U.S. patent application number 09/747755 was filed with the patent office on 2002-04-11 for omega-3 fatty acid desaturase.
Invention is credited to Browse, John A., Spychalla, James P..
Application Number | 20020042933 09/747755 |
Document ID | / |
Family ID | 26699930 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020042933 |
Kind Code |
A1 |
Browse, John A. ; et
al. |
April 11, 2002 |
Omega-3 fatty acid desaturase
Abstract
Recombinant expression of fat-1 gene of Caenorhabditis elegans
in a wide variety of cells, including cells of Arabidopsis thaliana
and Saccharomyces cerevisiae, produces a polypeptide having
.omega.-3 desaturase activity.
Inventors: |
Browse, John A.; (Palouse,
WA) ; Spychalla, James P.; (Antigo, WI) |
Correspondence
Address: |
KLARQUIST SPARKMAN CAMPBELL
LEIGH & WHINSTON, LLP
One World Trade Center, Suite 1600
121 S. W. Salmon Street
Portland
OR
97204
US
|
Family ID: |
26699930 |
Appl. No.: |
09/747755 |
Filed: |
December 20, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09747755 |
Dec 20, 2000 |
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09025578 |
Feb 18, 1998 |
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6194167 |
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60038409 |
Feb 18, 1997 |
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Current U.S.
Class: |
800/298 ;
435/419; 435/468 |
Current CPC
Class: |
C12N 15/8247 20130101;
C12N 9/0014 20130101; C12N 9/0083 20130101 |
Class at
Publication: |
800/298 ;
435/419; 435/468 |
International
Class: |
A01H 005/00; C12N
015/82 |
Goverment Interests
[0002] This invention was made with Government support under
Research Grant 95-37301-2287 from the USDA-NRICGP. The Government
has certain rights to this invention.
Claims
What is claimed is:
1. A cell comprising a recombinant FAT-1 polypeptide that
desaturates an .omega.-6 fatty acid to a corresponding .omega.-3
fatty acid.
2. The cell of claim 1 wherein the .omega.-6 fatty acid has a
carbon chain of at least 18 carbons.
3. The cell of claim 2 wherein the .omega.-6 fatty acid is a
20-carbon .omega.-6 fatty acid or a 22-carbon .omega.-6 fatty
acid.
4. The cell of claim 1 wherein the .omega.-6 fatty acid has a
double bond at one or more positions selected from the group
consisting of .DELTA.4, .DELTA.5, .DELTA.6, .DELTA.7, and
.DELTA.8.
5. The cell of claim 1 having a proportion of .omega.-3 fatty acids
that is at least 10% greater than an otherwise similar cell lacking
the recombinant FAT-1 polypeptide.
6. The cell of claim 5 having a proportion of .omega.-3 fatty acids
that is at least 20% greater than an otherwise similar cell lacking
the recombinant FAT-1 polypeptide.
7. The cell of claim 6 having a proportion of .omega.-3 fatty acids
that is at least 50% greater than an otherwise similar cell lacking
the recombinant FAT-1 polypeptide.
8. The cell of claim 1 wherein the .omega.-6 fatty acid has a
carbon chain of at least 20 carbons and at least 25% of the
.omega.-6 fatty acid is desaturated to the corresponding .omega.-3
fatty acid.
9. The cell of claim 1 wherein the recombinant FAT-1 polypeptide
has at least 60% amino acid sequence identity with the FAT-1
polypeptide of SEQ ID NO:1.
10. The cell of claim 1 wherein the recombinant FAT-1 polypeptide
has at least 70% amino acid sequence identity with the FAT-1
polypeptide of SEQ ID NO:1.
11. The cell of claim 10 wherein the recombinant FAT-1 polypeptide
has at least 80% amino acid sequence identity with the FAT-1
polypeptide of SEQ ID NO:1.
12. The cell of claim 10 wherein the recombinant FAT-1 polypeptide
has at least 90% amino acid sequence identity with the FAT-1
polypeptide of SEQ ID NO:1.
13. The cell of claim 1 wherein the recombinant FAT-1 polypeptide
has only conservative amino acid substitutions to the FAT-1
polypeptide of SEQ ID NO:1.
14. The cell of claim 1 wherein the recombinant FAT-1 polypeptide
has 100% amino acid sequence identity with the FAT-1 polypeptide of
SEQ ID NO:1.
15. The cell of claim 1 wherein the recombinant FAT-1 polypeptide
is encoded by a polynucleotide that comprises a sequence having at
least 70%s nucleotide sequence identity with the fat-1
polynucleotide sequence of SEQ ID NO:1.
16. The cell of claim 15 wherein the polynucleotide comprises a
sequence having at least 80% nucleotide sequence identity with the
fat-1 polynucleotide sequence of SEQ ID NO:1.
17. The cell of claim 15 wherein the polynucleotide comprises a
sequence having at least 90% nucleotide sequence identity with the
fat-1 polynucleotide sequence of SEQ ID NO:1.
18. The cell of claim 15 wherein the polynucleotide comprises a
sequence having 100% nucleotide sequence identity with the fat-1
polynucleotide sequence of SEQ ID NO:1.
19. The cell of claim 1 wherein the polypeptide is encoded by a
polynucleotide comprising a full-length native fat-1 protein-coding
region.
20. A cell of claim 1 of an organism selected from the group
consisting of a bacterium, a cyanobacterium, a phytoplankton, an
alga, a fungus, a plant, and an animal.
21. A lipid from the cell of claim 1.
22. The lipid of claim 21 wherein at least 25% of an .omega.-6
fatty acid of the cell having a carbon chain of at least 20 carbons
has been converted to a corresponding .omega.-3 fatty acid by the
FAT-1 polypeptide.
23. A transgenic plant comprising a fat-1 polynucleotide that is
expressible in at least a part of the plant.
24. The transgenic plant of claim 23 wherein the fat-1
polynucleotide is expressible in a seed of the plant.
25. A seed of the transgenic plant of claim 23.
26. A lipid from the transgenic plant of claim 23 that has a higher
proportion of .omega.-3 fatty acids than a control lipid obtained
from an otherwise similar plant lacking the fat-1
polynucleotide.
27. A method of desaturating an .omega.-6 fatty acid to a
corresponding .omega.-3 fatty acid comprising the steps of: (a)
providing a cell that comprises a recombinant FAT-1 polypeptide;
and (b) growing the cell under conditions under which the FAT-1
polypeptide desaturates an .omega.-6 fatty acid to produce a
corresponding .omega.-3 fatty acid.
28. The method of claim 27 wherein the .omega.-6 fatty acid is an
.omega.-6 fatty acid having a carbon chain of at least 18
carbons.
29. The method of claim 28 wherein the .omega.-6 fatty acid is a
20-carbon .omega.-6 fatty acid or a 22-carbon .omega.-6 fatty
acid.
30. The method of claim 27 wherein the .omega.-6 fatty acid
comprises a double bond at one or more positions selected from the
group consisting of .DELTA.4, .DELTA.5, .DELTA.6, .DELTA.7, and
.DELTA.8.
31. The method of claim 27 wherein the recombinant polypeptide has
at least 60% amino acid sequence identity with the FAT-1
polypeptide of SEQ ID NO:1.
32. The method of claim 27 wherein the recombinant polypeptide has
at least 70% amino acid sequence identity with the FAT-1
polypeptide of SEQ ID NO:1.
33. The method of claim 32 wherein the recombinant polypeptide has
at least 80% amino acid sequence identity with the FAT-1
polypeptide of SEQ ID NO:1.
34. The method of claim 32 wherein the recombinant polypeptide has
at least 90% amino acid sequence identity with the FAT-1
polypeptide of SEQ ID NO:1.
35. The method of claim 32 wherein the recombinant polypeptide has
only conservative amino acid substitutions to the FAT-1 polypeptide
of SEQ ID NO:1.
36. The method of claim 32 wherein the recombinant polypeptide has
100% amino acid sequence identity with the FAT-1 polypeptide of SEQ
ID NO:1.
37. The method of claim 27 wherein the FAT-1 polypeptide is encoded
by a recombinant polynucleotide that comprises a sequence having at
least 70% nucleotide sequence identity with the fat-1
polynucleotide sequence of SEQ ID NO:1.
38. The method of claim 37 wherein the polynucleotide comprises a
sequence having at least 80% nucleotide sequence identity with the
fat-1 polynucleotide sequence of SEQ ID NO:1.
39. The method of claim 37 wherein the polynucleotide comprises a
sequence having at least 90% nucleotide sequence identity with the
fat-1 polynucleotide sequence of SEQ ID NO:1.
40. The method of claim 37 wherein the polynucleotide comprises a
sequence having 100% nucleotide sequence identity with the fat-1
polynucleotide sequence of SEQ ID NO:1.
41. The method of claim 27 wherein the polypeptide is encoded by a
polynucleotide that comprises a full-length native fat-1
protein-coding region.
42. A method of claim 27 wherein the cell is a cell of an organism
selected from the group consisting of a bacterium, a
cyanobacterium, a phytoplankton, an alga, a fungus, a plant, and an
animal.
43. The method of claim 42 wherein the cell is a plant cell.
44. The method of claim 42 wherein the cell is a yeast cell.
45. A method of producing a lipid comprising an .omega.-3 fatty
acid comprising the steps of: (a) providing a lipid that comprises
an .omega.-6 fatty acid; and (b) desaturating at least some of the
.omega.-6 fatty acid to a corresponding .omega.-3 fatty acid with a
recombinant FAT-1 polypeptide.
46. The method of claim 45 wherein the lipid comprises an .omega.-6
fatty acid having a carbon chain of at least 20 carbons, the method
comprising desaturating at least 25% of the .omega.-6 fatty acid to
the corresponding .omega.-3 fatty acid.
47. The method of claim 45 comprising the step of expressing a
recombinant fat-1 polynucleotide in a cell comprising the lipid,
thereby producing the recombinant FAT-1 polypeptide in the cell.
Description
CROSS REFERENCE TO RELATED CASE
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/038,409, filed Feb. 18, 1997, which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] This invention relates to fatty acid metabolism, in
particular to fatty acid desaturases.
[0004] Polyunsaturated fatty acids are important as structural
components of membrane glycerolipids and as precursors of families
of signaling molecules including prostaglandins, thromboxanes, and
leukotrienes (Needleman et al., Annu. Rev. Biochem. 55:69-102,
1986; Smith and Borgeat, in Biochemistry of Lipids and Membranes,
eds. Vance and Vance, Benjamin/Cummings, Menlo Park, Calif., 1986,
pp. 325-360).
[0005] The principal fatty acid precursors of these signaling
molecules are arachidonic acid (.DELTA.5, 8, 11, 14-20:4),
providing an .omega.-6 substrate that is responsible for the major
synthesis of these compounds, and eicosapentanenoic acid (.DELTA.5,
8, 11, 14, 17-20:5), an .omega.-3 substrate that is responsible for
the parallel synthesis of many eicosanoids having an additional
double bond. An important class of enzymes involved in the
synthesis of polyunsaturated fatty acids is the fatty acid
desaturases, which catalyze the introduction of double bonds into
the hydrocarbon chain.
[0006] In vertebrates, desaturases are known to act at the
.DELTA.4, 5, 6, 8 and 9 positions (Holloway, In: The Enzymes, ed.
Boyer, Academic Press, New York, vol. 16, 1983, pp. 63-83). The
18:0-CoA .DELTA.9 desaturase from rat liver has been characterized
biochemically (Strittmatter et al., Proc. Natl. Acad. Sci. USA
71:4565-4569, 1974; Thiede et al., J. Biol. Chem. 260:14459-14463,
1985), and the corresponding gene has been cloned (Thiede et al.,
J. Biol. Chem. 261:13230-13235, 1986). However, the remaining four
enzymes have remained recalcitrant to purification and genes that
encode them have not been isolated. Based on available information,
and by analogy to the 18:0-CoA desaturase, it is likely that the
remaining four enzymes are integral membrane proteins that require
other membrane components (cytochrome b.sub.5 and NADH:cytochrome
b.sub.5 reductase) for activity (Strittmatter et al., Proc. Natl.
Acad. Sci. USA 71:4565-4569, 1974), and it is these features that
have limited progress in studying the biochemistry and molecular
genetics of these important synthetic reactions.
[0007] Biochemical studies of membrane-bound fatty acid desaturases
in plants have proven equally difficult, and only one enzyme has
been purified to homogeneity (Schmidt et al., Plant Mol. Biol.
26:631-642, 1994). Higher plants produce many different unsaturated
fatty acids (Hilditch and Williams, The Chemical Constituents of
Natural Fats, Chapman and Hall, London, 4th Ed., 1964), but in
membrane lipids the major locations for double bonds are at the
.DELTA.9, 12 and 15 (.omega.-3) positions of 18-carbon acyl chains
and the corresponding .DELTA.7, 10 and 13 (.omega.-3) positions of
16-carbon chains (Browse and Somerville, Ann. Rev. Plant Physiol.
Plant Mol. Biol. 42:467-5069, 1991).
SUMMARY OF THE INVENTION
[0008] According to one embodiment of the invention, a cell is
provided that includes a recombinant FAT-1 polypeptide that
desaturates an .omega.-6 fatty acid of the cell to a corresponding
.omega.-3 fatty acid. FAT-1 is capable of desaturating .omega.-6
fatty acids having carbon chains of at least 18 carbons (e.g.,
20-to 22-carbon fatty acids), and is significantly more efficient
than FAD3, for example, at desaturating .omega.-6 fatty acids
having carbon chains of 20 carbons or longer, producing lipids
having at least 25% of 20-carbon .omega.-6 fatty acids desaturated
to the corresponding .omega.-3 fatty acid. FAT-1 can desaturate
double bonds at positions .DELTA.4, .DELTA.5, .DELTA.6, .DELTA.7,
and .DELTA.8, for example. The expression of the FAT-1 polypeptide
in a cell permits the cell to have a greater proportion of the
.omega.-3 fatty acid than an otherwise similar cell lacking the
FAT-1 polypeptide, including cells from a wide variety of
organisms, such as bacteria, cyanobacteria, phytoplankton, algae,
fungi, plants, and animals.
[0009] According to another aspect of the invention, the
recombinant FAT-1 polypeptide has at least 60% amino acid sequence
identity with the FAT-1 polypeptide shown in FIG. 1 (SEQ ID NO:1
and 2). In preferred embodiments, the recombinant FAT-1 polypeptide
has only conservative amino acid substitutions to the FAT-1
polypeptide of FIG. 1.
[0010] According to another aspect of the invention, the
recombinant FAT-1 polypeptide is encoded by a polynucleotide that
includes a sequence having at least 70% nucleotide sequence
identity with the fat-1 polynucleotide sequence of FIG. 1. For
example, according to one embodiment, such a polynucleotide
includes a full-length native fat-1 protein-coding region, e.g.,
the protein-coding region of the fat-1 polynucleotide sequence of
FIG. 1.
[0011] According to another aspect of the invention, lipids are
provided that are produced from such cells.
[0012] According to another aspect of the invention, transgenic
plants are provided that include a fat-1 polynucleotide that is
expressible in at least a part of the plant, e.g., in seeds of the
plant. Also provided are seeds of such transgenic plants. Also
provided are lipids from such transgenic plants that have higher
proportions of .omega.-3 fatty acids than control lipids obtained
from otherwise similar plants lacking the fat-1 polynucleotide.
[0013] According to another aspect of the invention, related
methods of desaturating an .omega.-6 fatty acid to a corresponding
.omega.-3 fatty acid are provided. Such methods comprise the steps
of: (a) providing a cell that comprises a recombinant FAT-1
polypeptide; and (b) growing the cell under conditions under which
the FAT-1 polypeptide desaturates an .omega.-6 fatty acid to
produce a corresponding .omega.-3 fatty acid.
[0014] According to another aspect of the invention, related
methods of producing a lipid comprising an .omega.-3 fatty acid are
provided that include the steps of: (a) providing a lipid that
includes an .omega.-6 fatty acid; and (b) desaturating at least
some of the .omega.-6 fatty acid to a corresponding .omega.-3 fatty
acid with a recombinant FAT-1 polypeptide. For example, such a
method can be practiced by expressing a recombinant fat-1 nucleic
acid in a cell, thereby producing a recombinant FAT-1 polypeptide
in the cell.
[0015] The foregoing and other aspects of the invention will become
more apparent from the following detailed description and
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows the nucleotide sequence of the fat-1 cDNA and
the deduced amino-acid sequence of the FAT-1 polypeptide encoded by
the cDNA.
DETAILED DESCRIPTION OF THE INVENTION
[0017] We have cloned fat-1, an .omega.-3 fatty acyl desaturase
gene from Caenorhabditis elegans. When expressed in a wide range of
host cells, the polypeptide encoded by fat-1 catalyzes the
introduction of an .omega.-3 double bond into 18-, 20-, and
22-carbon fatty acids.
[0018] The C. elegans fat-1 gene encodes the first animal
representative of a class of glycerolipid desaturases that have
previously been characterized in plants and cyanobacteria. The
FAT-1 protein is an .omega.-3 fatty acyl desaturase that recognizes
a range of 18-, 20-, and 22-carbon .omega.-6 substrates. When
expressed in a wide range of host cells, FAT-1 catalyzes the
introduction of an .omega.-3 double bond into 18-, 20-, and
22-carbon fatty acids. The efficiency of FAT-1 in desaturating 20-
and 22-carbon substrates appears to be much greater than FAD3
desaturase of Arabidopsis, for example.
[0019] A recombinant .omega.-3 fatty acyl desaturase polypeptide,
e.g., a FAT-1 polypeptide, is useful for producing lipids having a
higher proportion of .omega.-3 fatty acids, whether by means of
recombinant expression in a cell or in an industrial processes
using purified FAT-1 polypeptide. Such lipids are useful as food
oils, as nutritional supplements, and as chemical feedstocks, for
example.
[0020] Definitions and Methods
[0021] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant art.
Definitions of common terms in molecular biology may also be found
in Rieger et al., Glossary of Genetics: Classical and Molecular,
5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V,
Oxford University Press: New York, 1994.
[0022] Nucleic Acids
[0023] "Polynucleotide".
[0024] A polynucleotide (or nucleic acid) sequence is a
naturally-occurring or chemically-synthesized DNA or RNA sequence.
A polynucleotide according to the invention may be single- or
double-stranded.
[0025] "Fat-1 Polynucleotide"; "Fat-1 Gene".
[0026] The terms "fat-1 polynucleotide" or "fat-1 gene" refer to a
native FAT-1-encoding polynucleotide or a fragment thereof, e.g., a
native C. elegans cDNA or genomic sequence or alleles, or fat-1
homologs from other species. The terms also encompass variant forms
of a native fat-1 polynucleotide sequence or fragment thereof as
discussed below, including polynucleotides that encodes a
polypeptide having FAT-1 biological activity.
[0027] Native fat-1 sequences can include 5'- and 3'-flanking
sequences or internal sequences operably linked to a native fat-1
polynucleotide sequence, including regulatory elements and/or
intron sequences.
[0028] "FAT-1 Biological Activity".
[0029] The term "FAT-1 biological activity" refers to a biological
activity characteristic of a native FAT-1 polypeptide.
[0030] "Native".
[0031] The term "native" refers to a naturally-occurring
("wild-type") polynucleotide or polypeptide.
[0032] "Homolog".
[0033] A "homolog" of fat-1 is a polynucleotide from a species
other than C. elegans that encodes a polypeptide that is
functionally similar to FAT-1 and that preferably has at least 60%
amino-acid sequence similarity, or more preferably, at least 60%
sequence identity, to FAT-1.
[0034] "Isolated".
[0035] An "isolated" polynucleotide is one that has been
substantially separated or purified away from other polynucleotide
sequences in the cell of the organism in which the polynucleotide
naturally occurs, i.e., other chromosomal and extrachromosomal DNA
and RNA, by conventional nucleic acid-purification methods. The
term also embraces recombinant polynucleotides and chemically
synthesized polynucleotides.
[0036] Fragments, Probes, and Primers.
[0037] A fragment of a fat-1 polynucleotide is a portion of a fat-1
polynucleotide that is less than full-length and comprises at least
a minimum length capable of hybridizing specifically with a native
fat-1 polynucleotide under stringent hybridization conditions. The
length of such a fragment is preferably at least 15 nucleotides,
more preferably at least 20 nucleotides, and most preferably at
least 30 nucleotides of a native fat-1 polynucleotide.
