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Originally published In Press as doi:10.1194/jlr.M400181-JLR200 on August 1, 2004

Papers In Press, published online ahead of print October 1, 2004
J. Lipid Res., doi:10.1194/jlr.M400181-JLR200
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Journal of Lipid Research, Vol. 45, 1899-1909, October 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis

Astrid Meyer*, Helene Kirsch*, Frédéric Domergue*, Amine Abbadi*, Petra Sperling*, Jörg Bauer{dagger}, Petra Cirpus{dagger}, Thorsten K. Zank{dagger}, Hervé Moreau§, Thomas J. Roscoe**, Ulrich Zähringer{dagger}{dagger} and Ernst Heinz1,*

* Biozentrum Klein Flottbek, Universität Hamburg, D-22609 Hamburg, Germany
{dagger} BASF Plant Science GmbH, D-67056 Ludwigshafen, Germany
§ Laboratoire Arago, Unité Mixte de Recherche 7628, Centre National de la Recherche Scientifique, F-66651 Banyuls sur mer, France
** Laboratoire de Génome et Développement des Plantes, Unité Mixte de Recherche 5096, Centre National de la Recherche Scientifique, F-66860 Perpignan, France
{dagger}{dagger} Forschungszentrum Borstel, D-23845 Borstel, Germany

The online version of this article (available at http://www.jlr.org) contains an additional figure. Back

Published, JLR Papers in Press, August 1, 2004. DOI 10.1194/jlr.M400181-JLR200

1 To whom correspondence should be addressed. e-mail: eheinz{at}botanik.uni-hamburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In algae, the biosynthesis of docosahexaenoic acid (22:6{omega}3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5{omega}3; EPA) to 22:5{omega}3, which is required as a substrate for the final {Delta}4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana, from the fish Oncorhynchus mykiss, from the frog Xenopus laevis, and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens. The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana: one specific for the elongation of ({Delta}6-)C18-PUFAs and one specific for ({Delta}5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss, X. laevis, and C. intestinalis accepted both C18- and C20-PUFAs. By coexpression of the {Delta}6- and {Delta}5-elongases from T. pseudonana and O. tauri, respectively, with the {Delta}5- and {Delta}4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast.

This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops.

Abbreviations: ALA, {alpha}-linolenic acid; ARA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; EST, expressed sequence tag; GLA, {gamma}-linolenic acid; LA, linoleic acid; LCPUFA, long-chain polyunsaturated fatty acid; ORF, open reading frame; STA, stearidonic acid

Supplementary key words Ciona intestinalis • Oncorhynchus mykiss • Ostreococcus tauri • polyunsaturated fatty acids • Thalassiosira pseudonana • Xenopus laevis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Docosahexaenoic acid (DHA) is a fatty acid with 22 carbon atoms and 6 methylene-interrupted Z-double bonds (22:6{Delta}4,7,10,13,16,19). Some human tissues such as brain, testis, and retina are characterized by membrane lipids carrying high proportions of this long-chain polyunsaturated fatty acid (LCPUFA), and many clinical studies have pointed out the crucial role of DHA in the development and functions of these tissues (1). In addition, DHA as well as the other LCPUFAs arachidonic acid (ARA; 20:4{Delta}5,8,11,14) and eicosapentaenoic acid (EPA; 20:5{Delta}5,8,11,14,17) are precursors of different classes of eicosanoid effectors involved in the regulation of many important functions in mammals. In view of the major roles attributed to LCPUFAs in human physiology, the reactions contributing to their biosynthesis have recently attracted growing interest (2). Because of its relevance for human nutrition, the biosynthetic sequence known in most detail is that realized in the mammalian liver and known as the Sprecher pathway (3). Moreover, LCPUFA biosynthesis was also studied in various organisms from phylogenetically divergent groups, such as algae, fungi, and lower plants. DHA is synthesized de novo in several microalgae as well as in some fungi, whereas in mammals its synthesis starts from the essential fatty acid {alpha}-linolenic acid (ALA; 18:3{Delta}9,12,15). Another route for DHA biosynthesis that will not be further described here is relying on polyketide synthase systems. It is found in some marine bacteria and primitive eukaryotes like the thraustochytrid protist Schizochytrium (4).

