U.S. patent application number 11/601563 was filed with the patent office on 2007-05-24 for delta-9 elongases and their use in making polyunsaturated fatty acids.
Invention is credited to Howard Glenn Damude, Brian McGonigle, Zhixiong Xue, Quinn Qun Zhu.
Application Number | 20070118929 11/601563 |
Document ID | / |
Family ID | 37836626 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070118929 |
Kind Code |
A1 |
Damude; Howard Glenn ; et
al. |
May 24, 2007 |
Delta-9 elongases and their use in making polyunsaturated fatty
acids
Abstract
Isolated nucleic acid fragments and recombinant constructs
comprising such fragments encoding delta-9 elongases along with a
method of making long-chain polyunsaturated fatty acids (PUFAs)
using these delta-9 elongases in plants.
Inventors: |
Damude; Howard Glenn;
(Hockessin, DE) ; McGonigle; Brian; (Wilmington,
DE) ; Zhu; Quinn Qun; (Wilmington, DE) ; Xue;
Zhixiong; (Chadds Ford, PA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
37836626 |
Appl. No.: |
11/601563 |
Filed: |
November 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60739989 |
Nov 23, 2005 |
|
|
|
Current U.S.
Class: |
800/281 ;
435/193; 435/419; 435/468; 536/23.2; 554/8; 800/312 |
Current CPC
Class: |
C12P 7/6472 20130101;
C11B 1/025 20130101; C12P 7/6427 20130101; C12P 7/6463 20130101;
C12N 9/1029 20130101; C12N 15/8247 20130101 |
Class at
Publication: |
800/281 ;
800/312; 435/468; 435/419; 435/193; 536/023.2; 554/008 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C11B 1/00 20060101 C11B001/00; C07H 21/04 20060101
C07H021/04; C12N 9/10 20060101 C12N009/10; C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04 |
Claims
1. An isolated polynucleotide comprising: (a) a nucleotide sequence
encoding a polypeptide having delta-9 elongase activity, wherein
the polypeptide has at least 70% amino acid identity, based on the
Clustal V method of alignment, when compared to an amino acid
sequence as set forth in SEQ ID NO:5 or SEQ ID NO:127; (b) a
nucleotide sequence encoding a polypeptide having delta-9 elongase
activity, wherein the nucleotide sequence has at least 70% sequence
identity, based on the BLASTN method of alignment, when compared to
a nucleotide sequence as set forth in SEQ ID NO:4, SEQ ID NO:90 or
SEQ ID NO:126; (c) a nucleotide sequence encoding a polypeptide
having delta-9 elongase activity, wherein the nucleotide sequence
hybridizes with a nucleotide sequence as set forth in SEQ ID NO:4,
SEQ ID NO:90 or SEQ ID NO:126 under the following stringent
hybridization conditions: 0.1.times.SSC, 0.1% SDS, 65.degree. C.
and washed with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC,
0.1% SDS; or (d) a complement of the nucleotide sequence of (a),
(b) or (c), wherein the complement and the nucleotide sequence
consist of the same number of nucleotides and are 100%
complementary.
2. The polynucleotide of claim 1 wherein the nucleotide sequence
comprises SEQ ID NO:4, SEQ ID NO:90 or SEQ ID NO:126.
3. A delta-9 elongase polypeptide wherein the amino acid sequence
of the polypeptide is selected from the group consisting of: (a) an
amino acid sequence as set forth in SEQ ID NO:5 or SEQ ID NO:127;
or (b) an amino acid sequence that differs from the amino acid
sequences in (a) by at least one conservative amino acid
substitution.
4. A recombinant construct comprising the polynucleotide of claim 1
or 2 operably linked to at least one regulatory sequence.
5. A plant cell comprising the recombinant construct of claim
4.
6. A method for transforming a cell, comprising transforming the
cell with the recombinant construct of claim 4 and selecting those
cells transformed with the recombinant construct of claim 4.
7. A method for producing a transgenic plant comprising
transforming a plant cell with at least one polynucleotide of claim
1 or 2 and regenerating a transgenic plant from the transformed
plant cell.
8. The method of claim 7 wherein the transgenic plant is a soybean
plant.
9. A transgenic seed comprising the recombinant construct of claim
4.
10. A transgenic seed obtained from the plant made by the method of
claim 7 or 8.
11. A method for making long-chain polyunsaturated fatty acids in a
plant cell comprising: (a) transforming a cell with the recombinant
construct of claim 4; (b) selecting those transformed cells that
make long-chain polyunsaturated fatty acids.
12. Oil obtained from the seed of claim 9.
13. Oil obtained from the seed of claim 10.
14. A method for producing at least one polyunsaturated fatty acid
in a soybean cell comprising: (a) transforming a soybean cell with
a first recombinant construct comprising an isolated polynucleotide
encoding at least one delta-9 elongase polypeptide, operably linked
to at least one regulatory sequence and at least one additional
recombinant construct comprising an isolated polynucleotide,
operably linked to at least one regulatory sequence, encoding a
polypeptide selected from the group consisting of a delta-4
desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-12
desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9
desaturase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a
C.sub.18/20 elongase and a C.sub.20/22 elongase; (b) regenerating a
soybean plant from the transformed soybean cell of step (a); and
(c) selecting those seeds obtained from the plants of step (b)
having an altered level of polyunsaturated fatty acids when
compared to the level in seeds obtained from a nontransformed
soybean plant.
15. An oilseed plant comprising the recombinant construct of claim
4.
16. An oilseed plant comprising: (a) a first recombinant construct
comprising an isolated polynucleotide encoding at least one delta-9
elongase polypeptide, operably linked to at least one regulatory
sequence; and (b) at least one additional recombinant construct
comprising an isolated polynucleotide, operably linked to at least
one regulatory sequence, encoding a polypeptide selected from the
group consisting of a delta4 desaturase, a delta-5 desaturase, a
delta-6 desaturase, a delta-12 desaturase, a delta-15 desaturase, a
delta-17 desaturase, a delta-9 desaturase, a C.sub.14/16 elongase,
a C.sub.16/18 elongase, a C.sub.18/20 elongase and a C.sub.20/22
elongase.
17. The oilseed plant of claim 15 or 16, wherein the oilseed plant
is selected from the group consisting of soybean, Brassica species,
sunflower, maize, cotton, flax and safflower.
18. The oilseed plant of claim 15 or 16 wherein the oilseed plant
is selected from the group consisting of soybean, Brassica species,
sunflower, maize, cotton, flax, and safflower and further wherein
the polyunsaturated fatty acid is at least one selected from the
group consisting of arachidonic acid, eicosadienoic acid,
eicosapentaenoic acid, eicosatetraenoic acid,.eicosatrienoic acid,
dihomo-gamma-linolenic acid, docosapentaenoic acid and
docosahexaemoic acid.
19. A seed obtained from the oilseed plant of claim 15.
20. A seed obtained from the oilseed plant of claim 16.
21. Oil obtained from the seed of claim 19.
22. Oil obtained from the seed of claim 20.
23. Oil obtained by the method of claim 11 or 14.
24. Food or feed which incorporates the oil of claim 21.
25. Food or feed which incorporates the oil of claim 22.
26. Food or feed which incorporates the oil of claim 23.
27. Food or feed comprising the seed of claim 19.
28. Food or feed comprising the seed of claim 20.
29. Food or feed comprising an ingredient derived from the
processing of the seeds of claim 19.
30. Food or feed comprising an ingredient derived from the
processing of the seed of claim 20.
31. An isolated nucleic acid fragment comprising a nucleic acid
sequence encoding a delta-9 elongase, excluding SEQ ID NO:27,
wherein the amino acid sequence comprising said elongase contains
at least one of the following amino acid sequence motifs selected
from the group consisting of: TABLE-US-00019 (SEQ ID NO:130) a) Y N
X (L or F) X X X X S X X S F; (SEQ ID NO:131) b) F Y X S K X X (E
or D) Y X D (T or S) X X L; (SEQ ID NO:132) c) L (Q or H) X F H H X
G A; (SEQ ID NO:133) d) M Y X Y Y X X X X X X X (K or R or N) F;
(SEQ ID NO:134) e) K X L (I or L or M) T X X Q; (SEQ ID NO:135) f)
W X F N Y X Y; and (SEQ ID NO:136) g) Y X G X V X X L F;
wherein X can be any amino acid.
32. Progeny plants obtained from the transgenic seed of claim
9.
33. Progeny plants obtained from the transgenic seed of claim
10.
34. Progeny plants obtained from the seed of claim 19 or 20.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/739,989, filed Nov. 23, 2005, the entire content
of which is herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention pertains to polynucleotide sequences encoding
delta-9 elongases and the use of these elongase in making
long-chain polyunsaturated fatty acids (PUFAs).
BACKGROUND OF THE INVENTION
[0003] Fatty acids (lipids) are water-insoluble organic
biomolecules that can be extracted from cells and tissues by
nonpolar solvents such as chloroform, ether or benzene. Lipids have
several important biological functions, serving as (1) structural
components of membranes; (2) storage and transport forms of
metabolic fuels; (3) a protective coating on the surface of many
organisms; and, (4) cell-surface components concerned in cell
recognition, species specificity and tissue immunity. More
specifically, polyunsaturated fatty acids (PUFAs) are important
components of the plasma membrane of the cell, where they may be
found in such forms as phospholipids and also can be found in
triglycerides. PUFAs also serve as precursors to other molecules of
importance in human beings and animals, including the
prostacyclins, leukotrienes and prostaglandins. There are two main
families of PUFAs (i.e., the omega-3 fatty acids and the omega-6
fatty acids).
[0004] The human body is capable of producing most of the PUFAs
which it requires to function. However, eicosapentaenoic acid (EPA;
20:5, delta-5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6,
delta-4,7,10,13,16,19) cannot be synthesized efficiently by the
human body and thus must be supplied through the diet. Since the
human body cannot produce adequate quantities of these PUFAs, they
are called essential fatty acids. Because of their important roles
in human health and nutrition, EPA and DHA are the subject of much
interest as discussed herein.
[0005] DHA is a fatty acid of the omega-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
(see FIG. 15). Production of DHA is important because of its
beneficial effect on human health. For example, increased intake of
DHA has been shown to be beneficial or have a positive effect in
inflammatory disorders (e.g., rheumatoid arthritis), Type II
diabetes, hypertension, atherosclerosis, depression, myocardial
infarction, thrombosis, some cancers and for prevention of the
onset of degenerative disorders such as Alzheimer's disease.
Currently the major sources of DHA are oils from fish and
algae.
[0006] EPA and arachidonic acid (AA or ARA; 20:4, delta-5,8,11,14)
are both delta-5 essential fatty acids. EPA belongs to the omega-3
series with five double bonds in the acyl chain, is found in marine
food, and is abundant in oily fish from the North Atlantic.
Beneficial or positive effects of increased intake of EPA have been
shown in patients with coronary heart disease, high blood pressure,
inflammatory disorders, lung and kidney diseases, Type II diabetes,
obesity, ulcerative colitis, Crohn's disease, anorexia nervosa,
burns, osteoarthritis, osteoporosis, attention
deficit/hyperactivity disorder and early stages of colorectal
cancer (see, for example, the review of McColl, J., NutraCos.
2(4):35-40 (2003)).
[0007] AA belongs to the omega-6 series with four double bonds. The
lack of a double bond in the omega-3 position confers on M
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 is recognized as the
principal omega-6 fatty acid found in the human brain and an
important component of breast milk and many infant formulas, based
on its role in early neurological and visual development. M can be
obtained from some foods (such as meat, fish, and eggs), but the
concentration is low.
[0008] Gamma-linolenic acid (GLA; 18:3, delta-6,9,12) is another
essential fatty acid found in mammals. GLA is the metabolic
intermediate for very long-chain omega-6 fatty acids and for
various active molecules. In mammals, formation of long-chain PUFAs
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).
[0009] As described above, research has shown that various omega
fatty acids reduce the risk of heart disease, have a positive
effect on children's development and on certain mental illnesses,
autoimmune diseases and joint complaints. However, although there
are many health benefits associated with a diet supplemented with
these fatty acids, it is recognized that different PUFAs exert
different physiological effects in the body (e.g., most notably,
the opposing physiological effects of GLA and AA). Thus, production
of oils using recombinant means is expected to have several
advantages over production from natural sources. For example,
recombinant organisms having preferred characteristics for oil
production can be used, since the naturally occurring fatty acid
profile of the host can be altered by the introduction of new
biosynthetic pathways in the host and/or by the suppression of
undesired pathways, thereby resulting in increased levels of
production of desired PUFAs (or conjugated forms thereof) and
decreased production of undesired PUFAs. Optionally, recombinant
organisms can provide PUFAs in particular forms which may have
specific uses; or, oil production can be manipulated such that the
ratio of omega-3 to omega-6 fatty acids so produced is modified
and/or a specific PUFA is produced without significant accumulation
of other PUFA downstream or upstream products (e.g., production of
oils comprising AA and lacking GLA).
[0010] The mechanism of PUFA synthesis frequently occurs via the
delta-6 desaturation pathway. For example, long-chain PUFA
synthesis in mammals proceeds predominantly by a delta-6
desaturation pathway, in which the first step is the delta-6
desaturation of linoleic acid (LA; 18:2, delta-9,12) and
alpha-linolenic acid (ALA; 18:3, delta-9,12,15) to yield
gamma-linolenic acid (GLA; 18:3, delta-6,9,12)) and stearidonic
acid (STA; 18:4, delta-6,9,12,15), respectively. Further fatty acid
elongation and desaturation steps give rise to arachidonic acid (AA
or ARA) and eicosapentaenoic acid (EPA). Accordingly, genes
encoding delta-6 desaturases, delta-6 elongase components (also
identified as C.sub.18/20 elongases) and delta-5 desaturases have
been cloned from a variety of organisms including higher plants,
algae, mosses, fungi, nematodes and humans. Humans can synthesize
long-chain PUFAs from the essential fatty acids, LA and ALA;
however biosynthesis of long-chain PUFAs is somewhat limited (they
are regulated by dietary and hormonal changes), and LA and ALA must
be obtained from the diet.
[0011] Elongases which have been identified in the past differ in
terms of the substrates upon which they act. They are present in
both animals and plants. Those found in mammals can act upon
saturated, monounsaturated and polyunsaturated fatty acids.
However, those found in plants are specific for saturated and
monounsaturated fatty acids. Thus, there is a need for a
PUFA-specific elongase to produce polyunsaturated fatty acids
(PUFAs) in plants.
[0012] The elongation process in plants involves a four-step
process initiated by the crucial step of condensation of malonate
and a fatty acid with release of a carbon dioxide molecule. The
substrates in fatty acid elongation are CoA-thioesters. The
condensation step is mediated by a 3-ketoacyl synthase, which is
generally rate limiting to the overall cycle of four reactions and
provides some substrate specificity. The product of one elongation
cycle regenerates a fatty acid that has been extended by two carbon
atoms (Browse et al., Trends in Biochemical Sciences 27(9):467-473
(September 2002); Napier, Trends in Plant Sciences 7(2): 51-54
(February 2002)).
[0013] WO 02/077213 (published Oct. 3, 2002) describes isolated
nucleic acid molecules encoding a fatty acid elongase with
specificity for linoleic acid or alpha-linolenic acid from
Isochrysis galbana (i.e., delta-9 elongase).
[0014] U.S. Pat. No. 6,403,349 (issued to Mukerji et al. on Jun.
11, 2002) concerns the identification of nucleotide and amino acid
sequences of an elongase gene derived from Mortierella alpina.
[0015] WO 02/26946 (published Apr. 4, 2002) describes isolated
nucleic acid molecules encoding FAD4, FAD5, FAD5-2 and FAD6 fatty
acid desaturase family members which are expressed in long-chain
PUFA-producing organisms, e.g., Thraustochytrium, Pythium
irregulars, Schizichytrium and Crypthecodinium. It is indicated
that 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 long-chain PUFAs.
[0016] WO 98/55625 (published Dec. 19, 1998) describes the
production of PUFAs by expression of polyketide-like synthesis
genes in plants.
[0017] WO 98/46764 (published Oct. 22, 1998) describes compositions
and methods for preparing long-chain fatty acids in plants, plant
parts and plant cells which utilize nucleic acid sequences and
constructs encoding fatty acid desaturases, including delta-5
desaturases, delta-6 desaturases and delta-12 desaturases.
[0018] U.S. Pat. No. 6,075,183 (issued to Knutzon et al. on Jun.
13, 2000) describes methods and compositions for synthesis of
long-chain PUFAs in plants.
[0019] U.S. Pat. No. 6,459,018 (issued to Knutzon et al. on Oct. 1,
2002) describes a method for producing STA in plant seed utilizing
a construct comprising a DNA sequence encoding a delta-6
desaturase.
[0020] Spychalla et al. (Proc. Natl. Acad. Sci. USA, 94:1142-1147
(1997)) describes the isolation and characterization of a cDNA from
Caenorhabditis elegans that, when expressed in Arabidopsis, encodes
a fatty acid desaturase which can catalyze the introduction of an
omega-3 double bond into a range of 18- and 20-carbon fatty
acids.
[0021] An alternate pathway for the biosynthesis of M and EPA
operates in some organisms (i.e., the delta-9 elongase/delta-8
desaturase pathway). Wherby LA and ALA are first elongated to
eicosadienoic acid (EDA; 20:2, delta-11,14) and eicosatrienoic acid
(EtrA; 20:3, delta-11,14,17), respectively, by a delta-9 elongase.
Subsequent delta-8 and delta-5 desaturation of these products
yields AA and EPA. The delta-8 pathway is present inter alia, in
euglenoid species where it is the dominant pathway for formation of
20-carbon PUFAs.
[0022] WO 2000/34439 (published Jun. 15, 2000) discloses amino acid
and nucleic acid sequences for delta-5 and delta-8 desaturase
enzymes. Based on the information presented herein, it is apparent
that the delta-8 nucleotide and amino acid sequences of WO
2000/34439 are not correct. However, the correct sequence is set
forth in corresponding U.S. Pat. No. 6,825,017 (issued to Browse et
al. on Nov. 30, 2004) that describes desaturases, in particular,
delta-5 and delta-8 desaturases and their use in synthesizing
PUFAs.
[0023] Applicants' Assignee's co-pending application having
application Ser. No. 11/166,003 filed Jun. 24, 2005 (Attorney
Docket No. 1547 USNA) discloses a delta-8 desaturase from Euglena
gracilis.
[0024] Wallis et al. (Arch. Biochem. and Biophys. 365(2):307-316
(May 1999)) describes the cloning of a gene that appears to encode
a delta-8 desaturase in Euglena gracilis. This sequence appears t6
be the same sequence disclosed in WO 2000/34439.
[0025] Qi et al. (Nat. Biotech. 22(6):73945 (2004)) describes the
production of long-chain PUFAs using, among other things, a delta-8
desaturase from Euglena gracilis; however, the complete sequence of
the delta-8 desaturase is not provided.
[0026] WO 2004/057001 (published Jul. 8, 2004) discloses amino acid
and nucleic acid sequences for a delta-8 desaturase enzyme from
Euglena gracilis.
[0027] An expansive study of PUFAs from natural sources and from
chemical synthesis are not sufficient for commercial needs.
Therefore, it is of interest to find alternative means to allow
production of commercial quantities of PUFAs. Biotechnology offers
an attractive route for producing long-chain PUFAs in a safe, cost
efficient manner in microorganisms and plants.
[0028] With respect to microorganisms, many algae, bacteria, molds
and yeast can synthesize oils in the ordinary course of cellular
metabolism. Thus, oil production involves cultivating the
microorganism in a suitable culture medium to allow for oil
synthesis, followed by separation of the microorganism from the
fermentation medium and treatment for recovery of the intracellular
oil. Attempts have been made to optimize production of fatty acids
by fermentative means involving varying such parameters as
microorganisms used, media and conditions that permit oil
production. However, these efforts have proved largely unsuccessful
in improving yield of oil or the ability to control the
characteristics of the oil composition produced.
[0029] One class of microorganisms that has not been previously
examined as a production platform for PUFAs (prior to work by the
Applicants' Assignee), however, are the oleaginous yeasts. These
organisms can accumulate oil up to 80% of their dry cell weight.
The technology for growing oleaginous yeast with high oil content
is well developed (for example, see EP 0 005 277B1; Ratledge, C.,
Prog. Ind. Microbiol. 16:119-206 (1982)), and may offer a cost
advantage compared to commercial micro-algae fermentation for
production of omega-3 or omega-6 PUFAs. Whole yeast cells may also
represent a convenient way of encapsulating omega-3 or omega-6
PUFA-enriched oils for use in functional foods and animal feed
supplements.
[0030] WO 2004/101757 and WO 2004/101753 (published Nov. 25, 2004)
concern the production of PUFAs in oleaginous yeasts and are
Applicants' Assignee's copending applications.
[0031] WO 2004/071467 (published Aug. 26, 2004) concerns the
production of PUFAs in plants, while WO 2004/071178 (published Aug.
26, 2004) concerns annexin promoters and their use in expression of
transgenes in plants; both are Applicants' Assignee's copending
applications.
[0032] Applicants' Assignee's copending applications also include
CL2698 (U.S. patent application Ser. No. 11/265761, filed Nov. 2,
2005), CL3136 (U.S. patent application Ser. No. 11/264784, filed
Nov. 1, 2005) and CL3160 (U.S. patent application Ser. No.
11/264737, filed Nov. 1, 2005) (methods of making EPA, ARA and DHA,
respectively, in Yarrowia lipolytica), each claiming benefit of the
earlier provisional filing date of CL2698 on Nov. 4, 2004.
SUMMARY OF THE INVENTION
[0033] The invention concerns an isolated polynucleotide
comprising: [0034] (a) a nucleotide sequence encoding a polypeptide
having delta-9 elongase activity, wherein the polypeptide has at
least 70% amino acid identity, based on the Clustal V method of
alignment, when compared to an amino acid sequence as set forth in
SEQ ID NO:5 or SEQ ID NO:127; [0035] (b) a nucleotide sequence
encoding a polypeptide having delta-9 elongase activity, wherein
the nucleotide sequence has at least 70% sequence identity, based
on the BLASTN method of alignment, when compared to a nucleotide
sequence as set forth in SEQ ID NO:4, SEQ ID NO:90 or SEQ ID
NO:126; [0036] (c) a nucleotide sequence encoding a polypeptide
having delta-9 elongase activity, wherein the nucleotide sequence
hybridizes with a nucleotide sequence as set forth in SEQ ID NO:4,
SEQ ID NO:90 or SEQ ID NO:126 under the following stringent
hybridization conditions: 0.1.times.SSC, 0.1% SDS, 65.degree. C.
and washed with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC,
0.1% SDS; or [0037] (d) a complement of the nucleotide sequence of
(a), (b) or (c), wherein the complement and the nucleotide sequence
consist of the same number of nucleotides and are 100%
complementary.
[0038] In a second embodiment, the invention concerns codon
optimization, specifically, an isolated nucleic acid molecule which
encodes a delta-9 elongase enzyme as set forth in SEQ ID NO:90
wherein at least 106 codons are codon-optimized for expression in
Yarrowia sp.
[0039] In a third embodiment, the invention concerns a recombinant
DNA construct comprising any of the polynucleotides of the
invention operably linked to at least one regulatory sequence.
[0040] In a fourth embodiment, the invention concerns a cell
comprising the recombinant DNA construct of the invention. Of
interest are cells selected from the group consisting of plants and
yeast.
[0041] In a fifth embodiment, the invention concerns a method for
transforming a cell, comprising transforming a cell with the
recombinant construct of the invention and selecting those cells
transformed with the recombinant construct of the invention.
[0042] In a sixth embodiment, the invention concerns a method for
producing a transformed plant comprising transforming a plant cell
with a polynucleotide of the invention and regenerating a plant
from the transformed plant cell. A preferred plant is soybean.
[0043] In an eighth embodiment, the invention concerns a seed
comprising the recombinant construct of the invention.
[0044] In a ninth embodiment, the invention concerns method for
making long-chain polyunsaturated fatty acids in a cell comprising:
[0045] (a) transforming a cell with the recombinant construct of
the invention; [0046] (b) selecting those transformed cells that
make long-chain polyunsaturated fatty acids.
[0047] In a tenth embodiment, the invention concerns oil obtained
from seed comprising the recombinant construct of the
invention.
[0048] In an eleventh embodiment, the invention concerns a method
for making long-chain polyunsaturated fatty acids in a plant cell
comprising: [0049] (a) transforming a cell with the recombinant
construct of the invention; and [0050] (b) selecting those
transformed cells that make long-chain polyunsaturated fatty
acids.
[0051] In a twelfth embodiment, the invention concerns a method for
producing at least one polyunsaturated fatty acid in a soybean cell
comprising: [0052] (a) transforming a soybean cell with a first
recombinant DNA construct comprising an isolated polynucleotide
encoding at least one delta-9 elongate polypeptide, operably linked
to at least one regulatory sequence and at least one additional
recombinant DNA construct comprising an isolated polynucleotide,
operably linked to at least one regulatory sequence, encoding a
polypeptide selected from the group consisting of a delta4
desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-12
desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9
desaturase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a
C.sub.18/20 elongase and a C.sub.20/22 elongase; [0053] (b)
regenerating a soybean plant from the transformed cell of step (a);
and [0054] (c) selecting those seeds obtained from the plants of
step (b) having an altered level of polyunsaturated fatty acids
when compared to the level in seeds obtained from a nontransformed
soybean plant.
[0055] In a thirteenth embodiment, the invention concerns an
oilseed plant comprising: [0056] (a) a first recombinant DNA
construct comprising an isolated polynucleotide encoding at least
one delta-9 elongase polypeptide, operably linked to at least one
regulatory sequence; and [0057] (b) at least one additional
recombinant DNA construct comprising an isolated polynucleotide,
operably linked to at least one regulatory sequence, encoding a
polypeptide selected from the group consisting of a delta-4
desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-12
desaturase, a delta-15 desaturase, a delta-17 desaturase, a delta-9
desaturase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a
C.sub.18/20 elongase and a C.sub.20/22 elongase.
[0058] Also of interest are seeds obtained from such oilseed plants
and oil obtained from these seeds.
[0059] In a fourteenth embodiment, the invention concerns food or
feed which incorporates oil of the invention.
[0060] In a fifteenth embodiment, the invention concerns food or
feed comprising an ingredient derived from the processing of the
seeds of the invention.
[0061] In a sixteenth embodiment, the invention concerns an
isolated nucleic acid fragment comprising a nucleic acid sequence
encoding a delta-9 elongase, excluding SEQ ID NO:27, wherein the
amino acid sequence comprising said elongase contains at least one
of the following amino acid sequence motifs selected from the group
consisting of: TABLE-US-00001 (SEQ ID NO:130) a) Y N X (L or F) X X
X X S X X S F; (SEQ ID NO:131) b) F Y X S K X X (E or D) Y X D (T
or S) X X L; (SEQ ID NO:132) c) L (Q or H) X F H H X G A; (SEQ ID
NO:133) d) M Y X Y Y X X X X X X X (K or R or N) F; (SEQ ID NO:134)
e) K X L (I or L or M) T X X Q; (SEQ ID NO:135) f) W X F N Y X Y;
and (SEQ ID NO:136) g) Y X G X V X X L F;
[0062] wherein X can be any amino acid.
[0063] In a seventeenth embodiment, the invention concerns an
isolated polynucleotide comprising a nucleotide sequence encoding a
polypeptide having delta-9 elongase activity, wherein the
nucleotide sequence has at least 70% sequence identity, based on
the BLASTN method of alignment, when compared to a nucleotide
sequence as set forth in SEQ ID NO:4, SEQ ID NO:90 ir SEQ ID
NO:126.
[0064] In an eighteenth embodiment, the invention concerns progeny
plants obtained from obtained from a plant made by the method of
the invention or an oilseed plant of the invention.
Biological Deposits
[0065] The following plasmids have been deposited with the American
Type Culture Collection (ATCC), 10801 University Boulevard,
Manassas, Va. 20110-2209, and bear the following designations,
Accession Numbers and dates of deposit (Table 1). TABLE-US-00002
TABLE 1 ATCC Deposits Plasmid Accession Number Date of Deposit
pKR72 PTA-6019 May 28, 2004 pKR275 PTA-4989 Jan. 30, 2003 pKR585
PTA-6279 Nov. 4, 2004 pKR578 PTA-6280 Nov. 4, 2004
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0066] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing, which form a part of this application.
[0067] A Sequence Listing is provided herewith on Compact Disk. The
contents of the Compact Disk containing the Sequence Listing are
hereby incorporated by reference in compliance with 37 C.F.R.
.sctn.1.52(e). The Compact Disks are submitted in triplicate and
are identical to one another. The disks are labeled "Copy
1--Sequence Listing", "Copy 2--Sequence Listing", and
"CRF--Sequence Listing". The disks contain the following file:
BB1562 US NA Sequence Listing.ST25 having the following size:
572,000 bytes and which were created Nov. 16, 2006.
[0068] The sequence descriptions summarize the Sequences Listing
provided herewith. The Sequence Listing contains one letter codes
for nucleotide sequence characters and the single and three letter
codes for amino acids as defined in the IUPAC-IUB standards
described in Nucleic Acids Research 13:3021-3030 (1985) and in the
Biochemical Journal 219(2):345-373 (1984).
[0069] FIG. 1 is a map of plasmid pY115.
[0070] FIG. 2 is a map of Yarrowia lipolytica Gateway.RTM.
destination vector pBY1.
[0071] FIG. 3 is a map of plasmid pBY2.
[0072] FIG. 4 is a map of plasmid pBY1-FAE.
[0073] FIG. 5 is a map of plasmid pY120.
[0074] FIG. 6 is a map of plasmid pY119.
[0075] FIG. 7 is a map of plasmid pKR912.
[0076] FIG. 8 is a map of plasmid pKR911.
[0077] FIG. 9 is a map of plasmid pKR913.
[0078] FIG. 10 is a map of plasmids pKR886 and pKR886r.
[0079] FIG. 11 is a map of plasmid pKR669.
[0080] FIG. 12 is a map of plasmid pKR873.
[0081] FIG. 13 shows a chromatogram of the lipid profile of an
Euglena gracilis cell extract as described in the Examples.
[0082] FIG. 14 are the results of functional analysis of the
Euglena gracilis delta-9 elongase in Saccharomyces cerevisiae.
[0083] FIG. 15 is a representative omega-3 and omega-6 fatty acid
pathway providing for the conversion of myristic acid through
various intermediates to docosahexaenoic acid (DHA).
[0084] FIG. 16 shows a Clustal V alignment (with default
parameters) of SEQ ID NO:5 (amino acid sequence of the delta-9
elongase of the instant invention) and SEQ ID NO:27 (amino acid
sequence of the long-chain PUFA elongation enzyme from Isochrysis
galbana (NCBI Accession No. ML37626 (GI 17226123)).
[0085] FIG. 17 is a map of plasmid pKR920.
[0086] FIG. 18 is a map of plasmid pKR926.
[0087] FIG. 19 is a map of plasmid pKR916.
[0088] FIG. 20 are the results of the fatty acid analysis of
transgenic somatic soybean embryos expressing pKR912.
[0089] FIG. 21 are the lipid profiles of the somatic soybean
embryos expressing the Euglena gracilis delta-9 elongase and the
Euglena gracilis delta-8 desaturase for the top 5 events.
[0090] FIG. 22 are the lipid profiles of T2 bulk seed seed for the
22 wild-type-transformed events as well as for untransformed
wild-type.
[0091] FIG. 23 are the lipid profiles of T2 bulk seed seed for the
16 fad3/fae1-transformed events as well as for untransformed
fad3/fae1.
[0092] FIG. 24 are the lipid profiles for ten single seeds for
wild-type pKR926-8 and ff pKR926-1.
[0093] FIG. 25A are the average fatty acid profiles for the ten
best EPA events (average of nine or ten individual embryos) for
pKR916 and pKR873.
[0094] FIG. 25B are the fatty acid profiles for the five best EPA
seed from two independent events.
[0095] FIGS. 26A and B show a comparison of the nucleotide
sequences of Euglena gracilis delta-9 elongase (EgD9e) (SEQ ID
NO:4) and the synthetic delta-9 elongase, derived from Euglena
gracilis and codon-optimized for expression in Yarrowia lipolytica
(EgD9eS) (SEQ ID NO:90).
[0096] FIG. 27 show a Clustal W alignment (with default parameters)
of SEQ ID NO:5 (amino acid sequence of the delta-9 elongase of the
instant invention) and SEQ ID NO:27 (amino acid sequence of the
long-chain PUFA elongation enzyme from Isochrysis galbana (NCBI
Accession No. AAL37626 (GI 17226123)).
[0097] FIG. 28 is a map of plasmid pFBAln-389Elo.
[0098] FIG. 29 shows a Clustal V alignment (with default
parameters) of the amino acid sequence of the Euglena gracilis
delta-9 elongase of the instant invention (SEQ ID NO:5), the amino
acid sequence of the Eutreptiella sp. CCMP389 delta-9 elongase of
the instant invention (SEQ ID NO:127) and the amino acid sequence
of the long-chain PUFA elongation enzyme from Isochrysis galbana
(NCBI Accession No. AAL37626 (GI 17226123)) (SEQ ID NO:27).
[0099] SEQ ID NO:1 is the 5' sequence of the cDNA insert from clone
eeg1c.pk001.n5.f.
[0100] SEQ ID NO:2 is the 3' sequence of the cDNA insert from clone
eeg1c.pk001.n5.f.
[0101] SEQ ID NO:3 is the sequence aligned from SEQ ID NO:1 and SEQ
ID NO:2 (full cDNA sequence excluding polyA tail).
[0102] SEQ ID NO:4 is nucleotide sequence of the CDS of the Euglena
gracilis delta-9 elongase of the instant invention (clone
eeg1c.pk001.n5.f (abbreviated EgD9e).
[0103] SEQ ID NO:5 is the deduced amino acid sequence of SEQ ID
NO:4 (Euglena gracilis delta-9 elongase of the instant
invention-clone eeg1c.pk001.n5.f.
[0104] SEQ ID NO:6 is the sequence of the ig-s primer.
[0105] SEQ ID NO:7 is the sequence of the ig-as primer.
[0106] SEQ ID NO:8 is the sequence of the oEugEL1-1 primer.
[0107] SEQ ID NO:9 is the sequence of the oEugEL1-2 primer.
[0108] SEQ ID NO:10 is the sequence of the Eg5-1 primer.
[0109] SEQ ID NO:11 is the sequence of the Eg3-3 primer.
[0110] SEQ ID NO:12 is the sequence of T7.
[0111] SEQ ID NO:13 is the sequence of M13-28Rev.
[0112] SEQ ID NO:14 is the sequence of Eg3-2.
[0113] SEQ ID NO:15 is the sequence of Eg5-2.
[0114] SEQ ID NO:16 is the nucleotide sequence for the Euglena
gracilis delta-8 desaturase (Eg5).
[0115] SEQ ID NO:17 is the amino acid sequence for the Euglena
gracilis delta-8 desaturase (Eg5) shown in SEQ ID NO:16.
[0116] SEQ ID NO:18 is the sequence of the KTi cassette 5' end MCS
for pKR457.
[0117] SEQ ID NO:19 is the sequence of the KTi cassette 3' end MCS
for pKR457 including the soy albumin transcription 3'
terminator.
[0118] SEQ ID NO:20 is the sequence of the oSalb-12 primer.
[0119] SEQ ID NO:21 is the sequence of the oSalb-13 primer.
[0120] SEQ ID NO:22 is the sequence of restriction sites added to
pKR287 to make pKR767.
