BACKGROUND OF THE DISCLOSURE
[0001] The present disclosure relates to isolated polynucleotides encoding a
delta-9 elongase, delta-9 elongases encoded by the isolated polynucleotides,
expression vectors comprising the isolated polynucleotides, host cells comprising the
expression vectors, and methods for producing delta-9 elongases and polyunsaturated
fatty acids.
[0002] Polyunsaturated fatty acids (PUFAs) play many roles in the proper
functioning of life forms. For example, PUFAs are important components of the
plasma membrane of a cell, where they are found in the form of phospholipids. They
also serve as precursors to mammalian prostacyclins, eicosanoids, leukotrienes and
prostaglandins. Additionally, PUFAs are necessary for the proper development of the
developing infant brain as well as for tissue formation and repair. In view of the
biological significance of PUFAs, attempts are being made to efficiently produce
them, as well as intermediates leading to their production.
[0003] A number of enzymes, most notably desaturases and elongases, are
involved in PUFA biosynthesis (see Figure 1). Desaturases catalyze the introduction
of unsaturations (e.g., double bonds) between carbon atoms within the fatty acid alkyl
chain of the substrate. Elongases catalyze the addition of a 2-carbon unit to a fatty
acid substrate. For example, linoleic acid (LA, 18:2n-6) is produced from oleic acid
(OA, 18:ln-9) by a A12-desaturase. Eicosadienoic acid (EDA, 20:2n-6) is produced
from linoleic acid (LA, 18:2n-6) by a A9-elongase. Dihomo-y-linolenic acid (DGLA,
20:3n-6) is produced from eicosadienoic acid (EDA, 20:2n-6) by a A8-desaturase.
Arachidonic acid (ARA, 20:4n-6) is produced from dihomo-y-linolenic acid (DGLA,
20:3n-6) by a A5-desaturase (see Figure 1).
[0004] Elongases catalyze the conversion of y-linolenic acid (GLA, 18:3n-6)
to dihomo-y-linolenic acid (DGLA, 20:3n-6) and the conversion of stearidonic acid
(SDA, 18:4n-3) to eicosatetraenoic acid (ETA, 20:4n-3). Elongase also catalyzes the
conversion of arachidonic acid (ARA, 20:4n-6) to adrenic acid (ADA, 22:4n-6) and
the conversion of eicosapentaenoic acid (EPA, 20:5n-3) to ©3-docosapentaenoic acid
-2-
(22:5n-3). A9-elongase elongates polyunsaturated fatty acids containing unsaturation
at the carbon 9 position. For example, A9-elongase catalyzes the conversion of
linoleic acid (LA, 18:2n-6) to eicosadienoic acid (EDA, 20:2n-6), and the conversion
of a-linolenic acid (ALA, 18:3n-3) to eicosatrienoic acid (ETrA, 20:3n-3). co3-ETrA
may then be converted to co3-ETA by a A8-desaturase. co3-ETA may then be utilized
in the production of other polyunsaturated fatty acids, such as co3-EPA, which may be
added to pharmaceutical compositions, nutritional compositions, animal feeds, as well
as other products such as cosmetics.
[0005] The elongases which have been identified in the past differ in terms
of the substrates upon which they act. Furthermore, they are present in both animals
and plants. Those found in mammals have the ability to act on saturated,
monounsaturated and polyunsaturated fatty acids. In contrast, those found in plants
are specific for saturated or monounsaturated fatty acids. Thus, in order to generate
polyunsaturated fatty acids in plants, there is a need for a PUFA-specific elongase.
[0006] In both plants and animals, the elongation process is believed to be
the result of a four-step mechanism (Lassner et al.. The Plant Cell 8:281-292 (1996)).
CoA is the acyl carrier. Step one involves condensation of malonyl-CoA with a longchain
acyl-CoA to yield carbon dioxide and a P-ketoacyl-CoA in which the acyl
moiety has been elongated by two carbon atoms. Subsequent reactions include
reduction to P-hydroxyacyl-CoA, dehydration to an enoyl-CoA, and a second
reduction to yield the elongated acyl-CoA. The initial condensation reaction is not
only the substrate-specific step but also the rate-limiting step.
[0007] It should be noted that animals cannot desaturate beyond the A9
position, and therefore cannot convert oleic acid (OA, 18:ln-9) into linoleic acid (LA,
18:2n-6). Likewise, a-linolenic acid (ALA, 18:3n-3) cannot be synthesized by
mammals, since they lack A15-desaturase activity. However, a-linolenic acid can be
converted to stearidonic acid (SDA, 18:4n-3) by a A6-desaturase (see WO 96/13591;
see also U.S. Pat. No. 5,552,306), followed by elongation to eicosatetraenoic acid
(ETA, 20:4n-3) in mammals and algae. This polyunsaturated fatty acid (i.e., ETA,
20:4n-3) can then be converted to eicosapentaenoic acid (EPA, 20:5-3) by a A5-
-3-
desaturase. Other eukaryotes, including fungi and plants, have enzymes which
desaturate at carbons 12 (see WO 94/11516 and U.S. Pat. No. 5,443,974) and 15 (see
WO 93/11245). The major polyunsaturated fatty acids of animals therefore are either
derived from diet and/or from desaturation and elongation of linoleic acid or alinolenic
acid. In view of the inability of mammals to produce these essential longchain
fatty acids, it is of significant interest to isolate genes involved in PUFA
biosynthesis from species that naturally produce these fatty acids and to express these
genes in a microbial, plant or animal system which can be altered to provide
production of commercial quantities of one or more PUFAs. Consequently, there is a
definite need for elongase enzymes, the genes encoding the enzymes, as well as
recombinant methods of producing the enzymes.
[0008] In view of the above discussion, a definite need also exists for oils
containing levels of PUFAs beyond those naturally present as well as those enriched
in novel PUFAs. Such oils can be made by isolation and expression of elongase
genes.
[0009] One of the most important long-chain PUFAs is eicosapentaenoic
acid (EPA). EPA is found in fiingi and also in marine oils. Docosahexaenoic acid
(DHA) is another important long-chain PUFA. DHA is most often found in fish oil
and can also be purified from mammalian brain tissue. Arachidonic acid (ARA) is a
third important long-chain PUFA. ARA is found in filamentous fiangi and can also be
purified from mammalian tissues including the liver and the adrenal glands.
[0010] ARA, EPA and/or DHA, for example, can be produced via either the
alternate A-8 desaturase/A9-elongase pathway or the conventional A6 pathway (see
Figure 1). Elongases, which are active on substrate fatty acids in the conventional A6
pathway for the production of long-chain PUFAs, particularly ARA, EPA and DHA,
have previously been identified. The conventional A6 pathway for converting LA to
DGLA and ALA to co3-ETA utilizes the A6-desaturase enzyme to convert LA to
GLA, and ALA to stearidonic acid (SDA), and the A6-elongase enzyme to convert
GLA to DGLA, and SDA to co3-ETA. However, in certain instances, the alternate
A8-desaturase/A9-elongase pathway may be preferred over the conventional A6
-4-
pathway. For example, if particular residual omega-6 or omega-3 fatty acid
intermediates, such as GLA or SDA, are not desired during production of DGLA, co3-
ETA, ARA, EPA, co3-docosapentaenoic acid, (o6-docosapentaenoic acid, ADA and/or
DHA, the alternate A8-desaturase/A9-elongase pathway may be used as an alternative
to the conventional A6 pathway, to bypass GLA and SDA formation.
[0011 ] In the present disclsure, a new source of A9-elongase has been
identified for the production of long-chain PUFAs, in particular DGLA, ETA, ARA,
EPA, co3-docosapentaenoic acid, a)6-docosapentaenoic acid, ADA and/or DHA. The
A9-elongase enzyme of the present disclosure converts, for example, LA to C06-EDA,
and ALA to co3-ETrA. The production of DGLA from a)6-EDA, and ARA from
DGLA, is then catalyzed by a A8-desaturase and a A5-desaturase, respectively.
SUMMARY OF THE DISCLOSURE
[0012] In one aspect, the present disclosure relates to an isolated nucleic acid
molecule or fragment thereof comprising or complementary to an isolated nucleotide
sequence encoding a polypeptide having elongase activity, wherein the amino acid
sequence of the polypeptide has at least 68% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 20.
[0013] In another aspect, the present disclosure relates to an isolated
nucleotide sequence or fragment thereof comprising or complementary to at least 68%
of a nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and
SEQ ID NO: 19. The isolated nucleotide sequence or fragment thereof encodes a
functionally active elongase which utilizes a polyunsaturated fatty acid as a substrate,
and in particular a functionally active A9-elongase.
[0014] The nucleotide sequence may be from, for example, a Euglenoid sp.,
and may specifically be isolated from, for example, Euglena deses Ehr. CCMP 2916.
[0015] In another aspect, the present disclosure relates to a purified
polypeptide encoded by the above-described isolated nucleotide sequence as well as a
purified polypeptide which elongates polyunsaturated fatty acids containing
-5-
unsaturation at the carbon 9 position and has at least 68% amino acid identity to an
amino acid sequence selected from the group consisting of SEQ ID NO: 18 and SEQ
ID NO: 20.
[0016] In still another aspect, the present disclosure relates to an expression
vector. The expression vector comprises a nucleotide sequence operably linked to a
regulatory sequence, wherein the nucleotide sequence comprises or si complementary
to at least 68% of a nucleotide sequence selected from the group consisting of SEQ ID
NO: 17 and SEQ ID NO: 19. The disclosure also relates to a host cell comprising this
expression vector. The host cell may be, for example, a eukaryotic cell or a
prokaryotic cell. Suitable eukaryotic cells and prokaryotic cells are set forth herein.
The disclosure also relates to a transgenic seed comprising the expression vector.
[0017] In another aspect, the present disclosure relates to a plant cell, plant
seed, plant or plant tissue comprising the above-described expression vector, wherein
expression of the nucleotide sequence of the expression vector results in production of
at least one polyunsaturated fatty acid by the plant cell, plant or plant tissue. The
polyunsaturated fatty acid may be, for example, selected from the group consisting of
C06-EDA and co3-ETrA, and combinations thereof The present disclosure also
includes one or more plant oils or fatty acids expressed by the above plant cell, plant
seed, plant or plant tissue.
[0018] Furthermore, the present disclosure relates to a method of producing
a A9-elongase. The method comprises the steps of: a) isolating a nucleotide sequence
comprising or complementary to at least 68% of a nucleotide sequence selected from
the group consisting of SEQ ID NO: 17 and SEQ ID NO: 19; b) constructing an
expression vector comprising: i) the isolated nucleotide sequence operably linked to
ii) a regulatory sequence; and c) introducing the expression vector into a host cell for
a time and under conditions sufficient for expression of the A9-elongase, as
appropriate. The host cell may be, for example, a eukaryotic cell or a prokaryotic
cell. In particular, the eukaryotic cell may be, for example, a mammalian cell, an
insect cell, a plant cell or a ftingal cell. The plant cell may be from an oilseed plant
-6-
selected from the group consisting of soybean, Brassica species, safflower, sunflower,
maize, cotton, and flax.
[0019] Additionally, the present disclosure relates to a method for producing
a polyunsaturated fatty acid comprising the steps of: a) isolating a nucleotide
sequence comprising or complementary to at least 68% of a nucleotide sequence
selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 19; b)
constructing an expression vector comprising the isolated nucleotide sequence
operably linked to a regulatory sequence; c) introducing the expression vector into a
host cell under time and conditions sufficient for expression of A9-elongase; and d)
exposing the expressed A9-elongase to a substrate polyunsaturated fatty acid in order
to convert the substrate polyunsaturated fatty acid to a first product polyunsaturated
fatty acid. The "substrate" polyunsaturated fatty acid is, for example, LA or ALA,
and the "first product" polyimsaturated fatty acid is, for example, co6-EDA or co3-
ETrA, respectively. This method may further comprise the step of exposing the first
product polyunsaturated fatty acid to at least one desaturase, at least one additional
elongase, or combinations thereof, in order to convert the first product
polyunsaturated fatty acid to a second or subsequent polyunsaturated fatty acid. The
second or subsequent product polyunsaturated fatty acid may be, for example, DGLA
or co3-ETA, ARA, EPA, DP A, DHA, or combinations thereof
[0020] In another aspect, the present disclosure relates to a method for
producing a polyunsaturated fatty acid in a host cell comprising the steps of: a)
isolating a nucleotide sequence comprising or complementary to at least 68% of a
nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and SEQ
ID NO: 19; b) constructing an expression vector comprising the isolated nucleotide
sequence operably linked to a regulatory sequence; c) introducing i) the expression
vector and ii) at least one additional recombinant DNA construct comprising an
isolated nucleotide sequence encoding a A8-desaturase and operably linked to at least
one regulatory sequence, into a host cell for a time and under conditions sufficient for
expression of a A9-elongase and the A8-desaturase; and d) exposing the expressed
A9-elongase and the A8-desaturase to a substrate polyunsaturated fatty acid selected
from the group consisting of LA, ALA, and combinations thereof in order to convert
-7-
the substrate polyunsaturated fatty acid to a first product polyunsaturated fatty acid.
The first product polyunsaturated fatty acid may be, for example, DGLA, coS-ETA, or
combinations thereof This method may fiirther comprise the step of exposing the
first product polyunsaturated fatty acid to at least one desaturase, at least one
additional elongase, or combinations thereof, in order to convert the first product
polyunsaturated fatty acid to a second or subsequent polyunsaturated fatty acid. The
second or subsequent product polyunsaturated fatty acid may be, for example, ARA,
EPA, DP A, DHA, or combinations thereof In one aspect, this method may fiirther
comprise introducing into the host cell a recombinant DNA construct comprising i) an
isolated nucleotide sequence encoding a A5-desaturase operably linked to ii) a
regulatory sequence. The host cell may be as described above.
[0021] In another aspect, the present disclosure relates to a method for
producing a transgenic plant comprising transforming a plant cell with at least one
isolated nucleotide sequence or fragment thereof comprising or complementary to at
least 68% of a nucleotide sequence selected from the group consisting of SEQ ID NO:
17 and SEQ ID NO: 19, and regenerating a transgenic plant from the transformed
plant cell. The plant cell may be from an oilseed plant selected from the group
consisting of soybean, Brassica species, safflower, sunflower, maize, cotton, and flax.
In another aspect, the present disclosure relates to a seed obtained from the transgenic
plant produced by this method.
[0022] It should also be noted that each nucleotide and amino acid sequence
referred to herein has been assigned a particular sequence identification number. The
Sequence Listing (which is attached hereto), incorporated herein by reference, lists
each such sequence and its corresponding number.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 shows the fatty acid biosynthetic pathway and the role of
A9-elongase in this pathway.
[0024] Figures 2 A and 2B show alignment of nucleotide sequences SEQ ID
NO: 26 and SEQ ID NO: 27, which are nucleotide sequences of Eug-MO7-ELO#10
-8-
and the Eug-M07-ELO#14 variant, respectively, cloned into the Bam Hi/Hind III
sites of vector pYX242, as discussed in Example 3. A box is drawn around variants.
[0025] Figures 3A and 3B show alignment of amino acid sequences of A9-
elongase from Euglena deses Ehr. CCMP 2916 (Eug-MO7-ELO-10) (SEQ ID NO:
18) with known A9-elongases from Euglena gracialis (SEQ ID NO: 4), Isochrysis
galbana (SEQ ID NO: 2); Mouse Elovl4 elongase (Accession # AAG47667; SEQ ID
NO: 21), human EL0VL2 elongase (Accession # NP_060240; SEQ ID NO: 22), and
C. elegans elongase (Ascession # AF244356; SEQ ID NO: 23). Invariant residues are
shaded.
