The availability of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is currently limited because they are produced mainly by marine fisheries that cannot keep pace with the demands of the growing market for these products. A sustainable non-animal source of EPA and DHA is needed. Metabolic engineering of the oleaginous yeast Yarrowia lipolytica resulted in a strain that produced EPA at 15% of dry cell weight. The engineered yeast lipid comprises EPA at 56.6% and saturated fatty acids at less than 5% by weight, which are the highest and the lowest percentages, respectively, among known EPA sources. Inactivation of the peroxisome biogenesis gene PEX10 was crucial in obtaining high EPA yields and may increase the yields of other commercially desirable lipid-related products. This technology platform enables the production of lipids with tailored fatty acid compositions and provides a sustainable source of EPA.

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

ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54–65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.

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