Biosynthesis of C₁₈ polyunsaturated fatty acids in microsomal membrane preparations from the filamentous fungus Mucor circinelloides

Abstract

The biosynthesis of C 18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides. Microsomal membrane preparations contained ∆9, ∆12 and ∆6 desaturase activities. The ∆9 desaturase exhibited characteristics similar to those of the animal and yeast ∆9 desaturases in being membrane bound and utilising stearoyl-CoA as substrate. Cytochrome b 5 (a soluble form lacking the 20-amino-acid hydrophobic C-terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes. A high ferricyanide reductase activity (indicative of NADH:cytochrome b 5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes. Time-course studies with radiolabelled oleoyl-CoA showed that the oleate [18:1(9)] was transferred to position sn-2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl-PtdCho. Removal of the excess oleoyl-CoA from the membranes prior to addition of reductant confirmed that oleoyl-PtdCho is a substrate for the ∆12 desaturase. The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso-PtdCho:acyl-CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl-CoA to lyso-PtdCho. Desaturation of oleate at the sn-1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway. Thus oleate at sn-1 and sn-2 positions served as substrate for the ∆12 desaturase and is consistent with observations in oil seed tissues. LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine: acyl-CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis. No desaturation on phosphatidylinositol could be demonstrated. ∆6 desaturase utilised linoleate at the sn-2 position of exogenously supplied PtdCho presented to the membranes in the presence of reductant. Thus, the entry of substrates into PtdCho via LPCAT and the synthesis of linoleate [18:2(9,12)] and γ-linolenate [18:3(6,9,12)] on this phospholipid is similar to that reported for oil seed membranes.Keywords : Mucor ; oil biosynthesis; polyunsaturated fatty acid ; desaturase substrate; phosphatidylcholine.The biosynthesis of polyunsaturated fatty acids in plants [1Ϫ that contain high levels of 18:3(6,9,12) [12Ϫ14] and have been 3] and animals [4,5] and their assembly to produce storage fats produced on an industrial scale [15]. We have studied previously and oils has been studied extensively. By comparison, and with the biosynthesis of 18:3(6,9,12) in higher plants and characterthe exception of oleaginous yeast [6,7], few detailed biochemi-ised the substrates and stereospecificity of the desaturases incal studies on lipid synthesis in fungi have been undertaken. Of volved in its formation [16Ϫ18]. In borage seed microsomes, particular commercial interest is the production of 'speciality the ∆12 desaturase utilised oleate esterified to positi...

Identification of bifunctional Δ12/ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants

“…All of these ⌬12 desaturase-related proteins have the three histidine boxes proposed to be involved in active-site domains and hydrophobic membranespanning regions of membrane-bound fatty acid desaturases. Their similarity to fungal ⌬12 desaturase reported to desaturate phospholipid-linked substrates (21) would argue that these also use phospholipid-linked fatty acid substrate.…”

Isolation of a Δ5-Fatty Acid Desaturase Gene fromMortierella alpina

“…The resultant DNA was packaged into phage particles to produce a library of 4ϫ10 6 plaque-forming units with an average insert size of 1.4 kilobases. The library was screened by standard techniques (16) using cloned PCR products as probes.…”

“…In all other respects, it is very similar to M. alpina (6). It thus has genes encoding ⌬ 9 -, ⌬ 12 -, and ⌬ 6 -desaturase activities and is likely to lack a ⌬ 5 -desaturase gene.…”

“…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.…”

Acyl Carriers Used as Substrates by the Desaturases and Elongases Involved in Very Long-chain Polyunsaturated Fatty Acids Biosynthesis Reconstituted in Yeast

“…VLCPUFAs are mainly found in fish, in some fungi and lower plants, as well as in a variety of microorganisms of the phytoplankton. With the exception of the anaerobically operating polyketide synthase-like systems found in some marine bacteria and primitive eukaryotes (4), VLC-PUFAs are synthesized by elongation and desaturation of linoleic acid (LA, 18:2 ⌬9,12 ) or ␣-linolenic acid (ALA, 18:3 ⌬9, 12,15 ) in the endoplasmic reticulum. Most algae, fungi, and lower plants producing VLCPUFAs possess the entire biosynthetic pathway to synthesize these fatty acids from acetate, whereas mammalia, which lack ⌬12-and ⌬15-desaturases, use as precursors LA and ALA that have to be supplied in their diet and thus are essential fatty acids.…”