Recombinant Oxidase from Armillaria tabescens as a Potential Tool for Aflatoxin B1 Degradation in Contaminated Cereal Grain.

Forage grain contamination with aflatoxin B1 (AFB1) is a global problem, so its detoxification with the aim of providing feed safety and cost-efficiency is still a relevant issue. AFB1 degradation by microbial enzymes is considered to be a promising detoxification approach. In this study, we modified an previously developed Pichia pastoris GS115 expression system using a chimeric signal peptide to obtain a new recombinant producer of extracellular AFB1 oxidase (AFO) from Armillaria tabescens (the yield of 0.3 g/L), purified AFO, and selected optimal conditions for AFO-induced AFB1 removal from model solutions. After a 72 h exposure of the AFB1 solution to AFO at pH 6.0 and 30 °C, 80% of the AFB1 was degraded. Treatments with AFO also significantly reduced the AFB1 content in wheat and corn grain inoculated with Aspergillus flavus. In grain samples contaminated with several dozen micrograms of AFB1 per kg, a 48 h exposure to AFO resulted in at least double the reduction in grain contamination compared to the control, while the same treatment of more significantly (~mg/kg) AFB1-polluted samples reduced their contamination by ~40%. These findings prove the potential of the tested AFO for cereal grain decontamination and suggest that additional studies to stabilize AFO and improve its AFB1-degrading efficacy are required.

Two distinct non-ribosomal peptide synthetase-independent siderophore synthetase gene clusters identified in Armillaria and other species in the Physalacriaceae.

Siderophores are important for ferric iron solubilization, sequestration, transportation, and storage, especially under iron-limiting conditions such as aerobic conditions at high pH. Siderophores are mainly produced by non-ribosomal peptide synthetase-dependent siderophore pathway, non-ribosomal peptide synthetase-independent siderophore synthetase pathway, or the hybrid non-ribosomal peptide synthetases/non-ribosomal peptide synthetases-independent siderophore pathway. Outcompeting or inhibition of plant pathogens, alteration of host defense mechanisms, and alteration of plant-fungal interactions have been associated with fungal siderophores. To understand these mechanisms in fungi, studies have been conducted on siderophore biosynthesis by ascomycetes with limited focus on the basidiomycetes. Armillaria includes several species that are pathogens of woody plants and trees important to agriculture, horticulture, and forestry. The aim of this study was to investigate the presence of non-ribosomal peptide synthetases-independent siderophore synthetase gene cluster(s) in genomes of Armillaria species using a comparative genomics approach. Iron-dependent growth and siderophore biosynthesis in strains of selected Armillaria spp. were also evaluated in vitro. Two distinct non-ribosomal peptide synthetases-independent siderophore synthetase gene clusters were identified in all the genomes. All non-ribosomal peptide synthetases-independent siderophore synthetase genes identified putatively encode Type A' non-ribosomal peptide synthetases-independent siderophore synthetases, most of which have IucA_IucC and FhuF-like transporter domains at their N- and C-terminals, respectively. The effect of iron on culture growth varied among the strains studied. Bioassays using the CAS assay on selected Armillaria spp. revealed in vitro siderophore biosynthesis by all strains irrespective of added FeCl3 concentration. This study highlights some of the tools that Armillaria species allocate to iron homeostasis. The information generated from this study may in future aid in developing molecular based methods to control these phytopathogens.

Honey Mushroom, Armillaria mellea (Agaricomycetes) and Its Fermentation Products Target Regulation of OAT1/OAT3 Proteins to Reduce Hyperuricemia in Mice.

