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Recent Patents on Biotechnology 2009, 3, 28-36

Biopesticide Production from Bacillus thuringiensis: An Environmentally Friendly Alternative

Ninfa M. Rosas-Garca*
Laboratorio de Biotecnologa Ambiental. Centro de Biotecnologa Genmica-IPN. Blvd. del Maestro s/n. Reynosa, Tamp. CP 88710 Mxico
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 pestimpacted 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 The bacterium Bacillus thuringiensis was discovered by Shigetane Ishiwata in 1901 [1], and rediscovered by Berliner ten years later. The bacterium was isolated from diseased larvae of Anagasta kuehniella, and this finding led to the establishment of B. thuringiensis as microbial insecticide. The first record of its application to control insects was in Hungary at the end of 1920, and in Yugoslavia at the beginning of 1930s, it was applied to control the European corn borer [2]. During the following two decades, several field tests were conducted to evaluate its effectiveness against lepidopterans, both in Europe and in the United States [3], and results favored the development of formulations against on this pathogen. Subsequently, the first commercial product was produced in 1938 by Libec in France. [4]. Unfortunately, the product was used only for a very short time, due to World War II [5]. After World War II, the Green Revolution provided great agricultural advantages via the use of agrochemicals, chemical fertilizers, highly productive cultivars, and mechanization. The result was a considerable decrease in a great variety of insect populations, and as a consequence, synthetic insecticidal compounds became popular due to the long residual action and the wide toxicity spectrum. However, fully synthetic chemical insecticides appeared in 1940, when organochlorinated and organophosphate insecticides were discovered. These insecticides were applied during all
*Address correspondence to this author at the Laboratorio de Biotecnologa Ambiental. Centro de Biotecnologa Genmica-IPN. Blvd. del Maestro s/n. Reynosa, Tamp. CP 88710 Mxico; Tel: 52-899-924-3627; Ext: 7721; Fax: 52-899-925-2889; E-mail: nrosas@ipn.mx; ninfarosasg@yahoo.com.mx 1872-2083/09 $100.00+.00

growing seasons to attack all the developmental stages of insect pests [6]. The indiscriminate use of these compounds caused, by 1950, a resurgence of pests, due to the elimination of their natural enemies and the appearance of pest populations showing resistance to insecticides. Also serious environmental and health issues began to be recognized by the presence of chemical residues in food, water, and air. To counteract this contamination, attention and efforts were directed to the use of biological control agents including insect pathogens [7]. However, an entomopathogenic organism must fulfill several requisites before being released to the environment as a potential control agent. It should be highly specific and effective against the target pest. The organism should demonstrate the potential to be successfully processed by continuous production technology. The control agent should be available in formulations with a reasonable shelf life, should be stable, and should be harmless to human and non-target flora and fauna [8]. As an entomopathogenic organism, B. thuringiensis fulfills all these requirements. Bacillus thuringiensis is a gram-positive spore-forming bacterium that produces crystalline proteins called deltaendotoxins during its stationary phase of growth [9]. The crystal is released to the environment after lysis of the cell wall at the end of sporulation, and it can account for 20 to 30% of the dry weight of the sporulated cells [9] Fig. (1). This bacterium is distributed worldwide. The soil has been 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 source for commercially used varieties, which include

