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I. INTRODUCTION Lipids form a vital component of many cell constituents and are an important source of energy. Oils and fats also contribute significantly as a functional ingredient in improving the sensory characteristics of numerous processed food products.

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About 70% of edible oils are derived from plant sources (about 50 million tonnes/annum), and there are three major groups of oil-producing crops. These are temperate annual oilseeds (soy, rapeseed, sunflower, and peanut), about 60% of the total vegetable oil production; perennial tropical crops (oil palm, coconut, and babassu nut), about 25% of total oil production; and crops such as cotton and corn where the embryo is a by-product of processing. This latter group accounts for about 10% of total vegetable oil production. The remaining 5% of total vegetable oils are derived from miscellaneous niche crops such as olive (3%), linseed (1%), and sesame (1%).

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Many oils are also used for nonfood applications (about 2% of total production). The plant oils most commonly used for industrial purposes include coconut, castor, linseed, and soy. The predominant source of industrial fatty acids, however, is tall oil, a by-product of the wood pulp and paper mill industry

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The utility of a given oil in a food or industrial application is determined primarily by acyl composition of the storage triacylglycerol (TAG). For most edible oils the acyl composition of the TAG is qualitatively the same as that of the membrane lipids. That is, the same five acyl groups palmitate (16:0), stearate (18:0), oleate (18:1), linoleate (18:2), and linolenate (18:3) occur in both lipid classes, often in the same molar ratios. The degree of fatty acid desaturation determines both the melting range and the thermal stability of the oil. Industrial oils such as castor, which is rich in hydroxy-fatty acids, often contain fatty acids with functional groups other than methylene-interrupted double bonds. These functional groups determine the reactivity and cross-linking ability of oils used for such applications as paints and coatings.


II. SHORT- AND MEDIUM-CHAIN SATURATEDFATTY ACIDS There are basically two classes of oilseed species with high levels of saturated fatty acids in their oil. The classes are divided by the presence or absence of saturated fats at the sn-2 position of their TAG. Coconut oil is the best known example of an oil with >90% of its acyl chains saturated (C8:0 to C16:0, predominantly C12:0) and thus containing saturated fats at the sn-2 position. In this species both the fatty acid elongation machinery and the acyl transferase (LAPAAT) that transfers acyl chains to the sn-2 position are modified. Other examples of these types of oils can be found in many plants including the Lauraceae, Myristicaceae, and Lythraceae, which have predominantly medium-chain (C10:0 to C14:0) acyl chains in all three sn TAG positions in their seed oil.

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In most oils saturates are not found at the sn-2 position of TAG because normal LPAAT cannot use saturated acyl chains as substrates. Thus, the second class of oilseed accumulates saturated acyl chains only up to about 60% of the total oil. In these oils the specificity of LPAAT has excluded16:0 and 18:0 acyl chains from the sn-2 position of TAG. Examples of these oils are cocoa butter (30% 16:0, 30% 18:0) and oil palm mesocarp (40% 16:0).

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The major biochemical determinant for seed oils containing medium chain (C8:0 to C16:0) fatty acids is now known to be the presence in the developing seed of a specialized acyl-acyl carrier protein (ACP) thioesterase that can redirect the common fatty acid synthase complex to produce medium- chain fatty acids, which are then incorporated into TAG formation. Calgene group, first purified and cloned a medium-chain fatty-acyl-ACP thioesterase from developing California bay seeds. Overexpression of this enzyme in seeds of both Arabidopsis and canola redirected fatty acid accumulation in the TAG of both seeds to accumulate up to 40% laurate. Complementary DNAs (cDNAs) encoding other mediumchain thioesterases have been cloned from a variety of plants whose seeds are enriched in medium-chain fatty acids, most notably Cuphea sp., coconut, and elm.

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It is now clear that all of these are members of the fat B gene subfamily. The production of medium-chain fatty acids (C8:0 to C14:0) in developing seeds of Cuphea is catalyzed by the seed- specific expression of these novel fat B enzymes. Beside fat B, other modifications are required to optimize short-chain production in vivo. In Cuphea sp. it has been shown that the fatty acid synthase was also modified to produce shorter chain fatty acids. In Cuphea wrightii, the short-chain ß-ketoacylsynthetase (kas A) somehow increased the efficiency of the medium-chain alkyl-ACP thioesterases. This hypothesis was supported by the work of Dehesh et al., who isolated embryo-specific has A cDNAs from C8:0- and C10:0-producing Cuphea species and functionally tested the encoded polypeptides in seeds of transgenic canola.


