C H A P T E R

2
The Biosynthesis of Cannabinoids
F. Degenhardt, F. Stehle, O. Kayser
Laboratory of Technical Biochemistry, Department of Biochemical and Chemical Engineering,
TU Dortmund University, Dortmund, Germany

SUMMARY POINTS • Two cannabigerol-like compounds were detected in

• This chapter focuses on the pathway which leads the aerial parts of Helichrysum umbraculigerum Less., a
to the biosynthesis of phytocannabinoids in plant common in the eastern parts of South Africa.
C. sativa L. • N-alkyl amides (cannabinomimetics), found in the
medicinal plants Echinaceae angustifolia and Echinaceae
• CBGA is the central precursor of
purpurea (purple cornflower), are known to interact
phytocannabinoid biosynthesis in Cannabis.
with the CB2 receptor.
• CBGAS, only three enzymes—THCAS, CBDAS,
and CBCAS—are involved in the biosynthesis of
phytocannabinoids in Cannabis plants. LIST OF ABBREVIATIONS
• Sequences of CBDAS and THCAS are known.
• The carboxyl group in CBGA seems to be AAE Acyl-activating enzyme
essential for the enzymatic reactions catalyzed by BBE Berberine bridge enzyme
CBDAS, CBCAS, and THCAS. CBC Cannabichromene
CBCA Cannabichromenic acid
• The diversity of more than 60 cannabinoids is the
CBCAS Cannabichromenic acid synthase
result of nonenzymatic modifications.
CBCVA Cannabichrovarinic acid
• Propyl cannabinoids occur by the prenylation of CBD Cannabidiol
divarinic acid (DA) with geranyl diphosphate CBDA Cannabidiolic acid
(GPP). CBDAS Cannabidiolic acid synthase
CBDV Cannabidivarin
CBDVA Cannabidivarinic acid
KEY FA C T S OF CBG Cannabigerol
PH YT OCA NNAB INOIDS —B ES IDE S CBGA Cannabigerolic acid (3-geranyl olivetolate)
C.   SAT I VA CBGAS Cannabigerolic acid synthase
• Phytocannabinoids are plant-derived natural compounds CBGVA Cannabigerovarinic acid
that act as ligands to cannabinoid receptors (CB1 and CB2) CBN Cannabinol
or share chemical similarity with cannabinoids. CBNRA Cannabinerolic acid (cis-CBGA)
• C. sativa L. is intensively investigated for the presence CHS Chalcone synthase
of phytocannabinoids. To date, only a few plants are CsAAE1 C. sativa hexanoyl-CoA synthetase 1
discovered that contain phytocannabinoids other than CsAAE3 C. sativa hexanoyl-CoA synthetase 2
the ones known from Cannabis. CsHCS1 C. sativa hexanoyl-CoA synthetase 1
• The New Zealand liverwort Radula marginata and CsHCS2 C. sativa hexanoyl-CoA synthetase 2
Japanese liverwort Radula perrottetii contain perrotteti- DA Divarinic acid
nene, a naturally occurring bibenzyl cannabinoid. DABB Dimeric α + β barrel
DMAPP Dimethylallyl diphosphate
Handbook of Cannabis and Related Pathologies. http://dx.doi.org/10.1016/B978-0-12-800756-3.00002-8
Copyright © 2017 Elsevier Inc. All rights reserved.
13
14 2.  The Biosynthesis of Cannabinoids

DOXP 1-Deoxy-d-xylulose-5-phosphate for the biosynthesis of cannabinoids, the terpenopheno-

