A Front-end Desaturase from Chlamydomonas reinhardtii Produces Pinolenic and Coniferonic Acids by ω13 Desaturation in Methylotrophic Yeast and Tobacco

Abstract

Pinolenic acid (PA; 18:3Δ^5,9,12) and coniferonic acid (CA; 18:4Δ^5,9,12,15) are Δ⁵-unsaturated bis-methylene-interrupted fatty acids (Δ⁵-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2Δ^9,12) and α-linolenic acid (ALA; 18:3Δ^9,12,15), respectively, by Δ⁵-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C.reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Δ⁵-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Δ⁵-desaturase activity on 18:1Δ⁹ but not 18:1Δ¹¹. Unexpectedly, CrDES also showed Δ⁷-desaturase activity on 20:2Δ^11,14 and 20:3Δ^11,14,17 to produce 20:3Δ^7,11,14 and 20:4Δ^7,11,14,17, respectively. Since both the Δ⁵ bond in C18 and the Δ⁷ bond in C20 fatty acids are ‘ω13’ double bonds, these results indicate that CrDES has ω13 desaturase activity for ω9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale.