Expression of a borage desaturase cDNA containing an N-terminal cytochrome <em>b</em>₅ domain results in the accumulation of high levels of Δ⁶-desaturated fatty acids in transgenic tobacco

Abstract

Δ^{-Desaturated fatty acids are of major importance in animal cells as they have roles in the maintenance of membrane structure and function, in the regulation of cholesterol synthesis and transport, in the prevention of water loss from the skin, and as precursors of eicosanoids, including prostaglandins and leucotrienes (}1). In animals, members of this class of fatty acids are synthesized from the essential fatty acid linoleic acid (C18:2 Δ^{), the first step being the desaturation to γ-linolenic acid (GLA; C18:3 Δ) catalyzed by a Δ-desaturase (}1). Decreased activity of this key enzyme, observed for example in aging, stress, diabetes, eczema, and some infections, or increased catabolism of GLA resulting from oxidation or more rapid cell division (e.g., in cancer or inflammation) may lead to a deficiency of GLA (reviewed in ref. 2). Clinical trials have shown that dietary supplementation with GLA may be effective in treating a number of such conditions (e.g., atopic eczema, mastalgia, diabetic neuropathy, viral infections, and some types of cancer; ref. 2). Oils containing GLA are therefore widely used as a general health supplement and have been registered for pharmaceutical use.

In the plant kingdom, GLA is an uncommon fatty acid (3). Only a small number of higher plant species synthesize GLA, and in many of these, the fatty acid is found exclusively in the seed. GLA is also present in some fungi (e.g., Mucor javanicus) and cyanobacteria (3). Major commercial sources of GLA (4) are evening primrose (Oenothera spp.), in which GLA accounts for about 8–10% of the seed oil and borage (starflower) (Borago officinalis L.) seeds that contain some 20–25% GLA. These plants, however, suffer from poor agronomic performance and low yield; borage, for example, produces 300–600 kg/ha in the United Kingdom (4) compared with about 3 t/ha for oilseed rape. There is therefore considerable interest in both increasing the GLA content of existing crops and the production of GLA in a conventional oil crop (such as high linoleate rape).

In the higher plant cell, the synthesis of saturated fatty acids with chain lengths up to C18 and monounsaturated fatty acids (generally with a double bond at the Δ ^{position) occurs in the plastid. Further desaturation can then occur either in the plastid or on the endoplasmic reticulum (ER; ref.} 5). The desaturase enzymes of the plastid require reduced ferredoxin as an electron donor and are either soluble enzymes acting on saturated acyl-ACP substrates or membrane-bound enzymes using unsaturated fatty acids esterified to complex lipids such as monogalactosyldialglycerol. In contrast, the ER-located Δ^{- and Δ-desaturases use fatty acids located at the} sn-2 position of phosphatidylcholine as substrates, and cytochrome b₅ as a cofactor (5, 6). The Δ^{-fatty acid desaturase in the developing cotyledons of borage is similar to the Δ- and Δ-desaturases in its location and substrate specificity (oleate/linoleate at the} sn-2 position of phosphatidylcholine), and is assumed to use cytochrome b₅ as its electron donor (7, 8). In addition, α-linolenic acid esterified to phosphatidylcholine may act as a substrate, resulting in the accumulation of octadecatetraenoic acid (OTA; C18:4 Δ^{) in borage leaves (}9).

We describe the isolation of a cDNA clone encoding the Δ^{-fatty acid desaturase from developing seeds of borage, using a PCR-based strategy. The identity of the cDNA has been confirmed by functional expression and analysis in transgenic tobacco plants. The encoded protein differs from other membrane-bound fatty acid desaturases of plants, such as those encoded by the FAD genes of} Arabidopsis (10, 11), in that the desaturase domain is preceded at the N terminus by a sequence that is related to cytochrome b₅ (12), the haemprotein involved in electron transport to other ER-located fatty acid desaturases (Δ^{) from higher plants (}8, 13).

Percentages were integrated from peak areas of GC traces shown in Fig. ​Fig.3.3. ND, not detected.

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