Subsequently, additional psilocybin bio-synthetic clusters were found in distant fungal species and provide some evolutionary evidence of the ecological role of psilocybin in influencing mycophagy in animals, which is to reduce their consumption from invertebrate predators.Thus, the bio-activity of 1 may provide a fitness advantage to natural producers over their competitors. Further, a recent preprint presents evidence of a new, diverged psilocybin cluster in Inocybe corydalina that contains a second methyltransferase that may produce the trimethylated, quaternary ammonium salt analogue of 1, aeruginascin.Since the elucidation of the psilocybin bio-synthetic pathway, engineering efforts for high-titer production of psilocybin 1 in various microbial hosts, such as the filamentous fungus A. nidulans, Baker’s yeast S. cerevisiae, and the model bacterium E. coli have been reported.Hoefgen et al. developed a polycistronic expression system in A. nidulans and used the psilocybin pathway as a proof-of-concept. They obtained 110 mg/L of 1 at 1.5% dry mycelial weight which is a titer comparable to native psilocybin producers. Adams et al. were able to combine heterologous expression and metabolic engineering strategies to achieve a titer of 1.16 g/L psilocybin 1 in E. coli in a 1.5-L bioreactor from 3.05 g/L of gradually supplied 4-hydroxyindole over several days. The exogenously supplied is first converted into 4-hydroxy-L-tryptophan by TrpB,plant growing stand an endogenous bacterial enzyme in the L-tryptophan bio-synthetic pathway that catalyzes the condensation of indole with serine to form .
PsiD, PsiK, and PsiM from P. cubensis were heterologously expressed under a single T7 promoter on a high copy plasmid, which facilitated the conversion of formed in situ into psilocybin 1. Endogenous levels of serine and SAM, required by TrpB and PsiM, respectively, were not sufficient for high-titer production and thus the media was supplemented with excess amounts of serine and methionine. MAOs, as the name suggest, catalyze the oxidative deamination of neurotransmitters and structurally similar compounds.This synergy is best demonstrated by ayahuasca, a psychoactive decoction commonly prepared from the vine Banisteriopsis caapi, containing MAO inhibiting harmala alkaloids, and some DMT containing species such as the shrub Psychotria viridis.Ayahuasca, derived from a Quechua term meaning “vine of the soul,” has been used as a spiritual medicine by indigenous groups in South America’s Amazon basin for at least one thousand years.During a ceremony in which the brew is ingested, practitioners experience several stages of visual and purgative experiences in order to heal physical, emotional, and spiritual imbalances.While there are no currently approved therapeutic uses for ayahuasca or its active metabolites, harmala alkaloids have shown promise as an antidepressant through brain plasticity modulation.The harmala alkaloids are compounds that contain a β-carboline scaffold with various methyl or methoxy substitutions and different degrees of unsaturation. The β-carbolinescaffold itself is characterized by a pyridine ring ortho-fused to indole resulting in a 6-5-6 tricycle with possible substitutions at the ortho position to the pyridine nitrogen.
The major harmala alkaloids that contribute to the MAOI activity are harmine , harmaline, and tetrahydroharmine . These compounds are abundant at ~ 0.05 – 0.1% of dried plant material in B. caapi .Thorough pharmacokinetic data is scarce, but psychotropic action of harmala alkaloids is expected to occur around 20–50 mg with a typical 100 mL ayahuasca brew containing between ~300–600 mg of harmala alkaloids and 20–60 mg of 29. Another example of a MAOI natural product cocktail is the recent isolation of and related β-carbolines from numerous hallucinogen-producing Psilocybe sp. as known as “magic mushrooms”.This serves as an interesting example of a single organism with diverged secondary metabolite scaffolds, where the bio-synthetic pathways of both compounds diverge at tryptamine but contribute to the same psychoactive effect.Initial feeding studies with radioactively labelled substrates into seedlings of the known harmala alkaloid producer, Peganum harmala, demonstrated that L-tryptophan and L-methionine are precursors in biosynthesis of harmala alkaloids.A later study demonstrated that radiolabeled could be converted into its dehydrogenated form, and that harmala alkaloid biosynthesis is likely compartmentalized across different tissues.While the complete set of bio-synthetic genes implicated in harmala alkaloid formation have yet to be determined, one proposal postulates the sequence shown in Fig. 17. As in the case of the other indole containing compounds described, is first decarboxylated to form . Next, pyruvic acid 22 is incorporated by a Mannich or Pictet-Spengler type reaction to form the β-carboline carboxylic acid. To determine the bio-synthetic origin of the C-1 β-carboline methyl, radiolabeled feeding of acetic acid and pyruvic acid was performed.