[0026] Figure 4A shows the A9-elongase amino acid sequence from
Pavlova salina (Accession # AAY15135; SEQ ID NO: 1).
[0027] Figure 4B shows the A9-elongase amino acid sequence from
Isochrysis galbana (Accession #AF390174; SEQ ID NO: 2).
[0028] Figure 4C shows the A9-elongase amino acid sequence from
Eutreptiella sp. (SEQ ID NO: 3).
[0029] Figure 4D shows the A9-elongase amino acid sequence from
Euglena gracialis (Accession # CAT16687; SEQ ID NO: 4).
[0030] Figure 4E shows the A9-elongase amino acid sequence from
Euglena anabena (SEQ ID NO: 5).
[0031] Figure 5A shows the nucleotide sequence (SEQ ID NO: 6) of clone
plate2_M07, obtained as described in Example 1.
[0032] Figure 5B shows the deduced amino acid sequence (SEQ ID NO: 7)
of clone plate2_M07, obtained as described in Example 1.
[0033] Figure 6A shows the nucleotide sequence (SEQ ID NO: 13) of the
plate2_M07 gene fragment putative 3'-end, obtained as described in Example 2.
-9-
[0034] Figure 6B shows the predicted amino acid sequence (SEQ ID NOs:
14 and 30-32) of the plate2_M07 gene fragment putative 3'-end, obtained as
described in Example 2. SEQ ID NOs: 14 and 30-32 are separated by an "*", which
represents a stop codon.
[0035] Figure 6C shows SEQ ID NO: 14.
[0036] Figure 6D shows SEQ ID NO: 30.
[0037] Figure 6E shows SEQ ID NO: 31.
[0038] Figure 6F shows SEQ ID NO: 32.
[0039] Figure 7A shows the nucleotide sequence (SEQ ID NO: 17) of the
putative A9-elongase from Euglena deses Ehr. CCMP 2916 (Eug-MO7-ELO#10),
obtained as described in Example 3.
[0040] Figure 7B shows the predicted amino acid sequence (SEQ ID NO:
18) encoded by the nucleotide sequence (SEQ ID NO: 17) of the putative A9-elongase
from Euglena deses Ehr. CCMP 2916 (Eug-MO7-ELO#10), obtained as described in
Example 3.
[0041 ] Figure 8A shows the nucleotide sequence (SEQ ID NO: 19) of a
variant A9-elongase from Euglena deses Ehr. CCMP 2916 (Eug-M07-ELO#14),
obtained as described in Example 3.
[0042] Figure 8B shows the predicted amino acid sequence (SEQ ID NO:
20) encoded by nucleotide sequence (SEQ ID NO: 19) of the variant A9-elongase
from Euglena deses Ehr. CCMP 2916 (Eug-M07-ELO#14), obtained as described in
Example 3.
[0043] Figure 9A shows the amino acid sequence from Mouse Elovl4
elongase (Accession # AAG47667; SEQ ID NO: 21).
[0044] Figure 9B shows the amino acid sequence from human EL0VL2
elongase (Accession # NP_060240; SEQ ID NO: 22).
-10-
[0045] Figure 9C shows the amino acid sequence from C elegans elongase
(Accession # AF244356; SEQ ID NO: 23).
DETAILED DESCRIPTION OF THE DISCLOSURE
[0046] The present disclosure is directed to the nucleotide (e.g., gene) and
translated amino acid sequences of a A9-elongase gene from Euglenoid sp., for
example, Euglena deses Ehr., specifically Euglena deses Ehr. CCMP 2916.
Furthermore, the present disclosure also includes uses of the gene and of the enzyme
encoded by the gene. For example, the gene and corresponding enzyme may be used
in the production of polyunsaturated fatty acids such as, for instance, co6-EDA, coBEtrA,
DGLA, co3-ETA, ARA, EPA, coB-docosapentaenoic acid, (B6-docosapentaenoic
acid, ADA, DHA, or any combinations thereof, which may be added to
pharmaceutical compositions, nutritional compositions and to other valuable products.
Definitions
[0047] As used herein, the singular forms "a" "an" and "the" include plural
referents unless the context clearly dictates otherwise. For the recitation of numeric
ranges herein, each intervening number there between with the same degree of
precision is explicitly contemplated. For example, for the range 6-9, the numbers 7
and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers
6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
[0048] Chimeric Construct: As used herein, the phrase "chimeric construct"
refers to a combination of nucleic acid molecules that are not normally found together
in nature. Accordingly, 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 normally found in nature.
[0049] Coding Sequence: As used herein, the term "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),
-11-
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, and polyadenylation recognition
sequences.
[0050] Complementarity: As used herein, the term "complementarity" refers
to the degree of relatedness between two DNA segments. It is determined by
measuring the ability of the sense strand of one DNA segment to hybridize with the
antisense strand of the other DNA segment, under appropriate conditions, to form a
double helix. In the double helix, adenine appears in one strand, thymine appears in
the other strand. Similarly, wherever guanine is found in one strand, cytosine is found
in the other. The greater the relatedness between the nucleotide sequences of two
DNA segments, the greater the ability to form hybrid duplexes between the strands of
the two DNA segments.
[0051] Encoded by. Hybridization, and Stringent Conditions: As used
herein, the phrase, "encoded by" refers to a nucleic acid sequence which codes for a
polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains
an amino acid sequence of at least 3 consecutive amino acids, more preferably at least
8 consecutive amino acids, and even more preferably at least 15 consecutive amino
acids from a polypeptide encoded by the nucleic acid sequence.
[0052] The present disclosure also encompasses an isolated nucleotide
sequence which encodes for an enzyme having PUFA elongase activity and that is
hybridizable, under moderately stringent conditions, to a nucleic acid having a
nucleotide sequence comprising or complementary to a nucleotide sequence
comprising SEQ ID NO: 17 or SEQ ID NO: 19 (shown in Figures 7A and 8A,
respectively). A nucleic acid molecule is "hybridizable" to another nucleic acid
molecule when a single-stranded form of the nucleic acid molecule can anneal to the
other nucleic acid molecule under the appropriate conditions of temperature and ionic
strength (See, Sambrook et al.. Molecular Cloning: A Laboratory Manual, Second
Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
-12-
York)). The conditions of temperature and ionic strength determine the "stringency"
of the hybridization. "Hybridization" requires that two nucleic acids contain
complementary sequences. However, depending on the stringency of the
hybridization, mismatches between bases may occur. The appropriate stringency for
hybridizing nucleic acids depends on the length of the nucleic acids and the degree of
complementation. Such variables are well known to those skilled in the art. More
specifically, the greater the degree of similarity or homology between two nucleotide
sequences, the greater the value of Tm for hybrids of nucleic acids having those
sequences. For hybrids of greater than 100 nucleotides in length, equations for
calculating Tm have been derived (See, Sambrook et al., supra). For hybridization
with shorter nucleic acids, the position of mismatches becomes more important, and
the length of the oligonucleotide determines its specificity (See, Sambrook et al.,
supra).
[0053] Typically, stringent conditions will be those in which the salt
concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion
concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about
30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long
probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved
with the addition of destabilizing agents such as formamide. An example of low
stringency conditions include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1
X to 2 X SSC (20 X SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C. An
example of moderate stringency conditions include hybridization in 40 to 45%
formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.5 X to 1 X SSC at 55 to
60°C. An example of high stringency conditions include hybridization in 50%)
formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65°C.
[0054] Exon: As used herein, the term "exon" refers to a portion of the
sequence of a gene that is transcribed and is found in the mature messenger RNA
derived from the gene, but is not necessarily a part of the sequence that encodes the
final gene product.
-13-
[0055] Expression, Antisense Inhibition, and Co-suppression: As used
herein, the term "expression", refers to the production of a functional end-product.
Expression of a gene involves transcription of the gene and translation of the mRNA
into a precursor or mature protein.
[0056] As used herein, the phrase "antisense inhibition" refers to the
production of antisense RNA transcripts capable of suppressing the expression of the
target protein.
[0057] As used herein, the term "co suppression" refers to the production of
sense RNA transcripts capable of suppressing the expression of identical or
substantially similar foreign or endogenous genes (See, U.S. Patent No. 5,231,020).
[0058] Fragment or Subfragment that is Functionally Equivalent: The terms
"fragment or subfragment that is functionally equivalent" and "ftinctionally
equivalent fragment or subfragment", used interchangeably herein, refer to a portion
or subsequence of an isolated nucleic acid molecule 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 constructs to produce the desired phenotype in a
transformed plant. Chimeric constructs can be designed for use in co-suppression or
antisense inhibition by linking a nucleic acid fragment or subfragment thereof,
whether or not it encodes an active enzyme, in the appropriate orientation relative to a
plant promoter sequence.
[0059] Gene, Native Gene. Foreign Gene, and Transgene: As used herein,
the term "gene" refers to a nucleic acid molecule that expresses a specific protein,
including regulatory sequences preceding (5' non-coding sequences) and following (3'
non-coding sequences) the coding sequence.
[0060] As used herein, the phrase "native gene" refers to a gene as found in
nature with its own regulatory sequences.
-14-
[0061] 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
constructs.
[0062] As used herein, the term "transgene" refers to gene that has been
introduced into the genome by a transformation procedure.
[0063] Gossvpium species: As used herein, the phrase "Gossypium species"
refers to any plants of Gossypium arboreum, Gossypium barbadense, Gossypium
herbaceum, Gossypium hirsutum, Gossypium hirsutum var hirsutum, Gossypium
hirsutum var marie-galante, Gossypium lapideum, Gossypium sturtianum, Gossypium
thuberi, Gossypium thurberi, Gossypium tomentosum or Gossypium tormentosum.
[0064] Homology: The terms "homology", "homologous", "substantially
similar" and "corresponding substantially" are used interchangeably herein and refer
to nucleic acid molecules wherein changes in one or more nucleotide bases does not
affect the ability of the nucleic acid molecule to mediate gene expression or produce a
certain phenotype. These terms also refer to modifications of the nucleic acid
molecules of the instant disclosure such as a deletion or insertion of one or more
nucleotides that do not substantially alter the fiinctional properties of the resulting
nucleic acid molecule relative to the initial, unmodified molecule. It is therefore
understood, as those skilled in the art will appreciate, that the disclosure encompasses
more than the specific exemplary sequences.
[0065] Host Cell: As used herein, the phrase "host cell" is meant a cell,
which comprises an isolated nucleic acid sequence or fi-agment thereof of the present
disclosure. Host cells may be prokaryotic cells (e.g. such as Escherichia coli,
cyanobacteria and Bacillus subtilis), or eukaryotic cells (e.g. such as fungal, insect,
plant or mammalian cells).
[0066] Examples of fungal cells that can be used are Saccharomyces spp.,
Candida spp., Lipomyces spp., Yarrowia spp., Kluyveromyces spp., Hamenula spp.,
-15-
Aspergillus spp., Penicillium spp., Neurospora spp., Trichoderma spp. and Pichia
spp. A particularly preferred fungal cell is Saccharomyces cerevisiae.
[0067] Plant cells can be monocotyledonous or dicotyledonous plant cells.
Particularly preferred plant cells are from oilseed plants such as Glycine max (e.g.,
soybean), a Brassica species, Carthamus tinctorius L. (e.g., safflower), Helianthus
annuus (e.g., sunflower), Zea mays (e.g., maize), a Gossypium species (cotton) and
Linum usitatissimum (e.g, flax).
[0068] Identity, Sequence Identity, and Percentage of Sequence Identity (%
Identity): The terms "identity" or "sequence identity," used interchangeably herein,
when used in the context of nucleotide or polypeptide sequences refer to the nucleic
acid bases or amino acid residues in two sequences that are the same when aligned for
maximum correspondence over a specified comparison window. Thus, identity is
defined as the degree of sameness, correspondence or equiyalence between the same
strands (either sense or antisense) of two DNA or polypeptide segments.
[0069] "Percentage of sequence identity" or "% identity" is calculated by
comparing two optimally aligned sequences oyer a particular region, determining the
number of positions at which the identical base occurs in both sequence in order to
yield the number of matched positions, dividing the number of such positions by the
total number of positions in the segment being compared and multiplying the result by
100. Optimal alignment of sequences may be conducted by the algorithm of Smith &
Waterman, Appl. Math. 2AS2 (1981), by the algorithm of Needleman & Wunsch, J.
Mol. Biol. 48:443 (1970), by the method of Pearson & Lipman, Proc. Natl. Acad Sci.
(USA) 85:2444 (1988) and by computer programs which implement the relevant
algorithms (e.g., Higgins et al, CABIOS. 5L151-153 (1989)), FASTDB
(Intelligenetics), BLAST (National Center for Biomedical Information; Altschul et al..
Nucleic Acids Research 25:3389-3402 (1997)), PILEUP (Genetics Computer Group,
Madison, WI) or GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics
Software Package Release 7.0, Genetics Computer Group, Madison, WI). (See, U.S.
Patent No. 5,912,120). Usefiil examples of percent sequence identities include, but
are not limited to, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
-16-
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%), 97%, 98%) or 99%. These identities can be determined using
any of the programs described herein.
[0070] Indirectly or Directly: As used herein, the term "indirectly" when
used in connection with the use of a gene and its corresponding enzyme in the
production of polyunsaturated fatty acids, encompasses the situation where a first acid
is converted to second acid (i.e., a pathway intermediate) by a first enzyme (e.g., LA
to C06-EDA, by, for example a A9-elongase) and then the second acid is converted to
third acid by use of a second enzyme (e.g., (06-EDA to DGLA by, for example, A8-
desaturase).
[0071] As used herein, the term "directly" when used in connection with the
use of a gene and its corresponding enzyme in the production of polyunsaturated fatty
acids encompasses the situation where the enzyme directly converts a first acid to a
second acid, wherein the second acid is then utilized in a composition (e.g., the
conversion of LA to a)6-EDA by, for example a A9-elongase or co3-ETrA to co3-ETA
by, for example a A8-desaturasae).
[0072] Intron: As used herein, the term "intron" refers to an intervening
sequence in a gene that does not encode a portion of the protein sequence. Thus, such
sequences are transcribed into RNA but are then excised and are not translated. The
term is also used for the excised RNA sequences.
[0073] Isolated: As used herein, the term "isolated" refers to a nucleic acid
molecule (DNA or RNA) or a protein or a biologically active portion thereof that is
removed from its naturally occurring envirormient or source using routine techniques
known in the art (e.g., from bacteria, algae, fungi, plants, vertebrates, mammals, etc.).
Isolated nucleic acid molecules or proteins are substantially or essentially free from
components that normally accompany or interact with the nucleic acid molecules or
proteins in their naturally occurring environment.
[0074] Isolated Nucleic Acid Fragment or Isolated Nucleic Acid Sequence:
As used herein, the phrase "isolated nucleic acid fragment" or "isolated nucleic acid
-17-
sequence" refers to a polymer of RNA or DNA that is single- or double-stranded,
optionally containing synthetic, non-natural or altered nucleotide bases. An isolated
nucleic acid fragment in the form of a polymer of DNA may be comprised of one or
more segments of cDNA, genomic DNA or synthetic DNA. (A "fragment" of a
specified polynucleotide refers to a polynucleotide sequence which comprises a
contiguous sequence of approximately at least about 6 consecutive nucleotides,
preferably at least about 8 consecutive nucleotides, more preferably at least about 10
consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 20
consecutive nucleotides, at least about 25 consecutive nucleotides, etc., identical or
complementary to a region of the specified nucleotide sequence.) Nucleotides
(usually found in their 5' monophosphate form) are referred to by their 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.
