Progress in Lipid Research 49 (2010) 108119

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Progress in Lipid Research

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Review

An alternative to sh oils: Metabolic engineering of oil-seed crops to produce omega-3 long chain polyunsaturated fatty acids
Mnica Venegas-Calern a,b, Olga Sayanova a, Johnathan A. Napier a,*
a b

Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK Instituto de la Grasa, CSIC, Av. Padre Garcia Tejero 4, E-41012 Seville, Spain

a r t i c l e

i n f o

a b s t r a c t
It is now accepted that omega-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA; 20:5D5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6D4,7,10,13,16,19) play important roles in a number of aspects of human health, with marine sh rich in these benecial fatty acids our primary dietary source. However, over-shing and concerns about pollution of the marine environment indicate a need to develop alternative, sustainable sources of very long chain polyunsaturated fatty acids (VLCPUFAs) such as EPA and DHA. A number of different strategies have been considered, with one of the most promising being transgenic plants reverse-engineered to produce these so-called sh oils. Considerable progress has been made towards this goal and in this review we will outline the recent achievements in demonstrating the production of omega-3 VLC-PUFAs in transgenic plants. We will also consider how these enriched oils will allow the development of nutritionally-enhanced food products, suitable either for direct human ingestion or for use as an animal feedstuff. In particular, the requirements of aquaculture for omega-3 VLC-PUFAs will act as a strong driver for the development of such products. In addition, biotechnological research on the synthesis of VLC-PUFAs has provided new insights into the complexities of acyl-channelling and triacylglycerol biosynthesis in higher plants. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 17 September 2009 Received in revised form 13 October 2009 Accepted 20 October 2009

Keywords: Polyunsaturated fatty acids Plants Omega-3 fatty acids Desaturases Elongases Transgenic plant

Contents 1. 2. 3. 4. 5. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Omega-3 long chain polyunsaturated fatty acids (x3 LC-PUFAs) in humans health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of VLC-PUFAs biosynthetic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic engineering to produce VLC-PUFAs in higher plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crucial issues: optimization the levels of LC-PUFA in transgenic plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. The identification of superior desaturases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Identification of a VLC-PUFA-specific acyl-exchange mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Maintenance of a continuous flux of substrates through the VLC-PUFA biosynthetic pathway without significant loss to TAG . . . . . . . 5.4. Optimizing the fatty acid elongase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Modulating the acyl-CoA pool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Co-ordinated expression of transgenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Appropriate localisation of transgene-derived activities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 109 110 112 115 115 115 115 116 116 116 116 116 117 117

6.

Abbreviations: ALA, a-linolenic acid; ARA, arachidonic acid; DAG, diacylglycerol; DGAT, diacylgylcerol acyltransferase; DHA, docosahexaenoic acid; ECR, enoyl-CoA reductase; EFA, essential fatty acid; EPA, eicosapentaenoic acid; GLA, c-linolenic acid; HCD, hydroxyacyl-CoA dehydratase; KCS, ketoacyl-CoA synthase; KCR, b-ketoacyl-CoA reductase; LA, linoleic acid; LPCAT, acyl-CoA: lyso-phosphatidylcholine acyltransferase; PDAT, phospholipid: diacylglycerol acyltransferase; SDA, stearidonic acid; TAG, triacylglycerol; VLC-PUFA, very long chain polyunsaturated fatty acid. * Corresponding author. Tel.: +44 (0) 1582 763133; fax: +44 (0) 1582 763010. E-mail address: johnathan.napier@bbsrc.ac.uk (J.A. Napier). 0163-7827/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2009.10.001

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1. Introduction Very long chain polyunsaturated fatty acids (VLC-PUFAs) are fatty acids of 20 carbons or more in length with three or more methylene-interrupted double bonds in the cis position. These fatty acids can be grouped into two main families, omega-6 (or n-6) and omega-3 (or n-3) families, depending on the position of the rst double bond proximal to the methyl end of the fatty acid. VLCLC-PUFAs are vital constituents of human metabolism. In particular, there is plentiful evidence (from epidemiology and dietary intervention studies) for the health-benecial properties to humans of dietary consumption of omega-3 VLC-PUFAs such as eicosapentaenoic acid (EPA; 20:5D5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6D4,7,10,13,16,19) [14]. This dietary requirement is almost certainly due to the fact that humans (like most animals) have a very limited capacity to synthesize these fatty acids from the essential precursor a-linolenic acid (ALA; 18:3D9,12,15) [5]; therefore dietary intake of these fatty acids is a key aspect of human nutrition [6]. The main source of EPA and DHA in the human diet is through the direct consumption of cold water marine sh (such as salmon, tuna, mackerel, and sardines) [6,7]. However, marine sh (like other animals) do not efciently metabolise ALA to VLC-PUFAs, but accumulate them as a result of their dietary acquisition (though it should be noted that freshwater sh appear to have a greater capacity to synthesize EPA and DHA from ALA) [7,8]. The primary de novo synthesis sources of VLC-PUFAs are marine microbes such as algae which form the base of an aquatic food web that culminates in the accumulation of these fatty acids in the lipids of the sh [9]. In these microorganisms, EPA and DHA are synthesized de novo by one of the two classes of biochemical pathway (reviewed on the text below). In addition, some fungi, mosses, bacteria and lower plants also have a capacity to synthesize significant amounts of VLC-PUFAs [10]. In higher plants these fatty acids are almost completely absent, although plants are rich in the two essential dietary fatty acids linoleic acid (LA; 18:2D9,12) and a-linolenic acid (ALA; 18:3D9,12,15) that serve as the metabolic precursors for VLC-PUFA biosynthesis in animals [11]. There is growing concern regarding the sustainability of global sh stocks (the predominant sources of omega-3 VLC-PUFA) because marine sh stocks are in severe decline as a result of decades of over-shing [12]. Moreover, environmental pollution of marine ecosystems has resulted in the accumulation of dioxins, heavy metals and polychlorinated biphenyls in sh, to the point of questioning the benets of sh consumption in human health [13]. Finally, the expansion of industrialised aquaculture exacerbates the overexploitation of natural marine resources, since farmed sh require omega-3 VLC-PUFA-containing feedstuffs. The marine oils used as aquaculture feedstocks are usually extracted from socalled trash species such as sand eels, which are specically harvested for this application (since they are not normally consumed by humans). However, the loss of these species from food-webs has a profound impact on the overall stability of ecosystems [14]. Aquaculture is certainly the largest consumer of sh-derived oils and currently even the most sophisticated husbandry of high value species such as salmon require the input of dietary sh oils to a level signicantly higher than that present in the nished product. Therefore aquaculture is (perhaps surprisingly, at least to the layperson) a net consumer of sh oils and as such, not operating in a sustainable manner. In view of all of these points, there is a very obvious requirement for an alternative and sustainable source of VLC-PUFA for their use in human nutrition [1517]. Perhaps the most obvious alternative to sh oils is via contained culture of the aquatic microbes which synthesize EPA and/or DHA. Approaches using microbiological sources to synthesize VLC-PUFA have been developed and are economically viable for specic high value applications (such as infant formula baby milk formula-