The currently best known routes for DHA biosynthesis are depicted in simplified form in Fig. 1 . In the liver of mammals, a {Delta}6-desaturase, a {Delta}6-elongase, and a {Delta}5-desaturase successively convert ALA via stearidonic acid (STA; 18:4{Delta}6,9,12,15) and 20:4{Delta}8,11,14,17 to EPA in the so-called {omega}3 pathway. In parallel, the same set of enzymes accepts the other essential fatty acid linoleic acid (LA; 18:2{Delta}9,12) to form ARA in the {omega}6 pathway. It is important to note that because of the absence of an {omega}3-desaturase in mammals, the intermediates of the {omega}6 and {omega}3 pathways are not interconvertible. In mammalian liver, EPA is then elongated twice without an intervening desaturation. Recent data suggest that the same elongase (ELOVL2) accepts EPA as well as {Delta}7-22:5 (i.e., the reaction product of its own first elongation cycle), leading to {Delta}9-C24:5 (5). At this point, the {Delta}6-desaturase that is responsible for the formation of {Delta}6-C18-PUFAs gets involved a second time and inserts a {Delta}6 double bond in {Delta}9-C24:5, leading to the synthesis of {Delta}6-C24:6 (5, 6). Finally, {Delta}6-C24:6 is transferred from the endoplasmic reticulum membranes to the peroxisomes for one round of ß-oxidative chain shortening and release of DHA. These final steps, from EPA to DHA, are attributable to the absence of a {Delta}4-desaturase activity in mammals and are characteristic for the Sprecher pathway. Labeling studies (7) have shown that this sequence is also present in fish, suggesting that this route for DHA biosynthesis is not restricted to mammals.



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  		Fig. 1. {omega}6 and {omega}3 pathways for the synthesis of long-chain polyunsaturated fatty acids in eukaryotes. {omega}6-18:2 and {omega}3-18:3 can be converted to docosahexaenoic acid (DHA) by the consecutive action of desaturases and elongases, either starting with a {Delta}6 desaturation (A) or with a {Delta}9 elongation (B). In mammals, the {Delta}4 double bond in DHA is the result of a more complicated series of reactions that involve the elongation to a C24 fatty acid, a second {Delta}6 desaturation, and the final chain shortening in peroxisomes (the so-called Sprecher pathway; C). {omega}3-desaturases (dashed arrows), which link the {omega}6 and {omega}3 pathways in many eukaryotes, are missing in mammals. Examples of cloned elongases involved in DHA synthesis have been included in the figure. Although some elongases are specific for a single step (e.g., the moss PpPSE1 and the fungal MaGLELO), others are nonspecific or multifunctional and show up at several steps (e.g., the human HsELOVL5 and the murine MmELOVL2; underlined).

 
Up to ARA and EPA, the biosynthetic pathways found in mosses, fungi, and algae are basically identical to the mammalian sequence. Also in these organisms, two front-end desaturases ({Delta}6 and {Delta}5) and one {Delta}6-elongase convert either LA or ALA into ARA or EPA, respectively, in the {omega}6 and {omega}3 pathways (Fig. 1A). In addition, some algae can use an alternative pathway, in which LA and ALA are elongated to the corresponding {Delta}11-C20-PUFA (Fig. 1B), necessitating a subsequent {Delta}8-desaturation (8). Because many of these organisms possess an {omega}3-desaturase, they do not show the strict separation of {omega}6 and {omega}3 pathways typical for mammals. In addition, these organisms may use acyl chains of phospholipids as substrates for the desaturation reactions, whereas acyl-CoA thioesters are required for elongation reactions (9). In contrast, in mammals all of the intermediates are kept in the form of acyl-CoA thioesters. Most importantly for this study, the primary de novo producers of DHA appear to follow a simplified route for DHA synthesis in which a {Delta}5-elongase and a {Delta}4-desaturase are responsible for the conversion of EPA to DHA (10–12).

Elongase complexes comprise four activities: the ß-ketoacyl-CoA synthase, the ketoacyl-CoA reductase, the hydroxyacyl-CoA dehydratase, and the enoyl-CoA reductase. From these, the first enzyme is considered to be rate limiting and specificity controlling with regard to chain length and pattern of double bonds. ELO-type sequences involved in LCPUFA biosynthesis were cloned from the moss Physcomitrella patens (13), the fungus Mortierella alpina (14), the nematode Caenorhabditis elegans (15), the alga Isochrysis alpina (8), and different mammals [for a recent review, see ref. (16)]. Because of their close relation to the enzymes ScELO1, ScELO2, and ScELO3 from Saccharomyces cerevisiae (17, 18), they are thought to code for ß-ketoacyl-CoA synthase activities and are often referred to as "elongases," although biochemical data supporting the actual condensing activity are still missing.

