Improving Human Nutrition through Genomics, Proteomics and Biotechnologies

Symposium: Improving Human Nutrition through
Genomics, Proteomics and Biotechnologies
Modification of Plant Lipids for Human Health: Development of Functional
Land-Based Omega-3 Fatty Acids1
Virginia M. Ursin2
Monsanto Company, Calgene Campus, Davis, CA
ABSTRACT We have remodeled canola seeds to accumulate the omega-3 fatty acid, stearidonic acid (SDA). In
doing so, we have demonstrated the feasibility of developing a land-based source of functional omega-3 fatty acids
on a large scale. Land-based omega-3 fatty acids represent a sustainable source of omega-3 fatty acids that can
be produced on large acreages and delivered to consumers in a wide variety of functional foods. And unlike
␣-linolenic acid, SDA can provide eicosapentaenoic acid equivalence at moderate intakes. Widely applied,
SDA-enriched foods could become a valuable tool for delivering recommended levels of omega-3 fatty acids to
large portions of the population. By obviating the need for dietary changes, SDA-enriched foods may facilitate
increased compliance with recommendations for daily omega-3 intakes. J. Nutr. 133: 4271– 4274, 2003.
KEY WORDS: ● canola oil
● biotechnology
●
stearidonic acid
●
PUFA
●
omega-6 fatty acids
●
omega-3 fatty acids
like eicosanoids. Dietary PUFA are almost exclusively plant
derived. In plants PUFA are derived from SFA. SFA are
progressively desaturated to form monosaturated fatty acid,
oleic acid (OA) [18:1(n-9)] and the PUFA, linoleic acid (LA)
and ␣-linolenic acid (ALA) [18:2(n-6) and 18:3(n-3), respectively]. Depending on the position of the first double bond in
the fatty acid molecule, polyunsaturates are classified as either
omega-6 (n-6) or omega-3 (n-3) fatty acids (Fig. 1). The
PUFA biosynthetic pathway occurs in virtually all plant cells,
hence, omega-6 and omega-3 fatty acids are present in varying
proportions in leaves, seeds and oil, and from there are incorporated into the diet. In the U.S. polyunsaturated fats constitute ⬃7% of total energy intake. LA compromises up to 89%
of total PUFA energy intake, whereas ALA typically comprises only about 10% total PUFA energy in adult diets (4).
This relative disparity in (n-3) to (n-6) intake is reflected in
the (n-3) to (n-6) ratio of the most widely consumed vegetable
oil in the U.S. diet, soybean oil, which constitutes ⬃83% of
the vegetable oil intake (5) (Fig. 2).
Omega-3 fatty acids, known to be essential for growth and
development, have been positively associated with health and
the prevention and treatment of heart disease, arthritis, inflammatory and autoimmune diseases and cancer (2). Accordingly, there are now dietary recommendations and guidelines
for omega-3 fatty acid intakes. For example, in a recent
Scientific Statement, the AHA Dietary Guidelines suggest
Americans consume at least two servings of fish per week,
and include in the diet vegetable oils rich in the omega-3
fatty acid, ALA (6). It also recommends that 1.3–2.7 g/d
total omega-3 fats be consumed. Despite these recommendations, it is estimated that actual dietary intakes of
omega-3 fatty acids, and eicosapentaenoic (EPA) and do-
Background
Dietary fats and oils represent a significant percentage of
the daily caloric intake in the United States comprising ⬎33%
of total calories (1). A large body of scientific evidence has
implicated the quantity and/or quality of dietary fats in the
development of several diseases, including cardiovascular disease (CVD),3 some cancers and arthritis (2). Not surprisingly,
organizations including the USDA, American Heart Association and National Academy of Sciences/Institute of Medicine
have made dietary recommendations recently that focus not
only on the quantity but also on the types of fats in the diet,
and generally recommend substituting monounsaturated and
polyunsaturated fats for saturated fatty acids (SFA) (3).
Because PUFA are not synthesized de novo in mammals,
they must be derived from the diet. Once ingested, they are
further metabolized and the resulting Long Chain PUFA populate cellular membranes and serve as precursors for hormone-
1
Presented at the Experimental Biology Meeting, April 11–15 2003, San
Diego, CA. The symposium was sponsored by The American Society for Nutritional Sciences and supported in part by an educational grant from Nestlé and a
USDA-NRI conference grant. The proceedings are published as a supplement to
The Journal of Nutrition. This supplement is the responsibility of the guest editors
to whom the Editor of The Journal of Nutrition has delegated supervision of both
technical conformity to the published regulations of The Journal of Nutrition and
general oversight of the scientific merit of each article. The opinions expressed in
this publication are those of the authors and are not attributable to the sponsors
or the publisher, editor or editorial board of The Journal of Nutrition. Guest Editors
for the symposium publication are Naima Moustaid-Moussa and Jay Whelan,
Department of Nutrition, The University of Tennessee, Knoxville, TN.
