Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production.
Interaction between microbes affects the growth, metabolism and differentiation of members of the microbial community. While direct and indirect competition, like antagonism and nutrient consumption have a negative effect on the interacting members of the population, microbes have also evolved in nature not only to fight, but in some cases to adapt to or support each other, while increasing the fitness of the community. The presence of bacteria and fungi in soil results in various interactions including mutualism. Bacilli attach to the plant root and form complex communities in the rhizosphere. Bacillus subtilis, when grown in the presence of Aspergillus niger, interacts similarly with the fungus, by attaching and growing on the hyphae. Based on data obtained in a dual transcriptome experiment, we suggest that both fungi and bacteria alter their metabolism during this interaction. Interestingly, the transcription of genes related to the antifungal and putative antibacterial defence mechanism of B. subtilis and A. niger, respectively, are decreased upon attachment of bacteria to the mycelia. Analysis of the culture supernatant suggests that surfactin production by B. subtilis was reduced when the bacterium was co-cultivated with the fungus. Our experiments provide new insights into the interaction between a bacterium and a fungus.
The psychotropic effectso fPsilocybe "magic" mushroomsa re caused by the l-tryptophan-derived alkaloid psilocybin.D espite their significance,t he secondary metabolome of these fungi is poorly understood in general. Our analysis of four Psilocybe speciesi dentified harmane, harmine, and ar ange of other l-tryptophan-derived b-carbolines as their natural products,w hichw as confirmed by 1D and 2D NMR spectroscopy.S table-isotope labeling with 13 C 11 -l-tryptophan verifiedt he b-carbolines as biosynthetic products of these fungi. In addition, MALDI-MS imaging showedt hat b-carbolines accumulate toward the hyphal apices.A sp otent inhibitors of monoamine oxidases, b-carbolines are neuroactive compounds and interfere with psilocybin degradation. Therefore, our findings represent an unprecedented scenario of natural product pathways that diverge from the same buildingb lock and produce dissimilar compounds, yet contribute directlyo ri ndirectly to the same pharmacological effects.
Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) is the main alkaloid of the fungal genus Psilocybe, the so-called "magic mushrooms." The pharmaceutical interest in this psychotropic natural product as a future medication to treat depression and anxiety is strongly re-emerging. Here, we present an enhanced enzymatic route of psilocybin production by adding TrpB, the tryptophan synthase of the mushroom Psilocybe cubensis, to the reaction. We capitalized on its substrate flexibility and show psilocybin formation from 4-hydroxyindole and l-serine, which are less cost-intensive substrates, compared to the previous method. Furthermore, we show enzymatic production of 7-phosphoryloxytryptamine (isonorbaeocystin), a non-natural congener of the Psilocybe alkaloid norbaeocystin (4-phosphoryloxytryptamine), and of serotonin (5-hydroxytryptamine) by means of the same in vitro approach.
The fungal genus Psilocybe and other genera comprise numerous mushroom species that biosynthesize psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine). It represents the prodrug to its dephosphorylated psychotropic analogue, psilocin. The colloquial term "magic mushrooms" for these fungi alludes to their hallucinogenic effects and to their use as recreational drugs. However, clinical trials have recognized psilocybin as a valuable candidate to be developed into a medication against depression and anxiety. We here highlight its recently elucidated biosynthesis, the concurrently developed concept of enzymatic in vitro and heterologous in vivo production, along with previous synthetic routes. The prospect of psilocybin as a promising therapeutic may entail an increased demand, which can be met by biotechnological production. Therefore, we also briefly touch on psilocybin's therapeutic relevance and pharmacology.
