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Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge
Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Review
Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge Yan Chen a, Dauenpen Meesapyodsuk a,b, Xiao Qiu a,b,n a b
Department of Food & Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8 National Research Council of Canada, Saskatoon, SK, Canada S7N 0W9
art ic l e i nf o
a b s t r a c t
Article history: Received 12 July 2013 Received in revised form 18 August 2013 Accepted 25 August 2013
Omega-3 very long chain polyunsaturated fatty acids (VLC-PUFAs or VLCPUFAs) such as eicosapentaenoic acid (EPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have important roles in human health. The current source of these fatty acids is oil from marine fish and oleaginous microorganisms. However, sustainability of this source is questionable due to the declining fish population in ocean as well as the high cost associated with the microbial culturing and oil extraction. Transgenic plants producing a high level of VLCPUFAs have been proposed to be a potential alternative source for these fatty acids. Detections of EPA and DHA in transgenic plants expressing heterologous desaturases and elongases as well as a PUFA synthase from VLCPUFA-producing microorganisms have indeed optimistically proven the concept. However, the yield of VLCPUFAs in transgenics is still low and the desirable composition of these fatty acids is not achieved. This mini-review discusses what has been done on the reconstitution of VLCPUFA-biosynthetic pathways in transgenics and what kind of challenges and possible solutions could be in producing VLCPUFAs in plants. & 2013 Published by Elsevier Ltd.
Keywords: Transgenic plants Very long chain polyunsaturated fatty acids Docosahexaenoic acid Eicosapentaenoic acid Omega-3 fatty acid
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Biosynthesis of VLCPUFAs in microorganisms . . . . . . . . . . 3. Reconstitution of microbial VLCPUFA pathways in plants . 4. Improving production of VLCPUFAs in plants . . . . . . . . . . 5. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Very long chain polyunsaturated fatty acids (VLCPUFAs) are fatty acids with 20 or more carbons and two or more double bonds. They can be classified into two families, omega-6 (ω6) and omega-3 (ω3) fatty acids, according to the position of the last double bond towards the methyl end (Fig. 1) (Abbadi et al., 2004). Omega-3 VLCPUFAs are important structural components of membrane phospholipids and precursors to eicosanoids and docosanoids which are hormone-like
n Corresponding author at: University of Saskatchewan, Department of Food and Bioproduct Sciences, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8. Tel.: þ 1 306 966 2181; fax: þ1 306 966 8898. E-mail address: [email protected] (X. Qiu).
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1 2 3 4 5 5
bioactive compounds helping maintain cell homeostasis (Tapiero et al., 2002; Venegas-Calerón et al., 2010). It has also been reported that ω3 VLCPUFAs play very important roles in brain development and in the prevention of cardiovascular and immunological diseases (Benatti et al., 2004; Poudyal et al., 2011). Among ω3 VLCPUFAs, eicosapentaenoic acid (EPA, 20:5-Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6-Δ4,7,10,13,16,19) have attracted much more attention from researchers because of their appealing benefits to human health. EPA possesses anti-inflammatory and anticachectic properties and prevents many human ailments such as cardiovascular disease and cancer (Abeywardena and Patten, 2011; Babcock et al., 2000; Beck et al., 1991). DHA is another important ω3 VLCPUFA that is involved in development of the brain and retina where a high level of DHA is accumulated (Koletzko, 1992; Ruiz-López et al., 2012;
1878-8181/$ - see front matter & 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bcab.2013.08.007
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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SanGiovanni et al., 2000). This health benefit has been recognized for several decades. Clinical research has shown that DHA can decrease fatty acid synthesis in the liver, increase red blood cell membrane fluidity and reduce blood pressure (Ikeda et al., 1998). It has also been shown that DHA can protect neural cells from stressinduced apoptosis and possess anticancer properties (Gleissman et al., 2010). Although ω3 VLCPUFAs, especially EPA and DHA are clearly beneficial to human health, human beings cannot de novo synthesize these fatty acids due to the lack of two critical enzymes, D12 desaturase and D15 desaturase ( Ruiz-López et al., 2012). A D12 desaturase (D12 Des) introduces a double bond at the 12th position of oleic acid (OA, 18:1-D9) to form linoleic acid (LA, 18:2D9,12), while a D15 desaturase (D15 Des) inserts a double bond at the 15th position of LA to form α-linolenic acid (ALA, 18:3D9,12,15) (Fig. 2). Therefore, LA and ALA are defined as the dietary essential fatty acids (EFAs), which are precursors for the synthesis of VLCPUFAs. Due to a limited capacity to convert two EFAs to their corresponding VLCPUFAs in animals, humans are encouraged to take VLCPUFA supplements through diets (Ganapathy, 2009; Hornung et al., 2005). Certain marine microorganisms can synthesize
11
14
/n
5
8
1
COOH
6 Arachidonic acid (ARA), 20:4n-6 5,8,11,14; 17
11
14
5
8
1
COOH
3
Eicosapentaenoic acid (EPA), 20:5n-3, 5,8,11,14,17; 19
16
13
10
4
7
COOH
3 Docosahexaenoic acid (DHA), 22:6n-3, 4,7,10,13,16,19;
Fig. 1. Nomenclature of VLCPUFAs. D-designation, carbon numbering starts from the carboxyl group; ω/n-designation, carbon numbering starts from the methyl end. ω3 family, fatty acids with the last double bond located three carbons away from the methyl end; ω6 family, fatty acids with the last double bond located six carbons away from the methyl end.
VLCPUFAs de novo (Domergue et al., 2005b) and these fatty acids are eventually accumulated in fish through the aquatic food chain. Thus, fish oil becomes our primary dietary source of VLCPUFAs for human nutrition (Domergue et al., 2005a). However, this source of VCLPUFAs is not sustainable due to dramatic dwindling of wild fish stock in oceans and possible contamination of fish oil.
2. Biosynthesis of VLCPUFAs in microorganisms Various microorganisms such as fungus, marine protists and micro-algae can effectively synthesize and accumulate VLCPUFAs so that these microbes are termed as VLCPUFA-producing microorganisms. There are two main pathways to synthesize VLCPUFAs in these microorganisms. The first pathway involves alternating desaturation and elongation, which mainly occurs in some eukaryotic microorganisms. This pathway is also known as the aerobic pathway, as it requires molecular oxygen for the desaturation process. The second pathway involves polyketide synthase (PKS)like polyunsaturated fatty acid synthase (PUFA synthase), which is widely found in bacteria and some eukaryotic microorganisms (Metz et al., 2001; Qiu, 2003). This pathway is also known as the anaerobic pathway, as it does not require the aerobic desaturation process to introduce double bonds. The biosynthetic process of VLCPUFAs in the aerobic pathway can be simply depicted in Fig. 2. As shown in the figure, the biosynthesis of VLCPUFAs is accomplished with alternating reactions of desaturation, introducing double bonds, and elongation, condensing a malonyl-CoA and the fatty acyl moiety to lengthen two carbon units of pre-formed C18 fatty acids. In the conventional D6 pathway, an ω6 VLCPUFA ARA and an ω3 VLCPUFA EPA, are synthesized respectively by utilizing LA and ALA as precursor substrates. The conventional D6 pathway is catalyzed by a D6 desaturase (D6 Des) and D6 elongase (D6 Elo) while the branched D8 pathway is catalyzed by a D9 elongase (D9 Elo) and a D8 desaturase (D8 Des). Both pathways can use a D5 desaturase (D5 Des) to synthesize D5 desaturated fatty acids. Interestingly, all these enzymes in the pathways can use both ω6 (LA, EDA, GLA,
Oleic acid (OA, 18:1-
Linoleic Acid (LA, 18:2-
- Linolenic Acid (ALA, 18:3) Elo
Elo Eicosadienoic acid (EDA, 20:2Des
Gamma linoleic Acid (GLA, 18:3)
Stearidonic Acid (SDA, 18:4-
Elo Dihomo -g- Linoleic Acid (DGLA, 20:3-
ARA
)
Elo
Eicosatrienoic acid (ERA, 20:3Des
Eicosatepentaenoic Acid (ETA, 20:4)
EPA Elo Elo Tetracosapentaenoic acid (TPA, 24:5-
Docosapentaenoic Acid (DPA, 22:5perxisomal -oxidation DHA
Tetracosahexaenoic acid (THA, 24:6-
Fig. 2. The aerobic biosynthetic pathway of VLCPUFAs. Des, desaturase; Elo, elongase. The dashed arrows indicate branched pathways.
