Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-02-18
2001-02-27
McElwain, Elizabeth F. (Department: 1638)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S419000, C435S440000, C435S252300, C435S468000, C800S281000
Reexamination Certificate
active
06194167
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to fatty acid metabolism, in particular to fatty acid desaturases.
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).
The principal fatty acid precursors of these signaling molecules are arachidonic acid (&Dgr;5,8,11, 14-20:4), providing an &ohgr;-6 substrate that is responsible for the major synthesis of these compounds, and eicosapentanenoic acid (&Dgr;5,8,11,14,17-20:5), an &ohgr;-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.
In vertebrates, desaturases are known to act at the &Dgr;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 &Dgr;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
5
and NADH:cytochrome b
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.
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 &Dgr;9, 12 and 15 (&ohgr;-3) positions of 18-carbon acyl chains and the corresponding &Dgr;7, 10 and 13 (&ohgr;-3) positions of 16-carbon chains (Browse and Somerville,
Ann. Rev. Plant Physiol. Plant Mol. Biol.
42:467-5069, 1991).
SUMMARY OF THE INVENTION
According to one embodiment of the invention, a cell is provided that includes a recombinant FAT-1 polypeptide that desaturates an &ohgr;-6 fatty acid of the cell to a corresponding &ohgr;-3 fatty acid. FAT-1 is capable of desaturating &ohgr;-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 &ohgr;-6 fatty acids having carbon chains of 20 carbons or longer, producing lipids having at least 25% of 20-carbon &ohgr;-6 fatty acids desaturated to the corresponding &ohgr;-3 fatty acid. FAT-1 can desaturate double bonds at positions &Dgr;4, &Dgr;5, &Dgr;6, &Dgr;7, and &Dgr;8, for example. The expression of the FAT-1 polypeptide in a cell permits the cell to have a greater proportion of the &ohgr;-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.
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
.
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
.
According to another aspect of the invention, lipids are provided that are produced from such cells.
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 &ohgr;-3 fatty acids than control lipids obtained from otherwise similar plants lacking the fat-1 polynucleotide.
According to another aspect of the invention, related methods of desaturating an &ohgr;-6 fatty acid to a corresponding &ohgr;-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 &ohgr;-6 fatty acid to produce a corresponding &ohgr;-3 fatty acid.
According to another aspect of the invention, related methods of producing a lipid comprising an &ohgr;-3 fatty acid are provided that include the steps of: (a) providing a lipid that includes an &ohgr;-6 fatty acid; and (b) desaturating at least some of the &ohgr;-6 fatty acid to a corresponding &ohgr;-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.
The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanying drawings.
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Arondel et al., “Map-Based Cloning of a Gene Controlling Omega-3 Fatty Acid Desaturation in Arabidopsis, ”Science 258:1353-1355 (1992).
Browse, Glycerolipid Synthesis: Biochemistry and RegulationAnnu. Rev. Plant Physiol. Plant Mol. Biol. 42:467-506 (1991).
Browse et al., in Arabidopsis, Meyerowitz et al. (ed.), Cold Spring Harbor Laboratory Press, New York, NY (1994).
Browse et al., “Mutants of Arabidopsis Deficient in the Synthesis of &agr;-Linolenate,”J. Biol. Chem. 268:16345-16351 (1993.
Grayburn et al., “Fatty Acid Alteration by a &Dgr;9 Desaturase in Transgenic Tobacco Tissue,”Bio/Tech. 10:675-677 (1992).
Jackson et al., “Identification of a Consensus Motif for Retention of Transmembrane Proteins in the Endoplasmic Reticulum,”EMBO J. 9:3153-3162 (1990).
McConn et al., “The Critical Requirement for Linolenic Acid is Pollen Development, Not Photosynthesis, in an Arbidopsis Mutant,”Plant Cell 8:403-416 (1996).
Miquel et al., “Arabidopsis Mutants Deficient in Poly-unsaturated Fatty Acid Synthesis,”J. Biol. Chem. 267:1502-1509 (1992).
Okuley et al., “Arabidopsis FAD2 Gene Encodes the Enzyme that is Essential for Polyunsaturated Lipid Synthesis,”Plant Cell 6:147-158 (1994).
Polashock et al., “Expression of the Yeast &Dgr;-9 Fatty Acid Desaturase inNicotiana tabacum, ” Plant Physiol. 100:894-902 (1992).
Shanklin et al., “Eight Histidine Residues are Catalytically Essential in a Membrane-Associa
Browse John A.
Spychalla James P.
Klarquist Sparkman Campbell & Leigh & Whinston, LLP
McElwain Elizabeth F.
Washington State University Research Foundation
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