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430 GENETIC MODIFICATION, APPLICATIONS / Cell Factories McKersie BD, Bowley SR, and Jones KS (1999) Winter survival

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genetic information which in some way alters the metabolism of the cell, resulting in the production of a valuable product. This could be a small metabolite, or even a large polymer. This approach is called metabolic engineering. The third is to introduce new genetic information into the cell which modies the properties of the plant in a new or desirable way, e.g., improving its nutritional qualities, changing the ower color, or prolonging the storage lifetime of its fruit. This approach can be called trait modication.

Engineering the Plant Cell Factory

Strategies for Plant Transformation

Cell Factories
R M Twyman, University of York, York, UK E Stoger, RWTH Aachen, Aachen, Germany P Christou, Fraunhofer Institute of Molecular Biology and Applied Ecology, Schmallenberg, Germany
Copyright 2003, Elsevier Ltd. All Rights Reserved.

Plant Cells as Factories

The plant cell can be viewed as a minute biomolecular factory, taking in simple molecules and assimilating them into complex ones. Human beings attempt to replicate this process by chemically synthesizing a variety of useful molecules, particularly to use as drugs, but we can achieve nothing like the specicity or selectivity of a living cell, especially when dealing with large and complex molecules. The alternative is therefore to harness the intricacy of the cell and exploit it for the production of specic desirable or useful molecules. There are three major ways in which the plant cell factory is exploited. The rst is to introduce new genetic information into the cell enabling it to synthesize an entirely new protein which is itself of value. This approach is called molecular farming. The second is to introduce new

Manipulation of the plant cell factory requires a stable and permanent genetic change in the host plant. Stable DNA transfer to plants can be achieved by two approaches. The rst is dependent on the soil pathogen Agrobacterium tumefaciens, which is known to induce tumors (crown galls) in certain plant species. The tumor results from the transfer of a small segment of DNA (the transferred DNA, T-DNA) from a resident tumor-inducing plasmid (Ti-plasmid) to the plant genome. The genes that cause tumor growth are contained in the T-DNA but they are not required for gene transfer. They can therefore be deleted and replaced with foreign DNA, leading to the production of transgenic plants carrying any foreign gene of interest. The second approach is called direct DNA transfer and encompasses several diverse techniques linked only by their ability to physically introduce DNA into plant cells. Such techniques include particle bombardment (where DNA-coated microprojectiles are accelerated into the cell), protoplast transformation mediated by polyethylene glycol, electroporation (where DNA is taken up through transient pores generated by a brief electric pulse), and whisker transformation (where silicon carbide whiskers perforate the cell wall allowing DNA to enter). Once inside the cell, some of the DNA nds its way to the nucleus where it may be expressed episomally. In rare cases, the DNA integrates into the genome, and these stably transformed cells can be selectively propagated if an appropriate marker gene is introduced into the cell along with any transgenes of interest.
Production of Novel Proteins

The simplest strategy for manipulating the cell factory is to express a new protein in the plant. This is always the case in molecular farming applications, where the protein itself is the desired molecule. For metabolic engineering, it is often necessary to express one or more new enzymes in order to divert


metabolic ux in a particular direction and achieve the synthesis of a desired product. Trait modication may involve the production of new proteins (e.g., insecticidal toxins) and/or incorporate aspects of metabolic engineering (e.g., the synthesis of b-carotene in rice (Oryza sativa) grains). In each case, the plant undergoes a gain of function when the introduced transgene is expressed. The success of this strategy relies principally on the design of the expression construct, which should normally include a strong promoter, elements to optimize the efciency of protein synthesis, and if necessary a modied coding region to match the codon preference of the host plant. For molecular farming applications it may be necessary to target the protein to a particular tissue, especially if the protein is toxic to the plant. Seed-specic promoters are often used for this purpose so that the recombinant protein does not accumulate in vegetative tissues and affect plant growth. Subcellular targeting may also be an important consideration, especially for the expression of complex proteins and glycoproteins. Transgene expression, and hence the accumulation of recombinant proteins, may also be inuenced by epigenetic phenomena and it may be necessary to screen a moderately sized population of plants to identify individual lines showing strong and stable transgene expression.
Interfering with Endogenous Gene Expression