2009 Bentham Science Publishers Ltd.

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toxin inserts itself into the membrane. During the intoxication 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 Hfte 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 sequence similarity to a known Cry protein. The diversity of B. thuringiensis is demonstrated in the almost 70 serotypes and the 92 subspecies described to date [26]. A full list of delta-endotoxins, including names and source strains can be found at http://www.lifesci.sussex.ac.uk/home/Neil_ Crickmore/Bt/toxins2.html The biological activity of B. thuringiensis strains or their products toward different target organisms has been subject to patent coverage for many years. Many of these patents belong to companies engaged in commercial endeavors, while others remain as a part of the basic research domain. It is well documented that many insects are susceptible to the toxic activity of B. thuringiensis; among them, lepidopterans have been exceptionally well studied, and many toxins have shown activity against them [27-33]. Order Lepidoptera encompasses the majority of susceptible species belonging to agriculturally important families such as Cossidae, Gelechiidae, Lymantriidae, Noctuidae, Pieridae, Pyralidae, Thaumetopoetidae, Tortricidae, and Yponomeutidae [11]. Recently, four novel B. thuringiensis strains, which are deposited at the BCCM-LMG under accession nos. LMG P-12592, LMG P-12593, LMG P-12594, and LMG P-13493, have been found to produce new crystal proteins during sporulation that are toxic to Lepidoptera, more particularly to Noctuidae such as Spodoptera spp. and Agrotis ipsilon, to Pyralidae such as Ostrinia nubilalis, and to Yponomeutidae such as Plutella xylostella. In addition, the genes that encode these novel proteins have also been studied [34]. Abad et al. [35] have patented an invention that describes nucleic acids, variants and fragments from B. thuringiensis strains that encode polypeptides having insecticidal activity against lepidopterans. This invention has been applied in methods for controlling plant pests. Baum et al. [36] also reported B. thuringiensis strains that produce novel crystal proteins exhibiting insecticidal activity against lepidopterans, as well as a method for using and making transgenic cells. A typical example of B. thuringiensis activity can be observed in Fig. (2). Dipterans are also important target pests, and many of them are highly susceptible to B. thuringiensis. This order includes the families Tephritidae, Culicidae, Muscidae,

Fig. (1). Scanning electron micrograph of spores (S) and crystals of Bacillus thuringiensis HD-125 strain. The strain synthesizes bypiramidal (B) and square (Sq) crystals. (The strain was kindly donated by the Bacillus Genetic Stock Center, Ohio, USA and is coded as 4L1). The photograph is provided by N.M. Rosas-Garca.

kurstaki, isolated from A. kuehniella; israelensis, isolated from mosquitoes, and tenebrionis, isolated from Tenebrio monitor larvae [10,11]. MODE OF ACTION The Cry proteins comprise at least 50 subgroups with more than 200 members [12]. The members belong to a three-domain family, and the larger group of Cry proteins is globular molecules with three structural domains connected by single linkers. The protoxins are characteristic of this family and have two different lengths. The C-terminal extension found in the long protoxins is necessary for toxicity and is believed to play a role in the formation of the crystal within the bacterium [13]. Their mode of action involves several events that must be completed several hours after ingestion in order to lead to insect death. Following ingestion, the crystals are solubilized by the alkaline conditions in the insect midgut and are subsequently proteolytically converted into a toxic core fragment [14]. During proteolytic activation, peptides from the N terminus and C terminus are cleaved from the full protein. Activated toxin binds to receptors located on the apical microvillus membranes of epithelial midgut cells. For Cry1A toxins, at least four different binding sites have been described in different lepidopteran insects: a cadherin-like protein (CADR), a glycosylphosphatidyl-inositol (GPI)-anchored aminopeptidase-N (APN), a GPI-anchored alkaline phosphatase (ALP) and a 270 kDa glycoconjugate [15-19]. After binding, toxin adopts a conformation allowing its insertion into the cell membrane. Subsequently, oligomerization occurs, and this oligomer forms a pore or ion channel induced by an increase in cationic permeability within the functional receptors contained on the brush borders membranes [20]. This allows the free flux of ions and liquids, causing disruption of membrane transport and cell lysis, and leading to insect death [14]. The complete nature of this process still remains unknown [21]; however, it is believed that toxin aggregation occurs at the membrane surface after receptor binding, or alternatively only after the

