AU711493B2 - Improved production of insecticidal proteins - Google Patents

Description

WO 97/39623 PCT/US97/14052 IMPROVED PRODUCTION OF INSECTICIDAL PROTEINS CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part application of U.S. Serial No.

08/588,492, filed January 18, 1996.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. 92- 37302-7603, awarded by the United States Department of Agriculture. The Government has certain rights in this invention.

FIELD OF THE INVENTION The present invention relates to a method of producing insecticidal endotoxins and recombinant cells that express such endotoxins.

BACKGROUND OF THE INVENTION Bacillus thuringiensis is characterized by its ability to produce crystalline inclusions, or parasporal bodies, during sporulation. These inclusions are composed of insecticidal endotoxins, for which at least 14 distinct genes have been identified (Hofte Whiteley, Microbiol. Rev. 53: 242-255 (1989)). These genes encode proteins that have varied yet specific insecticidal activity against members of lepidopteran, dipteran, and coleopteran species. The endotoxin genes are divided into two families, the cry genes and the cyt genes, on the basis of structural relationships. The cry family is further divided into four major classes of genes based on the insecticidal activity of the encoded proteins: Lepidoptera-specific Lepidoptera- and Diptera-specific Coleopteraspecific (III), and Diptera-specific (IV).

A number of microbial insecticides based on B. thuringiensis endotoxins are now available to control insect pests and disease vectors. The first subspecies of B.

thuringiensis developed as a commercial insecticide was B. thuringiensis subsp. kurstaki (Frankenhuyzen, in Bacillus thuringiensis, An Environmental Biopesticide: Theory and WO q7/396(23 Pr"T/US[')7/1 dAf4 2 Practice (Entwistle et al., eds. 1993)). The HD-1 strain of this subspecies is used in a variety of commercial products marketed to control caterpillar pests that attack vegetables, field crops, and forests. Bioinsecticides that make use of naturally occurring pathogens to controls insects have a number advantages over their synthetic counterparts: they are highly specific in their host range and non-toxic to other organisms such as birds, fish, other vertebrates, and mammals, including humans. However, because of the relatively high cost of these bioinsecticides, methods of enhancing endotoxin yields would be useful.

B. thuringiensis subsp. israelensis serves as a basis for products designed to control mosquitos and blackfly larve (Federici, J. Amer. Mosquito Control Assn. 11: 260-268 (1995)). This subspecies also produces small amounts of the 20 kDa protein, which is the second protein encoded by the crylVD endotoxin operon (Adams et al., J.

Bacteriol. 171: 521-530 (1989); Visick Whiteley, J. Bacteriol. 173: 1748-1756 (1991)). Studies have shown that in E. coli and B. thuringiensis, the 20 kDa protein gene must be expressed to produce significant amounts of CytA, which is the most prominent endotoxin expressed by B. thuringiensis subsp. israelensis (Adams et al., supra; Visick Whiteley, supra; Wu Federici, J. Bacteriol. 175: 5276-5280 (1993)).

Subsequently, the gene encoding the crylVD operon 20 kDa protein ("20 kDa protein gene") was found to enhance production of a member of the structurally unrelated cry family, CrylVD (Visick Whiteley, supra; Wu Federici (1993), supra; Wu Federici, Appl. Microbiol. Biotechnol. 42: 697-702 (1995)). The 20 kDa protein may enhance production of some endotoxins by acting as a possible chaperonin (Crickmore Ellar, Mol. Microbiol. 6: 1533-1537 (1992); Crickmore et al., Aspects Appl. Biol. 24: 17-24 (1990)). However, further studies have shown that expression of the 20 kDa protein gene in E. coli enhances the production of some endotoxin genes, but not others (Yoshisue et al., Biophys. Biotechnol. Biochem. 56: 1429-1433 (1992)). The ability of the 20 kDa protein to enhance production of insecticidal endotoxins is therefore unpredictable.

SUMMARY OF THE INVENTION The 20 kDa protein gene surprisingly can be used to enhance the production of Lepidoptera-specific proteins (CryIB and CryIC), Lepidoptera- and Diptera-specific proteins (CryllA), and Coleoptera-specific proteins (CryIIIA). Thus, the present WO 97/39623 PCT/TSO7/1 A43 3 invention provides a method of enhancing production of the insecticidal endotoxins CryIIA, CryIIIA, CryIB, and CryIC by introducing 20 kDa protein gene into cells that are competent to express these insecticidal endotoxins.

