Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: A target for antifungals.

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement toward the catalytic pocket by the N terminus upon ligand binding and identifies residues required for substrate binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved among Cryptococcal species and closely related basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.

A positive feedback to climate change: The effect of temperature on the respiration of key wood-decomposing fungi does not decline with time.

Heterotrophic soil microorganisms are responsible for ~50% of the carbon dioxide released by respiration from the terrestrial biosphere each year. The respiratory response of soil microbial communities to warming, and the control mechanisms, remains uncertain, yet is critical to understanding the future land carbon (C)-climate feedback. Individuals of nine species of fungi decomposing wood were exposed to 90 days of cooling to evaluate the medium-term effect of temperature on respiration. Overall, the effect of temperature on respiration increased in the medium term, with no evidence of compensation. However, the increasing effect of temperature on respiration was lost after correcting for changes in biomass. These results indicate that C loss through respiration of wood-decomposing fungi will increase beyond the direct effects of temperature on respiration, potentially promoting greater C losses from terrestrial ecosystems and a positive feedback to climate change.

Genome editing tools based improved applications in macrofungi.

Macrofungi commonly referred to as Mushrooms are distributed worldwide and well known for their nutritional, medicinal, and organoleptic properties. Strain improvement in mushrooms is lagging due to paucity of efficient genome modification techniques. Thus, for advanced developments in research and commercial or economical viability and benefit, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) emerged as an efficient genome editing tool. The higher efficiency and precision of the desired genetic modification(s) are the most valuable attributes of this recent technology. The present review comprehensively summarizes various conventional methods utilized for strain improvement in mushrooms including hybridization, protoplast fusion, and di-mon mating. Furthermore, the problems associated with these techniques have been discussed besides providing the potential recluses. The significance of CRISPR/Cas9 strategies employed for improvement in various mushroom genera has been deliberated, as these strategies will paves the way forward for obtaining improved strain and effective cultivation methods for enhancing the yield and quality of the fruit bodies.

Basidiomycota strains as whole-cell biocatalysts for the synthesis of high-value natural benzaldehydes.

Substituted benzaldehydes are the most commonly used natural-occurring flavours in the world. The consumer's preference for 'natural or organic' aromas has increased the request for flavours possessing the 'natural' status. The resulting shortage of aromatic aldehydes of extractive origin, such as vanillin, veratraldehyde and piperonal, can be offset by developing a new biotechnological synthesis method. Here, we report a study on the microbiological reduction of five natural benzoic acid derivatives, namely p-anisic, vanillic, veratric, piperonylic and eudesmic acids, to produce the corresponding fragrant aldehydes. We found that different Basidiomycota strains can efficiently perform this transformation, with good chemical selectivity and tolerance to the toxicity of substrates and products. Besides confirming the carboxylic acid reductase activity of the already studied fungi Pycnoporus cinnabarinus, we discovered that other species such as Pleurotus eryngii, Pleurotus sapidus and Laetiporus sulphureus as well as the non-ligninolytic fungi Lepista nuda are valuable microorganisms for the synthesis of anisaldehyde, vanillin, veratraldehyde, piperonal and 3,4,5-trimethoxybenzaldehyde from the corresponding acids. According to our findings, we propose a reliable process for the preparation of the above-mentioned aldehydes, in natural form. KEY POINTS: • Fragrant benzaldehydes were obtained by biotransformation. • Basidiomycota strains reduced substituted benzoic acid to the corresponding aldehydes. • Anisaldehyde, vanillin, veratraldehyde, piperonal and 3,4,5-trimethoxybenzaldehyde were prepared in natural form.

First report of silverleaf caused by Chondrostereum purpureum in hazelnut (Corylus avellana) in Chile.