GOT Geranylpyrophosphate:olivetolate
geranyltransferase
GPP Geranyl diphosphate
HTAL Hexanoyltriacetic acid lactone
IPP Isopentenyl diphosphate
MEP 2C-methyl-d-erythritol-4-phosphate
MVA Mevalonate
NPP Neryl diphosphate
OA Olivetolic acid
OAC Olivetolic acid cyclase
OLS Olivetol synthase
PKS Polyketide synthase
SNP Single nucleotide polymorphism
STS Stilbene synthase
THC Tetrahydrocannabinol
THCA Tetrahydrocannabinolic acid
THCAS Tetrahydrocannabinolic acid synthase
THCV Tetrahydrocannabivarin
THCVA Tetrahydrocannabivarinic acid
INTRODUCTION
Cannabis sativa L. (hemp) is one of the oldest do-
mestic plants in the history of mankind, and has been
cultivated for at least 10,000  years (Schultes, Klein,
Plowman, & Lockwood,  1974). Together with Humulus
lupulus (hop), C. sativa belongs to the small family of
Cannabaceae. Cannabis is an annual, usually dioecious,
wind-pollinated herb, with both male and female flow-
ers growing on separate plants. The plant is well known
lic constituents that show psychoactive effects. But since
other plants also have secondary metabolites that inter-
act with the human cannabinoid receptors, a new defini-
tion had to be made. Hence, phytocannabinoids are now
defined as any plant-derived natural compound that
can act as a ligand to human cannabinoid receptors (CB1
and CB2) or share chemical similarity with cannabinoids
(Gertsch, Pertwee, & Di Marzo,  2010). Interestingly, all
parts of the Cannabis plant, with the exception of seeds,
can contain cannabinoids, but they mainly accumulate
in the glandular trichomes of female flowers (Gagne
et al., 2012; van Bakel et al., 2011).
The following chapter focuses on the pathway that
leads to the enzymatic biosynthesis of cannabinoids. For
a long time, it was postulated that the key intermedi-
ate is cannabidiol (CBD) or cannabidiolic acid (CBDA),
both resulting from a condensation of a monoterpene,
and olivetol or olivetolic acid (OA), respectively. In 1964,
Gaoni and Mechoulam postulated cannabigerol (CBG)
as the key intermediate, the condensation product of ge-
ranyl diphosphate (GPP), and olivetol or OA. Based on
this, they concluded that the cannabinoids CBD, tetrahy-
drocannabinol (THC) and cannabinol (CBN) are all de-
rived from CBG, and just differ in the way of cyclization
(Gaoni & Mechoulam, 1964). Finally, incorporation stud-
ies with 13C-labeled glucose have shown that GPP and
OA are indeed the precursors for formation of cannabig-
erolic acid (CBGA). Thus, the general structure of canna-
binoids is assembled by two parts: (1) a diphenol (resor-
cin) carrying an alkyl chain (OA); and (2) a monoterpene
moiety (GPP) (Fig.  2.1). Subsequently, Fellermeier and

FIGURE 2.1  General structure of cannabinoids and their precursors, olivetolic acid, and geranyl diphosphate. Cannabinoids are composed
of two parts: a cyclic monoterpene part (red), and a diphenol (resorcin) part, carrying an alkyl chain (blue). The dibenzopyran-numbering system
is used.

