Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant
Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant
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DOI：
10.1007/s00253-008-1851-1
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发表时间：
2009-01
影响因子：
5
通讯作者：
Takashi Nemoto;Taisuke Watanabe;Yutaka Mizogami;J. Maruyama;K. Kitamoto
中科院分区：
文献类型：
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作者：
Takashi Nemoto;Taisuke Watanabe;Yutaka Mizogami;J. Maruyama;K. Kitamoto
米曲霉（Aspergillusoryzae）作为外源蛋白生产的宿主菌，因其分泌能力强、安全性高而受到广泛关注。然而，关于构建该生物体以改善其作为生产宿主的性质的报道很少。我们先前报道了蛋白酶基因（tppA，pepE）的双破坏剂提高人溶菌酶（HLY）的生产。然而，在这种双破坏剂中，HLY表达质粒由于其随机整合到基因组中而不能被去除。在本研究中，我们重新构建了tppA pepE干扰子作为异源蛋白生产的宿主。通过使用tppA pepE破坏剂，牛凝乳酶（CHY）的产量提高了1.9倍。此外，我们还利用标记技术从tppA pepE突变体中筛选出了HLY高产菌株，并利用基于HLY活性的晕圈法筛选出了HLY高产突变株。随后，通过阳性选择从突变体中消除用于HLY生产的基于theniaD的质粒。治愈菌株（命名为AUT菌株）的HLY和CHY产量水平分别比野生型菌株高2.6倍和3.2倍。因此，AUT菌株有望成为获得更高产量的各种异源蛋白的良好宿主。
Aspergillus oryzaehas 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 thetppA pepEdisruptant as a host for heterologous protein production. By the use of thetppA pepEdisruptant, bovine chymosin (CHY) production was enhanced by 1.9-fold. Moreover, we generated HLY-producing strain from thetppA pepEdisruptant by curableniaDmarker, and then isolated HLY-hyperproducing mutants using the halo assay based on HLY activity. Subsequently, theniaD-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.