HK1085615B

HK1085615B - Fatty acid desaturases from fungi

Info

Publication number: HK1085615B
Application number: HK06105720.0A
Authority: HK
Inventors: Virginia M. Ursin; Toni Voelker; Byron Froman
Original assignee: Monsanto Technology Llc
Priority date: 2002-05-22
Filing date: 2003-05-21
Publication date: 2011-12-16

Description

Fatty acid dehydrogenases from fungi

Background

This application claims priority from U.S. provisional patent application No. 60/382,391 filed on day 5/22/2002 and U.S. provisional patent application No. 60/453,125 filed on day 3/7/2003. The disclosure of each of the above applications is specifically incorporated by reference into this application in its entirety.

1. Field of the invention

The present invention relates generally to dehydrogenases that modulate the number and position of double bonds in long chain polyunsaturated fatty acids (LC-PUFA's), and methods and compositions for their use. In particular, the invention relates to the use of dehydrogenases for improving the properties of fatty acids and nucleic acids which have been identified in fungi and which code for these dehydrogenases.

2. Description of the related Art

In most organisms, the major products of fatty acid biosynthesis are 16-and 18-carbon compounds. The relative ratios of chain lengths of these fatty acids to the degree of unsaturation vary widely from species to species. For example, mammals produce primarily saturated and mono-saturated fatty acids, whereas most higher plants produce fatty acids containing one, two or three double bonds, the latter two including polyunsaturated fatty acids (PUFA's).

The two major classes of PUFA's are omega-3 fatty acids (also represented by "n-3" fatty acids), exemplified by eicosapentaenoic acid (EPA, 20:4, n-3); and omega-6 fatty acids (also represented by "n-6" fatty acids), exemplified by arachidonic acid (ARA, 20:4, n-6). PUFAs are important components of the cytoplasmic membrane and adipose tissue, where they are present in the form of phospholipids and triglycerides, respectively. PUFAs are essential for the normal development of mammals, particularly the development of the infant brain and the formation and repair of tissues.

Fatty acids can treat several diseases. Supplementation with PUFAs has been shown to reduce the rate of restenosis following angioplasty. The beneficial effects of certain edible omega-3 fatty acids on cardiovascular disease and rheumatoid arthritis have also been well reported in the literature (Simopoulos, 1997; James et al, 2000). In addition, PUFAs have been suggested for the treatment of asthma and psoriasis. There is evidence that PUFAs may be involved in calcium metabolism, suggesting that PUFAs may be useful in the treatment or prevention of osteoporosis and kidney or urinary tract stones. The main evidence of health benefits comes from long chain omega-3 fats, EPA and DHA in fish and fish oils. Based on these evidences, Health agencies and nutritionists in Canada (scientific review Committee, 1990, Nutrition Recommendations, Minister of national Health and Welfare, Canada, Ottowa), Europe (de Decker et al, 1998), The United kingdom (The British Nutrition Foundation, 1992, unsetatedfat-acids-Nutrition and physiologial design: The report of The British Nutrition's Task Force, Chapman and Hall, London) and The United states (Simopoulos et al, 1999) have suggested increasing The amounts of these PUFAs in The diet.

PUFAs can also be used to treat diabetes (U.S. Pat.No.4,826, 877; Horrobin et al, 1993). It has been demonstrated that fatty acid metabolism and composition are altered in diabetic animals. This suggests that these changes are associated with long-term complications from some forms of diabetes, including retinopathy, neuropathy, nephropathy, and damage to the reproductive system. Cerasus serrulata oil containing GLA has been shown to be able to prevent and treat diabetic nerve damage.

PUFAs, such as linoleic acid (LA, 18:2,. DELTA.9, 12) and alpha-linoleic acid (ALA, 18:3,. DELTA.9, 12, 15), are considered essential fatty acids in the diet because mammals lack the ability to synthesize these acids. However, mammals have the ability to metabolize the ingested LA and ALA into the n-6 and n-3 forms of long chain polyunsaturated fatty acids (LC-PUFAs). These LC-PUFA's are important cellular components that confer membrane fluidity and functionality as precursors to the bioactive eicosanoids that regulate normal physiological functions, such as prostaglandins, prostacyclins, and leukotrienes.

In mammals, the rate-limiting step in the formation of LC-PUFA is Δ 6 dehydrogenation, in which LA is converted to γ -linoleic acid (GLA, 18:3, Δ 6, 9, 12) and ALA is converted to SDA (18:4, Δ 6, 9,12, 15). A number of physiological and pathological conditions have been shown to inhibit this metabolic step and ultimately the production of LC-PUFA. However, bypassing Δ 6 dehydrogenation by dietary supplementation with EPA or DHA may be effective in alleviating many pathological conditions associated with low PUFA levels. However, as set forth in more detail below, the current use of sources of PUFAs is unsatisfactory for a variety of reasons. The need for a reliable and economical source of PUFA's has led to interest in alternative sources of PUFA's.

Important long-chain PUFAs mainly comprise docosahexaenoic acid (DHA, 22:6, n-3) and EPA, which are mainly found in different types of fish oils, and arachidonic acid (ARA, 20:4, n-6), which is found in filamentous fungi. For DHA, a number of sources exist for commercial production including a variety of marine organisms, oil and egg yolk fractions of cold water marine fish. Sources for commercial production of SDA include Trichodesma and Echium genera. However, commercial production of PUFAs from natural sources has several disadvantages. Natural sources of PUFAs, such as animals and plants, often have a very high composition of different types of oils. For example, the oil obtained from Echinnm seeds, except SDA, contains almost a comparable level of the omega-6 fatty acid GLA. The oils obtained from these sources therefore require further purification to isolate the target PUFAs or to produce oils enriched in one or more PUFAs.

PUFAs of natural origin also suffer from uncontrollable fluctuations in availability. The fish population may experience natural fluctuations or may be depleted by over-fishing. Furthermore, even though there is a great deal of evidence of their therapeutic efficacy, recommendations for omega-3 fatty acid diets have not drawn attention. Fish oils have unpleasant tastes and odors that cannot be economically removed from the target product, making these products unacceptable as food additives. Animal oils, particularly fish oils, can cause the accumulation of environmental pollutants. Food may be supplemented with fish oil, but again, this addition is problematic due to cost and worldwide fish population attenuation. There is also a problem in that it is difficult to consume and ingest whole fish. Nevertheless, if health information that increases the amount of food consumed by fish is accepted by society, meeting the needs of fish is likely to be a problem. Furthermore, the problem is that the continued growth of this industry depends heavily on the wild fish population used for aquaculture (Naylor et al, 2000).

Other natural limitations have prompted the search for a new method for producing omega-3 fatty acids. Climate and disease can cause fluctuations in yield depending on fish and plant resources. Farmlands for alternate oil-producing crop growth are facing competition from a continuing expansion of the population and a corresponding increase in the demand for food production on existing arable land. Crops do produce PUFAs, such as borage, but are not amenable to commercial production and are not well suited for single planting. Planting such crops is not economically competitive with more profitable, easier to plant crops. The cost of large scale fermentation of organisms such as Mortierella is also expensive. Natural animal tissue contains low amounts of ARA and is difficult to process. Microorganisms such as Porphyidium and Mortierella are difficult to culture on a commercial scale.

Many enzymes are involved in PUFA biosynthesis. LA (18:2, Δ 9, 12) is produced from oleic acid (OA, 18:1, Δ 9) by Δ 12-dehydrogenase, while ALA (18:3) is produced from LA by Δ 15-desaturase. SDA (18:4, Δ 6, 9,12, 15) and GLA (18:3, Δ 6, 9, 12) are produced from LA and ALA by Δ 6 desaturases. However, as noted above, mammals cannot dehydrogenate at positions further than the Δ 9 position, and thus cannot convert oleic acid to LA. Similarly, ALA cannot be synthesized by mammals. Other eukaryotes, including fungi and plants, contain enzymes that dehydrogenate at the carbon 12 and carbon 15 positions. Thus most of the polyunsaturated fatty acids in animals are from the diet and obtained by subsequent dehydrogenation and elongation of LA and ALA in the diet.

U.S. Pat. No.5,952,544 describes a nucleic acid fragment encoding a fatty acid dehydrogenase isolated and cloned from Brassica napus. Expression of the nucleic acid fragment of the' 544 patent is carried out in plants, resulting in accumulation of ALA. However, in transgenic plants expressing plant Δ 15-dehydrogenase, a large amount of LA is still not transformed by the dehydrogenase. It would be advantageous to have a more active enzyme that could convert more LA into ALA. Increasing the conversion of LA to ALA may result in larger amounts of ALA. When co-expressed with a nucleic acid encoding a.DELTA.15-dehydrogenase, elevated levels of ALA will cause a.DELTA.6-dehydrogenase to act on ALA, resulting in higher levels of SDA. Due to the many beneficial effects of using SDA, there is a need to substantially increase SDA production. Nucleic acids from various sources have been explored to increase SDA production. However, there is still a great deal of innovation that is expected to improve the commercial production of crops on land (see e.g., Reed et al, 2000). Furthermore, the use of dehydrogenase polynucleotides derived from Caenorhabditis elegans (meesapayodsuk et al, 2000) is not ideal for commercial production of enriched plant seed oils.

Nucleic acids encoding a.DELTA.15-dehydrogenase have been isolated from several species of the order cyanobacteria and plants, including Arabidopsis, soybean, and parsley. Without being bound by any theory, the deduced amino acid sequences of these dehydrogenases show a high degree of similarity, most notably in the 3 histidine-rich motif regions thought to be involved in iron binding. However, no Δ 15-dehydrogenase was isolated from any of the fungi. Furthermore, even if the genomes of several fungi were sequenced, no Δ 15-dehydrogenase was found using a complex algorithm using known Δ 15-dehydrogenase cDNA and amino acid sequences aligned in Aspergillus and neurospora dna databases.

Thus, it would be advantageous to have genetic material involved in PUFA biosynthesis and to express the isolated material in plant systems, particularly terrestrial crop systems grown on the ground, which can provide commercial yields of one or more PUFA's. Due to the need to increase omega-3 fat intake in humans and animals, there is a need to provide a variety of omega-3 rich foods and food additives that allow people to select feeds, feed ingredients, foods and food ingredients to meet their daily dietary habits. Currently, only one omega-3 fatty acid, ALA, is available in vegetable oils. However, only a small amount of the ingested ALA is converted into long chain omega-3 fatty acids like EPA and DHA. This is demonstrated in pending U.S. application 10/384,639 "treatment and prevention of inflammatory disorders" by increasing the human average ALA intake from 1 g/day to 14 g/day with linseed oil, but only moderately increasing the level of the cytosolic phospholipid EPA. A14-fold increase in ALA uptake only resulted in a 2-fold increase in the cytosolic phospholipid EPA (Manzioris et al, 1994).

Therefore, there is a need for efficient and commercially viable production of PUFAs using fatty acid dehydrogenases, genes encoding them and recombinant methods for producing them. There is a need to produce oils, food compositions and additives containing them that contain higher relative proportions and/or are enriched in specific PUFA's. There is also a need to find reliable and economical methods for producing specific PUFA's.

Despite the low efficiency and low yield described above, the production of omega-3 fatty acids by the terrestrial food chain, particularly SDA, is a public health benefit. As mentioned above, only small amounts of ALA are converted to EPA and hence SDA is of particular importance. This is because during these three enzymatic reactions (requiring Δ 6, Δ 12, and Δ 15), the starting enzyme Δ 6-dehydrogenase is very low in activity in humans, limiting speed. Evidence for the rate of limiting Δ 6-dehydrogenase activity arises from studies demonstrating a lower efficiency of conversion of its substrate ALA than of its product SDA to EPA in mice and rats (Yamazaki et al, 1992; Huang, 1991).

