Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus 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.
Kanamasa
Shin
S
JST Innovation Satellite Shizuoka, Japan Science and Technology Agency, 3-5-1 Johoku, Naka-ku, Hamamatsu, 432-8561, Japan.
Dwiarti
Lies
L
Okabe
Mitsuyasu
M
Park
Enoch Y
EY
D000156:Aconitic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013386:Succinates / Q000378:metabolism
D005656:Fungal Proteins; D013386:Succinates; D000156:Aconitic Acid; D002262:Carboxy-Lyases; C005229:itaconic acid