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Biotechnol. J. 2006, 1, 1419–1427

Review

DOI 10.1002/biot.200600117

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Bioreactor technology: A novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots

Ganapathy Sivakumar

Biotech Genomics, ENEA, Casaccia, Rome, Italy

Plants are the richest source for different bioactive molecules. Because of the vast number of side effects associated with synthetic pharmaceuticals, medical biotechnologists turned to nature to provide new promising therapeutic molecules from plant biofactories. The large-scale availability of the disease- and pesticide-free raw material is, however, restricted in vivo. Many bioactive plant secondary metabolites are accumulated in roots. Engineered plants can also produce human ther- apeutic proteins. Vaccines and diagnostic monoclonal antibodies can be won from their roots, so that engineered plants hold immense potential for the biopharmaceutical industry. To obtain suf- ficient amounts of the plant bioactive molecules for application in human therapy, adventitious and hairy roots have to be cultured in in vitro systems. High-tech pilot-scale bioreactor technolo- gy for the establishment of a long-term adventitious root culture from biopharmaceutical plants has recently been established. In this review, I briefly discuss a technology for cultivating bioactive molecule-rich adventitious and hairy roots from plants using a high-tech bioreactor system, as well as the principles and application of genome-restructuring mechanisms for plant-based biophar- maceutical production from roots. High-tech bioreactor-derived bioactive phytomolecules and biopharmaceuticals hold the prospect of providing permanent remedies for improving human well-being.

Received

7 July 2006

Revised

5 October 2006

Accepted

6 October 2006

Keywords: Adventitious roots · Bioactive molecules · Biopharmaceuticals · Bioreactor · Plantibodies

1

Introduction

Plant molecules have a higher bioactivity, and are safer and better for human health than synthetic drugs. This is so because human beings have co-evolved with plants over the past few million years. For 5.1 billion people worldwide, natural plant-based remedies are used for both acute and chronic health problems, from treating common colds to controlling blood pressure and choles- terol. Today, plants are the raw source materials for as many as 40% of the pharmaceuticals in use in the United

Correspondence: Dr. Ganapathy Sivakumar, Biotech Genomics, ENEA – Centro Ricerche, Casaccia, Via Anguillarese 301, 00060 Rome, Italy E-mail: sivakumar@libero.it

Abbreviations: OUR, oxygen uptake rate; CER, carbon dioxide evolution rate

States and Europe. Some commercial medicines, such as the cancer drugs taxol from Taxus brevifolia, colchicine from Gloriosa superba, camptothecin from Camptotheca acuminate, ginsenosides from Panax ginseng, vinblastine from Catharanthus roseus, the anti-malarial drugs qui- nine from Cinchona pubescens, and artemisin from Artemisia annua, are manufactured from plants. Synthet- ic drugs often act in the body as irritants and toxins, up- setting the balance of whole systems, and producing side effects that can be lethal. The relative potency of stereoisomers of natural and synthetic drugs differs for each effect, which may result in differences in therapeu- tic and adverse effects and in the benefit to risk ratio. Thus, pharmacologists consider drug products that con- sist of different stereoisomers as different drugs rather than different formulations of the same drug. It is impos- sible to obtain equally high bioactive stereoisomers through chemical synthesis. For these reasons, plant