tions [18]) in controlled culture systems. However, such systems are expensive to maintain and have limited exibility for signicant scale-up and requiring the appropriate microbiological facilities (such as fermenters) [19]. It is noteworthy that such fermentation-based systems are also sensitive to disruptions of power-supplies and have a signicant environmental footprint. In view of all these factors, there is an obvious need for an alternative, sustainable source of these important fatty acids. One attractive option is the use of transgenic plants to synthesize these fatty acids. Because no higher plant oilseeds produce VLC-PUFAs such as EPA and DHA, they must be reverse-engineered (the discovery of technological principles through deconstruction and analysis of component parts) [20] with this biosynthetic capacity by the introduction of this metabolic pathway from a suitable microbial source [16,17,20]. During the last ten years, genes encoding the primary enzymes involved in biosynthesis of these fatty acids have been successfully isolated from a range of VLC-PUFAsynthesising organisms with a number of these being heterologously expressed (singly or in combination) in oil-seed crops [21,22] the promise and the prospects of these new transgenic crops will be considered in this review.

2. Omega-3 long chain polyunsaturated fatty acids (x3 LC-PUFAs) in humans health All animals have lost the capacity to synthesize VLC-PUFAS due to the genetic absence of D12 and D15-desaturase activities and, as a consequence, cannot produce linolenic acid (LA; 18:2D9,12 n-6) and a-linolenic acid (ALA; 18:3D9,12,15 n-3) respectively from the precursor oleic acid (18:1D9) [23]. However, they do have a limited ability to synthesize ARA and EPA from these two dietaryessential fatty acids (EFAs) LA and ALA through a series of desaturation and elongation reactions (Fig. 1) [24]. Most dietary LA and ALA are b-oxidized to provide energy and only a small portion of them are converted to VLC-PUFAs [25]. It is estimated that the % conversion of ALA to EPA is 510%, whereas conversion of ALA to DHA is <1% [9]. Therefore, this route is unlikely to provide sufcient levels of VLC-PUFA for optimal human health and nutrition, indicating the requirement for supplementation via dietary intake of VLC-PUFAs. In addition, the dietary EFAs are not inter-convertible, meaning that omega-6 LA (and metabolites) cannot be metabolised to omega-3 fatty acids (due to the absence of the specic x3/D15-desaturase activity). This also means that the balance of omega-6 and omega-3 VLC-PUFAs can be inuenced by the dietary ratio and levels of EFAs because the D6-desaturase (rst desaturation and limiting step in VLC-PUFA biosynthesis Fig 1) can utilize both EFA as substrates. In most Western societies, the diets are dominated by vegetable oils and processed foods which contain excessive levels of omega-6 fatty acids such as LA, and are generally under-represented in levels of omega-3 fatty acids [26]. It has been estimated that our ancestral dietary fat composition showed an omega-6/omega-3 ratio of 2:1, this same ratio is today in excess of 10:1 [27]. Increasing epidemiological evidence indicates that functional deciencies or imbalances involving omega3 VLC-PUFA have been associated with a range of disorders including, cardiovascular disease [28,29], cancer, and inammatory and autoimmune disease [30,31]. In consequence, dietary recommendations now include not only LA and ALA but also EPA and DHA for optimal nutrition, in the range of 0.51.8 g/day (or the consumption of at least two serving of sh per week) [32,33]. It is an interesting diversion to speculate on the evolutionary basis for the loss in animals of desaturases involved in the synthesis of EFAs [11,24], the most logical reason being that genetic drift in these genes was effectively compensated by dietary intake of LA and ALA. It is also worth noting that the D12 and D15-desaturases

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Animals

Plants
18:0 Stearic acid (SA)

Conventional 6-pathway
3 Des

9 Des 12 Des
18:2 9,12 Linoleico acid (LA)

18:1 9 Oleico acid (OA)

15 Des

18:3 9,12,15 -Linolenic acid (ALA)

Alternative 8-pathway

Alternative 8-pathway

Micobial 4 pathway

Mammalian Sprecher pathway

Fig. 1. Aerobic VLC-PUFA biosynthetic pathways. The various routes for synthesis of arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are shown, as mediated by the consecutive action of desaturases and elongases. The predominant D6-pathway is shown, as is the alternative D8-pathway. Two routes for DHA synthesis are shown, microbial D4-pathway and mammalian Sprecher pathway. Des = desaturase, Elo = elongase.

are ubiquitous in higher plant and play a crucial role in the synthesis of membrane lipids for the support of photosynthesis and also the precursors for the oxylipin jasmonic acid required for male fertility [22]. The presence of these EFA desaturases has been observed in some lower animals, most notably Caenorhabditis elegans. Genetic analysis of the role of these enzymes in C. elegans indicates that they play a vital role in development [34] and such studies indicate the usefulness of this nematode in studies on the role of EFAs and VLC-PUFAs in multicellular organisms [35]. VLC-PUFAs are functional components that can modulate membrane uidity and permeability. As a consequence they play crucial roles in human metabolism, not only playing structural roles in phospholipid bilayers but also acting as precursors to bioactive molecules. For example, both omega-6 and omega-3 C20 fatty acids are precursors of the eicosanoids, oxygenated VLC-PUFA metabolites involved in the regulation of inammation, plaque aggregation, and vasoconstriction/dilation. Both EPA and ARA serve as substrates for the common cyclooxygenase and lipoxygenase enzymes; while omega-6 ARA produces more potent inammatory, pro-aggregatory and inmuno-active eicosanoids (series-2), eicosanoids derived from omega-3 fatty acids (series-1 and series-3) are anti-inammatory and modulate plaque aggregation and immune-reactivity [36,37]. Unsurprisingly, research has demonstrated that there are considerable health benets to be gained from having a diet rich in VLC-PUFA, and in particular EPA and DHA. For example, the VLC-PUFAs ARA and DHA play an important role in neonatal health and development [3840], in particular the

acquisition of ocular vision and brain development: it is for this reason that both these fatty acids are recommended for inclusion in infant formula milks [18]. Clinical trials have demonstrated protective roles for EPA and DHA in the prevention of cardiovascular disease and there is also emerging evidence of these VLC-PUFAs protecting against metabolic syndrome and related disease states, such as obesity and type-2 diabetes [31,41]. More recently, protective effects have been clinically studied for cancer [42], atherosclerosis, cognitive impairment, and various mental illness, in particular depression [29], childhood and attention-decit hyperactivity disorder (ADHD) [43,44] and dementia [45]. Finally, there are epidemiological studies which extend the benecial effects of omega-3 VLC-PUFA to the immune system (including diseased states such as rheumatoid arthritis) [46], the reproductive system, skin barrier function [47] and other exciting emerging roles such as inammation-resolution [48]. 3. Characterization of VLC-PUFAs biosynthetic pathways Over the last decade, all the primary genes involved in VLC-PUFA biosynthesis have been identied from a range of different species, including animals, fungi, plants and aquatic organisms. These genes can be classied into the two distinct enzymatic reactions that catalyse the primary biosynthetic process. The rst of these are the microsomal fatty acid desaturases, so-called front end PUFA desaturases which belong to the N-terminal cytochrome b5-fusion superfamily, rstly identied in 1997 by Sayanova et al. [49]. The