[0121] SEQ ID NO:23 is the sequence of the oSAlb-9 primer.
[0122] SEQ ID NO:24 is the sequence of the oSAlb-2 primer.
[0123] SEQ ID NO:25 is the sequence of pZUF17.
[0124] SEQ ID NO:26 is the sequence of pDMW237.
[0125] SEQ ID NO:27 amino acid sequence of the long-chain PUFA
elongation enzyme from Isochrysis galbana (NCBI Accession No.
AAL37626 (GI 17226123), locus AAL37626, CDS AF390174).
[0126] SEQ ID NO:28 is the sequence of the M13F universal
primer.
[0127] SEQ ID NO:29 is the nucleotide sequence of the long-chain
PUFA elongation enzyme from Isochrysis galbana (NCBI Accession No.
AAL37626 (GI 17226123), locus AAL37626, CDS AF390174) shown in SEQ
ID NO:27.
[0128] SEQ ID NO:30 is the sequence of pY115 (see FIG. 1).
[0129] SEQ ID NO:31 is the sequence of pBY1 (see FIG. 2).
[0130] SEQ ID NO:32 is the sequence of pBY2 (see FIG. 3).
[0131] SEQ ID NO:33 is the sequence of pBY1-FAE (see FIG. 4).
[0132] SEQ ID NO:34 is the sequence of pY120(see FIG. 5).
[0133] SEQ ID NO:35 is the sequence of pY119 (see FIG. 6).
[0134] SEQ ID NO:36 is the sequence of pKR72.
[0135] SEQ ID NO:37 is the sequence of pKR912 (see FIG. 7).
[0136] SEQ ID NO:38 is the sequence of pKS102.
[0137] SEQ ID NO:39 is the sequence of pKR197.
[0138] SEQ ID NO:40 is the sequence of pKR911 (see FIG. 8).
[0139] SEQ ID NO:41 is the sequence of pKS121.
[0140] SEQ ID NO:42 is the sequence of pKR457.
[0141] SEQ ID NO:43 is the sequence of pKR680.
[0142] SEQ ID NO:44 is the sequence of pKR913 (see FIG. 9).
[0143] SEQ ID NO:45 is the sequence of pKR767.
[0144] SEQ ID NO:46 is the sequence of pKR328.
[0145] SEQ ID NO:47 is the sequence of pKR886 (see FIG. 10).
[0146] SEQ ID NO:48 is the sequence of pKR886r (see FIG. 10).
[0147] SEQ ID NO:49 is the sequence of pKR271.
[0148] SEQ ID NO:50 is the sequence of pKR226.
[0149] SEQ ID NO:51 is the sequence of pKR275.
[0150] SEQ ID NO:52 is the sequence of pKR329.
[0151] SEQ ID NO:53 is the sequence of pKR585.
[0152] SEQ ID NO:54 is the sequence of pKR578.
[0153] SEQ ID NO:55 is the sequence of pKR667.
[0154] SEQ ID NO:56 is the sequence of pKR873 (see FIG. 12).
[0155] SEQ ID NO:57 is the sequence of pKR132.
[0156] SEQ ID NO:58 is the sequence of pKR160.
[0157] SEQ ID NO:59 is the sequence of pKR124.
[0158] SEQ ID NO:60 is the sequence of pKR163.
[0159] SEQ ID NO:61 is the sequence of pY34.
[0160] SEQ ID NO:62 is the sequence of pKR863.
[0161] SEQ ID NO:63 is the sequence of pKR869.
[0162] SEQ ID NO:64 is the sequence of pKR270.
[0163] SEQ ID NO:65 is the nucleotide sequence for the synthetic
delta-9 elongase derived from Isochrysis galbana codon-optimized
for expression in Yarrowia lipolytica.
[0164] SEQ ID NOs:66-81 correspond to primers IL3-1A, IL3-1B,
IL3-2A, IL3-2B, IL3-3A, IL3-3B, IL3-4A, IL3-4B, IL3-5A, IL3-5B,
IL3-6A, IL3-6B, IL3-7A, IL3-7B, IL3-8A, IL3-8A and IL3-8B,
respectively, used for amplification as described in Example 4.
[0165] SEQ ID NOs:82-85 correspond to primers IL3-1F, IL3-4R,
IL3-5F and IL3-8R, respectively, used for amplification as
described in Example 4.
[0166] SEQ ID NO:86 is the 417 bp NcoI/PstI fragment described in
Example 4.
[0167] SEQ ID NO:87 is the 377 bp PstI/Not1 fragment described in
Example 4.
[0168] SEQ ID NO:88 is the sequence of the Mortierella alpina
delta-5 desaturase.
[0169] SEQ ID NO:89 is the sequence of pDMW263.
[0170] SEQ ID NO:90 is the nucleotide sequence for the synthetic
delta-9 elongase derived from Euglena gracilis codon-optimized for
expression in Yarrowia lipolytica.
[0171] SEQ ID NO:91 is the sequence of pKR920 (see FIG. 17).
[0172] SEQ ID NO:92 is the sequence of cal a24-4.
[0173] SEQ ID NO:93 is the sequence of primer oCal-15.
[0174] SEQ ID NO:94 is the sequence of primer oCal-6.
[0175] SEQ ID NO:95 is the sequence of pKR53B
[0176] SEQ ID NO:96 is the sequence of pKR85.
[0177] SEQ ID NO:97 is the sequence of primer oKR85-1.
[0178] SEQ ID NO:98 is the sequence of primer oKR85-2.
[0179] SEQ ID NO:99 is the sequence of pPCR85.
[0180] SEQ ID NO:100 is the sequence of pKR91.
[0181] SEQ ID NO:101 is the sequence of pKR92.
[0182] SEQ ID NO:102 is the sequence of pKR926 (see FIG. 18)
[0183] SEQ ID NO:103 is the sequence of pKR767.
[0184] SEQ ID NO:104 is the sequence of pKR916 (see FIG. 19)
[0185] SEQ ID NO:105 is the sequence of pZuFmEgD9ES.
[0186] SEQ ID NO:106 is the sequence of pZuFmEgD9E.
[0187] SEQ ID NO:107 is the sequence of the SMART.TM. IV
oligonucleotide.
[0188] SEQ ID NO:108 is the sequence of the CDSIII/3' PCR
primer.
[0189] SEQ ID NO:109 is the sequence of the 5'-PCR primer.
[0190] SEQ ID NO:110 is the sequence of pFBAIN-389Elo.
[0191] SEQ ID NO:111 is the sequence of the Eutreptiella sp.
CCMP389 (E389D9e) complete assembled contig.
[0192] SEQ ID NO:112 is the nucleotide sequence of degenerate
primer EuEF3.
[0193] SEQ ID NO:113 is the deduced amino acid sequence of SEQ ID
NO:112.
[0194] SEQ ID NO:114 is the nucleotide sequence of degenerate
primer EuER3.
[0195] SEQ ID NO:115 is the deduced amino acid sequence of SEQ ID
NO:114.
[0196] SEQ ID NO:116 is the sequence of the 389Elo-5-1 primer.
[0197] SEQ ID NO:117 is the sequence of the 389Elo-5-2 primer.
[0198] SEQ ID NO:118 is the sequence of the DNR CDS 5'-2
primer.
[0199] SEQ ID NO:119 is the sequence of the Eutreptiella sp.
CCMP389 (E389D9e) 5' cDNA fragment #1.
[0200] SEQ ID NO:120 is the sequence of the 389Elo-5-4 primer.
[0201] SEQ ID NO:121 is the sequence of the Eutreptiella sp.
CCMP389 (E389D9e) 5' cDNA fragment #2.
[0202] SEQ ID NO:122 is the sequence of the 389Elo-3-1 primer.
[0203] SEQ ID NO:123 is the sequence of the 389Elo-3-2 primer.
[0204] SEQ ID NO:124 is the sequence of the Eutreptiella sp.
CCMP389 (E389D9e) 3' cDNA fragment.
[0205] SEQ ID NO:125 is the sequence of the Eutreptiella sp.
CCMP389 (E389D9e) complete assembled contig.
[0206] SEQ ID NO:126 is the nucleotide sequence of the Eutreptiella
sp. CCMP389 (E389D9e) delta-9 elongase.
[0207] SEQ ID NO:127 is the deduced amino acid sequence of SEQ ID
NO:126 (Eutreptiella sp. CCMP389 (E389D9e) delta-9 elongase).
[0208] SEQ ID NO:128 is the sequence of pFBAIN-MOD-1.
[0209] SEQ ID NO:129 is the sequence of the Eutreptiella sp.
CCMP389 (E389D9e) internal cDNA fragment.
[0210] SEQ ID NOs:130-136 are the motif sequences associated with a
delta-9 elongase.
DETAILED DESCRIPTION OF THE INVENTION
[0211] All patents, patent applications, and publications cited
herein are incorporated by reference in their entirety.
[0212] In the context of this disclosure, a number of terms shall
be utilized.
[0213] The term "fatty acids" refers to long-chain aliphatic acids
(alkanoic acids) of varying chain lengths, from about C.sub.12 to
C.sub.22 (although both longer and shorter chain-length acids are
known). The predominant chain lengths are between C.sub.16 and
C.sub.22. Additional details concerning the differentiation between
"saturated fatty acids" versus "unsaturated fatty acids",
"monounsaturated fatty acids" versus "polyunsaturated fatty acids"
(or "PUFAs"), and "omega-6 fatty acids" (.omega.-6 or n-6) versus
"omega-3 fatty acids" (.omega.-3 or n-3) are provided in WO
2004/101757.
[0214] Fatty acids are described herein by a simple notation system
of "X:Y", wherein the number before the colon indicates the number
of carbon atoms in the fatty acid and the number after the colon is
the number of double bonds that are present. The number following
the fatty acid designation indicates the position of the double
bond from the carboxyl end of the fatty acid with the "c" affix for
the cis-configuration of the double bond (e.g., palmitic acid
(16:0), stearic acid (18:0), oleic acid (18:1, 9c), petroselinic
acid (18:1, 6c), LA (18:2, 9c,12c), GLA (18:3, 6c,9c,12c) and ALA
(18:3, 9c,12c,15c)). Unless otherwise specified 18:1, 18:2 and 18:3
refer 5 to oleic, LA and linolenic fatty acids. If not specifically
written as otherwise, double bonds are assumed to be of the cis
configuration. For instance, the double bonds in 18:2 (9,12) would
be assumed to be in the cis configuration.
[0215] A representative pathway is illustrated in FIG. 15,
providing for the conversion of myristic acid through various
intermediates to DHA, which demonstrates how both omega-3 and
omega-6 fatty acids may be produced from a common source.
[0216] Nomenclature used to describe PUFAs in the present
disclosure is shown below in Table 2. In the column titled
"Shorthand Notation", the omega-reference system is used to
indicate the number of carbons, the number of double bonds and the
position of the double bond closest to the omega carbon, counting
from the omega carbon (which is numbered 1 for this purpose). The
remainder of the Table summarizes the common names of omega-3 and
omega-6 fatty acids, the abbreviations that will be used throughout
the remainder of the specification, and each compounds' chemical
name. TABLE-US-00003 TABLE 2 Nomenclature of Polyunsaturated Fatty
Acids Common Shorthand Name Abbreviation Chemical Name Notation
oleic -- cis-9-octadecenoic 18:1 linoleic LA
cis-9,12-octadecadienoic 18:2 .omega.-6 gamma- GLA cis-6,9,12- 18:3
.omega.-6 linolenic octadecatrienoic eicosadienoic EDA
cis-11,14-eicosadienoic 20:2 .omega.-6 dihomo- DGLA or
cis-8,11,14-eicosatrienoic 20:3 .omega.-6 gamma- HGLA linolenic
arachidonic AA or ARA cis-5,8,11,14- 20:4 .omega.-6
eicosatetraenoic alpha-linolenic ALA cis-9,12,15- 18:3 .omega.-3
octadecatrienoic stearidonic STA cis-6,9,12,15- 18:4 .omega.-3
octadecatetraenoic eicosatrienoic ETrA or ERA cis-11,14,17- 20:3
.omega.-3 eicosatrienoic eicosatetraenoic ETA cis-8,11,14,17- 20:4
.omega.-3 eicosatetraenoic eicosapentaenoic EPA cis-5,8,11,14,17-
20:5 .omega.-3 eicosapentaenoic docosapentaenoic DPA
cis-7,10,13,16,19- 22:5 .omega.-3 docosapentaenoic docosahexaenoic
DHA cis-4,7,10,13,6,19- 22:6 .omega.-3 docosahexaenoic
[0217] Other abbreviations that may be used are as follows (the
terms and abbreviations may be used interchangeably): [0218]
EgD9e=Euglena gracilis delta-9 elongase (wild-type) [0219]
EgD9eS=synthetic delta-9 elongase, derived from Euglena gracilis
and codon-optimized for expression in Yarrowia lipolytica [0220]
E389D9e=Eutreptiella sp. CCMP389delta-9 elongase (wild-type) [0221]
E389D9eS=synthetic delta-9 elongase, derived from Eutreptiella sp.
CCMP389 and codon-optimized for expression in Yarrowia
lipolytica
[0222] The term "essential fatty acid" refers to a particular PUFA
that an organism must ingest in order to survive, being unable to
synthesize the particular essential fatty acid de novo. For
example, mammals can not synthesize the essential fatty acid LA.
Other essential fatty acids include, but are not limited to, GLA,
DGLA, AA, EPA and DHA.
[0223] The term "fat" refers to a lipid substance that is solid at
25.degree. C. and usually saturated.
[0224] The term "oil" refers to a lipid substance that is liquid at
25.degree. C. and usually polyunsaturated. PUFAs are found in the
oils of some algae, oleaginous yeasts and filamentous fungi.
"Microbial oils" or "single cell oils" are those oils naturally
produced by microorganisms during their lifespan. Such oils can
contain long-chain PUFAs.
[0225] The term "PUFA biosynthetic pathway" refers to a metabolic
process that converts oleic acid to LA, EDA, GLA, DGLA, AA, ALA,
STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in
the literature (e.g., see WO 2005/003322 and WO 2006/052870).
Simplistically, this process involves elongation of the carbon
chain through the addition of carbon atoms and desaturation of the
molecule through the addition of double bonds, via a series of
special desaturation and elongation enzymes (i.e., "PUFA
biosynthetic pathway enzymes") present in the endoplasmic reticulim
membrane. More specifically, "PUFA biosynthetic pathway enzymes"
refer to any of the following enzymes (and genes which encode said
enzymes) associated with the biosynthesis of a PUFA, including: a
delta4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a
delta-12 desaturase, a delta-15 desaturase, a delta-17 desaturase,
a delta-9 desaturase, a delta-8 desaturase, a delta-9 elongase, a
C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20
elongase and/or a C.sub.20/22 elongase.
[0226] "Desaturase" is a polypeptide which can desaturate one or
more fatty acids to produce a mono- or poly-unsaturated fatty acid
or precursor which is of interest. Of particular interest herein
are delta-8 desaturases that will desaturate a fatty acid between
the eighth and ninth carbon atom numbered from the
carboxyl-terminal end of the molecule and that can, for example,
catalyze the conversion of EDA to DGLA and/or ETrA to ETA. Other
useful fatty acid desaturases include, for example, (1) delta-5
desaturases that catalyze the conversion of DGLA to AA and/or ETA
to EPA; (2) delta-6 desaturases that catalyze the conversion of LA
to GLA and/or ALA to STA; (3) delta-4 desaturases that catalyze the
conversion of DPA to DHA; (4) delta-12 desaturases that catalyze
the conversion of oleic acid to LA; (5) delta-15 desaturases that
catalyze the conversion of LA to ALA and/or GLA to STA; (6)
delta-17 desaturases that catalyze the conversion of M to EPA
and/or DGLA to ETA; and (7) delta-9 desaturases that catalyze the
conversion of palmitate to palmitoleic acid (16:1) and/or stearate
to oleic acid (18:1).
[0227] The term "elongase system" refers to a suite of four enzymes
that are responsible for elongation of a fatty acid carbon chain to
produce a fatty acid that is two carbons longer than the fatty acid
substrate that the elongase system acts upon. More specifically,
the process of elongation occurs in association with fatty acid
synthase, whereby CoA is the acyl carrier (Lassner et al., Plant
Cell 8:281-292 (1996)). In the first step, which has been found to
be both substrate-specific and also rate-limiting, malonyl-CoA is
condensed with a long-chain acyl-CoA to yield carbon dioxide
(CO.sub.2) and a .beta.-ketoacyl-CoA (where the acyl moiety has
been elongated by two carbon atoms). Subsequent reactions include
reduction to .beta.-hydroxyacyl-CoA, dehydration to an enoyl-CoA
and a second reduction to yield the elongated acyl-CoA. Examples of
reactions catalyzed by elongase systems are the conversion of GLA
to DGLA, STA to ETA and EPA to DPA.
[0228] For the purposes herein, an enzyme catalyzing the first
condensation reaction (i.e., conversion of malonyl-CoA to
.beta.-ketoacyl-CoA) will be referred to generically as an
"elongase". In general, the substrate selectivity of elongases is
somewhat broad but segregated by both chain length and the degree
of unsaturation. Accordingly, elongases can have different
specificities. For example, a C.sub.14/16 elongase will utilize a
C.sub.14 substrate (e.g., myristic), a C.sub.16/18 elongase will
utilize a C.sub.16 substrate (e.g., palmitate), a C.sub.18/20
elongase will utilize a C.sub.18 substrate (e.g., GLA, STA) and a
C.sub.20/22 elongase will utilize a C.sub.20 substrate (e.g., EPA).
In like manner, and of particular interest herein, a "delta-9
elongase" is able to catalyze the conversion of LA and ALA to EDA
and ETrA. It is important to note that some elongases have broad
specificity and thus a single enzyme may be capable of catalyzing
several elongase reactions (e.g., thereby acting as both a
C.sub.16/18elongase and a C.sub.18/20 elongase). In preferred
embodiments, it is desirable to empirically determine the
specificity of a fatty acid elongase by transforming a suitable
host with the gene for the fatty acid elongase and determining its
effect on the fatty acid profile of the host.
[0229] The term "delta-9 elongase/delta-8 desaturase pathway"
refers to a biosynthetic pathway for production of long-chain
PUFAs. This pathway, at a minimum, comprises a delta-9 elongase and
a delta-8 desaturase, thereby enabling biosynthesis of DGLA and/or
ETA from LA and ALA, respectively.
[0230] The term "delta-9 elongase" refers to an enzyme that is
capable of catalyzing at least one elongase reaction such as the
elongaton of linoleic or alpha-linolenic acid to EDA or ETrA,
respectively. It may act as both a C.sub.16/18 elongase and a
C.sub.18/20 elongase.
[0231] The term "amino acid" will refer to the basic chemical
structural unit of a protein or polypeptide. Amino acids are
identified by either the one-letter code or the three-letter codes
for amino acids, in conformity with the IUPAC-IYUB standards
described in Nucleic Acids Research, 13:3021-3030 (1985) and in the
Biochemical Journal, 219 (2):345-373 (1984), which are herein
incorporated by reference.
[0232] The term "conservative amino acid substitution" refers to a
substitution of an amino acid residue in a given protein with
another amino acid, without altering the chemical or functional
nature of that protein. For example, it is well known in the art
that alterations in a gene that result in the production of a
chemically equivalent amino acid at a given site (but that do not
affect the structural and functional properties of the encoded,
folded protein) are common. For the purposes of the present
invention, "conservative amino acid substitutions" are defined as
exchanges within one of the following five groups: [0233] 1. small
aliphatic, nonpolar or slightly polar residues: Ala [A], Ser [S],
Thr [T] (Pro [P], Gly [G]); [0234] 2. polar, negatively charged
residues and their amides: Asp [D], Asn [N], Glu [E], Gln [Q];
[0235] 3. polar, positively charged residues: His [H], Arg [R], Lys
[K]; [0236] 4. large aliphatic, nonpolar residues: Met [M], Leu
[L], lie [I], Val M (Cys [C]); and, [0237] 5. large aromatic
residues: Phe [F], Tyr [Y], Trp [W]. Conservative amino acid
substitutions generally maintain: 1) the structure of the
polypeptide backbone in the area of the substitution; 2) the charge
or hydrophobicity of the molecule at the target site; or 3) the
bulk of the side chain. Additionally, in many cases, alterations of
the N-terminal and C-terminal portions of the protein molecule
would also not be expected to alter the activity of the
protein.
[0238] The term "non-conservative amino acid substitution" refers
to an amino acid substitution that is generally expected to produce
the greatest change in protein properties. Thus, for example, a
non-conservative amino acid substitution would be one whereby: 1.)
a hydrophilic residue is substituted for/by a hydrophobic residue
(e.g., Ser or Thr for/by Leu, Ile, Val); 2.) a Cys or Pro is
substituted for/by any other residue; 3.) a residue having an
electropositive side chain is substituted for/by an electronegative
residue (e.g., Lys, Arg or His for/by Asp or Glu); or, 4.) a
residue having a bulky side chain is substituted for/by one not
having a side chain (e.g., Phe for/by Gly). Sometimes,
non-conservative amino acid substitutions between two of the five
groups will not affect the activity of the encoded protein.
[0239] The terms "polynucleotide", "polynucleotide sequence",
"nucleic acid sequence", "nucleic acid fragment" and "isolated
nucleic acid fragment" are used interchangeably herein. These terms
encompass nucleotide sequences and the like. A polynucleotide may
be a polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic
DNA, or mixtures thereof. Nucleotides (usually found in their
5'-monophosphate form) are referred to by a single letter
designation as follows: "A" for adenylate or deoxyadenylate (for
RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate,
"G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for
deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C
or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and
"N" for any nucleotide.
[0240] The terms "subfragment that is functionally equivalent" and
"functionally equivalent subfragment" are used interchangeably
herein. These terms refer to a portion or subsequence of an
isolated nucleic acid fragment in which the ability to alter gene
expression or produce a certain phenotype is retained whether or
not the fragment or subfragment encodes an active enzyme. For
example, the fragment or subfragment can be used in the design of
chimeric genes to produce the desired phenotype in a transformed
plant. Chimeric genes can be designed for use in suppression by
linking a nucleic acid fragment or subfragment thereof, whether or
not it encodes an active enzyme, in the sense or antisense
orientation relative to a plant promoter sequence.
[0241] The term "conserved domain" or "motif" means a set of amino
acids conserved at specific positions along an aligned sequence of
evolutionarily related proteins. While amino acids at other
positions can vary between homologous proteins, amino acids that
are highly conserved at specific positions indicate amino acids
that are essential in the structure, the stability, or the activity
of a protein. Because they are identified by their high degree of
conservation in aligned sequences of a family of protein
homologues, they can be used as identifiers, or "signatures", to
determine if a protein with a newly determined sequence belongs to
a previously identified protein family.
[0242] The terms "homology", "homologous", "substantially similar"
and "corresponding substantially" are used interchangeably herein.
They refer to nucleic acid fragments wherein changes in one or more
nucleotide bases do not affect the ability of the nucleic acid
fragment to mediate gene expression or produce a certain phenotype.
These terms also refer to modifications of the nucleic acid
fragments of the instant invention such as deletion or insertion of
one or more nucleotides that do not substantially alter the
functional properties of the resulting nucleic acid fragment
relative to the initial, unmodified fragment. It is therefore
understood, as those skilled in the art will appreciate, that the
invention encompasses more than the specific exemplary
sequences.
[0243] Moreover, the skilled artisan recognizes that substantially
similar nucleic acid sequences encompassed by this invention are
also defined by their ability to hybridize (under moderately
stringent conditions, e.g., 0.5.times.SSC, 0.1% SDS, 60.degree. C.)
with the sequences exemplified herein, or to any portion of the
nucleotide sequences disclosed herein and which are functionally
equivalent to any of the nucleic acid sequences disclosed herein.
Stringency conditions can be adjusted to screen for moderately
similar fragments, such as homologous sequences from distantly
related organisms, to highly similar fragments, such as genes that
duplicate functional enzymes from closely related organisms.
Post-hybridization washes determine stringency conditions.
[0244] The term "selectively hybridizes" includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to a specified nucleic acid target sequence
to a detectably greater degree (e.g., at least 2-fold over
background) than its hybridization to non-target nucleic acid
sequences and to the substantial exclusion of non-target nucleic
acids. Selectively hybridizing sequences typically have about at
least 80% sequence identity, or 90% sequence identity, up to and
including 100% sequence identity (i.e., fully complementary) with
each other.
[0245] The term "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will selectively hybridize to its target sequence. Stringent
conditions are sequence-dependent and will be different in
different circumstances. By controlling the stringency of the
hybridization and/or washing conditions, target sequences can be
identified which are 100% complementary to the probe (homologous
probing). Alternatively, stringency conditions can be adjusted to
allow some mismatching in sequences so that lower degrees of
similarity are detected (heterologous probing). Generally, a probe
is less than about 1000 nucleotides in length, optionally less than
500 nucleotides in length. Typically, stringent conditions will be
those in which the salt concentration is less than about 1.5 M Na
ion, typically about 0.01 to 1.0 M Na ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g., greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. Exemplary low
stringency conditions include hybridization with a buffer solution
of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate)
at 37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and a wash in 0.1.times.SSC at 60 to 65.degree. C. Other exemplary
stringent hybridization conditions include 0.1.times.SSC, 0.1% SDS,
65.degree. C. and washed with 2.times.SSC, 0.1% SDS followed by
0.1.times.SSC, 0.1% SDS.
[0246] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth et al.,
Anal. Biochem. 138:267-284 (1984): T.sub.m=81.5.degree. C.+16.6
(log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of
monovalent cations, % GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, hybridization and/or wash conditions can be adjusted to
hybridize to sequences of the desired identity. For example, if
sequences with .gtoreq.90% identity are sought, the T.sub.m can be
decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent
conditions can utilize a hybridization and/or wash at 1, 2, 3, or
4.degree. C. lower than the thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting
point (T.sub.m); low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C.
lower than the thermal melting point (T.sub.m). Using the equation,
hybridization and wash compositions, and desired T.sub.m, those of
ordinary skill will understand that variations in the stringency of
hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a T.sub.m of less than
45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution) it is preferred to increase the SSC concentration so that
a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Laboratory
Techniques in Biochemistry and Molecular Biology-Hybridization with
Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of
hybridization and the strategy of nucleic acid probe assays",
Elsevier, New York (1993); and Current Protocols in Molecular
Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and
Wiley-Interscience, New York (1995). Hybridization and/or wash
conditions can be applied for at least 10, 30, 60, 90, 120, or 240
minutes.
[0247] "Sequence identity" or "identity" in the context of nucleic
acid or polypeptide sequences refers to the nucleic acid bases or
amino acid residues in the two sequences that are the same when
aligned for maximum correspondence over a specified comparison
window.
[0248] Thus, "percentage of sequence identity" refers to the valued
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the results by 100 to yield the percentage of sequence
identity. Useful examples of percent sequence identities include,
but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95%, or any integer percentage from 50% to 100%, such as
such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. These
identities can be determined using any of the programs described
herein.
[0249] Sequence alignments and percent identity or similarity
calculations may be determined using a variety of comparison
methods designed to detect homologous sequences including, but not
limited to, the MegAlign.TM. program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences are performed using the Clustal
V method of alignment (Higgins, D. G. and Sharp, P. M. (1989)
Comput. Appl. Biosci. 5:151-153; Higgins, D. G. et al. (1992)
Comput. Appl. Biosci. 8:189-191) with the default parameters (GAP
PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise
alignments and calculation of percent identity of protein sequences
using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and
DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,
GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.
[0250] It is well understood by one skilled in the art that many
levels of sequence identity are useful in identifying polypeptides,
from other species, wherein such polypeptides have the same or
similar function or activity. Useful examples of percent identities
include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, or 95%, or any integer percentage from 50% to 100%, such
as such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
Indeed, any integer amino acid identity from 50% to 100% may be
useful in describing the present invention. Also, of interest is
any full-length or partial complement of this isolated nucleotide
fragment.
[0251] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Chimeric gene" refers to any
gene that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature. A
"foreign" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene
that has been introduced into the genome by a transformation
procedure.
[0252] A "codon-optimized gene" is a gene having its frequency of
codon usage designed to mimic the frequency of preferred codon
usage of the host cell.
[0253] An "allele" is one of several alternative forms of a gene
occupying a given locus on a chromosome. When all the alleles
present at a given locus on a chromosome are the same that plant is
homozygous at that locus. If the alleles present at a given locus
on a chromosome differ that plant is heterozygous at that
locus.
[0254] "Coding sequence" refers to a DNA sequence that codes for a
specific amino acid sequence. "Regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include, but are not limited to:
promoters, translation leader sequences, introns, polyadenylation
recognition sequences, RNA processing sites, effector binding sites
and stem-loop structures.
[0255] "Promoter" refers to a DNA sequence capable of controlling
the expression of a coding sequence or functional RNA. The promoter
sequence consists of proximal and more distal upstream elements,
the latter elements often referred to as enhancers. Accordingly, an
"enhancer" is a DNA sequence that can stimulate promoter activity,
and may be an innate element of the promoter or a heterologous
element inserted to enhance the level or tissue-specificity of a
promoter. Promoters may be derived in their entirety from a native
gene, or be composed of different elements derived from different
promoters found in nature, or even comprise synthetic DNA segments.
It is understood by those skilled in the art that different
promoters may direct the expression of a gene in different tissues
or cell types, or at different stages of development, or in
response to different environmental conditions. It is further
recognized that since in most cases the exact boundaries of
regulatory sequences have not been completely defined, DNA
fragments of some variation may have identical promoter activity.
Promoters that cause a gene to be expressed in most cell types at
most times are commonly referred to as "constitutive promoters".
New promoters of various types useful in plant cells are constantly
being discovered; numerous examples may be found in the compilation
by Okamuro, J. K., and Goldberg, R. B. Biochemistry of Plants
15:1-82 (1989).
[0256] "Translation leader sequence" refers to a polynucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner, R. and Foster, G. D., Mol. Biotechnol. 3:225-236
(1995)).
[0257] "3' non-coding sequences", "transcription terminator" or
"termination sequences" refer to DNA sequences located downstream
of a coding sequence and include polyadenylation recognition
sequences and other sequences encoding regulatory signals capable
of affecting mRNA processing or gene expression. The
polyadenylation signal is usually characterized by affecting the
addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht, I. L., et al. Plant Cell 1:671-680
(1989).
[0258] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript. A RNA transcript is
referred to as the mature RNA when it is a RNA sequence derived
from post-transcriptional processing of the primary transcript.
"Messenger RNA" or "mRNA" refers to the RNA that is without introns
and that can be translated into protein by the cell. "cDNA" refers
to a DNA that is complementary to, and synthesized from, a mRNA
template using the enzyme reverse transcriptase. The cDNA can be
single-stranded or converted into double-stranded form using the
Klenow fragment of DNA polymerase I. "Sense" RNA refers to RNA
transcript that includes the mRNA and can be translated into
protein within a cell or in vitro. "Antisense RNA" refers to an RNA
transcript that is complementary to all or part of a target primary
transcript or mRNA, and that blocks the expression of a target gene
(U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA
may be with any part of the specific gene transcript, i.e., at the
5' non-coding sequence, 3' non-coding sequence, introns, or the
coding sequence. "Functional RNA" refers to antisense RNA, ribozyme
RNA, or other RNA that may not be translated but yet has an effect
on cellular processes. The terms "complement" and "reverse
complement" are used interchangeably herein with respect to mRNA
transcripts, and are meant to define the antisense RNA of the
message.
[0259] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is regulated by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of regulating the expression of that coding sequence (i.e.,
the coding sequence is under the transcriptional control of the
promoter). Coding sequences can be operably linked to regulatory
sequences in a sense or antisense orientation. In another example,
the complementary RNA regions of the invention can be operably
linked, either directly or indirectly, 5' to the target mRNA, or 3'
to the target mRNA, or within the target mRNA, or a first
complementary region is 5' and its complement is 3' to the target
mRNA.
[0260] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning:
A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring
Harbor, N.Y. (1989). Transformation methods are well known to those
skilled in the art and are described below.
[0261] "PCR" or "polymerase chain reaction" is a technique for the
synthesis of large quantities of specific DNA segments and consists
of a series of repetitive cycles (Perkin Elmer Cetus Instruments,
Norwalk, Conn.). Typically, the double-stranded DNA is heat
denatured, the two primers complementary to the 3' boundaries of
the target segment are annealed at low temperature and then
extended at an intermediate temperature. One set of these three
consecutive steps is referred to as a "cycle".
[0262] The term "recombinant" refers to an artificial combination
of two otherwise separated segments of sequence, e.g., by chemical
synthesis or by the manipulation of isolated segments of nucleic
acids by genetic engineering techniques.
[0263] The terms "plasmid", "vector" and "cassette" refer to an
extra chromosomal element often carrying genes that are not part of
the central metabolism of the cell, and usually in the form of
circular double-stranded DNA fragments. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Transformation
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that facilitates
transformation of a particular host cell. "Expression cassette"
refers to a specific vector containing a foreign gene and having
elements in addition to the foreign gene that allow for enhanced
expression of that gene in a foreign host.
[0264] The terms "recombinant construct", "expression construct",
"chimeric construct", "construct", and "recombinant DNA construct"
are used interchangeably herein. A recombinant construct comprises
an artificial combination of nucleic acid fragments, e.g.,
regulatory and coding sequences that are not found together in
nature. For example, a chimeric construct may comprise regulatory
sequences and coding sequences that are derived from different
sources, or regulatory sequences and coding sequences derived from
the same source, but arranged in a manner different than that found
in nature. Such a construct may be used by itself or may be used in
conjunction with a vector. If a vector is used, then the choice of
vector is dependent upon the method that will be used to transform
host cells as is well known to those skilled in the art. For
example, a plasmid vector can be used. The skilled artisan is well
aware of the genetic elements that must be present on the vector in
order to successfully transform, select and propagate host cells
comprising any of the isolated nucleic acid fragments of the
invention. The skilled artisan will also recognize that different
independent transformation events will result in different levels
and patterns of expression (Jones et al., EMBO J. 4:2411-2418
(1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)),
and thus that multiple events must be screened in order to obtain
lines displaying the desired expression level and pattern. Such
screening may be accomplished by Southern analysis of DNA, Northern
analysis of mRNA expression, immunoblotting analysis of protein
expression, or phenotypic analysis, among others.
[0265] The term "expression", as used herein, refers to the
production of a functional end-product (e.g., a mRNA or a protein
[either precursor or mature]).
[0266] The term "expression cassette" as used herein, refers to a
discrete nucleic acid fragment into which a nucleic acid sequence
or fragment can be moved. "Mature" protein refers to a
post-translationally processed polypeptide (i.e., one from which
any pre- or propeptides present in the primary translation product
have been removed). "Precursor" protein refers to the primary
product of translation of mRNA (i.e., with pre- and propeptides
still present). Pre- and propeptides may be but are not limited to
intracellular localization signals.
[0267] "Stable transformation" refers to the transfer of a nucleic
acid fragment into a genome of a host organism, including both
nuclear and organellar genomes, resulting in genetically stable
inheritance. In contrast, "transient transformation" refers to the
transfer of a nucleic acid fragment into the nucleus, or
DNA-containing organelle, of a host organism resulting in gene
expression without integration or stable inheritance. Host
organisms containing the transformed nucleic acid fragments are
referred to as "transgenic" organisms.
[0268] "Antisense inhibition" refers to the production of antisense
RNA transcripts capable of suppressing the expression of the target
protein. "Co-suppression" refers to the production of sense RNA
transcripts capable of suppressing the expression of identical or
substantially similar foreign or endogenous genes (U.S. Pat. No.
