Available via license: CC BY 4.0
Content may be subject to copyright.
Review Article
Transgenic plants as a sustainable, terrestrial source of
fish oils
Johnathan A. Napier
1
, Sarah Usher
1
, Richard P. Haslam
1
, Noemi Ruiz-Lopez
2
and Olga Sayanova
1
1
Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, UK
2
Instituto de la Grasa (CSIC), Seville, Spain
An alternative, sustainable source of omega-3 long chain polyunsaturated fatty acids is widely recognized
as desirable, helping to reduce pressure on current sources (wild capture fisheries) and providing a de
novo source of these health beneficial fatty acids. This review will consider the efforts and progress to
develop transgenic plants as terrestrial sources of omega-3 fish oils, focusing on recent developments and
the possible explanations for advances in the field. We also consider the utility of such a source for use in
aquaculture, since this industry is the major consumer of oceanic supplies of omega-3 fish oils. Given the
importance of the aquaculture industry in meeting global requirements for healthy foodstuffs, an
alternative source of omega-3 fish oils represents a potentially significant breakthrough for this
production system.
Keywords: Aquaculture / GM plants / Omega-3 / Plant biotechnology
Received: March 4, 2015 / Revised: April 21, 2015 / Accepted: April 23, 2015
DOI: 10.1002/ejlt.201400452
1 Introduction
The aquaculture (fish farming) industry is heavily dependent
on fish oil as a commodity ingredient for the formulation of
fish feeds—this is based on the nutritional requirements of
marine and salmonid species for omega-3 long chain
polyunsaturated fatty acids (LC-PUFAs) in their diets,
and also to ensure that the final product destined for the
consumer contains these health beneficial fish oils [1].
Counter-intuitively, most farmed fish species are unable to
synthesise omega-3 fish oils such as eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA), and are entirely
dependent on dietary sources [2]. Thus, the predominant
farmed species such as salmon and trout represent major
consumers of oceanically-sourced fish oils (harvested from
the so-called reduction fisheries containing species such as
anchovies, menhaden, and capelin), and collectively the
aquafeed industry consumes in excess of 7 50 000 metric tons
of fish oils per annum [3]. When this demand is overlaid with
the continuing expansion of fish-farming (on average 6% per
annum for the last two decades) [4] and the ever-growing
human population, there are a number of significant issues.
Firstly, fish-farming is the most effective system for
producing animal protein for human consumption—the
input/output ratios for aquaculture are superior compared to
all terrestrial animal production systems [5]. Secondly,
farmed fish is now near-ubiquitous in many countries—for
example, 95% of all salmon sold in the UK is farmed [6].
Thirdly, the finished products have well-defined levels of
health-beneficial omega-3s [7], though feed inclusion rates
are showing a downward trend, most likely as a consequence
of price-sensitivity for raw materials such as fish oil [6, 8].
Thus, all of these factors need to be considered in the
continued attempts to ensure that fish farming remains an
economically-viable and environmentally-sustainable means
by which to ensure human health and nutrition. However,
the continued expansion of the aquaculture industry, and its
ability to deliver a nutritionally—important product, are
constrained by the sustainable availability of omega-3 fish oils
with which to formulate feeds. Reduced wild fish stocks and
associated quotas to protect these limit the annual global
harvests of fish oils to 1 million metric tons, of which <20%
is used for direct human nutrition [7]. Thus, the limited
availability of fish oils represents the critical bottleneck in this
Correspondence: Dr. Johnathan A. Napier, Rothamsted Research,
Harpenden, Herts AL5 2JQ, UK
E-mail: johnathan.napier@rothamsted.ac.uk
Fax: þ44 1582 763010
Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapentaenoic
acid; GM, genetically modified; LC-PUFAs, long chain polyunsaturated
fatty acids
Eur. J. Lipid Sci. Technol. 2015, 117, 1317–1324 1317
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com
This is an open access article under the terms of the Creative Commons Attribution License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
production system, and one that all major aquafeed
companies in this field are trying to overcome. Attempts to
replace fish oils in aquafeed diets with conventional vegetable
oils (which are devoid ofthe critical fatty acids such as EPA
and DHA) result in finished products that lack the health-
beneficial omega-3 fish oils and run the risk of losing
consumer-confidence in oily fish as a healthy foodstuff [8]. It
is for all of these reasons that we set out to develop a new,
sustainable, source of fish oils via their de novo synthesis in
transgenic (genetically modified; GM) plants. This review
will consider the approach we have adopted, contrast other
alternative systems, and also examine the steps that still need
to be taken to ensure that this research is translated into a
tangible outcome.
