14 Transgenic Plants for Production of Enzymes

Anne S. Ponstein
Syngenta Mogen, Leiden, The Netherlands

Rob F. Beudeker
DSM Food Specialties, Delft, The Netherlands

Jan Pen
PlantZyme, Leiden, The Netherlands

I.

INTRODUCTION

In terms of cost-effectiveness, plants can compete with any other production system. All that is required is a suitable plant, sunlight, mineral salts from the soil or fertilizers, and water. The overall costs of production in plants is largely determined by the existing and very efcient infrastructure and facilities in agriculture for growing, harvesting, storing, and processing of crops. Furthermore, traditional breeding has resulted in plant varieties optimized for a high yield of harvestable produce (1). Crops grown in the eld can yield bulk quantities of biomass. Millions of tons of carbohydrates, lipids, and proteins for the food, feed, or processing industries are produced annually from the harvested biomass. Biotechnology enables us to produce compounds of commercial interest in domesticated crops that were previously available only from exotic plant species, from other organisms or in limited quantities. Transgenic plants are an attractive and cost-effective alternative for the production of these biomolecules, as demonstrated by the steadily increasing number of carbohydrates (2), fatty acids (2), high-value pharmaceutical (poly)peptides (24), industrial enzymes (2

38), and biodegradable plastics (2) successfully produced in plants using this molecular farming approach. Plants are gradually earning their place among other systems for production of proteins and peptides. Although research focused initially on the production of high-value pharmaceutical (poly)peptides (2), plants are equally suited as a competitive source of nonpharmaceutical enzymes (238). The activity and interest in this area are clearly increasing as demonstrated by existing collaborations between small plant biotechnology boutique and established enzyme or agrochemical companies, such as between Prodigene and Genencor, Sembiosys and Dow Agrosciences, Zeneca MOGEN (formerly MOGEN International) and DSM Food Specialties (formerly Gist-brocades), Agracetus (now part of Monsanto), and FinnFeeds (part of Cultor; Cultor was recently acquired by Danisco). This chapter will summarize the method to generate transgenic plants, give an overview on what has been achieved in this area, compare plants with other methods of enzyme production, describe unique application methods for enzymes produced in plants, and nally present an illustrative example of an enzyme, phytase, applied in animal feed.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

II. A.

GENERATION OF TRANSGENIC PLANTS EXPRESSING ENZYMES Transformation and Regeneration of Plants

In general, expression of (poly)peptides relies on stable integration of the encoding genes in the plant genome. An advantage of this approach is that identical offspring can be obtained by self-fertilization of the transgenic crops and consequently the stable inheritance of the trait. An alternative is production by transient expression in plants via genetically engineered viruses as CaMV (39), TMV (4042), or CPMV (4345). Stable integration of genes generally consists of three phases: (a) introduction of foreign DNA into tissue amenable to transformation by methods as PEG-mediated transformation, particle bombardment, Whiskers, and Agrobacterium tumefaciens-mediated transformation; (b) selection of transformed material in the nontransgenic background by either selecting for tolerance to antibiotics, herbicides or another toxic chemical and/or by screening for expression of visual markers like luciferase, anthocyanin and GFP; (c) regeneration of shoots and roots after the successful selection of transgenic cells. This last phase may be quite cumbersome as cells amenable to transformation (competent cells) may not have the ability to regenerate. Transformation of plants has developed over the last decades into a routine procedure for many crop species, including tobacco, tomato, potato, canola, soybean, alfalfa, sugar beet, lettuce, carrot, cotton, apple, rubber, rice, corn, wheat, and banana. Preference for Agrobacterium tumefaciensmediated transformation exists as this generally leads to precise integration of only a limited number of copies of the gene of interest into the plant genome. However, cereals have appeared very recalcitrant to this method. Hence, the extensive use in the past of particle bombardment. With the recent development of a commercial transformation procedure for rice and the initial successes with the method in corn, wheat, and barley, it is likely that Agrobacterium tumefaciens will become the general vehicle for DNA delivery into plant species. B. Expression of Enzymes in Plants

Even when using an identical expression cassette, the expression levels obtained in plants will differ from gene to gene (7, 28, 46), just as in other production systems. However, some general principles apply to achieve high-level expression of proteins in plants,

like the use of promoters, enhancers, leader sequences, optimizations in codon usage, removal of mRNAdestabilizing sequences, etc. Other methods to increase the levels of heterologous proteins in plants can be found in the use of, e.g., integration-independent expression, enhancement of correct folding by coexpression of disulde isomerases or chaperone proteins and production of polyproteins for stabilization of especially small peptides. Reduction of protein degradation by, e.g., expression in tissues, as seeds, where the level of protease inhibitors is high, by expression in tissues where the activity of the degrading enzymes is low as in seeds owing to the low water content, by targeting the enzyme to the ER or by avoiding the presence of susceptible protease-cleavage sites in the enzyme will also result in an increase of the expressed enzyme. A unique aspect of expression in higher eukaryotes is the choice in the species, variety, and the location and timing for expression. Firstly, there is a choice in the plant species to be used for expression. A choice no longer severely limited by transformation technology (see above). If the protein content of a crop (tissue) would correlate with the accumulation of the transgene protein, crops with higher protein contents would be more cost-effective for production. However, as far as we are aware, no proof is available that this is indeed the case. Moreover, the argument may be turned aroundi.e., crops that are already accumulating high levels of endogenous protein may have little capacity left for the accumulation of heterologous protein. Secondly, there may even appear to be differences among varieties of one and the same species. It is well known that different germplasms differ in agronomic characteristics as yield, disease resistance, etc. It would therefore not be surprising if they would also differ in their ability to accumulate heterologous proteins. Thirdly, the enzyme can be expressed in various organs or tissues using appropriate promoters. Fourthly, expression in the plant, tissues, or organs can be limited to certain periods of development using promoters active only during these stages. Finally, the enzyme can be targeted to specic cellular compartments as the vacuoles, the protein bodies (in seed), the chloroplasts, the mitochondria, and the ER or to the apoplast using appropriate targeting signals described in the literature (47). Expression by integration in the plastid genome is another way to express enzymes. This can lead to dramatic increases in expression levels as demonstrated by the expression of -glucuronidase (GUS) that accumulated to 2.530% of total soluble protein (20).

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Combination of all these possibilities gives an almost unlimited choice. As these cellular compartments differ with regard to pH, ionic strength, and other environmental factors, these choices in fact allow the choice for a location within a plant species or a variety where active enzyme can be accumulated to the highest possible level. No study describing the application of a range of these possibilities for enhancing the expression level has been published; there are still ample opportunities for optimization. Step increases in expression of proteins in plants can be expected, as was the case with microbial systems in their early stages of use. Competing systems are much more mature and improvements in expression levels will therefore be much more modest for the latter expression system. The fact that in its present state plants can already be competitive shows that they should have a bright future as hosts for enzyme production.

D.

Exploitation of Plant Characteristics

C.

Downstream Processing

If purication is mandatory (which is not always the case, as shown below), rapid and economical downstream processing of the recombinant protein is essential. The benets of the plant-based production should not be offset by increased downstream processing costs. Any decrease in these costs will increase the competitive advantage of plants. The economic feasibility of protein purication from plant biomass has not been investigated in any detail, and if at all, mainly on a lab scale, except for Austin et al. (6), who focus on extraction and purication of industrial enzymes from alfalfa; by van Rooijen et al. (48; see below), Wilcox et al. (27), who puried bovine lysozyme from transgenic tobacco to 93% homogeneity in an easily scalable process; and by Kusnadi et al. (49), who described purication of GUS and avidin from transgenic corn. Downstream processing from plant biomass is generally assumed to be difcult and expensive. One reason for this assumption is the low ratio of recombinant protein to total biomass. Concentration and purication of the protein in the rst step would reduce this assumed disadvantage considerably. As only few studies have been published, no nal verdict is available regarding the cost of downstream processing. With step increases in expression level still feasible (see above), the economics of production will improve. In turn this will make it easier to use plants for production, even in cases where costly purication is mandatory.

Various production systems have been developed that are based on specic plant characteristics, on the use of plant regulatory DNA sequences, or on the exploitation of existing plant processing systems. Prodigene (College Station, TX) used a ubiquitin promoter that preferentially directs expression of proteins to the germ portion of corn seeds. The crude protein is present in the waste fraction of the corn wet-milling process, and thus essentially produced free of charge in that process. The only costs involved in production are downstream costs for extraction and purication of the protein. A comparison of the costs for production in the germ versus the whole seed is given by Howard (50). Similarly, starch or oil crops may be interesting as hosts for production, because the enzymes may be produced as byproducts in addition to the primary product (starch or oil), thus reducing the production costs. A very ingenious way to reduce these downstream costs was found in the use of plant seed oil bodies as carriers for proteins and peptides (48). Nonplant (poly)peptides were fused to the structural oil body protein oleosin and expressed in Brassica napus. The lipophilic nature of oleosins forms the basis for a highly efcient purication system for (poly)peptides. The feasibility of this approach has been demonstrated with -glucuronidase (GUS; 19, 48) and as a rst commercial example the expression and purication of the thrombin inhibitor hirudin (51). Alternatively, this system may be used to immobilize industrial enzymes as oleosin fusion proteins on the surface of oil bodies (19). Expression during germination of seeds, using germination-specic promoters, has the advantage that a well-established process, available equipment, and factories of malt production for, e.g., the brewing industry can be used. An additional regulatory advantage is obtained, because the actual expression takes place in a contained environment; i.e., seeds are allowed to germinate in a malting factory (52, 53). Using wound-inducible HMG2-derived promoters, expression can be effected postharvest by mechanical wounding of the leaves. An advantage is that growth of the plants is separated from the expression of the protein, which allows expression of proteins that would normally have a detrimental effect on the plant during growth or development. As the proteins are expressed in a contained environment postharvest, this approach could have regulatory advantages as well (54).

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An elegant way to produce and harvest proteins is described by Boffey et al. (55). Proteins are expressed in the easily harvestable latex fraction of the rubber tree, constituting a continuous source of the protein. The relatively simple composition of the latex would allow easy purication of the protein from the tapped latex. A disadvantage may be the long generation time and consequently the long time to develop this into a commercially operating system. Extracellular expression of proteins in the roots of hydroponically grown tobacco plants, termed rhizosecretion, is yet another way to optimize expression, as well as to facilitate purication (37).

III.

ENZYMES PRODUCED IN PLANTS

A substantial number of enzymes have meanwhile been produced in plants (see Table 1) (238). Expression of an enzyme in the vicinity of its potential substrate does not necessarily have a negative effect on the agronomic characteristics of the plant. Endogenous cell wall degrading enzymes like glucanases, polygalacturonases, and pectin esterases are present at the same location as their substrates without a detrimental effect on the plant. Also, extracellular expression of a vacuolar glucanasei.e., an enzyme that normally does not have access to the glucan substratedid not result in abberations in growth or morphology (56). Xylanase can be expressed in tobacco or Pisum sativum without an effect on plant phenotype (9, 16, 35). In one publication (35), the authors suggest that the high temperature optimum of the enzyme prevents substantial degradation at ambient temperatures. As the enzyme exhibits considerable residual activity at this temperature, a more likely explanation may be that, for unknown reasons, the apoplastically expressed enzyme does not, or does only to a very limited degree, degrade the xylans in the plant cell wall. Active (1-3,1-4)glucanases can be expressed in tobacco without phenotypic alterations despite their apoplastic location (16). A thermostable (1-3,1-4)glucanase, consisting of parts from two Bacillus spp. expressed in barley aleurone protoplasts and in transgenic barley under control of the germination-specic high-pI barley -amylase promoter yielded fertile plants (17). In contrast, no viable transgenic tobacco plants could be obtained after transformation with a endo-(1-4)glucanase gene construct. This was most likely due to degradation of cell wall -glucans, because transient expression in tobacco protoplasts showed that cell wall synthesis was never resumed

(14). A cellulase from Thermomonospora fusca was expressed in alfalfa, tobacco, wheat, and maize (10, 11). In one of these studies (11), the gene was put under the control of a chemically inducible promoter to prevent detrimental effects of the cellulase. Production of manganese-dependent lignin peroxidase in alfalfa severely affected plant growth and development. Affected plants showed yellowing foliage. However, they survived and set seed. In eld trials the dry matter content was reduced, as was the plant height. The enzyme was most likely directed to the ER (6, 8). GUS (-glucuronidase) is used in molecular biological experiments as a reporter for visualizing gene expression in plants. GUS, produced in transgenic corn seed and rapeseed (1820), is the rst enzyme produced in plants that is commercially available from Sigma. The enzyme is produced by Prodigene (College Station, TX). Sigma is also selling another protein, avidin, produced by Prodigene in corn (57). Alcohol and aldehyde dehydrogenases produced in corn (5) can nd application in prevention of alcohol toxicity when taken before social drinking. They should be formulated with NAD to ascertain activity in a pill, paste, or foodstuff. Although cyclodextrin glycosyl transferase (CGTase) was produced in potato tubers for the in planta production of cyclodextrins (13), these transgenic plants could nd application as a source of the enzyme as well, provided the enzyme levels can be increased considerably. Hen egg white and bovine and T-4 bacteriophage lysozymes have been expressed in tobacco (2427). This research was partially driven by the desire to create plants resistant to bacterial pathogens. However, lysozymes produced in this way can nd application as preservative in food, feed, cosmetics, and agriculture. Other enzymes expressed in plants include lipase (22, 23), -galactosidase (21), -amylase (3, 69), amylase (38), and phytase (2834 this chapter).

IV.

