海洋破囊壶菌Thraustochytrium sp. FJN-10 DHA生物合成 途径相关延长酶的克隆与表达

Cloning and expression of two elongase genes involved in the biosynthesis of docosahexaenoic acid in Thraustochytrium sp. FJN-10

摘要:

二十二碳六烯酸(Docosahexaenoic acid, DHA)是具有各种重要生理功能的高度不饱和脂肪酸。以海洋真菌Thraustochytrium sp. FJN-10为研究对象, 利用RT-PCR结合RACE, 获得了两个碳链延长酶(TFD6和TFD5)的完整基因, 其中TFD6 cDNA全长816 bp, 编码271个氨基酸; TFD5 cDNA全长831 bp, 编码276个氨基酸。将TFD6、TFD5酶切后分别连接到HindⅢ/XbaⅠ处理过的pYES2载体, 醋酸锂法转化酿酒酵母感受态细胞, 成功构建了延长酶酵母表达系统。气相色谱分析表明TFD6可延长C18:3n-6至C20:3n-6, TFD5可延长C20:5n-3至C22:5n-3。

Abstract:

Docosahexaenoic acid (DHA C22:6n-3), a typical long chain polyunsaturated fatty acids (PUFAs) has many positive effects on diseases such as artherosclerosis, hypertriglyceridemia, hypertension and cancers. Marine fungi, especially Thraustochytrium spp. producing much DHA can serve as model organisms for explaining the mechanism on the biosynthesis of PUFA. We described two elongase genes (TFD6 and TFD5) involved in the biosynthesis of DHA in Thraustochytrium sp. FJN-10 was cloned by using reverse transcription PCR and rapid amplification of cDNA ends. TFD6 cDNA was 816 bp in length and encoded a protein of 271 amino acids. TFD5 cDNA was 831 bp in length and encoded a protein of 276 amino acids. Transmembrane analysis revealed that TFD6 contained five transmembrane domains while TFD5 contained seven. Tertiary structures of TFD6, TFD5 elongases were predicted by HHMMSTR (Hidden markov model for local sequence-structure) model and Rosetta program. Alignment of TFD6, TFD5 with other elongases showed that both of them shared an HXXHH conserved histidine-rich motif. Phylogenetic analysis showed that TFD6 was the closest to Thraustochytrium Δ6 elongase, while TFD5 was the closest to Thraustochytrium sp. Δ5 elongase. TFD6 and TFD5 were subcloned into the Hind Ⅲ/Xba Ⅰrestriction site of pYES2 vector respectively. Recombined plasmids were transformed into Saccharomyces cerevisiae using lithium acetate method. Gas chromatography analysis showed that TFD6 could elongate C18:3n-3 to C20:3n-3 while TFD5 could elongate C20:5n-3 to C22:5n-3.