2 Context and progress to date
The accumulation of LC-PUFA EPA and DHA in the
marine environment is predominantly via the biosynthetic
activities of unicellular organisms such as microalgae,
diatoms, and some bacteria; organisms that form the base
of the marine foodweb. In general, most trophic levels above
these microbes have limited to zero capacity to make EPA
and DHA, most likely as a consequence of their environ-
ments being so omega-3 rich and hence providing a
continuous dietary source.
Several different biosynthetic pathways for EPA and
DHA have been identified in marine microbes, and the
biochemical and molecular nature of these have been well
described previously [9–11], but for clarity we will summa-
rize these briefly. The predominant route by which micro-
algae and diatoms synthesise omega-3 LC-PUFAs is via a
series of sequential desaturation and elongation reactions—
this is an aerobic process, and can be further sub-divided into
two forms—the predominant “D6-pathway,” in which the
first committed step is the introduction of a D6-desaturation
into a C18 substrate, followed by C2 elongation and further
D5-desaturation. The much less common route, the so-called
alternative or “D8-pathway,” initiates with the C2 elongation
of the C18 substrate, followed by two successive desaturation
reactions (D8, D5) [12]. If DHA is also synthesized, this
occurs by the C2 elongation of EPA and further (D4)
desaturation. These two pathways are represented schemati-
cally in Fig. 1. Multiple genes for all of these activities have
been identified in the last decade from a range of different
microbial sources.
An entirely different route favored by some bacteria and a
few examples of unicellular marine eukaryotes is the
biosynthesis of EPA or DHA via an anaerobic pathway
which uses a processive polyketide synthase-like enzymatic
system to convert malonyl-CoA to omega-3 LC-PUFAs
without any fatty acid intermediates. The genes for this
pathway have also been identified and functionally
characterized [13].
Collectively, all these different genes and the enzymatic
activities they encode, represent a toolkit by which the
metabolic engineer/synthetic biologist can attempt to
reconstitute the capacity to synthesise EPA and DHA in a
heterologous host. However, this is not as straight-forward as
Figure 1. Schematic representation of the aerobic pathways for the biosynthesis of omega-3 long chain polyunsaturated fatty acids. The
relevant desaturase and elongase activities, and their associated substrates, are indictated, as is the variant form (the alternative pathway)
observed in some limited examples.
1318 J. A. Napier et al. Eur. J. Lipid Sci. Technol. 2015, 117, 1317–1324
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com
it might appear, for a number of reasons. Firstly, in the case of
the aerobic pathways, the substrate for the constituent
enzymes is pre-existing C18 di- or tri-unsaturated fatty acids.
In the case of the anaerobic PKS-like pathway, the substrate
is malonyl-CoA, which is a low-abundance primary metab-
olite. Secondly, the successful reconstitution of the complete
pathway requires the coordinated expression and activity of
multiple genes, which is non-trivial in many transgenic
systems. Thirdly, by definition, the host into which one is
introducing the capacity to synthesise EPA and DHA has
limited (or no) endogenous capability to make these fatty
acids, meaning that native enzymes and pathways will be
unfamiliar with these fatty acids—this could result in either
selective exclusion of EPA/DHA from desired pathways
(such as neutral lipid synthesis) or alternatively, the
“trapping” of biosynthetic intermediates in undesired
metabolic dead-ends through the inclusive promiscuous
activity of house-keeping lipid enzymes. Finally, the under-
lying biochemical structural and biophysical properties of
these enzymes are only just starting to emerge, mainly due to
the membrane-associated nature which precludes crystal
structure-based studies [14]. Thus, although the genes with
which to attempt the heterologous expression of omega-3
LC-PUFA biosynthetic pathways have been in hand for over
10 years, the first successes are only now beginning to
emerge [15, 16].
In the context of the focus of this review, we will mainly
consider the potential of genetically engineered oilseed crop
plants to produce EPA and DHA, since it is likely that only
such agricultural systems will have the capacity (in terms of
scalability) to produce, in an economically viable manner,
sufficient omega-3 LC-PUFAs for “high volume/low value”
markets such as the aquafeed industry. However, we will also
briefly consider attempts to produce these fatty acids in
transgenic yeasts and diatoms, and how these contrast with
the situation observed in plants.
2.1 Engineering the accumulation of EPA and DHA in
transgenic plants—first attempts
As mentioned above, examples of all the biosynthetic genes
required for the accumulation of EPA and DHA biosynthesis
were identified by 2004 [9, 17], facilitating their combined
introduction into transgenic plants. Prior to that, individual
genes from this pathway had been evaluated in plants,
notably the D6- and D5-desaturases—these data gave
confidence that such heterologous activities were active in
plant cells, pointing the way for subsequent endeavors [18].
