Biosynthesis and Synthetic Biology of

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Biosynthesis and synthetic biology of

psychoactive natural products
Cite this: DOI: 10.1039/d1cs00065a
Cooper S. Jamieson, †a Joshua Misa, †b Yi Tang *ab and
John M. Billingsley *bc
Psychoactive natural products play an integral role in the modern world. The tremendous structural
complexity displayed by such molecules confers diverse biological activities of significant medicinal
value and sociocultural impact. Accordingly, in the last two centuries, immense effort has been devoted
towards establishing how plants, animals, and fungi synthesize complex natural products from simple
metabolic precursors. The recent explosion of genomics data and molecular biology tools has enabled
the identification of genes encoding proteins that catalyze individual biosynthetic steps. Once fully
elucidated, the ‘‘biosynthetic pathways’’ are often comparable to organic syntheses in elegance and
yield. Additionally, the discovery of biosynthetic enzymes provides powerful catalysts which may be
repurposed for synthetic biology applications, or implemented with chemoenzymatic synthetic
approaches. In this review, we discuss the progress that has been made toward biosynthetic pathway
elucidation amongst four classes of psychoactive natural products: hallucinogens, stimulants, cannabinoids,
Received 18th January 2021 and opioids. Compounds of diverse biosynthetic origin – terpene, amino acid, polyketide – are identified,
DOI: 10.1039/d1cs00065a and notable mechanisms of key scaffold transforming steps are highlighted. We also provide a description
of subsequent applications of the biosynthetic machinery, with an emphasis placed on the synthetic biology
rsc.li/chem-soc-rev and metabolic engineering strategies enabling heterologous production.
1 Introduction
a
Department of Chemistry and Biochemistry, University of California, Los Angeles, The consumption of psychoactive natural products predates
Los Angeles, CA, USA. E-mail: yitang@ucla.edu
b
recorded history.1 For millennia, our ancestors subsisted by
Department of Chemical and Biomolecular Engineering, University of California,
Los Angeles, Los Angeles, CA, USA. E-mail: johnbillingsley@ucla.edu
consuming materials foraged from the natural world. Over
c
Invizyne Technologies, Inc., Monrovia, CA, USA time, innumerable person-hours of trial and error resulted
† These authors contributed equally. in a keen understanding of the expected physiological and
Cooper S. Jamieson was born in Joshua Misa was born and raised
New York, NY and raised in San in Riverside, CA. He received his
Luis Obispo, CA. In 2016, he BS in Chemical Engineering with
received a BA in Chemistry and a an emphasis in Biochemical
BA in Art from Lewis & Clark Engineering from the University
College in Portland, OR. He of California, Riverside (UCR) in
moved to far-West Marfa, Texas 2018, graduating magna cum
and worked in art conservation at laude. At UCR he worked in
the Chinati Foundation. Now, Prof. Ian Wheeldon’s lab on the
Cooper has returned to academia development of CRISPR tools for
and is in Los Angeles, CA finishing engineering non-conventional yeasts
his PhD under the direction of Prof. as a Chancellor’s Research Fellow.
Cooper S. Jamieson K. N. Houk and Prof. Yi Tang at Joshua Misa Joshua is currently a PhD candidate
UCLA on pericyclases and pericyclic working under Prof. Yi Tang on
reactions in nature. developing yeast-based platforms for production of plant natural
products and new, biosynthetic analogues.
This journal is © The Royal Society of Chemistry 2021 Chem. Soc. Rev.
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psychological effects upon ingestion of specific plants, animals, has been made in the elucidation of the metabolic pathways
and fungi.2 This information propagated initially as traditional underpinning the chemical composition of psychoactive substances.
knowledge, forming the basis of valuable cultural practices and In the field of natural product biosynthesis, scientists investigate the
efficacious traditional medicine.3 The myriad of ethical concerns biosynthetic logic that enables Nature to synthesize psychoactive
around the appropriation of indigenous knowledge, exploitation natural products with high efficiencies and selectivities.15 Identifi-
of slave labor, as well as inequitable access to natural product cation and reconstitution of key enzymatic steps uncovers Nature’s
cultivation, sale, and use, typically go unanswered by mainstream synthetic schema towards complex molecular scaffolds from simple
science, and we encourage the reader to consult a selection of metabolic precursors. The accumulation of such biosynthetic infor-
responsibly written articles on these subject matters.4–8 Scientists mation is driven in part by advancements in synthetic biology;
are beginning to recognize that natural products have mediated emerging biotechnologies promise to outperform traditional
intimate evolutionary relationships between plants, animals, and synthetic methods in cost, safety, efficiency, and sustainability.
fungi.9 For instance, over centuries, winemakers selected grapes Thus, significant achievements have been made in the heterologous
harboring high-alcohol producing Crabtree-positive yeast, expression of natural product pathways towards consumer products.
enabling the co-domestication of a plant-fungal symbiont
pair.10 An additional, highly speculative example known as the 1.1 Four categories of psychoactive natural products
‘‘Stoned Ape Hypothesis’’ posits that the consumption of psy- Only in the last half of a century have scientists begun to
chedelic mushrooms may have played a role in rapid increase of investigate the molecular mechanisms of psychoactivity – the
brain size in early hominids.11 alterations in perception, consciousness, and behavior, asso-
This push–pull relationship of humans with natural products ciated with such small molecules.16 Prior to the 1950s, most
continues to this day, as the adoption of single molecule scientists believed that synaptic activity was dictated entirely
constituents by Western culture has triggered the expansion of through electrical impulses, and little evidence existed on the
traditional cultivation practices to meet global demands.12 role of chemical signaling.17 Our current understanding of
Isolation of and characterization of organic plant extracts psychopharmacology has been directly facilitated by the use
marked the beginnings of both organic chemistry and Western of natural products. The extraordinary protein receptor binding
medicine. Prior to 20th century prohibition, efforts towards the affinities of psychoactive natural products allowed scientists to
total synthesis of commodified natural products provided a deduce the role of neurotransmitters in the central nervous
foundation for generations of organic chemists. Sir Robert system.18 We now know that neuroreceptors are the key signal
Robinson’s 1917 route to the cocaine precursor tropinone is transducers able to integrate chemical signals into biological
widely lauded as a classic in total synthesis,13 while Woodward’s systems. It is the selective receptor binding and activation by
innumerable contributions to the field of natural product total native and non-native chemical ligands that causes modulation
synthesis included a route to the lysergic acid diethylamide of neural pathways, resulting in altered perception.19 These
(LSD) precursor lysergic acid.14 Incorporation of this knowledge receptors are differentially expressed in different populations
into semi-syntheses prompted researchers to think of biological of neurons, and may exist as splice variants or exhibit single-
materials as chemical factories, and beg the question: how do nucleotide polymorphisms between individuals.20 Further, differen-
organisms synthesize natural products? Extraordinary progress tial activation of receptor subtypes by a given ligand makes it difficult
Yi Tang received his undergraduate John M. Billingsley grew up in

degree in Chemical Engineering and Cambria, NY, and attended the
Material Science from Penn State University at Buffalo pursuing a
University. He received his PhD in BS in Chemical Engineering.
Chemical Engineering from There, John developed a budding
California Institute of Technology interest in natural product
in 2002. After NIH postdoctoral biosynthesis during an internship
training in Chemical Biology at in Prof. Andrew Gulick’s lab at the
Stanford University, he started his Hauptmann-Woodward Medical
independent career at the University Research Institute. John received a
of California Los Angeles in 2004. PhD in Chemical Engineering from
He is currently Professor in the UCLA in 2019 under the guidance
Yi Tang Department of Chemical and John M. Billingsley of Yi Tang, where they investigated
Biomolecular Engineering at UCLA, and engineered the biosynthesis of
and holds joint appointments in the Department of Chemistry and plant and fungal alkaloids. John currently lives in West Hollywood, is a
Biochemistry; and Department of Bioengineering. His lab is interested in Visiting Scientist at UCLA and Principal Scientist at Invizyne
natural product biosynthesis, biocatalysis and protein engineering. Technologies in Monrovia, CA.
Chem. Soc. Rev. This journal is © The Royal Society of Chemistry 2021
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Chem Soc Rev Review Article
to categorize psychoactive drugs based strictly on the physiological Nicotine 5 and cocaine 6, two other well-known alkaloidal
target. For example, activation of m-opioid receptors (MORs) by stimulants, exhibit high potential for dependence, but are each
agonists like morphine (Section 5.2) results in analgesia and approved for specific medicinal indications.29,30 While the legal
sedation,21 whereas activation of k-opioid receptors (KORs) by the status of Cannabis is currently in flux, the primary constituents
potent ligand salvinorin A (Section 2.9) results in dissociation.22 tetrahydrocannabinol (THC) 7 and cannabidiol (CBD) 8 are FDA
Thus, while formally an opioid, the consumer of Salvia divinorum approved medications.31 State-by-state deregulation has resulted
would classify the shrub as a bona fide hallucinogen based on in the ongoing cannabinoid boon driving academia and industry
perceived psychological effect. As a result, psychoactive drugs to discover additional applications for THC, CBD, and other rare
have traditionally been categorized based simply on the experi- cannabinoids. Finally, opioid analgesics are included on the
ence of the user, as opposed to complex molecular mechanisms World Health Organization’s List of Essential Medicines. Despite
of psychoactivity. The natural products discussed herein fall the ongoing opioid crisis, morphine 9 plays a critical role in pain
within one of four well-recognized classes: hallucinogens, management and palliative care.32 Kratom, which contains the
stimulants, cannabinoids, and opioids (Fig. 1). potent MOR agonist mitragynine 10, has emerged recently as
The utility of psychoactive natural products, if used safely, an alternative to opium-derived substances. Given its potential
cannot be questioned. Selective, potent binding of a ligand to a for abuse, additional epidemiological studies of kratom are
target is a hallmark feature of a pharmaceutical agent. While warranted.33 As opioid dependence soars, public health organiza-
immense pharmaceutical potential has been ascribed to many tions have described the importance of research into pain
psychoactive natural products, evidence-based drug development management and addiction. We advocate for an unbiased,
campaigns are largely hindered by regulatory status.23 Natural evidence-based evaluation of the risks and benefits of psychoactive
products in the Schedule I Controlled Substance category have natural product use in order to maximize societal value.
been designated as having no accepted medical use, hindering
clinical trials, even though many compounds on the list exhibit 1.2 Overview of biosynthesis of psychoactive compounds
great potential for clinical success. For example, evidence impli- As with most natural products isolated from microorganisms
cates psilocybin 1 as a promising candidate for treatment-resistant and plants, the psychoactive compounds discussed in this review
depression24 and post-traumatic stress disorder,25 whereas the are biosynthesized from simple, primary metabolites such as
alkaloid ibogaine 2 has undergone development as anti-addictive acetate, isoprene, and amino acids.15 With the exception of
agent.26 Meanwhile, a recent meta-analysis concluded that the cannabinoids and a few others, most of the compounds covered
natural product derivative lysergic acid diethylamide (LSD) 3 has are alkaloids derived from the decarboxylation of a small set of
strong potential in the treatment of alcoholism.27 These three amino acids. For example, L-tryptophan 11 is the precursor to
compounds fall into the category of hallucinogenic natural ibogaine 2 and psilocybin 3; L-tyrosine 12 is the precursor to
products, invoking psychedelic, introspective effects. Alkaloidal mescaline (Section 2.6) and morphine 10; while the nonproteino-
stimulants are also of great societal value, and include the genic amino acid L-ornithine 13 is the precursor to nicotine 5 and
world’s most widely consumed psychoactive drug, caffeine 4.28 cocaine 6. The decarboxylation of amino acids is catalyzed by an
Fig. 1 Four categories of psychoactive natural products or derivatives described in this review and representative members.
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enzyme family known as amino acid decarboxylase (AADC), while the para-hydroxybenzene side chain in L-tyrosine 12 can be
which uses pyridoxal-5 0 -phosphate (PLP) as a cofactor. A few of found in mescaline (Section 2.6) and morphine 9. The terminal
the compounds contain isoprenoid building blocks, such as the amine-containing L-lysine and L-ornithine 13 are also used as
C5 prenyl unit in lysergic acid (Section 2.5) and the C10 geranyl precursors. Relevant to this review, the four-carbon side chain of
unit in cannabinoids (Section 4.2). The C–C bonds between the L-ornithine 13 is required for the formation of pyrrolidines
isoprenes and the rest of the molecules in these compounds are and tropanes. The first step in the utilization of these amino
catalyzed by a group of enzymes known as prenyltransferases. acids for alkaloid biosynthesis is decarboxylation to give the
Prenyltransferases are one type of group transfer enzyme used by corresponding primary amines, although in lysergic acid bio-
nature to transfer functional groups from thermodynamically synthesis L-tryptophan is used without decarboxylation. The
activated carriers to natural product biosynthetic intermediates. decarboxylation products of L-tryptophan, L-tyrosine and L-ornithine
Other group transfer enzymes include acyltransferases and are tryptamine 14, tyramine 15, and putrescine 16, respectively
S-adenosylmethionine (SAM) dependent methyltransferases, which (Fig. 2A). In the case of tyramine 14, hydroxylation of one of the
are frequently found in biosynthetic pathways. Nature also uses meta positions in the para-phenol ring gives the metabolite dopa-
redox reactions extensively to modify the natural products to their mine 17. Dopamine 17 is a natural product building block, but also a
final, bioactive forms. The enzymes catalyzing these reactions are neurotransmitter in mammals. The chemical logic for the early
collectively referred to as oxidoreductases, and include examples decarboxylation is straightforward: to facilitate intra- and inter-
such as cytochrome P450s, ketoreductases and amine oxidases.34 molecular Mannich reactions with aldehydes and ketones using
The enzymology of these enzymes has been well-studied and the the nucleophilic amine (see Section 1.2.2). This decarboxylation-
reader can refer to other reviews for more information.35,36 Here we Mannich two step rapidly sets up the (poly)-heterocyclic scaffold
will briefly summarize a few enzyme-catalyzed or enzyme-mediated of many alkaloidal natural products.
reactions that will be found throughout the review. The decarboxylation reactions are catalyzed by dedicated
1.2.1 Decarboxylation of amino acids. The aromatic amino amino acid decarboxylases. For example, in the case of L-tryptophan,
acids L-tryptophan 12, L-tyrosine 13 and to a less extent, L-phenyl- a tryptophan decarboxylase is involved. These enzymes typically use
alanine, are commonly used precursors for alkaloid natural the PLP cofactor, as expected for many enzymes that perform Ca, Cb
product biosynthesis. For example, the indole ring in L-tryptophan and Cg modifications on amino acids.37 The mechanism of the
11 is preserved in compounds such as psilocybin 1 and ibogaine 2; reaction is shown in Fig. 2B. The aldehyde of PLP modifies an active
Fig. 2 PLP-Dependent amino acid decarboxylase. (A) Three amino acids are decarboxylated to give primary amines that are building blocks for alkaloids;
(B) mechanism of the PLP-dependent tryptophan decarboxylase.
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site lysine to form the resting aldimine in the decarboxylase active nucleophile, such as the acidic Ca of a carbonyl to form the
site. A transaldimination step takes place next in which the amine of b-amino-carbonyl product. Two examples of an intramolecular
the substrate amino acid attacks the aldimine and forms the Mannich reaction can be found in the formation of the tropane
amino acid–PLP aldimine. The PLP then serves as an electron unit in cocaine 6 (Section 3.4).39,40 Starting from putrescine 16,
sink in the enzyme-catalyzed cleavage of the Ca–COO bond via methylation of one of the primary amines gives the intermediate
a quinonoid species. Reprotonation of the Ca then generates the N-methylputrescine 18; oxidation and hydrolysis of the other
product aldimine, which can undergo another transaldimination amine yields N-methylaminobutanal 19, which is in equilibrium
with the active site lysine to release the product amine and regen- with the cyclic N-methylpyrrolinium 20. Attack of the imine by
erate the resting aldimine. the enolized 3-oxo-glutaric acid 21 yields the adduct pyrrolidine
1.2.2 Mannich/Pictet–Spengler reactions. Following decarboxy- tropane scaffold precursor (Fig. 3A). A subsequent dehydrogenation
lation of the amino acids to the corresponding primary amines, generates a new pyrrolinium species that can be attacked with Ca of
a common next step is the Mannich reaction involving the the 1,3-diketo unit in a second Mannich reaction (Section 3.4).
primary amine. The Mannich reaction is a two-step reaction One variation of the Mannich reaction that is central to the
that yields an alkylated amine.38 In the first step, the primary biosynthesis of plant alkaloids is the Pictet–Spengler (PS) reaction
amine reacts with either an aldehyde or a ketone to form the involving b-arylethylamines such as tryptamine 14 and dopamine
Schiff base. The CQN double bond is then attacked by a carbon 17. In the PS reaction, after the amine reacts with an aldehyde or
Fig. 3 Mannich reactions in alkaloid biosynthesis. (A) Formation of the pyrrolinium intermediate on pathway to tropane alkaloids; (B) the Pictet–Spengler
reaction involving tryptamine to form tetrahydro-b-carboline intermediates; (C) the Pictet–Spengler reaction involving dopamine to form tetrahy-
droisoquinoline on pathway to morphine.
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ketone to form the Schiff base, a carbanion resonance structure of example can be found in the psilocybin pathway (see Section 2.3).
the indole in tryptamine or the para-hydroxy phenol ring in Acetyltransferases catalyze the transfer of acetyl groups from the
dopamine can attack the imine to form the new C–C bond. This acetyl-CoA thioester to a variety of O and N nucleophiles (Fig. 4A).
can be followed by rearrangements to form the stable tricyclic SAM-dependent methyltransferases use S-adenosylmethionine
tetrahydro-b-carboline or bicyclic tetrahydroisoquinoline, respec- to transfer a methyl group from the trivalent sulfonium group
tively. The tryptamine-derived tetrahydro-b-carboline is found in to C, O, N, and S nucleophiles in an SN2 type substitution
harmala alkaloids (Section 2.4) and iboga alkaloids (Section 2.8). reaction (Fig. 4B). This reaction can be found in the majority of
To generate the harmala family of compounds, tryptamine 14 biosynthetic pathways described herein. For example, iterative
is condensed with pyruvic acid 22, followed by attack of the N-methylation of tryptamine yields the psychoactive molecule
imine by C3 from the indole ring to form a spirocycle, which N,N-dimethyltryptamine 29 (DMT, see Section 2.2). UDP-glucose is
collapses via single bond migration to complete the PS reaction an activated glucose donor in cells for the assembly of oligo-
(Fig. 3B).41 Similarly, the condensation between the aldehyde saccharides and polysaccharides. UDP-glucose is thermodyna-
donor secologanin 24 and tryptamine 14 is catalyzed by a mically activated but kinetically stable in the absence of
dedicated Pictet-Spenglerase, yielding strictosidine, the uni- glucosyltransferases.44 In the presence of glucosylating
versal precursor to monoterpene indole alkaloids (MIAs) enzymes, UDP dissociates via cleavage of the C–O bond in an
including ibogaine.42 In the biosynthesis of benzylisoquino- SN1 fashion to yield a C1 oxocarbonium ion, which can be
line alkaloids (BIAs) such as morphine 9, the PS reaction takes attacked by incoming nucleophiles (Fig. 4C). A notable example
place between dopamine 17 and 4-hydroxyphenylacetaldehyde of substrate glucosylation is in the biosynthetic pathway of
26, both oxidation products of tyramine 15, to form the key strictosidine 25, the precursor to ibogaine (Section 2.8). The
intermediate S-norcoclaurine 27, precursor to R-reticuline 28 enzyme 7DLGT glucosylates the hemiacetal in 7-deoxyloganetic
and morphine 9 (Fig. 3C).43 acid 30 to give 7-deoxyloganic acid 31.45 The glucose moiety
1.2.3 Common group transfer reactions. Group transfer serves as a protecting group to prevent formation of the
reactions are widely used by Nature in the biosynthesis of aldehyde, and remains in strictosidine 25. In order to transform
natural products. Functional groups that are frequently trans- strictosidine 25 into different scaffolds, a glucosidase removes
ferred from donor molecules to biosynthetic intermediates the glucose moiety, unmasking the aldehyde and leading to
include methyl, acetyl, small, medium and long alkyl- subsequent rearrangements towards structurally diverse mono-
substituted acyl chains, isoprenyl, glucosyl, etc. These reactions terpene indole alkaloids.
serve a multitude of purposes, including (i) increasing the size The final group transfer reaction that is relevant to this
and complexity of the molecules; (ii) changing the lipophilicity review is the transfer of prenyl groups from isoprenyl pyrophosphate
of molecules; (iii) altering the reactivity of functional groups; to different nucleophiles in small molecules. These reactions are
(iv) serving as a transient chemical protection group for down- catalyzed by a family of enzymes known as prenyltransferases. The
stream modifications; (v) acting as leaving groups in elimination prenyl unit that is transferred from the pyrophosphorylated donor
reactions; and (vi) changing the biological properties of the to the substrate can be small, as in the five-carbon dimethylallyl
natural product. Hence, these reactions are indispensable to (most common), or the more elongated oligoprenyl groups such as
the structural diversity of natural products that have been the ten-carbon geranyl, fifteen-carbon farnesyl, etc. In the enzyme
isolated to date. active site, the D2-prenyl pyrophosphate donors can undergo C–O
The donor molecules, those that ‘‘carry’’ the groups to be bond cleavage to yield the C1 carbocation, which is stabilized by
transferred, are kinetically stable and thermodynamically activated: delocalization of the positive charge. Attack of the carbocation by a
the molecules are high in energy and therefore releasing the groups nucleophile carbon forges the new bond and completes the prenyl
is a highly exergonic reaction; yet the molecules are stable under transfer reaction (Fig. 4D). Electron rich aromatic rings, such as
cellular conditions and enzyme catalysis is required to overcome the hydroxybenzenes and indoles can serve as nucleophiles to attack the
kinetic barriers. We recently reviewed eight such molecules that allyl cation to perform in essence an electrophilic aromatic
power cellular metabolism, which include ATP, NAD(P)H, substitution. Two examples in this review illustrate this reaction.
acetyl-CoA, SAM, carbamoyl phosphate, isoprenyl pyrophos- The first is the dimethylallyl tryptophan synthase (DMATS) in
phate, UDP-glucose and molecular oxygen.44 NAD(P)H and lysergic acid biosynthesis, which prenylates the C4 position in
molecular oxygen are involved in the redox reactions and will L-tryptophan 11 to give 4-dimethylallyl-L-tryptophan (4-DMAT,
be summarized in the next section. Among the remaining six, Section 2.4).46 This modification introduces an olefin-containing
carbamoyl-phosphate is involved in nitrogen metabolism and five carbon unit into L-tryptophan, which can be further oxidized
is not directly involved in natural product biosynthesis. and cyclized into the hallucinogenic lysergic acid. The mechanism
The remaining five, however, are frequently used group of this reaction has been thoroughly studied, and is likely a
transfer donor molecules, and examples can be found through- two-step reaction.47 The C3 position of the indole ring is
out the review. ATP, the universal cellular energy currency, is the most nucleophilic due to resonance with the indole nitro-
the donor in the transferring of phosphate groups to nucleophilic gen lone pair. Attack on the allyl cation can occur at either
oxygen in the presence of a phosphotransferase. This reaction is C1 or C3; this attack is proposed to take place at the more
ubiquitous in primary metabolism but is quite rare in natural stable C3 position of the allyl cation. This generates a
product biosynthesis (or secondary metabolism). One such ‘‘reverse’’-prenylated product that is proposed to undergo a
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Fig. 4 Enzyme catalyzed group transfer reactions in biosynthesis. (A) acetyltransferase-catalyzed acetyltransfer; (B) methyltransferase-catalyzed methyl
transfer; (C) glucosyltransferase-catalyzed glucosyl transfer.; and (D) prenyltransferase-catalyzed prenyl transfer.
non-enzymatic sigmatropic Cope rearrangement to yield the resorcinol derivative, the aromatic prenyltransferase transfers
‘‘forward’’-prenylated 4-DMAT. In addition to serving as the the ten-carbon geranyl group from geranyl pyrophosphate to
starting point for lysergic acid (Section 2.5), indole prenylation the C3 position in the ring to give cannabigerolic acid (CBGA, 33).
of early pathway intermediates is commonly observed in the As in the lysergic acid example, the introduced ten-carbon unit
biosynthesis of other fungal indole alkaloids.48–52 can undergo oxidative intramolecular cyclization, providing a
The second notable pathway that involves prenyl transfer is variety of cannabinoids (Section 4.2).
in cannabinoid biosynthesis (Section 4.2).53 Starting with the 1.2.4 Oxidative and reductive reactions. Natural product
first intermediate in the pathway, olivetolic acid 32 which is a biosynthetic pathways employ powerful redox enzymes to modify
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the intermediates en route to the final product. The redox feature of some biosynthetic P450s is the ability to iteratively
modification can directly modify the molecular scaffolds, or oxidize a substrate, either at a single carbon or at nearby atoms.
trigger rearrangement cascades, to introduce considerable For example, it is not uncommon to find a single P450 that can
structural complexities.34 On the reductive side, the NAD(P)H perform the six-electron oxidation of a methyl group into a
utilizing enzymes dominate as one would expect. These include carboxylic acid in both fungal and plant biosynthetic pathways.
ketoreductases, short-chain dehydrogenase/reductases (SDRs), One notable example of P450 catalysis in this review is the
ene-reductases, and imine reductases, etc. The two-electron secologanin synthase (SLS) found in the strictosidine biosynthetic
reduction of CQC, CQO or CQN bonds are initiated through pathway that ultimately leads to ibogaine (Section 2.8).55,56 The
the attack by a hydride equivalent from either the dihydropyridine substrate is loganin 34 which contains the iridoid core. SLS
ring of NAD(P)H or the hydroquinone form of flavin adenine performs hydrogen abstraction followed by oxygen rebound at
dinucleotide (FADH2). On the oxidative side, aerobic organisms the methyl group on the cyclopentanol ring to give a primary
use an assortment of enzymes and molecular oxygen as the hydroxyl group. This species then undergoes a Grob frag-
oxidant to perform a dazzling array of chemical modifications.15 mentation-like reaction to cleave the C–C bond which reveals
Both single electron (radical) and two electron manifolds are both an aldehyde and a terminal olefin in the product secolo-
used by enzymes. These enzymes include the large family of ganin 24 (Fig. 5A).57 This aldehyde then participates in the
heme-dependent cytochrome P450 monooxygenases that are aforementioned Pictet–Spengler reaction with tryptamine 14 to
abundant in plants and fungi; nonheme, iron and a-ketoglutarate give strictosidine 25. Hence, although this example illustrates
dependent oxygenases, copper-dependent oxidases (such as the a ‘‘standard’’ P450 reaction, the hydroxylation modification
amine oxidase mentioned above), and flavin-dependent monooxy- triggers a significant skeletal rearrangement.
genases and oxidases. In two-electron oxidation of substrates A second example that illustrates oxidation without oxygen
catalyzed by oxidases, molecular oxygen is reduced to hydrogen incorporation is found in the morphine biosynthetic pathway,
peroxide. In monooxygenases where oxygen is reduced fully to in which the salutaridine synthase catalyzes the phenyl coupling
water (four electron reduction), the substrate undergoes a two- in R-reticuline 28 to yield salutaridine 35 (Fig. 5B).58 A radical
electron oxidation, while NADPH is oxidized to NADP+. Here, addition mechanism is currently favored for this reaction:
the substrate can incorporate one of the oxygen atoms via hydrogen abstraction from one of the phenol group generates
hydroxylation or epoxidation, or alternatively the substrate can an oxygen radical that is delocalized throughout the aromatic
be oxidized without incorporation of oxygen atoms. Hence, ring. The carbon radical then adds into the isoquinoline ring
depending on the mechanism of the redox enzyme, the outcome and recombines with the second radical that is generated by the
of the reaction can be very different. This topic has been extensively P450 through the second hydrogen abstraction step. This forms
reviewed in the literature,15,34,54 and will not be discussed in detail a C–C bond that couples the two phenolic rings and gives rise to
here. However, we will highlight two reactions to illustrate the the rigidified morphinan scaffold of salutaridine 35 that is
enzymatic prowess of the P450s, a staple of the plant bio- found in morphine 9 and related opioids.
synthetic pathways.
P450 enzymes use heme as a coenzyme to bind molecular 1.3 Synthetic biology of psychoactive natural products
oxygen. The coordinated iron is reduced to the Fe(II) state by an The psychoactivity of a given plant or fungi is often attributed
associated cytochrome P450 reductase (CPR). Binding of mole- to a short list of molecules. In reality, psychoactive natural
cular oxygen and electron transfer from the Fe(II) and CPR leads products are produced as complex mixtures of metabolites and
to a hydroperoxy Fe(III)–O–O–H species. Cleavage of the O–O frequently have partially undefined compositions.59 Variability in
bond and the loss of water generates the high valent Fe(IV)QO growth conditions, in addition to pests, disease, agrochemicals,
porphyrin cation radical, which is also referred to as compound I. and climate may introduce further inconsistencies in product
This is a highly oxidizing species that can abstract hydrogen composition.60 In the event that a single psychoactive constituent
from substrate C, O, and N atoms to generate substrate is desired by the consumer and isolation from the native host
radicals, including ‘‘unactivated’’ sp3 carbons. This generates is costly, total synthesis may be one strategy to establish a
the Fe(IV)–OH species also known as compound II. Radical OH robust supply chain. In the last two decades, advances in DNA
transfer to the substrate carbon radical produces the hydro- technologies have resulted in the development of an alternative
xylated product in a process known as oxygen rebound. In many production strategy: synthetic biology.61,62 Synthetic biologists
P450-catalyzed reactions in biosynthesis, the substrate radical use genetic tools to build designed biological systems with
can migrate to other atoms in the molecule through internal useful functionality. Whether or not synthetic biology can
reactions and delocalization through p-bonds. This can lead to produce a viable process depends on the economic, environ-
rearrangement of the carbon skeleton, as well as oxygen atom mental, and societal cost of alternative production strategies.
incorporation at distal positions from the initial abstraction However, as novel DNA-related technologies continue to arise,
site. In some cases, the Fe(IV)–OH can abstract a second capabilities of molecular biologists are expected to expand. In
hydrogen atom from the substrate to generate a second radical 2010, Gibson assembly,63 DNA microarrays,64 and zinc-finger
in the substrate that can recombine with the first one to nucleases65 were considered state-of-the-art. A PhD student that
terminate the reaction cycle. In this scenario, no oxygen atom graduated in 2020, however, would have witnessed cost-efficient
is incorporated yet molecular oxygen is consumed. An additional gene synthesis,66 RNA-seq,67 and CRISPR/Cas968 emerge as routine.
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Fig. 5 Two examples of P450 catalyzed oxidative modifications in biosynthesis of plant natural products. (A) Secologanin synthase in biosynthesis of
monoterpene indole alkaloids; (B) salutaridine synthase in biosynthesis of morphine family of opioids.
The substantial unrealized potential of synthetic biology is an appropriate biosynthetic chassis must be selected. Finally,
evidenced by continued investments across industry and academia. the engineer must iterate through the design, build, test, learn
As these technologies expand, successful refactoring of a bio- (DBTL) cycle until sufficiently high titers, production rates,
synthetic pathway relies on the use of well-characterized ‘‘genetic and yields are reached.
parts’’ – these DNA-based elements permit coordinated expression 1.3.1 Pathway elucidation and design. Biocatalytic production
of genes of interest in a heterologous host.69 Following the methods benefit greatly from fully elucidated biosynthetic path-
standardization of genetic engineering protocols and genetic ways; a single missing biosynthetic step may completely
parts, reliable metabolic engineering techniques have been derail heterologous production efforts. Identification of natural
established that enable improvements in engineered systems. product biosynthetic logic is the primary focus of Sections 2–5.
The general methodology for synthetic biology-based hetero- Early biosynthetic investigations involved demonstrating that
logous production of natural products is outlined in Fig. 6. isotope labeled precursors could be site-specifically incorporated
First, a biosynthetic pathway must be elucidated such that a into final products, which provided connections between primary
heterologous production strategy can be envisaged. Second, metabolism and natural product biogenesis. Now, genomic
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sequencing and synthetic biology toolkits permit gene knockouts (multistep) are commonly used to refer to this type of hybrid
in the native host or expression in a heterologous host for synthetic approach, which has been leveraged in the biosyn-
functional analysis. ‘‘Reconstitution’’ of the activity of a recom- thesis of psilocybin81 and an ibogaine precursor.82 Lastly,
binantly expressed enzyme activity in vitro affords the most many in silico pathway design algorithms have been described
unequivocal evidence of a biosynthetic sequence. It should be in recent years, which perform automated retrobiosynthetic
mentioned that availability of transcriptomics data has provided analyses to predict novel or optimized pathways.83,84 This
a quantum leap in the ability to identify candidate enzymes, approach has been successfully applied to primary metabolic
particularly in unclustered plant pathways. Whereas bacterial products, highlighting the demand for continued investigation
and fungal biosynthetic pathways are frequently colocalized in a of secondary metabolic pathways.85–87 Machine-learning tech-
‘‘gene cluster,’’ examples of clustered plant pathways are nologies linked to databases of reactions using automated DBTL
scarce.70–72 Meanwhile, the differential abundance of RNA are predicted to play a role in the future of natural product
across plant tissues and cultivars gives metabolic engineers biomanufacturing.88
precise spatiotemporal gene expression data, which can be 1.3.2 Chassis selection. A critical parameter in the success-
mined for information about biosynthetic pathways. In recent ful refactoring of a natural product pathway is the selection of a
years, RNA-Seq has been used to identify a wide range of plant suitable biosynthetic chassis. Five representative biosynthetic
natural product biosyntheses, including a number of key chasses are shown in Fig. 6. The model bacterium Escherichia
conversions in psychoactive natural product pathways.45,73 coli has become a foundation of biotechnology as a DNA
For instance, Facchini and coworkers utilized RNA-Seq to bearing model organism. E. coli laboratory strains have been
discover neopinone isomerase, which catalyzes a reaction customized for plasmid propagation and protein expression.
previously believed to occur spontaneously in morphine Production of drugs with relatively short biosynthetic pathways
biosynthesis.74 As an additional example, Luo et al. identified has been shown,81,89 with stepwise mixed-strain cultures lever-
a functional prenyltransferase enabling cannabinoid production aged for longer pathways.90 Saccharomyces cerevisiae (Brewer’s
in S. cerevisiae by interrogating Cannabis sativa transcriptome yeast) was initially the subject of genetic studies, but has become
data.75 a favorite organism in academia to demonstrate heterologous
In some cases, a biosynthetic step from the native organism production of an impressive variety of plant or fungus-derived
cannot be identified, or functional expression of a known psychoactive drugs.73,75,77,91,92 The model ascomycete Aspergillus
pathway gene may not be feasible in a given organism. In this event, nidulans has also been used for the production of bioactive
bioprospecting or mining the genomes of alternative organisms to molecules due to its robust secondary metabolism and ability
identify functional proteins that carry out key reactions has been to splice fungal introns.93–95 Nicotiana benthamiana has proven
successfully applied. For example, incorporation of genes from useful in characterizing and reconstituting difficult plant path-
Gallus gallus (chicken) and Rattus norvegicus (rat) in place of missing ways, and is particularly attractive due to the well-established and
or non-functional yeast metabolic steps was a crucial advancement modular transient gene expression technologies.96–99 The fifth
in the development of MIA and BIA producing strains.76,77 chassis is synthetic biochemistry, wherein long-lived ‘‘cell-free’’
Alternatively, protein engineering strategies may be employed enzymatic reactions have enabled high-titer flux through lengthy
to alter the regiospecificity or substrate specificity of other well- biosynthetic pathways.53,100–102
characterized proteins in order to generate de novo suitable One must carefully consider the features of a given pathway
replacements for missing or nonfunctional steps. Dueber and before deciding if a particular chassis meets the biosynthetic
coworkers employed this method to engineer a L-tyrosine hydro- requirements. Many natural product pathways evolved in the
xylase, which normally requires a cofactor not produced in yeast, context of highly specialized organelles, cells, or tissues.103 In
and used the evolved enzyme to produce a morphine precursor.78 this case, pathway compartmentalization may be required in
The field of directed evolution is now well established,79 which order to sequester reactive biosynthetic intermediates from
can be implemented prior to DBTL or integrated into the DBTL endogenous metabolism. Currently, sub-cellular localization is
pipeline. possible through the use of organelle-targeting peptide signals
Following partial or complete pathway elucidation, a biosynthetic fused to the N-terminus of pathway enzymes, or the use of
strategy may be designed. For many psychoactive natural products, intracellular protein scaffolds.104,105 The recent production of
especially those which can be easily constructed from primary tropane alkaloids in yeast required extensive localization across
metabolites, de novo production from minimal media will provide six sub-cellular locations.73 Tissue specific pathway localization
the most cost-efficient route to a final product. Stephanopoulos in multicellular model organisms has yet to be employed but
and coworkers recently highlighted an alternative approach: the will require the implementation of intercellular metabolite
use of a late-stage pathway entry point to circumvent troublesome transport. Special attention must be given to enzymes that are
early biosynthetic steps.80 Such ‘‘mixed carbon’’ feeding strategies membrane associated, including the cytochrome P450s.106 Even in
may prove useful if an intermediate is commercially available or the most appropriate chassis, functional expression of trafficked
accessible via facile chemical synthesis. Efficient uptake of proteins may require extensive engineering. Galanie et al. employed
the late-stage entry point is another requirement, as transport a protein chimera strategy to ameliorate improper processing
limitations may prevent efficient substrate incorporation. The of a P450 for opioid biosynthesis in yeast.77 Solubilization of
terms biotransformation (single step) and bioconversion membrane anchored P450s has been successfully demonstrated,
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Fig. 6 Strategies in synthetic biology.
but a general strategy guaranteeing functional soluble expression industrially ‘‘robust’’ organisms may also be utilized. These
of P450s is still a major technological hurdle.107 It is also important may be proprietary strains that outperform laboratory strains,
to consider the primary metabolite building blocks required for but oftentimes lack the synthetic biology toolkit characteristic of
construction of the secondary metabolite to be produced. Indivi- the previously described model organisms. Proprietary methods
dual organisms exhibit variable fluxes towards given metabolic may be developed for rational engineering, or random mutagen-
pools, dictating initial maximum titers prior to strain engineering. esis may be employed for nonrational diversity generation.
To address this limitation, ‘‘metabolic chassis strains’’ – strains Additional properties of robust chasses are faster growth, resis-
with increased flux towards dedicated natural product building tance to contamination, and a tailored metabolic profile.
blocks – have been developed. Microbial chasses for the production Predictable scalability and ease of downstream purification
of N-methylpyrrolinium 20,108 strictosidine 25,76 (R)-reticuline costs should also be considered when assessing platform
28,90,109 and a number of other psychoactive natural product commercialization.110 For academic purposes, however, it is
precursors have been established in the last decade. most common to recapitulate biosynthetic pathways in model
The availability of a robust synthetic biology toolkit is another organisms as a proof-of-concept.
important factor to consider when selecting a production host. 1.3.3 Design, build, test, learn. Iterative design methodologies
An ideal suite of molecular biology tools permits accurate and are now commonplace in deploying synthetic biology-based
rapid genomic edits, precisely controlled gene expression, and engineering. In natural product production chasses, first
diversity generation using libraries of genetic parts. More generation strain prototypes almost never produce compounds
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in sufficient quantities to compete with alternative production are constructed, integration into the heterologous host is the final
strategies. As a result, many iterations of design, build, test, and hurdle in the ‘‘build’’ phase. The recent discovery of CRISPR/Cas9
learn (DBTL) are required before a process is cost competitive. has ameliorated this challenge. Cas9, an RNA-guided DNA endo-
The industrial feasibility of a bioprocess is often measured by nuclease, enables genomic modifications with unprecedented
titer (mass per volume), rate (mass per volume per time), and precision, greatly accelerating strain construction.68
yield (mass product per mass substrate) as these metrics relate Following the ‘‘build’’ phase, a screening approach is
to cost of goods sold (COGS).111 In addition to improving titers on required in order to ‘‘test’’ the performance of synthetic constructs.
the strain engineering front, large improvements in productivity can Screening throughput is dependent on the strategy used to
be made through bioprocess engineering, which has benefitted quantify production of a natural product. Direct measurement of
immensely from automated design of experiment methodologies. product titer using chromatography, mass spectrometry, and
The ability to iterate through the DBTL process is dependent on the spectrophotometry and comparison to an authentic standard
biosynthetic chassis, engineering strategy, and screening strategy, is the most accurate quantification method. Advancements in
among other factors. Novel metabolic engineering approaches instrumentation have increased the throughput and accuracy
aim to reduce the cost or duration of some aspect of the DBTL while decreasing costs, however these methods are still considered
cycle.112,113 As previously mentioned, ‘‘automated design’’ and low-to-medium throughput, requiring 1 minute–1 hour per
‘‘machine learning’’ technologies have only recently been sample. Meanwhile, indirect measurements of product titer
deployed in metabolic engineering studies. Thus, we focus employing biological readouts have enabled high-throughput
below on methodologies which streamline the ‘‘build’’ and testing of strains. So called ‘‘biosensors’’ transduce chemical
‘‘test’’ phases of iterative design. inputs into physiological outputs in order to establish a correlation
Within the DBTL cycle, synthetic biology toolkits have had between a titer and a selectable phenotype. Biosensors enable
the greatest impact on the ‘‘build’’ phase. Rapid and precise screening of constructs on the order of seconds or less per sample.
diversity generation, including the construction and integration In rare circumstances, a natural product is produced in sufficient
of expression assemblies into a platform, is a vital prerequisite quantities and has a unique enough absorbance spectrum to
to screening. Libraries of well characterized genetic parts provide function directly as the selectable chromophore. More typically, a
metabolic engineers with a set of elements that can precisely genetically-encoded biosensor must be engineered that robustly
control the expression of a pathway gene. To this end, vector sets, actuates a signal that can be correlated to the metabolite’s
promoter sets, terminator sets, and signal peptide sets are the concentration. Biosensors consist of a sensor-actuator pair and
most common control elements used. A vector is a circular are either RNA-based or protein-based. The sensor-input consists
fragment of DNA that harbors pathway genes, a selection marker, of binding of the biosensor to the secondary metabolite. Then,
and an origin of replication which dictates copy number and an actuator-output is generated resulting in modulation of
plasmid stability. Integration of synthetic biology constructs transcription or translation of a selectable protein. The genetic
directly into the genome may obfuscate the use vectors, however circuit may also encode Boolean logic in order to improve
shuttle vectors for cloning of constructs are generally still biosensor properties such as dynamic range or sensitivity.114
employed. Promoters are regulatory elements directly upstream Selection is then performed either in situ (cell viability) or ex situ
of a gene of interest, which recruit transcriptional elements for (high-throughput cell sorting). For example, a cell viability screen
gene expression. Promoters may be constitutive (always on) or can be established by tying a biosensor output to expression of an
inducible (turned on by a condition). The promoter ‘‘strength’’ antibiotic resistance gene or complementation of an auxotroph.
correlates to the copy number of mRNA upon induction; On the other hand, biosensor-dependent expression of a fluor-
promoters are often referred to as tight (no basal expression) escent protein enables high-throughput fluorescence-activated
or leaky (measurable basal expression). Terminators are the cell sorting (FACS) for rapid analysis of entire populations of
regulatory elements downstream of the protein coding sequence, cells. Microbial opioid production has benefited greatly from the
signaling transcriptional termination, and impact the half-life of use of biosensors, as both RNA and protein based metabolite
mRNA. Signal peptides may be employed to direct expression to sensors have been reported for benzylisoquinoline alkaloid path-
an organelle for localization or secretion. Prior to use, these way intermediates.78,115 Adaptive laboratory evolution (ALE) has
genetic parts must be assembled into a single contiguous DNA also emerged as an efficient method to circumvent traditional
fragment. Sequence independent cloning techniques such as DBTL strain construction. ALE employs natural selection and
Gibson assembly and yeast homologous recombination have in vivo diversity generation for population-wide engineering, and
replaced traditional methods such as digestion-ligation.63 has been primarily applied to primary metabolic products.116
Furthermore, gene fragments can now be affordably synthe- Although several generalizable biosensor development platforms
sized, circumventing strain procurement and DNA isolation.66 A have been proposed, research towards rapid expansion of the
once tedious and unpredictable process, heterologous gene variety of sensed metabolites is ongoing.
expression has been streamlined using reliably functional Compared to organic synthesis and biochemical engineering,
elements; gene expression is now definitively ‘‘engineerable’’. synthetic biology is a relatively nascent applied science. Despite
As we gain a more comprehensive understanding of sophisti- this, immense progress has been made in the last 20 years, and a
cated cellular programs, we will be able to assemble even more number of recent success stories illustrate the field’s potential.
robust and dynamic synthetic biology circuits. Once such systems Research groups now routinely refactor pathways with more than
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10 steps in A. nidulans and N. benthamiana, and pathways with the modulation of sensory perception, mood, cognition, memory,
more than 20 steps have been reconstituted using both S. cerevisiae and more through the peripheral and central nervous systems
and synthetic biochemistry. The ongoing challenge for these plat- (Fig. 7).121 There are many subtypes, and with the exception of
forms is to improve titers and reduce costs sufficiently to compete 5-HT3 which is a ligand-gated ion channel, the rest are G-protein-
with traditional production methods. General strategies range coupled receptors, each with unique spatial distribution and
from improving flux through pathway bottlenecks to ameliorating localization in the brain.122 Phylogenetic analysis and low
growth defects from metabolic burden or toxicity, however a more sequence identity (B25% between the major subtypes) demon-
nuanced engineering approach is often required. In depth strates early divergence, implicating 5-HT receptors as one of
discussions of the engineering strategies enabling benchmark the oldest receptor systems.121
production of the psychoactive natural products described in The relationship between 5-HT receptors was first determined
this review accompany the biosynthetic pathway descriptions. through testing of LSD 3. While hallucinogenic compounds like 3
(Fig. 8) have been shown to target multiple 5-HT receptors, the
5-HT2A receptor is most commonly associated with the majority of
2. Hallucinogenic natural products psychotropic effects.123 Previously, structure–activity relationship
Of all the psychoactive compounds that are either isolated as studies between 5-HT2A and numerous psychoactive compound
natural products or produced synthetically, hallucinogens may scaffolds have demonstrated that hallucinogenic potency is not
impart the most dramatic shifts in one’s psyche. This broad class necessarily a function of affinity, and likely due to more nuanced
of substances can induce potent alterations to consciousness, mechanisms of functional selectivity.124 However, a recent crystal
mood, and perception resulting in vivid visual hallucinations, structure of 3 complexed with 5-HT2B (a model system for 5-HT2A)
synesthesia, and a warped sense of time and space.117 The precise was reported and combined with molecular dynamic simulations,
mixture of perceptual and somatic effects of hallucinogens is identified a molecular basis for the particular potency of 3.125 The
highly compound specific and thus has led to many debates on authors demonstrate that the diethylamide side chain of 3 adopts
accurate nomenclature. There is yet to be a consensus with terms a restrictive conformation when bound to 5-HT2B that increases
such as ‘‘psychedelic’’ and ‘‘entheogen’’ often used interchange- residence time and improves b-arrestin translocation to the cell
ably with ‘‘hallucinogen’’ in different contexts. membrane. This enhanced b-arrestin translocation results in
Natural sources of hallucinogens famously include ‘‘magic desensitization of the cell to stimuli by uncoupling G-proteins
mushrooms’’ of the Psilocybe genus and other fungi such as from receptors and could explain the long duration of action of 3.
ergot and fly agaric. Other well-known sources of hallucinogens
2.2 N,N-Dimethyltryptamine
are from the spineless cactus, peyote, the psychoactive brew
ayahuasca, and with a recent resurgence, nutmeg.118 Most N,N-Dimethyltryptamine (DMT) 29 (Fig. 9) is likely the most
natural hallucinogens are alkaloids derived from amino acids pervasive psychoactive compound across species and is found
such a L-tryptophan 11, L-tyrosine 12, and L-glutamic acid 36 in dozens of plant and animal species, including humans.126
(Fig. 7), with one notable exception being the terpenoid salvinorin Root, bark, and leaf preparations from plants such as Psychotria
A 37. Numerous extensive reviews exist on the history, pharma- viridis, containing DMT and its structural analogs (Fig. 9) have
cology, and potential as therapeutics of hallucinogens which we been used in shamanic ritual practices for at least 1000 years.127
recommend.117,119,120 Interestingly, in addition to plants, structural analogs 5-methoxy-
N,N-dimethyltryptamine 39 and bufotenin 40, are also found in
2.1 Serotonin receptors the toxin of the Colorado River toad Incilius alvarius, formerly
The serotonin or 5-hydroxytryptamine (5-HT) receptors, named known as Bufo alvarius, whose remains have been found as a
for their native ligand, serotonin 38, have been implicated in part of Olmec ritual ceremonies dating back to pre-Columbian
Mesoamerica (Fig. 10).128,129 Referred to colloquially as the
‘‘Psychedelic Toad of the Sonoran Desert,’’ exudates from the
amphibian’s specialized glands may contain up to fifteen per-
centage dry weight 39, representing the most notable example of
a psychoactive natural product of animal origin.130 DMT 29 was
first isolated from the shrub Mimosa tenuiflora in 1946 by
Oswaldo Gonçalves de Lima,131 but its hallucinogenic effects
were not discovered for another decade.132 29, like all L-tryptophan
derived hallucinogens, is a serotonin receptor agonist. While the
functional selectivity of 29 towards the 5HT2A receptor is believed
to be necessary for its effects, 29 can bind to many serotonin
receptors that may also contribute to its psychoactivity.126
While the precise role of endogenous 29 in humans has yet
to be ascertained,133 one study speculates it may have a role in
Fig. 7 Amino acid building blocks for hallucinogens that target serotonin protecting from hypoxia.134 Further, 29 has shown promise as a
receptors. therapeutic anti-depressive agent and is known to promote
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Fig. 8 Overview of hallucinogenic natural products. *Note that LSD 3 is a semisynthetic compound derived from lysergic acid (Section 2.5).
14 by an aromatic amino acid decarboxylase (AADC) (Fig. 11, and

