高山被孢霉Δ^6-脂肪酸脱氢酶基因在毕赤酵母中的胞内表达

Expression of Δ~6-fatty Acid Desaturase Gene from Mortierella alpina in Pichia pastoris

γ 亚麻酸 (GLA)作为人体必需的不饱和脂肪酸 ,具有重要的营养和药用价值。Δ6 脂肪酸脱氢酶是γ 亚麻酸合成途径中的关键酶。为了在毕赤酵母中建立一种新的合成γ 亚麻酸的表达体系 ,将高山被孢霉Δ6 脂肪酸脱氢酶基因与胞内表达载体pPIC3 5K连接 ,SacⅠ线性化后电击法转化毕赤酵母SMD116 8,获得的转化子经PCR鉴定目的基因已整合到毕赤酵母的基因组中。用甲醇诱导表达 ,通过脂肪酸气相色谱和气相色谱 质谱 (GC MS)联用分析表明高山被孢霉Δ6 脂肪酸脱氢酶基因在毕赤酵母中获得表达 ,γ 亚麻酸含量占总脂肪酸的 16 2 6 %。

linolenic acid (GLA,C18:3Δ6,9,12), an essential polyunsaturated fatty acid, plays an important role in hormone regulation and fatty acid metabolization. Δ6-fatty acid desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic acid (C18:2Δ 9,12)in the production ofγ-linolenic acid. A deficiency of GLA may have occurred when Δ6-fatty acid desaturase activity decreases in aging,stress,diabetes,eczema,and some infections. To establish a new expression system for Δ6-fatty acid desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding Δ6-fatty acid desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoRⅠ and NotⅠ and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA 6.The resulting vector was linearized by SacⅠ and electroporated into P. pastoris SMD1168(his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30℃ for three days until colonies appeared.Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5'AOX and 3'AOX. The PCR product of Mut recombinants was shown as a band of 1.38kb of D6D gene and the product of 2.2kb of AOX1 gene, while the product of Muts transformants only was shown as a band of 1.38kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular fatty acid composition by GC. The resultant chromatograms of fatty acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control,. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the fatty acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GC-MS analyses. Thus, the active Δ6-fatty acid desaturase was expressed intracellularly in P. pastoris and γ-linolenic acid reached 16.26% of the total fatty acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.