I.  Setting the scene, botanical, general and international aspects

 Cannabinoid precursor biosynthesis 15
coworkers postulated CBGA as the central cannabinoid
precursor (Fellermeier, Eisenreich, Bacher, & Zenk, 2001;
Fellermeier & Zenk, 1998). Interestingly, free OA has nev-
er been detected in Cannabis plant material until now.
It is worthy to note that, although more than 60 can-
nabinoids are known, only three enzymes, besides can-
nabigerolic acid synthase (CBGAS), namely tetrahydro-
cannabinolic acid synthase (THCAS), cannabidiolic acid
synthase (CBDAS), and cannabichromenic acid synthase
(CBCAS), are involved in cannabinoid biosynthesis. The
resulting acidic cannabinoids are the most abundant ones
accumulating in Cannabis. The neutral and psychoactive
forms are the results of nonenzymatic decarboxylation
during storage, heat or sunlight; explaining the heating
of plant material (ie, smoking or baking), during Canna-
bis consumption (Fischedick, Hazekamp, Erkelens, Choi,
& Verpoorte, 2010; Taura et al., 2007a). Thus, the broad
diversity of the different cannabinoids is mainly due to
nonenzymatic transformation or degradation of both
acidic and neutral cannabinoids by the effects of light
(UV irradiation) and auto-oxidation (Crombie, Ponsford,
Shani, Yagnitinsky, & Mechoulam,  1968; Razdan,
Puttick, Zitko, & Handrick, 1972). It is still unclear if all
these forms are present in living plants as natural or ar-
tefacts, due to storage and sample preparation (ElSohly
& Slade, 2005).
CANNABINOID PRECURSOR
BIOSYNTHESIS
Polyketide Pathway Toward Olivetolic Acid
The origin of hexanoate in trichomes has not been
elucidated so far. Suzuki, Kurano, Esumi, Yamaguchi, and
Doi (2003) showed that the side-chain moiety of alkyl-
resorcinols is formed by fatty acid units, but it remains
unclear if the moiety is the result of biosynthesis or deg-
radation of fatty acids. Studies regarding the incorpora-
tion of 13C-labels into cannabinoids indicate that hexano-
ate is synthesized from acetyl-CoA as a starter unit, and
five molecules of malonyl-CoA. These building blocks
are precursors of the fatty acid biosynthesis (Fellermeier
et al., 2001).
Based on this, two pathways are feasibly possible,
after analysis of a cDNA/EST library generated from
female flowers (glands) of C. sativa. First, the hexanoyl
residue could be obtained by an early termination of the
fatty acid biosynthesis. Subsequently, the hexanoyl moi-
ety of the resulting hexanoyl-ACP would be cleaved by a
thioesterase or transferred to CoA by an ACP-CoA trans-
acylase. Finally, acyl-CoA synthetase would catalyze the
conversion of the obtained n-hexanol to hexanoyl-CoA
(Marks et  al.,  2009). Second, n-hexanol could be pro-
duced by the breakdown of C18 unsaturated fatty acids
I.  Setting the scene, botanical, general and international aspects
via the lipoxygenase pathway (Marks et al., 2009; Stout,
Boubakir, Ambrose, Purves, & Page, 2012). Nevertheless,
further studies are necessary to clarify the origin of the
hexanol moiety.
Hexanoyl-CoA is a medium-chain fatty acyl-CoA that
can be detected in high amounts in Cannabis flowers
(Stout et al., 2012). It is synthesized by an acyl-activating
enzyme (AAE) called hexanoyl-CoA synthetase (Marks
et  al.,  2009; Page & Stout,  2013). AAEs can use short,
medium, long as well as very long-chain fatty acids as
carboxylic acid substrates. Two novel enzymes were
identified, C. sativa hexanoyl-CoA synthetase 1 (CsHCS1
or CsAAE1) and C. sativa hexanoyl-CoA synthetase 2
(CsHCS2 or CsAAE3) that are capable of producing
hexanoyl-CoA using hexanoate and CoA as substrates.
Based on transcript levels, CsHCS1 seems to be tri-
chome-specific. Although CsHCS2 exhibits lower tran-
script levels, in comparison to CsHCS1, it is abundant in
all tissues. The gene of CsHCS1 consists of a 2163-nucle-
otide open reading frame, and encodes a 720-amino acid
polypeptide chain. The gene of CsHCS2 is composed of
a 1632-nucleotide open reading frame, and encodes a
543-amino acid polypeptide chain. Both CsHCSs gener-
ally require divalent cations for activity. This was shown
by adding Mg2+, Mn2+, and Co2+ to the enzyme assays.
Thus, CsHCS1 preferentially accepts Mg2+, and CsHCS2
Co2+. The highest enzyme activity was detected at 40°C
and pH 9 for both enzymes. Furthermore, both enzymes
can be inhibited by high concentrations of CoA (Page &
Stout, 2013; Stout et al., 2012).
Taken together, the published data suggest that
CsHCS1 is the enzyme involved in the biosynthesis of
cannabinoids: (1) it is the most abundant AAE in tri-
chomes; (2) it is highly specific for short-chain fatty acyl-
CoA, particularly hexanoate (KM value in the nM range);
and (3) it is localized in the cytosol, as suggested for the
olivetol synthase (see later). In contrast, CsHCS2 is lo-
calized in the peroxisomes and accepts a broad range of
substrates, while showing a KM value for hexanoate in
the mM range (Page & Stout, 2013; Stout et al., 2012).
The alkylresorcinol moiety of cannabinoids is de-
rived from OA, the product of polyketide synthases
(PKSs) that catalyze the aldol condensation of hexanoyl-
CoA with three molecules of malonyl-CoA (Fellermeier
et  al.,  2001; Raharjo, Chang, Choi, Peltenburg-Looman,
& Verpoorte, 2004) (Fig. 2.2). The second precursor mal-
onyl-CoA is predominantly derived from acetyl-CoA
