How Fungi Biosynthesize 3-Nitropropanoic Acid: The Simplest yet Lethal Mycotoxin.

We uncovered the biosynthetic pathway of the lethal mycotoxin 3-nitropropanoic acid (3-NPA) from koji mold Aspergillus oryzae. The biosynthetic gene cluster (BGC) of 3-NPA, which encodes an amine oxidase and a decarboxylase, is conserved in many fungi used in food processing, although most of the strains have not been reported to produce 3-NPA. Our discovery will lead to efforts that improve the safety profiles of these indispensable microorganisms in making food, alcoholic beverages, and seasoning.

Profile of Phenolic Compounds and Antioxidant Activity of Celery (Apium graveolens) Juices Obtained from Pulp after α-Amylase Treatment from Aspergillus oryzae.

The purpose of this study was to determine the content of certain phenolic compounds, antioxidant activity, pressing efficiency, extract content, and sugars in celeriac juices obtained from the pulp after α-amylase treatment from Aspergillus oryzae. The test material consisted of peeled and unpeeled celery pulp kept at a temperature of 25 °C with and without the enzyme for a period of 30 and 60 min. The juices obtained from them were analyzed for the content of selected phenolic acids and flavonoids using the UPLC-PDA-ESI-MS/MS method, for antioxidant activity measured using the ABTS˙+ and DPPH˙ method, and for the total polyphenol content using the F-C method. Additionally, the juice pressing efficiency, the extract content using the refractometer method, and the sugar content using the HPLC method were checked. Significantly higher antioxidant activity, pressing yield, and average content of caffeic acid glucoside, quinic acid, kaempferol-3,7-di-O-glucoside, and chrysoeriol-7-O-apiosylglucoside were obtained in juices from peeled celery. Maceration of the pulp with amylase resulted in a significant reduction in antioxidant activity compared to control samples. An is-total increase of 17-41% in total flavonoid content was observed in all juices tested after treatment with the enzyme for 30 and 60 min, and the phenolic acid content increased by 4-41% after treatment of the pulp with amylase for 60 min. The 60 min holding of the pulp at 25 °C, including with the enzyme, was shown to decrease the antioxidant activity and the content of quinic acid, ferulic acid, and chrysoriol-7-O-apiose-glucoside in the juices tested compared to the samples held for 30 min, while the content of other phenolic acids and flavonoids increased. In addition, after 60 min of enzymatic maceration, the pressing yield of the juices increased.

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit.

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

Metagenomics and untargeted metabolomics analyses to unravel the formation mechanism of characteristic metabolites in Cantonese soy sauce during different fermentation stages.

Cantonese soy sauce (CSS) is an important Chinese condiment due to its distinctive flavor. Microorganisms play a significant role in the flavor formation of CSS during fermentation. However, the correlation between microbes and flavor compounds as well as the potential fermentation mechanism remained poorly uncovered. Here we revealed the dynamic changes of microbial structure and characteristics metabolites as well as their correlation of CSS during the fermentation process. Metagenomics sequencing analysis showed that Tetragenococcus halophilus, Weissella confusa, Weissella paramesenteroides, Aspergillus oryzae, Lactiplantibacillus plantarum, Weissella cibaria were top six dominant species from day 0 to day 120. Sixty compounds were either positively or tentatively identified through untargeted metabolomics profile and they were 27 peptides, amino acids and derivatives, 8 carbohydrates and conjugates, 14 organic acids and derivatives, 5 amide compounds, 3 flavonoids and 3 nucleosides. Spearman correlation coefficient indicated that Tetragenococcus halophilus, Zygosaccharomyces rouxii, Pediococcus pentosaceus and Aspergillus oryzae were significantly related with the formation of taste amino acids and derivatives, peptides and functional substances. Additionally, the metabolisms of flavor amino acids including 13 main free amino acids were also profiled. These results provided valuable information for the production practice in the soy sauce industry.

Identification of cheese rancidity-related lipases in Aspergillus oryzae AHU 7139.

The adjunct product with enzymatic activity from Aspergillus oryzae is beneficial for flavor enrichment in the ripened cheese. However, an excessive lipolytic reaction leads to the release of volatile free fatty acids. Accordingly, a strong off-flavor (i.e., rancidity) has been detected when A. oryzae AHU 7139 is used. To identify the rancidity-related lipase from this strain, we evaluated the substrate specificity and lipase distribution using five mutants cultured on a whey-based solid medium under different initial pH conditions. The results showed a higher diacylglycerol lipase activity than triacylglycerol lipase activity. Moreover, an initial pH of 6.5 for the culture resulted in higher lipolytic activity than a pH of 4.0, and most of the activity was found in the extracellular fraction. Based on the gene expression analysis by real-time polymerase chain reaction and location and substrate specificity, five genes (No. 1, No. 19, mdlB, tglA, and cutL) were selected among 25 annotated lipase genes to identify the respective knockout strains. Because ΔtglA and ΔmdlB showed an outstanding involvement in the release of free fatty acids, these strains were applied to in vitro cheese curd experiments. In conclusion, we posit that triacylglycerol lipase (TglA) plays a key role as the trigger of rancidity and the resulting diglycerides have to be exposed to diacylglycerol lipase (MdlB) to stimulate rancidity in cheese made with A. oryzae AHU 7139. This finding could help screen suitable A.oryzae strains as cheese adjuncts to prevent the generation of the rancid-off flavor.

Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Overexpression of the DHA1 family, ChlH and ChlK, leads to enhanced dicarboxylic acids production in koji fungi, Aspergillus luchuensis mut. kawachii and Aspergillus oryzae.

The white koji fungus Aspergillus luchuensis mut. kawachii secretes substantial amounts of citric acid through the expression of the citric acid exporter CexA, a member of the DHA1 family. In this study, we aimed to characterize 11 CexA homologs (Chl proteins) encoded in the genome of A. luchuensis mut. kawachii to identify novel transporters useful for organic acid production. We constructed overexpression strains of chl genes using a cexA disruptant of the A. luchuensis mut. kawachii as the host strain, which prevented excessive secretion of citric acid into the culture supernatant. Subsequently, we evaluated the effects of overexpression of chl on producing organic acids by analyzing the culture supernatant. All overexpression strains did not exhibit significant citric acid accumulation in the culture supernatant, indicating that Chl proteins are not responsible for citric acid export. Furthermore, the ChlH overexpression strain displayed an accumulation of 2-oxoglutaric and fumaric acids in the culture supernatant, while the ChlK overexpression strain exhibited the accumulation of 2-oxoglutaric, malic and succinic acids. Notably, the ChlH and ChlK overexpression led to a substantial increase in the production of 2-oxoglutaric acid, reaching approximately 25 mM and 50 mM, respectively. Furthermore, ChlH and ChlK overexpression also significantly increased the secretory production of dicarboxylic acids, including 2-oxoglutaric acid, in the yellow koji fungus, Aspergillus oryzae. Our study demonstrates that overexpression of DHA1 family gene results in enhanced secretion of organic acids in koji fungi of the genus Aspergillus.

Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae.

Nitrogen source assimilation is important for the biological functions of fungi, and its pathway has been deeply studied. Aspergillus oryzae mutants defective in nitrogen source assimilation are known to grow poorly on Czapek-Dox (CD) medium. In this study, we found an industrial strain of A. oryzae that grew very poorly on a CD medium containing sodium nitrate as a nitrogen source. We used media with various nitrogen components to examine the steps affecting the nitrogen source assimilation pathway of this strain. The strain grew well on the CD medium supplied with nitrite salt or ammonium salt, suggesting that the strain was defective in nitrate assimilation step. To ascertain the gene causing the defect of nitrate assimilation, a gene expression vector harboring either niaD or crnA of A. oryzae RIB40 was introduced into the industrial strain. The industrial strain containing the crnA vector recovered its growth. This is the first report that a mutation of crnA causes poor growth on CD medium in an industrial strain of A. oryzae, and crnA can be used as a transformation marker for crnA deficient strains.

Origin of the 6/5/6/5 Tetracyclic Cyclopiazonic Acids.

The natural product α-cyclopiazonic acid (α-CPA) is a very potent Ca2+-ATPase inhibitor. The CPA family of compounds comprise over 80 chemical entities with at least five distinct skeletons. While α-CPA features a canonical 6/5/6/5/5 skeleton, the 6/5/6/5 skeleton is the most prevalent among the CPA family. However, the origin of the unique tetracyclic skeleton remains unknown. The 6/5/6/5-type CPAs may derive from a precursor of acetoacetyl-l-tryptophan (AATrp) generated from a hypothetic thioesterase-like pathway. Alternatively, cleavage of the tetramic acid ring would also result in the formation of the 6/5/6/5 scaffold. Aspergillus oryzae HMP-F28 is a marine sponge-associated filamentous fungus known to produce CPAs that act as primary neurotoxins. To elucidate the origin of this subfamily of CPAs, we performed homologous recombination and genetic engineering experiments on strain HMP-F28. Our results are supportive of the ring cleavage pathway through which the tetracyclic 6/5/6/5-type CPAs are generated from 6/5/6/5/5-type pentacyclic CPAs.

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.

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.

Construction of self-cloning Aspergillus oryzae strains with high production of multiple biomass-degrading enzymes on solid-state culture.