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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Table 1 The strategies used to produce VLCPUFAs in plants. Strategy
Reference
Plant species
Tissue
D8 alternative pathway
Qi et al. (2004) Abbadi et al. (2004) Abbadi et al. (2004)
Arabidopsis thaliana Nicotiana tabacum Linum usitatissimum
Leaves Seed Seed
Kinney et al. (2004) Wu et al. (2005) Ruiz-Lopez et al. (2009) Cheng et al. (2010) Petrie et al. (2012) Robert et al. (2005) Hoffmann et al. (2008) Petrie et al. (2010) Metz et al. (2006)
Glycine max Brassica juncea Linum usitatissimum Brassica carinata A. thaliana A. thaliana A. thaliana Nicotiana benthamiana A. thaliana
Embryo Seed Seed Seed Seed Seed Seed Leaves Seed
D6 conventional pathway
acyl-CoA desaturase PKS system
DGLA, ARA) and ω3 fatty acid substrates (ALA, ERA, SDA, ETA, EPA and DPA). Conversion of 18-carbon and 20-carbon ω6 fatty acids to their corresponding ω3 fatty acids is catalyzed by a D15 Des and D17 desaturase (D17 Des), respectively. Elongation of EPA is catalyzed by a D5 elongase (D5 Elo) and further D4 desaturation by a D4 desaturase (D4 Des). Therefore, ARA synthesis starts with a D6 desaturation of LA into γ-linolenic acid (GLA, 18:3), followed by a C2 elongation of GLA into dihomo γ-linolenic acid (DGLA, 20:3) and D5 desaturation of DGLA into ARA. Alternatively, ARA can also be synthesized from the D8 pathway with sequential enzymatic reactions catalyzed by D9 Elo, D8 Des and D5 Des. Similarly, ALA can be converted to EPA through D6 or D8 biosynthetic pathways, as in the ARA biosynthesis. In addition, EPA can also be synthesized from ARA by ω3 desaturase, also known as D17 Des. With the availability of EPA, DHA can be synthesized by two different ways aerobically in eukaryotes. In a D4 desaturation-dependent pathway, D5 Elo and D4 Des are required to convert EPA to DHA. This pathway generally occurs in certain eukaryotic VLCPUFAproducing microorganisms, such as Thraustochytrium (Qiu et al., 2001). However, in D4 desaturation-independent pathway in animals, DHA synthesis involves retro-conversion of a 24-carbon D6 fatty acid via a peroxisomal β-oxidation (Voss et al., 1991). In the pathway, elongation of DPA to TPA is catalyzed by a Δ5 Elo and then the desaturation catalyzed by the Δ6 Des. Conversion of THA to DHA occurs in the peroxisome through two-carbon chain shortening. It is noted that DHA can also be synthesized through the PUFA synthase pathway anaerobically, which has been reported in protist Schizochytrium sp. (Metz et al., 2001) and bacteria (Okuyama et al., 2007). The synthesis differs from the aerobic pathway in that it does not require the oxygen-dependent desaturation process to introduce double bonds along the acyl chain. Instead, double bonds are introduced during the process of fatty acid extension, as seen in the biosynthesis of unsaturated fatty acids in Escherichia coli. Similar to Type I PKS, the huge PUFA synthase (20–30 kb) (Venegas-Calerón et al., 2010) contains multiple catalytic domains and uses malonate as the substrate and acyl carrier protein (ACP) as the covalent attachment for the chain extension, proceeding with reiterative cycles. The full cycle of the biosynthesis also comprises four reactions: condensation of an acyl-ACP with a malonyl-ACP to produce a ketoacyl-ACP, ketoreduction to convert ketoacyl-ACP to hydroxyacyl-ACP, dehydration to remove a water molecule from hydroxylacyl-ACP resulting in an unsaturated enoyl-ACP, and reduction of enoyl-ACP to a saturated acyl chain. However, unlike Type I fatty acid synthesis, the synthesis of DHA by a PUFA synthase often omit the last step of the full cycle, as such, multiple double bonds can be retained in the acyl chain. Among various VLCPUFA-biosynthetic pathways mentioned above, the pathway involving alternation of desaturation and elongation has attracted much more attention of the scientific
25
Abbadi et al. (2004)
20
Wu et al. (2005) Cheng et al. (2010) Petrie et al. (2012)
15
Robert et al. (2005) Metz et al. (2006)
10
5
0 ARA
ETA
EPA
DPA
DHA
Fig. 3. The level of VLCPUFAs produced in transgenic plant seeds (%).
community because the genes encoding front-end desaturases and PUFA elongases are small and easier to clone and functionally characterize in a heterologous system compared to the large and complex PUFA synthase gene. Genes encoding front-end desaturases and PUFA elongases have been cloned from many different species (Napier, 2007). The desaturase usually contains a cytochrome b5 fusion domain at the N-terminus (Napier et al., 1999; Sperling and Heinz, 2001). The elongase is a condensing enzyme, one of four discrete subunits in the elongation complex consisting of so called acyl-CoA elongase, a ketoacyl-CoA reductase, a hydroxyl acyl-CoA dehydratase and an enoyl-CoA reductase (Leonard et al., 2004). Indirect evidence suggests that the rate-limiting step in the elongation reaction lies in activity of the condensing enzyme (elongase) rather than the other three enzymes. However, the detailed mechanism for the phenomenon still remains to be elucidated (Leonard et al., 2004). In terms of their substrate use, it is generally believed that the desaturase from animals prefers acyl-CoAs as substrates and the desaturase from fungus tends to use phosphatidylcholine (PC)-linked substrates, whereas, the elongase usually only use acyl-CoA as substrates, thus the elongation reaction occurs in the acyl-CoA pool.
3. Reconstitution of microbial VLCPUFA pathways in plants Although progress has been made in commercial production of ω3 VLCPUFAs through fermentation in some of the above mentioned microorganisms, attempts of producing these fatty acids through metabolic engineering of microbial pathways in oilseed crops to meet the market needs for VLCPUFAs have met with limited success (Table 1 and Fig. 3). The first “proof-of-concept” study was carried out in Arabidopsis thaliana via overexpression of three microbial enzymes (an Isochrysis galbana D9 Elo, an Euglena
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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gracilis D8 Des and a Mortierella alpina D5 Des), resulting in the accumulation of EPA and ARA to 3 and 6.6% of the total fatty acids in leaf tissues (Qi et al., 2004). The attempt to produce C20 VLCPUFAs in seed oil was more challenging when the D9 Elo/Δ8 Des pathway was constitutively expressed in Arabidopsis (Fraser et al., 2004). Consequently, the seed-specific expression of D5, D6 desaturases from diatom Phaeodactylum tricornutum and a D6 Elo from moss Physcomitrella patens in tobacco and linseed was undertaken, which resulted in an accumulation of EPA (1.6%) and ARA (2.7%) in the seed oil, as well as D6 desaturated C18 fatty acids such as GLA and SDA (totally 33%) (Abbadi et al., 2004). Using the similar approach, Kinney et al. (2004) reconstituted the EPA pathway in soybean somatic embryos using five different enzymes, a D6 Des, a D6 Elo and a D5 Des from M. alpina, a D15 Des from Arabidopsis and a D17 Des from Saprolegnia diclina. Transgenic somatic embryos accumulated 19.6% EPA. In addition, they also reconstituted the DHA pathway using an additional D4 Des from Schizochytrium aggregatum and D5 Elo from Pavlova salina. Transgenic somatic embryos produced a low level of DHA (2.0–3.3% of the total fatty acids). The entire DHA biosynthetic pathway was later reconstituted in oilseed crop Brassica juncea by stepwise metabolic engineering. Transgenic plants produced up to 25% ARA and 15% EPA, as well as up to 1.5% DHA in seeds (Wu et al., 2005). Another attempt to produce DHA in plants made by Robert et al. (2005) introduced four genes in Arabidopsis, which resulted in production of 0.2–0.5% of DHA in seeds. Recently, Kajikawa et al. (2008) co-expressed a D6 Des, a D6 Elo and a D5 Des from Marchantia polymorpha in tobacco, which resulted in an accumulation of 15.5% ARA and 4.9% EPA in transgenics. Hoffmann et al. (2008) reconstituted the EPA pathway in Arabidopsis using two acyl-CoA desaturases from Mantoniella squamata and an elongase from P. patens, however the yield of EPA in transgenic T2 seeds reached only 0.45% on overage. Cheng et al. (2010) co-expressed a 18C ω3 desaturase from Claviceps purpurea and a 20C ω3 desaturase from Pythium irregular along with a Δ6 Des, a Δ6 Elo and a Δ5 Elo in zeroerucic acid Brassica carinata, resulting in up to 25% of EPA in seeds. Petrie et al. introduced two yeast genes and five microalgal genes encoding desaturases/elongases involved in the DHA biosynthesis starting from oleic acid in Nicotiana benthamiana, resulting in DHA being accumulated up to 15.9% in the leaf triacylglycerols (TAGs). Up to 15% DHA level was produced in Arabidopsis seed oil when the construct was transformed into Arabidopsis (Petrie et al., 2012).
4. Improving production of VLCPUFAs in plants Although the biosynthesis of ω3 VLCPUFAs has been successfully reconstituted in plants using desaturases and elongases from microorganisms, the desirable level and composition of these fatty acids produced in oilseed crops, particularly for DHA, has not been achieved. Therefore, to improve the production of these fatty acids in plants, additional factors other than desaturases and elongases per se in the biosynthesis and accumulation of VLCPUFAs would deserve our deliberation when the microbial pathway is implemented in plants. Production of specialty fatty acids in the storage lipids involves many coordinated biochemical processes and each process involves many intricate enzymatic reactions. It has been shown that acyl-lipid metabolism in Arabidopsis involves more than 120 enzymatic reactions and more than 600 genes encoding the proteins and regulatory factors (Li-Beisson et al., 2010). For producing specialty fatty acid in TAGs of transgenic plants, it is essential to know the co-evolved enzymes involved in the biosynthesis and TAG assembly, as synthesis of VLCPUFAs and channelling of these fatty acids to TAGs engage many different enzymes which can be located in different organelles or in different domains of an organelle.
The first “bottleneck” in the biosynthesis of ω3 VLCPUFAs in plants is the “substrate dichotomy”, which derives from the difference of acyl-substrate requirement of two key enzymes, fatty acid desaturase and elongase in VLCPUFA biosynthesis (Napier et al., 2004; Napier, 2007). It is believed that most front-end desaturases prefer acyl-PC as their substrates, while elongases can only use acyl-CoAs as their substrates (Domergue et al., 2003). A detail metabolic analysis showed that after Δ6 desaturation on PC, Δ6 desaturated C18 fatty acids were preferentially channeled to TAGs rather than to the acyl-CoA pool for subsequent elongation. Thus, the absence of Δ6 desaturated acyl-CoA substrate for further elongation resulted in the limited synthesis of elongated C20 fatty acids (Abbadi et al., 2004). The inefficiency of acyl exchanges between PCs and acyl-CoA pool is a main factor responsible for the limited sequential elongation, which has a significant influence on the yield of VLCPUFAs. In transgenic oilseed plants with a reconstituted D6 pathway of VLCPUFAs, a high level of Δ6 desaturated products, GLA or SDA, were found in PCs or TAGs rather than in the acyl-CoA pool implying that the elongation step is limited due to absence of immediate substrates (Abbadi et al., 2004; Cheng et al., 2010; Domergue et al., 2003; Wu et al., 2005). On the other hand, the reconstitution of a Δ8 alternative pathway of VLCPUFAs showed that a significant amount of elongated C20 fatty acid products (20:2 and 20:3) were found in the acyl-CoA pool of transgenic Arabidopsis lines (Sayanova et al., 2006), while the PC form of substrates available for the subsequent desaturation might be limited. Utilization of acyl-CoA dependent desaturases is believed as one of effective approaches to overcome the so called “substrate dichotomy” bottleneck (Graham et al., 2007). Several front-end desaturases that can use acyl-CoA as substrate have thus been identified in microalgae (Domergue et al., 2005b; Hoffmann et al., 2008). Co-expression of an acyl-CoA D6 Des from Ostreococcus tauri with a D6 Elo and a D5 Des resulted in production of 4.5% of ARA and 4.7% of EPA in yeast, which was about 20 times higher than their previous experiments using a PC-preferred D6 Des (Domergue et al., 2005b). Co-expression of an acyl-CoA dependent D6 Des from Micromonas pusilla with D6 Elo and D5 Des resulted in accumulation of 26% of EPA in leaves of N. benthamiana (Petrie et al., 2010). However, a high level of VLCPUFAs in transgenic oilseeds using acyl-CoA dependent desaturase has not yet been reported. Lysophosphatidylcholine acyltransferase (LPCAT) has also been viewed as a potential target in helping overcome the problem, since it can not only catalyze the fatty acid incorporation into PC from acyl-CoA, but also remove fatty acids from the sn-2 position of PC and release them to the acyl-CoA pool in its reverse reaction (Furukawa-Stoffer, et al., 2003; Stymne and Stobart, 1984). However, so far there has been no experiment involving this enzyme in production of VLCPUFAs in plants. Another “bottleneck” in the VLCPUFA biosynthesis is the channelling of VLCPUFAs from the acyl-PC and acyl-CoA pools to TAGs. Ideally, all the forms of VLCPUFAs such as VLCPUFA-CoA and VLCPUFA-PC should be efficiently used for TAG synthesis. However, as a matter of fact, a certain amount of VLCPUFAs remains in PCs and other phospholipid classes in transgenic plants (Abbadi et al., 2004; Wu et al., 2005), suggesting that the VLCPUFA flux from phospholipids to TAGs is also limited for the VLCPUFA production. A possible solution to this might be the use of TAG biosynthetic enzymes such as glycerol-phosphate acyltranserase (GPAT), lysophosphatidic acid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) from VLCPUFA-producing microorganisms which have a substrate preference towards VLCPUFAs in transgenic plants where these fatty acids in the acyl-CoA form will be used by these enzymes to synthesize TAGs. An alternative solution is the use of acyl-CoA-independent enzyme such as phospholipid: diacylglycerol acyltransferase (PDAT) (Dahlqvist et al., 2000; Stahl et al., 2004) for mobilizing VLCPUFAs from PC to TAGs.
