Abstract
In order to identify novel genes encoding enzymes involved in the biosynthesis of nutritionally important omega-3 long chain polyunsaturated fatty acids, a database search was carried out in the genomes of the unicellular photoautotrophic green alga Ostreococcus RCC809 and cold-water diatom Fragilariopsis cylindrus. The search led to the identification of two putative “front-end” desaturases (Δ6 and Δ4) from Ostreococcus RCC809 and one Δ6-elongase from F. cylindrus. Heterologous expression of putative open reading frames (ORFs) in yeast revealed that the encoded enzyme activities efficiently convert their respective substrates: 54.1% conversion of α-linolenic acid for Δ6-desaturase, 15.1% conversion of 22:5n-3 for Δ4-desaturase and 38.1% conversion of γ-linolenic acid for Δ6-elongase. The Δ6-desaturase from Ostreococcus RCC809 displays a very strong substrate preference resulting in the predominant synthesis of stearidonic acid (C18:4Δ6,9,12,15). These data confirm the functional characterization of omega-3 long chain polyunsaturated fatty acid biosynthetic genes from these two species which have until now not been investigated for such activities. The identification of these new genes will also serve to expand the repertoire of activities available for metabolically engineering the omega-3 trait in heterologous hosts as well as providing better insights into the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in marine microalgae.
Highlights
It is well accepted that omega-3 long chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6Δ4,7,10,13,16,19) are vital for human health and nutrition and play a crucial role in preventing cardiovascular diseases and associated precursor conditions such as metabolic syndrome and obesity [1,2]
We examined the fatty acid profile of two marine microalgae, the unicellular photoautotrophic green alga Ostreococcus RCC809 and the cold-water diatom Fragilariopsis cylindrus, both of which have previously only been subject to limited investigation as to the nature of their synthesis and accumulation of omega-3 LC-PUFAs
To explore the LC-PUFA pathway operating in Ostreococcus RCC809 we analyzed by GC-Flame Ionization Detector (FID) and GC-MS the fatty acid methyl esters (FAMEs) of total lipids from Ostreococcus RCC809 cultures growing at stationary phase
Summary
It is well accepted that omega-3 long chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6Δ4,7,10,13,16,19) are vital for human health and nutrition and play a crucial role in preventing cardiovascular diseases and associated precursor conditions such as metabolic syndrome and obesity [1,2]. The first step in this pathway is the Δ6-desaturation of both linoleic acid (LA; 18:2Δ9,12) and ALA, resulting in the synthesis of γ-linolenic acid (GLA; 18:3Δ6,9,12) and stearidonic acid (SDA; 18:4Δ6,9,12,15), respectively This step is followed by a Δ6-specific C2 elongation, yielding di-homo γ-linolenic acid (DGLA; 20:3Δ8,11,14) and eicosatetraenoic acid (ETA; 20:4Δ8,11,14,17). Mantoniella squamata [10], Micromonas pusilla [11] and Ostreococcus lucimarinus [12] In this present study, we examined the fatty acid profile of two marine microalgae, the unicellular photoautotrophic green alga Ostreococcus RCC809 and the cold-water diatom Fragilariopsis cylindrus, both of which have previously only been subject to limited investigation as to the nature of their synthesis and accumulation of omega-3 LC-PUFAs. We used on-going genomic sequencing projects for these two organisms to identify and functionally characterize three examples of genes involved in the biosynthesis of EPA and DHA. One particular enzyme (Δ6-desaturase from Ostreococcus RCC809) showed a strong preference of ω-3 substrates against ω-6
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