5,231,020). Co-suppression constructs in plants previously have
been designed by focusing on overexpression of a nucleic acid
sequence having homology to an endogenous mRNA, in the sense
orientation, which results in the reduction of all RNA having
homology to the overexpressed sequence (Vaucheret et al., Plant J.
16:651-659 (1998); Gura, Nature 404:804-808 (2000)). The overall
efficiency of this phenomenon is low, and the extent of the RNA
reduction is widely variable. Recent work has described the use of
"hairpin" structures that incorporate all, or part, of an mRNA
encoding sequence in a complementary orientation that results in a
potential "stem-loop" structure for the expressed RNA (WO 99/53050,
published Oct. 21, 1999; WO 02/00904, published Jan. 3, 2002). This
increases the frequency of co-suppression in the recovered
transgenic plants. Another variation describes the use of plant
viral sequences to direct the suppression, or "silencing", of
proximal mRNA encoding sequences (WO 98/36083, published Aug. 20,
1998). Both of these co-suppressing phenomena have not been
elucidated mechanistically, although genetic evidence has begun to
unravel this complex situation (Elmayan et al., Plant Cell
10:1747-1757 (1998)).
[0269] The term "oleaginous" refers to those organisms that tend to
store their energy source in the form of lipid (Weete, In: Fungal
Lipid Biochemistry, 2.sup.nd Ed., Plenum, 1980). Generally, the
cellular oil content of these microorganisms follows a sigmoid
curve, wherein the concentration of lipid increases until it
reaches a maximum at the late logarithmic or early stationary
growth phase and then gradually decreases during the late
stationary and death phases (Yongmanitchai and Ward, Appl. Environ.
Microbiol. 57:419-25 (1991)).
[0270] The term "oleaginous yeast" refers to those microorganisms
classified as yeasts that make oil. It is not uncommon for
oleaginous microorganisms to accumulate in excess of about 25% of
their dry cell weight as oil. Examples of oleaginous yeast include,
but are no means limited to, the following genera: Yarrowia,
Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon
and Lipomyces.
[0271] The "Clustal V method of alignment" corresponds to the
alignment method labeled Clustal V (described by Higgins and Sharp,
CABIOS. 5:151-153 (1989)) and found in the MegAlign.TM. program of
the LASERGENE bioinformatics computing suite (DNASTAR Inc.,
Madison, Wis.). The "default parameters" are the parameters preset
by the manufacturer of the program. For multiple alignments, they
correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10; and, for
pairwise alignments, they are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and
DIAGONALS SAVED=5. After alignment of the sequences using the
Clustal V program, it is possible to obtain a "percent identity" by
viewing the "sequence distances" table in the same program.
[0272] "BLASTN method of alignment" is an algorithm provided by the
National Center for Biotechnology Information (NCBI) to compare
nucleotide sequences using default parameters.
[0273] The present invention concerns an isolated polynucleotide
comprising: [0274] (a) a nucleotide sequence encoding a polypeptide
having delta-9 elongase activity, wherein the polypeptide has at
least 70% amino acid identity, based on the Clustal V method of
alignment, when compared to an amino acid sequence as set forth in
SEQ ID NO:5 or SEQ ID NO:127; [0275] (b) a nucleotide sequence
encoding a polypeptide having delta-9 elongase activity, wherein
the nucleotide sequence has at least 70% sequence identity, based
on the BLASTN method of alignment, when compared to a nucleotide
sequence as set forth in SEQ ID NO:4, SEQ ID NO:90 or SEQ ID
NO:126; [0276] (c) a nucleotide sequence encoding a polypeptide
having delta-9 elongase activity, wherein the nucleotide sequence
hybridizes with a nucleotide sequence as set forth in SEQ ID NO:4,
SEQ ID NO:90 or SEQ ID NO:126 under the following stringent
hybridization conditions: 0.1.times.SSC, 0.1% SDS, 65.degree. C.
and washed with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC,
0.1% SDS; or [0277] (d) a complement of the nucleotide sequence of
(a), (b) or (c), wherein the complement and the nucleotide sequence
consist of the same number of nucleotides and are 100%
complementary.
[0278] In another aspect this invention concerns an isolated
nucleic acid fragment comprising a nucleic acid sequence encoding a
delta-9 elongase, excluding SEQ ID NO:27, wherein the amino acid
sequence comprising said elongase contains at least one of the
following amino acid sequence motifs selected from the group
consisting of: TABLE-US-00004 (SEQ ID NO:130) a) Y N X (L or F) X X
X X S X X S F; (SEQ ID NO:131) b) F Y X S K X X (E or D) Y X D (T
or S) X X L; (SEQ ID NO:132) c) L (Q or H) X F H H X G A; (SEQ ID
NO:133) d) M Y X Y Y X X X X X X X (K or R or N) F; (SEQ ID NO:134)
e) K X L (I or L or M) T X X Q; (SEQ ID NO:135) f) W X F N Y X Y;
and (SEQ ID NO:136) g) Y X G X V X X L F;
[0279] wherein X can be any amino acid.
[0280] The underlined amino acids may be unique to delta-9
elongases. FIG. 16 (see also FIG. 29) sets forth a comparison of
the delta-9 elongase of the invention with a delta-9 elongase from
Isochrysis galbana using Clustal V alignment (with default
parameters). Specifically, SEQ ID NO:5 (amino acid sequence of the
delta-9 elongase of the instant invention) and SEQ ID NO:27 (amino
acid sequence of the long-chain PUFA elongation enzyme from
Isochrysis galbana (NCBI Accession No. AAL37626 (GI 17226123)) were
compared.
[0281] In still another aspect, this invention concerns an isolated
polynucleotide comprising a nucleotide sequence encoding a
polypeptide having delta-9 elongase activity, wherein the
nucleotide sequence has at least 84% sequence identity, based on
the BLASTN method of alignment, when compared to a nucleotide
sequence as set forth in SEQ ID NO:4.
[0282] It was found that a comparison of SEQ ID NO:4 and SEQ ID
NO:90 using the BLASTN method of alignment with default parameters
showed that these sequences had at least 84% sequence identity.
[0283] This delta-9 elongase may be used alone or in combination
with other desaturase and elongase components to produce various
omega-6 and omega-3 PUFAs, including e.g., DGLA, ETA, AA, EPA, DPA
and/or DHA (FIG. 15). One skilled in the art will recognize the
appropriate combinations of the delta-9 elongase of the invention
herein in conjunction with a delta-4 desaturase, a delta-5
desaturase, a delta-6 desaturase, a delta-12 desaturase, a delta-15
desaturase, a delta-17 desaturase, a delta-9 desaturase, a delta-8
desaturase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a
C.sub.18/20 elongase and/or a C.sub.20/22 elongase, based on the
particular host cell (and its native PUFA profile and/or desaturase
and/or elongase profile), the availability of substrate, and the
desired end product(s).
[0284] In another embodiment, this invention concerns a recombinant
construct comprising the polynucleotide of the invention operably
linked to at least one regulatory sequence.
[0285] As was noted above, a promoter is a DNA sequence that
directs cellular machinery of a plant to produce RNA from the
contiguous coding sequence downstream (3') of the promoter. The
promoter region influences the rate, developmental stage, and cell
type in which the RNA transcript of the gene is made. The RNA
transcript is processed to produce mRNA which serves as a template
for translation of the RNA sequence into the amino acid sequence of
the encoded polypeptide. The 5' non-translated leader sequence is a
region of the mRNA upstream of the protein coding region that may
play a role in initiation and translation of the mRNA. The 3'
transcription termination/polyadenylation signal is a
non-translated region downstream of the protein coding region that
functions in the plant cell to cause termination of the RNA
transcript and the addition of polyadenylate nucleotides to the 3'
end of the RNA.
[0286] The origin of the promoter chosen to drive expression of the
coding sequence is not important as long as it has sufficient
transcriptional activity to accomplish the invention by expressing
translatable mRNA for the desired nucleic acid fragments in the
desired host tissue at the right time. Either heterologous or
non-heterologous (i.e., endogenous) promoters can be used to
practice the invention. For example, suitable promoters include,
but are not limited to: the alpha prime subunit of beta conglycinin
promoter, Kunitz trypsin inhibitor 3 promoter, annexin promoter,
Gly1 promoter, beta subunit of beta conglycinin promoter, P34/Gly
Bd m 30K promoter, albumin promoter, Leg A1 promoter and Leg A2
promoter.
[0287] The annexin, or P34, promoter is described in WO 2004/071178
(published Aug. 26, 2004). The level of activity of the annexin
promoter is comparable to that of many known strong promoters, such
as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol.
37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538
(1990); Holtorf et al., Plant Mol. Biol. 29:637-646 (1995);
Jefferson et al., EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant
Mol Biol. 28:949-955 (1995)); (2) the Arabidopsis oleosin promoters
(Plant et al., Plant Mol. Biol. 25:193-205 (1994); Li, Texas
A&M University Ph.D. dissertation, pp. 107-128 (1997)); (3) the
Arabidopsis ubiquitin extension protein promoters (Callis et al., J
Biol. Chem. 265(21):12486-93 (1990)); (4) a tomato ubiquitin gene
promoter (Rollfinke et al., Gene. 211(2):267-76 (1998)); (5) a
soybean heat shock protein promoter (Schoffl et al., Mol Gen Genet.
217(2-3):246-53 (1989)); and, (6) a maize H3 histone gene promoter
(Atanassova et al., Plant Mol Biol. 37(2):275-85 (1989)).
[0288] Another useful feature of the annexin promoter is its
expression profile in developing seeds. The annexin promoter is
most active in developing seeds at early stages (before 10 days
after pollination) and is largely quiescent in later stages. The
expression profile of the annexin promoter is different from that
of many seed-specific promoters, e.g., seed storage protein
promoters, which often provide highest activity in later stages of
development (Chen et al., Dev. Genet. 10:112-122 (1989); Ellerstrom
et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et al., Plant
Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)).
The annexin promoter has a more conventional expression profile but
remains distinct from other known seed specific promoters. Thus,
the annexin promoter will be a very attractive candidate when
overexpression, or suppression, of a gene in embryos is desired at
an early developing stage. For example, it may be desirable to
overexpress a gene regulating early embryo development or a gene
involved in the metabolism prior to seed maturation.
[0289] Following identification of an appropriate promoter suitable
for expression of a specific coding sequence, the promoter is then
operably linked in a sense orientation using conventional means
well known to those skilled in the art.
[0290] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook, J. et al., In Molecular Cloning: A Laboratory Manual;
2.sup.nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, N.Y., 1989 (hereinafter "Sambrook et al., 1989" ) or
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,
J. G., Smith, J. A. and Struhl, K., Eds.; In Current Protocols in
Molecular Biology, John Wiley and Sons: New York, 1990 (hereinafter
"Ausubel et al., 1990").
[0291] Once the recombinant construct has been made, it may then be
introduced into a plant cell of choice by methods well known to
those of ordinary skill in the art (e.g., transfection,
transformation and electroporation). Oilseed plant cells are the
preferred plant cells. The transformed plant cell is then cultured
and regenerated under suitable conditions permitting expression of
the long-chain PUFA which is then optionally recovered and
purified.
[0292] The recombinant constructs of the invention may be
introduced into one plant cell; or, alternatively, each construct
may be introduced into separate plant cells.
[0293] Expression in a plant cell may be accomplished in a
transient or stable fashion as is described above.
[0294] The desired long-chain PUFAs can be expressed in seed. Also
within the scope of this invention are seeds or plant parts
obtained from such transformed plants.
[0295] The term "plant" refers to whole plants, plant organs, plant
tissues, seeds, plant cells, seeds and progeny of the same. Plant
cells include, without limitation, cells from seeds, suspension
cultures, embryos, meristematic regions, callus tissue, leaves,
roots, shoots, gametophytes, sporophytes, pollen and
microspores.
[0296] "Progeny" comprises any subsequent generation of a
plant.
[0297] Plant parts include differentiated and undifferentiated
tissues including, but not limited to the following: roots, stems,
shoots, leaves, pollen, seeds, tumor tissue and various forms of
cells and culture (e.g., single cells, protoplasts, embryos and
callus tissue). The plant tissue may be in plant or in a plant
organ, tissue or cell culture.
[0298] The term "plant organ" refers to plant tissue or group of
tissues that constitute a morphologically and functionally distinct
part of a plant. The term "genome" refers to the following: (1) the
entire complement of genetic material (genes and non-coding
sequences) is present in each cell of an organism, or virus or
organelle; (2) a complete set of chromosomes inherited as a
(haploid) unit from one parent.
[0299] Thus, this invention also concerns a method for transforming
a cell, comprising transforming a cell with the recombinant
construct of the invention and selecting those cells transformed
with the recombinant construct of Claim 5.
[0300] Also of interest is a method for producing a transformed
plant comprising transforming a plant cell with the polynucleotide
of the instant invention and regenerating a plant from the
transformed plant cell.
[0301] Methods for transforming dicots (primarily by use of
Agrobacterium tumefaciens) and obtaining transgenic plants have
been published, among others, for: cotton (U.S. Pat. Nos.
5,004,863; 5,159,135); soybean (U.S. Pat. Nos. 5,569,834;
5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et
al. Plant Cell Rep. 15:653-657 (1996); McKently et al. Plant Cell
Rep. 14:699-703 (1995)); papaya (Ling, K. et al. Bio/technology
9:752-758 (1991)); and pea (Grant et al. Plant Cell Rep. 15:254-258
(1995)). For a review of other commonly used methods of plant
transformation see Newell, C. A. (Mol. Biotechnol. 16:53-65
(2000)). One of these methods of transformation uses Agrobacterium
rhizogenes (Tepfler, M. and Casse-Delbart, F. Microbiol. Sci.
4:24-28 (1987)). Transformation of soybeans using direct delivery
of DNA has been published using PEG fusion (WO 92/17598),
electroporation (Chowrira, G. M. et al. Mol. Biotechnol. 3:17-23
(1995); Christou, P. et al. Proc. Natl. Acad. Sci. U.S.A.
84:3962-3966 (1987)), microinjection, or particle bombardment
(McCabe, D. E. et. al. Bio/Technology 6:923 (1988); Christou et al.
Plant Physiol. 87:671-674 (1988)).
[0302] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated. The regeneration, development and
cultivation of plants from single plant protoplast transformants or
from various transformed explants is well known in the art
(Weissbach and Weissbach, In: Methods for Plant Molecular Biology,
(Eds.), Academic: San Diego, Calif. (1988)). This regeneration and
growth process typically includes the steps of selection of
transformed cells and culturing those individualized cells through
the usual stages of embryonic development through the rooted
plantlet stage. Transgenic embryos and seeds are similarly
regenerated. The resulting transgenic rooted shoots are thereafter
planted in an appropriate plant growth medium such as soil.
Preferably, the regenerated plants are self-pollinated to provide
homozygous transgenic plants. Otherwise, pollen obtained from the
regenerated plants is crossed to seed-grown plants of agronomically
important lines. Conversely, pollen, from plants of these important
lines is used to pollinate regenerated plants. A transgenic plant
of the present invention containing a desired polypeptide is
cultivated using methods well known to one skilled in the art.
[0303] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant DNA fragments and
recombinant expression constructs and the screening and isolating
of clones. See, for example: Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor: N.Y. (1989); Maliga et al.,
Methods in Plant Molecular Biology, Cold Spring Harbor: N.Y.
(1995); Birren et al., Genome Analysis: Detecting Genes, Vol. 1,
Cold Spring Harbor: N.Y. (1998); Birren et al., Genome Analysis:
Analyzing DNA, Vol. 2, Cold Spring Harbor: N.Y. (1998); Plant
Molecular Biology: A Laboratory Manual, eds. Clark, Springer: N.Y.
(1997).
[0304] Examples of oilseed plants include, but are not limited to,
soybean, Brassica species, sunflower, maize, cotton, flax and
safflower.
[0305] Examples of polyunsaturated fatty acids having at least
twenty carbon atoms and five or more carbon-carbon double bonds
include, but are not limited to, omega-3 fatty acids such as EPA,
DPA and DHA. Seeds obtained from such plants are also within the
scope of this invention as well as oil obtained from such
seeds.
[0306] In one embodiment this invention concerns an oilseed plant
comprising: a) a first recombinant DNA construct comprising an
isolated polynucleotide encoding a delta-9 elongase polypeptide,
operably linked to at least one regulatory sequence; and b) at
least one additional recombinant DNA construct comprising an
isolated polynucleotide, operably linked to at least one regulatory
sequence, encoding a polypeptide selected from the group consisting
of a delta-4 desaturase, a delta-5 desaturase, a delta-6
desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-12
desaturase, a delta-15 desaturase, and a delta-17 desaturase, a
C.sub.18/20 elongase and a C.sub.20/22 elongase.
[0307] Such desaturases are discussed, for example, in U.S. Pat.
Nos. 6,075,183, 5,968,809, 6,136,574, 5,972,664, 6,051,754,
6,410,288 and WO 98/46763, WO 98/46764, WO 00/12720 and WO
00/40705.
[0308] The choice of combination of cassettes used depends in part
on the PUFA profile and/or desaturase/elongase profile of the
oilseed plant cells to be transformed and the long-chain PUFA which
is to be expressed.
[0309] In another aspect, this invention concerns a method for
making long-chain polyunsaturated fatty acids in a plant cell
comprising: [0310] (a) transforming a cell with the recombinant
construct of the invention; and [0311] (b) selecting those
transformed cells that make long-chain polyunsaturated fatty
acids.
[0312] In still another aspect, this invention concerns a method
for producing at least one polyunsaturated fatty acid in a soybean
cell comprising: [0313] (a) transforming a soybean cell with a
first recombinant DNA construct comprising an isolated
polynucleotide encoding a delta-9 elongase polypeptide, operably
linked to at least one regulatory sequence and at least one
additional recombinant DNA construct comprising an isolated
polynucleotide, operably linked to at least one regulatory
sequence, encoding a polypeptide selected from the group consisting
of a delta4 desaturase, a delta-5 desaturase, a delta-6 desaturase,
a delta-8 desaturase, a delta-9 desaturase, a delta-1 2 desaturase,
a delta-15 desaturase, and a delta-17 desaturase, a C.sub.18/20
elongase and a C.sub.20/22 elongase; [0314] (b) regenerating a
soybean plant from the transformed cell of step (a); and [0315] (c)
selecting those seeds obtained from the plants of step (b) having
an altered level of polyunsaturated fatty acids when compared to
the level in seeds obtained from a nontransformed soybean
plant.
[0316] Methods of isolating seed oils are well known in the art:
(Young et al., Processing of Fats and Oils, In The Lipid Handbook,
Gunstone et al., eds., Chapter 5 pp 253-257; Chapman & Hall:
London (1994)). For example, soybean oil is produced using a series
of steps involving the extraction and purification of an edible oil
product from the oil-bearing seed. Soybean oils and soybean
byproducts are produced using the generalized steps shown in Table
3. TABLE-US-00005 TABLE 3 Generalized Steps for Soybean Oil and
Byproduct Production Process Impurities Removed and/or Step Process
By-Products Obtained # 1 soybean seed # 2 oil extraction meal # 3
degumming lecithin # 4 alkali or physical refining gums, free fatty
acids, pigments # 5 water washing soap # 6 bleaching color, soap,
metal # 7 (hydrogenation) # 8 (winterization) stearine # 9
deodorization free fatty acids, tocopherols, sterols, volatiles #
10 oil products
[0317] More specifically, soybean seeds are cleaned, tempered,
dehulled and flaked, thereby increasing the efficiency of oil
extraction. Oil extraction is usually accomplished by solvent
(e.g., hexane) extraction but can also be achieved by a combination
of physical pressure and/or solvent extraction. The resulting oil
is called crude oil. The crude oil may be degummed by hydrating
phospholipids and other polar and neutral lipid complexes that
facilitate their separation from the nonhydrating, triglyceride
fraction (soybean oil). The resulting lecithin gums may be further
processed to make commercially important lecithin products used in
a variety of food and industrial products as emulsification and
release (i.e., antisticking) agents. Degummed oil may be further
refined for the removal of impurities (primarily free fatty acids,
pigments and residual gums). Refining is accomplished by the
addition of a caustic agent that reacts with free fatty acid to
form soap and hydrates phosphatides and proteins in the crude oil.
Water is used to wash out traces of soap formed during refining.
The soapstock byproduct may be used directly in animal feeds or
acidulated to recover the free fatty acids. Color is removed
through adsorption with a bleaching earth that removes most of the
chlorophyll and carotenoid compounds. The refined oil can be
hydrogenated, thereby resulting in fats with various melting
properties and textures. Winterization (fractionation) may be used
to remove stearine from the hydrogenated oil through
crystallization under carefully controlled cooling conditions.
Deodorization (principally via steam distillation under vacuum) is
the last step and is designed to remove compounds which impart odor
or flavor to the oil. Other valuable byproducts such as tocopherols
and sterols may be removed during the deodorization process.
Deodorized distillate containing these byproducts may be sold for
production of natural vitamin E and other high-value pharmaceutical
products. Refined, bleached, (hydrogenated, fractionated) and
deodorized oils and fats may be packaged and sold directly or
further processed into more specialized products. A more detailed
reference to soybean seed processing, soybean oil production and
byproduct utilization can be found in Erickson, Practical Handbook
of Soybean Processing and Utilization, The American Oil Chemists'
Society and United Soybean Board (1995).
[0318] Soybean oil is liquid at room temperature because it is
relatively low in saturated fatty acids when compared with oils
such as coconut, palm, palm kernel and cocoa butter. Many processed
fats (including spreads, confectionary fats, hard butters,
margarines, baking shortenings, etc.) require varying degrees of
solidity at room temperature and can only be produced from soybean
oil through alteration of its physical properties. This is most
commonly achieved through catalytic hydrogenation.
[0319] Hydrogenation is a chemical reaction in which hydrogen is
added to the unsaturated fatty acid double bonds with the aid of a
catalyst such as nickel. High oleic soybean oil contains
unsaturated oleic, LA and linolenic fatty acids and each of these
can be hydrogenated. Hydrogenation has two primary effects. First,
the oxidative stability of the oil is increased as a result of the
reduction of the unsaturated fatty acid content. Second, the
physical properties of the oil are changed because the fatty acid
modifications increase the melting point resulting in a semi-liquid
or solid fat at room temperature.
[0320] There are many variables which affect the hydrogenation
reaction, which in turn alter the composition of the final product.
Operating conditions including pressure, temperature, catalyst type
and concentration, agitation and reactor design are among the more
important parameters that can be controlled. Selective
hydrogenation conditions can be used to hydrogenate the more
unsaturated fatty acids in preference to the less unsaturated ones.
Very light or brush hydrogenation is often employed to increase
stability of liquid oils. Further hydrogenation converts a liquid
oil to a physically solid fat. The degree of hydrogenation depends
on the desired performance and melting characteristics designed for
the particular end product. Liquid shortenings (used in the
manufacture of baking products, solid fats and shortenings used for
commercial frying and roasting operations) and base stocks for
margarine manufacture are among the myriad of possible oil and fat
products achieved through hydrogenation. A more detailed
description of hydrogenation and hydrogenated products can be found
in Patterson, H. B. W., Hydrogenation of Fats and Oils: Theory and
Practice. The American Oil Chemists' Society (1994).
[0321] Hydrogenated oils have also become controversial due to the
presence of trans-fatty acid isomers that result from the
hydrogenation process. Ingestion of large amounts of trans-isomers
has been linked with detrimental health effects including increased
ratios of low density to high density lipoproteins in the blood
plasma and increased risk of coronary heart disease.
[0322] Compared to other vegetable oils, the oils of the invention
are believed to function similarly to other oils in food
applications from a physical standpoint. Partially hydrogenated
oils, such as soybean oil, are widely used as ingredients for soft
spreads, margarine and shortenings for baking and frying.
[0323] In general, lipid accumulation in oleaginous microorganisms
is triggered in response to the overall carbon to nitrogen ratio
present in the growth medium. This process, leading to the de novo
synthesis of free palmitate (16:0) in oleaginous microorganisms, is
described in detail in WO 2004/101757. Palmitate is the precursor
of longer-chain saturated and unsaturated fatty acid derivates,
which are formed through the action of elongases and desaturases.
For example, palmitate is converted to its unsaturated derivative
(palmitoleic acid (16:1)) by the action of a delta-9 desaturase.
Similarly, palmitate is elongated by a C.sub.16/18 fatty acid
elongase to form stearic acid (18:0), which can be converted to its
unsaturated derivative by a delta-9 desaturase to thereby yield
oleic acid (18:1).
[0324] Triacylglycerols (the primary storage unit for fatty acids)
are formed by the esterification of two molecules of acyl-CoA to
glycerol-3-phosphate to yield 1,2-diacylglycerol phosphate
(commonly identified as phosphatidic acid). The phosphate is then
removed, by phosphatidic acid phosphatase, to yield
1,2-diacylglycerol. Triacylglycerol is formed upon the addition of
a third fatty acid by the action of a diacylglycerol-acyl
transferase.
[0325] Many microorganisms, including algae, bacteria, molds and
yeasts, can synthesize PUFAs and omega fatty acids in the ordinary
course of cellular metabolism. Particularly well-studied are fungi
including Schizochytrium aggregatm, species of the genus
Thraustochytrium and Morteriella alpina. Additionally, many
dinoflagellates (Dinophyceaae) naturally produce high
concentrations of PUFAs. As such, a variety of genes involved in
oil production have been identified through genetic means and the
DNA sequences of some of these genes are publicly available. See,
for example, AY131238, Y055118, AY055117, AF296076, AF007561,
L11421, NM.sub.--031344, AF465283, AF465281, AF110510, AF465282,
AF419296, AB052086, AJ250735, AF126799, AF126798 (delta-6
desaturases); AF199596, AF226273, AF320509, AB072976, AF489588,
AJ510244, AF419297, AF07879, AF067654, AB022097 (delta-5
desaturases); AAG36933, AF110509, AB020033, AAL13300, AF417244,
AF161219, AY332747, AAG36933, AF110509, X86736, AF240777, AB007640,
AB075526, AP002063 (delta-12 desaturases); NP.sub.--441622, BAA1
8302, BAA02924, ML36934 (delta-1 5 desaturases); AF338466,
AF438199, E11368, E11367, D83185, U90417, AF085500, AY504633,
NM.sub.--069854, AF230693 (delta-9 desaturases); AF390174 (delta-9
elongase); AF139720 and CQ831420 (delta-8 desaturase); and
AX464731, NM.sub.--11961, NM.sub.--134255, NM.sub.--134383,
NM.sub.--134382, NM.sub.--068396, NM.sub.--068392, NM.sub.--070713,
NM.sub.--068746, NM.sub.--064685 (elongases).
[0326] Additionally, the patent literature provides many additional
DNA sequences of genes (and/or details concerning several of the
genes above and their methods of isolation) involved in PUFA
production (e.g., WO 02/077213 (delta-9 elongases); WO 00/34439, WO
04/057001 and U.S. Pat. No. 6,825,017 (delta-8 desaturases); U.S.
Pat. No. 5,968,809 (delta-6 desaturases); U.S. Pat. No. 5,972,664
and U.S. Pat. No. 6,075,183 (delta-5 desaturases); WO 94/11516,
U.S. Pat. No. 5,443,974, WO 03/099216 and WO 05/047485 (delta-12
desaturases); WO 93/11245 (delta-15 desaturases); WO 91/13972 and
U.S. Pat. No. 5,057,419 (delta-9 desaturases); U.S. Patent
Application Publication No. 2003/0196217 A1 (delta-17 desaturase);
and WO 00/12720 and WO 2002/077213, U.S. Pat. No. 6,403,349, U.S.
Pat. No. 6,677,145, and U.S. Patent Application Publication No.
2004/0111763 (C.sub.14/16, C.sub.16/18 and C.sub.18/20 elongases)).
Each of these patents and applications are herein incorporated by
reference in their entirety.
[0327] As will be obvious to one skilled in the art, the particular
functionalities required to be introduced into a microbial host
organism for production of a particular PUFA final product will
depend on the host cell (and its native PUFA profile and/or
desaturase/elongase profile), the availability of substrate and the
desired end product(s). LA, GLA, EDA, DGLA, M, ALA, STA, ETrA, ETA,
EPA, DPA and DHA may all be produced in oleaginous yeasts, by
introducing various combinations of the following PUFA enzyme
functionalities: a delta-4 desaturase, a delta-5 desaturase, a
delta-6 desaturase, a delta-8 desaturase, a delta-12 desaturase, a
delta-15 desaturase, a delta-17 desaturase, a delta-9 desaturase, a
delta-9 elongase, a C.sub.14/16 elongase, a C.sub.16/18 elongase, a
C.sub.18/20 elongase and/or a C.sub.20/22 elongase. One skilled in
the art will be able to identify various candidate genes encoding
each of the above enzymes, according to publicly available
literature (e.g., GenBank), the patent literature, and experimental
analysis of microorganisms having the ability to produce PUFAs. The
sequences may be derived from any source, e.g., isolated from a
natural source (from bacteria, algae, fungi, plants, animals,
etc.), produced via a semi-synthetic route or synthesized de novo.
In some embodiments, manipulation of genes endogenous to the host
is preferred; for other purposes, it is necessary to introduce
heterologous genes.
[0328] Although the particular source of the desaturase and
elongase genes introduced into the host is not critical to the
invention, considerations for choosing a specific polypeptide
having desaturase or elongase activity include (1) the substrate
specificity of the polypeptide, (2) whether the polypeptide or a
component thereof is a rate-limiting enzyme, (3) whether the
desaturase or elongase is essential for synthesis of a desired
PUFA, and/or (4) co-factors required by the polypeptide. The
expressed polypeptide preferably has parameters compatible with the
biochemical environment of its location in the host cell. For
example, the polypeptide may have to compete for substrate with
other enzymes in the host cell. Analyses of the KM and specific
activity of the polypeptide are therefore considered in determining
the suitability of a given polypeptide for modifying PUFA
production in a given host cell. The polypeptide used in a
particular host cell is one that can function under the biochemical
conditions present in the intended host cell but otherwise can be
any polypeptide having desaturase or elongase activity capable of
modifying the desired PUFA.
[0329] In some cases, the host organism in which it is desirable to
produce PUFAs will possess endogenous genes encoding some PUFA
biosynthetic pathway enzymes. For example, oleaginous yeast can
typically produce 18:2 fatty acids (and some have the additional
capability of synthesizing 18:3 fatty acids); thus, oleaginous
yeast typically possess native delta-12 desaturase activity and may
also have delta-15 desaturases. In some embodiments, therefore,
expression of the native desaturase enzyme is preferred over a
heterologous (or "foreign") enzyme since (1) the native enzyme is
optimized for interaction with other enzymes and proteins within
the cell, and (2) heterologous genes are unlikely to share the same
codon preference in the host organism. Additionally, advantages are
incurred when the sequence of the native gene is known, as it
permits facile disruption of the endogenous gene by targeted
disruption. In many instances, however, the appropriate desaturases
and elongases are not present in the host organism of choice to
enable production of the desired PUFA products. Thus, it is
necessary to introduce heterologous genes. In one embodiment of the
present invention, work was conducted toward the goal of the
development of an oleaginous yeast that accumulates oils enriched
in long-chain omega-3 and/or omega-6 fatty acids via expression of
a delta-9 elongase/delta-8 desaturase pathway, to enable production
of EDA, DGLA, ARA, ALA, ETrA, ETA, EPA, DPA and/or DHA.
[0330] In order to express genes encoding the delta-9
elongase/delta-8 desaturase pathway for the biosynthesis of
long-chain PUFAs (e.g., AA and EPA) in these organisms, it was
therefore necessary to (1) identify a suitable delta-9 elongase and
delta-8 desaturase that functioned relatively efficiently in
oleaginous yeast based on substrate-feeding trials, and, (2)
subject the delta-9 elongase and delta-9 desaturase gene to
codon-optimization techniques (infra) to further enhance the
expression of the heterologous enzymes in the alternate oleaginous
yeast host, to thereby enable maximal production of omega-3 and/or
omega-6 fatty acids.
[0331] It will be obvious to one of skill in the art that
heterologous genes will be expressed with variable efficiencies in
an alternate host. Thus, omega-3 and/or omega-6 PUFA production may
be optimized by selection of a particular desaturase or elongase
whose level of expression in a heterologous host is preferred
relative to the expression of an alternate desaturase or elongase
in the host organism of interest. Furthermore, it may be desirable
to modify the expression of particular PUFA biosynthetic pathway
enzymes to achieve optimal conversion efficiency of each, according
to the specific PUFA product composition of interest. A variety of
genetic engineering techniques are available to optimize expression
of a particular enzyme. Two such techniques include codon
optimization and gene mutation, as described below. Genes produced
by, for example, either of these two methods, having desaturase
and/or elongase activity(s) would be useful in the invention herein
for synthesis of omega-3 and/or omega-6 PUFAs.
[0332] As will be appreciated by one skilled in the art, it is
frequently useful to modify a portion of the codons encoding a
particular polypeptide that is to be expressed in a foreign host,
such that the modified polypeptide uses codons that are preferred
by the alternate host. Use of host-preferred codons can
substantially enhance the expression of the foreign gene encoding
the polypeptide.
[0333] In general, host-preferred codons can be determined within a
particular host species of interest by examining codon usage in
proteins (preferably those expressed in the largest amount) and
determining which codons are used with highest frequency. Then, the
coding sequence for a polypeptide of interest having desaturase or
elongase activity can be synthesized in whole or in part using the
codons preferred in the host species. All (or portions) of the DNA
also can be synthesized to remove any destabilizing sequences or
regions of secondary structure that would be present in the
transcribed mRNA. All (or portions) of the DNA also can be
synthesized to alter the base composition to one more preferable in
the desired host cell.
[0334] In the present invention, it is desirable to modify a
portion of the codons encoding the polypeptide having delta-9
elongase activity, to enhance the expression of the gene in a host
organism including, but not limited to, a plant, plant parts and/or
oleaginous yeast Yarrowia lipolytica. The nucleic acid sequence of
the native gene (i.e., the Euglena gracilis delta-9 elongase
defined herein as SEQ ID NOs:3 and 4) is modified to employ
host-preferred codons. This wildtype elongase has 258 amino acids
(SEQ ID NO:5); in the codon-optimized gene (SEQ ID NO:90), 117 bp
of the 777 bp coding region (15.1%) and 106 codons) are
codon-optimized (41.1%) and the translation initiation site is
modified.
[0335] The skilled artisan will appreciate that modulation of the
Euglena gracilis delta-9 elongase as well as numerous other
heterologous delta-9 elongases from variable sources can be
codon-optimized to improve their expression in an oleaginous yeast
host (e.g, see Example 4 herein, wherein a synthetic
codon-optimized delta-9 elongase derived from Isochrysis galbana
was created for expression in Yarrowia lipolytica). The present
invention comprises the complete sequence of the synthetic
codon-optimized gene as reported in the accompanying Sequence
Listing (SEQ ID NO:90), the complement of those complete sequences,
and substantial portions of those sequences. Furthermore, the
codon-optimization method described in WO 2004/101753 and described
herein for optimization of the Euglena gracilis delta-9 elongase is
equally applicable to other genes in the omega-3/omega-6 fatty acid
biosynthetic pathway.
