毛霉△6-脂肪酸脱氢酶随机突变文库的构建与分析

Construction and analysis of a random mutation library of △6-desaturase gene in Mucor sp. EIM-10

摘要:

毛霉Mucor sp. EIM-10 △6-脂肪酸脱氢酶是γ-亚麻酸合成途径的关键酶。为提高脂肪酸脱氢酶的活性以及研究该酶一级结构对酶活性的影响, 利用易错PCR对毛霉△6-脂肪酸脱氢酶基因(mcd6)进行随机突变, 将PCR产物与大肠杆菌-酿酒酵母穿梭表达载体PYMD6PMCD6连接, 获得随机突变质粒PYTBMCD6, 转化酿酒酵母 Saccharomyces cerevisiae, 构建了原始库容为4.6×104 CFU的△6-脂肪酸脱氢酶的随机突变表达文库。随机突变表达文库的构建与分析为定点突变等

Abstract:

△6-desaturase plays an important role in the biosynthesis of γ-linolenic acid in Mucor sp. EIM-10. To improve enzymatic activity and confirm the effect of primary structure on the △6-desaturase activity, a random mutation library was constructed. The △6-desaturase gene (mcd6, GenBank accession No. EU717846) was mutated by error-prone PCR strategy. The PCR products were ligated into the yeast expression vector PYMD6PMCD6. The resulting plasmid was named PYTBMCD6 and transformed into Saccharomyces cerevisiae strain INVSc1. A random △6-desaturase gene mutation library was constructed in Saccharomyces cerevisiae. The titers of random mutation library were up to 4.6×104 CFU. The random mutation library of △6-desaturase gene could be used for screening the mutants with change in catalytic activity and the subsequent site-directed mutagenesis.