Hazelnut is among the most important nut crops in Chile, currently covering 46,000 ha. In 2023, the country exported 30,000-ton. In recent years the incidence of plants with internal discoloration, cankers and dieback has been increasing. In some cases, the trees died and had to be removed and, after a year, purple resupinate fruiting bodies were observed growing from the stumps. To determine the etiology of the symptoms and signs, wood samples (n=318) were collected since 2020, from 38 symptomatic orchards from Maule to La Araucanía Regions, primarily from the cvs. Tonda di Giffoni and Lewis. Wood sections 0.5 cm diameter were cut from the symptomatic tissues, disinfected using a sodium hypochlorite (10%) solution, and plated on a quarter-strength acidified potato dextrose agar (aPDA1/4). The plates were incubated and purified on PDA. Subsequently, isolates were identified by morphological and molecular means. Almost half of the isolates (47%) were preliminarily identified as basidiomycetes, based on mycelial features such as the presence of clamp connections, with 45% of them exhibiting abundant whitish cottony fast-growth mycelia, resembling Chondrostereum purpureum (Grinbergs et al., 2020). DNA was extracted and the 500-bp fragment, located between 5S and 18S ribosomal regions, was amplified using APN1 specific primers (Becker et al. 1999), identifying the isolates as C. purpureum. In addition, 5.8S gene of RGM1 (35°13'40.9"S 71°25'14.1"W), RGM2 (36°31'27.95"S 71°46'58.31"W), RGM3 (37°10'54.8"S 72°03'39.6"W), RGM4 (35°19'25.2"S 71°19'54.7"W) and RGM5 (36°35'30.8"S 72°05'18.8"W) isolates, representing different locations within the hazelnut growing area, was amplified using ITS1/ITS4 primers (White et al., 1990). The PCR product was sequenced, and the analysis showed 100% homology among isolates (Genebank codes: PP839283, PP839284, PP839285, PP839286 and PP839287, respectively). To determine the pathogenicity of the isolates, 30-cm healthy cuttings cv. Lewis were inoculated with mycelial plugs, while control shoots were inoculated with sterile agar plugs. Cuttings were vertically arranged in pots with 3-cm water and incubated for 60-d at 22°C. In addition, fresh cuts of 3-y potted plants cv. Lewis were inoculated with mycelial plugs and incubated for 137-d in a shadehouse. After incubation, bark was removed from inoculated cuttings and the length of necrotic lesions was measured. Although discoloration was reproduced by all the isolates in both pathogenicity tests, RGM1 isolate was the most aggressive, causing the complete discoloration of the cuttings and the death of the inoculated plants. To our knowledge this is the first report of C. purpureum causing wood disease in hazelnut. These findings are significant because the disease may not only reduce orchard longevity but also decrease fruit yield and quality, as observed in other fruit crops (Grinbergs et al., 2021).

Discovery and characterisation of a broderol-like illudin, omphaderol in the mycelial extracts of Omphalotus mexicanus (Omphalotaceae) using UPLC-QTOF-MS and NMR spectroscopy.

INTRODUCTION: The genus Omphalotus, in particular the "Jack-O'Lantern mushrooms" Omphalotus illudens and Omphalotus olearius, are famous for the production of the DNA-alkylating illudins. A lesser-known species, Omphalotus mexicanus, native to Central America, also produces cytotoxic illudins S and M, but its minor secondary metabolites are yet to be investigated. OBJECTIVE: To identify, isolate, and elucidate the structure of novel secondary metabolites of the illudin family in mycelial extracts of O. mexicanus from submerse cultivation. METHODOLOGY: A fermentation of the fungus in 15 L stirred tank bioreactors is described. Mycelial extracts were separated using a combination of flash chromatography with preparative RP-C18 high-performance liquid chromatography (HPLC). Analysis of metabolites was done using an ultrahigh-performance liquid chromatography ultraviolet diode array detector (UPLC-UV-DAD) system coupled to an electrospray ionisation quadrupole time-of-flight (ESI-QTOF) mass spectrometer. Structures were elucidated using one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance spectroscopy (NMR) techniques followed by comparison of experimental and simulated electronic circular dichroism (ECD) spectra to determine absolute configurations. RESULTS: Two novel illudin derivatives, for which we propose the names omphaderol (1) and illudaneol B (2), as well as illudaneol (3) and the unusual cyclobutylcyclopentane illudosin (4), were isolated from the mycelia and characterised. CONCLUSION: Particularly the illudaneol derivatives with their high titers may be potential building blocks for an alternative semisynthetic route to new illudin derivatives with improved medical properties. Additionally, the findings improve the knowledge of minor illudin compounds in the mycelial extract of this fungus and may be of significance for future biosynthetic studies of the illudins.

