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Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi.

Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is a promising microbial cell factory to biosynthesize various products in industry. However, it remains unclear for whether it is also a suitable host for synthesizing abundant L-malic acid. In this study, we developed a convenient and efficient double-gene-editing system in A. nidulans strain TN02A7 based on the CRISPR–Cas9 and Cre-loxP systems. Using this gene-editing system, we made a L-malic acid-producing strain, ZQ07, derived from TN02A7, by deleting or overexpressing five genes (encoding Pyc, pyruvate carboxylase; OahA, oxaloacetate acetylhydrolase; MdhC, malate dehydrogenase; DctA, C4-dicarboxylic acid transporter; and CexA, citric acid transporter). The L-malic acid yield in ZQ07 increased to approximately 9.6 times higher (up to 30.7 g/L titer) than that of the original unedited strain TN02A7, in which the production of L-malic acid was originally very low. The findings in this study not only demonstrate that A. nidulans could be used as a potential host for biosynthesizing organic acids, but also provide a highly efficient gene-editing strategy in filamentous fungi.

Fungal pigments, including melanin, are recognized as promising materials for biomedical, environmental, and technological applications. In previous studies, we have demonstrated that the DOPA-melanin produced by the MEL1 mutant of Aspergillus nidulans exhibits antioxidant, anti-inflammatory, and antimicrobial activities without any cytotoxic or mutagenic effects, suggesting its potential use in pharmaceuticals. In order to increase the yield of this pigment and reduce the costs of its large-scale production, the present study aimed to evaluate agro-industrial by-products, sugarcane molasses, vinasse, and corn steep liquor as inexpensive substrates for fungal growth using experimental design methodology. According to the results obtained, the optimal composition of the culture medium was 0.81% (v/v) vinasse and 1.62% (w/v) glucose, which promoted a greater production of melanin (225.39 ± 4.52 mg g−1 of biomass), representing a 2.25-fold increase compared with the condition before optimization (100.32 mg.g−1 of biomass). Considering the amount of biomass obtained in the optimized condition, it was possible to obtain a total melanin production of 1 g L−1. Therefore, this formulation of a less complex and low-cost culture medium composition makes the large-scale process economically viable for future biotechnological applications of melanin produced by A. nidulans.

Both catalase and peroxiredoxin show high activities of H2O2 decomposition and coexist in the same organism; however, their division of labor in defense against H2O2 is unclear. We focused on the major peroxiredoxin (PrxA) and catalase (CatB) in Aspergillus nidulans at different growth stages to discriminate their antioxidant roles. The dormant conidia lacking PrxA showed sensitivity to high concentrations of H2O2 (>100 mM), revealing that PrxA is one of the important antioxidants in dormant conidia. Once the conidia began to swell and germinate, or further develop to young hyphae (9 h to old age), PrxA-deficient cells (ΔprxA) did not survive on plates containing H2O2 concentrations higher than 1 mM, indicating that PrxA is an indispensable antioxidant in the early growth stage. During these early growth stages, absence of CatB did not affect fungal resistance to either high (>1 mM) or low (

Pectinolytic enzymes have diverse industrial applications. Among these, pectate lyases act on the internal α-1,4-linkage of the pectate backbone, playing a critical role in pectin degradation. While most pectate lyases characterized thus far are of bacterial origin, fungi can also be excellent sources of pectinolytic enzymes. In this study, we performed biochemical characterization of the pectate lyase AnPL9 belonging to the polysaccharide lyase family 9 (PL9) from the filamentous fungus Aspergillus nidulans. Recombinant AnPL9 was produced using a Pichia pastoris expression system and purified. AnPL9 exhibited high activity on homogalacturonan (HG), pectin from citrus peel, pectin from apple, and the HG region in rhamnogalacturonan-I. Although digalacturonic acid and trigalacturonic acid were not degraded by AnPL9, tetragalacturonic acid was converted to 4,5-unsaturated digalacturonic acid and digalacturonic acid. These results indicate that AnPL9 degrades HG oligosaccharides with a degree of polymerization 4. Furthermore, AnPL9 was stable within a neutral-to-alkaline pH range (pH 6.0-11.0). Our findings suggest that AnPL9 is a candidate pectate lyase for biotechnological applications in the food, paper, and textile industries. This is the first report on a fungal pectate lyase belonging to the PL9 family.

Building strains of filamentous fungi for stable long-term heterologous expression of large biosynthetic pathways is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase-mediated cassette exchange. We built A. nidulans strains harboring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21 kb DNA fragment in a single step. We further evaluated the integration at two loci by analyzing the expression of a fluorescent reporter and the production of a heterologous polyketide metabolite. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterized aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.

The bZIP transcription factors (TFs) govern regulation of development, secondary metabolism, and various stress responses in filamentous fungi. In this work, we carried out genome-wide expression studies employing Illumina RNAseq to understand the roles of the two bZIP transcription factors AtfA and AtfB in Aspergillus nidulans. Comparative analyses of transcriptomes of control, ΔatfA, ΔatfB, and ΔatfAΔatfB mutant strains were performed. Dependence of a gene on AtfA (AtfB) was decided by its differential downregulation both between the reference and ΔatfA (ΔatfB) strains and between the ΔatfB (ΔatfA) and the ΔatfAΔatfB strains in vegetatively grown cells (mycelia) and asexual spores (conidia) of menadione sodium bisulfite (MSB)-treated or untreated cultures. As AtfA is the primary bZIP TF governing stress-response in A. nidulans, the number of differentially expressed genes for ΔatfA was significantly higher than for ΔatfB in both mycelial and conidial samples, and most of the AtfB-dependent genes showed AtfA dependence, too. Moreover, the low number of genes depending on AtfB but not on AtfA can be a consequence of ΔatfA leading to downregulation of atfB expression. Conidial samples showed much higher abundance of atfA and atfB mRNAs and more AtfA- and AtfB-affected genes than mycelial samples. In the presence of MSB, the number of AtfB- (but not of AtfA-) affected genes decreased markedly, which was accompanied with decreased mRNA levels of atfB in MSB-treated mycelial (reference strain) and conidial (ΔatfA mutant) samples. In mycelia, the overlap between the AtfA-dependent genes in MSB-treated and in untreated samples was low, demonstrating that distinct genes can be under AtfA control under different conditions. Carbohydrate metabolism genes were enriched in the set of AtfA-dependent genes. Among them, AtfA-dependence of glycolytic genes in conidial samples was the most notable. Levels of transcripts of certain secondary metabolitic gene clusters, such as the Emericellamide cluster, also showed AtfA-dependent regulation. Genes encoding catalase and histidine-containing phosphotransfer proteins showed AtfA-dependence under all experimental conditions. There were 23 AtfB-dependent genes that did not depend on AtfA under any of our experimental conditions. These included a putative α-glucosidase (agdB), a putative α-amylase, calA, which is involved in early conidial germination, and an alternative oxidase. In summary, in A. nidulans there is a complex interaction between the two bZIP transcription factors, where AtfA plays the primary regulatory role.

Filamentous fungi have been used for studying long-distance transport of cargoes driven by cytoplasmic dynein. Aspergillus nidulans is a well-established genetic model organism used for studying dynein function and regulation in vivo. Here, we describe how we grow A. nidulans strains for live-cell imaging and how we observe the dynein-mediated distribution of early endosomes and secretory vesicles. Using an on-stage incubator and culture chambers for inverted microscopes, we can image fungal hyphae that naturally attach to the bottom of the chambers, using wide-field epifluorescence microscopes or the new Zeiss LSM 980 (with Airyscan 2) microscope. In addition to methods for preparing cells for imaging, a procedure for A. nidulans transformation is also described.

For many years, two central, unanswered questions in cytoskeleton research were how microtubule assembly is nucleated and microtubule polarity established. The discoveries of γ-tubulin and γ-tubulin ring complexes were key advances that allowed these questions to be substantially answered. The discovery of γ-tubulin was the product of a genetic screen in Aspergillus nidulans for genes important for microtubule function. γ-Tubulin is a member of the tubulin superfamily of proteins, closely related to α- and β-tubulin but distinct from both. It is ubiquitous in eukaryotes, and in many organisms there are small families of γ-tubulin genes. γ-Tubulin and associated proteins form ring-like complexes that localize to microtubule-organizing centers (MTOCs) and play an important role in the nucleation of microtubule assembly from MTOCs and the establishment of microtubule polarity.

58 p.-8 fig., The genus Aspergillus includes industrially, medically and agriculturally important species. All of them, as do fungi in general, disperse to new niches principally by means of asexual spores. Regarding the genetic/molecular control of asexual development, Aspergillus nidulans is the main reference. In this species, two pathways control the production of conidiophores, the structures bearing asexual spores (conidia). The Upstream Developmental Activation (UDA) pathway transduces environmental signals, determining whether the Central Developmental Pathway (CDP) and the required morphological changes are induced. The transcriptional regulator BrlA links both pathways as loss-of-function mutations in flb (UDA) genes block brlA transcription and, consequently, conidiation. However, the aconidial phenotype of specific flb mutants is reverted under salt-stress conditions. Previously, we generated a collection of ΔflbB mutants unable to conidiate on culture medium supplemented with NaH2PO4 (0.65M). Here, we identified a Gly347Stop mutation within flpA as responsible for the FLIP57 phenotype. The putative cyclin FlpA and the remaining putative components of the C-terminal domain kinase-1 (CTDK-1) complex are necessary for proper germination, growth and developmental patterns in both A. nidulans and A. fumigatus. Cellular localization and functional interdependencies of the three proteins are also analyzed. Overall, this work links the putative CTDK-1 complex of aspergilli with growth and developmental control., Work at O.E. s lab has been supported by UPV/EHU (GIU19/014 to O.E.) and the Basque Government (PIBA-PUE PIBA_2020_1_0032; Elkartek KK-2021/43 and KK 2022/00107; and GIC IT1662-22, to O.E.). Work at CIB Margarita Salas-CSIC has been supported by MICIU/AEI (RTI2018-094263-B-100, to E.A.E.). A.O. hold a Margarita Salas grant (MARSA21/69), funded by Next-Generation EU, at the UPV/EHU. A.F was a degree student with a collaboration grant by the Spanish Ministry of Education (21/19070). Z.A was a Master Thesis student at O.E.´s lab, held an Ikertalent Fellow funded by the Basque Government (PIF21/003) at the Basque Culinary Center, and is now a PhD student at O.E.´s lab with funds of the KK 2022/00107 project. Work at J.R.F.´s lab has been supported by NIH grants R01-AI158442 and R01-AI143197.

Nutrient acquisition is essential for all organisms. Fungi regulate their metabolism according to environmental nutrient availability through elaborate transcription regulatory programs. In filamentous fungi, a highly conserved GATA transcription factor AreA and its co-repressor NmrA govern expression of genes involved in extracellular breakdown, uptake, and metabolism of nitrogen nutrients. Here, we show that the Aspergillus nidulans PnmB protease is a moonlighting protein with extracellular and intracellular functions for nitrogen acquisition and metabolism. PnmB serves not only as a secreted protease to degrade extracellular nutrients, but also as an intracellular protease to control the turnover of the co-repressor NmrA, accelerating AreA transcriptional activation upon nitrogen starvation. PnmB expression is controlled by AreA, which activates a positive feedback regulatory loop. Hence, we uncover a regulatory mechanism in the well-established controls determining the response to nitrogen starvation, revealing functional evolution of a protease gene for transcriptional regulation and extracellular nutrient breakdown., Li et al. report the PnmB protease as a moonlighting protein in the fungus Aspergillus nidulans as both a secreted protease that degrades extracellular nutrients and an intracellular protease that controls the turnover of the co-repressor NmrA. These results reveal a regulatory mechanism of the drivers of the nitrogen starvation response, as well as the functional evolution of a protease gene for transcriptional regulation and extracellular nutrient breakdown.

