Note: Descriptions are shown in the official language in which they were submitted.
CA 02608848 2007-07-17
DEMANDES OU BREVETS VOLUMINEUX
LA PRtSENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME DE _2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
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THAN ONE VOLUME.
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NOTE: For additional volumes please contact the Canadian Patent Office.
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FAD4, FADS, FAD5-2, AND FAD6, NOVEL FAT'I'Y ACID DESATURASE
FAMMY MEMBERS AND USES THEREOF
.5 This application is a divisional application of Serial No. 2,421,267
entitled
"FAD4, FAD5, FAD5-2, AND FAD6, NOVEL FATTY ACID DESATURASE FAMILY
MEMBERS AND USES THEREOF", having an International filing date of September
28, 2001,
and a PCT Serial No. of PCT/IBO1/02346.
Backaround of the Invention
Fatty acids' are carboxylic acids with long-chain hydrocarbon side groups
and play a fundamental role in many biological processes. Fatty acids are
rarely free in
nature but, rather, occur in esterified form as the major component of lipids.
Lipids/fatty
acids are sources of energy (e.g., b-oxidation) and are an integral part of
cell membranes
which are indispensable for processing biological or biochemical information.
Fatty acids can be divided into two groups: the saturated fatty acids and
the unsaturated fatty acids which contain one or more carbon double bond in
cis-
configuration. Unsaturated fatty acids are produced by tenni.nal desaturases
that belong
to the class of nonheme-iron enzyrnes. Each of these enzymes are part of a
electron-
transport system that contains two other proteins, namely cytochrome b5 and
NADH-
cytochrome bs reductase. Specifically, such enzymes catalyze the formation of
double
bonds between the carbon atoms of a fatty acid molecule. Human and other
mammals
have a limited spectrum of these desaturases that are required for the
formation of
particular double bonds in unsaturated fatty acids. Thus, humans have to take
up some
fatty acids through their diet. Such essential fatty acids, for example, are
linoleic acid
(C 18:2); linolenic acid (C 18:3), arachidonic acid (C20:4). In contrast,
insects and plants
are able to synthesize a much larger variety of unsaturated fatty acids and
their
derivatives.
Long chain polyunsaturated fatty acids (LCPUFAs) such as
docosahexaenoic acid (DHA, 22:6(4,7,10,13,16,19)) are essential components of
cell
membranes of various tissues=and organelles in mamrnals (nerve, retina, brain
and
immune cells). For example, over 30% of fatty acids in brain phospholipid are
22:6
(n-3) and 20:4 (n-6). (Crawford, M.A., et al., (1997) Am. J. Clin. Nutr.
66:1032S-1041S). In retina, DHA accounts for more than 60% of the total fatty
acids in
the rod outer segment, the photosensitive part of the photoreceptor cell.
(Giusto, N.M.,
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}
-2-
et al. (2000) Prog. Lipid Res. 39:315-391). Clinical studies have shown that
DHA is
essential for the growth and development of the brain in infants, and for
maintenance of
normal brain function in adults (Martinetz, M. (1992) J. Pediatr. 120:S 129-S
138). DHA
also has significant effects on photoreceptor function involved in the signal
transduction
process, rhodopsin activation, and rod and cone development (Giusto, N.M., et
al.
(2000) Prog. Lipid Res. 39:315-391). In addition, some positive effects of DHA
were
also found on diseases such as hypertension, ar*itis, atherosclerosis,
depression,
thrombosis and cancers (Horrocks, L.A. and Yeo, Y.K. (1999) Pharmacol. Res.
40:211-215). Therefore, the appropriate dietary supply of the fatty acid is
important for
humans to remain healthy. It is particularly important for infant, young
children and
senior citizens to adequately intake these fatty acids from the diet since
they cannot be
efficiently synthesized in their body and must be supplemented by food
(Spector, A.A.
(1999) Lipids 34:S1-S3).
DHA. is a fatty acid of the n-3 series according to the location of the last
double bond in the methyl end. It is synthesized via alternating steps of
desaturation and
elongation. Starting with 18:3 (9,12,15), biosynthesis of DHA involves 06
desaturation
to 18:4 (6,9,12,15), followed by elongation to 20:4 (8,11,14,17). and A5
desaturation to
20:5 (5,8,11,14,17). Beyond this point, there are some controversies about the
biosynthesis. The conventional view is that 20:5 (5,8,11,14,17) is elongated
to 22:5
(7,10,13,16,19) and then converted to 22:6 (4,7,10,13,16,19) by the final A4
desaturation
(Horrobin, D.F. (1992) Prog. Lipid Res. 31:163-194). However, Sprecher et al.
recently
suggested an alternative pathway for DHA biosynthesis, which is independent of
04
desaturase, involving two consecutive elongations; a A6 desaturation and a two-
carbon
shortening via limited (3-oxidation in peroxisome (Sprecher, H., et al. (1995)
J. Lipid
Res. 36:2471-2477; Sprecher, H., et al. (1999) Lipids 34:S153-S156).
Production of DHA is important because of its beneficial effect on human
health. Currently the major sources of DHA are oils from fish and algae. Fish
oil is a
major and traditional source for this fatty acid, however, it is usually
oxidized by the
time it is sold. In addition, the supply of the oil is highly variable and its
source is in
jeopardy with the shrinking fish populations while the algal source is
expensive due to
low yield and the high costs of extraction.
EPA and AA are both A5 essential fatty acids. They form a unique class
of food and feed constituents for humans and animals. EPA belongs to the n-3
series
with five double bonds in the acyl chain, is found in marine food, and is
abundant in
3.5 oily fish from North Atlantic. AA belongs to the n-6 series with four
double bonds. The
lack of a double bond in the ca-3 position confers on AA different properties
than those
found in EPA. The eicosanoids produced from AA have strong inflammatory and
CA 02608848 2007-07-17
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platelet aggregating properties, whereas those derived from EPA have anti-
inflammatory
and anti-platelet aggregating properties. AA can be obtained from some foods
such as
meat; fish, and eggs, but the concentration i-; low.
Gamma-linolenic acid (GLA) is another essential fatty acid found in
mammals. GLA is the metabolic intermediate for very long chain n-6 fatty acids
and. for
various active molecules. In mammals, formation of long chain polyunsaturated
fatty
acids is rate-limited by 06 desaturation. Many physiological and pathological'
conditions such as aging, stress, diabetes, eczema, and some infections have
been shown
to depress the A6 desaturation step. In addition; GLA is readily catabolized
from the
oxidation and rapid cell division associated with certain disorders, e.g.,
cancer or
inflammation. Therefore, dietary supplementation with GLA can reduce the risks
of
these disorders. Clinical studies have shown that dietary supplementation with
GLA is
effective in treating some pathological conditions such as atopic eczema,
premenstrual
syndrome, diabetes, hypercholesterolemia, and inflammatory and cardiovascular
disorders.
The predominant sources of GLA are oils from plants such as evening
primrose (Oenothera biennis), borage (Borago officinalis L.), black currant
(Ribes
nigrum), and from microorganisms such as Mortierella sp., Mucor sp., and
Cyarr'abacteria. However, these GLA sources are not ideal for dietary
supplementation
due tb'large fluctuations in availability and costs associated with extraction
processes:
Summary of the Invention
The biosynthesis of fatty acids is a major activity of plants and
microorganisms. However, humans have a limited capacity for synthesizing
essential
325 fatty acids, e.g., long chain polyunsatura.ted fatty acids (LCPUFAs).
Biotechnology has
long been considered an efficient way to manipulate the process of producing
fatty acids
in plants and microorganisms. It is cost-effective and renewable with little
side effects.
Thus; tremendous industrial effort directed to the production of various
compounds
including speciality fatty acids and pharmaceutical polypeptides through the
manipulation
'30 of plant; animal, and microorganismal cells has ensued. Accordingly,
biotechnology is an
attractive route for producing unsaturated fatty acids, especially LCPUFAs, in
a safe,
cost-efficient manner so as to gamer the maximum therapeutic value from these
fatty
acids.
The present invention is based, at least in part, on the discovery of a
35 family of nucleic acid molecules encoding novel desaturases. In particular,
the present
inventors have identified the Fad 4(A4 desaturase), Fad5 and FadS-2 (d5
desaturase),
and Fad6 (A6 desaturase) which are involved in the biosynthesis of long chain
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polyunsaturated fatty acids DHA (docosahexaenoic acid, 22:6, n-3) and DPA
(docosapentaenoic acid, 22:5, n-6); more specifically, Fad4 desaturases 22:5
(n-3) and
22:4 (n-6) resulting in DHA and DPA; Fad5 and Fad5-2 desaturases 20:4 (n-3)
and
20:3(n-6) resulting in EPA and AA; and Fad6 desaturases 18:2 (n-6) and 18:3(n-
3)
resulting in GLA (gamma-linolenic acid) and, SDA (stearidonic acid).
In one embodiment, the invention features an isolated nucleic acid
molecule that includes the nucleotide sequence set forth in SEQ ID NO:I, SEQ
ID
NO:3, SEQ ID NO:5, or SEQ ID NO:7. In another embodiment, the invention
features
an isolated nucleic acid molecule that encodes a polypeptide including the
amino acid
sequence set forth in SEQ ID NO:2, 4, 6, or 8.
In still other embodiments, the invention features isolated nucleic acid
molecules including nucleotide sequences that are substantially identical
(e.g., 70%
identical) to the nucleotide sequence set forth as SEQ ID NO:1, SEQ ID NO:3,
SEQ ID
NO:5, or SEQ ID NO:7. The invention further features isolated nucleic acid
molecules
including at least 30 contiguous nucleotides of the nucleotide sequence set
forth as SEQ
ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. In another embodiment, the
invention-features isolated.nucleic acid molecules which encode a polypeptide
including
an amino acid sequence that is substantially identical (e.g., 50% identical)
to the amino
acid sequence set forth as SEQ ID NO:2, 4, 6, or 8. A1so featured are nucleic
acid
molecules which encode allelic variants of the polypeptide having the amino
acid
sequence set forth as SEQ ID NO: 2, 4, 6, or 8. In addition to isolated
nucleic acid
molecules encoding full-length polypeptides, the present invention also
features nucleic
acid molecules which encode fragments, for example, biologically active
fragments, of
the full-length polypeptides of the present invention (e.g., fragments
including at least
10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 2,
4, 6, or
8). In still other embodiments, the invention features nucleic acid molecules
that are
complementary to, or hybridize under stringent conditions to the isolated
nucleic acid
molecules described herein.
In a related aspect, the invention provides vectors including the isolated
nucleic acid molecules described herein (e.g., desaturase-encoding nucleic
acid
molecules). Also featured are host cells including such vectors (e.g., host
cells including
vectors suitable for producing desaturase.nucleic acid molecules and
polypeptides).
In another aspect, the invention features isolated desaturase polypeptides
and/or biologically active fragments thereof. Exemplary embodiments feature a
polypeptide including the amino acid sequence set forth as SEQ ID NO: 2, 4, 6,
or 8, a
polypeptide including an amino acid sequence at least 50% identical to the
amino acid
sequence set forth as SEQ ID NO: 2, 4, 6, or 8, a polypeptide encoded by a
nucleic acid
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molecule including a nucleotide sequence at least 70% identical to the
nucleotide
sequence set forth as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.
Also featured are fragments of the full-lengt'1 polypeptides described herein
(e.g.,
fragments including at least 10 contiguous amino acid residues of the sequence
set forth
as SEQ ID NO: 2, 4, 6, or 8) as well as allelic variants of the polypeptide
having the
amino acid sequence set forth as SEQ ID NO: 2, 4, 6, or 8.
In one embodiment, a desaturase polypeptide or fragment thereof has a
desaturase activity. In another embodiment, a desaturase polypeptide, or
fragment
thereof, has an N-terminal heme-binding motif, e.g., a cytochrome b5-like
domain found
-10 in front-end desaturases. In another embodiment, a desaturase polypeptide,
or fragment
thereof, has at least two, preferably about three, conservative histidine
motifs found in
all microsomal desaturases and, optionally, has.a desaturase activity. In a
preferred
embodiment, the desaturase polypeptide, or fragment thereof, has about three
histidine
motifs.
The constructs containing the desaturase genes can be used in any
expibssion system including plants, animals, and microorganisms for the
production of
cells:-capable of producing LCPUFAs such as DHA, EPA, AA, SDA, and GLA.
Examples of plants used for expressing the desaturases of the present
invention include,
among others, plants and plant seeds from oilseed crops, e.g., flax (Linum
sp.), rapeseed
(Brassica sp.), soybean (Glycine and Soja sp.); sunflower (Heliantlius sp.),
corron
(Gossypiunz sp.), corn (Zea mays), olive (Olea sp.), safflower (Cartlurmus
sp.), cocoa
(Theobronia cacoa), and peanut (Arachis sp.).
In a related aspect, the present invention provides new and improved
methods of producing unsaturated fatty acids, e.g., LCPUFAs, and other key
compounds
-25 of the unsaturated fatty acid biosynthetic pathway using cells, e.g.,
plant cells; animal
cells, and/or microbial cells in which the unsaturated fatty acid biosynthetic
pathway has
been manipulated such that LCPUFAs or other desired unsaturated fatty acid
compounds are produced.
The new and improved methodologies of the present invention include
methods of producing unsaturated fatty acids (e.g., DHA) in cells having at
least one
fatty acid desaturase of the unsaturated fattracid biosynthetic pathway
manipulated
such that unsaturated fatty acids are produced (e.g., produced at an increased
level). For
example, the invention features methods of producing an unsaturated fatty acid
(e.g.,
DHA) in cells comprising at least one isolated desaturase nucleic acid
molecule, e.g.,
Fad4, Fad5, Fad5-2, and/or Fad6, or a portion thereof, as described above,
such that an
unsaturated fatty acid, e.g., LCPUFA, e.g., DHA, is produced. Such methods can
fiarther comprise the step of recovering the LCPUFA.
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In another embodiment, the present invention provides methods of
producing unsaturated fatty acids, e.g., LCPUFAs, e.g., DHA, comprising
contacting a
composition comprising at least one desatur?se target molecule, as defined
herein, with
at least one isolated desaturase polypeptide, e.g., Fad4, Fad5, Fad5-2, and/or
Fad6, or a
portion thereof, as described above, under conditions such that an unsaturated
fatty acid,
e.g., LCPUFA, e.g., DHA, is produced. Such methods can further,comprise the
step of
recovering the LCPUFA.
The nucleic acids, proteins, and vectors described above are particularly
useful in the methodologies of the present invention. In particular, the
invention
features methods of enhancing unsaturated fatty acid production (e.g., DHA
production)
that include culturing a recombinant plant, animal, and/or microorganism
comprising a
desaturase nucleic acid, e.g., Fad4, Fad5, Fad5-2, andlor Fad6, under
conditions such
that fatty acid production is enhanced.
In another embodiment, the present invention features methods of
producing a cell capable of producing unsaturated fatty acids. Such methods
include
introducing into a cell, e.g., a plant cell, an isolated nucleic acid molecule
which encodes
a protein having an activity of catalyzing the formation of a double bond in a
fatty acid
molecule.
In another embodiment, the present invention features methods for
modulating the production of fatty -acids comprising culturing a cell
comprising an
isolated nucleic acid molecule which encodes a polypeptide having an activity
of
catalyzing the formation of a double bond, such that modulation of fatty acid
production
occurs.
In another embodiment, the present invention includes compositions
which comprise the unsaturated fatty acids nucleic acids or polypeptides
described
herein. Compositions of the present invention can also comprise the cells
capable of
producing such fatty acids, as described above, and, optionally, a
pharmaceutically
acceptable carrier.
In another embodiment, the compositions of the present invention are
used as a dietary supplement, e.g., in animal feed or as a neutraceutical. The
compositions of the present invention are also.used to treat a patient having
a disorder,
comprising administering the composition such that the patient is treated.
Disorders
encompassed by such methods include, for example, stress, diabetes, cancer,
inflammatory disorders, and cardiovascular disorders.
Other features and advantages of the invention will be apparent from the
following detailed description and claims.
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Brief Description of the Drawings
Figure 1 shows the DNA and protein sequence of Fad4 from Tliraustochytrium
sp.; (A) the cDNA sequence of the open reading frame (SEQ ID NO: 1); and (B)
the translated
protein sequence (SEQ ID NO:2).
Figure 2 shows the DNA and protein sequence of Fad5 from Thraustochytrium
sp; (A) the cDNA sequence of the open reading frame (SEQ ID NO:3); and (B) the
translated
protein sequence (SEQ ID NO:4).
Figure 3 shows a comparison of Fad4 and Fad5 protein sequences from
Thraustochytrium sp. (SEQ ID NO:2 and 4, respectively). The vertical bar
indicates amino
acid identity. The conserved motifs such as the cytochrome b5 heme-binding and
the histidine-
rich motifs are highlighted. The two arrows indicate the binding locations of
the two degenerate
primers.
Figure 4 shows the DNA and protein sequence of Fad5-2 from Pythium
irregulare; (A) the cDNA sequence of the open reading frame (SEQ ID NO:5); and
(B) the
translated protein sequence (SEQ ID NO:6).
Figure 5 shows the DNA and protein sequence of Fad6 of Pythium irregulare;
(A) the cDNA sequence of the open reading frame (SEQ ID NO:7); and (B) the
translated
protein sequence (SEQ ID NO:8).
Figure 6 shows a comparison of Fad5-2 and Fad6 protein sequences from
Pythium irregulare (SEQ ID NO:6 and 8, respectively). The vertical bar
indicates amino acid
identity. The conserved motifs such as the cytochrome b5 heme-binding and the
histidine-rich
motifs are highlighted. The two arrows indicate the binding locations of the
two degenerate
primers.
Figure 7 is a gas chromatographic (GC) analysis of fatty acid methyl esters
(FAMEs) from yeast strain Invsc2 expressing Fad4 with exogenous substrate 22:5
(n-3).
Figure 8 is a gas chromatographic/mass spectroscopy (MS) analysis of FAMEs
of the new peak in Figure 7; (A) the Fad4 product; (B) the DHA (22:6, n-3)
standard.
Figure 9 is a GC analysis of FAMEs from yeast strain Invsc2 expressing Fad4
with exogenous substrate 22:4 (n-6).
Figure 10 is a GC/MS analysis FAMEs of the new peak in Figure 9; (A) the
Fad4 product; (B) the DPA (22:5, n-6) standard.
Figure 11 is a GC analysis of FAMEs from yeast strain Invsc2 expressing Fad5
with exogenous substrate 20:3 (n-6).
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Figure 12 is a GC/MS analysis of FAMES of the new peak in Figure 11;
(A) the Fad5 product; (B) the AA (20:4-5,8,11,14) standard.
Figure 13 is a GC analysis of FAMES from yeast strain AMY2a
expressing Fad5-2 with exogenous substrate 18:1-9 (the upper panes) and 18:1-
11 (the
lov.rer panel), respectively.
Figure 14 is a GC analysis of FAMEs from yeast strain Invsc2
expressing Fad6 with exogenous substrate 18:2 (9,12).
Figure 15 is a MS analysis of the derivative of the new peak from Figure
14. The structure of the diethylamide of the new fatty acid is shown with m/z
values for
ions that include the amide moiety. The three pairs of ions at m/z, 156/168,
196/208,
and 236/248 are diagnostic for double bonds at the 06, A9, and A 12 position,
respectively.
Figure 16 is a GC analysis of FAMEs from leaves of Brassicajuncea
expressing Fad4 under the control of 35S promoter with exogenously supplied
substrate
22:5 (n-3).
Figure 17 shows the fatty acid composition of vegetative tissues (leaves,
stems, and roots) of one transgenic T1 line with Fad5-2 under the control of
the 35S
promoter. The fatty acid levels are shown as the weight percentage of total
fatty acids in
B. juficea.
Figure 18 is a GC analysis of root FAMEs of B. juncea expressing Fad5-
2 with exogenous substrate homo-y-linolenic acid (HGLA, 20:3-8,11,14).
Figure 19 is a GC analysis of FAMEs prepared from seeds of B. juncea
expressing Fad5-2 under the control of the napin promoter.
Figure 20 is a GC analysis of seed FAMEs from B. juncea expressing
Fad6. Three new peaks indicate three A6 desaturated fatty acids in transgenic
seeds.
Figiire 21 shows the weight percentage of GLA (y-linolenic acid) and
SDA (stearidonic acid) accumulating in Fad6 transgenic seeds of B. juncea.
Figure 22 shows the fattyacid compositions of the seed lipids from five
transgenic lines expressing Fad6; SA=stearic acid;.OA=oleic acid; LA=linoleic
acid;
GLA=y-linolenic acid; ALA=a-linolenic acid; SDA=stearidonic acid.
Figure 23 is a table showing the fatty acid profile of Thraustochytrium
sp.
Figure 24 is a table showing the fatty acid profile of Pythium irregulare.
Figure 25 is a table showing the conversion of exogneous fatty acids in
yeast AMY-2a/pFad5-2.
Figure 26 is a table showing the accumulation of A5-unsaturated
polymethylene-interrupted fatty acids (A5-UPIFAs) in transgenic flaxseeds
expressing
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Fad5-2 under the control of napin (Napin) and flax seed-specific (Cln)
promoters. The
fatty acid levels are shown as the weight percentage of the total fatty acids.
Figure 27 is a table showing ihe accumulation of A6 desaturated fatty
acids in transgenic flaxseeds (Solin and Normandy) expressing Fad6 under the
control
of the napin promoter. The fatty acid levels are shown as the weight
percentage of the
total fatoy acids.
Detailed Description of the Invention
The present invention is based; at least in part, on the discovery of novel
fatty acid desaturase family members, referred to interchangeably herein as
"desaturases" or "desaturase" nucleic acid and protein molecules (e.g., Fad4,
Fad5,
Fad5-2, and Fad6). These novel molecules are members of the fatty acid
desaturase
family and are expressed in LCPUFAs-producing organisms, e.g.,
Thraustochytrium,
Pythium irregulare, Schizichytrium, and Crythecodinium.
As used herein, the term "fatty acids" is art recognized and includes a
long-chain hydrocarbon based carboxylic acid. Fatty acids are components of
many
lipids including glycerides. The most common naturally occurring fatty acids
are
monocarboxylic acids which have an even number of carbon atoms (16 or 18) and
which may be saturated or unsaturated. "Unsaturated" fatty acids contain cis
double
bonds between the carbon atoms. Unsaturated fatty acids encompassed by the
present
invention include, for example, DHA, GLA, and SDA. "Polyunsaturated" fatty
acids
contain more than one double bond and the double bonds are arranged in a
methylene
interrupted system (-CH-CH-CH2-CH=CH-).
Fatty acids are described herein by a numbering system in which-the
number before the colon indicates the number of carbon atoms in the fatty
acid, whereas
the number after the colon is the number of double bonds that are present. In
the case of
unsaturated fatty acids, this is followed by a number in parentheses that
indicates the
position of the double bonds. Each number in parenthesis is the lower numbered
carbon
atom of the two connected by the double bond. For example; oleic acid can be
described as 18:4 (9) and linoleic acid can be-described as 18:2(9, 12)
indicating 18
carbons, one double bond at carbon 9, two double bonds at carbons 9 and 12,
respectively.
The controlling steps in the production of unsaturated fatty acids, i.e., the
unsaturated fatty acid biosynthetic pathway, are catalyzed by membrane-
associated fatty
acid desaturases, e.g., Fad4, Fad5, Fad5-2, and/or Fad6. Specifically, such
enzymes
catalyze the forrnation of double bonds between the carbon atoms of a fatty
acid
molecule. As used herein, the term "unsaturated fatty acid biosynthetic
pathway" refers
CA 02608848 2007-07-17
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to a series of chemical reactions leading to the synthesis of an unsaturated
fatty acid
either in vivo or in vitro. Such a pathway includes a series of desaturation
and
elongation steps which generate unsaturated fatty acids and ultimately, long
chain
polyunsaturated fatty acids. Such unsaturated fatty acids can include, GLA
18:3
(6,9,12), SDA 18:4 (6,9,12,15), AA 20:4 (5,8,11,14), EPA 20:5 (5,8,11,14,17),
and DPA
22:5 (4,7,10,13,16), and DHA 22:6 (4,7,10,13,16,19).
Desaturases can contain a heme-binding motif and/or about three
conservative histidine motifs, although additional domains may be present.
Members of
the fatty acid desaturase family convert saturated fatty acids to unsaturated
fatty acids,
e.g., long chain polyunsaturated fatty acids (LCPUFAs), which are components
of cell
membranes of various tissues and organelles in mammals (nerve, retina, brain
and
immune cells). Examples of LCPUFA include, among others, docosahexaenoic acid
(DHA, 22:6(4,7,10,13,16,19)). Clinical studies have shown that DHA is
essential for
the growth and development of the brain in infants, and for maintenance of
nonnal brain
fwiction in adults (Martinetz, M. (1992) J. Pediatr..120:SI29-S138). DHA also
has
effects on photoreceptor function involved in the signal transduction process,
rhodopsin
activation, and xod and cone development (Giusto, N.M., et al. (2000) Prog..
Lipid Res.
39:315-391). In addition, positive effects of DHA were also found in the
treatnient of
diseases such as hypertension, arthritis, atherosclerosis, depression,
thrombosis and
cancers (Horrocks, L.A. and Yeo, Y.K. (1999) Pharmacol. Res. 40:211-215).
Thus, the
desaturase molecules can be used to produce the LCPUFAs useful in treating
disorders
characterized by aberrantly regulated growth, proliferation, or
differentiation. Such
disorders include cancer, e.g., carcinoma; sarcoma, or leukemia; tumor
angiogenesis and
metastasis; skeletal dysplasia; hepatic disorders; myelodysplastic syndromes;
and
hematopoietic and/or myeloproliferative disorders. Other disorders related to
angiogenesis and which are, therefore, desaturase associated disorders include
hereditary
hemorrhagic telangiectasia type 1, fibrodysplasia ossificans progressiva,
idiopathic
pulmonary fibrosis, and Klippel-Trenaunay-Weber syndrome.
The term "family" when referring to the protein and nucleic. acid
molecules of the present invention is intended to mean two or more proteins or
nucleic
acid molecules having a common structural domain or motif and having
sufficient
amino acid or nucleotide sequence homology as defined herein. Such family
members
can be naturally or non-naturally occurring and can be from either the same or
different
species. For example, a family can contain a first protein of human origin as
well as
other distinct proteins of human origin or alternatively, can contain
homologues of non-
human origin, e.g., rat or mouse proteins. Members of a family can also have
common
functional characteristics.
CA 02608848 2007-07-17
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For example, the family of desaturase proteins of the present invention
comprises one cytochrome b5 heme-binding motif. As used herein, the term "heme-
binding
rnotif' is an N-terminal extension of the cytochrome b5-like domain found in
front-end
desaturases.
In another embodiment, members of the desaturase family of proteins include a
"histidine-motifs" in the protein, preferably, about three or four histidine
motifs. As used
herein, the term "histidene motif' includes a protein domain having at least
about two histidine
amino acid residues, preferably about three or four histidine amino acid
residues, and is
typically found in all microsomal desaturases as the third conservative
histidine motif.
Examples of cytochrome b5 heme-binding motifs and histidine motifs include
amino acid residues 41-44, 182-186, 216-233, and 453-462 of SEQ ID NO:2, amino
acid
residues 40-43,171-175, 207-213, and 375-384 of SEQ ID NO:4, amino acid
residues 40-45,
171-176, 208-213, and 395-400 of SEQ ID NO:6, and amino acid residues 42-47,
178-183, 215-
220, and 400-405 of SEQ ID NO:8, as shown in Figures 3 and 6.
Isolated desaturase proteins of the present invention have an amino acid
sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, 4,
6, or 8 or are
encoded by a nucleotide sequence sufficiently homologous to SEQ ID NO: l, 3,
5, or 7. As used
herein, the term "sufficiently homologous" refers to a first amino acid or
nucleotide sequence
which contains a sufficient or minimum number of identical or equivalent
(e.g., an amino acid
residue which has a similar side chain) amino acid residues or nucleotides to
a second amino
acid or nucleotide sequence such that the first and second amino acid or
nucleotide sequences
share common structural domains or motifs and/or a common functional activity.
For example,
amino acid or nucleotide sequences which share common structural domains
having at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or
more
homology or identity across the amino acid sequences of the domains and
contain at least one
and preferably two structural domains or motifs, are defined herein as
sufficiently homologous.
Furthermore, amino acid or nucleotide sequences which share at least 50%, 55%,
60%, 65%,
70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or
identity
and share a common functional activity are defined herein as sufficiently
holologous.
In a preferred embodiment, a desaturase protein includes at least one or more
of the
following domains or motifs: a heme-binding motif and/or a histidine motif and
has an amino
acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%,
95%,
96%, 97%, 98%, 99% or more holologous or identical to the
CA 02608848 2007-07-17
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amino acid sequence of SEQ ID NO:2, 4, 6, or 8. In yet another preferred
embodiment,
a desaturase protein includes at least one or more of the following domains: a
heme-
binding motif and/or a histidine motif, and is encoded by a nucleic acid
molecule having
a nucleotide sequence which hybridizes under stringent hybridization
conditions to a
complement of a nucleic acid molecule comprising the nucleotide sequence of
SEQ ID
NO: 1, 3, 5, or 7. In another preferred embodiment, a desaturase protein
includes at least
one heme-binding motif and/or at least about three histidine motifs, and has a
desaturase
activity.
As used interchangeably herein, a "desaturase activity," "biological
activity of a desaturase," or "functional activity of a desaturase," includes
an activity
exerted or mediated by a desaturase protein, polypeptide or nucleic acid
molecule on a
desaturase responsive cell or on a desaturase substrate, as determined in vivo
or in vitro,
according to standard techniques. In one embodiment, a.desaturase activity is
a direct
activity such as an association with a desaturase target molecule. As used
herein, a
"target molecule" or "binding partner" is a molecule e.g., a molecule involved
in the
synthesis of unsaturated fatty acids, e.g., an intermediate fatty acid, with
which a
desaturase protein binds or interacts in nature such that a desaturase-
mediated function
is achieved. A desaturase direct activity also includes the formation of a
double bond
between.the carbon atoms of a fatty acid molecule to form an unsaturated fatty
acid
molecule.
The nucleotide sequence of the isolated Thraustochytriurra sp. A4
desaturase, Fad4, cDNA and the predicted amino acid sequence encoded by the
Fad4
cDNA are shown in Figure 1 and in SEQ ID NOs:l and 2, respectively. The
Thr4ustochytriuni sp. Fad4 gene (the open reading frame), which is
approximately 1560
nucleotides in length, encodes a protein having a molecular weight of
approximately
59.1 kD and which is approximately 519 amino acid residues in length.
The nucleotide sequence of the Thraustochytrium sp. AS desaturase,
Fad5, cDNA and the predicted amino acid sequence encoded by the Fad5 cDNA are
shown in Figure 2 and in SEQ ID NOs:3 and 4, respectively. The
Thraustochytrium sp.
