Aspergillus oryzae PrtR alters transcription of individual peptidase genes in response to the growth environment.

Aspergillus oryzae PrtR is an ortholog of the transcription factor PrtT, which positively regulates the transcription of extracellular peptidase genes in Aspergillus niger and Aspergillus fumigatus. To identify the genes under the control of PrtR and elucidate its regulatory mechanism in A. oryzae, prtR gene disruption mutants were generated. The control strain clearly showed a halo on media containing skim milk as the nitrogen source, whereas the ΔprtR strain formed a smaller halo. Measurement of acid peptidase activity revealed that approximately 84% of acidic endopeptidase and 86% of carboxypeptidase activities are positively regulated by PrtR. As the transcription of the prtR gene varied depending on culture conditions, especially with or without a protein substrate, it was considered that its transcription would be regulated in response to a nitrogen source. In addition, contrary to previous expectations, PrtR was found to act both in promoting and repressing the transcription of extracellular peptidase genes. The mode of regulation varied from gene to gene. Some genes were regulated in the same manner in both liquid and solid cultures, whereas others were regulated in different ways depending on the culture conditions. Furthermore, PrtR has been suggested to regulate the transcription of peptidase genes that are closely associated with other transcription factors. KEY POINTS: • Almost all peptidase genes in Aspergillus oryzae are positively regulated by PrtR • However, several genes are regulated negatively by PrtR • PrtR optimizes transcription of peptidase genes in response to culture conditions.

Physiological ER stress caused by amylase production induces regulated Ire1-dependent mRNA decay in Aspergillus oryzae.

Regulated Ire1-dependent decay (RIDD) is a feedback mechanism in which the endoribonuclease Ire1 cleaves endoplasmic reticulum (ER)-localized mRNAs encoding secretory and membrane proteins in eukaryotic cells under ER stress. RIDD is artificially induced by chemicals that generate ER stress; however, its importance under physiological conditions remains unclear. Here, we demonstrate the occurrence of RIDD in filamentous fungus using Aspergillus oryzae as a model, which secretes copious amounts of amylases. α-Amylase mRNA was rapidly degraded by IreA, an Ire1 ortholog, depending on its ER-associated translation when mycelia were treated with dithiothreitol, an ER-stress inducer. The mRNA encoding maltose permease MalP, a prerequisite for the induction of amylolytic genes, was also identified as an RIDD target. Importantly, RIDD of malP mRNA is triggered by inducing amylase production without any artificial ER stress inducer. Our data provide the evidence that RIDD occurs in eukaryotic microorganisms under physiological ER stress.

A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme.

Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, 2 enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The 2 enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

Corrigendum: A Glycosylphosphatidylinositol-Anchored α-Amylase Encoded by amyD Contributes to a Decrease in the Molecular Mass of Cell Wall α-1,3-Glucan in Aspergillus nidulans.

[This corrects the article DOI: 10.3389/ffunb.2021.821946.].

Oryzapsins, the orthologs of yeast yapsin in Aspergillus oryzae, affect ergosterol synthesis.

