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The atromentin synthetase gene cluster is responsible for catalyzing the precursor pigment atromentin, which is further catalyzed into hundreds of different pigments that span different taxa in the Basidiomycota and is a distinguished feature of Boletales. Previous work identified co-transcription of the two essential clustered atromentin genes (the atromentin synthetase (NPS) and the aminotransferase) by inducible pigment conditions and also conserved genetic elements in the promoter regions (motifs). For this work, we found that the NPS and its promoter motif appeared to follow the same evolutionary path as the mushrooms’. The NPS appears to predate Boletales and originate in Agaricomycetes, and with convergent/parallel evolution that allowed ascomycetes to produce atromentin. Additionally, a consensus of the intron-exon gene structure for basidiomycetous, atromentin-catalyzing NPSs was identified whereby a significant deviation occurred in the paraphyletic group, Paxillaceae. This gene structure was not present in NPSs in Aspergilli. Lastly, we found a putative TATA box adjacent to the palindromic motif of NPS, indicating (co-)transcriptional control by a TATA(-like) binding transcription factor. Combined with previous decades’ worth of research, our results support that not only can atromentin derivatives be used for chemo-taxonomy, but also atromentin’s genetic basis. Future work using the putative promoter motif will provide new insight into which (co-)transcription factor may be responsible for the transcriptional control of atromentin synthetases.

Fourteen new compounds including thirteen drimane – phthalide derivatives (fendlerals A – C, fendlerins A – D, fendlerols A – B, fendleric acids A – C, fendlerinine G) and one terphenyl derivative (fendleryl E) along with eight known compounds, fendlerinine A, rickenyls C – D, fendleryls C – D, atromentin, tetramethyl atromentin, and (±)-microsphaerophthalide F, were isolated from the wood fungus Hypoxylon fendleri BCC32408. Compared with the prior work, the results indicated the agitation effect on the production of bioactive drimane – phthalides. The chemical structures were determined based upon spectroscopic analyses and the absolute configurations were verified by comparison of the ECD spectral data with the calculated ECD spectra of the related compounds. Compounds 1–3 exhibited antimicrobial activity against Plasmodium falciparum (IC50 4.15–4.39 μM), Colletotrichum capsici (MIC 6.25–12.5 μg/mL), and Bacillus cereus (MIC 1.56–3.13 μg/mL). All tested compounds displayed broad cytotoxicity against cancerous (MCF-7, KB, and NCI-H187) and non-cancerous (Vero) cells.

Summary Basidiomycete fungi are characterized ecologically for their vital functional role in ecosystem carbon recycling and chemically for their capacity to produce a diverse array of small molecules. Chromophoric natural products derived from the quinone precursor atromentin, such as variegatic acid and involutin, have been shown to function in redox cycling. Yet, in the context of an inter-kingdom natural system these pigments are still elusive. Here, we co-cultured the model saprotrophic basidiomycete Serpula lacrymans with an ubiquitous terrestrial bacterium, either Bacillus subtilis, Pseudomonas putida, or Streptomyces iranensis. For each, there was induction of the gene cluster encoding a non-ribosomal peptide synthetase-like enzyme (atromentin synthetase) and an aminotransferase which together produce atromentin. Correspondingly during co-culturing there was an increase in secreted atromentin-derived pigments, i.e., variegatic, xerocomic, isoxerocomic and atromentic acid. Bioinformatic analyses from 14 quinone synthetase genes, twelve of which are encoded in a cluster, identified a common promoter motif indicating a general regulatory mechanism for numerous basidiomycetes. This article is protected by copyright. All rights reserved.

Summary Non-ribosomal peptide synthetase (NRPS)-like enzymes catalyze the non-oxidative homodimerization of aromatic α-keto acids, but the exact reaction mechanism is unknown. The furanone-forming thioesterase domain of the Aspergillus terreus aspulvinone E synthetase MelA displays a predicted quinone-forming motif, whereby its catalytic triad contains an essential cysteine indicating an unusual thioester intermediate. To convert MelA into a quinone-forming atromentin synthetase its thioesterase domain was replaced with that from a Paxillus involutus or A. terreus atromentin synthetase. Phylogenetic proximity of donor and acceptor seems important, as only replacement with the A. terreus thioesterase was functional. Heterologous expression of atromentin synthetases in Aspergillus niger and Aspergillus oryzae revealed host-dependent product formation whereby cross-chemistry directed atromentin biosynthesis in A. niger toward atrofuranic acid. Screening of aspergilli from section Nigri identified an atromentin synthetase in Aspergillus brasiliensis that produced atrofuranic acid in the homologous host. Therefore, cross-chemistry on quinone cores appears common to section Nigri.