Stolle et al. observed specific incorporation of the radiolabeled C-2 and C-3 carbons of pyruvic acid into the pyridine ring of the β-carboline scaffold, while radiolabeled acetic acid carbons were non-specifically incorporated throughout as a result of primary metabolism. 1-methyl β-carboline is then formed by oxidative decarboxylation, followed by subsequent hydroxylation and O-methylation reactions to form harmaline . Formation of harmine or tetrahydroharmine takes places through either oxidation, or reduction of , respectively. Lysergic acid diethylamide 3 was first synthesized from lysergic acid by Albert Hofmann in 1938. Like other 5HT2A receptor agonists, ingestion of 3 results in altered states of consciousness and visual hallucinations.While 3 has not been observed to occur naturally, its precursor, is a natural product belonging to a class of diverse molecules broadly known as ergot alkaloids is isolated from many fungi with the ergot fungus, Claviceps purpurea being the most notable.Ergot alkaloids are commonly associated with the disease ergotism, known colloquially as Saint Anthony’s Fire, caused by eating rye or other cereal crops contaminated with ergot fungi.In addition to the vasoconstrictive and convulsive symptoms of the disease, mania and psychosis have been observed, underlining the psychoactivity of ergot alkaloids.Ergot alkaloids, derived from L-tryptophan 11, are characterized by a unique tetracyclic ergoline skeleton where the indole comprises the A and B rings. The C and D rings of the ergoline scaffold are derived from a cyclization of dimethylallyl pyrophosphate with the Ltryptophan amino group.There are three main ergot alkaloid classes, clavines, ergoamides , and ergopeptides, with 3 belonging to the ergoamide class.Ergoamides contain a C8-amide linkage on the D ring of the ergoline scaffold and is a common point of derivatization for drug development.Modifications on the amide can greatly affect bio-activity and in the case of 3, the diethylamide moiety is crucial for its prolonged psychoactivity.Isotope labeling studies during the 1950s and 1960s determined that a mevalonate acid-derived isoprenoid,plant grow table a methionine-derived methyl group and L-tryptophan 11 were key precursors to ergot alkaloid biosynthesis.The first enzymatic study in Claviceps sp. was the purification and characterization of 4- dimethylallyl- L-tryptophan synthetase that catalyzes the first committed step in ergot alkaloid biosynthesis: C-prenylation of L-tryptophan 11 with dimethylallylpyrophosphate at the indole C4 position to form 4-dimethylallyl-L-tryptophan.Recently, many laboratories have focused on characterizing prenyltransferases, of which DMATS is the original member of a new superfamily of prenyltransferase enzymes. Since the discovery of the DMATS, prenyltransferases that can regioselectively transfer allylic prenyl groups to almost every position on the indole ring have been identified.Members of the DMATS super family also have broad substrate scopes while maintaining regioselectivity which has aided in their development as tools for chemoenzymatic syntheses of natural and unnatural prenylated compounds, including the cannabinoid family .