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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 sequence of the toxin and the nucleotide sequence encoding the toxin were determined. With this new isolate, some nematodes such as Haemonchus, Nematodirus, soil nematodes and vegetable parasites can be controlled through the use of the toxin [43]. Another toxin with nematicidal activity is a truncated form of the Cry6A protein. Although this toxin exhibited particular toxic activity against the corn rootworm, a coleopteran, the truncated toxin also proved useful for nematode control. Specifically, truncated Cry6A, with a molecular mass about 40-50 kDa, is produced from genes that encode for the highly active truncated protein toxin, and it lacks of amino acid sequences removed from the Nterminus and the C-terminus [44]. Protozoans have also been discovered as target organisms susceptible to Cry toxins. The protein toxin produced by a novel isolate designated PS81F exhibited killing capabilities, allowing it to be used to treat humans and animals hosting parasitic protozoans. In addition, the gene encoding the toxin can be transferred to a suitable host via a biological vector, such as a plasmid or virus [45]. Furthermore, a pharmaceutical product for use in treating a mammalian host infected with a protozoan organism was manufactured with the same strain PS81F for control of Giardia, Toxoplasma, Frankelia, Hammondia, Isospora, Besnoita, Eimeria, Entamoeba, Trichomonas, and Pentatrichomonas [46]. Another interesting protein derived from a B. thuringiensis strain is the one that possess antitrichomonal activity; however, this protein had no insecticidal activity [47]. Aroian and Li [48] prevented, inhibited or treated parasitic infections of plants and vertebrates with crystals derived from cry5 genes. Also discussed was use of transgenic plants for expressing these proteins, as well as methods for using them. Other pathogenic organisms such as fungi can also be controlled by B. thuringiensis. Maeda et al. [49] used this bacterium to control the pathogenic fungus Fusarium solani present in farmed fish and shellfish, satisfying requirements for food safety while reducing installation operating costs. A chitinolytic isolate of B. thuringiensis subsp. dendrolimus (HD-548) exhibits an increased activity against plant pathogenic fungi, as well as insect control properties, including insecticidal activity against certain lepidopteran pests. The DNA sequences that encode the chitinase activity of the strain were determined, and the applications of the strain for protection of plants from fungal and insect damage were disclosed [50]. Thus, the chitinolytic enzymes produced by B. thuringiensis are also important. Although the Cry proteins play the main role in toxic activity, chitinases can increase the toxicity of this bacterium because

Fig. (2). Photograph shows lepidopteran larvae susceptible to B. thuringiensis toxic activity. A) Nine day-old, healthy larvae, not exposed to B. thuringiensis. B) Nine-day old larvae exposed to sublethal concentration of B. thuringiensis. Larvae exhibit a smaller size due to their poor feeding. C) Larvae exposed to lethal concentration of B. thuringiensis. The main characteristic after death is the black color of the larvae. The photographs are provided by N.M. Rosas-Garca.

Simuliidae, and Tipulidae. Lysyk et al. [37] developed a method for controlling insects of this order by application of B. thuringiensis strain LRC3 or its products. This strain contains a plasmid carrying genes which encode deltaendotoxins Cry1A, Cry1B, Cry1F, Cry1H, Cry1I, Cry1K, and Cry2. The strain is deposited as ATCC PTA-6248. The same authors also revealed methods for preparing the strain, spores, crystals, and mutants. Coleopterans are important pests in agriculture and forestry. Several families such as Chrysomelidae, Curculionidae, Tenebrionidae, and Scarabeidae have recently been found to be susceptible to toxic activity of the crystals. Some DNA sequences derived from B. thuringiensis strains encode for delta-endotoxins with pesticidal activity against these pests. These sequences were further mutagenized to improve pesticidal activity or altered pest specificity when applied to plants for the control of insect pests [38]. Furthermore, new classes of pesticidal proteins of approximately 40-50 kDa and of approximately 10-15 kDa were found and used for controlling corn rootworms. These proteins were obtained from the B. thuringiensis isolates designated as PS80JJ1, PS149B1, and PS167H2. [39]. In addition sequences of cry8A and cry8B, were reported to provide protection to plants against corn rootworm and Colorado potato beetle, both coleopterans infesting agricultural crops [40]. Novel B. thuringiensis isolates that produce toxins with hymenopteran activity were obtained from genes or gene fragments cloned from novel B. thuringiensis isolates with formicidal activity. These isolates are useful for control of ant populations after transformation into suitable hosts [41]. Continuing research on the toxic capabilities of B. thuringiensis has allowed the identification of strains able to kill organisms once considered less susceptible to this pathogen due to their feeding habits or their midgut pH conditions. The most important insects considered among these are the sucking insects, which feed on sap and not from the superficial areas of the plant where B. thuringiensis is generally applied. However, the discovery of novel B. thuringiensis strains containing parasporal crystal proteins having pesticidal properties against whiteflies, aphids,

