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Recent Advances in Food Biotechnology Research

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Jube S, and Borthakur D (2006) Recent advances in food biotechnology research. In: Hui YH, Nip W-K, Nollet LML, Paliyath G,
Sahlstrøm S, and Simpson BK (eds) Food Biochemistry and Food Processing. pp 35-70, Blackwell Publishing, Oxford, U.K.

3
Recent Advances in Food
Biotechnology Research
S. Jube and D. Borthakur*

Introduction INTRODUCTION
Bioengineered plants
Essential vitamins
Vitamin A Modern biotechnology involves molecular techniques
Vitamin C that use whole or parts of living organisms to produce
Vitamin E or improve commercial products and processes. It is a
Essential minerals relatively new and rapidly evolving branch of
Iron molecular biology, which started with the creation of
Essential amino acids the first recombinant gene 30 years ago. These
Lysine
techniques are, in many different ways, changing the
Methionine and tyrosine
Essential phytochemicals
way we live by improving the foods we eat, the
Isoflavonoids beverages we drink, the clothes we wear, and the
Bioengineered animals medicines we take. They also have enhanced other
Modified milk in transgenic dairy cattle aspects of our lives through the development of new
Increased muscle growth in cattle detection methods for early diagnosis of many
Reduced fat content in transgenic swine diseases such as arteriosclerosis, cancer, diabetes,
Transgenic poultry egg as bioreactor Parkinson’s and, Alzheimer’s. The application of
Bioengineered fish biotechnology methods in the food and agricultural
Improving fish growth rate
industry is one of the many aspects of biotechnology
Increasing antifreeze property in fish
Bioengineered microorganisms
that has great impact on society. By the year 2050, it
Elimination of carcinogenic compounds is expected that more than 10 billion people will be
Inhibition of pathogenic bacteria living on this planet, and it is also believed that there
Natural sweetener produced by microorganisms may not be enough resources to feed the world
Production of carotenoids in microorganisms population (UNFPA, 1995). Hunger and malnutrition
Detection methods in food biotechnology already claims 24,000 lives a day in the developing
Transgene detection countries of Asia, Africa, and Latin America (James,
Food pathogen detection 2003). Malnutrition however, is not exclusive to
Bovine spongiform encephalopathy detection
developing nations. Many people in industrialized
Conclusion
References
countries, although mostly well fed, still suffer from
lack of proper nourishment.
Biotechnology is the scientific field that offers the
greatest potential to stop hunger today and help avoid
*Corresponding contributor. Mailing address:
Department of Molecular Biosciences & mass starvation in the future. Through biotechnology,
Bioengineering, University of Hawaii at Manoa, 1955 scientists can enhance a crop’s resistance to diseases
East-West Road, Honolulu, HI 96822. Phone: (808) and environmental stresses, allowing crops to be
956-6600. Fax: (808) 956-3542. E-mail:
[email protected].
35
36 Part I: Principles

grown in relatively unproductive and unsuitable land. phytochemicals) and enhance their availability in
Recent developments in biotechnology will allow the plants. There are two main methods to transfer genes
production of more nutritious, safer, tastier, and into plants for production of transgenic plants:
healthier food. Advances in genetic engineering are Agrobacterium-mediated transformation and
revolutionizing the way we produce and consume microprojectile bombardment. In the Agrobacterium-
food, and it is quite possible that in the next decade a mediated transformation method, a genetically
large percentage of the food we eat will be engineered strain of Agrobacterium tumefaciens is
bioengineered. used to transfer the transgene into the plants. Some
In this review, the recent advances, methods, and strains of A. tumefaciens have the natural ability to
applications of biotechnology in the manufacture of transfer a segment of their own DNA into plants for
food products from transgenic plants, animals, and inducing crown-gall tumors. These crown gall-
microorganisms have been summarized. This article is inducing wild-type strains of A. tumefaciens have a Ti
not, by any means, a documentation of every (tumor inducing) plasmid that carries the genes for
application of biotechnology in the food industry, but tumor induction. During the process of infection,
a comprehensive review, which include the most Agrobacterium transfers a segment of Ti plasmid,
relevant examples. The results of important scientific known as T-DNA, to plant cells (Willmitzer et al.
research trying to improve the nutritional value of 1983). The Ti plasmid can be engineered into a two-
staple crops such as rice, potatoes, and soybeans will plasmid (binary) system containing a “disarmed” Ti
be discussed. This improvement can be achieved plasmid in which the T-DNA has been deleted, and a
through the introduction of genes that encode for small plasmid, which is referred to as a binary
enzymes in the biosynthetic pathway of vitamins, cloning vector, containing an “engineered” T-DNA
essential amino acids, essential elements, and segment. The disarmed Ti plasmid, which is
micronutrient binding proteins. We will describe maintained in an A. tumefaciens strain, serves as a
examples of genetic engineering of cattle, swine, helper, providing the transfer function for the
poultry, and fish, with the purpose of improving milk engineered T-DNA, which contains a target gene and
quality, decreasing fat content, increasing a plant selectable marker gene inserted between the
productivity/growth, and providing tolerance to T-DNA left and right borders. When the A.
freezing temperatures. The use of the mammary gland tumefaciens containing the disarmed Ti plasmid and
and egg as bioreactors for the production of important the binary cloning vector is grown in the presence of
proteins will also be addressed. The third part of this acetosyringone, the Agrobacterium vir (virulence)
article will focus on the role of microorganisms for gene proteins are produced, which help to transfer the
the betterment of food products through the engineered T-DNA region of the binary cloning
elimination of carcinogenic compounds from vector to the plant cells (Zambryski 1988). The
beverages, inhibition of pathogenic bacteria from Agrobacterium-mediated transformation is the most
starter cultures, the production of healthier natural commonly used method for genetic engineering of
sweeteners, and synthesis of beneficial compounds plants.
such as carotenoids. Finally, some examples on the The microprojectile bombardment method, also
use of biotechnology techniques in the detection of known as the gene gun or biolistic transformation
transgenic material and harmful pathogens in food method, involves the delivery and expression of
products will be described. Other recent reviews of foreign DNA in individual plant cells directly (Klein
specific aspects of food biotechnology will et al. 1987). It has been proven to be a powerful
complement the information provided in this article method for transforming a large number of plant
(Giddings et al. 2000; Kleter et al. 2001; Daniell and species, including monocots, which are often difficult
Dhingra 2002; Dove 2002; Sharma et al. 2002; Taylor to transform using A. tumefaciens (Vain et al. 1995).
and Hefle 2002; Vasil 2003). In this method, tungsten or gold spherical particles,
approximately 4 µm in size, are coated with DNA and
BIOENGINEERED PLANTS accelerated to high speed into plant cells using a
biolistic particle delivery system or a gene gun. Once
Genetic engineering methods have been extensively the DNA gets inside a cell, it integrates into the plant
used to increase the quantity of different nutrients DNA through some unknown process. It is not known
(vitamins, essential amino acids, minerals, and whether integration of DNA into the
 3 Recent Advances 37

chromosome requires the delivery of the Ingo Potrykus from the Swiss Federal Institute
microprojectiles into the plant nucleus. The of Technology, Zurich, Switzerland and Peter Beyer
microprojectile bombardment method has been used from the University of Freiburg recently developed
to transfer genes into various plant sections used in transgenic rice, expressing genes for ß-carotene
tissue culture regeneration, calli, cell suspensions, biosynthesis in rice grains (Potrykus 2001). Rice
immature embryos, and pollens in a wide range of endosperm naturally contains geranlylgeranyl
plant species. This method can also be used to pyrophosphate (GGPP), which is a precursor of the
transfer genes into chloroplasts and mitochondria, pathway for β-carotene biosynthesis. GGPP can be
which cannot be accomplished by the A. tumefaciens- converted into β-carotene in four steps (Bartley et al.
mediated gene transfer (Southgate et al. 1995). 1994) (Fig. 3.2). The bacterial enzyme phytoene
desaturase (EC 1.14.99.30) encoded by the crtI gene
ESSENTIAL VITAMINS can substitute the functions of both phytoene
desaturase and ζ-carotene desaturase (EC 1.14.99.30)
Vitamins play a crucial role in human health by in plants (Armstrong 1994). To reduce the number of
controlling metabolism and assisting the biochemical genes transformed into rice for the β-carotene
processes that release energy from foods. They are pathway, the researchers used the crtI gene from the
important in the formation of hormones, blood cells, bacterium Erwinia uredovora (Ye et al. 2000). The
nervous-system chemicals, and genetic material. psy gene encoding phytoene synthase (EC 2.5.1.32)
Vitamins combine with proteins to create and the lcy gene encoding lycopene β-cyclase used
metabolically active enzymes important in many for transformation originated from the plant daffodil.
chemical reactions. Out of the 13 well-known The plant psy gene (cDNA) and the bacterial crtI
vitamins, the body can only manufacture vitamin D; gene were placed under the control of the
all others, such as vitamin A, C, and E, must be endosperm-specific rice glutelin (Gt1) promoter and
derived from the diet. Insufficient vitamin intake may the 35S CaMV promoter, respectively, and
cause a variety of health problems. Through introduced in the binary plasmid pZPsC. Another
biotechnology, scientists can increase the content of plasmid, pZLcyH, was constructed by inserting the
vitamins in certain crops, allowing a wider range of lcy gene from daffodil under the control of rice Gt1
the world population to make use of their health promoter and the aphIV gene, for hygromycin
benefits. resistance, under the control of 35S CaMV promoter.
Plasmids pZPsC and pZLCyH were co-transformed
Vitamin A into immature rice embryos by Agrobacterium-
Nearly two-thirds of the world population depends mediated transformation (Ye et al. 2000). All
on rice as their major staple, and among them an hygromycin-resistant transformants were screened
estimated 300 million suffer from some degree of for the presence of the psy, crtI, and lcy genes by
vitamin A deficiency (WHO 1997). This is a serious Southern hybridization. A few of the transformed
public health problem in a number of countries, plants produced β-carotene in the endosperm, which
including highly populated areas of Asia, Africa, and caused the kernel to appear yellow. The selected line
Latin America. The rice endosperm (the starchy contained 1.6-µg β-carotene per gram of endosperm,
interior part of the rice grain) does not contain any ß- and was established as “golden rice.”
carotene, which is the precursor for vitamin A.
Vitamin A is a component of the visual pigments of Vitamin C
rod and cone cells in the retina, and its deficiency Vitamin C or ascorbic acid, found in many plants, is
causes symptoms ranging from night blindness to an important component in human nutrition. It has
total blindness. In Southeast Asia, it is estimated that antioxidant properties, improves immune cell and
a quarter of a million children go blind each year cardiovascular functions, prevents diseases linked to
because of this nutritional deficiency. Plant foods the connective tissue (Davey et al. 2000), and is
such as carrots and many other vegetables contain ß- required for iron utilization (Hallberg et al. 1989).
carotene. Each ß-carotene molecule is oxidatively Most animals and plants are able to synthesize
cleaved in the intestine to yield two molecules of ascorbic acid, but humans do not have the enzyme,
retinal, which can be then reduced to form retinol or L-gulono-1,4-lactone oxidoreductase (EC 1.1.3.8),
vitamin A (Fig. 3.1).
38 Part I: Principles

