Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L

Kotaro Kirimura, Masashi Yoda, Shoji Usami

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    Abstract

    A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5'-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger.

    Original languageEnglish
    Pages (from-to)472-477
    Number of pages6
    JournalCurrent Genetics
    Volume34
    Issue number6
    DOIs
    Publication statusPublished - 1999

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    Keywords

    • Alternative oxidase
    • Aspergillus niger
    • Cyanide-insensitive respiration
    • Mitochondria
    • Salicylhydroxamic acid

    ASJC Scopus subject areas

    • Genetics

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