RESUMO
Enfumafungin-type antibiotics, represented by enfumafungin and fuscoatroside, belong to a distinct group of triterpenoids derived from fungi. These compounds exhibit significant antifungal properties with ibrexafungerp, a semisynthetic derivative of enfumafungin, recently gaining FDA's approval as the first oral antifungal drug for treating invasive vulvar candidiasis. Enfumafungin-type antibiotics possess a cleaved E-ring with an oxidized carboxyl group and a reduced methyl group at the break site, suggesting unprecedented C-C bond cleavage chemistry involved in their biosynthesis. Here, we show that a 4-gene (fsoA, fsoD, fsoE, fsoF) biosynthetic gene cluster is sufficient to yield fuscoatroside by heterologous expression in Aspergillus oryzae. Notably, FsoA is an unheard-of terpene cyclase-glycosyltransferase fusion enzyme, affording a triterpene glycoside product that relies on enzymatic fusion. FsoE is a P450 enzyme that catalyzes successive oxidation reactions at C19 to facilitate a C-C bond cleavage, producing an oxidized carboxyl group and a reduced methyl group that have never been observed in known P450 enzymes. Our study thus sets the important foundation for the manufacture of enfumafungin-type antibiotics using biosynthetic approaches.
Assuntos
Antifúngicos , Antifúngicos/química , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Aspergillus oryzae/enzimologia , Aspergillus oryzae/metabolismo , Família Multigênica , Triterpenos/química , Triterpenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismoRESUMO
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.
Assuntos
Aspergillus oryzae , Endocitose , Proteínas Fúngicas , Glucose , Proteínas de Transporte de Monossacarídeos , Ubiquitina-Proteína Ligases , Ubiquitinação , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Aspergillus oryzae/enzimologia , Glucose/metabolismo , Endocitose/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Maltose/metabolismo , Proteólise , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genéticaRESUMO
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.
Assuntos
Aspergillus oryzae , Aspergillus oryzae/metabolismo , Estresse do Retículo Endoplasmático , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/metabolismoRESUMO
We previously reported autophagy-mediated degradation of nuclei, nucleophagy, in the filamentous fungus Aspergillus oryzae. In this study, we examined whether nuclei are degraded as a whole. We generated A. oryzae mutants deleted for orthologs of Saccharomyces cerevisiae YPT7 and ATG15 which are required, respectively, for autophagosome-vacuole fusion and vacuolar degradation of autophagic bodies. Degradation of histone H2B-EGFP under starvation conditions was greatly decreased in the ΔAoypt7 and ΔAoatg15 mutants. Fluorescence and electron microscopic observations showed that autophagosomes and autophagic bodies surrounding the entire nuclei were accumulated in the cytoplasm of ΔAoypt7 and the vacuole of ΔAoatg15, respectively. These results indicate that nuclei are engulfed in the autophagosomes as a whole and transported/released into the vacuolar lumen where they are degraded.
Assuntos
Aspergillus oryzae , Autofagossomos , Proteínas Fúngicas , Vacúolos , Vacúolos/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Autofagossomos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Deleção de Genes , Proteínas rab de Ligação ao GTPRESUMO
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.
Assuntos
Aspergillus oryzae , Indóis , Indóis/química , Aspergillus oryzae/metabolismoRESUMO
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.
Assuntos
Aflatoxinas , Aspergillus oryzae , Aspergillus flavus/metabolismo , Filogenia , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Aflatoxinas/genética , GenômicaRESUMO
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.
Assuntos
Aspergillus oryzae , Fermentação , Proteínas Filagrinas , Oryza , Aspergillus oryzae/metabolismo , Oryza/química , Oryza/metabolismo , Humanos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Queratinócitos/metabolismo , Queratinócitos/efeitos dos fármacos , Células HaCaT , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/efeitos dos fármacos , Higiene da Pele/métodos , Pele/metabolismoRESUMO
BACKGROUND: During the brewing of soy sauce, the conversion of multiple substances is driven by various microorganisms and their secreted enzyme systems. Soy sauce mash is an important source of enzyme systems during moromi fermentation, but the changes of enzyme systems in soy sauce mash during moromi fermentation are poorly understood. In order to explore the predominant enzyme systems existing during moromi fermentation and to explain the characteristics of the enzyme system changes, an enzymatic activities assay and 4D-label-free proteomics analysis were conducted on soy sauce mash at different stages of fermentation. RESULTS: The activities of hydrolytic enzymes in soy sauce mash decreased continuously throughout the fermentation process, while most of the characteristic physicochemical substances in soy sauce mash supernatant had already accumulated at the early stage of fermentation. Four hydrolytic enzymes were found to be positively correlated with important physicochemical indexes by principal component analysis and Pearson correlation analysis. The proteomics analysis revealed three highly upregulated enzymes and two enzymes that were present in important metabolic pathways throughout the fermentation process. Furthermore, it was found that Aspergillus oryzae was able to accumulate various nutrients in the soy sauce mash by downregulating most of its metabolic pathways. CONCLUSION: Enzymes present with excellent properties during the moromi fermentation period could be obtained from these results. Meanwhile, the characterization of the metabolic pathways of microorganisms during the moromi fermentation period was revealed. The results provide a basis for more scientific and purposeful improvement of moromi fermentation in the future. © 2024 Society of Chemical Industry.
