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Biopesticide Production from Bacillus thuringiensis: An Environmentally

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28 Recent Patents on Biotechnology 2009, 3, 28-36

Biopesticide Production from Bacillus thuringiensis: An Environmentally

Friendly Alternative
Ninfa M. Rosas-García*
Laboratorio de Biotecnología Ambiental. Centro de Biotecnología Genómica-IPN. Blvd. del Maestro s/n. Reynosa,
Tamp. CP 88710 México

Received: October 29, 2008; Accepted: November 26, 2008; Revised: November 28, 2008
Abstract: Since its discovery as a microbial insecticide, Bacillus thuringiensis has been widely used to control insect
pests important in agriculture, forestry, and medicine. The wide variety of formulations based on spore-crystal complexes
intended for ingestion by target insects, are the result of many years of research. The development of a great variety of
matrices for support of the spore-crystal complex enables many improvements, such as an increase in toxic activity,
higher palatability to insects, or longer shelf lives. These matrices use many chemical, vegetable or animal compounds to
foster contact between crystals and insect midguts, without harming humans or the environment.
Biotechnology companies are tasked with the production of these kinds of bioinsecticides. These companies must not only
provide formulations tailored to specific crops and the insect pests, but they must also search for and produce
bioinsecticides based on new strains of high potency, whether wild or genetically improved. It is expected that new
products will appear on the market soon, providing an increased activity spectrum and applicability to many other pest-
impacted crops. These products may help develop a more organic agriculture. This review article discusses recent patents
related to bioinsecticides.
Keywords: Bioinsecticides, formulations, insect pests, biological control, encapsulation, polymers, matrices, insecticidal
preparations, delta-endotoxin, cry genes, insecticidal crystal, toxic activity, lepidopterans, coleopterans, dipterans.

INTRODUCTION growing seasons to attack all the developmental stages of

insect pests [6]. The indiscriminate use of these compounds
The bacterium Bacillus thuringiensis was discovered by caused, by 1950, a resurgence of pests, due to the
Shigetane Ishiwata in 1901 [1], and rediscovered by Berliner
elimination of their natural enemies and the appearance of
ten years later. The bacterium was isolated from diseased
pest populations showing resistance to insecticides. Also
larvae of Anagasta kuehniella, and this finding led to the
serious environmental and health issues began to be
establishment of B. thuringiensis as microbial insecticide.
recognized by the presence of chemical residues in food,
The first record of its application to control insects was in water, and air. To counteract this contamination, attention
Hungary at the end of 1920, and in Yugoslavia at the and efforts were directed to the use of biological control
beginning of 1930s, it was applied to control the European agents including insect pathogens [7]. However, an
corn borer [2]. During the following two decades, several entomopathogenic organism must fulfill several requisites
field tests were conducted to evaluate its effectiveness before being released to the environment as a potential
against lepidopterans, both in Europe and in the United control agent. It should be highly specific and effective
States [3], and results favored the development of against the target pest. The organism should demonstrate the
formulations against on this pathogen. Subsequently, the first potential to be successfully processed by continuous
commercial product was produced in 1938 by Libec in production technology. The control agent should be available
France. [4]. Unfortunately, the product was used only for a in formulations with a reasonable shelf life, should be stable,
very short time, due to World War II [5]. and should be harmless to human and non-target flora and
fauna [8]. As an entomopathogenic organism, B.
After World War II, the Green Revolution provided great
agricultural advantages via the use of agrochemicals, chemi- thuringiensis fulfills all these requirements.
cal fertilizers, highly productive cultivars, and mechani- Bacillus thuringiensis is a gram-positive spore-forming
zation. The result was a considerable decrease in a great bacterium that produces crystalline proteins called delta-
variety of insect populations, and as a consequence, synthetic endotoxins during its stationary phase of growth [9]. The
insecticidal compounds became popular due to the long crystal is released to the environment after lysis of the cell
residual action and the wide toxicity spectrum. However, wall at the end of sporulation, and it can account for 20 to
fully synthetic chemical insecticides appeared in 1940, when 30% of the dry weight of the sporulated cells [9] Fig. (1).
organochlorinated and organophosphate insecticides were This bacterium is distributed worldwide. The soil has been
discovered. These insecticides were applied during all described as its main habitat; however it has also been
isolated from foliage, water, storage grains, and dead insects,
etc. Isolation of strains from dead insects has been the main
*Address correspondence to this author at the Laboratorio de Biotecnología
Ambiental. Centro de Biotecnología Genómica-IPN. Blvd. del Maestro s/n. source for commercially used varieties, which include
Reynosa, Tamp. CP 88710 México; Tel: 52-899-924-3627; Ext: 7721; Fax:
52-899-925-2889; E-mail: [email protected]; [email protected]

1872-2083/09 $100.00+.00 © 2009 Bentham Science Publishers Ltd.

