Tailoring seed oil composition in the real world: optimising omega-3 long chain polyunsaturated fatty acid accumulation in transgenic Camelina sativa

Abstract

There is considerable interest in the de novo production of omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), not least of all given the importance of these fatty acids in both aquaculture and human nutrition. Previously we have demonstrated the feasibility of using metabolic engineering in transgenic plants (Camelina sativa) to modify the seed oil composition to now include EPA and/or DHA. In this study, we further tailored the seed oil profile to reduce the omega-6 content, and evaluated the performance of such GM plants under field conditions (i.e. environmental releases), in terms of agronomic performance and also the lipidomic profile of seed oil. We used MALDI- mass spectrometry imaging to identify discrete tissue-types in the seed in which these non-native fatty acids preferentially accumulated. Collectively, these data provide new insights into the complexity of plant lipid metabolism and the challenges associated with predictive manipulation of these pathways. However, this study identified the likely dispensable nature of a Δ12-desturase activity in our omega-3 metabolic engineering rationales for Camelina.

Figure 1

Production of EPA and DHA in the seeds of C. sativa. (a) The conventional Δ6 biosynthetic pathway for the production of omega-3 LC-PUFAs EPA and DHA, with the various desaturase and elongase enzyme activities shown in different colours [also used in (b)]. (b) Simplified maps of vectors EPA _B4_1 and DHA_33_13 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; Ot∆6, ∆6-desaturase from O. tauri; Tc∆5, a Δ5-desaturase from Thraustochytrium sp.; Piw3, ω3-desaturase from Phytophthora infestans; Ps∆12, a Δ12-desaturase from Phytophthora sojae; EhΔ4, ∆4-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 laboratory and field-grown wild-type and engineered C. sativa seeds. Distribution of FAMEs in wildtype C. sativa grown (2015) in the field (a) and laboratory (b); the EPA producing line EPA_B4_1 grown (2015) in the field (c) and laboratory (d); and a comparison of the DHA producing line, DHA5_33, grown in the field in 2014 (e) and 2015 (f). An average of 75 seeds analysed by GC-FID (flame ionisation detection). 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

The accumulation of EPA and DHA in transformed field-grown seeds of C. sativa. The levels of specific n-3 EPA and DHA are shown in (a) and (b) for the individual sub plots of EPA_B4_1 and DHA5_33, respectively. The results are based on single seed analysis (n = 75); values for EPA are shown in yellow and DHA represented in orange.

Figure 4

Seed yield and agronomic performance. (a) Total seed oil (n = 6+/− SE); (b) carbon (n = 6+/− SE); (c) water (n = 19+/− SE) and (d) nitrogen (n = 6+/− SE) content was determined and presented for each sub-plot of the field grown C. sativa.

Figure 5

Analysis of triacylglycerols from mature C. sativa seeds. TAG molecular species were characterised from wild-type, EPA_B4_1 and DHA5_33 using a ESI-MS/MS neutral loss survey scan with each TAG species represented by the total number of fatty acid carbon atoms:desaturations. A Students T-test was used to independently compare C. sativa wildtype with EPA-B4_1 and DHA5_33. Those TAG molecular species with significantly (p 0.05) different accumulations are shown (a). Further analysis of the mass spectrometry data acquired for the engineered lines identified those significant TAG species containing either EPA (b) or DHA (c).

Figure 6

Representative images of TAG metabolites in mature field-grown seed of C. sativa. Cross-sections of embryos show the spatial distribution of specific TAG molecular species in wildtype (a), EPA_B4_1 (b) and DHA5_33_13 (c). Compare to bright-field images for orientation. False-colour images are converted from mol% of class amounts with red as the highest relative amount.