In vivo characterization of the first acyl-CoA Δ6-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri
In vivo characterization of the first acyl-CoA Δ6-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri
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DOI：
10.1042/bj20050111
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发表时间：
2005-07-15
影响因子：
4.1
通讯作者：
Heinz, E
中科院分区：
文献类型：
--
作者：
Domergue, F;Abbadi, A;Heinz, E
利用全序列海洋单细胞浮游植物Ostreococcus tauri的基因组DNA，扩增了一个典型的多不饱和脂肪酸合成中的前端去饱和酶的基因编码。在酿酒酵母中的异源表达显示出非常高的去饱和活性和Δ（6）-区域选择性。短时间动力学实验表明，去饱和酶产品中检测到的酰基辅酶A池5分钟后，加入外源性底物的酵母培养基和长时间之前，其外观的总脂肪酸。当该去饱和酶与来自小立碗藓的酰基-CoA Δ（6）-延伸酶和来自三角褐指藻的脂质连接的Δ（5）-去饱和酶共表达时，获得了高比例的花生四烯酸或二十碳五烯酸，因为几乎所有的Δ（6）-去饱和产物都被延伸。此外，对于Δ（6）-去饱和酶或对于酰基-CoA Δ（6）-延伸酶，在每种甘油脂质中计算的产物/离析物比率在大约相同的范围内，而在脂质连接的Δ（5）-去饱和酶的情况下，该比率显示出非常不均匀的分布。最后，基于序列的比较所有的功能特征的Δ（6）-去饱和酶表明，这种酶是不相关的任何先前描述的序列。总之，我们的数据表明，这种去饱和酶从O。tauri是一种酰基-CoA δ（6）-去饱和酶，是第一个从光合活性生物体中克隆的酶。
Genomic DNA of Ostreococcus tauri, a fully sequenced marine unicellular alga from the phytoplankton, was used to amplify a gene coding for a typical front-end desaturase involved in polyunsaturated fatty acid biosynthesis. Heterologous expression in Saccharomyces cerevisiae revealed very high desaturation activity with Delta(6)-regioselectivity. Short-time kinetic experiments showed that the desaturase product was detected in the acyl-CoA pool 5 min after addition of the exogenous substrate to the yeast medium and long before its appearance in the total fatty acids. When this desaturase was co-expressed with the acyl-CoA Delta(6)-elongase from Physcomitrella patens and the lipid-linked Delta(5)-desaturase from Phaeodactylum tricornutum, high proportions of arachidomic or eicosapentaenoic acid were obtained, because nearly all of the Delta(6)-desaturated products were elongated. Furthermore, the product/educt ratios calculated in each glycerolipid for the Delta(6)-desaturase or for the acyl-CoA Delta(6)-elongase were in about the same range, whereas this ratio showed a very uneven profile in the case of the lipid-linked Delta(5)-desaturase. Finally, a sequence-based comparison of all the functionally characterized Delta(6)-desaturases showed that this enzyme was not related to any previously described sequence. Altogether, our data suggest that this desaturase from O. tauri is an acyl-CoA Delta(6)-desaturase, the first one cloned from a photosynthetically active organism.