RESUMEN
Depending on their phosphorylation status, derivatives of phosphatidylinositol play important roles in vesicle identity, recognition and intracellular trafficking processes. In eukaryotic cells, phosphatidylinositol-4 phosphate pools generated by specific kinases are key determinants of the conventional secretion pathways. Earlier work in yeast has classified phosphatidylinositol-4 kinases in two types, Stt4p and Pik1p belonging to type III and Lsb6p to type II, with distinct cellular localizations and functions. Eurotiomycetes appear to lack Pik1p homologues. In Aspergillus nidulans, unlike homologues in other fungi, AnLsb6 is associated to late Golgi membranes and when heterologously overexpressed, it compensates for the thermosensitive phenotype in a Saccharomyces cerevisiae pik1 mutant, whereas its depletion leads to disorganization of Golgi-associated PHOSBP-labelled membranes, that tend to aggregate dependent on functional Rab5 GTPases. Evidence provided herein, indicates that the single type II phosphatidylinositol-4 kinase AnLsb6 is the main contributor for decorating secretory vesicles with relevant phosphatidylinositol-phosphate species, which navigate essential cargoes following the route of apical polarization via endocytic recycling.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa , Endocitosis , Aparato de Golgi , Saccharomyces cerevisiae , 1-Fosfatidilinositol 4-Quinasa/metabolismo , 1-Fosfatidilinositol 4-Quinasa/genética , Aspergillus nidulans/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/enzimología , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Aparato de Golgi/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Genetic code expansion technology allows the incorporation of unnatural amino acids (UAAs) into proteins, which is useful in protein engineering, synthetic biology, and gene therapy. Despite its potential applications in various species, filamentous fungi remain unexplored. This study aims to address this gap by developing these techniques in Aspergillus nidulans. We introduced an amber stop codon into a specific sequence within the reporter gene expressed in A. nidulans and replaced the anticodon of the fungal tRNATyr with CUA. This resulted in the synthesis of the target protein, confirming the occurrence of amber suppression in the fungus. When exogenous E. coli tRNATyrCUA (Ec. tRNATyrCUA) and E. coli tyrosyl-tRNA (Ec.TyrRS) were introduced into A. nidulans, they successfully synthesized the target protein via amber suppression and were shown to be orthogonal to the fungal translation system. By replacing the wild-type Ec.TyrRS with a mutant with a higher affinity for the UAA O-methyl-L-tyrosine, the fungal system was able to initiate the synthesis of the UAA-labeled protein (UAA-protein). We further increased the expression level of the UAA-protein through several rational modifications. The successful development of a genetic code expansion technique for A. nidulans has introduced a potentially valuable approach to the study of fungal protein structure and function.
Asunto(s)
Aminoácidos , Aspergillus nidulans , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Código Genético , Ingeniería de Proteínas/métodos , Codón de Terminación/genética , Codón/genética , Escherichia coli/genética , Escherichia coli/metabolismo , ARN de Transferencia de Tirosina/genética , ARN de Transferencia de Tirosina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMEN
Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans.
Asunto(s)
Sistemas CRISPR-Cas , Penicillium , Metabolismo Secundario , Penicillium/genética , Penicillium/metabolismo , Metabolismo Secundario/genética , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Familia de Multigenes , Marcación de Gen/métodos , Regulación Fúngica de la Expresión Génica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Vías Biosintéticas/genética , Ingeniería Metabólica/métodos , Expresión GénicaRESUMEN
The importance of fungi in ecological systems and pathogenicity hinges on their ability to search for nutrients, substrates, and hosts. Despite this, the question of whether fungal hyphae exhibit chemotropism toward them remains largely unresolved and requires close examination at the cellular level. Here, we designed a microfluidic device to assess hyphal chemotropism of Aspergillus nidulans in response to carbon and nitrogen sources, as well as pH. Within this device, hyphae could determine their growth direction in a two-layer flow with distinct compositions that were adjacent but non-mixing. Under conditions with and without a carbon source, hyphae changed growth direction to remain in the presence of a carbon source, but it was still difficult to distinguish between differences in growth and chemotropism. Although nitrogen sources such as ammonia and nitrate are important for growth, the hyphae indicated negative chemotropism to avoid them depending on the specific transporters. This fungus grows equally well at the colony level in the pH range of 4 to 9, but the hyphae exhibited chemotropism to acidic pH. The proton pump PmaA is vital for the chemotropism to acid pH, while the master regulatory for pH adaptation PacC is not involved, suggesting that chemotropism and adaptive growth via gene expression regulation are distinct regulatory mechanisms. Despite various plasma membrane transporters are distributed across membranes except at the hyphal tip, the control of growth direction occurs at the tip. Finally, we explored the mechanisms linking these two phenomena, tip growth and chemotropism.