[0075] Mature and Precursor: As used herein, the term, "matiire" when used
in connection with the term "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. As used herein, the term "precursor" when
used in connection with the term "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.
[0076] 3' Non-Coding Sequences: Asusedherein, the phrase "3'noncoding
sequences" refers 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 et al., (1989) Plant
Ce//1:671 680.
-18-
[0077] Non-Naturally Occurring: As used herein, the phrase, "non-naturally
occurring" refers to something that is artificial, not consistent with what is normally
found in nature.
[0078] Operably Linked: As used herein, the phrase "operably linked"
refers to the association of nucleic acid sequences on a single nucleic acid molecule 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., that 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 disclosure 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.
[0079] Plant: As used herein, the term "plant" refers to whole plants, plant
organs, plant tissues, seeds, plant cells, seeds and progeny of the same. Plant cells
include, without limitation, cells from seeds, suspension cultures, embryos,
meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes,
pollen and microspores.
[0080] Polymerase Chain Reaction or PCR: As used herein, the phrase
"Polymerase Chain Reaction" or "PCR" refers to a technique for the synthesis of
large quantities of specific DNA segments, consists of a series of repetitive cycles
(Perkin Elmer Cetus Instruments, Norwalk, CT). 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.
[0081 ] PCR is a powerful technique used to amplify DNA millions of fold,
by repeated replication of a template, in a short period of time (Mullis et al, Cold
Spring Harbor Symp. Quant. Biol. 51:263 273 (1986); Erlich et al, European Patent
Application 50,424; European Patent Application 84,796; European Patent
-19-
Application 258,017, European Patent Application 237,362; MuUis, European Patent
Application 201,184, Mullis et al U.S. Patent No. 4,683,202; Erlich, U.S. Patent No.
4,582,788; and Saiki et al, U.S. Patent No. 4,683,194). The process utilizes sets of
specific in vitro synthesized oligonucleotides to prime DNA synthesis. The design of
the primers is dependent upon the sequences of DNA that are desired to be analyzed.
The technique is carried out through many cycles (usually 20 50) of melting the
template at high temperature, allowing the primers to anneal to complementary
sequences within the template and then replicating the template with DNA
polymerase. The products of PCR reactions are analyzed by separation in agarose
gels followed by ethidium bromide staining and visualization with UV
transillumination. Alternatively, radioactive dNTPs can be added to the PCR in order
to incorporate label into the products. In this case the products of PCR are visualized
by exposure of the gel to x-ray film. The added advantage of radiolabeling PCR
products is that the levels of individual amplification products can be quantitated.
[0082] Promoter and Enhancer: As used herein, the term "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.
[0083] As used herein, the term "enhancer" refers to a DNA sequence which
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.
Promoter sequences can also be located within the transcribed portions of genes,
and/or downstream of the transcribed sequences. 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. Promoters which
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
-20-
by Okamuro and Goldberg, (1989) Biochemistry of Plants 15:1 82. It is further
recognized that since in most cases the exact boundaries of regulatory sequences have
not been completely defined, DNA molecules of some variation may have identical
promoter activity.
[0084] Recombinant: As used herein, 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.
[0085] Recombinant Construct, Expression Construct, and Recombinant
Expression Construct: The phrases "recombinant construct", "expression construct"
and "recombinant expression construct" are used interchangeably herein and refer to a
functional unit of genetic material that can be inserted into the genome of a cell using
standard methodology well known to one skilled in the art. Such construct may be
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 plants 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 molecules of the disclosure. The skilled artisan will
also recognize that different independent transformation events will result in different
levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411 2418; De
Almeida et al., (1989) Mol. Gen. Genetics 218:78 86), 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. Western analysis of protein expression, or
phenotypic analysis.
[0086] RNA transcript. Messenger RNA, cDNA, Functional RNA, and
Endogenous RNA: As used herein, the phrase, "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
-21-
referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional
processing of the primary transcript and is referred to as the mature
RNA.
[0087] As used herein, the phrase "messenger RNA (mRNA)" refers to the
RNA that is without introns and that can be translated into protein by the cell.
[0088] As used herein, the term "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 the doublestranded
form using the Klenow molecule 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. Patent 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.
[0089] As used herein, the phrase, "functional RNA" refers to antisense
RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect
on cellular processes.
[0090] 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.
[0091] As used herein, the phrase "endogenous RNA" refers to any RNA
which is encoded by any nucleic acid sequence present in the genome of the host prior
to transformation with the recombinant construct of the present disclosure, whether
naturally-occurring or non-naturally occurring, i.e., introduced by recombinant means,
mutagenesis, etc.
[0092] Similarity: The term "similarity," when referring to the "similarity"
between two amino acid sequences, proteins or polypeptides, refers to the presence of
-22-
a series of identical as well as conserved amino acid residues in both sequences. The
higher the degree of similarity between two amino acid sequences, the higher the
correspondence, sameness or equivalence of the two sequences.
[0093] Stable Transformation, Transient Transformation, and
Transformation: As used herein, the phrase "stable transformation" refers to the
transfer of a nucleic acid molecule into a genome of a host organism, including both
nuclear and organellar genomes, resulting in genetically stable inheritance.
[0094] In contrast, as used herein, the phrase "transient transformation"
refers to the transfer of a nucleic acid molecule 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 molecules
are referred to as "transgenic" organisms. The preferred method of cell
transformation of rice, com and other monocots is the use of particle-accelerated or
"gene gun" transformation technology (Klein et al,, (1987) Nature (London) 327:70
73; U.S. Patent No. 4,945,050), or an Agrobacterium-mediated method using an
appropriate Ti plasmid containing the transgene (Ishida Y. et al., (1996) Nature
Biotech. 14:745 750).
[0095] As used herein, the term "transformation" refers to both stable
transformation and transient transformation.
[0096] Translation Leader Sequence: As used herein, the phrase "translation
leader sequence" refers to a DNA 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. (1995) Molecular Biotechnology 3:225).
[0097] All patents, patent publications and priority documents cited herein
are hereby incorporated by reference in their entirety.
-23-
The A9-Elongase Gene and Enzyme Encoded Thereby
[0098] The enzyme encoded by the A9-elongase gene of the present
disclosure is essential in the production, yia the alternate A8-desaturase/A9-elongase
pathway, of long-chain polyunsaturated fatty acids (LC-PUFAs), having a length of
20 or greater carbons. The nucleotide sequence of the isolated Euglena deses Ehr.
CCMP 2916 A9-elongase gene is shovm in Figure 7A, and the predicted amino acid
sequence of the corresponding protein is shown in Figure 7B.
[0099] The conversion of LA to DGLA and ALA to (o3-ETA using a A9-
elongase enzyme and a A8-desaturase enzyme is referred to as the alternate A8-
desaturase/A9-elongase pathway. The conventional A6 pathway for converting LA to
DGLA and ALA to co3-ETA utilizes a A6-desaturase enzyme to convert LA to GLA,
and ALA to SDA, and a A6-elongase to convert GLA to DGLA, and SDA to 0)3-
ETA, respectively. In either pathway, the production of ARA or EPA is then
catalyzed by, for example, a A5-desaturase. DHA, for example, may be produced
upon the conversion of EPA to co3-docosapentaenoic acid (DPA), and co3-
docosapentaenoic acid to DHA, utilizing, for example, a A5-elongase and a A4-
desaturase, respectively.
[00100] Although, for example, DGLA, co3-ETA, ARA, EPA, co3-
docosapentaenoic acid, co6-docosapentaenoic acid, ADA and/or DHA can be
produced via either the alternate A8-desaturase/A9-elongase pathway or the
conventional A6 pathway, in certain instances, the alternate A8-desaturase/A9-
elongase pathway may be preferred over the conventional A6 pathway. For example,
if particular residual omega-6 or omega-3 fatty acid intermediates, such as GLA or
SDA, are not desired during production of DGLA, co3-ETA, ARA, EPA, ©3-
docosapentaenoic acid, co6-docosapentaenoic acid, ADA and/or DHA, the alternate
A8-desaturase/A9-elongase pathway may be used as an alternative to the conventional
A6 pathway, to bypass GLA and SDA formation.
[00101 ] As discussed above, A9-elongase is a necessary enzyme in the
alternate A8-desaturase/A9-elongase pathway. EPA, for example, cannot be
-24-
synthesized via the alternate A8-desaturase/A9-elongase pathway without the A9-
elongase gene and enzyme encoded thereby. As shown in Figure 1, the isolated A9-
elongase enzyme of the present disclosure converts, for example, ALA to (o3-ETrA
and LA to C06-EDA. The production of co3-ETA from co3-ETrA, and EPA from ©3-
ETA is then catalyzed by, for example, a A8-desaturase and a A5-desaturase,
respectively. As a result of using the alternate A8-desaturase/A9-elongase pathway,
the intermediate GLA and SDA fatty acids are bypassed.
[00102] It should be noted that the present disclosure also encompasses
nucleotide sequences (and the corresponding encoded proteins) having sequences
comprising, consisting of, or complementary to at least 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the
nucleotides in sequence (i.e., having sequence identity to) SEQ ID NO: 17 (i.e., the
isolated nucleotide sequence of the A9-elongase gene ofEuglena deses Ehr. CCMP
2916) or SEQ ID NO: 19 (i.e., a variant A9-elongase gene ofEuglena deses Ehr.
CCMP 2916). Such sequences may be from human sources as well as other nonhuman
sources (e.g., C. elegans or mouse).
[00103] Furthermore, the present disclosure also encompasses fragments and
derivatives comprising or consisting of the nucleotide sequence of SEQ ID NO: 17
(shown in Figure 7A) or SEQ ID NO: 19 (shown in Figure 8 A)), as well as of the
sequences from other sources, and having the above-described complementarity or
correspondence. Functional equivalents of the above-sequences (i.e., sequences
having A9-elongase activity) are also encompassed by the present disclosure.
[00104] Fragments derived from SEQ ID NO: 17 or SEQ ID NO: 19 can
have a length comprising or consisting of 10 to about 780 nucleotides, 10 to about 700
nucleotides, 10 to about 650 nucleotides, 10 to about 500 nucleotides, 10 to about 250
nucleotides, 10 to about 100 nucleotides, 10 to about 50 nucleotides, or 15 to 40
nucleotides. In one aspect, the fragments of SEQ ID NO: 17 and SEQ ID NO: 19
encode a polypeptide having A9-elongase activity. In another aspect, fragments of the
SEQ ID NO: 17 and SEQ ID NO: 19 can be used as primers and probes. Methods of
-25-
making primers and probes are well known to those skilled in the art. Such primers
and probes can have a length of 10 to 50 nucleotides, preferably from 15 to 40
nucleotides.
[00105] Variants of the nucleotide sequence of SEQ ID NO: 17 or SEQ ID
NO: 19 are also contemplated herein. Such variants may contain one or more base
pair additions, substitutions, or deletions. Non-limiting examples of nucleotide
variants of SEQ ID NO: 17 encompassed by the present disclosure are shown in Table
A below. One specific example of a variant of SEQ ID NO: 17 is SEQ ID NO: 19
(see Figure 8A).
Table A
Sequence Substitution (SEQ ID NO: 17 =>
SEQ ID NO: 19)
GCT24 => GCC24
GCgsC => GTgaC
G232TA =:> A232TA
A301TG => T301TG
C310TC => A310TC
ACA630 =0 ACT630
AAA750 => AAG750
[00106] The present disclosure also encompasses nucleotide sequences from
other sources, and having the above-described complementarity or correspondence to
SEQ ID NO: 17 or SEQ ID NO: 19. Functional equivalents of SEQ ID NO: 17 or
SEQ ID NO: 19 (i.e., sequences having A9-elongase activity) are also encompassed
by the present disclosure.
[00107] The present disclosure also encompasses nucleotide sequences or
fragments thereof encoding a polypeptide having A9-elongase activity, wherein the
-26-
amino acid sequence of said polypeptide has at least 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence
identity to an amino acid sequence comprising SEQ ID NO: 18 or SEQ ID NO: 20.
Such sequences may be from human sources as well as other non-human sources
(e.g., C. elegans or mouse).
[00108] The disclosure also includes an isolated and/or purified polypeptide
which elongates polyunsaturated fatty acids containing unsaturation at the carbon 9
position (i.e., has A9-elongase activity) and has at least 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
similarity or identity to the amino acid sequence (i.e., SEQ ID NO: 18 (shown in
Figure 7B) or SEQ ID NO: 20 (shown in Figure 8B)). Specifically, the present
disclosure includes a purified polypeptide having an amino acid sequence of SEQ ID
NO: 18 or SEQ ID NO: 20.
[00109] Fragments of the polypeptide having the sequence of SEQ ID NO:
18 or SEQ ID NO: 20 are also contemplated herein. Such fragments can have a
length comprising or consisting of 10 to about 260 consecutive amino acids, 10 to
about 200 consecutive amino acids, 10 to about 100 consecutive amino acids, 10 to
about 50 consecutive amino acids, 10 to about 40 consecutive amino acids, 10 to
about 30 consecutive amino acids, or 10 to about 20 consecutive amino acids.
[00110] Variants of the polypeptide having the sequence of SEQ ID NO: 18
or SEQ ID NO: 20 are also contemplated herein. Such variants may contain one or
more amino acid additions, substitutions, or deletions. Non-limiting examples of
amino acid variants of SEQ ID NO: 18 encompassed by the present disclosure are
shown in Table B below. One specific example of a variant of SEQ ID NO: 18 is
SEQ ID NO: 20 (see Figure 8B).
Amino Acid Substitution
(SEQ ID NO: 18 =>SEQ ID
-27-
NO: 20)
A28 => V28
V78 => lyg
Mioi => Lioi
L104 => I104
Production of the A9-elongase enzyme
[00111] Once the nucleic acid (e.g., gene) encoding the A9-elongase enzyme
has been isolated and/or purified, it may then be introduced into either a prokaryotic
or eukaryotic host cell through the use of a vector or construct. The vector, for
example, a bacteriophage, cosmid, or plasmid, may comprise the nucleotide sequence
encoding the A9-elongase enzyme, as well as any regulatory sequence (e.g., promoter)
which is functional in the host cell and is able to elicit expression of the A9-elongase
encoded by the nucleotide sequence. The regulatory sequence is in operable
association with or operably linked to the nucleotide sequence. (As noted above,
regulatory is said to be "operably linked" with a coding sequence if the regulatory
sequence affects transcription or expression of the coding sequence.) Suitable
promoters include, for example, those fi^om genes encoding alcohol dehydrogenase,
glyceraldehyde-3 -phosphate dehydrogenase, phosphoglucoisomerase,
phosphoglycerate kinase, acid phosphatase, T7, TPI, lactase, metallothionein,
cytomegalovirus immediate early, whey acidic protein, glucoamylase, and promoters
activated in the presence of galactose, for example, GALl and GAL 10. Additionally,
nucleotide sequences which encode other proteins, oligosaccharides, lipids, etc. may
also be included within the vector as well as other regulatory sequences such as a
polyadenylation signal (e.g., the poly-A signal of SV-40T-antigen, ovalalbimiin or
bovine growth hormone). The choice of sequences present in the construct is
dependent upon the desired expression products as well as the nature of the host cell.