Figure 3.1. Formation of vitamin A from β-carotene.

which is the immediate precursor of ascorbic acid

necessary for the final step in ascorbic acid (Fig. 3.3; Wheeler et al. 1998; Smirnoff et al. 2001).
biosynthesis. For this reason, ascorbic acid needs to Researchers in Spain (Agius et al. 2003) isolated and
be consumed from dietary sources, especially from characterized galUR, a gene in strawberry that
plants (Davey et al. 2000). The recent identification encodes the enzyme D-galacturonic acid reductase.
of ascorbic acid pathway in plants opened the way to The galUR gene was amplified by PCR as a 956-bp
manipulating its biosynthesis and allowed the design fragment and cloned into a binary vector behind a
of bioengineered plants that produce ascorbic acid at 35S CaMV promoter. The resulting plasmid was
significantly higher levels. The biosynthetic pathway transformed into E. coli and delivered to
of ascorbic acid differs from animals to plants. In Agrobacterium by triparental mating. Finally, the
plants, vitamin C biosynthesis can be accomplished GalUR gene was introduced into Arabidopsis
in two ways. First, D-galacturonic acid, which is thaliana plants via Agrobacterium-mediated
released upon the hydrolysis of pectin (a major cell transformation. The expression of the strawberry
wall component), is converted into L-galactonic acid GalUR gene in A. thaliana allowed the
with the help of the enzyme D-galacturonic acid bioengineered plants to increase the biosynthesis of
reductase (EC 2.7.1.44). L-galactonic acid is then ascorbic acid by two to three times compared with
readily converted into L-galactono-1,4-lactone, the wild-type plants (Agius et al. 2003).
 3 Recent Advances 39

Plants

O P P
Geranylgeranyl pyrophosphate (GGPP)

Phytoene synthase ( psy)

Phytoene

Phytoene desaturase ( crtB) Bacteria

Phytoene
desaturase
ζ−Carotene (crtI)
Phytoene desaturase ( crtP)

Lycopene

Lycopene cyclase ( lcy)

ß-Carotene

Figure 3.2. Biosynthetic pathway of β-carotene in plants and bacteria.

The second way by which plants synthesize could increase ascorbic acid synthesis, because a
vitamin C is through the recycling of used ascorbic more efficient ascorbate recycling process would be
acid (Fig. 3.4). During the first step of this recycling, achieved (Chen et al. 2003). To test their hypothesis,
ascorbic acid is oxidized forming a radical called they isolated DHAR cDNA from wheat and
monodehydroascorbate (MDHA). Once MDHA is expressed the gene in tobacco and maize plants.
formed, it can be readily converted back into ascorbic Tobacco plants were transformed by using
acid by the enzyme monodehydroascorbate reductase Agrobacterium. A His tag was added to DHAR,
(MDHAR) (EC 1.6.5.4), or further oxidized forming which was then introduced in the binary vector
dehydroascorbate (DHA). DHA can then undergo pBI101, behind a 35S CaMV promoter. For maize, a
irreversible hydrolysis or be recycled to ascorbic acid DHAR without a His tag was placed under the
by the enzyme dehydroascorbate reductase (DHAR) control of the maize ubiquitin (Ub) promoter or the
(EC 1.8.5.1), which uses the reductant glutathione Shrunken 2 (Sh2) promoter in the pACH18 vector.
(GSH) (Washko et al. 1992; Wheeler et al. 1998; Transgenic maize was generated by particle
Smirnoff and others 2001). Researchers from the bombardment of the embryogenic callus.
University of California, Riverside, hypothesized that
by enhancing the expression of DHAR in plants, they
 40 Part I: Principles

DHAR expression was amplified up to 32 times in

Pectin tobacco, and up to 100 times in maize, resulting in
an increased ascorbic acid levels of up to four-fold
Hydrolysis
in the bioengineered plants (Chen et al. 2003).

Vitamin E
D-galacturonic acid Vitamin E is a broad term used to describe a group of
eight lipid-soluble antioxidants in the tocotrienol and
D-galacturonic acid reductase
tocopherol families that are synthesized by
(GalUR gene) (EC 2.7.1.44)
photosynthetic organisms, mainly plants (Hess 1993).
Both tocotrienol and tocopherol families can be
L-galactonic acid distinguished into four different forms each (α, β, γ,
δ), based on the number and position of methyl
Aldono-lactonase groups in the aromatic ring (Kamal-Eldin and
(EC 3.1.1.17) Appelqvist 1996). Tocotrienols and tocopherols
protect plants against oxidative stresses and the
L-galactono-1,4-lactone antioxidant property of these molecules adds
functional qualities to food products (Andlauer and
L-galactono-1,4-lactone Furst 1998). Vitamin E is an important component of
dehydrogenase mammalian diet, and excess intake has been shown to
(EC 1.1.3.8) produce many beneficial therapeutic properties,
including reduction of cholesterol levels, inhibition of
Ascorbic acid breast cancer cell growth in vitro, decrease risk of
cardiovascular diseases, and decrease incidence of
Figure 3.3. Biosynthetic pathway of vitamin C in plants. many human degenerative disorders (Theriault et al.
1999).

L-galactono-1,4-lactone

Ascorbic Acid

MDHAR GSH

MDHA DHAR

GSSG

DHA

Figure 3.4. Oxidative pathway of vitamin C recycling.

 3 Recent Advances 41

Tocotrienols have more powerful antioxidant different precursors. Tocotrienols are produced from
properties than tocopherols but are not absorbed as the condensation of HGA and geranylgeranyl
readily. The predominant forms of vitamin E in diphosphate (GGDP), catalyzed by HGA
leaves and seeds are α-tocopherol and γ-tocotrienol, geranylgeranyl transferase (HGGT) (EC 2.5.1.32),
respectively (Munne´-Bosch and Alegre 2002). and tocopherols are formed from the condensation of
While the biosynthesis of tocopherols and HGA and phytyl diphosphate (PDP), catalyzed by
tocotrienols has been known for many years, the HGA phytyl transferase (HPT) (EC 2.5.1.62) (Fig.
particular genes that encode for the different 3.5) (Soll et al. 1980; Schultz et al. 1985; Collakova
enzymes in the pathway have only recently been and DellaPenna 2001). Researchers from the Institute
discovered. Researchers are trying to develop plants of Botany in Germany described the effects of
with increased vitamin E levels and some positive constitutive expression of HPPD cDNA from barley
results have already been achieved. (Hordeum vulgare) in tobacco plants. The HPPD
The first step in the pathway for the biosynthesis gene was cloned into the pBinAR binary vector, in a
of both tocopherols and tocotrienols is the formation SmaI cloning site located between the 35S CaMV
of homogentisic acid (HGA) from p-hydroxyphenyl- promoter and the octopine synthase (EC 1.5.1.11)
pyruvate, catalyzed by the enzyme p-hydroxyphenyl- polyadenylation signal. The construct was then
pyruvate dioxygenase (HPPD) (EC 1.13.11.27) (Fig. introduced into Agrobacterium GV3101, which was
3.5) (Grusack and DellaPenna 1999). Tocotrienol and
tocopherol biosynthesis in plants originates from two

p-hydroxyphenyl-pyruvate

HPPD
Geranylgeranyl-PP Phytyl-PP

(Condensation) Homogentisic Acid (Condensation)

(HGA)

HGGT HPT

2-methyl-6-geranylgeranylbenzoquinol 2-methyl-6-phytylbenzoquinol

γ-Tocotrienol γ-Tocopherol

γ-TMT γ-TMT

α-Tocotrienol α-Tocopherol

Figure 3.5. Biosynthetic pathway of vitamin E (α-tocotrienol and α-tocopherol). (Adapted from Cahoon et al. 2003)
42 Part I: Principles

used to transform tobacco explants. The results their diet. Minerals are inorganic ions found in
showed that transgenic lines had a greater capacity nature and cannot be made by living organisms.
for overall biosynthesis of homogentisic acid and They can be divided into two classes: macronutrients
produced a two-fold increase in the amount of and micronutrients. Macronutrients are the minerals
vitamin E in the seeds. Vitamin E content in leaves that we need in large quantity, including calcium,
was not affected (Falk et al. 2003). phosphorus, sodium, magnesium, chlorine, sulfur,
In another approach towards vitamin E and silicon. Micronutrients, or trace minerals, are the
enhancement, Cahoon et al. (2003) reported the minerals that are required in small amounts, of which
identification and isolation of a novel monocot gene iron is the most prevalent, followed by fluorine, zinc,
that encodes HGGT, which is so far the only known copper, cobalt, iodine, selenium, manganese,
enzyme specific for the synthesis of tocotrienols. molybdenum, and chromium. Although a balanced
These researchers found that the expression of the consumption of plant-based foods should naturally
barley HGGT enhanced the tocotrienol synthesis by provide these nutrients, mineral deficiency,
10- to 15-fold in the leaves of A. thaliana and by six- especially of iron, is widespread among the world
fold in the seeds of corn. The barley HGGT cDNA population.
was placed under the control of the 35S CaMV
promoter and the nopaline synthase terminator. The Iron
construct was inserted into the binary vector pZS199
to generate plasmid pSH24. The plasmid was then
Even though iron is required in trace amounts, it is
introduced into Agrobacterium for transformation
the most widespread nutrient deficiency worldwide.
into tobacco and A. thaliana (Cahoon et al. 2003).
It is believed that about 30% of the world population
A third way by which vitamin E content in
suffers from serious nutritional problems caused by
plants can be manipulated involves the last enzyme
insufficient intake of iron (WHO 1992). Iron is an
in the final step of tocotrienols and tocopherols
important constituent of hemoglobin, the oxygen-
biosynthetic pathway, in which γ-tocotrienol and γ- carrying component of the blood, and is also a part of
tocopherol are converted to α-tocotrienol and α- myoglobin, which helps muscle cells to store oxygen.
tocopherol, respectively. This step is catalyzed by the Low iron levels can cause the development of iron
enzyme γ-tocopherol methyltransferase (γ-TMT) (EC deficiency anemia. In an anemic person the blood
2.1.1.95) (Fig. 3.5) (Shintani and DellaPenna 1998). contains a low level of oxygen, which result in many
α-tocopherol has the highest oxidative property health problems including infant retardation (Walter
among the members of the vitamin E family (Kamal- et al. 1986), pregnancy complication (Murphy et al.
Eldin and Appelqvist 1996). Unfortunately, plant 1986), low immune function (Murakawa et al. 1987),
oils, which are the main dietary source of vitamin E, and tiredness (Basta et al. 1979). Iron is present in
contain only a fraction amount of α-tocopherol but a food in both inorganic (ferric and ferrous) and
high level of its precursor, γ-tocopherol. Shintani and organic (heme and nonheme) forms. Heme iron,
DellaPenna overexpressed endogenous A. thaliana γ- which is highly bioavailable, is derived primarily
TMT to enhance conversion of γ-tocopherol into α- from the hemoglobin and myoglobin of flesh foods
tocopherol. They introduced the γ-TMT cDNA such as meats, fish, and poultry (Taylor et al. 1986).
construct under the control of a 35S CaMV promoter In humans, reduced iron (ferrous) is taken up more
in a binary vector into A. thaliana plants by readily than oxidized (ferric) iron. Several
Agrobacterium-mediated transformation. α- approaches have been used in the fight against iron
tocopherol content of bioengineered seeds was nine- deficiency including nutraceutical supplementation,
fold greater than that of the wild-type seeds (Shintani food fortification, and different methods of food
and DellaPenna 1998). preparation and processing (Maberly et al. 1994). So
far, none of these approaches have been successful in
eradicating iron deficiency, especially in developing
countries. A new tool in the fight against nutrient
deficiency is the use of biotechnology to improve
ESSENTIAL MINERALS essential mineral nutrition in staple crops.
At this time, there are basically two ways in
To maintain a well functioning, healthy body, which genetic engineering can be used for this
humans require 17 different essential minerals in purpose: (1) by increasing the concentration of the
 3 Recent Advances 43

