Regulation of the glucoamylase-encoding gene (glaB), expressed in solid-state culture (koji) of Aspergillus oryzae

Aspergillus oryzae has two glucoamylase-encoding genes, glaA and glaB, the patterns of expression of which are different. Expression of the glaB gene is marked in solid-state culture (koji), but low in submerged culture. To elucidate the induction mechanism of the glaB promoter in solid-state culture (koji), we employed a fusion gene system using the glaA or glaB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). The expression of glaB-GUS was induced by starch or maltooligosaccharides in a similar manner to that glaA-GUS, but other physical factors were found to be required for the maximal expression of the glaB gene in solid-state culture (koji). The time-course of glaB-GUS expression in solid-state culture (rice-koji making) suggested that its expression is induced by low water activity (Aw) of the medium and high temperature. When mycelia grown on a membrane under standard conditions were transferred to low-Aw and high-temperature conditions (membrane-transfer culture, MTC), glaB expression was markedly induced, but that of glaA was not. Additionally, glaB-GUS production was induced in MTC using a membrane with smaller pore size, suggesting that a physical barrier against hyphal extension could regulate glaB expression. Under conditions found to induce glaB expression, namely, starch, low-Aw, high-temperature and physical barriers, approximately 6300 U/mg-protein was obtained, equivalent to that in solid-state culture (koji). In conclusion, glucoamylase production under these induction conditions achieved in MTC reached 274 U/ml-broth, which was equivalent to the level observed in solid-state culture (koji). Northern blot analysis indicated that glaB expression was induced at the level of transcription 4 h after the transfer to the inducible conditions described above.