Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae.

Citrate exporter CexA plays a key role in the production of citric acid in fungi; however, its role in intracellular metabolism has remained unclear. In this study, we comparably characterized homologous cexA genes in the white koji fungus Aspergillus luchuensis mut. kawachii and the yellow koji fungus Aspergillus oryzae, which exhibit high and low abilities, respectively, to produce citric acid. Disruption of cexA caused a significant decline of both extracellular and intracellular citric acid accumulation in Aspergillus kawachii, while overexpression of the A. kawachii cexA gene (AkcexA) into A. oryzae significantly enhanced both extracellular and intracellular citric acid accumulation in A. oryzae to a level comparable to that of A. kawachii. In addition, overexpression of two intrinsic cexA homologs (AocexA and AocexB) in A. oryzae also enhanced its extracellular and intracellular citric acid accumulation. Comprehensive analysis of intracellular metabolites from an AkcexA-overexpressing strain of A. oryzae compared with its control strain identified metabolic changes associated with intracellular citric acid accumulation via the glycolytic pathway, pentose phosphate pathway, and tricarboxylic acid cycle. Our results indicate that citric acid export enhances not only extracellular citric acid accumulation but also intracellular metabolic fluxes to generate citric acid.

Solar activation of fungus coated in photothermal cloth.

Bioorthogonal reactions based on manipulating the physicochemical and biological behavior of natural cells have gained tremendous attention for meeting the demands for multifunctional microorganisms without decreasing cell viability. Described herein is a novel bioorthogonal method for microorganism (Aspergillus oryzae) modification which coats the microorganism with a photothermal conversion cloth for staying bioactive in cold environments. Two steps, including ferric ions primarily binding to the microorganism cell surface, followed by in situ polymerization of pyrrole, are adopted to actualize highly efficient polypyrrole modification on the microorganism surfaces. The production of α-amylase by Aspergillus oryzae and α-amylase catalytic ability are two representative indexes of cold adaptation as confirmed by a starch decomposition test. This strategy for coating microorganisms with photothermal cloth is biocompatible and cost-effective, and can achieve non-contact modulation, which also offers great promise for generating living cell-polymer hybrid structures based on other microorganism systems for low-temperature environmental adaptation.

Influence of α-1,3-glucan synthase gene agsE on protoplast formation for transformation of Aspergillus luchuensis.

Aspergillus luchuensis NBRC4314 recently underwent genome sequencing. We have not used the frequently used protoplast-polyethylene glycol (PEG) method but have used agrobacterium-mediated transformation (AMT) to genetically engineer this strain because it was difficult to generate protoplasts using commercial cell wall lytic enzymes. In this study, we initially investigated the various conditions for protoplast formation in A. luchuensis. We found that A. luchuensis protoplasts could be generated using a minimal medium for the preculture medium, a static culture for the preculture condition, and Yatalase and α-1,3-glucanase as cell-wall lytic enzymes. These protoplasts could then be transformed with the protoplast-PEG method. Because α-1,3-glucanase was needed to form protoplasts in A. luchuensis, we investigated the role of the α-1,3-glucan synthase gene agsE in protoplast formation, one of five α-1,3-glucan synthase genes in A. luchuensis and a homolog of the major α-1,3-glucan synthase agsB in Aspergillus nidulans. We disrupted agsE in A. luchuensis (ΔagsE) with AMT and found that protoplast formation in ΔagsE was comparable with protoplast formation in Aspergillus oryzae with Yatalase. The ΔagsE protoplasts were also competent for transformation with the protoplast-PEG method. Hence, agsE appears to inhibit protoplast formation in A. luchuensis.

Development of koji by culturing Aspergillus oryzae on nori (Pyropia yezoensis).

