The advent of plant genetic engineering over two decades ago hastened new thinking on breeding crops with novel traits.
In particular, the concept of modifying seed oil composition to alter and improve its enduse was exemplified by pioneering studies by researchers at Calgene Inc. (Davis, CA), who modified plant oils with “gain-of-function” transgenes.1 Through those and similar studies, the “designer oilseed” concept was born—the ability to manipulate and modify, in a predictable manner, the composition of a plant oil.2
Fatty acids found in the plant kingdom collectively display considerable chemical diversity, in excess of 400 different fatty acids, with most of the so-called unusual fatty acids compartmentalized in seed storage oils.2,3 And although many unusual fatty acids in plants have potentially useful applications, ranging from industrial biolubricants and petrochemical replacements to human nutrition, most wild plant species are unsuitable for modern agricultural practices. Use of genetic engineering to transfer the “trait” for unusual fatty acids between species is limited by the identification of the gene(s) underlying the unusual fatty acid trait.1-3
Enormous progress has been made in understanding genetic components required for the biosynthesis and modification of fatty acids in plants, thus facilitating the identification of sequences responsible for unusual fatty acid accumulation. Perhaps surprisingly, many early attempts to engineer the fatty acid composition of transgenic plants produced much lower levels of target lipids than found in native species.2 Such observations elucidate the complexity of plant lipid metabolism. Consequently, employing iterative “learning–by-doing” approaches to engineering novel oil traits is advancing real progress in this area.4
Omega-3 Fatty Acids and Human Health
One promising area of research pertains to the production of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), the so called fish oils, in transgenic plants. There is compelling evidence for the health benefits of a diet rich in fish oils, e.g., in providing for optimal development of the unborn child and protection against cardiovascular disease. However, animals have only a limited capacity to synthesize these fatty acids, so dietary intake is important.
Unfortunately, a number of factors limit our consumption of omega-3 polyunsaturated fatty acids.3-5 First, the over-exploitation of wild fish stocks has reduced their sustainability, exacerbated by the increasing demands of aquaculture for fish oils and meal. Second, the environmental pollution of marine food webs has raised concerns over the ingestion of toxic substances such as heavy metals, PCBs, and dioxins. Finally, shifts in food production methods and changes to the historical patterns of food consumption have culminated in diets dominated by omega-6 fatty acids, as opposed to the (more health-protective) omega-3s.
To address these issues, we and others are focusing on producing omega-3 long-chain polyunsaturated fatty acids in transgenic plants to provide a safe and sustainable source of these important oils.3-5 Therefore, the goal is not the direct replication of the fatty acid profile found in marine microbes (the primary biosynthetic source of omega-3 LC-PUFA) or fish, but rather the nutritional enhancement of vegetable oils by the inclusion of specific marine fatty acids not normally synthesized by higher plants. In such a scenario, the dietary intake of these healthy fats would be achieved by consumption
of omega-3 LC-PUFA-enhanced vegetable oils, without a need for increased consumption of fish or supplements.
Transgenic Synthesis of Omega-3s
Considerable effort has focused on the transgenic synthesis in plants of two particular omega-3 LC-PUFAs: eicosapentaenoic
acid (EPA20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6Δ4,7,10,13,16,19). EPA and DHA are the predominant fatty acids in fish oils, and there is strong evidence based on epidemiological, clinical, and genetic studies of their cardiovascular system-protective role. However, the conversion (through genetic engineering) of a plant C18 fatty acid such as α-linolenic acid (ALA; 18:3Δ9,12,15) to EPA requires three separate enzyme activities (two desaturations interspersed with a two-carbon chain elongation) and conversion of ALA to DHA requires five different enzyme activities (desaturation, elongation x2, desaturation).
Despite recent progress in achieving useful levels (10 – 25% of total fatty acids) of EPA in transgenic seed oils,5,6 coordination of the expression and activity of multiple enzyme activities that act in a sequential manner is an on-going metabolic engineering challenge: this is even more true for DHA, where the levels achieved in transgenic plants are still relatively low (<5% of total fatty acids).5,6 Consequently, attention has also been drawn to the C18 omega-3 fatty acid stearidonic acid (SDA; 18:4Δ6,9,12,15), which is synthesized by the a single (Δ6-)desaturation reaction of ALA7. SDA shares many of the health benefits of EPA, presumably because it is a biosynthetic intermediate of that fatty acid; thus, consumption of SDA boosts EPA levels by bypassing our limited endogenous ability to desaturate ALA.
SDA is an example of an unusual plant fatty acid found in only a few species, predominantly members of the Boraginaceae family. Only one species (Echium spp.) is grown commercially for SDA, though this may change given increasing interest in this fatty acid. It should also be noted that Echium oil not only contains ~14% SDA, but also has significant levels of the omega-6 C18 γ-linolenic acid (GLA; 18:3Δ6,9,12). GLA, like other omega-6 fatty acids, does not deliver the health benefits seen with SDA or other omega-3 PUFAs, such as EPA and DHA.
Production of Stearidonic Acid in Transgenic Plants
On paper, engineering plants to accumulate high levels (>10%) of total fatty acids should be straightforward through the seed-specific expression of a suitable Δ6-desaturase isolated from borage or similar species. However, enhanced expression of Δ6-desaturase also produces the co-synthesis of GLA, since most Δ6-desaturases have no substrate preference for omega-6 (LA) or omega-3 (ALA).
