U.S. patent application number 15/173799 was filed with the patent office on 2016-12-22 for fad4, fad5, fad5-2, and fad6, novel fatty acid desaturase family members and uses thereof.
The applicant listed for this patent is Bioriginal Food & Science Corp. Invention is credited to Haiping HONG, XIAO QIU.
Application Number | 20160369289 15/173799 |
Document ID | / |
Family ID | 26929655 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160369289 |
Kind Code |
A1 |
QIU; XIAO ; et al. |
December 22, 2016 |
FAD4, FAD5, FAD5-2, AND FAD6, NOVEL FATTY ACID DESATURASE FAMILY
MEMBERS AND USES THEREOF
Abstract
The invention provides isolated nucleic acid molecules which
encode novel fatty acid desaturase family members. The invention
also provides recombinant expression vectors containing desaturase
nucleic acid molecules, host cells into which the expression
vectors have been introduced, and methods for large-scale
production of long chain polyunsaturated fatty acids (LCPUFAs),
e.g., DHA.
Inventors: |
QIU; XIAO; (Saskatoon,
CA) ; HONG; Haiping; (Morrisville, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bioriginal Food & Science Corp |
Saskatoon |
|
CA |
|
|
Family ID: |
26929655 |
Appl. No.: |
15/173799 |
Filed: |
June 6, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13339428 |
Dec 29, 2011 |
9359597 |
|
|
15173799 |
|
|
|
|
13150656 |
Jun 1, 2011 |
8088906 |
|
|
13339428 |
|
|
|
|
12538227 |
Aug 10, 2009 |
7977469 |
|
|
13150656 |
|
|
|
|
11342731 |
Jan 30, 2006 |
7671252 |
|
|
12538227 |
|
|
|
|
09967477 |
Sep 28, 2001 |
7087432 |
|
|
11342731 |
|
|
|
|
60236303 |
Sep 28, 2000 |
|
|
|
60297562 |
Jun 12, 2001 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/04 20180101;
C12Y 114/19003 20130101; A61P 9/10 20180101; A61P 9/12 20180101;
C12N 9/0071 20130101; Y02P 20/52 20151101; A61P 3/02 20180101; A61K
38/00 20130101; A61P 9/00 20180101; A61P 43/00 20180101; A23V
2002/00 20130101; C12N 15/8247 20130101; A61P 29/00 20180101; A61P
11/00 20180101; A61P 7/02 20180101; A61P 1/16 20180101; A61P 35/00
20180101; A61P 27/02 20180101; A61K 31/202 20130101; C12P 7/6472
20130101; A61P 19/08 20180101; A61P 19/02 20180101; A61P 7/00
20180101; A61P 25/18 20180101; A23K 20/158 20160501; A23L 33/12
20160801; A61P 25/24 20180101; A61P 25/28 20180101; A61P 3/10
20180101; C12P 7/6427 20130101; A61P 35/02 20180101; C12N 9/0083
20130101; A61P 35/04 20180101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A23K 20/158 20060101 A23K020/158; C12P 7/64 20060101
C12P007/64; A23L 33/12 20060101 A23L033/12; A61K 31/202 20060101
A61K031/202; C12N 9/02 20060101 C12N009/02 |
Claims
1. (canceled)
2. A method for producing high levels of gamma-linolenic acid (GLA)
in a plant, comprising: a) selecting a first plant line having high
content of linoleic acid (LA) relative to a second plant line of
the same plant species; and b) providing at least one .DELTA.6
desaturase in said first plant line, wherein the level of GLA
produced in said first plant line is higher than the level of GLA
produced in the second plant line expressing the .DELTA.6
desaturase.
3. The method of claim 2, wherein said at least one .DELTA.6
desaturase comprises a cytochrome b5-like domain
4. The method of claim 3, wherein said at least one .DELTA.6
desaturase further comprises at least two histidine motifs.
5. The method of claim 3, wherein said at least one .DELTA.6
desaturase comprises three histidine motifs.
6. The method of claim 2, wherein said at least one .DELTA.6
desaturase has at least 70% sequence identity to the amino acid
sequence of SEQ ID NO: 8.
7. The method of claim 2, wherein said at least one .DELTA.6
desaturase comprises: a) the amino acid sequence of SEQ ID NO: 8;
or b) an amino acid sequence encoded by the nucleotide sequence of
SEQ ID NO: 7.
8. The method of claim 2, wherein the plant species is a Brassica
species.
9. The method of claim 2, wherein the plant species is a Brassica
species, and wherein the first plant line produces GLA at a level
of 30% to 38% of the total fatty acids in seeds.
10. The method of claim 9, wherein the first plant line further
produces stearidonic acid (SDA) at a level of 3% to 10% of the
total fatty acids in seeds.
11. The method of claim 2, wherein the plant species is a Brassica
species, and wherein the level of LA in the first plant line is
reduced to less than 10%.
12. The method of claim 2, wherein the plant species is a Brassica
species, and wherein the level of linolenic acid in the first plant
line is reduced to 5%.
13. The method of claim 2, wherein the plant species is a Brassica
species, and wherein the level of LA in the first plant line is
reduced to less than 10% and the level of linolenic acid in the
first plant line is reduced to 5%.
14. A method for the production of unsaturated fatty acids,
comprising: a) producing high levels of GLA in a plant according to
the method of claim 2; and b) isolating unsaturated fatty acids
from said plant.
15. The method of claim 14, wherein said unsaturated fatty acids
are isolated from seeds of said plant.
16. The method of claim 14, wherein said unsaturated fatty acids
are isolated in form of lipids.
17. A composition comprising the unsaturated fatty acids produced
in claim 14.
18. A dietary supplement, animal feed, a neutraceutical, or a
pharmaceutical composition comprising the unsaturated fatty acids
produced in claim 14.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/339,428, filed Dec. 29, 2011, now U.S. Pat. No. 9,359,597,
which is a divisional of U.S. application Ser. No. 13/150,656,
filed Jun. 1, 2011, now U.S. Pat. No. 8,088,906, which is a
divisional of U.S. application Ser. No. 12/538,227, filed Aug. 10,
2009, now U.S. Pat. No. 7,977,469, which is a divisional of U.S.
application Ser. No. 11/342,731, filed Jan. 30, 2006, now U.S. Pat.
No. 7,671,252, which is a divisional of U.S. application Ser. No.
09/967,477, filed Sep. 28, 2001, now U.S. Pat. No. 7,087,432, which
claims priority to U.S. Provisional Application No. 60/236,303
filed on Sep. 28, 2000 and U.S. Provisional Application No.
60/297,562 filed on Jun. 12, 2001. The entire contents of each of
these applications are hereby incorporated by reference herein in
their entirety. The entire contents of all references cited therein
also are expressly incorporated by reference and are intended to be
part of the present application.
Submission of Sequence Listing
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is
Sequence_Listing_074008_1323_01. The size of the text file is 40
KB, and the text file was created on Jun. 6, 2016.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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 terminal desaturases that belong to the class
of nonheme-iron enzymes. Each of these enzymes are part of a
electron-transport system that contains two other proteins, namely
cytochrome b.sub.5 and NADH-cytochrome b.sub.5 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 (C18:2); linolenic acid (C18:3), arachidonic acid
(C20:4). In contrast, insects and plants are able to synthesize a
much larger variety of unsaturated fatty acids and their
derivatives.
[0005] 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 mammals (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., 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:S129-S138). 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, arthritis, 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).
[0006] 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
.DELTA.6 desaturation to 18:4 (6, 9, 12, 15), followed by
elongation to 20:4 (8, 11, 14, 17) and .DELTA.5 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
.DELTA.4 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
.DELTA.4 desaturase, involving two consecutive elongations, a
.DELTA.6 desaturation and a two-carbon shortening via limited
n-oxidation in peroxisome (Sprecher, H., et al. (1995) J. Lipid
Res. 36:2471-2477; Sprecher, H., et al. (1999) Lipids
34:S153-S156).
[0007] 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.
[0008] EPA and AA are both .DELTA.5 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 oily
fish from North Atlantic. AA belongs to the n-6 series with four
double bonds. The lack of a double bond in the .omega.-3 position
confers on AA different properties than those found in EPA. The
eicosanoids produced from AA have strong inflammatory and 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 is low.
[0009] 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 .DELTA.6 desaturation. Many physiological and pathological
conditions such as aging, stress, diabetes, eczema, and some
infections have been shown to depress the .DELTA.6 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.
[0010] 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 Cyanobacteria. However, these GLA
sources are not ideal for dietary supplementation due to large
fluctuations in availability and costs associated with extraction
processes.
SUMMARY OF THE INVENTION
[0011] The biosynthesis of fatty acids is a major activity of
plants and microorganisms. However, humans have a limited capacity
for synthesizing essential fatty acids, e.g., long chain
polyunsaturated 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 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 garner the maximum therapeutic value
from these fatty acids.
[0012] The present invention is based, at least in part, on the
discovery of a family of nucleic acid molecules encoding novel
desaturases. In particular, the present inventors have identified
the Fad 4 (.DELTA.4 desaturase), Fad5 and Fad5-2 (.DELTA.5
desaturase), and Fad6 (.DELTA.6 desaturase) which are involved in
the biosynthesis of long chain 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).
[0013] In one embodiment, the invention features an isolated
nucleic acid molecule that includes the nucleotide sequence set
forth in SEQ ID NO:1, 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.
[0014] 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. Also 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.
[0015] 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).
[0016] 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 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-length
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.
[0017] 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 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.
[0018] The constructs containing the desaturase genes can be used
in any expression 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 (Helianthus sp.), corron (Gossypium sp.), corn (Zea
mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa
(Theobroma cacoa), and peanut (Arachis sp.).
[0019] In a related aspect, the present invention provides new and
improved methods of producing unsaturated fatty acids, e.g.,
LCPUFAs, and other key compounds 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.
[0020] 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
fatty acid 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 further comprise the step of recovering
the LCPUFA.
[0021] 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
desaturase 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.
[0022] 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,
and/or Fad6, under conditions such that fatty acid production is
enhanced.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIGS. 1A and 1B show the DNA and protein sequence of Fad4
from Thraustochytrium sp.; FIG. 1A shows the cDNA sequence of the
open reading frame (SEQ ID NO:1); and FIG. 1B shows the translated
protein sequence (SEQ ID NO:2).
[0029] FIGS. 2A and 2B show the DNA and protein sequence of Fad5
from Thraustochytrium sp.; FIG. 2A shows the cDNA sequence of the
open reading frame (SEQ ID NO:3); and FIG. 2B shows the translated
protein sequence (SEQ ID NO:4).
