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Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus

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Abstract

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.

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Acknowledgment

We acknowledge the financial support by the comprehensive support program for the creation of regional innovation in the Japanese Science and Technology Agency (JST).

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Authors and Affiliations

  1. JST Innovation Satellite Shizuoka, Japan Science and Technology Agency, 3–5–1 Johoku, Naka-ku, Hamamatsu, 432-8561, Japan

    Shin Kanamasa

  2. Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Lies Dwiarti, Mitsuyasu Okabe & Enoch Y. Park

  3. Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Enoch Y. Park

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  1. Shin Kanamasa
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  2. Lies Dwiarti
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  3. Mitsuyasu Okabe
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  4. Enoch Y. Park
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Correspondence to Enoch Y. Park.

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Kanamasa, S., Dwiarti, L., Okabe, M. et al. Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus . Appl Microbiol Biotechnol 80, 223–229 (2008). https://doi.org/10.1007/s00253-008-1523-1

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  • DOI: https://doi.org/10.1007/s00253-008-1523-1

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