2
To whom correspondence should be addressed.
E-mail: virginia.ursin@monsanto.com.
3
Abbreviations used: ALA, ␣-linolenic acid; CVD, cardiovascular disease;
DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; GLA, ␥-linolenic acid;
LA, linoleic acid; OA, oleic acid; SDA, stearidonic acid; SFA, saturated fatty acids.
0022-3166/03 $8.00 © 2003 American Society for Nutritional Sciences.
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SYMPOSIUM
the same supply pressures that are facing wild stocks of fish will
likely impact farmed fish (10). In addition to issues of sustainability, there is increasing alarm over levels of methyl mercury
in some species of long-lived fish, that has prompted warnings
to limit consumption of certain fish species, including swordfish, mackerel and shark, and for some at risk groups, avoid
consumption all together (Environmental Protection Agency;
National Academy of Sciences; Institutes of Medicine). Oil
from fermentation of microalgae can be very high in EPA or
DHA. Once extracted, it is suitable for enrichment of food,
however, it is not currently being produced in sufficient quantities for wide-scale impact.
Stearidonic aid
FIGURE 1
PUFA biosynthesis pathway. The arrows represent
desaturation reactions. The enzymes catalyzing the reaction are listed
above or beside each arrow. The portion of the pathway depicted in the
solid box represents steps occurring principally in planta. The portion in
dashed box represents enzymatic steps of the PUFA biosynthesis
occurring principally in mammals after intake of dietary PUFA. The dark
arrows represent the enzymatic reactions that were targeted for development of SDA-canola.
cosahexaenoic acids (DHA) specifically, are as low as onetenth of these levels.
There is consensus for the need for increased dietary
omega-3 intakes among populations where intakes are below
recommended levels. However, it remains unknown how to
achieve this for large sectors of the population, especially
where dietary preferences exclude fish. The concept of enriching a wide variety of foods with omega-3 fatty acids, so that
consumers can chose omega-3 enriched foods that suit their
individual preferences, is logical and has been proven in concept as an effective means to increase omega-3 intakes on a
wide scale (7). Presently, sources of omega-3 fats suitable to
enrich foods include vegetable and nut oils, which can contain
up to 50% ALA, marine oils and EPA and DHA in varying
amounts (4) and single-cell oils that are derived from the
fermentation of microalgae, which contain DHA and/or EPA.
However, there are limitations to each of these sources. The
bioconversion of dietary ALA to EPA, which is necessary for
the therapeutic and preventative benefits of omega-3 fats, is
extremely inefficient with as little as 0.2% of plasma ALA
undergoing conversion to EPA (8). Nonhydrogenated fish oils,
on the other hand, provide EPA and DHA, the most effective
forms of dietary omega-3 fatty acids for decreasing CVD risk
and improving overall health. However, persistent questions
exist about the sustainability of global fisheries. It is estimated
that to achieve the recommended levels of EPA and DHA, a
fourfold increase in fish consumption in the United States is
necessary (4). Yet, yields from global fisheries have been reported to be stagnant or declining (9), and although aquaculture is a rapidly growing source of fish, the dietary requirements of omega-3 containing farmed fish for EPA and DHA
requires fish meal and fish oil be provided in their diets. Hence,
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We have previously proposed that the omega-3 fatty acid,
stearidonic acid (SDA) [18:4(n-3)], a metabolic intermediate
between ALA and EPA, could effectively bridge the gap
between the sustainability of a land-based oil and the effectiveness of fish oil (11). SDA is an intermediate between ALA
and EPA and can represent up to 4% of the total fatty acids in
fish oil (12), and thus has been present in the human diet for
as long as fish, such as menhaden, have been in the diet. SDA
represents the ⌬6 desaturation product of ALA and as such,
bypasses a rate-limiting step in the conversion of dietary ALA
to EPA. The relative effectiveness with which dietary SDA is
bioconverted to EPA is thus dependent upon regulation of the
subsequent desaturation and elongation reactions (Fig. 1).