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cytochrome b5 domain is assumed to be involved in the electron transport chain required for acyl-desaturation, and currently all known examples of microsomal VLC-PUFA desaturases contain this N-terminal extension. This is in contrast to the D12 and D15-desaturases found in plants, algae and some fungi which lack any such cytochrome b5 domain. Another characteristic of the front end desaturases is the substitution of histidine by glutamine in the third histidine box (consensus sequence QX[23]HH). The presence of diagnostic motifs such as a cytocrome b5 domain and this variant His box have greatly facilitated the identication of candidate front end desaturases from animals, fungi, and algae, and also from the few plant species (borage, evening primrose, black currant and Echium) that carry out D6-desaturation of LA and ALA [50]. One crucial observation regarding the microsomal desaturases from lower eukaryotes is that very many of these enzymes utilize glycerolipid-linked substrates, in particular fatty acids esteried to the sn-2 position of glycerolipids. This is not the case in animals, where the substrates for these enzyme activities are generally believed to be acyl-CoAs [5153]. The second key enzymatic reaction in the synthesis of VLCPUFA is elongation, which also occurs in the ER. The fatty acid elongation reaction consists of four sequencial activities: condensation of the substrate fatty acid with malonyl-CoA (b-ketoacyl-CoA synthase; KCS), reduction (b-ketoacyl-CoA reductase; KCR), dehydration (hydroxyacyl-CoA dehydratase; HCD), and a second reduction (enoyl-CoA reductase; ECR) [54] and resulting in a two-carbon chain elongation of the input substrate fatty acid. The condensing enzymes are considered to be rate-limiting and the regulators of substrate-specicity with regard to chain length and pattern of double bonds. Perhaps unexpectedly, the expression of sequences encoding b-ketoacyl-CoA synthase activities alone are able to reconstitute a heterologous elongating activity without requirement for the co-expression of any other components of the elongase [5557]. It is therefore for this reason that KCSs are often (semantically incorrectly) referred to as elongases. It is assumed that the ability of heterologously expressed KCSs to apparently direct the elongation of substrate fatty acids is due to the interaction between endogenous core elongase components (KCR, HCD, ECD) and the exogenous KCS in the absence of any of these three latter components, elongation cannot occur. It should be noted that KCS condensing enzymes can be divided into two distinct groups. A rst group comprises the so-called ELO-like sequences (named after the yeast ELO genes, which are required for the synthesis of saturated very long chain fatty acids found in sphingolipids [58]) some of which involved in VLC-PUFA biosynthesis, which have been cloned from a number of species including mammals, fungi (e.g. Mortierella alpina) [59], and aquatic algae (e.g. Isochrysis galbana) [60]. A second class of unrelated plant-specic KCS activities are known as FAE1-like enzymes (so-called after the founding member of this family, FAE1 fatty acid elongation1, an Arabidopsis gene required for the synthesis of VLCFAs found in seed triacylglycerols), involved in the biosynthesis of saturated and monounsaturated fatty acids with C1822+ chain length, [61]. Until very recently, it was believed that FAE-like activities were restricted to only being involved in the synthesis of saturated and monounsaturated VLCFAs for use in wax and storage lipid synthesis. However, there is now evidence that this FAE-like class is also involved in the synthesis of VLC-PUFAs a PUFA-FAE was functionally characterised from the parasitic protozoa Perkinsus marinus [62]. Interestingly, this KCS was in a small gene cluster with two cytochrome b5-fusion desaturases, and when all three open reading frames (ORFs) were heterologously expressed in yeast, the synthesis of ARA and EPA was achieved. As noted above, the heterologous activity of any KCS, be it ELO-like or FAE1-like, is dependent on the presence of the core elongase components. Although FAE1-like KCSs are structurally quite different to ELO-like (500aa, 2 TMs ver-

sus <300aa, 4+ TMs, respectively) plant KCSs are able to functionally complement yeast strains which are entirely lacking in ELO genes [63,64] and also physically interact with the other elongase components. Thus, the elongase is a multi-protein complex made up of (at the very least) the core elongase components (KCR, HCD, ECR) and a KCS (ELO or FAE). In contrast to microsomal desaturation, all microsomal elongation described, both ELO-type and FAE1-like, utilize substrates exclusively in the form of acyl-CoAs [65]. The availability of these many genes encoding the primary VLCPUFA biosynthetic activities provides a toolkit with which to attempt to reconstitute VLC-PUFA biosynthesis in a heterologous host [16,17,21]. It is now also clear that there are a number of different congurations of the biosynthetic pathway for C20 LC-PUFAs. In particular, two different aerobic routes exist for the biosynthesis of EPA and its omega-6 counterpart, ARA (Fig 1) [66]. The dominant form, the so-called D6-pathway, is widely spread throughout eukaryotes. This pathway commences with the D6-desaturation of LA and ALA, to yield GLA (18:3D6,9,12) and SDA (18:4D6,9,12,15) respectively. These two fatty acids are then chain-elongated by two carbons via a D6-specic elongase to generate di-homo-c-linolenic acid (DHGLA; 20:3D8,11,14) and eicosatetraenoic acid (ETetA; 20:4D8,11,14,17). Subsequently a second desaturation by a D5-desaturase occurs to produce nally ARA and EPA. In some species from algae such as Pavlova, Isochrysis (Prymesiophyceae) and protists such as Euglena (Euglenophyceae), Acanthamoeba castellanii and P. marinus a second pathway is used; this is the so-called alternative D8-pathway, in which ALA is rst elongated by a specic D9-elongase to eicosatrienoic acid (ERA; 20:3 D11,14,17), followed by D8-desaturation to ETA [62,6770]. As in the prevalent D6-pathway, these fatty acids are then desaturated to ARA and EPA by a D5-desaturase. Although ARA is generally considered to be the end point in the synthesis of the omega-6 class of VLC-PUFA, further modication of the omega-3 EPA to synthesize the nal product DHA can occur by two distinct routes [71]. In mammals EPA undergoes two rounds of elongation, generating rst DPA and then tetracosahexaenoic acid (THA; 24:5, n-3) which then is subject to D6-desaturation yielding 24:6 n-3. This C24 PUFA is then subjected to limited peroxisomal b-oxidation, by which it is chain-shortened by two carbons to yield the nal product DHA: this is known as the Sprecher pathway, in recognition of its identication by Howard Sprecher [72]. The D6-desaturase enzyme that carries out this desaturation is the same one as produces GLA and SDA, although the basis and mechanism for this substrate selectivity is unclear. In DHA-accumulating microbes, the synthesis of DHA is a simpler biochemical process where EPA is elongated to docosapentaenoic acid (DPA; 22:5 n-3) by a specic D5-elongase, with DPA then converted to DHA by the action of a D4-desaturase [73]. These activities (D5-elongase, D4-desaturase) have been isolated and functionally characterized from several organisms and are closely related to the ORFs required for the synthesis of ARA and EPA [21,74]. In conclusion, all the primary biosynthetic components required for the synthesis of the C20 PUFA ARA and EPA, as well as the C22 PUFA DHA have now been identied. All these components can be categorized as either cytochrome b5-fusion front-end desaturases or ELO-like elongating activities (with the unique exception of the FAE1-like KCS from P. marinus). (A schematic representation of the biosynthetic pathways is shown in Fig. 1). It should also be mentioned that an unrelated anaerobic VLC-PUFA biosynthetic system is present in a few diverse lower marine organisms, and does not require PUFA-specic aerobic desaturases and elongases [75]. Instead, EPA or DHA is synthesized by a processive polyketide synthase (PKS)-like reaction from malonyl-CoA [76]. This system is similar to the fatty acid synthase (FAS) pathway in that PUFA biosynthesis is initiated by the condensation between a