Basidiomycetes Polysaccharides Regulate Growth and Antioxidant Defense System in Wheat.

Higher-fungi xylotrophic basidiomycetes are known to be the reservoirs of bioactive metabolites. Currently, a great deal of attention has been paid to the exploitation of mycelial fungi products as an innovative alternative in crop protection. No data exist on the mechanisms behind the interaction between xylotrophic mushrooms' glycopolymeric substances and plants. In this study, the effects of basidiomycete metabolites on the morphophysiological and biochemical variables of wheat plants have been explored. Wheat (Triticum aestivum L. cv. Saratovskaya 29) seedlings were treated with extracellular polysaccharides (EPSs) isolated from the submerged cultures of twenty basidiomycete strains assigned to 13 species and 8 genera. The EPS solutions at final concentrations of 15, 40, and 80 mg/L were applied to wheat seedlings followed by their growth for 10 days. In the plant samples, the biomass, length of coleoptile, shoot and root, root number, rate of lipid peroxidation by malondialdehyde concentration, content of hydrogen peroxide, and total phenols were measured. The peroxidase and superoxide dismutase activity were defined. Most of the EPS preparations improved biomass yields, as well as the morphological parameters examined. EPS application enhanced the activities of antioxidant enzymes and decreased oxidative damage to lipids. Judging by its overall effect on the growth indices and redox system of wheat plants, an EPS concentration of 40 mg/L has been shown to be the most beneficial compared to other concentrations. This study proves that novel bioformulations based on mushroom EPSs can be developed and are effective for wheat growth and antioxidative response. Phytostimulating properties found for EPSs give grounds to consider extracellular metabolites produced in the xylotrophic basidiomycete cultures as an active component capable of inducing plant responses to stress.

Nonribosomal peptide synthetase gene clusters and characteristics of predicted NRPS-dependent siderophore synthetases in Armillaria and other species in the Physalacriaceae.

Fungal secondary metabolites are often pathogenicity or virulence factors synthesized by genes contained in secondary metabolite gene clusters (SMGCs). Nonribosomal polypeptide synthetase (NRPS) clusters are SMGCs which produce peptides such as siderophores, the high affinity ferric iron chelating compounds required for iron uptake under aerobic conditions. Armillaria spp. are mostly facultative necrotrophs of woody plants. NRPS-dependent siderophore synthetase (NDSS) clusters of Armillaria spp. and selected Physalacriaceae were investigated using a comparative genomics approach. Siderophore biosynthesis by strains of selected Armillaria spp. was evaluated using CAS and split-CAS assays. At least one NRPS cluster and other clusters were detected in the genomes studied. No correlation was observed between the number and types of SMGCs and reported pathogenicity of the species studied. The genomes contained one NDSS cluster each. All NDSSs were multi-modular with the domain architecture (ATC)3(TC)2. NDSS clusters of the Armillaria spp. showed a high degree of microsynteny. In the genomes of Desarmillaria spp. and Guyanagaster necrorhizus, NDSS clusters were more syntenic with NDSS clusters of Armillaria spp. than to those of the other Physalacriaceae species studied. Three A-domain orthologous groups were identified in the NDSSs, and atypical Stachelhaus codes were predicted for the A3 orthologous group. In vitro biosynthesis of mainly hydroxamate and some catecholate siderophores was observed. Hence, Armillaria spp. generally contain one highly conserved, NDSS cluster although some interspecific variations in the products of these clusters is expected. Results from this study lays the groundwork for future studies to elucidate the molecular biology of fungal phyto-pathogenicity.

Genomic and Secretomic Analyses of the Newly Isolated Fungus Perenniporia fraxinea SS3 Identified CAZymes Potentially Related to a Serious Pathogenesis of Hardwood Trees.

