Enzymes for transgenic biosynthesis of long-chain polyunsaturated fatty acids

Biochimie 86 (2004) 793–798 www.elsevier.com/locate/biochi

Enzymes for transgenic biosynthesis of long-chain polyunsaturated fatty acids Yung-Sheng Huang *, Suzette L. Pereira, Amanda E. Leonard Strategic Research, Ross Products Division, Abbott Laboratories, 625 Cleveland Avenue, Columbus, OH 43215, USA Received 26 August 2004; accepted 27 September 2004 Available online 19 October 2004

Abstract Polyunsaturated fatty acids (PUFAs) are important for the normal development and function of all organisms, and are essential in maintaining human health. Impaired PUFA metabolism is thought to be associated with pathogenesis of many chronic diseases. Dietary supplementation of PUFAs, such as c-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which bypass the defective or dysfunctional steps of the biosynthetic pathway has been found to significantly alleviate the symptoms of the disease. These findings have drawn a great deal of interest from general public and food manufacturers. As the demand of these beneficial PUFAs has drastically increased in recent years, there are also increasing efforts in finding the alternate sources of PUFAs that are more economical and sustainable. One option is to modify the oil-seed crops to produce PUFAs through genetic engineering technique. This review examines the isolation, identification and expression of genes encoding the enzymes required for the biosynthesis of the above mentioned PUFAs in plants. © 2004 Published by Elsevier SAS. Keywords: Long-chain polyunsaturated fatty acid; Enzyme; Transgenic; Biosynthesis

1. Introduction Polyunsaturated fatty acids (PUFAs) are fatty acids of 18 carbons or more in length with two or more methyleneinterrupted double bonds in the cis position. PUFAs can be grouped into two main families, x6 (or n-6) and x3 (or n-3) families, depending on the position of the first double bond proximate to the methyl end of fatty acids. In mammals, PUFAs are important structural components that modulate membrane fluidity and permeability [1]. For example, docosahexaenoic acid (DHA, C22:6n-3), a long-chain n-3 PUFA, and arachidonic acid (ARA, C20:4n-6), a long-chain n-6 PUFA, are found in high proportions in neuronal tissues such as brain and retina, and testis [2,3]. PUFAs also serve as precursors for a number of biologically active molecules, such as eicosanoids, growth regulators and hormones [4]. Thus, PUFAs have profound effects on human health [5]. The x6- and x3-PUFAs are derived from linoleic acid (LA, C18:2n-6) and a-linolenic acid (ALA, C18:3n-3), respectively. Human are incapable of synthesizing these two fatty * Corresponding author. Tel.: +1-614-624-3501; fax: +1-614-624-0008. E-mail address: [email protected] (Y.-S. Huang). 0300-9084/$ - see front matter © 2004 Published by Elsevier SAS. doi:10.1016/j.biochi.2004.09.019

acids due to lack of the D12 and D15-desaturases. However, humans can metabolize these two fatty acids obtained from the diet to form longer and more unsaturated PUFAs through a series of desaturation and elongation steps (Fig. 1) [6]. Normally, most of dietary LA and ALA in healthy subjects, are b-oxidized to provide energy and only a small portion of them (3.0% and 1.5%, respectively) are converted to longer PUFAs to meet the metabolic needs [7]. Often, this process of PUFA formation is further hampered

Fig. 1. Biosynthesis of long-chain PUFAs.

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by certain components in diet, hormonal imbalance [8] and the presence of chronic diseases, such as cancer and diabetes [9–14]. Indeed, deficiency of long-chain PUFAs is often seen in patients with chronic diseases, such as diabetes, hypercholesterolemia, rheumatoid arthritis, autoimmune disorders, Crohn’s disease, and cancer [15,16]. Increasing clinical evidence has shown that dietary supplementation of PUFAs, such as c-linolenic acid (GLA, C18:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and DHA (C22:6n-3) can provide beneficial effects and alleviate many of the symptoms associated with chronic diseases [17–22]. Inclusion of ARA and DHA in infant formula has also been shown to be beneficial to the growth and visual development in pre-term infants [23–26]. These findings have prompted the use of these long-chain PUFAs as supplements in the past few years.