III. LONG CHAIN SATURATED FATTY ACIDS Two major targets for fatty acid modification of edible oils are increased monunsaturates (18:1) with a concomitant decrease in polyunsaturates (18:2, 18:3), combined with a reduction in total saturates (16:0, 18:0). This would yield more a chemically stable oil (high 18:1) with reduced total saturated fat content. Another important target has been to increase the total 18:0 and 18:1 content of the plant oil so that it has an acceptable solid fat functionality for use in margarine and other confectionery applications but without the perceived deleterious health effects of partially hydrogenated oils rich in trans-fatty acids.

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It has been shown that the fat B class of acyl:ACP thioesterases control the release of 16:0 into the cytoplasm, making it available for TAG biosynthesis. Thus, inactivation of fat B should give a low 16:0 phenotype. Conversely, overexpression of fat B should give a high 16:0 phenotype. The insertion of the first double bond into fatty acyl chains is a plastidic reaction catalyzed by a soluble desaturase enzyme, stearoyl-ACP desaturase (AAD1). Inactivation of the aad 1 gene should result in a high 18:0-coenzyme A (CoA) pool in the cytoplasm and a resultant high 18:0 content of seed TAG.

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Oleoyl-CoA is also incorporated into membrane phosphatidylcholine (PC), where it is desaturated to linoleoyl-PtdCho by a membrane-bound 5-12 desaturase encoded by a fad 2 gene. Inactivation of the fad 2 gene should give a high 18:1 phenotype.

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High-oleic mutants of corn, peanut canola, and sunflower have been described, with an 18:1 ranging from 60 to 90%. The high-oleic sunflower is particularly noteworthy because it is a dominant mutation in a structural fad 2 gene. Transgenic soy lines in which the seed specific expression of fad 2 has been inactivated result in a consistently high oleic (>80%) content of the seed oil. Low-saturate mutants of soy also exist, primarily due to altered fat B activity. Transgenic soy lines in which the fat B thioesterase activities are suppressed have a 50% reduction in total 16:0 and are dominant. Transgenic soybean plants overexpressing fat B also produce >40% 16:0 in the seed oil but as a dominant trait.


IV. MONOUNSATURATED FATTY ACIDS Oleic acid (?9 18:1) is a component of most common plant oils, ranging from about 20% in soybean to over 80% in some mutant varieties of canola and sunflower. The ? 9 desaturase enzyme normally inserts a double bond at the ?9 position of stearoyl-ACP to form oleoyl- ACP. There are, however, a number of naturally occurring oils that contain monounsaturated fatty acids with double bonds in positions other than the ? 9 carbon from the carboxyl group. For, example, seeds of the Umbelliferae species such as carrot and coriander contain oils rich in petroselenic acid (?6 18:1). This unusual monounsaturate is the result of the activity of a seed-specific plastidial ? 4 desaturase that converts palmitoyl-ACP to ? 4 hexadecanoyl-ACP, which is then elongated to petroselenoyl-ACP. Thus, this soluble desaturase has both a different substrate (chain length) specificity and different region specificity from the canonical stearoyl-ACP desaturase, yet based on its primary amino acid sequence it is clearly a member of the same gene family.

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Gene family (aad 7) includes a number of other members that vary from the standard ? 9 18:0-ACP in their substrate specificity, regiospecificity, or both. Additional examples include the ? 6 18:0-ACP desaturase from Thunbergia alata and ? 9 16:0-ACP desaturases from Doxantha spp. and from Asclepia syriaca, all of which have >70% amino acid sequence similarity to ? 9 18:0-ACP desaturases. It has been shown that mutations in as little as five amino acids can produce changes in both substrate specificity and regiospecificity of an acyl-ACP desaturase, for example, converting a ? 6 16:0-ACP desaturase into a ? 9 18:0-ACP desaturase. Despite this strong conservation of primary sequence in the aad 1 gene family, scientists failed to express the coriander ? 4 palmitoyl- ACP desaturase in a standard oilseed (e.g., soybean, sunflower) in order to produce large amounts of petroselenic acid instead of oleic acid. This is because the Umbelliferae species producing this fatty acid have a modified fatty acid biosynthetic pathway. Adaptations include a modified has A gene product resulting in a condensing enzyme with specificity for ? 4 hexadecanoyl-ACP and a modified fat B gene encoding a petroselenyl-ACP thioesterase, plus other additional variants including acyltransferases, ferredoxins, and possibly ACPs. These observations demonstrate both the complexity of the evolutionary process that has resulted in divergent oils and the technical challenges of producing novel fatty acids in oilseed plants.

