We report an annotated draft genome of Heterobasidion occidentale, a fungus (Basidiomycota, Agaricomycetes) that has pathogenic and saprophytic lifestyles. This fungus belongs to the H. annosum (Fr.) Bref. sensu lato species complex that comprises several root rot pathogens. Heterobasidion occidentale causes annosus root and butt rot primarily in true fir (Abies spp.) and spruce (Picea spp.) species throughout western North America.

Laccase isoforms from basidiomycetes exhibit a superior redox potential compared to commercially available laccases obtained from ascomycete fungi, rendering them more reactive toward mono-substituted phenols and polyphenolic compounds. However, basidiomycetes present limitations for large-scale culture in liquid media, restraining the current availability of laccases from this fungal class. To advance laccase production from basidiomycetes, a newly designed 14-L low-shear aerated and agitated bioreactor provided enzyme titers up to 23.5 IU/mL from Trametes versicolor cultures. Produced enzymes underwent ultrafiltration and LC/MS-MS characterization, revealing the predominant production of only two out of the ten laccases predicted in the T. versicolor genome. Process simulation and economic analysis using SuperPro designer® suggested that T. versicolor laccase could be produced at US$ 3.60/kIU in a 200-L/batch enterprise with attractive economic parameters and a payback period of 1.7 years. The study indicates that new bioreactors with plain design help to produce low-cost enzymes from basidiomycetes.

A sporeless strain is an important breeding target in the mushroom industry. However, basidiospore production in the oyster mushroom Pleurotus ostreatus has been shown to be impaired by single-gene mutations in only two meiosis-related genes, mer3 and msh4. This study proposed a strategy for identifying the genes essential for basidiospore formation after meiotic division to determine new targets for molecular breeding. RNA-seq analysis was performed to identify P. ostreatus genes that are specifically expressed in the gill tissue of fruiting bodies, where basidiospore formation occurs. Transcriptome data during fruiting development of Coprinopsis cinerea, in which the meiotic steps progress synchronously, were then used to identify genes that are active in the postmeiotic stages. Based on these comparative analyses, five P. ostreatus genes were identified. Plasmids containing expression cassettes for hygromycin B-resistance screening, Cas9, and single-guide RNA targeting each gene were introduced into the protoplasts of dikaryotic strain, PC9×#64, to generate dikaryotic gene disruptants. Among the obtained transformants, three dikaryotic pcl1 disruptants and two cro6c disruptants did not produce basidiospores. Microscopic analyses indicated that spore formation was arrested at particular stages in these gene disruptants. These results indicate that these two genes are essential for mature spore formation in this fungus.

Hormographiella aspergillata is a basidiomycete exceptionally involved in invasive fungal infections (IFI). We report a case of H. aspergillata pulmonary infection in a 30-year-old female in a context of pancytopenia and relapsed of acute myeloid leukemia (AML). She presented with fever, thoracic pain, left pleural effusion and pneumonia, diagnosed on chest X-ray and CT-scan. Direct examination of a bronchoalveolar lavage (BAL) specimen performed on day (d) 10 was negative, while the culture was positive on d30. H. aspergillata was suspected, considering macroscopic and microscopic examination. Its identification was confirmed using Microflex® Bruker mass spectrometry and pan-fungal (PF)-PCR assay followed by DNA sequencing. After this initial diagnosis, the patient was monitored for 2.8 years. She was treated with liposomal amphotericin B and/or voriconazole until switching to isavuconazole on d298 due to side-effects. This antifungal treatment was maintained until d717 and then discontinued, the patient being considered as cured. Over this follow-up period, the patient was submitted to recurrent pulmonary sampling. Each time, cultures were negative, while PF - PCR assays and DNA sequencing confirmed the presence of H. aspergillata. The present case-report is the 32nd observation of H. aspergillata invasive infection showing that this IFI is still infrequent. Fifteen have occurred in patients with AML, which appears as the most frequent underlying disease favoring this IFI. Six recent case-reports in addition to ours highlight PF-PCR assays and DNA sequencing as relevant diagnostic tools that must be included in routine diagnosis and monitoring of IFI, specifically those due to rare basidiomycetes.