iron-binding protein ferritin and (2) by reducing the the rice grain endosperm, and its ability to induce
amount of iron-absorption inhibitor phytic acid. ferritin at a high level. The ferritin cDNA was
Although iron intake is important for human health, it isolated from soybean cotyledons, inserted into the
can be toxic, so the ability to store and release iron in binary vector pGPTV-35S-bar, and transferred into
a controlled manner is crucial. The 450 kDa ferritin rice using Agrobacterium. The iron content of the
protein, found in animals, plants, and bacteria, can rice seed in the transgenic plants was three times
accumulate up to 4500 atoms of iron (Andrews et al. greater than that of the untransformed wild-type
1992). This protein consists of 24 subunits assembled plants.
into a hollow spherical structure within which iron is Phytic acid, or phytate, is the major inhibitor of
stored as a hydrous ferric oxide mineral core (Fig. many essential minerals, including iron, zinc, and
3.6). The two main functions of ferritin in living magnesium, and is believed to be directly responsible
organisms are to supply iron for the synthesis of for the problem of iron deficiency (Ravindran et al.
proteins such as ferredoxin and cytochromes and to 1995). In cereal grains, phytic acid is the primary
prevent free radicals damage to cells. Studies have phosphate storage and it is deposited in the aleurone
shown that ferritin can be orally administrated and is storage vacuoles (Lott 1984). During seed
effective for treatment of rat anemia (Beard et al. germination, phytic acid is catalyzed into inorganic
1996), suggesting that increasing ferritin content of phosphorous, by the action of the hydrolytic enzyme
cereals may solve the problem of dietary iron phytase (EC 3.1.3.8) (Fig. 3.7). There is little or no
deficiency in humans. Japanese researchers (Goto et phytase activity in the dry seeds or in the digestive
al. 1999) introduced soybean ferritin cDNA into rice tract of monogastric animals (Gibson and Ullah 1990;
plants, under the control of a seed specific promoter, Lantzsch et al. 1992). In a recent study, it has been
GluB-1, from the rice seed-storage protein gene shown that phytase activity can be reestablished in
encoding glutelin. The two advantages of this mature dry seeds under optimum pH and temperature
promoter are the accumulation of iron specifically in conditions (Brinch-Pedersen et al. 2002).

Iron stored as
mineral inside
ferretin
3-fold
Iron channel
4-fold
Iron channel

3-fold
Iron channel

3-fold
Iron channel

Figure 3.6. Iron binding protein ferritin.

3-fold
Iron channel
44 Part I: Principles

OPO3H2 OPO3H2

OPO3H2

OPO3H2
H2PO3O

OPO3H2

Phytic Acid

OH OH Phytase OH OPO3H2

OH OH

OH OH
HO HO
OH
OH

Inositol Inositol Monophosphate

Figure 3.7. Phytic acid is degraded during seed germination by a specific enzyme called phytase [myo-inositol-(1,2,3,4,5,6)-
hexakisphosphate phosphohydrolase] (EC 3.1.3.8).

A reduction in the amount of phytic acid in staple foods enzyme that is thermostable and maintains high activity
is likely to result in a much greater bioavailability of in plant tissues.
iron and other essential minerals. Lucca et al. (2002)
inserted a fungal (Aspergillus fumigatus) phytase cDNA ESSENTIAL AMINO ACIDS
into rice to increase the degradation of phytic acid. Rice
suspension cells, derived from immature zygotic Proteins are organic molecules formed by amino acids.
embryos, were used for biolistic transformation with the The digestive system breaks down proteins into single
A. fumigatus phytase gene. Phytase from A. fumigatus amino acids so that they can enter into the bloodstream.
was the enzyme of choice because it is heat stable and Cells then use the amino acids as building blocks to
thus can refold into an active form after heat form enzymes and structural proteins. There are two
denaturation (Wyss et al. 1998). The main purpose of types of amino acids, essential and nonessential.
this research was to increase phytase activity during Essential amino acids cannot be synthesized by animals,
seed germination and to retain the enzyme activity in including humans, therefore, need to be acquired in the
the seed after food processing and in the human diet. The nine essential amino acids are histidine,
digestive tract. Although the researchers achieved high isoleucine, leucine, lysine, methionine, phenylalanine,
expression levels of phytase in the rice endosperm, by threonine, tryptophan, and valine. The body can
placing it under the control of the strong tissue-specific synthesize nonessential amino acids as long as there is a
globulin promoter, the thermotolerance of the proper intake of essential amino acids and calories.
transgenic rice was not as high as expected. It has been Proteins are present in foods in varying amounts, some
speculated that the reason for this unexpected low foods have all nine essential amino acids in them, and
thermostability of the A. fumigatus phytase in they are referred to as complete proteins. Most animal
transgenic rice is due to the interference of the cellular products (meat, milk, eggs) provide a good source of
environment of the endosperm to maintain the enzyme complete proteins. Vegetables sources, on the other
in an active configuration (Holm et al. 2002). Further hand, are usually low on or missing certain essential
studies are needed to develop an endogenous phytase amino acids.
 3 Recent Advances 45

For instance, grains tend to lack lysine while pulses starch and alcohol (Chakraborty et al. 2000). Potato
are short in methionine (Miflin et al. 1999). In order is a good source of potassium, iron, vitamin C and B,
to provide better nutrition from plant sources, it is but it is not a rich protein source. Potato proteins are
essential to increase the content of essential amino limited in nutritive value for the lack of the amino
acids in seed and tuber proteins. This is particularly acids lysine, methionine, and tyrosine (Jaynes et al.
important for countries where certain crops, such as 1986). A lack of methionine in a person’s diet may
rice, potatoes, and corn, are the main dietary source. result in an imbalanced uptake of other amino acids,
as well as retardation in growth and development.
Methionine is also the main supplier of sulfur, which
Lysine prevents disorders of the hair, skin, and nails, helps
lower cholesterol levels by increasing the liver’s
Rice is one of the most important staple crops and is production of the phospholipid lecithin, and is a
consumed by 65% of the world population on a daily natural chelating agent for heavy metals (Cooper
basis (Lee et al. 2003). It is a good source of 1996).
essential nutrients such as vitamins B1 (thiamin), B2 Scientists from the National Center for Plant
(riboflavin), B3 (niacin), but it is low in the essential Genome Research in India isolated and cloned a
amino acids, lysine and isoleucine (Fickler 1995). gene that encodes for a seed-specific protein from
Adequate intake of lysine is essential because it Amaranthus hypocondriacus called amaranth seed
serves many important functions in the body albumin (AmA1) (Chakraborty et al. 2000). The
including aiding calcium absorption, collagen advantages of using the AmA1 seed-protein to
formation, and the production of antibodies, improve crops nutritional value are that (1) it is well-
hormones, and enzymes. A deficiency in lysine may balanced in the composition of all essential amino
result in tiredness, inability to concentrate, acids, (2) it is a non-allergenic protein, and (3) it is
irritability, bloodshot eyes, retarded growth, hair encoded by a single gene AmAl. This gene was
loss, anemia, and reproductive problems (Cooper cloned into a binary vector, under the control of a
1996). Zheng et al. (1995) developed a transgenic constitutive 35S CaMV promoter (plasmid pSB8)
rice with enhanced lysine content. They and a tuber specific granule-bound starch synthase
accomplished this by expressing the seed storage (EC 2.4.1.21) promoter (plasmid pSB8G). The AmAl
protein β-phaseolin from the common bean gene constructs from these two binary plasmids were
(Phaseolus vulgaris) in the grain of transgenic rice. introduced into potato through Agrobacterium-
The genomic and cDNA sequences of the β- mediated transformation. The amino acid contents in
phaseolin gene from P. vulgaris was placed under the pSB8-transgenic potato showed a 2.5- to 4-fold
the control of either a rice seed-specific glutelin Gt1 increase in lysene, methionine, and tyrosine, while
promoter or the native β-phaseolin promoter. The the tissue-specifc pSB8G-transgenic potatoes
vectors containing the β-phaseolin gene were showed a four to eight-fold increase in these amino
transferred into the rice chromosome by protoplast- acids (Fig.3.8).
mediated transformation. Four percent of total
endosperm protein in the transgenic rice was
phaseolin, which resulted in a significant increase in ESSENTIAL PHYTOCHEMICALS
the lysine content in rice (Zheng et al. 1995).
Besides being a major supplier of essential nutrients
such as vitamins, amino acids, and minerals, plants
Methionine and Tyrosine are also an important source of phytochemicals that
are known to be beneficial for health. Some examples
In terms of global food production, potato (Solanum of phytochemicals include indoles, isothiocyanates,
tuberosum) is only behind rice, wheat, and corn on and sulforaphane, found in vegetables such as
the list of the crop species that are most important for broccoli; allylic sulfides, found in onions and garlic;
human nutrition worldwide (Chakraborty et al. and isoflavonoids found mainly in soybeans. Since
2000). There are four main purposes for the the intake of these phytochemicals is not always
production of potatoes: for the fresh food market, for sufficient, scientists are trying to enhance the
animal feed, for the food processing industry, and for nutritional quality of plants through genetic
nonfood industrial uses such as manufacture of engineering.
46 Part I: Principles

1400
Wild-type

pSB8
1200
pSB8G
Micromoles of AA/g of protein
1000

800

600

400

200

0
Methionine Tyrosine Lysine
Amino acids

Figure 3.8. Comparison of amino acid composition from transgenic and wild-type potatoes. (From Chakraborty et al. 2000).