by carboxylation. The ATP-dependent reaction is cata-
lyzed by an acetyl-CoA carboxylase (EC 6.4.1.2). The en-
zyme utilizes the first step in the fatty acid biosynthesis
(Chen, Kim, Weng, & Browse, 2011; Konishi, Shinohara,
Yamada, & Sasaki, 1996). Taura et al. (2009) discovered
a plant type III PKS in flowers and rapidly expanding
leaves of C. sativa. The gene of olivetol synthase (OLS)
encodes a 385-amino acid polypeptide chain that does
16 2.  The Biosynthesis of Cannabinoids
TABLE 2.1 Enzymes Involved In Cannabinoid Biosynthesis in C. sativa L
Enzyme
Olivetol synthase OLS
Olivetolic acid cyclase OAC
Cannabigerolic acid synthase CBGAS
Cannabichromenic acid synthase CBCAS
Cannabidiolic acid CBDAS
synthase
Tetrahydrocannabinolic acid synthase THCAS
The table lists the enzymes and the corresponding GenBank accession numbers involved in biosynthesis of C. sativa phytocannabinoids.
a
GenBank.
b
Patent number.
not contain a signal peptide (Table 2.1). The OLS protein
has a theoretical molecular mass of 43 kDa, as confirmed
by SDS-PAGE analysis. However, size-exclusion chro-
matography experiments revealed a molecular mass of
about 89 kDa, indicating a homodimeric enzyme (Gagne
et al., 2012; Taura et al., 2009). OLS (PKS-1) was prelimi-
narily crystallized by Taguchi et al. (2008) and the struc-
ture was finally published by Yang et al. (2016). It is of
interest that the enzyme does not produce OA, but olive-
tol, triketide pyrone, and tetraketide pyrone. Analysis of
the amino acid sequence displayed a high similarity with
those of Medicago sativa chalcone synthase (CHS), and
other plant PKSs (60–70%). Additionally, the catalytic tri-
ade residues of CHS (Cys164-His303-Asn336) are conserved
(Taura et al., 2009). Since CHSs catalyze intramolecular
C6 → C1 Claisen condensations, Raharjo, and coworkers
were the first to suggest in 2004 (Raharjo et al., 2004) that
OLS could be a stilbene synthase (STS). These enzymes
catalyze C2 → C7 aldol condensations, followed by a
decarboxylation step. Additionally, studies by Austin,
Bowman, Ferrer, Schröder, & Noel (2004) showed that
the cyclization reaction can be changed from a Claisen-
type (CHS) to an aldol-type (STS) by substitution of a
few amino acids in CHS (= aldol switch).
Nevertheless, since OLS alone is not capable to form
OA, another enzyme/PKS might be involved in the
biosynthesis. The missing enzyme should catalyze a C2
→ C7 intramolecular aldol condensation upon which
the carboxylate moiety is preserved. This is important
since CBGAS does not accept olivetol as a prenyl do-
nor (Fellermeier & Zenk, 1998). Gagne et al. (2012) iso-
lated a gene encoding a 101-amino acid polypeptide
chain. This small protein (12 kDa) shows similarities to
a polyketide cyclase that belongs to the dimeric α + β
barrel (DABB)-type protein family. Furthermore, the
identified gene exhibits high expression levels in glan-
dular trichomes. Together, this made the polyketide
cyclase a promising candidate for the missing olivetolic
acid cyclase (OAC).
I.  Setting the scene, botanical, general and international aspects
Accession no.a EC no. References
AB164375 2.3.1.206 Taura et al. (2009)
AFN42527.1 4.4.1.26 Gagne et al. (2012)
US2012/0144523 2.5.1.102 Fellermeier and Zenk (1998);
A1b Page and Boubakir (2012)
1.3.3.- Morimoto et al. (1998)
AB292682 1.21.3.8 Taura et al. (2007a)
AB057805 1.21.3.7 Sirikantaramas et al. (2004)
Finally, using both OLS and OAC with hexanoyl-CoA
and malonyl-CoA in one assay, the formation of OA,
pentyldiacetic acid (triketide pyrone), and hexanoyltri-
acetic acid lactone (HTAL; tetraketide pyrone) could be
demonstrated (Page & Gagne,  2013) (Fig.  2.2). It is as-
sumed that OLS catalyzes the formation of an interme-
diate that is subsequently converted into OA by OAC
(Gagne et al., 2012; Taguchi et al., 2008).
Biosynthesis of Geranyl Diphosphate
The monoterpene moiety of cannabinoids (Fig. 2.2) is
derived from GPP. Its precursors, isopentenyl diphos-
phate (IPP), and dimethylallyl diphosphate (DMAPP),
are predominantly (>98%) biosynthesized via the
2C-methyl-d-erythritol-4-phosphate (MEP) pathway
[also termed as nonmevalonate pathway or 1-deoxy-d-
xylulose-5-phosphate (DOXP) pathway] (Fellermeier
et al., 2001). These results are supported by Marks et al.
(2009). They isolated RNA from the glands of a tetra-
hydrocannabinolic acid (THCA)-producing Cannabis
strain and generated a cDNA library. After sequencing,
they were able to identify all but one enzyme involved
in the MEP pathway. Additionally, Stout et  al. (2012)
found high expression of MEP pathway genes in Can-
nabis flowers. Furthermore, in higher plants the MEP
pathway, mainly involved in secondary metabolism,
is localized in plastids (described in detail elsewhere,
for example, Eisenreich, Bacher, Arigoni, & Rohdich,
(2004), or Hunter (2007), whereas the mevalonate (MVA)
pathway, predominantly contributing to primary me-
tabolism, is localized in the cytosol. The compartmental
separation between these two pathways is not absolute.
The metabolites of both pathways can be transported bi-
directionally across the plastid membranes (Eisenreich
et al., 2004).
Subsequently, the head-to-tail condensation of IPP
and DMAPP to form GPP is catalyzed by geranyl di-
phosphate synthase (Fig. 2.2) (Burke et al., 1999).
 Cannabinoid precursor biosynthesis 17

