U.S. patent application number 12/431813 was filed with the patent office on 2009-10-22 for delta-8 desaturase and its use in making polyunsaturated fatty acids.
This patent application is currently assigned to E.I. DU PONT DE NEMOURS AND COMPANY. Invention is credited to Howard Glenn Damude, Quinn Qun Zhu.
Application Number | 20090264666 12/431813 |
Document ID | / |
Family ID | 34980122 |
Filed Date | 2009-10-22 |
United States Patent
Application |
20090264666 |
Kind Code |
A1 |
Damude; Howard Glenn ; et
al. |
October 22, 2009 |
DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY
ACIDS
Abstract
Isolated nucleic acid fragments and recombinant constructs
comprising such fragments encoding a delta-8 desaturase along with
a method of making long chain polyunsaturated fatty acids (PUFAs)
using this delta-8 desaturase in plants and oleaginous yeast.
Inventors: |
Damude; Howard Glenn;
(Hockessin, DE) ; Zhu; Quinn Qun; (West Chester,
PA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1122B, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Assignee: |
E.I. DU PONT DE NEMOURS AND
COMPANY
Wilmington
DE
|
Family ID: |
34980122 |
Appl. No.: |
12/431813 |
Filed: |
April 29, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11166003 |
Jun 24, 2005 |
7550651 |
|
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12431813 |
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60583041 |
Jun 25, 2004 |
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60624812 |
Nov 4, 2004 |
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Current U.S.
Class: |
554/9 ;
536/23.2 |
Current CPC
Class: |
C12P 7/6427 20130101;
A23D 9/00 20130101; A23L 33/12 20160801; C12N 15/8247 20130101;
C12P 7/6472 20130101; A23K 20/158 20160501; C12N 9/0083 20130101;
A23V 2002/00 20130101; A23V 2002/00 20130101; A23V 2300/21
20130101 |
Class at
Publication: |
554/9 ;
536/23.2 |
International
Class: |
C11B 1/00 20060101
C11B001/00; C07H 21/04 20060101 C07H021/04 |
Claims
1. An isolated polynucleotide comprising: (a) a nucleotide sequence
encoding a polypeptide having delta-8 desaturase activity, wherein
the polypeptide has an amino acid sequence consisting essentially
of SEQ ID NOs:2 or 113; or, (b) a complement of the nucleotide
sequence, wherein the complement and the nucleotide sequence
consist of the same number of nucleotides and are 100%
complementary.
2-38. (canceled)
39. Oil obtained from a seed obtained from a plant comprising a
recombinant construct comprising an isolated polynucleotide
comprising: (a) a nucleotide sequence encoding a polypeptide having
delta-8 desaturase activity, wherein the polypeptide has an amino
acid sequence consisting essentially of SEQ ID NOs:2; or, (b) a
complement of the nucleotide sequence, wherein the complement and
the nucleotide sequence consist of the same number of nucleotides
and are 100% complementary.
40. Oil obtained from a seed obtained from a plant made by a method
for producing a transformed plant comprising transforming a plant
cell with an isolated polynucleotide comprising: (a) a nucleotide
sequence encoding a polypeptide having delta-8 desaturase activity,
wherein the polypeptide has an amino acid sequence consisting
essentially of SEQ ID NOs:2; or, (b) a complement of the nucleotide
sequence, wherein the complement and the nucleotide sequence
consist of the same number of nucleotides and are 100%
complementary and (c) regenerating a plant from the transformed
plant cell.
41. The oil of claim 39, wherein the recombinant construct is
operably linked to at least one regulatory sequence.
42. The oil of claim 39, wherein the plant is an oilseed plant.
43. Oil obtained from a seed obtained from a plant made by a method
for producing a transformed plant comprising: a) transforming a
plant cell with an isolated polynucleotide comprising with: (i) a
nucleotide sequence encoding a polypeptide having delta-8
desaturase activity, wherein the polypeptide has an amino acid
sequence consisting essentially of SEQ ID NOs:2; or, (ii) a
complement of the nucleotide sequence, wherein the complement and
the nucleotide sequence consist of the same number of nucleotides
and are 100% complementary, and b) regenerating a plant from the
transformed plant cell.
44. The oil of claim 43, wherein the plant is a soybean plant.
45. Oil obtained from a seed obtained from an oilseed plant
comprising a recombinant construct comprising: a) a first
recombinant DNA construct comprising an isolated polynucleotide
encoding a delta-8 desaturase 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, a
delta-5, a delta-6, a delta-9, a delta-12, a delta-15, and a
delta-17 desaturase, a delta-9 elongase, a C18 to C22 elongase and
a C20 to C24 elongase.
46. Oil obtained by a method for making long chain fatty acids in a
plant cell comprising: a) transforming a cell with a recombinant
construct comprising an isolated polynucleotide comprising: (i) a
nucleotide sequence encoding a polypeptide having delta-8
desaturase activity, wherein the polypeptide has an amino acid
sequence consisting essentially of SEQ ID NOs:2; or, (ii) a
complement of the nucleotide sequence, wherein the complement and
the nucleotide sequence consist of the same number of nucleotides
and are 100% complementary, and b) selecting those transformed
cells that make long chain polyunsaturated fatty acids.
47. Oil obtained by a method for producing at least one
polyunsaturated fatty acid in a soybean cell comprising: a)
transforming a soybean cell with a recombinant DNA construct
comprising an isolated polynucleotide encoding a delta-8 desaturase
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 delta-4, a delta-5, a delta-6, a delta-9, a delta-12, a
delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to
C22 elongase and a C20 to C24 elongase; b) regenerating a soybean
plant from the transformed cell of step a); and c) selecting those
seeds obtained from the plants of step b), wherein the seeds have
an altered level of polyunsaturated fatty acids when compared to
the level in seeds obtained from a nontransformed soybean plant.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/583,041, filed Jun. 25, 2004, and U.S.
Provisional Application No. 60/624812, filed Nov. 4, 2004, the
entire contents of which are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention pertains to a polynucleotide sequence
encoding a delta-8 desaturase and the use of this desaturase in
making long chain polyunsaturated fatty acids (PUFAs).
BACKGROUND OF THE INVENTION
[0003] Lipids/fatty acids 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.
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 AA
different properties than those found in EPA. The eicosanoids
produced from AA have strong inflammatory and platelet aggregating
properties, whereas those derived from EPA have anti-inflammatory
and anti-platelet aggregating properties. AA 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. AA 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 ARA 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 LA and ALA to yield GLA and stearidonic acid (STA;
18:4, delta-6,9,12,15), respectively. Further fatty acid elongation
and desaturation steps give rise to AA and 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, linoleic acid (LA;
18:2, delta-9,12) and alpha-linolenic acid (ALA; 18:3,
delta-9,12,15); LA and ALA must be obtained from the diet. However,
biosynthesis of long chain PUFAs is somewhat limited and is
regulated by dietary and hormonal changes.
[0011] 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
irregulare, 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.
[0012] WO 98/55625 (published Dec. 19, 1998) describes the
production of PUFAs by expression of polyketide-like synthesis
genes in plants.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] Spychalla et al. (Proc. Natl. Acad. Sci. USA, 94:1142-1147
(1997)) describes the isolation and characterization of a cDNA from
C. 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.
[0017] An alternate pathway for the biosynthesis of AA and EPA
operates in some organisms (i.e., the delta-9 elongase/delta-8
desaturase pathway). Here 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.
[0018] 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.
[0019] Wallis et al. (Archives of Biochemistry and Biophysics,
365(2):307-316 (May 15, 1999)) describes the cloning of a gene that
appears to encode a delta-8 desaturase in Euglena gracilis. This
appears to be the same sequence disclosed in WO 2000/34439.
[0020] Qi et al. (Nature Biotechnology, 22(6):739-45 (2004))
describes the production of long chain PUFAs using, among other
things, a delta-8 desaturase from E. gracilis; however, the
complete sequence of the delta-8 desaturase is not provided.
[0021] WO 2004/057001 (published Jul. 8, 2004) discloses amino acid
and nucleic acid sequences for a delta-8 desaturase enzyme from E.
gracilis.
[0022] 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.
[0023] 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 fermentive 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. 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.
[0024] 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.
[0025] 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.
SUMMARY OF THE INVENTION
[0026] This invention concerns an isolated polynucleotide
comprising:
[0027] (a) a nucleotide sequence encoding a polypeptide having
delta-8 desaturase activity, wherein the polypeptide has an amino
acid sequence consisting essentially of SEQ ID NOs: 2 or 113;
or,
[0028] (b) a complement of the nucleotide sequence, wherein the
complement and the nucleotide sequence consist of the same number
of nucleotides and are 100% complementary.
[0029] In a second embodiment, this invention concerns a
recombinant construct comprising SEQ ID NOs:1 or 112 operably
linked to at least one regulatory sequence.
[0030] In a third embodiment, this invention concerns a cell
comprising the recombinant construct of the invention.
[0031] In a fourth embodiment, this invention concerns a method for
transforming cells, plants and yeast with the recombinant construct
of the invention.
[0032] In a fifth embodiment, this invention concerns seeds
obtained from such plants and oil obtained from such seeds.
[0033] In a sixth embodiment, this invention concerns a method for
making polyunsaturated fatty acids in a cell.
[0034] In a seventh embodiment, this invention concerns an oilseed
plant comprising a first recombinant DNA construct comprising an
isolated polynucleotide encoding a delta-8 desaturase 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 delta-4, a delta-5, delta-6, a delta-9, a delta-12, a
delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to
C22 elongase and a C20 to C24 elongase.
[0035] In still another aspect, this invention concerns a method
for producing at least one polyunsaturated fatty acid in a soybean
cell comprising:
[0036] (a) transforming a soybean cell with a first recombinant DNA
construct comprising an isolated polynucleotide encoding a delta-8
desaturase 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 delta-4, a delta-5, delta-6, a delta-9, a
delta-12, a delta-15, and a delta-17 desaturase, a delta-9
elongase, a C18 to C22 elongase and a C20 to C24 elongase.
[0037] (b) regenerating a soybean plant from the transformed cell
of step (a); and
[0038] (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.
[0039] In an eighth emodiment this invention concerns an oilseed
plant selected from the group consisting of soybean, Brassica
species, sunflower, maize, cotton, flax, and safflower.
[0040] In a ninth embodiment this invention concerns oilseed plants
wherein the polyunsaturated fatty acid is selected from the group
consisting of AA, EDA, EPA, ETA, EtrA, DGLA, DPA, DHA,
[0041] Further embodiments include seeds and oil obtained from the
plants transformed with the isolated polynucleotides of the instant
invention.
[0042] Additional embodiments concern food, feed and ingredients
derived from the processing of the seeds obtained from the plants
transformed with the isolated polynucleotides of the instant
invention.
BIOLOGICAL DEPOSITS
[0043] 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-US-00001 Plasmid Accession Number Date of Deposit pKR681 ATCC
PTA-6046 Jun. 4.sup.th, 2004 pKR685 ATCC PTA-6047 Jun. 4.sup.th,
2004 pY89-5 ATCC PTA-6048 Jun. 4.sup.th, 2004 pKR274 ATCC PTA-4988
Jan. 30.sup.th, 2003 PKR669 Jun. 13, 2005 PKR786 Jun. 13, 2005
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0044] 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.
[0045] The sequence descriptions summarize the Sequences Listing
attached hereto. 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).
[0046] FIG. 1 shows a chromatogram of the lipid profile of an
Euglena gracilis cell extract as described in Example 10.
[0047] FIG. 2 shows an alignment of the claimed delta-8 desaturase
polypeptide sequence from Euglena gracilis (SEQ ID NO:2), a version
of a delta-8 desaturase with reduced activity (SEQ ID NO:4) and
published non-functional versions of delta-8 desaturase sequences
set forth in gi:5639724 (GenBank Accession No. AAD45877 and SEQ ID
NO:6) and in WO 00/34439 or Wallis et al. (Archives of Biochem.
Biophys, 365:307-316 (1999)) (SEQ ID NO:7). The method of alignment
used corresponds to the "Clustal V method of alignment".
[0048] FIG. 3 provides plasmid maps for the following: (A) yeast
expression vector pY89-5 as described in Example 5; and, (B)
soybean expression vector pKR681 as described in Example 6.
[0049] FIG. 4 provides plasmid maps for the following: (A) soybean
expression vector pKR685 as described in Example 8; and, (B)
expression vector pKR274 as described in Example 9.
[0050] FIG. 5 provides plasmid maps for the following: (A) yeast
expression vector pDMW240 as described in Example 1; (B) yeast
expression vector pDMW255 as described in Example 1; (C) yeast
expression vector pDMW261 as described in Example 1; and, (D)
vector pKUNFmKF2 as described in Example 14.
[0051] FIG. 6 provides plasmid maps for the following: (A) yeast
expression vector pDMW277 as described in Example 14; (B) vector
pZF5T-PPC as described in Example 14; (C) yeast expression vector
pDMW287 as described in Example 14; and, (D) yeast expression
vector pDMW287F as described in Example 14.
[0052] FIG. 7 provides plasmid maps for the following: (A) vector
pZUF17 as described in Example 15; (B) yeast expression vector
pDMW237 as described in Example 15; (C) yeast expression vector
pKUNT2 as described in Example 16; and, (D) yeast expression vector
pDMW297 as described in Example 16.
[0053] FIG. 8 provides plasmid maps for the following: (A) soybean
expression vector pKR682 as described in Example 17; (B) soybean
expression vector pKR786 as described in Example 18; and, (C)
soybean expression vector pKR669 as described in Example 19.
[0054] FIG. 9 is a representative PUFA biosynthetic pathway.
[0055] FIG. 10 shows a chromatogram of the lipid profile of a
soybean embryo extract as described in Example 22.
[0056] SEQ ID NO:1 represents the 1271 bp of the Euglena gracilis
sequence containing the ORF (nucleotides 4-1269 (Stop)) of the
delta-8 desaturase gene.
[0057] SEQ ID NO:2 is the amino acid sequence encoded by
nucleotides 4-1269 of SEQ ID NO:1.
[0058] SEQ ID NO:3 represents the 1271 bp of the Euglena gracilis
sequence containing the ORF (nucleotides 4-1269 (Stop)) of the
delta-8 desaturase gene containing a guanine for adenine
substitution at position 835, as compared to the sequence of SEQ ID
NO:1.
[0059] SEQ ID NO:4 is the deduced amino acid sequence encoded by
nucleotides 4-1269 of SEQ ID NO:3, which contains an alanine for
threonine substitution at position 278, when compared to the
polypeptide sequence of SEQ ID NO:2.
[0060] SEQ ID NO:5 represents 1275 bp of the Euglena gracilis
sequence set forth in gi:5639724 (GenBank Accession No. AAD45877),
containing the ORF (nucleotides 14-1273 (Stop)) of a non-functional
version of the delta-8 desaturase gene.
[0061] SEQ ID NO:6 is the deduced amino acid sequence encoded by
nucleotides of SEQ ID NO:5 and set forth in gi:5639724.
[0062] SEQ ID NO:7 is the amino acid sequence of a non-functional
version of the delta-8 desaturase disclosed in Wallis et al.
(Archives of Biochem. Biophys., 365:307-316 (1999) and WO
00/34439).
[0063] SEQ ID NO:8 is the forward primer used for amplification of
the delta-8 desaturase from Euglena gracilis in Example 3.
[0064] SEQ ID NO:9 is the reverse primer used for amplification of
the delta-8 desaturase from Euglena gracilis in Example 3.
[0065] SEQ ID NO:10 is the forward primer used for sequencing a
delta-8 desaturase clone as described in Example 3.
[0066] SEQ ID NO:11 is the reverse primer used for sequencing a
delta-8 desaturase clone as described in Example 3.
[0067] SEQ ID NO:12 is the forward primer used for sequencing a
delta-8 desaturase clone as described in Example 3.
[0068] SEQ ID NO:13 is the reverse primer used for sequencing a
delta-8 desaturase clone as described in Example 3.
[0069] SEQ ID NO:14 is the multiple restriction enzyme site
sequence introduced in front of the beta-conglycinin promoter as
described in Example 6.
[0070] SEQ ID NO:15 is the forward primer used for amplification of
the elongase.
[0071] SEQ ID NO:16 is the reverse primer used for amplification of
the elongase.
[0072] SEQ ID NO:17 is the multiple restriction enzyme site
sequence introduced upstream of the Kti promoter as described in
Example 6.
[0073] SEQ ID NO:18 sets forth the sequence of the soy albumin
transcription terminator with restriction enzyme sites as described
in Example 6.
[0074] SEQ ID NO:19 is the primer oSalb-12 used for amplification
of the albumin transcription terminator.
[0075] SEQ ID NO:20 is primer oSalb-13 used for amplification of
the albumin transcription terminator.
[0076] SEQ ID NO:21 is primer GSP1 used for the amplification of
the soybean annexin gene.
[0077] SEQ ID NO:22 is primer GSP2 used for the amplification of
the soybean annexin gene.
[0078] SEQ ID NO:23 is primer GSP3 used for the amplification of
soybean BD30.
[0079] SEQ ID NO:24 is primer GSP4 used for the amplification of
soybean BD30.
[0080] SEQ ID NO:25 sets forth the soybean BD30 promoter
sequence.
[0081] SEQ ID NO:26 sets forth the soybean Glycinin Gy1 promoter
sequence.
[0082] SEQ ID NO:27 is the forward primer used for amplification of
the soybean Glycinin Gy1 promoter sequence.
[0083] SEQ ID NO:28 is the reverse primer used for amplification of
the soybean Glycinin Gy1 promoter sequence.
[0084] SEQ ID NO:29 sets forth the soybean annexin promoter
sequence.
[0085] SEQ ID NO:30 is the forward primer used for amplification of
the soybean annexin promoter sequence.
[0086] SEQ ID NO:31 is the reverse primer used for amplification of
the soybean annexin promoter sequence.
[0087] SEQ ID NO:32 is the forward primer used for amplification of
the soybean BD30 promoter sequence.
[0088] SEQ ID NO:33 is the reverse primer used for amplification of
the soybean BD30 promoter sequence.
[0089] SEQ ID NO:34 is primer oKTi5 used for amplification of the
Kti/NotI/Kti 3' cassette.
[0090] SEQ ID NO:35 is primer oKTi6 used for amplification of the
Kti/NotI/Kti 3' cassette.
[0091] SEQ ID NO:36 is primer oSBD30-1 used for amplification of
the soybean BD30 3' transcription terminator.
[0092] SEQ ID NO:37 is primer oSBD30-2 used for amplification of
the soybean BD30 3' transcription terminator.
[0093] SEQ ID NO:38 is primer oCGR5-1 used for amplification of the
M. alpina delta-6 desaturase.
[0094] SEQ ID NO:39 is primer oCGR5-2 used for amplification of the
M. alpina delta-6 desaturase.
[0095] SEQ ID NO:40 is primer oSGly-1 used for amplification of the
glycinin Gy1 promoter.
[0096] SEQ ID NO:41 is primer oSGly-2 used for amplification of the
glycinin Gy1 promoter.
[0097] SEQ ID NO:42 is primer LegPro5' used for amplification of
the legA2 promoter sequence.
[0098] SEQ ID NO:43 is primer LegPro3' used for amplification of
the legA2 promoter sequence.
[0099] SEQ ID NO:44 is primer LegTerm5' used for amplification of
the leg2A transcription terminator.
[0100] SEQ ID NO:45 is primer LegTerm3' used for amplification of
the leg2A transcription terminator.
[0101] SEQ ID NO:46 is primer CGR4forward used for the
amplification of the M. alpina desaturase.
[0102] SEQ ID NO:47 is primer CGR4reverse used for the
amplification of the M. alpina desaturase.
[0103] SEQ ID NO:48 is the Euglena gracilis sequence, set forth in
nucleotides 14-1275 of SEQ ID NO:5, optimized for codon usage in
Yarrowia lipolytica.
[0104] SEQ ID NOs:49-74 correspond to primers D8-1A, D8-1A, D8-1B,
D8-2A, D8-2B, D8-3A, D8-3B, D8-4A, D8-4B, D8-5A, D8-5B, D8-6A,
D8-6B, D8-7A, D8-7B, D8-8A, D8-8B, D8-9A, D8-9B, D8-10A, D8-10B,
D8-11A, D8-11B, D8-12A, D8-12B, D8-13A and D8-13B, respectively,
used for amplification as described in Example 1.
[0105] SEQ ID NOs:75-82 correspond to primers D8-1F, D8-3R, D8-4F,
D8-6R, D8-7F, D8-9R, D8-10F and D8-13R, respectively, used for
amplification as described in Example 1.
[0106] SEQ ID NO:83 is the 309 bp Nco/BgIII fragment described in
Example 1.
[0107] SEQ ID NO:84 is the 321 bp BgIII/XhoI fragment described in
Example 1.
[0108] SEQ ID NO:85 is the 264 bp XhoI/SacI fragment described in
Example 1.
[0109] SEQ ID NO:86 is the 369 bp Sac1/Not1 fragment described in
Example 1.
[0110] SEQ ID NO:87 is primer ODMW390 used for amplification as
described in Example 1.
[0111] SEQ ID NO:88 is primer ODMW391 used for amplification as
described in Example 1.
[0112] SEQ ID NO:89 is the chimeric gene described in Example
1.
[0113] SEQ ID NO:90 is the chimeric gene described in Example
1.
[0114] SEQ ID NO:91 is primer ODMW392 used for amplification as
described in Example 1.
[0115] SEQ ID NO:92 is primer ODMW393 used for amplification as
described in Example 1.
[0116] SEQ ID NO:93 is the synthetic delta-8 desaturase described
in Example 1.
[0117] SEQ ID NO:94 is primer ODMW404 used for amplification as
described in Example 14.
[0118] SEQ ID NO:95 is the Kpn/NotI fragment described in Example
14.
[0119] SEQ ID NOs:96-111 correspond to primers YL521, YL522, YL525,
YL526, YL527, YL528, YL529, YL530, YL531, YL532, YL533, YL534,
YL535, YL536, YL537 and YL538, respectively, used for amplification
as described in Example 14.
[0120] SEQ ID NO:112 is the nucleotide sequence for the synthetic
delta-8 desaturase codon-optimized for expression in Yarrowia
lipolytica.
[0121] SEQ ID NO:113 is the amino acid sequence encoded by
nucleotides 2-1270 of SEQ ID NO:112.
[0122] SEQ ID NO:114 is the DNA sequence (995 bp) of the Yarrowia
lipolytica fructose-bisphosphate aldolase promoter containing a
Yarrowia intron (FBAIN).
[0123] SEQ ID NO:118 is the nucleotide sequence for the synthetic
delta-9 elongase codon-optimized for expression in Yarrowia
lipolytica.
[0124] SEQ ID NO:119 is the DNA sequence of the Isochrysis galbana
delta-9 elongase (792 bp), while SEQ ID NO:120 is the amino acid
sequence of the Isochrysis galbana delta-9 elongase (263 AA).
[0125] SEQ ID NOs:121-136 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 and IL3-8B, respectively,
used for amplification as described in Example 15.
[0126] SEQ ID NOs:137-140 correspond to primers IL3-1F, IL3-4R,
IL3-5F and IL3-8R, respectively, used for amplification as
described in Example 15.
[0127] SEQ ID NO:141 is the 417 bp NcoI/PstI fragment described in
Example 15.
[0128] SEQ ID NO:142 is the 377 bp PstI/Not1 fragment described in
Example 15.
[0129] SEQ ID NO:146 is the DNA sequence of the Yarrowia lipolytica
delta-12 desaturase (1936 bp), while SEQ ID NO:147 is the amino
acid sequence of the Yarrowia lipolytica delta-12 desaturase (419
AA).
[0130] SEQ ID NO:149 is primer oIGsel1-1 used for amplifying a
delta-9 elongase as described in Example 17.
[0131] SEQ ID NO:150 is primer oIGsel1-2 used for amplifying a
delta-9 elongase as described in Example 17.
[0132] SEQ ID NO:151 is the fragment described in Example 18.
[0133] SEQ ID NOs:115, 116, 117, 143, 144, 145 and 148 are plasmids
as identified in Table 1.
TABLE-US-00002 TABLE 1 Summary of Plasmid SEQ ID Numbers Plasmid
SEQ ID NO Length pY54PC 115 8,502 bp pKUNFmkF2 116 7,145 bp
pZF5T-PPC 117 5,553 bp pZUF17 143 8,165 bp pDMW237 144 7,879 pKUNT2
145 6,457 bp pDMW297 148 10,448 bp
DETAILED DESCRIPTION OF THE INVENTION
[0134] All patents, patent applications, and publications cited
herein are incorporated by reference in their entirety.
[0135] In the context of this disclosure, a number of terms shall
be utilized.
[0136] Definitions
[0137] 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
WO2004/101757.
[0138] 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 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.
[0139] A representative pathway is illustrated in FIG. 9, providing
for the conversion of stearic acid through various intermediates to
DHA, which demonstrates how both .omega.-3 and .omega.-6 fatty
acids may be produced from a common source.
[0140] 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 Linoleic
LA cis-9,12-octadecadienoic 18:2 .omega.-6 .gamma.-Linoleic GLA
cis-6,9,12- 18:3 .omega.-6 octadecatrienoic Eicosadienoic EDA
cis-11,14-eicosadienoic 20:2 .omega.-6 Dihomo-.gamma.- DGLA
cis-8,11,14-eicosatrienoic 20:3 .omega.-6 Linoleic 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 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-4,7,10,13,16,19- 22:6 .omega.-3 docosahexaenoic
[0141] 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 GLA, DGLA, ARA, EPA and
DHA.
[0142] The term "fat" refers to a lipid substance that is solid at
25.degree. C. and usually saturated.
[0143] 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.
[0144] The term "PUFA biosynthetic pathway" refers to a metabolic
process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA,
STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in
the literature (e.g., see WO2005/003322). 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 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.
[0145] "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 8.sup.th and 9.sup.th 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 ARA 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 ARA 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).
[0146] 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 2 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., The
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
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.
[0147] 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.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, a delta-9 elongase is able to catalyze the conversion of LA
and ALA to EDA and ETrA, respectively (see WO 2002/077213). 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.16/18 elongase and
a C.sub.18/20 elongase).
[0148] The term "delta-9 elongase/delta-8 desaturase pathway"
refers to a biosynthetic pathway for production of long chain
PUFAs, said pathway minimally comprising a delta-9 elongase and a
delta-8 desaturase and thereby enabling biosynthesis of DGLA and/or
ETA from LA and ALA, respectively. This pathway may be advantageous
in some embodiments, as the biosynthesis of GLA and/or STA is
excluded.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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. One set
of preferred conditions involves a series of washes starting with
6.times.SSC, 0.5% SDS at room temperature for 15 min, then repeated
with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30 min, and then
repeated twice with 0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30
min. A more preferred set of stringent conditions involves the use
of higher temperatures in which the washes are identical to those
above except for the temperature of the final two 30 min washes in
0.2.times.SSC, 0.5% SDS was increased to 60.degree. C. Another
preferred set of highly stringent conditions involves the use of
two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0153] "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 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. 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.
[0154] 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.
[0155] "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.
[0156] "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).
[0157] "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)).
[0158] "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).
[0159] "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.
[0160] 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.
[0161] 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.
[0162] "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".
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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]).
[0167] 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.
[0168] "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.
[0169] "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.
[0170] "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)).
[0171] 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)).
[0172] 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.
[0173] 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 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.
[0174] The present invention concerns an isolated polynucleotide
comprising: [0175] (a) a nucleotide sequence encoding a polypeptide
having delta-8 desaturase activity, wherein the polypeptide has an
amino acid sequence consisting essentially of SEQ ID NOs:2 or 113;
or, [0176] (b) a complement of the nucleotide sequence, wherein the
complement and the nucleotide sequence consist of the same number
of nucleotides and are 100% complementary.
[0177] This delta-8 desaturase 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, ARA, EPA, DPA
and/or DHA (FIG. 9). One skilled in the art will recognize the
appropriate combinations of the delta-8 desaturase of the invention
herein in conjunction with 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-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, 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). In another embodiment, this invention concerns a
recombinant construct comprising the polynucleotide of the
invention operably linked to at least one regulatory sequence.
[0178] Plant Expression Systems, Cassettes and Vectors
[0179] 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.
[0180] 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.
[0181] 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)).
[0182] 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.
[0183] 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.
[0184] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory:
Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); by
Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with
Gene Fusions, Cold Spring
[0185] Harbor Laboratory Cold Spring Harbor, N.Y. (1984); and by
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
published by Greene Publishing Assoc. and Wiley-Interscience
(1987).
[0186] Plant Transformation
[0187] 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.
[0188] The recombinant constructs of the invention may be
introduced into one plant cell; or, alternatively, each construct
may be introduced into separate plant cells.
[0189] Expression in a plant cell may be accomplished in a
transient or stable fashion as is described above.
[0190] 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.
[0191] Plant parts include differentiated and undifferentiated
tissues including, but not limited to: 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.
[0192] 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.
[0193] 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 4.
[0194] 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.
[0195] Methods for transforming dicots (primarily by use of
Agrobacterium tumefaciens) and obtaining transgenic plants have
been published, among others, for: cotton (U.S. Pat. No. 5,004,863;
U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834; U.S.
Pat. No. 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)).
[0196] 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.
[0197] 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: NY (1989); Maliga et al.,
Methods in Plant Molecular Biology, Cold Spring Harbor: NY (1995);
Birren et al., Genome Analysis: Detecting Genes, Vol. 1, Cold
Spring Harbor: NY (1998); Birren et al., Genome Analysis: Analyzing
DNA, Vol. 2, Cold Spring Harbor: NY (1998); Plant Molecular
Biology: A Laboratory Manual, eds. Clark, Springer: NY (1997).
[0198] Examples of oilseed plants include, but are not limited to,
soybean, Brassica species, sunflower, maize, cotton, flax,
safflower.
[0199] 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.
[0200] In one embodiment this invention concerns an oilseed plant
comprising: a) a first recombinant DNA construct comprising an
isolated polynucleotide encoding a delta-8 desaturase 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, a delta-5, delta-6, a delta-9, a delta-12, a
delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to
C22 elongase and a C20 to C24 elongase.
[0201] Such desaturases are discussed 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, WO 00/40705.
[0202] The choice of combination of cassettes used depends in part
on the PUFA profile and/or desaturase profile of the oilseed plant
cells to be transformed and the LC-PUFA which is to be
expressed.
[0203] In another aspect, this invention concerns a method for
making long chain polyunsaturated fatty acids in a plant cell
comprising: [0204] (a) transforming a cell with the recombinant
construct of the invention; and [0205] (b) selecting those
transformed cells that make long chain polyunsaturated fatty
acids.
[0206] In still another aspect, this invention concerns a method
for producing at least one polyunsaturated fatty acid in a soybean
cell comprising: [0207] (a) transforming a soybean cell with a
first recombinant DNA construct comprising an isolated
polynucleotide encoding a delta-8 desaturase 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 delta-4, a delta-5, delta-6, a delta-9, a delta-12, a
delta-15, and a delta-17 desaturase, a delta-9 elongase, a C18 to
C22 elongase and a C20 to C24 elongase. [0208] (b) regenerating a
soybean plant from the transformed cell of step (a); and [0209] (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.
[0210] Plant Seed Oils: Isolation and Hydrogenation
[0211] 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 the
Table below.
TABLE-US-00004 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
[0212] In general, 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 the diagram
below.
TABLE-US-00005 Impurities Removed/ Process Byproducts Obtained
##STR00001## Meal ##STR00002## Lecithin ##STR00003## Gums, Free
Fatty Acids, Pigments ##STR00004## Soap ##STR00005## Color, Soap,
Metal ##STR00006## Stearine ##STR00007## FFA, Tocopherols, Sterols,
Volatiles
[0213] 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).
[0214] 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.
[0215] 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.
[0216] 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).
[0217] 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.
[0218] 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.
[0219] Examples of food products or food analogs into which altered
seed oils or altered seeds of the invention may be incorporated
include a meat product such as a processed meat product, a cereal
food product, a snack food product, a baked goods product, a fried
food product, a health food product, an infant formula, a beverage,
a nutritional supplement, a dairy product, a pet food product,
animal feed or an aquaculture food product. 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.
[0220] There can be mentioned meat analogs, cheese analogs, milk
analogs and the like.
[0221] Meat analogs made from soybeans contain soy protein or tofu
and other ingredients mixed together to simulate various kinds of
meats. These meat alternatives are sold as frozen, canned or dried
foods. Usually, they can be used the same way as the foods they
replace. Meat alternatives made from soybeans are excellent sources
of protein, iron and B vitamins. Examples of meat analogs include,
but are not limited to, ham analogs, sausage analogs, bacon
analogs, and the like.
[0222] Food analogs can be classified as imitiation 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.
[0223] Milk analogs or nondairy food products include, but are not
limited to, imitation milk, nondairy frozen desserts such as those
made from soybeans and/or soy protein products.
[0224] 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
processes 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.
[0225] 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.
[0226] 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, 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.
[0227] A snack food product comprises any of the above or below
described food products.
[0228] A fried food product comprises any of the above or below
described food products that has been fried.
[0229] A health food product is any food product that imparts a
health benefit. Many oilseed-derived food products may be
considered as health foods.
[0230] The beverage can be in a liquid or in a dry powdered
form.
[0231] For example, there can be mentioned non-carbonated drinks;
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).
[0232] Infant formulas are liquids or reconstituted powders fed to
infants and young children. They serve as substitutes for human
milk. Infant formulas have a special role to play in the diets of
infants because they are often the only source of nutrients for
infants. Although breast-feeding is still the best nourishment for
infants, infant formula is a close enough second that babies not
only survive but thrive. Infant formula is becoming more and more
increasingly close to breast milk.
[0233] 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 yoghurt or sour milk, cream, butter,
condensed milk, dehydrated milk, coffee whitener, coffee creamer,
ice cream, cheese, etc.
[0234] A pet food product is a product intended to be fed to a pet
such as a dog, cat, bird, reptile, fish, rodent and the like. 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, including but not limited to alfalfa, timothy, oat or
brome grass, vegetables and the like.
[0235] Animal feed is a product intended to be fed to animals such
as turkeys, chickens, cattle and swine and the like. 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.
[0236] Aqualculture feed is a product intended to be used in
aquafarming which concerns the propagation, cultivation or farming
of aquatic organisms, animals and/or plants in fresh or marine
waters.
[0237] Microbial Biosynthesis of Fatty Acids
[0238] The process of de novo synthesis of palmitate (16:0) in
oleaginous microorganisms is described in WO 2004/101757. This
fatty acid 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 to form stearic acid (18:0), which can be converted to
its unsaturated derivative by a delta-9 desaturase to thereby yield
oleic (18:1) acid.
[0239] 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.
[0240] Genes Involved in Omega Fatty Acid Production
[0241] 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, AB020033, AAL13300,
AF417244, AF161219, X86736, AF240777, AB007640, AB075526, AP002063
(delta-12 desaturases); NP.sub.--441622, BAA18302, BAA02924,
AAL36934 (delta-15 desaturases); AF338466, AF438199, E11368,
E11367, D83185, U90417, AF085500, AY504633, NM.sub.--069854,
AF230693 (delta-9 desaturases); AF390174 (delta-9 elongase); and
AX464731, NM.sub.--119617, 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).
[0242] 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., 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 and WO 03/099216
(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.
2003/0196217 A1 (delta-17 desaturase); and, WO 00/12720, WO
2002/077213 and U.S. 2002/0139974A1 (elongases)].
[0243] 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, ARA, 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
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.
[0244] 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 K.sub.M 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.
[0245] 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.
[0246] 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. In order to express genes
encoding the delta-9 elongase/delta-8 desaturase pathway for the
biosynthesis of ARA and EPA in these organisms, it was therefore
necessary to: (1) identify a suitable desaturase that functioned
relatively efficiently in oleaginous yeast based on
substrate-feeding trials; and, (2) subject the desaturase gene to
codon-optimization techniques (infra) to further enhance the
expression of the heterologous enzyme in the alternate oleaginous
yeast host, to thereby enable maximal production of omega-3 and/or
omega-6 fatty acids.
[0247] Optimization of Omega Fatty Acid Genes for Expression in
Particular Organisms
[0248] Although the particular source of a PUFA desaturase or
elongase is not critical in the invention herein, 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 e.g.,
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.
[0249] Codon Optimization: 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.
[0250] 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.
[0251] In the present invention, it was desirable to modify a
portion of the codons encoding the polypeptide having delta-8
desaturase activity, to enhance the expression of the gene in the
oleaginous yeast Yarrowia lipolytica. The nucleic acid sequence of
the native gene (e.g., the Euglena gracilis delta-8 desaturase
defined herein as Eg5) was modified to employ host-preferred
codons. This wildtype desaturase has 421 amino acids (SEQ ID NO:2);
in the codon-optimized gene created herein (SEQ ID NO:112), 207 bp
of the 1263 bp coding region (corresponding to 192 codons) were
codon-optimized and the translation initiation site was modified.
The skilled artisan will appreciate that this optimization method
will be equally applicable to other genes in the omega-3/omega-6
fatty acids biosynthetic pathway (see for example, WO 2004/101753,
herein incorporated entirely by reference). Furthermore, modulation
of the E. gracilis delta-8 desaturase is only exemplary; numerous
other heterologous delta-8 desaturases from variable sources could
be codon-optimized to improve their expression in an oleaginous
yeast host. The present invention comprises the complete sequences
of the synthetic codon-optimized gene as reported in the
accompanying Sequence Listing, the complement of those complete
sequences, and substantial portions of those sequences.
[0252] Gene Mutation: 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 (Feb. 15, 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.
[0253] Microbial Production of Omega-3 and/or Omega-6 Fatty
Acids
[0254] 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.
[0255] 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 ARA, EPA and/or DHA via the delta-9
elongase/delta-8 desaturase pathway, thereby avoiding synthesis of
GLA and/or STA).
[0256] Microbial Expression Systems, Cassettes and Vectors
[0257] 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.
