Download Transgenic Plants Produce Omega-3 And Omega

ISB NEWS REPORT
JULY 2004
TRANSGENIC PLANTS PRODUCE OMEGA-3 AND OMEGA-6 FATTY ACIDS
Baoxiu Qi
Recently, the importance of fish and fish oils in the diet has become significantly more recognized. It is known
that they play very important roles in human health and nutrition and are important building blocks in neonatal
retinal and brain development, as well as being important factors in cardiovascular health and disease prevention1-2. The active ingredients in fish and fish oils are omega-3 very long chain polyunsaturated fatty acids
(VLCPUFAs), such as eicosapentaenoic acid (EPA, C20:5 ω-3) and docosahexaenoic acid (DHA, C22:6 ω−3).
The realization of their importance started in the 1970s from the epidemiological finding that Greenland
Eskimos only had one-eighth of the fatal coronary heart disease of Eskimos who lived in Denmark. The reason
is that the Greenland Eskimos have a traditional ‘marine’ diet rich in very long chain ω-3 PUFAs. The diet of
most modern societies is nowadays relatively low in ω3 PUFAs with a concomitant increased level of ω6
PUFAs intake, largely resulting from a preference for plant-seed oils and food products from intensively bred
animals. In addition, some communities do not have access to fish supplies, due to geographic or economic
reasons; some people are allergic to fish, others choose not to eat fish because of their vegetarian life style.
Global fish stocks are declining due to general overfishing and overly efficient fishing methods, and the oils
derived from fish are sometimes contaminated with a range of pollutants, heavy metals, and toxins. Therefore,
alternative sources of these VLCPUFAs are clearly desirable, and the concept of obtaining them from higher
plants in commercial and sustainable quantities is particularly attractive. However, no oil-seed species produces
such products naturally, so there is considerable interest recently in genetically engineering the capacity to
synthesize these fatty acids in agronomically viable oil-seed species.
Humans can synthesize omega-6 and omega-3 VLCPUFAs from the so-called essential fatty acids, linoleic acid
(LA, C18:2 ω-6) and a-linolenic acid (ALA, C18:3 ω-3) by a D6-desaturation pathway in which the characteristic first step is the D6-desaturation of LA and ALA to yield -linolenic acid (GLA, C18:3 ω-6) and stearidonic
acid (STA, C18:4 ω-3), respectively. Further fatty acid elongation and desaturation steps give rise to arachidonic acid (AA, C20:4 ω-6) and EPA. DHA is the product of further elongation and desaturation of EPA.
However, these pathways are very inefficient, and to obtain these VLCPUFAs directly from the diet is considered necessary.
An alternative pathway for the biosynthesis of AA and EPA operates in some organisms. Here, LA and ALA
are first elongated specifically to eicosadienoic acid (EDA, C20:2 ω-6) and eicosatrienoic acid (EtrA, C20:3 ω3), respectively. Subsequent D8 and D5 desaturation of these products yields AA and EPA. We recently reported
the reconstitution of these D8-desaturation pathways for VLCPUFA synthesis in Arabidopsis thaliana, and the
accumulation of appreciable quantities of AA and EPA in the transgenic plants3 by sequential transfer and
expression of three genes encoding a D9-specific elongating activity from Isochrysis galbana (IgASE1)4, a D8desaturase from Euglena gracilis (EuD8)5, and a D5-desaturase from Mortierella alpina (MortD5)6, respectively. For expression in Arabidopsis, the coding regions of these three genes were placed in CaMV 35S
promoter-nos terminator expression cassettes of the plant binary vectors pCB302-1, pBECKS, and pCAMBIA
1300, respectively. Arabidopsis thaliana ecotype Columbia 4 was subjected to Agro-bacterium-mediated
transformation via floral dipping. Basta herbicide, kanamycin, and hygromycin were used to select single,
double, and triple transformants, respectively. Homozygous plants with a single copy of the transgenes were
selected by two rounds of self-fertilization after each transformation and subjected to further transformation
with the subsequent gene. Total fatty acids in the leaf tissues of wild type, single transgenic plants expressing
IgASE1, double transgenic plants expressing both IgASE1 and EuD8, and triple transgenic plants expressing
IgASE1, EuD8, as well as MortD5 were extracted, methylated, and analyzed by gas chromatography (GC).
The GC profile of total fatty acids extracted from leaves of wild type and transgenic plants is shown in Figure
1. LA and ALA, the substrates for the Isochrysis C18-D9-elongating activity IgASE1, are major fatty acids in
the leaves of wild type plants (Fig. 1a). Two additional fatty acid species are apparent in the single transgenic
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Figure 1. GC profiles of Arabidopsis leaf fatty acid methyl esters extracted from (a) an untransformed plant; (b), a single-transgenic plant
expressing the I. galbana D9-elongating activity IgASE1; (c), a double transgenic plant expressing IgASE1 and the E. gracilis D8-desaturase
(EuD8); (d) a triple transgenic plant expressing IgASE1, EuD8 and the M. alpina D5-desaturase (MortD5). The peaks in the boxed region are
designated 1 to 8 in the order of increasing retention time; peaks 4 and 8 are 20:4 and 20:5, respectively.
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plants expressing IgASE1 (Fig. 1b). These were identified as EDA and EtrA. C20:2 and C20:3 accumulated to
8.4 mol% and 10.4 mol% of total fatty acids, representing conversions of 51% and 22% of their C18 substrates, respectively. Two additional peaks are apparent in the GC profile of the leaf fatty acids of the double
transgenic line expressing both IgASE1 and EuD8 (Fig. 1c) compared to its single transgenic parent (Fig. 1b).