FIGURE 2.2  Biosynthesis of cannabigerolic acid (CBGA). The biosynthesis of the central intermediate CBGA is colored in dark green. The
minor products CBNRA and CBGVA are shaded in light green. The precursor pathways are highlighted in light blue (GPP) and blue (OA). MEP,
2C-methyl-d-erythritol-4-phosphate; DOXP, 1-deoxy-d-xylulose-5-phosphate; MVA, mevalonate (Burke, Wildung, & Croteau,  1999; de Meijer
et al., 2009; Fellermeier & Zenk, 1998; Page & Gagne, 2013; Taura et al., 2009).

Cannabigerolic Acid Biosynthesis by mass spectrometry (MS) measurements as CBGA and

Cannabigerolic acid synthase (CBGAS) or geranylpy
rophosphate:olivetolate geranyltransferase (GOT) pre-
dominantly catalyzes the C-prenylation of OA by GPP
to form CBGA (Fig.  2.2). CBGA is presumed to be the
central precursor for cannabinoid biosynthesis, since dif-
ferent cyclization of the prenyl moiety leads to THCA
or its isomers cannabichromenic acid (CBCA) and CBDA
(Page & Boubakir,  2012; Sirikantaramas, Morimoto, &
Shoyama, 2007).
Fellermeier and Zenk (1998) detected the enzyme in
crude homogenates of rapidly expanding young leaves
of C. sativa. This part of the plant contains the later en-
zymes of the THCA biosynthetic pathway (Morimoto,
Komatsu, Taura, & Shoyama, 1997; Taura et al., 1995a).
There are indications that CBGAS, like other prenyl-
transferases, is a membrane-bound prenyltransferase
(Yamamoto, Kimata, Senda, & Inoue,  1997). However,
Fellermeier and Zenk (1998) could not detect any enzyme
activity in particulate fractions, but in the soluble fraction
of the crude extract. Two major products were identified
its cis-isomer cannabinerolic acid (CBNRA; Fellermeier
and Zenk, 1998, used CBNA instead of CBNRA). The en-
zyme activity was found to be Mg2+-dependent. CBGAS
seems to be specific for OA as a prenyl acceptor, but also
accepts different prenyl donors like GPP and, to a lesser
degree, neryl diphosphate (NPP) (Fig. 2.2). The produc-
tion ratio of CBGA/CBNRA changes from 2:1 to 1:1
when NPP is used as a prenyl donor instead of GPP.
However, the aromatic prenyltransferase CBGAS
seems to be a soluble enzyme, but Fellermeier and Zenk
(1998) could not completely exclude a membrane-bound
activity. Besides, two soluble hop prenyltransferases,
involved in the biosynthesis of hop bitter acids, are de-
scribed by Zuurbier, Fung, Scheffer, & Verpoorte (1998).
Nevertheless, these are the only descriptions of soluble
plant C-prenylating enzymes; until now, it was not pos-
sible to get the sequence information or to isolate the cor-
responding genes or enzymes.
Contradictorily, all known sequences of plant aro-
matic prenyltransferases belong to membrane-bound

