Liu L, Li M C, Hu G W, Ge J, Zhang L, Cheng Z H, Xing L J
Department of Microbiology, NanKai University, Tianjin 300071, China.
Sheng Wu Gong Cheng Xue Bao. 2001 Mar;17(2):161-4.
delta 6-fatty acid desaturase is the rate-limiting enzyme of the desaturation of linoleic acid in the production of an essential fatty acid, gamma-linolenic acid. The 1.4 kb fragment in plasmid pTMACL6 encoding delta 6-fatty acid desaturase from Mortieralla alpina ATCC16266 was subcloned into the yeast-E. coli shuttle vector pYES2.0, thus an expression recombinant plasmid pYMAD6 containing target gene was constructed and obtained in the SC-Ura media. The pYMAD6 was introduced into defective mutant INCSc1 of Saccharomyces cerevisiae by LiAc method. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing delta 6-fatty acid desaturase activity under appropriate media and temperature condition, the level of gamma-linolenic acid reached 31.6% of the total yeast fatty acids by GC-MS detecting, which is the highest report of delta 6-fatty-acid desaturase gene in S. cerevisiae.
Δ6-脂肪酸去饱和酶是在必需脂肪酸γ-亚麻酸的生产过程中亚油酸去饱和的限速酶。来自高山被孢霉ATCC16266的编码Δ6-脂肪酸去饱和酶的质粒pTMACL6中的1.4 kb片段被亚克隆到酵母-大肠杆菌穿梭载体pYES2.0中,从而构建了含靶基因的表达重组质粒pYMAD6,并在SC-Ura培养基中获得。通过醋酸锂法将pYMAD6导入酿酒酵母的缺陷型突变体INCSc1中。当在合适的培养基和温度条件下,将亚油酸作为外源底物提供给表达Δ6-脂肪酸去饱和酶活性的酵母培养物时,通过气相色谱-质谱检测,γ-亚麻酸的水平达到酵母总脂肪酸的31.6%,这是酿酒酵母中Δ6-脂肪酸去饱和酶基因的最高报道。