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Enzyme and Microbial Technology 30 (2002) 279 –283 www.elsevier.

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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 remain soluble. Fungi produce glycoproteins that can be


secreted and are relatively easy to purify. However, in some
Proteins for industrial applications include those used as cases, mis-folding of proteins in bacteria and hyper-glyco-
purification and diagnostic tools as well as enzymes for sylation of proteins in fungi may occur [4 –7], making
industrial processes that may be as diverse as food process- alternative production systems necessary.
ing and paper and pulp bleaching. Enzymes were initially Animal cell culture and transgenic animal production of
obtained as natural products from animal, plant and/or mi- foreign proteins are currently being explored in academic
crobial tissues. Originally, the largest number of such en- and industrial laboratories. The major advantage of these
zymes were obtained from plant and animal sources, but as systems is that the sugars at the glycosylation sites more
microbial fermentation became more cost-effective, this nearly resemble those of the native protein in animals.
system provided the bulk of commercial enzymes from These systems are expensive and increasing the size of the
natural sources. However, natural sources often have disad- herd of transgenic animals is quite slow. Therefore, trans-
vantages as source material for enzyme production for a genic animals and cells are most often only cost effective
variety of reasons including limitations in the amount of that for high value proteins such as pharmaceuticals in which
material, geographical availability of that material, and cost sialic acid is required.
of the material. Therefore, foreign protein production sys-
tems were developed as sources of important industrial
products. Xenogenic protein production has been accom- 2. The advantages of plant production systems
plished in bacteria, fungi, cultured animal cells, transgenic
animals and of highest interest here, plants [1–3]. Bacteria Transgenic plants provide a viable technology for pro-
and fungi are relatively simple systems; however, these ducing protein products [1,2,8 –10]. They have many ad-
microbial systems require a large capital outlay initially for vantages including low cost of production, stability of pro-
fermentation equipment. Bacteria may efficiently synthesize tein products in storage tissues such as seeds, ease and speed
and secrete proteins and enzymes that are not glycosylated. of scale-up and the possibility of direct addition of plant
In many cases, this is acceptable. However, if glycosylation material to industrial processes. In addition, plant systems
is required, the bacterial system is not appropriate. The have expressed proteins with integrity across a wide range
bacterial-sourced proteins may be easily purified if they of conformations and molecular weights, including proteins
such as trypsin and a laccase isozyme (see below) that were
* Corresponding author. Tel.: ⫹1-979-690-8537; fax: ⫹1-979-690-
not successfully expressed in fermentation systems.
9527. The production cost of enzymes from any source in-
E-mail address: eehood@prodigene.com (E.E. Hood). cludes several factors such as raw materials, processing and

0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 5 0 2 - 6
280 E.E. Hood / Enzyme and Microbial Technology 30 (2002) 279 –283

