Expression of a borage desaturase cDNA containing an N-terminal cytochrome b(5) domain results in the accumulation of high levels of Delta(6)-desaturated fatty acids in transgenic tobacco

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Sayanova, O. V., Smith, M. A., Lapinskas, P., Stobart, A. K., Dobson, G., Christie, W. W., Shewry, P. R. and Napier, J. A. 1997. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b(5) domain results in the accumulation of high levels of Delta(6)-desaturated fatty acids in transgenic tobacco. Proceedings of the National Academy of Sciences of the United States of America. 94 (8), pp. 4211-4216.

AuthorsSayanova, O. V., Smith, M. A., Lapinskas, P., Stobart, A. K., Dobson, G., Christie, W. W., Shewry, P. R. and Napier, J. A.
Abstract

γ-Linolenic acid (GLA; C18:3 Δ6,9,12) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a Δ6-fatty acid desaturase using linoleic acid (C18:2 Δ9,12) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the Δ6-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 Δ6,9,12,15) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage Δ6-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b5. Δ6-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 Δ9,12), the first step being the desaturation to γ-linolenic acid (GLA; C18:3 Δ6,9,12) catalyzed by a Δ6-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 Δ9 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 Δ12- and Δ15-desaturases use fatty acids located at the sn-2 position of phosphatidylcholine as substrates, and cytochrome b5 as a cofactor (5, 6). The Δ6-fatty acid desaturase in the developing cotyledons of borage is similar to the Δ12- and Δ15-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 Δ6,9,12,15) in borage leaves (9). We describe the isolation of a cDNA clone encoding the Δ6-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 (Δ12,15) from higher plants (8, 13).

KeywordsRRES175; 175_Plant sciences; 175_Genetics
Year of Publication1997
JournalProceedings of the National Academy of Sciences of the United States of America
Journal citation94 (8), pp. 4211-4216
Digital Object Identifier (DOI)doi:10.1073/pnas.94.8.4211
Open accessPublished as bronze (free) open access
Publisher's version4211.full.pdf
ISSN00278424
PublisherNational Academy of Sciences

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