[0258] 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.
[0259] 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 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 ARA 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-8 desaturase of the invention permits the direct synthesis of
DGLA and ETA (when provided EDA and ETrA, respectively, as
substrate). Thus for example, the present invention is drawn to a
method of producing either DGLA or ETA, respectively, comprising:
[0260] a) providing an oleaginous yeast comprising: (i) a gene
encoding a delta-8 desaturase polypeptide as set forth in SEQ ID
NO:112; and [0261] (ii) a source of desaturase substrate consisting
of either EDA or ETrA, respectively; and, [0262] b) growing the
yeast of step (a) in the presence of a suitable fermentable carbon
source wherein the gene encoding a delta-8 desaturase polypeptide
is expressed and EDA is converted to DGLA or ETrA is converted to
ETA, respectively; and, [0263] c) optionally recovering the DGLA or
ETA, respectively, of step (b).
[0264] 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 elongase, delta-5 desaturase, delta-17
desaturase and delta-4 desaturase activity would enable a host cell
that naturally produces GLA, to instead produce DHA (such that GLA
is converted to DGLA by an elongase; DGLA may then be converted to
ARA by a delta-5 desaturase; ARA is then converted to EPA by a
delta-17 desaturase, which may in turn be converted to DPA by an
elongase; and DPA would be converted to DHA by a delta-4
desaturase). In a related manner, expression of the delta-8
desaturase of the invention enables the indirection production of
ARA, 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.
[0265] 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 and a region 3' of the DNA fragment that controls
transcriptional termination. 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.
[0266] 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. 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 O-acyltransferase (see U.S. Patent Application
No. 60/610,060), 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),
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.
[0267] 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.
[0268] 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.
[0269] 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.
[0270] Transformation of Microbial Hosts
[0271] 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.
[0272] In the present invention, the preferred 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.
[0273] 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 Y.
lipolytica, thereby permitting high-level gene expression.
Unfortunately, however, not all strains of Y. lipolytica possess
zeta regions (e.g., the strain identified as ATCC #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. AJ001300), the Pox3 gene locus
(Pox3: GenBank Accession No. XP.sub.--503244; or, Aco3: GenBank
Accession No. AJ001301), the delta-12 desaturase gene locus (SEQ ID
NO:23), the Lip1 gene locus (GenBank Accession No. Z50020) and/or
the Lip2 gene locus (GenBank Accession No. AJ012632).
[0274] 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.
[0275] 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.
[0276] 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. No. 4,880,741 and U.S. Pat.
No. 5,071,764 and Chen, D. C. et al. (Appl Microbiol Biotechnol.
48(2):232-235 (1997)).
[0277] 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.
[0278] The transformed host cell can be identified by various
selection techniques, as described in WO2004/101757. 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.
[0279] 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.
[0280] Metabolic Engineering of Omega-3 and/or Omega-6 Fatty Acid
Biosynthesis in Microbes
[0281] 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.
[0282] 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.
[0283] 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-15
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.
[0284] 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).
[0285] 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.
[0286] Techniques to Up-Regulate Desirable Biosynthetic
Pathways
[0287] 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.
[0288] Techniques to Down-Regulate Undesirable Biosynthetic
Pathways
[0289] 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. 171:4617-4622 (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)).
[0290] 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.
[0291] 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).
[0292] 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.
[0293] Preferred Microbial Hosts for Recombinant Production of
Omega-3 and/or Omega-6 Fatty Acids
[0294] 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.
[0295] 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).
[0296] Most preferred is the oleaginous yeast Yarrowia lipolytica;
and, in a further embodiment, most preferred are the Y. lipolytica
strains designated as ATCC #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)).
[0297] Historically, various strains of Y. lipolytica have been
used for the manufacture and production of: isocitrate lyase
(DD259637); lipases (SU1454852, WO2001083773, DD279267);
polyhydroxyalkanoates (WO2001088144); citric acid (RU2096461,
RU2090611, DD285372, DD285370, DD275480, DD227448, PL160027);
erythritol (EP770683); 2-oxoglutaric acid (DD267999);
.gamma.-decalactone (U.S. Pat. No. 6,451,565, FR2734843);
.gamma.-dodecalatone (EP578388); and pyruvic acid (JP09252790).
[0298] Microbial Fermentation Processes for PUFA Production
[0299] 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.)).
[0300] 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.
[0301] Nitrogen may be supplied from an inorganic (e.g.,
(NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or
glutamate). 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)).
[0302] 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.
[0303] 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.
[0304] Purification of Microbial PUFAs
[0305] 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)).
[0306] 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.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0307] The ultimate goal of the work described herein was the
identification of a delta-8 desaturase suitable to enable
expression of the delta-9 elongase/delta-8 desaturase pathway in
plants and oleaginous yeast. Thus, initial work performed herein
attempted to codon-optimize the delta-8 desaturase of Euglena
gracilis (GenBank Accession No. AAD45877; WO 00/34439) for
expression in Yarrowia lipolytica. Despite synthesis of three
different codon-optimized genes (i.e., "D8S-1", "D8S-2" and
"D8S-3"), none of the genes were capable of desaturating EDA to
DGLA (Example 1). On the basis of these results, it was
hypothesized that the previously published delta-8 desaturase
sequences were incorrect.
[0308] Isolation of the delta-8 desaturase from Euglena gracilis
directly, following mRNA isolation, cDNA synthesis and PCR
(Examples 2 and 3) was attempted as described below. This resulted
in two similar sequences, identified herein as Eg5 (SEQ ID NOs:1
and 2) and Eg12 (SEQ ID NOs:3 and 4), both of which possessed
significant differences when compared to the previously published
delta-8 desaturase sequences (Example 4). Eg5 and Eg12 were each
cloned into a Saccharomyces cerevisiae yeast expression vector
(Example 5) for functional analysis via substrate feeding trials
(Example 11). This demonstrated that both Eg5 and Eg12 were able to
desaturase EDA and ETrA to produce DGLA and ETA, respectively; Eg5
had significantly greater activity than Eg12.
[0309] Based on the confirmed delta-8 desaturase activity of Eg5
(SEQ ID NO:1 and 2), the sequence of Eg5 was codon-optimized for
expression in Yarrowia lipolytica (Example 14), thereby resulting
in the synthesis of a synthetic, functional codon-optimized delta-8
desaturase designated as "D8SF" (SEQ ID NOs:112 and 113).
Co-expression of the codon-optimized delta-8 desaturase of the
invention in conjunction with a codon-optimized delta-9 elongase
(derived from Isochrysis galbana (GenBank Accession No. 390174)) in
Y. lipolytica enabled synthesis of 6.4% DGLA, with no co-synthesis
of GLA (Example 16).
[0310] A number of expression constructs were then created to
enable synthesis of various PUFAs in soybean, using the confirmed
delta-8 desaturase sequence of Eg5, the Yarrowia lipolytica
codon-optimized Isochrysis galbana delta-9 elongase or the
Mortierella alpina elongase, the Mortierella alpina delta-5
desaturase, the Fusarium delta-15 desaturase, and the Saprolegnia
diclina delta-17 desaturase and combinations thereof (Examples 17
through 22). Expression of these constructs resulted in production
of up to about 29.9% DGLA and up to about 29.4% EPA (Examples 21
and 22 respectively).
EXAMPLES
[0311] 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.
[0312] 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), "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).
Transformation and Cultivation of Yarrowia lilpolytica
[0313] Yarrowia lipolytica strains ATCC #20362, #76982 and #90812
were purchased from the American Type Culture Collection
(Rockville, Md.). Y. lipolytica strains were usually grown at
28.degree. C. on YPD agar (1% yeast extract, 2% bactopeptone, 2%
glucose, 2% agar).
[0314] 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 hr. 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 hr 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.
[0315] 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).
[0316] 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.
Fatty Acid Analysis of Yarrowia lipolytica
[0317] 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.
[0318] 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
Synthesis and Expression of a Codon-Optimized Delta-8 Desaturase
Gene in Yarrowia lipolytica
[0319] In order to express the delta-8 desaturase gene of Euglena
gracilis (SEQ ID NOs:5 and 6, GenBank Accession No. AAD45877) in
Yarrowia lipolytica, the codon usage of the delta-8 desaturase gene
was optimized for expression in Y. lipolytica. A codon-optimized
delta-8 desaturase gene (designated "D8S-1", SEQ ID NO:48) was
designed, based on the published sequence of Euglena gracilis (SEQ
ID NO:5), according to the Yarrowia codon usage pattern (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,
200 bp of the 1260 bp coding region were modified (15.9%). None of
the modifications in the codon-optimized gene changed the amino
acid sequence of the encoded protein (SEQ ID NO:6) except the
second amino acid from `K` to `E` to add the NcoI site around the
translation initiation codon.
[0320] In Vitro Synthesis of a Codon-Optimized delta-8 Desaturase
Gene for Yarrowia
[0321] The codon-optimized delta-8 desaturase gene was synthesized
as follows. First, thirteen pairs of oligonucleotides were designed
to extend the entire length of the codon-optimized coding region of
the E. gracilis delta-8 desaturase gene (e.g., D8-1A, D8-1B, D8-2A,
D8-2B, D8-3A, D8-3B, D8-4A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B,
D8-7A, D8-7B, D8-8A, D8-8B, D8-9A, D8-9B, D8-10A, D8-10B, D8-11A,
D8-11B, D8-12A, D8-12B, D8-13A and D8-13B, corresponding to SEQ ID
NOs:49-74). 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 D8-1A, D8-3B, D8-7A,
D8-9B and D8-13B (SEQ ID NOs:49, 54, 60, 65 and 74) also introduced
NcoI, BgIII, Xho1, SacI and Not1 restriction sites, respectively,
for subsequent subcloning.
[0322] Each oligonucleotide (100 ng) was phosphorylated at
37.degree. C. for 1 hr 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,
D8-1A (SEQ ID NO:49) was annealed to D8-1B (SEQ ID NO:50) to
produce the double-stranded product "D8-1AB". Similarly, D8-2A (SEQ
ID NO:51) was annealed to D8-2B (SEQ ID NO:52) to produce the
double-stranded product "D8-2AB", etc.
[0323] Four separate pools of annealed, double-stranded
oligonucleotides were then ligated together, as shown below: (a)
Pool 1: comprised D8-1AB, D8-2AB and D8-3AB; (b) Pool 2: comprised
D8-4AB, D8-5AB and D8-6AB; (c) Pool 3: comprised D8-7AB, D8-8AB,
and D8-9AB; and, (d) Pool 4; comprised D8-10AB, D8-11AB, D8-12AB
and D8-13AB. 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.
[0324] 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 (i.e., D8-1AB, D8-2AB and
D8-3AB) as template, and oligonucleotides D8-1F (SEQ ID NO:75) and
D8-3R (SEQ ID NO:76) as primers, the first portion of the
codon-optimized delta-8 desaturase gene was amplified by PCR. The
PCR amplification was carried out in a 50 .mu.l total volume,
comprising PCR buffer containing 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2 mM
MgSO.sub.4, 0.1% Triton X-100, 100 .mu.g/mL BSA (final
concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10
pmole of each primer and 1 .mu.l of PfuTurbo DNA polymerase
(Stratagene, San Diego, Calif.). Amplification was carried out as
follows: initial denaturation at 95.degree. C. for 3 min, followed
by 35 cycles of the following: 95.degree. C. for 1 min, 56.degree.
C. for 30 sec, 72.degree. C. for 40 sec. A final extension cycle of
72.degree. C. for 10 min was carried out, followed by reaction
termination at 4.degree. C. The 309 bp PCR fragment was subcloned
into the pGEM-T easy vector (Promega) to generate pT8(1-3).
[0325] Using the ligated "Pool 2" mixture (i.e., D8-4AB, D8-5AB and
D8-6AB) as the template, and oligonucleotides D8-4F (SEQ ID NO:77)
and D8-6R (SEQ ID NO:78) as primers, the second portion of the
codon-optimized delta-8 desaturase gene was amplified similarly by
PCR and cloned into pGEM-T-easy vector to generate pT8(4-6). Using
the ligated "Pool 3" mixture (i.e., D8-7AB, D8-8AB and D8-9AB) as
the template and oligonucleotides D8-7F (SEQ ID NO: 79) and D8-9R
(SEQ ID NO:80) as primers, the third portion of the codon-optimized
delta-8 desaturase gene was amplified similarly by PCR and cloned
into pGEM-T-easy vector to generate pT8(7-9). Finally, using the
"Pool 4" ligation mixture (i.e., D8-10AB, D8-11AB, D8-12AB and
D8-13AB) as template, and oligonucleotides D8-10F (SEQ ID NO: 81)
and D8-13R (SEQ ID NO:82) as primers, the fourth portion of the
codon-optimized delta-8 desaturase gene was amplified similarly by
PCR and cloned into pGEM-T-easy vector to generate pT8(10-13).
[0326] E. coli was transformed separately with pT8(1-3), pT8(4-6),
pT8(7-9) and pT8(10-13) and the plasmid DNA was isolated from
ampicillin-resistant transformants. Plasmid DNA was purified and
digested with the appropriate restriction endonucleases to liberate
the 309 bp NcoI/BgIII fragment of pT8(1-3) (SEQ ID NO:83), the 321
bp BgIII/XhoI fragment of pT8(4-6) (SEQ ID NO:84), the 264 bp
XhoI/SacI fragment of pT8(7-9) (SEQ ID NO:85) and the 369 bp
Sac1/Not1 fragment of pT8(10-13) (SEQ ID NO:86). These fragments
were then combined and directionally ligated together with
Nco1/Not1 digested pY54PC (SEQ ID NO:115; WO2004/101757) to
generate pDMW240 (FIG. 5A). This resulted in a synthetic delta-8
desaturase gene ("D8S-1", SEQ ID NO:48) in pDMW240.
[0327] Compared with the published delta-8 desaturase amino acid
sequence (SEQ ID NO:6) of E. gracilis, the second amino acid of
D8S-1 was changed from `K` to `E` in order to add the NcoI site
around the translation initiation codon. Another version of the
synthesized gene, with the exact amino acid sequence as the
published E. gracilis delta-8 desaturase sequence SEQ ID NO:6), was
constructed by in vitro mutagenesis (Stratagene, San Diego, Calif.)
using pDMW240 as a template and oligonucleotides ODMW390 (SEQ ID
NO:87) and ODMW391 (SEQ ID NO:88) as primers. The resulting plasmid
was designated pDMW255 (FIG. 5B). The synthetic delta-8 desaturase
gene in pDMW255 was designated as "D8S-2" and the amino acid
sequence is exactly the same as the sequence depicted in SEQ ID
NO:5.
[0328] Yarrowia lipolytica strainATCC #76982(Leu-) was transformed
with pDMW240 and pDMW255, respectively, as described in the General
Methods. Yeast containing the recombinant constructs pDMW240 and
pDMW255 (i.e., containing D8S-1 and D8S-2 respectively) were grown
in MM supplemented with EDA, 20:2(11,14). Specifically, single
colonies of transformant Y. lipolytica containing either pDMW240 or
pDMW255 were grown in 3 mL MM at 30.degree. C. to an
OD.sub.600.about.1.0. For substrate feeding, 100 .mu.l of cells
were then subcultured in 3 mL MM containing 10 .mu.g of EDA
substrate for about 24 hr at 30.degree. C. 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.
[0329] Neither transformant produced DGLA from EDA and thus D8S-1
and D8S-2 were not functional and could not desaturate EDA. The
chimeric D8S-1::XPR terminator and D8S-2::XPR terminator genes are
shown in SEQ ID NOs:89 and 90, respectively.
[0330] A three amino acid difference between the protein sequence
of the delta 8-desaturase deposited in GenBank (Accession No.
AAD45877) and in WO 00/34439 or Wallis et al. (Archives of Biochem.
Biophys, 365:307-316 (1999)) (SEQ ID NO:7 herein) was found.
Specifically, three amino acids appeared to be missing in GenBank
Accession No. AAD45877. Using pDMW255 as template and ODMW392 (SEQ
ID NO:91) and ODMW393 (SEQ ID NO:92) as primers, 9 bp were added
into the synthetic D8S-2 gene by in vitro mutagenesis (Stratagene,
San Diego, Calif.), thus producing a protein that was identical to
the sequence described in WO 00/34439 and Wallis et al. (supra)
(SEQ ID NO:7). The resulting plasmid was called pDMW261 (FIG. 5C).
The synthetic delta-8 desaturase gene in pDMW261 was designated as
"D8S-3" (SEQ ID NO:93). Following transformation of the pDMW261
construct into Yarrowia, a similar feeding experiment using EDA was
conducted, as described above. No desaturation of EDA to DGLA was
observed with D8S-3.
Example 2
Euglena gracilis Growth Conditions, Lipid Profile and mRNA
Isolation
[0331] 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) and 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
(Catalog #15-3790, Carolina Biological Supply Company, Burlington,
N.C.) was aseptically added to produce the final Eg medium. Euglena
gracilis cultures were grown at 23.degree. C. with a 16 hr light, 8
hr dark cycle for 2 weeks with no agitation.
[0332] 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 (Catalog #24152, Supelco Inc.). 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 (Catalog #U-99-A,
Nu-Chek Prep, Inc.) and the resulting chromatogram is shown in FIG.
1.
[0333] 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 3
cDNA Synthesis and PCR of Euglena gracilis Delta-8 Desaturase
[0334] cDNA was Synthesized from 765 ng of mRNA (Example 2) 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.
[0335] The Euglena gracilis delta-8 desaturase was amplified from
cDNA with oligonucleotide primers Eg5-1 (SEQ ID NO:8) and Eg3-3
(SEQ ID NO:9) using the conditions described below.
[0336] cDNA (1 .mu.L) from the reaction described above was
combined with 50 .mu.mol of Eg5-1, 50 .mu.mol of Eg5-1, 1 .mu.L of
PCR nucleotide mix (10 mM, Promega, Madison, Wis.), 5 .mu.L of
10.times.PCR buffer (Invitrogen), 1.5 .mu.L of MgCl.sub.2 (50 mM,
Invitrogen), 0.5 .mu.L of Taq polymerase (Invitrogen) 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 the DNA band was 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:10), M13-28Rev (SEQ ID NO:11), Eg3-2 (SEQ ID NO:12) and
Eg5-2 (SEQ ID NO:13).
[0337] Thus, two classes of DNA sequences were obtained, Eg5 (SEQ
ID NO:1) and Eg12 (SEQ ID NO:3), that differed in only a few bp.
Translation of Eg5 and Eg12 gave rise to protein sequences that
differed in only one amino acid, SEQ ID NO:2 and 4, respectively.
Thus, the DNA and protein sequences for Eg5 are set forth in SEQ ID
NO:1 and SEQ ID NO:2, respectively; the DNA and protein sequences
for Eg12 are set forth in SEQ ID NO:3 and SEQ ID NO:4,
respectively.
Example 4
Comparison of the Polypeptide Sequences Set Forth in SEQ ID NOs:2
and 4 to Published Euglena gracilis Delta-8 Desaturase
Sequences
[0338] An alignment of the protein sequences set forth in SEQ ID
NO:2 and SEQ ID NO:4 with the protein sequence from GenBank
Accession No. AAD45877 (gi: 5639724) and with the published protein
sequences of Wallis et al. (Archives of Biochem. Biophys.,
365:307-316 (1999); WO 00/34439) is shown in FIG. 2.
[0339] Amino acids conserved among all 4 sequences are indicated
with an asterisk (*). Dashes are used by the program to maximize
alignment of the sequences. The putative cytochrome b.sub.5 domain
is underlined. A putative His box is shown in bold.
[0340] Clearly, there are significant differences between the
sequences of this invention and those described previously.
Specifically, the N-terminus has multiple amino acid changes. As
compared to SEQ ID NO:2, the published amino acid sequences have an
extra serine between L9 and P10 and amino acids from position
T12-T16 are completely different (`TIDGT` to `QLMEQ`). These
changes result from multiple insertions in the DNA sequence of the
published sequence and this causes 3 shifts in frame in this
region. These changes are only 10 amino acids away from the
putative cytochrome b.sub.5 domain (`HPGG`).
[0341] In addition to this, there are seven other single amino acid
changes (S50 to F, S67 to F, W177 to C, L203 to P, S244 to C, T278
to A, S323 to P) with the change at W177 being only 4 amino acids
away from the second putative His box (`HNAHH`). Surprisingly, the
published GenBank protein sequence is missing 3 amino acids (S20,
A21, W22) as compared to that for either SEQ ID NO:2, SEQ ID NO:4
or WO 00/34439. The DNA sequence shown in WO 00/34439 codes for a
protein that is identical to AAD45877 (i.e., missing these 3 amino
acids) and not for the protein sequence described in WO 00/34439.
Interestingly, the protein sequence set forth in SEQ ID NO:4 has a
single amino acid change as compared to SEQ ID NO:2 (T278 to A). In
Table 4 percent identities between the functional delta-8
desaturase protein sequence from Euglena gracilis claimed in this
invention (SEQ ID NO:2) and the published sequences (SEQ ID NOs:6
and 7) are shown.
TABLE-US-00006 TABLE 4 Percent Identity Of The Amino Acid Sequences
Of Delta-8 Desaturase From Euglena gracilis And Homologous
Polypeptides From Euglena gracilis % Identity to % Identity to SEQ
ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 2 95.5 96.2 * "% Identity" is
defined as the percentage of amino acids that are identical between
the two proteins.
[0342] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989))
with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).
Default parameters for pairwise alignments using the Clustal method
were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Example 5
Cloning the Euglena gracilis Delta-8 Desaturase into a Yeast
Expression Vector
[0343] The yeast episomal plasmid (YEp)-type vector pRS425
(Christianson et al., Gene, 110:119-22 (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 II SK+. The S. cerevisiae
strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD)
promoter was cloned between the SaclI and SpeI sites of pRS425 in
the same way as described in Jia et al. (Physiological Genomics,
3:83-92 (2000)) to produce PGPD-425. A NotI site was introduced
into the BamHI site of pGPD-425 thus producing a NotI site flanked
by BamHI sites, thereby resulting in plasmid pY-75. Eg5 (SEQ ID
NO:1) and Eg12 (SEQ ID NO:3) were released from the PGEM.RTM.-T
Easy vectors described in Example 2 by digestion with NotI and
cloned into the NotI site of pY-75 to produce pY89-5 and pY89-12,
respectively. In this way, the delta-8 desaturases (i.e., Eg5 [SEQ
ID NO:1] and Eg12 [SEQ ID NO:3]) were cloned behind a strong
constitutive promoter for expression in S. cerevisiae. A map of
pY89-5 is shown in FIG. 3A.
Example 6
Cloning the Euglena gracilis Delta-8 Desaturase into a Soybean
Expression Vector and Co-Expression with a Mortierella alpina
Elongase
[0344] A starting plasmid pKS123 (WO 02/08269, 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, pKS123 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.
pKS123 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, J. J. 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.
[0345] Vector pKR72 is a derivative of pKS123, wherein the HindIII
fragment containing the .beta.-conglycinin/NotI/phaseolin cassette
has been inverted and a sequence (SEQ ID NO:14) containing SbfI,
FseI and BsiWI restriction enzyme sites was introduced between the
HindIII and BamHI sites in front of the .beta.-conglycinin
promoter.
[0346] The gene for the Mortierella alpina elongase was amplified
from pRPB2 (WO 00/12720) using primers RPB2forward (SEQ ID NO:15)
and RPB2reverse (SEQ ID NO:16) which were designed to introduce
NotI restriction enzyme sites at both ends of the elongase. The
resulting PCR fragment was digested with NotI and cloned into the
NotI site of pKR72 to produce pKR324.
[0347] Vector pKS121 (WO 02/00904) 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 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 downstream of the Kti terminator to
lengthen and strengthen termination of transcription. In pKR457,
the BamHI site upstream of the Kti promoter in pKS121 was removed
and a new sequence (SEQ ID NO:17) added containing a BsiWI, SalI,
SbfI and HindIII site with the BsiWI site being closest the 5' end
of the Kti promoter.
[0348] In addition, the SalI site downstream of the Kti terminator
in pKS121 was removed and a new sequence (SEQ ID NO:18) was added
containing a XbaI (closest to 3' end of Kti terminator), a BamHI
site, the soy albumin transcription terminator sequence, a BsiWI
site and another BamHI site.
[0349] The albumin transcription terminator was previously
amplified from soy genomic DNA using primer oSalb-12 (SEQ ID NO:19;
designed to introduce BamHI, XbaI and BsiWI sites at the 3' end of
the terminator), and primer oSalb-13 (SEQ ID NO:20; designed to
introduce BamHI sites at the 5' end of the terminator). After PCR,
sites at ends were modified by sub-cloning through various
intermediate vectors to finally produce the sequence shown in SEQ
ID NO:5.
[0350] Eg5 (SEQ ID NO:1) was released from the PGEM.RTM.-T Easy by
digestion with NotI and cloned into the NotI site of pKR457 to
produce pKR680. Plasmid pKR680 was then digested with BsiWI and the
fragment containing Eg5 (SEQ ID NO:1) was cloned into the BsiWI
site of pKR324 (WO 2004/071467) to produce pKR681. Thus, the
delta-8 desaturase (Eg5; SEQ ID NO:1) could be co-expressed with
the Mortierella alpina elongase behind strong, seed-specific
promoters. A map of pKR681 is shown in FIG. 3B.
Example 7
Isolation of Soybean Seed-Specific Promoters
[0351] The soybean annexin and BD30 promoters were isolated with
the Universal GenomeWalker system (Clontech) according to its user
manual (PT3042-1). To make soybean GenomeWalker libraries, samples
of soybean genomic DNA were digested with DraI, EcoRV, PvuII and
StuI separately for two hrs. After DNA purification, the digested
genomic DNAs were ligated to the GenomeWalker adaptors AP1 and
AP2.
[0352] Two gene specific primers (i.e., GSP1 [SEQ ID NO:21] and
GSP2 [SEQ ID NO:22]) were designed for the soybean annexin gene
based on the 5' annexin cDNA coding sequences available in an EST
database (E.I. duPont de Nemours and Co., Inc., Wilmington,
Del.).
[0353] The AP1 and the GSP1 primers were used in the 1.sup.st round
PCR using the conditions defined in the GenomeWalker system
protocol. Cycle conditions were 94.degree. C. for 4 min; 94.degree.
C. for 2 sec and 72.degree. C. for 3 min, 7 cycles; 94.degree. C.
for 2 sec and 67.degree. C. for 3 min, 32 cycles; 67.degree. C. for
4 min. The products from the first run PCR were diluted 50-fold.
One microliter of the diluted products were used as templates for
the 2.sup.nd PCR with primers AP2 and GSP2. Cycle conditions were
94.degree. C. for 4 min; 94.degree. C. for 2 sec and 72.degree. C.
for 3 min, 5 cycles; 94.degree. C. for 2 sec and 67.degree. C. for
3 min, 20 cycles; 67.degree. C. for 3 min. A 2.1 kB genomic
fragment was amplified and isolated from the EcoRV-digested
GenomeWalker library. The genomic fragment was digested with BamH I
and Sal I and cloned into Bluescript KS.sup.+ vector for
sequencing. The DNA sequence of this 2012 bp soybean annexin
promoter fragment is set forth in SEQ ID NO:29. Based on this
sequence, two oligonucleotides with either BamH I or NotI sites at
the 5' ends were designed to re-amplify the promoter (i.e., SEQ ID
NOs:30 and 31).
[0354] Two gene specific primers (GSP3 [SEQ ID NO:23] and GSP4 [SEQ
ID NO:24]) were designed to amplify the soybean BD30 promoter based
on the 5' BD30 cDNA coding sequences in GenBank (Accession No.
J05560). The AP1 and the GSP3 primers were used in the 1.sup.st
round PCR using the same conditions defined in the GenomeWalker
system protocol; however, the cycle conditions used for soybean
annexin promoter did not work well for the soybean BD30 promoter. A
modified touchdown PCR protocol was used, wherein cycle conditions
were: 94.degree. C. for 4 min; 94.degree. C. for 2 sec and
74.degree. C. for 3 min, 6 cycles in which annealing temperature
drops 1.degree. C. every cycle; 94.degree. C. for 2 sec and
69.degree. C. for 3 min, 32 cycles; 69.degree. C. for 4 min. The
products from the 1.sup.st run PCR were diluted 50-fold. One
microliter of the diluted products were used as templates for the
2.sup.nd PCR with primers AP2 and GSP4. Cycle conditions were:
94.degree. C. for 4 min; 94.degree. C. for 2 sec and 74.degree. C.
for 3 min, 6 cycles in which annealing temperature drops 1.degree.
C. every cycle; 94.degree. C. for 2 sec and 69.degree. C. for 3
min, 20 cycles; 69.degree. C. for 3 min. A 1.5 kB genomic fragment
was amplified and isolated from the PvuII-digested GenomeWalker
library. The genomic fragment was digested with BamHI and SalI and
cloned into Bluescript KS.sup.+ vector for sequencing. DNA
sequencing determined that this genomic fragment contained a 1408
bp soybean BD30 promoter sequence (SEQ ID NO:25). Based on the
sequence of the cloned soybean BD30 promoter, two oligonucleotides
with either BamHI or Not I sites at the 5' ends were designed to
re-amplify the BD30 promoter (i.e., SEQ ID NOs:32 and 33).
[0355] The re-amplified annexin and BD30 promoter fragments (supra)
were digested with BamHI and NotI, purified and cloned into the
BamHI and NotI sites of plasmid pZBL115 to produce pJS88 and pJS89,
respectively. The pZBL115 plasmid contains the origin of
replication from pBR322, the bacterial HPT hygromycin resistance
gene driven by a T7 promoter and T7 terminator, and a 35S
promoter-HPT-Nos3' gene to serve as a hygromycin resistant plant
selection marker. The M. alpina delta-6 desaturase gene was cloned
into the NotI site of pJS88 and pJS89, in the sense orientation, to
make plant expression cassettes pJS92 and pJS93, respectively.
[0356] Based on the sequences of the soybean Glycinin Gy1 promoter
sequence in GenBank (Accession No. X15121), the oligonucleotides
set forth in SEQ ID NOs:27 and 28 were designed to amplify the
soybean Glycinin Gy1 promoter (SEQ ID NO:26), wherein the primers
had either BamHI or NotI sites at the 5' ends. The amplified
soybean glycinin Gy1 promoter fragment was digested with BamHI and
NotI, purified and cloned into the BamHI and NotI sites of plasmid
pZBL115 (supra) to produce pZBL117.
Example 8
Cloning the Euglena gracilis Delta-8 Desaturase into a Soybean
Expression Vector and Co-Expression with EPA Biosynthetic Genes
(Delta-8 Desaturase and Delta-17 Desaturase)
[0357] Plasmid pKR325 was generated from pKR72 (Example 5) by
digestion with HindIII to remove the .beta.con/NotI/Phas3'
cassette. Plasmid pKR680 (Example 5) was digested with BsiWI and
the fragment containing Eg5 (SEQ ID NO:1) was cloned into the BsiWI
site of pKR325 to produce pKR683.
[0358] The KTi/NotI/KTi3' cassette from pKS121 was PCR-amplified
using primers oKTi5 (SEQ ID NO:34) and oKTi6 (SEQ ID NO:35),
designed to introduce an XbaI and BsiWI site at both ends of the
cassette. The resulting PCR fragment was subcloned into the XbaI
site of the cloning vector pUC19 to produce plasmid pKR124, thus
adding a PstI and SbfI site at the 3' end of the Kti transcription
terminator.
[0359] The SalI fragment of pJS93 containing soy BD30 promoter (WO
01/68887) was combined with the SalI fragment of pUC19 to produce
pKR227, thus adding a PstI and SbfI site at the 5' end of the BD30
promoter.
[0360] The BD30 3' transcription terminator was PCR-amplified from
soy genomic DNA using primer oSBD30-1 (SEQ ID NO:36; designed to
introduce an NotI site at the 5' end of the terminator) and primer
oSBD30-2 (SEQ ID NO:37; designed to introduce a BsiWI site at the
3' end of the terminator). The resulting PCR fragment was subcloned
into the intermediate cloning vector pCR-Script AMP SK(+)
(Stratagene) according the manufacturer's protocol to produce
plasmid pKR251r. The EcoRI/NotI fragment from pKR251r, containing
the BD30 3' transcription terminator, was cloned into the
EcoRI/NotI fragment of intermediate cloning vector pKR227 to
produce pKR256.
[0361] The annexin promoter from pJS92 (Example 7) was released by
BamHI digestion and the ends were filled. The resulting fragment
was ligated into the filled BsiWI fragment from the vector backbone
of pKR124 in a direction which added a PstI and SbfI site at the 5'
end of the annexin promoter to produce pKR265. The annexin promoter
was released from pKR265 by digestion with SbfI and NotI and was
cloned into the SbfllNotI fragment of pKR256 (containing the BD30
3' transcription terminator, an ampicillin resistance gene and a
bacterial ori region) to produce pKR268.
[0362] The gene for the Saprolegnia diclina delta-17 desaturase was
released from pKS203 (Pereira et al., Biochem. J. 378:665-671
(2004)) by partial digestion with NotI, and was cloned into the
NotI site of pKR268 to produce pKR271. In this way, the delta-17
desaturase was cloned as an expression cassette behind the annexin
promoter with the BD30 transcription terminator.
[0363] Plasmid pKR271 was then digested with PstI and the fragment
containing the Saprolegnia diclina delta-17 desaturase was cloned
into the SbfI site of pKR683 to produce pKR685. In this way, the
delta-8 desaturase could be co-expressed with the S. diclina
delta-17 desaturase behind strong, seed-specific promoters. A map
of pKR685 is shown in FIG. 4A.
Example 9
Assembling EPA Biosynthetic Pathway Genes for Expression in Somatic
Soybean Embryos and Soybean Seeds (Delta-6 Desaturase, Elongase and
Delta-5 Desaturase)
[0364] The M. alpina delta-6 desaturase (U.S. Pat. No. 5,968,809),
M. alpina elongase (WO 00/12720) and M. alpina delta-5 desaturase
(U.S. Pat. No. 6,075,183) were cloned into plasmid pKR274 (FIG. 4B)
behind strong, seed-specific promoters allowing for high expression
of these genes in somatic soybean embryos and soybean seeds. All of
these promoters exhibit strong tissue specific expression in the
seeds of soybean. Plasmid pKR274 also contains the hygromycin B
phosphotransferase gene (Gritz, L. and Davies, J. Gene 25:179-188
(1983)) cloned behind the T7 RNA polymerase promoter and followed
by the T7 terminator (T7prom/HPT/T7term cassette) for selection of
the plasmid on hygromycin B in certain strains of E. coli (e.g.,
NovaBlue(DE3) (Novagen, Madison, Wis.), a strain that is lysogenic
for lambda DE3 and carries the T7 RNA polymerase gene under lacUV5
control). In addition, plasmid pKR274 contains a bacterial origin
of replication (ori) functional in E. coli from the vector pSP72
(Stratagene).
[0365] More specifically, the delta-6 desaturase was cloned behind
the promoter for the .alpha.' subunit of .beta.-conglycinin (Beachy
et al., EMBO J. 4:3047-3053 (1985)) followed by the 3'
transcription termination region of the phaseolin gene (Doyle, J.
J. et al. J. Biol. Chem. 261:9228-9238 (1986))
(.beta.con/Mad6/Phas3' cassette).
[0366] The delta-5 desaturase was cloned behind the Kunitz soybean
Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell
1:1079-1093 (1989)), followed by the KTi 3' termination region, the
isolation of which is described in U.S. Pat. No. 6,372,965
(KTi/Mad5/KTi3' cassette).
[0367] The elongase was cloned behind the glycinin Gy1 promoter
followed by the pea leguminA2 3' termination region
(Gy1/Maelo/legA2 cassette).
[0368] The gene for the M. alpina delta-6 desaturase was
PCR-amplified from pCGR5 (U.S. Pat. No. 5,968,809) using primers
oCGR5-1 (SEQ ID NO:38) and oCGR5-2 (SEQ ID NO:39), which were
designed to introduce NotI restriction enzyme sites at both ends of
the delta-6 desaturase and an NcoI site at the start codon of the
reading frame for the enzyme. The resulting PCR fragment was
subcloned into the intermediate cloning vector pCR-Script AMP SK(+)
(Stratagene) according the manufacturer's protocol to produce
plasmid pKR159. The NotI fragment of pKR159, containing the M.
alpina delta-6 desaturase gene, was cloned into NotI site of
pZBL117 (Example 7) in the sense orientation to produce plant
expression cassette pZBL119.
[0369] Vector pKR197 was constructed by combining the AscI fragment
from plasmid pKS102 (WO 02/00905), containing the T7prom/hpt/T7term
cassette and bacterial ori, with the Asci fragment of plasmid pKR72
(Example 5), containing the .beta.con/NotI/Phas cassette. Plasmid
pKR159 was digested with NotI to release the M. alpina delta-6
desaturase, which was, in turn, cloned into the NotI site of the
soybean expression vector pKR197 to produce pKR269.
[0370] The glycinin Gy1 promoter was amplified from pZBL119 using
primer oSGly-1 (SEQ ID NO:40; designed to introduce an SbfI/PstI
site at the 5' end of the promoter) and primer oSGly-2 (SEQ ID
NO:41; designed to introduce a NotI site at the 3' end of the
promoter). The resulting PCR fragment was subcloned into the
intermediate cloning vector pCR-Script AMP SK(+) (Stratagene)
according to the manufacturer's protocol to produce plasmid
pSGly12.
