an alternative to fish oils metabolic engineering of oil-seed crops to produce

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Review

An alternative to fish oils: Metabolic engineering of oil-seed crops to produceomega-3 long chain polyunsaturated fatty acids

Mnica Venegas-Calern a,b, Olga Sayanova a, Johnathan A. Napier a,*

a Department of Biological Chemistry, Rothamsted Research, Harpenden, Herts AL5 2JQ, UKb 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

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

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 die-

tary 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 (VLC-

PUFAs) such as EPAand 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 aquacul-

ture 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.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

2. Omega-3 long chain polyunsaturated fatty acids (x3 LC-PUFAs) in humans health. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1093. Characterization of VLC-PUFAs biosynthetic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

4. Metabolic engineering to produce VLC-PUFAs in higher plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

5. Crucial issues: optimization the levels of LC-PUFA in transgenic plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

5.1. The identification of superior desaturases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

5.2. Identification of a VLC-PUFA-specific acyl-exchange mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

5.3. Maintenance of a continuous flux of substrates through the VLC-PUFA biosynthetic pathway without significant loss to TAG . . . . . . . 115

5.4. Optimizing the fatty acid elongase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

5.5. Modulating the acyl-CoA pool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

5.6. Co-ordinated expression of transgenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

5.7. Appropriate localisation of transgene-derived activities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1166. Conclusions and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

0163-7827/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.plipres.2009.10.001

Abbreviations: ALA, a-linolenic acid; ARA, arachidonic acid; DAG, diacylglycerol; DGAT, diacylgylcerol acyltransferase; DHA, docosahexaenoic acid; ECR, enoyl-CoAreductase; EFA, essential fatty acid; EPA, eicosapentaenoic acid; GLA, c-linolenic acid; HCD, hydroxyacyl-CoA dehydratase; KCS, ketoacyl-CoA synthase; KCR, b-ketoacyl-CoAreductase; 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).

Progress in Lipid Research 49 (2010) 108119

Contents lists available at ScienceDirect

Progress in Lipid Research

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http://dx.doi.org/10.1016/j.plipres.2009.10.001mailto:[email protected]://www.sciencedirect.com/science/journal/01637827http://www.elsevier.com/locate/plipreshttp://www.elsevier.com/locate/plipreshttp://www.sciencedirect.com/science/journal/01637827mailto:[email protected]://dx.doi.org/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 first double bond proximal to the methyl end of the fatty acid.

VLCLC-PUFAs are vital constituents of human metabolism. In par-

ticular, there is plentiful evidence (from epidemiology and dietary

intervention studies) for the health-beneficial properties to hu-

mans of dietary consumption of omega-3 VLC-PUFAs such as eico-

sapentaenoic 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 hu-

man nutrition[6]. The main source of EPA and DHA in the human

diet is through the direct consumption of cold water marine fish

(such as salmon, tuna, mackerel, and sardines) [6,7]. However,

marine fish (like other animals) do not efficiently metabolise ALA

to VLC-PUFAs, 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)

[7,8]. The primary de novo synthesis sources of VLC-PUFAs are mar-

ine 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 these microorganisms, EPA and DHA are

synthesized de novoby one of the two classes of biochemical path-

way (reviewed on the text below). In addition, some fungi, mosses,

bacteria and lower plants also have a capacity to synthesize signif-

icant 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) anda-lin-olenic acid (ALA; 18:3D9,12,15) that serve as the metabolic precur-

sors for VLC-PUFA biosynthesis in animals[11].There is growing concern regarding the sustainability of global

fish stocks (the predominant sources of omega-3 VLC-PUFA) be-

cause marine fish stocks are in severe decline as a result of decades

of over-fishing[12]. Moreover, environmental pollution of marine

ecosystems has resulted in the accumulation of dioxins, heavy

metals and polychlorinated biphenyls in fish, to the point of ques-

tioning the benefits of fish consumption in human health [13]. Fi-

nally, the expansion of industrialised aquaculture exacerbates the

overexploitation of natural marine resources, since farmed fish re-

quire omega-3 VLC-PUFA-containing feedstuffs. The marine oils

used as aquaculture feedstocks are usually extracted from so-

called trash species such as sand eels, which are specifically har-

vested for this application (since they are not normally consumed

by humans). However, the loss of these species from food-webs hasa profound impact on the overall stability of ecosystems [14].