BACKGROUND: Ganoderma boninense is a phytopathogen of oil palm, causing basal and upper stem rot diseases.
METHODS: The genome sequence was used as a reference to study gene expression during growth in a starved carbon (C) and nitrogen (N) environment with minimal sugar and sawdust as initial energy sources. This study was conducted to mimic possible limitations of the C-N nutrient sources during the growth of G. boninense in oil palm plantations.
RESULTS: Genome sequencing of an isolate collected from a palm tree in West Malaysia generated an assembly of 67.12 Mb encoding 19,851 predicted genes. Transcriptomic analysis from a time course experiment during growth in this starvation media identified differentially expressed genes (DEGs) that were found to be associated with 29 metabolic pathways. During the active growth phase, 26 DEGs were related to four pathways, including secondary metabolite biosynthesis, carbohydrate metabolism, glycan metabolism and mycotoxin biosynthesis. G. boninense genes involved in the carbohydrate metabolism pathway that contribute to the degradation of plant cell walls were up-regulated. Interestingly, several genes associated with the mycotoxin biosynthesis pathway were identified as playing a possible role in pathogen-host interaction. In addition, metabolomics analysis revealed six metabolites, maltose, xylobiose, glucooligosaccharide, glycylproline, dimethylfumaric acid and arabitol that were up-regulated on Day2 of the time course experiment.
CONCLUSIONS: This study provides information on genes expressed by G. boninense in metabolic pathways that may play a role in the initial infection of the host.

A comprehensive analysis to survey heme-binding proteins produced by the white-rot fungus Phanerochaete chrysosporium was achieved using a biotinylated heme-streptavidin beads system. Mitochondrial citrate synthase (PcCS), glyceraldehyde 3-phosphate dehydrogenase (PcGAPDH), and 2-Cys thioredoxin peroxidase (mammalian HBP23 homolog) were identified as putative heme-binding proteins. Among these, PcCS and PcGAPDH were further characterized using heterologously expressed recombinant proteins. Difference spectra of PcCS titrated with hemin exhibited an increase in the Soret absorbance at 414 nm, suggesting that the axial ligand of the heme is a His residue. The activity of PcCS was strongly inhibited by hemin with Ki oxaloacetate of 8.7 μM and Ki acetyl-CoA of 5.8 μM. Since the final step of heme biosynthesis occurred at the mitochondrial inner membrane, the inhibition of PcCS by heme is thought to be a physiological event. The inhibitory mode of the heme was similar to that of CoA analogues, suggesting that heme binds to PcCS at His347 at the AcCoA-CoA binding site, which was supported by the homology model of PcCS. PcGAPDH was also inhibited by heme, with a lower concentration than that for PcCS. This might be caused by the different location of these enzymes. From the integration of these phenomena, it was concluded that metabolic regulations by heme in the central metabolic and heme synthetic pathways occurred in the mitochondria and cytosol. This novel pathway crosstalk between the central metabolic and heme biosynthetic pathways, via a heme molecule, is important in regulating the metabolic balance (heme synthesis, ATP synthesis, flux balance of the tricarboxylic acid (TCA) cycle and cellular redox balance (NADPH production) during fungal aromatic degradation. KEY POINTS: • A comprehensive survey of heme-binding proteins in P. chrysosporium was achieved. • Several heme-binding proteins including CS and GAPDH were identified. • A novel metabolic regulation by heme in the central metabolic pathways was found.

Sesquiterpenes are a type of abundant natural product with widespread applications in several industries. They are biosynthesized by sesquiterpene synthases (STSs). As valuable and abundant biological resources, mushroom-forming fungi are rich in new sesquiterpenes and STSs, which remain largely unexploited. In the present study, we collected information on 172 STSs from mushroom-forming fungi with experimentally characterized products from the literature and sorted them to develop a dataset. Furthermore, we analyzed and discussed the phylogenetic tree, catalytic products, and conserved motifs of STSs. Phylogenetic analysis revealed that the STSs were clustered into four clades. Furthermore, their cyclization reaction mechanism was divided into four corresponding categories. This database was used to predict 12 putative STS genes from the edible fungi Flammulina velutipes. Finally, three FvSTSs were selected to experimentally characterize their functions. FvSTS03 predominantly produced Δ-cadinol and FvSTS08 synthesized β-barbatene as the main product; these findings were consistent with those of the functional prediction analysis. A product titer of 78.8 mg/L β-barbatene was achieved in Saccharomyces cerevisiae via metabolic engineering. Our study findings will help screen or design STSs from fungi with specific product profiles as functional elements for applications in synthetic biology.