I.  Setting the scene, botanical, general and international aspects

18 2.  The Biosynthesis of Cannabinoids

enzymes (Yamamoto et  al.,  1997; Yamamoto, Senda, &
Inoue,  2000; Zhao, Inoue, Kouno, & Yamamoto,  2003).
This is in accordance with the second report dealing with
the CBGAS (Page & Boubakir, 2012). They published a
sequence of CBGAS that was mainly expressed in glan-
dular trichomes of female flowers and young leaves
of Cannabis plants. The gene encodes a 395-amino acid
polypeptide chain showing a membrane-bound type of
prenyltransferases. They were able to express the recom-
binant CBGAS in Sf9 insect as well as in Saccharomyces
cerevisiae cells, and verified the CBGAS activity in the
microsomal fractions. Using MS measurements, CBGA
(3-geranyl olivetolate; comparison with CBGA stan-
dard) was identified as the major product, and 5-geranyl
olivetolate (identification only by LC-MS analysis) as the
minor product. Furthermore, Page and Boubakir (2012)
showed that CBGAS is specific only to GPP as a prenyl
donor, and approves OA, olivetol, phlorisovalerophe-
none, naringenin, and resveratrol as prenyl acceptor. Ad-
ditionally, the enzyme reaction is dependent on divalent
cations, whereas the highest enzyme activity was ob-
tained by using Mg2+ (Page & Boubakir, 2012).
CANNABINOID PATHWAY
CBGA, the central precursor of cannabinoid biosyn-
thesis, is converted by three enzymes (Fig. 2.3): CBDAS,
CBCAS, and THCAS. They predominantly use CBGA
as substrate, and catalyze the stereoselective, oxida-
tive cyclization of the monoterpene moiety of CBGA to
CBDA, CBCA, or THCA, respectively. The THCAS and
CBDAS reactions are oxygen-dependent, producing hy-
drogen peroxide proportional to either CBDA or THCA
(Sirikantaramas et  al.,  2004; Taura et  al., 2007b). Re-
markably, the CBCAS reaction is oxygen independent,
and can be inhibited by hydrogen peroxide. Thus, the
enzyme seems not to be an oxygenase or a peroxidase
(Morimoto, Komatsu, Taura, & Shoyama,  1998). Fur-
thermore, all three enzymes also convert CBNRA, the

FIGURE 2.3  Biosynthesis of cannabinoids. The enzymatically catalyzed reactions are highlighted in dark green. All nonenzyme-dependent
modifications reactions are colored in light green. Biosynthesis of C3-cannabinoids starting from cannabigerovarinic acid (CBGVA) is carried out
by the same enzymes and for better clarity not shown (Crombie et al., 1968; de Meijer, 2011; Morimoto et al., 1998; Shoyama, Fujita, Yamauchi, &
Nishioka, 1968; Shoyama, Oku, Yamauchi, & Nishioka, 1972; Taura et al., 1995a; 1996).

