Progress in Lipid Research 49 (2010) 108–119Contents lists available at ScienceDirect Progress in Lipid Research journal homepage: www.elsevier.com/locate/plipres Review An alternative to fish oils: Metabolic engineering of oil-seed crops to produce omega-3 long chain polyunsaturated fatty acids Mónica Venegas-Calerón 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 fish rich in these beneficial fatty acids our primary dietary source. However, over-fishing 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 fish 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. Ó 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:
[email protected] (J.A. Napier). 0163-7827/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2009.10.001 indicating the requirement for supplementation via dietary intake of VLC-PUFAs. It is estimated that the % conversion of ALA to EPA is 5–10%. 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. Venegas-Calerón et al. This also means that the balance of omega-6 and omega-3 VLC-PUFAs can be influenced by the dietary ratio and levels of EFAs because the D6-desaturase (first desaturation and limiting step in VLC-PUFA biosynthesis – Fig 1) can utilize both EFA as substrates.7. sustainable source of these important fatty acids.17.8]. environmental pollution of marine ecosystems has resulted in the accumulation of dioxins. 2. It has been estimated that our ancestral dietary fat composition showed an omega-6/omega-3 ratio of 2:1. cancer. / Progress in Lipid Research 49 (2010) 108–119 109 1. Perhaps the most obvious alternative to fish oils is via contained culture of the aquatic microbes which synthesize EPA and/or DHA. VLCLC-PUFAs are vital constituents of human metabolism.19) [1–4]. since farmed fish require omega-3 VLC-PUFA-containing feedstuffs. there is plentiful evidence (from epidemiology and dietary intervention studies) for the health-beneficial properties to humans of dietary consumption of omega-3 VLC-PUFAs such as eicosapentaenoic acid (EPA. 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. mosses. meaning that omega-6 LA (and metabolites) cannot be metabolised to omega-3 fatty acids (due to the absence of the specific x3/D15-desaturase activity). Approaches using microbiological sources to synthesize VLC-PUFA have been developed and are economically viable for specific high value applications (such as infant formula – baby milk – formula- tions [18]) in controlled culture systems. However. bacteria and lower plants also have a capacity to synthesize significant amounts of VLC-PUFAs [10].12) and a-linolenic acid (ALA. dietary recommendations now include not only LA and ALA but also EPA and DHA for optimal nutrition. The main source of EPA and DHA in the human diet is through the direct consumption of cold water marine fish (such as salmon. in the range of 0. In addition. Moreover.5–1.12.8. heavy metals and polychlorinated biphenyls in fish.15) that serve as the metabolic precursors for VLC-PUFA biosynthesis in animals [11].11. the diets are dominated by vegetable oils and processed foods which contain excessive levels of omega-6 fatty acids such as LA. Increasing epidemiological evidence indicates that functional deficiencies or imbalances involving omega3 VLC-PUFA have been associated with a range of disorders including.M. It is noteworthy that such fermentation-based systems are also sensitive to disruptions of power-supplies and have a significant environmental footprint. These fatty acids can be grouped into two main families.15 n-3) respectively from the precursor oleic acid (18:1D9) [23]. 18:3D9. 1) [24].17) and docosahexaenoic acid (DHA. but accumulate them as a result of their dietary acquisition (though it should be noted that freshwater fish appear to have a greater capacity to synthesize EPA and DHA from ALA) [7.33]. 18:2D9. 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. depending on the position of the first double bond proximal to the methyl end of the fatty acid.24]. Finally. 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 fish [9]. In consequence. 18:3D9. this same ratio is today in excess of 10:1 [27].10. Therefore. Most dietary LA and ALA are b-oxidized to provide energy and only a small portion of them are converted to VLC-PUFAs [25].12. Because no higher plant oilseeds produce VLC-PUFAs such as EPA and DHA. 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. However. marine fish (like other animals) do not efficiently metabolise ALA to VLC-PUFAs. as a consequence.12 n-6) and a-linolenic acid (ALA. cardiovascular disease [28. cannot produce linolenic acid (LA. EPA and DHA are synthesized de novo by one of the two classes of biochemical pathway (reviewed on the text below). One attractive option is the use of transgenic plants to synthesize these fatty acids. In these microorganisms. 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. whereas conversion of ALA to DHA is <1% [9].13. and sardines) [6. some fungi. such systems are expensive to maintain and have limited flexibility for significant scale-up and requiring the appropriate microbiological facilities (such as fermenters) [19]. the expansion of industrialised aquaculture exacerbates the overexploitation of natural marine resources.8 g/day (or the consumption of at least two serving of fish per week) [32. 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. the dietary EFAs are not inter-convertible. Aquaculture is certainly the largest consumer of fish-derived oils and currently even the most sophisticated husbandry of high value species such as salmon require the input of dietary fish oils to a level significantly higher than that present in the finished product. However. to the point of questioning the benefits of fish consumption in human health [13]. In most Western societies. 18:2D9. It is also worth noting that the D12 and D15-desaturases . 20:5D5. the most logical reason being that genetic drift in these genes was effectively compensated by dietary intake of LA and ALA. there is a very obvious requirement for an alternative and sustainable source of VLC-PUFA for their use in human nutrition [15–17]. which are specifically harvested for this application (since they are not normally consumed by humans). and inflammatory and autoimmune disease [30. tuna.20]. 