small interfering RNA to induce RNA interference, and the expression of antibodies or dominant negative alleles to interfere with the activity of endogenous proteins. When it comes to increasing levels of an endogenous gene product, one simple strategy is to introduce extra copies of the gene by transformation, usually under the control of a strong constitutive promoter. However, as discussed above, the overproduction of sense transcripts can trigger cosuppression, resulting in the opposite effect to that desired. Another way in which increased levels of gene expression have been achieved is activation tagging, where a T-DNA carrying a strong, outward-facing promoter integrates adjacent to an endogenous gene and enhances its expression level. Other strategies that have been explored include the introduction of a transgene encoding a transcription factor that positively regulates the target gene. This is a particularly useful approach in metabolic engineering because genes acting in a single metabolic pathway are often coregulated by the same transcription factors, so this is a convenient method of controlling the activity of several relevant genes in one experiment.

Cell Factories: Applications

Molecular Farming

Metabolic engineering and trait modication sometimes require that the expression of certain endogenous genes is either reduced or increased. First we consider ways in which endogenous genes can be inactivated, resulting in a loss of function in the host plant. Conceptually the simplest way to achieve this is to mutate the gene, which can be carried out by homologous recombination with a suitable targeting construct (a procedure called gene knockout). Unfortunately, while it is an efcient process in some plant systems (particularly the moss Physcomitrella patens and in chloroplast genomes) homologous recombination occurs with such low efciency in the nuclear genomes of higher plants that it cannot be used as a routine approach for manipulating the cell factory. The alternative approaches each work by reducing gene expression, either at the RNA or protein levels. The methods include blocking transcription and/or protein synthesis through the expression of antisense RNA, destroying messenger RNA (mRNA) through the expression of antisense RNA joined to ribozymes, overproduction of sense transcripts to provoke cosuppression of endogenous gene expression, the use of double-stranded RNA or

Molecular farming involves the manipulation of the cell factory to produce a valuable protein, often with therapeutic potential in humans. Human proteins are popular targets because the use of proteins isolated from natural sources risks contamination with viruses or prions that are wholly absent from plants. Plants have similar protein synthesis and modication pathways to mammals, so recombinant proteins expressed in plants are generally soluble and functional, which is a signicant advantage over microbial expression systems. However, plants can also be used to produce industrial enzymes, technical proteins used in research, food and feed additives, and biopolymers. A selection of proteins that have been expressed in plants is listed in Table 1. The rst human protein to be expressed in plants was growth hormone, and this was produced in transgenic tobacco (Nicotiana tabacum) and sunower (Helianthus annuus) as a fusion with the Agrobacterium nopaline synthase enzyme. Tobacco plants have been widely used for molecular farming because the yield of recombinant proteins is high, and there is an established infrastructure for agriculture and downstream processing. However, alternative expression hosts such as cereals, legumes, and plants with edible fruit are becoming more popular


Table 1 A selection of recombinant proteins that have been produced by molecular farming in plants, including pharmaceutical proteins, industrial/processing enzymes, food additives (nutriceuticals), technical proteins, and biopolymers Species Recombinant protein1

Pharmaceutical proteins: human blood products, enzymes, hormones, and growth factors Tobacco (leaves, chloroplasts, seeds), sunower Growth hormone Tobacco, potato (Solanum tuberosum) Serum albumin Tobacco Epidermal growth factor Rice a-Interferon Rice (cell suspension cultures) a1-Antitrypsin Tobacco Erythropoietin Pharmaceutical proteins: recombinant antibodies Tobacco Tobacco Tobacco (cell suspension culture) Soybean (Glycine max) Tobacco, rice, wheat (Triticum aestivum), pea (Pisum sativum) (whole plants and culture systems) Tobacco Tobacco (virus-infected plants) Pharmaceutical/veterinary proteins: vaccines Tobacco Tomato (Lycopersicon esculentum) Potato, tobacco Alfalfa (Medicago sativa), Arabidopsis thaliana Tobacco, potato Industrial/processing enzymes Alfalfa, barley (Hordeum vulgare), potato, tobacco Barley, canola (Brassica napus), tobacco Canola, rice, tobacco, wheat Tobacco, bean (Phaseolus vulgaris), pea Food additives (nutriceuticals) Potato Potato Technical proteins Maize (Zea mays) Maize Maize Biopolymers Tobacco Tobacco Tobacco, potato