[0336] Methods for synthesizing sequences and bringing sequences
together are well established in the literature. For example, in
vitro mutagenesis and selection, site-directed mutagenesis, error
prone PCR (Melnikov et al., Nucleic Acids Research, 27(4):1056-1062
(February 1999)), "gene shuffling" or other means can be employed
to obtain mutations of naturally occurring desaturase or elongase
genes (wherein such mutations may include deletions, insertions and
point mutations, or combinations thereof). This would permit
production of a polypeptide having desaturase or elongase activity,
respectively, in vivo with more desirable physical and kinetic
parameters for function in the host cell such as a longer half-life
or a higher rate of production of a desired PUFA. Or, if desired,
the regions of a polypeptide of interest (i.e., a desaturase or an
elongase) important for enzymatic activity can be determined
through routine mutagenesis, expression of the resulting mutant
polypeptides and determination of their activities. An overview of
these techniques are described in WO 2004/101757. All such mutant
proteins and nucleotide sequences encoding them that are derived
from the codon-optimized gene described herein are within the scope
of the present invention.
[0337] Microbial production of omega-3 and/or omega-6 fatty acids
has several advantages. For example, (1) many microbes are known
with greatly simplified oil compositions compared with those of
higher organisms, making purification of desired components easier,
(2) microbial production is not subject to fluctuations caused by
external variables, such as weather and food supply, (3)
microbially produced oil is substantially free of contamination by
environmental pollutants, (4) microbes can provide PUFAs in
particular forms which may have specific uses, and (5) microbial
oil production can be manipulated by controlling culture
conditions, notably by providing particular substrates for
microbially expressed enzymes, or by addition of compounds/genetic
engineering to suppress undesired biochemical pathways.
[0338] In addition to these advantages, production of omega-3
and/or omega-6 fatty acids from recombinant microbes provides the
ability to alter the naturally occurring microbial fatty acid
profile by providing new biosynthetic pathways in the host or by
suppressing undesired pathways, thereby increasing levels of
desired PUFAs, or conjugated forms thereof, and decreasing levels
of undesired PUFAs. For example, it is possible to modify the ratio
of omega-3 to omega-6 fatty acids so produced, produce either
omega-3 or omega-6 fatty acids exclusively while eliminating
production of the alternate omega fatty acid, or engineer
production of a specific PUFA without significant accumulation of
other PUFA downstream or upstream products (e.g., enable
biosynthesis of AA, EPA and/or DHA via the delta-9 elongase/delta-8
desaturase pathway, thereby avoiding synthesis of GLA and/or
STA).
[0339] The genes and gene products described herein may be produced
in heterologous microbial host cells, particularly in the cells of
oleaginous yeasts (e.g., Yarrowia lipolytica). Expression in
recombinant microbial hosts may be useful for the production of
various PUFA pathway intermediates, or for the modulation of PUFA
pathways already existing in the host for the synthesis of new
products heretofore not possible using the host.
[0340] Microbial expression systems and expression vectors
containing regulatory: sequences that direct high level expression
of foreign proteins are well known to those skilled in the art. Any
of these could be used to construct chimeric genes for production
of any of the gene products of the preferred desaturase and/or
elongase sequences. These chimeric genes could then be introduced
into appropriate microorganisms via transformation to provide
high-level expression of the encoded enzymes.
[0341] Accordingly, it is expected that introduction of chimeric
genes encoding a PUFA biosynthetic pathway, under the control of
the appropriate promoters will result in increased production of
omega-3 and/or omega-6 fatty acids. It is contemplated that it will
be useful to express various combinations of these PUFA desaturase
and elongase genes together in a host microorganism. It will be
obvious to one skilled in the art that the particular genes
included within a particular expression cassette(s) will depend on
the host cell, its ability to synthesize PUFAs using native
desaturases and elongases, the availability of substrate and the
desired end product(s). For example, it may be desirable for an
expression cassette to be constructed comprising genes encoding one
or more of the following enzymatic activities: a delta-4
desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8
desaturase, a delta-12 desaturase, a delta-15 desaturase, a
delta-17 desaturase, a delta-9 desaturase, a delta-9 elongase, a
C.sub.14/16 elongase, a C.sub.16/18 elongase, a C.sub.18/20
elongase and/or a C.sub.20/22 elongase. As such, the present
invention encompasses a method of producing PUFAs comprising
exposing a fatty acid substrate to the PUFA enzyme(s) described
herein, such that the substrate is converted to the desired fatty
acid product. Thus, each PUFA gene and corresponding enzyme product
described herein (e.g., a wildtype, codon-optimized, synthetic
and/or mutant enzyme having appropriate desaturase or elongase
activity) can be used directly or indirectly for the production of
PUFAs. Direct production of PUFAs occurs wherein the fatty acid
substrate is converted directly into the desired fatty acid product
without any intermediate steps or pathway intermediates. For
example, production of AA would occur in a host cell which produces
or which is provided DGLA, by adding or introducing into said cell
an expression cassette that provides delta-5 desaturase activity.
Similarly, expression of the delta-9 elongase of the invention
permits the direct synthesis of EDA and ETrA (when provided LA and
ALA, respectively, as substrate). Thus, for example, the present
invention may encompass a method of producing either EDA or ETrA,
respectively, comprising: [0342] a) providing a host organism
including, but not limited to, an oleaginous yeast comprising: (i)
a gene encoding a delta-9 elongase polypeptide as set forth in SEQ
ID NO:5 or SEQ ID NO:127; and [0343] (ii) a source of elongase
substrate consisting of either LA or ALA, respectively; and, [0344]
b) growing the yeast of step (a) in the presence of a suitable
fermentable carbon source wherein the gene encoding a delta-9
elongase polypeptide is expressed and LA is converted to EDA or ALA
is converted to ETrA, respectively; and, [0345] c) optionally
recovering the EDA or ETrA, respectively, of step (b).
[0346] In some preferred embodiments, the nucleotide sequence of a
gene encoding a delta-9 elongase polypeptide is set forth in SEQ ID
NO:90 wherein at least 106 codons have been optimized for
expression in Yarrowia.
[0347] In contrast, multiple genes encoding the PUFA biosynthetic
pathway may be used in combination, such that a series of reactions
occur to produce a desired PUFA. For example, expression
cassette(s) encoding delta-9 elongase, delta-8 desaturase, delta-5
desaturase and delta-17 desaturase activity would enable a host
cell that naturally produces LA, to instead produce ARA (such that
LA is converted to EDA by delta-9 elongase; EDA may then be
converted to DGLA by a delta-8 desaturase; DGLA is then converted
to ARA by a delta-5 desaturase). In a related manner, expression of
the delta-9 elongase of the invention enables the indirection
production of ETA, EPA, DPA and/or DHA as down-stream PUFAs, if
subsequent desaturase and elongation reactions are catalyzed. In a
preferred embodiment, wherein the host cell is an oleaginous yeast,
expression cassettes encoding each of the enzymes necessary for
PUFA biosynthesis will need to be introduced into the organism,
since naturally produced PUFAs in these organisms are limited to
18:2 fatty acids (i.e., LA), and less commonly, 18:3 fatty acids
(i.e., ALA). Alternatively, substrate feeding may be required.
[0348] Vectors or DNA cassettes useful for the transformation of
suitable microbial host cells are well known in the art. The
specific choice of sequences present in the construct is dependent
upon the desired expression products (supra), the nature of the
host cell and the proposed means of separating transformed cells
versus non-transformed cells. Typically, however, the vector or
cassette contains sequences directing transcription and translation
of the relevant gene(s), a selectable marker and sequences allowing
autonomous replication or chromosomal integration. Suitable vectors
comprise a region 5' of the gene that controls transcriptional
initiation (e.g., a promoter) and a region 3' of the DNA fragment
that controls transcriptional termination (i.e., a terminator). It
is most preferred when both control regions are derived from genes
from the transformed host cell, although it is to be understood
that such control regions need not be derived from the genes native
to the specific species chosen as a production host.
[0349] Initiation control regions or promoters which are useful to
drive expression of desaturase and/or elongase ORFs in the desired
microbial host cell are numerous and familiar to those skilled in
the art. Virtually any promoter capable of directing expression of
these genes in the selected host cell is suitable for the present
invention. Expression in a microbial host cell can be accomplished
in a transient or stable fashion. Transient expression can be
accomplished by inducing the activity of a regulatable promoter
operably linked to the gene of interest, alternatively, stable
expression can be achieved by the use of a constitutive promoter
operably linked to the gene of interest. As an example, when the
host cell is yeast, transcriptional and translational regions
functional in yeast cells are provided, particularly from the host
species. The transcriptional initiation regulatory regions can be
obtained, for example, from (1) genes in the glycolytic pathway,
such as alcohol dehydrogenase,
glyceraldehyde-3-phosphate-dehydrogenase (WO 2005/003310),
phosphoglycerate mutase (WO 2005/003310), fructose-bisphosphate
aldolase (WO 2005/049805), phosphoglucose-isomerase,
phosphoglycerate kinase, glycerol-3-phosphate 0-acyltransferase
(see U.S. patent application Ser. No. 11/225354), etc.; or (2)
regulatable genes such as acid phosphatase, lactase,
metallothionein, glucoamylase, the translation elongation factor
EF1-.alpha.(TEF) protein (U.S. Pat. No. 6,265,185), ribosomal
protein S7 (U.S. Pat. No. 6,265,185), ), ammonium transporter
proteins (U.S. patent application Ser. No. 11/185301), export
proteins, etc. Any one of a number of regulatory sequences can be
used, depending upon whether constitutive or induced transcription
is desired, the efficiency of the promoter in expressing the ORF of
interest, the ease of construction and the like.
[0350] Nucleotide sequences surrounding the translational
initiation codon `ATG` have been found to affect expression in
yeast cells. If the desired polypeptide is poorly expressed in
yeast, the nucleotide sequences of exogenous genes can be modified
to include an efficient yeast translation initiation sequence to
obtain optimal gene expression. For expression in yeast, this can
be done by site-directed mutagenesis of an inefficiently expressed
gene by fusing it in-frame to an endogenous yeast gene, preferably
a highly expressed gene. Alternatively, as demonstrated in the
invention herein in Yarrowia lipolytica, one can determine the
consensus translation initiation sequence in the host and engineer
this sequence into heterologous genes for their optimal expression
in the host of interest.
[0351] The termination region can be derived from the 3' region of
the gene from which the initiation region was obtained or from a
different gene. A large number of termination regions are known and
function satisfactorily in a variety of hosts (when utilized both
in the same and different genera and species from where they were
derived). The termination region usually is selected more as a
matter of convenience rather than because of any particular
property. Preferably, the termination region is derived from a
yeast gene, particularly Saccharomyces, Schizosaccharomyces,
Candida, Yarrowia or Kluyveromyces. The 3'-regions of mammalian
genes encoding .gamma.-interferon and .alpha.-2 interferon are also
known to function in yeast. Termination control regions may also be
derived from various genes native to the preferred hosts.
Optionally, a termination site may be unnecessary; however, it is
most preferred if included.
[0352] As one of skill in the art is aware, merely inserting a gene
into a cloning vector does not ensure that it will be successfully
expressed at the level needed. In response to the need for a high
expression rate, many specialized expression vectors have been
created by manipulating a number of different genetic elements that
control aspects of transcription, translation, protein stability,
oxygen limitation and secretion from the host cell. More
specifically, some of the molecular features that have been
manipulated to control gene expression include: (1) the nature of
the relevant transcriptional promoter and terminator sequences; (2)
the number of copies of the cloned gene and whether the gene is
plasmid-borne or integrated into the genome of the host cell; (3)
the final cellular location of the synthesized foreign protein; (4)
the efficiency of translation in the host organism; (5) the
intrinsic stability of the cloned gene protein within the host
cell; and (6) the codon usage within the cloned gene, such that its
frequency approaches the frequency of preferred codon usage of the
host cell. Each of these types of modifications are encompassed in
the present invention, as means to further optimize expression of
the PUFA biosynthetic pathway enzymes.
[0353] Once the DNA encoding a desaturase or elongase polypeptide
suitable for expression in an oleaginous yeast has been obtained,
it is placed in a plasmid vector capable of autonomous replication
in a host cell; or, it is directly integrated into the genome of
the host cell. Integration of expression cassettes can occur
randomly within the host genome or can be targeted through the use
of constructs containing regions of homology with the host genome
sufficient to target recombination within the host locus. Where
constructs are targeted to an endogenous locus, all or some of the
transcriptional and translational regulatory regions can be
provided by the endogenous locus.
[0354] A method of expressing genes in Yarrowia lipolytica is by
integration of linear DNA into the genome of the host; and,
integration into multiple locations within the genome can be
particularly useful when high level expression of genes are
desired. Toward this end, it is desirable to identify a sequence
within the genome that is present in multiple copies.
[0355] Schmid-Berger et al. (J. Bact. 176(9):2477-2482 (1994))
discovered the first retrotransposon-like element Ylt1 in Yarrowia
lipolytica. This retrotransposon is characterized by the presence
of long terminal repeats (LTRs; each approximately 700 bp in
length) called zeta regions. Ylt1 and solo zeta elements were
present in a dispersed manner within the genome in at least 35
copies/genome and 50-60 copies/genome, respectively; both elements
were determined to function as sites of homologous recombination.
Further, work by Juretzek et al. (Yeast 18:97-113 (2001))
demonstrated that gene expression could be dramatically increased
by targeting plasmids into the repetitive regions of the yeast
genome (using linear DNA with LTR zeta regions at both ends), as
compared to the expression obtained using low-copy plasmid
transformants. Thus, zeta-directed integration can be ideal as a
means to ensure multiple integration of plasmid DNA into Yarrowia
lipolytica, thereby permitting high-level gene expression.
Unfortunately, however, not all strains of Yarrowia lipolytica
possess zeta regions (e.g., the strain identified as ATCC Accession
No. #20362). When the strain lacks such regions, it is also
possible to integrate plasmid DNA comprising expression cassettes
into alternate loci to reach the desired copy number for the
expression cassette. For example, preferred alternate loci include:
the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene
locus (GenBank Accession No. AF260230), the Lys5 gene (GenBank
Accession No. M34929), the Aco2 gene locus (GenBank Accession No.
AJ001 300), the Pox3 gene locus (Pox3: GenBank Accession No.
XP.sub.--503244; or, Aco3: GenBank Accession No. AJ001301), the
delta-12 desaturase gene locus, the Lip1 gene locus (GenBank
Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession
No. AJ012632).
[0356] Advantageously, the Ura3 gene can be used repeatedly in
combination with 5-fluoroorotic acid (5-fluorouracil-6-carboxylic
acid monohydrate; "5-FOA") selection (infra), to readily permit
genetic modifications to be integrated into the Yarrowia genome in
a facile manner.
[0357] Where two or more genes are expressed from separate
replicating vectors, it is desirable that each vector has a
different means of selection and should lack homology to the other
constructs to maintain stable expression and prevent reassortment
of elements among constructs. Judicious choice of regulatory
regions, selection means and method of propagation of the
introduced construct can be experimentally determined so that all
introduced genes are expressed at the necessary levels to provide
for synthesis of the desired products.
[0358] Constructs comprising the gene of interest may be introduced
into a host cell by any standard technique. These techniques
include transformation (e.g., lithium acetate transformation
[Methods in Enzymology, 194:186-187 (1991)]), protoplast fusion,
bolistic impact, electroporation, microinjection, or any other
method that introduces the gene of interest into the host cell.
More specific teachings applicable for oleaginous yeasts (i.e.,
Yarrowia lipolytica) include U.S. Pat. Nos. 4,880,741 and 5,071,764
and Chen, D. C. et al. (Appl Microbiol Biotechnol. 48(2):232-235
(1997)).
[0359] For convenience, a host cell that has been manipulated by
any method to take up a DNA sequence (e.g., an expression cassette)
will be referred to as "transformed" or "recombinant" herein. The
transformed host will have at least one copy of the expression
construct and may have two or more, depending upon whether the gene
is integrated into the genome, amplified or is present on an
extrachromosomal element having multiple copy numbers.
[0360] The transformed host cell can be identified by various
selection techniques, as described in WO 04/101757. Preferred
selection methods for use herein are resistance to kanamycin,
hygromycin and the amino glycoside G418, as well as ability to grow
on media lacking uracil, leucine, lysine, tryptophan or histidine.
In alternate embodiments, 5-FOA is used for selection of yeast
Ura-mutants. The compound is toxic to yeast cells that possess a
functioning URA3 gene encoding orotidine 5'-monophosphate
decarboxylase (OMP decarboxylase); thus, based on this toxicity,
5-FOA is especially useful for the selection and identification of
Ura.sup.- mutant yeast strains (Bartel, P. L. and Fields, S., Yeast
2-Hybrid System, Oxford University: New York, v. 7, pp 109-147,
1997). More specifically, one can first knockout the native Ura3
gene to produce a strain having a Ura-phenotype, wherein selection
occurs based on 5-FOA resistance. Then, a cluster of multiple
chimeric genes and a new Ura3 gene could be integrated into a
different locus of the Yarrowia genome to thereby produce a new
strain having a Ura+phenotype. Subsequent integration would produce
a new Ura3-strain (again identified using 5-FOA selection), when
the introduced Ura3 gene is knocked out. Thus, the Ura3 gene (in
combination with 5-FOA selection) can be used as a selection marker
in multiple rounds of transformation.
[0361] Following transformation, substrates suitable for the
recombinantly expressed desaturases and/or elongases (and
optionally other PUFA enzymes that are expressed within the host
cell) may be produced by the host either naturally or
transgenically, or they may be provided exogenously.
[0362] Methods for manipulating biochemical pathways are well known
to those skilled in the art; and, it is expected that numerous
manipulations will be possible to maximize omega-3 and/or omega-6
fatty acid biosynthesis in oleaginous yeasts, and particularly, in
Yarrowia lipolytica. This may require metabolic engineering
directly within the PUFA biosynthetic pathway or additional
manipulation of pathways that contribute carbon to the PUFA
biosynthetic pathway.
[0363] In the case of manipulations within the PUFA biosynthetic
pathway, it may be desirable to increase the production of LA to
enable increased production of omega-6 and/or omega-3 fatty acids.
Introducing and/or amplifying genes encoding delta-9 and/or
delta-12 desaturases may accomplish this.
[0364] To maximize production of omega-6 unsaturated fatty acids,
it is well known to one skilled in the art that production is
favored in a host microorganism that is substantially free of ALA.
Thus, preferably, the host is selected or obtained by removing or
inhibiting delta-15 or omega-3 type desaturase activity that
permits conversion of LA to ALA. The endogenous desaturase activity
can be reduced or eliminated by, for example, (1) providing a
cassette for transcription of antisense sequences to the delta-1 5
desaturase transcription product, (2) disrupting the delta-15
desaturase gene through insertion, substitution and/or deletion of
all or part of the target gene; or (3) using a host cell which
naturally has [or has been mutated to have] low or no delta-15
desaturase activity. Inhibition of undesired desaturase pathways
can also be accomplished through the use of specific desaturase
inhibitors such as those described in U.S. Pat. No. 4,778,630.
[0365] Alternatively, it may be desirable to maximize production of
omega-3 fatty acids (and minimize synthesis of omega-6 fatty
acids). Thus, one could utilize a host microorganism wherein the
delta-12 desaturase activity that permits conversion of oleic acid
to LA is removed or inhibited, using any of the means described
above (see also e.g., WO 2004/104167, herein incorporated entirely
by reference). Subsequently, appropriate expression cassettes would
be introduced into the host, along with appropriate substrates
(e.g., ALA) for conversion to omega-3 fatty acid derivatives of ALA
(e.g., STA, ETrA, ETA, EPA, DPA, DHA).
[0366] Beyond the immediate PUFA biosynthetic pathway, it is
expected that manipulation of several other enzymatic pathways
leading to the biosynthesis of precursor fatty acids may contribute
to the overall net biosynthesis of specific PUFAs. Identification
and manipulation of these related pathways will be useful in the
future.
[0367] Additional copies of desaturase and elongase genes may be
introduced into the host to increase the output of omega-3 and/or
omega-6 fatty acid biosynthetic pathways. Expression of the
desaturase or elongase genes also can be increased at the
transcriptional level through the use of a stronger promoter
(either regulated or constitutive) to cause increased expression,
by removing/deleting destabilizing sequences from either the mRNA
or the encoded protein, or by adding stabilizing sequences to the
mRNA (U.S. Pat. No. 4,910,141). Yet another approach to increase
expression of the desaturase or elongase genes, as demonstrated in
the instant invention, is to increase the translational efficiency
of the encoded mRNAs by replacement of codons in the native gene
with those for optimal gene expression in the selected host
microorganism.
[0368] Conversely, biochemical pathways competing with the omega-3
and/or omega-6 fatty acid biosynthetic pathways for energy or
carbon, or native PUFA biosynthetic pathway enzymes that interfere
with production of a particular PUFA end-product, may be eliminated
by gene disruption or down-regulated by other means (e.g.,
antisense mRNA). For gene disruption, a foreign DNA fragment
(typically a selectable marker gene) is inserted into the
structural gene to be disrupted in order to interrupt its coding
sequence and thereby functionally inactivate the gene.
Transformation of the disruption cassette into the host cell
results in replacement of the functional native gene by homologous
recombination with the non-functional disrupted gene (see, for
example: Hamilton et al. J. Bacteriol. 171:46174622 (1989); Balbas
et al. Gene 136:211-213 (1993); Gueldener et al. Nucleic Acids Res.
24:2519-2524 (1996); and Smith et al. Methods Mol. Cell. Biol.
5:270-277 (1996)).
[0369] Antisense technology is another method of down-regulating
genes when the sequence of the target gene is known. To accomplish
this, a nucleic acid segment from the desired gene is cloned and
operably linked to a promoter such that the anti-sense strand of
RNA will be transcribed. This construct is then introduced into the
host cell and the antisense strand of RNA is produced. Antisense
RNA inhibits gene expression by preventing the accumulation of mRNA
that encodes the protein of interest. The person skilled in the art
will know that special considerations are associated with the use
of antisense technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
antisense genes may require the use of different chimeric genes
utilizing different regulatory elements known to the skilled
artisan.
[0370] Although targeted gene disruption and antisense technology
offer effective means of down-regulating genes where the sequence
is known, other less specific methodologies have been developed
that are not sequence-based (e.g., mutagenesis via UV
radiation/chemical agents or use of transposable
elements/transposons; see WO 2004/101757).
[0371] Within the context of the present invention, it may be
useful to modulate the expression of the fatty acid biosynthetic
pathway by any one of the methods described above. For example, the
present invention provides methods whereby genes encoding key
enzymes in the biosynthetic pathways are introduced into oleaginous
yeasts for the production of omega-3 and/or omega-6 fatty acids. It
will be particularly useful to express these genes in oleaginous
yeasts that do not naturally possess omega-3 and/or omega-6 fatty
acid biosynthetic pathways and coordinate the expression of these
genes, to maximize production of preferred PUFA products using
various means for metabolic engineering of the host organism.
[0372] Microbial host cells for production of omega fatty acids may
include microbial hosts that grow on a variety of feedstocks,
including simple or complex carbohydrates, organic acids and
alcohols, and/or hydrocarbons over a wide range of temperature and
pH values.
[0373] Preferred microbial hosts, however, are oleaginous yeasts.
These organisms are naturally capable of oil synthesis and
accumulation, wherein the oil can comprise greater than about 25%
of the cellular dry weight, more preferably greater than about 30%
of the cellular dry weight, and most preferably greater than about
40% of the cellular dry weight. Genera typically identified as
oleaginous yeast include, but are not limited to: Yarrowia,
Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon
and Lipomyces. More specifically, illustrative oil-synthesizing
yeasts include: Rhodosporidium toruloides, Lipomyces starkeyii, L.
lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C.
utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R.
graminis and Yarrowia lipolytica (formerly classified as Candida
lipolytica).
[0374] Most preferred is the oleaginous yeast Yarrowia lipolytica;
and, in a further embodiment, most preferred are the Yarrowia
lipolytica strains designated as ATCC Accession Nos. #20362, ATCC
#8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S.,
and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)).
[0375] The transformed microbial host cell is grown under
conditions that optimize desaturase and elongase activities and
produce the greatest and the most economical yield of the preferred
PUFAs. In general, media conditions that may be optimized include
the type and amount of carbon source, the type and amount of
nitrogen source, the carbon-to-nitrogen ratio, the oxygen level,
growth temperature, pH, length of the biomass production phase,
length of the oil accumulation phase and the time of cell harvest.
Microorganisms of interest, such as oleaginous yeast, are grown in
complex media (e.g., yeast extract-peptone-dextrose broth (YPD)) or
a defined minimal media that lacks a component necessary for growth
and thereby forces selection of the desired expression cassettes
(e.g., yeast nitrogen base (Difco Laboratories, Detroit,
Mich.)).
[0376] Fermentation media in the present invention must contain a
suitable carbon source. Suitable carbon sources may include, but
are not limited to: monosaccharides (e.g., glucose, fructose),
disaccharides (e.g., lactose, sucrose), oligosaccharides,
polysaccharides (e.g., starch, cellulose or mixtures thereof, sugar
alcohols (e.g., glycerol) or mixtures from renewable feedstocks
(e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses,
barley malt). Additionally, carbon sources may include alkanes,
fatty acids, esters of fatty acids, monoglycerides, diglycerides,
triglycerides, phospholipids and various commercial sources of
fatty acids including vegetable oils (e.g., soybean oil) and animal
fats. Additionally, the carbon source may include one-carbon
sources (e.g., carbon dioxide, methanol, formaldehyde, formate and
carbon-containing amines) for which metabolic conversion into key
biochemical intermediates has been demonstrated. Hence it is
contemplated that the source of carbon utilized in the present
invention may encompass a wide variety of carbon-containing sources
and will only be limited by the choice of the host organism.
Although all of the above mentioned carbon sources and mixtures
thereof are expected to be suitable in the present invention,
preferred carbon sources are sugars and/or fatty acids. Most
preferred is glucose and/or fatty acids containing between 10-22
carbons.
[0377] Nitrogen may be supplied from an inorganic (e.g.,
(NH.sub.4).sub.2SO.sub.4) or organic (e.g., urea or glutamate)
source. In addition to appropriate carbon and nitrogen sources, the
fermentation media must also contain suitable minerals, salts,
cofactors, buffers, vitamins and other components known to those
skilled in the art suitable for the growth of the microorganism and
promotion of the enzymatic pathways necessary for PUFA production.
Particular attention is given to several metal ions (e.g.,
Mn.sup.+2, Co.sup.+2, Zn.sup.+2, Mg.sup.+2) that promote synthesis
of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. Single Cell
Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
[0378] Preferred growth media in the present invention are common
commercially prepared media, such as yeast nitrogen base (Difco
Laboratories, Detroit, Mich.). Other defined or synthetic growth
media may also be used and the appropriate medium for growth of the
particular microorganism will be known by one skilled in the art of
microbiology or fermentation science. A suitable pH range for the
fermentation is typically between about pH 4.0 to pH 8.0, wherein
pH 5.5 to pH 7.0 is preferred as the range for the initial growth
conditions. The fermentation may be conducted under aerobic or
anaerobic conditions, wherein microaerobic conditions are
preferred.
[0379] Typically, accumulation of high levels of PUFAs in
oleaginous yeast cells requires a two-stage process, since the
metabolic state must be "balanced" between growth and
synthesis/storage of fats. Thus, most preferably, a two-stage
fermentation process is necessary for the production of PUFAs in
oleaginous yeast. This approach is described in WO 2004/101757, as
are various suitable fermentation process designs (i.e., batch,
fed-batch and continuous) and considerations during growth.
[0380] The PUFAs may be found in the host microorganism as free
fatty acids or in esterified forms such as acylglycerols,
phospholipids, sulfolipids or glycolipids, and may be extracted
from the host cell through a variety of means well-known in the
art. One review of extraction techniques, quality analysis and
acceptability standards for yeast lipids is that of Z. Jacobs
(Critical Reviews in Biotechnology 12(5/6):463-491 (1992)). A brief
review of downstream processing is also available by A. Singh and
O. Ward (Adv. Appl. Microbiol. 45:271-312 (1997)).
[0381] In general, means for the purification of PUFAs may include
extraction with organic solvents, sonication, supercritical fluid
extraction (e.g., using carbon dioxide), saponification and
physical means such as presses, or combinations thereof. One is
referred to the teachings of WO 2004/101757 for additional
details.
[0382] The market place currently supports a large variety of food
and feed products, incorporating omega-3 and/or omega-6 fatty acids
(particularly ARA, EPA and DHA). It is contemplated that the plant
oils of the invention and the yeast oils of the invention
comprising long-chain PUFAs will function in food and feed products
to impart the health benefits of current formulations. More
specifically, oils of the invention containing omega-3 and/or
omega-6 fatty acids will be suitable for use in a variety of food
and feed products including, but not limited to food analogs, meat
products, cereal products, baked foods, snack foods and dairy
products.
[0383] Additionally the present oils may be used in formulations to
impart health benefit in medical foods including medical
nutritionals, dietary supplements, infant formula as well as
pharmaceutical products. One of skill in the art of food processing
and food formulation will understand how the amount and composition
of the present oils may be added to the food or feed product. Such
an amount will be referred to herein as an "effective" amount and
will depend on the food or feed product, the diet that the product
is intended to supplement or the medical condition that the medical
food or medical nutritional is intended to correct or treat.
[0384] A "food analog" is a food-like product manufactured to
resemble its food counterpart, whether meat, cheese, milk or the
like, and is intended to have the appearance, taste, and texture of
its counterpart. Thus, the term "food" as used herein also
encompasses food analogs. Food analogs can be made use processes
well known to those skilled in the art. U.S. Pat. Nos. 6,355,296 B1
and 6,187,367 B1 describe emulsified meat analogs and emulsified
meat extenders. U.S. Pat. No. 5,206,050 B1 describes soy protein
curd useful for cooked food analogs (also can be used as a process
to form a curd useful to make food analogs). U.S. Pat. No.
4,284,656 to Hwa describes a soy protein curd useful for food
analogs. U.S. Pat. No. 3,988,485 to Hibbert et al. describes a
meat-like protein food formed from spun vegetable protein fibers.
U.S. Pat. No. 3,950,564 to Puski et al. describes a process of
making a soy based meat substitute and U.S. Pat. No. 3,925,566 to
Reinhart et al. describes a simulated meat product. For example,
soy protein that has been processed to impart a structure, chunk or
fiber for use as a food ingredient is called "textured soy protein"
(TSP). TSPs are frequently made to resemble meat, seafood, or
poultry in structure and appearance when hydrated.
[0385] Food analogs can be classified as imitation or substitutes
depending on their functional and compositional characteristics.
For example, an imitation cheese need only resemble the cheese it
is designed to replace. However, a product can generally be called
a substitute cheese only if it is nutritionally equivalent to the
cheese it is replacing and meets the minimum compositional
requirements for that cheese. Thus, substitute cheese will often
have higher protein levels than imitation cheeses and be fortified
with vitamins and minerals.
[0386] Milk analogs or nondairy food products include, but are not
limited to, imitation milks and nondairy frozen desserts (e.g.,
those made from soybeans and/or soy protein products).
[0387] Meat products encompass a broad variety of products. In the
United States "meat" includes "red meats" produced from cattle,
hogs and sheep. In addition to the red meats there are poultry
items which include chickens, turkeys, geese, guineas, ducks and
the fish and shellfish. There is a wide assortment of seasoned and
processed meat products: fresh, cured and fried, and cured and
cooked. Sausages and hot dogs are examples of processed meat
products. Thus, the term "meat products" as used herein includes,
but is not limited to, processed meat products.
[0388] A cereal food product is a food product derived from the
processing of a cereal grain. A cereal grain includes any plant
from the grass family that yields an edible grain (seed). The most
popular grains are barley, corn, millet, oats, quinoa, rice, rye,
sorghum, triticale, wheat and wild rice. Examples of a cereal food
product include, but are not limited to: whole grain, crushed
grain, grits, flour, bran, germ, breakfast cereals, extruded foods,
pastas, and the like.
[0389] A baked goods product comprises any of the cereal food
products mentioned above and has been baked or processed in a
manner comparable to baking (i.e., to dry or harden by subjecting
to heat). Examples of a baked good product include, but are not
limited to: bread, cakes, doughnuts, bars, pastas, bread crumbs,
baked snacks, mini-biscuits, mini-crackers, mini-cookies, and
mini-pretzels. As was mentioned above, oils of the invention can be
used as an ingredient.
[0390] A snack food product comprises any of the above or below
described food products.
[0391] A fried food product comprises any of the above or below
described food products that has been fried.
[0392] The beverage can be in a liquid or in a dry powdered
form.
[0393] For example, there can be mentioned non-carbonated drinks
such as fruit juices, fresh, frozen, canned or concentrate;
flavored or plain milk drinks, etc. Adult and infant nutritional
formulas are well known in the art and commercially available
(e.g., Similac.RTM., Ensure.RTM., Jevity.RTM., and Alimentum.RTM.
from Ross Products Division, Abbott Laboratories).
[0394] Infant formulas are liquids or reconstituted powders fed to
infants and young children. "Infant formula" is defined herein as
an enteral nutritional product which can be substituted for human
breast milk in feeding infants and typically is composed of a
desired percentage of fat mixed with desired percentages of
carbohydrates and proteins in an aquous solution (e.g., see U.S.
Pat. No. 4,670,285). Based on the worldwide composition studies, as
well as levels specified by expert groups, average human breast
milk typically contains about 0.20% to 0.40% of total fatty acids
(assuming about 50% of calories from fat); and, generally the ratio
of DHA to ARA would range from about 1:1 to 1:2 (see, e.g.,
formulations of Enfamil LIPIL.TM. (Mead Johnson & Company) and
Similac Advance.TM. (Ross Products Division, Abbott Laboratories)).
Infant formulas have a special role to play in the diets of infants
because they are often the only source of nutrients for infants;
and, although breast-feeding is still the best nourishment for
infants, infant formula is a close enough second that babies not
only survive but thrive.
[0395] A dairy product is a product derived from milk. A milk
analog or nondairy product is derived from a source other than
milk, for example, soymilk as was discussed above. These products
include, but are not limited to: whole milk, skim milk, fermented
milk products such as yogurt or sour milk, cream, butter, condensed
milk, dehydrated milk, coffee whitener, coffee creamer, ice cream,
cheese, etc.
[0396] Additional food products into which the long-chain
PUFA-containing oils of the invention could be included are, for
example, chewing gums, confections and frostings, gelatins and
puddings, hard and soft candies, jams and jellies, white granulated
sugar, sugar substitutes, sweet sauces, toppings and syrups, and
dry-blended powder mixes.
[0397] A health food product is any food product that imparts a
health benefit and include functional foods, medical foods, medical
nutritionals and dietary supplements. Additionally, the oils of the
invention may be used in standard pharmaceutical compositions
(e.g., the long-chain PUFA containing oils could readily be
incorporated into the any of the above mentioned food products, to
thereby produce a functional or medical food). For example, more
concentrated formulations comprising ARA, EPA or DHA include
capsules, powders, tablets, softgels, gelcaps, liquid concentrates
and emulsions which can be used as a dietary supplement in humans
or animals other than humans.
[0398] Animal feeds are generically defined herein as products
intended for use as feed or for mixing in feed for animals other
than humans. The long-chain PUFA containing oils of the invention
can be used as an ingredient in various animal feeds.
[0399] More specifically, although not limited therein, it is
expected that the oils of the invention can be used within pet food
products, ruminant and poultry food products and aquacultural food
products. Pet food products are those products intended to be fed
to a pet (e.g., dog, cat, bird, reptile, rodent). These products
can include the cereal and health food products above, as well as
meat and meat byproducts, soy protein products, grass and hay
products (e.g., alfalfa, timothy, oat or brome grass, vegetables).
Ruminant and poultry food products are those wherein the product is
intended to be fed to an animal (e.g., turkeys, chickens, cattle,
swine). As with the pet foods above, these products can include
cereal and health food products, soy protein products, meat and
meat byproducts, and grass and hay products as listed above.
