少根根霉Δ_-~6脂肪酸脱氢酶基因在毕赤酵母中的表达(英文)

Heteroexpression of Rhizopus arrhizus Δ_-~6Fatty AcidDesaturase Gene in Pichia pastoris

Δ6- 脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶。在前期工作中,通过RT PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码Δ6- 脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的Δ6- 脂肪酸脱氢酶基因。把少根根霉Δ6- 脂肪酸脱氢酶基因(RAD6)亚克隆到表达载体pPIC3.5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达。提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱质谱连用分析表明,目的基因的编码产物能将C16∶1、C17∶1、C18∶1、亚油酸和α- 亚麻酸在Δ6和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性。此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平。综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析Δ6 脂肪酸脱氢酶基因的功能。

Abstract Δ^6-fatty acid desaturase is a membrane-bound enzyme, which is rate-limiting for the biosynthesis of polyunsaturated fatty acids. A cDNA sequence putatively encoding a Δ^6-fatty acid desaturase was isolated from Rhizopus arrhizus NK300037 using RT-PCR and RACE methods in our previous work. Sequence and function analysis indicated that this sequence was a novel Δ^6-fatty acid desaturase gene which had an open reading frame of 1377bp coding 458 amino acids of 52kD. The methylotrophic yeast Pichia pastoris, has been developed into a highly successful system for the production of a variety of heterologous proteins during the past 20 years. In this work, the Rhizopus arrhizus Δ^6-fatty acid desaturase gene （RAD6） was subcloned into expression vector pPIC3.5K to generate a recombinant plasmid pPICRAD6, which was subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. Total fatty acids were extracted from the induced cells and methylated. The resultant fatty acid methyl esters （FAME） were analyzed by gas chromatography （GC） and gas chromatography-mass spectrometry （GC-MS）. Fatty acids analysis showed that the coding product introduced a new double bond at Δ6 position of appropriate fatty acid substrates including C16 ： 1, C17 ： 1, C18 ： 1, linoleic acid and α-linolenic acid without chain length specificity of fatty acids. Furthermore, modification of sequence flanking AUG codon of this Δ^6-fatty acid desaturase gene increased the expression of target gene in P. pastoris. All of these results suggest that P. pastoris is an optimal expression system of Δ^6-fatty acid desaturase gene.