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all insects and fungi have a chitin layer covering their bodies, which is susceptible to degradation by these enzymes. The combination of these proteins could enhance the potential of this bacterium in biological control programs. Another novel B. thuringiensis strain produced by mutagenesis and selection showed enhanced activity against certain plant pathogenic fungi. Such fungi include: Botrytis cinerea; Alternaria solani; and Aspergillus, etc. Additionally the strain retains the insecticidal activity normally associated with the wild-type strain. The strain designated AU634 protects plants against damage by the lesser cornstalk borer and other lepidopteran pests that are ususally susceptible to Cry1Ab and Cry1Ac toxins [50]. Cockroaches, belonging to Order Orthoptera, are common house pests, and they cause serious problems in hospitals, food industry and agriculture. These pests are important because their body surface is a vector for several microorganisms, many of which are potentially dangerous to human beings. In many cases, cockroach infestations can elicit allergic reactions in humans. An alternative for their control involves the use of activated toxins of B. thuringiensis var. israelensis. In particular, the strain B.t. PS123D1 is effective for cockroach control, and a truncated form of a toxin obtained from PS123D1, having particular activity to cockroaches, is also indicated for use in controlling the pest. [51]. Mite pests are susceptible to the action of B. thuringiensis. Isolates designated B.t. PS50C, B.t. PS86A1, 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 and B.t. PS74G1 or their mutants were found to be particularly active against the two-spotted spider mite, Tetranychus urticae. Furthermore, genes encoding novel delta-endotoxins can be cloned from these isolates and transferred to other host microbes or plants. Expression of the delta-endotoxins in microbe hosts has resulted in the control of mite pests, whereas transformed plants become resistant to these pests [52]. BACILLUS LATIONS THURINGIENSIS-BASED FORMU-

required. The bioinsecticide must exhibit stability in storage, requiring improvement of its biological and physical properties. The use of additives is required in order to reduce evaporation and avoid formulation loss, and to provide a more extended coverage on and high adherence to foliage, improved dispersion, and a long residual effect. A great variety of ingredients have been employed to prepare formulations, including liquid or solid carriers, surfactants, coadjuvants, fluidity agents, adherents, dispersants, stabilizers, moisturizers, attractants, and protective agents among others [54]. An interesting and recent example of these kind of inert ingredients is the superabsorbent starch graft copolymer, which combined with a B. thuringiensis strain among many other pesticides constitutes a novel formulation that could be applied in an agricultural environment [55]. The biological activity of one particular bioinsecticide was enhanced when the antibiotic zwittermycin was added. This combination was also successful in pest control [56]. Other compounds with such a synergism have also been used, in amounts effective for enhancing the pesticidal activity of a B. thuringiensis-based biopesticide. This can help reduce the amount of B. thuringiensis needed for a commercially effective bioinsecticide [57]. Hobbs [58] patented a pesticidal composition comprising a B. thuringiensis biopesticide present at ineffective levels, and a surfactant present at effective levels in order to control pests or to reduce the amount of the B. thuringiensis normally needed for commercially effective use. Bacillus thuringiensis-based products are classified according to their formulation. All products containing a blend of spores and crystals from a native strain are classified as first-generation products. These products constitute the majority of commercial products. The secondgeneration products are based on spores and crystals from a B. thuringiensis strain bearing artificially introduced genes coding for delta-endotoxins from several strains, in order to increase the activity spectrum against other insect pests. Formulations containing dead recombinant Pseudomonas fluorescens cells transformed with genes coding for deltaendotoxins are classified as third-generation products [3]. An insecticide can be introduced into the market in different forms, depending on the insect to be controlled, the kind of crop, and the application method (Table 1). In addition, the form of any formulation for release or dispensing into the environment is an important consideration. It depends on the pest to be controlled and the plant to which it will be applied. An interesting example comes from laboratory research to control mosquito larvae [59]. The procedure was patented in 1999, and four years later, this invention, containing B. thuringiensis subsp. israelesis suspended in ice granules, was applied to control Aedes vexans larvae. The ice granules containing the toxic agent were prepared in a special ice machine and applied on the water surface where they melted and released the toxic crystals [60]. This clean technology resulted in a very economic and convenient means for the control of an important disease vector. For other kinds of dipterans, a solution proposed by Gomes Sanchez et al. [61]

