Isolation of a Δ5-Fatty Acid Desaturase Gene fromMortierella alpina

Abstract

Arachidonic acid (C20:4 ⌬ 5,8,11,14 ) is a polyunsaturated fatty acid synthesized by the ⌬ 5 -fatty acid desaturation of di-homo-␥-linolenic acid (C20:3 ⌬ 8,11,14 ). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the ⌬ 5 -fatty acid desaturase from M. alpina via a polymerase chain reaction-based strategy using primers designed to the conserved histidine box regions of microsomal desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina ⌬ 5 -desaturase is the first example of a cloned ⌬ 5 -desaturase, and differs from other fungal desaturases previously characterized by the presence of an N-terminal domain related to cytochrome b 5 .The filamentous fungus M. alpina is unusual in that it can produce a wide range of polyunsaturated fatty acids. It differs from higher plants in its fatty acid unsaturation as it is able to produce arachidonic acid (C20: 4 ⌬ 5,8,11,14 ) and eicosapentaenoic acid (C20: 5 ⌬ 5,8,11,14,17 ) (1). In filamentous fungi, polyunsaturated fatty acids are present both in the phospholipids and the triacylglycerols, indicating that they have a role in membrane structure and as storage oils (2). In animals, arachidonic acid is a precursor of the short-lived regulatory molecules, the eicosanoids, which comprise the prostaglandins, the leukotrienes, and the thromboxanes (3). All mammalian cells except erythrocytes synthesize eicosanoids, and among their many functions they are involved in inflammatory response, reproductive function, and regulation of blood pressure (4).In fungi, unsaturated fatty acids are formed in the endoplasmic reticulum by the action of integral membrane-bound fattyacid desaturase enzymes, which sequentially insert double bonds into the acyl chain (5, 6). The ⌬ 5 -desaturase is responsible for the conversion of di-homo-␥-linolenic acid (C20:3 ⌬ 8,11,14 ) to arachidonic acid. It is thought to function like the other microsomal desaturases from higher plants and yeast, catalyzing aerobic reactions requiring cytochrome b 5 as a cofactor. Electrons are transferred from NADH-dependent cytochrome b 5 reductase, via the heme-containing cytochrome b 5 molecule to the fatty acid desaturase (7,8).Biochemical characterization of membrane-bound desaturases has been limited because they are difficult to purify due to their hydrophobic nature. Hence, molecular genetic approaches, particularly the use of Arabidopsis mutants, have provided much information on desaturation reactions (9, 10). Analysis of the predicted protein sequences for the higher plant desaturases together with those from cyanobacteria, yeast, and mammals revealed the presence of eight highly conserved histidine residues (11). Mutagenesis studies on the microsomal ⌬ 9 -desaturase from rat and the ⌬ 12 -desaturase from Synechocystis ...

Insights into the phylogenetic and molecular evolutionary histories of Fad and Elovl gene families in Actiniaria

“…In clade A, a functionally characterized Δ6 Fad protein with the ability to desaturate PUFAs from the plant species, Borago officinalis , is present (Sayanova et al., 1997). In clade C, a functionally characterized Δ5 Fad from the oleaginous fungus, Mortierella alpina , was found (Michaelson, Lazarus, Griffiths, Napier, & Stobart, 1998). The fatty acid profile of M. alpina has been found to have high levels of ARA and also EPA when conditions are optimal, but lack > C 20 PUFAs, such as DHA (Knutzon et al., 1998; Michaelson et al., 1998).…”

“…The fatty acid profile of M. alpina has been found to have high levels of ARA and also EPA when conditions are optimal, but lack > C 20 PUFAs, such as DHA (Knutzon et al., 1998; Michaelson et al., 1998). Furthermore, M. alpina has been shown to encode an additional Δ6 desaturase enzyme; however, this sequence is not present in the Swiss‐prot database and therefore was not included in this analysis (Knutzon et al., 1998; Michaelson et al., 1998). …”

Zebrafish cDNA Encoding Multifunctional Fatty Acid Elongase Involved in Production of Eicosapentaenoic (20:5n-3) and Docosahexaenoic (22:6n-3) Acids

“…This is in contrast to all other vertebrate and eukaryotic systems studied in which desaturase activities are encoded by disparate genes (Aki et al, 1999;Cho et al, 1999a,b;Leonard et al, 2000a;Michaelson et al, 1998;Napier et al, 1998;Watts and Browse, 1998).…”

Characterization and comparison of fatty acyl Δ6 desaturase cDNAs from freshwater and marine teleost fish species

“…Full-length cDNAs for Δ6 desaturases have been isolated from the filamentous fungus Mortierella alpina (Huang et al, 1999), the nematode Caenorhabditis elegans (Napier et al, 1998), rat (Aki et al, 1999), mouse and human (Cho et al, 1999a). Fatty acid Δ5 desaturase genes have been isolated from M. alpina (Michaelson et al, 1998a), C. elegans (Michaelson et al, 1998b;Watts and Browse, 1999) and human (Cho et al, 1999b;Leonard et al, 2000). Using extant sequence information, we isolated a cDNA from the freshwater zebrafish (Danio rerio) with high similarity to mammalian Δ6 desaturase genes (Hastings et al, 2001).…”