Cloning and Characterization of Unusual Fatty Acid Desaturases from Anemone leveillei: Identification of an Acyl-Coenzyme A C20 Δ5-Desaturase Responsible for the Synthesis of Sciadonic Acid

摘要

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Δ^5,11,14; SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C20 Δ^5cis-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Δ⁵-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C18 Δ⁹-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Δ^5,11,14,17). Thus, AL10 acted only on C20 polyunsaturated fatty acids in a manner analogous to “front-end” desaturases. However, neither AL10 nor AL21 contain the cytochrome b5 domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Δ⁵-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Δ⁵-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Δ⁵, 18:1Δ⁵, and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Δ⁵-desaturases using acyl-CoA substrates.