Abstract Cytochrome P450 monooxygenases (P450s) are considered nature’s most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. Key points •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

Cellular functions such as autophagy, cell signaling and vesicular trafficking involve the retrograde transport of motor-driven cargo along microtubules. Typically, newly formed cargo engages in slow diffusive movement from its point of origin before attaching to a microtubule. In some cell types, cargo destined for delivery to the perinuclear region relies on capture at dynein-enriched loading zones located near microtubule plus-ends. Such systems include extended cell regions of neurites and fungal hyphae, where the efficiency of the initial diffusive loading process depends on the axial distribution of microtubule plus-ends relative to the initial cargo position. We use analytic mean first passage time calculations and numerical simulations to model diffusive capture processes in tubular cells, exploring how the spatial arrangement of microtubule plus-ends affects the efficiency of retrograde cargo transport. Our model delineates the key features of optimal microtubule ar-rangements that minimize mean cargo capture times. Namely, we show that configurations with a single long microtubule and broad distribution of additional microtubule plus-ends allow for efficient capture in a variety of different scenarios for retrograde transport. Live-cell imaging of microtubule plus-ends in Aspergillus nidulans hyphae indicates that their distributions exhibit these optimal qualitative features. Our results highlight important coupling effects between microtubule length distribution and retrograde cargo transport, providing guiding principles for the spatial arrangement of microtubules within tubular cell regions.

Hepatocellular carcinoma is among the leading causes of cancer-related deaths worldwide and needs efficient and feasible approach of treatment. Present study focuses on exploring the anticancer activity of a secondary metabolite called siderophore of Aspergillus nidulans against hepatocellular carcinoma cell line HepG2. These small peptides are produced by microorganisms including fungi for scavenging iron from its surroundings. Fungi including Aspergillus spp. are known to produce siderophores under iron-limited conditions. Siderophores have high affinity towards iron and are classified into various types. In the present study, siderophore isolated and purified from fungal cultures was confirmed to be of hydroxamate type by chrome azurol sulfonate and Atkin's assay. HPLC analysis confirmed purity while LC-ESI-MS revealed that the siderophore is triacetyl fusigen. Cancerous cells, HepG2, grown under siderophore treatment showed inhibition in growth and proliferation in a dose- and time-dependent manner. Reduction in viability and metabolic activity was evident upon treatment as seen in trypan blue, MTT and WST assay. Fluorescent staining using PI and DAPI confirmed the same while DCFDA staining revealed increased reactive oxygen species production which might have led to cell death and deterioration. Such increase in ROS has been correlated with iron accumulation by assessing intracellular iron level through ICP-MS. To assess the effect of siderophore treatment on normal cells, WRL-68, same assays were carried out but the effect was mostly non-significant up to 48 h. Thus, present work suggests that an optimum dose of siderophore purified from A. nidulans culture might prove a useful anticancer agent.

Fungal endophytes are living inside plants without any harmful effects; the prospecting about them is increased day by day because they can produce bioactive compounds which can be used in different applications. Herein, the current study was aimed to isolate the endophytic fungi from the Ocimum basilicum plant as safe microorganisms and evaluate their biological activities. The results illustrated that three endophytic fungal strains were isolated and identified morphologically and genetically as Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus flavus and deposited in gene bank under accession numbers MZ045561, MZ045562, and MZ045563 respectively. Moreover, cell-free filtrates of endophytic fungal strains were extracted using ethyl acetate, where these crude extracts exhibited promising antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumonia, and Candida albicans at a concentration of 1000 µg/mL. Furthermore, these endophytic strains exhibited a potential antioxidant activity where IC50 of the crude extract of A. nidulans, A. fumigatus, and A. flavus were (166.3, 68.4, and 347.1 µg/mL) and (151.2, 77.9, and 246.3 µg/mL) using DPPH and ABTS methods, respectively. Furthermore, the ethyl acetate crude extracts of these endophytic fungi did not exhibit any cytotoxic effect against Vero and Wi 38 normal cells. GC–MS analysis of the crude extract of A. nidulans, A. fumigatus, and A. flavus indicated the presence of 22, 22, and 20 active compounds, respectively. The major compounds in the fungal extracts are belonging to fatty acids, fatty acid esters, tetrahydrofurans, and sterols. In conclusion, the isolated endophytic A. nidulans, A. fumigatus, and A. flavus from Ocimum basilicum are promising sources for bioactive compounds.

Fungi are specifically dangerous as they exhibit a significant tolerance to environmental changes and can proliferate under low relative humidity, unlike bacteria. They produce spores that are easily dispersed by air hence they are ubiquitous. The study aimed at identifying the fungal isolates present in the bathrooms located on the three floors of the hostel, University of Benin, Benin City. Samples were collected from the walls of the bathrooms using sterile swab sticks and were identified using standard microbiological techniques. The isolated fungi were Aspergillus nidulans, A. niger, A. tamarii, A. flavus, Candida albicans, Penicillium cyclopium, P. oxalicum, Mucor mucedo, Trichophyton rubrum and Rhodotorula species. From the ground floor bathrooms, Candida albicans (23.40%) were most frequently isolated, Aspergillus nidulans (55.56%) were mostly isolated from the first floor and Mucor mucedo (56.00%) were the most isolated from the second floor. After washing the bathrooms, Mucor mucedo was scarcely isolated from the walls of the bathrooms. The findings were processed statistically using the two-tailed test to detect the significant difference between the groups of means for the fungal counts from each floor. A significant difference (p

Heterologous expression of the flavipucine biosynthetic gene cluster in Aspergillus nidulans led to the production of flavipucine (1) and dihydroisoflavipucine (3), as well as six unusual flavipucine related products containing three classes of heterocycles. This combined with gene inactivation, chemical complementation, and transcriptome analysis demonstrated unprecedented ways to form 2-pyridone and 2-pyrone structures by the oxidative rearrangements of pyrrolinone precursors as well as provided insights into the biosynthesis of this important class of natural products.