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short chain acyl-CoA and an unit of malonyl-CoA, followed by successive rounds of reduction, dehydration, reduction, and condensation, with the acyl chain growing by two-carbon units with each round. A dehydratase/isomerase from this PKS complex catalyze the trans- to cis-conversion of the double bonds to form EPA and DHA. The domains encoding these activities are arranged sequentially on long (2030 kb) open reading frames (ORFs) in bacteria such as Shewanella and Vibrio, and marine protist, as Schizochytrium. Their expression in yeast and plants has been reported [77] providing an alternative to the aerobic desaturase/elongase system for transgenic VLC-PUFA production in plants. Interestingly, this PKSlike pathway generates EPA or DHA as free fatty acids, meaning that these products will most likely require activation to CoA to facilitate their incorporation into lipids [78]. It is also worthy of note that genomic and biochemical analysis of lipid biosynthesis in Schizochytrium indicated that, in addition to the PKS-like pathway, this organism also possessed desaturase and elongase activities of the D6-pathway, but crucially lacked the D12-desaturase activity (analogous to higher eukaryotes) [79]. The retention of this defective aerobic pathway was suggested by the authors to represent a scavenging mechanism by which fatty acids prematurely released from the PKS-like system might undergo further modications [79]. 4. Metabolic engineering to produce VLC-PUFAs in higher plants Higher plants lack the capacity to synthesize LC-PUFAs, though a few taxonomically unrelated phyla can synthesize D6-desaturated fatty acids (the rst step on the D6-pathway) such as omega-6 c-linolenic acid (GLA; 18:3D6,9,12) and omega-3 stearidonic acid (SDA; 18:4D6,9,12,15) [80]. The possibility of using transgenic plants that have been engineered to synthesize and accumulate VLC-PUFAs in their storage seed oils has been thoroughly investigated over the last 15 years. The conversion of native plant fatty acids such as LA and ALA to VLC-PUFAs requires a minimum of three sequential non-native enzymatic reactions (e.g. two desaturations and acylCoA elongation) to generate C20 PUFAs such as ARA and EPA. The last few years have seen considerable progress in the identication from diverse sources (algae, fungi, mosses, plants and mammals) of genes encoding the primary VLC-PUFA biosynthetic activities, effectively completing the rst stage of attempts to reverse-engineer this trait into a heterologous host such as a transgenic plant. Proof-ofconcept demonstration that the VLC-PUFA pathway could function in a transgenic system was rst provided by expression in yeast, with initial data showing the low accumulation of ARA and EPA [56,57,81,82] and subsequent experiments (with additional genes) to generate DHA [59]. Such experiments indicated the feasibility of transgenic expression of the LC-PUFA biosynthetic pathway in plants. The possibility of producing VLC-PUFAs in transgenic plants also became clear from the earliest attempts to accumulate GLA and SDA in oil-seed crops by expression of an individual gene, the D6-desaturase. Although the rst expression of a cyanobacterial D6-desaturase in transgenic tobacco plants [83] resulted in the accumulation of low levels of these fatty acids, considerable progress has been made reaching high levels (up to 40%) in several transgenic plants [49,84,85]. In 2004, three different reports by Qi et al. [86], Abbadi et al. [87] and Kinney et al. [88] demonstrated , VLC-PUFA biosynthesis in transgenic plants by reverse engineering although each utilized distinct strategies toward the efcient reconstitution of the process. Apart from the important biotechnological breakthroughs, they also provide some new insights into the biochemical pathways under manipulation and provide useful new tools for the dissection of the underlying enzymatic reactions. A diagrammatic summary of the results obtained from the studies discussed below is shown in Fig. 2. The rst study utilizing the alternative pathway was the expression of the Isochrysis C18 D9-elongase [89] in leaves of Arabidopsis

using the constitutive CaMV 35S promoter and resulted in the synthesis of signicant levels of EDA and ETriA (15% of total FA) [90]. To fully reconstitute the alternative VLC-PUFA biosynthesis pathway for ARA and EPA, transgenic Arabidopsis lines expressing the Isochrysis D9-elongase were sequential transformed with the Euglena D8-desaturase and the M. alpina D5-desaturase [91] under the control of the constitutive 35S promoter [86]. ARA and EPA products were accumulated to a combined level of 10% (3% EPA and 6.6% ARA) of total fatty acids in leaf tissues; these data represented an important proof-of-concept demonstration [86,92]. Detailed analyses of leaf lipids [90] have conrmed that both D9C18-PUFAs were efciently elongated, accumulating to very high levels in the acyl-CoA pool of transgenic plants [93]. This indicated the inefcient transfer of these non-native fatty acids from the acyl-CoA pool into extra-plastidial phospholipids for their subsequent desaturation. In addition to accumulation of ARA and EPA, several other C20 PUFA were also detected and identied as sciadonic acid (20:3D5,11,14) and juniperonic acid (20:4D5,11,14,17) [86]. These two non-methylene-interrupted PUFA appear to have arisen from the promiscuous activity of the D5-desaturase on substrates that might be expected to undergo D8-desaturation, due to a competition between enzymes for the elongated product. The M. alpina D5-desaturase used in the reconstitution of the alternative VLC-PUFA biosynthetic pathway was previously observed to utilize unexpected substrates when individually expressed in transgenic canola, resulting in the accumulation of the unusual D5-desaturated C18 FA, taxoleic and pinolenic acids [91]. It remains to be seen how well the alternative pathway performs when it is expressed in seeds, as opposed to vegetative tissue. Complementary studies were described by Abbadi et al. [87] on the expression of the conventional D6-desaturase pathway in linseed (Linum usitatissimum) and tobacco by coexpressing the D5and D6-desaturases from the diatom Phaeodactylum tricornutum [82] together with the D6-elongase from the moss Physcomitrella patens under the control of seed-specic promoters and introduced as a single integration event. Transgenic lines accumulated relatively low levels, only 1.6% EPA and 2.7% ARA. However, whilst these C20 LC-PUFA were low, very high levels (>25% of total fatty acids) of D6-desaturated fatty acids (GLA and SDA) were observed. This indicated that whilst the rst desaturation in the VLC-PUFA biosynthetic pathway was functioning efciently, the elongation , described as step was severely limited. There was a bottleneck substrate dichotomy [22], as a result of poor acyl-exchange of GLA and SDA from the phospholipid species from where they were generated to their acyl-CoA derivatives. The authors suggested that the linseed acyl-CoA:lyso-phosphatidylcholine acyltransferase (LPCAT), the enzyme believed to be primarily responsible in mediating acyl-exchange between phosphatidylcholine and the acylCoA pool [94], discriminates against D6-desaturated acyl groups as substrates. Detailed biochemical and metabolic analysis conrmed that this poor exchange resulted in the incorporation of GLA away from the VLC-PUFA biosynthetic activities, instead being directly incorporated into TAG in an acyl-CoA-independent manner. This is most likely to result from the direct conversion of phosphatidylcholine (PC)-containing GLA to TAG, presumptively via a strong action of a phospholipid: diacylglycerol acyltransferase (PDAT)-like activity [87,95]. In respect to the substrate dichotomy bottleneck observed in linseed, this was analogous to that observed for the D8-alternative pathway in Arabidopsis [86], with the buildup of the product of the rst enzyme in the pathway. This presumptively occurred as a result of poor acyl-exchange between the two metabolic pools (phospholipids, acyl-CoA) through which VLC-PUFA biosynthesis progresses, and highlights the importance of acyl-exchange in both the forward (acyl-CoA ? PC; required after the rst reaction of the D8-alternative pathway) and reverse (PC ? acyl-CoA; required after the rst reaction of the