Recently, the relative efficiencies of dietary SDA, ALA and
EPA in elevating plasma EPA was assessed in a clinical study
(11). In this study, dietary SDA was found to increase plasma
EPA between threefold and fourfold more efficiently than
comparable levels of ALA and was approximately one-third as
effective as dietary EPA (Fig. 3). At this relative rate of
metabolic conversion of SDA to EPA, moderate intakes of
SDA-containing oil could positively impact plasma EPA to an
extent that would be expected to confer the cardiovascular
benefits associated with consumption of EPA. Through incorporation into a variety of widely consumed foods, SDA could
significantly increase omega-3 intakes to such an extent that
the health benefits of a diet containing adequate omega-3 fats
can be realized by a larger sector of the population.
Natural sources of SDA do exist, but are limited such that
wide-scale enrichment of foods is currently impossible. In
FIGURE 2 Fatty acid composition of the major vegetable oils. The
approximate percentage of saturated fatty acids: SFA; monosaturated
fatty acids, oleic acid; the PUFA, linoleic and ␣-linolenic acid; as weight
percentage of total seed oil. Following each bar is the percentage of
total fat intake for each vegetable oil in the U.S. * Ref. 5.
LAND-BASED OMEGA-3 FATTY ACIDS
addition to its occurrence in fish oil, SDA is also found in the
seed oil and leaves of a number of plants, including evening
primrose (Oenothera biennis), borage (Borago officinalis), black
currant (Ribes nigrum) and Echium plantagineum. However,
none of these species represent crops adapted to wide scale
production and yields of oil are low and variable and not
economically competitive. One approach to achieving SDA
production on a large scale would be to implement an intensive breeding program for a species such as Echium plantagineum for adaptation to commercial scale agriculture, although
the feasibility of this is questionable. An alternative approach,
and one we have attempted successfully, is to engineer the
capacity to produce SDA in to a high oil-yielding oilseed crop
where existing commercial cropping and oil extraction technologies are utilized. That approach is presented below.
Genetic engineering of plant lipids
The genetic modification of oilseed crops provides an opportunity to tailor the composition of seed oils for optimal
dietary or processing characteristics. Until recently, modifications of oil composition could be achieved through traditional
plant breeding, where natural diversity within closely related
species could be exploited, or through mutagenesis. Transgenic
technology widens the scope of modifications achievable in oil
composition by allowing the introduction of a wider range of
genetic elements than is otherwise possible. There has been
considerable focus on the identification and analysis of enzymes and underlying genes involved in plant lipid biosynthesis (13). Coupled with efficient plant transformation systems
for the key oilseed crops, canola and soybean, metabolic engineering of seed lipids in these two crops has become feasible.
As a result there has been successful development of a variety
of novel oils in which the fatty acid composition has been
optimized for improved processing characteristics (14), healthfulness (15) or industrial applications (16).
Development of SDA-canola
In conventional canola oil, ⬃97% of the lipids are represented in three classes: SFA (5.25%, 16:0 and 18:0), monosaturated fatty acids (66%, 18:1) and PUFA (26%, 18:2 and
18:3) (Fig. 4), with the PUFA containing an ⬃2:1 ratio of LA
to ALA. SDA, which is not normally present in canola oil,
represents the ⌬6 desaturation product of ALA. Because
FIGURE 3 Effects of dietary ␣-linolenic acid (ALA), stearidonic
acid (SDA) and eicosapentaenoic acid (EPA) on erythrocyte phospholipid EPA. Bars represent EPA at initiation of the study (⫺3 wk), after
3-wk dietary run-in period (0 wk), 3 wk of 0.75 g/d fatty acid intake (3
wk), and 3 wk of 1.5 g/d fatty acid intake (from Ref. 11). Asterisks
indicates statistically significant differences, P ⬍ 0.05, 2-way repeated
measures ANOVA with Tukey’s post-hoc test.
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FIGURE 4 Fatty acid composition oil of conventional, nontransgenic and transgenic stearidonic acid (SDA)-canola oils. Sections of
each bar represent the weight percentage of each fatty acid class in
total lipids from a single seed. Top bar is conventional, nontransgenic
canola, Middle bar is transgenic canola line transformed with a triplegene vector containing the M. alpina ⌬6 and ⌬12 desaturases and B.
napus ⌬15 desaturase. Bottom bar is from an F1 hybrid between
transgenic high ␥-linolenic acid (GLA) canola line containing the M.
alpina ⌬6 and ⌬12 desaturases, and transgenic high ␣-linolenic acid
(ALA) canola line containing the B. napus ⌬15 desaturase. Abbreviation:
OA, oleic acid.