Aquacultural food products (or "aquafeeds") are those products
intended to be used in aquafarming which concerns the propagation,
cultivation or farming of aquatic organisms and/or animals in fresh
or marine waters.
EXAMPLES
[0400] The present invention is further defined in the following
Examples, in which parts and percentages are by weight and degrees
are Celsius, unless otherwise stated. It should be understood that
these Examples, while indicating preferred embodiments of the
invention, are given by way of illustration only. From the above
discussion and these Examples, one skilled in the art can ascertain
the essential characteristics of this invention, and without
departing from the spirit and scope thereof, can make various
changes and modifications of the invention to adapt it to various
usages and conditions. Thus, various modifications of the invention
in addition to those shown and described herein will be apparent to
those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
[0401] The meaning of abbreviations is as follows: "sec" means
second(s), "min" means minute(s), "h" means hour(s), "d" means
day(s), ".mu.l" means microliter(s), "mL" means milliliter(s), "L"
means liter(s), ".mu.M" means micromolar, "mM" means millimolar,
"M" means molar, "mmol" means millimole(s), "pmole" mean
micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "ng"
means nanogram(s), "U" means unit(s), "bp" means base pair(s) and
"kB" means kilobase(s).
[0402] Transformation and Cultivation of Yarrowia lipolytica
[0403] Yarrowia lipolytica strains with ATCC Accession Nos. #20362,
#76982 and #90812 were purchased from the American Type Culture
Collection (Rockville, Md.). Yarrowia lipolytica strains were
typically grown at 28.degree. C. on YPD agar (1% yeast extract, 2%
bactopeptone, 2% glucose, 2% agar).
[0404] Transformation of Yarrowia lipolytica was performed
according to the method of Chen, D. C. et al. (Appl. Microbiol.
Biotechnol. 48(2):232-235 (1997)), unless otherwise noted. Briefly,
Yarrowia was streaked onto a YPD plate and grown at 30.degree. C.
for approximately 18 h. Several large loopfuls of cells were
scraped from the plate and resuspended in 1 mL of transformation
buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of
2 M Li acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 .mu.g sheared
salmon sperm DNA. Then, approximately 500 ng of linearized plasmid
DNA was incubated in 100 .mu.L of resuspended cells, and maintained
at 39.degree. C. for 1 h with vortex mixing at 15 min intervals.
The cells were plated onto selection media plates and maintained at
30.degree. C. for 2 to 3 days.
[0405] For selection of transformants, minimal medium ("MM") was
generally used; the composition of MM is as follows: 0.17% yeast
nitrogen base (Difco Laboratories, Detroit, Mich.) without ammonium
sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1).
Supplements of uracil were added as appropriate to a final
concentration of 0.01% (thereby producing "MMU" selection media,
prepared with 20 g/L agar).
[0406] Alternatively, transformants were selected on 5-fluoroorotic
acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate)
selection media, comprising: 0.17% yeast nitrogen base (Difco
Laboratories, Detroit, Mich.) without ammonium sulfate or amino
acids, 2% glucose, 0.1% proline, 75 mg/L uracil, 75 mg/L uridine,
900 mg/L FOA (Zymo Research Corp., Orange, Calif.) and 20 g/L
agar.
[0407] Fatty Acid Analysis of Yarrowia lipolytica
[0408] For fatty acid analysis, cells were collected by
centrifugation and lipids were extracted as described in Bligh, E.
G. & Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)).
Fatty acid methyl esters were prepared by transesterification of
the lipid extract with sodium methoxide (Roughan, G., and Nishida
I. Arch Biochem Biophys. 276(1):38-46 (1990)) and subsequently
analyzed with a Hewlett-Packard 6890 GC fitted with a 30
m.times.0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The
oven temperature was from 170.degree. C. (25 min hold) to
185.degree. C. at 3.5.degree. C./min.
[0409] For direct base transesterification, Yarrowia culture (3 mL)
was harvested, washed once in distilled water, and dried under
vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 .mu.L of
1%) was added to the sample, and then the sample was vortexed and
rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 .mu.L
hexane, the sample was vortexed and spun. The upper layer was
removed and analyzed by GC as described above.
Example 1
Euglena gracilis Growth Conditions, Lipid Profile and mRNA
Isolation
[0410] Euglena gracilis was obtained from Dr. Richard Triemer's lab
at Michigan State University (East Lansing, Mich.). From 10 mL of
actively growing culture, a 1 mL aliquot was transferred into 250
mL of Euglena gracilis (Eg) Medium in a 500 mL glass bottle. Eg
medium was made by combining 1 g of sodium acetate, 1 g of beef
extract (U126-01, Difco Laboratories, Detroit, Mich.), 2 g of
Bacto.RTM. tryptone (0123-17-3, Difco Laboratories), 2 g of
Bacto.RTM.) yeast extract (0127-17-9, Difco Laboratories) in 970 mL
of water. After filter sterilizing, 30 mL of soil-water supernatant
(15-3790, Carolina Biological Supply Company, Burlington, N.C.,)
was aseptically added to give the final Eg medium. Euglena gracilis
cultures were grown at 23.degree. C. with a 16 h light, 8 h dark
cycle for 2 weeks with no agitation.
[0411] After 2 weeks, 10 mL of culture was removed for lipid
analysis and centrifuged at 1,800.times.g for 5 min. The pellet was
washed once with water and re-centrifuged. The resulting pellet was
dried for 5 min under vacuum, resuspended in 100 .mu.L of
trimethylsulfonium hydroxide (TMSH) and incubated at room
temperature for 15 min with shaking. After this, 0.5 mL of hexane
was added and the vials were incubated for 15 min at room
temperature with shaking. Fatty acid methyl esters (5 .mu.L
injected from hexane layer) were separated and quantified using a
Hewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320
fused silica capillary column (Supelco Inc., Cat. No. 24152). The
oven temperature was programmed to hold at 220.degree. C. for 2.7
min, increase to 240.degree. C. at 20.degree. C./min and then hold
for an additional 2.3 min. Carrier gas was supplied by a Whatman
hydrogen generator. Retention times were compared to those for
methyl esters of standards commercially available (Nu-Chek Prep,
Inc. Cat. No. U-99-A) and the resulting chromatogram is shown in
FIG. 13.
[0412] The remaining 2 week culture (240 mL) was pelleted by
centrifugation at 1,800.times.g for 10 min, washed once with water
and re-centrifuged. Total RNA was extracted from the resulting
pellet using the RNA STAT-60.TM. reagent (TEL-TEST, Inc.,
Friendswood, Tex.) and following the manufacturer's protocol
provided (use 5 mL of reagent, dissolved RNA in 0.5 mL of water).
In this way, 1 mg of total RNA (2 mg/mL) was obtained from the
pellet. The mRNA was isolated from 1 mg of total RNA using the mRNA
Purification Kit (Amersham Biosciences, Piscataway, N.J.) following
the manufacturer's protocol provided. In this way, 85 .mu.g of mRNA
was obtained.
Example 2
Euglena gracilis cDNA Synthesis, Library Construction and
Sequencing
[0413] A cDNA library was generated using the Cloneminer.TM. CDNA
Library Construction Kit (Cat. No. 18249-029, Invitrogen
Corporation, Carlsbad, Calif.) and following the manufacturer's
protocol provided (Version B, 25-0608). Using the non-radiolabeling
method, cDNA was synthesized from 3.2 .mu.g of mRNA (described
above) using the Biotin-attB2-Oligo(dT) primer. After synthesis of
the first and second strand, the attb1 adapter was added, ligated
and the cDNA was size fractionated using column chromatography. DNA
from fractions 7 and 8 (size ranging from .about.800-1500 bp) were
concentrated, recombined into PDONR T222 and transformed into E.
coli ElectroMAX.TM. DH10B.TM. T1 Phage-Resistant cells (Invitrogen
Corporation). The Euglena gracilis library was named eeg1 c.
[0414] For sequencing, clones first were recovered from archived
glycerol cultures grown/frozen in 384-well freezing media plates,
and replicated with a sterile 384 pin replicator (Genetix, Boston,
Mass.) in 384-well microtiter plates containing LB +75 .mu.g/mL
Kanamycin (replicated plates). Plasmids then were isolated, using
the Templiphi DNA sequencing template amplification kit method
(Amersham Biosciences) following the manufacturer's protocol.
Briefly, the Templiphi method uses bacteriophage (.phi.29 DNA
polymerase to amplify circular single-stranded or double-stranded
DNA by isothermal rolling circle amplification (Dean et al., Genome
Res. 11:1095-1099 (2001); Nelson et al., Biotechniques 32:S44-S47
(2002)). After growing 20 h at 37.degree. C., cells from the
replicated plate were added to 5 .mu.L of dilution buffer and
denatured at 95.degree. C. for 3 min to partially lyse cells and
release the denatured template. 5 .mu.L of Templiphi premix then
were added to each sample and the resulting reaction mixture was
incubated at 30.degree. C. for 16 h, then at 65.degree. C. for 10
min to inactivate the .phi.29 DNA polymerase activity. DNA
quantification with the PicoGreen.RTM. dsDNA Quantitation Reagent
(Molecular Probes) was performed after diluting the amplified
samples 1:3 in distilled water.
[0415] The amplified products then were denatured at 95.degree. C.
for 10 min and end-sequenced in 384-well plates, using the M13F
universal primer (SEQ ID NO:28), and the ABI BigDye version 3.1
Prism Sequencing Kit. For the sequencing reaction, 100-200 ng of
templates and 6.4 pmol of primers were used, and the following
reaction conditions were repeated 25 times: 96.degree. C. for 10
sec, 50.degree. C. for 5 sec and 60.degree. C. for 4 min. After
ethanol-based cleanup, cycle sequencing reaction products were
resolved and detected on Perkin-Elmer ABI 3730xl automated
sequencers.
Example 3
Identification of Long-Chain Polyunsaturated Fatty Acid Elongation
Enzyme Homologs from Euglena gracilis cDNA Library eeg1c
[0416] cDNA clones encoding long-chain polyunsaturated fatty acid
elongation enzyme homologs (LC-PUFA ELO homologs or delta-9
elongases) were identified by conducting BLAST (Basic Local
Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410
(1993)) searches for similarity to sequences contained in the BLAST
"nr" database (comprising all non-redundant GenBank CDS
translations, sequences derived from the 3-dimensional structure
Brookhaven Protein Data Bank, the last major release of the
SWISS-PROT protein sequence database, EMBL, and DDBJ databases).
The cDNA sequences obtained in Example 2 were analyzed for
similarity to all publicly available DNA sequences contained in the
"nr" database using the BLASTN algorithm provided by the National
Center for Biotechnology Information (NCBI). The DNA sequences were
translated in all reading frames and compared for similarity to all
publicly available protein sequences contained in the "nr" database
using the BLASTX algorithm (Gish and States, Nat. Genet. 3:266-272
(1993)) provided by the NCBI. For convenience, the P-value
(probability) of observing a match of a cDNA sequence to a sequence
contained in the searched databases merely by chance as calculated
by BLAST are reported herein as "plog" values, which represent the
negative of the logarithm of the reported P-value. Accordingly, the
greater the pLog value, the greater the likelihood that the cDNA
sequence and the BLAST "hit" represent homologous proteins.
[0417] The BLASTX search using the nucleotide sequences from clone
eeg1c.pk001.n5.f revealed similarity of the protein encoded by the
cDNA to the long-chain PUFA elongation enzyme from Isochrysis
galbana (SEQ ID NO:27) (NCBI Accession No. AAL37626 (GI 17226123),
locus AAL37626, CDS AF390174; Qi et al., FEBS Lett. 510(3):159-165
(2002)). The sequence of a portion of the cDNA insert from clone
eeg1c.pk001.n5.f is shown in SEQ ID NO:1 (5' end of cDNA insert).
Additional sequence was obtained from the 3' end of the cDNA insert
of eeg1c.pk001.n5.1 as described above, but using the poly(A)
tail-primed WobbleT oligonucleotides. Briefly, the WobbleT primer
is an equimolar mix of 21mer poly(T)A, poly(T)C, and poly(T)G, used
to sequence the 3' end of CDNA clones.
[0418] The 3' end sequence is shown in SEQ ID NO:2. Both the 5' and
3' sequences were aligned using Sequencher.TM. (Version 4.2, Gene
Codes Corporation, Ann Arbor, Mich.) and the resulting sequence for
the cDNA is shown in SEQ ID NO:3. Sequence for the coding sequence
from the cDNA in eeg1c.pk001.n5.f and the corresponding deduced
amino acid sequence is shown in SEQ ID NO:4 and SEQ ID NO:5,
respectively.
[0419] The amino acid sequence set forth in SEQ ID NO:5 was
evaluated by BLASTP, yielding a pLog value of 38.70 (E value of
2e-39) versus the Isochrysis galbana sequence (SEQ ID NO:27). The
Euglena gracilis delta-9 elongase is 39.4% identical to the
Isochrysis galbana delta-9 elongase sequence using the Jotun Hein
method. Sequence percent identity calculations performed by the
Jotun Hein method (Hein, J. J., Meth. Enz. 183:626-645 (1990)) were
done using the MegAlign.TM. v6.1 program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with
the default parameters for pairwise alignment (KTUPLE=2). The
Euglena gracilis delta-9 elongase is 31.8% (SEQ ID NO:5) identical
to the Isochrysis galbana delta-9 elongase (SEQ ID NO:27) sequence
using the Clustal V method (see FIG. 16 and FIG. 29). Sequence
percent identity calculations performed by the Clustal V method
(Higgins, D. G. and Sharp, P. M., Comput. Appl. Biosci. 5:151-153
(1989); Higgins et al., Comput. Appl. Biosci. 8:189-191 (1992))
were done using the MegAlign.TM. v6.1 program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) with
the default parameters for pairwise alignment (KTUPLE=1, GAP
PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5 and GAP LENGTH
PENALTY=10). BLAST scores and probabilities indicate that the
instant nucleic acid fragment (SEQ ID NO:3) encode an entire
Euglena gracilis delta-9 elongase.
Example 4
Synthesis and Functional Expression of a Codon-Optimized Delta-9
Elongase Gene (Derived from Isochrysis galbana) in Yarrowia
lipolytica
[0420] The codon usage of the delta-9 elongase gene of Isochrysis
galbana (SEQ ID NO:27, GenBank Accession No. AF390174) was
optimized for expression in Yarrowia lipolytica. According to the
Yarrowia codon usage pattern, the consensus sequence around the ATG
translation initiation codon, and the general rules of RNA
stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23
(2001)), a codon-optimized delta-9 elongase gene was designed,
affording SEQ ID NO:65, based on the DNA sequence of Isochrysis
galbana (SEQ ID NO:29). In addition to modification of the
translation initiation site, 127 bp of the 792 bp coding region
were modified, and 122 codons were optimized. None of the
modifications in the codon-optimized gene changed the amino acid
sequence of the encoded protein (GenBank Accession No. AF390174;
SEQ ID NO:27).
In Vitro Synthesis of a Codon-Optimized Delta-9 Elongase Gene for
Yarrowia lipolytica:
[0421] Eight pairs of oligonucleotides were designed to extend the
entire length of the codon-optimized coding region of the
Isochrysis galbana delta-9 elongase gene (e.g., IL3-1A, IL3-1B,
IL3-2A, IL3-2B, IL3-3A, IL3-3B, IL3-4A, IL3-4B, IL3-5A, IL3-5B,
IL3-6A, IL3-6B, IL3-7A, IL3-7B, IL3-8A, IL3-8B, corresponding to
SEQ ID NOs:66-81). Each pair of sense (A) and anti-sense (B)
oligonucleotides were complementary, with the exception of a 4 bp
overhang at each 5'-end. Additionally, primers IL3-1F, IL3-4R,
IL3-5F and IL3-8R (SEQ ID NOs:82-85) also introduced NcoI, PstI,
PstI and Not1 restriction sites, respectively, for subsequent
subcloning. Each oligonucleotide (100 ng) was phosphorylated at
37.degree. C. for 1 h in a volume of 20 .mu.L containing 50 mM
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 10 mM DTT, 0.5 mM spermidine,
0.5 mM ATP and 10 U of T4 polynucleotide kinase. Each pair of sense
and antisense oligonucleotides was mixed and annealed in a
thermocycler using the following parameters: 95.degree. C. (2 min),
85.degree. C.(2 min), 65.degree. C. (15 min), 37.degree. C. (15
min), 24.degree. C. (15 min) and 4.degree. C. (15 min). Thus,
IL3-1A (SEQ ID NO:66) was annealed to IL3-1 B (SEQ ID NO:67) to
produce the double-stranded product "IL3-1AB". Similarly, IL3-2A
(SEQ ID NO:68) was annealed to IL3-2B (SEQ ID NO:69) to produce the
double-stranded product "IL3-2AB", etc.
[0422] Two separate pools of annealed, double-stranded
oligonucleotides were then ligated together, as shown below: Pool 1
(comprising IL3-1AB, IL3-2AB, IL3-3AB and IL3-4AB); and Pool 2
(comprising IL3-5AB, IL3-6AB, IL3-7AB and IL3-8AB). Each pool of
annealed oligonucleotides was mixed in a volume of 20 .mu.L with 10
U of T4 DNA ligase and the ligation reaction was incubated
overnight at 16.degree. C.
[0423] The product of each ligation reaction was then used as
template to amplify the designed DNA fragment by PCR. Specifically,
using the ligated "Pool 1" mixture (e.g., IL3-1AB, IL3-2AB, IL3-3AB
and IL34AB) as template, and oligonucleotides IL3-1F and IL3-4R
(SEQ ID NOs:82 and 83) as primers, the first portion of the
codon-optimized delta-9 elongase gene was amplified by PCR. The 417
bp PCR fragment was subcloned into the PGEM-T easy vector (Promega)
to generate pT9(1-4) (SEQ ID NO:86).
[0424] Using the ligated "Pool 2" mixture (e.g. IL3-5AB, IL3-6AB,
IL3-7AB and IL3-8AB) as the template, and oligonucleotides IL3-5F
and IL3-8R (SEQ ID NOs:84 and 85) as primers, the second portion of
the codon-optimized delta-9 elongase gene was amplified similarly
by PCR and cloned into the pGEM-T-easy vector to generate pT9(5-8)
(SEQ ID NO:87).
[0425] E. coli was transformed separately with pT9(1-4) (SEQ ID
NO:86) and pT9(5-8) (SEQ ID NO:87) and the plasmid DNA was isolated
from ampicillin-resistant transformants. Plasmid DNA was purified
and digested with the appropriate restriction endonucleases to
liberate the 417 bp Ncol/PstI fragment of pT9(1-4) (SEQ ID NO:86)
and the 377 bp PstI/Not1 fragment of pT9(5-8) (SEQ ID NO:87). These
two fragments were then combined and directionally ligated together
with Nco1/Not1 digested pZUF17 (SEQ ID NO:25) to generate pDMW237
(SEQ ID NO:26). The DNA sequence of the resulting synthetic delta-9
elongase gene ("IgD9e") in pDMW237 was exactly the same as the
originally designed codon-optimized gene (e.g., SEQ ID NO:65) for
Yarrowia lipolytica.
Example 5
Construction of pDMW263
[0426] Plasmid pY5-30 (which was previously described in PCT
Publication No. WO 05/003310 (the contents of which are hereby
incorporated by reference),) is a shuttle plasmid that can
replicate both in E. coli and Yarrowia lipolytica. Plasmid pY5-30
contains the following: a Yarrowia autonomous replication sequence
(ARS18); a ColE1 plasmid origin of replication; an
ampicillin-resistance gene (Amp.sup.R), for selection in E. coli; a
Yarrowia LEU2 gene, for selection in Yarrowia; and a chimeric
TEF::GUS::XPR gene. Plasmid pDMW263 (SEQ ID NO:89) was created from
pY5-30, by replacing the TEF promoter with the Yarrowia lipolytica
FBAINm promoter (PCT Publication No. WO 05/049805) using techniques
well known to one skilled in the art. Briefly, this promoter refers
to a modified promoter which is located in the 5' upstream
untranslated region in front of the `ATG` translation initiation
codon of the fructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13)
encoded by the fba1 gene and that is necessary for expression, plus
a 5 portion of 5' coding region that has an intron, wherein FBAINm
has a 52 bp deletion between the ATG translation initiation codon
and the intron of the FBAIN promoter (thereby including only 22
amino acids of the N-terminus) and a new translation consensus
motif after the intron. Table 4 summarizes the components of
pDMW263. TABLE-US-00006 TABLE 4 Components of Plasmid pDMW263 RE
Sites and Nucleotides Within Description of Fragment and SEQ ID NO:
89 Chimeric Gene Components 4992-4296 ARS18 sequence (GenBank
Accession No. A17608) SalI/SaclI FBAINm::GUS::XPR, comprising:
(8505-2014) FBAINm: FBAINm promoter (WO2005/049805) GUS: E. coli
gene encoding .beta.-glucuronidase (Jefferson, R. A. Nature.
14:342: 837-838 (1989) XPR: .about.100 bp of the 3' region of the
Yarrowia Xpr gene (GenBank Accession No. M17741) 6303-8505 Yarrowia
Leu2 gene (GenBank Accession No. AF260230)
Example 6
Construction of Yarrowia lipolytica Expression Vector pY1 15 and
Gateway.RTM. Destination Vector pBY1
[0427] The NcoI/SalI DNA fragment from pDMW263 (see construction in
Example 5), containing the Yarrowia lipolytica FBAINm promoter, was
cloned into the NcoI/SalI DNA fragment of pDMW237 (see construction
in Example 4), containing 20 the synthetic delta-9 elongase gene
(IgD9e), to produce pY 115 (SEQ ID NO:30; FIG. 1).
[0428] Plasmid pY115 (SEQ ID NO:30) was digested with NcoI/NotI and
the resulting DNA ends were filled using Klenow. After filling to
form blunt ends, the DNA fragments were treated with calf
intestinal alkaline phosphatase and separated using agarose gel
electrophoresis. The 6989 bp fragment containing the Yarrowia
lipolytica FBAINm promoter was excised from the agarose gel and
purified using the QIAquick.RTM. Gel Extraction Kit (Qiagen Inc.,
Valencia, Calif.) following the manufacturer's protocol. The
purified 6989 bp fragment was ligated with cassette rfA using the
Gateway Vector Conversion System (Cat. No. 11823-029, Invitrogen
Corporation) following the manufacturer's protocol to form Yarrowia
lipolytica Gateway.RTM. destination vector pBY1 (SEQ ID NO:31; FIG.
2).
Example 7
Construction of Yarrowia lipolytica Expression Vectors PBY2 and
pBY1-FAE
[0429] Plasmid was purified from eeg1c.pk001.n5.f using the
QIAprep.RTM. Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.)
following the manufacturer's protocol. Using the Gateway.RTM. LR
Clonase.TM. II enzyme mix (Cat. No. 11791-020, Invitrogen
Corporation) and following the manufacturer's protocol, the cDNA
from eeg1c.pk001.n5.f was transferred to pBY1 to form pBY2 (SEQ ID
NO:32; FIG. 3). Since sequencing was performed with the WobbleT
primer, the full sequence of the 3' end of eeg1c.pk001.n5.f,
containing the polyA tail, was not known. Based on restriction
digest and agarose gel analysis, the poly A tail appeared to be
less than 100 bp long. pBY2 (SEQ ID NO:32) was transformed into E.
coli DH10BTM (Invitrogen Corporation), cells were grown and pBY2
was isolated using the QIAprep.RTM. Spin Miniprep Kit (Qiagen Inc.,
Valencia, Calif.) as above.
[0430] The Isochrysis galbana synthetic delta-9 elongase gene
(IgD9e) was amplified from pY115 (SEQ ID NO:30) with
oligonucleotide primers ig-s (SEQ ID NO:6) and ig-as (SEQ ID NO:7)
using the AccuPrime.TM. Taq Polymerase High Fidelity (Cat. No.
12346-086, Invitrogen Corporation) following the manufacturer's
protocol. The resulting DNA fragment was cloned into
pENTRT.TM./D-TOPO.RTM. using the PENTRTM Directional TOPO.RTM.
Cloning Kit (Invitrogen Corporation), following the manufacturer's
protocol, to produce pENTR-FAE. Plasmid pENTR-FAE was purified
using the QIAprep.RTM. Spin Miniprep Kit (Qiagen Inc., Valencia,
Calif.) following the manufacturer's protocol as above. Using the
Gateway.RTM. LR Clonase.TM. II enzyme mix (Cat. No. 11791-020,
Invitrogen Corporation) and following the manufacturer's protocol,
the CDS for the Isochrysis galbana synthetic delta-9 elongase gene
(IgD9e) was transferred to pBY1 to form pBY1-FAE (SEQ ID NO:33;
FIG. 4). pBY1-FAE was transformed into E. coli DH10B.TM.
(Invitrogen Corporation), cells were grown and pBY1-FAE was
isolated using the QIAprep.RTM. Spin Miniprep Kit (Qiagen Inc.,
Valencia, Calif.) as above.
Example 8
Construction of Yarrowia lipolytica Expression Vector pY120
[0431] The Euglena gracilis delta-9 elongase was amplified from
eeg1c.pk001.n5.f with oligonucleotide primers oEugEL1-1 (SEQ ID
NO:8) and oEugEL1-2 (SEQ ID NO:9) using the VentR.RTM. DNA
Polymerase (Cat. No. M0254S, New England Biolabs Inc., Beverly,
Mass.) following the manufacturer's protocol. The resulting DNA
fragment was cloned into the pCR-Blunt.RTM. cloning vector using
the Zero Blunt.RTM. PCR Cloning Kit (Invitrogen Corporation),
following the manufacturer's protocol, to produce pKR906. The
NcoI/NotI DNA fragment from pKR906, containing the Eulgena gracilis
delta-9 elongase, was cloned into the NcoI/NotI DNA fragment from
pY115, containing the Yarrowia lipolytica FBAINm promoter, to
produce pY120 (SEQ ID NO:34; FIG. 5).
Example 9
Cloning the Euglena gracilis Delta-9 Elongase into a Yeast
Expression Vector
[0432] The yeast episomal plasmid (YEp)-type vector pRS425
(Christianson et al., Gene 110:119-122 (1992)) contains sequences
from the Saccharomyces cerevisiae 2.mu. endogenous plasmid, a LEU2
selectable marker and sequences based on the backbone of a
multifunctional phagemid, pBluescript 11 SK(+). The Saccharomyces
cerevisiae strong, constitutive glyceraldehyde-3-phosphate
dehydrogenase (GPD) promoter was cloned between the SacII and SpeI
sites of pRS425 in the same way as described by Jia et al.
(Physiol. Genomics, 3, 83-92 (2000)) to produce pGPD-425. A NotI
site was introduced into the BamHI site of pGPD425, thus giving a
NotI site flanked by BamHI sites, and this plasmid was called
pY-75. The Euglena gracilis delta-9 elongase was released from
pKR906 (see Example 8) by digestion with NotI and cloned into the
NotI site of pY-75 to produce pY119 (SEQ ID NO:35; FIG. 6).
Example 10
Cloning the Euglena gracilis Delta-9 Elongase into a Soybean
Expression Vector
[0433] A starting plasmid pKR72 (ATCC Accession No. PTA-6019; SEQ
ID NO:36, 7085 bp sequence), a derivative of pKS123 which was
previously described in PCT Publication No. WO 02/008269 (the
contents of which are hereby incorporated by reference), contains
the hygromycin B phosphotransferase gene (HPT) (Gritz, L. and
Davies, J., Gene 25:179-188 (1983)), flanked by the T7 promoter and
transcription terminator (T7prom/hpt/T7term cassette), and a
bacterial origin of replication (ori) for selection and replication
in bacteria (e.g., E. Coli). In addition, pKR72 also contains the
hygromycin B phosphotransferase gene, flanked by the 35S promoter
(Odell et al., Nature 313:810-812 (1985)) and NOS 3' transcription
terminator (Depicker et al., J. Mol. Appl. Genet. 1:561-570 (1982))
(35S/hpt/NOS3' cassette) for selection in plants such as soybean.
pKR72 also contains a NotI restriction site, flanked by the
promoter for the .alpha.' subunit of .beta.-conglycinin (Beachy et
al., EMBO J. 4:3047-3053 (1985)) and the 3' transcription
termination region of the phaseolin gene (Doyle et al., J. Biol.
Chem. 261:9228-9238 (1986)), thus allowing for strong
tissue-specific expression in the seeds of soybean of genes cloned
into the NotI site.
[0434] The gene for the Euglena gracilis delta-9 elongase was
released from pKR906 (see Example 8) by digestion with NotI and
cloned into the NotI site of pKR72 to produce pKR912 (SEQ ID
NO:37). A schematic depiction of pKR912 is shown in FIG. 7.
Example 11
Cloning the Euglena qracilis Delta-9 Elongase into an Intermediate
Cloning Vector
[0435] Vector pKS102 (SEQ ID NO:38), previously described in PCT
Publication No. WO 02/00905 (the contents of which are hereby
incorporated by reference), contains the hygromycin B
phosphotransferase gene (HPT) (Gritz, L. and Davies, J., Gene
25:179-188 (1983)), flanked by the T7 promoter and transcription
terminator (T7prom/hpt/T7term cassette), and a bacterial origin of
replication (ori) for selection and replication in bacteria (e.g.,
E. coli).
[0436] Vector pKR197 (SEQ ID NO:39), previously described in PCT
Publication No. WO 04/071467 (the contents of which are hereby
incorporated by reference) was constructed by combining the AscI
fragment from plasmid pKS102 (SEQ ID NO:38), containing the
T7prom/hpt/T7term cassette and bacterial ori, with the AscI
fragment of plasmid pKR72, containing the .beta.con/NotI/Phas
cassette.
[0437] The gene for the Euglena gracilis delta-9 elongase was
released from pKR906 (see Example 6) by digestion with NotI and
cloned into the Noti site of pKR197 to produce intermediate cloning
vector pKR911 (SEQ ID NO:40). A schematic depiction of pKR911 is
shown in FIG. 8.
Example 12
cDNA Synthesis and PCR of Euglena qracilis Delta-8 Desaturase
[0438] cDNA was synthesized from 765 ng of mRNA (described in
Example 1 above) using the SuperScript.TM. Choice System for cDNA
synthesis (Invitrogen.TM. Life Technologies, Carlsbad, Calif.) with
the provided oligo(dT) primer according to the manufacturer's
protocol. The synthesized cDNA was dissolved in 20 .mu.L of
water.
[0439] The Euglena gracilis delta-8 desaturase was amplified from
cDNA with oligonucleotide primers Eg5-1 (SEQ ID NO:10) and Eg3-3
(SEQ ID NO:11) using the conditions described below.
[0440] cDNA (1 .mu.L) from the reaction described above was
combined with 50 pmol of Eg5-1 (SEQ ID NO:10), 50 pmol of Eg5-3
(SEQ ID NO:11), 1 .mu.L of PCR nucleotide mix (10 mM, Promega,
Madison, Wis.), 5 .mu.L of 10.times. PCR buffer (Invitrogen
Corporation), 1.5 .mu.L of MgCl.sub.2 (50 mM, Invitrogen
Corporation), 0.5 .mu.L of Taq polymerase (Invitrogen Corporation)
and water to 50 .mu.L. The reaction conditions were 94.degree. C.
for 3 min followed by 35 cycles of 94.degree. C. for 45 sec,
55.degree. C. for 45 sec and 72.degree. C. for 1 min. The PCR was
finished at 72.degree. C. for 7 min and then held at 4.degree. C.
The PCR reaction was analyzed by agarose gel electrophoresis on 5
.mu.L and a DNA band with molecular weight around 1.3 kb was
observed. The remaining 45 .mu.L of product was separated by
agarose gel electrophoresis and DNA band purified using the
Zymoclean.TM. Gel DNA Recovery Kit (Zymo Research, Orange, Calif.)
following the manufacturer's protocol. The resulting DNA was cloned
into the pGEM.RTM.-T Easy Vector (Promega) following the
manufacturer's protocol. Multiple clones were sequenced using T7
(SEQ ID NO:12), M13-28Rev (SEQ ID NO:13), Eg3-2 (SEQ ID NO:14) and
Eg5-2 (SEQ ID NO:15).
[0441] Thus, a DNA sequence for the Euglena gracilis delta-8
desaturase (Eg5) was obtained (SEQ ID NO:16). Translation of Eg5
gave rise to the protein sequence set forth in SEQ ID NO:17.
Example 13
Cloning the Euglena gracilis Delta-8 Desaturase with the Euglena
gracilis Delta-9 Elongase
[0442] Vector pKS121 (SEQ ID NO:41), which was previously described
in PCT Publication No. WO 02/00904 (the contents of which are
hereby incorporated by reference), contains a NotI site flanked by
the Kunitz soybean Trypsin Inhibitor (KTi) promoter (Jofuku et al.,
Plant Cell 1:1079-1093 (1989)) and the KTi 3' termination region,
the isolation of which is described in U.S. Pat. No. 6,372,965
(KTi/NotI/KTi3' cassette). Vector pKR457 (SEQ ID NO:42), which was
previously described in PCT Publication No. WO 05/047479 (the
contents of which are hereby incorporated by reference), is a
derivative of pKS121 where the restriction sites upstream and
downstream of the KTi/NotI/KTi3' cassette have been altered through
a number of subcloning steps. Vector pKR457 also contains the soy
albumin transcription terminator, which was previously described in
PCT Publication No. WO 04/071467 (the contents of which are hereby
incorporated by reference), downstream of the KTi terminator to
lengthen and strengthen termination of transcription. In pKR457,
the BamHI site upstream of the KTi promoter in the KTi/NotI/KTi3'
cassette was removed and a new sequence (SEQ ID NO:18) added
containing a BsiWI, SalI, SbfI and HindIII site with the BsiWI site
being closest the 5' end of the KTi promoter.
[0443] In addition, the SalI site downstream of the KTi terminator
in the KTi/NotI/KTi3' cassette from pKS121 was removed and a new
sequence (SEQ ID NO:19) added containing an Xbal (closest to 3' end
of KTi terminator), a BamHI site, the soy albumin transcription
terminator sequence, a BsiWI site and another BamHI site. The
albumin transcription terminator had been previously amplified from
soy genomic DNA using primer oSaIb-12 (SEQ ID NO:20), designed to
introduce a BsiWI site at the 3' end of the terminator, and primer
oSaIb-13 (SEQ ID NO:21), designed to introduce a BamHI site at the
5' end of the terminator.
[0444] Eg5 (SEQ ID NO:16) was released from the pGEM.RTM.-T Easy
Vector described in Example 12 by digestion with NotI and cloned
into the NotI site of pKR457 to produce pKR680 (SEQ ID NO:43).
Plasmid pKR680 was then digested with BsiWI and the fragment
containing Eg5 (SEQ ID NO:16) was cloned into the BsiWI site of
pKR911 (SEQ ID NO:40) to produce pKR913 (SEQ ID NO:44). A schematic
depiction of pK913 is shown in FIG. 9.
Example 14
Cloning the Euglena gracilis Delta-8 Desaturase into a Soybean
Expression Vector and Co-expression with the Euglena gracilis
Delta-9 Elongase
[0445] Plasmid pKR680 (SEQ ID NO:43) is digested with BsiWI and the
fragment containing Eg5 (SEQ ID NO:16) is cloned into the BsiWI
site of pKR912 (SEQ ID NO:37). In this way, the Euglena gracilis
delta-8 desaturase is co-expressed with the Euglena gracilis
delta-9 elongase behind strong, seed-specific promoters.