The first successful (proof-of-concept) demonstration of the
feasibility of making EPA in a transgenic plant was published
by Qi et al [19], expressing algal components of the
alternative pathway in the leaves of Arabidopsis. Interest-
ingly, this not only generated moderate amounts of EPA, but
also the C20 omega-6 LC-PUFA arachidonic acid (ARA).
Although this study was the first to generate EPA in a
transgenic plant, the approach taken was via sequential
transformation of individual genes, which was less than
optimal (since it means that the three transgenes are
genetically unlinked and prone to segregation in subsequent
generations). Moreover, although vegetative accumulation of
EPA was achieved, this was predominantly in phospholipids,
whereas the target lipid species would ideally be neutral
storage lipids such as triacylglycerols found in seeds.
The first demonstration of the seed-specificaccumu-
lation of EPA was also achieved in the same year by Abbadi
et al. [20], who expressed genes of the conventional D6-
pathway in transgenic linseed, under the control of seed-
specific promoters. Analysis of the resulting transgenic plants
and their seeds confirmed the presence of low levels of EPA,
but also very high levels of C18 D6-desaturation products.
This lead the authors to hypothesize that this unwanted
build-up of a biosynthetic intermediate was as a consequence
of poor acyl-exchange between different metabolic pools,
and this concept has been further defined as “substrate
dichotomy” [18]. In this situation, a bottleneck is generated
as a result of the differing substrate requirements for the
sequential reactions present in the aerobic biosynthesis of
LC-PUFAs—specifically, most lower eukaryotic desaturases
require their fatty acid substrate to be linked to a glycerolipid
backbone, whereas fatty acid elongases require the substrate
in the form of acyl-CoAs.
These initial first steps formed the basis for further
iterations to drive up the accumulation of target fatty acids
(EPA, DHA) and reduce the levels of undesired biosynthetic
intermediates (such as the D6-desaturation product g-
linolenic acid; GLA). These studies have confirmed first
the ability to make significant levels of EPA, albeit with
attendant levels of GLA, [21, 22] and then also DHA [21,
23]. The critical breakthroughs in optimizing the accumu-
lation of target fatty acids were achieved by: (i) the use of
omega-3 specific desaturases, which prevented the accumu-
lation of unwanted omega-6 fatty acids; (ii) the use of acyl-
CoA-dependent desaturases for the first committed (D6) step
on the pathway [24], breaking the substrate-dichotomy
bottleneck and reducing the accumulation of GLA [22, 24–
27]. As a consequence, it is now technically possible to
accumulate fish oil-like levels of omega-3 LC-PUFAs in the
seed oils of transgenic plants similar to that found in fish oils,
in which EPA and DHA accumulate to 20% of total fatty
acids. A comparison of the fatty acid composition of fish oil,
vegetable oil (GM and native), and also algal oil is shown in
Fig. 2.
2.2 Approaches to successful accumulation of target
fatty acids
Two discrete paths were adopted to ultimately produce EPA
and DHA in the seeds of transgenic plants, though both
started from the same position of having a “toolkit” of
suitable biosynthetic genes isolated from microalgae and
Eur. J. Lipid Sci. Technol. 2015, 117, 1317–1324 A sustainable source of fish oils from GM plants 1319
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com
other omega-3 accumulators, validated by heterologous
expression in yeast. The first approach, exemplified by
Petrie and colleagues at CSIRO (Canberra, Australia), took
advantage of Agrobacterium-mediated transient expression
systems which allow the rapid evaluation of multiple gene
combinations (via co-infection) in the leaves of Nicotiana
benthamiana [28]. This allowed the identification of an
optimal set of non-native genes which directed the efficient
synthesis of EPA and DHA in plants, albeit non-seed tissues.
However, by the further co-expression in leaves of a “master
regulator” transcription factor LEC2, the authors were able
to “reprogramme” lipid metabolism in this tissue so that it
now more closely resembled that of seeds, with the specific
capacity to synthesize seed oil in the form of triacylglycerol.
Thus, the host leaf cells under evaluation came to more
closely resemble the metabolic context observed in seeds. In
addition, an added benefit of this transcription factor-
mediated reprogramming was that instead of having to use
constitutive promoters to drive the expression of the non-
native genes, it was now possible to use seed-specific
promoters (since they were now active as a result of the
LEC2 expression), meaning that both regulatory elements
and biosynthetic enzymes were validated in a plant host. The
resulting study [29] allowed for the selection of the optimal
cassettes for subsequent stable transformation into plants
(discussed below).
The alternative approach, adopted by ourselves and
colleagues at the University of Hamburg, Germany was to
directly assess and iterate gene combinations and their
associated regulatory elements in the seeds of stably trans-
formed transgenic plants [20, 21]. The first example of
this [21], demonstrated the potential of this approach,
although the resulting seed oil profile still was suboptimal.