Koji is a traditional fermentation culture medium, based on Aspergillus oryzae, which is commonly used in the manufacture process of Japanese fermented products such as soy sauce, miso, and sake, and promote enzymatic degradation. Koji is usually prepared by culturing a mold on cereals such as wheat flour, soybean, or rice, but that cultured on seaweeds has not been developed yet. This study prepared the koji by culturing A. oryzae on seaweed nori (dried piece of Pyropia yezoensis), and, then, characterized on this nori koji. The nori koji contained 0.85 µg N-acetylglucosamine, estimated as 6.1 µg mold cells, per gram dry matter and showed various kind of enzymatic activities in glycosidase, protease, and phosphatase as well as traditional soy sauce koji and rice koji. The suitability of these characteristics for degradation of nori was tested on nori sauce culture with and without the addition of the nori koji. After 167 days of culture, the fermentation tank with the nori koji showed over 74% recovery of supernatant while that without the nori koji had less than 57% recovery. The supernatant of culture mashes contained more than two times larger quantity of total nitrogen compounds in nori koji test group against control group. The present study prepared koji on seaweed nori for the first time and demonstrated its advantages to shorten the culture period and increase taste value in nori sauce manufacture. Development of seaweed koji enables a method to prepare cereal allergen free fermented sauces from seaweeds.

Production of milk peptides with antimicrobial and antioxidant properties through fungal proteases.

Bioactive peptides can provide health benefits due to different mechanisms. The aims of the present study are to produce bioactive peptides from bovine and goat milk subjected to the proteolytic activity of Aspergillus oryzae and Aspergillus flavipes enzymes, as well as to assess their putative antimicrobial and antioxidant activity. Bioactive peptides were successfully generated from proteases of fungi cultivated in solid-state fermentation. The generated peptides were effective against all tested bacteria and fungi. There was antioxidant activity, up to 92.5% DPPH reduction and ORAC stabilization at 52.5 µmol µL-1 of Trolox Equivalent. The generation of milk-specific sequences peptides in the samples was obtained through 2D-PAGE fractioning followed by mass spectrometry (ESI-MS/MS). Based on results in the present study, milk bioactive peptides presenting broad antimicrobial action and antioxidant activity spectra can be cost-effectively produced through solid-state fermentation. The herein addressed approach can be valuable for the pharmaceutical and food industries.

BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.

Early endosome motility mediates α-amylase production and cell differentiation in Aspergillus oryzae.

Recent research in filamentous fungi has revealed that the motility of an endocytic organelle early endosome (EE) has a versatile role in many physiological functions. Here, to further examine the motility of EEs in the industrially important fungus Aspergillus oryzae, we visualized these organelles via the Rab5 homolog AoRab5 and identified AoHok1, a putative linker protein between an EE and a motor protein. The Aohok1 disruptant showed retarded mycelial growth and no EE motility, in addition to an apical accumulation of EEs and peroxisomes. We further demonstrated that the Aohok1 disruptant exhibited less sensitivity to osmotic and cell wall stresses. Analyses on the protein secretory pathway in ΔAohok1 cells showed that, although distribution of the endoplasmic reticulum and Golgi was not affected, formation of the apical secretory vesicle cluster Spitzenkörper was impaired, probably resulting in the observed reduction of the A. oryzae major secretory protein α-amylase. Moreover, we revealed that the transcript level of α-amylase-encoding gene amyB was significantly reduced in the Aohok1 disruptant. Furthermore, we observed perturbed conidial and sclerotial formations, indicating a defect in cell differentiation, in the Aohok1 disruptant. Collectively, our results suggest that EE motility is crucial for α-amylase production and cell differentiation in A. oryzae.

Cell wall α-1,3-glucan prevents α-amylase adsorption onto fungal cell in submerged culture of Aspergillus oryzae.

