Polyporic acid

Light induces the production of ink-blue pentacyclic natural products, the corticin pigments, in the cobalt crust mushroom Terana caerulea. Here, we describe the genetic locus for corticin biosynthesis and provide evidence for a light-dependent dual transcriptional/cotranscriptional regulatory mechanism. Light selectively induces the expression of the corA gene encoding the gateway enzyme, the first described mushroom polyporic acid synthetase CorA, while other biosynthetic genes for modifying enzymes necessary to complete corticin assembly are induced only at lower levels. The strongest corA induction was observed following exposure to blue and UV light. A second layer of regulation is provided by the light-dependent splicing of the three introns in the pre-mRNA of corA. Our results provide insight into the fundamental organization of how mushrooms regulate natural product biosynthesis. IMPORTANCE The regulation of natural product biosyntheses in mushrooms in response to environmental cues is poorly understood. We addressed this knowledge gap and chose the cobalt crust mushroom Terana caerulea as our model. Our work discovered a dual-level regulatory mechanism that connects light as an abiotic stimulus with a physiological response, i.e., the production of dark-blue pigments. Exposure to blue light elicits strongly increased transcription of the gene encoding the gateway enzyme, the polyporic acid synthetase CorA, that catalyzes the formation of the pigment core structure. Additionally, light is a prerequisite for the full splicing of corA pre-mRNA and, thus, its proper maturation. Dual transcriptional/cotranscriptional light-dependent control of fungal natural product biosynthesis has previously been unknown. As it allows the tight control of a key metabolic step, it may be a much more prevalent mechanism among these organisms.

Mushroom poisoning is a significant and increasing form of toxin-induced-disease. Existing classifications of mushroom poisoning do not include more recently described new syndromes of mushroom poisoning and this can impede the diagnostic process. We reviewed the literature on mushroom poisoning, concentrating on the period since the current major classification published in 1994, to identify all new syndromes of poisoning and organise them into a new integrated classification, supported by a new diagnostic algorithm. New syndromes were eligible for inclusion if there was sufficient detail about both causation and clinical descriptions. Criteria included: identity of mushrooms, clinical profile, epidemiology, and the distinctive features of poisoning in comparison with previously documented syndromes. We propose 6 major groups based on key clinical features relevant in distinguishing between poisoning syndromes. Some clinical features, notably gastrointestinal symptoms, are common to many mushroom poisoning syndromes. Group 1 - Cytotoxic mushroom poisoning. Syndromes with specific major internal organ pathology: (Subgroup 1.1; Primary hepatotoxicity); 1A, primary hepatotoxicity (amatoxins); (Subgroup 1.2; Primary nephrotoxicity); 1B, early primary nephrotoxicity (amino hexadienoic acid; AHDA); 1C, delayed primary nephrotoxicity (orellanines). Group 2 - Neurotoxic mushroom poisoning. Syndromes with primary neurotoxicity: 2A, hallucinogenic mushrooms (psilocybins and related toxins); 2B, autonomic-toxicity mushrooms (muscarines); 2C, CNS-toxicity mushrooms (ibotenic acid/muscimol); 2D, morel neurologic syndrome (Morchella spp.). Group 3 - Myotoxic mushroom poisoning. Syndromes with rhabdomyolysis as the primary feature: 3A, rapid onset (Russula spp.); 3B, delayed onset (Tricholoma spp.). Group 4 - Metabolic, endocrine and related toxicity mushroom poisoning. Syndromes with a variety of clinical presentations affecting metabolic and/or endocrine processes: 4A, GABA-blocking mushroom poisoning (gyromitrins); 4B, disulfiram-like (coprines); 4C, polyporic mushroom poisoning (polyporic acid); 4D, trichothecene mushroom poisoning (Podostroma spp.); 4E, hypoglycaemic mushroom poisoning (Trogia venenata); 4F, hyperprocalcitoninemia mushroom poisoning (Boletus satanas); 4G, pancytopenic mushroom poisoning (Ganoderma neojaponicum). Group 5 - Gastrointestinal irritant mushroom poisoning. This group includes a wide variety of mushrooms that cause gastrointestinal effects without causing other clinically significant effects. Group 6 - Miscellaneous adverse reactions to mushrooms. Syndromes which do not fit within the previous 5 groups: 6A, Shiitake mushroom dermatitis; 6B, erythromelagic mushrooms (Clitocybe acromelagia); 6C, Paxillus syndrome (Paxillus involutus); 6D, encephalopathy syndrome (Pleurocybella porrigens).

Background: Non-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides. Results: Here, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation. Conclusion: This study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin.