Functional differentiation and selective inactivation of multiple <Emphasis Type="Italic"> Saccharomyces cerevisiae </Emphasis>genes involved in very-long-chain fatty acid synthesis

While de novo fatty acid synthesis uses acetyl-CoA, fatty acid elongation uses longer-chain acyl-CoAs as primers. Several mutations that interfere with fatty acid elongation in yeast have already been described, suggesting that there may be different elongases for medium- and long-chain acyl-CoA primers. In the present study, an experimental approach is described that allows differential characterization of the various yeast elongases in vitro. Based on their characteristic primer specificities and product patterns, at least three different yeast elongases are defined. Elongase I extends C12-C16 fatty acyl-CoAs to C16-C18 fatty acids. Elongase II elongates palmitoyl-CoA and stearoyl-CoA up to C22 fatty acids, and elongase III synthesizes 20-26-carbon fatty acids from C18-CoA primers. Elongases I, II and III are specifically inactivated in, respectively, elo1, elo2 and elo3 mutants. Elongases II and III share the same 3-ketoacyl reductase, which is encoded by the YBR159w gene. Inactivation of YBR159w inhibits in vitro fatty acid elongation after the first condensation reaction. Although in vitro elongase activity is absent, the mutant nevertheless contains 10-30% of normal VLCFA levels. On the basis of this finding, an additional elongating activity is inferred to be present in vivo. ybr159Delta cells show synthetic lethality in the presence of cerulenin, which inactivates fatty acid synthase. An involvement of FAS in VLCFA synthesis may account for these findings, but remains to be demonstrated directly. Alternatively, a vital role for C18 and C20 hydroxyacids, which are dramatically overproduced in ybr159Delta cells, may be postulated.     

Biochemical and molecular characterization of corn (Zea mays L.) root elongases

Root surfaces are protected against the soil environment by the deposition of lignin and suberin. In order to obtain more insight into the regulation of root suberin biosynthesis, elongases from primary roots of corn (Zea mays L.) seedlings were characterized. Elongase activities (acyl-CoA and ATP-dependent) were located in the microsomal fraction of the root cells. C(20), C(22) and C(24) fatty acids were detected as primary products of elongases. Preferred substrates of the acyl-CoA elongases were C(18:0)-CoA and C(20:0)-CoA. Applying a molecular approach, using PCR and degenerate primers derived from the sequences of known leaf and seed 3-ketoacyl-CoA synthases (KCSs), catalysing the first step of very-long-chain fatty acid synthesis, the cDNA of a putative root KCS was obtained showing high homology to known leaf and seed KCSs at the DNA and amino acid levels. Thus, our approach provides the first direct evidence for the presence and the activity of root elongases in Z. mays. Ongoing research is focusing on the molecular analysis and the regulation of KCS expression in roots in reaction to different environmental stimuli.     

Effect of fatty-acyl-CoAs on the elongation of saturated fatty acid in porcine aorta microsomes

The microsomal elongation system from porcine aorta for longchain fatty-acyl-CoAs was investigated. Palmitoleoyl-CoA (16:1-CoA), oleoyl-CoA (18:1-CoA), and eicosenoyl-CoA (20:1-CoA) remarkably depressed the elongation activity for 16:0-CoA in aorta microsomes by 44.8, 52.4, and 43.7% of the control activity, respectively. Saturated and polyunsaturated fatty-acyl-CoAs had little effect on the 16:0-CoA elongation activity. These results indicate that monounsaturated long-chain fatty acyl-CoAs can regulate the synthesis of saturated fatty acids in the vessel walls.     