[00112] As noted above, once the vector has been constructed, it may then
be introduced into the host cell of choice by methods known to those of ordinary skill
-28-
in the art including, for example, transfection, transformation and electroporation (see
Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, ed. Sambrook et al.,
Cold Spring Harbor Laboratory Press (1989)). The host cell is then cultured under
suitable conditions permitting expression of the genes leading to the production of the
desired PUFA, which is then recovered and purified using routine techniques known
in the art.
[00113 ] Examples of suitable prokaryotic host cells include, but are not
limited to, bacteria such as Escherichia coli. Bacillus subtilis as well as cyanobacteria
such as Spirulina spp. (i.e., blue-green algae). The eukaryotic cell may be, for
example, a mammalian cell, an insect cell, a plant cell or a fungal cell. The fungal
cell may be, for example, Saccharomyces spp., Candida spp., Lipomyces spp.,
Yarrowia spp., Aspergillus spp., Penicillium spp., Neurospora spp., Kluyveromyces
spp., Hansenula spp., Trichoderma spp., or Pichia spp. In particular, the fungal cell
may be a yeast cell such as, for example, Saccharomyces spp., Candida spp.,
Hansenula spp. and Pichia spp. The yeast cell may also be Saccharomyces
cerevisiae. The plant cell includes, but is not limited to, plant cells from oilseed
plants such as Glycine max (e.g., soybean), a Brassica species, Carthamus tinctorius
L. (e.g., safflower), Helianthus annus (e.g., sunflower), Zea mays (e.g., maize), a
Gossypium species (e.g., cotton), and Linum usitatissimum (e.g., flax).
[00114] Expression in a host cell can be accomplished in a transient or stable
fashion. Transient expression can occur from introduced constructs which contain
expression signals functional in the host cell, but which constructs do not replicate
and rarely integrate in the host cell, or where the host cell is not proliferating.
Transient expression also can be accomplished by inducing the activity of a
regulatable promoter operably linked to the gene of interest, although such inducible
systems frequently exhibit a low basal level of expression. Stable expression can be
achieved by introduction of a construct that can integrate into the host genome or that
autonomously replicates in the host cell. Stable expression of the gene of interest can
be selected for through the use of a selectable marker located on or transfected with
the expression construct, followed by selection for cells expressing the marker. When
stable expression results from integration, the site of the construct's integration can
-29-
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 with 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.
[00115] A transgenic mammal may also be used in order to express the A9-
elongase enzyme and ultimately the PUFA(s) of interest. More specifically, once the
above-described construct is created, it may be inserted into the pronucleus of an
embryo. The embryo may then be implanted into a recipient female. Alternatively, a
nuclear transfer method could also be utilized (Schnieke, et al., Science 278:2130-
2133 (1997)). Gestation and birth are then permitted (see, e.g., U.S. Patent No.
5,750,176 and U.S. Patent No. 5,700,671). Milk, tissue or other fluid samples from
the offspring should then contain altered levels of PUFAs, as compared to the levels
normally foxmd in the non-transgenic animal. Subsequent generations may be
monitored for production of the altered or enhanced levels of PUFAs and thus
incorporation of the gene encoding the desired desaturase enzyme into their genomes.
The mammal utilized as the host may be selected from the group consisting of, for
example, a mouse, a rat, a rabbit, a pig, a goat, a sheep, a horse and a cow. However,
any mammal may be used provided it has the ability to incorporate DNA encoding the
enzyme of interest into its genome.
[00116] For expression of a A9-elongase polypeptide, fimctional
transcriptional and translational initiation and termination regions are operably linked
to the DNA encoding the elongase polypeptide. Transcriptional and translational
initiation and termination regions are derived from a variety of nonexclusive sources,
including the DNA to be expressed, genes known or suspected to be capable of
expression in the desired system, expression vectors, chemical synthesis, or from an
endogenous locus in a host cell. Expression in a plant tissue and/or plant part presents
certain efficiencies, particularly where the tissue or part is one which is harvested
early, such as seed, leaves, fruits, flowers, roots, etc. Expression can be targeted to
that location with the plant by utilizing specific regulatory sequence such as those of
-30-
U.S. Patent Nos. 5,463,174, 4,943,674, 5,106,739, 5,175,095, 5,420,034, 5,188,958,
and 5,589,379.
[00117] Alternatively, the expressed protein can be an enzyme which
produces a product which may be incorporated, either directly or upon further
modifications, into a fluid fraction from the host plant. Expression of a A9-elongase
gene, or antisense A9-elongase transcripts, can alter the levels of specific PUFAs, or
derivatives thereof, found in plant parts and/or plant tissues.
[00118] The A9-elongase polypeptide coding region may be expressed either
by itself or with other genes (e.g., a gene encoding a A8-desaturase, a gene encoding a
A5-desaturase, a gene encoding a A17-desaturase, a gene encoding a A5-elongase,
and/or a gene encoding a A4-desaturase), in order to produce tissues and/or plant parts
containing higher proportions of desired PUFAs or in which the PUFA composition
more closely resembles that of human breast milk (see WO 95/24494). The
termination region may 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 to and have been foxmd to be satisfactory in a variety
of hosts from the same and different genera and species. The termination region
usually is selected as a matter of convenience rather than because of any particular
property.
[00119] As noted above, a plant (e.g., Glycine max (soybean) or Brassica
napus (canola)) or plant tissue may also be utilized as a host or host cell, respectively,
for expression of the A9-elongase enzyme which may, in turn, be utilized in the
production of polyunsaturated fatty acids. More specifically, desired PUFAS can be
expressed in seed. Methods of isolating seed oils are known in the art. Thus, in
addition to providing a source for PUFAs, seed oil components may be manipulated
through the expression of the A9-elongase gene, as well as perhaps desaturase genes
(e.g., A8-desaturase, A17-desaturase, A5-desaturases, A4-desaturase, etc.) and other
elongase genes (e.g., A5-elongase, etc.), in order to provide seed oils that can be
added to nutritional compositions, pharmaceutical compositions, animal feeds and
cosmetics. Once again, a vector which comprises a DNA sequence encoding the A9-
-31-
elongase operably linked to a promoter, will be introduced into the plant tissue or
plant for a time and under conditions sufficient for expression of the A9-elongase
gene. The vector may also comprise one or more genes that encode other enzymes,
for example, A4-desaturase, A5-desaturase, A6-desaturase, AlO-desaturase, A12-
desaturase, A15-desaturase, A17-desaturase, A19-desaturase, A6-elongase, and/or A5-
elongase. The plant tissue or plant may produce the relevant substrate upon which the
enzymes act or a vector encoding enzymes which produce such substrates may be
introduced into the plant tissue, plant cell or plant. In addition, substrate may be
sprayed on plant tissues expressing the appropriate enzymes. Using these various
techniques, one may produce PUFAs by use of a plant cell, plant tissue or plant. It
should also be noted that the disclosure also encompasses a transgenic plant
comprising the above-described vector, wherein expression of the nucleotide
sequence of the vector results in production of a polyunsaturated fatty acid in, for
example, the seeds of the transgenic plant.
[00120] The regeneration, development, and cultivation of plants from
single plant protoplast transformants or fi-om various transformed explants is well
known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular
Biology, (Eds.), Academic Press, Inc. San Diego, CA, (1988)). This regeneration and
growth process typically includes the steps of selection of transformed cells, 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.
[00121] The development or regeneration of plants containing the foreign,
exogenous gene that encodes a protein of interest is well known in the art. 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 disclosure containing a desired polypeptide is cultivated using methods well
known to one skilled in the art.
-32-
[00122] 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.
[00123] Methods for transforming dicots, primarily by use of Agrobacterium
tumefaciens, and obtaining transgenic plants have been published for cotton (U.S.
Patent No. 5,004,863, U.S. Patent No. 5,159,135, U.S. Patent No. 5,518, 908);
soybean (U.S. Patent No. 5,569,834, U.S. Patent No. 5,416,011, McCabe et. al.,
BiolTechnology 6:923 (1988), Christou et al.. Plant Physiol. 87:671 674 (1988));
Brassica (U.S. Patent 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; and pea
(Grant et al.. Plant Cell Rep. 15:254 258, (1995)).
[00124] Transformation of monocotyledons using electroporation, particle
bombardment, and Agrobacterium have also been reported. Transformation and plant
regeneration have been achieved in asparagus (Bytebier et al., Proc. Natl. Acad. Sci.
(USA) 84:5354, (1987)); barley (Wan and Lemaux, Plant Physiol 104:37 (1994));
Zea mays (Rhodes et al.. Science 240:204 (1988), Gordon-Kamm et al., Plant Cell
2:603 618 (1990), Fromm et al., BiolTechnology 8:833 (1990), Koziel et al.,
BiolTechnology 11: 194, (1993), Armstrong et al.. Crop Science 35:550 557 (1995));
oat (Somers et al., BiolTechnology 10: 15 89 (1992)); orchard grass (Horn et al.. Plant
Cell Rep 7:469 (1988)); rice (Toriyama et al., TheorAppl. Genet. 205:34, (1986); Part
et al.. Plant Mol Biol. 32:1135 1148, (1996); Abedinia et al., Aust. J. Plant Physiol.
24:133 141 (1997); Zhang and Wu, Theor. Appl. Genet. 76:835 (1988); Zhang et al.
Plant Cell Rep. 7:379, (1988); Battraw and Hall, P/awrSd. 86:191 202(1992);
Christou et al., Bio/Technology 9:957 (1991)); rye (De la Pena et al.. Nature 325:274
(1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992)); tall fescue (Wang et al.,
BiolTechnology 10:691 (1992)), and wheat (Vasil et al, Bio/Technology 10:667
(1992); U.S. Patent No. 5,631,152).
[00125] Assays for gene expression based on the transient expression of
cloned nucleic acid constructs have been developed by introducing the nucleic acid
molecules into plant cells by polyethylene glycol treatment, electroporation, or
-33-
particle bombardment (Marcotte et al, Nature 335:454 457 (1988); Marcotte et al.,
Plant Cell 1:523 532 (1989); McCarty et al., Cell 66:895 905 (1991); Hattori et al.,
Genes Dev. 6:609 618 (1992); Goff et al., EMBOJ. 9:2517 2522 (1990)).
[00126] Transient expression systems may be used to fiinctionally dissect
gene constructs (see generally, Maliga et al.. Methods in Plant Molecular Biology,
Cold Spring Harbor Press (1995)). It is understood that any of the nucleic acid
molecules of the present disclosure can be introduced into a plant cell in a permanent
or transient manner in combination with other genetic elements such as vectors,
promoters, enhancers etc.
[00127] 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 organisms and the
screening and isolating of clones, (see for example, Sambrook et al.. Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989); Maliga et al..
Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995); Birren et al..
Genome Analysis: Detecting Genes, 1, Cold Spring Harbor, New York (1998); Birren
et al., Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, New York (1998);
Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer, New York
(1997)).
[00128] The substrates which may be produced by the host cell either
naturally or transgenically, as well as the enzymes which may be encoded by DNA
sequences present in the vector which is subsequently introduced into the host cell,
are shown in Figure 1.
[00129] In view of the above, the present disclosure encompasses a method
of producing the A9-elongase enzyme comprising the steps of: 1) isolating a
nucleotide sequence comprising or complementary to at least 68% of the nucleotide
sequence encoding the A9-elongase enzyme (e.g., a nucleotide sequence selected from
the group consisting of SEQ ID NO: 17 and SEQ ID NO: 19); 2) constructing an
-34-
expression vector comprising the nucleotide sequence operably linked to a regulatory
sequence; and 3) introducing the vector into a host cell lander time and conditions
sufficient for the production of the A9-elongase enzyme.
[00130] The present disclosure also encompasses a method of producing
polyunsaturated fatty acids. In one aspect, the method involves: 1) isolating a
nucleotide sequence comprising or complementary to at least 68% of the nucleotide
sequence encoding the A9-elongase enzyme (e.g., a nucleotide sequence selected from
the group consisting of SEQ ID NO: 17 and SEQ ID NO: 19); 2) constructing an
expression vector comprising the nucleotide sequence operably linked to a regulatory
sequence; 3) introducing the expression vector into a host cell under time and
conditions sufficient for the production of a A9-elongase enzyme; and 4) exposing the
expressed A9-elongase to a substrate polyunsaturated fatty acid in order to convert the
substrate polyunsaturated fatty acid to a first product polyunsaturated fatty acid.
Examples of substrate PUFAs include LA, ALA, and combinations thereof.
Examples of first product polyunsaturated fatty acid that can be produced by this
method are CD6-EDA, co3-ETrA, or both co6-EDA and co3-ETrA. For example, when
LA is exposed to a A9-elongase enzyme, it is converted to CD6-EDA. In another
example, when ALA is exposed to a A9-elongase enzyme, it is converted to co3-ETrA.
[00131 ] The method can further involve the step(s) of exposing the first
product polyunsaturated fatty acid to at least one desaturase, at least one additional
elongase, or combinations thereof, and optionally repeating this step (i.e., exposing
the second or subsequent product polyunsaturated fatty acid to a desaturase or
elongase (which can be the same or different fi-om any previously used desaturase or
elongase) to convert the first product polyunsaturated fatty acid to a second or
subsequent (e.g., third, fourth, fifth, sixth, etc.) product polyimsaturated fatty acid).
This step can be repeated as many times as necessary until the desired product
polyunsaturated fatty acid is obtained. For example, if the first product
polyunsaturated fatty acid is ©6-EDA, the method can fiirther comprise exposing ©6-
EDA to, for example, A8-desaturase which converts the co6-EDA to DGLA (a second
product polyunsaturated fatty acid). The DGLA then may optionally be converted to
-35-
ARA (a third product polyunsaturated fatty acid) by exposing the DGLA to, for
example, A5-desaturase. The ARA can then be exposed to a A17-desaturase to
produce EPA (a fourth product polyunsaturated fatty acid). Still further, optionally
the EPA can be exposed to a A5-elongase to produce DPA (a fifth product
polyunsaturated fatty acid). The DPA can then optionally be exposed to a A4-
desaturase to produce DHA (a sixth product polyunsaturated fatty acid). In another
example, if the first product polyunsaturated fatty acid is (o3-ETrA, the method can
fiarther comprise exposing the co3-ETrA to, for example, A8-desaturase which
converts the co3-ETrA to ETA (a second product polyimsaturated fatty acid). The
ETA may then be converted to EPA (a third product polyimsaturated fatty acid) by
exposing the ETA to, for example, A5-desaturase. The EPA may be further converted
to other polyunsaturated fatty acids as described above.
[00132] In another aspect, the method involves: 1) isolating a nucleotide
sequence comprising or complementary to at least 68% of a nucleotide sequence
encoding the A9-elongase enzyme (e.g., a nucleotide sequence selected from the
group consisting of SEQ ID NO: 17 and SEQ ID NO: 19); 2) constructing an
expression vector comprising the isolated nucleotide sequence operably linked to a
regulatory sequence; 3) introducing the expression vector and at least one additional
recombinant DNA construct comprising an isolated nucleotide sequence encoding a
A8-desaturase and operably linked to at least one regulatory sequence into a host cell
for a time and under conditions sufficient for expression of a A9-elongase and the A8-
desaturase; and 4) exposing the expressed A9-elongase and the A8-desaturase to a
substrate polyunsaturated fatty acid selected fi"om the group consisting of LA, ALA,
and combinations thereof, in order to convert the substrate polyunsaturated fatty acid
to a first product polyunsaturated fatty acid. Examples of the first product
polyunsaturated fatty acid include DGLA, co3-ETA, and combinations thereof.