[0371] The legA2 promoter was amplified from pea genomic DNA using
primer LegPro5' (SEQ ID NO:42; designed to introduce XbaI and BsiWI
sites at the 5' end of the promoter) and primer LegPro3' (SEQ ID
NO:43; designed to introduce a NotI site at the 3' end of the
promoter). The legA2 transcription terminator was amplified from
pea genomic DNA using primer LegTerm5' (SEQ ID NO:44; designed to
introduce NotI site at the 5' end of the terminator) and primer
LegTerm3' (SEQ ID NO:45; designed to introduce BsiWI and XbaI sites
at the 3' end of the terminator). The resulting PCR fragments were
then combined and re-amplified using primers LegPro5' and
LegTerm3', thus forming a legA2/NotI/legA23' cassette. The
legA2/NotI/legA23' cassette PCR fragment was subcloned into the
intermediate cloning vector pCR-Script AMP SK(+) (Stratagene)
according to the manufacturer's protocol to produce plasmid
pKR140.
[0372] Plasmid pKR142 was constructed by cloning the BsiWI fragment
of pKR140 (containing the legA2/NotI/legA23' cassette) into the
BsiWI site of pKR124 (containing a bacterial ori and ampicillin
resistance gene). The PstI/NotI fragment from plasmid pKR142 was
then combined with the PstI/NotI fragment of plasmid pSGly12
(containing the glycininGy1 promoter) to produce pKR263. The gene
for the M. alpina delta-5 desaturase was amplified from pCGR4 (U.S.
Pat. No. 6,075,183) using primers CGR4foward (SEQ ID NO:46) and
CGR4reverse (SEQ ID NO:47) which were designed to introduce NotI
restriction enzyme sites at both ends of the desaturase. The
resulting PCR fragment was digested with NotI and cloned into the
NotI site of vector pKR124 (Example 6) to produce pKR136.
[0373] The NotI fragment containing the M. alpina elongase (Example
5) was cloned into the NotI site of vector pKR263 to produce
pKR270. The Gy1/Maelo/legA2 cassette was released from plasmid
pKR270 by digestion with BsiWI and SbfI and was cloned into the
BsiWI/SbfI sites of plasmid pKR269 (containing the delta-6
desaturase, the T7prom/hpt/T7term cassette and the bacterial ori
region). This was designated as plasmid pKR272. The KTi/Mad5/KTi3'
cassette, released from pKR136 by digestion with BsiWI, was then
cloned into the BsiWI site of pKR272 to produce pKR274 (FIG.
4B).
Example 10
Assembling EPA Biosynthetic Pathway Genes for Expression in Somatic
Soybean Embryos and Soybean Seeds (Delta-17 Desaturase and Delta-5
Desaturase)
[0374] In a manner similar to that described in Example 9, the
delta-17 desaturase from S. diclina could be cloned into a soy
expression vector along with the delta-5 desaturase from M. alpina.
The annexin/delta17/BD30 cassette of pKR271 could be released by
digestion with a suitable restriction enzyme such as PstI and
cloned into a soy expression vector already carrying the M. alpina
delta-5 desaturase behind a suitable promoter and a suitable
selection marker such as hygromycin. The M. alpina delta-5
desaturase could be part of any suitable expression cassette
described here. For instance, the NotI fragment containing the M.
alpina delta-5 desaturase described above could be cloned into the
NotI site of the Gy1/NotI/legA2 cassette of pKR263. This
Gy1/delta5/legA2 cassette could then be cloned into a vector
containing a suitable selectable marker for soy transformation.
Such a vector could be co-transformed into soy with pKR681 (Example
6) and transformants expression genes from both plasmids selected.
In this way, EPA could be produced using the delta-8 pathway
independent of a delta-6 desaturase.
Example 11
Functional Analysis of the Euglena gracilis Delta-8 Desaturase in
Saccharomyces cerevisiae
[0375] Plasmids pY89-5 (comprising the Eg5 sequence; see FIG. 3A
and ATCC PTA-6048), pY89-12 (identical to pY89-5, with the
exception that the Eg12 sequence was inserted instead of Eg5) and
pY-75 (Example 5, negative control cloning vector [lacking Eg5 or
Eg12]) were transformed into Saccharomyces cerevisiae BY4741 (ATCC
#201388) 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 grown for 1 day at 30.degree. C., after which 0.5 mL
was transferred to the same medium supplemented with either EDA or
EtrA to 1 mM. These were incubated overnight at 30.degree. C., 250
rpm, pellets were obtained by centrifugation and dried under
vacuum. Pellets were transesterified with 50 .mu.L of TMSH and
analyzed by GC as described in Example 1. Two clones for pY-75
(i.e., clones 75-1 and 75-2) and pY89-5 (i.e., clones 5-6-1 and
5-6-2) were analyzed, while two sets of clones for pY89-12 (i.e.,
clones 12-8-1, 12-8-2, 12-9-1 and 12-9-2) from two independent
transformations were analyzed.
[0376] The lipid profile obtained by GC analysis of clones fed EDA
are shown in Table 5; and the lipid profile obtained by GC analysis
of clones fed EtrA are shown in Table 6.
TABLE-US-00007 TABLE 5 20:3 %20:2 Clone 16:0 16:1 18:0 18:1 20:2
(8, 11, 14) Converted 75-1 14 32 5 38 10 0 0 75-2 14 31 5 41 9 0 0
5-6-1 14 32 6 40 6 2 24 5-6-2 14 30 6 41 7 2 19 12-8-1 14 30 6 41 9
1 7 12-8-2 14 32 5 41 8 1 8 12-9-1 14 31 5 40 9 1 8 12-9-2 14 32 5
41 8 1 7
TABLE-US-00008 TABLE 6 20:3 20:4 %20:3 Clone 16:0 16:1 18:0 18:1
(11, 14, 17) (8, 11, 14, 17) Converted 75-1 12 25 5 33 24 0 0 75-2
12 24 5 36 22 1 5 5-6-1 13 25 6 34 15 7 32 5-6-2 13 24 6 34 17 6 27
12-8-1 12 24 5 34 22 2 8 12-8-2 12 25 5 35 20 2 9 12-9-1 12 24 5 34
22 2 9 12-9-2 12 25 6 35 20 2 9
[0377] The data in Tables 4 and 5 showed that the cloned Euglena
delta-8 desaturase is able to desaturate EDA and EtrA. The sequence
set forth in SEQ ID NO:4 has one amino acid change compared to the
sequence set forth in SEQ ID NO:2 and has reduced delta-8
desaturase activity.
[0378] The small amount of 20:4(8,11,14,17) generated by clone 75-2
in Table 6 had a slightly different retention time than a standard
for 20:4(8,11,14,17). This peak was more likely a small amount of a
different fatty acid generated by the wild-type yeast in that
experiment.
Example 12
Cloning Other Delta-8 Desaturases or Elongases into Soybean
Expression Vectors
[0379] In addition to the delta-8 desaturase from Euglena gracilis,
other delta-8 desaturases can be cloned into the soybean expression
vectors such as those described in Example 6 and Example 8. For
instance, a suitable delta-8 desaturase from an organism other than
Euglena gracilis can be cloned using methods similar to, but not
limited to, the methods described in Example 2 and Example 3. 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 cloned
into a soybean expression vector such as pKR457. Further
sub-cloning into other vectors as described in Example 6 or Example
8 would yield vectors suitable for expression and co-expression of
the delta-8 desaturase in soybean.
[0380] Likewise, in addition to the elongase from Mortierella
alpina, other elongases can be cloned into the soybean expression
vectors such as those described in Example 6 and Example 8.
Specifically, elongases with specificity for linoleic acid or
alpha-linolenic acid such as that from Isochrysis galbana (WO
2002/077213) can be used. For instance, a suitable elongase from an
organism other than Mortierella alpina can be cloned using methods
similar to, but limited not to, the methods described in Example 2
and Example 3. PCR primers designed to introduce NotI sites at the
5' and 3' ends of the elongase can be used to amplify the gene. The
resulting PCR product can then be digested with NotI and cloned
into soybean expression vectors such as pKR72 or pKR263. Further
sub-cloning into other vectors as described in Example 6 or Example
8 would yield vectors suitable for expression and co-expression of
the elongase in soybean.
Example 13
Transformation of Somatic Soybean Embryo Cultures
[0381] Culture Conditions: Soybean embryogenic suspension cultures
(cv. Jack) can be 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 are 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).
[0382] 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, 327:70 (1987))
using a DuPont Biolistic PDS1000/HE instrument (helium retrofit)
for all transformations.
[0383] Soybean Embryogenic Suspension Culture Initiation: Soybean
cultures are initiated twice each month with 5-7 days between each
initiation.
[0384] 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 (i.e., 95 mL of autoclaved distilled water
plus 5 mL Clorox and 1 drop of soap, mixed 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 8 weeks. After this time secondary embryos are cut
and placed into SB196 liquid media for 7 days.
[0385] Preparation of DNA for Bombardment: 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 and pKR685 or pKR681 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 AscI (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 could be separated by
gel electrophoresis on 1% SeaPlaque 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 digesting enzyme
following the manufacturer's protocol. Alternatively, whole
plasmids or a combination of whole plasmid with fragment could be
used.
[0386] A 50 .mu.l aliquot of sterile distilled water containing 3
mg of gold particles 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 particles per
bombardment (i.e., per disk).
[0387] Tissue Preparation and Bombardment with DNA: Approximately
150-200 mg of 7 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 1 or 2 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.
[0388] Selection of Transformed Embryos: Transformed embryos are
selected either using hygromycin (when the hygromycin
phosphotransferase, HPT, gene was used as the selectable marker) or
chlorsulfuron (when the acetolactate synthase, ALS, gene was used
as the selectable marker). Specifically, 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 either a selection agent of 30 mg/L hygromycin or
a 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 to generate new, clonally
propagated, transformed embryogenic suspension cultures.
[0389] Regeneration of Soybean Somatic Embryos into Plants: In
order to obtain whole plants from embryogenic suspension cultures,
the tissue must be regenerated.
[0390] Embryo Maturation: Embryos can be cultured for 4-6 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 hr photoperiod with light intensity of 90-120
.mu.E/m.sup.2s. After this time embryo clusters are removed to a
solid agar media, SB166, for 1-2 weeks. Clusters are then
subcultured to medium SB103 for 3 weeks. During this period,
individual embryos can be removed from the clusters and screened
for alterations in their fatty acid compositions as described in
Example 11. It should be noted that any detectable phenotype,
resulting from the expression of the genes of interest, could be
screened at this stage. This 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.
[0391] Embryo Desiccation and Germination: Matured individual
embryos can be desiccated by placing them into an empty, small
petri dish (35.times.10 mm) for approximately 4-7 days. The plates
are sealed with fiber tape (creating a small humidity chamber).
Desiccated embryos can be planted into SB71-4 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 trays, covered with clear plastic domes. After 2 weeks
the dome is removed and plants hardened off for a further week. If
plantlets look hardy they are transplanted to 10'' pots of
Redi-Earth with up to 3 plantlets per pot. After 10 to 16 weeks,
mature seeds can be harvested, chipped and analyzed for fatty acids
as described above.
Media Recipes
TABLE-US-00009 [0392] 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 g (NH.sub.4).sub.2SO.sub.4
0.463 g Asparagine 1.0 g Sucrose (1%) 10 g pH 5.8
TABLE-US-00010 FN Lite Stock Solutions Stock # 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 pkg. MS salts (Catalog
#11117-066, Gibco/BRL) 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 pkg. MS salts (Catalog #11117-066,
Gibco/BRL) 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 pkg. MS salts (Catalog #11117-066,
Gibco/BRL) 1 mL B5 vitamins 1000X stock 60 g maltose 750 mg
MgCl.sub.2 hexahydrate pH 5.7 2 g gelrite SB 71-4 solid medium (per
liter) 1 bottle Gamborg's B5 salts with sucrose (Catalog
#21153-036, Gibco/BRL) pH 5.7 5 g TC agar 2,4-D stock: obtained
premade from Phytotech, Catalog #D 295; concentration is 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 in 0.01 N ammonium hydroxide
[0393] 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 6-10 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.
[0394] 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 hour day/night schedule. Cultures
are subcultured every two weeks by inoculating approximately 35 mg
of tissue into 35 mL of liquid medium.
[0395] Soybean embryogenic suspension cultures may then be
transformed 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 PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0396] 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 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.
[0397] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): .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 min, 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.
[0398] 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 5-10 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.
[0399] 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 14
Further Modification of the Delta-8 Desaturase Gene Codon-Optimized
for Yarrowia lipolytica
[0400] The amino acid sequence of the synthetic codon-optimized
D8S-3 gene in pDMW261 (Example 1) was corrected according to the
amino acid sequence of the functional Euglena delta-8 desaturase
(SEQ ID NOs:1 and 2). Using pDMW261 as a template and
oligonucleotides ODMW404 (SEQ ID NO:94) and D8-13R (SEQ ID NO:36),
the DNA fragment encoding the synthetic D8S-3 desaturase gene was
amplified. The resulting PCR fragment was purified with Bio101's
Geneclean kit and subsequently digested with Kpn1 and Not1 (primer
ODMW404 introduced a KpnI site while primer D8-13R introduced a
NotI site). The Kpn1/Not1 fragment (SEQ ID NO:95) was cloned into
Kpn1/Not1 digested pKUNFmKF2 (FIG. 5D; SEQ ID NO:116) to produce
pDMW277 (FIG. 6A).
[0401] Oligonucleotides YL521 (SEQ ID NO:96) and YL522 (SEQ ID
NO:97), which were designed to amplify and correct the 5' end of
the D8S-3 gene, were used as primers in another PCR reaction where
pDMW277 was used as the template. The primers introduced into the
PCR fragment a Nco1 site and BgIII site at its 5' and 3' ends,
respectively. The 318 bp PCR product was purified with Bio101's
GeneClean kit and subsequently digested with Nco1 and BgIII. The
digested fragment, along with the 954 bp BgIII/NotI fragment from
pDMW277, was used to exchange the NcoI/NotI fragment of pZF5T-PPC
(FIG. 6B; SEQ ID NO:117) to form pDMW287 (FIG. 6C). In addition to
correcting the 5' end of the synthetic D8S-3 gene, this cloning
reaction also placed the synthetic delta-8 desaturase gene under
control of the Yarrowia lipolytica fructose-bisphosphate aldolase
promoter containing a Yarrowia intron (FBAIN; SEQ ID NO:114; see WO
2005/049805).
[0402] The first reaction in a final series of site-directed
mutagenesis reactions was then performed on pDMW287. The first set
of primers, YL525 (SEQ ID NO:98) and YL526 (SEQ ID NO:99), was
designed to correct amino acid from F to S (position #50) of the
synthetic D8S-3 gene in pDMW287. The plasmid resulting from this
mutagenesis reaction then became the template for the next
site-directed mutagenesis reaction with YL527 (SEQ ID NO:100) and
YL528 (SEQ ID NO:101) as primers. These primers were designed to
correct the amino acid from F to S (position #67) of the D8S-3 gene
and resulted in creation of plasmid pDMW287/YL527.
[0403] To complete the sequence corrections within the second
quarter of the gene, the following reactions were carried out
concurrently with the mutations on the first quarter of the gene.
Using pDMW287 as template and oligonucleotides YL529 (SEQ ID
NO:102) and YL530 (SEQ ID NO:103) as primers, an in vitro
mutagenesis reaction was carried out to correct the amino acid from
C to W (position #177) of the synthetic D8S-3 gene. The product
(i.e., pDMW287NY529) of this mutagenesis reaction was used as the
template in the following reaction using primers YL531 (SEQ ID
NO:104) and YL532 (SEQ ID NO:105) to correct the amino acid from P
to L (position #213). The product of this reaction was called
pDMW287NYL529-31.
[0404] Concurrently with the mutations on the first and second
quarter of the gene, reactions were similarly carried out on the 3'
end of the gene. Each subsequent mutagenesis reaction used the
plasmid product from the preceding reaction. Primers YL533 (SEQ ID
NO:106) and YL534 (SEQ ID NO:107) were used on pDMW287 to correct
the amino acid from C to S (position #244) to create pDMW287NYL533.
Primers YL535 (SEQ ID NO:108) and YL536 (SEQ ID NO:109) were used
to correct the amino acid A to T (position #280) in the synthetic
D8S-3 gene of pDMW287/YL533 to form pDMW287/YL533-5. Finally, the
amino acid P at position of #333 was corrected to S in the
synthetic D8S-3 gene using pDMW287/YL533-5 as the template and
YL537 (SEQ ID NO:110) and YL538 (SEQ ID NO:111) as primers. The
resulting plasmid was named pDMW287/YL533-5-7.
[0405] The BgIII/XhoI fragment of pDMW287/YL529-31, and the
XhoI/NotI fragment of pDMW287/YL533-5-7 was used to change the
BgIII/NotI fragment of pDMW287/YL257 to produce pDMW287F (FIG. 6D)
containing the completely corrected synthetic delta-8 desaturase
gene, designated "D8SF" and set forth in SEQ ID NO:112. SEQ ID
NO:113 sets forth the amino acid sequence encoded by nucleotides
2-1270 of SEQ ID NO:112, which is essentially the same as the
sequence set forth in SEQ ID NO:2, except for an additional valine
following the start methionine.
Example 15
Synthesis and Functional Expression of a Codon-Optimized Delta-9
Elongase Gene in Yarrowia lipolytica
[0406] In order to express the delta-9 elongase/delta-8 desaturase
pathway in Yarrowia lipolytica, it was necessary to obtain an
appropriate delta-9 elongase that could be co-expressed with the
synthetic codon-optimized delta-8 desaturase from Example 14. Thus,
the codon usage of the delta-9 elongase gene of Isochrysis galbana
(GenBank Accession No. AF390174) was optimized for expression in Y.
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 (SEQ ID NO:118), based on the DNA sequence of
Isochrysis galbana; SEQ ID NO:119. In addition to modification of
the translation initiation site, 126 bp of the 792 bp coding region
were modified, and 123 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:120).
In Vitro Synthesis of a Codon-Optimized Delta-9 Elongase Gene for
Yarrowia
[0407] The method used to synthesize the codon-optimized delta-9
elongase gene was the same as that used for synthesis of the
delta-8 desaturase gene (Example 1). First, eight pairs of
oligonucleotides were designed to extend the entire length of the
codon-optimized coding region of the I. 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:121-136). 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:137-140) also
introduced NcoI, PstI, PstI and Not1 restriction sites,
respectively, for subsequent subcloning.
[0408] Each oligonucleotide (100 ng) was phosphorylated at
37.degree. C. for 1 hr 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:121) was annealed to IL3-1B (SEQ ID NO:122) to
produce the double-stranded product "IL3-1AB". Similarly, IL3-2A
(SEQ ID NO:123) was annealed to IL3-2B (SEQ ID NO:124) to produce
the double-stranded product "IL3-2AB", etc.
[0409] 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.
[0410] 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 (i.e., IL3-1AB, IL3-2AB, IL3-3AB
and IL3-4AB) as template, and oligonucleotides IL3-1F and IL3-4R
(SEQ ID NOs:137 and 138) as primers, the first portion of the
codon-optimized delta-9 elongase gene was amplified by PCR (as
described in Example 1). The 417 bp PCR fragment was subcloned into
the PGEM-T easy vector (Promega) to generate pT9(1-4).
[0411] Using the ligated "Pool 2" mixture (i.e. IL3-5AB, IL3-6AB,
IL3-7AB and IL3-8AB) as the template, and oligonucleotides IL3-5F
and IL3-8R (SEQ ID NOs:139 and 140) 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).
[0412] E. coli was transformed separately with pT9(1-4) and
pT9(5-8) 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
NcoI/PstI fragment of pT9(1-4) (SEQ ID NO:141) and the 377 bp
PstI/NotI fragment of pT9(5-8) (SEQ ID NO:142). These two fragments
were then combined and directionally ligated together with
NcoI/NotI digested pZUF17 (SEQ ID NO:143; FIG. 7A) to generate
pDMW237 (FIG. 7B; SEQ ID NO:144). 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 (i.e., SEQ
ID NO:118) for Yarrowia.
Generation of Y. lipolytica Strain Y2031 (A Ura-Derivative of ATCC
#20362)
[0413] Strain Y2031 was generated by integration of the
TEF::Y..DELTA.12::Pex20 chimeric gene of plasmid pKUNT2 (FIG. 7C)
into the Ura3 gene locus of Yarrowia lipolytica ATCC #20362, to
thereby generate the Ura-genotype of strain Y2031.
[0414] Specifically, plasmid pKUNT2 contained the following
components:
TABLE-US-00011 TABLE 7 Description of Plasmid pKUNT2 (SEQ ID NO:
145) RE Sites And Nucleotides Within SEQ ID Description Of NO: 145
Fragment And Chimeric Gene Components AscI/BsiWI 784 bp 5' part of
Yarrowia Ura3 gene (3225-3015) (GenBank Accession No. AJ306421)
SphI/PacI 516 bp 3' part of Yarrowia Ura3 gene (5933-13) (GenBank
Accession No. AJ306421) EcoRI/BsiWI TEF::Y..DELTA.12::Pex20,
comprising: (6380-8629) TEF: TEF promoter (GenBank Accession No.
AF054508) Y..DELTA.12: Yarrowia delta-12 desaturase gene (SEQ ID
NO: 146; see also WO 2004/104167) Pex20: Pex20 terminator sequence
from Yarrowia Pex20 gene (GenBank Accession No. AF054613)
[0415] The pKUNT2 plasmid was digested with AscI/SphI, and then
used for transformation of wild type Y. lipolytica ATCC #20362
according to the General Methods. The transformant cells were
plated onto 5-fluoroorotic acid ("FOA"; also
5-fluorouracil-6-carboxylic acid monohydrate) selection media
plates and maintained at 30.degree. C. for 2 to 3 days.
Specifically, FOA selection media comprised: 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. The FOA resistant colonies were picked and streaked onto
MM and MMU selection plates. The colonies that could grow on MMU
plates but not on MM plates were selected as Ura-strains. Single
colonies (5) of Ura-strains were then inoculated into liquid MMU at
30.degree. C. and shaken at 250 rpm/min for 2 days.
[0416] 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. GC analyses showed that there were about
45% LA in two Ura--strains (strains #2 and #3), compared to about
20% LA in the wild type ATCC #20362. Transformant strain #2 was
designated as strain "Y2031".
Expression of the Codon-Optimized Delta-9 Elongase Gene in Y.
lipolytica
[0417] Construct pDMW237 (comprising the chimeric
FBAIN::IgD9e::Pex20 gene) was transformed into Yarrowia lipolytica
strain Y2031, as described in the General Methods. Three
transformants of Y2031 with pDMW237 were grown individually in MM
media for two 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.
[0418] The GC results showed that there were about 7.1%, 7.3% and
7.4% EDA produced in these transformants with pDMW237. These data
demonstrated that the synthetic IgD9e could convert the C18:2 to
EDA. The "percent (%) substrate conversion" or "conversion
efficiency" of the codon-optimized gene was determined to be about
13%, wherein 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. This term refers to the efficiency by
which the particular enzyme can convert substrate to product.
Example 16
Delta-9 Elongase/Delta-8 Desaturase Pathway Expression to Produce
DGLA in Yarrowia lipolytica
[0419] The present Example describes DGLA biosynthesis and
accumulation in Yarrowia lipolytica that was transformed to express
the delta-9 elongase/delta-8 desaturase pathway. Thus, this
required co-synthesis of the synthetic codon-optimized delta-9
elongase (SEQ ID NO:118; Example 15) and the synthetic
codon-optimized delta-8 desaturase (SEQ ID NO:112; Example 14).
[0420] Specifically, the ClaI/PacI fragment comprising the chimeric
FBAIN::D8SF::Pex16 gene of construct pDMW287F (FIG. 6D) was
inserted into the ClaI/PacI sites of pDMW237 (FIG. 7B) to generate
the construct pDMW297 (FIG. 7D). Thus, plasmid pDMW297 contained
the following components:
TABLE-US-00012 TABLE 8 Description of Plasmid pDMW297(SEQ ID NO:
148) RE Sites And Nucleotides Within SEQ ID Description Of NO: 148
Fragment And Chimeric Gene Components EcoRI/ClaI ARS18 sequence
(GenBank Accession No. A17608) (9053-10448) ClaI/PacI
FBAIN::.DELTA.8S::Pex16, comprising: (1-2590) FBAIN: FBAIN promoter
(SEQ ID NO: 114) .DELTA.8S: codon-optimized delta-8 desaturase gene
(SEQ ID NO: 112), derived from Euglena gracilis (GenBank Accession
No. AF139720) Pex16: Pex16 terminator sequence of Yarrowia Pex16
gene (GenBank Accession No. U75433) PacI/SalI Yarrowia Ura3 gene
(GenBank Accession No. (2590-4082) AJ306421) SalI/BsiWI
FBAIN::.DELTA.9ES::Pex120, comprising: (4082-6257) FBAIN: FBAIN
promoter (SEQ ID NO: 114) .DELTA.9ES: codon-optimized delta-9
elongase gene (SEQ ID NO: 118), derived from Isochrysis galbana
(GenBank Accession No. 390174) Pex20: Pex20 terminator sequence of
Yarrowia Pex20 gene (GenBank Accession No. AF054613)
[0421] The pDMW297 plasmid was then used for transformation of
strain Y2031 (Example 15) according to the General Methods. The
transformant cells were plated onto MM selection media plates and
maintained at 30.degree. C. for 2 to 3 days. A total of 8
transformants grown on the MM plates were picked and re-streaked
onto fresh MM plates. Once grown, these strains were individually
inoculated into 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.
[0422] GC analyses showed that DGLA was produced in all of the
transformants analyzed. One strain produced about 3.2%, 4 strains
produced 4.3-4.5%, two strains produced 5.5-5.8% and one strain
produced 6.4% DGLA (designated herein as strain "Y0489"). The
"percent (%) substrate conversion" of the codon-optimized D8SF gene
in strain Y0489 was determined to be 75% (using the formula of
Example 15).
[0423] It will be obvious to one of skill in the art that other
chimeric genes could be co-expressed with the D8SF and IgD9e genes
in engineered Yarrowia to enable production of various other PUFAs.
For example, in addition to the codon-optimized delta-9 elongase
and delta-8 desaturase genes, one could readily express: (1) a
delta-15 desaturase to enable production of ETA; (2) a delta-5
desaturase to enable production of ARA; (3) a delta-17 desaturase
to enable production of ETA; (4) a delta-5 desaturase and a
delta-17 desaturase to enable production of EPA; (6) a delta-5
desaturase, a delta-17 desaturase and a C.sub.20/22 elongase to
enable production of DPA; or (7) a delta-5 desaturase, a delta-17
desaturase, a C.sub.20/22 elongase and a delta-4 desaturase to
enable production of DHA (FIG. 9).
Example 17
Cloning the Euglena gracilis Delta-8 Desaturase into a Soybean
Expression Vector and Co-Expression with an Isochrysis galbana
Elongase
[0424] The gene for the Isochrysis galbana elongase was amplified
from pDMW237 (FIG. 7B; SEQ ID NO:144) using primers olGsel1-1 (SEQ
ID NO:149) and olGsel1-2 (SEQ ID NO:150) which were designed to
introduce NotI restriction enzyme sites at both ends of the
elongase. The resulting PCR fragment was digested with NotI and
cloned into the NotI site of pKR72 to give pKR607.
[0425] Plasmid pKR680 was digested with BsiWI and the fragment
containing Eg5 (SEQ ID NO:1) was cloned into the BsiWI site of
pKR607 to give pKR682. Thus, the delta-8 desaturase (Eg5; SEQ ID
NO:1) could be co-expressed with the Isochrysis galbana elongase
behind strong, seed-specific promoters. A map of pKR682 is shown in
FIG. 8A.
Example 18
Assembling EPA Biosynthetic Pathway Genes with the Euglena gracilis
Delta-8 Desaturase and Isochrysis galbana Elongase for Expression
in Somatic Soybean Embryos and Soybean Seeds
[0426] An soybean expression vector (pKR786) containing the Euglena
gracilis delta-8 desaturase, the Isochrysis galbana delta-9
elongase and the Mortierella alpina delta-5 desaturase (all under
control of strong seed specific promoters) was constructed in the
following way.
[0427] Through a number of sub-cloning steps, a sequence of DNA
(SEQ ID NO:151) was effectively added into the SmaI site of vector
pKR287 (WO 2004/071467 A2) to produce pKR767. In this way, a SbfI
restriction site was added to the 3' end of the leg1A transcription
terminator of the Gy1/Mad5/legA2 cassette.
[0428] The AscI fragment of pKR682 was cloned into the AscI site of
pKR277 (WO 2004/071467 A2) to produce pKR769.
[0429] The Gy1/Mad5/legA2 cassette was released from pKR767 by
digestion with SbfI and the resulting fragment was cloned into the
SbfI site of pKR769 to produce pKR786. A map of pKR786 is shown in
FIG. 8B.
Example 19
Cloning the Fusarium Delta-15 Desaturase into a Soybean Expression
Vector and Co-Expression with EPA Biosynthetic Genes (Delta-15
Desaturase, Delta-17 Desaturase)
[0430] The Kti3 promoter:Fm .DELTA.15 desaturase ORF:Kti3
terminator cassette was released from plasmid pKR578 (WO
2005/047479) by digestion with BsiWI and was cloned into the BsiWI
site of plasmid pKR226 (WO 2004/071467 A2), containing the ALS gene
for selection, the T7prom/hpt/T7term cassette and the bacterial ori
region, to produce pKR667.
[0431] Plasmid pKR271 was digested with PstI and the fragment
containing the Saprolegnia diclina delta-17 desaturase was cloned
into the SbfI site of pKR667 to produce pKR669. In this way, the
delta-15 desaturase could be co-expressed with the Saprolegnia
diclina delta-17 desaturase behind strong, seed-specific promoters.
A map of pKR669 is shown in FIG. 8C.
Example 20
Analysis of Somatic Soy Embryos Containing the Euglena gracilis
Delta-8 Desaturase and Mortierella alpina Elongase Genes
(pKR681)
[0432] 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 (Example 3 in WO
02/00904). Most importantly, the model system is also predictive of
the fatty acid composition of seeds from plants derived from
transgenic embryos.
[0433] Transgenic somatic soybean embryos containing the constructs
described above were analyzed in a similar way. For this, fatty
acid methyl esters were prepared from single, matured, somatic soy
embryos by transesterification. Embryos were placed in a vial
containing 50 .mu.L of trimethylsulfonium hydroxide (TMSH) and 0.5
mL of hexane and incubated for 30 min at room temperature while
shaking. Fatty acid methyl esters (5 .mu.L injected from hexane
layer) are 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 2.7 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 (Catalog #U-99-A,
Nu-Chek Prep, Inc.). Routinely, 6-10 embryos per event were
analyzed by GC, using the methodology described above.
[0434] More specifically, embryo fatty acid profiles for .about.6
lines containing pKR681 are shown in Table 9. The best line (i.e.,
1618-1-1-1) had embryos with an average DGLA content of 8.9% and an
average ETA content of 3.1%. For lines 1618-1-8-1, 1618-3-6-1 and
1618-4-1-1, only the elongase appeared to be functioning. The best
elongase line (i.e., 1618-4-1-1) had embryos with an average EDA
content of 10.6% and an average EtrA content of 6.5%. Calculated %
elongation, % desaturation and elongation and desaturation ratios
are shown in Table 10. In line 1618-1-1-1, the delta-8 desaturase
converts an average of 76.3% of the elongated C20 fatty acids to
product with the best embryo converting 82.1% to product. The
delta-8 desaturase appears to utilize EDA and EtrA equally well as
the ratio of their respective % desaturations is around 1.0. In
line 1618-4-1-1, the Mortiella alpina elongase converts an average
of 23% of the C18 fatty axcids to product with the best embryo
converting 30.2% to product. The elongase appears to have a slight
preference for ALA as the ratio of their respective % elongations
is around 0.6. Expression of only the elongase in these lines
likely resulted from fragmentation of the construct during the
transformation procedure or due to positional insertion effects
differentially affecting expression of the delta-8.
TABLE-US-00013 TABLE 9 Accumulation Of Long Chain PUFAs In Lines
Transformed With pKR681 Line 16:0 18:0 18:1 LA GLA ALA EDA DGLA
EtrA ETA 1618-1-1-1 13.9 6.8 7.1 40.2 0.0 14.4 3.0 10.2 1.1 3.3 -2
14.5 10.0 6.2 38.9 0.0 11.0 4.1 10.5 1.2 3.0 -3 14.1 4.9 4.7 42.2
0.0 21.5 2.0 7.2 1.0 2.5 -4 14.1 7.1 6.2 42.7 0.0 13.3 3.3 9.1 1.1
2.8 -5 12.0 5.0 5.8 46.3 0.0 16.0 2.2 8.1 1.0 3.2 -6 12.1 4.7 5.7
42.0 0.0 20.9 1.7 8.5 0.9 3.5 Ave 13.5 6.4 6.0 42.1 0.0 16.2 2.7
8.9 1.0 3.1 1618-1-2-1 11.7 4.3 4.6 46.8 0.0 17.4 3.9 5.7 2.4 3.1
-2 12.2 6.0 4.7 45.5 0.0 14.9 5.1 5.8 2.9 2.9 -3 12.7 5.0 7.1 44.7
0.0 17.5 4.2 4.1 2.4 2.3 -4 12.4 6.3 6.8 43.0 0.0 13.9 6.6 4.9 3.9
2.3 -5 13.4 8.7 5.2 39.6 0.0 13.2 6.3 7.2 3.2 3.1 -6 12.8 5.5 6.2
45.7 0.0 15.6 4.3 5.2 2.2 2.5 Ave 12.5 6.0 5.8 44.2 0.0 15.4 5.1
5.5 2.8 2.7 1618-1-8-1 8.7 3.5 6.7 53.3 0.0 17.2 6.9 0.0 3.6 0.0 -2
9.2 2.9 12.0 49.0 0.0 18.9 4.7 0.0 3.3 0.0 -3 11.2 2.8 7.7 48.6 0.0
22.4 4.1 0.0 3.2 0.0 -4 12.0 3.6 13.6 46.7 0.0 16.0 4.8 0.0 3.2 0.0
-5 9.1 3.6 5.0 52.6 0.0 16.5 8.5 0.0 4.8 0.0 -6 9.3 2.8 12.7 47.2
0.0 20.0 4.6 0.0 3.4 0.0 Ave 9.9 3.2 9.6 49.6 0.0 18.5 5.6 0.0 3.6
0.0 1618-3-6-1 11.8 2.4 8.3 42.1 0.0 28.2 3.3 0.0 3.9 0.0 -2 10.6
4.2 12.8 43.7 0.0 17.6 6.7 0.0 4.3 0.0 -3 10.3 4.9 5.6 45.7 0.0
18.4 8.7 0.0 6.3 0.0 -4 11.8 5.2 21.2 39.5 0.0 15.2 4.4 0.0 2.8 0.0
-5 10.3 3.0 9.2 47.8 0.0 21.5 4.7 0.0 3.5 0.0 -6 9.4 2.5 9.4 47.9
0.0 23.2 4.0 0.0 3.7 0.0 Ave 10.7 3.7 11.1 44.4 0.0 20.7 5.3 0.0
4.1 0.0 1618-4-1-1 15.4 9.2 6.5 38.1 0.0 9.9 13.8 0.0 7.0 0.0 -2
11.1 5.6 5.7 43.3 0.0 16.8 10.2 0.0 7.4 0.0 -3 10.5 5.0 6.6 45.4
0.0 15.4 10.1 0.0 6.9 0.0 -4 10.2 5.8 6.5 45.1 0.0 12.9 12.3 0.0
7.2 0.0 -5 11.4 4.4 10.1 45.3 0.0 16.1 7.4 0.0 5.2 0.0 -6 10.7 5.2
13.6 42.9 0.0 12.6 9.6 0.0 5.3 0.0 Ave 11.5 5.9 8.2 43.4 0.0 14.0
10.6 0.0 6.5 0.0 Fatty acid compositions listed in Table 9 are
expressed as wt. %. 16:0 = Palmitic acid, 18:0 = Stearic acid, 18:1
= Oleic acid, LA = Linoleic acid, GLA = .gamma.-Linoleic acid, ALA
= alpha-Linolenic acid, EDA = Eicosadienoic acid, DGLA =
Dihomo-.gamma.-Linoleic, EtrA = Eicosatrienoic acid, ETA =
Eicosa-tetraenoic acid.
