Molecular cloning and characterization of the genes encoding a microsomal oleate Δ12 desaturase (CsFAD2) and linoleate Δ15 desaturase (CsFAD3) from Camelina sativa

Camelina sativa produces oil that is rich in polyunsaturated linoleic (18:2(Delta 9,12)) and linolenic (18:3(Delta 9,12,15)) acids. These fatty acids are obtained by the successive desaturation of oleic (18:1(Delta 9)) acid, which is catalyzed in the endoplasmic reticulum by two different microsomal desaturases: oleate Delta(12) desaturase (FAD2) and linoleate Delta(15) desaturase (FAD3). The objective of the present study is to investigate the contribution of these two desaturases to the composition and properties of C. sativa seed oil, and look over the effect that temperature exerts on their activity. Three different copies of both these genes were identified, which when analysed contained three histidine rich motifs (HXCGHX, HRXHH and HVXHH) and six highly conserved transmembrane domains. Comparing their sequences, the CsFAD2 copies accommodated four conservative changes (Glu-36-Asp, Arg-48-His, Val-97-Ala and Ala-177-Pro) and two semi-conservative ones (Val-63-Ile and Leu-249-Met), whereas only one semi-conservative change (Ala-327-Ser) was detected in CsFAD3 but with two extra amino acids (His-147 and Gly-148). The CsFAD2 and CsFAD3 cDNAs were heterologously expressed in Saccharomyces cerevisiae to confirm that they were active enzymes and their dependence on temperature was investigated by growing the recombinant yeast cells at low (22 degrees C) and optimal (30 degrees C) temperatures. The conversion of 18:1(Delta 9) to 18:2(Delta 9,12) by CsFAD2 was slightly better at 30 degrees C than at 22 degrees C, and it was also able to desaturate palmitoleic acid (16:1 9) to hexadecadienoic acid (16:2 9,12). By contrast, the conversion driven by CsFAD3 was enhanced 5-fold at 22 degrees C compared to 30 C, although it was not able to desaturate 16:2(Delta 9,12). The distribution of these microsomal desaturases was also studied in C sativa, which were expressed most strongly in expanding leaves and developing seeds. (C) 2016 Elsevier B.V. All rights reserved.