Formation of conjugated Δ8,Δ10-double bonds by Δ12-oleic-acid desaturase-related enzymes -: Biosynthetic origin of calendic acid

Divergent forms of the plant Delta (12)-oleic-acid desaturase (FADS) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta (11),Delta (13)-double bonds by modification of an existing Delta (12)-double bond in C-18 fatty acids. Here, we report a class of FADS-related enzymes that modifies a Delta (9)-double bond to produce the conjugated trans-hs,trans-Delta (10)-double bonds found in calendic acid (18:3 Delta (8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis, Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-8 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) Of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18: 2 Delta (9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta (9)-double bond of linoleic acid. Regiospecificity for he-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.