BACKGROUND: Disorders of purine metabolism are the main cause of hyperuricemia. Current drugs for the treatment of hyperuricemia usually cause a degree of cardiovascular damage. METHODS: This study aimed to investigate the therapeutic effects of Armillaria mellea fruiting body (AFB), Armillaria rhizomorph (AR) and Armillaria mellea fermentation product (after rhizomorphs removal) (AFP) on hyperuricemic mice. The hyperuricemia mouse model was established by oral administration of potassium oxonate 0.9 g⋅kg-1 and hypoxanthine 0.5 g⋅kg-1 for two weeks. Starting from the third week, the intragastric administration of the intervention drug group was as follows: Allopurinol 0.013 g⋅kg-1, AFB (3.9 and 7.8 g⋅kg-1), AR (3.9 and 7.8 g⋅kg-1), AFP (1.95 and 3.9 g⋅kg-1) once daily for 14 days. RESULTS: Results showed that AFB, AR, and AFP reduced the contents of serum uric acid, serum creatinine, and blood urea nitrogen in hyperuricemic mice and the mechanism of action might be through up-regulation of the expression levels of organic anion transporter 1/organic anion transporter 3 proteins in kidney tissue. AR and AFP both exhibited better uric acid-lowering effects than AFB, which may be due to the higher purine content of AFB. CONCLUSIONS: Armillaria mellea and its fermentation products can treat hyperuricemia by up-regulating OAT1 protein and OAT3 protein, reducing uric acid content in mice.

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria.

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.

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.

Armillaria root rot fungi host single-stranded RNA viruses.

Species of Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named "ambiviruses" with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae).

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm-isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)-like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria-colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high-affinity sucrose-specific proton-dependent importer. Plasma-membrane/tonoplast localization of GeSUT4-GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant-microbe interactions.

Do the Main Components of the Sclerotia of Umbrella Polypore Mushroom, Polyporus umbellatus (Agaricomycetes), Correlate with Armillaria Associates?

Polyporus umbellatus is a traditional Chinese medicinal mushroom. The growth of P. umbellatus sclerotia requires the rhizomorphs of Armillaria spp. to supply nutrition. Whether the main components (MC) of sclerotia of P. umbellatus are related to the phylogeny of Armillaria associates or other environmental factors is largely unknown. In this study, we collected 17 sclerotia and soil samples from northeast to southwest China. In total, 17 Armillaria associates were isolated, and sclerotial MC contents and soil characteristics (total N, P, K, and organic matter) were determined. The analysis revealed that the MC content of P. umbellatus did not resemble a Brownian motion process in phylogeny of Armillaria associates, but were significantly influenced by the total N content of the soil. These results provide clear evidence that sclerotia of P. umbellatus associating with phylogenetic related Armillaria associates possess differing MC content. The mechanisms of nutrient exchange in P. umbellatus-Armillaria associations now require further elucidation.

Insight to shape of soil microbiome during the ternary cropping system of Gastradia elata.

BACKGROUND: The ternary cropping system of Gastradia elata depends on a symbiotic relationship with the mycorrhizal fungi Armillaria mellea, which decays wood to assimilate nutrition for the growth of G. elata. The composition of microbe flora as key determinants of rhizoshere and mycorrhizoshere soil fertility and health was investigated to understand how G. elata and A. mellea impacted on its composition. The next generation pyrosequencing analysis was applied to assess the shift of structure of microbial community in rhizoshere of G. elata and mycorrhizoshere of A. mellea compared to the control sample under agriculture process. RESULTS: The root-associated microbe floras were significantly impacted by rhizocompartments (including rhizoshere and mycorrhizoshere) and agriculture process. Cropping process of G. elata enhanced the richness and diversity of the microbial community in rhizoshere and mycorrhizoshere soil. Furthermore, planting process of G. elata significantly reduced the abundance of phyla Basidiomycota, Firmicutes and Actinobacteria, while increased the abundance of phyla Ascomycota, Chloroflexi, Proteobacteria, Planctomycetes, and Gemmatimonadetes in rhizoshere and mycorrhizoshere. Besides, A. mellea and G. elata significantly enriched several members of saprophytoic and pathogenic fungus (i.e., Exophiala, Leptodontidium, Cosmospora, Cercophora, Metarhizium, Ilyonectria, and Sporothrix), which will enhance the possibility of G. elata disease incidence. At the same time, the ternary cropping system significantly deterred several members of beneficial ectomycorrhizal fungus (i.e., Russula, Sebacina, and Amanita), which will reduce the ability to protect G. elata from diseases. CONCLUSIONS: In the ternary cropping system of G. elata, A. mellea and G. elata lead to imbalance of microbial community in rhizoshere and mycorrhizoshere soil, suggested that further studies on maintaining the balance of microbial community in A. mellea mycorrhizosphere and G. elata rhizosphere soil under field conditions may provide a promising avenue for high yield and high quality G. elata.