Chain elongation in the formation of polyunsaturated fatty acids by brain: Some properties of the microsomal system

Chain elongation of polyunsaturated acids has been investigated using microsomes from developing rat brain. With 18:3(n ? 6) in 0.05% detergent as an acceptor and [2-¹⁴C]malonyl-coenzyme A (CoA) as a two-carbon donor, incorporation of radioactivity into 20:3 was optimal (and incorporation into other acyl chains was minimal) in the presence of 100 μm substrate, 200 μmp-bromophenacylbromide and 10 mm KCN. Up to 30% of the labeled products were incorporated into phospholipids and triacylglycerol. Maximal microsomal elongation activity was observed at 3–4 weeks of age. Several other fatty acid or acyl-CoA acceptors tested in this system were elongated at slower rates compared to 18:3(n ? 6) [e.g., 16:0-CoA, 75%; 20:4(n ? 6), 57%; 18:3(n ? 3), 13%; 18:2(n ?6), 10%; 20:3(n ? 6), 6%]. The rate of elongation of chemically synthesized 18:3-CoA was only 50% of the detergent-suspended acid and was optimal at 6 μm substrate; inhibition above 6 μm 18:3-CoA was reduced by bovine serum albumin, but incorporation of label into palmitate was greatly stimulated. CoA markedly inhibited elongation of 18:3(n ? 6) or 18:3-CoA; N-ethylmaleimide at equimolar amounts reversed this CoA inhibition but did not alter the inhibition caused by concentrations of 18:3-CoA above 6 μm. ATP was absolutely required for elongation of either the free acid or the acyl-CoA derivative, whereas exogenous MgCl₂ had little effect.     

Fatty acyl-CoA elongation in Blatella germanica integumental microsomes

Insect cuticular hydrocarbons are synthesized de novo in integumental tissue through the concerted action of fatty acid synthases (FASs), fatty acyl-CoA elongases, a reductase, and a decarboxylase to produce hydrocarbons and CO2. Elongation of fatty acyl-CoAs to very long chain fatty acids was studied in the integumental microsomes of the German cockroach, Blatella germanica. Incubation of [1-14C]palmitoyl-CoA, malonyl-CoA, and NADPH resulted in the production of 18-CoA with minor amounts of C20, C22, C24, C30, and C32 labeled acyl-CoA moieties. Similar experiments with [1-14C]stearoyl-CoA rendered C20-CoA as the major product, and lesser amounts of C22 and C24-CoAs were also detected. After solubilization of the microsomal FAS, kinetic parameters were determined radiochemically or by measuring NADPH consumption. The reaction velocity was linear for up to 3 min incubation time, and with a protein concentration up to 0.025 microg/microl. The effect of the chain length on the reaction velocity was compared for palmitoyl-CoA, stearoyl-CoA, and eicosanoyl-CoA. The optimal substrate concentration was 10 microM for C16-CoA, between 8 and 12 microM for C18-CoA, and close to 3 microM for C20-CoA. In vivo hydrocarbon biosynthesis was inhibited from 55.5 to 72.5% in the presence of 1 mM trichloroacetic acid, a known inhibitor of elongation reactions.     

Identification of two mammalian reductases involved in the two-carbon fatty acyl elongation cascade

The de novo synthesis of fatty acids occurs in two distinct cellular compartments. Palmitate (16:0) is synthesized from acetyl-CoA and malonyl-CoA in the cytoplasm by the enzymes acetyl-CoA carboxylase 1 and fatty acid synthase. The synthesis of fatty acids longer than 16 carbons takes place in microsomes and utilizes malonyl-CoA as the carbon source. Each two-carbon addition requires four sequential reactions: condensation, reduction, dehydration, and a final reduction to form the elongated fatty acyl-CoA. The initial condensation reaction is the regulated and rate-controlling step in microsomal fatty acyl elongation. We previously reported the cDNA cloning and characterization of a murine long chain fatty acyl elongase (LCE) . Overexpression of LCE in cells resulted in the enhanced addition of two-carbon units to C12-C16 fatty acids, and evidence was provided that LCE catalyzed the initial condensation reaction of long chain fatty acid elongation. The remaining three enzymes in the elongation reaction have not been identified in mammals. Here, we report the identification and characterization of two mammalian enzymes that catalyze the 3-ketoacyl-CoA and trans-2,3-enoyl-CoA reduction reactions in long and very long chain fatty acid elongation, respectively.     

