The very long-chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid (ARA, 20:4ω6), is a component of neuron tissues such as brain and retina cells and an important component of the human diet. ARA is a primary substrate for the biosynthesis of eicosanoids, including the 2-group prostaglandins, 4-group leukotrienes, thromboxanes and lipoxins that serve as biological effectors involved in inflammatory and immune responses and cell signaling. Being an important and dominant VLC-PUFA in human breast milk, ARA needs to be externally supplied for normal development of preterm babies if they are not breast-fed. Due to its beneficial properties there is a growing interest in the production of ARA for baby formulae. At present, the major commercial source of ARA is the filamentous fungus Mortierella alpina. 
Microalgae are the most efficient producers and one of the richest sources of VLC-PUFAs. Furthermore, algae can be used as sources of genes for the implementation of VLC-PUFA biosynthesis in genetically engineered oil crops. The genetic information on enzymes involved in the biosynthesis of VLC-PUFA in some algae led to in vivo applications of VLC-PUFA production in seed oil. The gene pool of the green freshwater microalga Parietochloris incisa (Trebouxiophyceae) is of special interest since it is the only known microalga able to accumulate extraordinary high amounts of ARA-rich triacylglycerols (TAG). When P. incisa is cultivated under nitrogen starvation, the condition triggering storage oil accumulation, ARA constitutes about 60 percent of total fatty acids (TFA) and over 95 percent of cellular ARA is deposited in TAG in cytoplasmic lipid bodies.
The biosynthesis of VLC-PUFAs in algae follows various pathways initiating from oleic acid exported from the chloroplast and employing polar extraplastidial lipids. In the ω6 and ω3 pathways, linoleic acid (LA; 18:2ω6) and α-linolenic acid (ALA; 18:30ω3) are successively converted by Δ6 desaturase, Δ6 elongase and Δ5 desaturase to ARA and eicosapentaenoic acid (EPA, 20:5ω3), respectively. E.g., In P. incisa, as well as the red microalga Porphyridium cruentum, ARA biosynthesis proceeds via the ω6 pathway.
Unusual elongations and desaturations leading to the biosynthesis of VLC-PUFA have been reported in the marine haptophyte Isochrysis galbana and the fresh water euglenophyte Euglena gracilis. In the alternative route, elongation of 18:2ω6 and 18:3ω3 by a C18-Δ9-specific fatty acid elongase to the respective C20 intermediates precedes sequential Δ8 and Δ5 desaturations to ARA and EPA, respectively. It is assumed that in E. gracilis EPA produced by the ω3-Δ8 pathway is further Δ4 desaturated and finally elongated to docosahexaenoic acid (DHA, 22:6ω3).
Fatty acid elongation is a multi-step process involving four sequential enzymatic reactions: rate limiting condensation (of malonyl-CoA and acyl-CoA), reduction, dehydration and enoyl reduction. Only the expression of the condensing enzyme component is required to reconstitute elongase activity in the heterologous host; there is no requirement for the co-expression of any other component of the elongase complex. Multiple microsomal elongation systems with different specificities to the acyl chain length exist in various organisms. Recent studies have identified and characterized PUFA-specific elongases, responsible for the elongation of PUFA in mammals, fish, algae, lower plants and fungi. The elongation of 18:3ω6 to 20:3ω6, the immediate precursor of ARA, was shown to be the rate limiting step in ARA biosynthesis in M. alpina. Functional expression of the PUFA elongase condensation component in yeast revealed enzymes of various specificities for C18 and C20 acyl substrates. Thus, two types of PUFA elongases engaged in DHA biosynthesis were cloned from the green microalga Ostreococcus tauri and the diatom Thalassiosira pseudonana: OtELO1 and TpELO1 are Δ6 C18-PUFA specific and involved in the elongation of the 18:3ω6 and 18:4ω3, while OtELO2 and TpELO2 are Δ5 C20-PUFA elongases involved in the elongation of 20:5ω3. Bifunctional PUFA elongases able to elongate both Δ6 and Δ5 PUFA, as well as elongases of wide substrate specificity utilizing both C20 and C22 PUFA substrates, were isolated from aquatic animals.
USPTO Backgrounds
USPTO Backgrounds