y. v. sheludko- recent advances in plant biotechnology and genetic engineering for production of...

ISSN 0095�4527, Cytology and Genetics, 2010, Vol. 44, No. 1, pp. 52–60. © Allerton Press, Inc., 2010.

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1 INTRODUCTION

One of the most important tasks for moderngenetic engineering, biotechnology and pharmacol�ogy is search or creation of systems for high�scaleobtaining of valuable natural products – complexorganic compounds produced by living organisms.Since the earliest time people used plants not merelyas food crops but additionally as sources of variouschemicals: pharmaceuticals, insecticides, food sup�plements, dyes etc. Currently plants remain an essen�tial provider of biologically�active compounds in spiteof development of chemical or microbial technologies.A number of inherent advantages make plants the cen�tral and highly perspective object in natural productbiosynthesis researches e.g. (a) ecological and phar�macological safety; (b) high native biosynthetic capa�bilities including multistep stereospecific synthesis ofcomplex organic molecules, eukaryotic type ofbiopolymer synthesis and processing; (c) possibilitiesof scaling up of valuable compound production usingnatural potential of plant systems; and (d) economicalvalues.

For primary classification of pharmacologicallyvaluable plant natural substances one can define agroup of mainly low molecular weight compounds,including first of all plant secondary metabolites, anda group of proteins and peptides with high molecularweight which are the products of heterological expres�sion of foreign genes in plant cells.

All biochemical processes in plant cell can be condi�tionally classified as primary and secondary metabo�lism. Compounds and processes which are necessary forgrowth, development and breeding belong to primarymetabolism. It includes mainly the metabolism of pro�teins, nucleic acids, carbohydrates and lipids. On the

1 The article is published in the original.

other hand, biosynthesis and catabolism of variable pig�ments, alkaloids, terpenes, phenolics belong to second�ary metabolism. All these substances considered to benot directly essential for plant cell life, and their func�tion in plants is not always clear [1, 2]. The majority ofsecondary compounds are considered to be participat�ing in plant�environment interactions: they defenseplants from pathogens, pests or herbivores, serve asattractants, have allelopathic, photo�protective or light�harvesting functions [1–4]. It is not surprising that mostof them have a strong influence upon animal andhuman organism. Numerous examples of pharmaceuti�cal applications of plant secondary metabolites aregiven in recent reviews [5–9].

People studied pharmacological properties of sec�ondary compounds since great antiquity: mentions ofmedicinal applications of alkaloid�containing plantwere found among Chinese, Mesopotamia and, later,India ancient sources dated 3000–1000 B. C. [10].Organic synthesis progress at the close of the 19 cen�tury and development of chromatographic separationprotocols in the first half of the 20 century allowed iso�lation and identification of numerous organic sub�stances responsible for pharmacological activities ofplant extracts. However, in spite of considerable suc�cess in modern organic synthesis, plants’ ability toform biologically active stereoisomers often makesthem the unique and essential source of pharmacolog�ically�valuable natural products. Moreover, consider�able part of synthetic pharmaceuticals has been devel�oped as modifications of natural substances of plantorigin. As the experts estimate, in USA nearly 50% ofdrugs for cancer chemotherapy are derivatives of plantextract components [11]. One should remark thatsearching of the optimal balance between drug effi�ciency and toxicity in the recent years brought scien�

Recent Advances in Plant Biotechnology and Genetic Engineering for Production of Secondary Metabolites1

Y. V. SheludkoInstitute of Cell Biology and Genetic Engineering, Kiev

e�mail: [email protected]

Abstract⎯For a long time people are using plants not only as crop cultures but also for obtaining of variouschemicals. Currently plants remain one of the most important and essential sources of biologically activecompounds in spite of progress in chemical or microbial synthesis. In our review we compare potentials andperspectives of modern genetic engineering approaches for pharmaceutical biotechnology and give examplesof actual biotechnological systems used for production of several promising natural compounds: artemisinin,paclitaxel and scopolamine.

DOI: 10.3103/S009545271001010X

CYTOLOGY AND GENETICS Vol. 44 No. 1 2010

RECENT ADVANCES IN PLANT BIOTECHNOLOGY AND GENETIC ENGINEERING 53

tists again to substances isolated from natural sources,first of all from plants [12].