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Fig. 2. Overview of oil composition in transgenic lines. The fatty acid compositions of published transgenic lines have been compared, with the levels of target products and biosynthetic intermediates shown. The different congurations used are indicated. For clarity, the endogenous fatty acids are not shown, since these vary on a species-byspecies basis. The studies compared are: Qi et al. (2004) [86], Abbadi et al. (2004) [87], Kinney et al. (2004) [88], Wu et al. 2005 [100], Robert et al. (2005) [102], Hoffmann et al. (2008) [104]. (A) Omega-3 VLC-PUFA (EPA, DHA) accumulation in transgenic plants. (B) Omega-6 VLC-PUFA (ARA) accumulation in transgenic plants.

D6-pathway) directions in heterologous VLC-PUFA biosynthesis. Given that such acyl-exchange is dependent on endogenous acyltransferases activities accepting non-native substrates (i.e. the

intermediates of VLC-PUFA pathway) it is also likely that different activities (e.g. lyso-phospholipid acyltransferases, PDAT etc.) have different afnities for these novel fatty acids [96]. Moreover, since

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many of these acyl-exchange enzymes can work in both forward and reverse directions, the pool sizes of individual metabolites is also likely to prove critical in determining the predominant enzyme activity. A third exemplication is described in the patent application by Kinney et al. [88] realized in soybean (Glycine max). These researchers used a similar approach to that of Abbadi et al. [87], expressing genes encoding components of the conventional D6desaturase pathway, a D6-desaturase (either from the oomycete fungus Saprolegnia diclina or M. alpina), a D6-elongase from M. alpina and nally a D5-desaturase from M. alpine, in transgenic soybean seeds and somatic embryos. However, to maximize the accumulation of omega-3 VLC-PUFAs such as EPA and DHA, an Arabidopsis FAD3 gene [97] and a S. diclina D17-desaturase [98] were also co-expressed to turn the omega-6 PUFA metabolites into their omega-3 counterparts. The expression of these ve enzymes yielded of 19.6% EPA in the transgenic somatic embryos, while almost no ARA intermediate was observed because of the presence of the highly efcient D17-desaturase used. Although the reasons for the high yields obtained in soybean as compared to linseed are not clear, the differences between the two studies may be a reection on the differing endogenous lipid metabolism present in linseed, though it should be noted that similar attempts to produce EPA in transgenic soybeans have been much less successful, for unknown reasons [99]. The lower accumulation of GLA in soybean oil, compared with tobacco and linseed, suggests a higher efcient transfer of GLA to the acyl-CoA pool for subsequent elongation by the reverse reaction of soybean LPCAT. Unexpectedly, up to 4.7% of x3-docosapentaenoic acid (DPA; 22:5D7,10,13,16, 19), a DHA precursor, was also detected in the high EPA lines as a result of the additional activity of the M. alpine D6 elongase toward the D5fatty acid EPA. This activity was not previously reported when the elongase was expressed in yeast [56], demonstrating a difference in substrate-specicities in plant and yeast. This promiscuity is worthy of further investigation, and maybe indicates problems in the correct assembly of the elongase (discussed below). As a result of the accumulation of DPA, Kinney et al. [88] carried out another co-transformation series with six cDNAs to produce DHA in somatic embryos. Additional genes for the D4-pathway, a D4desaturase from the fungus Schizochytrium aggregatum and a specic D5-elongase from the alga Pavlova sp. were expressed in addition to the activities required to generate EPA. However, only low levels of DHA (2.03.3% of total fatty acids) were obtained, most likely due to the generic problems of substrate dichotomy discussed above, resulting in inefcient acyl-exchange between the D5-elongase and the D4-desaturase. Two additional studies have demonstrated the accumulation of DHA and EPA in oilseeds. Firstly, Wu et al. [100] described expression of the D6-pathway in Brassica juncea using a similar approach to that used by Kinney et al. [88] but involving more transgenes. A D17desaturase from Phytophtora infestans was introduced to convert omega-6 substrates to omega-3 counterparts, a D12-desaturase from Calendula ofcinalis [101] to increase the ux through the entire transgenic pathway and nally a gene encoding a LPCAT from Thraustochytrium sp. was also co-expressed to increase exchange of D6unsaturated acyl groups from acyl-phospholipids to acyl-CoAs for elongation. As a result of the genes co-expression transgenic B. juncea plants accumulated up to 25% ARA or 15% EPA [100]. This high level of EPA allowed the introduction of the additional genes required for DHA synthesis (D5-elongase, D4-desaturase) to attempt the conversion of EPA to DHA, resulting in low but signicant amounts of DHA (0.21.5% of fatty acids in the seed lipids). These data indicate that B. juncea is a highly efcient host for the synthesis of ARA and EPA to high levels (comparable to that observed in soybean) but capable only of low level synthesis of DHA, reecting an apparent block in the conversion of EPA to the C22 PUFA, DHA. This would also

