Benzylisoquinoline alkaloid biosynthesis in opium poppy

14 Pages·2014·3.37 MB·English

Planta (2014) 240:19–32
DOI 10.1007/s00425-014-2056-8
RevIew
Benzylisoquinoline alkaloid biosynthesis in opium poppy
Guillaume A. W. Beaudoin · Peter J. Facchini
Received: 6 December 2013 / Accepted: 5 March 2014 / Published online: 27 March 2014
© Springer-verlag Berlin Heidelberg 2014
Abstract Opium poppy (Papaver somniferum) is one of Introduction
the world’s oldest medicinal plants and remains the only
commercial source for the narcotic analgesics morphine, Opium poppy (Papaver somniferum) is an ancient medici-
codeine and semi-synthetic derivatives such as oxycodone nal plant that remains the only commercial source of the
and naltrexone. The plant also produces several other ben- narcotic analgesics morphine, codeine and semi-synthetic
zylisoquinoline alkaloids with potent pharmacological analogs including oxycodone, hydrocodone, buprenorphine
properties including the vasodilator papaverine, the cough and naltrexone (Berényi et al. 2009). Other pharmaceuti-
suppressant and potential anticancer drug noscapine and cally important benzylisoquinoline alkaloids (BIAs) found
the antimicrobial agent sanguinarine. Opium poppy has in opium poppy include the antimicrobial agent sanguinar-
served as a model system to investigate the biosynthesis ine, the muscle relaxant papaverine and the cough-suppres-
of benzylisoquinoline alkaloids in plants. The application sant and potential anticancer drug noscapine. The ~2,500
of biochemical and functional genomics has resulted in a known BIAs represent a diverse group of nitrogen-contain-
recent surge in the discovery of biosynthetic genes involved ing specialized metabolites found mainly in the basal eud-
in the formation of major benzylisoquinoline alkaloids in icot order Ranunculales, in particular the families Papaver-
opium poppy. The availability of extensive biochemical aceae, Ranunculaceae, Berberidaceae and Menispermaceae
genetic tools and information pertaining to benzylisoqui- (Liscombe et al. 2005). Many BIAs of pharmaceutical
noline alkaloid metabolism is facilitating the study of a importance possess one or more chiral centers, which pre-
wide range of phenomena including the structural biology clude chemical synthesis as an economically viable option
of novel catalysts, the genomic organization of biosynthetic for commercial production. In addition to its pharmaceuti-
genes, the cellular and sub-cellular localization of biosyn- cal importance, opium poppy is also cultivated as an illicit
thetic enzymes and a variety of biotechnological applica- source of morphine, which is easily converted to O,O-dia-
tions. In this review, we highlight recent developments and cetylmorphine, or heroin. Opium poppy is a classic exam-
summarize the frontiers of knowledge regarding the bio- ple of dual-use technology whereby compounds of both
chemistry, cellular biology and biotechnology of benzyliso- great positive and negative value to humanity are produced.
quinoline alkaloid biosynthesis in opium poppy. The biochemistry and physiology of BIA metabolism in
opium poppy have long been investigated. Major advances
Keywords Biosynthetic gene discovery · Cellular were achieved through (1) the application of radiotracer
compartmentalization · Functional genomics · Laticifer · techniques in the 1960s, (2) enzyme isolation and charac-
Papaver somniferum · Sieve element · Specialized terization methodologies in the 1980s and (3) recombinant
metabolism DNA technologies in the 1990s (Hagel and Facchini 2013).
The recent applications of next-generation DNA sequenc-
ing systems and powerful functional genomics meth-
G. A. w. Beaudoin · P. J. Facchini (*)
ods have expedited the rate of discovery. The cumulative
Department of Biological Sciences, University of Calgary,
knowledge acquired over the past half-century provides a
Calgary, AB T2N 1N4, Canada
e-mail: [email protected] remarkable platform for understanding BIA metabolism
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in opium poppy. This review summarizes recent develop- Fig. 1 Biosynthetic pathways leading to major benzylisoqui-▸
ments and current frontiers in our understanding that define noline alkaloids accumulating in opium poppy and other plant spe-
cies. Pathways: pink 1-benzylisoquinoline; green, morphinan;
the status of opium poppy as a model system for the study
dark blue, papaverine; purple, phthalideisoquinoline; orange,
of alkaloid biosynthesis in plants. benzo[c]phenanthridine; light blue, protoberberine; brown, bisben-
zylisoquinoline; dark pink, aporphine. enzymes for which corre-
sponding genes have been isolated from opium poppy or from other
plants are shown in green and blue, respectively. enzymes for which
Biosynthesis of the major alkaloids in opium poppy
corresponding genes have not been isolated are shown in black.
Chemical conversions catalyzed by each enzyme are shown in red.
