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

Abstract

The health benefits attributed to very long-chain polyunsaturated fatty acids and the long term goal to produce them in transgenic oilseed crops have led to the cloning of all the genes coding for the desaturases and elongases involved in their biosynthesis. The encoded activities have been confirmed in vivo by heterologous expression, but very little is known about the actual acyl substrates involved in these pathways. Using a ⌬6-elongase and front-end desaturases from different organisms, we have reconstituted in Saccharomyces cerevisiae the biosynthesis of arachidonic acid from exogenously supplied linoleic acid in order to identify these acyl carriers. Acyl-CoA measurements strongly suggest that the elongation step involved in polyunsaturated fatty acids biosynthesis is taking place within the acyl-CoA pool. In contrast, detailed analyses of lipids revealed that the two desaturation steps (⌬5 and ⌬6) occur predominantly at the sn-2 position of phosphatidylcholine when using ⌬5-and ⌬6-desaturases from lower plants, fungi, worms, and algae. The specificity of these ⌬6-desaturases for the fatty acid acylated at this particular position as well as a limiting re-equilibration with the acyl-CoA pool result in the accumulation of ␥-linolenic acid at the sn-2 position of phosphatidylcholine and prevent efficient arachidonic acid biosynthesis in yeast. We confirm by using a similar experimental approach that, in contrast, the human ⌬6-desaturase uses linoleoyl-CoA as substrate, which results in high efficiency of the subsequent elongation step. In addition, we report that ⌬12-desaturases have no specificity toward the lipid polar headgroup or the sn-position.

A Bifunctional Δ12,Δ15-Desaturase from Acanthamoeba castellanii Directs the Synthesis of Highly Unusual n-1 Series Unsaturated Fatty Acids

“…This system uses the principles and methodologies established by Domergue et al (7) in which the presence of an acyl-CoA desaturase is detected by its ability to provide substrate for acyl-CoAdependent fatty acid elongation, whereas phospholipid-dependent desaturation does not directly generate such a substrate. A. castellanii AcD12 was co-expressed in yeast with the C 18 ⌬9-elongase from Isochrysis galbana (32).…”

“…where the fatty acid substrate is esterified to a glycerol backbone) (7). This distinction appears particularly marked in the case of microsomal desaturases; in animals such enzymes are predominantly acyl-CoA-dependent, whereas in higher plants, microsomal desaturases use phosphoglyceride substrates (7,8). Relatively little is known about the processes by which desaturases "measure" regiospecificity (i.e.…”

“…Equally, mechanistic properties can be used to separate desaturases into those requiring acyl-CoA substrates, and those which utilize glycerolipid-linked substrates (i.e. where the fatty acid substrate is esterified to a glycerol backbone) (7). This distinction appears particularly marked in the case of microsomal desaturases; in animals such enzymes are predominantly acyl-CoA-dependent, whereas in higher plants, microsomal desaturases use phosphoglyceride substrates (7,8).…”

Switching desaturase enzyme specificity by alternate subcellular targeting

“…Fatty acids from purified MGDG or lyso-MGDG were directly methylated by using NaOCH 3 as described in ref. 23. Free fatty acids were methylated by using 1 ml of 2% (vol͞vol) H 2 SO 4 in methanol and incubating for 30 min at 80°C.…”

Molecular analysis of ∆6 desaturase and ∆6 elongase from Conidiobolus obscurus in the biosynthesis of eicosatetraenoic acid, a ω3 fatty acid with nutraceutical potentials

“…ARA and EPA are two 5 desaturated fatty 382 acids in the n-6 and n-3 pathways. The ∆5 desaturation producing these two fatty acids is 383 generally believed to occur in phospholipids (Domergue et al 2003). Therefore, a high 384 level VLCPUFAs accumulated in phospholipids rather than in triacylglycerols may imply 385 that C. obscurus has developed an active system to synthesize these VLCPUFAs, but lack 386 an efficient mechanism to channel these fatty acids to the storage lipids.…”