[0038] Polynucleotide probes and primers can be prepared based on a
native fat-1 polynucleotide. A "probe" is an isolated
polynucleotide to which is attached a conventional detectable label
or reporter molecule, e.g., a radioactive isotope, ligand,
chemiluminescent agent, or enzyme. A "primer" is an isolated
polynucleotide that can be annealed to a complementary target DNA
strand by nucleic acid hybridization to form a hybrid between the
primer and the target polynucleotide strand, then extended along
the target polynucleotide strand by a polymerase, e.g., a DNA
polymerase. Primer pairs can be used for amplification of a
polynucleotide sequence, e.g., by the polymerase chain reaction
(PCR) or other conventional amplification methods.
[0039] Probes and primers are generally 15 nucleotides or more in
length, preferably 20 nucleotides or more, more preferably 25
nucleotides, and most preferably 30 nucleotides or more. Such
probes and primers hybridize specifically to a native C. elegans
fat-1 polynucleotide under high stringency hybridization conditions
and hybridize specifically to a native fat-1 sequence of another
species under at least moderately stringent conditions. Preferably,
probes and primers according to the present invention have complete
sequence identity with the native C. elegans fat-1 sequence.
[0040] Methods for preparing and using probes and primers are
described, for example, in Molecular Cloning: A Laboratory Manual,
2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989 (hereinafter,
"Sambrook et al., 1989"); Current Protocols in Molecular Biology,
ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New
York, 1992 (with periodic updates) (hereinafter, "Ausubel et al.,
1992); and Innis et al., PCR Protocols: A Guide to Methods and
Applications, Academic Press: San Diego, 1990. PCR-primer pairs can
be derived from a known sequence, for example, by using computer
programs intended for that purpose such as Primer.TM. (Whitehead
Institute for Biomedical Research, Cambridge, Mass.).
[0041] Primers and probes based on the native fat-1 sequence
disclosed herein can be used to confirm (and, if necessary, to
correct) the disclosed fat-1 nucleotide sequence by conventional
methods, e.g., by-re-cloning and sequencing a fat-1 cDNA or genomic
sequence.
[0042] Nucleotide Sequence Identity.
[0043] Nucleotide sequence "identity" or "similarity" is a measure
of the degree to which two polynucleotide sequences have identical
nucleotide bases at corresponding positions in their sequence when
optimally aligned (with appropriate nucleotide insertions or
deletions). Preferably, a fat-nucleotide sequence as defined herein
has at least about 75% nucleotide sequence identity, preferably at
least about 80% identity, more preferably at least about 85%
identity, and most preferably at least about 90% identity with the
C. elegans fat-1 cDNA sequence (SEQ ID NO:1). Such a degree of
nucleotide sequence identity is considered "substantial" nucleotide
sequence identity. Sequence identity can be determined by comparing
the nucleotide sequences of two polynucleotides using sequence
analysis software such as the Sequence Analysis Software Package of
the Genetics Computer Group, University of Wisconsin Biotechnology
Center, Madison, Wis.
[0044] Alternatively, two polynucleotides are substantially similar
if they hybridize under stringent conditions, as defined below.
[0045] "Operably Linked".
[0046] A first nucleic-acid sequence is "operably" linked with a
second nucleic-acid sequence when the first nucleic-acid sequence
is placed in a functional relationship with the second nucleic-acid
sequence. For instance, a promoter is operably linked to a coding
sequence if the promoter affects the transcription or expression of
the coding sequence. Generally, operably linked DNA sequences are
contiguous and, where necessary to join two protein coding regions,
in reading frame.
[0047] "Recombinant".
[0048] A "recombinant" polynucleotide is made by an artificial
combination of two otherwise separated segments of sequence, e.g.,
by chemical synthesis or by the manipulation of isolated segments
of polynucleotides by genetic engineering techniques.
[0049] Techniques for nucleic-acid manipulation are well-known
(see, e.g., Sambrook et al., 1989, and Ausubel et al., 1992).
Methods for chemical synthesis of polynucleotides are discussed,
for example, in Beaucage and Carruthers, Tetra. Letts.
22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc.
103:3185, 1981. Chemical synthesis of polynucleotides can be
performed, for example, using commercial automated oligonucleotide
synthesizers.
[0050] Preparation of Recombinant or Chemically Synthesized
Polynucleotides; Vectors, Transformation, Host Cells.
[0051] Natural or synthetic polynucleotides according to the
present invention can be incorporated into recombinant nucleic-acid
constructs, typically DNA constructs, capable of being introduced
into, replicating in, and expressing a FAT-1 polypeptide in a host
cell.
[0052] For the practice of the present invention, conventional
compositions and methods for preparing and using vectors and host
cells are employed, as discussed, inter alia, in Sambrook et al.,
1989, or Ausubel et al., 1992.
[0053] A cell, tissue, organ, or organism into which has been
introduced a foreign polynucleotide, such as a recombinant vector,
is considered "transformed", "transfected", or "transgenic." A
"transgenic" or "transformed" cell or organism also includes
progeny of the cell or organism and progeny that carries the
transgene, including, for example, progeny of sexual crosses
between a transgenic parent and a non-transgenic parent that
exhibit an altered phenotype resulting from the presence of a fat-1
polynucleotide construct.
[0054] Such a construct preferably is a vector that includes a
replication system and sequences that are capable of transcription
and translation of a polypeptide-encoding sequence in a given host
cell. Expression vectors may include, for example, any well-known
origin of replication or autonomously replicating sequence (ARS),
expression control sequence, promoter, enhancer, secretion signal,
ribosome-binding site, RNA splice site, polyadenylation site,
transcriptional terminator sequence, mRNA stabilizing sequence,
etc., that is operable in a given host. Such DNA constructs are
prepared and introduced into a host cell(s) by conventional
methods.
[0055] Expression and cloning vectors preferably also include a
selectable or screenable marker appropriate for a given host cell
or organism. Typical selection genes encode proteins that, for
example (a) confer resistance to antibiotics or other toxic
substances, e.g. ampicillin, neomycin, methotrexate, etc.; (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media.
[0056] The vectors containing the polynucleotides of interest can
be introduced into a host cell by any well-known method, including
electroporation; transfection employing calcium chloride, rubidium
chloride calcium phosphate, DEAE-dextran, or other substances;
microprojectile bombardment; lipofection; infection (where the
vector is an infectious agent, such as T-DNA of Agrobacterium for
plant cell transformation or a retroviral genome for animal cell
transformation); etc.
[0057] The fat-1 gene is derived from C. elegans and, when
expressed in plants and yeast, the gene product is biologically
active. Therefore, it is expected that fat-1 can be successfully
expressed and that the expressed FAT-1 polypeptide will be active
in a wide variety of prokaryotic and eukaryotic hosts, including,
but not limited to: bacteria, including Gram negative bacteria such
as Escherichia coli and Gram-positive bacteria such as Bacillus
(e.g., B. subtilis), cyanobacteria, phytoplankton, algae, fungi
(including, but not limited to, yeast such as Saccharomyces
cerevisiae and filamentous fungi), plants (including monocots and
dicots), and animals (e.g., insect, avian, and mammalian species
and marine organisms such as Schizochytrium spp.).
[0058] If a host cell does not naturally produce a substrate for
FAT-1, one or more substrate molecules can be provided exogenously
to cells transformed with an expressible fat-1 polynucleotide, or
fat-1 can be co-expressed in cells together with one or more cloned
genes that encode polypeptides that can produce substrate compounds
from compounds available in such cells.
[0059] A recombinant fat-1 polynucleotide expression vector in a
cell can be used to produce a recombinant FAT-1 polypeptide that is
functional in the cell to desaturate an .omega.-6 fatty acid, which
is naturally produced by the cell or that is provided exogenously
to the cell, to a corresponding .omega.-3 fatty acid. In this way,
a cell can be produced that has a higher proportion of .omega.-3
fatty acids than an otherwise similar cell lacking the recombinant
FAT-1 polypeptide. Alternatively, for example, an extracted lipid
that includes an .omega.-6 fatty acid can be treated with a FAT-1
polypeptide to desaturate an .omega.-6 fatty acid to a
corresponding .omega.-3 fatty acid Preferably at least 10% of an
.omega.-6 fatty acid is desaturated to the corresponding .omega.-3
fatty acid, more preferably at least 20%, and most preferably at
least 50%.
[0060] Nucleic-acid Hybridization; "Stringent Conditions";
"Specific".
[0061] The nucleic-acid probes and primers of the present invention
hybridize under stringent conditions to a target DNA sequence,
e.g., to a native fat-1 polynucleotide.
[0062] The term "stringent conditions" is functionally defined with
regard to the hybridization of a nucleic-acid probe to a target
polynucleotide (i.e., to a particular nucleic-acid sequence of
interest) by the specific hybridization procedure discussed in
Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al.,
1989 at 9.47-9.52, 9.56-9.58; Kanehisa, Nucl. Acids Res.
12:203-213, 1984; and Wetmur and Davidson, J. Mol. Biol.
31:349-370, 1968.
[0063] Regarding the amplification of a target nucleic-acid
sequence (e.g., by PCR) using a particular amplification primer
pair, "stringent conditions" are conditions that permit the primer
pair to hybridize substantially only to the target nucleic-acid
sequence to which a primer having the corresponding wild-type
sequence (or its complement) would bind so as to produce a unique
amplification product.
[0064] For hybridization of a probe or primer to a polynucleotide
of another plant species in order to identify fat-1 homologs,
preferred hybridization and washing conditions are as discussed in
Sambrook et al., 1989 at 9.47-9.57, wherein "high stringency
hybridization conditions" include hybridization at 65.degree. C. in
a hybridization solution that includes 6.times.SSC and washing for
1 hour at 65.degree. C. in a wash solution that includes
0.5.times.SSC, 0.5% SDS. "Moderate stringency" conditions are
similar except that the temperature for the hybridization and
washing steps are performed at a lower temperature at which the
probe is specific for a target sequence, preferably at least
42.degree. C., more preferably at least 50.degree. C., more
preferably at 55.degree. C, and most preferably at least 60.degree.
C.
[0065] The term "specific for (a target sequence)" indicates that a
probe or primer hybridizes under given hybridization conditions
substantially only to the target sequence in a sample comprising
the target sequence. It is expected that hybridization of a C.
elegans fat-1 probe or primer to genomic DNA or cDNA of another
species will identify more than one hybridizing sequence in many
cases, including fat-1 homologs and other sequences having
substantial sequence identity with C. elegans fat-1, particularly
other desaturase genes.
[0066] Nucleic-acid Amplification.
[0067] As used herein, "amplified DNA" refers to the product of
nucleic-acid amplification of a target nucleic-acid sequence.