18:3D9. at least to the layperson) a net consumer of fish oils and as such. 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. The marine oils used as aquaculture feedstocks are usually extracted from socalled ‘‘trash” species such as sand eels. there is an obvious need for an alternative. In view of all these factors. although plants are rich in the two essential dietary fatty acids linoleic acid (LA.22] – the promise and the prospects of these new transgenic crops will be considered in this review. In view of all of these points.12.7]. In addition.14. the loss of these species from food-webs has a profound impact on the overall stability of ecosystems [14]. However. In particular. There is growing concern regarding the sustainability of global fish stocks (the predominant sources of omega-3 VLC-PUFA) because marine fish stocks are in severe decline as a result of decades of over-fishing [12]. this route is unlikely to provide sufficient levels of VLC-PUFA for optimal human health and nutrition. omega-6 (or n-6) and omega-3 (or n-3) families.29]. not operating in a sustainable manner.16. In higher plants these fatty acids are almost completely absent. mackerel. and are generally under-represented in levels of omega-3 fatty acids [26]. 22:6D4. Therefore aquaculture is (perhaps surprisingly. therefore dietary intake of these fatty acids is a key aspect of human nutrition [6].31]. During the last ten years.15) [5]. Unsurprisingly. all the primary genes involved in VLC-PUFA biosynthesis have been identified from a range of different species. eicosapentaenoic acid (EPA). Des = desaturase. 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]. These genes can be classified into the two distinct enzymatic reactions that catalyse the primary biosynthetic process. pro-aggregatory and inmuno-active eicosanoids (series-2). Elo = elongase. and vasoconstriction/dilation. / Progress in Lipid Research 49 (2010) 108–119 Animals ∆ Plants 18:0 Stearic acid (SA) Conventional ∆6-pathway ω 3 Des ∆ 9 Des 12 Des ∆ γ 18:2 ∆ 9. 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.12 Linoleico acid (LA) 18:1 ∆ 9 Oleico acid (OA) ∆ 15 Des 18:3 ∆ 9.44] and dementia [45]. Characterization of VLC-PUFAs biosynthetic pathways Over the last decade. so-called ‘‘front end” PUFA desaturases which belong to the N-terminal cytochrome b5-fusion superfamily. in particular depression [29]. most notably Caenorhabditis elegans. as is the alternative D8-pathway. atherosclerosis.37]. research has demonstrated that there are considerable health benefits to be gained from having a diet rich in VLC-PUFA. Two routes for DHA synthesis are shown. The various routes for synthesis of arachidonic acid (ARA).12. oxygenated VLC-PUFA metabolites involved in the regulation of inflammation. 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]. Genetic analysis of the role of these enzymes in C. VLC-PUFAs are functional components that can modulate membrane fluidity and permeability. The first of these are the microsomal fatty acid desaturases.15 α-Linolenic acid (ALA) ∆ ω ∆ ∆ Alternative ∆8-pathway γ ω Alternative ∆8-pathway ω ω ω β Micobial ∆4 pathway Mammalian “Sprecher” pathway Fig. and various mental illness. microbial D4-pathway and mammalian ‘‘Sprecher” pathway. plaque aggregation. fungi. Finally. while omega-6 ARA produces more potent inflammatory. eicosanoids derived from omega-3 fatty acids (series-1 and series-3) are anti-inflammatory and modulate plaque aggregation and immune-reactivity [36. cognitive impairment. there are epidemiological studies which extend the beneficial effects of omega-3 VLC-PUFA to the immune system (including diseased states such as rheumatoid arthritis) [46]. For example. 1. Venegas-Calerón et al. Both EPA and ARA serve as substrates for the common cyclooxygenase and lipoxygenase enzymes. As a consequence they play crucial roles in human metabolism. The . and docosahexaenoic acid (DHA) are shown. the VLC-PUFAs ARA and DHA play an important role in neonatal health and development [38–40]. such as obesity and type-2 diabetes [31. protective effects have been clinically studied for cancer [42]. the reproductive system. not only playing structural roles in phospholipid bilayers but also acting as precursors to bioactive molecules. both omega-6 and omega-3 C20 fatty acids are precursors of the eicosanoids. More recently.110 M. plants and aquatic organisms. and in particular EPA and DHA. as mediated by the consecutive action of desaturases and elongases. 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].41]. Aerobic VLC-PUFA biosynthetic pathways. childhood and attention-deficit hyperactivity disorder (ADHD) [43. 3. The presence of these EFA desaturases has been observed in some lower animals. [49]. skin barrier function [47] and other exciting emerging roles such as inflammation-resolution [48]. including animals. firstly identified in 1997 by Sayanova et al. For example. The predominant D6-pathway is shown. 20:3 D11. It should be noted that KCS condensing enzymes can be divided into two distinct groups. As noted above. FAE1 – fatty acid elongation1. utilize substrates exclusively in the form of acyl-CoAs [65]. all the primary biosynthetic components required for the synthesis of the C20 PUFA ARA and EPA. the so-called D6-pathway. the synthesis of ARA and EPA was achieved. and does not require PUFA-specific aerobic desaturases and elongases [75]. involved in the biosynthesis of saturated and monounsaturated fatty acids with C18–22+ chain length. HCD. It is therefore for this reason that KCSs are often (semantically incorrectly) referred to as ‘‘elongases”. Venegas-Calerón et al. KCS).17. is dependent on the presence of the core elongase components. The dominant form. two different aerobic routes exist for the biosynthesis of EPA and its omega-6 counterpart. As in the prevalent D6-pathway.15) respectively. Isochrysis (Prymesiophyceae) and protists such as Euglena (Euglenophyceae). The presence of diagnostic motifs such as a cytocrome b5 domain and this variant His box have greatly facilitated the identification of candidate ‘‘front end” desaturases from animals. 