I.  Setting the scene, botanical, general and international aspects

 Cannabinoid pathway 19
cis-isomer of CBGA, with a lower specificity (Morimoto
et  al.,  1998; Shoyama et  al.,  2012; Sirikantaramas,
Morimoto, & Shoyama, 2007; Taura et al., 1995b; Taura,
Morimoto, & Shoyama, 1996). Since no enzymatic activ-
ity was detectable using the neutral cannabinoid CBG,
the carboxyl group in CBGA seems to be essential for the
enzymatic reactions catalyzed by THCAS, CBDAS, and
CBCAS (Morimoto et al., 1997; Taura et al., 1995a; Taura
et al., 1996).
Besides, it was postulated that THCA is biosyn-
thesized and stored in the storage cavity of the glan-
dular ­trichomes of Cannabis plants (Sirikantaramas,
­Morimoto, & Shoyama, 2007).
Tetrahydrocannabinolic Acid Synthase
The THCAS gene encodes a 545-amino acid polypep-
tide chain (Table 2.1). According to Sirikantaramas et al.
(2004), a 28 amino acid long signal peptide is cleaved
in the processed THCAS, leading to a protein of 517
amino acids. The mature THCAS has a theoretical mo-
lecular mass of 59 kDa. An actual mass of about 75 kDa
was detected using SDS-PAGE (Taura et  al., 1995b).
This could be explained by posttranslational modifica-
tions, since eight possible Asn glycosylation sites were
confirmed (Sirikantaramas et  al.,  2004). Furthermore,
deglycosylated THCAS indeed showed a molecular
mass of 59 kDa, and remained fully active (Taura et al.,
2007b). THCAS is a monomeric enzyme with the high-
est activity between pH 5.5 and pH 6.0 (Taura et  al.,
1995b). Sequence comparison identified similarities
to the berberine bridge enzyme (BBE) of Eschscholzia
californica (Shoyama et  al.,  2012). BBE belongs to the
family of oxidoreductases and has a covalently bound
FAD (Kutchan & Dittrich, 1995). The THCAS amino acid
sequence revealed a flavinylation consensus sequence
(Arg110-Ser-Gly-Gly-His114) in which His114 is probably
the FAD-binding site (Sirikantaramas et al., 2004). This
could be confirmed by X-ray crystallography (PDB ID:
3VTE) at a resolution of 2.75Å. The results show that
the enzyme is composed of two domains and one FAD
binding pocket present in between. Besides His114, a
second residue, Cys176, could be identified to be cova-
lently bound to the FAD (Shoyama et al., 2012). Based
on X-ray structure data and mutational analysis of
THCAS, a possible catalytic reaction mechanism of
THCAS was proposed by Shoyama et al. (2012), assign-
ing a central role to Tyr484 in the catalytic mechanism
(Fig. 2.5). Nevertheless, since the crystal structure was
published without substrate analoga, further studies
are necessary to verify the suggested mechanism.
Cannabis plants can be divided into two groups:
“fiber-type” and “drug-type” plants (see dictionary).
Alignment of THCAS coding sequences from “fiber-
type” and “drug-type” plants showed 37 major amino
I.  Setting the scene, botanical, general and international aspects
acid substitutions. These substitutions seem to be the rea-
son for decreased THCAS activity in “fiber-type” strains
(Kojoma, Seki, Yoshida, & Muranaka,  2006). Minise-
quencing of samples from both types of Cannabis plants
showed three different single nucleotide polymorphism
(SNP) genotypes. “Fiber-type” plants are homozygous
for the inactive THCAS form. “Drug-type” plants are
either homozygous or heterozygous for the active form
of THCAS. It seems that only a single copy of the gene
encoding the active THCAS form is necessary for the
biosynthesis of THCA (Rotherham & Harbison, 2011).
Cannabidiolic Acid Synthase
The gene of the wildtype CBDAS encodes a 544-amino
acid polypeptide (Table  2.1). According to Taura et  al.
(2007b), processed CBDAS consists of 517 amino ac-
ids following cleavage of the 28 amino acid long signal
peptide. The mature CBDAS has a theoretical molecu-
lar mass of 59 kDa. An actual mass of about 74 kDa was
detected by SDS-PAGE that is possibly caused by post-
translational glycosylation of seven Asn residues (Taura
et al., 1996; Taura et al., 2007b). CBDAS is a monomeric
enzyme with a pH optimum of 5.0 (Taura et  al.,  1996).
A comparison between THCAS and CBDAS revealed a
sequence similarity of 84% (Taura et al., 2007b) (Figs. 2.4
and 2.5). Like THCAS, CBDAS is a flavinated enzyme in
which His114 and Cys176 are most likely the FAD-binding
sites. Since CBDAS exhibits structural and biochemical
properties related to those of THCAS, it is probable that
the reaction mechanism of CBDAS is similar to that of
THCAS (Taura et al., 2007b).
Cannabichromenic Acid Synthase (CBCAS)
The sequence of CBCAS is still unknown. Morimoto
et al. (1998) purified the enzyme to apparent homogene-
ity, but its sequence is not yet available in public databas-
es. According to van Bakel et al. (2011), possible CBCAS
candidates are currently analyzed biochemically.
However, CBCAS was isolated and partially purified
from young leaves of C. sativa (Morimoto et  al.,  1997;
1998). In contrast to CBDAS and THCAS, CBCAS seems
to be homodimer with a determined native molecular
mass of 136  kDa and a maximum activity at pH 6.5. A
molecular mass of 71 kDa was estimated for the mono-
mers using SDS-PAGE. According to kinetic data, CBCAS
has a higher affinity for CBGA than THCAS and CBCAS
(Morimoto et al., 1998). CBCA and its neutral form CBC
are both racemic. Studies of Morimoto et al. (1997) sug-
gested that both enantiomers of CBCA are formed by
a CBCAS catalyzed reaction with a molar ratio of 5:1.
But it is still unknown which of the two isomers is the
major product (Gaoni & Mechoulam,  1971; Morimoto
et al., 1997; Taura et al., 2007a).
20 2.  The Biosynthesis of Cannabinoids

FIGURE 2.4  Alignment of amino acid sequences of THCA synthase and CBDA synthase. The alignment was performed with CLUSTAL W
using the BLOSUM 62 matrix (Henikoff & Henikoff, 1992; Larkin et al., 2007). The signal peptide cleavage sites are indicated by a triangle. Second-
ary elements (α-alpha-helices; β-beta-sheets; TT-turns; η-310 helix) are shown for the THCAS. Fully conserved residues are shaded in black. The
sequences show an overall identity of 84%. The figure was made with ESPript 3.0 (Robert & Gouet, 2014). THCAS, tetrahydrocannabinolic acid
synthase; CBDAS, cannabidiolic acid synthase.