Nucleic-acid amplification can be accomplished by any of the
various nucleic-acid amplification methods known in the art,
including the polymerase chain reaction (PCR). A variety of
amplification methods are known in the art and are described, inter
alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR
Protocols: A Guide to Methods and Applications, ed. Innis et al.,
Academic Press, San Diego, 1990.
[0068] Nucleotide- and Amino-acid Sequence Variants.
[0069] Using the fat-1 nucleotide and amino-acid sequences
disclosed herein, those skilled in the art can create
polynucleotides and polypeptides that have minor sequence
variations from the corresponding native sequence.
[0070] "Variant" polynucleotides are polynucleotides containing
minor changes in a native fat-1 polynucleotide sequence, i.e.,
changes in which one or more nucleotides of a native fat-1
polynucleotide is deleted, added, and/or substituted, preferably
while substantially maintaining a biological activity of FAT-1.
Variant polynucleotides can be produced, for example, by standard
DNA mutagenesis techniques or by chemically synthesizing the
variant polynucleotide molecule or a portion thereof. Such variants
preferably do not change the reading frame of the protein-coding
region of the polynucleotide and preferably encode a polypeptide
having no change, only a minor reduction, or an increase in FAT-1
biological activity.
[0071] Amino-acid substitutions are preferably substitutions of
single amino-acid residues. Insertions are preferably of about 1 to
10 contiguous nucleotides and deletions are preferably of about 1
to 30 contiguous nucleotides. Insertions and deletions are
preferably insertions or deletions from an end of the
protein-coding or non-coding sequence and are preferably made in
adjacent base pairs. Substitutions, deletions, insertions or any
combination thereof can be combined to arrive at a final
construct.
[0072] Preferably, variant polynucleotides according to the present
invention are "silent" or "conservative" variants. "Silent"
variants are variants of a native fat-1 sequence or a homolog
thereof in which there has been a substitution of one or more base
pairs but no change in the amino-acid sequence of the polypeptide
encoded by the polynucleotide. "Conservative" variants are variants
of the native fat-1 polynucleotide or an allele or homolog thereof
in which at least one codon in the protein-coding region of the
polynucleotide has been changed, resulting in a conservative change
in one or more amino-acid residues of the polypeptide encoded by
the polynucleotide, i.e., an amino acid substitution. A number of
conservative amino acid substitutions are listed below. In
addition, one or more codons encoding cysteine residues can be
substituted for, resulting in a loss of a cysteine residue and
affecting disulfide linkages in the FAT-1 polypeptide.
1 TABLE 1 Original Residue Conservative Substitutions Ala ser Arg
lys Asn gln, his Asp glu Cys ser Gln asn Glu asp Gly pro His asn;
gln Ile leu, val Leu ile; val Lys arg; gln; glu Met leu; ile Phe
met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu
[0073] Substantial changes in function are made by selecting
substitutions that are less conservative than those listed above,
e.g., causing changes in: (a) the structure of the polypeptide
backbone in the area of the substitution; (b) the charge or
hydrophobicity of the polypeptide at the target site; or (c) the
bulk of an amino acid side chain. Substitutions generally expected
to produce the greatest changes in protein properties are those in
which: (a) a hydrophilic residue, e.g., seryl or threonyl, is
substituted for (or by) a hydrophobic residue, e.g., leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histadyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine.
[0074] Polypeptides
[0075] "FAT-1 Polypeptide".
[0076] The term "FAT-1 polypeptide" (or protein) refers to a
polypeptide encoded by a fat-1 polynucleotide, including alleles,
homologs, and variants of a native fat-1 polynucleotide. A FAT-1
polypeptide can be produced by the expression of a recombinant
fat-1 polynucleotide or can be chemically synthesized. Techniques
for chemical synthesis of polypeptides are described, for example,
in Merrifield, J. Amer. Chem. Soc. 85:2149-2156, 1963.
[0077] Polypeptide Sequence Identity and Similarity.
[0078] FAT-1 polypeptides encompassed by the present invention have
at least about 60% amino acid sequence similarity to the C. elegans
FAT-1 polypeptide (SEQ ID NO:2), more preferably at least about 70%
similarity, more preferably at least about 80% similarity, and most
preferably at least about 95% similarity. Even more preferable are
similar degrees of amino acid sequence identity. Such similarity
(or identity) is considered to be "substantial" similarity (or
identity), although more important than shared amino-acid sequence
similarity can be the common possession of characteristic
structural features and the retention of biological activity that
is characteristic of FAT-1.
[0079] Amino acid sequence "identity" (or "homology") is a measure
of the degree to which aligned amino acid sequences possess
identical amino acids at corresponding positions. Amino acid
sequence "similarity" is a measure of the degree to which aligned
amino acid sequences possess identical amino acids or conservative
amino acid substitutions at corresponding positions.
[0080] Amino acid sequence similarity and identity is typically
analyzed using sequence analysis software such as the Sequence
Analysis Software Package of the Genetics Computer Group,
University of Wisconsin Biotechnology Center, Madison, Wis.).
Polypeptide sequence analysis software matches homologous sequences
using measures of homology assigned to various substitutions,
deletions, substitutions, and other modifications.
[0081] "Isolated," "Purified," "Homogeneous" Polypeptides.
[0082] A polypeptide is "isolated" if it has been separated from
the cellular components (nucleic acids, lipids, carbohydrates, and
other polypeptides) that naturally accompany it. Such a polypeptide
can also be referred to as "pure" or "homogeneous" or
"substantially" pure or homogeneous. Thus; a polypeptide that is
chemically synthesized or recombinant (i.e., the product of the
expression of a recombinant polynucleotide, even if expressed in a
homologous cell type) is considered to be isolated. A monomeric
polypeptide is isolated when at least 60% by weight of a sample is
composed of the polypeptide, preferably 90% or more, more
preferably 95% or more, and most preferably more than 99%. Protein
purity or homogeneity is indicated, for example, by polyacrylamide
gel electrophoresis of a protein sample, followed by visualization
of a single polypeptide band upon staining the polyacrylamide gel;
high performance liquid chromatography; or other conventional
methods.
[0083] It is expected that purified FAT-1 polypeptide will be
useful for enzymatic conversion of .omega.-6 fatty acids to
corresponding .omega.-3 fatty acids under conditions (e.g.,
detergent, salt, pH, temperature) suitable for FAT-1 enzymatic
activity, for example, with FAT-1 polypeptide incorporated into
liposomes or free in solution.
[0084] Protein Purification.
[0085] The polypeptides of the present invention can be purified by
any of the means known in the art. Various methods of protein
purification are described, e.g., in Guide to Protein Purification,
ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990;
and Scopes, Protein Purification: Principles and Practice, Springer
Verlag, New York, 1982.
[0086] Variant and Modified Forms of FAT-1 Polypeptides.
[0087] Encompassed by the FAT-1 polypeptides of the present
invention are variant polypeptides in which there have been
substitutions, deletions, insertions or other modifications of a
native FAT-1 polypeptide. The variants substantially retain
structural characteristics and biological activities of a
corresponding native FAT-1 polypeptide and are preferably silent or
conservative substitutions of one or a small number of contiguous
amino acid residues.
[0088] A native FAT-1 polypeptide sequence can be modified by
conventional methods, e.g., by acetylation, carboxylation,
phosphorylation, glycosylation, ubiquitination, and labeling,
whether accomplished by in vivo or in vitro enzymatic treatment of
a FAT-1 polypeptide or by the synthesis of a FAT-1 polypeptide
using modified amino acids.
[0089] Labeling.
[0090] FAT-1 polypeptides can be labeled using conventional methods
and reagents. Typical labels include radioactive isotopes, ligands
or ligand receptors, fluorophores, chemiluminescent agents, and
enzymes. Methods for labeling and guidance in the choice of labels
appropriate for various purposes are discussed, e.g., in Sambrook
et al., 1989 and Ausubel et al., 1992.
[0091] Polypeptide Fragments.
[0092] The present invention also encompasses fragments of a FAT-1
polypeptide that lacks at least one residue of a native full-length
FAT-1 polypeptide. Preferably, such a fragment retains FAT-1
desaturase activity, possession of a characteristic functional
domain, or an immunological determinant characteristic of a native
FAT-1 polypeptide. Immunologically active fragments typically have
a minimum size of 7 to 17 or more amino acids. Fragments retaining
substantial desaturase activity are preferred, and can be obtained
by deleting one or more amino acids from the N-terminus or the
C-terminus of the polypeptide, for example.
[0093] Fusion Polypeptides.
[0094] The present invention also provides fusion polypeptides
including, for example, heterologous fusion polypeptides in which a
FAT-1 polypeptide sequence is joined to a well-known fusion
partner. Such fusion polypeptides can exhibit biological properties
(such as substrate or ligand binding, enzymatic activity, antigenic
determinants, etc.) derived from each of the fused sequences.
Fusion polypeptides are preferably made by the expression of
recombinant polynucleotides that include sequences for each of the
fusion partners joined in frame.
[0095] Polypeptide Sequence Determination.
[0096] The sequence of a polypeptide of the present invention is
determined by any conventional method.
[0097] Antibodies
[0098] The present invention also encompasses polyclonal and/or
monoclonal antibodies capable of specifically binding to a FAT-1
polypeptide and/or fragments thereof. Such antibodies are raised
against a FAT-1 polypeptide or fragment thereof and are capable of
distinguishing a FAT-1 polypeptide from other polypeptides, i.e.,
they are FAT-1-specific.
[0099] For the preparation and use of antibodies according to the
present invention, including various immunoassay techniques and
applications, see, e.g., Goding, Monoclonal Antibodies: Principles
and Practice, 2d ed, Academic Press, New York, 1986; and Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1988. FAT-1-specific
antibodies are useful, for example in: purifying a FAT-1
polypeptide from a biological sample, such as a host cell
expressing a recombinant FAT-1 polypeptide; in cloning a fat-1
allele or homolog from an expression library; as antibody probes
for protein blots and immunoassays; etc.
[0100] Such antibodies can be labeled by any of a variety of
conventional methods. Suitable labels include, but are not limited
to, radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent agents, chemiluminescent agents, magnetic particles,
etc.