Isoflavonoids

Flavonoids, which include anthocyanins, condensed They help prevent the buildup of arterial plaque,
tannins, and isoflavonoids, are a class of which reduces the risk of coronary heart disease and
phytochemicals that perform a range of important stroke (FDA 1999); help reduce breast cancer
functions for the plants including pigmentation, feed (Peterson et al. 1991); help prevent prostate cancer by
deterrence, wood protection, fungi and insects delaying cell growth (Messina and Barnes 1991);
defense, and induction of genes for root nodulation fight osteoporosis by stimulating bone formation
(Buchanan et al. 2001). Isoflavonoids (or isoflavones) (Civitelli 1997); and even relieve some menopausal
are a type of phytoestrogen, or plant hormone, that symptoms (Nestel et al. 1999). The main source of
have a chemical structure similar to human estrogen. isoflavonoids in human diet comes from the
The health benefits believed to be provided by consumption of soybean and its products. Although
isoflavonoids come from the weak estrogenic activity present in high concentration in unprocessed soybean,
of these molecules in the human body (Jung et al. isoflavonoid levels can decrease by 50% during seed
2000). Isoflavonoids are found in soybeans, processing for traditional soy foods (Wang and
chickpeas, and many other legumes; however, Murphy 1996). Increasing isoflavonoid
soybeans are unique because they have the highest concentrations in soybean could solve this problem.
concentration of the two most beneficial Another way to take advantage of isoflavonoids’
isoflavonoids, genistein and daidzein (Eldrige and health benefits is through the development of other
Kwolek 1983, Tsukamoto et al. 1995). In the studies crops that can produce this powerful compound,
conducted so far, isoflavonoids show great potential thereby widening their consumption.
to fight many types of diseases.
 3 Recent Advances 47

p-Coumaroyl-CoA

CHS + CHR CHS

4,2’,4’- 4,2’,4’,6’-
trihydroxychalcone tetrahydroxychalcone

CHI CHI

Liquiritigenin Naringenin

IFS IFS

Daidzein Genistein
Figure 3.9. Biosynthetic pathway for the soybean isoflavonoids: daidzein and genistein. (Adapted from Jung et al. 2000).

Isoflavonoids are synthesized by a branch in the species have become possible by transferring genes
degradation pathway of the amino acid from related or unrelated species. Genetic
phenylalanine, and its first committed step is improvement through biotechnology can be achieved
catalyzed by the enzyme isoflavone synthase (EC in one generation, instead of several generations
1.14.13.53) (Fig. 3.9). Jung et al. (2000) identified required for traditional animal breeding methods.
two soybean genes encoding isoflavone synthase, Although several methods of gene transfer have been
IFS1/IFS2, and expressed these genes in A. thaliana, developed, four methods are used today in the
triggering the synthesis of the isoflavonoid genistein. production of most transgenic animals: nuclear
Although A. thaliana does not synthesize transfer, microinjection, viral vector infection, and
isoflavonoids, it does have the substrate naringenin, embryonic stem cell transfer. The method of nuclear
which is an intermediate of the anthocyanin transfer entails inserting the entire genetic material
biosynthetic pathway. Naringenin can then be from the nucleus of a donor cell into a mature
converted to the isoflavonoid genistein by a foreign unfertilized egg whose nucleus has been removed.
isoflavone synthase. The soy isoflavone synthase After that, the embryo is transferred into a foster
gene IFS1 was cloned in the plasmid pOY204 under mother, where it will develop into an animal that is
the control of the 35S CaMV promoter, and genetically identical to the donor cell (Wolf et al.
transferred into A. thaliana by Agrobacterium- 2001). In microinjection, a segment of foreign DNA
mediated transformation. The introduced ISF1 gene carrying one or more genes is injected into the male
expressed and produced active isoflavone synthase in pronucleus of a fertilized egg. The egg needs to be in
the transformed plant. The amount of genistein a single-cell stage to ensure that all somatic cells in
produced was approximately 2ng/µg of fresh plant the animal contain the transgene. The embryo is then
weight (Jung et al. 2000). transferred to the uterus of a surrogate mother (Wall
2002). In the retroviral infection technique, gene is
transferred with the help of a viral vector.
BIOENGINEERED ANIMALS
Retroviruses are frequently used in the process of
DNA transfer due to their natural ability to infect
With the development of transgenic technology, cells (Cabot et al. 2001). In the stem cell transfer
improvements in commercially important livestock technique, embryonic stem cells are collected from
48 Part I: Principles

blastocysts and grown in culture. The cultured cells the micelle resulting in improved heat stability. β-
are then injected into the inner cell mass of the caseins are highly phosphorylated and bind to
embryo in a blastocyst stage, which is implanted into calcium phosphate, thus influencing milk calcium
the foster mother, resulting in the production of a levels (Dalgleish et al. 1989; Jimenez Flores and
chimeric animal (Hochedlinger and Jaenisch 2003). Richardson 1988).
It is important to remember that these methods do Research on modification of milk composition
not create new species, but only offer tools for to improve nutritional or functional properties has
producing new strains of animals that carry novel been mostly done in transgenic mice. Mice are good
genetic information. Some examples of genetically models for the study of protein expression in
engineered animals include transgenic cows that mammary glands, but they do not always reflect the
produce milk with improved composition and same protein expression levels as ruminants (Colman
transgenic swine that produce meat with lower fat 1996). Brophy et al. (2003), using nuclear transfer
content. The main goal of livestock genetic technology, produced transgenic cows carrying extra
engineering programs is to increase production copies of the genes CSN2 and CSN3, which encode
efficiency while delivering healthier animal food bovine β- and κ-caseins, respectively. Genomic
products. clones containing CSN2 and CSN3 were isolated
from a bovine genomic library. Previous studies
MODIFIED MILK IN TRANSGENIC DAIRY conducted with mice revealed that CSN3 had very
low expression levels (Persuy et al. 1995). In order
CATTLE
to enhance expression of CSN3, the researchers
created a CSN2/3-fusion construct, in which the
Bovine milk has been described as an almost perfect CSN3 gene was fused with the CSN2 promoter. The
food, because it is a rich source of vitamins, calcium, CSN2 genomic clone and the CSN2/3-fusion
and essential amino acids (Karatzas and Turner construct were co-transfected into bovine fetal
1997). Some of the vitamins found in milk include fibroblast (BFF) cells, where the two genes showed
vitamin A, B, C, and D. Milk has greater calcium coordinated expression. The transgenic cells became
content than any other food source, and daily the donor cells in the process of nuclear transfer,
consumption of two servings of milk or other dairy generating nine fully healthy and functional cows.
products supplies all the calcium requirements of an Overexpression of CSN2 and CSN2/3 in the
adult person (Rinzler et al. 1999). Caseins represent transgenic cows resulted in an 8-20% increase in β-
about 80% of the total milk protein and have high casein and 100% increase in κ-casein levels (Brophy
nutritional value and functional property (Brophy et et al. 2003).
al. 2003). The caseins have a strong affinity for
cations such as calcium, magnesium, iron, and zinc.
There are four types of naturally occurring caseins in INCREASED MUSCLE GROWTH IN CATTLE
milk, αS1, αS2, β, and κ (Brophy et al. 2003). They
are clumped in large micelles, which determine the Myostatin, also known as growth and differentiation
physicochemical properties of milk. Even small factor 8 (GDF-8), is a member of the transforming
variations in the ratio of the different caseins growth factor β (TGF-β) family, which is responsible
influence micelles structure, which in turn can for negative regulation of skeletal muscle mass in
change the milk’s functional properties. The amount mice, cattle, and possibly other vertebrates.
of caseins in milk is an important factor for cheese Myostatin is expressed in embryo myoblast and
manufacturing, since greater casein content results in developing adult skeletal muscle; it is produced as a
greater cheese yield and improved nutritional quality 375-amino-acid precursor molecule that is further
(McMahon and Brown 1984). It has been estimated processed by enzymatic cleavage of the N-terminus
that by enhancing the casein content in milk by 20% prodomain segment. The remaining C terminus 109-
would result in an increase in cheese production, amino-acid segment is the myostatin protein (Gleizes
generating an additional $190 million/year for the et al. 1997). The processed protein forms dimmers
dairy industry (Wall et al. 1997). Dairy cattle have that are biologically active. McPherron and Lee
only one copy of the genes that encode α (s1/s2), β, (1997), through alignment of myostatin amino acid
and κ-casein proteins, and out of the four caseins, κ sequences from baboon, bovine, chicken, human,
and β are the most important (Bawden et al. 1994). murine, ovine, porcine, rat, turkey, and zebrafish,
Increased milk κ-casein content reduces the size of determined that the myostatin gene is highly
 3 Recent Advances 49

conserved among vertebrates, which suggests that

conserved among vertebrates, which suggests that its
function may be conserved as well. The researchers
also demonstrated that the increase in muscle mass
(double-muscling) phenotype observed in some
breeds of cattle, such as Belgian Blue and
Piedmontese, is due to mutations in the myostatin
gene. In myostatin-null mice that had the myostatin
gene knocked-out by gene targeting, individual
muscles weighted on average two to three times
more than those in wild-type mice, and body weight
was 30% higher. This difference was not due to an
increase in fat amount, but to an increase in the
cross-sectional area of the muscle fibers
(hypertrophy) and an increase in the number of
muscle fibers (hyperplasia) (McPherron and Lee
1997).
It is believed that myostatin prodomain may
bind non-covalently with the mature myostatin, Figure 3.10. Comparison between wild-type (right) and
transgenic mice (left) overexpressing Myostatin prodomain.
resulting in inhibition of the biological activity of (Courtesy of Dr. J. Yang, Univ. of Hawaii.)
myostatin (Thies et al. 2001). Based on the general
molecular model of TGF proteins, Yang et al. (2001)
hypothesized that overexpression of the prodomain times greater than the energy obtained from
segment would interfere with mature myostatin, carbohydrates and proteins, most of the energy that is
resulting in the promotion of muscle development. stored in the body is in the form of fat. Fats are a
The myostatin prodomain DNA was cloned into a group of chemical compounds that contain fatty
pMEX-NMCS2 vector, which contained a rat acids. The most common fatty acids found in animal
myosin light chain 1 (MLC1) promoter, a SV40 poly fats are palmitic acid, stearic acid, and oleic acid. The
adenylation sequence and a MLC enhancer. This human body is able to synthesize these fats, but there
construct was then inserted into mouse genome using is one more class of fatty acids called the essential
the pronuclear microinjection technique. In fatty acids (linolenic acid, linoleic acid, and
comparison to wild-type mice, overexpression of the arachidonic acid), which the body cannot produce;
myostatin prodomain in the transgenic mice resulted they therefore must be obtained from diet (Campbell
in a 17-30% increase in body weight; a 22-44% and Reece 2002). There are two main types of
increase in total carcass weight at 9 weeks of age naturally occurring fatty acids: saturated and
(Fig. 3.10), and a significant decrease in epididymal unsaturated. Saturated fatty acids (SFA) are mainly
fat pad weight, which is an indicator of body fat animal fats. They are called saturated because all the
mass (Yang et al. 2001). No undesirable phenotypes carbon chains are completely filled with hydrogen
or health or reproductive problems were observed in and there are no double bonds formed between the
the transgenic animals. These results indicated that carbon atoms. Saturated fatty acids are believed to be
this same approach can be used to develop farm “bad” fats since they raise both high-density
animals with enhanced growth performance, lipoproteins (HDL) and low-density lipoproteins
increased muscle mass, and decreased fat content, (LDL) cholesterol (Keys et al. 1965, NRC 1988).
which would equate to a healthier meat product for Unsaturated fats are found mainly in products derived
consumers (Yang et al. 2001). from plant sources and are divided into two
categories: monounsaturated fatty acids (MUFA),
REDUCED FAT CONTENT IN TRANSGENIC which have one double bond; and polyunsaturated
SWINE fatty acids (PUFA), which have two or more double
bonds in the carbon chain. It has been observed that
In human diet, ingested exogenous fats serve as the the increased consumption of these “good” fats
raw material for the synthesis of fat, cholesterol, and actually reduces LDL levels and enhances HDL
many phospholipids. Since fat energy content is two levels (Grundy 1986, NRC 1988). It is now well
50 Part I: Principles