COMPARISON OF HOSTS FOR ENZYME PRODUCTION

Bacterial and fungal cells selected for high-level expression have traditionally been used for production of industrial enzymes (58). Extraction from these nonrecombinant sources will, for some enzymes, remain the preferred method of production. For the production of commercially interesting proteins, with the use of recombinant DNA techniques, various host organisms

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Table 1 Plants as Bioreactors for the Production of Enzymes Enzyme Alcohol dehydrogenase Aldehyde dehydrogenase Alpha-amylase Beta-amylase Cellulase Chymosin Cyclodextrin glycosyltransferase (CGTase) Glucanase Origin of gene(s) Human Human Bacillus licheniformis Barley Thermomonospora fusca Calf Klebsiella pneumoniae Trichodema reesei, Hybrid from two Bacillus spp., Ruminococcus avefaciens Escherichia coli Escherichia coli Dog gastric preduodenal, Rhizopus niveus Hen egg white, bovine, bacteriophage T4 Phanerochaete chrysosporium Aspergillus niger Application Prevention alcohol toxicity Prevention alcohol toxicity Liquefaction of starch Brewing Ethanol production, paper and pulp Dairy industry Production cyclodextrins Brewery, feed Plant species Corn Corn Tobacco, alfalfa, Vicia narbonensis, Pisum sativum Barley Alfalfa, tobacco, corn, wheat Tobacco, potato Potato Barley cells Ref 5 5 3, 69 38 10, 11 12 13 1417

GUS (-glucuronidase) Lactase (-galactosidase) Lipase Lysozyme Manganese-dependent lignin peroxidase Phytase

Molecular biology tool Lactose intolerance Food, medical, fatty acid production Food preservation Bleaching and pulping of paper Feed

Corn, canola Maize, sunower Tobacco, corn, canola, tomato Tobacco, potato Alfalfa

1820 21 22, 23 2427 6, 8

Xylanase

Clostridium thermocellum, Cryptococcus albidus, Ruminococcus avefaciens

Feed, brewing, paper and pulp, bakery

2834 Alfalfa, tobacco, canola, soybean, soybean cell suspension cultures, wheat Tobacco, Pisum sativum 9, 16, 3537

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are currently used: bacteria (in particular Escherichia coli and Bacillus subtilis), lamentous fungi, yeast, mammalian cells (in particular Chinese hamster ovary [CHO] cells), insect cells, transgenic animals and transgenic plants, as well as a number of other organisms that are less important. The rst tier of biotechnology companies have mainly used lamentous fungi, yeast, and bacteria for production of enzymes. Well-known industrial problems in developing systems for the production of heterologous proteins included instability and insolubility of the product, disappointing production rates, instability of selected cell lines, difculties in scaling up, purication of the recombinant product, and complicated registration procedures (59). A specic problem for industrial enzymes is the production costs, because of the small margins on these products. Economic factors inuencing the choice of production system are the rate of biomass production, equipment costs, medium composition and costs, processes for protein recovery and purication, product yields, and the potential hazard of contamination. A lot of development work is driven by the intended commercial application and by regulations set by the FDA and other organizations. A suitable host for production is selected based on its ability to fulll all the technical and economic requirements set by the protein of interest and its application. The production of proteins, by whatever system, remains an empirical process, making it impossible to predict the expression level. Such predictability may come with an increase in understanding of the molecular biology, biochemistry, and physiology of the expression systems and of the expressed proteins. Because expression in each organism is gene dependent, and this dependency differs from organism to organism, some proteins will express well in one system and not well in another. This argues strongly for the availability of a multitude of production systems, each having its own (dis)advantages. Plants will earn their place among these systems. A comparison of different hosts for production of enzymes is given in Table 2 (60).

from bacterial and fungal origins nd wide application in e.g. the starch processing and alcohol industry for liquefaction, in the brewing industry for production of low-calorie beer, in bakery for increasing bread volume, in the juice and wine industries for clarication and in the detergent industry for removal of starch stains (6164). The -amylase from B. licheniformis is the most commonly used enzyme in starch liquefaction, because of its extreme heat stability and its activity over a wide pH range (61, 65). A. Production and Purication from Plants (Fig. 1a)

Bacillus licheniformis -amylase was expressed extracellularly in tobacco at a level of maximally 0.5% of soluble protein in leaves (7, 66). The properties of the enzyme as produced in tobacco were very similar to the bacterial one. However, the protein is, in obvious contrast with the bacterial protein, glycosylated. The enzyme produced in tobacco was active and exhibited extreme thermostability, similar to the Bacillus enzyme. No apparent effect of the presence of the enzyme on plant phenotype was observed. Purication from tobacco has not yet been carried out for bacterial -amylase. B. Process-Dependent Conversion (Fig. 1b)

V.

UNIQUE APPLICATION METHODS FOR ENZYMES PRODUCED IN PLANTS

The feasibility of different methods for application of enzymes produced in plants as described below can be nicely demonstrated with -amylase from Bacillus licheniformis expressed in tobacco (7). Alpha-amylases

Enzymes produced in crops can be expressed at a sequestered location in the crop that also harbors its substrate. Alternatively, the enzyme may be expressed at the same location as the substrate, but in an active form. The conversion thus becomes process dependent. During processing of the crop, enzyme and substrate are brought into contact (or the enzyme is activated), resulting in conversion of the substrate into product. In this approach, the plant becomes equipped with the enzyme having optimal characteristics for conversion of its endogenous substrate. In planta conversion of the substrate into the desired product (Fig. 1d), by expression of active enzyme at the same location as the substrate, will have similar advantages as processdependent conversion, but will not be suitable if the enzymatic activity has a negative effect on the plants phenotype or its agronomic characteristics. The feasibility of this concept is exemplied by extracellular expression of -amylase. Extracellular expression of -amylase does not have an effect on endogenous starch, as is expected from the separated locations of enzyme and substrate. Moreover, the transgenic plants do not show any abberations in their phenotype.

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Table 2 Qualitative Comparison of Hosts for Production of Industrial Enzymes Transgenic plants Research and development Development time Feasibility improvements (state of the art) Process characteristics Reproducibility production Downstream processing costs Logistics Crude nonformulated product: Stability Storage period Upscaling time Upscaling cost (capital costs) Production volume Product characteristicsAuthenticity Glycosylation High-mannose Complex Human Other modications Folding capabilities Safety, regulations, and public acceptance Food use Containment level Presence mammalian pathogens Public acceptance level Intermediate Very high ? Seed Indenite Fast Low Unlimited Yes Yes Yes No Transgenic animals Long High Milk Limited Fast Intermediate Unlimited Yes Yes Yes Yes CHO cells Intermediate Limited Medium Limited Intermediate High Limited Yes Yes Yes Yes Insect cells Intermediate Intermediate Medium Limited Intermediate High Limited Yes Yes Yes/No No Fungi Short Limited Medium Limited Intermediate High Limited Yes Yes No No Yeast Short Limited Medium Limited Intermediate High Limited Yes Yes No No Bacteria Short Limited Medium Limited Intermediate High Limited No No No No

Source: Ref. 60. Key: : absent; : very low or doubtful; : low; : intermediate; : high

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Figure 1 Application methods of industrial enzymes produced in transgenic crops. (a) Production and purication: The enzyme is puried from the plant, formulated and subsequently applied in the industrial process. (b) Process-dependent conversion; Expression of the enzyme in the crop sequestered from its substrate. Conversion of the substrate occurs during processing. (c) Seed-formulated enzymes: The transgenic seeds are directly, without any purication of the enzyme, applied in the industrial process. (d) In planta conversion: Expression of the enzyme in planta at the same location as its substrate results in conversion of the substrate and consequently accumulation of product in planta.

Homogenization of transgenic leaf material, followed by incubation at 95 C, resulted in conversion of endogenous starch by the transgene product (Table 3). In starch potatoes, the -amylase levels as obtained in tobacco (7, 66) would be sufcient to hydrolyze all the starch present in the tuber. Similar applications for -amylase and other enzymes may be found in other industrial processes, like for instance the application of -amylase in the production of bioethanol, heat-stable -glucanases for the degradation of cell wall material in the brewing industry, and the production of fatty acids in oil plants (23). In this approach the aim is not producing enzymes in crops per se, but creating either a higher-quality input resource or an enabling process through expression of the enzyme inside the plant. This application has unique advantages. Firstly, enzyme manufacturing costs will be low, because there is no need for purication and formulation of the enzymes. Secondly, it obviates the necessity for separate production of enzymes. Thirdly, expression in the vicinity of the substrate assures better access to its substrate than is achieved

by in vitro addition of the enzyme. In some cases as wood pulping, in vitro addition has limited success because the enzymes do not have sufcient access to the substrate. Finally, expression in the crop guarantees an optimal dispersion of the enzyme throughout the biomass; i.e., an optimal homogeneous mixture of enzyme and substrate is obtained. This is less easily achieved by in vitro addition of enzymes. C. Seed-Formulated Enzymes (Fig. 1c)

Another concept for application is the direct use of engineering plant material in the processing, food, or feed industry. The transgenic plant material serves as novel formulation for the expressed enzyme. Other plant material can be used, depending on the process, but in general seeds may be the plant organ of choice, because these create a stable environment for longterm storage of the enzymes. The potential of plants as production vehicles for enzymes is enormously increased by producing the enzyme in seeds and directly applying these in the industrial process.

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Table 3 Process-Dependent Conversion of Leaf Starch by Expressed -Amylase Incubation time (min) Sample Control tobacco Control tobacco 0:02 T.A.U. B. licheniformis -amylase/mg Control tobacco 0:2 T.A.U. B. licheniformis -amylase/mg Control tobacco 2 T.A.U. B. licheniformis-amylase/mg Transgenic tobacco line MOG227.3 0 30 = = 60 =

Homogenization of transgenic leaf material containing Bacillus licheniformis -amylase, followed by incubation at 95 C for 30 min hydrolyzed starch present in the leaves. Addition of 2 T.A.U./mg Bacillus licheniformis -amylase to the nontransgenic control leaf homogenate for 30 min or of 0.2 T.A.U./mg for 60 min had the same effect, while incubation of nontransgenic control leaf homogenate had no visible effect on the endogenous starch during the time of the experiment. The presence of starch, as demonstrated by I2 =KI-staining is indicated with , intermediate staining with = and absence with .

Compared with the production followed by purication, this concept has additional advantages over traditional production systems. Firstly, the formulation of the enzyme as obtained by genetic engineering of the crop is convenient; i.e., it does not require an extra step in the manufacturing process, and it guarantees stable and safe storage of the enzyme. No allergenic problems due to the enzymes are to be expected, because these are contained in the seeds. Secondly, the manufacturing costs will be lower, because purication and formulation of the enzyme are no longer required. This concept is most easily applied in, but certainly not limited to, cases where enzymes are required for application in (processed) food or feed. The enzyme can then be expressed in the plants and plant organs that are the natural component in this animal feed and food. The transgenic plant material containing the enzyme is used as delivery vehicle for the protein with the same advantages as described above. The transgenic seeds are a novel specialty enzyme product to be added in small quantities into the industrial process, provided the expression level is sufciently high (additive route). This creates exibility, because various enzymes can be engineered into plants in the same manner and used and combined according to the needs of the user. The total premium for production, transportation, etc., of transgenic plant material will be lower than in case the transgene is built into the commodity crop. Production, transportation, and marketing can be strictly and simply controlled, preventing the mingling of transgenic with nontransgenic seeds. Alternatively, the enzyme can be expressed at low levels in all (or a large part of the) seeds used in the application (bulk route). A comparison between these two approaches in various aspects is given in Table 4.

This enzymes-in-seed approach is exemplied by the direct application of milled transgenic -amylase seeds in liquefaction of corn and potato starch (Fig. 2). Seeds of a transgenic plant line were milled and used without any further purication in the liquefaction of potato and corn starch. From the nature and the quality of the hydrolysis products obtained from corn and potato starch with the transgenic seeds it can be concluded that the enzyme as produced in tobacco is as suited for liquefaction of starch as the Bacillus licheniformis amylase. As shown in Table 5, the dextrose equivalent (DE) values, which were calculated from the HPLC patterns, were found to be similar to those obtained with the commercial preparations and within the commercially acceptable range (DE-12, preferably DE16; 67). Seeds containing -amylase maintained full enzyme stability for at least a year. When seeds are the only organs used, seed-specic expression is preferred, because it will minimize the chance of effects on agronomic characteristics.

Table 4 Comparison of Bulk and Additive Application of Enzymes in Seed Additive Expression level Research costs Amount of plant material Importance host Agronomic performance Development costs IP production Level of control Maintenance added value High Medium Low Low Less critical Low High High Bulk Low Low High High Critical High ? Low Low

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Table 5 Dextrose Equivalent (DE) Values Obtained from Hydrolysis of Corn and Potato Starch DE values Transgenic tobacco seeds (MOG227.3) Nontransgenic tobacco seeds Bacillus licheniformis amylase Bacillus amyloliquefaciens amylase Potato starch 16 0 18 15 Corn starch 13 0 16 18

Figure 2 Liquefaction of potato and corn starch. Oligosaccharide HPLC patterns obtained from hydrolysis of corn starch by milled transgenic seeds of plant line MOG227.3 expressing -amylase (left), Bacillus licheniformis -amylase (middle), or Bacillus amyloliquefaciens -amylase (right). The degree of polymerization (DP) of glucose is indicated by the numbers. The products obtained with the transgenic seeds and with Bacillus licheniformis amylase are virtually identical and clearly different from those obtained with B. amyloliquefaciens amylase. The main difference distinguishing the two specicities is the amount of DP5 (indicated in black).

improve digestibility and utilization of nutrients has become common practice. Examples are industrial enzymes such as -glucanase and endoxylanase which are added to diets containing high contents of barley, triticale, and wheat to improve growth, quality of eggs, and feed conversion ratio in poultry (68). Favorable effects of the inclusion of these enzymes in the diets of pigs have been reported as well. Another example is the enzyme phytase which is used to optimize phosphorus utilization in monogastric animals such as pigs, sh, and poultry. B. Phytase

Plants will often still be a competitive source, because of the low-cost production of biomass. The novel concepts of seed-formulated enzymes and process-dependent conversion will nd application in various markets. For process-dependent conversion the increased access to the substrate will for some industrial processes create the required competitive advantage. For seed-formulated enzymes, application will depend on the compatibility of the seeds with the industrial process. A rst commercial example of the latter concept is described below. VI. A. AN EXAMPLE: PHYTASEED Feed Enzymes