Based on these studies, it was shown that in commercial oilseed crops, such as canola, soybean, corn, sunflower, safflower or flax, converting a certain fraction of the mono-and polyunsaturated fatty acids typical of their seed oils into SDA requires seed-specific expression of a variety of dehydrogenases, including Δ 6-and Δ 12 and enzymes with Δ 15-dehydrogenase activity. Oils from plants with increased expression levels of Δ 6, Δ 12, and Δ 15-dehydrogenases are enriched in SDA and other omega-3 fatty acids. These oils can be used to produce food and food additives rich in omega-3 fatty acids, and consumption of such food products can be effective in increasing the levels of EPA and DHA in the tissue. Food and food materials such as milk, margarine and sausages made and prepared entirely with omega-3 rich oil will yield therapeutic benefits. It has been shown that omega-3 equivalent to at least 1.8 grams of EPA and DHA per day can be ingested with omega-3 fatty acid rich food products without altering the eating habits of people (Naylor, supra.). Thus, there is a great need for novel Δ 15-dehydrogenase nucleic acids for use in transgenic crops for the production of oils rich in PUFAs. There is a need for new plant seed oils rich in PUFAs, in particular omega-3 fatty acids such as stearidonic acid.

Summary of The Invention

In one aspect, the invention provides an isolated nucleic acid encoding a polypeptide (Δ 15-dehydrogenase) capable of dehydrogenation at carbon 15 of a fatty acid molecule. These can be used to transform cells or to improve the fatty acid composition of plants or of oils produced by plants. One embodiment of the invention is an isolated polynucleotide sequence isolated from a fungal species having unique dehydrogenase activity. The isolated polynucleotide is preferably isolated from a fungal species belonging to one of the phyla selected from the group consisting of zygomycota, basidiomycota and ascomycota. In certain examples, the isolated polynucleotide is isolated from a fungal species selected from the group consisting of neurospora crassa (neurospora crassa), aspergillus nidulans (aspergillus nidulans), and botrytis cinerea (botrytis cinerea).

In another aspect, the invention provides an isolated polynucleotide selected from the group consisting of: (a) encodes the amino acid sequence of SEQ ID NO: 3. SEQ ID NO: 5 or SEQ ID NO: 34; (b) comprises the amino acid sequence shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4 or SEQ ID NO: 33; (c) and SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4 or SEQ ID NO: 33 or a complement thereof, a polynucleotide that hybridizes under 5 x SSC, 50% formamide, and 42 ℃; and (d) a fungal polynucleotide encoding a polypeptide having at least one of the following amino acid motifs:

TrpILeLeu AlaHisGluCysGlyHisGlyAlaSerPhe (WILAHECGHGASF) (SEQ ID NO: 6); LeuAlaHisGluCysGlyHis (LAHECGH) (SEQ ID NO: 7); HisSerPheLeu Leu ValProTyrPheSerTrLys (HSFLLVPYFSWK) (SEQ ID NO: 8); LeuLeuValProTyrPheSerTrLys (LLVPYFSLK) (SEQ ID NO: 9); his (His/Ala) ArgHisHisArg (Phe/Tyr) ThrThr (H (H/A) RHHR (F/Y) TT) (SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21); TrpValHisTrpLeu ValAlaIleThrTyrLeu (His/Gln) HisThrHis (WVHHWLVAITYL (H/Q) HTH) (SEQ ID NO: 11); AlaIleThrTyrLeu (His/Gln) HisThr (AITYL (H/Q) HT) (SEQ ID NO: 12); GlyAlaLeuAlaThrValAspArg (GALAVDR) (SEQ ID NO: 13) or HisValValHisHisLeuPheXaaArgIlePheTyr (HVVHLFXRIPFY) (SEQ ID NO: 14 or SEQ ID NO: 22).

In yet another aspect, the invention provides a recombinant vector comprising an isolated polynucleotide of the invention. The term "recombinant vector" as used herein includes any recombinant segment of DNA which it is desired to introduce into a host cell, tissue and/or organism, and specifically includes expression cassettes which have been isolated from the starting polynucleotide. The recombinant vector may be linear or circular. In various aspects, the recombinant vector may comprise at least one additional sequence selected from the group consisting of: a regulatory sequence operably linked to the polynucleotide; a selectable marker operably linked to the polynucleotide; a marker sequence operably linked to the polynucleotide; a purification moiety operably linked to the polynucleotide; and a target sequence to which the polynucleotide is operably linked.

In another aspect, the invention provides cells, e.g., mammalian, plant, insect, yeast and bacterial cells, transformed with a polynucleotide of the invention. In a further embodiment, cells are transformed with a recombinant vector containing a structural and tissue-specific promoter in addition to the polynucleotide of the invention. In certain particular embodiments of the invention, the cells are further defined as transformed with a nucleic acid sequence encoding a polypeptide having dehydrogenase activity for dehydrogenation at the 6-carbon position of a fatty acid.

In another aspect, the invention provides polypeptides, including fragments and proteins, having dehydrogenase activity for dehydrogenation at carbon 15 of a fatty acid molecule. In one embodiment of the invention, the polypeptide comprises at least one of the following amino acid motifs:

In a further embodiment, the polypeptide is further defined as containing all of the above amino acid motifs. The invention also provides a polypeptide comprising SEQ ID NO: 3. SEQ ID NO: 5, or SEQ ID NO: 34, or a fragment thereof, having dehydrogenase activity for dehydrogenation at carbon 15 of a fatty acid molecule.

In another aspect, the present invention provides a method for producing seed oil containing omega-3 fatty acids from plant seeds comprising the steps of (a) obtaining seeds of a plant of the present invention; and (b) extracting oil from the seeds. These plant seeds include canola, soybean, rapeseed, sunflower, cotton, cocoa, peanut, safflower, coconut, flax, oil palm, oilseed rape and corn. Preferred methods for transforming these plant cells include the use of Ti and Ri plasmids of Agrobacterium, electroporation and high-speed ballistic bombardment.

In another aspect, methods are provided for producing a plant comprising seed oil having altered omega-3 fatty acid levels comprising introducing into an oil-producing plant a recombinant vector of the present invention. In this method, introducing the recombinant vector may include cultivating a plant and the steps of: (a) transforming a plant cell with the recombinant vector; and (b) regenerating a plant from the plant cell, wherein the omega-3 fatty acid level of the plant is altered. In this method, the plant may be selected from the group consisting of, for example, arabidopsis, oilseed brassica, rapeseed, sunflower, safflower, canola, corn, soybean, cotton, flax, jojoba, chinese tallow tree, tobacco, cacao beans, peanuts, fruit trees, citrus plants, nut and berry producing plants. The plant can be further defined as transformed with a nucleic acid sequence encoding a polypeptide having dehydrogenase activity for dehydrogenation at carbon 6 of the fatty acid molecule, and the plant has increased SDA content. The method may also further comprise introducing the recombinant vector into a plurality of oil-producing plants, and selecting for plants or progeny plants that inherit a plant recombinant vector having the desired omega-3 fatty acid profile.

In another aspect, the present invention provides an endogenous canola oil having a SDA content of from about 8% to about 27%, and an oleic acid content of from about 40% to about 70%. In certain examples, canola oil may be further defined as containing less than 10% ALA acid, LA and GLA in admixture. The oil may further be defined as having an SDS content of from about 10% to about 20%, including from about 12% to about 20%, about 15% to about 20%, about 10% to about 17%, about 12% to about 17%. In further embodiments of the present invention, the oleic acid content of canola oil may be further defined as from about 45% to about 65%, including from about 50% to about 65%, about 50% to about 60%, about 55% to about 65%. In still further embodiments of the present invention, the SDA content is further defined as from about 12% to about 17%, and the oleic acid content is further defined as from about 55% to about 65%. In a specific example, the canola oil is derived from Brassica napus (Brassica napus) or Brassica rapa (Brassica rapa) seeds. In certain examples, oils are provided in which the ratio of omega-6 to omega-3 fatty acids is from about 1: 1 to about 1: 4, including from about 1: 2 to about 1: 4.

In another aspect, the present invention provides a method of increasing the nutritional value of an edible product for human and animal consumption comprising adding the canola oil provided by the present invention to the edible product. In a particular example, the product is a food product for humans and/or animals. The edible product may also be an animal feed and/or food additive. In this process, canola oil can increase the SDA content of the edible product and/or decrease the ratio of omega-6 to omega-3 fatty acids in the edible product. The edible food product may lack SDA prior to the addition of canola oil.

In another aspect, the present invention provides a method for producing food or feed comprising adding the canola oil provided by the present invention to raw food or feed ingredients to produce food or feed. In certain examples, the method is further defined as a method of producing a food and/or feed. The invention also provides food or feed prepared by the method.

In another aspect, the invention includes a method of providing SDA to humans and animals comprising administering the canola oil of claim 1 to the humans or animals. In the present method, canola oil may be administered in the form of an edible composition, including food or feed. Examples of food products include beverages, impregnated foods, sauces, dressings, salad dressings, juices, syrups, desserts, icings and fillings, soft frozen products, desserts or intermediate foods. The edible composition may be substantially liquid or solid. The edible composition may also be a food additive and/or a nutraceutical. In the present method, canola oil may be administered to humans and/or animals. Examples of animals to which the oil is administered may include livestock or poultry.

Brief Description of Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific examples set forth herein. The invention will be more fully understood from the following description of the figures:

FIG. 1 shows the coding region of the fungal Δ 15-dehydrogenase NcD15D in the pCR2.1 cassette (pMON 67004).

FIG. 2 shows the coding region of the fungal Δ 15-dehydrogenase NcD15D in the yeast expression vector pYES2.1(pMON 77208).

Fig. 3 shows the levels of ALA in 200 half seeds (the seeds are split in half), in order from lowest to highest ALA.

FIG. 4 shows a flow chart or plasmid map for obtaining plasmids pMON77214 and pMON 77217.

Figure 5 shows a typical dendrogram of dehydrogenase polypeptides including n.

Figure 6 shows the sequence alignment of a typical dehydrogenase polypeptide with n.

FIGS. 7A-7H show plasmid maps for the preparation of constructs.

Detailed Description

The present invention overcomes the limitations of the prior art by providing methods and compositions for producing plants with improved PUFA content. Modulation of fatty acid content in organisms such as plants results in a number of benefits, including beneficial improvements in nutrition and health. Adjusting the fatty acid content can be used to obtain desired beneficial levels or profiles of PUFA's in plants, parts of plants, and plant products, including plant seed oils. For example, when desired PUFA's are produced in seed tissue of plants, the oil can be isolated from the particular seed, resulting in oils having high levels of desired PUFAs or oils having desired fatty acid contents or profiles, which can then be used to provide beneficial properties to food materials and other products. The present invention specifically provides endogenous canola oil having SDA and beneficial oleic acid content.

Various aspects of the invention include methods and compositions for modulating the PUFA content of cells, for example, modulating the PUFA content of plant cells. Compositions related to the present invention include novel isolated polynucleotide sequences, polynucleotide constructs, and plants and/or plant parts transformed with a polynucleotide of the present invention. The isolated polynucleotide may encode a fungal fatty acid dehydrogenase and in particular may encode a fungal Δ 15-dehydrogenase. Host cells can be used to express polynucleotides encoding dehydrogenase polypeptides that catalyze the dehydrogenation of fatty acids.

Aspects of the invention include a wide variety of dehydrogenase polypeptides and polynucleotides encoding same. Various embodiments of the invention allow for the combined use of dehydrogenase polynucleotides and their encoded host cell-specific polypeptides, substrate availability, and end product of interest. "dehydrogenase" refers to a polypeptide that dehydrogenates or catalyzes the formation of double bonds between consecutive carbon atoms of one or more fatty acids to produce a mono-or poly-unsaturated fatty acid or a precursor thereof. Of particular note are polypeptides capable of catalyzing the conversion of stearic acid to oleic acid, oleic acid to LA, LA to ALA, or ALA to SDA, including enzymes that dehydrogenate at the 12, 15, or 6 positions. The term "polypeptide" refers to any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The selection of a particular polypeptide having dehydrogenase activity is made taking into account, but not limited to, the pH optimum of the polypeptide, whether the polypeptide is the rate-limiting enzyme or component thereof, whether the dehydrogenase employed is necessary for synthesis of the desired PUFA, and/or what cofactors are required for the polypeptide. The expressed polypeptide preferably has properties compatible with its biochemical environment at the location of the host cell. For example, the polypeptide may have to compete for the substrate.