Fig. 2).140 The PLP-dependent AADCs in most species display a broad
substrate scope, operating on multiple aromatic amino acids and
derivatives.140 Tryptamine 14 is then methylated sequentially by an
iterative N-methyltransferase (INMT) to first form the secondary
amine, then 29, using SAM (Fig. 2B) as a methyl donor.141,142
2.3 Psilocybin
Fig. 9 Psychotria viridis is one of the common sources of DMT for ritual Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) 1, one of
purposes. Image on the left courtesy of Paulo Pedro P. R. Costa via. CC-
the major natural products from hallucinogenic Psilocybe sp.
4.0. https://upload.wikimedia.org/wikipedia/commons/0/01/Psychotriavir
idisFrutoDSC75.jpg. (‘‘magic mushrooms’’), was first isolated from Psilocybe mexicana
by Albert Hofmann in 1958 (Fig. 12).143 The description of ‘‘magic
mushrooms’’ in scientific literature and the subsequent isolation
neural plasticity.135,136 Interestingly, brominated forms of DMT and characterization of their psychoactive metabolites was the
such as, 5-bromo-N,N-dimethyltryptamine 41, have been iso- culmination of decades of effort to identify the sacred mushroom
lated from marine sponges137,138 and show particular promise that the South American Aztecs referred to as teonanacatl,
as anti-depressives.139 Finally, 29 has limited neurotoxicity and meaning ‘‘god’s flesh.’’144 Psilocybin 1 itself is not psychoactive,
only exhibits cardiovascular effects when taken intravenously in but rather exists as a prodrug. After ingestion, psilocybin 1 is
large doses, furthering its therapeutic potential.126 metabolized through dephosphorylation and becomes psilocin
2.2.1 Biosynthesis of DMT. The biosynthesis of DMT 29 is (4-hydroxy-N,N-dimethyltryptamine) 42, a potent psychotropic
the shortest pathway described in this review, requiring just 5HT2A receptor agonist.145,146 In addition to its psychoactivity,
two enzymes. Biogenesis begins with the decarboxylation of the 1 has shown some promise as a therapeutic for treating depres-
proteinogenic amino acid L-tryptophan 11 to form tryptamine sion, anxiety and tobacco addiction.147–149
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Fig. 12 Psilocybe mexicana contains B1% psilocybin. Image on left

courtesy of Alan Rockefeller via CC-3.0. https://upload.wikimedia.org/
wikipedia/commons/4/46/Psilocybe_mexicana_53960.jpg.
by Fricke et al. (Fig. 13).151 The authors first sequenced the

genomes of both Psilocybe sp. Then, using a combination of a
methyltransferase, a hydroxylase, and a kinase as queries,
a putative biosynthetic cluster present in both species was
identified and characterized. Fricke et al. determined that the
iterative N-methylation was the terminal step of psilocybin
biosynthesis by enzymatic action of PsiM whose sequence is
unrelated to the well-characterized mammalian indolethylamine-
Fig. 10 Incilius alvarius’s skin and exudates contain 5-methoxy-N,N- N-methyltransferases, and thus revised the hypothesis that DMT
ditryptamine and bufotenin. Image on top courtesy of Wildfeurer via. 29 is an intermediate in psilocybin biosynthesis. Starting from
CC-3.0. https://upload.wikimedia.org/wikipedia/commons/4/4f/2009-
L-tryptophan 11, PsiD catalyzes a decarboxylation reaction to
03-13Bufo_alvarius067.jpg.
yield 14. The amino acid sequence for PsiD diverges from the
more common PLP-dependent aromatic amino acid decarboxy-
lases and instead shares similarity with the PLP-independent
Fig. 11 Biosynthesis of DMT.
2.3.1 Biosynthesis of psilocybin. A biosynthetic pathway

for psilocybin was proposed based on isotope feeding studies as
early as 1968.150 Agurell et al. hypothesized that following
decarboxylation, L-tryptophan 11, now tryptamine 14, would be
methylated iteratively to form the psychoactive dimethyltryptamine
29. This was a reasonable hypothesis because indolethylamine-
(tryptamine)-N-methyltransferases were a popular enzyme for
study at the time following their discovery in rat, rabbit, and
human tissues.
Recently, a psilocybin biosynthetic cluster from Psilocybe
cubensis and Psilocybe cyanescens was identified and characterized Fig. 13 Biosynthetic pathway of psilocybin and psilocin from L-tryptophan.
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phosphatidylserine decarboxylases. PsiH, a P450 monooxygenase,

then hydroxylates the indole C4 to yield 4-hydroxytryptamine 43.
Next PsiK, a predicted kinase, catalyzes the phosphorylation
of 4-hydroxytryptamine 43 into norbaeocystin 44 using ATP as
the phosphate donor. Phosphoryltransferase (or kinases) are
relatively uncommon in natural product biosynthesis. Recent
examples include the biosynthesis of calyculin protoxins and

the lasso peptide paeninodin, in which phosphorylation plays a
role in self-immunity which could highlight the importance of
dedicated kinases.152,153 Lastly, PsiM methylates the terminal
amine in 44 in an iterative fashion using SAM as a methyl
donor to give 1. PsiM only methylates phosphorylated trypta-
mine 44, indicating that psilocybin biosynthesis is nearly
linear. In water, 1 undergoes spontaneous hydrolysis of the
phosphate group to form 42, but PsiK accepts psilocin as a
substrate and readily phosphorylates to reform psilocybin 1. As
previously mentioned, this hydrolysis results in the psychoactive
form, 42, upon ingestion by vertebrates.
Subsequently, additional psilocybin biosynthetic clusters were
found in distant fungal species and provide some evolutionary
evidence of the ecological role of psilocybin in influencing myco-
phagy in animals, which is to reduce their consumption from
invertebrate predators.154 Thus, the bioactivity of 1 may provide a
fitness advantage to natural producers over their competitors.
Further, a recent preprint presents evidence of a new, diverged
psilocybin cluster in Inocybe corydalina that contains a second
methyltransferase that may produce the trimethylated, quaternary Fig. 14 Engineered production of psilocybin in E. coli.81
ammonium salt analogue of 1, aeruginascin.155
2.3.2 Heterologous production of psilocybin. Since the
elucidation of the psilocybin biosynthetic pathway, engineering Engineered de novo production of psilocybin 1 was recently
efforts for high-titer production of psilocybin 1 in various reported in a fully integrated S. cerevisiae strain with a titer of
microbial hosts, such as the filamentous fungus A. nidulans, 627 140 mg L1 of psilocybin 1 and 580 276 mg L1 of
Baker’s yeast S. cerevisiae, and the model bacterium E. coli have psilocin 1 from 1 L scale fed-batch fermentation over B9 days
been reported.81,91,94 Hoefgen et al. developed a polycistronic (Fig. 15). Psilocybin pathway genes from P. cubensis were expressed
expression system in A. nidulans and used the psilocybin path- under strong, constitutive promoters. Instead of expressing the
way as a proof-of-concept. They obtained 110 mg L1 of 1 at pathway decarboxylase PsiD, Milne et al. expressed a tryptophan
1.5% dry mycelial weight which is a titer comparable to native decarboxylase from Catharanthus roseus, CrTDC, which was pre-
psilocybin producers. viously shown to have high catalytic efficiency when expressed in
Adams et al. were able to combine heterologous expression yeast.76 Additionally, the authors expressed a cytochrome P450
and metabolic engineering strategies to achieve a titer of 1.16 g L1 reductase (CPR) from P. cubensis to improve activity of PsiH.
psilocybin 1 in E. coli in a 1.5 L bioreactor from 3.05 g L1 of Matching a P450 with its cognate reductase partner has been
gradually supplied 4-hydroxyindole 45 (Fig. 14) over several days. demonstrated to be important for functional heterologous
The exogenously supplied 45 is first converted into 4-hydroxy-L- expression and is an effective technique to improve hetero-
tryptophan 46 by TrpB, an endogenous bacterial enzyme in the logous expression of P450 enzymes.106,156 To increase endogenous
L-tryptophan biosynthetic pathway that catalyzes the condensation
L-tryptophan levels, the authors overexpressed ARO1 and ARO2,
of indole with serine to form 46. PsiD, PsiK, and PsiM from which are involved in combining erythrose 4-phosphate 47 and
P. cubensis were heterologously expressed under a single T7 phosphoenolpyruvic acid 48 to form chorismic acid 49 in the
promoter on a high copy plasmid, which facilitated the conversion shikimate pathway leading to L-tryptophan biosynthesis. This,
of 44 formed in situ into psilocybin 1. Endogenous levels of serine combined with knockout of a shikimate pathway regulator,
and SAM, required by TrpB and PsiM, respectively, were not RIC1, were effective towards elevating L-tryptophan supply.
sufficient for high-titer production and thus the media was
supplemented with excess amounts of serine and methionine.
The native E. coli enzyme MetK is able to anabolize the exogenous 2.4 Ayahuasca
methionine into SAM while the E. coli enzymes Mtn, LuxS, and Most hallucinogens are rapidly metabolized in vivo following
MetE are able to recycle the by-product S-adenosylhomocysteine ingestion by the action of monoamine oxidases (MAO) for
(SAH) into more methionine. eventual renal elimination, resulting in many hallucinogens
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Fig. 15 Engineered production of psilocybin and psilocin in yeast.91
being orally inactive. MAOs, as the name suggest, catalyze the

oxidative deamination of neurotransmitters and structurally
similar compounds.157 It follows that ingestion of MAO inhibitors
(MAOIs) concurrently with hallucinogens can increase their bio-
availability. This synergy is best demonstrated by ayahuasca, a
psychoactive decoction commonly prepared from the vine
Banisteriopsis caapi, containing MAO inhibiting harmala alkaloids,
and some DMT 29 containing species such as the shrub Psychotria
viridis.158,159 Ayahuasca, derived from a Quechua term meaning
‘‘vine of the soul,’’ has been used as a spiritual medicine by
indigenous groups in South America’s Amazon basin for at least
one thousand years.127 During a ceremony in which the brew is
ingested, practitioners experience several stages of visual and
purgative experiences in order to heal physical, emotional, and
spiritual imbalances.160 While there are no currently approved
therapeutic uses for ayahuasca or its active metabolites, harmala
alkaloids have shown promise as an antidepressant through brain
plasticity modulation.161
The harmala alkaloids are compounds that contain a b-carboline
scaffold with various methyl or methoxy substitutions and different
degrees of unsaturation. The b-carboline scaffold itself is
Fig. 16 Banisteriopsis caapi contains many compounds with the b-
characterized by a pyridine ring ortho-fused to indole resulting carboline scaffold, including harmine. Image on left courtesy Forest and
in a 6-5-6 tricycle with possible substitutions at the ortho Kim Starr via CC-2.0. https://upload.wikimedia.org/wikipedia/commons/1/
position to the pyridine nitrogen. The major harmala alkaloids 17/Starr-140222-0335-Banisteriopsis_caapi-leaves-Haiku-
that contribute to the MAOI activity are harmine 23, harmaline Maui_%2825240510635%29.jpg.
50, and tetrahydroharmine 51. These compounds are produced
at B0.05–0.1% of dried plant material in B. caapi (Fig. 16).162
Thorough pharmacokinetic data is scarce, but psychotropic with a typical 100 mL ayahuasca brew containing between B300–
action of harmala alkaloids is expected to occur around 20–50 mg 600 mg of harmala alkaloids and 20–60 mg of 29.163
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Another example of a MAOI natural product cocktail is the the radiolabeled C-2 and C-3 carbons of pyruvic acid 22 into the
recent isolation of 23 and related b-carbolines from numerous pyridine ring of the b-carboline scaffold, while radiolabeled
hallucinogen-producing Psilocybe sp. as known as ‘‘magic acetic acid carbons were non-specifically incorporated through-
mushrooms’’.164 This serves as an interesting example of a out as a result of primary metabolism. 1-Methyl b-carboline 53
single organism with diverged secondary metabolite scaffolds, is then formed by oxidative decarboxylation, followed by subsequent
where the biosynthetic pathways of both compounds diverge at hydroxylation and O-methylation reactions to form harmaline 50.
tryptamine 14 but contribute to the same psychoactive effect. Formation of harmine 23 or tetrahydroharmine 51 takes places
2.4.1 Biosynthesis of harmala alkaloids. Initial feeding through either oxidation, or reduction of 50, respectively.
studies with radioactively labelled substrates into seedlings of
the known harmala alkaloid producer, Peganum harmala, 2.5 Lysergic acid and LSD
demonstrated that L-tryptophan and L-methionine are precursors
Lysergic acid diethylamide (LSD) 3 was first synthesized from
in the biosynthesis of harmala alkaloids.165 A later study demon-
lysergic acid 54 by Albert Hofmann in 1938. Like other 5HT2A
strated that radiolabeled 26 could be converted into its dehydro-
receptor agonists, ingestion of 3 results in altered states of
genated form, 50, and that harmala alkaloid biosynthesis is likely
consciousness and visual hallucinations.168 While 3 has not
compartmentalized across different tissues.166
been observed to occur naturally, its precursor, 54, is a natural
While the complete set of biosynthetic genes implicated in
product belonging to a class of diverse molecules broadly
harmala alkaloid formation have yet to be determined, one
known as ergot alkaloids. 54 is isolated from many fungi with
proposal167 postulates the sequence shown in Fig. 17. As in the
the ergot fungus, Claviceps purpurea (Fig. 18) being the most
case of the other indole containing compounds described, 11 is
notable.169,170 Ergot alkaloids are commonly associated with
first decarboxylated to form 14 (Fig. 17). Next, pyruvic acid 22 is
the disease ergotism, known colloquially as Saint Anthony’s
incorporated by a Mannich or Pictet–Spengler type reaction to
Fire, caused by eating rye or other cereal crops contaminated
form the b-carboline carboxylic acid 52 (also see Fig. 2). To
with ergot fungi.171 In addition to the vasoconstrictive and
determine the biosynthetic origin of the C-1 b-carboline methyl,
convulsive symptoms of the disease, mania and psychosis
radiolabeled feeding of acetic acid and pyruvic acid was
have been observed, underlining the psychoactivity of ergot
performed.165 Stolle et al. observed specific incorporation of
alkaloids.171
Ergot alkaloids, derived from L-tryptophan 11, are characterized
by a unique tetracyclic ergoline skeleton where the indole com-
prises the A and B rings. The C and D rings of the ergoline scaffold
are derived from a cyclization of dimethylallyl pyrophosphate
with the L-tryptophan amino group.172 There are three main
ergot alkaloid classes, clavines, ergoamides (lysergamides), and
ergopeptides, with 3 belonging to the ergoamide class.173
Ergoamides contain a C8-amide linkage on the D ring of the
ergoline scaffold and is a common point of derivatization for
drug development.174 Modifications on the amide can greatly
affect bioactivity and in the case of 3, the diethylamide moiety is
crucial for its prolonged psychoactivity.125
2.5.1 Biosynthesis of lysergic acid. Isotope labeling studies
during the 1950s and 1960s determined that a mevalonate-
derived isoprenoid, a methionine-derived methyl group and
L-tryptophan 11 were key precursors to ergot alkaloid bio-
synthesis.175 The first enzymatic study in Claviceps sp. was the
Fig. 18 Claviceps purpurea (ergot fungus) infecting Dactylis glomerata