In one aspect, this invention provides a method of enhancing production of an insecticidal endotoxin in a host, which is carried out in two steps: First, the host cell is transformed with the 20 kDa protein gene. The host cell is competent to express cryIA, cryllIA, crylB, or crylC. Second, the 20 kDa protein gene is expressed in the host cell. Expression of the 20 kDa protein gene in the host cell enhances production of the specific endotoxin as compared to a host cell that is not transformed with the 20 kDa protein gene.

In one embodiment, the host cell is B. thuringiensis or E. coli. In another embodiment, the host cell is a commercial strain of B. thuringiensis, and in a further embodiment, the host cell is B. thuringiensis subsp. kurstaki, B. thuringiensis subsp.

tenebrionis, or B. thuringiensis subsp. israelensis strain 4Q7.

In one embodiment, the insecticidal endotoxin is expressed endogenously in B. thuringiensis. In a further embodiment, the endogenously expressed insecticidal endotoxin is CryIIA or CryIIIA.

In one embodiment, the host cell is transformed with a gene encoding an insecticidal endotoxin, and in a further embodiment the endotoxin is CryIIA or CryIIIA.

In another embodiment, the host cell transformed with an exogenous endotoxin gene is B. thuringiensis or E. coli.

In one embodiment, the 20 kDa protein gene is operably linked to a crylA promoter.

In another aspect, the method of enhancing production of an insecticidal endotoxin in B. thuringiensis is composed of two steps: First, transforming B.

thuringiensis with a 20 kDa protein gene operably linked to a crylA(c) promoter. The transformed B. thuringiensis is competent to express CryllA, CryIIIA, CryIB, or CryIC.

Second, the 20 kDa protein gene is expressed, so that production of the specific endotoxin is enhanced as compared to B. thuringiensis that is not transformed with the kDa protein gene.

In another aspect, the invention provides a method of controlling insects by applying to a locus an insecticidally effective amount of insecticidal endotoxin prepared by the methods of the invention as summarized above.

WO 97/39623 PCT/ITS97/1 4 In another aspect, the invention provides a recombinant bacterium that is transformed with the 20 kDa protein gene and has enhanced production of insecticidal endotoxins, prepared by the methods of the invention as summarized above.

BRIEF DESCRIPTION OF THE DRAWINGS Not applicable.

DETAILED DESCRIPTION I. Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); and Hale Marham, The Harper Collins Dictionary of Biology (1991). Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

"Polynucleotide" and "nucleic acid" refer to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic nonnaturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs. It will be understood that when a nucleotide sequence is represented by a DNA sequence A, T, G, this also includes an RNA sequence A, U, G, C) in which replaces "Recombinant" refers to polynucleotides synthesized or otherwise manipulated in vitro ("recombinant polynucleotides") and to methods of using recombinant polynucleotides to produce gene products encoded by those polynucleotides in cells or other biological systems. For example, an cloned polynucleotide may be inserted into a suitable expression vector, such as a bacterial plasmid, and the plasmid WO9 7/3962 PCT/US97/14052 can be used to transform a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a "recombinant host cell" or a "recombinant bacterium." The gene is then expressed in the recombinant host cell to produce, a "recombinant protein." A recombinant polynucleotide may serve a non-coding function promoter, origin of replication, ribosome-binding site, etc.) as well.

A "heterologous polynucleotide sequence" or a "heterologous nucleic acid" is a relative term referring to a polynucleotide that is functionally related to another polynucleotide, such as a promoter sequence, in a manner so that the two polynucleotide sequences are not arranged in the same relationship to each other as in nature.

Heterologous polynucleotide sequences include, a promoter operably linked to a heterologous nucleic acid, and a polynucleotide including its native promoter that is inserted into a heterologous vector for transformation into a recombinant host cell.

Heterologous polynucleotide sequences are considered "exogenous" because they are introduced to the host cell via transformation techniques. However, the heterologous polynucleotide can originate from a foreign source or from the same source.

Modification of the heterologous polynucleotide sequence may occur, by treating the polynucleotide with a restriction enzyme to generate a polynucleotide sequence that can be operably linked to a regulatory element. Modification can also occur by techniques such as site-directed mutagenesis.

The term "expressed endogenously" refers to polynucleotides that are native to the host cell and are naturally expressed in the host cell.