Many reactions from a limited number of enzyme families TYDC tyrosine/DOPA decarboxylase, 3OHase tyrosine/tyramine
3-hydroxylase, 4HPPDC 4-hydroxyphenylpuruvate decarboxylase,
NCS norcoclaurine synthase, 6OMT norcoclaurine 6-O-methyltrans-
enzymes involved in BIA metabolism belong to a relatively
ferase, CNMT coclaurine N-methyltransferase, NMCH N-methylco-
limited number of protein families, including cytochromes claurine 3′-hydroxylase, 4′OMT2 3′-hydroxyl-N-methylcoclaurine
P450 (CYPs), S-adenosylmethionine-dependent O- and 4′-O-methyltransferase (isoform 2), BBE berberine bridge enzyme,
N-methyltransferases, four distinct groups of NADPH- SOMT1 scoulerine 9-O-methyltransferase, CAS canadine synthase,
TNMT tetrahydroprotoberberine N-methyltransferase, CYP82Y1
dependent dehydrogenases/reductases, FAD-linked oxi-
N-methylcanadine 1-hydroxylase, NOS noscapine synthase, STOX
doreductases (FADOXs), and more restricted numbers of (S)-tetrahydroxyprotoberberineoxidase, CoOMT columbamine
acetyl-CoA-dependent O-acetyltransferases, 2-oxoglutar- O-methyltransferase, CFS cheilanthifoline synthase, SPS stylopine
ate/Fe(II)-dependent dioxygenases (ODDs) and carboxy- synthase, MSH N-methylstylopine 14-hydroxylase, P6H protopine
6-hydroxylase, DBOX dihydrosanguinarine oxidase, SanR sanguinar-
lesterases. The monophylogeny of BIA biosynthesis in the
ine reductase, SalSyn salutaridine synthase, SalR salutaridine reduc-
Ranunculales (Liscombe et al. 2005) facilitates the identi- tase, SalAT salutaridinol 7-O-acetyltransferase, T6ODM thebaine
fication of novel biosynthetic genes based on amino acid 6-O-demethylase, COR codeinone reductase, CODM codeine O-dem-
sequence similarity with previously characterized enzymes. ethylase, N7OMT norreticuline 7-O-methyltransferase, 3′OHase
uncharacterized 3′-hydroxylase, 3′OMT uncharacterized 3′-O-meth-
Additional enzyme families, including UDP-dependent
yltransferase
glucosyltransferases, are undoubtedly involved in unchar-
acterized aspects of alkaloid metabolism based on the
occurrence of signature metabolites, such as glucosylated BIA subgroups, but appear to primarily target 1-benzyliso-
BIAs produced by these enzymes (Liscombe et al. 2009). quinolines and protoberberines (Dang and Facchini 2012).
The remarkable structural diversity of BIAs is achieved N-methyltransferases involved in BIA biosynthesis display
initially through modicfi ation of the 1-benzylisoquinoline substantial sequence similarity and catalyze the transfer
backbone, but multiplies extensively by the addition of of a methyl group to the nitrogen on the isoquinoline ring.
various functional groups. Remodeling of the 1-benzyliso- Despite the occurrence of a similar S-adenosylmethio-
quinoline scaffold is mediated by oxidative enzymes (i.e. nine binding domain, it is still not known whether O- and
CYPs, FADOXs and ODDs). CYPs catalyze a wide variety N-methyltransferases have a common ancestor (Ibrahim
of reactions ranging from hydroxylations to C–C and C–O and Muzac 2000).
couplings and are responsible for the formation of most BIA
structural subgroups with the exception of protoberberines. (S)-Reticuline
In BIA metabolism, FADOXs have only been implicated in
the formation of C–C and C–N bonds, the most notewor- BIA biosynthesis begins with the condensation of two
thy being the berberine bridge enzyme (BBe), which gen- l-tyrosine derivatives, 4-hydroxyphenylacetaldehyde
erates the protoberberine scaffold (Dittrich and Kutchan (4HPAA) and dopamine, via decarboxylation, meta-hydrox-
1991; Facchini et al. 1996; winkler et al. 2006). In contrast, ylation and transamination yielding the precursor to all other
FADOX homologs appear to only catalyze the formation BIAs, (S)-norcoclaurine (Fig. 1). In the formation of 4HPAA,
of C–O bonds in the formation of non-alkaloid specialized l-tyrosine can undergo transamination by l-tyrosine ami-
metabolites in other plants (Custers et al. 2004; Sirikanta- notransferase (TyrAT) (Lee and Facchini 2011) and subse-
ramas et al. 2004). Although ODDs are known to perform quent decarboxylation by an unidentiefi d enzyme isolated as
a variety of hydroxylations, epoxidations and desaturations 4-hydroxyphenylpyruvate decarboxylase (4HPPDC) (Ruef-
in a variety of plant metabolic pathways (Prescott and John fer and Zenk 1987). meta-Hydroxylation of l-tyrosine and/
1996), enzymes from this family have only been shown to or tyramine by another uncharacterized enzyme (referred
catalyze the dealkylation of certain BIA subgroups (Hagel to as 3′OHase in Fig. 1) yields l-dihydroxyphenylalanine
and Facchini 2010; Farrow and Facchini 2013). (DOPA) and dopamine, respectively. Alternatively, l-tyros-
A collection of diverse O-methytransferases catalyze ine and DOPA are converted to tyramine and dopamine by
the regiospecific transfer of a methyl group from S-aden- tyrosine decarboxylase (TYDC) (Facchini and De Luca
osylmethionine to a free hydroxyl moiety on a variety of 1994). The stereoselective Pictet–Spengler condensation of
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(S)-norcoclaurine from 4HPAA and dopamine is catalyzed catalyze the formation of (S)-norcoclaurine in Coptis japon-
by norcoclaurine synthase (NCS), which is a member of the ica (Minami et al. 2007). However, the actual physiological
pathogenesis-related (PR)10/Bet v 1 protein family (Lis- role of this enzyme is not known.