Assuntos
Fermentação , Proteômica , Alimentos de Soja , Alimentos de Soja/análise , Alimentos de Soja/microbiologia , Proteínas Fúngicas/metabolismo , Aspergillus oryzae/metabolismo , Aspergillus oryzae/enzimologiaRESUMO
BACKGROUND: Solid-state fermentation (SSF) has been widely used in the processing of sorghum grain (SG) because it can produce products with improved sensory characteristics. To clarify the influence of different microbial strains on the SSF of SG, especially on the polyphenols content and composition, Lactiplantibacillus plantarum, Saccharomyces cerevisiae, Rhizopus oryzae, Aspergillus oryzae, and Neurospora sitophila were used separately and together for SSF of SG. Furthermore, the relationship between the dynamic changes in polyphenols and enzyme activity closely related to the metabolism of polyphenols has also been measured and analyzed. Microstructural changes observed after SSF provide a visual representation of the SSF on the SG. RESULTS: After SSF, tannin content (TC) and free phenolic content (FPC) were decreased by 56.36% and 23.48%, respectively. Polyphenol oxidase, ß-glucosidase and cellulase activities were increased 5.25, 3.27, and 45.57 times, respectively. TC and FPC were negatively correlated with cellulase activity. A positive correlation between FPC and xylanase activity after 30 h SSF became negative after 48 h SSF. The SG surface was fragmented and porous, reducing the blocking effect of cortex. CONCLUSION: Cellulase played a crucial role in promoting the degradation of tannin (antinutrient) and phenolic compounds. Xylanase continued to release flavonoids while microbial metabolism consumed them with the extension of SSF time. SSF is an effective way to improve the bioactivity and processing characteristics of SG. © 2024 Society of Chemical Industry.
Assuntos
Catecol Oxidase , Fermentação , Polifenóis , Saccharomyces cerevisiae , Sorghum , Sorghum/química , Sorghum/metabolismo , Polifenóis/metabolismo , Polifenóis/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Catecol Oxidase/metabolismo , Rhizopus/metabolismo , Rhizopus/enzimologia , Taninos/metabolismo , Taninos/análise , Taninos/química , Aspergillus oryzae/metabolismo , Aspergillus oryzae/enzimologia , Celulase/metabolismo , Celulase/química , Neurospora/metabolismo , Manipulação de Alimentos/métodos , beta-Glucosidase/metabolismo , Sementes/química , Sementes/metabolismo , Sementes/microbiologia , Bactérias/metabolismo , Bactérias/classificação , Bactérias/enzimologia , Bactérias/isolamento & purificação , Fenóis/metabolismo , Fenóis/química , Fenóis/análiseRESUMO
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.
Assuntos
Aspergillus oryzae , Aspergillus oryzae/metabolismo , Biomassa , Regiões Promotoras Genéticas , Clonagem MolecularRESUMO
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.
Assuntos
Aspergillus oryzae , Aspergillus , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Ácidos Dicarboxílicos , Ácidos Cetoglutáricos , Ácido Cítrico/metabolismoRESUMO
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.
Assuntos
Aspergillus oryzae , Nitratos , Nitratos/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , RNA Complementar , Nitrogênio/metabolismo , MutaçãoRESUMO
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.
Assuntos
Aspergillus oryzae , Biologia Sintética , Biologia Sintética/métodos , Edição de Genes , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Micélio/genética , Heme/metabolismoRESUMO
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.
Assuntos
Aspergillus oryzae , Micotoxinas , Micotoxinas/metabolismo , Nitrocompostos , Propionatos , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismoRESUMO
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.
Assuntos
Aspergillus oryzae , Queijo , Lipase Lipoproteica/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Lipase/genética , Lipase/metabolismoRESUMO
Stale bread is a waste product with a potential to be recycled. One way to manage this waste material is to process it by fermentation for the purpose of food production. This paper proposes the use of stale wheat and rye bread as ingredients in amazake, a liquid dessert traditionally obtained from rice by fermentation with the koji mould Aspergillus oryzae, followed by liquefaction by the action of fungal enzymes. The stale bread was introduced instead of rice at both the koji stage (wheat bread) and the liquefaction stage (wheat and rye bread). The resulting products had an extended volatile compound profile, from 5 to 15 compounds identified, and modified sensory parameters, compared to the traditional version. Amazake containing bread had an increased protein content, from 1.10 to 6.4 g/100 g, and were more abundant in dietary fibre (up to a maximum of 1.8 g/100 g), additionally enriched with a soluble fraction. The proposed procedure of obtaining of new formula amazake can be directly applied in households to reduce the amount of discarded bread. Due to its simplicity, it also has the potential for further modification in terms of production scale and product parameters.