Asunto(s)
Aspergillus nidulans , Hifa , Aspergillus nidulans/metabolismo , Aspergillus nidulans/fisiología , Hifa/metabolismo , Hifa/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Nitrógeno/metabolismo , Nutrientes/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Carbono/metabolismo , Regulación Fúngica de la Expresión Génica , Amoníaco/metabolismoRESUMEN
Fungal secondary metabolite (SM) biosynthetic gene clusters (BGCs) containing dimethylallyltryptophan synthases (DMATSs) produce structurally diverse prenylated indole alkaloids with wide-ranging activities that have vast potential as human therapeutics. To discover new natural products produced by DMATSs, we mined the Department of Energy Joint Genome Institute's MycoCosm database for DMATS-containing BGCs. We found a DMATS BGC in Aspergillus homomorphus CBS 101889, which also contains a nonribosomal peptide synthetase (NRPS). This BGC appeared to have a previously unreported combination of genes, which suggested the cluster might make novel SMs. We refactored this BGC with highly inducible promoters into the model fungus Aspergillus nidulans. The expression of this refactored BGC in A. nidulans resulted in the production of eight tryptophan-containing diketopiperazines, six of which are new to science. We have named them homomorphins A-F (2, 4-8). Perhaps even more intriguingly, to our knowledge, this is the first discovery of C4-prenylated tryptophan-containing diketopiperazines and their derivatives. In addition, the NRPS from this BGC is the first described that has the ability to promiscuously combine tryptophan with either of two different amino acids, in this case, l-valine or l-allo-isoleucine.
Asunto(s)
Aspergillus nidulans , Aspergillus , Dicetopiperazinas , Péptido Sintasas , Triptófano , Triptófano/metabolismo , Triptófano/química , Dicetopiperazinas/química , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Aspergillus/química , Péptido Sintasas/metabolismo , Péptido Sintasas/genética , Estructura Molecular , Familia de Multigenes , Alcaloides Indólicos/química , Alcaloides Indólicos/metabolismo , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/genéticaRESUMEN
The great variety and fascinating complexity of terpenoid skeletons are achieved through different cyclizations catalyzed by terpene cyclases. Here, we report a sesquiterpene cyclase (MfdS) from Aspergillus ustus for the formation of malfilanol D, a member of the group of biochemically less investigated sesquiterpenes with a bicyclo[5.4.0]undecane skeleton. Feeding 13C-labeled acetates in Aspergillus nidulans with the mfdS sequence provides evidence for a C-1 to C-10 cyclization with subsequent 1,2-alkyl and 1,2-hydride shifts in the formation of the 6/7-fused rings.
Asunto(s)
Aspergillus , Sesquiterpenos , Aspergillus/química , Aspergillus/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Estructura Molecular , Ciclización , Alcanos/química , Alcanos/metabolismo , Aspergillus nidulans/metabolismo , Aspergillus nidulans/química , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/metabolismoRESUMEN
Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi and are involved in the regulation of mRNA expression of genes. In this study, we identified and characterized one MYB-like protein in the model organism Aspergillus nidulans. We screened the mRNA levels of genes encoding MYB-like proteins containing two MYB repeats in conidia and found that the mRNA levels of four genes including flbD, cicD, and two uncharacterized genes, were high in conidia. To investigate the roles of two uncharacterized genes, AN4618 and AN10944, deletion mutants for each gene were generated. Our results revealed that AN4618 was required for fungal development. Therefore, we further investigated the role of AN4618, named as mylA, encoding the MYB-like protein containing two MYB repeats. Functional studies revealed that MylA was essential for normal fungal growth and development. Phenotypic and transcriptomic analyses demonstrated that deletion of mylA affected stress tolerance, cell wall integrity, and long-term viability in A. nidulans conidia. In addition, the germination rate of the mylA deletion mutant conidia was decreased compared with that of the wild-type conidia. Overall, this study suggests that MylA is critical for appropriate development, conidial maturation, dormancy, and germination in A. nidulans.