B. thuringiensis Based-Biopesticides Recent Patents on Biotechnology 2009, Vol. 3, No. 1 29

toxin inserts itself into the membrane. During the into-

xication process, in lepidopterans as in coleopterans, many
histopathological changes have been described, including
swelling and disruption of the microvilli, vacuolization of
the cytoplasm, hypertrophy of epithelial cells and necrosis of
the nuclei [22-24].
TARGET ORGANISMS
In the past decades, B. thuringiensis Cry toxins were
classified according to the target pest they attacked [14];
however, due to the dual toxic activity exhibited by some cry
genes and the inconsistencies in the original classification
proposed by Höfte and Whiteley [14], Crickmore et al. in
1998 [25] proposed a revision of the nomenclature for
insecticidal crystal proteins, based on the ability of a crystal
protein to exhibit some experimentally verifiable toxic effect
in a target organism, or any protein that has obvious
Fig. (1). Scanning electron micrograph of spores (S) and crystals of sequence similarity to a known Cry protein. The diversity of
Bacillus thuringiensis HD-125 strain. The strain synthesizes B. thuringiensis is demonstrated in the almost 70 serotypes
bypiramidal (B) and square (Sq) crystals. (The strain was kindly and the 92 subspecies described to date [26]. A full list of
donated by the Bacillus Genetic Stock Center, Ohio, USA and is delta-endotoxins, including names and source strains can be
coded as 4L1). The photograph is provided by N.M. Rosas-García. found at http://www.lifesci.sussex.ac.uk/home/Neil_
Crickmore/Bt/toxins2.html
kurstaki, isolated from A. kuehniella; israelensis, isolated The biological activity of B. thuringiensis strains or their
from mosquitoes, and tenebrionis, isolated from Tenebrio
products toward different target organisms has been subject
monitor larvae [10,11].
to patent coverage for many years. Many of these patents
MODE OF ACTION belong to companies engaged in commercial endeavors,
while others remain as a part of the basic research domain.
The Cry proteins comprise at least 50 subgroups with
more than 200 members [12]. The members belong to a It is well documented that many insects are susceptible to
three-domain family, and the larger group of Cry proteins is the toxic activity of B. thuringiensis; among them,
globular molecules with three structural domains connected lepidopterans have been exceptionally well studied, and
by single linkers. The protoxins are characteristic of this many toxins have shown activity against them [27-33].
family and have two different lengths. The C-terminal Order Lepidoptera encompasses the majority of susceptible
extension found in the long protoxins is necessary for species belonging to agriculturally important families such
toxicity and is believed to play a role in the formation of the as Cossidae, Gelechiidae, Lymantriidae, Noctuidae, Pieridae,
crystal within the bacterium [13]. Their mode of action Pyralidae, Thaumetopoetidae, Tortricidae, and Yponomeu-
involves several events that must be completed several hours tidae [11]. Recently, four novel B. thuringiensis strains,
after ingestion in order to lead to insect death. Following which are deposited at the BCCM-LMG under accession
ingestion, the crystals are solubilized by the alkaline nos. LMG P-12592, LMG P-12593, LMG P-12594, and
conditions in the insect midgut and are subsequently LMG P-13493, have been found to produce new crystal
proteolytically converted into a toxic core fragment [14]. proteins during sporulation that are toxic to Lepidoptera,
During proteolytic activation, peptides from the N terminus more particularly to Noctuidae such as Spodoptera spp. and
and C terminus are cleaved from the full protein. Activated Agrotis ipsilon, to Pyralidae such as Ostrinia nubilalis, and
toxin binds to receptors located on the apical microvillus to Yponomeutidae such as Plutella xylostella. In addition,
membranes of epithelial midgut cells. For Cry1A toxins, at the genes that encode these novel proteins have also been
least four different binding sites have been described in studied [34].
different lepidopteran insects: a cadherin-like protein Abad et al. [35] have patented an invention that describes
(CADR), a glycosylphosphatidyl-inositol (GPI)-anchored nucleic acids, variants and fragments from B. thuringiensis
aminopeptidase-N (APN), a GPI-anchored alkaline strains that encode polypeptides having insecticidal activity
phosphatase (ALP) and a 270 kDa glycoconjugate [15-19]. against lepidopterans. This invention has been applied in
After binding, toxin adopts a conformation allowing its methods for controlling plant pests. Baum et al. [36] also
insertion into the cell membrane. Subsequently, oligomeri- reported B. thuringiensis strains that produce novel crystal
zation occurs, and this oligomer forms a pore or ion channel proteins exhibiting insecticidal activity against lepidopterans,
induced by an increase in cationic permeability within the as well as a method for using and making transgenic cells. A
functional receptors contained on the brush borders typical example of B. thuringiensis activity can be observed
membranes [20]. This allows the free flux of ions and in Fig. (2).
liquids, causing disruption of membrane transport and cell
lysis, and leading to insect death [14]. The complete nature Dipterans are also important target pests, and many of
of this process still remains unknown [21]; however, it is them are highly susceptible to B. thuringiensis. This order
believed that toxin aggregation occurs at the membrane includes the families Tephritidae, Culicidae, Muscidae,
surface after receptor binding, or alternatively only after the
30 Recent Patents on Biotechnology 2009, Vol. 3, No. 1 Ninfa M. Rosas-García

