3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a recreational drug under clinical investigation for the treatment of central nervous system disorders, including post-traumatic stress disorder. Chemical synthesis of MDMA mainly relies on the use of safrole and piperonal as starting materials. We report a novel strategy integrating bioconversion and biocatalysis in the bioproduction of MDMA and other methamphetamine derivatives. For the initial step, a yeast-based bioconversion system was used to produce phenylacetylcarbinol (PAC) derivatives from ring-substituted benzaldehyde precursors, including piperonal, using variants of pyruvate decarboxylase (PDC). Among seven wildtype enzymes tested, Candida tropicalis PDC (CtPDC) showed the highest yield from piperonal, and a CtPDC1-I479A mutant further improved the titer. Five of sixteen ring-substituted benzaldehyde analogs (i.e., piperonal, 6-chloropiperonal, 4-acetylbenzaldehyde, 2-fluoro-4-methoxybenzaldehyde, and 2-fluoro-4-propoxybenzaldehyde) yielded corresponding PAC derivatives with yields between 20 and 70 ​%, which allowed the purification of multiple milligram quantities of each product. Three stereoselective ω-transaminases were evaluated for their ability to catalyze the transamination of PAC derivatives, with the (R)-selective enzyme ATA-117-Rd11 able to convert all five isolated PAC derivatives. Isolated transamination products were subsequently N-methylated using human phenylethanolamine N-methyltransferase. Chemical reduction facilitated the final production of MDMA and its analog 6-chloro-MDMA. Our work represents the first reported bioproduction method leading to MDMA and other methamphetamine derivatives, suitable for future pathway and strain optimization.

Drug screening programs targeting novel indolethylamines with pharmacological properties suitable for the treatment of psychiatric and central nervous system disorders benefit from the availability of large compound libraries normally prepared using synthetic chemistry. Bioproduction strategies based on microbial metabolic engineering and fermentation generally fail to achieve the throughput, scale, or versatility of synthetic chemistry owing, in part, to a lack of efficient and promiscuous enzymes. Moreover, synthetic biology rarely extends to the purification of targeted products, which is an essential component of synthetic chemistry and drug screening regimes. A lattice of biosynthetic routes beginning with endogenous tryptophan or exogenous indole derivatives were engineered in Escherichia coli using heterologous genes encoding enzymes sourced from plants, mushrooms, microbes and animals. Twelve tryptophan decarboxylase candidates were screened and highly versatile top-performers from Bacillus atrophaeus and the gut microbiome species Clostridium sporogenes were identified. Seven halogenases, three tryptophan synthase β-subunits, six N-methyltransferases, five regioselective prenyltransferases, a cytochrome P450 oxidoreductase 5-hydroxylase, an N-acetyltransferase, a 4-O-kinase and various accessory proteins were also tested. These enzymes were used in various combinations and permutations to build E. coli strains capable of 344 putative biotransformations, which resulted in the formation of 279 products with only 63 targeted compounds not detected. A set of 17 novel N-acetylated derivatives were selected for upscaled culturing and purification to ≥95% from 0.5 to 1 L of the fermentation broth, which yielded ∼6-80 mg of each molecule. The potential of each compound for bioactivity at 14 different receptors or transporters with established or purported involvement in neuropsychiatric diseases was tested using a single ligand concentration. Nearly all the N-acetylated compounds showed interaction with the melatonin (MT1) receptor, and several molecules showed interaction with serotonergic receptors 5-HT2B, 5-HT2C, and 5-HT7. Overall, we show that bio-fermentation is useful in the large-scale screening of molecules with potential in drug development.

The recent relaxation of psychedelic drug regulations has prompted extensive clinical investigation into their potential use to treat diverse mental health conditions including anxiety, depression, post-traumatic stress, and substance-abuse disorders. Most clinical trials have relied on a small number of known molecules found in nature, such as psilocybin, or long-known synthetic analogs of natural metabolites, including lysergic acid diethylamide (LSD). Elucidation of biosynthetic pathways leading to several psychedelic compounds has established an opportunity to use synthetic biology as a complement to synthetic chemistry for the preparation of novel derivatives with potentially superior pharmacological properties compared with known drugs. Herein we review the metabolic biochemistry of pathways from plants, fungi and animals that yield the medicinally important hallucinogenic specialized metabolites ibogaine, mescaline, psilocybin, lysergic acid, and N,N-dimethyltryptamine (DMT). We also summarize the reconstitution of these pathways in microorganisms and comment on the integration of native and non-native enzymes to prepare novel derivatives.

The evolution of morphinan alkaloid biosynthesis in plants of the genus Papaver includes permutation of several processes including gene duplication, fusion, neofunctionalization, and deletion resulting in the present chemotaxonomy. A critical gene fusion event resulting in the key bifunctional enzyme reticuline epimerase (REPI), which catalyzes the stereochemical inversion of (S)-reticuline, was suggested to precede neofunctionalization of downstream enzymes leading to morphine biosynthesis in opium poppy (Papaver somniferum). The ancestrally related aldo-keto reductases 1,2-dehydroreticuline reductase (DRR), which occurs in some species as a component of REPI, and codeinone reductase (COR) catalyze the second and penultimate steps, respectively, in the pathway converting (S)-reticuline to morphine. Orthologs for each enzyme isolated from the transcriptomes of 12 Papaver species were shown to catalyze their respective reactions in species that capture states of the metabolic pathway prior to key evolutionary events, including the gene fusion event leading to REPI, thus suggesting a patchwork model for pathway evolution. Analysis of the structure and substrate preferences of DRR orthologs in comparison with COR orthologs revealed structure-function relationships underpinning the functional latency of DRR and COR orthologs in the genus Papaver, thus providing insights into the molecular events leading to the evolution of the pathway. Conservation across the Papaver genus of two aldo-keto reductases catalyzing the second and penultimate steps in morphine biosynthesis reveals the latent activity of several enzymes and suggests a patchwork model of pathway evolution in opium poppy.

The psychedelic prodrug psilocybin has shown therapeutic benefits for the treatment of numerous psychiatric conditions. Despite positive clinical end points targeting depression and anxiety, concerns regarding the duration of the psychedelic experience produced by psilocybin, associated with enduring systemic exposure to the active metabolite psilocin, pose a barrier to its therapeutic application. Our objective was to create a novel prodrug of psilocin with similar therapeutic benefits but a reduced duration of psychedelic effects compared with psilocybin. Here, we report the synthesis and functional screening of 28 new chemical entities. Our strategy was to introduce a diversity of cleavable groups at the 4-hydroxy position of the core indole moiety to modulate metabolic processing. We identified several novel prodrugs of psilocin with altered pharmacokinetic profiles and reduced pharmacological exposure compared with psilocybin. These candidate prodrugs have the potential to maintain the long-term benefits of psilocybin therapy while attenuating the duration of psychedelic effects.

Several proteins from plant pathogenesis-related family 10 (PR10) are highly abundant in the latex of opium poppy and have recently been shown to play diverse and important roles in the biosynthesis of benzylisoquinoline alkaloids (BIAs). The recent determination of the first crystal structures of PR10-10 showed how large conformational changes in a surface loop and adjacent β-strand are coupled to the binding of BIA compounds to the central hydrophobic binding pocket. A more detailed analysis of these conformational changes is now reported to further clarify how ligand binding is coupled to the formation and cleavage of an intermolecular disulfide bond that is only sterically allowed when the BIA binding pocket is empty. To decouple ligand binding from disulfide-bond formation, each of the two highly conserved cysteine residues (Cys59 and Cys155) in PR10-10 was replaced with serine using site-directed mutagenesis. Crystal structures of the Cys59Ser mutant were determined in the presence of papaverine and in the absence of exogenous BIA compounds. A crystal structure of the Cys155Ser mutant was also determined in the absence of exogenous BIA compounds. All three of these crystal structures reveal conformations similar to that of wild-type PR10-10 with bound BIA compounds. In the absence of exogenous BIA compounds, the Cys59Ser and Cys155Ser mutants appear to bind an unidentified ligand or mixture of ligands that was presumably introduced during expression of the proteins in Escherichia coli. The analysis of conformational changes triggered by the binding of BIA compounds suggests a molecular mechanism coupling ligand binding to the disruption of an intermolecular disulfide bond. This mechanism may be involved in the regulation of biosynthetic reactions in plants and possibly other organisms.

