酿酒酵母中少根根霉△~6-脂肪酸脱氢酶对α-亚麻酸催化活性的研究  

Study on the Conversion of α-Linolenic Aicd by Rhizopus Arrhizus Δ~6-Fatty Acid Desaturase in Saccharomyces Cerevisiae

作　　者：张琦[1] 李明春[1] 张飙[2] 孙颖[1] 蔡易[1] 邢来君[1] 

机构地区：[1]南开大学微生物学系,天津300071 [2]天津中医学院,天津300193

出　　处：《南开大学学报（自然科学版）》2005年第5期31-35,共5页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金资助项目(30200176);教育部高等学校骨干教师资助计划项目

摘　　要：多不饱和脂肪酸(po lyunsaturated fatty ac ids,PU FA s)可分为n-6和n-3两个系列,而Δ6-脂肪酸脱氢酶是这些PU FA s合成途径中的限速酶.将少根根霉Δ6-脂肪酸脱氢酶基因转化酿酒酵母营养缺陷型菌株INV S-cl,在添加外源性底物α-亚麻酸,经半乳糖诱导后,通过气相色谱(GC)和气相色谱/质谱(GC-M S)联用分析细胞脂肪酸表明,少根根霉Δ6-脂肪酸脱氢酶催化α-亚麻酸转化成十八碳四烯酸,所生成十八碳四烯酸的含量占酵母细胞总脂肪酸的7.67%,而在空载体pYES2.0转化的酵母中没有检测到.同时,参照K ozak序列,我们把少根根霉Δ6-脂肪酸脱氢酶基因的转译起始密码子周边序列进行适当的改变,并转化INV Scl进行分析,结果显示修改后的序列同样能催化α-亚麻酸转化成十八碳四烯酸,而且表达量提高到细胞总脂肪酸的11.23%,表明在酿酒酵母中,改变转移起始密码子周边序列可提高少根根霉Δ6-脂肪酸脱氢酶基因催化α-亚麻酸转合成十八碳四烯酸的水平.Polyunsaturated fatty acids （PUFAs） can be classified into n-6 and n-3 families. △^6-fatty acid desaturase is a rate-limiting enzyme in the biosynthesis of PUFAs. The coding sequence （RAD6） of Rhizopus arrhizus △^6-fatty acid desaturase was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYRAD6, whcih was subsequently transformed into Saccharomyces cerevisiae strain INVScl using lithium-acetate method. Heterologous expression of RAD6 was induced under transcriptional control of the yeast GAL1 promoter, supplemented with 0. 5 mol/L ALA and 2% galactose. The resultant fatty acid methyl esters （FAME） were analyzed by gas chromatography （GC） and Gas chromatographymass spectromentry（GC-MS）. A novel peak corresponding to OTA methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector pYES2.0. The percentage of this new fatty acid to total fatty acids was 7.67%. Furthermore, based on the Kozak sequence, the sequence flanking AUG codon of R. arrhizus △^6-fatty acid desaturase gene was modified. The resultant recombinant vector pYRAD6-1 was then transformed into S, cerevisiae for expression. Gas chromatogram （GC） analysis of its fatty acid methyl esters indicated that OTA was synthesized, corresponding to the novel peak in pYRAD6-transformed yeasts. The percentage of OTA to total fatty acids reached 11. 23%. which demonstrated that the expression level of R. arrhizus △^6-fatty acid desaturase gene was increased by the modification of sequences flanking AUG codon in S. cerevisiae.

关 键 词：少根根霉 △^6-脂肪酸脱氢酶基因 酿酒酵母 Α-亚麻酸 十八碳四烯酸 KOZAK序列 

分 类 号：Q344.13[生物学—遗传学]