Chromosome walking using the gene encoding DMATS as a step-off point led to the identification of an ergot alkaloid bio-synthetic gene cluster in the fungus C. purpurea. Sequence alignment revealed an N-methyltransferase, EasF which was proposed to convert 4-dimethylallyl-L-tryptophan into 4-dimethylallyl-L-abrine using SAM as a methyl donor. Thorough characterization of a homologous enzyme in an Aspergillus fumigatus ergot gene cluster, FgaMT, supported this hypothesis.Conversion of into the cyclized chanoclavine-I is facilitated by the FAD-linked oxidoreductase EasE and EasC, which was initially annotated as a catalase. Knock-out studies in both C. purpurea and the homologous cluster in A. fumigatus confirmed that both enzymes are necessary for production of 57. Subsequent pathway reconstitution studies in Aspergillus nidulans and Saccharomyces cerevisiae further supported the essential roles of EasE and EasC in biosynthesis.Until recently, however, the precise mechanisms of EasE and EasC were not resolved. Lorenz et al. initially postulated that EasE catalyzes the oxidative diene formation from followed by decarboxylation through an epoxide intermediate to yield chanoclvaine-I , with EasC serving as a scavenger of hydrogen peroxide generated from EasE.A recent pathway reconstitution in A. nidulans enabled isolation of the a previously unknown intermediate, pre-chanoclavine diene , which verified the diene formation activity of EasE.Subsequent incubation of 58 with EasC recombinantly purified from E. coli led to the formation of 57 via a proposed radical addition mechanism using O2 as an oxidant.Hence, EasC is an essential redox enzyme in the main pathway to 54. A short-chain reductase , FgaDH, was identified in an A. fumigatus gene cluster that produces a related ergot alkaloid fumigaclavine C.193 In vitro assays using recombinantly expressed enzyme determined that FgaDH catalyzes the oxidation of the allylic alcohol on 57 to an aldehyde to form chanoclavine-I aldehyde 59, strictly using NAD+ as the electron acceptor.A homologous SDR was subsequently identified in the lysergic acid bio-synthetic gene cluster in C. purpurea and named EasD.The next steps in the pathway represent a branching point for ergot alkaloids. Functional differences in a conserved flavin-dependent old yellow enzyme known as EasA from C. purpurea and FgaOx3 from A. fumigatus and P. commune represent a mechanistic branching point in D-ring formation.Here we will focus on the formation of agroclavine 61 from 59 towards the psychoactive lysergic acid amides in C. purpurea. EasA performs a hydride mediated isomerization of the α,β-unsaturated carbonyl from the E-alkene geometry to the Z-configuration through an enolate intermediate.This rearrangement positions the carbonyl for an intramolecular cyclization with the secondary amine resulting in the formation of the D-ring.Following ring closure, the iminium intermediate agroclavinium then undergoes NADPH-dependent reduction by the oxidoreductase EasG to form. Assays of microsomal fractions from C. purpurea determined that undergoes a 2-electron oxidation of the methyl group to an alcohol to form elymoclavine by an unidentified cytochrome P450 monooxygenase.The only P450 enzyme in the bio-synthetic gene cluster, CloA, does not catalyze this transformation and instead performs the 4-electron oxidation of to paspalic acid as suggested from two knock-out studies.In ΔcloA mutants, was still detected and supports the likelihood of an additional P450 enzyme in the host that can perform the first 2-electron oxidation. Finally, allylic isomerization of forms the product lysergic acid. This transformation can occur spontaneously, but it remains possible that that an unidentified isomerase can catalyze this reaction as enzymecatalyzed allylic rearrangements have been observed in other pathways. Itself serves as a branching point for the formation of many ergopeptines or ergoamides. These derivatives are formed by a non-ribosomal peptide synthase enzyme complex of two synthetases, LPS1 and LPS2.One of these lysergic acid derivatives from Ipomoea purpurea , ergine is psychoactive. The pathway leading to formation of 64, while unconfirmed, could involve amidation by an NRPS or degradation of another NRPS product.Peoples indigenous to North America have consumed the cactus, peyote, for over one thousand years as a part of their religious practices.Peyote, Lophophora williamsii , is a small, spineless cactus with a crown consisting of round buttons that, among other cacti species, contain the hallucinogen, mescaline. The psychoactive effects have been described to be similar to LSD, but with a significantly lower potency at a ratio of about 1:2500 mescaline:LSD.Despite peyote’s status as a Schedule I controlled substance in the United States, it remains legal as an important part of religious practices by the Native American Church and other religious organizations who are protected by the American Indian Religious Freedom Act. The natural products, elemicin and myristicin from nutmeg, or Myristica fragrans, are tetrasubstituted benzenes and structurally related to 65. Despite not being psychoactive, and are believed to be prodrugs as they are metabolized in the liver into 3-methoxy-4,5-methylenedioxyamphetamine, also known as MMDA.MMDA and its analogs were first synthesized from 65 by Alexander Shulgin, and similar to 65, MMDA is a 5HT2A receptor agonist, but with almost double the potency.Shulgin would later detail his extensive clandestine investigations into the syntheses and effects of substituted phenethylamines and tryptamines, earning him the title “godfather of psychedelics.”Before the discovery of the mammalian iterative methyltransferase that catalyzes N-methylation of tryptamine 14 and serotonin 38 into hallucinogenic compounds,Axelrod and Tomchick identified another neurotransmitter methyltransferase, catechol O-methyltransferase .