Example 15
Cloning the Mortierella alpina Delta-5 Desaturase into a Soybean
Expression Vector and Co-expression with the Euglena gracilis
Delta-9 Elongase and the Euglena gracilis Delta-8 Desaturase
[0446] A soybean expression vector containing the Euglena gracilis
delta-8 desaturase (SEQ ID NO:16), the Euglena gracilis delta-9
elongase (SEQ ID NO:4) and the Mortierella alpina delta-5
desaturase (SEQ ID NO:88), which is described in U.S. Pat. No.
6,075,183 and PCT Publication Nos. WO 04/071467 and WO 05/0479479
(the contents of which are hereby incorporated by reference), all
under the control of strong seed-specific promoters, is constructed
in the following way.
[0447] Through a number of sub-cloning steps, a sequence of DNA
(SEQ ID NO:22) is effectively added into the Smal site of vector
pKR287 (which is described in PCT Publication No. WO 04/071467, the
contents of which are hereby incorporated by reference), to produce
pKR767 (SEQ ID NO:45). In this way, a SbfI restriction site is
added to the 3' end of the leg1A transcription terminator of the
Gy1/Mad5/legA2 cassette, which is described in PCT Publication Nos.
WO 04/071467 and WO 05/0479479.
[0448] The Gy1/Mad5/legA2 cassette is released from pKR767 by
digestion with SbfI and the resulting fragment is cloned into the
SbfI site of the vector described in Example 14 to produce a new
vector that co-expresses all three genes under control of strong
seed-specific promoters.
Example 16
Co-expressing the Mortierella alpina Delta-5 Desaturase, the
Euglena gracilis Delta-9 Elongase and the Euglena gracilis Delta-8
Desaturase with the Saprolegnia diclina Delta-17 Desaturase
[0449] The soybean expression vector described in Example 15 is
co-transformed along with other vectors expressing multiple
different seed-specific promoter/LCPUFA-biosynthetic gene
combinations. Whole plasmids or purified AscI fragments from the
plasmids, containing the appropriate gene combinations, are used,
as could any combination of either fragment of plasmid.
[0450] For instance, the vector described in Example 15 could be
co-transformed with pKR328 (SEQ ID NO:46, described in PCT
Publication No. WO 04/071467) containing the Saprolegnia diclina
delta-17 desaturase under control of the annexin promoter and
having a hygromycin resistance gene for selection in plants.
[0451] Similarly, the vector described in Example 15, could be
co-transformed with pKR886 or pKR886r (FIG. 10), two vectors
similar to pKR328 but having the SAMS/ALS/ALS3' cassette (which is
described in PCT Publication No. WO 04/071467) for selection in
plants. Vectors pKR886 (SEQ ID NO:47) and pKR886r (SEQ ID NO:48)
are made by cloning the PstI fragment, containing the
Ann/Sdd17/BD30 cassette from pKR271 (SEQ ID NO:49, which is
described in PCT Publication No. WO 04/071467) into the SbfI site
of pKR226 (SEQ ID NO:50, which is described in PCT Publication No.
WO 04/071467).
Example 17
Co-expressing the Mortierella alpina Delta-5 Desaturase, the
Euglena gracilis Delta-9 Elongase and the Euglena gracilis Delta-8
Desaturase with the Saprolegnia diclina Delta-17 Desaturase and
Arabidopsis Fad3
[0452] Alternatively, the vector described in Example 15 could be
co-transformed into soybeans with either pKR275 (SEQ ID NO:51,
which is described in PCT Publication No. WO 04/071467 and has ATCC
Accession Number PTA4989) or pKR329 (SEQ ID NO:52, which is
described in PCT Publication No. WO 04/07146). Plasmids pKR275 and
pKR329 have ALS or hygromycin selection, respectively, and contain
the KTi/Fad3/KTi3' gene cassette (which is described in PCT
Publication No. WO 04/071467) in addition to the Ann/Sdd17/BD30
cassette. In this way, the Arabidopsis Fad3 gene could be
co-expressed with the Saprolegnia diclina delta-17 desaturase
behind strong, seed-specific promoters.
Example 18
Co-expressing the Mortierella alpina Delta-5 Desaturase, the
Euglena gracilis Delta-9 Elongase and the Euglena gracilis Delta-8
Desaturase with the Saprolegnia diclina Delta-17 Desaturase and
Fusarium moniliforme Delta-15 Desaturase
[0453] The vector described in Example 15 could be co-transformed
into soybeans with pKR585 (SEQ ID NO:53, which is described in PCT
Publication No. WO 05/0479479 and has ATCC Accession No. PTA-6019),
having hygromycin selection and containing the Fusarium moniliforme
delta-15 desaturase under control of the KTi promoter.
[0454] The vector described in Example 15 could also be
co-transformed into soybeans with pKR669, having ALS selection and
containing the Fusarium moniliforme delta-15 desaturase under
control of the KTi promoter in addition to the Ann/Sdd17/BD30
cassette. Plasmid pKR669 is produced in the following way. The KTi
promoter:Fm delta-15 desaturase ORF:KTi terminator cassette is
released from plasmid pKR578 (SEQ ID NO:54, which is described in
PCT Publication No. WO 05/0479479 and has ATCC Accession No.
PTA-6280) by digestion with BsiWI and is cloned into the BsiWI site
of plasmid pKR226 (SEQ ID NO:50, which is described in PCT
Publication No. WO 04/071467), containing the ALS gene for
selection, the T7prom/hpt/T7term cassette and the bacterial ori
region, to produce pKR667 (SEQ ID NO:55). Plasmid pKR271 (SEQ ID
NO: 49, which is described in PCT Publication No. WO 04/071467) is
digested with PsfI and the fragment containing the Saprolegnia
diclina delta-17 desaturase is cloned into the SbfI site of pKR667
to produce pKR669. In this way, the Fusarium moniliforme delta-15
desaturase could be co-expressed with the Saprolegnia diclina
delta-17 desaturase behind strong, seed-specific promoters. A
schematic depiction of pKR669 is shown in FIG. 11.
[0455] The vector described in Example 15 could also be
co-transformed into soybeans with pKR873 (SEQ ID NO:56), having ALS
selection and containing the Fusarium moniliforme delta-15
desaturase under control of the soy albumin promoter (which is
described in PCT Publication No. WO 04/071467) in addition to the
Ann/Sdd17/BD30 cassette. Plasmid pKR873 is produced in the
following way. The SA/NotI/SA3' cassette is amplified from plasmid
pKR132 (SEQ ID NO:57, which is described in PCT Publication No. WO
04/071467) using PCR. Primer oSAIb-9 (SEQ ID NO:23) is designed to
introduce XbaI and BsiWI sites at the 5' end of the promoter and
primer oSAIb-2 (SEQ ID NO:24) is designed to introduce BsiWI and
XbaI sites at the 3' end of the terminator. The resulting PCR
fragment is subsequently cloned into pCR-Script AMP SK(+)
(Stratagene Company, San Diego, Calif.) to produce pKR160 (SEQ ID
NO:58). Plasmid pKR160 is then digested with BsiWI and the
SA/NotI/SA3' cassette ligated into the BsiWI site of pKR124 (SEQ ID
NO:59, which is described in PCT Publication No. WO 05/0479479) to
produce pKR163 (SEQ ID NO:60). The NotI fragment from pY34 (SEQ ID
NO:61, which is described in PCT Publication No. WO 05/0479479),
containing the Fusarium moniliforme delta-15 desaturase, is cloned
into the NotI site of pKR163 (SEQ ID NO:60) to produce pKR863 (SEQ
ID NO:62). The SA/Fusd15/SA3' cassette is released from plasmid
pKR863 by digestion with BsiWI and is cloned into the BsiWI site of
plasmid pKR226 (SEQ ID NO:50, which is described in PCT Publication
No. WO 04/071467), containing the ALS gene for selection, the
T7prom/hpt/T7term cassette and the bacterial ori region, to produce
pKR869 (SEQ ID NO:63). Plasmid pKR271 (SEQ ID NO:49, which is
described in PCT Publication No. WO 04/071467) is digested with
PstI and the fragment containing the Saprolegnia diclina delta-17
desaturase is cloned into the SbfI site of pKR869 (SEQ ID NO:63) to
produce pKR873 (SEQ ID NO:56). In this way, the Fusarium
moniliforme delta-15 desaturase could be co-expressed with the
Saprolegnia diclina delta-17 desaturase behind strong,
seed-specific promoters. A schematic depiction of pKR873 is shown
in FIG. 12. Subsequent to the writing of this Example in the
Provisional Application, the work describing the preparation of
pKR873 was done and is described in Example 29 below.
Example 19
Co-expressing the Mortierella alpina Delta-5 Desaturase, the
Euglena gracilis Delta-9 Elongase and the Euglena gracilis Delta-8
Desaturase with the Saprolegnia diclina Delta-17 Desaturase and
Mortierella alpina Elongase
[0456] The vector described in Example 15 could also be
co-transformed into soybeans with a vector having ALS selection and
containing the Mortierella alpina elongase (which is described in
PCT Publication Nos. WO 04/071467 and WO 00/12720) under control of
the soy albumin promoter (which is described in PCT Publication No.
WO 04/071467) in addition to the Ann/Sdd17/BD30 cassette. This
plasmid could be produced in a similar way as to those described
supra. For instance, the Notl fragment from pKR270 (SEQ ID NO:64,
which is described in PCT Publication No. WO 04/071467), containing
the Mortierella alpina elongase, could be cloned into the Notl site
of pKR163 (SEQ ID NO:60) to produce a vector having the
SA/Maelo/SA3' cassette. The SA/Maelo/SA3' cassette could be
released from that plasmid by digestion with BsiWI and could be
cloned into the BsiWI site of plasmid pKR226 (SEQ ID NO:50, which
is described in PCT Publication No. WO 04/071467), containing the
ALS gene for selection, the T7prom/hpt/T7term cassette and the
bacterial ori region, to produce a new plasmid. Plasmid pKR271 (SEQ
ID NO:49, which is described in PCT Publication No. WO 04/071467)
could then be digested with PstI and the fragment containing the
Saprolegnia diclina delta-17 desaturase could be cloned into the
SbfI site of the new plasmid containing the SA/MaeIo/SA3' cassette.
In this way, the Mortierella alpina elongase could be co-expressed
with the Saprolegnia diclina delta-17 desaturase behind strong,
seed-specific promoters.
Example 20
Functional Analysis of the Euglena qracilis Delta-9 Elongase in
Saccharomyces cerevisiae
[0457] Plasmids pY119 and pY-75 (Example 9, cloning vector used to
insert the Euglena gracilis delta-9 elongase yielding pY 119) were
transformed into Saccharomyces cerevisiae INVSC1 (Invitrogen
Corporation) using standard lithium acetate transformation
procedures. Transformants were selected on DOBA media supplemented
with CSM-leu (Qbiogene, Carlsbad, Calif.). Transformants from each
plate were inoculated into 2 mL of DOB medium supplemented with
CSM-leu (Qbiogene) and 0.2% tergitol. Cells were grown for 1 day at
30 .degree. C. after which, 0.1 mL was transferred to 3 mL of the
same medium supplemented with either linoleic acid [LA-18:2(9,12)],
.alpha.-linolenic acid [ALA-1 8:3(9,12,15)], .gamma.-linolenic acid
[GLA-18:3(6,9,12)], stearidonic acid [STA-18:4(6,9,12,15,)],
arachiconic acid [AA-20:4(5,8,11,14)] or eicosapentaenoic acid
[EPA-20:5(5,8, 11,14,17)] to 0.175 mM. These were incubated for 16
h at 30 .degree. C., 250 rpm and then pellets were obtained by
centrifugation. Cells were washed once with water, pelleted by
centrifugation and air dried. Pellets were transesterified
(Roughan, G., and Nishida I. Arch Biochem Biophys. 276(1):3846
(1990)) with 500 .mu.L of 1% sodium methoxide for 30 min. at 50
.degree. C. after which 500 .mu.L of 1 M sodium chloride and 100
.mu.L of heptane were added. After thorough mixing and
centrifugation, fatty acid methyl esters (FAMEs) were analyzed by
GC as described supra. Results for feeding cells containing pY75
(vector control) or pY119 (3 independent transformants; pY119-5,
pY119-6, pY119-8) are shown in FIG. 14. The elongation efficiency
(% Elo) was calculated for each fatty acid fed as [%
FA.sub.product/(% FA.sub.product+%
FA.sub.substrate).times.100].
[0458] The data in FIG. 14 demonstrated that the cloned Euglena
gracilis delta-9 elongase efficiently elongated linoleic acid
[LA-18:2(9, 12)] and .alpha.-linolenic acid [ALA-18:3(9, 12, 15)]
to eicosadienoic acid [EDA-20:2(11, 14)] and eicosatrienoic acid
[ETrA-20:3(11, 14, 17)], respectively.
[0459] Additionally, FAMEs from cells where no fatty acid had been
fed were analyzed by GC using slightly different temperature
profiles in order to achieve separation of oleic acid [OA-18:1(9)]
and vaccenic acid [VA-18:1(11)], the elongation product of
palmitoleic acid-[PA-16:1(9)] elongation. Fatty acid methyl esters
(3 .mu.L injected from hexane layer) were separated and quantified
using a Hewlett-Packard 6890 Gas Chromatograph fitted with an
Omegawax 320 fused silica capillary column (Supelco Inc., Cat. No.
24152). The oven temperature was programmed to hold at 200 .degree.
C. for 2.7 min, increase to 240 .degree. C. at 20 .degree. C./min
and then hold for an additional 2.3 min. Results are shown in Table
5. TABLE-US-00007 TABLE 5 Lipid Profiles Having No Exogenous Fatty
Acid Added Sample 16:1 18:1 18:1 % Elo % Elo Name 16:0 (9) 18:0 (9)
(11) 16:0 16:1 pY75 13.1 54.7 3.5 27.6 1.2 20.9 2.1 pY119-5 12.9
55.6 3.6 26.0 1.8 21.6 3.2 pY119-6 13.4 54.0 3.6 27.3 1.6 21.2 3.0
pY119-8 12.7 53.3 3.5 29.0 1.5 21.7 2.8
Example 21
Functional Analysis of the Euglena gracilis Delta-9 Elongase in
Yarrowia lipolytica
[0460] A uracil ura3 auxotrophic strain of Yarrowia lipolytica
(strain Y2224) was used for functional assays. Yarrowia lipolytica
(ATCC Accession No. 20362) cells from a YPD plate were streaked
onto a minimal medium plate (75 mg/L each of uracil and uridine,
6.7 g/L YNB with ammonia sulfate, without amino acid, and 20 g/L
glucose) containing 250 mg/L 5-FOA (Zymo Research). Plates were
incubated at 28 .degree. C. and four of the resulting colonies were
patched separately onto minimal medium plates containing 200 mg/mL
5-FOA and minimal medium plates lacking uracil and uridine to
confirm uracil ura3 auxotrophy.
[0461] Yarrowia lipolytica strain Y2224 was grown at 28.degree. C.
on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2%
agar). For selection of transformants, minimal medium (0.17% yeast
nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium
sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1) was used.
Supplements of adenine, leucine, lysine and/or uracil are added to
a final concentration of 0.01%.
Transformation of Yarrowia lipolytica
[0462] pBY1-FAE, containing the Isochrysis galbana delta-9 elongase
(SEQ ID NO:27), and pBY2, containing the Euglena gracilis delta-9
elongase were transformed into Yarrowia lipolytica strain Y2224 as
described in the General Methods.
[0463] Briefly, Yarrowia lipolytica Strain #2224 was streaked onto
a YPD plate and grown at 30.degree. C. for approximately 18 h.
Several large loopfuls of cells were scraped from the plate and
resuspended in 1 mL of transformation buffer containing: [0464]
2.25 mL of 50% PEG, average MW 3350; [0465] 0.125 mL of 2 M Li
acetate, pH 6.0; [0466] 0.125 mL of 2M DTT; and [0467] 50 pg
sheared salmon sperm DNA. About 500 .mu.g of pBY1-FAE or pBY2
plasmid DNA was incubated in 100 .mu.L of resuspended cells, and
maintained at 39.degree. C. for 1 h with vortex mixing at 15 min
intervals. The cells were plated onto minimal media plates lacking
uracil and maintained at 30.degree. C. for 2 to 3 days
[0468] Single colonies of transformant Yarrowia lipolytica
containing pBY1-FAE or pBY2 were grown in 3 mL minimal media
lacking uracil at 30.degree. C. to an OD.sub.600.about.1.0. Y2224
was also grown in a similar way on minimal media supplemented with
uracil.
[0469] Cells were subsequently washed with water, collected by
centrifugation and lipids transesterified as described supra. FAMEs
from cells containing pBY1-FAE or pBY2 were analyzed by GC as were
those for cells containing pYl 19 in Example 13. Results for the
average of three replications of each are shown in Table 6.
TABLE-US-00008 TABLE 6 Comparison of Lipid Profiles of Yarrowis
Expressing Delta-9 Elongases % Sample 16:1 17.1 18:1 Elo Name 16:0
(9) (9) 18:0 (9) LA EDA LA Y2224-1 13.4 12.6 0.8 2.8 43.1 27.2 0.1
0.2 Y2224-2 12.2 12.3 0.8 2.3 46.1 26.2 0.1 0.2 Y2224-3 11.7 10.8
1.1 2.8 48.4 25.0 0.1 0.2 pBY1-FAE-1 11.9 11.9 0.8 3.1 50.6 20.2
1.6 7.5 pBY1-FAE-2 12.9 11.4 0.9 3.6 46.7 23.0 1.4 5.9 pBY1-FAE-3
12.1 12.5 0.8 3.2 50.0 19.8 1.6 7.4 pBY2-1 12.3 11.7 0.8 3.4 48.4
21.1 2.2 9.5 pBY2-2 12.1 12.5 0.8 3.2 50.1 19.1 2.3 10.6 pBY2-3
12.1 12.2 0.8 3.3 50.0 19.4 2.1 9.9
Example 22
Cloning other Delta-8 Desaturases or Elongases into Soybean
Expression Vectors
[0470] In addition to the delta-8 desaturase or delta-9 elongase
from Euglena gracilis, other delta-8 desaturases or delta-9
elongases can be cloned into the soybean expression vectors
described in the preceding Examples. For instance, a suitable
delta-8 desaturase or delta-9 elongase from an organism other than
Euglena gracilis can be cloned using methods similar to, but not
limited to, the methods described herein. PCR primers designed to
introduce NotI sites at the 5' and 3' ends of the delta-8
desaturase can be used to amplify the gene. The resulting PCR
product can then be digested with NotI and can be cloned into a
suitable soybean expression vector containing a NotI site flanked
by a strong seed-specific promoter and a transcription terminator.
Further sub-cloning into other vectors such as those described
herein, or in WO 04/071467 or WO 05/047479, but not limited to
these, should yield vectors suitable for expression and
co-expression of the delta-8 desaturase and or delta-9 elongase in
soybean.
Example 23
Co-expressing Delta-5 Elongases and Delta4 Desaturases
[0471] Delta4 desaturases or delta-5 elongases can also be
co-expressed in soybean expression vectors similar to those
described herein. For instance, a delta-4 desaturase from
Schizochytrium aggregatum (WO 02/090493) or a delta-5 elongase (EPA
elongase or C.sub.20/22 elongase) from Paviova (WO 04/071467), can
be cloned into suitable soybean expression vectors such as those
described in WO 04/071467. PCR primers designed to introduce NotI
sites at the 5' and 3' ends of the delta-4 desaturase or delta-5
elongase can be used to amplify the gene. The resulting PCR product
can then be digested with NotI and can be cloned into a suitable
soybean expression vector containing a NotI site flanked by a
strong seed-specific promoter and a transcription terminator.
Further sub-cloning into other vectors such as those described
herein, or in WO 04/071467 or WO 05/047479, but not limited to
these, should yield vectors suitable for expression and
co-expression of the delta-4 desaturase and or delta-5 elongase in
soybean.
Example 24
Transformation of Somatic Soybean Embryo Cultures
Please also see Example 30.
Culture Conditions:
[0472] Soybean embryogenic suspension cultures (cv. Jack) can be
maintained in 35 mL liquid medium SB196 (see recipes below) on
rotary shaker, 150 rpm, 26.degree. C. with cool white fluorescent
lights on 16:8 h day/night photoperiod at light intensity of 60-85
pE/m2/s. Cultures are subcultured every seven days to two weeks by
inoculating approximately 35 mg of tissue into 35 mL of fresh
liquid SB196 (the preferred subculture interval is every seven
days).
[0473] Soybean embryogenic suspension cultures can be transformed
with the plasmids and DNA fragments described earlier by the method
of particle gun bombardment (Klein et al., Nature (London)
327:70-73 (1987); U.S. Pat. No. 4,945,050). A DuPont Biolistic.TM.
PDS1000/HE instrument (helium retrofit) would be used for all
transformations.
Soybean Embryogenic Suspension Culture Initiation:
[0474] Soybean cultures are initiated twice each month with
five-seven days between each initiation. Pods with immature seeds
from available soybean plants 45-55 days after planting are picked,
removed from their shells and placed into a sterilized magenta box.
The soybean seeds are sterilized by shaking them for 15 min in a 5%
Clorox solution with 1 drop of ivory soap (95 mL of autoclaved
distilled water plus 5 mL Clorox and 1 drop of soap--mix well.
Seeds are rinsed using 2 1-liter bottles of sterile distilled water
and those less than 4 mm were placed on individual microscope
slides. The small end of the seed is cut and the cotyledons pressed
out of the seed coat. Cotyledons are transferred to plates
containing SB1 medium (25-30 cotyledons per plate). Plates are
wrapped with fiber tape and stored for eight weeks. After this time
secondary embryos are cut and placed into SB196 liquid media for
seven days.
Preparation of DNA for Bombardment:
[0475] Either an intact plasmid or a DNA plasmid fragment
containing the genes of interest and the selectable marker gene can
be used for bombardment. Fragments from plasmids such pKR274 (ATCC
Accession No. PTA-4988) and pKR685 (ATCC Accession No. PTA-6047) or
pKR681 (ATCC Accession No. PTA-6046) and/or other expression
plasmids can be obtained by gel isolation of digested plasmids. In
each case, 100 .mu.g of plasmid DNA can be used in 0.5 mL of the
specific enzyme mix described below. Plasmids could be digested
with AcsI (100 units) in NEBuffer 4 (20 mM Tris-acetate, 10 mM
magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, pH
7.9), 100 pg/mL BSA, and 5 mM beta-mercaptoethanol at 37 .degree.
C. for 1.5 h. The resulting DNA fragments could be separated by gel
electrophoresis on 1% SeaPlaque.RTM. GTG agarose (BioWhitaker
Molecular Applications) and the DNA fragments containing EPA
biosynthetic genes could be cut from the agarose gel. DNA can be
purified from the agarose using the GELase.RTM. digesting enzyme
following the manufacturer's protocol. Alternatively, whole
plasmids or a combination of whole plasmid with fragment could be
used.
[0476] A 50 .mu.L aliquot of sterile distilled water containing 3
mg of gold particles (3 mg gold) can be added to 5 .mu.L of a 1
.mu.g/.mu.L DNA solution (either intact plasmid or DNA fragment
prepared as described above), 50 .mu.L 2.5M CaCl.sub.2 and 20 .mu.L
of 0.1 M spermidine. The mixture is shaken 3 min on level 3 of a
vortex shaker and spun for 10 sec in a bench microfuge. After a
wash with 400 .mu.L 100% ethanol the pellet is suspended by
sonication in 40 .mu.L of 100% ethanol. Five .mu.L of DNA
suspension is dispensed to each flying disk of the Biolistic
PDS1000/HE instrument disk. Each 5 .mu.L aliquot contained
approximately 0.375 mg gold per bombardment (e.g., per disk).
Tissue Preparation and Bombardment with DNA:
[0477] Approximately 150-200 mg of seven day old embryonic
suspension cultures are placed in an empty, sterile 60.times.15 mm
petri dish and the dish is covered with plastic mesh. Tissue is
bombarded one or two shots per plate with membrane rupture pressure
set at 1100 PSI and the chamber is evacuated to a vacuum of 27-28
inches of mercury. Tissue is placed approximately 3.5 inches from
the retaining/stopping screen.
Selection of Transformed Embryos:
[0478] Transformed embryos are selected either using hygromycin
(when the hygromycin B phosphotransferase (HPT) gene was used as
the selectable marker) or chlorsulfuron (when the acetolactate
synthase (ALS) gene was used as the selectable marker).
Hygromycin (HPT) Selection:
[0479] Following bombardment, the tissue is placed into fresh SB196
media and cultured as described above. Six days post-bombardment,
the SB196 is exchanged with fresh SB196 containing a selection
agent of 30 mg/L hygromycin. The selection media is refreshed
weekly. Four to six weeks post selection, green, transformed tissue
may be observed growing from untransformed, necrotic embryogenic
clusters. Isolated, green tissue is removed and inoculated into
multiwell plates to generate new, clonally propagated, transformed
embryogenic suspension cultures.
Chlorsulfuron (ALS) Selection:
[0480] Following bombardment, the tissue is divided between two
flasks with fresh SB196 media and cultured as described above. Six
to seven days post-bombardment, the SB196 is exchanged with fresh
SB196 containing selection agent of 100 ng/mL chlorsulfuron. The
selection media is refreshed weekly. Four to six weeks post
selection, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated, green
tissue is removed and inoculated into multiwell plates containing
SB196 to generate new, clonally propagated, transformed embryogenic
suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants:
[0481] In order to obtain whole plants from embryogenic suspension
cultures, the tissue needs to be regenerated.
Embryo Maturation:
[0482] Embryos can be cultured for four-six weeks at 26.degree. C.
in SB196 under cool white fluorescent (Phillips cool white
Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40
watt) on a 16:8 h photoperiod with light intensity of 90-120
.mu.E/m2/s. After this time embryo clusters are removed to a solid
agar media, SB166, for one-two weeks. Clusters are then subcultured
to medium SB103 for three weeks. During this period, individual
embryos can be removed from the clusters and screened for
alterations in their fatty acid compositions as described supra. It
should be noted that any detectable phenotype, resulting from the
expression of the genes of interest, could be screened at this
stage. This detectable phenotype would include, but not be limited
to, alterations in fatty acid profile, protein profile and content,
carbohydrate content, growth rate, viability, or the ability to
develop normally into a soybean plant.
Embryo Desiccation and Germination:
[0483] Matured individual embryos can be desiccated by placing them
into an empty, small petri dish (35.times.10 mm) for approximately
four-seven days. The plates are sealed with fiber tape (creating a
small humidity chamber). Desiccated embryos can be planted into
SB714 medium where they are left to germinate under the same
culture conditions described above. Germinated plantlets are
removed from germination medium and rinsed thoroughly with water
and then planted in Redi-Earth in 24-cell pack tray, covered with
clear plastic dome. After two weeks the dome is removed and plants
hardened off for a further week. If plantlets look hardy they are
transplanted to a 10 inch pot of Redi-Earth with up to 3 plantlets
per pot. After ten to sixteen weeks, mature seeds can be harvested,
chipped and analyzed for fatty acids as described above.
[0484] Media Recipes: TABLE-US-00009 SB 196 - FN Lite Liquid
Proliferation Medium (per liter) MS FeEDTA - 100x Stock 1 10 mL MS
Sulfate - 100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL
FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL
2,4-D (10 mg/L final concentration) 1.0 mL KNO.sub.3 2.83 gm
(NH.sub.4).sub.2SO.sub.4 0.463 gm asparagine 1.0 gm sucrose (1%) 10
gm pH 5.8 FN Lite Stock Solutions Stock Number 1000 mL 500 mL 1 -
MS Fe EDTA 100x Stock Na.sub.2 EDTA* 3.724 g 1.862 g
FeSO.sub.4--7H.sub.2O 2.784 g 1.392 g *Add first, dissolve in dark
bottle while stirring 2 - MS Sulfate 100x stock
MgSO.sub.4--7H.sub.2O 37.0 g 18.5 g MnSO.sub.4 --H.sub.2O 1.69 g
0.845 g ZnSO.sub.4--7H.sub.2O 0.86 g 0.43 g CuSO.sub.4--5H.sub.2O
0.0025 g 0.00125 g 3 - FN Lite Halides 100x Stock
CaCl.sub.2--2H.sub.2O 30.0 g 15.0 g KI 0.083 g 0.0715 g
CoCl.sub.2--6H.sub.2O 0.0025 g 0.00125 g 4 - FN Lite P, B, Mo 100x
Stock KH.sub.2PO.sub.4 18.5 g 9.25 g H.sub.3BO.sub.3 0.62 g 0.31 g
Na.sub.2MoO.sub.4--2H.sub.2O 0.025 g 0.0125 g SB1 Solid Medium (per
liter) 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5
vitamins 1000X stock 31.5 g sucrose 2 mL 2,4-D (20 mg/L final
concentration) pH 5.7 8 g TC agar SB 166 Solid Medium (per liter) 1
package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5 vitamins
1000X stock 60 g maltose 750 mg MgCl.sub.2 hexahydrate 5 g
activated charcoal pH 5.7 2 g gelrite SB 103 Solid Medium (per
liter) 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1 mL B5
vitamins 1000X stock 60 g maltose 750 mg MgCl2 hexahydrate pH 5.7 2
g gelrite SB 71-4 Solid Medium (per liter) 1 bottle Gamborg's B5
salts w/ sucrose (Gibco/BRL - Cat. No. 21153-036) pH 5.7 5 g TC
agar 2,4-D Stock Obtain premade from Phytotech Cat. No. D 295 -
concentration 1 mg/mL B5 Vitamins Stock (per 100 mL) Store aliquots
at -20.degree. C. 10 g myo-inositol 100 mg nicotinic acid 100 mg
pyridoxine HCl 1 g thiamine If the solution does not dissolve
quickly enough, apply a low level of heat via the hot stir plate.
Chlorsulfuron Stock 1 mg/mL 0.01 N Ammonium Hydroxide
[0485] To induce somatic embryos, cotyledons, 3-5 mm in length
dissected from surface sterilized, immature seeds of the soybean
cultivar A2872, can be cultured in the light or dark at 26.degree.
C. on an appropriate agar medium for six-ten weeks. Somatic
embryos, which produce secondary embryos, are then excised and
placed into a suitable liquid medium. After repeated selection for
clusters of somatic embryos which multiplied as early, globular
staged embryos, the suspensions are maintained as described
below.
[0486] Soybean embryogenic suspension cultures can be maintained in
35 mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C.
with florescent lights on a 16:8 h day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0487] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et
al., Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont
Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be used
for these transformations.
[0488] A selectable marker gene which can be used to facilitate
soybean transformation is a recombinant DNA construct composed of
the 35S promoter from Cauliflower Mosaic Virus (Odell et al.,
Nature 313:810-812 (1985)), the hygromycin B phosphotransferase
gene from plasmid pJR225 (from E. coli; Gritz et al., Gene
25:179-188 (1983)) and the 3' region of the nopaline synthase gene
from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The
seed expression cassette comprising the phaseolin 5' region, the
fragment encoding the instant polypeptide and the phaseolin 3'
region can be isolated as a restriction fragment. This fragment can
then be inserted into a unique restriction site of the vector
carrying the marker gene.
[0489] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.L
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 sec and the supernatant removed. The DNA-coated particles
are then washed once in 400 .mu.L 70% ethanol and resuspended in 40
.mu.L of anhydrous ethanol. The DNA/particle suspension can be
sonicated three times for one sec each. Five .mu.L of the
DNA-coated gold particles are then loaded on each macro carrier
disk.
[0490] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately five-ten plates of tissue
are normally bombarded. Membrane rupture pressure is set at 1100
psi and the chamber is evacuated to a vacuum of 28 inches mercury.
The tissue is placed approximately 3.5 inches away from the
retaining screen and bombarded three times. Following bombardment,
the tissue can be divided in half and placed back into liquid and
cultured as described above.
[0491] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post
bombardment with fresh media containing 50 mg/mL hygromycin. This
selective media can be refreshed weekly. Seven to eight weeks post
bombardment, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated green tissue
is removed and inoculated into individual flasks to generate new,
clonally propagated, transformed embryogenic suspension cultures.
Each new line may be treated as an independent transformation
event. These suspensions can then be subcultured and maintained as
clusters of immature embryos or regenerated into whole plants by
maturation and germination of individual somatic embryos.
Example 25
Synthesis and Functional Expression of a Codon-Optimized Delta-9
Elongase Gene (derived from Euglena gracilis) in Yarrowia
lipolytica
[0492] The codon usage of the delta-9 elongase gene of Euglena
gracilis (SEQ ID NOs:3 and 4) is optimized for expression in
Yarrowia lipolytica, in a manner similar to that described in
Example 4 (supra) and PCT Publication No. WO 2004/101753.
Specifically, a codon-optimized delta-9 elongase gene (designated
"EgD9S"), SEQ ID NO:90) is designed, based on the coding sequence
of the delta-9 elongase of the instant invention (clone
eeg1c.pk001.n5.f), according to the Yarrowia codon usage pattern
(PCT Publication No. WO 2004/101753), the consensus sequence around
the `ATG` translation initiation codon, and the general rules of
RNA stability (Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23
(2001)). In addition to the modification of the translation
initiation site, 117 bp of the 777 bp coding region are modified
(15.1%) and 106 codons are optimized (40.9%). None of the
modifications in the codon-optimized gene change the amino acid
sequence of the encoded protein (SEQ ID NO:5). The designed EgD9
gene can be synthesized by GenScript Corporation (Piscataway, N.J.)
and can be cloned into pUC57 (GenBank Accession No. Y14837) to
generate pEgD9S.
[0493] Subsequent to the writing of this Example in the Provisional
Application, the work describing the preparation codon-optimized
Euglena gracilis delta-9 elongase gene (designated "EgD9S") was
done and is described in Example 36 below.
Example 26
Cloning the Euglena gracilis Delta-8 Desaturase into a Soybean
Expression Vector and Co-expression with the Euglena gracilis
Delta-9 Elonqase
[0494] Plasmid pKR680 (SEQ ID NO:43) was digested with BSiWI and
the fragment containing Euglena gracilis delta-8 desaturase (Eg5)
(SEQ ID NO:16) was cloned into the BSiWI site of pKR912 (SEQ ID
NO:37) to produce pKR920 (SEQ ID NO:91). A schematic depiction of
pK920 is shown in FIG. 17. In this way, the Euglena gracilis
delta-8 desaturase (called eug d8-sq5 in FIG. 17) was co-expressed
with the Euglena gracilis delta-9 elongase (called eug el1 in FIG.
17) behind strong, seed-specific promoters.
Example 27
Cloning the Euglena qracilis Delta-9 Elongase into an Arabidopsis
thaliana Binary Expression Vector (pKR926)
[0495] Various restriction sites were added, through a number of
cloning steps, to the ends of the Bcon/NotI/Phas3' cassette from
KS123, which was previously described in PCT Publication No. WO
02/008269 (the contents of which are hereby incorporated by
reference). Briefly, a DNA fragment (cal a24-4; SEQ ID NO:92) was
amplified from plasmid CalFad2-2 (described in PCT Publication No.
WO 01/12800) using primers oCal-15 (SEQ ID NO:93) and oCal-6 (SEQ
ID NO:94). DNA fragment cal a24-4 was digested with BglII and BamHI
and cloned into the BamHI site of pKS123 to give pKR53B (SEQ ID
NO:95). The XbaI/SbfI fragment of pKR53B, containing the
Bcon/NotI/Phas3' cassette was cloned into the XbaI/SbfI fragment of
pKR72, containing the bacterial hygromycin phosphotransferase gene,
to give pKR85 (SEQ ID NO:96).
