Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant

  • Takashi Nemoto
  • Taisuke Watanabe
  • Yutaka Mizogami
  • Jun-ichi Maruyama
  • Katsuhiko Kitamoto
Applied Genetics and Molecular Biotechnology
First Online:
Received:
Revised:
Accepted:

Abstract

Aspergillus oryzae has attracted much attention as a host for heterologous protein production because of its high secretion ability and safety. However, there have been only a few reports on construction of this organism to improve its properties as a production host. We previously reported that the double disruptant of the proteinase gene (tppA, pepE) improved human lysozyme (HLY) production. In this double disruptant, however, the HLY expression plasmid cannot be removed due to its random integration into the genome. In this study, we re-constructed the tppA pepE disruptant as a host for heterologous protein production. By the use of the tppA pepE disruptant, bovine chymosin (CHY) production was enhanced by 1.9-fold. Moreover, we generated HLY-producing strain from the tppA pepE disruptant by curable niaD marker, and then isolated HLY-hyperproducing mutants using the halo assay based on HLY activity. Subsequently, the niaD-based plasmid for HLY production was cured from the mutants by positive selection. The cured strains (named AUT strains) showed production levels of HLY and CHY that were 2.6- and 3.2-fold higher than those of the wild-type strain, respectively. Thus, the AUT strains are expected to be good hosts for obtaining higher production levels of various heterologous proteins.

Keywords

Aspergillus oryzae Heterologous protein production Protease Mutant Lysozyme Chymosin 
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Notes

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research (S) and Grant-in-Aid for Scientific Research on Priority Areas ‘‘Applied Genomics’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN of Japan).

Supplementary material

253_2008_1851_MOESM1_ESM.doc (216 kb)
ESM Table S1 List of up-regulated genes (DOC 215 kb)
253_2008_1851_MOESM2_ESM.doc (272 kb)
ESM Table S2 List of down-regulated genes (DOC 272 kb)

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Takashi Nemoto
    • 1
  • Taisuke Watanabe
    • 1
  • Yutaka Mizogami
    • 1
  • Jun-ichi Maruyama
    • 1
  • Katsuhiko Kitamoto
    • 1
  1. 1.Department of BiotechnologyThe University of TokyoTokyoJapan

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