Comparative metabolomics implicates threitol as a fungal signal supporting colonization of Armillaria luteobubalina on eucalypt roots.

Armillaria root rot is a fungal disease that affects a wide range of trees and crops around the world. Despite being a widespread disease, little is known about the plant molecular responses towards the pathogenic fungi at the early phase of their interaction. With recent research highlighting the vital roles of metabolites in plant root-microbe interactions, we sought to explore the presymbiotic metabolite responses of Eucalyptus grandis seedlings towards Armillaria luteobuablina, a necrotrophic pathogen native to Australia. Using a metabolite profiling approach, we have identified threitol as one of the key metabolite responses in E. grandis root tips specific to A. luteobubalina that were not induced by three other species of soil-borne microbes of different lifestyle strategies (a mutualist, a commensalist, and a hemi-biotrophic pathogen). Using isotope labelling, threitol detected in the Armillaria-treated root tips was found to be largely derived from the fungal pathogen. Exogenous application of d-threitol promoted microbial colonization of E. grandis and triggered hormonal responses in root cells. Together, our results support a role of threitol as an important metabolite signal during eucalypt-Armillaria interaction prior to infection thus advancing our mechanistic understanding on the earliest stage of Armillaria disease development. Comparative metabolomics of eucalypt roots interacting with a range of fungal lifestyles identified threitol enrichment as a specific characteristic of Armillaria pathogenesis. Our findings suggest that threitol acts as one of the earliest fungal signals promoting Armillaria colonization of roots.

Melleolides impact fungal translation via elongation factor 2.

Melleolides from the honey mushroom Armillaria mellea represent a structurally diverse group of polyketide-sesquiterpene hybrids. Among various bioactivites, melleolides show antifungal effects against Aspergillus and other fungi. This bioactivity depends on a Δ2,4-double bond present in dihydroarmillylorsellinate (DAO) or arnamial, for example. Yet, the mode of action of Δ2,4-unsaturated, antifungal melleolides has been unknown. Here, we report on the molecular target of DAO in the fungus Aspergillus nidulans. Using a combination of synthetic chemistry to create a DAO-labelled probe, protein pulldown assays, MALDI-TOF-based peptide analysis and western blotting, we identify the eukaryotic translation elongation factor 2 (eEF2) as a binding partner of DAO. We confirm the inhibition of protein biosynthesis in vivo with an engineered A. nidulans strain producing the red fluorescent protein mCherry. Our work suggests a binding site dissimilar from that of the protein biosynthesis inhibitor sordarin, and highlights translational elongation as a valid antifungal drug target.

Inducing Secondary Metabolite Production by Co-culture of the Endophytic Fungus Phoma sp. and the Symbiotic Fungus Armillaria sp.

Co-culturing the endophytic fungus Phoma sp. YUD17001 from Gastrodia elata with Armillaria sp. in liquid nutrient medium resulted in the production of five new secondary metabolites, including two phenolic compounds, phexandiols A and B (1 and 2), three aliphatic ester derivatives, phomesters A-C (3-5), and a known fatty acid (6). The structures and absolute configurations of these compounds were elucidated by the interpretation of data from detailed spectroscopic analysis, Mosher's method, and electronic circular dichroism spectra, together with consideration of the biogenetic origins. None of the five new compounds were detected in single-strain cultures under identical fermentation conditions. The results of this work indicated that the production of 1-5 involved a complicated interaction process. None of the new compounds possessed significant cytotoxicity or antimicrobial activities.

Microbial Asymmetric Functionalization of ß-Cyclocitral-Derived Tetramethyl-Substituted γ-Lactone.

