Formation of conjugated Δ¹¹Δ¹³‐double bonds by Δ¹²‐linoleic acid (1,4)‐acyl‐lipid‐desaturase in pomegranate seeds

Abstract

For the biosynthesis of punicic acid (18:3 D9Z,11E,13Z) a (11,14)-linoleoyl desaturase activity has been proposed. To isolate this acyl-lipid-desaturase, PCR-based cloning was used. This approach resulted in the isolation of two complete cDNAs. The first isolated full-length cDNA harbors a sequence of 1350 bp encoding a protein of 395 amino acids. The second cDNA was 1415 bp long encoding a protein of 387 amino acids. For functional identification proteins encoded by the cDNAs were expressed in Saccharomyces cerevisiae, and formation of newly formed fatty acids was analyzed by gas chromatography-free induction decay (GC-FID) and GC/MS. The expression of the heterologous enzymes resulted in the first case in a significant amount of linoleic acid and in the second case, after linoleic acid supplementation, in formation of punicic acid. The results presented here identify one cDNA coding for a classical D 12-acyl-lipid-desaturase. The other one codes for a new type of (1,4)-acyl-lipid-desaturase that converts a cis double bond located in the D 12-position of linoleic acid or c-linolenic acid, but not in a-linolenic acid, into a conjugated cis-trans double bond system.

Δ12-Oleate Desaturase-related Enzymes Associated with Formation of Conjugated trans-Δ11, cis-Δ13 Double Bonds

“…Very recently cDNA encoding PuFADX, an enzyme identical to PgFac, was isolated from P. granatum (10 (Fig. 3).…”

“…Two types of conjugases associated with the formation of conjugated double bonds in trans-configuration have been identified: one catalyzes the conversion of a cis-⌬12 double bond into the conjugated trans-⌬11, trans-⌬13 double bonds found in ␣-eleostearic acid (7), and the other modifies a cis-⌬9 double bond into the trans-⌬8, trans-⌬10 double bonds of calendic acid (8,9). Very recently a class of conjugase associated with the formation of cis-⌬11, trans-⌬13 double bonds of punicic acid was identified (10). Although the primary structures of these conjugases are similar, each enzyme specifically catalyzes the formation of ␣-eleostearic, punicic, or calendic acids.…”

A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

“…Fatty acids were transesterified with sodium methoxide in methanol or methanol/toluol solution (26). Fatty acid methyl esters (FAMEs) were then analyzed by gas-liquid chromatography.…”

Activity of Brassica napus and Perilla frutescens microsomal ω-3 desaturases expressed in yeast (Saccharomyces cerevisiae)

“…E. coli competent DH5α cells were used for the transformation of the ligation reaction yields, and then minipreps using the QIAprep Spin Miniprep system (QIAGEN) were carried out. Double digestion with BamH1 and EcoR1 restriction enzymes was followed by loading 5 µL of each the reaction products on a 0.9% agarose gel as a verification of successful cloning (Elledge et al, 1991;Hornung et al, 2002). Constructions obtained of yeast expression plasmid were used for yeast transformation and expression experiments.…”

“…In addition, yeast can take up and incorporate many kinds of fatty acids from the growth medium; it has a low level of β-oxidation and it has the carbon sources needed for the incorporation of the exogenously provided substrate and any fatty acid that results from the heterologous expression of plant desaturases (Kunau et al, 1987;Covello and Reed, 1996;Watts and Browse, 1999;Cahoon et al, 2002). For instance, exogenous feeding of oleic acid to yeast transformed with a Δ-12 desaturase successfully results in the production of linoleate (Covello and Reed, 1996;Hornung et al, 2002). In plants, ω-3 fatty acid desaturation is found in 2 different cell compartments on 2 different substrate classes (Somerville and Browse, 1991;Los and Murata, 1998).…”