Perenniporia fraxinea can colonize living trees and cause severe damage to standing hardwoods by secreting a number of carbohydrate-activate enzymes (CAZymes), unlike other well-studied Polyporales. However, significant knowledge gaps exist in understanding the detailed mechanisms for this hardwood-pathogenic fungus. To address this issue, five monokaryotic P. fraxinea strains, SS1 to SS5, were isolated from the tree species Robinia pseudoacacia, and high polysaccharide-degrading activities and the fastest growth were found for P. fraxinea SS3 among the isolates. The whole genome of P. fraxinea SS3 was sequenced, and its unique CAZyme potential for tree pathogenicity was determined in comparison to the genomes of other nonpathogenic Polyporales. These CAZyme features are well conserved in a distantly related tree pathogen, Heterobasidion annosum. Furthermore, the carbon source-dependent CAZyme secretions of P. fraxinea SS3 and a nonpathogenic and strong white-rot Polyporales member, Phanerochaete chrysosporium RP78, were compared by activity measurements and proteomic analyses. As seen in the genome comparisons, P. fraxinea SS3 exhibited higher pectin-degrading activities and higher laccase activities than P. chrysosporium RP78, which were attributed to the secretion of abundant glycoside hydrolase family 28 (GH28) pectinases and auxiliary activity family 1_1 (AA1_1) laccases, respectively. These enzymes are possibly related to fungal invasion into the tree lumens and the detoxification of tree defense substances. Additionally, P. fraxinea SS3 showed secondary cell wall degradation capabilities at the same level as that of P. chrysosporium RP78. Overall, this study suggested mechanisms for how this fungus can attack the cell walls of living trees as a serious pathogen and differs from other nonpathogenic white-rot fungi. IMPORTANCE Many studies have been done to understand the mechanisms underlying the degradation of plant cell walls of dead trees by wood decay fungi. However, little is known about how some of these fungi weaken living trees as pathogens. P. fraxinea belongs to the Polyporales, a group of strong wood decayers, and is known to aggressively attack and fell standing hardwood trees all over the world. Here, we report CAZymes potentially related to plant cell wall degradation and pathogenesis factors in a newly isolated fungus, P. fraxinea SS3, by genome sequencing in conjunction with comparative genomic and secretomic analyses. The present study provides insights into the mechanisms of the degradation of standing hardwood trees by the tree pathogen, which will contribute to the prevention of this serious tree disease.

High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae.

BACKGROUND: Orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid, OA) and its structural analog o-Orsellinaldehyde, have become widely used intermediates in clinical drugs synthesis. Although the research on the biosynthesis of such compounds has made significant progress, due to the lack of suitable hosts, there is still far from the industrial production of such compounds based on synthetic biology. RESULTS: With the help of genome mining, we found a polyketide synthase (PKS, HerA) in the genome of the Hericium erinaceus, which shares 60% amino acid sequence homology with ArmB from Armillaria mellea, an identified PKS capable of synthesizing OA. To characterize the function of HerA, we cloned herA and heterologously expressed it in Aspergillus oryzae, and successfully detected the production of OA. Subsequently, the introduction of an incomplete PKS (Pks5) from Ustilago maydis containing only three domains (AMP-ACP-R), which was into herA-containing A. oryzae, the resulted in the production of o-Orsellinaldehyde. Considering the economic value of OA and o-Orsellinaldehyde, we then optimized the yield of these compounds in A. oryzae. The screening showed that when maltose was used as carbon source, the yields of OA and o-Orsellinaldehyde were 57.68 mg/L and 15.71 mg/L respectively, while the yields were 340.41 mg/Kg and 84.79 mg/Kg respectively in rice medium for 10 days. CONCLUSIONS: Herein, we successfully expressed the genes of basidiomycetes using A. oryzae heterologous host. As a fungus of ascomycetes, which not only correctly splices genes of basidiomycetes containing multiple introns, but also efficiently produces their metabolites. This study highlights that A. oryzae is an excellent host for the heterologous production of fungal natural products, and has the potential to become an efficient chassis for the production of basidiomycete secondary metabolites in synthetic biology.

BASIDIN as a New Protein Effector of the Phytopathogen Causing Witche's Broom Disease in Cocoa.