(cat grass). Image on the left courtesy of Bildoj via CC-3.0. https://upload.
Fig. 17 Proposed biosynthesis of harmala alkaloids.167 wikimedia.org/wikipedia/commons/c/c4/Dactylis_026.JPG.
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Fig. 19 Biosynthesis of lysergic acid from L-tryptophan.
purification and characterization of 4-dimethylallyl-L-tryptophan which was initially annotated as a catalase. Knock-out studies
synthetase (DMATS) that catalyzes the first committed step in in both C. purpurea and the homologous cluster in A. fumigatus
ergot alkaloid biosynthesis: C-prenylation of L-tryptophan 11 with confirmed that both enzymes are necessary for production of
dimethylallylpyrophosphate at the indole C4 position to form 57.187,188 Subsequent pathway reconstitution studies in Aspergillus
4-dimethylallyl-L-tryptophan 55 (Fig. 19, also see Fig. 4D).176 nidulans and Saccharomyces cerevisiae further supported the
Recently, many laboratories have focused on characterizing essential roles of EasE and EasC in biosynthesis.189,190 Until
prenyltransferases, of which DMATS is the original member of recently, however, the precise mechanisms of EasE and EasC
a new superfamily of prenyltransferase enzymes. Since the dis- were not resolved. Lorenz et al. initially postulated that EasE
covery of the DMATS, prenyltransferases that can regioselectively catalyzes the oxidative diene formation from 56 followed by
transfer allylic prenyl groups to almost every position on the decarboxylation through an epoxide intermediate to yield
indole ring have been identified.48,177–181 Members of the DMATS chanoclvaine-I 57, with EasC serving as a scavenger of hydrogen
superfamily also have broad substrate scopes while maintaining peroxide generated from EasE.188 A recent pathway reconstitution
regioselectivity which has aided in their development as tools for in A. nidulans enabled isolation of the a previously unknown
chemoenzymatic syntheses of natural and unnatural prenylated intermediate, pre-chanoclavine diene 58, which verified the diene
compounds, including the cannabinoid family (see 4.2.2).47,53,182,183 formation activity of EasE.191,192 Subsequent incubation of 58 with
Chromosome walking using the gene encoding DMATS as a EasC recombinantly purified from E. coli led to the formation of
step-off point led to the identification of an ergot alkaloid bio- 57 via a proposed radical addition mechanism using O2 as an
synthetic gene cluster in the fungus C. purpurea.184,185 Sequence oxidant.192 Hence, EasC is an essential redox enzyme in the
alignment revealed an N-methyltransferase, EasF which was pro- main pathway to 54.
posed to convert 4-dimethylallyl-L-tryptophan 55 into 4-dimethylallyl- A short-chain reductase (SDR), FgaDH, was identified in an
L-abrine 56 using SAM as a methyl donor. Thorough characterization A. fumigatus gene cluster that produces a related ergot alkaloid
of a homologous enzyme in an Aspergillus fumigatus ergot gene fumigaclavine C.193 In vitro assays using recombinantly expressed
cluster, FgaMT, supported this hypothesis.186 enzyme determined that FgaDH catalyzes the oxidation of the
Conversion of 56 into the cyclized chanoclavine-I 57 is allylic alcohol on 57 to an aldehyde to form chanoclavine-I
facilitated by the FAD-linked oxidoreductase EasE and EasC, aldehyde 59, strictly using NAD+ as the electron acceptor.193
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A homologous SDR was subsequently identified in the lysergic

acid biosynthetic gene cluster in C. purpurea and named EasD.194
The next steps in the pathway represent a branching point
for ergot alkaloids. Functional differences in a conserved flavin-
dependent old yellow enzyme known as EasA (an isomerase)
from C. purpurea and FgaOx3 (a reductase) from A. fumigatus
and P. commune represent a mechanistic branching point in

D-ring formation.195–197 Here we will focus on the formation of
Fig. 20 Lophophora williamsii, one of the many cacti species that contain
agroclavine 61 from 59 towards the psychoactive lysergic acid mescaline. Image on the left courtesy of Peter A. Mansfeld via CC-3.0. https://
amides in C. purpurea. EasA performs a hydride mediated upload.wikimedia.org/wikipedia/commons/6/69/Lophophora_williamsii_
isomerization of the a,b-unsaturated carbonyl from the E-alkene pm.jpg.
geometry to the Z-configuration through an enolate intermediate.196
This rearrangement positions the carbonyl for an intramolecular
cyclization with the secondary amine resulting in the formation of 66 and 67 are believed to be prodrugs as they are metabolized in
the D-ring.196 Following ring closure, the iminium intermediate the liver into 3-methoxy-4,5-methylenedioxyamphetamine, also
agroclavinium 60 then undergoes NADPH-dependent reduction by known as MMDA.206,207 MMDA and its analogs were first synthe-
the oxidoreductase EasG to form 61.198 sized from 65 by Alexander Shulgin, and similar to 65, MMDA is a
Assays of microsomal fractions from C. purpurea determined 5HT2A receptor agonist, but with almost double the potency.208
that 61 undergoes a 2-electron oxidation of the methyl group to Shulgin would later detail his extensive clandestine investigations
an alcohol to form elymoclavine 62 by an unidentified cyto- into the syntheses and effects of substituted phenethylamines and
chrome P450 monooxygenase.199 The only P450 enzyme in the tryptamines, earning him the title ‘‘godfather of psychedelics.’’209,210
biosynthetic gene cluster, CloA, does not catalyze this transfor- 2.6.1 Biosynthesis of mescaline. Before the discovery of the
mation and instead performs the 4-electron oxidation of 62 to mammalian iterative methyltransferase that catalyzes N-methylation
paspalic acid 63 as suggested from two knock-out studies.200,201 of tryptamine 14 and serotonin 38 into hallucinogenic
In DcloA mutants, 62 was still detected and supports the like- compounds,141 Axelrod and Tomchick identified another neuro-
lihood of an additional P450 enzyme in the host that can perform transmitter methyltransferase, catechol O-methyltransferase
the first 2-electron oxidation. Finally, allylic isomerization of 63 (COMT).211 COMT, along with monoamine oxidase, modified
forms the product lysergic acid 54. This transformation can occur the L-tyrosine-derived catecholamine neurotransmitter dopamine
spontaneously, but it remains possible that that an unidentified 17 (Fig. 21) for excretion in the urine.212 In the years following,
isomerase can catalyze this reaction as enzyme-catalyzed allylic similar to the case of endogenous DMT biosynthesis, several
rearrangements have been observed in other pathways.202,203 studies identified enzymes in mammalian tissues that could
54 itself serves as a branching point for the formation of catalyze the chemical transformations of dopamine-related meta-
many ergopeptines or ergoamides. These derivatives are formed bolites 3-methoxytyramine 68 into 3-methoxy-5-hydroxytyramine
by a non-ribosomal peptide synthase (NRPS) enzyme complex of 69 and 3,5-dimethoxytyramine 70 into 65, although no endogenous
two synthetases, LPS1 and LPS2.173 One of these lysergic acid 65 could be identified from mammalian organisms.213,214 Several
derivatives from Ipomoea purpurea (Morning Glory), ergine 64 mechanisms for 65 biosynthesis in peyote and related cacti have
(lysergic acid amide, LSA) is psychoactive. The pathway leading been proposed by metabolite isolation and radiolabeled
to formation of 64, while unconfirmed, could involve amidation feeding studies.215–219 One proposed pathway by Lundström
by an NRPS or degradation of another NRPS product.204 is shown in Fig. 21.219
The proposed biosynthesis begins with hydroxylation of
2.6 Peyote L-tyrosine 12 to 3-hydroxy-L-tyrosine (L-DOPA, 71) by tyrosine
Peoples indigenous to North America have consumed the cactus, hydroxylase (TH), followed by decarboxylation catalyzed by
peyote, for over one thousand years as a part of their religious DOPA decarboxylase (DDC) to yield 17. Alternatively, 12 may
practices.205 Peyote, Lophophora williamsii (Fig. 20), is a small, also be converted to tyramine 15 through a decarboxylation
spineless cactus with a crown consisting of round buttons that, catalyzed by tyrosine decarboxylase (TyrDC), followed by aro-
among other species of cacti, contain the hallucinogen, mescaline matic hydroxylation to 17 by an unknown enzyme. From either
65.205 The psychoactive effects have been described to be similar to route, 17 can be converted into 3-methoxytyramine 68, which
LSD, but with a significantly lower potency at a ratio of about has been isolated from mescaline producing plants, by the
1 : 2500 mescaline:LSD.117 Despite peyote’s status as a Schedule I enzyme catechol O-methyltransferase (COMT) using SAM as the
controlled substance in the United States, it remains legal as an methyl donor. The final intermediates towards mescaline production
important part of religious practices by the Native American 3-methoxy-5-hydroxytyramine 69 and 3,5-dimethoxytyramine 70 have
Church and other religious organizations who are protected by been determined to be naturally occurring in mescaline producing
the American Indian Religious Freedom Act. plants by inverse isotope dilution, but neither have been isolated
The natural products, elemicin 66 and myristicin 67 (Fig. 8) from plants. These are likely to be on pathway intermediates since
from nutmeg, or Myristica fragrans, are tetrasubstituted benzenes they are incorporated into mescaline to a greater extent than other
and structurally related to 65. Despite not being psychoactive, possible intermediates.219
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Fig. 22 Amanita muscaria contains about B100–1000 ppm of ibotenic

acid and muscimol.
processing and motor function, respectively.223 A. muscaria is

classified as poisonous, which can in part be attributed to the
neurotoxicity of 72. Its structural similarity to L-glutamic acid
36 allows 72 to act as an agonist towards the N-methyl-D-
aspartate (NMDA) receptor resulting in electrolytic lesions in
the brain.224
72 and 73 naturally occur in low concentrations (B100–1000
ppm) in the cap and stem of A. muscaria.225 Minimal dosage for
psychedelic effects are estimated as low as 6 mg for 46 and 30–
60 mg for 72.226 Interestingly, A. muscaria and its constituents
are not regulated by the United States federal government, in
contrast to 1 and 42 from Psilocybe sp.
While 72 was first isolated over 50 years ago, its biosynthesis
remained elusive.227 Recently, Obermaier and Muller identified
a gene cluster encoding 72 and 73 biosynthesis in A. mus-
caria.228 The key to locating this cluster was the identification
of a glutamate hydroxylase, an enzyme first implicated in 72
Fig. 21 Proposed biosynthesis of mescaline.221 biosynthesis over 50 years ago, but never found. This enzyme, a
nonheme, iron and a-ketoglutarate-dependent dioxygenase
named IboH, hydroxylates L-glutamate 36 at the C3 position
While the biosynthesis of 65 in peyote has yet to be eluci- resulting in the formation of 3-hydroxy-L-glutamic acid 74.
dated, Ibarra-Laclette et al. recently generated two cDNA 2.7.1 Biosynthesis of ibotenic acid. Obermaier and Muller
libraries of the L. williamsii transcriptome, one for button and proposed two pathways (A and B) for ibotenic acid biosynthesis
one for root, using RNA-seq.220 From this data set, the authors diverging from 74 (Fig. 23). One proposal (pathway A) is that 74
identified putative genes that may encode biosynthetic undergoes a condensation reaction catalyzed by IboA, an ade-
enzymes for mescaline production including DOPA decarbox- nylating enzyme, with ammonia from an unidentified donor to
ylases, hydroxylases, and O-methyltransferases based on phy- form 3-hydroxyglutamine 75. A likely amine source is glutamine
logenetic analysis.220 Careful in vitro experiments will be which is the amine donor in various metabolic reactions. IboF,
required to finally ascertain the mescaline biosynthetic a flavin-dependent monooxygenase, would then catalyze N-
pathway. oxidation of the terminal amide to form 3-hydroxyglutamine
hydroxamic acid 76. Next, either IboG1 or IboG2, PLP-
2.7 Fly agaric dependent paralogs found in the biosynthetic gene cluster,
Ibotenic acid 72, a nonproteinogenic amino acid with a hydro- catalyzes the intramolecular cyclization of the hydroxamic acid
xylated isoxazole ring, and its decarboxylated form, muscimol with the hydroxyl group at the C3 position to form the five-
73, are the main psychoactive constituents of the toadstool, membered heterocycle tricholomic acid 77. Alternatively, path-
Amanita muscaria, commonly known as fly agaric (Fig. 22).164 way B involves N–O bond formation between an unidentified,
Similar to Psilocybe sp., recreational consumption of Amanita hydroxylamine 78 with the C3 hydroxyl group on 74 by IboG1/
sp. rose in popularity in the 1960s. However, contrary to other G2 to form a 3-hydroxy-L-glutamic acid derivative 79. In this
fungal psychoactives that target the serotonin receptor, these pathway, the external hydroxylamine could derive from hydro-
compounds are g-aminobutyric acid type A (GABAA) receptor xylation of an external amine 80 catalyzed by IboF. IboA would
agonists.222 GABAA receptors are found in multiple regions of then facilitate cyclization of the hydroxylamine with the C-5
the brain and thus 72 and 73 can alter the activity of the carbonyl of the 3-hydroxy-L-glutamic acid derivative 79 to form
cerebral cortex and cerebellum leading to alterations in sensory 50. From tricholomic acid 77, IboC, a cytochrome P450,
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Fig. 23 Biosynthesis of ibotenic acid and muscimol from L-glutamic acid.228
catalyzes the desaturation of the 3-oxoisoxazolidine ring to

form ibotenic acid 72. IboD, a PLP-dependent decarboxylase
can catalyze the further decarboxylation of 72 to form the other
major psychoactive compound, muscimol 73.
2.8 Iboga alkaloids

Root and bark from the iboga tree, Tabernanthe iboga, has been
used for both therapeutic and spiritual ritual purposes in West Fig. 24 Tabernanthe iboga in fruit.
Central Africa for hundreds of years.229 T. iboga is rich in
L-tryptophan derived-monoterpene indole alkaloids (MIAs), an
expansive class of over 3000 plant natural products starting indoloazepine-isoquinuclidine junction lead to different family
from the universal MIA precursor, strictosidine 25.230,231 Many members within this class. Interestingly, 2 is the only known
molecules of this class have broad bioactivities that include compound with the iboga scaffold to have hallucinogenic
anti-cancer,221 anti-malarial,232 anti-addiction233 and more.234 properties, which raises questions about the structure–activity
The potent MIA cancer therapeutics vincristine and vinblastine relationship between 2 and 5-HT receptors. According to a
from Catharanthus roseus are listed on the World Health recent study, iboga scaffolds lacking the isoquinuclidine ring
Organization’s List of Essential Medicines, underlining the resulting in an indole-tetrahydroazepine tricycle lost their
value of MIAs as human therapeutics. One of the MIAs from hallucinogenic properties but maintained their ability to pro-
iboga roots is the psychedelic ()-ibogaine 2 that has multiple mote neural plasticity, the mechanism that may be the key to
neurotransmitter interactions including the k- and m-opioid its anti-addiction properties.26
receptors and the serotonin transporter, which collectively 2.8.1 Biosynthesis of iboga alkaloids. Given that strictosidine
results in a feeling of a dream-like state of consciousness.229 25 is the central metabolite in the MIA biosynthetic pathways in
Additionally, 2 and some of its derivatives have shown promise plants, there has been intense efforts to understand how nature
as anti-addictive agents.233,235 transforms the simple geranyl (C10) precursor that combines with
The iboga alkaloid scaffold is characterized by a 6-5-7-6-6 tryptamine 14 to yield the complex 25. These efforts from different
ring system comprised of indole and tertrahydroazepine fused labs have fully elucidated the pathway to 25. In recent years, further
to an isoquinuclidine ring to form a pentacyclic skeleton with efforts have led to the complete mapping of the downstream
a tertiary amine serving as the bridgehead (Fig. 24). The enzymatic transformation to vinblastine in C. roseus, comprised of
addition of a C5 methoxy group on the indole ring within over 30 enzymes starting from primary metabolites.45,236–243 Shortly
the iboga scaffold provides 3. Variable substitutions on the after, the ()-ibogaine biosynthetic pathway from 25 was also
indole ring and the presence of a carbomethoxy group at the elucidated, as well as other complex MIA compounds.244
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Fig. 25 Biosynthesis of secologanin from geranyl pyrophosphate (GPP).
The first committed step in the seco-iridoid pathway towards molecule in the pathway that has the iridoid structure. In plants
the monoterpene scaffold in 25 is the formation of geraniol 81 such as Nepeta, 85 can be oxidized to neptalactone, which is the cat
(Fig. 25). While it was predicted that 81 was hydrolyzed from the attractant produced by these plants.249 In the MIA pathway 85
mevalonate pathway intermediate, geranyl pyrophosphate (GPP) undergoes a 4-electron oxidation catalyzed by the P450 iridoid
82,245,246 the enzymatic basis of its formation was unknown oxidase (IO) to install an a,b-unsaturated carboxylic acid in
until the discovery of geraniol synthase (GES) from sweet basil 7-deoxyloganetic acid 30. The next step is glucosylation by
(Ocimum basilicum) decades later.193 Since then, many GES 7-deoxyloganetic acid glucosyl transferase (7DLGT) with UDP-
homologs have been discovered from various plants. The activity glucose to form 7-deoxyloganic acid 31 (see Fig. 3C). Glucosylation
of GES, which is to hydrolyze 82 to 81, represents a divergence of the hemiacetal presumably stabilizes the compound and
point between primary and secondary terpene metabolism in prevents spontaneous ring opening. The third P450 in the
plants. In primary metabolism, GPP is further elongated to pathway, 7-deoxyloganic acid hydroxylase (7DLH), catalyzes hydro-
farnesyl pyrophosphate (FPP), which is central towards the synthesis xylation of the cyclopentane ring in 31 to form loganic acid 86.
of steroids and coenzyme Q. By hydrolyzing the pyrophosphate in Expression data revealed that the next two genes in the seco-
GPP, GES commits the geraniol group for MIA biosynthesis and iridoid pathway encoding for loganic acid O-methyltransferase
siphons GPP away from primary metabolism. In the MIA pathway, (LAMT) and secologanin synthase (SLS) are part of a separate
geraniol 81 is then hydroxylated by the P450 enzyme geraniol regulon from the early pathway.250,251 The seco-iridoid pathway is
8-hydroxylase (G8H) to form 8-hydroxygeraniol 83.247 also spatially segmented between the internal phloem associated
The next four biosynthetic steps were all discovered from parenchyma (IPAP) cells for iridoid production and leaf epidermis
analysis of the C. roseus transcriptome.45 8-Hydroxygeraniol cells for the remaining steps towards production of strictosidine
oxidoreductase (GOR) iteratively oxidizes the two alcohols in 25.252 86 is first transported from the cytosol of the IPAP cells into
83 to yield 8-oxogeranial 84, a dialdehyde that is poised for the cytosol of epidermic cells by a nitrate/peptide family (NPF)
intramolecular cyclization. It was initially believed that iridoid transporter.253 The cytosolic LAMT subsequently converts 86 into
synthase (ISY) was an NAD(P)H-dependent cyclase.248 However, loganin 34.250 The fourth P450 in the pathway, SLS then catalyzes
a recent report demonstrated that ISY is a reductase that can oxidative cleavage of the cyclopentanol ring of 34 to unveil the
reduce 84 to an enol intermediate.249 A previously undiscovered reactive aldehyde handle in secologanin 24 (see Fig. 5A).56
cyclase, major latex protein-like (MLPL), then facilitates the To form strictosidine 25, 24 and 14 are condensed through a
cyclization of the reduced enol to form cis–trans nepetalactol 85 stereospecific Pictet–Spengler reaction catalyzed by strictosidine
via a non-cofactor dependent mechanism.243 85 is the first synthase (STR) (Fig. 26, and see Fig. 3).254 This mechanism had
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Fig. 26 Biosynthesis of ibogaine from tryptamine and secologanin.
been long proposed before the discovery of STR, modeled after herbivores mirroring the activation of the related phenolic
the formation of L-benzylisoquinolines alkaloids.255 Considering secoiridoid glycoside, oleuropein, from the privet tree, Ligustrum
the synthetic challenges associated with accessing 25, STR has obstusifolium following tissue damage.261
become an attractive enzyme for the chemoenzymatic and bio- From 25, different branches of the MIA family can be
transformative syntheses of analogs of 25.256–258 The regulation accessed. The first step is the deglucosylation of 25 by the
and complexity of MIA biosynthesis is further highlighted by the enzyme strictosidine-O-b-glucosidase (SGD).262 Whereas 25 is
transient sub-cellular compartmentalization of 25 formation in the relatively stable and benign to the host, removal of the glucose
vacuole of epidermis cells followed by immediate export towards group which essentially serves to mask the hemiacetal, leads to
the nucleus.259 It is believed that the spatial isolation of STR and the dialdehyde 4,21-dehydrogeissoschizine 87 that is prone to
its substrates prevents accumulation of the highly-reactive stricto- cross-linking. 87 exists in equilibrium with the more stable
sidine aglycone intermediate, 4,21-dehydrogeissoschzine 87 (vide epimers cathenamine and epicathenamine.263 Each of these agly-
infra), a dialdehyde which leads to toxic protein cross-linking.260 It cone intermediates represents a divergence point towards different
is hypothesized that this is a plant defense mechanism from terminal alkaloids.241,264 From 87, the next two transformations to
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form 19(E)-geissoschizine 88 and preakuammicine 89 catalyzed by pathway genes did not result in detectable production of pathway
geissoscizine synthase (GS) and geissoschizine oxidase (GO), intermediates, the authors employed a series of metabolic
respectively, were characterized by Tatsis et al.242 87 is converted engineering steps to boost precursor titers, reduce nonproductive
into 88 through iminium reduction catalyzed by GS.241 88 then shunt product formation, and increase P450 activity.
undergoes an oxidative rearrangement catalyzed by the P450 Towards increasing precursor titers, a truncated yeast
GO to yield an unstable intermediate, preakuammicine 89, 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) was expressed
which can undergo spontaneous rearrangement and tandem to increase the reduction of 3-hydroxy-3-methylglutaryl-CoA 96 to
enzyme-catalyzed reductions to form the stable stemmadenine form mevalonate 97. Since GPP 82 is not a native yeast metabolite,
90. Reactive intermediates that form between 88 and 90 exist expression of a GPP synthase (AgGPPS1) from Abies grandis
transiently can spontaneously undergo chemical transformations combined with expression of a mutated farnesyl pyrophosphate
that diverge towards different MIAs including corynanthean (see synthase (mFPS144) with partial GPP synthase activity from the
mitragynine, Section 5.3.1), strychnos, iboga, and aspidosperma avian Gallus gallus resulted in 82 biosynthesis. Maintaining some
skeletons.236 From 90, stemmadenine O-acetyltransferase (SAT) level of essential yeast metabolite farnesyl pyrophosphate (FPP)
catalyzed acetylation forms stemmadenine acetate 91. biosynthesis with mFPS144 was required since yeast FPP synthase,
A series of redox transformations and divergent cycloaddition ERG20, was knocked-out to shift mevalonate pathway flux from
reactions take place to transform 91 into catharanthine, taberso- FPP to 82. Balancing concentrations of isomers isopentenyl
nine, and ()-coronardine 92. Catharanthine and tabersonine are pyrophosphate 98 and dimethylallyl pyrophosphate 99 required
both on-pathway intermediates to vinblastine, while 92 has for 82 formation was achieved through expression of a second copy
essentially the same carbon skeleton as ibogaine 2. These trans- of the yeast isomerase IDI1. To further increase 81 titers, the
formations have recently been characterized through analysis of authors overexpressed MAF1, a negative tRNA biosynthesis regu-
transcriptome datasets from T. iboga and subsequent biochem- lator, which reduced the amount of 98 utilized for tRNA formation.
ical characterizations.244,265 First, a tandem amine oxidation- Geraniol 81 can be rapidly metabolized by yeast enzymes to
iminium reduction cascade catalyzed by precondylocarpine acet- form esterified and reduced shunt products.268 ATF1, an alcohol
ate (PAS) and dihydroprecondylocarpine acetate synthase (DPAS), acetyltransferase, and OYE2, an NADPH-dependent oxidoreductase,
respectively, would generate the enamine dihydroprecondylocar- were knocked-out to reduce nonproductive shunt product for-
pine acetate 93. The net outcome from 92 to 93 is migration of mation. A later study by Billingsley et al. identified more yeast
the olefin to set up the subsequent [4+2]-Diels–Alder reactions.237 enzymes that when knocked-out, further attenuate shunt product
In ibogaine biosynthesis, TiDPAS would promote the deacetoxylation formation from 8-hydroxygeraniol 83 and channel additional flux
with concomitant carbon–carbon bond cleavage, and NADPH- of 83 towards iridoid biosynthesis.82
dependent tautomerization to generate the iminium dehydro- The strictosidine biosynthetic pathway contains four P450
secodine 94. The enzyme coronaridine synthase (CS) would then enzymes which require reductase partners to facilitate the electron
catalyze a formal [4+2]-Diels–Alder to form ()-coronaridine 92. shuttling during catalysis. The C. roseus P450 reductase partners,
In the biosynthesis of catharanthine and tabersonine, a corres- cytochrome P450 reductase (CPR) and cytochrome b5 (CYB5), were
ponding pair of DPAS and cyclization enzyme (catharanthine expressed in yeast along with a putative alcohol dehydrogenase that
synthase and tabersonine synthase, respectively) are involved to was identified from MIA biosynthesis coexpression profiles
forge the different connectivities via cycloadditions. A recent (CYPADH) to increase P450 activity. Since these P450s also all
study by the O’Connor group reports the structural basis for the require NADPH as an electron donor, ZWF1 which is yeast glucose-
divergence in regio- and stereo-selectivity of the Diels-Alderases 6-phosphate dehydrogenase, was overexpressed to increase intra-
found in iboga and aspidosperma alkaloid biosynthesis.266 cellular NADPH concentrations. SAM2, a yeast SAM synthetase,
From 92, the P450 enzyme ibogaine 10-hydroxylase (I10H) was also overexpressed to increase SAM availability for LAMT.
catalyzes hydroxylation at the C-5 position of the indole ring, Overall, the metabolic engineering and synthetic biology
followed by noribogaine 10-O-methyltransferase (N10OMT)- strategies employed to create this yeast platform illustrates
catalyzed O-methylation to yield ()-voacangine 95.265 Both 92 the possibility of an alternative pipeline for MIA production.
and 95 have shown promise as acetylcholinesterase inhibitors.267 Biotransformation of 25 into ibogaine 2 is another twelve
In the last step, 92 undergoes decarboxylation to form ()-ibogaine enzymatic steps. While a de novo ibogaine biosynthesis yeast
2. This process can occur nonenzymatically under heat, but it is platform has yet to be published, many of the enzymes down-
possible that there is an unidentified decarboxylase that facilitates stream of 53 biosynthesis have been demonstrated to be func-
this step in planta. tional in yeast which is promising for providing sustainable
2.8.2 Heterologous production of iboga alkaloids. De novo access to 2 and potential derivatives.236,244
production of strictosidine 25 in S. cerevisiae was demonstrated
by Brown et al. in a landmark achievement of synthetic biology in 2.9 Salvia
2015 (Fig. 27). The authors’ engineered yeast strain comprised of Salvia divinorum colloquially known as the ‘‘sage of the diviners’’
twenty-one genome integrated genes, three genome-deletions and was introduced to western academics by the indigenous people of
expression of a high-copy plasmid encoding a codon-optimized the Sierra Mazateca in Mexico. While the botanical history of this
G8H gene. The host produced B0.5 mg L1 of extracellular plant has remained elusive, the hallucinogenic properties of
strictosidine after 6 days. Since simple expression of the required salvia are currently employed by Mazatec shamans to facilitate
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Fig. 27 Heterologous production of strictosidine in S. cerevisiae.76
visions of curing and divination. The active constituent,

salvinorin A 37, was first isolated by Ortega and coworkers in
1982 and further characterized by Valdes et al. two years later
(Fig. 28).269,270 The potent hallucinogenic properties of the
isolated 37 were confirmed by ethnobotanist Daniel Siebert,
who noted that a dose of 200 mg could product effects identical
to that of whole herb ingestion.271 Unlike other known halluci-
nogenic natural products, salvia does not function as a serotonin
receptor agonist. Instead, 37 is a selective opioid agonist, binding
strongly to k-opioid receptors (KORs), and was identified as the
first non-nitrogenous opioid receptor ligand.22,272 Given these
unique properties, numerous therapeutic uses for salvinorins
have been proposed, including anti-inflammatory, analgesic, Fig. 28 Salvia divinorum contains salvinorin A, a structurally unique
and anti-addiction treatments.273,274 Salvia is consumed terpene hallucinogen. Image on the left courtesy of Eric Hunt via CC-2.5.
primarily as a recreational drug inducing powerful, sometimes https://upload.wikimedia.org/wikipedia/commons/3/35/Salvia_divinorum_-1.jpg.
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disorienting hallucinations and has a legal status that is highly has been proposed.279 However, the exact series of oxidative
contested. decorations and cyclizations leading formation of the rare furyl-
2.9.1 Biosynthesis of salvinorins. 37 is a modified neo- d-lactone moiety will be of interest to biosynthetic chemists and
clerodane type diterpenoid featuring a unique furyl-d-lactone metabolic engineers alike.
fragment. Structural-activities relationship studies of 37 analogues
with modifications to the of furanyl group, as well as molecular
3. Alkaloidal stimulants
modelling have implicated the furan ring in selective KOR

binding.275 In 2015, Gupta et al. reported collybolide, a fungal
Alkaloidal stimulants may be regarded as the most culturally
sesquiterpene bearing a similar furyl-d-lactone, exhibiting KOR
pervasive secondary metabolites; consumption of plants con-
agonism similar to salvinorin.276 Investigations into the biosyn-
taining the alkaloidal stimulant caffeine 4 may have occurred
thetic route to 37 are still in their infancy. Produced and stored
as early as 2500 BC in China. By the 1600s, alkaloid containing
in the leaf trichomes,277 tissue culture of S. divinorum grown on
plants were distributed as luxury commodities along every
isotopically labelled substrate confirmed that the diterpene core of
major trade route. Alkaloid consumption was a key driver of
salvinorins arises via the deoxyxylulose phosphate (DXP) pathway.278
the Euro-American slave trade, which occurred from the 16th to
This information aided the trichome-specific transcriptomics
the 19th centuries and enabled early efforts to characterize
studies that investigators have used to identify pathway genes.
active constituents.281 Indeed, the alkaloid caffeine is currently
In 2016, two research groups simultaneously identified and
world’s most widely consumed psychoactive drug; although
characterized the first enzyme involved in biosynthesis of 37,
global consumption statistics have been difficult to estimate,
the ()-kolavenyl diphosphate synthase (KPS) (Fig. 29). KPS is a
more than 85% of adults in the U.S. regularly consume caffeine
class II diterpene synthase, performing cycloisomerization of
at an average rate of 0.2 grams per person per day.28 While an
geranylgeranyl pyrophosphate 100 through a carbocation inter-
exhaustive list of natural product stimulants would encompass
mediate to form ()-kolavenyl pyrophosphate 101.279,280 Hardwickiic
molecules that are of diverse biosynthetic origins, the well-known
acid 102 has been proposed as an on-pathway intermediate based on
members covered in this review fall within three major categories
co-localization and structural similarity to the salvinorins.
– the purine alkaloids, pyridine alkaloids, and tropane alkaloids.
Based on more than a dozen salvinorin-like molecules that have
In addition to these alkaloids for which the biosyntheses have
been isolated, a hypothetical downstream biosynthetic pathway
been well-studied, stimulants from a number of other plants
including khat, areca, and ephedra are increasing in notoriety.
Investigations into the biosynthesis, safety, and efficacy of these
alkaloidal stimulants remain in their infancy.
3.1 Catecholamine neurotransmitters