In recent years an intensive work has been carriedout on screening of biological activity and structuraldiversity of secondary metabolites. Nevertheless thebiosynthetic potential of plant cells is considered to benot even half exhausted �a total amount of substancesproduced by plants was estimated in range about500 thousands [13, 14]. Actual models suggest correla�tion between evolution of secondary metabolism inplants and reciprocal adaptation of pests or pathogensleading to divergence and stimulating biodiversity inthe both groups [1].

The majority of secondary biosynthetic pathwaysare multistep enzymatic processes with complex anddelicate regulation mechanisms on transcriptionaland/or posttranscriptional level. Segregation of inter�mediates inside of single plant cell or their transportbetween different parts of the whole plant oftenoccurs. All these factors make investigation and, espe�cially, controlled biotechnological production of sec�ondary compounds an extremely complicated task.

Classification of secondary metabolites may bebased on the chemical structure or biological charac�teristics of substances. In general, three big groups ofsecondary compounds can be assigned: terpenes, phe�nolics and alkaloids, which include the main part ofcurrently identified compounds. Their number is esti�mated to be from more than 50 000 structures to about100 000 [2, 14–16].

Many terpenes exhibit strong pharmacologicalactivities against a number of human diseases. Amongthem we can mention cardenolides of Digitalis sp.[17], glycyrrhizin extracted from the licorice root andcalanolides from Calophyllum with anti�HIV activities[18], antibacterial shikonin from Lithospermum eryth�rorhizon [19], monoterpenoid alkaloid camptothecinisolated from Camptotheca acuminate and Nothapodytesfoetida [20,21], artemisinin from Artemisia annua usedfor malaria treatment and having additionally cyto�toxic features [22, 23], and many others. In recentpublications Morimoto et al. reported about success�ful studies of cannabinoid biosynthesis: 5 enzymeswere characterized and the corresponding genes werecloned [24, 25]. Heterologous expression of tetrahy�drocannabinolic acid synthase gene resulted in forma�tion of tetrahydrocannabinolic acid (precursor of tet�rahydrocannabinol) from cannabigerolic acid [24].This gene was later expressed in Pichia pastoris cells.High level of enzymatic activity (app. 1.3 nkat/L) wasdetected in culture medium [26].

In spite of impressive scope and wide range ofresearches, only several secondary biosynthetic path�ways have been studied in details on the enzymatic andgene levels. In our manuscript we will focus on theseexamples. Evidently, the frame of this publication doesnot allow performing a thorough review of all plantsecondary metabolism research areas. Therefore wewill discuss here biotechnological systems developed

for production of certain valuable and perspective nat�ural products.

Artemisinin production. Artemisinin from A. annuais currently one of the most effective antimalarial drugsrecommended by WHO during short�course artemisi�nin�based combination therapy [27]. Low content ofartemisinin in plants (0.01–1% DW) and ever�growingdemand for artemisinin�containing pharmaceuticalsstimulated studies on biosynthetic pathway of this com�pound formation and attempts to enhance its accumu�lation in plant systems [28]. Total organic synthesis ofartemisinin was found to be very difficult and costlyprocess [29]. More perspective were approaches onimprovement of artemisinin production in plant tissueunder salinity stress conditions [30].

Numerous studies have been carried out in order toobtain artemisinin from plant cell culture systems byselection of a highly productive line, supplying withprecursors or elicitation [28, 31]. Additionally a hairyroot culture of A. annua was established [32].

Cloning of several terpene biosynthesis genes likecotton farnesyl diphosphate synthase and its overex�pression in A. annua hairy roots resulted in three� tofour�fold higher yield of artemisinin [33]. Redirectionof amorpha�4,11�diene synthase and farnesyl diphos�phate synthase to the plastids in transgenic Nicotianatabacum allowed to enhance considerably accumula�tion of one of the artemisinin precursors, amorpha�4,11�diene [34].