[0496] The Bcon/NotI/Phas3' cassette was amplified from plasmid
pKR85 using primers oKR85-1 (SEQ ID NO:97) and oKR85-2 (SEQ ID
NO:98) and the resulting DNA fragment was cloned into
PCR-Script.RTM. (Stratgene) following the manufacture's protocol,
to give pPCR85 (SEQ ID NO:99).
[0497] The EcoRI/BglII fragment of pPCR85, containing the
Bcon/NotI/Phas3' cassette was cloned into the EcoRI/BamHI fragment
of plasmid pZS199 (PCT Publication No. WO 93/11245 (also U.S. Pat.
No. 5,952,544) which was published on Jun. 10, 1993, the
disclosures of which are hereby incorporated by reference),
containing the Arabidopsis binary vector backbone to produce pKR91
(SEQ ID NO:100).
[0498] The Bcon/NotI/Phas3' cassette was removed from pKR91 by
digestion with AcsI and the re-ligated binary vector containing a
unique AcsI cloning site was produced called pKR92 (SEQ ID
NO:101).
[0499] The AscI fragment of pKR911 (SEQ ID NO:40; Example 11),
containing the Euglena gracilis delta-9 elongase was cloned into
the AscI site of pKR92 to give pKR926 (SEQ ID NO:102). A schematic
depiction of pK926 is shown in FIG. 18. In this way, the Euglena
gracilis delta-9 elongase (called eug el1 in FIG. 18) was expressed
in Arabidopsis behind the soybean beta-conglycinin promoter. The
soybean beta-conglycinin promoter functions as a strong,
seed-specific promoter in Arabidopsis (see U.S. application Ser.
No. 11/258,704).
Example 28
Cloning the Mortierella alpina Delta-5 Desaturase into a Soybean
Expression Vector and Co-expression with the Euglena qracilis
Delta-9 Elongase and the Euglena gracilis Delta-8 Desaturase
(pKR916)
[0500] A soybean expression vector containing the Euglena gracilis
delta-8 desaturase (SEQ ID NO:16), the Euglena gracilis delta-9
elongase (SEQ ID NO:4) and the Mortierella alpina delta-5
desaturase (SEQ ID NO:88), which is described in U.S. Pat. No.
6,075,183 and PCT Publication Nos. WO 04/071467 and WO 05/0479479
(the contents of which are hereby incorporated by reference), all
under the control of strong seed-specific promoters, was
constructed in the following way.
[0501] The Gy1/Mad5/legA2 cassette was released from pKR767 (SEQ ID
NO:103) by digestion with SbfI and the resulting fragment was
cloned into the SbfI site of pKR913 (SEQ ID NO:44; Example 13) to
produce pKR916 (SEQ ID NO:104). A schematic depiction of pKR916 is
shown in FIG. 19. In this way, the Euglena gracilis delta-9
elongase (called eug el1 in FIG. 19) was co-expressed with the
Euglena gracilis delta-8 desaturase (called eug d8-sq5 in FIG. 19)
and the Mortierella alpina delta-5 desaturase (called DELTA 5
DESATURASE M ALPINA in FIG. 19) behind strong, seed specific
promoters.
Example 29
Co-expressing the Saprolegnia diclina Delta-17 Desaturase with the
Fusarium moniliforme Delta-15 Desaturase (pKR873)
[0502] Plasmid pKR873 (SEQ ID NO:56; FIG. 12) was produced in the
following way. The SA/NotI/SA3' cassette was amplified from plasmid
pKR132 (SEQ ID NO:57, which is described in PCT Publication No. WO
04/071467) using PCR. Primer oSAIb-9 (SEQ ID NO:23) was designed to
introduce XbaI and BsiWI sites at the 5' end of the promoter and
primer oSAIb-2 (SEQ ID NO:24) was designed to introduce BsiWI and
XbaI sites at the 3' end of the terminator. The resulting PCR
fragment was subsequently cloned into pCR-Script AMP SK(+)
(Stratagene Company, San Diego, Calif.) to produce pKR160 (SEQ ID
NO:58).
[0503] Plasmid pKR160 was then digested with BsiWl and the
SA/NotI/SA3' cassette ligated into the BsiWI site of pKR124 (SEQ ID
NO:59, which is described in PCT Publication No. WO 05/0479479) to
produce pKR163 (SEQ ID NO:60). The NotI fragment from pY34 (SEQ ID
NO:61, which is described in PCT Publication No. containing the
Fusarium moniliforme delta-15 desaturase, was cloned into the NotI
site of pKR163 (SEQ ID NO:60) to produce pKR863 (SEQ ID NO:62). The
SA/Fusd15/SA3' cassette was released from plasmid pKR863 by
digestion with BsiWI and was cloned into the BsiWI site of plasmid
pKR226 (SEQ ID NO:50, which is described in PCT Publication No. WO
04/071467), containing the ALS gene for selection, the
T7prom/hpt/T7term cassette and the bacterial ori region, to produce
pKR869 (SEQ ID NO:63). Plasmid pKR271 (SEQ ID NO:49, which is
described in PCT Publication No. WO 04/071467) was digested with
PstI and the fragment containing the Saprolegnia diclina delta-17
desaturase was cloned into the SbfI site of pKR869 (SEQ ID NO:63)
to produce pKR873 (SEQ ID NO:56). In this way, the Fusarium
moniliforme delta-15 desaturase was co-expressed with the
Saprolegnia diclina delta-17 desaturase behind strong,
seed-specific promoters. A schematic depiction of pKR873 is shown
in FIG. 12.
Example 30
Transformation of Somatic Soybean Embryo Cultures with Soybean
Expression Vectors
Culture Conditions:
[0504] Soybean embryogenic suspension cultures (cv. Jack) were
maintained in 35 mL liquid medium SB196 (infra) on a rotary shaker,
150 rpm, 26.degree. C. with cool white fluorescent lights on 16:8
hr day/night photoperiod at light intensity of 60-85 .mu.E/m2/s.
Cultures were subcultured every 7 days to two weeks by inoculating
approximately 35 mg of tissue into 35 mL of fresh liquid SB196 (the
preferred subculture interval is every 7 days).
[0505] Soybean embryogenic suspension cultures were transformed
with the soybean expression plasmids by the method of particle gun
bombardment (Klein et al., Nature, 327:70 (1987)) using a DuPont
Biolistic PDS1000/HE instrument (helium retrofit) for all
transformations.
Soybean Embryogenic Suspension Culture Initiation:
[0506] Soybean cultures were initiated twice each month with 5-7
days between each initiation. Pods with immature seeds from
available soybean plants were picked 45-55 days after planting.
Seeds were removed from the pods and placed into a sterilized
magenta box. The soybean seeds were sterilized by shaking them for
15 min in a 5% Clorox solution with 1 drop of ivory soap (i.e., 95
mL of autoclaved distilled water plus 5 mL Clorox and 1 drop of
soap, mixed well). Seeds were rinsed using 2 1-liter bottles of
sterile distilled water and those less than 4 mm were placed on
individual microscope slides. The small end of the seed was cut and
the cotyledons pressed out of the seed coat. When cultures were
being prepared for production transformation, cotyledons were
transferred to plates containing SB1 medium (25-30 cotyledons per
plate). Plates were wrapped with fiber tape and were maintained at
26.degree. C. with cool white fluorescent lights on 16:8 h
day/night photoperiod at light intensity of 60-80 .mu.E/m2/s for
eight weeks, with a media change after 4 weeks. When cultures were
being prepared for model system experiments, cotyledons were
transferred to plates containing SB199 medium (25-30 cotyledons per
plate) for 2 weeks, and then transferred to SB1 for 2-4 weeks.
Light and temperature conditions were the same as described above.
After incubation on SB1 medium, secondary embryos were cut and
placed into SB196 liquid media for 7 days.
Preparation of DNA for Bombardment:
[0507] Either an intact plasmid or a DNA plasmid fragment
containing the genes of interest and the selectable marker gene
were used for bombardment. Fragments from soybean expression
plasmids, the construction of which is described herein, were
obtained by gel isolation of digested plasmids. In each case, 100
.mu.g of plasmid DNA was used in 0.5 mL of the specific enzyme mix
described below. Plasmids were digested with AcsI (100 units) in
NEBuffer 4 (20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM
potassium acetate, 1 mM dithiothreitol, pH 7.9), 100 .mu.g/mL BSA,
and 5 mM beta-mercaptoethanol at 37.degree. C. for 1.5 hr. The
resulting DNA fragments were separated by gel electrophoresis on 1%
SeaPlaque GTG agarose (BioWhitaker Molecular Applications) and the
DNA fragments containing gene cassettes were cut from the agarose
gel. DNA was purified from the agarose using the GELase digesting
enzyme following the manufacturer's protocol.
[0508] A 50 .mu.L aliquot of sterile distilled water containing 3
mg of gold particles (3 mg gold) was added to 30 .mu.L of a 10
ng/.mu.L DNA solution (either intact plasmid or DNA fragment
prepared as described herein), 25 .mu.L 5M CaCl.sub.2 and 20 .mu.L
of 0.1 M spermidine. The mixture was shaken 3 min on level 3 of a
vortex shaker and spun for 10 sec in a bench microfuge. The
supernatant was removed, followed by a wash with 400 .mu.L 100%
ethanol and another brief centrifugation. The 400 .mu.l ethanol was
removed and the pellet was resuspended in 40 .mu.L of 100% ethanol.
Five .mu.L of DNA suspension was dispensed to each flying disk of
the Biolistic PDS1000/HE instrument disk. Each 5 .mu.L aliquot
contained approximately 0.375 mg gold per bombardment (e.g., per
disk).
[0509] For model system transformations, the protocol was identical
except for a few minor changes (ie, 1 mg of gold particles was
added to 5 .mu.L of a 1 .mu.g/.mu.L DNA solution, 50 .mu.L of a
2.5M CaCl.sub.2 was used and the pellet was ultimately resuspended
in 85 .mu.L of 100% ethanol thus providing 0.058 mg of gold
particles per bombardment).
Tissue Preparation and Bombardment with DNA:
[0510] Approximately 150-200 mg of seven day old embryogenic
suspension cultures was placed in an empty, sterile 60.times.15 mm
petri dish and the dish was covered with plastic mesh. The chamber
was evacuated to a vacuum of 27-28 inches of mercury, and tissue
was bombarded one or two shots per plate with membrane rupture
pressure set at 1100 PSI. Tissue was placed approximately 3.5
inches from the retaining/stopping screen. Model system
transformation conditions were identical except 100-150 mg of
embryogenic tissue was used, rupture pressure was set at 650 PSI
and tissue was place approximately 2.5 inches from the retaining
screen.
Selection of Transformed Embryos:
[0511] Transformed embryos were selected either using hygromycin
(when the hygromycin B phosphotransferase (HPT) gene was used as
the selectable marker) or chlorsulfuron (when the acetolactate
synthase (ALS) gene was used as the selectable marker).
[0512] Following bombardment, the tissue was placed into fresh
SB196 media and cultured as described above. Six to eight days
post-bombardment, the SB196 was exchanged with fresh SB196
containing either 30 mg/L hygromycin or 100 ng/mL chlorsulfuron,
depending on the selectable marker used. The selection media was
refreshed weekly. Four to six weeks post-selection, green,
transformed tissue was observed growing from untransformed,
necrotic embryogenic clusters. Isolated, green tissue was removed
and inoculated into multiwell plates to generate new, clonally
propagated, transformed embryogenic suspension cultures.
Embryo Maturation:
[0513] Transformed embryogenic clusters from production
transformation were cultured for four-six weeks (one-three weeks
for model system) in multiwell plates as described above at
26.degree. C. in SB196 under cool white fluorescent (Phillips cool
white Econowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs
(40 watt) on a 16:8 hr photoperiod with light intensity of 90-120
.mu.E/m.sup.2s. After this time embryo clusters were removed to a
solid agar media, SB166, for one-two weeks (1 week for model
system) and then subcultured to SB103 medium for 34 weeks to mature
embryos. After maturation on plates in SB103, individual embryos
were removed from the clusters, dried and screened for alterations
in their fatty acid compositions as described supra. When desired,
plants were obtained from some events as described below.
[0514] Alternatively, in some model system experiments, embryos
were matured in soybean histodifferentiation and maturation liquid
medium (SHaM liquid media; Schmidt et al., Cell Biology and
Morphogenesis 24:393 (2005)) using a modified procedure. Briefly,
after 4 weeks of selection in SB196 as described above, embryo
clusters were removed to 35 mL of SB228 (SHaM liquid media) in a
250 mL . Erlenmeyer flask. Tissue was maintained in SHaM liquid
media on a rotary shaker at 130 rpm and 26.degree. C. with cool
white fluorescent lights on a 16:8 hr day/night photoperiod at a
light intensity of 60-85 .mu.E/m2/s for 2-3 weeks as embryos
matured. Embryos grown for 2-3 weeks in SHaM liquid media were
equivalent in size and fatty acid content to embryos cultured on
SB166/SB103 for 5-8 weeks.
[0515] After maturation in SHaM liquid media, individual embryos
were removed from the clusters, dried and screened for alterations
in their fatty acid compositions as described supra. When desired,
plants were obtained from some events as described below.
Embryo Desiccation and Germination:
[0516] Matured individual embryos were desiccated by placing them
into an empty, small petri dish (60.times.15 mm) for approximately
four-seven days. The plates were sealed with fiber tape (creating a
small humidity chamber). Desiccated embryos were planted into
SB71-4 medium where they were left to germinate under the same
culture conditions described above. Germinated plantlets were
removed from germination medium and rinsed thoroughly with water
and then planted in Redi-Earth in a 24-cell pack tray, and covered
with a clear plastic dome. After one-two weeks the dome was removed
and plants hardened off for a further week. If plantlets look hardy
they were transplanted to a 10 inch pot of Redi-Earth with up to 3
plantlets per pot. After ten to sixteen weeks, mature seeds were
harvested, chipped and analyzed for fatty acids as described
herein.
[0517] Media Recipes: TABLE-US-00010 SB 196 - FN Lite Liquid
Proliferation Medium (per liter) MS FeEDTA - 100x Stock 1 10 mL MS
Sulfate - 100x Stock 2 10 mL FN Lite Halides - 100x Stock 3 10 mL
FN Lite P, B, Mo - 100x Stock 4 10 mL B5 vitamins (1 mL/L) 1.0 mL
2,4-D (10 mg/L final concentration) 1.0 mL KNO.sub.3 2.83 gm
(NH.sub.4).sub.2SO.sub.4 0.463 gm asparagine 1.0 gm sucrose (1%) 10
gm pH 5.8 FN Lite Stock Solutions Stock Number 1000 mL 500 mL 1 MS
Fe EDTA 100x Stock Na.sub.2 EDTA* 3.724 g 1.862 g
FeSO.sub.4-7H.sub.2O 2.784 g 1.392 g *Add first, dissolve in dark
bottle while stirring 2 MS Sulfate 100x stock MgSO.sub.4--7H.sub.2O
37.0 g 18.5 g MnSO.sub.4--H.sub.2O 1.69 g 0.845 g
ZnSO.sub.4--7H.sub.2O 0.86 g 0.43 g CuSO.sub.4--5H.sub.2O 0.0025 g
0.00125 g 3 FN Lite Halides 100x Stock CaCl.sub.2--2H.sub.2O 30.0 g
15.0 g KI 0.083 g 0.0715 g CoCl.sub.2--6H.sub.2O 0.0025 g 0.00125 g
4 FN Lite P, B, Mo 100x Stock KH.sub.2PO.sub.4 18.5 g 9.25 g
H.sub.3BO.sub.3 0.62 g 0.31 g Na.sub.2MoO.sub.4--2H.sub.2O 0.025 g
0.0125 g SB1 Solid Medium (per liter) 1 package MS salts (Gibco/BRL
- Cat. No. 11117-066) 1 mL B5 vitamins 1000X stock 31.5 g glucose 2
mL 2,4-D (20 mg/L final concentration) pH 5.7 8 g TC agar SB199
Solid Medium (per liter) 1 package MS salts (Gibco/BRL - Cat. No.
11117-066) 1 mL B5 vitamins 1000X stock 30 g Sucrose 4 ml 2,4-D (40
mg/L final concentration) pH 7.0 2 gm Gelrite SB 166 Solid Medium
(per liter) 1 package MS salts (Gibco/BRL - Cat. No. 11117-066) 1
mL B5 vitamins 1000X stock 60 g maltose 750 mg MgCl.sub.2
hexahydrate 5 g activated charcoal pH 5.7 2 g gelrite SB 103 Solid
Medium (per liter) 1 package MS salts (Gibco/BRL - Cat. No.
11117-066) 1 mL B5 vitamins 1000X stock 60 g maltose 750 mg MgCl2
hexahydrate pH 5.7 2 g gelrite SB 71-4 Solid Medium (per liter) 1
bottle Gamborg's B5 salts w/ sucrose (Gibco/BRL - Cat. No.
21153-036) pH 5.7 5 g TC agar 2,4-D Stock Obtain premade from
Phytotech Cat. No. D 295 - concentration 1 mg/mL B5 Vitamins Stock
(per 100 mL) Store aliquots at -20.degree. C. 10 g myo-inositol 100
mg nicotinic acid 100 mg pyridoxine HCl 1 g thiamine If the
solution does not dissolve quickly enough, apply a low level of
heat via the hot stir plate. SB 228- Soybean Histodifferentiation
& Maturation (SHaM) (per liter) DDI H.sub.2O 600 mL FN-Lite
Macro Salts for SHaM 10X 100 mL MS Micro Salts 1000x 1 mL MS FeEDTA
100x 10 mL CaCl 100x 6.82 mL B5 Vitamins 1000x 1 mL L-Methionine
0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mL pH 5.8
Autoclave Add to cooled media (.ltoreq.30.degree. C.): *Glutamine
(final concentration 30 mM) 4% 110 mL *Note: Final volume will be
1010 mL after glutamine addition. Since glutamine degrades
relatively rapidly, it may be preferable to add immediately prior
to using media. Expiration 2 weeks after glutamine is added; base
media can be kept longer w/o glutamine. FN-lite Macro for SHAM 10X-
Stock #1 (per liter) (NH.sub.4)2SO.sub.4 (ammonium sulfate) 4.63 g
KNO.sub.3 (potassium nitrate) 28.3 g MgSO.sub.4*7H.sub.20
(magnesium sulfate heptahydrate) 3.7 g KH.sub.2PO.sub.4 (potassium
phosphate, monobasic) 1.85 g Bring to volume Autoclave MS Micro
1000X- Stock #2 (per 1 liter) H.sub.3BO.sub.3 (boric acid) 6.2 g
MnSO.sub.4*H.sub.2O (manganese sulfate monohydrate) 16.9 g
ZnSO4*7H20 (zinc sulfate heptahydrate) 8.6 g Na.sub.2MoO.sub.4*2H20
(sodium molybdate dihydrate) 0.25 g CuSO.sub.4*5H.sub.20 (copper
sulfate pentahydrate) 0.025 g CoCl.sub.2*6H.sub.20 (cobalt chloride
hexahydrate) 0.025 g KI (potassium iodide) 0.8300 g Bring to volume
Autoclave FeEDTA 100X- Stock #3 (per liter) Na.sub.2EDTA* (sodium
EDTA) 3.73 g FeSO.sub.4*7H.sub.20 (iron sulfate heptahydrate) 2.78
g *EDTA must be completely dissolved before adding iron. Bring to
Volume Solution is photosensitive. Bottle(s) should be wrapped in
foil to omit light. Autoclave Ca 100X- Stock #4 (per liter)
CaCl.sub.2*2H.sub.20 (calcium chloride dihydrate) 44 g Bring to
Volume Autoclave B5 Vitamin 1000X- Stock #5 (per liter)
Thiamine*HCl 10 g Nicotinic Acid 1 g Pyridoxine*HCl 1 g
Myo-Inositol 100 g Bring to Volume Store frozen 4% Glutamine- Stock
#6 (per liter) DDI water heated to 30.degree. C. 900 mL L-Glutamine
40 g Gradually add while stirring and applying low heat. Do not
exceed 35.degree. C. Bring to Volume Filter Sterilize Store frozen*
*Note: Warm thawed stock in 31.degree. C. bath to fully dissolve
crystals.
Example 31
Functional Analysis of the Euglena gracilis Delta-9 Elongase in
Somatic Soybean Embryos Transformed with Soybean Expression Vector
pKR912
[0518] Mature somatic soybean embryos are a good model for zygotic
embryos. While in the globular embryo state in liquid culture,
somatic soybean embryos contain very low amounts of triacylglycerol
or storage proteins typical of maturing, zygotic soybean embryos.
At this developmental stage, the ratio of total triacylglyceride to
total polar lipid (phospholipids and glycolipid) is about 1:4, as
is typical of zygotic soybean embryos at the developmental stage
from which the somatic embryo culture was initiated. At the
globular stage as well, the mRNAs for the prominent seed proteins,
.alpha.'-subunit of .beta.'-conglycinin, kunitz trypsin inhibitor
3, and seed lectin are essentially absent. Upon transfer to
hormone-free media to allow differentiation to the maturing somatic
embryo state, triacylglycerol becomes the most abundant lipid
class. As well, mRNAs for .alpha.'-subunit of .beta.'-conglycinin,
kunitz trypsin inhibitor 3 and seed lectin become very abundant
messages in the total mRNA population. On this basis, the somatic
soybean embryo system behaves very similarly to maturing zygotic
soybean embryos in vivo, and is thus a good and rapid model system
for analyzing the phenotypic effects of modifying the expression of
genes in the fatty acid biosynthesis pathway (see PCT Publication
No. WO 2002/00904, Example 3). Most importantly, the model system
is also predictive of the fatty acid composition of seeds from
plants derived from transgenic embryos.
Fatty Acid Analysis of Transgenic Somatic Soybean Embryos
Expressing pKR912
[0519] Individual single, matured, somatic soybean embryos that had
been transformed with pKR912 (SEQ ID NO:37; FIG. 7) and matured in
the model system on SB103 plates as described in Example 30, with
hygromycin as selection, were picked into glass GC vials and fatty
acid methyl esters were prepared by transesterification. For
transesterification, 50 .mu.L of trimethylsulfonium hydroxide
(TMSH) and 0.5 mL of hexane were added to the embryos in glass
vials and incubated for 30 min at room temperature while shaking.
Fatty acid methyl esters (5 .mu.L injected from hexane layer) were
separated and quantified using a Hewlett-Packard 6890 Gas
Chromatograph fitted with an Omegawax 320 fused silica capillary
column (Catalog #24152, Supelco Inc.). The oven temperature was
programmed to hold at 220.degree. C. for 2.6 min, increase to
240.degree. C. at 20.degree. C./min and then hold for an additional
2.4 min. Carrier gas was supplied by a Whatman hydrogen generator.
Retention times were compared to those for methyl esters of
standards commercially available (Nu-Chek Prep, Inc.). Routinely,
5-10 embryos per event were analyzed by GC, using the methodology
described above.
[0520] Individual embryo fatty acid profiles for each event (5
embryos each) containing pKR912 were obtained from a total of 44
events. Of the 44 events, 37 events had at least 1 embryo with
greater than 1% EDA and/or ERA. The lipid profiles of somatic
soybean embryos expressing the Euglena gracilis delta-9 elongase
for the top 5 events are shown in FIG. 20. Fatty acids are
identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1 (oleic
acid), LA, ALA, EDA, and ERA; and, fatty acid compositions listed
in FIG. 20 are expressed as a weight percent (wt. %) of total fatty
acids. The activity of the Euglena gracilis delta-9 elongase is
expressed as percent delta-9 elongation (% Elong), calculated
according to the following formula:
([product]/[substrate+product])*100.
[0521] More specifically, the combined percent elongation for LA
and ALA is shown as "delta-9% Elong", determined as:
([EDA+ERA]/[LA+ALA+EDA+ERA])*100. This elongation is also referred
to as the overall % elongation. The individual omega-6 delta-9
elongation ("LA % Elong") was calculated as: ([EDA]/[LA+EDA])*100.
Similarly, the individual omega-3 delta-9 elongation ("ALA %
Elong") was calculated as: ([ERA]/[ALA+ERA])*100. The ratio of
delta-9 elongation for omega-6 versus omega-3 substrates ("ratio
[LA/ALA] % Elong") was obtained by dividing the LA % delta-9
elongation by the ALA % delta-9 elongation.
[0522] In summary of FIG. 20, the Euglena gracilis delta-9 elongase
functioned in soybean to convert both LA and ALA to EDA and ERA,
respectively. The line with the highest average EDA content (i.e.,
1936-6-26) had embryos with an average EDA content of 36.1% and an
average ERA content of 6.7%. The highest EDA and ERA content for an
individual embryo from this line was 44.0% and 10.5%, respectively.
The highest average overall % delta-9 elongation was 67.4% with the
highest overall % delta-9 elongation for an individual embryo being
75.7%. When broken down into % delta-9 elongation for the omega-6
and omega-3 substrates, the highest average % delta-9 elongation
was 67.3% and 67.1% for LA and ALA, respectively. The highest %
delta-9 elongation for an individual embryo was 74.7% and 80.0% for
LA and ALA, respectively. In this example, the Euglena gracilis
delta-9 elongase may have a slight preference for ALA over LA, with
the average desaturation ratio ranging from 0.8 to 1.0.
Example 32
Functional Analysis of the Euglena qracilis Delta-9 Elongase
Co-expressed with the Euglena gracilis Delta-8 desaturase in
Somatic Soybean Embryos Transformed with Soybean Expression Vector
pKR920
[0523] Individual single, matured, somatic soybean embryos that had
been transformed with pKR920 (SEQ ID NO:91) and matured in the
model system on SB103 plates as described in Example 30, with
hygromycin as selection, were picked into glass GC vials and fatty
acid methyl esters were prepared by transesterification and
analyzed as described in Example 31.
[0524] Individual embryo fatty acid profiles for each event (six
embryos each) containing pKR920 were obtained from a total of 48
events. Of the 48 events, 40 events had at least one embryo with
greater than 1% C20 fatty acids (sum of EDA, ERA, DGLA and ETA) and
29 of these also had a functional Euglena gracilis delta-8
desaturase (at least one embryo with greater than 1% DGLA and/or
ETA). The lipid profiles of somatic soybean embryos expressing the
Euglena gracilis delta-9 elongase and the Euglena gracilis delta-8
desaturase for the top 5 events are shown in FIG. 21. Fatty acids
are identified as 16:0 (palmitate), 18:0 (stearic acid), 18:1
(oleic acid), LA, ALA, EDA, HGLA, ERA and ETA; and, fatty acid
compositions listed in FIG. 21 are expressed as a weight percent
(wt. %) of total fatty acids. The activity of the Euglena gracilis
delta-9 elongase is expressed as percent delta-9 elongation (%
Elong), calculated according to the following formula:
([product]/[substrate+product])*100.
[0525] More specifically, the combined percent elongation for LA
and ALA is shown as "Total delta-9% Elong", determined as:
([EDA+HGLA+ERA+ETA]/[LA+ALA +EDA+HGLA+ERA+ETA])*100. This
elongation is also referred to as the overall % elongation. The
individual omega-6 delta-9 elongation ("LA % Elong") was calculated
as: ([EDA+HGLA]/[LA+EDA+HGLA])*100. Similarly, the individual
omega-3 delta-9 elongation ("ALA % Elong") was calculated as:
([ERA+ETA]/[ALA+ERA+ETA])*1 00. The ratio of delta-9 elongation for
omega-6 versus omega-3 substrates ("ratio [LA/ALA] % Elong") was
obtained by dividing the LA % delta-9 elongation by the ALA %
delta-9 elongation.
[0526] In summary of FIG. 21, the Euglena gracilis delta-9 elongase
functioned in soybean to convert both LA and ALA to EDA and ERA,
respectively, and these were further converted to HGLA and ETA,
respectively, when the Euglena gracilis delta-8 desaturase was
functional. The line with the highest average overall % delta-9
elongation (i.e., 1919-6-8) had embryos with an average total C20
fatty acid (EDA+HGLA+ERA+ETA) content of 42.2%. The highest total
C20 fatty acid (EDA+HGLA+ERA+ETA) content for an individual embryo
from this line was 50.1%. The highest average overall % delta-9
elongation was 65.5% with the highest overall % delta-9 elongation
for an individual embryo being 80.8%. When broken down into %
delta-9 elongation for the omega-6 and omega-3 substrates, the
highest average % delta-9 elongation in this event was 64.7% and
69.3% for LA and ALA, respectively. The highest % delta-9
elongation for an individual embryo in this event was 80.4% and
83.8% for LA and ALA, respectively.
Example 33
Transformation of Arabidopsis
[0527] Transformed Arabidopsis plants were created by whole plant
Agrobacterium transformation. Binary vector pKR926 (SEQ ID NO:102)
was transformed into Agrobacterium tumefaciens NTL4 (Luo et al.,
Molecular Plant-Microbe Interactions 14(1):98-103 (2001)) by
electroporation. Briefly, 1 .mu.g plasmid DNA was mixed with 100
.mu.L of electro-competent cells on ice. The cell suspension was
transferred to a 100 .mu.L electro oration curette (1 mm gap width)
and electro orated using a BIORAD electro orator set to 1 kV,
400.OMEGA. and 25 .mu.F. Cells were transferred to 1 mL LB medium
and incubated for 2 h at 30.degree. C. Cells were plated onto LB
medium containing 50 .mu.g/mL kanamycin. Plates were incubated at
30.degree. C. for 60 h. Recombinant agrobacterium cultures (500 mL
LB, 50 .mu.g/mL kanamycin) were inoculated from single colonies of
transformed Agrobacterium cells and grown at 30 .degree. C. for 60
h.
[0528] Cells were harvested by centrifugation (5000.times.g, 10
min) and resuspended in 1 L of 5% (W/V) sucrose containing 0.05%
(V/V) Silwet L-77 (OSI Specialties, Inc). Arabidopsis plants were
grown in soil at a density of 10 plants per 100 cm.sup.2 pot in
metromix 360 soil mixture for 4 weeks (22.degree. C., 16 h light/8
h dark, 100 .mu.E m-.sup.-2s.sup.-1). At early bolting, Arabidopsis
plants were dipped into the Agrobacterium suspension. Two days
later, the same plants were dipped again with the same
Agrobacterium strain in sucrose/Silwet. Plants were grown for three
to four weeks under standard plant growth conditions described
above and plant material was harvested and dried for one week at
ambient temperatures in paper bags. Seeds were harvested using a
0.425 mm mesh brass sieve.
[0529] Cleaned Arabidopsis seeds (2 grams, corresponding to about
100,000 seeds) were sterilized by washes in 45 mL of 80% ethanol,
0.01% triton X-100, followed by 45 mL of 30% (V/V) household bleach
in water, 0.01% triton X-100 and finally by repeated rinsing in
sterile water. Aliquots of 20,000 seeds were transferred to square
plates (20.times.20 cm) containing 150 mL of sterile plant growth
medium comprised of 0.5.times.MS salts, 1.0% (W/V) sucrose, 0.05
MES/KOH (pH 5.8), 200 .mu.g/mL timentin, and 50 .mu.g/mL kanamycin
solidified with 10 g/L agar. Homogeneous dispersion of the seed on
the medium was facilitated by mixing the aqueous seed suspension
with an equal volume of melted plant growth medium. Plates were
incubated under standard growth conditions for fourteen days.
Kanamycin-resistant seedlings were transferred to soil and grown to
maturity as described above. T2 seed was obtained from these
individual transformants.
Example 34
Functional Analysis of the Euglena qracilis Delta-9 Elongase in
Arabidopsis Seed Transformed with Arabidopsis Expression Vector
pKR926
[0530] Wild-type Arabidopsis thaliana (Columbia ecotype) and a
fad3/fae1 double mutant (Smith et al., Planta 217:507-516 (2003))
were transformed with pKR926 (SEQ ID NO:102) as described in
Example 33 and segregating T2 seed was obtained from a number of
individual events for each. Bulk T2 seed lipid profiles for each
event were obtained by transesterification with TMSH as described
in Example 31 with the following modificiations. For each event, a
small scoopful of seeds (approximately 25-50 seed each scoopful)
was crushed in 50 .mu.L of TMSH in a 1.5 mL eppendorf tube. After
shaking in TMSH for 15 min., 400 .mu.L of heptane was added and the
tubes were vortexed well, shaken for an additional 15 min and
centrifuged at 13,000.times.g for 1 min. After shaking, the heptane
layer was removed into glass GC vials and the fatty acid methyl
esters were analyzed as described in Example 31.
[0531] Bulk T2 seed fatty acid profiles were obtained for 22 events
where wild-type Arabidopsis was transformed with pKR926 (SEQ ID
NO:102) and for 16 events where the fad3/fae1 mutant was
transformed. The lipid profiles of T2 bulk seed seed for the 22
wild-type-transformed events as well as for untransformed wild-type
are shown in FIG. 22. The lipid profiles of T2 bulk seed seed for
the 16 fad3/fae1-transformed events as well as for untransformed
fad3/fae1 are shown in FIG. 23. Fatty acids are identified as 16:0
(palmitate), 18:0 (stearic acid), 18:1 (oleic acid), LA, ALA, 20:0
(arachidic acid), 20:1 (eicosenoic acid), EDA and ERA; and, fatty
acid compositions listed in FIG. 22 and FIG. 23 are expressed as a
weight percent (wt. %) of total fatty acids.
[0532] Individual T2 seed lipid profiles (ten seed per event) for
one representative wild-type- and fad3/fae1-transformed event each
(i.e., wt pKR926-8 and ff pKR926-1) were obtained by
transesterification with TMSH as described in Example 31 with the
following modifications. For each event, one seed was crushed in 10
.mu.L of TMSH in a 1.5 mL eppendorf tube. After shaking in TMSH for
15 min, 75 .mu.L of heptane was added and the tubes were vortexed
well, shaken for an additional 15 min and centrifuged at
13,000.times.g for 1 min. After centrifugation, the heptane layer
was removed into glass GC vials containing 200 .mu.L inserts and
the fatty acid methyl esters were analyzed as described in Example
31. In addition to having a representative T2 bulk seed fatty acid
profile, each event chosen for single seed analysis also segregated
both for resistance to kanamycin and for phenotype as a single loci
insertion (i.e., 3:1).
[0533] The lipid profiles for ten single seeds for wt pKR926-8 and
ff pKR926-1 are shown in FIG. 24. The activity of the Euglena
gracilis delta-9 elongase is expressed as percent delta-9
elongation (% Elong), calculated according to the following
formula: ([product]/[substrate+product])*100 as described in
Example 31. Since the seed are T2 and are segregating, some of the
seed have a wt or ff phenotype, respectively and these are
indicated with shading (rows 1, 7-8,12, 16-17 and 20).
Example 35
Co-expression of the Euglena gracilis Delta-9 Elongase with the
Euglena gracilis Delta-8 Desaturase, the Mortierella alpina Delta-5
Desaturase, the Saprolegnia diclina Delta-1 7 Desaturase and the
Fusarium moniliforme Delta-15 Desaturase in Soybean Embryos
Transformed with Soybean Expression Vectors pKR916 and pKR873
[0534] Soybean embryogenic suspension culture (cv. Jack) was
transformed with the AcsI fragments of pKR916 (SEQ ID NO:104; FIG.
19) and pKR873 (SEQ ID NO:56; FIG. 12) (fragments containing the
expression cassettes), as described for production in Example 30. A
subset of soybean embryos generated from each event (ten embryos
per event) were harvested and analyzed for fatty acid composition
as described in Example 31. Fatty acids were identified by
comparison of retention times to those for authentic standards.