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drugs can provide better natural bioactive medicine than synthetic drugs without harmful side effect. Most of the bioactive molecules, such as campto- thecin, vinblastine, and ginsenosides, are of root origin. Harvesting roots is destructive for the plants and hence there has been increasing interest in developing in vitro cultures of adventitious and hairy roots from several me- dicinal plant species. The gap between discovery and commercialization can be bridged when biotechnologists and bioengineers join forces and integrate their research disciplines to develop bioreactor technology for the pro- duction of natural therapeutic molecules. Recently, the adventitious and hairy root culture technology for the pro- duction of pharmaceuticals has reached from small-scale laboratory to large-scale industrial production: the Ger- man company ROOTec has described commercial-scale cultures of hairy roots, producing camptothecin and podophyllotoxin for medicine. Normally, adventitious root cultures need an exogenous phytohormone supply for growth [1–4]. However, the transgenic hairy roots can grow actively without phytohormone [5]. The ability to manipulate roots at a cellular and molecular level shows great potential for commercial production of bioactive molecules. In the future, demand for existing biopharma- ceuticals (e.g., erythropoietin to treat anemia and insulin to treat diabetes), as well as new therapeutic proteins dis- covered through genomics efforts, is expected to rise con- siderably. It is prudent, therefore, to evaluate alternative plant biopharmaceutical production systems and deter- mine how the future availability of safe recombinant bio- pharmaceuticals can be ensured in a cost-effective man- ner. Producing human therapeutic proteins in plants has many economic and qualitative benefits, including re- duced health risks from pathogen contamination, com- paratively high yields, and production in roots or other storage organs [6]. In addition, plants have a big advan- tage over mammalian cell systems, in that plant viruses are not pathogenic to humans unlike mammalian viruses. In just 25 years, the biopharmaceutical industry has boomed, from a few early companies to today’s $ 50-bil- lion market [7]. Currently, more than 300 plant-made bio- pharmaceuticals are under various stages of discovery, development and production at various academic and company sites around the world. Several plant-derived biopharmaceutical proteins are reaching the late stages of commercial development. These products include an- tibodies, vaccines, human blood products, hormones and growth regulators [8]. Basically, the genetic engineering process involves incorporating the desired foreign gene into the genome of the plant to create a biopharmaceuti- cal-producing plant, or introducing the gene through ex- ternal means into the plant and just transforming that plant to make the desired product. The biopharmaceuti- cal hairy root system offers tremendous potential for in- troducing additional genes, along with the Ri T-DNA genes, for alteration of metabolic pathways and produc-

tion of useful human therapeutic molecules. The hairy roots continuously secrete recombinant proteins into a liquid medium or accumulated them in the intercellular spaces, thus simplifying the purification process. Globally, the field cultivation of biopharmaceuticals plants requires the application of pesticides, which re- sults in the serious problem of pesticide residues. Fur- thermore, major problems in biopharmaceutical plant cultivation are that geographical constraints and higher production costs. To overcome these problems, we estab- lished high-tech bioreactor technology for commercial- scale production of plant biopharmaceuticals, creating an alternative way to produce the corresponding bioactive secondary metabolites and vaccines through a root cul- ture system. High-tech bioreactors are essential in ad- ventitious and hairy root engineering, not only because they provide an aseptic environment mimicking con- trolled conditions for the growth of roots, but also because they enable high-tech systematic development of the responses of higher root biomass and biopharmaceutical production.

  • 2 Bioreactor technology

Caring for a better world, the major challenge is how to translate the laboratory-scale product designs into large- scale production of high-tech biopharmaceuticals from plant roots that are reproducible, safe, and economically competitive. Bioreactor culture is the key step towards commercial production of bioactive therapeutics by plant biotechnology. The engineering of adventitious and hairy roots represents one of todays fastest-growing biotech- nology areas of world pharmaceutical and nutraceutical economy. The in vitro cultivation of adventitious and hairy roots in the bioreactor that supports efficient nutrition of cells, possibly combined with the application of mechan- ical stimulation to direct cellular activity, differentiation and function, is an important step towards the develop- ment of high-tech products. At the heart of bioreactor control is the ability to mon- itor important process variables such as pH, temperature, dissolved oxygen, oxygen uptake rate (OUR), carbon diox- ide evolution rate (CER), temperature and specific gravi- ty. Mixing, oxygen transfer and shear stress remain the biggest challenges as far as the scale-up to industrial-size high-tech bioreactors is concerned. These parameters are generally linked, and compromises need to be made, for instance, in aeration to avoid excessive shear stress. The development of bubble-free bioreactor systems without conventional agitation technologies is needed to address this problem. At initial stages in a bioreactor oxygen transfer is not difficult as the medium contains enough dissolved oxygen to support the growth of the plant root inoculum. Mixing is a very important factor because it serves the dual purpose of supplying dissolved oxygen