Asunto(s)
Aspergillus nidulans , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Esporas Fúngicas , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus nidulans/metabolismo , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In the filamentous ascomycete Aspergillus nidulans, at least three high hierarchy transcription factors are required for growth at extracellular alkaline pH: SltA, PacC and CrzA. Transcriptomic profiles depending on alkaline pH and SltA function showed that pacC expression might be under SltA regulation. Additional transcriptional studies of PacC and the only pH-regulated pal gene, palF, confirmed both the strong dependence on ambient pH and the function of SltA. The regulation of pacC expression is dependent on the activity of the zinc binuclear (C6) cluster transcription factor PacX. However, we found that the ablation of sltA in the pacX- mutant background specifically prevents the increase in pacC expression levels without affecting PacC protein levels, showing a novel specific function of the PacX factor. The loss of sltA function causes the anomalous proteolytic processing of PacC and a reduction in the post-translational modifications of PalF. At alkaline pH, in a null sltA background, PacC72kDa accumulates, detection of the intermediate PacC53kDa form is extremely low and the final processed form of 27 kDa shows altered electrophoretic mobility. Constitutive ubiquitination of PalF or the presence of alkalinity-mimicking mutations in pacC, such as pacCc14 and pacCc700, resembling PacC53kDa and PacC27kDa, respectively, allowed the normal processing of PacC but did not rescue the alkaline pH-sensitive phenotype caused by the null sltA allele. Overall, data show that Slt and PacC/Pal pathways are interconnected, but the transcription factor SltA is on a higher hierarchical level than PacC on regulating the tolerance to the ambient alkalinity in A. nidulans.
Asunto(s)
Aspergillus nidulans , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Factores de Transcripción/metabolismo , Cationes/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Fungal linear polyketides, such as α-pyrones with a 6-alkenyl chain, have been a rich source of biologically active compounds. Two new (1 and 2) and four known (3-6) 6-alkenylpyrone polyketides were isolated from a marine-derived strain of the fungus Arthrinium arundinis. Their structures were determined based on extensive spectroscopic analysis. The biosynthetic gene cluster (alt) for alternapyrones was identified from A. arundinis ZSDS-F3 and validated by heterologous expression in Aspergillus nidulans A1145 ΔSTΔEM, which revealed that the cytochrome P450 monooxygenase Alt2' could convert the methyl group 26-CH3 to a carboxyl group to produce 4 from 3. Another cytochrome P450 monooxygenase, Alt3', catalyzed successive hydroxylation, epoxidation, and oxidation steps to produce 1, 2, 5, and 6 from 4. Alternapyrone G (1) not only suppressed M1 polarization in lipopolysaccharide (LPS)-stimulated BV2 microglia but also stimulated dendrite regeneration and neuronal survival after Aß treatment, suggesting alternapyrone G may be utilized as a privileged scaffold for Alzheimer's disease drug discovery.