Using transgenic Arabidopsis, we subsequently systemati-
cally evaluated 22 genes in 9 different combinations, and in 2
different genetic backgrounds [30, 31]. For each construct,
multiple independent transgenic lines were subject to
detailed lipidomic analysis to define the incorporation of
non-native fatty acids into different lipid classes, which was
then used to inform subsequent iterations. This systematic
approach also identified an optimal suite of (different) genes
for introduction into crop species [30, 31].
2.3 The transition from model to crop
As indicated above, different approaches resulted in the
identification of a combination of algal and other lower
eukaryotic activities which were capable of directing the
synthesis and accumulation of EPA and DHA in model
systems such as Arabidopsis or tobacco. However, this was
still one step removed from the demonstration of the
accumulation of these fatty acids in a suitable oilseed crop
Figure 2. Comparison of fatty acid composition in different sources of omega-3 LC-PUFAs. The major fatty acids present in either fish
oil, vegetable oil, GM vegetable oil, or algal oil are presented. Note that non-GM vegetable oil lacks any EPA and DHA.
1320 J. A. Napier et al. Eur. J. Lipid Sci. Technol. 2015, 117, 1317–1324
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com
plant. Given the significant volumes of fish oils being
annually removed from the oceans, it is only via the use of
agricultural crops (and the scalability that brings) can one
hope to address the challenges associated with generating a
potential substitute.
To date, two oilseed crop species have been identified as
potential hosts for the omega-3 LC-PUFA biosynthetic trait
—canola, a cultivar of rapeseed (Brassica napus L.) and
(Camelina sativa). In the case of canola, there are no peer-
reviewed data yet in the public domain to allow an accurate
assessment. However, for Camelina, both the Rothamsted
and CSIRO groups have demonstrated the suitability of this
crop to act as an efficient host for the accumulation of EPA
and DHA, although interestingly, the fatty acid profile
obtained varies between the two studies. In the first publically
available data [32], we built on our iterative studies in
Arabidopsis to test two optimized constructs in transgenic
Camelina—one directing the synthesis of just EPA, and one
making both EPA and DHA. Based on the total fatty acid
composition of seeds from stably transformed lines, the
average level of EPA alone was 24%, and for EPA and DHA,
11 and 8%, respectively [32]. Very low levels of undesired
C18 biosynthetic intermediates such as GLA and stearidonic
acid were observed. Interestingly, for both the EPA and
EPAþDHA lines, the endogenous levels of a-linolenic acid
(ALA) were markedly decreased (presumptively since this
fatty acid serves as direct precursor for the synthesis of EPA),
whereas linoleic acid was effectively unchanged, most likely
due to the transgene presence of a FAD2 D12-desaturase.
Surprisingly, levels of oleic acid were also strongly reduced
for both constructs. Overall, the total level of target omega-3
LC-PUFAs was 19% (EPAþDHA), though this was found
to be greater than 25% in some single seeds.
In the CSIRO study [33], a construct (GA7) previously
evaluated in Arabidopsis (Petrie et al., 2012) was introduced
into Camelina, and the performance compared with two
additional constructs (mod-F, mod-G) in which minor
changes were made. Significant levels of DHA (up to 12.4%
for mod-F) were obtained for all three constructs, though the
levels of EPA were markedly low (maximally 3.3% for mod-
F). In contrast to the results observed by Ruiz-Lopez
et al., [32], levels of ALA were only moderately reduced,
whereas LA was down from 18.1 to 8.4% (mod-F), whereas
accumulation of the C18 D6-desaturation product stear-
idonic acid was noticeably higher. The observed differences
between the various constructs almost certainly resulted from
the contribution of a number of different factors, including
but not limited to: (i) precise expression of seed-specific
promoters; (ii) superior examples of the same enzyme
activity; (iii) construct design and orientation of elements;
and (iv) site of integration in the Camelina genome. Perhaps
the most striking and relevant difference between the two
studies is the variation in the level of EPA—although
the levels of DHA are broadly comparable (>10%), Petrie
et al. [33] reported only 0.8–3.3% EPA for the three
constructs, in contrast to the >11% observed by Ruiz-Lopez
et al. [32]. The most likely explanation for this is the use of a
highly active D5-elongase in the former study, which delivers
increased levels of DHA at the expense of EPA.
One additional interesting observation from these studies
relates to the apparent utility (or not) of using a model system
to predict the subsequent outcome in crop systems. Based
on both sets of results obtained in Camelina, it appears as
though previous studies in Arabidopsis underestimated the
levels of target fatty acids obtainable in the crop, though
obviously these earlier studies formed an important part of
the proof-of-concept. For example, in the earlier Arabidopsis
study by Ruiz-Lopez et al. [30, 31], maximal levels of EPA
observed were 8%, whereas in Camelina this was 24% [32].
