not intended to limit the
scope of the invention in any way.
EXAMPLE 1
Protocol for Initiation of Vanilla Embryo Culture
[0076] Green vanilla beans (from Indonesia), 2 to 8 months after
pollination, were washed with cold water, then with mild detergent
and water, and were next held for 30 minutes in a water solution
containing 20% bleach and a drop of Tween-80. The beans were then
rinsed in sterile water and dried. Seeds from the washed beans were
scraped and placed on a petri plate containing solid medium
("G-medium") as follows. [0077] Gamborg's B-5 basal medium [0078]
2% sucrose [0079] vitamins (see attached list) [0080]
antibiotics--cefotaxime sodium and vancomycin sodium at 100 mg/l
each; [0081] optionally, tetracycline or chloramphenicol at 50 mg/l
each; [0082] 0.8% agar Beans were dissected transversely or
longitudinally and the tissue containing the seeds planted on the
agar.
[0083] To break seed dormancy and to accelerate germination, we
applied the following: [0084] 10 ppm ethylene [0085] 100% oxygen
[0086] 1-10 .mu.M urea [0087] 1-10 .mu.M abscisic acid [0088] Heat
shock (37.degree. C. for 3 hours) [0089] Cold shock (2-3.degree. C.
for 48 hours) After 2 to 6 months, seeds germinated and were
transferred to fresh agar medium. When germinating shoots reached
about 10 mm, they were dissected in half and transferred to fresh
agar medium. This process was repeated every two weeks for three
months. The agar-cultured embryo tissue was transferred to liquid
G-medium without agar. The liquid culture was maintained on an
orbital shaker at 130 rpm. Embryo culture was subcultured every two
weeks by collecting embryos on a sieve and dissecting the growing
embryos into 2 to 4 pieces, depending on the size.
[0090] Some embryos were maintained on solid medium and some were
kept on rafts (Sigma, St. Louis, Mo.). All cultures were held in
light (80 .mu.E/sec/cm) at 25-28.degree. C.
[0091] The protocol for initiation of cell suspensions from embryo
cultures of V. planifolia was as follows. Established embryo
cultures were transferred from petri plates containing G-medium to
the same medium, supplemented with 1 .mu.M 2,4-D, then subcultured
for two weeks. After callus was initiated, it was transferred to
solid medium containing 0.5 .mu.M 2,4-D. After 2 to 6 additional
subcultures, the resulting soft callus was transferred to liquid
G-medium with 0.5 .mu.M 2,4-D and maintained as cell
suspension.
EXAMPLE 2
Scheme for Vanillin Biosynthetic Pathway
[0092] We derived a scheme for the vanillin biosynthetic pathway by
analysis of metabolites in cultured embryos and by experiments with
feeding of precursors and intermediates. This scheme is shown in
FIG. 1 and the experiments are described below.
[0093] Embryo cultures of Vanilla planifolia were established as
described in Example 1. The procedures used to extract phenolics
from the cultured embryo cells and to analyze the extracts by high
pressure liquid chromatography (HPLC) were as described generally
by Havkin-Frenkel et al., Plant Cell, Tissue and Organ Culture 45:
133-136 (1996).
[0094] In the extraction procedure, 3 ml of 0.05 M sodium acetate
buffer, pH 5.5, was added to about 1 gram of fresh culture. Samples
were placed in boiling water for three minutes, then chilled. The
cells were next homogenized in a Polytron blender for 1 minute at
medium speed. A .beta.-glucosidase solution was added to the
homogenized cells to give a final concentration of 0.2%. Each
sample was then incubated at 37.degree. C. for 5 hr. Next, 17 ml of
95% ethanol was added, after which incubation at 37.degree. C. was
continued for an additional 24 hrs. The extract was then filtered
and the ethanol evaporated. The filtrate was extracted twice using
ethyl acetate, then extracted twice with ethyl acetate acidified to
pH 3 with HCl. The extracts were combined and the ethyl acetate
evaporated. The residue was dissolved in 1 ml of acidified methanol
and filtered with a 0.45 .mu.m syringe filter for HPLC
analysis.
[0095] Extractions of metabolites from culture medium was similar
to the cell extraction protocol. Five ml of spent medium was
incubated in a .beta.-glucosidase solution at 37.degree. C. for 24
hrs. The medium was then extracted with ethyl acetate and the
organic portions combined and evaporated, as for the cell
extracts.
[0096] Metabolite levels were measured with a Hewlett Packard 1090L
or a Waters HPLC with a UV detector at 280 nm. The Waters HPLC was
also equipped with a diode array detector to confirm the identities
of the various intermediates, and the identities of the various
intermediates were further confirmed by mass spectrometry. The
column was a Supelco C-18 DB column of dimensions 250 mm.times. 4.6
mm and a particle size of 5 .mu.m. The mobile phase contained
methanol and water, each of which was acidified with 1.25% acetic
acid. The flow rate was 1 ml/min, with a solvent gradient as
follows: TABLE-US-00001 Time (min) % Water 0-10 85 20-25 80 30-42
50 42-end 85
[0097] HPLC analyses of tissue extracts from cultured embryos
revealed threshold levels of p-coumaric acid, p-hydroxybenzoic acid
and p-hydroxybenzaldehyde; usually high levels of p-hydroxybenzyl
alcohol, trace levels of 3,4-dihydroxybenzyl aldehyde (Pro-ald),
vanillin and vanillyl alcohol.
[0098] It is known that coumaric acid (CA) is derived from the
deaminization of phenylalanine or tyrosine. The acid, a C6-C3
compound, is converted by chain shortening to p-hydroxybenzaldehyde
(BA), a C6-C1 compound. Feeding experiments with CA revealed that
exogenously applied CA is immediately converted to BA. We examined
if benzoic acid or p-hydroxybenzyl aldehyde may be intermediates in
the conversion of CA to BA, but a definitive answer has not yet
been reached. However, it is clear that at least one chain
shortening enzyme is involved in the conversion from CA to BA, and
that this step does not appear to be rate-limiting in cultured
cells. However, some evidence indicates that it is the
rate-limiting step in intact vanilla beans.
[0099] A key juncture in the pathway in cultured cells appears to
be the hydroxylation of HBA to 3,4-dihydroxybenzyl aldehyde or
alcohol and subsequently, vanillin and vanillyl alcohol. Feeding of
3,4-dihydroxybenzyl aldehyde resulted in the rapid methylation and
conversion to vanillin or vanillyl alcohol, indicating that these
steps are not limiting. The constraint appears to be in the
hydroxylation of HBA, for the following reasons:
[0100] 1. HBA is usually found in higher levels than other
intermediates, suggesting a block in further turnover of the
compound.
[0101] 2. Feeding of CA resulted in the accumulation of HBA but
only trace amounts of other compounds.
[0102] 3. Chemical stresses that induce the enzymatic turnover of
HBA resulted in the disappearance of the compound and the
simultaneous increase in dihydroxybenzyl alcohol or aldehyde, as
well as vanillin and vanillyl alcohol.
[0103] 4. In tissue homogenates where enzyme and substrate are
accessible to each other, HBA was rapidly metabolized to
dihydroxybenzyl aldehyde. Feeding the homogenates with HBA
increased accumulation of dihydroxybenzyl aldehyde, which was the
final product since methylation requires intact tissue or the
addition of S-adenosyl methionine (SAM).
[0104] 5. Cytochrome P450 enzymes are normally inducible enzymes
that become active at certain stages of development or
differentiation. Since the embryo culture is composed of
undifferentiated cells, this explains why Cyt p450 activity is not
observed.
[0105] Thus, our data suggest unhindered metabolite flux to and
from HBA in cultured vanilla embryo cells. Hydroxylation of the
compound induced by chemical or genetic means is expected to lead
to augmented production of vanillin and related compounds in
cultured cells.
EXAMPLE 3
Use of Vanilla Tissue Culture for the Bio-Conversion of
3,4-Dihydroxybenzaldehyde to Vanillin and Vanillyl Alcohol
[0106] The following procedure was used for the bio-conversion of
3,4-dihydroxybenzaldehyde ("proaldehyde") to vanillin and vanillyl
alcohol in V. planifolia tissue culture. The medium used for
cultures was G-medium as described above. Pro-ald solutions were
prepared in G-medium, to final concentrations of 0.01 to 5 mM.
[0107] Cultures used for the bio-conversion were (1) clusters, (2)
embryo culture, and (3) tissue homogenates of the above cultures.
Cultures were of varying ages ranging from 0 to 1 month old. The
cultures were allowed to remain under normal culture conditions for
0 to 15 days. As controls, untreated cultures were extracted and
analyzed as described in Example 2.
[0108] Proaldehyde at different concentrations was added to the
medium, either alone or in combination with the following
treatments: [0109] malic acid (0.01-3.0%) [0110] varying pH of the
medium [0111] varying ascorbic acid concentration [0112] varying
temperatures, including cold and heat. Bioreactor-grown cultures
were used for the bio-conversions. Different kinds of impeller
designs were used to increase or decrease shear stress on the cells
prior to the addition of pro-ald. As shown in Example 5, it was
found that addition of 1-5 mM pro-ald increased the production of
vanillin/vanillyl alcohol by several hundred fold.
[0113] In another experiment, the effect of daily refreshing of the
pro-ald containing culture medium was examined. Cultures were
transferred to medium containing 5 mM pro-ald. Control cultures
were left in this medium for the duration of the experiment. For
test cultures, the medium was removed daily and replaced with fresh
medium containing pro-ald. Cultures subjected to this daily medium
change were improved in their appearance and growth, as compared
with cultures remaining in the same medium.
EXAMPLE 4
Malic Acid-Induced Vanillin and Vanillyl Alcohol Production
[0114] Application of malic acid to vanilla tissue culture induced
the production/accumulation of vanillin and vanillyl alcohol. Malic
acid in concentrations of 0.1 to 3% was applied as a disodium salt
to the growing medium. The culture was maintained for 1 to 15 days,
then was extracted as described in Example 2. Results are shown in
Table 6 in the following example.
