Characterization of amylolytic enzyme overproducing mutant of <i>Aspergillus luchuensis</i> obtained by ion beam mutagenesis

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  • Kojo Toshihiro
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Kadooka Chihiro
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Komohara Marisa
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Onitsuka Shiori
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Tanimura Miya
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Muroi Yukiko
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Kurazono Shugo
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Shiraishi Yohei
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University Bio’c, Co., Ltd
  • Oda Ken
    National Research Institute of Brewing
  • Iwashita Kazuhiro
    National Research Institute of Brewing
  • Onoue Masahira
    Natural Science Centre for Research and Education, Kagoshima University
  • Okutsu Kayu
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Yoshizaki Yumiko
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Takamine Kazunori
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Futagami Taiki
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University
  • Mori Kazuki
    Computational Bio-Big Data Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology
  • Tamaki Hisanori
    Education and Research Centre for Fermentation Studies, Faculty of Agriculture, Kagoshima University

Abstract

<p>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.</p>

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