ISOLATION OF THREE NOVEL LONG‐CHAIN POLYUNSATURATED FATTY ACID Δ9‐ELONGASES AND THE TRANSGENIC ASSEMBLY OF THE ENTIRE PAVLOVA SALINA DOCOSAHEXAENOIC ACID PATHWAY IN NICOTIANA BENTHAMIANA1

The transgenic aerobic synthesis of long‐chain polyunsaturated fatty acids (LC‐PUFA) will in most land plants commence with either a Δ6‐desaturation or a Δ9‐elongation. Numerous Δ6‐desaturases have been characterized, but only one Δ9‐elongase has been reported in peer‐reviewed literature. In the present study, we describe the isolation of three additional Δ9‐elongases from the class Haptophyceae and demonstrate that the Δ9‐elongase group contains highly conserved regions, which differentiate them from other ELO‐type elongases. One such important difference is the presence of an LQxFHH motif instead of the usual LHxYHH motif, a feature that should simplify further gene discovery efforts in this group of enzymes. Moreover, the identification of the Pavlova salina (N. Carter) J. C. Green Δ9‐elongase completes the isolation of the entire P. salina docosahexaenoic acid (DHA) pathway, and we describe the assembly of this pathway in Nicotiana benthamiana. Finally, we comment on possible explanations for the widespread presence of the Δ6‐desaturated fatty acid stearidonic acid (SDA, 18:4^Δ6,9,12,15) in the plastidial lipids of organisms using the Δ9‐elongase pathway.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Biosynthesis of saturated very long chain fatty acids by purified membrane fractions from leek epidermal cells

Elongation of fatty acids by microsomal fractions obtained from leek epidermal cells was measured by the incorporation of [1-¹⁴C]stearate, [1-¹⁴C]stearyl CoA, and [1-¹⁴C]stearyl ACP in the presence of malonyl CoA and NADPH. Stearoyl CoA appears to be the primer of the elongase (s) rather than stearoyl ACP. There are at least two elongases, the first elongating C₁₈ to C₂₀, the second synthesizing C₂₂ to C₃₀ fatty acids from C₂₀. The main site of the elongase (s) is a subcellular fraction enriched in endoplasmic reticulum. The plasma membrane-enriched fraction, which contains large amounts of saturated very long chain fatty acids, synthesizes only minor amounts of them.     

Oleoyl-CoA is not an immediate substrate for fatty acid elongation in developing seeds of Brassica napus

The substrate specificity of fatty acid elongase was studied using an oil body fraction from developing seeds of Brassica napus. ATP was essential for high rates of elongase activity, but there was no apparent requirement for oleoyl-CoA, oleic acid (18:1) or CoA. Furthermore, ¹⁴C from 18:1-CoA was incorporated into eicosenoic (20:1) and erucic (22:1) acids at a much slower rate than ¹⁴C from malonyl-CoA. Incubation of [¹⁴C]18:1-CoA with the oil body fraction resulted in a rapid loss of [¹⁴C]18:1-CoA into several lipid fractions whether in the absence or presence of ATP, but the loss of 18:1-CoA had a comparatively small effect on the overall rate of elongation. Acyl-CoAs were derivatized to their respective acylbutylamide and analyzed by gas chromatography-mass spectrometry. This analysis of acyl-CoAs demonstrated that there was no detectable 20:1-CoA or 22:1-CoA at 0 min incubation, while newly synthesized 20:1-CoA and 22:1-CoA were present at 10 min. Analysis of the %¹⁴C of the substrates and products of the elongation reaction revealed that the endogenous pool of 18:1-CoA is quite small in elongase preparations. In addition, [¹⁴C]18:1-CoA added to the incubation, although incorporated into lipids, was not significantly diluted by turnover or new synthesis. In contrast, the %¹⁴C of the 20:1-CoA was two- to threefold less than that of the 18:1-CoA. Taken together, these results indicate that the [¹⁴C]18:1 from the [¹⁴C]18:1-CoA was diluted in an intermediate 18:1 pool and that the 18:1-CoA was not the major donor of the acyl group to the elongase reaction.     

Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid

The fatty acid elongase [often designated FAE or beta-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium beta-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.     