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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Choline
Ethanolamine
CK
P-Choline
EK
P-Ethanolamine
CT
G3P
GPAT
LPA
LPAT
PA
PAP
CDP-Choline
DAG DGAT
Acyl-CoA
PDCT /PLC
ET
CDP-Ethanolamine
CPT
PDAT
EPT
CMP
PC
PEMT
PE
5
et al., 1994; Vogel and Browse, 1996). However, a CPT (PiCPT1) recently cloned from Phytophthora infestans was confirmed to have substrate specificity towards VLCPUFAs (Chen et al., 2013). As the channeling of DAG to PC and then to TAG represents the major bottleneck in the production of unusual fatty acids in transgenic plants, PiCPT1 may play an important role in converting VLCPUFAcontaining DAG to PC during the assembly of VLCPUFAs in the oomycete.
(desaturation)
TAG LPCAT
Fig. 4. Interconnection of phospholipid and neutral lipid biosynthesis. TAG biosynthesis follows Kennedy pathway, starting from G3P. Biosyntheses of phospholipids, PC and PE, are catalyzed by three enzymes, CK/EK, CT/ET and CPT/EPT. G3P, glycerol-3-phosphate; LPA, lyso- phosphatidic acid; PA, phosphatidic acid; GPAT, acyl-CoA:glycerol-3-phosphate acyltransferase; LPAT, acyl-CoA: lyso-phosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase; PLC, phospholipase C; CK, Choline kinase; CT, CTP/phosphocholine cytidyltransferase; EK, ethanolamine kinase; ET, CTP/phosphoethanolamine cytidylyltransferase; PEMT, phosphatidylethanolamine methyl transferase; PDAT, phosphocholine diacylglycerol acyltransferase; LPCAT, lysophosphatidylcholine acyltransferase.
Recent metabolic labeling experiments demonstrated that the relative flux of de novo diacylglycerol (DAG) to PC is over 14 times the rate of the direct conversion from de novo DAG to TAG in wild type Arabidopsis and the major bottleneck for the accumulation of unusual fatty acids in TAGs is the step in the flux of DAG to PC (Bates and Browse, 2011; Bates et al., 2009). This result implies that factors involved in the flux of special fatty acids to TAGs through PC are very important in VLCPUFA accumulation in transgenic plants. In TAG synthesis, DGAT catalyzes the terminal and only committed step using DAG and fatty acyl CoA as substrates (Cases et al., 1998). Therefore, availability of VLCPUFADAGs and/or acyl-CoA form of VLCPUFAs is crucial for synthesizing VLCPUFA-TAGs. There are two main pathways to synthesize DAGs (Fig. 4). The first pathway, so called the Kennedy pathway, is for de novo synthesis of DAG, involves sequential actions of GPAT and LPAT to esterify fatty acids onto the sn-1 and sn-2 positions of glycerol-3 phosphate (G3P), producing lysophosphatidic acid (LPA), phosphatidyl acid (PA), respectively. Phosphate group of PA is removed by phosphatidic acid phosphatase (PAP) to produce de novo DAG. DAG can also be synthesized through PC by the removal of phosphocholine group on the sn-3 position of PC. The de novo PC synthesis involves the utilization of de novo DAG as substrate and catalytic reaction of cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT). Because PC is the site of fatty acid desaturation, the fatty acids in de novo DAG are different from those in PC-derived DAG produced either by the reverse action of CPT or phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) or phospholipase C (PLC) (Fig. 4). PDCT was first identified from Arabidopsis where the null mutant (AtROD1) reduces 18:2 and 18:3 accumulation in seed TAG by 40% due to the lack of interconversion between DAG and PC (Lu et al., 2009). CPT, on the other hand, has been long well known for its role in the de novo synthesis of PC (Kennedy and Weiss, 1956), however, contribution of this enzyme to the synthesis of TAGs with specialty fatty acids has not been appreciated until recently. As PC-derived DAG contributes mostly to oil synthesis, the fatty acid profile of PC is mostly reflective of that of TAGs. Thus, substrate selectivity of CPT could have a huge impact on the fatty acid composition of the final oil. Although the substrate preference of CPT for DAG species has been studied in several eukaryotes, no or little substrate preference towards DAG species for this enzyme has been observed (Henneberry et al., 2000, 2002; Hjelmstad
5. Concluding remarks The demand for ω3 VLCPUFAs such as EPA and DHA is increasing in the market place due to the public awareness of potential health benefits of these fatty acids. Traditional sources for VLCPUFAs are oceanic fish and oleaginous microbes. However, these sources are either unsustainable due to declining of the fish population in ocean or expensive due to the high cost in growing and extracting oil from VLCPUFA-producing microbes. Producing VLCPUFAs in plants by metabolic engineering is viewed as an attractive alternative which has recently drawn much attention from lipid research community and nutraceutical industries. Since plants are unable to de novo synthesize VLCPUFAs, a genetic engineering approach employed to produce these fatty acids in plants has to be built on our understanding of fatty acid and TAG biosynthesis in plants as well as in VLCPUFA-producing microorganisms. Numerous desaturases and elongases involved in the biosynthesis of VLCPUFAs have been cloned from microbial species and successfully introduced into oilseed plants to produce VLCPUFAs, however, the level and composition of the fatty acids in transgenics is not desirable for the viable commercialization. To improve the VLCPUFA production, limiting factors or bottlenecks in the metabolic pathway reconstituted in transgenic plants have to be thoroughly examined. As mentioned above, one of the main issues in reconstituting the VLCPUFA pathway in plant is how to increase VLCPUFA trafficking between PC, DAG and TAG. Commercial oilseed crops produce only a simple fatty acid profile in their oils and possess an enzymatic system for effective use of their own endogenous fatty acids, thus may lack effective mechanisms to channel heterologous VLCPUFAs to TAGs. PDAT, DGAT, PDCT and CPT are enzymes involved in the acyl flux among PC, DAG and TAG pools. Therefore, studying these enzymes from VLCPUFA-producing microbes is very important for us to understand the shuffling process between them during the biosynthesis and assembly of VLCPUFAs, thereby designing an effective strategy to improve the production of VLCPUFAs in transgenic plants. References Abbadi, A., Domergue, F., Bauer, J., Napier, J.A., Welti, R., Zahringer, U., Cirpus, P., Heinz, E., 2004. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell 16, 2734–2748. Abeywardena, M.Y., Patten, G.S., 2011. Role of n-3 long chain polyunsaturated fatty acids in reducing cardio-metabolic risk factors. Endocrine, Metabolic and Immune Disorders Drug Targets 11, 232–246. Babcock, T., Helton, W.S., Espat, N.J., 2000. Eicosapentaenoic acid (EPA): an antiinflammatory ω-3 fat with potential clinical applications. Nutrition, 16; , pp. 1116–1118. Bates, P.D., Browse, J., 2011. The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds. Plant Journal 68, 387–399. Bates, P.D., Durrett, T.P., Ohlrogge, J.B., Pollard, M., 2009. Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean embryos. Plant Physiology 150, 55–72. Beck, S.A., Smith, K.L., Tisdale, M.J., 1991. Anticachectic and antitumor effect of eicosapentaenoic acid and its effect on protein turnover. Cancer Research 51, 6089–6093. Benatti, P., Peluso, G., Nicolai, R., Calvani, M., 2004. Polyunsaturated fatty acids: biochemical, nutritional and epigenetic properties. Journal of the American College of Nutrition 23, 281–302.