Bacillus thuringiensis-based biopesticide production depends on high quality, and high-efficiency formulation processes. Formulations must be safe and effective products, must be easy to use, and should have a long shelf life. The active ingredient in commercial formulations is the sporecrystal complex, which is more effective to use and cheaper to obtain than the crystals alone, which are frequently used in experimental tests. The spore-crystal complex must be carried by suitable excipients that can function to protect the spore-crystal complex or to increase palatability to insects. In this sense, many studies reveal the inclusion of some kinds of components, related to enhance toxicity, or to enhance the attraction of insects. A good example is a formulation developed for killing fire ants that included a purified and activated Cry toxin from a novel strain of B. thuringiensis, along with an attractant consisting of a biodegradable, environmentally sound glycoprotein [53]. Many biodegradable compounds can be considered for use as inert carriers, depending on the type of formulation

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Table 1. Types of Bacillus thuringiensis Formulations and Their Applications for Insect Pest Control
Formulation Emulsions Encapsulations Wettable powders Granules Powders Briquettes Application Agriculture and forestry Agriculture and forestry Gardens and agriculture Agriculture and forestry Forestry Aquatic systems

as fluidity and dispersion play an important role. To this end, a patent was granted to Suenaga et al. [62] for improving dispersibility, suspensibility and hydration tendency in an agrochemical preparation that included a product derived from B. thuringiensis var. aizawai (K strain), an inorganic salt (ammonium sulfate) and/or a saccharide (refined sugar), a surfactant (sodium lignosulfonate), and a binder (silica, talc, or kaolin). Today, a great variety of B. thuringiensis-based bioinsecticides is commercially available for the control of a wide variety of agriculture and forestry pests, including disease vectors [54] Table 2. Many formulations already exist in the market; however, many patents relate to the use of different components that can further improve their insecticidal activity. In many cases, these products provide interesting advantages over previously developed insecticides [63-65]. Fig. (3) shows the abundance of intellectual property developed in B. thuringiensis around the world during the last 22 years. GENES, NUCLEOTIDES SEQUENCES, TOXINS, AND OTHERS The majority of the patents in B. thuringiensis relate to the discovery of novel genes associated with toxicity, or to the characterization of nucleotide sequences that could be

was a bioinsecticide formulation dispensed as a dry powder or tablet. Formulations contained B. thuringiensis var. israelensis and chemical dryers, dispersing agents, binding agents and moisturizing agents, protectors against sunlight; and optionally, diluents, lubricant and neutralizing agents. The bioinsecticide obtained was considered to be an ecologically safe product. As mentioned above, physical properties must always be considered when appropriate formulations are being developed. For successful field applications, properties such

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

Company Certis Certis Certis Certis Commercial Name Agree WG Condor CoStar Crymax Active Ingredient B. thuringiensis v. aizawai B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki Genetically engineered Bt strain derived from Bt v. kurstaki and Bt v. aizawai B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki Target Pest Lepidopterans Lepidopterans Lepidopterans Lepidopterans

Certis Certis Certis Certis Certis AFA Environment Inc. Valent Biosciences Corp. Valent Biosciences Corp.

Deliver Jackpot WP Javelin/Delfin Lepinox WDG Turix WP/Agree WP Agribac DiPel XenTari

Lepidopterans Lepidopterans Lepidopterans Lepidopterans Lepidopterans More than 30 insect species Lepidopterans Particularly effective against Spodoptera ssp. and Plutella xilostella Lepidopterans Coleopterans Mosquito and fly larvae Mosquito and black fly larvae Larval stage of Sciarid mushroom flies Lepidopterans Lepidopterans and certain leaf-eating worms

Valent Biosciences Corp. Valent Biosciences Corp. Valent Biosciences Corp. Valent Biosciences Corp. Valent Biosciences Corp. Valent Biosciences Corp. Valent Biosciences Corp.*

Biobit Novodor VectoBac Teknar GnatrolDG Foray Thuricide

B. thuringiensis v. kurstaki B. thuringiensis v. tenebrionis B. thuringiensis v. israelensis B. thuringiensis v. israelensis B. thuringiensis v. israelensis B. thuringiensis v. kurstaki B. thuringiensis v. kurstaki

*Valent BioSciences has also acquired exclusive marketing rights to Thuricide biological insecticide. Thuricide is a registered trademark of Certis, USA.