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 le-

vel significantly higher than that present in the finished product.

Therefore aquaculture is (perhaps surprisingly, at least to the lay-

person) a net consumer of fish 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 fish 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 specific highvalue applications (such as infant formula baby milk formula-

tions [18]) in controlled culture systems. However, such systems

are expensive to maintain and have limited flexibility for signifi-

cant scale-up and requiring the appropriate microbiological facili-

ties (such as fermenters) [19]. It is noteworthy that such

fermentation-based systems are also sensitive to disruptions of

power-supplies and have a significant environmental footprint.

In view of all these factors, there is an obvious need for an alter-

native, sustainable source of these important fatty acids. Oneattractive 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 dis-

covery 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 encod-

ing the primary enzymes involved in biosynthesis of these fatty

acids have been successfully isolated from a range of VLC-PUFA-

synthesising organisms with a number of these being heterolo-

gously 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 ofD12 and D15-desaturase activities and, as

a consequence, cannot produce linolenic acid (LA; 18:2D9,12n-6)

anda-linolenic acid (ALA; 18:3D9,12,15 n-3) respectively from theprecursor oleic acid (18:1D9) [23]. However, they do have a lim-

ited ability to synthesize ARA and EPA from these two dietary-

essential fatty acids (EFAs) LA and ALA through a series of desatu-

ration 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 ALAto DHA is


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are ubiquitous in higher plant and play a crucial role in the synthe-

sis of membrane lipids for the support of photosynthesis and also

the precursors for the oxylipin jasmonic acid required for male fer-

tility [22]. The presence of these EFA desaturases has been ob-

served 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 mem-

brane fluidity and permeability. As a consequence they play crucial

roles in human metabolism, not only playing structural roles inphospholipid 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 inflammation, 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 inflamma-

tory, pro-aggregatory and inmuno-active eicosanoids (series-2),

eicosanoids derived from omega-3 fatty acids (series-1 and ser-

ies-3) are anti-inflammatory and modulate plaque aggregation

and immune-reactivity[36,37]. Unsurprisingly, research has dem-

onstrated that there are considerable health benefits 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 importantrole 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 pro-

tective 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, protec-

tive effects have been clinically studied for cancer[42], atheroscle-

rosis, cognitive impairment, and various mental illness, in

particular depression[29], childhood and attention-deficit hyper-

activity disorder (ADHD)[43,44]and dementia[45]. Finally, there

are epidemiological studies which extend the beneficial effects ofomega-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

inflammation-resolution[48].

3. Characterization of VLC-PUFAs biosynthetic pathways

Over the last decade, all the primary genes involved in VLC-PUFA

biosynthesis have been identified from a range of different species,

including animals, fungi, plants and aquatic organisms. These genes

can be classifiedinto the twodistinct enzymatic reactions that cata-

lyse the primary biosynthetic process. The first of these are the

microsomal fatty acid desaturases, so-called front end PUFA

desaturases which belong to the N-terminal cytochrome b5-fusionsuperfamily, firstly identified in 1997 by Sayanova et al. [49]. The

18:2 9,12Linoleico acid (LA)

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

3 Des

15 Des18:1 9

Oleico acid (OA)

12 Des

18:0

Stearic acid (SA)

9 Des

Plants

Animals

Micobial 4 pathwayMammalian Sprecher

pathway

Conventional 6-pathway

Alternative

8-pathwayAlternative

8-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 DHAsynthesis are shown, microbial D4-pathway and mammalian Sprecher pathway. Des = desaturase, Elo = elongase.

110 M. Venegas-Calern et al. / Progress in Lipid Research 49 (2010) 108119


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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 theD12 and D15-desat-

urases found in plants, algae and some fungi which lack any such

cytochrome b5 domain. Another characteristic of the front end

desaturases is thesubstitutionof histidine by glutamine in the third

histidine box (consensus sequence QX[23]HH). The presence

of diagnostic motifs such as a cytocromeb5 domain and this variant

His box have greatly facilitated the identification of candidate

front end desaturases from animals, fungi, and algae, and also

from the few plant species (borage, evening primrose, black currant

andEchium) that carry out D6-desaturationof LA and ALA [50]. One

crucial observation regarding the microsomal desaturases from

lower eukaryotes is that very many of these enzymes utilize glycer-

olipid-linked substrates, in particular fatty acids esterified 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 VLC-

