Unlocking the biosynthesis of psychedelic-inspired indolethylamines.

A recent report by Chen et al. describes the discovery of RmNMT, a highly active and promiscuous tryptamine N-methyltransferase from the cane toad, Rhinella marina. N,N-dimethyltryptamine derivatives produced by this enzyme were then evaluated for their potential to serve as next-generation treatments for mental health disorders.

Simultaneous glucose and xylose utilization by an Escherichia coli catabolite repression mutant.

As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation of the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutant of the gene encoding the cyclic AMP receptor protein, crp*, has been shown to disable CCR and improve the co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site-specific crp* chromosomal mutant in E. coli BL21 star (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomics further showed that glucose was routed to the C5 carbon utilization pathways to support both de novo nucleotide synthesis and energy production in the crp* mutant strain. Metabolite analyses further show that overflow metabolism contributes to the slower growth in the crp* mutant. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass.IMPORTANCEAs the need for renewable biofuel and biochemical production processes continues to grow, there is an associated need for microbial technology capable of utilizing cheap, widely available, and renewable carbon substrates. This work details the construction and characterization of the first B-lineage Escherichia coli strain with mutated cyclic AMP receptor protein, Crp*, which deregulates the carbon catabolite repression (CCR) system and enables the co-utilization of multiple sugar sources in the growth medium. In this study, we focus our analysis on glucose and xylose utilization as these two sugars are the primary components in lignocellulosic biomass hydrolysate, a promising renewable carbon feedstock for industrial bioprocesses. This strain is valuable to the field as it enables the use of mixed sugar sources in traditional fed-batch based approaches, whereas the wild-type carbon catabolite repression system leads to biphasic growth and possible buildup of non-preferential sugars, reducing process efficiency at scale.

"In vivo biosynthesis of N,N-dimethyltryptamine, 5-MeO-N,N-dimethyltryptamine, and bufotenine in E.coli".

N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and 5-hydroxy-N,N-dimethyltryptamine (bufotenine) are psychedelic tryptamines found naturally in both plants and animals and have shown clinical potential to help treat mental disorders, such as anxiety and depression. Advances in both metabolic and genetic engineering make it possible to engineer microbes as cell factories to produce DMT and its aforementioned derivatives to meet demand for ongoing clinical study. Here, we present the development of a biosynthetic production pathway for DMT, 5-MeO-DMT, and bufotenine in the model microbe Escherichia coli. Through the application of genetic optimization techniques and process optimization in benchtop fermenters, the in vivo production of DMT in E. coli was observed. DMT production with tryptophan supplementation reached maximum titers of 74.7 ± 10.5 mg/L under fed batch conditions in a 2-L bioreactor. Additionally, we show the first reported case of de novo production of DMT (from glucose) in E. coli at a maximum titer of 14.0 mg/L and report the first example of microbial 5-MeO-DMT and bufotenine production in vivo. This work provides a starting point for further genetic and fermentation optimization studies with the goal to increase methylated tryptamine production metrics to industrially competitive levels.

"Biosynthesis of psilocybin and its nonnatural derivatives by a promiscuous psilocybin synthesis pathway in Escherichia coli".

Traditional psychedelics are undergoing a transformation from recreational drugs, to promising pharmaceutical drug candidates with the potential to provide an alternative treatment option for individuals struggling with mental illness. Sustainable and economic production methods are thus needed to facilitate enhanced study of these drug candidates to support future clinical efforts. Here, we expand upon current bacterial psilocybin biosynthesis by incorporating the cytochrome P450 monooxygenase, PsiH, to enable the de novo production of psilocybin as well as the biosynthesis of 13 psilocybin derivatives. The substrate promiscuity of the psilocybin biosynthesis pathway was comprehensively probed by using a library of 49 single-substituted indole derivatives, providing biophysical insights to this understudied metabolic pathway and opening the door to the in vivo biological synthesis of a library of previously unstudied pharmaceutical drug candidates.

In vivo production of psilocybin in E. coli.

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 1.16 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.

Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli.

Robust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering.

Deciphering flux adjustments of engineered E. coli cells during fermentation with changing growth conditions.