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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 encoding toxins, and of the toxins themselves. Many patents disclose new families of toxins. For example a recent patent revealed a novel insecticidal crystal protein gene cry7Bal from B. thuringiensis subsp. huazhongensis YBT-978, which encodes a novel crystal protein with toxic activity against lepidopteran insects [66]. Bermudez et al. [67] patented insecticidal polypeptides related to Cry2 proteins, including the nucleic acids which encode them, and the methods for using them to enhance resistance of plants to insect feeding. Other nucleic acids, variants and fragments obtained from B. thuringiensis strains, encoding polypeptides with toxic activity against insect pests including coleopterans, have been patented [68]. Novel isolates, insecticidal toxins, genes, nucleotide probes and primers for the identification of genes provided entirely new families of toxins [69-74]. The isolation and characterization of nucleotide sequences encoding novel insecticidal proteins includes new DNA sequences encoding proteins designated as Cry9Fa, Cry1Jd, and Cry1Bf, useful to protect plants from insect damage [75], recombinant DNA sequences encoding pesticidal proteins designated as ISLP proteins, and toxic fragments [76]. Carozzi et al. [77] and Abad et al. [78, 79] have described representative patents in which the insecticidal activity has been directed toward different insect pests, particularly to protect plants from pest damage [80, 81]. Novel insecticidal compounds derived from B. thuringiensis strains and new proteins designated Cry2Ae, Cry2Af, and Cry2Ag [82], lepidopteran toxic proteins designated as CryET4 and CryET5 from strain EG5847 [83], delta-endotoxins polypeptides designed CryET10 active against lepidopteran and coleopteran pests as well as methods for controlling an insect population, such as the Western corn rootworm and Colorado potato beetle, and for conferring to a plant population resistance to the target insect

species [84], are some of the patents that relate to the discovery of novel genes with insecticidal activity. In addition, compositions, methods, and other procedures are requisite for the study of these bacteria. Any novel component can be intellectually protected, as demonstrated by the following patents: Carozzi et al. [85] patented compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds, as well as a coding sequence for a delta-endotoxin to be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria, and delta-endotoxinassociated polypeptides. Adams et al. [86] patented another method for producing an integrant(s) of B. thuringiensis that produces a larger quantity of a crystal delta-endotoxin with greater pesticidal activity as compared to the crystal deltaendotoxin produced by the corresponding parental strain. The crystal delta-endotoxin produced by the integrant B. thuringiensis will have an activity directed towards the same pest(s) as its parent B. thuringiensis crystal delta-endotoxin. The invention further relates to such integrants, compositions comprising such integrants, as well as methods for controlling pests using these compositions. Another similar patent by Adams et al. [87] discloses an integrant of B. thuringiensis that produces a larger quantity of a crystal delta-endotoxin with greater pesticidal activity and optionally a larger crystal size as a result of gene amplification or hyperexpression, as compared to the corresponding parental strain. The integrant strain will have an activity directed towards the same pest as the corresponding parental strain delta-endotoxin. CHIMERIC CRYSTAL PROTEINS In recent years, hybrid delta-endotoxins have arisen as proteins with potential for enhanced toxic activity or improved properties. Recent advances in molecular methodologies have allowed gene fusions and chimeric