established that a high fat diet (specially of SFA) not transgenic pigs expressing BGH had a significant
only increases the risk of heart disease but also the decrease in the levels of specific fatty acids
risk of breast, colon, and prostate cancer. Many compared to nontransgenic pigs in the control group
health agencies, including the American Dietetic (Fig. 3.11). These results indicate that consumers
Association, the American Diabetes Association, and might greatly benefit from a pork product with a low
the American Heart Association, recommend that fat fat content if regulation of BGH secretion levels can
intake should be no more than 30% of the total daily be precisely controlled during the fast growth stage
calories. of young pigs (Solomon et al. 1994).
In research conducted by the United State
Department of Agriculture (USDA), scientists TRANSGENIC POULTRY: EGG AS
introduced a recombinant bovine growth hormone BIOREACTOR
(rBGH) gene into pigs, with the purpose of
understanding the relationship between rBGH
expression and the amount of fatty acids in the Mammals and birds have been the focus of intense
animal (Solomon et al. 1994). Bovine growth research for their possible use as bioreactors. The use
hormone (BGH), also known as bovine of mammals as bioreactors became possible with the
somatotropin, which is produced in the pituitary creation of transgenic mice and the isolation of
gland, stimulates growth in immature cattle and tissue-specific promoters (Gordon et al. 1980, Swift
enhances milk production in lactating cows (Leury et et al. 1984). Clark et al. (1987) were the first to
al. 2003). BGH is a protein hormone, and as such, it propose the use of transgenic livestock mammary
is broken down during digestion in the glands for the production of biopharmaceutical
gastrointestinal tract, making it biologically inactive proteins in milk. Although expression of foreign
in humans (Etherton 1991). In 1993, based on protein in milk is high and milk production is large,
rigorous scientific investigations, the U.S. Food and there are some problems associated with the use of
Drug Administration (FDA) concluded that products mammary glands as bioreactors, including the long
from transgenic-BGH and supplemented-BGH time required to establish a stable line of transgenic
animals are safe for human consumption. founder animals and the high cost to purify foreign
Bovine growth hormone has been shown to protein from milk (Ivarie 2003). Researchers have
decrease fat content of transgenic pigs expressing a also long envisioned using chicken eggs for the
rBGH gene (Pursel et al. 1989). The transgenic pigs expression of exogenous proteins. There are many
used in this study were created by pronuclear advantages associated with the use of eggs as
microinjection technique. The gene encoding rBGH bioreactors, including the fact that a single ovalbumin
was introduced into the pig genome under the control gene, controls most of the proteins in egg white
of the mouse metallothionein-I (MT) promoter. After (Gilbert 1984). Also, egg white has relatively high
rBGH transgenic lines of pigs were established, protein content, is naturally sterile, and has a long
successive generations were produced by artificial shelf life (Tranter and Board 1982; Harvey et al.
insemination of nontransgenic females with sperms 2002).
collected from rBGH transgenic males. To determine
the effect of rBGH in the pigs’ carcass composition, Table 3.1. Comparison of Total Carcass Fat
transgenic and nontransgenic (control) pigs were (g/100g) between rBGH Transgenic and
raised under the same conditions and fed the same Control Pigs, Measured at Different Live
type of diet. The animals were processed at five Weights
different live weights: 14, 28, 48, 68, and 92 kg. The
entire left side of each carcass was ground, and Total Fat, g/100g
random samples of tissue collected and analyzed for Weight Group, kg Transgenic Control
fatty acid and cholesterol content. The researchers 14 6.19 10.04
observed that as live body weight increased, 28 7.62 12.32
carcasses from transgenic pigs showed a constant 48 8.16 16.58
decline in the amount of total fat compared to control 68 5.97 26.78
pigs (Table 3.1). Although the results did not 92 4.49 29.07
demonstrate a difference in the cholesterol content of Source: Adapted from Solomon et al. 1994.
transgenic and control pigs, it was shown that
 3 Recent Advances 51

100%

14kg
28kg
48kg
80%
68kg
92kg
% of Fatty Acids

60%

40%

20%

0%
SFA MUFA PUFA
Types of Fatty Acids

Figure 3.11. Fatty acids differences in the carcass composition of rBGH transgenic and wild-type pigs. (From Solomon et al.
1994.)

There is an already established infrastructure for the These results demonstrate that it is technically
production, harvesting, and processing of chicken possible to express and secret foreign proteins in the
eggs (Ivarie 2003). chicken’s egg, making it an attractive candidate for a
Recently, a group of researchers from the bioreactor. The main work that needs to be done with
biotech company AviGenics, Athens, Georgia, the chicken model is to develop more efficient non-
successfully introduced, expressed, and secreted a viral based methods for creation of transgenic
bacterial gene in the egg white of transgenic chicken chicken and to identify, isolate, and characterize
(Harvey et al. 2002). The transgene chosen was the gene enhancers and promoters that have high activity
E. coli β-lactamase (EC 3.5.2.6) reporter gene and drive tissue-specific expression of proteins in
because it is easily secreted and assayed from adult oviducts (Harvey et al. 2002).
eukaryotic cells. A replication-deficient retroviral
vector, named NLB, from the avian leucosis virus BIOENGINEERED FISH
(ALV) was used to express the transgene. The β-
lactamase coding sequence was inserted into the Out of all the transgenic, domesticated animals that
pNLB-CMV-BL viral vector and placed under the have been produced so far, fish are considered to be
control of the ubiquitous cytomegalovirus (CMV) the safest for human consumption and are expected to
promoter. The protein β-lactamase was found to be be the first transgenic animal to be approved as a
biologically active and was secreted in the blood and food item (Niiler 2000). The company AquaBounty
egg white, and its expression levels remained has an application under review with the FDA for the
constant across four generations of transgenic hens. commercialization of Atlantic salmon carrying a
52 Part I: Principles

growth hormone (GH) gene from Chinook salmon date, two types of these proteins that have been
(Zbikowska 2003). The main obstacle to be characterized are the antifreeze proteins (AFPs) and
overcome for the achievement of this goal is to better antifreeze glycoproteins (AFGs) (Davies and Sykes
understand the potential risks involved with the 1997). AFPs and AFGs lower the freezing
release of transgenic fish in the wild, and at this temperature of the fish’s serum and therefore protect
point, not enough research has been conducted to the fish from freezing by attaching themselves to the
answer these concerns (Muir and Howard 1999). ice surface, inhibiting ice crystal formation (DeVries
One of the options to avoid proliferation of 1984). There are four types of AFPs (I, II, III, IV)
transgenic fish in the wild is to sterilize all transgenic and at least one type of AFG identified at this time
fish, but a reliable method for 100% sterilization has (Davies and Hew 1990, Deng et al. 1997). Most
not yet been achieved (Razak et al. 1999). Some of aquaculture-important species of fish, such as the
the transgenic strategies that are being developed to Atlantic salmon and the tilapia, do not naturally
improve growth rate and increase antifreeze property produce any type of antifreeze proteins, therefore
are described below. they cannot survive and be raised in areas of the
world where water reaches sub-zero temperatures,
IMPROVING FISH GROWTH RATE making it a major problem for sea cage farming in
the northern Atlantic coast (Hew et al. 1995). The
The fish growth hormone gene has been cloned and production of commercially important transgenic
characterized from a number of fishes, including fish, especially salmon, that are freeze tolerant would
many salmon species (Du et al. 1992, Devlin et al. greatly expand the area for fish farms, increase
1994). Researchers from the University of productivity, and reduce price for consumers.
Southampton in the United Kingdom (Rahman et al. Flounder AFPs are small polypeptides that are
1998) developed transgenic tilapia fish part of the Type-I AFP, which has two different
(Oreochromis niloticus) transformed with growth isoforms, “skin-type” and “liver-type.” Skin-type
hormone genes of several salmonids. Rahman et al. AFPs are intracellular, mature proteins expressed in
used different types of constructs in their experiment, several peripheral tissues; liver-type AFPs are
but the one that gave the best results was the immature proteins that need to be further processed
construct with a Chinook salmon GH gene under the before being secreted into circulation and are found
control of the ocean pout antifreeze promoter. The mainly in the liver tissue (Hew et al. 1986, Gong et
method used for the insertion of the transgene al. 1996). Hew et al. (1999) used the liver-type AFP
construct was the cytoplasmic microinjection of gene from winter flounder to generate a transgenic
fertilized fish egg. The researchers reported the stable line of Atlantic salmon (Salmo salar) that
successful genomic integration of the construct in the demonstrated freeze tolerance capacity. By injecting
founder (G0) tilapia, and subsequent transfer of the the genes into the fertilized eggs, a single copy of the
transgene to G1 and G2 generations. Transgenic AFP gene was inserted and integrated in the salmon
tilapia expressing the transgene showed a growth rate chromosome, generating transgenic founder fish with
three times greater than the wild-type tilapia and had stable AFP expression and biologically active
a 33% higher food conversion ratio, which would protein. The same levels of expression and protein
reduce farmers’ production cost. This transgenic activity were observed up to three subsequent
tilapia also showed infertility at mature age, which is generations of transgenic salmon. Expression of AFP
a desirable trait for commercial transgenic fish was liver specific and demonstrated seasonal
(Rahman et al. 1998). variation similar to those in winter flounder, but the
levels of AFP in the blood of these fish were low
INCREASING ANTIFREEZE PROPERTY IN FISH (250 µg/ml) compared to natural AFP concentrations
in winter flounder (10-20 mg/ml) and therefore
Many species of fish, such as ocean pout
insufficient to provide freeze resistance to the salmon
(Macrozoarces americanus) and winter flounder
(Hew et al. 1999). The focus of current research has
(Pleuronectes americanus), that inhabit bellow
been to design gene constructs that will increase the
freezing water in the northern regions produce and
copy number of the transgene and therefore enhance
secret specific proteins in their plasma to protect their
expression levels of AFP in appropriate tissues,
body from freezing (Davies and Hew 1990). To this
 3 Recent Advances 53

