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Chapter 8
Biosynthesis and Regulation of Alkaloids
G. Guirimand, V. Courdavault, B. St-Pierre, and V. Burlat
8.1 Introduction
Plant secondary metabolites, also commonly named plant natural products, nowadays
encompassed about 100,000 chemically identified, low molecular weight compounds.
These molecules are commonly synthesised in a plant-, organ- and even cell-specific
manner. Alkaloids constitute a very chemically diverse group of secondary metabo-
lites with an estimated 12,000 different molecules sharing as a unique common
feature the presence of a nitrogen atom within a heterocyclic ring. Many alkaloids
are toxic, in agreement with some function in plant defence. This toxicity has long
been understood by humans, and some alkaloids are very well known for their human
health benefit, leading to the development of many recent research projects towards
understanding the architecture and the regulation of their long and complex biosyn-
thetic pathways. The purpose of this chapter is to give an overview of some of the
major advances obtained recently with the most prominent model species, including
the architecture and the spatial organisation of biosynthetic pathways, the crystal-
lisation and modelling of alkaloid biosynthetic enzymes and the transcription factor
regulatory networks of alkaloid biosynthesis. The ultimate goal of such research is the
application of the discoveries to develop metabolic engineering strategies to over-
come the usually very low yield of production for these biomolecules. In the mean-
time, some peculiarities of plant physiology have been elucidated in these highly
G. Guirimand, V. Courdavault, and B. St-Pierre
Universite
´Franc¸ois Rabelais de Tours, EA 2106 “Biomole
´cules et Biotechnologies Ve
´ge
´tales”,
IFR 135, “Imagerie Fonctionnelle”, 37200, Tours, France
e-mail: vincent.courdavault@univ-tours.fr
V. Burlat
Surfaces Cellulaires et Signalisation chez les Ve
´ge
´taux, UMR 5546 CNRS - UPS - Universite
´de
Toulouse, Po
ˆle de Biotechnologie Ve
´ge
´tale, 24 chemin de Borde-Rouge, BP 42617 Auzeville,
31326, Castanet-Tolosan, France
Universite
´Franc¸ois Rabelais de Tours, EA 2106 “Biomole
´cules et Biotechnologies Ve
´ge
´tales”,
IFR 135, “Imagerie Fonctionnelle”, 37200, Tours, France
e-mail: burlat@scsv.ups-tlse.fr
E‐C. Pua and M.R. Davey (eds.),
Plant Developmental Biology – Biotechnological Perspectives: Volume 2,
DOI 10.1007/978-3-642-04670-4_8, #Springer-Verlag Berlin Heidelberg 2010
139
specialised plant species, illustrating the fundamental interest of studying such sec-
ondary metabolic pathways beyond their primary interest for industrial application.
8.2 Chemical Diversity and Biosynthesis
Alkaloids are classified in several families that present totally different biosynthetic
pathways. Four major families, for which the biosynthesis and the regulation are
more particularly studied, are discussed in this chapter, these being monoterpene
indole alkaloids (MIA), benzylisoquinoline alkaloids (BIA), tropane and nicotine
alkaloids (TNA) and purine alkaloids (PA; Fig. 8.1, Table 8.1). Despite their
chemical diversity, alkaloids share the fact that they originate commonly from
primary metabolites such as amino acids or bases (Fig. 8.1). Except for Nicotiana
tabacum, no genome sequencing project exists for the major alkaloid-producing
plants. Therefore, most of the enzymatic steps have been identified using classical
biochemical and molecular biology studies. The ongoing elucidation of some
biosynthetic pathways illustrates the recruitment of enzymes belonging to recurrent
multigene families such as cytochrome P450 monooxygenases, acetyl transferases
or methyltransferases. Recent expressed sequence tags (EST) transcriptomic pro-
jects focusing on MIA-producing species (Murata et al. 2006,2008; Rischer et al.
2006; Shukla et al. 2006), BIA-producing species (Morishige et al. 2002; Carlson
et al. 2006; Ziegler et al. 2006; Kato et al. 2007; Zulak et al. 2007) and TNA-
producing species (Li et al. 2006) have been helpful for the identification of some
missing enzymatic steps, and should accelerate this process in the near future
(Table 8.2). Metabolic profiling studies of the major alkaloid-producing species
have also been performed recently (Dra
¨ger 2002; Yamazaki et al. 2003; Schmidt
et al. 2007; Hagel et al. 2008), sometimes in association with transcriptomic studies
(Rischer et al. 2006; Ziegler et al. 2006; Zulak et al. 2007; Hagel and Facchini
2008). Together, these approaches now allow tremendous progress in understand-
ing these complex alkaloid biosynthetic pathways.
The purpose of this section is more to give an overlook and a link to references,
including major recent reviews on the different biosynthetic pathways, than to give
an exhaustive detailed description of all these complicated biochemical processes.