In earlier studies, we identified Δ6-desaturases from Primula spp. that showed strong substrate preference for ALA; thus, by using the Primula Δ6-desaturase, SDA can be generated without producing GLA. Previous attempts to generate transgenic plants high in SDA but low in GLA had some success, most notably by Eckert et al.,8 who co-expressed borage Δ6-desaturase with a Δ15-desaturase (to convert GLA to SDA) in transgenic soybean. This resulted in an SDA concentration between 10% – 29%, but the soybeans still contained significant GLA (7.2% – 12.4%).
We sought to avoid the complications of using multiple enzyme activities by expressing the Primula vialii Δ6-desaturase in transgenic Arabidopsis and linseed, under control of a seed specific promoter.7 Thus, the same construct was introduced by Agrobacterium-mediated transformation into either a model plant or an actual oilseed crop. This very straightforward experiment immediately provided some interesting results, most strikingly, the disparity in the achieved levels of SDA between the two plant species: whereas linseed accumulated ~12% SDA as part of total seed fatty acids, Arabidopsis seed oils only contained ~3% SDA.7 Attempts to elevate Arabidopsis SDA, by increasing the amount of substrate ALA to equal that found in linseed, also failed to improve this low yield. Thus, it seems clear that even simple transgenic traits for modified plant lipid metabolism are context-dependent, meaning that the configuration of endogenous
biochemical pathways for seed oil synthesis, modification, and accumulation significantly influences the efficacy of any transgene-derived heterologous activity.
The acyl composition of plant oils varies markedly between species, even though they share a common set of biosynthetic enzymes. Subtle differences in the level and temporal expression of enzyme activities during seed development, which varies from species to species,3,4 largely determine the final composition of any given seed oil.7 Therefore, the “cut and paste” method of using heterologous transgene-derived acyl-modifying activities needs to incorporate the additional qualities of the host, which requires a detailed biochemical characterization of the pathways of oil synthesis.
Linseed as Novel SDA-Enriched Omega-3 Crop
Though Arabidopsis proved a poor accumulator of SDA, linseed accumulates levels of SDA which are equivalent to the native Echium species.7 Perhaps more importantly, the ALA-specific Δ6-desaturase derived from Primula synthesized SDA without any co-synthesis of omega-6 GLA. Thus, transgenic SDA-containing linseed differed from both Echium and transgenic soybean oil by being essentially devoid of GLA.7,8 Detailed analysis of different lipid classes in developing and mature linseed lines confirmed the preferential accumulation of SDA in triacylglycerols (neutral storage lipids) as opposed to phospholipids (the actual site of synthesis of SDA).7 Thus, linseed efficiently channeled SDA from phospholipids to triacylglycerols through an acyl-CoA-independent pathway. This result confirms our observations from earlier attempts to synthesize EPA in transgenic linseed in which C20 LC-PUFAs generation was blocked by the absence of substrates (such as SDA) in the acyl-CoA pool.9
Transgenic linseed engineered to accumulate SDA is a potentially valuable novel oilseed. The oil of this SDA-enriched linseed contains 60% omega-3 fatty acids, of which approximately one quarter is SDA (14%). In addition, not only are levels of the native omega-6 LA relatively low (12%), the oil contains only trace levels of omega-6 GLA (0.3%). The absence of GLA is important, as this Δ6-desaturated fatty acid can (in animals) be converted to the C20 omega-6 arachidonic acid, which in turn is a precursor for pro-inflammatory eicosanoids. Thus, for “heart-healthy” applications, transgene-derived SDA-enriched linseed oil may be superior for enhanced omega-3 nutrition.
In conclusion, these studies illustrate both the power of genetic engineering to generate novel oil traits and the need for a deeper understanding of plant lipid metabolism. It is hoped that the combination of these factors will, in the near future, deliver further beneficial oilseed crops carrying new transgene-derived traits, as well help to improve knowledge of the underlying biochemical processes.
References
1. Yuan L, Knauf VC. (1997) Modification of plant components. Curr Opin Biotechnol. 8, 227-233
2. Murphy DJ. (1999) Production of novel oils in plants. Curr Opin Biotechnol. 10, 175-180
3. Napier JA. (2007) The production of unusual fatty acids in transgenic plants. Annu Rev Plant Biol. 58, 295-319
4. Graham IA, Larson T, Napier JA. (2007) Rational metabolic engineering of transgenic plants for biosynthesis of omega-3 polyunsaturates. Curr Opin Biotechnol. 18, 142-147
5. Damude HG, Kinney AJ. (2008) Enhancing plant seed oils for human nutrition. Plant Physiol. 147, 962-968
6. Cheng B et al. (2009) Towards the production of high levels of eicosapentaenoic acid in transgenic plants: the effects of different host species, genes and promoters. Transgenic Res. in press DOI 10.1007/s11248-009-9302-z
7. Ruiz-López N, et al. (2009) The synthesis and accumulation of stearidonic acid in transgenic plants: a novel source of ‘heart-healthy’ omega-3 fatty acids. Plant Biotechnol J. 7, 704-716
8. Eckert H, et al. (2006) Co-expression of the borage Delta 6 desaturase and the Arabidopsis Delta 15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean. Planta. 224, 1050-1057
9. Abbadi A, et al. (2004) Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell. 16, 2734-2748
