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Basidiomycete fungi are a rich source of natural products with a diverse array of potentially exploitable bioactivities. Two dimeric sesquiterpenes, bovistol B (1) and D (2), and one monomeric sesquiterpene, strossmayerin (7), were isolated from the culture filtrate of the basidiomycete fungus Coprinopsis strossmayeri. The structures were determined through a combination of MS and 1D/2D NMR spectroscopic techniques. Likely monomeric precursors, identified on the basis of HRMS analysis, allow a plausible biosynthetic pathway to be proposed for the biosynthesis of 1 and 2, involving the dimerisation of the monomer through a hetero-Diels-Alder mechanism. A gene cluster, including a putative sesquiterpene 1-11 cyclase, was identified through phylogenetic and RNA-seq analysis, and is proposed to be responsible for the biosynthesis of 1 and 2., Competing Interests: The authors have declared that no competing interests exist.

Naematolin is a biologically active sesquiterpene produced by Hypholoma species. Low titres and complex structure constrain the exploitation of this secondary metabolite. Here, we de novo sequenced the H. fasciculare genome to identify a candidate biosynthetic gene cluster for production of naematolin. Using Aspergillus oryzae as a heterologous host for gene expression, the activity of several sesquiterpene synthases were investigated, highlighting one atypical sesquiterpene synthase apparently capable of catalysing the 1,11 and subsequent 2,10 ring closures, which primes the synthesis of the distinctive structure of caryophyllene derivatives. Co-expression of the cyclase with an FAD oxidase adjacent within the gene cluster generated four oxidised caryophyllene-based sesquiterpenes: 5β,6α,8β-trihydroxycariolan, 5β,8β-dihydroxycariolan along with two previously unknown caryophyllene derivatives 2 and 3. This represents the first steps towards heterologous production of such basidiomycete-derived caryophyllene-based sesquiterpenes, opening a venue for potential novel antimicrobials via combinatorial biosynthesis.

The basidiomycete fungi Hypholoma fasciculare and H. sublateritium are both prolific producers of sesquiterpenes and triterpenes, some of which have relevant pharmaceutical properties. Although H. sublateritium has been transformed in the past, the low reported efficiencies highlighted the need for establishing an effective simple transformation system for these valuable species. We have optimized Agrobacterium tumefaciens-mediated transformation through testing various parameters in these two Hypholoma species, showing that a mixture of homogenized mycelia and Agrobacterium (strain LBA4404) co-cultivated for 84h at 25°C is optimal for efficient transformation in these basidiomycetes. This study also reveals the requirements for transgene expression, with the first report of GFP expression in these Hypholoma, the need for an intron for such transgene expression, and further demonstrates the functionality of the expression vector by its use in Clitopilus passeckerianus. This development of transformation system and expression constructs, can facilitate further genetic investigation such as gene functionality in these fungi., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

This paper describes the optimisation of an existing basidiomycete molecular toolkit through the development of new versatile vectors. These vectors enable the straightforward and rapid construction of gene expression and silencing cassettes by allowing the easy exchange of promoters, coding regions and terminator elements. The constructs contain multiple cloning sites (MCS) allowing any gene to be inserted using a range of restriction sites, with the option of a 5' integral intron for efficient gene expression. We describe the testing of these vectors through marker gene expression in Coprinopsis cinerea. This work also extends the range of marker genes available for use in C. cinerea with the first report of DsRed and monomeric red fluorescent protein (mRFP) expression in C. cinerea and further demonstrates the requirement for an intron in the expression cassette for some marker genes. However, analysis of transformants containing either beta-glucuronidase (GUS) or luciferase (LUC) genes, with and without an intron revealed no detectable marker gene expression. The inclusion of an intron does therefore not guarantee expression and other genetic factors may be involved.

We describe efficient polyethylene glycol (PEG)-mediated and Agrobacterium-mediated transformation systems for a pharmaceutically important basidiomycete fungus, Clitopilus passeckerianus, which produces pleuromutilin, a diterpene antibiotic. Three dominant selectable marker systems based on hygromycin, phleomycin, and carboxin selection were used to study the feasibility of PEG-mediated transformation of C. passeckerianus. The PEG-mediated transformation of C. passeckerianus protoplasts was successful and generated hygromycin-resistant transformants more efficiently than either phleomycin or carboxin resistance. Agrobacterium-mediated transformation with plasmid pBGgHg containing hph gene under the control of the Agaricus bisporus gpdII promoter led to hygromycin-resistant colonies and was successful when homogenized mycelium and fruiting body gill tissue were used as starting material. Southern blot analysis of transformants revealed the apparently random integration of the transforming DNA to be predominantly multiple copies for the PEG-mediated system and a single copy for the Agrobacterium-mediated system within the genome. C. passeckerianus actin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression in C. passeckerianus, but only when constructs contained a 5' intron, demonstrating that the presence of an intron is prerequisite for efficient transgene expression. The feasibility of RNA interference-mediated gene silencing was investigated using gfp as a target gene easily scored in C. passeckerianus. Upon transformation of gfp antisense constructs into a highly fluorescent strain, transformants were recovered that exhibited either reduced or undetectable fluorescence. This was confirmed by Northern blotting showing depletion of the target mRNA levels. This demonstrated that gene silencing is a suitable tool for modulating gene expression in C. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.

Pleuromutilin is a broad-spectrum antibiotic that has been used in veterinary medicine for over 20 years, but is now gaining interest as a human therapeutic. The compound is a fungal secondary metabolite, but there is some degree of confusion within the literature concerning which species may produce pleuromutilin, with several differently named fungi reported to make the compound. Here, we describe a taxonomic survey of publicly available cultures known to produce pleuromutilin, and other similar species. The pleuromutilin production of these strains was assessed and a phylogenetic assessment was carried out based on the sequence of the nuclear rRNA internal transcribed spacer region. Eleven strains were confirmed as being pleuromutilin producers and all of these isolates appear to fall within a discrete clade of the genus Clitopilus. The phylogenetic analysis also highlights the need for a revision of the taxonomic status of these fungi.

The Agaricus bisporus serine proteinase 1 (SPR1) appears to be significant in both mycelial nutrition and senescence of the fruiting body. We report on the construction of an SPR promoter::green fluorescent protein (GFP) fusion cassette, pGreen_hph1_SPR_GFP, for the investigation of temporal and developmental expression of SPR1 in homobasidiomycetes and to determine how expression is linked to physiological and environmental stimuli. Monitoring of A. bisporus pGreen_hph1_SPR_GFP transformants on media rich in ammonia or containing different nitrogen sources demonstrated that SPR1 is produced in response to available nitrogen. In A. bisporus fruiting bodies, GFP activity was localized to the stipe of postharvest senescing sporophores. pGreen_hph1_SPR_GFP was also transformed into the model basidiomycete Coprinopsis cinerea. Endogenous C. cinerea proteinase activity was profiled during liquid culture and fruiting body development. Maximum activity was observed in the mature cap, while activity dropped during autolysis. Analysis of the C. cinerea genome revealed seven genes showing significant homology to the A. bisporus SPR1 and SPR2 genes. These genes contain the aspartic acid, histidine, and serine residues common to serine proteinases. Analysis of the promoter regions revealed at least one CreA and several AreA regulatory motifs in all sequences. Fruiting was induced in C. cinerea dikaryons, and fluorescence was determined in different developmental stages. GFP expression was observed throughout the life cycle, demonstrating that serine proteinase can be active in all stages of C. cinerea fruiting body development. Serine proteinase expression (GFP fluorescence) was most concentrated during development of young tissue, which may be indicative of high protein turnover during cell differentiation.