[0115] Malic acid was applied to the following: (1) intact roots,
(2) intact shoots, (3) embryo cultures, (4) cluster cultures, and
(5) cuttings. The age of the cultures were between 0 and 1 month.
Malic acid was applied alone or in combination with the following:
starvation without sugar (sucrose); shear stress induced by
bioreactor impeller; citric acid; varying concentrations of oxygen
andethylene; oxaloacetic acid (sodium salt); ascorbic acid; pyruvic
acid; glutamic acid; succinic acid; or salt stress.
[0116] Adding proaldehyde for a few days, followed by addition of
malic acid, was found to increase production of vanillin and
vanillyl alcohol. If sucrose is omitted from the malic acid
treatment (i.e. starvation due to lack of sucrose), the onset of
vanillyl alcohol production occurs more quickly.
[0117] Shear stress had a significant effect on vanillin
production. The bioreactor with a marine impeller (5 liters,
110-120 rpm, airspeed 250 ml/min) was used to culture embryo and
cluster cultures for about 21 days. Addition of malic acid after
this time resulted in the highest production of vanillin and
vanillyl alcohol.
EXAMPLE 5
Results of Selected Feeding Experiments
[0118] Results of selected experiments in which precursors or
elicitors were added to vanilla cultures are set forth below. These
experiments were performed in accordance with the procedures set
forth in Examples 1-4.
[0119] The following abbreviations are used:
[0120] CA or PC=p-coumaric acid
[0121] HY=p-hydroxybenzoic acid
[0122] BA=p-hydroxybenzaldehyde
[0123] HBA=p-hydroxybenzyl alcohol
[0124] Pro-aid=3,4-dihydroxybenzaldehyde
[0125] HMBA and Vn. Alc.=vanillyl alcohol
[0126] Vn=vanillin
[0127] FA=ferulic acid
[0128] CAF=caffeic acid
[0129] The table below shows results of experiments in which
vanillin precursors were fed to vanilla embryo cultures.
TABLE-US-00002 TABLE 1 Feeding Vanilla Embryo Culture with Vanillin
Precursors (mg/100 g dry wt.) PRECURSORS CA HBA PRO-ALD VN ALC VN
CONTROL 108 9300 13 53 0.01 CA (1 mM) 220 13000 10 10 0.01 FA (25
mM) 136 11007 4.8 23.8 11.8 CAF (2 mM) 189 11350 21 32 0.5 BA (1
mM) 125 10305 5.8 12.3 0.015 HBA (1 mM) 158 13100 4.6 9.6 0.001
PRO-ALD (1 mM) 285 8950 433 286.8 16.7 VN.ALC. (2 mM) 206 10350 1.1
882 8.5
[0130] In an experiment with intact plant material, whole green
vanilla beans (6 months post-pollination) were infiltrated with
various vanillin precursors. The precursors (1.0 mM each in 0.1 M
mannitol) were infiltrated by submerging the beans under vacuum
into the solutions for 15 minutes, removing and drying the beans,
then measuring amounts of precursors daily, for 5 days. The table
below shows the results of one such experiment. TABLE-US-00003
TABLE 2 Feeding green vanilla beans with vanillin precursors
PRECURSOR CA BA HY HBA PRO-ALD Vn Van. Alc CA + ++ ++ ++ BA ++ HY
++ HBA -+ ++ ++ ++ PRO-ALD ++ +++ Vn +++ Van Alc -+ +++
[0131] The table below shows the results of experiments in which
Fusarium cell walls were added to vanilla embryo cultures as an
elicitor of vanillin production. The results show that Fusarium
cell walls stimulate production of various precursors of vanillin,
up to the apparently rate-limiting step of HBA to pro-ald.
TABLE-US-00004 TABLE 3 Effect of Fusarium Cell Wall on Flavor
Production in Vanilla planifolia Embryo Culture TREATMENT/ mg/100 g
Dry Weight COMPOUND HBA HY BA PC Control/No Additions 3700 35 65 52
27 mg. dry cell wall 4300 50 67 127 50 mg. dry cell wall 6700 128
198 389 Culture conditions: Cells were grown for 2 days at
28.degree. C. at 180 RPM
[0132] The table below shows the results of experiments testing the
effect of chilling temperature on vanillin precursors in vanilla
cluster cultures. These results indicate that chilling stress
stimulates production of vanillin precursors, up to the
rate-limiting conversion of HBA to pro-ald. TABLE-US-00005 TABLE 4
Effect of Chilling Temperature on Vanillin Precursors in Vanilla
planifolia Cluster Culture mg/100 g Dry Weight HBA HY BA PC 15 Hrs.
at 13.degree. C. 144.0 2.1 15.9 7.2 15 Hrs. at 13.degree. C. 17
Hrs. at 28.degree. C. 232.0 1.8 26.4 7.4 15 Hrs. at 13.degree. C. 7
Days at 28.degree. C. 586.0 7.4 52.4 15.7 Control 111.0 0.74 20.3
5.5 7 Days at 28.degree. C.
