Plant oil TAGs are known to accumulate more than 300 unusual fatty acids (UFAs) in addition to the five fatty acids common in almost plant tissues and the main components of most commodity oils (van de Loo et al., 1993). However, in most cases sources of these UFAs cannot be produced economically on a commercial scale. Hydroxy, epoxy, conjugated, acetylenic, very long chain and branched chain fatty acids and liquid waxes are among the industrial targets of greatest interest. Oils high in hydroxy fatty acids can be produced from castor and Lesquerella, but they could be produced more economically on a large scale currently with canola or soybeans engineered with genes for hydroxy fatty acid accumulation. This is due principally to the value of the meal co-product and better developed agronomic properties providing higher yields and greater ease of planting and harvest. Genes for most of these UFAs have been cloned and good reviews have been written on this subject, including that of Napier (2007). It is easy to produce UFAs in transgenic oilseeds with the genes encoding enzymes for UFA biosynthesis, but it has been very difficult to achieve accumulation of UFAs to more than 10% of the total lipids. This is in contrast to the accumulation of as much of 95% of the seed oil TAG being composed of a single UFA such as the hydroxy fatty acid ricinoleate in castor oil, the epoxy fatty acid vernoleate in Bernardia pulchella oil, and the short-chain fatty acid caproate in Cuphea koehneana oil. In the cases where the details of the biosynthesis of the UFA are known they are made from phosphatidyl choline (PC) in the ER, but then selectively accumulate in seed oil TAG. They do not accumulate in membrane lipids such as the starting PC in the plants that accumulate high levels in TAG, but do not show such selective distribution in transgenic oilseeds with UFA biosynthetic genes alone. This has led to studies on whether TAG biosynthetic enzymes might have selectivity for such fatty acids that accumulate in the TAG.
In order to induce large changes in oil composition, LPAT has been considered an important target enzyme because of its selective discrimination ability (Franzosi et al., 1998). Rapeseed (Brassica napus) and meadowfoam (Limnanthes) have 60% and 90% erucic acid in their TAGs. In meadowfoam, erucic acid is present in the sn2 position of TAGs, whereas it is excluded in rapeseed. This difference was attributed to the substrate specificity of LPAT in the two species (Cao et al., 1990). To alter the stereochemical composition of rapeseed oil, a cDNA encoding Limnanthes seed-specific LPAT was expressed in Brassica napus plants using a napin expression cassette. In the transgenic plants, 22.3% erucic acid was present at the sn2 position leading to the production of trierucin. However, alteration of erucic acid at the sn2 position did not affect the total erucic acid content. It may be that the meadowfoam LPAT does not increase the erucic acid content of rapeseed (Lassner et al., 1995) because of the limited pool size of the 22:1 coenzyme A in the maturing embryos of B. napus. The metabolism of laurate was found to be different in transgenic Brassica napus lines (transformed with a California bay lauroyl-acyl carrier protein thiosesterase cDNA driven by napin promoter) and the natural laurate accumulators coconut, oil palm and Cuphea wrightii. When tested at the mid-stage of embryo development, the PC had up to 26 mol% of laurate in the transgenic rapeseed high laurate line, whereas in other species it ranged between 1 to 4 mol%. The laurate in the Brassica TAG was almost totally confined to the outer sn1 and sn3 positions, whereas the laurate in coconut and Cuphea was highest in the sn2 position. Very low amounts of laurate were found in the sn2 position in DAG and PC of the rapeseed lipids, indicating that no arrangement of laurate between the outer and sn2 positions occurred in any of the lipids. There was an enhanced activity of lauroyl-PC metabolising enzymes in the laurate-producing rapeseed when embryos were fed with 14C-lauroyl-PC and 14C-palmitoyl-PC. The data indicated that DAG was preferentially utilised from natural laurate accumulators like oil palm, coconut and Cuphea (Wiberg et al., 1997). Transgenic rapeseed oil expressing California bay thioesterase produced 60% saturated FA, with laurate alone comprising 48%. In these plants laurate was present only at the sn1 and sn3 positions. When these plants were crossed with transgenic lines expressing coconut LPAT laurate was present at the sn2 position along with sn1 and sn3 positions in the resulting hybrids. An overall increase in the oil content was also observed.