8.2.1 Biosynthesis of Monoterpene Indole Alkaloids (MIA)
The approximately 2,000 MIA chemical structures described so far are widespread
in a large number of plant species (Ziegler and Facchini 2008). Some of these
molecules are of interest to human health, such as the anticancer drugs vinblastine
and vincristine and the antihypertensive drug ajmalicine specifically produced in
Catharanthus roseus, the anti-arrythmic ajmaline produced in Rauvolfia serpen-
tina, or the anticancer compound camptothecin produced mostly in Camptotheca
acuminata (Fig. 8.1, Table 8.1). These molecules are part of the large array of MIA
140 G. Guirimand et al.
OPO3--
OH
OH
H3C
OH
NH2
N
H
COOH
N
N
H
CO2CH3
CO2CH3
HN
NO-COCH3
HO
H3CH
H
MeO
OH
NH2
COOH
HO
O
O
O
O
N+
CH3
HO
O
HO
HNCH3
H
NH2
N
H
NH
COOH
H2N
O
HCH2OH
O
N
CH3
O
N
N
CH3
H
O
HOH2C
OH OH
N
N
N
H
O
O
N
N
HN
HN
N
O
O
CH3
CH3
CH3
N
N
N
N
O
O
CH3
H3C
MEP argininetryptophan tyrosine
xanthosine
theobromine
caffeinescopolamine nicotinesanguinarine morphinevinblastine
MIA BIA TNA PA
Fig. 8.1 Chemical structures of precursors and examples of alkaloids from the four families where the major advances in the elucidation and regulation of the
biosynthetic pathways have been obtained. MIA, monoterpene indole alkaloids; BIA, benzylisoquinoline alkaloids; TNA, tropane and nicotine alkaloids; PA, purine
alkaloids; MEP, 2-C-methyl-D-erythritol 4-phosphate
8 Biosynthesis and Regulation of Alkaloids 141
Table 8.1 Sources, chemical diversity and biological activity of four major families of alkaloids
Alkaloid family Family (plant
species)
Active compounds Pharmacological activity Target
Monoterpene indole alkaloids
(MIA)
Apocynaceae
Catharanthus roseus Vinblastine,
vincristine
Anticancer Tubulin
Ajmalicine Antihypertensive aAdrenergic receptor
Rauvolfia serpentina Ajmaline Anti-arrythmic Na
+
channels
Nyssaceae
Camptotheca
acuminata
Camptothecin Anticancer DNA topoisomerase I
Benzylisoquinoline alkaloids
(BIA)
Papaveraceae
Papaver somniferum Codeine Antitussive, analgesic Nicotinic acetylcholine (nACh) receptor
Morphine Analgesic, narcotic m3 Opioid receptor
Papaverine Spasmolytic, vasodilators c-AMP phosphodiesterase
Eschscholzia
californica
Sanguinarine Antibacterial, proapoptotic FtsZ (bacterial cytokinesis), mitoch.
pathway
Ranunculaceae
Thalictrum flavum Berberine Antibacterial, antimicrobial DNA
Coptis japonica Berberine,
sanguinarine
Tropane and nicotine alkaloids
(TNA)
Solanaceae
Hyoscyamus niger Hyoscyamine Anticholinergic, narcotic,
myorelaxant
Muscarinic receptor
Datura stramonium Scopolamine Anticholinergic, narcotic,
myorelaxant
Muscarinic receptor
Atropa belladonna Hyoscyamine Anticholinergic, narcotic,
myorelaxant
Muscarinic receptor
Nicotiana tabacum Nicotine Neurostimulant, insecticide nACh receptor
Purine alkaloids (PA) Coffeeae
Coffea arabica Caffeine Central nervous system
stimulant
Adenosine A
1
&A
2A
receptors;
phosphodiesterase
Theaceae
142 G. Guirimand et al.
Camellia sinensis Caffeine,
theophylline
Central nervous system
stimulant
Adenosine A
1
&A
2A
receptors;
phosphodiesterase
Byttnerioideae
Theobroma cacao Theobromine,
caffeine
Central nervous system
stimulant
Adenosine A
1
&A
2A
receptors;
phosphodiesterase
8 Biosynthesis and Regulation of Alkaloids 143
Table 8.2 Molecular resources for alkaloid biosynthesis
Family (plant species) NCBI hints
(nt seq)
NCBI hints
(EST)
Alkaloid biosynthetic enzymes (GenBank access numbers, full enzyme name and
abbreviation)
Alkaloid family: monoterpene indole alkaloids (MIA)
Apocynaceae
Catharanthus roseus 20,820 19,899 25 [CAA09804(1-deoxyxylulose 5-phosphate synthase: dxs); ABI35993(dxs2);
AAF65154(1-deoxy-D-xylulose-5-phosphate reductoisomerase: dxr); ACI16377 (4-
diphosphocytidyl-methylerythritol 2-phosphate synthase: cms); ABI35992(4-
diphosphocytidyl-2-C-methyl-D-erythritol kinase: cmk); AAF65155(2C-methyl-D-
erythritol 2,4-cyclodiphosphate synthase: mecs); AAO24774(4-hydroxy-3-methyl-2-
butenyl diphosphate synthase: hds); ABI30631(1-hydroxy-2-methyl-butenyl 4-
diphosphate reductase: hdr); ABW98669(isopentenyl pyrophosphate:dimethylallyl
pyrophosphate isomerase: idi); ACC77966(geranyl pyrophosphate synthase: gpps);
CAC80883(CYP76B6=geraniol 10-hydroxylase: g10h); Q05001(NADPH
cytochrome P450 reductase: cpr); AAQ55962(10-hydroxygeraniol oxidoreductase:
10hgo); ABW38009(loganic acid methyltransferase: lamt); AAA33106
(CYP72A1=secologanin synthase: sls); CAC29060(anthranilate synthase asubunit:
asa); AAA33109(tryptophan decarboxylase: tdc); CAA43936(strictosidine synthase:
str); AAF28800(strictosidine b-D-glucosidase: sgd); AAO13736(minovincinine 19-
hydroxy-O-acetyltransferase: mat); CAB56503(CYP71D12=tabersonine 16-
hydroxylase: t16h); ABR20103(16-hydroxytabersonine O-methyltransferase:
16omt); O04847(desacetoxyvindoline-4-hydroxylase: d4h); AAC99311
(deacetylvindoline 4-O-acetyltransferase: dat); CAJ84723 (peroxidase 1: prx1)]
Rauvolfia serpentina 17 - 7 [CAA44208(strictosidine synthase: str1); CAC83098(strictosidine b-D-glucosidase:
sgd1); AAF22288(polyneuridine aldehyde esterase: pnae); Q70PR7(vinorine
synthase: vs); AAF03675(raucaffricine b-D-glucosidase: rg); AAW88320
(acetylajmalan acetylesterase: aae); AAX11684(perakine reductase: pr)]
Nyssaceae
Camptotheca acuminata 31 - 10 [ABC86579(1-deoxy-D-xylulose-5-phosphate reductoisomerase: dxr); O48964
(isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase: idi1); O48965
(idi2); AAV64030(5-enolpyruvylshikimate 3-phosphate synthase: epsps);
AAU84988(anthranilate synthase asubunit: asa1); AAU84989(asa2); AAB97526
(tryptophan synthase bsubunit: tsb); AAB39708(tryptophan decarboxylase: tdc1);
AAB39709(tdc2); AAQ20892(10-hydroxygeraniol oxidoreductase: 10hgo)]
144 G. Guirimand et al.
Alkaloid family: benzylisoquinoline alkaloids (BIA)
Papaveraceae
Papaver somniferum 20,458 20,340 16 [P54768(tyrosine/DOPA decarboxylase: tydc1); P54770(tydc3); AAX56303(S-
norcoclaurine synthase: ncs1); AAX56304.(ncs2); AAQ01669(norcoclaurine 6-O-
methyltransferase: 6omt); AAP45316(coclaurine N-methyltransferase: cnmt);
AAF61400(CYP80B1 renamed CYP80B3=N-methylcoclaurine 30-hydroxylase);
AAP45313(30-hydroxy-N-methylcoclaurine 40-O-methyltransferase: 40omt1);
AAP45314(40omt2); AAQ01668(reticuline 7-O-methyltransferase: 7omt); P93479
(berberine bridge enzyme: bbe); AAY79177(tetrahydroprotoberberine-cis-N-
methyltransferase: tnmt); ABR14720(salutaridine synthase: ss); ABC47654
(salutaridine reductase: salr); Q94FT4(salutaridinol 7-O-acetyltransferase: salat);
AAF13738(codeinone reductase: cor1)]
Eschscholzia californica 9,161 9,083 5 [P30986(berberine bridge enzyme: bbe1); BAE79723(reticuline-7-O-
methyltransferase: 7omt); O64899(CYP80B1 renamed CYP80B3=N-
methylcoclaurine 30-hydroxylase); AAC39454(CYP82B1=N-methylcoclaurine 30-
hydroxylase); O64900(CYP80B2=N-methylcoclaurine 30-hydroxylase)]
Ranunculaceae
Thalictrum flavum
10 - 9 [AAG60665(tyrosine/dopa decarboxylase: tydc1); AAR22502(norcoclaurine
synthase: ncs); AAU20765(norcoclaurine 6-O-methyltransferase: 6omt); AAU20766
(coclaurine N-methyltransferase: cnmt); AAU20767(CYP80B3=N-
methylcoclaurine 30-hydroxylase); AAU20768(30-hydroxy-N-methylcoclaurine 40-
O-methyltransferase: 40omt); AAU20769(berberine bridge enzyme: bbe);
AAU20770(scoulerine 9-O-methyltransferase: somt); AAU20771
(CYP719A1=canadine synthase)]
Coptis japonica 135 37 9 [BAF45337(norcoclaurine synthase: ncs); Q9LEL6(norcoclaurine 6-O-
methyltransferase: 6omt); Q948P7(coclaurine N-methyltransferase: cnmt); Q9LEL5
(30-hydroxy-N-methylcoclaurine 40-O-methyltransferase: 40omt); Q39522(scoulerine
9-O-methyltransferase: somt); BAB68769(CYP719A=methylenedioxy bridge-
forming enzyme); Q8H9A8(columbamine O-methyltransferase: coomt); BAB12433
(CYP80B2=N-methylcoclaurine-30-hydroxylase); BAF80448
(CYP80G2=corytuberine synthase)]
(continued)
8 Biosynthesis and Regulation of Alkaloids 145
Table 8.2 (continued)
Family (plant species) NCBI hints
(nt seq)
NCBI hints
(EST)
Alkaloid biosynthetic enzymes (GenBank access numbers, full enzyme name and
abbreviation)
Alkaloid family: tropane and nicotine alkaloids (TNA)
Solanaceae
Hyoscyamus niger 23 - 5 [BAA82263(putrescine N-methyltransferase: pmt); BAA85844(tropinone reductase:
tr1); P50164(tr2); ABD39696(CYP80F1=littorine mutase/monooxygenase); P24397
(hyoscyamine 6-b-hydroxylase: h6h)]
Atropa belladonna 58 - 3 [BAA82264(putrescine N-methyltransferase: pmt1); BAA82262(pmt2); BAA78340
(hyoscyamine 6-b-hydroxylase: h6h)]
Datura stramonium 31 - 6 [P50134(ornithine decarboxylase: odc); CAB64599(arginine decarboxylase: adc1);
CAE47481(putrescine N-methyltransferase: pmt); P50162(tropinone reductase: tr1);
P50163(tr2); P50165(trh)]
Nicotiana tabacum 1,673,039 240,440 4 [AAQ14852(ornithine decarboxylase: odc); AAF14881(putrescine N-
methyltransferase: pmt); ABI93948(methylputrescine oxidase: mpo1); DQ131886
(CYP82E4v1=nicotine N-demethylase)]
Coffeeae
Coffea arabica 6,079 1,577 10 [BAB39215(xanthosine methyltransferase: xmt1); BAC75665(xmt2); BAB39216(7-
methylxanthine N-methyltransferase: mxmt1); BAC75664(mxmt2); BAC75663(3,7-
dimethylxanthine N-methyltransferase: dxmt1); BAC43756(theobromine synthase:
cts1); BAC43757(cts2); BAC43755(7-methylxanthosine synthase: xrs1); BAC43760
(caffeine synthase: ccs1); BAC43761(ctcs7)]
Camellia sinensis 3,732 3,288 1 [ABP98983(caffeine synthase: tcs)]
Byttnerioideae
Theobroma cacao 7,469 6,790 1 [BAE79730(caffeine synthase: bcs1)]