We have previously reported that α-amylase (Taka-amylase A, TAA) activity disappears in the later stage of submerged Aspergillus oryzae culture as a result of TAA adsorption onto the cell wall. Chitin, one of the major components of the cell wall, was identified as a potential factor that facilitates TAA adsorption. However, TAA adsorption only occurred in the later stage of cultivation, although chitin was assumed to be sufficiently abundant in the cell wall regardless of the submerged culture period. This suggested the presence a factor that inhibits TAA adsorption to the cell wall in the early stage of cultivation. In the current study, we identified α-1,3-glucan as a potential inhibiting factor for TAA adsorption. We constructed single, double, and triple disruption mutants of three α-1,3-glucan synthase genes (agsA, agsB, and agsC) in A. oryzae. Growth characteristics and cell wall component analysis of these disruption strains showed that AgsB plays a major role in α-1,3-glucan synthesis. In the ΔagsB mutant, TAA was adsorbed onto the mycelium in all stages of cultivation (early and later), and the ΔagsB mutant cell walls had a significantly high capacity for TAA adsorption. Moreover, the α-1,3-glucan content of the cell wall prepared from the wild-type strain in the later stage of cultivation was markedly reduced compared with that in the early stage. These results suggest that α-1,3-glucan is a potential inhibiting factor for TAA adsorption onto the cell wall component, chitin, in the early stage of submerged culture in A. oryzae.

Carbon and nitrogen depletion-induced nucleophagy and selective autophagic sequestration of a whole nucleus in multinucleate cells of the filamentous fungus Aspergillus oryzae.

Autophagy is a conserved cellular degradation process in eukaryotes, in which cytoplasmic components and organelles are digested in vacuoles/lysosomes. Recently, autophagic degradation of nuclear materials, termed "nucleophagy", has been reported. In the multinucleate filamentous fungus Aspergillus oryzae, a whole nucleus is degraded by nucleophagy after prolonged culture. While developing an H2B-EGFP processing assay for the evaluation of nucleophagy in A. oryzae, we found that nucleophagy is efficiently induced by carbon or nitrogen depletion. Microscopic observations in a carbon depletion condition clearly demonstrated that autophagosomes selectively sequester a particular nucleus, despite the presence of multiple nuclei in the same cell. Furthermore, AoNsp1, the A. oryzae homolog of the yeast nucleoporin Nsp1p, mainly localized at the nuclear periphery, but its localization was restricted to the opposite side of the autophagosome being formed around a nucleus. In contrast, the perinuclear ER visualized with the calnexin AoClxA was not morphologically affected by nucleophagy. The findings of nucleophagy-inducing conditions enabled us to characterize the morphological process of autophagic degradation of a whole nucleus in multinucleate cells.

High-efficiency extracellular release of free fatty acids from Aspergillus oryzae using non-ionic surfactants.

Free fatty acids (FFAs) are useful for generating biofuel compounds and functional lipids. Microbes are increasingly exploited to produce FFAs via metabolic engineering. However, in many microorganisms, FFAs accumulate in the cytosol, and disrupting cells to extract them is energy intensive. Thus, a simple cost-effective extraction technique must be developed to remove this drawback. We found that FFAs were released from cells of the filamentous fungus Aspergillus oryzae with high efficiency when they were cultured or incubated with non-ionic surfactants such as Triton X-100. The surfactants did not reduce hyphal growth, even at 5% (w/v). When the faaA disruptant was cultured with 1% Triton X-100, more than 80% of the FFAs synthesized de novo were released. When the disruptant cells grown without surfactants were incubated for 1h in 1% Triton X-100 solution, more than 50% of the FFAs synthesized de novo were also released. Other non-ionic surfactants in the same ether series, such as Brij 58, IGEPAL CA-630, and Tergitol NP-40, elicited a similar FFA release. The dry cell weight of total hyphae decreased when grown with 1% Triton X-100. The decrement was 4.9-fold greater than the weight of the released FFAs, implying release of other intracellular compounds. Analysis of the culture supernatant showed that intracellular lactate dehydrogenase was also released, suggesting that FFAs are not released by a specific transporter. Therefore, ether-type non-ionic surfactants probably cause non-specific release of FFAs and other intracellular compounds by increasing cell membrane permeability.

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.

AoAtg26, a putative sterol glucosyltransferase, is required for autophagic degradation of peroxisomes, mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae.

Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus Aspergillus oryzae AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast Pichia pastoris. Deletion of Aoatg26 caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient A. oryzae strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the Aoatg26 deletion strain, indicating that autophagic process is impaired. Indeed, the Aoatg26 deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in A. oryzae, which may physiologically contribute to the differentiation in filamentous fungi.

Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function. Using an enhanced green fluorescent protein (EGFP)-tagged AoACBP construct we showed that AoACBP localized to punctate structures in the cytoplasm, some of which moved inside the cells in a microtubule-dependent manner. Further microscopic analyses showed that AoACBP-EGFP co-localized with the autophagy marker protein AoAtg8 tagged with red fluorescent protein (mDsRed). Expression of AoACBP-EGFP in disruptants of autophagy-related genes revealed aggregation of AoACBP-EGFP fluorescence in the cytoplasm of Aoatg1, Aoatg4 and Aoatg8 disruptant cells. However, in cells harboring disruption of Aoatg15, which encodes a lipase for autophagic body, puncta of AoACBP-EGFP fluorescence accumulated in vacuoles, indicating that AoACBP is transported to vacuoles via the autophagy machinery. Collectively, these results suggest the existence of a regulatory mechanism between AoACBP localization and autophagy.

Functional analysis of AoAtg11 in selective autophagy in the filamentous fungus Aspergillus oryzae.

Autophagy is a highly conserved cellular degradation process in eukaryotes and consists of both non-selective and selective types. Selective autophagic processes include pexophagy, mitophagy, and the cytoplasm-to-vacuole targeting (Cvt) pathway of yeast, in which particular vacuolar proteins, such as aminopeptidase I (Ape1), are selectively transported to vacuoles. Although selective autophagy has been mainly studied in the yeasts Saccharomyces cerevisiae and Pichia pastoris, there is evidence for selective autophagy in filamentous fungi; however, the details are poorly understood. In S. cerevisiae, Atg11 is a selective autophagy-specific protein that recognizes and transports substrates to the pre-autophagosomal structure (PAS). Here, we first identified an ATG11 homologue in the filamentous fungus Aspergillus oryzae and analyzed the localization of the corresponding protein, designated AoAtg11, fused to enhanced green fluorescent protein (EGFP). Imaging analysis revealed that AoAtg11-EGFP was localized to PAS-like structures. We next constructed an Aoatg11 disruptant of A. oryzae and showed that AoAtg11 is involved in pexophagy and mitophagy. In addition, AoAtg11 was found to be dispensable for non-selective autophagy and for transporting AoApe1 to vacuoles. Taken together, these results suggest that AoAtg11 is a selective autophagy-specific protein in A. oryzae, and has distinct molecular functions from that of S. cerevisiae Atg11.

Cell biology of the Koji mold Aspergillus oryzae.

Koji mold, Aspergillus oryzae, has been used for the production of sake, miso, and soy sauce for more than one thousand years in Japan. Due to the importance, A. oryzae has been designated as the national micro-organism of Japan (Koku-kin). A. oryzae has been intensively studied in the past century, with most investigations focusing on breeding techniques and developing methods for Koji making for sake brewing. However, the understanding of fundamental biology of A. oryzae remains relatively limited compared with the yeast Saccharomyces cerevisiae. Therefore, we have focused on studying the cell biology including live cell imaging of organelles, protein vesicular trafficking, autophagy, and Woronin body functions using the available genomic information. In this review, I describe essential findings of cell biology of A. oryzae obtained in our study for a quarter of century. Understanding of the basic biology will be critical for not its biotechnological application, but also for an understanding of the fundamental biology of other filamentous fungi.

Molecular characterization of intergeneric hybrid between Aspergillus oryzae and Trichoderma harzianum by protoplast fusion.

