Emilianiahuxleyi是一种海洋微藻,在海洋中起着重要的生态和生物地球化学作用。它可以产生几种多不饱和脂肪酸(PUFA),如二十二碳六烯酸(DHA,22:6-4,7,10,13,16,19)和十八碳五烯酸(OPA,18:5-3,6,9,12,15),为海洋食物链中营养重要的ω3PUFA提供主要来源。然而,这些PUFA在该生物体中的生物合成还没有很好地理解。在这项研究中,从该藻类中克隆了全长质体ω3去饱和酶cDNA(EhN3)。有和没有叶绿体靶向肽(cTP)的EhN3在蓝藻蓝藻中的异源表达表明,它对C18-ω6PUFA具有很高的去饱和活性,亚油酸(LA,18:2-9,12),γ-亚麻酸(GLA,18:3-6,9,12),和C20-ω6PUFA,二高-γ-亚麻酸(DGLA,20:3-8,11,14)和花生四烯酸(ARA,20:4-5,8,11,14)是外源供应的。在一个时间过程中,去饱和效率可以达到几乎100%。另一方面,当在酿酒酵母中表达时,有和没有cTP的EhN3没有表现出任何活性。表达EhN3的聚球菌转化体的脂质分析显示其利用半乳糖脂作为底物。转录表达分析表明,随着生长温度的降低,该基因的表达量增加,这与ω3-PUFA的产量增加有关,尤其是OPA。这是来自微藻的质体ω3去饱和酶的首次报道,该酶可以有效地将ω3双键引入C18-ω6和C20-ω6PUFA中。EhN3也可能是通过质体有氧途径参与胡氏大肠杆菌OPA生物合成的关键酶之一。
Emiliania huxleyi is a marine microalga playing a significant ecological and biogeochemical role in oceans. It can produce several polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and octadecapentaenoic acid (OPA, 18:5-3,6,9,12,15), providing a primary source for nutritionally important ω3 PUFAs in the marine food chain. However, the biosynthesis of these PUFAs in this organism is not well understood. In this study, a full length plastidial ω3 desaturase cDNA (EhN3) was cloned from this alga. Heterologous expression of EhN3 with and without the chloroplast targeting peptide (cTP) in cyanobacterium Synechococcus elongatus showed that it possessed high desaturation activity toward C18-ω6 PUFAs, linoleic acid (LA, 18:2-9,12), γ-linolenic acid (GLA, 18:3-6,9,12), and C20-ω6 PUFAs, dihomo-γ-linolenic acid (DGLA, 20:3-8,11,14) and arachidonic acid (ARA, 20:4-5,8,11,14) that were exogenously supplied. Desaturation efficiency could reach almost 100% in a time course. On the other hand, when expressed in Saccharomyces cerevisiae, EhN3 with and without cTP did not exhibit any activity. Lipid analysis of Synechococcus transformants expressing EhN3 showed that it utilized galactolipids as substrates. Transcriptional expression analysis revealed that the expression of the gene increased while the growth temperature decreased, which was correlated with the increased production of ω3-PUFAs, particularly OPA. This is the first report of a plastidial ω3 desaturase from microalgae that can effectively introduce an ω3 double bond into both C18-ω6 and C20-ω6 PUFAs. EhN3 might also be one of the key enzymes involved in the biosynthesis of OPA in E. huxleyi through the plastidial aerobic pathway.