conferring better antifreeze properties in farm fish. sake, Kitamoto et al. (1991) developed a transgenic
yeast strain in which the CAR1 gene is inactivated.
The researchers constructed the mutant yeast strain
BIOENGINEERED by introducing an ineffective CAR1 gene, flanked by
MICROORGANISMS DNA sequence homologous to regions of the
arginase gene. Through homologous recombination,
the ineffective gene was integrated into the active
For over 5000 years, mankind has, knowingly and CAR1 gene in the yeast chromosome, interrupting its
unknowingly, made use of spontaneous fermentation function (Fig. 3.12). As a result, urea was eliminated
of a variety of food items, which include bread, and ethylcarbamate was no long formed during sake
alcoholic beverages, dairy products, vegetable fermentation. This same procedure can be used to
products, and meat products. But it was more eliminate ethylcarbamate from other alcoholic
recently, just in the last century, that scientists beverages including wine (Kitamoto et al. 1991).
realized that the process of fermentation was done by
the action of microorganisms and that each
microorganism responsible for a specific food
processing could be isolated and identified. Now, INHIBITION OF PATHOGENIC BACTERIA
with advanced bioengineering techniques, it is
possible to characterize with high precision To increase the safety, hygiene and efficiency in the
important food strains, isolate and improve genes production of fermented foods, the use of starter and
involved in the process of fermentation, and transfer protective bacterial cultures is a common practice in
desirable traits between strains or even between the food industry today (Gardner et al. 2001). Starter
different organisms. culture is a liquid consisting of a blend of selected
microorganisms, used to start a commercial
fermentation. The difference between starter and
ELIMINATION OF CARCINOGENIC COMPOUNDS
protective cultures is that starter cultures give the
Brewer’s yeast (Saccharomyces cerevisiae) is one of food a desired aroma or texture, while protective
the most important and widely used microorganisms culture do not change the food property, but inhibit
in the food industry. This microorganism is cultured the growth of undesirable pathogenic
not only for the end products it synthesizes during microorganisms (Geisen and Holzapfel 1996). For
fermentation, but also for the cells and the cell the purpose of practicality during the food
components (Aldhous 1990). Today, yeast is mainly processing, the same microorganism should be used
used in the fermentation of bread and of alcoholic for both starter and protective cultures, but
beverages. Recombinant DNA technologies has unfortunately this is not always possible. Methods of
made possible to introduce new properties into yeast, genetically engineering help to improve available
as well as to eliminate undesirable by-products. One strains of microorganisms used in starter and
of the undesirable by-products formed during yeast protective cultures, so that new characteristics can be
fermentation of foods and beverages is added and undesirable properties eliminated (Hansen
ethylcarbamate or urethane, which is a potential 2002).
carcinogenic substance (Ough 1976). For this reason, Genetic engineering research aimed at
the alcoholic beverage industry has dedicated a large optimizing starter cultures is focused on three main
amount of its resources to funding research oriented goals: to enhance process stability; to increase
to the reduction of ethylcarbamate in its products efficiency; and to improve product safety (Geisen and
(Dequin 2001). Ethylcarbamate is synthesized by the Holzapfel 1996). During the production of some
spontaneous reaction between ethanol and urea, fermented food, such as mould-ripened cheese, pH
which is produced from the degradation of arginine, level rises in the culture due to lactic acid degradation
found in large amount in grapes. Yeasts, used in by fungal activity. This alkaline media offers an ideal
wine fermentation, possess the enzyme arginase that condition for the proliferation of food-borne
catalyzes degradation of arginine. If this enzyme can pathogenic microorganisms such as Listeria
be blocked, arginine will no longer be degraded into monocytogenes (Lewus and others 1991). The safety
urea, which in turn will not react with ethanol to of food products could be greatly improved by the use
form ethylcarbamate. In industrial yeast, the gene of starter cultures that can also serve as protective
CAR1 encodes the enzyme arginase (EC 3.5.3.1) cultures and inhibit the growth of such harmful
(Dequin 2001). To reduce the formation of urea in
54 Part I: Principles

Truncated
CAR1 gene

chr. chr.
DNA DNA
Target Vector
Homologous
Recombination

chr. CAR1 gene chr.

DNA DNA
Yeast
Chromosome

chr. chr.
DNA DNA Yeast with
disrupted
Inactive Integrated CAR1 gene
Inactive
Gene Vector Gene
Figure 3.12. Gene disruption by homologous recombination.

microorganisms. The enzyme lysozyme (EC flavor production and enhancement. Today, many of
3.2.1.17) can be an effective agent for the inhibition the techniques for flavoring food and beverages
of Listeria in food. Van de Guchte et al. (1992) make use of synthetic chemicals (Vanderhaegen et
integrated the gene responsible for lysozyme al. 2003). With increased public concern about the
formation in a strain of the bacterium Lactococcus danger of using synthetic chemicals, flavors
lactis. After genetic transformation, this bacterial produced by biological methods, also called
strain was able to express and secret lysozyme at bioflavors, are becoming more popular with
high levels. The researchers cloned lysozyme- consumers (Armstrong and Yamazaki 1986,
encoding genes from E. coli bacteriophages T4 and Cheetham 1993). The flavor and fragrance industry
lambda in wide-host-range vectors and expressed in is estimated worldwide at $10 billion per year; and
L. lactis. Biologically active lysozyme were although thousands of natural volatile and synthetic
produced and secreted by the transgenic L. lactis fragrances are known, only a few hundred are
strains, suggesting that these bacteria can be used regularly used and manufactured on an industrial
both as a starter and protective culture (Van de scale (Somogyi 1996). There are several methods for
Guchte et al. 1992). the production of bioflavors including (1) product
extraction from plant materials and (2) the use of
NATURAL SWEETENER PRODUCED BY specific bioengineered microorganisms for their
MICROORGANISMS biosynthesis. Biotechnological production of
bioflavors using microorganisms has certain
Techniques to enhance flavor in food have been advantages such as large-scale production with low
known for a long time, but only recently it has been cost, non-dependence on plant material, and
recognized that microorganisms can also be used in preservation of natural resources (Krings and Berger
1998).
 3 Recent Advances 55
Xylitol, also called wood sugar, is made from 1995). There is an increased interest in extracting
xylose, which is found in the cell walls of most land large amount of carotenoids from natural sources.
plants (Nigam and Singh 1995). Pure xylitol is a The 1999 world market for carotenoids was $800
white crystalline substance that looks and tastes like million, with projections for $1 billion in 2005
sugar, making it important for the food industry as a (Business Communications Co. 2000). Although
sweetener. One of the main advantages of xylitol researchers have found certain microalgae such as
over other sweeteners is that it can be used by Haematococcus pluvialis that produce high amounts
diabetic patients, since its utilization is not dependent of the carotenoid astaxanthin, extraction of
on insulin (Pepper and Olinger 1988). Xylitol is carotenoids from these microalgae is difficult
believed to reduce tooth decay rates by inhibiting because of their thick cell wall. For this reason,
Streptococcus mutans, the main bacteria responsible genetic engineering methods have been applied to
for cavities. Because xylitol is slowly absorbed and produce carotenoids in other microorganisms. The
only partially utilized in the human body, it contains edible yeast Candida utilis is a good candidate for
40% less calories than regular sugar and other commercial carotenoid production. It is a “generally
carbohydrates. In the United States, xylitol has been recognized as safe” (GRAS) organism, and large-
used since the 1960s, and it is approved as an scale production of peptides, such as glutathione, has
additive for foods with special dietary purposes already being successfully achieved in C. utilis (Boze
(Emodi 1978). Yeast (S. cerevisiae) is considered the et al. 1992).
ideal microorganism for commercial production of In microorganisms and plants, carotenoids are
xylitol from xylose because of its well-established synthesized from the precursor farnesyl
use in the fermentation industry. South African pyrophosphate (Fig. 3.13). Miura et al. (1998)
researchers (Govinden et al. 2001) isolated a xylose developed a de novo biosynthesis of the carotenoids
reductase (EC 1.1.1.21) gene (XYL1) from Candida
shehatae and introduced it into S. cerevisiae. The
XYL1 gene from Candida was cloned into the yeast FPP
expression vector pJC1, behind the PGK1 promoter, crtE
and the construct was transformed into yeast by
electroporation. Xylitol production from xylose by GGPP
the transformant was evaluated in the presence of crtB
different cosubstrates including glucose, galactose,
and maltose. The highest xylitol yield (15g/L from phytoene
50 g/L of xylose) was obtained with glucose as
crtI
cosubstrate.
lycopene
PRODUCTION OF CAROTENOIDS IN crtY
MICROORGANISMS
β-carotene
Carotenoids are structurally diverse pigments found
crtZ
in microorganisms and plants. These pigments have a crtW
variety of biological functions, such as coloration, β-cryptoxanthin echinenone
photo protection, light harvesting, and hormone crtZ crtW
production (Campbell and Reece 2002). Carotenoids
are used as food colorants, animal feed supplements, zeaxanthin canthaxanthin
and more recently, as nutraceuticals in the
crtW crtZ
pharmaceutical industry. Recent studies have
suggested many health benefits from the
consumption of carotenoids. Carotenoids such as adonixanthin phoenicoxanthin
astaxanthin, β-carotene, and lycopene have high anti- crtW crtZ
oxidant properties, which may protect against many
types of cancers, enhance the immune system, and astaxanthin
help relieve the pain and inflammation of arthritis Figure 3.13. Biosynthetic pathway of the carotenoids lycopene,
β-carotene, and astaxanthin.
(Miki 1991, Jyocouchi et al. 1991, Giovannucci et al.
56 Part I: Principles

crtE crtI crtB

A.

pCLR1EBI-3 (lycopene)

crtY crtI crtE crtB

B.

pCRAL10EBIY-3 (β-carotene)

crtZ crtE crtI crtY crtB crtW

C. D.

pCLEIZ1 (astaxanthin) pCLBWY1 (astaxanthin)