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed Km and Vmax of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD+. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD+ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

Nitric oxide (NO) is a signaling molecule in eukaryotic and prokaryotic organisms. NO levels transiently boost upon induction of conidiation in Aspergillus nidulans. Only one pathway for NO synthesis involving nitrate reductase has been reported in filamentous fungi so far, but this does not satisfy all the NO produced in fungal cells. Here we provide evidence for at least one additional biosynthetic pathway in A. nidulans involving L-arginine or an intermediate metabolite as a substrate. Under certain growth conditions, the addition of L-arginine to liquid media elicited a burst of nitric oxide that was not dependent on any of the urea cycle genes. The NO levels were controlled by the metabolically available arginine, which was regulated by mobilization from the vacuoles and during development. In vitro assays with protein extracts and amino acid profiling strongly suggested the existence of an arginine-dependent NO pathway analogous to the mammalian nitric oxide synthase. Addition of polyamines induced NO synthesis, and mutations in the polyamine synthesis genes puA and spdA reduced the production of NO. In conclusion, here we report an additional pathway for the synthesis of NO in A. nidulans using urea cycle intermediates. This article is protected by copyright. All rights reserved.

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.

The VosA-VelB hetero-dimeric complex plays a pivotal role in regulating development and secondary metabolism in

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus

Four emestrin hybrid polymers, asperemestrins A-D (

The monomer, 3-hydroxybutyrate (3HB), plays an interesting role as a precursor for antibiotics, vitamins, and bioplastics such as polyhydroxybutyrates (PHB). The biotechnological production of both compounds in Escherichia coli has seen increased interest in the last decade. The central metabolite in the 3HB production pathway is acetyl-CoA, the derivative of coenzyme A (CoA). Enriching the intracellular pool of these cofactors could improve 3HB titers. In our study, we opted to increase CoA titers by upregulating pantothenate kinase (PanK), the rate limiting step in CoA biosynthetic pathway. To this end, 4 PanKs genes of different taxonomic origins (mammalian, fungal, and bacterial) were individually expressed and evaluated in 3HB producing E. coli cells. In shake flask studies, strains expressing Aspergillus nidulans PankII and Mus musculus PanK1β achieved the highest 3HB titers. In a bioreactor fermentation, the strain harboring murine PanK1β resulted in 7.6 g/L compared to 5.4 g/L of 3HB in the control strain. Although structurally different from the bacterial PankI, our study showed that eukaryotic Panks can supplement the kinase activity in prokaryotes. Expressing the exogenous PanKs offer several advantages over the host’s enzyme; PanKII is only inhibited by acetyl-CoA, for which the 3HB-production system would provide a constant metabolic sink. Additionally, PanK1β is weakly regulated by acetyl-CoA, and its activity is stimulated by free CoA. Overexpressing eukaryotic PanKs constitutes a suitable strategy for improving 3HB titers in E. coli.

Introduction: Aspergillus exhibits a wide variation of susceptibility against antifungals according to genetic and environmental factors. Identification to the species level is necessary for appropriate treatment. Our objective was to determine the Aspergillus species involved in invasive pulmonary aspergillosis (IPA) among ICU patients in Jakarta, Indonesia. Methodology: The incidence of IPA in ICU patients at six hospitals in Jakarta from October 2012 – January 2015 was investigated. It involved a collection of endotracheal aspirates (ETA), nasal swabs and environmental samples around the hospitals, phenotypic screening, molecular characterization, and antifungal susceptibility testing. Results: Of the 405 patients investigated, 31 patients (7.7%) were diagnosed with putative IPA, from whom 45 Aspergillus isolates were collected. Aspergillus isolates were identified from pulmonary secretions in 24 patients, from nasal swabs in 7 patients and from both pulmonary secretions and nasal swabs in 7 patients. The phenotypic method showed 33 isolates of Aspergillus flavus (73.4%), nine Aspergillus fumigatus (20%), two Aspergillus niger (4.4%), and one Aspergillus nidulans (2.2%) isolate. Molecular identification showed 27 isolates of A. flavus (60.0%), eight isolates of A. fumigatus (17.8%), two isolates of A. niger (4.4%) and one isolate of A. nidulans (2.2%), while seven isolates (15.6%) were cryptic species or mixed isolates. Conclusions: Susceptibility testing showed all isolates were susceptible to amphotericin B, azoles and micafungin. Aspergillus flavus was the main causative organism in IPA cases in Jakarta, followed by A. fumigatus.

Expression of a nonribosomal peptide synthetase-nonreducing polyketide synthase hybrid gene pcr10109 from Penicillium crustosum PRB-2 in Aspergillus nidulans led to the accumulation of 4-hydroxy-6-(4-hydroxyphenyl)-α-pyrone (1). Adding para-hydroxybenzoic acid into the medium in which the overexpressing mutant is growing increased the product yield up to 5-fold. This strategy could be helpful for heterologous gene expression experiments requiring special substrates for product formation.