In livestock farming of pigs and poultry, the supplementation of animal feed with industrial enzymes to

Phytases (myoinositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) are widely distributed in all classes of organisms. The enzyme catalyzes the conversion of phytate into myoinositol and phosphorus. The substrate of this reaction, phytate, is a salt of phytic acid and divalent cations of calcium, magnesium, iron, or zinc. In plant seeds up to 80% of the total phosphorus has been reported to be present in the form of phytate (69). During the germination process phytate is rapidly converted into myoinositol and phosphorus for growth and development of the seedlings. Seeds of crops like wheat, corn, soybean, and barley are used in large amounts in animal feed. The amount of phosphorus present in these seeds would be more than sufcient to meet the animals requirements for optimal growth. However, monogastric animals, such as pigs and chickens, are only able to use a small portion of the phosphorus present in these seeds, because the remaining part is present in the form of phytate. These animals cannot convert phytate into myoinositol and inorganic phosphate. Therefore, inorganic feed phosphate has to be added to feed. As a consequence the manure of the animals contains a huge amount of phosphate, which, released into the environment, causes pollution through eutrophication of surface waters. In many countries, especially those with inten-

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sive livestock farming, there is a strong tendency to reduce the amount of phosphate. For example, in the Netherlands, farmers are taxed based on the amount of phosphate released into the environment. In Singapore, pig production is banned because of the related environmental problem. The amount of manure secreted into the environment is enormous. Even in as small a country as the Netherlands, 215 million kg of phosphate (P2 O5 ) from animal manure is added to the soil per year. Application of the enzyme phytase will optimize phosphorus utilization by pigs and poultry (7074). Feed phosphate can be substituted by the enzyme phytase, thus reducing the environmental problems caused by phosphate considerably. The phytase gene has been cloned successfully from A. niger var. van Tieghem (75, 76). The enzyme is produced on an industrial scale by means of fermentation of A. niger. The phytase gene is overexpressed by placing the gene under control of the A. niger amyloglucosidase promoter. Phytase is secreted as a large percentage of total protein by this lamentous fungus during growth in a fermenter. After fermentation, the enzyme is separated from the broth through a series of ltration steps. The nal ultraltrate is either stabilized and sold as a liquid product or dried and formulated to a free-owing product. The unique pH optima at 2 and 5.5 allow application of the enzyme in feed for monogastric animals because of the optimal compatibility with the environment in the gastrointestinal tract of these animals and the crop of poultry. The microbial phytase from Aspergillus niger is marketed by the alliance of DSM Food Specialties (former Gist-brocades) and BASF under the brand name Natuphos for use in animal feed. C. Expression of Phytase in Tobacco

after transfer of the plants to the greenhouse, was 1.7% of total soluble protein in leaves and 1% of total soluble protein in seeds. In leaves, phytase accumulated during further maturation of the plants up to a maximum level of 14.4% of soluble protein $ 7 weeks after transfer to the greenhouse, showing the extreme stability of phytase in the extracellular space of leaves (33). The molecular mass of tobacco-derived phytase from leaves was $ 70 kDa and from seeds $ 68 kDa, compared to $ 80 kDa for the Aspergillus enzyme. The difference in molecular weight between phytase expressed in seeds and leaves and between plantderived and Aspergillus phytase was found to be caused by differences in glycosylation, which was not unexpected because a total of 10 potential asparaginelinked sites are present in the primary structure and the Aspergillus niger enzyme is known to be heavily glycosylated (75). Deglycosylation experiments showed that both leaf- and seed-derived phytase contain highmannose, as well as complex-type, carbohydrate chains. Tissue-dependent differential glycosylation of proteins, as found here for seeds and leaves, has been described before (79). Despite the differences in glycosylation, the specic activity of puried leaf-derived phytase was found to be identical with that of the puried Aspergillus niger enzyme. D. PhytaSeed1

By expressing the phytase gene in plants instead of in A. niger, we are substituting an existing production process by a novel and innovative way of producing industrial enzymes. To demonstrate this concept, tobacco was transformed with a gene construct (28), encoding phytase from Aspergillus niger (75). The construct contains a DNA sequence encoding the tobacco PR-S signal peptide that will give export of the enzyme to the extracellular space (77). The cauliower mosaic virus (CaMV) 35S promoter (78) was used that gives expression of phytase in all parts of the plants. A total of 72 independent transgenic tobacco plants were analyzed for phytase expression in leaves and seeds. The highest expression level, measured 3 weeks

For the development of a commercially viable product, the fact that plants and plant organs are natural components of animal feed was fully exploited. PhytaSeed, the enzyme formulated by genetic engineering in canola seed, is used directly without any other postharvest treatment than milling. In other words, the seeds are used as the packaging material and the delivery vehicle for the protein. Aspergillus niger phytase was expressed extracellularly under control of the seed-specic cruciferin promoter in canola (Brassica napus cv. Westar) seeds. Canola is commonly used in animal feed, albeit in low amounts. Therefore, transgenic phytase-containing canola seeds can be simply incorporated. Transformation of canola is simple and routine, so many independent transgenic lines can be obtained. As PhytaSeed, canola seeds containing phytase, is envisioned to be marketed as an enzyme product, and since expression levels are high (see below), there is no need to incorporate the enzyme in seeds of crops that are major constituents of animal feed like soybean, corn, wheat, or barley. Small amounts of canola seed containing sufcient enzyme for the conversion of the phy-

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tate present in the feed mixture will be added. The nutritional value of the seeds will be a bonus to the enzyme formulation. A total of 95 independent transgenic canola lines were generated and grown in the greenhouse, and mature seeds were tested for phytase activity. The average phytase expression level was found to be 0.85% of total soluble protein. The highest phytase expression level obtained was 9.3%, sufcient for further development into the commercial product PhytaSeed. The application experiments described below were carried out using transgenic tobacco seeds, but similar results (to be reported elsewhere) were obtained with PhytaSeed (8082).

E.

Application of Transgenic Seeds Containing Phytase

Phytase packaged in seeds by genetic engineering techniques was found to be extremely stable. Even milled seeds did not show loss of enzyme activity when stored at room temperature for a period of a year (Fig. 3). This characteristic, brought about by the stable seed environment, is commercially very important because it generates a reliable supply to customers, as a reduced seed yield in a particular year can be replenished with

stored seeds from the previous year. Packaging of the enzyme in seeds also makes it more resistant to thermomechanical processing, as for example during feed pelleting (83). The effect of addition of milled transgenic seeds on the liberation of inorganic phosphate from phytate in animal fodder was tested by rst incubating a standard poultry feed sample under conditions simulating chicken crop and stomach conditions. Milled transgenic seeds released inorganic phosphate from the fodder, whereas milled control seeds had no activity (Fig. 4). This experiment shows that phytase as produced in plant seeds has retained the essential characteristics to exert its activity in the gastrointestinal tract and crop of monogastrics animals. The effect of transgenic seeds containing phytase on animal growth was tested in vivo by milling and subsequent addition to the basal diet of broilers. Diets supplemented with nontransgenic seeds and diets with and without added phosphate were used as controls. In addition, the commercial phytase preparation from Aspergillus niger, Natuphos, was used for comparison. Growth during the 4-week course of the experiment was used as a measure of the effect of each supplementation. Diets supplemented with transgenic seeds resulted in signicantly higher growth rates

Figure 3 Stability of phytase packaged in tobacco seeds. Unmilled transgenic tobacco seeds (MOG 413.25) stored at 4 C (&) and room temperature ( ), and milled seeds stored at room temperature (&) were analyzed for phytase activity (vertical axis) at different time points during storage (horizontal axis). Milled transgenic seeds stored at 20 C were not analyzed at the rst time point.

Figure 4 In vitro testing of transgenic phytase tobacco seeds under conditions simulating the digestive tract of poultry. Seeds containing phytase from a transgenic tobacco line (MOG 413.32) and from nontransgenic tobacco were used. For comparison, Aspergillus phytase was used in doses of 500 and 750 FTU/kg feed. During the incubation period samples were taken and analyzed for the amount of phosphate released.

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than those with control seeds or without any addition (Fig. 5). Growth rates obtained with transgenic seeds were not signicantly different from those obtained with Aspergillus phytase as a supplement or those with inorganic phosphate addition. This feeding experiment conclusively demonstrates that transgenic seeds containing phytase, added to animal feed without prior extraction of the enzymes, can compensate for the lower amount of inorganic phosphorus available to the animals.

G.

Other Potential Benets of PhytaSeed

F.

Low-Phytate Mutants

Low-phytic acid mutants have been generated in several crop species such as corn and barley by Victor Raboy and coworkers (84). These mutants might be considered to become an alternative to the use of phytase for optimizing phosphorus utilization from feed components if the agronomic performance is unaltered. However, application of phytase in low-phytate corn-based diets still results in a marked improvement in the availability of phytate phosphorous from these diets in poultry. It seems likely that there is an opportunity for both technologies to reduce the phosphate contents in manure from monogastric animals.

In the processing industries, where seed and/or plant materials are used, seed-formulated enzymes may be simply incorporated in existing processes. Phytate present in corn kernels precipitates during the wet-milling process in the concentrated steep water. This causes problems in handling, transportation, and storing of the corn steep liquor. These problems may be avoided by using phytase during steeping. The addition of phytase also results in a reduction in steeping time and in an increase in starch yield (85). An efcient and elegant manner to achieve the same goal would be to package phytase in corn kernels by genetic engineering techniques. The application of transgenic phytase seeds in food and feed may have other desirable effects. Phytate is considered to be a nutritional factor, because it chelates essential minerals like calcium, iron, and zinc, reducing their availability to monogastrics (71, 86). An increase in calcium availability by the application of microbial phytase has been demonstrated in pigs and poultry, and degradation of phytate in cereals has been shown to increase the availability of iron in vitro. Dietary addition of milled transgenic seeds containing phytase to feed or food will reduce the amount of phytate and consequently increase mineral absorption in animals as well as humans. This is especially important for infants and elderly people. Phytate is well known to bind to proteins in general and to some digestive enzymes such as proteases and amylases in particular (8789). Application of PhytaSeed may prevent the formation of phytateprotein complexes and thus improve digestion of feed and food.

VII.

CONCLUSION

Figure 5 Effect of phytase-containing transgenic tobacco seeds on the growth of broilers. Milled transgenic seeds of line MOG413.25 was used in a concentration of 295 FTU/kg. As controls, diets supplemented with feed phosphate, nontransgenic seeds, and A. niger phytase (400 FTU/kg) were included. Growth was measured after 2, and 4 weeks. Growth observed for diets supplemented with A. niger phytase, feed phosphate, or transgenic seeds was not statistically different but better than for those with nontransgenic seeds or with no addition. This demonstrates that phytase-enriched seeds improve phosphorus utilization and can substitute for phosphate as a feed supplement.

This example, as well as the other examples referred to, clearly demonstrate the advantages of using plants as a production system for enzymes. As many other applications can be envisioned, plants will certainly earn a prominent place among the various methods for producing enzymes.

REFERENCES
1. GC Whitelam, B Cockburn, AR Gandecha, MRL Owen. Heterologous protein production in transgenic plants. Biotechnol Genet Eng Rev 11:129, 1993.

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2. 3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

OJM Goddijn, J Pen. Plants as bioreactors. Trends Biotechnol 13:379387, 1995. K Herbers, U Sonnewald. Production of new/modied proteins in transgenic plants. Curr Opin Biotechnol 10:163168, 1999. AR Kusnadi, ZL Nikolov, JA Howard. Production of recombinant proteins in transgenic plants: practical considerations. Biotechnol Bioeng 56:473484, 1997. S Hickmann, JD Loike, D Holzer. Mammalian alcohol dehydrogenase and aldehyde dehydrogenase production in plants. WO patent application 98/41616, 1998. S Austin, ET Bingham, RG Koegel, DE Mathews, MN Shahan, RJ Straub, RR Burgess. An overview of a feasibility study for the production of industrial enzymes in transgenic alfalfa. Ann NY Acad Sci 721:234244, 1999. J Pen, L Molendijk, WJ Quax, PC Sijmons, AJJ van Ooyen, PJM van den Elzen, K Rietveld, A Hoekema. Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction. Bio/Technology 10:292296, 1992. S Austin, ET Bingham, DE Mathews, MN Shahan, J Will, RR Burgess. Production and eld performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase. Euphytica 85:381393, 1995. L Saalbach, J Hofemeister, H Baumlein. Plant seeds as bioreactors: use of a heat-stable, bacterial amylase in transgenic seeds of legumes for liquefaction of seed starch. In: Schriftenreihe des Bundesministeriums fur Ernahrung, Landwirtschaft und Forsten. Reihe A, Angewandte Wissenschaft. Munster-Hiltrup: Landwirtschaftverlag GmbH, 1997, pp 182184. S Austin-Phillips, RR Burgess, TL German, T Ziegelhoffer. Transgenic plants as an alternative source of lignocellulosic-degrading enzymes. WO patent application 98/16651, 1998. E Lebel, P Heifetz, E Ward, S Uknes. Transgenic plants expressing cellulolytic enzymes. WO patent application 98/11235, 1998. L Willmitzer, U Sonnewald, M Roeber, SK Carlsen. Transgenic plants expressing industrial enzymes. Methods for commercial production and extraction of protein from seed. WO patent application 92/ 01042, 1992. JV Oakes, CK Shewmaker, DM Stalker. Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato plants. Bio/Technology 9:982986, 1991. K Aspegren, L Mannonen, A Ritala, R Puupponen Pimia, U Kurten, M Salmenkallio-Marttila, V Kauppinen, TH Teeri. Secretion of a heat-stable fungal -glucanase form transgenic, suspension-cultured barley cells. Mol Breed 1:9199, 1995.

15.

16.

17.

18.

19.

20.

21. 22.

23.

24.

25.

26.

27.

BA Phillipson. Expression of a hybrid (1-3,1-4)--glucanase in barley protoplasts. Plant Sci 91:195206, 1993. K Herbers, HJ Flint, U Sonnewald. Apoplastic expression of the xylanase and (1-3,1-4) glucanase domains of the xynD gene from Ruminococcus avefaciens leads to functional polypeptides in transgenic tobacco plants. Mol Breed 2:8187, 1996. LG Jensen, O Olsen, O Kops, N Wolf, KK Thomsen, D von Wettstein. Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)--glucanase during germination. Proc Natl Acad Sci USA 93:34873491, 1996. DR Witcher, EE Hood, D Peterson, M Bailey, D Bond, A Kusnadi, R Evangelista, Z Nikolov, C Wooge, R Mehigh, W Kappel, J Register, JA Howard. Commercial production of -glucuronidase (GUS): a model system for the production of proteins in plants. Mol Breed 4:301312, 1998. B Kuhnel, LA Holbrook, MM Moloney, GJH van Rooijen. Oil bodies of transgenic Brassica napus as a source of immobilized -glucuronidase. JAOCS 73:15331538, 1996. KE McBride, DJ Schaaf, M Daley, DM Stalker. Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. Proc Natl Acad Sci USA 91:73017305, 1994. J Howard, KW Culver. Method of treating lactose intolerance. WO patent application 97/48810, 1997. P Lenee, V Gruber, S Baudino, C Benicourt, C Cudrey. Recombinant preduodenal lipases and polypeptide derivatives produced by plants, processes for obtaining them and their uses. WO patent application 96/33277, 1996. G Alibert, Z Mouloungui, A Boudet. Method for producing fatty acids or derivatives thereof from oil plants. European patent application 0770134, 1996. A Kato, S Nakamura, H Ibrahim, T Matsumi, C Tsumiyama, M Kato. Production of genetically modied lysozymes having extreme heat stability and antimicrobial activity against Gram negative bacteria in yeast and in plant. Nahrung 42:128130, 1998. J Trudel, C Potvin, A Asselin. Expression of active hen egg white lysozyme in transgenic tobacco. Plant Sci 87:5567, 1992. K During, P Porsch, M Fladung, H Lorz. Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora. Plant J 3:587598, 1993. CP Wilcox, AK Weissinger, RC Long, LC Fitzmaurice, TE Mirkov, HE Swaisgood. Production and purication of an active bovine lysozyme in tobacco (Nicotiana tabacum): utilization of valueadded crop plants traditionally grown under intensive agriculture. J Agric Food Chem 45:27932797, 1997.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

J Pen, TC Verwoerd, PA van Paridon, RF Beudeker, PJM van den Elzen, K Geerse, JD van der Klis, HAJ Versteegh, AJJ van Ooyen, A Hoekema. Phytase-containing transgenic seeds as a novel feed additive for improved phosphorus utilization. Bio/Technology 11:811814, 1993. J Li, CE Hegeman, RW Hanlon, GH Lacy, DM Denbow, EA Grabau. Secretion of active recombinant phytase from soybean cell-suspension cultures. Plant Physiol 1141:11031111, 1997. DM Denbow, EA Grabau, GH Lacy, ET Kornegay, DR Russell, PF Umbeck. Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers. Poultry Sci 77:878881, 1998. S Austin-Phillips, M Cook, RG Koegel, RJ Straub. Animal feed composition for poultry, sh and livestock, containing the juice of transgenic alfalfa. US patent 5,900,525. H Brinch-Pedersen, PB Holm, A Olesen, SR Rasmussen. Generation of transgenic wheat (Triticum aestivum L.) for obtaining elevated levels of phytase activity in the grain. Plant Mol Biol (Abstr. 305) Vol 15(3), 1997. TC Verwoerd, PA van Paridon, AJJ van Ooyen, JWM van Lent, A Hoekema, J Pen. Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol 109:11991205, 1995. J Pen, A Hoekema, PC Sijmons, AJJ van Ooijen, KR Rietveld, TC Verwoerd. The expression of phytase in plants. European patent application 0449 375, 1991. K Herbers, I Wilke, U Sonnewald. A thermostable xylanase from Clostridium thermocellum expressed at high levels in the apoplast of transgenic tobacco has no detrimental effects and is easily puried. Bio/ Technology 13:6366, 1995. J-F Laliberte, O Nicolas, S Durand, R Morsoli. The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants. Plant Mol Biol 18:447451, 1992. NV Borisjuk, LG Borisjuk, S Logendra, F Petersen, Y Gleba, I Raskin. Production of recombinant proteins in plant root exudates. Nature Biotechnol 17:466469, 1999. Y Okada, N Yoshigi, K Ito, M Kihara. Tissue-specic promoter. European patent application 0 781 849, 1996. GA de Zoeten, JR Penswick, MA Horisberger, P Ahl, M Schultze, T Hohn. The expression, localization and effect of human interferon in plants. Virology 172:213222, 1989. LK Grill. Tobacco mosaic virus as a gene expression vector. Agron Food Indust Hi Technol Nov/Dec:20 23, 1993. H Hamamoto, Y Sugiyama, N Nakagawa, E Hashida, Y Matsunaga, S Takemoto, Y Watanabe, Y Okada. A new tobacco mosaic virus vector and its use for the

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52. 53.

54.

55.

systemic production of angiotensin-l-converting enzyme inhibitor in transgenic tobacco and tomato. Bio/Technology 11:930932, 1993. MH Kumagai, TH Turpen, N Weinzettl, G DellaCioppa, AM Turpen, J Donson, ME Hilf, GL Grantham, WO Dawson, TP Chow, M Piatak Jr, LK Grill. Rapid, high-level expression of biologically active -trichosanthin in transfected plants by an RNA viral vector. Proc Natl Acad Sci USA 90:427 430, 1993. GP Lomonossoff. Plant viruses and pharmaceuticals. Agron Food Indust Hi Technol March/April:711, 1995. C Porta, VE Spall, J Loveland, JE Johnson, PJ Barker, GP Lomonossoff. Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides. Virology 202:949955, 1994. R Usha, JB Rohll, VE Spall, M Shanks, AJ Maule, JE Johnson, GP Lomonossoff. Expression of an animal virus antigenic site on the surface of a plant virus particle. Virology 197:366-374, 1993. PC Sijmons, BMM Dekker, B Schrammeijer, TC Verwoerd, PJM van den Elzen, A Hoekema. Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 8:217221, 1990. AR Kermode. Mechanisms of intracellular protein transport and targeting in plant cells. Crit Rev Plant Sci 15:285423, 1996. GJH van Rooijen, MM Moloney. Plant seed oilbodies as carriers for foreign proteins. Bio/ Technology 13:7277, 1995. AR Kusnadi, EE Hood, DR Witcher, JA Howard, ZL Nikolov. Production and purication of two recombinant proteins from transgenic corn. Biotechnol Prog 14:149155, 1998. J Howard. Methods for commercial production and extraction of protein from seed. WO patent application 98/39461, 1998. DL Parmenter, JG Boothe, GJH van Rooijen, EC Yeung, MM Moloney. Production of biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol Biol 29:11671180, 1995. RL Rodriguez. Process for protein production in plants. WO patent application 95/14099, 1995. H-G Sarx, T Diefenthal, N Wolf. Process for the production of degradation and/or conversion products of storage substances present in transgenic plant material with the help of a malting process. WO patent application 97/32986, 1997. CL Cramer, DL Weissenborn. HMG2 promoter expression system and post-harvest production of gene products in plants and plant cells. WO patent application 95/03690, 1995. SA Boffey, H Jones, RJ Slater, P Arokiaraj, KF Cheong, WYWA Rahaman, HY Yeang. Method for

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

56.

57.

58. 59.

60.

61.

62.

63.

64. 65.

66.

67.

68.

the production of proteins in plant uids. US patent application 5,580,768, 1996. LS Melchers, MB Sela-Buurlage, SA Vloemans, CP Woloshuk, JSC van Roekel, J Pen, PJM van den Elzen, BJC Cornelissen. Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and 1,3-glucanase in transgenic plants. Plant Mol Biol 21:583593, 1993. EE Hood, DR Witcher, S Maddock, T Meyer, C Baszczynski, M Bailey, P Flynn, J Register, L Marshall, D Bond, E Kulisek, A Kusnadi, R Evangelista, Z Nikolov, C Wooge, RJ Mehigh, R Hernan, WK Kappel, D Ritland, CP Li, JA Howard. Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purication. Mol Breed 3:291306, 1998. J Hodgson. Expression systems: a users guide. Bio/ Technology 11:887-893, 1993. JBL Damm. Application of glycobiology in the biotechnological production of pharmaceuticals. Pharm Technol Eur Sept:2834, 1995. J Pen. Comparison of host systems for the production of recombinant proteins. In: J Pen, MRL Owen, eds. Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins. Chichester: John Wiley and Sons, 1996, pp 149167. HJ Peppler, G Reed. Enzymes in food and feed processing. In: JF Kennedy, ed. Biotechnology, Vol 7A. Weinheim, Germany: VCH, 1987, pp 547603. JF Kennedy, VM Cabalda, CA White. Enzymic starch utilization and genetic engineering. TIBTECH 6:184 189, 1988. E Schwardt. Production and use of enzymes degrading starch and some other polysaccharides. Food Biotechnol 4:337351, 1990. JR Whitaker. New and future uses of enzymes in food processing. Food Biotechnol 4:669697, 1990. T Yuuki, T Nomura, H Tezuka, A Tsuboi, H Yamagata, N Tsukagoshi, S Udaka. Complete nucleotide sequence of a gene coding for heat- and pH-stable -amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying -amylases deduced from the cDNA sequences. J Biochem 98:11471156, 1985. J Pen, AJJ van Ooyen, PJM van den Elzen, K Rietveld, A Hoekema. Direct screening for high-level expression of an introduced -amylase gene in plants. Plant Mol Biol 18:11331139, 1992. PJ Reilly. Enzymic degradation of starch. In: GMA van Beynum, JA Roels eds. Starch Conversion Technology. New York: Marcel Dekker, 1985, pp 101142. D Cowan. Advances in feed enzyme technology. Agron Food Indust Hi Technol May/June:9, 1992.

69. 70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

M Lolas, P Markakis. Phytase of navy beans. J Food Sci 42:10941097, 1977. R Beudeker, C Geerse, GJ Verschoor. Biotechnological products for the compound feed industry. In: K North, ed. Biotechnology International. London: Century Press, 1991, pp 340 343. PCM Simons, HAJ Versteegh, AW Jongbloed, PA Kemme, P Slump, KD Bos, MGE Wolters, RF Beudeker, GJ Verschoor. Improved phosphorus availability by microbial phytase in broilers and pigs. Br J Nutr 64:525540, 1990. M Nasi. Microbial phytase supplementation for improving availability of plant phosphorus in the diet of growing pigs. J Agr Sci Finland 62:435442, 1990. H Vogt. Einsatz von Phytase im Broilermastfutter mit unterschiedlichem Phosphorgehalt. Arch Geugelk 56:9398, 1992. J von Pallauf, D Hohler, G Rimbach, H Neusser. Einub einer Zulage an mikrobieller Phytase zu einer Mais-Soja-Diat auf die scheinbare Absorption von Phosphor und Calcium beim Ferkel. J Anim Physiol Anim Nutr 67:3040, 1992. W van Hartingsveldt, CMJ van Zijl, GM Harteveld, RJ Gouka, MEG Suykerbuyk, RGM Luiten, PA van Paridon, GCM Selten, AE Veenstra, RFM van Gorcom, CAMJJ van Hondel. Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger. Gene 127:8794, 1993. RF van Gorcom, W van Hartingsveldt, PA van Paridon, AE Veenstra, RGM Luiten, GCM Selten. Cloning and expression of microbial phytase. European patent application 0420 358, 1991. BJC Cornelissen, RAM Hooft van Huijsduijnen, JF Bol. A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin. Nature 321:531532, 1986. H Guiley, RK Dudley, G Jonard, E Balazs, KE Richards. Transcription of cauliower mosaic virus DNA, detection of promoter sequences and characterization of transcripts. Cell 30:763773, 1982. R Kornfeld, S Kornfeld. Assembly of asparaginelinked oligosaccharides. Annu Rev Biochem 54:631 664, 1985. DR Ledoux, JH Broomhead, JD Firman, AJ Bermudez. Efcacy of PhytaSeed, a phytase containing transgenic canola, to improve phytase phosphorus utilization from corn-soybean meal diets fed to turkey poults from day 1 to 35. Poultry Science 77:54, 1998. ZB Zhang, HP Veit, ET Kornegay. Comparison of Natuphos and PhytaSeed phytase for young pigs (1998). Report from the Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University at Blacksburg.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

82.

83.

84.

ZB Zhang, HP Veit, CT Larsen, DM Denbow, ET Kornegay. Comparison of phytase in Natuphos and PhytaSeed for young broilers (1998). Report from the Department of Animal and Poultry Sciences Virginia Polytechnic Institute and State University at Blacksburg, V. RF Beudeker. Stable compositions comprising transgenic plant materials. WO patent application 97/ 16981, 1997. V Raboy. The genetics of seed storage phosphorus pathways. In: JP Lynch, J Deikman, eds. Phosphorus in Plant Biology: Regulatory Roles in Molecular, Cellular, Organismic and Ecosystem Processes. Am Soc Plant Physiol 1998, pp 192203.

85.

86.

87.

88.

89.

A Caransa, M Simell, A Lehmussari, M Vaara, T Vaara. A novel enzyme application of corn wet milling. Starch/Starke 40 S409, 1988. XS Zhu, PA Seib, GL Allee, YT Liang. Preparation of a low-phytate feed mixture and its bioavailability to chicks. Anim Feed Sci Technol 27:341351, 1990. SS Desphande, M Cheryan. Effect of phytic acid, divalent cations and their interaction on alpha amylase activity. J Food Sci 49:516519, 1984. BE Knuckles, DD Kuzmickuy, MR Cumbermann, AA Berschart. Effect of myoinositol phosphate esters on in vitro and in vivo digestion of protein. J Food Sci 54:13481349, 1989. H-J Ingelmann, G Rimbach, J Pallauf. Phytinsaure ein antinutritiver Faktor? Ernahrungs-Umschau 40:400404, 1993.

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