Alteration of the Fatty Acid Composition of Vegetable Oils

Considerable progress has been made in the genetic alteration of the relative amounts of palmitic, stearic, oleic, linoleic, and α-linolenic acids in seed oils of crop plants. These modifications have been achieved primarily by either up- or downregulating expression of genes for fatty acid desaturases or acyl-ACP thioesterases. The production of seed oils with high levels of oleic acid represents the most significant commercial achievement to date in the metabolic engineering of seed oil composition. This oil modification was achieved in numerous crop species by blocking expression of FAD2 genes, through both transgenic and chemical mutagenic approaches. The major remaining target is development of temperate crops with seed oils that are solid at room temperature and, therefore, do not require hydrogenation for use in margarine production. Achieving this target will require the engineering of seeds to produce high levels of saturated fatty acids that are sequestered in TAG, but not accumulated in membrane lipids such as PC. The enrichment of saturated fatty acids in PC and other phospholipids likely compromises membrane integrity, especially when seeds are subjected to low germination temperatures (A&a0cute; lvarez-Ortega et al., 1997; Knutzon et al., 1992; Liu et al., 2002).

Metabolic engineering of novel fatty acid synthesis and accumulation in seeds of transgenic plants has met with limited success. cDNAs for numerous divergent fatty acid modification and biosynthetic enzymes have been identified. These include variant forms of acyl-ACP desaturases, acyl-ACP thioesterases, acyl-CoA desaturases, Δ¹²-oleic acid desaturases, fatty acid elongases, cytochrome P450s, and cytochrome b₅-fusion desaturases (Voelker and Kinney, 2001). The availability of these cDNAs offers numerous possibilities for the metabolic engineering of seeds with enhanced nutritional, industrial, and animal feed properties. The development of canola seeds with high lauric acid content for detergent applications and the development of canola and soybean seeds with high GLA content for nutraceutical applications are perhaps the most notable technical successes in this research area (Del Vecchio, 1996; Sato et al., 2004).

Despite these accomplishments, most metabolic engineering efforts have resulted in the development of seeds with only low to moderate levels of unusual fatty acids. In general terms, a major limitation on unusual fatty acid accumulation in transgenic seeds appears to be the inefficient flux from the site of synthesis to the final deposition in oil bodies as a component of TAG (Cahoon et al., 2007). In the case of divergent forms of FAD2, such as epoxygenases, conjugases, acetylenases, and hydroxylases, the modification reaction occurs while the fatty acid substrate is bound to the membrane lipid PC. The unusual fatty acid product then must be efficiently removed from PC and mobilized onto glycerol backbones for storage as TAG in lipid bodies. This movement or channeling of novel fatty acids between PC and TAG likely involves specialized forms of metabolic enzymes such as acyltransferases and phospholipases that are absent from seeds of transgenic plants. The lack of these specialized enzymes could result in the aberrant accumulation of novel fatty acids in membrane phospholipids in seeds of transgenic plants, as has been observed for the production of acetylenic and conjugated fatty acids (Thomaeus et al., 2001; Cahoon et al., 2006). The accumulation of medium-chain length fatty acids also appears to be limited by inefficient incorporation onto glycerol backbones for TAG production in seeds of transgenic plants. In this case, the synthesis of decanoic and lauric acids in plastids of transgenic B. napus seeds by the activity of divergent acyl-ACP thioesterases resulted in the enrichment of decanoyl-CoA and lauroyl-CoA, relative to CoA esters of common fatty acids, in acyl-CoA pools (Larson et al., 2002). A similar enrichment was not observed in seeds of Cuphea hookeriana, which naturally accumulate high levels of these fatty acids. In addition, seeds of developing B. napus that have been engineered to produce decanoic (10:0) and lauric (12:0) acids contained elevated amounts of these fatty acids in phospholipids, relative to seeds that naturally accumulate these fatty acids (Wiberg et al., 2000). These results suggest that specialized forms of metabolic enzymes such as acyltransferases are also important for the storage of unusual medium-chain length fatty acids generated in seed plastids. Inefficient incorporation of novel fatty acids into TAG, as evidenced by their enrichment in acyl-CoA pools, may ultimately induce b-oxidation for the breakdown of these fatty acids. Such a phenomenon has been observed in seeds that have been engineered to produce mediumchain- length fatty acids and epoxy-fatty acids (Eccleston and Ohlrogge, 1998; Moire et al., 2004).

Also poorly characterized is the intracellular organization of enzymes associated with the synthesis and metabolism of unusual fatty acids. These enzymes might be closely associated in specific physical or metabolic domains. The synthesis of petroselinic acid, for example, appears to involve the association of at least three enzymes in a biosynthetic complex or metabolon, and it cannot be ruled out that specialized plastids have evolved for the synthesis of this fatty acid (Cahoon and Ohlrogge, 1994; Schultz et al., 1996). In addition, it is possible that variant FAD2s (e.g., hydroxylases, epoxygenases, conjugases) (Fig. 7.4 and Table 7.5) naturally function in discrete domains of the ER that contain specialized forms of acyltransferases and phospholipases that efficiently metabolize novel fatty acids following their synthesis on PC.

In addition, aspects of gene expression and protein accumulation have not been well characterized in most attempts to produce unusual fatty acids in seeds of transgenic plants. Promoters for seed storage protein genes are typically used to mediate the expression of transgenes in these experiments. Such promoters might not provide the proper timing or levels of gene expression in the engineered seeds, particularly when compared to seeds that naturally accumulate large amounts of unusual fatty acids. It is also possible that certain enzymes associated with unusual fatty acid synthesis and metabolism are prone to high rates of turnover in transgenic plants, which may affect levels of unusual fatty acid accumulation.

Clearly, a basic understanding of the underlying enzymology, cell biology, and gene expression associated with unusual fatty acid synthesis and metabolism is essential in facilitating efforts to produce novel vegetable oils in existing crop plants, while maintaining the agronomic viability of the engineered seeds.