Probing the limits of very long chain polyunsaturated fatty acid accumulation in transgenic Brassica napus

Transgenic oilseeds capable of producing high levels of very long chain polyunsaturated fatty acids (VLCPUFA) represent a promising alternative to marine oils as a source of nutritionally important fatty acids such as arachidonic acid (ARA, 20:4Delta5,8,11,14) and eicosapentaenoic acid (EPA, 20:5Delta5,8,11,14,17). Previous studies demonstrated that in transgenic oilseeds, elongation of fatty acids from C18 to C20 is a major bottleneck in VLCPUFA accumulation, likely due to limited availability of polyunsaturated C18 acyl-CoA substrates for elongation. The objective of the current study was to determine the underlying biochemical basis for the absence of C18-PUFA in the acyl-CoA pool of transgenic Brassica napus engineered to produce ARA and EPA. A comparison of acyl-CoA dependent acyltransferase activities in developing seeds suggested that the high activity of lysophosphatidylcholine acyltransferase (LPCAT) throughout seed development may support the rapid channeling of acyl-CoAs into phosphatidylcholine (PC). However, both 14C-acetate and fatty acid feeding experiments with zygotic embryos of B. napus showed that fatty acids are incorporated into triacylglycerol (TAG), with no substantial short- or long-term accumulation in phosphatidylcholine (PC). Additionally, it was demonstrated application of exogenous gamma-linolenic acid (GLA, 18:3Delta6,9,12 ) to cultured zygotic embryos increases its availability in the acyl-CoA pool and results in a significant increase in dihomo-gamma-linolenic acid (DGLA, 20:3Delta,8,11,14). Attempts to manipulate the availability of cytosolic malonyl-CoA through citrate, malonate or acetate supplementation did not result in a further increase in GLA elongation, suggesting malonyl-CoA availability does not limit overall elongation efficiency in transgenic B. napus. Finally, preliminary experiments investigating the fate of acyl groups at the sn-2 position of PC revealed considerable movement of acyl groups from PC to phosphatidic acid (PA), suggesting the presence of a highly active phospholipase D. The contribution of this enzyme to acyl group trafficking in the context of VLCPUFA accumulation remains to be determined.;In summary, this study effectively ruled out the hypothesis that acyl-CoA availability for elongation is limited by competition and rapid removal by other acyl-CoA dependent activities, and supports the hypothesis that newly desaturated PUFA are channeled into storage lipids through acyl-CoA independent routes, thereby limiting their availability for elongation.