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and driving away the carbon dioxide. The OUR by a unit of root biomass in a set time is known as the oxygen trans- fer coefficient. Other dissolved gaseous metabolites, i.e., carbon dioxide and ethylene, also affect the overall pro- ductivity of the roots. Although cells at the core of the roots would be exposed to adequate oxygen tensions, high oxygen levels near the surfaces of the roots may be- come toxic to peripheral cells and cause localized oxida- tive stress [9]. High-tech bioreactor systems typically em- ploy mathematical models that describe the process be- ing controlled via a software sensor [10]. To enable the ki- netic analysis of bioreactors, Wu et al. [11] developed a black-box transfer function model, and were able to iden- tify the system parameters from input-output data. They obtained excellent agreement between the measured and estimated values of the OUR and CER within seconds fol- lowing a system perturbation. A stirred tank bioreactor is not suitable for mass pro- duction of adventitious and hairy roots because of shear stress and high electric charge. Among the various kinds of bioreactors, airlift and bubble column bioreactor are commercially successful for adventitious and hairy root culture because of low shear stress and the simplicity of their design and construction. This reactor comprises a main body, air bubbling device, steam generator for ster- ilization, air inlet, air vent system and various control sys- tem for checking temperature, oxygen, and pH, and pipeline systems for transferring steam, air, medium and root masses [1]. Bioreactors with computer control systems theoreti- cally offer various advantages over conventional culture procedures due to possibilities of automation, saving la- bor, reduction of production costs and high-tech. The bioreactor culture system is more advanced than the tra- ditional tissue culture system because the culture condi- tions in a bioreactor can be optimized by on-line manipu- lation of temperature, pH, concentrations of oxygen, car- bon dioxide and nutrients in the medium. Nutrient uptake of roots can also be enhanced by continuous medium cir- culation. Furthermore, cell proliferation and regeneration rates can be increased. Thus, production cost and time can be substantially reduced, product quality can be con- trolled and standardized, products can be free of pesticide contamination, and production can be conducted all year round without geographical constraints [1]. In this regard, high-tech bioreactor technology is useful for the large- scale cultivation of biopharmaceuticals from plant roots.

  • 3 Adventitious root culture

Several secondary metabolites of pharmaceutical interest are accumulated in adventitious roots. According to our experience, the adventitious root cultures (Fig. 1) consti- tute a good biological material for stable commercial pro- duction of higher secondary metabolites without foreign

genes under sterile conditions. Indeed, compared to cell cultures, adventitious roots have a higher stability in the physical chemical conditions and accumulate large quan- tities of secondary metabolites on intercellular spaces, which can be more easily isolated. Adventitious roots, once established, can be grown in a medium with low in- oculum with a high growth rate. The change in carbon as- similation mode has dramatic effects on the patterns of phytochemicals produced by adventitious root cultures, thus providing a system to study the coordination be- tween primary and secondary metabolism. Adventitious roots can also express their photosyn- thetic abilities in vitro. We have induced green adventi- tious roots under phototropic condition. Chloroplast-de- pendent reactions are a vital part of certain metabolic pathways and could result in a novel pattern of com- pounds produced by adventitious roots. Green adventi- tious roots are known to produce certain metabolites that are normally synthesized in green parts of the plant. For example, in plants tocopherol biosynthesis takes place in the plastid, and the enzymes are associated with the plas- tidial envelop [12]. Homogentisic acid derived from aro- matic amino acid metabolism, and phytyldiphosphate de- rived from the plastidic methylerythritol phosphate path- way [13], serve as precursors for tocopherol biosynthesis [14]. α-Tocopherol is the major vitamin E compound found in leaf chloroplast, where it is located in the chloroplast envelope, thylakoid membranes and plastoglobuli [15]. Natural α-tocopherol contains three stereogenic centers (RRR), and thus exists as a mixture of eight stereoisomers in all racemic forms that are provided in most commer- cially available vitamin preparations. The arrangement of atoms influences the properties and the biological activi- ty of a substance. The natural α-tocopherol isomer (RRR) disappears more quickly from the liver after supplemen- tation than the enantiomer (SSS) [16]. This implies that the stereoisomers of α-tocopherol are discriminated from

