ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use

The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.     

The Evolution of Fatty Acid Desaturases and Cytochrome b5 in Eukaryotes

Desaturases that introduce double bonds into the fatty acids are involved in the adaptation of membrane fluidity to changes in the environment. Besides, polyunsaturated fatty acids (PUFAs) are increasingly recognized as important pharmaceutical and nutraceutical compounds. To successfully engineer organisms with increased stress tolerance or the ability to synthesize valuable PUFAs, detailed knowledge about the complexity of the desaturase family as well as understanding of the coevolution of desaturases and their cytochrome b5 electron donors is needed. We have constructed phylogenies of several hundred desaturase sequences from animals, plants, fungi and bacteria and of the cytochrome b5 domains that are fused to some of these enzymes. The analysis demonstrates the existence of three major desaturase acyl-CoA groups that share few similarities. Our results indicate that the fusion of Δ⁶-desaturase-like enzymes with their cytochrome b5 electron donor was a single event that took place in the common ancestor of all eukaryotes. We also propose the Δ⁶-desaturase-like enzymes as the most probable donor of the cytochrome b5 domain found in fungal Δ⁹-desaturases and argue that the recombination most likely happened soon after the separation of the animal and fungal ancestors. These findings answer some of the previously unresolved questions and contribute to the quickly expanding field of research on desaturases.     

Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activity with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.     

The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans

Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n − 9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans.     

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.     

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.     

Identification of Primula “front-end” desaturases with distinct n−6 or n−3 substrate preferences

cDNA clones encoding cytochrome b₅ fusion desaturases were isolated from Primula cortusoides L. and Primula luteola Ruprecht, species previously shown to preferentially accumulate either n−6 or n−3 Δ6-desaturated fatty acids, respectively. Functional characterisation of these desaturases in yeast revealed that the recombinant Primula enzymes displayed substrate preferences, resulting in the predominant synthesis of either γ-linolenic acid (n−6) or stearidonic acid (n−3). Independent expression of the two Primula desaturases in transgenic Arabidopsis thaliana confirmed these results, with γ-linolenic acid and stearidonic acid accumulating in both leaf and seed tissues to different levels, depending on the substrate specificity of the desaturase. Targeted lipid analysis of transgenic Arabidopsis lines revealed the presence of Δ6-desaturated fatty acids in the acyl-CoA pools of leaf but not seed tissue. The implications for the transgenic synthesis of C₂₀ polyunsaturated fatty acids via the elongation of Δ6-desaturated fatty acids are discussed, as is the potential of using Primula desaturases in the synthesis of C₁₈ n−3 polyunsaturated fatty acids such as stearidonic acid.     

Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(5,11,14); SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(5)-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(5)-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.     

Identification of a fatty acid Delta11-desaturase from the microalga Thalassiosira pseudonana

A set of genomic DNA sequences putatively encoding front-end desaturases were identified by in silico analysis of the draft genome of the marine microalga Thalassiosira pseudonana. Among these candidate genes, an open reading frame named TpdesN was found to be full-length, intronless, and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpDESN, exhibited typical features of desaturases involved in the production of polyunsaturated fatty acids (PUFAs) in algae, i.e. a cytochrome b5-like domain at the N-terminus and three conserved histidine-rich motifs in the desaturase domain. Expression of TpDESN in Saccharomyces cerevisiae revealed that this enzyme was not involved in PUFA synthesis, but specifically desaturated palmitic acid 16:0 to 16:1Delta11. To our knowledge, until this report, Delta11-desaturase activity had only been detected in insect cells.     

Mutagenesis and heterologous expression in yeast of a plant Delta6-fatty acid desaturase.

Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' desaturases. These enzymes are also characterized by the presence of a cytochrome b5 domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a 'front end' desaturase, the borage Delta6-fatty acid desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue.     

The role of cytochrome b5 in delta 12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.)

The electron donors for the membrane-bound fatty acid desaturases of higher plants have not previously been identified. In order to assess the participation of cytochrome b5 in microsomal fatty acid desaturation, the cytoplasmic domain of microsomal cytochrome b5 was purified from Brassica oleracea, and murine polyclonal antibodies were prepared. The IgG fraction from ascites fluid inhibited 62% of NADH-dependent cytochrome c reduction in safflower (Carthamus tinctorius L.) microsomes. These antibodies also blocked desaturation of oleic acid to linoleic acid in lipids of C. tinctorius microsomes by 93%, suggesting that cytochrome b5 is the electron donor for the delta 12 desaturase.     

The alternative pathway C20 Delta8-desaturase from the non-photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5-fusion desaturase

A cDNA encoding a C20 Delta8-desaturase was isolated from the free-living soil amoeba, Acanthamoeba castellanii and functionally characterised by heterologous expression. The open reading frame of the A. castellanii C20 Delta8-desaturase showed similarity to other microsomal front-end desaturases, but the N-terminal domain contained a variant form of the conserved heme-binding motif in which H-P-G-G is replaced by H-P-A-G. Co-expression of the A. castellani Delta8-desaturase with the Isochrysis galbana Delta9-elongase in transgenic Arabidopsis plants confirmed the activity observed in yeast and its role in the alternative pathway for C20 polyunsaturated fatty acid synthesis. Acyl-CoA profiles of these transgenic plants revealed an unexpected accumulation of C20 fatty acids in the acyl-CoA pool. This is the first report of an alternative pathway C20 Delta8-desaturase from a non-photosynthetic organism, and also the first report of a front-end desaturase lacking the canonical cytochrome b5 domain.     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

“自我剪切”2A肽介导的Δ-12和ω-3脂肪酸脱氢酶以及过氧化氢酶在转基因小鼠肌肉表达研究

哺乳动物因为缺乏Δ-12和ω-3脂肪酸脱氢酶,不能自身合成必需的多不饱和脂肪酸．目前,通过转基因技术在哺乳动物体内表达ω-3脂肪酸脱氢酶,能将长链的n-6多不饱和脂肪酸转化成n-3多不饱和脂肪酸,造成体内长链的n-6多不饱和脂肪酸含量显著减低．本研究通过自我剪切2A肽介导Δ-12和ω-3脂肪酸脱氢酶(FAT-2和FAT-1)以及人过氧化氢酶(human catalase,hCAT)在小鼠的肌肉同时表达．结果表明,转基因小鼠肌肉中长链n-3多不饱和脂肪酸含量提高2.6倍,长链n-6多不饱和脂肪酸含量没有显著变化,而n-6/n-3比例显著降低(P < 0.01)．同时蛋白质印迹检测到人过氧化氢酶hCAT在小鼠的肌肉组织中表达,且过氧化氢酶活性比野生型小鼠显著提高(P < 0.01)．     

A second functional delta5 fatty acid desaturase in the cellular slime mould Dictyostelium discoideum.

A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www. csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the Delta5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain-of-function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced amino-acid sequence comprised 467 amino-acid residues containing an N-terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the Delta5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5-containing Delta5 fatty acid desaturase. As we have already reported one other Delta5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional Delta5 fatty acid desaturase genes. The substrate specificities of the two functional Delta5 desaturases of D. discoideum were also examined.     

Polychlorinated biphenyls increase fatty acid desaturation in the proliferating endoplasmic reticulum of pigeon and rat livers

1. Polychlorinated biphenyls (PCB) are abundant and persistent pollutants in the ecosystem. Commercial mixtures (e.g. Aroclor 1254) can contain up to 80 different isomers and congeners, many of which accumulate in biological systems by the ingestion of PCB-contaminated lipid components of food chains. 2. Commercial mixtures of PCB induce, in hepatic microsomal membranes in vivo, a variety of different forms of the cytochrome P-450 components of enzyme systems involved in the metabolism of drugs and other xenobiotics, and can also induce the proliferation of this membrane. Since these microsomal enzyme systems share a number of the requirements of microsomal fatty acid desaturases, we have investigated whether the induction by PCB in vivo of cytochrome-P-450-linked enzymes in the proliferating hepatic microsomal membrane of the pigeon and the rat is accompanied by increased proportions of polyunsaturated fatty acids in this membrane. 3. The most striking changes observed 120 h after treating pigeons and rats with 1.5 mmol Aroclor 1254/kg body mass were 2.2-fold and 1.6-fold increases, respectively, in the proportion of arachidonic acid in the hepatic microsomal membrane. When the effects of this treatment on the proliferation of this membrane and increase in liver mass are taken into account, the amount of arachidonic acid in the total microsomal membrane of pigeon and rat livers increased 6.7-fold and 1.9-fold, respectively. 4. These changes were accompanied by very significant increases in pigeons and rats of the concentration of hepatic microsomal cytochrome P-450, and in the activity in microsomal protein of a wide range of cytochrome P-450-dependent enzyme involved in the metabolism of drugs and other xenobiotics. 5. This effect of PCB, of increasing in vivo the degree of unsaturation of fatty acids of hepatic microsomal membrane, appears to be a novel finding, and does not seem to have been investigated for other drugs and xenobiotics. Preliminary results have shown that the effect is accompanied by substantial increases in the total activity of delta 6 and delta 5 microsomal fatty acid desaturases converting 18:2 (9, 12) (linoleic acid) to 20:4 (5, 8, 11, 14) (arachidonic acid) [Borlakoglu, J.T., Dils, R.R., Edwards-Webb, J.D. & Walker, C.H. (1988) Biochem. Soc. Trans. 16, 1072]. 6. It is postulated that there is a significant link between increased fatty acid desaturation and the induction of cytochrome-P-450-linked enzymes, and this is discussed in terms of the mechanisms involved in the metabolism of foreign compounds.