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Very long chain monounsaturated fatty acids (VLCFAs, 20-26 carbons) are found in the storage oils of some plants, such as members of the Cruciferae family, and in epicuticular and storage wax esters. Most unsaturated VLCFAs are the result of the elongation of oleoyl-CoA by a membrane- bound elongase complex. Waxes, esters of long-chain alcohols and fatty acids, are abundant in plants as a cell wall component to give hydrophobicity, resistance to fungal attack, and light absorption and reflection. Jojoba (Simmondsia chinensis) is the only angiosperm known to accumulate liquid waxes in its seeds as an energy store. Up to 60% of the seed dry weight consists of linear wax esters of ?-9 monounsaturated C20, C22, and C24 fatty acids and alcohols. TAG is absent. Acyl-CoA-based chain elongation was responsible for the long-chain acyl chains of jojoba wax.

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Calgene group cloned cDNA of all three components, a jojoba ß-ketoacyl-CoA synthase, a fatty acyl reductase, and a wax synthase proposed as necessary for wax synthesis. In transgenic Arabidopsis seed expressing all three of these cDNAs, large quantities of short-chain liquid wax accumulated, representing up to 70% of the seed oil. An Arabidopsis gene (fae 1) related to the jojoba condensing enzyme (ß-ketoacyl-CoA synthase) has been cloned by insertional mutagenesis. Fae 1 was shown to complement Arabidopsis mutants that have reduced 20- C fatty acids and increased oleic acid and to produce VLCFAs when expressed in tobacco. It seems probable based on these observations that a single condensing enzyme controls the elongation of CIS to C20, C22 acyl CoAs in VLCFA-producing seed.


V. POLYUNSATURATED FATTY ACIDS The 18-carbon polyunsaturated fatty acids (PUFAs) linoleic and ?-linolenic acids are essential components of plant membranes (18:2, 18:3). PUFAs are also substrates for the lipoxygenase-mediated production of various volatile compounds including methyl jasmonate –signaling agents in response to pathogen attack and as flavoring components in the area of food and fragrance chemistry. The PUFAs ?-linoleic and linolenic acids are widely distributed in nature. In addition, ?-linolenic acid (GLA) is found in the seeds of a few plant species (borage, evening primrose, black currant) as well as cyanobacteria and fungi. Longer chain PUFAs such as arachidonic acid (ARA 20:4) are found in microorganisms such as the fungus Morteriella alpina and the marine diatom Porphyridiom cruentum. The related LC-PUFAS eicosapentanoic acid (EPA 20:5n) and docosahexanoic acid (DHA 22:6n) are found in marine microorganisms and in fish oil.

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Both linoleic and ?-linolenic acids are essential fatty acids in the human diet because the ?12 and n-3 desaturase activities necessary to convert oleic acid to these fatty acids are lacking in most mammalian microsomes. The dietary PUFAs (18:2, 18:3) are used primarily in mammals as precursors for the eicosanoids including prostaglandins and leukotrienes. Because of the known beneficial effects associated with the intake of LC-PUFAs in the diet of both infants and adults and the limited natural sources commercially available, there has been much interest lately in the possibility of producing ARA, EPA, and DHA in plant seed oils, where they do not normally occur.

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In higher eukaryotes, the major long-chain PUFA arachidonate (ARA 20:4n-6) is derived from linoleic acid (18:2, and EPA (20:5n-3) and DHA (22:6n-3) from ?-linolenic acid (ALA). The metabolic pathways for longchain PUFA formation have been known for some time, but until recently none of the proteins had been identified

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cDNA databases of PUFA-containing organisms using conserved desaturase and elongase domains was identified. Similarly ?5 and ? 6 desaturases and the 18:3 n-6 elongase from the ARA-accumulating filamentous fungus Morteriella alpina were identified. Using similar approaches, orthologues of the ? 5 desaturase and the 18:3n-6 elongase have been identified from C. elegans and human cDNA EST databases respectively. Rapid functional identification of putative cDNA clones was carried out by expression in yeast. This technique enabled identification of individual positive cDNA clones and led to the possibility of producing these very long chain PUFAs in the oil of transgenic Plants.

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Coexpression of M. alpina ?6 and ? 12 desaturases in canola seed resulted in a GLA content of about 40% of the seed oil fatty acids. Similarly, seed-specific expression of the borage ? 6 desaturase in transgenic soy seed resulted in a similar total GLA content in the seed oil. With the cloning of all of the individual components of the eukaryotic pathway of LC-PUFA formation, the technical challenges remaining are those of metabolic pathway engineering. For example, it will be necessary to co-express up to six different enzymes simultaneously in a developing oilseed in order to produce DHA from ?-linolenic acid in transgenic plants. Finally, it should be noted that some marine microorganisms (bacteria and diatoms) have a completely different pathway of LC-PUFA formation using polyketide synthases.

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