Noemi Ruiz-Lopez

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega-3 (also called n-3) long-chain polyunsaturated fatty acids (≥C20; LC-PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega-3 LC-PUFAs, i.e. eicosapentaenoic acid (20:5 n-3, EPA) and docosahexaenoic acid (22:6 n-3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega-3 LC-PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non-native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.

Metabolic engineering of biomass for high energy density: oilseed‐like triacylglycerol yields from plant leaves

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.

Reconstitution of EPA and DHA biosynthesis in Arabidopsis: Iterative metabolic engineering for the synthesis of n−3 LC-PUFAs in transgenic plants

An iterative approach to optimising the accumulation of non-native long chain polyunsaturated fatty acids in transgenic plants was undertaken in Arabidopsis thaliana. The contribution of a number of different transgene enzyme activities was systematically determined, as was the contribution of endogenous fatty acid metabolism. Successive iterations were informed by lipidomic analysis of neutral, polar and acyl-CoA pools. This approach allowed for a four-fold improvement on levels previously reported for the accumulation of eicosapentaenoic acid in Arabidopsis seeds and also facilitated the successful engineering of the high value polyunsaturated fatty acid docosahexaenoic acid to 10-fold higher levels. Our studies identify the minimal gene set required to direct the efficient synthesis of these fatty acids in transgenic seed oil.

The synthesis and accumulation of stearidonic acid in transgenic plants: a novel source of 'heart-healthy' omega-3 fatty acids

Dietary omega-3 polyunsaturated fatty acids have a proven role in reducing the risk of cardiovascular disease and precursor disease states such as metabolic syndrome. Although most studies have focussed on the predominant omega-3 fatty acids found in fish oils (eicosapentaenoic acid and docosahexaenoic acid), recent evidence suggests similar health benefits from their common precursor, stearidonic acid. Stearidonic acid is a Delta6-unsaturated C18 omega-3 fatty acid present in a few plant species (mainly the Boraginaceae and Primulaceae) reflecting the general absence of Delta6-desaturation from higher plants. Using a Delta6-desaturase from Primula vialii, we generated transgenic Arabidopsis and linseed lines accumulating stearidonic acid in their seed lipids. Significantly, the P. vialiiDelta6-desaturase specifically only utilises alpha-linolenic acid as a substrate, resulting in the accumulation of stearidonic acid but not omega-6 gamma-linolenic acid. Detailed lipid analysis revealed the accumulation of stearidonic acid in neutral lipids such as triacylglycerol but an absence from the acyl-CoA pool. In the case of linseed, the achieved levels of stearidonic acid (13.4% of triacylglycerols) are very similar to those found in the sole natural commercial plant source (Echium spp.) or transgenic soybean oil. However, both those latter oils contain gamma-linolenic acid, which is not normally present in fish oils and considered undesirable for heart-healthy applications. By contrast, the stearidonic acid-enriched linseed oil is essentially devoid of this fatty acid. Moreover, the overall omega-3/omega-6 ratio for this modified linseed oil is also significantly higher. Thus, this nutritionally enhanced linseed oil may have superior health-beneficial properties.

Enhancing the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic Arabidopsis thaliana via iterative metabolic engineering and genetic crossing

The synthesis and accumulation of long chain polyunsaturated fatty acids such as eicosapentaenoic acid has previously been demonstrated in the seeds of transgenic plants. However, the obtained levels are relatively low, indicating the need for further studies and the better definition of the interplay between endogenous lipid synthesis and the non-native transgene-encoded activities. In this study we have systematically compared three different transgenic configurations of the biosynthetic pathway for eicosapentaenoic acid, using lipidomic profiling to identify metabolic bottlenecks. We have also used genetic crossing to stack up to ten transgenes in Arabidopsis. These studies indicate several potential approaches to optimize the accumulation of target fatty acids in transgenic plants. Our data show the unexpected channeling of heterologous C20 polyunsaturated fatty acids into minor phospholipid species, and also the apparent negative metabolic regulation of phospholipid-dependent Δ6-desaturases. Collectively, this study confirms the benefits of iterative approaches to metabolic engineering of plant lipid synthesis.