Fad5 gene, which is approximately 1320 nucleotides in length, encodes a
protein having
a molecular weight of approximately 49.8 kD and which is approximately 439
amino
acid residues in length.
The nucleotide sequence of the Pythium irregulare AS desaturase, Fad5-
2, cDNA and.the predicted amino acid sequence encoded by the Fad5-2 cDNA are
shown in Figure 4 and in SEQ ID NOs:5 and 6, respectively. The Pythium
irregulare
Fad5-2 gene, which is approximately 1371 nucleotides in length, encodes a
protein
having approximately 456 amino acid residues in length.
CA 02608848 2007-07-17
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The nucleotide sequence of the Pythium irregulare A6 desaturase, Fad6,
cDNA and the predicted amino acid sequence encoded by the Fad6 cDNA are shown
in
Figure 5 and in SEQ ID NOs:7 and 8, respe.;tively. The Pythiuni irregulare
Fad6 gene,
which is approximately 1383 nucleotides in length, encodes a protein having
approximately 460 amino acid residues in length.
Various aspects of the invention are described in further detail in the
following subsections:
I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules
that encode desaturase proteins or biologically active portions thereof, as
well as nucleic
acid fragments sufficient for use as hybridization probes to identify
desaturase-encoding
nucleic acid molecules (e.g., desaturase mRNA) and fragments for use as PCR
primers
for the ar!iplification or mutation of desaturase nucleic acid molecules. As
used herein,
the teriii "nucleic acid molecule" is intended to include DNA molecules (e.g.,
cDNA or
genoinic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA
generated using nucleotide analogs. The nucleic acid.molecule can be single-
stranded or
doubie-siranded, but preferably is double-stranded DNA.
The term "isolated nucleic acid molecule" includes nucleic acid
moiecules which are separated from other nucleic acid molecules which are
present in
the natural source of the nucleic acid: For example, with regards to genomic
DNA, the
term "isolated" includes nucleic. acid molecules which are separated from the
chromosome with which the genomic DNA is natura.lly associated. Preferably, an
"isolated" nucleic acid is free of sequences which naturally flank the nucleic
acid Q. e:,
sequences located at the 5' and 3' ends of the nucleic acid) in the genomic
DNA of the
organism. from which the nucleic acid is derived. For example, in various
embodiments,
the isolated desaturase nucleic acid molecule ean contain less than about 5
kb, 4kb, 3kb,
2kb, 1 kb, 0.5 kb orØ1 kb of nucleotide sequences which naturally flank the
nucleic
acid molecule in genomic DNA of the cell from which the nucleic acid is
derived.
Moreover, an "isolated" nucleic acid molecule;-such as a eDNA molecule, can be
substantially free of other cellular material, or culture medium when produced
by
recombinant techniques, or substantially free of chemical precursors or other
chemicals
when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, or a portion
thereof,
can be isolated using standard molecular biology techniques and the sequence
CA 02608848 2007-07-17
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information provided herein. Using all or a portion of the nucleic acid
sequence of SEQ
ID NO:1, 3, 5, or 7, as hybridization probes, desaturase nucleic acid
molecules can be
isolated using standard hybridization and c!oning techniques (e.g., as
described in
Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2"d ed., Cold
Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989).
Moreover, a nucleic acid molecule encompassing all or a portion of SEQ
ID NO:1, 3, 5, or 7, can be isolated by the polymerase chain reaction (PCR)
using
synthetic oligonucleotide primers designed based upon the sequence of SEQ ID
NO: 1,
3,5,or7.
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively, genomic DNA, as a template and appropriate oligonucleotide
primers .
according to standard PCR amplification techniques. The nucleic acid so
amplified can
be cloned into an appropriate vector and characterized by DNA sequence
analysis.
Furthermore, oligonucleotides corresponding to desaturase nucleotide sequences
can be
prepared by standard synthetic techniques, e.g., using an automated DNA
synthesizer.
In another embodiment, the nucleic acid molecule consists of the
nucleotide sequence set forth as SEQ ID NO: 1, 3, 5, or 7.
In still another embodiment, an isolated nucleic acid molecule of the
invention comprises a nucleic acid molecule which is a complement of the
nucleotide
sequence shown in SEQ ID NO: 1, 3, 5, or 7, or a portion of any of these
nucleotide
sequences. A nucleic acid molecule which is complementary to the nucleotide
sequence
shown in SEQ ID NO:1, 3, 5, or. 7 is one which is sufficiently complementary
to the
nucleotide seqt.ience shown in SEQ ID NO: 1, 3, 5, or 7, such that it can
hybridize to the
nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7, thereby forming a
stable
duplex.
In still another embodiment, an isolated nucleic acid molecule of the
present invention comprises a nucleotide sequence which is at least about 50%,
55%,
60%, 65%, 70%, 75%, 80%, 85%, 90 l0, 95%, 96%, 97%, 98%, 99% or more identical
to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7(e.g.; to the
entire length
of the nucleotide sequence), or a portion or complement of any of these
nucleotide
sequences. In one embodiment, a nucleic acid molecule of the present invention
comprises a nucleotide sequence which is at least (or no greater than) 50-100,
100-250,
250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000-
2250, 2250-2500, 2500-2750, 2750-3000, 3250-3500, 3500-3750 or more
nucleotides in
length and hybridizes under stringent hybridization conditions to a complement
of a
nucleic acid molecule of SEQ ID NO:1, 3, 5, or 7.
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Moreover, the nucleic acid molecule of the invention can comprise only a
portion of the nucleic acid sequence of SEQ ID NO:1, 3, 5, or 7, for example,
a
fragment which can be used as a probe or pr.:ner, or a fragment encoding a
portion of a
desaturase protein, e.g., a biologically active portion of a desaturase
protein. The
nucleotide sequence determined from the cloningõof the desaturase gene allows
for the
generation of probes and primers designed for use in identifying and/or
cloning other
desaturase family members, as well as desaturase homologues from other
species. The
probe/primer (e.g., oligonucleotide) typically comprises substantially
purified
oligonucleotide. The oligonucleotide typically comprises a region of
nucleotide sequence that
hybridizes under stringent conditions to at least about 12 or 15, preferably
about 20 or 25, more
preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides
of a sense sequence of
SEQ ID NO:1, 3, 5, or 7 of an anti-sense sequence of SEQ ID NO:1, 3, 5, or 7,
or of a naturally
occurring allelic variant or mutant of SEQ ID NO: 1, 3, 5, or 7.
Exemplary probes or primers are at least (or no greater than) 12 or 15, 20
or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides in length
and/or
comprise consecutive nucleotides of an isolated nucleic acid molecule
described herein.
Also included within the scope of the present invention are probes or primers
comprising contiguous or consecutive nucleotides of an isolated nucleic acid
molecule
described herein, but for the difference of l, 2, 3, 4, 5, 6, 7, 8, 9 or 10
bases within the
probe or primer sequence. Probes based on the desaturase nucleotide sequences
can be
used to detect (e.g., specifically detect) transcripts or genomic sequences
encoding the
same or homologous proteins. In preferred embodiments, the probe further
comprises a
label group attached thereto, e.g., the label group can be a radioisotope, a
fluorescent
compound, an enzyme, or an enzyme co-factor.,. In another embodiment a set of
primers
is provided, e.g., primers suitable for use in a PCR, which can be used to
amplify a
selected region of a desaturase sequence, e.g., a: domain, region, site or
other sequence
described herein. The primers should be at least 5, 10, or 50 base pairs in
length and
less than 100, or less than 200, base pairs in length. The primers should be
identical, or
differ by no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9pr 10 bases when compared
to a sequence
disclosed herein or to the sequenee of a naturally occurring variant. Such
probes can be
used as a part of a diagnostic test kit for identibring cells or tissue which
misexpress a
desaturase protein, such as by measuring a leveL of a desaturase-encoding
nucleic acid in
a sample of cells from a subject, e.g., detecting desaturase mRNA levels or
determining
whether a genomic desaturase gene has been mutated or deleted.
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A nucleic acid fragment encoding a "biologically active portion of a
desaturase protein" can be prepared by isolating a portion of the nucleotide
sequence of
SEQ ID NO: 1, 3, 5, or 7, which encodes a polypeptide having a desaturase
biological
activity (the biological activities of the desaturase proteins are described
herein),
expressing the encoded portion of the desaturase protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded portion of the
desaturase
protein. In an exemplary embodiment, the nucleic acid molecule is at least 50-
100, 100-
250, 250-500, 500-700, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000,
2000-2250, 2250-2500, 2500-2750, 2750-3000, 3250-3500, 3500-3750 or more
nucleotides in length and encodes a protein having a desaturase activity (as
described
herein).
The invention further encompasses nucleic acid molecules that differ.
from the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7 due to
degeneracy of
the genetic code and thus encode the same desaturase proteins as those encoded
by the
nucleotide sequence shown in SEQ ID NO: l, 3, 5, or 7. In another embodiment,
an
isolated nucleic acid molecule of the invention has a nucleotide sequence
encoding a
protein having an amino acid sequence which differs by at least 1, but no
greater than 5,
10; 20, 50 or 100 amino acid residues from the amino acid sequence shown in
SEQ ID
NO:2, 4, 6; or 8. In yet another embodiment, the nucleic acid molecule encodes
the
aniino acid'sequence of human desaturase. If an alignment is needed for this
comparison, the sequences should be aligned for maximum homology.
Nucleic acid variants can be naturally occurring, such as allelic variants
(same locus), homologues (different lociis), and orthologues (different
organism) or can be
non naturally occurring. Non-natutally occurring variants can be made by
mutagenesis
techniques, including those applied to polynucleotides, cells, or organisms.
The variants
can contain nucleotide substitutions, deletions, inversions and insertions.
Variation can
occur in either or both the coding and non-coding regions. The variations can
produce both
conservative and non-conservative amino acid substitutions (as compared in the
encoded
product).
Allelic variants result, for example, from DNA sequence polymorphisms
within a population (e.g., the human population) that lead to changes in the
amino acid
sequences of the desaturase proteins. Such genetic polymorphism in the
desaturase
genes may exist among individuals within a population due to natural allelic
variation.
As used herein, the terms "gene" and "recombinant gene" refer to nucleic
acid molecules which include an open reading frame encoding a desaturase
protein, e.g.,
oilseed desaturase protein, and can further include non-coding regulatory
sequences, and
introns.
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Accordingly, in one embodiment, the invention features isolated nucleic
acid molecules which encode a naturally occurring allelic variant of a
polypeptide
comprising the amino acid sequence of SEC ID:NO:2, 4, 6, or 8, wherein the
nucleic
acid molecule hybridizes to a complement of a nucleic acid molecule comprising
SEQ
ID NO: 1, 3, 5, or 7, for example, under stringent hybridization conditions.
Allelic variants of desaturase, e.g; Fad4, Fad5, Fad5-2, or Fad6, include
both functional and non=functional desaturase proteins. Functional allelic
variants are
naturally occurring amino acid sequence variants of the desaturase protein
that maintain
the ability to, e.g., (i) interact with a desaturase substrate or target
molecule (e.g., a fatty
acid, e.g., DHA); and/or (ii) form a double bond'between carbon atoms in a
desaturase
substrate or target molecule. The fatty acids produced by the nucleic acid and
protein
molecules of the present invention are also useful in treating disorders such
as aging,
stress, diabetes, cancer, inflammatory disorders (e.g., arthritis, eczema),
and
cardiovascular disorders. Functional allelic variants will typically contain
only a
conservative substitution of one or more amino acids of SEQ ID NO:2, 4, 6, or
8, or a
substitution, deletion or insertion of non-critical residues in non-critical
regions of the
protein.
Non-functional allelic variants are =naturally occurring amino acid
sequehce variants of the desaturase protein, e.g., Fad4, Fad5, Fad5-2, or
Fad6, that do
not ha've the ability to, e.g., (i) interact with a desaturase substrate or
target molecule
(e.g.; an intermediate fatty acid, such as 18:4 (6,9,12,15)); and/or (ii) form
a double bond
between carbon atoms in a desaturase substrate or target molecule. Non-
functional
allelic variants will typically contain a non-conservative substitution, a
deletion, or
insertion, or premature truncation of the amino acid sequence of SEQ ID NO:2,
4, 6, or
8, or a substitution, insertion, or deletion in critical residues or critical
regions of the
protein.
The present invention further provides orthologues (e.g., human
orthologues of the desaturase proteins). Orthologues of the Thraustochytrium
sp. and
Pythiuin irregulare desaturase proteins are proteins that are isolated from
other
organisms and possess the same desaturase substrate or target molecule binding
mechanisms, double bond forming mechanisms, modulating mechanisms of growth
and
development of the brain in infants, maintenance mechanisms of normal brain
function
in adults, ability to affect photoreceptor function involved in the signal
transduction
process, ability to affect rhodopsin activation, development mechanisms of
rods and/or
cones, and/or modulating mechanisms of cellular growth and/or proliferation of
the non-
human desaturase proteins. Orthologues of the Thraustochytrium sp. and Pythium
CA 02608848 2007-07-17
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irregulare desaturase proteins can readily be identified as comprising an
amino acid
sequence that is substantially homologous to SEQ ID NO:2, 4, 6, or 8.
Moreover, nucleic acid mo1 ~-.ules encoding other desaturase family
members and, thus, which have a nucleotide sequence which differs from the
desaturase
sequences of SEQ ID NO: 1, 3, 5, or 7 are intended to be within the scope of
the
invention. For example, another desaturase cDNA can be identified based on the
nucleotide sequence of Fad4, Fad5,. Fad5-2, or Fad6. Moreover, nucleic acid
molecules
encoding desaturase proteins from different species, and wh.ich, thus, have a
nucleotide
sequence which differs from the desaturase sequences of SEQ ID NO: 1, 3, 5, or
7 are
intended to be within the scope of the invention. For example, Schizochytrium
or
Crythecodinium desaturase cDNA can be identified based on the nucleotide
sequence of
a Fad4, Fad5, Fad5-2, or Fad6.
Nucleic acid molecules corresponding to natural allelic variants and
homologues of the desaturase eDNAs of the invention can be isolated based on
their
homology to the desaturase nucleic acids disclosed herein using the
cDNAs.disclosed
herein, or a portion thereof, as a hybridization probe according to standard
hybridiza.tion
techniques under stringent hybridization conditions.
Orthologues, homologues and allelic variants can be identified using
methods known in the art (e.g., by hybridization to an isolated nucleic acid
molecule of
the present invention, for example, under stringent hybridization conditions).
In one
embodiment, an isolated nucleic acid molecule of the invention is at least 15,
20, 25, 30
or, more nucleotides in length and hybridizes under stringent conditions to
the nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, or.7.
In other
embodiment, the nucleic acid is at least 50-100, 100-250, 250-500, 500-700;
750-1000,
1000-1250, 1250-1500, 1500-1750, 1750-2000, 2000-2250, 2250-2500,2500-2750,
2750-3000, 3250-3 500, 3 500-3750 or more nucleotides in length.
As used herein, the term "hybridizes under stringent conditions" is
intended to describe conditions for hybridization and washing under which
nucleotide
sequences that are significantly identical or homologous to each other- remain
hybridized to each other. Preferably, the conditions are such that sequences
at least
about 70%, more preferably at least about 80%, even more preferably at least
about 85%
or 90% identical to each other remain hybridized to each other. Such stringent
conditions are known to those skilled in the art and can be found in Current
Protocols in
Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),
sections 2, 4,
and 6. Additional stringent conditions can be found in Molecular Cloning: A
Laboratory Manual, Sambrook et a1., Cold Spring Harbor Press, Cold Spring
Harbor,
NY (1989), chapters 7, 9, and 11. A preferred, non-limiting example of
stringent
CA 02608848 2007-07-17
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hybridization conditions includes hybridization in 4X sodium chloride/sodium
citrate
(SSC), at about 65-70 C (or alternatively hybridization in 4X SSC plus 50%
formamide
at about 42-50 C) followed by one or more washes in 1X SSC, at about 65-70 C.