[0030] FIG. 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.
[0031] FIGS. 4A and 4B show the DNA and protein sequence of Fad5-2
from Pythium irregulare; FIG. 4A shows the cDNA sequence of the
open reading frame (SEQ ID NO:5); and FIG. 4B shows the translated
protein sequence (SEQ ID NO:6).
[0032] FIGS. 5A and 5B show the DNA and protein sequence of Fad6 of
Pythium irregulare; FIG. 5A shows the cDNA sequence of the open
reading frame (SEQ ID NO:7); and FIG. 5B shows the translated
protein sequence (SEQ ID NO:8).
[0033] FIG. 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.
[0034] FIG. 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).
[0035] FIGS. 8A and 8B are gas chromatographic/mass spectroscopy
(MS) analysis of FAMEs of the new peak in FIG. 7; FIG. 8A shows the
Fad4 product; FIG. 8B shows the DHA (22:6, n-3) standard.
[0036] FIG. 9 is a GC analysis of FAMEs from yeast strain Invsc2
expressing Fad4 with exogenous substrate 22:4 (n-6).
[0037] FIGS. 10A and 10B are GC/MS analysis FAMEs of the new peak
in FIG. 9; FIG. 10A shows the Fad4 product; FIG. 10B shows the DPA
(22:5, n-6) standard.
[0038] FIG. 11 is a GC analysis of FAMEs from yeast strain Invsc2
expressing Fad5 with exogenous substrate 20:3 (n-6).
[0039] FIGS. 12A and 12B are GC/MS analysis of FAMES of the new
peak in FIG. 11; FIG. 12A shows the Fad5 product; FIG. 12B shows
the AA (20:4-5, 8, 11, 14) standard.
[0040] FIG. 13 is a GC analysis of FAMES from yeast strain
AMY2.alpha. expressing Fad5-2 with exogenous substrate 18:1-9 (the
upper panes) and 18:1-11 (the lower panel), respectively.
[0041] FIG. 14 is a GC analysis of FAMEs from yeast strain Invsc2
expressing Fad6 with exogenous substrate 18:2 (9, 12).
[0042] FIG. 15 is a MS analysis of the derivative of the new peak
from FIG. 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 .DELTA.6, .DELTA.9,
and .DELTA.12 position, respectively.
[0043] FIG. 16 is a GC analysis of FAMEs from leaves of Brassica
juncea expressing Fad4 under the control of 35S promoter with
exogenously supplied substrate 22:5 (n-3).
[0044] FIG. 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.
juncea.
[0045] FIG. 18 is a GC analysis of root FAMEs of B. juncea
expressing Fad5-2 with exogenous substrate homo-.gamma.-linolenic
acid (HGLA, 20:3-8, 11, 14).
[0046] FIG. 19 is a GC analysis of FAMEs prepared from seeds of B.
juncea expressing Fad5-2 under the control of the napin
promoter.
[0047] FIG. 20 is a GC analysis of seed FAMEs from B. juncea
expressing Fad6. Three new peaks indicate three .DELTA.6
desaturated fatty acids in transgenic seeds.
[0048] FIG. 21 shows the weight percentage of GLA
(.gamma.-linolenic acid) and SDA (stearidonic acid) accumulating in
Fad6 transgenic seeds of B. juncea.
[0049] FIG. 22 shows the fatty acid compositions of the seed lipids
from five transgenic lines expressing Fad6; SA=stearic acid;
OA=oleic acid; LA=linoleic acid; GLA=.gamma.-linolenic acid;
ALA=.alpha.-linolenic acid; SDA=stearidonic acid.
[0050] FIG. 23 is a table showing the fatty acid profile of
Thraustochytrium sp.
[0051] FIG. 24 is a table showing the fatty acid profile of Pythium
irregulare.
[0052] FIG. 25 is a table showing the conversion of exogneous fatty
acids in yeast AMY-2.alpha./pFad5-2.
[0053] FIG. 26 is a table showing the accumulation of
.DELTA.5-unsaturated polymethylene-interrupted fatty acids
(.DELTA.5-UPIFAs) in transgenic flaxseeds expressing 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.
[0054] FIG. 27 is a table showing the accumulation of .DELTA.6
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 fatty acids.
DETAILED DESCRIPTION OF THE INVENTION
[0055] 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.
[0056] 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.dbd.CH--CH.sub.2--CH.dbd.CH--).
[0057] 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
18:1(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.
[0058] 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 formation of double bonds between the carbon atoms of
a fatty acid molecule. As used herein, the term "unsaturated fatty
acid biosynthetic pathway" refers 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).
[0059] 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
normal brain function in adults (Martinetz, M. (1992) J. Pediatr.
120:S129-S138). DHA also has 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, positive effects of DHA were
also found in the treatment 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.
[0060] 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.
[0061] 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 motif" is an N-terminal
extension of the cytochrome b5-like domain found in front-end
desaturases.
[0062] 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
"histidine 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.
[0063] Examples of cytochrome b5 heme-binding motifs and histidine
motifs include amino acid residues 41-44, 182-186, 216-223, 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 FIGS. 3 and 6.
[0064] 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:1, 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 homologous.
[0065] 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 homologous or identical
to the 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.
[0066] 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.
[0067] The nucleotide sequence of the isolated Thraustochytrium sp.
.DELTA.4 desaturase, Fad4, cDNA and the predicted amino acid
sequence encoded by the Fad4 cDNA are shown in FIGS. 1A and 1B and
in SEQ ID NOs:1 and 2, respectively. The Thraustochytrium 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.
[0068] The nucleotide sequence of the Thraustochytrium sp. .DELTA.5
desaturase, Fad5, cDNA and the predicted amino acid sequence
encoded by the Fad5 cDNA are shown in FIGS. 2A and 2B 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.
[0069] The nucleotide sequence of the Pythium irregulare .DELTA.5
desaturase, Fad5-2, cDNA and the predicted amino acid sequence
encoded by the Fad5-2 cDNA are shown in FIGS. 4A and 4B 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.
[0070] The nucleotide sequence of the Pythium irregulare .DELTA.6
desaturase, Fad6, cDNA and the predicted amino acid sequence
encoded by the Fad6 cDNA are shown in FIGS. 5A and 5B and in SEQ ID
NOs:7 and 8, respectively. The Pythium irregulare Fad6 gene, which
is approximately 1383 nucleotides in length, encodes a protein
having approximately 460 amino acid residues in length.
[0071] Various aspects of the invention are described in further
detail in the following subsections:
I. Isolated Nucleic Acid Molecules
[0072] 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 amplification or
mutation of desaturase nucleic acid molecules. As used herein, the
term "nucleic acid molecule" is intended to include DNA molecules
(e.g., cDNA or genomic 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 double-stranded,
but preferably is double-stranded DNA.
[0073] The term "isolated nucleic acid molecule" includes nucleic
acid molecules 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 naturally associated.
Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank the nucleic acid (i.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 can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1
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
cDNA 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.
[0074] 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 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
cloning techniques (e.g., as described in Sambrook, J. et al.
Molecular Cloning: A Laboratory Manual. 2.sup.nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989).
[0075] 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, or
7.
[0076] 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.
[0077] In another embodiment, the nucleic acid molecule consists of
the nucleotide sequence set forth as SEQ ID NO: 1, 3, 5, or 7.
[0078] 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 sequence 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.
[0079] 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%, 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.
[0080] 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 primer 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 cloning of 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.
[0081] 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 1, 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, 9
or 10 bases when compared to a sequence disclosed herein or to the
sequence of a naturally occurring variant. Such probes can be used
as a part of a diagnostic test kit for identifying 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.
[0082] 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).
[0083] 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: 1, 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 amino acid sequence of human desaturase. If an
alignment is needed for this comparison, the sequences should be
aligned for maximum homology.
[0084] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus), homologues (different locus), and
orthologues (different organism) or can be non naturally occurring.
Non-naturally 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).
[0085] 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.
[0086] 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 SEQ 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.
[0087] 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., 22:5(n-3)); 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.
[0088] Non-functional allelic variants are naturally occurring
amino acid sequence variants of the desaturase protein, e.g., Fad4,
Fad5, Fad5-2, or Fad6, that do not have 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.
[0089] The present invention further provides orthologues (e.g.,
human orthologues of the desaturase proteins). Orthologues of the
Thraustochytrium sp. and Pythium 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 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.
[0090] Moreover, nucleic acid molecules 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
which, 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.
[0091] Nucleic acid molecules corresponding to natural allelic
variants and homologues of the desaturase cDNAs 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
hybridization techniques under stringent hybridization
conditions.
[0092] 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, 3250-3500,
3500-3750 or more nucleotides in length.
[0093] 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 al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7,
9, and 11. A preferred, non-limiting example of stringent
hybridization conditions includes hybridization in 4.times. sodium
chloride/sodium citrate (SSC), at about 65-70.degree. C. (or
alternatively hybridization in 4.times.SSC plus 50% formamide at
about 42-50.degree. C.) followed by one or more washes in
1.times.SSC, at about 65-70.degree. C. A preferred, non-limiting
example of highly stringent hybridization conditions includes
hybridization in 1.times.SSC, at about 65-70.degree. C. (or
alternatively hybridization in 1.times.SSC plus 50% formamide at
about 42-50.degree. C.) followed by one or more washes in
0.3.times.SSC, at about 65-70.degree. C. A preferred, non-limiting
example of reduced stringency hybridization conditions includes
hybridization in 4.times.SSC, at about 50-60.degree. C. (or
alternatively hybridization in 6.times.SSC plus 50% formamide at
about 40-45.degree. C.) followed by one or more washes in
2.times.SSC, at about 50-60.degree. C. Ranges intermediate to the
above-recited values, e.g., at 65-70.degree. C. or at 42-50.degree.
C. are also intended to be encompassed by the present invention.
SSPE (1.times.SSPE is 0.15M NaCl, 10 mM NaH.sub.2PO.sub.4, and 1.25
mM EDTA, pH 7.4) can be substituted for SSC (1.times.SSC is 0.15M
NaCl and 15 mM 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.degree. C. less than the melting temperature MO of the
hybrid, where T.sub.m is determined according to the following
equations. For hybrids less than 18 base pairs in length,
T.sub.m(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For
hybrids between 18 and 49 base pairs in length, T.sub.m(.degree.
C.)=81.5+16.6(log.sub.10[Na.sup.+]) 0.41(% G+C)-(600/N), where N is
the number of bases in the hybrid, and [Na.sup.+] is the
concentration of sodium ions in the hybridization buffer
([Na.sup.+] for 1.times.SSC=0.165 M). It will also be recognized by
the skilled practitioner that additional reagents may be added to
hybridization and/or wash buffers to decrease non-specific
hybridization of nucleic acid molecules 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, PVP
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
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.
