Enhanced Production of Bovine Chymosin by Autophagy Deficiency in the Filamentous Fungus <i>Aspergillus oryzae</i>

Abstract

<div><p><i>Aspergillus oryzae</i> has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, <i>A. oryzae</i> 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 (<i>Aoatg</i>) gene disruptants of <i>A. oryzae</i>. We transformed <i>A. oryzae</i> gene disruptants of <i>Aoatg1</i>, <i>Aoatg13</i>, <i>Aoatg4</i>, <i>Aoatg8</i>, or <i>Aoatg15</i>, 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 <i>Aoatg</i> gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed <i>Aoatg</i> gene-conditional expression strains in which the promoter region of the <i>Aoatg</i> gene was replaced with the thiamine-controllable <i>thiA</i> 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 <i>Aoatg</i> gene disruptants. Consequently, we succeeded in the construction of <i>A. oryzae</i> 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 <i>A. oryzae</i>.</p></div

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