Biotechnol. J. 2006, 1, 1419–1427 www.biotechnology-journal.com and driving away the carbon dioxide. The OUR by a

Figure 1. High-Tech bioreactor cultivation of hazelnut adventitious roots for the production of human therapeutic molecules.

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each other and suggests that there is an interaction with another chiral entity, likely a macromolecule. It is impos- sible to obtain equally highly bioactive α-tocopherol through chemical synthesis [17]. Thus, we produced RRR- α-tocopherol through a green adventitious root culture system. Furthermore, glucosinolates can be derived from amino acids and contain a sulfate and thioglucose residue. Upon tissue damage, glucosinolates are brought into contact with myrosinases and hydrolyze into unsta- ble aglycons. These are further converted into a range of biologically active compounds like isothiocyanates and nitriles that possess antimicrobial, cancer-preventing, goitrogenic or inflammatory activities, depending on the nature of the glucosinolate precursor amino acid and the type of degradation product formed. Sulforaphane is one of a class of bioactive molecule called isothiocyanates. Sulforaphane predominantly found in broccoli, may help to reduced risk of developing several types of cancers [18]. It is an indirect antioxidant; it does not neutralize free rad- icals directly, but rather boosts Phase 2 enzymes that trig- ger ongoing and long-lasting antioxidant activity [19]. Sulforaphane is a very expensive molecule, costing ap- proximately 72 Euro/5 mg. Sulforaphane from chemical synthesis requires several highly toxic substances, and fi- nal products from these reactions still contain toxic residues and require further purification. This disadvan- tage limits the use of synthesized sulforaphane as food ad- ditives. Thus, natural sulforaphane from plants is more fa- vorable for the consumer [20]. We therefore produced sul- foraphane through an adventitious root culture system. Many traditional strategies can be used to increase the production of bioactive secondary metabolites, but jas- monic acid elicitation is usually one of the most commer- cially successful.

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Elicitation

An elicitor can be defined as any compound or mixture of compounds that induces a plant defense reaction, which may enhance secondary metabolism in plant cell and root cultures. Besides, biogenetic precursors, salicylic acid, chitosan and heavy metals, the jasmonic acid elicitation has been commercially successful. Jasmonic acid and its volatile derivative methyl jasmonate (MeJA) are collec- tively called jasmonates. Jasmonic acid is an important plant stress signaling molecule. It induces the biosynthe- sis of defense proteins and protective secondary metabo- lites. Jasmonates are fatty acid derivatives with a 12-car- bon backbone that are synthesized from 18-carbon inter- mediates via the so-called octadecanoid pathway [21]. The induction of secondary metabolite accumulation is an important stress response that depends on jasmonates as a regulatory signal [22, 23]. The effect of jasmonates on secondary metabolism have been studied in detail for al- kaloid biosynthesis. In alkaloid metabolism, jasmonate