Analysis of the Km and specific activity of the polypeptides can be used to determine whether a given polypeptide is suitable for modulating the production, level or status of PUFAs in a given host cell. The polypeptide used in a particular case is one which can function specifically under the conditions already present in the intended host cell, however in other aspects any dehydrogenase polypeptide having the desired properties or capable of modifying the corresponding production, level or properties of the desired PUFAs(s) or any other desired trait can also be explored herein. The substrate for the expressed enzyme may be produced by the host cell or provided exogenously. To achieve expression, the polypeptides of the invention are encoded by the following polynucleotides.

The present inventors have isolated and prepared enzymes of fungal origin that exhibit Δ 15-dehydrogenase activity. Fungal sources include, but are not limited to, Aspergillus (Aspergillus), such as Aspergillus nidulans; botrytis (Botrytis), such as Botrytis cinerea; the genus of streptosporium (Neurospora), such as Neurospora crassa; and other fungi that exhibit Δ 15-dehydrogenase activity.

Of particular interest are Neurospora crassa and/or Aspergillus nidulans.DELTA.15-dehydrogenase. The amino acid sequence of crassa Δ 15-dehydrogenase consists of SEQ ID NO: 3, set forth by SEQ ID NO: 1 and SEQ ID NO: 2 and has a molecular weight of about 49,123.37 daltons. The sequence consists of 429 amino acids; 32 of which are strongly basic amino acids (lysine, arginine); 35 of them are strongly acidic amino acids (aspartic acid, glutamic acid); 170 hydrophobic amino acids (alanine, isoleucine, leucine, phenylalanine, tryptophan, valine); and 100 polar amino acids (asparagine, cysteine, glutamine, serine, threonine, tyrosine). SEQ ID NO: 3 has an isoelectric point of 7.187; a charge of 1.634 at pH 7.0; davis, Botsein, Roth melting temperature of 89.65 deg.C, Wallace temperature of 5098.00.

Amino acid sequence of nidulans Δ 15-dehydrogenase consisting of SEQ ID NO: 5, the nucleic acid sequence encoding it consists of SEQ ID NO: list 4, determined to have a molecular weight of approximately 46,300 daltons. The sequence consists of 401 amino acids; of these 31 are strongly basic (lysine, arginine); 34 are strongly acidic (aspartic acid, glutamic acid); 161 hydrophobic amino acids (alanine, isoleucine, leucine, phenylalanine, tryptophan, valine); and 100 polar amino acids (asparagine, cysteine, glutamine, serine, threonine, tyrosine). SEQ ID NO: the isoelectric point of 5 was 6.83.

The sequences encoding Neurospora crassa and/or Aspergillus nidulans.DELTA.15-dehydrogenase may be expressed in transgenic plants, microorganisms or animals, while the synthesis of LA to ALA is increased, as is SDA. Other polynucleotides may also be used, provided that they are substantially identical to the n.crassa and/or a.nidulans Δ 15-dehydrogenase polynucleotides, or that they encode polypeptides substantially identical to the n.crassa and/or a.nidulans Δ 15-dehydrogenase polypeptides. By "substantially the same" is meant that the amino acid sequence or nucleic acid sequence exhibits at least 80%, 90% or 95% identity, preferably in increasing order, with the n.crassa and/or a.nidulans Δ 15-dehydrogenase amino acid sequence or nucleic acid sequence encoding the amino acid sequence. The polypeptides or polynucleotides may be compared using sequence analysis software, for example, the sequence analysis software packages GCG Wisconsin package (Accelrys, San Diego, Calif.), MEGAlign (DNASAR, Inc., 1228S, Park St., Madison, Wis.56-3715), and Mac vector (Oxford Molecular Group, 2105 S.Bascom, Suite 200, Campbell, Calif, 95008). These software match similar sequences by determining the degree of similarity or agreement.

The invention encompasses related dehydrogenases from the same or other related organisms. These related dehydrogenases include variants of the known Δ 15-dehydrogenases which occur naturally in the same or different fungal species. The corresponding dehydrogenase can be identified by its ability to function substantially identically to known dehydrogenases; namely, LA can be effectively converted into ALA, and GLA can be effectively converted into SDA. The corresponding dehydrogenases can also be identified by screening sequence databases for sequences homologous to known dehydrogenases, by hybridization of probes based on known dehydrogenases with libraries constructed from the original organism, or by RT-PCR using mRNA from the organism and primers based on known dehydrogenases.

Certain aspects of the invention include fragments and variants of fungal Δ 15-dehydrogenase polypeptides, as well as nucleotides encoding those polypeptides having dehydrogenase activity. Another aspect of the invention is a vector comprising a nucleic acid or fragment thereof, comprising a promoter, a.DELTA.15-dehydrogenase encoding sequence, and a termination region, which vector is transferable to an organism in which the promoter and terminator function. The invention accordingly provides organisms which produce recombinant Δ 15-dehydrogenases. Another aspect of the invention provides an isolated Δ 15-dehydrogenase enzyme that can be purified from recombinant organisms by standard protein purification methods (see, e.g., Ausubel et al, 1987).

Various aspects of the invention include the nucleic acid sequence encoding a dehydrogenase. Nucleic acids can be isolated from fungi including, but not limited to, Neurospora crassa, Aspergillus nidulans, Botrytis cinerea, and the like. The chromosomes of these fungi have been sequenced, each of which is known to be ala-rich, cloning strategies based on oligonucleotide primers for amplifying sequences that are potential fatty acid dehydrogenases and BLAST searches of the n.crassa genomic DNA database can be used to determine the sequence of individual clones. These clones can then be functionally characterized.

The nucleic acid construct may be provided by being integrated into the genome of the host cell or autonomously replicating (e.g., episomal replication) in the host cell. For the production of ALA and/or SDA, commonly used expression cassettes (i.e. protein-encoding polynucleotides, which are operably linked to a nucleic acid sequence, directing polynucleotide expression) comprise expression cassettes which provide for the expression of a polynucleotide encoding a Δ 15-dehydrogenase. In certain examples, the host cell can have a content of wild-type oleic acid.

Methods and compositions for constructing expression vectors for expressing fungal dehydrogenase will be apparent to those of ordinary skill in the art in view of the teachings provided herein. The expression vector is a DNA or RNA molecule designed for controlled expression of a polynucleotide of interest, such as a polynucleotide encoding a.DELTA.15-dehydrogenase. Examples of vectors include plasmids, phages, cosmids or viruses. Shuttle plasmids such as (Wolk et al, 1984; Butots et al, 1991) are also contemplated according to the invention. A review of vectors and methods for preparing vectors for use can be found in Sambrook et al (1989), Goeddel (1990), and Perbal (1988). Sequence elements effective for expression of a polynucleotide include promoters, enhancers, upstream activating sequences, transcription termination signals, and polyadenylation sites.

The polynucleotide encoding the dehydrogenase may be placed under the transcriptional control of a strong promoter. In some cases, this results in an increase in the amount of dehydrogenase expressed and an increase in the production of fatty acids produced in conjunction with the enzyme-catalyzed reaction. A variety of plant promoter sequences are available to facilitate tissue-specific expression of a polynucleotide encoding a dehydrogenase in a transgenic plant. For example, the napin promoter and acetyl carrier protein promoter have previously been used to modulate the composition of seed oil by expressing antisense forms of dehydrogenases (Knutzon et al, 1999). Similarly, promoters of the β -subunit of soybean β -conglobulin have shown high activity and result in tissue-specific expression in transgenic plants of species other than soybean (Bray et al, 1987). Arondel et al (1992) increased linolenic acid content in transgenic plant southbound tissues by placing the endoplasmic reticulum-localized fad3 gene under the transcriptional control of a strong, constitutive cauliflower mosaic virus 35S promoter (18: 3).

One of ordinary skill can determine the appropriate vector and regulatory elements (including operably linked promoter and coding regions) for expression in a particular host cell. By "operably linked" herein is meant that the promoter and terminator sequences are effective to function in regulating transcription. As a further example, a vector suitable for expression of a.DELTA.15-dehydrogenase in a transgenic plant may include a seed-specific promoter sequence derived from sunflower methyl-aurantiol, napin or glycinin operably linked to a.DELTA.15-dehydrogenase encoding region and further operably linked to a seed storage protein termination signal or a nopaline synthase termination signal. As a further example, a vector for expressing a.DELTA.15-dehydrogenase in a plant may comprise a constitutive promoter or a tissue specific promoter operably linked to a.DELTA.15-dehydrogenase encoding region and further operably linked to a constitutive or tissue specific terminator or a nopaline synthase termination signal.

Modifications of the nucleotide sequences having the functions described herein or of the regulatory factors disclosed herein are within the scope of the invention. Such modifications include insertions, substitutions and deletions as well as specific substitutions which reflect the degeneracy of the genetic code.

Standard techniques for constructing such recombinant vectors are well known to those skilled in the art, and reference may be made, for example, to Sambrook et al (1989), or any of the widely used protocols for recombinant DNA technology. Various strategies for joining DNA fragments should be selected based on the nature of the DNA fragment ends. It is further contemplated according to the present invention that nucleotide sequence elements for cloning, expression or processing, such as a sequence encoding a signal peptide, a sequence encoding KDEL for protein retention in the endoplasmic reticulum or a sequence encoding a transit peptide directing the transport of a.DELTA.15-dehydrogenase into chloroplasts, are introduced into the nucleic acid vector. These sequences are known to those of ordinary skill in the art. A preferred transporter is described, for example, by Van den Broeck et al (1985). Prokaryotic and eukaryotic signal sequences are disclosed, for example, by Michaelis et al (1982).

In certain embodiments, the expression cassette may comprise a cassette for providing Δ 6-and/or Δ 15-dehydrogenase activity, particularly in a host cell that produces or ingests LA or ALA, respectively. Host organisms that are unable to produce ALA are advantageous for the production of omega-6 type unsaturated fatty acids such as LA. Host ALA production may be eliminated, reduced and/or inhibited by inhibiting Δ 15-dehydrogenase activity. This can be accomplished using standard screening methods that provide Δ 15-dehydrogenase antisense expression cassettes, disrupting the target Δ 15-dehydrogenase gene by insertion, deletion, substitution of part or all of the target gene, or by addition of a Δ 15-dehydrogenase inhibitor. Similarly, microorganisms or animals having Δ 6-dehydrogenase activity are made more favorable for the production of LA or ALA by disruption of the Δ 6-dehydrogenase gene, or by using a Δ 6-dehydrogenase inhibitor, using expression cassettes for Δ 6 antisense transcription.

A polynucleotide encoding a dehydrogenase of interest can be identified by a variety of methods. For example, a source of the dehydrogenase of interest is screened, e.g., with a genomic or cDNA library derived from Neurospora using a detectable enzymatic reaction or chemically synthesized probe, which can be prepared from DNA, RNA, or non-naturally occurring nucleotides, or mixtures thereof. Probes can be enzymatically synthesized from known dehydrogenase polynucleotides for use in standard or reduced stringency hybridization methods. Oligonucleotide probes based on known dehydrogenase sequences, including conserved sequences in known dehydrogenases, or peptide sequences obtained based on purified proteins of interest, can also be used to screen sources. Oligonucleotide probes based on amino acid sequence can degenerate to include degeneracy of the genetic code, or can be preferred to the preferred codons of the organism. Oligonucleotides may also be used as PCR primers to reverse transcribe mRNA from known or suspected sources; the PCR product may be a full-length cDNA or may be used to prepare a probe to obtain a full-length cDNA of interest. Alternatively, the protein of interest may be sequenced in its entirety and the DNA encoding the polypeptide synthesized in its entirety.

