Note: Descriptions are shown in the official language in which they were submitted.
CA 02455163 2004-O1-26
Method for producing arachidonic acid in transgenic organisms
The invention relates to a method for the production of arachidonic acid in
transgenic
organisms, especially in transgenic plants and yeasts. The invention also
relates to DNA
sequences, which code for a protein having the enzymatic activity of a d5-
desaturase from
Phytophthora megasperma. The invention further relates to transgenic plants
and plant cells
and transgenic yeasts, containing a nucleic acid molecule comprising a DNA
sequence
according to the present invention and having, on the basis thereof, an
increased arachidonic
acid synthesis in comparison with wild-type cells. The invention also relates
to harvest
products and propagating material of transgenic plants.
Unsaturated fatty acids are essential components, which are required for a
normal cellular
function. Unsaturated fatty acids fulfill many different functions; they, for
example contribute
to membrane fluidity, or serve as signal molecules. A particular class of
fatty acids, the
polyunsaturated fatty acids (PUFA), have attracted great interest as
pharmaceutical and
nutritechnical compounds. PUFA can be defined as fatty acids with a length of
I8 or more
carbon atoms containing two or more double bonds. These double bonds are
introduced by
fatty acid-specific desaturase enzymes.
PUFA can be classified into two groups, n-6 or n-3, depending on the position
(n) of the
double bond that is closest to the methyl terminus of the fatty acid. Thus, y-
linolenic acid
(18:305° 9r2) is classified as 18:3, n-6 PUFA, whereas a-linolenic acid
(18:309° ~2> is) is a 18:3, ,P
n-3 PUFA. Many PUFAs are also essential fatty acids that have to be present in
the food in
order to enable normal development in mammals that are unable to synthesize
the primary
essential PUFA fatty acid linoleic acid (18:2, n-6). The C20-fatty acid
arachidonic acid (20:4,
n-6) has been shown to be important for the health of neonates, including
brain and eye
development, and has recently attracted much attention as well.
Arachidonic acid (5,8,11,14-eicosatetraenic acid), as well as linoleic acid
and linolenic acid,
are counted among the essential fatty acids. The enzymatic oxidation of
arachidonic acid
leads to a number of biochemically important compounds, such as the
prostaglandins,
thromboxanes, prostacyclins, Ieukotrienes and lipoxins, all of which are
called eicosanoids.
CA 02455163 2004-O1-26
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C20-fatty acids such as 20:4, n-6, are synthesized in the food from 18:2, n-6
by successive
desaturation at d6, subsequent elongation to C-20 and further desaturation at
~5.
Figure 1 shows an outline of the biosynthesis of PUFAs in animals. Primary n-6
and n-3 fatty
acids (such as linoleic acid and a-linolenic acid) have to be provided in the
food, since
animals are unable to further desaturate oleic acid. The enzyme activities
that are required in
this synthesis pathway are shown here.
Whereas arachidonic acid is the main metabolite of the n-6 biosynthesis
pathway in
mammals, the main end products of the n-3 pathway are eicosapentaenic acid
(20:5, n-3) and
docosahexaenic acid (22:6, n-3).
Arachidonic acid is present in fairly large amounts in the liver and in the
adrenal glands,
among others. It is also synthesized in the filamentous fungus Mortierella
alpina and in the
red alga Porphyridium cruentrum. Eicosapentaenic acid is found in fish oil,
and in other
marine organisms. However, many of these sources are not easily available for
human use.
Since plant oils are currently the greatest source of PUFAs in human
nutrition, the modi-
fication of fatty acid biosynthesis pathways by genetic manipulation for
production of the
desired PUFA in an oil seed plant may provide an economic source of these
important fatty
acids. In recent years, fatty acid desaturase genes from various organisms
have been cloned
(Napier et al., Curr. Opin. Plant Biol. (1999) 2:123-127).
However, the DS-desaturase enzymes described in the art are not specific for
dihomo-Y-
linolenic acid (DGLA; 20:3, n-6). The work that has been conducted so far
using relevant
genes or gene products shows that the 05-desaturase enzymes known in the art
not only
convert 20:3- but also 20:2-, 20:1-, and in some cases also C 18-fatty acids
(see for example
Beaudoin et al., Proc. Natl. Acad. Sci. USA (2000) 97: 6421-6426; Cho et al.,
J. Biol. Chem.
(1999) 274: 37335-37339; Knutzon et al., J. Biol. Chem. (1998) 273: 29360-
29366; Saito et
al., Eur. J. Biochem. (2000) 267: 1813-1818). From this, important
disadvantages arise which
should be avoided, if possible. Due to the lack of specificity of the enzymes
known in the art,
only fatty acid mixtures are always generated, but it is desired to avoid
this. As a result, not
only defined long-chain PUFAs are generated, but also by-products that are not
known in the
CA 02455163 2004-O1-26
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literature, or that are without value, such as picolinic acid (see Knutzon et
al., Saito et al., vide
supra). Such enzymes are thus not suited, for example, for application in the
food industry.
An enzyme is desired which enables the specific production and accumulation of
one or a few
fatty acids in a transgenic organism. By providing an enzyme having substrate
specificity for
20:3 fatty acids, particularly dihomo-~y-linolenic acid, the disadvantages in
the art could be
overcome, and 20:4 fatty acids, particularly arachidonic acid, could be
specifically produced
in transgenic organisms.