acts by coordinate activation of the expression of multiple biosynthesis genes. However, how the jasmonic acid sig- nal is transduced to affect gene expression is largely un- known. Two regions were identified that dictated elicitor and jasmonate responsive reporter gene activation: the so-called BA region and a region close to the TATAbox called jasmonate and elicitor responsive element (JERE). The JERE interacts with two jasmonic acid responsive transcription factors called octadecanoid responsive Catharanthus AP2-domain proteins (ORCAs). In ter- penoid indole alkaloid metabolism and primary precursor pathways, jasmonate induces gene expression and me- tabolism via ORCAs, which are members of the AP2/ERF- domain family of plant transcription factors. Other jas- monate-regulated secondary metabolic pathways might also be controlled by ORCA-like AP2/ERF-domain tran- scription factors. [24]. We assume that it could be a mech- anism in adventitious root cultures, as MeJA treatment increased accumulation of bioactive secondary metabo- lites. This treatment consists in applying chemical stress- es to the adventitious cultures that triggers the higher production of secondary metabolites. This offers the pos- sibility of obtaining adventitious roots with improved nu- tritional value and anti-carcinogenic properties through natural molecules without foreign genes [25].

  • 5 Hairy root culture

In the past few decades, a lot of attention has been paid to hairy root research for the production of important sec- ondary metabolites. Hairy roots also offer a valuable source of root-derived phytochemicals that are useful as pharmaceuticals and cosmetics [5]. Hairy roots develop as the consequence of the interaction between Agrobacteri- um rhizogenes, a gram-negative soil bacterium, and the host plant. To generate hairy roots, wounded plant tissues are inoculated with A. rhizogenes, which transfers the T- DNA comprising the loci between the T R and T L regions of the Ri plasmid into the plant genome. The rhizogenic strains contain a single copy of a large Ri plasmid [26]. The T-DNA carries the rol and aux genes. The rol genes are re- sponsible for the phenotype of hairy roots and the aux genes direct auxin synthesis, involved in root induction [27, 28]. A. rhizogenes A4 type (A4, ATCC, 15834, 1855, TR105, etc.) can synthesis both agropine and mannopine. A. rhizogenes 8196 type (TR7, TR101, etc) synthesize the mannopine only. The vir region is about 35 kb in the Ri plasmid, and encodes six transcriptional loci, vir A–G, which have important functions in gene transfer. Genes involved in agropine and auxin synthesis are located in the T R DNA region. Genes of Ri T L -DNA such as rolA, rolB, rolC and rolD direct the synthesis of a substance that stimulates hairy root differentiations under the influence of endogenous auxin synthesis. These four genes cor- respond to ORF10, ORF11, ORF12 and ORF15 of the

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eighteen ORFs of T L -DNA, respectively. ORFs of these se- quences were named c-rol genes. Ngrol (NgrolB, NgrolC, NgORF13 and NgORF14) and trol (trolC, torf 13 -1 and torf 13 -2 ) genes are c-rol genes contained in the genome of Nicotiana glauca and N. tabacum, respectively. Introduc- tion of a DNA fragment that contained RirolC, RiORF13 and RiORF14 resulted in more intense and earlier root for- mation than that of RirolB. Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) in the genome of Nicotiana glauca are similar in sequence to Rirol genes [29]. The cul- ture requirements and applications of hairy roots have been extensively investigated in recent years [5]. Hairy roots induced by rol genes are usually characterized by extensive secondary branching and an increase in the number of root hairs with a higher ratio of metabolically active meristem tissues. In addition, hairy root is an ex- cellent starting material for the production of bioactive secondary metabolites and plant-based biopharmaceuti- cals. In contrast to ordinary root cultures, which often re- quire a balanced supplement of auxin and cytokinin to maintain growth and phenotype, transformed roots are auxoautotrophic and so vigorous, stable cultures can be maintained on hormone-free media. Also, fast growing hairy roots can be used as a continuous source for the pro- duction of valuable human therapeutic molecules.

