Cloning and molecular characterization of the Δ6‐desaturase from two Echium plant species: Production of GLA by heterologous expression in yeast and tobacco

Abstract

The synthesis of GLA (delta6,9,12-1-8:3) is carried out in a number of plant taxa by introducing a double bond at the delta6 position of its precursor, linoleic acid (delta9,12-18:2), through a reaction catalyzed by a delta6-desaturase enzyme. We have cloned genes encoding the delta6-desaturase (D6DES) from two different Macaronesian Echium species, E. pitardii and E. gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds. The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b(5) domain at the N-terminus. Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other delta6-desaturases in a different cluster from that of the highly related delta8-desaturases. Expression analysis carried out in E. pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant. Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves. Functional characterization of the D6DES gene from E. gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae. In both cases, overexpression of the gene led to the synthesis of GLA. Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.

Functional characterisation of two cytochrome�b5-fusion desaturases from Anemone leveillei: the unexpected identification of a fatty acid ?6-desaturase

“…The sphingolipid LCB D 8 desaturases insert a double bond six carbons from the last oxygen-carrying carbon atom of the alkyl chain in the LCBs and this common regio-specificity is also indicative of an apparent paralogous relationship with the D 6 -desaturases (Sperling et al 1998;Napier et al 1999). Whilst several fatty acid D 6 -desaturases have previously been characterised from higher plants, these have all been isolated from the borage family and, as such, display a very high level of sequence identity (Sayanova et al 1997;Garcia-Maroto et al 2002). The (unexpected) identification of an Anemone D 6 -desaturase from the taxonomically unrelated Ranunculaceae provides new information to facilitate the identification of functionally conserved amino acid residues within this higher-plant enzyme.…”

Identification of Primula “front-end” desaturases with distinct n−6 or n−3 substrate preferences

“…The resultant PCR products of the expected size (»870 bp) were cloned and sequenced. The cloned PCR products isolated from these two Primula species were found to encode polypeptides similar to known fatty acid 6 desaturases from higher plants (Borago oYcinalis, Echium spp, Primula spp, Anemone leveillei; Sayanova et al 1997Sayanova et al , 2003Garcia-Maroto et al 2002;Whitney et al 2003) and also to plant sphingolipid 8-desaturases (Sperling et al 1998;Libisch et al 2000). These partial sequences were used to design 5Ј-RACE primers to amplify the 5Ј ends of two putative desaturases.…”

Δ6-Desaturase sequence evidence for explosive Pliocene radiations within the adaptive radiation of Macaronesian Echium (Boraginaceae)

“…This gene encodes an acyl-desaturase enzyme catalyzing the synthesis of polyunsaturated fatty acids (Alonso and Maroto, 2000). The functional D 6 -desaturase gene (D6DES) has been cloned from different Macaronesian Echium species (García-Maroto et al, 2002, and Southern blotting analysis revealed a single-copy in their genomes (García-Maroto et al, 2002). The inverse PCR technique allowed us to isolate the D6DES promoter region (D6DES-p) even from distant continental Echium species.…”