Now, a gene for an as yet unknown protein having the enzymatic activity of a
QS-desaturase
has been successfully isolated from the fungus Phytophthora megasperma. In
contrast to the
enzymes with DS-desaturase activity described in the art, this enzyme is
specific for dihomo-
~y-linolenic acid. For this reason, the gene described herein may be used
especially advan-
tageously for the expression of the OS-desaturase and thus for the enzyme-
catalyzed pro-
duction of arachidonic acid in transgenic plants and yeast cells.
Regarding the substrate specificity, the enzymes according to the invention
are clearly
different from the OS-desaturases described in the art. Whereas the known
enzymes also
convert other fatty acids besides 20:3 fatty acids, the enzymes of the
invention solely,convert
20:3. This high substrate specificity was completely unexpected in view of the
specificities of
other enzymes known in the art. Using simple substrate specificity analyses,
e.g. expression in
yeast, the enzymes of the invention can be distinguished from the enzymes
known in the art.
The present invention thus relates to DNA sequences that code for a protein
having the
enzymatic activity of a OS-desaturase, with the activity being specific for
dihomo-y-linolenic
acid.
Within the scope of the invention, a dihomo-y-linolenic acid-specific activity
means that the
proteins having the enzymatic activity of a 05-desaturase and encoded by the
DNA sequences
according to the invention are essentially exclusively specific for dihomo-Y-
linolenic acid
(20:3, DHLA) and essentially do not convert other fatty acids. Particularly
preferably,
specificity for DHLA means that the 05-desaturase exclusively converts DHLA as
a
substrate. In other words, DHLA-specificity in the sense of the invention also
means that the
CA 02455163 2004-O1-26
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enzyme does not convert 20:2-, 20:1-, or C18-fatty acids in appreciable
amounts, and
preferably not at all.
In contrast to this, the OS-desaturases from Mortierella alpina and
Dictyostelium discoideum
described in the art also convert other fatty acids, which is associated with
the disadvantages
described above.
An expert can easily determine if a QS-desaturase has the DHLA-specificity
according to the
invention, and if the enzyme is thus a DS-desaturase in the sense of the
present invention. For
example, an expert can recognize from simple standard feeding experiments if
an enzyme
possesses the high substrate specificity for DHLA (see, for example, Figures 3
and 4). If the
DHLA-specificity according to the invention is present, only DHLA is
significantly converted
to arachidonic acid, when, for example, various C20-fatty acids such as
20:201'' ~'~,
20:308° ~'° 14 (DHLA) and 20:3d~ ~~ ~4° ~? are offered as
substrates in a feeding experiment.
Furthermore, the invention relates to recombinant nucleic acid molecules,
comprising the
following elements:
a) regulatory sequences of a promoter that is active in the target organism;
b) operatively linked thereto a DNA sequence that codes for a protein having
the
enzymatic activity of a ~S-desaturase that is specific for dihomo-'y-linolenic
acid;
c) optionally, operatively linked thereto regulatory sequences, which can
serve as
transcription, termination and/or polyadenylation signals in the target
organism.
Preferably, the coding DNA sequence is the sequence from Phytophthora
megasperma given
in SEQ ID NO. 1. The target organism is preferably a plant or a plant cell, or
a yeast cell, but
other organisms such as fungi, algae may be considered as well.
The nucleic acid sequence given in SEQ ID NO. 1 shows sequence homologies to
OS-desaturases from Dictyostelium discoideum (58%) and from Mortierella alpina
(54%) as
well as to D6-desaturases from Synchocystis sp. (46%) and Homo sapiens (43%),
which are
known in the art; and from which sequence homologies an expert would have been
unable to
distinguish between DS- or D6-acyl-lipid- or even 08-sphingolipid-desaturases
(the deter-
CA 02455163 2004-O1-26
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urination of the sequence similarity was performed using the alignment program
HUSAR
BLAST X2).
The DNA sequence that codes for a protein having the enzymatic activity of a
dihomo-Y-
linolenic acid-specific DS-desaturase may be isolated from natural sources or
may be
synthesized according to conventional procedures. Using common molecular
biological
techniques (see for example Sambrook et al. (1989) Molecular Cloning: A
Laboratory
Manual, 2"d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York) it is
possible to prepare or generate desired constructs for the transformation of
plant cells or yeast
cells. The methods of cloning, mutagenesis, sequence analysis, restriction
analysis, and
further biochemical or molecular biological methods commonly used for gene
technological
manipulation of prokaryotic cells are well known to an average person skilled
in the art. Thus,
not only suitable chimeric gene constructs containing the desired fusion of
promoter and ~5-
desaturase DNA sequence according to the invention, and, optionally, further
regulatory
and/or signal sequences, can be generated; rather, the person skilled in the
art can, if desired,
additionally introduce various mutations into the DNA sequence which codes for
the OS-
desaturase according to the invention using routine techniques, resulting in
the synthesis of
proteins with possibly altered biological properties. For example, here it is
possible to
generate deletion mutants which permit the synthesis of correspondingly
shortened proteins
by progressive deletion from the 5'- or the 3'-end of the coding DNA sequence.
Furthermore,
it is possible to specifically produce enzymes that, by addition of suitable
signal sequences,
are localized in certain compartments of the plant cell or yeast cell. Such
sequences are ,,.
described in the literature and are well known to the person skilled in the
art. Furthermore, the
introduction of point mutations at positions in which a modification of the
amino acid
sequence affects for example the enzymatic activity or the regulation of the
enzyme may be
considered. In this way, mutants may be generated, for example, which are not
subject
anymore to the regulatory mechanisms that are usually present in the cell,
such as allosteric
regulation or covalent modification. Furthermore, mutants may be generated
which show an
altered substrate or product specificity. In addition, mutants may be
generated which have an
altered profile of activity, temperature, and/or pH.
