Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L

Kotaro Kirimura, Masashi Yoda, Ikuyo Ko, Yuichi Oshida, Kouichiro Miyake, Shoji Usami

    Research output: Contribution to journalArticle

    14 Citations (Scopus)

    Abstract

    The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction 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 was isolated and sequenced, within which an ORF of 1425 bp encoding a protein of 475 aa with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted aa sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.

    Original languageEnglish
    Pages (from-to)237-243
    Number of pages7
    JournalJournal of Bioscience and Bioengineering
    Volume88
    Issue number3
    DOIs
    Publication statusPublished - 1999 Sep

    Fingerprint

    Citrate (si)-Synthase
    Aspergillus niger
    Cloning
    Aspergillus
    DNA Sequence Analysis
    Organism Cloning
    DNA
    Complementary DNA
    Genes
    Glutamic Acid
    Clone Cells
    Phenotype
    Neurospora crassa
    DNA Primers
    Polymerase chain reaction
    DNA sequences
    Staphylococcal Protein A
    Proteins
    Gene Library
    Codon

    Keywords

    • Aspergillus niger
    • Citrate synthase
    • Citric acid production
    • Heterologous gene expression
    • TCA cycle

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering

    Cite this

    Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L. / Kirimura, Kotaro; Yoda, Masashi; Ko, Ikuyo; Oshida, Yuichi; Miyake, Kouichiro; Usami, Shoji.

    In: Journal of Bioscience and Bioengineering, Vol. 88, No. 3, 09.1999, p. 237-243.

    Research output: Contribution to journalArticle

    Kirimura, Kotaro ; Yoda, Masashi ; Ko, Ikuyo ; Oshida, Yuichi ; Miyake, Kouichiro ; Usami, Shoji. / Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L. In: Journal of Bioscience and Bioengineering. 1999 ; Vol. 88, No. 3. pp. 237-243.
    @article{225f7ff8d11b4e0faa7fe62337e6d53c,
    title = "Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L",
    abstract = "The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction 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 was isolated and sequenced, within which an ORF of 1425 bp encoding a protein of 475 aa with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted aa sequence was 82, 68, and 65{\%} homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.",
    keywords = "Aspergillus niger, Citrate synthase, Citric acid production, Heterologous gene expression, TCA cycle",
    author = "Kotaro Kirimura and Masashi Yoda and Ikuyo Ko and Yuichi Oshida and Kouichiro Miyake and Shoji Usami",
    year = "1999",
    month = "9",
    doi = "10.1016/S1389-1723(00)80003-1",
    language = "English",
    volume = "88",
    pages = "237--243",
    journal = "Journal of Bioscience and Bioengineering",
    issn = "1389-1723",
    publisher = "Elsevier",
    number = "3",

    }

    TY - JOUR

    T1 - Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L

    AU - Kirimura, Kotaro

    AU - Yoda, Masashi

    AU - Ko, Ikuyo

    AU - Oshida, Yuichi

    AU - Miyake, Kouichiro

    AU - Usami, Shoji

    PY - 1999/9

    Y1 - 1999/9

    N2 - The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction 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 was isolated and sequenced, within which an ORF of 1425 bp encoding a protein of 475 aa with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted aa sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.

    AB - The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction 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 was isolated and sequenced, within which an ORF of 1425 bp encoding a protein of 475 aa with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted aa sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.

    KW - Aspergillus niger

    KW - Citrate synthase

    KW - Citric acid production

    KW - Heterologous gene expression

    KW - TCA cycle

    UR - http://www.scopus.com/inward/record.url?scp=0033199941&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=0033199941&partnerID=8YFLogxK

    U2 - 10.1016/S1389-1723(00)80003-1

    DO - 10.1016/S1389-1723(00)80003-1

    M3 - Article

    C2 - 16232605

    AN - SCOPUS:0033199941

    VL - 88

    SP - 237

    EP - 243

    JO - Journal of Bioscience and Bioengineering

    JF - Journal of Bioscience and Bioengineering

    SN - 1389-1723

    IS - 3

    ER -