Yeast and animal cells were used for the expression of most of the elongases catalyzing the elongation steps shown in Fig. 1. Most of the sequences characterized to date are of mammalian origin, but few of them have been studied in sufficient detail to answer all questions regarding regioselectivity and chain length selectivity. Therefore, it remains unclear which substrates other than those indicated in Fig. 1 could also be accepted by these enzymes. Among the few sequences of nonmammalian origin studied in more detail, none was shown to be specific for the elongation of EPA. The {Delta}5-elongases cloned to date were all isolated from mammals and rather unspecific, in that they carried out multiple elongation reactions not restricted to C20-PUFAs, as shown in Fig. 1.

As mentioned above, the synthesis of LCPUFAs in mammals depends on the dietary supply of LA and ALA, and {omega}6- and {omega}3-LCPUFAs are each precursors of antagonistic eicosanoid effectors. Typical Western diets are characterized by very high ratios of LA/ALA that are far above the recommended value of ~5 and thus favor the synthesis of ARA at the expense of EPA and DHA (19). In addition, because ALA appears to be rapidly degraded by ß-oxidation, it seems best to include an appropriate mixture of LCPUFAs in the diet. As this growing demand cannot be met by farmed or landed fish (20) and none of the oilseeds produces LCPUFAs, the implementation of LCPUFA biosynthesis into oilseed crops by modern biotechnology would provide a truly sustainable source of these valuable fatty acids. Because the biosynthesis of DHA according to the Sprecher pathway is clearly too complicated to be reconstituted by gene technology, the alternative route relying on the use of a {Delta}5-elongase and a {Delta}4-desaturase is most promising. Whereas all of the desaturases, including the {Delta}4-desaturase, and the {Delta}6-elongase have already been isolated from various organisms, an elongase specific for the conversion of a C20-PUFA to a C22-PUFA has not been cloned yet. To reconstitute the simpler pathway of DHA biosynthesis, we started experiments to clone a specific C20-PUFA-elongase restricted in its action to a single elongation cycle to produce a C22-PUFA. Such an activity may become particularly relevant in transgenic plants, in which other potential substrates may be present in excess, leading to complicated mixtures of elongated products.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Identification and cloning of putative elongase sequences
To identify novel elongases, we used the {Delta}6-elongase sequence from P. patens (PpPSE1) as the query in a tBLASTn (21) search and identified putative elongase expressed sequence tag (EST) clones from Xenopus laevis (GenBank accession number BC044967), Ciona intestinalis (GenBank accession number AK112719), and Oncorhynchus mykiss (GenBank accession number CA350786). The C. intestinalis clone was kindly provided by S. Fujiwara (22), the clone from O. mykiss was a gift from C. E. Rexroad (United States Department of Agriculture, Agricultural Research Service, National Center for Cool and Cold Water Aquaculture), and the X. laevis clone was purchased from the American Type Culture Collection (ATCC 6844054). The open reading frames (ORFs) were amplified by PCR (primers XlELO, CiELO, and OmELO; Table 1) and cloned into the yeast expression vector pYES2.1/V5-His-TOPO (XlELO and CiELO) or pYES3CT (OmELO) from Invitrogen, yielding pXlELO, pCiELO, and pOmELO.


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  		TABLE 1. PCR primers used in this work

 
A bacterial artificial chromosome library of Ostreococcus tauri OTHH0595 was constructed and sequenced as previously described (23) and used for tBLASTn with PpPSE1 as the query. Two ORFs showing significant similarity to the elongase and apparently lacking introns were amplified by PCR with specific primers (OtELO1 and OtELO2). Fresh cells that were grown in K medium (Sigma-Aldrich) at 23°C in the light and frozen once were used as template. The PCR products were cloned into the pYES2.1/V5-His-TOPO yeast expression vector, yielding pOtELO1 and pOtELO2, respectively.

PpPSE1 was also used to blast a genomic library of Thalassiosira pseudonana (Department of Energy Joint Genome Institute), yielding two putative elongase clones (PQI68798 and PQI119277). T. pseudonana SAG 1020-1b (Sammlung für Algenkulturen, Göttingen, Germany) was grown in the light at 23°C in f/2 medium, and the genomic DNA was isolated using the DNeasy Kit (Qiagen). The genomic DNA was used for PCR (primers Tp687GENOMIC and Tp119GENOMIC), and the PCR products were cloned into pGEM-T (Promega) and sequenced. GENESCAN (Arabidopsis algorithm) identified in each clone two exons separated by one intron. The ORF of PQI68798 was amplified by PCR, whereby the 5' primer was constructed in such a way that it was complementary to exon I (91 bp) and to the 5' end of exon II (23 bp); therefore, it was missing the part coding for the 142 bp intron (primer Tp687ELO). The 5' primer of exon I was altered to approximate optimal yeast codon usage without changing the translated sequence. The putative ORF of PQI119277 was also constructed by PCR, taking into account the yeast codon usage. In a first step, both exons were amplified with primers (Tp119EI and Tp119EII) that created a 54 bp overlapping sequence. The overlapping products subsequently served as templates for the second PCR using the 5' primer of exon I and the 3' primer of exon II. The ORFs of PQI68798 and PQI119277 were cloned into pYES2.1/V5-His-TOPO, yielding pTpELO1 and pTpELO2, respectively.