The fungus Moniliophthora perniciosa secretes protein effectors that manipulate the physiology of the host plant, but few effectors of this fungus have had their functions confirmed. We performed functional characterization of a promising candidate effector of M. perniciosa. The inoculation of rBASIDIN at 4 µmol L-1 in the mesophyll of leaflets of Solanum lycopersicum caused symptoms of shriveling within 6 h without the presence of necrosis. However, when sprayed on the plant at a concentration of 11 µmol L-1, it caused wilting symptoms only 2 h after application, followed by necrosis and cell death at 48 h. rBASIDIN applied to Theobroma cacao leaves at the same concentration caused milder symptoms. rBASIDIN caused hydrogen peroxide production in leaf tissue, damaging the leaf membrane and negatively affecting the photosynthetic rate of Solanum lycopersicum plants. Phylogenetic analysis indicated that BASIDIN has orthologs in other phytopathogenic basidiomycetes. Analysis of the transcripts revealed that BASIDIN and its orthologs are expressed in different fungal species, suggesting that this protein is differentially regulated in these basidiomycetes. Therefore, the results of applying BASIDIN allow the inference that it is an effector of the fungus M. perniciosa, with a strong potential to interfere in the defense system of the host plant.

Activity of aurisin A isolated from Neonothopanus nambi against methicillin-resistant Staphylococcus aureus strains.

Mycopharmaceuticals from basidiomycetes represent a promising source of new antimicrobials to overcome the challenges of multidrug-resistant bacteria. Here we report for the first time the in vitro activity of aurisin A, a dimeric sesquiterpenoid isolated from wild bioluminescent basidiomycetes Neonothopanus nambi DSM 24013, against methicillin-resistant Staphylococcus aureus (MRSA). Aurisin A revealed strong anti-MRSA activity with minimum inhibitory concentration 7.81 µg/mL against ATCC 33591 and ATCC 43300 reference strains, and BD 16876 and BD 15358 clinical strains. Activity against the clinical strains is 10- to 40-fold higher than that of the antibiotic fusidic acid. Furthermore, aurisin A proved to be more potent (MIC 3.91 µg/mL) in inhibiting growth of vancomycin-intermediate S. aureus (VISA) ATCC 700699 and displayed a rapid time-dependent bactericidal activity against MRSA (complete killing within 1 h). Additionally, aurisin A and oxacillin combination displayed synergy with notable decrease in the MICs of both compounds against MRSA. Notable synergism was also observed in combinations with linezolid and fusidic acid. Our findings indicate that aurisin A is a promising candidate for developing therapeutic agents against multidrug-resistant S. aureus and warrants further investigation.

Structure Analysis of the MatA Locus of Sexual Compatibility in the Edible Mushroom Pleurotus ostreatus.

The edible oyster mushroom Pleurotus ostreatus is one of the most cultivated species worldwide. Morphogenesis associated with the maturation of fruit bodies is controlled by two unlinked loci of sexual compatibility matA and matB with multiple alleles (tetrapolar system of sexual compatibility). Quantitative analysis of the alleles of mating compatibility loci in 17 natural isolates collected in the Moscow region was performed in mon-mon (monokaryons-monokaryon) and di-mon (dikaryon-monokaryon) crossings. Four monokaryotic testers strains which were heteroallelic at both mating type loci were obtained for each of the five natural mushroom isolates by using original technique of sterile spore prints on Petri dishes and mon-mon crossing. Twelve natural isolates were crossed via di-mon mating with the four monokaryotic testers M-38. Genetic analysis of the alleles of sexual compatibility loci in 17 natural isolates revealed multiple alleles at both loci: at least ten alleles at matA locus and eight alleles at matB locus. Structural organization analysis of the matA locus was performed in silico for homokaryotic strains PC9 and PC15 based on the whole-genome sequencing data available at DOE Joint Genome Institute. The matA locus has an extremely divergent structure: there are one copy of the homeodomain gene hd1 and one copy of the hd2 gene in the PC9 strain, whereas the matA locus of the PC15 strain is composed by two copies of hd1.1 and hd1.2 genes (class HD1 homeodomain proteins) and one copy of hd2 gene (class HD2 proteins). Comprehensive analysis of amino acid sequences of HD1 and HD2 homeodomain proteins demonstrated that the proteins have a globular structure with the nuclear localization and contain a variable N-terminus and a more conserved DNA-binding domain with a specific conserved motif  WFXNXR in the third ɑ-helix. The results suggest that multiple alleles of the matA locus of sexual compatibility in basidiomycete fungi is achieved due to both different copy number of the coding hd genes within the locus and the variability of the coding gene sequences.