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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Review
Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge Yan Chen a, Dauenpen Meesapyodsuk a,b, Xiao Qiu a,b,n a b
Department of Food & Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8 National Research Council of Canada, Saskatoon, SK, Canada S7N 0W9
art ic l e i nf o
a b s t r a c t
Article history: Received 12 July 2013 Received in revised form 18 August 2013 Accepted 25 August 2013
Omega-3 very long chain polyunsaturated fatty acids (VLC-PUFAs or VLCPUFAs) such as eicosapentaenoic acid (EPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have important roles in human health. The current source of these fatty acids is oil from marine fish and oleaginous microorganisms. However, sustainability of this source is questionable due to the declining fish population in ocean as well as the high cost associated with the microbial culturing and oil extraction. Transgenic plants producing a high level of VLCPUFAs have been proposed to be a potential alternative source for these fatty acids. Detections of EPA and DHA in transgenic plants expressing heterologous desaturases and elongases as well as a PUFA synthase from VLCPUFA-producing microorganisms have indeed optimistically proven the concept. However, the yield of VLCPUFAs in transgenics is still low and the desirable composition of these fatty acids is not achieved. This mini-review discusses what has been done on the reconstitution of VLCPUFA-biosynthetic pathways in transgenics and what kind of challenges and possible solutions could be in producing VLCPUFAs in plants. & 2013 Published by Elsevier Ltd.
Keywords: Transgenic plants Very long chain polyunsaturated fatty acids Docosahexaenoic acid Eicosapentaenoic acid Omega-3 fatty acid
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Biosynthesis of VLCPUFAs in microorganisms . . . . . . . . . . 3. Reconstitution of microbial VLCPUFA pathways in plants . 4. Improving production of VLCPUFAs in plants . . . . . . . . . . 5. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Very long chain polyunsaturated fatty acids (VLCPUFAs) are fatty acids with 20 or more carbons and two or more double bonds. They can be classified into two families, omega-6 (ω6) and omega-3 (ω3) fatty acids, according to the position of the last double bond towards the methyl end (Fig. 1) (Abbadi et al., 2004). Omega-3 VLCPUFAs are important structural components of membrane phospholipids and precursors to eicosanoids and docosanoids which are hormone-like
n Corresponding author at: University of Saskatchewan, Department of Food and Bioproduct Sciences, 51 Campus Drive, Saskatoon, SK, Canada S7N 5A8. Tel.: þ 1 306 966 2181; fax: þ1 306 966 8898. E-mail address: [email protected] (X. Qiu).
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bioactive compounds helping maintain cell homeostasis (Tapiero et al., 2002; Venegas-Calerón et al., 2010). It has also been reported that ω3 VLCPUFAs play very important roles in brain development and in the prevention of cardiovascular and immunological diseases (Benatti et al., 2004; Poudyal et al., 2011). Among ω3 VLCPUFAs, eicosapentaenoic acid (EPA, 20:5-Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6-Δ4,7,10,13,16,19) have attracted much more attention from researchers because of their appealing benefits to human health. EPA possesses anti-inflammatory and anticachectic properties and prevents many human ailments such as cardiovascular disease and cancer (Abeywardena and Patten, 2011; Babcock et al., 2000; Beck et al., 1991). DHA is another important ω3 VLCPUFA that is involved in development of the brain and retina where a high level of DHA is accumulated (Koletzko, 1992; Ruiz-López et al., 2012;
1878-8181/$ - see front matter & 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bcab.2013.08.007
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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SanGiovanni et al., 2000). This health benefit has been recognized for several decades. Clinical research has shown that DHA can decrease fatty acid synthesis in the liver, increase red blood cell membrane fluidity and reduce blood pressure (Ikeda et al., 1998). It has also been shown that DHA can protect neural cells from stressinduced apoptosis and possess anticancer properties (Gleissman et al., 2010). Although ω3 VLCPUFAs, especially EPA and DHA are clearly beneficial to human health, human beings cannot de novo synthesize these fatty acids due to the lack of two critical enzymes, D12 desaturase and D15 desaturase ( Ruiz-López et al., 2012). A D12 desaturase (D12 Des) introduces a double bond at the 12th position of oleic acid (OA, 18:1-D9) to form linoleic acid (LA, 18:2D9,12), while a D15 desaturase (D15 Des) inserts a double bond at the 15th position of LA to form α-linolenic acid (ALA, 18:3D9,12,15) (Fig. 2). Therefore, LA and ALA are defined as the dietary essential fatty acids (EFAs), which are precursors for the synthesis of VLCPUFAs. Due to a limited capacity to convert two EFAs to their corresponding VLCPUFAs in animals, humans are encouraged to take VLCPUFA supplements through diets (Ganapathy, 2009; Hornung et al., 2005). Certain marine microorganisms can synthesize
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8
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6 Arachidonic acid (ARA), 20:4n-6 5,8,11,14; 17
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Eicosapentaenoic acid (EPA), 20:5n-3, 5,8,11,14,17; 19
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3 Docosahexaenoic acid (DHA), 22:6n-3, 4,7,10,13,16,19;
Fig. 1. Nomenclature of VLCPUFAs. D-designation, carbon numbering starts from the carboxyl group; ω/n-designation, carbon numbering starts from the methyl end. ω3 family, fatty acids with the last double bond located three carbons away from the methyl end; ω6 family, fatty acids with the last double bond located six carbons away from the methyl end.
VLCPUFAs de novo (Domergue et al., 2005b) and these fatty acids are eventually accumulated in fish through the aquatic food chain. Thus, fish oil becomes our primary dietary source of VLCPUFAs for human nutrition (Domergue et al., 2005a). However, this source of VCLPUFAs is not sustainable due to dramatic dwindling of wild fish stock in oceans and possible contamination of fish oil.
2. Biosynthesis of VLCPUFAs in microorganisms Various microorganisms such as fungus, marine protists and micro-algae can effectively synthesize and accumulate VLCPUFAs so that these microbes are termed as VLCPUFA-producing microorganisms. There are two main pathways to synthesize VLCPUFAs in these microorganisms. The first pathway involves alternating desaturation and elongation, which mainly occurs in some eukaryotic microorganisms. This pathway is also known as the aerobic pathway, as it requires molecular oxygen for the desaturation process. The second pathway involves polyketide synthase (PKS)like polyunsaturated fatty acid synthase (PUFA synthase), which is widely found in bacteria and some eukaryotic microorganisms (Metz et al., 2001; Qiu, 2003). This pathway is also known as the anaerobic pathway, as it does not require the aerobic desaturation process to introduce double bonds. The biosynthetic process of VLCPUFAs in the aerobic pathway can be simply depicted in Fig. 2. As shown in the figure, the biosynthesis of VLCPUFAs is accomplished with alternating reactions of desaturation, introducing double bonds, and elongation, condensing a malonyl-CoA and the fatty acyl moiety to lengthen two carbon units of pre-formed C18 fatty acids. In the conventional D6 pathway, an ω6 VLCPUFA ARA and an ω3 VLCPUFA EPA, are synthesized respectively by utilizing LA and ALA as precursor substrates. The conventional D6 pathway is catalyzed by a D6 desaturase (D6 Des) and D6 elongase (D6 Elo) while the branched D8 pathway is catalyzed by a D9 elongase (D9 Elo) and a D8 desaturase (D8 Des). Both pathways can use a D5 desaturase (D5 Des) to synthesize D5 desaturated fatty acids. Interestingly, all these enzymes in the pathways can use both ω6 (LA, EDA, GLA,
Oleic acid (OA, 18:1-
Linoleic Acid (LA, 18:2-
- Linolenic Acid (ALA, 18:3) Elo
Elo Eicosadienoic acid (EDA, 20:2Des
Gamma linoleic Acid (GLA, 18:3)
Stearidonic Acid (SDA, 18:4-
Elo Dihomo -g- Linoleic Acid (DGLA, 20:3-
ARA
)
Elo
Eicosatrienoic acid (ERA, 20:3Des
Eicosatepentaenoic Acid (ETA, 20:4)
EPA Elo Elo Tetracosapentaenoic acid (TPA, 24:5-
Docosapentaenoic Acid (DPA, 22:5perxisomal -oxidation DHA
Tetracosahexaenoic acid (THA, 24:6-
Fig. 2. The aerobic biosynthetic pathway of VLCPUFAs. Des, desaturase; Elo, elongase. The dashed arrows indicate branched pathways.