pXlELO, pCiELO, pOmELO, pOtELO1, pOtELO2, pTpELO1, and pTpELO2 were then used for the transformation of S. cerevisiae 334 (24) or INVSc1 (Invitrogen).

Expression in yeast
For functional expression of the elongases, precultures were grown at 30°C in minimal medium with 2% raffinose lacking the respective amino acid or base for vector selection. Five milliliters of the medium were inoculated with precultures (2 days old) to an optical density at 600 nm of 0.05, and expression was induced with 2% galactose. Expressions were carried out for 4 days at 20°C in the presence of exogenously supplied fatty acids of commercial origin (250–500 µM). Pinolenic acid (18:3{Delta}5,9,12) was part of a fatty acid mixture isolated from Larix decidua seeds (25). For the coexpression of elongases and desaturases, the yeasts were additionally transformed with the {Delta}5-desaturase from Phaeodactylum tricornutum (26) and the {Delta}4-desaturase from Euglena gracilis (11). Yeasts harboring the empty vectors (pYES2, pYES3CT, and pESC-Leu) were used as controls.

Fatty acid analysis
Yeast cells were sedimented by centrifugation and directly used for transmethylation of fatty acids. Fatty acid methyl esters were routinely analyzed by gas liquid chromatography as described previously (11), whereas detailed structural identities of new fatty acids were determined by GC-MS (27).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of elongase genes from Xenopus, Ciona, Oncorhynchus, Ostreococcus, and Thalassiosira
The majority of elongase sequences available in databases originate from mammals (i.e., human, mouse, and rat), and among those functionally characterized, none was shown to be specific for the elongation of {Delta}6-C18- or {Delta}5-C20-PUFAs. Because the PpPSE1 gene from the moss P. patens is known to code for a specific {Delta}6-elongase, we decided to use its translated sequence as the query in a tBLASTn search to identify PUFA-elongases in EST and genomic databases of nonmammalian organisms. The fish O. mykiss, the frog X. laevis, the sea squirt C. intestinalis, and the two DHA-producing algae O. tauri and T. pseudonana were selected as candidate organisms. Among several identified ORFs of interest, we could amplify seven putative elongase clones. The actual numbers of amino acids (272–358) representing the various ORFs as well as their identity compared with the other elongases cloned in the present study are summarized in Table 2. Most of the proteins were only 19–26% identical to PpPSE1, with the exception of OtELO1, which showed a significantly higher identity (42%). Interestingly, the identity between OtELO1 and OtELO2 is only 20%. It should be mentioned that TpELO1 and TpELO2 from T. pseudonana were constructed by PCR after a GENESCAN analysis to delete putative introns and, therefore, may not represent the ORFs translated in the alga. The deduced amino acid sequences of the newly cloned proteins all contained seven to nine putative transmembrane helices as well as the various motifs (16) that are typical for this group of elongases (KxxE/DxxDT, the extended histidine box QxxFLHxYHH, the tyrosine box NxxxHxxMYxYY, and TxxQxxQ) (Fig. 2) . Lysine residues close to the C terminus that may function as endoplasmic reticulum retention signals were clearly seen in three sequences (XlELO, OtELO1, and TpELO1). The phylogenetic alignment of the currently cloned elongases together with previously characterized enzymes will be discussed below.


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  		TABLE 2. Elongase genes cloned in this work

 


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  		Fig. 2. Amino acid sequences of elongases characterized in the present study. The alignment was obtained using the CLUSTAL X program (gap opening 10, gap extension 0.05). Conserved amino acids are shown on a gray background, whereas conserved motifs found in all ELO-type sequences are framed. The nucleotide sequence data have been submitted to the DDBJ/EMBL/GenBank sequence data bank (see Table 2).