indicate that whilst endogenous B. juncea acyltransferases can facilitate the exchange of acyl-intermediates on the pathway to EPA, the longer, more unsaturated forms of the DHA pathway are only very poorly utilized. Thus, the successful accumulation of DHA may require the co-expression of suitable acyl-exchange activities. A similar approach was carried out by Robert et al. [102] expressing a bifunctional D6/D5-desaturase from zebrash (Danio rerio) [103] in conjunction with the D6-elongase PEA-1 from C. elegans [57] to generate EPA. To generate DHA, two additional activities (D5-elongase and D4-desaturase) from the algae Pavlova salina were co-expressed [69]. Based on the observations of Abbadi et al. [87] and subsequent studies, Robert et al. hypothesised that the use of the (putative) acyl-CoA-dependent desaturase from zebrash might overcome substrate dichotomy bottlenecks prior to D4-desaturation. However, whilst this study did demonstrate a proof-of-concept accumulation of ARA, EPA and DHA in Arabidopsis seeds, the levels achieved were relatively low: ARA and EPA (4.2% of total lipids) and 0.20.5% of DHA. These results could be explained by low substrate levels of LA-CoA and ALA-CoA in the acyl-CoA pool, which then rate-limits the levels of D6-desaturation products and all subsequent metabolites. Alternatively, the codon usage of the two animal genes (C. elegans PEA-1 D6-ELO, D. rerio desaturase) may have resulted in the inefcient translation of these enzyme activities. Finally, it remains to be demonstrated that the D. rerio desaturase is indeed a bona de acyl-CoA dependent activity. These additional studies on the heterologous expression of desaturase/elongase combinations in different host plant species demonstrated that minor differences in host plant biochemistry can be of vital importance on the successful synthesis of ARA and EPA. Endogenous acyltransferases activities from transgenic soybean and Brassica presumptively have a broader substrate-specicity than linseed or tobacco and can partially overcome the substrate dichotomy problem. A further attempt to avoid the acyl-exchange bottleneck in transgenic plants by using acyl-CoA-dependent desaturases has been recently described [104]. These authors isolated and characterized two cDNAs from the microalga Mantoniella squamata which encoded for D6- and D5-desaturases with predicted acyl-CoA-substrate dependence (as described previously for a D6desaturase from Ostreococcus tauri [105]. These desaturases were co-expressed under the control of a seed-specic promoter with the D6-elongase PSE1 of the moss P. patens [106] in Arabidopsis. Transgenic plants accumulated low but representative amounts of EPA, and crucially lacked the accumulation of D6-desaturation products previously observed in earlier studies. Thus, Hoffmann et al. [104] conrmed the potential of using acyl-CoA-dependent activities to overcome the problems associated with substrate dichotomy. However, it is perhaps surprising that this optimal conguration of the VLC-PUFA pathway yields only low levels of the target fatty acids (<0.5% EPA). This is again possibly due to problems with codon usage of the algal genes and/or lack of sufcient substrate acyl-CoAs. In this latter respect, it is worth also considering this with regard to the alternative D8-pathway, since this conguration also requires the same substrates as the acyl-CoAdependent D6-pathway (i.e. LA-CoA and ALA-CoA). Sayanova et al. [93] observed the high level accumulation of C20 elongation products in both the acyl-CoA pool and phospholipids in transgenic Arabidopsis plants expressing the Isochrysis D9-ELO. These observations would indicate that (in leaves) acyl-CoA substrates are not limiting for the synthesis of VLC-PUFAs, though it would be interesting to conrm this by the similar expression of the O. tauri D6-desaturase. Finally, the anerobic PKS-like pathway has been described by Metz et al. [107] for DHA production in plants using a PKS pathway system. The genes expressed in Arabidopsis seeds were the three subunits (ORFA, B, C) of a Schizochytrium PKS with a phosphopantetheinyl transferase (PPT) from Nostoc, essential for activating the acyl

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carrier protein (ACP) domains of the DHA synthase PKS. These seeds accumulated up to 0.8% DHA with an additional 1.7% DPA n-6. Further optimization of this pathway in commercial oilseeds is ongoing. 5. Crucial issues: optimization the levels of LC-PUFA in transgenic plants Although the effective biosynthesis of ARA, EPA and to some extent DHA has been demonstrated using different approaches in transgenic plants, the resultant fatty acid compositions and levels are not equivalent to that found in sh oil. Moreover, in most current examples such transgenic plants also contain high levels of omega-6 and omega-3 metabolic intermediaries. Marine oils, rich in EPA and/or DHA, are almost completely devoid of omega-6 fatty acids such as GLA and DHGLA. The goal now is to generate a vegetal oil substitute for sh oils optimizing the accumulation of VLC-PUFAs. Several approaches (discussed below) have been suggested as a result of the studies described in this article. In addition, these data also provide new insights into our understanding of plant lipid biochemistry, in particular the channelling of FA into various different lipids. Outlined below are logical approaches which might be expected to enhance the accumulation of VLC-PUFAs in transgenic plants. 5.1. The identication of superior desaturases The rst approach is to identify highly active acyl-CoA dependent desaturases from a lower eukaryote, as has already been described for the D6-desaturase from O. tauri [105] or M. squamata [104]. With such enzyme activities, both the desaturation and elongation reactions utilize acyl-CoA substrates and avoid the requirement for acyl-exchange with PC. As noted above, published examples of the use of an acyl-CoA dependent route have resulted in the signicant reduction in the accumulation of biosynthetic intermediates (most notably omega-6 GLA, linked to PC) but the actual levels of target VLC-PUFAs such as EPA and DHA are disappointingly low [102,104]. This may be due to a number of problems (substrate availability, use of non-optimized sequences) or reect additional (undened) metabolic bottlenecks. It remains to be demonstrated that an exclusively acyl-CoA dependent pathway delivers signicant improvement to yields of EPA or DHA levels, thought it could also be argued that the unambiguous identication of an acyl-CoA dependent D5-desaturase from lower eukaryotes is currently lacking. Very recently, the molecular identication of an acyl-CoA dependent D12-desaturase was reported from insects [108] and it will be of interest to see if the co-expression of this activity would enhance (through the generation of LA-CoA) the activity of the algal acyl-CoA D6-desaturases in transgenic plants. A second modication based around desaturation is to ensure the conversion of omega-6 fatty acids to their omega-3 equivalents is through the use of x3-desaturases: such enzyme activities have been demonstrated to be pivotal in the production of elevated levels of EPA in Brassica juncea [100]. Such x3-desaturases ideally have a high preference for C20 substrates (such as ARA) and have been identied from a number of fungal species [17]. An alternative iteration is to identify VLC-PUFA desaturases with strong preferences for omega-3 substrates such as have been identied from M. squamata, Primula and Echium [104,109,110]. In the case of the Primula D6-desaturase, expression of this activity in linseed has recently been shown to result in the signicant accumulation of SDA without the concomitant accumulation of GLA [85]. Another potentially very useful enzyme activity, a bifunctional D12- and D15-desaturase, has also recently been described from a number of organisms by several different groups. Damude et al. [111] identied such a bifunctional desaturase from Fusarium moniliforme and demonstrated that the co-expression of this activity with the