The oryzapsin genes opsA and opsB in Aspergillus oryzae encoding glycosylphosphatidylinositol (GPI)-anchored aspartic endopeptidase are homologs of Saccharomyces cerevisiae yapsins. We recently found another homolog, opsC, in the A. oryzae genome database, which was suggested to be a pseudogene. However, the profiles and roles of the proteins encoded by these genes have not yet been clarified. Toward this end, we first produced opsA- and opsB-overexpression strains and performed enzymatic analyses, revealing that OpsA and OpsB can attack sites other than the carboxyl-terminal peptide bonds of basic amino acids. Moreover, OpsA and OpsB were confirmed to bind to the cell membrane with a GPI anchor. Second, opsA and opsB single-deletion and double-deletion strains (ΔopsA, ΔopsB, and ΔopsAΔopsB) were constructed to explore the expected roles of oryzapsins in cell wall synthesis, similar to the role of yapsins. The transcription level of mpkA in the cell wall integrity pathway was increased in ΔopsB and ΔopsAΔopsB strains, suggesting that OpsB might be involved in processing cell wall synthesis-related proteins. Treatment with an ergosterol biosynthesis inhibitor reduced the growth of the ΔopsAΔopsB strain. Moreover, the mRNA levels of Aoerg1, Aoerg3-1, Aoerg3-2, Aoerg7b, Aoerg11, and Aohmg1,2 showed a decreasing tendency in the ΔopsAΔopsB strain, and the ergosterol content in the membrane was reduced in the ΔopsAΔopsB strain. These results suggest that oryzapsins exist in the cell membrane and play roles in the formation of cell membranes. This is the first report of the involvement of GPI-anchored aspartic endopeptidases in ergosterol biosynthesis.Key points• The oryzapsins have wider substrate specificity than yaspins in S. cerevisiae.• Unlike the yapsins, the oryzapsins might not be involved in the main structure synthesis of the cell wall.• The oryzapsins would be involved in ergosterol biosynthesis.

Japanese Traditional Miso and Koji Making.

Miso is a traditional Japanese seasoning paste produced by fermenting soybeans using the power of koji mold. A recent Japanese cohort study has shown that increased consumption of fermented soybean products is associated with a reduced risk of death in both men and women. In this review, we briefly explain what miso means in the Japanese culture and food industry, varieties of miso available today, and steps involved in miso making. Then, we review early and latest scientific researches in koji mold species, their safety, and beneficial enzymes they produce during fermentation and maturation processes, which play a major part in determining the quality and sensory profile of miso.

Downregulation of the ypdA Gene Encoding an Intermediate of His-Asp Phosphorelay Signaling in Aspergillus nidulans Induces the Same Cellular Effects as the Phenylpyrrole Fungicide Fludioxonil.

Many eukaryotic histidine-to-aspartate (His-Asp) phosphorelay systems consist of three types of signal transducers: a His-kinase (HK), a response regulator (RR), and a histidine-containing phosphotransfer intermediate (HPt). In general, the HPt acts as an intermediate between the HK and the RR and is indispensable for inducing appropriate responses to environmental stresses. In a previous study, we attempted but were unable to obtain deletion mutants of the ypdA gene in order to characterize its function in the filamentous fungus Aspergillus nidulans. In the present study, we constructed the CypdA strain in which ypdA expression is conditionally regulated by the A. nidulans alcA promoter. We constructed CypdA strains with RR gene disruptions (CypdA-sskAΔ, CypdA-srrAΔ, and CypdA-sskAΔsrrAΔ). Suppression of YpdA induced by ypdA downregulation activated the downstream HogA mitogen-activated protein kinase cascade. YpdA suppression caused severe growth defects and abnormal hyphae, with features such as enhanced septation, a decrease in number of nuclei, nuclear fragmentation, and hypertrophy of vacuoles, both regulated in an SskA-dependent manner. Fludioxonil treatment caused the same cellular responses as ypdA suppression. The growth-inhibitory effects of fludioxonil and the lethality caused by ypdA downregulation may be caused by the same or similar mechanisms and to be dependent on both the SskA and SrrA pathways.

A Glycosylphosphatidylinositol-Anchored α-Amylase Encoded by amyD Contributes to a Decrease in the Molecular Mass of Cell Wall α-1,3-Glucan in Aspergillus nidulans.

α-1,3-Glucan is one of the main polysaccharides in the cell wall of Aspergillus nidulans. We previously revealed that it plays a role in hyphal aggregation in liquid culture, and that its molecular mass (MM) in an agsA-overexpressing (agsAOE) strain was larger than that in an agsB-overexpressing (agsBOE) strain. The mechanism that regulates its MM is poorly understood. Although the gene amyD, which encodes glycosylphosphatidylinositol (GPI)-anchored α-amylase (AmyD), is involved in the biosynthesis of α-1,3-glucan in A. nidulans, how it regulates this biosynthesis remains unclear. Here we constructed strains with disrupted amyD (ΔamyD) or overexpressed amyD (amyDOE) in the genetic background of the ABPU1 (wild-type), agsAOE, or agsBOE strain, and characterized the chemical structure of α-1,3-glucans in the cell wall of each strain, focusing on their MM. The MM of α-1,3-glucan from the agsBOE amyDOE strain was smaller than that in the parental agsBOE strain. In addition, the MM of α-1,3-glucan from the agsAOE ΔamyD strain was greater than that in the agsAOE strain. These results suggest that AmyD is involved in decreasing the MM of α-1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions.

