Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

Abstract

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega-3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl-CoA substrates in the presence of malonyl-CoA. To distinguish between enzymes that desaturate CoA- and phosphatidylcholine-linked fatty acid substrates, we developed a novel in vitro method. We show that a delta-12 desaturase from Phytophthora sojae, an omega-3 desaturase from Phytophthora infestans and a delta-4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine-linked acyl substrates with comparatively low use of acyl-CoA substrates. To further validate our method, a delta-9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl-CoA as substrate, but could not use phosphatidylcholine-linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s11745-017-4235-4) contains supplementary material, which is available to authorized users.

Comparison of the Substrate Preferences of ω3 Fatty Acid Desaturases for Long Chain Polyunsaturated Fatty Acids

“…The desaturases Pi-ω3D and Sd-ω3D obtained from EPA-rich fungi and the animal desaturase Ce-ω3D revealed high activity levels for the C20 substrates, 20:3ω6 (DGLA) and 20:4ω6 (ARA), and higher still for the C22 substrates, 22:4ω6 (DTA) and 22:5ω6 (DPAω6). Recently, Yilmaz et al [13] also demonstrated the activities of Pi-ω3D for C18, C20 and C22 fatty acid substrates in yeast, but in contrast to our study in leaf, observed lower activities to C22 than C20 fatty acids. On the other hand, Sd-ω3D, when expressed in yeast, exclusively desaturated C20 fatty acid substrates (5% conversion of 20:3ω6 to 20:4ω3 and 26% conversion of 20:4ω6 to 20:5ω3) [11].…”

“…In contrast, the ω3D from an EPA-rich fungus, Saprolgenia diclina , when expressed in yeast, did not recognize C18 substrates and was active only on C20 ω6 fatty acid substrates [11]. More broadly, the ω3D from another EPA-rich fungus, Phytophthora infestans [12], had greater activity on C20 and C22 fatty acid substrates than on C18 substrates but was active on all of those tested [13]. The FAT-1 ω3D of the EPA-containing animal species Caenorhabditis elegans [14] was highly active on C18 fatty acids as well as the C20 substrate, dihomo-gamma-linolenic acid (20:3ω6) [15].…”

Genome‐wide characterization of Phytophthora infestans metabolism: a systems biology approach

“…In fact, the BRENDA database holds information on an omega‐3 desaturase able to convert arachidonic acid into EPA (EC 1.14.19.25). This exact enzyme of P. infestans has recently been proven to be capable of catalysing this reaction in yeast (Yilmaz et al ., ). We also revealed one unannotated tyrosine decarboxylase gene in the P. infestans genome, emphasizing the fact that the genome annotation of the reference genome published by Haas et al .…”

Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil

“…For example, the acyl-CoA desaturation ability of the M. pusilla 6-desaturase was described in Petrie et al (2010). In addition, Yilmaz et al (2017) demonstrated that the P. lutheri 4-desaturase, a gene FIGURE 6 | Fatty acid profiles of (A) Brassica napus AV-Jade cultivar (parental), and (B) GA7_mod-B transgenic B. napus seeds after being stored for six months at 4 • C, 24 • C, or 32 • C. T0 = time zero (no storage). Data were analyzed by One Way Analysis of Variance (ANOVA) using Sigmaplot (v13) by the Holm-Sidak method.…”