An engineered oilseed crop produces oil enriched in two very long chain polyunsaturated fatty acids with potential health-promoting properties.

Very long chain polyunsaturated fatty acids (VLCPUFAs) are well recognized for their health benefits in humans and animals. Here we report that identification and characterization of a gene (EhELO1) encoding the first functional ELO type elongase (3-ketoacyl-CoA synthase) in higher plants that is involved in the biosynthesis of two VLCPUFAs docosadienoic acid (DDA, 22:2n-6) and docosatrienoic acid (DTA, 22:3n-3) that possess potential health-promoting properties. Functional analysis of the gene in yeast indicated that this novel enzyme could elongate a wide range of polyunsaturated fatty acids with 18-22 carbons and effectively catalyze the biosynthesis of DDA and DTA by the sequential elongations of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata showed that the transgenic plants produced the level of DDA and DTA at approximately 30% of the total fatty acids in seeds, and the amount of the two fatty acids remained stable over four generations. The oilseed crop producing a high and sustained level of DDA and DTA provides an opportunity for high value agricultural products for nutritional and medical uses.

Phytophthora infestans cholinephosphotransferase with substrate specificity for very-long-chain polyunsaturated fatty acids.

The effective flux between phospholipids and neutral lipids is critical for a high level of biosynthesis and accumulation of very-long-chain polyunsaturated fatty acids (VLCPUFAs), such as arachidonic acid (ARA; 20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3). Here we describe a cDNA (PiCPT1) from Phytophthora infestans, a VLCPUFA-producing oomycete, that may have a role in acyl trafficking between diacylglycerol (DAG) and phosphatidylcholine (PC) during the biosynthesis of VLCPUFAs. The cDNA encodes a polypeptide of 393 amino acids with a conserved CDP-alcohol phosphotransferase motif and approximately 27% amino acid identity to the Saccharomyces cerevisiae cholinephosphotransferase (ScCPT1). In vitro assays indicate that PiCPT1 has high cholinephosphotransferase (CPT) activity but no ethanolaminephosphotransferase (EPT) activity. Substrate specificity assays show that it prefers VLCPUFA-containing DAGs, such as ARA DAG and DHA DAG, as substrates. Real-time PCR analysis reveals that expression of PiCPT1 was upregulated in P. infestans organisms fed with exogenous VLCPUFAs. These results lead us to conclude that PiCPT1 is a VLCPUFA-specific CPT which may play an important role in shuffling VLCPUFAs from DAG to PC in the biosynthesis of VLCPUFAs in P. infestans.

A peroxygenase pathway involved in the biosynthesis of epoxy fatty acids in oat.

While oat (Avena sativa) has long been known to produce epoxy fatty acids in seeds, synthesized by a peroxygenase pathway, the gene encoding the peroxygenase remains to be determined. Here we report identification of a peroxygenase cDNA AsPXG1 from developing seeds of oat. AsPXG1 is a small protein with 249 amino acids in length and contains conserved heme-binding residues and a calcium-binding motif. When expressed in Pichia pastoris and Escherichia coli, AsPXG1 catalyzes the strictly hydroperoxide-dependent epoxidation of unsaturated fatty acids. It prefers hydroperoxy-trienoic acids over hydroperoxy-dienoic acids as oxygen donors to oxidize a wide range of unsaturated fatty acids with cis double bonds. Oleic acid is the most preferred substrate. The acyl carrier substrate specificity assay showed phospholipid and acyl-CoA were not effective substrate forms for AsPXG1 and it could only use free fatty acid or fatty acid methyl esters as substrates. A second gene, AsLOX2, cloned from oat codes for a 9-lipoxygenase catalyzing the synthesis of 9-hydroperoxy-dienoic and 9-hydroperoxy-trienoic acids, respectively, when linoleic (18:2-9c,12c) and linolenic (18:3-9c,12c,15c) acids were used as substrates. The peroxygenase pathway was reconstituted in vitro using a mixture of AsPXG1 and AsLOX2 extracts from E. coli. Incubation of methyl oleate and linoleic acid or linolenic acid with the enzyme mixture produced methyl 9,10-epoxy stearate. Incubation of linoleic acid alone with a mixture of AsPXG1 and AsLOX2 produced two major epoxy fatty acids, 9,10-epoxy-12-cis-octadecenoic acid and 12,13-epoxy-9-cis-octadecenoic acid, and a minor epoxy fatty acid, probably 12,13-epoxy-9-hydroxy-10-transoctadecenoic acid. AsPXG1 predominately catalyzes intermolecular peroxygenation.

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

Conidiobolus obscurus, an entomopathogenic fungus able to infect aphids, was previously reported to produce substantial amounts of very long chain polyunsaturated fatty acids (VLCPUFAs) that may mediate the insect infection. However, the genes involved in the biosynthesis of these VLCPUFAs from the order Entomophthorales have yet to be identified. Using degenerate reverse transcriptase-polymerase chain reaction and rapid amplification of the cDNA end methods, we cloned a ∆6 desaturase cDNA (CoD6) and a ∆6 elongase cDNA (CoE6) from C. obscurus. Expression of CoD6 and CoE6 in Saccharomyces cerevisiae revealed CoD6 could introduce a Δ6 double bond into α-linolenic acid (18:3n-3), and CoE6 preferentially elongated 18-carbon Δ6 desaturated fatty acid stearidonic acid (18:4n-3). When the fungus was grown under a temperature shift from 20 °C to 10 °C, the transcript level of CoD6 and CoE6 increased, whereas when the fungal culture was shifted from 20 °C to 30 °C, the transcript level of both genes decreased. The entire eicosatetraenoic acid biosynthetic pathway was reconstituted in yeast using four genes, CoD6 and CoE6 from C. obscurus, CpDes12 (a Δ12 desaturase) and CpDesX (a ω3 desaturase) from Claviceps purpurea. Yeast transformants expressing the four genes produced ten new fatty acids including the final product eicosatetraenoic acid (ETA). This represents the reconstitution of the entire ETA pathway in yeast without supplementation of any exogenous fatty acids.