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Bioengineering

Metabolic engineering now offers new possibilities to im- prove the metabolic pathway of specific molecules in hairy root for the production of human therapeutic mole- cules. Metabolic engineering is based on integrating genes that encode enzymes of a given plant biosynthetic pathway between the T-borders of the Ri plasmid, then transferring this construct into plant genome. For exam- ple, the Hyoscyamus niger gene for hyoscyamine 6β-hy- droxylase has been placed under the control of the 35S CaMV promoter and introduced into hyoscyamine-rich Atropa belladonna plants by a binary vector system using A. rhizogenes. The resulting hairy roots showed increased levels of hydroxylase activity and contained up to fivefold higher concentrations of scopolamine than control [30]. Putrescine:SAM N-methyltransferase (PMT) catalyzes the N-methylation of the diamine putrescine to form N- methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present to- gether in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector sys- tem using the disarmed A. tumefaciens strain C58C1 car- rying the rooting plasmid pRiA4. The N-methylputrescine levels of the resulting engineered hairy roots increased fourfold [31]. The cDNA encoding farnesyl diphosphate synthase (FDS) placed under a CaMV35S promoter was

transferred into Artemisia annua using A. rhizogenes strain ATCC15834. As a result a fourfold higher accumu- lation of artemisinin was found compared to control [32]. Hairy root cultures of C. acuminata were transformed with A. rhizogenes strains ATCC 15834 and R-1000. The hairy roots produced and secreted camptothecin as well as the more potent and less toxic natural derivative, 10-hydrox- ycamptothecin (HCPT) into the medium. Remarkably, the hairy roots were able to synthesize higher alkaloids than in vivo roots, i.e., 1.0 and 0.15 mg/g dry weight for camp- tothecin and the 10-hydroxycamptothecin, respectively

[33].

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Biopharmaceuticals

Besides bioactive secondary metabolites, hairy roots can also produce human therapeutic proteins, vaccines and diagnostic monoclonal antibodies from engineered roots, and thereby hold immense potential for the biopharma- ceutical. Plants are capable of carrying out acetylation, phosphorylation, and glycosylation as well as other post- translational protein modifications required for the bio- logical activity of many eukaryotic proteins. Plants have now been modified, on the basis of peptide epitopes of pathogens, to produce vaccines against a variety of hu- man and animal diseases [8]. Indeed, numerous heterolo- gous (recombinant) proteins have been produced in plant leaves, fruits, roots, tubers, and seeds, and targeted to dif- ferent subcellular compartments, such as cytoplasm, en- doplasmic reticulum (ER), or apoplastic space [34]. Sever- al biotechnology companies are now actively developing, and patenting hairy root expression systems, while clini- cal trials are proceeding on the first biopharmaceuticals derived from them. Producing therapeutic proteins in hairy roots has many economical and practical benefits, including reduced health risks from pathogen contamina- tion and comparatively high yields. The bioreactor culti- vation, harvesting, storage, and processing of hairy roots can also use an existing high tech infrastructure. Hairy roots are potentially a cheap source of recombinant prod- ucts, and it has been estimated that the cost of producing recombinant proteins in roots could be 10- to 15-fold low- er than producing the same protein by E. coli fermenta- tion. Biopharmaceuticals produced in animal cell culture systems have to be purified from the culture supernatant, an expensive process. Hairy roots can be made to store proteins in intercellular or extracellular spaces, from where they can be easily extracted [35]. Borisjuk et al. [34] demonstrated that root secretion can be successfully ex- ploited for the continuous production of recombinant pro- teins in a process called “rhizosecretion.” Recombinant proteins targeted to the root intercellular space through the secretion pathway would also be secreted continu- ously by the roots [36]. Root-derived recombinant bio- pharmaceutical proteins, whether purified or not, are less