Systematic screening of morphine pathway intermediates in engineered yeast revealed key biosynthetic enzymes displaying potent feedback inhibition: 3'-hydroxy-N-methylcoclaurine 4'-methyltransferase (4'OMT), which yields (S)-reticuline, and the coupled salutaridinol-7-O-acetyltransferase (SalAT) and thebaine synthase (THS2) enzyme system that produces thebaine. The addition of deuterated reticuline-d1 to a yeast strain able to convert (S)-norcoclaurine to (S)-reticuline showed reduced product accumulation in response to the feeding of all four successive pathway intermediates. Similarly, the addition of deuterated thebaine-d3 to a yeast strain able to convert salutaridine to thebaine showed reduced product accumulation from exogenous salutaridine or salutaridinol. In vitro analysis showed that reticuline is a noncompetitive inhibitor of 4'OMT, whereas thebaine exerts mixed inhibition on SalAT/THS2. In a yeast strain capable of de novo morphine biosynthesis, the addition of reticuline and thebaine resulted in the accumulation of several pathway intermediates. In contrast, morphine had no effect, suggesting that circumventing the interaction of reticuline and thebaine with 4'OMT and SalAT/THS2, respectively, could substantially increase opiate alkaloid titers in engineered yeast.

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant specialized metabolites found mainly in members of the order Ranunculales, including opium poppy (Papaver somniferum), for which BIA biosynthetic pathways leading to the critical drugs morphine, noscapine, and sanguinarine have been elucidated. Sacred lotus (Nelumbo nucifera), in the order Proteales, accumulates medicinal BIAs in the proaporphine, aporphine, and bisbenzylisoquinoline structural subgroups with a prevalence of R enantiomers, opposed to the dominant S configuration occurring in the Ranunculales. Nevertheless, distinctive BIA biosynthetic routes in sacred lotus have not been explored. In planta labeling experiments and in vitro assays with recombinant enzymes and plant protein extracts showed that dopamine and 4-hydroxyphenylacetaldehyde derived from L-tyrosine serve as precursors for the formation of (R,S)-norcoclaurine in sacred lotus, whereas only (R)-norcoclaurine byproducts are favored in the plant by action of R-enantiospecific methyltransferases and cytochrome P450 oxidoreductases (CYPs). Enzymes responsible for the R-enantiospecific formation of proaporphine (NnCYP80Q1) and bisbenzylisoquinoline (NnCYP80Q2) scaffolds, and a methylenedioxy bridge introduction on aporphine substrates (NnCYP719A22) were identified, whereas additional aspects of the biosynthetic pathways leading to the distinctive alkaloid profile are discussed. This work expands the availability of molecular tools that can be deployed in synthetic biology platforms for the production of high-value alkaloids.

Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include ‘magic mushrooms’, plants used to prepare ahayuasca, and toads. Owing to the occurrence of indolethylamines such as N,N -dimethyltryptamine, toad skin and parotid gland preparations have a history of ceremonial use, yet their biosynthesis remains uncharacterized. We report the isolation of an indolethylamine N -methyltransferase (RmNMT) from cane toad ( Rhinella marina ). Unlike functionally similar mammalian and fungal enzymes, the efficiency and promiscuity of RmNMT made it suitable for the bioproduction of new-to-nature indolethylamine derivatives. N,N -Dimethylated indolethylamines showed reduced affinity and engagement at 5-HT 1A and 5-HT 2A receptors respectively, compared with primary amine equivalents. Despite reduced 5-HT 2A receptor engagement of tertiary amines, only these molecules induced hallucinogenic behavior in mice underscoring the importance of metabolic stability. The discovery of RmNMT facilitated the establishment of a bioproduction platform for the isolation and pharmacological screening of novel indolethylamines.

Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include 'magic mushrooms' (Psilocybe spp.), plants used to prepare ayahuasca, and toads. The skin and parotid glands of certain toads accumulate a variety of specialized metabolites including toxic guanidine alkaloids, lipophilic alkaloids, poisonous steroids, and hallucinogenic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin. The occurrence of psychedelics has contributed to the ceremonial use of toads, particularly among Mesoamerican peoples. Yet, the biosynthesis of psychedelic alkaloids has not been elucidated. Herein, we report a novel indolethylamine N-methyltransferase (RmNMT) from cane toad (Rhinella marina). The RmNMT sequence was used to identify a related NMT from the common toad, Bufo bufo. Close homologs from various frog species were inactive, suggesting a role for psychedelic indolethylamine biosynthesis in toads. Enzyme kinetic analyses and comparison with functionally similar enzymes showed that recombinant RmNMT was an effective catalyst and not product inhibited. The substrate promiscuity of RmNMT enabled the bioproduction of a variety of substituted indolethylamines at levels sufficient for purification, pharmacological screening, and metabolic stability assays. Since the therapeutic potential of psychedelics has been linked to activity at serotonergic receptors, we evaluated binding of derivatives at 5-HT1A and 5-HT2A receptors. Primary amines exhibited enhanced affinity at the 5-HT1A receptor compared with tertiary amines. With the exception of 6-substituted derivatives, N,N-dimethylation also protected against catabolism by liver microsomes.

Peyote (Lophophora williamsii) is an entheogenic and medicinal cactus native to the Chihuahuan desert. The psychoactive and hallucinogenic properties of peyote are principally attributed to the phenethylamine derivative mescaline. Despite the isolation of mescaline from peyote over 120 years ago, the biosynthetic pathway in the plant has remained undiscovered. Here, we use a transcriptomics and homology-guided gene discovery strategy to elucidate a near-complete biosynthetic pathway from l-tyrosine to mescaline. We identified a cytochrome P450 that catalyzes the 3-hydroxylation of l-tyrosine to l-DOPA, a tyrosine/DOPA decarboxylase yielding dopamine, and four substrate-specific and regiospecific substituted phenethylamine O-methyltransferases. Biochemical assays with recombinant enzymes or functional analyses performed by feeding putative precursors to engineered yeast (Saccharomyces cerevisiae) strains expressing candidate peyote biosynthetic genes were used to determine substrate specificity, which served as the basis for pathway elucidation. Additionally, an N-methyltransferase displaying broad substrate specificity and leading to the production of N-methylated phenethylamine derivatives was identified, which could also function as an early step in the biosynthesis of tetrahydroisoquinoline alkaloids in peyote.

Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.

Opium poppy is the only commercial source of the narcotic analgesics morphine and codeine, and semi-synthetic derivatives of the natural opiate precursor thebaine, including oxycodone and the opioid antagonist naloxone. The plant also accumulates the vasodilator and antitussive agents papaverine and noscapine, respectively, which together with morphine, codeine and thebaine comprise the major benzylisoquinoline alkaloids (BIAs) in opium poppy. A majority of enzymes involved in the highly branched BIA metabolism in opium poppy have now been discovered, with many specifically localized to sieve elements of the phloem based on immunofluorescence labeling techniques. Transcripts corresponding to sieve element-localized biosynthetic enzymes were detected in companion cells, as expected. The more recent application of shotgun proteomics has shown that several enzymes operating late in the morphine and noscapine biosynthetic pathways occur primarily in laticifers that are adjacent or proximal to sieve elements. BIA biosynthesis and accumulation in opium poppy involves three phloem cell types and implicates the translocation of key pathway intermediates between sieve elements and laticifers. The recent isolation of uptake transporters associated with laticifers supports an apoplastic rather than a symplastic route for translocation. In spite of the extensive elucidation of BIA biosynthetic enzymes in opium poppy, additional transporters and other auxiliary proteins are clearly necessary to support the complex spatial organization and dynamics involved in product formation and sequestration. In this review, we provide an update of BIA metabolism in opium poppy with a focus on the role of phloem in the biosynthesis of the major alkaloids.

Plants produce many compounds used by humans as medicines, including alkaloids of the benzylisoquinoline (BIA), monoterpene indole (MIA) and tropane classes. The biosynthetic pathways of these pharmaceutical alkaloids are complex and spatially segregated across several tissues, cell-types and organelles. This review discusses the origin of primary metabolic inputs required by these specialized biosynthetic pathways and considers aspects relevant to their spatial organization. These factors are important for alkaloid production both in the native plants and for synthetic biology pathway reconstruction in microorganisms.