Furthermore, the method can further involve the step(s) of exposing the first product
polyunsaturated fatty acid to at least one additional desaturase or at least one
additional elongase and, optionally, repeating this step (namely, exposing the second
or subsequent product polyunsaturated fatty acid to a desaturase or elongase (which
can be the same or different from any desaturase or elongase used previously)) to
-36-
convert the first product polyunsaturated fatty acid (e.g., DGLA and/or co3-ETA) to a
second or subsequent (e.g., third, fourth, fifth, sixth, etc.) product polyunsaturated
fatty acid. This step can be repeated as many times as necessary until the desired
product polyunsaturated fatty acid is obtained. In one aspect, the method further
includes introducing into the host cell a recombinant DNA construct comprising an
isolated nucleotide sequence encoding a A5-desaturase operably linked to a regulatory
sequence.
[00133] Thus, A9-elongase may be used in the production of
polyunsaturated fatty acids which may be used, in turn, for particular beneficial
purposes, or may be used in the production of other PUFAs.
Uses of the A9-Elongase Gene
[00134] As noted above, the A9-isolated elongase gene and the A9-elongase
enzyme encoded thereby have many uses. For example, the gene and corresponding
enzyme may be used indirectly or directly in the production of polyunsaturated fatty
acids, for example, A9-elongase may be used in the production of co6-EDA, co3-ETrA,
DGLA, co3-ETA, ARA, EPA, a)3-docosapentaenoic acid, co6-docosapentaenoic acid,
ADA and/or DHA. "Directly" is meant to encompass the situation where the enzyme
directly converts the acid to another acid, the latter of which is utilized in a
composition (e.g., the conversion of LA to co6-EDA). "Indirectly" is meant to
encompass the situation where an acid is converted to another acid (i.e., a pathway
intermediate) by the enzyme (e.g., LA to co6-EDA) and then the latter acid is
converted to another acid by use of a non-elongase enzyme (e.g., co6-EDA to DGLA
by, for example, A8-desaturase. These polyunsaturated fatty acids (i.e., those
produced either directly or indirectly by activity of the A9-elongase enzyme) may be
added to, for example, nutritional compositions, pharmaceutical compositions,
cosmetics, and animal feeds, all of which are encompassed by the present disclosure.
These uses are described, in detail, below.
Nutritional Compositions
-37-
[00135] The present disclosure includes nutritional compositions. Such
compositions, for purposes of the present disclosure, include any food or preparation
for human consumption including for enteral or parenteral consumption, which when
taken into the body (a) serve to nourish or build up tissues or supply energy and/or (b)
maintain, restore or support adequate nutritional status or metabolic function.
[00136] The nutritional composition of the present disclosure comprises at
least one oil or acid produced directly or indirectly by use of the A9-elongase gene, as
described herein, and may either be in a solid or liquid form. Additionally, the
composition may include edible macronutrients, vitamins and minerals in amounts
desired for a particular use. The amount of such ingredients will vary depending on
whether the composition is intended for use with normal, healthy infants, children or
adults having specialized needs such as those which accompany certain metabolic
conditions (e.g., metabolic disorders).
[00137] Examples of macronutrients which may be added to the
composition include but are not limited to edible fats, carbohydrates and proteins.
Examples of such edible fats include but are not limited to coconut oil, soy oil, and
mono- and diglycerides. Examples of such carbohydrates include but are not limited
to glucose, edible lactose and hydrolyzed search. Additionally, examples of proteins
which may be utilized in the nutritional composition of the disclosure include but are
not limited to soy proteins, electrodialysed whey, electrodialysed skim milk, milk
whey, or the hydrolysates of these proteins.
[00138] With respect to vitamins and minerals, the following may be added
to the nutritional compositions of the present disclosure: calcium, phosphorus,
potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium,
iodine, and Vitamins A, E, D, C, and the B complex. Other such vitamins and
minerals may also be added.
[00139] The components utilized in the nutritional compositions of the
present disclosure will be of semi-purified or purified origin. By semi-purified or
-38-
purified is meant a material which has been prepared by purification of a natural
material or by synthesis.
[00140] Examples of nutritional compositions of the present disclosure
include but are not limited to infant formulas, dietary supplements, dietary substitutes,
and rehydration compositions. Nutritional compositions of particular interest include
but are not limited to those utilized for enteral and parenteral supplementation for
infants, specialty infant formulas, supplements for the elderly, and supplements for
those with gastrointestinal difficulties and/or malabsorption.
[00141 ] The nutritional composition of the present disclosure may also be
added to food even when supplementation of the diet is not required. For example,
the composition may be added to food of any type including but not limited to
margarines, modified butters, cheeses, milk, yogurt, chocolate, candy, snacks, salad
oils, cooking oils, cooking fats, meats, fish and beverages.
[00142] In a preferred embodiment of the present disclosure, the nutritional
composition is an enteral nutritional product, more preferably, an adult or pediatric
enteral nutritional product. This composition may be administered to adults or
children experiencing stress or having specialized needs due to chronic or acute
disease states. The composition may comprise, in addition to polyvmsaturated fatty
acids produced in accordance with the present disclosure, macronutrients, vitamins
and minerals as described above. The macronutrients may be present in amoimts
equivalent to those present in human milk or on an energy basis, i.e., on a per calorie
basis.
[00143] Methods for formulating liquid or solid enteral and parenteral
nutritional formulas are well known in the art.
[00144] The enteral formula, for example, may be sterilized and
subsequently utilized on a ready-to-feed (RTF) basis or stored in a concentrated liquid
or powder. The powder can be prepared by spray drying the formula prepared as
indicated above, and reconstituting it by rehydrating the concentrate. Adult and
pediatric nutritional formulas are well known in the art and are commercially
-39-
available (e.g., Similac®, Ensure®, Jevity® and Alimentum® from Ross Products
Division, Abbott Laboratories, Columbus, Ohio). An oil or acid produced in
accordance vs^ith the present disclosure may be added to any of these formulas.
[00145] The energy density of the nutritional compositions of the present
disclosure, when in liquid form, may range from about 0.6 Kcal to about 3 Kcal per
ml. When in solid or powdered form, the nutritional supplements may contain from
about 1.2 to more than 9 Kcals per gram, preferably about 3 to 7 Kcals per gm. In
general, the osmolality of a liquid product should be less than 700 mOsm and, more
preferably, less than 660 mOsm.
[00146] The nutritional formula may include macronutrients, vitamins, and
minerals, as noted above, in addition to the PUFAs produced in accordance with the
present disclosure. The presence of these additional components helps the individual
ingest the minimum daily requirements of these elements. In addition to the provision
of PUFAs, it may also be desirable to add zinc, copper, folic acid and antioxidants to
the composition. It is believed that these substance boost a stressed immune system
and will therefore provide further benefits to the individual receiving the composition.
A pharmaceutical composition may also be supplemented with these elements.
[00147] In a more preferred embodiment, the nutritional composition
comprises, in addition to antioxidants and at least one PUFA, a source of
carbohydrate wherein at least 5 weight percent of the carbohydrate is indigestible
oligosaccharide. In a more preferred embodiment, the nutritional composition
additionally comprises protein, taurine, and carnitine.
[00148] As noted above, the PUFAs produced in accordance with the
present disclosure, or derivatives thereof, may be added to a dietary substitute or
supplement, particularly an infant formula, for patients undergoing intravenous
feeding or for preventing or treating malnutrition or other conditions or disease states.
As background, it should be noted that human breast milk has a fatty acid profile
comprising from about 0.15% to about 0.36% as DHA, from about 0.03% to about
0.13% as EPA, from about 0.30% to about 0.88% as ARA, from about 0.22% to about
-40-
0.67% as DGLA, and from about 0.27% to about 1.04% as GLA. Thus, fatty acids
such as ARA, EPA and/or DHA, produced in accordance with the present disclosure,
can be used to alter, for example, the composition of infant formulas in order to better
replicate the PUFA content of human breast milk or to alter the presence of PUFAs
normally found in a non-human mammal's milk. In particular, a composition for use
in a pharmacologic or food supplement, particularly a breast milk substitute or
supplement, will preferably comprise one or more of ARA, EPA, DGLA, and DHA.
More preferably, the oil will comprise from about 0.3 to 30% ARA, and from about
0.2 to 30% DGLA.
[00149] Parenteral nutritional compositions comprising from about 2 to
about 30 weight percent fatty acids calculated as triglycerides are encompassed by the
present disclosure. Other vitamins, particularly fat-soluble vitamins such as vitamin
A, D, E and L-camitine can optionally be included. When desired, a preservative
such as alpha-tocopherol may be added in an amount of about 0.1%) by weight.
[00150] In addition, the ratios of ARA and DGLA can be adapted for a
particular given end use. When formulated as a breast milk supplement or substitute,
a composition which comprises one or more of ARA, DGLA and GLA will be
provided in a ratio of about 1:19:30 to about 6:1:0.2, respectively. For example, the
breast milk of animals can vary in ratios of ARA:DGLA:GLA ranging from 1:19:30
to 6:1:0.2, which includes intermediate ratios which are preferably about 1:1:1,1:2:1,
1:1:4. When produced together in a host cell, adjusting the rate and percent of
conversion of a precursor substrate such as EDA and DGLA to ARA can be used to
precisely control the PUFA ratios. For example, a 5% to 10%) conversion rate of
DGLA to ARA can be used to produce an ARA to DGLA ratio of about 1:19,
whereas a conversion rate of about 75%) to 80%) can be used to produce an ARA to
DGLA ratio of about 6:1. Therefore, whether in a cell culture system or in a host
animal, regulating the timing, extent and specificity of elongase expression, as well as
the expression of desaturases (such as but not limited to A8-desaturases) and other
elongases, can be used to modulate PUFA levels and ratios. The PUFAs/acids
produced in accordance with the present disclosure (e.g., ARA and EPA) may then be
-41-
combined with other PUFAs/acids (e.g., DGLA) in the desired concentrations and
ratios.
[00151 ] Additionally, PUFA produced in accordance with the present
disclosure or host cells containing them may also be used as animal food supplements
to alter an animal's tissue or milk fatty acid composition to one more desirable for
human or animal consumption.
[00152] Examples of some of the nutritional supplements, infant
formulations, nutritional substitutes and other nutritional solutions that employ the
polyunsaturated fatty acids produced pursuant to the present disclosure are described
below.
[00153] Infant Formulations: Examples of infant formulations include, but
are not limited to, Isomil® Soy Formula with Iron, Isomil® DF Soy Formula For
Diarrhea, Isomil® Advance® Soy Formula with Iron, Isomil® Advance® 20 Soy
Formula With Iron Ready To Feed, Similac® Infant Formula, Similac® Advance®
Infant Formula with Iron, Similac® NeoSure® Advance® Infant Formula With Iron,
Similac Natural Care Advance Low-Iron Human Milk Fortifier Ready To Use, all
commercially available from Abbott Nutrition (Colimibus, OH). The various PUF As
of the present disclosure can be substituted and/or added to the infant formulae
described herein and to other infant formulae known to those in the art.
[00154] Nutritional Formulations: Examples of nutritional formulations
include, but are not limited to, ENSURE®, ENSURE® HIGH PROTEIN, ENSURE
PLUS®, ENSURE® POWDER, ENSURE® PUDDING, ENSURE® WITH FIBER,
Oxepa''"'^ Nutritional Product, all conmiiercially available fi-om Abbott Nutrition
(Columbus, OH). The various nutritional supplements described above and known to
others of skill in the art can be substituted and/or supplemented with the PUF As
produced in accordance with the present disclosure.
Pharmaceutical Compositions
-42-
[00155] The present disclosure also encompasses a pharmaceutical
composition comprising one or more of the acids and/or resulting oils produced using
the A9-elongase genes described herein, in accordance with the methods described
herein. More specifically, such a pharmaceutical composition may comprise one or
more of the acids and/or oils as well as a standard, well-known, non-toxic
pharmaceutically acceptable carrier, adjuvant or vehicle such as, for example,
phosphate buffered saline, water, ethanol, polyols, vegetable oils, a wetting agent or
an emulsion such as a water/oil emulsion. The composition may be in either a liquid
or solid form. For example, the composition may be in the form of a tablet, capsule,
ingestible liquid or powder, injectible, or topical ointment or cream. Proper fluidity
can be maintained, for example, by the maintenance of the required particle size in the
case of dispersions and by the use of surfactants. It may also be desirable to include
isotonic agents, for example, sugars, sodium chloride and the like. Besides such inert
diluents, the composition can also include adjuvants, such as wetting agents,
emulsifying and suspending agents, sweetening agents, flavoring agents and
perfuming agents.
[00156] Suspensions, in addition to the active compounds, may comprise
suspending agents such as, for example, ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar and tragacanth or mixtures of these substances.
[00157] Solid dosage forms such as tablets and capsules can be prepared
using techniques well known in the art. For example, PUFAs produced in accordance
with the present disclosure can be tableted with conventional tablet bases such as
lactose, sucrose, and cornstarch in combination with binders such as acacia,
cornstarch or gelatin, disintegrating agents such as potato starch or alginic acid, and a
lubricant such as stearic acid or magnesium stearate. Capsules can be prepared by
incorporating these excipients into a gelatin capsule along with antioxidants and the
relevant PUFA(s). The antioxidant and PUFA components should fit within the
guidelines presented above.
-43-
[00158] For intravenous administration, the PUFAs produced in accordance
with the present disclosure or derivatives thereof may be incorporated into
commercial formulations such as Intralipids''"**. The typical normal adult plasma fatty
acid profile comprises 6.64 to 9.46% ARA, 1.45 to 3.11% of DGLA, and 0.02 to
0.08% of GLA. These PUFAs or their metabolic precursors can be administered
alone or in combination with other PUFAs in order to achieve a normal fatty acid
profile in a patient. Where desired, the individual components of the formulations
may be provided individually, in kit form, for single or multiple use. A typical dosage
of a particular fatty acid is from 0.1 mg to 20 g (up to 100 g) daily and is preferably
from 10 mg to 1, 2, 5 or 10 g daily.
[00159] Possible routes of administration of the pharmaceutical
compositions of the present disclosure include, for example, enteral (e.g., oral and
rectal) and parenteral. For example, a liquid preparation may be administered, for
example, orally or rectally. Additionally, a homogenous mixture can be completely
dispersed in water, admixed under sterile conditions with physiologically acceptable
diluents, preservatives, buffers or propellants in order to form a spray or inhalant.
[00160] The route of administration will, of course, depend upon the desired
effect. For example, if the composition is being utilized to treat rough, dry, or aging
skin, to treat injured or burned skin, or to treat skin or hair affected by a disease or
condition, it may perhaps be applied topically.
[00161] The dosage of the composition to be administered to the patient may
be determined by one of ordinary skill in the art and depends upon various factors
such as weight of the patient, age of the patient, immune status of the patient, etc.
[00162] With respect to form, the composition may be, for example, a
solution, a dispersion, a suspension, an emulsion or a sterile powder which is then
reconstituted.
[00163] The present disclosure also includes the treatment of various
disorders by use of the pharmaceutical and/or nutritional compositions described
herein. In particular, the compositions of the present disclosure may be used to treat
-44-
restenosis after angioplasty. Furthermore, symptoms of inflammation, rheumatoid
arthritis, asthma and psoriasis may also be treated with the compositions of the
disclosure. Evidence also indicates that PUFAs may be involved in calcium
metabolism; thus, the compositions of the present disclosure may, perhaps, be utilized
in the treatment or prevention of osteoporosis and of kidney or urinary tract stones.