Microbial fermentation conditions are dynamic, due to transcriptional induction, nutrient consumption, or changes to incubation conditions. In this study, 13C-metabolic flux analysis was used to characterize two violacein-producing E. coli strains with vastly different productivities, and to profile their metabolic adjustments resulting from external perturbations during fermentation. The two strains were first grown at 37°C in stage 1, and then the temperature was transitioned to 20°C in stage 2 for the optimal expression of the violacein synthesis pathway. After induction, violacein production was minimal in stage 3, but accelerated in stage 4 (early production phase) and 5 (late production phase) in the high producing strain, reaching a final concentration of 1.5mmol/L. On the contrary, ~0.02mmol/L of violacein was obtained from the low producing strain. To have a snapshot of the temporal metabolic changes in each stage, we performed 13C-MFA via isotopomer analysis of fast-turnover free metabolites. The results indicate strikingly stable flux ratios in the central metabolism throughout the early growth stages. In the late stages, however, the high producer rewired its flux distribution significantly, which featured an upregulated pentose phosphate pathway and TCA cycle, reflux from acetate utilization, negligible anabolic fluxes, and elevated maintenance loss, to compensate for nutrient depletion and drainage of some building blocks due to violacein overproduction. The low producer with stronger promoters shifted its relative fluxes in stage 5 by enhancing the flux through the TCA cycle and acetate overflow, while exhibiting a reduced biomass growth and a minimal flux towards violacein synthesis. Interestingly, the addition of the violacein precursor (tryptophan) in the medium inhibited high producer but enhanced low producer's productivity, leading to hypotheses of unknown pathway regulations (such as metabolite channeling).

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.

Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation.

Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc.

Naringenin-responsive riboswitch-based fluorescent biosensor module for Escherichia coli co-cultures.

The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture. Our efforts helped identify a number of key operating parameters that affect biosensor performance, including the selection of promoter and linker elements within the sensor-actuator domain, and the effect of host strain, fermentation time, and growth medium on sensor dynamic range. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin titer produced by the second member of the synthetic co-culture system. This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified. The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design. Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for re-optimization of the producer module when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114: 2235-2244. © 2017 Wiley Periodicals, Inc.

Experimental and computational optimization of an Escherichia coli co-culture for the efficient production of flavonoids.

Metabolic engineering and synthetic biology have enabled the use of microbial production platforms for the renewable production of many high-value natural products. Titers and yields, however, are often too low to result in commercially viable processes. Microbial co-cultures have the ability to distribute metabolic burden and allow for modular specific optimization in a way that is not possible through traditional monoculture fermentation methods. Here, we present an Escherichia coli co-culture for the efficient production of flavonoids in vivo, resulting in a 970-fold improvement in titer of flavan-3-ols over previously published monoculture production. To accomplish this improvement in titer, factors such as strain compatibility, carbon source, temperature, induction point, and inoculation ratio were initially optimized. The development of an empirical scaled-Gaussian model based on the initial optimization data was then implemented to predict the optimum point for the system. Experimental verification of the model predictions resulted in a 65% improvement in titer, to 40.7±0.1mg/L flavan-3-ols, over the previous optimum. Overall, this study demonstrates the first application of the co-culture production of flavonoids, the most in-depth co-culture optimization to date, and the first application of empirical systems modeling for improvement of titers from a co-culture system.

Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering.

Reconstruction of highly efficient biosynthesis pathways is essential for the production of valuable plant secondary metabolites in recombinant microorganisms. In order to improve the titer of green tea catechins in Escherichia coli, combinatorial strategies were employed using the ePathBrick vectors to express the committed catechin pathway: flavanone 3ß-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR). Three F3H, three DFR, and two LAR genes originating from different plant species were selected and synthesized, to create 18 pathway variants to be screened in E. coli. Constructs containing F3H(syn) originally from Camellia sinensis, DFR(syn) from Anthurium andraeanum, C. sinensis, or Fragaria ananass, and LAR(syn) from Desmodium uncinatum (p148, p158 and p168) demonstrated high conversion efficiency with either eriodictyol or naringenin as substrate. A highly efficient construct was created by assembling additional copies of DFR(syn) and LAR(syn) enabling a titer of 374.6 ± 43.6 mg/L of (+)-catechin. Improving the NADPH availability via the ΔpgiΔppc mutation, BLΔpgiΔppc-p148 produced the highest titer of catechin at 760.9 ± 84.3 mg/L. After utilizing a library of scaffolding proteins, the strain BLΔpgiΔppc-p168-759 reached the highest titer of (+)-catechin of 910.9 ± 61.3 mg/L from 1.0 g/L of eriodictyol in batch culture with M9 minimal media. The impact of oxygen availability on the biosynthesis of catechin was also investigated.

Effect of oral tryptamines on the gut microbiome of rats-a preliminary study.