 
Functional expression in yeast
The functions of the proteins encoded by the isolated genes were verified by expression studies in S. cerevisiae comprising incubations with exogenous fatty acids followed by GC-MS analysis of total fatty acid methyl esters. To identify which ORF could encode a {Delta}5-C20-specific elongase, each ORF was expressed in the presence of either {Delta}6- or {Delta}5-polyunsaturated fatty acids (STA or EPA, respectively). Low basal elongation of STA, but no elongation of EPA, was obtained with yeast transformed with the empty vectors (data not shown). All cloned elongases were active in yeast and produced novel fatty acids whose structures were confirmed by GC-MS. Using either STA or EPA as exogenously supplied substrates, the seven elongases could be separated into three different groups: {Delta}6-C18-PUFA-elongases (TpELO1 and OtELO1), {Delta}5-C20-PUFA-elongases (TpELO2 and OtELO2), and bifunctional enzymes accepting both C18- and C20-PUFAs (OmELO, XlELO, and CiELO). Results obtained with one member of each group are given in Fig. 3 . On expression of TpELO1 (and of OtELO1; data not shown, but see Fig. 4) , STA was very efficiently converted to 20:4{omega}3, whereas EPA was not elongated. In contrast, when OtELO2 (and TpELO2; data not shown, but see Fig. 4) was expressed, EPA was very efficiently elongated, whereas STA elongation did not exceed that of the control strain. When OmELO was expressed, STA was successively elongated to 20:4{omega}3 and 22:4{omega}3, whereas EPA was elongated to 22:5{omega}3. Yeast expressing XlELO or CiELO were also able to convert both STA and EPA (data not shown, but see Fig. 4). As shown in Fig. 3, OmELO was in addition active on 16:1{Delta}9, 18:1{Delta}9, and 18:1{Delta}11, as indicated by the increase/presence of 18:1{Delta}11, 20:1{Delta}11, and 20:1{Delta}13. Starting from 16:1{Delta}9, OmELO was in fact capable of five successive elongation steps leading to the synthesis of 26:1{Delta}19.



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  		Fig. 3. Elongation of stearidonic acid (STA) and eicosapentaenoic acid (EPA) in transgenic yeast expressing the elongases TpELO1, OtELO2, or OmELO. Transgenic yeasts were incubated for 4 days in the presence of exogenously supplied STA or EPA. Fatty acid profiles were analyzed by gas-liquid chromatography. These fatty acid profiles reflect results obtained in three to five independent expression experiments. FID, flame ionization detector.

 


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  		Fig. 4. Specificities of functionally expressed elongase enzymes. All transformed yeasts were separately incubated with 250–500 µM of the C18, C20, and C22 fatty acids listed on the abscissa for 4 days, except for pinolenic acid [18:3{Delta}5,9,12 (18:3Pi)], which was part of a fatty acid mixture isolated from Larix seeds. Fatty acid profiles were analyzed by gas-liquid chromatography. The elongation is given as [product/(substrate + product) x 100]. Each value is the mean ± SD from three to five independent experiments.

 
Substrate specificity of the different elongases
We then investigated to what extent these regioselective specificities persisted when fatty acid substrates of different structures, regarding both chain length and position of double bonds, were supplied. The results (Fig. 4) show that TpELO1 and OtELO1 were exclusively active with C18-PUFAs. Although the activity of TpELO1 was restricted to the {Delta}6-unsaturated fatty acids {gamma}-linolenic acid (GLA) and STA, OtELO1 elongated GLA and STA very efficiently but also to a minor extent {Delta}9-C18-PUFAs (LA and ALA) and a {Delta}5-C18-PUFA (pinolenic acid). The activity of the {Delta}5-C20-PUFA-elongases (TpELO2 and OtELO2) was restricted to C20-PUFAs, with EPA being most efficiently elongated in both cases. None of these enzymes elongated {Delta}8-20:3{omega}6, but both TpELO2 and OtELO2 were active on {Delta}5-20:4{omega}3 (2% and 13% conversion, respectively). The most active clone, OtELO2, also displayed activity with {Delta}8-20:3{omega}3 (7% conversion), indicating that the presence of an {omega}3 double bond of the C20-PUFAs may play an important role in substrate recognition. For example, OtELO2 elongated 11% of ARA but 56% of EPA.

