高山被孢霉Δ5去饱和酶基因的克隆及异源表达研究

Cloning and Heterologous Expression of Δ5 Desaturase Gene from Mortiella alpina

通过RT-RCR的方法从高山被孢霉W15菌株中扩增到完整的Δ5去饱和酶基因,其全长为1341bp,编码446个氨基酸。对推导的氨基酸序列进行分析表明,其N末端存在细胞色素b5结构域,在序列中还存在3个保守的组氨酸富集区。将该基因连接到表达载体pPIC3.5K上,转化毕赤酵母细胞,利用G418筛选高拷贝数的转化子。以双高γ-亚麻酸为底物,在甲醇诱导的条件下,Δ5去饱和酶可催化底物脱氢生成花生四烯酸,其含量占总脂肪酸含量的2.85%。说明克隆的Δ5去饱和酶基因能在毕赤酵母中进行功能性的表达。

Arachidonic acid, an essential fatty acid for humans, plays important physiological functions in the intelligence development and vision development of infants. In this study, a complete Δ5 desaturase gene was cloned from Mortierella alpina W15 by RT-PCR amplification. The gene with 1341 bp in length encoded 446 amino acids. The deduced amino acid sequence demonstrated the cytochrome b5 domain in the N terminal and three conserved histidine boxes in the sequence. Then the gene was inserted into the expression vector pPIC3.5K and transformed into Pichia pastoris GS 115. Transformants containing multi-copy Δ5 desaturase genes were screened by Geneticin （G418）. When dihomo-γ-1inolenic acid was provided as the exogenous substrate to the culture induced by methanol, the dehydrogenation of the substrate was catalyzed by Δ5 desaturase to produce arachidonic acid reaching 2.85% in the total fatty acids. This result indicated that the cloned gene encoded functional Δ5 desaturase in P. pastoris GS 115.