The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.

In mammalian cells, Sprecher has proposed that the synthesis of long-chain PUFA from the 20-carbon substrates involves two consecutive elongation steps, a delta6-desaturation step followed by retroconversion (Sprecher, H., Biochim. Biophys. Acta 1486, 219-231, 2000). We searched the database using the translated sequence of human elongase ELOVL5, whose encoded enzyme elongates monounsaturated and polyunsaturated FA, as a query to identify the enzyme(s) involved in elongation of very long chain PUFA. The database search led to the isolation of two cDNA clones from human and mouse. These clones displayed deduced amino acid sequences that had 56.4 and 58% identity, respectively, to that of ELOVL5. The open reading frame of the human clone (ELOVL2) encodes a 296-amino acid peptide, whereas the mouse clone (Elovl2) encodes a 292-amino acid peptide. Expression of these open reading frames in baker's yeast, Saccharomyces cerevisiae, demonstrated that the encoded proteins were involved in the elongation of both 20- and 22-carbon long-chain PUFA, as determined by the conversion of 20:4n-6 to 22:4n-6, 22:4n-6 to 24:4n-6, 20:5n-3 to 22:5n-3, and 22:5n-3 to 24:5n-3. The elongation activity of the mouse Elovl2 was further demonstrated in the transformed mouse L cells incubated with long-chain (C20- and C22-carbon) n-6 and n-3 PUFA substrates by the significant increase in the levels of 24:4n-6 and 24:5n-3, respectively. This report demonstrates the isolation and identification of two mammalian genes that encode very long chain PUFA specific elongation enzymes in the Sprecher pathway for DHA synthesis.

Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited delta12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid. The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited delta6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the delta12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the delta6-desaturase activity, the level of gamma-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the delta6- and delta12-desaturase cDNA resulted in the endogenous production of gamma-linolenic acid. The yields of gamma-linolenic acid reached as high as 8% of total fatty acids in yeast.

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of ␥-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two ⌬6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina ⌬5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

A DNA fragment with homology to ⌬6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known ⌬6-desaturase genes. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M. alpina library. Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had ⌬5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant ⌬5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina ⌬5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (⌬5,9 -18:2) and pinolenic acid (⌬5,9,12-18: 3), which are the ⌬5-desaturation products of oleic and linoleic acids, respectively.

Long-chain n-3 PUFAs (polyunsaturated fatty acids) such as EPA (eicosapentaenoic acid; 20:5 n-3) have important therapeutic and nutritional benefits in humans. In plants, cyanobacteria and nematodes, omega3-desaturases catalyse the formation of these n-3 fatty acids from n-6 fatty acid precursors. Here we describe the isolation and characterization of a gene ( sdd17 ) derived from an EPA-rich fungus, Saprolegnia diclina, that encodes a novel omega3-desaturase. This gene was isolated by PCR amplification of an S. diclina cDNA library using oligonucleotide primers corresponding to conserved regions of known omega3-desaturases. Expression of this gene in Saccharomyces cerevisiae, in the presence of various fatty acid substrates, revealed that the recombinant protein could exclusively desaturate 20-carbon n-6 fatty acid substrates with a distinct preference for ARA (arachidonic acid; 20:4 n-6), converting it into EPA. This activity differs from that of the known omega3-desaturases from any organism. Plant and cyanobacterial omega3-desaturases exclusively desaturate 18-carbon n-6 PUFAs, and a Caenorhabditis elegans omega3-desaturase preferentially desaturated 18-carbon PUFAs over 20-carbon substrates, and could not convert ARA into EPA when expressed in yeast. The sdd17 -encoded desaturase was also functional in transgenic somatic soya bean embryos, resulting in the production of EPA from exogenously supplied ARA, thus demonstrating its potential for use in the production of EPA in transgenic oilseed crops.