[0101] Obtaining Alleles and Homologs of fat-1
[0102] Based upon the availability of the fat-1 cDNA sequence
disclosed herein, genomic clones and alleles and homologs of the
disclosed fat-1 sequence can be obtained by conventional methods,
e.g., by screening a cDNA or genomic library with a probe that
specifically hybridizes to a native fat-1 polynucleotide under at
least moderately stringent conditions, by PCR or another
amplification method using a primer or primers that specifically
hybridize to a native fat-1 polynucleotide under at least
moderately stringent conditions, or by identification of fat-1
alleles or homologs in an expression library using FAT-1-specific
antibodies. The identity of fat-1 alleles or homologs can be
confirmed by application of an exogenous .omega.-6 fatty acid
substrate to cells (e.g., bacterial, yeast, or plant cells) in
which the cloned fat-1 gene is expressed, followed by gas
chromatography analysis to determine whether the cloned gene
converts the .omega.-6 substrate to the corresponding .omega.-3
fatty acid, for example.
[0103] Probes and primers based on the fat-1 sequence disclosed
herein can also be used to obtain closely related genes having
substantial nucleotide sequence identity to fat-1, e.g., other
desaturase genes, including other .omega.-3 fatty acyl desaturase
genes, by conventional methods.
[0104] Plant Transformation and Regeneration
[0105] Nucleic-acid constructs that include a fat-1 polynucleotide
are useful for producing transgenic plants that are capable of
efficiently converting .omega.-6 fatty acids, including fatty acids
having a carbon chain of greater than 18 carbons (e.g., 20, 22, or
24 carbons), to the corresponding .omega.-3 fatty acids, thus
producing plant cells and lipids obtained therefrom that have an
altered fatty acid profile. Such plants include plants that are
commonly grown for oil production, including, but not limited to,
rapeseed, corn, canola, safflower, soybean, sunflower, peanut,
etc.
[0106] A number of vectors suitable for stable transfection of
plant cells or for the establishment of transgenic plants have been
described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory
Manual, 1985, supp. 1987); Weissbach and Weissbach, Methods for
Plant Molecular Biology, Academic Press, 1989; and Gelvin et al.,
Plant Molecular Biology Manual, Kluwer Academic Publishers,
1990.
[0107] Examples of constitutive plant promoters useful for
expressing fat-1 polynucleotides include but are not limited to:
the cauliflower mosaic virus (CaMV) 35S promoter, which confers
constitutive, high-level expression in most plant tissues (see,
e.g., Odel et al., Nature 313:810, 1985), including monocots (see,
e.g., Dekeyser et al., Plant Cell 2:591, 1990; Terada and
Shimamoto, Mol. Gen. Genet. 220:389, 1990); the nopaline synthase
promoter (An et al., Plant Physiol. 88:547, 1988) and the octopine
synthase promoter (Fromm et al., Plant Cell 1:977, 1989).
[0108] A variety of plant gene promoters that are regulated in
response to environmental, hormonal, chemical, and/or developmental
signals, also can be used for expression of a fat-1 polynucleotide
in plant cells. Seed-specific promoters are preferred, but such
regulated promoters may also include promoters regulated by: (1)
heat (Callis et al., Plant Physiol. 88:965, 1988); (2) light (e.g.,
pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell 1:471, 1989;
maize rbcS promoter, Schaffner and Sheen, Plant Cell 3:997, 1991;
or chlorophyll a/b-binding protein promoter, Simpson et al., EMBO
J. 4:2723, 1985); (3) hormones, such as abscisic acid (Marcotte et
al., Plant Cell 1:969, 1989); (4) wounding (e.g., wunI, Siebertz et
al., Plant Cell 1:961, 1989); or (5) chemicals such as methyl
jasmonate, salicylic acid, or safeners.
[0109] In addition, vectors for plant expression can include
additional regulatory sequences from the 3'-untranslated region of
plant genes (Thornburg et al., Proc. Natl. Acad. Sci. USA 84:744
(1987); An et al., Plant Cell 1:115 (1989), e.g., a 3' terminator
region to increase mRNA stability of the mRNA, such as the PI-II
terminator region of potato or the octopine or nopaline synthase 3'
terminator regions.
[0110] Useful dominant selectable marker genes include genes
encoding antibiotic resistance genes (e.g., resistance to
hygromycin, kanamycin, bleomycin, G418, streptomycin or
spectinomycin); and herbicide resistance genes (e.g.,
phosphinothricin acetyltransferase).
[0111] Any well-known method can be employed for plant cell
transformation, culture, and regeneration in the practice of the
present invention with regard to a particular plant species.
Conventional methods for introduction of foreign DNA into plant
cells include, but are not limited to: (1) Agrobacterium-mediated
transformation (Lichtenstein and Fuller, in: Genetic Engineering,
Vol 6, Rigby, ed., London, Academic Press, 1987; and Lichtenstein
and Draper, in: DNA Cloning, Vol II, Glover, ed., Oxford, IRI
Press, 1985); (2) particle delivery (see, e.g., Gordon-Kamm et al.,
Plant Cell 2:603, 1990; or BioRad Technical Bulletin 1687); (3)
microinjection (see, e.g., Green et al., Plant Tissue and Cell
Culture, Academic Press, New York, 1987); (4) polyethylene glycol
(PEG) procedures (see, e.g., Draper et al., Plant Cell Physiol.
23:451, 1982); Zhang and Wu, Theor. Appl. Genet. 76:835, 1988); (5)
liposome-mediated DNA uptake (see, e.g., Freeman et al., Plant Cell
Physiol. 25:1353, 1984); (6) electroporation (see, e.g., Fromm et
al., Nature 319:791, 1986); and (7) vortexing methods (see, e.g.,
Kindle, Proc. Natl. Acad. Sci. USA 87:1228, 1990).
[0112] The term "plant" encompasses any higher plant and progeny
thereof, including monocots (e.g., lily, corn, rice, wheat, barley,
etc.), dicots (e.g., tomato, potato, soybean, cotton, tobacco,
etc.), and includes parts of plants, including reproductive units
of a plant (e.g., seeds, fruit, flowers, etc.)
[0113] A "reproductive unit" of a plant is any totipotent part or
tissue of the plant from which one can obtain a progeny of the
plant, including, for example, seeds, cuttings, tubers, buds,
bulbs, somatic embryos, cultured cells (e.g., callus or suspension
cultures), etc.
[0114] Transformation of Algal Cells
[0115] Nucleic-acid constructs that include a fat-1 polynucleotide
are also useful for recombinant expression in algal cells,
including plankton, that are capable of efficiently converting
.omega.-6 fatty acids to the corresponding .omega.-3 fatty acids.
Manipulation of algal cells in general is discussed, for example,
in Dunahay et al., Appl. Biochem. Biotechnol. 57/58:223-231, 1996
and Dunahay et al., J. Phycol. 31:1004-1012, 1995.
[0116] The invention will be better understood by reference to the
following Examples, which are intended to merely illustrate the
best mode now known for practicing the invention. The scope of the
invention is not to be considered limited thereto.
EXAMPLES
Example 1
[0117] The Cloning and Sequencing of a cDNA Encoding fat-1, an
Animal Omega-3 Desaturase
[0118] In Arabidopsis there are seven membrane desaturases. The
biochemistry and function of these membrane desaturases has been
facilitated by the availability of mutants with altered fatty acid
compositions (Browse et al., Science 227:763-765, 1985) (Browse and
Somerville, In: Arabidopsis, Cold Spring Harbor Press, New York,
1994, pp. 881-912) Genes encoding desaturases in Arabidopsis have
been cloned (Arondel et al., Science 258:1353-1355, 1992; Yadav et
al., Plant Physiol. 103:467-476, 1993; Yadav et al. in:
Biochemistry and Molecular Biology of Membrane and Storage Lipids
of Plants, ed. Murala and Somerville, American Society of Plant
Physiologists, 1993, pp. 60-66; Okuley et al., Plant Cell
6:147-158, 1994). All the plant membrane-bound desaturases use
complex glycerolipids rather than acyl-CoAs as substrates. This
finding, together with data indicating that the .DELTA.5 desaturase
from rat liver acts on glycerolipids (Pugh and Kates, J. Biol.
Chem. 252:68-73, 1977), suggested the possibility that the majority
of animal desaturases also catalyze glycerolipid-linked
desaturation. However, there has been little progress in
characterizing other animal desaturases. In animals, fatty acid
desaturases catalyze key reactions in the synthesis of arachidonic
acid and other polyunsaturated fatty acids.
[0119] C. elegans elaborates a wide range of polyunsaturated fatty
acids, including arachidonic (20:4, .omega.-6) and eicosapentaenoic
acids (20:5, .omega.3) from very simple precursors available in the
diet of the organism (Hutzell and Krusberg, Comp. Biochem. Physiol.
73B:517-520, 1982; Satouchi et al., Lipids 28:837-840, 1993). The
.omega.-3 fatty acids (.DELTA.9, 12, 15-18:3; .DELTA.8, 11, 14,
17-20:4 and .DELTA.5, 8, 11, 14, 17-20:5) account for 17% of the
total fatty acids in C. elegans (Hutzell and Krusberg, Comp.
Biochem. Physiol. 73B:517-520, 1982) and 20:5 .omega.-3 is the
major fatty acid in phosphatidylcholine from this organism
(Satouchi et al., Lipids 28:837-840, 1993). These lipids are
produced even when the worms are grown exclusively on E. coli,
which provides only saturated and monounsaturated fatty acids
(Satouchi et al., Lipids 28:837-840, 1993) Evidently, C. elegans
must contain all the enzymes required for the synthesis of these
highly unsaturated acyl groups.
[0120] We searched the National Center for Biotechnology
Information's (NCBI) peptide sequence data base using a BLAST
server with the peptide sequences of the Arabidopsis thaliana FAD2,
FAD6 and FAD7 fatty acid desaturases as queries. (The GenBank
accession numbers for the corresponding Arabidopsis cDNAs are
L2629G, U09503 and L22931, respectively.) The highest scoring C.
elegans expressed sequence tag (EST) clones were NCBI-5443,
NCBI-5881 and NCBI-5049. (The corresponding GenBank accession
numbers were Z14935, M88884 and Z14543, respectively.) An alignment
of cDNA sequences revealed a common identity of 301 bp among all
three clones, indicating that the three ESTs originated from a
single gene. The greatest amount of sequence data (486 bp) was
available from EST clone NCBI-5881. This clone was requested from
its origin at the C. elegans genome Sequencing Center, Washington
University School of Medicine, St. Louis, Mo., with its original
source identifier (CEL10e11). Upon receipt of CEL10e11, its
identity was confirmed by partial sequencing.
