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.

Rapid generation of O 2 Ϫ and H 2 O 2 , which is reminiscent of the oxidative burst in neutrophils, is a central component of the resistance response of plants to pathogen challenge. Here, we report that the Arabidopsis rbohA ( for respiratory burst oxidase homolog A ) gene encodes a putative 108-kD protein, with a C-terminal region that shows pronounced similarity to the 69-kD apoprotein of the gp91 phox subunit of the neutrophil respiratory burst NADPH oxidase. The RbohA protein has a large hydrophilic N-terminal domain that is not present in gp91 phox . This domain contains two Ca 2 ϩ binding EF hand motifs and has extended similarity to the human RanGTPase-activating protein 1. rbohA , which is a member of a divergent gene family, generates transcripts of 3.6 and 4.0 kb that differ only in their polyadenylation sites. rbohA transcripts are most abundant in roots, with weaker expression in aerial organs and seedlings. Antibodies raised against a peptide near the RbohA C terminus detected a 105-kD protein that, unlike gp91 phox , does not appear to be highly glycosylated. Cell fractionation, two-phase partitioning, and detergent extraction indicate that RbohA is an intrinsic plasma membrane protein. We propose that plants have a plasma membrane enzyme similar to the neutrophil NADPH oxidase but with novel potential regulatory mechanisms for Ca 2 ϩ and G protein stimulation of O 2 Ϫ and H 2 O 2 production at the cell surface. INTRODUCTIONRecognition of avirulent pathogens causes a burst of oxidative metabolism generating superoxide (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ) (Baker and Orlandi, 1995). Reactive oxygen intermediates are direct protective agents and also drive cross-linking of the cell wall before the activation of transcription-dependent defenses (Lamb and Dixon, 1997). Moreover, the oxidative burst can trigger the collapse of challenged host cells at the onset of the hypersensitive response and generate signals for defense gene induction (Levine et al., 1994; Jabs et al., 1997).The kinetics and defense functions of O 2 Ϫ and H 2 O 2 generation are reminiscent of the oxidative burst during neutrophil activation ( Tenhaken et al., 1995;Lamb and Dixon, 1997). The neutrophil oxidative burst involves the reaction O 2 ϩ NADPH → O 2 Ϫ ϩ NADP ϩ ϩ H ϩ catalyzed by a plasma membrane oxidase, followed by dismutation of O 2 Ϫ to H 2 O 2 (Taylor et al., 1993). The NADPH oxidase consists of two plasma membrane proteins, gp91 phox and p22 phox ( phox for phagocyte oxidase), which together form heterodimeric flavocytochrome b Ϫ 558 and three cytosolic regulatory proteins, p40 phox , p47 phox , and p67 phox , which translocate to the plasma membrane after stimulation to form the active complex (Bokoch, 1994). The small cytosolic GTPase Rac2 and possibly other G proteins also appear to be required for activation of the oxidase.A membrane-bound enzyme resembling the neutrophil NADPH oxidase may contribute to the pathogen-induced oxidative burst in plants. Thus, O 2 Ϫ generation can be observed in microsomal preparat...

Rapid generation of O 2 and H 2 O 2 , which is reminiscent of the oxidative burst in neutrophils, is a central component of the resistance response of plants to pathogen challenge. Here, we report that the Arabidopsis rbohA (for respiratory burst oxidase homolog A) gene encodes a putative 108-kD protein, with a C-terminal region that shows pronounced similarity to the 69-kD apoprotein of the gp91 phox subunit of the neutrophil respiratory burst NADPH oxidase. The RbohA protein has a large hydrophilic N-terminal domain that is not present in gp91 phox. This domain contains two Ca 2 binding EF hand motifs and has extended similarity to the human RanGTPase-activating protein 1. rbohA , which is a member of a divergent gene family, generates transcripts of 3.6 and 4.0 kb that differ only in their polyadenylation sites. rbohA transcripts are most abundant in roots, with weaker expression in aerial organs and seedlings. Antibodies raised against a peptide near the RbohA C terminus detected a 105-kD protein that, unlike gp91 phox , does not appear to be highly glycosylated. Cell fractionation, two-phase partitioning, and detergent extraction indicate that RbohA is an intrinsic plasma membrane protein. We propose that plants have a plasma membrane enzyme similar to the neutrophil NADPH oxidase but with novel potential regulatory mechanisms for Ca 2 and G protein stimulation of O 2 and H 2 O 2 production at the cell surface.

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