Once the genome or cDNA of interest is isolated, it can be sequenced by known methods. It is recognized in the art that these methods are error-prone, so multiple sequencing of the same region is necessary, and that a certain proportion of errors in the resulting deduced sequence is expected to remain, particularly in regions with repetitive regions, large secondary structures, or unusual base composition, e.g. high GC base content. When a difference occurs, sequencing can be performed again and a special method can be used. Particular methods include varying the sequencing conditions used: different temperatures, different enzymes, proteins that alter the ability of oligonucleotides to form higher order structures; altered nucleotides such as ITP or methylated dGTP, different gel compositions such as addition of formamide, different primers or primers at different distances from the region of interest or different templates, e.g.single stranded DNAs. mRNA can also be sequenced.

Some or all of the sequences encoding polypeptides having dehydrogenase activity may be obtained from natural sources. In some cases, however, it may be desirable to modify all or a portion of the codons, for example, to enhance expression by using host-biased codons. Host-preferred codons are determined by the codons that occur with the highest frequency when proteins are expressed in large amounts in a particular target host species. Thus, the sequence encoding the polypeptide having dehydrogenase activity may be wholly or partially synthesized. It is also possible to synthesize the entire DNA or a part thereof to remove any unstable sequence or secondary structure region occurring in the transcribed mRNA. It is also possible to synthesize all or part of the DNA to alter the base composition to make it more preferable in the intended host cell. The methods for synthesizing sequences and bringing sequences together are well documented in the literature. In vitro mutagenesis and screening, site-directed mutagenesis, or other means can be used to obtain a mutant gene of a naturally occurring dehydrogenase gene for use in producing a polypeptide having dehydrogenase activity with improved physical and kinetic functional parameters in a host cell, such as for example, a polyunsaturated fatty acid with a longer half-life or higher yield.

Once the polynucleotide encoding the dehydrogenase polypeptide is obtained, it can be placed into a vector capable of replication in a host cell, or amplified in vitro by PCR or long PCR techniques. Replication vectors may include plasmids, phages, viruses, cosmids, and the like. Desirable vectors include those that facilitate mutation of the gene of interest or expression of the gene of interest in a host cell. Long PCR techniques have made it possible to amplify large constructs in vitro, so that modifications to the gene of interest, such as mutations or addition of expression signals and amplification-generated constructs, can be performed entirely in vitro without the use of replicating vectors or host cells.

To express the dehydrogenase polypeptide, functional transcriptional and translational initiation and termination regions are operably linked to a polynucleotide encoding the dehydrogenase polypeptide. Expression of the polypeptide coding region may occur in vitro or in a host cell. Transcriptional and translational initiation and termination regions are obtained from a variety of non-single sources, including the polynucleotide to be expressed, a gene known to be either constitutive or expected to be expressed in a desired system, an expression vector, chemically synthesized, or from an endogenous site in the host cell.

Expression in a host cell can be transient or stably accomplished. Transient expression occurs when the introduced construct, which contains an expression signal functional in the host, cannot replicate and is hardly integrated into the host cell, or the host cell cannot proliferate. Transient expression can also be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest, although such inducible systems oftentimes exhibit low levels of background expression. Stable expression is accomplished by introducing a construct that is capable of integrating into the host genome or that can autonomously replicate in the host cell. Stable expression of the gene of interest can be selected by using a localized selectable marker or by transfection with an expression construct followed by selection of cells expressing the marker. Where stable expression is produced by integration, integration of the construct into the host genome may occur randomly or by using a construct containing regions effective for targeted recombination with the host site and homology to the host genome. Where the construct is targeted to an endogenous site, all or part of the transcriptional and translational regulatory regions may be provided by the endogenous site.

When increased expression of the dehydrogenase polypeptide in an organism is desired, several methods can be used. Additional genes encoding dehydrogenase polypeptides can be introduced into the host organism. Expression from the native dehydrogenase site can be increased by homologous recombination, for example by inserting a stronger promoter in the host genome, by deleting the host genome information to remove unstable sequences from the mRNA or encoded protein, or by adding stabilizing sequences to the mRNA (u.s.pat. No.4,910,141).

It is contemplated that multiple polynucleotides encoding one dehydrogenase or polynucleotides encoding multiple dehydrogenases may be introduced and amplified in a host cell by using episomes or integrated expression vectors. When two or more genes are expressed from separate replicating vectors, it is desirable that each vector has different means of replication. Each introduced construct, whether integrated or not, should have a different screening method and lack homology to other constructs to maintain stable expression and to prevent redistribution of elements in the construct. Judicious selection of the regulatory regions, screening means and amplification methods of the introduced constructs can be determined experimentally so that all introduced polynucleotides are expressed at the necessary levels to satisfy the synthesis of the product of interest.

When transformation is desired, the Δ 15-dehydrogenase encoding sequence of the invention can be inserted into a plant transformation vector, such as the binary vector described by Bevan (1984). Plant transformation vectors can be obtained by modifying the natural gene transfer system Agrobacterium tumefaciens. The natural system comprises a large Ti (tumor-inducing) -plasmid containing a large fragment, known as T-DNA, which can be transferred into transformed plants. Another Ti plasmid fragment, the vir region, was used for T-DNA transfer. The T-DNA region is contiguous with the terminal repeat. In the modified binary vector, the tumor-inducing gene has been deleted, and the function of the vir region is used to transfer foreign DNA bordered by T-DNA border sequences. The T-region also contains a selection marker for resistance to antibiotics, and a multiple cloning site for insertion of the transfer sequence. These engineered strains, designated "disarmed" a. tumefaciens strains, are capable of efficiently transferring sequences delimited by the T-region into the nuclear genome of plants.

The present invention finds many applications. It was found that probes based on the polynucleotides of the invention can be used in a method for isolating related molecules or for detecting organisms expressing dehydrogenases. When used as a probe, the polynucleotide or oligonucleotide must be detectable. This is usually achieved by attaching a label at an internal site or at the 5 'or 3' end, for example by incorporating a modified residue. Such labels may be detected directly, may be bound to a detectably labeled secondary molecule, or may be bound to an unlabeled secondary molecule and a detectably labeled tertiary molecule; this process can be continued as long as no unacceptable background signal is present during the procedure and a satisfactory detectable signal is obtained. Secondary, tertiary, or bridge systems may include the use of antibodies directed against any other molecule, including labels or other antibodies; or may comprise any molecule that can bind to each other, such as the biotin-streptavidin/avidin system. Typical detectable labels include radioisotopes, chemically synthesized or enzymatically produced or luminescent-altering molecules, enzymes that produce detectable reaction products, magnetic molecules, fluorescent molecules or molecules that alter the fluorescent or luminescent properties upon binding. An example of a labeling method can be found in U.S. Pat. No.5,011,770. Either by isothermal titration calorimetry to measure the change in heat of solution when the probe is bound to the target, or by coating the probe or target on a surface and detecting the change in surface light scattering produced by the target or probe binding, each of which can be accomplished by the BIAcore system.

The construct comprising the gene of interest may be introduced into the host cell by conventional techniques. For convenience, host cells containing the DNA sequences or constructs treated by any method are designated herein as "transformed" or "recombinant. The target host will contain at least one copy of the expression construct, and may also contain two or more copies, depending, for example, on whether the gene is integrated into the genome, amplified, or present with additional chromosomal elements at multiple copy numbers.

Transformed host cells can be identified by the presence of a selectable marker on the introduced construct. Alternatively, a separate marker construct may be introduced with the construct of interest, just as many transformation techniques introduce many DNA molecules into a host cell. Typically, transformed hosts can be selected for their ability to grow on a selection medium. The selection medium may incorporate antibiotics or lack factors necessary for growth of the untransformed host, such as trophic factors or growth factors. The introduced marker gene may therefore confer antibiotic resistance, or encode an essential growth factor or enzyme, and be capable of growing on selective media when expressed by the transformed host. When the expressed marker protein is detected, screening of the transformed host may be performed directly or indirectly. The marker protein may be expressed alone or in fusion with other proteins. The marker protein can be detected based on the enzymatic activity; for example, β -galactosidase can convert the substrate X-gal to a colored product, and luciferase can convert luciferin to a light emitting product. The marker protein may be detected by its light-generating or modifying properties; for example, the Aequorea Victoria green fluorescent protein fluoresces when illuminated with blue light. Antibodies can be used to detect marker proteins or molecular tags on the protein of interest. Cells expressing the marker protein or tag can be screened, for example, by visual inspection, or by techniques such as FACS or panning using antibodies. Of note are the tolerance to kanamycin and the aminoglycoside G418, and the ability to grow in media lacking uracil, leucine, lysine or tryptophan.

Of particular interest is Δ 15-dehydrogenase-mediated production of PUFA's in eukaryotic host cells. Eukaryotic cells include plant cells, such as those from oil-producing crops, and other genetically manipulated cells including fungal cells. The cells may be cultured to become or form part or all of an organic host including a plant. In a preferred embodiment, the host is a plant cell that produces and/or can take up an exogenously supplied Δ 15-dehydrogenase substrate, and preferably produces a large amount of one or more substrates.

The transformed host cell is grown under suitable conditions for the desired end product. For growth of the host cells in culture, conditions are optimized to give the maximum or most economical production of PUFA's, which correlates with the dehydrogenase activity screened. Conditions of the medium that can be optimized include: carbon source, nitrogen source, added substrate, final concentration of added substrate, form of added substrate, aerobic or anaerobic growth, growth temperature, inducer, induction temperature, growth phase at induction, growth phase at harvest, pH, density and maintenance of screening.

Another aspect of the invention provides a transgenic plant or plant progeny comprising an isolated DNA of the invention. Both monocotyledonous and dicotyledonous plants are contemplated. Plant cells are transformed with an isolated DNA encoding a.DELTA.15-dehydrogenase using any of the plant transformation methods described above. Transformed plant cells are usually in a callus culture medium or on leaf disks, which can be regenerated into fully transgenic plants by methods known to those of ordinary skill in the art (e.g., Horsch et al, 1985). In one example, the transgenic plant is selected from the group consisting of arabidopsis thaliana, canola, soybean, rapeseed, sunflower, cotton, cacao, peanut, safflower, coconut, flax, oil palm, oil seed vine, corn, jojoba, chinese tallow tree, tobacco, fruit trees, citrus plants, or nut and berry producing plants. Since progeny of the transformed plant inherit the polynucleotide encoding the Δ 15-dehydrogenase, either the seed or the cuttings of the transformed plant can be used to maintain the transgenic plant line.

The invention further provides methods for conferring increased ALA and/or SDA content to a transgene. The method comprises, for example, introducing a DNA encoding a.DELTA.15-dehydrogenase into plant cells which lack or have low levels of ALA or SDA but contain LA, and regenerating plants with increased ALA and/or SDA content from the transgenic cells. In certain embodiments of the invention, DNA encoding a.DELTA.6-and/or a.DELTA.12-dehydrogenase may be introduced into a plant cell. These plants may or may not also comprise endogenous Δ 6-and/or Δ 12-dehydrogenase activity. In certain embodiments, engineered, commercially planted crops are contemplated as transgenic organisms, including, but not limited to, Arabidopsis, canola, soybean, rapeseed, sunflower, cotton, cacao, peanut, safflower, coconut, flax, oil palm, oilseed rape and corn, jojoba, plants that are Sapium sebiferum, tobacco, fruit trees, citrus, or plants that produce nuts and berries.

The invention further provides methods by which transgenic plants may be made to contain increased levels of ALA and/or SDA, wherein the increased levels are greater than those present in untransformed plants. The method may comprise introducing one or more polynucleotides encoding a Δ 15-dehydrogenase into a plant lacking or having low levels of ALA but containing LA. Expression vectors containing DNA encoding a.DELTA.15-dehydrogenase or a.DELTA.15-dehydrogenase and a.DELTA.6-dehydrogenase can be constructed by recombinant techniques known to those of ordinary skill in the art (Sambrook et al, 1989). In particular, commercially grown crops are contemplated as transgenic organisms, including, but not limited to, Arabidopsis, canola, soybean, rapeseed, sunflower, cotton, cocoa, peanut, safflower, coconut, flax, oil palm, oilseed rape and corn.