[0121] In order to obtain a full-length cDNA corresponding to
CEL10e11, the cDNA insert was released by double digestion with
HindIII and SacI, gel purified, and labeled with .sup.32P-dCTP
using a random priming kit (Promega, Madison, Wis.). The denatured
probe was used to screen a mixed stage C. elegans cDNA lambda phage
(Uni-Zap XR) library (Stratagene, La Jolla, Calif.). Nucleic acid
hybridizations and high stringency washes were performed as
described (Amasino, Anal. Biochem. 152:304-307, 1986). Thirteen
hybridizing plaques were visualized by autoradiography. The longest
eight clones were all approximately 1.4 kb in length as judged by
agarose gel electrophoresis. Positive clones were isolated and
excised from the phage vector according to the manufacturer's
protocol to yield pBluescript.TM. plasmids.
[0122] The plasmid clone with the longest insert, pCES, was fully
sequenced in both directions and found to contain a 1,410 bp cDNA
insert. Sequence analysis was carried out using the programs
available in the GCG package (Devereaux et al., Nucl. Acids Res.
12:387-395, 1984) using default settings for parameters unless
otherwise indicated. This sequence was deposited in GenBank under
Accession Number L41807.
[0123] The nucleotide sequence of the fat-1 cDNA (1391 nt) and the
deduced FAT-1 amino-acid sequence are shown in FIG. 1 (and SEQ ID
NO:1 and 2).
[0124] The DNA sequence corresponding to the open reading frame of
the fat-1 cDNA was used to search the database of the C. elegans
genome sequencing project using the BLAST server (Waterston and
Sulston, Proc. Natl. Acad. Sci. USA 92:10836-10840, 1995). No
homologous sequence was found (highest BLAST score: 145; p=0.034),
indicating that the fat-1 genomic sequence had not yet been
included in this project.
[0125] The cDNA insert in pCE8 contained an open reading frame that
would be expected to encode a protein of 402 amino acids, with a
molecular mass of 46.4 kD. Sequence comparisons were made using the
programs of the Genetics Computer Group Package (Devereaux et al.,
Nucl. Acids Res. 12:387-395, 1984). The predicted amino acid
sequence of the protein showed several regions of common homology
with the predicted sequences of the FAD2 and FAD3 desaturases of
Arabidopsis. Alignment with FAD2 revealed 35% sequence identity and
61% similarity (i.e., including both identical amino acid residues
and conservative substitutions). Alignment with FAD3 indicated 32%
sequence identity and 54% similarity. The FAD2 and FAD3 genes are
known to encode enzymes that desaturate oleate (FAD2) or linoleate
(FAD3) esterified to phosphatidylcholine of the endoplasmic
reticulum (Arondel et al., Science 258:1353-1355, 1992; okuley et
al., Plant Cell 6:147-158, 1994). Within a tripartite alignment of
the three sequences are 69 residues common to all three sequences,
including eight histidines (amino acids 123, 127, 159, 162 163,
324, 327 and 328 in the C. elegans sequence) whose presence and
locations are highly conserved among all the membrane desaturases
(Okuley et al., Plant Cell 6:147-158, 1994). These findings
strongly indicate that the gene represented by the pCE8 cDNA
encodes a fatty acid desaturase or a related enzyme function. We
have designated this gene fat-1 (Fatty Acid Metabolism-1).
[0126] It is somewhat surprising that the C. elegans gene shows a
similar amino acid sequence homology to each of the two Arabidopsis
desaturases, especially in view of the fact that the FAD2 and FAD3
sequences are relatively divergent, with only 37% common amino acid
identity (Okuley et al., Plant Cell 6:147-158, 1994). From these
comparisons alone, it is difficult to deduce whether the fat-1 gene
is likely to represent a .DELTA.12 desaturase (like FAD2), an
.omega.-3 desaturase (like FAD3), or a more distantly related
enzyme.
[0127] In contrast with the identity found between the deduced FAT
1 polypeptide sequence and FAD2 and FAD3 of Arabidopsis, there was
only 17-23% homology to the yeast and rat genes that encode
18:0-CoA desaturases, only slightly above the level from
comparisons with entirely unrelated genes. For this reason, it is
unlikely that a database search using an 18:0-CoA desaturase as the
query could have identified the fat-1 sequence.
[0128] The predicated protein sequence of the fat-1 gene product
includes the three histidine-rich sequences that are highly
conserved among all the membrane-bound fatty acid desaturases and
that are believed to be the residues that coordinate the diiron-oxo
structure at the active site of these enzymes (Shanklin et al.,
Biochemistry 33:12787-12794, 1994; Stukey et al., J. Biol. Chem.
265:20144-20149, 1990). Furthermore, two long stretches (>40
residues each) of hydrophobic residues are present (80 to 124 and
229 to 284). The length of these stretches and their positions
relative to the conserved histidine sequences are similar to other
desaturases. Therefore, the FAT-1 protein could conform with the
model proposed by Stukey et al. (Stukey et al., J. Biol. Chem.
265:20144-20149, 1990), in which the bulk of the protein is exposed
on the cytosolic face of the endoplasmic reticulum, while two
membrane-traversing loops (each comprised of two membrane-spanning,
.alpha.-helical segments) lock the protein into the bilayer. In
common with many, though not all, of the proposed endoplasmic
reticulum desaturases, the FAT-1 protein contains a
carboxy-terminal motif (KAKAK) that conforms to a consensus
retention signal for transmembrane proteins in the endoplasmic
reticulum (Jackson et al., EMBO J. 9:3153-312, 1990).
[0129] These features are consistent with FAT-1 being a member of
the membrane-bound desaturase/hydroxylase family of diiron-oxo
proteins (Shanklin et al., Biochemistry 33:12787-12794, 1994).
However, the FAT-1 sequence shows equal homology to both the
.DELTA.12 glycerolipid desaturase encoded by FAD2 and the .omega.-3
glycerolipid desaturase encoded by FAD3.
Example 2
[0130] Expression of fat-1 in Arabidopsis and Characterization of
its Function
[0131] To determine which class of reaction is catalyzed by FAT-1
and to explore the substrate chainlength and regiochemical
specificities of the enzyme it was necessary to use heterologous
expression of a fat-1 cDNA in a host that contained potential fatty
acid substrates. Both Escherichia coli and Saccharomyces
cerevisiae, which are two common laboratory hosts for heterologous
expression, possess a very limited range of endogenous desaturation
activities and hence fatty acid compositions. By contrast, plants
possess both a wider range of desaturase activities and fatty acids
that are potential substrates for a desaturase of unknown function.
In addition, the lipid and fatty acid metabolism of plants,
especially Arabidopsis, have been well characterized. These
features make Arabidopsis a more attractive host for transgenic
studies of putative eukaryotic fatty acid desaturases than either
E. coli or S. cerevisiae.
[0132] In higher plants, desaturases have been characterized from
two cellular compartments. Enzymes localized to the chloroplast (or
plastid) use soluble ferredoxin as the electron donor for the
reaction (Schmidt et al., Plant Mol. Biol. 26:631-642, 1994; Heinz,
in Lipid Metabolism in Plants, ed., CRC Press, Boca Raton, Fla.,
1993, pp. 33-89). Enzymes localized to the endoplasmic reticulum
(including the FAD2 and FAD3 gene products) are similar to known
yeast and animal desaturases inasmuch as they rely on cytochrome
b.sub.5 and cytochrome b.sub.5 reductase to supply electrons from
NAD(P)H (Heinz, in Lipid Metabolism in Plants, ed., CRC Press, Boca
Raton, Fla., 1993, pp. 33-89). Mutants deficient in each of the
major desaturases are available in Arabidopsis (Browse and
Somerville, in Arabidopsis, ed. Cold Spring Harbor Press, New York,
pp. 881-912, 1994). Genes that encode the 18:0-CoA desaturases from
yeast and mammals have been expressed in plants and shown to alter
the fatty acid compositions of the plant tissues (Polashok et al.,
Plant Physiol. 100:894-901, 1992; Grayburn et al., Bio/Technology
10:675-677, 1992). These considerations indicated that Arabidopsis
is a suitable heterologous system to study the expression and
function of the fat-1 gene.
[0133] In order to produce a fat-1 gene construct for plant
expression, the cauliflower mosaic virus (CaMV) 35S
promoter/nopaline synthase terminator cassette of Baulcombe et al.
(Baulcombe et al., Nature 321:446-449, 1986) was cloned into the
XbaI/EcoRI sites of pBIN400 (Spychalla and Bevan, In: Plant Tissue
Culture Manual: Fundamentals and Applications, ed. Lindsay, Kluwer
Academic Publishers, Dordrecht, Vol. B11, 1993, pp. 1-18) to make
the binary transformation vector pBIN420. The cDNA insert of pCE8
was released with a EcoRI/KpnI double digest, end-filled with
Klenow fragment, and blunt-ligated into the SmaI site of pBIN420 to
make pBIN420-CE8. These vectors contain the NPTII gene within their
T-DNA, thus conferring kanamycin resistance to transgenic
plants.
[0134] The Columbia ecotype of the wild-type line of Arabidopsis
thaliana (L.) Heynh. was used for plant transformation. The binary
vector pBIN420-CE8 was introduced into the Agrobacterium strain
PC2760 by the freeze-thaw method (Holsters et al., Mol. Gen. Genet.
163:181-187, 1978). Agrobacterium-mediated transformation was
accomplished with the in planta vacuum-infiltration method (Bouchez
et al., C. R. Acad. Sci. Paris 316:1188-1193, 1993). Primary
generation transformed seeds were selected on plates containing
Murashige and Skoog basal salts (4.3 g/L), 1% (w/v) sucrose, 0.8%
(w/v) Bacto-Agar, 200 mg/L carbenicillin, and 50 mg/L kanamycin,
and was adjusted to pH 5.8 with KOH. In vitro roots were grown from
sterilized seeds placed on vertical plates at 23.degree. C. under
continuous illumination (50-100 micromol quanta m.sup.-2s.sup.-1).
The media for in vitro roots contained Gamborg B5 salts (3.1 g/L),
2% (w/v) glucose, and 0.2% Phytagel.TM. (Sigma, St. Louis, Mo.),
and was adjusted to pH 5.8 with KOH.
[0135] Five individual transformants were obtained and allowed to
set seed. Lines #9.7 and #10.5 were selected for further analysis
by Southern and Northern blotting using the HindIII/SacI fragment
of pCE8 as a probe on the RNA and DNA blots. Probe labeling,
hybridizations and washings were as described above for cDNA
library screens.