TABLE-US-00014 TABLE 10 Comparison Of % Desaturation And %
Elongation In Lines Transformed With pKR681 Ratio C20 Ratio EDA
EtrA (EDA/EtrA) C18 % delta- LA ALA (LA/ALA) % delta- % delta-
de;ta-8 Line % Elong 8 desat % Elong % Elong Elong 8 desat 8 desat
seat 1618-1-1-1 24.4 76.6 24.7 23.5 1.1 77.1 75.0 1.0 -2 27.3 71.9
27.2 27.7 1.0 72.1 71.1 1.0 -3 16.6 76.4 17.9 14.0 1.3 78.1 72.0
1.1 -4 22.5 73.3 22.4 22.7 1.0 73.5 72.6 1.0 -5 18.9 77.7 18.3 20.7
0.9 78.4 76.0 1.0 -6 18.8 82.1 19.6 17.4 1.1 83.1 79.9 1.0 Ave 21.4
76.3 21.7 21.0 1.1 77.0 74.4 1.0 1618-1-2-1 19.1 58.1 17.0 24.0 0.7
59.0 56.6 1.0 -2 21.7 52.2 19.3 28.0 0.7 53.2 50.4 1.1 -3 17.3 49.2
15.8 21.0 0.8 49.5 48.5 1.0 -4 23.7 40.6 21.0 30.8 0.7 42.6 36.9
1.2 -5 27.3 51.8 25.5 32.2 0.8 53.3 48.6 1.1 -6 18.9 54.1 17.3 23.1
0.7 54.6 53.1 1.0 Ave 21.3 51.0 19.3 26.5 0.7 52.0 49.0 1.1
1618-1-8-1 13.0 11.5 17.5 0.7 -2 10.6 8.8 14.8 0.6 -3 9.4 7.9 12.5
0.6 -4 11.4 9.3 16.8 0.6 -5 16.2 13.9 22.7 0.6 -6 10.7 9.0 14.6 0.6
Ave 11.9 10.1 16.5 0.6 1618-3-6-1 9.3 7.4 12.0 0.6 -2 15.2 13.3
19.5 0.7 -3 19.0 15.9 25.6 0.6 -4 11.6 10.0 15.6 0.6 -5 10.6 9.0
14.0 0.6 -6 9.8 7.7 13.9 0.6 Ave 12.6 10.5 16.8 0.6 1618-4-1-1 30.2
26.6 41.4 0.6 -2 22.6 19.0 30.5 0.6 -3 21.9 18.2 31.0 0.6 -4 25.2
21.5 35.8 0.6 -5 17.1 14.1 24.4 0.6 -6 21.1 18.3 29.6 0.6 Ave 23.0
19.6 32.1 0.6
The C18% Elongation (C18% Elong) in Table 10 was calculated by
dividing the sum of the wt. % for EDA, DGLA, EtrA and ETA (Table 9)
by the sum of the wt. % for LA, ALA, EDA, DGLA, EtrA and ETA (Table
9) and multiplying by 100 to express as a %. The C20% A8
desaturation (C20% A8 desat. Table 10) was calculated by dividing
the sum of the wt. % for DGLA and ETA (Table 9) by the sum of the
wt. % for EDA, DGLA, EtrA and ETA (Table 9) and multiplying by 100
to express as a %. The individual elongations (LA % Elong or ALA %
Elong) or A8 desaturations (EDA % .DELTA.8 desat or EtrA % .DELTA.8
desat) shown in Table 10 were calculated in a similar way but only
using either the .omega.-6 substrates/products or the .omega.-3
substrates/products for each. The Ratio elongation for LA and ALA
was obtained by dividing the LA % Elongation by the ALA %
elongation. Similarly, the Ratio delta-8 desaturation was obtained
by dividing the EDA % delta-8 desaturation by the EtrA % delta-8
desaturation.
Example 21
Analysis of Somatic Soy Embryos Containing the Euglena gracilis
Delta-8 Desaturase and Isochrysis galbana Elongase Genes
(pKR682)
[0435] Embryo fatty acid profiles for 9 lines containing pKR682 are
shown in Table 11. Calculated % elongation, % desaturation and
elongation and desaturation ratios are shown in Table 12. The best
line (1619-6-7) had embryos with an average DGLA content of 21.8%
and an average ETA content of 4.1%. As can be seen from Table 12,
in this line, the delta-8 desaturase converts an average of 91.6%
of the elongated C20 fatty acids to product and, in the best embryo
(1619-6-7-1), 95.1% of the elongated fatty acids are converted to
product. As for pKR681, the delta-8 desaturase appears to utilize
EDA and EtrA equally well with the ratio of their respective %
desaturations being around 1.0 (Table 12). In these lines, the
average % conversion of C18 fatty acids to C20 fatty acids ranges
from 40.0% to 49.5%. As seen with pKR681, there are lines
(1619-6-4, 1619-8-4) where only the elongase is functioning (Table
11). Again, this is likely due to positional effects or
fragmentation of DNA. In lines where the delta-8 desaturase is not
functioning, the best elongase line (1619-6-4) had embryos with an
average EDA content of 24.1% and an average ETrA content of 8.7%.
The best embryo analyzed had 27.4% EDA and 10.3% ETrA. Average
elongation in this line is 49.5% with the best embryo (1619-6-4-6)
having 58.9% elongation (Table 12). In these lines, the elongase
appears to have no preference for LA or ALA as the ratio of their
respective % elongations is around 1.0. Interestingly, in lines
that also express the delta-8 desaturase, there seems to be a
slight preference of the elongase for LA and the average elongation
ratio is as high as 2.3 in line 1617-16-2-7. In many of the lines,
a small amount of a fatty acid that runs with retention time
identical to GLA is present when the delta-8 desaturase is
functioning well.
TABLE-US-00015 TABLE 11 Accumulation Of Long Chain PUFAs In Lines
Transformed With pKR682 16:0 18:0 18:1 LA GLA ALA EDA DGLA EtrA ETA
1617-16-2-7 20.1 2.7 5.9 18.0 2.0 12.8 5.1 26.5 2.4 4.4 -8 19.3 1.3
5.0 22.7 1.7 23.4 3.7 16.8 1.6 4.5 -9 20.4 2.4 4.7 13.7 2.3 15.3
5.2 26.2 3.5 6.3 -10 17.0 1.7 6.2 19.9 1.7 27.0 2.5 17.4 1.1 5.5
-11 16.4 1.3 5.1 21.5 3.3 28.2 2.8 15.2 2.1 4.1 -12 26.7 2.4 6.1
0.0 4.1 20.2 6.5 26.9 2.2 5.0 -13 17.5 1.5 5.8 21.6 2.6 20.3 3.9
19.9 1.8 5.1 -14 20.2 2.4 8.9 24.6 1.6 17.9 4.3 14.7 1.4 4.2 Ave
19.7 2.0 6.0 17.7 2.4 20.6 4.2 20.4 2.0 4.9 1619-6-4-1 18.5 1.7 9.4
25.0 0.0 8.2 27.4 0.0 9.7 0.0 -2 14.5 2.0 15.9 26.8 0.0 7.8 24.5
0.0 8.5 0.0 -3 23.6 3.8 12.2 19.3 0.0 8.7 23.8 0.0 8.8 0.0 -4 15.5
1.2 12.6 34.5 0.0 14.7 15.4 0.0 6.0 0.0 -5 15.2 1.6 15.7 25.5 0.0
6.7 26.4 0.0 8.9 0.0 -6 15.8 2.2 18.0 19.1 0.0 7.2 27.4 0.0 10.3
0.0 Ave 17.2 2.1 14.0 25.0 0.0 8.9 24.1 0.0 8.7 0.0 1619-6-5-1 22.1
2.1 6.2 23.9 3.9 10.5 4.2 21.1 1.3 4.8 -2 17.4 1.6 9.5 32.3 1.5
11.0 3.0 18.0 0.7 4.6 -3 17.5 2.6 9.9 32.9 0.5 11.3 5.8 15.3 0.6
3.3 -4 17.2 2.0 12.1 29.5 0.6 10.6 6.0 13.9 2.8 4.7 -5 24.2 3.1 7.1
25.4 2.0 10.7 5.2 16.8 1.9 3.4 -6 17.9 1.6 5.9 30.8 2.4 12.3 5.7
18.2 1.6 3.7 Ave 19.4 2.2 8.5 29.2 1.8 11.1 5.0 17.2 1.5 4.1
1619-6-7-1 19.1 1.7 4.8 32.0 1.1 21.7 1.0 16.3 0.0 2.3 -2 19.0 1.3
5.0 40.1 1.2 17.9 1.4 11.9 0.2 2.0 -3 17.8 1.2 6.2 26.6 2.0 13.1
2.1 24.6 0.4 5.9 -4 19.4 1.3 8.1 29.4 1.2 12.6 3.0 20.5 0.5 4.1 -5
19.9 1.4 9.2 19.6 3.1 8.8 2.1 29.9 0.5 5.4 -6 20.1 1.6 6.9 25.0 2.9
8.4 2.5 27.6 0.4 4.6 Ave 19.2 1.4 6.7 28.8 1.9 13.7 2.0 21.8 0.3
4.1 1619-7-3-1 15.4 1.9 9.4 34.7 0.6 12.1 11.9 6.7 4.3 3.0 -2 15.2
1.5 9.6 37.4 0.0 17.0 9.9 3.7 3.6 1.9 -3 17.0 3.0 14.5 26.6 0.5 9.9
11.0 10.5 3.1 3.9 -4 18.5 3.4 8.6 17.7 1.3 4.2 21.7 16.9 4.0 3.7 -5
16.5 2.4 10.2 25.8 0.8 7.0 15.1 12.6 4.7 5.0 -6 16.9 2.2 10.3 24.4
0.4 6.8 22.7 6.3 7.3 2.6 Ave 16.6 2.4 10.4 27.8 0.6 9.5 15.4 9.5
4.5 3.4 1619-7-7-1 21.2 1.5 12.8 17.6 1.3 8.8 6.8 23.1 2.1 4.8 -2
15.1 1.2 19.9 27.7 0.7 11.2 7.4 12.1 1.7 3.0 -3 17.4 2.1 16.4 23.8
0.6 9.1 10.4 15.2 1.9 3.2 -4 17.2 1.5 18.3 21.4 0.9 9.2 9.3 16.6
1.8 3.9 -5 16.4 1.0 13.4 24.2 1.2 15.9 6.5 16.2 2.1 3.3 -6 20.3 2.3
7.5 19.5 1.3 9.5 8.9 22.9 2.4 5.1 Ave 17.9 1.6 14.7 22.4 1.0 10.60
8.2 17.7 2.0 3.9 1619-7-8-1 19.2 1.8 5.7 21.7 2.1 11.1 12.1 17.5
4.0 4.9 -2 15.0 1.1 10.6 28.5 1.0 16.2 10.3 10.8 3.5 3.1 -3 17.0
1.6 11.3 20.6 1.2 8.6 12.4 17.8 4.3 5.2 -4 16.5 1.5 10.3 25.4 1.0
13.6 11.1 13.4 3.3 3.8 -5 16.0 1.3 10.0 29.0 0.8 12.6 4.7 19.0 1.2
5.5 -6 15.6 1.6 12.2 28.7 1.0 9.9 9.4 15.6 1.7 4.6 Ave 16.6 1.5
10.0 25.6 1.2 12.0 1.0.0 15.7 3.0 4.5 1619-8-1-1 20.4 1.7 5.8 24.4
2.7 15.0 4.3 20.3 1.5 3.9 -2 17.2 1.9 14.5 24.2 0.0 9.3 5.0 20.9
1.1 5.9 -3 16.4 1.8 13.0 23.5 1.4 11.6 6.9 19.9 1.3 4.1 -4 17.2 1.5
15.3 22.2 1.1 7.2 7.7 21.5 1.2 4.9 -5 19.4 1.3 9.9 21.7 2.8 13.8
5.1 20.2 1.7 4.1 -6 17.9 1.3 10.5 23.4 1.5 9.9 6.5 22.8 1.4 4.8 Ave
18.1 1.6 11.5 23.3 1.6 11.1 5.9 20.9 1.4 4.6 1619-8-4-1 15.7 1.1
6.7 50.5 0.0 18.1 6.2 0.0 1.8 0.0 -2 15.0 1.8 8.2 38.6 0.0 28.4 5.8
0.0 2.2 0.0 -3 18.1 2.9 7.2 35.6 0.0 32.2 2.7 0.0 1.3 0.0 -4 18.0
2.7 9.7 40.7 0.0 18.2 8.7 0.0 1.9 0.0 -5 16.0 1.5 6.9 50.4 0.0 20.8
3.0 0.0 1.4 0.0 -6 15.3 0.9 7.7 50.8 0.0 20.6 3.6 0.0 1.2 0.0 Ave
16.4 1.8 7.7 44.4 0.0 23.00 5.0 0.0 1.6 0.0 Fatty acid compositions
listed in Table 11 are expressed as wt. %. 16:0 = Palmitic acid,
18:0 = Stearic acid, 18:1 = Oleic acid, LA = Linoleic acid, GLA =
.gamma.-Linoleic acid, ALA = alpha-Linolenic acid, EDA =
Eicosadienoic acid, DGLA = Dihomo-.gamma.-Linoleic, EtrA =
Eicosatrienoic acid, ETA = Eicosatetraenoic acid.
TABLE-US-00016 TABLE 12 Comparison Of % Desaturation And %
Elongation In Lines Transformed With pKR682 Ratio C20 Ratio EDA
EtrA (EDA/EtrA) C18 % delta- LA ALA (LA/ALA) % delta- % delta-
de;ta-8 Line % Elong 8 desat % Elong % Elong Elong 8 desat 8 desat
seat 1617-16-2-7 55.5 80.5 63.8 34.7 1.8 83.9 65.0 1.3 -8 36.6 80.2
47.4 20.8 2.3 82.1 73.6 1.1 -9 58.8 78.8 69.6 39.3 1.8 83.4 64.1
1.3 -10 36.0 86.6 49.9 19.5 2.6 87.5 83.9 1.0 -11 32.8 79.6 45.6
18.1 2.5 84.3 66.1 1.3 -12 66.7 78.6 100.0 26.1 3.8 80.6 69.5 1.2
-13 42.3 81.3 52.4 25.4 2.1 83.5 74.0 1.1 -14 36.6 76.7 43.5 23.8
1.8 77.4 74.4 1.0 Ave 45.7 80.3 59.0 26.0 2.3 82.8 71.3 1.2
1619-6-4-1 52.8 52.3 54.2 1.0 -2 48.8 47.7 52.1 0.9 -3 53.8 55.2
50.3 1.1 -4 30.3 30.8 29.1 1.1 -5 52.3 50.9 57.0 0.9 -6 58.9 58.9
58.9 1.0 Ave 49.5 49.3 50.3 1.0 1619-6-5-1 47.6 82.5 51.3 36.7 1.4
83.4 78.5 1.1 -2 37.8 85.8 39.4 32.3 1.2 85.5 86.9 1.0 -3 36.0 74.5
39.0 25.5 1.5 72.6 84.7 0.9 -4 40.6 67.7 40.2 41.7 1.0 69.6 62.5
1.1 -5 43.1 74.0 46.4 33.2 1.4 76.3 64.6 1.2 -6 40.4 74.9 43.7 30.1
1.5 76.0 70.1 1.1 Ave 40.9 76.6 43.3 33.2 1.3 77.3 74.6 1.0
1619-6-7-1 26.8 95.1 35.1 9.6 3.6 94.4 100.0 0.9 -2 21.2 89.5 25.0
11.1 2.3 89.4 90.1 1.0 -3 45.4 92.4 50.1 32.4 1.5 92.0 94.1 1.0 -4
40.1 87.6 44.4 26.5 1.7 87.2 89.9 1.0 -5 57.2 93.2 62.0 40.1 1.5
93.4 92.3 1.0 -6 51.2 91.9 54.6 37.4 1.5 91.8 92.9 1.0 Ave 40.3
91.6 45.2 26.2 2.0 91.4 93.2 1.0 1619-7-3-1 35.6 37.6 34.9 37.6 0.9
36.0 41.5 0.9 -2 26.1 29.4 26.8 24.5 1.1 27.3 34.6 0.8 -3 43.9 50.4
44.7 41.4 1.1 48.8 55.2 0.9 -4 67.8 44.5 68.5 64.6 1.1 43.8 48.1
0.9 -5 53.3 47.0 51.8 58.0 0.9 45.4 51.6 0.9 -6 55.5 23.0 54.3 59.2
0.9 21.8 26.5 0.8 Ave 47.0 38.7 46.8 47.6 1.0 37.2 42.9 0.9
1619-7-7-1 58.3 75.7 63.0 44.0 1.4 77.1 -2 38.4 62.4 41.3 29.8 1.4
61.9 86.9 1.0 -3 48.3 60.0 51.8 36.0 1.4 59.3 84.7 0.9 -4 50.7 64.9
54.6 38.2 1.4 64.2 62.5 1.1 -5 41.1 69.5 48.3 25.4 1.9 76.3 64.6
1.2 -6 57.5 71.1 62.0 44.0 1.4 76.0 70.1 1.1 Ave 49.0 67.2 53.5
36.2 1.5 77.3 74.6 1.0 1619-7-8-1 54.0 58.2 57.7 44.4 1.3 59.2 54.9
1.1 -2 38.2 50.1 42.6 28.8 1.5 51.2 46.6 1.1 -3 57.6 57.9 59.4 52.2
1.1 58.9 54.6 1.1 -4 44.8 54.5 49.1 34.5 1.4 54.8 53.5 1.0 -5 42.2
80.7 45.0 34.7 1.3 80.3 82.1 1.0 -6 44.7 64.6 46.5 38.8 1.2 62.4
73.0 0.9 Ave 46.9 61.0 50.1 38.9 1.3 61.1 60.8 1.0 1619-8-1-1 43.2
80.7 50.2 26.4 1.9 82.6 72.1 1.1 -2 49.5 81.5 51.7 42.7 1.2 80.7
84.7 1.0 -3 47.9 74.5 53.3 31.8 1.7 74.2 76.0 1.0 -4 54.6 74.1 56.8
45.8 1.2 73.7 79.6 0.9 -5 46.6 78.3 53.8 29.4 1.8 80.0 70.9 1.1 -6
51.5 77.8 55.6 38.3 1.5 77.8 77.9 1.0 Ave 48.9 77.9 53.6 35.7 1.6
78.2 76.9 1.0 1619-8-4-1 10.5 11.0 9.1 1.2 -2 10.7 13.1 7.1 1.8 -3
5.6 7.1 3.8 1.9 -4 15.3 17.7 9.5 1.8 -5 5.8 5.7 6.1 0.9 -6 6.3 6.6
5.4 1.2 Ave 9.0 10.2 6.8 1.5
The C18% Elongation (C18% Elong) in Table 12 was calculated by
dividing the sum of the wt. % for EDA, DGLA, EtrA and ETA (Table
11) by the sum of the wt. % for LA, ALA, EDA, DGLA, EtrA and ETA
(Table 11) and multiplying by 100 to express as a %. The C20%
.DELTA.8 desaturation (C20% .DELTA.8 desat. Table 12) was
calculated by dividing the sum of the wt. % for DGLA and ETA (Table
11) by the sum of the wt. % for EDA, DGLA, EtrA and ETA (Table 11)
and multiplying by 100 to express as a %. The individual
elongations (LA % Elong or ALA % Elong) or .DELTA.8 desaturations
(EDA % .DELTA.8 desat or EtrA % .DELTA.8 desat) shown in Table 12
were calculated in a similar way but only using either the
.omega.-6 substrates/products or the .omega.-3 substrates/products
for each. The Ratio elongation for LA and ALA was obtained by
dividing the LA % Elongation by the ALA % elongation. Similarly,
the Ratio delta-8 desaturation was obtained by dividing the EDA %
delta-8 desaturation by the EtrA % delta-8 desaturation.
Example 22
Analysis of Somatic Soy Embryos Containing the Euglena gracilis
Delta-8 Desaturase, Isochrysis galbana Elongase and Other EPA
Biosynthetic Genes (pKR786, pKR669)
[0436] Plasmid pKR786 and pKR669 were digested with AscI and the
DNA fragments containing ALS selection and EPA biosynthetic genes
were transformed into soy as described previously. Fatty acids from
ten embryos for each line obtained containg pKR786 and pKR669 were
analyzed by GC as described.
[0437] Ten embryos were analyzed for each individual transformation
event. Fatty acids were identified by comparison of retention times
to those for authentic standards. In this way, 169 events were
analyzed. From the 169 lines analyzed, 25 were identified that
produced EPA (average of 10 individual embryos) at a relative
abundance greater than 10% of the total fatty acids. The ten best
EPA-producing events are shown in Table 13 and Table 14. The
results for 10 embryos from the best event are shown in Tables 15
and 16. The best line analyzed averaged 21.2% EPA with the best
embryo of this line having 29.4% EPA (Table 16). A chromatogram for
the embryo is shown in FIG. 10
[0438] Fatty acids in Table 13 and Table 14 are defined as X:Y
where X is the fatty acid chain length and Y is the number of
double bonds. In addition, fatty acids from Table 13 and Table 14
are further defined as follows where the number in parentheses
corresponds to the position of the double bonds from the carboxyl
end of the fatty acid: 18:1=18:1(9), 18:2=18:2(9,12),
GLA=18:3(6,9,12), 18:3=18:3(9,12,15), STA=18:4(6,9,12,15),
DGLA=20:3(8,11,14), ARA=20:4(5,8,11,14), ETA=20:4(8,11,14,17),
EPA=20:5(5,8,11,14,17) and DPA=22:5(7,10,13,16,19). Fatty acids
listed as "others" include: 18:2(6,9), 20:0, 20:1(11), 20:2(8,11)
and 20:3 (5,11,14). Each of these fatty acids is present at a
relative abundance of less than 2% of the total fatty acids. In all
of the top lines, GLA is not present or is present at levels less
than 0.2%.
TABLE-US-00017 TABLE 13 Accumulation Of Long Chain PUFAs In Lines
Transformed With pKR786 And pKR669 (Averages of 10 embryos per
line) Line 16:0 18:0 18:1 LA GLA ALA STA EDA DGLA ARA AFS 4314-2-1
17.0 2.6 15.6 16.8 0.1 20.4 0.3 2.7 1.2 0.1 AFS 4310-1-2 16.7 2.4
14.9 15.5 0.1 17.0 0.9 4.4 1.4 0.4 AFS 4310-5-6 15.7 3.0 15.7 17.6
0.0 10.1 0.7 7.4 1.7 0.2 AFS 4310-1-8 15.2 2.7 16.4 15.2 0.1 17.2
0.8 4.7 1.6 0.5 AFS 4314-6-1 14.0 3.1 12.3 17.0 0.1 8.5 0.7 11.2
2.4 0.4 AFS 4314-5-6 15.6 2.7 12.9 4.6 0.0 28.0 1.2 1.7 1.0 0.7 AFS
4310-7-5 17.3 1.9 9.6 16.0 0.0 22.9 0.8 2.4 2.1 1.9 AFS 4310-1-9
14.8 2.8 13.8 12.8 0.0 17.2 0.7 4.8 1.2 0.2 AFS 4314-3-4 16.1 2.5
12.6 14.9 0.1 18.6 0.3 3.4 1.4 0.2 AFS 4310-5-2 15.8 2.5 8.5 15.8
0.1 17.5 0.6 4.2 2.4 0.8 Fatty acid compositions listed in Table 13
are expressed as wt. %. 16:0 = Palmitic acid, 18:0 = Stearic acid,
18:1 = Oleic acid, LA = Linoleic acid, GLA = .gamma.-Linoleic acid,
ALA = alpha-Linolenic acid, STA = Stearidonic acid, EDA =
Eicosadienoic acid, DGLA = Dihomo-.gamma.-Linoleic, ARA =
Arachidonic acid.
TABLE-US-00018 TABLE 14 Accumulation Of Long Chain PUFAs In Lines
Transformed With pKR786 And pKR669 (Averages of 10 embryos per
line) Line EtrA 20:4(5, 11, 14, 17) ETA EPA DPA Other AFS 4314-2-1
2.4 2.1 4.0 13.6 0.1 1.0 AFS 4310-1-2 3.6 3.6 3.1 14.1 0.4 1.6 AFS
4310-5-6 3.8 2.8 4.2 15.1 0.4 1.4 AFS 4310-1-8 2.9 2.7 3.1 15.3 0.2
1.6 AFS 4314-6-1 4.8 3.0 4.8 15.6 0.2 1.9 AFS 4314-5-6 5.3 3.1 5.1
16.2 0.3 1.5 AFS 4310-7-5 2.4 1.6 4.0 16.6 0.1 0.5 AFS 4310-1-9 4.5
4.8 3.6 16.8 0.3 1.8 AFS 4314-3-4 3.3 3.2 4.6 16.9 0.4 1.6 AFS
4310-5-2 3.0 2.1 4.6 21.2 0.2 0.7 Fatty acid compositions listed in
Table 14 are expressed as wt. %. EtrA = Eicosatrienoic acid, ETA =
Eicosa-tetraenoic acid, EPA = Eicosa-pentaenoic acid, DPA =
Docosa-pentaenoic acid
TABLE-US-00019 TABLE 15 Accumulation Of Long Chain PUFAs In Line
AFS 4310-5-2 Transformed With pKR786 And pKR669 Embryo # 16:0 18:0
18:1 LA GLA ALA STA EDA DGLA ARA 1 16.9 2.1 8.4 20.4 0.0 18.1 0.0
2.0 1.9 0.5 2 16.3 1.8 5.3 15.6 0.1 22.3 0.4 2.2 1.9 0.9 3 17.4 3.6
10.9 14.4 0.1 21.4 0.4 5.3 2.1 0.2 4 18.3 2.6 7.9 11.5 0.2 20.4 0.9
6.9 2.9 2.0 5 13.8 3.8 11.1 14.5 0.1 15.0 0.7 7.7 3.0 1.0 6 15.3
3.0 11.8 15.5 0.0 18.6 0.9 4.9 1.6 0.5 7 14.3 2.0 7.6 16.4 0.2 15.9
0.6 3.2 2.1 0.3 8 15.7 2.0 5.0 17.4 0.2 12.5 0.6 3.0 3.1 1.0 9 15.1
2.2 8.1 16.3 0.2 17.0 1.1 2.6 2.8 1.3 10 15.1 1.5 8.5 15.8 0.2 13.7
0.8 4.3 2.3 0.5 Ave 15.8 2.5 8.5 15.8 0.1 17.5 0.6 4.2 2.4 0.8
Fatty acid compositions listed in Table 13 are expressed as wt. %.
16:0 = Palmitic acid, 18:0 = Stearic acid, 18:1 = Oleic acid, LA =
Linoleic acid, GLA = .gamma.-Linoleic acid, ALA = alpha-Linolenic
acid, STA = Stearidonic acid, EDA = Eicosadienoic acid, DGLA =
Dihomo-.gamma.-Linoleic, ARA = Arachidonic acid.
TABLE-US-00020 TABLE 16 Accumulation Of Long Chain PUFAs In Line
AFS 4310-5-2 Transformed With pKR786 And pKR669 Embryo # EtrA
20:4(5, 11, 14, 17) ETA EPA DPA Other 1 1.9 2.1 4.5 21.1 0.0 0.0 2
2.5 2.0 5.1 22.8 0.3 0.6 3 2.9 1.0 4.7 14.7 0.0 0.6 4 5.6 1.9 4.1
13.6 0.1 1.0 5 3.7 2.4 3.3 18.5 0.1 1.2 6 4.1 2.5 3.6 16.4 0.1 1.1
7 2.7 2.6 5.6 25.7 0.3 0.6 8 2.0 2.3 4.6 29.4 0.6 0.7 9 1.8 2.0 4.2
24.0 0.3 0.9 10 2.9 2.2 5.9 25.6 0.2 0.6 Ave 3.0 2.1 4.6 21.2 0.2
0.7 Fatty acid compositions listed in Table 14 are expressed as wt.
%. EtrA = Eicosatrienoic acid, ETA = Eicosa-tetraenoic acid, EPA =
Eicosa-pentaenoic acid, DPA = Docosa-pentaenoic acid
Sequence CWU 1
1
15111271DNAEuglena gracilismisc_feature(4)..(1269)Eg5 delta-8
desaturase 1gaaatgaagt caaagcgcca agcgcttccc cttacaattg atggaacaac
atatgatgtg 60tctgcctggg tcaatttcca ccctggtggt gcggaaatta tagagaatta
ccaaggaagg 120gatgccactg atgccttcat ggttatgcac tctcaagaag
ccttcgacaa gctcaagcgc 180atgcccaaaa tcaatcccag ttctgagttg
ccaccccagg ctgcagtgaa tgaagctcaa 240gaggatttcc ggaagctccg
agaagagttg atcgcaactg gcatgtttga tgcctccccc 300ctctggtact
catacaaaat cagcaccaca ctgggccttg gagtgctggg ttatttcctg
360atggttcagt atcagatgta tttcattggg gcagtgttgc ttgggatgca
ctatcaacag 420atgggctggc tttctcatga catttgccac caccagactt
tcaagaaccg gaactggaac 480aacctcgtgg gactggtatt tggcaatggt
ctgcaaggtt tttccgtgac atggtggaag 540gacagacaca atgcacatca
ttcggcaacc aatgttcaag ggcacgaccc tgatattgac 600aacctccccc
tcttagcctg gtctgaggat gacgtcacac gggcgtcacc gatttcccgc
660aagctcattc agttccagca gtactatttc ttggtcatct gtatcttgtt
gcggttcatt 720tggtgtttcc agagcgtgtt gaccgtgcgc agtttgaagg
acagagataa ccaattctat 780cgctctcagt ataagaagga ggccattggc
ctcgccctgc actggacctt gaagaccctg 840ttccacttat tctttatgcc
cagcatcctc acatcgctgt tggtgttttt cgtttcggag 900ctggttggcg
gcttcggcat tgcgatcgtg gtgttcatga accactaccc actggagaag
960atcggggact cagtctggga tggccatgga ttctcggttg gccagatcca
tgagaccatg 1020aacattcggc gagggattat cacagattgg tttttcggag
gcttgaatta ccagattgag 1080caccatttgt ggccgaccct ccctcgccac
aacctgacag cggttagcta ccaggtggaa 1140cagctgtgcc agaagcacaa
cctgccgtat cggaacccgc tgccccatga agggttggtc 1200atcctgctgc
gctatctggc ggtgttcgcc cggatggcgg agaagcaacc cgcggggaag
1260gctctataag g 12712421PRTEuglena gracilis 2Met Lys Ser Lys Arg
Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr1 5 10 15Tyr Asp Val Ser
Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile 20 25 30Ile Glu Asn
Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met 35 40 45His Ser
Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn 50 55 60Pro
Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu65 70 75
80Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp
85 90 95Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly
Leu 100 105 110Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met
Tyr Phe Ile 115 120 125Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln
Met Gly Trp Leu Ser 130 135 140His Asp Ile Cys His His Gln Thr Phe
Lys Asn Arg Asn Trp Asn Asn145 150 155 160Leu Val Gly Leu Val Phe
Gly Asn Gly Leu Gln Gly Phe Ser Val Thr 165 170 175Trp Trp Lys Asp
Arg His Asn Ala His His Ser Ala Thr Asn Val Gln 180 185 190Gly His
Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu 195 200
205Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe
210 215 220Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe
Ile Trp225 230 235 240Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu
Lys Asp Arg Asp Asn 245 250 255Gln Phe Tyr Arg Ser Gln Tyr Lys Lys
Glu Ala Ile Gly Leu Ala Leu 260 265 270His Trp Thr Leu Lys Thr Leu
Phe His Leu Phe Phe Met Pro Ser Ile 275 280 285Leu Thr Ser Leu Leu
Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe 290 295 300Gly Ile Ala
Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile305 310 315
320Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His
325 330 335Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe
Phe Gly 340 345 350Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro
Thr Leu Pro Arg 355 360 365His Asn Leu Thr Ala Val Ser Tyr Gln Val
Glu Gln Leu Cys Gln Lys 370 375 380His Asn Leu Pro Tyr Arg Asn Pro
Leu Pro His Glu Gly Leu Val Ile385 390 395 400Leu Leu Arg Tyr Leu
Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro 405 410 415Ala Gly Lys
Ala Leu 42031271DNAEuglena gracilismisc_feature(4)..(1269)Eg12
delta-8 desaturase 3gaaatgaagt caaagcgcca agcgcttccc cttacaattg
atggaacaac atatgatgtg 60tctgcctggg tcaatttcca ccctggtggt gcggaaatta
tagagaatta ccaaggaagg 120gatgccactg atgccttcat ggttatgcac
tctcaagaag ccttcgacaa gctcaagcgc 180atgcccaaaa tcaatcccag
ttctgagttg ccaccccagg ctgcagtgaa tgaagctcaa 240gaggatttcc
ggaagctccg agaagagttg atcgcaactg gcatgtttga tgcctccccc
300ctctggtact catacaaaat cagcaccaca ctgggccttg gagtgctggg
ttatttcctg 360atggttcagt atcagatgta tttcattggg gcagtgttgc
ttgggatgca ctatcaacag 420atgggctggc tttctcatga catttgccac
caccagactt tcaagaaccg gaactggaac 480aacctcgtgg gactggtatt
tggcaatggt ctgcaaggtt tttccgtgac atggtggaag 540gacagacaca
atgcacatca ttcggcaacc aatgttcaag ggcacgaccc tgatattgac
600aacctccccc tcttagcctg gtctgaggat gacgtcacac gggcgtcacc
gatttcccgc 660aagctcattc agttccagca gtactatttc ttggtcatct
gtatcttgtt gcggttcatt 720tggtgtttcc agagcgtgtt gaccgtgcgc
agtttgaagg acagagataa ccaattctat 780cgctctcagt ataagaagga
ggccattggc ctcgccctgc actggacctt gaaggccctg 840ttccacttat
tctttatgcc cagcatcctc acatcgctgt tggtgttttt cgtttcggag
900ctggttggcg gcttcggcat tgcgatcgtg gtgttcatga accactaccc
actggagaag 960atcggggact cagtctggga tggccatgga ttctcggttg
gccagatcca tgagaccatg 1020aacattcggc gagggattat cacagattgg
tttttcggag gcttgaatta ccagattgag 1080caccatttgt ggccgaccct
ccctcgccac aacctgacag cggttagcta ccaggtggaa 1140cagctgtgcc
agaagcacaa cctgccgtat cggaacccgc tgccccatga agggttggtc
1200atcctgctgc gctatctggc ggtgttcgcc cggatggcgg agaagcaacc
cgcggggaag 1260gctctataag g 12714421PRTEuglena gracilis 4Met Lys
Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr1 5 10 15Tyr
Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile 20 25
30Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met
35 40 45His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile
Asn 50 55 60Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala
Gln Glu65 70 75 80Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr
Gly Met Phe Asp 85 90 95Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser
Thr Thr Leu Gly Leu 100 105 110Gly Val Leu Gly Tyr Phe Leu Met Val
Gln Tyr Gln Met Tyr Phe Ile 115 120 125Gly Ala Val Leu Leu Gly Met
His Tyr Gln Gln Met Gly Trp Leu Ser 130 135 140His Asp Ile Cys His
His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn145 150 155 160Leu Val
Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr 165 170
175Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln
180 185 190Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp
Ser Glu 195 200 205Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys
Leu Ile Gln Phe 210 215 220Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile
Leu Leu Arg Phe Ile Trp225 230 235 240Cys Phe Gln Ser Val Leu Thr
Val Arg Ser Leu Lys Asp Arg Asp Asn 245 250 255Gln Phe Tyr Arg Ser
Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu 260 265 270His Trp Thr
Leu Lys Ala Leu Phe His Leu Phe Phe Met Pro Ser Ile 275 280 285Leu
Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe 290 295
300Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys
Ile305 310 315 320Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val
Gly Gln Ile His 325 330 335Glu Thr Met Asn Ile Arg Arg Gly Ile Ile
Thr Asp Trp Phe Phe Gly 340 345 350Gly Leu Asn Tyr Gln Ile Glu His
His Leu Trp Pro Thr Leu Pro Arg 355 360 365His Asn Leu Thr Ala Val
Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys 370 375 380His Asn Leu Pro
Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile385 390 395 400Leu
Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro 405 410
415Ala Gly Lys Ala Leu 42051275DNAEuglena
gracilismisc_feature(14)..(1273)non-functional delta-8 desaturase
5attttttttc gaaatgaagt caaagcgcca agcgctatcc cccttacaat tgatggaaca
60aacatatgat gtggtcaatt tccaccctgg tggtgcggaa attatagaga attaccaagg
120aagggatgcc actgatgcct tcatggttat gcactttcaa gaagccttcg
acaagctcaa 180gcgcatgccc aaaatcaatc ccagttttga gttgccaccc
caggctgcag tgaatgaagc 240tcaagaggat ttccggaagc tccgagaaga
gttgatcgca actggcatgt ttgatgcctc 300ccccctctgg tactcataca
aaatcagcac cacactgggc cttggagtgc tgggttattt 360cctgatggtt
cagtatcaga tgtatttcat tggggcagtg ttgcttggga tgcactatca
420acagatgggc tggctttctc atgacatttg ccaccaccag actttcaaga
accggaactg 480gaacaacctc gtgggactgg tatttggcaa tggtctgcaa
ggtttttccg tgacatgttg 540gaaggacaga cacaatgcac atcattcggc
aaccaatgtt caagggcacg accctgatat 600tgacaacctc ccccccttag
cctggtctga ggatgacgtc acacgggcgt caccgatttc 660ccgcaagctc
attcagttcc agcagtacta tttcttggtc atctgtatct tgttgcggtt
720catttggtgt ttccagtgcg tgttgaccgt gcgcagtttg aaggacagag
ataaccaatt 780ctatcgctct cagtataaga aggaggccat tggcctcgcc
ctgcactgga ccttgaaggc 840cctgttccac ttattcttta tgcccagcat
cctcacatcg ctgttggtgt ttttcgtttc 900ggagctggtt ggcggcttcg
gcattgcgat cgtggtgttc atgaaccact acccactgga 960gaagatcggg
gacccagtct gggatggcca tggattctcg gttggccaga tccatgagac
1020catgaacatt cggcgaggga ttatcacaga ttggtttttc ggaggcttga
attaccagat 1080tgagcaccat ttgtggccga ccctccctcg ccacaacctg
acagcggtta gctaccaggt 1140ggaacagctg tgccagaagc acaacctgcc
gtatcggaac ccgctgcccc atgaagggtt 1200ggtcatcctg ctgcgctatc
tggcggtgtt cgcccggatg gcggagaagc aacccgcggg 1260gaaggctcta taagg
12756419PRTEuglena gracilis 6Met Lys Ser Lys Arg Gln Ala Leu Ser
Pro Leu Gln Leu Met Glu Gln1 5 10 15Thr Tyr Asp Val Val Asn Phe His
Pro Gly Gly Ala Glu Ile Ile Glu 20 25 30Asn Tyr Gln Gly Arg Asp Ala
Thr Asp Ala Phe Met Val Met His Phe 35 40 45Gln Glu Ala Phe Asp Lys
Leu Lys Arg Met Pro Lys Ile Asn Pro Ser 50 55 60Phe Glu Leu Pro Pro
Gln Ala Ala Val Asn Glu Ala Gln Glu Asp Phe65 70 75 80Arg Lys Leu
Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp Ala Ser 85 90 95Pro Leu
Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu Gly Val 100 105
110Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile Gly Ala
115 120 125Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser
His Asp 130 135 140Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp
Asn Asn Leu Val145 150 155 160Gly Leu Val Phe Gly Asn Gly Leu Gln
Gly Phe Ser Val Thr Cys Trp 165 170 175Lys Asp Arg His Asn Ala His
His Ser Ala Thr Asn Val Gln Gly His 180 185 190Asp Pro Asp Ile Asp
Asn Leu Pro Pro Leu Ala Trp Ser Glu Asp Asp 195 200 205Val Thr Arg
Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe Gln Gln 210 215 220Tyr
Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp Cys Phe225 230
235 240Gln Cys Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn Gln
Phe 245 250 255Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala
Leu His Trp 260 265 270Thr Leu Lys Ala Leu Phe His Leu Phe Phe Met
Pro Ser Ile Leu Thr 275 280 285Ser Leu Leu Val Phe Phe Val Ser Glu
Leu Val Gly Gly Phe Gly Ile 290 295 300Ala Ile Val Val Phe Met Asn
His Tyr Pro Leu Glu Lys Ile Gly Asp305 310 315 320Pro Val Trp Asp
Gly His Gly Phe Ser Val Gly Gln Ile His Glu Thr 325 330 335Met Asn
Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly Gly Leu 340 345
350Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg His Asn
355 360 365Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys
His Asn 370 375 380Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu
Val Ile Leu Leu385 390 395 400Arg Tyr Leu Ala Val Phe Ala Arg Met
Ala Glu Lys Gln Pro Ala Gly 405 410 415Lys Ala Leu 7422PRTEuglena
gracilis 7Met Lys Ser Lys Arg Gln Ala Leu Ser Pro Leu Gln Leu Met
Glu Gln1 5 10 15Thr Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly
Gly Ala Glu 20 25 30Ile Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp
Ala Phe Met Val 35 40 45Met His Phe Gln Glu Ala Phe Asp Lys Leu Lys
Arg Met Pro Lys Ile 50 55 60Asn Pro Ser Phe Glu Leu Pro Pro Gln Ala
Ala Val Asn Glu Ala Gln65 70 75 80Glu Asp Phe Arg Lys Leu Arg Glu
Glu Leu Ile Ala Thr Gly Met Phe 85 90 95Asp Ala Ser Pro Leu Trp Tyr
Ser Tyr Lys Ile Ser Thr Thr Leu Gly 100 105 110Leu Gly Val Leu Gly
Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe 115 120 125Ile Gly Ala
Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu 130 135 140Ser
His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn145 150
155 160Asn Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser
Val 165 170 175Thr Cys Trp Lys Asp Arg His Asn Ala His His Ser Ala
Thr Asn Val 180 185 190Gln Gly His Asp Pro Asp Ile Asp Asn Leu Pro
Pro Leu Ala Trp Ser 195 200 205Glu Asp Asp Val Thr Arg Ala Ser Pro
Ile Ser Arg Lys Leu Ile Gln 210 215 220Phe Gln Gln Tyr Tyr Phe Leu
Val Ile Cys Ile Leu Leu Arg Phe Ile225 230 235 240Trp Cys Phe Gln
Cys Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp 245 250 255Asn Gln
Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala 260 265
270Leu His Trp Thr Leu Lys Ala Leu Phe His Leu Phe Phe Met Pro Ser
275 280 285Ile Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val
Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe Met Asn His Tyr
Pro Leu Glu Lys305 310 315 320Ile Gly Asp Pro Val Trp Asp Gly His
Gly Phe Ser Val Gly Gln Ile 325 330 335His Glu Thr Met Asn Ile Arg
Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350Gly Gly Leu Asn Tyr
Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365Arg His Asn
Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln 370 375 380Lys
His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val385 390
395 400Ile Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys
Gln 405 410 415Pro Ala Gly Lys Ala Leu 420819DNAArtificial
SequencePrimer Eg5-1 8gaaatgaagt caaagcgcc 19919DNAArtificial
SequencePrimer Eg3-3 9ccttatagag ccttccccg 191022DNAArtificial
SequencePrimer T7 10gtaatacgac tcactatagg gc 221119DNAArtificial
SequencePrimer M13-28Rev 11ggaaacagct atgaccatg 191219DNAArtificial
SequencePrimer Eg3-2 12ttggcaatgg tctgcaagg 191319DNAArtificial
SequencePrimer Eg5-2 13aatgttcatg gtctcatgg
191437DNAArtificial SequencePrimer 14ggatctcctg caggatctgg
ccggccggat ctcgtac 371524DNAArtificial SequencePrimer RPB2forward
15gcggccgcat ggagtcgatt gcgc 241624DNAArtificial SequencePrimer
RPB2reverse 16gcggccgctt actgcaactt cctt 241734DNAArtificial
SequencePrimer 17aagcttgcat gcctgcaggt cgactcgacg tacg
3418280DNAArtificial Sequencesoybean albumin transcription
terminator 18tctagaggat ccaaggccgc gaagttaaaa gcaatgttgt cacttgtcgt
actaacacat 60gatgtgatag tttatgctag ctagctataa cataagctgt ctctgagtgt
gttgtatatt 120aataaagatc atcactggtg aatggtgatc gtgtacgtac
cctacttagt aggcaatgga 180agcacttaga gtgtgctttg tgcatggcct
tgcctctgtt ttgagacttt tgtaatgttt 240tcgagtttaa atctttgcct
ttgcgtacgt gggcggatcc 2801932DNAArtificial SequencePrimer oSalb-12
19tttggatcct ctagacgtac gcaaaggcaa ag 322036DNAArtificial
SequencePrimer oSalb-13 20aaaggatcca aggccgcgaa gttaaaagca atgttg
362124DNAArtificial SequencePrimer GSP1 21gccccccatc ctttgaaagc
ctgt 242234DNAArtificial SequencePrimer GSP2 22cgcggatccg
agagcctcag catcttgagc agaa 342327DNAArtificial SequencePrimer GSP3
23ggtccaatat ggaacgatga gttgata 272435DNAArtificial SequencePrimer
GSP4 24cgcggatccg ctggaactag aagagagacc taaga 35251408DNAGlycine
maxmisc_featureBD30 promoter 25aactaaaaaa agctctcaaa ttacattttg
agttgtttca ggttccattg ccttattgct 60aaaactccaa ctaaaataac aaatagcaca
tgcaggtgca aacaacacgt tactctgatg 120aaggtgatgt gcctctagca
gtctagctta tgaggctcgc tgcttatcaa cgattcatca 180ttccccaaga
cgtgtacgca gattaaacaa tggacaaaac ttcaatcgat tatagaataa
240taattttaac agtgccgact tttttctgta aacaaaaggc cagaatcata
tcgcacatca 300tcttgaatgc agtgtcgagt ttggaccatt tgagtacaaa
gccaatattg aatgattttt 360cgattttaca tgtgtgaatc agacaaaagt
gcatgcaatc acttgcaagt aaattaagga 420tactaatcta ttcctttcat
tttatatgct ccacttttat ataaaaaaat atacattatt 480atatatgcat
tattaattat tgcagtatta tgctattggt tttatggccc tgctaaataa
540cctaaatgag tctaactatt gcatatgaat caaatgaagg aagaatcatg
atctaaacct 600gagtacccaa tgcaataaaa tgcgtcctat tacctaaact
tcaaacacac attgccatcg 660gacgtataaa ttaatgcata taggttattt
tgagaaaaga aaacatcaaa agctctaaaa 720cttcttttaa ctttgaaata
agctgataaa aatacgcttt aaatcaactg tgtgctgtat 780ataagctgca
atttcacatt ttaccaaacc gaaacaagaa tggtaacagt gaggcaaaaa
840tttgaaaaat gtcctacttc acattcacat caaattaatt acaactaaat
aaataaacat 900cgtgattcaa gcagtaatga aagtcgaaat cagatagaat
atacacgttt aacatcaatt 960gaattttttt ttaaatggat atatacaagt
ttactatttt atatataatg aaaattcatt 1020ttgtgttagc acaaaactta
cagaaagaga taaattttaa ataaagagaa ttatatccaa 1080ttttataatc
caaaataatc aaattaaaga atattggcta gatagaccgg ctttttcact
1140gcccctgctg gataatgaaa attcatatca aaacaataca gaagttctag
tttaataata 1200aaaaagttgg caaactgtca ttccctgttg gtttttaagc
caaatcacaa ttcaattacg 1260tatcagaaat taatttaaac caaatatata
gctacgaggg aacttcttca gtcattacta 1320gctagctcac taatcactat
atatacgaca tgctacaagt gaagtgacca tatcttaatt 1380tcaaatcata
aaattcttcc accaagtt 140826690DNAGlycine maxmisc_featureGlycinin Gy1
promoter 26tagcctaagt acgtactcaa aatgccaaca aataaaaaaa aagttgcttt
aataatgcca 60aaacaaatta ataaaacact tacaacaccg gatttttttt aattaaaatg
tgccatttag 120gataaatagt taatattttt aataattatt taaaaagccg
tatctactaa aatgattttt 180atttggttga aaatattaat atgtttaaat
caacacaatc tatcaaaatt aaactaaaaa 240aaaaataagt gtacgtggtt
aacattagta cagtaatata agaggaaaat gagaaattaa 300gaaattgaaa
gcgagtctaa tttttaaatt atgaacctgc atatataaaa ggaaagaaag
360aatccaggaa gaaaagaaat gaaaccatgc atggtcccct cgtcatcacg
agtttctgcc 420atttgcaata gaaacactga aacacctttc tctttgtcac
ttaattgaga tgccgaagcc 480acctcacacc atgaacttca tgaggtgtag
cacccaaggc ttccatagcc atgcatactg 540aagaatgtct caagctcagc
accctacttc tgtgacgttg tccctcattc accttcctct 600cttccctata
aataaccacg cctcaggttc tccgcttcac aactcaaaca ttctcctcca
660ttggtcctta aacactcatc agtcatcacc 6902736DNAArtificial
SequencePrimer 27cgcggatcct agcctaagta cgtactcaaa atgcca
362841DNAArtificial SequencePrimer 28gaattcgcgg ccgcggtgat
gactgatgag tgtttaagga c 41292012DNAGlycine maxmisc_featureannexin
promoter 29atcttaggcc cttgattata tggtgtttag atggattcac atgcaagttt
ttatttcaat 60cccttttcct ttgaataact gaccaagaac aacaagaaaa aaaaaaaaag
aaaaggatca 120ttttgaaagg atatttttcg ctcctattca aatactgtat
ttttaccaaa aaaactgtat 180ttttcctaca ctctcaagct ttgtttttcg
cttcgactct catgatttcc ttcatatgcc 240aatcactcta tttataaatg
gcataaggta gtgtgaacaa ttgcaaagct tgtcatcaaa 300agcttgcaat
gtacaaatta atgtttttca tgcctttcaa aattatctgc accccctagc
360tattaatcta acatctaagt aaggctagtg aattttttcg aatagtcatg
cagtgcatta 420atttccccgt gactattttg gctttgactc caacactggc
cccgtacatc cgtccctcat 480tacatgaaaa gaaatattgt ttatattctt
aattaaaaat attgtccctt ctaaattttc 540atatagttaa ttattatatt
acttttttct ctattctatt agttctattt tcaaattatt 600atttatgcat
atgtaaagta cattatattt ttgctatata cttaaatatt tctaaattat
660taaaaaaaga ctgatatgaa aaatttattc tttttaaagc tatatcattt
tatatatact 720ttttcttttc ttttctttca ttttctattc aatttaataa
gaaataaatt ttgtaaattt 780ttatttatca atttataaaa atattttact
ttatatgttt tttcacattt ttgttaaaca 840aatcatatca ttatgattga
aagagaggaa attgacagtg agtaataagt gatgagaaaa 900aaatgtgtta
tttcctaaaa aaaacctaaa caaacatgta tctactctct atttcatcta
960tctctcattt catttttctc tttatctctt tctttatttt tttatcatat
catttcacat 1020taattatttt tactctcttt attttttctc tctatccctc
tcttatttcc actcatatat 1080acactccaaa attggggcat gcctttatca
ctactctatc tcctccacta aatcatttaa 1140atgaaactga aaagcattgg
caagtctcct cccctcctca agtgatttcc aactcagcat 1200tggcatctga
ttgattcagt atatctattg catgtgtaaa agtctttcca caatacataa
1260ctattaatta atcttaaata aataaaggat aaaatatttt tttttcttca
taaaattaaa 1320atatgttatt ttttgtttag atgtatattc gaataaatct
aaatatatga taatgatttt 1380ttatattgat taaacatata atcaatatta
aatatgatat ttttttatat aggttgtaca 1440cataatttta taaggataaa
aaatatgata aaaataaatt ttaaatattt ttatatttac 1500gagaaaaaaa
aatattttag ccataaataa atgaccagca tattttacaa ccttagtaat
1560tcataaattc ctatatgtat atttgaaatt aaaaacagat aatcgttaag
ggaaggaatc 1620ctacgtcatc tcttgccatt tgtttttcat gcaaacagaa
agggacgaaa aaccacctca 1680ccatgaatca ctcttcacac catttttact
agcaaacaag tctcaacaac tgaagccagc 1740tctctttccg tttcttttta
caacactttc tttgaaatag tagtattttt ttttcacatg 1800atttattaac
gtgccaaaag atgcttattg aatagagtgc acatttgtaa tgtactacta
1860attagaacat gaaaaagcat tgttctaaca cgataatcct gtgaaggcgt
taactccaaa 1920gatccaattt cactatataa attgtgacga aagcaaaatg
aattcacata gctgagagag 1980aaaggaaagg ttaactaaga agcaatactt ca
20123037DNAArtificial SequencePrimer 30cgcggatcca tcttaggccc
ttgattatat ggtgttt 373143DNAArtificial SequencePrimer 31gaattcgcgg
ccgctgaagt attgcttctt agttaacctt tcc 433241DNAArtificial
SequencePrimer 32cgcggatcca actaaaaaaa gctctcaaat tacattttga g
413344DNAArtificial SequencePrimer 33gaattcgcgg ccgcaacttg
gtggaagaat tttatgattt gaaa 443429DNAArtificial SequencePrimer oKTi5
34atctagacgt acgtcctcga agagaaggg 293522DNAArtificial
SequencePrimer oKTi6 35ttctagacgt acggatataa tg 223617DNAArtificial
SequencePrimer oSBD30-1 36tgcggccgca tgagccg 173732DNAArtificial
SequencePrimer oSBD30-2 37acgtacggta ccatctgcta atattttaaa tc
323832DNAArtificial SequencePrimer oCGR5-1 38ttgcggccgc aaaccatggc
tgctgctccc ag 323924DNAArtificial SequencePrimer oCGR5-2
39aagcggccgc ttactgcgcc ttac 244029DNAArtificial SequencePrimer
oSGly-1 40ttcctgcagg ctagcctaag tacgtactc 294121DNAArtificial
SequencePrimer oSGly-2 41aagcggccgc ggtgatgact g
214236DNAArtificial SequencePrimer LegPro5' 42tttctagacg tacgtccctt
cttatctttg atctcc 364334DNAArtificial SequencePrimer LegPro3'
43gcggccgcag ttggatagaa tatatgtttg tgac 344441DNAArtificial
SequencePrimer LegTerm5' 44ctatccaact gcggccgcat ttcgcaccaa
atcaatgaaa g 414538DNAArtificial SequencePrimer LegTerm3'
45aatctagacg tacgtgaagg ttaaacatgg tgaatatg 384624DNAArtificial
SequencePrimer CGR4forward 46gcggccgcat gggaacggac caag
244724DNAArtificial SequencePrimer CGR4reverse 47gcggccgcct
actcttcctt ggga 24481270DNAArtificial SequenceD8S-1 Synthetic gene
codon-optimized for expression in Yarrowia lipolytica 48ccatggagtc
caagcgacag gctctgtctc ccctccagct gatggaacag acctacgacg 60tcgtgaactt
ccaccctggt ggagctgaaa tcattgagaa ctaccaggga cgagatgcta
120ctgacgcctt catggttatg cactttcagg aagccttcga caagctcaag
cgaatgccca 180agatcaaccc ctcctttgag ctgcctcccc aggctgccgt
caacgaagct caggaggatt 240tccgaaagct ccgagaagag ctgatcgcca
ctggcatgtt tgacgcctct cccctctggt 300actcgtacaa gatctccacc
accctgggtc ttggcgtgct tggatacttc ctgatggtcc 360agtaccagat
gtacttcatt ggtgctgtgc tgctcggtat gcactaccag caaatgggat
420ggctgtctca tgacatctgc caccaccaga ccttcaagaa ccgaaactgg
aataacctcg 480tgggtctggt ctttggcaac ggactccagg gcttctccgt
gacctgttgg aaggacagac 540acaacgccca tcattctgct accaacgttc
agggtcacga tcccgacatt gataacctgc 600ctcccctcgc ctggtccgag
gacgatgtca ctcgagcttc tcccatctcc cgaaagctca 660ttcagttcca
acagtactat ttcctggtca tctgtattct cctgcgattc atctggtgtt
720tccagtgcgt gctgaccgtt cgatccctca aggaccgaga caaccagttc
taccgatctc 780agtacaagaa agaggccatt ggactcgctc tgcactggac
tctcaaggct ctgttccacc 840tcttctttat gccctccatc ctgacctcgc
tcctggtgtt ctttgtttcc gagctcgtcg 900gtggcttcgg aattgccatc
gtggtcttca tgaaccacta ccctctggag aagatcggtg 960atcccgtctg
ggacggacat ggcttctctg tgggtcagat ccatgagacc atgaacattc
1020gacgaggcat cattactgac tggttctttg gaggcctgaa ctaccagatc
gagcaccatc 1080tctggcccac cctgcctcga cacaacctca ctgccgtttc
ctaccaggtg gaacagctgt 1140gccagaagca caacctcccc taccgaaacc
ctctgcccca tgaaggtctc gtcatcctgc 1200tccgatacct ggccgtgttc
gctcgaatgg ccgagaagca gcccgctggc aaggctctct 1260aagcggccgc
127049104DNAArtificial SequencePrimer D8-1A 49atggagtcca agcgacaggc
tctgtctccc ctccagctga tggaacagac ctacgacgtc 60gtgaacttcc accctggtgg
agctgaaatc attgagaact acca 10450104DNAArtificial SequencePrimer
D8-1B 50tccctggtag ttctcaatga tttcagctcc accagggtgg aagttcacga
cgtcgtaggt 60ctgttccatc agctggaggg gagacagagc ctgtcgcttg gact
10451102DNAArtificial SequencePrimer D8-2A 51gggacgagat gctactgacg
ccttcatggt tatgcacttt caggaagcct tcgacaagct 60caagcgaatg cccaagatca
acccctcctt tgagctgcct cc 10252102DNAArtificial SequencePrimer D8-2B
52ctggggaggc agctcaaagg aggggttgat cttgggcatt cgcttgagct tgtcgaaggc
60ttcctgaaag tgcataacca tgaaggcgtc agtagcatct cg
10253101DNAArtificial SequencePrimer D8-3A 53ccaggctgcc gtcaacgaag
ctcaggagga tttccgaaag ctccgagaag agctgatcgc 60cactggcatg tttgacgcct
ctcccctctg gtactcgtac a 10154101DNAArtificial SequencePrimer D8-3B
54atcttgtacg agtaccagag gggagaggcg tcaaacatgc cagtggcgat cagctcttct
60cggagctttc ggaaatcctc ctgagcttcg ttgacggcag c
10155101DNAArtificial SequencePrimer D8-4A 55ccaccaccct gggtcttggc
gtgcttggat acttcctgat ggtccagtac cagatgtact 60tcattggtgc tgtgctgctc
ggtatgcact accagcaaat g 10156101DNAArtificial SequencePrimer D8-4B
56atcccatttg ctggtagtgc ataccgagca gcacagcacc aatgaagtac atctggtact
60ggaccatcag gaagtatcca agcacgccaa gacccagggt g
10157104DNAArtificial SequencePrimer D8-5A 57ggatggctgt ctcatgacat
ctgccaccac cagaccttca agaaccgaaa ctggaataac 60ctcgtgggtc tggtctttgg
caacggactc cagggcttct ccgt 10458104DNAArtificial SequencePrimer
D8-5B 58ggtcacggag aagccctgga gtccgttgcc aaagaccaga cccacgaggt
tattccagtt 60tcggttcttg aaggtctggt ggtggcagat gtcatgagac agcc
10459101DNAArtificial SequencePrimer D8-6A 59gacctgttgg aaggacagac
acaacgccca tcattctgct accaacgttc agggtcacga 60tcccgacatt gataacctgc
ctcccctcgc ctggtccgag g 10160101DNAArtificial SequencePrimer D8-6B
60tcgtcctcgg accaggcgag gggaggcagg ttatcaatgt cgggatcgtg accctgaacg
60ttggtagcag aatgatgggc gttgtgtctg tccttccaac a
1016195DNAArtificial SequencePrimer D8-7A 61tcactcgagc ttctcccatc
tcccgaaagc tcattcagtt ccaacagtac tatttcctgg 60tcatctgtat tctcctgcga
ttcatctggt gtttc 956295DNAArtificial SequencePrimer D8-7B
62actggaaaca ccagatgaat cgcaggagaa tacagatgac caggaaatag tactgttgga
60actgaatgag ctttcgggag atgggagaag ctcga 956389DNAArtificial
SequencePrimer D8-8A 63cagtgcgtgc tgaccgttcg atccctcaag gaccgagaca
accagttcta ccgatctcag 60tacaagaaag aggccattgg actcgctct
896489DNAArtificial SequencePrimer D8-8B 64gtgcagagcg agtccaatgg
cctctttctt gtactgagat cggtagaact ggttgtctcg 60gtccttgagg gatcgaacgg
tcagcacgc 896585DNAArtificial SequencePrimer D8-9A 65gcactggact
ctcaaggctc tgttccacct cttctttatg ccctccatcc tgacctcgct 60cctggtgttc
tttgtttccg agctc 856685DNAArtificial SequencePrimer D8-9B
66cgacgagctc ggaaacaaag aacaccagga gcgaggtcag gatggagggc ataaagaaga
60ggtggaacag agccttgaga gtcca 856791DNAArtificial SequencePrimer
D8-10A 67gtcggtggct tcggaattgc catcgtggtc ttcatgaacc actaccctct
ggagaagatc 60ggtgatcccg tctgggacgg acatggcttc t 916891DNAArtificial
SequencePrimer D8-10B 68acagagaagc catgtccgtc ccagacggga tcaccgatct
tctccagagg gtagtggttc 60atgaagacca cgatggcaat tccgaagcca c
916992DNAArtificial SequencePrimer D8-11A 69ctgtgggtca gatccatgag
accatgaaca ttcgacgagg catcattact gactggttct 60ttggaggcct gaactaccag
atcgagcacc at 927092DNAArtificial SequencePrimer D8-11B
70agagatggtg ctcgatctgg tagttcaggc ctccaaagaa ccagtcagta atgatgcctc
60gtcgaatgtt catggtctca tggatctgac cc 927193DNAArtificial
SequencePrimer D8-12A 71ctctggccca ctctgcctcg acacaacctc actgccgttt
cctaccaggt ggaacagctg 60tgccagaagc acaacctccc ctaccgaaac cct
937293DNAArtificial SequencePrimer D8-12B 72gcagagggtt tcggtagggg
aggttgtgct tctggcacag ctgttccacc tggtaggaaa 60cggcagtgag gttgtgtcga
ggcagagtgg gcc 937390DNAArtificial SequencePrimer D8-13A
73ctgccccatg aaggtctcgt catcctgctc cgatacctgg ccgtgttcgc tcgaatggcc
60gagaagcagc ccgctggcaa ggctctctaa 907490DNAArtificial
SequencePrimer D8-13B 74ccgcttagag agccttgcca gcgggctgct tctcggccat
tcgagcgaac acggccaggt 60atcggagcag gatgacgaga ccttcatggg
907538DNAArtificial SequencePrimer D8-1F 75tttccatgga gtccaagcga
caggctctgt ctcccctc 387637DNAArtificial SequencePrimer D8-3R
76tttagatctt gtacgagtac cagaggggag aggcgtc 377741DNAArtificial
SequencePrimer D8-4F 77acaagatctc caccaccctg ggtcttggcg tgcttggata
c 417843DNAArtificial SequencePrimer D8-6R 78tttctcgagt gacatcgtcc
tcggaccagg cgaggggagg cag 437932DNAArtificial SequencePrimer D8-7F
79tcactcgagc ttctcccatc tcccgaaagc tc 328029DNAArtificial
SequencePrimer D8-9R 80cgacgagctc ggaaacaaag aacaccagg
298139DNAArtificial SequencePrimer D8-10F 81tttgagctcg tcggtggctt
cggaattgcc atcgtggtc 398236DNAArtificial SequencePrimer D8-13R
82tttgcggccg cttagagagc cttgccagcg ggctgc 3683309DNAArtificial
Sequence309 bp NcoI/BglII fragment of pT8(1-3) 83catggagtcc
aagcgacagg ctctgtctcc cctccagctg atggaacaga cctacgacgt 60cgtgaacttc
caccctggtg gagctgaaat cattgagaac taccagggac gagatgctac
120tgacgccttc atggttatgc actttcagga agccttcgac aagctcaagc
gaatgcccaa 180gatcaacccc tcctttgagc tgcctcccca ggctgccgtc
aacgaagctc aggaggattt 240ccgaaagctc cgagaagagc tgatcgccac
tggcatgttt gacgcctctc ccctctggta 300ctcgtacaa 30984321DNAArtificial
Sequence321 bp BglII/XhoI fragment of pT8(4-6) 84gatctccacc
accctgggtc ttggcgtgct tggatacttc ctgatggtcc agtaccagat 60gtacttcatt
ggtgctgtgc tgctcggtat gcactaccag caaatgggat ggctgtctca
120tgacatctgc caccaccaga ccttcaagaa ccgaaactgg aataacctcg
tgggtctggt 180ctttggcaac ggactccagg gcttctccgt gacctgttgg
aaggacagac acaacgccca 240tcattctgct accaacgttc agggtcacga
tcccgacatt gataacctgc ctcccctcgc 300ctggtccgag gacgatgtca c
32185264DNAArtificial Sequence264 bp XhoI/SacI fragment of pT8(7-9)
85tcgagcttct cccatctccc gaaagctcat tcagttccaa cagtactatt tcctggtcat
60ctgtattctc ctgcgattca tctggtgttt ccagtgcgtg ctgaccgttc gatccctcaa
120ggaccgagac aaccagttct
accgatctca gtacaagaaa gaggccattg gactcgctct 180gcactggact
ctcaaggctc tgttccacct cttctttatg ccctccatcc tgacctcgct
240cctggtgttc tttgtttccg agct 26486369DNAArtificial Sequence369 bp
Sac1/Not1 fragment of pT8(10-13) 86cgtcggtggc ttcggaattg ccatcgtggt
cttcatgaac cactaccctc tggagaagat 60cggtgatccc gtctgggacg gacatggctt
ctctgtgggt cagatccatg agaccatgaa 120cattcgacga ggcatcatta
ctgactggtt ctttggaggc ctgaactacc agatcgagca 180ccatctctgg
cccaccctgc ctcgacacaa cctcactgcc gtttcctacc aggtggaaca
240gctgtgccag aagcacaacc tcccctaccg aaaccctctg ccccatgaag
gtctcgtcat 300cctgctccga tacctggccg tgttcgctcg aatggccgag
aagcagcccg ctggcaaggc 360tctctaagc 3698734DNAArtificial
SequencePrimer ODMW390 87aagaatcatt caccatgaag tccaagcgac aggc
348834DNAArtificial SequencePrimer ODMW391 88gcctgtcgct tggacttcat
ggtgaatgat tctt 34891852DNAArtificial Sequencechimeric D8S-1::XPR
terminator gene 89cgatcaggag agaccgggtt ggcggcgtat ttgtgtccca
aaaaacagcc ccaattgccc 60caattgaccc caaattgacc cagtagcggg cccaaccccg
gcgagagccc ccttcacccc 120acatatcaaa cctcccccgg ttcccacact
tgccgttaag ggcgtagggt actgcagtct 180ggaatctacg cttgttcaga
ctttgtacta gtttctttgt ctggccatcc gggtaaccca 240tgccggacgc
aaaatagact actgaaaatt tttttgcttt gtggttggga ctttagccaa
300gggtataaaa gaccaccgtc cccgaattac ctttcctctt cttttctctc
tctccttgtc 360aactcacacc cgaaatcgtt aagcatttcc ttctgagtat
aagaatcatt caccatggag 420tccaagcgac aggctctgtc tcccctccag
ctgatggaac agacctacga cgtcgtgaac 480ttccaccctg gtggagctga
aatcattgag aactaccagg gacgagatgc tactgacgcc 540ttcatggtta
tgcactttca ggaagccttc gacaagctca agcgaatgcc caagatcaac
600ccctcctttg agctgcctcc ccaggctgcc gtcaacgaag ctcaggagga
tttccgaaag 660ctccgagaag agctgatcgc cactggcatg tttgacgcct
ctcccctctg gtactcgtac 720aagatctcca ccaccctggg tcttggcgtg
cttggatact tcctgatggt ccagtaccag 780atgtacttca ttggtgctgt
gctgctcggt atgcactacc agcaaatggg atggctgtct 840catgacatct
gccaccacca gaccttcaag aaccgaaact ggaataacct cgtgggtctg
900gtctttggca acggactcca gggcttctcc gtgacctgtt ggaaggacag
acacaacgcc 960catcattctg ctaccaacgt tcagggtcac gatcccgaca
ttgataacct gcctcccctc 1020gcctggtccg aggacgatgt cactcgagct
tctcccatct cccgaaagct cattcagttc 1080caacagtact atttcctggt
catctgtatt ctcctgcgat tcatctggtg tttccagtgc 1140gtgctgaccg
ttcgatccct caaggaccga gacaaccagt tctaccgatc tcagtacaag
1200aaagaggcca ttggactcgc tctgcactgg actctcaagg ctctgttcca
cctcttcttt 1260atgccctcca tcctgacctc gctcctggtg ttctttgttt
ccgagctcgt cggtggcttc 1320ggaattgcca tcgtggtctt catgaaccac
taccctctgg agaagatcgg tgatcccgtc 1380tgggacggac atggcttctc
tgtgggtcag atccatgaga ccatgaacat tcgacgaggc 1440atcattactg
actggttctt tggaggcctg aactaccaga tcgagcacca tctctggccc
1500accctgcctc gacacaacct cactgccgtt tcctaccagg tggaacagct
gtgccagaag 1560cacaacctcc cctaccgaaa ccctctgccc catgaaggtc
tcgtcatcct gctccgatac 1620ctggccgtgt tcgctcgaat ggccgagaag
cagcccgctg gcaaggctct ctaagcggcc 1680gccaccgccg agattccggc
ctcttcggcc gccaagcgac ccgggtggac gtctagaggt 1740acctagcaat
taacagatag tttgccggtg ataattctct taacctccca cactcctttg
1800acataacgat ttatgtaacg aaactgaaat ttgaccagat attgtgtccg cg
1852901898DNAArtificial Sequencechimeric D8S-2::XPR terminator gene
90cgatcaggag agaccgggtt ggcggcgtat ttgtgtccca aaaaacagcc ccaattgccc
60caattgaccc caaattgacc cagtagcggg cccaaccccg gcgagagccc ccttcacccc
120acatatcaaa cctcccccgg ttcccacact tgccgttaag ggcgtagggt
actgcagtct 180ggaatctacg cttgttcaga ctttgtacta gtttctttgt
ctggccatcc gggtaaccca 240tgccggacgc aaaatagact actgaaaatt
tttttgcttt gtggttggga ctttagccaa 300gggtataaaa gaccaccgtc
cccgaattac ctttcctctt cttttctctc tctccttgtc 360aactcacacc
cgaaatcgtt aagcatttcc ttctgagtat aagaatcatt caccatgaag
420tccaagcgac aggctctgtc tcccctccag ctgatggaac agacctacga
cgtcgtgaac 480ttccaccctg gtggagctga aatcattgag aactaccagg
gacgagatgc tactgacgcc 540ttcatggtta tgcactttca ggaagccttc
gacaagctca agcgaatgcc caagatcaac 600ccctcctttg agctgcctcc
ccaggctgcc gtcaacgaag ctcaggagga tttccgaaag 660ctccgagaag
agctgatcgc cactggcatg tttgacgcct ctcccctctg gtactcgtac
720aagatctcca ccaccctggg tcttggcgtg cttggatact tcctgatggt
ccagtaccag 780atgtacttca ttggtgctgt gctgctcggt atgcactacc
agcaaatggg atggctgtct 840catgacatct gccaccacca gaccttcaag
aaccgaaact ggaataacct cgtgggtctg 900gtctttggca acggactcca
gggcttctcc gtgacctgtt ggaaggacag acacaacgcc 960catcattctg
ctaccaacgt tcagggtcac gatcccgaca ttgataacct gcctcccctc
1020gcctggtccg aggacgatgt cactcgagct tctcccatct cccgaaagct
cattcagttc 1080caacagtact atttcctggt catctgtatt ctcctgcgat
tcatctggtg tttccagtgc 1140gtgctgaccg ttcgatccct caaggaccga
gacaaccagt tctaccgatc tcagtacaag 1200aaagaggcca ttggactcgc
tctgcactgg actctcaagg ctctgttcca cctcttcttt 1260atgccctcca
tcctgacctc gctcctggtg ttctttgttt ccgagctcgt cggtggcttc
1320ggaattgcca tcgtggtctt catgaaccac taccctctgg agaagatcgg
tgatcccgtc 1380tgggacggac atggcttctc tgtgggtcag atccatgaga
ccatgaacat tcgacgaggc 1440atcattactg actggttctt tggaggcctg
aactaccaga tcgagcacca tctctggccc 1500accctgcctc gacacaacct
cactgccgtt tcctaccagg tggaacagct gtgccagaag 1560cacaacctcc
cctaccgaaa ccctctgccc catgaaggtc tcgtcatcct gctccgatac
1620ctggccgtgt tcgctcgaat ggccgagaag cagcccgctg gcaaggctct
ctaagcggcc 1680gccaccgcgg cccgagattc cggcctcttc ggccgccaag
cgacccgggt ggacgtctag 1740aggtacctag caattaacag atagtttgcc
ggtgataatt ctcttaacct cccacactcc 1800tttgacataa cgatttatgt
aacgaaactg aaatttgacc agatattgtg tccgcggtgg 1860agctccagct
tttgttccct ttagtgaggg ttaattaa 18989145DNAArtificial SequencePrimer
ODMW392 91gaacagacct acgacgtctc cgcttgggtg aacttccacc ctggt
459245DNAArtificial SequencePrimer ODMW393 92accagggtgg aagttcaccc
aagcggagac gtcgtaggtc tgttc 45931269DNAArtificial SequenceD8S-3
synthetic delta 8-desaturase gene codon-optimized for Yarrowia
lipolytica in pDMW261 93atgaagtcca agcgacaggc tctgtctccc ctccagctga
tggaacagac ctacgacgtc 60tccgcttggg tgaacttcca ccctggtgga gctgaaatca
ttgagaacta ccagggacga 120gatgctactg acgccttcat ggttatgcac
tttcaggaag ccttcgacaa gctcaagcga 180atgcccaaga tcaacccctc
ctttgagctg cctccccagg ctgccgtcaa cgaagctcag 240gaggatttcc
gaaagctccg agaagagctg atcgccactg gcatgtttga cgcctctccc
300ctctggtact cgtacaagat ctccaccacc ctgggtcttg gcgtgcttgg
atacttcctg 360atggtccagt accagatgta cttcattggt gctgtgctgc
tcggtatgca ctaccagcaa 420atgggatggc tgtctcatga catctgccac
caccagacct tcaagaaccg aaactggaat 480aacctcgtgg gtctggtctt
tggcaacgga ctccagggct tctccgtgac ctgttggaag 540gacagacaca
acgcccatca ttctgctacc aacgttcagg gtcacgatcc cgacattgat
600aacctgcctc ccctcgcctg gtccgaggac gatgtcactc gagcttctcc
catctcccga 660aagctcattc agttccaaca gtactatttc ctggtcatct
gtattctcct gcgattcatc 720tggtgtttcc agtgcgtgct gaccgttcga
tccctcaagg accgagacaa ccagttctac 780cgatctcagt acaagaaaga
ggccattgga ctcgctctgc actggactct caaggctctg 840ttccacctct
tctttatgcc ctccatcctg acctcgctcc tggtgttctt tgtttccgag
900ctcgtcggtg gcttcggaat tgccatcgtg gtcttcatga accactaccc
tctggagaag 960atcggtgatc ccgtctggga cggacatggc ttctctgtgg
gtcagatcca tgagaccatg 1020aacattcgac gaggcatcat tactgactgg
ttctttggag gcctgaacta ccagatcgag 1080caccatctct ggcccaccct
gcctcgacac aacctcactg ccgtttccta ccaggtggaa 1140cagctgtgcc
agaagcacaa cctcccctac cgaaaccctc tgccccatga aggtctcgtc
1200atcctgctcc gatacctggc cgtgttcgct cgaatggccg agaagcagcc
cgctggcaag 1260gctctctaa 12699429DNAArtificial SequencePrimer
ODMW404 94cctggtacca tgaagtccaa gcgacaggc 29951272DNAArtificial
Sequencechimeric gene 95catgaagtcc aagcgacagg ctctgtctcc cctccagctg
atggaacaga cctacgacgt 60ctccgcttgg gtgaacttcc accctggtgg agctgaaatc
attgagaact accagggacg 120agatgctact gacgccttca tggttatgca
ctttcaggaa gccttcgaca agctcaagcg 180aatgcccaag atcaacccct
cctttgagct gcctccccag gctgccgtca acgaagctca 240ggaggatttc
cgaaagctcc gagaagagct gatcgccact ggcatgtttg acgcctctcc
300cctctggtac tcgtacaaga tctccaccac cctgggtctt ggcgtgcttg
gatacttcct 360gatggtccag taccagatgt acttcattgg tgctgtgctg
ctcggtatgc actaccagca 420aatgggatgg ctgtctcatg acatctgcca
ccaccagacc ttcaagaacc gaaactggaa 480taacctcgtg ggtctggtct
ttggcaacgg actccagggc ttctccgtga cctgttggaa 540ggacagacac
aacgcccatc attctgctac caacgttcag ggtcacgatc ccgacattga
600taacctgcct cccctcgcct ggtccgagga cgatgtcact cgagcttctc
ccatctcccg 660aaagctcatt cagttccaac agtactattt cctggtcatc
tgtattctcc tgcgattcat 720ctggtgtttc cagtgcgtgc tgaccgttcg
atccctcaag gaccgagaca accagttcta 780ccgatctcag tacaagaaag
aggccattgg actcgctctg cactggactc tcaaggctct 840gttccacctc
ttctttatgc cctccatcct gacctcgctc ctggtgttct ttgtttccga
900gctcgtcggt ggcttcggaa ttgccatcgt ggtcttcatg aaccactacc
ctctggagaa 960gatcggtgat cccgtctggg acggacatgg cttctctgtg
ggtcagatcc atgagaccat 1020gaacattcga cgaggcatca ttactgactg
gttctttgga ggcctgaact accagatcga 1080gcaccatctc tggcccaccc
tgcctcgaca caacctcact gccgtttcct accaggtgga 1140acagctgtgc
cagaagcaca acctccccta ccgaaaccct ctgccccatg aaggtctcgt
1200catcctgctc cgatacctgg ccgtgttcgc tcgaatggcc gagaagcagc
ccgctggcaa 1260ggctctctaa gc 12729680DNAArtificial SequencePrimer
YL521 96tttccatggt gaagtccaag cgacaggctc tgcccctcac catcgacgga
actacctacg 60acgtctccgc ttgggtgaac 809730DNAArtificial
SequencePrimer YL522 97tggagatctt gtacgagtac cagaggggag
309837DNAArtificial SequencePrimer YL525 98ccttcatggt tatgcactct
caggaagcct tcgacaa 379937DNAArtificial SequencePrimer YL526
99ttgtcgaagg cttcctgaga gtgcataacc atgaagg 3710038DNAArtificial
SequencePrimer YL527 100ccaagatcaa cccctcctcc gagctgcctc cccaggct
3810138DNAArtificial SequencePrimer YL528 101agcctgggga ggcagctcgg
aggaggggtt gatcttgg 3810237DNAArtificial SequencePrimer YL529
102gggcttctcc gtgacctggt ggaaggacag acacaac 3710337DNAArtificial
SequencePrimer YL530 103gttgtgtctg tccttccacc aggtcacgga gaagccc
3710438DNAArtificial SequencePrimer YL531 104acattgataa cctgcctctg
ctcgcctggt ccgaggac 3810538DNAArtificial SequencePrimer YL532
105gtcctcggac caggcgagca gaggcaggtt atcaatgt 3810638DNAArtificial
SequencePrimer YL533 106tcatctggtg tttccagtct gtgctgaccg ttcgatcc
3810738DNAArtificial SequencePrimer YL534 107ggatcgaacg gtcagcacag
actggaaaca ccagatga 3810839DNAArtificial SequencePrimer YL535
108ctgcactgga ctctcaagac cctgttccac ctcttcttt 3910939DNAArtificial
SequencePrimer YL536 109aaagaagagg tggaacaggg tcttgagagt ccagtgcag
3911037DNAArtificial SequencePrimer YL537 110ctggagaaga tcggtgattc
cgtctgggac ggacatg 3711137DNAArtificial SequencePrimer YL538
111catgtccgtc ccagacggaa tcaccgatct tctccag 371121272DNAArtificial