Production of basidiomycete atromentin-derived pigments like variegatic acid (pulvinic acid-type) and involutin (diarylcyclopentenone) from the brown-rotter Serpula lacrymans and the ectomycorrhiza-forming Paxillus involutus, respectively, is induced by complex nutrition, and in the case of S. lacrymans, bacteria. Pigmentation in S. lacrymans was stimulated by 13 different bacteria and cell-wall-damaging enzymes (lytic enzymes and proteases), but not by lysozyme or mechanical damage. The use of protease inhibitors with Bacillus subtilis or heat-killed bacteria during co-culturing with S. lacrymans significantly reduced pigmentation indicating that enzymatic hyphal damage and/or released peptides, rather than mechanical injury, was the major cause of systemic pigment induction. Conversely, no significant pigmentation by bacteria was observed from P. involutus. We found additional putative transcriptional composite elements of atromentin synthetase genes in P. involutus and other ectomycorrhiza-forming species that were absent from S. lacrymans and other brown-rotters. Variegatic and its precursor xerocomic acid, but not involutin, in return inhibited swarming and colony biofilm spreading of Bacillus subtilis, but did not kill B. subtilis. We suggest that dissimilar pigment regulation by fungal lifestyle was a consequence of pigment bioactivity and additional promoter motifs. The focus on basidiomycete natural product gene induction and regulation will assist in future studies to determine global regulators, signalling pathways and associated transcription factors of basidiomycetes.

SummaryThe symbiotic fungus Paxillus involutus serves a critical role in maintaining forest ecosystems, which are carbon sinks of global importance. P. involutus produces involutin and other 2,5-diarylcyclopentenone pigments that presumably assist in the oxidative degradation of lignocellulose via Fenton chemistry. Their precise biosynthetic pathways, however, remain obscure. Using a combination of biochemical, genetic, and transcriptomic analyses, in addition to stable-isotope labeling with synthetic precursors, we show that atromentin is the key intermediate. Atromentin is made by tridomain synthetases of high similarity: InvA1, InvA2, and InvA5. An inactive atromentin synthetase, InvA3, gained activity after a domain swap that replaced its native thioesterase domain with that of InvA5. The found degree of multiplex biosynthetic capacity is unprecedented with fungi, and highlights the great importance of the metabolite for the producer.

Non-ribosomal peptide synthetases (NRPSs) are key enzymes in microorganisms for the assembly of peptide backbones of biologically and pharmacologically active natural products. The monomodular NRPS-like enzymes comprise often an adenylation (A), a thiolation (T), and a thioesterase (TE) domain. In contrast to the NRPSs, they do not contain any condensation domain and usually catalyze a dimerization of α-keto carboxylic acids and thereby provide diverse scaffolds for further modifications. In this study, we established an expression system for NRPS-like genes in Saccharomyces cerevisiae. By expression of four known genes from Aspergillus terreus, their predicted function was confirmed and product yields of up to 35 mg per liter culture were achieved. Furthermore, expression of ATEG_03090 from the same fungus, encoding for the last uncharacterized NRPS-like enzyme with an A-T-TE domain structure, led to the formation of the benzoquinone derivative atromentin. All the accumulated products were isolated and their structures were elucidated by NMR and MS analyses. This study provides a convenient system for proof of gene function as well as a basis for synthetic biology, since additional genes encoding modification enzymes can be introduced.

The genus Hypoxylon , a member of the family Xylariaceae, has been known to produce significant secondary metabolites in terms of chemical diversity. Moreover, the compounds isolated can also be used as chemotaxonomic characters for differentiation among the two sections, which are sect. Annulata and sect. Hypoxylon . In our continuing chemical screening programme for novel compounds, the crude extracts of H. fendleri BCC32408 gave significant chemical profiles in HPLC analyses. Thus, the chemical investigation of these crude extracts was then carried out. The investigation led to the isolation of ten previously undescribed compounds including three terphenylquinones (fendleryls A – C), one terphenyl (fendleryl D), and six novel drimane – phthalide-type lactone/isoindolinones derivatives (fendlerinines A – F) along with seven known compounds (2- O -methylatromentin, rickenyl E, atromentin, rickenyls C – D, (+)-ramulosin, and O -hydroxyphenyl acetic acid). The chemical structures were determined on the basis of spectroscopic analyses, including 1D, 2D NMR and high-resolution mass spectrometry, as well as chemical transformations. In addition, these isolated compounds were assessed for antimicrobial activity including antimalarial (against Plasmodium falciparum , K-1 strain), antifungal (against Candida albicans ), antibacterial (against Bacillus cereus ) activities. Cytotoxicity against both cancerous (KB, MCF-7, NCI-H187) and non-cancerous (Vero) cells of these compounds were also evaluated.