combe et al. 2005; Samanani et al. 2004; Lee and Facchini Many BIAs are derived from the central pathway inter-
2010). In addition to a PR10-type NCS, a second enzyme mediate (S)-reticuline, which is formed via 3′-hydroxyla-
(CjNCS1) sharing similarity with ODDs was reported to tion and a series of O- and N-methylations (Fig. 1). The
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conversion order was inferred by the substrate preference of N7OMT, which is specific to the N-desmethyl pathway,
of the relevant enzymes although recent evidence suggests decreased papaverine levels. In contrast, suppression of
that the pathway is not linear, but rather operates as a grid 7OMT transcript levels did not affect papaverine accumula-
(Desgagné-Penix and Facchini 2012). (S)-Norcoclaurine tion in further support of the N-desmethyl pathway as the
is first converted by norcoclaurine-6-O-methyltransferase major route to papaverine. However, a minor contribution
(6OMT) to (S)-coclaurine (Morishige et al. 2000; Ouna- of the N-methyl pathway to papaverine biosynthesis cannot
roon et al. 2003), which is accepted by coclaurine N-meth- be ruled out.
yltransferase (CNMT) (Choi et al. 2001). N-Methyl- Another study used a transcriptomics approach to com-
coclaurine is hydroxylated by (S)-N-methylcoclaurine pare high and low papaverine cultivars (Patahnk et al.
3′-hydroxylase (NMCH) (Pauli and Kutchan 1998; Frick 2013). Similarly, transcripts encoding enzymes in the
et al. 2007) to 3′-hydroxy-N-methylcoclaurine, which is N-desmethyl pathway were upregulated, whereas tran-
converted by 3′-hydroxy-N-methylcoclaurine 4′-O-methyl- scripts corresponding to 7OMT were downregulated.
transferase (4′OMT) (Morishige et al. 2000) to (S)-reticu- The final step of papaverine biosynthesis involves oxida-
line. Interestingly, only NMCH has been reported to show tion of the fully O-methylated and N-desmethyl compound
strict substrate and stereoisomer specificity, accepting tetrahydropapaverine by dihydrobenzophenanthridine oxi-
(S)-N-methylcoclaurine and not the corresponding (R)- dase (DBOX), an enzyme that also catalyzes the final step
N-methylcoclaurine or N-desmethyl compounds. In con- in the formation of the quaternary benzo[c]phenanthridine
trast, the O- and N-methyltransferases typically accept a sanguinarine (Hagel et al. 2012). Interestingly, DBOX tran-
variety of (R)- and (S)-tetrahydroisoquinolines. Reticuline scripts occur exclusively in the roots of opium poppy sug-
can be further methylated by reticuline 7-O-methyltrans- gesting the possible systemic translocation of papaverine
ferase (7OMT) (Ounaroon et al. 2003) yielding laudanine, from roots to aerial organs.
which can be fully O-methylated to laudanosine by an uni-
dentified 3′-O-methyltransferase (indicated as 3′OMT in Morphine
Fig. 1).
Morphine biosynthesis has only been described in a few
Papaverine plant species restricted to the Papaveraceae, although the
tetracyclic promorphinan alkaloid salutaridine and sev-
Two routes for papaverine biosynthesis have been pro- eral derivatives have been reported in some members of
posed: (1) an N-methyl pathway involving (S)-reticuline the euphorbiaceae (Theuns et al. 1986). The biosynthesis
and the N-demethylation of an unspecified intermediate of morphine (Fig. 1) requires the epimerization of (S)-reti-
by a hypothetical enzyme and (2) an N-desmethyl pathway culine to (R)-reticuline. The reaction mechanism has been
involving (S)-norreticuline and precluding the requirement proposed to proceed via the dehydrogenation of (S)-reticu-
for N-demethylation (Fig. 1). The efficient incorporation of line to a 1,2-dehydroreticulinium ion (Hirata et al. 2004),
radiolabeled N-desmethyl compounds (Brochmann-Hans- which is subsequently reduced to (R)-reticuline. Although
sen et al. 1975; Uprety et al. 1975) and the identification the 1,2-dehydroreticuline synthase (DRS) catalyzing the
of a norreticuline 7-O-methyltransferase (N7OMT), which first conversion is not known, a 1,2-dehydroreticuline
accepts only norreticuline yielding norlaudanine (Pienkny reductase (DRR) potentially involved in the second step has
et al. 2009), provides biochemical support for the N-des- been purified to homogeneity and partially characterized
methyl pathway. A recent study using mass spectrometry (De-eknamul and Zenk 1992). Although (S)-tetrahydroiso-
and heavy stable-isotope labeling of (S)-reticuline, (S)- quinoline intermediates are assumed as the major route to
laudanine and (S)-laudanosine suggested that N-methylated (R,S)-reticuline, most of the enzymes involved accept both
1-benzylisoquinolines are incorporated into papaverine at S and R epimers. In some plants, (R)-tetrahydroisoquino-
low levels (Han et al. 2010), providing some support for the lines are required for the synthesis of BIA structural sub-
N-methyl pathway. However, most of the labeled (S)-reticu- groups other than morphinan alkaloids, including the for-
line was incorporated into morphinan alkaloids. mation of the bisBIA berbamunine in Berberis stolonifera
The use of virus-induced gene silencing (vIGS) further via the C–O coupling of (S)- and (R)-N-methylcoclaurine
demonstrated that the major route to papaverine is likely by the CYP berbamunine synthase (CYP80A1) (Fig. 1)
through the N-desmethyl pathway and showed that nor- (Stadler and Zenk 1993; Kraus and Kutchan 1995).