[00164] Additionally, the compositions of the present disclosure may also be
used in the treatment of cancer. Malignant cells have been shown to have altered fatty
acid compositions. Addition of fatty acids has been shown to slow their growth,
cause cell death and increase their susceptibility to chemotherapeutic agents.
Moreover, the compositions of the present disclosure may also be useful for treating
cachexia associated with cancer.
[00165] The compositions of the present disclosure may also be used to treat
diabetes (see U.S. Patent No. 4,826,877 and Horrobin et al.. Am. J. Clin. Nutr. 1993)
Vol. 57 (Suppl.) 732S-737S). Altered fatty acid metabolism and composition have
been demonstrated in diabetic animals.
[00166] Furthermore, the compositions of the present disclosure, comprising
PUFAs produced either directly or indirectly through the use of the A9-elongase
enzyme, may also be used in the treatment of eczema, in the reduction of blood
pressure, and in the improvement of mathematics examination scores. Additionally,
the compositions of the present disclosure may be used in inhibition of platelet
aggregation, induction of vasodilation, reduction in cholesterol levels, inhibition of
proliferation of vessel wall smooth muscle and fibrous tissue (Brenner et al.. Adv.
Exp. Med. Biol. (1976) Vol. 83, p.85-101), reduction or prevention of gastrointestinal
bleeding and other side effects of non-steroidal anti-inflammatory drugs (see U.S.
Patent No. 4,666,701), prevention or treatment of endometriosis and premenstrual
syndrome (see U.S. Patent No. 4,758,592), and treatment of myalgic
encephalomyelitis and chronic fatigue after viral infections (see U.S. Patent No.
5,116,871).
-45-
[00167] Further uses of the compositions of the present disclosiire include
use in the treatment of AIDS, multiple sclerosis, and inflammatory skin disorders, as
well as for maintenance of general health.
[00168] Additionally, the composition of the present disclosure may be
utilized for cosmetic purposes. It may be added to pre-existing cosmetic
compositions such that a mixture is formed or may be used as a sole composition.
Veterinary Applications
[00169] It should be noted that the above-described pharmaceutical and
nutritional compositions may be utilized in connection with animals (i.e., domestic or
non-domestic), as well as humans, as animals experience many of the same needs and
conditions as humans. For example, the oil or acids of the present disclosure may be
utilized in animal or aquaculture feed supplements, animal feed substitutes, animal
vitamins or in animal topical ointments.
[00170] The present disclosure may be illustrated using the following nonlimiting
examples.
Example 1: cDNA Library Construction from Euslena deses Ehr. CCMP 2916 and
Sequence Analysis to Isolate Putative A9 Elongase Candidates
[00171] Analysis of the fatty acid composition of some marine algae
revealed the presence of a considerable amount of docosahexaenoic acid (DHA, 22:6
n-3) (15% by weight of total lipids) in Euglena deses Ehr. CCMP 2916 (see Table 1).
In addition, this organism displayed intermediates of the alternate A8-desaturase/A9-
elongase pathway (see Figure 1), indicating that this pathway is active in this
organism. Thus, it is predicted that this organism would contain an active A9-
elongase capable of converting linoleic acid (LA, 18:2 n-6) to co6-Eicosadienoic acid
(o)6-EDA, 20:2 n-6), or a-linolenic acid (ALA, 18:3, n-3) to co3-Eicosatrienoic acid
(co3-ETrA, 20:3n-3), as well as an active A8-desaturase that would convert (o6-
Eicosadienoic acid (co6-EDA, 20:2 n-6) to Dihomo-y-linolenic acid (DGLA, 20:3 n-
-46-
6), or co3-Eicosatrienoic acid (o)3-EtrA, 20:3n-3) to to3-Eicosatetraenoic acid (co3-
ETA, 20:4n-3) (see Figure 1).
Table 1: Fatty acid profile of Euglena deses Ehr. CCMP 2916
Fatty Acid I % Total Lipid
Steraric Acid I isio ' 0.529
Oleic Acid 18:1 n-9 1^663
Linoleic Acid (LA) 18:2 n-6 3.137
y Linolenic Acid (GLA) 18:3 n-6 0.096
a-LinoIenic Acid (ALA) 18:3 n-3 16.515
Stearidonic Acid (SDA) 18:4 n-3 0.126
co6-Eicosadienoic Acid (EDA) 20:2 n-6 4.149
Dihomo-Y-linoleic acid (DGLA) 20:3 n-6 0.442
Arachidonic Acid (ARA) 20:4 n-6 3.719
co3-Eicosatrienoic acid (ci>3-ETrA) 20:3 n-3 1.984
(o3-Eicosatetraenoic Acid (to3-ETA) 20:4 n-3 0.496
Eicosapentaenoic acid (EPA) 20:5 n-3 7.104
Adrenic Acid (ADA) 22:4 n-6 0.841
co6-Docosapentaenoic acid (a)6-DPA) 22:5 n-6 5.775
a)3-Docosapentaenoic acid (co3-DPA) 22:5 n-3 1.176
Docosahexaenoic Acid (DHA) 22:6 n-3 15.239
[00172] The goal of this study was to isolate the full-length A9-elongase
gene from Euglena deses Ehr. CCMP 2916 and to characterize its enzymatic activity
by expression in a heterologous host, Saccharomyces cerevisiae.
[00173] To isolate full-length genes from Euglena deses Ehr. CCMP 2916, a
micro-cDNA library was constructed using total RNA isolated from the organism.
For this, cell pellets of the Euglena deses Ehr. CCMP 2916 were obtained from
Provasoli-Guillard-National Center for Marine Phytoplankton (CCMP-Bigelow
Laboratories, West Boothbay, Maine), and total RNA was isolated from it using the
Qiagen RNeasy Maxi kit (Qiagen, Valencia, CA) as per manufacturer's protocol.
-47-
Briefly, frozen cell pellets were crushed in liquid nitrogen using a mortar and pestle,
suspended in RLT buffer (Qiagen RNeasy Plant Mini kit), and passed through a
QiaShredder. The RNA was purified using RNeasy maxi columns as per
manufacturer's protocol.
[00174] The micro-cDNA library was constructed by Agencourt Biosciences
(Waltham, MA), using 50 ^g of RNA from Euglena deses Ehr. CCMP 2916 by
proprietary technology. Agencourt uses several unique and proprietary steps during
first strand that ultimately yields a 25 to 30% increased efficiency over commonly
used techniques. During the proprietary process, the RNA is reverse transcribed into
ssDNA using conditions designed to reduce or eliminate internal priming events. The
combination of this and a specialized cycling program increases the number of fulllength
clones. Following second strand synthesis, the cDNA clones are then size
selected at greater than 1.2kb to decrease preferential cloning of small, truncated
cDNAs. For the large insert library, the insert size selected is >4kb to enhance for the
larger insert clones. Follov^ng size selection, cDNA ends are polished and the
cDNAs are digested using the rare cutting enzyme. A "rare-cutter" restriction
enzyme, the site for which is introduced into the clones during the cDNA priming
step, is then used to prepare the clones for directional cloning into the pAGEN vector.
The "rare-cutter" restriction enzyme is 20 times less likely to cut within the cDNA
clones, thus yielding many more full-length clones versus other cDNA library
construction processes, which utilize more common restriction enzymes that cut at
random intervals along the clone. The result is an insert with a 5' blunt end and a 3'
overhang created from the rare cutting restriction enzyme. Because of this process,
no additional adapter ligation is required to ensure directional cloning. This improves
the overall efficiency of the cloning process. The vector is specially engineered for
directional cloning without the use of 5' adaptors, further enhancing the
transformation efficiency due to a reduced number of manipulations of the cDNA
during cloning. After the primary cDNA library is complete, it is tested for the
number of independent clones, the percentage of recombinant clones and the average
insert size.
-48-
[00175] The clones are then transformed into DHIOB E. coli (Tl phage
resistant bacterial cells). The titer of the resulting library was 3.2 x 10^cfu/ml,with
3.52 X 10*" number of independent colonies with an average insert size of 1.3 kb.
[00176] 4224 clones from this cDNA library were sequenced, and vectortrimmed
sequences were analyzed using BLAST to identify sequences with homology
to known A9-elongase sequences. BLAST analysis revealed five putative hits from
the Euglena deses Ehr. CCMP 2916 cDNA library with homology to known A9-
elongase sequences from Pavlova salina (Accession # AAY15135; SEQ ID NO: 1;
Figure 4A), Isochrysis galbana (Accession #AF390174; SEQ ID NO: 2; Figure 4B),
Eutreptiella sp. (see WO 2007/061845 A2; SEQ ID NO: 3; Figure 4C), Euglena
gracialis (Accession # CAT16687; SEQ ID NO: 4; Figure 4D), and Euglena anabena
(see WO 2008/0194685 Al; SEQ ID NO: 5; Figure 4E).
[00177] One EST clone designated 'plate2_M07' (SEQ ID NO: 6; Figure
5A), obtained from sequencing clones from the Euglena deses Ehr. CCMP 2916
cDNA library, showed high sequence homology to previously identified A9-
elongases. This DNA fragment was 744 bp in length, and its deduced amino acid
sequence (SEQ ID NO: 7; Figure 5B) displayed highest sequence identity (66%
amino acid sequence identity) with the A9-elongase from Euglena gracialis (SEQ ID
NO: 4). The plate2_M07 gene fragment appeared to contain the 'ATG' start site of
the gene based upon alignment with other A9-elongases, but did not contain the 3'-
end of the putative Euglena deses Ehr. CCMP 2916 A9-elongase.
Example 2: Isolation of the 3'-end of the plate2 M07 elongase from Euslena deses
Ehr. ecu? 2916
[00178] The plate2_M07 clone sequence from Example 1 was used as a
template to isolate its 3'- end.
[00179] First strand cDNA was synthesized using the SMARTTM RACE kit
(BD Biosciences) according to the manufacturer's instructions. For synthesis of 3'
RACE-ready cDNA, 1.5 ^ig total RNA from Euglena deses Ehr. CCMP 2916 and 1 ^1
of 3' CDS primer (5'-AAGCAGTGGTATCAACGCAGAGTAC(T)3oVN-3', wherein
-49-
N = A, C, G, or T; and V = A, G, or C (SEQ ID NO: 8)) (12 nM) were mixed in a
total volume of 5 |al in a nuclease-free PCR tube, incubated at 70°C for 2 minutes, and
snap-chilled on ice. After brief centrifiigation, 2 \il of 5X first strand buffer [250 mM
Tris-HCl (pH-8.3), 375 mM KCl and 30 mm MgCy, 1 ^l of 0.1 M DTT and 1 ^1 of
10 mM dNTP mix was added to the tubes. After incubation at 42°C for 2 minutes, 1
|j,l of reverse transcriptase (PowerScript'""'^ RT, BD Biosciences) was added to the tube
and incubated at 42°C for 90 minutes. The first strand cDNA was diluted in 100 |il of
Tricine-EDTA buffer [10 mM Tricine-KOH (pH 8.5), 1.0 mM EDTA] and enzymes
heat inactivated at 72°C for 7 minutes.
[00180] To isolate the 3'- end of the Euglenoid sp. elongase gene fragment
(i.e., the plate2_M07 clone sequence), primers were designed based on the sequence
information from the partial gene sequence of plate2_M07. Primary PCR
amplification was carried out using the 3'- RACE ready cDNA as a template and the
following primers: Eug Elo MO-7 FPl (gene specific primer) (5'-AGG CGC TGT
GGA TCT TCG TCT TCC -3') (SEQ ID NO: 9), in combination with RACE primer
Universal Primer Mix A (UPM, BD Biosciences):
[00181] Long primer (0.4 ^iM): 5'- CTA ATA CGA CTC ACT ATA GCA
AGC AGT GGT ATC AAC GCA GAG T-3' (SEQ ID NO: 10); and
[00182] Short primer (2 ^M): 5'- CTA ATA CGA CTC ACT ATA GGG C -
3'(SEQ ID NO: 11).
[00183] Amplification was carried out using 0.25 ^il (100 mM) of the gene
specific primer, 0.25 \i\ (100 mM) of the UPM primer, 2.5 |al of cDNA template, 2.5
1^1 of 2.5 mM dNTP, 5 |il of 5X PCR Buffer (Advantage® GC II polymerase buffer
(Clontech), 200 mM Tricine-KOH (pH 9.2), 75 mM potassium acetate, 17.5 mM
magnesium acetate, 25% DMSO, 18.75 ^g/ml BSA, 0.005 % Tween 20, 0.005%
Nonidet-P40), 2.5 ^1 GC MeU Reagent (Clontech), 0.5 ^1 of SOX Advantage® GC I
polymerase (Clontech), and 11.5 |iL Milli-Q® water (Millipore), in a final reaction
volume of 25 \x\. Samples were denatured initially at 94°C for 3 minutes, followed by
2 cycles of 94°C for 30 seconds, 64°C for 30 seconds, and 68°C for 1.3 minutes; 3
-50-
cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 68°C for 1.30 minutes; 4
cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 68°C for 1.30 minutes; and
26 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 1.30 minutes. A
final extension cycle at 68°C for 10 minutes was carried out before the reaction was
terminated at 4°C.
[00184] Analysis of the PCR products revealed very faint bands, which were
likely due to low levels of the elongase gene transcripts in the cell. Hence, a nested
PCR reaction was carried out using 1 \il of the product from the above-described
primary PCR reaction as a template. Primers used for the nested PCR were Bug Elo
MO-7 FP2 (a gene-specific primer): 5'- TCC CCG TGC CGA AGT CGT TCA TCA
CC -3' (SEQ ID NO: 12), and the Universal Primer Mix A (UPM) primers (SEQ ID
NOs: 10 and 11). PCR reaction conditions and cycling parameters were same as used
for the primary PCR reaction.
[00185] A 548 bp amplicon (SEQ ID NO: 13; Figure 6A), obtained by
nested PCR, was gel purified using the Qiagen Gel Purification kit (Qiagen), and was
cloned into pTZ57R/T vector (T/A cloning vector, MBI Fermentas) and sequenced.
Sequencing revealed that this fi-agment (SEQ ID NO: 13) was contained the complete
3'- end of the plate2_M07 elongase fi-agment along with the 'TAG' stop codon and
downstream region containing the polyA tail. The predicted amino acid sequence of
this fragment (SEQ ID NOs: 14 and 30-32) is shown in Figure 6B. The first asterisk
denotes the stop site of the plate 2_M07 encoded protein.
Example 3: Isolation of the Full-Length plate2 M07 Elongase Gene from Euslena
desesEhr. CCMP2916
[00186] The fiill-length gene sequence of plate2_M07 elongase was isolated
by PCR amplification using the Euglena deses Ehr. cDNA library as the template, and
primers that were designed to contain the 5'- and 3'- ends of the plate2_M07 gene
based upon sequence information obtained in Example 1 and Example 2. In addition,
BamHI/Hindlll sites were incorporated into the primers (underlined) to facilitate
-51-
cloning of the gene into the BamHI/Hindlll sites of the yeast expression vector,
pYX242. The following primer sequences were used:
[00187] MO7-EI0 forward primer: 5'- CAC CAT GGA TCC ATG GAC
GTC GCG ACT ACG CTG G -3' (SEQ ID NO: 15), and
[00188] MO7-EI0 reverse primer: 5'- ACG CGT AAGCTT CTA GTC CAC
TTT CTT CTC ATC CTT C-3' (SEQ ID NO: 16).