leafhoopers, and possibly other sucking insects of agronomic

importance, extended the potential applications of this
bacterium. The peptide sequences of these proteins were also
patented [42]. However, the novel toxic activities found in
this noble bacterium are not limited only to insects, as some
novel strains produce crystals with activity against
nematodes, protozoans, flukes, collembolans, mites, and
worms, among others [11].
For example, a new isolate of B. thuringiensis expressing
a toxin with nematicidal activity was found. The amino acid
Fig. (2). Photograph shows lepidopteran larvae susceptible to B. sequence of the toxin and the nucleotide sequence encoding
thuringiensis toxic activity. A) Nine day-old, healthy larvae, not the toxin were determined. With this new isolate, some
exposed to B. thuringiensis. B) Nine-day old larvae exposed to nematodes such as Haemonchus, Nematodirus, soil nema-
sublethal concentration of B. thuringiensis. Larvae exhibit a smaller todes and vegetable parasites can be controlled through the
size due to their poor feeding. C) Larvae exposed to lethal use of the toxin [43]. Another toxin with nematicidal activity
concentration of B. thuringiensis. The main characteristic after is a truncated form of the Cry6A protein. Although this toxin
death is the black color of the larvae. The photographs are provided exhibited particular toxic activity against the corn rootworm,
by N.M. Rosas-García. a coleopteran, the truncated toxin also proved useful for
nematode control. Specifically, truncated Cry6A, with a
Simuliidae, and Tipulidae. Lysyk et al. [37] developed a molecular mass about 40-50 kDa, is produced from genes
method for controlling insects of this order by application of that encode for the highly active truncated protein toxin, and
B. thuringiensis strain LRC3 or its products. This strain it lacks of amino acid sequences removed from the N-
contains a plasmid carrying genes which encode delta- terminus and the C-terminus [44].
endotoxins Cry1A, Cry1B, Cry1F, Cry1H, Cry1I, Cry1K,
Protozoans have also been discovered as target organisms
and Cry2. The strain is deposited as ATCC PTA-6248. The
susceptible to Cry toxins. The protein toxin produced by a
same authors also revealed methods for preparing the strain,
novel isolate designated PS81F exhibited killing capabilities,
spores, crystals, and mutants.
allowing it to be used to treat humans and animals hosting
Coleopterans are important pests in agriculture and parasitic protozoans. In addition, the gene encoding the toxin
forestry. Several families such as Chrysomelidae, Curculio- can be transferred to a suitable host via a biological vector,
nidae, Tenebrionidae, and Scarabeidae have recently been such as a plasmid or virus [45].
found to be susceptible to toxic activity of the crystals. Some
Furthermore, a pharmaceutical product for use in treating
DNA sequences derived from B. thuringiensis strains encode
a mammalian host infected with a protozoan organism was
for delta-endotoxins with pesticidal activity against these manufactured with the same strain PS81F for control of