reticuline and other N-desmethyled 1-benzylisoquinolines (R)-Reticuline is converted by the CYP salutaridine
are natural metabolites in opium poppy (Desgagné-Penix synthase (CYP719B1) to salutaridine (Gesell et al. 2009),
and Facchini 2012). The vIGS-mediated suppression of which is then converted by the short-chain dehydroge-
CNMT transcript levels in opium poppy plants increased nase/reductase salutaridine reductase (SalR) (Ziegler
papaverine accumulation, whereas reduced transcript levels et al. 2006). Subsequently, salutaridinol is O-acetylated
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by salutaridinol 7-O-acetyltransferase (SalAT) (Grothe enzyme operating downstream of (R)-reticuline (wijekoon
et al. 2001; Lenz and Zenk 1995). Interestingly, salutari- and Facchini 2012).
dinol 7-O-acetate undergoes spontaneous cyclization to the
first pentacyclic morphinan alkaloid thebaine at pH 8–9, Protoberberines, protopines and sanguinarine
whereas a dibenz[d,f]azonine alkaloid is formed at pH 6–7,
suggesting compartmentalization of the intermediate in a (S)-Scoulerine, the branch-point intermediate leading to
basic environment or the involvement of an uncharacter- protoberberine alkaloids, is formed from (S)-reticuline by
ized enzyme (Fisinger et al. 2007). A reduced derivative, BBe (Dittrich and Kutchan 1991; Facchini et al. 1996;
neodihydrothebaine has only been reported in Papaver Kutchan and Dittrich 1995; winkler et al. 2006). (S)-
bracteatum (Theuns et al. 1984). Scoulerine is converted via methylenedioxy bridge for-
The morphine pathway bifurcates at thebaine with mation and O-methylation to a variety of protoberberines
the initial step in the major route catalyzed by thebaine including (S)-canadine, (S)-stylopine and (S)-sinactine.
6-O-demethylase (T6ODM) yielding neopinone, which (S)-Canadine, the precursor to the protopine allocryptopine
undergoes purportedly spontaneous rearrangement to and the phthalideisoquinoline noscapine, is formed sequen-
codeinone (Hagel and Facchini 2010). Codeinone is then tially from (S)-scoulerine by (1) scoulerine 9-O-methyl-
reduced by the aldo–keto reductase codeinone reductase transferase (SOMT1) (Dang and Facchini 2012; Takeshita
(COR) (Unterlinner et al. 1999) to codeine, which is con- et al. 1995) yielding tetrahydrocolumbamine and (2) the
verted to morphine by codeine O-demethylase (CODM). methylenedioxy bridge forming enzyme canadine synthase
Alternatively, the CODM catalyzes the first step in the (CAS), which is a member of the CYP719A subfamily
minor route, which involves the 3-O-demethylation of (Díaz Chávez et al. 2011; Ikezawa et al. 2003; winzer et al.
thebaine to oripavine. T6ODM then converts oripavine 2012). Similarly, (S)-sinactine is formed from (S)-scouler-
to morphinone, which is reduced by COR to morphine ine by (1) the addition of a methylenedioxy bridge by
(Fig. 1). cheilanthifoline synthase, also a member of the CYP719A
The physiological role of BIA biosynthetic enzymes has subfamily (Díaz Chávez et al. 2011; Ikezawa et al. 2009),
been investigated using a variety of gene suppression and followed by (2) 2-O-methylation of cheilanthifoline by an
over-expression methods in opium poppy. The RNAi-medi- enzyme not yet characterized from opium poppy. A proto-
ated suppression of COR transcript levels resulted in the berberine 2-O-methyltransferase, columbamine O-meth-
accumulation of reticuline and methylated 1-benzylisoqui- yltransferase, has been reported from meadow rue (Coptis
noline derivatives with a corresponding decrease in morph- japonica) (Morishige et al. 2002). Alternatively, cheilanthi-
inan alkaloid content (Allen et al. 2004). In support of a foline can be oxidized to stylopine by another CYP719A,
role for COR in the modulation of morphine biosynthesis, stylopine synthase (SPS) (Ikezawa et al. 2007; Díaz Chávez
over-expression of COR resulted in a significant increase et al. 2011).
in morphinan alkaloid levels (Larkin et al. 2007). Simi- Protopine alkaloids are formed via the 14-hydroxyla-
larly, over-expression of SalAT also increased morphinan tion of quaternary protoberberine alkaloids, which leads
alkaloid content, whereas suppression of SalAT transcript to ring tautomerization through C–N bond cleavage and
levels caused the accumulation of salutaridine, but did formation of a C14 keto moiety. Quaternary protoberber-
not alter the abundance or profile of morphinan alkaloids ines are formed by (S)-tetrahydroprotoberberine N-methyl-
(Allen et al. 2008). The physiological roles of T6ODM transferase (TNMT) and 14-hydroxylation is catalyzed by
and CODM were suggested in the opium poppy mutant (S)-cis-N-methylstylopine 14-hydroxylase (MSH). Opium
top1 (Millgate et al. 2004), which accumulates high levels poppy TNMT was shown to accept a variety of protober-
of thebaine and oripavine at the expense of codeine and berine substrates, but displays a preference for stylopine
morphine. The absence of T6ODM transcripts in a high- and canadine (Liscombe and Facchini 2007). MSH is a
thebaine/oripavine, low-codeine/morphine opium poppy member of the CYP82 N subfamily and accepts a variety
chemotype was compared to three high-codeine/morphine of quaternary protoberberines (Rueffer and Zenk 1987;
varieties to identify the corresponding gene using a micro- Beaudoin and Facchini 2013). The protopines allocrypto-
array-based approach (Hagel and Facchini 2010). Suppres- pine, cryptopine and protopine are formed from canadine,
sion of T6ODM transcript levels resulted in the accumula- sinactine and stylopine, respectively. T6ODM, CODM and
tion of thebaine and oripavine and substantially reduced other ODDs are potentially involved in regulating proto-
levels of codeine and morphine. Similarly, suppression pine alkaloid biosynthesis through the oxidative dealkyla-
of CODM transcript levels caused the accumulation of tion of methylenedioxy bridges or methoxy groups (Farrow
codeine at the expense of morphine (Hagel and Facchini and Facchini 2013).
