RESUMO
Psilocybin, a drug most commonly recognized as a recreational psychedelic, is quickly gaining attention as a promising therapy for an expanding range of neurological conditions, including depression, anxiety, and addiction. This growing interest has led to many recent advancements in psilocybin synthesis strategies, including multiple in vivo fermentation-based approaches catalyzed by recombinant microorganisms. In this work, we show that psilocybin can be produced in biologically relevant quantities using a recombinant E. coli strain in a homebrew style environment. In less than 2 days, we successfully produced approximately 300 mg/L of psilocybin under simple conditions with easily sourced equipment and supplies. This finding raises the question of how this new technology should be regulated as to not facilitate clandestine biosynthesis efforts, while still enabling advancements in psilocybin synthesis technology for pharmaceutical applications. Here, we present our homebrew results, and suggestions on how to address the regulatory concerns accompanying this new technology.
Assuntos
Escherichia coli/metabolismo , Alucinógenos/metabolismo , Engenharia Metabólica/métodos , Preparações Farmacêuticas/metabolismo , Psilocibina/biossíntese , Escherichia coli/crescimento & desenvolvimento , Fermentação , HumanosRESUMO
Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) is an indole-based secondary metabolite produced by numerous species of mushrooms. South American Aztec Indians referred to them as teonanacatl, meaning "god's flesh," and they were used in religious and healing rituals. Spanish missionaries in the 1500s attempted to destroy all records and evidence of the use of these mushrooms. Nevertheless, a 16th century Spanish Franciscan friar and historian mentioned teonanacatl in his extensive writings, intriguing 20th century ethnopharmacologists and leading to a decades-long search for the identity of teonanacatl. Their search ultimately led to a 1957 photo-essay in a popular magazine, describing for the Western world the use of these mushrooms. Specimens were ultimately obtained, and their active principle identified and chemically synthesized. In the past 10-15 years several FDA-approved clinical studies have indicated potential medical value for psilocybin-assisted psychotherapy in treating depression, anxiety, and certain addictions. At present, assuming that the early clinical studies can be validated by larger studies, psilocybin is poised to make a significant impact on treatments available to psychiatric medicine.
Assuntos
Alucinógenos/história , Psilocibina/história , Agaricales/química , Alucinógenos/isolamento & purificação , História do Século XV , História do Século XX , Humanos , Psilocibina/análogos & derivados , Psilocibina/biossíntese , Psilocibina/síntese química , Psilocibina/isolamento & purificaçãoRESUMO
Psilocybin is a tryptamine-derived psychoactive alkaloid found mainly in the fungal genus Psilocybe, among others, and is the active ingredient in so-called "magic mushrooms". Although its notoriety originates from its psychotropic properties and popular use as a recreational drug, clinical trials have recently recognized psilocybin as a promising candidate for the treatment of various psychological and neurological afflictions. In this work, we demonstrate the de novo biosynthetic production of psilocybin and related tryptamine derivatives in Saccharomyces cerevisiae by expression of a heterologous biosynthesis pathway sourced from Psilocybe cubensis. Additionally, we achieve improved product titers by supplementing the pathway with a novel cytochrome P450 reductase from P. cubensis. Further rational engineering resulted in a final production strain producing 627 ± 140 mg/L of psilocybin and 580 ± 276 mg/L of the dephosphorylated degradation product psilocin in triplicate controlled fed-batch fermentations in minimal synthetic media. Pathway intermediates baeocystin, nor norbaeocystin as well the dephosphorylated baeocystin degradation product norpsilocin were also detected in strains engineered for psilocybin production. We also demonstrate the biosynthetic production of natural tryptamine derivative aeruginascin as well as the production of a new-to-nature tryptamine derivative N-acetyl-4-hydroxytryptamine. These results lay the foundation for the biotechnological production of psilocybin in a controlled environment for pharmaceutical applications, and provide a starting point for the biosynthetic production of other tryptamine derivatives of therapeutic relevance.