pests. These sequences were further mutagenized to improve
Giardia, Toxoplasma, Frankelia, Hammondia, Isospora,
pesticidal activity or altered pest specificity when applied to
Besnoita, Eimeria, Entamoeba, Trichomonas, and Penta-
plants for the control of insect pests [38].
trichomonas [46]. Another interesting protein derived from a
Furthermore, new classes of pesticidal proteins of B. thuringiensis strain is the one that possess antitrichomonal
approximately 40-50 kDa and of approximately 10-15 kDa activity; however, this protein had no insecticidal activity
were found and used for controlling corn rootworms. These [47]. Aroian and Li [48] prevented, inhibited or treated
proteins were obtained from the B. thuringiensis isolates parasitic infections of plants and vertebrates with crystals
designated as PS80JJ1, PS149B1, and PS167H2. [39]. In derived from cry5 genes. Also discussed was use of
addition sequences of cry8A and cry8B, were reported to transgenic plants for expressing these proteins, as well as
provide protection to plants against corn rootworm and methods for using them.
Colorado potato beetle, both coleopterans infesting Other pathogenic organisms such as fungi can also be
agricultural crops [40].
controlled by B. thuringiensis. Maeda et al. [49] used this
Novel B. thuringiensis isolates that produce toxins with bacterium to control the pathogenic fungus Fusarium solani
hymenopteran activity were obtained from genes or gene present in farmed fish and shellfish, satisfying requirements
fragments cloned from novel B. thuringiensis isolates with for food safety while reducing installation operating costs.
formicidal activity. These isolates are useful for control of A chitinolytic isolate of B. thuringiensis subsp.
ant populations after transformation into suitable hosts [41].
dendrolimus (HD-548) exhibits an increased activity against
Continuing research on the toxic capabilities of B.
plant pathogenic fungi, as well as insect control properties,
thuringiensis has allowed the identification of strains able to
including insecticidal activity against certain lepidopteran
kill organisms once considered less susceptible to this
pests. The DNA sequences that encode the chitinase activity
pathogen due to their feeding habits or their midgut pH
of the strain were determined, and the applications of the
conditions. The most important insects considered among strain for protection of plants from fungal and insect damage
these are the sucking insects, which feed on sap and not from
were disclosed [50]. Thus, the chitinolytic enzymes
the superficial areas of the plant where B. thuringiensis is
produced by B. thuringiensis are also important. Although
generally applied. However, the discovery of novel B.
the Cry proteins play the main role in toxic activity,
thuringiensis strains containing parasporal crystal proteins
chitinases can increase the toxicity of this bacterium because
having pesticidal properties against whiteflies, aphids,
B. thuringiensis Based-Biopesticides Recent Patents on Biotechnology 2009, Vol. 3, No. 1 31