(see e.g., Church and Gilbert (1984)Proc. Natl. Acad. Sci. USA
81:1991-1995), or alternatively 0.2.times.SSC, 1% SDS.
[0094] Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:1, 3, 5, or 7 corresponds to a
naturally-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).
[0095] 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 unamenable 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 amenable to
alteration.
[0096] 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, 5, 8, or 11.
[0097] 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 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, 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 side 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.
[0098] 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
[0099] 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.
[0100] 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 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 preparations of
desaturase protein having less than about 80%, 70%, 60%, 50%, 40%,
or 30% (by dry weight) of non-desaturase protein (also referred to
herein as a "contaminating protein"), more preferably less than
about 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 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.
[0101] 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-desaturase 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.
[0102] 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-desaturase 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.
[0103] In one embodiment, a biologically active portion of a
desaturase protein comprises a heme-binding motif and/or at least
one histidine 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.
[0104] 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.
[0105] 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 molecule consisting of 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, or a complement
thereof.
[0106] 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 NO:2 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 amino 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 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.
[0107] 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, 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, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package, 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.
[0108] 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 2.0U), using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4.
[0109] 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 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.
III. Methods of Producing Unsaturated Fatty Acids
[0110] 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
[0111] 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
sequence(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 contents of which are incorporated herein
by reference.
[0112] 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 Fad6 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
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.
[0113] Preferred regulatory sequences include promoters, enhancers,
termination signals and other expression control elements (e.g.,
binding sites for transcriptional and/or translational 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, N.Y., 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.
[0114] 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.
[0115] 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.
[0116] 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 of the host organism). For
example, amyE sequences can be used as homology targets for
recombination into the host chromosome.
[0117] 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.
[0118] 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 manipulation of the cell
or in a comparable cell 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 not been manipulated. Genetic
manipulation can include, but is not limited to, altering or
modifying regulatory sequences or sites associated with expression
of a particular gene (e.g., by adding strong promoters, inducible
promoters or multiple promoters or by removing regulatory sequences
such that expression is constitutive), modifying the chromosomal
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 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).
[0119] In another embodiment, the cell can be physically or
environmentally manipulated to overexpress a level of gene product
greater than that expressed prior to manipulation of the cell 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.
[0120] 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 a biosynthetic pathway in which more than one gene
that encodes an enzyme in a biosynthetic pathway is altered 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".
[0121] 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 and/or 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 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., regulatory 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 antisense 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.
[0122] 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 naturally found in a microorganism 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 ID
NO:5, or SEQ ID NO:7).
[0123] The methodologies of the present invention feature
recombinant cells which 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 least one of Thrauschytrium sp. .DELTA.4 or
.DELTA.5 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 encoded by the nucleic acid sequence of SEQ ID NO:1 or 3).
[0124] In another embodiment, a recombinant cell of the present
invention has been genetically engineered to overexpress a Pythium
irregulare .DELTA.5 or .DELTA.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).
[0125] In another embodiment, the invention features a cell (e.g.,
a microbial cell) that has been transformed with a vector
comprising 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).
[0126] Another aspect of the present invention features a method of
modulating the production of fatty acids comprising culturing cells
transformed by the nucleic acid molecules of the present invention
(e.g., a desaturase) such that modulation of fatty acid 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 modulate 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, optionally,
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.
[0127] 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 necessary
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),
acetone-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.
[0128] An important aspect of the present invention involves
growing the recombinant plant or culturing 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 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 the
invention is cultured in media (e.g., a sterile, liquid media)
comprising nutrients essential or beneficial to the maintenance
and/or growth of 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 potassium 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).
[0129] Preferably, microorganisms of the present invention are
cultured under controlled pH. The term "controlled pH" includes any
pH which results in production of the desired product (e.g., an
unsaturated fatty acid). In one embodiment, microorganisms 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.
[0130] Also preferably, microorganisms of the present invention are
cultured under controlled aeration. The term "controlled 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). Also preferably, microorganisms of the
present invention are cultured without excess foaming (e.g., via
addition of antifoaming agents).
[0131] 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.degree. C. and 95.degree. C. In another embodiment, controlled
temperatures include temperatures between 15.degree. C. and
70.degree. C. Preferred temperatures are between 20.degree. C. and
55.degree. C., more preferably between 30.degree. C. and 45.degree.
C. or between 30.degree. C. and 50.degree. C.
[0132] 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.
[0133] 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
time sufficient 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
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 acids are 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.
[0134] In producing unsaturated fatty acids, it may further 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.
[0135] 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 resin (e.g., anion or cation exchange resin, non-ionic
adsorption resin, etc.), treatment with a 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, concentration, 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 exchange 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).
[0136] 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 compound
is an unsaturated fatty acid that has been derivatized to a salt,
the compound is preferably further 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 an alcohol, the compound is preferably further
free (e.g., substantially free) of chemical contaminants associated
with the formation of the alcohol.
[0137] 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
supernatant) can be used as a source of product (e.g., crude
product). In one embodiment, the plant or culture (or culture
supernatant) supernatant is used without modification. In another
embodiment, the plant or culture (or culture supernatant) is
concentrated. In yet another embodiment, the plant or culture (or
culture supernatant) is pulverized, dried, or lyophilized.
B. High Yield Production Methodologies
[0138] 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 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 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 10 g/L. 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 manipulated 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 g/L.
[0139] 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 microorganism under conditions such that a sufficiently elevated
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/L in 36 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 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 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 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
[0140] 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 of the present invention include, e.g.,
compositions for use as animal feed, compositions for use as
neutraceuticals (e.g., dietary supplements), and pharmaceutical
compositions suitable for administration. Such pharmaceutical
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.
[0141] 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
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity 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
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0142] 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 EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition 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 the case of dispersion
and by the use of 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.
[0143] 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 enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0148] 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 acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0149] 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.
[0150] Toxicity and therapeutic efficacy of such compounds can be
determined 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.
[0151] The data obtained from the cell culture assays and animal
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 within 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-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0152] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg 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 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 subject, 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.
[0153] 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 1 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 will 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.
[0154] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0155] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the figures, are
incorporated herein by reference.
EXAMPLES
Materials
[0156] Thraustochytrium s.p ATCC 21685 and Pythium irregulare were
purchased from American type culture collection (12301 Parklawn
Drive, Rockville, Md., 20852 USA) and grown in a medium (Weete, J.
D., et al. (1997) Lipids 32:839-845) at 24.degree. C. for 7 days.
After then biomass were harvested by centrifugation and used for
RNA isolation.
Example 1
Construction and Screening of cDNA Library
[0157] 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 II
reverse transcriptase from Gibco-BRL. The second strand cDNA was
synthesized by DNA polymerase I from Stratagene. After size
fractionation, cDNA inserts larger than 1 kb were ligated into
.lamda. Uni-Zap XR vector (Stratagene). The recombinant DNAs were
then packed with Gigapack III Gold packaging extract (Stratagene)
and plated on NZY plates. The resulting library represented more
than 5.times.106 independent clones. Screening of the cDNA library
was performed according to standard methods (Sambrook, J, Fritseh,
E. F., Maniatis, T. (1989) Molecular cloning--A laboratory manual.
(Cold Spring Harbor, N.Y., USA.)
Example 2
RT-PCR
[0158] The single strand cDNA was synthesized by superscript II
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/TCCNGGXGG (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.degree.
C., 1.5 min at 55.degree. C. and 2 min at 72.degree. C. followed by
an extension step at 72.degree. 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.RTM. 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
[0159] The open reading frames of Fad4, Fad5, Fad5-2, and Fad6 were
amplified by PCR using the Precision Plus enzyme (Stratagene) and
cloned into a TA cloning vector (pCR.RTM.2.1, Invitrogen). Having
confirmed that the PCR products were identical to the original
cDNAs by sequencing, the 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 GAL1.
[0160] Yeast strains InvSc2 (Invitrogen) was transformed with the
expression constructs using the lithium acetate method and
transformants were selected on minimal medium plates lacking uracil
(Gietz, D., et al. (1992) Nucleic Acids Res. 20:1425; Covello, P.
S. and Reed, D. W. (1996) Plant Physiol. 111:223-226).
[0161] Transformants were first grown in minimal medium lacking
uracil and containing glucose at 28.degree. 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 with the yeast transformant cell
suspension and incubated at 20.degree. C. for three days, and then
15.degree. C. for another three days.
Example 4
Fatty Acid Analysis
[0162] Thraustochytrium, Pythium 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.degree. 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 HCl 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.degree. C. for 2 hours. After cooling to room temperature, 1 mL
0.9% NaCl was added and the mixture extracted twice with 2.times.2
mL hexane. 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).
[0163] GC/MS analysis was performed in standard EI mode using a
Fisons VG 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.times.0.25 mm i.d.,
0.25 .mu.m film thickness, J&W Scientific, Folsom, Calif.) that
was temperature-programmed at 180.degree. C. for 1 min, then 4
C/min to 240.degree. 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
[0164] The hypocotyls of 5-6 day seedlings of B. juncea and flax
were used as explants for inoculation with the Agrobacterium
tumefaciens that hosts binary vectors with the full-length cDNAs
under the control of the different promoters. 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 cut 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, Minn.) was added. The plate was then incubated
at 24.degree. C. for 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 Thrauschytrium Sp
[0165] Thraustochytrium and Pythium irregulare have recently drawn
scientific attention due to its ability in production of LCPUFAs
such as DHA, AA, EPA and DPA. FIGS. 23 and 24 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 .DELTA.4 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 and 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.
326:425-430). The 24-carbon fatty acid may not be involved in in
vivo synthesis of 22-carbon .DELTA.4 fatty acids such as DHA and
DPA in Thraustochytrium sp.
Example 7
Identification of cDNAs Coding for the "Front-End" Desaturase
[0166] To identify genes coding for desaturases involved in
biosynthesis of LCFUFAs in Thraustochytrium sp. and Pythium
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
Thraustochytrium sp. and Pythium irregulare, should have similar
primary structure as other front-end desaturases, i.e. N-terminal
extension of cyt b5-like domain in the desaturase. Four cDNAs
fragments were identified from Thraustochytrium sp. and Pythium
irregulare that encode fusion proteins containing cyt b5-like
domain in the N-terminus.
[0167] To isolate full-length cDNA clones, the four inserts were
used as probes to screen cDNA libraries of Thraustochytrium 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 (FIGS.