2010). The isolation of all biosynthetic genes facilitated a Formation of the root-specific benzo[c]phenanthridine
coordinated confirmation of the physiological role of each alkaloid dihydrosanguinarine (Fig. 1) from protopine is
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catalyzed by protopine 6-hydroxylase (P6H), another Based on the isolation of purported pathway intermedi-
member of the CYP82 N subfamily (Tanahashi and Zenk ates and the identification of the enzymes involved in the
1990; Takemura et al. 2012). The resulting 6-hydroxylation formation of N-methylcanadine, a metabolic scheme for
results in a spontaneous rearrangement of 6-hydroxyproto- noscapine biosynthesis was proposed (Fig. 1); (Facchini
pine to dihydrosanguinarine, which is oxidized to sangui- et al. 2007). The scheme involves sequential hydroxyla-
narine by the FAD-linked enzyme dihydrobenzophenan- tion of the isoquinoline ring, oxidative cleavage of the
thridine oxidase (DBOX) (Hagel et al. 2012). Interestingly, berberine bridge and two subsequent oxidations to form
sanguinarine can be reduced to dihydrosanguinarine by the characteristic phthalideisoquinoline lactone ring. Fol-
sanguinarine reductase (SanR) (vogel et al. 2010; weiss lowing the identification of relevant O-methyltransferases
et al. 2006). The physiological role of SanR has been sug- (Dang and Facchini 2012), a gene cluster proposed to
gested as the mitigation of the cytotoxic effects of sangui- encode all noscapine biosynthetic enzymes was identi-
narine via conversion to the less-toxic dihydrosanguinarine. fied in opium poppy. The functions for some gene cluster
Further oxidized protoberberines are common in some members were suggested using vIGS to show correlations
members of the Papaveraceae and are widespread in related between the suppression of a specific gene and the accumu-
plant families. In particular, berberine is formed from (S)- lation of compounds purported as pathway intermediates
canadine by the FAD-linked enzyme (S)-tetrahydropro- (winzer et al. 2012). The function of metabolic gene clus-
toberberine oxidase (STOX) (Fig. 1) (Amann et al. 1984; ters in plants has been suggested as a mechanism to regu-
Gesell et al. 2011; Matsushima et al. 2012). However, ber- late expression at the epigenetic level (wegel et al. 2009)
berine and other modified protoberberines have generally and to facilitate complete inheritance of a specialized meta-
not been reported in opium poppy (Shamma and Moniot bolic pathway (Swaminathan et al. 2009). Recently, CAS
1978; Shulgin and Perry 2002). Moreover, protoberber- (Dang and Facchini 2014a), N-methylcanadine 1-hydroxy-
ines in which the berberine bridge has formed between lase (CYP82Y1) (Dang and Facchini 2014b) and noscapine
the N-methyl and the 6′ carbons, as opposed to the 2′ posi- synthase (NOS) (Chen and Facchini 2014) were isolated
tion (i.e. yielding scoulerine), have been reported in other and characterized from opium poppy. NOS was character-
plants, but are not detected in opium poppy (Tsai and Lee ized as a NAD -dependent, short-chain dehydrogenase/
+
2010). reductase catalyzing the irreversible conversion of nar-
It is interesting to note that other species accumulate cotinehemiacetal to noscapine.
a variety of benzo[c]phenanthridine alkaloids derived
from corresponding protoberberine alkaloids with differ- Other alkaloids
ential methylation patterns. However, the only abundant
benzo[c]phenanthridine alkaloids in opium poppy are dihy- The rhoeadine alkaloids, some of which are also known as
drosanguinarine and sanguinarine, which suggests that papaverrubine alkaloids, are found exclusively in members
P6H might exhibit relatively narrow substrate specificity of the Papaveraceae, including opium poppy (Montgomery
in opium poppy. Alternatively, the sanguinarine pathway et al. 1983). Radiolabeling studies in Persian poppy (Papa-
could involve inter- or intra-cellular compartmentalization ver bracteatum) showed that the precursors to the rhoeadine
restricting the availability of substrates, other than proto- alpinigenine were the protoberberines tetrahydropalmatine
pine, to P6H. (Rönsch 1972), N-tetrahydropalamatine and the protopine
muramine (Rönsch 1977). A route through protopines was
Noscapine confirmed in Papaver rhoeas by feeding protopine, which
was converted to rhoeadine (Tani and Tagahara 1977).
