Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger

Kotaro Kirimura, Satoshi Ogawa, Takasumi Hattori, Kuniki Kino

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    24 Citations (Scopus)

    Abstract

    In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc1 to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.

    Original languageEnglish
    Pages (from-to)210-214
    Number of pages5
    JournalJournal of Bioscience and Bioengineering
    Volume102
    Issue number3
    DOIs
    Publication statusPublished - 2006 Sep

    Fingerprint

    Aspergillus niger
    Aspergillus
    Citric acid
    Citric Acid
    Mitochondria
    Genes
    Proteins
    Fluorescence
    Antimycin A
    Cytochromes
    Glucose
    Fusion reactions
    Electron Transport Complex III
    Gene Fusion
    Mycelium
    Cyanides
    Cytochromes c
    Fungi
    alternative oxidase
    enhanced green fluorescent protein

    Keywords

    • alternative oxidase
    • Aspergillus niger
    • citric acid
    • enhanced green fluorescent protein
    • respiration

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering

    Cite this

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    title = "Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger",
    abstract = "In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc1 to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.",
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    author = "Kotaro Kirimura and Satoshi Ogawa and Takasumi Hattori and Kuniki Kino",
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    T1 - Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger

    AU - Kirimura, Kotaro

    AU - Ogawa, Satoshi

    AU - Hattori, Takasumi

    AU - Kino, Kuniki

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    N2 - In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc1 to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.

    AB - In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc1 to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.

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