In the case of food additives, the purified PUFAs, transformed plants or plant parts, or derivatives thereof, may be incorporated into cooking oils, fats or synthetic margarines in amounts which are desired by the consumer in normal use. PUFAs can also be incorporated into infant formulas, nutritional supplements or other food products, and can also be used as anti-inflammatory or cholesterol lowering agents.

As used herein, "edible composition" is defined as a composition that can be ingested by a mammal, such as food materials, nutritional substances, and pharmaceutical compositions. As used herein, "foodstuff" refers to a substance that can be used in or to prepare a food for a mammal, including substances that may be used in the preparation of a food (e.g., frying oil) or food additive. For example, food materials include animals for human consumption or products derived therefrom, such as eggs. Typical food ingredients include, but are not limited to, beverages (e.g., soft drinks, sodas, beverages for blending), pickled foods (e.g., fruits and vegetables), sauces, dressings, salad dressings, juices, syrups, confections (e.g., puddings, gelatins, icings and fillings, baked goods and frozen desserts such as ice creams and sherbet), soft frozen products (e.g., soft frozen creams, soft frozen ice creams and yogurts, dressings over soft frozen toppings such as dairy or non-dairy whipped toppings), oils and emulsified products (e.g., shortenings, margarines, mayonnaise, butter, cooking oils and salad dressings), and intermediate moist foods (e.g., rice and dog foods).

In addition, the edible compositions described herein may also be ingested as additives or supplements contained in foods and beverages. These can be formulated with nutritional substances such as various vitamins and minerals and incorporated into substantially liquid compositions, such as nutritional beverages, soy milk, and soups; in a substantially solid composition; and gelatin or incorporation into various food products in powdered form. The content of the active ingredient in such functional or healthy foods may be similar to the dose contained in a typical pharmaceutical agent.

Purified PUFAs, transformed plants or plant parts may also be incorporated into the feed of animals, in particular livestock. Thus, the animal itself may benefit from a PUFA-rich diet, as may human consumers consuming food products made from such livestock. It is expected that in some instances SDA will be converted to EPA in animals, so that animals may benefit from increased EPA by consumption of SDA.

For pharmaceutical use (human or veterinary), the compositions are generally administered orally, but may be administered by any route which results in successful absorption, for example parenterally (i.e. subcutaneously, intramuscularly or intravenously), rectally, vaginally or topically, for example as a skin patch or lotion. The transformed plants or plant parts of PUFAs of the present invention may be administered alone or in combination with a pharmaceutically acceptable carrier or excipient. Gelatin capsules are the preferred dosage form for oral administration that can be used. The dietary supplements mentioned above may also be administered orally. The unsaturated acids of the present invention may be administered in combination, or as a salt, ester, amide or prodrug of a fatty acid. The present invention includes any pharmaceutically acceptable salt; sodium, potassium or lithium salts are particularly preferred. Also included are N-alkylpolyhydroxy amine salts, such as N-methyl glutamine, see PCT publication WO 96/33155. The preferred ester is ethyl ester. As solid salts, PUFAs may also be administered in the form of tablets. For intravenous administration, PUFAs or derivatives thereof may also be incorporated into commercial formulations such as Intralipids.

The regions of the dehydrogenase polypeptide which are important for the dehydrogenase activity can be determined by conventional mutagenesis, followed by expression of the resulting mutant polypeptides and determination of their activity. Mutations may include substitutions, deletions, insertions, and site mutations, or combinations thereof. Substitutions may be made on the basis of conserved hydrophobicity or hydrophilicity (Kyte and Doolittle, 1982), or on the basis of the ability to form similar secondary structures of polypeptides (Chou and Fasman, 1978). A typical functional analysis begins with deletion mutations to determine the N-and C-terminal limitations of functionally essential proteins, followed by internal deletions, insertions or site mutations to further determine functionally essential regions. Other techniques such as cassette mutagenesis or total synthesis may also be used. Deletion mutations are accomplished, for example, by using an exonuclease to sequentially remove 5 'or 3' coding regions. Such techniques may be performed using a kit. After deletion, the coding region is completed by ligating oligonucleotides containing the start or stop codon to the deleted coding region following the 5 'or 3' deletion. Alternatively, oligonucleotides encoding start or stop codons can be inserted into the coding region by a variety of methods including site-directed mutagenesis, mutagenic PCR or by ligation with DNA digested at the restriction sites present.

Similarly, internal deletions can be accomplished by a variety of methods, including the use of restriction sites present in the DNA, by site directed mutagenesis using mutagenic primers, or by mutagenic PCR. Insertions are made by methods such as ligation-scanning mutagenesis, site-directed mutagenesis or mutagenic PCR. Point mutation is accomplished by methods such as site-directed mutagenesis or mutagenic PCR. Chemical mutations can also be used to identify regions of the dehydrogenase polypeptide that are critical to activity. These structural functional analyses can determine which regions can be deleted, which regions can tolerate insertions, and which site mutations cause the mutein to function essentially like a native dehydrogenase. All such muteins and nucleotide sequences encoding them are included in the scope of the present invention.

As mentioned above, certain embodiments of the present invention relate to plant transformation constructs. For example, one aspect of the invention is a plant transformation vector comprising one or more dehydrogenase genes or cdnas. Representative coding sequences for use in the present invention include the Neurospora crassa gene Δ 15-dehydrogenase NcD15D (SEQ ID NO: 1 AnD SEQ ID NO: 2) AnD Aspergillus nidulans Δ 15-dehydrogenase AnD15D (SEQ ID NO: 4). In certain instances, antisense dehydrogenase sequences may also be used in the invention. Typical dehydrogenase-encoding nucleic acids can also be used, including at least 20, 40, 80, 120, 300 and up to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4 or SEQ ID NO: 33, is full length. In certain particular aspects, the nucleic acid can encode 1, 2,3, 4, or more dehydrogenases. As a specific example, the nucleic acid can encode a.DELTA.6 and a.DELTA.15-dehydrogenase.

In certain examples of the invention, a coding sequence is provided that is operably linked, in sense or antisense orientation, to a heterologous promoter. Expression constructs comprising these sequences are also provided, and the construction of constructs (which can be used in conjunction with plant transformation) using these or other sequences of the invention is known to those of ordinary skill in the art in light of the present disclosure (see, e.g., Sambrook et al, 1989, Gelvin et al, 1990). Thus, the techniques of the present invention are not limited to any particular nucleic acid sequence.

One use of the sequences provided herein is to alter the phenotype of a plant, such as the composition of oil, by genetic transformation with a dehydrogenase gene, a particular example being a fungal Δ 15-dehydrogenase. Other sequences may also be used in conjunction with the dehydrogenase gene. When an expressible coding region (not necessarily a marker coding region) is used in combination with a marker coding region, separate coding regions on the same or different DNA fragments may be used for transformation. In the latter case, different vectors are delivered simultaneously to the recipient cells to achieve maximum co-transformation.

The additional elements used with the dehydrogenase-encoding sequence are often selected for the purpose of transformation. One of the main objectives of transforming crops is to increase the commercially desirable and agronomically important characteristics of plants. PUFAs are known to confer a number of beneficial health effects, with a concomitant increase in SDA production being beneficial, which can be obtained by expression of a fungal Delta 15-dehydrogenase. In certain examples of the invention, such an increase in SDA can comprise expression of a Δ 6 and/or Δ 12 dehydrogenase, including a fungal or plant Δ 6 and/or Δ 12 dehydrogenase.

Vectors used for plant transformation may include, for example, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), or any other suitable cloning system, as well as DNA fragments derived therefrom. Thus, when the term "vector" or "expression vector" is used, all of the aforementioned vector types and nucleotide sequences isolated therefrom are included therein. It is contemplated that large DNA sequences containing more than one specific gene can be introduced using cloning systems with strong insertion capabilities. According to the invention, this can be used to introduce various dehydrogenase-encoding nucleic acids. These sequences can be introduced by using bacterial or yeast artificial chromosomes (BACs or YACs, respectively), or even plant artificial chromosomes. For example, Hamilton et al (1996) disclose the use of BACs for Agrobacterium-mediated transformation.

Expression cassettes isolated from these vectors are particularly useful for transformation. Of course, the DNA segment used to transform a plant cell will typically contain cDNA, a gene, or a gene that is desired to be introduced and expressed in the host cell. These DNA fragments may further include a desired structure such as a promoter, enhancer, polylinker, or regulatory gene. The DNA segment or gene selected for cellular introduction will often encode a protein that is expressed in the resulting recombinant cell to form a screenable or selectable trait, and/or a protein that confers an improved phenotype on the resulting transgenic plant. However, this is not always the case and the invention also encompasses transgenic plants which have inserted a transgene which is not expressed. Preferred components that may be included in the vectors used in the present invention are as follows.

In one embodiment of the invention a specific promoter is used. Examples of promoters useful in the present invention include, but are not limited to, 35SCaMV (cauliflower mosaic virus), 34SFMV (figwort mosaic virus) (see, e.g., U.S. Pat. No.5,378,619, the disclosure of which is incorporated herein by reference in its entirety), Napin (from brassica), 7S (from soybean), Glob, and Lec (from corn). The 35SCaMV promoter and promoters regulated during plant seed maturation are of particular interest in the application of the present invention. It is contemplated that the use of all such promoters and transcription regulatory elements, alone or in combination, in replicable expression vectors is known to those of ordinary skill in the art.

For example, the CaMV 35S promoter is described by Restrepto et al (1990). Genetically transformed and mutated regulatory sequences resulting in seed specific expression may also be used to produce improved seed oil compositions. Such modifications described herein will be apparent to those of ordinary skill in the art.

DNA sequences between the transcription start site and the start of the coding sequence, such as untranslated leader sequences, may also affect gene expression. Thus, specific leader sequences may be used in the transformation constructs of the invention. Preferred leader sequences to be considered are those which contain a sequence predicted to direct optimal expression of the attached gene, for example including a preferred consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The selection of such sequences is known to those of ordinary skill in the art in light of the present disclosure. Sequences obtained from genes typically highly expressed in plants are preferred.

Typically, transformation constructs made according to the invention include a 3' terminal DNA sequence that serves as a transcription termination signal and enables polyadenylation of the resulting mRNA by a sequence operably linked to a dehydrogenase gene (e.g., cDNA). In one embodiment of the invention, the terminator of the native dehydrogenase gene is used. Alternatively, the heterologous 3' end may enhance expression of the dehydrogenase encoding region. Examples of sources of terminators which are considered useful include the nopaline synthase gene of Agrobacterium tumefaciens (nos3 ' end) (Bevan et al, 1983), the T7 transcription terminator of the octopine synthase gene of Agrobacterium tumefaciens, the 3 ' end of the protease inhibitor I or II gene of potato or tomato and the CaMV 35S terminator (tml3 '). Regulatory elements such as an Adh intron (Callis et al, 1987), a sucrose synthase intron (Vasil et al, 1989) or a TMV.OMEGA.element (Gallie et al, 1989) may also be included if desired.

The ability to identify transformants can be provided or enhanced by the use of selectable or screenable marker proteins. A "marker gene" is a gene that confers a unique phenotype on cells expressing the marker protein, which allows transformed cells to be distinguished from cells that do not have the marker. The gene may encode a selectable or screenable marker depending on whether the marker confers a property that can be screened chemically, for example by use of a selective agent (e.g., herbicide, antibiotic, etc.), or whether it is a property that can only be identified by observation or testing, for example by "screening" (e.g., green fluorescent protein). Of course, many examples of suitable marker proteins are known in the art and may be practically used in the present invention.