The VosA-VelB heterocomplex governs expression of several genes associated with fungal development and secondary metabolism. In this study, we have investigated the functions of one of the VosA-VelB-activated developmental genes vadJ in development and production of the mycotoxin sterigmatocystin in the model fungus Aspergillus nidulans. The vadJ gene is predicted to encode a 957-amino acid length protein containing a highly conserved sensor histidine kinase domain. The deletion of vosA or velB resulted in decreased mRNA levels of vadJ throughout the life cycle, suggesting that VosA and VelB are necessary for proper expression of vadJ. Nullifying vadJ led to highly restricted colony growth, lowered formation of asexual spores, and about two-fold reduction in conidial viability. Conversely, the deletion of vadJ resulted in elevated production of sexual fruiting bodies and sterigmatocystin. These suggest that VadJ is necessary for proper coordination of asexual and sexual development, and sterigmatocystin production. In accordance with this idea, the deletion of vadJ led to elevated mRNA levels of the two key sexual developmental activators esdC and nsdD. In summary, the putative sensor histidine kinase VadJ represses sexual development and sterigmatocystin production, but activates asexual development in A. nidulans.

Regulation of signal transduction pathways is crucial for the maintenance of cellular homeostasis and organismal development in fungi. Transcription factors are key elements of this regulatory network. The basic-region leucine zipper (bZIP) domain of the bZIP-type transcription factors is responsible for DNA binding while their leucine zipper structural motifs are suitable for dimerization with each other facilitiating the formation of homodimeric or heterodimeric bZIP proteins. This review highlights recent knowledge on the function of fungal orthologs of the Schizosaccharomyces pombe Atf1, Aspergillus nidulans AtfA, and Fusarium verticillioides FvAtfA, bZIP-type transcription factors with a special focus on pathogenic species. We demonstrate that fungal Atf1-AtfA-FvAtfA orthologs play an important role in vegetative growth, sexual and asexual development, stress response, secondary metabolite production, and virulence both in human pathogens, including Aspergillus fumigatus, Mucor circinelloides, Penicillium marneffei, and Cryptococcus neoformans and plant pathogens, like Fusarium ssp., Magnaporthe oryzae, Claviceps purpurea, Botrytis cinerea, and Verticillium dahliae. Key points • Atf1 orthologs play crucial role in the growth and development of fungi. • Atf1 orthologs orchestrate environmental stress response of fungi. • Secondary metabolite production and virulence are coordinated by Atf1 orthologs.

Endophytic fungi are a substantial source of bioactive secondary metabolites. Present studies on Aspergillus nidulans―an endophytic fungus from Nyctanthes arbor-tristis Linn. afforded one new 1, 5-dihydroxy-3-methylxanthone-6-carboxylic acid methyl ester (1) and one known 5, 10-dihydro-phenazine-1-carboxylic acid (2) compound. The structures were elucidated by spectral techniques (Mass, 1D and 2D NMR), and their urease, carbonic anhydrase and prolyl endopeptidase inhibitory activities were evaluated. Compound 1 showed notable prolyl endopeptidase inhibition (IC50 = 3.13 ± 0.09 µM) and compound 2 showed significant carbonic anhydrase (IC50 =14.3 ± 1.05 µM) and urease (IC50 = 24.3 ± 1.15 µM) inhibition.

Fungal melanins represent a resource for important breakthroughs in industry and medicine, but the characterization of their composition, synthesis, and structure is not well understood. Raman spectroscopy is a powerful tool for the elucidation of molecular composition and structure. In this work, we characterize the Raman spectra of wild-type Aspergillus fumigatus and Cryptococcus neoformans and their melanin biosynthetic mutants and provide a rough “map” of the DHN (A. fumigatus) and DOPA (C. neoformans) melanin biosynthetic pathways. We compare this map to the Raman spectral data of Aspergillus nidulans wild-type and melanin biosynthetic mutants obtained from a previous study. We find that the fully polymerized A. nidulans melanin cannot be classified according to the DOPA pathway, nor can it be solely classified according to the DHN pathway, consistent with mutational analysis and chemical inhibition studies. Our approach points the way forward for an increased understanding of, and methodology for, investigating fungal melanins.

Aspergillus nidulans snxA, an ortholog of Saccharomyces cerevisiae Hrb1/Gbp2 messenger RNA shuttle proteins, is—in contrast to budding yeast—involved in cell cycle regulation, in which snxA1 and snxA2 mutations as well as a snxA deletion specifically suppress the heat sensitivity of mutations in regulators of the CDK1 mitotic induction pathway. snxA mutations are strongly cold sensitive, and at permissive temperature snxA mRNA and protein expression are strongly repressed. Initial attempts to identify the causative snxA mutations revealed no defects in the SNXA protein. Here, we show that snxA1/A2 mutations resulted from an identical chromosome I–II reciprocal translocation with breakpoints in the snxA first intron and the fourth exon of a GYF-domain gene, gyfA. Surprisingly, a gyfA deletion and a reconstructed gyfA translocation allele suppressed the heat sensitivity of CDK1 pathway mutants in a snxA+ background, demonstrating that 2 unrelated genes, snxA and gyfA, act through the CDK1–CyclinB axis to restrain the G2–M transition, and for the first time identifying a role in G2–M regulation for a GYF-domain protein. To better understand snxA1/A2-reduced expression, we generated suppressors of snxA cold sensitivity in 2 genes: (1) loss of the abundant nucleolar protein Nsr1/nucleolin bypassed the requirement for snxA and (2) loss of the Set2 histone H3 lysine36 (H3K36) methyltransferase or a nonmethylatable histone H3K36L mutant rescued hypomorphic snxA mutants by restoring full transcriptional proficiency, indicating that methylation of H3K36 acts normally to repress snxA transcription. These observations are in line with known Set2 functions in preventing excessive and cryptic transcription of active genes.