Four groups of male Long-Evans rats were reared artificially from postnatal d 5 to 18 by being fed through a gastrostomy tube with rat milk substitutes containing oils providing 10% linoleic acid and 1% alpha-linolenic acid (g/100 g fat); with the use of a 2 x 2 design, they were fed one of two levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) (0.0 and 2.5 g/100 g of fatty acids). A fifth artificially reared group was fed a diet high in saturated fat, and a sixth group was reared by dams fed a standard AIN-93M diet. The pups were weaned onto modified AIN-93G diets, with a fat composition similar to that fed during the artificial rearing period. Behavioral testing was conducted between 6 and 9 wk of age; brain lipid composition was then assessed. Relative to the unsupplemented group (0.0 g/100 g AA and DHA), dietary supplementation resulted in a wide range of AA (84-103%) and particularly DHA (86-119%) levels in forebrain membrane phospholipids. AA supplementation increased AA levels and decreased DHA levels, and DHA supplementation increased DHA levels and decreased AA levels, with the magnitude of these effects dependent on the level of the other fatty acid. DHA levels were very low in the saturated fat group. The groups did not differ on the place or cued version of the Morris water-maze, but on a test of working memory, the saturated fat group was impaired relative to the suckled control group. Further correlational analyses in the artificially reared animals did not support a relationship between the wide range of DHA and AA levels in the forebrain and working-memory performance.

The seed oil from a genetically transformed canola (Brassica napus) containing 43% (w/w) of γ-linolenic acid (G, 18:3n-6), 22% linoleic acid (L, 18:2n-6), and 16% oleic acid (O, 18:1n-9) was evaluated. In this high γ-linolenic acid canola oil (HGCO), the predominant 18:3n-6-containing triacylglycerol (TG) molecular species were GGL (23%), GLO (20%), and GGG (11%). In the total TG, approximately 75% of the 18:3n-6 was located at the sn-1,3 positions, while only 34% of linoleic acid was at the sn-1,3 positions. The GGL molecular species of HGCO contained approximately equal amounts of GLG and GGL positional isomers, while the GLO molecular species had 95% GOL and 5% GLO isomers. The general characteristics and the tocopherol and phytosterol contents were mostly similar between HGCO and nontransformed canola oil. No detectable amounts of amino acids and nucleotides were observed in the HGCO.Dietary supplementation of γ-linolenic acid (G, or ∆6,9,12-18:3, or 18:3n-6) is beneficial in many physiological and pathological conditions such as inflammatory diseases, cancer, and diabetes (1-4). Gamma (γ)-linolenic acid is commercially available mainly from the seed oils of borage, black currant, and evening primrose. It is also found in some fungi and algae such as Mortierella spp. and Spirulina spp. However, γ-linolenic acid-containing oils are not widely used due to their high production cost, fluctuation in availability, or safety concerns. In order to develop an economical, reliable, and wholesome source of γ-linolenic acid, we have genetically transformed a low α-linolenic acid canola plant into strains that can produce high levels of γ-linolenic acid (5). The nontransformed canola plants produce seed oils with large amounts of oleic (O, 18:1n-9) and linoleic (L, ∆9,12-18:2) acids. By introducing the ∆6-desaturase (converts L to G) and the ∆12-desaturase (converts O to L) genes from the fungus M. alpina, within a seed-specific expression vector, into canola plant cells, a high level of 18:3n-6 was produced in seeds of the genetically transformed canola plant. The seeds from the fifth generation field trial were crushed, and the oil was extracted and refined in a commercial facility. The oil is the first kind of genetically transformed canola oil that contains a high level of 18:3n-6. In this study, we evaluated the fatty acid composition, triacylglycerol (TG) molecular species, positional distribution of n-6 fatty acids, unsaponifiable matter (phytosterols and tocopherols), general quality, and nonlipid constituents of this high-18:3n-6 canola oil (HGCO) and compared them with properties of the oils from borage, black currant, evening primrose, and the nontransformed canola. EXPERIMENTAL PROCEDURESCanola oils. In this study, the seed oils from transgenic and nontransformed canola (Brassica napus) were analyzed. The method of cloning ∆12-and ∆6-desaturases from M. alpina and recombinant production of 18:3n-6 in plants was previously reported (5). Briefly, two cDNA clones with homology to known desaturase genes were is...