高山被孢霉ATCC16266△^6—脂肪酸脱氢酶基因在酿酒酵母中的表达

△^6－脂肪酸脱氢酶基因是形成γ－亚麻酸的关键酶。从含有高山被孢霉△^6－脂肪酸脱氢酶基因的重组质粒pT-MACL6中，酶切出1.4kb的目的片段，亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0，在大肠杆菌中筛选到含有目的基因的重组质粒pYMAD6，用醋酸昔方法转化到酿洒酵母的缺陷型菌株INCSc1中，在SC－Ura合成培养基中，选择得到酿酒酵母工程株YMAD6。在合适的培养基及培养条件下，加入外源底物亚油酸，经半乳糖诱导后,收集菌体。通过GC－MS对酵母工程株进行脂肪酸色谱分析，结果表明，产生了31.6％的γ－亚麻酸,边是迄今为止，国内外△^6－脂肪酸脱氢酶基因在酿酒酵母中表达量最高的报道。

Expression of

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.