possibly purification. For an enzyme that requires purifica- Table 1


tion, the raw material can be a minor part of the overall cost Yield of target protein per generational increase in seed yield
of the product—in some cases as little as 10% depending on Generation Yield of target protein
purity. If a plant extract and fungal fermentation broth offer @ 200⫻ @ 1000⫻
a protein at the same concentration, then no difference is
seen in the final cost of production. However, if an extract 0 1g 1g
1 2000 g 1 kg
is 10 fold more concentrated, the cost advantage for a 2 40 kg 1000 kg
partially purified product is significantly better, because 3 8000 kg 106 kg
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 an advantage. An additional direct influence on cost is the
concentration than fermentation broth in the initial extract ability to achieve high expression of the protein in the
because of the expression levels that can be achieved. This production material. Plants are particularly advantageous
will not be true for all enzymes, of course, but particularly here, as several crops have promoters, targeting sequences
for those enzymes that do not express or are poorly ex- and other molecular components available that allow high
pressed in fermentation cultures. We have current examples expression to occur.
of proteins expressed at 1% of dry weight. Moreover, the Another characteristic of an ideal crop is one that has a
technology for plant expression is very young and the po- large-scale production capability already established. This
tential for improvement is very large. is true for major crops such as corn, soybeans, canola and
An additional advantage of a seed-based production sys- alfalfa. Scale-up time for protein production from identified
tem is the ability to apply the product directly to industrial transgenic lines is short and inexpensive, mostly involving
processes, minimizing handling and enzyme manipulation planting of increased acreage. For example, corn can in-
and preparation. Direct addition of transgenic plant material crease anywhere from 200 to 1000-fold per generation de-
to industrial processes, including those in the food industry, pending on the quality of the hybrid material (Table 1). The
is not done today, but is probable in the USA. When using protein yield potential from such a system is obvious.
an edible seed product such as maize as the carrier of a Finally, plant-produced products are safe to humans be-
transgenic protein, the question of direct addition to a food cause of their lack of human pathogens.
or food process can be addressed as a food additive. If
defined as a food additive or established as GRAS (gener-
ally recognized as safe), then the products to be added are 3. The process of plant production of xenogenic
regulated but the proteins would not have to be purified proteins
away from the genetic material producing those products.
Precedence in this regard has been set by approval of the Plant production of proteins involves many steps. The
direct addition of genetically modified yeasts that can be gene itself may require engineering so that the signals are
used in bread, beer, wine and cheese, among other products. recognized by the plant transcription and translation ma-
In other industries where the transgenic material will not be chinery. The signals required include a promoter, a subcel-
consumed as food, direct addition of the transgenic corn lular targeting signal, and a terminator (Fig. 1). The vector
flour may be advantageous for cost as well as synergy with of choice depends on the transformation protocol being
the process. An example of this could be amylases for starch employed. ProdiGene is currently using Agrobacterium tu-
break down. The enzymes would be produced in the same mefaciens to transform maize, so binary vectors are used.
corn grain used for the production of ethanol. For biolistic transformation, E. coli vectors can be used.
The ideal crop for molecular farming has several distinct For plant systems to compete with production by fer-
characteristics. One of the most important requirements of mentation of similar products, the expression levels must be
industrial enzymes is that they are inexpensive. In this high enough, usually 0.01– 0.1% of tissue weight, to justify
regard, stable storage of the protein in production material is 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.
E.E. Hood / Enzyme and Microbial Technology 30 (2002) 279 –283 281

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

Gene Transformation Copy Promoter Target T1 high Tn ear


method # seed bulk

Avidina (17 kDa) Biolistics 2 Constitutive CW 100 ng/mg 2 ␮g/mg (T8)


GUSb (68 kDa) Biolistics 1 Constitutive Cyto 20 ng/mg 200 ng/mg (T5)
Trypsin (24 kDa) Agrobacterium ? Seed preferred CW 300 ng/mg 50 ng/mg (T2)
Laccase (63 kDa) Agrobacterium ? Seed preferred CW 35 ng/mg 65 ng/mg (T4)
a
Reference 17.
b
Reference 18.

high expression drives technology development in this in- mation, ProdiGene’s genetics group back-crosses our trans-
dustry, where it takes many forms. New promoters are being genic lines for several generations into inbred lines that
isolated from all plant systems being utilized today. These when combined, produce high yielding hybrids. Yield per
promoters take advantage of tissue-type specificity as well acre of bushels of seed and of protein are critical to attain
as preferred sinks in the plant or seed. Targeting experi- the cost targets required for industrial enzymes. Thus ex-
ments designed to take advantage of different subcellular perimentation with multiple germplasms may reveal lines,
compartments to accumulate protein are being conducted as e.g. those with higher oil content, that are more conducive
well. In many cases, genes are resynthesized to reflect to high expression and high yield per acre in the presence of
codon usage in the host species, as well as to minimize the transgene. ProdiGene has an active breeding program to
sequences that may destabilize the RNA in its new host. achieve these goals.
When combined, these parameters can have a major positive Grain is produced for protein purification and process
influence on expression of the foreign gene. engineering experiments at various points during the pro-
The expression vector is introduced into Agrobacterium cess of generating high yielding hybrids. Strict adherence to
by electroporation [11] and the resulting strain is used to USDA guidelines is followed for growing of transgenic
transfer genes to maize immature embryos [12]. Embryos grain. ProdiGene’s confinement system is documented with
are cultured to recover transgenic events and regenerated to grower contracts and Standard Operating Procedures
recover plants [13]. Seed from To plants is produced in the (SOPs). Processing of the grain produces whole grain or
greenhouse, using pollen donors that are selected inbreds fractionated flour that can either be directly used in an
from our genetics program. Analytical assays are developed industrial process, or extracted and formulated into a final
for each protein —preferably ELISAs or enzyme assays, product. Often costs can be recovered through sales of unused
and western blots are performed on a subset of samples to grain fractions i.e. through by by-product credits [14].
confirm protein integrity and concentration estimates from
activity assays. Protein purification and characterization are 4. Examples of industrial proteins from transgenic
performed on transgenic events selected for commercializa- plants
tion. These events are also analyzed at the molecular level
for integrity of the insert. ProdiGene has expressed and produced four industrial
Because we employ HiII maize lines [13] for transfor- proteins in transgenic maize (Table 2). The transformation