Background: Psilocybin and related tryptamines have come into the spotlight in recent years as potential therapeutics for depression. Research on the mechanisms of these effects has historically focused on the direct effects of these drugs on neural processes. However, in addition to such neural effects, alterations in peripheral physiology may also contribute to their therapeutic effects. In particular, substantial support exists for a gut microbiome-mediated pathway for the antidepressant efficacy of other drug classes, but no prior studies have determined the effects of tryptamines on microbiota. Methods: To address this gap, in this preliminary study, male Long Evans rats were treated with varying dosages of oral psilocybin (0.2 or 2 mg/kg), norbaeocystin (0.25 or 2.52 mg/kg), or vehicle and their fecal samples were collected 1 week and 3 weeks after exposure for microbiome analysis using integrated 16S ribosomal DNA sequencing to determine gut microbiome composition. Results: We found that although treatment with neither psilocybin nor norbaeocystin significantly affected overall microbiome diversity, it did cause significant dose- and time-dependent changes in bacterial abundance at the phylum level, including increases in Verrucomicrobia and Actinobacteria, and decreases in Proteobacteria. Conclusion and Implications: These preliminary findings support the idea that psilocybin and other tryptamines may act on the gut microbiome in a dose- and time-dependent manner, potentially identifying a novel peripheral mechanism for their antidepressant activity. The results from this preliminary study also suggest that norbaeocystin may warrant further investigation as a potential antidepressant, given the similarity of its effects to psilocybin.

Evaluation of TrpM and PsiD substrate promiscuity reveals new biocatalytic capabilities.

N-methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N-dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for mental health disorders. The clinical relevance of these compounds has driven scientists to develop biosynthetic production routes to a number of tryptamine drug candidates, and efforts are ongoing to expand and further develop these biosynthetic capabilities. To that end, we have further characterized the substrate preferences of two enzymes involved in tryptamine biosynthesis: TrpM, a tryptophan N-methyltransferase from Psilocybe serbica, and PsiD, the gateway decarboxylase of the psilocybin biosynthesis pathway. Here, we show that TrpM can N-methylate the non-native amino acid substrate, 4-hydroxytryptophan, a key intermediate in the Escherichia coli-based recombinant psilocybin biosynthesis pathway. However, the ability to incorporate TrpM into a functional psilocybin biosynthesis pathway was thwarted by PsiD's inability to use N,N-dimethyl-4-hydroxytryptophan as substrate, under the culturing conditions tested, despite demonstrating activity on N-methylated and 4-hydroxylated tryptophan derivatives individually. Taken together, this work expands upon the known substrates for TrpM and PsiD, further increasing the diversity of tryptamine biosynthetic products.

Pharmacological and behavioural effects of tryptamines present in psilocybin-containing mushrooms.

BACKGROUND AND PURPOSE: Demand for new antidepressants has resulted in a re-evaluation of the therapeutic potential of psychedelic drugs. Several tryptamines found in psilocybin-containing "magic" mushrooms share chemical similarities with psilocybin. Early work suggests they may share biological targets. However, few studies have explored their pharmacological and behavioural effects. EXPERIMENTAL APPROACH: We compared baeocystin, norbaeocystin and aeruginascin with psilocybin to determine if they are metabolized by the same enzymes, similarly penetrate the blood-brain barrier, serve as ligands for similar receptors and modulate behaviour in rodents similarly. We also assessed the stability and optimal storage and handling conditions for each compound. KEY RESULTS: In vitro enzyme kinetics assays found that all compounds had nearly identical rates of dephosphorylation via alkaline phosphatase and metabolism by monoamine oxidase. Further, we found that only the dephosphorylated products of baeocystin and norbaeocystin crossed a blood-brain barrier mimetic to a similar degree as the dephosphorylated form of psilocybin, psilocin. The dephosphorylated form of norbaeocystin was found to activate the 5-HT2A receptor with similar efficacy to psilocin and norpsilocin in in vitro cell imaging assays. Behaviourally, only psilocybin induced head twitch responses in rats, a marker of 5-HT2A-mediated psychedelic effects and hallucinogenic potential. However, like psilocybin, norbaeocystin improved outcomes in the forced swim test. All compounds caused minimal changes to metrics of renal and hepatic health, suggesting innocuous safety profiles. CONCLUSIONS AND IMPLICATIONS: Collectively, this work suggests that other naturally occurring tryptamines, especially norbaeocystin, may share overlapping therapeutic potential with psilocybin, but without causing hallucinations.

Effect of psilocybin on decision-making and motivation in the healthy rat.

Psilocybin and its active metabolite psilocin are hallucinogenic serotonergic agonists with high affinity for several serotonin receptors. In addition to underlying the hallucinogenic effects of these compounds, serotonin receptor activation also has important effects on decision-making and goal-directed behaviors. The impact of psilocybin and psilocin on these cognitive systems, however, remains unclear. This study investigated the effects of psilocybin treatment on decision-making and motivation in healthy male and female rats. We compared probability and delay discounting performance of psilocybin treated (1 mg/kg) to vehicle rats (n = 10/sex/group), and further assessed motivation in each group using a progressive ratio task. We also confirmed drug action by assessing head twitch responses after psilocybin treatment (1 mg/kg). Results from this study demonstrated that exposure to 1 mg/kg psilocybin did not affect decision-making in the probability and delay discounting tasks and did not reduce response rates in the progressive ratio task. However, psilocybin treatment did cause the expected increase in head twitch responses in both male and female rats, demonstrating that the drug was delivered at a pharmacologically relevant dosage. Combined, these results suggest that psilocybin may not impair or improve decision-making and motivation. Considering recent interest in psilocybin as a potential fast-acting therapeutic for a variety of mental health disorders, our findings also suggest the therapeutic effects of this drug may not be mediated by changes to the brain systems underlying reward and decision-making. Finally, these results may have important implications regarding the relative safety of this compound, suggesting that widespread cognitive impairments may not be seen in subjects, even after chronic treatment.