A
preferred, non-liiniting example of highly stringent hybridization conditions
includes
hybridization in 1X SSC, at about 65-70 C (or alternatively hybridization in
1X SSC
plus 50% formamide at about 42-50 C) followed by one or more washes in 0.3X
SSC,
at about 65-70 C. A preferred, non-limiting example of reduced stringency
hybridization conditions includes hybridization in 4X SSC,. at about 50-60 C
(or
alternatively hybridization in 6X SSC plus 50% formamide at about 40-45 CJ
followed
by one or more washes in 2X SSC, at about 50-60 C. Ranges intermediate to the
above-
recited values, e.g., at 65-70 C or at 42=50 C are also intended to be
encompassed by
the present invention. SSPE (1xSSPE is 0.15M NaCI, l0mM NaH2PO4i and 1.25mM
EDTA, pH 7.4) can be substituted for SSC (IX SSC is 0.15M NaCI and 15mM sodium
citrate) in the hybridization and wash buffers; washes are performed for 15
minutes each
after hybridization is complete. The hybridization temperature for hybrids
anticipated to
be less than 50 base pairs in length should be 5-10 C less than the melting
temperature
{Tm) of the hybrid, where T. is determined according to the following
equations. For
hybrids less than 18 base pairs in length, Tm( C) = 2(# of A + T bases) + 4(#
of G + C
btases). For hybrids between 18 and 49 base pairs in length, Tm( C) = 81.5 +
-16:60og, o(Na ]) + 0.41(%G+C) -(600/N), where N is the number of bases in the
hybrid,
and [Nal is the concentration of sodium ions in the hybridization buffer
([Na+] for 1X
SSC =-0.165 M). It will also be recognized by the sldlled practitioner that
additional
reagents may be added to hybridization and/or wash buffers to decrease non-
specific
hybridization of nucleic acid molecales to membranes, for example,
nitrocellulose or
nylon membranes,.including but not limited to blocking agents (e.g., BSA or
salmon or
herring sperm carrier DNA), detergents (e.g.; SDS), chelating agents (e.g.;
EDTA),
Ficoll, FVP and the like. When using nylon membranes, in particular, an
additional
preferred, non-limiting example of stringent hybridization conditions is
hybridization in
0.25-0.5M NaH2PO4, 7% SDS at about 65 C; followed by one or more washes at
0.02M
NaH2PO4, 1% SDS at 65 C (see e.g., Chur&and Gilbert (1984) Proc. Natl. Acad.
Sci.
USA 81:1991-1995), or alternatively 0.2X SSC, 1% SDS.
Preferably, an isolated nucleidiacid molecule of the invention that
hybridizes under stringent conditions to the sbquence o: SEQ ID NO: 1, 3, 5,
or 7
corresponds to a nati.irally-occurring nucleic acid molecule. As used herein,
a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule
having
a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
CA 02608848 2007-07-17
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In addition to naturally-occurring allelic variants of the desaturase
sequences
that may exist in the population, the skilled artisan will further appreciate
that changes can be
introduced by mutation into the nucleotide sequences of SEQ ID NO:1, 3, 5, or
7, thereby
leading to changes in the amino acid sequence of the encoded desaturase
proteins, without
altering the functional ability of the desaturase proteins. For example,
nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid residues can
be made in the
sequence of SEQ ID NO: 1, 3, 5, or 7. A "non-essential amino acid residue is a
residue that can
be altered from the wild-type sequence of Fad4, Fad5, Fad5-2, or Fad6 (e.g.,
the sequence of
SEQ ID NO:2, 4, 6, or 8) without altering the biological activity, whereas an
"essential" amino
acid residue is required for biological activity. For example, amino acid
residues that are
conserved among the desaturase proteins of the present invention, e.g., those
present in a heme-
binding motif or a histidine motif, are predicted to be particularly
unamendable to alteration.
Furthermore, additional amino acid residues that are conserved between the
desaturase proteins
of the present invention and other members of the fatty acid desaturase family
are not likely to
be amendable to alteration.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding desaturase proteins that contain changes in amino acid residues that
are not essential
for activity. Such desaturase proteins differ in amino acid sequence from SEQ
ID NO:2, 4, 6, or
8, yet retain biological activity. In one embodiment, the isolated nucleic
acid molecule
comprises a nucleotide sequence encoding a protein, wherein the protein
comprises an amino
acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or more homologous to SEQ ID NO:2, 4, 6, or 8, e.g., to the
entire length of SEQ ID NO:2, 4, 6, or 8.
An isolated nucleic acid molecule encoding a desaturase protein
homologous to the protein of SEQ ID NO:2, 4, 6, or 8 can be created by
introducing one
or more nucleotide substitutions, additions or deletions into the nucleotide
sequence of
SEQ ID NO: 1, 3, 5, or 7, such that one or more amino acid substitutions,
additions or
deletions are introduced into the encoded protein. Mutations can be introduced
into
SEQ ID NO: 1, 3, 5, or 7 by standard techniques, such as site-directed
mutagenesis and
PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions
are made
at one or more predicted non-essential amino acid residues. A "conservative
amino acid
substitution" is one in which the amino acid residue is replaced with an amino
acid
residue having a similar side chain. Families of amino acid residues having
similar side
chains have been defined in the art. These families include amino acids with
basic side
CA 02608848 2007-07-17
- 20a -
chains (e.g., lysine, arginine, histidine), acidic side chains (e.g, aspartic
acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine,
serine,
CA 02608848 2007-07-17
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threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.,
alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side
chains (e.g.,
threonine, valine, isoleucine) and aromatic ~:de. chains (e.g., tyrosine,
phenylalanine,
tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a
desaturase
protein is preferably replaced with another amino acid residue from the same
side chain
family. Alternatively, in another embodiment, mutations can be introduced
randomly
.along all or part of a desaturase coding sequence, such as by saturation
mutagenesis, and
the resultant mutants can be screened for desaturase biological activity to
identify
mutants that retain activity. Following mutagenesis of SEQ ID NO:1, 3, 5, or
7, the
encoded protein can be expressed recombinantly and the activity of the protein
can be
determined.
In a preferred embodiment, a mutant desaturase protein can be assayed
for the ability to (i) interact with a desaturase substrate or target molecule
(e.g., an
intermediate fatty acid); and/or (ii) form a double bond between carbon atoms
in a
desaturase substrate or target molecule.
II_ ;Isolated Desaturase Proteins
One aspect of the invention pertains to isolated or recombinant desaturase
proteins and polypeptides, and biologically active portions thereof. In one
embodiment,
native desaturase proteins can be isolated from cells or tissue sources by an
appropriate
purification scheme using standard protein purification techniques. In another
embodiment, desaturase proteins are produced by recombinant DNA techniques.
Alternative to recombinant expression, a desaturase protein or polypeptide can
be
synthesized chemically using standard peptide synthesis techniques.
25- An "isolated" or "purified" protein or biologically active portion thereof
is substantially free of cellular material or other contaminating proteins
from the cell or
tissue source from which the desaturase protein is derived, or substantially
free from
chemical precursors or other chemicals when chemically synthesized. The
language
"substantially free of cellular material" includes preparations of desaturase
protein in
~30 ~which the protein is separated from cellular.components of the cells from
which it is
isolated or recombinantly produced. In one~embodiment, the language
"substantially
free of cellular material" includes preparatigns of desaturase protein having
less than
about 80%, 70%, 60%, 50%o, 40%, or 30% (by dry weight) of non-desaturase
protein
(also referred to herein as a "contaminating protein"), more preferably less
than about
35 20% of non-desaturase protein, still more preferably less than about 10% of
non-
desaturase protein, and most preferably less than about 5% non-desaturase
protein.
When the desaturase protein or biologically active portion thereof is
recombinantly
CA 02608848 2007-07-17
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produced, it is also preferably substantially free of culture medium, i.e.,
culture medium
represents less than about 20%, more preferably less than about 10%, and most
preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other
chemicals" includes preparations of desaturase protein in which the protein is
separated
from chemical precursors or other chemicals which are involved in the
synthesis of the
protein. In one embodiment, the language "substantially free of chemical
precursors or
other chemicals" includes preparations of desaturase protein having less than
about 30%
(by dry weight) of chemical precursors or non-desaturase chemicals, more
preferably
less than about 20% chemical precursors or non-desaXurase chemicals, still
more
preferably less than about 10% chemical precursors or non-desaturase
chemicals, and
most preferably less than about 5% chemical precursors, or non-desaturase
chemicals. It
should be understood that the proteins or this invention can also be in a form
which is
different than their corresponding naturally occurring proteins and/or which
is still in
association with at least some cellular components. For example, the protein
can be
associated with a cellular membrane.
As used herein, a "biologically active portion" of a desaturase protein
includes a fragment of a desaturase protein which participates in an
interaction between
a desaturase molecule and a non 4esaturase molecule (e.g., a desaturase
substrate such
as fatty acid). Biologically active portions of a desaturase protein include
peptides comprising amino
acid sequences sufficiently homologous to or derived from the desaturase amino
acid sequences,
e.g., the amino acid sequences shown in SEQ ID NO:2, 4, 6, or 8 which include
sufficient amino
acid residues to exhibit at least one activity of a desaturase protein.
Typically, biologically active
portions comprise a domain or motif with at least one activity of the
desaturase protein; the ability to
(i) interact with a desaturase substrate or target molecule (e.g., an
intermediate fatty acid); and/or (ii)
form a double bond between carbon atoms in a desaturase substrate or target
molecule. A
biologically active portion of a desaturase protein can be a polypeptide which
is, for example, 10,
25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 750, 800, 850,
900, 950, 1000, 1050, 1100, 1150, 1200 or more amino acids in length.
In one embodiment, a biologically active portion of a desaturase protein
comprises a heme-binding motif and/or at least one hi tidine motifs,
preferably about
three histidine motifs. Moreover, other biologically active portions, in which
other
regions of the protein are deleted, can be .prepared by recombinant techniques
and
evaluated for one or more of the functional activities of a native desaturase
protein.
CA 02608848 2007-07-17
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In a preferred embodiment, a desaturase protein has an amino acid
sequence shown in SEQ ID NO: 2, 4, 6, or 8. In other embodiments, the
desaturase
protein is substantially identical to SEQ ID NO: 2, 4, 6, or 8 and retains the
functional
activity of the protein of SEQ ID NO: 2, 4, 6, or 8, yet differs in amino acid
sequence
due to natural allelic variation or mutagenesis, as described in detail in
subsection I
above. In another embodiment, the desaturase protein is a protein which
comprises an
amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%,
95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2, 4, 6, or 8:
In another embodiment, the invention features a desaturase protein which
is encoded by a nucleic acid molecule consisting of a nucleotide sequence at
least about
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more
identical to a nucleotide sequence of SEQ ID NO:1, 3, 5, or 7, or a complement
thereof.
This invention further features a desaturase protein which is encoded by a
nucleic acid
moleeule consisting of a nucleotide sequence which hybridizes under stringent
hybridization conditions to a complement of a nucleic acid molecule camprising
the
n-icleotide sequence of SEQ ID NO:1, 3, 5, or 7, or a complement thereof.
To determine the percent identity of two amino acid sequences or of two
nucleic acid sequences, the sequences are aligned for optimal comparison
purposes (e.g.,
gaps can be introduced in one or both of a first and a second amino acid or
nucleic acid
sequence for optimal alignment and non-homologous sequences can be disregarded
for
comparison purposes). In a preferred embodiment, the length of a reference
sequence
aligned for comparison purposes is at least 30%, preferably at least 40%, more
preferably at least 50%, even more preferably at least 60%, and-even more
preferably at
least 70%, 80%, or 90% of the length of the reference sequence (e.g., when
aligning a
'second sequence to the Fad4 amino acid sequence of SEQ ID N0:1 having 519
amino
acid residues, at least 156, preferably at least 208, more preferably at least
260, even
more preferably at least 311, and even more preferably at least 363, 415, or
467 amino
acid residues are aligned; when aligning a second sequence to the Fad5 amino
acid
sequence of SEQ ID NO:4 having 439 amind acid residues, at least 132,
preferably at
least 176, more preferably at least 220, even-more preferably at least 263,
and even more
preferably at least 307, 351, or 395 amino acid residues are aligned; when
aligning a
second sequence to the Fad5-2 amino acid sequence of SEQ ID NO:6 having 456
amino
acid residues, at least 137, preferably at least 182, more preferably at least
228, even
more preferably at least 273, and even more preferably at least 319, 365, or
419 amino
acid residues are aligned; when aligning a second sequence to the Fad6 amino
acid
sequence of SEQ ID NO:8 having 460 amino acid residues, at least 138,
preferably at
least 184, more preferably at least 230, even more preferably at least 276,
and even more
CA 02608848 2007-07-17
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preferably at least 322, 368, or 414 amino acid residues are aligned). The
amino acid residues
or nucleotides at corresponding amino acid positions or nucleotide positions
are then compared.
When a position in the first sequence is occupied by the same amino acid
residue or nucleotide
as the corresponding position in the second sequence, then the molecules are
identical at that
position (as used herein amino acid or nucleic acid "identity" is equivalent
to amino acid or
nucleic acid "homology"). The percent identity between the two sequences is a
function of the
number of identical positions shared by the sequences, taking into account the
number of gaps,
and the length of each gap, which need to be introduced for optimal alignment
of the two
sequences.
The comparison of sequences and determination of percent identity between
two sequences can be accomplished using a mathematical algorithm. In a
preferred
embodiment, the percent identity between two amino acid sequences is
determined using the
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has
been
incorporated into the GAP program in the GCG software package (available from
Accelrys Inc.,
9685 Scranton Road, San Diego, CA 92121-3752, U.S.A.), using either a Blossum
62 matrix or
a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1,
nucleotide sequences is determined using the GAP program in the GCG software
package
(available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752,
U.S.A.), using
a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2,
3, 4, 5, or 6. A preferred non-limiting example of parameters to be used in
conjunction with the
GAP program include a Blosum 62 scoring matrix with a gap penalty of 12, a gap
extend
penalty of 4, and a frameshift gap penalty of 5.
In another embodiment, the percent identity between two amino acid or
nucleotide sequences is determined using the algorithm of Meyers and Miller
(Comput. Appl.
Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program
(version 2.0 or
version 2.OU), using a PAM 120 weight residue table, a gap length penalty of
12 and a gap
penalty of 4.
The nucleic acid and protein sequences of the present invention can further be
used as a "query sequence" to perform a search against public databases to,
for example,
identify other family members or related sequences. Such searches can be
performed using the
NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol.
Biol. 215:403-
10. BLAST nucleotide searches can be performed with the NBLAST program, score
= 100,
wordlength =12 to obtain nucleotide sequences homologous to desaturase nucleic
acid
molecules of the invention. BLAST protein searches can be performed with the
XBLAST
program, score = 50, wordlength = 3 to obtain amino acid sequences homologous
to desaturase
CA 02608848 2007-07-17
-25-
protein molecules of the invention. To obtain gapped alignments for comparison
purposes, Gapped
BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids
Res. 25(17):3389-3402.
When utilizing BLAST and Gapped BLAST programs, the default parameters of the
respective
programs (e.g., XBLAST and NBLAST) can be used. See National Center for
Biotechnology
Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda,
MD 20894, U.S.A.