Asunto(s)
Lipopolisacáridos , Fármacos Neuroprotectores , Pironas , Pironas/farmacología , Pironas/química , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Estructura Molecular , Lipopolisacáridos/farmacología , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/química , Biología Marina , Microglía/efectos de los fármacos , Microglía/metabolismo , Ascomicetos/química , Policétidos/farmacología , Policétidos/química , Policétidos/aislamiento & purificación , Aspergillus nidulans , Ratones , Familia de Multigenes , Sistema Enzimático del Citocromo P-450/metabolismoRESUMEN
The potential of natural products as pharmaceutical and agricultural agents is based on their large structural diversity, resulting in part from modifications of the backbone structure by tailoring enzymes during biosynthesis. Flavin-dependent monooxygenases (FMOs), as one such group of enzymes, play an important role in the biosynthesis of diverse natural products, including cyclodipeptide (CDP) derivatives. The FMO PboD was shown to catalyze C-3 hydroxylation at the indole ring of cyclo-l-Trp-l-Leu in the biosynthesis of protubonines, accompanied by pyrrolidine ring formation. PboD substrate promiscuity was investigated in this study by testing its catalytic activity toward additional tryptophan-containing CDPs in vitro and biotransformation in Aspergillus nidulans transformants bearing a truncated protubonine gene cluster with pboD and two acetyltransferase genes. High acceptance of five CDPs was detected for PboD, especially of those with a second aromatic moiety. Isolation and structure elucidation of five pyrrolidine diketopiperazine products, with two new structures, proved the expected stereospecific hydroxylation and pyrrolidine ring formation. Determination of kinetic parameters revealed higher catalytic efficiency of PboD toward three CDPs consisting of aromatic amino acids than of its natural substrate cyclo-l-Trp-l-Leu. In the biotransformation experiments with the A. nidulans transformant, modest formation of hydroxylated and acetylated products was also detected.
Asunto(s)
Aspergillus , Dicetopiperazinas , Aspergillus/enzimología , Aspergillus/química , Aspergillus nidulans/enzimología , Aspergillus nidulans/metabolismo , Dicetopiperazinas/química , Dicetopiperazinas/metabolismo , Flavinas/metabolismo , Hidroxilación , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/química , Estructura Molecular , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Especificidad por SustratoRESUMEN
Kinesin-14s, a subfamily of the large superfamily of kinesin motor proteins, function mainly in spindle assembly and maintenance during mitosis and meiosis. KlpA from Aspergillus nidulans and GiKIN14a from Giardia intestinalis are two types of kinesin-14s. Available experimental results puzzlingly showed that while KlpA moves preferentially toward the minus end in microtubule-gliding setups and inside parallel microtubule overlaps, it moves preferentially toward the plus end on single microtubules. More puzzlingly, the insertion of an extra polypeptide linker in the central region of the neck stalk switches the motility direction of KlpA on single microtubules to the minus end. Prior experimental results showed that GiKIN14a moves preferentially toward the minus end on single microtubules in either tailless or full-length forms. The tail not only greatly enhances the processivity but also accelerates the ATPase rate and velocity of GiKIN14a. The insertion of an extra polypeptide linker in the central region of the neck stalk reduces the ATPase rate of GiKIN14a. However, the underlying mechanism of these puzzling dynamical features for KlpA and GiKIN14a is unclear. Here, to understand this mechanism, the dynamics of KlpA and GiKIN14a were studied theoretically on the basis of the proposed model, incorporating potential changes between the kinesin head and microtubule, as well as the potential between the tail and microtubule. The theoretical results quantitatively explain the available experimental results and provide predicted results. It was found that the elasticity of the neck stalk determines the directionality of KlpA on single microtubules and affects the ATPase rate and velocity of GiKIN14a on single microtubules.
Asunto(s)
Cinesinas , Microtúbulos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Cinesinas/metabolismo , Cinesinas/química , Microtúbulos/metabolismo , Modelos Moleculares , Giardia lamblia/genética , Giardia lamblia/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Although tyrosol is a quorum-sensing molecule of Candida species, it has antifungal activity at supraphysiological concentrations. Here, we studied the effect of tyrosol on the physiology and genome-wide transcription of Aspergillus nidulans to gain insight into the background of the antifungal activity of this compound. Tyrosol efficiently reduced germination of conidia and the growth on various carbon sources at a concentration of 35 mM. The growth inhibition was fungistatic rather than fungicide on glucose and was accompanied with downregulation of 2199 genes related to e.g. mitotic cell cycle, glycolysis, nitrate and sulphate assimilation, chitin biosynthesis, and upregulation of 2250 genes involved in e.g. lipid catabolism, amino acid degradation and lactose utilization. Tyrosol treatment also upregulated genes encoding glutathione-S-transferases (GSTs), increased specific GST activities and the glutathione (GSH) content of the cells, suggesting that A. nidulans can detoxify tyrosol in a GSH-dependent manner even though this process was weak. Tyrosol did not induce oxidative stress in this species, but upregulated "response to nutrient levels", "regulation of nitrogen utilization", "carbon catabolite activation of transcription" and "autophagy" genes. Tyrosol may have disturbed the regulation and orchestration of cellular metabolism, leading to impaired use of nutrients, which resulted in growth reduction.