An "expression cassette" refers to a series of polynucleotide elements that permit transcription of a gene in a host cell. Typically, the expression cassette includes a promoter and a heterologous or native polynucleotide sequence that is transcribed.

Expression cassettes may also include, transcription termination signals, polyadenylation signals, and enhancer elements.

A "promoter" is an array of nucleic acid control sequences, e.g, the crylA(c) promoter from B. thuringiensis, that direct transcription of an associated polynucleotide, which may be a heterologous or native polynucleotide. A promoter includes nucleic acid sequences near the start site of transcription, such as a polymerase binding site. The promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.

WO 97/39623 PCTIS97/14052 6 The term "operably linked" refers to a functional relationship between two parts in which the activity of one part the ability to regulate transcription) results in an action on the other part transcription of the sequence). Thus, a polynucleotide is "operably linked to a promoter" when there is a functional linkage between a polynucleotide expression control sequence (such as a promoter or other transcription regulation sequences) and a second polynucleotide sequence a native or a heterologous polynucleotide), where the expression control sequence directs transcription of the polynucleotide.

"Bacillus thuringiensis" refers to a gram positive soil bacterium characterized by its ability to produce crystalline inclusions during sporulation. The inclusions include insecticidal endotoxins.

A "commercial strain" of Bacillus thuringiensis refers to a strain that has been used or developed as an insecticide, Bacillus thuringiensis subsp. kurstaki, Bacillus thuringiensis subsp. tenebrionis, and Bacillus thuringiensis subsp. aizawai.

An insecticidal endotoxin refers to a family of genes encoding endotoxin proteins that exhibit insecticidal activity, also known as crystal proteins, CryIIA, CryIIIA, CryIB, CryIC (see Hofte Whiteley, Microbiol. Rev. 53: 242-255 (1989)).

Such insecticidal endotoxins are produced by Bacillus thuringiensis and are toxic to insects, particularly insect larvae.

An "insecticidally effective amount" of an insecticidal endotoxin is a unit dose amount that provides insecticidal activity when applied to a plant, soil, or another "locus," e.g, site or location.

The "gene encoding the crylVD operon 20 kDa protein" (20 kDa protein gene) refers to the gene in the cryIVD operon that encodes a protein of approximately kDa (as described in Frutos et al., Biochem. Sys. and Ecol. 19: 599-609 (1991); see Frutos et al. Figure 4 for nucleotide and amino acid sequence).

"Enhancing production" refers to an activity of a first protein, such as the crylV operon 20 kDa protein, that increases the net amount of a second protein, such as an insecticidal endotoxin, in a host cell.

"Competent to express" refers to a host cell that provides a sufficient cellular environment for expression of endogenous and/or exogenous polynucleotides.

WO 97/39623 PCTfUS97/14052 7 II. 20 kDa protein gene and insecticidal endotoxin genes In the method of the present invention, host cells are transformed with the kDa protein gene, which encodes a known protein (Frutos et al., supra; Visick Whitely, supra), to enhance the production of specific insecticidal endotoxins. In order to practice the invention, recombinant vectors that express the 20 kDa protein gene are provided. The 20 kDa protein gene is isolated and sequenced from two subspecies of B.

thuringiensis (Frutos et al., Biochem. Syst. and Ecol. 19: 599-609 (1991)). The level of expression of the 20 kDa protein has been characterized in cells transformed with the kDa protein gene (Adams et al., supra; Visick et al., supra). Using methods and sequence information described herein, the 20 kDa protein gene can be isolated by those skilled in the art and used to construct recombinant expression vectors for transformation of a host cell.

The host cells transformed with the 20 kDa protein gene are competent to express specific insecticidal endotoxins. The cells may express the endotoxins endogenously, or the cells may be transformed with exogenous endotoxin expression vectors. Thus, recombinant vectors that express specific endotoxins are provided. The genes for cryllA, crylIA, crylB, and crylC have been isolated and sequenced (see Hofte Whiteley, supra, and references cited therein). This sequence information can be used by one skilled in the art, along with the methods described herein, to construct recombinant vectors for transformation of a host cell with cryllA, crylllA, crylB, or crylC. CryllA and cryllA are preferred insecticidal endotoxin genes.

III. Preparation and transformation of recombinant vectors A recombinant expression vector for transformation of a host cell is prepared by first isolating the constituent polynucleotide sequences, as discussed herein.

The polynucleotide sequences, the 20 kDa protein gene or an insecticidal endotoxin gene, are then ligated to create a recombinant expression vector suitable for transformation of a host cell. Methods for isolating and preparing recombinant polynucleotides are know to those skilled in the art (see Sambrook et al., Molecular Cloning. A Laboratory Manual (2d ed. 1989); Ausubel et al., Current Protocols in Molecular Biology (1995)).

WO 97/39623 PCT/US9714052 8 One preferred method for obtaining specific polynucleotides combines the use of synthetic oligonucleotide primers with polymerase extension or ligation on a mRNA or DNA template. Such a method, RT, PCR, or LCR, amplifies the desired nucleotide sequence (see U.S. Patents 4,683,195 and 4,683,202). Restriction endonuclease sites can be incorporated into the primers. Amplified polynucleotides are purified and ligated to form an expression cassette. Alterations in the natural gene sequence can be introduced by techniques such as in vitro mutagenesis and PCR using primers that have been designed to incorporate appropriate mutations. Another preferred method of isolating polynucleotide sequences uses known restriction endonuclease sites to isolate nucleic acid fragments from plasmids. In a preferred embodiment of the invention, the 20 kDa protein gene is initially isolated as a restriction enzyme fragment from the plasmid pM1 (Galjart et al., Curr. Microbiol. 16: 171-177 (1987); see Example I and Wu Federici (1993), supra). The 20 kDa protein gene can also be isolated by one of skill in the art using primers based on the known gene sequence (Frutos et al., supra, Figure 4).

The isolated polynucleotide sequence of choice, the 20 kDa protein gene or an insecticidal endotoxin gene, is inserted into an "expression vector," "cloning vector," or "vector," terms which usually refer to plasmids or other nucleic acid molecules that are able to replicate in a chosen host cell. Expression vectors can replicate autonomously, or they can replicate by being inserted into the genome of the host cell. Often, it is desirable for a vector to be usable in more than one host cell, e.g., in E. coli for cloning and construction, and in B. thuringiensis for expression.

Additional elements of the vector can include, for example, selectable markers, e.g., tetracycline resistance or hygromycin resistance, which permit detection and/or selection of those cells transformed with the desired polynucleotide sequences (see, U.S.

Patent 4,704,362). The particular vector used to transport the genetic information into the cell is also not particularly critical. Any suitable vector used for expression of recombinant proteins host cells can be used. A preferred vector of the invention is pHT3101, which is an E. coli-B. thuringiensis shuttle vector (Lereclus et al., FEMS Microbiol. Lett. 60: 211-218 (1989)).

Expression vectors typically have an expression cassette that contains all the elements required for the expression of the polynucleotide of choice in a host cell. A typical expression cassette contains a promoter operably linked to the polynucleotide WO 97/39623 PCT/US97/14052 9 sequence of choice. The promoter used to direct expression of the nucleic acid depends on the particular application, for example, it may be operably linked to a heterologous polynucleotide or to its native polynucleotide. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function. Often the promoter used to drive the polynucleotide is a crystal protein promoter. Other promoters include any promoter suitable for driving the expression of a heterologous gene in a host cell, including those typically used in standard expression cassettes, e.g., SV40 and CMV promoters. In one embodiment of the invention, described in Example I, the 20 kDa protein gene is operably linked to the BtI and.BtII promoters ("the crylA(c) promoter") of the crylA gene, creating a heterologous nucleic acid operably linked to a promoter. The crylA(c) promoter is highly active in growth conditions that induce sporulation. In another embodiment, described in Example II, the crylllA gene is operably linked to its native promoter.

An expression cassette may also include enhancer elements that can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers include, for example, the SV40 early gene enhancer, which is suitable for many cell types. Additional enhancer combinations that are suitable for the present invention include those derived from polyoma virus, and human or murine cytomegalovirus (see Enhancers and Eukaryotic Expression (1983)).

Polyadenylation sequences are also commonly present in expression cassettes. Termination and polyadenylation signals that are suitable for the present invention are derived from standard sources and can include native 20 kDa protein and endotoxin sequences. Other suitable sequences include polyadenylation and termination sequences derived from SV40, or a partial genomic copy of a gene already resident on the expression vector. In one embodiment of the invention, the 20 kDa protein gene expression cassette includes a native hairpin transcription terminator (see Example I, plasmid pWP41), and in another embodiment, the hairpin transcription terminator has been deleted from the 20 kDa protein gene expression cassette (see Example I, plasmid pWP61).

