Aspergillus oryzae α-l-rhamnosidase: Crystal structure and insight into the substrate specificity.

The subsequent biochemical and structural investigations of the purified recombinant α-l-rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild-type was higher toward hesperidin and narirutin, where the l-rhamnose residue was α-1,6-linked to ß-d-glucoside, than toward neohesperidin and naringin with an α-1,2-linkage to ß-d-glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that Km values for neohesperidin, naringin, and rutin were lower compared to those for hesperidin and narirutin. kcat values for hesperidin and narirutin were higher than those for neohesperidin, naringin, and rutin. High catalytic efficiency (kcat /Km ) toward hesperidin and narirutin was a result of a considerably high kcat value, while Km values for hesperidin and narirutin were higher than those for naringin, neohesperidin, and rutin. The crystal structure of rAoRhaA revealed that the catalytic domain was represented by an (α/α)6 -barrel with the active site located in a deep cleft and two ß-sheet domains were also present in the N- and C-terminal sites of the catalytic domain. Additionally, five asparagine-attached N-acetylglucosamine molecules were observed. The catalytic residues of AoRhaA were suggested to be Asp254 and Glu524, and their catalytic roles were confirmed by mutational studies of D254N and E524Q variants, which lost their activity completely. Notably, three aspartic acids (Asp117, Asp249, and Asp261) located at the catalytic pocket were replaced with asparagine. D117N variant showed reduced activity. D249N and D261N variants activities drastically decreased.

Unveiling the reaction mechanism of arginine decarboxylase in Aspergillus oryzae: Insights from crystal structure analysis.

Agmatine, a natural polyamine also known as 4-aminobutyl-guanidine, is biosynthesized from arginine by decarboxylation. Aspergillus oryzae contains high amounts of agmatine, suggesting highly active arginine decarboxylase (ADC) in this organism. However, genome analysis revealed no ADC homolog in A. oryzae. A. oryzae strain RIB40 has six homologs of phosphatidylserine decarboxylase (PSD), an enzyme that synthesizes phosphatidyl ethanolamine from phosphatidylserine. We previously discovered that one of these homologs, AO090102000327, encodes arginine decarboxylase, which we named ADC1. In the present study, we determined the crystal structures of ligand-free, arginine-treated, and agmatine-treated ADC1 each at 1.9-2.15 Å resolution. Each structure contained four ADC1 molecules (chains A-D) in the asymmetric unit of the cell. Each ADC1 molecule is a heterodimer consisting of the N-terminal region (Asn60-Gly441) and C-terminal region (Ser442-Thr482). In the ligand-free ADC1, the N-terminus of Ser442 was modified to form a pyruvoyl group. In the arginine-treated ADC1, arginine was converted to agmatine, with the pyruvoyl group covalently bound to agmatine by forming a Schiff base. The same structure was observed in agmatine-treated ADC1. These results indicate that ADC1 is a pyruvoyl-dependent decarboxylase and unveils the reaction mechanism of ADC from A. oryzae.

Glucose-induced endocytic degradation of the maltose transporter MalP is mediated through ubiquitination by the HECT-ubiquitin ligase HulA and its adaptor CreD in Aspergillus oryzae.

In the filamentous fungus Aspergillus oryzae, large amounts of amylolytic enzymes are inducibly produced by isomaltose, which is converted from maltose incorporated via the maltose transporter MalP. In contrast, the preferred sugar glucose strongly represses the expression of both amylolytic and malP genes through carbon catabolite repression. Simultaneously, the addition of glucose triggers the endocytic degradation of MalP on the plasma membrane. In budding yeast, the signal-dependent ubiquitin modification of plasma membrane transporters leads to selective endocytosis into the vacuole for degradation. In addition, during glucose-induced MalP degradation, the homologous of E6AP C-terminus-type E3 ubiquitin ligase (HulA) is responsible for the ubiquitin modification of MalP, and the arrestin-like protein CreD is required for HulA targeting. Although CreD-mediated MalP internalization occurs in response to glucose, the mechanism by which CreD regulates HulA-dependent MalP ubiquitination remains unclear. In this study, we demonstrated that three (P/L)PxY motifs present in the CreD protein are essential for functioning as HulA adaptors so that HulA can recognize MalP in response to glucose stimulation, enabling MalP internalization. Furthermore, four lysine residues (three highly conserved among Aspergillus species and yeast and one conserved among Aspergillus species) of CreD were found to be necessary for its ubiquitination, resulting in efficient glucose-induced MalP endocytosis. The results of this study pave the way for elucidating the regulatory mechanism of MalP endocytic degradation through ubiquitination by the HulA-CreD complex at the molecular level.

