Heterologous Reconstitution of Omega-3 Polyunsaturated Fatty Acids in Arabidopsis

Abstract

Reconstitution of nonnative, very-long-chain polyunsaturated fatty acid (VLC-PUFA) biosynthetic pathways in Arabidopsis thaliana was undertaken. The introduction of three primary biosynthetic activities to cells requires the stable coexpression of multiple proteins within the same cell. Herein, we report that C22 VLC-PUFAs were synthesized from C18 precursors by reactions catalyzed by Δ(6)-desaturase, an ELOVL5-like enzyme involved in VLC-PUFA elongation, and Δ(5)-desaturase. Coexpression of the corresponding genes (McD6DES, AsELOVL5, and PtD5DES) under the control of the seed-specific vicilin promoter resulted in production of docosapentaenoic acid (22:5 n-3) and docosatetraenoic acid (22:4 n-6) as well as eicosapentaenoic acid (20:5 n-3) and arachidonic acid (20:4 n-6) in Arabidopsis seeds. The contributions of the transgenic enzymes and endogenous fatty acid metabolism were determined. Specifically, the reasonable synthesis of omega-3 stearidonic acid (18:4 n-3) could be a useful tool to obtain a sustainable system for the production of omega-3 fatty acids in seeds of a transgenic T3 line 63-1. The results indicated that coexpression of the three proteins was stable. Therefore, this study suggests that metabolic engineering of oilseed crops to produce VLC-PUFAs is feasible.

Figure 1

Metabolism of the two series of polyunsaturated fatty acids (PUFAs). Eicosapentaenoic acid (EPA, 20:5 n-3) is the main n-3 VLC-PUFA derived from the essential precursor α-linolenic acid (ALA, 18:3 n-3), and arachidonic acid (ARA, 20:4 n-6) is the main n-6 VLC-PUFA derived from the essential precursor linoleic acid (LA, 18:2 n-6). VLC-PUFAs are synthesized by successive elongations and desaturations. The key enzymes involved in this work are indicated in the pathway with large bold arrows.

Figure 2

Schematic of constructs used for coexpression of the genes McD6DES, AsELOVL5, and PtD5DES in seeds of Arabidopsis. (a) The intermediate vector pGEM7Zf, used for individual seed-specific expression constructs. (b) The seed-specific expression vector D6ELD5, designed for easy coexpression of multiple genes. Vicilin P: vicilin promoter; OCS: octopine synthase terminator; bar, used to confer Basta resistance to plants under the control of a CaMV 35S promoter (P35S).

Figure 3

Analysis of pCAM::D6ELD5 transcript accumulation in seeds of Arabidopsis. The genes McD6DES, AsELOVL5, and PtD5DES were assayed for transcript abundance. Data were normalized to ACTIN1 mRNA levels and expressed as 2^−ΔΔCT. Mean values obtained from three independent experiments are shown by the line.