Generally speaking, a stimulant may be defined as a substance
that increases the activity of the central nervous system (CNS).
Most stimulants function by increasing the synaptic concentrations
of catecholamine neurotransmitters – namely dopamine 17,
epinephrine 103, and norepinephrine 104.282 Produced by adrenal
glands, catecholamines act as signaling molecules to activate the
sympathetic nervous system. Increases in synaptic catecholamine
levels are primarily achieved via blocking their reuptake or stimu-
lating their efflux, however there are notable examples of stimu-
lants with more indirect modes of CNS activation. As a result, a
description of the physiological targets of natural products
described in this section is provided alongside the individual
stimulant. Despite disparate mechanisms of achieving their
effects, all known natural product stimulants are alkaloidal in
nature (Fig. 30). Alkaloids are commonly defined as molecules
possessing one or more basic nitrogen atoms; this chemical
property facilitated early isolation via acid–base extraction,
making alkaloidal stimulants some of the first natural products
to undergo biosynthetic investigation.
3.2 Caffeine
In addition to early reports of human consumption of caffeine 4
(Fig. 31) in the Yunnan Province of China, caffeine containing
Fig. 29 Proposed biosynthetic pathway for salvinorin A.279 plants were independently discovered in Africa and South America,
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methylation patterns of the purine heterocycle exhibit variable

potencies and include theobromine 107, theophylline 108, and
xanthine 109.
3.2.1 Biosynthesis of purine alkaloids. Caffeine 4 was first
isolated in 1819 by the German chemist Friedlieb Ferdinand
Runge.288 By the end of the century, Fisher devised a synthesis
from theobromine 107 (Fig. 32) which employed methyl iodide

for base-catalyzed N-alkylation, thus establishing caffeine’s
structure and formula.289 Given the widespread occurrence of
caffeine across the plant kingdom, the biosynthesis of caffeine
and related PuAs has been of interest from both a secondary
metabolism and evolutionary perspective. The biosynthesis has
been studied primarily in Camellia sinensis (tea plant) and
Coffea arabica (coffee plant). As early as 1962, the feeding of
14
C-labeled precursors confirmed that PuAs originate from the
primary purine metabolite xanthosine in Coffea.290 Direct evidence
for the conversion of xanthosine 110 to 7-methylxanthosine 111
was first shown by Negishi et al. using plant extracts.291 Elucidation
of the subsequent hydrolysis step by a nonspecific N-methyl nucleo-
sidase was frustrated by contaminating nucleosidase activity in
crude enzyme extracts, but eventually confirmed using advanced
chromatography methods.292 Finally, tedious preparation of tea leaf
enzymatic extracts in 1975 provided direct evidence for the transfer
of methyl groups from SAM in the conversion of 7-methylxanthine
Fig. 30 Alkaloidal stimulants as structural mimics of neurotransmitters. 111 via theobromine 107 to caffeine 4.293 Development of methods
for recombinant protein production enabled Ashihara, Fujimura,
and others to provide conclusive in vitro evidence for the
biosynthetic route from xanthosine shown in Fig. 32A, with
the genes encoding the responsible enzymes identified in both
coffee and tea.294,295
Several routes to the primary metabolite xanthosine 110
have been elucidated, however efficient incorporation of adenine
113 implicated adenosine monophosphate (AMP) 114 as a
prominent source of purine equivalents.296 Caffeine production
Fig. 31 Coffea arabica (the dominant coffee cultivar) contains B1.2
from AMP 114 begins with deamination to inosine monophos-
percent dry weight caffeine.
phate 115, oxidation to xanthosine monophosphate 116, and
hydrolysis to xanthosine 110 by AMP deaminase (AMPD),
where they were consumed for their energizing, anti-fatigue effects. IMP dehydrogenase (IMPDH), and 5 0 -nucleotidase (XMPN),
Caffeine belongs to the purine alkaloid (PuA) family of natural respectively.297 The resulting xanthosine 110 is methylated by a
products, which are defined by their 3,7-dihydropurine-2,6-dione xanthosine methyltransferase (XMT) and hydrolyzed by N-methyl-
core. Despite their structurally simplicity, at least 80 plant species in nucleosidase (NS) to give 7-methylxanthine 112. Iterative methylation
13 orders of the kingdom are known to produce PuAs, indicating of 112 in tea has been confirmed by isolation of a caffeine synthase
important biological function.283 Bitter in taste PuAs are primarily (CsTCS1) exhibiting both N3 and N1 methylation activity.294 Ortho-
involved in plant defense as an antifeedant, comprising between logous genes in coffee have been identified which exhibit either
1–3 percent dry weight in most producing organisms.60,284 theobromine synthase (CaMXMT1) or caffeine synthase (CaDXMT1)
Additional research suggests that caffeine may function as an activity, using 112 and 107 as a substrates.298,299
allelopathic signaling molecule,285 or even a conditioning molecule In addition to the major pathway described above, caffeine
to train plant pollinators.286 In humans, PuAs work as antagonists biosynthesis evolved independently at least five times during
of adenosine A2AG protein-coupled receptors. During the course of flowering plant history, a striking example of convergent evolution
the day, adenosine 105 accumulates in the neuronal synapse; towards a secondary metabolite.300 Analysis of the enzymes
subsequent binding results a negative regulation of CNS activity recruited by distantly related plants to carry out identical reactions
causing drowsiness. As structural analogues of adenosine, PuAs has provided strong evidence for the ‘‘patchwork hypothesis’’ as a
bind tightly (caffeine KD = 2.4 mM) but do not activate adenosine model to describe pathway evolution. Additional studies aimed at
receptors.287 The resulting excitation of specific regions of the brain unravelling pathway regulation in the plant have given further
causes accumulation of stimulatory dopamine 17 and acetylcholine insight into the ‘‘provider pathways’’ used by plants to increase
106, facilitating wakefulness. Other purine alkaloids with varying xanthosine 110 pools. In 2001, Koshiishi et al. unexpectedly
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Fig. 32 Caffeine biosynthesis and microbial engineering strategies. (A) Major caffeine biosynthetic route identified in Camellia sinensis and Coffea
arabica. (B) SAH-derived adenosine may be funneled into purine metabolism in tea leaves following methyl transfer. (C) Xanthine recycle pathway utilized
during heterologous production in yeast. (D) Novel xanthine-to-caffeine conversion pathway leveraged for caffeine production in E. coli.
observed incorporation of SAM-derived adenosine 105 into the efforts in plant and microbial engineering, facilitating the
purine ring using cell free extracts of tea leaves.301 As shown in creation of caffeine (and caffeine-free) biotechnologies. Knock-
Fig. 32B, SAH-equivalents released upon substrate methylation down of the CaMXMT1 encoding theobromine synthase using
with SAM could be funneled into purine metabolism, providing RNA interference resulted in a 70% reduction of caffeine content,
an alternative pathway to the well-established de novo adenosine highlighting the possibility to circumvent costly decaffeination
production routes. Alternative guanosine recycling pathways protocols using genetic engineering of Coffea.304 Recent efforts
have also been identified via incorporation of [8-14C]guano- in microbial engineering for de novo production of xanthine
sine.297 Sub-cellular localization of the caffeine biosynthetic alkaloids have also garnered moderate success, with benchmark
pathway has also been examined. Like many plant secondary titers of 0.27 mg L1 and 21 mg L1 in S. cerevisiae and E. coli
metabolites, caffeine accumulates in the vacuole,302 whereas respectively.89,92 In both studies, low levels of endogenous
several enzymes involved in the biosynthesis associate with the xanthosine represented a key hurdle that was approached using
chloroplasts303 or cytosol.301 two different methods. McKeague et al. devised a xanthine 109
3.2.2 Heterologous production of purine alkaloids. Extensive salvage pathway in yeast, using xanthine phosphoribosyltrans-
biosynthetic investigations provided a foundation for numerous ferase (XPT) to revert flux towards 116 (Fig. 32C). A combination
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of genomic integration and low copy expression of the biosynthetic 3.3.1 Biosynthesis of pyridine alkaloids. Initially isolated
and salvage pathways using strong constitutive promoters provided as tobacco’s active constituent in 1828, nicotine was structurally
a maximum caffeine titer of 0.031 mg L1 following 6 days of characterized in 1893 and synthesized by Pictet in 1904.311 Efforts
culture. In the same study, a key observation was made that to isolate nicotine and oxidation products by Weidel also led to the
xanthine could be accepted by caffeine synthase, which enabled discovery of an aspartatic acid 120-derived nicotinic acid (niacin,
construction of a theophylline production strain. Bench scale 121), precursor to the universal cellular redox currency nicotina-
fermentations of customized strains permitted improved mide adenine dinucleotide cofactors.312 Investigations into the
production titers of caffeine 4 (0.27 mg L1), theophylline 108 biosynthesis commenced rapidly in the late 1950s with the dis-
(0.06 mg L1), and 3-methylxanthine 117 (3.71 mg L1). covery that metabolic precursors could be incorporated into
In E. coli, a xanthosine-to-caffeine conversion pathway was nicotine via sterile root cultures. Tandem feeding studies by
leveraged, taking advantage of background xanthine methylation Byerrum et al. indicated that 2-14C-labeled L-glutamic acid313
activity exhibited by the CsTCS1 (Fig. 32D). Li et al. employed 36 and L-ornithine314 13 were incorporated into two different
plasmid-based expression using inducible promoters to enhance positions of nicotine 5, sparking interest in a proposed ‘‘sym-
xanthine and SAM biosynthesis.89 Following bioprospecting, metrical intermediate.’’ Leete was the first to propose the
codon optimization, and media optimization, a 4-day shake flask diamine putrescine 16, derived via L-ornithine decarboxylation,
culture enabled production of caffeine at 21 mg L1. Despite as a pyridine alkaloid precursor.315 Later studies confirmed
these efforts, microbes lack the optimized flavor profiles and incorporation of putrescine 16,316 N-methylputrescine 18,317 and
titers of caffeine plant products. In each of these studies, how- N-methylpyrrolinium 20.318 Tamaki and coworkers subsequently
ever, accumulation of monomethylated xanthines was observed, identified putrescine N-methyltransferase319 and N-methyl-
indicating the potential for metabolic engineers to produce putrescine oxidase320 activity in tobacco roots, confirming the
valuable pathway intermediates of low natural abundance. pathway to 20 shown in Fig. 34A.
Genes in tobacco encoding the responsible enzymes were
3.3 Nicotine later identified by comparison to a low-nicotine mutant and
The pyridine alkaloids (PyAs) are comprised of the highly- cloned into E. coli for functional characterization.321 L-Glutamic
addictive stimulant nicotine 5, along with the structurally acid 36 derived L-ornithine 13 is first decarboxylated by an
related anabasine 118 and nornicotine 119 (Fig. 33 and 34). ornithine decarboxylate (ODC), which is subsequently methylated
Nicotine 5 is produced by numerous members of the Solanaceae by putrescine N-methyltransferase (PMT). Sequence analysis
(nightshade) family of flowering plants, and like the xanthine indicates that PMTs are closely related to spermidine synthases
alkaloids, pyridine alkaloids are bitter antifeedants. In fact, the (SPDSs), which utilize decarboxylated SAM as the coenzyme to
nicotine scaffold served as inspiration for the controversial neoni- transfer an aminopropyl group onto putrescine. PMTs likely evolved
cotinoid insecticides, the use of which has been linked to honey-bee via gene duplication and neofunctionalization, as mutational studies
health and colony collapse disorder.305,306 Most of the nightshades, indicated that changing only a few amino acids resulted in the
including potatoes, tomatoes, and eggplant, produce PyAs in trace generation of PMT activity in SPDS proteins.322 Next, oxidative
amounts (B0.00001 percent dry weight);307 selective breeding has deamination of N-methyl putrescine 18 to N-methylaminobutanal
been used to generate tobacco cultivars containing up to 3.0 percent 19 is carried out by N-methylputrescine oxidase (NMO), which was
dry weight nicotine.308 Discovered by the native people of Mesoa- functionally verified using E. coli expression.323 NMO belongs to the
merica and South America, tobacco was traditionally used in superfamily of copper-containing amine oxidases, which employ a
spiritual ceremonies as well as for its medicinal properties, owing covalently bound topaquinone (TPQ) cofactor generated via autoox-
to its analgesic effects when smoked.128 Binding of nicotine to idation of a conserved tyrosine residue (Fig. 34A). The Cu-TPQ
nicotinic acetylcholine receptors results in activation of the meso- complex enables radical-based oxidative deamination consisting of
limbic pathway and subsequent release of dopamine; at higher two steps. Following formation of a Schiff base between the TPQ and
concentrations, the activity shifts from stimulant to sedative via substrate amine, proton abstraction and hydrolysis result in
dampening of neural activity.309 Recent evidence suggests that aldehyde release. Then, reduced TPQ is reoxidized with molecular
cotinine, a metabolic degradation product of 5, is responsible for oxygen through two sequential single electron transfers via a Cu(I)-
at least some of tobacco’s psychoactive effects.310 semiquinone radical intermediate, releasing H2O2 and NH4+. A
homodimer, NMO exhibits 7-fold greater catalytic efficiency
towards 18 compared to 16, achieved via a substantial decrease in
KM. The NMO product N-methylaminobutanal 19 spontaneously
cyclizes to give the Schiff base N-methylpyrrolinium cation, 20.
Surprisingly, the identity of the final stereospecific enzyme
in the pathway, nicotine synthase, has yet to be elucidated.
Little progress has been made since Frieson and Leete demon-
strated formation of nicotine from N-methylpyrrolinium cation
20 and [2-3H]-labeled-121 using crude extracts.324 The loss of
Fig. 33 Nicotiana tabacum leaves contain 2 to 8 percent dry weight the C-6 hydrogen suggested a hydride mediated formation of the
nicotine. 3,6-dihydronicotinic acid, which would readily decarboxylate to
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Fig. 34 Summary of the nicotine biosynthetic pathway, including known and proposed enzymatic steps. (A) N-methylpyrrolinium formation via the
polyamine pathway. (B) Proposed reduction of nicotinic acid via A622. (C) Proposed oxidation of condensation products via BBL towards nicotine,
nornicotine.
give the 1,2-dihydropyridine 122 (Fig. 34B). Hashimoto and Following reduction of aspartic acid 120 derived niacin 121,
coworkers utilized RNAi knockdown of the A622 gene in a proposed nucleophilic attack of N-methylpyrrolinium 20 by
tobacco (belonging to the PIP family of NADPH-dependent 122 is believed to occur either spontaneously or enzymatically
reductases), which resulted in a decrease in the formation of in a Mannich-like reaction, providing the dihydropyridine
nicotine and accumulation of nicotinic acid N-glucoside 123, a precursor to the final product nicotine 5. Subsequent RNAi
presumed detoxification product.325 While additional studies targeting of a vacuolar berberine bridge enzyme-like (BBL)
have confirmed the involvement of A622 in nicotine bio- protein resulted in accumulation of a new nicotine-related
synthesis, more biochemical evidence is needed to ascertain metabolite, dihydrometanicotine 124 (Fig. 34C).326 Direct con-
its catalytic function. version of the ring-open 124 by the BBL protein would explain
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the enantiomeric purity of (S)-nicotine 5 (whereas the (R)-enantiomer use can be traced to indigenous populations in the Andes. As
only accounts for just 0.2% of the total nicotine). However, in early as 1000 BC, the leaves of coca were chewed for religious and
experiments using recombinant BBL protein as well as crude medicinal purposes. At present, Columbia is the largest producer
tobacco cell extracts, oxidative conversion of 124 was not observed. of cocaine; hundreds of metric tons are extracted and exported
Enantioselective demethylation of (R)-nicotine 125 by several annually.332 The product may be refined and distributed as the
P450s (CYP82E4, CYP82E5v2, and CYP82E10) has been postulated cocaine hydrochloride salt, or neutralized and distributed as the
to explain how tobacco maintains the (S)-nicotine 5 and inhalable free base, a subtle difference which has resulted in
(R)-nornicotine 119 pools. Further studies are required in order extreme sentencing discrepancies.333 Despite its current Schedule II
to definitively establish the identity and mechanism of nicotine status in the United States, a myriad of medicinal uses for cocaine
synthase, as well as the predicted anabasine 118 synthase. have been described, and in 2020 a cocaine hydrochloride formula-
3.3.2 Heterologous production of pyridine alkaloids. Com- tion was approved for use as a topical anesthetic. The principal
plete reconstitution of the nicotine pathway in a heterologous mechanism of action is to block monoamine transporters,
host has not been possible due to missing steps in the bio- resulting in the accumulation of dopamine 17 in the synaptic
synthesis. However, identification of genes involved in nicotine cleft and a strong sympathomimetic effect.334 In addition to cocaine,
precursor formation have been used in genome mining and the tropane alkaloids include the hallucinogenic scopolamine 126 as
N-methylpyrrolinium platform engineering. Complete pathway well as catuabines and calystegines.
elucidation will enable the use of such chassis strains for the 3.4.1 Biosynthesis of tropane alkaloids. Tropane structure
production of rare or unnatural pyridine alkaloids. Now an and biosynthesis has been a topic of intense investigation for
undisputed model organism and biotechnological chassis, over a century. Structural confirmation of the tropane core
Nicotiana plants have been extensively engineered for applications in cocaine accompanied Willstätter’s lengthy synthesis, which
ranging from production of biopharmaceuticals to heterologous warranted a 1915 Nobel Prize in Chemistry.335 Two years later,
natural product biosynthesis.327,328 Synthetic biology tools analo- Robinson published a one-pot tropinone formation by the
gous to those developed for microbial engineering have been addition of succinaldehyde to an acetone-dicarboxylic equivalent,
extended to N. tabacum and N. benthamiana; the future of such which is widely regarded as the first biomimetic synthesis.13
Nicotiana ‘‘plant biofactories’’ has been recently reviewed.329 This elegant method stimulated Robinson’s 1955 proposal of an
Identification and silencing of the nicotine N-demethylase using analogous biosynthesis involving condensation of a pyrrolidine
RNAi led to suppression of P450-mediated conversion of (R)-nicotine ring with an ‘‘acetone equivalent.’’ Indeed, work by Leete con-
125 to the carcinogenic nornicotine 119.330 Recently, the CRISPR- firmed incorporation of L-ornithine 13 into tropanes,336 establish-
mediated simultaneous knockout of the BBL-family protein and five ing a pathway identical to the N-methylpyrrolinium 20 formation
additional isoforms was shown to reduce the amount of nicotine described in nicotine biosynthesis. Feeding of labeled acetate and
produced in tobacco by 499%, providing additional evidence for advanced intermediates hinted that condensation of 20 with
the involvement of this BBL protein in nicotine formation.331 malonate units occurs via a polyketide synthase (PKS).337 Subse-
Optimizing the alkaloid profiles of ‘‘nonfood’’ industrial crops such quent advancements in molecular biology and genomics have
as tobacco expands the capabilities of an additional chassis for rapidly facilitated the near complete pathway elucidation and
industrial chemical manufacture. engineering of tropane alkaloids.
Using differential transcriptomics of plant tissues, Bedewitz
3.4 Cocaine et al. identified a type III PKS (AbPYKS) expressed in the roots of
Tropane alkaloids encompass the third class of nitrogen-containing Atropa belladonna, confirming its involvement in tropinone
stimulants discussed, and are defined by their characteristic biosynthesis using virus-induced gene silencing.338 Recombinant
8-methyl-8-azabicyclo(3.2.1)octane (‘‘tropane’’) ring system. expression of AbPYKS in E. coli indicated direct use of the N-methyl-
While just a few tropane alkaloids are known to be psychoactive, pyrrolinium cation 20 as a starter substrate prior to incorporation of
this family of molecules includes the well-known illicit stimulant two malonyl-CoA 105 units, forming 4-(1-methyl-2-pyrrolidinyl)-3-
cocaine 6 (Fig. 35). Named for Erythroxylum coca (endogenous to oxobutanoic acid 106 (Fig. 36). Subsequently, Huang and coworkers
South America, Mexico, Indonesia, and the West Indies), cocaine proposed an alternative route to 106 following additional crystal-
lographic and mechanistic studies.339 In the absence of 20, AbPYKS
was shown to produce 3-oxo-glutaric acid 21; this compound under-
goes non-enzymatic condensation with 20 via an intermolecular
Mannich reaction, the kinetics of which were unaffected by the
presence of AbPYKS.339 The resulting racemic 128 is thought to be
the divergence point between the tropinone 129 pathway (leading to
scopolamine – resolved, Fig. 37) and methylecognone 130 pathway
(leading to cocaine – unresolved, Fig. 38).
Bedewitz et al. also hypothesized that a P450 may be
Fig. 35 Erythroxylum coca leaves contain B0.7 percent dry weight
responsible for the cyclization of nascent 128 via amine oxidation.
cocaine. Image on left courtesy of Danna Lizeth Guevara Prieto via CC- Pathway reconstitution of candidate P450s identified via tran-
4.0. https://www.inaturalist.org/photos/22483426. scriptomics indicated that AbCYP82M3 encodes a tropinone
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Fig. 36 Formation of tropine, pseudotropine.
Fig. 37 Scopolamine biosynthesis from phenylalanine and tropine.
synthase (TS), which was directly confirmed by conversion of 128 to aromatic amino acid aminotransferase (AT4)342 and reduced by a
129 using yeast microsomes.338 The proposed mechanism involves phenylpyruvic acid reductase (PPAR)343 to provide phenyllactic
hydroxylation and dehydration of the pyrrolidinyl to generate the acid 136. This compound is subsequently glucosylated by
pyrrolinium intermediate. Oxidation of 128 sets up the intra- phenyllactate UDP-glycosyltransferase (UGT1).344 The resulting
molecular Mannich cyclization to produce ecgonone 131, establish- phenylacetylglucose 137 is then used by littorine synthase (LS)
ing the tropane skeleton; subsequent nonenzymatic decarboxylation to acylate 132, forming littorine.344 The longstanding mystery
produces 129. As discussed in Section 1.2.2, iminium formation and around rearrangement of littorine was solved in 2006, wherein
intramolecular Mannich-cyclization is a common cascade observed 134 was converted into hyoscamine aldehyde 138 by CYP80F1
in the biogenesis of diverse plant alkaloid scaffolds.340 Two different via a benzylic carbocation intermediate.345,346 A recently identified
tropinone reductases (TPI and TPII) were identified in Datura hyoscyamine dehydrogenase (HDH) then reduces 138 to hyos-
stramonium of high sequence identity (64% identity), each perform- camine 139 followed by epoxidation catalyzed by an a-keto-
ing stereospecific reduction of 129 to either tropine 132 (TPI) or glutarate-dependent hydroxylase/dioxygenase (DsH6H) to complete
pseudotropine 133 (TPII), the precursor to the calystegines.341 the biosynthetic pathway to scopolamine 126.73
The phenylacetate unit required for littorine 134 biosynthesis The majority of the pathway towards cocaine 6 has been
is derived from phenylalanine 135, which is transaminated by an established, with the exception of the enzymes responsible for
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production of the precursor methylecognone 130. Evidence confirmed on pathway metabolite methylecognone 130. Product
suggests a sequence analogous to tropinone 129 formation methylation is believed to take place before cyclization, otherwise
starting from a PKS product. During tropinone 129 biogenesis, rapid decarboxylation of the putative b-keto acid would occur. This
the spontaneous decarboxylation following cyclization permits hypothesis is supported by a feeding study in which a low but
the use of either stereoisomer of 128. The retention of the observable amount of the methyl ester of 128 painted on coca
carboxymethyl in the methylecognone 130 scaffold, however, leaves was incorporated into cocaine.347 Following cyclization,
necessitates incorporation of the (S)-enantiomer. The decarbox- methylecognine 141 is formed via methylecognine reductase
ylation product of 128, hygrine 140, is known to racemize (MecgoR).348 MecgoR belongs to the aldo-keto reductase family
rapidly at physiological conditions. The proposed mechanism of enzymes, indicating tropine ester formation evolved indepen-
involves a retro-aza-Michael addition (Fig. 38A). Stereospecific dently in E. coca and A. belladonna. The final enzyme, cocaine
incorporation of (S)-128 into cocaine may involve selective synthase, is a BAHD acyltransferase which condenses methylecog-
methylation and cyclization, facilitated by spontaneous or nine with activated benzoyl-CoA 142.349
enzyme catalyzed stereoinversion of (R)-128. A proposed methyla- 3.4.2 Heterologous production of tropane alkaloids. Extensive
tion of (S)-128 followed by a P450-mediated Mannich-cyclization by engineering efforts by Srinivasan and Smolke allowed for the first
an enzyme homologous to tropinone synthase would yield the reported de novo production of hyoscyamine 139 (10.3 mg L1) and
scopolamine 126 (0.87 mg L1) in yeast (Fig. 39).73 This synthetic
biology achievement builds upon previous works to reconstitute
segments of the tropane alkaloid biosynthetic pathway in E. coli
and yeast.108,350,351 The fully integrated yeast strain contains 26
additional genes from yeast, E. coli and five different plants
along with disruption of 8 native yeast genes for a total of 34
chromosomal modifications (Fig. 39). The authors organized
the biosynthetic pathway with five modules, each comprised of
a distinct pathway segment.
Module I is dedicated to putrescine 16 production and
contains heterologous plant (AsADC) and bacterial (SpeB)
putrescine pathway genes as well as additional copies of native
yeast putrescine biosynthesis genes (Arg2, Fms1, Car1, Spe1) to
maximize putrescine 16 accumulation. The authors also dis-
rupted two yeast genes MEU1 and OAZ1 involved in off-pathway
polyamine formation that reduce putrescine 16 accumulation.
Module II then contains the genes encoding for the enzymes
required to transform putrescine 16 into tropine 132 along
with disruptions of five endogenous aldehyde dehydrogenases
(Ald2-5 and Hfd1) that were previously determined to decrease
N-methylaminobutanal 19 titers.108 These two modules were a
part of the platform strain from previous work by Srinivasan
et al. that were leveraged to produce the non-canonical tropane
alkaloid, cinnamoyltropine, from the acyl donor cinnamoyl-
CoA.351 This acyl donor is also used in the biogenesis of the
polyketide-derived kavalactones, which are the anxiolytic seda-
tives found in the kava plant, Piper methysticum.98
The next module, Module III, contains the genes required
for biotransformation of phenylalanine 135 into the acyl donor,
phenylacetyl glucose 137. The pathway intermediate phenyllactic
acid 136 is likely produced non-specifically by action of an
endogenous yeast lactate dehydrogenase. However, the authors
determined that expression of a phenylpyruvic acid reductase
from the fungus Wickerhamia fluorescens increased phenyllactic
acid 136 titers by nearly 80-fold. Yeast glucosidase Egh1 was
disrupted to prevent hydrolysis of the heterologous glucoside,
phenylacetyl glucose 137.
Module IV contains the genes encoding enzymes to trans-
Fig. 38 Cocaine biosynthesis. (A) Racemization of the cocaine pathway
form the TA scaffold into medicinal alkaloids, hyoscyamine 139
intermediate decarboxylation product hygrine. (B) Proposed biosynthesis and scopolamine 126, including a newly identified hyoscyamine
of methylecognone and subsequent formation of cocaine. dehydrogenase (HDH) that was discovered by manually screening
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Fig. 39 Production of tropane alkaloids in yeast.73
12 putative HDHs genome mined from available A. belladonna activity in vivo. To allow ample supply of tropine 132 and
transcriptome datasets. The final module, Module V, contains phenyllactic acid glucoside 137 into the vacuole to access the
genes that encode for the vacuole transporter NtJAT1 and an sorted AbLS, several vacuole transporters from plants were tested
engineered, chimeric AbLS to form littorine 134 in the yeast and expression of the transporter NtJAT1 resulted in the highest
vacuole. Initial expression of AbLS resulted in growth defects and titers of the AbLS product, littorine 134.
no activity in vivo which the authors attributed to difficulties in Like heterologous production of iboga alkaloids, a yeast-
post-translational processing stemming from differences in based platform for medicinal tropane alkaloids demonstrates
glycosylation pattern recognition and transport factors between the potential of a more sustainable and reliable pipeline for
yeast and plants. Srinivasan et al. determined AbLS may be production. While titers on the microgram scale do not make
stalled in the secretion pathway upstream of the trans-Golgi this platform competitive to current processes for obtaining
network based on an N-terminal signal peptide and designed a tropane alkaloids, further host engineering combined with
chimera with DsRed linked to the N-terminus of AbLS to mask fermentation optimization could result in an economically
said signal peptide. This modification allowed the chimeric viable strain. In recent years, sub-cellular localization has been
AbLS to be properly sorted to the yeast vacuole and restored a popular method to greatly improve product titers and cellular
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fitness via forming enzymatic cascades, accessing rich chemical