In some opium poppy chemotypes, the phthalideisoquino- Labeling studies also showed that the pro-S-13-hydrogen
line alkaloid noscapine accounts for a majority of the total of C13 is stereospecifically removed, suggesting that the
alkaloid content in latex (Frick et al. 2005). Noscapine also cyclization is enzyme-mediated and not spontaneous (Bat-
occurs in other members of the Papaveraceae and Men- tersby and Staunton 1974). Many rhoeadines isolated from
ispermaceae. Owing to its cough-suppressing activity and opium poppy are 10-O-desmethylated (Montgomery et al.
potential as an anticancer drug (Mahmoudian and Rahimi- 1983). The vIGS-mediated suppression of gene transcripts
Moghaddam 2009), the biosynthesis of noscapine has been encoding CODM, which catalyzes the O-demethylation
the subject of renewed interest (Dang and Facchini 2012; and/or O,O-demethylenation of morphinan and protopine
Facchini et al. 2007; winzer et al. 2012). The formation alkaloids, resulted in elevated levels of rhoeadine alkaloids,
of noscapine has not been as extensively investigated as possibly owing to the increased accumulation of crypto-
other alkaloids, such as morphine. For decades, the role of pine and 10-O-desmethylcryptopine (Farrow and Facchini
scoulerine as a pathway intermediate was the extent of our 2013). enzymes involved in the conversion of protopines to
knowledge concerning noscapine biosynthesis. rhoeadines are not known.
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Planta (2014) 240:19–32 25
Regulation of alkaloid metabolism an ABC transporter performs a similar function in opium
poppy. The major sites of BIA biosynthetic gene transcript
Cellular localization and transport and enzyme localization and the possible points of translo-
cation within the morphine pathway are summarized sche-
The biosynthesis and storage of BIAs in opium poppy has matically in Fig. 2.
been extensively investigated and shown to involve three
distinct cell types (Facchini and De Luca 1995; Bird et al. Subcellular compartmentalization and trafficking
2003; Samanani et al. 2006; Lee and Facchini 2010). In
situ hybridization and immunofluorescence labeling dem- In opium poppy, the antimicrobial alkaloid sanguinar-
onstrated the localization of biosynthetic gene transcripts ine is not normally present in the latex, but accumulates
and enzymes to companion cells and sieve elements, constitutively in roots. The subcellular localization of
respectively. Since all characterized genes and enzymes most enzymes involved in the biosynthesis of sanguinar-
involved in the formation of (S)-reticuline, and some func- ine has been investigated in cultured opium poppy cells
tioning in various branch pathways, were initially local- using microprojectile bombardment followed by imaging
ized to companion cells and sieve elements, respectively, of green fluorescent protein fusions (Hagel and Facchini
laticifers were proposed to function only in the storage of 2012). As shown previously by density gradient fractiona-
alkaloids. The model proposed that BIA biosynthetic genes tion (Amann et al. 1986; Rueffer and Zenk 1987; Tanahashi
are expressed in companion cells and the corresponding and Zenk 1990; Bauer and Zenk 1991), and using immu-
enzymes are subsequently translocated to sieve elements. nogold localization (Alcantara et al. 2005), BBe and CYPs
Alkaloids synthesized in sieve elements are finally trans- associate with the endoplasmic reticulum (eR). Opium
ported to nearby laticifers for storage in large cytoplasmic poppy BBe was also shown to contain a 25-amino acid,
vesicles. N-terminal signal peptide directing the enzyme to the eR,
However, SalAT and COR transcripts have also been and an adjacent vacuolar-sorting determinant, resulting
reported in the cytoplasm, or latex, of laticifers (Decker in its final translocation to the vacuole (Bird and Facchini
et al. 2000; Larkin et al. 2007; Allen et al. 2008), although 2001). Interestingly, the intracellular translocation of NCS
in situ hybridization and immunofluorescence labeling was similar to that reported for BBe, predicting the exten-
failed to detect SalAT and COR mRNA and corresponding sive subcellular trafficking of sanguinarine biosynthetic
enzymes in laticifers (Bird et al. 2003; weid et al. 2004; pathway intermediates. The compartmentalization of NCS
Samanani et al. 2006). Recently, the application of shot- in the eR lumen requires the concomitant translocation of
gun proteomics to determine the relative abundance of dopamine and 4HPAA, as depicted schematically in Fig. 3.
