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.