Similarly, Petrie et al. [23] reported an average of 5.5% DHA
in T2 Arabidopsis seeds, compared with 9.6% DHA in T2
Camelina seeds [33]. Bearing in mind that in both studies,
the same identical constructs (EPA-A5.1; GA7, respectively)
were introduced into the two different hosts, these data
would imply that, at least for the omega-3 LC-PUFA trait,
Arabidopsis may underestimate the outcome in more
complex (genomically and biochemically) crops. In that
respect, it may be that in addition to the four influencing
factors described above, an additional consideration should
be the “metabolic context” into which the heterologous
pathway is superimposed [34]. Since the transgene-derived
activities need to biochemically integrate with endogenous
lipid metabolism to reconstitute the efficient synthesis of
EPA and DHA, it may be beneficial to have multiple copies
of such native genes, each with perhaps minor differences
in expression pattern, substrate-specificity, or efficiency.
Thus, working in a hexaploid crop such as Camelina provides
more opportunities (as a result of increased variation
via homeologues) for the smoother incorporation of the
heterologous pathway, and hence, higher synthesis and
accumulation of target fatty acids.
2.4 Utility and application of novel plant oils
The availability of these modified Camelina oils is a very
recent development, so there is currently only limited data on
their properties and applications of them. However, two
studies are of note. Firstly, Mansour et al. [35] examined the
detailed composition of Camelina oil derived from the GA7
construct, looking at both glycerolipid composition and also
sterols. Interestingly, the maximum level of DHA present in
TAG (the dominant neutral lipid) was 6.8%, and basal levels
of EPA (0.4%). There was significantly less DHA (1.6%)
in phospholipids, though the residual protein meal also
contained DHA (5.4%) though the overall lipid content in
this fraction was very low (0.3% of starting seed weight).
Although this preliminary study did not indicate which
generation or event for the GA7 construct was examined, it is
noteworthy that the levels of DHA in TAG are somewhat
reduced compared to the original study (6.8% vs. 9.6%;
Eur. J. Lipid Sci. Technol. 2015, 117, 1317–1324 A sustainable source of fish oils from GM plants 1321
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com
respectively) [33, 35]. There was no significant difference
between the GA7 line and wildtype Camelina with regards to
levels or profiles of sterols, indicating that the presence of
non-native omega-3 LC-PUFAs did not alter this pathway.
More recently, the first study on the application of these
novel oils has been published—Betancor et al. [36] used
the EPA-containing Camelina oil described by Ruiz-Lopez
et al. [32] to determine if this oil could substitute for fish oil in
a salmon feeding trial. In this study, juvenile (post-smolt)
Atlantic salmon were fed diets containing fish oil, EPA-
containing Camelina oil, or wild type Camelina oil. All three
diets were isocalorific, and also contained low levels of
DHA (via the deliberate use of fish meal as a protein source)
to ensure no deficiency for this fatty acid. The fish were fed
the different diets for seven weeks, after which they had all
doubled in weight and displayed no indicators for ill-health.
Fatty acid analysis of the flesh and the liver indicated the
expected accumulation of EPA, and in the latter tissue,
some conversion to DHA [36]. Transcriptomic analyses of
these tissues also indicated the up-regulation of genes
involved in omega-LC-PUFA metabolism, though both
Camelina treatments (WT, GM) induced transcriptome
signatures more similar to each other than the fish oil
treatment—this most likely reflects the fact that the GM oil
still contains fatty acids normally associated with vegetable
oils, but not observed in fish oils (such as linoleic acid), and
also the common presence of (phyto) sterols, which are
known to induce the up-regulation of the cholesterol pathway
in fish [37]. In that respect, the EPA-containing GM
Camelina oil can be considered a “hybrid” oil, representing
an in vivo blend of plant and marine fatty acids (see also Fig. 2
for graphical representation). Given that most aquafeed diet
formulations are now a mixture of fi sh oil and vegetable oil,
this GM Camelina oil is well-suited for direct use in
aquaculture. Moreover, this study, for the first time,
demonstrates the utility of using novel oils derived from a
GM plant as a safe and effective replacement for fish oils.
2.5 Alternative production systems for the synthesis
of omega-3 LC-PUFAs
Currently, two additional approaches also contribute to the
de novo production of omega-3 LC-PUFAs, via either the
fermentation of a GM yeast strain, or via the cultivation of
native strains of microalgae, and as such, may have the
capacity to augment piscine sources of these fatty acids. In
the latter case of algae cultivation such production systems
use a diverse range of different algal strains, and play an
important role in providing suitable nutrients to the earlier
stages of the life cycle of various species used in fish-farming,
though the actual levels of omega-3 LC-PUFAs produced by
this process are very modest (<1% of annual fish oil
production rates). Currently, there are no examples of
genetically modified algal strains being used in this way,
though recently it has been reported that a GM strain of
Phaeodactylum tricornutum had been produced which had
increased levels of DHA [38].