The gene greA was cloned from the genome of the basidiomycete Suillus grevillei. It encodes a monomodular natural product biosynthesis protein composed of three domains for adenylation, thiolation, and thioesterase and, hence, is reminiscent of a nonribosomal peptide synthetase (NRPS). GreA was biochemically characterized in vitro. It was identified as atromentin synthetase and therefore represents one of only a limited number of biochemically characterized NRPS-like enzymes which accept an aromatic α-keto acid. Specificity-conferring amino acid residues--collectively referred to as the nonribosomal code--were predicted for the primary sequence of the GreA adenylation domain and were an unprecedented combination for aromatic α-keto acids. Plausible support for this new code came from in silico simulation of the adenylation domain structure. According to the model, the predicted residues line the active site and, therefore, very likely contribute to substrate specificity.

Among the first documented studies on the chemistry of fungal natural products were descriptions of quinoid pigments, i.e., the L-tyrosine- and L-phenylalanine-derived terphenylquinones atromentin and polyporic acid, respectively. The isolation of these compounds from mushroom fruiting bodies (basidiomes) was published around 1877 by Stahlschmidt and Thorner. Ever since, organic chemists embraced basidiomycetes as a prolific source of bioactive compounds and investigated these fungi with regard to compound isolation, structure elucidation, and synthesis (Gill and Steglich, 1987; Zhou and Liu, 2010; De Silva et al., 2013 and previous reviews referenced therein, Lorenzen and Anke, 1998; Richter et al., in press). Mushrooms seem to be particularly talented in producing unique terpenoids, and the molecular background behind the biosynthesis of some of those compounds has only recently been elucidated (Quin et al., 2014). Prominent examples of basidiomycete metabolites for lead structures in agrochemistry and drug research are, among others, the strobilurins, i.e., agriculturally used s-methoxyacrylate fungicides from cultures of Mycena, Oudemansiella, Strobilurus, Xerula and several other basidiomycete genera (Sauter et al., 1999, Figure 1). Other examples are the pleuromutilins, the illudins, and the omphalotins (Figure ​(Figure1).1). The pleuromutilins from cultures of species that are now placed in the genera Clitopilus and Omphalina served as scaffold for the development of the semisynthetic antibacterial antibiotic retapamulin (Kirst, 2013) which is clinically used for topical treatment of infections with Staphylococcus aureus. The illudins from Lampteromyces and Omphalotus species (Omphalotaceae) are sesquiterpenes featuring an unusual cyclopropane ring and are currently developed as anticancer drugs (Tanasova and Sturla, 2012). The omphalotins are cyclopeptides with pronounced nematicidal activites against root knot nematodes (Buchel et al., 1998), which are also exclusively found in the Omphalotaceae. Recently, the blazeispirols from Agaricus subrufescens were discovered as strong and selective agonists of the Liver X receptor (LXR alpha). Concurrently, relevant in vivo effects of blazeispirols in a mouse model were observed which might give rise to the development of a new anti-hypercholesterolemic agent from cultures of a medicinal mushroom (Grothe et al., 2011).

The genus Thelephora is widely distributed in East Asia, Australia, and America. Thelephora aurantiotincta belongs to the Thelephora genus. It is a mushroom that grows in symbiosis with pine trees found in Yunnan Province, China. T. aurantiotincta is also one of the most prized edible mushrooms and is sold in a mixture with Thelephora ganbajun. In previous studies, some p-terphenyl derivatives, such as aurantiotinin A, ganbajunins C and E, atromentin, thelephorin A, and thelephantins A–H, have been obtained from the fungus [1–3].

Polyporic acid, atromentin, bovinone, and oosporein are common metabolic products of a number of species of fungi. The related compound cochliodinol and its congeners are produced by several Chaetomium spp. These quinonoid metabolites have been shown to inhibit the growth and metabolism of a range of bacterial genera. The antibiotic activity of the quinones depends on the substituents at the 3 and 6 positions of the 2,5-dihydroxy-1,4-benzoquinone ring; in aerobic systems the activity appears to be inversely proportional to the polarity of the metabolite. It has been shown that reduction of the quinone to the hydroquinone changes the antibiotic activity of these metabolites but does not abolish it. Contrary to previous reports, the activity of these hydroquinones is not reversed by cysteine.

