Cloning and expression of the cyanide-insensitive alternative oxidase gene ( aox1) from a citric acid-producing strain Aspergillus niger WU-2223L.

Masashi Yoda, Kohtaro Kirimura, Kiyotake Kamigaki, Kuniki Kino, and Shoji Usam

Author address: 

Department of Applied Chemistry, School of Science and Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku-ku, Tokyo, 169-8555, Japan

Abstract: 

In Aspergillus niger WU-2223L, a cyanide (CN)- and antimycin A- insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists and is catalyzed by the alternative oxidase (AOX), besides the CN- sensitive cytochrome pathway. Both the CN-insensitive and SHAM-sensitive respiration and the activity of AOX, as duroquinol oxidase activity, were shown to be localized in mitochondria. Such respiration and specific activity in purified mitochondria increased by addition of methanol, an inducer of citric acid production, or antimycin A, both accompanying the increase of citric acid productivity. On the other hand, when WU-2223L was cultivated with SHAM, the AOX activity decreased accompanying the decrease of citric acid production drastically although mycelial growth was not affected. The complementary DNA (cDNA) and chromosomal DNA encoding the AOX were cloned. One full-length cDNA was obtained and sequenced to reveal that the clone contained an ORF encoding a polypeptide of 351 amino acid. The deduced amino acid sequences revealed that there are two long hydrophobic regions regarded as membrane-spanning regions of the protein and two iron-binding motifs as a reactive center. When the whole ORF was introduced and expressed in Escherichia coli, the transformant harboring pKAOX1 containing the ORF gene showed cyanide-insensitive and SHAM- sensitive respiration, and the expression was induced to two folds by addition of IPTG. The chromosomal DNA encoding AOX gene (aox1) was also cloned from a chromosomal DNA library of A. niger and a 2856 bp-long DNA fragment was isolated. The aox1 contains two introns, and two TATA boxes in untranslated regions. Southern hybridization analysis with the cDNA and aox1 as probes revealed that there is only one copy of aox1 in the chromosome of A. niger WU-2223L. The motifs for carbon catabolite repressor (CREA) and two nitrogen metabolite repressor (AREA) binding sites were also found in upstream region of the ORF, suggesting that the carbon catabolism and nitrogen metabolism regulation might be involved in the transcription of aox1. The Northern blot analysis was done on the total RNA extracted from the mycelia cultivated under citric acid producing-conditions with 2% methanol (v/v). The transcription activity was highest for the mycelia cultivated for 2 days, the lowest for 4 days, and maintained at almost constant level through 6 to 8 days, and the correlation between the mRNA levels and AOX activities was observed.
2001

abstract No: 

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Full conference title: 

21st Fungal Genetics Conference
    • Fungal Genetics Conference 21st (2000)