Figure 3.14. Lycopene, β-carotene, and astaxanthin plasmid constructs.

lycopene, β-carotene, and astaxanthin in C. utilis

using cloned bacterial genes that encode enzymes of
DETECTION METHODS IN FOOD
the biosynthetic pathway. They used four genes BIOTECHNOLOGY
(crtE, crtB, crtI, crtY) from Erwinia uredovora and
two genes (crtZ, crtW) from Agrobacterium Biotechnology plays an important role in maintaining
aurantiacum for construction of four different the safety of the food supply. The development of
plasmids. The plasmid pCLR1EBI-3 contained crtE, reliable methods to ensure traceability of genetic
crtI, and crtB, required for the production of material in the food chain is of great value to food
lycopene (Fig. 3.14a). For synthesis of β-carotene, manufacturers and consumers. Consumer confidence
plasmid pCRAL10EBIY-3 contained the genes crtY, in food biotechnology will increase if better
crtI, crtE, and crtB (Fig. 3.14b). A dual plasmid traceability methods are in place. Modern
system with pCLEIZ1 containing crtE, crtI, and crtZ biotechnology tools are also applied to develop
and pCLBWY1 containing crtW, crtY, and crtB, was sensitive, reliable, fast, and inexpensive methods for
used to produce astaxanthin (Fig. 3.14c,d). In order to detection of harmful pathogenic organisms such as E.
integrate these genes into yeast chromosome, the coli O157:H7 and the infectious agent for mad cow
plasmids were linearized by restriction digest and disease.
transformed into C. utilis by electroporation. The
resultant transgenic yeast produced significant TRANSGENE DETECTION
amounts of lycopene (1.1 mg/g dry weight), β-
carotene (0.4 mg/g dry weight), and astaxanthin (0.4 Accomplishments in food biotechnology require
mg/g dry weight), in quantity similar to amounts continuous development of new products and their
found in microorganisms that naturally produce these successful commercialization through consumer
carotenoids (Miura et al. 1998). These results indicate acceptance. One of the greatest demands made by
that C. utilis has a great potential for use in large- consumer groups as a prerequisite for their support of
scale production of commercially important transgenic plant use, is the development of reliable
carotenoids. methods of detection of the transgene in human food
products (James 2001). But as the number of
genetically modified organisms (GMOs) approved
 3 Recent Advances 57

for cultivation and commercialization grows, there is information is encoded will be cloned between
also an increased risk of transgenic material conserved sequences that contain primer-binding
contamination in nontransgenic food products. One domains. To read the DNA-encoded information,
such well-publicized event took place in October one only needs to perform PCR and sequence the
2000, when Safeway and Taco Bell recalled corn fragment.
products because they were contaminated with small Another PCR-based method for GMO detection
amounts of genetically engineered corn. For this and involves the use of unique genomic sequences
other reasons, the future success and acceptance of flanking the transgene. Hernandez et al. (2003),
GMOs will depend on mechanisms for containment working with Monsanto’s transgenic maize line
and proper detection of transgenic material. Among MON810, which contains a gene encoding for the
different detection methods of transgenic material insecticide CryIA(b) endotoxin, identified a genomic
that are in use today, real-time quantitative PCR is sequence adjacent to the 3´-integration site of the
the most powerful, accessible, and cost efficient transgenic plant by using a thermal asymmetric
(Higuchi et al. 1992). The main concern for the interlaced (TAIL)-PCR approach. PCR amplification
implementation of reliable detection methods is to of target DNA and real-time PCR product
determine what type of unique gene sequence should quantification are the two most used techniques for
be amplified during the PCR screening. Signature accurate DNA quantification. Real-time quantitative
sequences, such as antibiotic resistance markers and PCR can be used with different quantitative tools
promoters, are the main elements used today for such as DNA-binding dyes (Morrison et al. 1998),
detection of GMOs, but they are not ideal since the fluorescent oligonucleotides (Whitcombe et al.
same signature sequences can be found in more than 1999), molecular beacons (Tyagi and Kramer 1996),
one type of GMO. Also, there is an unproven fluorescence resonance energy transfer (FRET)
concern that these signature sequences, especially probes (Wittwer et al. 1997), and TaqMan probes
antibiotic resistance markers, may cause health and (Heid et al. 1996). The main advantage of the
environmental problems. To address this concern, TaqMan system is that it is highly specific because it
the European Union, which has adopted stringent uses three oligonucleotides in the PCR reaction. This
regulation on GMOs, banned the use of antibiotic detection system consists of two primers that are
gene as markers for transformation selection, by the responsible for product amplification, and the
year 2004. The European Union also established TaqMan probe, a fluorogenic oligonucleotide, that
mandatory labeling of GMO foods with a 1% will anneal to the product. During amplification, Taq
threshold level for the presence of transgenic polymerase releases a 5’ fluorescent tag from the
material, which in turn encouraged more aggressive annealed TaqMan probe, which gives off a
research on highly specific, precise, and sensitive quantifiable fluorescence light. Higher light intensity
methods for detection and quantification of GMOs in translates into a greater amount of target gene
food products (European Commission 2000). present in the food sample.
Researchers for the German company Icon
Genetics developed a novel idea for universal
identification of GMOs (Marillonet et al. 2003). FOOD PATHOGEN DETECTION
They proposed the creation of a standardized
procedure in which nontranscribed DNA-based Food poisoning may occur due to contamination of
technical information can be added to the transgene food by certain toxin-producing bacteria such as
before it is inserted in the organism’s genome. This Salmonella, Vibrio, Listeria, and E. coli. The strain
artificial coding would be based on nucleotide O157:H7 is the deadliest among all E. coli strains; it
triplets, just like amino acids codons, and each triplet produces toxins called Shiga, which are encoded by
would encode for one of the 26 Latin alphabetic two genes, stx1 and stx2. Shiga toxins (Stx1 and
letters, an Arabic numeral from 0 to 9, and one space Stx2) damage the lining of the large intestine, causing
character, giving a total of 37 characters (Table 3.2) severe diarrhea and dehydration; and if absorbed into
(Marillonet et al. 2003). With these characters, the the bloodstream, the toxins can harm other organs
researchers could insert biologically neutral, non- such as the kidney (Riley et al. 1983). In North
genetic coding sequences that translate into unique America, the O157:H7 strain is found mostly in the
information such as the name of the company, intestines of healthy cattle. The Center for Disease
production date, place of production, product model, Control (CDC) estimated that Shiga toxin-producing
and serial number. The variable region where the E. coli (STEC), such as O157:H7, causes 73,480
58 Part I: Principles

Table 3.2. Artificial Triplet Codons Encoding for Specific Alphabetical and Numeric
Characters, Used in the Identification of GM Organisms

Characters Codons Characters Codons

1 TTA CCT H CAC AGA

2 TTG CCC I CAA AGG

3 CTT ACC J GTC
4 CTC ACA K GTA
5 CTA ACG L CAG AGT
6 CTG ACT M GTG
7 ATT GCA N AAT GGA
8 ATC GCG O AAC GGG
9 ATA GCT P TCT
0 TTC CCG GAG Q TCC
space TTT CCA AAG R AAA GGT
S GAT TGC
A TAT CGA T GAC TGA
B ATG U GAA TGG
C TAC CGG V TCA
D TAA CGT W TCG
E TAG CGC X GCC
F GTT Y TGT
G CAT AGC Z GGC
Source: Adapted from Marillonnet et al. 2003.

illness and 61 deaths per year in the United States Maryland, and from the Center for Food Safety and
alone and that 85% of these cases are attributed to Applied Nutrition, Food and Drug Administration,
foodborne transmission, especially from ground beef, Washington, D.C., developed a simple, rapid, large-
unpasteurized milk, and roast beef (Mead et al. scale method for the analysis of PCR products with
1999). As reports of E. coli O157:H7 outbreaks the use of enzyme-linked immunosorbent assay
become more common, greater effort has been made (ELISA) (Ge et al. 2002). This PCR-ELISA approach
for the development of fast and reliable methods for for the detection of E. coli O157:H7 and other STEC
its detection. PCR has become the method of choice in food was based on the incorporation of
for pathogen detection because, contrary to culture digoxigenin-labeled dUTP and a biotin-labeled
isolation and serological tests, PCR methods provide primer specific for stx1 and stx2 genes during PCR
fast, accurate, and highly sensitive results. Among amplification. In this method, the biotin-labeled PCR
the genes currently used as target for PCR products were bound to microtiter plate wells coated
amplification of O157:H7 are Shiga toxins (stx) with streptavidin and then detected by ELISA using
(Brian et al. 1992), intimin (Gannon et al. 1993), an anti-DIG-peroxidase conjugate (Fig. 3.15). To
enterohemorrhagic E. coli hemolysin (Hall et al. establish the specificity of the primers used in this
1998), and β-glucuronidase (EC 3.2.1.31) (Feng PCR method, 39 different bacterial strains including
1993). In traditional PCR methods for detection of STEC and non-STEC strains, such as Salmonella,
pathogens, which include gel electrophoresis, the were used. All of the STEC strains were positive and
number of samples that can be analyzed during one all non-STEC organisms were negative. The
electrophoresis run is very small. For this reason, researchers observed that in comparison with the
researchers from the Department of Nutrition and traditional gel electrophoresis with ethidium bromide
Food Science, University of Maryland, College Park, staining method, the ELISA system enhanced the
 Biotin-labeled
primer
A.
B

B
PCR reaction containing
Biotin-labeled
DIG-labeled dUTP primer

DIG DIG DIG

B. U U U
B

B
U U
DIG DIG

Biotin-labeled PCR product binds to

streptavidin on the microtiter plate

DIG

streptavidin U
C. B
microtiter plate

anti-digoxigenin-enzyme
E
Plate is exposed to anti-digoxigenin-
enzyme conjugate, which binds to DIG-
labeled dUTP
DIG

streptavidin U
D. B
microtiter plate

anti-digoxigenin-enzyme
E
Substrate is added to the plate, and
enzyme converts substrate into light-
DIG emitting product

streptavidin U
B
E. microtiter plate

Figure 3.15. Diagram of digoxygenin-ELISA (DIG-ELISA) method.