VH domain, substance P IgG, scFv, human creatine kinase ScFv-immunotoxin, CD-40 Humanized IgG, herpes simplex virus ScFv, carcinoembryonic antigen IgG, IgA, Streptococcus mutans adhesin ScFv, 38C13 murine B-cell lymphoma

Hepatitis B virus surface antigen Rabies virus glycoprotein Cholera toxin B subunit Foot-and-mouth disease virus VP1 Norwalk virus capsid protein

1,4-b-D-endoglucanase Xylanase Phytase a-Amylase

b-Casein Lactoferrin

Avidin Aprotinin b-Glucuronidase

Synthetic elastin Human collagen Synthetic spider silk

1 The table lists selected recombinant proteins that have been expressed in plants. Under recombinant antibodies, the description indicates the type of antibody/antibody derivative and the corresponding antigen. Under vaccines, the description indicates the pathogen for which the vaccine was developed.

because recombinant proteins produced in such crops can be consumed directly without processing. This is particularly relevant in the case of vaccines and antibodies designed for passive immunotherapy. Furthermore, different plants have advantages for particular applications. Recombinant proteins produced in cereal grains, for example, are stable at ambient temperatures for years without loss of stability or functionality. The production of antibodies in transgenic plants is a particularly useful example of molecular farm-

ing, since antibodies are multisubunit glycoproteins that are used diagnostically and therapeutically in humans. Such proteins thus test molecular farming to its limit; they must not only be expressed at high enough levels to be economically puried from the host plant, but they must also be correctly folded (to preserve their antigen-binding capability), correctly glycosylated (to avoid inducing an immune response when administered to human subjects), and correctly assembled from individual chains. A typical monoclonal immunoglobulin G (IgG) comprises two heavy


chains and two light chains. The corresponding transgenes must be introduced into the plant either at the same time (by cotransformation) or into separate plant lines followed by crossing to generate hybrids. Both these strategies have been used successfully, but an alternative is to express singlechain Fv antibodies, which contain the antigenbinding regions of the heavy and light chains joined by a exible peptide linker. A number of principles have been learned from the expression of antibodies in transgenic plants. These include the importance of subcellular targeting to the secretory pathway, which ensures correct glycosylation and improves stability compared to antibodies accumulating in the cytosol. Furthermore, the inclusion of a retrieval signal that returns the antibody to the lumen of the endoplasmic reticulum signicantly increases the yield of recombinant protein. Remarkably, it has proven possible to express secretory immunoglobulin A (IgA) antibodies in transgenic plants. These have four separate components which must assemble correctly in a single plant cell. Even in mammals, two different cell types are required to assemble these molecules. A plant-derived recombinant sIgA against the oral pathogen Streptococcus mutans has reached phase II clinical trials as the drug CaroRxTM, which should help to reduce the incidence of dental caries.
Metabolic Engineering

Metabolic engineering involves the use of gene transfer technology to modify or extend metabolic pathways in the plant cell. In some cases, plants that produce a particular metabolite in small quantities are modied so that ux along that particular pathway is increased resulting in the accumulation of larger amounts of the product. In other cases, existing pathways are extended or entirely new pathways are imported so that the plant produces one or more novel metabolites. First, we consider how to increase the production of a particular metabolite already synthesized in the plant cell. Plant secondary metabolism produces an extremely diverse array of small molecules, many of which are therapeutically relevant in humans. For example, alkaloids are isolated from plants and used as drugs even though they are produced in only minute quantities. Isolation is technically demanding and very expensive, but because the molecules are too complex to produce by direct chemical synthesis, it is currently the only feasible approach. Metabolic engineering offers the prospect of producing designer plants or plant cell cultures that produce specic alkaloids in large amounts. This has been achieved in a few cases, the most obvious examples being the

