Cloning and functional analysis of the genes from entomopathogenic fungi involved in the biosynthesis of eicosatetraenoic acid (ETA)

Abstract

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA, 20:4ù6), eicosapentaenoic acid (EPA, 20:5ù3) and docosahexaenoic acid (DHA, 22:6ù3) have been shown to have many health benefits, some of which include lowering blood pressure, providing protection against cardiovascular diseases and improving brain and eye functions. Entomopathogenic fungi, a group of fungal pathogens able to infect insects, were previously reported to produce substantial amounts of VLCPUFAs, however the genes involved in the biosynthesis of these fatty acids have yet to be identified. This research started with fatty acid analysis of five entomopathagenic fungi, of which Conidiobolus obscurus and Conidiobolus thromboides were found to produce high levels of VLCPUFAs such as ARA and EPA. Thus, these two fungal species were selected as potential gene sources for the enzymes involved in the biosynthesis of VLCPUFAs. Using degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) methods; we cloned two full-length putative ∆6 desaturase cDNAs (CoD6 and CtD6) from the two fungi.

Functional expression of CoD6 in Saccharomyces cerevisiae showed it codes for a functional Ä6 desaturase, which can introduce a Ä6 double bond into linoleic acid and á-linolenic acid, respectively. However, expression of CtD6 in S. cerevisiae showed it does not have any Ä6 desaturase activity. Using degenerate RT-PCR and RACE, we also cloned two full-length ∆6 elongase cDNAs (CoE6 and CtE6) from the C. obscurus and C. thromboides species. Functional expression of these genes in S. cerevisiae showed CoE6 and CtE6 code for functional ∆6 elongase. Substrate specificity analysis indicated that both preferentially elongate 18-carbon Ä6 desaturated fatty acids, such as ã-linolenic acid and stearidonic acid. In addition, CtE6 can also elongate 20-carbon VLCPUFAs, such as ARA and EPA. The entire eicosatetraenoic acid (ETA, 20:4ù3) biosynthetic pathway was reconstituted in yeast using four genes, CoD6 (a ∆6 desaturase) and CoE6 (a ∆6 elongase) from Conidiobolus obscurus, CpDes12 (a Ä12 desaturase) and CpDesX (a ù3 desaturase) from Claviceps purpurea. Yeast transformant expressing the four genes produced several new fatty acids. Among them, eicosatetraenoic acid (ETA) accounts for approximately 0.1% of the total fatty acids. Although the level of ETA in the transformant is low, this represents the first report describing the reconstitution of the entire ETA pathway in yeast without exogenous supplementation of any fatty acids.