 60 Part I: Principles

sensitivity of the PCR assay by up to 100-fold (Ge et Scientists believe that BSE can spread among
al. 2002). In the future, the use of robotic equipment cattle through contaminated feed containing the
will result in automation of the PCR-ELISA disease-inducing form of prion. It is a common
procedure, allowing for fast, sensitive, accurate, and practice in many countries to feed cattle with the
large-scale screening of microorganisms that produce remains of other farm animals as a source of protein.
the Shiga toxins. This method also can be applied for The hypothesis for the spread of BSE in cattle is that
detection of any other food pathogens, using specific body parts of sheep infected with scrapie were
biotin-labeled PCR primers. included in the feed and PrPSc jumped species to
infect bovines (Bruce et al. 1997). The disease
spread throughout the world when England sold
BSE-contaminated cattle feed to other countries.
BOVINE SPONGIFORM ENCEPHALOPATHY Since it first appeared in 1986, the risk of BSE
DETECTION infection resulted in the destruction of 3.7 million
animals in the United Kingdom. In humans, CJD is
The transmissible spongiform encephalopathies an inherited disease caused by a mutation in the
(TSEs), are a group of neurodegenerative diseases prion protein gene, PRNP, and affects one in a
affecting many animals, including humans, and are million people. It is believed that the human victims
characterized by the formation of microscopic may have contracted a new variant of CJD (nv-CJD)
“holes” in the brain tissue. Members of the TSE from eating meat products contaminated with BSE.
family include (1) the diseases that afflict humans: The pathogenic prion protein that causes BSE is
kuru, Gertsmann-Sträussler-Scheinker (GSS), fatal nearly identical to the prion that causes nv-CJD
familial insomnia (FFI), and Creutzfeldt-Jakob (Johnson and Gibbs 1998). Between 1996 and 2003,
disease (CJD); and (2) the diseases that afflict 156 cases of nv-CJD have been suspected or
animals: scrapie (sheep and goats), wasting disease confirmed in many countries, mainly in Great
(elk and deer), mink encephalopathy (mink), feline Britain.
spongiform encephalopathy (cats), and bovine One of the main problems in dealing and
spongiform encephalopathy (BSE) (Prusiner 1998). containing the BSE epidemic is that there are no tests
Bovine spongiform encephalopathy affects cattle and for detection of the disease in a live animal. Prions
is commonly known as mad cow disease. The do not trigger any detectable specific immune
symptoms associated with BSE are weight loss, response and levels of abnormal prions in other parts
drooling, head waving, aggressive behavior, and of the body, such as the blood, are too low to detect.
eventually death. All TSEs, including BSE, are The only means of diagnosis in live animals is by
caused by a new infectious agent called a “prion observing BSE symptoms in the animal. Because of
protein” (PrP) (Prusiner 1982). Dr. Stanley Prusiner the lack of reliable live-detection methods, all
discovered prions in 1984 and was awarded the 1997 animals in a herd that may have been in contact with
Medicine Nobel Prize for his research. Prions are BSE-contaminated feed must be destroyed, causing
endogenous glycoproteins found abundantly in the great losses for the cattle industry.
brain tissue of all mammals and may function as a The primary laboratory method used to confirm
neuron helper. They can manifest in two different a diagnosis of BSE is the microscopic examination
protein conformations: (1) PrPc, which is the normal of brain tissue after death of the animal. In addition
form, non-pathogenic, protease sensitive and high in to microscopic examination, there are several
α-helical content, and (2) PrPSc, which is the techniques used to detect the PrPSc, among which,
misfolded form, disease-inducing, protease resistant, Western blot test and immunocytochemistry
and high in β-sheet content. PrPSc has infection (developed by Prionics AG, Switzerland) are the
properties and when it comes in contact with PrPc, most commonly used (Kübler et al. 2003). Other
starts a chain reaction, transforming PrPc into PrPSc currently approved BSE tests include (1) a test
(Horiuchi et al. 1999). Prions are not completely developed by CEA, a research group in France, in
destroyed by sterilization, autoclaving, disinfectants, which a sandwich immunoassay technique for PrPSc
radiation, or cooking. They are totally degraded only is done following denaturation and concentration
with incineration at temperatures greater than steps and (2) a test developed by Enfer Scientific, in
1000ºC or treatment with strong sodium hydroxide Ireland, in which polyclonal antibodies and an
solutions (Dormont 1999). enzyme-coupled secondary antibody are used, and
 3 Recent Advances 61

detection is done by ELISA-chemiluminescence producers. The creation of pathogen-, insect-, and

(Moynagh and Schimmel 2000). Although these tests herbicide-resistant transgenic plants have led to
are 100% reliable, they can only be done in post- increased productivity and decreased costs of
mortem brain tissue. producing many food crops. Similarly, the
To improve the sensitivity of current detection introduction of foreign genes in transgenic salmon
methods and enable live-animal BSE detection, enabled it to grow three times faster then wild-type
intense research has been done in developing assays salmon. The benefits of the next generation of
that take advantage of the infectious capacity of the biotechnology research will be directed towards the
PrPSc to convert the normal prion protein into consumers and will entail the creation of designer
pathogenic ones. A team of researchers from Serono foods with enhanced characteristics such as better
Pharmaceutical Research Institute in Geneva nutrition, taste, quality, and safety. A good example
cultured mutated prions in vitro for the first time and of such research is the use of biotechnology to
have devised a method to replicate them to high decrease the amount of saturated fatty acids in
enough levels to detect the disease at an earlier stage vegetable oils.
(Saborio et al. 2001). The technique, called protein- Among the many possibilities available in the
misfolding cyclic amplification (PMCA), is similar field of food biotechnology, future research in the
to a PCR reaction, in which amplification of small area of plant genetic engineering is aimed at
amounts of the pathogenic protein in a sample is increasing the shelf life of fresh fruits and
achieved through multiple cycles of PrPSc vegetables, creating plants that produce sweet
incubation in the presence of excess PrPc, followed proteins, developing caffeine-free coffee and tea, and
by disruption of the aggregates through sonication in improving the flavor of fruits and vegetables. Great
the presence of detergents (Fig 3.16). This new progress has been achieved in using plants as a
technique has the potential to improve BSE and other bioreactor for manufacturing and as a delivery
TSEs detection methods, to give a better system for vaccines. A variety of crops such as
understanding of prion diseases, and to help in the tobacco, potato, tomato, and banana have been used
search of drug targets for brain diseases. More to produce experimental vaccines against infectious
recently, researchers from Chronix Biomedical in diseases. Advances in genetic engineering will
San Francisco and the Institute of Veterinary enable the creation of transgenic plants that produce
Medicine in Göttingen, Germany, claim to have proteins essential for the production of
developed the first BSE test that can be performed pharmaceuticals, such as growth hormones, antigens,
on live animals (Urnovitz 2003). This detection antibodies, enzymes, collagen, and blood proteins.
method, called the surrogate marker living test for The applications of genetic engineering in the
BSE, is a real-time PCR test based on in vitro food industry are not limited only to the
detection of specific RNA in the bovine serum. It manipulation of plant genome. Animals and
uses blood samples from cows for the identification microorganisms also have been extensively
of a microvesicle RNA and it is considered a researched to produce better food products. In the
surrogate marker test because it detects blood RNA area of animal bioengineering, the main focus has
and not prion proteins. RNA is found in the blood been on the use of mammary gland and egg as
fraction that contains primarily microvesicles and bioreactors in the manufacturing of
while microvesicles are found in both healthy and biopharmaceuticals. So far, the focus of animal
diseased animals, their RNA contents appear to be bioengineering research has been directed toward the
different. Cattle that show reactivity to this test benefit of the producers. In the future, transgenic
should be subjected to a second more specific test for animal research will be shifted towards the
conclusive diagnosis of mad cow disease (Urnovitz production of healthier meat products, with
2003). decreased amounts of fat and cholesterol.
Microorganisms, especially yeast, have been
vital to the food processing industry for centuries.
Among their many qualities, yeast plays an
CONCLUSION important role in the production of fermented foods,
enzymes, and proteins. Future research on genetic
Research on transgenic organisms for the food engineering of microorganisms may be focused on
industry has been intended mostly to benefit the the large-scale production of biologically active
 62 Part I: Principles

sonication

PrPc PrPSc old new

PrPSc PrPSc

incubation conversion PrPSc

+ aggregates

amplification

PrPSc
PrPc

incubation

+
sonication conversion

new and Growing number of

old PrPSc pathogenic protein (PrPSc)

amplification

Figure 3.16. Diagram of protein-misfolding cyclic amplification (PMCA). (From Saborio et al. 2001.)

components that provide health benefits, but are the scope of the use of microorganism in the food and
found in low quantities in plants. Among such pharmaceutical industry.
components that have anticancer properties include Besides the production of novel compounds and
lycopenes found in tomato, glucosinolates found in improvement of existing ones, biotechnology plays
broccoli, ellagic acids found in strawberry, and an important role in food safety. Microbial
isoflavonoids found in soybean. Further contamination is a major concern in the food
biotechnology advances will enhance the value and industry. New biotechnology methods are being
 3 Recent Advances 63

developed to help decrease the amount of microbes evolution of ferritins. J Inorg Biochem 47:161-
on animal and plant products and thereby improve 174.
the safety of the food supply. In addition, Armstrong DW, Yamazaki H. 1986. Natural flavours
biotechnology is providing many tools to detect production: a biotechnological approach. Trends
microorganisms and their toxins. Some new and old Biotechnol 4:264-268.
detection methods such as ELISA tests, polymerase Armstrong GA. 1994. Eubacteria show their true
chain reaction (PCR), protein-misfolding cyclic colors; Genetics of carotenoid pigment
amplification (PMCA), DNA probes, and biosensors biosynthesis from microbes to plants. J Bacteriol
have been developed to detect the presence of 176:4795-4802.
infectious pathogenic agents such as bacteria, virus, Bartley GE, Scolnik PA, Giuliano G. 1994.
fungi, and prions. A recent improvement in the Molecular biology of carotenoid biosynthesis in
detection method is the development of the real-time plants. Ann. Rev. Plant Physiol. Plant Mol Biol
PCR technology, which provides a sensitive and 45:287-301.
more reliable tool for identification of pathogens in Basta SS, Soekirman, Karyadi D, Scrimshaw NS.
food products. Another way by which biotechnology 1979. Iron deficiency anemia and the productivity
helps to improve the safety of food products for of adult males in Indonesia. Am J Clin Nutr
human consumption is through rapid identification 32:916-925.
and extraction of allergenic proteins in food items Bawden WS, Passey, RJ, MacKinlay, AG. 1994. The
such as shellfishes, peanuts, and soybeans. genes encoding the major milk-specific proteins
Despite the benefits provided by modern and their use in transgenic studies and protein
biotechnology, this relatively new science is not yet engineering. Biotechnol. Genet Eng Rev 12:89.
fully embraced by the general population. There are Beard JL, Burton JW, Theil EC. 1996. Purified
still many issues of safety, reliability, and efficacy ferritin and soybean meal can be sources of iron
that must be overcome before scientists can for treating iron deficiency in rats. J Nutr 126:154-
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time, there is a very strong polarization on the issues Boze H, Moulin G, Glazy P. 1992. Production of
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food products to all people at a more affordable Brophy B, Smolenski G, Wheeler T, Wells D,
price. L'Huillier P, Laible G. 2003. Cloned transgenic
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