Rigidoporus corticola Colonization and Invasive Fungal Disease in Immunocompromised Patients, United States.

We report 2 cases of Rigidoporus corticola (Oxyporus corticola) infection in humans in the United States. Clinical manifestations consisted of angioinvasive fungal sinusitis in 1 patient and pulmonary intracavitary fungus ball in the other patient. These cases illustrate previously undescribed clinicopathologic manifestations of infection by this filamentous basidiomycete in humans.

An endophytic Schizophyllum commune possessing antioxidant activity exhibits genoprotective and organprotective effects in fresh water fish Channa punctatus exposed to bisphenol A.

BACKGROUND: Oxidative stress is responsible for the onset of several chronic and degenerative diseases. Exogenous supply of antioxidants is reported to neutralize the effects of oxidative stress. Several synthetic antioxidants suffer from various side effects which necessitates the exploration of antioxidant compounds from natural sources. Endophytic fungi residing in the plants are gaining the attention of researchers as a source of novel antioxidants. Majority of the research conducted so far on endophytic fungi has been restricted to the members of phylum ascomycota. Basidiomycota, inspite of their immense bioactive potential remain relatively unexploited. This study aimed to assess the ameliorative effects of an endophytic Schizophyllum commune (basidiomycetous fungus) against oxidative stress associated altered antioxidant levels, genotoxicity and cellular damage to different organs in bisphenol A exposed fresh water fish Channa punctatus. RESULTS: Good antioxidant and genoprotective potential was exhibited by S. commune extract in in vitro studies conducted using different antioxidant, DNA damage protection, and cytokinesis blocked micronuclei assays. In vivo studies were performed in fresh water fish Channa punctatus exposed to bisphenol A. A significant decrease in the considered parameters for DNA damage (% micronuclei and comet assay) were recorded in fish treated with S. commune extract on comparison with untreated bisphenol A exposed group. The S. commune extract treated fish also exhibited an increase in the level of antioxidant enzymes viz. catalase, superoxide dismutase and glutathione reductase as well as histoprotective effect on various organs. GC-MS analysis revealed the presence of 3-n-propyl-2,4-pentanedione, n-heptadecanol-1, trans-geranylgeraniol, 3-ethyl-2-pentadecanone, 1-heneicosanol and squalene as some of the compounds in S. commune extract. CONCLUSION: The study highlights the significance of an endophytic basidiomycetous fungus S. commune as a source of antioxidant compounds with possible therapeutic potential.

Onset and stepwise extensions of recombination suppression are common in mating-type chromosomes of Microbotryum anther-smut fungi.

Sex chromosomes and mating-type chromosomes can display large genomic regions without recombination. Recombination suppression often extended stepwise with time away from the sex- or mating-type-determining genes, generating evolutionary strata of differentiation between alternative sex or mating-type chromosomes. In anther-smut fungi of the Microbotryum genus, recombination suppression evolved repeatedly, linking the two mating-type loci and extended multiple times in regions distal to the mating-type genes. Here, we obtained high-quality genome assemblies of alternative mating types for four Microbotryum fungi. We found an additional event of independent chromosomal rearrangements bringing the two mating-type loci on the same chromosome followed by recombination suppression linking them. We also found, in a new clade analysed here, that recombination suppression between the two mating-type loci occurred in several steps, with first an ancestral recombination suppression between one of the mating-type locus and its centromere; later, completion of recombination suppression up to the second mating-type locus occurred independently in three species. The estimated dates of recombination suppression between the mating-type loci ranged from 0.15 to 3.58 million years ago. In total, this makes at least nine independent events of linkage between the mating-type loci across the Microbotryum genus. Several mating-type locus linkage events occurred through the same types of chromosomal rearrangements, where similar chromosome fissions at centromeres represent convergence in the genomic changes leading to the phenotypic convergence. These findings further highlight Microbotryum fungi as excellent models to study the evolution of recombination suppression.

The earth-star basidiomycetous mushroom Astraeus odoratus produces polyhydroxyalkanoates during cultivation on malt extract.