Modifying the lipid content and composition of plant seeds: engineering the production of LC-PUFA

Omega-3 fatty acids are characterized by a double bond at the third carbon atom from the end of the carbon chain. Latterly, long chain polyunsaturated omega-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexanoic acid (DHA; 22:6 Δ4,7,10,13,16,19), which typically only enter the human diet via the consumption of oily fish, have attracted much attention. The health benefits of the omega-3 LC-PUFAs EPA and DHA are now well established. Given the desire for a sustainable supply of omega-LC-PUFA, efforts have focused on enhancing the composition of vegetable oils to include these important fatty acids. Specifically, EPA and DHA have been the focus of much study, with the ultimate goal of producing a terrestrial plant-based source of these so-called fish oils. Over the last decade, many genes encoding the primary LC-PUFA biosynthetic activities have been identified and characterized. This has allowed the reconstitution of the LC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate omega-3 LC-PUFA to levels similar to that found in fish oil. In this review, we will describe the most recent developments in this field and the challenges of overwriting endogenous seed lipid metabolism to maximize the accumulation of these important fatty acids.

Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues.

A method for plant tissue digestion and triacylglycerol (TAG) extraction followed by transmethylation of TAGs to produce the fatty acid methyl esters (FAMEs) from small storage tissue samples is presented. The method allows the analysis of both TAGs and FAMEs from the same sample. Several reagent mixtures and different experimental conditions were tested on sliced sunflower seeds. The best results were obtained using a mixture that was 33.3% a solution of NaCl (0.17 M) in methanol and 66.6% heptane by volume. The TAGs in the heptane solution were transmethylated with a mixture containing methanol:toluene:dimethoxypropane:H(4)SO(2) (39:20:5:2, by vol). The method was also tested on other oil seed storage tissue (soybean) and fruit tissues from olive and acorn. In all cases, sunflower, soybean, olive, and acorn, the TAGs and FAMEs composition data obtained by this method were quite similar to data from a standard analysis method. In samples with high protein content, such as soybean and sunflower seeds, the TAG extraction was incomplete. The water content of fruit samples did not interfere with TAG extraction obtained by this method.

Transgenic plants as a sustainable, terrestrial source of fish oils

1 An alternative, sustainable source of omega‐3 long chain polyunsaturated fatty acids is widely recognized as desirable, helping to reduce pressure on current sources (wild capture fisheries) and providing a de novo source of these health beneficial fatty acids. This review will consider the efforts and progress to develop transgenic plants as terrestrial sources of omega‐3 fish oils, focusing on recent developments and the possible explanations for advances in the field. We also consider the utility of such a source for use in aquaculture, since this industry is the major consumer of oceanic supplies of omega‐3 fish oils. Given the importance of the aquaculture industry in meeting global requirements for healthy foodstuffs, an alternative source of omega‐3 fish oils represents a potentially significant breakthrough for this production system. Transgenic Camelina seeds engineered to accumulate the omega‐3 fatty acids EPA and DHA, represent a sustainable alternative to fish oils.

An alternative pathway for the effective production of the omega-3 long-chain polyunsaturates EPA and ETA in transgenic oilseeds

Summary The synthesis and accumulation of omega‐3 long‐chain polyunsaturated fatty acids in transgenic Camelina sativa is demonstrated using the so‐called alternative pathway. This aerobic pathway is found in a small number of taxonomically unrelated unicellular organisms and utilizes a C18 Δ9‐elongase to generate C20 PUFAs. Here, we evaluated four different combinations of seed‐specific transgene‐derived activities to systematically determine the potential of this pathway to direct the synthesis of eicosapentaenoic acid (EPA) in transgenic plants. The accumulation of EPA and the related omega‐3 LC‐PUFA eicosatetraenoic acid (ETA) was observed up to 26.4% of total seed fatty acids, of which ETA was 9.5%. Seed oils such as these not only represent an additional source of EPA, but also an entirely new source of the bona fide fish oil ETA. Detailed lipidomic analysis of the alternative pathway in Camelina revealed that the acyl‐substrate preferences of the different activities in the pathway can still generate a substrate‐dichotomy bottleneck, largely due to inefficient acyl‐exchange from phospholipids into the acyl‐CoA pool. However, significant levels of EPA and ETA were detected in the triacylglycerols of transgenic seeds, confirming the channelling of these fatty acids into this storage lipid.