PUFA is elongation, which also occurs in the ER. The fatty acid elon-

gation reaction consists of four sequencial activities: condensation

of the substrate fatty acid with malonyl-CoA (b-ketoacyl-CoA syn-

thase; KCS), reduction (b-ketoacyl-CoA reductase; KCR), dehydra-

tion (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-specificity 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 as-

sumed that the ability of heterologously expressed KCSs to appar-

ently direct the elongation of substrate fatty acids is due to theinteraction 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 first 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 biosyn-

thesis, 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-specific

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 inseed 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 synthe-

sis. However, there is now evidence that this FAE-like class is also

involved in the synthesis of VLC-PUFAs a PUFA-FAE was function-

ally 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 synthe-

sis 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-likeKCSs are structurallyquite different to ELO-like (500aa, 2 TMs ver-

sus


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short chain acyl-CoA and an unit of malonyl-CoA, followed by suc-

cessive rounds of reduction, dehydration, reduction, and condensa-

tion, with the acyl chain growing by two-carbon units with each

round. A dehydratase/isomerase from this PKS complex catalyze

the trans- tocis-conversion of the double bonds to form EPA and

DHA. The domains encoding these activities are arranged sequen-

tially on long (2030 kb) open reading frames (ORFs) in bacteria

such as Shewanella andVibrio, and marine protist, as Schizochytrium.

Their expression in yeast and plants has been reported [77] provid-

ing an alternative to the aerobic desaturase/elongase system for

transgenic VLC-PUFA production in plants. Interestingly, this PKS-

like pathway generatesEPA or DHA as free fattyacids, meaning that

these products will most likely requireactivation to CoAto facilitate

their incorporation into lipids [78]. It is also worthy of note that

genomic andbiochemical analysis of lipid biosynthesis in Schizochy-

trium indicated that, in addition to thePKS-like pathway, this organ-

ism 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 defec-

tive aerobic pathway was suggested by the authors to represent a

scavenging mechanism by which fatty acids prematurely released

fromthe PKS-like system might undergo further modifications [79].

4. Metabolic engineering to produce VLC-PUFAs in higher plants

Higher plants lack the capacity to synthesize LC-PUFAs, thougha

few taxonomically unrelated phyla can synthesize D6-desaturated

fatty acids (thefirststepon theD6-pathway) such as omega-6c-lin-olenic 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 acyl-

CoA elongation) to generate C20 PUFAs such as ARA and EPA. The

last few years have seen considerable progress in the identificationfrom diverse sources (algae, fungi, mosses, plants and mammals) of

genes encoding theprimary VLC-PUFA biosynthetic activities, effec-

tively completingthe first stage of attempts to reverse-engineer this

trait into a heterologous host such as a transgenic plant. Proof-of-

concept demonstration that the VLC-PUFA pathway could function

in a transgenic system was first 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 fromthe earliest attempts to accumulateGLA and

SDA in oil-seed crops by expression of an individual gene, the

D6-desaturase. Although the first expression of a cyanobacterialD6-desaturase in transgenic tobacco plants [83] resulted in the

accumulation of low levels of these fatty acids, considerable pro-

gress 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-PUFAbiosynthesis in transgenicplantsby reverseengineering,

although eachutilized distinctstrategies towardthe efficient recon-

stitution of the process. Apart from the important biotechnological

breakthroughs, they also provide some new insights into the bio-

chemical 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 stud-

ies discussed below is shown inFig. 2.

The first study utilizing the alternative pathway was the expres-sion of theIsochrysisC18 D9-elongase[89]in leaves ofArabidopsis

using the constitutive CaMV 35S promoter and resulted in the syn-

thesis of significant levels of EDA and ETriA (15% of total FA) [90].

To fully reconstitute the alternative VLC-PUFA biosynthesis path-

way for ARA and EPA, transgenic Arabidopsis lines expressing the

Isochrysis D9-elongase were sequential transformed with the Eu-

glena 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 repre-

sented an important proof-of-concept demonstration [86,92].

Detailed analyses of leaf lipids[90]have confirmed that both D9-

C18-PUFAs were efficiently elongated, accumulating to very high

levels in the acyl-CoA pool of transgenic plants[93]. This indicated

the inefficient transfer of these non-native fatty acids from the

acyl-CoA pool into extra-plastidial phospholipids for their subse-

quent desaturation. In addition to accumulation of ARA and EPA,

several other C20 PUFA were also detected and identified as scia-

donic 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 alter-

native 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 re-

mains 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 lin-

seed (Linum usitatissimum) and tobacco by coexpressing the D5-

and 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. Transgenic lines accumulated rela-

tively low levels, only 1.6% EPA and 2.7% ARA. However, whilstthese C20 LC-PUFA were low, very high levels (>25% of total fatty

acids) ofD6-desaturated fatty acids (GLA and SDA) were observed.

This indicated that whilst the first desaturation in the VLC-PUFA

biosynthetic pathway was functioning efficiently, the elongation

step was severely limited. There was a bottleneck, described as

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 medi-

ating acyl-exchange between phosphatidylcholine and the acyl-

CoA pool [94], discriminates against D6-desaturated acyl groups

as substrates. Detailed biochemical and metabolic analysis con-

firmed that this poor exchange resulted in the incorporation ofGLA away from the VLC-PUFA biosynthetic activities, instead being

directly incorporated into TAG in an acyl-CoA-independent man-

ner. This is most likely to result from the direct conversion of phos-

phatidylcholine (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 build-

up of the product of the first 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 first reaction of the D8-alternative pathway) andreverse (PC? acyl-CoA; required after the first reaction of the



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D6-pathway) directions in heterologous VLC-PUFA biosynthesis.

Given that such acyl-exchange is dependent on endogenous acyl-transferases 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.) havedifferent affinities for these novel fatty acids[96]. Moreover, since

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 configurations used are indicated. For clarity, the endogenous fatty acids are not shown, since these vary on a species-by-

species 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.

M. Venegas-Calern et al./ Progress in Lipid Research 49 (2010) 108119 113


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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 en-

zyme activity.

A third exemplification 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 D6-

desaturase pathway, a D6-desaturase (either from the oomycete

fungusSaprolegnia diclinaorM. alpina), a D6-elongase from M. alp-

ina and finally a D5-desaturase fromM. alpine, in transgenic soy-

bean seeds and somatic embryos. However, to maximize the

accumulation of omega-3 VLC-PUFAs such as EPA and DHA, anAra-

bidopsis FAD3 gene[97]and aS. diclina D17-desaturase[98]were

also co-expressed to turn the omega-6 PUFA metabolites into their

omega-3 counterparts. The expression of these five enzymes

yielded of 19.6% EPA in the transgenic somatic embryos, while al-

most no ARA intermediate was observed because of the presence of

the highly efficient 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 reflection

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 un-

known reasons [99]. The lower accumulation of GLA in soybean

oil, compared with tobacco and linseed, suggests a higher efficient

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 pre-cursor, was also detected in the high EPA lines as a result of the

additional activity of the M. alpine D6 elongase toward the D5-

fatty acid EPA. This activity was not previously reported when

the elongase was expressed in yeast [56],demonstrating a differ-

ence in substrate-specificities 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 re-

sult of the accumulation of DPA, Kinney et al. [88]carried out an-other co-transformation series with six cDNAs to produce DHA in

somatic embryos. Additional genes for the D4-pathway, a D4-

desaturase from the fungusSchizochytrium aggregatum and a spe-

cific D5-elongase from the alga Pavlovasp. were expressed in addi-

tion 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 dis-

cussed above, resulting in inefficient acyl-exchange between the

D5-elongase and the D4-desaturase.

Two additional studies have demonstrated the accumulation of

DHAandEPA inoilseeds. Firstly,Wu et al.[100] describedexpression

oftheD6-pathwayin Brassica junceausing a similarapproachto that

used by Kinney et al.[88]but involving more transgenes. A D17-

desaturase from Phytophtora infestans was introduced to convertomega-6 substrates to omega-3 counterparts, a D12-desaturase

from Calendula officinalis [101] toincrease theflux through theentire

transgenicpathway andfinallya gene encoding a LPCAT from Thrau-

stochytrium sp. was also co-expressed to increase exchange of D6-

unsaturated acyl groups from acyl-phospholipids to acyl-CoAs for

elongation. As a result of the genes co-expression transgenic B. jun-

cea plants accumulated upto 25%ARA or 15%EPA [100]. This highle-

vel of EPAallowed the introduction of the additional genes required

forDHA synthesis(D5-elongase, D4-desaturase) to attempt thecon-

version of EPA to DHA, resulting in low but significant amounts of

DHA (0.21.5% of fatty acids in the seed lipids). These data indicate

thatB. junceais 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, reflecting an apparentblockin the conversionof EPA tothe C22 PUFA, DHA.Thiswouldalso

