Metabolic Engineering of High and Low Saturated Fatty Acid Vegetable Oils

Palmitic acid (16:0) and stearic acid (18:0) are the primary saturated fatty acid components of the seed oil of most crops. Considerable research effort has been devoted to either increasing or decreasing the content of these fatty acids in seed oils for specific commercial applications. For example, the reduction of saturated fatty acids is generally believed to result in vegetable oils with improved cardiovascular health properties. Conversely, enhancement of saturated fatty acid content results in oils with improved oxidative stability and increased melting point. The latter property is especially important for confectionary applications and margarine production. The use of conventional vegetable oils in margarine production requires chemical hydrogenation to reduce the double bonds of polyunsaturated fatty acids. The resulting oil is solid at room temperature, but contains trans-fatty acids that have been increasingly linked with elevated total- and low density lipoprotein (LDL)-cholesterol levels in humans (Hu et al., 2001). As a result, increased emphasis has been placed on metabolic engineering of oilseeds to produce high levels of saturated fatty acids, especially stearic acid, so that the oil does not require hydrogenation for use in margarine production.

Seed oils with increased or decreased amounts of palmitic acid have been achieved through alteration of the expression levels of genes for the FatB acyl-ACP thioesterase. As described previously, this enzyme releases 16:0 and, to a lesser extent, 18:0, from ACP in plastids. By enhancing the expression of FatB genes using strong seed-specific promoters, oils that contain 30–40% 16:0 have been generated in seeds of a number of plants including A. thaliana, canola, and soybean (Do¨rmann et al., 2000; Kinney, 1996). In contrast, 16:0 typically comprises 5–15% of the seed oil of most plant species. Downregulation of FatB expression in seeds has the opposite effect on the 16:0 content. Using transgenic approaches, and through the development of mutants, seed oils with as little as 2–5% 16:0 have been achieved in a variety of plants (Do¨rmann et al., 2000; Kinney, 1996; Li et al., 2002; Schnebly et al., 1994). In addition, an A. thaliana T-DNA-insertion mutant of the FatB1 locus was described that contained reduced 16:0 content throughout the plant, including <4% 16:0 in seeds (Bonaventure et al., 2003). Interestingly, this mutant has reduced vegetative growth, indicating an essential role of 16:0 in plant growth and development.

Genetic enhancement of the stearic acid (18:0) content of seeds has been achieved by downregulating or blocking expression of the gene for the stearoyl- ACP desaturase, the enzyme that catalyzes the conversion of 18:0 into the monounsaturated 18:1Δ9 (Table 7.4). Transgenic and mutagenic approaches have proven successful for suppressing expression of genes for the stearoyl-ACP desaturase in seeds. An early example of the transgenic production of high stearic acid seeds was achieved through antisense suppression of stearoyl-ACP desaturase genes in canola and turnip rape (Knutzon et al., 1992). RNAi methods were used to generate cotton seeds with high 18:0 content. In both cases, seeds were obtained with oils containing 30–40% 18:0 (Liu et al., 2002). The resulting seeds, however, germinated poorly, particularly at low temperature. Growth temperature has also been shown to have a major impact on 18:0 accumulation in seeds of a sunflower high stearic acid mutant (Fernández-Moya et al., 2002). When these plants were maintained at day/night temperatures of 35/25 °C, stearic acid comprised nearly 35% of the seed oil. In contrast, when the plants were grown at day/night temperatures of 20/10 °C, levels of 18:0 in seeds decreased to ~8% of the total fatty acids, essentially the same amounts present in the nonmutant parental seeds. These results reflect the plant cell’s ability to adjust fatty acid composition in order to maintain the fluidity of membranes. At low temperatures, the presence of high levels of 18:0, which has a melting point of 70 °C, likely disrupts the integrity of membranes and perhaps oil bodies and, as a result, compromises the viability of cells. It should be noted that in seeds engineered to produce high levels of 18:0, the content of this fatty acid is not only increased in the storage oil but also in microsomal membranes (A´ lvarez-Ortega et al., 1997).

From a metabolic engineering standpoint, one would predict that an 18:0 content of 70–80% should be attainable in seeds by suppression of stearoyl-ACP desaturase expression. These levels of oleic acid are routinely obtained by suppression of FAD2 expression in seeds of transgenic plants. Instead, stearic acid levels in transgenic or mutant seeds rarely exceed 30–40% of the total fatty acids. The likely explanation for this apparent shortfall is that seeds are not viable if stearic acid accumulates to high levels, especially under conditions of low to moderate growth temperatures. It is notable that a limited number of tropical and subtropical plants produce seed oils with >65% 18:0, suggesting that transgenic crop seeds accumulating comparable levels are a viable goal. Perhaps, a successful engineering strategy for high levels of stearic acid accumulation will include the introduction of a metabolic mechanism to exclude stearic acid from membrane lipids, coupled with the cultivation of these genetically enhanced crops in warm climates.

An alternative, but quantitatively less successful approach for producing seeds with elevated 18:0 content has involved the transgenic expression of divergent forms of the oleoyl-ACP thioesterase or FatA. The FatA enzyme typically displays a strong substrate preference for oleoyl-ACP; however, a divergent form of this enzyme with increased activity for stearoyl-ACP has been identified in seeds of Garcinia mangostana or mangosteen (Hawkins and Kridl, 1998). Stearic acid comprises about 55% of the oil of these seeds. Expression of the mangosteen cDNA in canola under control of a strong seed specific promoter permitted accumulation of 18:0 to ~22% of the seed oil (Hawkins and Kridl, 1998).