In the case of microbial fermentation, work from DuPont
demonstrated the feasibility of using the oleaginous yeast
Yarrowia lipolytica as a heterologous host for the production
of EPA. This tour-de-force of metabolic engineering
introduced components of the alternative pathway into the
yeast to enable it synthesize e EPA, with very high levels (up
to 15% dry weight) of this fatty acid being obtained.
Serendipitously, part of this high level of accumulation arose
as a result of the insertional mutagenesis (via integration of
the transgenes) of a gene (PXA10) involved in peroxisomal
biogenesis, resulting in decreased levels of fatty acid
catabolism by beta-oxidation [39]. The utility and applica-
tions of this source of EPA are discussed in a recent
review [40].
3 Conclusions
As reviewed above, it is clear that the promise of a plant-
based source of omega-3 LC-PUFAs has now been
deomonstrated, at least in the case of Camelina. However,
there still remain a number of hurdles to clear before such a
crop becomes a commercial reality and contributes to the
pressing need for an alternative, sustainable source of omega-
3 LC-PUFAs. Given the progressive and forward-looking
nature of the aquafeed industry, it is hoped that such novel
ingredients can, subject to appropriate regulatory and safety
approval, become incorporated into a wide range of fish diets,
and in turn, relieve pressure on the oceanic stocks of the
reduction fisheries. Since Camelina as an oilseed crop can
easily yield 0.75 ton of oil/ha, then a GM oil containing
similar levels of EPA and DHA to that found in fish oils could
make a significant contribution to off-setting oceanic sources.
For example, 2 00 000 ha of GM Camelina could produce
1 50 000 MT of oil which could serve as a direct replacement
for fish oils in aquafeed, representing 15% of the global
oceanic harvest of these oils. And although 2 00 000 ha might
seem like a large area, in agricultural terms, it is a quite
modest scale—for comparison, the current annual Canadian
sowing of related oilseeds such as canola is in excess of 7
million ha. Thus, the proposed 2 00 000 ha of Camelina
represents only a small (<3%) area of land currently given
over to vegetable oil production in one country, yet could
significantly increase the de novo synthesis of omega-3
LC-PUFAs.
Converting this hypothetical concept into reality will
require a number of different factors to be dealt with,
including seeking and obtaining regulatory approval to grow
a novel GM crop, better understanding of the agronomy and
processing of Camelina, use and formulation of the novel oil
by end-users in the feed sector, and ultimately acceptance by
the consumer. None of these steps are trivial or should be
taken for granted, and will most likely require significant
1322 J. A. Napier et al. Eur. J. Lipid Sci. Technol. 2015, 117, 1317–1324
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com
resources and organizational focus. However, given the
increasing demand for omega-3 LC-PUFAs, and the
demonstrated examples of “proof-of-concept” described in
this article, it is hoped that a terrestrial, GM oilseed source of
EPA and DHA might be available by the end of this decade.
Rothamsted Research receives grant-aided support from the
Biotechnology and Biological Sciences Research Council of
the U.K. Some of the work described in this review was supported
by BBSRC grant BB/J00166X/1.
The authors confirm that they have no conflict of interest.
References
[1] Saravanan, P., Davidson, N. C., Schmidt, E. B., Calder,
P. C., Cardiovascular effects of marine omega-3 fatty acids.
Lancet 2010, 376, 540–550.
[2] Tacon, A. G., Metian, M., Global overview on the use of fish
meal and fish oil in industrially compounded aquafe eds
trends and future prospects. Aquaculture 2008, 285, 146–
158.
[3] Hixson, S. M., Fish nutrition and current issues in
aquaculture: The balance in providing safe and nutritious
seafood, in an environmentally sustainable manner. J. Aquac.
Res. Devel. 2014, 5,1–10.
[4] Bostock, J., McAndrew, B., Richards, R., Jauncey, K., et al.,
Aquaculture: Global status and trends. Philos. Trans. R. Soc.
Lond. B 2010, 365, 2897–2912.
[5] Salem, N., Eggersdorfer, M., Is the world supply of omega-3
fatty acids adequate for optimal human health?. Curr. Opin.
Clin. Nutr. Metab. Care 2015, 18, 147–154.
[6] Tacon, A. G., Metian, M., Fish matters: Importance of
aquatic foods in human nutrition and global food supply.
Rev. Fisheries Sci. 2013, 21,22–38.