primary VLC-PUFA biosynthetic enzymes resulted in signicant enhancement of the levels of EPA in both yeast (Y. lipolytica) and plants (soybean). Similarly, bifunctional desaturases were characterised from the free living amoeba A. castellanii [70], Claviceps purpurea [112] and Coprinus cinereus [113]. Thus, such activities have the potential to generate considerable omega-3 substrates for conversion to LC-PUFAs. 5.2. Identication of a VLC-PUFA-specic acyl-exchange mechanism Metabolic bottlenecks appear to limit the full potential of oilseed crops to accumulate economically sufcient amounts of these novel fatty acids. Such factors are likely to be represented by enzymes involved in the channelling and partitioning of fatty acids between the different metabolic pools involved in lipid synthesis and compartmentation this can take the form of spatial separation of different organelles (as is the case for TAG-containing oil bodies) or exchange between different substrate pools. Central to that latter problem is the so-called substrate dichotomy, where desaturation uses acyl-substrates linked to phospholipids whereas elongation requires acyl-CoA substrates. It is predicted that the enzyme LPCAT, responsible for catalysing bidirectional exchange between these two pools, could help alleviate this bottleneck (represented schematically in Fig. 3). Very recently, genes encoding LPCAT have been functionally characterised from yeast and animals (reviewed in [114]), though in all cases only the forward reaction of LPCAT was demonstrated (i.e. acyl-CoA-dependent acylation of lyso-PC) (e.g. [115,116]). Interestingly, two Arabidopsis genes which showed strong homology to animal LPCATs were shown to encode predominantly LPEAT activities (acyl-CoA-dependent acylation of lyso-PE) [117]. Thus, the identication of a plant or algal form of LPCAT remains to be demonstrated, as does the role of the reverse reaction (release of a fatty acid from the sn-2 positions of PC and activation to acyl-CoA) and its utility in transgenic synthesis of VLC-PUFAs. 5.3. Maintenance of a continuous ux of substrates through the VLC-PUFA biosynthetic pathway without signicant loss to TAG Technological modications to endogenous lipid metabolism to overcome such problem are not obvious, not least of all since such acyl-channelling represents the sum of multiple different acylexchange activities. In addition, it is highly likely that each plant

Acyl-CoA pool

Lyso-PC
LPCAT LPCAT

PC pool
PDAT

G3P
G3PAT

LPA
LPAAT

PA
PAP

DAG

CPT

PC

CPT

DAG

DGAT

TAG

Fig. 3. Schematic representation of triacylglycerol synthesis in plants. The acylCoA-dependent (Kennedy) pathway is shown as the central route for TAG synthesis. The primary activities are shown: acyl-CoA:glycerol-3-phosphate acyltransferases (G3PAT); acyl-CoA: lyso-phosphatidic acid acyltransferases (LPAAT); phosphatidic acid phosphatise (PAP); acyl-CoA:diacylglycerol acyltranserase (DGAT). Also shown are the acyl-CoA-independent activities such as the acyl-transfer between PC and DAG to generate TAG (catalysed by PDAT this reaction also generates lyso-PC) and also acyl-exchange between PC and DAG catalysed by cholinephosphotransferase (CPT). The acyl-CoA pool is generated by export of fatty acids from the plastid, and represents the sum of this activity plus reverse acyl-exchange from extra-plastidial phospholipids.

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species has a different combination of such activities (perhaps evidenced by the huge disparity in the composition of plant seed oils), making a generic intervention unlikely if not impossible. A hypothetical solution might be the identication of TAG biosynthetic enzymes (such as diacylglycerol acyltransferases, DGAT or LPAT) which have a very strong substrate preference for EPA or DHA. However, such activities are generally acyl-CoA-dependent, meaning that the substrate VLC-PUFA must be present in the acyl-CoA pool. The converse solution to this requirement is to use acyl-CoA-independent enzymes such PDAT [95], which catalyses the generation of TAG through the removal of fatty acids from the sn-2 position of phospholipids and then acylates them to DAG. Such an activity has the advantage of removing desaturation products from their site of synthesis, but it appears that PDATs are not restricted to PC as substrates. Genes for PDAT have been identied in plants, but little evidence has been found to date to suggest that these enzymes play a major quantitative or qualitative role in seed TAG metabolism. However, it may be possible to identify TAG biosynthetic enzymes from VLC-PUFA-synthesising lower eukaryotes which display the desired activities. Interestingly, recent evidence from plants has shown that the DGAT2 class of DGAT displays a more precise range of substrate-specicities, compared with DGAT1-type enzymes [118]. 5.4. Optimizing the fatty acid elongase Microsomal fatty acid elongation occurs as a result of four sequential enzymatic reactions: condensation, ketoreduction, dehydration and enoyl reduction, although for transgenic is possible through heterologous expression of just the initial condensing enzyme [55,64]. It has been assumed that the contribution of the other elongase components to VLC-PUFA synthesis is neutral, since the condensing enzyme acts in a trans-dominant manner. However, it is conceivable (if not likely) that the physical and biochemical interactions between a non-native condensing enzyme and the other three endogenous elongase components may be sub-optimal. Perhaps of signicance is the fact that in higher plants, unlike yeast or animals, the predominant microsomal KCS activities take the form of FAE1-like enzymes, whereas the transgenic VLC-PUFA elongating activity is of the ELO-like form [63]. In that respect the atypical FAE1-like alternative pathway D9-elongating activity isolated from P. marinus [62] may warrant further evaluation, as might the search for additional examples of FAE1-like VLC-PUFA elongating activities. Alternatively, the use of (ELO-like) activities from VLCPUFA-synthesising lower plants such as Marchantia polymorpha may prove of benet initial studies indicate that M. polymorpha activities perform well in transgenic plants [119]. Currently, our understanding of the regulation, organisation and assembly of the elongase complex is limited. Overexpression of the elongase ketoreductase resulted in an increase in the accumulation of VLC monosaturated fatty acids in yeast, presumably by either increasing ux through the elongase or increasing the absolute number of elongase complexes, challenging the concept that the core elongase components have a neutral role in determining the levels of VLCFAs [120]. Thus, for all of the reasons outlined above, it may be that to obtain maximal elongation of target fatty acids, the additional core components of the elongases may need to be isolated from suitable EPA- or DHA-accumulating organisms and co-expressed with the transgene condensing enzyme from the same species. 5.5. Modulating the acyl-CoA pool As discussed above, the use of acyl-CoA dependent desaturases is predicted to bypass the metabolic bottleneck generated by substrate dichotomy between the desaturase and the elongase. The success of this approach is dependent on signicant levels of sub-