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. This C24 PUFA is then subjected to limited peroxisomal b-oxidation.12. an Arabidopsis gene required for the synthesis of VLCFAs found in seed triacylglycerols).9. where the substrates for these enzyme activities are generally believed to be acyl-CoAs [51–53]. which also occurs in the ER. Mortierella alpina) [59]. all microsomal elongation described.12) and SDA (18:4D6. Another characteristic of the ‘‘front end” desaturases is the substitution of histidine by glutamine in the third histidine box (consensus sequence Q–X[2–3]–H–H). These activities (D5-elongase. Perhaps unexpectedly.74]. 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.g. ECR) [54] and resulting in a two-carbon chain elongation of the input substrate fatty acid.21]. and also from the few plant species (borage.17). marinus a second pathway is used. A first group comprises the so-called ELO-like sequences (named after the yeast ELO genes. Thus.14. (A schematic representation of the biosynthetic pathways is shown in Fig.14) and eicosatetraenoic acid (ETetA. with DPA then converted to DHA by the action of a D4-desaturase [73]. It is now also clear that there are a number of different configurations of the biosynthetic pathway for C20 LC-PUFAs. and when all three open reading frames (ORFs) were heterologously expressed in yeast. The condensing enzymes are considered to be rate-limiting and the regulators of substrate-specificity with regard to chain length and pattern of double bonds. 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. algae and some fungi which lack any such cytochrome b5 domain. 20:4D8. fungi (e. This is not the case in animals. to yield GLA (18:3D6. The D6-desaturase enzyme that carries out this desaturation is the same one as produces GLA and SDA. A second class of unrelated plant-specific KCS activities are known as FAE1-like enzymes (so-called after the founding member of this family. HCD. followed by D8-desaturation to ETA [62. by which it is chain-shortened by two carbons to yield the final product DHA: this is known as the Sprecher pathway. the synthesis of DHA is a simpler biochemical process where EPA is elongated to docosapentaenoic acid (DPA. ARA (Fig 1) [66]. One crucial observation regarding the microsomal desaturases from lower eukaryotes is that very many of these enzymes utilize glycerolipid-linked substrates. In contrast to microsomal desaturation. be it ELO-like or FAE1-like. Acanthamoeba castellanii and P. dehydration (hydroxyacyl-CoA dehydratase.11. Interestingly. HCD). However. and aquatic algae (e. fungi. This system is similar to the fatty acid synthase (FAS) pathway in that PUFA biosynthesis is initiated by the condensation between a . Instead. 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 [55–57]. further modification of the omega-3 EPA to synthesize the final product DHA can occur by two distinct routes [71]. the heterologous activity of any KCS. The second key enzymatic reaction in the synthesis of VLCPUFA is elongation. the elongase is a multi-protein complex made up of (at the very least) the core elongase components (KCR. [61]. 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. It should also be mentioned that an unrelated anaerobic VLC-PUFA biosynthetic system is present in a few diverse lower marine organisms.M. EPA or DHA is synthesized by a processive polyketide synthase (PKS)-like reaction from malonyl-CoA [76]. marinus).9. reduction (b-ketoacyl-CoA reductase. in particular fatty acids esterified to the sn-2 position of glycerolipids. The fatty acid elongation reaction consists of four sequencial activities: condensation of the substrate fatty acid with malonyl-CoA (b-ketoacyl-CoA synthase. 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. both ELO-type and FAE1-like.14. 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. In mammals EPA undergoes two rounds of elongation. In particular. Subsequently a second desaturation by a D5-desaturase occurs to produce finally ARA and EPA. In DHA-accumulating microbes. This pathway commences with the D6-desaturation of LA and ALA. Until very recently. and a second reduction (enoyl-CoA reductase. respectively) plant KCSs are able to functionally complement yeast strains which are entirely lacking in ELO genes [63.11. evening primrose.67–70]. although the basis and mechanism for this substrate selectivity is unclear. Isochrysis galbana) [60]. KCR). and currently all known examples of microsomal VLC-PUFA desaturases contain this N-terminal extension. 4+ TMs.17). 1).64] and also physically interact with the other elongase components. in recognition of its identification by Howard Sprecher [72]. 22:5 n-3) by a specific D5-elongase. ECR) and a KCS (ELO or FAE). These two fatty acids are then chain-elongated by two carbons via a D6-specific elongase to generate di-homo-c-linolenic acid (DHGLA. elongation cannot occur. Although ARA is generally considered to be the end point in the synthesis of the omega-6 class of VLC-PUFA. in which ALA is first elongated by a specific D9-elongase to eicosatrienoic acid (ERA. 24:5. as well as the C22 PUFA DHA have now been identified. 2 TMs ver- sus <300aa. This is in contrast to the D12 and D15-desaturases found in plants. generating first DPA and then tetracosahexaenoic acid (THA. which have been cloned from a number of species including mammals. is widely spread throughout eukaryotes. In conclusion. 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]. these fatty acids are then desaturated to ARA and EPA by a D5-desaturase. black currant and Echium) that carry out D6-desaturation of LA and ALA [50]. / Progress in Lipid Research 49 (2010) 108–119 111 cytochrome b5 domain is assumed to be involved in the electron transport chain required for acyl-desaturation.g. and algae. this KCS was in a small gene cluster with two cytochrome b5-fusion desaturases. this is the so-called alternative D8-pathway. n-3) which then is subject to D6-desaturation yielding 24:6 n-3. In some species from algae such as Pavlova. 20:3D8. Although FAE1-like KCSs are structurally quite different to ELO-like (500aa. ECD) and the exogenous KCS – in the absence of any of these three latter components. and highlights the importance of acyl-exchange in both the ‘‘forward” (acyl-CoA ? PC.85]. the enzyme believed to be primarily responsible in mediating acyl-exchange between phosphatidylcholine and the acylCoA pool [94]. this was analogous to that observed for the D8-alternative pathway in Arabidopsis [86]. Transgenic lines accumulated relatively low levels. [87] and Kinney 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-specific promoters and introduced as a single integration event. these data represented an important ‘‘proof-of-concept” demonstration [86. [88] demonstrated ´. The first 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 significant levels of EDA and ETriA (15% of total FA) [90]. though a few taxonomically unrelated phyla can synthesize D6-desaturated fatty acids (the first step on the D6-pathway) such as omega-6 c-linolenic acid (GLA. [86]. 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.84. described as step was severely limited. instead being directly incorporated into TAG in an acyl-CoA-independent manner. reduction. There was a ‘bottleneck ‘‘substrate dichotomy” [22].7% ARA. plants and mammals) of genes encoding the primary VLC-PUFA biosynthetic activities. ARA and EPA products were accumulated to a combined level of 10% (3% EPA and 6.57.17) [86]. / Progress in Lipid Research 49 (2010) 108–119 short chain acyl-CoA and an unit of malonyl-CoA. Venegas-Calerón et al.95]. in addition to the PKS-like pathway. This indicated that whilst the first desaturation in the VLC-PUFA biosynthetic pathway was functioning efficiently. accumulating to very high levels in the acyl-CoA pool of transgenic plants [93]. two desaturations and acylCoA elongation) to generate C20 PUFAs such as ARA and EPA.12) and omega-3 stearidonic acid (SDA. It is also worthy of note that genomic and biochemical analysis of lipid biosynthesis in Schizochytrium indicated that. due to a competition between enzymes for the elongated product. with the acyl chain growing by two-carbon units with each round. A dehydratase/isomerase from this PKS complex catalyze the trans. 18:4D6. effectively completing the first stage of attempts to reverse-engineer this trait into a heterologous host such as a transgenic plant.82] and subsequent experiments (with additional genes) to generate DHA [59]. 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. with initial data showing the low accumulation of ARA and EPA [56. alpina D5-desaturase [91] under the control of the constitutive 35S promoter [86]. The last few years have seen considerable progress in the identification from diverse sources (algae. transgenic Arabidopsis lines expressing the Isochrysis D9-elongase were sequential transformed with the Euglena D8-desaturase and the M.to cis-conversion of the double bonds to form EPA and DHA. the elongation ´ . A diagrammatic summary of the results obtained from the studies discussed below is shown in Fig. This is most likely to result from the direct conversion of phosphatidylcholine (PC)-containing GLA to TAG. 2. Detailed biochemical and metabolic analysis confirmed that this poor exchange resulted in the incorporation of GLA away from the VLC-PUFA biosynthetic activities. Interestingly. taxoleic and pinolenic acids [91]. whilst these C20 LC-PUFA were low. This indicated the inefficient transfer of these non-native fatty acids from the acyl-CoA pool into extra-plastidial phospholipids for their subsequent desaturation.14) and juniperonic acid (20:4D5.6% EPA and 2. The domains encoding these activities are arranged sequentially on long (20–30 kb) open reading frames (ORFs) in bacteria such as Shewanella and Vibrio.9.6% ARA) of total fatty acids in leaf tissues.g. acyl-CoA) through which VLC-PUFA biosynthesis progresses. resulting in the accumulation of the unusual D5-desaturated C18 FA. very high levels (>25% of total fatty acids) of D6-desaturated fatty acids (GLA and SDA) were observed. only 1. discriminates against D6-desaturated acyl groups as substrates. with the buildup of the product of the first enzyme in the pathway. Apart from the important biotechnological breakthroughs. The M. 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.81. required after the first reaction of the D8-alternative pathway) and ‘‘reverse” (PC ? acyl-CoA. Although the first expression of a cyanobacterial D6-desaturase in transgenic tobacco plants [83] resulted in the accumulation of low levels of these fatty acids. In respect to the substrate dichotomy bottleneck observed in linseed. 4. In addition to accumulation of ARA and EPA. meaning that these products will most likely require activation to CoA to facilitate their incorporation into lipids [78]. as Schizochytrium. fungi. In 2004. presumptively via a strong action of a phospholipid: diacylglycerol acyltransferase (PDAT)-like activity [87. This presumptively occurred as a result of poor acyl-exchange between the two metabolic pools (phospholipids. 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.11. VLC-PUFA biosynthesis in transgenic plants by ‘reverse engineering although each utilized distinct strategies toward the efficient reconstitution of the process.12. dehydration. However. Proof-ofconcept demonstration that the VLC-PUFA pathway could function in a transgenic system was first provided by expression in yeast. mosses. Detailed analyses of leaf lipids [90] have confirmed that both D9C18-PUFAs were efficiently elongated. this PKSlike pathway generates EPA or DHA as free fatty acids.15) [80]. The authors suggested that the linseed acyl-CoA:lyso-phosphatidylcholine acyltransferase (LPCAT).92]. 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. required after the first reaction of the . To fully reconstitute the alternative VLC-PUFA biosynthesis pathway for ARA and EPA. Metabolic engineering to produce VLC-PUFAs in higher plants Higher plants lack the capacity to synthesize LC-PUFAs. 18:3D6. three different reports by Qi et al. the D6-desaturase. Such experiments indicated the feasibility of transgenic expression of the LC-PUFA biosynthetic pathway in plants. 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. and condensation. 