卷枝毛霉Δ12-脱饱和酶基因的克隆及其在酿酒酵母中的高效表达
Cloning and high expression of Δ12- desaturase genes from Mucor circinelloides EIM-10
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摘要: Δ12-脱饱和酶是亚油酸合成的关键酶,为提高Δ12-脱饱和酶的活性,本研究利用RT-PCR对Mucor sp.EIM-10的Δ12-脱饱和酶c DNA进行克隆,并导入p YES2.0质粒中,构建重组表达载体。运用醋酸锂转化法将重组质粒导入酿酒酵母,成功构建p YMD12酿酒酵母表达系统。为了进一步提高Δ12-脱饱和酶的表达水平,用GAP启动子替代p YES2.0质粒自带的启动子,成功构建p YGAPMD12重组菌。最终产物经GC-MS检测显示,p YGAPMD12重组菌转化C18∶1的转化率为69.172%,比p YMD12重组菌提高32.771%。本文为进一步提高Δ12-脱饱和酶的表达水平提供参考依据。Abstract: Δ12- desaturase plays an important role in the biosynthesis of linoleic acid in Mucor sp EIM- 10. To improve enzymatic on the Δ12- desaturase activity, a recombinant expression vector was constructed. The Δ12-desaturase gene was cloned by RT- PCR technology. The PCR products were ligated into p YES2.0 plasmid. And then the recombinant plasmid ( p YMD12) was transformed into Saccharomyces cerevisiae strain INVSc1 by lithium acetate method. To improve enzymatic activity, built- in promoter of p YES2.0 was replaced by GAP promoter, another new recombinant expression ( p YGAPMD12) were constructed in Saccharomyces cerevisiae strain INVSc1.Gas chromatography analysis showed that conversion efficiency of p YGAPMD12 was 69.172% and the conversion efficiency of p YMD12 was 36.401%. This paper was provided important reference for further stay of Δ12-desaturase.
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