[0133] The table below shows the results of experiments testing the
effect of the glycosylated lysozyme elicitor proteins described in
U.S. Pat. No. 5,552,307 on vanillin precursors in vanilla embryo
cultures. As can be seen, these proteins were effective in
stimulating vanillin production in the cultured cells.
TABLE-US-00006 TABLE 5 Effect of Elicitor Protein on Vanillin
Precursors in Vanilla planifolia Embryo Cultures Treatment/ mg/100
g Dry Weight Compound HBA HMBA PROALD HY BA VN CA Control/ 1990.8
38.1 9.7 76.0 77.7 3.6 151.0 No Addt 30 .mu.g/ml 20006.3 30.7 137.0
83.4 79.7 8.4 152.0 Elicitor Protein Added Each point represents an
average of 5 flasks. Culture conditions: Cells were grown for 7
days at 25.degree. C. at 180 RPM
[0134] The table below shows the results of HPLC analysis of
intermediary metabolites induced by malic acid elicitation in
embryo culture and grown under standard conditions, respectively.
Cultures were grown in medium containing 3% malic acid by weight,
for 7 days. These results show that malic acid stimulates vanillin
production in embryo cultures more than tenfold. TABLE-US-00007
TABLE 6 HPLC Analysis of Intermediary Metabolites Induced by 3%
Malic Acid in Vanilla planifolia Embryo Culture Growth HBA VN HMBA
PROALD BA HY PC conditions (Percent of Dry Weight) Standard 11.07
0.005 0.023 0.024 0.03 0.05 0.20 Malic 3.60 0.072 0.700 0.025 0.02
0.050 0.20 Acid
EXAMPLE 6
Purification and Characterization of Hydroxybenzaldehyde Synthase
from Vanilla Planifolia Green Embryo Culture
[0135] Conversion of p-coumaric acid to p-hydroxybenzyl alcohol in
vanilla is catalyzed by at least one chain-shortening enzyme. The
rate of conversion as catalyzed by this enzyme is shown in FIG. 2.
Characteristics of p-coumaric acid chain shortening enzyme, also
referred to as hydroxybenzaldehyde Synthase, are described in this
example. It should also be noted that ferulic acid is converted to
vanillin by one or more other chain shortening enzymes, which are
believed to be distinct from the p-coumaric acid chain shortening
enzyme.
Plant Material
[0136] The embryos were cultivated on sterile Gamborg B-5 liquid
medium (3% inoculum,) containing microelements, vitamins,
supplemented with 2% of sucrose. The culture was grown at room
temperature, under constant illumination (2.times. OSRAM-DULUX EI
GLOBE, 100 W each) on rotary shaker (150 rev./min) and subcultured
every 3-4 weeks. The conversion of p-coumaric acid to
p-hydroxybenzaldehyde as catalyzed by the enzyme(s) is shown in
FIG. 3.
Crude Enzyme Extraction
[0137] Sterile plant material (2 g) 2-3 weeks after subculture, was
homogenized in a cooled Potter glass homogenizer with 4 ml of 0.1 M
HEPES buffer, pH 8.0, containing 10 mM DTT. The homogenate was next
centrifuged at 4.degree. C. at 15.000.times.g for 15 min. Resultant
supernatant (4 ml) was filtered through Sephadex G-25 column (void
volume 3-4 ml.) Equilibrated with Tris/HCL buffer pH7, containing
10 mM DTT. The column washed with the same buffer and 2 ml fraction
following the void volume was collected. This fraction was used as
the crude enzyme source.
[0138] The crude enzyme was subjected to SDS polyacrylamide gel
electrophoresis. A major band was observed at about 150-200
kDa.
Determination of the Enzyme Activity
[0139] The p-HO-aldehyde synthase activity was determined in the
following mixture: TABLE-US-00008 crude enzyme extract 10.mu.
substrate 100 .mu.l (1.8 mM p- coumarate in 0.1 M Tris/HCl, pH 8.0
containing 10 mM DTT)
[0140] The mixture was incubated for 10-60 min (for longer
incubation times, the activity was not proportional with time) at
35.degree. C. and next, the reaction stopped by addition of 200
.mu.l of acidified methanol (10% acetic acid in methanol). The
slurry was passed through 0.45 .mu.m filter and the filtrate (50
.mu.l) injected into HPLC Bio-sil 18 HL 90-5 column (Reversed
phase, 250 mm.times.4.6 mm). The HPLC column mobile phase was
methanol:water (15:85) acidified with acetic acid (1.25%) at flow
rate 1 ml/min. The eluate was monitored at 280 nm and retention
time of the reaction product was compared to retention time of the
standard compounds (benzaldehyde, p-HO-benzaldehyde, vanillin,
protocatechuic aldehyde, p-coumaric acid, caffeic acid, ferulic
acid, coniferyl aldehyde). The results were quantified using LKB
Bromma 2221 Integrator. TABLE-US-00009 TABLE 7 Enzymatic Activity
of Crude Preparation of p-hydroxybenzaldehyde synthase from V.