[00189] Amplification was carried out using 0.5 ^1 (100 ^M) of each primer,
1 |j,l (~ 1 lOng) of the Euglena deses Ehr. cDNA library plasmid pool as the template,
5 \x\ of 2.5 mM dNTP, 10 ^1 of 5X Phusion GC Buffer (Finnzymes), 5 ^L of DMSO,
0.5 i^L (1 U) of Phusion polymerase (Finnzymes), and 27.5 i^L of Milli-Q® water
(Millipore). Samples were denatured initially at 98°C for 3 minutes, followed by 2
cycles of 98°C for 8 seconds, 60°C for 12 seconds, and 72°C for 45 seconds; and 28
cycles of 98°C for 8 seconds, 58°C for 12 seconds, and 72°C for 45 seconds. A final
extension cycle at 72°C for 3 minutes was carried out before the reaction was
terminated at 4°C.
[00190] PCR resulted in an -789 bp product, which was cloned into the Bam
HI/Hind III sites of pYX242 vector and transformed into E.coli DH5a (Invitrogen).
Plasmid DNA thus obtained was sequenced to obtain the fiiU-length gene sequence of
the 789 bp gene, designated 'Eug-MO7-ELO#10' (SEQ ID NO: 17; Figure 7A). SEQ
ID NO: 17 was deposited with the American Type Culture Collection, 10801
University Boulevard, Manassas, VA 20110-2209, on July 10, 2009, under the terms
of the Budapest Treaty, and was accorded deposit number ATCC . This gene
was thought to encode the putative A9-elongase from Euglena deses Ehr. CCMP
2916, with a predicted length of 262 amino acids (SEQ ID NO: 18; Figure 7B). This
gene was used for expression studies to characterize its enzymatic activity.
[00191] In addition to the Eug-M07-EL0# 10 clone, additional variant
clones were identified during sequencing that displayed some sequence variations in
certain regions across the full-length gene. These sequence variations probably arose
during the process of PCR amplification. Sequence analysis of one such variant, Eug-
-52-
M07-EL0 #14 revealed a number of nucleotide and corresponding amino acid
changes when compared to the original Eug-MO7-ELO#10 clone (see Table 2 and
Figures 2A and 2B). The nucleotide (SEQ ID NO: 19) and predicted amino acid
sequence (SEQ ID NO: 20) of Eug-M07-EL0 #14 are shown in Figures 8A and 8B,
respectively. Both the original Eug-MO7-ELO#10 clone and the variant Eug-M07-
ELO #14 were used for expression analysis.
Table 2: Nucleotide and amino acid changes in the variant clone Eug-M07-
ELO#14 in comparison to the original clone Eug-MO7-ELO#10
Nucleotide Changes Corresponding Amino Acid
Eug-MO7-ELO#10 (SEQ ID NO: 17) => Eug- ^"^^-^es (SEQ ID NO: 18
M07-ELO#14 (SEQ ID NO: 19) ^^^^Q °^ ^^^ 2«>
GCT24 => GCC24 Silent mutation
GC83C => GTgsC A28 => V28
G232TA => A232TA V78 => I78
A301TG => T301TG Mioi => Lioi
C310TC => A310TC L104 => I104
ACA630 => ACT630 Silent mutation
AAA750 =* AAG750 ^'•^"t mutation
[00192] Blast searches, using Eug-M07-EL0 #10 as query, for similarity to
sequences contained in the BLAST 'nr' database revealed that the predicted amino
acid sequence encoded by Eug-MO7-ELO#10 (SEQ ID NO: 18) displayed highest
amino acid sequence identity (36% sequence identity) with the Isochrysis galbana
A9-elongase (SEQ ID NO: 2). Pair wise alignment of SEQ ID NO: 18 to the known
A9-eIongase from Euglena gracialis (Accession # CAT 16687; SEQ ID NO: 4)
revealed a much higher amino acid sequence identity (66% identity). Here the default
parameters of Vector NTI®AlignX program were used for pair wise alignment. Pair
-53-
wise alignment with the Pavlova salina A9-elongase (SEQ ID NO: 1) revealed only
-15% sequence identity.
[00193] Unlike desaturases, the elongase enzymes display very few highly
conserved motifs. These enzymes are highly hydrophobic proteins containing four to
five hydrophobic stretches that are predicted to be membrane-spanning region. In
addition a highly conserved histidine box (HXXHH) (SEQ ID NO: 28) is found
embedded in the fourth membrane spanning region and is essential for enzymatic
activity (see Leonard, et al., "Elongation of long-chain fatty acids," Prog Lipid Res.
(2004) Vol.43, p. 36-54). In some elongases, the first histidine residue of the
'HXXHH' motif (SEQ ID NO: 28) is replaced with a Glutamine (Q) resuhing in
'QXXHH' (SEQ ID NO: 29) as the conserved motif. This QXXHH (SEQ ID NO: 29)
motif is found in most of the A9-elongases including Eug-MO7-ELO#10. In addition,
the Eug-MO7-ELO#10 elongase contains other invariant residues that are present in
most elongases to date, as described by Leonard, et al., "Elongation of long-chain
fatty acids," Prog Lipid Res. (2004) Vol. 43, p. 36-54.
[00194] Figures 3 A and 3B depict an alignment of the amino acid sequence
from Eug-MO7-ELO#10 elongase with other known elongases that have varying
substrate specificity. These include the Mouse Elovl4 elongase (Accession #
AAG47667; SEQ ID NO: 21; Figure 9A), human EL0VL2 elongase (Acession #
NP_060240; SEQ ID NO: 22; Figure 9B), and C. elegans elongase (Accession #
AF244356; SEQ ID NO: 23), in addition to the A9-elongases from Euglena gracialis
(SEQ ID NO: 4) and Isochrysis galbana (SEQ ID NO: 2). Invariant amino acids in
the alignment are shaded. It is assumed that these invariant residues are important
determinants for fiinctionality of these elongating enzymes due to high degree of
conservation across species. Aligrmient was carried out using Vector NTI software
that uses a modified ClustalW algorithm.
Example 4: Characterization of the enzvmatic activity of the putative A9-elongase
encoded by the gene Eug-MO7-ELO#10
-54-
[00195] The Eug-MO7-ELO#10 and Eug-M07-ELO#14 variant encoding a
putative A9-elongase were cloned into BamHI/Hindlll sites of the yeast expression
vector, pYX242 (Novagen), respectively. These constructs were transformed into
competent Saccharomyces cerevisiae strain SC334 cells. Yeast transformation was
carried out using the Alkali-Cation Yeast Transformation Kit (QBioGene) according
to conditions specified by the manufacturer. Transformants were selected for leucine
auxotrophy on media lacking leucine (DOB [-Leu]).
[00196] To characterize the elongase activity of the enzymes encoded by
Eug-MO7-ELO#10 and Eug-M07-ELO#14, transformants were grown in the
presence of 50 ^iM specific fatty acid substrates (listed below) and conversion to
specific product was used to determine substrate specificity:
[00197] For A9-elongase activitv:
[00198] Linoleic acid (18:2 n-6) => Eicosadienoic acid (EDA, 20:2 n-6)
[00199] Alpha-linolenic acid (18:3 n-3) => Eicosatrienoic acid (ETrA, 20:3
n-3)
[00200] For C^s-elongase activitv:
[00201] Gamma-linolenic acid (GLA, 18:3 n-6) => Dihomo-y-linolenic acid
(DGLA, 20:3 n-6)
[00202] Stearidonic acid (SDA, 18:4 n-3) => co3-Eicoastetraenoic acid (co3-
ETA, 20:4 n-3)
[00203] For C2n-elongase activitv:
[00204] Arachidonic acid (ARA, 20:4 n-6) ^ Adrenic acid (Q)6-ADA, 22:4
n-6)
[00205] Eicosapentaenoic acid (EPA, 20:5 n-3) => co3-Docosapentaenoic
acid (a)3-DPA, 22:5 n-3)
-55-
[00206] The negative control strain consisted of pYX242 vector expressed
in S. cerevisiae 334.
[00207] The transformed colonies isolated from selective DOB [-Leu]
media were grown overnight in 10 ml of YPD liquid broth at 30°C, with vigorous
agitation. 5 ml of this overnight culture was then added to 45 ml of selective media
(DOB [-Leu]) containing 50 jiM (final concentration) of various fatty acid substrates
(as specified), and these were vigorously agitated (250 rpm) for 48 to 72 hours (as
indicated) at 24°C.
[00208] For total lipid extraction, yeast cells were spun down at 2000 rpm
for 15 minutes and 0.5 ml water was added, samples vortexed, followed by addition
of 10 ml methanol with gentle swirling. 20 ml chloroform was then added, samples
were vortexed for 1 minute at high speed and allowed to stand for 2 hours at room
temperature. 6 ml saline was then added to the sample followed by centrifugation at
2200 rpm for 10 minutes. The upper chloroform layer was removed to a clean/dry 30
ml vial and chloroform evaporated to dryness at 40°C under a stream of nitrogen.
Once the solvents had completely evaporated, 2 ml chloroform was added to each vial
and samples were derivatized.
[00209] For derivitization of lipids to Fatty acid methyl esters (FAME), each
tube was spiked with 100 \x\ internal standard (17.216 fig/100 \x\) Triheptadecanoin.
Chloroform was evaporated to dryness under nitrogen at 40°C, 2 ml Boron Trifluoride
in 14% Methanol was added, followed by addition of 2 drops (~50 i^l) Toluene. Each
vial was flushed with nitrogen, and heated for 15 minutes at 95°C. After vials had
cooled, 2 ml saline was added and lipids extracted with 4 ml hexane by vigorously
vortexing for 1 minute. The hexane extract was then transferred into a 20 ml
clean/dry screw-cap tube, 5 ml di-H20 was added and sample vortexed, and
centrifuged at 1500 rpm for 4 minutes. The washed hexane was then transferred into
a 20 ml reagent tube. Hexane was evaporated to dryness and each sample
reconstituted with 0.5 ml fresh hexane. The reconstituted final hexane was vortexed
to disperse the lipids. The entire sample was then loaded into the GC auto sampler
-56-
vials and 4 \i\ was injected for analysis. The GC was calibrated with the NuChek Std.
461.
[00210] The percent conversion of substrate to product was calculated using
the formula:
fproductl xlOO
[product] + [substrate]
[00211 ] Table 3 represents the enzyme activity of the Eug-M07-EL0# 10-
and Eug-M07-ELO#14 encoded proteins based on the percent conversion of substrate
added. Eug-MO7-ELO#10 encoded protein converted 10.5% of LA (18:2n-6) to
EDA (20:2 n-6), and 23.2% of ALA (18:3n-3) to ETrA (20:3n-3). This indicated that
the Eug-MO7-ELO#10 gene encodes a A9-elongase that can recognize both n-6 and
n-3 fatty acid substrates. The variant clone, Eug-M07-ELO#14 encoded protein also
displayed A9 elongase activity, converting converted 7.84%) of LA (18:2n-6) to EDA
(20:2 n-6), and 17.15% of ALA (18:3n-3) to ETrA (20:3n-3). However this activity
was lower that that of the original Eug-MO7-ELO#10 encoded protein. This indicates
that the residues that differ between Eug-M07-ELO#14 and Eug-MO7-ELO#10 are
important determinants of A9-elongating activity of this enzyme.
[00212] Very low backgroimd (non-specific conversion of substrate) activity
was detected with the vector-only control (see Table 3). Both Eug-MO7-ELO#10 &
Eug-M07-ELO#14 encoded enzymes did not have activity on any of the other PUFA
substrates tested (see Table 4), indicating that this enzyme is specific for substrates
involved in the alternate A8-desaturase/A9-elongase pathway (see Figure 1).
Table 3: A9-eIongase activity of Eug-MO7-ELO#10 and Eug-M07-ELO#14
encoded proteins expressed in Saccharomyces cerevisiae strain SC334
% Total Fatty Acid I Eug-M07- I Eug-M07- \ Vector
ELO#10 ELO#14 Control
LA (18:211-6)" %M 12.575 1065
EDA(20:2n-6,All,14)'' LOSS L066 0.0985
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% LA -> EDA Conversion' \ lois I 7^84 I 091
ALA (18:311-3)" 8J88 io!89 i3!96
ETrA (20:3 n-3,Al 1,14,17)" 2!665 2l98 oT66
% ALA -> ETrA Conversion' 23J 17.15 TilZ
^ Cultures grown in presence of 50 [xM substrate at 24°C for 48 hours. Numbers
represent an average of 2 different experiments.
'' Amount of product formed
'^ % Conversion = ([product] / {[product] + [substrate]}) x 100
Table 4: Specificity of Elongase Activity of Eug-MO7-ELO#10 & Eug-M07-
EL0#14 encoded proteins expressed in Saccharomyces cerevisiae strain SC334
% Total Fatty Acid I Eug-M07- I Eug-M07- I Vector
ELO#10 ELO#14 Control
GLA (18:311-6)" 12^90 14^23 14^49
DGLA (20:311-6)" 0.171 0.194 0.164
% GLA -> DGLA Conversion' LSI \M L12
ARA (20:411-6)" 27.645 25.044 22.711
Adrenic Acid (22:4 n-6)" Oio OO 0.019
% ARA -^ Adrenic Acid Conversion' 0 0 008
SDA(18:4n-3)" 6.899 8.335 8.642
C03-ETA (20:4n-3)" O077 0047 07l98
% SDA^ Q)3-ETA Conversion' LIO 056 2^24
EPA (20:511-3)" 18.84 13.351 12.016
C03-DPA (22:5 n-3)" 0.131 0.093 0.083
% EPA ^ (03-DPA Conversion' 069 069 069
* Cultures grown in presence of 50 ^iM substrate at 24°C for 48 hours. Numbers
represent an average of 2 different experiments.
Amount of product formed
'^ % Conversion = ([product] / { [product] + [substrate]}) x 100
Example 5: Expression of the A9-elongase 'Eug-MO7-ELO#10' in plant seeds
[00213 ] The coding sequence of the Eug-M07-EL0# 10 elongase was
amplified by PCR from a plasmid containing the corresponding gene with the
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following sense and antisense oligonucleotide primers (added restriction enzyme sites
are underlined):
[00214] 5'- TATAGAATTCAAATGGACGTCGCGACTACGCTG-3'
(SEQ ID NO: 24), and
[00215] 5'- TATTCTCGAGTTCTAGTCCACTTTCTTCTCATCCTTC-3'
(SEQ ID NO: 25).
[00216] The PCR reaction was conducted with high-fidelity Phusion
polymerase (New England Biolabs). The PCR amplified gene was digested with
restriction enzymes EcoKi. a n d ^ o l , and the resulting product was linked on its 5'-
end to the seed-specific glycinin-1 promoter fi-om soybean and on its 3'-end to the
glycinin-1 3' untranslated region in the binary vector p0308-DsRed to generate the
plasmid 'pEugELO'. The glycinin-1 regulatory elements have been previously
described by Nielsen, et al., "Characterization of the glycinin gene family in
soybean," Plant Cell (1989) Vol. 1, p. 313-328. This vector also contains a Ds-Red
transgene under control of the cassava mosaic virus promoter for selection of
transformed seeds by fluorescence and a kanamycin resistance marker for bacterial
selection. As a control for these experiments, the Isochrysis galbana A9-elongase
gene (SEQ ID NO: 2) was also cloned as an EcoRUXhol fragment under control of
the glycinin-1 promoter in p0308-Ds-Red to generate the plasmid 'pIsoD9'.