2. Source of commercial PUFAs Currently, the richest sources of x3-PUFAs, such as EPA and DHA are oils extracted from marine fish such as mackerel, herring, salmon, and sardines. These fish obtain their long-chain PUFAs (LC-PUFAs) by consuming the LCPUFA-rich microalage and phytoplankton. Commercially, fish oils are available in the form of gelatin capsules or oily preparations, which contain 20% to 30% EPA and DHA [27]. EPA and DHA oils can also be obtained from single cell organisms like microalgae and fungi. Diatoms such as Nitzschia, are good producers of EPA, and dinoflagellates such as Crypthecodinium cohnii are used for commercial production of DHA [28]. Marine protists such as the Thraustochytrids can also make large amounts of DHA and are a potential source of DHA for human consumption [28,29]. x6-PUFAs such as GLA can be found in plant oils derived from borage (23%), evening primrose (9%), and black currant (16%) [30]. ARA is commercially produced by fermentation of oleagenous fungi such as Mortierella alpina, which contains approximately 40% (by wt.) of ARA in their oils [31,32]. Generally, the cost of production of the above mentioned commercial oils is very high. For example, the increasing cost involved in processing, refining, and stabilizing the fish oils, and the decreased yields due to over-fishing, have continuously driven up the cost of fish oils [33]. The recent finding of toxic chemicals in fish oil has further raised the safety concern on consumption of fish oil [34]. With respect to the x6-rich plant seed oils, the costs of production are also high. In addition, the cost associated with fermentation and extraction of oils from single cell sources is extremely high. Although strain-improvement and cultivation-optimization may raise the levels of the desired fatty acids in the oils, these processes are time-consuming and labor-demanding. The increasing demand has raised the interest in obtaining these PUFAs from alternate sources that are more economical and sustainable. One attractive option is to genetically

engineer the oil-seed crops like soybean, canola, and others to produce long-chain PUFAs such as GLA, ARA, EPA, and DHA. During the past few years, genes encoding the enzymes involved in biosynthesis of these LC-PUFAs, such as desaturases and elongases have been successfully isolated from LC-PUFA-rich organisms, and transgenically expressed in oil-seed crops. The present review will focus on the transgenic expression of non-mammalian sources of LCPUFA biosynthetic genes in crop plants for the production of PUFA-enriched transgenic seed oils. 2.1. Transgenic production of GLA GLA-enriched oils from borage, evening primrose, and black currant [35] are expensive due to high costs of cultivation, seed harvesting and oil extraction. There are on-going efforts to produce GLA in an oil-seed crop using the transgenic technique. The key step in GLA production is to insert a double bond between carbon #6 and #7 of LA to generate GLA (Fig. 1), a reaction mediated by the D6-desaturase. D6-desaturases has been isolated from M. alpina [36], Mucor rouxii [37], Phytium irregulare [38], Physcomitrella patens [39], borage [40], Echium plant sp. [41], Primula sp. [42], and Synechcocystis [40,43]. Generally, the D6-desaturases isolated from the microbes and plants act exclusively on the phospholipid-linked LA substrate [44], whereas those from mammalian sources recognize CoA-linked LA substrates [45]. To date, most of the D6-desaturases have been functionally expressed in yeast, and some have also been tested in oil-seed crop plants [36,38,40,41,46]. In our laboratory, we have co-expressed the M. alpina D6-desaturase and D12desaturase genes in a low-linolenic acid variety of Brassica napus resulting in the generation of GLA at a level of greater than 40% [47,48]. In other laboratory, expression of the P. irregulare D6-desaturase gene in Brassica juncea has also generated 25–40% GLA in the transgenic seed. However, due to the high initial content of ALA in this plant line, 2–10% stearidonic acid (SDA, 18:4n-3) was also reported to be formed [38]. 2.2. Transgenic production of ARA The production of the C20-PUFA ARA from LA involves the desaturation of LA to GLA, followed by the elongation of GLA to dihomo-c-linolenic acid (DGLA, C20:3n-6), and a subsequent desaturation of DGLA to ARA (Fig. 1). Thus, three major enzymes are involved in this process: a D6desaturase, a C18-PUFA-specific elongase, and a D5desaturase. Since use of the D6-desaturase for the GLA production has just been discussed in the previous section, this section will focus only on the C18-PUFA-specific elongase and the D5-desaturase. The first C18-PUFA-specific elongase was isolated and identified in our laboratory from the ARA-rich fungus, M. alpina [47]. This enzyme when tested in bakers’ yeast, spe-