1A and 1B). Fad5 is 1230 bp in length and codes for 439 amino acids
with molecular weight of 49.8 kDa (FIGS. 2A and 2B). 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 (FIG. 3). The open reading frame of Fad5-2 from Pythium
irregulare is 1371 bp and codes for 456 amino acids (FIGS. 4A and
4B). Fad6 from Pythium irregulare is 1383 bp in length and codes
for 460 amino acids (FIGS. 5A and 5B). Sequence comparison of the
two sequences from Pythium irregulare showed over 39% similarity
between the deduced proteins (FIG. 6).
[0168] A BLASTP.TM. search of the protein database revealed the
following hits for each of the four proteins, Fad4, Fad5, Fad5-2,
and Fad6:
TABLE-US-00001 Fad 4 (519 amino acid residues) Blastp nr Accession
No. Organism Description Length % Identity AF067654 Mortierella
.DELTA.5 fatty acid 509 29 alpina desaturase AF054824 Mortierella
.DELTA.5 microsomal 509 28 alpina desaturase AB022097 Dictyostelium
.DELTA.5 fatty acid 507 27 discoideum desaturase AB029311
Dictyostelium fatty acid 519 26 discoideum desaturase L11421
Synechocystis sp. .DELTA.6 desaturase 410 25 D90914
TABLE-US-00002 Fad 5 (439 amino acid residues) Blastp nr Accession
No. Organism Description Length % Identity AF139720 Euglena
gracilis .DELTA.8 fatty acid 404 29 desaturase AF007561 Borago
officinalis .DELTA.6 desaturase 421 27 U79010 Borago officinalis
.DELTA.6 desaturase 421 27 AF309556 Danio rerio .DELTA.6 fatty acid
422 26 desaturase AF110510 Mortierella .DELTA.6 fatty acid 463 25
alpina desaturase
TABLE-US-00003 Fad 5-2 (456 amino acid residues) Blastp nr
Accession No. Organism Description Length % Identity AB029311
Dictostelium Fatty acid 443 41 discoideum desaturase AB022097
Dictostelium .DELTA.5 fatty acid 445 39 discoideum desaturase
AF067654 Mortierella .DELTA.5 fatty acid 441 38 alpina desaturase
AF054824 Mortierella .DELTA.5 microsomal 441 38 alpina desaturase
L11421 Synechocystis sp. .DELTA.6 desaturase 361 28 D90914
TABLE-US-00004 Fad 6 (459 amino acid residues) Blastp nr Accession
No. Organism Description Length % Identity AF110510 Mortierella
.DELTA.6 fatty acid 437 38 alpina desaturase AB020032 Mortierella
.DELTA.6 fatty acid 437 38 alpina desaturase AF306634 Mortierella
.DELTA.6 fatty acid 437 38 isabellina desaturase AF307940
Mortierella .DELTA.6 fatty acid 438 38 alpina desaturase AJ250735
Ceratodon .DELTA.6 fatty acid 438 36 purpureus desaturase
Example 8
Expression of Fad4, Fad5, Fad5-2, and Fad6 in Yeast
[0169] To confirm the function of Fad4, the full-length cDNA was
expressed in the yeast strain InvSc2 under the control of the
inducible promoter. FIG. 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 DHA 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 confirmed
that the spectrum of the peak is identical to that of the DHA
standard (FIGS. 8A and 8B). These results indicate that Fad4 is a
.DELTA.4 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 .DELTA.4 desaturated fatty acid, DHA (22:6-4, 7, 10,
13, 16, 19).
[0170] To further study the substrate 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 (FIG. 9) to produce another
.DELTA.4 desaturated fatty acid, DPA (22:5-4, 7, 10, 13, 16) (FIGS.
10A and 10B). The rest of the fatty acids examined were not
effective substrates.
[0171] To confirm the function of Fad5 and Fad5-2, the S.
cerevisiae Invsc2 was transformed with plasmids, which contain the
open reading frame 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 (FIG. 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 confirm the
regiochemistry of the products, the FAMEs were analyzed by GC/MS.
FIGS. 12A and 12B indicate 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.
[0172] To further study the substrate specificity of Fad5-2, the
plasmid containing Fad5-2 was transferred into another yeast strain
AMY-2a where ole1, a .DELTA.9 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(11Z,14Z) and 20:3(11Z,14Z,17Z) were tested.
Results indicated that Fad5-2 could desaturate unsaturated fatty
acids with .DELTA.9 ethylenic and .DELTA.11 ethylenic double bonds,
as well as the fatty acid with .DELTA.8 ethylenic double bond
(20:3-8, 11, 14). As shown in FIG. 13, Fad5-2 effectively converted
both 18:1(9Z) and 18:1(11Z) substrates into their corresponding
.DELTA.5 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).
[0173] FIG. 25 is a comparison of substrate preference of Fad5-2
for fatty acid substrates tested in the yeast strain AMY-2.alpha..
The relative proportions of the substrates and the products
accumulated are a useful indicator of substrate preference of the
enzyme. As shown in FIG. 25, Fad5-2 prefers fatty acids with
20-carbon as substrates, such as 20:3(8Z,11Z,14Z),
20:3(11Z,14Z,17Z) and 20:2(11Z,14Z). Whereas, the shorter chain
fatty acid is a relatively weaker substrate for the enzyme in
yeast.
[0174] 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, GAL1. 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 (FIG. 14). A comparison of
the chromatogram with that of the standards revealed that the peak
had a retention time identical to the gamma-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. FIG. 15 shows that the
new peak is indeed GLA with three double bonds at the .DELTA.6,
.DELTA.9, and .DELTA.12 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 .DELTA.6,
.DELTA.9, and .DELTA.12 positions, respectively. These results
demonstrate that Fad6 is a .DELTA.6 desaturase that converts
linoleic acid (18:2) to GLA in yeast.
Example 9
Expression of Fad4 in B. Juncea
[0175] To determine whether Traustochytrium Fad4 is functional in
oilseed crops, B. juncea were transformed with the construct
containing Fad4 under the control of a constitutive promoter. Eight
independent transgenic plants were obtained. In B. juncea there is
no .DELTA.4 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
only in 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
.DELTA.4 desaturated fatty acid (22:6, n-3) was produced in a range
of 3-6% of the total fatty acids (FIG. 16). These results indicate
that the .DELTA.4 fatty acid desaturase from Traustochytrium is
functional in oilseed crops.
Example 10
Expression of Fad5-2 in B. Juncea
[0176] 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. FIG. 17 shows the fatty acid
composition of three-week-old seedling plants from one T.sub.1
line. Compared with wild type, all transgenic plant tissues have an
altered fatty acid profile containing four additional peaks which
were identified as four different .DELTA.5-undesaturated
polymethylene-interrupted fatty acids (.DELTA.5-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 .DELTA.5 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 .DELTA.5 desaturated fatty acids. The roots produced
the highest amount of the .DELTA.5-UPIFAs, representing more than
20% of the total fatty acids, followed by 6% in stems and 5% in
leaves (FIG. 17).
[0177] 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 aqueous solution of sodium
homo-gamma-linolenate. It was found that exogenously applied
substrates were readily taken up by roots, stem, and leaves of
transgenic plants and converted into AA in plants (FIG. 18).
[0178] There was no observable phenotypic effect on the growth and
development in the transgenic B. juncea, although the
.DELTA.5-UPIFAs accumulated in all parts of the plant. Growth and
differentiation of vegetative tissues such as the leaves, stems,
and roots were indistinguishable from the corresponding wild
type.
[0179] To produce .DELTA.5 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
transgenics compared with the wild type control (FIG. 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. Accumulation of .DELTA.5-UPIFAs has no
significant effect on the oleic acid content compared with the
untransformed control.
Example 11
Expression of Fad5-2 in Flax
[0180] To produce .DELTA.5 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 FIG. 26, transgenic
plants containing the napin promoter produced one .DELTA.5
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 .DELTA.5
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 .DELTA.5 desaturated fatty acids in
transgenic seeds has significant impact on the composition of other
fatty acids, especially the oleic acid level. Accumulation of
.DELTA.5-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
[0181] To produce .DELTA.6 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 whose retention times
correspond to GLA and SDA and they constitute 0.1 to 4.3% of the
total fatty acids (FIG. 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 .alpha.-linolenic
acid is a major fatty acid in Normandy seeds.
Example 13
Expression of Fad6 in B. Juncea
[0182] To produce .DELTA.6 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. napus
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 (FIG. 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
.DELTA.6 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 component is SDA, which
accounts for 3-10% of the total fatty acids in several transgenic
lines (FIG. 21).
[0183] The fatty acid compositions of transgenic seeds are shown in
FIG. 22. It is clear that the high level production of .DELTA.6
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%.
[0184] 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 acids in transgenic seeds
are the substrates of the .DELTA.6 desaturase, and the reduction is
the cost for producing two corresponding .DELTA.6 desaturated fatty
acids.
EQUIVALENTS
[0185] 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.