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likely to be contaminated with human pathogenic mi- croorganisms than those derived from animal cells, be- cause hairy roots do not act as hosts for human infectious agents. Using recombinant approaches, biotechnologist be- gan turning DNA sequences into protein drugs or bio- pharmaceuticals. The design and construct used to ex- press the recombinant protein is a key factor in determin- ing the yield. Promoter choice affects the yield by deter- mining the rate of transcription [37]. Regulated promoters can be used in preference to constitutive promoters be- cause these often have both practical and biosafety ad- vantages. The rate of translation can be optimized by en- suring that any mRNA instability sequences are eliminat- ed from the transgene construct, and that the translation- al start-site matches the Kozak consensus for plants. It might be necessary to modify codon usage in some trans- genes, not only to maximize the rate of protein synthesis, but also to eliminate cryptic introns and instability se- quences [38]. Plant viral proteins offer new opportunities to activate immunity against linked T cell epitopes to at- tack cancer [39]. Genes were expressed from either the strong constitutive cauliflower mosaic virus (CaMV) 35S promoter, or the strong modified mannopine synthase (mas2’) promoter from Agrobacterium, preferentially ac- tive in plant roots [40]. The two host-virus systems most frequently used are tobacco with tobacco mosaic virus (TMV) or cowpeas with cowpea mosaic virus (CPMV). Many forms of recombinant antibodies (plantibodies) can be produced in biopharmaceutical plants and the variety ranges from single chain antibody fragments to full-size antibodies and antibody complexes, as well as antibody fusion proteins. Vectors derived from TMV have been used to produce oral anti-hypertensive peptide (an- giotensin-1-converting enzyme inhibitor) in tomato and tobacco [41] and an inhibitor of HIV replication (a-tri- chosanthin) in N. bethamiana [42]. Chimeric CPMV parti- cles, displaying human rhinovirus-14 and HIV-1, have been purified from infected cowpea plants [43]. They were found to elicit antibody production and, in the case of the HIV chimera, to neutralize the infection of T cells by HIV- 1 in vivo. Recombinant plantibodies include fully assem- bled whole immunoglobulins [44], antigen-binding frag- ments of immunoglobulins, and synthetic single-chain variable fragment gene fusions [45]. Single-chain Fv plan- tibodies are encoded by an artificial gene made by joining together light- and heavy-chain variable sequences [46]. A novel Tobravirus-based system [47] for root expression was reported, and new vector technologies continue to be developed. For example, internal ribosome entry sites (IRES) have been used to direct the expression of bi- cistronic mRNA [48]. Hairy root cultures have been shown to produce recombinant proteins [49]. Recently, hairy root cultures of potato were transformed with A. rhizogenes strains ATCC 15834 with a 681-bp BamHI fragment from pBSHER containing the hepatitis B surface antigen (HB-

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sAg) ‘s’ gene. These hairy roots expressed higher levels of HBsAg [50]. In the case of hairy root cultures expressing monoclonal antibodies, it was shown that antibodies were not only associated with the root tissue, but were re- leased extracellularly [51, 52]. Sharp and Doran [53] re- ported that expression of a murine immunoglobulin (Ig) G1 in hairy root cultures led to secretion of the recombi- nant antibodies into the medium. Assembly and post- translational modifications in the ER are important for the synthesis of full-size antibodies and Fab fragments, and these molecular forms must be directed to the secretory pathway. Peeters et al. [54] demonstrated that an Fab fragment was efficiently secreted to the apoplast of roots. Secretion of recombinant proteins into tobacco root exu- dates was developed for the production of human secret- ed alkaline phosphatase and has recently been used for the secretion of recombinant antibodies [55]. Green fluo- rescent protein (GFP) was genetically fused to ricinB and expressed in tobacco hairy root cultures. Tobacco-syn- thesized ricinB:GFP hairy root media was used for nasal immunization and oral gavage of mice and systemic and mucosal immune responses against GFP [56]. Plantibodies could be of particular benefit for topical immunotherapy. Plantibodies derived from roots have a multitude of applications, including binding to pathogen- ic organisms, binding to serum or body fluid effector pro- teins, binding to tumor antigens to deliver imaging or anti-tumor agents, or binding to a cellular receptor site to up- or down regulate receptor function. The ability to as- semble immunoglobulins is a major advantage that plants have over bacterial expression systems [6]. More remark- ably, plants produce functional secretory plantibodies, which are dimers of the typical serum immunoglobulins and have two additional polypeptide components, mak- ing ten separate polypeptides in total [57]. Two different cell types are required to assemble such antibodies in mammals [58]. The biggest component of the cost in- volved in production of plantibodies will be for purifica- tion. However, expression roots opens up the possibility of oral administration of some therapeutic antibodies without the need for expensive purification. The expres- sion of antigens in hairy roots has opened up a new av- enue for the development of oral vaccines. Oral delivery of vaccines is an attractive alternative to injection, largely for reasons of low cost and easy administration. Therefore, oral vaccines are particularly applicable to combating dis- eases that affect very large populations. Furthermore, oral root-based vaccines are stable during storage at ambient temperatures. However, silencing of introduced trans- genes has frequently been observed in roots, constituting a major commercial problem. Post-transcriptional gene si- lencing is a sequence-specific RNA degradation mecha- nism that is widespread in eukaryotic organisms. It is of- ten associated with methylation of the transcribed region of the silenced gene and with the accumulation of small RNA molecules homologous to the silenced gene. Efforts