Changes of free fatty acids and acyl-CoAs in rat brain hippocampal slice with tetraethylammonium-induced long-term potentiation

We investigated the role of acyl-CoAs during induction and maintenance of long-term potentiation in rat brain hippocampus. Changes of acyl-CoA and free fatty acids (FFA) in hippocampus were measured during tetraethylammonium (TEA)-induced LTP. Results indicated that concentrations of acyl-CoAs and FFAs in slices were changed during TEA-induced LTP and 16:0-CoA and 18:0-CoA were increased in the early phase of stimulation, whereas free fatty acids in this phase were rather decreased. The increase of 20:4-CoA was delayed more than saturated acyl-CoAs. To examine the role of acyl-CoA in LTP of evoked transmitter release, we measured the glutamate release from hippocampal slice with the addition of acyl-CoA using glutamate electrode. Acyl-CoA (16:0-, 18:1-, and 20:4-CoA) could enhance glutamate release in hippocampal slice. It is suggested that saturated acyl-CoAs may play a functional role in the early phase of LTP.     

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-¹⁴C]18:0)-CoA was synthesized from [1-¹⁴C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-¹⁴C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-¹⁴C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [¹⁴C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.     

Functional analysis of a fatty acid elongase from arachidonic acid-producing <Emphasis Type="Italic">Mortierella alpina</Emphasis> 1S-4

We describe the isolation and characterization of a gene (MAELO) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4. Although the homologous MAELO gene had already been isolated from M. alpina ATCC 32221, its function had not yet been identified. The MAELO gene from M. alpina 1S-4 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae. Analysis of the fatty acid composition of the yeast transformant revealed the accumulation of 22-, 24-, and 26-carbon saturated fatty acids. On the other hand, RNA interference of the MAELO gene in M. alpina 1S-4 was carried out. The gene-silenced strain obtained on RNA interference exhibited low contents of 20-, 22-, and 24-carbon saturated fatty acids and a high content of stearic acid (18 carbons), compared with those in the wild strain. The enzyme encoded by the MAELO gene was demonstrated to be involved in the biosynthesis of 20-, 22-, and 24-carbon saturated fatty acids in M. alpina 1S-4.     

Characterization of the Bubblegum acyl-CoA synthetase of Microchloropsis gaditana

The metabolic pathways of glycerolipids are well described in cells containing chloroplasts limited by a two-membrane envelope but not in cells containing plastids limited by four membranes, including heterokonts. Fatty acids (FAs) produced in the plastid, palmitic and palmitoleic acids (16:0 and 16:1), are used in the cytosol for the synthesis of glycerolipids via various routes, requiring multiple acyl-Coenzyme A (CoA) synthetases (ACS). Here, we characterized an ACS of the Bubblegum subfamily in the photosynthetic eukaryote Microchloropsis gaditana, an oleaginous heterokont used for the production of lipids for multiple applications. Genome engineering with TALE-N allowed the generation of MgACSBG point mutations, but no knockout was obtained. Point mutations triggered an overall decrease of 16:1 in lipids, a specific increase of unsaturated 18-carbon acyls in phosphatidylcholine and decrease of 20-carbon acyls in the betaine lipid diacylglyceryl–trimethyl–homoserine. The profile of acyl-CoAs highlighted a decrease in 16:1-CoA and 18:3-CoA. Structural modeling supported that mutations affect accessibility of FA to the MgACSBG reaction site. Expression in yeast defective in acyl-CoA biosynthesis further confirmed that point mutations affect ACSBG activity. Altogether, this study supports a critical role of heterokont MgACSBG in the production of 16:1-CoA and 18:3-CoA. In M. gaditana mutants, the excess saturated and monounsaturated FAs were diverted to triacylglycerol, thus suggesting strategies to improve the oil content in this microalga.

A heterokont Bubblegum acyl-CoA synthetase (ACSBG), or lipidosin, is essential in Microchloropsis (Nannochloropsis) gaditana and thio-esterifies 16:1 and 18:3 fatty acids to Coenzyme A in vivo.     

