Chaperone complex formation of the transcription factor MalR involved in maltose utilization and amylolytic enzyme production in Aspergillus oryzae.

The Zn2Cys6-type transcription factor MalR controls the expression of maltose-utilizing (MAL) cluster genes and the production of amylolytic enzymes in Aspergillus oryzae. In the present study, we demonstrated that MalR formed a complex with Hsp70 and Hsp90 chaperones under non-inducing conditions similar to the yeast counterpart Mal63 and that the complex was released from the chaperone complex after the addition of the inducer maltose. The MalR protein was constitutively localized in the nucleus and mutation in both the putative nuclear localization signals (NLSs) located in the zinc finger motif and the C-terminal region resulted in the loss of nuclear localization. This result indicated the involvement of NSLs in the MalR nuclear localization. However, mutation in both NLSs did not affect the dissociation mode of the MalR-Hsp70/Hsp90 complex, suggesting that MalR activation induced by maltose can occur regardless of its intracellular localization.

Distinct mechanism of activation of two transcription factors, AmyR and MalR, involved in amylolytic enzyme production in Aspergillus oryzae.

The production of amylolytic enzymes in Aspergillus oryzae is induced in the presence of starch or maltose, and two Zn2Cys6-type transcription factors, AmyR and MalR, are involved in this regulation. AmyR directly regulates the expression of amylase genes, and MalR controls the expression of maltose-utilizing (MAL) cluster genes. Deletion of malR gene resulted in poor growth on starch medium and reduction in α-amylase production level. To elucidate the activation mechanisms of these two transcription factors in amylase production, the expression profiles of amylases and MAL cluster genes under carbon catabolite derepression condition and subcellular localization of these transcription factors fused with a green fluorescent protein (GFP) were examined. Glucose, maltose, and isomaltose induced the expression of amylase genes, and GFP-AmyR was translocated from the cytoplasm to nucleus after the addition of these sugars. Rapid induction of amylase gene expression and nuclear localization of GFP-AmyR by isomaltose suggested that this sugar was the strongest inducer for AmyR activation. In contrast, GFP-MalR was constitutively localized in the nucleus and the expression of MAL cluster genes was induced by maltose, but not by glucose or isomaltose. In the presence of maltose, the expression of amylase genes was preceded by MAL cluster gene expression. Furthermore, deletion of the malR gene resulted in a significant decrease in the α-amylase activity induced by maltose, but had apparently no effect on the expression of α-amylase genes in the presence of isomaltose. These results suggested that activation of AmyR and MalR is regulated in a different manner, and the preceding activation of MalR is essential for the utilization of maltose as an inducer for AmyR activation.