[0136] For Southern blots, genomic DNA was isolated from lines #9.7
and #10.5 according to the method of Dellaporta et al. (Dellaporta
et al., Plant Mol. Biol. Rep. 1:19-21, 1983), restricted with
BamHI, separated by agarose gel-electrophoresis, and alkaline
blotted to nylon membranes. The Southern blot confirmed the
presence of at least one copy of the transgene in line #9.7 and at
least two copies in line #10.5.
[0137] For Northern blots, total RNA was extracted from leaves
according to the method of Verwoerd et al. (Verwoerd et al., Nucl.
Acids Res. 17:2362, 1989). Twenty-five micrograms of total RNA was
separated on 1.2% agarose-formaldehyde gels and blot transferred to
nylon membranes. Northern blots of total RNA from plants of the two
lines and wild-type Arabidopsis showed that the appropriate fat-1
transcript accumulated in both transgenic lines. Plants from line
#9:7 consistently produced higher transcript levels than line
#10.5.
[0138] Characterization of Arabidopsis lipid mutants has indicated
that lesions in the fad2 and fad3 genes are partly masked in leaf
tissue by action of the chloroplast desaturases (encoded by FAD6,
FAD7 and FAD8). For this reason, a first attempt to determine the
function of the fat-1 gene product was made by analyzing the
overall fatty-acid composition of root tissues from wild-type and
fat-1 transgenic plants. The data in Table 2 show very large
increases in the proportion of 18:3 in both transgenic lines
compared with wild-type Arabidopsis. These increases were
accompanied by concomitant decreases in the proportion of 18:2 but
no significant changes in the levels of any other fatty acid. The
alterations in root fatty-acid composition induced by expression of
the C. elegans fat-1 gene are comparable to those observed by
overexpression of the plant FAD3 gene (Arondel et al., Science
258:1353-1355, 1992). The FAT1 protein is thus operating as an
efficient .omega.-3 desaturase in Arabidopsis.
2TABLE 2 Composition (mol %) of total fatty acids from in vitro
grown roots of the wild type (WT) and two transgenic lines (#9.7
and #10.5) of Arabidopsis expressing a C. elegans fat-1 cDNA.sup.1
Genotype 16:0 16:1(c) 18:0 18:1 18:2 18:3 WT 16.8a 1.2a 1.4a 22.7a
39.3a 18.0b #9.7 17.8a 1.2a 2.2a 21.7a 22.7b 33.7a #10.5 17.5a 1.0a
1.6a 20.9a 23.7b 34.5a .sup.1Values are means of quadruplicate
measurements. Values for each fatty acid with the same letter do
not differ significantly (p < 0.01).
[0139] In untreated Arabidopsis, linolenic acid (.DELTA.9, 12,
15-18:3) is the only significant product resulting from fat-1
expression. However, C. elegans contains a wider range of PUFAs
than does Arabidopsis. Three .omega.-3 fatty acids are present in
the membrane lipids of the worm, of which linolenic acid is the
least abundant (0.15% of total fatty acids). The 20-carbon fatty
acids .DELTA.8, 11, 14, 17-eicosatetraenoic acid (an isomer of
arachidonic acid) and .DELTA.5, 8, 11, 14, 17-eicosapentaenoic acid
(the expected product of .omega.-3 desaturation of arachidonic
acid) account for 7.7% and 8.7% of total fatty acids, respectively
(Hutzell and Krusberg, Comp. Biochem. Physiol. 73B:517-520,
1982).
[0140] Exogenous fatty acids applied to Arabidopsis leaves as
sodium soaps are readily taken up and incorporated into membrane
glycerolipids to levels that correspond to 2-5% of the total leaf
lipids (McConn and Browse, Plant Cell 8:403-416, 1996). To test
whether the fat-1-encoded desaturase is likely to be involved in
synthesis of the 20-carbon .omega.-3 fatty acids in C. elegans,
wild-type and transgenic Arabidopsis plants were sprayed once a day
for twenty days with solutions of the sodium salts of arachidonic
acid (.DELTA.5, 8, 11, 14-20:4) or a homogamma linolenic acid
(.DELTA.8, 11, 14-20:3).
[0141] For exogenous fatty-acid treatments, plants were grown in a
growth chamber at 20.degree. C. on a 12 h day/night cycle.
Fatty-acid treatments began when the plant rosettes reached
approximately 2 cm in diameter. Sodium soaps of homogamma-linolenic
acid (.DELTA.8, 11, 14-20:3) and arachidonic acid (.DELTA.5, 8, 11,
14-20:4) (NuCheck Prep, Elysian, Minn.) were made to a 0.1% aqueous
solution and frozen in 5 mL aliquots. Plants were sprayed daily at
the beginning of the dark period using a perfume atomizer. Groups
of fifteen plants were sprayed with 5 mL of soap solution for 20
consecutive days.
[0142] Methods for extraction and separation of lipids, and for the
preparation of fatty acid methyl esters have been described
previously (Miquel and Browse, J. Biol. Chem. 267:1502-1509, 1992).
Analysis of fatty acid methyl esters by gas chromatography was
carried out using a 15 m.times.0.53 mm Supelcowax column (Supelco,
Bellefonte, Pa.) with flame ionization detection. The initial
column temperature of 160.degree. C. was held for 1 min, then
raised at 20.degree. C./min to 190.degree. C., followed by a ramp
of 5.degree. C./min to 230.degree. C. The final temperature was
held for 5 min. When wild-type and fat-1 transgenic plants were
sprayed with exogenous fatty acids, the peaks for the .omega.-6
substrates, .DELTA.8, 11, 14-20:3, .DELTA.5, 8, 11, 14-20:4 and of
the .omega.-3 desaturation products .DELTA.8, 11, 14, 17-20:4 and
.DELTA.5, 8, 11, 14, 17-20:5 were identified based on their
coelution with authentic standards (NuCheck Prep, Elysian, Minn.)
and on the results of gas chromatography-mass spectrometry (GC-MS)
analysis. For this analysis, fatty acid methyl esters derived from
phosphatidylcholine were separated on a 30 m.times.0.2 mm AT1000
column (Alltech Assoc., Deerfield, Ill.) in a HP6890 Instrument
(Hewlett-Packard, Avondale, Pa.). Oven temperature at injection was
50.degree. C. and this was increased at 5.degree. C./min to
230.degree. C., then held at 230.degree. C. for 10 min. Criterion
for identification of .DELTA.5, 8, 11, 14, 17-20:5 in
phosphatidylcholine from fat1 transgenic plants were: (1) the
identification of a mass peak at m/z=316, which corresponds to the
expected molecular ion, and (2) a retention time (36.11 min) and
fragmentation pattern identical to those of the authentic .DELTA.5,
8, 11, 14, 17-20:5 standard. No commercial standard was available
for .DELTA.8, 11, 14, 17-20:4. A fatty acid methyl ester present in
fat-1 transgenic plants sprayed with .DELTA.8, 11, 14-20:3, but not
in wild-type control plants, had a retention time of 35.74 min
during GC-MS. This compound showed a mass peak at m/z=318 (the
expected molecular ion for 20:4) and a fragmentation pattern very
similar to that of the authentic .DELTA.5, 8, 11, 14-20:4 standard.
The retention time of .DELTA.5, 8, 11, 14-20:4 was 35.04 min for
both the authentic standard and for the methyl esters recovered
from plants sprayed with soaps of this isomer. Therefore, it was
concluded that the new compound detected only in fat-1 transgenic
plants sprayed with .DELTA.8, 11, 14-20:3 was an isomer of 20:4 and
most probably .DELTA.8, 11, 14, 17-20:4.
[0143] Analyses of total leaf lipids indicated that the exogenously
supplied fatty acids were incorporated at levels of 1-3% of the
total fatty acids. There was extensive incorporation into
phosphatidylcholine, which is the major lipid of the endoplasmic
reticulum and the major substrate for the plant 18:1 and 18:2
desaturases (Miquel and Browse, J. Biol. Chem. 267:1502-1509, 1992;
Browse et al., J. Biol. Chem. 268:16345-16351, 1993). In wild-type
leaves, the peak corresponding to .DELTA.5, 8, 11, 14-20:4 or
.DELTA.8, 11, 14-20:3 accounted for approximately 3-5% of the total
fatty acids in phosphatidylcholine, but there was no detectable
conversion of either of these fatty acids to their .omega.-3
unsaturated derivatives. By contrast, in leaves from plants
expressing the fat-1 cDNA, the peaks corresponding to the
exogenously-supplied .omega.-6 fatty acids were substantially
replaced by peaks that correspond to the expected .omega.-3
desaturated products, .DELTA.5, 8, 11, 14, 17-20:5 and .DELTA.8,
11, 14, 17-20:4. Thus, in contrast to the Arabidopsis FAD3 gene
product, the C. elegans FAT-1 protein is a desaturase that acts on
a range of .omega.-6 fatty acid substrates. These results
demonstrate that the fat-1 gene encodes an .omega.-3 desaturase
that is able to carry out the final step in the synthesis of all
these fatty acids.
Example 3
[0144] Relative Efficiencies of the FAT-1 and FAD-3 Desaturases
[0145] A fat-1 cDNA confers to Arabidopsis plants the ability to
desaturate 20:3, .omega.-6 and 20:4, .omega.-6 fatty acyl groups to
the corresponding .omega.-3 products (.DELTA.8, 11, 14, 17-20:4 and
.DELTA.5, 8, 11, 14, 17-20:5, respectively). The absence of
detectable levels of these .omega.-3 fatty acids from untransformed
Arabidopsis tissues suggests that the endogenous plant .omega.-3
desaturases (the FAD3, FAD7 and FAD8 enzymes in Arabidopsis) have
little or no ability to desaturate 20-carbon substrates. However,
the fat-1 cDNA is highly expressed in the transgenic plant line
#9.7.
[0146] To more accurately compare the relative efficiencies of the
FAT-1 and FAD3 desaturases, we used a transgenic Arabidopsis line
(wild-type:pTiDES3) in which the FAD3 gene is overexpressed to a
high degree (Arondel et al., Science 258:1353-1355, 1992).
