Jiang Mulan, Guo Bing, Wan Xia, Gong Yangmin, Zhang Yinbo, Hu Chuanjiong
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, China.
Mar Drugs. 2014 Mar 7;12(3):1317-34. doi: 10.3390/md12031317.
The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host.
三角褐指藻能够积累高达总脂肪酸30%的二十碳五烯酸（EPA）。该物种已成为分离长链多不饱和脂肪酸（LC-PUFA）代谢工程中编码去饱和酶和延长酶基因的目标。在此，我们首次报道了三角褐指藻中Δ5-延长酶基因的克隆与特性分析。一个全长cDNA序列，命名为PhtELO5，显示其含有一个1110 bp的开放阅读框，编码一个369个氨基酸的多肽。推测的蛋白质含有七个跨膜区域以及FLHXYHH和MYSYY这两个延长酶特征基序，后者是微藻Δ5-延长酶的典型基序。系统发育分析表明，PhtELO5属于ELO5组，与拟菱形藻的对应物紧密聚类。PhtELO5在毕赤酵母中的异源表达证实它编码一种能够延长花生四烯酸和二十碳五烯酸的特异性Δ5-延长酶。PhtELO5和IsFAD4（来自球等鞭金藻的一种∆4-去饱和酶）的共表达表明，在转基因酵母中组装了二十二碳六烯酸的高效生物合成途径。底物竞争表明，PhtELO5对n-3多不饱和脂肪酸的活性高于n-6多不饱和脂肪酸。据推测，三角褐指藻ELO5可能优先通过酵母宿主中的n-3途径参与转基因LC-PUFA的生物合成。
