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

Appl Microbiol Biotechnol. 2008 Aug;80(2):223-9. doi: 10.1007/s00253-008-1523-1. Epub 2008 Jun 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aconitic Acid / metabolism
  • Amino Acid Sequence
  • Aspergillus / chemistry
  • Aspergillus / enzymology*
  • Aspergillus / genetics
  • Base Sequence
  • Carboxy-Lyases / chemistry
  • Carboxy-Lyases / genetics*
  • Carboxy-Lyases / isolation & purification
  • Carboxy-Lyases / metabolism
  • Cloning, Molecular*
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics*
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism
  • Gene Expression
  • Molecular Sequence Data
  • Open Reading Frames
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Succinates / metabolism

Substances

  • Fungal Proteins
  • Succinates
  • Aconitic Acid
  • Carboxy-Lyases
  • itaconic acid