Interrogation of an EST database for Clitopilus passeckerianus identified a putative homolog to the unusual stress response gene from yeast; ddr48, as being upregulated under pleuromutilin production conditions. Silencing of this gene, named cprp, produced a population of transformants which demonstrated significantly reduced pleuromutilin production. Attempts to complement a Saccharomyces cerevisiae ddr48 mutant strain (strain Y16748) with cprp were hampered by the lack of a clearly identifiable mutant phenotype, but interestingly, overexpression of either ddr48 or cprp in S. cerevisiae Y16748 led to a conspicuous and comparable reduction in growth rate. This observation, combined with the known role of DDR48 proteins from a range of fungal species in nutrient starvation and stress responses, raises the possibility that this family of proteins plays a role in triggering oligotrophic growth. Localization studies via the production of a Cprp:GFP fusion protein in C. passeckerianus showed clear localization adjacent to the hyphal septa and, to a lesser extent, cell walls, which is consistent with the identification of DDR48 as a cell wall-associated protein in various yeast species. To our knowledge this is the first study demonstrating that a DDR48-like protein plays a role in the regulation of a secondary metabolite, and represents the first DDR48-like protein from a basidiomycete. Potential homologs can be identified across much of the Dikarya, suggesting that this unusual protein may play a central role in regulating both primary and secondary metabolism in fungi.

Laccase is a superfamily of ligninolytic enzymes known to degrade a wide variety of xenobiotics, including synthetic dyes. Congo Red (CR) has a diazo dye function, carcinogenic and mutagenic potential, and is currently applied in clinical analysis. The objective of this work was to produce and characterize the crude extract of Lentinus sp. in semi-solid fermentation (FSS) and perform in vitro and in silico studies to assess the potential of the crude extract to discolor the CR dye. Laccase activity was determined using ABTS as substrate and characterized. The in vitro discoloration was carried out using experimental design 22 at room temperature and monitored at 340 nm for 24h. Molecular docking and molecular dynamics simulations were performed between laccase and CR. The maximum laccase activity production was 29.63 U L-1 with six days of FSS. The optimal temperature and pH were 50 °C and 3.0, respectively. Discoloration of the CR dye was obtained only in tests containing CuSO4. Laccase formed stable complexes with the dye, presenting negative binding energy values ranging from -70.94 to -63.16 kcal mol-1 and the occurrence of seven hydrogen bonds. Molecular dynamics results showed the stability of the system (RMSD ranging from 1.0 to 2.5 Ä) and protein-ligand interaction along simulation. RMSF values pointed residues at the end of chains A (residues 300 to 305, 480 to 500) and B (residues 650 to 655 and 950 to 1000) as the most flexible regions of the laccase. This study highlighted the enzymatic action in the bioremediation of CR in vitro in agreement with the in silico simulations that demonstrate the enzyme potential.Communicated by Ramaswamy H. Sarma.

BACKGROUND: The termite-fungus symbiosis is an ancient stable mutualism of two partners that reproduce and disperse independently. With the founding of each termite colony the symbiotic association must be re-established with a new fungus partner. Complementarity in the ability to break down plant substrate may help to stabilize this symbiosis despite horizontal symbiont transmission. An alternative, non-exclusive, hypothesis is that a reduced rate of evolution may contribute to stabilize the symbiosis, the so-called Red King Effect.
METHODS: To explore this concept, we produced the first linkage map of a species of Termitomyces, using genotyping by sequencing (GBS) of 88 homokaryotic offspring. We constructed a highly contiguous genome assembly using PacBio data and a de-novo evidence-based annotation. This improved genome assembly and linkage map allowed for examination of the recombination landscape and its potential effect on the mutualistic lifestyle.
RESULTS: Our linkage map resulted in a genome-wide recombination rate of 22 cM/Mb, lower than that of other related fungi. However, the total map length of 1370 cM was similar to that of other related fungi.
CONCLUSIONS: The apparently decreased rate of recombination is primarily due to genome expansion of islands of gene-poor repetitive sequences. This study highlights the importance of inclusion of genomic context in cross-species comparisons of recombination rate.