Metabolic Engineering of High Oleic Acid Vegetable Oils

The most significant achievement in the metabolic engineering of oilseed crops has been the alteration of the unsaturated fatty acid content of vegetable oils. Anotable example is the development of vegetable oils with oleic acid content exceeding 70% of the total fatty acids (Kinney, 1996). Such oils have high oxidative stability (or increased shelf life) and have beneficial health properties, especially compared to o-6 rich oils such as those obtained from soybean seeds. The high oleic acid trait has been developed in most of the major oilseed crops through either transgenic or mutagenic approaches (Auld et al., 1992; Bruner et al., 2001; Buhr et al., 2002; Liu et al., 2002; Norden et al., 1987; Soldatov, 1976). In all reported cases, these oils result from the suppressed expression of FAD2, the ER D12-oleic acid desaturase that converts monounsaturated oleic acid to polyunsaturated linoleic acid (Table 7.4 and Fig. 7.4). In the transgenic approaches, downregulation of FAD2 gene expression has been achieved by sense and antisense suppression, or byRNAinterference (RNAi) (Kinney, 1996; Liu et al., 2002; Smith et al., 2000). This is typically conducted using seed-specific promoters, which help to ensure that the biological and physical properties of membranes are not compromised in vegetative parts of the plant.

High oleic acid lines of most of the major oilseed crops have been developed by screening of chemically mutagenized seed populations (Auld et al., 1992; Bruner et al., 2001; Norden et al., 1987; Soldatov, 1976). This approach has proven to be especially effective for the generation of high oleic acid lines of sunflower and peanut that also have acceptable agronomic properties. In contrast, the oleic acid content of seeds from FAD2 mutants of crops such as soybean typically varies in response to environmental conditions, particularly temperature (Carver et al., 1986; Kinney, 1994). This property has precluded commercialization of high and midoleic acid mutants of these crops. The environmental instability of the oleic acid content of soybeanmutants is likely due to the presence of at least three FAD2 genes, designated GmFAD2–1a, GmFAD2–1b, and GmFAD2–2, combined with the known influence of temperature on FAD2 activity (Cheesbrough, 1989; Heppard et al., 1996; Tang et al., 2005).GmFAD2–1a and b are expressed primarily in seeds, andmutations in these genes likely account for the majority of the oleic acid phenotype in high oleic acid mutants (Heppard et al., 1996; Kinney, 1996). The expression levels of these genes are not significantly affected by temperature (Heppard et al., 1996; Tang et al., 2005). Instead, the activities of the corresponding enzymes appear to be differentially regulated through posttranslational mechanisms in response to temperature (Cheesbrough, 1989; Tang et al., 2005). The GmFAD2–1a and b polypeptides, for example, display different turnover rates when expressed in heterologously in yeast at various growth temperatures (Tang et al., 2005). In addition, because at least three FAD2 genes are expressed in soybean seeds, the achievement of a high oleic phenotype would require mutations in each of these genes, including GmFAD2–2, which is also expressed in vegetative organs. Seedlings from such mutants would likely be poorly equipped to respond to low temperatures by increasing membrane unsaturation. Even A. thaliana lines with mutations in the single FAD2 gene display reduced seed germination and seedling vigor at low temperatures (Miquel and Browse, 1994). These examples illustrate the types of difficulties that can arise with the agronomic development of mutants for genes, such as FAD2, that are critical to plant growth and development, as well as the difficulties associated with the breeding of phenotypes controlled by multigene families.