The Endoplasmic reticulum-associated maize GL8 protein is a component of the acyl-coenzyme A elongase ivolved in the production of cuticular waxes

The gl8 gene is required for the normal accumulation of cuticular waxes on maize (Zea mays) seedling leaves. The predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of a ketone group to a hydroxyl group. Polyclonal antibodies raised against the recombinant Escherichia coli-expressed GL8 protein were used to investigate the function of this protein in planta. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum membranes. Furthermore, polyclonal antibodies raised against the partially purified leek (Allium porrum) microsomal acyl-coenzyme A (CoA) elongase can react with the E. coli-expressed GL8 protein. In addition, anti-GL8 immunoglobulin G inhibited the in vitro elongation of stearoyl-CoA by leek and maize microsomal acyl-CoA elongase. In combination, these findings indicate that the GL8 protein is a component of the acyl-CoA elongase. In addition, the finding that anti-GL8 immunoglobulin G did not significantly inhibit the 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA dehydrase, and (E) 2,3-enoyl-CoA reductase partial reactions of leek or maize acyl-CoA elongase lends further support to our previous hypothesis that the GL8 protein functions as a beta-ketoacyl reductase during the elongation of very long-chain fatty acids required for the production of cuticular waxes.     

Fatty acid elongases in mammals: their regulation and roles in metabolism

A significant amount of the fatty acids synthesized by the cytosolic enzyme complex fatty acid synthase (FAS) or taken up by the diet are further elongated into very long chain fatty acids (VLCFA) in a four-step reaction cycle by membrane-bound enzymes predominantly located in the endoplasmic reticulum. Members of the Elovl (elongation-of-very-long-chain-fatty acids) gene family encode for enzymes (elongases), which are believed to perform the first, regulatory, step (condensation) in the elongation cycle in mammals. The family of enzymes consists of at least six members in mouse and human, believed to carry out substrate-specific elongation with fatty acids of different lengths and degrees of unsaturation. The ability to synthesize VLCFA is a ubiquitous system found in different organs and cell types. However, VLCFAs seldom occur unesterified. Instead, they are joined either by an ester or amide linkage to a broad variety of different lipid species. VLCFA are most commonly found as building blocks in sphingolipids, although they are also important constituents of glycerophospholipids, triacylglycerols, sterol- and wax-esters. To generalize, the fatty acid elongases can be divided into two major groups: (a) enzymes which are suggested to be involved in the elongation of saturated and monounsaturated VLCFA (ELOVL1, 3 and 6) and (b) enzymes which are elongases of polyunsaturated fatty acids (PUFA) (ELOVL2, 4 and 5). All the elongases exhibit specific spatial and temporal expression. In this review, we will present and discuss the regulation of the mammalian fatty acid elongases and their potential role in lipid metabolism. We will consider both the biochemical functions of the proteins, as well as their role in a more physiological context.     

Cloning and functional characterisation of polyunsaturated fatty acid elongases of marine and freshwater teleost fish

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids are key to the biosynthesis of the highly unsaturated fatty acids, arachidonic, eicosapentaenoic and docosahexaenoic acids from linoleic and alpha-linolenic acids. A Mortierella alpina cDNA polyunsaturated fatty acid elongase sequence identified mammalian, amphibian, zebrafish and insect expressed sequence tags (ESTs) in GenBank. Consensus primers were designed in conserved motifs and used to isolate full length cDNA from livers of several fish species by Rapid Amplification of cDNA Ends (RACE). The amplified cDNAs encoded putative open reading frames (ORFs) of 288-294 amino acids that were highly conserved among the fish species. Heterologous expression in yeast, Saccharomyces cerevisiae, demonstrated that all of the ORFs encoded elongases with the ability to lengthen polyunsaturated fatty acid substrates with chain lengths from C18 to C22 and also monounsaturated fatty acids, but not saturated fatty acids. There were differences in the functional competence of the elongases from different fish species. Most of the fish elongases showed a pattern of activity towards different fatty acid substrates in the rank order C18>C20>C22, although the tilapia and turbot elongases had similar activity towards 18:4n-3 and 20:5n-3. The fish elongases generally showed greater activity or similar activities with n-3 than with n-6 homologues, with the exception of the cod enzyme which was more active towards n-6 fatty acids.