environments, and sequestering toxic intermediates.105,352,353
Here, the recapitulation of the endogenous plant vacuole sort-
ing and intermediate transport system in yeast is an innovative
approach to sub-cellular localization that is a promising strategy
that may benefit other yeast systems heterologously expressing
complex, spatially-organized plant pathways.
4. Cannabinoids
Cannabis indica, C. sativa (Fig. 40), and C. ruderalis are traditional
plants that have been used as medicine, recreationally, and as a
fiber for all of recorded history.354 This plant treats epilepsy,355
inflammatory bowel disorder,356 fibromyalgia357 and holds promise
in treating cancer,358 psychiatric disorders,359 multiple sclerosis,360
basal ganglia disorders,361 and others.362 Despite this lengthy
history and myriad of medicinal applications, the usage of this
plant has remained controversial.363 For example, in the 1930s,
Harry Anslinger demonized usage of Cannabis for his own political
benefit and reshaped the American consensus on the plant.363
Because of this, today, previous colloquialisms such as ‘marijuana’
are currently being depopularized, as the term was used to
purposely force negative associations with the Latino community.
This is reestablishing the scientific name and ‘‘pot,’’ ‘‘weed,’’ or
‘‘bud’’ as common names.
Arguably, the Cannabis plant is unparalleled in morphological
and biochemical diversity as well as its gamut of bioactive
constituents – producing more than 80 biologically active Fig. 41 Structural motifs and examples of isolated natural products from
compounds.354,365 Cannabinoids covered in this section are the Cannabis plant.
psychoactive Cannabis plant-based hybrid meroterpenoid natural
products, containing terpene and polyketide fragments. These
structures all contain a 5-pentylbenzene-1,3-diol (olivetol) substi- in C. indica.367 Fascinatingly, 8 is reported to act against adverse
tuted with a dimethyl octadiene chain (geranyl) (Fig. 41). The effects of 7.368 Some reports refer to 8 as non-psychoactive as it
geranyl diene arm undergoes various intramolecular reactions is not intoxicating nor euphoric, but this compound does alter
with the polyketide core to form classic cannabinoid tetrahydro- brain behavior369–371 and therefore referred to herein as psy-
benzochromene, vinyl biphenyl, and related chromene scaffolds. choactive. The pharmacology of 8 is complex as it interacts with
Tetrahydrocannabinol (THC, 7) is the major psychoactive many types of receptors and enzymes.372–374
cannabinoid and is metabolized to cannabinol (CBN, 143) Cannabichromene (CBC, 144) is a natural chromene racemate
(Fig. 41).366 7 and 143 define the classic cannabinoid benzo- that functions through non-cannabinoid receptor mechanisms
chromene skeleton. Typically, Cannabis plants contain, depending activating the ankyrin transient receptor potential channels 1
on variety, between 5–16% 7 content, but in some cases can be as (TRPA1).375 144 has also been reported to modulate 7 activity.376
high as 18% with a theoretical maximum of 54%.354 Cannabicyclol (CBL, 145) is the photochemical formal [2+2]
Cannabidiol (CBD, 8) has a vinyl biphenyl skeleton and is a cycloadduct of 144 – this process is nonenzymatic and depen-
non-euphoric compound that is produced in a 1 : 1 ratio with 7 dent on storing the plant material in light.377 Formation of
145 likely occurs during medicinal and recreational smoking of
the Cannabis plant. However, there is no published pharmaco-
logical data on 145.
Natural products 7, 8, and 143–145 all derive from cannabi-
gerolic acid (CBGA, 33) and cannabinerolic acid (CBNRA, 146).
This pathway was originally hypothesized in 1964 upon isolation of
the decarboxylated product cannabigerol (CBG, 147).378 Until
recently, 147 has not been studied, and like 8, was believed to
be non-psychoactive despite modulating multiple receptors.369,379
Previously, the cannabinoid natural product scaffolds were
Fig. 40 The Cannabis sativa plant typically contains 5–16% tetrahydro- thought to be exclusively produced by the Cannabis plant.
cannabinol (7).364 With an increase in interest in such compounds and modern
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Fig. 42 Exemplary structurally related cannabinoid-like natural products

isolated from other plant and fungal sources (italics). Structural deviations
highlighted in red. Amorfrutin 2 (B) (148) is a 148 derivative,380 ()-cis-
perrottetinene (149) is a 7 derivative,381 machaeridiol (150) is a 8 derivative,382
and 6-chloro-cannabiorchichromene (151) is a 144 derivative.383
characterization techniques, related scaffolds have been dis-

covered in rhododendrons (Rhododendron dauricum), liverworts
(Radula perrottetii), indigo bushes (Amphora fruticosa), and even
Fig. 43 CB1 and CB2 activity for 7, 8, 147, the natural endocannabinoid
fungi (Cylindrocarpon olidum) (Fig. 42).380–383 The key difference
arachidonylcyclopropylamide, and synthetic analogue JWH-133.
in many of these scaffolds is the alkyl chain substituent
changing from an alkyl to an aralkyl (148, 149) or b-aralkyl
substituent (150). In other cases, this alkyl chain is truncated been found to selectively potentiate the cannabinoid receptors
(151). Intriguingly, some of these compounds were shown to (Fig. 43).
exhibit similar bioactivities to classical cannabinoids.381 Over the years, the structure–activity relationships between
cannabinoids and receptors have been established. A key dis-
4.1 Cannabinoid receptors covery was that molecule potency is proportional to the C3
Isolation of 7, 8, and 143 and then chemical synthesis of 7 chain length.389 Cannabinoid analogues have been isolated and
facilitated the discovery and characterization of the G protein- synthesized with C3 alkyl chain lengths ranging from 1–7
coupled receptors (GPCR) named the cannabinoid receptor carbons; the CB1 and CB2 inhibitory activities of propyl and
type 1 and 2 (CB1 and CB2).384–386 CB1 and CB2 cooperatively heptyl-substituted analogs are highlighted in Fig. 44. The propyl-
function with heterotrimeric G protein alpha subunits (Gi/o) to substituted THC and CBD derivatives, tetrahydrocannabivarin
inhibit adenylyl cyclase activity and activate mitogen-activated (THCV, 152) and cannabidivarin (CBDV, 153), have weaker inhibi-
protein kinase (MAPK).387 The CB1 and CB2 receptors are tory activities as the alkyl chain cannot adequately fill the hydro-
involved in achieving homeostasis after exposure to physical phobic channel of CB1 and CB2.390–392 This means that THCV and
or mental stimuli, and therefore are attractive as therapeutic CBDV have a more subtle or even no psychoactive effect, giving
targets to treat various pathologies.388 The CB1 receptor is these molecules other therapeutic potentials.393,394 Recently,
primarily found in the central nervous system at the terminals tetrahydrocannabiphorol (THCP, 154) and cannabidiphorol
of central and peripheral neurons. The location of the CB1 (CBDP, 155) were isolated from C. sativa L. that feature a C3
correlates receptor activation with effects on motor function, heptyl-substituent and are currently the most potent natural
cognition and memory, and analgesia. The CB2 is found in the CB1 and CB2 modulators.395
immune system cells and affects immune cell migration. These Shortly after the discovery of CB1 and CB2 as targets of
GPCR receptors share 44% sequence homology overall, and cannabinoids, Mechoulam et al. discovered the entourage
68% homology between transmembrane domains.385 The functional effect.396,397 When biologically inactive compounds are administered
equivalence of CB1 and CB2 is indicated by cannabinoids’ half with THC (7), these ‘entourage’ compounds modulate the observed
maximal inhibitory concentration (IC50), where typically these small psychoactivity. This effect is observed in vivo with fatty acid amides,
molecules inhibit the receptors at near similar concentrations terpenes, cannabinoids, and other compounds.396,398 Before naming
(Fig. 43). Synthetic analogues and some natural cannabinoids have this effect, other researchers have noted similar properties, for
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marked the first example of a type III PKS and polyketide

cyclase acting in concert to form cyclic polyketides in planta.402
The biosynthesis starts with incorporation of the hexanoyl unit as
the starter unit for TKS, followed by three rounds of decarbox-
ylative chain extension with malonyl-CoA to form a tetraketide.
OAC then catalyzes the Claisen-like cyclization to form the
dihydroxybenzene ring, followed by hydrolytic release of 32.

The next step in cannabinoid biosynthesis is the electro-
philic addition of a terpene unit to C6 of 32 (also see Fig. 4D).
The aromatic prenyltransferase (APT) enzyme403 selectively
prenylates C6 of 32 to form either CBGA 33 or CBNRA 157.404 These
molecules 33 and 157 are (E) and (Z) isomers and are derived from
geranyl or neryl pyrophosphate (82 or 158), respectively. The activity
of APT is dependent on the carboxylic acid of 32404 as the reaction
does not occur with a decarboxylated olivetol substrate. This
indicates that the decarboxylation to form cannabigerol 147 and
cannabinerol 159 occurs after prenylation. Despite this C2 substi-
tuent requirement, APT is actually quite promiscuous and able to
accommodate varying alkyl-chains at the neighboring C3 position.75
The diverse psychoactive cannabinoid skeletons all diverge
Fig. 44 CB1 and CB2 activity of THC (7) with varying C3 alkyl chain
from 33 and 157 as shown in Fig. 46. The aptly named tetra-
lengths, propyl (varin, 152) and heptyl (phorol, 154). CBD alkyl chain length
derivatives also shown for clarity. hydrocannabinolic acid synthase (THCAS),405 cannabidiolic
acid synthase (CBDAS),406 and cannabichromenic acid synthase
(CBCAS)407 catalyze the oxidative cyclizations to form tetrahydro-
example a Cannabis extract produced a psychoactive effect 2 to cannabinolic acid 160, cannabidiolic acid 161, and cannabi-
4-fold greater than pure 7.399 The entourage effect could explain chromenic acid 162 respectively. These compounds can transform
why consumers of Cannabis might prefer to smoke or vaporize nonenzymatically to further generate structural diversity, either at
the plant material versus taking single, purified compounds. elevated temperatures or in sunlight. Subsequent modifications
can lead to the decarboxylated 7, 8 and 144, aromatized 143 and
4.2 Biosynthesis of cannabinoids cannabinodiol (CBND, 163), and further cyclized products 145,
Cannabinoid biosynthesis begins with the Claisen and aldol cannabielsoin (CBE, 164), and isotetrahydrocannabinol (ITHC,
condensations of malonyl- and hexanoyl-CoA (127 and 156) – 165). There are other known, further functionalized cannabinoid
which is produced by the acyl activating enzyme (AAE1)400 – to skeletons, but all of them – including 145, 164, and 165 – are
form the polyketide olivetolic acid 32 (Fig. 45). Taura et al. proposed to form nonenzymatically due to environmental stimuli.
discovered a type III polyketide synthase (tetraketide synthase, However, many of these molecules have no published pharmaco-
TKS) and proposed its function in cannabinoid biosynthesis, logical data. Despite our chemical interest in these structures, we
but at the time were unable to produce olivetolic acid in vitro.401 do not speculate on the potential herein.
Later, Page et al. showed that this TKS enzyme cooperatively Biochemical characterization and crystal structures of the
functions with olivetolic acid cyclase (OAC) to form 32 – this cyclization enzymes have revealed the likely mechanism
Fig. 45 Biosynthesis of cannabigerol (147) and cannabinerol (159) from hexanoyl-CoA 156 and malonyl-CoA 127.
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Fig. 46 Biosynthesis of tetrahydrocannabinol (7), cannabidiol (8), cannabichromene (144), and further nonenzymatic derivatized products.
through which 33 is modified into the more advanced been observed.408 The carboxylic acid is likely an electronic
cannabinoids.407–409 These transformations are catalyzed by FAD- requirement for reactivity with FAD, as when the acid is protonated
dependent berberine bridge enzymes (BBEs). Data indicates that the pKa of the C5 phenolic hydrogen will decrease and when
upon binding of 33, oxidation likely occurs by an active site deprotonated the electron density at C6 will increase. In either
tyrosine-484 deprotonating the resorcinol C5 proton followed by protonation state, the carboxylic acid makes the FAD-catalyzed
FAD-catalyzed dehydrogenation of the exocyclic methylene to form dehydrogenation more facile.
a key quinone methide intermediate 166 (Fig. 47A). Interestingly, From this key quinone methide intermediate 166, all three
enzyme activity likely requires the C2 carboxylic acid to be present cannabinoid scaffolds (160, 161, and 162) can be formed by
in the substrate 33, as cyclization of the decarboxylated 147 has not hetero-Diels–Alder, Alder-ene, or electrocyclization reactions,
Fig. 47 Key proposed step in biosynthesis of cannabis natural products converting cannabigerolic or cannabinerolic acid to THCA (160). (A) Enzymatic
dehydrogenation reaction leads to a reactive quinone methide intermediate 166 that can undergo various pericyclic reactions to yield all cannabis scaffolds.
Flavin adenine dinucleotide (FAD), R = C16H26N5O13P2. (B) Related enzymatic transformations by CBCAS and CBDAS form CBCA (162) and CBDA (161).
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respectively (Fig. 47A and B). This proposed mechanism biosynthesis. Despite efforts to incorporate APT and catalyze
indicates that these enzymes THCAS, CBDAS, and CBCAS can the electrophilic prenylation to form 33, no activity could be
be considered as multifunctional pericyclases – enzymes that observed when expressed in yeast. The authors searched Can-
catalyze pericyclic reactions.410 Very recently, the plant BBE MaDa nabis transcriptomes for enzymes that share homology with
that shares 45% identity with THCAS has been characterized to well-functioning aromatic prenyl transferases, such as NphB
catalyze the Diels–Alder reaction.411 Our laboratory has also shown (vide infra) of Streptomyces sp., and discovered the enzyme
enzymes groups that share 470% homology catalyze stereo- CsPT4 – which not only efficiently catalyzes the reaction, but is
selective dehydrations and concomitant pericyclic reactions – clustered with other prenyltransferases in Cannabis. Incorporation
either hetero-Diels–Alder or Alder-ene.412 These findings point us of all genes above led to a 1.4 mgL1 titer of 33. To functionally
back to the THCAS, CBDAS, and CBCAS enzymes and led us to reconstitute the final oxidative cyclization by THCAS or CBDAS in
ask: are these reactions pericyclic? Another aspect of this yeast, the N-terminal domain of THCAS and CBDAS were replaced
transformation that warrants further investigation is the 33 with a vacuolar localization tag. In total, integrating all genes
substrate D8,9-alkene configuration. 33 is in the (E) configuration, into a single strain and culturing with galactose yielded titers of
but the products of THCAS and CBDAS are in the (Z) configuration. 8.0 mg L1 160 or 4.2 mg L1 161.
Authors have shown that THCAS can convert either cannabigerolic Due to the substrate promiscuity of OAC, Keasling et al. also used
acid (33) or cannabinerolic acid 157 into 160.407 This implies this platform to produce cannabinoid C3 alkyl chain derivatives.
that the enzyme facilitates isomerization upon quinone methide Starting from various fatty acids, 32, 33 and 160 could be produced
formation and before cyclization, but there is no evidence for with a propyl, butyl, pentenyl, 3-methylpentyl, hexyl, and hexynyl C3
the mechanism of isomerization. Further research needs to be substituents. This heterologous expression showcases the feasibility
conducted in order to fully understand the mechanism in which of complete cannabinoid and cannabinoid derivative production in
the psychoactive cannabinoid skeletons are forged. yeast. Improvements to this method for microbial cannabinoid
production methods are currently being pursued by different
4.3 Heterologous production of cannabinoids synthetic biology startup companies.
Keasling and coworkers realized heterologous production of Cell-free platforms for cannabinoid production have also
160 and 161 in Saccharomyces cerevisiae from galactose garnered much interest and success. As geranyl pyrophosphate
(Fig. 48).75 In order to produce cannabinoids in yeast, it was 82 levels are challenging to optimize in cells, cell-free methods
crucial to optimize the flux of geranyl pyrophosphate 82 and circumvent inherent issues of forming prenylated natural pro-
hexanoyl-CoA 156 by introducing an upregulated mevalonate ducts in large quantities. The Bowie laboratory has successfully
pathway, a mutant (F96W, N127W) of the endogenous farnesyl used cell-free platforms to produce CBGA 33 with a 1.25 g L1
pyrophosphate synthase (ERG20), and incorporation of an acyl titer53 and, most recently, using a far simpler and more cost-
activating enzyme from Cannabis sativa to form hexanoyl-CoA effective system were able to realize a 0.48 0.12 g L1 titer.102
156. The use of the mutant ERG20 is to attenuate the conver- Perhaps a more important discovery than this titer improve-
sion of GPP to FPP, as discussed in Section 2.8 in strictosidine ment was the implementation and engineering of promiscuous
Fig. 48 Heterologous production of tetrahydrocannabinolic acid (160) and cannabidiolic acid (161).
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bacterial prenyltransferases to catalyze the electrophilic addition of discovered route417,418 in order to minimize the number of
a geranyl pyrophosphate to 32 and 32 derivatives.53,413,414 This expensive adenosine triphosphate (ATP) and coenzyme A (CoA)
strategy avoids the native integral membrane bound Cannabis molecules required for cell-free synthesis. As shown Fig. 49A, first
prenyltransferase that is intrinsically difficult to work with both acetic acid is phosphorylated by AckA and then thioesterified to
in vivo and in cell-free systems.403 Previous work by Kuzuyama and form acetyl-CoA. The CoA group of acetyl-CoA is then transferred
coworkers showed that the enzyme NphB could prenylate a wide to malonic acid to form malonyl-CoA 127 which continues on the
variety of substrates including olivetol to form 148.413,415,416 Wild canonical pathway to form olivetolic acid 32. The authors used
type NphB prenylates olivetolic acid nonspecifically generating ThiM to phosphorylate isoprenol and then a subsequent phos-
a mixture of the desired cannabigerolic acid 33 and undesired phorylation by IPK.417,418 Typically ThiM, a hydroxyethylthiazole
O-prenylated product with a very low kcat (0.002 min1). Bowie kinase, phosphorylates 2-hydroxyethyl thiazoles. Here they have
and coworkers expanded on this work by using Rosetta to used this enzyme to catalyze the same reaction on a simpler acyclic
computationally redesign NphB.53 The endpoint was a soluble, starting material. The following steps to form geranyl pyrophosphate
easy-to-work-with enzyme – named M23 – that was highly were reported previously101 using typical isopentyl-diphosphate
selective for the desired electrophilic prenylation of C6 to form delta-isomerase (IDI) and a modified farnesyl pyrophosphate
33 and exhibited a 1000-fold increase in kcat from the wild-type synthase (FPPS) enzyme that generates the C10 dimethylallyl
enzyme. A variant (M31) was also designed to function with derived geranyl pyrophosphate. Ultimately, this strategy is a
divarinic acid 167 (the C3 propyl derivative of olivetolic acid 32). highly modular method to make various malonyl-CoA products
Now, NphB and its variants can be expressed heterologously or and useful for making high-titers of geranylated natural products.
used in cell-free systems to produce 33 and derivatives thereof.
Cell-free systems for divarinic and olivetolic acid (32 and
163) production are becoming fairly effective in producing large
5. Opioids
titers. Recently, Bowie and coworkers used a six-enzyme system Western medicine was born from the poppy plant Papaver
to produce 32 and 163 (Fig. 49A) as well as further develop somniferum. Opium has been scraped from the P. somniferum
their platform for geranyl pyrophosphate production (Fig. 49B). bulb and used both recreationally and medicinally for all of
These methods are generalizable and applicable to a wide written history.419,420 For example, in the 16th century, people
variety of molecules. The authors build off of a previously used the botanical tincture laudanum,421 which is mixture of
ambergris, musk, alcohol and opium, for the promise of good
health. In the 1800s, morphine (9), the major component of
opium, was isolated and sold as the first single-molecule drug
(Fig. 50).422 This began the contemporary 150-year medicinal
tradition of prescribing single-molecule drugs versus botanical
tinctures.
5.1 Opioid receptors

There are four class A GPCR opioid receptors, m, q, K, and N.424
The m opioid receptor (MOP) is named for binding morphine.

The q opioid receptor (DOP) is named for being expressed in
the vas deferens. The K opioid receptor (KOP) is named for
binding the synthetic ligand ketocyclazocine. The N opioid
receptor (NOP) is named after the endogenous mammalian
peptide nociceptin.
These names are not truly informative and
have been subject to debate. Opioid receptors are distributed
throughout the central nervous system and partially in the vas
deferens, joints, and immune system. MOP, DOP, and KOP are
Fig. 49 Cell-free system for improved olivetolic acid, divarinic acid, and Fig. 50 Image of bulbs and bloom of the poppy plant, Papaver somni-
geranyl pyrophosphate production. ferum. On average, poppy bulbs contain 16% by weight morphine 9.423
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sometimes referred to as classical opioid receptors. Whereas These discoveries led chemists to develop related compounds, i.e.
NOP is ‘less’ classical; NOP was discovered later and now is heroin 169, that were more potent, safer (minimizes hypoventila-
considered an opioid receptor as well. As the ligand was not tion), and touted as ‘‘free from abuse liability.’’419 This claim
originally known for NOP, the receptor is sometimes still marked the first falsification of opioids being safe to use without
referred to as an opioid-like receptor or an orphan receptor. risk of addiction, which were most recently repeated by the
Other opioid-like receptors have been identified based on Sacklers at Purdue Pharma. The morphinan alkaloids are potent
binding, however these are not bona fide opioid receptors. analgesics that have been used for thousands of years as opium
The s receptor (named for binding SKF10047) binds opioids mixtures and now as isolated pure compounds.424 Natural
as well as other drugs of abuse like phenylcyclidine. Other products oripavine 170 and thebaine 171 do not exhibit safe
receptors that do exhibit related pharmacology to MOP, DOP, pharmacology, but are highly useful morphinan alkaloids as
KOP, or NOP have been identified but are not fully character- synthetic building blocks for derivatization.
ized; for example, the z receptor is an opioid growth factor The reticulines are key early pathway intermediates that
receptor, and the l receptor and e binding site have been many families of opioid scaffolds diverge from. These structures
proposed in b-endorphin binding. are simple benzylisoquinolines (Fig. 52). (S)-Reticuline 172 can
The structure function relationship of opioid receptors is epimerize to (R)-reticuline 28 and continue to morphinan bio-
relatively well understood.425–427 MOP, DOP, KOP, and NOP synthesis or directly undergo a C–C coupling reaction and lead to
share B60% homology with highly conserved fingerprints of the phthalide isoquinolines and protoberberines families of
class A GPCRs and a homologous binding cavity. The small opioids. (S)-norcoclaurine 27 is formed from dopamine 17 and
molecule ligands like morphine bind to the conserved receptor 4-hydroxyphenylacetaldehyde 26 by a Pictet-Spenglerase and
residues in the homologous binding cavities. marks the first dedicated step in opioid biosynthesis (also see
Fig. 3). Though these compounds are not known to be psychoac-
5.2 Opium alkaloids tive, they are the building blocks to form many psychoactive
The morphinan alkaloids are the most colloquial family of natural products. Of note, the oxidation of the isoquinoline to
opium alkaloids as morphine (9) is the flagship molecule of the form papaverine 173 alters the pharmacological properties as 173
family. This family features a tetracyclic phenanthrene fused is an approved antispasmodic drug.
piperidine core, a so-called morphinan scaffold (Fig. 51). In the The phthalide isoquinolines class of opioids encompass two
early 1800s, Friedrich Sertürner isolated 9 (Fig. 51) from Papaver general structures in which the isoquinoline is either intact or
somniferum.428 In the following years, Pierre-Jean Robiquet isolated open as a dimethyl amino sidechain, as exemplified by nosca-
the O-methylated morphine derivative, codeine 168 (Fig. 51).429 pine 174 and narceine 175. 174 and 175 are non-narcotic,
antitussives with minor hypnotic, euphoric, and analgesic
properties. 174 has a lengthy history as a pharmaceutical with
its isolation in 1817 and initial use as an anti-malarial drug
until 1930.430 Now, many are rediscovering 174 as an anti-
cancer drug candidate.431,432 Such compounds are produced by
many species of the Papveraceae poppy plant (Fig. 53).
Aporphine opioids are C–C phenol coupled benzylisoquino-
lines that feature a functionalized aporphine structure (Fig. 54).
Fig. 51 Structures of natural morphinan opioids and synthetic compound Fig. 52 Structures of simple benzyl isoquinolines that play key roles in
diacetylmorphine (heroin, 169). opioid biosynthesis (172, 28, 27) and as antispasmodic drugs (173).
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5.2.1 Biosynthesis of opium alkaloids. Biosynthesis of mor-

phinan opioids requires more than 10 enzymatic steps starting
from dopamine 17 and 4-hydroxyphenylacetaldehyde 26.
The elucidation of this route has taken more than 30 years
of research and is condensed into a single figure, Fig. 56. The
first dedicated step in opioid biosynthesis is a Pictet–Spengler
reaction catalyzed by norcoclaurine synthase (NCS) to forge the

tetrahydroisoquinoline core of (S)-norcoclaurine 27 – which is a
Fig. 53 Phthalide isoquinoline opioid natural products, noscapine (174)
simple example of the benzylisoquinoline alkaloid natural product
and narceine (175).
family.433,434 There have been many mechanistic studies of this
enzyme that are not discussed herein (also see Fig. 3).43,435,436
(S)-Reticuline 172 is formed from 27 by two hydroxylations,
an N-methylation, and an O-methylation by the enzymes norco-
claurine 6-O-methyltransferase (6OMT), coclaurine N-methyl-
transferase (CNMT), N-methylcoclaurine 3 0 -hydroxylase (NMCH
CYP80B1), and 3 0 -hydroxy-N-methylcoclaurine 4 0 -O-methyltrans-
ferase (4 0 OMT), respectively.437–440 These steps can occur in a
variety of orders with similar efficiencies and are drawn in a
typical order in Fig. 56. 172 is a key branch point in opioid
biosynthesis from which many benzylisoquinoline scaffolds can
form. In 2015, the subsequent epimerase enzyme was discovered
independently by three different groups. Termed STORR reticuline
epimerase (REPI) and 1,2-dehydroreticuline synthase-1,2-dehydro-
reticuline reductase (DRS-DRR), the fusion protein was found to
convert (S)- to (R)-reticuline by dehydrogenation at C1 to form
an iminium cation which is hydrogenated from the opposing
face.441,442 In order to identify these genes, laboratories inves-
tigated RNA interface mediated silencing of the codeinone
Fig. 54 Aporphine opioids corytuberine (176), natural (S)-glaucine (177) reductase (COR) gene. Silencing COR, which operates several
and unnatural (R)-glaucine (178). steps downstream from the epimerization of reticuline, results
in accumulation of (S)-reticuline versus the substrate codeinone
181. This could occur due to off-target co-silencing of the
These natural products have a range of activity from anti- homologous reductase that catalyze the second step in the
convulsant (corytuberine, 176) to antinociceptive ((S)-glaucine, epimerization of (S) to (R) reticuline. Using this strategy, a
177). The unnatural (R)-isomer of glaucine 178 is known to be a fusion protein REPI and DRS-DRR was identified that was able
potent hallucinogen that modulates the 5-HT2A receptor and is to catalyze the crucial epimerization reaction.
sometimes used recreationally. Such compounds can be iso- In order to discover the enzyme without access to the opium
lated from a variety of Papaveraceae species such as Glaucium poppy, the Smolke lab searched the 1000 Plants Project and
flavum and Corydalis yanhusuo. PhytoMetaSyn databases for COR-like enzymes in Papaver species.
Lastly, the berberine opioids are pentacycles with a dibenzo- This revealed several genes that encoded for a two-domain enzyme
quinizolium core. These molecules are quaternary ammonium with P450 82Y1-like and COR-like domains. From these two
salts. Berberine 179 is a traditional natural yellow dye and domains it was reasonable to hypothesize that the (S)-reticuline
sanguinarine 180 is an escharotic toxin that also causes epidemic 172 could be oxidized to an isoquinilonium (P450) and then
dropsy. The berberine opioids have been isolated from Papaver reduced to (R)-reticuline 28 by the COR domain. One of the gene
somniferum and Macleaya cordata (Fig. 55). candidates from P. somniferum, named DRS-DRR was cultured
with P450 reductases, CNMT, 6OMT and 1 mM norlaudanosoline
182, a desmethoxy derivative of reticuline, for 72 hours, and 450%
conversion to (R)-reticuline 28 was observed.
(R)-Reticuline 28 is the substrate for the salutaridine
synthase (SalSyn) CYP8719B1-catalyzed oxidative phenol coupling
reaction that forms a carbon–carbon bond between C20 and C4a
yielding salutaridine 183.58,443 This reaction is proposed to occur by
the iron oxo heme compound I abstracting the hydrogen from the
C30 hydroxyl of 28 to generate compound II, which then abstracts
Fig. 55 Examples of berberine opioids, berberine (179) and sanguinarine the remaining phenol hydrogen to facilitate the cyclization (also see
(180). Fig. 5B).58 The direct di-keto product readily enolizes to form 183.
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Fig. 56 Biosynthesis of the morphine opioids from dopamine 17 and 4-hydroxyphenylacetaldehyde 26.
Salutaridine 183 is converted to thebaine 171 in three enzymatic In 2012, a 10-gene cluster responsible for noscapine 174
steps. First, salutaridine reductase (SalR) reduces the quinone biosynthesis was discovered.451 Noscapine 174 biosynthesis
ketone to form salutaridinol 184 which is then acylated by the acyl diverges from 9 biosynthesis after (S)-reticuline 172 formation
transferase enzyme salutaridinol 7-O-acetyltransferase (SalAT) and (Fig. 57). First, 172 is transformed to (S)-scoulerine 186 by a
was believed to slowly cyclizes nonenzymatically to form thebaine berberine bridge enzyme (BBE).452,453 This BBE catalyzes an
171.444–446 Recently, thebaine synthase (THS) was discovered and FAD-dependent dehydrogenation of the N-methyl group to form
isolated in Papaver somniferum opium poppy latex and found to a methylene isoquinolinium. This reactive intermediate then
accelerate this cyclization to form the morphine skeleton of 171.447 undergoes C–C bond formation between the methylene and C2 0
The final four enzymatic steps in morphine 9 biosynthesis that is facilitated by glutamate sidechain hydrogen bonding to
are two O-demethylations, an isomerization and ketone reduction the C3 0 phenolic hydrogen. Mechanistic studies have proposed
that are catalyzed by codeine O-demethylase (CODM), thebaine that complete proton transfer is not required,452 but the C3 0
6-O-demthylase (T6ODM), neopinone isomerase (NISO), and codei- hydroxyl – which increases the nucleophilicity of C2 0 – is
none reductase (COR), respectively.74,448,449 There are two estab- required for catalysis.454 Multiple alkaloid classes derive from
lished paths that differ in the first O-demethylation, which can 186; for example, the protoberbines, benzophenanthridines,
lead to either oripavine 170 or codeinone 181. In 2018, the crystal protopines, and the phthalideisoquinolines.
structure for T6ODM was solved, but the mechanism for the (S)-Canadine 187, an antioxidant, is formed by subsequent
O-demethylation is still unknown.450 Further O-demethylation O-methylation and etherification of (S)-scoulerine 186 by the
and isomerization (a formal 1,5-hydrogen shift) produces morphi- scoulerine 9-O-methyltransferase (S9OMT) and canadine synthase
none 185 which is, finally, reduced to form morphine 9. (CAS) enzymes, respectively.455,456 187 undergoes N-methylation by
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Fig. 57 Noscapine biosynthesis from (S)-reticuline.
tetrahydroprotoberberine N-methyltransferase (TNMT) to form the oxidized to the lactone (narcotoline, 193) by the enzyme
isoquinolinium core of (S)-N-methylcanadine 188 that can undergo SDR1.460,461 Lastly, noscapine 170 is formed by the O-methylation
dihydroxylation by CYP82Y1 to form (S)-1,13-dihydroxy-N- by N40 OMT.455 Of note, these last two steps can occur in either
methylcanadine 189.457–459 order; N40 OMT O-methylation can precede SDR1 lactonization.
The noscapine core is formed by the oxidative ring opening 5.2.2 Heterologous production of opium alkaloids. There have
and cyclization to yield narcotoline hemiacetal 190. These been many efforts in heterologous production of opioids.109,462–467
transformations begin with acetylation of (S)-1,13-dihydroxy- These pathways, at the time, were the longest biosynthetic pathways
N-methylcanadine 189 to form (S)-1-hydroxy-13-O-acetyl-N-canadine reconstituted in yeast.466 However, almost all studies stopped at
191 by the acetyltransferase AT1 (see Fig. 4B).460 This enzymatically- (S)-reticuline 172 or begin at highly functionalized opioids, like
installed acetyl group is essential for CYP82X1 hydroxylation activity thebaine 171. This had to do with the fact that the crucial
and has been proposed to function as a protecting group to epimerase that forms (R)-reticuline 28 was not characterized
alleviate from precocious hemiacetalization.460 Following acetyla- until 2015. At this time, Smolke’s laboratory had already rea-
tion, CYP82X1 installs a hydroxyl ortho to the nitrogen that lized heterologous production of thebaine 171 and hydrocodone
facilitates a spontaneous oxidative ring opening to form (S)-4 0 -O- 194 in yeast (Fig. 58).77 To complete biosynthetic reconstitution,
desmethyl-3-O-acetylpapaveroxine 192. the laboratory had to overcome two main challenges: (1) discover
(S)-4 0 -O-Desmethyl-3-O-acetylpapaveroxine 192 undergoes an enzyme that racemizes (S)-reticuline 172 to (R)-reticuline 28;
three final enzymatic transformations to form noscapine 174: and (2) engineer the aryl coupling P450 SalSyn to be fully
hemiacetalization, oxidation and O-methylation. The enzyme functional when expressed in yeast. A further challenge was
CXE1 catalyzes the hemiacetalization to form the phthalideiso- implicit in the task; simply expressing 420 genes and obtaining
quinoline core of narcotoline hemiacetal 190 which is then high efficiency with each enzymatic transformation. In spite of
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Fig. 58 Heterologous production of thebaine and hydrocodone from sugar in yeast.
these challenges, Galanie et al. engineered a fully integrated yeast 12 to form L-DOPA 71 catalyzed by mammalian tyrosine hydroxylase
strain that produced 6.4 0.3 mg L1 of thebaine 171 and with (TyrH) and is not native to yeast. 6-Pyruvoyl-tetrahydropterin (PTPS)
additional downstream enzymes, B0.3 mg L1 of hydrocodone and sepiapterin reductase (SepR) are used to produce BH4 from
194 in a culmination of decades of research.78,109 dihydroneopterin, a yeast metabolite. Quinonoid dihydropteridine
The engineered strain contained 19 heterologously expressed reductase (QDHPR) and pterin carbinolamine dehydratase (PCD)
mammalian, bacterial, and plant enzymes, two modified yeast are then used to recycle BH4 back to its active form.
enzymes, two overexpressed native yeast enzymes and one inacti- Module III uses bacterial, plant, and mammalian enzymes to
vated enzyme for a total of 24 chromosomal modifications. These catalyze formation of the first BIA scaffold. Dihydrofolate reductase
modifications were split between seven modules for both pathway (DHFR) is another BH4 salvage enzyme that works with TyrHWT, a
and chromosomal organization. mutant that is more inhibition resistant. Following hydroxylation,
Module I consists of overexpression of two modified shikimate L-DOPA 71 undergoes decarboxylation catalyzed by DOPA decarb-
pathway enzymes and two native yeast genes. The Q166K point oxylase (DoDC) to form dopamine 17 followed by a Pictet–Spengler
mutation in Aro4p, which catalyzes the aldol condensation of reaction between 4-hydroxyphenylacetaldehyde 26 and 17 by
erythrose 4-phosphate 47 and phosphoenolpyruvic acid 48 to form norcoclaurine synthase (NCS) to form (S)-norcoclaurine 27.
3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate 195, renders The remaining modules consist of the biosynthetic pathway
the enzyme feedback inhibition resistant. Similarly, the T226I enzymes towards thebaine 171 and hydrocodone 194 and the
mutation in Aro7p, which is one of the enzymes involved in the discovered enzyme for (S)-reticuline epimerization. The native
biotransformation of 195 into 4-hydroxyphenolpyruvic acid 196, P450 enzyme SalSyn had low activity when initially expressed in
makes the enzyme feedback resistant. Overexpression of Aro10p yeast. This was hypothesized to be due to incorrect translocation
and Tkl1 resulted in shifting metabolic flux towards the pathway. of nascent SalSyn to the endoplasmic reticulum (ER) lumen as
The next module (II) focuses on producing and recycling the opposed to correct anchoring to the outer ER membrane based
mammalian redox cofactor, tetrahydrobiopterin (BH4). This on nonnative N-glycosylation patterns. Mistranslocation could
cofactor is essential for the selective C3 hydroxylation of L-tyrosine stem from a poorly recognized N-terminus and thus the authors
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replaced the N-terminus portion of SalSyn with that from a

