Cloning and Expression of Aspergillus niger icdA Gene Encoding Mitochondrial NADP^+-Specific Isocitrate Dehydrogenase

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概要

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• The complementary DNA (cDNA) and chromosomal DNA (icdA) encoding the NADP^+-specific isocitrate dehydrogenase (EC 1.1.1.42) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Two cDNA clones (cDNA-1, 2.0 kb; cDNA-2, 1.5 kb) were obtained and sequenced, and an ORF of 1494 base pairs (bp) encoding a protein of 498 amino acids (aa) was identified in cDNA-1. The predicted amino acid sequence showed 73% and 67% sequence identities with those of the mitochondrial NADP^+-ICDHs from Saccharomyces cerevisiae and pig, respectively. The sequence analysis of cDNA-1 and -2 revealed that the cDNA-2 lacks a 500-bp fragment from cDNA-1 which contains a mitochondrial targeting motif. A peroxisomal targeting motif at the C-terminus was found on the aa sequences of cDNA-1 and cDNA-2, but the cDNA-2 product seemed to be localized in the cytoplasm since the peroxisomes were not found in the mycelia of WU-2223L cultivated under the conditions of citric acid production. The expression of both cDNAs in Escherichia coli DEK2004, an isocitrate dehydrogenase-deficient mutant, revealed that both cDNAs complemented the glutamate-requiring phenotype, and that the transformants retamed NADP^+-ICDH activities. Therefore, it was clarified that both of the cDNA-1 and -2 products are fully functional. The chromosomal DNA, icdA, was cloned to correspond to cDNA-1, and its nucleotide sequence revealed that it contains seven introns. Southern hybridization using cDNA-1 and cDNA-2 indicated that there is only one copy of icdA on the chromosomes of A. niger WU-2223L. Northern hybridization analysis as for total RNA of WU-2223L revealed that two mRNAs of different sizes, 2.0 kb and 1.5 kb, were hybridized to the ORF of cDNA-1 used as a probe. Therefore, it was found that approximately 1500-nt and 2000-nt mRNAs were transcribed from only one icdA chromosomal gene in A. niger. Such a transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.

• 社団法人日本生物工学会の論文
• 2002-02-25

著者

• Kino Kuniki

  Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University

• Usami Shoji

  Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima Uni

• Izumi Yoshikazu

  Dep. Of Biotechnology Tottori Univ.

• KIRIMURA Kohtaro

  Department of Applied Chemistry, School of Science and Engineering, Waseda University

• Kino K

  Department Of Applied Chemistry School Of Science And Engineering Waseda University

• Kino Kuniki

  Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University

• Kino Kuniki

  Department Of Applied Chemistry School Of Science And Engineering Waseda University

• Ishii Yoshitaka

  Department of Applied Chemistry, School of Science and Engineering, Waseda University

• YODA MASASHI

  Department of Applied Chemistry, School of Science and Engineering, Waseda University

• KUMATANI MASAKI

  Department of Applied Chemistry, School of Science and Engineering, Waseda University

• Usami S

  Waseda Univ. Tokyo Jpn

• Usami S

  Waseda Univ.

• Usami Shoji

  Dept. Biol. Fac. Sci. Nagoya Univ.

• Kino Kuniki

  Dep. Of Applied Chemistry Fac. Of Sci. And Engineering Waseda Univ. 3-4-1 Ohkubo Shinjuku-ku Tokyo 1

• Ishii Yoshitaka

  Institute For Biomedical Engineering Consolidated Research Institute For Advanced Science And Medica

• Kirimura Kohtaro

  Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University

• Usami Shoji

  Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University

• Yoda Masashi

  Department Of Applied Chemistry School Of Science And Engineering Waseda University

• Ishii Yoshitaka

  Bio-refining Process Laboratory Advanced Technology And Research Institute Petroleum Energy Center

• Kumatani Masaki

  Department Of Applied Chemistry School Of Science And Engineering Waseda University

• Ishii Yoshitaka

  Department Of Applied Chemistry Faculty Of Science And Engineering Waseda University

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