146 G. Guirimand et al.
that a single plant species is able to produce. For example, there are more than 130
MIA in C. roseus (van der Heijden et al. 2004). The elucidation of MIA biosyn-
thetic pathways in the three mentioned species has undergone major recent progress
with the identification of 42 clones corresponding to 31 enzymatic steps (Table 8.2).
In C. roseus alone, 27 enzymatic steps have been studied (25 cDNA clones and two
additional enzymatic activities with no assigned clone). In these species, the path-
ways share a common origin with strictosidine synthase (STR), catalysing the
condensation of the indole precursor tryptamine with the terpenoid precursor
secologanin to form the first MIA, strictosidine. The upstream biosynthesis of the
indole precursor derived from the shikimate pathway via tryptophan, and of the
terpenoid precursor originating from the methyl erythritol phosphate (MEP) path-
way, is also shared within these plant species. Strictosidine b-glucosidase (SGD),
catalysing the deglucosylation of strictosidine, is the last common enzyme for the
biosynthesis of 2,000 MIA, since the resulting aglycon is the starting point for many
different species-specific lateral MIA pathways, with the observed possibility for a
given species to harbour more than one of these pathways (e.g. C. roseus, reviewed
in van der Heijden et al. 2004). Many enzymatic steps are yet to be discovered.
More details on these pathways and the identified enzymatic steps are available in
recent reviews (Lorence and Nessler 2004; van der Heijden et al. 2004; Sto
¨ckigt and
Panjikar 2007; Mahroug et al. 2007; Ziegler and Facchini 2008).
8.2.2 Biosynthesis of Benzylisoquinoline Alkaloids (BIA)
BIA constitute a diverse class of more than 2,500 compounds with, for some of
them, potent pharmacological properties and socio-economic importance. In opium
poppy (Papaver somniferum) alone, more than 80 alkaloids have been identified.
BIA have also been widely studied in other members of the Papaveraceae, such as
Eschscholzia californica, or members of the Ranunculaceae such as Thalictrum
flavum and Coptis japonica (Table 8.1). The biosynthesis of BIA starts with the
condensation of two tyrosine derivatives to produce (S)-norcoclaurine. Four char-
acterised enzymatic steps are necessary to produce (S)-reticuline, the central
precursor of the five major BIA subpathways, leading to palmatine, berberine,
sanguinarine, laudanine and codeine/morphine respectively. Overall, regardless of
the plant model species, a total of 39 clones and 19 enzymatic steps have been
characterised (Table 8.2), making the BIA biosynthetic pathway one of the best
characterised plant natural product complex pathways (reviewed in Ziegler and
Facchini 2008).
8.2.3 Biosynthesis of Tropane and Nicotine Alkaloids (TNA)
TNA are widely used in medicine as nonselective muscarinic antagonists affecting
peripheral and central nervous systems (Table 8.1). This alkaloid family is found
8 Biosynthesis and Regulation of Alkaloids 147
mainly in Solanaceae species, such as Hyoscyamus niger,Datura stramonium,
Atropa belladonna or Nicotiana tabacum, and accounts for more than 200 different
compounds (Dra
¨ger 2002,2006; Oksman-Caldentey 2007). TNA are amongst the
most studied alkaloids and their pharmacological effects are well documented.
However, their biosynthetic pathways are still only partially understood. TNA are
derived from the amino acids ornithine and arginine (Fig. 8.1), and the early
biosynthetic steps leading to N-methylputrescine formation have been elucidated
in several species (Table 8.2). The oxidative deamination of N-methylputrescine
leads to N-methylpyrrolium cation, which constitutes a branching point towards
tropane alkaloids and nicotine alkaloids respectively. The final steps of both path-
ways are partially characterised, but molecular information concerning the central
steps is still missing. Overall, seven enzymatic steps of the tropane alkaloid
biosynthetic pathway and four enzymatic steps of the nicotinic alkaloid biosyn-
thetic pathway have been characterised. More details on these pathways and on the
identified enzymatic steps are available in recent reviews (Dra
¨ger 2006; Oksman-
Caldentey 2007).
