Characterization of amylolytic enzyme overproducing mutant of Aspergillus luchuensis obtained by ion beam mutagenesis

Aspergillus luchuensis is a kuro (black) koji fungus that has been used as a starch degrader for the awamori- and shochu-making industries in Japan. In this study, we investigated the effect of ion beam irradiation on A. luchuensis RIB2601 and obtained a high starch-degrading mutant strain U1. Strain U1 showed reduced growth rate, whereas it showed higher α-amylase, glucoamylase, and α-glucosidase activities on a mycelial mass basis than the wild type (wt) strain both on agar plates and in rice koji. In addition, strain U1 showed higher N-acetylglucosamine content in the cell wall and higher sensitivity to calcofluor white, suggesting a deficiency in cell wall composition. Interestingly, produced protein showed higher expression of acid-labile α-amylase (AmyA) and glucoamylase (GlaA) in strain U1, although real-time RT-PCR indicated no significant change in the transcription of the amyA or glaA gene. These results suggested that the high amylolytic activity of strain U1 is attributable to a high AmyA and GlaA production level, but the elevated production is not due to transcriptional regulation of the corresponding genes. Furthermore, RNA-seq analysis indicated that strain U1 shows transcriptional changes in at least 604 genes related to oxidation-reduction, transport, and glucosamine-containing compound metabolic processes, which may be involved in the deficient cell wall composition of strain U1.