planifolia Incubation time activity (nmoles of P--HO-- (min)
benzaldehyde/g fr wt./hr) 10 215 20 342 50 946 60 984 120 1022
Optimum pH
[0141] Extraction efficiency was checked for pH condition from 3 to
9 using citrate and Tris buffers. A broad optimum was found with
maximum at pH 8.0. The enzyme pH optimum activity was located
between pH 7 and pH 9 and corresponded to optimum of the enzyme
extraction. TABLE-US-00010 TABLE 8 Effect of pH of Extraction on
Enzymatic Activity pH of extraction activity (nmoles/gfw/h) 3 199 4
214 5 258 6 306 8 350 9 205
[0142] TABLE-US-00011 TABLE 9 Effect of pH of Reaction on Enzymatic
Activity pH of reaction activity (nmoles/gfw/h) 4 29 5 29 7 171 8
548 9 479
Stability of the Crude Enzyme Preparation
[0143] Samples of the G-25 Sephadex filtered enzyme were stored up
to 11 days at 5.degree. C. and frozen at minus 17.degree. C.:
TABLE-US-00012 TABLE 10 Stability of Crude Enzyme Preparation
5.degree. C. 17.degree. C. Days of storage % of activity 0 100 100
1 55 60 4 30 41 6 27 34 11 0 20
Ammonium Sulfate Fractionation
[0144] HEPES pH 8.0 enzyme extract was subjected to ammonium
sulfate fractionation (Salt grinded into fine powder, ice bath).
TABLE-US-00013 TABLE 11 Activity in Various Ammonium Sulfate
Fractions Amm. Sulf. Protein total activity sp. activity %
saturation content (.mu.g) (nmol/h) (mnol/mg/hr) control 7100 7800
1098 0-30 4800 465 97 30-60 450 4710 10466 60-95 100 3510 35100
These results demonstrate no loss in recovery of total activity and
about 35.times. purification in fraction precipitated between 60
and 95% ammonium sulfate saturation. Substrate Specificity
[0145] As a potential substrates for chain shortening enzyme the
following compounds were tested: TABLE-US-00014 TABLE 12 Substrate
Specificity of p-hydroxybenzaldehyde Synthetase Compound Expected
product Result t-cinnamic acid benzaldehyde negative p-coumaric
acid p-HO-benzaldehyde positive caffeic acid protocatechuic
aldehyde negative ferulic acid vanillin negative
4-HO-3-metoxycinnamyl vanillin negative aldehyde (coniferyl
aldehyde)
These results indicate very high specificity of the tested enzyme
towards p-coumaric acid.
EXAMPLE 7
Strategies for Cloning a cDNA Encoding the Cyt P450 Monooxygenase
that Catalyzes the Rate-Limiting Step in Vanillin Biosynthesis
I. PCR-Based Method
[0146] One object of the present invention is the cloning of the
cytochrome P450 that catalyzes the 3-hydroxylation of
p-hydroxybenzyl alcohol. Although the activity of this enzyme is
apparent from the precursor feeding studies described above, the
general liability and low abundance of plant cytochrome P450
enzymes probably rules out cloning by classical enzyme
purification. Therefore, an alternative strategy is to use a
polymerase chain reaction (PCR) based method, using RNA isolated
from a system in which the enzyme activity is highly induced,
namely vanilla cell cultures exposed to an elicitor, such as malic
acid. This strategy is facilitated by the recent appearance of many
plant P450 sequences in the gene data bases, as this allows the
design of primers that can be used for PCR amplification of unknown
P450 sequences. According to the latest review on plant cytochrome
P450s, ninety different sequences have been listed that appear to
encode such enzymes. However, most of these have no known function
as yet.
[0147] The genes encoding cytochrome P450s are highly divergent at
the nucleotide sequence level. Nevertheless, these enzymes do
contain conserved sequence motifs in their open reading frames
sufficient for the design of PCR-based cloning strategies.
Specifically, a highly conserved motif (F-G-R-C-G), that includes
the cysteine residue which binds the heme group necessary for
catalysis by this class of enzyme, is present in all known P450s,
and is located near the carboxyl end of the protein. Forward and
reverse oligonucleotide primers are constructed for PCR
amplification. These are based on sequence motifs surrounding
nucleotides 500, 1050, and 1400 (the heme binding region) of the
alfalfa cinnamate 4-hydroxylase cytochrome P450, one of the best
characterized plant cytochromes P450. Degenerate primers are
constructed. In particular, inclusion of each of the 4 nucleotides
(A, T, G, C) at the 3 end, optimizes the amplification of novel
P450 sequences. The PCR primers also contain restriction
endonuclease sites at their ends to facilitate cloning of the PCR
products.
[0148] The template for PCR amplification, as described above, is
double-stranded DNA produced by reverse transcription of RNA from
vanilla cell culture exposed to an elicitor, such as malic acid.
After separation of PCR products by gel electrophoresis, the
amplified band containing multiple P450s is cut out from the gel,
and cloned into E. coli. The inserts in individual clones are
analyzed by gel electrophoresis to determine insert size, followed
by restriction enzyme analysis in order to place the clones into
classes. Central to our experimental design, we then use the
various P450 inserts as labeled probes for northern blot
hybridization to RNA isolated from elicited and unelicited
cultures. P450s that are present in the elicited but not the
unelicited culture are taken to the final stage of the analysis,
the functional expression for enzymatic activity.