Asunto(s)
Antifúngicos , Aspergillus nidulans , Alcohol Feniletílico/análogos & derivados , Antifúngicos/farmacología , Antifúngicos/metabolismo , Transcriptoma , Glutatión/genética , Glutatión/metabolismo , Glutatión/farmacología , Carbono/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMEN
Glycosides make up a biomedically important class of secondary metabolites. Most naturally occurring glycosides were isolated from plants and bacteria; however, the chemical diversity of glycosylated natural products in fungi remains largely unexplored. Herein, we present a paradigm to specifically discover diverse and bioactive glycosylated natural products from fungi by combining tailoring enzyme-guided genome mining with mass spectrometry (MS)-based metabolome analysis. Through in vivo genes deletion and heterologous expression, the first fungal C-glycosyltransferase AuCGT involved in the biosynthesis of stromemycin was identified from Aspergillus ustus. Subsequent homology-based genome mining for fungal glycosyltransferases by using AuCGT as a probe revealed a variety of biosynthetic gene clusters (BGCs) containing its homologues in diverse fungi, of which the glycoside-producing capability was corroborated by high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. Consequently, 28 fungal aromatic polyketide C/O-glycosides, including 20 new compounds, were efficiently discovered and isolated from the three selected fungi. Moreover, several novel fungal C/O-glycosyltransferases, especially three novel α-pyrone C-glycosyltransferases, were functionally characterized and verified in the biosynthesis of these glycosides. In addition, a proof of principle for combinatorial biosynthesis was applied to design the production of unnatural glycosides in Aspergillus nidulans. Notably, the newly discovered glycosides exhibited significant antiviral, antibacterial, and antidiabetic activities. Our work demonstrates the promise of tailoring enzyme-guided genome-mining approach for the targeted discovery of fungal glycosides and promotes the exploration of a broader chemical space for natural products with a target structural motif in microbial genomes.
Asunto(s)
Aspergillus nidulans , Productos Biológicos , Glicosiltransferasas/metabolismo , Metaboloma , Espectrometría de Masas , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Glicósidos , Familia de MultigenesRESUMEN
BACKGROUND: Surveillance studies are crucial for updating trends in Aspergillus species and antifungal susceptibility information. OBJECTIVES: Determine the Aspergillus species distribution and azole resistance prevalence during this 3-year prospective surveillance study in a Spanish hospital. MATERIALS AND METHODS: Three hundred thirty-five Aspergillus spp. clinical and environmental isolates were collected during a 3-year study. All isolates were screened for azole resistance using an agar-based screening method and resistance was confirmed by EUCAST antifungal susceptibility testing. The azole resistance mechanism was confirmed by sequencing the cyp51A gene and its promoter. All Aspergillus fumigatus strains were genotyped using TRESPERG analysis. RESULTS: Aspergillus fumigatus was the predominant species recovered with a total of 174 strains (51.94%). The rest of Aspergillus spp. were less frequent: Aspergillus niger (14.93%), Aspergillus terreus (9.55%), Aspergillus flavus (8.36%), Aspergillus nidulans (5.37%) and Aspergillus lentulus (3.28%), among other Aspergillus species (6.57%). TRESPERG analysis showed 99 different genotypes, with 72.73% of the strains being represented as a single genotype. Some genotypes were common among clinical and environmental A. fumigatus azole-susceptible strains, even when isolated months apart. We describe the occurrence of two azole-resistant A. fumigatus strains, one clinical and another environmental, that were genotypically different and did not share genotypes with any of the azole-susceptible strains. CONCLUSIONS: Aspergillus fumigatus strains showed a very diverse population although several genotypes were shared among clinical and environmental strains. The isolation of azole-resistant strains from both settings suggest that an efficient analysis of clinical and environmental sources must be done to detect azole resistance in A. fumigatus.