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Lipid production by robust Aspergillus oryzae BCC7051 and a mathematical model describing its growth and lipid phenotypic traits.

AIMS: To identify the promising oleaginous Aspergillus oryzae strain and leverage its lipid and biomass production through a mathematical model. METHODS AND RESULTS: Comparative profiling of the cell growth and total fatty acid (TFA) content among 13 strains of A. oryzae was performed to explore the discrimination in their lipid productions. The oleaginicity of A. oryzae was found to be strain dependent, where the fungal strain BCC7051 exhibited superior performance in producing lipid-rich biomass by submerged fermentation. The TFA contents of the strain BCC7051 were comparable when cultivated at a range of pH values (pH 3.5-6.5) and temperatures (24-42°C). The mathematical model was generated, well describing and predicting the fungal growth and lipid phenotypic traits at various temperatures and carbon substrates. CONCLUSION: The A. oryzae strain BCC7051 was a robust cell factory, acquiring economically feasible options for producing valuable lipid-based products.

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.

Class VI G protein-coupled receptors in Aspergillus oryzae regulate sclerotia formation through GTPase-activating activity.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in eukaryotes that sense and transduce extracellular signals into cells. In Aspergillus oryzae, 16 canonical GPCR genes are identified and classified into nine classes based on the sequence similarity and proposed functions. Class VI GPCRs (AoGprK-1, AoGprK-2, and AoGprR in A. oryzae), unlike other GPCRs, feature a unique hybrid structure containing both the seven transmembrane (7-TM) and regulator of G-protein signaling (RGS) domains, which is not found in animal GPCRs. We report here that the mutants with double or triple deletion of class VI GPCR genes produced significantly increased number of sclerotia compared to the control strain when grown on agar plates. Interestingly, complementation analysis demonstrated that the expression of the RGS domain without the 7-TM domain is sufficient to restore the phenotype. In line with this, among the three Gα subunits in A. oryzae, AoGpaA, AoGpaB, and AoGanA, forced expression of GTPase-deficient mutants of either AoGpaA or AoGpaB caused an increase in the number of sclerotia formed, suggesting that RGS domains of class VI GPCRs are the negative regulators of these two GTPases. Finally, we measured the expression of velvet complex genes and sclerotia formation-related genes and found that the expression of velB was significantly increased in the multiple gene deletion mutants. Taken together, these results demonstrate that class VI GPCRs negatively regulate sclerotia formation through their GTPase-activating activity in the RGS domains. KEY POINTS: • Class VI GPCRs in A. oryzae regulate sclerotia formation in A. oryzae • RGS function of class VI GPCRs is responsible for regulation of sclerotia formation • Loss of class VI GPCRs resulted in increased expression of sclerotia-related genes.

Plant polysaccharide degradation-related enzymes in Aspergillus oryzae.

Plants synthesize large amounts of stored and structural polysaccharides. Aspergillus oryzae is used in traditional Japanese fermentation and produces many types of plant polysaccharide degradation-related enzymes. The carbohydrate-active enzymes of A. oryzae are important in the fermentation process and biotechnological applications. Because plant polysaccharides have a complex structure, cooperative and synergistic actions of enzymes are crucial for the degradation of plant polysaccharides. For example, the cooperative action of isoprimeverose-producing oligoxyloglucan hydrolase, ß-galactosidase, and α-xylosidase is important for the degradation of xyloglucan, and A. oryzae coordinates these enzymes at the expression level. In this review, I focus on the plant polysaccharide degradation-related enzymes identified in A. oryzae.

Evaluation of antifungal activity of cerium oxide nanoparticles and associated cellular responses.

Cerium oxide (CeO2) nanoparticles, as a metal oxide nanomaterial, are increasingly used for various industrial and biomedical applications. Although their cytotoxicity to bacteria and the associated mechanisms have attracted particular attention, the mechanisms behind their antifungal effects have remained unclear. This study investigated the antifungal properties of CeO2, focusing on Aspergillus oryzae. CeO2 inhibited fungal spore germination on solid substrates, and the effect was fungistatic rather than fungicidal. CeO2 inhibited fungal growth, especially under UV irradiation, and induced reactive oxygen species (ROS) production. Tocopherol reduced the intracellular ROS levels and the growth-inhibitory effects of CeO2, suggesting that ROS are involved in these growth-inhibitory effects. Transcriptomic analysis revealed upregulated expression of genes related to phospholipases and phosphate metabolism. CeO2 affected phosphate ion concentration in the medium, potentially influencing cellular responses. This research provided valuable insights into the antifungal effects of CeO2 application, which differ from those of conventional photocatalysts like TiO2.

