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

Abstract

We report the identification of bifunctional ⌬12͞ 3 desaturases from Fusarium moniliforme, Fusarium graminearum, and Magnaporthe grisea. The bifunctional activity of these desaturases distinguishes them from all known ⌬12 or 3 fatty acid desaturases. The 3 desaturase activity of these enzymes also shows a broad 6 fatty acid substrate specificity by their ability to convert linoleic acid (LA), ␥-linolenic acid, di-homo-␥-linolenic acid, and arachidonic acid to the 3 fatty acids, ␣-linolenic acid (ALA), stearidonic acid, eicosatetraenoic acid, and eicosapentaenoic acid (EPA), respectively. Phylogenetic analysis suggests that 3 desaturases arose by independent gene duplication events from a ⌬12 desaturase ancestor. Expression of F. moniliforme ⌬12͞ 3 desaturase resulted in high ALA content in both Yarrowia lipolytica, an oleaginous yeast naturally deficient in 3 desaturation, and soybean. In soybean, seed-specific expression resulted in 70.9 weight percent of total fatty acid (%TFA) ALA in a transformed seed compared with 10.9%TFA in a null segregant seed and 53.2%TFA in the current best source of ALA, linseed oil. The ALA͞LA ratio in transformed seed was 22.3, a 110-and 7-fold improvement over the null segregant seed and linseed oil, respectively. Thus, these desaturases have potential for producing nutritionally desirable 3 longchain polyunsaturated fatty acids, such as EPA, with a significantly improved ratio of 3͞ 6 long-chain polyunsaturated fatty acids in both oilseeds and oleaginous microbes. polyunsaturated fatty acids ͉ Yarrowia ͉ soybean

A Bifunctional Δ12,Δ15-Desaturase from Acanthamoeba castellanii Directs the Synthesis of Highly Unusual n-1 Series Unsaturated Fatty Acids

“…This separation between AcD12 and other desaturases may indicate that the bifunctional enzyme from A. castellanii represent a new lineage in the evolution of desaturases. Interestingly, the bifunctional desaturase identified from G. fujikuroi (synonym, Fusarium moniliforme) appears to act predominantly as a -3 desaturase and is restricted to C 18ϩ substrates (40). In that respect, the fungal desaturase does not generate 16:2(n-4) or 16:3(n-1) (40), highlighting the difference between that enzyme and the A. castellanii bifunctional desaturase described in this 3 W. W. Christie, unpublished data.…”

“…We found no evidence for the use of acyl-CoA substrates by this enzyme, which indicates that phosphoglycerolipid-linked desaturation, rather than acyl-CoA-dependent activities, exists in protist species like A. castellanii (17). The substrate preference of the G. fujikuroi bifunctional desaturase has not been described (40).…”

The repertoire of desaturases and elongases reveals fatty acid variations in 56 eukaryotic genomes

“…Recent reports also show the independent divergence of D12 and D15 in several organisms, such as Mortierella alpina and Saprolegnia diclina (49,50). It should be noted that the first diversification occurred in the old common ancestor, whereas the second occurred independently in each lineage.…”

Atypical Biosynthetic Properties of a Δ 12 /ν+3 Desaturase from the Model Basidiomycete Phanerochaete chrysosporium

“…Despite the sheer number of plant ⌬ 12 -desaturases, the refractory nature of these membrane-bound enzymes to purification has left structure/function relationships ill defined (33). Consequently, only the rough classification of the FAD2 enzymes into distinct functional or evolutionary classes has occurred, largely using genetic and in vivo functional characterization (12,38,42).While the ⌬ 12 -or FAD2 desaturases, which form a 12,13-double bond, are best known from plants, fungal fad2 homologs have been found in zygomycetes and ascomycetes (8,52,59). As suggested by molecular clock data, Basidiomycota and Ascomycotina diverged approximately 550 million years ago (3), indicating that metabolic basidiomycete genes may differ significantly from those of other fungal subtypes.…”