Assuntos
Pão , Reciclagem , Triticum , Pão/análise , Reciclagem/métodos , Fermentação , Aspergillus oryzae/metabolismo , Fibras na Dieta/análise , Valor Nutritivo , Oryza , Resíduos/análise , PaladarRESUMO
Filamentous fungi are eukaryotic microorganisms that differentiate into diverse cellular forms. Recent research demonstrated that phospholipid homeostasis is crucial for the morphogenesis of filamentous fungi. However, phospholipids involved in the morphological regulation are yet to be systematically analyzed. In this study, we artificially controlled the amount of phosphatidylcholine (PC), a primary membrane lipid in many eukaryotes, in a filamentous fungus Aspergillus oryzae, by deleting the genes involved in PC synthesis or by repressing their expression. Under the condition where only a small amount of PC was synthesized, A. oryzae hardly formed aerial hyphae, the basic structures for asexual development. In contrast, hyphae were formed on the surface or in the interior of agar media (we collectively called substrate hyphae) under the same conditions. Furthermore, we demonstrated that supplying sufficient choline to the media led to the formation of aerial hyphae from the substrate hyphae. We suggested that acyl chains in PC were shorter in the substrate hyphae than in the aerial hyphae by utilizing the strain in which intracellular PC levels were controlled. Our findings suggested that the PC levels regulate hyphal elongation and differentiation processes in A. oryzae and that phospholipid composition varied depending on the hyphal types.
Assuntos
Aspergillus oryzae , Hifas , Fosfatidilcolinas , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Fosfatidilcolinas/metabolismo , Aspergillus oryzae/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/crescimento & desenvolvimento , Colina/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genéticaRESUMO
Rice wine, well known for its unique flavor, rich nutritional value, and health benefits, has potential for extensive market development. Rhizopus and Aspergillus are among several microorganisms used in rice wine brewing and are crucial for determining rice wine quality. The strains were isolated via Rose Bengal and starch as a combined separation medium, followed by oenological property and sensory evaluation screening. The strain exhibiting the best performance can be screened using the traditional rice wine Qu. The strains YM-8, YM-10, and YM-16, which exhibited strong saccharification and fermentation performance along with good flavor and taste, were obtained from traditional rice wine Qu. Based on ITS genetic sequence analysis, the YM-8, YM-10, and YM-16 strains were identified as Rhizopus microsporus, Rhizopus arrhizus, and Aspergillus oryzae. The optimum growth temperature of each of the three strains was 30°C, 32°C, and 30°C, and the optimum initial pH was 6.0, 6.5, and 6.5, respectively. The activities of α-amylase, glucoamylase, and protease of YM-16 were highest at 220.23±1.88, 1,269.04±30.32, and 175.16±1.81 U/g, respectively. The amino acid content of rice wine fermented in a 20-L bioreactor with the three mold strains was higher than that of the control group, except for arginine, which was significantly lower than that of the control group. The total amino acid content and the total content of each type of amino acid were ranked as YM-16 > YM-8 > YM-10 > control group, and the amino acid content varied greatly among the strains. The control group had a higher content, whereas YM-8 and YM-16 had lower contents of volatile aroma components than the control group and had the basic flavor substances needed for rice wine, which is conducive to the formation of rice wine aroma. This selected strain, YM-16, has strong saccharification and fermentation ability, is a rich enzyme system, and improves the flavor of rice wine, thereby demonstrating its suitability as a production strain for brewing.
Assuntos
Reatores Biológicos , Fermentação , Oryza , Vinho , Vinho/análise , Vinho/microbiologia , Oryza/microbiologia , Oryza/metabolismo , Reatores Biológicos/microbiologia , Rhizopus/metabolismo , Paladar , Aspergillus oryzae/metabolismo , Aspergillus oryzae/genética , Concentração de Íons de HidrogênioRESUMO
Fungi produce various bioactive secondary metabolites (SMs) as protective and weaponized tools to enhance survival in shared ecological niches. By mimicking a competitive ecosystem, cocultivation has been proven to be particularly successful in stimulating SM discovery. Here, we reported the identification of four novel metabolites, epiclactones A and B, epioxochromane and aoergostane, from the coculture of two biotechnologically important strains, Aspergillus oryzae and Epicoccum dendrobii. Transcriptome and metabolome analyses revealed widespread silent gene activation during fungal-fungal interaction. The majority of differentially expressed gene clusters were summarized for both strains. Based on these highly activated biosynthetic pathways, we suggested that a bidirectional chemical defense occurred under cocultivation. E. dendrobii enhanced the production of the spore inhibitor, fumigermin. Moreover, A. oryzae highly accumulated the antifungal agent kojic acid with a yield of up to 1.10 g/L. This study provides an excellent example for the discovery of hidden natural products by cocultivation.
Assuntos
Ascomicetos , Aspergillus oryzae , Técnicas de Cocultura , Aspergillus oryzae/metabolismo , Aspergillus oryzae/genética , Ascomicetos/metabolismo , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Metabolismo Secundário , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genéticaRESUMO
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.