Copper ion (Cu2+) is involved in the transcription of the tyrosinase-encoding melB gene of Aspergillus oryzae in solid-state culture.

In Aspergillus oryzae, the tyrosinase-encoding gene melB causes undesirable browning of sake and sake lees. This gene is known to be expressed specifically in solid-state culture; however, its expression mechanisms remain unknown. Here, we evaluated the possible factors affecting the transcription of melB and found that the copper ion (Cu2+) significantly enhanced the transcription level of melB in solid-state culture.

Transcriptional and post-transcriptional regulation of genes encoding secretory proteins in Aspergillus oryzae.

Aspergillus oryzae, also known as the yellow koji mold, produces various hydrolytic enzymes that are widely used in different industries. Its high capacity to produce secretory proteins makes this filamentous fungus a suitable host for heterologous protein production. Amylolytic gene promoter is widely used to express heterologous genes in A. oryzae. The expression of this promoter is strictly regulated by several transcription factors, whose activation involves various factors. Furthermore, the expression levels of amylolytic and heterologous genes are post-transcriptionally regulated by mRNA degradation mechanisms in response to aberrant transcriptional termination or endoplasmic reticulum stress. This review discusses the transcriptional and post-transcriptional regulatory mechanisms controlling the expression of genes encoding secretory proteins in A. oryzae.

A key component Rxt3 in the Rpd3L histone deacetylase complex regulates development, stress tolerance, amylase production and kojic acid synthesis in Aspergillus oryzae.

Rpd3L is a highly conserved histone deacetylase complex in eukaryotic cells and participates in various cellular processes. However, the roles of the Rpd3L component in filamentous fungi remain to be delineated ultimately. In this study, we constructed two knockout mutants of Rpd3L's Rxt3 subunit and characterized their biological functions in A. oryzae. Phenotypic analysis showed that AoRxt3 played a positive role in hyphal growth and conidia formation. Deletion of Aorxt3 resulted in augmented tolerance to multiple stresses, including cell wall stress, cell membrane stress, endoplasmic reticulum stress, osmotic stress and oxidative stress. Noteworthily, we found that Aorxt3-deleting mutants showed a higher kojic acid production than the control strain. However, the loss of Aorxt3 led to a significant decrease in amylase synthesis. Our findings lay the foundation for further exploring the role of other Rpd3L subunits and provide a new strategy to improve kojic acid production in A. oryzae.

Comparative pangenome analysis of Aspergillus flavus and Aspergillus oryzae reveals their phylogenetic, genomic, and metabolic homogeneity.

Aspergillus flavus and Aspergillus oryzae are closely related fungal species with contrasting roles in food safety and fermentation. To comprehensively investigate their phylogenetic, genomic, and metabolic characteristics, we conducted an extensive comparative pangenome analysis using complete, dereplicated genome sets for both species. Phylogenetic analyses, employing both the entirety of the identified single-copy orthologous genes and six housekeeping genes commonly used for fungal classification, did not reveal clear differentiation between A. flavus and A. oryzae genomes. Upon analyzing the aflatoxin biosynthesis gene clusters within the genomes, we observed that non-aflatoxin-producing strains were dispersed throughout the phylogenetic tree, encompassing both A. flavus and A. oryzae strains. This suggests that aflatoxin production is not a distinguishing trait between the two species. Furthermore, A. oryzae and A. flavus strains displayed remarkably similar genomic attributes, including genome sizes, gene contents, and G + C contents, as well as metabolic features and pathways. The profiles of CAZyme genes and secondary metabolite biosynthesis gene clusters within the genomes of both species further highlight their similarity. Collectively, these findings challenge the conventional differentiation of A. flavus and A. oryzae as distinct species and highlight their phylogenetic, genomic, and metabolic homogeneity, potentially indicating that they may indeed belong to the same species.

Green preparation and evaluation of the anti-psoriatic activity of vesicular elastic nanocarriers of kojic acid from Aspergillus oryzae N12: Repurposing of a dermo-cosmetic lead.

