Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1997-06-03
2001-10-23
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S281000, C435S419000, C536S023600
Reexamination Certificate
active
06307128
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to fatty acid elongase complexes and nucleic acids encoding elongase proteins. More particularly, the invention relates to nucleic acids encoding &bgr;-keto acyl synthase proteins that are effective for producing very long chain fatty acids, polypeptides produced from such nucleic acids and transgenic plants expressing such nucleic acids.
BACKGROUND OF THE INVENTION
Plants are known to synthesize very long chain fatty acids (VLCFAs). VLCFAs are saturated or unsaturated monocarboxylic acids with an unbranched even-numbered carbon chain that is greater than 18 carbons in length. Many VLCFAs are 20-32 carbons in length, but VLCFAs can be up to 60 carbons in length. Important VLCFAs include erucic acid (22:1, i.e., a 22 carbon chain with one double bond), nervonic acid (24:1), behenic acid (22:0), and arachidic acid (20:0).
Plant seeds accumulate mostly 16- and 18-carbon fatty acids. VLCFAs are not desirable in edible oils. Oilseeds of the Crucifereae (e.g., rapeseed) and a few other plants, however, accumulate C20 and C22 fatty acids (FAs). Although plant breeders have developed rapeseed lines that have low levels of VLCFAs for edible oil purposes, even lower levels would be desirable. On the other hand, vegetable oils having elevated levels of VLCFAs are desirable for certain industrial uses, including uses as lubricants, fuels and as a feedstock for plastics, pharmaceuticals and cosmetics.
The biosynthesis of saturated fatty acids up to an 18-carbon chain occurs in the chloroplast. C2 units from acyl thioesters are linked sequentially, beginning with the condensation of acetyl Coenzyme A (CoA) and malonyl acyl carrier protein (ACP) to form a C4 acyl fatty acid. This condensation reaction is catalyzed by a &bgr;-ketoacyl synthase III (KASIII). &bgr;-ketoacyl moieties are also referred to as 3-ketoacyl moieties.
The enzyme &bgr;-ketoacyl synthase I (KASI) is involved in the addition of C2 groups to form the C6 to C16 saturated fatty acids. KASI catalyzes the stepwise condensation of a fatty acyl moiety (C4 to C14) with malonyl-ACP to produce a 3-ketoacyl-ACP product that is 2 carbons longer than the substrate. The last condensation reaction in the chloroplast, converting C16 to C18, is catalyzed by &bgr;-ketoacyl synthase II (KASII).
Each elongation cycle involves three additional enzymatic steps in addition to the condensation reaction as discussed above. Briefly, the &bgr;-ketoacyl condensation product is reduced to &bgr;-hydroxyacyl-ACP, dehydrated to the enoyl-ACP, and finally reduced to a fully reduced acyl-ACP. The fully reduced fatty acyl-ACP reaction product then serves as the substrate for the next cycle of elongation.
The C18 saturated fatty acid (stearic acid, 18:0) can be transported out of the chloroplast and converted to the monounsaturate C18:1 (oleic acid), and the polyunsaturates C18:2 (linoleic acid) and C18:3 (&agr;-linolenic acid). C18:0 and C18:1 can also be elongated outside the chloroplast to form VLCFAs. The formation of VLCFAs involves the sequential condensation of two carbon groups from malonyl CoA with a C18:0 or C18:1 fatty acid substrate. Elongation of fatty acids longer than 18 carbons depends on the activity of a fatty acid elongase complex to carry out four separate enzyme reactions similar to those described above for fatty acid synthesis in the chloroplast. Fehling, Biochem. Biophys. Acta 1082:239-246 (1991). In plants, elongase complexes are distinct from fatty acid synthases since elongases are extraplastidial and membrane bound.
Mutations have been identified in an Arabidopsis gene associated with fatty acid elongation. This gene, designated the FAE1 gene, is involved in the condensation step of an elongation cycle. See, WO 96/13582, incorporated herein by reference. Plants carrying a mutation in FAE1 have significant decreases in the levels of VLCFAs in seeds. Genes associated with wax biosynthesis in jojoba have also been cloned and sequenced (WO 95/15387, incorporated herein by reference).
Very long chain fatty acids are key components of many biologically important compounds in animals, plants, and microorganisms. For example, in animals, the VLCFA arachidonic acid is a precursor to many prostaglandins. In plants VLCFAs are major constituents of triacylglycerols in many seed oils, are essential precursors for cuticular wax production, and are utilized in the synthesis of glycosylceramides, an important component of the plasma membrane.
Obtaining detailed information on the biochemistry of KAS enzymes has been hampered by the difficulties encountered when purifying membrane bound enzymes. Although elongase activities have been partially purified from a number of sources, or studied using cell fractions, the elucidation of the biochemistry of elongase complexes has been hampered by the complexity of the membrane fractions used as the enzyme source. For example, until recently, it was unclear as to whether plant elongase complexes were composed of a multifunctional polypeptide similar to the FAS found in animals and yeast, or if the complexes existed as discrete and dissociable enzymes similar to the FAS of plants and bacteria. Partial purification of an elongase KAS, immunoblot identification of the hydroxy acyl dehydrase, and the recent cloning of a KAS gene (FAEL) suggest that the enzyme activities of elongase complexes exist on individual enzymes.
SUMMARY OF THE INVENTION
The invention disclosed herein relates to an isolated polynucleotide selected from one of the following: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; an RNA analog of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15; and a polynucleotide having a nucleic acid sequence complementary to one of the above. The polynucleotide can also be a nucleic acid fragment of one of the above sequences that is at least 15 nucleotides in length and that hybridizes under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.
Also disclosed herein is an isolated polypeptide that has an amino acid sequence substantially identical to one of the following: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14. Also disclosed are isolated polynucleotides encoding polypeptides substantially identical in their amino acid sequence to: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.
The invention also relates to a transgenic plant containing a nucleic acid construct. The nucleic acid construct comprises a polynucleotide described above. The construct further comprises a regulatory element operably linked to the polynucleotide. The regulatory element may a tissue-specific promoter, for example, an epidermal cell-specific promoter or a seed-specific promoter. The regulatory element may be operably linked to the polynucleotide in sense or antisense orientation. The plant has altered levels of very long chain fatty acids in tissues where the polynucleotide is expressed, compared to a parental plant lacking the nucleic acid construct.
A method is disclosed for altering the levels of very long chain fatty acids in a plant. The method comprises the steps of creating a nucleic acid construct and introducing the construct into the plant. The construct includes a polynucleotide selected from one of the following: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; an RNA analog of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15; and a polynucleotide having a nucleic acid sequence complementary to one of the above. The polynucleotide can also be a nucleic acid fragment of one of the above that is at least 15 nucleotides in length and that hybridizes under stringent conditions to genomic DNA encoding the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14. The polynucleotide is effective for altering the levels of very long chain fatty acids i
Jaworski Jan G.
Post-Beittenmiller Martha Ann
Todd James
Fish & Richardson P.C. P.A.
McElwain Elizabeth F.
Miami University
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