De novo Biosynthesis of Salvianolic Acid B in Saccharomyces cerevisiae Engineered with the Rosmarinic Acid Biosynthetic Pathway.

Salvianolic acid B (SAB), also named lithospermic acid B, belongs to a class of water-soluble phenolic acids, originating from plants such as Salvia miltiorrhiza. SAB exhibits a variety of biological activities and has been clinically used to treat cardio- and cerebrovascular diseases and also has great potential as a health care product and medicine for other disorders. However, its biosynthetic pathway has not been completely elucidated. Here, we report the de novo biosynthesis of SAB in Saccharomyces cerevisiae engineered with the heterologous rosmarinic acid (RA) biosynthetic pathway. The created pathway contains seven genes divided into three modules on separate plasmids, pRS424-FjTAL-Sm4CL2, pRS425-SmTAT-SmHPPR or pRS425-SmTAT-CbHPPR, and pRS426-SmRAS-CbCYP-CbCPR. These three modules were cotransformed into S. cerevisiae, resulting in the recombinant strains YW-44 and YW-45. Incubation of the recombinant strains in a basic medium without supplementing any substrates yielded 34 and 30 µg/L of SAB. The findings in this study indicate that the created heterologous RA pathway cooperates with the native metabolism of S. cerevisiae to enable the de novo biosynthesis of SAB. This provides a novel insight into a biosynthesis mechanism of SAB and also lays the foundation for the production of SAB using microbial cell factories.

Thermodynamics contributes to high limonene productivity in cyanobacteria.

Terpenoids are a large group of secondary metabolites with broad industrial applications. Engineering cyanobacteria is an attractive route for the sustainable production of commodity terpenoids. Currently, a major obstacle lies in the low productivity attained in engineered cyanobacterial strains. Traditional metabolic engineering to improve pathway kinetics has led to limited success in enhancing terpenoid productivity. In this study, we reveal thermodynamics as the main determinant for high limonene productivity in cyanobacteria. Through overexpressing the primary sigma factor, a higher photosynthetic rate was achieved in an engineered strain of Synechococcus elongatus PCC 7942. Computational modeling and wet lab analyses showed an increased flux toward both native carbon sink glycogen synthesis and the non-native limonene synthesis from photosynthate output. On the other hand, comparative proteomics showed decreased expression of terpene pathway enzymes, revealing their limited role in determining terpene flux. Lastly, growth optimization by enhancing photosynthesis has led to a limonene titer of 19 mg/L in 7 days with a maximum productivity of 4.3 mg/L/day. This study highlights the importance of enhancing photosynthesis and substrate input for the high productivity of secondary metabolic pathways, providing a new strategy for future terpenoid engineering in phototrophs.

Development of an E. coli-based norbaeocystin production platform and evaluation of behavioral effects in rats.

Interest in the potential therapeutic efficacy of psilocybin and other psychedelic compounds has escalated significantly in recent years. To date, little is known regarding the biological activity of the psilocybin pathway intermediate, norbaeocystin, due to limitations around sourcing the phosphorylated tryptamine metabolite for in vivo testing. To address this limitation, we first developed a novel E. coli platform for the rapid and scalable production of gram-scale amounts of norbaeocystin. Through this process we compare the genetic and fermentation optimization strategies to that of a similarly constructed and previously reported psilocybin producing strain, uncovering the need for reoptimization and balancing upon even minor genetic modifications to the production host. We then perform in vivo measurements of head twitch response to both biosynthesized psilocybin and norbaeocystin using both a cell broth and water vehicle in Long-Evans rats. The data show a dose response to psilocybin while norbaeocystin does not elicit any pharmacological response, suggesting that norbaeocystin and its metabolites may not have a strong affinity for the serotonin 2A receptor. The findings presented here provide a mechanism to source norbaeocystin for future studies to evaluate its disease efficacy in animal models, both individually and in combination with psilocybin, and support the safety of cell broth as a drug delivery vehicle.

Homebrewed psilocybin: can new routes for pharmaceutical psilocybin production enable recreational use?

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.