[7] Henriques, J., Dick, J. R., Tocher, D. R., Bell, J. G.,
Nutritional quality of salmon products available from major
retailers in the UK: Content and composition of n-3 long-
chain PUFA. Br. J. Nutr. 2014, 112, 964–975.
[8] Nichols, P. D., Glencross, B., Petrie, J. R., Singh, S. P.,
Readily available sources of long-chain omega-3 oils: Is
farmed Australian seafood a better source of the good oil than
wild-caught seafood? Nutrients 2014, 6, 1063–1079.
[9] Sayanova, O. V., Napier, J. A., Eicosapentaenoic acid:
Biosynthetic routes and the potential for synthesis in
transgenic plants. Phytochemical 2004, 65, 147–158.
[10] Venegas-Caler
on, M., Sayanova, O., Napier, J. A., An
alternative to fish oils: Metabolic engineering of oil-seed
crops to produce omega-3 long chain polyunsaturated fatty
acids. Prog. Lipid Res. 2010, 49, 108–119.
[11] Petrie, J. R., Singh, S. P., Expanding the docosahexaenoic
acid food web for sustainable production: Engineering lower
plant pathways into higher plants. AoB Plants 2011, plr 011.
[12] Ruiz-Lopez, N, Haslam, RP, Usher, S, Napier, JA,
Sayanova, O., An alternative pathway for the effective
production of the omega -3 long-chain polyunsaturates EPA
and ETA in transgenic oilseeds. Plant Biotechnol. J. 2015, Jan
30 DOI: 10.1111/pbi.12328
[13] Metz, J. G., Roessler, P., Facciotti, D., Levering, C., et al.,
Production of polyunsaturated fatty acids by polyketide
synthases in both prokaryotes and eukaryotes. Science 2001,
293, 290–293.
[14] McCartney, A. W., Dyer, J. M., Dhanoa, P. K., Kim, P. K.,
et al., Membrane-bound fatty acid desaturases are inserted
co-translationally into the ER and contain different ER
retrieval motifs at their carboxy termini. Plant J. 2004, 37,
156–173.
[15] Cahoon, E. B., Shockey, J. M., Dietrich, C. R., Gidda, S. K.,
et al., Engineering oilseeds for sustainable production of
industrial and nutritional feedstocks: Solving bottlenecks in
fatty acid flux. Curr. Opin. Plant Biol. 2007, 3, 236–244.
[16] Ruiz-Lopez, N., Usher, S., Sayanova, O. V., Napier, J. A.,
Haslam, R. P., Modifying the lipid content and composition
of plant seeds: Engineering the production of LC-PUFA.
Appl. Microbiol. Biotechnol. 2015, 99, 143–154.
[17] Kinney, A. J., Metabolic engineering in plants for human
health and nutrition. Curr. Opin. Biotechnol. 2006, 17, 130–
138.
[18] Napier, J. A., The production of unusual fatty acids in
transgenic plants. Annu. Rev. Plant Biol. 2007, 58, 295–319.
[19] Qi, B., Fraser, T., Mugford, S., Dobson, G., et al.,
Production of very long chain polyunsaturated v-3 and v-
6 fatty acids in plants. Nat. Biotech. 2004, 22, 739–745.
[20] Abbadi, A., Domergue, F., Bauer, J., Napier, J. A., et al.,
Biosynthesis of very-long-chain polyunsaturated fatty acids
in transgenic oilseeds: Constraints on their accumulation.
Plant Cell. 2004, 16, 2734–2748.
[21] Wu, G., Truksa, M., Datla, N., Vrinten, P., et al., Stepwise
engineering to produce high yields of very long-chain
polyunsaturated fatty acids in plants. Nat. Biotechnol.
2005, 23, 1013–1017.
[22] Cheng, B., Wu, G., Vrinten, P., Falk, K., et al., Towards the
production of high levels of eicosapentaenoic acid in
transgenic plants: The effects of different host species, genes
and promoters. Transgenic Res. 2010, 19, 221
–229.
[23] Petrie, J. R., Shrestha, P., Zhou, X. R., Mansour, M. P.,
et al., Metabolic engineering plant seeds with fish oil-like
levels of DHA. PLoS ONE 2012, 7, 49165.
[24] Domergue, F., Abbadi, A., Z
€
ahringer, U., Moreau, H.,
Heinz, E., In vivo characterization of the first acyl-CoA D6-
desaturase from a member of the plant kingdom, the
microalga Ostreococcus tauri. Biochem. J. 2005, 389, 483–
490.
[25] Hoffmann, M., Wagner, M., Abbadi, A., Fulda, M.,
Feussner, I., Metabolic engineering of v-3 very long chain
polyunsaturated fatty acid production by an exclusively acyl-
CoA-dependent pathway. J. Biol. Chem. 2008, 283, 22352–
22362.