[0149] Initially, the P450s will be expressed in E. coli. For such
a method to be successful, it is usual to co-express, in trans, a
NADPH cytochrome P450 reductase. This could be from various
sources, but the P450 reductase from the bacterium Pseudomonas, or
the plant Arabidopsis is used initially. Enzymatic assay in
bacteria is facilitated by a staining method for colonies
expressing an enzyme capable of forming ortho-dihydroxyphenols, as
described by Yabannavar and Zylstra (1995). In an alternative
strategy, the DNA is expressed in yeast, using the pYEUra3
expression vector that has been successfully used for yeast
expression of the alfalfa cinnamate 4-hydroxylase P-450 (Fahrendorf
and Dixon, 1993).
[0150] Once cloned and expressed, the cyt P450 that catalyzes the
hydroxylation p-hydroxybenzyl alcohol to 3,4-dihydroxybenzyl
alcohol is further analyzed in order to determine its substrate
specificity. For example, is the enzyme promiscuous in its
specificity, or does it only hydroxylate p-hydroxybenzyl
alcohol.
[0151] The 3-hydroxylase cDNA is cloned into a suitable expression
vector for vanilla transformation. Initially, the cDNA is expressed
constitutively, driven by the rice actin or the maize ubiquitin
promoters. These promoters are very effective in monocots.
Transformation of vanilla is described in detail in a later
Example.
II. Use of Auxotrophs
[0152] A major technical problem in the isolation of a particular
plant gene is the method of screening cloned libraries containing
many thousands of DNA or cDNA sequences for the one desired gene
sequence. Recently, the method of functional complementation has
been applied to the screening of libraries of cloned eukaryotic
cDNA sequences. While this is one of the many technologies of gene
isolation, it is particularly appealing in its power and
simplicity. In this method, a mutant bacterial strain with a
selectable phenotype is transformed with a higher plant cDNA
library which carries full-length copies of messenger RNA molecules
in the expressible form. The desired cDNA sequences are actually
selected in this method, which is more powerful than screening
methods in finding very rare sequences in the library. For example,
mutations resulting in nutrient auxotrophies in the test bacteria
have been readily used to identify the homologous gene from higher
plants. The test bacteria can only grow and form colonies if they
have received the homologous gene from the library which restores
their nutritional deficiency. However, this method is not limited
to mutant bacterial strains alone. Indeed, any selectable phenotype
can be used for the complementation test and a wide variety of test
organisms are possible. The best selectable phenotype is growth of
the test cells. In the case of HBA-hydroxylating activity, a test
strain may only be able to use HBA as a carbon source if this
substance is first hydroxylated. For example, recently, the pathway
of HBA metabolism has been studied in the fungus Aspergillus
fumigatus (ATCC 28282) as part of the metabolism of p-cresol. The
data indicate that HBA metabolism requires the hydroxylation by a
monooxagenase enzyme for further metabolism. As this organism grows
on p-cresol as a sole source of carbon and energy, the development
of a mutant strain suitable for complementation testing for a plant
HBA monooxagenase activity is expected to be successful. In this
instance, cDNA clones encoding higher plant HBA-hydroxylating
enzymes, such as the enzyme involved in the vanillin biosynthetic
pathway, are cloned by selecting for microorganisms that are
capable of growth on medium containing HBA as the sole carbon
source.
EXAMPLE 8
Purification and Characterization of Vanillyl Alcohol Dehydrogenase
(VAD), Cloning of VAD-Encoding cDNA and Gene, and Regulation of VAD
Gene Expression
[0153] Application of vanillin to the growing medium of embryo
culture results in the rapid uptake and reduction of the applied
compound to vanillyl alcohol as illustrated in FIG. 3. Further
more, application of 3,4-dihydroxy-benzaldehyde, a vanillin
precursor also results in the accumulation of vannilyl alcohol
indicating that the tissue has high capacity for the reduction of
vanillin to produce vanillyl alcohol. Similarly, vanilla embryo
culture that can be elicited by various elicitors to produce
vanillin accumulates vanillyl alcohol as a final product. It is
important to state at this stage that all the intermediates in the
pathway are found mainly as glucosides.
[0154] We have purified and characterized the activity of VAD that
catalyze the reduction of vanillin to vanillyl alcohol. The enzyme
was identified as an NADH or NADPH-dependent alcohol dehydrogenase.
The purification protocol was as set forth below.
[0155] Crude enzyme extract: Tissue was homogenized in 0.05 M
actetate-Na buffer pH 4.0 (in proportion, 1 g of the tissue and 5
ml of the buffer) in an ice bath, using Polytrone homogenizer, at
20,000 revolutions of the blade per min. The homogenate was
centrifuged at 13000 g for 15 min at 4.degree. C. and the
supernatant served as the crude enzyme source.