homologous, non-glycosylated P450, Cheilanthifoline synthase,
that shares 61% identity and exhibits high activity in yeast.468
The engineered chimeric SalSyn enzyme exhibited nearly 6-fold
improvement in conversion of (R)-reticuline 28 to salutaridine
183 compared to the wild type enzyme.
After establishing B6 mg L1 thebaine 171 production with

their platform, the authors sought to introduce downstream
enzymes towards hydrocodone 194 production. Upon coexpression
of two more enzymes, MorB and T6ODM and supplementation
with 50 mM oxoglutarate, the strain produced 0.3 mg L–1 194. The Fig. 59 Mitragyna speciosa cultivars may contain up to one percent dry
weight mitragynine. Image on left courtesy of Thor Porre via CC-3.0.
Smolke lab previously used MorB, an NADH-dependent morphi-
https://commons.wikimedia.org/wiki/File:Kratom_tree.jpg.
none reductase from a bacteria Pseudomonas putida M10 that was
originally discovered in an opium poppy processing factory, for
production of natural and semi-synthetic opioids.465,469 Expression 5.3.1 Biosynthesis of mitragynine. Kratom alkaloids belong
of such a long pathway required careful codon-optimization of to the MIA family, and are presumed to be derived from the
multiple enzymes and led to proof-of-concept titers that highlight universal MIA precursor strictosidine. The 12-step pathway leading
the potential of chassis species for pharmaceutical production. to the formation of strictosidine 25 from the primary metabolites
In 2018, the Smolke lab modified this pathway to produce L-tryptophan 11 and isopentenyl pyrophosphate 98 has been
noscapine 174.470 The new work branches at (S)-reticuline 172, elucidated in C. roseus and is discussed in Section 2.8. While the
using the BBE to produce (S)-scoulerine 186. Therein, more remaining biosynthetic steps leading to the formation of mitragy-
than 30 enzymes were heterologously expressed, including five nine are largely unknown, we have proposed the pathway shown in
plant P450s which are notoriously difficult to express in yeast. Fig. 60 based on a number of biochemical observations. It is
To overcome challenges in P450 activity and other pathway known that following deglucosylation of 25 by strictosidine-O-b-
bottlenecks, the authors (i) deleted the first 24 amino acids of glucosidase (SGD) and subsequent rearrangement, a reductase
NCS corresponding to an N-terminal signal vacuole translocation converts the reactive aglycone 87 isomer into a more stable path-
peptide to avoid detrimental sorting of the nascent peptide,471 way intermediate.476 Examples from literature include tetrahy-
(ii) codon optimized the TyrH R37E, R38E, W166Y (TyrHWR), droalstonine synthase, geissoschizine synthase, and vitrosamine
(iii) incorporated an NADPH regenerating system, (iv) and lastly, synthase, which are all NADPH-dependent reductases.242,477,478
optimized media and fermentation conditions which led to the Moreover, O’Connor and coworkers recently identified a dihydro-
largest gain (B300-fold) in production. Overall, the combined corynantheine aldehyde synthase (CpDCS) from Cinchona
strategies resulted in a noscapine 174 titer of 2.21 mg L1 in pubescens involved in quinine biosynthesis.479 CpDCS performs
72 h. Finally, Li et al. demonstrated the versatility of their yeast iterative reduction of geissoschizine 87 to provide a demethyl-
platform by generating halogenated BIA derivatives through corynantheidine 200 isomer. The authors identified an orthologue
feeding modified L-tyrosines. in Mitragyna speciosa named MsDCS, postulating that following
deglycosylation of 25, two successive reductions of the conjugated
5.3 Kratom iminium 87 would provide the stable demethylcorynantheidine
In addition to the opium alkaloids, more than 50 kratom 199. Reduction of conjugated iminiums has also been demon-
alkaloids have been isolated from the Mitragyna speciosa plant, strated in the formation of other late stage MIAs including
several of which exhibit opioid-like properties.472 Native to tabersonine and catharanthine.236,237 Additionally, production
Southeast Asia, kratom (Mitragyna speciosa) has been used in of 199 has been reported in Uncaria rhynchophylla, which like
traditional Thai medicine for centuries. The use in the United kratom belongs to the family Rubiaceae.480 Methylation of the
States has increased rapidly since early 2000s, both recreation- putative 199 would provide corynantheidine 200, which has
ally and to relieve chronic pain or opioid withdrawal symptoms. been isolated from Mitragyna and differs from mitragynine by
Compared to conventional opium alkaloids, kratom alkaloids one methoxy group.472 Following aromatic hydroxylation and
exhibit ‘‘unique binding and functional profiles’’ suggesting that methylation, mitragynine 10 is likely further hydroxylated to
plant extracts may be effective alternative to the benzylisoquinoline- 7-hydroxymitragynine 197. The P450-mediated conversion of 10
based pain treatments.473 However, similar to opium alkaloids, to 197 has been demonstrated in both mouse and human liver
repeated use of kratom may lead to addiction, and the FDA has preparations.238 A semi-pinacol rearrangement to provide the
not approved kratom for any medical use; as a result, the DEA lists mitragynine pseudoindoxyl 198 may occur either sponta-
kratom as a Drug of Concern. The first reported and most abundant neously or enzymatically481 as has been described in analogous
kratom alkaloid is mitragynine 10, comprising up to 66% of the transformations by FAD-dependent oxidases in fungal alkaloid
alkaloid content in Thai cultivars (Fig. 59).474 The less abundant pathways.482,483 Identification of the M. speciosa biosynthetic
7-hydroxymitragynine 197 and its rearrangement product mitra- enzymes will provide biocatalytic tools necessary for hetero-
gynine pseudoindoxyl 198 are potent partial agonists of human logous production of kratom alkaloids in existing seco-iridoid
m-opioid receptors at nanomolar concentrations.27,475 producing yeast platforms.76
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Fig. 60 Proposed biosynthetic route to kratom alkaloids.
6. Conclusions and perspective information. Given the rapid expansion of molecular biology
techniques and prominent early successes in pathway refactoring,
The natural products described in this review run the gamut of such synthetic biology technologies will very likely play a role in
metabolic origin, psychoactive effect, and biological source. 21st century biomanufacturing. Major questions around the cost,
While most of the compounds discussed have been isolated ethics, and legality of synthetic-biology-based production of psy-
from plants, we have highlighted several well-known psychoactive choactive substances must be answered in the very near future.
natural products produced by fungi and one of animal origin. At present, the natural products covered in this review are
Given the immense structural diversity exhibited by such mole- either regulated or unregulated, however this legal binary is
cules, the wide array of psychoactivities is not surprising. We currently being traversed by Cannabis products. Popular culture
have noted that the majority of the compounds originate from and the media have sensationalized cannabinoid research.
amino acid metabolism, however prominent examples of com- Despite the wide interest in cannabinoids, the research is still
pound biogenesis via terpenoid and polyketide metabolism highly controversial and difficult to fund. This is rapidly changing
have been provided. Moreover, we have featured a number of as the World realizes there is ‘money in cannabinoids.’ Western
remarkable enzymatic transformations that not only provide medicine prefers pure, single molecule therapeutics to botanical
inspiration for biomimetic syntheses, but have been directly extracts. In 1996, California legalized botanical cannabis for
used in chemoenzymatic applications; these include comple- medicinal use.484 This goes against the western medicine doctrine
tely stereoselective nucleophilic additions, tightly controlled and begs the question: as pharmacology, chemistry, and biochem-
scaffold rearrangements, and regioselective group transfer istry all advance, will western medicine move towards curated
reactions on deprotected substrates. We have also chosen to complex mixtures of small molecules that emulate botanical
outline biosynthetic pathways ranging from fully elucidated to tinctures? Perhaps, the cannabinoids will represent a case study
almost entirely incomplete. Ongoing efforts towards total pathway that other scheduled substances will follow. Evidentiary develop-
elucidation are necessitated by the multitude of synthetic biology ments indicate that Cannabis components such as 8 modulate
applications that benefit from a complete set of biosynthetic adverse effects of 7, a phenomenon commonly described as the
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entourage effect.396,397 However the majority of cannabinoid com- 3 E. J. Buenz, R. Verpoorte and B. A. Bauer, Annu. Rev.
pounds do not have published pharmacological data. And, the Pharmacol. Toxicol., 2018, 58, 509–530.
molecules that are well-studied are still controlled substances. 4 T. Efferth, M. Banerjee, N. W. Paul, S. Abdelfatah, J. Arend,
For example, 8 is a non-euphoric compound with a safe pharma- G. Elhassan, S. Hamdoun, R. Hamm, C. Hong, O. Kadioglu,
cokinetic profile, yet it is a controlled substance. As Di Marzo and J. Naß, D. Ochwangi, E. Ooko, N. Ozenver, M. E. M. Saeed,
coworkers say, ‘‘This anomaly makes clear that, despite consider- M. Schneider, E. J. Seo, C. F. Wu, G. Yan, M. Zeino, Q. Zhao,
able scientific evidence, talks about legalization, and the many M. S. Abu-Darwish, K. Andersch, G. Alexie, D. Bessarab,
industrial and medical uses of the plant, stigma around cannabis D. Bhakta-Guha, V. Bolzani, E. Dapat, F. V. Donenko,
still hinders the conclusive assessment of the therapeutic potential of M. Efferth, H. J. Greten, L. Gunatilaka, A. A. Hussein,
the plant’s most abundant components. Further education is needed A. Karadeniz, H. E. Khalid, V. Kuete, I. S. Lee, L. Liu,
to reduce the negative impact of these factors on research.’’485 J. Midiwo, R. Mora, H. Nakagawa, O. Ngassapa, C. Noysang,
Undoubtedly, this paradigm extends beyond cannabinoids, L. K. Omosa, F. H. Roland, A. A. Shahat, A. Saab, E. M. Saeed,
as immense untapped therapeutic potential exists in regards to L. Shan and S. J. J. Titinchi, Phytomedicine, 2016, 23, 166–173.
the other compounds described. As Western medicine has 5 J. R. George, T. I. Michaels, J. Sevelius and M. T. Williams,
long cannibalized indigenous discoveries, however, we must J. Psychedelic Stud., 2019, 4, 4–15.
prioritize the rights of practitioners of traditional medicine as 6 P. Gootenberg, Am. Anthropol., 2005, 107, 152–153.
we unpack the potential applications of these natural products. 7 S. Kean, Science, 2019, 364, 16–20.
Ironically, the same reductionist vision of single molecule thera- 8 D. M. Provine, Annu. Rev. Law Soc. Sci., 2011, 7, 41–60.
peutics has resulted in a persistent rejection of holistic approaches 9 R. J. Sullivan and E. H. Hagen, Addiction, 2002, 97, 389–400.
to medicine. However, the tides are changing, and practitioners of 10 A. Martini, J. Wine Res., 1993, 4, 165–176.
science are more readily acknowledging the limitations of reduc- 11 G. S. L. Brito, Cad. Saude Publica, 1994, 10, 403–405.
tionist frameworks. In the same way that we have reduced the 12 D. A. Dias, S. Urban and U. Roessner, Metabolites, 2012, 2,
extraordinary complexity of metabolic networks into linear biosyn- 303–336.
thetic pathways, reductionism should be used to complement 13 R. Robinson, J. Chem. Soc., Trans., 1917, 111, 762–768.
holism. From this perspective, medicine, culture, and technology 14 E. C. Kornfeld, E. J. Fornefeld, G. Bruce Kline, M. J. Mann,
can all be beneficiaries of a comprehensive understanding of D. E. Morrison, R. G. Jones and R. B. Woodward, J. Am.
Nature’s biosynthetic routes to psychoactive natural products. Chem. Soc., 1956, 78, 3087–3114.
15 C. T. Walsh and Y. Tang, Natural Product Biosynthesis:
Chemical and Enzymatic Logic, Royal Society of Chemistry,
Conflicts of interest 2017.
The authors declare the following competing financial interest(s): 16 T. Pluskal and J. K. Weng, Chem. Soc. Rev., 2018, 47, 1592–1637.
John Billingsley is an employee of Invizyne Technologies, Inc. 17 E. S. Valenstein, Brain Cogn., 2002, 49, 73–95.
(Monrovia, CA, USA), a company seeking to commercialize synthetic 18 K. S. Murnane, Progress in Brain Research, 2018, vol. 242,
biochemistry. pp. 25–67.
19 F. Nadim and D. Bucher, Curr. Opin. Neurobiol., 2014, 29,
48–56.
Acknowledgements 20 L. Bracco and J. Kearsey, Trends Biotechnol., 2003, 21,
346–353.
Related work in the Tang lab is supported by NIH 1R01AT010001. 21 C. Contet, B. L. Kieffer and K. Befort, Curr. Opin. Neuro-
C.S. Jamieson is grateful for additional funding from the Saul biol., 2004, 14, 370–378.
Winstein fellowship, the Foote fellowship, and a UCLA Disserta- 22 B. L. Roth, K. Baner, R. Westkaemper, D. Siebert, K. C.
tion Year Fellowship. J. Misa is supported by NIGMS-funded Rice, S. A. Steinberg, P. Ernsberger and R. B. Rothman,
predoctoral fellowship T32 GM136614. The authors wish to Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 11934–11939.
acknowledge the indigenous peoples whose immense knowledge 23 D. Nutt, PLoS Biol., 2015, 13, e1002047.
of the natural world has facilitated the study of psychoactive 24 R. L. Carhart-Harris, M. Bolstridge, C. M. J. Day, J. Rucker,
natural products. We recognize that many of the scientific dis- R. Watts, D. E. Erritzoe, M. Kaelen, B. Giribaldi, M. Bloomfield,
coveries described in this review would not be possible were it not S. Pilling, J. A. Rickard, B. Forbes, A. Feilding, D. Taylor,
for the expert observations of indigenous people of the Sierra H. V. Curran and D. J. Nutt, Psychopharmacology, 2018, 235,
Mazateca, indigenous groups in the Amazon basin, indigenous 399–408.
populations in the Andes, indigenous Bwiti practitioners in Gabon, 25 R. L. Carhart-Harris, R. Leech, T. M. Williams, D. Erritzoe,
and countless others to whom we pay our respects. N. Abbasi, T. Bargiotas, P. Hobden, D. J. Sharp, J. Evans,
A. Feilding, R. G. Wise and D. J. Nutt, Br. J. Psychiatry, 2012,
References 200, 238–244.
26 L. P. Cameron, R. J. Tombari, J. Lu, A. J. Pell, Z. Q. Hurley,
1 M. D. Merlin, Econ. Bot., 2003, 57, 295–323. Y. Ehinger, M. V. Vargas, M. N. McCarroll, J. C. Taylor,
2 E. Guerra-Doce, Time Mind, 2015, 8, 91–112. D. Myers-Turnbull, T. Liu, B. Yaghoobi, L. J. Laskowski,
View Article Online
E. I. Anderson, G. Zhang, J. Viswanathan, B. M. Brown, 52 H. C. Lin, G. Chiou, Y. H. Chooi, T. C. McMahon, W. Xu,

M. Tjia, L. E. Dunlap, Z. T. Rabow, O. Fiehn, H. Wulff, N. K. Garg and Y. Tang, Angew. Chem., Int. Ed., 2015, 54,
J. D. McCorvy, P. J. Lein, D. Kokel, D. Ron, J. Peters, Y. Zuo 3004–3007.
and D. E. Olson, Nature, 2021, 589, 474–479. 53 M. A. Valliere, T. P. Korman, N. B. Woodall, G. A. Khitrov,
27 V. Salim, F. Yu, J. Altarejos and V. De Luca, Plant J., 2013, R. E. Taylor, D. Baker and J. U. Bowie, Nat. Commun., 2019,
76, 754–765. 10, 565.
28 J. J. Barone and H. R. Roberts, Food Chem. Toxicol., 1996, 54 C. T. Walsh and T. A. Wencewicz, Nat. Prod. Rep., 2013, 30,
34, 119–129. 175–200.
29 M. C. Fiore, S. S. Smith, D. E. Jorenby and T. B. Baker, 55 H. Yamamoto, N. Katano, A. Ooi and K. Inoue, Phytochem-
JAMA, J. Am. Med. Assoc., 1994, 271, 1940–1947. istry, 2000, 53, 7–12.
30 L. Grinspoon and J. B. Bakalar, J. Ethnopharmacol., 1981, 3, 56 S. Irmler, G. Schröder, B. St-Pierre, N. P. Crouch, M. Hotze,
149–159. J. Schmidt, D. Strack, U. Matern and J. Schröder, Plant J.,
31 M. R. Amin and D. W. Ali, Advances in Experimental 2000, 24, 797–804.
Medicine and Biology, 2019, vol. 1162, pp. 151–165. 57 S. Kishimoto, M. Sato, Y. Tsunematsu and K. Watanabe,
32 A. Rosenblum, L. A. Marsch, H. Joseph and R. K. Portenoy, Molecules, 2016, 21(8), 1098.
Exp. Clin. Psychopharmacol., 2008, 16, 405–416. 58 A. Gesell, M. Rolf, J. Ziegler, M. L. D. Chávez, F. C. Huang
33 Y. Iijima, D. R. Gang, E. Fridman, E. Lewinsohn and and T. M. Kutchan, J. Biol. Chem., 2009, 284, 24432–24442.
E. Pichersky, Plant Physiol., 2004, 134, 370–379. 59 K. A. Shipkowski, J. M. Betz, L. S. Birnbaum, J. R. Bucher,
34 M.-C. Tang, Y. Zou, K. Watanabe, C. T. Walsh and Y. Tang, P. M. Coates, D. C. Hopp, D. MacKay, H. Oketch-Rabah,
Chem. Rev., 2017, 117, 5226–5333. N. J. Walker, C. Welch and C. V. Rider, Food Chem. Toxicol.,
35 C. T. Walsh and B. S. Moore, Angew. Chem., Int. Ed., 2019, 2018, 118, 963–971.
58, 6846–6879. 60 R. G. Hollingsworth, J. W. Armstrong and E. Campbell,
36 C.-I. Lin, R. M. McCarty and H. Liu, Angew. Chem., Int. Ed., Ann. Appl. Biol., 2003, 142, 91–97.
2017, 56, 3446–3489. 61 E. Leonard, W. Runguphan, S. O’Connor and K. J. Prather,
37 C. T. Walsh and Y. Tang, The Chemical Biology of Human Nat. Chem. Biol., 2009, 5, 292–300.
Vitamins, Royal Society of Chemistry, 2018. 62 A. Cravens, J. Payne and C. D. Smolke, Nat. Commun., 2019,
38 F. F. Blicke, Org. React., 2011, 303–341. 10, 2142.
39 E. Leete, Planta Med., 1990, 56, 339–352. 63 D. G. Gibson, L. Young, R. Y. Chuang, J. C. Venter, C. A.
40 K. Kohnen-Johannsen and O. Kayser, Molecules, 2019, Hutchison and H. O. Smith, Nat. Methods, 2009, 6, 343–345.
24, 796. 64 D. Gresham, M. J. Dunham and D. Botstein, Nat. Rev.
41 J. R. F. Allen and B. R. Holmstedt, Phytochemistry, 1980, 19, Genet., 2008, 9, 291–302.
1573–1582. 65 F. D. Urnov, E. J. Rebar, M. C. Holmes, H. S. Zhang and
42 J. J. Maresh, L.-A. Giddings, A. Friedrich, E. A. Loris, P. D. Gregory, Nat. Rev. Genet., 2010, 11, 636–646.
S. Panjikar, B. L. Trout, J. Stöckigt, B. Peters and S. E. 66 S. Kosuri and G. M. Church, Nat. Methods, 2014, 11,
O’Connor, J. Am. Chem. Soc., 2008, 130, 710–723. 499–507.
43 A. Ilari, S. Franceschini, A. Bonamore, F. Arenghi, B. Botta, 67 Z. Wang, M. Gerstein and M. Snyder, Nat. Rev. Genet., 2009,
A. Macone, A. Pasquo, L. Bellucci and A. Boffi, J. Biol. 10, 57–63.
Chem., 2009, 284, 897–904. 68 P. D. Hsu, E. S. Lander and F. Zhang, Cell, 2014, 157,
44 C. T. Walsh, B. P. Tu and Y. Tang, Chem. Rev., 2018, 118, 1262–1278.
1460–1494. 69 A. S. Khalil and J. J. Collins, Nat. Rev. Genet., 2010, 11,
45 K. Miettinen, L. Dong, N. Navrot, T. Schneider, V. Burlat, 367–379.
J. Pollier, L. Woittiez, S. Van Der Krol, R. Lugan, T. Ilc, 70 A. Osbourn, Plant Physiol., 2010, 154, 531–535.
R. Verpoorte, K. M. Oksman-Caldentey, E. Martinoia, 71 B. Field, A. S. Fiston-Lavier, A. Kemen, K. Geisler, H. Quesneville
H. Bouwmeester, A. Goossens, J. Memelink and D. Werck- and A. E. Osbourn, Proc. Natl. Acad. Sci. U. S. A., 2011, 108,
Reichhart, Nat. Commun., 2014, 5, 1–12. 16116–16121.
46 U. Metzger, C. Schall, G. Zocher, I. Unsöld, E. Stec, S.-M. Li, 72 Z. Liu, J. Cheema, M. Vigouroux, L. Hill, J. Reed, P. Paajanen,
L. Heide and T. Stehle, Proc. Natl. Acad. Sci. U. S. A., 2009, L. Yant and A. Osbourn, Nat. Commun., 2020, 11, 5354.
106, 14309–14314. 73 P. Srinivasan and C. D. Smolke, Nature, 2020, 585, 614–619.
47 M. E. Tanner, Nat. Prod. Rep., 2015, 32, 88–101. 74 M. Dastmalchi, X. Chen, J. M. Hagel, L. Chang, R. Chen,
48 A. Grundmann and S. M. Li, Microbiology, 2005, 151, S. Ramasamy, S. Yeaman and P. J. Facchini, Nat. Chem.
2199–2207. Biol., 2019, 15, 384–390.
49 H. Kato, T. Yoshida, T. Tokue, Y. Nojiri, H. Hirota, T. Ohta, 75 X. Luo, M. A. Reiter, L. d’Espaux, J. Wong, C. M. Denby,
R. M. Williams and S. Tsukamoto, Angew. Chem., Int. Ed., A. Lechner, Y. Zhang, A. T. Grzybowski, S. Harth, W. Lin,
2007, 46, 2254–2256. H. Lee, C. Yu, J. Shin, K. Deng, V. T. Benites, G. Wang,
50 S. M. Li, Nat. Prod. Rep., 2010, 27, 57–78. E. E. K. Baidoo, Y. Chen, I. Dev, C. J. Petzold and J. D.
51 C. T. Walsh, ACS Chem. Biol., 2014, 9, 2718–2728. Keasling, Nature, 2019, 567, 123–126.
View Article Online
76 S. Brown, M. Clastre, V. Courdavault and S. E. O’Connor, 99 R. S. Nett, W. Lau and E. S. Sattely, Nature, 2020, 584,
Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 3205–3210. 148–153.
77 S. Galanie, K. Thodey, I. J. Trenchard, M. F. Interrante and 100 P. H. Opgenorth, T. P. Korman and J. U. Bowie, Nat. Chem.
C. D. Smolke, Science, 2015, 349, 1095–1100. Biol., 2016, 12, 393–395.
78 W. C. Deloache, Z. N. Russ, L. Narcross, A. M. Gonzales, 101 T. P. Korman, P. H. Opgenorth and J. U. Bowie, Nat.
V. J. J. Martin and J. E. Dueber, Nat. Chem. Biol., 2015, 11, Commun., 2017, 8, 15526.
465–471. 102 M. A. Valliere, T. P. Korman, M. A. Arbing and J. U. Bowie,

79 M. S. Packer and D. R. Liu, Nat. Rev. Genet., 2015, 16, Nat. Chem. Biol., 2020, 16, 1427–1433.
379–394. 103 L. V. Roze, A. Chanda and J. E. Linz, Fungal Genet. Biol.,
80 N. Liu, S. Santala and G. Stephanopoulos, Curr. Opin. 2011, 48, 35–48.
Biotechnol., 2020, 62, 15–21. 104 J. E. Dueber, G. C. Wu, G. R. Malmirchegini, T. S. Moon,
81 A. M. Adams, N. A. Kaplan, Z. Wei, J. D. Brinton, C. S. C. J. Petzold, A. V. Ullal, K. L. J. Prather and J. D. Keasling,
Monnier, A. L. Enacopol, T. A. Ramelot and J. A. Jones, Nat. Biotechnol., 2009, 27, 753–759.
Metab. Eng., 2019, 56, 111–119. 105 S. K. Hammer and J. L. Avalos, Nat. Chem. Biol., 2017, 13,
82 J. M. Billingsley, A. B. DeNicola, J. S. Barber, M. C. Tang, 823–832.
J. Horecka, A. Chu, N. K. Garg and Y. Tang, Metab. Eng., 106 H. Renault, J. E. Bassard, B. Hamberger and D. Werck-
2017, 44, 117–125. Reichhart, Curr. Opin. Plant Biol., 2014, 19, 27–34.
83 W. B. Copeland, B. A. Bartley, D. Chandran, M. Galdzicki, 107 E. Leonard and M. A. G. Koffas, Appl. Environ. Microbiol.,
K. H. Kim, S. C. Sleight, C. D. Maranas and H. M. Sauro, 2007, 73, 7246–7251.
Metab. Eng., 2012, 14, 270–280. 108 Y. Ping, X. Li, B. Xu, W. Wei, W. Wei, G. Kai, Z. Zhou and
84 W. Finnigan, L. J. Hepworth, S. L. Flitsch and N. J. Turner, Y. Xiao, ACS Synth. Biol., 2019, 8, 257–263.
Nat. Catal., 2021, 4, 98–104. 109 K. M. Hawkins and C. D. Smolke, Nat. Chem. Biol., 2008, 4,
85 M. J. Sheppard, A. M. Kunjapur, S. J. Wenck and 564–573.
K. L. J. Prather, Nat. Commun., 2014, 5, 5031. 110 J. U. Bowie, S. Sherkhanov, T. P. Korman, M. A. Valliere,
86 B. O. Bachmann, Nat. Chem. Biol., 2010, 6, 390–393. P. H. Opgenorth and H. Liu, Trends Biotechnol., 2020, 38,
87 A. Casini, F. Y. Chang, R. Eluere, A. M. King, E. M. Young, 766–778.
Q. M. Dudley, A. Karim, K. Pratt, C. Bristol, A. Forget, 111 J. M. Clomburg, A. M. Crumbley and R. Gonzalez, Science,
A. Ghodasara, R. Warden-Rothman, R. Gan, A. Cristofaro, 2017, 355, aag0804.
A. E. Borujeni, M. H. Ryu, J. Li, Y. C. Kwon, H. Wang, 112 V. Kumar, A. Bhalla and A. S. Rathore, Biotechnol. Prog.,
E. Tatsis, C. Rodriguez-Lopez, S. O’Connor, M. H. Medema, 2014, 30, 86–99.
M. A. Fischbach, M. C. Jewett, C. Voigt and D. B. Gordon, 113 J. Gilman, L. Walls, L. Bandiera and F. Menolascina, ACS
J. Am. Chem. Soc., 2018, 140, 4302–4316. Synth. Biol., 2021, 10, 1–18.
88 P. Carbonell, R. Le Feuvre, E. Takano and N. S. Scrutton, 114 P.-F. Xia, H. Ling, J. L. Foo and M. W. Chang, Biotechnol.
Synth. Biol., 2020, 5, ysaa020. Adv., 2019, 37, 107393.
89 M. Li, Y. Sun, S. A. Pan, W. W. Deng, O. Yu and Z. Zhang, 115 M. N. Win, J. S. Klein and C. D. Smolke, Nucleic Acids Res.,
RSC Adv., 2017, 7, 56382–56389. 2006, 34, 5670–5682.
90 A. Nakagawa, E. Matsumura, T. Koyanagi, T. Katayama, 116 V. A. Portnoy, D. Bezdan and K. Zengler, Curr. Opin.
N. Kawano, K. Yoshimatsu, K. Yamamoto, H. Kumagai, Biotechnol., 2011, 22, 590–594.
F. Sato and H. Minami, Nat. Commun., 2016, 7, 10390. 117 D. E. Nichols, Pharmacol. Rev., 2016, 68, 264–355.
91 N. Milne, P. Thomsen, N. Mølgaard Knudsen, P. Rubaszka, 118 C. O’Neill-Dee, H. A. Spiller, M. J. Casavant, S. Kistamgari,
M. Kristensen and I. Borodina, Metab. Eng., 2020, 60, 25–36. T. Chounthirath, G. A. Smith, C. O’Neill-Dee, H. A. Spiller,
92 M. McKeague, Y. H. Wang, A. Cravens, M. N. Win and M. J. Casavant, S. Kistamgari, T. Chounthirath and
C. D. Smolke, Metab. Eng., 2016, 38, 191–203. G. A. Smith, Clin. Toxicol., 2020, 58, 813–820.
93 E. Skellam, Trends Biotechnol., 2019, 37, 416–427. 119 F. Tylš, T. Pálenı́ček and J. Horáček, Eur. Neuropsychophar-
94 S. Hoefgen, J. Lin, J. Fricke, M. C. Stroe, D. J. Mattern, macol., 2014, 24, 342–356.
J. E. Kufs, P. Hortschansky, A. A. Brakhage, D. Hoffmeister 120 A. L. Halberstadt, Behav. Brain Res., 2015, 277, 99–120.
and V. Valiante, Metab. Eng., 2018, 48, 44–51. 121 D. E. Nichols and C. D. Nichols, Chem. Rev., 2008, 108,
95 M. Ohashi, F. Liu, Y. Hai, M. Chen, M. Tang, Z. Yang, 1614–1641.
M. Sato, K. Watanabe, K. N. Houk and Y. Tang, Nature, 122 V. Beliveau, M. Ganz, L. Feng, B. Ozenne, L. Højgaard,
2017, 549, 502. P. M. Fisher, C. Svarer, D. N. Greve and G. M. Knudsen,
96 J. Reed, M. J. Stephenson, K. Miettinen, B. Brouwer, A. Leveau, J. Neurosci., 2017, 37, 120–128.
P. Brett, R. J. M. Goss, A. Goossens, M. A. O’Connell and 123 J. F. López-Giménez and J. González-Maeso, Current Topics
A. Osbourn, Metab. Eng., 2017, 42, 185–193. in Behavioral Neurosciences, Springer Verlag, 2017, vol. 36,
97 W. Lau and E. S. Sattely, Science, 2015, 349, 1224–1228. pp. 45–73.
98 T. Pluskal, M. P. Torrens-Spence, T. R. Fallon, A. De Abreu, 124 D. E. Nichols, Wiley Interdiscip. Rev.: Membr. Transp. Sig-
C. H. Shi and J. K. Weng, Nat. Plants, 2019, 5, 867–878. naling, 2012, 1, 559–579.
View Article Online
125 D. Wacker, S. Wang, J. D. McCorvy, R. M. Betz, A. J. 152 T. Wakimoto, Y. Egami, Y. Nakashima, Y. Wakimoto,
Venkatakrishnan, A. Levit, K. Lansu, Z. L. Schools, T. Che, T. Mori, T. Awakawa, T. Ito, H. Kenmoku, Y. Asakawa,
D. E. Nichols, B. K. Shoichet, R. O. Dror and B. L. Roth, Cell, J. Piel and I. Abe, Nat. Chem. Biol., 2014, 10, 648–655.
2017, 168, 377–389.e12. 153 S. Zhu, J. D. Hegemann, C. D. Fage, M. Zimmermann,
126 T. M. Carbonaro and M. B. Gatch, Brain Res. Bull., 2016, X. Xie, U. Linne and M. A. Marahiel, J. Biol. Chem., 2016,
126, 74–88. 291, 13662–13678.
127 M. J. Miller, J. Albarracin-Jordan, C. Moore and J. M. 154 H. T. Reynolds, V. Vijayakumar, E. Gluck-Thaler, H. B.