III. Methods of Producing Unsaturated Fatty Acids
The present invention provides new and improved methods of producing
unsaturated fatty acids, e.g., LCPUFAs, such as, DHA (docosahexaenoic acid,
22:6 (n-6)), DPA
(docosapentaenoic acid, 22:5 (n-6)), AA (Arachidonic acid, 20:4 (n-6)) and EPA
(eicosapentaenioc
acid, 20:5(n-3)).
A. Recombinant Cells and Methods for Culturing Cells
The present invention further features recombinant vectors that include
nucleic acid
sequences that encode the gene products as described herein, preferably Fad4,
Fad5, Fad5-2, and
Fad6 gene products. The term recombinant vector includes a vector (e.g.,
plasmid) that has been
altered, modified or engineered such that it contains greater, fewer or
different nucleic acid
sequences than those included in the native vector or plasmid. In one
embodiment, a recombinant
vector includes the nucleic acid sequence encoding at least one fatty acid
desaturase enzyme
operably linked to regulatory sequences. The phrase "operably linked to
regulatory sequences(s)"
means that the nucleotide sequence of interest is linked to the regulatory
sequence(s) in a manner
which allows for expression (e.g., enhanced, increased, constitutive, basal,
attenuated, decreased or
repressed expression) of the nucleotide sequence, preferably expression of a
gene product encoded
by the nucleotide sequence (e.g., when the recombinant vector is introduced
into a cell). Exemplary
vectors are described in further detail herein as well as in, for example,
Frascotti et al., U.S. Pat. No.
5,721,137.
The term "regulatory sequence" includes nucleic acid sequences which affect
(e.g.,
modulate or regulate) expression of other (non-regulatory) nucleic acid
sequences. In one
embodiment, a regulatory sequence is included in a recombinant vector in a
similar or identical
position and/or orientation relative to a particular gene of interest as is
observed for the regulatory
sequence and gene of interest as it appears in nature, e.g., in a native
position and/or orientation. For
example, a gene of interest (e.g., a Fad4, Fad5, Fad5-2, or Fad 6 gene) can be
included in a
recombinant vector operably linked to a regulatory sequence which accompanies
or is adjacent to
the gene in the natural organism (e.g., operably linked to "native" Fad4,
Fad5, Fad5-2, or Fad6
CA 02608848 2007-07-17
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regulatory sequence (e.g., to the "native" Fad4, Fad5, Fad5-2, or Fad6
promoter).
Alternatively, a gene of interest (e.g., a Fad4, Fad5, Fad5-2, or Fad6 gene)
can be
included in a recombinant vector operably linked to a regulatory sequence
which
accompanies or is adjacent to another (e.g., a different) gene in the natural
organism.
For example, a Fad4, Fad5, Fad5-2, or Fad6 gene can be included in a vector
operably
linked to non-Fad4, Fad5, Fad5-2, or Fad6 regulatory sequences. Alternatively,
a gene
of interest (e.g., a Fad4, Fad5, Fad5-2, or Fad6 gene) can be included in a
vector
operably linked to a regulatory sequence from another organism. For example,
regulatory sequences from other microbes (e.g., other bacterial regulatory
sequences,
bacteriophage regulatory sequences and the like) can be operably linked to.a
particular
gene of interest.
Preferred regulatory sequences include promoters, enhancers, termination
signals and other expression control elements (e.g, binding sites for
transcriptional
and/or translationa.l regulatory proteins, for example, in the transcribed
mRNA). Such
regulatory sequences are described, for example, in Sambrook, J., Fritsh, E.
F.,and
Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor
Laboratory, Cold- Spring Harbor Laboratory Press, Cold Spring- Harbor, NY,
1989.
Regulatory sequences include those which direct constitutive expression of a
nucleotide
sequence in a cell (e:g., constitutive promoters, and strong constitutive
promoters), those
which direct inducible expression of a nucleotide.sequence in a cell (e.g.,
inducible
promoters, for example, xylose inducible promoters) and those which attenuate
or
repress -expression of a nucleotide sequence in a cell (e.g., attenuation
signals or
repressor sequences). It is also within the scope of the present invention to
regulate
expression of a gene of interest by removing or deleting regulatory sequences.
For
example, sequences-involved in the negative regulation of transcription can be
removed
such that expression of a gene of interest is-enhanced.
In one embodiment, a recombinant vector of the present invention
includes nucleic acid sequences that encode at least one gene product (e.g.,
Fad4, Fad5,
Fad5-2, or Fad6) operably linked to a promoter or promoter sequence.
In yet another embodiment, a recombinant vector of the present invention
includes a terminator sequence or terminator sequences (e.g.,
transcription.terminator
sequences). The term "terminator sequences" includes regulatory sequences
which
serve to terminate transcription of mRNA. Terminator sequences (or tandem
transcription terminators) can further serve to stabilize mRNA (e.g., by
adding structure
to mRNA), for example, against nucleases.
CA 02608848 2007-07-17
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In yet another embodiment, a recombinant vector of the present invention
includes antibiotic resistance sequences. The term "antibiotic resistance
sequences"
includes sequences which promote or confer resistance to antibiotics on the
host
organism. In one embodiment, the antibiotic resistance sequences are selected
from the
group consisting of cat (chloramphenicol resistance), tet (tetracycline
resistance)
sequences, erm (erythromycin resistance) sequences, neo (neomycin resistance)
sequences and spec (spectinomycin resistance).sequences. Recombinant vectors
of the
present invention can further include homologous recombination sequences
(e.g.,
sequences designed to allow recombination of the gene of interest into the
chromosome
10' of the host organism). For example, amyE sequences can be used as homology
targets
for recombination into the host chromosome.
The term "manipulated cell" includes a cell that has been engineered
(e.g., genetically engineered) or modified such that the cell has at least one
fatty acid
desaturase, e.g.; Fad4, Fad5, Fad5-2, and/or Fad6, such that an unsaturated
fatty acid is
produced. Modification or engineering of such microorganisms can be according
to any
methodology described herein including, but not limited to, deregulation of a
biosynthetic pathway and/or overexpression of at least one biosynthetic
enzyme. A
"manipulated" enzyme (e.g., a "manipulated" biosynthetic enzyme) includes an
enzyme,
the.expression or production of which has been altered or modified such that
at least one
upstream or downstream precursor, substrate or product of the enzyme is
altered or
modified, for example, as compared to a corresponding wild-type or naturally
occurring
enzyme.
The term "overexpressed" or "overexpression" includes expression of a
gene product (e.g., a fatty acid desaturase) at a level greater than that
expressed prior to
25manipulation of the cell or in a comparable. ce11 which has not been
manipulated. In one
embodiment, the cell can be genetically manipulated (e.g., genetically
engineered) to
overexpress a level of gene product greater than that expressed prior to
manipulation of
the cell or in a comparable cell which has not4been manipulated. Genetic
manipulation
can include, but is not limited to, altering or modif'ying regulatory
sequences or sites
associated with expression of a particular gene:(e.g., by adding strong
promoters,
inducible promoters or multiple promoters %:by removing regulatory sequences
such
that expression is constitutive), modifying thpohromosomal location of a
particular
gene, altering nucleic acid sequences adjacent to a particular gene such as a
ribosome
binding site or transcription terminator, increasing the copy number of a
particular gene,
modifying proteins (e.g., regulatory proteins, suppressors, enhancers,
transcriptional
activators and the like) involved in transcription of a particular gene and/or
translation of
a particular gene product, or any other conventional means of deregulating
expression of
CA 02608848 2007-07-17
-2s-
a particular gene routine in the art (including but not limited to use of
antisense nucleic
acid molecules, for example, to block expression of repressor proteins).
In another embodiment, he cell can be physically or environmentally
manipulated to overexpress a level of gene product greater than that expressed
prior to
manipulatiotn of the celi or in a comparable cell which has not been
manipulated. For
example, a cell can be treated with or cultured in the presence of an agent
known or
suspected to increase transcription of a particular gene and/or translation of
a particular
gene product such that transcription and/or translation are enhanced or
increased.
Alternatively, a cell can be cultured at a temperature selected to increase
transcription of
a particular gene and/or translation of a particular gene product such that
transcription
and/or translation are enhanced or increased.
The term "deregulated" or "deregulation" includes the alteration or
modification of at least one gene in a cell that encodes,an enzyme in a
biosynthetic
pathway, such that the level or activity of the biosynthetic enzyme in the
cell is altered
or modified. Preferably, at least one gene that encodes an enzyme in a.
biosynthetic
pathway is altered or modified such that the gene product is enhanced or
increased. The
phrase "deregulated pathway" can also include abiosynthetic pathway in which
more
than one gene that encodes an enzyme in a biosynthetic pathway is aitered.or
modified
such that the level or activity of more than one biosynthetic enzyme is
altered or
modified. The ability to "deregulate" a pathway (e.g., to simultaneously
deregulate
more than one gene in a given biosynthetic pathway) in a cell arises from the
particular
phenomenon of cells in which more than one enzyme (e.g., two or three
biosynthetic
enzymes) are encoded by genes occurring adjacent to one another on a
contiguous piece
of genetic material termed an "operon".
The term "operon" includes a coordinated unit of gene expression that
contains a promoter and possibly a regulatory element associated with one or
more,
preferably at least two, structural genes (e.g., genes encoding enzymes, for
example,
biosynthetic enzymes). Expression of the structural genes can be coordinately
regulated,
for example, by regulatory proteins binding to the regulatory element or by
anti-
termination of transcription. The structural genes can be transcribed to give
a single
mRNA that encodes all of the structural proteins. Due to the coordinated
regulation of
genes included in an operon, alteration or modification of the single promoter
andlor
regulatory element can result in alteration or modification of each gene
product encoded
by the operon. Alteration or modification of the regulatory element can
include, but is
not limited to removing the endogenous promoter and/or regulatory element(s),
adding
strong promoters, inducible promoters or multiple promoters or removing
regulatory
sequences such that expression of the gene products is modified, modifying the
CA 02608848 2007-07-17
-29-
chromosomal location of the operon, altering nucleic acid sequences adjacent
to the
operon or within the operon such as a ribosome binding site, increasing the
copy number
of the operon, modifying proteins (e.g., regu] itory proteins, suppressors,
enhancers,
transcriptional activators and the like) involved in transcription of the
operon and/or
translation of the gene products of the operon, or any other conventional
means of
deregulating expression of genes routine in the art (including but not limited
to use of
14ntisense nucleic acid molecules, for example, to block expression of
repressor
proteins). Deregulation can also involve altering the coding region of one or
more genes
to yield, for example, an enzyme that is feedback resistant or has a higher or
lower
specific activity.
A particularly preferred "recombinant" cell of the present invention has
'been genetically engineered to overexpress a plant-derived gene or gene
product or an
microorganismally-derived gene or gene product. The term "plant-derived,"
"microorganismally-derived," or "derived-from," for example, includes a gene
which is
natutally found in a niicroorganism or a plant, e.g., an oilseed plant, or a
gene product
(e:g -; Fad4, Fad5, Fad5-2, or Fad6) or which is encoded by a plant gene or a
gene from a
microorganism (e.g., encoded SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, or SEQ ID
NO:7).
The methodologies of the present invention feature recombinant cells
whidli.overexpress at least one fatty acid desaturase. In one embodiment, a
recombinant
cell of the present invention has been genetically engineered to overexpress a
Thrauschytrium sp. fatty acid desaturase (e.g., has been engineered to
overexpress at
leastone of Thrau.rchytrium sp: 04 or d5 desaturase (the Fad4. or Fad5 gene
product)
(e.g., a fatty acid desaturase having the amino acid sequence of SEQ ID NO:2
or 4 or
25' ~~e'tleoded by the nucleic acid sequence of SEQ.JD NO:1 or 3).
In another embodiment, a recombinant cell of the present invention has been
genetically engineered to overexpress a Pythium irregulare 4-5 or A-6
desaturase (the Fad5-2 or
Fad6 gene product) (e.g., a fatty acid desaturase having the amino acid
sequence of SEQ ID NO:6 or
8 or encoded by a nucleic acid molecule having the nucleotide sequence of SEQ
ID NO:5 or 7).
In another embodiment, the invention features a cell (e.g , a microbial
cell) that has been transformed with a vector"mprising a fatty acid desaturase
nucleic
acid sequence (e.g., a fatty acid desaturase nucleic acid sequence as set
forth in SEQ ID
NO:1, 3, 5, or. 7).
Another aspect of the present invention features a method of modulating
the production of fatty acids comprising culturing eells transformed by the
nucleic acid
molecules of the present invention (e.g., a desaturase) such that modulation
of fatty acid
CA 02608848 2007-07-17
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production occurs (e.g., production of unsaturated fatty acids is enhanced).
The method
of culturing cells transformed by the nucleic acid molecules of the present
invention
(e.g.,Fad4, Fad5, Fad5-2, and Fad6) to modrlate the production of fatty acids
is referred
to herein as "biotransformation." The biotransformation processes can utilize
recombinant cells and/or desaturases described herein. The term
"biotransformation
process," also referred to herein as "bioconversion processes," includes
biological
processes which result in the production (e.g., transformation or conversion)
of any
compound (e.g., substrate, intermediate, or product) which is upstream of a
fatty acid
desaturase to a compound (e.g., substrate, intermediate, or product) which is
downstream of a fatty acid desaturase, in particular, an unsaturated fatty
acid. In one
embodiment,.the invention features a biotransformation process for the
production of an
unsaturated fatty acid comprising contacting. a cell which overexpresses at
least one fatty
acid desaturase with at least one appropriate substrate under conditions such
that an
unsaturated fatty acid is produced and, optionally, recovering the fatty acid.
In a
preferred embodiment, the invention features a biotransformation process for
the
production of unsaturated fatty acids comprising contacting a cell which
overexpresses
Fad4, Fad5; Fad5-2, or Fad6 with an appropriate substrate (e.g., an
intermediate fatty
acid) under conditions such that an unsaturated fatty acid (e.g., DHA, SDA, or
GLA) is
produced and, aptionally,recovering the unsaturated fatty acid. Conditions
under which
an unsaturated fatty acid is produced can include any conditions which result
in the
desired production of an unsaturated fatty acid.
The cell(s) and/or enzymes used in the biotransformation reactions are in
a form allowing them to perform their intended function (e.g., producing a
desired fatty
acids). The cells can be whole cells, or can be only those portions of the
cells neclessary
to obtain the desired end result. The cells can be suspended (e.g., in an
appropriate
solution such as buffered solutions or media), rinsed (e. g. , rinsed free of
media from
culturing the cell), aeetone-dried, immobilized (e.g., with polyacrylamide gel
or k-
carrageenan or on synthetic supports, for example, beads, matrices and the
like), fixed,
cross-linked or permeablized (e.g., have permeablized membranes and/or walls
such that
compounds, for example, substrates, intermediates or products can more easily
pass
through said membrane or wall). The type of cell can be any cell capable of
being used
within the methods of the invention, e.g., plant, animal, or microbial cells.
An important aspect of the present invention involves growing the
recombinant plant or cttlturing the recombinant microorganisms described
herein, such
that a desired compound (e.g., a desired unsaturated fatty acid) is produced.
The term
"culturing" includes maintaining and/or growing a living microorganism of the
present
invention (e.g., maintaining and/or growing a culture or strain). In one
embodiment, a
CA 02608848 2007-07-17
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microorganism of the invention is cultured in liquid media. In another
embodiment, a
microorganism of the invention is cultured in solid media or semi-solid media.