In contrast, the three elongases from animals elongated almost all LCPUFA substrates offered, although with different efficiencies. The X. laevis XlELO mainly converted {Delta}6-C18- and {Delta}5-C20-PUFAs (e.g., 24% of STA and 10% of EPA), but it was also very active with the {Delta}11 fatty acid 20:3{omega}3 (13% conversion). CiELO from the sea squirt was most active with pinolenic acid (38% conversion; see supplemental data) and least active with STA. OmELO was the most efficient among the three enzymes and elongated most substrates by more than 20% (Fig. 3). However, OmELO did not elongate ARA or EPA to C24-PUFAs; therefore, it does not seem to be involved in the Sprecher pathway.

Phylogenetic relationships
Several elongase phylograms have been constructed before, but because of the limited number of functionally assigned sequences, only a few questions regarding the branching pattern could be addressed (13, 16, 28). The elongase enzymes studied here significantly increase the number of available sequences with known specificities. To see how the resulting branching correlates with substrate specificity and phylogenetic relationships of the organisms, we created an unrooted phylogenetic tree comprising all of the elongases cloned and functionally characterized to date by the neighbor-joining method using Tree View. Figure 5 shows that two major groups (with exceptions that will be discussed below) could be identified. The first group comprises elongases involved in saturated fatty acid (SFA) and MUFA elongation and does not contain any sequence from the present study. The second group contains the three new animal elongase sequences (XlELO, OmELO, and CiELO; marked by asterisks) together with mammalian elongase sequences involved in PUFA metabolism. A representative member of this group is the murine MmELOVL2, which elongates {Delta}6-C18-, {Delta}5-C20-, and {Delta}7-C22-PUFAs (29). The three new elongases expand the substrate specificity to include {Delta}8, {Delta}9, and {Delta}11 coverage, suggesting that the members of this group generally display multiple regioselectivities. The remaining one-step PUFA-elongases do not fall into one of the two groups mentioned, but the algal elongases TpELO1 and OtELO1 together with the moss PpPSE1 and the fungal MaGLELO may form a putative third group of elongases restricted to the elongation of {Delta}6-C18-PUFAs (encircled by a dotted line in Fig. 5). Among these, TpELO1 shows the deepest separation from the other members. The two algal enzymes specific for the elongation of C20-PUFAs (TpELO2 and OtELO2) are not closely related to each other, and despite their identical specificity, it is questionable whether they can be considered members of a common branch. Similarly, at present it is not possible to conclude that the unique {Delta}9-elongase from Isochrysis galbana (IgASE1) (8) forms a separate branch. Although there are some exceptions in some of these groups (see Discussion), Fig. 5 suggests that a first approximation of the substrate specificity of elongases cloned in the future may be deduced from their mapping in this phylogram.



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  		Fig. 5. Unrooted phylogram of functionally characterized ELO-type sequences. The alignment was generated by the CLUSTAL X program, and the unrooted phylogram was constructed by the neighbor-joining method using Tree View. The DDBJ/EMBL/GenBank accession numbers of the different protein sequences are as follows: (1) AAF71789, (2) NP_013476, (3) NP_012339, (4) NP_009963, (5) AAG17875, (6) NP_031729, (7) NP_076995, (8) AAL14239, (9) BAB69888, (10) NP_503114, (11) AAF70462, (12) XP_002040, (13) NP_062295, (14) NP_060240, (15) NP_062296, (16) NP_068586, (17) NP_599209, (18) NP_073563, (19) AAG47667, (20) AAF70417, (21) AAL84174, and (22) AAL37626. The enzymes characterized in the present study are marked by asterisks. The branch comprising the four {Delta}6-specific, single-step elongases is marked by a dotted line to show its preliminary delineation.

 
Synthesis of DHA in yeast
Because the production of DHA was the major reason for the isolation of new elongase genes, we tried to implement its biosynthesis into yeast. For this purpose, we coexpressed one of the new {Delta}6-elongases, the {Delta}5-desaturase from the diatom P. tricornutum (26), a new {Delta}5-elongase, and the {Delta}4-desaturase from E. gracilis (11) in a single strain that was grown in the presence of STA. To compare the relevance of using multifunctional or highly specific elongases for the production of DHA, we used either the OmELO elongase (which can catalyze both elongation steps) or the two most active and specific elongases, TpELO1 and OtELO2. DHA synthesis was observed in both yeast strains (Fig. 6) . STA was first elongated to 20:4{omega}3 either by OmELO or TpELO1; the elongated product was subsequently {Delta}5-desaturated to EPA (20:5{omega}3), which was then further elongated by the {Delta}5 elongation activity of OmELO or OtELO2. The resulting 22:5{omega}3 was finally converted to DHA by the {Delta}4-desaturase. The DHA structure was confirmed by GC-MS analysis (data not shown). The only by-product observed in the strain expressing the two algal elongases was 22:4{omega}3, which can be ascribed to the "leaky" substrate specificity of OtELO2 (Fig. 4). On the other hand, the strain expressing the bifunctional OmELO showed a dramatic increase in 18:1{Delta}11 as well as significant proportions of 20:1{Delta}11, 20:1{Delta}13, and 26:1{Delta}19. They all were most likely formed by OmELO-catalyzed elongation of the yeast monounsaturated fatty acids (Fig. 3).



