From green plants to industrial enzymes

Enzyme and Microbial Technology 30 (2002) 279 –283

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From green plants to industrial enzymes Elizabeth E. Hood* ProdiGene, 101 Gateway Blvd., Suite 100, College Station, TX 77845, USA

Abstract Transgenic plants provide a viable technology for producing industrial proteins. Advantages of plant production systems include low cost of goods, stable protein storage in seeds, ease and speed of scale-up and the possibility of direct addition of plant material to industrial processes. Using transgenic plants for production of proteins includes several steps designed to result in high expression of foreign proteins—from gene manipulation to breeding. Characteristics of four protein examples are presented —␤ glucuronidase, avidin, laccase and trypsin. These proteins represent a range of molecular weights, activities, and localizations, demonstrating the versatility of the system. The benefits of transgenic plant technology for industrial enzyme production include replacement of chemical processes that cause environmental pollution. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction Proteins for industrial applications include those used as purification and diagnostic tools as well as enzymes for industrial processes that may be as diverse as food processing and paper and pulp bleaching. Enzymes were initially obtained as natural products from animal, plant and/or microbial tissues. Originally, the largest number of such enzymes were obtained from plant and animal sources, but as microbial fermentation became more cost-effective, this system provided the bulk of commercial enzymes from natural sources. However, natural sources often have disadvantages as source material for enzyme production for a variety of reasons including limitations in the amount of that material, geographical availability of that material, and cost of the material. Therefore, foreign protein production systems were developed as sources of important industrial products. Xenogenic protein production has been accomplished in bacteria, fungi, cultured animal cells, transgenic animals and of highest interest here, plants [1–3]. Bacteria and fungi are relatively simple systems; however, these microbial systems require a large capital outlay initially for fermentation equipment. Bacteria may efficiently synthesize and secrete proteins and enzymes that are not glycosylated. In many cases, this is acceptable. However, if glycosylation is required, the bacterial system is not appropriate. The bacterial-sourced proteins may be easily purified if they * Corresponding author. Tel.: ⫹1-979-690-8537; fax: ⫹1-979-6909527. E-mail address: [email protected] (E.E. Hood).

remain soluble. Fungi produce glycoproteins that can be secreted and are relatively easy to purify. However, in some cases, mis-folding of proteins in bacteria and hyper-glycosylation of proteins in fungi may occur [4 –7], making alternative production systems necessary. Animal cell culture and transgenic animal production of foreign proteins are currently being explored in academic and industrial laboratories. The major advantage of these systems is that the sugars at the glycosylation sites more nearly resemble those of the native protein in animals. These systems are expensive and increasing the size of the herd of transgenic animals is quite slow. Therefore, transgenic animals and cells are most often only cost effective for high value proteins such as pharmaceuticals in which sialic acid is required.

2. The advantages of plant production systems Transgenic plants provide a viable technology for producing protein products [1,2,8 –10]. They have many advantages including low cost of production, stability of protein products in storage tissues such as seeds, ease and speed of scale-up and the possibility of direct addition of plant material to industrial processes. In addition, plant systems have expressed proteins with integrity across a wide range of conformations and molecular weights, including proteins such as trypsin and a laccase isozyme (see below) that were not successfully expressed in fermentation systems. The production cost of enzymes from any source includes several factors such as raw materials, processing and

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possibly purification. For an enzyme that requires purification, the raw material can be a minor part of the overall cost of the product—in some cases as little as 10% depending on purity. If a plant extract and fungal fermentation broth offer a protein at the same concentration, then no difference is seen in the final cost of production. However, if an extract is 10 fold more concentrated, the cost advantage for a partially purified product is significantly better, because processing costs are directly related to concentration of the starting material. The advantage that plants have in this regard is that seed can offer proteins at a much higher concentration than fermentation broth in the initial extract because of the expression levels that can be achieved. This will not be true for all enzymes, of course, but particularly for those enzymes that do not express or are poorly expressed in fermentation cultures. We have current examples of proteins expressed at 1% of dry weight. Moreover, the technology for plant expression is very young and the potential for improvement is very large. An additional advantage of a seed-based production system is the ability to apply the product directly to industrial processes, minimizing handling and enzyme manipulation and preparation. Direct addition of transgenic plant material to industrial processes, including those in the food industry, is not done today, but is probable in the USA. When using an edible seed product such as maize as the carrier of a transgenic protein, the question of direct addition to a food or food process can be addressed as a food additive. If defined as a food additive or established as GRAS (generally recognized as safe), then the products to be added are regulated but the proteins would not have to be purified away from the genetic material producing those products. Precedence in this regard has been set by approval of the direct addition of genetically modified yeasts that can be used in bread, beer, wine and cheese, among other products. In other industries where the transgenic material will not be consumed as food, direct addition of the transgenic corn flour may be advantageous for cost as well as synergy with the process. An example of this could be amylases for starch break down. The enzymes would be produced in the same corn grain used for the production of ethanol. The ideal crop for molecular farming has several distinct characteristics. One of the most important requirements of industrial enzymes is that they are inexpensive. In this regard, stable storage of the protein in production material is

Table 1 Yield of target protein per generational increase in seed yield Generation

0 1 2 3

Yield of target protein @ 200⫻

@ 1000⫻

1g 2000 g 40 kg 8000 kg

1g 1 kg 1000 kg 106 kg

an advantage. An additional direct influence on cost is the ability to achieve high expression of the protein in the production material. Plants are particularly advantageous here, as several crops have promoters, targeting sequences and other molecular components available that allow high expression to occur. Another characteristic of an ideal crop is one that has a large-scale production capability already established. This is true for major crops such as corn, soybeans, canola and alfalfa. Scale-up time for protein production from identified transgenic lines is short and inexpensive, mostly involving planting of increased acreage. For example, corn can increase anywhere from 200 to 1000-fold per generation depending on the quality of the hybrid material (Table 1). The protein yield potential from such a system is obvious. Finally, plant-produced products are safe to humans because of their lack of human pathogens.

3. The process of plant production of xenogenic proteins Plant production of proteins involves many steps. The gene itself may require engineering so that the signals are recognized by the plant transcription and translation machinery. The signals required include a promoter, a subcellular targeting signal, and a terminator (Fig. 1). The vector of choice depends on the transformation protocol being employed. ProdiGene is currently using Agrobacterium tumefaciens to transform maize, so binary vectors are used. For biolistic transformation, E. coli vectors can be used. For plant systems to compete with production by fermentation of similar products, the expression levels must be high enough, usually 0.01– 0.1% of tissue weight, to justify their use from an economic standpoint. Thus, the need for

Fig. 1. Representative vector used to transform a plant with a foreign gene, using the Agrobacterium tumefaciens system. Gene transfer to the plant occurs directionally from the right border (RB) and ends at the left border (LB) although the LB is usually less precise than the RB. Because transfer is directional, the selectable marker gene, in this case the “pat” gene for herbicide resistance, is placed downstream of the gene of interest.

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Table 2 Examples of industrial proteins produced by ProdiGene in transgenic maize Gene

Transformation method

Copy #

Promoter

Target

T1 high seed

Tn ear bulk

Avidina (17 kDa) GUSb (68 kDa) Trypsin (24 kDa) Laccase (63 kDa)

Biolistics Biolistics Agrobacterium Agrobacterium

2 1 ? ?

Constitutive Constitutive Seed preferred Seed preferred

CW Cyto CW CW

100 ng/mg 20 ng/mg 300 ng/mg 35 ng/mg

2 ␮g/mg (T8) 200 ng/mg (T5) 50 ng/mg (T2) 65 ng/mg (T4)

a b

Reference 17. Reference 18.

high expression drives technology development in this industry, where it takes many forms. New promoters are being isolated from all plant systems being utilized today. These promoters take advantage of tissue-type specificity as well as preferred sinks in the plant or seed. Targeting experiments designed to take advantage of different subcellular compartments to accumulate protein are being conducted as well. In many cases, genes are resynthesized to reflect codon usage in the host species, as well as to minimize sequences that may destabilize the RNA in its new host. When combined, these parameters can have a major positive influence on expression of the foreign gene. The expression vector is introduced into Agrobacterium by electroporation [11] and the resulting strain is used to transfer genes to maize immature embryos [12]. Embryos are cultured to recover transgenic events and regenerated to recover plants [13]. Seed from To plants is produced in the greenhouse, using pollen donors that are selected inbreds from our genetics program. Analytical assays are developed for each protein —preferably ELISAs or enzyme assays, and western blots are performed on a subset of samples to confirm protein integrity and concentration estimates from activity assays. Protein purification and characterization are performed on transgenic events selected for commercialization. These events are also analyzed at the molecular level for integrity of the insert. Because we employ HiII maize lines [13] for transfor-

mation, ProdiGene’s genetics group back-crosses our transgenic lines for several generations into inbred lines that when combined, produce high yielding hybrids. Yield per acre of bushels of seed and of protein are critical to attain the cost targets required for industrial enzymes. Thus experimentation with multiple germplasms may reveal lines, e.g. those with higher oil content, that are more conducive to high expression and high yield per acre in the presence of the transgene. ProdiGene has an active breeding program to achieve these goals. Grain is produced for protein purification and process engineering experiments at various points during the process of generating high yielding hybrids. Strict adherence to USDA guidelines is followed for growing of transgenic grain. ProdiGene’s confinement system is documented with grower contracts and Standard Operating Procedures (SOPs). Processing of the grain produces whole grain or fractionated flour that can either be directly used in an industrial process, or extracted and formulated into a final product. Often costs can be recovered through sales of unused grain fractions i.e. through by by-product credits [14]. 4. Examples of industrial proteins from transgenic plants ProdiGene has expressed and produced four industrial proteins in transgenic maize (Table 2). The transformation

Table 3 Physical characteristics of recombinant GUS and avidin from maize Biochemical properties

Native E. coli GUS

Maize-derived GUS

Egg white avidin

Maize-derived avidin

Molecular weight Km Binding stoichiometry

68,000 Da 0.21 ⫾ 0.04 nM N.A.

68,000 Da 0.19 ⫾ 0.05 nM N.A.

Vmax Isoelectric point Ki Heat stable Antigenic similarity Glycosylated N-terminal sequence

3.2 ⫻ 105 ⫾ 3.3 ⫻ 104 nmoles/hr 4.8–5.0 N.T. Yes Identical No Native

1.5 ⫻ 105 ⫾ 3.8 ⫻ 104 nmoles/hr 4.8–5.0 N.T. Yes Identical No Identical except for initial methionine

17,700 N.A. Binds one biotin per subunit N.A. 10 3.16 ␮M ? Identical Yes Native

16,800 N.A. Binds one biotin per subunit N.A. 10 3.34 ␮M ? Identical Yes Identical

N.A. ⫽ not applicable. N.T. ⫽ not tested.

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Table 4 Comparison of physical characteristics of laccase from two sources Property

Fungal laccase

Maize-derived laccase

Kinetics PI N-terminal sequence Glycosylated Molecular weight

Similar 5–7 Native Yes 66 and 55 kDa

Similar 5–7 Identical Yes 63 and 59 kDa

method used to produce GUS and avidin events was biolistics, while Agrobacterium-mediated transformation was used for trypsin and laccase events. Each gene is present in one or very few copies per genome. The expression level in the highest T1 seed ranged from 20 ng per mg seed dry weight for GUS up to 300 for trypsin, and is not correlated with promoter or targeting sequence. Through breeding and selection, these levels are present in an ear bulk at values