Psilocybin and its direct precursor baeocystin are indole alkaloids of psychotropic Psilocybe mushrooms. The pharmaceutical interesti np silocybin as at reatment optiona gainst depression and anxiety is currently being investigated in advanced clinicalt rials. Here, we reportabiocatalytic route to synthesize 6-methylated psilocybin and baeocystin from 4-hydroxy-6methyl-l-tryptophan, which was decarboxylated and phosphorylated by the Psilocybec ubensis biosynthesis enzymes PsiD and PsiK. N-Methylation was catalyzed by PsiM. We further present an in silico structural model of PsiM that revealed aw ell-conserved SAM-binding core along with peripheraln onconserved elements that likely govern substrate preferences.The indole nucleus is ac ommon motif with bioactiven atural products that is, in numerous instances, modified duringb iosynthesis. Modifications include, for example, 7-chlorination in the case of rebeccamycin, prenyl transfer to various positions to make asterriquinones, or hydroxylation at C-5 and C-6 of serotonin and amanitins,r espectively.R eserpine, ibogaine, and harmaline are methoxy-substituted att hesec arbons. Dissimilar from the above positionsa nd substituents, psilocybin( 1, Scheme 1) features aunique 4-phosphoryloxy group.Compound 1 is the principal metabolite of Psilocybe carpophores ("magic mushrooms"), whereas baeocystin (2)a nd norbaeocystin (3), the immediate metabolic precursors, are present in minor quantities. [1] The dephosphorylated analogue of 1, psilocin (4), represents the pharmacologically active, that is, psychotropic compound as it is ap artial agonist at the human 5HT 2A receptor. [2] The pharmaceutical value of 1 in the treat-ment of end-of-life anxiety and therapy refractory depression has been documentedi nv ariousa dvanced clinicalt rials. [3] Syntheses of 1 congeners date back to the late 1950s. Following the discovery of 1 by Albert Hofmann and co-workers, various series of derivatives were synthesized,w hich also contained the 6-phosphoryloxy and 6-hydroxy isomerso f1 and 4,a long with a6 -methylated, side-chain-alkylated tryptamine. [4] Subsequent efforts were made to install bromine or af ormyl group at C-5-or C-7, vary the substituents at the w-N-atom of 4,o r add various substituentst ot he aromatic ring of N,N-dimethyltryptamine (5), to produce its 6-and 7-methyl analogues, among others. [5] Despite theser esultsa nd despite the synthesis of 2-methyl derivatives of 5 and N,N-diethyltryptamine, [6] none of the previouse fforts, using synthetic chemistry,h ad focused on introducing am ethyl substituent to the indole of 1 or 2.We report the in vitro synthesis of 6-methylpsilocybin (6, Scheme1)a nd, as main products,i ts immediate precursors 6methylnorbaeocystin (7)a nd 6-methylbaeocystin (8). We address the previous lack of ring-methylated 1 analogues and simultaneously provide am odel of the PsiM structure for insight into the origin of its methylating activity on 3 and 7.W ei dentified al oop structure as criticalf or methyl acceptors ubstrate selectivity.C oncu...
Psilocybe mushrooms are best known for their l-tryptophan-derived psychotropic alkaloid psilocybin. Dimethylation of norbaeocystin, the precursor of psilocybin, by the enzyme PsiM is a critical step during the biosynthesis of psilocybin. However, the "magic" mushroom Psilocybe serbica also mono- and dimethylates l-tryptophan, which is incompatible with the specificity of PsiM. Here, a second methyltransferase, TrpM, was identified and functionally characterized. Mono- and dimethylation activity on l-tryptophan was reconstituted in vitro, whereas tryptamine was rejected as a substrate. Therefore, we describe a second l-tryptophan-dependent pathway in Psilocybe that is not part of the biosynthesis of psilocybin. TrpM is unrelated to PsiM but originates from a retained ancient duplication event of a portion of the egtDB gene that encodes an ergothioneine biosynthesis enzyme. During mushroom evolution, this duplicated gene was widely lost but re-evolved sporadically and independently in various genera. We propose a new secondary metabolism evolvability mechanism, in which weakly selected genes are retained through preservation in a widely distributed, conserved pathway.
Bacterial-fungal interactions (BFIs) influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Using a simple BFI system encompassing the Gram-positive bacterium Bacillus subtilis and the black mould fungus Aspergillus niger, we established a co-culture experimental evolution method to investigate bacterial adaptation to the presence of a fungus. In the evolving populations, B. subtilis was rapidly selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles and RlmA-dependent cell wall stress induction. Increased surfactin production typically enhances the competitive success of bacteria against fungi, which likely explains the primary adaption path in the presence of A. niger.
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