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High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds

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Abstract

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova™400.

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Acknowledgments

This work was conducted under an existing agreement between SemBioSys Genetics Inc. and Arcadia Biosciences for commercial applications protected under US patent application 20070067870 filed by Arcadia Biosciences.

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  1. SemBioSys Genetics Inc., 110, 2985-23 Ave NE, Calgary, AB, T1Y 7L3, Canada

    Cory L. Nykiforuk, Indra Harry, Olga P. Yurchenko, Mei Zhang, Catherine Reed, Gunamani S. Oinam, Steve Zaplachinski, Joseph G. Boothe & Maurice M. Moloney

  2. BluGoose Consulting, Woodland, CA, 95776, USA

    Christine Shewmaker

  3. Arcadia Biosciences Inc., 202 Cousteau Place, Suite 200, Davis, CA, 95618, USA

    Ana Fidantsef

  4. Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, UK

    Maurice M. Moloney

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  1. Cory L. Nykiforuk
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11248_2011_9543_MOESM1_ESM.pdf

Figure S1. Plant binary vectors and strategies employed to accumulate GLA in safflower. (a) The plant binary expression vectors designed to express Δ6-fatty acid desaturase from Saprolegnia diclina (pSBS 4119), Δ6-fatty acid desaturase from Mortierella alpina (pSBS4763) or the double expression cassette encoding for both Δ6-fatty acid desaturase and Δ12-fatty acid desaturase from M. alpina (pSBS4766) under the transcriptional control of Arabidopsis oleosin promoter (OleoP) and terminator (OleoT) to ensure tightly controlled expression during seed development. Other genetic elements of the TDNA including RB, right border; UbiP, ubiquitin promoter, PAT, phosphinothricin acetyltransferase gene; UbiT, ubiquitin terminator; LB, left border are described in Experimental procedures. (b) Schematic of recombinant Δ6-fatty acid desaturase (Δ6 FAD) and Δ12-fatty acid desaturase (Δ12 FAD) activities introduced into high oleic (S317) and high linoleic (Centennial) backgrounds by Agrobacterium-mediated transformation as described in Experimental procedures within the context of endogenous Δ9-fatty acid desaturase (Δ9 FAD) and Δ12-fatty acid desaturase (Δ12 FAD) activity present in safflower and further described below. Supplementary material 1 (PDF 115 kb)

11248_2011_9543_MOESM2_ESM.pdf

Figure S2. Representative GLC chromatograms following the separation of FAMES on the HP-Innowax column (as described in Experimental procedures). (a) The identification of FAMES was performed using NuChek 502 GLC standard in combination with a retention time established with (b) mGLA standard (C18:3Δ6,9,12 cis) also purchased from NuChek (Elysian, MN, USA). Representative chromatograms are also provided for transgenic seed in the high linoleic acid variety (c) expressing pSBS4119 (S. diclina Δ6-FAD) in comparison to a (d) pSBS4119 null segregant (NS) and (e) expressing pSBS4763 (M. alpina Δ6-FAD) in comparison to a (f) pSBS4763 null segregant (NS). (g) A representative chromatogram of wild type Centennial (WT CENT) is also provided for comparisons between non-transformed seed, null segregants and transgenic seed. Representative chromatograms presented for transgenic seed in the high oleic acid variety (h) expressing the double expression cassette pSBS4766 (M. alpina Δ12-FAD and Δ6-FAD) in comparison to (i) a pSBS4766 null segregant (NS) and (j) non-transformed wild type S317 seed are also provided. Supplementary material 2 (PDF 83 kb)

11248_2011_9543_MOESM3_ESM.pdf

Figure S3. FAMEs analysis of the different classes of lipids between seed accumulating high-levels of GLA from pSBS4119 (S. diclina Δ6-FAD) transformations in comparison to the non-transformed wild-type Centennial seed in the separated lipid classes consisting of phospholipids (PL) and triacylgylcerol (TAG) separated by TLC prior to transmethylation as described in experimental procedures. Each assessment is the mean plus or minus one standard deviation (n = 3) and the *horizontal line with hanging arrows represents P values of less than 0.05 between LA (C18:2Δ9,12 cis) and GLA (C18:3Δ6,9,12 cis) for the PL analysis and OA (C18:1Δ9 cis), LA (C18:2Δ9,12 cis) and GLA (C18:3Δ6,9,12 cis) for the TAG analysis. Supplementary material 3 (PDF 15 kb)

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Nykiforuk, C.L., Shewmaker, C., Harry, I. et al. High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds. Transgenic Res 21, 367–381 (2012). https://doi.org/10.1007/s11248-011-9543-5

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