The most promising way to scale up the productionof artemisinin was cloning and heterologous expres�sion of genes coding for several consequent enzymes ofmevalonate pathway (amorpha�4,11�diene synthase,cytochrome P450 monooxygenase (CYP71AV1),cytochrome P450 oxidoreductase) from A. annua inSaccharomyces cerevisiae strain (Fig. 1). As a result,100 mg/L of artemisinic acid, direct precursor of arte�misinin, were synthesized in the course of three�stepreaction from native yeast intermediate metabolitefarnesyl pyrophosphate. Its further conversion to arte�misinin is not complex [35, 36]. Production of arte�misinic acid from S. cerevisiae in bioreactor increasedrecently 25�fold and reached up to 2.5 g/L [37]. Thisexample demonstrates efficiency of the present strat�egy of secondary pathway genetic engineering com�prising characterization and cloning of respectivegenes, regulator elements and correct choice of heter�ologous expression system.

Paclitaxel production. Perhaps one of the mostfamous cytotoxic natural compounds discovered dur�ing the last decades was diterpene amid paclitaxel alsoknown as taxol. Its antitumour activity as a componentof Taxus brevifolia extract is known since 1965; in 1972the chemical structure of taxol was elucidated [38]. In1992 Taxol® was registered and appeared in the worldpharmaceutical market. Numerous clinical trialsproved its efficiency against several types of cancercurrently making taxol one of the most perspectiveanticancer drugs. Cytotoxic effect of paclitaxel is

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HO

O

H

H

based on cell division blocking by microtubules stabi�lization [39, 40].

Ever�growing demand for paclitaxel and its lowcontent in wood of slowly growing yew�trees (about0.03% d. w. in T. brevifolia� bark of several hundredthousands of yew�trees needs to be extracted to supplyworld year demand for paclitaxel) stimulatedresearches on chemical and biotechnology synthesis ofthis compound. More than 300 relative compoundshave been isolated and characterised from differentTaxus species up to now [41].

The total chemical synthesis of paclitaxel wasfound to be very complex process too expensive forcommercial production. Partial biosynthesis of pacli�taxel and its more active derivatives like Taxotere®

from precursors (for example, baccatin III) appearedmore perspective. Baccatin III was isolated from yewneedles that did not destroy trees and extended thesource of raw materials [40, 42].

High value of paclitaxel and its extremely low nat�ural supply became a prerequisite for numerousprojects on selection of highly productive Taxus celllines and enhancing of paclitaxel biosynthesis in cellcultures. Results of these studies were summarized inrecent publications [43, 44]. Manipulation with cul�tural medium composition in combination with effi�cient selection allowed in a number of cases accumu�lation of paclitaxel in cells up to 0.03–0.05% d. w. thatis comparable or even surpasses the metabolite level inT. brevifolia bark [45, 46]. Further investigationproved efficiency of elicitation for taxoid biosynthesisstimulation because a number of important enzymesof terpene pathway (for instance geranylgeranyldiphosphate synthase and taxadiene synthase) are jas�monate inducible [47, 48].

Tabata reported that development of Taxus cell sus�pension selection, cultivation and elicitation protocolresulted in stable paclitaxel production up to 295 mg/L[49]. Multiple jasmonate treatments in bioreactorincreased taxoid yield in cell suspensions up to612 mg/L [50]. Companies of Phyton Catalytic Inc.(USA) and Samyang Genex (South Korea) informedabout commercial isolation of paclitaxel from cell cul�tures [16, 39].

Two alternative pathways of terpene biosynthesishave been described at present time. Both pathways leadto production of common terpene precursors (dimeth�ylallyl diphosphate (DMAPP) and isopentenyl diphos�phate (IPP)) which can be transformed in more com�plex molecules in the course of further conversions. Theclassic mevalonate pathway (MYA) which functions inthe cytosol initially was assumed to be the sole source ofthe terpenoid precursors IPP and DMAPP. It suppliesthe precursors for production of sesquiterpenes and trit�erpenes. Alternative pathway named after the first com�mitted precursor, 2�C�methyl�D�erythritol�4�phos�phate (MEP) is localized in plastids and is generallyused to supply precursors for the production of monot�erpenoids, diterpenoids and tetraterpenoids [1, 51].Moreover, recent studies showed possibilities for inter�changes between intermediates of the both pathways[52]. Experiments on inhibition of IPP transport fromcytoplasm to plastids demonstrated that some IPP frommevalonate pathway might be transfered from the cyto�plasm to the plastids in the course of taxol and baccatinIII biosynthesis. It was also presumed that different IPPbiosynthesis pathways occur during different growthphases in Taxus cells [53].