Assuntos
Engenharia Metabólica/métodos , Psilocibina/análogos & derivados , Psilocibina/biossíntese , Saccharomyces cerevisiae/metabolismo , Triptaminas/biossíntese , Escherichia coli/metabolismo , Fermentação , NADPH-Ferri-Hemoproteína Redutase/biossíntese , NADPH-Ferri-Hemoproteína Redutase/genética , Psilocybe/genética , Psilocybe/metabolismo , Psilocibina/metabolismo , Triptofano/metabolismoRESUMO
Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment-resistant depression and major depressive disorder. The psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot-scale protocol for recombinant PsiK that yielded 150â mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost.
Assuntos
Agaricales/química , Transtorno Depressivo Maior/tratamento farmacológico , Psilocybe/química , Psilocibina/análogos & derivados , Psilocibina/química , Triptaminas/química , Biocatálise , Humanos , Psilocibina/biossíntese , Triptaminas/metabolismoRESUMO
Psychotropic Psilocybe mushrooms biosynthesize their principal natural product psilocybin in five steps, among them a phosphotransfer and two methyltransfer reactions, which consume one equivalent of 5'-adenosine triphosphate (ATP) and two equivalents of S-adenosyl-l-methionine (SAM). This short but co-substrate-intensive pathway requires nucleoside cofactor salvage to maintain high psilocybin production rates. We characterized the adenosine kinase (AdoK) and S-adenosyl-l-homocysteine (SAH) hydrolase (SahH) of Psilocybe cubensis. Both enzymes are directly or indirectly involved in regenerating SAM. qRT-PCR expression analysis revealed an induced expression of the genes in the fungal primordia and carpophores. A one-pot in vitro reaction with the N-methyltransferase PsiM of the psilocybin pathway demonstrates a concerted action with SahH to facilitate biosynthesis by removal of accumulating SAH.
Assuntos
Adenosina Quinase/metabolismo , Adenosina/metabolismo , Adenosil-Homocisteinase/metabolismo , Psilocybe/enzimologia , Psilocibina/biossíntese , S-Adenosilmetionina/metabolismo , Adenosina Quinase/genética , Adenosil-Homocisteinase/genética , Perfilação da Expressão Gênica , Psilocybe/genéticaRESUMO
Psilocybin, the prodrug of the psychoactive molecule psilocin, has demonstrated promising results in clinical trials for the treatment of addiction, depression, and post-traumatic stress disorder. The development of a psilocybin production platform in a highly engineerable microbe could lead to rapid advances towards the bioproduction of psilocybin for use in ongoing clinical trials. Here, we present the development of a modular biosynthetic production platform in the model microbe, Escherichia coli. Efforts to optimize and improve pathway performance using multiple genetic optimization techniques were evaluated, resulting in a 32-fold improvement in psilocybin titer. Further enhancements to this genetically superior strain were achieved through fermentation optimization, ultimately resulting in a fed-batch fermentation study, with a production titer of 1.16â¯g/L of psilocybin. This is the highest psilocybin titer achieved to date from a recombinant organism and a significant step towards demonstrating the feasibility of industrial production of biologically-derived psilocybin.