Die Kremplinge Paxillus atrotomentosus und P. panuoides produzieren in geringen Mengen orangegelbe Flavomentine und violette Spiromentine. Bei den Flavomentinen 1–3 handelt es sich um Ester des Atromentins (6) mit (2Z,4E)-2,4-Hexadiensaure oder (2Z,4S,5S)-4,5-Epoxy-2-hexensaure, auserdem kommt als weitere Verbindung dieses Typs 3-O-Acetylatromentin (4) vor. Die Spiromentine 8, 11, 12 und 13 besitzen eine ungewohnliche Spirostruktur, in der ein 4,5-Dihydroxy-1,2-benzochinon mit einem Lacton acetalartig verknupft ist. Die spektroskopisch abgeleiteten Strukturen der Farbstoffe konnten durch die Synthese von Modellverbindungen und die biomimetische Uberfuhrung von Flavomentin B (2) in die Spiromentine B (11) und C (12) gesichert werden. Fungal Pigments, 61. – Flavomentins and Spiromentins, Novel Terphenylquinone Derivatives from Paxillus atrotomentosus and P. panuoides (Boletales) The gilled toadstools Paxillus atrotomentosus and P. panuoides produce small amounts of orange-yellow flavomentins and violet spiromentins. The flavomentins 1–3 constitute mono- and diesters of atromentin (6) with (2Z,4E)-2,4-hexadienoic acid or (2Z,4S,5S)-4,5-epoxy-2-hexenoic acid. In addition, 3-O-acetylatromentin (4) has been isolated from P. atrotomentosu. The spiromentins 8,11,12, and 13 possess a unique spiro structure in which a 4,5-dihydroxy-1,2-benzoquinone is linked to a lactone acetal unit. The structures of the pigments were confirmed by the synthesis of model compounds and the biomimetic conversion of flavomentin B (2) into the spiromentins B (11) and C (12).

Die Blauverfarbung verletzter Fruchtkorper von Gyroporus cyanescens (Boletales) beruht auf der Oxidation von Gyrocyanin (3a) zum blauen Anion 5a. 5a wird beim Ansauern zu Gyroporin (7) hydratisiert. Die Darstellung der Cyclopentan-Derivate 5 und 7 gelingt auch durch oxidative Ringverengung von Atromentin (8a), moglicherweise ein Modell fur die Biosynthese dieser Verbindungen. Die Struktur des Involutins16) wird durch das NMR-Spektrum im Sinne von Formel 11a festgelegt. Fungus Pigments, XVII. Gyrocyanin, the Blueing Principle of Gyroporus cyanescens, and an Oxidative Ring Contraction of Atromentin The blueing or injured sporophores of Gyroporus cyanescens (Boletales) is due to the oxidation of gyrocyanin (3a) to the blue anion 5a. Hydration of 5a by acidification leads to gyroporin (7). The cyclopentane derivatives 5 and 7 may also be obtained by oxidative ring contraction of atromentin (8a), a possible model for the biosynthesis of these compounds. N.m.r. evidence for the structure 11a of involutin16) is presented.

Two procedures were employed for the isolation of aurantiacin from an ether extract of the basidiomycete Hydnellum caeruleurn. Identification of the compound was based on its melting point, spectral properties (infrared, visible, and ultraviolet), and alkaline degradation to atromentin and benroic acid.

Atromentin was found to be present in both 30- and 45-day-old cultures of Clitocybe subilludens Murr., whereas thelephoric acid was present in the 30-, 45-, and 60-day-old cultures. These cultures were grown on 6% malt extract agar under continuous light conditions at 29° for 15, 30, 45, and 60 days. Cultures grown in the absence of light failed to produce detectable quantities of either terphenylquinone. Examination of all cultures for diphenyl-substituted tetronic acids failed to demonstrate detectable amounts. It is postulated from these observations that atromentin may act as a precursor for thelephoric acid, since the disappearance of the former coincides with the appearance of the latter terphenyl-quinone.

Two styrylpyrone, three terphenylquinone, and six diphenyl-substituted tetronic acid derivatives were tested for in vitro antimicrobial activity. The best inhibitory responses were seen with hispidin, bisnoryangonin, and vulpinic acid against Gram-positive organisms, including the acid-fast Mycobacterium smegmatis, and with atromentin against the acid-fast bacterium, the Gram-negative organisms, and Candida albicans. However, all of the observed activities were low relative to those normally associated with clinically useful antibiotics.

The sporophore of Boletus (Suillus) bovinus(Linn. ex Fr.) contains a new benzoquinone as the major metabolite which has been identified as 2,5-dihydroxy-3-geranylgeranyl-1,4-benzoquinone (I). Atromentin, amitenone, and variegatic acid have also been identified as minor constituents.