Proportions of 18:2 and 18:3 in the root fatty acid composition
produced in this line are altered to a slightly greater degree than
in line #9.7, indicating that the FAD3-overexpressing line contains
a somewhat higher activity for .omega.-3 desaturation of 18:2 fatty
acyl groups. However, when plants of the wild-type:pTiDES3 line
were supplied with exogenous 20:4, .omega.-6 fatty acids using the
protocol described above, less than 25% of this compound was
converted to 20:5, .omega.-3 as judged by fatty acid analysis of
phosphatidylcholine purified from leaf tissue of the sprayed
plants. The low extent of conversion confirms that the enzyme
encoded by the fat-1 cDNA is considerably more efficient than the
plant FAD3 enzyme when 20:4, .omega.-6, is the substrate for
.omega.-3 desaturation.
[0147] The ability of Arabidopsis plants to take up exogenous fatty
acids provided us with a means to extend the biochemical
characterization of the FAT-1 desaturase by showing that all the
18- and 20-carbon .omega.-6 fatty acids normally present in C.
elegans are recognized as its substrates, as well as 22:5,
.omega.-6 (i.e., .DELTA.4, 7, 10, 13, 16-22:5). The FAD2 and FAD3
desaturases are known to use membrane glycerolipids, not acyl-CoAs,
as substrates (Miquel and Browse, J. Biol. Chem. 267:1502-1509,
1992; Browse et al., J. Biol. Chem. 268:16345-16351, 1993). The
high efficiency with which the FAT-1 enzyme desaturates the 18:2 of
Arabidopsis membrane lipids and the high homology of FAT-1 to FAD2
and FAD3 strongly suggest that FAT-1 is also a glycerolipid
desaturase. In this respect the enzyme is similar to the .DELTA.5
desaturase activity described in rat liver (Pugh and Kates, J.
Biol. Chem. 252:68-73, 1977). Other desaturases required for the
synthesis of arachidonic acid in mammals may also use membrane
phospholipids as their substrates.
Example 4
[0148] fat-1 Transgenic Arabidopsis Plants Desaturate .DELTA.4, 7,
10, 13, 16-22:5 to .DELTA.4, 7, 10, 13, 16, 19-22:6
[0149] The 22-carbon fatty acids docosapentaenoic acid (22:5,
.omega.-6) and docosahexaenoic acid (22:6, .omega.-3) also have
important dietary and pharmaceutical uses. For many applications,
22:6, .omega.-3 is the more desirable product. Most sources of
22-carbon highly unsaturated fatty acids contain both 22:5,
.omega.-6 and 22:6, .omega.-3. We therefore investigated whether
FAT-1 could desaturate 22:5, .omega.-6 to the .omega.-3
product.
[0150] For this purpose, plants of the transgenic Arabidopsis line
#9.7, which express FAT-1, were grown together with control
wild-type plants at 24.degree. C. with continuous illumination
under fluorescent lights (150 .mu.mol quanta/m.sup.2/s). Sets of 15
leaves from 20-day-old wild-type or fat-1 transgenic plants were
harvested and placed in 2-inch diameter petri dishes. To each petri
dish was added either 4 mL of an aqueous solution containing 1%
(v/v) dimethylsulfoxide and 0.025% (wt/v) of the potassium soap of
22:5 .omega.-6 fatty acid or 4 mL of a similar solution lacking the
22:5 .omega.-6 soap. Each dish was covered with a single layer of
absorbent tissue to ensure good contact between the solution and
the leaves, closed, covered with aluminum foil, and incubated in
the dark for four hours. After the solution was removed, the leaves
were rinsed several times with distilled water, then covered with 4
mL of water and incubated under fluorescent lights (150 .mu.mol
quanta/m.sup.2/s) for an additional 24 hours before lipid
extraction.
[0151] Methods for extraction and separation of lipid classes and
for the preparation of fatty acid methyl esters have been described
previously (Miquel and Browse, J. Biol. Chem. 267:1502-1509, 1992).
Analysis of fatty acid methyl esters by gas chromatography was
carried out using a 15 m.times.0.5 mm Supelcowax column (Supelco,
Bellefonte, Pa.) with flame ionization detection. The initial
column temperature of 160.degree. C. was held for 0.5 min, then
raised at 20.degree. C./min to 190.degree. C. and thereafter at
5.degree. C./min to 215.degree. C. This final temperature was held
for 10 min.
[0152] The 22:5, .omega.-6 fatty acid was prepared from lipids of
the marine organism Schizochytrium. Schizochytrium lipids (100 mg)
were dissolved in 1 mL tetrahydrofuran and converted to fatty acid
methyl esters as described (Miquel and Browse, J. Biol. Chem.
267:1502-1509, 1992). The 22:5 methyl ester was separated from
other components by chromatography on silica gel G plates that had
been dipped in a solution of 5% AgNO.sub.3+0.01% rhodamine B in
acetonitrile using a solvent containing hexane:diethyl ether 40:60
(v/v). Fatty acid methyl ester bands were visualized under
ultraviolet light and the 22:5 (second band from the bottom of each
plate) was scraped into a screw-cap tube. Water (4 mL), methanol
(10 mL) and chloroform (10 mL) were added to the silica gel. The
mixture was filtered through glass wool and the silica gel rinsed
with 5 mL of chloroform:methanol:water (1:1:0.1, v/v/v). The
combined filtrate was separated into two phases by addition of 4 mL
H.sub.2O. The 22:5-methyl ester was recovered with the lower phase,
reduced to dryness under a stream of nitrogen, redissolved in
hexane, and stored under argon at -20.degree. C. To prepare the
sodium soap of 22:5, a sample of the hexane solution was dried-in a
screw-cap tube under a stream of nitrogen, redissolved in 0.3 mL of
1 M KOH in 95% aqueous methanol, sealed under argon, and heated to
80.degree. C. for 90 minutes. After cooling, the preparation was
diluted with 2 mL of water and extracted with 2 mL of hexane to
remove non-saponifiable lipids. After removal of the hexane phase,
the aqueous solution was titrated to pH 10 using 0.1 M HCl. Fatty
acid methyl esters from a sample of this solution were analyzed by
gas chromatography to confirm the identity of the 22:5 potassium
soap and to calculate the concentration of the soap.
[0153] There was extensive incorporation of the exogenous 22:5,
.omega.-6 fatty acid into phosphatidylcholine and
phosphatidylethanolamine, which are the major phospholipids of the
endoplasmic reticulum and other extrachloroplast membranes of plant
cells. Phosphatidylcholine is the major lipid substrate for the
plant 18:1 and 18:2 desaturases (Miquel and Browse, J. Biol. Chem.
278:1502-1509, 1992; Browse et al., J. Biol. Chem. 268:16345-16351,
1993). In wild-type leaves, the peak corresponding to .DELTA.4, 7,
10, 13, 16-22:5 accounted for approximately 2-4% of the total fatty
acids in phosphatidylcholine, but there was no detectable
conversion of this fatty acid to the .omega.-3 unsaturated
.DELTA.4, 7, 10, 13, 16, 19-22:6. By contrast, in leaves of plants
expressing the fat-1 cDNA, the peak corresponding to 22:5,
.omega.-6 was substantially replaced by a peak corresponding to the
expected .omega.-3 unsaturated product, .DELTA.4, 7, 10, 13, 16,
19-22:6. Data for three independent experiments are shown in Table
3. The identity of the 22:6, .omega.-3 product was confirmed by
GC-MS as described above to show that the retention time, molecular
ion peak (m/z=342), and fragmentation pattern of the fatty acid
isolated from fat-1 transgenic plants corresponded to those of
genuine .DELTA.4, 7, 10, 13, 16, 19-22:6.
[0154] These results demonstrate that the FAT-1 protein, expressed
in a heterologous host from a fat-1 cDNA, efficiently converts
.DELTA.4, 7, 10, 13, 16-22:5 to .DELTA.4, 7, 10, 13, 16,
19-22:6.
[0155] We also supplied 22:5, .omega.-6 (0.025% w/v as the
potassium soap in aqueous solution containing 1% v/v
dimethylsulfoxide) to live Caenorhabditis elegans growing in liquid
culture. The C. elegans incorporated the exogenous fatty acid into
phospholipids and converted it to 22:6, .omega.-3.
3TABLE 3 Metabolism of 22:5, .omega.-6 fatty acid in wild-type
Arabidopsis and transgenic Arabidopsis of line #9.7 expressing a
fat-1 cDNA. Results are the amounts of 22:5, .omega.-6 and 22:6,
.omega.-3 in phosphatidylcholine expressed as a percentage of the
total fatty acids in this lipid. 22:5 22:6 Total % conversion*
Experiment 1 wild-type water control 0.00 0.00 0.00 -- fat-1 water
control 0.00 0.00 0.00 -- wild-type +22:5 3.25 0.00 3.25 0 fat-1
+22:5 1.35 1.82 3.17 57 Experiment 2 wild-type +22:5 2.67 0.00 2.67
0 fat-1 +22:5 0.57 2.31 2.88 80 Experiment 3 wild-type +22:5 2.09
0.00 2.09 0 fat-1 0.36 1.67 2.03 82 *Percent conversion is
calculated as 22:6/(22:5 + 22:6).
Example 5
[0156] Expression of FAT-1 in Yeast
[0157] A fat-1 cDNA was incorporated into the pYX232 yeast
expression vector (Novagen Inc., 597 Science Dr., Madison, Wis.
53711) in a sense orientation at the multicloning site (to create
plasmid pYX232:fat-1) so that it could be expressed in yeast
(Saccharomyces cerevisiae) cells under control of the
triosephosphate isomerase promoter. The fat-1 vector construct was
transformed into yeast cells (strain YRP685), in which an .omega.-6
fatty acyl substrate, .DELTA.9, 12-18:2, was available. Gas
chromatography analysis of yeast cells derived from this
transformation experiment contained approximately 11 of their total
fatty acids as 18:3 compared with less than 0.5% in control cells
that did not contain pYX232:fat-1. This increase in accumulation of
18:3 indicates that the fat-1 cDNA encodes a product that is able
to act as an .omega.-3 fatty acid desaturase in yeast. One can
readily increase the level of .omega.-3 desaturation in yeast cells
using the same fat-1 coding sequence but employing, for example,
different combinations of well-known promoters and yeast
strains.
[0158] This invention has been detailed both by example and by
direct description. It should be apparent that one having ordinary
skill in the relevant art would be able to surmise equivalents to
the invention as described in the claims which follow but which
would be within the spirit of the foregoing description. Those
equivalents are to be included within the scope of this invention.
Sequence CWU 1
1
* * * * *