canola accumulates primarily OA in its oil, the production of
significant quantities of SDA in canola required increased flux
through the PUFA pathway from OA to ALA and then the
addition of ⌬6 desaturase activity (Fig. 1). Genes encoding the
fatty acid desaturase enzymes that catalyze these reactions
have been identified and characterized from diverse sources
including higher plants and fungi (17). We generated transgenic canola lines that expressed in seeds the ⌬6 and ⌬12 fatty
acid desaturases isolated from the commercially grown fungus,
Mortierella alpina, and the ⌬15 fatty acid desaturase from
canola (Brassica napus). Seed oil from independent transformants accumulated SDA, as predicted (18). SDA accumulated
up to 23% of the oil by weight, although the amount of SDA
and the ratio of SDA to other seed lipids was influenced by the
strategy utilized. For example, in one approach, the ⌬6, ⌬15
and ⌬12 desaturases were combined on the same transformation vector. Resulting canola seeds were evaluated for seed
fatty acid composition in the first transgenic generation and
selected lines were self-pollinated and evaluated in the subsequent generation. The fatty acid composition of one line is
shown in Figure 4. In this line, SDA accumulated to ⬃16% of
the total fatty acids. The total omega-3 content in the seed
lipids (ALA ⫹ SDA) was ⬎60% of the fatty acids whereas the
total omega-6 fatty acid content of the seed lipids (GLA ⫹
LA) was ⬃22%. OA was reduced from 60% of the seed lipids
to ⬃12%. In an alternative approach, a crossing strategy was
utilized wherein transgenic lines containing only the M. alpina
⌬6 and ⌬12 desaturase genes were hybridized with independently produced lines containing only the B. napus ⌬15 desaturase gene. Seed lipids were evaluated in the F1 and F2
generations from several independent crosses. In these progeny, SDA accumulated up to 23% of the lipids F1 seed (Fig. 4).
The total omega-3 content in the seed lipids (ALA ⫹ SDA)
exceeded 55% of the seed lipids whereas the total omega-6
fatty acid content of the seed lipids (GLA ⫹ LA) was ⬃22%
of the seed lipids.
CONCLUSIONS
The enhancement of vegetable oil quality is an exciting
and valuable application of biotechnology. We have produced
prototype canola lines that have been engineered to accumu-
SYMPOSIUM
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TABLE 1
Total omega-3 and n-6 to n-3 ratios of non-transgenic
vegetable oil and genetically modified, SDA-canola oil
mation, greenhouse and analytical staff for the excellent work that
enabled this project. The author thanks Toni Voelker and Michael
James for reviewing this manuscript.
LITERATURE CITED
Vegetable Oil
Source
Total Omega-3
(% fatty acids)
n-6:n-3 ratio
Sunflower
Corn
Soybean
Canola
SDA-Canola*
0
1
7
12
50
69:1
61:1
7:1
2.6:1
1:2
* Based on composition of F2 Seed, Reported in Knutzon, 1999 (ref.
24). SDA, stearidonic acid.
late in the seed oil, the omega-3 fatty acid, SDA. Oil from
these lines would be suitable for incorporation into a range of
foods, and by comparison to conventional canola oil and
soybean oil, have significantly higher levels of effective long
chain omega-3 fatty acid precursors and an improved omega-3
to omega-6 ratio (Table 1.) Additionally, because SDA is
approximately one-third as efficient as EPA, modest intakes of
SDA-containing vegetable oil can be expected to provide the
health benefits of recommended fish intakes. A wide variety of
SDA-enriched foods, if available, could supply a significant
portion of the recommended daily intakes of omega-3 fatty
acids to large numbers of consumers. In addition, specific
populations exist that could realize even greater benefit from
the availability of SDA-enriched foods. These include vegetarians and vegans, whose omega-3 status may border on
biochemical deficiency (19) and pregnant women who are
faced with contradictory recommendations that include increasing omega-3 intake and decreasing intake of several species of fatty fish including shark, swordfish and king mackerel
because of elevated levels of methyl mercury (20).
A large nutritional gap exists between recommended and
actual daily intakes of omega-3 fatty acids (2) and it is widely
accepted within the scientific and nutritional communities
that this contributes to a variety of health consequences. If
current advice to increase omega-3 intakes is heeded, demand
for omega-3 fatty acids will increase as will the need for
alternative sources to fish and fish oil. Biotechnology may
provide a truly unique solution to the supply of dietary
omega-3 fats by simultaneously solving the existing intrinsic
problems of availability and sustainability, as well as that of
bioactivity.
ACKNOWLEDGMENTS
The author thanks Debbie Knutzon for the development of the
SDA canola lines described in this paper and the Calgene transfor-
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