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Received 14 Jul. 2004 Accepted 22 Oct. 2004

Supported by the Opening Fund from Key Laboratory for Biotechnology on Medicinal Plant of Jiangsu Province in China (KJS03080).

* These authors contributed equally to this work.

**Author for correspondence. Tel (Fax): +86 (0)21 2507 0394; E-mail: .

Journal of Integrative Plant Biology

FormerlyActa Botanica Sinica 2005, 47 (2): 136143

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Lei ZHANG1, 3*, Guo-Yin KAI2*, Bei-Bei LU1, Han-Ming ZHANG1, Ke-Xuan TANG2,

Ji-Hong JIANG3 and Wan-Sheng CHEN1**

(1. School of Pharmacy, The Second Military Medical University,Shanghai 200433, China;

2. Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-Shanghai Jiaotong University-Nottingham

Plan t Biotechnology Rese arch and Development Cent er, School of Life Sciences and Technology ,

Shanghai Jiaotong University,Shanghai 200030, China;

3. Key Laboratory for Biotechnology on Medi cina l Plan t of Jiangsu Province, Xuzhou Normal University,Xuzhou 221116, China)

Abstract: Over the past decade, the evolving commercial importance of so-called plant secondary

metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to

enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute oneof the largest groups of natural products, provide many pharmacologically active compounds. Several

genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering

of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased

by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encod-

ing regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been

used to block competitive pathways. These investigations have opened up new, promising perspectives for

increased production in plants or plant cell culture. Recent achievements have been made in the metabolic

engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.

Key words: biosynthesis pathway; genetic transformation; hyoscyamine; plant secondary metabolic

engineering; scopolamine; tropane alkaloids.

Plants produce a wide variety of so-called second-

ary metabolites, which play an important role in the

survival of the plants in their ecosystem. Thus, plant

secondary metabolites (PSM) are involved in resistance

against pests and diseases, the attraction of pollinators,

the interaction with symbiotic microorganisms etc.

(Dixon 2001; Harborne 2001). Approximately 100 000

PSM are already known, with approximately 4 000 new

ones being discovered every year (Verpoorte et al.

2000). The largest group of PSM consists of

isoprenoids, comprising more than one-third of all

known compounds. The second largest group is formed

by alkaloids, which comprise many drugs and poisons

(Verpoorte et al. 1999). Plant secondary metabolism

has multiple functions throughout the life cycle of

plants. Plant secondary metabolites are also of interest

because they determine the quality of food (color, taste,

and aroma) and ornamental plants (flower color, smell).

More recently, various health-improving effects and

disease-preventing activities of PSM have come to light,

such as anti-oxidative and cholesterol-lowering

properties. In addition to these aspects, a number of

PSM isolated from plants are commercially available

as fine chemicals; for example, drugs, dyes, flavors,

fragrances, and insecticides. Some of these

phytochemicals are quite expensive because of their

low abundance in natural plants.

Over the past years, there has been a rapidly in-

creasing interest in plant secondary metabolism. In

particular, the possibilities of genetic modification have

http://www.blackwell-synergy.com

http://www.chineseplantscience.com

.Review.

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Lei ZHANG et al.: Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants 137

opened the way to genetic engineering to alter various

traits of plants, such as increasing resistance against

pests or diseases and improving yields and the quality

traits of plants (Verpoorte et al. 2000). The introduc-

tion of new genes into plants by particle bombardment

orAgrobacterium-mediated transformation has become

increasingly routine and has demonstrated consider-

able potential for the expoitation of the biosynthetic ca-

pacity of plants and plant cells(Verpoorte et al. 2000).

Plant alkaloids constitute one of the largest groups

of natural products, providing many pharmacologically

active compounds. A few genera of the plant family

Solanaceae, includingHyoscyamus,Duboisia,Atropa,

and Scopolia, are able to produce biologically active

nicotine and tropane alkaloids simultaneously (Endo et

al. 1991; Christen et al. 1993; Hashimoto and Yamada

1994). Tropanealkaloids, such as hyoscyamine and

scopolamine, are widely used as parasympatholytics

that competitively antagonize acetylcholine. Consider-

able research has already been performed on the ge-

netic engineering of tropane alkaloids. An in-depth un-

derstanding of the biosynthetic pathways at the level

of intermediates and enzymes, along with an increasein the number of genes cloned involved in biosynthesis,

has enabled the exploration of metabolic engineering as

a potential effective approach to increase the yield of

specific metabolites by enhancing the rate-limiting steps

or by blocking competitive pathways. In particular, the

conversion of hyoscyamine to much more valuable

scopolamine is the major goal. Recently, we have suc-

cessfully used transgenic hairy root lines expressing

bothpmtand h6h to produce significantly higher levels

of scopolamine compared with the wild type andtransgenic lines harboring a single gene (pmtorh6h) in

Hyoscyamus nigerL. (Zhang et al. 2004), suggesting

that this approach is very effective for the large-scale

commercial production of scopolamine by plants. In

the present review, we focus on describing the recent

advances in the metabolic engineering of the tropane

alkaloid biosynthetic pathway and propose some fur-

ther prospects in this field.

1 Mapping the Biosynthetic Pathway of Tro-

pane Alkaloids

The biosynthesis of tropane alkaloids, such as hyos-cyamine and scopolamine, has been elucidated at

the enzyme level (Fig. 1). The generally accepted

hypothesis for the formation of these alkaloids begins

with the formation of theN-methyl-1-pyrrolinium ion

from the amino acids ornithine and arginine (Stephen

and David 2002), but 1-methylpyrrolidine-2-acetic acid

is not an efficient precursor for tropane alkaloids (Marcy

et al. 1996). Both tropane and pyridine alkaloid biosyn-

thetic pathways share a common polyamine metabo-

lism in their early steps. Putrescine is a common pre-cursor of both polyamines, such as spermidine and

spermine, and tropane/pyridine alkaloids (Hashimoto and

Yamada 1986, 1987; Hibi et al. 1992). PutrescineN-

methyltransferase (PMT; EC 2.1.1.53) is the enzyme

involved in the removal of putrescine from the

polyamine pool and this enzyme catalyses theN-me-

thylation of the diamine to formN-methylputrescine.

Because both the tropane ring moiety of the tropane

alkaloids and the pyrrolidine ring of nicotine are de-

rived from putrescine by way ofN-methylputrescine

synthesis, theN-methylation of putrescine catalysed

by PMT is the first committed step in the biosynthesis

of these alkaloids (Matsuda et al. 1991). The biosyn-

thesis of tropic acid, the ester moiety of the tropanes

hyoscyamine and scopolamine, has been of interest for

many years. It has been demonstrated by isotopic la-

beling studies in transformed root culture ofDatura

stramonium L. that the last step in the biosynthesis of

tropane alkaloids is the carbon skeleton rearrangement

of littorine to hyoscyamine (Marcy et al. 1996).

Hyoscymaine is stable to in vivo oxidation and is not

derived from littorine via a vicinal interchange process

(Stephen and David 2002). Scopolamine, which is the

6,7--epoxide of hyoscyamine, is formed from hyos-

cyamine by means of 6-hydroxyhyoscyamine. Hyos-

cyamine 6-hydroxylase (H6H; EC 1.14.11.11), a 2-oxo-

glutarate-dependent dioxygenase, catalyses the hydroxy-

lation of hyoscyamine to 6-hydroxyhyoscyamine, as

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Journal of Integrative Plant Biology (FormerlyActa Botanica Sinica) Vol. 47 No. 2 2005138

well as the epoxidation of 6-hydroxyhyoscyamine to

scopolamine (Hashimoto and Yamada 1986; Matsuda

et al. 1991; Fig. 1).

In pathway mapping, one should keep in mind that

certain enzymes may be capable of catalysing two

reactions. The conversion of hyoscyamine via

6-hydroxyhyoscyamine to scopolamine by the enzyme

H6H is such an example (Matsuda et al. 1991). Deter-

mining enzyme selectivity is an important aspect of

pathway mapping. In the biosynthesis of tropane alka-

loids in H. niger, two reductases play an important role

in channeling tropinone into two different pathways.

Fig. 1. Biosynthetic pathway of nicotine and tropane alkaloids. ArgDC, arginine decarboxylase; DAO, diamine oxidase;

H6H, hyoscyamine 6-hydroxylase; OrnDC, ornithine decarboxylase; PMT, putrescineN-methyltransferase; TR, tropinone

reductase. Adapted with the permission of Annual Reviews Inc., from Hashimoto and Yamada (1994).

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Each of the reductases produces only one stereoiso-

mer of tropinol (Hashimoto et al. 1992). Tropine is the

precursor for the tropane alkaloids hyoscyamine and

scopolamine, whereas pseudotropine is channeled to

the polyhydroxylated calystegines.