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. Complementary studies were described by Abbadi et al. several other C20 PUFA were also detected and identified as sciadonic acid (20:3D5. It remains to be seen how well the alternative pathway performs when it is expressed in seeds. considerable progress has been made reaching high levels (up to 40%) in several transgenic plants [49. but crucially lacked the D12-desaturase activity (analogous to higher eukaryotes) [79].112 M.9.14.11. 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. Abbadi et al. this organism also possessed desaturase and elongase activities of the D6-pathway. 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 modifications [79]. and marine protist. as opposed to vegetative tissue. followed by successive rounds of reduction. with the levels of target products and biosynthetic intermediates shown. (2004) [87]. (2004) [88]. 2. (2005) [102].M. For clarity. since these vary on a species-byspecies basis. (A) Omega-3 VLC-PUFA (EPA. DHA) accumulation in transgenic plants. PDAT etc. Venegas-Calerón et al. D6-pathway) directions in heterologous VLC-PUFA biosynthesis. The different configurations used are indicated. 2005 [100]. (B) Omega-6 VLC-PUFA (ARA) accumulation in transgenic plants. lyso-phospholipid acyltransferases. / Progress in Lipid Research 49 (2010) 108–119 113 Fig. Overview of oil composition in transgenic lines.e. Kinney et al. The studies compared are: Qi et al. The fatty acid compositions of published transgenic lines have been compared. (2004) [86].) have different affinities for these novel fatty acids [96]. Given that such acyl-exchange is dependent on endogenous acyltransferases activities accepting non-native substrates (i. Hoffmann et al. since . the intermediates of VLC-PUFA pathway) it is also likely that different activities (e. Wu et al. Robert et al. the endogenous fatty acids are not shown.g. Moreover. (2008) [104]. Abbadi et al. the successful accumulation of DHA may require the co-expression of suitable acyl-exchange activities. it is perhaps surprising that this ‘‘optimal” configuration of the VLC-PUFA pathway yields only low levels of the target fatty acids (<0. This activity was not previously reported when the elongase was expressed in yeast [56]. However. suggests a higher efficient transfer of GLA to the acyl-CoA pool for subsequent elongation by the reverse reaction of soybean LPCAT. a D6-elongase from M. a DHA precursor. This would also indicate that whilst endogenous B. hypothesised that the use of the (putative) acyl-CoA-dependent desaturase from zebrafish might overcome substrate dichotomy bottlenecks prior to D4-desaturation. This promiscuity is worthy of further investigation. and crucially lacked the accumulation of D6-desaturation products previously observed in earlier studies. To generate DHA. / Progress in Lipid Research 49 (2010) 108–119 many of these acyl-exchange enzymes can work in both forward and reverse directions. [88] carried out another co-transformation series with six cDNAs to produce DHA in somatic embryos. These results could be explained by low substrate levels of LA-CoA and ALA-CoA in the acyl-CoA pool. Wu et al. EPA and DHA in Arabidopsis seeds.2% of total lipids) and 0. the differences between the two studies may be a reflection on the differing endogenous lipid metabolism present in linseed. Finally. 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. However. D4-desaturase) to attempt the conversion of EPA to DHA.13. expressing genes encoding components of the conventional D6desaturase pathway. As a result of the accumulation of DPA. it remains to be demonstrated that the D.e. [104] confirmed the potential of using acyl-CoA-dependent activities to overcome the problems associated with substrate dichotomy. elegans [57] to generate EPA. This high level of EPA allowed the introduction of the additional genes required for DHA synthesis (D5-elongase. Finally. Kinney et al. 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. Hoffmann et al. These researchers used a similar approach to that of Abbadi et al. B. though it should be noted that similar attempts to produce EPA in transgenic soybeans have been much less successful. Based on the observations of Abbadi et al.3% of total fatty acids) were obtained. LA-CoA and ALA-CoA). This is again possibly due to problems with codon usage of the algal genes and/or lack of sufficient substrate acyl-CoAs. alpine D6 elongase toward the D5fatty acid EPA. only low levels of DHA (2. Thus. Endogenous acyltransferases activities from transgenic soybean and Brassica presumptively have a broader substrate-specificity than linseed or tobacco and can partially overcome the substrate dichotomy problem. juncea plants accumulated up to 25% ARA or 15% EPA [100]. A D17desaturase from Phytophtora infestans was introduced to convert omega-6 substrates to omega-3 counterparts. resulting in inefficient acyl-exchange between the D5-elongase and the D4-desaturase. since this configuration also requires the same substrates as the acyl-CoAdependent D6-pathway (i.114 M.5% of DHA. Unexpectedly. while almost no ARA intermediate was observed because of the presence of the highly efficient D17-desaturase used. two additional activities (D5-elongase and D4-desaturase) from the algae Pavlova salina were co-expressed [69]. the longer. 22:5D7. essential for activating the acyl . and maybe indicates problems in the correct assembly of the elongase (discussed below). The lower accumulation of GLA in soybean oil. alpina). juncea is a highly efficient 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. [107] for DHA production in plants using a PKS pathway system. Two additional studies have demonstrated the accumulation of DHA and EPA in oilseeds. D. Sayanova et al. it is worth also considering this with regard to the alternative D8-pathway. rerio desaturase) may have resulted in the inefficient translation of these enzyme activities. The expression of these five enzymes yielded of 19. compared with tobacco and linseed. Thus.5% of fatty acids in the seed lipids). As a result of the gene’s co-expression transgenic B. These desaturases were co-expressed under the control of a seed-specific promoter with the D6-elongase PSE1 of the moss P. whilst this study did demonstrate a proof-of-concept accumulation of ARA. the codon usage of the two animal genes (C. the anerobic PKS-like pathway has been described by Metz et al. up to 4. alpina and finally a D5-desaturase from M. [102] expressing a bifunctional D6/D5-desaturase from zebrafish (Danio rerio) [103] in conjunction with the D6-elongase PEA-1 from C.and D5-desaturases with predicted acyl-CoA-substrate dependence (as described previously for a D6desaturase from Ostreococcus tauri [105]. 