[00217] pEugELO and pIsoD9 were introduced into Agrobacterium
tumefaciens strain C58 MP90 by electroporation. Kanamycin-resistant agrobacterium
was then used for transformation of Arabidopsis thaliana ecotype Col-0 by the floral
dip method (Clough, et al., "Floral dip: a simplified method for Agrobacteriummediated
transformation of Arabidopsis thaliana," Plant J, (1998) Vol. 16, p. 735-
743). Following the agrobacterium floral dip, plants were maintained at 22°C with 16
hour day length until reaching maturity and dry down. For these experiments, a
facB/fael mutant of Arabidopsis was used that contains low levels of a-linolenic acid
and very-long chain fatty acids (>C20) but elevated levels of linoleic acid in its seed
oil (Cahoon, et al., "Conjugated fatty acids accumulate to high levels in phospholipids
-59-
of metabolically engineered soybean and Arabidopsis seeds," Phytochemistry (2006)
Vol. 67, p. 1166-1176). This genetic background approximates the fatty acid profile
of seed oils from crops such as safflower and low linolenic acid soybean. Transgenic
seeds obtained from the agrobacterium-dipped Arabidopsis plants were identified by
fluorescence of the DsRed marker protein using the methodology described by
Pidkowich, et al., "Modulating seed beta-ketoacyl-acyl carrier protein synthase II
level converts the composition of a temperate seed oil to that of a palm-like tropical
oil," Proc Natl Acad Sci USA (2007) Vol. 104, p. 4742-4747. Single transgenic and
non-transgenic control seeds were subjected to direct transesterification of the
constituent lipids, including triacylglycerols, by use of trimethylsulfonium hydroxide
(TMSH) reagent as described by Cahoon and Shanklin, "Substrate-dependent mutant
complementation to select fatty acid desaturase variants for metabolic engineering of
plant seed oils," Proc Natl Acad Sci USA (2000) Vol. 97, p. 12350-12355. Fatty
acid methyl esters obtained from the single seeds were analyzed by gas
chromatography with flame ionization detection by use of an Agilent 6890 gas
chromatograph fitted with an INNOWax column (30 m length x 0.25 mm irmer
diameter) and oven temperature programming from 185°C (1 minute hold) to 230°C
(2 minute hold) at 7°C/minute. Component fatty acid methyl esters were identified
based on their retention times relative to fatty acid methyl esters of known identity
from seeds of wild-type Arabidopsis thaliana Col-0 and by comparison of retention
times with those of standard fatty acid methyl esters.
[00218] Shown in Table 5 are the fatty acid compositions of single Tl seeds
from six independent transformation events from plants transformed with pEugELO
construct. Also shown are the fatty acid compositions of single Tl seeds representing
independent events from plants transformed with pIsoD9 construct, the control A9
elongase (see Table 6). The major change in the fatty acid composition of transgenic
seeds from the pEugELO transformation relative to non-transformedya^//ael seeds
(see Table 7) was the presence of high levels of EDA (20:2n-6, Al 1,14). In these
seeds, relative amounts of 20:2 ranged from 40% to 49% (w/w) of the total fatty
acids. By comparison 20:2 accoimted for >0.5% of the total fatty acids of nontransgenic
yafiGZ/oel seeds (see Table 7). This was accompanied by concomitant
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decreases in relative amounts of LA (18:2n-6, A9, 12) from approximately 50% in
non-transgenicya^//ael seeds (see Table 7) to as low as 14% in the pEugELO. This
is consistent with 18:2 serving as the primary substrate for 20:2 synthesis conferred
by the Eug-MO7-ELO#10 elongase. Amounts of Eicosenoic acid (20:1A11) and
Eicosanoic acid (20:0) were also elevated in the pEugELO-transformed seeds relative
to non-transgenic fad3/fae\ seeds, but each of these fatty acids composed <3% of the
total fatty acids in the transgenic seeds. These findings indicate that Eug-M07-
ELO#10 elongase has substrate preference in plants for Cig PUFAs such as LA
(18:2n-6) and is an effective enzyme for the production of 20:2 in seeds that are
enriched in LA (18:2n-6). For comparison, seeds engineered to express the Isochrysis
galbana A9-EL0 (pIsoD9) accumulated 20:2 to amounts of 30 to 40% of the total
fatty acids and 20:0 and 20:1 each to amounts of <3% of the total fatty acids (see
Table 6).
Table 5*: Fatty acid composition of single Tl transgenic ArabidopsisyarfJ^ae/
seeds expressing Eug-MO7-ELO#10.
Fatty Line 1 Line 2 Line 3 Line 4 Line 5 Line 6
acid
16:0 9,5 9A 82 1_A 82 8.0
18:0 15 16 4J 14 12 3.5
18:1 18,5 17,5 19.1 20.3 12/7 16.5
18:2 21.3 143 14J 15.6 19.9 21.5
18:3 09 L3 >0.1 >0.1 >0.1 0.7
20:0 LO LO L2 09 0,8 0.9
20:1 L3 L2 L5 23 2JO 2.3
20:2 42.3 49.4 48.6 47.2 49.3 44.3
other I 1.7 | 2.6 | 2.4 | 2.1 | 2.1 | 1.8
^ Each seed represents an independent transgenic event. Values shown are the wt% of
the total fatty acids in the seed.
Table 6^: Fatty acid composition of single Ti transgenic Arabidopsis/arfiz/aei
seeds expressing the Isochrysis galbana ELO.
Fatty Line 1 Line 2 Line 3 Line 4 Line 5
acid
16:0 7J %A_ 6J 7.4 7.2
18:0 19 33 4,0 33 4.3
18:1 I 22.8 I 19.5 | 15.9 | 15.2 | 20.8
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18:2 I 26.3 I 23.9 I 29.2 I 25.3 I 26.2
18:3 LO L2 04 0,6 0.9
20:0 U 09 L2_ LO^ 1.1
20:1 2,0 L6 2^ 22 2.2
20:2 34J 30 3X1 40.2 36.1
other I 1.1 I 2.3 | 2.2 | 2.9 | 1.1
" Each seed represents an independent transgenic event. Values shown are the wt% of
the total fatty acids in the seed.
Table T: Fatty acid composition of single Arabidopsis fadS^ael control seeds.
Fatty Line 1 Line 2 Line 3 Line 4 Line 5
acid
16:0 7,9 84 6,9 8,9 8.0
18:0 4,9 3,9 32 53 3.8
18:1 28,6 34/7 406 32,5 31.1
18:2 533 49,6 46,8 ^09 53.6
18:3 2,6 1,8 LO 1.3 1.5
20:0 L3 07 08 LO 08
20:1 09 04 04 02 05
20:2 ^01 ^01 ^01 ^01 >0.1
other I 0.1 | 0.2 | 0.1 | 01 | 0.5
* Values shown are the wt% of the total fatty acids in the seed.
Example 6: Coexpression of the A9-elongase 'Eug-MO7-ELO#10 with a A8-
desaturase
[00219] It is possible to co-express Eug-M07-EL0# 10 along with a A8-
desaturase to reconstruct the alternate A8-desaturase/A9-elongase pathway leading to
ARA production. In addition it will be possible to coexpress three genes, the A9-
elongase 'Eug-MO7-ELO#10' along with a A8-desaturase and a A5-desaturase in a
heterologous host such as oilseed plants or oleaginous yeast to reconstruct the ARA
biosynthesis pathway with will result in ARA production in these heterologous hosts.
[00220] In view of the above, it will be seen that the several objects of the
disclosure are achieved and other advantageous results attained.
[00221] As various changes could be made in the above matter without
departing from the scope of the disclosure, it is intended that all matter contained in
the above description shall be interpreted as illustrative and not in a limiting sense.

lAVE CLAIM:
1. An isolated nucleic acid or fragment thereof comprising or complementary to an
isolated nucleotide sequence encoding a polypeptide having elongase activity, wherein the
amino acid sequence of the polypeptide has at least 68% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 20.
2. An isolated nucleotide sequence or fragment thereof comprising or
complementary to at least 75% of a nucleotide sequence selected from the group consisting
ofSEQIDNO: 17 and SEQ ID NO: 19.
3. The isolated nucleotide sequence of claim 1 or 2 wherein the isolated nucleotide
sequence encodes a functionally active elongase which utilizes a polyunsaturated fatty acid
as a substrate.
4. The isolated nucleotide sequence of claim 1 or 2 wherein the isolated nucleotide
sequence is from a Euglenoid sp.
5. The isolated nucleotide sequence of claim 4 wherein the isolated nucleotide
sequence is from Euglena deses Ehr. CCMP 2916.
6. A purified polypeptide encoded by the isolated nucleotide sequence of claim 1 or
2.
7. A purified polypeptide which elongates polyunsaturated fatty acids containing
unsaturation at the carbon 9 position and has at least 68% amino acid identity to an amino
acid sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 20.
8. An expression vector comprising a nucleotide sequence operably linked to a
regulatory sequence, wherein the nucleotide sequence comprises or is complementary to at
least 75% of a nucleotide sequence selected from the group consisting of SEQ ID NO: 17
and SEQ ID NO: 19.
9. A host cell comprising the expression vector of claim 8.
10. The host cell of claim 9 wherein the host cell is selected from the group
consisting of a eukaryotic cell and a prokaryotic cell.
63
11. The host cell of claim 10 wherein the eukaryotic cell is selected from the group
consisting of: a mammalian cell, an insect cell, a plant cell, and a fungal cell.
12. The host cell of claim 11 wherein the plant cell is from an oilseed plant selected
from the group consisting of: soybean, Brassica species, safflower, sunflower, maize,
cotton, and flax.
13. A plant cell, plant seed, plant, or plant tissue comprising the expression vector
of claim 8, wherein expression of the nucleotide sequence of the expression vector results
in production of at least one polyunsaturated fatty acid by the plant cell, plant seed, plant,
or plant tissue.
14. The plant cell, plant seed, plant or plant tissue of claim 13 wherein the
polyunsaturated fatty acid is selected from the group consisting of a)6-eicosadienoic acid
(co6-EDA), co3-eicosatrienoic acid (co3-ETrA), and combinations thereof.
15. A method of producing a A9-elongase, the method comprising the steps of:
a) isolating a nucleotide sequence comprising or complementary to at least 75% of
a nucleotide sequence selected from the group consisting of: SEQ ID NO: 17 and SEQ ID
NO: 19;
b) constructing an expression vector comprising i) the isolated nucleotide sequence
operably linked to ii) a regulatory sequence; and
c) introducing the expression vector into a host cell for a time and under conditions
sufficient for production of the A9-elongase.
16. The method of claim 15 wherein the host cell is selected from the group
consisting of a eukaryotic cell and a prokaryotic cell.
17. The method of claim 16 wherein the eukaryotic cell is selected from the group
consisting of a mammalian cell, an insect cell, a plant cell, and a fungal cell.
18. The method of claim 17 wherein the plant cell is from an oilseed plant selected
from the group consisting of soybean, Brassica species, safflower, sunflower, maize,
cotton, and flax.
19. A method for producing a polyunsaturated fatty acid comprising the steps of:
64
a) isolating a nucleotide sequence comprising or complementary to at least 75% of
a nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID
NO: 19;
b) constructing an expression vector comprising i) the isolated nucleotide sequence
operably linked to ii) a regulatory sequence;
c) introducing the expression vector into a host cell for a time and under conditions
sufficient for expression of a A9-elongase; and
d) exposing the expressed A9-elongase to a substrate polyunsaturated fatty acid in
order to convert the substrate polyunsaturated fatty acid to a first product polyunsaturated
fatty acid.
20. The method of claim 19 wherein the substrate polyunsaturated fatty acid is
linoleic acid (LA) and the first product polyunsaturated fatty acid is co6-eicosadienoic acid
(Q)6-EDA).
21. The method of claim 19 wherein the substrate polyunsaturated fatty acid is alinolenic
acid (ALA) and the first product polyunsaturated fatty acid is (o3-eicosatrienoic
acid (co3-ETrA).
22. The method of claim 19 further comprising the step of exposing the first
product polyunsaturated fatty acid to at least one desaturase, at least one additional
elongase, or combinations thereof, in order to convert the first product polyunsaturated
fatty acid to a second or subsequent product polyunsaturated fatty acid.
23. The method of claim 22 wherein the second or subsequent product
polyunsaturated fatty acid is selected from the group consisting of dihomo-y-linolenic acid
(DGLA), co3-eicosatetraenoic acid (co3-ETA), arachidonic acid (ARA), eicosapentaenoic
acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), and
combinations thereof.
24. A method for producing a polyunsaturated fatty acid in a host cell comprising
the steps of:
65
a) isolating a nucleotide sequence comprising or complementary to at least 75% of
a nucleotide sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID
NO: 19;
b) constructing an expression vector comprising i) the isolated nucleotide sequence
operably linked to ii) a regulatory sequence;
c) introducing i) the expression vector and ii) at least one additional recombinant
DNA construct comprising an isolated nucleotide sequence encoding a A8-desaturase and
operably linked to at least one regulatory sequence, into a host cell for a time and under
conditions sufficient for expression of a A9-elongase and the A8-desaturase; and
d) exposing the expressed A9-elongase and the A8-desaturase to a substrate
polyunsaturated fatty acid selected from the group consisting of linoleic acid (LA), alinolenic
acid (ALA), and combinations thereof, in order to convert the substrate
polyunsaturated fatty acid to a first product polyunsaturated fatty acid.
25. The method of claim 24 wherein the first product polyunsaturated fatty acid is
selected from the group consisting of dihomo-y-linolenic acid (DGLA), co3-
eicosatetraenoic acid (co3-ETA), and combinations thereof
26. The method of claim 24 further comprising the step of exposing the first
product polyimsaturated fatty acid to at least one additional desaturase or to at least one
additional elongase in order to convert the first product polyunsaturated fatty acid to a
second or subsequent polyunsaturated fatty acid.
27. The method of claim 26 wherein the second or subsequent polyunsaturated fatty
acid is selected from the group consisting of arachidonic acid (ARA), eicosapentaenoic
acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), and
combinations thereof
28. The method of claim 24 wherein the host cell is selected from the group
consisting of a prokaryotic cell and a eukaryotic cell.
29. The method of claim 28 wherein the eukaryotic cell is selected from the group
consisting of a mammalian cell, an insect cell, a plant cell, and a fungal cell.
66
30. The method of claim 29 wherein the plant cell is from an oilseed plant selected
from the group consisting of soybean, Brassica species, safflower, sxmflower, maize,
cotton, and flax.
31. The method of claim 24 further comprising introducing into the host cell a
recombinant DNA construct comprising i) an isolated nucleotide sequence encoding a A5-
desaturase operably linked to ii) a regulatory sequence.
32. A method for producing a transgenic plant comprising transforming a plant cell
with at least one isolated nucleotide sequence or fragment thereof of claim 2 and
regenerating a transgenic plant from the transformed plant cell.
33. The method of claim 32 wherein the plant cell is from an oilseed plant selected
from the group consisting of soybean, Brassica species, safflower, sunflower, maize,
cotton, and flax.
34. A transgenic seed obtained from the transgenic plant made by the method of
claim 32.
35. A transgenic seed comprising the expression vector of claim 8.
Date 16 January 2012