In a
preferred embodiment, a microorganism of ;he>invention is cultured in media
(e.g., a
sterile, liquid media) comprising nutrients essential or beneficial to the
glaintenance
and/or growthof the microorganism (e.g., carbon sources or carbon substrate,
for
example complex carbohydrates such as bean or grain meal, starches, sugars,
sugar
alcohols, hydrocarbons, oils, fats, fatty acids, organic acids and alcohols;
nitrogen
sources, for example, vegetable proteins, peptones, peptides and amino -acids
derived
from grains, beans and tubers, proteins, peptides and amino acids derived form
animal
sources such as meat, milk and animal byproducts such as peptones, meat
extracts and
casein hydrolysates; inorganic nitrogen sources such as urea, ammonium-
sulfate,
ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus
sources,
for example, phosphoric acid, sodium and pbtassium salts thereof; trace
elements, for
example; magnesium, iron, manganese, calcium, copper, zinc, boron, molybdenum,
-and/or cobalt salts; as well as growth factors such as amino acids, vitamins,
growth
promoters and the like).
Preferably, microorganisms of the present invention are cultured under
controlled pH. The term "controlled pH" includes any pH which results in
production of
the.~iesired product (e.g:, an unsaturated iatty acid). In one embodiment,
micxoorganisms are cultured at a pH of about 7. In another embodiment,
microorganisms are cultured at a pH of between 6.0 and 8.5. The desired pH may
be
maintained by any number of methods known to those skilled in the art.
Also preferably, microorganisms of the present invention are cultured
under controlled aeration. The term '.'controiled aeration" includes
sufficient aeration
(e.g., oxygen)~to result in production of the desired product (e.g:, an
unsaturated fatty
acid). In one-embodiment, aeration is controlled by regulating oxygen levels
in the
culture, for.example, by regulating the amount of oxygen dissolved in culture
media.
Preferably, aeration of the culture is controlled by agitating the culture.
Agitation may
be provided by a propeller or similar mechanical agitation equipment, by
revolving or
shaking the growth vessel (e.g., fermentor). or by various pumping equipment.
Aeration
may be further- controlled by the passage of sterile air or oxygen through the
medium
(e.g., through the fermentation mixture). Als~q preferably, microorganisms of
the present
invention are cultured without excess foaming (e.g., via addition of
antifoaming agents).
Moreover, plants or microorganisms of the present invention can be
cultured under controlled temperatures. The term "controlled temperature"
includes any
temperature which results in production of the desired product (e.g., an
unsaturated fatty
acid). In one embodiment, controlled temperatures include temperatures between
15 C
CA 02608848 2007-07-17
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and 95 C. In another embodiment, controlled temperatures include temperatures
between 15 C and 70 C. Preferred temperatures are between 20 C and 55 C, more
preferably between 30 C and 45 C or between 30 C and 50 C.
Microorganisms can be cultured (e.g., maintained and/or grown) in liquid
media and preferably are cultured, either continuously or intermittently, by
conventional
culturing methods such as standing culture, test tube culture, shaking culture
(e.g., rotary
shaking culture, shake flask culture, etc.), aeration spinner culture, or
fermentation. In a
preferred embodiment, the microorganisms are cultured in shake flasks. In a
more
preferred embodiment, the microorganisms are cultured in a fermentor (e.g., a
fermentation process). Fermentation processes of the present invention
include, but are
not limited to, batch, fed-batch and continuous methods of fermentation. The
phrase
"batch process" or "batch fermentation" refers to a closed system in which the
composition of media, nutrients, supplemental additives and the like is set at
the
beginning of the fermentation and not subject to alteration during the
fermentation,
however, attempts may be made to control such factors as pH and oxygen
concentration
to prevent excess media acidification and/or microorganism death. The phrase
"fed-
batch process" or."fed-batch" fermentation refers to a batch fermentation with
the
exception that one or more substrates or supplements are added (e.g., added in
increments or continuously) as the.fermentation progresses. The phrase
'.'continuous
process" or "continuous fermentation" refers to a system in which a defined
fermentation media is added continuously to a fermentor and an equal amount of
used or
"conditioned" media is simultaneously removed, preferably for recovery of the
desired
product (e.g., an unsaturated fatty acid). A variety of such processes have
been
developed and are well-known in the art.
The phrase "culturing under conditions such that a desired compound '
(e.g., an unsaturated fatty acid, for example, DHA) is produced" includes
maintaining
and/or growing plants or microorganisms under conditions (e.g., temperature,
pressure,
pH, duration, etc.) appropriate or sufficient to obtain production of the
desired
compound or to obtain desired yields of the particular compound being
produced. For
example, culturing is continued for a timesufficient to produce the desired
amount of a
unsaturated fatty acid (e.g., DHA). Preferably, culturing is continued for a
time
sufficient to substantially reach maximal production of the unsaturated fatty
acid. In one
embodiment, culturing is continued for about 12 to 24 hours. In another
embodiment,
culturing is continued for about 24 to 36 hours, 36 to 48 hours, 48 to 72
hours, 72 to 96
hours, 96 to 120 hours, 120 to 144 hours, or greater than 144 hours. In
another
embodiment, culturing is continued for a time sufficient to reach production
yields of
unsaturated fatty acids, for example, cells are cultured such that at least
about 15 to 20
CA 02608848 2007-07-17
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g/L of unsaturated fatty acids are produced, at least about 20 to 25 g/L
unsaturated fatty
acids are produced, at least about 25 to 30 g/L unsaturated fatty acids are
produced, at
least about 30 to 35 g/L unsaturated fatty acidsare produced, at least about
35 to 40 g/L
unsaturated fatty acids are produced (e.g., at least about 37 g/L unsaturated
fatty acids )
or at least about 40 to 50 g/L unsaturated fatty acids are produced. In yet
another
embodiment, microorganisms are cultured under conditions such that a preferred
yield
of unsaturated-.fatty acids, for example, a yield.within a range set forth
above, is
produced in about 24 hours, in about 36 hours, in about 48 hours, in about 72
hours, or
in about 96 hours.
-In producing unsaturated fatty acids, it may fiuther be desirable to culture
cells of the present invention in the presence of supplemental- fatty acid
biosynthetic
substrates. The_term "supplemental fatty acid biosynthetic substrate"
includes. an agent
or compound which, when brought into contact with a cell or included in the
culture
medium of a cell, serves to enhance or increase unsaturated fatty acid
biosynthesis.
Supplemental fatty acid biosynthetic substrates of the present invention can
be added in
the form of a concentrated solution or suspension (e.g., in a suitable solvent
such as
water or buffer) or in the form of a solid (e.g., in the form of a powder).
Moreover,
supplemental fatty acid biosynthetic substrates of the present invention can
be added as
a single aliquot, continuously or intermittently over a given period of time.
The methodology of the present invention can further include a step of
recovering a desired compound (e.g., an unsaturated fatty acid). The term
"recovering"
a desired compound includes extracting, harvesting, isolating or purifying the
compound
from culture media. Recovering the compound can be performed according to any
conventional isolation or purification methodology- known in the art
including, but not
limited to; treatment with a conventional resip (e.g., ariion or
cationexchange resin, non-
ionie adsorption resin, etc.), treatment with aa;conventional adsorbent (e,g ;
activated
charcoal, silicic acid, silica gel, cellulose, alumina, etc.); alteration of
pH, solvent
extraction (e.g., with a conventional solvent such as an alcohol, ethyl
acetate, hexane
and the like), dialysis, filtration, coneentratian, crystallization,
recrystallization, pH
adjustment, lyophilization and the like. For:example, a compound can be
recovered
from culture media by first removing the microorganisms from the culture.
Media is
then passed through or over a cation exchanp resin to remove unwanted cations
and
then through or over an anion exchange resin to remove unwanted inorganic
anions and
organic acids having stronger acidities than the unsaturated fatty acid of
interest (e.g.,
DHA).
CA 02608848 2007-07-17
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Preferably, a desired compound of the present invention is "extracted,"
"isolated" or "purified" such that the resulting preparation is substantially
free of other
components (e.g., free of media components and/or fermentation byproducts).
The
language "substantially free of other components" includes preparations of
desired
compound in which the compound is separated (e.g., purified or partially
purified) from..
media components or fermentation byproducts of the culture from which it is
produced.
In one embodiment, the preparation has greater than about 80% (by dry weight)
of the
desired compound (e.g., less than about 20% of other media components or
fermentation
byproducts), more preferably greater than about 90% of the desired compound
(e.g:, less
than about 10% of other media components or fermentation byproducts),, still
more
preferably greater than about 95% of the desired compound (e.g., less than
about 5% of
other media components or fermentation byproducts), and most preferably
greater than
about 98-99% desired compound (e.g., less than about 1-2% other media
components or
fermentation byproducts). When the desired compotmd is an unsaturated fatty
acid that
has been derivatized to a salt, the compound is preferably fiuther free (e.g.,
substantially
free) of chemical contaminants associated with the formation of the. salt..
When the
desired compound is an unsaturated fatty acid that has been derivatized to
analcohol,.
the compound is preferably further free (e.g., substantially free) of chemical
contaminants associated with the formation of the alcohol.
In an alternative embodiment, the desired unsaturated fatty acid is not
purified from the plant or microorganism, for example, when the plant or
microorganism
is biologically non-hazardous (e.g., safe). For example, the entire plant or
culture (or
culture supematant) can be used as a source of product (e.g., crude product);
In one
embodiment, the plant or culture (or culture supernatant) supematant is used
without
modification. In another embodiment, the plant or culture (or culture
supematant) is
concentrated. In yet another embodiment, the plant or culture (or culture
supematant) is
pulverized, dried, or lyophilized.
B. High Yield Production Methodologies
A particularly preferred embodiment of the present.invention is a high
yield production method for producing unsaturated fatty acids, e.g., DHA,
comprising
culturing a manipulated plant or microorganism under conditions such that the
unsaturated fatty acid is produced at a significantly high yield. The phrase.
"high yield
production method," for example, a high yield production method for producing
a
desired compound (e.g., for producing an unsaturated fatty acid) includes a
method that
results in production of the desired compound at a level which is elevated or
above what
is usual for comparable production methods. Preferably, a high yield
production method
CA 02608848 2007-07-17
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results in production of the desired compound at a significantly high yield.
The phrase
"significantly high yield" includes a level of production or yield which is
sufficiently
elevated or above what is usual for comparable production methods, for
example, which
is elevated to a level sufficient for commercial production of the desired
product (e.g.,
production of the product at a commercially feasible cost). In one embodiment,
the
invention features a high yield production method of producing unsaturated
fatty acids
that includes culturing a manipulated plant or microorganism under conditions
such that
an unsaturated fatty acid is produced at a level greater than 2 g/L. In
another
embodiment, the invention features a high yieldproduction method of producing
unsaturated fatty-'acids that includes culturing amanipulated plant or
microorgarusm
under conditions such that an unsaturated fatty acid is produced at a level
greater than 10
gfL. In another:embodiment, the invention features a high yield production
method of
producing unsaturated fatty acids that includes culturing a manipulated, plant
or
microorganism under conditions such that an unsaturated fatty acid is produced
at a
level greater than 20 g/L. In yet another embodiment, the invention features a
high yield
production method of producing unsaturated fatty acids that includes culturing
a
rrianipulated plant or microorganism under conditions such that an unsaturated
fatty acid
is produced at a level greater than 30 g/L. In yet another.embodiment, the
invention
features a high yield production method of producing unsaturated fatty acids
that
includes culturing a manipulated plant or microorganism under conditions such
that an
unsaturated fatty acid is produced at a level greater than 40 gfL.
The invention further features a high yield production method for
producing a desired compound (e.g., for producing an unsaturated fatty
acid):that
involves culturing a manipulated plant or microorganismunder conditions such
that a
'25 sufficientlyelevated level of compound is produced within a commercially
desirable
period of time:' -In an exemplary embodiment, the invention features a high
yield
production method of producing unsaturated fatty acids that includes culturing
a
manipulated plant or microorganism under conditions such that an unsaturated
fatty acid
is produced at a level greater than 15-20 g/I.; in 36 hours. In another
embodiment, the
invention featcires a high yield production method of producing unsaturated
fatty acids
that includes culturing a manipulated plant pr microorganism under- conditions
such that
an unsaturated fatty acids produced at a level; greater than 25-30 g/L in 48
hours. In
another embodiment, the invention features'a high yield production method of
producing unsaturated fatty acids that includes culturing a manipulated.plant
or
microorganism under conditions such that an unsaturated fatty acids produced
at a level
greater than 35-40 g/L in 72 hours, for example, greater that 37 g/L in 72
hours. In
another embod'unent, the invention features a high yield production method of
= CA 02608848 2007-07-17
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producing unsaturated fatty acids that includes culturing a manipulated plant
or
microorganism under conditions such that an unsaturated fatty acid is produced
at a
level greater than 30-40 g!L in 60 hours, for example, greater that 30, 35 or
40 g/L in 60
hours. Values and ranges included and/or intermediate within the ranges set
forth herein
are also intended to be within the scope of the present invention. For
example,
unsaturated fatty acid production at levels of at least 31, 32, 33, 34, 35,
36, 37, 38 and 39
g/L in 60 hours are intended to be included within the range of 30-40 g/L in
60 hours.
In another example, ranges of 30-35 g/L or 35-40 g/L are intended to be
included within
the range of 30-40 g/L in 60 hours. Moreover, the skilled artisan will
appreciate that
culturing a manipulated microorganism to achieve a production level of, for
example,
"30-40 g/L in 60 hours" includes culturing the microorganism for additional
time
periods (e.g., time periods longer than 60, hours), optionally resulting in
even higher
yields of an unsaturated fatty acid being produced.
IV. Compositions
The desaturase nucleic acid molecules, proteins, and fragments thereof,
of the invention can be used to produce unsaturated fatty acids which can be
incorporated into compositions: Compositions ofthe present invention include,
e.g.,
compositions for use as animal feed, compositions for use as neutraceuticals
(e.g.,
dietary supplements), and phannaceutical compositions suitable for
administration.
Such pharmaeeutical compositions typically comprise an unsaturated
fatty acid and a pharmaceutically acceptable carrier. As used herein the
language
"pharmaceutically acceptable carrier" is intended to include any and all
solvents,
dispersion media, coatings, antibacterial and antifungal agents; isotonic and
absorption
delaying'agents, and the like, compatible with pharmaceutical administration.
The use
of such media and agents for pharmaceutically active substances is well known
in the
art. Except insofar as any conventional media or agent is incompatible
with.the active
compound, use thereof in the compositions is contemplated: Supplementary
active
compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be
compatible with its intended route'of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral
(e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous
application
can include the following components: a sterile diluent such as water for
injection,
saline solution, fixed oi1s, polyethylene glycols, glycerine, propylene glycol
or other
synthetic solvents; antibacterial agents such as benzyl alcohol or methyl
parabens;
CA 02608848 2007-07-17
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antioxidants such as ascorbic acid or sodiutn bisulfite; chelating agents such
as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
phosphates and
agents for the adjustment of toriicity such as sodium chloride or dextrose, pH
can be
adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The
parenteral preparation can be enclosed in ampouies, disposable syringes or
multiple dose
vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersion. For
intravenous administration, suitable carriers include physiological saline,-
bacteriostatic
water, Cremophor ELTM (BASF, Parsippany; NJ) or phosphate buffered saline
(PBS).