Psoriasis is a skin disorder characterized by impaired epidermal differentiation that is regularly treated by systemic drugs with undesirable side effects. Based on its anti-inflammatory, antiproliferative and anti-melanoma attributes, the fungal metabolite kojic acid represents an attractive candidate for anti-psoriatic research. The present work aims to investigate an efficient topical bio-friendly vesicular system loaded with kojic acid isolated from Aspergillus oryzae as an alternative way for the management of psoriasis to avoid systemic toxicity. Kojic acid-loaded spanlastics were prepared by ethanol injection technique, employing span 60 along with brij 35 and cremophor rh40 as edge activators, with the complete in vitro characterization of the developed nanoplatform. The selected formulation displayed a spherical morphology, an optimum particle size of 234.2 ± 1.65 nm, high entrapment efficiency (87.4% ± 0.84%) and significant sustained drug release compared with the drug solution. In vivo studies highlighted the superior relief of psoriasis symptoms and the ability to maintain healthy skin with the least changes in mRNA expression of inflammatory cytokines, achieved by the developed nanoplatform compared to kojic acid solution. Moreover, the in vivo histopathological studies confirmed the safety of the topically applied spanlastics. In addition, the molecular mechanism was approached through in vitro assessment of cathepsin S and PDE-4 inhibitory activities and in silico investigation of kojic acid docking in several anti-psoriatic drug targets. Our results suggest that a topically applied vesicular system loaded with kojic acid could lead to an expansion in the dermo-cosmetic use of kojic acid as a natural bio-friendly alternative for systemic anti-psoriatic drugs.

Study on the Skincare Effects of Red Rice Fermented by Aspergillus oryzae In Vitro.

Red rice, a variety of pigmented grain, serves dual purposes as both a food and medicinal resource. In recent years, we have witnessed an increasing interest in the dermatological benefits of fermented rice extracts, particularly their whitening and hydrating effects. However, data on the skincare advantages derived from fermenting red rice with Aspergillus oryzae remain sparse. This study utilized red rice as a substrate for fermentation by Aspergillus oryzae, producing a substance known as red rice Aspergillus oryzae fermentation (RRFA). We conducted a preliminary analysis of RRFA's composition followed by an evaluation of its skincare potential through various in vitro tests. Our objective was to develop a safe and highly effective skincare component for potential cosmetic applications. RRFA's constituents were assessed using high-performance liquid chromatography (HPLC), Kjeldahl nitrogen determination, the phenol-sulfuric acid method, and enzyme-linked immunosorbent assay (ELISA). We employed human dermal fibroblasts (FB) to assess RRFA's anti-aging and antioxidative properties, immortalized keratinocytes (HaCaT cells) and 3D epidermal models to examine its moisturizing and reparative capabilities, and human primary melanocytes (MCs) to study its effects on skin lightening. Our findings revealed that RRFA encompasses several bioactive compounds beneficial for skin health. RRFA can significantly promote the proliferation of FB cells. And it markedly enhances the mRNA expression of ECM-related anti-aging genes and reduces reactive oxygen species production. Furthermore, RRFA significantly boosts the expression of Aquaporin 3 (AQP3), Filaggrin (FLG), and Hyaluronan Synthase 1 (HAS1) mRNA, alongside elevating moisture levels in a 3D epidermal model. Increases were also observed in the mRNA expression of Claudin 1 (CLDN1), Involucrin (IVL), and Zonula Occludens-1 (ZO-1) in keratinocytes. Additionally, RRFA demonstrated an inhibitory effect on melanin synthesis. Collectively, RRFA contains diverse ingredients which are beneficial for skin health and showcases multifaceted skincare effects in terms of anti-aging, antioxidant, moisturizing, repairing, and whitening capabilities in vitro, highlighting its potential for future cosmetic applications.

Upgrading the Bioactive Potential of Hazelnut Oil Cake by Aspergillus oryzae under Solid-State Fermentation.