19). [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. which then rate-limits the levels of D6-desaturation products and all subsequent metabolites. was also detected in the high EPA lines as a result of the additional activity of the M. [88] but involving more transgenes. [100] described expression of the D6-pathway in Brassica juncea using a similar approach to that used by Kinney et al. [88] realized in soybean (Glycine max). resulting in low but significant amounts of DHA (0. [87] and subsequent studies. These observations would indicate that (in leaves) acyl-CoA substrates are not limiting for the synthesis of VLC-PUFAs. Alternatively. However. the pool sizes of individual metabolites is also likely to prove critical in determining the predominant enzyme activity. demonstrating a difference in substrate-specificities in plant and yeast.16. juncea acyltransferases can facilitate the exchange of acyl-intermediates on the pathway to EPA.2–1. in transgenic soybean seeds and somatic embryos.0–3. reflecting an apparent block in the conversion of EPA to the C22 PUFA. A further attempt to avoid the acyl-exchange bottleneck in transgenic plants by using acyl-CoA-dependent desaturases has been recently described [104].5% EPA). DHA. These authors isolated and characterized two cDNAs from the microalga Mantoniella squamata which encoded for D6.2–0. Transgenic plants accumulated low but representative amounts of EPA. A similar approach was carried out by Robert et al. the levels achieved were relatively low: ARA and EPA (4.10. C) of a Schizochytrium PKS with a phosphopantetheinyl transferase (PPT) from Nostoc. a D4desaturase from the fungus Schizochytrium aggregatum and a specific D5-elongase from the alga Pavlova sp. Additional genes for the D4-pathway. However. most likely due to the generic problems of substrate dichotomy discussed above. for unknown reasons [99]. A third exemplification is described in the patent application by Kinney et al. patens [106] in Arabidopsis. rerio desaturase is indeed a bona fide acyl-CoA dependent activity. was also co-expressed to increase exchange of D6unsaturated acyl groups from acyl-phospholipids to acyl-CoAs for elongation. to maximize the accumulation of omega-3 VLC-PUFAs such as EPA and DHA. Firstly. These data indicate that B.6% EPA in the transgenic somatic embryos. In this latter respect. Robert et al.7% of x3-docosapentaenoic acid (DPA. The genes expressed in Arabidopsis seeds were the three subunits (ORFA. [87]. alpine. Although the reasons for the high yields obtained in soybean as compared to linseed are not clear. were expressed in addition to the activities required to generate EPA. Venegas-Calerón et al. though it would be interesting to confirm this by the similar expression of the O. tauri D6-desaturase. a D6-desaturase (either from the oomycete fungus Saprolegnia diclina or M. elegans PEA-1 D6-ELO. a D12-desaturase from Calendula officinalis [101] to increase the flux through the entire transgenic pathway and finally a gene encoding a LPCAT from Thraustochytrium sp. more unsaturated forms of the DHA pathway are only very poorly utilized. lipolytica) and plants (soybean). Identification of a VLC-PUFA-specific acyl-exchange mechanism Metabolic´ bottlenecks‘ appear to limit the full potential of oilseed crops to accumulate economically sufficient amounts of these novel fatty acids. responsible for catalysing bidirectional exchange between these two pools. In addition. 3. acyl-CoA-dependent acylation of lyso-PC) (e. 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]. could help alleviate this bottleneck (represented schematically in Fig. tauri [105] or M. 3). The primary activities are shown: acyl-CoA:glycerol-3-phosphate acyltransferases (G3PAT). use of non-optimized sequences) or reflect additional (undefined) metabolic bottlenecks. Claviceps purpurea [112] and Coprinus cinereus [113]. 5. Maintenance of a continuous flux of substrates through the VLC-PUFA biosynthetic pathway without significant loss to TAG Technological modifications to endogenous lipid metabolism to overcome such problem are not obvious. In addition. Very recently. Very recently. Central to that latter problem is the so-called ‘‘substrate dichotomy”. has also recently been described from a number of organisms by several different groups. and represents the sum of this activity plus reverse acyl-exchange from extra-plastidial phospholipids. as has already been described for the D6-desaturase from O. 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. [115. Venegas-Calerón et al. The identification of superior desaturases The first approach is to identify highly active acyl-CoA dependent desaturases from a lower eukaryote. It remains to be demonstrated that an exclusively acyl-CoA dependent pathway delivers significant improvement to yields of EPA or DHA levels.110]. phosphatidic acid phosphatise (PAP).g. Several approaches (discussed below) have been suggested as a result of the studies described in this article. squamata [104]. 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). 