8.2.4 Biosynthesis of Purine Alkaloids (PA)
PA are natural products derived from purine nucleotides (Fig. 8.1; Ashihara et al.
2008). The main PA are caffeine and theobromine that affect the central nervous
system as neurostimulants and are synthesised by several plants, including Coffea
arabica,Camellia sinensis or Theobroma cacao (Table 8.1). The initial precursor
of PA is xanthosine, which is supplied by at least four different pathways. The main
caffeine biosynthetic pathway has four enzymatic steps, comprising three charac-
terised S-adenosylmethionine-dependant N-methylation reactions and one unchar-
acterised nucleosidase reaction (Table 8.2). However, according to structural
studies of N-methyltransferases involved in caffeine biosynthesis, it has been
suggested that the ribose hydrolysis could be performed by xanthosine 7 N-methyl-
transferase (McCarthy and McCarthy 2007). The detail of this pathway has been
reviewed recently (Ashihara et al. 2008).
8.3 Spatial Organisation of Alkaloid Biosynthesis
The spatial organisation of alkaloid biosynthesis has been recently investigated
extensively using in situ hybridisation and immunocytochemistry methods
(reviewed in De Luca and St-Pierre 2000; Kutchan 2005; Mahroug et al. 2007;
Ziegler and Facchini 2008). An astonishing complexity has been uncovered
showing multicellular organisations as a recurrent common feature. These types
of organisation implicate the necessity of intercellular translocation processes. In
C. roseus, a series of publications showed the sequential involvement of internal
phloem associated parenchyma (IPAP), epidermis and laticifers-idioblasts during
148 G. Guirimand et al.
a
c
ef
d
b
Fig. 8.2 Examples of multicellular organisation of alkaloid biosynthesis implicating the necessity
of intercellular translocation events. Six models of spatial organisation of alkaloid biosynthesis in
higher plants are presented in a–f.Ina–c, the green–orange–red traffic light signal colour code
8 Biosynthesis and Regulation of Alkaloids 149
MIA biosynthesis in aerial organs (Fig. 8.2a; St-Pierre et al. 1999; Irmler et al.
2000; Burlat et al. 2004; Oudin et al. 2007). The IPAP cells harbour the expression
of genes involved in early steps of monoterpenoid biosynthesis, i.e. four MEP
pathway genes and geraniol 10-hydroxylase (G10H, CYP76B6) encoding the first
committed enzyme in monoterpenoid biosynthesis (Burlat et al. 2004; Oudin et al.
2007; Guirimand et al. 2009). The intermediate steps leading to the synthesis of the
two MIA precursors, tryptamine and secologanin, and to their subsequent conden-
sation to form the first MIA strictosidine, occur within the epidermis (St-Pierre et al.
1999; Irmler et al. 2000). Finally, the last two steps in the biosynthesis of vindoline,
one of the monomeric MIA precursors of the dimeric MIA vinblastine, are localised
to specialised laticifer-idioblast cells (St-Pierre et al. 1999). Recently, these results
were elegantly completed by an RT-PCR analysis of laser capture microdissected
C. roseus leaf cells (Murata and De Luca 2005) and by an EST analysis study of
epidermis-enriched fractions obtained using an original carborundum abrasion
technique (Levac et al. 2008; Murata et al. 2008). Altogether these results suggest
that, to ensure a continuity in the metabolic flux along the MIA pathway, it is
necessary to consider the translocation of an unknown monoterpenoid intermediate
from IPAP to epidermis, and the translocation of an unknown MIA intermediate
from epidermis to laticifer-idioblast cells (Fig. 8.2a). The identification of these
shuttling intermediates will necessitate the localisation of two subsequent enzy-
matic steps within two different cell types. Similarly, in the roots of several
solanaceous species, the shuttle of intermediate TNA between the pericycle and
the endodermis is suggested by the specific localisation of early and late enzymatic
steps (putrescine N-methyltransferase and hyoscyamine 6-hydroxylase respec-
tively) to the pericycle, and the specific expression of an intermediate enzymatic
step (tropinone reductase 1) within the neighbouring endodermis (Fig. 8.2b;
Fig. 8.2 (continued) illustrates (respectively) early, intermediate and late biosynthetic steps
sequentially occurring in different cell types along a given pathway. These three models implicate
the intercellular translocation of metabolic intermediates. In d–f, the yellow, magenta and blue
colour code shows the common localisation of all the mRNAs (yellow), enzymes (magenta) and
alkaloids (blue) from a given pathway in different cell types. The Facchini model (d) implicates
the translocation of enzymes from companion cells to sieve elements and the translocation of
alkaloids from sieve elements to laticifers. The models in d–fillustrate that a same pathway can
be localised to different cell types according to the plant species and/or to the organ considered.
IPAP, internal phloem associated parenchyma; Ep, epidermis; Lat, laticifers; Pal Id, palisade
idioblasts; Sp Id, spongy parenchyma idioblasts; Pal Par, palisadic parenchyma; Sp par, spongy
parenchyma; ICP, internal conducting phloem; ECP, external conducting phloem; Xy, xylem; Pi,
pith; Per, pericycle; End, endodermis; Co, cortex; CC, companion cells; SE, sieve elements; Co
Par/Id, cortex parenchyma/Idioblasts; PAP, phloem associated parenchyma; PD, protoderm;
SAM, shoot apical meristem; VB, vascular bundles; RAM, root apical meristem; Dev End,
developing endodermis; Mat End, mature endodermis. These models were drawn from results
taken from the reports by aSt-Pierre et al. (1999), Irmler et al. (2000), Burlat et al. (2004) and
Oudin et al. (2007); bHashimoto et al. (1991), Nakajima and Hashimoto (1999) and Suzuki et al.
