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Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives - PubMed

Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives

N Milne et al. Metab Eng. 2020 Jul.

Abstract

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.

Keywords: Aeruginascin; Baeocystin; Metabolic engineering; Norbaeocystin; Psilocybe cubensis; Psilocybin; Saccharomyces cerevisiae; Tryptamine derivatives.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of competing interest NM and IB are co-inventors on a patent application related to this research.

Figures

Fig. 1
Fig. 1

Psilocybin route of administration in humans. After biosynthesis in Psilocybe mushroom species (or in this case S. cerevisiae), psilocybin is typically administered by oral ingestion. Upon consumption, psilocybin acts as a prodrug where alkaline phosphatases, non-specific esterases, or the acidic conditions in the stomach convert the molecule to the bioactive psilocin. Psilocin then exerts its psychotropic or therapeutic effect by crossing the blood-brain barrier and interacting with serotonin receptors. Psilocin is eventually removed from the body via glucuronidation and excretion through the kidneys (Manevski et al., 2010).

Fig. 2
Fig. 2

Psilocybin biosynthesis in S. cerevisiae. The heterologous biosynthetic pathway begins with the native production of tryptophan, which itself is derived from metabolites produced via glycolysis, the pentose phosphate pathway, and the shikimate pathway. DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate; EPSP, 5-enolpyruvoyl-shikimate 3-phosphate; Glyc. 3-P, glyceraldehyde 3-phosphate; Ala, alanine; Glu, glutamate; Tyr, tyrosine; Phe, phenylalanine. Multiple arrows represent multiple enzymatic reactions grouped for simplicity.

Fig. 3
Fig. 3

De novo psilocybin production in S. cerevisiae (A). LC-MS chromatograms confirming psilocybin, psilocin and tryptamine production in ST9327 (psilocybin biosynthetic pathway) compared to wild-type control strain ST9326 using authentic analytical standards. (B). Corresponding mass spectra for psilocybin, psilocin and tryptamine peaks in ST9327.

Fig. 4
Fig. 4

Improved De novo psilocybin biosynthesis in S. cerevisiae. (A). Introduction of the heterologous biosynthesis pathway and corresponding final titers in micro-titer plate cultivation. ST9326, wild-type parental strain; ST9327, psilocybin biosynthetic pathway (CrTdc, PcPsiH, PcPsiK, PcPsiM); ST9649, psilocybin biosynthetic pathway + NCP1 expressed from TEF1 promoter; ST9330, psilocybin biosynthetic pathway + A. thaliana CPR (AtAtr2) expressed from TEF1 promoter; ST9329, psilocybin biosynthetic pathway + P. cubensis CPR expressed from TEF2 promoter (pTEF2→PcCpr); ST9328, psilocybin biosynthetic pathway + P. cubensis CPR expressed from TEF1 promoter (pTEF1→PcCpr). (B). Iterative strain improvement to increase tryptophan availability and overcome rate-limiting reactions with resulting final titers in micro-titer plate cultivation. Gene names represent genes that were expressed from strong constitutive promoters. Strains were cultivated in synthetic media with 20 g/L glucose for 5 days and subjected to acetonitrile extraction and analysis by LC-MS. Media was supplemented with uracil when required. Data is presented as averages and standard deviations from biological duplicates. *; Not detected. Heterologous pathway; Strain expressing Crtdc, PcpsiH, PcpsiK, PcpsiM and Pccpr from the TEF1 promoter.

Fig. 5
Fig. 5

Controlled fed-batch fermentation results in higher titers. Production data from fed-batch fermentations of ST9482. Data is presented as averages from triplicate fermentations with standard deviations presented in shaded colours.

Fig. 6
Fig. 6

Production of 4-hydroxytryptamine derivatives and accumulation of psilocybin pathway intermediates in engineered S. cerevisiae strains. LC-MS chromatograms and corresponding mass spectra for (A) Norbaeocystin, (B) Baeocystin, (C) Norpsilocin, (D) Dephosphorylated aeruginascin, and (E) N-acetyl-4-hydroxytryptamine produced in engineered S. cerevisiae strains ST9326 (Wild-type control), ST9346 (4-hydroxytryptamine control) ST9328 (Crtdc, PcpsiH, Pccpr, PcpsiK, PcpsiM), ST9335 (Crtdc, PcpsiH, Pccpr, PcpsiK, PcpsiM multi-copy), ST9442 (Crtdc, PcpsiH, Pccpr, BtAANAT multi-copy).

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References

    1. Adams A.M., Kaplan N.A., Wei Z., Brinton J.D., Monnier C.S., Enacopol A.L., Ramelot T.A., Jones J.A. In vivo production of psilocybin in E . coli. Metab. Eng. 2019;56:111–119. doi: 10.1016/j.ymben.2019.09.009. - DOI - PubMed
    1. Averesch N.J.H., Krömer J.O. Metabolic engineering of the shikimate pathway for production of aromatics and derived compounds—present and future strain construction strategies. Front. Bioeng. Biotechnol. 2018;6 doi: 10.3389/fbioe.2018.00032. - DOI - PMC - PubMed
    1. Bigwood J., Beug M.W. Variation of psilocybin and psilocin levels with repeated flushes (harvests) of mature sporocarps of Psilocybe cubensis (earle) singer. J. Ethnopharmacol. 1982;5:287–291. doi: 10.1016/0378-8741(82)90014-9. - DOI - PubMed
    1. Bogenschutz M.P., Forcehimes A.A., Pommy J.A., Wilcox C.E., Barbosa P., Strassman R.J. Psilocybin-assisted treatment for alcohol dependence: a proof-of-concept study. J. Psychopharmacol. 2015;29:289–299. doi: 10.1177/0269881114565144. - DOI - PubMed
    1. Borodina I., Nielsen J. Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals. Biotechnol. J. 2014;9:609–620. doi: 10.1002/biot.201300445. - DOI - PubMed

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