indicatethat whilst endogenous B. juncea acyltransferases can facil-

itate 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 re-

quire 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 zebrafish (Danio

rerio)[103]in conjunction with theD6-elongase PEA-1 from C. ele-

gans [57]to generate EPA. To generate DHA, two additional activi-

ties (D5-elongase and D4-desaturase) from the algae Pavlova

salinawere 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 zeb-

rafish might overcome substrate dichotomy bottlenecks prior to

D4-desaturation. However, whilst this study did demonstrate a

proof-of-concept accumulation of ARA, EPAand DHAin Arabidopsis

seeds, the levels achieved were relativelylow: ARA and EPA (4.2%of

total lipids) and 0.20.5% of DHA. These results could be explained

by lowsubstrate levels of LA-CoAand ALA-CoA in theacyl-CoA pool,

which then rate-limits the levels ofD6-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 inefficient translation of these enzyme

activities. Finally, it remains to be demonstrated that the D. rerio

desaturase is indeed abona fide 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 soy-

bean and Brassica presumptively have a broader substrate-specific-

ity than linseed or tobacco and can partially overcome thesubstrate

dichotomy problem. A further attempt to avoid the acyl-exchange

bottleneck in transgenicplants by using acyl-CoA-dependent desat-

uraseshasbeen recently described [104]. These authors isolatedand

characterized two cDNAs from the microalga Mantoniella squamata

which encoded for D6- and D5-desaturases with predictedacyl-CoA-substrate dependence (as described previously for a D6-

desaturase from Ostreococcus tauri [105]. These desaturases were

co-expressed under the control of a seed-specific 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]confirmed the potential of using acyl-CoA-dependent

activities to overcome the problems associated with substrate

dichotomy. However, it is perhaps surprising that this optimal

configuration of the VLC-PUFA pathway yields only low levels of

the target fatty acids (


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carrier protein(ACP) domains of theDHAsynthase PKS. These seeds

accumulated up to 0.8% DHA with an additional 1.7% DPA n-6. Fur-

ther optimizationof thispathway 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 ex-

tent DHA has been demonstrated using different approaches in

transgenic plants, the resultant fatty acid compositions and levels

are not equivalent to that found in fish oil. Moreover, in most cur-

rent 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 veg-

etal oil substitute for fish oils optimizing the accumulation of

VLC-PUFAs. Several approaches (discussed below) have been sug-

gested as a result of the studies described in this article. In addi-

tion, 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-PU-

FAs in transgenic plants.

5.1. The identification of superior desaturases

The first approach is to identify highly active acyl-CoA depen-

dent desaturases from a lower eukaryote, as has already been de-

scribed for the D6-desaturase fromO. tauri [105]or M. squamata

[104]. With such enzyme activities, both the desaturation and elon-

gation reactions utilize acyl-CoA substrates and avoid the require-

ment for acyl-exchange with PC. As noted above, 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, linked to PC) but the ac-

tual levels of target VLC-PUFAssuch as EPAand DHAare disappoint-

ingly low [102,104]. This may be due to a number of problems(substrate availability, use of non-optimized sequences) or reflect

additional (undefined) metabolic bottlenecks. It remains to be dem-

onstrated that an exclusively acyl-CoA dependent pathway delivers

significant improvement to yields of EPA or DHA levels, thought it

could also be argued that the unambiguous identification of an

acyl-CoA dependent D5-desaturase from lower eukaryotes is cur-

rently lacking. Very recently, 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.

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 ofx3-desaturases: such enzyme activities havebeen demonstrated to be pivotal in the production of elevated lev-

els of EPA in Brassica juncea [100]. Such x3-desaturases ideallyhave a high preference for C20 substrates (such as ARA) and have

been identified from a number of fungal species[17]. An alterna-

tive iteration is to identify VLC-PUFA desaturases with strong pref-

erences for omega-3 substrates such as have been identified 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 significant 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]iden-

tified such a bifunctional desaturase fromFusarium moniliformeand 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. lipolytica) and

plants (soybean). Similarly, bifunctional desaturases were charac-

terised from the free living amoeba A. castellanii [70], Claviceps

purpurea [112] andCoprinus cinereus [113]. Thus, such activities

have the potential to generate considerable omega-3 substrates

for conversion to LC-PUFAs.

