The Plant Cell, Vol. 7, 1059-1070, July 1995 O 1995 American Society of Plant Physiologists

Alkaloid Biosynthesis -The Basis for Metabolic Engineering of Medicinal Plants

Toni M. Kutchan Laboratorium für Molekulare Biologie, Universitat München, Karlstrasse 29, 80333 Munich, Germany

INTRODUCTION

Alkaloids-the term is linguistically derived from the Arabic word a/-qali, the plant from which soda was first obtained- are nitrogenous compounds that constitute the pharmacolog- ically active “basic principles” of predominantly, although not exclusively, flowering plants. Since the identification of the first alkaloid, morphine, from the opium poppy, Papaver somnife- rum, by Sertürner in 1806, .ulO,OOO alkaloids have been isolated and their structures elucidated (Southon and Buckingham, 1989). Historically, the use of alkaloid-containing plant extracts as potions, medicines, and poisons can be traced back almost to the start of civilization. Famed examples include Socrates’ death in 399 B.C. by consumption of coniine-containing hem- lock (Conium maculatum) and Cleopatra’s use during the last century B.C. of atropine-containing extracts of Egyptian hen- bane (Hyoscyamus muficus) to dilate her pupils and thereby appear more alluring. Medieval European women utilized ex- tracts of deadly nightshade, Atropa belladonna, for the same purpose, hence the name belladonna. Although coniine is too toxic to find therapeutic use today outside of homeopathy, tropicamide, an anticholinergic that is a synthetic derivative of atropine, is routinely used in eye examinations to dilate the pupil. Tropicamide has also recently shown promise as an early diagnostic tool in the detection of Alzheimer’s disease (Scinto et al., 1994). A tonic prepared from the bark of Cinchona officinalis that contains the antimalarial drug quinine greatly facilitated European exploration and inhabitation of the trop- ics during the past two centuries.

In total, -~13,000 plant species are known to have been used as drugs throughout the world (Tyler, 1994). Approximately 25% of contemporary materia medica is derived from plants and used either as pure compounds (such as the narcotic analge- sic morphine, the analgesic and antitussive codeine, and the chemotherapeutic agents vincristine and vinblastine; Figure 1) oras teas and extracts. Plant constituents have also served as models for modern synthetic drugs, such as atropine for tropicamide, quinine for chloroquine, and cocaine for procaine and tetracaine. In fact, active plant extract screening programs continue to result in new drug discoveries. The most recent examples of anticancer alkaloids are taxo1 (clinically available since 1994) from the western yew, Taxus brevifolia, and camp- tothecin (and derivatives currently in clinical trials) from the Chinese “happy tree,” xi shu (Camptotheca acuminata), both

of which were originally isolated and assayed for biological activity in the 1960s in the laboratory of M.E. Wall. In other areas, there are intense searches for nove1 antivirals and antimalarials.

We also encounter alkaloids as the stimulants caffeine in coffee and tea and nicotine in cigarettes. Although a wealth of information is available on the pharmacological effects of these compounds, surprisingly little is known about how plants synthesize these substances, and almost nothing is known about how this synthesis is regulated. This is due, in part, to the complex chemical structures of many alkaloids, which con- tain multiple asymmetric centers. For example, although nicotine (one asymmetric center) was discovered in 1828, its structure was not known until it was synthesized in 1904, and the structure of morphine (five asymmetric centers) was not unequivocally elucidated until 1952, 146 years after its isola- tion. Beginning in the late 1950s, radiolabeled precursors were fed to plants and the resultant radioactive alkaloids were chem- ically degraded to identify the position of the label. This opened the field of alkaloid biosynthesis to experimentation. As ana- lytical instrumentation became more sophisticated, precursors labeled with stable isotopes were fed to plants and the prod- ucts analyzed by nuclear magnetic resonance spectroscopy. No real progress was made in identifying alkaloid biosynthetic enzymes until the use of plant cell cultures as experimental systems was introduced in the 1970s. Since then, on the or- der of 80 new enzymes that catalyze steps in the biosynthesis of the indole, isoquinoline, tropane, pyrrolizidine, and purine classes of alkaloids have been discovered and partially characterized.

Alkaloids belong to the broad category of secondary me- tabolites. This class of molecule has historically been defined as naturally occurring substances that are not vital to the or- ganism that produces them. Alkaloids have traditionally been of interest only due to their pronounced and various physio- logical activities in animals and humans. A picture has now begun to emerge that alkaloids do have important ecochemi- cal functions in the defense of the plant against pathogenic organisms and herbivores or, as in the case of pyrrolizidine alkaloids, as pro-toxins for insects, which further modifiy the alkaloids and then incorporate them into their own defense secretions (reviewed in Hartmann, 1991). Alkaloids have now

1060 The Plant Cell

HO :NCH,

Morphine, CodeinePapever somnllerum L

StrychnineStrychnos nux-vomica L.

ConilneCon/urn maculatum L.

MeIM

NicotineNicotiana tabacum L.

OAc

TaxolTaxua brevi/olia Nuttall

.. ^ OCOCH,| HO COjCHjCH3

VinblastineCatharanthus roaeua (L.)G.Don.

AjmalineRauvolfla serpentine (L.)Benth.

Hi

PilocarpinePilocarpus jaborandi Holmes

NxCH«

|/ *A CHjOH

OOCCH

CH2OH °«H»

OOCCHC6HS . CH30

jfScopolamine, AtroplneHyoscyamus niger L.

.COOCH,

N'Quinine

Cinchona olficlnalls L

HCocaine

Erytnroxylcn coca Lam.

EmetineUragoga ipecacuanha (Brot.)Balll.

SanguinarlneEschacholtzla calllornica Cham.

(+)-Tubocura/)lChondodendmn tomentoaum R.&P.

CamptotheclnCamptotheca acumlntta Descne.

Figure 1. Some Physiologically Active Alkaloids and Plants That Produce Them.

Alkaloids and Their Biosynthesis 1061

been isolated from such diverse organisms as frogs, ants (pheromones), butterflies (defense), marine bacteria, sponges, fungi, spiders (venom neurotoxin), beetles (defense), and mam- mals, although is not yet clear whether de novo alkaloid biosynthesis occurs in each organism.

With the introduction of molecular biology into the plant al- kaloid field, induction of alkaloid biosynthesis in response to exposure to wounding or to elicitors can be analyzed at the leve1 of gene activation, and gene expression patterns in the plant can be determined and interpreted as a first indication of possible function. We also now have the capability to alter the pattern of alkaloid accumulation in plants for the purpose of studying the biological function of alkaloids, for engineer- ing tailor-made plants that accumulate increased quantities of desired pharmaceuticals, or for producing foodstuff plants with lower alkaloid content (for example, coffee without caf- feine). Plants are some of nature’s very best chemists, and sophisticated structures such as codeine, vinblastine, taxol, and camptothecin remain well beyond the reach of commer- cially feasible total chemical syntheses. With the ability to express alkaloid biosynthetic enzymes heterologously in or- ganisms with better fermentation characteristics than plants, we can achieve unlimited quantities of these “biocatalysts” for use in syntheses of important drugs. In this review, I dis- cuss the progress that has been made in these areas as a result of the fusion of alkaloid chemistry, enzymology, and molecular biology, as well as some perspectives for future de- velopments in this field.

MONOTERPENOID INDOLE ALKALOIDS

The monoterpenoid indole alkaloids comprise a large family of alkaloids, with over 1800 members of rich structural diver- sity. Many of these natural products are physiologically active in mammals. Among the monoterpenoid indole alkaloid phar- maceuticals that are still commercially isolated from plant material are the antimalarial drug quinine from C. officinalis, the antineoplastic drug camptothecin from C. acuminata, the rat poison and homeopathic drug strychnine from Strychnos nux- vomica, and the antineoplastic chemotherapeutic agents vin- cristine and vinblastine from Catharanthus m e u s (periwinkle)

(Figure 1). Total chemical syntheses of these complex alkaloids would be of academic interest but due to low yields are not likely to be applied commercially. To develop nove1 sources of these drugs, two options are available. The cDNAs for en- zymes that catalyze those biosynthetic steps that are difficult to achieve by chemical means can be isolated and heterolog- ously expressed for use in biornimetic syntheses. Alternatively, instead of single transformation steps, microorganisms could be engineered to express short pathways, thus producing an end-product alkaloid of interest. Alkaloid biosynthetic pathways that are to0 long to be introduced into a single microorganism could be modified in the parent plant using antisense or co- suppression technologies such that a desired alkaloid can be accumulated by blocking side pathways or catabolic steps (schematically depicted in Figure 2).

All of these approaches require thorough knowledge of the alkaloid biosynthetic pathway and the enzymes that catalyze the individual transformation steps. Progress toward identify- ing the enzymes of monoterpenoid indole alkaloid biosynthesis has been made primarily in the laboratory of J. Stockigt using Rauvolfia serpentina cell suspension cultures in studies of the biosynthesis of the antiarrythmic drug ajmaline and in the lab- oratory of V. De Luca using C. Toseus cell suspension cultures and plants to study the biosynthesis of vindoline, a precursor to the antineoplastics vincristine and vinblastine (reviewed in Herbert, 1994; Kutchan, 1994). The first successful cDNA clon- ing experiments in the alkaloid field were achieved with two cDNAs encoding enzymes that catalyze early steps in the bio- synthetic pathway that leads to all monoterpenoid indole alkaloids, tryptophan decarboxylase (De Luca et al., 1989) and strictosidine synthase (Kutchan et al., 1988) (Figure 3).

Tryptophan Decarboxylase

Tryptophan decarboxylase (aromatic L-amino-acid decarbox- ylase; EC 4.1.1.28) catalyzes the decarboxylation of the amino acid L-tryptophan to the protoalkaloid tryptamine. Tryptamine can then serve as substrate for the enzyme strictosidine syn- thase (Stockigt and Zenk, 1977), which catalyzes the first committed step in monoterpenoid indole alkaloid biosynthe- sis. The cDNA clone encoding tryptophan decarboxylase was

Figure 1. (continued).

Ajmaline, antiarrythmic that functions by inhibition of glucose uptake by heart tissue mitochondria; atropine ([*I-hyoscyamine), anticholinergic, antidote to nerve gas poisoning; caffeine, widely used central nervous system stimulant; camptothecin, potent anticancer agent; cocaine, topical anesthetic, potent central nervous system stimulant, and adrenergic blocking agent, drug of abuse; codeine, relatively nonaddictive analgesic and antitussive; coniine, first alkaloid to be synthesized, extremely toxic, causes paralysis of motor nerve endings, used in homeopathy; emetine, orally active emetic, amoebicide; morphine, powerful narcotic analgesic, addictive drug of abuse; nicotine, highly toxic, causes respiratory paraly- sis, horticultural insecticide; pilocarpine, peripheral stimulant of the parasympathetic system, used to treat glaucoma; quinine, traditional antimalarial, important in treating Plasmodium falcipafum strains that are resistant to other antimalarials; sanguinarine, antibacterial showing antiplaque activ- ity, used in toothpastes and oral rinses; scopolamine, powerful narcotic, used as a sedative for motion sickness; strychnine, violent tetanic poison, rat poison, used in homeopathy; taxol, antitumor agent; (+)-tubocurarine, nondepolarizing muscle relaxant producing paralysis, adjuvant to anesthesia; vinblastine, antineoplastic that is used to treat Hodgkin’s disease and other lymphomas.

1062 The Plant Cell

MicroorganismsPlant pathway: >^ F

D •=> EG, G3

Single geneexpression forbiomimetic syntheses

C D

HeterologousGene

A

I

Nc1

Plants

NormalPattiway

Antisense orCosuppression

1E

\

A

IB

1C

i

A

IBIC

D\iE

Expression ofshort pathways

Figure 2. Potential Biotechnological Exploitation of Alkaloid Biosynthetic Genes.

Plant alkaloid genes can be functionally expressed in microorganisms to produce either single biotransformation steps or short biosynthetic path-ways. Likewise, using overexpression or antisense or cosuppression technologies, medicinal plants can be tailored to produce important pharmaceuticalalkaloids by introducing side pathways, eliminating side pathways, or accumulating biosynthetic intermediates. For example, a known plant biosyn-thetic pathway contains an enzyme encoded by gene G3 that catalyzes a transformation step, of compound C to alkaloid D, which is difficultto achieve by chemical synthesis. The G3 gene can be heterologously expressed in a microorganism and the gene product used in a biomimeticsynthesis of alkaloid D (that is, the microorganism is supplied with compound C and produces alkaloid D). Likewise, a short pathway consistingof enzymes encoded by genes G,, G2, 63, and G,, could be expressed in a microorganism to produce alkaloid E directly from precursor A. Manyalkaloid biosynthetic pathways involve 20 to 30 enzymes. Our current knowledge of these pathways indicates that the genes encoding the biosyn-thetic enzymes are neither clustered nor coordinately controlled by one operon. Expression of an entire, long alkaloid pathway in a single microorganismis currently beyond our technical capability. In this case, it may be possible to alter the pathway in the plant cell and produce the desired alkaloideither in culture or in the field. For example, to accumulate alkaloid Z, which is not normally produced in a particular plant species, a transgene(from another plant or a microorganism) can be introduced. To accumulate alkaloid E, a side pathway that also uses precursor D may have tobe blocked. If intermediate alkaloid C is the target for accumulation, catabolism to D could be interrupted.

isolated from C. roseus by antibody screening of a cDNA ex-pression library prepared from poly(A)+ RNA of developingseedlings (De Luca et al., 1989). The tryptophan decarboxy-lase amino acid sequence shows similarities with an aromaticL-amino acid decarboxylase from Drosophila melanogastar andwith L-amino acid decarboxylases from diverse animal origins.The tryptophan decarboxylase transcript was shown to ac-cumulate in C. roseus cell suspension cultures exposed to avariety of biotic elicitors (Pasquali et al., 1992; Roewer et al.,1992). Auxin reduces transcription of the gene, as demon-strated by run-off transcription experiments with C. roseusnuclei (Goddijn et al., 1992).

A main point of interest in C. roseus—and the likely reasonfor the recent increase in the number of researchers workingon this plant- is that it produces the dimeric alkaloids vinblastine

and vincristine. It has been well established that cell culturesof C. roseus do not produce these commercially interestingand valuable alkaloids because the cultures lack vindoline,one component of both dimers. Induction of the last two en-zymes of vindoline biosynthesis in C. roseus seedlings,2-oxoglutarate-/V(1)-methyl-16-methoxy-23-dihydro-3-hydroxyta-bersonine 4-hydroxylase and 17-O-deacytylvindoline O-acetyl-transferase, appears to be regulated by phytochrome (Aertsand De Luca, 1992), but red light-induced accumulation ofat least the acetyl transferase has not been observed in cellcultures. The biotechnologically most important experiments,those that may potentially result in the production of the anti-neoplastic dimeric alkaloids in cell culture, thus await theisolation of these final genes of vindoline biosynthesis.

The tryptophan decarboxylase cDNA from C. roseus has

Monoterpenoid Indole Alkaloids

3n(S)-Slnclosidme

Benzylisoquinoline Alkaloids

Enzyme

OCH3

(S)-Reticuline (S)-Scoulerine

Bisbenzylisoquinoline AlkaloidsH3CO

Berberine Alkaloids

Protopine Alkaloids

BenzophenanthridineAlkalloids

ObamegineBerbamine

Alkaloids and Their Biosynthesis 1063

Tropane and Nicotine Alkaloids

Putrescine HaN NHS

PutrescmeA/-Methyltransferase

W-Methyl- J [putrescine H;N NHCH3

*^

^N'CH,

1-Melhyl-i'Nicotinic

acid

/pyrrolinium /

cation / Tropinone/ Reduclase-l

nN

NicotineHO

Tropine

CH,OH^

HOOC-C^Q

Tropic acid

CH,OH

Hyoscyamine ][ H [I0

IHyoscyamme6fi-Hydroxylase

,(fl)-W -Methylcoclaurine R, • pH, R2 • CH3(S)-N -Methylcoclaurine R, - aH, R; - CH3

Figure 3. Reactions Catalyzed by Alkaloid Biosynthetic Enzymes for which cDNAs Have Been Isolated.

been heterologously expressed in tobacco plants (Songstadet al., 1990,1991), and it increased their levels of tryptamineand tyramine, the product of u-tyrosine decarboxylation. A fineexample of what metabolic engineering can achieve in second-ary metabolism has been provided by the transformation of8rass/ca napus with the C. roseus tryptophan decarboxylasecDNA (Chavadej et al., 1994). The seed of this oil-producingcrop has limited use as animal feed due in part to the presenceof indole glucosinolates, which make the protein meal less pal-atable. The introduced cDNA for tryptophan decarboxylaseredirects tryptophan pools away from indole glucosinolate pro-duction and into tryptamine. The mature seed of the transgenic8. napus plants contain reduced levels of indole glucosinatesbut no tryptamine, achieving a potentially economically use-ful product.

Strictosidine Synthase

Strictosidine synthase (EC 4.3.3.2) catalyzes the stereospecificcondensation of the primary amino group of tryptamine andthe aldehyde moiety of the iridoid glucoside secologanin toform the first monoterpenoid indole alkaloid, 3a(S)-strictosidine.Strictosidine synthase is of biotechnological interest in the bio-mimetic syntheses of monoterpenoid indole alkaloids because,

although the condensation of tryptamine and secologanin canbe achieved chemically, the reaction product is a mixture ofthe diastereomers vincoside and Strictosidine. Only Strictosi-dine with the 3a(S) configuration, the exclusive product of theenzymatic reaction, can serve as a precursor to the monoterpe-noid indole alkaloids (Stockigt and Zenk, 1977). The enzymeis stable, requires no cofactor addition, and is readily immobi-lized (Pfitzner and Zenk, 1987) for producing Strictosidine.

The cDNA encoding Strictosidine synthase was first isolatedfrom R. serpentine (Kutchan et al., 1988), a plant used todayas the commercial source of antihypertensive indole alkaloidssuch as ajmaline and ajmalicine; a Strictosidine synthase cDNAwas subsequently isolated from C. roseus (McKnight et al.,1990). These two enzymes show 80% amino acid homology,which probably reflects the fact that both plants are membersof the Apocynaceae. DMA gel blot analysis using the R. ser-pentina cDNA as a probe shows that not all species that containthe enzyme contain a similar gene, possibly due to differencesin codon usage (Kutchan, 1993a). Strictosidine synthase fromR. serpentine has been functionally expressed in Escherichiaco// and Saccharomyces cerevisiae, and in insect cells usinga baculovirus vector (Kutchan, 1989; Kutchan et al., 1994). TheC. roseus enzyme has been expressed in tobacco and £ co//(McKnight et al., 1991; Roessner et al., 1992). A comparisonof these as production systems has been reviewed by Kutchan(1994).

1064 The Plant Cell

The gene for strictosidine synthase, strl, has been isolated from R. serpentina (Bracher and Kutchan, 1992). A single gene appears to code for the enzyme in both R. serpentina and C. mseus(Pasqua1i et al., 1992). In both species, the transcript accumulates predominantly in the roots and leaves of mature plants, although it can also be detected in flowers and stems. Levels of C. mseus strictosidine synthase transcript can be increased in cell culture (Pasquali et al., 1992; Roewer et al., 1992) and during a very narrow time period in developing seed- lings (Aerts et al., 1994) by treatment with various elicitors. However, the high levels of transcript present in mature plants suggest that sfrl is not one of the regulating genes of indole alkaloid biosynthesis.

TROPANE AND NlCOTlNE ALKALOIDS

The tropane class of alkaloids, which are found mainly in the Solanaceae, contains the anticholinergic drugs hyoscyamine (the racemate of which is called atropine) and scopolamine. Solanaceous plants have been used traditionally for their me- dicinal, hallucinogenic, and poisonous properties, which are due to their tropane alkaloids. The narcotic topical anesthetic and central nervous system stimulant cocaine is a tropane al- kaloid found outside of the Solanaceae in Erythmxylon coca. What is a useful medicinal in small, controlled doses is poten- tially lethal when abused. Hence, scopolamine isolated from Duboisia is commonly used today in the form of a transdermal patch to combat motion sickness, but Datura leaves are smoked for the hallucinogenic effects of this alkaloid. Although cocaine has served as a “lead,” a starting structure that medicinal chemists modify to design an optimized drug, for the develop- ment of synthetic topical anesthetics, this alkaloid is illicitly applied to mucus membranes for its addictive stimulatory ef- fects. Metabolic engineering of the plants that serve as commercial sources of nicotine or scopolamine could enhance classic breeding in the effort to develop plants with optimal alkaloid patterns, that is, that serve as improved sources of pharmaceuticals.

A study of the biosynthesis of tropane alkaloids implies a study of nicotine biosynthesis also, because the N-methyl-A’- pyrrolinium cation is a precursor to both classes of alkaloids. Analyses of nicotine biosynthesis in tobacco and cocaine bio- synthesis in coca were pioneered in the laboratory of E. Leete. The enzymology and molecular biology of scopolamine bio- synthesis in Hyoscyamus have been studied principally by T. Hashimoto and Y. Yamada. The cDNAs encoding hyoscya- mine 6p-hydroxylase (Matsuda et ai., 1991) and tropinone reductase (Nakajima et al., 1993), both of which are enzymes of scopolamine biosynthesis in Hyoscyamus niger, have been cloned (Figure 3). In addition, a tobacco cDNA for putrescine N-methyltransferase, an enzyme involved in the biosynthesis of the N-methyl-A’-pyrrolinium cation, has been isolated (Hibi et al., 1994).

Putrescine N-Methyltransferase

Cuban cigar tobacco varieties discovered in the 1930s to have low nicotine content were used in backcrosses to establish a genetically stable breeding line with a low alkaloid content for producing low nicotine cigarettes. This commercial low nico- tine variety, LA Burley 21, and the parenta1 strain, Burley 21, which is isogenic at all but two low nicotine biosynthesis loci, were used to isolate a cDNA for an enzyme of nicotine biosyn- thesis, putrescine N-methyltransferase (EC 2.1.1.53). This enzyme catalyzes the transfer of a methyl group from S-ade- nosyl-L-methionine to an amino group of putrescine, which is the first committed step in the biosynthesis of nicotine and tro- pane alkaloids. The availability of the near-isogenjc Burley Strains made it possible to use subtractive hybridization to iso- late a cDNA encoding this enzyme (Hibi et al., 1994). The deduced amino acid sequence of one of the clones obtained by subtractive hybridization is homologous to spermidine syn- thase from human (73% identical), mouse (70% identical), and E. coli(58% identical). Heterologous expression of the tobacco cDNA in an E. colideletion mutant lacking the spermidine syn- thase gene resulted in accumulation of N-methyl putrescine, confirming the identity of the cDNA. Transcripts for putrescine N-methyltransferase accumulate predominantly, if not exclu- sively, in root tissue of the wild-type tobacco plant, suggesting that this organ is the major site of nicotine biosynthesis. This corresponds to the site of tropane alkaloid biosynthesis in other members of the Solanaceae (Hashimoto et al., 1991). The log- ical progression of this work isto isolate a putrescine N-methyl- transferase gene from a plant that produces tropane alkaloids.

Tropinone Reductase

Along the biosynthetic pathway that leads specifically to the tro- pane alkaloid scopolamine, tropinone reductase I (EC 1.1.1.236) converts the 3-keto group of tropinone to the 3a-hydroxyl of tropine. The cDNA encoding this enzyme was isolated from Datura stramonium (jimsonweed) by screening a cDNA library prepared from hairy roots with oligonucleotides based on pep- tide amino acid sequences from purified native tropinone reductase I (Nakajimaet al., 1993). The cDNA was expressed as a p-galactosidase fusion protein in E. coli and found to have tropinone reductase I activity. DNA gel blot analysis identified homologous DNA fragments in the nuclear DNA of the tro- pane alkaloid-producing species H. niger and A. belladonna, but not in tobacco, a species in which tropane alkaloids are not biosynthesized.

A cDNA for a second reductase, tropinone reductase II, was also isolated in these experiments. Tropinone reductase I1 reduces tropinone with a stereospecificity opposite to that of tropinone reductase I. Hence, the 3-keto group of tropinone is converted to the 3p-hydroxyl of pseudotropine. The deduced amino acid sequences of the two cDNAs are 64% identical. In a series of elegant experiments, various domains of these

Alkaloids and Their Biosynthesis 1065

two reductases were exchanged and the chimeric enzymes were expressed in E. coli to identify the domain that confers the stereospecificity of the reaction (Nakajima et al., 1994). In this manner, it was demonstrated that a 120-amino acid resi- due peptide at the C terminus of each reductase determines the stereospecificity of the reaction it catalyzes.

Hyoscyamine 6p-Hydroxylase

The final two steps in the biosynthetic pathway leading from hyoscyamine to the medicinally important alkaloid scopolamine is catalyzed by a 2-oxoglutarate-dependent enzyme, hyoscya- mine 6P-hydroxylase (hyoscyamine [6S]-dioxygenase; EC 1.14.11.11). This dioxygenase first hydroxylates hyoscyamine in the 6s position and subsequently catalyzes epoxide for- mation. Molecular cloning and heterologous expression of hyoscyamine 6P-hydroxylase have demonstrated that this enzyme catalyzes both the hydroxylation reaction and the intra- molecular epoxidation reaction (Matsuda et al., 1991). RNA gel blot analysis of the hyoscyamine 6P-hydroxylase gene has corroborated the results obtained by immunohistochemical lo- calization of the enzyme (Hashimoto et al., 1991), which showed that hyoscyamine 6P-hydroxylase is localized to the root peri- cycle in scopolamine-producing species of Hyoscyamus, Atropa, and Duboisia. Similarly, the hyoscyamine 6P-hydroxy- lase transcript is localized to roots and cultured roots of H. niger but is not found in stem, leaves, or cultured cells of this same species. These results explain why it has not been possible to produce substantial quantities of tropane alkaloids in cell culture. The gene encoding hyoscyamine 6P-hydroxylase should help with the elucidation of the underlying mechanisms of tissue- and cell-specific expression of tropane alkaloids in the Solanaceae.

The current commercial source of scopolamine is Duboisia, which was cultivated originally in Australia. Certain tropane alkaloid-producing species, such as Atropa, accumulate hyoscyamine instead of scopolamine as the major alkaloid. To ask whether expression of a transgene in a medicinal plant could alter the alkaloid pattern such that more of the phar- maceutically useful alkaloid, scopolamine, could be produced, the cDNA encoding hyoscyamine 6P-hydroxylase from H. niger was introduced into A. belladonna using either Agrobacferium tumefaciens- or Agrobacterium rhizogenes-mediated trans- formation. The resultant transgenic plants (Yun et al., 1992) and hairy roots (Hashimoto et al., 1993) each contained elevated levels of scopolamine.

Each of these two successful transformation experiments has a distinct implication for the future of metabolic engineer- ing of medicinal plants. First, the transgenic A. belladonna plants, although not necessarily commercially useful, provide the first example of how medicinal plants can be successfully altered using molecular genetic techniques to produce in- creased quantities of a medicinally important alkaloid. The future of this field is completely dependent upon a thorough

knowledge of the alkaloid biosynthetic pathway at the enzyme level so that meaningful transformation experiments can be designed. It is also limited by our ability to transform and regenerate medicinal plants. To date, expertise in this impor- tant area lags well behind that for tobacco, petunia, and cereal crops, among others. For example, in the area of tropane alkaloids, transformation and regeneration of Duboisia, a plant for which plantation, harvesting, and purification techniques have already been commercially established, will have to be developed before any potential commercialization. The sec- ond implication is that metabolic engineering may make it possible to produce alkaloids in cultured cells. The produc- tion of alkaloids in tissue and cell culture, such as in hairy roots, is an area that received much attention in the past because it promised an alternative source of pharmaceuticals that would be independent of weather, blight, and politics. It has become clear with time, however, that many important compounds, such as vincristine, vinblastine, pilocarpine, morphine, and codeine, are not synthesized to any appreciable extent in culture. The reason is thought to be tissue-specific expression of alkaloid biosynthetic genes, because in some cases, plants regener- ated from nonproducing callus cells contain the same alkaloid profile as the parent plant. For certain costly alkaloids (for ex- ample, taxol), cell culture production at a commercial level would be worthwhile establishing. The increased accumula- tion of scopolamine in hairy roots that contain an hyoscyamine 6P-hydroxylase transgene is an elegant demonstration that this approach remains a viable one.

BENZYLISOQUINOLINE ALKALOIDS

The benzylisoquinoline alkaloids are avery large and diverse class of alkaloids. This family contains such varied physiolog- ically active members as emetine (an antiamoebic), colchicine (a microtubule disrupter and gout suppressant), berberine (an antimicrobial against eye and intestinal infections), morphine (a narcotic analgesic), codeine (a narcotic analgesic and an- titussive), and sanguinarine (an antimicrobial used in oral hygeine). As with the pharmacologically active members of the indole and tropane classes of alkaloids, those of phar- maceutical interest from the benzylisoquinoline class are largely still isolated from plants, again due to the complexity of their structures. One notable exception to this is the ben- zylisoquinoline alkaloid berberine, which is isolated in Japan from cell suspension cultures of Copfisjaponica (summarized in Zenk et al., 1988).

Benzo(c)phenanthridine alkaloids, a subclass of the benzyl- isoquinolines, are produced in a numberof species within the Papaveraceae, incl uding Sanguinaria canadensis (bloodroot), I? somniferum (opium poppy), and Eschscholtzia californica (California poppy, used as a sedative by Native Americans). Extracts of bloodroot that are rich in the intensely red ben- zophenanthridine alkaloid sanguinarine are currently added

1066 The Plant Cell

to toothpastes and oral rinses in'the United States because of this alkaloid's antiplaque properties. Benzophenanthridine alkaloids have been shown to accumulate in cell cultures of E. californica in response to funga1 attack and to treatment with numerous elicitor substances (Schumacher et al., 1987). This phenomenon, when exploited in cell suspension cultures, pro- vided the main experimental system with which the entire biosynthetic pathway leading from two molecules of L-tyrosine to the most highly oxidized benzophenanthridine alkaloid, macarpine, was elucidated in the laboratory of M.H. Zenk. This pathway, encompassing 21 conversions, 20 of which are enzymatic and one of which is a spontaneous skeletal rear- rangement, is the single longest secondary metabolic pathway to have been completely elucidated at the enzyme leve1 (sum- marized in Kutchan and Zenk, 1993; Kammerer et al., 1994). A cDNA encoding one of seven inducible enzymes from this pathway, the berberine bridge enzyme (Figure 3), has been isolated (Dittrich and Kutchan, 1991).

Berberine Bridge Enzyme

The berberine bridge enzyme ([SI-reticu1ine:oxygen oxidore- ductase [methylene bridge forming]; EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl group of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine. There are two main reasons why this particular enzyme is of interest. First, the reaction catalyzed by the berberine bridge enzyme is very elegant from the chemist's point of view. The direct con- version of the N-methyl group into a methylene bridge moiety is not presently achievable by synthetic chemical methods and exists nowhere else in nature. It is of biochemical interest to know how an enzyme catalyzes a reaction that we as chemists cannot. The second point of interest is that the E. californica berberine bridge enzyme is elicitor inducible. This implies that regulation of berberine bridge enzyme transcript accumula- tion may regulate benzophenanthridine alkaloid accumulation. From an analysis of the promoter of the bbel gene, details can therefore be learned about how alkaloid biosynthesis is regu- lated in response to pathogen attack.

The cDNA encoding the berberine bridge enzyme has been isolated from a cDNA bank prepared from elicited cell sus- pension cultures of E. californica using oligonucleotides based on peptide amino acid sequences of the purified native en- zyme (Dittrich and Kutchan, 1991). Translation of the nucleotide sequence confirmed the presence of a signal peptide (by com- parison with the experimentally determined sequence of the mature protein N terminus) that directs the enzyme into the endoplasmic reticulum and then into the smooth vesicles in which it accumulates (Amann et al., 1986). Heterologous ex- pression of this cDNA in insect cell culture using a baculovirus expression vector resulted in the production of sufficient quan- tities (4 mg of homogeneous enzyme per liter of insect cell culture medium) for a biochemical analysis of the berberine bridge enzyme (Kutchan et al., 1994). A series of spectral determinations and modified-substrate conversion-rate mea- surements has demonstrated that the berberine bridge enzyme

is covalently flavinylated and that closure of the ring system to form (S)-scoulerine during the course of the enzymatic reac- tion proceeds via an ionic mechanism with the methylene iminium ion as a reaction intermediate (T.M. Kutchan, unpub- lished data).

An analysis of the time course of the elicitation process using the cDNA clone as a hybridization probe in RNA gel blot ex- periments revealed that the berberine bridge enzyme transcript reaches maximal levels within 6 hr after addition of a yeast elicitor to E. californica cultures. Enzyme activity increases until 17 to 22 hr after elicitation, and total benzophenanthridine alkaloids continue to accumulate for severa1 days after elicita- tion (Dittrich and Kutchan, 1991). This scenario is indicative of de novo transcription of phytoalexin biosynthetic genes, which also occurs during stress-induced phenylpropanoid me- tabolism (see Dixon and Paiva, 1995, this issue). Methyl jasmonate induces the accumulation of low molecular weight compounds in a large number of plant species in culture (Gundlach et al., 1992), and both methyl jasmonate and the jasmonic acid biosynthetic precursor 124x0-phytodienoic acid induce transcription of the single bbel gene in E. californica cell cultures (Kutchan, 1993b; Kutchan and Zenk, 1993). It has recently been shown that the Pseudomonas syringae von Hall phytotoxin, coronatine, mimics 124x0-phytodienoic acid in its ability to induce a series of physiological responses such as tendril coiling, secondary metabolite accumulation in cell cul- ture, and bbel transcription in E. californica (Weiler et al., 1994). Unlike various abiotic and biotic elicitors (Gundlach et al., 1992; Mueller et al., 1993), coronatine affects gene transcription without inducing endogenous 12-0~0-phytodienoic acid and jasmonic acid accumulation. lnduction of the berberine bridge enzyme transcript with trihomo-jasmonate has demonstrated that P-oxidation is not necessary for gene activation (Blechert et al., 1995). Analysis of the cis and rrans elements necessary for elicitor-induced transcription of bbel and of other induc- ible genes along the benzophenanthridine alkaloid biosynthetic pathway should help to elucidate the complex defense re- sponse signal transduction chain.

BISBENZYLISOQUINOLINE ALKALOIDS

The bisbenzylisoquinoline class of alkaloids contains over 270 members, all of which are dimers of tetrahydrobenzylisoquino- lines connected by one to three ether linkages formed by phenol coupling. The structure of a prototype of these natural prod- ucts, (+)-tubocurarine, which is the muscle relaxant isolated from tube-curare (a preparation of Chondodendmn romenro- sum in wooden tubes), contains two ether linkages with the tetrahydrobenzylisoquinoline moieties combined in a head-to- tail orientation (Figure 1). Tube-curare has been traditionally used by South American lndians as an arrow poison. In modern medicine, tubocurarine chloride is used as a neuromuscular blocking agent to secure muscular relaxation in surgical oper- ations. It is of interest that only one plant, C. romenrosum, is known to contain (+)-tubocurarine.

Alkaloids and Their Biosynthesis 1067

The bisbenzylisoquinoline family of alkaloids is rich in phar- macologically active constituents that range in activity from cytotoxins to antihypertensives to antimalarials. The structural variations in these alkaloids include substitutions on the phenyl rings, the regiospecificity of the ether linkages, and the stereospecificity of the isoquinoline moieties. The best under- stood biosynthesis is that of the dimeric alkaloids berbamunine and guattegaumerine, which are produced in cell suspension cultures of Berberis stolonifera (barberry). The single most im- portant step in the biosynthesis of this category of alkaloids is ether linkage formation through phenol coupling. Any bio- technological production of the pharmaceutically important bisbenzylisoquinolines requires correct stereo- and regio- selectivity of ether bond formation. It has only recently been shown that the enzymes that catalyze either carbon-carbon or carbon-oxygen phenol coupling in plants are not nonspecific peroxidases or laccases but rather highly regio- and stereo- selectke substrate-specific cytochrome P-450-dependent oxidases (Zenk et al., 1989; Stadler and Zenk, 1993). The reac- tion catalyzed by these enzymes is nove1 for cytochromes P-450 in that it proceeds without incorporation of oxygen into the prod- uct. In other words, this cytochrome P-450 functions as an oxidase rather than as a monooxygenase. It would be mecha- nistically interesting to discern how these enzymes act on molecular oxygen differently from the more common hydrox- ylases. Studies of the enzymatic mechanism require the ability to isolate large quantities of functional enzyme and to alter the enzyme's structure and observe the types of changes that occur in the enzymatic reaction. These approaches are, of course, dependent on molecular genetic manipulations. TO this end, the first cDNA known to encode a plant cytochrome P-450 unequivocally involved in alkaloid biosynthesis has recently been cloned. This cDNA encodes berbamunine synthase (Fig- ure 3) (Kraus and Kutchan, 1995).

Berbamunine Synthase

Berbamunine synthase (N-methylcoclaurine, NADPH:oxygen oxidoreductase [carbon-oxygen phenol coupling]; EC 1.1.3.34; CYP80) catalyzes formation of the ether linkage between one molecule of (R)-N-methylcoclaurine and one molecule of (S)-N-methylcoclaurine to form the bisbenzylisoquinoline al- kaloid berbamunine. This cytochrome P-450 enzyme alSO couples two molecules of (R)-N-methylcoclaurine to form guat- tegaumerine. The cDNA encoding berbamunine synthase was isolated from a B. stolonifera cell suspension culture cDNA library (Kraus and Kutchan, 1995). An oligonucleotide primer based on the N-terminal amino acid sequence of the purified native oxidase was used as a screening probe. Translation of the nucleotide sequence of the clone revealed at least one notable amino acid residue difference in berbamunine syn- thase as compared with the sequences of cloned cytochrome P-450 monooxygenases. Structure-function studies on bac- teria1 P-450cam suggest that three amino acid residues of the dista1 helix (helix I), Gly-248, Gly-249, and Thr-252, are essen- tia1 for formation of the molecular oxygen binding pocket

(Poulos et al., 1985). Mutation of Thr-252 to Ala or Val abol- ishes insertion of oxygen into the substrate (Imai et al., 1989). Thr-252 is present in berbamunine synthase, although there is no monooxygenation reaction in the carbon-oxygen phe- no1 coupling process. However, the essential Ala or Gly at the position corresponding to 248 in P-450cam is replaced by a Pro in berbamunine synthase. Because this amino acid resi- due is one of three highly conserved residues of the oxygen binding pocket that are thought to be required for insertion of oxygen into the substrate, this may be a first indication of how berbamunine synthase functions as an oxidase.

Berbamunine synthase has been expressed in functional form in insect cell culture using a baculovirus expression vec- tor (Kraus and Kutchan, 1995). The oxidase accepts electrons from either insect cell, porcine, or Berberis cytochrome P-450 reductases. Heterologously expressed berbamunine synthase can be obtained in near-homogeneous form and in large quan- tities (5 mgll) after insect cell microsome solubilization followed by a single column-chromatography fractionation step. To ob- tain this amount of enzyme from B. stolonifera cells, 18,000 L of suspension culture would have to be extracted. A single gene probably codes for berbamunine synthase in the B. stolo- nifera genome, although two more weakly hybridizing DNA fragments are also present that may represent genes encod- ing other phenol coupling cytochromes.

ECOCHEMICAL FUNCTION OF ALKALOIDS

Their medicinal and toxicological properties clearly make alkaloids important to people, but their role in plants has been a longstanding question. Why should a plant invest so much nitrogen in synthesizing such a large number of alkaloids of such diverse structure? C, meus alone contains over 100 differ- ent monoterpenoid indole alkaloids. That many alkaloids are cytotoxic provides insight into their potential function in the chemical defense arsenal of the plant. It needs to be system- atically addressed whether alkaloids function as phytoalexins, as nitrogen storage forms, as UV protectants, or in any combi- nation of these and other potential functions. The question of alkaloid function in plants and the relevance of that function have been reviewed (Hartmann, 1991; Caporale, 1995).

One alkaloid whose ecochemical functions have been thor- oughly analyzed in recent years is nicotine, a highly toxic alkaloid that has been identified in the leaves, stems, and roots of a number of Nicotiana species. Nicotine sulfate, a byproduct of the tobacco industry, serves commercially as a very effec- tive insecticide and fumigant. To date, no insect has evolved a resistance mechanism against nicotine. In mammals, inges- tion of nicotine results in nausea, vomiting, diarrhea, mental confusion, convulsions, and respiratory paralysis. With such strong and varied physiological responses, nicotine would seem to be an effective deterrent against insect attack and herbivore grazing. This is indeed the case. A series of studies in the laboratory of I.T. Baldwin has demonstrated that nico- tine, which is known to be synthesized in roots and transported

1068 The Plant Cell

to leaves, is synthesized de novo from (15N)-nitrate in re- sponse to wounding of leaves of hydroponically grown N. sy/- vesfris (Baldwin et al., 1994a). The amount of alkaloid that can be accumulated in the leaf is sufficient to reduce larva1 growth of the tobacco hornworm Manduca sexta (Baldwin, 1988). Most importantly, field tests with native populations of the summer annual N. artenuata demonstrated a systemic alkaloid induc- tion in response to simulated leaf herbivory and browsing (Baldwin and Ohnmeiss, 1993).

Nicotine is clearly synthesized in response to wounding, but does the alkaloid have additional functions, for example, as a nitrogen storage form oras a UV protectant? Although exog- enously administered nicotine is catabolized to nornicotine and myosmine, most likely as a result of a detoxification mecha- nism, de novo-synthesized nicotine is not turned over. The plant does not recover nicotine nitrogen and reinvest it in other metabolic processes, even under conditions of nitrogen-limited growth, implying that nicotine is not used as a nitrogen stor- age form (Baldwin and Ohnmeiss, 1994). Nicotine supplied exogenously to D. sframonium, a species that accumulates tro- pane alkaloids but not nicotine, does not result in increased UV protection, although nicotine has a high molar extinction coefficient (2695 M-l cm-l) at 262 nm (Baldwin and Huh, 1994). Taken together, these results suggest that nicotine func- tions as a phytoalexin in tobacco and has no additional role as a storage form of nitrogen or as a filter for UV radiation.

These results raise an important question: How does leaf wounding induce nicotine biosynthesis in the root? Jasmonates are known to induce accumulation of secondary metabolites in plant cell culture, and endogenous jasmonate pools increase rapidly in response to treatment of cells with a yeast elicitor (Gundlach et al., 1992). Leaf damage to N. sy/vesfris produces a similar increase in endogenous jasmonic acid pools in shoots within 30 min, and root pools of this signaling molecule in- crease within 2 hr (Baldwin et al., 1994b). In addition, application of methyl jasmonate as a lanolin paste to leaves results in increases in both endogenous jasmonic acid in roots and de novo nicotine biosynthesis. Jasmonate and 12-0x0- phytodienoic acid function as inducers of alkaloid biosynthe- sis both in culture and in plants. The next question that needs to be addressed is whether jasmonate is the systemic signal- ing molecule that is transported from the wounded leaf to roots, where it then induces transcription of the alkaloid biosynthetic genes.

PERSPECTIVES

Although the alkaloid field is a very old one, it is still in its in- fancy with regard to being fully understood, and our exploitation of the biotechnological potential of alkaloid biosynthesis has only just begun. We are still a long way from understanding how most alkaloids are synthesized in plants and how this bio- synthesis is regulated. We also have much to learn about the chemical ecology of alkaloids so that we can better understand

why these sophisticated and diverse structures evolved. What is clear is that alkaloids as integral components of medicinal plants have enjoyed a long and important history in traditional medicine. Our first drugs originated in the form of plant extracts, and some of our most important contemporary pharmaceuti- cals are either still isolated from plants or structurally derived from natural products. New antineoplastic, antiviral, and an- timalarial alkaloids are still being discovered in plants. As we accumulate the tools (biochemical knowledge, clones, and transformation and regeneration techniques) necessary for fu- ture developments in this field, we can look forward to genetically engineered microorganisms and eukaryotic cell cul- tures that produce medicinal alkaloids, to medicinal plants with opitimized alkaloid spectra, and even to the production of im- portant pharmaceuticals in transgenic plant cell cultures.

ACKNOWLEDGMENTS

Our molecular genetic research on strictosidine synthase, the berber- ine bridge enzyme, and berbamunine synthase has been supported by a grant from the Bundesminister fijr Forschung und Technologie, Bonn, and by the Deutsche Forschungsgemeinschaft (Grant No. SFB 369), Bonn.

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DOI 10.1105/tpc.7.7.1059 1995;7;1059-1070Plant Cell

T. M. KutchanAlkaloid Biosynthesis[mdash]The Basis for Metabolic Engineering of Medicinal Plants.

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