Filamentous fungi produce numerous industrially important enzymes. Among them, Aspergillus oryzae-derived enzymes are widely used in various fermentation applications. In this study, we constructed self-cloning strains that overproduce multiple biomass-degrading enzymes under the control of a strong promoter of α-amylase-coding gene (amyB) using the industrial strain A. oryzae AOK11. Two strains (strains 2-4 and 3-26) were introduced with different combinations of genes encoding xylanase (xynG1), phytase (phyA), pectin lyase (pelA), and polygalacturonase (pgaB). These strains had at least one copy of each enzyme gene derived from the expression cassette in the genome. The transcription levels of enzyme-coding genes introduced were more than 100-fold higher than those in the parent strain. Reflecting the high transcription levels, the activities of the enzymes derived from the expression cassettes of these two strains were significantly higher than those of the parent strain in both liquid and solid-state cultures. Even in ventilated solid-state cultures that were scaled up using mechanical equipment for practical applications, the two strains showed significantly higher enzyme activity than the parent strain. These results indicate that these strains constructed using a safe self-cloning technique represent industrially valuable practical strains that can be used in the food and livestock industries.

Impacts of Aspergillus oryzae 3.042 on the flavor formation pathway in Cantonese soy sauce koji.

To investigate the mechanism of flavor formation during the traditional preparation Cantonese soy sauce koji (TP), the changes of microorganisms, physicochemical properties, and flavor compounds in TP were comprehensively and dynamically monitored by absolute quantitative methods. Results demonstrated that inoculating Aspergillus oryzae 3.042 in TP was crucial role in enhancing enzyme activity properties. Absolute quantification of flavor combined with multivariate statistical analysis yielded 5 organic acids, 15 amino acids, and 2 volatiles as significantly different flavors of TP. Amplicon sequencing and RT-qPCR revealed that the dominant genera were Staphylococcus, Weissella, Enterobacter, Lactic streptococci, Lactobacillus, and Aspergillus, which exhibited a increasing trend in TP. Correlation analysis exhibited that Staphylococcus and Aspergillus were the pivotal genera contributing to the enzyme activities and flavor of TP. The flavor formation network involved lipid and protein degradation, carbohydrate metabolism and other pathways. Simultaneously, TP can appropriately increase the fermentation time to improve product quality.

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.

Monaspin B, a Novel Cyclohexyl-furan from Cocultivation of Monascus purpureus and Aspergillus oryzae, Exhibits Potent Antileukemic Activity.

Natural products are a rich resource for the discovery of innovative drugs. Microbial cocultivation enables discovery of novel natural products through tandem enzymatic catalysis between different fungi. In this study, Monascus purpureus, as a food fermentation strain capable of producing abundant natural products, was chosen as an example of a cocultivation pair strain. Cocultivation screening revealed that M. purpureus and Aspergillus oryzae led to the production of two novel cyclohexyl-furans, Monaspins A and B. Optimization of the cocultivation mode and media enhanced the production of Monaspins A and B to 1.2 and 0.8 mg/L, respectively. Monaspins A and B were structurally elucidated by HR-ESI-MS and NMR. Furthermore, Monaspin B displayed potent antiproliferative activity against the leukemic HL-60 cell line by inducing apoptosis, with a half-maximal inhibitory concentration (IC50) of 160 nM. Moreover, in a mouse leukemia model, Monaspin B exhibited a promising in vivo antileukemic effect by reducing white blood cell, lymphocyte, and neutrophil counts. Collectively, these results indicate that Monaspin B is a promising candidate agent for leukemia therapy.

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.

Cyclic extraction of phosphate from soybean meal using immobilized Aspergillus oryzae SBS50 phytase.

Phytase enzyme found in plants, animals, and microorganisms is mainly involved in catalyzing the systematic removal of a phosphate group from phytic acid. Enzyme immobilization is one of the cost-effective methods for the wide usage of enzymes in the industrial sector. This paper reports the covalent immobilization of phytase on glutaraldehyde-activated aluminum oxide beads. The immobilization yield, efficiency, and activation energy were found to be 47.8%, 71.5%, and 15.78 J/mol, respectively. The bound enzyme displayed a shift in pH optima from 5.5 to 4.5, which is more beneficial to increase digestibility in comparison with the free enzyme. Immobilized phytase retained 42.60% of its activity after 1.0 h incubation at 80 °C, whereas free enzyme retained only 4.20% of its activity. Thermodynami increase in half-lives, D-values, enthalpy and free energy change after covalent immobilization could be credited to the enhanced stability. Immobilized phytase could be reused for five consecutive cycles retaining 51% of its initial activity with sodium phytate. The immobilized phytase was also found effective to hydrolyze the soybean meal, thus increasing the digestibility of poultry feed. The hydrolyzing reaction of soybean meal was carried out for six consecutive cycles and immobilized phytase retained nearly 50% of activity till the fifth cycle. The amount of phosphorus released after treatment with immobilized phytase was far higher than that from free phytase. Immobilization on this support is significant, as this support can sustain high mechanical resistance at high pH and temperature. This considerable stability and reusability of the bound enzyme may be advantageous for its industrial application.

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.

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.

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.