Sequence CWU 1
1
1011560DNAThraustochytrium sp.CDS(1)...(1560)VARIANT462Xaa = Gly
1atg acg gtc ggc tac gac gag gag atc ccg ttc gag cag gtc cgc gcg
48Met Thr Val Gly Tyr Asp Glu Glu Ile Pro Phe Glu Gln Val Arg Ala1
5 10 15cac aac aag ccg gat gac gcc tgg tgc gcg atc cac ggg cac gtg
tac 96His Asn Lys Pro Asp Asp Ala Trp Cys Ala Ile His Gly His Val
Tyr 20 25 30gat gtg acc aag ttc gcg agc gtg cac ccg ggc ggc gac att
atc ctg 144Asp Val Thr Lys Phe Ala Ser Val His Pro Gly Gly Asp Ile
Ile Leu 35 40 45ctg gcc gca ggc aag gag gcc acc gtg ctg tac gag act
tac cat gtg 192Leu Ala Ala Gly Lys Glu Ala Thr Val Leu Tyr Glu Thr
Tyr His Val 50 55 60cgg ggc gtc tcg gac gcg gtg ctg cgc aag tac cgc
atc ggc aag ctg 240Arg Gly Val Ser Asp Ala Val Leu Arg Lys Tyr Arg
Ile Gly Lys Leu65 70 75 80ccg gac ggc caa ggc ggc gcg aac gag aag
gaa aag cgg acg ctc tcg 288Pro Asp Gly Gln Gly Gly Ala Asn Glu Lys
Glu Lys Arg Thr Leu Ser 85 90 95ggc ctc tcg tcg gcc tcg tac tac acg
tgg aac agc gac ttt tac agg 336Gly Leu Ser Ser Ala Ser Tyr Tyr Thr
Trp Asn Ser Asp Phe Tyr Arg 100 105 110gta atg cgc gag cgc gtc gtg
gct cgg ctc aag gag cgc ggc aag gcc 384Val Met Arg Glu Arg Val Val
Ala Arg Leu Lys Glu Arg Gly Lys Ala 115 120 125cgc cgc gga ggc tac
gag ctc tgg atc aag gcg ttc ctg ctg ctc gtc 432Arg Arg Gly Gly Tyr
Glu Leu Trp Ile Lys Ala Phe Leu Leu Leu Val 130 135 140ggc ttc tgg
agc tcg ctg tac tgg atg tgc acg ctg gac ccc tcg ttc 480Gly Phe Trp
Ser Ser Leu Tyr Trp Met Cys Thr Leu Asp Pro Ser Phe145 150 155
160ggg gcc atc ctg gcc gcc atg tcg ctg ggc gtc ttt gcc gcc ttt gtg
528Gly Ala Ile Leu Ala Ala Met Ser Leu Gly Val Phe Ala Ala Phe Val
165 170 175ggc acg tgc atc cag cac gac ggc aac cac ggc gcc ttt gcc
cag tcg 576Gly Thr Cys Ile Gln His Asp Gly Asn His Gly Ala Phe Ala
Gln Ser 180 185 190cga tgg gtc aac aag gtt gcc ggg tgg acg ctc gac
atg atc ggc gcc 624Arg Trp Val Asn Lys Val Ala Gly Trp Thr Leu Asp
Met Ile Gly Ala 195 200 205agc ggc atg acg tgg gag ttc cag cac gtc
ctg ggc cac cat ccg tac 672Ser Gly Met Thr Trp Glu Phe Gln His Val
Leu Gly His His Pro Tyr 210 215 220acg aac ctg atc gag gag gag aac
ggc ctg caa aag gtg agc ggc aag 720Thr Asn Leu Ile Glu Glu Glu Asn
Gly Leu Gln Lys Val Ser Gly Lys225 230 235 240aag atg gac acc aag
ctg gcc gac cag gag agc gat ccg gac gtc ttt 768Lys Met Asp Thr Lys
Leu Ala Asp Gln Glu Ser Asp Pro Asp Val Phe 245 250 255tcc acg tac
ccg atg atg cgc ctg cac ccg tgg cac cag aag cgc tgg 816Ser Thr Tyr
Pro Met Met Arg Leu His Pro Trp His Gln Lys Arg Trp 260 265 270tac
cac cgt ttc cag cac att tac ggc ccc ttc atc ttt ggc ttc atg 864Tyr
His Arg Phe Gln His Ile Tyr Gly Pro Phe Ile Phe Gly Phe Met 275 280
285acc atc aac aag gtg gtc acg cag gac gtc ggt gtg gtg ctc cgc aag
912Thr Ile Asn Lys Val Val Thr Gln Asp Val Gly Val Val Leu Arg Lys
290 295 300cgg ctc ttc cag att gac gcc gag tgc cgg tac gcg agc cca
atg tac 960Arg Leu Phe Gln Ile Asp Ala Glu Cys Arg Tyr Ala Ser Pro
Met Tyr305 310 315 320gtg gcg cgt ttc tgg atc atg aag gcg ctc acg
gtg ctc tac atg gtg 1008Val Ala Arg Phe Trp Ile Met Lys Ala Leu Thr
Val Leu Tyr Met Val 325 330 335gcc ctg ccg tgc tac atg cag ggc ccg
tgg cac ggc ctc aag ctg ttc 1056Ala Leu Pro Cys Tyr Met Gln Gly Pro
Trp His Gly Leu Lys Leu Phe 340 345 350gcg atc gcg cac ttt acg tgc
ggc gag gtg ctc gca acc atg ttc att 1104Ala Ile Ala His Phe Thr Cys
Gly Glu Val Leu Ala Thr Met Phe Ile 355 360 365gtg aac cac atc atc
gag ggc gtc tcg tac gct tcc aag gac gcg gtc 1152Val Asn His Ile Ile
Glu Gly Val Ser Tyr Ala Ser Lys Asp Ala Val 370 375 380aag ggc acg
atg gcg ccg ccg aag acg atg cac ggc gtg acg ccc atg 1200Lys Gly Thr
Met Ala Pro Pro Lys Thr Met His Gly Val Thr Pro Met385 390 395
400aac aac acg cgc aag gag gtg gag gcg gag gcg tcc aag tct ggc gcc
1248Asn Asn Thr Arg Lys Glu Val Glu Ala Glu Ala Ser Lys Ser Gly Ala
405 410 415gtg gtc aag tca gtc ccg ctc gac gac tgg gcc gtc gtc cag
tgc cag 1296Val Val Lys Ser Val Pro Leu Asp Asp Trp Ala Val Val Gln
Cys Gln 420 425 430acc tcg gtg aac tgg agc gtc ggc tcg tgg ttc tgg
aat cac ttt tcc 1344Thr Ser Val Asn Trp Ser Val Gly Ser Trp Phe Trp
Asn His Phe Ser 435 440 445ggc ggc ctc aac cac cag att gag cac cac
ctg ttc ccc ggr ctc agc 1392Gly Gly Leu Asn His Gln Ile Glu His His
Leu Phe Pro Xaa Leu Ser 450 455 460cac gag acg tac tac cac att cag
gac gtc ttt cag tcc acc tgc gcc 1440His Glu Thr Tyr Tyr His Ile Gln
Asp Val Phe Gln Ser Thr Cys Ala465 470 475 480gag tac ggc gtc ccg
tac cag cac gag cct tcg ctc tgg acc gcg tac 1488Glu Tyr Gly Val Pro
Tyr Gln His Glu Pro Ser Leu Trp Thr Ala Tyr 485 490 495tgg aag atg
ctc gag cac ctc cgt cag ctc ggc aat gag gag acc cac 1536Trp Lys Met
Leu Glu His Leu Arg Gln Leu Gly Asn Glu Glu Thr His 500 505 510gag
tcc tgg cag cgc gct gcc tga 1560Glu Ser Trp Gln Arg Ala Ala
5152519PRTThraustochytrium sp.VARIANT462Xaa = Gly 2Met Thr Val Gly
Tyr Asp Glu Glu Ile Pro Phe Glu Gln Val Arg Ala1 5 10 15 His Asn
Lys Pro Asp Asp Ala Trp Cys Ala Ile His Gly His Val Tyr 20 25 30
Asp Val Thr Lys Phe Ala Ser Val His Pro Gly Gly Asp Ile Ile Leu 35
40 45 Leu Ala Ala Gly Lys Glu Ala Thr Val Leu Tyr Glu Thr Tyr His
Val 50 55 60 Arg Gly Val Ser Asp Ala Val Leu Arg Lys Tyr Arg Ile
Gly Lys Leu65 70 75 80 Pro Asp Gly Gln Gly Gly Ala Asn Glu Lys Glu
Lys Arg Thr Leu Ser 85 90 95 Gly Leu Ser Ser Ala Ser Tyr Tyr Thr
Trp Asn Ser Asp Phe Tyr Arg 100 105 110 Val Met Arg Glu Arg Val Val
Ala Arg Leu Lys Glu Arg Gly Lys Ala 115 120 125 Arg Arg Gly Gly Tyr
Glu Leu Trp Ile Lys Ala Phe Leu Leu Leu Val 130 135 140 Gly Phe Trp
Ser Ser Leu Tyr Trp Met Cys Thr Leu Asp Pro Ser Phe145 150 155 160
Gly Ala Ile Leu Ala Ala Met Ser Leu Gly Val Phe Ala Ala Phe Val 165
170 175 Gly Thr Cys Ile Gln His Asp Gly Asn His Gly Ala Phe Ala Gln
Ser 180 185 190 Arg Trp Val Asn Lys Val Ala Gly Trp Thr Leu Asp Met
Ile Gly Ala 195 200 205 Ser Gly Met Thr Trp Glu Phe Gln His Val Leu
Gly His His Pro Tyr 210 215 220 Thr Asn Leu Ile Glu Glu Glu Asn Gly
Leu Gln Lys Val Ser Gly Lys225 230 235 240 Lys Met Asp Thr Lys Leu
Ala Asp Gln Glu Ser Asp Pro Asp Val Phe 245 250 255 Ser Thr Tyr Pro
Met Met Arg Leu His Pro Trp His Gln Lys Arg Trp 260 265 270 Tyr His
Arg Phe Gln His Ile Tyr Gly Pro Phe Ile Phe Gly Phe Met 275 280 285
Thr Ile Asn Lys Val Val Thr Gln Asp Val Gly Val Val Leu Arg Lys 290
295 300 Arg Leu Phe Gln Ile Asp Ala Glu Cys Arg Tyr Ala Ser Pro Met
Tyr305 310 315 320 Val Ala Arg Phe Trp Ile Met Lys Ala Leu Thr Val
Leu Tyr Met Val 325 330 335 Ala Leu Pro Cys Tyr Met Gln Gly Pro Trp
His Gly Leu Lys Leu Phe 340 345 350 Ala Ile Ala His Phe Thr Cys Gly
Glu Val Leu Ala Thr Met Phe Ile 355 360 365 Val Asn His Ile Ile Glu