The pharmaceutical properties of plant‐derived compounds are widely used in medicine. Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are closely related BIAs with particularly useful analgesic properties. The aldo‐keto reductase (AKR) codeinone reductase (COR) catalyzes the final step and the penultimate step in biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). Using X‐ray crystallography, we have determined the structure of apo‐COR (2.4 Å resolution) for the first time, using the structure of chalcone reductase as a search model. The structure of apo‐COR reveals a three‐dimensional framework for understanding BIA substrate recognition and catalysis. Structural comparisons of COR to closely related plant AKR's and more distantly related homologues reveal a novel conformation in the β1α1 loop adjacent to the BIA binding pocket. The proximity of this loop several highly conserved active site residues and the expected location of the nicotinamide ring of the NADPH cofactor suggest the importance of several key residues in BIA substrate recognition. The effects of mutations in residues in this loop and at other nearby positions help to define specific roles in substrate recognition and catalysis. Combined with the structure of COR, these findings from mutagenesis also help to suggest structure‐function relationships in a second AKR critical to BIA biosynthesis, the 1,2 dehydroreticuline reductase domain in the critical enzyme reticuline epimerase.

Benzylisoquinoline alkaloids (BIAs) have profound implications on human health owing to their potent pharmacological properties. Notable naturally occurring BIAs are the narcotic analgesics morphine, the cough suppressant codeine, the potential anticancer drug noscapine, the muscle relaxant papaverine, and the antimicrobial sanguinarine, all of which are produced in opium poppy (Papaver somniferum). Thebaine, an intermediate in the biosynthesis of codeine and morphine, is used in the manufacture of semisynthetic opiates, including oxycodone and naloxone. As the only commercial source of pharmaceutical opiates, opium poppy has been the focus of considerable research to understand BIA metabolism in the plant. The elucidation of several BIA biosynthetic pathways has enabled the development of synthetic biology platforms aimed at the alternative commercial production of valuable phytochemicals in microorganisms. The detection and identification of BIA pathway products and intermediates in complex extracts is essential for the continuing advancement of research in plant specialized metabolism and microbial synthetic biology. Herein, we report the use of liquid chromatography coupled with linear trap quadrupole and high-resolution Orbitrap multistage mass spectrometry to characterize 44 authentic BIAs using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and pulsed Q collision-induced dissociation (PQD) MS2 fragmentation, with MS2 CID followed by MS3 and MS4 fragmentation. Our deep library of diagnostic spectral data constitutes a valuable resource for BIAs identification. In addition, we identified 22 BIAs in opium poppy latex and roots extracts.

Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the β1α1 loop adjacent to the BIA-binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway.

Methyltransferases (MTs) are a diverse family of enzymes responsible for the addition of a methyl group from a donor molecule (typically S-adenosyl methionine) to target substrates. Methyltransferases are found widely across nature and are critical for many cellular activities, including central metabolism, DNA and RNA processing, signal transduction, and protein localization. The ubiquity of MTs in multiple contexts highlights the importance of methyl transfer for many biological processes. In addition to their intrinsic importance to biology, the diversity of sequences and 3-dimensional structures of MTs, particularly in the domain responsible for recognizing methyl acceptor substrates, underlines the importance of characterizing the broad range of molecular mechanisms for substrate recognition and catalysis used within this enzyme family.

The pharmaceutical properties of plant-derived compounds are widely used in modern day medicine. Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the penultimate step in morphine biosynthesis in opium poppy (Papaver somniferum). After extensive optimization of crystal growth conditions, we have determined the structure of apo-COR (2.4 Å resolution) for the first time, using the structure of chalcone reductase as the search model for molecular replacement. The structure of apo-COR reveals a three-dimensional framework for understanding BIA substrate recognition and catalysis. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveals a novel conformation in the β1α1 loop adjacent to the BIA binding pocket. The proximity of this loop to several highly conserved active site residues and the expected location of the nicotinamide ring of the NADPH cofactor suggest the importance of several key residues in BIA substrate recognition. The effects of mutations to residues in this loop and at other nearby positions help to define specific roles in substrate recognition and catalysis. Combined with the structure and COR, these findings from mutagenesis also help to suggest structure-function relationships in a second AKR critical to BIA biosynthesis, the 1,2 dehydroreticuline reductase domain in the critical enzyme reticuline epimerase.

Ephedra sinica is a high-value medicinal plant that produces important phenylpropylamino alkaloids pseudoephedrine and ephedrine. Few genomics resources exist for E . sinica, which has been characterized as a tetraploid with a monoploid genome size of 8.56 Gb. Here we reported a partial genome assembly of E. sinica (12.8 Gb) based on Illumina short-read sequencing at low coverage.