I.  Setting the scene, botanical, general and international aspects

 Mini-dictionary 21

FIGURE 2.5  X-ray structure of the active center of THCAS. The backbone is shown as cartoon diagram (PDB ID: 3VTE). The FAD molecule
(orange) is covalently attached to His114 and Cys176 (yellow). The active site residues are highlighted in green (Shoyama et al., 2012). The close-up of
active center was prepared with PyMOL. THCAS, tetrahydrocannabinolic acid synthase.

Cannabinoids with Propyl Side Chains
In contrast to the classic C5-phytocannabinoids,
which contain an n-pentyl side chain, cannabinoids with
an n-propyl side chain are called C3-phytocannabinoids
or propyl cannabinoids. The C-prenylation of divarinic
acid (DA) instead of OA by GPP yields in cannabiger-
ovarinic acid (CBGVA) (Fig. 2.2) (de Meijer, Hammond,
& Micheler, 2009). The formation of propyl cannabinoids
does not occur by shortening the side chain of pentyl can-
nabinoids (Kajima & Piraux, 1982). CBGVA is the central
branch-point intermediate in the biosynthesis of C3-can-
nabinoid acids, like CBGA for pentyl cannabinoid acids.
The enzymes CBDAS, CBCAS, and THCAS are not se-
lective for the length of the alkyl side chain, and can use
both as a substrate. The resulting cannabinoids are called
cannabidivarinic acid (CBDVA), cannabichrovarinic acid
(CBCVA), and tetrahydrocannabivarinic acid (THCVA).
The diverse amount of 2-carboxylic acids in different
Cannabis strains is caused by dissimilar enzyme specifici-
ties at the level of CBGA or CBGA-analogs formation (de
Meijer et al., 2009; Shoyama, Hirano, & Nishioka, 1984).
Relatively high amounts of tetrahydrocannabivarin
(THCV) and/or cannabidivarin (CBDV) are usually only
detectable in Cannabis indica (de Zeeuw, 1972).
MINI-DICTIONARY
Cannabinoids  Cannabinoids are a group of terpenophenolic
compounds. They show affinities to cannabinoid receptors (CB1,
CB2) or are structurally related to tetrahydrocannabinol (THC).
Cannabinoids can be differentiated into phytocannabinoids,
synthetic cannabinoids, and endocannabinoids.
CBGA  Cannabigerolic acid (CBGA) is the central precursor of
phytocannabinoid biosynthesis. It is nonpsychoactive.
“Drug-type” plants  These are THCA-rich plants. The THCA
is converted into psychoactive ∆9-THC by a nonenzymatic
decarboxylation that enables the plants to be labelled as THC-rich.
“Fiber-type” plants  “Fiber-type” plants are also known as
“nondrug” plants. These plants have a low (<0.2%) or no THCA
content, but they contain a high amount of cannabidiolic acid
(CBDA).
Phytocannabinoids  Phytocannabinoids are a unique group
of secondary metabolites (cannabinoids) occurring naturally
in plants. Other names include natural cannabinoids or herbal
cannabinoids (see Key facts).
∆8-THC  In Cannabis plants, ∆8-tetrahydrocannabinol (∆8-THC)
is detectable in low amounts (<1% of the present ∆9-THC). Like

I.  Setting the scene, botanical, general and international aspects

22 2.  The Biosynthesis of Cannabinoids
∆9-THC, it is also psychoactive. Maybe ∆8-THC is an artefact of
extraction and/or analysis process. The term THC includes a
combination of ∆8-THC and ∆9-THC.
∆9-THC  ∆9-Tetrahydrocannabinol (∆9-THC) and ∆1-THC
describe the same compound, differing in the numbering system
used (dibenzopyran-numbering and monoterpene-numbering
system, respectively). ∆9-THC is responsible for the psychoactive
effects of Cannabis products. It binds to the human cannabinoid
receptors located in the central and peripheral nervous system.
Misleadingly, ∆9-THC is termed as the main component of drug-
type Cannabis plants (see THCA).
∆9-THCA  ∆9-Tetrahydrocannabinolic acid (THCA), not THC,
is the main component of drug-type Cannabis plants. It is
nonpsychoactive. THCA is converted into neutral psychoactive ∆9-
THC by a nonenzymatic decarboxylation during heating or storage.
Acknowledgment
We gratefully acknowledge Parijat Kusari for critically reading this
manuscript.
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