SequenceD8SF synthetic delta-8 desaturase (codon-optimized for
Yarrowia lipolytica) 112catggtgaag tccaagcgac aggctctgcc cctcaccatc
gacggaacta cctacgacgt 60ctccgcttgg gtgaacttcc accctggtgg agctgaaatc
attgagaact accagggacg 120agatgctact gacgccttca tggttatgca
ctctcaggaa gccttcgaca agctcaagcg 180aatgcccaag atcaacccct
cctccgagct gcctccccag gctgccgtca acgaagctca 240ggaggatttc
cgaaagctcc gagaagagct gatcgccact ggcatgtttg acgcctctcc
300cctctggtac tcgtacaaga tctccaccac cctgggtctt ggcgtgcttg
gatacttcct 360gatggtccag taccagatgt acttcattgg tgctgtgctg
ctcggtatgc actaccagca 420aatgggatgg ctgtctcatg acatctgcca
ccaccagacc ttcaagaacc gaaactggaa 480taacctcgtg ggtctggtct
ttggcaacgg actccagggc ttctccgtga cctggtggaa 540ggacagacac
aacgcccatc attctgctac caacgttcag ggtcacgatc ccgacattga
600taacctgcct ctgctcgcct ggtccgagga cgatgtcact cgagcttctc
ccatctcccg 660aaagctcatt cagttccaac agtactattt cctggtcatc
tgtattctcc tgcgattcat 720ctggtgtttc cagtctgtgc tgaccgttcg
atccctcaag gaccgagaca accagttcta 780ccgatctcag tacaagaaag
aggccattgg actcgctctg cactggactc tcaagaccct 840gttccacctc
ttctttatgc cctccatcct gacctcgctc ctggtgttct ttgtttccga
900gctcgtcggt ggcttcggaa ttgccatcgt ggtcttcatg aaccactacc
ctctggagaa 960gatcggtgat tccgtctggg acggacatgg cttctctgtg
ggtcagatcc atgagaccat 1020gaacattcga cgaggcatca ttactgactg
gttctttgga ggcctgaact accagatcga 1080gcaccatctc tggcccaccc
tgcctcgaca caacctcact gccgtttcct accaggtgga 1140acagctgtgc
cagaagcaca acctccccta ccgaaaccct ctgccccatg aaggtctcgt
1200catcctgctc cgatacctgg ccgtgttcgc tcgaatggcc gagaagcagc
ccgctggcaa 1260ggctctctaa gc 1272113422PRTArtificial SequenceD8SF
synthetic delta-8 desaturase (codon-optimized for
Yarrowialipolytica) 113Met Val Lys Ser Lys Arg Gln Ala Leu Pro Leu
Thr Ile Asp Gly Thr1 5 10 15Thr Tyr Asp Val Ser Ala Trp Val Asn Phe
His Pro Gly Gly Ala Glu 20 25 30Ile Ile Glu Asn Tyr Gln Gly Arg Asp
Ala Thr Asp Ala Phe Met Val 35 40 45Met His Ser Gln Glu Ala Phe Asp
Lys Leu Lys Arg Met Pro Lys Ile 50 55 60Asn Pro Ser Ser Glu Leu Pro
Pro Gln Ala Ala Val Asn Glu Ala Gln65 70 75 80Glu Asp Phe Arg Lys
Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe 85 90 95Asp Ala Ser Pro
Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly 100 105 110Leu Gly
Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe 115 120
125Ile Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu
130 135 140Ser His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn
Trp Asn145 150 155 160Asn Leu Val Gly Leu Val Phe Gly Asn Gly Leu
Gln Gly Phe Ser Val 165 170 175Thr Trp Trp Lys Asp Arg His Asn Ala
His His Ser Ala Thr Asn Val 180 185 190Gln Gly His Asp Pro Asp Ile
Asp Asn Leu Pro Leu Leu Ala Trp Ser 195 200 205Glu Asp Asp Val Thr
Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln 210 215 220Phe Gln Gln
Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile225 230 235
240Trp Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp
245 250 255Asn Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly
Leu Ala 260 265 270Leu His Trp Thr Leu Lys Thr Leu Phe His Leu Phe
Phe Met Pro Ser 275 280 285Ile Leu Thr Ser Leu Leu Val Phe Phe Val
Ser Glu Leu Val Gly Gly 290 295 300Phe Gly Ile Ala Ile Val Val Phe
Met Asn His Tyr Pro Leu Glu Lys305 310 315 320Ile Gly Asp Ser Val
Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile 325 330 335His Glu Thr
Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350Gly
Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360
365Arg His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln
370 375 380Lys His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly
Leu Val385 390 395 400Ile Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg
Met Ala Glu Lys Gln 405 410 415Pro Ala Gly Lys Ala Leu
420114995DNAYarrowia lipolytica 114agtgtacgca gtactataga ggaacaattg
ccccggagaa gacggccagg ccgcctagat 60gacaaattca acaactcaca gctgactttc
tgccattgcc actagggggg ggccttttta 120tatggccaag ccaagctctc
cacgtcggtt gggctgcacc caacaataaa tgggtagggt 180tgcaccaaca
aagggatggg atggggggta gaagatacga ggataacggg gctcaatggc
240acaaataaga acgaatactg ccattaagac tcgtgatcca gcgactgaca
ccattgcatc 300atctaagggc ctcaaaacta cctcggaact gctgcgctga
tctggacacc acagaggttc 360cgagcacttt aggttgcacc aaatgtccca
ccaggtgcag gcagaaaacg ctggaacagc 420gtgtacagtt tgtcttaaca
aaaagtgagg gcgctgaggt cgagcagggt ggtgtgactt 480gttatagcct
ttagagctgc gaaagcgcgt atggatttgg ctcatcaggc cagattgagg
540gtctgtggac acatgtcatg ttagtgtact tcaatcgccc cctggatata
gccccgacaa 600taggccgtgg cctcattttt ttgccttccg cacatttcca
ttgctcggta cccacacctt 660gcttctcctg cacttgccaa ccttaatact
ggtttacatt gaccaacatc ttacaagcgg 720ggggcttgtc tagggtatat
ataaacagtg gctctcccaa tcggttgcca gtctcttttt 780tcctttcttt
ccccacagat tcgaaatcta aactacacat cacacaatgc ctgttactga
840cgtccttaag cgaaagtccg gtgtcatcgt cggcgacgat gtccgagccg
tgagtatcca 900cgacaagatc agtgtcgaga cgacgcgttt tgtgtaatga
cacaatccga aagtcgctag 960caacacacac tctctacaca aactaaccca gctct
9951158502DNAArtificial SequencePlasmid pY54PC 115ggccgccacc
gcggcccgag attccggcct cttcggccgc caagcgaccc gggtggacgt 60ctagaggtac
ctagcaatta acagatagtt tgccggtgat aattctctta acctcccaca
120ctcctttgac ataacgattt atgtaacgaa actgaaattt gaccagatat
tgtgtccgcg 180gtggagctcc agcttttgtt ccctttagtg agggttaatt
aatcgagctt ggcgtaatca 240tggtcatagc tgtttcctgt gtgaaattgt
tatccgctca caattccaca caacatacga 300gccggaagca taaagtgtaa
agcctggggt gcctaatgag tgagctaact cacattaatt 360gcgttgcgct
cactgcccgc
tttccagtcg ggaaacctgt cgtgccagct gcattaatga 420atcggccaac
gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
480actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
ctcaaaggcg 540gtaatacggt tatccacaga atcaggggat aacgcaggaa
agaacatgtg agcaaaaggc 600cagcaaaagg ccaggaaccg taaaaaggcc
gcgttgctgg cgtttttcca taggctccgc 660ccccctgacg agcatcacaa
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 720ctataaagat
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
780ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
gctttctcat 840agctcacgct gtaggtatct cagttcggtg taggtcgttc
gctccaagct gggctgtgtg 900cacgaacccc ccgttcagcc cgaccgctgc
gccttatccg gtaactatcg tcttgagtcc 960aacccggtaa gacacgactt
atcgccactg gcagcagcca ctggtaacag gattagcaga 1020gcgaggtatg
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
1080agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
aaaaagagtt 1140ggtagctctt gatccggcaa acaaaccacc gctggtagcg
gtggtttttt tgtttgcaag 1200cagcagatta cgcgcagaaa aaaaggatct
caagaagatc ctttgatctt ttctacgggg 1260tctgacgctc agtggaacga
aaactcacgt taagggattt tggtcatgag attatcaaaa 1320aggatcttca
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
1380tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
tatctcagcg 1440atctgtctat ttcgttcatc catagttgcc tgactccccg
tcgtgtagat aactacgata 1500cgggagggct taccatctgg ccccagtgct
gcaatgatac cgcgagaccc acgctcaccg 1560gctccagatt tatcagcaat
aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 1620gcaactttat
ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
1680tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
ggtgtcacgc 1740tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
gatcaaggcg agttacatga 1800tcccccatgt tgtgcaaaaa agcggttagc
tccttcggtc ctccgatcgt tgtcagaagt 1860aagttggccg cagtgttatc
actcatggtt atggcagcac tgcataattc tcttactgtc 1920atgccatccg
taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
1980tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
taccgcgcca 2040catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
cttcggggcg aaaactctca 2100aggatcttac cgctgttgag atccagttcg
atgtaaccca ctcgtgcacc caactgatct 2160tcagcatctt ttactttcac
cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 2220gcaaaaaagg
gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
2280tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
tgaatgtatt 2340tagaaaaata aacaaatagg ggttccgcgc acatttcccc
gaaaagtgcc acctgacgcg 2400ccctgtagcg gcgcattaag cgcggcgggt
gtggtggtta cgcgcagcgt gaccgctaca 2460cttgccagcg ccctagcgcc
cgctcctttc gctttcttcc cttcctttct cgccacgttc 2520gccggctttc
cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
2580ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
tgggccatcg 2640ccctgataga cggtttttcg ccctttgacg ttggagtcca
cgttctttaa tagtggactc 2700ttgttccaaa ctggaacaac actcaaccct
atctcggtct attcttttga tttataaggg 2760attttgccga tttcggccta
ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 2820aattttaaca
aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt
2880gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
gggggatgtg 2940ctgcaaggcg attaagttgg gtaacgccag ggttttccca
gtcacgacgt tgtaaaacga 3000cggccagtga attgtaatac gactcactat
agggcgaatt gggtaccggg ccccccctcg 3060aggtcgacgg tatcgataag
cttgatatcg aattcatgtc acacaaaccg atcttcgcct 3120caaggaaacc
taattctaca tccgagagac tgccgagatc cagtctacac tgattaattt
3180tcgggccaat aatttaaaaa aatcgtgtta tataatatta tatgtattat
atatatacat 3240catgatgata ctgacagtca tgtcccattg ctaaatagac
agactccatc tgccgcctcc 3300aactgatgtt ctcaatattt aaggggtcat
ctcgcattgt ttaataataa acagactcca 3360tctaccgcct ccaaatgatg
ttctcaaaat atattgtatg aacttatttt tattacttag 3420tattattaga
caacttactt gctttatgaa aaacacttcc tatttaggaa acaatttata
3480atggcagttc gttcatttaa caatttatgt agaataaatg ttataaatgc
gtatgggaaa 3540tcttaaatat ggatagcata aatgatatct gcattgccta
attcgaaatc aacagcaacg 3600aaaaaaatcc cttgtacaac ataaatagtc
atcgagaaat atcaactatc aaagaacagc 3660tattcacacg ttactattga
gattattatt ggacgagaat cacacactca actgtctttc 3720tctcttctag
aaatacaggt acaagtatgt actattctca ttgttcatac ttctagtcat
3780ttcatcccac atattccttg gatttctctc caatgaatga cattctatct
tgcaaattca 3840acaattataa taagatatac caaagtagcg gtatagtggc
aatcaaaaag cttctctggt 3900gtgcttctcg tatttatttt tattctaatg
atccattaaa ggtatatatt tatttcttgt 3960tatataatcc ttttgtttat
tacatgggct ggatacataa aggtattttg atttaatttt 4020ttgcttaaat
tcaatccccc ctcgttcagt gtcaactgta atggtaggaa attaccatac
4080ttttgaagaa gcaaaaaaaa tgaaagaaaa aaaaaatcgt atttccaggt
tagacgttcc 4140gcagaatcta gaatgcggta tgcggtacat tgttcttcga
acgtaaaagt tgcgctccct 4200gagatattgt acatttttgc ttttacaagt
acaagtacat cgtacaacta tgtactactg 4260ttgatgcatc cacaacagtt
tgttttgttt ttttttgttt tttttttttc taatgattca 4320ttaccgctat
gtatacctac ttgtacttgt agtaagccgg gttattggcg ttcaattaat
4380catagactta tgaatctgca cggtgtgcgc tgcgagttac ttttagctta
tgcatgctac 4440ttgggtgtaa tattgggatc tgttcggaaa tcaacggatg
ctcaaccgat ttcgacagta 4500ataatttgaa tcgaatcgga gcctaaaatg
aacccgagta tatctcataa aattctcggt 4560gagaggtctg tgactgtcag
tacaaggtgc cttcattatg ccctcaacct taccatacct 4620cactgaatgt
agtgtacctc taaaaatgaa atacagtgcc aaaagccaag gcactgagct
4680cgtctaacgg acttgatata caaccaatta aaacaaatga aaagaaatac
agttctttgt 4740atcatttgta acaattaccc tgtacaaact aaggtattga
aatcccacaa tattcccaaa 4800gtccacccct ttccaaattg tcatgcctac
aactcatata ccaagcacta acctaccaaa 4860caccactaaa accccacaaa
atatatctta ccgaatatac agtaacaagc taccaccaca 4920ctcgttgggt
gcagtcgcca gcttaaagat atctatccac atcagccaca actcccttcc
4980tttaataaac cgactacacc cttggctatt gaggttatga gtgaatatac
tgtagacaag 5040acactttcaa gaagactgtt tccaaaacgt accactgtcc
tccactacaa acacacccaa 5100tctgcttctt ctagtcaagg ttgctacacc
ggtaaattat aaatcatcat ttcattagca 5160gggcagggcc ctttttatag
agtcttatac actagcggac cctgccggta gaccaacccg 5220caggcgcgtc
agtttgctcc ttccatcaat gcgtcgtaga aacgacttac tccttcttga
5280gcagctcctt gaccttgttg gcaacaagtc tccgacctcg gaggtggagg
aagagcctcc 5340gatatcggcg gtagtgatac cagcctcgac ggactccttg
acggcagcct caacagcgtc 5400accggcgggc ttcatgttaa gagagaactt
gagcatcatg gcggcagaca gaatggtggc 5460aatggggttg accttctgct
tgccgagatc gggggcagat ccgtgacagg gctcgtacag 5520accgaacgcc
tcgttggtgt cgggcagaga agccagagag gcggagggca gcagacccag
5580agaaccgggg atgacggagg cctcgtcgga gatgatatcg ccaaacatgt
tggtggtgat 5640gatgatacca ttcatcttgg agggctgctt gatgaggatc
atggcggccg agtcgatcag 5700ctggtggttg agctcgagct gggggaattc
gtccttgagg actcgagtga cagtctttcg 5760ccaaagtcga gaggaggcca
gcacgttggc cttgtcaaga gaccacacgg gaagaggggg 5820gttgtgctga
agggccagga aggcggccat tcgggcaatt cgctcaacct caggaacgga
5880gtaggtctcg gtgtcggaag cgacgccaga tccgtcatcc tcctttcgct
ctccaaagta 5940gatacctccg acgagctctc ggacaatgat gaagtcggtg
ccctcaacgt ttcggatggg 6000ggagagatcg gcgagcttgg gcgacagcag
ctggcagggt cgcaggttgg cgtacaggtt 6060caggtccttt cgcagcttga
ggagaccctg ctcgggtcgc acgtcggttc gtccgtcggg 6120agtggtccat
acggtgttgg cagcgcctcc gacagcaccg agcataatag agtcagcctt
6180tcggcagatg tcgagagtag cgtcggtgat gggctcgccc tccttctcaa
tggcagctcc 6240tccaatgagt cggtcctcaa acacaaactc ggtgccggag
gcctcagcaa cagacttgag 6300caccttgacg gcctcggcaa tcacctcggg
gccacagaag tcgccgccga gaagaacaat 6360cttcttggag tcagtcttgg
tcttcttagt ttcgggttcc attgtggatg tgtgtggttg 6420tatgtgtgat
gtggtgtgtg gagtgaaaat ctgtggctgg caaacgctct tgtatatata
6480cgcacttttg cccgtgctat gtggaagact aaacctccga agattgtgac
tcaggtagtg 6540cggtatcggc tagggaccca aaccttgtcg atgccgatag
cgctatcgaa cgtaccccag 6600ccggccggga gtatgtcgga ggggacatac
gagatcgtca agggtttgtg gccaactggt 6660aaataaatga tgactcaggc
gacgacggaa ttcctgcagc ccatcgatgc agaattcagg 6720agagaccggg
ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc cccaattgac
6780cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttcacc
ccacatatca 6840aacctccccc ggttcccaca cttgccgtta agggcgtagg
gtactgcagt ctggaatcta 6900cgcttgttca gactttgtac tagtttcttt
gtctggccat ccgggtaacc catgccggac 6960gcaaaataga ctactgaaaa
tttttttgct ttgtggttgg gactttagcc aagggtataa 7020aagaccaccg
tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca
7080cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaccatgg
ctgctgctcc 7140cagtgtgagg acgtttactc gggccgaggt tttgaatgcc
gaggctctga atgagggcaa 7200gaaggatgcc gaggcaccct tcttgatgat
catcgacaac aaggtgtacg atgtccgcga 7260gttcgtccct gatcatcccg
gtggaagtgt gattctcacg cacgttggca aggacggcac 7320tgacgtcttt
gacacttttc accccgaggc tgcttgggag actcttgcca acttttacgt
7380tggtgatatt gacgagagcg accgcgatat caagaatgat gactttgcgg
ccgaggtccg 7440caagctgcgt accttgttcc agtctcttgg ttactacgat
tcttccaagg catactacgc 7500cttcaaggtc tcgttcaacc tctgcatctg
gggtttgtcg acggtcattg tggccaagtg 7560gggccagacc tcgaccctcg
ccaacgtgct ctcggctgcg cttttgggtc tgttctggca 7620gcagtgcgga
tggttggctc acgacttttt gcatcaccag gtcttccagg accgtttctg
7680gggtgatctt ttcggcgcct tcttgggagg tgtctgccag ggcttctcgt
cctcgtggtg 7740gaaggacaag cacaacactc accacgccgc ccccaacgtc
cacggcgagg atcccgacat 7800tgacacccac cctctgttga cctggagtga
gcatgcgttg gagatgttct cggatgtccc 7860agatgaggag ctgacccgca
tgtggtcgcg tttcatggtc ctgaaccaga cctggtttta 7920cttccccatt
ctctcgtttg cccgtctctc ctggtgcctc cagtccattc tctttgtgct
7980gcctaacggt caggcccaca agccctcggg cgcgcgtgtg cccatctcgt
tggtcgagca 8040gctgtcgctt gcgatgcact ggacctggta cctcgccacc
atgttcctgt tcatcaagga 8100tcccgtcaac atgctggtgt actttttggt
gtcgcaggcg gtgtgcggaa acttgttggc 8160gatcgtgttc tcgctcaacc
acaacggtat gcctgtgatc tcgaaggagg aggcggtcga 8220tatggatttc
ttcacgaagc agatcatcac gggtcgtgat gtccacccgg gtctatttgc
8280caactggttc acgggtggat tgaactatca gatcgagcac cacttgttcc
cttcgatgcc 8340tcgccacaac ttttcaaaga tccagcctgc tgtcgagacc
ctgtgcaaaa agtacaatgt 8400ccgataccac accaccggta tgatcgaggg
aactgcagag gtctttagcc gtctgaacga 8460ggtctccaag gctacctcca
agatgggtaa ggcgcagtaa gc 85021167145DNAArtificial SequencePlasmid
pKUNFmkF2 116catggcgtcc acttcggctc tgcccaagca gaaccctgcg cttagacgca
ccgtcacctc 60aactactgtg acggattctg agtctgccgc cgtctctcct tcagactctc
cccgccactc 120ggcctcttcc acatcgctct cgtccatgtc cgaggttgat
atcgccaagc ccaagtccga 180gtatggtgtc atgctcgaca cctacggcaa
ccagttcgag gttcccgact ttaccatcaa 240ggacatctac aatgccatcc
ctaagcactg cttcaagcgc tccgctctca agggatacgg 300ttatatcctc
cgcgacattg tcctcctgac taccactttc agcatctggt acaactttgt
360gacccccgaa tatatcccct ccacccccgc ccgcgctggt ctgtgggccg
tgtacaccgt 420tcttcagggt cttttcggta ctggtctctg ggttattgcc
catgagtgcg gtcacggtgc 480tttctccgat tctcgcatca tcaacgacat
tactggctgg gttcttcact cttccctcct 540tgtcccctac ttcagctggc
aaatctccca ccgaaagcac cacaaggcca ctggcaacat 600ggagcgtgac
atggtcttcg ttccccgaac ccgcgagcag caggctactc gtctcggaaa
660gatgacccac gagctcgctc atcttactga gnnnntcgtn ggctggccca
actacctcat 720caccaatgtt accggccaca actaccacga gcgccagcgt
gagggtcgcg gcaagggcaa 780gcataacggc ctcggcggtg gtgttaacca
cttcgatccc cgcagccctc tgtacgagaa 840cagtgacgct aagctcatcg
tcctcagcga tattggtatc ggtctgatgg ccactgctct 900gtacttcctc
gttcagaagt tcggtttcta caacatggcc atctggtact ttgttcccta
960cctctgggtt aaccactggc tcgttgccat caccttcctc cagcacaccg
accctaccct 1020tccccactac accaacgacg agtggaactt cgtccgtggt
gccgctgcta ccattgaccg 1080tgagatgggc ttcatcggcc gccaccttct
ccacggcatc atcgagactc atgtcctcca 1140ccactacgtc agcagcatcc
ccttctacaa cgcggacgag gccaccgagg ccattaagcc 1200catcatgggc
aagcactacc gggctgatgt ccaggatggt cctcgtggct tcatccgcgc
1260catgtaccgc agtgcgcgta tgtgccagtg ggttgagccc agcgctggtg
ccgagggtgc 1320tggtaagggt gttctgttct tccgcaaccg caacaacgtg
ggcacccccc ccgctgttat 1380caagcccgtt gcttaagtag gcgcggccgc
tatttatcac tctttacaac ttctacctca 1440actatctact ttaataaatg
aatatcgttt attctctatg attactgtat atgcgttcct 1500ctaagacaaa
tcgaaaccag catgtgatcg aatggcatac aaaagtttct tccgaagttg
1560atcaatgtcc tgatagtcag gcagcttgag aagattgaca caggtggagg
ccgtagggaa 1620ccgatcaacc tgtctaccag cgttacgaat ggcaaatgac
gggttcaaag ccttgaatcc 1680ttgcaatggt gccttggata ctgatgtcac
aaacttaaga agcagccgct tgtcctcttc 1740ctcgatcgat ggtcatagct
gtttcctgtg tgaaattgtt atccgctcac aattccacac 1800aacgtacgaa
gtcgtcaatg atgtcgatat gggttttgat catgcacaca taaggtccga
1860ccttatcggc aagctcaatg agctccttgg tggtggtaac atccagagaa
gcacacaggt 1920tggttttctt ggctgccacg agcttgagca ctcgagcggc
aaaggcggac ttgtggacgt 1980tagctcgagc ttcgtaggag ggcattttgg
tggtgaagag gagactgaaa taaatttagt 2040ctgcagaact ttttatcgga
accttatctg gggcagtgaa gtatatgtta tggtaatagt 2100tacgagttag
ttgaacttat agatagactg gactatacgg ctatcggtcc aaattagaaa
2160gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa aatgtgatca
tgatgaaagc 2220cagcaatgac gttgcagctg atattgttgt cggccaaccg
cgccgaaaac gcagctgtca 2280gacccacagc ctccaacgaa gaatgtatcg
tcaaagtgat ccaagcacac tcatagttgg 2340agtcgtactc caaaggcggc
aatgacgagt cagacagata ctcgtcgacc ttttccttgg 2400gaaccaccac
cgtcagccct tctgactcac gtattgtagc caccgacaca ggcaacagtc
2460cgtggatagc agaatatgtc ttgtcggtcc atttctcacc aactttaggc
gtcaagtgaa 2520tgttgcagaa gaagtatgtg ccttcattga gaatcggtgt
tgctgatttc aataaagtct 2580tgagatcagt ttggcgcgcc agctgcatta
atgaatcggc caacgcgcgg ggagaggcgg 2640tttgcgtatt gggcgctctt
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 2700gctgcggcga
gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
2760ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
accgtaaaaa 2820ggccgcgttg ctggcgtttt tccataggct ccgcccccct
gacgagcatc acaaaaatcg 2880acgctcaagt cagaggtggc gaaacccgac
aggactataa agataccagg cgtttccccc 2940tggaagctcc ctcgtgcgct
ctcctgttcc gaccctgccg cttaccggat acctgtccgc 3000ctttctccct
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
3060ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
agcccgaccg 3120ctgcgcctta tccggtaact atcgtcttga gtccaacccg
gtaagacacg acttatcgcc 3180actggcagca gccactggta acaggattag
cagagcgagg tatgtaggcg gtgctacaga 3240gttcttgaag tggtggccta
actacggcta cactagaaga acagtatttg gtatctgcgc 3300tctgctgaag
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
3360caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
gaaaaaaagg 3420atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagtgga acgaaaactc 3480acgttaaggg attttggtca tgagattatc
aaaaaggatc ttcacctaga tccttttaaa 3540ttaaaaatga agttttaaat
caatctaaag tatatatgag taaacttggt ctgacagtta 3600ccaatgctta
atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
3660tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
ctggccccag 3720tgctgcaatg ataccgcgag acccacgctc accggctcca
gatttatcag caataaacca 3780gccagccgga agggccgagc gcagaagtgg
tcctgcaact ttatccgcct ccatccagtc 3840tattaattgt tgccgggaag
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 3900tgttgccatt
gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
3960ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
aaaaagcggt 4020tagctccttc ggtcctccga tcgttgtcag aagtaagttg
gccgcagtgt tatcactcat 4080ggttatggca gcactgcata attctcttac
tgtcatgcca tccgtaagat gcttttctgt 4140gactggtgag tactcaacca
agtcattctg agaatagtgt atgcggcgac cgagttgctc 4200ttgcccggcg
tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
4260cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
tgagatccag 4320ttcgatgtaa cccactcgtg cacccaactg atcttcagca
tcttttactt tcaccagcgt 4380ttctgggtga gcaaaaacag gaaggcaaaa
tgccgcaaaa aagggaataa gggcgacacg 4440gaaatgttga atactcatac
tcttcctttt tcaatattat tgaagcattt atcagggtta 4500ttgtctcatg
agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
4560gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac
agatgcgtaa 4620ggagaaaata ccgcatcagg aaattgtaag cgttaatatt
ttgttaaaat tcgcgttaaa 4680tttttgttaa atcagctcat tttttaacca
ataggccgaa atcggcaaaa tcccttataa 4740atcaaaagaa tagaccgaga
tagggttgag tgttgttcca gtttggaaca agagtccact 4800attaaagaac
gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
4860actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
aagcactaaa 4920tcggaaccct aaagggagcc cccgatttag agcttgacgg
ggaaagccgg cgaacgtggc 4980gagaaaggaa gggaagaaag cgaaaggagc
gggcgctagg gcgctggcaa gtgtagcggt 5040cacgctgcgc gtaaccacca
cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat 5100tcgccattca
ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
5160cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
gccagggttt 5220tcccagtcac gacgttgtaa aacgacggcc agtgaattgt
aatacgactc actatagggc 5280gaattgggcc cgacgtcgca tgcagtggtg
gtattgtgac tggggatgta gttgagaata 5340agtcatacac aagtcagctt
tcttcgagcc tcatataagt ataagtagtt caacgtatta 5400gcactgtacc
cagcatctcc gtatcgagaa acacaacaac atgccccatt ggacagatca
5460tgcggataca caggttgtgc agtatcatac atactcgatc agacaggtcg
tctgaccatc 5520atacaagctg aacaagcgct ccatacttgc acgctctcta
tatacacagt taaattacat 5580atccatagtc taacctctaa cagttaatct
tctggtaagc ctcccagcca gccttctggt 5640atcgcttggc ctcctcaata
ggatctcggt tctggccgta cagacctcgg ccgacaatta 5700tgatatccgt
tccggtagac atgacatcct caacagttcg gtactgctgt ccgagagcgt
5760ctcccttgtc gtcaagaccc accccggggg tcagaataag ccagtcctca
gagtcgccct 5820taattaattt gaatcgaatc gatgagccta aaatgaaccc
gagtatatct cataaaattc 5880tcggtgagag gtctgtgact gtcagtacaa
ggtgccttca ttatgccctc aaccttacca 5940tacctcactg aatgtagtgt
acctctaaaa atgaaataca gtgccaaaag ccaaggcact 6000gagctcgtct
aacggacttg atatacaacc aattaaaaca aatgaaaaga aatacagttc
6060tttgtatcat ttgtaacaat taccctgtac aaactaaggt attgaaatcc
cacaatattc 6120ccaaagtcca cccctttcca aattgtcatg cctacaactc
atataccaag cactaaccta 6180ccgtttaaac agtgtacgca gatctactat
agaggaacat ttaaattgcc ccggagaaga 6240cggccaggcc gcctagatga
caaattcaac aactcacagc tgactttctg ccattgccac 6300tagggggggg
cctttttata tggccaagcc aagctctcca cgtcggttgg gctgcaccca
6360acaataaatg ggtagggttg caccaacaaa gggatgggat ggggggtaga
agatacgagg 6420ataacggggc tcaatggcac aaataagaac gaatactgcc
attaagactc gtgatccagc 6480gactgacacc attgcatcat ctaagggcct
caaaactacc tcggaactgc tgcgctgatc 6540tggacaccac agaggttccg
agcactttag gttgcaccaa atgtcccacc aggtgcaggc 6600agaaaacgct
ggaacagcgt gtacagtttg tcttaacaaa aagtgagggc gctgaggtcg
6660agcagggtgg tgtgacttgt tatagccttt agagctgcga aagcgcgtat
ggatttggct 6720catcaggcca gattgagggt ctgtggacac atgtcatgtt
agtgtacttc aatcgccccc 6780tggatatagc cccgacaata ggccgtggcc
tcattttttt gccttccgca catttccatt 6840gctcgatacc cacaccttgc
ttctcctgca cttgccaacc
ttaatactgg tttacattga 6900ccaacatctt acaagcgggg ggcttgtcta
gggtatatat aaacagtggc tctcccaatc 6960ggttgccagt ctcttttttc
ctttctttcc ccacagattc gaaatctaaa ctacacatca 7020cagaattccg
agccgtgagt atccacgaca agatcagtgt cgagacgacg cgttttgtgt
7080aatgacacaa tccgaaagtc gctagcaaca cacactctct acacaaacta
acccagctct 7140ggtac 71451175553DNAArtificial SequencePlasmid
pZF5T-PPC 117ggccgcattg atgattggaa acacacacat gggttatatc taggtgagag
ttagttggac 60agttatatat taaatcagct atgccaacgg taacttcatt catgtcaacg
aggaaccagt 120gactgcaagt aatatagaat ttgaccacct tgccattctc
ttgcactcct ttactatatc 180tcatttattt cttatataca aatcacttct
tcttcccagc atcgagctcg gaaacctcat 240gagcaataac atcgtggatc
tcgtcaatag agggcttttt ggactccttg ctgttggcca 300ccttgtcctt
gctgtctggc tcattctgtt tcaacgcctt ttaattaatc gagcttggcg
360taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
tccacacaac 420atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
aatgagtgag ctaactcaca 480ttaattgcgt tgcgctcact gcccgctttc
cagtcgggaa acctgtcgtg ccagctgcat 540taatgaatcg gccaacgcgc
ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 600tcgctcactg
actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
660aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
catgtgagca 720aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
tgctggcgtt tttccatagg 780ctccgccccc ctgacgagca tcacaaaaat
cgacgctcaa gtcagaggtg gcgaaacccg 840acaggactat aaagatacca
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 900ccgaccctgc
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
960tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc 1020tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
tatccggtaa ctatcgtctt 1080gagtccaacc cggtaagaca cgacttatcg
ccactggcag cagccactgg taacaggatt 1140agcagagcga ggtatgtagg
cggtgctaca gagttcttga agtggtggcc taactacggc 1200tacactagaa
ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
1260agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt 1320tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
aagatccttt gatcttttct 1380acggggtctg acgctcagtg gaacgaaaac
tcacgttaag ggattttggt catgagatta 1440tcaaaaagga tcttcaccta
gatcctttta aattaaaaat gaagttttaa atcaatctaa 1500agtatatatg
agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
1560tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
gtagataact 1620acgatacggg agggcttacc atctggcccc agtgctgcaa
tgataccgcg agacccacgc 1680tcaccggctc cagatttatc agcaataaac
cagccagccg gaagggccga gcgcagaagt 1740ggtcctgcaa ctttatccgc
ctccatccag tctattaatt gttgccggga agctagagta 1800agtagttcgc
cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
1860tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
aaggcgagtt 1920acatgatccc ccatgttgtg caaaaaagcg gttagctcct
tcggtcctcc gatcgttgtc 1980agaagtaagt tggccgcagt gttatcactc
atggttatgg cagcactgca taattctctt 2040actgtcatgc catccgtaag
atgcttttct gtgactggtg agtactcaac caagtcattc 2100tgagaatagt
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
2160gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
ggggcgaaaa 2220ctctcaagga tcttaccgct gttgagatcc agttcgatgt
aacccactcg tgcacccaac 2280tgatcttcag catcttttac tttcaccagc
gtttctgggt gagcaaaaac aggaaggcaa 2340aatgccgcaa aaaagggaat
aagggcgaca cggaaatgtt gaatactcat actcttcctt 2400tttcaatatt
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
2460tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
agtgccacct 2520gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
tggttacgcg cagcgtgacc 2580gctacacttg ccagcgccct agcgcccgct
cctttcgctt tcttcccttc ctttctcgcc 2640acgttcgccg gctttccccg
tcaagctcta aatcgggggc tccctttagg gttccgattt 2700agtgctttac
ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
2760ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
ctttaatagt 2820ggactcttgt tccaaactgg aacaacactc aaccctatct
cggtctattc ttttgattta 2880taagggattt tgccgatttc ggcctattgg
ttaaaaaatg agctgattta acaaaaattt 2940aacgcgaatt ttaacaaaat
attaacgctt acaatttcca ttcgccattc aggctgcgca 3000actgttggga
agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
3060gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
cgacgttgta 3120aaacgacggc cagtgaattg taatacgact cactataggg
cgaattgggt accgggcccc 3180ccctcgaggt cgacgtttaa acagtgtacg
cagtactata gaggaacatc gattgccccg 3240gagaagacgg ccaggccgcc
tagatgacaa attcaacaac tcacagctga ctttctgcca 3300ttgccactag
gggggggcct ttttatatgg ccaagccaag ctctccacgt cggttgggct
3360gcacccaaca ataaatgggt agggttgcac caacaaaggg atgggatggg
gggtagaaga 3420tacgaggata acggggctca atggcacaaa taagaacgaa
tactgccatt aagactcgtg 3480atccagcgac tgacaccatt gcatcatcta
agggcctcaa aactacctcg gaactgctgc 3540gctgatctgg acaccacaga
ggttccgagc actttaggtt gcaccaaatg tcccaccagg 3600tgcaggcaga
aaacgctgga acagcgtgta cagtttgtct taacaaaaag tgagggcgct
