Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis
Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis
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基于序列截断和定点诱变，对来自高山被孢霉的新鉴定的角鲨烯合酶进行定向优化。
DOI：
10.1007/s10295-015-1668-8
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发表时间：
2015-10-01
影响因子：
3.4
通讯作者：
Liu, Bin
中科院分区：
文献类型：
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作者：
Huang, Di;Yao, Yongpeng;Liu, Bin
萜类化合物是一类异戊二烯类化合物，通常从植物中分离出来，一直被用作商品香料和抗癌药物。角鲨烯(SQ)是合成三萜类和甾醇的重要前体，由两个法尼基二磷酸分子合成的角鲨烯合成酶(SQS)催化合成角鲨烯。高山被孢霉是一种工业菌株，经常用于生产不饱和脂肪酸，如γ-亚麻酸和花生四烯酸，在这项工作中，我们鉴定并鉴定了参与甾醇合成的关键基因SQS。生物信息学分析表明，MaSQS含有416个氨基酸残基，涉及4个高度保守的区域。系统发育分析表明，MaSQS与灵芝和曲霉的亲缘关系最近，也属于该真菌的一员。随后，重组蛋白在大肠杆菌BL21(DE3)中进行了表达，并进行了SDS-PAGE检测。为了改善蛋白质的表达和溶解性，在C末端删除了17或27个氨基酸。基于气相色谱-质谱法的体外活性研究表明，两种截短酶均能功能性地催化从FPP到SQ的反应，且在37℃、pH 7.2时酶活性最高。此外，在定点突变的基础上，突变酶mMaSQS Delta C17(E186K)的催化效率(k(Cat)/K(M))比对照提高了3.4倍。这是首次报道了高山木霉SQS的性质和修饰，为研究该真菌中类异戊二烯的生物合成提供了便利。工程合成的mMaSQS Delta C17(E186K)可能是合成生物学中合成萜类和类固醇的潜在候选者。
Terpenoids, a class of isoprenoids usually isolated from plants, are always used as commercial flavor and anticancer drugs. As a key precursor for triterpenes and sterols, biosynthesis of squalene (SQ) can be catalyzed by squalene synthase (SQS) from two farnesyl diphosphate molecules. In this work, the key SQS gene involved in sterols synthesis by Mortierella alpine, an industrial strain often used to produce unsaturated fatty acid such as gamma-linolenic acid and arachidonic acid, was identified and characterized. Bioinformatic analysis indicated that MaSQS contained 416 amino acid residues involved in four highly conserved regions. Phylogenetic analysis revealed the closest relationship of MaSQS with Ganoderma lucidum and Aspergillus, which also belonged to the member of the fungus. Subsequently, the recombinant protein was expressed in Escherichia coli BL21(DE3) and detected by SDS-PAGE. To improve the expression and solubility of protein, 17 or 27 amino acids in the C-terminal were deleted. In vitro activity investigation based on gas chromatography-mass spectrometry revealed that both the truncated enzymes could functionally catalyze the reaction from FPP to SQ and the enzymatic activity was optimal at 37 A degrees C, pH 7.2. Moreover, based on the site-directed mutagenesis, the mutant enzyme mMaSQS Delta C17 (E186K) displayed a 3.4-fold improvement in catalytic efficiency (k (cat)/K (m)) compared to the control. It was the first report of characterization and modification of SQS from M. alpine, which facilitated the investigation of isoprenoid biosynthesis in the fungus. The engineered mMaSQS Delta C17 (E186K) can be a potential candidate of the terpenes and steroids synthesis employed for synthetic biology.