Cloning of a Higher-Plant Plastid [omega]-6 Fatty Acid Desaturase cDNA and Its Expression in a Cyanobacterium

Abstract

Oligomers based on amino acids conserved between known plant w-3 and cyanobacterium w -6 fatty acid desaturases were used to screen an Arabidopsis cDNA library for related sequences. An identified clone encoding a novel desaturase-like polypeptide was used to isolate its homologs from Glycine mar and Brassica napus. The plant deduced amino acid sequences showed less than 27% similarity to known plant w -6 and w-3 desaturases but more than 48% similarity to cyanobacterial w-6 desaturase, and they contained putative plastid transit sequences. Thus, we deduce that the plant cDNAs encode the plastid w-6 desaturase. l h e identity was supported by expression of the B. napus cDNA in cyanobaderium.Synechococcus transformed with a chimeric gene that contains a prokaryotic promoter fused to the rapeseed cDNA encoding all but the first 7 3 amino acids partially converted its oleic acid fatty acid to linoleic acid, and the 161(9c) fatty acid was converted primarily to 162(9c,12) in vivo. lhus, the plant w -6 desaturase, which utilizes 16:1(7c) in plants, can utilize 161(9c) in the cyanobaderium. l h e plastid and cytosolic homologs of plant w-6 desaturases are much more distantly related than those of w-3 desaturases.In leaf tissue there are two distinct pathways for the biosynthesis of the polyunsaturated fatty acids 1 8 2 and 18:3, one located in cytosolic membranes and the other located in plastid membranes. In Arabidopsis thaliana, cytosolic and plastid w-6 fatty acid desaturations that result in the production of diene fatty acids are controlled by the FAD 2 and FAD 6 loci, respectively. Cytosolic and plastid w-3 desaturations that result in the production of triene fatty acids are controlled by FAD 3 and FAD 7, respectively (Lemieux et al., 1990;Browse and Somerville 1991). It has been postulated that these loci correspond to structural genes for the desaturase enzymes. For FAD 3, proof of this postulate has come from the cloning of a cDNA encoding a desaturase corresponding to the FAD 3 locus (Arondel et al., 1992; Yadav et al., 1993).Cytosolic w-3 fatty acid desaturase cDNAs have been used to isolate their plastid homologs by screening plant cDNA libraries under low-stringency hybridization conditions, and the amino acid sequences of the higher-plant 0-3 desaturases show significant similarity (68% or greater) both among different plant species and between the microsomal and plastid homologs within each species (Iba et al., 1993; Yadav et al., 1993). However, this screening did not result in the isolation of cDNAs encoding other than w-3 desaturases.* Corresponding author; fax 1-302-695-4296.Recently, the T-DNA tagging method was used to isolate the Arabidopsis microsomal w-6 fatty acid desaturase (FAD 2) and it showed only 36% similarity at the amino acid leve1 with the w-3 desaturases (Okuley et al., 1994). In contrast to the results with w-3 desaturases, low-stringency screening of an Arabidopsis cDNA library with the Arabidopsis microsomal w-6 desaturase cDNA did not result in the identification of its plastid...

Identification of bifunctional Δ12/ω3 fatty acid desaturases for improving the ratio of ω3 to ω6 fatty acids in microbes and plants

“…It will be interesting to study how these ⌬12͞ 3 enzymes exert their dual positional specificity, because the regioselectivity of ⌬12 and 3 desaturases is Јv ϩ 3Ј (i.e., three carbons away from an existing double bond) and Ϫ3 (i.e., three carbons from the methyl end), respectively (30,31). Fatty acid feeding studies demonstrated that the fungal 3 desaturases also showed a broad catalytic promiscuity with respect to 3 desaturation on both C18-and C20-carbon 6 substrates.…”

Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria

“…The 15 histidine residues in the four conserved domains are found in all the A12 desaturases. The cDNAs and genes for A12 acyl-lipid desaturases have also been isolated from higher plants [40,41]. Comparisons of amino acid sequences of cyanobacterial A12 desaturases with those of higher plant A12 desaturases reveals that histidine residues are highly conserved in these desaturases also.…”

Lipid biosynthesis.

“…fAD3 was isolated by both map-based chromosome walking (Arondel et al, 1992) and T-DNA tagging. The FADG (Hitz et al, 1994), FAD7 (Iba et al, 1993;Yadav et al, 1993), and FADB (Gibson et al, 1994) genes were all cloned based on their homology to FADP and FAD8 The homology of these genes with gene products from other organisms that have been characterized at the biochemical level indicates that the FAD loci represent the structural genes for desaturases rather than proteins that control the desaturation reactions in any other way. The power of the approaches available to Arabidopsis researchers is evidenced by the fact that clones are available now for more than six different desaturases, whereas research in animal lipid metabolism has yielded only a single, membrane-bound desaturase clone.…”