all insects and fungi have a chitin layer covering their required. The bioinsecticide must exhibit stability in storage,
bodies, which is susceptible to degradation by these requiring improvement of its biological and physical
enzymes. The combination of these proteins could enhance properties. The use of additives is required in order to reduce
the potential of this bacterium in biological control evaporation and avoid formulation loss, and to provide a
programs. Another novel B. thuringiensis strain produced by more extended coverage on and high adherence to foliage,
mutagenesis and selection showed enhanced activity against improved dispersion, and a long residual effect. A great
certain plant pathogenic fungi. Such fungi include: Botrytis variety of ingredients have been employed to prepare
cinerea; Alternaria solani; and Aspergillus, etc. Additionally formulations, including liquid or solid carriers, surfactants,
the strain retains the insecticidal activity normally associated coadjuvants, fluidity agents, adherents, dispersants,
with the wild-type strain. The strain designated AU634 stabilizers, moisturizers, attractants, and protective agents
protects plants against damage by the lesser cornstalk borer among others [54]. An interesting and recent example of
and other lepidopteran pests that are ususally susceptible to these kind of inert ingredients is the superabsorbent starch
Cry1Ab and Cry1Ac toxins [50]. graft copolymer, which combined with a B. thuringiensis
strain among many other pesticides constitutes a novel
Cockroaches, belonging to Order Orthoptera, are
formulation that could be applied in an agricultural
common house pests, and they cause serious problems in
environment [55].
hospitals, food industry and agriculture. These pests are
important because their body surface is a vector for several The biological activity of one particular bioinsecticide
microorganisms, many of which are potentially dangerous to was enhanced when the antibiotic zwittermycin was added.
human beings. In many cases, cockroach infestations can This combination was also successful in pest control [56].
elicit allergic reactions in humans. An alternative for their Other compounds with such a synergism have also been
control involves the use of activated toxins of B. used, in amounts effective for enhancing the pesticidal
thuringiensis var. israelensis. In particular, the strain B.t. activity of a B. thuringiensis-based biopesticide. This can
PS123D1 is effective for cockroach control, and a truncated help reduce the amount of B. thuringiensis needed for a
form of a toxin obtained from PS123D1, having particular commercially effective bioinsecticide [57]. Hobbs [58]
activity to cockroaches, is also indicated for use in patented a pesticidal composition comprising a B.
controlling the pest. [51]. thuringiensis biopesticide present at ineffective levels, and a
surfactant present at effective levels in order to control pests
Mite pests are susceptible to the action of B.
thuringiensis. Isolates designated B.t. PS50C, B.t. PS86A1, or to reduce the amount of the B. thuringiensis normally
needed for commercially effective use.
B.t. PS69D1, B.t. PS72L1, B.t. PS75J1, B.t. PS83E5, B.t.
PS45B1, B.t. PS24J, B.t. PS94R3, B.t. PS17, B.t. PS62B1 Bacillus thuringiensis-based products are classified
and B.t. PS74G1 or their mutants were found to be according to their formulation. All products containing a
particularly active against the two-spotted spider mite, blend of spores and crystals from a native strain are
Tetranychus urticae. Furthermore, genes encoding novel classified as first-generation products. These products
delta-endotoxins can be cloned from these isolates and constitute the majority of commercial products. The second-
transferred to other host microbes or plants. Expression of generation products are based on spores and crystals from a
the delta-endotoxins in microbe hosts has resulted in the B. thuringiensis strain bearing artificially introduced genes
control of mite pests, whereas transformed plants become coding for delta-endotoxins from several strains, in order to
resistant to these pests [52]. increase the activity spectrum against other insect pests.
Formulations containing dead recombinant Pseudomonas
BACILLUS THURINGIENSIS-BASED FORMU- fluorescens cells transformed with genes coding for delta-
LATIONS
endotoxins are classified as third-generation products [3].
Bacillus thuringiensis-based biopesticide production An insecticide can be introduced into the market in
depends on high quality, and high-efficiency formulation different forms, depending on the insect to be controlled, the
processes. Formulations must be safe and effective products, kind of crop, and the application method (Table 1). In addi-
must be easy to use, and should have a long shelf life. The tion, the form of any formulation for release or dispensing
active ingredient in commercial formulations is the spore- into the environment is an important consideration. It
crystal complex, which is more effective to use and cheaper depends on the pest to be controlled and the plant to which it
to obtain than the crystals alone, which are frequently used will be applied.
in experimental tests. The spore-crystal complex must be
carried by suitable excipients that can function to protect the An interesting example comes from laboratory research
spore-crystal complex or to increase palatability to insects. to control mosquito larvae [59]. The procedure was patented
In this sense, many studies reveal the inclusion of some in 1999, and four years later, this invention, containing B.
kinds of components, related to enhance toxicity, or to thuringiensis subsp. israelesis suspended in ice granules,
enhance the attraction of insects. A good example is a was applied to control Aedes vexans larvae. The ice granules
formulation developed for killing fire ants that included a containing the toxic agent were prepared in a special ice
purified and activated Cry toxin from a novel strain of B. machine and applied on the water surface where they melted
thuringiensis, along with an attractant consisting of a and released the toxic crystals [60]. This clean technology
biodegradable, environmentally sound glycoprotein [53]. resulted in a very economic and convenient means for the
control of an important disease vector. For other kinds of
Many biodegradable compounds can be considered for dipterans, a solution proposed by Gomes Sanchez et al. [61]
use as inert carriers, depending on the type of formulation
32 Recent Patents on Biotechnology 2009, Vol. 3, No. 1 Ninfa M. Rosas-García