Mechanosensitive ion channels are integral membrane proteins ubiquitously present in bacteria, archaea, and eukarya. They act as molecular sensors of mechanical stress to serve vital functions such as touch, hearing, osmotic pressure, proprioception and balance, while their malfunction is often associated with pathologies. Amongst them, the structurally distinct MscL and MscS channels from bacteria are the most extensively studied. MscS-like channels have been found in plants and

An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best studied elevator transporters is the UapA uric acid-xanthine/H+ symporter of the filamentous fungus Aspergillus nidulans. Here, we present a novel genetic analysis for deciphering the role of transmembrane segments (TMS) 5 and 12 in UapA transport function. We show that specific residues in both TMS5 and TMS12 control, negatively or positively, the dynamics of transport, but also substrate binding affinity and specificity. More specifically, mutations in TMS5 can lead to increased rate of transport, but also to an inactive transporter due to high-affinity substrate-trapping, whereas mutations in TMS12 lead to apparently uncontrolled sliding, and thus broadened specificity and UapA-mediated purine toxicity. Our findings shed new light on how elevator transporters function or how their transport characteristics might be altered genetically or have been modified in the course of evolution.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells’ ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.

Kae1 is a subunit of the highly evolutionarily conserved KEOPS/EKC complex, which is involved in universal (t6A

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes found in viruses, archaea, and bacteria as well as eukaryotes, such as fungi, algae and insects, actively contributing to the degradation of different polysaccharides. In Aspergillus nidulans, LPMOs from family AA9 (AnLPMO9s), along with an AA3 cellobiose dehydrogenase (AnCDH1), are cosecreted upon growth on crystalline cellulose and lignocellulosic substrates, indicating their role in the degradation of plant cell wall components. Functional analysis revealed that three target LPMO9s (AnLPMO9C, AnLPMO9F and AnLPMO9G) correspond to cellulose-active enzymes with distinct regioselectivity and activity on cellulose with different proportions of crystalline and amorphous regions. AnLPMO9s deletion and overexpression studies corroborate functional data. The abundantly secreted AnLPMO9F is a major component of the extracellular cellulolytic system, while AnLPMO9G was less abundant and constantly secreted, and acts preferentially on crystalline regions of cellulose, uniquely displaying activity on highly crystalline algae cellulose. Single or double deletion of AnLPMO9s resulted in about 25% reduction in fungal growth on sugarcane straw but not on Avicel, demonstrating the contribution of LPMO9s for the saprophytic fungal lifestyle relies on the degradation of complex lignocellulosic substrates. Although the deletion of AnCDH1 slightly reduced the cellulolytic activity, it did not affect fungal growth indicating the existence of alternative electron donors to LPMOs. Additionally, double or triple knockouts of these enzymes had no accumulative deleterious effect on the cellulolytic activity nor on fungal growth, regardless of the deleted gene. Overexpression of AnLPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of AnLPMO9G to improve lignocellulose saccharification. IMPORTANCE Fungal lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that boost plant biomass degradation in combination with glycoside hydrolases. Secretion of LPMO9s arsenal by Aspergillus nidulans is influenced by the substrate and time of induction. These findings along with the biochemical characterization of novel fungal LPMO9s have implications on our understanding of their concerted action, allowing rational engineering of fungal strains for biotechnological applications such as plant biomass degradation. Additionally, the role of oxidative players in fungal growth on plant biomass was evaluated by deletion and overexpression experiments using a model fungal system.

RNA-binding proteins are involved in RNA metabolism and posttranscriptional regulation of various fundamental biological processes. The PUF family of RNA-binding proteins is highly conserved in eukaryotes, and its members regulate gene expression, mitochondrial biogenesis, and RNA processing. However, their biological functions in Aspergillus species remain mostly unknown in filamentous fungi. Here we have characterized the puf genes in the model organism Aspergillus nidulans. We generated deletion mutant strains for the five putative puf genes present in the A. nidulans genome and investigated their developmental phenotypes. Deletion of pufA or pufE affected fungal growth and asexual development. pufA mutants exhibited decreased production of asexual spores and reduced mRNA expression of genes regulating asexual development. The pufE deletion reduced colony growth, increased formation of asexual spores, and delayed production of sexual fruiting bodies. In addition, the absence of pufE reduced both sterigmatocystin production and the mRNA levels of genes in the sterigmatocystin cluster. Finally, pufE deletion mutants showed reduced trehalose production and lower resistance to thermal stress. Overall, these results demonstrate that PufA and PufE play roles in the development and sterigmatocystin metabolism in A. nidulans.

Aims Echinocandin B (ECB) is a kind of lipopeptide antifungal antibiotic, as well as the key precursor of antifungal drug Anidulafungin. Its efficient bioproduction plays an important role in promoting the industrial production of Anidulafungin. Methods and results In this study, methyl oleate and Tween 80 were firstly used to enhance the ECB fermentation by Aspergillus nidulans, the results showed that the ECB titre was significantly enhanced with the addition of methyl oleate and Tween 80. Among the lipids, methyl oleate was found to play a pivotal role in increasing the ECB titre to 2123 mg l-1 , which was more than five times higher than that of the control. The addition of Tween 80 in the medium resulted in ECB titre increased to 2584 mg l-1 . The scanning electron microscope (SEM) and N-phenyl-1-naphthylamine (NPN) assay indicated that Tween 80 could influence the cell membrane permeability of A. nidulans, and enhance the intracellular and extracellular substance exchange, therefore lead to the increasing of ECB titre. Conclusions Methyl oleate and Tween 80 are optimal carbon sources and surfactants for efficient ECB biosynthesis respectively. Significance and impact of the study Surfactant was used in ECB fermentation for the first time, which provided feasible ideas for optimizing the fermentation process of other fungi.