Capriles, Proc. Natl. Acad. Sci. U. S. A., 2019, 166, 11207–11212. Korotkin, P. B. Matheny and J. C. Slot, Evol. Lett., 2018, 2,
128 F. J. Carod-Artal, Neurol. (Engl. Ed.), 2015, 30, 42–49. 88–101.
129 A. T. Weil and W. Davis, J. Ethnopharmacol., 1994, 41, 1–8. 155 A. R. Awan, J. M. Winter, D. Turner, W. M. Shaw, L. M. Suz,
130 A. Most, Bufo alvarius: the psychedelic toad of the Sonoran A. J. Bradshaw, T. Ellis and B. T. M. Dentinger, bioRxiv,
Desert, Venom Press, 1984. 2018, 374199.
131 O. G. de Lima, Arq. Inst. Pesqui. Agron., 1946, 4, 45–80. 156 C. J. Paddon, P. J. Westfall, D. J. Pitera, K. Benjamin,
132 S. Szára, Experientia, 1956, 12, 441–442. K. Fisher, D. McPhee, M. D. Leavell, A. Tai, A. Main,
133 S. A. Barker, Front. Neurosci., 2018, 12, 536. D. Eng, D. R. Polichuk, K. H. Teoh, D. W. Reed, T. Treynor,
134 A. Szabo, A. Kovacs, J. Riba, S. Djurovic, E. Rajnavolgyi and J. Lenihan, H. Jiang, M. Fleck, S. Bajad, G. Dang, D. Dengrove,
E. Frecska, Front. Neurosci., 2016, 10, 423. D. Diola, G. Dorin, K. W. Ellens, S. Fickes, J. Galazzo,
135 L. P. Cameron, C. J. Benson, L. E. Dunlap and D. E. Olson, S. P. Gaucher, T. Geistlinger, R. Henry, M. Hepp, T. Horning,
ACS Chem. Neurosci., 2018, 9, 1582–1590. T. Iqbal, L. Kizer, B. Lieu, D. Melis, N. Moss, R. Regentin,
136 C. Ly, A. C. Greb, L. P. Cameron, J. M. Wong, E. V. S. Secrest, H. Tsuruta, R. Vazquez, L. F. Westblade, L. Xu,
Barragan, P. C. Wilson, K. F. Burbach, S. Soltanzadeh M. Yu, Y. Zhang, L. Zhao, J. Lievense, P. S. Covello, J. D.
Zarandi, A. Sood, M. R. Paddy, W. C. Duim, M. Y. Keasling, K. K. Reiling, N. S. Renninger and J. D. Newman,
Dennis, A. K. McAllister, K. M. Ori-McKenney, J. A. Gray Nature, 2013, 496, 528–532.
and D. E. Olson, Cell Rep., 2018, 23, 3170–3182. 157 S. Osamu, Y. Katsumata and O. Masakazu, Biochem. Phar-
137 P. Djura, D. B. Stierle, B. Sullivan, D. J. Faulkner, E. Arnold macol., 1981, 30, 1353–1358.
and J. Clardy, J. Org. Chem., 1980, 45, 1435–1441. 158 D. McKenna and J. Riba, Current Topics in Behavioral
138 A. Longeon, B. R. Copp, E. Quévrain, M. Roué, B. Kientz, Neurosciences, Springer Verlag, 2018, vol. 36, pp. 283–311.
T. Cresteil, S. Petek, C. Debitus and M. L. Bourguet- 159 E. A. Estrella-Parra, J. C. Almanza-Pérez and F. J. Alarcón-
Kondracki, Mar. Drugs, 2011, 9, 879–888. Aguilar, Nat. Products Bioprospect., 2019, 9, 251–265.
139 A. J. Kochanowska, K. V. Rao, S. Childress, A. El-Alfy, 160 F. Ayala Flores and W. H. Lewis, Econ. Bot., 1978, 32,
R. R. Matsumoto, M. Kelly, G. S. Stewart, K. J. Sufka and 154–156.
M. T. Hamann, J. Nat. Prod., 2008, 71, 186–189. 161 J. A. Morales-Garcı́a, M. De La Fuente Revenga, S. Alonso-
140 W. Lovenberg, H. Weissbach and S. Udenfriend, J. Biol. Gil, M. I. Rodrı́guez-Franco, A. Feilding, A. Perez-Castillo
Chem., 1962, 237, 89–93. and J. Riba, Sci. Rep., 2017, 7, 1–13.
141 J. Axelrod, Science, 1961, 134, 343. 162 J. C. Callaway, G. S. Brito and E. S. Neves, J. Psychoactive
142 M. A. Thompson and R. M. Weinshilboum, J. Biol. Chem., Drugs, 2005, 37, 145–150.
1998, 273, 34502–34510. 163 A. M. Brito-da-costa, D. Dias-da-silva, N. G. M. Gomes,
143 A. Hofmann, R. Heim, A. Brack, H. Kobel, A. Frey, H. Ott, R. J. Dinis-oliveira and Á. Madureira-carvalho, Pharmaceuticals,
T. Petrzilka and F. Troxler, Helv. Chim. Acta, 1959, 42, 2020, 13, 1–39.
1557–1572. 164 F. Blei, S. Dörner, J. Fricke, F. Baldeweg, F. Trottmann,
144 D. E. Nichols, J. Antibiot., 2020, 73, 679–686. A. Komor, F. Meyer, C. Hertweck and D. Hoffmeister, Chem. –
145 F. Hasler, D. Bourquin, R. Brenneisen and F. X. Vollenweider, Eur. J., 2020, 26, 729–734.
J. Pharm. Biomed. Anal., 2002, 30, 331–339. 165 K. Stolle and D. Gröger, Arch. Pharm., 1968, 301, 561–571.
146 F. Hasler, U. Grimberg, M. A. Benz, T. Huber and F. X. 166 L. Nettleship and M. Slaytor, Phytochemistry, 1974, 13, 735–742.
Vollenweider, Psychopharmacology, 2004, 172, 145–156. 167 P. M. Dewick, Medicinal Natural Products: A Biosynthetic
147 A. Mahapatra and R. Gupta, Ther. Adv. Psychopharmacol., Approach: Third Edition, Second., 2009.
2017, 7, 54–56. 168 D. E. Nichols, Pharmacol. Ther., 2004, 101, 131–181.
148 C. S. Grob, A. L. Danforth, G. S. Chopra, M. Hagerty, C. R. 169 N. Lorenz, T. Haarmann, S. Pažoutová, M. Jung and
McKay, A. L. Halberstad and G. R. Greer, Arch. Gen. Psychiatry, P. Tudzynski, Phytochemistry, 2009, 70, 1822–1832.
2011, 68, 71–78. 170 C. Wallwey and S. M. Li, Nat. Prod. Rep., 2011, 28, 496–510.
149 M. W. Johnson, A. Garcia-Romeu, M. P. Cosimano and 171 P. W. J. van Dongen and A. N. J. A. de Groot, Eur. J. Obstet.
R. R. Griffiths, J. Psychopharmacol., 2014, 28, 983–992. Gynecol. Reprod. Biol., 1995, 60, 109–116.
150 S. Agurell and J. L. Nilsson, Acta Chem. Scand., 1968, 22, 172 D. Jakubczyk, J. Z. Cheng and S. E. O’Connor, Nat. Prod.
1210–1218. Rep., 2014, 31, 1328–1338.
151 J. Fricke, F. Blei and D. Hoffmeister, Angew. Chem., Int. Ed., 173 N. Gerhards, L. Neubauer, P. Tudzynski and S. M. Li,
2017, 56, 12352–12355. Toxins, 2014, 6, 3281–3295.
View Article Online
174 N. Sharma, V. Sharma, H. Manikyam and A. Krishna, 201 S. L. Robinson and D. G. Panaccione, Appl. Environ. Micro-
European J. Med. Plants, 2016, 14, 1–17. biol., 2014, 80, 6465–6472.
175 D. Gröcer and H. G. Floss, Alkaloids Chem. Biol., 1998, 50, 202 C. L. Schardl, D. G. Panaccione and P. Tudzynski, Alkaloids
171–218. Chem. Biol., 2006, 63, 45–86.
176 S. L. Lee, H. G. Floss and P. Heinstein, Arch. Biochem. 203 J. M. Schwab and B. S. Henderson, Chem. Rev., 1990, 90,
Biophys., 1976, 177, 84–94. 1203–1245.
177 W. B. Yin, A. Grundmann, J. Cheng and S. M. Li, J. Biol. 204 J.-J. J. Chen, M.-Y. Y. Han, T. Gong, J.-L. L. Yang and P. Zhu,
Chem., 2009, 284, 100–109. Recent progress in ergot alkaloid research, Royal Society of
178 J. Winkelblech and S. M. Li, ChemBioChem, 2014, 15, Chemistry, 2017, vol. 7.
1030–1039. 205 J. G. Bruhn, J. E. Lindgren, B. Holmstedt and J. M.
179 A. W. Schultz, C. A. Lewis, M. R. Luzung, P. S. Baran and Adovasio, Science, 1978, 199, 1437–1438.
B. S. Moore, J. Nat. Prod., 2010, 73, 373–377. 206 U. Braun and D. Kalbhen, Pharmacology, 1973, 9, 312–316.
180 X. Yu, Y. Liu, X. Xie, X. D. Zheng and S. M. Li, J. Biol. Chem., 207 U. Stein, H. Greyer and H. Hentschel, Forensic Sci. Int.,
2012, 287, 1371–1380. 2001, 118, 87–90.
181 A. Fan, H. Chen, R. Wu, H. Xu and S. M. Li, Appl. Microbiol. 208 A. T. Shulgin, Nature, 1964, 201, 1120–1121.
Biotechnol., 2014, 98, 10119–10129. 209 A. T. Shulgin and A. Shulgin, Pihkal: a chemical love story,
182 S. M. Li, Appl. Microbiol. Biotechnol., 2009, 84, 631–639. Transform Press, Berkeley, CA, 1991.
183 T. Mori, J. Nat. Med., 2020, 74, 501–512. 210 A. T. Shulgin and A. Shulgin, Tihkal: the continuation,
184 P. Tudzynski, K. Hölter, T. Correia, C. Arntz, N. Grammel Transform Press, Berkeley, CA, 1997.
and U. Keller, Mol. Gen. Genet., 1999, 261, 133–141. 211 J. Axelrod and R. Tomchick, J. Biol. Chem., 1958, 233, 702–705.
185 T. Haarmann, C. Machado, Y. Lübbe, T. Correia, C. L. 212 I. J. Kopin, Catecholamines, Springer Berlin Heidelberg,
Schardl, D. G. Panaccione and P. Tudzynski, Phytochemistry, Berlin, Heidelberg, 1972, pp. 270–282.
2005, 66, 1312–1320. 213 F. Benington and R. D. Morin, Experientia, 1968, 24, 33–34.
186 O. Rigbers and S. M. Li, J. Biol. Chem., 2008, 283, 26859–26868. 214 A. J. Friedhoff, J. W. Schweitzer and J. Miller, Nature, 1972,
187 K. E. Goetz, C. M. Coyle, J. Z. Cheng, S. E. O’Connor and 237, 454–455.
D. G. Panaccione, Curr. Genet., 2011, 57, 201–211. 215 S. Agurell, J. Lundström and F. Sandberg, Tetrahedron Lett.,
188 N. Lorenz, J. Olšovská, M. Šulc and P. Tudzynski, Appl. 1967, 8, 2433–2435.
Environ. Microbiol., 2010, 76, 1822–1830. 216 G. J. Kapadia, Y. N. Vaishnav and M. B. E. Fayez, J. Pharm.
189 K. L. Ryan, C. T. Moore and D. G. Panaccione, Toxins, 2013, Sci., 1969, 58, 1157–1159.
5, 445–455. 217 J. Lundström and S. Agurell, Tetrahedron Lett., 1969, 10,
190 C. A. Nielsen, C. Folly, A. Hatsch, A. Molt, H. Schröder, 3371–3374.
S. E. O’Connor and M. Naesby, Microb. Cell Fact., 2014, 218 J. Lundström, Acta Pharm. Suec., 1970, 7, 651–666.
13, 95. 219 J. Lundström, Acta Chem. Scand., 1971, 25, 3489–3499.
191 K. L. Chen, C. Y. Lai, M. T. Pham, R. J. Chein, Y. Tang and 220 E. Ibarra-Laclette, F. Zamudio-Hernández, C. A. Pérez-
H. C. Lin, Org. Lett., 2020, 22, 3302–3306. Torres, V. A. Albert, E. Ramı́rez-Chávez, J. Molina-Torres,
192 Y. Yao, C. An, D. Evans, W. Liu, W. Wang, G. Wei, N. Ding, A. Fernández-Cortes, C. Calderón-Vázquez, J. L. Olivares-
K. N. Houk and S. S. Gao, J. Am. Chem. Soc., 2019, 141, Romero, A. Herrera-Estrella and L. Herrera-Estrella, BMC
17517–17521. Genomics, 2015, 16, 657.
193 Y. Iijima, D. R. Gang, E. Fridman, E. Lewinsohn and 221 J. Lundström, Acta Chem. Scand., 1971, 25, 3489–3499.
E. Pichersky, Plant Physiol., 2004, 134, 370–379. 222 P. Krogsgaard-Larsen, H. Hjeds, D. R. Curtis, D. Lodge and
194 C. Wallwey, C. Heddergott, X. Xie, A. A. Brakhage and G. A. R. Johnston, J. Neurochem., 1979, 32, 1717–1724.
S. M. Li, Microbiology, 2012, 158, 1634–1644. 223 D. Chandra, L. M. Halonen, A. M. Linden, C. Procaccini,
195 C. M. Coyle, J. Z. Cheng, S. E. O’Connor and D. G. K. Hellsten, G. E. Homanics and E. R. Korpi, Neuropsycho-
Panaccione, Appl. Environ. Microbiol., 2010, 76, 3898–3903. pharmacology, 2010, 35, 999–1007.
196 J. Z. Cheng, C. M. Coyle, D. G. Panaccione and S. E. 224 P. Winn, A. Tarbuck and S. B. Dunnett, Neuroscience, 1984,
O’Connor, J. Am. Chem. Soc., 2010, 132, 12835–12837. 12, 225–240.
197 D. Jakubczyk, L. Caputi, A. Hatsch, C. A. F. Nielsen, 225 K. Tsujikawa, K. Kuwayama, H. Miyaguchi, T. Kanamori,
M. Diefenbacher, J. Klein, A. Molt, H. Schröder, J. Z. Y. Iwata, H. Inoue, T. Yoshida and T. Kishi, J. Chromatogr.
Cheng, M. Naesby and S. E. O’Connor, Angew. Chem., B: Anal. Technol. Biomed. Life Sci., 2007, 852, 430–435.
2015, 127, 5206–5210. 226 K. Stebelska, Ther. Drug Monit., 2013, 35, 420–442.
198 M. Matuschek, C. Wallwey, X. Xie and S. M. Li, Org. Biomol. 227 T. Takemoto, T. Nakajima and T. Yokobe, J. Pharm. Soc.
Chem., 2011, 9, 4328–4335. Jpn., 1964, 84, 1232–1233.
199 W. Maier, B. Schumann and D. Gröger, J. Basic Microbiol., 228 S. Obermaier and M. Müller, Angew. Chem., Int. Ed., 2020,
1988, 28, 83–93. 59, 12432–12435.
200 T. Haarmann, I. Ortel, P. Tudzynski and U. Keller, Chem- 229 K. R. Alper, Alkaloids: Chemistry and Biology, Academic
BioChem, 2006, 7, 645–652. Press, 2001, vol. 56, pp. 1–38.
View Article Online
230 S. E. O’Connor and J. J. Maresh, Nat. Prod. Rep., 2006, 250 J. Murata, J. Roepke, H. Gordon and V. De Luca, Plant Cell,
23, 532. 2008, 20, 524–542.
231 Q. Pan, N. R. Mustafa, K. Tang, Y. H. Choi and R. Verpoorte, 251 G. Guirimand, A. Guihur, O. Ginis, P. Poutrain, F. Héricourt,
Phytochem. Rev., 2016, 15, 221–250. A. Oudin, A. Lanoue, B. St-Pierre, V. Burlat and V. Courdavault,
232 T. P. Chierrito, A. C. Aguiar, I. M. De Andrade, I. P. Ceravolo, FEBS J., 2011, 278, 749–763.
R. A. Gonçalves, A. J. De Oliveira and A. U. Krettli, Malar. J., 252 V. Courdavault, N. Papon, M. Clastre, N. Giglioli-Guivarc’h,
2014, 13, 142. B. St-Pierre and V. Burlat, Curr. Opin. Plant Biol., 2014, 19,
233 R. MačiIulaitis, V. Kontrimavičiute, F. M. M. Bressolle and 43–50.
V. Briedis, Hum. Exp. Toxicol., 2008, 27, 181–194. 253 B. Larsen, V. L. Fuller, J. Pollier, A. Van Moerkercke,
234 S. Li, J. Long, Z. Ma, Z. Xu, J. Li and Z. Zhang, Curr. Med. F. Schweizer, R. Payne, M. Colinas, S. E. O’Connor,
Res. Opin., 2004, 20, 409–415. A. Goossens and B. A. Halkier, Plant Cell Physiol., 2017,
235 K. R. Alper, H. S. Lotsof and C. D. Kaplan, J. Ethnopharmacol., 58, 1507–1518.
2008, 115, 9–24. 254 D. Bracher and T. M. Kutchan, Arch. Biochem. Biophys.,
236 Y. Qu, M. E. A. M. Easson, R. Simionescu, J. Hajicek, 1992, 294, 717–723.
A. M. K. Thamm, V. Salim and V. De Luca, Proc. Natl. 255 A. R. Battersby, Alkaloids, 2007, vol. 1, pp. 31–47.
Acad. Sci. U. S. A., 2018, 115, 3180–3185. 256 E. McCoy and S. E. O’Connor, J. Am. Chem. Soc., 2006, 128,
237 L. Caputi, J. Franke, S. C. Farrow, K. Chung, R. M. E. Payne, 14276–14277.
T. D. Nguyen, T. T. T. Dang, I. S. Teto Carqueijeiro, 257 P. Bernhardt, E. McCoy and S. E. O’Connor, Chem. Biol.,
K. Koudounas, T. D. De Bernonville, B. Ameyaw, D. M. 2007, 14, 888–897.
Jones, I. J. Curcino Vieira, V. Courdavault and S. E. O’Connor, 258 W. Runguphan, X. Qu and S. E. O’Connor, Nature, 2010,
Science, 2018, 360, 1235–1239. 468, 461–467.
238 V. Salim, F. Yu, J. Altarejos and V. De Luca, Plant J., 2013, 259 R. M. E. Payne, D. Xu, E. Foureau, M. I. S. Teto Carquei-
76, 754–765. jeiro, A. Oudin, T. D. De Bernonville, V. Novak, M. Burow,
239 K. Asada, V. Salim, S. Masada-Atsumi, E. Edmunds, C. E. Olsen, D. M. Jones, E. C. Tatsis, A. Pendle,
M. Nagatoshi, K. Terasaka, H. Mizukami and V. De Luca, B. A. Halkier, F. Geu-Flores, V. Courdavault, H. H. Nour-
Plant Cell, 2013, 25, 4123–4134. Eldin and S. E. O’Connor, Nat. Plants, 2017, 3, 16208.
240 Y. Qu, M. L. A. E. Easson, J. Froese, R. Simionescu, 260 G. Guirimand, V. Courdavault, A. Lanoue, S. Mahroug,
T. Hudlicky and V. DeLuca, Proc. Natl. Acad. Sci. U. S. A., A. Guihur, N. Blanc, N. Giglioli-Guivarc’h, B. St-Pierre and
2015, 112, 6224–6229. V. Burlat, BMC Plant Biol., 2010, 10, 182.
241 Y. Qu, A. M. K. Thamm, M. Czerwinski, S. Masada, K. H. 261 K. Konno, C. Hirayama, H. Yasui and M. Nakamura, Proc.
Kim, G. Jones, P. Liang and V. De Luca, Planta, 2018, 247, Natl. Acad. Sci. U. S. A., 1999, 96, 9159–9164.
625–634. 262 T. J. C. Luijendijk, L. H. Stevens and R. Verpoorte, Plant
242 E. C. Tatsis, I. Carqueijeiro, T. Dugé De Bernonville, J. Franke, Physiol. Biochem., 1998, 36, 419–425.
T. T. T. Dang, A. Oudin, A. Lanoue, F. Lafontaine, A. K. 263 A. Geerlings, M. Martinez-Lozano Ibañez, J. Memelink,
Stavrinides, M. Clastre, V. Courdavault and S. E. O’Connor, R. Van Der Heijden and R. Verpoorte, J. Biol. Chem.,
Nat. Commun., 2017, 8, 1–10. 2000, 275, 3051–3056.
243 B. R. Lichman, G. T. Godden, J. P. Hamilton, L. Palmer, 264 I. Gerasimenko, Y. Sheludko, X. Ma and J. Stöckigt, Eur.
M. O. Kamileen, D. Zhao, B. Vaillancourt, J. C. Wood, J. Biochem., 2002, 269, 2204–2213.
M. Sun, T. J. Kinser, L. K. Henry, C. Rodriguez-Lopez, 265 S. C. Farrow, M. O. Kamileen, J. Meades, B. Ameyaw, Y. Xiao
N. Dudareva, D. E. Soltis, P. S. Soltis, C. Robin Buell and and S. E. O’Connor, J. Biol. Chem., 2018, 293, 13821–13833.
S. E. O’Connor, Sci. Adv., 2020, 6, eaba0721. 266 L. Caputi, J. Franke, K. Bussey, S. C. Farrow, I. J. C. Vieira,
244 S. C. Farrow, M. O. Kamileen, L. Caputi, K. Bussey, J. E. A. C. E. M. Stevenson, D. M. Lawson and S. E. O’Connor, Nat.
Mundy, R. C. McAtee, C. R. J. Stephenson and S. E. O’Connor, Chem. Biol., 2020, 16, 383–386.
J. Am. Chem. Soc., 2019, 141, 12979–12983. 267 Z. J. Zhan, Q. Yu, Z. L. Wang and W. G. Shan, Bioorg. Med.
245 R. Croteau, Chem. Rev., 1987, 87, 929–954. Chem. Lett., 2010, 20, 6185–6187.
246 H. J. Bouwmeester, J. Gershenzon, M. C. J. M. Konings and 268 D. Steyer, C. Erny, P. Claudel, G. Riveill, F. Karst and
R. Croteau, Plant Physiol., 1998, 117, 901–912. J. L. Legras, Food Microbiol., 2013, 33, 228–234.
247 G. Collu, N. Unver, A. M. G. Peltenburg-Looman, R. Van der 269 A. Ortega, J. F. Blount and P. S. Manchand, J. Chem. Soc.,
Heijden, R. Verpoorte and J. Memelink, FEBS Lett., 2001, Perkin Trans. 1, 1982, 2505.
508, 215–220. 270 L. J. Valdes, W. M. Butler, G. M. Hatfield, A. G. Paul and
248 F. Geu-Flores, N. H. Sherden, W. S. Glenn, S. E. O’connor, M. Koreeda, J. Org. Chem., 1984, 49, 4716–4720.
V. Courdavault, V. Burlat, E. Nims, C. Wu and Y. Cui, 271 D. J. Siebert, J. Ethnopharmacol., 1994, 43, 53–56.
Nature, 2012, 492, 138–142. 272 T. E. Prisinzano, Life Sci., 2005, 78, 527–531.
249 B. R. Lichman, M. O. Kamileen, G. R. Titchiner, G. Saalbach, 273 U. Coffeen and F. Pellicer, J. Pain Res., 2019, 12, 1069–1076.
C. E. M. Stevenson, D. M. Lawson and S. E. O’Connor, Nat. 274 B. M. Kivell, A. W. M. Ewald and T. E. Prisinzano, Advances
Chem. Biol., 2019, 15, 71–79. in Pharmacology, 2014, vol. 69, pp. 481–511.
View Article Online
275 F. Yan, P. D. Mosier, R. B. Westkaemper, J. Stewart, J. K. 304 S. Ogita, H. Uefuji, Y. Yamaguchi, K. Nozomu and H. Sano,
Zjawiony, T. A. Vortherms, D. J. Sheffler and B. L. Roth, Nature, 2003, 423, 823.
Biochemistry, 2005, 44, 8643–8651. 305 C. Lu, K. M. Warchol and R. A. Callahan, Bull. Insectol.,
276 A. Gupta, I. Gomes, E. N. Bobeck, A. K. Fakira, N. P. 2014, 67, 125–130.
Massaro, I. Sharma, A. Cavé, H. E. Hamm, J. Parello, 306 N. Tsvetkov, O. Samson-Robert, K. Sood, H. S. Patel,
L. A. Devi and S. H. Snyder, Proc. Natl. Acad. Sci. U. S. A., D. A. Malena, P. H. Gajiwala, P. Maciukiewicz, V. Fournier
2016, 113, 6041–6046. and A. Zayed, Science, 2017, 356, 1395–1397.