Polyhydroxyalkanoates (PHAs) including poly-3-hydroxybutyrate (P3HB) as secondary metabolisms were in vitro produced by the edible basidiomycetous mushroom Astraeus odoratus during its growth on malt agar extract. Various carbon and nitrogen sources containing cellulose, glucose, glycerol, rice straw powder, soybean meal and peptone were investigated for the growth of basidiomycetous mushrooms. During cultivation, the A. odoratus culture exudated the considerably extracellular fluid up to approx. 2.3 ml on 2% malt extract agar plate within 7 days. The chemical compounds of the exudated fluid were further investigated by Fourier-transform infrared spectroscopy (FTIR) and gas chromatography/mass spectrometry (GC/MS); and its morphology of the lyophilized sample was observed by scanning electron microscope (SEM). FTIR results showed the characteristic bands of OH at 3445 cm-1, CH/CH2/symmetric CH3 (stretch) at 2923 and 2852 cm-1, C=O at 1730 cm-1, asymmetric CH3 (bend) at 1454 and 1414 cm-1, C-O of COO- at 1396 cm-1 and C-O-C at 1223, 1160, 1116, 1058 and 1019 cm-1 which were similar to the absorptive characteristics of P3HB. Methyl ester derivatives of GC/MS results identified 7 compounds including: 3-hydroxybutanoic (monomer of PHB), aminobenzoic, salicylic, hexadecenoic, octadecadienoic, octadecenoic and octadecanoic acids. SEM images revealed a fibriform and porous materials. Hence, the occurrence of PHAs was first described in a basidiomycetous mushroom A. odoratus. Thus, PHAs could be found not only in bacteria and but also in basidiomycetous mushroom, which can be promising target for bioplastics and green environmental studies.

Genomic and proteomic analysis of Tausonia pullulans reveals a key role for a GH15 glucoamylase in starch hydrolysis.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

A convenient separation strategy for fungal anthraquinones by centrifugal partition chromatography.

As recently shown, some fungal pigments exhibit significant photoactivity turning them into promising agents for the photodynamic treatment of microbial infections or malignant diseases. In the present study, a separation strategy for fungal anthraquinones was developed based on centrifugal partition chromatography. A suitable method was explored employing a methanolic extract of the fruiting bodies of Cortinarius sanguineus (Agaricales, Basidiomycota). An excellent fractionation was achieved using a biphasic solvent system comprising chloroform/ethyl acetate/methanol/water/acetic acid (3:1:3:2:1, v/v/v/v/v) operating in ascending mode. Experiments on an analytical scale with extracts of closely related Cortinarius species exhibited broad applicability of the devised system. Up to six pigments could be purified directly from the crude extract. Preparative-scale fractionation of the methanol extracts of C. malicorius and C. sanguineus demonstrated that up-scaling was possible without compromising selectivity.

A Robust Fermentation Process for Natural Chocolate-like Flavor Production with Mycetinis scorodonius.

Submerged fermentation of green tea with the basidiomycete Mycetinis scorodonius resulted in a pleasant chocolate-like and malty aroma, which could be a promising chocolate flavor alternative to current synthetic aroma mixtures in demand of consumer preferences towards healthy natural and 'clean label' ingredients. To understand the sensorial molecular base on the chocolate-like aroma formation, key aroma compounds of the fermented green tea were elucidated using a direct immersion stir bar sorptive extraction combined with gas chromatography-mass spectrometry-olfactometry (DI-SBSE-GC-MS-O) followed by semi-quantification with internal standard. Fifteen key aroma compounds were determined, the most important of which were dihydroactinidiolide (odor activity value OAV 345), isovaleraldehyde (OAV 79), and coumarin (OAV 24), which were also confirmed by a recombination study. Furthermore, effects of the fermentation parameters (medium volume, light protection, agitation rate, pH, temperature, and aeration) on the aroma profile were investigated in a lab-scale bioreactor at batch fermentation. Variation of the fermentation parameters resulted in similar sensory perception of the broth, where up-scaling in volume evoked longer growth cycles and aeration significantly boosted the concentrations yet added a green note to the overall flavor impression. All findings prove the robustness of the established fermentation process with M. scorodonius for natural chocolate-like flavor production.