proteins in isolated latex, or in whole stem including latex, As expected, O- and N-methyltransferases were detected as
revealed that the final three steps in morphine biosynthesis, cytosolic enzymes; thus, the subsequent enzyme (6OMT)
catalyzed by T6ODM, COR and CODM, occur primarily predicts the immediate export of (S)-norcoclaurine from
in laticifers (Onoyovwe et al. 2013). Other BIA biosyn- the eR. The biosynthesis of (S)-reticuline could involve a
thetic enzymes abundant in laticifers include NOS (Chen metabolic channel anchored to the cytosolic face of the eR
and Facchini 2014) and 7OMT (Onoyovwe et al. 2013), by NMCH, although no evidence is yet available to support
indicating that the final steps in the formation of noscapine interactions among the relevant enzymes. The co-compart-
and 7-O-methylated derivatives of reticuline also occur in mentalization of BBe in the eR lumen implies that (S)-reti-
latex and are spatially separated from upstream enzymes. culine is also imported from the cytosol for conversion to
Top candidates for transport from sieve elements to latic- (S)-scoulerine, which is promptly exported. Four of the five
ifers include thebaine and narcotinehemiacetal in the mor- subsequent enzymes are CYPs with active domains on the
phine and noscapine pathways, respectively. However, cytosolic face of the eR, with a cytosolic enzyme (TNMT)
the detection of most pathway intermediates in isolated operating in the middle. Although not localized as a GFP
latex suggests a relatively non-specific transport process. fusion along with the other enzymes (Hagel and Facchini
Although the mechanism of translocation is unknown, the 2012), DBOX contains a predicted N-terminal signal pep-
occurrence of plasmodesmata between opium poppy sieve tide (Hagel et al. 2012) and related STOX enzymes have
elements and laticifers (Facchini and DeLuca 2008) sug- been associated with the eR (Amann et al. 1988); thus, a
gests that both symplastic and apoplastic transport routes third reentry of a pathway intermediate into the eR lumen
are possible. An ATP-binding cassette (ABC) transporter is predicted. Oxidation of dihydrosanguinarine in the eR
has been implicated in the uptake (Sakai et al. 2002) and would facilitate the vesicle-mediated transport of sangui-
efflux (Terasaka et al. 2003) of berberine in Coptis japon- narine to the central vacuole of cultured opium poppy cells
ica, and the corresponding gene has been isolated (Shi- (Alcantara et al. 2005). The proposed subcellular traffick-
tan et al. 2003, 2012). However, it is not known whether ing features of sanguinarine biosynthesis might also occur
1 3
2 6 Planta (2014) 240:19–32
Fig. 2 Cellular localization
model showing the roles of
companion cells, sieve elements
and laticifers in the biosyn-
thesis of morphine in opium
poppy. Genes expressed in each
cell type are shown in italics,
whereas enzymes are indicated
in red. The horizontal black
arrow suggests thebaine as
the major pathway intermedi-
ate translocated between sieve
elements and laticifers. Font
size is generally proportional to
relative transcript and enzyme
levels. Abbreviations are pro-
vided in the legend of Fig. 1
Fig. 3 Subcellular localization
model showing the proposed
compartmentalization of sangui-
narine biosynthetic enzymes in
cultured opium poppy cells. The
red arrow tracks the intracel-
lular transport of pathway
intermediates from the precur-
sors dopamine and 4-hydroxy-
phenylacetaldehyde (4HPAA) to
sanguinarine. The green arrow
shows the proposed trafficking
of sanguinarine from the lumen
of the endoplasmic reticulum to
the central vacuole. Abbrevia-
tions are provided in the legend
of Fig. 1
1 3
Planta (2014) 240:19–32 27
in other BIA branch pathways, although no empirical evi- The first transcriptional regulator of BIA metabolism
dence is available. vacuolar transport in Coptis japonica was identified from C. japonica using RNAi to silence five
has been reported to depend on a H /berberine antiporter putative regulatory factors showing reduced expression in a
+
(Otani et al. 2005), but the process in opium poppy has not cultured cell line with relatively low BIA biosynthetic gene
been investigated. transcript levels (Kato et al. 2007). The transient silencing
of one candidate (CjWRKY1) resulted in a marked decrease
Hormonal regulation in berberine biosynthetic gene transcripts. In contrast, over-
expression of CjWRKY1 increased berberine biosynthetic
The phytohormonal activation of BIA metabolism has gene transcript levels. A bHLH transcriptional regulator
been used to identify biosynthetic genes in plants related involved in berberine biosynthesis was isolated using a sim-
to opium poppy. Addition of cytokinin to Thalictrum minus ilar approach (Yamada et al. 2011). In contrast, wounding
cell cultures caused a marked increase in the accumulation and other stimuli to elicit a defense response was used to
of berberine resulting from increased expression of 6OMT identify expressed sequence tags corresponding to induced
(Hara et al. 1994) and STOX (Hara et al. 1995). ethylene gene transcripts in opium poppy plants (Mishra et al.
was also shown to promote berberine biosynthesis in T. 2013). A putative wKRY transcription factor was isolated
minus cell cultures, and the potent ethylene inhibitor silver and shown to bind conserved wRKY binding elements in
thiosulfate caused a substantial decrease in berberine accu- promoters of certain biosynthetic genes in vitro and using
mulation (Kobayashi et al. 1991). a yeast one-hybrid approach. Another wRKY transcrip-
Industry-sponsored research has focused on increas- tion factor was identified from opium poppy T-DNA inser -
ing the levels of morphinan pathway intermediates at the tion lines displaying altered BIA accumulation (Kawano
expense of morphine through the application of specific et al. 2012). Moreover, the ectopic expression of an Arabi-
phytohormones or phytohormone catabolic inhibitors. dopsis thaliana wRKY transcription factor in California
The application of trinexapac-ethyl, an inhibitor of ODDs poppy (Eschscholzia californica) caused the induction of
involved in gibberellin (GA) biosynthesis, with or without BBE and NMCH transcripts, and a substantial increase in
methyl jasmonate results in the introduction of a putative the accumulation of benzo[c]phenanthridine alkaloids,
catabolic bottleneck leading to an increase in the relative including sanguinarine (Apuya et al. 2008). Interestingly,
abundance of thebaine and codeine and a decrease in the several putative wRKY elements occur within or near the
accumulation of morphine (Cotterill 2010). Interestingly, promoter regions of biosynthetic genes in the reported
trinexapac-ethyl and other acylcyclohexanediones do not noscapine gene cluster (winzer et al. 2012), suggesting that
inhibit T6ODM and CODM directly (Hagel and Facchini noscapine biosynthesis might also be regulated by wRKY
2010). exogenous addition of GA was reported to increase factors.