The velvet regulators VosA and VelB are primarily involved in spore maturation and dormancy. Previous studies found that the VosA-VelB hetero-complex coordinates certain target genes that are related to fungal differentiation and conidial maturation in Aspergillus nidulans. Here, we characterized the VosA/VelB-inhibited developmental gene vidD in A. nidulans. Phenotypic analyses demonstrated that the vidD deleted mutant exhibited defect fungal growth, a reduced number of conidia, and delayed formation of sexual fruiting bodies. The deletion of vidD decreased the amount of conidial trehalose, increased the sensitivity against heat stress, and reduced the conidial viability. Moreover, the absence of vidD resulted in increased production of sterigmatocystin. Together, these results show that VidD is required for proper fungal growth, development, and sterigmatocystin production in A. nidulans.

The microtubule-associated protein tau promotes the stabilization of the axonal cytoskeleton in neurons. In several neurodegenerative diseases, such as Alzheimer's disease, tau has been found to dissociate from microtubules, leading to the formation of pathological aggregates that display an amyloid fibril-like structure. Recent structural studies have shown that the tau filaments isolated from different neurodegenerative disorders have structurally distinct fibril cores that are specific to the disease. These "strains" of tau fibrils appear to propagate between neurons in a prion-like fashion that maintains their initial template structure. In addition, the strains isolated from diseased tissue appear to have structures that are different from those made by the most commonly used in vitro modeling inducer molecule, heparin. The structural differences among strains in different diseases and in vitro-induced tau fibrils may contribute to recent failures in clinical trials of compounds designed to target tau pathology. This study identifies an isoquinoline compound (ANTC-15) isolated from the fungus Aspergillus nidulans that can both inhibit filaments induced by arachidonic acid (ARA) and disassemble preformed ARA fibrils. When compared to a tau aggregation inhibitor currently in clinical trials (LMTX, LMTM, or TRx0237), ANTC-15 and LMTX were found to have opposing inducer-specific activities against ARA and heparin in vitro-induced tau filaments. These findings may help explain the disappointing results in translating potent preclinical inhibitor candidates to successful clinical treatments.

Biogas is the product of anaerobic digestion (AD) of organic waste and is considered to be one of the most valuable natural renewable energy carriers. Plant biomass represents the most abundant eco-friendly energy reservoir on Earth. However, the tenacious and heterogeneous structure of the lignocellulose-rich elements makes it difficult for the involved microbes to digest the recalcitrant substrates. Both the degradation process and the biogas production yield can be enhanced by appropriate pre-treatment of lignocellulosic materials. Filamentous fungi have been known as proficient colonizers of lignocellulosic plant tissues and have been recognized as producers of exceptionally rich and diverse hydrolytic enzymes. We tested Aspergillus nidulans, Trichoderma reesei, Rhizomucor miehei and Gilbertella persicaria filamentous fungal strains for pre-treatment of various agricultural lignocellulosic wastes. During the pre-treatment phase, the β-glucosidase and endoglucanase activity was measured spectrophotometrically. In the AD step, methane production was monitored by gas chromatography. The preliminary results showed that all the applied strains (Aspergillus nidulans, Trichoderma reesei, Rhizomucor miehei and Gilbertella persicaria) were highly effective in producing both β-glucosidase and endo-(1,4)-β-D-glucanase enzymes, which might explain the greatly improved AD results. Pre-treatment with the above-mentioned filamentous fungi positively affected the biogas production, although the effect strongly depended on the selection of the fungal partner for any given biomass substrate. Depending on the used substrate and the pre-treatment strain, overall methane yields were elevated two-fold relative to the controls.

pH, as one of the most important environmental factors, affects various biological processes in pathogenic fungi. Sensing and responding to fluctuations in ambient pH are essential for these fungi to complete their life cycle. Fungi have evolved a complicated and conserved system, the so-called Pal-pH pathway, to regulate genes and adapt to alterations in ambient pH. PacC is the dominant transcription factor in the Pal-pH pathway and regulates various biological processes. The regulatory mode of PacC has been extensively studied in Aspergillus nidulans and is generally conserved in other fungal species, including numerous phytopathogenic fungi. However, species-specific alterations have been reported. This review summarizes recent advances in the regulatory mechanisms of PacC and its role in controlling development, pathogenicity, and mycotoxin biosynthesis in phytopathogenic fungi. Potential applications of these findings and some unresolved questions are also discussed.

L-rhamnose is found in nature mainly as a component of structural plant polysaccharides and can be used as a carbon source by certain microorganisms. Catabolism of this sugar in bacteria, archaea and fungi occurs by two routes involving either phosphorylated or non-phosphorylated intermediates. Unlike the corresponding pathway in yeasts, the metabolic details of the non-phosphorylated pathway in filamentous fungi are not fully defined. The first three genes (lraA, lraB and lraC) of the non-phosphorylated pathway in Aspergillus nidulans have recently been studied revealing dependence on lraA function for growth on L-rhamnose and α-L-rhamnosidase production. In the present work two genes encoding the subsequent steps catalysed by L-2-keto-3-deoxyrhamnonate (L-KDR) aldolase (AN9425) and L-lactaldehyde dehydrogenase (AN0554) are identified. Loss-of-function mutations cause adverse growth effects on L-rhamnose. Akin to genes lraA-C and those encoding rhamnosidases (rhaA, rhaE), their expression is induced on L-rhamnose via the transcriptional activator RhaR. Interestingly, the aldolase belongs to the family of bacterial L-KDR aldolases (PF03328/COG3836) and not that of yeasts (PF00701/COG0329). In addition, AN0554 corresponds to the previously characterized aldA gene (encodes aldehyde dehydrogenase involved in ethanol utilization) thus revealing a previously unknown role for this gene in the catabolism of L-rhamnose. This article is protected by copyright. All rights reserved.