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to understand and eliminate factors responsible for trans- gene silencing are therefore useful. The greatest advan- tages of biopharmaceuticals in roots are that recombinant proteins are synthesized in an aseptic non-toxic aqueous environment and are stable, resulting in high yields.

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Conclusion

Plant biotechnology can lead to the commercial produc- tion of pharmacologically important proteins which are, in many cases, fully functional and nearly identical to their mammalian counterparts. Biopharmaceutical production in roots necessitates a series of careful decisions regard- ing two critical areas: the gene expression system to be used and the type of plant to be used. The use of roots as factories for the production of novel plant-based bioactive molecules, vaccines, antibodies and other therapeutic proteins will undoubtedly continue to develop. With a growing number of antibodies moving into clinical evalu- ation, the utility of biopharmaceutical production will be evaluated alongside plant root culture for the production of therapeutic antibodies. Biopharmaceuticals derived from engineered roots will have to meet the standards of performance and safety of biopharmaceuticals originat- ing from other systems. Many companies, such as Phy- tomedics and Photosynthetic in the US, are now produc- ing commercial-scale plant based on bioactive molecules and vaccines through root system. Biopharmaceutical in roots from high-tech pilot-scale bioreactor system hold great potential for the safe production of relatively inex- pensive plant-based pharmaceutical bioactive human therapeutic molecule and vaccines that would open prospects for improving world health. Easy scale-up of production is a major advantage of biopharmaceutical root system, thus in terms of cost-effectiveness the full po- tential of roots may be realized best at higher production requirements. Long-term targets for high-tech bioreac- tors may therefore encompass high-volume, low-cost an- tibodies, which do not require extensive purification [59]. Roots have many advantages over established production technologies for the large-scale expression of recombi- nant proteins, but several challenges remain to be ad- dressed in terms of improving yields and product quality. A small number of root-derived bioactive molecules are approaching commercialization, but these are the minor- ity that have met the technological challenges, cleared the regulatory hurdles and overcome inertia in the biotechnology industry. Furthermore, high-tech bioreac- tor technology would, of course, block certain safety ‘locks’ to prevent the release of biopharmaceutical plant materials into the open environment and to protect the operating personnel. Thus, we are at an important plat- form in global health where rapid advances are possible. In the future plant root bioactive molecules and vaccines from high-tech bioreactor system may be the premier ex-

pression system for diagnostic and therapeutic safe drugs for human well-being.

The authors thank Dr. Loretta Bacchetta for helpful suggestions during the course of this project and for bio- reactor facilities. This work was supported in part by research grants from ENEA, Rome, Italy.

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