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

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酶蛋白的理性设计奠定基础。

△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 li-brary 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.