Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop

Abstract

Omega-3 (also called n-3) long-chain polyunsaturated fatty acids (≥C20; LC-PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega-3 LC-PUFAs, i.e. eicosapentaenoic acid (20:5 n-3, EPA) and docosahexaenoic acid (22:6 n-3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega-3 LC-PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non-native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.

Keywords: Camelina sativa; desaturase; elongase; omega-3; polyunsaturated fatty acids; transgenic plants.

Figure 1

Production of EPA and DHA in C. sativa. (a) Biosynthetic pathway for the production of the LC-PUFAs EPA and DHA, with the various enzyme activities shown in different colours [also used in (b)]. (b) Simplified maps of vectors p5_EPA and p7_DHA used for transformation of C. sativa. Abbreviations: Cnl, conlinin 1 promoter for the gene encoding the flax 2S storage protein conlinin; USP, promoter region of the unknown seed protein of Vicia faba; SBP, sucrose binding protein 1800 promoter; NP, napin; OtΔ6, Δ6-desaturase from O. tauri; TcΔ5, a Δ5-desaturase from Thraustochytrium sp.; Piw3, ω3-desaturase from Phytophthora infestans; PsΔ12, a Δ12-desaturase from Phytophthora sojae; EhΔ4, Δ4-desaturase from E. huxleyi; PSE1, a Δ6-elongase from P. patens; OtElo5, Δ5-elongase from O. tauri; OCS, 35S, E9 and CatpA represent terminators.

Figure 2

Total fatty acid composition (mol%) of wild-type and engineered C. sativa oilseeds. Distribution of FAMEs in (a) wild-type C. sativa, (b) bulk fish oil, (c) the RRes_EPA line, (d) the highest EPA seed, (e) the RRes_DHA line and (f) the highest DHA seed. Endogenous fatty acids are shown in shades of green; intermediates of the introduced biosynthetic pathway are shown in shades of red, and the key target fatty acids (EPA and DHA) are shown in shades of yellow.

Figure 3

Distribution of EPA and/or DHA content (mol% of total fatty acids) in approximately 145 single seeds of RRes_EPA and RRes_DHA lines. FAMEs of single seeds derived from selected T₂ events for either RRes_EPA (n = 8) or RREs_DHA (n = 12) were analysed by GC-FID (flame ionization detection), and the mol% for either EPA (a) or EPA and DHA (b) is shown. See also Table S1.

Figure 4

Stereospecific analysis of PC and TAG in RRes_EPA and RRes_DHA lines. The positional distribution of fatty acids in TAG (sn-2 and sn-1/3) and PC (sn-1 and sn-2) was determined in wild-type C. sativa (a), RRes_EPA (b) and RRes_DHA (c).