深黄被孢霉M_(6-22) Δ~6-脂肪酸脱氢酶基因在酿酒酵母中的表达

IDENTIFICATION OF MORTIERALLA ISABELLINA M_(6-22) Δ~6-FATTY ACID DESATURASEBY HETEROLOGOUS EXPRESSION IN SACCHAROMYCES CEREVISIAE

应用PCR技术 ,从含有深黄被孢霉Δ6 脂肪酸脱氢酶基因的重组质粒pTMICL6中 ,扩增出 1 38kb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pYMID6,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INVSc1中 ,在SC Ura合成培养基中 ,选择到酵母工程株YMID6。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对酵母工程株所含的全部脂肪酸进行色谱分析 ,结果表明 ,γ 亚麻酸的含量占酵母总脂肪酸的 8 69%。

Using plasmid pTMICL6 containing Δ 6\|fatty\|acid desaturase gene from Mortieralla isabellina M 6-22 as a template,1.38kb DNA fragment was amplified by PCR.The fragment was subcloned into the yeast\| E.coli shuttle vector pYES2.0,then an expression recombinant plamid pYMID6 containing target gene was constructed.The pYMID6 was transformed into Saccharomyces cerevisiae for expression by LiAc method.It was found to exhibit Δ 6\|fatty acid desaturase activity in the recombinant S.cerevisiae YMID6 in the presence of exogenous fatty acid substrate linoleic acid under introduction of GAL.Expression of the Δ 6\|fatty acid desaturase gene under appropriate media and temperature conditions led to the production of γ-linolenic acid reached 8.69% of the total yeast fatty acid by GC\|MS detection.It is the first report about expression of M.isabellina D6D gene in S.cerevisiae.