Because of diterpenoid origin of paclitaxel, the spe�cial attention was paid to the investigation of MEP

Fig. 1. Part of artemisinin biosynthesis pathway in S. cerevisiae (strain expressing amorphadiene synthase gene (ADS), cyto�chrome P450 monooxygenase (CYP71AV1) and NADPH: cytochrome P450 oxidoreductase (CPR) [35]: (1) famesyl pyrophos�phate; (2) amorpha�4,11�diene; (3) artemisinic acid; (4) artemisinin.

H

O

H

H

HO

HO

H

H

PPOH

H

H

H

HO

ADS

CYP71AV1/CPR

CYP71AV1/CPR

NON�ENZYMATIC

OOH

OO

H

H

HO

CYP71AV1/CPR

1 2

34 SEMI�SYNTHESIS



pathway regulation and cloning the appropriate genes.In general, 15 consequent secondary enzymatic reac�tions should be accomplished to form baccatin III thekey precursor of paclitaxel [54] (Fig. 2). Recent reviewsreported cloning and characterization of 10 genes oftaxane biosynthesis [43, 51 and references cited therein,54, 55]. In particular, 2�C�methyl�D�erythritol2,4�cyclodiphosphate synthase gene, which is the 5�thenzyme of the MEP pathway, was cloned fromT. medium [56].

The efficiency of Agrobacterium transformation ofyew cells is low and successful transformation protocolof Taxus cell suspensions was developed not long ago[57]. Because of this the majority of cloned genes werefunctionally expressed in E. coli and Saccharomycescerevisiae [54, 58]. In the last case, 5 genes coding for5 consequent reaction enzymes from primary metabo�lism to the intermediate taxadien�5�α�acetoxy�10�β�olwere installed in a single yeast host. It was shown thatenzymes encoded by introduced heterologous genesutilized yeast isoprenoid precursors. However biosyn�thesis was blocked at the first cytochrome P450hydroxylation step [54]. In order to enhance thehydroxylation activity, coexpression of cytochromeP450 reductase with cytochrome P450 oxygenase wassuccessfully performed in yeast cells [59].

Among plant species, A. thaliana was transformedwith recombinant T. baccata taxadiene synthase genecoding for plastid localized enzyme of one of earlystages of paclitaxel biosynthesis catalyzing conversionof geranylgeranyl diphosphate to taxadiene. It led toaccumulation of taxadiene in Arabidopsis cells [60].This experiment demonstrated the perspective ofapproaches based on engaging of natural terpenoid

precursors of plant host in taxane biosynthesis payth�way. However, constitutive production of the full�length His�tagged enzyme in A. thaliana plants causedgrowth retardation and decreased the levels of photo�synthetic pigments. Although these effects may bedriven by a toxic taxadiene, the lower accumulation ofendogenous plastid isoprenoids such as carotenoidsand chlorophylls in transgenic plants also suggestedthe alteration of the balance of geranylgeranyl diphos�phate pool. Using of inducible transgene expressionsystem allowed optimization of taxadiene productionwhich reached 30�fold higher levels than those inplants constitutively expressing the transgene [60],Even higher taxadiene accumulation was observedafter expression of taxadiene synthase in tomato fruitsdue to redirection of carotenoid metabolites: about160 mg of taxadiene was extracted from 1 kg of freezedried fruits [61].

Except for higher plants, taxadiene synthase wasexpressed in a moss Physcomitrella patens [62] and inthe yeast S. cerevisiae [63]. Transgenic moss accumu�lated taxadiene up to 0.05% of fresh weight. Transgeneexpression did not affect significantly the amounts ofthe endogenous diterpenoids. In contrast to othertransgenic plants expressing heterologous taxadienesynthase, transgenic P. patens did not exhibit anygrowth inhibition due to the alteration of diterpenoidmetabolic pools that suggests the perspective of thisobject for the biotechnological production of pacli�taxel and its precursors.

Introduction of T. chinensis taxadiene synthasealone in S. cerevisiae did not increase the taxadienelevels because of insufficient levels of the universalditerpenoid precursor geranylgeranyl diphosphate. In

Fig. 2. Selected stages of paclitaxel biosynthesis: (1) geranylgeranyl pyrophosphate; (2) taxa�4(5), 11(12)�diene; (3) baccatin III;(4) paclitaxel; TS – taxadiene synthase. Numerous arrows indicate more than one step.

OHOHO

AcO OH

OOAc

O

O

OH OO

AcO OH

OOAc

O

O

ONH

O

MEPpathway

OPP

TS

4

1 2 3

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order to attain a high level of taxadiene and its inter�mediate metabolites, geranylgeranyl diphosphate syn�thase from Sulfolobus acidocaldarius and codon opti�mized T. chinensis taxadiene synthase gene were intro�duced into yeast genome. It resulted in 40�foldincrease in taxadiene to app. 8.7 mg/L as well as signif�icant amounts of geranyl�geraniol (app. 33.1 mg/L),suggesting possibility for further increase of taxadienelevel [63].

Scopolamine production. The anticholinergic tro�pane alkaloids hyosciamine, its racemic form atro�pine, and scopolamine have been known among theoldest drugs in the medicine because of their effect onparasympathetic nervous system. Currently they arewidely used in pharmacology as muscle relaxants.These substances together with a number of other tro�pane alkaloids were isolated mainly from Solanaceaespecies, although tropane alkaloids were additionallydetected in plants of several other families [64]. Hyos�cyamine is normally the more abundant alkaloid inSolanacea species while scopolamine (which is morephysiologically active and valuable) is produced ingreater quantities only in Duboisia spp and Daturametel [64, 65]. As it was also shown for other naturalproducts mentioned above, the chemical synthesis ofthese alkaloids has proved to be difficult and not eco�nomically feasible so that plant material is their onlysource. World demand for scopolamine was estimatedto be about 10 times greater than that of hyoscyaminetogether with atropine [66]. This provoked the interestto tropane alkaloid biosynthesis pathway and biotech�nological production of scopolamine. Because it wasshown that undifferentiated systems such as calluses orcell cultures have low productivity [67], hairy rootscaused by the infection of plants with A. rhizogeneshave been chosen as an object for attempts to enhancescopolamine production. Owing to their stable andhigh productivity, hairy root cultures have been inves�tigated for several decades for biotechnological pro�duction of the valuable metabolites (progress in under�standing of secondary biosynthesis mechanisms inhairy root cultures was reflected in the recent reviews[68–71].

Hairy roots of Hyoscyamus muticus may producehigh contents of hyoscyamine, but in many cases onlytrace amounts of scopolamine [72]. Sevon et al.

described obtaining and analysis of hairy roots in morethan 15 species. Amounts of scopolamine in the stud�ied cultures varied from 0.2 to 32 mg/g DW Laboriousselection of the more productive clones and optimiza�tion of the growth conditions was often necessary toreach these levels of scopolamine accumulation [71].Thus it is obvious that metabolic engineering of thisbiosynthetic pathway or its single steps could help toimprove scopolamine production. In particular, theconversion of hyoscyamine to the much more valuablescopolamine could be regarded as the major goal ofthese studies.

Early stages of nicotine and tropane alkaloid bio�synthesis are coinciding and discussed together withfurther reactions in the recent reviews [65, 73]. Thefirst committed step of both pyridine and tropanealkaloid metabolism is S�adenosylmethionine(SAM)�dependent methylation of putrescine cataly�sed by putrescine N�methyltransferase, formingN�methylputrescine. The overexpression ofN. tabacum putrescine N�methyltransferase (PMT)gene in scopolamine�rich Duboisia hybrids, Daturametel, Atropa belladonna and H. muticus causedincreasing in accumulation of the direct metaboliteN�methylputrescine (2�4�fold compared to wild typeroots) [74], but there was no significant increase ineither tropane or pyridine�type alkaloids [74–76] orthe effect on the alkaloid level was only marginal.However, regulation of the expression of this gene canbe crucial for alkaloid production in several species: insome transgenic N. sylvestris lines overexpression ofpmt gene increased the nicotine content, whereas sup�pression of endogenous PMT activity severelydecreased the nicotine content and induced abnormalmorphologies [75].

