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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.

Studies of the barium salts of bromanilic acid, nitranilic acid and polyporic acid show that they are not superior to barium chloranilate as reagents for determining sulfate. Improved syntheses of polyporic acid and atromentin are presented; two new syntheses of barium bromanilate, and one new synthesis of barium nitranilate and of barium polyporate are also presented. Ultraviolet absorption spectra show that polyporic acid forms a dimer in dimethylsulfoxide solution.

Gross screening revealed that Hydnellum diabolus Banker contained a parenterally effective anticoagulant, while H. aurantiacum , H. caeruleum , and Hydnum laevigatum lacked activity. A 70 per cent ethanolic extract of H. diabolus contained all anticoagulant activity as well as pressor-leiomyotonic principles. The anticoagulant activity grossly resembled that of heparin, except that in vivo activity was not reversed by protamine injection. In vivo , 1 mg. of the ethanol extract was equivalent to 0.58 u. of heparin. Selective solvent extraction of the mushroom with diethyl ether removed all anticoagulant activity, and from this extract atromentin was isolated. In vitro , 1 mg. of atromentin was equivalent to 5.1 u. of heparin and 2.3 mg. of reference 70 per cent ethanol extract. Synthetic dimethylatromentin, 2,5-diphenylbenzoquinone, and polyporic acid were without anticoagulant properties.

Chromatographic studies, using several solvent systems with thin‐layer polyamide and silica gel G plates, indicated that Paxillus panuoides (Fr.) Fr. and P. atrotomentosus (Batsch) Fr. contain several common pigments in both carpophores and surface cultures. Atromentin was isolated and identified from the carpophores of P. panuoides , and atromentic and xerocomic acids were recovered and identified from the cultures of this species. These pigments were reported previously from the corresponding growth forms of P. atrotomentosus . Identification of the pigments was based on comparisons of chromatographic properties and spectral data (IR, UV, and high‐resolution mass spectra) with authentic materials. Preliminary interpretation of the complex mass spectrum of atromentin suggested the involvement of at least three basic fragmentation pathways. The pKa values and fluorescent spectra (before and after exposure to radiant energy) of atromentic, pulvinic, and xerocomic acids were determined, and the response of xerocomic acid to thermal energy during sublimation was clarified.

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.