strate fatty acids (LA, ALA) being present in the extra-plastidial acyl-CoA pool; given that the acyl-CoA pool in most plant cells is considered to be lower than that found in yeast or animals [121], this also indicates the requirement for a strong ux of fatty acids into this metabolic pool. One proven method for altering the prole of fatty acids present in the acyl-CoA pool is via the use of plastidial thioesterases which prematurely release fatty acids from the fatty acid synthase such approaches have been shown to generate increased levels of medium chain acyl-CoAs on expression of a Cuphea thioesterase [121]. However, it is not obvious how such approaches would directly result in the enhanced synthesis of VLC-PUFAs. Alternatively, it has recently been shown that blocking the peroxisomal ABC transporter CTS (required for beta-oxidation) results in elevated levels of cytosolic acyl-CoAs and their incorporation into storage lipid [122]. Whilst a total blockade of beta-oxidation results in abnormal plant development and impaired germination, it may be possible to use developmentally-regulated silencing of such activities to modulate the acyl-CoA pool, increasing both the substrates available for VLC-PUFA biosynthesis and also the accumulation of target fatty acids in storage triacylglycerols. It must also be noted that our understanding of the ux of fatty acids into the acylCoA is partial, with very recent studies questioning the established model in which the products of the plastidial fatty acid synthase (16:0, 18:0, 18:1) are directly exported from the plastid into the cytosolic acyl-CoA pool [123]. Similarly, detailed kinetic analysis of the channelling of fatty acids into soybean embryo triacylglycerols indicates the central of acyl-exchange between PC and the acyl-CoA pool [124]. Thus, further research on the biosynthesis and homeostasis of the acyl-CoA pool is required. 5.6. Co-ordinated expression of transgenes All of the examples described above of the production of VLC-PUFAs in transgenic plants have relied on the simultaneous co-expression of desaturases and elongases in developing seeds. In most cases, the promoters used to drive this seed-specic expression were derived not from genes involved in oil biosynthesis but more often instead from storage protein synthesis. Thus, it may be possible to enhance the overall levels of target VLC-PUFAs through the use of promoters whose activity coincides with maximal oil synthesis and accumulation. Certainly the choice of appropriate promoter has been postulated to play a key role in the wide variation in VLCPUFAs levels observed in transgenic soybeans [17,99]. 5.7. Appropriate localisation of transgene-derived activities It is now believed likely that many microsomal biochemical reactions occur in discrete sub-domains of the endomembrane system. Such sub-domains could be generated by local variation in lipid compositions (such as so-called lipid rafts [125]) or via proteinprotein interaction to nucleate higher-order structures [126]. In either scenario, it is envisaged that multiple enzyme activities for a particular biochemical pathway are co-located, resulting in the minimal loss of intermediates and the optimal channelling of products to their intended metabolic pool. One possible reason for limited production of non-native fatty acids such as VLC-PUFAs could be due to lack of sub-domain co-location for critical activities this could be either primary biosynthetic enzymes or those involved in the generation of a strong ux (i.e. acyltransferases such as DGAT [126]. 6. Conclusions and future perspectives It is obvious from the studies described in this article that heterologous reconstitution of VLC-PUFA synthesis in transgenic

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plants has been successfully demonstrated. This has been achieved by the reverse engineering of the primary biosynthetic enzymes, and has yielded signicant levels of nutritionally important VLCPUFA such as ARA, EPA and (to a lesser extent) DHA. These data have also demonstrated that our understanding of the biochemical pathways involved in the synthesis of these FA is still incomplete. In particular, acyl-channelling between the PC, acyl-CoA and TAG pools is not yet fully understood. In that respect, the large numbers of different iterations of transgenic VLC-PUFA biosynthesis provide a powerful and valuable resource with which to study these fundamental processes. Given the complexity of the pathways under study, it would seem prudent to adopt a holistic (systems) approach to the deconvolution of key factors in the accumulation of VLC-PUFAs in the seed oils of transgenic plants. On the other hand, although the target VLC-PUFAs have been produced in crop plants, if the primary goal is to obtain a plant-based sh oil substitute, there remain further obstacles to be overcome in order to produce a transgenic oil which is substantially equivalent to that from marine sources. This is because current iterations of VLC-PUFAaccumulating plants synthesize a mixture of omega-6 and omega-3 fatty acids and intermediates that are not normally found in sh oil. Therefore, it is not only desirable to elevate the levels of EPA and particularly DHA closer to that found in marine oils but, perhaps more crucially, decrease the levels of omega-6 fatty acids. This may be possible through the use of x3-desaturase activities and omega-3-specic enzymes, and may also take advantage of a number of host oil-seed crops which are very high in omega-3 fatty acids. The demonstration that oil-seed crops can synthesize signicant levels of VLC-PUFA, especially EPA, signify a progress toward the provision of an alternative source of VLC-PUFA, in particular as a replacement to the diminishing natural reserves of sh oils, in the form of transgenic plants. These plants represent the second wave of plant biotechnology, by which output traits of benet to the consumer or end-user are delivered. The benets of producing VLC-PUFAs in transgenic plants should be obvious, in that they would provide a sustainable and non-contaminated source of these important fatty acids for human nutrition. Thus, not only do these fatty acids help to improve chronic and acute human diseases (cardiovascular disease, obesity, type 2 diabetes and metabolic syndrome) thus consequently reducing public expenditure on the healthcare system, but also contribute to correct development of neonates and infants. However, there are still signicant issues which need to be considered before any VLC-PUFA trait can progress towards the marketplace. Firstly, the trait has to be moved into elite germplasm and demonstrated not to impact on the overall performance (in terms of resistance to stress and in yield) of the crop plant. Then such lines would need to undergo the rigorous and time-consuming process of regulatory approval [127] which is extremely costly and could serve as a barrier to commercialisation. In addition, the nal product and appropriate end-users must be clearly identied. With that in mind, two distinct approaches can be envisaged as routes to deliver GM VLC-PUFAs into our diet. Firstly, by direct ingestion of transgene-derived plant oils, most likely in the form of dairy product formulated with these oils (such as yogurt, margarine, and spreads). However, taking into consideration the antipathy of European consumers to GM foods, such an option, despite the human health and environmental benets offered by transgenic plant derived sh oils is currently not immediately likely. However, it should be remembered that Europe represents only a portion of the global marketplace, and many other (larger) economies do not share the same views on the direct consumption of GM foodstuff, most notably North America and China. A second approach to the delivery of transgenic VLC-PUFAs into the human food chain is via an indirect route, in which the GM plant oils are used as animal feedstuffs. In this scenario, transgene-

derived VLC-PUFA-enriched oils are used to formulate animal feeds, resulting in the accumulation of these benecial fatty acids in the animal products (e.g. meat, milk, eggs etc.). Perhaps the most obvious route is through aquaculture, which as noted above has a strong demand for omega-3 VLC-PUFAs. Thus, farmed sh would be provided with a diet in which marine-derived oils are replaced by terrestrial modied plant oils [128]. Whatever the scenario for the uptake and utilization of transgene-derived VLC-PUFAs, it will be important to ensure that the end-user and consumers are fully informed of the underlying science and benets of the resulting products with such an approach, the consumer can make an informed decision and also not feel coerced into purchasing any particular product. Given the very real current concerns over sh stocks, human health and food security, we believe that transgene-derived sh oils have an important role to play in securing the sustainability of future sh stocks and ensuring optimal nutrition. Acknowledgements Rothamsted Research receives grant-aided support from the BBSRC. Our research in this area was partially supported by Lipgene, an EU Sixth Framework Programme Integrated Project (Project Number: FOOD-CT-2003-505944). We thank BASF Plant Sciences for their input and support. References
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