3
the yield of morphine (Khan et al. 2007), suggesting a sub-
stantial role for phytohormone control in morphinan alka-
loid metabolism. The developmental and phytohormonal Metabolic engineering
control of BIA biosynthesis in opium poppy is a key area
for future research, which could have direct consequences Plants
on the commercial production of opiate pharmaceuticals.
Metabolic engineering has been used to modulate alkaloid
Transcriptional regulation composition and yield in opium poppy plants, with mixed
results. Opium poppy plants engineered with constitutively
The transcriptional regulation of BIA biosynthesis has expressed or antisense-suppressed NMCH showed substan-
recently been investigated in opium poppy (Kawano et al. tial modulations in overall alkaloid content, but the BIA
2012; Mishra et al. 2013), but has been more extensively profile was not altered (Frick et al. 2007). In contrast, over-
studied in Coptis japonica (Kato et al. 2007; Yamada et al. expression of COR (Larkin et al. 2007) and SalAT (Allen
2011). The importance of transcriptional control in BIA et al. 2008) specifically resulted in increased morphinan
metabolism is particularly evident from (1) the cell type- alkaloid accumulation. Surprisingly, the RNAi-mediated
specific localization of biosynthetic gene transcripts (Bird silencing of COR (Allen et al. 2004) did not result in the
et al. 2003; Samanani et al. 2006; Lee and Facchini 2010; accumulation of codeine, but rather caused a substan-
Onoyovwe et al. 2013) and (2) the coordinated induc- tial accumulation of (S)-reticuline and a concomitant
tion of all expressed biosynthetic gene transcripts (Zulak decrease in the levels of morphine pathway intermediates
et al. 2007) and corresponding proteins (Zulak et al. 2009; downstream of salutaridine. The biochemical basis for the
Desgagné-Penix et al. 2010) in opium poppy cell cultures accumulation of an intermediate several enzymatic steps
after the addition of a fungal-derived elicitor. upstream of the silenced gene is not known, but suggestions
1 3
2 8 Planta (2014) 240:19–32
include the possible occurrence of (1) feedback regulation protoberberine alkaloids and salutaridine (Hawkins and
and (2) metabolic channels (Allen et al. 2004). The occur- Smolke 2008).
rence of multienzyme complexes in morphine biosynthesis
is supported by the suppression of SalAT in using RNAi Future prospects
(Allen et al. 2008). SalAT-silenced plants showed increased
levels of salutaridine, which is not normally abundant in Our knowledge of alkaloid biosynthesis in opium poppy
opium poppy. The accumulation of salutaridine was unex- has undergone two periods of major advancement: the first,
pected since the SalAT enzymatic substrate (salutaridinol) several decades ago, involving elucidation of the chemis-
did not accumulate. Thus, salutaridine might be channeled try of some pathways and the second, over the past decade,
to thebaine through an enzyme complex that includes SalR highlighting the isolation of most key biosynthetic genes
and SalAT. Finally, over-expression of A. thaliana tran- largely facilitated by rapid developments in genomics. The
scriptional regulators in opium poppy resulted in increased availability of complete pathways at the genetic level will
thebaine and codeine accumulation associated with the up- facilitate renewed advances in plant breeding and meta-
regulation of several BIA biosynthetic enzymes (Apuya bolic engineering and could lead to the establishment of
et al. 2008). alternative production systems, perhaps the most revolu-
tionary being microbes. Beyond the isolation and charac-
Microbes terization of enzymes, aspects of alkaloid biosynthesis that
still require considerable research include (1) pathway reg-
Microbes engineered to express genes encoding BIA-bio- ulation, at the levels of both gene expression and metabolic
synthetic enzymes provide a novel approach for the devel- biochemistry, and (2) cellular biology, especially the inter-
opment of scalable manufacturing processes. The avail- and intra-cellular transport processes involved in the forma-
ability of large numbers of BIA biosynthetic genes from tion and storage of compounds. The dedicated efforts of a
opium poppy and related plants has facilitated the recon- relatively small number of research groups over the past
stitution of several pathways leading to the production half-century have generated an impressive body of infor-
of (S)-reticuline and (R,S)-reticuline in E. coli (Minami mation and elevated opium poppy to the status of a model
et al. 2008) and Saccharomyces cerevisiae (Hawkins and system in the study of alkaloid biosynthesis in plants.
Smolke 2008), respectively. Production of (S)-reticuline
in E. coli was initially achieved by supplementing the Acknowledgments Research in the laboratory of the correspond-
ing author was supported through funding from Genome Canada,
bacterial culture medium with dopamine, some of which
Genome Alberta, the Government of Alberta, the Canada Founda-
was converted to 3,4-dihydroxyphenylacetaldehyde (3,4-
tion for Innovation Leaders Opportunity Fund, and the Natural Sci-
DHPAA) by a heterologously expressed bacterial mono- ences and engineering Research Council of Canada. P.J.F. holds the
amine oxidase (Minami et al. 2008). PR10-type NCS from Canada Research Chair in Plant Metabolic Processes Biotechnology.
G.A.w.B. is the recipient of scholarships from the Natural Sciences
C. japonica catalyzed the condensation of dopamine and
and engineering Research Council of Canada, Alberta Innovates
3,4-DHPAA to (S)-norlaudanosoline, which underwent
Technology Futures, and the fonds québécois de la recherche sur la
successive methylations via 6OMT, CNMT and 4′OMT to nature et les technologies.
yield (S)-reticuline. Recent modifications of this platform
include the de novo synthesis of dopamine by the expres-
sion of two additional enzymes, tyrosinase and DOPA
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