For the gene technological manipulation in prokaryotic cells, the recombinant
nucleic acid
molecules according to the invention or parts thereof, may be introduced into
plasmids that
CA 02455163 2004-O1-26
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allow for mutagenesis or a sequence alteration by recombination of DNA
sequences. Using
standard methods (see e.g. Sambrook et al. (1989), supra), bases may be
exchanged or natural
or synthetic sequences may be added. In order to link the DNA fragments with
each other,
adapters or linkers may be attached to the fragments where necessary. In
addition, suitable
restriction sites maybe provided, or unnecessary DNA or restriction sites may
be removed
using enzymatic or other manipulations. Where insertions, deletions or
substitutions may be
possible, in vitro mutagenesis, "primer repair", restriction or ligation may
be used. Sequence
analysis, restriction analysis, and other biochemical-molecular biological
methods are
generally carried out as analytical methods.
In a preferred embodiment, the DNA sequence, which codes for a protein having
the enzy-
matic activity of a ~5-desaturase that is specific for dihomo-y-linolenic acid
is selected from
the group consisting of
a) - DNA sequences, comprising a nucleotide sequence that codes for the amino
acid
sequence identified in SEQ ID No. 2 or fragments thereof, the length of the
fragments
being sufficient to be enzymatically active;
b) DNA sequences comprising the coding nucleotide sequence given in SEQ ID No.
1 or
fragments thereof, the length of the fragments being sufficient to code for an
enzy-
matically active protein;
c) DNA sequences comprising a nucleotide sequence or fragments of said
nucleotide
sequence, that hybridize to a complementary strand of the nucleotide sequence
from a)
or b), the length of the fragments being sufficient to code for an
enzymatically active ,. ~.
protein;
d) DNA sequences comprising a nucleotide sequence or fragments of said
nucleotide
sequence, which is degenerate to a nucleotide sequence from c), the length of
the
fragments being sufficient to code for an enzymatically active protein;
e) DNA sequences representing a derivative, analogue, or fragment of a
nucleotide
sequence from a), b), c) or d), the length of the fragment being sufficient to
code for an
enzymatically active protein.
Within the context of this invention, the term "hybridization" means
hybridization under
conventional hybridization conditions, preferably under stringent conditions,
such as the ones
described for example in Sambrook et al. (1989, vide supra).
CA 02455163 2004-O1-26
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According to the invention, hybridization is always performed in vitro under
conditions that
are stringent enough to ensure specific hybridization. Such stringent
hybridization conditions
are known to the expert, and may be derived from the literature (Sambrook et
al. (2001 ),
Molecular cloning: A laboratory manual, 3rd edition, Cold Spring Harbor
Laboratory Press).
Generally, "to hybridize~specifically" means that a molecule preferentially
binds to a specific
nucleotide sequence under stringent conditions, when this sequence is present
in a complex
mixture of (e.g. total) DNA or RNA. The term "stringent conditions" generally
stands for
conditions, under which a nucleic acid sequence preferentially hybridizes to
its target
sequence, and to a noticeably lesser extent, or not at all, to other
sequences. Stringent
conditions are partly sequence-dependent, and will vary depending on the
different
circumstances. Longer sequences hybridize specifically at higher temperatures.
Generally,
stringent conditions are selected in such a way that the temperature is
approx. 5°C below the
thermal melting point (Tm) for the specific sequence at a defined ionic
strength and a defined
pH. The Tm represents the temperature (at a defined ionic strength, pH and
nucleic acid
concentration) at which 50% of the molecules complementary to the target
sequence hybri-
dize to the target sequence in the state of equilibrium. Typically, stringent
conditions are
conditions at which the salt concentration is at least about 0.01 to 1.0 M
sodium ion concen-
tration (or another salt) at a pH between 7.0 and 8.3, and the temperature is
at least about
30°C for short molecules (i.e. for example 10-SO nucleotides).
Additionally, stringent
conditions, as already described above, may be obtained by addition of
destabilizing agents
such as for example formamide.
DNA sequences that hybridize to DNA sequences coding for a protein having the
enzymatic
activity of a OS-desaturase with the specificity for dihomo-y-linolenic acid,
may be isolated
e.g. from genomic or cDNA libraries of any organism that naturally contains
the
DS-desaturase coding DNA sequences of the invention. The identification and
isolation of
such DNA sequences may be conducted e.g. using DNA sequences having exactly or
essentially the nucleotide sequence given in SEQ ID No. 1 or fragments
thereof, or the
reversely complementary sequences of these DNA sequences, e.g. by
hybridization according
to standard procedures (see, e.g. Sambrook et al. (1989), vide supra). The
fragments used as
hybridization probe may also be synthetic fragments that have been generated
using common
CA 02455163 2004-O1-26
-8
synthesis techniques, the sequence of which essentially corresponds to one of
the above
mentioned OS-desaturase DNA sequences, or to a fragment thereof. DNA sequences
according to the invention may of course be isolated using other procedures
such as e.g. PCR
as well.
