海洋破囊壶菌Δ~4-脂肪酸脱饱和酶基因在酿酒酵母中的表达

Expression of Thraustochytrium sp.FJN-10 Δ~4-Desaturase Gene in Saccharomyces cerevisiae

以质粒pGEM-TFAD4为模板,扩增获得1.6kb的Δ4-脂肪酸脱饱和酶基因(FAD4)。将FAD4酶切后连接到HindⅢ/XbaⅠ处理过的pYES2.0载体,构建重组表达质粒pYFAD4。转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子。添加外源脂肪酸C22:5底物,半乳糖诱导表达。气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22:6(占酵母总脂肪含量的41.13%),Δ4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达。

1.6 kb △^4-desatursse gene（ FAD4）was amplified by PCR using plasmid pGEM-TFAD4 as template. The fragment was subeloned into the HindⅢ/Xba Ⅰ restriction site of pYES2.0 vector. Recombinant plusmid pYFAD4 was transformed into Soccharomyces ceresisiae strain INVScl for expression. It was found to exhibit As-fatty acid desaturese activity in the recombinant S. cerevisiae YFAD4 in the presence of exogenous fatty acid substrate docesapentaenoic acid（100μmol/L） under introduction of GALl. Expression of the FAD4 under appropriate media and temperature conditions led to the production of DHA and it reached 41.13% of the total yeast fatty acid by GC detection. It was suggested that the protein encoded by FAD4 could specifically catalyze DPA into DHA.