277 D. J. Siebert, Ann. Bot., 2004, 93, 763–771. 307 B. Siegmund, E. Leitner and W. Pfannhauser, J. Agric. Food
278 L. Kutrzeba, F. E. Dayan, J. Howell, J. Feng, J. L. Giner and Chem., 1999, 47, 3113–3120.
J. K. Zjawiony, Phytochemistry, 2007, 68, 1872–1881. 308 R. S. Lewis, K. E. Drake-Stowe, C. Heim, T. Steede, W. Smith
279 X. Chen, A. Berim, F. E. Dayan and D. R. Gang, J. Exp. Bot., and R. E. Dewey, Front. Plant Sci., 2020, 11, 368.
2017, 68, 1109–1122. 309 N. L. Benowitz, Annu. Rev. Pharmacol. Toxicol., 2009, 49,
280 K. A. Pelot, R. Mitchell, M. Kwon, D. M. Hagelthorn, J. F. 57–71.
Wardman, A. Chiang, J. Bohlmann, D. K. Ro and P. Zerbe, 310 V. E. Moran, Front. Pharmacol., 2012, 3, 173.
Plant J., 2017, 89, 885–897. 311 A. Pictet and A. Rotschy, Berichte der Dtsch. Chem.
281 N. Nunn and N. Qian, J. Econ. Perspect., 2010, 24, 163–188. Gesellschaft, 1904, 37, 1225–1235.
282 J. J. DeVido, Absolute Addiction Psychiatry Review, 2020, 312 H. Weidel, Justus Liebigs Ann. Chem., 1873, 165,
pp. 185–203. 328–349.
283 H. Ashihara and T. Suzuki, Front. Biosci., 2004, 9, 1864–1876. 313 J. Z. Cheng, C. M. Coyle, D. G. Panaccione and S. E. O’Connor,
284 J. A. Nathanson, Science, 1984, 226, 184–187. J. Am. Chem. Soc., 2010, 132, 12835–12837.
285 C. H. Chou and G. R. Waller, J. Chem. Ecol., 1980, 6, 643–654. 314 B. L. Lamberts, L. J. Dewey and R. U. Byerrum, BBA,
286 G. A. Wright, D. D. Baker, M. J. Palmer, D. Stabler, J. A. Biochim. Biophys. Acta, 1959, 33, 22–26.
Mustard, E. F. Power, A. M. Borland and P. C. Stevenson, 315 E. Leete, Alkaloids: chemical and biological perspectives, The
Science, 2013, 339, 1202–1204. Society, 1983, vol. 1, pp. 85–151.
287 B. Carpenter and G. Lebon, Front. Pharmacol., 2017, 8, 898. 316 N. J. Walton, R. J. Robins and M. J. C. Rhodes, Plant Sci.,
288 S. R. Waldvogel, Angew. Chem., Int. Ed., 2003, 42, 604–605. 1988, 54, 125–131.
289 E. Fischer, Berichte der Dtsch. Chem. Gesellschaft, 1898, 31, 317 E. Leete and J. A. McDonell, J. Am. Chem. Soc., 1981, 103,
3266–3277. 658–662.
290 L. Anderson and M. Gibbs, J. Biol. Chem., 1962, 237, 318 S. Mizusaki, T. Kisaki and E. Tamaki, Plant Physiol., 1968,
1941–1944. 43, 93–98.
291 O. Negishi, T. Ozawa and H. Imagawa, Agric. Biol. Chem., 319 S. Mizusaki, Y. Tanabe, M. Noguchi and E. Tamaki, Plant
1985, 49, 887–890. Cell Physiol., 1971, 12, 633–640.
292 O. Negishi, T. Ozawa and H. Imagawa, Agric. Biol. Chem., 320 S. Mizusaki, Y. Tanabe, M. Noguchi and E. Tamaki, Phy-
1988, 52, 169–175. tochemistry, 1972, 11, 2757–2762.
293 M. Kato, K. Mizuno, T. Fujimura, M. Iwama, M. Irie, A. Crozier 321 N. Hibi, S. Higashiguchi, T. Hashimoto and Y. Yamada,
and H. Ashihara, Plant Physiol., 1999, 120, 579–586. Plant Cell, 1994, 6, 723–735.
294 M. Kato, K. Mizuno, A. Crozier, T. Fujimura and 322 S. Biastoff, N. Reinhardt, V. Reva, W. Brandt and B. Dräger,
H. Ashihara, Nature, 2000, 406, 956–957. FEBS Lett., 2009, 583, 3367–3374.
295 K. Mizuno, M. Kato, F. Irino, N. Yoneyama, T. Fujimura 323 S. Moriyama, H. Tanaka, M. Uwataki, M. Muguruma and
and H. Ashihara, FEBS Lett., 2003, 547, 56–60. K. Ohta, J. Biosci. Bioeng., 2003, 96, 324–331.
296 H. Ashihara, A. M. Monteiro, F. M. Gillies and A. Crozier, 324 J. Brent Friesen and E. Leete, Tetrahedron Lett., 1990, 31,
Plant Physiol., 1996, 111, 747–753. 6295–6298.
297 O. Negishi, T. Ozawa and H. Imagawa, Biosci., Biotechnol., 325 M. Kajikawa, N. Hirai and T. Hashimoto, Plant Mol. Biol.,
Biochem., 1992, 56, 499–503. 2009, 69, 287–298.
298 K. Mizuno, A. Okuda, M. Kato, N. Yoneyama, H. Tanaka, 326 M. Kajikawa, T. Shoji, A. Kato and T. Hashimoto, Plant
H. Ashihara and T. Fujimura, FEBS Lett., 2003, 534, 75–81. Physiol., 2011, 155, 2010–2022.
299 M. Ogawa, Y. Herai, N. Koizumi, T. Kusano and H. Sano, 327 M.-C. Goulet, L. Gaudreau, M. Gagné, A.-M. Maltais, A.-C.
J. Biol. Chem., 2001, 276, 8213–8218. Laliberté, G. Éthier, N. Bechtold, M. Martel, M.-A. D’Aoust,
300 R. Huang, A. J. O’Donnell, J. J. Barboline and T. J. Barkman, A. Gosselin, S. Pepin and D. Michaud, Front. Plant Sci., 2019,
Proc. Natl. Acad. Sci. U. S. A., 2016, 113, 10613–10618. 10, 735.
301 C. Koshiishi, A. Kato, S. Yama, A. Crozier and H. Ashihara, 328 S. Romanowski and A. S. Eustáquio, Curr. Opin. Chem.
FEBS Lett., 2001, 499, 50–54. Biol., 2020, 58, 137–145.
302 S. S. M. Waldhauser and T. W. Baumann, Phytochemistry, 329 F. J. Molina-Hidalgo, M. Vazquez-Vilar, L. D’Andrea, O. C.
1996, 42, 985–996. Demurtas, P. Fraser, G. Giuliano, R. Bock, D. Orzáez and
303 A. Kato, A. Crozier and H. Ashihara, Phytochemistry, 1998, A. Goossens, Trends Biotechnol., DOI: 10.1016/j.tibtech.2020.
48, 777–779. 11.012.
View Article Online
330 R. S. Lewis, A. M. Jack, J. W. Morris, V. J. M. Robert, 356 S. Lal, N. Prasad, M. Ryan, S. Tangri, M. S. Silverberg,
L. B. Gavilano, B. Siminszky, L. P. Bush, A. J. Hayes and A. Gordon and H. Steinhart, Eur. J. Gastroenterol. Hepatol.,
R. E. Dewey, Plant Biotechnol. J., 2008, 6, 346–354. 2011, 23, 891–896.
331 J. Schachtsiek and F. Stehle, Plant Biotechnol. J., 2019, 17, 357 J. Fiz, M. Durán, D. Capellà, J. Carbonell and M. Farré,
2228–2230. PLoS One, 2011, 6, e18440.
332 United Nations, World Drug Report 2018, 2018. 358 D. I. Abrams and M. Guzman, Clin. Pharmacol. Ther., 2015,
333 L. S. Walker and B. Mezuk, BMC Int. Health Hum. Rights, 97, 575–586.
2018, 18, 43. 359 J. Sarris, J. Sinclair, D. Karamacoska, M. Davidson and
334 E. Nestler, Sci. Pract. Perspect., 2005, 3, 4–10. J. Firth, BMC Psychiatry, 2020, 20, 24.
335 A. J. Humphrey and D. O’Hagan, Nat. Prod. Rep., 2001, 18, 360 J. Corey-Bloom, T. Wolfson, A. Gamst, S. Jin, T. D. Marcotte,
494–502. H. Bentley and B. Gouaux, Can. Med. Assoc. J., 2012, 184,
336 E. Leete, L. Marion and I. D. Spenser, Nature, 1954, 174, 1143–1150.
650–651. 361 K. R. Müller-Vahl, H. Kolbe, U. Schneider and H. M. Emrich,
337 R. Duran-Patron, D. O’Hagan, J. T. G. Hamilton and Complement. Med. Res., 1999, 6(suppl 3), 23–27.
C. W. Wong, Phytochemistry, 2000, 53, 777–784. 362 D. Wei, D. Dinh, D. Lee, D. Li, A. Anguren, G. Moreno-Sanz,
338 M. A. Bedewitz, A. D. Jones, J. C. D’Auria and C. S. Barry, C. M. Gall and D. Piomelli, Cannabis Cannabinoid Res.,
Nat. Commun., 2018, 9, 5281. 2016, 1, 81–89.
339 J.-P. Huang, C. Fang, X. Ma, L. Wang, J. Yang, J. Luo, 363 R. Solomon, Cannabis Cannabinoid Res., 2020, 5, 2–5.
Y. Yan, Y. Zhang and S.-X. Huang, Nat. Commun., 2019, 364 E. B. Russo, Chem. Biodiversity, 2007, 4, 1614–1648.
10, 4036. 365 N. E. Sharpless, Remarks by Dr. Sharpless at the FDA Public
340 B. R. Lichman, Nat. Prod. Rep., 2021, 38, 103–129. Hearing on Scientific Data and Information about Products
341 B. Dräger, Phytochemistry, 2006, 67, 327–337. Containing Cannabis or Cannabis-Derived Compounds,
342 E. Gonzales-Vigil, E. Góngora-Castillo, J. B. Uebler, White Oak, MD, 2019.
E. Gonzales-Vigil, K. E. Wiegert-Rininger, K. L. Childs, 366 Y. Gaoni and R. Mechoulam, J. Am. Chem. Soc., 1971, 93,
J. P. Hamilton, B. Vaillancourt, Y. S. Yeo, J. Chappell, D. P. 217–224.
Della, A. Daniel Jones, C. Robin Buell and C. S. Barry, Plant 367 R. Mechoulam and Y. Shvo, Tetrahedron, 1963, 19,
Cell, 2014, 26, 3745–3762. 2073–2078.
343 D. E. Nichols and C. D. Nichols, Chem. Rev., 2008, 108, 368 R. J. M. Niesink and M. W. van Laar, Front. Psychiatry,
1614–1641. 2013, 4, 130.
344 F. Qiu, J. Zeng, J. Wang, J. P. Huang, W. Zhou, C. Yang, 369 L. De Petrocellis, A. Ligresti, A. S. Moriello, M. Allarà,
X. Lan, M. Chen, S. X. Huang, G. Kai and Z. Liao, New T. Bisogno, S. Petrosino, C. G. Stott and V. Di Marzo, Br.
Phytol., 2020, 225, 1906–1914. J. Pharmacol., 2011, 163, 1479–1494.
345 R. Li, D. W. Reed, E. Liu, J. Nowak, L. E. Pelcher, J. E. Page 370 R. B. Laprairie, A. M. Bagher, M. E. M. Kelly and E. M.
and P. S. Covello, Chem. Biol., 2006, 13, 513–520. Denovan-Wright, Br. J. Pharmacol., 2015, 172, 4790–4805.
346 P. Nasomjai, D. W. Reed, D. J. Tozer, M. J. G. Peach, 371 A. Thomas, G. L. Baillie, A. M. Phillips, R. K. Razdan,
A. M. Z. Slawin, P. S. Covello and D. O’Hagan, ChemBio- R. A. Ross and R. G. Pertwee, Br. J. Pharmacol., 2007, 150,
Chem, 2009, 10, 2382–2393. 613–623.
347 E. Leete, J. A. Bjorklund, M. M. Couladis and S. H. Kim, 372 E. M. Rock, D. Bolognini, C. L. Limebeer, M. G. Cascio,
J. Am. Chem. Soc., 1991, 113, 9286–9292. S. Anavi-Goffer, P. J. Fletcher, R. Mechoulam, R. G. Pertwee
348 J. Jirschitzka, G. W. Schmidt, M. Reichelt, B. Schneider, and L. A. Parker, Br. J. Pharmacol., 2012, 165, 2620–2634.
J. Gershenzon and J. C. D’Auria, Proc. Natl. Acad. Sci. U. S. 373 E. B. Russo, A. Burnett, B. Hall and K. K. Parker, Neuro-
A., 2012, 109, 10304–10309. chem. Res., 2005, 30, 1037–1043.
349 G. W. Schmidt, J. Jirschitzka, T. Porta, M. Reichelt, K. Luck, 374 S. Yamaori, M. Kushihara, I. Yamamoto and K. Watanabe,
J. C. P. Torre, F. Dolke, E. Varesio, G. Hopfgartner, J. Gershenzon Biochem. Pharmacol., 2010, 79, 1691–1698.
and J. C. D’auria, Plant Physiol., 2015, 167, 89–101. 375 L. De Petrocellis, V. Vellani, A. Schiano-Moriello, P. Marini,
350 Y. Ping, X. Li, W. You, G. Li, M. Yang, W. Wei, Z. Zhou and P. C. Magherini, P. Orlando and V. Di Marzo, J. Pharmacol.
Y. Xiao, ACS Synth. Biol., 2019, 8, 1257–1262. Exp. Ther., 2008, 325, 1007–1015.
351 P. Srinivasan and C. D. Smolke, Nat. Commun., 2019, 376 G. T. DeLong, C. E. Wolf, A. Poklis and A. H. Lichtman,
10, 3634. Drug Alcohol Depend., 2010, 112, 126–133.
352 I. Wheeldon, S. D. Minteer, S. Banta, S. C. Barton, 377 L. O. Hanuš, S. M. Meyer, E. Muñoz, O. Taglialatela-Scafati
P. Atanassov and M. Sigman, Nat. Chem., 2016, 8, 299–309. and G. Appendino, Nat. Prod. Rep., 2016, 33, 1357–1392.
353 W. C. DeLoache, Z. N. Russ and J. E. Dueber, Nat. Commun., 378 Y. Gaoni and R. Mechoulam, Proc. Chem. Soc., 1964, 73.
2016, 7, 1–11. 379 M. G. Cascio, L. A. Gauson, L. A. Stevenson, R. A. Ross and
354 E. B. Russo, Chem. Biodiversity, 2007, 4, 1614–1648. R. G. Pertwee, Br. J. Pharmacol., 2010, 159, 129–141.
355 C. A. Press, K. G. Knupp and K. E. Chapman, Epilepsy 380 L. Fuhr, M. Rousseau, A. Plauth, F. C. Schroeder and
Behav., 2015, 45, 49–52. S. Sauer, J. Nat. Prod., 2015, 78, 1160–1164.
View Article Online
381 A. Chicca, M. A. Schafroth, I. Reynoso-Moreno, R. Erni, 406 F. Taura, S. Sirikantaramas, Y. Shoyama, K. Yoshikai,
V. Petrucci, E. M. Carreira and J. Gertsch, Sci. Adv., 2018, Y. Shoyama and S. Morimoto, FEBS Lett., 2007, 581, 2929–2934.
4, eaat2166. 407 S. Morimoto, K. Komatsu, F. Taura and Y. Shoyama,
382 I. Muhammad, X.-C. Li, M. R. Jacob, B. L. Tekwani, D. C. Phytochemistry, 1998, 49, 1525–1529.
Dunbar and D. Ferreira, J. Nat. Prod., 2003, 66, 804–809. 408 Y. Shoyama, T. Tamada, K. Kurihara, A. Takeuchi, F. Taura,
383 K. Quaghebeur, J. Coosemans, S. Toppet and F. Compernolle, S. Arai, M. Blaber, Y. Shoyama, S. Morimoto and R. Kuroki,
Phytochemistry, 1994, 37, 159–161. J. Mol. Biol., 2012, 423, 96–105.

384 L. A. Matsuda, S. J. Lolait, M. J. Brownstein, A. C. Young 409 B. Zirpel, O. Kayser and F. Stehle, J. Biotechnol., 2018, 284,
and T. I. Bonner, Nature, 1990, 346, 561–564. 17–26.
385 S. Munro, K. L. Thomas and M. Abu-Shaar, Nature, 1993, 410 C. S. Jamieson, M. Ohashi, F. Liu, Y. Tang and K. N. Houk,
365, 61–65. Nat. Prod. Rep., 2019, 36, 698–713.
386 R. Mechoulam, N. Lander, T. H. Varkony, I. Kimmel, 411 L. Gao, C. Su, X. Du, R. Wang, S. Chen, Y. Zhou, C. Liu, X. Liu,
O. Becker, Z. Ben-Zvi, H. Edery and G. Porath, J. Med. R. Tian, L. Zhang, K. Xie, S. Chen, Q. Guo, L. Guo, Y. Hano,
Chem., 1980, 23, 1068–1072. M. Shimazaki, A. Minami, H. Oikawa, N. Huang, K. N. Houk,
387 A. C. Howlett, Prostaglandins Other Lipid Mediat., 2002, 68– L. Huang, J. Dai and X. Lei, Nat. Chem., 2020, 12, 620–628.
69, 619–631. 412 M. Ohashi, C. S. Jamieson, Y. Cai, D. Tan, D. Kanayama,
388 R. G. Pertwee, A. C. Howlett, M. E. Abood, S. P. H. M.-C. Tang, S. M. Anthony, J. V. Chari, J. S. Barber, E. Picazo,
Alexander, V. Di Marzo, M. R. Elphick, P. J. Greasley, T. B. Kakule, S. Cao, N. K. Garg, J. Zhou, K. N. Houk and
H. S. Hansen, G. Kunos, K. Mackie, R. Mechoulam and Y. Tang, Nature, 2020, 586, 64–69.
R. A. Ross, Pharmacol. Rev., 2010, 62, 588–631. 413 T. Kuzuyama, J. P. Noel and S. B. Richard, Nature, 2005,
389 E. W. Bow and J. M. Rimoldi, Perspect. Med. Chem., 2016, 8, 435, 983–987.
17–39. 414 B. Zirpel, F. Degenhardt, C. Martin, O. Kayser and
390 L. Vollner, D. Bieniek and F. Korte, Tetrahedron Lett., 1969, F. Stehle, J. Biotechnol., 2017, 259, 204–212.
10, 145–147. 415 T. Kumano, S. B. Richard, J. P. Noel, M. Nishiyama and
391 F. W. H. M. Merkus, Nature, 1971, 232, 579–580. T. Kuzuyama, Bioorg. Med. Chem., 2008, 16, 8117–8126.
392 P. Linciano, C. Citti, L. Luongo, C. Belardo, S. Maione, 416 T. Kumano, T. Tomita, M. Nishiyama and T. Kuzuyama,
M. A. Vandelli, F. Forni, G. Gigli, A. Laganà, C. M. Montone J. Biol. Chem., 2010, 285, 39663–39671.
and G. Cannazza, J. Nat. Prod., 2020, 83, 88–98. 417 A. O. Chatzivasileiou, V. Ward, S. M. Edgar and
393 M. G. Cascio, E. Zamberletti, P. Marini, D. Parolaro and G. Stephanopoulos, Proc. Natl. Acad. Sci. U. S. A., 2019,
R. G. Pertwee, Br. J. Pharmacol., 2015, 172, 1305–1318. 116, 506–511.
394 E. M. Rock, M. A. Sticht, M. Duncan, C. Stott and L. A. 418 S. Lund, R. Hall and G. J. Williams, ACS Synth. Biol., 2019,
Parker, Br. J. Pharmacol., 2013, 170, 671–678. 8, 232–238.
395 C. Citti, P. Linciano, F. Russo, L. Luongo, M. Iannotta, 419 M. J. Brownstein, Proc. Natl. Acad. Sci. U. S. A., 1993, 90,
S. Maione, A. Laganà, A. L. Capriotti, F. Forni, M. A. Vandelli, 5391–5393.
G. Gigli and G. Cannazza, Sci. Rep., 2019, 9, 20335. 420 A. Singh, I. M. Menéndez-Perdomo and P. J. Facchini,
396 S. Ben-Shabat, E. Fride, T. Sheskin, T. Tamiri, M.-H. Rhee, Phytochem. Rev., 2019, 18, 1457–1482.
Z. Vogel, T. Bisogno, L. De Petrocellis, V. Di Marzo and 421 H. E. Sigerist, Paracelsus in the light of four hundred years,
R. Mechoulam, Eur. J. Pharmacol., 1998, 353, 23–31. Columbia University Press, 1941.
397 R. Mechoulam and S. Ben-Shabat, Nat. Prod. Rep., 1999, 16, 422 I. Jurna, Schmerz, 2003, 17, 280–283.
131–143. 423 L. Krenn, S. Glantschnig and U. Sorgner, Chromatographia,
398 E. B. Russo, Br. J. Pharmacol., 2011, 163, 1344–1364. 1998, 47, 21–24.
399 E. A. Carlini, I. G. Karniol, P. F. Renault and C. R. Schuster, 424 M. Waldhoer, S. E. Bartlett and J. L. Whistler, Annu. Rev.
Br. J. Pharmacol., 1974, 50, 299–309. Biochem., 2004, 73, 953–990.
400 J. M. Stout, Z. Boubakir, S. J. Ambrose, R. W. Purves and 425 R. M. Quock, T. H. Burkey, E. Varga, Y. Hosohata, K. Hosohata,
J. E. Page, Plant J., 2012, 71, 353–365. S. M. Cowell, C. A. Slate, F. J. Ehlert, W. R. Roeske and
401 F. Taura, S. Tanaka, C. Taguchi, T. Fukamizu, H. Tanaka, H. I. Yamamura, Pharmacol. Rev., 1999, 51, 503–532.
Y. Shoyama and S. Morimoto, FEBS Lett., 2009, 583, 2061–2066. 426 M. Minami and M. Satoh, Neurosci. Res., 1995, 23, 121–145.
402 S. J. Gagne, J. M. Stout, E. Liu, Z. Boubakir, S. M. Clark and 427 I. D. Pogozheva, A. L. Lomize and H. I. Mosberg, Biophys. J.,
J. E. Page, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 1998, 75, 612–634.
12811–12816. 428 R. Schmitz, Pharm. Hist., 1985, 27, 61–74.
403 J. E. Page and Z. Boubakir, US9765308B2, 2009. 429 P.-J. Robiquet, Ann. Chem., 1833, 5, 82–111.
404 M. Fellermeier and M. H. Zenk, FEBS Lett., 1998, 427, 430 P. C. G. Rida, D. Livecche, A. Ogden, J. Zhou and R. Aneja,
283–285. Med. Res. Rev., 2015, 35, 1072–1096.
405 S. Sirikantaramas, S. Morimoto, Y. Shoyama, Y. Ishikawa, 431 K. Ye, Y. Ke, N. Keshava, J. Shanks, J. A. Kapp, R. R. Tekmal,
Y. Wada, Y. Shoyama and F. Taura, J. Biol. Chem., 2004, J. Petros and H. C. Joshi, Proc. Natl. Acad. Sci. U. S. A., 1998,
279, 39767–39774. 95, 1601–1606.
View Article Online
432 Y. Ke, K. Ye, H. E. Grossniklaus, D. R. Archer, H. C. Joshi 455 T. T. T. Dang and P. J. Facchini, Plant Physiol., 2012, 159,
and J. A. Kapp, Cancer Immunol. Immunother., 2000, 49, 618–631.
217–225. 456 T. T. T. Dang and P. J. Facchini, FEBS Lett., 2014, 588,
433 N. Samanani and P. J. Facchini, J. Biol. Chem., 2002, 277, 198–204.
33878–33883. 457 D. K. Liscombe and P. J. Facchini, J. Biol. Chem., 2007, 282,
434 D. K. Liscombe, B. P. MacLeod, N. Loukanina, O. I. Nandi 14741–14751.
and P. J. Facchini, Phytochemistry, 2005, 66, 2501–2520. 458 D. E. Lang, J. S. Morris, M. Rowley, M. A. Torres,
435 B. R. Lichman, M. C. Gershater, E. D. Lamming, T. Pesnot, V. A. Maksimovich, P. J. Facchini and K. K. S. Ng, J. Biol.
A. Sula, N. H. Keep, H. C. Hailes and J. M. Ward, FEBS J., Chem., 2019, 294, 14482–14898.
2015, 282, 1137–1151. 459 T. T. T. Dang and P. J. Facchini, J. Biol. Chem., 2014, 289,
436 B. R. Lichman, A. Sula, T. Pesnot, H. C. Hailes, J. M. Ward 2013–2026.
and N. H. Keep, Biochemistry, 2017, 56, 5274–5277. 460 T. T. T. Dang, X. Chen and P. J. Facchini, Nat. Chem. Biol.,
437 H. H. Pauli and T. M. Kutchan, Plant J., 1998, 13, 793–801. 2015, 11, 104–106.
438 P. J. Facchini and S. U. Park, Phytochemistry, 2003, 64, 461 X. Chen and P. J. Facchini, Plant J., 2014, 77, 173–184.
177–186. 462 A. Nakagawa, H. Minami, J. S. Kim, T. Koyanagi, T. Katayama,
439 T. Gurkok, E. Ozhuner, I. Parmaksiz, S. Özcan, M. Turktas, F. Sato and H. Kumagai, Nat. Commun., 2011, 2, 326.
A. İpek, I. Demirtas, S. Okay and T. Unver, Front. Plant Sci., 463 A. Nakagawa, H. Minami, J. S. Kim, T. Koyanagi, T. Katayama,
2016, 7, 98. F. Sato and H. Kumagai, Bioeng. Bugs, 2012, 3, 49–53.
440 M. R. Bennett, M. L. Thompson, S. A. Shepherd, M. S. 464 J. S. Kim, A. Nakagawa, Y. Yamazaki, E. Matsumura, T. Koyanagi,
Dunstan, A. J. Herbert, D. R. M. Smith, V. A. Cronin, H. Minami, T. Katayama, F. Sato and H. Kumagai, Biosci.,
B. R. K. Menon, C. Levy and J. Micklefield, Angew. Chem., Biotechnol., Biochem., 2013, 77, 2166–2168.
Int. Ed., 2018, 57, 10600–10604. 465 K. Thodey, S. Galanie and C. D. Smolke, Nat. Chem. Biol.,
441 S. C. Farrow, J. M. Hagel, G. A. W. Beaudoin, D. C. Burns 2014, 10, 837–844.
and P. J. Facchini, Nat. Chem. Biol., 2015, 11, 728–732. 466 E. Fossati, A. Ekins, L. Narcross, Y. Zhu, J. P. Falgueyret,
442 T. Winzer, M. Kern, A. J. King, T. R. Larson, R. I. Teodor, G. A. W. Beaudoin, P. J. Facchini and V. J. J. Martin, Nat.
S. L. Donninger, Y. Li, A. A. Dowle, J. Cartwright, R. Bates, Commun., 2014, 5, 3283.
D. Ashford, J. Thomas, C. Walker, T. A. Bowser and 467 M. E. Pyne, K. Kevvai, P. S. Grewal, L. Narcross, B. Choi,
I. A. Graham, Science, 2015, 349, 309–312. L. Bourgeois, J. E. Dueber and V. J. J. Martin, Nat. Commun.,
443 C. P. Wijekoon and P. J. Facchini, Plant J., 2012, 69, 2020, 11, 3337.
1052–1063. 468 I. J. Trenchard and C. D. Smolke, Metab. Eng., 2015, 30, 96–104.
444 R. Lenz and M. H. Zenk, J. Biol. Chem., 1995, 270, 31091–31096. 469 C. E. French and N. C. Bruce, Biochem. J., 1994, 301(Pt 1),
445 T. Grothe, R. Lenz and T. M. Kutchan, J. Biol. Chem., 2001, 97–103.
276, 30717–30723. 470 Y. Li, S. Li, K. Thodey, I. Trenchard, A. Cravens and C. D.
446 J. Ziegler, W. Brandt, R. Geißler and P. J. Facchini, J. Biol. Smolke, Proc. Natl. Acad. Sci. U. S. A., 2018, 115, E3922–E3931.
Chem., 2009, 284, 26758–26767. 471 J. Li, E.-J. Lee, L. Chang and P. J. Facchini, Sci. Rep., 2016,
447 X. Chen, J. M. Hagel, L. Chang, J. E. Tucker, S. A. Shiigi, 6, 39256.
Y. Yelpaala, H. Y. Chen, R. Estrada, J. Colbeck, M. Enquist- 472 L. Flores-Bocanegra, H. A. Raja, T. N. Graf, M. Augustinović,
Newman, A. B. Ibáñez, G. Cottarel, G. M. Vidanes and E. D. Wallace, S. Hematian, J. J. Kellogg, D. A. Todd, N. B.
P. J. Facchini, Nat. Chem. Biol., 2018, 14, 738–743. Cech and N. H. Oberlies, J. Nat. Prod., 2020, 83, 2165–2177.
448 S. C. Farrow and P. J. Facchini, J. Biol. Chem., 2013, 288, 473 D. A. Todd, J. J. Kellogg, E. D. Wallace, M. Khin, L. Flores-
28997–29012. Bocanegra, R. S. Tanna, S. McIntosh, H. A. Raja, T. N. Graf,
449 B. Unterlinner, R. Lenz and T. M. Kutchan, Plant J., 1999, S. E. Hemby, M. F. Paine, N. H. Oberlies and N. B. Cech,
18, 465–475. Sci. Rep., 2020, 10, 19158.
450 A. Kluza, E. Niedzialkowska, K. Kurpiewska, Z. Wojdyla, 474 H. Takayama, Chem. Pharm. Bull., 2004, 52, 916–928.
M. Quesne, E. Kot, P. J. Porebski and T. Borowski, J. Struct. 475 L. T. Yamamoto, S. Horie, H. Takayama, N. Aimi, S. I.
Biol., 2018, 202, 229–235. Sakai, S. Yano, J. Shan, P. K. T. Pang, D. Ponglux and
451 T. Winzer, V. Gazda, Z. He, F. Kaminski, M. Kern, K. Watanabe, Gen. Pharmacol., 1999, 33, 73–81.
T. R. Larson, Y. Li, F. Meade, R. Teodor, F. E. Vaistij, 476 A. Stavrinides, E. C. Tatsis, L. Caputi, E. Foureau, C. E. M.
C. Walker, T. A. Bowser and I. A. Graham, Science, 2012, Stevenson, D. M. Lawson, V. Courdavault and S. E. O’Connor,
336, 1704–1708. Nat. Commun., 2016, 7, 12116.
452 T. M. Kutchan and H. Dittrich, J. Biol. Chem., 1995, 270, 477 A. Stavrinides, E. C. Tatsis, E. Foureau, L. Caputi,
24475–24481. F. Kellner, V. Courdavault and S. E. O’Connor, Chem. Biol.,
453 P. J. Facchini, C. Penzes, A. G. Johnson and D. Bull, Plant 2015, 22, 336–341.
Physiol., 1996, 112, 1669–1677. 478 A. K. Stavrinides, E. C. Tatsis, T. T. Dang, L. Caputi,
454 H. M. Gaweska, K. M. Roberts and P. F. Fitzpatrick, Bio- C. E. M. Stevenson, D. M. Lawson, B. Schneider and
chemistry, 2012, 51, 7342–7347. S. E. O’Connor, ChemBioChem, 2018, 19, 940–948.
View Article Online
479 F. Trenti, K. Yamamoto, B. Hong, C. Paetz, Y. Nakamura 483 Y. Ye, L. Du, X. Zhang, S. A. Newmister, M. McCauley, J. V.
and S. E. O’Connor, Org. Lett., 2021, 23, 1793–1797. Alegre-Requena, W. Zhang, S. Mu, A. Minami, A. E. Fraley,
480 F. Kong, Q. Ma, S. Huang, S. Yang, L. Fu, L. Zhou, H. Dai, M. L. Adrover-Castellano, N. A. Carney, V. V. Shende, F. Qi,
Z. Yu and Y. Zhao, Nat. Prod. Res., 2017, 31, 1403–1408. H. Oikawa, H. Kato, S. Tsukamoto, R. S. Paton, R. M.
481 S. H. Kamble, F. León, T. I. King, E. C. Berthold, C. Lopera- Williams, D. H. Sherman and S. Li, Nat. Catal., 2020, 3,
Londonõ, K. Siva Rama Raju, A. J. Hampson, A. Sharma, 497–506.
B. A. Avery, L. R. McMahon and C. R. McCurdy, ACS 484 M. O. Bonn-Miller, M. A. ElSohly, M. J. E. Loflin,

Pharmacol. Transl. Sci., 2020, 3, 1063–1068. S. Chandra and R. Vandrey, Int. Rev. Psychiatry, 2018, 30,
482 Y. Tsunematsu, N. Ishikawa, D. Wakana, Y. Goda, 277–284.
H. Noguchi, H. Moriya, K. Hotta and K. Watanabe, Nat. 485 L. Cristino, T. Bisogno and V. Di Marzo, Nat. Rev. Neurol.,
Chem. Biol., 2013, 9, 818–825. 2020, 16, 9–29.

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Biosynthesis and synthetic biology of

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Yi Tang received his undergraduate John M. Billingsley grew up in

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Review Article Chem Soc Rev

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Fig. 6 Strategies in synthetic biology.

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Chem Soc Rev Review Article

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14 by an aromatic amino acid decarboxylase (AADC) (Fig. 11, and

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Fig. 12 Psilocybe mexicana contains B1% psilocybin. Image on left

by Fricke et al. (Fig. 13).151 The authors first sequenced the

Fig. 11 Biosynthesis of DMT.

2.3.1 Biosynthesis of psilocybin. A biosynthetic pathway

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phosphatidylserine decarboxylases. PsiH, a P450 monooxygenase,

examples include the biosynthesis of calyculin protoxins and

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Fig. 15 Engineered production of psilocybin and psilocin in yeast.91

being orally inactive. MAOs, as the name suggest, catalyze the

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Fig. 18 Claviceps purpurea (ergot fungus) infecting Dactylis glomerata

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Fig. 19 Biosynthesis of lysergic acid from L-tryptophan.

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A homologous SDR was subsequently identified in the lysergic

and P. commune represent a mechanistic branching point in

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Fig. 22 Amanita muscaria contains about B100–1000 ppm of ibotenic

processing and motor function, respectively.223 A. muscaria is

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Fig. 23 Biosynthesis of ibotenic acid and muscimol from L-glutamic acid.228

catalyzes the desaturation of the 3-oxoisoxazolidine ring to

2.8 Iboga alkaloids

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Fig. 25 Biosynthesis of secologanin from geranyl pyrophosphate (GPP).

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Fig. 26 Biosynthesis of ibogaine from tryptamine and secologanin.

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Fig. 27 Heterologous production of strictosidine in S. cerevisiae.76

visions of curing and divination. The active constituent,

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modelling have implicated the furan ring in selective KOR

3.1 Catecholamine neurotransmitters

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methylation patterns of the purine heterocycle exhibit variable

from theobromine 107 (Fig. 32) which employed methyl iodide

Chem Soc Rev Review Article