Methods suitable for transforming plants or other cells of the invention include virtually any method of introducing DNA into a cell, for example, direct delivery of DNA by PEG-mediated protoplast transformation (Omirulleh et al, 1993), by drying/inhibition mediated DNA uptake (Potrykus et al, 1985), by electroporation (U.S. Pat. No.5,384,253, expressly incorporated herein by reference in its entirety), by agitation with silicon carbide fibers (Kaeppler et al, 1990; U.S. patent No.5,302,523, expressly incorporated herein by reference in its entirety; U.S. patent No.5,464,765, expressly incorporated herein by reference in its entirety), by Agrobacterium-mediated transformation (U.S. patent No.5,591,616 and U.S. patent No.5,563,055; expressly incorporated herein by reference in its entirety) and by acceleration of DNA-coated particles (U.S. patent No.5,550,318; U.S. patent No.5,538,877; and U.S. patent No.5,538,880; each expressly incorporated herein by reference in its entirety), and the like. By applying these techniques, cells of virtually any plant can be stably transformed and these cells will develop into transgenic plants.

After efficient delivery of exogenous DNA to recipient cells, the next step is generally directed to the identification of transformed cells for further culture and plant regeneration. In order to improve the ability to identify transformants, a selectable or screenable marker gene carried by the transformation vector prepared according to the present invention should be used. In such cases, it is often followed by analysis of the potentially transformed cell population by exposure of the cells to a selective agent, or by screening for cells with the desired marker gene characteristics.

Cells that survive exposure to the selective agent or that are positive in the screening assay should be cultured in media that maintains plant regeneration. In a typical example, MS and N6 media were modified by the addition of additional substances such as growth regulators. These growth regulators used were dicamba or 2, 4-D. However, other growth regulators may be used including NAA, NAA +2, 4-D or picloram. It has been found that media modified in this or a similar manner facilitates cell growth at a particular developmental stage of the cell. The tissue may be continued to grow in minimal medium containing growth regulatory factors until sufficient tissue is formed to initiate plant regeneration, or then manually selected repeatedly until the tissue morphology is suitable for regeneration, usually for at least 2 weeks, and then transferred to a medium conducive to embryo maturation. Cultures were transferred to the medium every two weeks. The emergence of shoots means that transfer to medium lacking growth regulators is possible.

To confirm the presence of foreign DNA or "transgenes" in the regenerated plants, various assays can be performed. Such assays include, for example, "molecular biology" assays, such as Southern and Northern blotting and PCR^TM(ii) a "biochemical" identification, e.g.detection of the presence of protein products, e.g.by immunological means (ELISAs and Western blotting) or by enzymatic function; identification of plant parts, such as leaf or root identification; it is also possible to analyze the phenotype of the whole regenerated plant.

In addition to direct transformation of a particular plant genotype with a construct prepared according to the present invention, transgenic plants can also be made by crossing a plant carrying the selected DNA of the present invention with another plant lacking this DNA. Plant breeding techniques can also be used to introduce multiple dehydrogenases, e.g., Δ 6, Δ 12, and/or Δ 15-dehydrogenases into individual plants. In this way, the Δ 15-dehydrogenase can be effectively up-regulated. By making plant homozygotes having Δ 15-dehydrogenase activity and/or other dehydrogenase activity (e.g., Δ 6-and/or Δ 12-dehydrogenase activity), beneficial metabolites in plants can be increased.

As described above, the selected dehydrogenase gene can be introduced into a particular plant species by crossing, without the need to directly transform a given plant species. Thus, the present invention encompasses not only directly transformed plants or plants regenerated from cells transformed according to the invention, but also the progeny of these plants. The term "progeny" as used herein refers to progeny of any generation of a parent plant made according to the present invention, wherein the progeny comprises a selected DNA construct made according to the present invention. "crossing" of a plant can provide a plant line having one or more additional transgenes or alleles relative to the starting cell line, as defined herein, by the introduction of a particular sequence into a plant line by crossing the starting line with a donor plant line containing a transgene or allele of the invention. For this purpose, for example, the following steps should be carried out: (a) growing seeds of a first (starter line) and a second (donor plant line containing the desired transgene or allele) parent plant; (b) growing seeds of the first and second parent plants into flowering plants; (c) pollinating a flower of the first parent plant with pollen from a plant of the second parent; and (d) harvesting seed produced on the parent plant containing the fertilized flower.

Backcrossing is defined herein as a process that includes the following steps: (a) crossing a plant of a first genotype containing a gene, DNA sequence or element of interest with a plant of a second genotype lacking said gene, DNA sequence or element of interest; (b) selecting one or more progeny plants comprising the gene, DNA sequence, or element of interest; (c) crossing the progeny plant with a second genotype plant; and (d) repeating steps (b) and (c) with the objective of transferring the DNA sequence of interest in the first genotype plant into the second genotype plant.

The incorporation of DNA element genes into the genotype of a plant is defined as the result of the backcross transformation process. The plant genotype incorporating the DNA sequence may be considered a backcross transition genotype, line, inbred, or hybrid. Similarly, a plant genotype lacking a DNA sequence of interest can be considered a genotype, line, inbred or hybrid without conversion.

Examples

The following examples are provided to illustrate embodiments of the present invention. It should be recognized by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Example 1

Strains and growth conditions

The neurospora crassa hybrid a-type and the aspergillus nidulans glasgow wild-type are from a fungal genetic storage center. Cultures were grown in Vogel's medium N (Case et al, neurospora Newsletter, 8: 25-26, 1965). Liquid cultures were inoculated with cystospores and shake-cultured at 100RPM for 3 days at 15 ℃. The mycelia were collected by filtration through Whatman1 filter paper in a Buchner funnel and stored at 80 ℃ for RNA isolation or fatty acid composition analysis by gas chromatography by direct freeze-drying. The saccharomyces cerevisiae strain used was INVSc1, a diploid strain auxotrophic for histidine, leucine, tryptophan and uracil (Invitrogen). The cells were stored at 30 ℃ in YPD medium.

Example 2

Isolation of fungal RNA

Total RNA was isolated from fungal mycelia of 3 strains described in example 1 using the guanidinium-phenol-chloroform hydrochloride method of Chomczynski and Sacchi (1987, Tri-Reagent, SIGMA). This procedure 500 mg of mycelium were ground in liquid nitrogen and then added to 7 ml of Tri-Reagent. Chloroform was added and the aqueous phase was separated from the organic phase. RNA was precipitated with isopropanol and then rinsed with 70% ethanol before being resuspended in deionized water

Example 3

Cloning of the crassa Δ 12 and Δ 15-dehydrogenase sequences

Gene specific primers were designed to amplify the full-length putative Δ 12-dehydrogenase (Nc111F2 and Nc111R3) and Δ 15-dehydrogenase (Nc94F6 and Nc94R8) coding regions, based on sequence alignment to the n. The forward primer was designed to have 3 nucleotides at the 5' end of the initiating methionine

Nc111F2：5’-AAGATGGCGTCCGTCTCCTCTGCCCTTCCC-3’(SEQ ID NO：15)

Nc111R3：5’-TTAGTTGGTTTTGGGGAGCTTGGCAGGCTTG-3’(SEQ ID NO：16)

Nc94F6：5’-AACATGACGGTCACCACCCGCAGCCA-3' (SEQ ID NO: 17). NotI sites added to the 5' end of the oligonucleotide are shown in italics.

Nc94R8：5’-TTACTGGGTGCTCTGAACGGTGTGCG-3' (SEQ ID NO: 18). The Sse8387I site added at the 5' end of the oligonucleotide is shown in italics.

Crassa cDNA was prepared using Marathon cDNA amplification kit (clontech laboratories). The putative dehydrogenases were amplified using these primers and 3' -RACE readycDNA under the recommended cycling conditions by gene amplification PCR System 9700(PE applied biosystems). The PCR product generated with oligonucleotides Nc94F6 and Nc94R8 was ligated to pCR2.1-TOPO (Invitrogen) and named pMON67004 (FIG. 1). The cDNA was sequenced and 3 "histidine-boxes" were found at the positions 124-128, 160-164 and 359-363, a conserved property of membrane-bound dehydrogenases.

The final "HXXHhistidine box motif was also found to be intact compared to other membrane-bound Δ 12 and Δ 15-dehydrogenases. The corresponding Δ 15-dehydrogenase (NcD15D) nucleotide and polypeptide sequences are set forth in SEQ ID NO: 2 and SEQ ID NO: 3, genomic clone set forth in SEQ ID NO: 1, listed in the table. pMON67004 was digested with EcoRI and ligated into the yeast expression vector pYES2/CT at EcoR1 to form pMON77208 (FIG. 2). For plant transformation vectors, pMON67004 was digested with EcoR1, followed by a filling-in reaction, and then digested with Sse 8387I. The gene fragment was ligated into the binary vector pMON73270, which had been digested with NotI, followed by a filling-in reaction, and then digested with Sse 8387I. In the vector pMON77214 (FIG. 4) thus produced, the Δ 15-dehydrogenase gene NcD15D is under the regulation of the seed-specific Napin promoter. The EcoRI/Sse 8387I-digested DNA fragment was also ligated to the binary vector pMON73273, thereby generating pMON77217 (FIG. 4) in which NcD15D is under the regulation of the constitutive 35S promoter.

The PCR product generated with oligonucleotides Nc111R3 and Nc111F2 was directly ligated to pYES2.1/V5-His-TOPO (Invitrogen) to form pMON67005 (FIG. 7A). The cDNA was sequenced and 3 "histidine-boxes" were found at the positions 158-, 162-, 194-, 198 and 394-398 of the amino acids. The final "HXXHl" histidine box motif was also found to be intact compared to other membrane-bound Δ 12 and Δ 15-dehydrogenases. The predicted nucleotide and polypeptide sequences of Δ 12-dehydrogenase (NcD12D) are set forth in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

Example 4

Yeast transformation and expression

Constructs of pMON67005 and pMON77208 were introduced into the host bacterium S.cerevisiae INVSC1 (uracil auxotrophy) using the PEG/Li Ac protocol described in Invitrogen's handbook for pYES2.1/V5-His-TOPO. Transformants were selected on plates made of SC minimal medium containing no uracil and 2% glucose. The transformant clones were inoculated into 5 ml of SC minimal medium containing 2% glucose without uracil and cultured overnight at 30 ℃. For induction, stationary phase yeast cells were pelleted by centrifugation, resuspended in SC minimal medium without uracil supplemented with 2% galactose and cultured at 15 ℃ for 3 days. When exogenous fatty acid was added to the medium, 0.01% LA (. DELTA.9, 12-18:2) and 0.1% emulsifier Tergitol were added. The culture was grown at 15 ℃ for 3 days, followed by centrifugation to collect the culture. The cell pellet was washed once with sterile TE buffer pH7.5, the medium was removed, and lyophilized for 24 hours. Host bacteria transformed with vectors containing the LacZ gene were used as negative controls in all experiments.

For fatty acid analysis, the extraction of yeast lipids was performed after the preceding procedure. Briefly, the lyophilized yeast cell pellet was extracted with 15 ml of methanol and 30 ml of chloroform containing 100. mu.g of trinecanoid. After extraction, the yeast lipids are first saponified to methylate the free fatty acids. The fatty acid methyl ester distribution was then analyzed by Gas Chromatography (GC) using a Hewlett-Packard5890H Plus gas chromatograph (Hewlett-Packard, Palo Alto, Calif.) equipped with a luminescence ionization detector and a fused silica capillary column (Supelcomega; 50 m. times.0.25 mm, ID., Supelco, Bellefonte, Pa.).

In yeast transformed with an expression vector containing LacZ as a control, no LA or ALA was detected in cell lines grown without the addition of LA (18: 3). In yeast transformed with the expression vector containing NcD15D or BnD15D, ALA was not accumulated without adding LA. In the yeast transformed with the NcD 12D-containing expression vector, LA accumulated to 22% of the fatty acid without adding LA, and showed D12D activity. When LA was added to the yeast line expressing NcD15D, ALA was reduced to 1% of the fatty acids. In yeast lines expressing the Brassica napus.DELTA.15-dehydrogenase (BnD15D), ALA was reduced to 0.2% of the fatty acids after the addition of LA. In the LacZ control, no ALA was detected after addition of LA.