Scopolamine is 6,7β�epoxide derivative of hyos�cyamine, formed from hyoscyamine in a two�step pro�cess via 6β�hydroxyhyoscyarnine [78] by enzymehyoscyamine 6β�hydroxylase (H6H) which can beclassified as 2�oxoglutarate�dependent dioxygenase(Fig. 3). The enzyme was purified and characterizedfrom H. niger [79]. The cDNA encoding H. niger H6Hhas been isolated by Matsuda et al. [80]. Additionally,H6H cDNA was cloned from several other scopola�mine�producing Solanaceae species e.g. A. baetica[81], A. belladonna [82], Anisodus tanguticus [83] etc.Additionally, tropinone reductase, which catalyzes anearlier reaction of scopolamine biosynthesis inH. niger, has been cloned [84]. H6H gene fromH. niger was placed under the control of 35S promoterand introduced to A. belladonna using A. rhizogenes.The obtained hairy roots contained up to five�foldhigher concentrations of scopolamine than wild�typecultures [85]. Hyoscyamine was almost completelyconverted to scopolamine in the leaves of transgenicA. belladonna plants expressing h6h gene. The level ofscopolamine in the leaves reached up to 1.2% DW[86]. Later, 35S�h6h gene was introduced into H. muti�cus producing high amounts of tropane alkaloids

NCH3

O

O

OH

NCH3

O

O

OHO

1

H6H

2

Fig. 3. Conversion of hyoscyamine (1) to scopolamine (2)by enzyme hyoscyamine 6p�hydroxylase (H6H).



(up to 6% of the dry weight in the leaves of matureplant). The best selected transgenic line produced17 mg/L scopolamine, although conversion of hyos�cyamine to scopolamine was still incomplete. In theseexamples overexpression of a single gene in the path�way has often led to an improved accumulation of themore valuable end product.

Further experiments included simultaneous over�expression of genes encoding PMT and the down�stream H6H in H. niger hairy root cultures. It resultedin accumulation of significantly higher amounts ofscopolamine (up to 411 mg/L,) in hairy root linesexpressing both pmt and h6h genes compared with thecontrol cultures (app. nine times more than that in thewild type) and transgenic lines harboring only one ofthe mentioned genes (more than two times higher levelof scopolamine as compared with the best single�genetransgenic lines) [87].

Biotransformation was reported to be an alternativeway for scopolamine production using non�hyos�cyamine�producing transgenic systems fed with pre�cursor hyoscyamine. Hairy roots of N. tabacum trans�formed with 35S�h6h gene have been studied for theproduction of scopolamine and nicotine alkaloidsafter feeding the cultures with hyoscyamine. In theoptimal conditions the most productive clones ofN. tabacum hairy roots converted up to 45% of exoge�nous hyoscyamine to scopolamine; up to 85% of thetotal scopolamine was released to the culture medium[88]. Recently, the protocol for bioconversion of hyos�cyamine into scopolamine in bioreactor withN. tabacum cell suspension cultures was reported [89].Functionally active H6H was obtained after heterolo�gous expression of h6h gene from Brugmansia candidain S. cerevisiae [90].

Conclusions and future perspectives. In conclusion,cloning and heterologous overexpression of genes cod�ing for several key enzymes of secondary metabolismoften allowed considerable increasing of the level ofvaluable end product. The next step on the way toobtaining the commercial amounts of metaboliteincluded correct choice of expression system andadaptation of the process to bioreactor scale. However,the efficient control of desired product synthesisrequires a complete knowledge of all the steps in bio�synthetic pathway, regulation mechanisms and clon�ing of the respective genes. It is difficult to forecast theresults of introduction into plant genome of a single orreduced number of genes. Their overexpression maycause appearance of multiple rate�limiting steps anddid not enhance production of desirable metabolite. Itis necessary to consider the processes involved in theregulation of the whole pathway and interconnectingcellular pathways. Alternatively, translocation of genecluster encoding the enzymes responsible for sequenceof biochemical conversation in non�plant expressionsystem can result in creation of highly efficient pro�ductive complex.

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