Gly Val Ser Tyr Ala Ser Lys Asp Ala Val 370 375 380 Lys Gly Thr Met
Ala Pro Pro Lys Thr Met His Gly Val Thr Pro Met385 390 395 400 Asn
Asn Thr Arg Lys Glu Val Glu Ala Glu Ala Ser Lys Ser Gly Ala 405 410
415 Val Val Lys Ser Val Pro Leu Asp Asp Trp Ala Val Val Gln Cys Gln
420 425 430 Thr Ser Val Asn Trp Ser Val Gly Ser Trp Phe Trp Asn His
Phe Ser 435 440 445 Gly Gly Leu Asn His Gln Ile Glu His His Leu Phe
Pro Xaa Leu Ser 450 455 460 His Glu Thr Tyr Tyr His Ile Gln Asp Val
Phe Gln Ser Thr Cys Ala465 470 475 480 Glu Tyr Gly Val Pro Tyr Gln
His Glu Pro Ser Leu Trp Thr Ala Tyr 485 490 495 Trp Lys Met Leu Glu
His Leu Arg Gln Leu Gly Asn Glu Glu Thr His 500 505 510 Glu Ser Trp
Gln Arg Ala Ala 515 31320DNAThraustochytrium sp.CDS(1)...(1320)
3atg ggc aag ggc agc gag ggc cgc agc gcg gcg cgc gag atg acg gcc
48Met Gly Lys Gly Ser Glu Gly Arg Ser Ala Ala Arg Glu Met Thr Ala1
5 10 15gag gcg aac ggc gac aag cgg aaa acg att ctg atc gag ggc gtc
ctg 96Glu Ala Asn Gly Asp Lys Arg Lys Thr Ile Leu Ile Glu Gly Val
Leu 20 25 30tac gac gcg acg aac ttt aag cac ccg ggc ggt tcg atc atc
aac ttc 144Tyr Asp Ala Thr Asn Phe Lys His Pro Gly Gly Ser Ile Ile
Asn Phe 35 40 45ttg acc gag ggc gag gcc ggc gtg gac gcg acg cag gcg
tac cgc gag 192Leu Thr Glu Gly Glu Ala Gly Val Asp Ala Thr Gln Ala
Tyr Arg Glu 50 55 60ttt cat cag cgg tcc ggc aag gcc gac aag tac ctc
aag tcg ctg ccg 240Phe His Gln Arg Ser Gly Lys Ala Asp Lys Tyr Leu
Lys Ser Leu Pro65 70 75 80aag ctg gat gcg tcc aag gtg gag tcg cgg
ttc tcg gcc aaa gag cag 288Lys Leu Asp Ala Ser Lys Val Glu Ser Arg
Phe Ser Ala Lys Glu Gln 85 90 95gcg cgg cgc gac gcc atg acg cgc gac
tac gcg gcc ttt cgc gag gag 336Ala Arg Arg Asp Ala Met Thr Arg Asp
Tyr Ala Ala Phe Arg Glu Glu 100 105 110ctc gtc gcc gag ggg tac ttt
gac ccg tcg atc ccg cac atg att tac 384Leu Val Ala Glu Gly Tyr Phe
Asp Pro Ser Ile Pro His Met Ile Tyr 115 120 125cgc gtc gtg gag atc
gtg gcg ctc ttc gcg ctc tcg ttc tgg ctc atg 432Arg Val Val Glu Ile
Val Ala Leu Phe Ala Leu Ser Phe Trp Leu Met 130 135 140tcc aag gcc
tcg ccc acc tcg ctc gtg ctg ggc gtg gtg atg aac ggc 480Ser Lys Ala
Ser Pro Thr Ser Leu Val Leu Gly Val Val Met Asn Gly145 150 155
160att gcg cag ggc cgc tgc ggc tgg gtc atg cac gag atg ggc cac ggg
528Ile Ala Gln Gly Arg Cys Gly Trp Val Met His Glu Met Gly His Gly
165 170 175tcg ttc acg ggc gtc atc tgg ctc gac gac cgg atg tgc gag
ttc ttc 576Ser Phe Thr Gly Val Ile Trp Leu Asp Asp Arg Met Cys Glu
Phe Phe 180 185 190tac ggc gtc ggc tgc ggc atg agc ggg cac tac tgg
aag aac cag cac 624Tyr Gly Val Gly Cys Gly Met Ser Gly His Tyr Trp
Lys Asn Gln His 195 200 205agc aag cac cac gcc gcg ccc aac cgc ctc
gag cac gat gtc gat ctc 672Ser Lys His His Ala Ala Pro Asn Arg Leu
Glu His Asp Val Asp Leu 210 215 220aac acg ctg ccc ctg gtc gcc ttt
aac gag cgc gtc gtg cgc aag gtc 720Asn Thr Leu Pro Leu Val Ala Phe
Asn Glu Arg Val Val Arg Lys Val225 230 235 240aag ccg gga tcg ctg
ctg gcg ctc tgg ctg cgc gtg cag gcg tac ctc 768Lys Pro Gly Ser Leu
Leu Ala Leu Trp Leu Arg Val Gln Ala Tyr Leu 245 250 255ttt gcg ccc
gtc tcg tgc ctg ctc atc ggc ctt ggc tgg acg ctc tac 816Phe Ala Pro
Val Ser Cys Leu Leu Ile Gly Leu Gly Trp Thr Leu Tyr 260 265 270ctg
cac ccg cgc tac atg ctg cgc acc aag cgg cac atg gag ttc gtc 864Leu
His Pro Arg Tyr Met Leu Arg Thr Lys Arg His Met Glu Phe Val 275 280
285tgg atc ttc gcg cgc tac att ggc tgg ttc tcg ctc atg ggc gct ctc
912Trp Ile Phe Ala Arg Tyr Ile Gly Trp Phe Ser Leu Met Gly Ala Leu
290 295 300ggc tac tcg ccg ggc acc tcg gtc ggg atg tac ctg tgc tcg
ttc ggc 960Gly Tyr Ser Pro Gly Thr Ser Val Gly Met Tyr Leu Cys Ser
Phe Gly305 310 315 320ctc ggc tgc att tac att ttc ctg cag ttc gcc
gtc agc cac acg cac 1008Leu Gly Cys Ile Tyr Ile Phe Leu Gln Phe Ala
Val Ser His Thr His 325 330 335ctg ccg gtg acc aac ccg gag gac cag
ctg cac tgg ctc gag tac gcg 1056Leu Pro Val Thr Asn Pro Glu Asp Gln
Leu His Trp Leu Glu Tyr Ala 340 345 350gcc gac cac acg gtg aac att
agc acc aag tcc tgg ctc gtc acg tgg 1104Ala Asp His Thr Val Asn Ile
Ser Thr Lys Ser Trp Leu Val Thr Trp 355 360 365tgg atg tcg aac ctg
aac ttt cag atc gag cac cac ctc ttc ccc acg 1152Trp Met Ser Asn Leu
Asn Phe Gln Ile Glu His His Leu Phe Pro Thr 370 375 380gcg ccg cag
ttc cgc ttc aag gaa atc agt cct cgc gtc gag gcc ctc 1200Ala Pro Gln
Phe Arg Phe Lys Glu Ile Ser Pro Arg Val Glu Ala Leu385 390 395
400ttc aag cgc cac aac ctc ccg tac tac gac ctg ccc tac acg agc gcg
1248Phe Lys Arg His Asn Leu Pro Tyr Tyr Asp Leu Pro Tyr Thr Ser Ala
405 410 415gtc tcg acc acc ttt gcc aat ctt tat tcc gtc ggc cac tcg
gtc ggc 1296Val Ser Thr Thr Phe Ala Asn Leu Tyr Ser Val Gly His Ser
Val Gly 420 425 430gcc gac acc aag aag cag gac tga 1320Ala Asp Thr
Lys Lys Gln Asp 4354439PRTThraustochytrium sp. 4Met Gly Lys Gly Ser
Glu Gly Arg Ser Ala Ala Arg Glu Met Thr Ala 1 5 10 15 Glu Ala Asn
Gly Asp Lys Arg Lys Thr Ile Leu Ile Glu Gly Val Leu 20 25 30 Tyr
Asp Ala Thr Asn Phe Lys His Pro Gly Gly Ser Ile Ile Asn Phe 35 40
45 Leu Thr Glu Gly Glu Ala Gly Val Asp Ala Thr Gln Ala Tyr Arg Glu
50 55 60 Phe His Gln Arg Ser Gly Lys Ala Asp Lys Tyr Leu Lys Ser
Leu Pro65 70 75 80 Lys Leu Asp Ala Ser Lys Val Glu Ser Arg Phe Ser
Ala Lys Glu Gln 85 90 95 Ala Arg Arg Asp Ala Met Thr Arg Asp Tyr
Ala Ala Phe Arg Glu Glu 100 105 110 Leu Val Ala Glu Gly Tyr Phe Asp
Pro Ser Ile Pro His Met Ile Tyr 115 120 125 Arg Val Val Glu Ile Val
Ala Leu Phe Ala Leu Ser Phe Trp Leu Met 130 135 140 Ser Lys Ala Ser
Pro Thr Ser Leu Val Leu Gly Val Val Met Asn Gly145 150 155 160 Ile
Ala Gln Gly Arg Cys Gly Trp Val Met His Glu Met Gly His Gly 165 170
175 Ser Phe Thr Gly Val Ile Trp Leu Asp Asp Arg Met Cys Glu Phe Phe
180 185 190 Tyr Gly Val Gly Cys Gly Met Ser Gly His Tyr Trp Lys Asn
Gln His 195 200 205 Ser Lys His His Ala Ala Pro Asn Arg Leu Glu His
Asp Val Asp Leu 210 215 220 Asn Thr Leu Pro Leu Val Ala Phe Asn Glu
Arg Val Val Arg Lys Val225 230 235 240 Lys Pro Gly Ser Leu Leu Ala
Leu Trp Leu Arg Val Gln Ala Tyr Leu 245 250 255 Phe Ala Pro Val Ser
Cys Leu Leu Ile Gly Leu Gly Trp Thr Leu Tyr 260 265 270 Leu His Pro
Arg Tyr Met Leu Arg Thr Lys Arg His Met Glu Phe Val 275 280 285 Trp
Ile Phe Ala Arg Tyr Ile Gly Trp Phe Ser Leu Met Gly Ala Leu 290 295
300 Gly Tyr Ser Pro Gly Thr Ser Val Gly Met Tyr Leu Cys Ser Phe
Gly305 310 315 320 Leu Gly Cys Ile Tyr
Ile Phe Leu Gln Phe Ala Val Ser His Thr His 325 330 335 Leu Pro Val
Thr Asn Pro Glu Asp Gln Leu His Trp Leu Glu Tyr Ala 340 345 350 Ala
Asp His Thr Val Asn Ile Ser Thr Lys Ser Trp Leu Val Thr Trp 355 360
365 Trp Met Ser Asn Leu Asn Phe Gln Ile Glu His His Leu Phe Pro Thr
