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
18:1Δ
9 (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).