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cifically recognized and elongated the n-6 and n-3 C18-PUFA substrates, GLA and SDA (C18:4n-3), respectively, whereas it demonstrated no activity towards monounsaturated or saturated fatty acid substrates [49–52]. Enzymes with similar elongating activity have also been isolated from Caenorhabditis elegans [53], P. patens [54], the marine protist, Thraustochytrium sp. [55], algae, Ostreococcus tauri and Thalassiosira pseudonada [56], and from mammalian sources [56–58]. These C18-PUFA-specific elongases are thought to recognize CoA-linked substrates [44] and different from the acyl carrier proteins (ACP)-linked substrate-specific plant elongases that are involved in elongation of very long-chain saturated and monounsaturated fatty acids, but not PUFAs [59,60]. An alternative pathway for the biosynthesis of DGLA is also found in some organisms [61–64]. In these organisms, LA is first elongated to form eicosadienoic acid (EDA, C20:2n-6), and subsequent desaturated at the D8-position to yield DGLA (Fig. 1). The PUFA-specific elongase involved in this pathway has been identified from the marine algae, Isochroysis [65] and a D8-desaturase gene has been isolated from the DHA-producing marine protist, Euglena gracilis [64]. The final step in the production of the C20-PUFAs, ARA is the introduction of a double bond at the D5-position of the DGLA to generate ARA. D5-Desaturase genes have been identified from fungi and algae such as M. alpina [66,67], Thraustochytrium sp. [46], and Phaeodactylum tricornutum [68], and these have been functionally characterized in yeast. Additional D5-desaturases have been identified from C. elegans [69], human [70], and rat [71]. When introduced into a low-linolenic variety of B. napus, the M. alpina D5desaturase was capable of desaturating oleic acid (OA, 18:1n-9) to taxoleic acid (D5,9–18:2), and LA to pinolenic acid (D5,9,12–18:3) [66]. Recently, Qi et al. [72] have coexpressed the genes encoding D9-specific elongase from I. galbana [65], D8-desaturase from E. gracilis [64] and a D5-desaturase from M. alpina and demonstrated production of low amounts of ARA in Arabidopsis. 2.3. Transgenic production of EPA The production of EPA from ALA involves the desaturation of ALA to SDA by a D6-desaturase, which is further elongated to x3-eicosatrienoic acid (ETA, C20:4n-3), and is subsequently desaturated at the D5-position to form EPA. Since the three enzymes involved in this process, i.e. D6desaturase, C18-PUFA-specific elongase and D5-desaturase function on both n-3 and n-6 pathway, and plant oils rich in ALA contains significant levels of LA, the transgenic expression of these three genes in an ALA-rich plant would result in synthesis of both ARA and EPA. To minimize the production of ARA during EPA biosynthesis, one approach involves shunting the n-6 PUFA metabolites to their n-3 counterparts by the action of a group of enzymes designated the x3desaturases (Fig. 1). These enzymes found in some plants,