Asunto(s)
Aspergilosis , Aspergillus nidulans , Humanos , Azoles/farmacología , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Aspergilosis/microbiología , Prevalencia , Estudios Prospectivos , Farmacorresistencia Fúngica , Aspergillus fumigatus , Hospitales , Proteínas Fúngicas/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Clinical and histologic examination of a 12-y-old client-owned Quarter Horse gelding with pituitary pars intermedia dysfunction revealed dermatitis, cellulitis, and osteomyelitis caused by Aspergillus nidulans, confirmed by a PCR assay. This novel presentation of a fungal disease in a horse was characterized by aggressive local invasion and failure to respond to all medical therapy attempted over a 1-y period. Treatments included systemic and topical antifungals, anti-inflammatories, and use of cellular matrices. Surgical excision was not attempted but should be strongly considered early in the disease process in similar cases if clean margins can be achieved. Postmortem findings were of locally aggressive disease with no dissemination.
Asunto(s)
Aspergillus nidulans , Dermatitis , Enfermedades de los Caballos , Osteomielitis , Enfermedades de la Hipófisis , Adenohipófisis Porción Intermedia , Caballos , Masculino , Animales , Celulitis (Flemón)/veterinaria , Enfermedades de los Caballos/diagnóstico , Enfermedades de los Caballos/patología , Enfermedades de la Hipófisis/diagnóstico , Enfermedades de la Hipófisis/veterinaria , Adenohipófisis Porción Intermedia/patología , Osteomielitis/diagnóstico , Osteomielitis/veterinaria , Dermatitis/patología , Dermatitis/veterinariaRESUMEN
Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in A. nidulans.
Asunto(s)
Aspergillus nidulans , Animales , Humanos , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , ARN Mensajero , Quitina/genéticaRESUMEN
Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.
Asunto(s)
Aspergillus nidulans , Fosfatos de Poliisoprenilo , Sesquiterpenos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Farnesil Difosfato Farnesil Transferasa/genética , Farnesil Difosfato Farnesil Transferasa/metabolismo , Escualeno , Terpenos/metabolismoRESUMEN
One of the four cutinases encoded in the Aspergillus nidulans genome, ANCUT1, is described here. Culture conditions were evaluated, and it was found that this enzyme is produced only when cutin is present in the culture medium, unlike the previously described ANCUT2, with which it shares 62% amino acid identity. The differences between them include the fact that ANCUT1 is a smaller enzyme, with experimental molecular weight and pI values of 22 kDa and 6, respectively. It shows maximum activity at pH 9 and 60 °C under assayed conditions and retains more than 60% of activity after incubation for 1 h at 60 °C in a wide range of pH values (6-10) after incubations of 1 or 3 h. It has a higher activity towards medium-chain esters and can modify long-chain length hydroxylated fatty acids constituting cutin. Its substrate specificity properties allow the lipophilization of alkyl coumarates, valuable antioxidants and its thermoalkaline behavior, which competes favorably with other fungal cutinases, suggests it may be useful in many more applications.
Asunto(s)
Aspergillus nidulans , Hidrolasas de Éster Carboxílico , Aspergillus nidulans/genética , Aspergillus nidulans/enzimología , Especificidad por Sustrato , Concentración de Iones de Hidrógeno , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Hidrolasas de Éster Carboxílico/química , Temperatura , Peso Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Estabilidad de Enzimas , Medios de Cultivo/químicaRESUMEN
Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.
Asunto(s)
Aspergillus nidulans , Dipéptidos , Ingeniería Metabólica , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Reactores Biológicos , Fenilalanina/genética , Fenilalanina/metabolismoRESUMEN
Three new furano-lactones, asperilactones A-C (1-3), and two known compounds silvaticol (4) and violaceic acid (5) were isolated from an ethanol extract of Aspergillus nidulans, a fungus isolated from the Annelida Whitmania pigra Whitman (Haemopidae). Their structures were elucidated by a combination of spectroscopy, ECD calculations, comparing optical rotation values, and single-crystal X-ray diffraction analyses. Asperilactone A (1) represented the first example of furano-lactone with an unusual 2-thia-6-oxabicyclo[3.3.0]octane ring system. Asperilactones A and B showed weak toxicity against the HL-60 and RKO.