Molecular biological characterization of inducible expression of filamentous fungal genes and its application

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Filamentous fungi represented by Aspergillus oryzae have been playd important roles in Japanese fermentation industries. In this resarch project, we aimed to utilize genes efficiently in filamentous fungi by characterizing at the molecular levels transcriptional activator and repressor ptoteins involved in the inducible expression of filamentous fungal genes using Taka-amylase A gene as a model gene.We have cloned and determined the nucleotide sequences of two genomic genes and a cDNA encoding Taka-amylaseA from Aspergillus oryzae JCM02239. By using A.nidulans as an intermediate host, the expression properties of Taka-amylase A gene have been examined, which showed that CCAAT sequence and cis element recognized by CREA protein in the promoter region appeared to regulate Taka-amylase A gene expression.In this project, AnCP,CCAAT binding factor, has been shown to recognize CCAAT sequence of amdS and gatA genes of A.nidulans in addition to Taka-amylase A gene, which indicates AnCP in one of wide domain regulatory proteins in A.nidulans. Although we have been trying to clone the gene coding for AnCP by various methods such as South-Western or hybridization using DNA binding region of human NF-YA gene, AnCP gene has not been yet cloned. At the same time, we are trying to purify AnCP factor from nuclei by using DNA affinity column. As for CREA repressor protein, CREA binding sequences in the promoter region have been modified and uesd to transform A.nidulans. In near future, we will get information which CREA binding sequences are important to exert to carbon catabolite repression. Furthermore, maltose binding proteins have been isolated by using maltose affinity column and two binding proteins, 35 and 80kDa proteins, were detected which are now under chemical characterization.