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lower eukaryotes, and cyanobacteria [73], are so called because they introduce a double bond at the third carbon atom counted from the methyl- (x-) end of the fatty acyl chain. All plant- and cyanobacterial x3-desaturases act exclusively on the C18-PUFA substrate, LA, converting to ALA (Fig. 1). However, these enzymes do not efficiently convert LA to ALA as shown by a high LA to ALA ratio in their seed oils [35]. Recently, a novel x3-desaturase capable of recognizing multiple n-6 PUFA substrates, including the C18-PUFAs, LA and GLA, as well as the C20-PUFA, DGLA, was identified from C. elegans [74,75], and shown to be functional in plants [74]. More recently, a novel fungal x3-desaturase that could specifically convert ARA to EPA was identified in our laboratory and successfully expressed in an oil-seed crop [76]. Thus, these x3-desaturases have potential applications for transgenic production of EPA by catalyzing the conversion of n-6 to n-3 pathway shunt. To date, the yields of ARA or EPA obtained by coexpression of the PUFA biosynthesis genes in yeast or plants have been poor [68,72]. These coexpression studies revealed an accumulation of the D6-desaturated fatty acids in the glycerolipid fractions and almost none in the acyl-CoA pool, and thus a decrease in flux through the pathway. This results from the fact that the fungal and algal D6-desaturase function on phospholipid-linked (mainly phosphatidylcholine-linked) LA or ALA substrates, whereas the PUFA-specific elongase requires its substrates to be present in the acyl-CoA pool. In the plant or yeast model systems tested, there appears to be an inefficient transfer of the D6-desaturated products from the phospholipids to the acyl-CoA pool due to lack of suitable acyl transferase in these organisms [44]. Thus, additional work is on going to identify enzymes that are involved in the transfer of phospholipid-linked PUFAs to the acyl-CoA pools. 2.4. Transgenic production of DHA In lower eukaryotes, DHA can be synthesized from EPA in a two-step process that involves: (a) the elongation of EPA to x3-DPA; and (b) the desaturation of x3-DPA by a D4desaturase to generate DHA (Fig. 1). A number of mammalian elongases have been identified that can recognize and elongate C20-PUFAs [56,58,70]. Expression of these genes in bakers yeast showed that those enzymes recognize multiple chain-length PUFAs, such as C18-, C20- and C22-PUFAs [77]. Recently, elongases specific for C20-PUFAs have been identified from lower eukaryotes and demonstrated to function in production of DHA [56,78]. The first D4-desaturase was identified from a marine protist, Thraustochytrium, rich in DHA [79]. This D4-desaturase is capable of introducing a double bond at carbon #4 of x3-DPA to form DHA, and of adrenic acid (ADA, C22:4n-6) to generate x6-DPA (C22:5n-6) (Fig. 1). Qiu et al. [79] have expressed this D4-desaturase gene in a oilseed crop, B. juncea, in the presence of exogenously supplied x3-DPA substrate and showed a production of 3–6% DHA in the leaves,

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stems and roots of the transgenic Brassica [79]. Recently, new D4-desaturase genes were identified from E. gracilis [80], Pavlova [81] and Isochroysis [78]. Although feasibility of production of DHA in yeast model system has been demonstrated by coexpression of the C18 elongase, D5desaturase, C20-elongase and a D4-desaturase [56], similar studies have not been carved out in the plants. Thus the production of transgenic DHA in plant seed oils would involve coexpressing the C20-PUFA elongase and D4desaturase in addition to the previously described genes needed for EPA production. 2.5. Alternate system for transgenic production of EPA/DHA—the PKS system In certain microbes, LC-PUFA biosynthesis is carried out through an alternate system that does not involve desaturation/elongation. For example, biosynthesis of EPA in bacteria such as Shewanella and Vibrio occurs via the polyketide synthase (PKS)-like system [82–85]. Similar system is also involved in DHA production in the marine protist, Schizochytrium [85]. This PKS pathway is similar to the fatty acid synthase (FAS) pathway in that PUFA biosynthesis is initiated by the condensation between a short chain acyl-CoA and an unit of malonyl-CoA, followed by successive rounds of reduction, dehydration, reduction, and condensation, with the acyl chain growing by two-carbon units with each round. A possible dehydratase/isomerase existed in this PKS complex thought to be responsible for catalyzing the trans- to cisconversion of the double bonds to form EPA and DHA [85]. The genes encoding these enzymes are known to be present sequentially on long (20–30 kb) open reading frames (ORFs) in bacteria. However, the identity of many regions within these ORFs are still not known [82,84,85]. The Shewanella PKS system has been expressed in E. coli and Synechococcus, resulting in low yields of EPA [83,85]. Currently, no information is available on the expression of PUFA-PKS genes in plants. Thus, the feasibility of using this PUFA-PKS system as an alternative to the desaturase/elongase system for production of EPA/DHA in plants remains to be determined.

alternative to naturally occurring oil and will help the increasing demands of the chemical, pharmaceutical and nutraceutical industry for therapeutic and prophylactic use.

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Fatty acids are critical for the normal development and function of all organisms, and in particular, very long-chain PUFAs are necessary for the health and maintenance in humans. Recent progress in the cloning and identification of genes encoding the PUFA biosynthetic enzymes from different organisms has provided a potential mean to produce long-chain PUFA-rich transgenic oils. However, there are still issues regarding the compatibility of genes isolated from difference organisms and accumulation of undesirable PUFA intermediate that remained to be solved. However, once successful, these transgenic oils will provide an economical

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