Gene Cloning of the Enzymes Related to Citric Acid Production in Fungi

1997 Fiscal Year Final Research Report Summary

• Project/Area Number: 07650965
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 生物・生体工学
• Research Institution: Waseda University
• Principal Investigator: USAMI Shoji School Sci.&Engineer., Dept.Appl.Chem., Professor, 理工学部, 教授 (50063508)
• Co-Investigator(Kenkyū-buntansha): KIRIMURA Kohtaro School Sci.&Engineer., Dept.Appl.Chem., Assoc.Professor, 理工学部, 助教授 (90195412)
• Project Period (FY): 1995 – 1997
• Keywords: isocitrate dehydrogenase / genetic engineering / citric acid production / citrate synthase / gene cloning / Aspergillus niger

Research Abstract

Genes encoding citrate synthase and NADP-isocitrate dehydrogenase of the citric acid-producing fungus, Aspergillus niger, were cloned and sequenced. In the cDNA clone encoding citrate synthase, an open reading frame of 1,425 bp encoding 475 amino acids with a molecular weight of 52,153 Da was found, and its N-terminal region consists of a typical mitochondrial-targeting motif. The chromosomal gene encoding citrate synthase is interrupted by six introns, and in the upstream of the coding region a CT rich region but not TATAAA nor CAAT motifs was found as a presumable promoter, In the chromosomal DNA and cDNA encoding NADP-isocitrate dehydrogenase, an open reading frame encoding the protein of 498 amino acids having a mitochondrial-targeting motif was found. The chromosomal DNA was interrupted by seven introns with sizes of 49 241 bp, and one intron before the starting codon ATG was also found, In the upstream of the coding region, two CAAT-like motifs were found. When each of the cDNA clones encoding the citrate synthase or the NADP-isocitrate dehydrogenase was expressed in the Escherichia coli mutant strain deficient of each enzyme, the enzyme activity corresponding to the cDNA expressed was detected in each transformant.