Searching for the new anticancer compounds we prepared three new ß-cyclocitral-derived hydroxyl-γ-lactones by microbial hydroxylation of tetramethyl-substituted bicyclic γ-lactone. The substrate was transformed by the enzymatic system of filamentous fungi. Three out of fifteen strains were selected as effective biocatalysts (Fusarium culmorum AM10, Armillaria mellea AM296, Trametes versicolor AM536). The hydroxylation processes were not only regioselective but also stereoselective. The hydroxylation products of each secondary carbon atom in the cyclohexane ring were obtained by the application of the selected fungal strains. The Fusarium culmorum AM10 introduced the hydroxy function at C-3 and C-4, Armillaria mellea AM296 incorporated the hydroxy function at C-3 and C-5 and Trametes versicolor AM536 transformed the substrate to the mixture of C-3, C-4 and C-5 hydroxylactones. The hydroxylactones obtained were enantiomericaly enriched (ee values in the range 17⁻99%). The in vitro antiproliferative activities of the functionalization products were also evaluated. Regardless of the hydroxy substituent location all tested lactones exhibited similar, significant activity towards selected cancer cell lines (IC50 in the range 22.8⁻33.9 µg/mL).

Scalable Biosynthesis of Melanin by the Basidiomycete Armillaria cepistipes.

Natural melanin features many interesting properties, including the ability to shield electromagnetic radiation, the ability to act as scavenger for radical and reactive oxygen species and the capacity to chelate different metal ions. For these reasons, melanin is becoming increasingly relevant for the development of functional materials with potential applications in cosmetics, drug delivery, and water purification. However, the extraction and purification of melanin from conventional sources (e.g., sepia ink, hair, and wool) is inefficient and not easily scalable, hence diverting its technological applications. Some fungal species, especially wood-decay basidiomycetes, can be regarded as promising sources of melanin. In the present study, we screened different fungi in regard to their melanin-biosynthesis abilities using l-tyrosine as a precursor, and we found that an Armillaria cepistipes strain (Empa 655) produced the highest yield of melanin (27.98 g L-1). Physicochemical characterization of the obtained fungal melanin revealed a typical eumelanin structure. The method for the biosynthesis of fungal melanin we propose is efficient, scalable, and sustainable and has the potential to provide support for further technological exploitation.

Why does the bioluminescent fungus Armillaria mellea have luminous mycelium but nonluminous fruiting body?

By determining the components involved in the bioluminescence process in luminous and nonluminous organs of the honey fungus Armillaria mellea, we have established causes of partial luminescence of this fungus. The complete set of enzymes and substrates required for bioluminescence is formed only in the mycelium and only under the conditions of free oxygen access. Since the synthesis of luciferin precursor (hispidin) and 3-hydroxyhispidin hydroxylase in the fruiting bodies is blocked, the formation of luciferin-the key component of fungal bioluminescent system-was not observed. That is why the fruiting body of Armillaria mellea is nonluminous despite the presence of luciferase, the enzyme that catalyzes the oxidation of luciferin with a photon emission.

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins.

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

Transcriptome analysis of genes involved in defence response in Polyporus umbellatus with Armillaria mellea infection.

Polyporus umbellatus, a species symbiotic with Armillaria mellea and it also exhibits substantial defence response to Armillaria mellea infection. There are no genomics resources databases for understanding the molecular mechanism underlying the infection stress of P. umbellatus. Therefore, we performed a large-scale transcriptome sequencing of this fungus with A. mellea infection using Illumina sequencing technology. The assembly of the clean reads resulted in 120,576 transcripts, including 38,444 unigenes. Additionally, we performed a gene expression profiling analysis upon infection treatment. The results indicated significant differences in the gene expression profiles between the control and the infection group. In total, 10933 genes were identified between the two groups. Based on the differentially expressed genes, a Gene Ontology annotation analysis showed many defence-relevant categories. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes pathway analysis uncovered some important pathways. Furthermore, the expression patterns of 13 putative genes that are involved in defence response resulting from quantitative real-time PCR were consistent with their transcript abundance changes as identified by RNA-seq. The sequenced genes covered a considerable proportion of the P. umbellatus transcriptome, and the expression results may be useful to strengthen the knowledge on the defence response of this fungus defend against Armillaria mellea invasion.