These were identified as dihomo- -linolenic acid (DGLA, C20:3 ω-6) and eicosatetraenoic acid (ETA, C20:4
ω-3), respectively. DGLA and ETA accounted for 8.7 and 6.5 mol% of the total fatty acids and represented a
conversion of 88% and 63% of their respective substrates. The total C20 PUFA content in this double transgenic line was ca. 20 mol% of the total fatty acids. Four other fatty acid peaks are apparent in the tripletransgenic plants expressing IgASE1, EuD8, and MortD5 compared to the double transgenic plants (Fig. 1d).
Two of these, peak 4 and 8, correspond to AA and EPA, the expected D5-desaturation products of DGLA and
ETA, respectively. The yields of AA and EPA were 6.6 and 3.0 mol% of the total fatty acids, representing 84%
conversion of substrate DGLA and 71% of ETA. Small amounts (3.2 mol% of total FAs) of two other C20
fatty acids were also present and these were identified as sciadonic acid, C20:3 D5,11,14, and juniperonic acid,
C20:4 D5,11,14,17. The total C20 PUFA content was ca. 22 mol% of total fatty acids. All transgenic plants were
phenotypically identical to wild type, which implies that production of significant amounts of C20 PUFAs did
not affect the plants’ normal development.
Our results demonstrate that both the ω3 D8- and ω6 D8-desaturation biosynthetic pathways for VLCPUFA
production can operate in higher plants, yielding the potentially valuable products AA and EPA in appreciable
amounts (6.6% and 3%, respectively). The key step would appear to be the C18-D9-PUFA-specific elongating
activity IgASE1 from Isochrysis, which specifically catalyses the elongation of the essential C18-D9-PUFAs,
LA and ALA.
These fatty acids are in abundance in green vegetative tissues of higher plants, and in Arabidopsis their elongation resulted in appreciable levels of EDA (20:2ω-6) and EtrA (20:3ω-3). The efficiency of this alternative
pathway may be because the first step is catalyzed by elongase, which uses LA-CoA and ALA-CoA as substrates, and these have previously been shown to be present in Arabidopsis leaf tissue. Moreover, the subsequent two desaturation steps can occur while the acyl groups remain on the phosphotidylcholine substrate. In
contrast, recent attempts to reconstitute the conventional ω6 and ω3 D6-desaturation pathways in yeast met
with limited success7. To explain their relatively low yield of AA in yeast, Domergue et al.7 argued that insufficient GLA substrate was made available from the site of D6-desaturation to the acyl-CoA pool for elongation,
and that this explained a similar poor activity of the reconstituted conventional pathway in transgenic plants.
The results reported here demonstrate that “pathway engineering” for the viable production of VLCPUFAs in
plants is possible. In order to exploit these observations more fully it will be necessary to express the genes in
question in a seed-specific manner and in a lipid background rich in the fatty acid substrates that are specific
for the elongase component, IgASE1. To this end, oilseed species such as soy and linseed should provide
appropriate experimental material for further study. Our data indicate that the use of these alternative D8desaturation pathways is likely to be more appropriate for genetic modification of oilseeds than the previously
described conventional D6-desaturase/elongase route. The triple-transgenic plants accumulate both AA and
EPA in amounts that suggest that, if their incorporation into seed storage oils can be achieved, the production
of these fatty acids in oil seed crops could become an economically viable proposition.
We have successfully reconstituted the D8-desaturation pathways for both ω6 and ω3 VLCPUFA biosynthesis
in a higher plant. This has been achieved by sequential transfer and expression of three genes in the model plant
A. thaliana. The accumulation of AA and EPA in substantial quantities is a significant breakthrough in the
search for alternative sustainable sources of these health promoting very long chain polyunsaturated fatty acids.
References
1. Crawford M. (2000) Placental delivery of arachidonic acid and docosahexaenoic acids: implication for the lipid
nutrition of preterm infants. Am. J. Clin. Nutr. 71: 275S-284S.
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2. Thies F et al. (2003) Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques: a
randomized controlled trial. The Lancet 361: 477-485.3. Qi B et al. (2004) Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nature Biotechnol. 22: 739-745.
4. Qi B et al. (2002) Identification of a cDNA encoding a novel C18-D9 polyunsaturated fatty acid-specific elongating
activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana. FEBS Lett. 510: 159-165.
5. Wallis JG and Browse J. (1999) The D8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20carbon poly-unsaturated fatty acids. Arch. Biochem. Biophys. 365: 307-316.
6. Michaelson LV et al. (1998) Isolation of a D5-fatty acid desaturase gene from Mortierella alpina. J. Biol. Chem. 273:
19055-19059.
7. Domergue F et al. (2003) Acyl carriers used as substrates by the desaturases and elongases involved in very longchain polyunsaturated fatty acids biosynthesis reconstituted in yeast. J. Biol. Chem. 278: 35115-35126.
Baoxiu Qi
School of Biology and Biochemistry
University of Bath, England, UK
[email protected] 1. GC profiles of Arabidopsis leaf fatty acid methyl esters extracted from (a) an untransformed plant; (b), a
single-transgenic plant expressing the I. galbana D9-elongating activity IgASE1; (c), a double transgenic plant expressing IgASE1 and the E.
gracilis D8-desaturase (EuD8); (d) a triple transgenic plant expressing IgASE1, EuD8 and the M. alpina D5-desaturase (MortD5). The peaks in
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