TABLE 1 Yeast expression data

Example 5

Transformation of Arabidopsis with NcD15D

This example describes the transformation and regeneration of transgenic Arabidopsis plants expressing a heterologous. DELTA.15-dehydrogenase coding sequence. Seeds were sown in 4-inch pots containing Reverse Osmosis Water (ROW) and saturated MetroMix 200(SCOTTS, Inc., Columbia, OH) to grow Arabidopsis plants. Plant development was artificially promoted by placing the pots in a wet-topped cell in a 4-7 ℃ culture room with 8 hours of light daily for 4-7 days. The unit was transferred to a 22 ℃ culture chamber with a relative humidity of 55% and an average brightness of about 160 and 200 Mol/sec in 16 hours of light per day. After germination, the roof was lifted 1 "backwards to ensure adequate circulation of non-dry air. The wet top was removed when the actual leaves were formed. Plant roots were irrigated with ROW, if necessary, until 2-3 weeks after germination. The roots were then irrigated, if necessary, with a PLANTEX 18-18-5 solution (Plantex Co., Ottawa, Canada) under 50ppm nitrogen. The pots were so thin that 1 plant per pot was maintained 2-3 weeks after germination. Once a plant begins bolting, the primary inflorescence should be trimmed to promote growth of axillary bolting.

Transformation vectors pMON77214 and pMON77217 were introduced into agrobacterium tumefaciens strain ABI using methods known in the art. Transgenic A.thaliana plants were obtained as described by Bent et al (1994) or Bechtold et al (1993). Briefly, cultures of Agrobacterium containing the binary vectors pMON77214 or pMON77217 were grown overnight in LB (10% Bacto tryptone, 5% yeast extract and 10% NaCl containing kanamycin (75 mg/L), chloramphenicol (25 mg/L) and spectinomycin (100 mg/L)). The pellet was centrifuged and resuspended in 5% sucrose +. 05% Silwet-77. Aerial parts of whole a.thaliana plants (about 5-7 weeks of age) were immersed in the resulting solution for 2-3 seconds. Excess solution was blotted off with a paper towel. The soaked plant side was placed on a covered unit and transferred to a culture chamber at 19 ℃. After 16 to 24 hours, the top was removed to allow the plants to stand upright. When the plant develops to maturity, the water is cut off for 2 to 7 days to harvest the seeds. The harvested seeds were screened through stainless steel mesh.

To select transformants, the seeds were plated on agar medium containing 50mg/L glyphosate. Green seedlings were picked and transplanted into 4 "pots and grown under the above conditions. Leaves were collected for fatty acid analysis when the plexus was in the 4-leaf state. After freeze-drying, the leaf fatty acids were analyzed as described above.

Example 6

Functional expression of crassa clones

To evaluate the functional specificity of the n.crassa D15D clone, the coding region of pMON67004 was cloned into a plant expression vector, where the constitutive 35S promoter drives expression of the transgene. The resulting construct pMON77217 was transformed into a. thaliana and leaves of the transformed T2 plant were analyzed for fatty acid composition. In the non-transformed lines about 20% of the fatty acids are LA and about 48% of the fatty acids are ALA. LA levels decreased to approximately 3% and 5% and ALA levels increased to 65% and 63%, respectively, in two separate a.thaliana transformation experiments, showing Δ 15-dehydrogenase activity in plants. These data are summarized in table 2. CONT was designated as control.

TABLE 2 fatty acid content of Arabidopsis leaves

To evaluate the functional specificity of the n.crassa D15D clone to promote ALA production in seeds, the coding region of pMON67004 was cloned into a seed-specific expression vector, where the Napin promoter drives expression of the transgene. The resulting construct pMON77214 was transformed into a. thaliana and seeds of the transformed T2 plants were subjected to fatty acid composition analysis. In the non-transformed lines, about 26% of the seed lipids appeared as LA and about 18% appeared as ALA. In two separate a.thaliana conversion experiments, LA levels were reduced to about 14% and 13%, and ALA levels were increased to 26% and 30%, respectively, showing Δ 15-dehydrogenase activity in the seeds. These data are shown in table 3.

TABLE 3 fatty acid content of Arabidopsis seeds

These results indicate that the protein encoded by the Neurospora NcD15D cDNA is a Δ 15-dehydrogenase functional in plants and is capable of directing ALA synthesis in leaves and seeds.

Example 7

Neurospora crassa delta 15-dehydrogenase activity in canola

Cell lines were transformed with construct pMON77214 containing Napin promoter-driven Neurospora Δ 15-dehydrogenase. Both Quantum and Ebony rape species were transformed, including controls for both. The data shown in Table 4 are from R₀Percentage of 18:2(LA) and 18:3(ALA) in a mixture of 20 seeds of the plant.

TABLE 4 toFrom R₀Percentage of PUFAs in a mixture of 20 seeds of a plant

ALA levels produced in these lines above 12% of the seed fatty acids indicate exogenous Δ 15-dehydrogenase activity. The highest ALA level observed from this conversion was present in the BN-G1395 line, which contained 30.17% ALA.

For several lines expressing pMON77214, fatty acids were assayed in individual seeds and the lines progressed to the next generation. As expected, ALA levels were increased nearly 2-fold in individual seeds relative to the mixture, showing homozygosity of the transgene for the individual segregants in each silique. In the BN-1296 line, the R1 seed mixture contained 25.04% ALA. Up to 48.2% ALA was found in individual seeds of this line (BN-G1296-14). In fig. 3 ALA levels in 200 half seeds are shown, in order of lowest to highest ALA.

Example 8

Cloning of the.DELTA.15-dehydrogenase sequence of nidulans and the.DELTA.12-and.DELTA.15-dehydrogenase sequences of B.cinerea

Gene-specific primers were designed to amplify the full-length coding region of the putative Δ 15-dehydrogenase (andd 15-F1 AnD andd 15-R1) based on sequence alignment with the a. nidulans genomic sequence. The forward primer was designed to contain 3 nucleotides at the 5' end of the initiating methionine

AnD15-F1：5’-AATATGGCTGCAACTGCAACAACCC-3’(SEQ IDNO：23)

AnD15-R1：5’-TTCCGCTTTGGCACCCTTCTTC-3’(SEQ IDNO：24)

Oligonucleotide primers BcD12F1 and BcD12R1 were designed based on partial genomic sequences (a partial gDNA clone owned by Monsanto found with BLASTALL) to amplify the full-length coding region of B.cinerea.DELTA.12-dehydrogenase. Degenerate primers D15D-R9 were designed to amplify any putative b.cinerea Δ 15-dehydrogenase in a 5' -RACE reaction. Oligonucleotide BCD15-F1 was designed for performing a 3' RACE reaction of the PCR products generated by oligonucleotide D15D-R9. Oligonucleotides BcD15F3 and BcD15R1F were designed to amplify the full-length coding region of the putative B.cinerea Δ 15-dehydrogenase.

BcD12F 1: 5'-GTCGACACCATGGCCTCTACCACTGCTCTC-3', 5 ' end contains SalI (SEQ ID NO: 25)

BcD12R 1: 5'-CTGCAGTGCCTTGAGCTTCATTGGTGGTGTA-3', 5 ' end containing PstI (SEQ ID NO: 26)

D15D-R9：5’-GCCRTGNCCRCAYTCRTGNGCANGDAT-3’(SEQID NO：27)

BcD15-F1：5’-ACGATGACTCTCGATTACACAAGTCACCCG-3’(SEQ ID NO：28)

BcD 15-F3: 5'-GTCGACACGATGACTCTCGATTACACAAGTCACC-3', 5 ' end contains SalI (SEQ ID NO: 29)

BcD15R 1: 5'-CTGCAGAATGCTTGAGCTATCAGCAGATCCCAA-3', 5 ' end containing PstI (SEQ ID NO: 30)

A. nidulans and b.cinerea dna were prepared using the GeneRacer kit (Invitrogen). These primers were used with the 3' -RACE ready cDNA to amplify the putative dehydrogenase under the recommended cycling conditions using the gene AmpPCR system 9700(PE APPLIED BIOSYSTEMS). The PCR product encoding A.nidulans.DELTA.15-dehydrogenase was amplified with oligonucleotides AnD15-F1 AnD AnD15-R1, ligated into pYES2.1-TOPO (Invitrogen) AnD designated pMON67010 (FIG. 7B). The cDNA was sequenced and found to have 3 "histidine-boxes" at the amino acid positions 93-97, 129-133 and 327-331, a conserved property in membrane-bound dehydrogenases. The corresponding Δ 15-dehydrogenase (andd 15D) nucleotide AnD polypeptide sequences are set forth in SEQ ID NO: 4 and SEQ ID NO: and 5, list.

cDNA encoding Δ 12-dehydrogenase in b.cinerea was PCR amplified with oligonucleotides BcD12F1 and BcD12R1, which were then directly ligated to pyes2.1/V5-His-topo (invitrogen) to form pMON67022 (fig. 7D). The cDNA was sequenced and 3 "histidine-boxes" were found at the 155-, 159-, 191-, 195-and 390-394 positions of amino acids, a conserved property in membrane-bound dehydrogenases. The predicted nucleotide and polypeptide sequences of Δ 12-dehydrogenase (BcD12D) are set forth in SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

To clone Δ 15-dehydrogenase from b.cinerea, degenerate oligonucleotides were generated according to alignment of the amino acid sequences of n.crassa and Aspergillus sp.Δ 12 and Δ 15-dehydrogenase. After cDNA synthesis, the 5' -end of the putative Δ 15-dehydrogenase was PCR amplified using degenerate oligonucleotide D15D-R9 and ligated to pCR2.1-TOPO. The resulting 742bp fragment was sequenced and determined by inferring similarity to the amino acid sequence of a.DELTA.15-dehydrogenase of other fungi. A664 bp fragment was amplified from the putative B.cinerea.DELTA.15-dehydrogenase 3 'end using a 3' -RACE reaction with oligonucleotide BcD15-F1 and ligated into pCR2.1-TOPO. Oligonucleotides BcD15F3 and BcD15R1 were designed based on the composite sequence of the 5 ' -and 3 ' -RACE products, and the full-length putative B.cinerea.DELTA.15-dehydrogenase cDNA was amplified by the 3 ' -RACE reaction and ligated to pYes 2.1-TOPO. The resulting plasmid was designated pMON67021 (FIG. 7C). The predicted corresponding nucleotide and polypeptide sequences of the Δ 15-dehydrogenase (BcD15D) are set forth in SEQ ID NO: 33 and SEQ ID NO: 34, respectively.

To evaluate the Δ 15-dehydrogenase activity of andd 15D predicted in the yeast expression assay, a substrate for the predicted Δ 15-dehydrogenase, LA, was provided to yeast expressing the enzyme, AnD ALA produced was then quantified. Data for ALA production by the n.crassa Δ 15-dehydrogenase pMON67023 were compared to data for a.nidulans Δ 15-dehydrogenase, as shown in table 5. pMON67023 (FIG. 7E) was constructed as follows:

Nc94F2：5’-AACATGACGGTCACCACCCGCAGCCACAAG-3’(SEQ ID NO：35)

Nc94R2：5’-CTGGGTGCTCTGAACGGTGTGCGCCCAAAT-3’(SEQ ID NO：36)

the region encoding NcD15D without a stop codon was amplified with primers Nc94F2 and Nc94R 2. The resulting fragment was ligated to pYES2.1-TOPO, resulting in an in-frame fusion between the NcD15D coding region and the V5 epitope and 6-histidine region contained in the pYES2.1 expression vector.

TABLE 5 preparation of ALA by Neurospora crassa Δ 15-dehydrogenase and Aspergillus nidulans Δ 15-dehydrogenase

These results indicate that a. nidulans dehydrogenase is about 2-fold more active than NcD15D in this expression system.

