海洋破囊壶菌Δ4-脂肪酸脱饱和酶基因在酿酒酵母中的表达

Expression of Thraustochytrium sp.FJN-10 Δ4-Desaturase Gene in Saccharomyces cerevisiae

摘要:

以质粒pGEM-TFAD4为模板，扩增获得1.6 kb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。

关键词:海洋破囊壶菌FJN-10，二十二碳六烯酸，Δ⁴-脂肪酸脱饱和酶，酿酒酵母

Abstract:

1.6 kb Δ⁴-desaturase gene(FAD4)was amplified by PCR using plasmid pGEM-TFAD4 as template. The fragment was subcloned into the HindⅢ/XbaⅠrestriction site of pYES2.0 vector. Recombinant plasmid pYFAD4 was transformed into Saccharomyces cerevisiae strain INVScl for expression.It was found to exhibit Δ⁴-fatty acid desaturase activity in the recombinant S.cerevisiae YFAD4 in the presence of exogenous fatty acid substrate docosapentaenoic acid(100μmol/L)under introduction of GAL1.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.

Key words:Thraustochytrium sp.FJN-10，DHA，Δ⁴-desaturase，Saccharomyces cerevisiae