Table 3
Physical characteristics of recombinant GUS and avidin from maize

Biochemical Native E. coli Maize-derived Egg white Maize-derived


properties GUS GUS avidin avidin

Molecular weight 68,000 Da 68,000 Da 17,700 16,800


Km 0.21 ⫾ 0.04 nM 0.19 ⫾ 0.05 nM N.A. N.A.
Binding stoichiometry N.A. N.A. Binds one biotin per Binds one biotin per
subunit subunit
Vmax 3.2 ⫻ 105 ⫾ 3.3 ⫻ 104 nmoles/hr 1.5 ⫻ 105 ⫾ 3.8 ⫻ 104 nmoles/hr N.A. N.A.
Isoelectric point 4.8–5.0 4.8–5.0 10 10
Ki N.T. N.T. 3.16 ␮M 3.34 ␮M
Heat stable Yes Yes ? ?
Antigenic similarity Identical Identical Identical Identical
Glycosylated No No Yes Yes
N-terminal sequence Native Identical except for initial methionine Native Identical

N.A. ⫽ not applicable.


N.T. ⫽ not tested.
282 E.E. Hood / Enzyme and Microbial Technology 30 (2002) 279 –283

Table 4 from less than the high T1 seed, to 20-fold greater than the
Comparison of physical characteristics of laccase from two sources single high T1 seed (Table 2). This is correlated with the
Property Fungal Maize-derived number of backcross generations in the material. More
laccase laccase backcrosses yield selections with higher expression.
Kinetics Similar Similar To the extent they have been tested, the recombinant
PI 5–7 5–7 proteins are functionally equivalent to the protein from
N-terminal sequence Native Identical native sources [8] (Tables 3 and 4). This is crucial if the
Glycosylated Yes Yes proteins are to be used for their activity. A slight difference
Molecular weight 66 and 55 kDa 63 and 59 kDa
in molecular weight is seen between avidin (Table 3), lac-
case (Table 4) and trypsin (Fig. 2B) derived from maize
versus their native sources. For avidin, this is due to differ-
method used to produce GUS and avidin events was biolis- ences in glycosylation, which is probably also the case for
tics, while Agrobacterium-mediated transformation was laccase. Trypsin differences in molecular weight are still
used for trypsin and laccase events. Each gene is present in under investigation.
one or very few copies per genome. The expression level in Laccase is a blue copper oxidase with applications in the
the highest T1 seed ranged from 20 ng per mg seed dry wood products and textile industries, depending on enzyme
weight for GUS up to 300 for trypsin, and is not correlated properties. We have expressed the gene for the laccase 1
with promoter or targeting sequence. Through breeding and isozyme from Trametes versicolor [15] at commercial lev-
selection, these levels are present in an ear bulk at values els 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 –283 283

levels show two bands at approximately 63 and 59 kDa (Fig. References


2A) with several smaller bands that react with the antibod-
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replacing formaldehyde or chlorite, respectively. As more
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