Hazelnut oil cake (HOC) has the potential to be bioactive component source. Therefore, HOC was processed with a solid-state fermentation (SSF) by Aspergillus oryzae with two steps optimization: Plackett-Burman and Box-Behnken design. The variables were the initial moisture content (X1: 30-50%), incubation temperature (X2: 26-37 °C), and time (X3: 3-5 days), and the response was total peptide content (TPC). The fermented HOC (FHOC) was darker with higher protein, oil, and ash but lower carbohydrate content than HOC. The FHOC had 6.1% more essential amino acid and benzaldehyde comprised 48.8% of determined volatile compounds. Fermentation provided 14 times higher TPC (462.37 mg tryptone/g) and higher phenolic content as 3.5, 48, and 7 times in aqueous, methanolic, and 80% aqueous methanolic extract in FHOC, respectively. FHOC showed higher antioxidant as ABTS+ (75.61 µmol Trolox/g), DPPH (14.09 µmol Trolox/g), and OH (265 mg ascorbic acid/g) radical scavenging, and α-glucosidase inhibition, whereas HOC had more angiotensin converting enzyme inhibition. HOC showed better water absorption while FHOC had better oil absorption activity. Both cakes had similar foaming and emulsifying activity; however, FHOC produced more stable foams and emulsions. SSF at lab-scale yielded more bioactive component with better functionality in FHOC.

Release of CM-12 from A2-type casein by the cleavage of Ser-Leu-Xaa at the C-terminus using Aspergillus oryzae alkaline protease.

BACKGROUND: Aspergillus oryzae protease can release the opioid peptide ß-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. RESULTS: A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with µ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. CONCLUSION: Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.

Large-Scale Production of Anti-RNase A VHH Expressed in pyrG Auxotrophic Aspergillus oryzae.

Nanobodies, also referred to as VHH antibodies, are the smallest fragments of naturally produced camelid antibodies and are ideal affinity reagents due to their remarkable properties. They are considered an alternative to monoclonal antibodies (mAbs) with potential utility in imaging, diagnostic, and other biotechnological applications given the difficulties associated with mAb expression. Aspergillus oryzae (A. oryzae) is a potential system for the large-scale expression and production of functional VHH antibodies that can be used to meet the demand for affinity reagents. In this study, anti-RNase A VHH was expressed under the control of the glucoamylase promoter in pyrG auxotrophic A. oryzae grown in a fermenter. The feature of pyrG auxotrophy, selected for the construction of a stable and efficient platform, was established using homologous recombination. Pull-down assay, size exclusion chromatography, and surface plasmon resonance were used to confirm the binding specificity of anti-RNase A VHH to RNase A. The affinity of anti-RNase A VHH was nearly 18.3-fold higher (1.9 nM) when expressed in pyrG auxotrophic A. oryzae rather than in Escherichia coli. This demonstrates that pyrG auxotrophic A. oryzae is a practical, industrially scalable, and promising biotechnological platform for the large-scale production of functional VHH antibodies with high binding activity.

Expression and characterization of recombinant IL-1Ra in Aspergillus oryzae as a system.

BACKGROUND: The interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae. RESULTS: We purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53 mg/L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Asp. IL-1Ra is N-glycosylated and approximately 17 kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life between Asp. IL-1Ra and E. coli IL-1Ra. Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5 nM. The in vitro half-life of Asp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96 h) and showed higher stability than E. coli IL-1Ra, despite exhibiting a 100-fold lower binding affinity (2 nM). CONCLUSION: This study reports the production of a functional Asp. IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed in A. oryzae. Our results suggest that Asp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative to E. coli IL-1Ra.

The C2H2-type zinc-finger regulator AoKap5 is required for the growth and kojic acid synthesis in Aspergillus oryzae.

Aspergillus oryzae is an important filamentous fungus widely used for the industrial production of fermented foods and secondary metabolites. The clarifying of the mechanism of the growth and secondary metabolites in A. oryzae is important for its industrial production and utilization. Here, the C2H2-type zinc-finger protein AoKap5 was characterized to be involved in the growth and kojic acid production in A. oryzae. The Aokap5-disrupted mutants were constructed by the CRISPR/Cas9 system, which displayed increased colony growth but decreased conidial formation. Deletion of Aokap5 enhanced the tolerance to cell-wall and oxidative but not osmotic stress. The transcriptional activation assay revealed that AoKap5 itself didn't have transcriptional activation activity. Disruption of Aokap5 resulted in the reduced production of kojic acid, coupled with the reduced expression of the kojic acid synthesis genes kojA and kojT. Meanwhile, overexpression of kojT could rescue the decreased production of kojic acid in Aokap5-deletion strain, indicating that Aokap5 serves upstream of kojT. Furthermore, the yeast one-hybrid assay demonstrated that AoKap5 could directly bind to the kojT promoter. These findings suggest that AoKap5 regulates kojic acid production through binding to the kojT promoter. This study provides an insight into the role of zinc finger protein in the growth and kojic acid biosynthesis of A. oryzae.