(1999a,b); cBock et al. (2002) and Weid et al. (2004); dBird et al. (2003) and Samanani et al.
(2005); e,fSamanani et al. (2005)
<
150 G. Guirimand et al.
Hashimoto et al. 1991; Nakajima and Hashimoto 1999; Suzuki et al. 1999a,b). In
this case, given the proximity of conducting tissues, it is hypothesised that the
precursor of TNA (arginine) could come from the phloem, and that final TNA
products, such as scopolamine, could flow to the aerial parts of the plant through the
xylem (Fig. 8.2b; Nakajima and Hashimoto 1999). Four models of the spatial
organisation of BIA synthesis in opium poppy and in a species (T. flavum)of
Ranunculaceae also illustrate the complexity of these compartmentations. In
opium poppy, two different models have been proposed (Fig. 8.2c, Kutchan
2005; Fig. 8.2d, Facchini and St-Pierre 2005). In one model, a rationale similar to
that described for MIA and TNA biosynthesis is proposed, since a cell-specific
separation occurs between early and late steps of different branches of the BIA
pathway. According to immunolocalisation studies performed on five biosynthetic
enzymes, this model proposed that BIA synthesis commonly starts in the phloem
parenchyma with the synthesis of (S)-reticuline. Different situations are then
observed for three BIA subpathways leading to laudanine, codeine/morphine and
scoulerine respectively. The synthesis of laudanine appears to occur within the
same phloem parenchyma cells, whereas the biosynthesis of scoulerine is localised
to leaf idioblasts and root cortex cells. Finally, an intermediate step in the codeine
pathway is also localised in phloem parenchyma, whereas the final step occurs in
laticifers (Fig. 8.2c; Bock et al. 2002; Weid et al. 2004; reviewed in Kutchan 2005).
However, Facchini and St-Pierre (2005) proposed a totally different type of com-
partmentation in the same species with a so-called tale of three cell types, implicat-
ing the transcription of seven BIA biosynthesis genes within companion cells,
the immunolocalisation of the corresponding enzymes to the sieve elements, and
the accumulation of BIA in neighbouring laticifers (Fig. 8.2d; Bird et al. 2003;
Samanani et al. 2006; reviewed in Ziegler and Facchini 2008). The discrepancy
between both models has been attributed tentatively to differences in cultivars and/
or in developmental stages. The last example of BIA synthesis in T. flavum
illustrates that several steps of a pathway may be compartmentalised in a different
manner within different plant species (compare P. somniferum, Fig. 8.2c, d and
T. flavum, Fig. 8.2e, f) and even within different organs (rhizomes versus roots) of
the same plant species (Fig. 8.2e, f; Samanani et al. 2005).
Alkaloid biosynthesis also involves subcellular compartmentation of biosyn-
thetic enzymes, as is the case for most natural product biosynthetic pathways.
Beside cytosol, organelles such as ER (either lumen or membranes), plastids (either
stroma or thylakoids), mitochondria and vacuoles have been implicated in various
alkaloid biosynthetic pathways (reviewed in Facchini and St-Pierre 2005; Mahroug
et al. 2007; Ziegler and Facchini 2008). These results are based on in silico analysis
of biosynthetic enzyme sequences and also on more formal experimental evidence.
Part of these experiments corresponded to density gradient analysis, and a few
examples of direct localisation of enzymes by immunogold (McKnight et al. 1991;
Bock et al. 2002; Alcantara et al. 2005; Samanani et al. 2006; Oudin et al. 2007;
Guirimand et al. 2009) and by GFP-fusion image analyses (Bird and Facchini 2001;
Costa et al. 2008; Guirimand et al. 2009) have been published. This emphasizes that
a systematic (re)evaluation of the subcellular localisation of all the enzymes
8 Biosynthesis and Regulation of Alkaloids 151
available in a given alkaloid pathway constitutes a future challenge that should
enable the drawing of more complete spatial compartmentation models that inte-
grate both cellular and subcellular levels.
Together, these results suggest the recurrent necessity of transmembrane and
intercellular translocation processes during alkaloid biosynthesis. Members of the
ATP binding cassette (ABC) transporter superfamily have been demonstrated to be
able to recruit various alkaloids such as vinblastine, hyoscyamine, scopolamine or
berberine (Kolaczkowski et al. 1996; Sakai et al. 2002; Goossens et al. 2003; Shitan
et al. 2003). The possibility that alkaloid intermediates and/or enzymes flow
through the symplasm should also be considered, even though no experimental
proofs are available. Finally, the organisation of clusters of biosynthetic enzymes in
metabolic channels has also been purported in BIA synthesis (Samanani et al.
2006).
8.3.1 Crystallisation and Three-Dimensional Structure
of Alkaloid Biosynthetic Enzymes
The molecular architecture of some alkaloid biosynthetic enzymes has recently
been investigated, providing details on their catalytic mechanism and opening new
perspectives on the production of alkaloid derivatives with improved properties
using enzyme engineering. The TNA biosynthetic pathway provides the first two
examples of elucidation of three-dimensional architecture of plant alkaloid biosyn-
thetic enzymes by determination of the crystal structures of two tropinone reduc-
tases from D. stramonium (Nakajima et al. 1998). Similarly, elucidation of the
structure of two N-methyltransferases from the caffeine (PA) biosynthetic pathway
of Coffea canephora led to the identification of critical residues for substrate
selectivity and catalysis (McCarthy and McCarthy 2007). The MIA biosynthetic
pathway shows the broadest characterisation of alkaloid biosynthetic enzyme
structure. In R. serpentina, in addition to the preliminary X-ray analysis performed
on crystals of raucaffricine glucosidase and perakine reductase (Rosenthal et al.