3660gaggtcgagc agggtggtgt gacttgttat agcctttaga gctgcgaaag
cgcgtatgga 3720tttggctcat caggccagat tgagggtctg tggacacatg
tcatgttagt gtacttcaat 3780cgccccctgg atatagcccc gacaataggc
cgtggcctca tttttttgcc ttccgcacat 3840ttccattgct cggtacccac
accttgcttc tcctgcactt gccaacctta atactggttt 3900acattgacca
acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct
3960cccaatcggt tgccagtctc ttttttcctt tctttcccca cagattcgaa
atctaaacta 4020cacatcacac aatgcctgtt actgacgtcc ttaagcgaaa
gtccggtgtc atcgtcggcg 4080acgatgtccg agccgtgagt atccacgaca
agatcagtgt cgagacgacg cgttttgtgt 4140aatgacacaa tccgaaagtc
gctagcaaca cacactctct acacaaacta acccagctct 4200ccatgggaac
ggaccaagga aaaaccttca cctgggaaga gctggcggcc cataacacca
4260aggacgacct actcttggcc atccgcggca gggtgtacga tgtcacaaag
ttcttgagcc 4320gccatcctgg tggagtggac actctcctgc tcggagctgg
ccgagatgtt actccggtct 4380ttgagatgta tcacgcgttt ggggctgcag
atgccattat gaagaagtac tatgtcggta 4440cactggtctc gaatgagctg
cccatcttcc cggagccaac ggtgttccac aaaaccatca 4500agacgagagt
cgagggctac tttacggatc ggaacattga tcccaagaat agaccagaga
4560tctggggacg atacgctctt atctttggat ccttgatcgc ttcctactac
gcgcagctct 4620ttgtgccttt cgttgtcgaa cgcacatggc ttcaggtggt
gtttgcaatc atcatgggat 4680ttgcgtgcgc acaagtcgga ctcaaccctc
ttcatgatgc gtctcacttt tcagtgaccc 4740acaaccccac tgtctggaag
attctgggag ccacgcacga ctttttcaac ggagcatcgt 4800acctggtgtg
gatgtaccaa catatgctcg gccatcaccc ctacaccaac attgctggag
4860cagatcccga cgtgtcgacg tctgagcccg atgttcgtcg tatcaagccc
aaccaaaagt 4920ggtttgtcaa ccacatcaac cagcacatgt ttgttccttt
cctgtacgga ctgctggcgt 4980tcaaggtgcg cattcaggac atcaacattt
tgtactttgt caagaccaat gacgctattc 5040gtgtcaatcc catctcgaca
tggcacactg tgatgttctg gggcggcaag gctttctttg 5100tctggtatcg
cctgattgtt cccctgcagt atctgcccct gggcaaggtg ctgctcttgt
5160tcacggtcgc ggacatggtg tcgtcttact ggctggcgct gaccttccag
gcgaaccacg 5220ttgttgagga agttcagtgg ccgttgcctg acgagaacgg
gatcatccaa aaggactggg 5280cagctatgca ggtcgagact acgcaggatt
acgcacacga ttcgcacctc tggaccagca 5340tcactggcag cttgaactac
caggctgtgc accatctgtt ccccaacgtg tcgcagcacc 5400attatcccga
tattctggcc atcatcaaga acacctgcag cgagtacaag gttccatacc
5460ttgtcaagga tacgttttgg caagcatttg cttcacattt ggagcacttg
cgtgttcttg 5520gactccgtcc caaggaagag taggcagcta agc
5553118792DNAArtificial SequenceIgD9e synthetic delta-9 elongase
(codon-optimized for Yarrowia lipolytica) 118atggctctgg ccaacgacgc
tggcgagcga atctgggctg ccgtcaccga tcccgaaatc 60ctcattggca ccttctccta
cctgctcctg aagcctctcc tgcgaaactc tggtctcgtg 120gacgagaaga
aaggagccta ccgaacctcc atgatctggt acaacgtcct cctggctctc
180ttctctgccc tgtccttcta cgtgactgcc accgctctcg gctgggacta
cggtactgga 240gcctggctgc gaagacagac cggtgatact ccccagcctc
tctttcagtg tccctctcct 300gtctgggact ccaagctgtt cacctggact
gccaaggcct tctactattc taagtacgtg 360gagtacctcg acaccgcttg
gctggtcctc aagggcaagc gagtgtcctt tctgcaggcc 420ttccatcact
ttggagctcc ctgggacgtc tacctcggca ttcgactgca caacgagggt
480gtgtggatct tcatgttctt taactcgttc attcacacca tcatgtacac
ctactatgga 540ctgactgccg ctggctacaa gttcaaggcc aagcctctga
tcactgccat gcagatttgc 600cagttcgtcg gtggctttct cctggtctgg
gactacatca acgttccctg cttcaactct 660gacaagggca agctgttctc
ctgggctttc aactacgcct acgtcggatc tgtctttctc 720ctgttctgtc
acttctttta ccaggacaac ctggccacca agaaatccgc taaggctggt
780aagcagcttt ag 792119792DNAIsochrysis galbanamisc_featuredelta-9
elongase 119atggccctcg caaacgacgc gggagagcgc atctgggcgg ctgtgaccga
cccggaaatc 60ctcattggca ccttctcgta cttgctactc aaaccgctgc tccgcaattc
cgggctggtg 120gatgagaaga agggcgcata caggacgtcc atgatctggt
acaacgttct gctggcgctc 180ttctctgcgc tgagcttcta cgtgacggcg
accgccctcg gctgggacta tggtacgggc 240gcgtggctgc gcaggcaaac
cggcgacaca ccgcagccgc tcttccagtg cccgtccccg 300gtttgggact
cgaagctctt cacatggacc gccaaggcat tctattactc caagtacgtg
360gagtacctcg acacggcctg gctggtgctc aagggcaaga gggtctcctt
tctccaggcc 420ttccaccact ttggcgcgcc gtgggatgtg tacctcggca
ttcggctgca caacgagggc 480gtatggatct tcatgttttt caactcgttc
attcacacca tcatgtacac ctactacggc 540ctcaccgccg ccgggtataa
gttcaaggcc aagccgctca tcaccgcgat gcagatctgc 600cagttcgtgg
gcggcttcct gttggtctgg gactacatca acgtcccctg cttcaactcg
660gacaaaggga agttgttcag ctgggctttc aactatgcat acgtcggctc
ggtcttcttg 720ctcttctgcc actttttcta ccaggacaac ttggcaacga
agaaatcggc caaggcgggc 780aagcagctct ag 792120263PRTIsochrysis
galbana 120Met Ala Leu Ala Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala
Val Thr1 5 10 15Asp Pro Glu Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu
Leu Lys Pro 20 25 30Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Lys Lys
Gly Ala Tyr Arg 35 40 45Thr Ser Met Ile Trp Tyr Asn Val Leu Leu Ala
Leu Phe Ser Ala Leu 50 55 60Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly
Trp Asp Tyr Gly Thr Gly65 70 75 80Ala Trp Leu Arg Arg Gln Thr Gly
Asp Thr Pro Gln Pro Leu Phe Gln 85 90 95Cys Pro Ser Pro Val Trp Asp
Ser Lys Leu Phe Thr Trp Thr Ala Lys 100 105 110Ala Phe Tyr Tyr Ser
Lys Tyr Val Glu Tyr Leu Asp Thr Ala Trp Leu 115 120 125Val Leu Lys
Gly Lys Arg Val Ser Phe Leu Gln Ala Phe His His Phe 130 135 140Gly
Ala Pro Trp Asp Val Tyr Leu Gly Ile Arg Leu His Asn Glu Gly145 150
155 160Val Trp Ile Phe Met Phe Phe Asn Ser Phe Ile His Thr Ile Met
Tyr 165 170 175Thr Tyr Tyr Gly Leu Thr Ala Ala Gly Tyr Lys Phe Lys
Ala Lys Pro 180 185 190Leu Ile Thr Ala Met Gln Ile Cys Gln Phe Val
Gly Gly Phe Leu Leu 195 200 205Val Trp Asp Tyr Ile Asn Val Pro Cys
Phe Asn Ser Asp Lys Gly Lys 210 215 220Leu Phe Ser Trp Ala Phe Asn
Tyr Ala Tyr Val Gly Ser Val Phe Leu225 230 235 240Leu Phe Cys His
Phe Phe Tyr Gln Asp Asn Leu Ala Thr Lys Lys Ser 245 250 255Ala Lys
Ala Gly Lys Gln Leu 260121101DNAArtificial SequencePrimer IL3-1A
121gccaacgacg ctggcgagcg aatctgggct gccgtcaccg atcccgaaat
cctcattggc 60accttctcct acctgctcct gaagcctctc ctgcgaaact c
101122101DNAArtificial SequencePrimer IL3-1B 122accagagttt
cgcaggagag gcttcaggag caggtaggag aaggtgccaa tgaggatttc 60gggatcggtg
acggcagccc agattcgctc gccagcgtcg t 101123100DNAArtificial
SequencePrimer IL3-2A 123tggtctcgtg gacgagaaga aaggagccta
ccgaacctcc atgatctggt acaacgtcct 60cctggctctc ttctctgccc tgtccttcta
cgtgactgcc 100124100DNAArtificial SequencePrimer IL3-2B
124cggtggcagt cacgtagaag gacagggcag agaagagagc caggaggacg
ttgtaccaga 60tcatggaggt tcggtaggct cctttcttct cgtccacgag
100125100DNAArtificial SequencePrimer IL3-3A 125accgctctcg
gctgggacta cggtactgga gcctggctgc gaagacagac cggtgatact 60ccccagcctc
tctttcagtg tccctctcct gtctgggact 100126100DNAArtificial
SequencePrimer IL3-3B 126ttggagtccc agacaggaga gggacactga
aagagaggct ggggagtatc accggtctgt 60cttcgcagcc aggctccagt accgtagtcc
cagccgagag 100127100DNAArtificial SequencePrimer IL3-4A
127ccaagctgtt cacctggact gccaaggcct tctactattc taagtacgtg
gagtacctcg 60acaccgcttg gctggtcctc aagggcaagc gagtgtcctt
100128100DNAArtificial SequencePrimer IL3-4B 128cagaaaggac
actcgcttgc ccttgaggac cagccaagcg gtgtcgaggt actccacgta 60cttagaatag
tagaaggcct tggcagtcca ggtgaacagc 10012989DNAArtificial
SequencePrimer IL3-5A 129ttccatcact ttggagctcc ctgggacgtc
tacctcggca ttcgactgca caacgagggt 60gtgtggatct tcatgttctt taactcgtt
8913089DNAArtificial SequencePrimer IL3-5B 130aatgaacgag ttaaagaaca
tgaagatcca cacaccctcg ttgtgcagtc gaatgccgag 60gtagacgtcc cagggagctc
caaagtgat 8913191DNAArtificial SequencePrimer IL3-6A 131cattcacacc
atcatgtaca cctactatgg actgactgcc gctggctaca agttcaaggc 60caagcctctg
atcactgcca tgcagatttg c 9113291DNAArtificial SequencePrimer IL3-6B
132actggcaaat ctgcatggca gtgatcagag gcttggcctt gaacttgtag
ccagcggcag 60tcagtccata gtaggtgtac atgatggtgt g
9113394DNAArtificial SequencePrimer IL3-7A 133cagttcgtcg gtggctttct
cctggtctgg gactacatca acgttccctg cttcaactct 60gacaagggca agctgttctc
ctgggctttc aact 9413494DNAArtificial SequencePrimer IL3-7B
134gcgtagttga aagcccagga gaacagcttg cccttgtcag agttgaagca
gggaacgttg 60atgtagtccc agaccaggag aaagccaccg acga
9413591DNAArtificial SequencePrimer IL3-8A 135acgcctacgt cggatctgtc
tttctcctgt tctgtcactt cttttaccag gacaacctgg 60ccaccaagaa atccgctaag
gctggtaagc a 9113691DNAArtificial SequencePrimer IL3-8B
136aagctgctta ccagccttag cggatttctt ggtggccagg ttgtcctggt
aaaagaagtg 60acagaacagg agaaagacag atccgacgta g
9113741DNAArtificial SequencePrimer IL3-1F 137tttccatggc tctggccaac
gacgctggcg agcgaatctg g 4113836DNAArtificial SequencePrimer IL3-4R
138tttctgcaga aaggacactc gcttgccctt gaggac 3613941DNAArtificial
SequencePrimer IL3-5F 139tttctgcagg ccttccatca ctttggagct
ccctgggacg t 4114042DNAArtificial SequencePrimer IL3-8R
140tttgcggccg ctaaagctgc ttaccagcct tagcggattt ct
42141417DNAArtificial Sequence417 bp NcoI/PstI fragment pT9(1-4)
141catggctctg gccaacgacg ctggcgagcg aatctgggct gccgtcaccg
atcccgaaat 60cctcattggc accttctcct acctgctcct gaagcctctc ctgcgaaact
ctggtctcgt 120ggacgagaag aaaggagcct accgaacctc catgatctgg
tacaacgtcc tcctggctct 180cttctctgcc ctgtccttct acgtgactgc
caccgctctc ggctgggact acggtactgg 240agcctggctg cgaagacaga
ccggtgatac tccccagcct ctctttcagt gtccctctcc 300tgtctgggac
tccaagctgt tcacctggac tgccaaggcc ttctactatt ctaagtacgt
360ggagtacctc gacaccgctt ggctggtcct caagggcaag cgagtgtcct ttctgca
417142377DNAArtificial Sequence377 bp PstI/Not1 fragment pT9(5-8)
142ggccttccat cactttggag ctccctggga cgtctacctc ggcattcgac
tgcacaacga 60gggtgtgtgg atcttcatgt tctttaactc gttcattcac accatcatgt
acacctacta 120tggactgact gccgctggct acaagttcaa ggccaagcct
ctgatcactg ccatgcagat 180ttgccagttc gtcggtggct ttctcctggt
ctgggactac atcaacgttc cctgcttcaa 240ctctgacaag ggcaagctgt
tctcctgggc tttcaactac gcctacgtcg gatctgtctt 300tctcctgttc
tgtcacttct tttaccagga caacctggcc accaagaaat ccgctaaggc
360tggtaagcag ctttagc 3771438165DNAArtificial SequencePlasmid
pZUF17 143gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
ctaactcaca 60ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
ccagctgcat 120taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
ttgggcgctc ttccgcttcc 180tcgctcactg actcgctgcg ctcggtcgtt
cggctgcggc gagcggtatc agctcactca 240aaggcggtaa tacggttatc
cacagaatca ggggataacg caggaaagaa catgtgagca 300aaaggccagc
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
360ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
gcgaaacccg 420acaggactat aaagatacca ggcgtttccc cctggaagct
ccctcgtgcg ctctcctgtt 480ccgaccctgc cgcttaccgg atacctgtcc
gcctttctcc cttcgggaag cgtggcgctt 540tctcatagct cacgctgtag
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600tgtgtgcacg
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
660gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
taacaggatt 720agcagagcga ggtatgtagg cggtgctaca gagttcttga
agtggtggcc taactacggc 780tacactagaa ggacagtatt tggtatctgc
gctctgctga agccagttac cttcggaaaa 840agagttggta gctcttgatc
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900tgcaagcagc
agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
960acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
catgagatta 1020tcaaaaagga tcttcaccta gatcctttta aattaaaaat
gaagttttaa atcaatctaa 1080agtatatatg agtaaacttg gtctgacagt
taccaatgct taatcagtga ggcacctatc 1140tcagcgatct gtctatttcg
ttcatccata gttgcctgac tccccgtcgt gtagataact 1200acgatacggg
agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
1260tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
gcgcagaagt 1320ggtcctgcaa ctttatccgc ctccatccag tctattaatt
gttgccggga agctagagta 1380agtagttcgc cagttaatag tttgcgcaac
gttgttgcca ttgctacagg catcgtggtg 1440tcacgctcgt cgtttggtat
ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500acatgatccc
ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
1560agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
taattctctt 1620actgtcatgc catccgtaag atgcttttct gtgactggtg
agtactcaac caagtcattc 1680tgagaatagt gtatgcggcg accgagttgc
tcttgcccgg
cgtcaatacg ggataatacc 1740gcgccacata gcagaacttt aaaagtgctc
atcattggaa aacgttcttc ggggcgaaaa 1800ctctcaagga tcttaccgct
gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860tgatcttcag
catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
1920aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
actcttcctt 1980tttcaatatt attgaagcat ttatcagggt tattgtctca
tgagcggata catatttgaa 2040tgtatttaga aaaataaaca aataggggtt
ccgcgcacat ttccccgaaa agtgccacct 2100gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160gctacacttg
ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
2220acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
gttccgattt 2280agtgctttac ggcacctcga ccccaaaaaa cttgattagg
gtgatggttc acgtagtggg 2340ccatcgccct gatagacggt ttttcgccct
ttgacgttgg agtccacgtt ctttaatagt 2400ggactcttgt tccaaactgg
aacaacactc aaccctatct cggtctattc ttttgattta 2460taagggattt
tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
2520aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc
aggctgcgca 2580actgttggga agggcgatcg gtgcgggcct cttcgctatt
acgccagctg gcgaaagggg 2640gatgtgctgc aaggcgatta agttgggtaa
cgccagggtt ttcccagtca cgacgttgta 2700aaacgacggc cagtgaattg
taatacgact cactataggg cgaattgggt accgggcccc 2760ccctcgaggt
cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct
2820tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt
ctacactgat 2880taattttcgg gccaataatt taaaaaaatc gtgttatata
atattatatg tattatatat 2940atacatcatg atgatactga cagtcatgtc
ccattgctaa atagacagac tccatctgcc 3000gcctccaact gatgttctca
atatttaagg ggtcatctcg cattgtttaa taataaacag 3060actccatcta
ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt
3120acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt
taggaaacaa 3180tttataatgg cagttcgttc atttaacaat ttatgtagaa
taaatgttat aaatgcgtat 3240gggaaatctt aaatatggat agcataaatg
atatctgcat tgcctaattc gaaatcaaca 3300gcaacgaaaa aaatcccttg
tacaacataa atagtcatcg agaaatatca actatcaaag 3360aacagctatt
cacacgttac tattgagatt attattggac gagaatcaca cactcaactg
3420tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt
tcatacttct 3480agtcatttca tcccacatat tccttggatt tctctccaat
gaatgacatt ctatcttgca 3540aattcaacaa ttataataag atataccaaa
gtagcggtat agtggcaatc aaaaagcttc 3600tctggtgtgc ttctcgtatt
tatttttatt ctaatgatcc attaaaggta tatatttatt 3660tcttgttata
taatcctttt gtttattaca tgggctggat acataaaggt attttgattt
3720aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg
taggaaatta 3780ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa
aatcgtattt ccaggttaga 3840cgttccgcag aatctagaat gcggtatgcg
gtacattgtt cttcgaacgt aaaagttgcg 3900ctccctgaga tattgtacat
ttttgctttt acaagtacaa gtacatcgta caactatgta 3960ctactgttga
tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat
4020gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta
ttggcgttca 4080attaatcata gacttatgaa tctgcacggt gtgcgctgcg
agttactttt agcttatgca 4140tgctacttgg gtgtaatatt gggatctgtt
cggaaatcaa cggatgctca atcgatttcg 4200acagtaatta attaagtcat
acacaagtca gctttcttcg agcctcatat aagtataagt 4260agttcaacgt
attagcactg tacccagcat ctccgtatcg agaaacacaa caacatgccc
4320cattggacag atcatgcgga tacacaggtt gtgcagtatc atacatactc
gatcagacag 4380gtcgtctgac catcatacaa gctgaacaag cgctccatac
ttgcacgctc tctatataca 4440cagttaaatt acatatccat agtctaacct
ctaacagtta atcttctggt aagcctccca 4500gccagccttc tggtatcgct
tggcctcctc aataggatct cggttctggc cgtacagacc 4560tcggccgaca
attatgatat ccgttccggt agacatgaca tcctcaacag ttcggtactg
4620ctgtccgaga gcgtctccct tgtcgtcaag acccaccccg ggggtcagaa
taagccagtc 4680ctcagagtcg cccttaggtc ggttctgggc aatgaagcca
accacaaact cggggtcgga 4740tcgggcaagc tcaatggtct gcttggagta
ctcgccagtg gccagagagc ccttgcaaga 4800cagctcggcc agcatgagca
gacctctggc cagcttctcg ttgggagagg ggactaggaa 4860ctccttgtac
tgggagttct cgtagtcaga gacgtcctcc ttcttctgtt cagagacagt
4920ttcctcggca ccagctcgca ggccagcaat gattccggtt ccgggtacac
cgtgggcgtt 4980ggtgatatcg gaccactcgg cgattcggtg acaccggtac
tggtgcttga cagtgttgcc 5040aatatctgcg aactttctgt cctcgaacag
gaagaaaccg tgcttaagag caagttcctt 5100gagggggagc acagtgccgg
cgtaggtgaa gtcgtcaatg atgtcgatat gggttttgat 5160catgcacaca
taaggtccga ccttatcggc aagctcaatg agctccttgg tggtggtaac
5220atccagagaa gcacacaggt tggttttctt ggctgccacg agcttgagca
ctcgagcggc 5280aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag
ggcattttgg tggtgaagag 5340gagactgaaa taaatttagt ctgcagaact
ttttatcgga accttatctg gggcagtgaa 5400gtatatgtta tggtaatagt
tacgagttag ttgaacttat agatagactg gactatacgg 5460ctatcggtcc
aaattagaaa gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa
5520aatgtgatca tgatgaaagc cagcaatgac gttgcagctg atattgttgt
cggccaaccg 5580cgccgaaaac gcagctgtca gacccacagc ctccaacgaa
gaatgtatcg tcaaagtgat 5640ccaagcacac tcatagttgg agtcgtactc
caaaggcggc aatgacgagt cagacagata 5700ctcgtcgact caggcgacga
cggaattcct gcagcccatc tgcagaattc aggagagacc 5760gggttggcgg
cgtatttgtg tcccaaaaaa cagccccaat tgccccggag aagacggcca
5820ggccgcctag atgacaaatt caacaactca cagctgactt tctgccattg
ccactagggg 5880ggggcctttt tatatggcca agccaagctc tccacgtcgg
ttgggctgca cccaacaata 5940aatgggtagg gttgcaccaa caaagggatg
ggatgggggg tagaagatac gaggataacg 6000gggctcaatg gcacaaataa
gaacgaatac tgccattaag actcgtgatc cagcgactga 6060caccattgca
tcatctaagg gcctcaaaac tacctcggaa ctgctgcgct gatctggaca
6120ccacagaggt tccgagcact ttaggttgca ccaaatgtcc caccaggtgc
aggcagaaaa 6180cgctggaaca gcgtgtacag tttgtcttaa caaaaagtga
gggcgctgag gtcgagcagg 6240gtggtgtgac ttgttatagc ctttagagct
gcgaaagcgc gtatggattt ggctcatcag 6300gccagattga gggtctgtgg
acacatgtca tgttagtgta cttcaatcgc cccctggata 6360tagccccgac
aataggccgt ggcctcattt ttttgccttc cgcacatttc cattgctcgg
6420tacccacacc ttgcttctcc tgcacttgcc aaccttaata ctggtttaca
ttgaccaaca 6480tcttacaagc ggggggcttg tctagggtat atataaacag
tggctctccc aatcggttgc 6540cagtctcttt tttcctttct ttccccacag
attcgaaatc taaactacac atcacacaat 6600gcctgttact gacgtcctta
agcgaaagtc cggtgtcatc gtcggcgacg atgtccgagc 6660cgtgagtatc
cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat gacacaatcc
6720gaaagtcgct agcaacacac actctctaca caaactaacc cagctctcca
tggctgagga 6780taagaccaag gtcgagttcc ctaccctgac tgagctgaag
cactctatcc ctaacgcttg 6840ctttgagtcc aacctcggac tctcgctcta
ctacactgcc cgagcgatct tcaacgcatc 6900tgcctctgct gctctgctct
acgctgcccg atctactccc ttcattgccg ataacgttct 6960gctccacgct
ctggtttgcg ccacctacat ctacgtgcag ggtgtcatct tctggggttt
7020ctttaccgtc ggtcacgact gtggtcactc tgccttctcc cgataccact
ccgtcaactt 7080catcattggc tgcatcatgc actctgccat tctgactccc
ttcgagtcct ggcgagtgac 7140ccaccgacac catcacaaga acactggcaa
cattgataag gacgagatct tctaccctca 7200tcggtccgtc aaggacctcc
aggacgtgcg acaatgggtc tacaccctcg gaggtgcttg 7260gtttgtctac
ctgaaggtcg gatatgctcc tcgaaccatg tcccactttg acccctggga
7320ccctctcctg cttcgacgag cctccgctgt catcgtgtcc ctcggagtct
gggctgcctt 7380cttcgctgcc tacgcctacc tcacatactc gctcggcttt
gccgtcatgg gcctctacta 7440ctatgctcct ctctttgtct ttgcttcgtt
cctcgtcatt actaccttct tgcatcacaa 7500cgacgaagct actccctggt
acggtgactc ggagtggacc tacgtcaagg gcaacctgag 7560ctccgtcgac
cgatcgtacg gagctttcgt ggacaacctg tctcaccaca ttggcaccca
7620ccaggtccat cacttgttcc ctatcattcc ccactacaag ctcaacgaag
ccaccaagca 7680ctttgctgcc gcttaccctc acctcgtgag acgtaacgac
gagcccatca ttactgcctt 7740cttcaagacc gctcacctct ttgtcaacta
cggagctgtg cccgagactg ctcagatttt 7800caccctcaaa gagtctgccg
ctgcagccaa ggccaagagc gactaagcgg ccgcaagtgt 7860ggatggggaa
gtgagtgccc ggttctgtgt gcacaattgg caatccaaga tggatggatt
7920caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg
atatttatgt 7980ttgacacttg agaatgtacg atacaagcac tgtccaagta
caatactaaa catactgtac 8040atactcatac tcgtacccgg gcaacggttt
cacttgagtg cagtggctag tgctcttact 8100cgtacagtgt gcaatactgc
gtatcatagt ctttgatgta tatcgtattc attcatgtta 8160gttgc
81651447879DNAArtificial SequencePlasmid pDMW237 144ggccgcaagt
gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60gatggatgga
ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac
120ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag
tacaatacta 180aacatactgt acatactcat actcgtaccc gggcaacggt
ttcacttgag tgcagtggct 240agtgctctta ctcgtacagt gtgcaatact
gcgtatcata gtctttgatg tatatcgtat 300tcattcatgt tagttgcgta
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360gagtgagcta
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
420tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
ttgcgtattg 480ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
ggtcgttcgg ctgcggcgag 540cggtatcagc tcactcaaag gcggtaatac
ggttatccac agaatcaggg gataacgcag 600gaaagaacat gtgagcaaaa
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660tggcgttttt
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
720agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
ggaagctccc 780tcgtgcgctc tcctgttccg accctgccgc ttaccggata
cctgtccgcc tttctccctt 840cgggaagcgt ggcgctttct catagctcac
gctgtaggta tctcagttcg gtgtaggtcg 900ttcgctccaa gctgggctgt
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960ccggtaacta
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
1020ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
ttcttgaagt 1080ggtggcctaa ctacggctac actagaagga cagtatttgg
tatctgcgct ctgctgaagc 1140cagttacctt cggaaaaaga gttggtagct
cttgatccgg caaacaaacc accgctggta 1200gcggtggttt ttttgtttgc
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260atcctttgat
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
1320ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
taaaaatgaa 1380gttttaaatc aatctaaagt atatatgagt aaacttggtc
tgacagttac caatgcttaa 1440tcagtgaggc acctatctca gcgatctgtc
tatttcgttc atccatagtt gcctgactcc 1500ccgtcgtgta gataactacg
atacgggagg gcttaccatc tggccccagt gctgcaatga 1560taccgcgaga
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
1620gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
attaattgtt 1680gccgggaagc tagagtaagt agttcgccag ttaatagttt
gcgcaacgtt gttgccattg 1740ctacaggcat cgtggtgtca cgctcgtcgt
ttggtatggc ttcattcagc tccggttccc 1800aacgatcaag gcgagttaca
tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860gtcctccgat
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
1920cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
actggtgagt 1980actcaaccaa gtcattctga gaatagtgta tgcggcgacc
gagttgctct tgcccggcgt 2040caatacggga taataccgcg ccacatagca
gaactttaaa agtgctcatc attggaaaac 2100gttcttcggg gcgaaaactc
tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160ccactcgtgc
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
2220caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
aaatgttgaa 2280tactcatact cttccttttt caatattatt gaagcattta
tcagggttat tgtctcatga 2340gcggatacat atttgaatgt atttagaaaa
ataaacaaat aggggttccg cgcacatttc 2400cccgaaaagt gccacctgac
gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460ttacgcgcag
cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
2520tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
cgggggctcc 2580ctttagggtt ccgatttagt gctttacggc acctcgaccc
caaaaaactt gattagggtg 2640atggttcacg tagtgggcca tcgccctgat
agacggtttt tcgccctttg acgttggagt 2700ccacgttctt taatagtgga
ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760tctattcttt
tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
2820tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
atttccattc 2880gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
cgggcctctt cgctattacg 2940ccagctggcg aaagggggat gtgctgcaag
gcgattaagt tgggtaacgc cagggttttc 3000ccagtcacga cgttgtaaaa
cgacggccag tgaattgtaa tacgactcac tatagggcga 3060attgggtacc
gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaattcat
3120gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga
gactgccgag 3180atccagtcta cactgattaa ttttcgggcc aataatttaa
aaaaatcgtg ttatataata 3240ttatatgtat tatatatata catcatgatg
atactgacag tcatgtccca ttgctaaata 3300gacagactcc atctgccgcc
tccaactgat gttctcaata tttaaggggt catctcgcat 3360tgtttaataa
taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt
3420atgaacttat ttttattact tagtattatt agacaactta cttgctttat
gaaaaacact 3480tcctatttag gaaacaattt ataatggcag ttcgttcatt
taacaattta tgtagaataa 3540atgttataaa tgcgtatggg aaatcttaaa
tatggatagc ataaatgata tctgcattgc 3600ctaattcgaa atcaacagca
acgaaaaaaa tcccttgtac aacataaata gtcatcgaga 3660aatatcaact
atcaaagaac agctattcac acgttactat tgagattatt attggacgag
3720aatcacacac tcaactgtct ttctctcttc tagaaataca ggtacaagta
tgtactattc 3780tcattgttca tacttctagt catttcatcc cacatattcc
ttggatttct ctccaatgaa 3840tgacattcta tcttgcaaat tcaacaatta
taataagata taccaaagta gcggtatagt 3900ggcaatcaaa aagcttctct
ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960aaaggtatat
atttatttct tgttatataa tccttttgtt tattacatgg gctggataca
4020taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc
agtgtcaact 4080gtaatggtag gaaattacca tacttttgaa gaagcaaaaa
aaatgaaaga aaaaaaaaat 4140cgtatttcca ggttagacgt tccgcagaat
ctagaatgcg gtatgcggta cattgttctt 4200cgaacgtaaa agttgcgctc
cctgagatat tgtacatttt tgcttttaca agtacaagta 4260catcgtacaa
ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg
4320tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact
tgtagtaagc 4380cgggttattg gcgttcaatt aatcatagac ttatgaatct
gcacggtgtg cgctgcgagt 4440tacttttagc ttatgcatgc tacttgggtg
taatattggg atctgttcgg aaatcaacgg 4500atgctcaatc gatttcgaca
gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560ctcatataag
tataagtagt tcaacgtatt agcactgtac ccagcatctc cgtatcgaga
4620aacacaacaa catgccccat tggacagatc atgcggatac acaggttgtg
cagtatcata 4680catactcgat cagacaggtc gtctgaccat catacaagct
gaacaagcgc tccatacttg 4740cacgctctct atatacacag ttaaattaca
tatccatagt ctaacctcta acagttaatc 4800ttctggtaag cctcccagcc
agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860ttctggccgt
acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc
4920tcaacagttc ggtactgctg tccgagagcg tctcccttgt cgtcaagacc
caccccgggg 4980gtcagaataa gccagtcctc agagtcgccc ttaggtcggt
tctgggcaat gaagccaacc 5040acaaactcgg ggtcggatcg ggcaagctca
atggtctgct tggagtactc gccagtggcc 5100agagagccct tgcaagacag
ctcggccagc atgagcagac ctctggccag cttctcgttg 5160ggagagggga
ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc
5220ttctgttcag agacagtttc ctcggcacca gctcgcaggc cagcaatgat
tccggttccg 5280ggtacaccgt gggcgttggt gatatcggac cactcggcga
ttcggtgaca ccggtactgg 5340tgcttgacag tgttgccaat atctgcgaac
tttctgtcct cgaacaggaa gaaaccgtgc 5400ttaagagcaa gttccttgag
ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460tcgatatggg
ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc
5520tccttggtgg tggtaacatc cagagaagca cacaggttgg ttttcttggc
tgccacgagc 5580ttgagcactc gagcggcaaa ggcggacttg tggacgttag
ctcgagcttc gtaggagggc 5640attttggtgg tgaagaggag actgaaataa
atttagtctg cagaactttt tatcggaacc 5700ttatctgggg cagtgaagta
tatgttatgg taatagttac gagttagttg aacttataga 5760tagactggac
tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg
5820tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag caatgacgtt
gcagctgata 5880ttgttgtcgg ccaaccgcgc cgaaaacgca gctgtcagac
ccacagcctc caacgaagaa 5940tgtatcgtca aagtgatcca agcacactca
tagttggagt cgtactccaa aggcggcaat 6000gacgagtcag acagatactc
gtcgactcag gcgacgacgg aattcctgca gcccatctgc 6060agaattcagg
agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc
6120cccggagaag acggccaggc cgcctagatg acaaattcaa caactcacag
ctgactttct 6180gccattgcca ctaggggggg gcctttttat atggccaagc
caagctctcc acgtcggttg 6240ggctgcaccc aacaataaat gggtagggtt
gcaccaacaa agggatggga tggggggtag 6300aagatacgag gataacgggg
ctcaatggca caaataagaa cgaatactgc cattaagact 6360cgtgatccag
cgactgacac cattgcatca tctaagggcc tcaaaactac ctcggaactg
6420ctgcgctgat ctggacacca cagaggttcc gagcacttta ggttgcacca
aatgtcccac 6480caggtgcagg cagaaaacgc tggaacagcg tgtacagttt
gtcttaacaa aaagtgaggg 6540cgctgaggtc gagcagggtg gtgtgacttg
ttatagcctt tagagctgcg aaagcgcgta 6600tggatttggc tcatcaggcc
agattgaggg tctgtggaca catgtcatgt tagtgtactt 6660caatcgcccc
ctggatatag ccccgacaat aggccgtggc ctcatttttt tgccttccgc
6720acatttccat tgctcggtac ccacaccttg cttctcctgc acttgccaac
cttaatactg 6780gtttacattg accaacatct tacaagcggg gggcttgtct
agggtatata taaacagtgg 6840ctctcccaat cggttgccag tctctttttt
cctttctttc cccacagatt cgaaatctaa 6900actacacatc acacaatgcc
tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc 6960ggcgacgatg
tccgagccgt gagtatccac gacaagatca gtgtcgagac gacgcgtttt
7020gtgtaatgac acaatccgaa agtcgctagc aacacacact ctctacacaa
actaacccag 7080ctctccatgg ctctggccaa cgacgctggc gagcgaatct
gggctgccgt caccgatccc 7140gaaatcctca ttggcacctt ctcctacctg
