Metabolic Evidence for the Involvement of a [delta]4-Palmitoyl-Acyl Carrier Protein Desaturase in Petroselinic Acid Synthesis in Coriander Endosperm and Transgenic Tobacco Cells

Abstract

We have previously demonstrated that the double bond of petroselinic acid ( 18:1Akis) in coriander (Coriandrum safivum L.) seed results from the activity of a 36-kD desaturase that is structurally related to the Ag-stearoyl-acyl carrier protein (ACP) desaturase (E.B. Cahoon, J. Shanklin, J.B. Ohlrogge [1992] Proc Natl Acad Sci USA 89: 11 184-1 1188). To further characterize the biosynthetic pathway of this unusual fatty acid, 14C-labeling experiments were conducted using developing endosperm of coriander. Studies were also performed using suspension cultures of transgenic tobacco (Nicotiana tabacum L.) that express the coriander 36-kD desaturase, and as a result produce petroselinic acid and A4-hexadecenoic acid. When supplied exogenously to coriander endosperm slices, [I -14C]palmitic acid and stearic acid were incorporated into glycerolipids but were not converted to petroselinic acid. This suggested that petroselinic acid is not formed by the desaturation of a fatty acid bound to a glycerolipid or by reactions involving acyl-coenzyme As (COA). Instead, evidence was most consistent with an acyl-ACP route of petroselinic acid synthesis. For example, the exogenous feeding of [l-'4C]lauric acid and myristic acid to coriander endosperm slices resulted in the incorporation of the radiolabels into long-chain fatty acids, including primarily petroselinic acid, presumably through acyl-ACP-associated reactions. In addition, using an in vitro fatty acid biosynthetic system, homogenates of coriander endosperm incorporated [2-'4C]malonyl-CoA into petroselinic acid, of which a portion was detected in a putative acyl-ACP fraction. Furthermore, analysis of transgenic tobacco suspension cultures expressing the coriander 36-kD desaturase revealed significant amounts of petroselinic acid and A4-hexadecenoic acid in the acyl-ACP pool of these cells. Also presented is evidence derived from [U-'4C]nonanoic acid labeling of coriander endosperm, which demonstrates that the coriander 36-kD desaturase positions double bonds relative to the carboxyl end of acyl-ACP substrates. l h e data obtained in these studies are rationalized in terms of a biosynthetic pathway of petroselinic acid involving the A4 desaturation of palmitoyl-ACP by the 36-kD desaturase followed by two-carbon elongation of the resulting A4-hexadecenoyl-ACP.

Biosynthesis and function of polyacetylenes and allied natural products

“…A Δ 6 -palmitoyl-ACP variant desaturase from the endosperm of Thunbergia alata is known [74]. Details of the Δ 4 -desaturation of palmitoyl-ACP by a coriander enzyme related to AAD1 and elongation of the product by a 16:1 4c -specific KASA condensing enzyme to 5R-ACP have been reported [75][76][77]. Structure-function studies have shown that within a series of Δ 9 -18:0, Δ 6 -16:0, and Δ 9 -16:0 desaturases, 2-5 residue substitutions can shift the regiochemistry or substrate preference to that of a different isozyme [34].…”

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

“…In addition, a number of other acyl-ACP DES have been identified that are associated with the synthesis of unusual monounsaturated fatty acids and have altered substrate and regio-specifities relative to the ⌬ 9 -18:0-ACP DES. Variant acyl-ACP DES identified to date include the ⌬ 4 -palmitoyl (16:0)-ACP DES from Umbelliferae seed (2,3), the ⌬ 6 -16:0-ACP DES from black-eyed Susan vine (Thunbergia alata) seed (4), the ⌬ 9 -myristoyl (14:0)-ACP DES from geranium (Pelargonium xhortorum) trichomes (5), and ⌬ 9 -16:0-ACP DES from cat's claw (Doxantha unguis-cati) (6) and milkweed (Asclepias syriaca) (7) seeds. These enzymes share Ն60% amino acid sequence identity with the ⌬ 9 -18:0-ACP DES.…”

REGLEMENTATION, EXPERTISE, MISE SUR LE MARCHE Les enjeux de la propriété intellectuelle : brevet, certificat d’obtention végétale

“…Celui-ci résulte de la désaturation du stéaroyl-ACP par une désaturase particulière [13] qui introduit une double liaison en •6.…”