In all cases; the;romposition must be sterile and should be fluid to the
extent that easy
syringeability exists. It must be stable under the conditions of manufacture
and storage
and must be preserved against the contaminating action of microorganisms such
as
bacteria and fungi. The carrier can be a solvent or dispersion medium
containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyetheylene glycol, and the like), and suitable mixtures thereof. The proper
fluidity
can be maintained, for example; by the use of a coating such as lecithin, by
the
maintenance of the required particle size in t1ie. case of dispersion and by
the use of
-20 surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable
to include
isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol,
sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be
brought about by including in the composition.;an agent which delays
absorption, for
example, aluminum monostearate and gelatin.:.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g., a LCPUFA, or a fragment thereof, produced by the nucleic acid
and
protein molecules of the present invention) in the required amount in an
appropriate
solvent with one or a combination of ingredients enuinerated above, as
required,
followed by filtered sterilization. Generally, dispersions are prepared by
incorporating
the active compound into a sterile vehicle whij5h contains a basic dispersion
medium and
the required other ingredients from those enumerated above. In the case of
sterile
powders for the preparation of sterile injectable solutions, the preferred
methods of
preparation are vacuum drying and freeze-drying which yields a powder of the
active
ingredient plus any additional desired ingredient from a previously sterile-
filtered
solution thereof.
CA 02608848 2007-07-17
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Oral compositions generally include an inert diluent or an edible carrier.
They can be enclosed in gelatin capsules or compressed into tablets. For the
purpose of
oral therapeutic administration, the active compound.can be incorporated with
excipients
and used in the form of tablets, troches, or capsules. oral compositions can
also be
prepared using a fluid carrier for use as a mouthwash, wherein the compound in
the fluid
carrier is applied orally and swished and expectorated or swallowed.
Pharmaceutically
compatible binding agents, and/or adjuvant materials can. be included as part
of the
composition. The tablets, pills, capsules, troches and the like can contain
any of the
following ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as
starch or
lactose, a disintegrating agent such as -alginic acid, Primogel, or corn
starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint,
methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the
form of an aerosol spray from pressured container or dispenser which contains
a-suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal
means. . For transmucosal or transdermal administration, penetrants
appropriate to the
barrier to -be permeated are used in the formulation. Such penetrants are
generally
known in the art, and include, for example, for transmucosal administration,
detergents,
bile salts, and fusidic acid derivatives. Transmucosal administration can be
accomplished through the use of nasal sprays or suppositories. For transdermal
administration, the active compounds are formulated into ointments, salves,
gels, or
creams as generally known in. the art.
The compounds can also be prepared in the form -of suppositories.(e.g.,
with con;entional suppository bases such as cocoa butter and other glycerides)
or
retention enemas for rectal delivery.
In one embodiment, the active. compounds are prepared with carriers that
will protect the compound against rapid elimination from the body, such as a
controlled
release formulation, including implants and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can. be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid.
Methods for preparation of such formulations will be apparent to those skilled
in the art.
The materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to
infected
cells with monoclonal antibodies to viral antigens) can also be used as
pharmaceutically
CA 02608848 2007-07-17
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acceptable carriers. These can be prepared according to methods known to those
skilled
in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions
in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit
form as used herein refers to physically discrete units suited as unitary
dosages for the
subject to be treated; each unit containing a predetermined quantity of active
compound
calculated to produce the desired therapeutic effect in association with the
required
pharmaceutical carrier. The specification for the dosage unit forms of the
invention are
dictated by and directly dependent on the unique characteristics of the active
compound
and the particular therapeutic effect to be achieved, and the limitations
inherent in'the art
of compounding'such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be deternuned
by standard pharmaceutical procedures in cell cultures -or experimental
animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and the ED50
(the dose
therapeutically effective in 50% of the population). The dose ratio between
toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio
LD50/ED50. Compounds which exhibit large therapeutic indices are preferred.
While
compounds that exhibit toxic side effects may be used, care should be taken to
design a
delivery system that targets such compounds to the site of affected tissue in
order to
minimize potential damage to uninfected cells and, thereby, reduce side
effects.
The data obtained from the cell culture assays and anim.al studies can be
used in formulating a range of dosage for use in humans. The dosage of such
compounds lies preferably within a range of circulating concentrations that
include the
ED50 with little or no toxicity. The dosage may vary wi'1~in this range
depending upon
the dosage form' employed and the route of administration utilized. For any
compound
used in the method of the invention, the therapeutically effective dose can be
estimated
initially from cell culture assays. A dose may be formulated in animal models
to
achieve a circulating plasma concentration range that includes the IC50 (i.e.,
the
concentration of the test compound which achieves a half-maxunal inhibition of
symptoms) as determined in cell culture. Suclf infonnation can be used to more
accurately determine useful doses in humans. tevels in plasma may be measured,
for
example, by high performance liquid chromatvgraphy.
;:õ
As defined herein, a therapeutically effective amount of protein or
polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kjg
body
weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about
0.1 to 20
mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8
mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will
appreciate
CA 02608848 2007-07-17
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that certain factors may influence the dosage required to effectively treat a
subject,
including but not limited to the severity of the disease or disorder, previous
treatments,
the general health and/or age of the subiect, and other diseases present.
Moreover,
treatment of a subject with a therapeutically effective amount of a protein,
polypeptide,
or antibody can include a single treatment or, preferably, can include a
series of
treatments.
In a preferred example, a subject is treated with a LCPUFA in the range
of between about 0.1 to 20 mg/kg body weight, one time per week for between
about I
to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3
to 7
weeks, and even more preferably for about 4, 5, or 6 weeks. It wiIl also be
appreciated
that the effective dosage of antibody, protein, or polypeptide used for
treatment may
increase or decrease over the course. of a particular treatment. 'Changes in
dosage may
result and become apparent from the results of diagnostic assays as described
herein.
The pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
This. invention is fiuther illustrated by the following examples which
should not be construed as limiting.
.EXAMPLES
Materials: Thraust.ochytrium s.p ATCC 21685 and Pytl:iun: irregulare were
purchased
from American type culture collection (12301 Parklawn nrive, Rockville,
Maryland,
20852 tJSA) and grown in a medium (Weete, J:D., et al. (1997) Lipids 32:839-
845)'at
24 C for 7days. After then biomass were harvested by centrifugation and used
for RNA
isolation.
EXAA:MPLE 1: CONSTRUCTION AND SCREENING OF eDNA LIBRARY
Total RNA was isolated from the above materials.according to Qiu and
Erickson (Qiu, X. and Eriekson, L. (1994) Plant Mol..Biol. Repr: 12:209-214).
The
cDNA library was constructed from the total RNA. The first strand cDNA was
synthesized by Superscript 11* reverse transcriptase from Gibco-BRL. The
second strand
cDNA was synthesized by DNA polymerase I from Stratagene. After size
&actionation,
cDNA inserts larger than I kb were ligated into X Uni 7ap XRvector
(Stratagene). The
recombinant - DNAs were then packed with Gigapack III Gold packaging extract
*Trade-mark
CA 02608848 2007-07-17
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(Stratagene) and plated on NZY plates. The resulting library represented more
than 5 x
106 independent clones. Screening of the eDNA library was performed according
to
standard methods (Sambrook, J, Fritseh, E.F . Maniatis, T. (1989) Molecular
cloning - A
laboratory manual. (Cold Spring Harbor, New York, USA.)
EXAMPLE 2: RT-PCR
The single strand cDNA was synthesized by superscript 11 reverse transcriptase
(Gibco-BRL) from total RNA and was then used as the template for PCR reaction
with two
degenerate primers (The forward primer: GCNCA/GANGANCAC/TCCNGGNGG (SEQ ID
NO:9)
and the reverse primer: ATNTG/TNGGA/GAANAG/AG/ATGG/ATG) (SEQ ID NO:10). The
PCR
amplification consisted of 35 cycles with 1 min at 94 C, 1.5 min at 55 C and 2
min at 72 C followed
by an extension step at 72 C for 10 min. The amplified products from 800 bp to
1000 bp were
isolated from agarose gel and purified by a kit (Qiaex II* gel purification,
Qiagen), and subsequently
cloned into the TA cloning vector pCR* 2.1 (Invitrogen). The cloned inserts
were then sequenced
by PRISM DyeDeoxy Terminator Cycle Sequencing System (Perkin Elmer/Applied
Biosystems).
EXAMPLE 3: EXPRESSION OF FAD4, FAD5, FAD5-2, AND FAD6 IN
YEAST
The open reading frames of Fad4, Fad5, Fad5-2, and Fad6 were
amplified by PCR using the Precision Plus .enzyme (Strazagene) and cloned into
a. TA
cloning vector (pGR 2.1, Invitrogen). HavingOnfirmed that the PCR products
were
identical to the original cDNAs by sequencing,-Xhe fragments were then
released by a
BarnHI-EcoRI double digestion and inserted into the yeast expression vector
pYES2 ~
(Invitrogen) under the control of the inducible promoter GALl.
Yeast strains InvSc2 (Invitroge4) was transformed with the expression
constructs using the lithium acetate method and txansformants were selected on
minirnal
medium plates lacking uracil (Gietz, D., et al..(1992) Nucleic Acids Res.
20:1425;
.~siol. 111:223-226).
Covello, P.S. and Reed, D.W. (1996) Plant Pliy
Transformants were first grown'in minimal medium l.a.clcing uracil and
containing glucose at 28 C. After overnight culture, the cells were spun down,
washed
and resuspended in distilled water. Minimal medium containing 2% galactose,
with or
without 0.3 mM substrate fatty acids in the presence of 0.1 % tergitol, was
inoculated
*Trade-mark
CA 02608848 2007-07-17
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with the yeast transformant cell suspension and incubated at 20 C for three
days, and
then 15 C for another three days.
EXAMPLE 4: FATTY ACID ANALYSIS
Thran$tochytrium, Pythiunz irregulare and yeast cells were harvested and
washed twice with distilled water. Then 2 mL methanolic KOH (7.5% w/v KOH in
95% methanol) was added to the materials and the mixture sealed in a 12 ml
glass
culture tube was heated to 80 C for 2 hours. 0.5 mL water was added and the
sample
was extracted twice with 2 mL hexane to remove the non-saponifiable lipids.
The
remaining aqueous phase was then acidified by adding 1 mL 6 N HCI and
extracted
twice with 2 mL hexane. The hexane. phases were.combined and dried under a
stream of
nitrogen. 2 mL 3 N methanolic HCl (SUPELCO, Supelco Park, Bellefonte, PA
16823-0048) was added and the mixture was heated at 80 C for 2 hours. After
cooling
to room temperature, 1 mL 0.9% NaCI was added and the mixture extracted twice
with
2 x 2 mL hexanP. The combined hexane was evaporated under nitrogen. The
resulting
fatty acid methyl esters (FAMEs) were analyzed by GC and GC-MS according to
Covello & Reed (Covello, P.S. and Reed, D.W. (1996) Plant Physiol. 111:223-
226).
GC/MS analysis was performed in standard El mode using a Fisons V G
TRIO 2000 mass spectrometer (VG Analytical, UK) controlled by Masslynx version
2.0
software, coupled to a GC 8000 Series gas chromatograph. A DB-23 column (30M x
0.25 mm i.d., 0.25 Ilm film thickness, J&W Scientific, Folsom, CA) that was
temperature-programmed at 180 C for 1 min, then 4 C/rnin to 240 C and held
for 15
minutes, was used for FAME analysis.
EXAMPLE 5: TRANSFORMATION OF BRASSICA JUNCEA AND
FLAX (LINUM USITATISSIMUM) AND EXOGENOUS
FATTY ACID TREATMENT
The hypocotyls of 5-6 day seedlings of B. juncea and flax were used as
explants for inoculation with the Agrobacterium tunzefaciens that hosts binary
vectors
with the full-Iength cDNAs under the control of the different proinoters. The
20-day
transgenic seedlings were used for exogenous fatty acid treatment. The
seedling was
divided into three parts: leaves, stems and roots. Each was eut into the small
pieces and
placed in a 24-well titer plate. To each well, 2 mL 0.05% sodium salt of
substrates
(NuCheck Prep Inc., Elysian, MN) was added. The plate was then incubated at 24
C for
CA 02608848 2007-07-17
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4 h with gentle shaking. After incubation, the plant tissues were washed three
times
with water and then used for fatty acid analysis.
EXAMPLE 6: FATTY ACID PROFILE OF THE THRAUSC.HYTRIUMSP.
Thraustochytrium and Pythium irregulare have recently drawn scientific
attention due to its ability in production of LCPI7FAs such as DHA, AA, EPA
and DPA.
Figures 15 and 16 show the fatty acid composition of the lipids isolated from
7 day
cultures of Thraustochytrium sp. and Pythium irregulare, respectively. As
shown in the
tables, the microorganisms contain a broad range of polyunsaturated fatty
acids, from
both n-3 and n-6 families, from 1 8-carbon A6 fatty acids (gamma-linolenic
acid and
steardonic acid),to 22-carbon A4 fatty acids (DHA, and DPA). The organisms,
especially Thraustochytrium sp., appear to contain a full-set of desaturation
and
elongation enzymes required for the DHA and DPA biosynthesis. The strain lacks
24-carbon polyunsaturated fatty acids, the proposed precursors for DHA aad DPA
synthesis in Precher' s pathway (Voss, A., et al. (1991) J. Biol. Chem.
266:19995-20000;
Mohammed, B.S., et al. (1997) Biochem. J. 326:425430). The 24-carbon fatty
acid
may not be involved in in vivo synthesis of 22-carbon A4 fatty acids such as
DHA and
DPA in Thraustochytrium sp.
'20
EXAMPLE 7: IDENTIFICATION OF cDNAs CODING FOR THE
"FRONT-END" DESATURASE
To identify genes coding for desaturases involved in biosynthesis of
LCFUFAs in Thraustochytrium sp. and Pythiz}'in irregulare, a PCR-based cloning
strategy was adopted. Two degenerate primers are designed to target the heme-
binding
motif of N-terminal extension of cyt b5-like domain in front-end desaturases
and the
third conservative histidine motif in all microsomal desaturases,
respectively. The
rational behind the design is that the desaturases involved in EPA and DHA
biosynthesis
in Thraustochytriunz sp. and Pythium irregulare, should have similar primary
structure
as other front-end desaturases, i.e. N terminalYextension of cyt b5-like
domain in the
desaturase. Four cDNAs fragments were identified from Tlzraustochytrium sp.
and
Pythiunz irregulare that encode fusion proteins containing cyt b5-like doimain
in the
N-terminus.
CA 02608848 2007-07-17
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To isolate full-length cDNA clones, the four inserts were used as probes
to screen cDNA libraries of Thraustochytriunn sp. and Pythium irregulare,
which
resulted in identification of several cDNA clones in each group. Sequencing of
all those
clones identified four full-length cDNAs which were named as Fad4, Fad5, Fad5-
2 and
Fad6. The open reading frame of Fad4 is 1560 bp and codes for 519 amino acids
with
molecular weight of 59.1 kDa (Figure 1). Fad5 is 1230 bp in length and codes
for 439
amino acids with molecular weight of 49.8 kDa (Figure 2). A sequence
comparison of
these two sequences from Thraustochytrium sp. showed only 16% amino acid
identity
between the deduced proteins. A detailed analysis revealed that Fad4 is 80
amino acids
longer than Fad5, occurring between the second and third conservative
histidine motifs
(Figure 3). The open reading frame of Fad5-2 &om Pythiunz irregulare is 1371
bp and
codes for 456 amino acids (Figure 4). Fad6 from Pythium irregulare is 1383 bp
in
length and codes for 460 amino acids (Figure 5). Sequence comparison of the
two
sequences from Pythiuni irregulare showed over 39% similarity between the
deduced
proteins (Figure 6).