TABLE 6 analysis of substrate utilization of AnD15D in Yeast

These results indicate that a. nidulans d15d is capable of dehydrogenating LA and GLA in this expression system.

Example 9

Codon optimization from A.nidulans and N.crassa. delta.15-dehydrogenase for soybean

The codon usage table was constructed from 8 highly expressed seed-specific proteins from soybean (conglycinin, glycinin, globulin) and 17 highly expressed seed-specific proteins from canola (cuciferin, napin, oleosin). The nucleotide sequences NcD15D AnD AnD15D, together with the codon usage table described above, were sent to Blue Heron Biotechnology, Inc. (Bothell, Wa), who then used all algorithms to obtain the final optimized codon sequence with the lowest free energy for RNA secondary structure formation. The optimized codon sequence of NcD15D was synthesized by Blue Heron Biotechnology, Inc. and named NcD15Dnno (SEQ ID NO: 37). The optimized codon sequence of AnD15D was synthesized by Midland (Midland, TX) AnD was designated AnD15Dnno (SEQ ID NO: 38).

Example 10

Activity of Neurospora DELTA 15-dehydrogenase in combination with Mortiella alpina DELTA 6 and DELTA 12-dehydrogenase

The activity of the combination of Neurospora Δ 15-dehydrogenase and Mortierella alpina Δ 6 and Δ 12-dehydrogenase was assessed by transforming canola with construct pMON77216 (FIG. 7G) containing 3 dehydrogenases under the control of the Napin promoter. However, in many of the lines obtained, it was found that the Δ 12-dehydrogenase was partially deleted. The fatty acid content of a mixture of 10 seeds from an individual R0 plant was determined. The levels of stearic acid (18:0) (SA), oleic acid (18:1) (OA), LA, ALA, SDA and GLA are shown in Table 6 below. The control line was Ebony. The seed mixtures from multiple transgenic experiments contained measurable SDA, with SDA accumulating more than 10% of the fatty acids in 8 experiments.

Table 6 relative area percent results (approximate weight percent) for R1 seed blends

Fatty acid data, including homozygotes and heterozygotes, from single seeds of experiment BN-G1824 are shown in table 7 below. In one example, 18.6% SDA, 17.8% ALA, 11.2% LA, 24% oleic acid, and 18.8% GLA were observed. This example is considered to be an example of high SDA/high GLA. 16.8% SDA, 7% ALA, 2% LA, 62.1% oleic acid and 3.1% GLA were observed in another seed in this example, which is considered to be a high SDA/low GLA line. Molecular data show that the Δ 12 coding sequence is not functional in the high SDA/low GLA lines. It was also shown in particular that the high SDA/low GLA line contained a single copy of a partial T-DNA insertion that lost all of the inserted DNA between the left border and the terminal 51 base pairs of the Mortierella Δ 12-dehydrogenase coding region (e.g., the last 51 bases of SEQ ID NO: 41). Notably, the levels of oleic acid were close to those of the wild type in the high SDA/low GLA lines, while the levels of oleic acid were about 2.5-fold lower in the high SDA/high GLA lines compared to the wild type. Lines showing a high SDA/high oleic phenotype are highlighted in grey.

TABLE 7 relative area percent results (approximate weight percent) for single R1 seed of BN _ G1190

To further evaluate N.crassa. Activity of the combination of Δ 15-dehydrogenase and M.alpina Δ 6-and Δ 12-dehydrogenases, a line homozygous for the construct pCGN5544 (containing M.alpina Δ 6-and Δ 12-dehydrogenases), containing up to 35% GLA in its seed oil, was again transformed with the construct pMON77214 containing NcD 15D. Analyzed the R from 11 species₀A mixture of 20 seeds of plants. The levels of LA, ALA, SDA and GLA in these lines are shown in table 8.

Table 8 analysis of relative area percent results (approximate weight percent) for R1 seed blends

Example 11

Neurospora crassa. Activity of a combination of Δ 15-dehydrogenase and Mortierella Δ 6-dehydrogenase

Evaluation of neurospora crassa was evaluated by transformation of canola with construct pMON77215 (fig. 7F) containing two dehydrogenases under the control of the Napin promoter. Δ 15-dehydrogenase and Mortierella alpina Δ 6-dehydrogenase. This vector was constructed by NotI digested pCGN5536(u.s. patent No. 6,459,018B 1) with Napin promoter driven m.alpina Δ 6-dehydrogenase (MaD6D) expression therein, followed by ligation of the expression cassette fragment to the NotI site of the binary vector pMON70660 to form pMON 77212. The pMON77215 plasmid was constructed by digesting pMON77214 with PmeI and AscI and then ligating the resulting Napin-NcD15D expression cassette fragment to the SwaI and AscI sites of pMON77212 to form a construct containing both MaD6D and NcD 15D.

The fatty acid content of a mixture of 10 seeds from a single R0 canola transformant was determined. The levels of SA, OA, LA, ALA, SDA and GLA are shown in Table 9 below. The control line was Ebony (SP 30052). The mixture of seeds produced from multiple transgenic experiments contained measurable SDA, with 25% of the experiments (10 out of 40) accumulating more than 10% of the fatty acids.

TABLE 9 relative area percent results (approximate weight percent) in R1 seed blends of pMON77215

Fatty acid data, including homozygotes and heterozygotes, from single seeds of experiment BN-G1860 are shown in table 10 below. In one example, up to 19% SDA, 10% ALA, 7% LA, 48% oleic acid, and 5% GLA were observed.

Table 10 area percent results (approximate weight percent) of pMON77215 single R1 seed relative for BN _ G1860

Example 12

Codon optimization of the delta 15-dehydrogenase sequence from n.crassa. for maize

Codon usage tables were constructed from 9 highly expressed seed-specific genes of maize (6 zeins and 3 oleosins). Using this table, two codons of NcD15D were mutated using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) and the resulting sequence was named NcFAD3m (SEQ ID NO: 42). The codon change operation was as follows: 1) a more preferred translation initiation site is obtained by altering the first base of the second codon (SEQ ID NO: 42) to GCG, such that SEQ ID NO: 2 by substitution of one alanine with threonine; and 2) to remove a rare codon, a valine codon was added at position 882 (SEQ ID NO: 42) change from GTA to GTG.

Example 13

EPA equivalent value

The standard for measuring seed oil for health quality is the EPA equivalence. This value reflects the rate of metabolic conversion to EPA. This value was obtained by dividing the percentage of ALA by the percentage of 14 and SDA by the sum of 4. The double low canola oil compositions obtained by the present inventors have high EPA equivalence values, indicating the property of having health benefits in humans and animals associated with increased EPA levels. The following analytical results were obtained by comparing conventional double low canola oil with the example consisting of a typical high SDA oil containing 10% ALA and 15% SDA. The traditional type of double low rape oil contains about 12% ALA and 0% SDA with an EPA equivalent of 12/14+0/4 to 0.8. In contrast, the EPA equivalent for the example of high SDA oil composition is 10/14+15/4 — 4.4. The corresponding values are shown below. This value is based on weight% and not on application. The great difference indicates the importance of SDA produced in double low rape oil.

TABLE 11EPA equivalence comparison

Vegetable oil	Total omega-3 (% fatty acids)	n-6: n-3 ratio (% fatty acid)	Corresponding EPA equivalence (weight% ALA + SDA)
				Double low rape	12	2.6∶1	0.8
SDA double low rape	50	1∶5	4.4

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Claims

1. An isolated polynucleotide whose nucleic acid sequence encodes a polypeptide having dehydrogenase activity for dehydrogenation at carbon 15 of a fatty acid molecule, wherein the isolated polynucleotide is selected from the group consisting of:

(a) encoding the amino acid sequence of SEQ ID NO: 3;

(b) the nucleic acid sequence is SEQ ID NO: 1 or SEQ ID NO: 2.

2. The isolated polynucleotide of claim 1, wherein the polynucleotide is from a gate selected from the group consisting of: selected from zygomycota, basidiomycota and ascomycota.

3. The isolated polynucleotide of claim 1, wherein the polynucleotide is from a species selected from the group consisting of: neurospora crassa, Aspergillus nidulans and Botrytis cinerea.

4. The isolated polynucleotide of claim 1, wherein the polynucleotide encodes the polypeptide of SEQ ID NO: 3.

5. The isolated polynucleotide of claim 1, wherein the polynucleotide comprises the sequence of seq id NO: 1. SEQ ID NO: 2.

6. A recombinant vector comprising the isolated polynucleotide of claim 1.

7. The recombinant vector of claim 6, further comprising at least one additional sequence selected from the group consisting of:

(a) a regulatory sequence operably linked to the polynucleotide;

(b) a selectable marker operably linked to the polynucleotide;

(c) a marker sequence operably linked to the polynucleotide;

(d) a purification moiety operably linked to the polynucleotide; and

(e) a target sequence operably linked to a polynucleotide.

8. The recombinant vector of claim 6, further defined as comprising a promoter operably linked to the isolated polynucleotide.

9. The recombinant vector of claim 8, wherein the promoter is a developmentally regulated, organelle-specific, tissue-specific, constitutive, or cell-specific promoter.

10. The recombinant vector of claim 8, wherein the promoter is selected from the group consisting of 35SCaMV, 34S FMV, Napin, 7S, Glob, and Lec.

11. The recombinant vector of claim 6, defined as an isolated expression cassette.

12. The recombinant vector of claim 6, further defined as comprising a nucleic acid sequence encoding a polypeptide having dehydrogenase activity for dehydrogenation at carbon 6 of the fatty acid molecule, and/or a nucleic acid sequence encoding a polypeptide having dehydrogenase activity for dehydrogenation at carbon 12 of the fatty acid molecule.

13. A fungal polypeptide comprising SEQ ID NO: 3.

14. Use of the recombinant vector of claim 6 for the preparation of a transgenic plant.

15. The use of claim 14, wherein the plant contains a nucleic acid sequence encoding a polypeptide having dehydrogenase activity for dehydrogenation at carbon 6 of a fatty acid molecule.

16. A host cell transformed with the recombinant vector of claim 6.

17. The host cell of claim 16, wherein said host cell expresses a protein encoded by said vector.

18. The host cell of claim 16, wherein the cell inherits the recombinant vector from an ancestor of the cell.

19. The host cell of claim 16, wherein the cell has been transformed with the recombinant vector.

20. The host cell of claim 16, wherein said host cell is a plant cell.

21. A method of preparing seed oil containing omega-3 fatty acids from plant seeds comprising the steps of:

(a) obtaining plant seeds according to claim 14; and

(b) extracting oil from the seeds.

22. A method of producing a plant comprising seed oil having an increased level of omega-3 fatty acids, comprising introducing the recombinant vector of claim 6 into an oil-producing plant.

23. The method of claim 22, wherein introducing the recombinant vector comprises growing a plant.

24. The method of claim 22, wherein introducing the recombinant vector comprises the steps of:

(a) transforming a plant cell with the recombinant vector of claim 6; and

(b) regenerating said plant from a plant cell, wherein the plant has an increased level of omega-3 fatty acids.

25. The method of claim 22, wherein the plant is selected from the group consisting of arabidopsis, oilseed brassica, rapeseed, sunflower, safflower, canola, corn, soybean, cotton, flax, jojoba, chinese tallow tree, tobacco, cacao beans, peanuts, fruit trees, citrus plants, nut and berry producing plants.

26. The method of claim 22, wherein the plant is further defined as transformed with a nucleic acid sequence encoding a polypeptide having dehydrogenase activity for dehydrogenation at carbon 6 of a fatty acid molecule.

27. The method of claim 26, wherein stearidonic acid is increased.

28. The method of claim 22, further defined as comprising introducing the recombinant vector of claim 6 into a plurality of oil-producing plants and selecting plants having a desired omega-3 fatty acid profile from the plants or progeny thereof from which the recombinant vector is inherited.