[0535] In this way, 169 events transformed with pKR916 and pKR873
were analyzed. From the 169 events analyzed, 127 were identified
that produced EPA in at least one embryo out of ten analyzed at a
relative abundance greater than 1.0% of the total fatty acids. Of
these, 49 were identified that produced EPA in at least one embryo
out of ten analyzed at a relative abundance greater than 10.0% of
the total fatty acids. The average fatty acid profile for the ten
best EPA events (average of nine or ten individual embryos) is
shown in FIG. 25A. Fatty acids are identified as 16:0 (palmitate),
18:0 (stearic acid), 18:1 (oleic acid), LA, GLA, ALA, EDA, HGLA,
ARA, ERA, JUN, ETA, EPA and DPA and, fatty acid compositions listed
in FIG. 25A are expressed as a weight percent (wt. %) of total
fatty acids. For FIG. 25A., fatty acids listed as "others" include:
18:2 (5,9), STA, 20:0, 20:1(11), 20:2 (7,11) or 20:2 (8,11) and
20:3 (5,11,14). Each of these fatty acids is present at a relative
abundance of less than 1% of the total fatty acids. The activity of
the Euglena gracilis delta-9 elongase is expressed as percent
delta-9 elongation (% Elong), calculated according to the following
formula: ([product]/[substrate+product])*100.
[0536] More specifically, the combined percent elongation for LA
and ALA is shown as "Total delta-9% Elong", determined as:
([EDA+HGLA+ARA+ERA+JUN+ETA+EPA+DPA]/[LA+ALA+EDA+HGLA+ARA+ERA+JUN+ETA+EPA+-
DPA])*100. This elongation is also referred to as the overall %
elongation.
[0537] In summary of FIG. 25A, the Euglena gracilis delta-9
elongase functioned in soybean to convert both LA and ALA to EDA
and ERA, respectively and these were further converted to other
LC-PUFAs. The high EPA line with the highest average overall %
delta-9 elongation (i.e., AFS 4697-7-5) had embryos with an average
total C20 fatty acid (EDA+HGLA+ARA+ERA+JUN+ETA+EPA+DPA) content of
38.2%. The highest total C20 fatty acid (EDA+HGLA+ARA+ERA+JUN+ETA
+EPA+DPA) content for a high EPA individual embryo was 51.4%
(embryo from AFS 4709-8-6) and in this embryo, EPA was 24.4%.
[0538] Four plants each from top EPA events were regenerated and
grown as described in Example 30. Seeds were harvested and a small
chip was taken from part of each seed (from directly opposite the
embryonic axis) using a razor blade. The seed chips were analyzed
for fatty acids as described above. Fatty acid profiles for five
seeds with highest EPA from 2 representative events (4697-6-1 and
6697-6-5) as well for a segregating wild-type seed for each are
shown in FIG. 25B. Seed names are designated by a five number
series separated by hyphens where the first three numbers indicate
a particular event, the fourth number indicates the plant and the
fifth number indicates the seed analyzed.
[0539] The seed with the highest total C20 fatty acid
(EDA+HGLA+ARA+ERA+JUN+ETA+EPA+DPA) content for a high EPA event had
total C20 fatty acids of 48.0% (seed number 4697-6-5-2-4) with a
overall % elongation of 77.1% and in this seed, EPA was 16.2%.
Example 36
Construction and Functional Analysis of Yarrowia lipolytica
Expression Vector pZuFmEqD9ES, Comprising a Synthetic Delta-9
Elongase Gene (Derived From Euglena gracilis), Codon-Optimized for
Expression in Yarrowia lipolytica (EqD9eS)
[0540] The present Example describes the expression of Yarrowia
lipolytica vector pZuFmEgD9ES, comprising a chimeric
FBAINm::EgD9ES::Pex20 gene, wherein EgD9eS is a synthetic delta-9
elongase derived from Euglena gracilis and codon-optimized for
expression in Yarrowia. This analysis included: (1) synthesis of
EgD9eS; (2) construction and transformation of pZuFmEgD9ES into
Yarrowia lipolytica strain Y2224; and (3) analysis of lipid
profiles within transformant organisms of Yarrowia lipolytica
strain Y2224 that were comprising pZuFmEgD9ES (expressing
EgD9eS).
Synthesis of EgD9eS
[0541] The codon usage of the delta-9 elongase gene of Euglena
gracilis (EgD9e; SEQ ID NOs:4 and 5) was optimized for expression
in Yarrowia lipolytica, in a manner similar to that described in
Example 4 and PCT Publication No. WO 2004/101753. Specifically, a
codon-optimized delta-9 elongase gene (designated "EgD9eS"; SEQ ID
NO:90) was designed, based on the coding sequence of EgD9e (i.e.,
from clone eeg1c.pk001.n5.f), according to the Yarrowia codon usage
pattern (PCT Publication No. WO 2004/101753), the consensus
sequence around the `ATG` translation initiation codon, and the
general rules of RNA stability (Guhaniyogiet al., Gene,
265(1-2):11-23 (2001)). In addition to modification of the
translation initiation site, 117 bp of the 777 bp coding region
were modified (15.1%) and 106 codons were optimized (40.9%). FIGS.
26A and B show a comparison of the nucleotide sequences of EgD9e
and EgD9eS. None of the modifications in the codon-optimized gene
changed the amino acid sequence of the encoded protein (SEQ ID
NO:5). The designed EgD9eS gene was synthesized by GenScript
Corporation (Piscataway, N.J.) and cloned into pUC57 (GenBank
Accession No. Y14837) to generate pEgD9S.
Generation of Construct pZuFmEgD9E (Comprising EgD9E) and
pZuFmEqD9ES (Comprising EqD9ES)
[0542] Plasmid pZuFmEgD9ES (SEQ ID NO:105), comprising a chimeric
FBAINm::EgD9ES::Pex20 gene, was constructed by replacing the
NcoI/NotI fragment of pZUF17 (SEQ ID NO:25) with the NcoI/NotI
fragment from pEgD9S comprising EgD9eS. The product of this
ligation was auto-replicating expression vector pZuFmEgD9ES, which
thereby contained the following components: TABLE-US-00011 TABLE 7
Components of Plasmid pZuFmEgD9ES (SEQ ID NO: 105) RE Sites and
Nucleotides Within SEQ ID NO: 105 Description of Fragment and
Chimeric Gene Components Swa I/BsiW I FBAINm::EgD9eS::Pex20,
comprising: (6067-318) FBAINm: Yarrowia lipolytica FBAINm promoter
(WO 2005/049805) EgD9eS: codon-optimized delta-9 elongase (SEQ ID
NO: 5, described herein as EgD9eS), derived from Euglena gracilis
Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank
Accession No. AF054613) 1354-474 ColE1 plasmid origin of
replication 2284-1424 Ampicillin-resistance gene (Amp.sup.R) for
selection in E. coli 3183-4487 Yarrowia autonomous replication
sequence (ARS18; GenBank Accession No. A17608) 6020-4533 Yarrowia
Ura 3 gene (GenBank Accession No. AJ306421)
[0543] Plasmid pZuFmEgD9E (SEQ ID NO:106), comprising a chimeric
FBAINm::EgD9E::Pex20 gene, was synthesized in a similar manner
using the pZUF17 plasmid backbone.
Functional Analysis of Yarrowia lipolytica Transformants Comprising
pZuFmEgD9E nd pZuFmEqD9ES
[0544] Plasmid pZuFmEgD9E and pZuFmEgD9ES (comprising a chimeric
FBAINm::EgD9e::Pex20 gene and FBAINm::EgD9eS::Pex20 gene,
respectively) were transformed into strain Y2224 (the FOA resistant
mutant from an autonomous mutation of the Ura3 gene of wildtype
Yarrowia strain ATCC #20362; Example 21), as described in the
General Methods. The transformants were selected on MM plates.
After 2 days growth at 30.degree. C., 3 transformants grown on the
MM plates were picked and re-streaked onto fresh MM plates. Once
grown, these strains were individually inoculated into 3 mL liquid
MM at 30.degree. C. and shaken at 250 rpm/min for 2 days. The cells
were collected by centrifugation, lipids were extracted, and fatty
acid methyl esters were prepared by trans-esterification, and
subsequently analyzed with a Hewlett-Packard 6890 GC.
[0545] GC analyses showed that there were about 3.2% EDA (C20:2) of
total lipids produced in all seven transformants with pZuFmEgD9E,
wherein the average conversion efficiency of LA (C18:2) to EDA in
these seven strains was determined to be about 18.3% (average;
calculated as described in Example 20).
[0546] In contrast, GC analyses showed that there were about 3.6%
EDA (C20:2) of total lipids produced in all seven transformants
with pZuFmEgD9ES, wherein the average conversion efficiency of LA
(C18:2) to EDA in these seven strains was determined to be about
20.1% (average). Thus, the experimental data demonstrated that the
synthetic Euglena gracilis delta-9 elongase codon-optimized for
expression in Yarrowia lipolytica (i.e., EgD9eS; SEQ ID NO:90) was
about 16.2% more efficient elongating LA to EDA than the wildtype
EgD9e gene (i.e., SEQ ID NO:4).
Example 37
Preparation of Eutreptiella sp. CCMP389 Genomic DNA, RNA And
cDNA
[0547] The present Example describes the preparation of genomic
DNA, RNA and cDNA from Eutreptiella sp. CCMP389, which had been
purchased from The Provasoli-Guillard National Center for Culture
of Marine Phytoplankton (CCMP) (Bigelow Laboratory for Ocean
Sciences, West Boothbay Harbor, Me.).
Preparation of RNA and Genomic DNA From Eutreptiella sp.
CCMP389
[0548] Total RNA and genomic DNA were isolated from 1 liter of
culture using Trizol reagent (Invitrogen, Carlsbad, Calif.),
according to the manufacturer's protocol. Specifically, the cell
pellet was resuspended in 0.75 mL of Trizol reagent, mixed with 0.5
mL of 0.5 mm glass beads, and homogenized in a Biospec mini
beadbeater (Bartlesville, Okla.) at the highest setting for 3 min.
The mixtures were centrifuged in an Eppendorf centrifuge for 30 sec
at 14,000 rpm to remove debris and glass beads. The supernatant was
extracted with 150 .mu.L of 24:1 chloroform:isoamyl alcohol
(Invitrogen). The upper aqueous phase was used for RNA isolation
and the lower organic phase was used for DNA isolation.
[0549] For RNA isolation, the aqueous phase was mixed with 0.375 mL
of isopropyl alcohol and allowed to incubate at room temperature
for 5 min. Precipitated RNA was collected by centrifugation at
8,000 rpm and 4.degree. C. for 5 min. The pellet was washed once
with 0.7 mL of 80% ethanol and air-dried. In this way, 360 .mu.g of
total RNA were obtained.
[0550] For genomic DNA isolation, the lower organic phase was mixed
with 75 .mu.L of ethanol and incubated at room temperature for 5
min. The sample was then centrifuged at 5,000 rpm for 2 min in an
Eppendorf centrifuge. The pellet was washed with 0.75 mL of 0.1 M
sodium citrate:10% ethanol twice. Each time, the sample was
incubated for 15 min at room temperature in the wash solution,
followed by centrifugation at 5,000 rpm for 5 min at 4.degree. C.
The pellet was air-dried and re-dissolved in 300 .mu.L of 8 mM
NaOH. The pH of the sample was adjusted to 7.5 with 1 M HEPES. The
genomic DNA was then further purified with a Qiagen PCR
purification kit (Valencia, Calif.) exactly as described in the
manufacturer's protocol. Thus, 40 .mu.g of genomic DNA was
isolated.
Preparation of cDNA from Eutreptiella sp. CCMP389
[0551] Double-stranded cDNA was generated, using the Creator.TM.
SMART.TM. cDNA Library Construction Kit from BD Bioscience Clontech
(Palo Alto, Calif.). Specifically, for first strand cDNA synthesis,
1 .mu.L of the total RNA sample (1.2 .mu.g) was mixed individually
with 1 .mu.L of SMART.TM. IV oligonucleotide (SEQ ID NO:107), 1
.mu.L CDSIII/3' PCR primer (SEQ ID NO:108) and 2 .mu.L of water.
The mixture was heated to 75.degree. C. for 5 min and cooled on ice
for 5 min. To the sample was added 2 .mu.L of 5.times. first strand
buffer, 1 .mu.L 20 mM DTT, 1 .mu.L of dNTP mix (10 mM each of dATP,
dCTP, dGTP and dTTP) and 1 .mu.L of PowerScript reverse
transcriptase. The sample was incubated at 42.degree. C. for 1
hr.
[0552] The first strand cDNA synthesis mixture was used as template
for amplification. Specifically, the reaction mixture contained 2
.mu.L of the above first strand cDNA sample, 80 .mu.L of water, 10
.mu.L of 10.times. Advantage 2 PCR buffer, 2 .mu.L 50.times.dNTP
mix (10 mM each of dATP, dCTP, dGTP and dTTP), 2 .mu.L of 5'-PCR
primer (SEQ ID NO:109), 2 .mu.L CDSIII/3'-PCR primer (SEQ ID
NO:108) and 2 .mu.L 50.times. Advantage 2 polymerase mix. PCR
amplification was performed using the following conditions:
95.degree. C. for 1 min, followed by 20 cycles of 95.degree. C. for
10 sec and 68.degree. C. for 6 min. Amplification products were
purified with a Qiagen PCR purification kit following the
manufacturer's protocol exactly. Purified products were eluted with
50 .mu.L of water.
Example 38
Isolation of the Full-length Delta-9 Elongase from Eutreptiella sp.
CCMP389
[0553] The present Example describes the identification of a
partial cDNA fragment encoding a delta-9 elongase from Eutreptiella
sp. CCMP389, by use of primers derived from conserved regions of
the Euglena gracilis (EgD9e; Example 3) and Isochrysis galbana
(IgD9e) delta-9 elongase sequences. Then, based on the sequence of
the partial cDNA fragment, the 5' and 3' ends of the gene were
isolated. This enabled assembly of a contig (SEQ ID NO:111),
extending 51 bases upstream of the Eutreptiella sp. CCMP389 delta-9
elongase translation initiation `ATG` codon and 662 bp beyond the
delta-9 elongase termination codon. Identification of a CDNA
Fragment Encoding a Partial Delta-9 Elongase From Eutreptiella sp.
CCMP389
[0554] It was assumed that a delta-9 elongase/delta-8 desaturase
pathway operated in Eutreptiella sp. CCMP389, based on the
Applicants' Assignee's previous identification of a delta-8
desaturase within the organism (co-pending U.S. Pat. application
Ser. No. 60/853,563; filed Oct. 23, 2006; Attorney Docket No.
BB1574). Design of degenerate primers suitable to isolate the
Eutreptiella sp. CCMP389 delta-9 elongase was based on the
identification of several stretches of conserved amino acid
sequences common to both EgD9e (SEQ ID NO:5) and IgD9e (SEQ ID
NO:27), when an alignment of the two elongases was produced using
the method of Clustal W (slow, accurate, Gonnet option; Thompson et
al., Nucleic Acids Res., 22:4673-4680 (1994)) of the MegAlign.TM.
program of DNASTAR software (see FIG. 27).
[0555] Based on this alignment, the following set of degenerate
oligonucleotides were designed to amplify a portion of the coding
region of the delta-9 elongase gene from Eutreptiella sp. CCMP389,
as shown in Table 8. TABLE-US-00012 TABLE 8 Degenerate
Oligonucleotides used to Amplify the Delta-9 Elongase Gene From
Eutreptiella sp. CCMP389 Position Within Amino Acid SEQ ID NO: 2
Primer Nucleotide Sequence Sequence (EgD9e) EuEF3 YTNCARTTYTTYCAYCA
LQFFHHL 150-156 YTT (SEQ ID NO: 112) (SEQ ID NO: 113) EuER3
TTRAAYTGDATDATYTG MQIIQFN 210-216 CAT (SEQ ID NO: 114) (SEQ ID NO:
115) [Note: The nucleic acid degeneracy code used for SEQ ID NOs:
112 and 114 was as follows: R = A/G; Y = C/T; D = G/A/T; and N =
A/C/T/G.]
[0556] The reaction mixture contained 1 .mu.L of 1:20 diluted cDNA,
5 .mu.L each of the forward and reverse primers (20 .mu.M), 14
.mu.L water and 25 .mu.L of TaKaRa ExTaq 2.times. premix (TaKaRa
Bio, Mountain View, Calif.). PCR amplification was performed using
the following parameters: 94.degree. C. for 1 min, then 35 cycles
of 94.degree. C. for 20 sec, 55.degree. C. for 20 sec, and
72.degree. C. for 1 min, followed by a final elongation cycle at
72.degree. C. for 5 min.
[0557] Agarose gel analysis of the PCR products showed that a
.about.200 bp fragment was obtained. The fragments were purified
with a Qiagen PCR purification kit, cloned into pCR2.1-TOPO
(Invitrogen) and sequenced. The resultant sequence (SEQ ID NO:129),
when translated, had homology with the known delta-9 elongase from
Isochrysis galbana (IgD9e; SEQ ID NO:27), based on BLAST program
analysis (Basic Local Alignment Search Tool; Altschul, S. F., et
al., J. Mol. Biol., 215:403-410 (1993)).
Isolation of the 5'-End Sequence of the Eutre Ptiella sp. CCMP389
Delta-9 Elongase
[0558] Double-stranded cDNA of Eutreptiella sp. CCMP389 (Example
37) was used as template in two separate rounds of PCR
amplification. In the first round of PCR amplification, the
oligonucleotide primers consisted of a gene specific
oligonucleotide (i.e., 389Elo-5-1 (SEQ ID NO:116)) and the generic
oligonucleotide 5'-PCR primer (SEQ ID NO:109) from the BD-Clontech
Creator.TM. SMART.TM. cDNA Library Kit. The PCR amplifications were
carried out in a 50 .mu.L total volume, comprising: 1 .mu.L of 1:10
diluted Eutreptiella sp. CCMP389 cDNA as template, 1 .mu.L of each
primer (20 .mu.M), 22 .mu.L water and 25 .mu.L TaKaRa ExTaq
2.times. premix. Amplification was carried out at 94.degree. C. for
90 sec, then 30 cycles of 94.degree. C. for 30 sec, 55.degree. C.
for 30 sec and 72.degree. C. for 1 min, followed by a final
elongation cycle at 72.degree. C. for 7 min.
[0559] The second round of PCR amplification used 1 .mu.L of
diluted product (1:50) from the first round PCR reaction as
template. Primers consisted of a gene specific oligonucleotide
(i.e., 389Elo-5-2 (SEQ ID NO:117)) and the oligonucleotide DNR CDS
5'-2 (SEQ ID NO:118). Amplification was conducted as described
above.
[0560] The products of the second round PCR reaction were
electrophoresed in 1% (w/v) agarose and appeared as a diffused band
spanning the size range of 200 to 800 bp. Products between 400 bp
to 600 bp were isolated using a Qiagen Gel purification kit
according to the manufacturer's protocol, cloned into pCR2.1-TOPO
(Invitrogen), and transformed into E. coli. Transformants were
selected on LB agar containing ampicillin (100 .mu.g/mL).
[0561] Sequence analysis of the plasmid DNA from one transformant
comprising the 5' region of the putative delta-9 elongase CDNA
revealed a fragment of 406 bp (i.e., 5'-cDNA fragment 1; SEQ ID
NO:1 19). This fragment extended to near the gene's `ATG`
translation initiation codon, but neither the start codon nor the
first 20 to 30 amino acids were included in SEQ ID NO:119.
[0562] An additional oligonucleotide (i.e., 389Elo-5-4 (SEQ ID
NO:120)) was then designed to obtain the complete 5' end of the
gene by PCR, based on the sequence of 5'-cDNA fragment 1 (SEQ ID
NO:119). The reaction mixture and amplification conditions were
identical to those used for the second round of PCR above, except
that primer 389Elo-5-2 was replaced with 389Elo-5-4. When analyzed
by agarose gel electrophoresis, PCR products again appeared as a
diffused band between 200 and 800 bp and fragments with a size of
200 to 500 bp were isolated, cloned and transformed as described
above.
[0563] Sequence analysis of the plasmid DNA from one transformant
comprising the 5' region of the putative delta-9 elongase cDNA
revealed a fragment of 197 bp (5'-cDNA fragment 2; SEQ ID NO:121).
This included the 5'-end of the cDNA and 51 bp of upstream
untranslated region.
Isolation of the 3'-End of the Eutreptiella sp. CCMP389 Delta-9
Elongase
[0564] The 3' end of the putative delta-9 delta elongase was also
isolated by PCR amplification using cDNA as template. The
methodology was as described above for isolation of the 5' end;
however, the primers used on both the first and second round of PCR
amplification were as shown below in Table 9 and were 10 .mu.M
instead of 20 .mu.M. Additionally, the final elongation cycle at
72.degree. C. was decreased from 7 min to 5 min. TABLE-US-00013
TABLE 9 Oligonucleotide Primers Used For 3' cDNA Isolation PCR Gene
Specific Amplification Oligonucleotide Generic Oligonucleotide
1.sup.st Round 389Elo-3-1 CDSIII/3' PCR primer (SEQ ID NO: 122)
(SEQ ID NO: 108) 2.sup.nd Round 389Elo-3-2 CDSIII/3' PCR primer
(SEQ ID NO: 123) (SEQ ID NO: 108) * CDSIII/3' PCR primer was
supplied in Clontech's Creator .TM. SMART .TM. cDNA Library
Construction Kit.
[0565] A .about.1 kB DNA fragment was generated from the 2.sup.nd
round PCR amplication, which was purified with a Qiagen PCR
purification kit, cloned into pCR2.1-TOPO, transformed and
sequenced. Sequence analysis of several clones showed that the
.about.1 kB DNA fragment contained the 3'-region of the putative
delta-9 elongase cDNA, including the polyA tail. The 920 bp
assembled contig sequence of the 3'-region is shown as SEQ ID
NO:124.
Assembly Of The Full-Length Delta-9 Elonqase Sequence From
Eutreptiella sp. CCMP389
[0566] Assembly of the original partial cDNA fragment (SEQ ID NO:1
16), the two 5' cDNA fragments (SEQ ID NOs:119 and 121) and 3'-cDNA
fragment (SEQ ID NO:124) resulted in the complete sequence of the
delta-9 elongase from Eutreptiella sp. CCMP389, plus 51 bp of 5'
untranslated region and 662 bp of 3' untranslated region (SEQ ID
NO:125; 1504 bp). The coding region is 792 bp long and encodes a
protein of 263 amino acids (SEQ ID NO:126). SEQ ID NO:127 is the
nucleotide sequence of the coding sequence of Eutreptiella sp.
CCMP389 delta-9 elongase (designated herein as E389D9e).
Comparison of the Delta-9 Elonqase Sequence of Eutreptiella sp.
CCMP389 (E389D9e) to Known Delta-9 Elongases
[0567] Identity of SEQ ID NO:127 (i.e., E389D9e) was determined by
conducting BLAST searches for similarity to sequences contained in
the BLAST "nr" database (Example 3). The results of the BLAST
comparison summarizing the sequence to which SEQ ID NO:127 has the
most similarity are reported according to the % identity, %
similarity, and Expectation value. "% Identity" is defined as the
percentage of amino acids that are identical between the two
proteins. "% Similarity" is defined as the percentage of amino
acids that are identical or conserved between the two proteins.
"Expectation value" estimates the statistical significance of the
match, specifying the number of matches, with a given score, that
are expected in a search of a database of this size absolutely by
chance.
[0568] Thus, the amino acid fragment described herein as SEQ ID
NO:126 shared 38% identity and 56% similarity with IgD9e, the
delta-9 elongase of Isochrysis galbana (SEQ ID NO:27), with an
expectation value of 2E43. Similarly, E389D9e is 33.1% identical to
IgD9e using the Clustal V method and E389D9e is 65.1% identical to
EgD9e using the Clustal V method (see FIG. 29). Sequence percent
identity calculations performed by the Clustal V method (Higgins,
D. G. and Sharp, P. M., Comput. Appl. Biosci., 5:151-153 (1989);
Higgins et al., Comput. Appl. Biosci., 8:189-191 (1992)) were done
using the MegAlign.TM. v6.1 program of the LASERGENE bioinformatics
computing suite with the default parameters for pairwise alignment
(KTUPLE=1, GAP PENALTY=3, WINDOW=5, DIAGONALS SAVED=5 and GAP
LENGTH PENALTY=10).
[0569] Additionally, pairwise alignment of SEQ ID NO:126 to the
EgD9e sequence of SEQ ID NO:5 using default parameters of Vector
NTI.RTM.'s AlignX program revealed 65% identity and 76.5%
similarity between the two proteins over a length of the 258 amino
acids of EgD9e.
Example 39
Construction and Functional Analysis of Yarrowia lipolytica
Expression Vector pFBAIN-389Elo (Comprising the Eutreptiella sp.
CCMP389 Delta-9 Elongase (E389D9e)) in Yarrowia lipolytica Strain
Y2224)
[0570] The present Example describes synthesis of Yarrowia
lipolytica expression vector pFBAIN-389Elo (comprising a chimeric
FBAINm::E389D9e::Pex20 gene). Delta-9 elongase activity of E389D9e
when expressed in Yarrowia lipolytica strain Y2224 was subsequently
determined.
Construction of Yarrowia lipolytica Expression Vector
pFBAIN-389Elo
[0571] Oligonucleotides 389Elo-F and 389Elo-R1 (SEQ ID NOs:1 16 and
117, respectively) were used as primers to amplify the full length
cDNA of E389D9e (SEQ ID NO:127). The PCR reactions, with
Eutreptiella sp. CCMP389 CDNA (Example 27) as template, were
individually carried out in a 50 .mu.L total volume comprising: 1
.mu.L each of 20 lM forward and reverse primers, 1 .mu.L cDNA, 10
.mu.L 5.times.PCR buffer, 1 .mu.L dNTP mix (10 .mu.M each), 35
.mu.L water and 1 .mu.L Phusion polymerase (New England Biolabs,
Inc., Ipswich, Mass.). Amplification was carried out at 98.degree.
C. for 1 min, then 30 cycles of 98.degree. C. for 10 sec,
55.degree. C. for 10 sec, and 72.degree. C. for 30 sec, followed by
a final elongation cycle at 72.degree. C. for 5 min. The PCR
product was digested with NcoI and EarI to generate a .about.210 bp
fragment that contained the 5' region of the delta-9 elongase cDNA.
It was also digested with EarI and NotI to generate a .about.600 bp
fragment that contained the 3' region of the cDNA. The NcoI/EarI
and the EarI/NotI digested fragments were purified following gel
electrophoresis in 1% (w/v) agarose.
[0572] The NcoI/EarI and the EarI/NotI delta-9 elongase digested
fragments were directionally ligated with NcoI/NotI digested
pFBAIN-MOD-1 (SEQ ID NO:128), such that the E389D9e gene was under
the control of the Yarrowia lipolytica FBAINm promoter and the
PEX20-3' terminator region. Specifically, the ligation reaction
contained: 10 .mu.L 2.times. ligation buffer, 1 .mu.L T4 DNA ligase
(Promega), 4 .mu.L each of the .about.210 bp and the .about.600 bp
fragment (.about.300 ng each), and 1 .mu.L pFBAIN-MOD-1 (.about.150
ng). The reaction mixture was incubated at room temperature for 2 h
and used to transform E. coli Top10 competent cells (Invitrogen).
Plasmid DNA from transformants was recovered using a Qiagen
Miniprep kit. Correct clones were identified by restriction mapping
and the final construct was designated "pFBAIN-389Elo".
[0573] Thus, pFBAIN-389Elo (FIG. 28; SEQ ID NO:110) thereby
contained the 15 following components: TABLE-US-00014 TABLE 10
Components of Plasmid pFBAIN-389Elo (SEQ ID NO: 110) RE Sites and
Nucleotides Within SEQ Description of Fragment and Chimeric Gene ID
NO: 110 Components BgIII-BsiWI FBAINm::E389D9e::Pex20, comprising:
(6040-301) FBAINm: Yarrowia lipolytica FBAINm promoter (PCT
Publication No. WO 2005/049805) E389D9e: Eutreptiella sp. CCMP389
delta-9 elongase (SEQ ID NO: 127 described herein) Pex20: Pex20
terminator sequence of Yarrowia Pex20 gene (Gen Bank Accession No.
AF054613) PacI-BgIII Yarrowia Ura 3 gene (GenBank Accession No.
(4533-6040) AJ306421) (3123-4487) Yarrowia autonomous replication
sequence (ARS18; GenBank Accession No. A17608) (2464-2864) f1
origin (1424-2284) Ampicillin-resistance gene (Amp.sup.R) for
selection in E. coli (474-1354) ColE1 plasmid origin of
replication
Functional Analysis of Yarrowia lipolytica Transformants Comprising
pFBAIN-389Elo
[0574] Five (5) individual clones of pFBAIN-389Elo (comprising
E389D9e) and control plasmid pFBAIN-MOD-1 were transformed into
Yarrowia lipolytica strain Y2224 (Example 20) as described in the
General Methods. The cells were plated onto MM plates lacking
uracil and maintained at 30.degree. C. for 2 to 3 days. Then, cells
from each plate were scraped off, lipids were extracted, and fatty
acid methyl esters were prepared by trans-esterification, and
subsequently analyzed with a Hewlett-Packard 6890 GC.
[0575] GC analyses showed that EDA was produced in all five of the
transformants comprising pFBAIN-389Elo, while no EDA was produced
in the control strain (Table 11). Fatty acids are identified as
18:2 (LA) and 20:2 (EDA); and the composition of each is presented
as a % of the total fatty acids. The conversion efficiency was
calculated according to the following formula:
([product]/[substrate+product])*100, where `product` includes the
immediate product and all products in the pathway derived from it.
TABLE-US-00015 TABLE 11 Lipid Composition in Yarrowia lipolytica
Strain Y2224 Engineered to Over-Express Eutreptiella sp. CCMP389
Delta-9 Elongase (E389D9e) Conversion Plasmid Clone C18:2 C20:2
Efficiency pFBAIN-MOD-1 1 17.4 0 0 pFBAIN-389Elo 1 13.49 2.16 13.80
2 13.16 1.79 11.97 3 14.11 1.92 11.98 4 15.55 0.78 4.78 5 13.24
1.79 11.91
[0576] The results shown above confirmed that the cloned cDNA from
Eutreptiella sp. CCMP389, described herein as SEQ ID NOs:126 and
127, efficiently desaturated LA to EDA and thus functioned as a
delta-9 elongase.
Example 40
Construction of Alternate Soybean Expression Vectors for Expression
of Other PUFA Genes
[0577] In addition to the genes, promoters, terminators and gene
cassettes described herein, one skilled in the art can appreciate
that other promoter/gene/terminator cassette combinations can be
synthesized in a way similar to, but not limited to, that described
herein for expression of EgD9e, EgD9eS, E389D9e or E389D9eS.
Similarly, it may be desirable to express other PUFA genes (such as
those described below in Table 14), for co-expression with any of
the delta-9 elongases of the present invention.
[0578] For instance, PCT Publication Nos. WO 2004/071467 and WO
2004/071178 describe the isolation of a number of promoter and
transcription terminator sequences for use in embryo-specific
expression in soybean. Furthermore, PCT Publication Nos. WO
2004/071467, WO 2005/047479 and WO 2006/012325 describe the
synthesis of multiple promoter/gene/terminator cassette
combinations by ligating individual promoters, genes and
transcription terminators together in unique combinations.
Generally, a NotI site flanked by the suitable promoter (such as
those listed in, but not limited to, Table 12) and a transcription
terminator (such as those listed in, but not limited to, Table 13)
is used to clone the desired gene. NotI sites can be added to a
gene of interest such as those listed in, but not limited to, Table
14 using PCR amplification with oligonucleotides designed to
introduce NotI sites at the 5' and 3' ends of the gene. The
resulting PCR product is then digested with NotI and cloned into a
suitable promoter/NotI/terminator cassette.
[0579] In addition, PCT Publication Nos. WO 2004/071467, WO
2005/047479 and WO 2006/012325 describe the further linking
together of individual gene cassettes in unique combinations, along
with suitable selectable marker cassettes, in order to obtain the
desired phenotypic expression. Although this is done mainly using
different restriction enzymes sites, one skilled in the art can
appreciate that a number of techniques can be utilized to achieve
the desired promoter/gene/transcription terminator combination. In
so doing, any combination of embryo-specific
promoter/gene/transcription terminator cassettes can be achieved.
One skilled in the art can also appreciate that these cassettes can
be located on individual DNA fragments or on multiple fragments
where co-expression of genes is the outcome of co-transformation of
multiple DNA fragments. TABLE-US-00016 TABLE 12 Seed-specific
Promoters Promoter Organism Promoter Reference .beta.-conglycinin
.alpha.'-subunit soybean Beachy et al., EMBO J. 4: 3047-3053 (1985)
kunitz trypsin inhibitor soybean Jofuku et al., Plant Cell 1:
1079-1093 (1989) annexin soybean WO 2004/071467 glycinin Gy1
soybean WO 2004/071467 albumin 2S soybean U.S. Pat. No. 6,177,613
legumin A1 pea Rerie et al., Mol. Gen. Genet. 225: 148-157 (1991)
.beta.-conglycinin .beta.-subunit soybean WO 2004/071467 BD30 (also
called P34) soybean WO 2004/071467 legumin A2 pea Rerie et al.,
Mol. Gen. Genet. 225: 148-157 (1991)
[0580] TABLE-US-00017 TABLE 13 Transcription Terminators
Transcription Terminator Organism Reference phaseolin 3' bean WO
2004/071467 kunitz trypsin inhibitor 3' soybean WO 2004/071467 BD30
(also called P34) 3' soybean WO 2004/071467 legumin A2 3' pea WO
2004/071467 albumin 2S 3' soybean WO 2004/071467
[0581] TABLE-US-00018 TABLE 14 PUFA Biosynthetic Pathway Genes Gene
Organism Reference delta-6 desaturase Saprolegnia diclina WO
2002/081668 delta-6 desaturase Mortierella alpina U.S. Pat. No.
5,968,809 elongase Mortierella alpina WO 2000/12720 U.S. Pat. No.
6,403,349 delta-5 desaturase Mortierella alpina U.S. Pat. No.
6,075,183 delta-5 desaturase Saprolegnia diclina WO 2002/081668
delta-15 desaturase Fusarium WO 2005/047479 moniliforme delta-17
desaturase Saprolegnia diclina WO 2002/081668 elongase
Thraustochytrium WO 2002/08401 aureum U.S. Pat. No. 6,677,145
elongase Pavlova sp. Pereira et al., Biochem. J. 384: 357-366
(2004) delta-4 desaturase Schizochytrium WO 2002/090493 aggregatum
delta-9 elongase Isochrysis galbana WO 2002/077213 delta-9 elongase
Euglena gracilis U.S. Provisional Application No. 60/739,989
delta-8 desaturase Euglena gracilis WO 2000/34439 U.S. Pat. No.
6,825,017 WO 2004/057001 WO 2006/012325 delta-8 desaturase
Acanthamoeba Sayanova et al., FEBS Lett. castellanii 580: 1946-1952
(2006) delta-8 desaturase Pavlova salina WO 2005/103253 delta-8
desaturase Pavlova lutheri U.S. Provisional Application No.
60/795,810 delta-8 desaturase Tetruetreptia U.S. Provisional
Application pomquetensis No. 60/853,563 CCMP1491 delta-8 desaturase
Eutreptiella sp. U.S. Provisional Application CCMP389 No.
60/853,563 delta-8 desaturase Eutreptiella cf U.S. Provisional
Application gymnastica No. 60/853,563 CCMP1594
[0582]
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070118929A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070118929A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References