2 Gene Cloning

So far, most genes involved in the tropane alkaloids

biosynthesis have been cloned by the classical approach

of identification and purification of the enzyme, fol-

lowed by isolation of the encoding gene.

The cDNAs encoding PMT, which catalyses the S-

adenosylmethionine-dependent

N-methylation of putrescine at the first committed

step in the biosynthetic pathways of tropane alkaloids,

were isolated fromAtropa belladonna L. andH. niger

(Hibi et al. 1992). It was found that AbPMT1 RNA

was much more abundant in the root ofA. belladonna

than AbPMT2 RNA. The 5'-flanking region of the

Abpmt1 gene was fused to the -glucuronidase (GUS)

reporter gene and transferred to A. belladonna. His-

tochemical analysis showed that GUS is expressed spe-

cifically in root pericycle cells and the 0.3 kb 5'-up-stream region was sufficient for pericycle-specific ex-

pression (Suzuki et al. 1999).

Two stereospecific NADPH-dependent reductases,

tropinone reductase TR-I and TR-II, constitute a

branching point in the biosynthesis of tropane alkaloids:

TR-I catalyses the stereospecific reduction of tropinone

to tropine, whereas TR-II reduces tropinone to pseudot-

ropine (Koelen and Gross 1982; Drageret al. 1988).

Hashimoto et al. (1992) previously characterized TRs

that had been purified from cultured roots ofH. nigerand showed that the two TRs had both common and

different biochemical and kinetic properties. Two TRs

from H. nigerhave 64% identical amino acids and,

hence, a common evolutionary origin (Nakajima et al.

1993, 1999a, 1999b). Immunoblot analyses revealed

that accumulation of both TRs was highest in the lat-

eral roots ofH. nigerthroughout its development. In

cultured roots, TR proteins were accumulated in the

abasal region, but not in the root apex (Nakajima and

Hashimoto 1999).

Matsuda et al. (1991) reported the isolation of cDNA

clones encoding H6H from a cDNA library made from

the mRNA of the cultured roots ofH. niger. Cultured

roots contained much more H6H mRNA than plant

roots. Based on the number of positive clones obtained

from the total number of cDNA clones screened

(approximately 30 000), it may be estimated that H6H

mRNA comprises approximately 0.3% of the total

mRNA in cultured roots. Such strong expression of

H6H in cultured roots is explained anatomically by the

specific localization of H6H protein in the pericycle, a

cell type present only in young roots without second-

ary growth, such as cultured roots(Hashimoto et al.

1991; Kanegae et al. 1994). This pericycle-specific

localization of scopolamine biosynthesis provides an

explanation for the tissue-specific biosynthesis of tro-

pane alkaloids and may be important for translocation

of tropane alkaloids from the roots to the aerial parts.

3 Genetic Engineering of the Tropane Alka-

loid Biosynthetic Pathway

In most cases, the natural yields of the tropane al-kaloids hyoscyamine and scopolamine are too low for

commercialization. There remains a need to increase

alkaloid production rates for commercial exploitation

(Moyano et al. 2002). Considerable research has al-

ready been undertaken into the genetic engineering of

pharmaceutically important tropane alkaloids (Oksman-

Caldentey and Arroo 2000). In particular, the conver-

sion from hyoscyamine to the much more valuable sco-

polamine is the major goal of these studies.

3.1 Engineering single stepIn the case of engineering individual steps for in-

creasing enzyme activity, overexpression of the endog-

enous gene, or the introduction of a more suitable het-

erologous gene, thereby overcoming specific rate-lim-

iting steps in the pathway, to shut down competitive

pathways, and to decrease catabolism of the produc-

tion of interest, can be considered. The heterologous

enzyme may have more favorable properties, such as

no feedback inhibition by downstream products, or a

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higher affinity for the substrate. Such an enzyme may

be from another source, but could also be engineered

(Buckland et al. 2000). Considerable work on engi-

neering single step has already been performed in plants,

with different degrees of success, as described in the

following examples.

It has been reported that overexpression of PMT in

transgenic plants ofNicotiana sylvestris (Sato et al.

2001) increases the nicotine content, whereas suppres-

sion of endogenous PMT activity severely decreases

nicotine content and induces abnormal morphologies.

In recent years, the overexpression of PMT inA. bel-

ladonna has not affected tropane alkaloid levels in ei-

ther transgenic plants or hairy roots (Sato et al. 2001).

Moyano et al. (2002) inserted the tobacco (N.

tabacum L.)pmtgene into the hairy roots of a hybrid

ofDuboisia and theN-methylputrescine levels of the

resulting engineered hairy roots increased (two- to

fourfold) compared with wild-type roots, but there was

no significant increase in either tropane or pyridine-

type alkaloids. Moyano et al. (2003) also introduced

the transferred-DNA (T-DNA) of the root inducing plas-

mid (Ri plasmid) together with the tobaccopmtgeneinto the genome ofDatura metelL. andHyoscyamus

muticus Linn. in order to influence tropane alkaloid

production. This was the first t ime that the

overexpression of the tobaccopmtgene had been dem-

onstrated to improve tropane alkaloid production in hairy

root cultures in a plant species-dependent manner. Hairy

root cultures overexpressing thepmtgene aged faster

and accumulated higher amounts of tropane alkaloids

than control hairy roots. The production of both hyos-

cyamine and scopolamine was improved in hairy rootcultures ofD. metel, whereas inH. muticus only hyos-

cyamine content was increased by pmt gene

overexpression (Moyano et al. 2003). The results indi-

cate that the same biosynthetic pathway in two related

plant species can be dif ferently regulated and that

overexpression of a given gene does not necessarily

lead to a similar accumulation pattern of secondary

metabolites. Furthermore, it may also indicate that the

transgene allows by-passing of the endogenous

control of metabolic flux to the alkaloids that would

take place at the level of the first committed enzymatic

step in their biosynthesis.

The h6h gene encodes a dioxygenase that first causes

the introduction of a hydroxy group at the C6 position,

followed by an epoxidation by the same enzyme

(Matsuda et al. 1991). The hydroxylase gene ofH.

nigerwas placed under the control of the cauliflower

mosaic virus 35S promoter and introduced to hyos-

cyamine-richA. belladonnaby a binary vector system

usingA. rhizogenes (Hashimoto et al. 1993). The pres-

ence of the transgene in kanamycin-resistant hairy roots

was confirmed by polymerase chain reaction (PCR)

analysis. The engineered belladonna hairy roots showed

increased amounts and enzyme activities of the hy-

droxylase and contained up to fivefold higher concen-

trations of scopolamine than wild-type hairy roots. The

6-hydroxyhyoscyamine content also increased in the

transformed roots. Such genetically engineered hairy

roots should be useful for enhancing scopolamine pro-

ductivity in in vitro root culture systems. At the same

time, Yun et al. (1992) also introduced h6h intoA. bel-

ladonna by use ofAgrobacterium tumefaciens. In theprimary transformation and its selfed progeny that in-

herited the transgene, the alkaloid content of the leaf

and stem was almost exclusively scopolamine. From a

pharmaceutical point of view, the work of Hashimoto

et al. (1992) on the cloning of the h6h gene and the

subsequent introduction of scopolamine in this plant is

an excellent illustration of the great potential of applying

metabolic engineering for trimming the plant cell factory.

Subsequently, Jouhikainen et al. (1999) reported that

the 35S-h6h transgene, which coded for the enzymeH6H, was introduced into H. muticus strain Cairo

(Egyptian henbane). The best clone produced 17 mg/L

scopolamine, which was over 100-fold more than that

produced by control clones. The expression ofh6h

was found to be proportional to scopolamine production.

Thus, the single-gene approach is an excellent way

to find where a limiting step occurs in a particular

pathway. In some cases, it may be the key to improv-

ing the productivity of the plant cell factory.

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3.2 Engineering two steps

Although a substantial increase in productivity is

feasible when a rate-limiting enzyme is targeted (Leech

et al. 1998), in most biosynthetic pathways for sec-

ondary metabolites single rate-limiting steps may not

exist. Overexpression of one enzyme often renders

subsequent reactions more rate limiting and, thus, the

effect of overexpression of a single enzyme may be

dampened. Strategies should include fortification of

multiple steps by overexpressing multiple biosynthetic

genes, manipulating regulatory genes that control the

expression of multiple pathway enzyme genes, or both

(Sato et al. 2001).

A good example is simultaneous introduction and

overexpression of genes encoding the rate-limiting up-

stream enzyme PMT and the downstream key enzyme

H6H of scopolamine biosynthesis in transgenic hen-

bane (H. niger) hairy root cultures (Zhang et al. 2004).

Transgenic hairy root lines expressing both pmtand

h6hproduced significantly higher levels of scopola-

mine compared with the wild type and transgenic lines

harboring a single gene (pmtorh6h). The best line pro-

duced 411 mg/L scopolamine, which was over nine-fold greater than that in the wild type (43 mg/L) and

more than twice the amount in the highest scopola-

mine-producing h6h single-gene transgenic line (184

mg/L). Overexpression of multiple biosynthetic genes

in the target bioengineering pathway is a promising strat-

egy to alter the accumulation of certain secondary

metabolic products.

By functional expressing of genes for TR-I (trI) and

hyoscyamine-6-hydroxylase (h6h) fromH. nigerin

N. tabacum, in addition to the expected TR-I and H6Hreaction products, acetylated forms of tropine were

generated in the transgenic plants, indicating that the

expression of alkaloid pathway enzymes in a transgenic

background can produce unexpected substances. Nico-

tine levels were approximately three- to 13-fold higher

in both the parental transgenic lines and T1 progeny

compared with levels in wild-type plants and in

transgenic plants carrying the aminnoglycoside-3'-

phosphotransferase II (nptII) transgene alone. In

addition, nicotine-related compounds, such as

anatabine, nornicotine, bipyridine, anabasine, and

myosmine, were identified in transgenic tobacco lines

and were below the detection limit in wild-type plants,

suggesting changes in the activity of the enzymes in

the nicotine biosynthetic pathway (Rocha et al. 2002).

These findings will be useful for the generation of new

compounds by pathway combinatorial or analogue-

feeding approaches.

4 Conclusions

Although we still know very little about plant sec-

ondary metabolism, its role, and its regulation, this lim-

ited knowledge has already been used quite extensively

to explore the possibilities of metabolic engineering.

Despite the success of recent work (significantly im-

proved scopolamine levels by cotransformation of two

key genes), it remains a challenging task to generate

desired, or to suppress undesired, alkaloid compounds

by highly controlled metabolic engineering of the tro-

pane biosynthesis pathway. Usually, there may be mul-

tiple rate-limiting steps in a specific pathway, so, in

general, it can be concluded that it is difficult to predictthe result of overexpression of a single gene. However,

conversely, it is very time consuming and difficult to

introduce many genes into plants synchronously. Re-

cent attempts have been made to change the expres-

sion of regulatory genes that control multiple biosyn-

thesis genes (Verpoorte and Memelink 2002).

Overexpression of octadecanoid-derivative responsive

CatharanthusAP2-domain protein 3 (ORCA3) resulted

in enhanced expression of several metabolite biosyn-

thetic genes and, consequently, in increased accumu-lation of terpenoid indole alkaloids (van der Fits and

Memelink 2000), implying that transcription factors can

act as other promising tools for metabolic engineering

in plants in the future. Transcription factors are se-

quence-specific DNA-binding proteins that interact with

the promoter regions of target genes and modulate the

rate of initiation of mRNA synthesis. The production

of secondary metabolites is under strict regulat ion in

pl an t ce lls owing to co or di na ted cont ro l of th e

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biosynthetic genes by transcription factors. The use of

specific transcription factors would avoid the time-con-

suming step of acquiring knowledge about all the en-

zymatic steps of a poorly characterized biosynthetic

pathway and transcription factors could be used to in-

crease the level of a series of enzymes in a pathway,

avoiding the need to overexpress each individual path-

way gene (Gantet and Memelink 2002). Until now, there

have been no reports regarding the cloning of tran-

scription factors involved in the tropane biosynthesis

pathway, primarily because of a lack of established

genetic and easily visible phenotypes. However, pro-

moter studies of single-pathway genes are a feasible

alternative approach for the isolation of transcription

factors. Recently, there have been some reports pub-

lished indicating that PMT is responsive to methyl

jasmonate (MJ) (Shoji et al. 2001), implying that a

jasmonate- and elicitor-responsive element (JERE) may

exist in its promoter and enable us to isolate the

promoter. Once the specific element involved inJERE

gene expression is obtained, we can isolate the tran-

scription factor using a yeast one-hybrid screening

system with this element as bait, which will be of con-siderable importance and interest for the metabolic en-

gineering of tropane. In addition, antisense genes have

been used to block competitive pathways, thereby in-

creasing the flux towards the desired secondary me-

tabolites (Chintapakorn and Hamill 2003).

Using the metabolic engineering procedure, when

the steps of the biosynthetic pathway are completely

known and the respective genes cloned, exact regula-

tion towards the desired medicinal product will be

possible. Metabolic engineering provides the means,either alone or in combination with traditional breeding,

to develop novel sources for the production of plants

with quantitatively and qualitatively improved pharma-

cological properties.

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