A BLASTP' search of the protein database revealed the following hits
for each of the four proteins, Fad4, Fad5, Fad5-2, and Fad6:
Fad 4 (519 amino acid residues)
Blastp nr
Accession No. Organism Description Length % Identity
AF067654 Mortierella alpina AS fatty acid 509 29.
desaturase
AF054824 Mortierella alpina 05 microsomal 509 28
desaturase
AB022097 Dictyostelium AS fatty acid 507 27
discoideunz desaturase
AB029311 Dictyostelium fatty acid 519 26
discoideum &saturase
L11421 Synechocystis sp. A6 desaturase 410 25
D90914
CA 02608848 2007-07-17
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Fad 5(439 aniino acid residues)
Blastp nr
Accession No. Organism Description Length % Identity
AF139720 Euglena gracilis 08 fatty acid 404 29
desaturase
AF007561 Borago officinalis A6 desaturase 421 27
U79010 Borago officinalis A6 desaturase 421 27
AF309556 Danio rerio A6 fatty acid 422 26
desaturase
AF110510 'Mortierella A6 fattyacid 463 25
alpina desaturase
Fad 5-2 (456 amino acid residues)
Blastp nr
Accession No. Organism Description Length % Identity
AB029311 Dictosteliuni Fatty acid 443 41
discoideum desaturase
AB022097 Dictostelium A5 fatty acid 445 39
discoideum desaturase
AF067654 Mortierella 05 fatty acid 441 38
alpina desaturase
AF054824 Mortierella A5 microsomal 441 38
alpina desaturase
L11421 Syfiechocystis sp. A6 desaturase 361 28
D90914
15
CA 02608848 2007-07-17
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Fad 6 (459 amino acid residues)
Blastp nr
Accession No. Organism Description Length % Identity.
AF110510 Mortierella A6 fatty acid 437 38
alpina desaturase
AB020032 Mortierella A6 fatty acid 437 -38
alpina desaturase
AF306634 Mortierella 06 fatty acid 437 38
isabellina desaturase
AF307940 Mortzerella A6 fatty acid 438 38
alpina desaturase
AJ250735 Ceratodon A6 fatty acid 438 36
purpureus desaturase
EXAMPLE 8: EXPRESSION OF.FAD4, FAD5, FADS-2, AND FAD6 IN
YEAST
To confum the function of Fad4, the full-length cDNA was expressed in
the yeast strain InvSc2 under the control of the inducible promoter. Figure 7
shows that
with supplementation of the medium with 22:5 (7,10,13,16,19), yeast cells
containing
Fad4 cDNA had an extra fatty acid as compared to the vector control. The peak
has a
retention time identical to the DI-Lk standard. LC/MS analysis of the free
fatty acid
showed that it yields deprotonated molecular ions (m/z = 279) identical to the
DHA
standard in negative ion electrospray. Moreover, GC/MS analysis of the FAME
confirnied that the spectrum of the peak is identical to that of the DHA
standard (Figure
8). These results indicate that Fad4 is a A4 fatty acid desaturase which is
able to
introduce a double bond at position 4 of the 22:5(7,10,13,16,19) substrate,
resulting in a
04 desaturated fatty acid, DHA (22:6-4,7,10,13,16,19).
To furkher study the subsirate specificity of the Fad4, a number of
substrates including 18:2(9,12), 18:3(9,12,15), 20:3(8,11,14) and
22:4(7,10,13,16) were
separately supplied to the yeast transformants. The results indicated Fad4
could also use
22:4 (7,10,13,16) as a substrate (Figure 9) to produce another A4 desaturated
fatty acid,
DPA (22:5-4,7,10,13,16) (Figure 10). The rest of the fatty acids examined were
not
effective substrates.
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To confirm the function of Fad5 and Fad5-2, the S. cerevisiae Invsc2
was transformed with plasmids, which contain the open reading fiame of the
Fad5 and
Fad5-2 respectively under the control of the galactose-inducible promoter.
When the
yeast transformants were induced by galactose in a medium containing homo-
gamma-
linolenic acid (HGLA, 20:3-8,11,14), an extra peak was observed in the
chromatogram
of FAMEs accumulating in the transformants compared with the control (Figure
11). A
comparison of the chromatogram with that of the standards revealed that the
fatty acid
had a retention time identical to the arachidonic acid standard (AA, 20:4-
5,8,11,14). To
further confiirm the regiochemistry of the products, the FAMEs were analyzed
by
GC/MS. Figure ;12 indicates that the mass spectra of the new fatty acid and
the AA
standard are identical. These results demonstrate that Fad5 and Fad5-2 convert
HGLA
(20:3-8,11,13) into AA (20:4-5,8,11,14) in yeast. To further study the
substrate
specificity of Fad5-2, the plasmid containing Fad5-2 was transferred into
another yeast
strain AMY-2a where olel, a A9 desaturase gene, is disrupted. The strain is
unable to
grow in minimal media without supplementation with mono-unsaturated fatty
acids. In
this experiment, the strain was grown in minimal medium supplemented with
17:1(10Z),
a non-substrate of Fad5-2, which enabled study of the specificity of the
enzyme towards
various substrates, especially monounsaturates. A number of possible
substrates
including 16:1(9Z), 18:1(9Z), 18:1(11Z), 18:1(11E), 18:1(12E), 18:1(15Z),
18:2(9Z,12Z), 18:3(9Z,12Z,15Z), 20:2(i.1Z,14Z) and 20:3(11Z,14Z,17Z) were
tested.
Results indicated that F05-2 could desaturate unsaturated fatty acids with A9
ethylenic
and Al 1 ethylenie double bonds, as well as the fatty acid with A8 ethylenic
double bond
(203-8,11,14). As shown in Figure 13, Fad5-2 effectively converted both
18:1(9Z) and
18:1(11Z) substrates into their corresponding A5 desaturated fatty acids, 18:2-
5,9 (the
retention time 10.34 min) and 18:1-5,11 (the retention time 10.44 min),
respectively.
Fad5-2 also desaturated trans fatty acid such as 18:1(11E) and 18:1(12E).
Figure 25 is a comparison of substrate preference of Fad5-2 for fatty acid
substrates tested in the yeast strain AMY-2a. ':' Phe relative proportions of
the substrates
and the products accumulated are a useful indicator of substrate preference-of
the
enzyme. As shown in Figure 25, Fad5-2 prefers fatty acids with 20-carbon as
substrates,
such as 20:3(8Z,11Z,14Z), 20:3 (11 Z,14Z,17Z) and 20:2(11Z,14Z). Whereas, the
shorter
chain fatty acid is a relatively weaker substrate for the enzyme in yeast.
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To confirm the function of Fad6, the S. cerevisiae host strain Invsc2 was
transformed with a plasmid containing the open reading frame of Fad6 under the
control
of the galactose-inducible promoter, GAL 1. When the yeast transformant was
induced
by galactose in a medium containing linoleic acid, an extra peak was observed
in the
chromatogram of the FAMEs accumulating in the transformants compared with the
control (Figure 14). A comparison of the chromatogram with that of the
standards
revealed that the peak had a retention time identical to the ganuna-linolenic
acid (GLA,
18:3-6,9,12) standard. To confirm the regioselectivity of the products, the
diethylamine
derivatives of fatty acids from the expressing strain were analyzed by GC-MS.
Figure
15 shows that the new peak is indeed GLA with three double bonds at the A6,
A9, and
A12 positions. Major fragments of n and n+1 carbons 'differing by 12-D are
diagnostic
of a double bond between carbon n+1 and n+2. Thus, the fragments at 156 and
168, 196
and 208, and 236 and 248, indicate double bonds at the 06, 09, and A 12
positions,
respectively. These results demonstrate that Fad6 is a A6 desaturase that
converts '
linoleic acid (18:2) to GLA in yeast.
EXA1VII'LE 9: EXPRESSION OF FAD4 IN B. JUIVCEA
To detennine whether Traustochytrium Fad4 is functional in oilseed
crops, B. juizcea were transformed with the constructcontaining Fad4 under the
control
of a constitutive promoter. Eight independent transgenic plants were obtained.
In B.
juncea there is no 04 fatty acid desaturase substrates available. Thus, to
examine the
activity of the transgenic enzyme in the plants, the 22:5 (n-3) substrate must
be
exogenously supplied. In this ~experiment, both wild type and transgenics were
applied
with an aqueous solution of sodium docosapentaeneate. It was found that
exogenously
applied substrates were readily taken up by roots, stem, and leaves of both
types of
plants, but converted into DHA onlyin transgenics. Leaves have a higher level
of
production of DHA than roots and stems., In leaves, the exogenous substrate
was
incorporated to a level of 10-20% of the total fatty acids and A4 desaturated
fatty acid
(22:6, n-3) was produced in a range of 3-6% of the total fatty acids (Figure
16). These
results indicate that the A4 fatty acid desaturase from Traustochytriuni is
functional in
oilseed crops.
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EXAMPLE 10: EXPRESSION OF FAD5-2 IN B. JUNCEA
To determine whether Fad5-2 is functional in oilseed crops and its
expression has any. effect on their growth and development, B. juncea were
transformed,
with a binary vector that contained Fad5-2 cDNA behind a constitutive promoter
(a
tandem cauliflower mosaic virus 35S promoter). Six independent primary
transgenic
plants were obtained and the fatty acid profile of lipids from different
tissues was
determined. Figure 17 shows the fatty acid composition of three-week-old
seedling
plants from one TIJine. Compared with wild type, all transgenic plant tissues
have an
altered fatty acid profile containing four additional peaks which were
identified as four
different A5-undesaturated polymethylene-interrupted fatty acids (A5-UPIFAs),
specifically; taxoleic (18:2-5,9); ephedrenic (18:2-5,11); pinolenic (18:3-
5,9,12), and
coniferonic acids (18:4-5,9,12,15). Thus B. juncea, like yeast, can
functionally express
the P. irregulare A5 desaturase to convert the endogenous substrates 18:1-9;
18:1-11;
18:2-9,12, and 18:3-9,12,15 to the corresponding 05 desaturated fatty acids.
The roots
produced the highest amount of the A5-UPIFAs, representing more than 20% of
the total
fatty acids, followed by 6% in stems and 5% in leaves (Figure 17).
In B. juncea there is no homo-gamma-linolenic acid (20:3-8,11,14)
substrate available. Thus, to examine whether the transgenic plant can produce
AA, the
substrate 20:3(8,11,14) was exogenously supplied. In this experiment, both
wild type
and transgenics Were applied with an aqueouR solution of sodium homo-gamma-
linolenate. It was found that exogenously applied substrates were readily
taken up by
roots, stem, and Ieaves of transgenic plants and converted into AA in plants
(Figure 18).
There was no observable phenptypic effect on the growth and
development in the transgenic B. juncea, although the A5-UPIFAs accumulated in
all
parts of the plant:' Growth and differentiatiori of vegetative tissues such as
the leaves,
stems, and roots were indistinguishable from~Ahe corresponding wild type.
To produce 05 desaturated fatty acids in seeds, B. juncea were
transformed with the construct containing Fad5-2 cDNA behind a heterologous
seed-
specific promoter (B. napus napin promoter). Fatty acid analysis of transgenic
seeds
showed that there were two new fatty acids appearing in the gas chromatogram
of
CA 02608848 2007-07-17
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transgenics compared with the wild type control (Figure 19). They were
identified as
taxoleic acid (18:2-5,9) and pinolenic acids (18:3-5,9,12). Together, these
fatty acids
represent 9.4 % of the seed fatty acids. Acci.unulation of 05-UPIFAs has no
significant
effect on the oleic acid content compared with the untransformed control.
EXAMPLE 11: EXPRESSION OF FAD5-2 IN FLAX
To produce 05 desaturated fatty acids in flax seeds, flax was transformed
with Fad5-2 under the control of two seed-specific promoters, a heterologous
B. napus
napin promoter, and a flax endogenous promoter. As shown in Figure 26,
transgenic
plants containing the napin promoter produced one 05 desaturated fatty acid,
taxoleic
acid in seeds at the level of less than 1% of the total fatty acids. Whereas
transgenic
plants containing the flax seed-specific promoter produced three A5
desaturated fatty
acids: taxoleic, pinolenic, and coniferonic acid. Of these, taxoleic (18:2-
5,9) was the
most abundant and accounted for more than 9% of the total fatty acids in a
elite line
(FN-10-1), followed by coniferonic and pinolenic acids. Surprisingly,
accumulation of
d5 desaturated fatty acids in transgenic seeds has significant impact on the
composition
of other fatty acids, especially the oleic acid level. Accumulation of A5-
UPIFAs was
accompanied by a huge increase of the oleic acids in both types of transgenic
plants
expressing Fad5-2 desaturase under the control of the different promoters. The
content
of oleic acid in transgenic plants with the napin and flax seed-specific
promoters, on the
average, reached 44.7% and 24.3% of the total fatty acids, respectively,
relative to the
untransformed control at '17.4%.
EXAMPLE 12: EXPRESSION OF FAD6 IN FLAX
To produce a6 desaturated fatty acids in flax seeds, two types of flax
were transformed with the construct that contains Fad6 cDNA under the control
of a
heterologous seed-specific promoter (B. napus napin promoter). Type I flax
(Normandy) is a traditional industrial oilseed crop, whereas Type II (Solin)
is an edible
oilseed crop derived from chemical mutagenesis of Type I. A total of twelve
transgenic
plants were produced. The majority of transgenics exhibited two novel fatty
acids
CA 02608848 2007-07-17
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whose retention times correspond to GLA and SDA and they constitute 0.1 to 4.3
% of
the total fatty acids (Figure 27). The level of GLA in transgenic Solin type
is higher
than that of SDA, while GLA in transgenic Normandy is lower than SDA. This is
understandable because linoleic acid is a major fatty acid in Solin type
linseed while a-
linolenic acid is a major fatty acid in Normandy seeds.
EXAMPLE 13: EXPRESSION OF FAD6 IN B. JUNCEA
To produce A6 desaturated fatty acids in seeds of B. juncea, B. juncea
were transformed with the same construct used in flax transformation, i.e.,
Fad6 under
the control of the B. turpus napin promoter. Fifteen,independent transgenic
plants were
obtained. Fatty acid analysis of the transgenic seeds showed that there were
three new
fatty acids in the gas chromatogram of most transgenics compared with the wild
type
control (Figure 20). The three fatty acids were identified as 18:2(6,9) and
18:3(6,9,12),
and 18:4(6,9,12,15). B. juncea, like flax, can also functionally express Fad6
from P.
irregulare, introducing a double bond at position 6 of endogenous substrate
18:1(9),
18:2.(9,12), and 18:3(6,9,12) resulting in production of three corresponding
d6 fatty
acids, in the transgenic seeds. Among the three new fatty acids produced in
transgenic
seeds, GLA is the most abundant one, with a level in transgenic seeds of 30%
to 38% of
the total fatty acids. The next most abundant componerit is SDA, which
accounts for 3-
10 % of the total fatty acids in several transgenic lines (Figure 21).
The fatty acid compositions of transgenic seeds are shown in Figure 22.
It is clear that the high level production of A6 desaturated fatty acids is at
the cost of two
major fatty acids, linoleic and linolenic acids. Proportions of oleic and
stearic acids in
transgenics are slightly reduced, but not significantly compared to those in
the wild type
control. The content of linoleic acid in the transgenics was dramatically
reduced. In the
untransformed wild type, linoleic acid accounts for more than 40% of the total
fatty
acids in seeds. In transgenics, the level was reduced to less than 10%.
As compared to the reduction of linoleic acid in transgenics, the decrease in
linolenic
acid in transgenics is less dramatic, but still significant. In the
untransformed wild type,
linolenic acid accounts for more than 10% of the total fatty acids in seeds
while in
transgenics the level was reduced to less than 5%. The two dramatically
reduced fatty
CA 02608848 2007-07-17
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acids in transgenic seeds are the substrates of the A6 desaturase, and the
reduction is the
cost for producing two corresponding A6 desaturated fatty acids.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
embodiments of the
invention described herein. Such equivalents are intended to be encompassed by
the
following claims.
CA 02608848 2007-07-17
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CECI EST LE TOME DE _2
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