When the yeast LPAT genes SLC1 and SLC1-1 (mutant form of yeast LPAT) were expressed in Brassica napus and Arabidopsis under the CaMV35S promoter, the TAG and VLCFA contents were increased by 56% and 80%, respectively (Zou et al., 1997). In the transgenic plants, seed weight increased indicating at least a partial contribution from enhanced oil content. In the total oil content, 60-75% consisted of VLCFAs and up to 40% that of non-VLCFAs such as palmitate, oleate, linoleate and linolenate. No increase in total oil content was reported in coconut or meadowfoam LPAT-transformed rapeseed. This could be due to different regulatory properties of the plant and yeast LPAT enzymes. The plant LPAT genes have 62% amino acid identity among themselves, whereas the yeast genes have 24% homology. In transgenic plants the high expression of SLC1-1 gene did not correlate with an increased oil content, indicating that even small levels of expression were sufficient to overcome the PA limitations during TAG biosynthesis. Although SLC1-1 levels were stronger in leaves than in seeds, no significant changes were observed in the fatty acid composition of leaves, indicating that the pools of available LPA and/or acyl-CoAs may be more tightly regulated in leaves (source) than in seeds (sink). There are preliminary data indicating a 1-2% increase in oil content of soybeans expressing the yeast SLC1 gene (Rao and Hildebrand, 2009).
In studies on the expression profiles of genes encoding TAG biosynthetic enzymes, it was found that DGAT1, unlike DGAT2 or PDAT, has an expression profile in different tissues of soybeans and Arabidopsis consistent with a role in seed oil synthesis. DGAT1 and DGAT2, in contrast, display expression consistent with a role in seed oil synthesis in high epoxy and hydroxy fatty acid accumulating plants, implicating DGATs, and particularly DGAT2, as playing an important role in the selective accumulation of UFA in TAG (Li et al., 2005, 2010a). As mentioned above, Zhang et al. (2009) have established that DGAT2 has no role in TAG biosynthesis in Arabidopsis. However, there is good evidence that DGAT2 from tung trees, Vernicia fordii, has specificity for the conjugated fatty acid that accumulates in tung oil, eleostearic acid (Shockey et al., 2006a,b). Coexpression of hydroxylase and DGAT2 from castor and epoxygenase + DGAT2 from Bernardia and Vernonia can increase the accumulation of hydroxy and epoxy fatty acids in seed oil up to five-fold over expression of the hydroxylase and epoxygenase genes alone (Burgal et al., 2008; Zhou et al., 2008; Li et al., 2010a). Li et al. (2010a) were the first to demonstrate this in a commercial oilseed going from about 5% epoxy fatty acid in seed lipids of soybeans expressing an epoxygenase, increasing to >10% in soybeans expressing an epoxygenase + a DGAT1 from a high epoxy fatty acid accumulating plant, and to >30% in soybeans expressing an epoxygenase + a DGAT2 from the same high epoxy fatty acid accumulating plant (unpublished data).
Global plant oil production is dominated by four main oil crops: palm, soybeans, rapeseed or canola, and sunflowers (Wilson and Hildebrand, 2010) (Figure 2.4). Plant oil production
in 2009 exceeded 120 million metric tons. Worldwide palm and soybean production has increased rapidly and rapeseed has also shown steady increases, and this trend is expected to continue with a projected global oil production of over 170 million metric tons in 2020 (Figure 2.4). Palm oil production has been dominated by Malaysia and Indonesia, although expansion in South America and Africa is feasible. The vast majority of soybeans are produced in the US, Brazil, China and Argentina, with further expansion possible in southern Africa and South America. Global oilseed production is dominated by soybeans followed by rapeseed as a distant second (Wilson and Hildebrand, 2010) (Figure 2.5). This is because among oilseeds, soybeans are low in oil and high in protein, making soybeans the dominant global protein source. Over 200 million metric tons of soybean seeds have been produced annually in recent years. Soybeans average ~20% oil and 40% protein on a dry weight basis, whereas rapeseed is ~50% oil, and palm fruit close to 90% oil. Oil palm fruit contains seeds from which palm kernel oil is derived. Palm fruit and kernel oils are quite different in fatty acid composition, with palm kernel oil being dominated by medium chain fatty acids similar to coconut oil. Palm fruit cannot be stored in large quantities or transported long distances, unlike most oilseeds.
Breeding for increased oilseed yield per unit land area continues to progress, with steady soybean yield increases being a good example (Egli, 2008a,b). This is often with little or no increased inputs, making renewable oil production from plants less expensive over time and progressively more competitive with petroleum as an industrial chemical feedstock. Further, it is becoming increasingly possible to alter hydrocarbon flux into oil in some oilseeds, increasing the oil yield per unit land area independent of yield increases. This is particularly true for low-oil oilseeds such as soybeans. A number of studies indicate that oil content
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