Yasuaki Kawarasaki

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 dipeptide/tripeptide transporter gene by degrading a 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 the 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 dipeptidyl-peptidase and tripeptidyl-peptidase genes and dipeptide/tripeptide transporter genes. This regulation was independent of PrtR, the transcription factor regulating a broad range of extracellular peptidase genes. Our data showed that UbrA is involved in the expression of various peptidase genes in concert with dipeptide/tripeptide transporter genes. IMPORTANCE: Peptidases 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 dipeptide/tripeptide 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 dipeptide/tripeptide 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 dipeptide/tripeptidyl peptidase genes and dipeptide/tripeptide transporter genes were reduced by ubrA disruption. These results suggest that UbrA regulates the expression of various peptidase genes to facilitate positive feedback of dipeptide/tripeptide transporter genes.

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. IMPORTANCE: Peptidases 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.

Yeast’s extracellular expression provides a cost-efficient means of producing recombinant proteins of academic or commercial interests. However, depending on the protein to be expressed, the production occasionally results in a poor yield, which is frequently accompanied with a deteriorated growth of the host. Here we describe our simple approach, high cell-density expression, to circumvent the cellular toxicity and achieve the production of a certain range of “difficult-to-express” secretory protein in preparative amount. The system features an ease of performing: (a) pre-cultivate yeast cells to the stationary phase in non-inducing condition, (b) suspend the cells to a small aliquot of inducing medium to form a high cell-density suspension or “a phalanx,” then (c) give a sufficient aeration to the phalanx. Factors and pitfalls that affect the system’s performance are also described.

Peptides inhibiting human dipeptidyl peptidase IV (hDPP-IV) have potential as functional food ingredients to help prevent type 2 diabetes. In the present study, we analyzed the hDPP-IV inhibitory dipeptides contained in the hydrolysates of green tea grounds. The IC50 values of 337 standard dipeptides were calculated for the hDPP-IV inhibition. Next, to reveal whether the top 17 dipeptides with IC50 values less than 0.1 mM were present in the hydrolysates, the tea grounds hydrolysates were analyzed by LC-MS. Among the 17 dipeptides, Val-Leu, Leu-Trp, Trp-Ile, Asn-His, Thr-His, Thr-Trp, Trp-Ala, and Trp-Val were identified in the hydrolysates as novel hDPP-IV inhibitory dipeptides. Val-Leu particularly inhibited hDPP-IV (IC50: 0.074 mM) and was the most abundant (558 µg/g). These results suggest that green tea grounds are a promising dietary source for generating inhibitory peptides of hDPP-IV.

Bitterness-masking agents support the intake of functional food ingredients, which taste bitter. However, evidence-based methodology to find bitterness-masking agents remains to be established. In the present study, focusing on the fact that a bitter tastant-binding compound is expected to have a bitterness-masking effect, several epigallocatechin gallate (EGCg)-binding peptides were identified from the amino acid sequence of ribulose 1,5-bisphosphate carboxylase/oxygenase using peptide array technology. Deletion analysis and alanine scanning analysis of these peptides revealed that electrostatic interaction contribute strongly to the binding mechanism. EGCg-binding peptides identified in this study, namely, MHFRVLAKALR and FTGLKSTSAFPVTRK, successfully suppressed the activation of the bitter-taste receptor hTAS2R39 by administration of EGCg. These results suggest that peptide array technology can be used to screen bitterness-masking peptides.