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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3
Table 1 The strategies used to produce VLCPUFAs in plants. Strategy
Reference
Plant species
Tissue
D8 alternative pathway
Qi et al. (2004) Abbadi et al. (2004) Abbadi et al. (2004)
Arabidopsis thaliana Nicotiana tabacum Linum usitatissimum
Leaves Seed Seed
Kinney et al. (2004) Wu et al. (2005) Ruiz-Lopez et al. (2009) Cheng et al. (2010) Petrie et al. (2012) Robert et al. (2005) Hoffmann et al. (2008) Petrie et al. (2010) Metz et al. (2006)
Glycine max Brassica juncea Linum usitatissimum Brassica carinata A. thaliana A. thaliana A. thaliana Nicotiana benthamiana A. thaliana
Embryo Seed Seed Seed Seed Seed Seed Leaves Seed
D6 conventional pathway
acyl-CoA desaturase PKS system
DGLA, ARA) and ω3 fatty acid substrates (ALA, ERA, SDA, ETA, EPA and DPA). Conversion of 18-carbon and 20-carbon ω6 fatty acids to their corresponding ω3 fatty acids is catalyzed by a D15 Des and D17 desaturase (D17 Des), respectively. Elongation of EPA is catalyzed by a D5 elongase (D5 Elo) and further D4 desaturation by a D4 desaturase (D4 Des). Therefore, ARA synthesis starts with a D6 desaturation of LA into γ-linolenic acid (GLA, 18:3), followed by a C2 elongation of GLA into dihomo γ-linolenic acid (DGLA, 20:3) and D5 desaturation of DGLA into ARA. Alternatively, ARA can also be synthesized from the D8 pathway with sequential enzymatic reactions catalyzed by D9 Elo, D8 Des and D5 Des. Similarly, ALA can be converted to EPA through D6 or D8 biosynthetic pathways, as in the ARA biosynthesis. In addition, EPA can also be synthesized from ARA by ω3 desaturase, also known as D17 Des. With the availability of EPA, DHA can be synthesized by two different ways aerobically in eukaryotes. In a D4 desaturation-dependent pathway, D5 Elo and D4 Des are required to convert EPA to DHA. This pathway generally occurs in certain eukaryotic VLCPUFAproducing microorganisms, such as Thraustochytrium (Qiu et al., 2001). However, in D4 desaturation-independent pathway in animals, DHA synthesis involves retro-conversion of a 24-carbon D6 fatty acid via a peroxisomal β-oxidation (Voss et al., 1991). In the pathway, elongation of DPA to TPA is catalyzed by a Δ5 Elo and then the desaturation catalyzed by the Δ6 Des. Conversion of THA to DHA occurs in the peroxisome through two-carbon chain shortening. It is noted that DHA can also be synthesized through the PUFA synthase pathway anaerobically, which has been reported in protist Schizochytrium sp. (Metz et al., 2001) and bacteria (Okuyama et al., 2007). The synthesis differs from the aerobic pathway in that it does not require the oxygen-dependent desaturation process to introduce double bonds along the acyl chain. Instead, double bonds are introduced during the process of fatty acid extension, as seen in the biosynthesis of unsaturated fatty acids in Escherichia coli. Similar to Type I PKS, the huge PUFA synthase (20–30 kb) (Venegas-Calerón et al., 2010) contains multiple catalytic domains and uses malonate as the substrate and acyl carrier protein (ACP) as the covalent attachment for the chain extension, proceeding with reiterative cycles. The full cycle of the biosynthesis also comprises four reactions: condensation of an acyl-ACP with a malonyl-ACP to produce a ketoacyl-ACP, ketoreduction to convert ketoacyl-ACP to hydroxyacyl-ACP, dehydration to remove a water molecule from hydroxylacyl-ACP resulting in an unsaturated enoyl-ACP, and reduction of enoyl-ACP to a saturated acyl chain. However, unlike Type I fatty acid synthesis, the synthesis of DHA by a PUFA synthase often omit the last step of the full cycle, as such, multiple double bonds can be retained in the acyl chain. Among various VLCPUFA-biosynthetic pathways mentioned above, the pathway involving alternation of desaturation and elongation has attracted much more attention of the scientific
25
Abbadi et al. (2004)
20
Wu et al. (2005) Cheng et al. (2010) Petrie et al. (2012)
15
Robert et al. (2005) Metz et al. (2006)
10
5
0 ARA
ETA
EPA
DPA
DHA
Fig. 3. The level of VLCPUFAs produced in transgenic plant seeds (%).
community because the genes encoding front-end desaturases and PUFA elongases are small and easier to clone and functionally characterize in a heterologous system compared to the large and complex PUFA synthase gene. Genes encoding front-end desaturases and PUFA elongases have been cloned from many different species (Napier, 2007). The desaturase usually contains a cytochrome b5 fusion domain at the N-terminus (Napier et al., 1999; Sperling and Heinz, 2001). The elongase is a condensing enzyme, one of four discrete subunits in the elongation complex consisting of so called acyl-CoA elongase, a ketoacyl-CoA reductase, a hydroxyl acyl-CoA dehydratase and an enoyl-CoA reductase (Leonard et al., 2004). Indirect evidence suggests that the rate-limiting step in the elongation reaction lies in activity of the condensing enzyme (elongase) rather than the other three enzymes. However, the detailed mechanism for the phenomenon still remains to be elucidated (Leonard et al., 2004). In terms of their substrate use, it is generally believed that the desaturase from animals prefers acyl-CoAs as substrates and the desaturase from fungus tends to use phosphatidylcholine (PC)-linked substrates, whereas, the elongase usually only use acyl-CoA as substrates, thus the elongation reaction occurs in the acyl-CoA pool.
