Conidiobolus obscurus, an entomopathogenic fungus able to infect aphids, was previously reported to produce substantial amounts of very long chain polyunsaturated fatty acids (VLCPUFAs) that may mediate the insect infection. However, the genes involved in the biosynthesis of these VLCPUFAs from the order Entomophthorales have yet to be identified. Using degenerate reverse transcriptase-polymerase chain reaction and rapid amplification of the cDNA end methods, we cloned a a dagger 6 desaturase cDNA (CoD6) and a a dagger 6 elongase cDNA (CoE6) from C. obscurus. Expression of CoD6 and CoE6 in Saccharomyces cerevisiae revealed CoD6 could introduce a Delta 6 double bond into alpha-linolenic acid (18:3n-3), and CoE6 preferentially elongated 18-carbon Delta 6 desaturated fatty acid stearidonic acid (18:4n-3). When the fungus was grown under a temperature shift from 20A degrees C to 10A degrees C, the transcript level of CoD6 and CoE6 increased, whereas when the fungal culture was shifted from 20A degrees C to 30A degrees C, the transcript level of both genes decreased. The entire eicosatetraenoic acid biosynthetic pathway was reconstituted in yeast using four genes, CoD6 and CoE6 from C. obscurus, CpDes12 (a Delta 12 desaturase) and CpDesX (a omega 3 desaturase) from Claviceps purpurea. Yeast transformants expressing the four genes produced ten new fatty acids including the final product eicosatetraenoic acid (ETA). This represents the reconstitution of the entire ETA pathway in yeast without supplementation of any exogenous fatty acids.