Table 1. Types of Bacillus thuringiensis Formulations and as fluidity and dispersion play an important role. To this end,
Their Applications for Insect Pest Control a patent was granted to Suenaga et al. [62] for improving
dispersibility, suspensibility and hydration tendency in an
Formulation Application agrochemical preparation that included a product derived
from B. thuringiensis var. aizawai (K strain), an inorganic
Emulsions Agriculture and forestry salt (ammonium sulfate) and/or a saccharide (refined sugar),
a surfactant (sodium lignosulfonate), and a binder (silica,
Encapsulations Agriculture and forestry talc, or kaolin).
Wettable powders Gardens and agriculture Today, a great variety of B. thuringiensis-based
Granules Agriculture and forestry bioinsecticides is commercially available for the control of a
wide variety of agriculture and forestry pests, including
Powders Forestry disease vectors [54] Table 2. Many formulations already
exist in the market; however, many patents relate to the use
Briquettes Aquatic systems
of different components that can further improve their
insecticidal activity. In many cases, these products provide
was a bioinsecticide formulation dispensed as a dry powder interesting advantages over previously developed insec-
or tablet. Formulations contained B. thuringiensis var. ticides [63-65]. Fig. (3) shows the abundance of intellectual
israelensis and chemical dryers, dispersing agents, binding property developed in B. thuringiensis around the world
agents and moisturizing agents, protectors against sunlight; during the last 22 years.
and optionally, diluents, lubricant and neutralizing agents.
The bioinsecticide obtained was considered to be an GENES, NUCLEOTIDES SEQUENCES, TOXINS, AND
ecologically safe product. OTHERS
As mentioned above, physical properties must always be The majority of the patents in B. thuringiensis relate to
considered when appropriate formulations are being the discovery of novel genes associated with toxicity, or to
developed. For successful field applications, properties such the characterization of nucleotide sequences that could be

Table 2. Most Common Commercial Bacillus thuringiensis-based Bioinsecticides [63-65]

Company Commercial Name Active Ingredient Target Pest

Certis Agree WG B. thuringiensis v. aizawai Lepidopterans

Certis Condor B. thuringiensis v. kurstaki Lepidopterans

Certis CoStar B. thuringiensis v. kurstaki Lepidopterans

Certis Crymax Genetically engineered Bt strain derived Lepidopterans

from Bt v. kurstaki and Bt v. aizawai

Certis Deliver B. thuringiensis v. kurstaki Lepidopterans

Certis Jackpot WP B. thuringiensis v. kurstaki Lepidopterans

Certis Javelin/Delfin B. thuringiensis v. kurstaki Lepidopterans

Certis Lepinox WDG B. thuringiensis v. kurstaki Lepidopterans

Certis Turix WP/Agree WP B. thuringiensis v. kurstaki Lepidopterans

AFA Environment Inc. Agribac B. thuringiensis v. kurstaki More than 30 insect species

Valent Biosciences Corp. DiPel B. thuringiensis v. kurstaki Lepidopterans

Valent Biosciences Corp. XenTari B. thuringiensis v. kurstaki Particularly effective against Spodoptera ssp.
and Plutella xilostella

Valent Biosciences Corp. Biobit B. thuringiensis v. kurstaki Lepidopterans

Valent Biosciences Corp. Novodor B. thuringiensis v. tenebrionis Coleopterans

Valent Biosciences Corp. VectoBac B. thuringiensis v. israelensis Mosquito and fly larvae

Valent Biosciences Corp. Teknar B. thuringiensis v. israelensis Mosquito and black fly larvae

Valent Biosciences Corp. GnatrolDG B. thuringiensis v. israelensis Larval stage of Sciarid mushroom flies

Valent Biosciences Corp. Foray B. thuringiensis v. kurstaki Lepidopterans

Valent Biosciences Corp.* Thuricide B. thuringiensis v. kurstaki Lepidopterans and certain leaf-eating worms
*Valent BioSciences has also acquired exclusive marketing rights to Thuricide biological insecticide. Thuricide is a registered trademark of Certis, USA.
B. thuringiensis Based-Biopesticides Recent Patents on Biotechnology 2009, Vol. 3, No. 1 33

Fig. (3). Number of patents granted and applications related to B. thuringiensis inventions in the last 22 years. Data were obtained from the
World Intellectual Property Organization, the United States Patent and Trademark Office, the European Patent Office and the Japan Patent
Office. Inventions related to B. thuringiensis transgenic plants are not included in this graphic.

