Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus
Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus
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DOI：
10.1007/s00253-008-1523-1
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发表时间：
2008-08-01
影响因子：
5
通讯作者：
Park, Enoch Y.
中科院分区：
文献类型：
--
作者：
Kanamasa, Shin;Dwiarti, Lies;Park, Enoch Y.
一种丝状真菌Aspergillus terreus产生衣康酸，据预测，衣康酸通过真菌碳代谢中的顺式乌头酸脱羧酶（CAD）催化而衍生自顺式乌头酸。为了阐明该酶的功能和生物合成途径，我们克隆了一个新的基因编码的酶。该基因的开放阅读框（CAD 1）由1，529 bp组成，编码490个氨基酸，并被单个内含子中断。在数据库中鉴定的蛋白质中，由CAD 1编码的蛋白质的一级结构与MmgE/PrpD蛋白家族共享高度的同一性，包括细菌的许多2-甲基柠檬酸酯酶。将不含内含子的克隆基因导入由ADH 1启动子控制的表达质粒pAUR-CAD 1中。以顺乌头酸为底物，通过直接检测其产物衣康酸，证实了酿酒酵母中的CAD活性。这一结果首次揭示了该基因编码CAD，CAD是A. terreus
A filamentous fungus Aspergillus terreus produces itaconic acid, which is predicted to be derived from cis-aconitic acid via catalysis by cis-aconitic acid decarboxylase (CAD) in the carbon metabolism of the fungus. To clarify the enzyme's function and a pathway for itaconic acid biosynthesis, we cloned a novel gene encoding the enzyme. The open reading frame of this gene (CAD1) consists of 1,529 bp encoding 490 amino acids and is interrupted by a single intron. Among the identified proteins in the database, the primary structure of the protein encoded by CAD1 shared high identity with the MmgE/PrpD family of proteins, including a number of 2-methylcitrate dehydratases of bacteria. The cloned gene excluding an intron was introduced into the expression plasmid pAUR-CAD1 controlled by the ADH1 promoter. The CAD activity in Saccharomyces cerevisiae was confirmed by directly detecting itaconic acid as a product from cis-aconitic acid as a substrate. This result reveals for the first time that this gene encodes CAD, which is essential for itaconic acid production in A. terreus.