The DNA sequences which code for a protein having the biological activity of a
DS-desaturase with specificity for dihomo-y-linolenic acid also comprise DNA
sequences,
the nucleotide sequences of which are degenerate to any of the previously
described DNA
sequences. Degeneracy of the genetic code offers the person skilled in the
art, among other
things, the possibility of adapting the nucleotide sequence of the DNA
sequence to the colon
preference (colon usage) of the target organism, i.e. the plant or plant cell
or yeast cell having
an altered content of arachidonic acid as a result of the expression of the
enzymatic activity
according to the invention, and thereby optimizing the expression.
The above described DNA sequences also comprise fragments, derivatives, and
allelic
variants of the above described DNA sequences which code for a protein having
the biologi-
cal activity of a DS-desaturase specific for dihomo-'y-linolenic acid. The
term "fragments" is to
be understood as parts of the DNA sequence that are long enough to code for
one of the
described proteins. The term "derivative" in this context means that the
sequences differ from
the above described DNA sequences in one or more positions, but still possess
a high degree
of homology to these sequences.
r ~.
In this respect, homology means a sequence identity of at least 60 percent, 64
percent,
68 percent, especially an identity of at least 70 percent, 72 percent, 74
percent, 76 percent,
78 percent, preferably of at least 80 percent, 82 percent, 84 percent, 86
percent, 88 percent,
particularly preferred of at least 90 percent, 92 percent, 94 percent, and
most preferably of
at least 95 percent, 97 percent, 99 percent.
The enzymes that are coded by these DNA sequences show a sequence identity to
the amino
acid sequence given in SEQ ID No. 2 of at least 60, 64, 68, 70, 74, 78
percent, especially of at
least 80, 82 percent, preferably of at least 84, 86, 88, 90 percent, and
particularly preferred of
at least 92, 94, 96, 98 percent. The deviations from the above described DNA
sequences may
have occurred for example due to deletion, substitution, insertion or
recombination.
CA 02455163 2004-O1-26
_g_
Degrees of homology or sequence identities are generally determined using
various alignment
programs such as e.g. CLUSTAL. Generally, algorithms that are suitable for the
determi-
nation of sequence identity/similarity are available to the expert, such as
the program, which
is accessible under http://www.ncbi.nlm.nih.govBLAST (e.g. the link "Standard
nucleotide-
nucleotide BLAST [blastn]").
The DNA sequences that are homologous to the above described sequences and are
derivatives of these sequences are normally variations of these sequences
which represent
modifications fulfilling the same biological function. These variations can be
both naturally
occurring variations, for example sequences from other organisms, or
mutations, whereby
these mutations can have been generated naturally, or may have been introduced
by targeted
mutagenesis. Furthermore, the variations can be synthetic sequences. The
allelic variants may
be both naturally occurring variants and synthetic variants or variants
generated by
recombinant DNA techniques.
In an especially preferred embodiment, the described DNA sequence which codes
for a
DS-desaturase that is specific for dihomo-y-linolenic acid is obtained from
Phytophthora
megasperma.
Furthermore, the invention relates to nucleic acid molecules that contain the
nucleic acid
sequences according to the invention, or that have been generated or derived
from these by . '.
naturally occurring processes or by gene technological or chemical processes
and synthesis
methods. These may be, for example, DNA or RNA molecules, cDNA, genomic DNA,
mRNA, etc.
The invention also relates to those nucleic acid molecules in which the
nucleic acid sequences
are linked to regulatory elements, which ensure the transcription and, if
desired, the trans-
lation in the transgenic cell.
For the expression of the DNA sequences contained in the recombinant nucleic
acid mole-
cules according to the invention in plant cells, basically any promoter can be
used that is
active in plant cells. Thus, the DNA sequences according to the invention may
be expressed
CA 02455163 2004-O1-26
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in plant cells for example under the control of constitutive, but also
inducible or tissue- or
development-specific regulatory elements, especially promoters. Whereas for
example the use
of an inducible promoter allows for the targeted induced expression of the DNA
sequences
according to the invention in plant cells, the use of tissue-specific, e.g.
leaf or seed-specific,
promoters for example offers the possibility of altering the content of
arachidonic acid in
specific tissues, such as leaf or seed tissues. Other suitable promoters
mediate for example
light-induced gene expression in transgenic plants. With respect to the plant
to be trans-
formed, the promoter may be homologous or heterologous.
Suitable promoters are for example the 35S RNA promoter of the cauliflower
mosaic virus
and the ubiquitin promoter from maize for constitutive expression. Suitable
seed specific
promoters are, for instance, the USP promoter (Baumlein et al., Mol. Gen.
Genet. ( 1991 ) 225:
459-467) or the hordein promoter (Brandt et al., Carlsberg Res. Commun. (1985)
S0:
333-345).
Within the scope of this invention, constitutive, germination-specific and
seed-specific
promoters are preferred, since they are particularly suitable for the targeted
increase of the
arachidonic acid content in transgenic seeds.
Anyway, the expert can gather suitable promoters from the literature, or can
isolate them
himself from any plant using routine methods.
This also applies to the expression of the DNA sequences according to the
invention in yeast
cells. Inducible promoters, or for example the OLE 1 promoter may be
preferably used in this
case.
Furthermore, transcription or termination sequences are present which allow
for correct
transcription termination, as well as for the addition of a poly-A tail to the
transcript to which
a function in the stabilization of transcripts is assigned. Such elements are
described in the
literature (e.g. Gielen, EMBOJ., (1989) 8:23-29), and are interchangeable in
any order, for
example the terminator of the octopin synthase gene from Agrobacterium
tumefaciens.