drugs paclitaxel and shikonin. Unfortunately, in many cases, secondary metabolic pathways are long and complex. They may have cell type-specic components, and they may be regulated by numerous overlapping feedback mechanisms. To produce more of a particular metabolite, a simple strategy would be to identify the rate-limiting step in the metabolic pathway and introduce a transgene encoding that enzyme, thereby removing the bottleneck and allowing increased ux down the pathway. However, where this has been tried a common result is that another form of feedback regulation then limits the activity of the pathway, leaving the investigator no better off. Even where ux is increased through the bottleneck, this approach generally only serves to reveal the next bottleneck, frequently at the next step in the pathway! As an example, let us consider production of the valuable antineoplastic alkaloids vinblastine and vincristine in the Madagascar periwinkle (Catharanthus roseus). These molecules are produced in very small amounts and are isolated at great expense, accounting for their current market value of over US $1 million per gram. Two pathways converge to make these alkaloids, the terpenoid pathway and the shikimate pathway (Figure 1). At the end of the shikimate pathway, tryptophan must be decarboxylated to generate tryptamine, and this is catalyzed by the enzyme tryptophan decarboxylase (TDC). Tryptamine is then condensed with the terpenoid molecule secologanin to produce strictosidine, the precursor of all terpenoid indole alkaloids. The enzyme strictosidine synthase (STR) is responsible for this reaction. TDC is a rate-limiting step in alkaloid biosynthesis, so the C. roseus tdc gene was overexpressed under the control of a strong and constitutive promoter. The resulting transgenic cell cultures produced high levels of tryptamine, but no alkaloids, because strictosidine synthase then became the rate-limiting step in the pathway. Multistep regulation is a possible solution to this problem. It has been shown that several of the biosynthetic genes in this pathway are coinduced by externally applied agents such as methyl jasmonate, including tryptophan decarboxylase (tdc), strictosidine synthase (str), and at least six others. Transcription factors have been identied that bind to jasmonate response elements in the promoters of these genes and have been shown to upregulate the genes coordinately when overexpressed. A more thorough understanding of the regulatory mechanisms controlling this pathway may eventually allow precise control over the levels of valuable metabolites such as vinblastine and vincristine produced in this plant.


Shikimate pathway Terpenoid pathway




NH 2 10-Hydroxygeraniol OH

Tryptophan decarboxylase (TDC)

H C Tryptamine N H NH 2 H CH3OOC

O H OGlu O Secologanin

Strictosidine synthase (STD)


NH H OGlu Strictosidine O

Terpenoid indole alkaloids

Figure 1 Convergence of the shikimate and terpenoid pathways for the biosynthesis of terpenoid indole alkaloids.

Now we consider how plants can be persuaded to manufacture entirely novel compounds. One example is the production of polyhydroxyalkanoates in plants by diverting metabolic ux from the fatty acid biosynthesis pathway. Such products are not known to be made naturally in any plant species, and the relevant transgenes were imported from bacteria. The polyhydroxyalkanoates can be used to make biodegradable thermoplastics. Another example is the production of scopolamine, an antihypertensive alkaloid drug, in Atropa belladonna. Scopalomine is synthesized from hyoscyamine in a two-step reaction carried out by the enzyme hyoscyamine-6-hydroxylase (H6H). This enzyme is present in the plant Hyoscyamus niger, which produces scopalomine, but not in A. belladonna, which accumulates hyoscyamine. However, when a H. niger cDNA encoding H6H was expressed in A. belladonna, the hyoscyamine pool was used to produce scopalomine, i.e., the endogenous metabolic pathway was extended beyond its natural endpoint in this species.

Trait Modication

At the simplest level, any manipulation of the plant genome which results in a new phenotype can be considered a trait modication. However, while gene transfer can be used to engineer disease resistance, herbicide resistance, pest resistance, or tolerance of abiotic stresses such as drought, cold, and waterlogging, the result is still a plant which looks, and where appropriate tastes, the same as normal. Other types of trait modication make more of an impact on the quality of the plant. In other cases, trait In some cases, trait modication overlaps with metabolic engineering. For example, modifying the ower color of petunia generates a striking phenotypic change, but the underlying theme is still one of increasing or decreasing the levels of various enzymes in the developing plant, in this case those involved in pigment biosynthesis. Nutritional improvement is another example. Cereal grains lack b-carotene (provitamin A), which is required in the


diet for vitamin A biosynthesis. A lack of vitamin A in childhood leads to blindness, and since almost two-thirds of the population of the world eat rice as their staple diet, improving the vitamin A content of this cereal would make a real impact on world health. Initially, rice plants were transformed with the phytoene synthase gene from the daffodil (Narcissus pseudonarcissus) and accumulated phytoene, an intermediate in the vitamin A biosynthesis pathway. More recently, rice plants were generated expressing two daffodil enzymes and one bacterial enzyme, recapitulating the entire b-carotene pathway and producing grains that were golden in color. In this example, an entire metabolic pathway was assembled and imported into rice. modication overlaps with molecular farming. The expression of a single heterologous protein, the iron-storage protein ferritin, can increase the levels of bioavailable iron in mineral-depleted foods, such as rice. In a slightly different approach, rice was transformed with a fungal gene encoding the enzyme phytase. This removes phytic acid from cereal grains, a molecule which inhibits iron absorption from the gut.

List of Technical Nomenclature

Activation tagging The isolation of genes through the integration of a DNA element containing a strong promoter that can activate genes adjacent to the insertion site. Complex nitrogen-containing heterocyclic organic compounds, mainly of plant origin, with potent pharmacological properties in humans. RNA that is complementary to a given mRNA. Expression of antisense RNA results in stoichiometric binding to the target message, blocking transcription and protein synthesis and leading to degradation. The frequency with which particular codons are used to specify the same amino acid in different species. A promoter that drives transcription continuously and in all cell types. Simultaneous transformation with multiple genes. Refers to phenomena that affect the phenotype of a plant without altering the nucleotide sequence of the genome. Includes factors such as DNA methylation, repression of gene expression or protein function, and environmental effects.


Antisense RNA

Codon preference Constitutive promoter Cotransformation Epigenetic

Future Directions
In other cases, trait The scope for manipulating the cell factory is limited only by the imagination of scientists and the availability of genes allowing desirable products to be synthesized. Molecular farming is the simplest application and improvements in this area will come mainly from the development of better expression constructs, particularly in terms of helping recombinant proteins to accumulate to high levels in transgenic plants. Metabolic engineering and trait modication often require more complex manipulations, which may involve the transfer of multiple genes into the same plant, or the modication of endogenous gene expression levels. Improvements in this area will come largely from a more thorough understanding of plant metabolism, particularly how the various steps in complex pathways are regulated. There is no doubt that the completion of the genome projects for several crop plants plus the increasing amounts of available expressed sequence tags (ESTs) and proteomic data will provide the basis for such research, and that new functional genomics tools such as high-throughput transcriptional proling, systematic mutagenesis, genome-wide protein interaction screening, and rapid determination of protein structures will be critical in the accumulation of knowledge about metabolic pathways.

Gain of function A new function conferred on a plant by gene transfer or mutation. Gene knockout A form of directed homologous recombination in which a functional allele is replaced by a nonfunctional (null) allele. A ubiquitous recombination process occurring between two DNA sequences with long regions of homology but no particular sequence specicity. Transcriptional or posttranscriptional epigenetic effect in which gene expression is reduced or eliminated, brought about by the presence of multiple copies of the same DNA sequence. The deliberate alteration of metabolism by gene manipulation, in order to modify the levels of a particular metabolite. An intermediate in elicitor-stimulated signaling which can also induce defense responses when applied to plant cells. The production, in transgenic plants or transformed plant cells, of valuable recombinant proteins.

Homologous recombination

Homologydependent gene silencing

Metabolic engineering (Methyl) jasmonate Molecular farming

436 GENETIC MODIFICATION, APPLICATIONS / Molecular Farming Monoclonal Describing an antibody produced by a clonal hybridoma cell line, which recognizes a single antigenic determinant. Genetic engineering to improve the nutritional content or quality of a particular crop plant. The effects of surrounding chromatin or genomic sequence context on the level of transgene expression. Repressive position effects lead to silencing even if the transgene is present as a single copy. A protein encoded by a transgene and produced by a transformed cell or transgenic plant. A catalytic RNA molecule that cleaves other RNAs. Can be targeted to specic mRNAs if linked to an appropriate antisense RNA molecule. The ability of double-stranded RNA to induce potent and specic silencing of a homologous gene by targeting the mRNA for degradation via the RNA induced silencing complex. The sum of metabolic pathways producing molecules with specialized functions, not involved in general homeostasis. An antibody with four component polypeptides, which is generally found in mucosal secretions. A derivatized antibody molecule in which the antigen-binding regions of the heavy and light chains are expressed as a single polypeptide. A transformation vector containing a homology region matching an endogenous gene, designed to stimulate homologous recombination and replace the endogenous gene with the sequence carried in the vector. The use of gene transfer technology to express one or more new proteins or alter endogenous metabolism in order to improve a certain trait in a crop plant.

Further Reading
Fischer R and Emans N (2000) Molecular farming for pharmaceutical proteins. Transgenic Research 9: 279299. Fray RG and Grierson D (1993) Molecular genetics of tomato fruit ripening. Trends in Genetics 9: 438443. Hiatt A (1990) Antibodies produced in plants. Nature 344: 469470. Lucca P, Hurrell R, and Potrykus I (2001) Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theoretical and Applied Genetics 102: 392397. Mason HS and Arntzen CJ (1995) Transgenic plants as vaccine production systems. Trends in Biotechnology 13: 388392. Murphy DJ (1999) Production of novel oils in plants. Current Opinion in Biotechnology 10: 175180. Primrose SB, Twyman RM, and Old RW (2001) Principles of Gene Manipulation, 6th edn. Oxford: Blackwell Science. Verpoorte R, van der Heijden R, ten Hoopen HJG, and Memelink J (1999) Metabolic engineering of plant secondary metabolic pathways for the production of ne chemicals. Biotechnology Letters 21: 467479. Verpoorte R, van der Heijden R, and Memelink J (2000) Engineering the plant cell factory for secondary metabolite production. Transgenic Research 9: 323343. Walmsey AM and Arntzen CJ (2000) Plants for delivery of edible vaccines. Current Opinion in Biotechnology 11: 126129. Ye XD, Al-Babili S, Kloti A, et al. (2000) Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287: 303305.

Nutritional improvement Position effects

Recombinant protein Ribozyme

RNA interference

Secondary metabolism

Secretory IgA

Single chain Fv

Molecular Farming
R M Twyman, University of York, York, UK E Stoger, RWTH Aachen, Aachen, Germany P Christou, Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg, Germany
Copyright 2003, Elsevier Ltd. All Rights Reserved.

Targeting construct

Trait modication

Molecular farming refers to the production, in transformed cells or transgenic organisms, of recombinant proteins with potential therapeutic or commercial value. Molecular farming began in the 1980s when the rst recombinant human proteins insulin and growth hormone were synthesized in the bacterium Escherichia coli. Since that time, a wide variety of alternative expression systems has been explored, including other species of bacteria, yeasts

See also: Genetic Modication: Gene Cloning, General Principles; Transformation, General Principles. Genetic Modication, Applications: Flower Color; Molecular Farming; Plantibodies. Genetic Modication of Secondary Metabolism: Alkaloids; Terpenoids. Tissue Culture: Secondary Metabolism in Plant Cell Cultures.