[0156] Ammonium sulfate fractionation and molecular sieving: Crude
enzyme extract (100 ml) was supplied with ammonium sulfate in an
ice bath up to 60% of saturation (44.4 g of Ammonium sulphate per
100 ml). The precipitate was centrifuged and discarded. The
supernatant was supplied with ammonium sulfate up to 90% of
saturation (total 64.6 g per 100 ml of crude extract). The
precipitate was collected (10 min centrifugation at 10,000 g),
dissolved in 7 ml of extraction buffer and subjected to molecular
sieving at Sephacryl S-300 High Load Column (LKB Pharmacia)
2.6.times.60 cm, in 0.1 M pH 4.0 acetate-Na buffer. The flow rate
was adjusted to 1 ml/min and 5 ml fractions were collected. The
protein elution profile was monitored at 280 nm. Five fractions (of
a total of 50) containing dehydrogenase activity (130-155 ml of the
column eluate) were collected.
[0157] Affinity chromatography on Red Sepharose CL
6B(LKB-Pharmacia): The active combined fractions from the Sephacryl
column (24 ml) were applied on 2.5.times.3 cm Red Sepharose CL 6B
column, equiluibrated with 0.05 M pH 4.0 acetate-Na buffer. The
column was developed with 0.5 M Tris/HCL buffer pH 7.4 in 0.0-1.0 M
NaCl gradient (total gradient volume--70 ml) and 2.5 ml fractions
were collected. Vanillyl alcohol dehydrogenase was released from
the column between 0.3-0.4 M NaCl. Fractions containing VAD
activity were dialysed overnight against 0.05 M caetate-Na buffer
pH 4.0 and concentrated up to 50 times using Minicon concentrating
filters (Amicon).
[0158] Polyacrylamide Gel Electrophoresis: Concentrated enzyme
extract was used for native and SDS polyacrylamide gel
electrophoresis. In native electrophoresis, two active bands
(corresponding to protein bands localized with Coomassie brilliant
blue) of molecular weight between 43 and 67 kDa were found. SDS
electrophoresis revealed 3 still active protein bands--of 20 kDa,
37 kDa and 40 kDa.
[0159] The pH optimum for the enzyme extraction was at 3.0 and
optimum activity was obtained at pH 4.0. The subunit molecular
weight determined on the basis of electrophoretic mobility in the
presence of SDS was around 43 kDa. Table 13 indicates that VAD
shows preference toward C.sub.6-C.sub.1 phenolic compounds and no
activity toward C.sub.6-C.sub.3 phenolics.
3,4-dihydroxy-benzaldehyde and vanillin appear to be the most
preferred substrates while affinity to other C.sub.6-C.sub.1
aldehydes or acetaldehyde is lower. TABLE-US-00015 TABLE 13
Substrate specificity of alcohol dehydrogenase from Vanilla
planifolia embryo culture. Activity (nmoles gfwt.sup.-1 min.sup.-1)
Substrate NADH NADPH 1. Acetaldehyde 0.26 -- 2. Benzaldehyde 0.33
0.18 3. 4-hydroxybenzaldehyde 0.32 0.14 4.
3,4-dihydroxybenzaldehyde 1.89 2.11 5.
4-hydroxy-3-methoxybenzaldehyde 1.26 0.97 6.
4-methoxy-3-hydroxybenzaldehyde 0.00 0.00 7.
4-hydroxy-3-ethoxybenzaldehyde 0.42 -- 8.
4-methoxy-3-ethoxybenzaldehyde 0.00 -- 9. Cinnamylaldehyde 0.00 --
10. 4-hydroxy-3-methoxycinnamylaldehyde 0.00 --
[0160] Sequence information from the purified VAD protein is used
to design primers to clone the gene encoding VAD, and the cloned
gene is used for the creation of a VAD antisense gene using
established methods (below). Vanilla tissue culture is transformed
with the antisense gene and the tissue assessed for an expected
attenuation in the activity of VAD and a corresponding reduction in
vanillyl alcohol accumulation concomitant with an increase in the
level of vanillin.
Sequencing of the VAD Protein
[0161] The purified VAD is purified further to homogeneity, using
conventional chromatographic approaches such as chromatofocusing
and hydrophobic interaction chromatography. Tryptic peptides from
the purified protein are sequenced by automated Edman degradation
and used to design oligonucleotide primers for PCR amplification
(He and Dixon, Arch. Biochem. Biophys. 336: 121-129, 1996). Since
the relative position of the tryptic fragments in the VAD sequence
may not be known, degenerate oligonucleotide primers based on
regions of minimal degeneracy in the genetic code are designed for
each peptide in both forward and reverse orientations, and the
various primer combinations evaluated. Oligonucleotide sequences
are synthesized as outlined by He and Dixon (1996).
Cloning of the VAD Gene
[0162] Production of cDNA Library. High levels of VAD are produced
constitutively in vanilla embryo culture. Embryo culture cells are
harvested on nylon mesh, frozen in liquid N2, and stored at -70 C.
A cNDA library for DNA probing and expression is constructed from
poly(A)+ RNA extracted from vanilla embryo culture cells using the
LambdaZAP system.