Phytochrome-dependent light signaling has been studied in several fungi. In Aspergillus nidulans light-stimulated phytochrome activates the high-osmolarity glycerol (HOG) signaling pathway and thereby controls the expression of a large number of genes, many of which are related to stress responses. In a genome-wide expression analysis in A. nidulans we found that phytochrome, fphA, is under strict expression control of the central regulator of the sulfur-starvation response, MetR. This transcriptional regulator is required for the expression of genes involved in inorganic sulfur assimilation. In the presence of organic sulfur, MetR is probably ubiquitinated and possibly degraded and the transcription of sulfur-assimilation genes, e.g., sulfate permease, is turned off. The expression analysis described here revealed, however, that MetR additionally controls the expression of hundreds of genes, many of which are required for secondary metabolite production. We also show that metR mutation phenocopies fphA deletion, and five other histidine-hybrid kinases are down-regulated in the metR1 mutant. Furthermore, we found that light and phytochrome regulate the expression of at least three carbon–sulfur hydrolases. This work is a further step towards understanding the interplay between light sensing and metabolic pathways.

Endoglucanases (EC 3.2.1.4) are important enzymes involved in the hydrolysis of cellulose, acting randomly in the β-1,4-glycosidic bonds present in the amorphous regions of the polysaccharide chain. These biocatalysts have been classified into 14 glycosyl hydrolase (GH) families. The GH7 family is of particular interest since it may act on a broad range of substrates, including cellulose, β-glucan, and xylan, an attractive feature for biotechnological applications, especially in the renewable energy field. In the current work, a gene from the thermophilic fungus Thermothielavioides terrestris, encoding an endoglucanase GH7 (TtCel7B), was cloned in the secretion vector pEXPYR and transformed into the high-protein-producing strain Aspergillus nidulans A773. Purified TtCel7B has a molecular weight of approximately 66 kDa, evidenced by SDS-PAGE. Circular dichroism confirmed the high β-strand content consistent with the canonical GH7 family β-jellyroll fold, also observed in the 3D homology model of TtCel7B. Biochemical characterization assays showed that TtCel7B was active over a wide range of pH values (3.5–7.0) and temperatures (45–70 °C), with the highest activity at pH 4.0 and 65 °C. TtCel7B also was stable over a wide range of pH values (3.5–9.0), maintaining more than 80% of its activity after 24 h. The KM and Vmax values in low-viscosity carboxymethylcellulose were 9.3 mg mL−1 and 2.5 × 104 U mg−1, respectively. The results obtained in this work provide a basis for the development of applications of recombinant TtCel7B in the renewable energy field.

Three fungal strains isolated from the oily sludge contaminated soil were identified as Trichoderma atroviride, Aspergillus nidulans and Aspergillus sydowii using 18s rDNA sequences and BLAST analy...

Previous studies showed that the FeII/α‐ketoglutarate dependent dioxygenase AsqJ induces a skeletal rearrangement in viridicatin biosynthesis in Aspergillus nidulans, generating a quinolone scaffold from benzo[1,4]diazepine‐2,5‐dione substrates. We report that AsqJ catalyzes an additional, entirely different reaction, simply by a change in substituent in the benzodiazepinedione substrate. This new mechanism is established by substrate screening, application of functional probes, and computational analysis. AsqJ excises H2CO from the heterocyclic ring structure of suitable benzo[1,4]diazepine‐2,5‐dione substrates to generate quinazolinones. This novel AsqJ catalysis pathway is governed by a single substituent within the complex substrate. This unique substrate‐directed reactivity of AsqJ enables the targeted biocatalytic generation of either quinolones or quinazolinones, two alkaloid frameworks of exceptional biomedical relevance., AsqJ catalyzes a complex rearrangement sequence in quinolone biosynthesis. We show the AsqJ biocatalytic potential to significantly exceed this natural function. An unprecedented reaction pathway leading to quinazolinones is uncovered, functionally and mechanistically characterized in detail, revealing a unique substrate‐dependent product selectivity in enzyme catalysis.

Hydrophobins are relatively small proteins produced naturally by filamentous fungi with interesting biotechnological and biomedical applications given their self-assembly capacity, efficient adherence to natural and artificial surfaces, and to introduce modifications on the hydrophobicity/hydrophilicity of surfaces. In this work we demonstrate the efficient expression on the S. cerevisiae cell surface of class II HFBI of Trichoderma reesei and class I DewA of Aspergillus nidulans, a hydrophobin not previously exposed, using the Yeast Surface Display a-agglutinin (Aga1-Aga2) system. We show that the resulting modifications affect surface properties, and also yeast cells’ resistance to several adverse conditions. The fact that viability of the engineered strains increases under heat and osmotic stress is particularly interesting. Besides, improved biocatalytic activity toward the reduction of ketone 1-phenoxypropan-2-one takes place in the reactions carried out at both 30 °C and 40 °C, within a concentration range between 0.65 and 2.5 mg/mL. These results suggest interesting potential applications for hydrophobin-exposing yeasts. • Class I hydrophobin DewA can be efficiently exposed on S. cerevisiae cell surfaces. • Yeast exposure of HFBI and DewA increases osmotic and heat resistance. • Engineered strains show modified biocatalytic behavior

The bisegmented genome of a novel double-stranded (ds) RNA mycovirus, named "Aspergillus nidulans partitivirus 1" (AnPV1), isolated from the fungus Aspergillus nidulans strain HJ5-47, was sequenced and analyzed. AnPV1 contains two segments, AnPV1-1 and AnPV1-2. AnPV1-1 has 1837 bp with an open reading frame (ORF) that potentially encodes a putative RNA-dependent RNA polymerase (RdRp) of 572 amino acids (aa). AnPV1-2 has 1583 bp with an ORF encoding a putative capsid protein (CP) of 488 aa. Phylogenetic analyses indicated that AnPV1 and related viruses clustered in a group that could represent a new unclassified genus in the family Partitiviridae.