2006; Ruppert et al. 2006), X-ray crystallography allowed the elucidation of STR
and SGD structures, as well as vinorine synthase located in the ajmaline branch of
MIA biosynthesis (Ma et al. 2005,2006; Barleben et al. 2007). STR represents a
novel six-bladed b-propeller fold protein that catalyses a Pictet-Spengler conden-
sation between secologanin and tryptamine in a highly substrate-specific manner.
The elucidation of the architecture of the substrate binding pockets and of catalytic
residues led to the development of structure-based engineering of STR aiming at a
redesign of the substrate binding pockets (Chen et al. 2006; Bernhardt et al. 2007;
Loris et al. 2007). Several STR variants generated by site-directed mutagenesis
displayed altered substrate specificity and accommodated analogs of both trypt-
amine and secologanin. The three-dimensional structure of SGD appears as a
typical (b/a)
8
barrel fold as encountered in the glucosidase family 1. Using
152 G. Guirimand et al.
site-directed mutagenesis and structural analysis, catalytic residues have been
identified that are involved in the deglucosylation of strictosidine and the confor-
mation of the catalytic pocket (Barleben et al. 2007). Furthermore, steady-state
kinetics of SGD with various strictosidine analogs enabled the identification of the
substrate preference of SGD at two positions of strictosidine, opening new oppor-
tunities for SGD redesign (Yerkes et al. 2008). Finally, crystal structure analysis of
vinorine synthase provides the first example of the three-dimensional organisation
of an enzyme of the BAHD superfamily (Ma et al. 2005). This is of particular
interest, since the vinorine synthase structure could be used for homology-based
modelling of other BAHD enzymes occurring in MIA and BIA biosynthetic path-
ways. Indeed, homology-based modelling has already been applied successfully to
the identification of catalytic residue of polyneuridine aldehyde esterase (PNAE),
an ab-hydrolase superfamily member from the ajmaline biosynthetic pathway of
R. serpentina, using the X-ray crystallographic structure of hydroxynitrile lyase,
another ab-hydrolase superfamily member (Mattern-Dogru et al. 2002). In Papaver
bracteatum, the structure of salutaridine reductase implicated in BIA biosynthesis
was also analysed by homology modelling using the X-ray structure of human
carbonyl reductase 1 (Geissler et al. 2007).
The elucidation of the structure of alkaloid biosynthetic enzymes using X-ray
crystallography and homology modelling, as well as the identification of key
residues for reaction catalysis and substrate specificity, open new enzyme redesign
perspectives for the production of large libraries of alkaloid analogs of potential
interest.
8.3.2 Transcription Factor Regulatory Networks of Alkaloid
Biosynthesis
Transcription factors (TF) are thought to play a key role in regulating fluxes through
alkaloid pathways by controlling the levels of pathway gene expression, transpor-
ters, and the differentiation of specialised cellular structures where the alkaloids are
synthesised and accumulate. TF usually exert their control by modulating the
expression level of multiple pathway genes. In several species, alkaloid accumula-
tion is preceded by the coordinated induction of several pathway genes, a result of
their regulation by specific TF. Methyl jasmonate (MeJA), one of the major internal
signal molecule in plant defence response, is known to induce a number of
secondary metabolisms, including nicotine (Shoji et al. 2008), MIA (van der Fits
and Memelink 2000) and BIA (Fa
¨rber et al. 2003). For instance, the majority of
MIA pathway genes tested are induced by MeJA in C. roseus cell cultures (van der
Fits and Memelink 2000; Oudin et al. 2007). Detailed analysis of the STR gene
promoter led to the identification of several TF involved in elicitor and jasmonate
responses. The jasmonate- and elicitor-responsive element (JERE) was found to
interact with two TF, octadecanoid-responsive Catharanthus AP2-domain proteins
8 Biosynthesis and Regulation of Alkaloids 153
(ORCAs; Menke et al. 1999; van der Fits and Memelink 2000). The expression of
the ORCA2 and ORCA3 genes themselves is rapidly induced by MeJA (Menke
et al. 1999; van der Fits and Memelink 2001). In nicotine biosynthesis, MeJA
response is controlled by homologs of the Arabidopsis MeJA-signal transduction
pathway (Chini et al. 2007; Thines et al. 2007; Katsir et al. 2008), namely the
NtCOI1 subunit of E3-ubiquitin ligase complex, the NtJAZ repressor family (Shoji
et al. 2008), as well as by the transcription factor NtORC1, an homolog of ORCA3,
and NtJAP1 (De Sutter et al. 2005). NtORC1 and NtJAP1 were shown to up-
regulate the promoter of the PMT gene encoding the first committed step in nicotine
biosynthesis (De Sutter et al. 2005). In BIA biosynthesis, a WRKY protein was
isolated recently as a transcriptional regulator of the berberine pathway in
C. japonica (Kato et al. 2007). Silencing of CjWRKY1 down-regulated the expres-
sion of several berberine pathway genes, whereas over-expression of CjWRKY1
up-regulated the berberine pathway but did not regulate primary metabolism genes,
including the tyrosine biosynthetic genes. Interestingly, over-expression of an
Arabidopsis WRKY TF was found to enhance BIA concentration up to 30-fold in
California poppy cell cultures (Apuya et al. 2008). This TF was isolated in a study
that involved screening of regulatory factors isolated from Arabidopsis to identify
those that modulate the expression of genes encoding enzymes involved in the
biosynthesis of BIA in P. somniferum and E. californica. In opium poppy, the over-
expression of selected regulatory factors was found to increase the concentrations
of codeinone reductase, 30-hydroxy-N-methylcoclaurine 40-O-methyltransferase
and norcoclaurine 6-O-methyltransferase transcripts 10- to 100-fold, and to
enhance BIA production up to tenfold (Apuya et al. 2008).