5. Another potentially very useful enzyme activity.7% DPA n-6. / Progress in Lipid Research 49 (2010) 108–119 115 carrier protein (ACP) domains of the DHA synthase PKS. Primula and Echium [104. expression of this activity in linseed has recently been shown to result in the significant accumulation of SDA without the concomitant accumulation of GLA [85]. both the desaturation and elongation reactions utilize acyl-CoA substrates and avoid the requirement for acyl-exchange with PC.8% DHA with an additional 1.M. in most current examples such transgenic plants also contain high levels of omega-6 and omega-3 metabolic intermediaries. the molecular identification 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. such activities have the potential to generate considerable omega-3 substrates for conversion to LC-PUFAs. linked to PC) but the actual levels of target VLC-PUFAs such as EPA and DHA are disappointingly low [102. Further optimization of this pathway in commercial oilseeds is ongoing. These seeds accumulated up to 0. in particular the channelling of FA into various different lipids. genes encoding LPCAT have been functionally characterised from yeast and animals (reviewed in [114]). a bifunctional D12. Interestingly. With such enzyme activities. The acyl-CoA pool is generated by export of fatty acids from the plastid. [111] identified 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 significant enhancement of the levels of EPA in both yeast (Y. these data also provide new insights into our understanding of plant lipid biochemistry. . Similarly. An alternative iteration is to identify VLC-PUFA desaturases with strong preferences for omega-3 substrates such as have been identified from M.1. 5. thought it could also be argued that the unambiguous identification of an acyl-CoA dependent D5-desaturase from lower eukaryotes is currently lacking. It is predicted that the enzyme LPCAT.and D15-desaturase. The acylCoA-dependent (Kennedy) pathway is shown as the central route for TAG synthesis. Thus. rich in EPA and/or DHA. A second modification 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].116]).2. In the case of the Primula D6-desaturase. The goal now is to generate a vegetal oil substitute for fish oils optimizing the accumulation of VLC-PUFAs. the resultant fatty acid compositions and levels are not equivalent to that found in fish oil. Moreover. Thus.104]. where desaturation uses acyl-substrates linked to phospholipids whereas elongation requires acyl-CoA substrates. 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.e. though in all cases only the forward reaction of LPCAT was demonstrated (i. bifunctional desaturases were characterised from the free living amoeba A. the identification of a plant or algal form of LPCAT remains to be demonstrated. This may be due to a number of problems (substrate availability. Outlined below are logical approaches which might be expected to enhance the accumulation of VLC-PUFAs in transgenic plants.109. squamata. are almost completely devoid of omega-6 fatty acids such as GLA and DHGLA. acyl-CoA:diacylglycerol acyltranserase (DGAT). Damude et al. not least of all since such acyl-channelling represents the sum of multiple different acylexchange activities. castellanii [70]. Crucial issues: optimization the levels of LC-PUFA in transgenic plants Although the effective biosynthesis of ARA. 5. published examples of the use of an acyl-CoA dependent route have resulted in the significant reduction in the accumulation of biosynthetic intermediates (most notably omega-6 GLA. 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. acyl-CoA: lyso-phosphatidic acid acyltransferases (LPAAT). Schematic representation of triacylglycerol synthesis in plants. EPA and to some extent DHA has been demonstrated using different approaches in transgenic plants. As noted above. Marine oils. Such x3-desaturases ideally have a high preference for C20 substrates (such as ARA) and have been identified from a number of fungal species [17]. 5. Similarly. 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 significance is the fact that in higher plants. 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. Alternatively.7. 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. Optimizing the fatty acid elongase Microsomal fatty acid elongation occurs as a result of four sequential enzymatic reactions: condensation. marinus [62] may warrant further evaluation. The success of this approach is dependent on significant levels of sub- strate fatty acids (LA. Overexpression of the elongase ketoreductase resulted in an increase in the accumulation of VLC monosaturated fatty acids in yeast. it may be that to obtain maximal elongation of target fatty acids. In most cases. this also indicates the requirement for a strong flux of fatty acids into this metabolic pool. It has been assumed that the contribution of the other elongase components to VLC-PUFA synthesis is neutral. In that respect the atypical FAE1-like alternative pathway D9-elongating activity isolated from P. but it appears that PDATs are not restricted to PC as substrates. making a generic intervention unlikely if not impossible. challenging the concept that the ‘‘core” elongase components have a neutral role in determining the levels of VLCFAs [120].116 M. the promoters used to drive this seed-specific expression were derived not from genes involved in oil biosynthesis but more often instead from storage protein synthesis. ketoreduction. the predominant microsomal KCS activities take the form of FAE1-like enzymes. Such sub-domains could be generated by local variation in lipid compositions (such as so-called lipid rafts [125]) or via protein–protein interaction to nucleate higher-order structures [126]. / Progress in Lipid Research 49 (2010) 108–119 species has a different combination of such activities (perhaps evidenced by the huge disparity in the composition of plant seed oils). Modulating the acyl-CoA pool As discussed above. acyltransferases such as DGAT [126]. resulting in the minimal loss of intermediates and the optimal channelling of products to their intended metabolic pool. the additional core components of the elongases may need to be isolated from suitable EPA. 18:1) are directly exported from the plastid into the cytosolic acyl-CoA pool [123]. although for transgenic is possible through heterologous expression of just the initial condensing enzyme [55.e. 5. meaning that the substrate VLC-PUFA must be present in the acyl-CoA pool. our understanding of the regulation. with very recent studies questioning the established model in which the products of the plastidial fatty acid synthase (16:0. 