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  		Fig. 6. Implementation of DHA synthesis in S. cerevisiae starting from STA. A: A yeast strain coexpressing the bifunctional elongase OmELO, the {Delta}5-desaturase from P. tricornutum (26), and the {Delta}4-desaturase from E. gracilis (11). B: A yeast strain coexpressing the same two desaturases used in A together with the elongases TpELO1 and OtELO2. The cultures were grown for 4 days in the presence of exogenously supplied STA. The fatty acid profile was analyzed by gas-liquid chromatography. These fatty acid profiles reflect results obtained in three independent expression experiments.

 
The enzyme combinations with the bifunctional fish elongase or the specific algal elongases were similarly efficient concerning DHA yield. DHA represented ~0.5% of the total fatty acids in both yeast strains. However, the formation of unwanted by-products (i.e., fatty acids that are not part of the {omega}3 pathway shown in Fig. 1A and that, therefore, lead to "dead end" products) was largely reduced in the strain expressing the specific elongases TpELO1 and OtELO2. The total fatty acid profiles presented in Fig. 6 suggest that the low yield of DHA was largely attributable to the low {Delta}5-desaturation activity. Because the activity of the P. tricornutum desaturase was shown to be restricted to fatty acids acylated at the sn-2 position of phosphatidylcholine (9), the substrate availability for the {Delta}5-desaturase may be the limiting factor. In the present experiments, the {Delta}5-desaturase substrate 20:4{Delta}8,11,14,17 results from the elongation of STA and is therefore produced as acyl-CoA ester before being transferred to the various lipids found in yeast (9). Although some of it will be acylated at the sn-2 position of phosphatidylcholine, all of the 20:4{Delta}8,11,14,17 that is channeled elsewhere, such as in the neutral lipids, remains unavailable for the {Delta}5-desaturase.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using the P. patens PpPSE1 sequence, which is known to code for a specific {Delta}6-elongase, as query in a tBLASTn search in EST and genomic databases, we were able to isolate seven PUFA-elongase clones from the fish O. mykiss (OmELO), the frog X. laevis (XlELO), the sea squirt C. intestinalis (CiELO), the prasinophyte alga O. tauri (OtELO1 and OtELO2), and the diatom T. pseudonana (TpELO1 and TpELO2). The deduced amino acid sequences were 19–42% identical to that of the moss elongase, with OtELO1 being the most similar. All seven elongases were active when expressed in yeast and enabled us to carry out a detailed analysis of the substrate specificities and to conclusively annotate OtELO1 and TpELO1 as {Delta}6-C18-PUFA-elongases, OtELO2 and TpELO2 as {Delta}5-C20-PUFA-elongases, and OmELO, XlELO, and CiELO as bifunctional C18/C20-PUFA-elongases. To our knowledge, OtELO2 and TpELO2 represent the first examples of {Delta}5-C20-PUFA-elongases.

The seven new elongases studied in this work originate from widely divergent organisms and display very diverse substrate specificities. In an unrooted phylogenetic tree of 29 functionally characterized elongases, the new enzymes grouped into two of three major clades. One clade comprises elongases specific for SFAs and MUFAs and includes the well-characterized elongases ScELO1–ScELO3 from S. cerevisiae (17, 18), MaELO from the oleaginous fungus M. alpina (GenBank accession number AAF71789), and two elongases from the nematode C. elegans, CeELO1 and CeELO2. CeELO2 is specific for C16:0 (30), whereas CeELO1 from the same organism has a preference for {Delta}6-C18-PUFAs (15). In the context of this clade, the CeELO1 enzyme represents a functional exception that may be ascribed to a local and independent invention of PUFA elongation after the duplication of an ancestral SFA/MUFA-elongase. Similarly, the {Delta}5- and {Delta}6-desaturases from C. elegans also group together, although the {Delta}5- and {Delta}6-desaturases from a single organism (such as M. alpina) are usually found in different clades (31).