370 375 380 Ala Pro Gln Phe Arg Phe Lys Glu Ile Ser Pro Arg Val Glu
Ala Leu385 390 395 400 Phe Lys Arg His Asn Leu Pro Tyr Tyr Asp Leu
Pro Tyr Thr Ser Ala 405 410 415 Val Ser Thr Thr Phe Ala Asn Leu Tyr
Ser Val Gly His Ser Val Gly 420 425 430 Ala Asp Thr Lys Lys Gln Asp
435 51371DNAPythium irregulareCDS(1)...(1371) 5atg acc gag aag gcg
agt gac gag ttc acg tgg cag gag gtc gcc aag 48Met Thr Glu Lys Ala
Ser Asp Glu Phe Thr Trp Gln Glu Val Ala Lys1 5 10 15cac aac acg gcc
aag agc gcg tgg gtg atc atc cgc ggc gag gtg tac 96His Asn Thr Ala
Lys Ser Ala Trp Val Ile Ile Arg Gly Glu Val Tyr 20 25 30gac gtg acc
gag tgg gcg gac aag cac ccg ggc ggc agc gag ctc atc 144Asp Val Thr
Glu Trp Ala Asp Lys His Pro Gly Gly Ser Glu Leu Ile 35 40 45gtc ctg
cac tcc ggt cgt gaa tgc acg gac acg ttc tac tcg tac cac 192Val Leu
His Ser Gly Arg Glu Cys Thr Asp Thr Phe Tyr Ser Tyr His 50 55 60ccg
ttc tcg aac cgc gcc gac aag atc ttg gcc aag tac aag atc ggc 240Pro
Phe Ser Asn Arg Ala Asp Lys Ile Leu Ala Lys Tyr Lys Ile Gly65 70 75
80aag ctc gtg ggc ggc tac gag ttc ccg gtg ttc aag ccg gac tcg ggc
288Lys Leu Val Gly Gly Tyr Glu Phe Pro Val Phe Lys Pro Asp Ser Gly
85 90 95ttc tac aag gaa tgc tcg gag cgc gtg gcc gag tac ttt aag acg
aac 336Phe Tyr Lys Glu Cys Ser Glu Arg Val Ala Glu Tyr Phe Lys Thr
Asn 100 105 110aat ctg gac cca aag gcg gcg ttc gcg ggt ctc tgg cgc
atg gtg ttc 384Asn Leu Asp Pro Lys Ala Ala Phe Ala Gly Leu Trp Arg
Met Val Phe 115 120 125gtg ttc gcg gtc gcc gcg ctc gcg tac atg ggc
atg aat gag ctc atc 432Val Phe Ala Val Ala Ala Leu Ala Tyr Met Gly
Met Asn Glu Leu Ile 130 135 140cct gga aac gtg tac gcg cag tac gcg
tgg ggc gtg gtg ttc ggt gtc 480Pro Gly Asn Val Tyr Ala Gln Tyr Ala
Trp Gly Val Val Phe Gly Val145 150 155 160ttc cag gcg ctg cca ttg
ctg cac gtg atg cac gac tcg tcg cac gcg 528Phe Gln Ala Leu Pro Leu
Leu His Val Met His Asp Ser Ser His Ala 165 170 175gca tgc tcg agc
agc cca gcg atg tgg cag atc atc ggt cgt ggt gtg 576Ala Cys Ser Ser
Ser Pro Ala Met Trp Gln Ile Ile Gly Arg Gly Val 180 185 190atg gac
tgg ttc gct ggc gcc agc atg gtg tcg tgg ttg aac cag cac 624Met Asp
Trp Phe Ala Gly Ala Ser Met Val Ser Trp Leu Asn Gln His 195 200
205gtt gtg ggc cac cac atc tac acg aac gtc gcg ggc gcg gac ccg gat
672Val Val Gly His His Ile Tyr Thr Asn Val Ala Gly Ala Asp Pro Asp
210 215 220ctc ccg gtc gac ttt gag agc gac gtg cgc cgc atc gtg cac
cgc cag 720Leu Pro Val Asp Phe Glu Ser Asp Val Arg Arg Ile Val His
Arg Gln225 230 235 240gtg ctg ctg ccg atc tac aag ttc cag cac atc
tac ctg cca ccg ctc 768Val Leu Leu Pro Ile Tyr Lys Phe Gln His Ile
Tyr Leu Pro Pro Leu 245 250 255tac ggc gtg ctg ggc ctc aag ttc cgc
atc cag gac gtg ttc gag acg 816Tyr Gly Val Leu Gly Leu Lys Phe Arg
Ile Gln Asp Val Phe Glu Thr 260 265 270ttc gtg tcg ctc acg aac ggc
ccg gtg cgt gtg aac ccg cac ccg gtg 864Phe Val Ser Leu Thr Asn Gly
Pro Val Arg Val Asn Pro His Pro Val 275 280 285tcg gac tgg gtg caa
atg atc ttc gcc aag gcg ttc tgg acg ttc tac 912Ser Asp Trp Val Gln
Met Ile Phe Ala Lys Ala Phe Trp Thr Phe Tyr 290 295 300cgc atc tac
atc ccg ttg gcg tgg ctc aag atc acg ccg tcg acg ttc 960Arg Ile Tyr
Ile Pro Leu Ala Trp Leu Lys Ile Thr Pro Ser Thr Phe305 310 315
320tgg ggc gtg ttt ttc ctc gcc gag ttc acc aca ggt tgg tac ctc gcg
1008Trp Gly Val Phe Phe Leu Ala Glu Phe Thr Thr Gly Trp Tyr Leu Ala
325 330 335ttc aac ttc cag gtg agc cac gtc tcg acc gag tgc gag tac
ccg tgc 1056Phe Asn Phe Gln Val Ser His Val Ser Thr Glu Cys Glu Tyr
Pro Cys 340 345 350ggt gat gcg ccg tcg gcc gag gtc ggt gac gag tgg
gcg atc tcg cag 1104Gly Asp Ala Pro Ser Ala Glu Val Gly Asp Glu Trp
Ala Ile Ser Gln 355 360 365gtc aag tcg tcg gtg gac tac gcg cac ggc
tcg ccg ctc gcg gcg ttc 1152Val Lys Ser Ser Val Asp Tyr Ala His Gly
Ser Pro Leu Ala Ala Phe 370 375 380ctc tgc ggc gcg ctc aac tac cag
gtg acc cac cac ttg tac ccg ggc 1200Leu Cys Gly Ala Leu Asn Tyr Gln
Val Thr His His Leu Tyr Pro Gly385 390 395 400atc tca cag tac cac
tac cct gcg atc gcg ccg atc atc atc gac gtg 1248Ile Ser Gln Tyr His
Tyr Pro Ala Ile Ala Pro Ile Ile Ile Asp Val 405 410 415tgc aag aag
tac aac atc aag tac acg gtg ctg ccg acg ttc acc gag 1296Cys Lys Lys
Tyr Asn Ile Lys Tyr Thr Val Leu Pro Thr Phe Thr Glu 420 425 430gcg
ctg ctc gcg cac ttc aag cac ctg aag aac atg ggc gag ctc ggc 1344Ala
Leu Leu Ala His Phe Lys His Leu Lys Asn Met Gly Glu Leu Gly 435 440
445aag ccc gtg gag atc cac atg ggt taa 1371Lys Pro Val Glu Ile His
Met Gly 450 4556456PRTPythium irregulare 6Met Thr Glu Lys Ala Ser
Asp Glu Phe Thr Trp Gln Glu Val Ala Lys1 5 10 15 His Asn Thr Ala
Lys Ser Ala Trp Val Ile Ile Arg Gly Glu Val Tyr 20 25 30 Asp Val
Thr Glu Trp Ala Asp Lys His Pro Gly Gly Ser Glu Leu Ile 35 40 45
Val Leu His Ser Gly Arg Glu Cys Thr Asp Thr Phe Tyr Ser Tyr His 50
55 60 Pro Phe Ser Asn Arg Ala Asp Lys Ile Leu Ala Lys Tyr Lys Ile
Gly65 70 75 80 Lys Leu Val Gly Gly Tyr Glu Phe Pro Val Phe Lys Pro
Asp Ser Gly 85 90 95 Phe Tyr Lys Glu Cys Ser Glu Arg Val Ala Glu
Tyr Phe Lys Thr Asn 100 105 110 Asn Leu Asp Pro Lys Ala Ala Phe Ala
Gly Leu Trp Arg Met Val Phe 115 120 125 Val Phe Ala Val Ala Ala Leu
Ala Tyr Met Gly Met Asn Glu Leu Ile 130 135 140Pro Gly Asn Val Tyr
Ala Gln Tyr Ala Trp Gly Val Val Phe Gly Val145 150 155 160 Phe Gln
Ala Leu Pro Leu Leu His Val Met His Asp Ser Ser His Ala 165 170 175
Ala Cys Ser Ser Ser Pro Ala Met Trp Gln Ile Ile Gly Arg Gly Val 180
185 190 Met Asp Trp Phe Ala Gly Ala Ser Met Val Ser Trp Leu Asn Gln
His 195 200 205 Val Val Gly His His Ile Tyr Thr Asn Val Ala Gly Ala
Asp Pro Asp 210 215 220 Leu Pro Val Asp Phe Glu Ser Asp Val Arg Arg
Ile Val His Arg Gln225 230 235 240 Val Leu Leu Pro Ile Tyr Lys Phe
Gln His Ile Tyr Leu Pro Pro Leu 245 250 255 Tyr Gly Val Leu Gly Leu
Lys Phe Arg Ile Gln Asp Val Phe Glu Thr 260 265 270 Phe Val Ser Leu
Thr Asn Gly Pro Val Arg Val Asn Pro His Pro Val 275 280 285 Ser Asp
Trp Val Gln Met Ile Phe Ala Lys Ala Phe Trp Thr Phe Tyr 290 295 300
Arg Ile Tyr Ile Pro Leu Ala Trp Leu Lys Ile Thr Pro Ser Thr Phe305
310 315 320 Trp Gly Val Phe Phe Leu Ala Glu Phe Thr Thr Gly Trp Tyr
Leu Ala 325 330 335 Phe Asn Phe Gln Val Ser His Val Ser Thr Glu Cys
Glu Tyr Pro Cys 340 345 350 Gly Asp Ala Pro Ser Ala Glu Val Gly Asp
Glu Trp Ala Ile Ser Gln 355 360 365 Val Lys Ser Ser Val Asp Tyr Ala
His Gly Ser Pro Leu Ala Ala Phe 370 375 380 Leu Cys Gly Ala Leu Asn
Tyr Gln Val Thr His His Leu Tyr Pro Gly385 390 395 400 Ile Ser Gln
Tyr His Tyr Pro Ala Ile Ala Pro Ile Ile Ile Asp Val 405 410 415 Cys
Lys Lys Tyr Asn Ile Lys Tyr Thr Val Leu Pro Thr Phe Thr Glu 420 425
430 Ala Leu Leu Ala His Phe Lys His Leu Lys Asn Met Gly Glu Leu Gly