The invention further relates to proteins having the biological activity of a
OS-desaturase that
r ~.
is specific for dihomo-'y-linolenic acid, or biologically active fragments
thereof, which are
CA 02455163 2004-O1-26
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coded by a nucleic acid sequence according to the invention or by a nucleic
acid molecule
according to the invention. Preferably, it is a fungal OS-desaturase,
particularly preferably it is
a protein having the amino acid sequence shown in SEQ ID NO. 2, or an active
fragment
thereof.
A further object of the invention is to provide vectors, the use of which
enables the production
of new plants, or plant cell or tissue cultures, or yeasts in which an altered
content of arachi-
donic acid can be achieved. This object is solved by providing the vectors
according to the
invention containing nucleic acid sequences, which code for enzymes having the
biological
activity of a d5-desaturase according to the invention.
Thus, the present invention also relates to vectors, especially plasmids,
cosmids, viruses,
bacteriophages, and other vectors that are common in gene technology
containing the
previously described nucleic acid molecules according to the invention, and
which may be
optionally used for the transfer of the nucleic acid molecules according to
the invention to
plants or to plant cells or yeast cells.
Optionally, the nucleic acid sequences of the invention can further contain
enhancer
sequences or other regulatory sequences. These regulatory sequences also
include, for
example, signal sequences, which enable the transport of the gene product to a
specific
compartment.
It is also an object of the invention to provide new transgenic plants, plant
cells, plant parts,
transgenic propagation material and transgenic harvest products having an
altered content of
arachidonic acid compared to wild-type plants or plant cells.
This object is solved by transfer of the nucleic acid molecules according to
the invention to
plants and their expression in plants. By providing the nucleic acid molecules
according to the
invention it is now possible to alter plant cells by gene technological
procedures, so that they
have a novel or altered DS-desaturase activity with the specificity for dihomo-
~y-linolenic acid
compared to wild-type cells, thus resulting in an alteration of the content of
arachidonic acid.
In one embodiment the invention thus relates to plants or their cells and
parts, having an
increased content of arachidonic acid compared to wild-type plants due to the
presence and
expression of the nucleic acid molecules according to the invention.
CA 02455163 2004-O1-26
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Further subjects of the invention are transgenic plant cells, or plants
comprising such plant
cells and their parts and products, in which the nucleic acid molecules
according to the
invention are integrated into the plant genome. Another subject of the
invention are plants, in
whose cells the nucleic acid sequence according to the invention is present in
replicated form,
i.e. the plant cell contains the foreign DNA on a separate nucleic acid
molecule (transient
expression).
Plants which are transformed with the nucleic acid molecules according to the
invention and
in which an altered amount of arachidonic acid is synthesized due to the
introduction of such
a molecule, can in principle be any plant. Preferably the plant is a
monocotyledonous or a
dicotyledonous useful plant.
Examples for monocotyledonous plants are plants belonging to the genera Avena
(oats),
Triticum (wheat), Secale (rye), Hordeum (barley), Oryza (rice), Panicum,
Pennisetum,
Setaria, Sorghum (millet), Zea (maize). DicotyIedonous useful plants are,
among others,
leguminous plants, and especially alfalfa, soy bean, rape, tomato, sugar beet,
potato,
ornamental plants or trees. Other useful plants may be e.g. fruits
(particularly apples, pears,
cherries, grapes, citrus fruits, pineapple and banana), oil palms, tea shrubs,
cacao and coffee
trees, tobacco, sisal, cotton, flax, sunflower as well as medicinal plants and
pastures as well as
fodder plants. Particularly preferred are wheat, rye, oat, barley, rice, maize
and millet, fodder
corns, sugar beet, rape, soy, tomato, potato, sweet grass, fodder grass, and
clover.
It goes without saying that the invention especially relates to common food or
fodder plants,
such as, in addition to the already mentioned plants, peanut, lentil, field
bean, mangel-wurzel,
buckwheat, carrot, sunflower, Jerusalem artichoke, turnip rape, white mustard,
rutabaga and
wild turnip.
Especially preferred are oil seeds, and here especially the so-called 18:2-
plants, i.e. soy bean,
flax and sunflower.
Subject of the invention are further propagation material and harvest products
of plants
according to the invention, such as seeds, fruits, cuttings, bulbs, rhizomes,
etc., as well as
parts of these plants, such as protoplasts, plant cells and calli.
CA 02455163 2004-O1-26
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However, the invention not only relates to the increase of the arachidonic
acid content in
plants with the purpose of increasing the nutritional value for the nutrition
of humans and
animals. Rather, the invention also relates to the production of arachidonic
acid in cell
cultures with the purpose of subsequently obtaining arachidonic acid from the
transgenic
cells. This may be plant cell cultures such as suspension cultures or callus
cultures, but also
yeast cultures that are suitable for the production of arachidonic acid and
subsequent isolation
of arachidonic acid. Of course, transgenic plants or parts from transgenic
plants may also be
used for the production of arachidonic acid in order to obtain the arachidonic
acid from these
plants or plant parts. As with plants or plant cultures, yeasts or yeast
cultures may not only be
used for isolation of the arachidonic acid from the transgenic, i.e.
arachidonic acid-producing
cells, but also they may be directly suitable as foods with increased
nutritional value.
If required, the transgenic, arachidonic acid-producing cells may be
supplemented with
dihomo-y-linolenic acid to provide sufficient starting material for the
synthesis of arachidonic
acid.
For obtaining the arachidonic acid from the transgenic cells, plants or cell
cultures, con-
ventional procedures are suitable, such as e.g. cold pressing, which is also
used for olive oil.
