Development of Aspergillus oryzae BCC7051 as a Robust Cell Factory Towards the Transcriptional Regulation of Protease-Encoding Genes for Industrial Applications

Enzyme-mediated protein degradation is a major concern in industrial fungal strain improvement, making low-proteolytic strains preferable for enhanced protein production. Here, we improved food-grade <i>Aspergillus oryzae</i> BCC7051 by manipulating the transcriptional regulation of protease-encoding genes. Genome mining of the transcription factor <i>AoprtR</i> and computational analysis confirmed its deduced amino acid sequence sharing evolutionary conservation across <i>Aspergillus</i> and <i>Penicillium</i> spp. The AoPrtR protein, which is classified into the Zn(II)2-Cys6-type transcription factor family, manipulates both intra- and extracellular proteolytic enzymes. Our transcriptional analysis indicated that the regulation of several protease-encoding genes was AoPrtR-dependent, with AoPrtR acting as a potent activator for extracellular acid-protease-encoding genes and a likely repressor for intracellular non-acid-protease-encoding genes. An indirect regulatory mechanism independent of PrtR may enhance proteolysis. Moreover, AoPrtR disruption increased extracellular esterase production by 2.55-fold, emphasizing its role in protein secretion. Our findings highlight the complexity of AoPrtR-mediated regulation by <i>A. oryzae</i>. Manipulation of regulatory processes through AoPrtR prevents secreted protein degradation and enhances the quantity of extracellular proteins, suggesting the low-proteolytic variant as a promising platform for the production of these proteins. This modified strain has biotechnological potential for further refinement and sustainable production of bio-based products in the food, feed, and nutraceutical industries.