Effect of surfactants and identification of metabolites on the biodegradation of fluoranthene by basidiomycetes fungal isolate Armillaria sp. F022.

The effects of structure and concentration of surfactants on the biodegradation of fluoranthene, a three rings polycyclic aromatic hydrocarbon in the aqueous phase, as well as their effects on the biodegradation and enzyme activity were investigated. The toxicity ranking of studied surfactants is: non-ionic Tween 80 4,500 mg/L) was showed by Tween 80 (10 mg/L) culture, manifesting that the non-ionic surfactant present in the culture were beneficial to the fungal growth. Laccase showed the highest enzymes activity in all surfactants culture. Non-ionic Tween 80 showed a significant result for laccase activity (1,902 U/L) in the Armillaria sp. F022 culture. The increased enzymes cumulative activity may stem directly from the rising fluoranthene biodegradability as addition of appropriate surfactants. The biotransformation of fluoranthene was greatly improved by Tween 80, and totally fluoranthene degradation was obtained as Tween 80 was 10 mg/L. Two fluoranthene metabolites were isolated from the culture medium and analyzed by a thin layer chromatography, UV visible spectrometer and gas chromatography-mass spectrometry (GC-MS). The oxidation of fluoranthene is initiated by oxygenation at the C-2,3 positions resulting 9-fluorenone. At the end of experiment, one metabolite was detected in the culture extract and identified as phthalic acid. Evidently, Armillaria sp. F022 seems efficient, high effective and deserves further application on the enhanced bioremediation technologies for the treatment of fluoranthene-contaminated soil.

Fluorene biodegradation and identification of transformation products by white-rot fungus Armillaria sp. F022.

A diverse surfactant, including the nonionic Tween 80 and Brij 30, the anionic sodium dodecyl sulphate, the cationic surfactant Tetradecyltrimethylammonium bromide, and biosurfactant Rhamnolipid were investigated under fluorine-enriched medium by Armilaria sp. F022. The cultures were performed at 25 °C in malt extract medium containing 1 % of surfactant and 5 mg/L of fluorene. The results showed among the tested surfactants, Tween-80 harvested the highest cell density and obtained the maximum specific growth rate. This due Tween-80 provide a suitable carbon source for fungi. Fluorane was also successfully eliminated (>95 %) from the cultures within 30 days in all flasks. During the experiment, laccase production was the highest among other enzymes and Armillaria sp. F022-enriched culture containing Non-ionic Tween 80 showed a significant result for laccase activity (1,945 U/L). The increased enzyme activity was resulted by the increased biodegradation activity as results of the addition of suitable surfactants. The biotransformation of fluorene was accelerated by Tween 80 at the concentration level of 10 mg/L. Fluorene was initially oxidized at C-2,3 positions resulting 9-fluorenone. Through oxidative decarboxylation, 9-fluorenone subjected to meta-cleavage to form salicylic acid. One metabolite detected in the end of experiment, was identified as catechol. Armillaria sp. F022 evidently posses efficient, high effective degrader and potential for further application on the enhanced bioremediation technologies for treating fluorene-contaminated soil.

Assembly of melleolide antibiotics involves a polyketide synthase with cross-coupling activity.

Little is known about polyketide biosynthesis in mushrooms (basidiomycota). In this study, we investigated the iterative type I polyketide synthase (PKS) ArmB of the tree pathogen Armillaria mellea, a producer of cytotoxic melleolides (i.e., polyketides esterified with various sesquiterpene alcohols). Heterologously produced ArmB showed orsellinic acid (OA) synthase activity in vitro. Further, we demonstrate cross-coupling activity of ArmB, which forms OA esters with various alcohols. Using a tricyclic Armillaria sesquiterpene alcohol, we reconstituted the biosynthesis of melledonol. Intermolecular transesterification reactions may represent a general mechanism of fungal PKSs to create structural diversity of small molecules. Phylogenetic network construction of thioesterase domains of both basidiomycetes and ascomycetes suggests that the fungal nonreducing PKS family has likely evolved from an ancient OA synthase and has gained versatility by adopting Claisen-like cyclase or transferase activity.