In order to obtain arachidonic acid from yeast cells or yeast cultures, steam
distillation may be
suitable as well. The expert can gather suitable methods from the art.
tr
For the cultivation of transgenic plant cells, common cell culture methods may
be used which
are known to the expert. The same applies to the cultivation of yeast cells.
In a further embodiment, the invention relates to host cells, especially
prokaryotic and
eukaryotic cells, that have been transformed or infected with an above
described nucleic acid
molecule or a vector, respectively, as well as cells, that are derived from
such host cells, and
that contain the described nucleic acid molecules or vectors. The host cells
may be bacteria,
viruses, algae, yeast cells and fungal cells, as well as plant or animal
cells.
It is also an object of the present invention to provide methods for the
production of plant
cells and plants characterized by an increased content of arachidonic acid.
CA 02455163 2004-O1-26
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This object is solved by methods rendering possible the production of new
plant cells and
plants having an increased content of arachidonic acid due to the transfer of
nucleic acid
molecules according to the invention that code for t15-desaturase that is
specific for dihomo-
y-linolenic acid. For the production of such new plant cells and plants,
various methods may
be used. Firstly, plants and plant cells may be modified by common gene
technological
transformation methods in such a way that the new nucleic acid molecules are
integrated into
the plant genome, i.e. stable transformants are generated. Secondly, a nucleic
acid molecule
according to the invention, the presence and the expression of which results
in an altered
biosynthesis performance in the plant cell, may be present in the plant cell
or in the plant as a
self replicating system. Thus, the nucleic acid molecules of the invention may
be present e.g.
in a virus, with which the plant or plant cells comes in contact.
According to the invention, plant cells and plants having an increased content
of arachidonic
acid due to the expression of a nucleic acid sequence according to the
invention, are produced
by a method comprising the following steps:
a) Producing a recombinant nucleic acid molecule, comprising the following
elements in
5'->3' orientation:
regulatory sequences of a promoter which is active in plant cells,
operatively linked thereto a nucleic acid sequence which codes for a protein
having the enzymatic activity of a OS-desaturase that is specific for dihomo-
~y-
Iinolenic acid, and
optionally, operatively linked thereto regulatory sequences, which can serve
as
transcription, termination and/or polyadenylation signals in plant cells.
b) transferring the nucleic acid molecule from a) to plant cells.
In the case of yeast cells, the above-described method is modified in such way
that regulatory
sequences of a promoter active in yeast cells and the corresponding
transcription, termination
and/or polyadenylation signals are used.
In order to prepare the introduction of foreign genes into higher plants or
their cells, a large
number of cloning vectors are available which contain a replication signal for
E. coli and a
CA 02455163 2004-O1-26
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marker gene for the selection of transformed bacterial cells. Examples of such
vectors are
pBR322, the pUC series, the Ml3mp series, pACYC184, etc.. The desired sequence
may be
introduced into the vector at a suitable restriction site. The obtained
plasmid is then used for
the transformation of E. coli cells. Transformed E. coli cells are grown in a
suitable medium,
and then harvested and lysed in order to recover the plasmid. Restriction
analyses, gel
electrophoresis, and other biochemical-molecular biological methods are
generally used as
analytic methods to characterize the plasmid DNA so obtained. After each
manipulation, the
plasmid DNA may be cleaved and the DNA fragments thus obtained can be linked
to other
DNA sequences.
A plurality of techniques is available for introducing DNA into a plant host
cell, and the
person skilled in the art will not have any difficulties in selecting a
suitable method in each
case. These techniques comprise the transformation of plant cells with T-DNA
using
Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transforming
agent, the
fusion of protoplasts, injection, electroporation or direct gene transfer of
isolated DNA into
protoplasts, introduction of DNA using biolistic methods, as well as other
possibilities which
have been well established for several years and which belong to the standard
repertoire of the
person skilled in the art in plant molecular biology or plant biotechnology
and in cell and
tissue cultures, and which are described in generally known review articles
and manuals
regarding the transformation of plants.
Once the introduced DNA has been integrated into the genome of the plant cell,
it is generally
stable there, and is maintained in the progeny of the originally transformed
cell as well. It
usually contains a selection marker which imparts the transformed plant cells
resistance to a
biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin,
methotrexate,
glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin, and
others. The
individually selected marker should therefore allow for the selection of
transformed cells from
cells lacking the introduced DNA. Alternative markers may also be suitable for
this purpose,
such as nutritive markers, screening markers (such as GFP, green fluorescent
protein). It
could also be done without any selection marker, although this would involve a
rather high
screening effort. If the selection marker used is to be removed after
transformation and
identification of successfully transformed cells and plants, the expert can
choose among
various strategies. For example, sequence-specific recombinases may be used,
for example by
CA 02455163 2004-O1-26
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performing retransformation of a recombinase-expressing parent line, followed
by outcrossing
of the recombinase after removal of the selection marker. The selection marker
may also be
removed by cotransformation followed by outcrossing.
If desired, transgenic plants are regenerated from transgenic plant cells by
conventional
regeneration methods using conventional culture media and phytohormones. If
desired, the
plants so obtained may be analyzed for the presence of the introduced DNA
which codes for a
protein having the enzymatic activity of a dihomo-'y-Iinolenic acid-specific
DS-desaturase, or
for the presence of enzyme activity attributed to the DS-desaturase using
conventional
methods including molecular biological methods, such as PCR, blot analyses, or
by bio-
chemical methods.
