Department of Biotechnology, The University of Tokyo, Tokyo, Japan.
PLoS One. 2013 Apr 29;8(4):e62512. doi: 10.1371/journal.pone.0062512. Print 2013.
Aspergillus oryzae has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (Aoatg) gene disruptants of A. oryzae. We transformed A. oryzae gene disruptants of Aoatg1, Aoatg13, Aoatg4, Aoatg8, or Aoatg15, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the Aoatg gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed Aoatg gene-conditional expression strains in which the promoter region of the Aoatg gene was replaced with the thiamine-controllable thiA promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the Aoatg gene disruptants. Consequently, we succeeded in the construction of A. oryzae strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in A. oryzae.
米曲霉已被用作异源蛋白生产的宿主，因为它具有高蛋白质分泌能力和食品安全特性。然而，由于各种潜在机制，包括自噬等降解过程，米曲霉通常产生的目标异源蛋白产量低于预期，这可能是蛋白质生产的一个重大瓶颈。在本研究中，我们研究了米曲霉几种自噬（Aoatg）基因敲除突变体中外源蛋白的生产。我们转化了 Aoatg1、Aoatg13、Aoatg4、Aoatg8 或 Aoatg15 的米曲霉基因敲除突变体，带有牛凝乳酶（CHY）表达构建体，发现 CHY 的产量与对照菌株相比增加了两倍。然而，值得注意的是，Aoatg 基因敲除突变体的分生孢子形成显著减少。由于大量的分生孢子是接种大规模培养所必需的，我们还构建了 Aoatg 基因条件表达菌株，其中 Aoatg 基因的启动子区域被硫胺素可控制的 thiA 启动子取代。在没有硫胺素的情况下，所得转化体的分生孢子形成明显增强，而自噬在存在硫胺素的情况下仍受到抑制。此外，这些转化体显示出增加的 CHY 生产能力，与 Aoatg 基因敲除突变体相当。因此，我们成功构建了由于自噬缺陷而能够产生高水平 CHY 的米曲霉菌株。我们的发现表明，自噬的条件调节是增加米曲霉异源蛋白生产的有效方法。
