^ A6-desaturase J-ôamma-linolenate Stearidonate
^ A5-desaturase ^
Figure 2 Higher plant pathway of long-chain PUFA formation from linoleic and a-linolenic acids. A simplified schematic showing the extraplastidic pathway of arachidonate and eicosa-pentaenoate from stearate. The minimum amounts of enzyme activities are shown with no cross talk between the two pathways.
The pathways of very long chain PUFA synthesis in fungi and marine algae involve a series of sequential reductions and elongations that resemble the production of long-chain fatty acids such as erucic acid in plants like Arabidopsis and rapeseed (30). With the advent of modern genomic techniques and the existence of known desaturase and elongase protein sequences, it was possible to search cDNA EST databases of PUFA-containing organisms using conserved desaturase and elongase domains. In this way, A5 and A6 desaturases and the 18:3 n-6 elongase from the ARA-accumu-lating filamentous fungus Morteriella alpina were identified (45-48). Using similar approaches, orthologues of the A5 desaturase and the 18:3n-6 elongase have been identified from C. elegans (49,50) and human cDNA EST databases respectively (51,52). Rapid functional identification of putative cDNA clones was carried out by expression in yeast (45-52). This technique enabled identification of individual positive cDNA clones and led to the possibility of producing these very long chain PUFAs in the oil of transgenic plants. Earlier work on the expression of a 5-6 desaturase from a cyanobac-terium in transgenic tobacco showed no accumuation of GLA or OTA in seeds (53). In contrast, coexpression of M. alpina A6 and A12 desaturases in canola seed resulted in a GLA content of about 40% of the seed oil fatty acids (54). Similarly, seed-specific expression of the borage A6 desaturase in transgenic soy seed resulted in a similar total GLA content in the seed oil (S. Coughlan, unpublished results). The differences between canola and soy seed may be related to the higher amount of 18:2 naturally present in soy oil (50%) compared with canola oil (20%). With the cloning of all of the individual components of the eukaryotic pathway of LC-PUFA formation, the technical challenges remaining are those of metabolic pathway engineering. For example, it will be necessary to co-express up to six different enzymes simultaneously in a developing oilseed in order to produce DHA from a-linolenic acid in transgenic plants. Finally, it should be noted that some marine microorganisms (bacteria and diatoms) have a completely different pathway of LC-PUFA formation using polyketide synthases (55).
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