It is understood that plant cells containing the nucleic acid molecules
according to the
invention, may also be further cultivated as plant cells (including
protoplasts, calli, suspension
cultures and the like). The invention also relates to the production of
arachidonic acid in plant
cultures.
According to the invention, the term transgenic plant comprises the plant as a
whole, as well
as all transgenic plant parts, in which a desaturase according to the
invention is expressed.
Such plant parts may for example be plant cells, plant seeds, leaves,
blossoms, fruits, storage
organs such as bulbs, and pollen. "Transgenic plant" according to the
invention is also
intended to mean the propagation material of transgenic plants according to
the invention, r '.
such as e.g. seeds, fruits, cuttings, bulbs, rhizomes, etc., whereby this
propagation material
optionally contains the above described transgenic plant cells, as well as
transgenic parts of
these transgenic plants such as protoplasts, plant cells and calli.
The transformation, expression and cultivation of yeast cells may be
accomplished according
to conventional protocols, e.g. as described in Ausubel et al., Current
Protocols in Molecular
Biology (2000). Examples for this are also given in the embodiments below.
The invention furthermore relates to a method of obtaining arachidonic acid
from plant cells
or yeast cells. The literature provides the expert with protocols for the
purification of C20-
fatty acids and especially for the purification of arachidonic acid.
CA 02455163 2004-O1-26
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The following examples are intended to illustrate the invention without
restricting it in any
way.
Examples
Isolation of a cDNA coding for a DS-desaturase from Phytophthora megasperma
Poly(A)+-RNA was isolated from 2 days old Phytophthora megasperma mycelia that
had
been cultivated on an oleate-containing medium (Oligotex-dT mRNA Maxi Kit,
Qiagen,
Hilden, Germany). cDNA synthesis was then performed using S ~g of the poly(A)+-
RNA and
the SuperscriptTM cDNA synthesis kit from LifeTechnologies (Eggenstein,
Germany). The
obtained cDNA has asymmetrical ends. The cDNA was then Iigated into the
plasmid
pSPORT-1 (LifeTechnologies, Eggenstein, Germany) that had already been cut
with the
restriction enzymes SaII and NotI. The ligation was performed using T4 ligase
according to
the manufacturer's protocol (LifeTechnologies).
Cells of the E. coli strain XL1-Blue were then transformed with the ligation
mixture, i.e. the
cDNA ligated into pSPORT-1 by electroporation, and plated on LB plates
containing
ampicillin, so that 10,000 to 15,000 colonies grew on each agar plate having a
diameter of
1 S cm. r ,.
The colonies were transferred from the agar plates to 2 nitrocellulose filters
each, and then
hybridization was conducted according to state of the art (Sambrook et al.
1989 supra) using
a-[32P]-dCTP labeled nucleotide sequences. Colonies that showed a
hybridization signal were
cultivated and sequenced.
The cDNA library was screened using the following nucleotide sequences:
Oligo 1 5'-GTG GCC AAG CAC AAC ACG GCC AAG AGC-3'
Oligo 2 S'-ACC ATC CGC GGC GTC GTC TAC GAC GTG ACC-3'
CA 02455163 2004-O1-26
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A positive cDNA clone is given in SEQ ID No. 1. This DNA sequence from P.
megasperma
codes for an enzyme having the activity of a OS-desaturase with 20:3-substrate
specificity.
Expression of the t15-desaturase cDNA clone in yeast
First of all, the cDNA clone was cloned into the yeast expression vector pYES2
(Invitrogen,
Groningen, Netherlands). This was carried out using the following
oligonuclotide primers:
5'-GGA TCC ATG GCC CCC ATC GAG ACT GTC AAA G3' (primer a)
and
5'-GCA TGC CCC GCG TTA TCC AGC CAA AGC TTA CC3' (primer b)
Primer a contains a restriction site for BamHI (underlined), primer b contains
a restriction site
for the restriction enzyme SphIb (underlined).
The PCR was carried out according to the following PCR protocol:
Set up for PCR reaction:
dNTP mix 1 ~1
5'-primer (primer a) 4 ~1
3'-primer (primer b) 4 ~1
template 1 ~.l plasmid DNA (10 ng)
polymerase 0.5 ~1 high fidelity, Roche Diagnostics
GmbH
x buffer 5 u1
water 34.5 ~l
total volume50 ~1
The PCR was performed under the following conditions:
- 2 min 94°C
- 10 cycles, each with: 30 seconds at 94°C, 30 seconds at 55°C,
1 minute at 72°C
CA 02455163 2004-O1-26
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15 cycles with a time increment of 5 seconds: 30 seconds at 94°C, 30
seconds
at 55°C, 1 minute at 72°C
- 5 minutes at 72°C
The obtained PCR fragment was then ligated into the vector pGEM-T (Promega,
Madison,
USA) for an intermediate cloning step. The ligation reaction was transformed
into XL1-Blue
cells.
The miniprep DNA obtained from the transformed cells (from 5 ml overnight
culture, 37°C,
processed with spin-prep kit from Macherey & Nagel, Duren, Germany) was
digested with
the restriction enzymes BamHI and PaeI from MBI Fermentas (St. Leon-Rot,
Germany).