used as probes and primers for identification of genes species [84], are some of the patents that relate to the
encoding toxins, and of the toxins themselves. Many patents discovery of novel genes with insecticidal activity. In
disclose new families of toxins. For example a recent patent addition, compositions, methods, and other procedures are
revealed a novel insecticidal crystal protein gene cry7Bal requisite for the study of these bacteria. Any novel
from B. thuringiensis subsp. huazhongensis YBT-978, which component can be intellectually protected, as demonstrated
encodes a novel crystal protein with toxic activity against by the following patents: Carozzi et al. [85] patented
lepidopteran insects [66]. Bermudez et al. [67] patented compositions and methods for conferring pesticidal activity
insecticidal polypeptides related to Cry2 proteins, including to bacteria, plants, plant cells, tissues and seeds, as well as a
the nucleic acids which encode them, and the methods for coding sequence for a delta-endotoxin to be used in DNA
using them to enhance resistance of plants to insect feeding. constructs or expression cassettes for transformation and
Other nucleic acids, variants and fragments obtained from B. expression in plants and bacteria, and delta-endotoxin-
thuringiensis strains, encoding polypeptides with toxic associated polypeptides. Adams et al. [86] patented another
activity against insect pests including coleopterans, have method for producing an integrant(s) of B. thuringiensis that
been patented [68]. Novel isolates, insecticidal toxins, genes, produces a larger quantity of a crystal delta-endotoxin with
nucleotide probes and primers for the identification of genes greater pesticidal activity as compared to the crystal delta-
provided entirely new families of toxins [69-74]. The endotoxin produced by the corresponding parental strain.
isolation and characterization of nucleotide sequences The crystal delta-endotoxin produced by the integrant B.
encoding novel insecticidal proteins includes new DNA thuringiensis will have an activity directed towards the same
sequences encoding proteins designated as Cry9Fa, Cry1Jd, pest(s) as its parent B. thuringiensis crystal delta-endotoxin.
and Cry1Bf, useful to protect plants from insect damage The invention further relates to such integrants, compositions
[75], recombinant DNA sequences encoding pesticidal comprising such integrants, as well as methods for
proteins designated as ISLP proteins, and toxic fragments controlling pests using these compositions. Another similar
[76]. Carozzi et al. [77] and Abad et al. [78, 79] have patent by Adams et al. [87] discloses an integrant of B.
described representative patents in which the insecticidal thuringiensis that produces a larger quantity of a crystal
activity has been directed toward different insect pests, delta-endotoxin with greater pesticidal activity and optio-
particularly to protect plants from pest damage [80, 81]. nally a larger crystal size as a result of gene amplification or
hyperexpression, as compared to the corresponding parental
Novel insecticidal compounds derived from B.
strain. The integrant strain will have an activity directed
thuringiensis strains and new proteins designated Cry2Ae,
towards the same pest as the corresponding parental strain
Cry2Af, and Cry2Ag [82], lepidopteran toxic proteins
delta-endotoxin.
designated as CryET4 and CryET5 from strain EG5847 [83],
delta-endotoxins polypeptides designed CryET10 active CHIMERIC CRYSTAL PROTEINS
against lepidopteran and coleopteran pests as well as
methods for controlling an insect population, such as the In recent years, hybrid delta-endotoxins have arisen as
Western corn rootworm and Colorado potato beetle, and for proteins with potential for enhanced toxic activity or
conferring to a plant population resistance to the target insect improved properties. Recent advances in molecular
methodologies have allowed gene fusions and chimeric
34 Recent Patents on Biotechnology 2009, Vol. 3, No. 1 Ninfa M. Rosas-García