435 440 445 Lys Pro Val Glu Ile His Met Gly 450 455 71380DNAPythium
irregulareCDS(1)...(1380) 7atg gtg gac ctc aag cct gga gtg aag cgc
ctg gtg agc tgg aag gag 48Met Val Asp Leu Lys Pro Gly Val Lys Arg
Leu Val Ser Trp Lys Glu1 5 10 15atc cgc gag cac gcg acg ccc gcg acc
gcg tgg atc gtg att cac cac 96Ile Arg Glu His Ala Thr Pro Ala Thr
Ala Trp Ile Val Ile His His 20 25 30aag gtc tac gac atc tcc aag tgg
gac tcg cac ccg ggt ggc tcc gtg 144Lys Val Tyr Asp Ile Ser Lys Trp
Asp Ser His Pro Gly Gly Ser Val 35 40 45atg ctc acg cag gcc ggc gag
gac gcc acg gac gcc ttc gcg gtc ttc 192Met Leu Thr Gln Ala Gly Glu
Asp Ala Thr Asp Ala Phe Ala Val Phe 50 55 60cac ccg tcc tcg gcg ctc
aag ctg ctc gag cag ttc tac gtc ggc gac 240His Pro Ser Ser Ala Leu
Lys Leu Leu Glu Gln Phe Tyr Val Gly Asp65 70 75 80gtg gac gaa acc
tcc aag gcc gag atc gag ggg gag ccg gcg agc gac 288Val Asp Glu Thr
Ser Lys Ala Glu Ile Glu Gly Glu Pro Ala Ser Asp 85 90 95gag gag cgc
gcg cgc cgc gag cgc atc aac gag ttc atc gcg tcc tac 336Glu Glu Arg
Ala Arg Arg Glu Arg Ile Asn Glu Phe Ile Ala Ser Tyr 100 105 110cgt
cgt ctg cgc gtc aag gtc aag ggc atg ggg ctc tac gac gcc agc 384Arg
Arg Leu Arg Val Lys Val Lys Gly Met Gly Leu Tyr Asp Ala Ser 115 120
125gcg ctc tac tac gcg tgg aag ctc gtg agc acg ttc ggc atc gcg gtg
432Ala Leu Tyr Tyr Ala Trp Lys Leu Val Ser Thr Phe Gly Ile Ala Val
130 135 140ctc tcg atg gcg atc tgc ttc ttc ttc aac agt ttc gcc atg
tac atg 480Leu Ser Met Ala Ile Cys Phe Phe Phe Asn Ser Phe Ala Met
Tyr Met145 150 155 160gtc gcc ggc gtg att atg ggg ctc ttc tac cag
cag tcc gga tgg ctg 528Val Ala Gly Val Ile Met Gly Leu Phe Tyr Gln
Gln Ser Gly Trp Leu 165 170 175gcg cac gac ttc ttg cac aac cag gtg
tgc gag aac cgc acg ctc ggc 576Ala His Asp Phe Leu His Asn Gln Val
Cys Glu Asn Arg Thr Leu Gly 180 185 190aac ctt atc ggc tgc ctc gtg
ggc aac gcc tgg cag ggc ttc agc gtg 624Asn Leu Ile Gly Cys Leu Val
Gly Asn Ala Trp Gln Gly Phe Ser Val 195 200 205cag tgg tgg aag aac
aag cac aac ctg cac cac gcg gtg ccg aac ctg 672Gln Trp Trp Lys Asn
Lys His Asn Leu His His Ala Val Pro Asn Leu 210 215 220cac agc gcc
aag gac gag ggc ttc atc ggc gac ccg gac atc gac acc 720His Ser Ala
Lys Asp Glu Gly Phe Ile Gly Asp Pro Asp Ile Asp Thr225 230 235
240atg ccg ctg ctg gcg tgg tct aag gag atg gcg cgc aag gcg ttc gag
768Met Pro Leu Leu Ala Trp Ser Lys Glu Met Ala Arg Lys Ala Phe Glu
245 250 255tcg gcg cac ggc ccg ttc ttc atc cgc aac cag gcg ttc cta
tac ttc 816Ser Ala His Gly Pro Phe Phe Ile Arg Asn Gln Ala Phe Leu
Tyr Phe 260 265 270ccg ctg ctg ctg ctc gcg cgc ctg agc tgg ctc gcg
cag tcg ttc ttc 864Pro Leu Leu Leu Leu Ala Arg Leu Ser Trp Leu Ala
Gln Ser Phe Phe 275 280 285tac gtg ttc acc gag ttc tcg ttc ggc atc
ttc gac aag gtc gag ttc 912Tyr Val Phe Thr Glu Phe Ser Phe Gly Ile
Phe Asp Lys Val Glu Phe 290 295 300gac gga ccg gag aag gcg ggt ctg
atc gtg cac tac atc tgg cag ctc 960Asp Gly Pro Glu Lys Ala Gly Leu
Ile Val His Tyr Ile Trp Gln Leu305 310 315 320gcg atc ccg tac ttc
tgc aac atg agc ctg ttt gag ggc gtg gca tac 1008Ala Ile Pro Tyr Phe
Cys Asn Met Ser Leu Phe Glu Gly Val Ala Tyr 325 330 335ttc ctc atg
ggc cag gcg tcc tgc ggc ttg ctc ctg gcg ctg gtg ttc 1056Phe Leu Met
Gly Gln Ala Ser Cys Gly Leu Leu Leu Ala Leu Val Phe 340 345 350agt
att ggc cac aac ggc atg tcg gtg tac gag cgc gaa acc aag ccg 1104Ser
Ile Gly His Asn Gly Met Ser Val Tyr Glu Arg Glu Thr Lys Pro 355 360
365gac ttc tgg cag ctg cag gtg acc acg acg cgc aac atc cgc gcg tcg
1152Asp Phe Trp Gln Leu Gln Val Thr Thr Thr Arg Asn Ile Arg Ala Ser
370 375 380gta ttc atg gac tgg ttc acc ggt ggc ttg aac tac cag atc
gac cat 1200Val Phe Met Asp Trp Phe Thr Gly Gly Leu Asn Tyr Gln Ile
Asp His385 390 395 400cac ctg ttc ccg ctc gtg ccg cgc cac aac ttg
cca aag gtc aac gtg 1248His Leu Phe Pro Leu Val Pro Arg His Asn Leu
Pro Lys Val Asn Val 405 410 415ctc atc aag tcg cta tgc aag gag ttc
gac atc ccg ttc cac gag acc 1296Leu Ile Lys Ser Leu Cys Lys Glu Phe
Asp Ile Pro Phe His Glu Thr 420 425 430ggc ttc tgg gag ggc atc tac
gag gtc gtg gac cac ctg gcg gac atc 1344Gly Phe Trp Glu Gly Ile Tyr
Glu Val Val Asp His Leu Ala Asp Ile 435 440 445agc aag gaa ttc atc
acc gag ttc cca gcg atg taa 1380Ser Lys Glu Phe Ile Thr Glu Phe Pro
Ala Met 450 4558459PRTPythium irregulare 8Met Val Asp Leu Lys Pro
Gly Val Lys Arg Leu Val Ser Trp Lys Glu 1 5 10 15 Ile Arg Glu His
Ala Thr Pro Ala Thr Ala Trp Ile Val Ile His His 20 25 30 Lys Val
Tyr Asp Ile Ser Lys Trp Asp Ser His Pro Gly Gly Ser Val 35 40 45
Met Leu Thr Gln Ala Gly Glu Asp Ala Thr Asp Ala Phe Ala Val Phe 50
55 60 His Pro Ser Ser Ala Leu Lys Leu Leu Glu Gln Phe Tyr Val Gly
Asp65 70 75 80 Val Asp Glu Thr Ser Lys Ala Glu Ile Glu Gly Glu Pro
Ala Ser Asp 85 90 95 Glu Glu Arg Ala Arg Arg Glu Arg Ile Asn Glu
Phe Ile Ala Ser Tyr 100 105 110 Arg Arg Leu Arg Val Lys Val Lys Gly
Met Gly Leu Tyr Asp Ala Ser 115 120 125 Ala Leu Tyr Tyr Ala Trp Lys
Leu Val Ser Thr Phe Gly Ile Ala Val 130 135 140 Leu Ser Met Ala Ile
Cys Phe Phe Phe Asn Ser Phe Ala Met Tyr Met145 150 155 160 Val Ala
Gly Val Ile Met Gly Leu Phe Tyr Gln Gln Ser Gly Trp Leu 165 170 175
Ala His Asp Phe Leu His Asn Gln Val Cys Glu Asn Arg Thr Leu Gly 180
185 190 Asn Leu Ile Gly Cys Leu Val Gly Asn Ala Trp Gln Gly Phe Ser
Val 195 200 205 Gln Trp Trp Lys Asn Lys His Asn Leu His His Ala Val
Pro Asn Leu 210 215 220 His Ser Ala Lys Asp Glu Gly Phe Ile Gly Asp
Pro Asp Ile Asp Thr225 230 235 240 Met Pro Leu Leu Ala Trp Ser Lys
Glu Met Ala Arg Lys Ala Phe Glu 245 250 255 Ser Ala His Gly Pro Phe
Phe Ile Arg Asn Gln Ala Phe Leu Tyr Phe 260 265 270 Pro Leu Leu Leu
Leu Ala Arg Leu Ser Trp Leu Ala Gln Ser Phe Phe 275 280 285 Tyr Val
Phe Thr Glu Phe Ser Phe Gly Ile Phe Asp Lys Val Glu Phe 290 295 300
Asp Gly Pro Glu Lys Ala Gly Leu Ile Val His Tyr Ile Trp Gln Leu305
310 315 320 Ala Ile Pro Tyr Phe Cys Asn Met Ser Leu Phe Glu Gly Val
Ala Tyr 325 330 335 Phe Leu Met Gly Gln Ala Ser Cys Gly Leu Leu Leu
Ala Leu Val Phe 340
345 350 Ser Ile Gly His Asn Gly Met Ser Val Tyr Glu Arg Glu Thr Lys
Pro 355 360 365 Asp Phe Trp Gln Leu Gln Val Thr Thr Thr Arg Asn Ile
Arg Ala Ser 370 375 380 Val Phe Met Asp Trp Phe Thr Gly Gly Leu Asn
Tyr Gln Ile Asp His385 390 395 400 His Leu Phe Pro Leu Val Pro Arg
His Asn Leu Pro Lys Val Asn Val 405 410 415 Leu Ile Lys Ser Leu Cys
Lys Glu Phe Asp Ile Pro Phe His Glu Thr 420 425 430 Gly Phe Trp Glu
Gly Ile Tyr Glu Val Val Asp His Leu Ala Asp Ile 435 440 445 Ser Lys
Glu Phe Ile Thr Glu Phe Pro Ala Met 450 455 923DNAArtificial
Sequencesynthetic construct 9gcncaganga ncactccngg ngg
231025DNAArtificial Sequencesynthetic construct 10atntgtngga
gaanagagat ggatg 25
* * * * *