Then, using T4 ligase from MBI Fermentas, it was ligated into the yeast
expression vector
pYES2 that had been already cut also with the restriction enzymes BamHI and
PaeI. The
ligation reaction was transformed into E. coli-XL1-Blue cells.
ml of overnight cultures from various clones were analyzed for their plasmid
DNA
(miniprep DNA isolation with the spin prep kit from Macherey & Nagel). The
desired clone
(in sense-orientation behind the galactose promoter Gal l ) was transformed
into the yeast
strain lNVSc1 (Invitrogen, Groningen, Netherlands) using the lithium acetate
method
(Ausubel et al. (2000) supra).
The transformed yeast cells were grown overnight at 30°C in SD medium
(Ausubel et al.
(2000) supra), containing glucose (2%) and amino acid solution, without
uracile. After the
pre-culture had been washed twice without sugar, a main culture was
subsequently cultivated
for 72 hours at 30°C in SD medium, containing galactose and amino acid
solution, without
uracile, and containing linoleic acid (20 rng, 0.02%) or dihomo-y-linolenic
acid (15 mg,
0.015%) and Tergitol (10%).
This main culture was then harvested by centrifugation, lyophilization of the
samples and
transmethylation of the samples using sodium methylate.
CA 02455163 2004-O1-26
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Expression inyeast cells
All yeast techniques are state of the art according to Ausubel et al., Current
Protocols in
Molecular Biology, 2000.
Pre-culture
20 ml SD medium + glucose + amino acid solution without the respective amino
acid for the
selection are inoculated with a single colony, incubated overnight at
30°C and shaken at
140 rpm.
Main culture
The pre-culture is washed twice (by centrifugation and resuspension) in SD
medium without
sugar, and the main culture is then inoculated to an ODboo of 0.1 - 0.3.
Cultivation of the main culture in SD medium + galactose + amino acid solution
without the
respective amino acid + fatty acid, Tergitol NP40 (10%) at 30°C for 72
h with shaking at
140 rpm.
Harvesting of the main culture by centrifugation in 50 ml sterile centrifuge
tubes.
L oy philization of the yeast cell pellet
Frozen cell pellets are lyophilized for approx. 18 h.
Transmethylation
Dry sample + 1.35 ml of methanolaoluene (2:1) + 0.5 ml Na-methoxide solution -
have to be
ground very finely with a glass rod
Incubate for 1 h at RT shaking on a belly dancer
Add 1.8 ml of 1M NaCI solution
Add 2 full pasteur pipettes of n-heptane
Extract for 10 min shaking on a belly dancer
Centrifuge (10 min, 400 rpm, 4°C)
CA 02455163 2004-O1-26
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Transfer heptane supernatant into a test tube
Evaporate solvents under NZ
Transfer with 3 x 0.3 ml of hexane into Eppendorf tube
Evaporate solvents under N2, and resuspend residue in MeCN.
GC analysis
7 ~l sample (in MeCN) in a sample tube
Inject 1 p1 into the GC
~l sample were evaporated under a nitrogen flow. The residue was taken up in
400 p1
MeOH and 10 p1 EDAC solution (10 ml EDAC/100 p1 MeOH) were added.
Then, it is shaken for 2 hours at room temperature. After the addition of 200
~.l tris buffer
(0.1 M, pH 7.4), it was extracted twice by shaking, each with 1 ml hexane. The
hexane phases
were combined, and evaporated under nitrogen. The residue was dissolved in 20
~l MeCN.
The GC analysis was conducted under the following conditions:
Column: HP-INNOWax (cross-linked PEG), 30 m x 0.32 mm x 0.5 ~m
Flow rate: 1.5 ml/min (constant flow) helium 150°C
Infection: 220°C
Oven: 150°C (1 min), to 200°C (15 K/min), to 250°C
(2 K/min), 250°C
(5 minutes)
Detection: FID 275°C
CA 02455163 2004-O1-26
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In feeding experiments it could be clearly confirmed that the desaturases
according to the
invention are highly specific for DGLA, i.e. these enzymes convert only this
substrate
detectably to arachidonic acid.
Within the scope of the feeding experiments, the yeast strain INVScI was
usually cultivated
for 3 days at 30°C on medium containing different C20-fatty acids
depending on the expe-
riment. The yeast strain INVScl containing the control vector pYES2 without
the desaturase
sequence was used as a control.
Description of the Figures
Fig. 1 shows an outline of PUFA biosynthesis in animals.
Fig. 2 shows the result of the GC analysis:
Fig. 2 a) yeast strain INVScI with pYES2 (control) and feeding with dihomo-y-
linolenic acid
(dihomo ~y-LEA);
Fig. 2 b) yeast strain INVScI with pYES2 + ~5-desaturase and feeding with
dihomo-y-
linolenic acid;
r~
Fig. 2 c) standard.
Fig. 3 shows the result of a GC analysis:
Top: Yeast strain INVScI with pYES2 (control) and feeding with dihomo-y-
linolenic acid (dihomo y-LEA)
Middle: Yeast strain INVScl with pYES2 + DS-desaturase and feeding with dihomo-
~y-
linolenic acid;
Bottom: Standard.
CA 02455163 2004-O1-26
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Fig. 4 shows the result of a GC analysis of a co-feeding experiment with three
different C20-
fatty acids:
Top: Yeast strain INVScl with pYES2 (control) and feeding with dihomo-y-
linolenic acid (dihomo y-LEA, 20:38' "' ~'~), 20:2013, ~6 and 20:3~~ ~' ~'~'
~~;
Middle: Yeast strain INVScI with pYES2 + DS-desaturase and feeding with dihomo-
y-
linolenic acid;
Bottom: Standard.
Only arachidonic acid is detectable as a product.