Strategy towards tailored donor tissue-specific pancreatic islet isolation.

BACKGROUND: Optimizing the collagenase G (ColG):collagenase H (ColH) ratio is a key strategy for achieving tailored donor-tissue specific islet isolation. Collagen V (Col V) and collagen III (Col III) are crucial target matrices of ColG and ColH, respectively. We herein investigated the relevance between the expression of target matrices in pancreatic tissues and influence of ColG:ColH ratio on islet isolation outcome. METHODS: Islet isolation was performed in Lewis and SD rats using different ColG:ColH ratios (5:1, 1:1 and 1:5; n = 7/group). The composition of Col III and Col V was examined using immunohistochemical staining, real-time polymerase chain reaction (PCR), Western blotting and mass spectrometry. Chain types in collagen I (Col I) were also assessed using mass spectrometry. RESULTS: No beneficial effects were observed by increasing the ColG amount, irrespective of the rat strain. In contrast, the islet yield in Lewis rats was considerably increased by high amounts of ColH but decreased in SD rats, suggesting that Lewis pancreas contains more Col III than SD pancreas. Neither immunohistochemical nor real-time PCR showed correlation with isolation outcome. However, Western blotting revealed that Lewis contained considerably higher amount of Col III than SD (p = 0.10). Likewise, Col-I(α1)/Col-III(α1) and Col-I(α2)/Col-III(α1) were significantly lower in Lewis than in SD rats (p = 0.007, respectively). Furthermore, the isolation outcome was considerably correlated with the composition of homotrimeric Col I. CONCLUSIONS: The Col III expression and the composition of homotrimeric Col I in pancreatic tissues determined using mass analyses appeared useful for optimizing the ColG:ColH ratio in islet isolation.

Deletion of admB gene encoding a fungal ADAM affects cell wall construction in Aspergillus oryzae.

Mammals possess a unique signaling system based on the proteolytic mechanism of a disintegrin and metalloproteinases (ADAMs) on the cell surface. We found two genes encoding ADAMs in Aspergillus oryzae and named them admA and admB. We produced admA and admB deletion strains to elucidate their biological function and clarify whether fungal ADAMs play a similar role as in mammals. The ∆admA∆admB and ∆admB strains were sensitive to cell wall-perturbing agents, congo red, and calcofluor white. Moreover, the two strains showed significantly increased weights of total alkali-soluble fractions from the mycelial cell wall compared to the control strain. Furthermore, ∆admB showed MpkA phosphorylation at lower concentration of congo red stimulation than the control strain. However, the MpkA phosphorylation level was not different between ∆admB and the control strain without the stimulation. The results indicated that A. oryzae AdmB involved in the cell wall integrity without going through the MpkA pathway.

Analysis of the ionic interaction between the hydrophobin RodA and two cutinases of Aspergillus nidulans obtained via an Aspergillus oryzae expression system.

Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. In the fungus Aspergillus oryzae, the hydrophobin RolA and the polyesterase CutL1 are co-expressed when the sole available carbon source is the biodegradable polyester polybutylene succinate-co-adipate (PBSA). RolA promotes the degradation of PBSA by attaching to the particle surface, changing its structure and interacting with CutL1 to concentrate CutL1 on the PBSA surface. We previously reported that positively charged residues in RolA and negatively charged residues in CutL1 are cooperatively involved in the ionic interaction between RolA and CutL1. We also reported that hydrophobin RodA of the model fungus Aspergillus nidulans, which was obtained via an A. oryzae expression system, interacted via ionic interactions with CutL1. In the present study, phylogenetic and alignment analyses revealed that the N-terminal regions of several RolA orthologs contained positively charged residues and that the corresponding negatively charged residues on the surface of CutL1 that were essential for the RolA-CutL1 interaction were highly conserved in several CutL1 orthologs. A PBSA microparticle degradation assay, a pull-down assay using a dispersion of Teflon particles, and a kinetic analysis using a quartz crystal microbalance revealed that recombinant A. nidulans RodA interacted via ionic interactions with two recombinant A. nidulans cutinases. Together, these results imply that ionic interactions between hydrophobins and cutinases may be common among aspergilli and other filamentous fungi.

Genome sequence of Aspergillus luchuensis NBRC 4314.

Awamori is a traditional distilled beverage made from steamed Thai-Indica rice in Okinawa, Japan. For brewing the liquor, two microbes, local kuro (black) koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae are involved. In contrast, that yeasts are used for ethanol fermentation throughout the world, a characteristic of Japanese fermentation industries is the use of Aspergillus molds as a source of enzymes for the maceration and saccharification of raw materials. Here we report the draft genome of a kuro (black) koji mold, A. luchuensis NBRC 4314 (RIB 2604). The total length of nonredundant sequences was nearly 34.7 Mb, comprising approximately 2,300 contigs with 16 telomere-like sequences. In total, 11,691 genes were predicted to encode proteins. Most of the housekeeping genes, such as transcription factors and N-and O-glycosylation system, were conserved with respect to Aspergillus niger and Aspergillus oryzae An alternative oxidase and acid-stable α-amylase regarding citric acid production and fermentation at a low pH as well as a unique glutamic peptidase were also found in the genome. Furthermore, key biosynthetic gene clusters of ochratoxin A and fumonisin B were absent when compared with A. niger genome, showing the safety of A. luchuensis for food and beverage production. This genome information will facilitate not only comparative genomics with industrial kuro-koji molds, but also molecular breeding of the molds in improvements of awamori fermentation.

Collagen V Is a Potential Substrate for Clostridial Collagenase G in Pancreatic Islet Isolation.

The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans.

A novel non-thermostable deuterolysin from Aspergillus oryzae.

Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database ( http://www.bio.nite.go.jp/dogan/project/view/AO ). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB.

Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae.

Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes ß-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the ß-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of ß-1,3-glucan polymerization.

Three extracellular dipeptidyl peptidases found in Aspergillus oryzae show varying substrate specificities.

Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.

A long natural-antisense RNA is accumulated in the conidia of Aspergillus oryzae.

Analysis of expressed sequence tag libraries from various culture conditions revealed the existence of conidia-specific transcripts assembled to putative conidiation-specific reductase gene (csrA) in Aspergillus oryzae. However, the all transcripts were transcribed with opposite direction to the gene csrA. The sequence analysis of the transcript revealed that the RNA overlapped mRNA of csrA with 3'-end, and did not code protein longer than 60 amino acid residues. We designated the transcript Conidia Specific Long Natural-antisense RNA (CSLNR). The real-time PCR analysis demonstrated that the CSLNR is conidia-specific transcript, which cannot be transcribed in the absence of brlA, and the amount of CSLNR was much more than that of the transcript from csrA in conidia. Furthermore, the csrA deletion, also lacking coding region of CSLNR in A. oryzae reduced the number of conidia. Overexpression of CsrA demonstrated the inhibition of growth and conidiation, while CSLNR did not affect conidiation.

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate.

Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation.

BACKGROUND: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1.

Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co-adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA-CutL1 interaction by using site-directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N-terminus of RolA, play crucial roles in the RolA-CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 (K(D) = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger KD values, particularly in the interaction between the double variant RolA-H32S/K34S and the triple variant CutL1-E31S/D142S/D171S (K(D) = 78.0 nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface.