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Home , Ephedrine, Methoxyphenamine, Phenylalanine, Pseudoephedrine, Selegiline, Synephrine

Review

Biosynthesis of analogs

in plants

1 2 3 2

Jillian M. Hagel , Raz Krizevski , Fre´ de´ ric Marsolais , Efraim Lewinsohn and

1

Peter J. Facchini

1

Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada

2

Department of Aromatic, Medicinal and Spice Crops, Newe Ya’ar Research Center, Agricultural Research Organization,

PO Box 1021, Ramat Yishay 30095, Israel

3

Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ont, N5 V 43T, Canada

Amphetamine analogs are produced by plants in the microorganisms for the de novo production of pseudoephed-

genus and by Catha edulis, and include the rine and . The potential acceptance of substituted

widely used and appetite suppressants precursors by such and the possible occurrence of

and ephedrine. A combination of catalysts from any organism with the serendipitous capacity

(Candida utilis or ) fermenta- to modify amphetamine analogs might provide an unprece-

tion and subsequent chemical modification is used for dented opportunity for the commercial production of some

the commercial production of these compounds. The pharmaceuticals in synthetic biosystems.

availability of certain plant biosynthetic genes would

facilitate the engineering of yeast strains capable of de Amphetamine analogs in plants

novo pseudoephedrine and ephedrine . is a flowering perennial shrub with a long history of

Chemical synthesis has yielded amphetamine analogs cultivation in East Africa and the Arabian Peninsula.

with myriad functional group substitutions and diverse Evidence suggests that the traditional practice of chewing

pharmacological properties. The isolation of enzymes khat leaves as part of social affairs dates back at least 1000

with the serendipitous capacity to accept novel sub- years [4] and might even pre-date the use of coffee [5]. The

strates could allow the production of substituted presence of (S)- in young khat leaves imparts a

in synthetic biosystems. Here, we review mild and stimulating effect, making khat a pop-

the biology, and biotechnological potential ular concentration aid for students and scholars. Although

of amphetamine analogs in plants. use of the plant has recently generated health concerns,

studies involving short- and long-term effects of khat

Plants and the biotechnological potential for chewing have been incomplete or controversial [6]. Khat

amphetamine analog biosynthesis possession is strictly illegal in many Western countries.

Plants produce a vast array of specialized nitrogenous Notable exceptions include the UK and Holland, where

metabolites, including a wide variety of and glu- khat use is entirely unregulated [4]. Ephedra sinica,

cosinolates [1,2]. Substituted amphetamines, which are also known as Ma Huang in Chinese , also has a long

called phenylpropylamino alkaloids, are a diverse group of history of traditional use, particularly in the treatment

-containing compounds that feature a phenethyla- of bronchial , coughs, , headaches

mine backbone with a at the a-position rela- and [7]. (1S,2S)-Pseudoephedrine is a common

tive to the nitrogen (Figure 1). Countless variation in ingredient in many over-the-counter cold or for-

1 1

functional group substitutions has yielded a collection of mulations, including Claritin D , Contac and various

1

synthetic with diverse pharmacological properties as Sudafed products. (1R,2S)-Ephedrine is marketed as

, empathogens and [3]. Although an anticongestant, aid and sports-enhancing ,

amphetamine (1-phenylpropan-2-) and most analogs although its availability is more restricted. (S)-Cathinone,

are synthetic, several well-known phenylpropylamino alka- (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine are sym-

loids are produced by Catha edulis, commonly known as pathomimetic agents acting with either, or a combination

khat, and by members of the genus Ephedra (Figure 1). of: (i) direct activity at receptors [8]; (ii)

Although the widely used pseudoephedrine indirect activity through the release of noradrenaline from

and the diet aid ephedrine occur in several Ephedra species, sympathetic neurons; or (iii) activity at currently uniden-

commercial production of these alkaloids involves a combi- tified receptors [9]. Ephedrine causes central

nation of and chemical synthesis. The forma- and release [10], which might contribute to its

tion of amphetamine analogs in plants has been partially purported addictive nature [11].

characterized at the biochemical level. The availability of (S)-Cathinone is the principal psychostimulant in

the key biosynthetic genes of amphetamine analog biosyn- khat and accumulates primarily in young leaves

thesis in plants could provide opportunities to engineer (Figure 1), whereas (1S,2S)-norpseudoephedrine ()

and (1R,2S)-norephedrine occur throughout all the aerial

Corresponding author: Facchini, P.J. ([email protected]). organs of the plant [12]. Ephedra species such as E. sinica

404 doi:10.1016/j.tplants.2012.03.004 Trends in Plant Science, July 2012, Vol. 17, No. 7

Review Trends in Plant Science July 2012, Vol. 17, No. 7

Ephedra spp. Catha edulis O

NH2 OH H α N β (S)-cathinone OH (1R,2S)-ephedrine NH2

OH H (1R,2S)-norephedrine 2 N 1 OH

NH2 (1S,2S)-pseudoephedrine

(1S,2S)-pseudonorephedrine

TRENDS in Plant Science

Figure 1. Naturally occurring substituted amphetamines in Ephedra species and Catha edulis (khat). (S)-Cathinone imparts a mild effect upon chewing young khat

leaves, whereas N-methylated alkaloids (1R,2S)-ephedrine and (1S,2S)-pseudoephedrine in plants such as Ephedra sinica are effective nasal decongestants.

and E. distachya have N-methylated versions of these com- a-carbon. Such structural similarities promoted the initial

pounds, including (1R,2S)-ephedrine and (1S,2S)-pseudo- hypothesis that amphetamine analogs, such as ephedrine,

ephedrine (Figure 1) [13]. The occurrence of a hydroxyl were derived directly from Phe [27] or by reaction of a Phe-

group at the b-carbon of norephedrine and ephedrine alka- derived with a formate-derived one-car-

loids forms a second chiral center, creating diastereomer bon unit [28]. However, early pulse-labeling studies using

pairs. In the mature stems of E. sinica, a second N-methyl C. edulis [29] and [30,31] established

group may be present, forming (1R,2S)-N-methylephedrine that only the benzylic (i.e. C6–C1) component of ephedrine

or (1S,2S)-N-methylpseudoephedrine. Ephedrine has also alkaloids is derived from Phe. Interestingly, this finding

been reported in , an important plant in implied that Phe first undergoes deamination and side-

Ayurvedic medicine [14,15], and other amphetamine ana- chain shortening, followed by chain lengthening and ream-

logs have been detected in Acacia berlandieri [16] and ination (Figure 2). In support of this model, authors

Acacia rigidula [17]. Related compounds found in plants showed that , benzoic acid and

include the stimulant , a popular diet aid and were each efficiently incorporated into ephedrine [30,31].

sports-enhancing drug present in ( aur- Nearly two decades later, it was shown that the remaining

antium) [18]; , a derived from the C2–C3 unit was derived from [32,33]

peyote cactus (Lophophora williamsii) [19]; and adrenalin, (Figure 2). Further tracer experiments supported benzoic

found in trace amounts in cacti and other plants [20]. acid (or benzoyl-CoA) over benzaldehyde as the C6–C1

Khat is a member of the Celestraceae, an angiosperm molecule undergoing carboligation with pyruvic acid

family of dicotyledonous vines, shrubs and small trees [21]. [34]. In other systems, such as yeast, the carboligation of

Conversely, the genus Ephedra is the sole member of the benzaldehyde and pyruvic acid, catalyzed by a thiamin

Ephedraceae family, which in turn represents one of three diphosphate (ThDP)-dependent , is a well-charac-

families in the order Gnetales. Although generally viewed terized reaction [35,36]. Whether benzoic acid (benzoyl-

as gymnosperms, members of the Gnetales share certain CoA) or benzaldehyde directly supplies the C6–C1 unit of

features with angiosperms, such as vessel elements, chla- ephedrine alkaloids remains an open question [37].

mys-surrounded ovules and double fertilization [22]. Such The molecular biochemical basis of substituted amphet-

characteristics gave rise to the ‘anthophyte’ hypothesis amine biosynthesis in khat and Ephedra species is largely

[23], which held that Gnetales were a sister taxa to flower- unknown. Only the initial step [the deamination of Phe by

ing plants. However, recent molecular-based evidence has lyase (PAL)] has been character-

consistently placed Gnetales within the division Coniferae ized at the molecular level in E. sinica [38]. Figure 3

(i.e. conifers) as a sister group to the Pinaceae, or pine illustrates the proposed biosynthesis of ephedrine alka-

family [24,25]. The restricted occurrence of substituted loids based on molecular evidence gathered from other

amphetamine biosynthesis in both angiosperms and gym- plant species and biochemical studies of khat or E. sinica

nosperms suggests the convergent recruitment of required [12,13,38]. Several enzymes involved in benzoic acid bio-

biosynthetic genes [26]. The independent evolution of key synthesis, including 4-coumaroyl-CoA ligase (4CL) [39],

enzymes or whole pathways within plant specialized me- benzoyl-CoA ligase (BZO1) [40], a 3-ketoacyl-CoA thiolase

tabolism is not uncommon. For example, purine alkaloids (KAT1) [41] and two distinct dehydrogenases from snap-

(e.g. ), pyrrolizidine alkaloids and various plant dragon (Antirrhinum majus) (BALDH) [42] and Arabidop-

pigments are likely products of convergent evolution [26]. sis (Arabidopsis thaliana) (AAO4) [43], have been cloned

and characterized from plants that produce floral volatiles

Biosynthesis of substituted amphetamines in plants or . Studies of benzoic acid biosynthesis in

The biosynthesis of naturally occurring substituted these plants have revealed at least two possible courses

amphetamines in plants begins with L-phenylalanine of Phe side-chain shortening: the b-oxidative and non-b-

(Phe) as the precursor. Similar to amphetamine, Phe com- oxidative routes [44] (Figure 3). Alternatively, benzalde-

prises a C6–C3 backbone, with an amino group at the hyde might be formed via phenylpyruvate, which is a

405

Review Trends in Plant Science July 2012, Vol. 17, No. 7

An enzyme catalyzing the condensation of benzoic acid,

benzoyl-CoA or benzaldehyde with pyruvate has not yet

been isolated from -producing

plants, although a ThDP-dependent pyruvate decarboxyl-

ase (PDC; EC 4.1.1.1) or an acetohydroxyacid synthase

(AHAS; EC 2.2.1.6) have been suggested [33]. These two

Deamination distantly related enzymes [47] share the common step of

pyruvate . However, whereas PDCs gener-

ally decompose pyruvate to and CO2 [48],

AHASs catalyze carboligation reactions, forming either

acetolactate or acetohydroxybutyrate products as part of

branched-chain biosynthesis [36]. Members of

β-Oxidation both enzyme classes have shown carboligase activity toward

benzaldehyde, yielding (R)- (R-PAC)

and a variety of other products [48,49]. The possibility that

such reaction products are formed in khat or Ephedra

species is acknowledged in Figure 3. It is currently postu-

lated that 1-phenylpropane-1,2-dione is a direct precursor to

amphetamine analogs in plants [12,13,34,37]. This diketone

Carboligation

has been isolated from khat leaves [12] and young E. sinica

stems [13]. 1-Phenylpropane-1,2-dione subsequently under-

goes transamination to yield (S)-cathinone, which is con-

verted to (1S,2S)-cathine or (1R,2S)-norephedrine by

NADH-dependent reduction (Figures 2 and 3). Although

the transamination step has not been characterized, (S)-

Transamination cathinone reductase activity was reported recently in E.

sinica stems [13] and khat leaves [12]. S-Adenosylmethio-

nine (SAM)-dependent N-methylation completes the path-

way in Ephedra species, yielding (1R,2S)-ephedrine and its

diastereomer. N-Methylation activity has been detected in

cell-free extracts of E. sinica stems [13]. By contrast, the end

products cathine and (1R,2S)-norephedrine do not undergo

Reduction

N-methylation in khat.

Beyond PAL, no genes encoding enzymes involved in

amphetamine analog biosynthesis have been isolated. For

the purposes of gene discovery, we recently established a

functional genomics platform enabling the identification of

key biosynthetic gene candidates from khat [37] and E.

N-methylation sinica, including deep sequence resources based on RNA-

seq technology [50]. The availability of sequence resources

will provide homologs or entirely novel enzymes potential-

ly involved in biosynthesis. For example, the

amino acid sequences of enzymes listed in Table 1 can

serve as queries to identify homologs in khat or E. sinica,

TRENDS in Plant Science and functional analyses can be used to establish their roles

in substituted amphetamine biosynthesis (Figure 3).

Figure 2. Schematic representation of substituted amphetamine biosynthesis.

Although begins with a C6–C3 backbone including a nitrogen (L- Understanding the pathway at the molecular biochemical

phenylalanine), deamination and chain shortening, followed by chain lengthening

level is critical for the development of novel biotechnologi-

and an amino transfer, are necessary to build an amphetamine framework.

cal tools with potential applications in the pharmaceutical

Deamination of phenylalanine yields cinnamic acid, which undergoes chain

shortening from C6–C3 to C6–C1 by either b-oxidative or non-b-oxidative industry.

pathways, generating benzoic acid or a derivative (Figure 3). Reforming of a C6–

C3 backbone is then achieved by carboligation with pyruvate, yielding 1-

Potential biotechnological applications

phenylpropane-1,2-dione. Nitrogen is reintroduced by transamination to

cathinone, followed by reduction to cathine and/or norephedrine and N- Three different methodologies exist for the commercial

methylation to pseudoephedrine/ephedrine. N-Methylation occurs in Ephedra

production of ephedrine and related alkaloids: (i) historic

species, but not in khat. Color coding: black, carbon; red, oxygen; blue, nitrogen.

practices have included extraction from plants such as

E. sinica, a method still used for herbal formulations

transamination product of Phe. This route occurs in lactic and various products containing Ma Huang. Alkaloids

acid [45], but has not been confirmed in plants. are obtained by an initial treatment with alkali, followed

Nonetheless, the transamination of Phe to phenylpyruvate by extraction with an organic solvent. However, extraction

has recently been demonstrated, which might lead to the from plant sources is laborious, time-consuming and rela-

formation of benzaldehyde in melon fruit [46]. tively expensive [51]; (ii) in addition to natural sources,

406

Review Trends in Plant Science July 2012, Vol. 17, No. 7

Table 1. Level of characterization for enzymes with putative or established roles in , benzoic acid and

amphetamine analog metabolism in plants

Enzyme activity Level of characterization in different plants Refs

Enzymatic Molecular genetic

Phenylalanine ammonia lyase Numerous species Numerous species, including Ephedra sinica [38]

a

Cinnamoyl-CoA ligase Numerous species Numerous species

Cinnamoyl-CoA hydratase Hypericum androsaemum Not characterized [93]

Retro-aldol cleavage H. androsaemum Not characterized [93]

Benzaldehyde dehydrogenase H. androsaemum Antirrhinum majus [42,43,93,94]

Sorbus aucuparia Arabidopsis thaliana

Benzoyl-CoA ligase Clarkia breweri A. thaliana [40,95]

3-Ketoacyl-CoA thiolase Not characterized Petunia hybrida [41]

ThDP-dependent carboligase Hypothesized in plants but well Not characterized [33,34,37]

established in microorganisms

Transaminase Not characterized Not characterized

Reductase Catha edulis Not characterized [12,13]

E. sinica

N-Methyltransferase E. sinica Not characterized [13]

a

Including 4-coumaroyl-CoA ligases with cinnamoyl-CoA ligase activity.

de novo chemical syntheses generating racemic mixtures with organic solvents from the fermentation broth and

have long been established [52]; and (iii) a third methodo- chemically converted to an imine intermediate by reaction

logy involves the enantioselective production of (1R,2S)- with (Figure 4a). Reduction of the imine to

ephedrine from R-PAC [51]. The current commercial man- (1R,2S)-ephedrine is completed using hydrogen and a

ufacture of (1R,2S)-ephedrine and (1S,2S)-pseudoephed- metal catalyst. A recent patent outlined a process wherein

rine relies largely on this semi-synthetic process. The idea reductive amination of R-PAC may be conducted in the

of using benzaldehyde-supplemented microbial fermenta- presence of supercritical carbon dioxide, thereby circum-

tion to produce R-PAC, followed by reductive amination to venting the use of conventional organic solvents [60].

yield ephedrine alkaloids, was patented nearly 80 years (1S,2S)-Pseudoephedrine is produced from (1R,2S)-ephed-

ago [53] and is still used today (Figure 4a). rine by Welsh rearrangement [51].

Modern industrial production of ephedrine and Emerging synthetic biosystems for industrial alkaloid

pseudoephedrine production

Many microbes have the capacity to perform a biotransfor- The fact that the industrial production of (1R,2S)-ephedrine

mation that yields R-PAC, although most processes have and (1S,2S)-pseudoephedrine is at least partially completed

relied on the use of either Candida utilis or Saccharomyces through fermentation raises the intriguing possibility of

cerevisiae [54]. To better understand biotransformation employing synthetic biology for the production of these

processes, in vitro studies with yeast extract and/or puri- alkaloids. In this context, synthetic biology would involve

fied PDC enzyme have been conducted. Such studies show the expression in microbes of biosynthetic genes derived

that, under anaerobic conditions, PDC (native or engi- from plants or other organisms for the production of key

neered) converts exogenously supplied benzaldehyde and intermediates or amphetamine end products. Parts or even

endogenously available pyruvate to R-PAC [55]. As a whole pathways may be transferred from plants to systems

potential alternative to whole-cell biotransformation, pro- such as yeast, which permits the use of scalable fermenta-

cesses involving purified PDC have been evaluated and tion [61]. For example, recent efforts toward the production

shown to produce R-PAC levels up to 28 g/L with conver- of benzylisoquinoline alkaloids (BIAs), such as and

sion rates of 90–95% [55]. Several undesired side-reactions , in microorganisms have led to the production of a

are known to occur, especially the oxidation of benzalde- key biosynthetic intermediate, reticuline, from a norlauda-

hyde to benzyl by native oxidoreductases. However, nosoline precursor by heterologously expressing four genes

the greatest obstacle is that carboligation of benzaldehyde derived from BIA-producing plants [62–64]. Furthermore,

with pyruvate is normally a minor side-reaction of micro- the use of a human (CYP2D6) to convert

bial PDCs; in fact, this reaction is generally not observed (R)-reticuline to the promorphinan alkaloid salutaridine

unless specific conditions are used (e.g. the presence of [63] highlighted the use of surrogate genes sourced from

large amounts of acetaldehyde, the predominant reaction non-plant organisms. Beyond BIAs, synthetic biology has

product) that subsequently elevate R-PAC production to been used for the production of precursors to important

1% [48]. Continued engineering of microbial PDCs over pharmaceuticals such as the antimalarial agent artemisinin

the past two decades has yielded enzyme variants with [65], the anticancer drug taxol [66] and other small meta-

altered product profiles, increased stability and enhanced bolites [67]. Additionally, heterologous gene expression in

carboligation potential [48,49,56–59]. Recently, an active microbes has been used for the production of dietary omega-

site variant of Zymomonas mobilis PDC was shown to 3 fatty acids [68,69] and biofuels [70]. Synthetic biology is

prefer carboligation over aldehyde formation by nearly also used for the production of halogenated or otherwise non-

100-fold [48]. Following its formation, R-PAC is extracted natural metabolites with potential clinical relevance [61].

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Review Trends in Plant Science July 2012, Vol. 17, No. 7

O

OH

NH2 L-phenylalanine β-Oxidative CoA-dependent pathway PAL

O O Non-β-oxidative CoA ligase CoA-independent pathway OH S-CoA

CoASH H2O trans-Cinnamic trans-Cinnamoyl-CoA acid H2O Cinnamoyl- CoA hydratase Hydration OH O OH O Retro-aldol OH cleavage S-CoA

3-Hydroxy-3-phenylpropionic acid 3-hydroxy-3-phenylpropionyl-CoA

O NAD Retro-aldol cleavage NADH Dehydrogenase OH O O O O

S-CoA S-CoA

Benzaldehyde 3-oxo-3-phenylpropionyl-CoA

Dehydrogenase Thiolase CoASH NAD Thioesterase O NADH O O CoA ligase S-CoA OH S-CoA

Benzoic acid CoASH Benzoyl-CoA

Pyruvate Pyruvate

ThDP-dependent enzyme CO ThDP-dependent enzyme CO2 2 O

HO H

O O 1-phenylpropane-1,2-dione (S)-phenylacetyl- carbinol Transaminase H OH O

NH2 O (R)-phenylacetyl- carbinol (S)-cathinone

O Reductase NADH Reductase

+ NAD HO H OH OH

NH2 NH2 (S)-2-hydroxy- propiophenone (1S,2S)-pseudonorephedrine (1R,2S)-norephedrine O (cathine) SAM SAM OH H SAH N-methyltransferase SAH N-methyltransferase

(R)-2-hydroxy- OH OH H H propiophenone N N

(1S,2S)-pseudoephedrine (1R,2S)-ephedrine

TRENDS in Plant Science

Figure 3. Proposed biosynthetic routes leading from L-phenylalanine to substituted amphetamines in khat and Ephedra species. A CoA-independent, non-b-oxidative

pathway of L-phenylalanine side-chain shortening is shown in blue, whereas a CoA-dependent, b-oxidative route is shown in purple. Red arrows indicate an alternative

CoA-dependent, non-b-oxidative route suggested to occur in some plants [44]. Benzoic acid or benzoyl-CoA undergoes condensation with pyruvate, a reaction putatively

catalyzed by a ThDP-dependent enzyme [34]. Benzaldehyde may also undergo carboligation, producing numerous possible products (pink), a reaction observed in

numerous microbes. It is not yet firmly established which carboligation products give rise to downstream alkaloids, although 1-phenylpropane-1,2-dione is thought to be an

408

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(a) O (b) O H N NH2 HO + O Pyruvate Benzaldehyde (R)- (S)-amphetamine (decongestant) (attention deficit hyperactivity Pyruvate disorder treatment) decarboxylase OH H N NH2 Fermentation

O (R)-phenylacetylcarbinolyla (vasoconstrictor) ( treatment)

Methylamine -H2O O O H N H OH N N

Cl Intermediateer () ()

Reduction H2 / Pt O OH CH H N C N N Chemical EphedrineEp (Parkinson’s disease treatment) (obesity treatment) H Welsh O H rearrangement N OH H2N H N NH2

H H Tranylcipromine Pseudoephedrine (chronic fatigue treatment) (antianxiety)

TRENDS in Plant Science

Figure 4. Industrial production of ephedrine and pseudoephedrine, and the structures of several synthetic amphetamine-type pharmaceuticals. (a) Industrial production of

(1R,2S)-ephedrine and (1S,2S)-pseudoephedrine relies on both fermentation and synthetic chemistry. Benzaldehyde fed to yeast cultured under anaerobic conditions

undergoes pyruvate decarboxylase (PDC)-catalyzed carboligation with endogenous pyruvate, yielding (R)-phenylacetylcarbinol (R-PAC). R-PAC is extracted from the

fermentation broth and subjected to reductive amination using methylamine in combination with hydrogen and a metal catalyst. (1S,2R)-pseudoephedrine is produced

from its diastereomer via Welsh rearrangement. (b) Examples of synthetic, pharmaceutically important substituted amphetamines. Functional genomics could potentially

provide new tools for the enzymatic production of ephedrine, pseudoephedrine and novel pharmaceuticals.

Halogenation, in particular, may impact the pharmacologi- analog substrates with up to sixfold greater product yields

cal profile of a compound [71] and expand possibilities for compared with wild-type enzyme [48].

novel drug development. Recently, chlorination biosynthet-

ic genes from soil bacteria were introduced into the medici- Potential for commercial ephedrine production using

nal plant Catharanthus roseus, a source of monoterpene synthetic biosystems

indole alkaloids (MIAs), such as the anticancer agent vin- The potential application of synthetic biology to (1R,2S)-

blastine, resulting in the production of several novel halo- ephedrine production would require the availability of

genated MIAs [72]. Enzyme engineering has led to catalytic specific molecular tools, sourced either from amphetamine

variants with altered substrate acceptance profiles, en- analog-producing plants, such as E. sinica, or other organ-

abling more facile halogenation of MIA precursors [73,74]. isms. For example, a transaminase capable of accepting

Notably, the approach of enzyme engineering has similarly hydroxyketone substrates, such as R-PAC, could generate

been applied in the context of R-PAC production. Recently, a (1R,2S)-norephedrine. Conversely, S-PAC, produced by

PDC variant was found to accept halogenated benzaldehyde certain PDC mutants [49], could be transaminated to

immediate precursor to (S)-cathinone via transamination [13]. (S)-Cathinone is reduced to (1S,2S)-cathine and (1R,2S)-norephedrine. N-Methylation occurs in Ephedra

species, but not in khat. Activity has been detected for enzymes highlighted in yellow, and corresponding genes are available for enzymes highlighted in green. Enzymes

highlighted in pink have not been detected. Note that PAL is the sole enzyme characterized at the molecular level in substituted amphetamine-producing plant species [38].

Abbreviations: CoA, Coenzyme A; PAL, phenylalanine ammonia lyase; ThDP, thiamine diphosphate.

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(1S,2S)-norpseudoephedrine. Although such transaminase raises the intriguing possibility of coupling fermentation-

activity has yet to be reported in plants, amino transfer based ephedrine production with the use of novel enzymes

reactions involving hydroxyketone substrates and ami- possessing natural or engineered functions, enabling the

noalcohol products have been reported in microorganisms partial or complete biosynthesis of existing or new pharma-

[75,76]. An N-methyltransferase step would complete the ceuticals. For example, engineered PDC from Zymomonas

formation of (1R,2S)-ephedrine and its diasteriomer. Al- mobilis was shown to accept chlorinated benzaldehyde deri-

though genes encoding a transaminase and an N-methyl- vatives [48], indicating potential for the downstream pro-

have yet to be cloned from khat or E. sinica, the duction of drugs with halogenated ring systems. Enzymes

current availability of functional genomics resources from catalyzing reductive dehydroxylation (a reaction hypotheti-

these plants provide a platform for the discovery of such cally required to generate key compounds from the ephed-

tools. Genes encoding numerous enzymes involved in benz- rine precursor) have been isolated from pathogenic [86] and

aldehyde biosynthesis have been isolated from other gut bacteria [87]. Several different enzymes catalyze aro-

plants, such as Arabidopsis and snapdragon, raising the matic ring , including ortho-hydroxylases

possibility of integrating these ‘upstream’ components into from plants [88]. The formation of C–C bonds toward the

a microbial-based system for the de novo production of production of functionalized amphetamine analogs is

ephedrine alkaloids [38–43]. Although de novo production uniquely challenging from a biochemical point of view,

of benzaldehyde in microorganisms is a rather ambitious although many examples of carboligating enzymes exist

goal with envisioned caveats such as potential toxicity [77], [89]. Although few enzymes are known to accept (1R,2S)-

such an approach would ultimately preclude the need for ephedrine and related alkaloids, the availability of such

chemical precursors. Furthermore, the use of heterologous- biocatalysts from plants or other organisms would be in-

ly expressed enzymes downstream of PAC would preclude valuable toward the metabolic engineering of amphetamine

the need for further chemical processing. analog biosynthesis.

Synthetic pathways for amphetamine analog Concluding remarks

biosynthesis in microbes Recent advances in the production of plant specialized

Beyond (1R,2S)-ephedrine and (1S,2S)-pseudoephedrine, metabolites in synthetic biosystems [63,90,91] have creat-

myriad other substituted amphetamines have important ed new opportunities for improving strategies for the

pharmaceutical applications. The at the production of pharmaceuticals and other high-value com-

a-carbon is often a key determinant of pharmacological pounds. The existing fermentation-based commercial prep-

activity, with (S)- being more potent. For ex- aration of pseudoephedrine and ephedrine could benefit

ample, (S)-amphetamine, commonly known as d-amphet- immensely from the isolation of plant genes involved in the

amine or , displays five times greater biosynthesis of these substituted amphetamines. Broader

psychostimulant activity compared with its (R)-isomer [78]. surveys targeting the identification of modifying enzymes

Most such molecules are produced exclusively through coupled with the possible acceptance of substituted pre-

chemical syntheses and many are prescribed widely in cursors by key plant enzymes would extend the bioproces-

modern medicine. For example, (S)-amphetamine sing potential to a wide range of presently synthetic

1 1

(Figure 4b), a key ingredient in and Dexedrine , amphetamine analogs.

is used to treat attention deficit hyperactivity disorder

(ADHD) [79]. N-Methylated amphetamine is known as Note added in the proof

methamphetamine, which is prescribed under the trade Recently, targeted metabolite profiling and comparative

1

name Desoxyn for the treatment of ADHD and exogenous biochemical analyses were used to show that benzaldehyde

obesity. The (R)- of methamphetamine is the is a direct precursor to ephedrine alkaloids in Ephedra

1

active ingredient in Vicks Vapor Inhaler , a nasal decon- sinica [92]. This finding was accompanied by the detection

gestant sold over-the-counter in the USA and other coun- of ThDP-dependent carboligase activity with benzaldehyde

tries [80] (Figure 4b). Bupropion is prescribed extensively as the substrate.

under a variety of different trade names as an antidepres-

Acknowledgments

sant [81] and, more recently, a smoking cessation aid [82].

This work was supported by a grant from the Binational Agricultural

Selegiline, a drug used for the treatment of early-stage

Research and Development Fund (CA-9117-09). PJF holds the Canada

Parkinson’s disease and senile dementia [83], acts as a

Research Chair in Plant Metabolic Processes Biotechnology.

inhibitor and neuroprotective agent.

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