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

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.

SUBMITTER: Milne N

PROVIDER: S-EPMC7232020 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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

Milne N N Thomsen P P Mølgaard Knudsen N N Rubaszka P P Kristensen M M Borodina I I

Metabolic engineering 20200326

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 ...[more]

PMID: 32224264

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Project description:BACKGROUND:(+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield. RESULTS:As the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to ?-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of ?-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing ?-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively. CONCLUSION:We successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.

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Dataset Information

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

Publications

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

Similar Datasets

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