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 flux (i. Whilst a total blockade of beta-oxidation results in abnormal plant development and impaired germination.4. 18:0. It must also be noted that our understanding of the flux of fatty acids into the acylCoA is partial. compared with DGAT1-type enzymes [118]. One proven method for altering the profile 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]. ALA) being present in the extra-plastidial acyl-CoA pool. it may be possible to identify TAG biosynthetic enzymes from VLC-PUFA-synthesising lower eukaryotes which display the desired activities. presumably by either increasing flux through the elongase or increasing the absolute number of elongase complexes. A hypothetical solution might be the identification of TAG biosynthetic enzymes (such as diacylglycerol acyltransferases.99]. 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].5. dehydration and enoyl reduction.or DHA-accumulating organisms and co-expressed with the transgene condensing enzyme from the same species. but little evidence has been found to date to suggest that these enzymes play a major quantitative or qualitative role in seed TAG metabolism. 6. it is envisaged that multiple enzyme activities for a particular biochemical pathway are co-located. 5. whereas the transgenic VLC-PUFA elongating activity is of the ELO-like form [63]. Venegas-Calerón et al. as might the search for additional examples of FAE1-like VLC-PUFA elongating activities. the use of (ELO-like) activities from VLCPUFA-synthesising lower plants such as Marchantia polymorpha may prove of benefit – initial studies indicate that M. the use of acyl-CoA dependent desaturases is predicted to bypass the metabolic bottleneck generated by substrate dichotomy between the desaturase and the elongase. 5. 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. Conclusions and future perspectives It is obvious from the studies described in this article that heterologous reconstitution of VLC-PUFA synthesis in transgenic . Such an activity has the advantage of removing desaturation products from their site of synthesis. 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. 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. The converse solution to this requirement is to use acyl-CoA-independent enzymes such PDAT [95]. In either scenario. such activities are generally acyl-CoA-dependent. However. polymorpha activities perform well in transgenic plants [119].6. increasing both the substrates available for VLC-PUFA biosynthesis and also the accumulation of target fatty acids in storage triacylglycerols. Interestingly.64]. Currently. Genes for PDAT have been identified in plants. for all of the reasons outlined above. since the condensing enzyme acts in a trans-dominant manner. Alternatively. DGAT or LPAT) which have a very strong substrate preference for EPA or DHA. it is not obvious how such approaches would directly result in the enhanced synthesis of VLC-PUFAs. Thus. 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]. further research on the biosynthesis and homeostasis of the acyl-CoA pool is required. However. it may be possible to use developmentally-regulated silencing of such activities to modulate the acyl-CoA pool. However. recent evidence from plants has shown that the DGAT2 class of DGAT displays a more precise range of substrate-specificities. Thus. Thus. given that the acyl-CoA pool in most plant cells is considered to be lower than that found in yeast or animals [121]. organisation and assembly of the elongase complex is limited. unlike yeast or animals. signify a progress toward the provision of an alternative source of VLC-PUFA. Meyer A. an EU Sixth Framework Programme Integrated Project (Project Number: FOOD-CT-2003-505944). Grattan L. J Appl Phycol 2001. [11] Burr GO. Mar Biotechnol 2006. We thank BASF Plant Sciences for their input and support. [17] Damude HG. Our research in this area was partially supported by Lipgene. Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies. Schmidt SL. In particular. Heinz E. Platt I. Gilbert JF. Suh M. Carr J. On the other hand. by which output traits of benefit to the consumer or end-user are delivered.94:367–74. human health and food security. However.431:502–4. [8] Tocher DR. Blasbalg TL. in that they would provide a sustainable and non-contaminated source of these important fatty acids for human nutrition. Lancet 1989. eggs etc. Burr MM. With that in mind. A systemic review of the roles of n-3 fatty acids in health and disease. Venegas-Calerón et al. [4] Burr ML. acyl-channelling between the PC.147:962–8. Domergue F. it will be important to ensure that the end-user and consumers are fully informed of the underlying science and benefits of the resulting products – with such an approach. EPA and (to a lesser extent) DHA. Nature 2004. Role of long-chain polyunsaturated fatty acids in the first year of life.200:69–73. [14] Dalton R. problems and first results. Riggs JA. Sperling P. the final product and appropriate end-users must be clearly identified. Healthy intakes of n-3 and n-6 fatty acids: estimations considering worldwide diversity. Given the complexity of the pathways under study. and spreads). Sargent JR. there are still significant issues which need to be considered before any VLC-PUFA trait can progress towards the marketplace. However. and many other (larger) economies do not share the same views on the direct consumption of ‘‘GM foodstuff”. in the form of transgenic plants. The demonstration that oil-seed crops can synthesize significant levels of VLC-PUFA. [15] Drexler H. and may also take advantage of a number of ‘‘host” oil-seed crops which are very high in omega-3 fatty acids. it should be remembered that Europe represents only a portion of the global marketplace. Comp Biochem Physiol B 1989. Plant Physiol 2008. especially EPA.42:544–68. Valente JG. Abbadi A. Relief for fish stocks: oceanic fatty acids in transgenic oilseeds. Environ Health: A Global Access Sci Sour 2003. farmed fish would be provided with a diet in which marine-derived oils are replaced by terrestrial modified plant oils [128]. Holliday RM.10:112–6. [9] Williams CM. 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