from less than the high T1 seed, to 20-fold greater than the single high T1 seed (Table 2). This is correlated with the number of backcross generations in the material. More backcrosses yield selections with higher expression. To the extent they have been tested, the recombinant proteins are functionally equivalent to the protein from native sources [8] (Tables 3 and 4). This is crucial if the proteins are to be used for their activity. A slight difference in molecular weight is seen between avidin (Table 3), laccase (Table 4) and trypsin (Fig. 2B) derived from maize versus their native sources. For avidin, this is due to differences in glycosylation, which is probably also the case for laccase. Trypsin differences in molecular weight are still under investigation. Laccase is a blue copper oxidase with applications in the wood products and textile industries, depending on enzyme properties. We have expressed the gene for the laccase 1 isozyme from Trametes versicolor [15] at commercial levels in transgenic maize. Maize seeds with high expression

Fig. 2. Western blot of maize seed extracts containing either laccase (A) or bovine trypsin (B). Molecular weight markers are as indicated. Control seed extract and control protein standards are as indicated. The fungal laccase was purified from a recombinant Aspergillus strain containing the laccase I gene from Trametes versicolor. The trypsin and trypsinogen standards were purchased from Sigma Chemical Co. Protein gels were electroblotted onto PVDF membranes, blocked with dry milk in PBST, incubated with dilutions of primary polyclonal antibodies and horseradish peroxidase conjugated secondary antibodies. Detection was with chemiluminescent substrate. Primary antibodies were prepared in rabbits that were pre-screened for a lack of serum cross-reactivity with corn seed proteins. LCG: a laccase expressing corn line in which a seed-preferred promoter is driving expression; TRF: a bovine trypsinogen expressing corn line in which a seed-preferred promoter is driving expression.

E.E. Hood / Enzyme and Microbial Technology 30 (2002) 279 –283

levels show two bands at approximately 63 and 59 kDa (Fig. 2A) with several smaller bands that react with the antibodies. The partially purified enzyme from maize has identical physical characteristics to the fungal-produced enzyme (Table 4). We are currently back-crossing high-expressing lines into parity-yielding germplasm. This particular isozyme is not well-suited to the textiles industry as a bleaching agent because of its activity profile. New commercial applications may now be possible in the wood products industry because of laccase enzyme availability from transgenic maize. Protease production in transgenic plants is best accomplished through zymogen production, preferably in a seedspecific manner. This concept is protected by U.S. Patent #6,087,558. Using this method, ProdiGene has produced transgenic lines in maize that express trypsin at high levels in single T1 seed and bulk ears from the T2 generation (Table 1). Even though seed extracts contain 2–3 bands as detected on a western blot (Fig. 2B), N-terminal sequence indicates that the bands all represent active trypsins, and not the zymogen form. The enzyme is active when extracted from seed, though it was translated as a zymogen (data not shown), presumably because tryspinogen is self-activating [16]. The genetic background of the maize inbreds in which trypsin is expressed has an impact on our ability to estimate its total amount, most likely due to the variation in level of corn trypsin inhibitor in these lines.

5. Conclusion Paradigms now exist for production of industrial proteins in plants. We have reached a level of expression (0.02– 0.2% of dry weight) that allows cost effective use of these proteins in industrial processes. Proteins for large-scale processes, demanding large amounts of enzyme are logical targets for plant production, because increasing supply is fast and inexpensive. In these cases, plants may out-compete fermentation investments. Transgenic plant technology has been under recent scrutiny. Much of the discussion about transgenic plants has centered around crops for food. The application of transgenic plants to industrial processes avoids their direct addition to the human food chain, and may offer benefits by providing replacement technology for polluting industries. An example of this would be the use of laccase as a glue in the wood products industry or as a bleach for paper, i.e. replacing formaldehyde or chlorite, respectively. As more products from transgenic plant technology come to market, especially including human pharmaceuticals and vaccines, public perception of the benefit of such technology may be improved.

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