AIMS: Protoplast fusion between Aspergillus oryzae and Trichoderma harzianum and application of fusant in degradation of shellfish waste. METHODS AND RESULTS: The filamentous chitinolytic fungal strains A. oryzae NCIM 1272 and T. harzianum NCIM 1185 were selected as parents for protoplast fusion. Viable protoplasts were released from fungal mycelium using enzyme cocktail containing 5 mg ml(-1) lysing enzymes from T. harzianum, 0.06 mg ml(-1) ß-glucuronidase from Helix pomatia and 1 mg ml(-1) purified Penicillium ochrochloron chitinase in 0.8 mol l(-1) sorbitol as an osmotic stabilizer. Intergeneric protoplast fusion was carried out using 60% polyethylene glycol as a fusogen. At optimum conditions, the regeneration frequency of the fused protoplasts on colloidal chitin medium and fusion frequency were calculated. Fusant showed higher rate of growth pattern, chitinase activity and protein content than parents. Fusant formation was confirmed by morphological markers, viz. colony morphology and spore size and denaturation gradient gel electrophoresis (DGGE). CONCLUSIONS: This study revealed protoplast fusion between A. oryzae and T. harzianum significantly enhanced chitinase activity which ultimately provides potential strain for degradation of shellfish waste. Consistency in the molecular characterization results using DGGE is the major outcome of this study which can be emerged as a fundamental step in fusant identification. SIGNIFICANCE AND IMPACT OF THE STUDY: Now it is need to provide attention over effective chitin degradation to manage shrimp processing issues. In this aspect, ability of fusant to degrade shellfish waste efficiently in short incubation time revealed discovery of potential strain in the reclamation of seafood processing crustacean bio-waste.

Regioselective synthesis of cytarabine monopropionate by using a fungal whole-cell biocatalyst in nonaqueous medium.

The utilization of a dehydrated fungal biocatalyst of Aspergillus oryzae cells was successfully performed to achieve efficient acylation modification of a polar nucleoside cytarabine (ara-C). Organic solvents showed evident influence on the reaction catalyzed by the A. oryzae whole-cells. Except for hexane-pyridine, the catalytic activity and regioselectivity of the whole-cells clearly increased with increasing the polarity of the hydrophobic organic solvents used. The effects of some crucial factors on the reaction were further examined. The best reaction medium, hydrophobic solvent concentration, vinyl propionate/ara-C ratio, reaction temperature and shaking speed were confirmed as isopropyl ether (IPE)-pyridine, 30% (v/v), 90, 30 °C and 140-180 rpm, respectively. The cell biocatalyst also showed good thermal stabilities in both IPE-pyridine and hexane-pyridine systems. In addition, the desired 3'-O-propional derivative of ara-C was synthesized with the yields of 88.3% and regioselectivity (>70%). The resulting biocatalytic system appears to be an effective alternative, and can thus be employed for application in highly regioselective modification of nucleoside analogues.

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.

Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalyst highly expressing Candida antarctica lipase B.

For enzymatic biodiesel production from plant oil hydrolysates, an Aspergillus oryzae whole-cell biocatalyst that expresses Candida antarctica lipase B (r-CALB) with high esterification activity was developed. Each of soybean and palm oils was hydrolyzed using Candida rugosa lipase, and the resultant hydrolysates were subjected to esterification where immobilized r-CALB was used as a catalyst. In esterification, r-CALB afforded a methyl ester content of more than 90% after 6 h with the addition of 1.5 M equivalents of methanol. Favorably, stepwise additions of methanol and a little water were unnecessary for maintaining the lipase stability of r-CALB during esterification. During long-term esterification in a rotator, r-CALB can be recycled for 20 cycles without a significant loss of lipase activity, resulting in a methyl ester content of more than 90% even after the 20th batch. Therefore, the presented reaction system using r-CALB shows promise for biodiesel production from plant oil hydrolysates.

Multiple modes for gatekeeping at fungal cell-to-cell channels.

Cell-to-cell channels appear to be indispensable for successful multicellular organization and arose independently in animals, plants and fungi. Most of the fungi obtain nutrients from the environment by growing in an exploratory and invasive manner, and this ability depends on multicellular filaments known as hyphae. These cells grow by tip extension and can be divided into compartments by cell walls that typically retain a central pore that allows intercellular transport and cooperation. In the major clade of filamentous Ascomycota, integrity of this coenocytic organization is maintained by Woronin body organelles, which function as emergency patches of septal pores. In this issue of Molecular Microbiology, Bleichrodt and co-workers show that Woronin bodies can also form tight reversible associations with the pore and further link this to variation in levels of compartmental gene expression. These data define an additional modality of Woronin body-dependent gatekeeping. This commentary focuses on the implications of this work and the potential role of different modes of pore gating in controlling the growth and development of fungal tissues.