Contribution of UbrA, a ubiquitin ligase essential for Arg/N-degron pathway, to peptidase gene expression in Aspergillus oryzae

Abstract

The degradation of intracellular proteins by N-degron pathways depends on their N-terminal amino acids. In budding yeast, the Arg/N-degron pathway controls the expression of di/tri-peptide transporter gene by degrading transcriptional repressor. However, there is no detailed information on the N-degron pathway in filamentous fungi, and its role in regulating microbial nitrogen metabolism is unclear. Here, we demonstrated that the E3 ubiquitin ligase, UbrA, which is required for the Arg/N-degron pathway, regulates peptidase gene expression in the filamentous fungus Aspergillus oryzae. Using ubiquitin-fused green fluorescent protein as a reporter, we showed that the Arg/N-degron pathway in A. oryzae is similar to that in budding yeast. Disruption of ubrA significantly reduced activities of acidic endopeptidase and carboxypeptidase in submerged culture using soy protein as the nitrogen source. In addition, ubrA disruption dramatically reduced the mRNA expression of the major endopeptidase and carboxypeptidase genes but increased alkaline peptidase production. Moreover, ubrA disruption reduced the expression levels of di peptidyl- and tripeptidyl- peptidase genes and di/tri-peptide transporter genes. This regulation was independent of PrtR, the transcription factor of major extracellular peptidase genes. Our data showed that UbrA is involved in the expression of various peptidase genes in concert with di/tri-peptide transporter genes. IMPORTANCEPeptidases produced by Aspergillus oryzae are important in the production of Japanese fermented foods and are used as industrial enzymes for various food-processing and pharmaceutical applications. The expression of di/tri-peptide transporter gene in budding yeast is controlled by a positive feedback mechanism through the dipeptide-mediated activation of the E3 ubiquitin ligase, Ubr1, which is essential for the Arg/N-degron pathway, which determines the lifetime of intracellular proteins. In this study, we demonstrated that A. oryzae UbrA (an ortholog of yeast Ubr1) regulates peptidase gene expression in addition to di/tri-peptide transporter genes. Disruption of ubrA decreases the expression of major acidic peptidase genes and increases the expression of alkaline peptidase gene. In addition, the expression levels of di/tri-peptidyl peptidase genes and di/tri-peptide transporter genes were reduced by ubrA disruption. These results suggest that UbrA regulates the expression of various peptidase genes to facilitate positive feedback of di/tri-peptide transporter genes.