ctcctgaagc ctctcctgcg aaactctggt 7200ctcgtggacg agaagaaagg
agcctaccga acctccatga tctggtacaa cgtcctcctg 7260gctctcttct
ctgccctgtc cttctacgtg actgccaccg ctctcggctg ggactacggt
7320actggagcct ggctgcgaag acagaccggt gatactcccc agcctctctt
tcagtgtccc 7380tctcctgtct gggactccaa gctgttcacc tggactgcca
aggccttcta ctattctaag 7440tacgtggagt acctcgacac cgcttggctg
gtcctcaagg gcaagcgagt gtcctttctg 7500caggccttcc atcactttgg
agctccctgg gacgtctacc tcggcattcg actgcacaac 7560gagggtgtgt
ggatcttcat gttctttaac tcgttcattc acaccatcat gtacacctac
7620tatggactga ctgccgctgg ctacaagttc aaggccaagc ctctgatcac
tgccatgcag 7680atttgccagt tcgtcggtgg ctttctcctg gtctgggact
acatcaacgt tccctgcttc 7740aactctgaca agggcaagct gttctcctgg
gctttcaact acgcctacgt cggatctgtc 7800tttctcctgt tctgtcactt
cttttaccag gacaacctgg ccaccaagaa atccgctaag 7860gctggtaagc
agctttagc 78791456457DNAArtificial SequencePlasmid pKUNT2
145tttgaatcga atcgatgagc ctaaaatgaa cccgagtata tctcataaaa
ttctcggtga 60gaggtctgtg actgtcagta caaggtgcct tcattatgcc ctcaacctta
ccatacctca 120ctgaatgtag tgtacctcta aaaatgaaat acagtgccaa
aagccaaggc actgagctcg 180tctaacggac ttgatataca accaattaaa
acaaatgaaa agaaatacag ttctttgtat 240catttgtaac aattaccctg
tacaaactaa ggtattgaaa tcccacaata ttcccaaagt 300ccaccccttt
ccaaattgtc atgcctacaa ctcatatacc aagcactaac ctaccgttta
360aacaccacta aaaccccaca aaatatatct taccgaatat acagatctgc
gacgacggaa 420ttcctgcagc ccatctgcag aattcaggag agaccgggtt
ggcggcgtat ttgtgtccca 480aaaaacagcc ccaattgccc caattgaccc
caaattgacc cagtagcggg cccaaccccg 540gcgagagccc ccttcacccc
acatatcaaa cctcccccgg ttcccacact tgccgttaag 600ggcgtagggt
actgcagtct ggaatctacg cttgttcaga ctttgtacta gtttctttgt
660ctggccatcc gggtaaccca tgccggacgc aaaatagact actgaaaatt
tttttgcttt 720gtggttggga ctttagccaa gggtataaaa gaccaccgtc
cccgaattac ctttcctctt 780cttttctctc tctccttgtc aactcacacc
cgaaatcgtt aagcatttcc ttctgagtat 840aagaatcatt caccatggat
tcgaccacgc agaccaacac cggcaccggc aaggtggccg 900tgcagccccc
cacggccttc attaagccca ttgagaaggt gtccgagccc gtctacgaca
960cctttggcaa cgagttcact cctccagact actctatcaa ggatattctg
gatgccattc 1020cccaggagtg ctacaagcgg tcctacgtta agtcctactc
gtacgtggcc cgagactgct 1080tctttatcgc cgtttttgcc tacatggcct
acgcgtacct gcctcttatt ccctcggctt 1140ccggccgagc tgtggcctgg
gccatgtact ccattgtcca gggtctgttt ggcaccggtc 1200tgtgggttct
tgcccacgag tgtggccact ctgctttctc cgactctaac accgtcaaca
1260acgtcaccgg atgggttctg cactcctcca tgctggtccc ttactacgcc
tggaagctga 1320cccactccat gcaccacaag tccactggtc acctcacccg
tgatatggtg tttgtgccca 1380aggaccgaaa ggagtttatg gagaaccgag
gcgcccatga ctggtctgag cttgctgagg 1440acgctcccct catgaccctc
tacggcctca tcacccagca ggtgtttgga tggcctctgt 1500atctgctgtc
ttacgttacc ggacagaagt accccaagct caacaaatgg gctgtcaacc
1560acttcaaccc caacgccccg ctgtttgaga agaaggactg gttcaacatc
tggatctcta 1620acgtcggtat tggtatcacc atgtccgtca tcgcatactc
catcaaccga tggggcctgg 1680cttccgtcac cctctactac ctgatcccct
acctgtgggt caaccactgg ctcgtggcca 1740tcacctacct gcagcacacc
gaccccactc tgccccacta ccacgccgac cagtggaact 1800tcacccgagg
agccgccgcc accatcgacc gagagtttgg cttcatcggc tccttctgct
1860tccatgacat catcgagacc cacgttctgc accactacgt gtctcgaatt
cccttctaca 1920acgcccgaat cgccactgag aagatcaaga aggtcatggg
caagcactac cgacacgacg 1980acaccaactt catcaagtct ctttacactg
tcgcccgaac ctgccagttt gttgaaggta 2040aggaaggcat tcagatgttt
agaaacgtca atggagtcgg agttgctcct gacggcctgc 2100cttctaaaaa
gtaggcggcc gcaagtgtgg atggggaagt gagtgcccgg ttctgtgtgc
2160acaattggca atccaagatg gatggattca acacagggat atagcgagct
acgtggtggt 2220gcgaggatat agcaacggat atttatgttt gacacttgag
aatgtacgat acaagcactg 2280tccaagtaca atactaaaca tactgtacat
actcatactc gtacccgggc aacggtttca 2340cttgagtgca gtggctagtg
ctcttactcg tacagtgtgc aatactgcgt atcatagtct 2400ttgatgtata
tcgtattcat tcatgttagt tgcgtacgaa gtcgtcaatg atgtcgatat
2460gggttttgat catgcacaca taaggtccga ccttatcggc aagctcaatg
agctccttgg 2520tggtggtaac atccagagaa gcacacaggt tggttttctt
ggctgccacg agcttgagca 2580ctcgagcggc aaaggcggac ttgtggacgt
tagctcgagc ttcgtaggag ggcattttgg 2640tggtgaagag gagactgaaa
taaatttagt ctgcagaact ttttatcgga accttatctg 2700gggcagtgaa
gtatatgtta tggtaatagt tacgagttag ttgaacttat agatagactg
2760gactatacgg ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg
gcgtcgcctt 2820tgccgacaaa aatgtgatca tgatgaaagc cagcaatgac
gttgcagctg atattgttgt 2880cggccaaccg cgccgaaaac gcagctgtca
gacccacagc ctccaacgaa gaatgtatcg 2940tcaaagtgat ccaagcacac
tcatagttgg agtcgtactc caaaggcggc aatgacgagt 3000cagacagata
ctcgtcgacc ttttccttgg gaaccaccac cgtcagccct tctgactcac
3060gtattgtagc caccgacaca ggcaacagtc cgtggatagc agaatatgtc
ttgtcggtcc 3120atttctcacc aactttaggc gtcaagtgaa tgttgcagaa
gaagtatgtg ccttcattga 3180gaatcggtgt tgctgatttc aataaagtct
tgagatcagt ttggcgcgcc agctgcatta 3240atgaatcggc caacgcgcgg
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 3300gctcactgac
tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
3360ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
tgtgagcaaa 3420aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct 3480ccgcccccct gacgagcatc acaaaaatcg
acgctcaagt cagaggtggc gaaacccgac 3540aggactataa agataccagg
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3600gaccctgccg
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
3660tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
agctgggctg 3720tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
tccggtaact atcgtcttga 3780gtccaacccg gtaagacacg acttatcgcc
actggcagca gccactggta acaggattag 3840cagagcgagg tatgtaggcg
gtgctacaga gttcttgaag tggtggccta actacggcta 3900cactagaaga
acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
3960agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
tttttgtttg 4020caagcagcag attacgcgca gaaaaaaagg atctcaagaa
gatcctttga tcttttctac 4080ggggtctgac gctcagtgga acgaaaactc
acgttaaggg attttggtca tgagattatc 4140aaaaaggatc ttcacctaga
tccttttaaa ttaaaaatga agttttaaat caatctaaag 4200tatatatgag
taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
4260agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
agataactac 4320gatacgggag ggcttaccat ctggccccag tgctgcaatg
ataccgcgag acccacgctc 4380accggctcca gatttatcag caataaacca
gccagccgga agggccgagc gcagaagtgg 4440tcctgcaact ttatccgcct
ccatccagtc tattaattgt tgccgggaag ctagagtaag 4500tagttcgcca
gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
4560acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
ggcgagttac 4620atgatccccc atgttgtgca aaaaagcggt tagctccttc
ggtcctccga tcgttgtcag 4680aagtaagttg gccgcagtgt tatcactcat
ggttatggca gcactgcata attctcttac 4740tgtcatgcca tccgtaagat
gcttttctgt gactggtgag tactcaacca agtcattctg 4800agaatagtgt
atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
4860gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
ggcgaaaact 4920ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
cccactcgtg cacccaactg 4980atcttcagca tcttttactt tcaccagcgt
ttctgggtga gcaaaaacag gaaggcaaaa 5040tgccgcaaaa aagggaataa
gggcgacacg gaaatgttga atactcatac tcttcctttt 5100tcaatattat
tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
5160tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
tgccacctga 5220tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
ccgcatcagg aaattgtaag 5280cgttaatatt ttgttaaaat tcgcgttaaa
tttttgttaa atcagctcat tttttaacca 5340ataggccgaa atcggcaaaa
tcccttataa atcaaaagaa tagaccgaga tagggttgag 5400tgttgttcca
gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
5460gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
aatcaagttt 5520tttggggtcg aggtgccgta aagcactaaa tcggaaccct
aaagggagcc cccgatttag 5580agcttgacgg ggaaagccgg cgaacgtggc
gagaaaggaa gggaagaaag cgaaaggagc 5640gggcgctagg gcgctggcaa
gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 5700gcttaatgcg
ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa
5760gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
atgtgctgca 5820aggcgattaa gttgggtaac gccagggttt tcccagtcac
gacgttgtaa aacgacggcc 5880agtgaattgt aatacgactc actatagggc
gaattgggcc cgacgtcgca tgcagtggtg 5940gtattgtgac tggggatgta
gttgagaata agtcatacac aagtcagctt tcttcgagcc 6000tcatataagt
ataagtagtt caacgtatta gcactgtacc cagcatctcc gtatcgagaa
6060acacaacaac atgccccatt ggacagatca tgcggataca caggttgtgc
agtatcatac 6120atactcgatc agacaggtcg tctgaccatc atacaagctg
aacaagcgct ccatacttgc 6180acgctctcta tatacacagt taaattacat
atccatagtc taacctctaa cagttaatct 6240tctggtaagc ctcccagcca
gccttctggt atcgcttggc ctcctcaata ggatctcggt 6300tctggccgta
cagacctcgg ccgacaatta tgatatccgt tccggtagac atgacatcct
6360caacagttcg gtactgctgt ccgagagcgt ctcccttgtc gtcaagaccc
accccggggg 6420tcagaataag ccagtcctca gagtcgccct taattaa
64571461936DNAYarrowia lipolyticaCDS(283)..(1539)delta-12
desaturase 146cgtagttata tacaagaggt agatgcgtgc tggtgttaga
ggggctctca ggattaggag 60gaaaatttga cattggccct caacatataa cctcgggtgt
gcctctgttt accctcagct 120tttgcttgtc cccaagtcag tcacgccagg
ccaaaaaggt tggtggattg acagggagaa 180aaaaaaaagc ctagtgggtt
taaactcgag gtaagacatt gaaatatata ccggtcggca 240tcctgagtcc
ctttctcgta ttccaacaga ccgaccatag aa atg gat tcg acc 294 Met Asp Ser
Thr 1acg cag acc aac acc ggc acc ggc aag gtg gcc gtg cag ccc ccc
acg 342Thr Gln Thr Asn Thr Gly Thr Gly Lys Val Ala Val Gln Pro Pro
Thr5 10 15 20gcc ttc att aag ccc att gag aag gtg tcc gag ccc gtc
tac gac acc 390Ala Phe Ile Lys Pro Ile Glu Lys Val Ser Glu Pro Val
Tyr Asp Thr 25 30 35ttt ggc aac gag ttc act cct cca gac tac tct atc
aag gat att ctg 438Phe Gly Asn Glu Phe Thr Pro Pro Asp Tyr Ser Ile
Lys Asp Ile Leu 40 45 50gat gcc att ccc cag gag tgc tac aag cgg tcc
tac gtt aag tcc tac 486Asp Ala Ile Pro Gln Glu Cys Tyr Lys Arg Ser
Tyr Val Lys Ser Tyr 55 60 65tcg tac gtg gcc cga gac tgc ttc ttt atc
gcc gtt ttt gcc tac atg 534Ser Tyr Val Ala Arg Asp Cys Phe Phe Ile
Ala Val Phe Ala Tyr Met 70 75 80gcc tac gcg tac ctg cct ctt att ccc
tcg gct tcc ggc cga gct gtg 582Ala Tyr Ala Tyr Leu Pro Leu Ile Pro
Ser Ala Ser Gly Arg Ala Val85 90 95 100gcc tgg gcc atg tac tcc att
gtc cag ggt ctg ttt ggc acc ggt ctg 630Ala Trp Ala Met Tyr Ser Ile
Val Gln Gly Leu Phe Gly Thr Gly Leu 105 110 115tgg gtt ctt gcc cac
gag tgt ggc cac tct gct ttc tcc gac tct aac 678Trp Val Leu Ala His
Glu Cys Gly His Ser Ala Phe Ser Asp Ser Asn 120 125 130acc gtc aac
aac gtc acc gga tgg gtt ctg cac tcc tcc atg ctg gtc 726Thr Val Asn
Asn Val Thr Gly Trp Val Leu His Ser Ser Met Leu Val 135 140 145cct
tac tac gcc tgg aag ctg acc cac tcc atg cac cac aag tcc act 774Pro
Tyr Tyr Ala Trp Lys Leu Thr His Ser Met His His Lys Ser Thr 150 155
160ggt cac ctc acc cgt gat atg gtg ttt gtg ccc aag gac cga aag gag
822Gly His Leu Thr Arg Asp Met Val Phe Val Pro Lys Asp Arg Lys
Glu165 170 175 180ttt atg gag aac cga ggc gcc cat gac tgg tct gag
ctt gct gag gac 870Phe Met Glu Asn Arg Gly Ala His Asp Trp Ser Glu
Leu Ala Glu Asp 185 190 195gct ccc ctc atg acc ctc tac ggc ctc atc
acc cag cag gtg ttt gga 918Ala Pro Leu Met Thr Leu Tyr Gly Leu Ile
Thr Gln Gln Val Phe Gly 200 205 210tgg cct ctg tat ctg ctg tct tac
gtt acc gga cag aag tac ccc aag 966Trp Pro Leu Tyr Leu Leu Ser Tyr
Val Thr Gly Gln Lys Tyr Pro Lys 215 220 225ctc aac aaa tgg gct gtc
aac cac ttc aac ccc aac gcc ccg ctg ttt 1014Leu Asn Lys Trp Ala Val
Asn His Phe Asn Pro Asn Ala Pro Leu Phe 230 235 240gag aag aag gac
tgg ttc aac atc tgg atc tct aac gtc ggt att ggt 1062Glu Lys Lys Asp
Trp Phe Asn Ile Trp Ile Ser Asn Val Gly Ile Gly245 250 255 260atc
acc atg tcc gtc atc gca tac tcc atc aac cga tgg ggc ctg gct 1110Ile
Thr Met Ser Val Ile Ala Tyr Ser Ile Asn Arg Trp Gly Leu Ala 265 270
275tcc gtc acc ctc tac tac ctg atc ccc tac ctg tgg gtc aac cac tgg
1158Ser Val Thr Leu Tyr Tyr Leu Ile Pro Tyr Leu Trp Val Asn His Trp
280 285 290ctc gtg gcc atc acc tac ctg cag cac acc gac ccc act ctg
ccc cac 1206Leu Val Ala Ile Thr Tyr Leu Gln His Thr Asp Pro Thr Leu
Pro His 295 300 305tac cac gcc gac cag tgg aac ttc acc cga gga gcc
gcc gcc acc atc 1254Tyr His Ala Asp Gln Trp Asn Phe Thr Arg Gly Ala
Ala Ala Thr Ile 310 315 320gac cga gag ttt ggc ttc atc ggc tcc ttc
tgc ttc cat gac atc atc 1302Asp Arg Glu Phe Gly Phe Ile Gly Ser Phe
Cys Phe His Asp Ile Ile325 330 335 340gag acc cac gtt ctg cac cac
tac gtg tct cga att ccc ttc tac aac 1350Glu Thr His Val Leu His His
Tyr Val Ser Arg Ile Pro Phe Tyr Asn 345 350 355gcc cga atc gcc act
gag aag atc aag aag gtc atg ggc aag cac tac 1398Ala Arg Ile Ala Thr
Glu Lys Ile Lys Lys Val Met Gly Lys His Tyr 360 365 370cga cac gac
gac acc aac ttc atc aag tct ctt tac act gtc gcc cga 1446Arg His Asp
Asp Thr Asn Phe Ile Lys Ser Leu Tyr Thr Val Ala Arg 375 380 385acc
tgc cag ttt gtt gaa ggt aag gaa ggc att cag atg ttt aga aac 1494Thr
Cys Gln Phe Val Glu Gly Lys Glu Gly Ile Gln Met Phe Arg Asn 390 395
400gtc aat gga gtc gga gtt gct cct gac ggc ctg cct tct aaa aag
1539Val Asn Gly Val Gly Val Ala Pro Asp Gly Leu Pro Ser Lys Lys405
410 415tagagctaga aatgttattt gattgtgttt taactgaaca gcaccgagcc
cgaggctaag 1599ccaagcgaag ccgaggggtt gtgtagtcca tggacgtaac
gagtaggcga tatcaccgca 1659ctcggcactg cgtgtctgcg ttcatgggcg
aagtcacatt acgctgacaa ccgttgtagt 1719ttccctttag tatcaatact
gttacaagta ccggtctcgt actcgtactg atacgaatct 1779gtgggaagaa
gtcaccctta tcagaccttc atactgatgt ttcggatatc aatagaactg
1839gcatagagcc gttaaagaag tttcacttaa tcactccaac cctcctactt
gtagattcaa 1899gcagatcgat aagatggatt tgatggtcag tgctagc
1936147419PRTYarrowia lipolytica 147Met Asp Ser Thr Thr Gln Thr Asn
Thr Gly Thr Gly Lys Val Ala Val1 5 10 15Gln Pro Pro Thr Ala Phe Ile
Lys Pro Ile Glu Lys Val Ser Glu Pro 20 25 30Val Tyr Asp Thr Phe Gly
Asn Glu Phe Thr Pro Pro Asp Tyr Ser Ile 35 40 45Lys Asp Ile Leu Asp
Ala Ile Pro Gln Glu Cys Tyr Lys Arg Ser Tyr 50 55 60Val Lys Ser Tyr
Ser Tyr Val Ala Arg Asp Cys Phe Phe Ile Ala Val65 70 75 80Phe Ala
Tyr Met Ala Tyr Ala Tyr Leu Pro Leu Ile Pro Ser Ala Ser 85 90 95Gly
Arg Ala Val Ala Trp Ala Met Tyr Ser Ile Val Gln Gly Leu Phe 100 105
110Gly Thr Gly Leu Trp Val Leu Ala His Glu Cys Gly His Ser Ala Phe
115 120 125Ser Asp Ser Asn Thr Val Asn Asn Val Thr Gly Trp Val Leu
His Ser 130 135 140Ser Met Leu Val Pro Tyr Tyr Ala Trp Lys Leu Thr
His Ser Met His145 150 155 160His Lys Ser Thr Gly His Leu Thr Arg
Asp Met Val Phe Val Pro Lys 165 170 175Asp Arg Lys Glu Phe Met Glu
Asn Arg Gly Ala His Asp Trp Ser Glu 180 185 190Leu Ala Glu Asp Ala
Pro Leu Met Thr Leu Tyr Gly Leu Ile Thr Gln 195 200 205Gln Val Phe
Gly Trp Pro Leu Tyr Leu Leu Ser Tyr Val Thr Gly Gln 210 215 220Lys
Tyr Pro Lys Leu Asn Lys Trp Ala Val Asn His Phe Asn Pro Asn225 230
235 240Ala Pro Leu Phe Glu Lys Lys Asp Trp Phe Asn Ile Trp Ile Ser
Asn 245 250 255Val Gly Ile Gly Ile Thr Met Ser Val Ile Ala Tyr Ser
Ile Asn Arg 260 265 270Trp Gly Leu Ala Ser Val Thr Leu Tyr Tyr Leu
Ile Pro Tyr Leu Trp 275 280 285Val Asn His Trp Leu Val Ala Ile Thr
Tyr Leu Gln His Thr Asp Pro 290 295 300Thr Leu Pro His Tyr His Ala
Asp Gln Trp Asn Phe Thr Arg Gly Ala305 310 315 320Ala Ala Thr Ile
Asp Arg Glu Phe Gly Phe Ile Gly Ser Phe Cys Phe 325 330 335His Asp
Ile Ile Glu Thr His Val Leu His His Tyr Val Ser Arg Ile 340 345
350Pro Phe Tyr Asn Ala Arg Ile Ala Thr Glu Lys Ile Lys Lys Val Met
355 360 365Gly Lys His Tyr Arg His Asp Asp Thr Asn Phe Ile Lys Ser
Leu Tyr 370 375 380Thr Val Ala Arg Thr Cys Gln Phe Val Glu Gly Lys
Glu Gly Ile Gln385 390 395 400Met Phe Arg Asn Val Asn Gly Val Gly
Val Ala Pro Asp Gly Leu Pro 405 410 415Ser Lys Lys
14810448DNAArtificial SequencePlasmid pDMW297 148cgattgcccc
ggagaagacg gccaggccgc ctagatgaca aattcaacaa ctcacagctg 60actttctgcc
attgccacta ggggggggcc tttttatatg gccaagccaa gctctccacg
120tcggttgggc tgcacccaac aataaatggg tagggttgca ccaacaaagg
gatgggatgg 180ggggtagaag atacgaggat aacggggctc aatggcacaa
ataagaacga atactgccat 240taagactcgt gatccagcga ctgacaccat
tgcatcatct aagggcctca aaactacctc 300ggaactgctg cgctgatctg
gacaccacag aggttccgag cactttaggt tgcaccaaat 360gtcccaccag
gtgcaggcag aaaacgctgg aacagcgtgt acagtttgtc ttaacaaaaa
420gtgagggcgc tgaggtcgag cagggtggtg tgacttgtta tagcctttag
agctgcgaaa 480gcgcgtatgg atttggctca tcaggccaga ttgagggtct
gtggacacat gtcatgttag 540tgtacttcaa tcgccccctg gatatagccc
cgacaatagg ccgtggcctc atttttttgc 600cttccgcaca tttccattgc
tcggtaccca caccttgctt ctcctgcact tgccaacctt 660aatactggtt
tacattgacc aacatcttac aagcgggggg cttgtctagg gtatatataa
720acagtggctc tcccaatcgg ttgccagtct cttttttcct ttctttcccc
acagattcga 780aatctaaact acacatcaca caatgcctgt tactgacgtc
cttaagcgaa agtccggtgt 840catcgtcggc gacgatgtcc gagccgtgag
tatccacgac aagatcagtg tcgagacgac 900gcgttttgtg taatgacaca
atccgaaagt cgctagcaac acacactctc tacacaaact 960aacccagctc
tccatggtga agtccaagcg acaggctctg cccctcacca tcgacggaac
1020tacctacgac gtctccgctt gggtgaactt ccaccctggt ggagctgaaa
tcattgagaa 1080ctaccaggga cgagatgcta ctgacgcctt catggttatg
cactctcagg aagccttcga 1140caagctcaag cgaatgccca agatcaaccc
ctcctccgag ctgcctcccc aggctgccgt 1200caacgaagct caggaggatt
tccgaaagct ccgagaagag ctgatcgcca ctggcatgtt 1260tgacgcctct
cccctctggt actcgtacaa gatctccacc accctgggtc ttggcgtgct
1320tggatacttc ctgatggtcc agtaccagat gtacttcatt ggtgctgtgc
tgctcggtat 1380gcactaccag caaatgggat ggctgtctca tgacatctgc
caccaccaga ccttcaagaa 1440ccgaaactgg aataacctcg tgggtctggt
ctttggcaac ggactccagg gcttctccgt 1500gacctggtgg aaggacagac
acaacgccca tcattctgct accaacgttc agggtcacga 1560tcccgacatt
gataacctgc
ctctgctcgc ctggtccgag gacgatgtca ctcgagcttc 1620tcccatctcc
cgaaagctca ttcagttcca acagtactat ttcctggtca tctgtattct
1680cctgcgattc atctggtgtt tccagtctgt gctgaccgtt cgatccctca
aggaccgaga 1740caaccagttc taccgatctc agtacaagaa agaggccatt
ggactcgctc tgcactggac 1800tctcaagacc ctgttccacc tcttctttat
gccctccatc ctgacctcgc tcctggtgtt 1860ctttgtttcc gagctcgtcg
gtggcttcgg aattgccatc gtggtcttca tgaaccacta 1920ccctctggag
aagatcggtg attccgtctg ggacggacat ggcttctctg tgggtcagat
1980ccatgagacc atgaacattc gacgaggcat cattactgac tggttctttg
gaggcctgaa 2040ctaccagatc gagcaccatc tctggcccac cctgcctcga
cacaacctca ctgccgtttc 2100ctaccaggtg gaacagctgt gccagaagca
caacctcccc taccgaaacc ctctgcccca 2160tgaaggtctc gtcatcctgc
tccgatacct ggccgtgttc gctcgaatgg ccgagaagca 2220gcccgctggc
aaggctctct aagcggccgc attgatgatt ggaaacacac acatgggtta
2280tatctaggtg agagttagtt ggacagttat atattaaatc agctatgcca
acggtaactt 2340cattcatgtc aacgaggaac cagtgactgc aagtaatata
gaatttgacc accttgccat 2400tctcttgcac tcctttacta tatctcattt
atttcttata tacaaatcac ttcttcttcc 2460cagcatcgag ctcggaaacc
tcatgagcaa taacatcgtg gatctcgtca atagagggct 2520ttttggactc
cttgctgttg gccaccttgt ccttgctgtc tggctcattc tgtttcaacg
2580ccttttaatt aagtcataca caagtcagct ttcttcgagc ctcatataag
tataagtagt 2640tcaacgtatt agcactgtac ccagcatctc cgtatcgaga
aacacaacaa catgccccat 2700tggacagatc atgcggatac acaggttgtg
cagtatcata catactcgat cagacaggtc 2760gtctgaccat catacaagct
gaacaagcgc tccatacttg cacgctctct atatacacag 2820ttaaattaca
tatccatagt ctaacctcta acagttaatc ttctggtaag cctcccagcc
2880agccttctgg tatcgcttgg cctcctcaat aggatctcgg ttctggccgt
acagacctcg 2940gccgacaatt atgatatccg ttccggtaga catgacatcc
tcaacagttc ggtactgctg 3000tccgagagcg tctcccttgt cgtcaagacc
caccccgggg gtcagaataa gccagtcctc 3060agagtcgccc ttaggtcggt
tctgggcaat gaagccaacc acaaactcgg ggtcggatcg 3120ggcaagctca
atggtctgct tggagtactc gccagtggcc agagagccct tgcaagacag
3180ctcggccagc atgagcagac ctctggccag cttctcgttg ggagagggga
ctaggaactc 3240cttgtactgg gagttctcgt agtcagagac gtcctccttc
ttctgttcag agacagtttc 3300ctcggcacca gctcgcaggc cagcaatgat
tccggttccg ggtacaccgt gggcgttggt 3360gatatcggac cactcggcga
ttcggtgaca ccggtactgg tgcttgacag tgttgccaat 3420atctgcgaac
tttctgtcct cgaacaggaa gaaaccgtgc ttaagagcaa gttccttgag
3480ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg tcgatatggg
ttttgatcat 3540gcacacataa ggtccgacct tatcggcaag ctcaatgagc
tccttggtgg tggtaacatc 3600cagagaagca cacaggttgg ttttcttggc
tgccacgagc ttgagcactc gagcggcaaa 3660ggcggacttg tggacgttag
ctcgagcttc gtaggagggc attttggtgg tgaagaggag 3720actgaaataa
atttagtctg cagaactttt tatcggaacc ttatctgggg cagtgaagta
3780tatgttatgg taatagttac gagttagttg aacttataga tagactggac
tatacggcta 3840tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg
tcgcctttgc cgacaaaaat 3900gtgatcatga tgaaagccag caatgacgtt
gcagctgata ttgttgtcgg ccaaccgcgc 3960cgaaaacgca gctgtcagac
ccacagcctc caacgaagaa tgtatcgtca aagtgatcca 4020agcacactca
tagttggagt cgtactccaa aggcggcaat gacgagtcag acagatactc
4080gtcgactcag gcgacgacgg aattcctgca gcccatctgc agaattcagg
agagaccggg 4140ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc
cccggagaag acggccaggc 4200cgcctagatg acaaattcaa caactcacag
ctgactttct gccattgcca ctaggggggg 4260gcctttttat atggccaagc
caagctctcc acgtcggttg ggctgcaccc aacaataaat 4320gggtagggtt
gcaccaacaa agggatggga tggggggtag aagatacgag gataacgggg
4380ctcaatggca caaataagaa cgaatactgc cattaagact cgtgatccag
cgactgacac 4440cattgcatca tctaagggcc tcaaaactac ctcggaactg
ctgcgctgat ctggacacca 4500cagaggttcc gagcacttta ggttgcacca
aatgtcccac caggtgcagg cagaaaacgc 4560tggaacagcg tgtacagttt
gtcttaacaa aaagtgaggg cgctgaggtc gagcagggtg 4620gtgtgacttg
ttatagcctt tagagctgcg aaagcgcgta tggatttggc tcatcaggcc
4680agattgaggg tctgtggaca catgtcatgt tagtgtactt caatcgcccc
ctggatatag 4740ccccgacaat aggccgtggc ctcatttttt tgccttccgc
acatttccat tgctcggtac 4800ccacaccttg cttctcctgc acttgccaac
cttaatactg gtttacattg accaacatct 4860tacaagcggg gggcttgtct
agggtatata taaacagtgg ctctcccaat cggttgccag 4920tctctttttt
cctttctttc cccacagatt cgaaatctaa actacacatc acacaatgcc
4980tgttactgac gtccttaagc gaaagtccgg tgtcatcgtc ggcgacgatg
tccgagccgt 5040gagtatccac gacaagatca gtgtcgagac gacgcgtttt
gtgtaatgac acaatccgaa 5100agtcgctagc aacacacact ctctacacaa
actaacccag ctctccatgg ctctggccaa 5160cgacgctggc gagcgaatct
gggctgccgt caccgatccc gaaatcctca ttggcacctt 5220ctcctacctg
ctcctgaagc ctctcctgcg aaactctggt ctcgtggacg agaagaaagg
5280agcctaccga acctccatga tctggtacaa cgtcctcctg gctctcttct
ctgccctgtc 5340cttctacgtg actgccaccg ctctcggctg ggactacggt
actggagcct ggctgcgaag 5400acagaccggt gatactcccc agcctctctt
tcagtgtccc tctcctgtct gggactccaa 5460gctgttcacc tggactgcca
aggccttcta ctattctaag tacgtggagt acctcgacac 5520cgcttggctg
gtcctcaagg gcaagcgagt gtcctttctg caggccttcc atcactttgg
5580agctccctgg gacgtctacc tcggcattcg actgcacaac gagggtgtgt
ggatcttcat 5640gttctttaac tcgttcattc acaccatcat gtacacctac
tatggactga ctgccgctgg 5700ctacaagttc aaggccaagc ctctgatcac
tgccatgcag atttgccagt tcgtcggtgg 5760ctttctcctg gtctgggact
acatcaacgt tccctgcttc aactctgaca agggcaagct 5820gttctcctgg
gctttcaact acgcctacgt cggatctgtc tttctcctgt tctgtcactt
5880cttttaccag gacaacctgg ccaccaagaa atccgctaag gctggtaagc
agctttagcg 5940gccgcaagtg tggatgggga agtgagtgcc cggttctgtg
tgcacaattg gcaatccaag 6000atggatggat tcaacacagg gatatagcga
gctacgtggt ggtgcgagga tatagcaacg 6060gatatttatg tttgacactt
gagaatgtac gatacaagca ctgtccaagt acaatactaa 6120acatactgta
catactcata ctcgtacccg ggcaacggtt tcacttgagt gcagtggcta
6180gtgctcttac tcgtacagtg tgcaatactg cgtatcatag tctttgatgt
atatcgtatt 6240cattcatgtt agttgcgtac gagccggaag cataaagtgt
aaagcctggg gtgcctaatg 6300agtgagctaa ctcacattaa ttgcgttgcg
ctcactgccc gctttccagt cgggaaacct 6360gtcgtgccag ctgcattaat
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 6420gcgctcttcc
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
6480ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
ataacgcagg 6540aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
cgtaaaaagg ccgcgttgct 6600ggcgtttttc cataggctcc gcccccctga
cgagcatcac aaaaatcgac gctcaagtca 6660gaggtggcga aacccgacag
gactataaag ataccaggcg tttccccctg gaagctccct 6720cgtgcgctct
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
6780gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
tgtaggtcgt 6840tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
cccgaccgct gcgccttatc 6900cggtaactat cgtcttgagt ccaacccggt
aagacacgac ttatcgccac tggcagcagc 6960cactggtaac aggattagca
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 7020gtggcctaac
tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
7080agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
ccgctggtag 7140cggtggtttt tttgtttgca agcagcagat tacgcgcaga
aaaaaaggat ctcaagaaga 7200tcctttgatc ttttctacgg ggtctgacgc
tcagtggaac gaaaactcac gttaagggat 7260tttggtcatg agattatcaa
aaaggatctt cacctagatc cttttaaatt aaaaatgaag 7320ttttaaatca
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
7380cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
cctgactccc 7440cgtcgtgtag ataactacga tacgggaggg cttaccatct
ggccccagtg ctgcaatgat 7500accgcgagac ccacgctcac cggctccaga
tttatcagca ataaaccagc cagccggaag 7560ggccgagcgc agaagtggtc
ctgcaacttt atccgcctcc atccagtcta ttaattgttg 7620ccgggaagct
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
7680tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
ccggttccca 7740acgatcaagg cgagttacat gatcccccat gttgtgcaaa
aaagcggtta gctccttcgg 7800tcctccgatc gttgtcagaa gtaagttggc
cgcagtgtta tcactcatgg ttatggcagc 7860actgcataat tctcttactg
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 7920ctcaaccaag
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
7980aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
ttggaaaacg 8040ttcttcgggg cgaaaactct caaggatctt accgctgttg
agatccagtt cgatgtaacc 8100cactcgtgca cccaactgat cttcagcatc
ttttactttc accagcgttt ctgggtgagc 8160aaaaacagga aggcaaaatg
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 8220actcatactc
ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
8280cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
gcacatttcc 8340ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta
agcgcggcgg gtgtggtggt 8400tacgcgcagc gtgaccgcta cacttgccag
cgccctagcg cccgctcctt tcgctttctt 8460cccttccttt ctcgccacgt
tcgccggctt tccccgtcaa gctctaaatc gggggctccc 8520tttagggttc
cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
8580tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
cgttggagtc 8640cacgttcttt aatagtggac tcttgttcca aactggaaca
acactcaacc ctatctcggt 8700ctattctttt gatttataag ggattttgcc
gatttcggcc tattggttaa aaaatgagct 8760gatttaacaa aaatttaacg
cgaattttaa caaaatatta acgcttacaa tttccattcg 8820ccattcaggc
tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
8880cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
agggttttcc 8940cagtcacgac gttgtaaaac gacggccagt gaattgtaat
acgactcact atagggcgaa 9000ttgggtaccg ggccccccct cgaggtcgat
ggtgtcgata agcttgatat cgaattcatg 9060tcacacaaac cgatcttcgc
ctcaaggaaa cctaattcta catccgagag actgccgaga 9120tccagtctac
actgattaat tttcgggcca ataatttaaa aaaatcgtgt tatataatat
9180tatatgtatt atatatatac atcatgatga tactgacagt catgtcccat
tgctaaatag 9240acagactcca tctgccgcct ccaactgatg ttctcaatat
ttaaggggtc atctcgcatt 9300gtttaataat aaacagactc catctaccgc
ctccaaatga tgttctcaaa atatattgta 9360tgaacttatt tttattactt
agtattatta gacaacttac ttgctttatg aaaaacactt 9420cctatttagg
aaacaattta taatggcagt tcgttcattt aacaatttat gtagaataaa
9480tgttataaat gcgtatggga aatcttaaat atggatagca taaatgatat
ctgcattgcc 9540taattcgaaa tcaacagcaa cgaaaaaaat cccttgtaca
acataaatag tcatcgagaa 9600atatcaacta tcaaagaaca gctattcaca
cgttactatt gagattatta ttggacgaga 9660atcacacact caactgtctt
tctctcttct agaaatacag gtacaagtat gtactattct 9720cattgttcat
acttctagtc atttcatccc acatattcct tggatttctc tccaatgaat
9780gacattctat cttgcaaatt caacaattat aataagatat accaaagtag
cggtatagtg 9840gcaatcaaaa agcttctctg gtgtgcttct cgtatttatt
tttattctaa tgatccatta 9900aaggtatata tttatttctt gttatataat
ccttttgttt attacatggg ctggatacat 9960aaaggtattt tgatttaatt
ttttgcttaa attcaatccc ccctcgttca gtgtcaactg 10020taatggtagg
aaattaccat acttttgaag aagcaaaaaa aatgaaagaa aaaaaaaatc
10080gtatttccag gttagacgtt ccgcagaatc tagaatgcgg tatgcggtac
attgttcttc 10140gaacgtaaaa gttgcgctcc ctgagatatt gtacattttt
gcttttacaa gtacaagtac 10200atcgtacaac tatgtactac tgttgatgca
tccacaacag tttgttttgt ttttttttgt 10260tttttttttt tctaatgatt
cattaccgct atgtatacct acttgtactt gtagtaagcc 10320gggttattgg
cgttcaatta atcatagact tatgaatctg cacggtgtgc gctgcgagtt
10380acttttagct tatgcatgct acttgggtgt aatattggga tctgttcgga
aatcaacgga 10440tgctcaat 1044814928DNAArtificial SequencePrimer
oIGsel1-1 149agcggccgca ccatggctct ggccaacg 2815025DNAArtificial
SequencePrimer oIGsel1-2 150tgcggccgct aaagctgctt accag
2515169DNAArtificial SequencePrimer 151catggtcaat caatgagacg
ccaacttctt aatctattga gacctgcagg tctagaaggg 60cggatcccc 69
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