[26] Petrie, J. R., Shrestha, P., Mansour, M. P., Nichols, P. D.,
et al., Metabolic engineering of omega-3 long-chain poly-
unsaturated fatty acids in plants using an acyl-CoA D6-
desaturase with omega3-preference from the marine micro-
alga Micromonas pusilla. Metab. Eng. 2010, 12, 233–240.
[27] Sayanova, O., Ruiz-Lopez, N., Haslam, R. P., Napier, J. A.,
The role of D6-desaturase acyl-carrier specificity in the
efficient synthesis of long-chain polyunsaturated fatty acids
in transgenic plants. Plant Biotechnol. J. 2012, 10, 195–206.
[28] Wood, C. C., Petrie, J. R., Shrestha, P., Mansour, M. P.,
et al., A leaf-based assay using interchangeable design
Eur. J. Lipid Sci. Technol. 2015, 117, 1317–1324 A sustainable source of fish oils from GM plants 1323
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com
principles to rapidly assemble multistep recombinant path-
ways. Plant Biotechnol. J. 2009, 7, 914–924.
[29] Petrie, J. R., Shrestha, P., Liu, Q., Mansour, M. P., et al.,
Rapid expression of transgenes driven by seed-specific
constructs in leaf tissue: DHA production. Plant Meth.
2010, 6,8.
[30] Ruiz-L
opez, N., Haslam, R. P., Venegas-Caler
on, M., Li,
T., et al., Enhancing the accumulation of omega-3 long chain
polyunsaturated fatty acids in transgenic Arabidopsis thaliana
via iterative metabolic engineering and genetic crossing.
Transgenic Res. 2012, 21, 1233–1243.
[31] Ruiz-Lopez, N., Haslam, R. P., Usher, S. L., Napier, J. A.,
Sayanova, O., Reconstitution of EPA and DHA biosynthesis
in Arabidopsis: iterative metabolic engineering for the
synthesis of n-3 LC-PUFAs in transgenic plants. Metab.
Eng. 2013, 17,30–41.
[32] Ruiz-Lopez, N., Haslam, R. P., Napier, J. A., Sayanova, O.,
Successful high-level accumulation of fish oil omega-3 long-
chain polyunsaturated fatty acids in a transgenic oilseed crop.
Plant J. 2014, 77, 198–208.
[33] Petrie, J. R., Shrestha, P., Belide, S., Kennedy, Y., et al.,
Metabolic engineering Camelina sativa with fish oil-like levels
of DHA. PLoS ONE 2014, 9, e85061.
[34] Napier, J. A., Haslam, R. P., Beaudoin, F., Cahoon, E. B.,
Understanding and manipulating plant lipid composition:
Metabolic engineering leads the way. Curr. Opin. Plant Biol.
2014, 19,68–75.
[35] Mansour, M. P., Shrestha, P., Belide, S., Petrie, J. R., et al.,
Characterization of oilseed lipids from “DHA-producing
Camelina sativa”: A new transformed land plant containing
long-chain omega-3 oils. Nutrients 2014, 6, 776–789.
[36] Betancor, M. B., Sprague, M., Usher, S., Sayanova, O.,
et al., A nutritionally-enhanced oil from transgenic Camelina
sativa effectively replaces fish oil as a source of eicosapen-
taenoic acid for fish. Sci. Rep. 2015, 5, 8104.
[37] Leaver, M. J., Villeneuve, L. A., Obach, A., Jensen, L., et al.,
Functional genomics reveals increases in cholesterol bio-
synthetic genes and highly unsaturated fatty acid biosyn-
thesis after dietary substitution of fish oil with vegetable oils
in Atlantic salmon (Salmo salar). BMC Genomics 2008, 24,
299.
[38] Hamilton, M. L., Haslam, R. P., Napier, J. A., Sayanova, O.,
Metabolic engineering of Phaeodactylum tricornutum for the
enhanced accumulation of omega-3 long chain polyunsatu-
rated fatty acids. Metab. Eng. 2014, 22,3–9.
[39] Xue, Z., Sharpe, P. L., Hong, S. P., Yadav, N. S., et al.,
Production of omega-3 eicosapentaenoic acid by metabolic
engineering of Yarrowia lipolytica. Nat. Biotechnol. 2013, 31,
734–740.
[40] Xie, D., Jackson, E. N., Zhu, Q., Sustainable source of
omega-3 eicosapentaenoic acid from metabolically engi-
neered Yarrowia lipolytica: From fundamental research to
commercial production. Appl. Microbiol. Biotechnol. 2015,
99, 1599–1610.
1324 J. A. Napier et al. Eur. J. Lipid Sci. Technol. 2015, 117, 1317–1324
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com