protein construction. This construction can include alteration hybrid toxin fragment comprises at its C-terminus domain III
of amino acid sequences, fusion of portions of two or more a first Cry protein, and at its N-terminus, a second Cry
proteins together into a single recombinant protein, or protein [95].
alteration of the genetic sequences encoding for proteins
An isolated B. thuringiensis Cry8Bb1 toxin polypeptide
with commercial application. Although this is not an easy with at least one engineered proteolytic protection site is not
process for Cry proteins, due to their complex nature, a
sensitive to a plant protease and protects the toxin
careful selection of target regions in the protein will allow
polypeptide from proteolytic inactivation in a plant. The
the production of engineered toxins with a high insecticidal
proteolytic protection site comprises the amino acid
activity. A number of advances have been made to improve
sequence NGSRNGSR and replaces a proteolytic site that
the desirable properties of bioinsecticides.
comprises the amino acid sequence FRRGFRRGH. This
In the continuous struggle against lepidopteran pests, the invention is used for controlling agriculturally significant
synergistic combination of a CryIF chimeric and CryIA(c) pests, such as the Western corn rootworm, Diabrotica
chimeric B. thuringiensis delta-endotoxin exhibited excellent virgifera virgifera; the Northern corn rootworm, D. longi-
toxic activity [88]. cornis barberi; the Southern corn rootworm, D. unde-
cimpunctata howardi; wireworms of the genera Melanotus,
A wider spectrum of toxicity is shown by mutant toxins,
which possess toxicity against a variety of insect genera, and Aeolus; the boll weevil Anthonomus grandis; the
Colorado potato beetle Leptinotarsa decemlineata; and the
particularly mosquitoes from the genera Culex, Aedes, and
alfalfa weevil Hypera nigrirostris [96].
Anopheles. The toxin Cry4Ba was modified by substitution
of the amino acid aspartic acid at position 454 with proline, CURRENT & FUTURE DEVELOPMENTS
glycine, alanine, threonine or serine; and by insertion of two
additional amino acids immediately after position 454, There is a great amount of scientific research on the
wherein the additional amino acids are chosen from the bacterium B. thuringiensis, involving aspects ranging from
group consisting of: glycine, alanine, valine, leucine, its molecular biology to its activity in a bioinsecticide.
isoleucine, methionine, proline, threonine, and serine. The Although B. thuringiensis has been studied for more than a
modified Cry4Ba protein exhibits enhanced toxicity to century, it is amazing to know that scientists always find
Culex, as compared to Cry4Ba protein. Cry19Aa has something new in it. The fascinating arrangement of its
undergone modifications or at one or more positions within genetic content, along with the high diversity of toxins
the amino acid sequence [89]. After studying the chimeric derived from it, makes this bacterium a unique organism.
gene segments and the methods for making synthetically New activities for it are discovered daily, and as a result,
modified chimerical crystal proteins, some chimeric crystal more pests can be controlled with environmentally clean
proteins provide a new tool to control insects, particularly technologies. The toxic activity of B. thuringiensis is highly
coleopteran, dipteran, and lepidopteran pests [90]. Other specific and it is practically harmless to human, vertebrates,
novel synthetically modified chimeric crystal proteins having beneficial insects and other organisms.
improved insecticidal activity and a broader insect host range The development of formulations with biodegradable
against coleopteran, dipteran and lepidopteran insects, as ingredients is a favored approach for the reduction of chemi-
well as the nucleic acid segments encoding these novel cal insecticide use, which can threaten the environment.
peptides, were disclosed [91].
Although there is an enormous possibility to control
Modified Cry35 proteins whose segments, domains, and many pests, mainly in agriculture and forestry, and to obtain
motifs were exchanged with other proteins showed enhanced more organic pest-control products, the ignorance about
insecticidal properties relative to the wild type Cry 35 advantages of use of B. thuringiensis products limits its
protein, and this invention provided good control against application; therefore, it is necessary to establish measures
plant pests including rootworms [92]. Another chimeric aimed at promoting the knowledge of biological control
protein, formed from delta-endotoxins Cry1Ea and Cty1Ca, programs. Intellectual property is the only tool that one can
resulted in an extraordinarily high insecticidal activity in possesses for the protection of his or her inventions, and said
recombinant plants [93]. inventions, as in the B. thuringiensis case, must be exploited
An interesting patent discloses new chimeric genes for their benefits to human health and environmental
encoding a Cry1C, Cry1B or Cry1D protein that are useful in friendliness.
the protection of plants from insect damage [94]. ACKNOWLEDGEMENTS
One particular hybrid crystal protein is composed of This work received financial support by the Secretaría de
proteins CryIC together with CryIE, CryIA, or CryIG. The Investigación y Posgrado del Instituto Politécnico Nacional,
nucleotide sequence of the CryIC gene is obtained from B. project No. 20080218.
thuringiensis subsp. entomocidus 60.5. The nucleotide
sequence of the CryIE gene derived from B. thuringiensis CONFLICT OF INTEREST
subsp kenyae 4FI. The nucleotide sequence of the CryIG The author has no conflict of interest to declare.
gene derived from another B. thuringiensis strain. These
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