Cloning and expression of Aspergillus niger icdA gene encoding mitochondrial NADP⁺-specific isocitrate dehydrogenase

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 retained 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. Sucha transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.