8.3.3 Metabolic Engineering of Alkaloid Biosynthesis
Progress in the knowledge of alkaloid pathways at the gene level allows several
attempts to improve alkaloid factories. Various objectives have been considered to
improve the economic value of medicinal plants or cell cultures, including yield
improvement of active or total alkaloids by enhancing flux in the pathway from
primary precursor, by overcoming the rate-limiting step, and by reducing flux to
competing pathways. Metabolic capacity to produce alkaloids has also been intro-
duced into new species by gene transfer or, in contrast, blocked to eliminate toxic or
undesired alkaloids.
Significant yield improvement has been achieved in opium poppy recently by
deregulating either an early or a late metabolic step. In an elite commercial line of
P. somniferum, over-expression of the CYP80B3 gene encoding the early-step
N-methylcoclaurine 30-hydroxylase resulted in an increase in morphine reaching
450% under greenhouse conditions (Frick et al. 2007). Over-expression of the
penultimate step, codeinone reductase, also resulted in an increase in total alkaloids
and morphine of up to 20% under field conditions (Larkin et al. 2007). In MIA
biosynthesis, attempts to deregulate the indole branch have been disappointing,
154 G. Guirimand et al.
probably owing to the dual origin of this class of alkaloids and the complexity of the
regulatory networks. Over-expression of tryptophan decarboxylase (TDC) in
C. roseus cell culture increased the concentration of only tryptamine (Whitmer
et al. 2002), while over-expression of both TDC and STR resulted in increased
tryptamine and MIA (Canel et al. 1998; Geerlings et al. 1999). Similarly, in
C. roseus hairy roots, inducible expression of both a feedback insensitive anthrani-
late synthase from Arabidopsis and TDC led to a marked increase in tryptophan and
tryptamine pools, but only modest change in alkaloid pools (Hong et al. 2006). The
terpenoid branch has been shown to be limiting in these transgenic lines by
precursor feeding experiments (Whitmer et al. 2002; Peebles et al. 2006), suggest-
ing that further improvement would need to target this part of the pathway.
Genetic manipulation of the tropane alkaloid pathway is a good example to
improve the metabolic profile of a medicinal plant. Compared to the more valuable
scopolamine, hyoscyamine usually accumulates to greater concentrations in vari-
ous solanaceous plants (e.g. Atropa,Datura,Hyoscyamus). Improved conversion of
the precursor hyoscyamine to scopolamine has been achieved by over-expression of
hyoscyamine 6-hydroxylase (Yun et al. 1992; Hashimoto et al. 1993). Recently,
biotransformation of hyoscyamine into scopolamine has been reported in transgenic
tobacco cell cultures by over-expressing the H6H gene from Hyoscyamus muticus
(Moyano et al. 2007). In some cases, incomplete knowledge of metabolic pathways
may be overcome by using a more conventional approach. For instance, chemical
mutagenesis of seeds has resulted in the isolation of a P. somniferum mutant, top1,
which accumulates thebaine, the starting material for several analgesic semi-
synthetic derivatives (Milgate et al. 2004).
Blocking an undesired production of alkaloid may be achieved by silencing an
essential enzymatic step early in the pathway. For instance, engineering of decaf-
feinated coffee plants is in progress in Coffea canophora, by silencing 7-methyl-
xanthine methyltransferase (Ashihara et al. 2006). Due to the high conservation of
the amino acid sequence, the two other N-methyltransferases were also down-
regulated. Conversely, new metabolic capacity for caffeine production was intro-
duced into tobacco by expressing the three N-methyltransferase genes. The
transgenic plants accumulated caffeine, which apparently deters insect pests and
enhances resistance to bacterial and viral infections (Uefuji et al. 2005; Kim and
Sano 2008). In tobacco, RNAi-induced suppression of nicotine demethylase activ-
ity was shown to reduce the levels of N0-nitrosonornicotine, a carcinogen in cured
tobacco leaves (Lewis et al. 2008).
8.4 Conclusions
Progress in metabolomic and transcriptomic analyses as well as system biology
approaches are likely to yield detailed analysis of bottlenecks in metabolic path-
ways and more rational strategies to improve alkaloid biosynthesis. The importance
of cellular and subcellular compartmentation, including metabolic channels, has
8 Biosynthesis and Regulation of Alkaloids 155
been so far overlooked in improving alkaloid yield. The targeting of enzymes to
proper compartments is likely to be an important factor. Engineering at the enzyme
structural level by site-directed mutagenesis will also likely be an important avenue
to create a new level of metabolic diversity by generating new semi-synthetic
alkaloid structures from synthetic precursors and modified enzymes with broad
substrate specificity.
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