The animal elongases OmELO, CiELO, and XlELO cloned in the present work fall into another clade comprising chordate elongases specific for PUFAs but displaying multiple regioselectivities. One apparent exception in this group is MmELOVL1 (Scc1) from mouse, which can complement yeast mutants deleted in the SFA/MUFA-elongase ELO3 (32). Because MmELOVL1 is most closely related to the PUFA-specific human HsELOVL1 (33), the activity displayed by MmELOVL1 in yeast mutants may represent a normally minor activity just sufficient for complementation, whereas its preferred substrates may be PUFAs, which has never been assayed to our knowledge. The inclusion of the elongase CiELO from the sea squirt C. intestinalis in this clade emphasizes that this branch is not restricted to PUFA-elongases from vertebrates but rather comprises animals from different chordate groups including, in addition to vertebrates, the primitive tunicate Ciona.

A third clade is made up of elongases specific for a single step in PUFA biosynthesis, the elongation of {Delta}6-C18-PUFAs. At present, this clade includes only four enzymes, the algal OtELO1 and TpELO1, the moss PpPSE1 (13), and the fungal MaGLELO (14). Additional sequences to be found in the future will show whether the delineation of this clade can persist. According to the phylogram, the {Delta}5-elongases of the two algae, OtELO2 and TpELO2, are not closely related to each other or to the {Delta}6-elongase of the same algae, pointing to ancient gene duplications.

The elongase sequences collected to date suggest that the evolution of C18-PUFA elongation up to DHA was realized by following two alternative strategies. In one case, two different and specific enzymes for the consecutive {Delta}6 and {Delta}5 elongations evolved that have only been detected in algae. Because elongation stopped at a chain length of C22, the development of a {Delta}4-desaturase was necessary. In the second group, exemplified by mammals, a single multifunctional elongase enzyme, accompanied by secondary copies, was developed. This development resulted in an extended "unspecificity" accepting in addition to C18- and C20- also C22-PUFA substrates, which in turn made DHA biosynthesis independent of a {Delta}4-desaturase (Sprecher pathway; Fig. 1C). This pathway is also realized by nonmammalian vertebrates such as fish, but it is not clear whether all chordates follow this pathway to produce DHA.

With regard to the biotechnological implementation of DHA production in oilseed crops, the pathway involving {Delta}4 desaturation is the preferred choice for several reasons. First, in contrast to the Sprecher pathway, this route does not lead to the synthesis of C24-PUFA intermediates and, therefore, does not depend on the peroxisomal ß-oxidation machinery. Second, various reasons regarding the future acceptance of transgenic foods require the use of nonmammalian organisms as gene sources to clone all of the sequences required for an assembly of LCPUFA biosynthesis in transgenic oilseeds. Furthermore, the broad substrate specificities of the mammalian elongases would favor the formation of unwanted by-products with unknown physiological effects. This is particularly unacceptable in the background of transgenic plants, in which several enzymes with different ranges of substrate specificities have to cooperate in a processive manner. The use of enzymes restricted to a single step in the DHA biosynthetic pathway might provide a means to significantly reduce these by-products.

The synthesis of ARA and EPA from GLA and ALA, respectively, has already been successfully demonstrated in transgenic yeast (15, 26), but the experimental proof of a reconstitution of DHA production was still missing. Using the {Delta}5-desaturase from P. tricornutum and the {Delta}4-desaturase from E. gracilis, together with the {Delta}6- and {Delta}5-elongases from O. tauri and T. pseudonana, we were able to implement DHA synthesis starting from STA in yeast using enzymes of exclusively algal origin. We could also compare the efficiency of this pathway with that involving the bifunctional elongating enzyme from the fish O. mykiss. Both combinations were able to convert STA via EPA into DHA. There was no difference in DHA yield, but several different fatty acids that are not intermediates of the pathway were found in the yeast expressing the fish elongase. The only detectable by-product in the strain expressing the algal elongases was 22:4{Delta}10,13,16,19, which resulted from the elongation of 20:4{Delta}8,11,14,17, underlining the advantage of using specific single-step elongases.

In summary, we present here the cloning and functional characterization of the first C20-specific PUFA-elongases. In addition, the successful reconstitution of DHA synthesis in yeast represents an encouraging step in the process of establishing transgenic, DHA-producing oilseed plants.


   ACKNOWLEDGMENTS
 
This research was supported financially by grants from the Bundesministerium für Bildung und Forschung (Napus 2000, FK 0312252F) and BASF Plant Science GmbH (Ludwigshafen, Germany). H.M. acknowledges the participation of the Génopole Languedoc-Roussillon in the genome sequencing program.

Manuscript received May 11, 2004 and in revised form July 20, 2004.


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