5.2. Identification of a VLC-PUFA-specific acyl-exchange mechanism

Metabolicbottlenecks appear to limit the full potential of oil-

seed crops to accumulate economically sufficient amounts of these

novel fatty acids. Such factors are likely to be represented by en-

zymes involved in thechannelling andpartitioning of fatty acids be-

tween 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

problemis theso-called substrate dichotomy, where desaturation

uses acyl-substrateslinkedto phospholipidswhereas elongation re-

quires 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 sche-

matically 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 predom-

inantly LPEAT activities (acyl-CoA-dependent acylation of lyso-PE)

[117]. Thus, the identification of a plant or algal form of LPCAT re-

mains 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 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, not least of all since such

acyl-channelling represents the sum of multiple different acyl-

exchange activities. In addition, it is highly likely that each plant

G3PATG3P LPA PA DAG

LPAAT PAPPC DAG

DGATTAG

PC pool

CPT CPT

Lyso-PC

LPCAT

Acyl-CoApool

PDAT

LPCAT

Fig. 3. Schematic representation of triacylglycerol synthesis in plants. The acyl-

CoA-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-plastidialphospholipids.



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species has a different combination of such activities (perhaps evi-

denced by the huge disparity in the composition of plant seed oils),

making a generic intervention unlikely if not impossible. A hypo-

thetical solution might be the identification of TAGbiosynthetic en-

zymes (such as diacylglycerol acyltransferases, DGAT or LPAT)

which have a very strong substrate preferencefor EPAor DHA. How-

ever, 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-inde-

pendent 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

hastheadvantage of removing desaturation products fromtheirsite

of synthesis, but it appears that PDATs are not restricted to PC as

substrates. Genes for PDAT have been identified 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-specificities, 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 andenoyl reduction, although for transgenic is possible

through heterologous expression of just the initial condensing en-

zyme [55,64]. It has been assumedthat 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 andbiochemical inter-

actions between a non-native condensing enzyme and the other

three endogenous elongase components may be sub-optimal. Per-

haps of significance is the fact that in higher plants, unlike yeastor animals, the predominant microsomal KCS activities take the

formof FAE1-like enzymes, whereas the transgenic VLC-PUFA elon-

gating activity is of the ELO-like form [63]. In that respect the atyp-

ical FAE1-like alternative pathway D9-elongating activity isolated

fromP. 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 VLC-

PUFA-synthesising lower plants such as Marchantia polymorpha

may prove of benefit 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 complexis limited. Overexpression of theelongase ketore-

ductaseresulted in an increasein theaccumulationof VLCmonosat-

urated fatty acids in yeast, presumably by either increasing fluxthrough theelongase or increasingthe absolutenumber of elongase

complexes, challenging the concept that the core elongase com-

ponents 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 sub-

strate dichotomy between the desaturase and the elongase. Thesuccess of this approach is dependent on significant 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 flux of fatty acids

into this metabolic pool. One proven method for alteringthe 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 in-

creased 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 peroxi-

somal 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 sub-

strates available for VLC-PUFA biosynthesis and also the accumula-

tion of target fatty acids in storage triacylglycerols. It must also be

noted that our understanding of the flux of fatty acids into the acyl-

CoA 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 triacylglyce-

rols 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 theexamples described above of theproduction of VLC-PU-

FAs in transgenic plants have relied on the simultaneous co-expres-

sion of desaturases and elongases in developing seeds. In most

cases, 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. Thus, it may be possi-

ble to enhance the overall levels of target VLC-PUFAs through the

use of promoterswhose activity coincides with maximal oil synthe-

sis and accumulation. Certainly the choice of appropriate promoter

has been postulated to play a key role in the wide variation in VLC-

PUFAs 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 sys-

tem. Such sub-domains could be generated by local variation in li-

pid 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 pos-

sible 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 en-

zymes or those involved in the generation of a strong flux (i.e. acyl-

transferases such as DGAT[126].

6. Conclusions and future perspectives

It is obvious from the studies described in this article that het-erologous reconstitution of VLC-PUFA synthesis in transgenic



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