3. Reconstitution of microbial VLCPUFA pathways in plants Although progress has been made in commercial production of ω3 VLCPUFAs through fermentation in some of the above mentioned microorganisms, attempts of producing these fatty acids through metabolic engineering of microbial pathways in oilseed crops to meet the market needs for VLCPUFAs have met with limited success (Table 1 and Fig. 3). The first “proof-of-concept” study was carried out in Arabidopsis thaliana via overexpression of three microbial enzymes (an Isochrysis galbana D9 Elo, an Euglena
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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gracilis D8 Des and a Mortierella alpina D5 Des), resulting in the accumulation of EPA and ARA to 3 and 6.6% of the total fatty acids in leaf tissues (Qi et al., 2004). The attempt to produce C20 VLCPUFAs in seed oil was more challenging when the D9 Elo/Δ8 Des pathway was constitutively expressed in Arabidopsis (Fraser et al., 2004). Consequently, the seed-specific expression of D5, D6 desaturases from diatom Phaeodactylum tricornutum and a D6 Elo from moss Physcomitrella patens in tobacco and linseed was undertaken, which resulted in an accumulation of EPA (1.6%) and ARA (2.7%) in the seed oil, as well as D6 desaturated C18 fatty acids such as GLA and SDA (totally 33%) (Abbadi et al., 2004). Using the similar approach, Kinney et al. (2004) reconstituted the EPA pathway in soybean somatic embryos using five different enzymes, a D6 Des, a D6 Elo and a D5 Des from M. alpina, a D15 Des from Arabidopsis and a D17 Des from Saprolegnia diclina. Transgenic somatic embryos accumulated 19.6% EPA. In addition, they also reconstituted the DHA pathway using an additional D4 Des from Schizochytrium aggregatum and D5 Elo from Pavlova salina. Transgenic somatic embryos produced a low level of DHA (2.0–3.3% of the total fatty acids). The entire DHA biosynthetic pathway was later reconstituted in oilseed crop Brassica juncea by stepwise metabolic engineering. Transgenic plants produced up to 25% ARA and 15% EPA, as well as up to 1.5% DHA in seeds (Wu et al., 2005). Another attempt to produce DHA in plants made by Robert et al. (2005) introduced four genes in Arabidopsis, which resulted in production of 0.2–0.5% of DHA in seeds. Recently, Kajikawa et al. (2008) co-expressed a D6 Des, a D6 Elo and a D5 Des from Marchantia polymorpha in tobacco, which resulted in an accumulation of 15.5% ARA and 4.9% EPA in transgenics. Hoffmann et al. (2008) reconstituted the EPA pathway in Arabidopsis using two acyl-CoA desaturases from Mantoniella squamata and an elongase from P. patens, however the yield of EPA in transgenic T2 seeds reached only 0.45% on overage. Cheng et al. (2010) co-expressed a 18C ω3 desaturase from Claviceps purpurea and a 20C ω3 desaturase from Pythium irregular along with a Δ6 Des, a Δ6 Elo and a Δ5 Elo in zeroerucic acid Brassica carinata, resulting in up to 25% of EPA in seeds. Petrie et al. introduced two yeast genes and five microalgal genes encoding desaturases/elongases involved in the DHA biosynthesis starting from oleic acid in Nicotiana benthamiana, resulting in DHA being accumulated up to 15.9% in the leaf triacylglycerols (TAGs). Up to 15% DHA level was produced in Arabidopsis seed oil when the construct was transformed into Arabidopsis (Petrie et al., 2012).
4. Improving production of VLCPUFAs in plants Although the biosynthesis of ω3 VLCPUFAs has been successfully reconstituted in plants using desaturases and elongases from microorganisms, the desirable level and composition of these fatty acids produced in oilseed crops, particularly for DHA, has not been achieved. Therefore, to improve the production of these fatty acids in plants, additional factors other than desaturases and elongases per se in the biosynthesis and accumulation of VLCPUFAs would deserve our deliberation when the microbial pathway is implemented in plants. Production of specialty fatty acids in the storage lipids involves many coordinated biochemical processes and each process involves many intricate enzymatic reactions. It has been shown that acyl-lipid metabolism in Arabidopsis involves more than 120 enzymatic reactions and more than 600 genes encoding the proteins and regulatory factors (Li-Beisson et al., 2010). For producing specialty fatty acid in TAGs of transgenic plants, it is essential to know the co-evolved enzymes involved in the biosynthesis and TAG assembly, as synthesis of VLCPUFAs and channelling of these fatty acids to TAGs engage many different enzymes which can be located in different organelles or in different domains of an organelle.
The first “bottleneck” in the biosynthesis of ω3 VLCPUFAs in plants is the “substrate dichotomy”, which derives from the difference of acyl-substrate requirement of two key enzymes, fatty acid desaturase and elongase in VLCPUFA biosynthesis (Napier et al., 2004; Napier, 2007). It is believed that most front-end desaturases prefer acyl-PC as their substrates, while elongases can only use acyl-CoAs as their substrates (Domergue et al., 2003). A detail metabolic analysis showed that after Δ6 desaturation on PC, Δ6 desaturated C18 fatty acids were preferentially channeled to TAGs rather than to the acyl-CoA pool for subsequent elongation. Thus, the absence of Δ6 desaturated acyl-CoA substrate for further elongation resulted in the limited synthesis of elongated C20 fatty acids (Abbadi et al., 2004). The inefficiency of acyl exchanges between PCs and acyl-CoA pool is a main factor responsible for the limited sequential elongation, which has a significant influence on the yield of VLCPUFAs. In transgenic oilseed plants with a reconstituted D6 pathway of VLCPUFAs, a high level of Δ6 desaturated products, GLA or SDA, were found in PCs or TAGs rather than in the acyl-CoA pool implying that the elongation step is limited due to absence of immediate substrates (Abbadi et al., 2004; Cheng et al., 2010; Domergue et al., 2003; Wu et al., 2005). On the other hand, the reconstitution of a Δ8 alternative pathway of VLCPUFAs showed that a significant amount of elongated C20 fatty acid products (20:2 and 20:3) were found in the acyl-CoA pool of transgenic Arabidopsis lines (Sayanova et al., 2006), while the PC form of substrates available for the subsequent desaturation might be limited. Utilization of acyl-CoA dependent desaturases is believed as one of effective approaches to overcome the so called “substrate dichotomy” bottleneck (Graham et al., 2007). Several front-end desaturases that can use acyl-CoA as substrate have thus been identified in microalgae (Domergue et al., 2005b; Hoffmann et al., 2008). Co-expression of an acyl-CoA D6 Des from Ostreococcus tauri with a D6 Elo and a D5 Des resulted in production of 4.5% of ARA and 4.7% of EPA in yeast, which was about 20 times higher than their previous experiments using a PC-preferred D6 Des (Domergue et al., 2005b). Co-expression of an acyl-CoA dependent D6 Des from Micromonas pusilla with D6 Elo and D5 Des resulted in accumulation of 26% of EPA in leaves of N. benthamiana (Petrie et al., 2010). However, a high level of VLCPUFAs in transgenic oilseeds using acyl-CoA dependent desaturase has not yet been reported. Lysophosphatidylcholine acyltransferase (LPCAT) has also been viewed as a potential target in helping overcome the problem, since it can not only catalyze the fatty acid incorporation into PC from acyl-CoA, but also remove fatty acids from the sn-2 position of PC and release them to the acyl-CoA pool in its reverse reaction (Furukawa-Stoffer, et al., 2003; Stymne and Stobart, 1984). However, so far there has been no experiment involving this enzyme in production of VLCPUFAs in plants. Another “bottleneck” in the VLCPUFA biosynthesis is the channelling of VLCPUFAs from the acyl-PC and acyl-CoA pools to TAGs. Ideally, all the forms of VLCPUFAs such as VLCPUFA-CoA and VLCPUFA-PC should be efficiently used for TAG synthesis. However, as a matter of fact, a certain amount of VLCPUFAs remains in PCs and other phospholipid classes in transgenic plants (Abbadi et al., 2004; Wu et al., 2005), suggesting that the VLCPUFA flux from phospholipids to TAGs is also limited for the VLCPUFA production. A possible solution to this might be the use of TAG biosynthetic enzymes such as glycerol-phosphate acyltranserase (GPAT), lysophosphatidic acid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) from VLCPUFA-producing microorganisms which have a substrate preference towards VLCPUFAs in transgenic plants where these fatty acids in the acyl-CoA form will be used by these enzymes to synthesize TAGs. An alternative solution is the use of acyl-CoA-independent enzyme such as phospholipid: diacylglycerol acyltransferase (PDAT) (Dahlqvist et al., 2000; Stahl et al., 2004) for mobilizing VLCPUFAs from PC to TAGs.
Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i
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Choline
Ethanolamine
CK
P-Choline
EK
P-Ethanolamine
CT
G3P
GPAT
LPA
LPAT
PA
PAP
CDP-Choline
DAG DGAT
Acyl-CoA
PDCT /PLC
ET
CDP-Ethanolamine
CPT
PDAT
EPT
CMP
PC
PEMT
PE
5
et al., 1994; Vogel and Browse, 1996). However, a CPT (PiCPT1) recently cloned from Phytophthora infestans was confirmed to have substrate specificity towards VLCPUFAs (Chen et al., 2013). As the channeling of DAG to PC and then to TAG represents the major bottleneck in the production of unusual fatty acids in transgenic plants, PiCPT1 may play an important role in converting VLCPUFAcontaining DAG to PC during the assembly of VLCPUFAs in the oomycete.
(desaturation)
TAG LPCAT
Fig. 4. Interconnection of phospholipid and neutral lipid biosynthesis. TAG biosynthesis follows Kennedy pathway, starting from G3P. Biosyntheses of phospholipids, PC and PE, are catalyzed by three enzymes, CK/EK, CT/ET and CPT/EPT. G3P, glycerol-3-phosphate; LPA, lyso- phosphatidic acid; PA, phosphatidic acid; GPAT, acyl-CoA:glycerol-3-phosphate acyltransferase; LPAT, acyl-CoA: lyso-phosphatidic acid acyltransferase; PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase; PLC, phospholipase C; CK, Choline kinase; CT, CTP/phosphocholine cytidyltransferase; EK, ethanolamine kinase; ET, CTP/phosphoethanolamine cytidylyltransferase; PEMT, phosphatidylethanolamine methyl transferase; PDAT, phosphocholine diacylglycerol acyltransferase; LPCAT, lysophosphatidylcholine acyltransferase.
Recent metabolic labeling experiments demonstrated that the relative flux of de novo diacylglycerol (DAG) to PC is over 14 times the rate of the direct conversion from de novo DAG to TAG in wild type Arabidopsis and the major bottleneck for the accumulation of unusual fatty acids in TAGs is the step in the flux of DAG to PC (Bates and Browse, 2011; Bates et al., 2009). This result implies that factors involved in the flux of special fatty acids to TAGs through PC are very important in VLCPUFA accumulation in transgenic plants. In TAG synthesis, DGAT catalyzes the terminal and only committed step using DAG and fatty acyl CoA as substrates (Cases et al., 1998). Therefore, availability of VLCPUFADAGs and/or acyl-CoA form of VLCPUFAs is crucial for synthesizing VLCPUFA-TAGs. There are two main pathways to synthesize DAGs (Fig. 4). The first pathway, so called the Kennedy pathway, is for de novo synthesis of DAG, involves sequential actions of GPAT and LPAT to esterify fatty acids onto the sn-1 and sn-2 positions of glycerol-3 phosphate (G3P), producing lysophosphatidic acid (LPA), phosphatidyl acid (PA), respectively. Phosphate group of PA is removed by phosphatidic acid phosphatase (PAP) to produce de novo DAG. DAG can also be synthesized through PC by the removal of phosphocholine group on the sn-3 position of PC. The de novo PC synthesis involves the utilization of de novo DAG as substrate and catalytic reaction of cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT). Because PC is the site of fatty acid desaturation, the fatty acids in de novo DAG are different from those in PC-derived DAG produced either by the reverse action of CPT or phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) or phospholipase C (PLC) (Fig. 4). PDCT was first identified from Arabidopsis where the null mutant (AtROD1) reduces 18:2 and 18:3 accumulation in seed TAG by 40% due to the lack of interconversion between DAG and PC (Lu et al., 2009). CPT, on the other hand, has been long well known for its role in the de novo synthesis of PC (Kennedy and Weiss, 1956), however, contribution of this enzyme to the synthesis of TAGs with specialty fatty acids has not been appreciated until recently. As PC-derived DAG contributes mostly to oil synthesis, the fatty acid profile of PC is mostly reflective of that of TAGs. Thus, substrate selectivity of CPT could have a huge impact on the fatty acid composition of the final oil. Although the substrate preference of CPT for DAG species has been studied in several eukaryotes, no or little substrate preference towards DAG species for this enzyme has been observed (Henneberry et al., 2000, 2002; Hjelmstad
5. Concluding remarks The demand for ω3 VLCPUFAs such as EPA and DHA is increasing in the market place due to the public awareness of potential health benefits of these fatty acids. Traditional sources for VLCPUFAs are oceanic fish and oleaginous microbes. However, these sources are either unsustainable due to declining of the fish population in ocean or expensive due to the high cost in growing and extracting oil from VLCPUFA-producing microbes. Producing VLCPUFAs in plants by metabolic engineering is viewed as an attractive alternative which has recently drawn much attention from lipid research community and nutraceutical industries. Since plants are unable to de novo synthesize VLCPUFAs, a genetic engineering approach employed to produce these fatty acids in plants has to be built on our understanding of fatty acid and TAG biosynthesis in plants as well as in VLCPUFA-producing microorganisms. Numerous desaturases and elongases involved in the biosynthesis of VLCPUFAs have been cloned from microbial species and successfully introduced into oilseed plants to produce VLCPUFAs, however, the level and composition of the fatty acids in transgenics is not desirable for the viable commercialization. To improve the VLCPUFA production, limiting factors or bottlenecks in the metabolic pathway reconstituted in transgenic plants have to be thoroughly examined. As mentioned above, one of the main issues in reconstituting the VLCPUFA pathway in plant is how to increase VLCPUFA trafficking between PC, DAG and TAG. Commercial oilseed crops produce only a simple fatty acid profile in their oils and possess an enzymatic system for effective use of their own endogenous fatty acids, thus may lack effective mechanisms to channel heterologous VLCPUFAs to TAGs. PDAT, DGAT, PDCT and CPT are enzymes involved in the acyl flux among PC, DAG and TAG pools. Therefore, studying these enzymes from VLCPUFA-producing microbes is very important for us to understand the shuffling process between them during the biosynthesis and assembly of VLCPUFAs, thereby designing an effective strategy to improve the production of VLCPUFAs in transgenic plants. References Abbadi, A., Domergue, F., Bauer, J., Napier, J.A., Welti, R., Zahringer, U., Cirpus, P., Heinz, E., 2004. Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell 16, 2734–2748. Abeywardena, M.Y., Patten, G.S., 2011. Role of n-3 long chain polyunsaturated fatty acids in reducing cardio-metabolic risk factors. Endocrine, Metabolic and Immune Disorders Drug Targets 11, 232–246. Babcock, T., Helton, W.S., Espat, N.J., 2000. Eicosapentaenoic acid (EPA): an antiinflammatory ω-3 fat with potential clinical applications. Nutrition, 16; , pp. 1116–1118. Bates, P.D., Browse, J., 2011. The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds. Plant Journal 68, 387–399. Bates, P.D., Durrett, T.P., Ohlrogge, J.B., Pollard, M., 2009. Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean embryos. Plant Physiology 150, 55–72. Beck, S.A., Smith, K.L., Tisdale, M.J., 1991. Anticachectic and antitumor effect of eicosapentaenoic acid and its effect on protein turnover. Cancer Research 51, 6089–6093. Benatti, P., Peluso, G., Nicolai, R., Calvani, M., 2004. Polyunsaturated fatty acids: biochemical, nutritional and epigenetic properties. Journal of the American College of Nutrition 23, 281–302.
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Please cite this article as: Chen, Y., et al., Transgenic production of omega-3 very long chain polyunsaturated fatty acids in plants: Accomplishment and challenge. Biocatal. Agric. Biotechnol. (2013), http://dx.doi.org/10.1016/j.bcab.2013.08.007i