[0163] PCR Screening. Template DNA for PCR amplification is
obtained by boiling a portion of a vanilla cDNA library as
previously described (Junghans et al., Plant Mol. Biol. 22:
239-253, 1993). Amplified fragments are cloned, and sequenced to
check that they contain sequences corresponding to one or more of
the cryptic peptides, along with sequence diagnostic for
dehydrogenases. The Lambda ZAP cDNA library from vanilla tissue are
autoexcised into p-Bluescript, and screened with PCR fragments that
had been 32-labeled by random priming. Positive plaques are
identified by autoradiography. Full length clones are sequenced on
both strands, and functional identification is performed by
expression in E. coli.
[0164] Immunoscreening. In another approach the expression library
containing cDNAs derived from transcripts from vanilla embryo
culture cells are screened with an antiserum raised against VAD. E.
coli (XL-1 Blue cells obtained from Stratagene) are infected with
the library, and positive clones selected be purified by several
rounds of screening and processed to homogeneity. Sequencing of VAD
cDNA clones is according to standard methods.
Regulation of Expression
[0165] Expression of VAD in Vanilla Tissues and Cell Cultures. The
expression pattern of VAD in vanilla beans and embryo cultures is
determined by northern blot hybridization, and the genomic
organization of VAD determined by Southern blot hybridization. RNA
is extracted as previously described (Logemann et al., Anal.
Biochem. 163: 16-20, 1987), total RNA separated and transferred to
and fixed onto cellulose membranes (Jorrin and Dixon, Plant
Physiol. 92: 447-455, 1989) and hybridized to an internal coding
fragment of VAD. Genomic DNA is isolated from vanilla culture,
digested with restriction enzymes, fractionated by electrophoresis
and hybridized to a labeled VAD using standard procedures.
[0166] Antisense. Double stranded full length VAD clones are
sequenced in both directions and used to construct a VAD antisense
gene.
[0167] Transformation of Vanilla Cultured Cells with VAD Antisense
Constructs. The VAD cDNA is cloned, in both sense and antisense
orientations, into a suitable expression vector for vanilla tissue
transformation. The idea is that some sense transformants may
exhibit highly reduced VAD activity due to epigenetic
co-suppression. Initial transformations focus on achieving
constitutive expression driven by the rice actin or the maize
ubiquitin promoters. The cDNA clone is introduced by particle
bombardment as described in a later Example.
[0168] Analysis of Transgenic Plants. Putative transgenic plants
are screened for the VAD antisense transgene by PCR using primers
designed to sequences within the selectable marker gene.
Transformation is confirmed by Southern border analysis using a VAD
cDNA probe. Screening for expression of VAD activity in transformed
vanilla culture is done spectrophotometrically based on NADH
oxidation as previously described (Biscak et al., Arch. Biochem.
Biophys. 215: 605-615, 1982, Longhurst et al., J. Food Biochem. 14:
421-433, 1990) and VAD transcripts determined by northern blot
hybridization. In addition, the culture is extracted and analyzed
by HPLC for the intermediates in the vanillin biosynthetic pathway
(Havkin-Frenkel et al., 1996), in particular the levels of vanillin
and vanillyl alcohol. The enzyme (VAD) is extracted as described
above.
EXAMPLE 9
Transformation of Vanilla Planifolia
[0169] Cultured Vanilla planifolia was transformed by the procedure
set forth below:
[0170] 1. Vanilla culture was grown on a basic medium agar plates
containing 2,4 dichloro-phenoxyacetic acid (2,4-D), was subcultured
3 times during 4 months according to the procedures described
above. Cultures were kept at 25.degree. C., 15% humidity, under
illumination of 80 .mu.E/sec/m.sup.2.
[0171] 2. Soft callus from the culture was chopped to very small
pieces.
[0172] 3. The pieces were washed with regular (basic) liquid media
(G medium as described above) and transferred to agar plates
containing basic media plus 1% polyvinylpyrrolidone (PVP) and to
plates containing basic media plus 1% charcoal.
[0173] 4. The green pieces from each plate were washed every day
for 5 days and transferred to new PVP/charcoal plates.
[0174] 5. Only the green pieces were collected and placed on a disk
in a basic medium plus 0.6 M mannitol agar plates for 4 hours.
[0175] 6. Particle bombardment was done according to standard
methods, using ACT 1 D plasmid (McElroy et al., Plant Cell 2:
163-171, 1990) having the rice actin promoter fused to a
beta-glucoronidase "GUS" coding sequence. The gun used was a Biored
PDS1000/He Biolistic Delivery System, used at 11,000 psi at a
distance of about 10 cm.
The samples were kept over night after bombardment.
[0176] 7. The samples were transferred to a basic media agar plates
for 24 hours.
[0177] 8. The tissue samples were stained for "GUS" activity using
X/Gluc, and incubated at 37.degree. C. over night.
[0178] Results of GUS staining demonstrated that the vanilla tissue
had taken up the plasmid and were able to express GUS. This
indicates a successful transformation of vanilla callus tissue
using the above-described procedure.
[0179] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
* * * * *