Assuntos
Técnicas de Cultura Celular por Lotes , Escherichia coli , Engenharia Metabólica , Psilocibina , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Psilocibina/biossíntese , Psilocibina/genéticaRESUMO
Aromatic l-amino acid decarboxylases (AAADs) are a phylogenetically diverse group of enzymes responsible for the decarboxylation of aromatic amino acid substrates into their corresponding aromatic arylalkylamines. AAADs have been extensively studied in mammals and plants as they catalyze the first step in the production of neurotransmitters and bioactive phytochemicals, respectively. Unlike mammals and plants, the hallucinogenic psilocybin mushroom Psilocybe cubensis reportedly employs an unrelated phosphatidylserine-decarboxylase-like enzyme to catalyze l-tryptophan decarboxylation, the first step in psilocybin biosynthesis. To explore the origin of this chemistry in psilocybin mushroom, we generated the first de novo transcriptomes of P. cubensis and investigated several putative l-tryptophan-decarboxylase-like enzymes. We report the biochemical characterization of a noncanonical AAAD from P. cubensis ( PcncAAAD) that exhibits substrate permissiveness toward l-phenylalanine, l-tyrosine, and l-tryptophan, as well as chloro-tryptophan derivatives. The crystal structure of PcncAAAD revealed the presence of a unique C-terminal appendage domain featuring a novel double-ß-barrel fold. This domain is required for PcncAAAD activity and regulates catalytic rate and thermal stability through calcium binding. PcncAAAD likely plays a role in psilocybin production in P. cubensis and offers a new tool for metabolic engineering of aromatic-amino-acid-derived natural products.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/química , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Cálcio/metabolismo , Psilocybe/enzimologia , Psilocibina/biossíntese , Aminoácidos Aromáticos/metabolismo , Descarboxilases de Aminoácido-L-Aromático/genética , Domínio Catalítico , Catharanthus/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Papaver/enzimologia , Filogenia , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Psilocybe/genética , Especificidade por Substrato , Transcriptoma , Leveduras/genéticaRESUMO
Heterologous expression of multi-gene biosynthetic pathways in eukaryotic hosts is limited by highly regulated individual monocistrons. Dissimilar to prokaryotes, each eukaryotic gene is strictly controlled by its own regulatory elements, such as promoter and terminator. Consequently, parallel transcription can occur only when a group of genes is synchronously activated. A strategy to circumvent this limitation is the concerted expression of multiple genes as a polycistron. By exploiting the "stop-carry on" mechanism of picornaviruses, we have designed a sophisticated, yet easy-to-assemble vector system to heterologously express multiple genes under the control of a single promoter. For facile selection of correctly transformed colonies by basic fluorescence microscopy, our vector includes a split gene for a fluorescent reporter protein. This method was successfully applied to produce the psychotropic mushroom alkaloid psilocybin in high yields by heterologous expression of the entire biosynthetic gene cluster in the mould Aspergillus nidulans.
Assuntos
Aspergillus nidulans , Expressão Gênica , Genes Reporter , Engenharia Genética/métodos , Proteínas de Fluorescência Verde , Regiões Promotoras Genéticas , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Psilocibina/biossíntese , Psilocibina/genéticaRESUMO
Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60â years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i)â PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii)â PsiK, which catalyzes the phosphotransfer step, iii)â the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv)â 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.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Alucinógenos/metabolismo , Metiltransferases/metabolismo , Oxigenases de Função Mista/metabolismo , Psilocybe/enzimologia , Psilocibina/biossíntese , 5-Hidroxitriptofano/química , Catálise , Cromatografia Líquida/métodos , Genes Fúngicos , Espectrometria de Massas/métodos , Psilocybe/genética , S-Adenosilmetionina/metabolismo , Especificidade por SubstratoAssuntos
Bufotenina/biossíntese , Psilocibina/análogos & derivados , Serotonina/análogos & derivados , Serotonina/biossíntese , Amanita/metabolismo , Animais , Anuros/metabolismo , Áustria , Basidiomycota/metabolismo , Brasil , Bufonidae/metabolismo , Bufotenina/história , Química , China , História do Século XVII , História do Século XIX , História do Século XX , História Antiga , Hidroxilação , Isomerismo , Israel , Mamíferos/metabolismo , Psilocibina/biossíntese , Cidade de Roma , Estados UnidosRESUMO
Sixty collections of ten species referred to three families of the Agaricales have been analyzed for the presence of baeocystin by thin-layer chromatography. Baeocystin was detected in collections of Psilocybe, Conocybe, and Panaeolus from the U.S.A., Canada, Mexico, and Peru. Laboratory cultivated fruitbodies of Psilocybe cubensis, P. semilanceata, and P. cyanescens were also studied. Intra-species variation in the presence of decay rate of baeocystin, psilocybin and psilocin are discussed in terms of age and storage factors. In addition, evidence is presented to support the presence of 4-hydroxytryptamine in collections of P. baeocystis and P. cyanescens. The possible significance of baeocystin and 4-hydroxytryptamine in the biosynthesis of psilocybin in these organisms is discussed.