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Ninfa M. Rosas-Garca

protein construction. This construction can include alteration of amino acid sequences, fusion of portions of two or more proteins together into a single recombinant protein, or alteration of the genetic sequences encoding for proteins with commercial application. Although this is not an easy process for Cry proteins, due to their complex nature, a careful selection of target regions in the protein will allow the production of engineered toxins with a high insecticidal activity. A number of advances have been made to improve the desirable properties of bioinsecticides. In the continuous struggle against lepidopteran pests, the synergistic combination of a CryIF chimeric and CryIA(c) chimeric B. thuringiensis delta-endotoxin exhibited excellent toxic activity [88]. A wider spectrum of toxicity is shown by mutant toxins, which possess toxicity against a variety of insect genera, particularly mosquitoes from the genera Culex, Aedes, and Anopheles. The toxin Cry4Ba was modified by substitution of the amino acid aspartic acid at position 454 with proline, glycine, alanine, threonine or serine; and by insertion of two additional amino acids immediately after position 454, wherein the additional amino acids are chosen from the group consisting of: glycine, alanine, valine, leucine, isoleucine, methionine, proline, threonine, and serine. The modified Cry4Ba protein exhibits enhanced toxicity to Culex, as compared to Cry4Ba protein. Cry19Aa has undergone modifications or at one or more positions within the amino acid sequence [89]. After studying the chimeric gene segments and the methods for making synthetically modified chimerical crystal proteins, some chimeric crystal proteins provide a new tool to control insects, particularly coleopteran, dipteran, and lepidopteran pests [90]. Other novel synthetically modified chimeric crystal proteins having improved insecticidal activity and a broader insect host range against coleopteran, dipteran and lepidopteran insects, as well as the nucleic acid segments encoding these novel peptides, were disclosed [91]. Modified Cry35 proteins whose segments, domains, and motifs were exchanged with other proteins showed enhanced insecticidal properties relative to the wild type Cry 35 protein, and this invention provided good control against plant pests including rootworms [92]. Another chimeric protein, formed from delta-endotoxins Cry1Ea and Cty1Ca, resulted in an extraordinarily high insecticidal activity in recombinant plants [93]. An interesting patent discloses new chimeric genes encoding a Cry1C, Cry1B or Cry1D protein that are useful in the protection of plants from insect damage [94]. One particular hybrid crystal protein is composed of proteins CryIC together with CryIE, CryIA, or CryIG. The nucleotide sequence of the CryIC gene is obtained from B. thuringiensis subsp. entomocidus 60.5. The nucleotide sequence of the CryIE gene derived from B. thuringiensis subsp kenyae 4FI. The nucleotide sequence of the CryIG gene derived from another B. thuringiensis strain. These proteins are toxic to lepidopterans, but within this order of insects, each protein has different specificity. CryIC, for example, is particularly active against Spodoptera exigua and Mamestra brassicae. The isolated B. thuringiensis

hybrid toxin fragment comprises at its C-terminus domain III a first Cry protein, and at its N-terminus, a second Cry protein [95]. An isolated B. thuringiensis Cry8Bb1 toxin polypeptide with at least one engineered proteolytic protection site is not sensitive to a plant protease and protects the toxin polypeptide from proteolytic inactivation in a plant. The proteolytic protection site comprises the amino acid sequence NGSRNGSR and replaces a proteolytic site that comprises the amino acid sequence FRRGFRRGH. This invention is used for controlling agriculturally significant pests, such as the Western corn rootworm, Diabrotica virgifera virgifera; the Northern corn rootworm, D. longicornis barberi; the Southern corn rootworm, D. undecimpunctata howardi; wireworms of the genera Melanotus, and Aeolus; the boll weevil Anthonomus grandis; the Colorado potato beetle Leptinotarsa decemlineata; and the alfalfa weevil Hypera nigrirostris [96]. CURRENT & FUTURE DEVELOPMENTS There is a great amount of scientific research on the bacterium B. thuringiensis, involving aspects ranging from its molecular biology to its activity in a bioinsecticide. Although B. thuringiensis has been studied for more than a century, it is amazing to know that scientists always find something new in it. The fascinating arrangement of its genetic content, along with the high diversity of toxins derived from it, makes this bacterium a unique organism. New activities for it are discovered daily, and as a result, more pests can be controlled with environmentally clean technologies. The toxic activity of B. thuringiensis is highly specific and it is practically harmless to human, vertebrates, beneficial insects and other organisms. The development of formulations with biodegradable ingredients is a favored approach for the reduction of chemical insecticide use, which can threaten the environment. Although there is an enormous possibility to control many pests, mainly in agriculture and forestry, and to obtain more organic pest-control products, the ignorance about advantages of use of B. thuringiensis products limits its application; therefore, it is necessary to establish measures aimed at promoting the knowledge of biological control programs. Intellectual property is the only tool that one can possesses for the protection of his or her inventions, and said inventions, as in the B. thuringiensis case, must be exploited for their benefits to human health and environmental friendliness. ACKNOWLEDGEMENTS This work received financial support by the Secretara de Investigacin y Posgrado del Instituto Politcnico Nacional, project No. 20080218. CONFLICT OF INTEREST The author has no conflict of interest to declare. REFERENCES
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