WO 97/39623 Pr'T/iTS'7/1 IV. Expression of the 20 kDa protein gene and enhanced crystal protein production After construction and isolation of the recombinant expression vector, it is used to transform a host cell for expression of the gene of interest, e.g, the 20 kDa protein gene or an insecticidal endotoxin gene. The particular procedure used to introduce the genetic material into the host cell for expression of a protein is not particularly critical. Any of the well known procedures for introducing foreign polynucleotide sequences into host cells can be used. Transformation methods, which vary depending on the type of host cell, include electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent); and other methods (see generally Sambrook et al., supra; Ausubel et al., supra). A preferred method of transforming B. thuringiensis is electroporation, as described in Wu et al., Mol. Microbiol. 13: 965-972 (1994).

Hosts for transformation with the 20 kDa protein gene include any suitable host competent to express an insecticidal endotoxin for enhanced production. Hosts that are transformed with the 20 kDa protein gene alone or co-transformed with an insecticidal endotoxin gene are useful recombinant bacteria for enhanced production of endotoxins and as insecticides. Such hosts include those that endogenously express insecticidal endotoxins, such as B. thuringiensis, as well as those that can express insecticidal endotoxins upon transformation with an expression vector, such as E. coli.

Suitable hosts for expression and enhanced production of an exogenous insecticidal endotoxin include organisms from the genera Bacillus, Pseudomonas, and Escherichia, E. coli, B. thuringiensis subsp. israelensis strain 4Q7 (acrystalliferous), B. subtilis, B. megaterium, and P. fluorescens. Suitable hosts for endogenous expression and enhanced production of insecticidal endotoxins include subspecies and commercial strains of Bacillus thuringiensis (see, Microbial Control of Pests and Plant Diseases 1970- 1989 (Burges ed., 1981)). Preferred subspecies of B. thuringiensis include, B.

thuringiensis subsp. kurstaki, B. thuringiensis subsp. aizawai, B. thuringiensis subsp.

israelensis, and B. thuringiensis subsp. tenebrionis. Preferred commercial strains include B. thuringiensis subsp. kurstaki strains HD-1 and NB75, and B. thuringiensis subsp.

tenebrionis strains NB176 and DSM 2803.

WO 97/39623 PCTTTSQ'7/14n5 11 After the host cell is transformed with the 20 kDa protein gene, the host cell is incubated under conditions suitable for expression of the 20 kDa protein gene and insecticidal endotoxin genes. Typically, the host will be grown under conditions that promote sporulation and expression of insecticidal endotoxin genes. Host cells may be prepared in any quantity required by fermenting an inoculum in standard media known to those skilled in the art. The media will, for example, generally contain a nitrogen source and a carbohydrate source, glucose. Suitable conditions for incubation include a temperature in the range of 15-45°C, preferably 30 0 C, and an approximately neutral pH.

Incubation may be conveniently carried out in batches, typically for a period of 3-5 days.

E. coli strains transformed with exogenous insecticidal endotoxin expression vectors may be prepared by growing cells to stationary phase on solid nutrient media, L-agar.

In one preferred embodiment, an inoculum from a stock host cell culture is grown on PWYE medium peptone, 0.1% yeast extract, 0.5% NaC1, pH Herrnstadt et al., Biotechnology 4: 305-308 (1986)). When the cells are in log-phase growth, the culture is diluted with either nutrient broth or G-Tris medium and incubated at 30°C in a shaker until sporulation and cell lysis were at least 95% complete, usually 3-4 days (see Example I).

Enhanced production of CryIIA, CryIIIA, CryIB, and CryIC is observed after host cells competent to express these genes are transformed with the 20 kDa protein gene and the cells are grown under suitable conditions. Enhanced production of specific insecticidal endotoxins may be observed by standard methods know to those skilled in the art. For example, parasporal inclusions of insecticidal endotoxins can be purified (see Wu Federici (1995), supra, harvested by centrifugation from lysed cultures (see Example or examined with microscopy (see Wu Federici (1995), supra).

Parasporal inclusions that have been harvested by centrifugation or purified may be separated using standard methods known in the art, for example, chromatography, immunoprecipitation, ELISA, bioassay, western analysis, or gel electrophoresis (see, Wu Federici (1995), supra; Ausubel et al., supra). Amounts of protein are quantified by suitable means, including width and intensity of stained bands, densitometry, bioactivity, and fluorescence. Enhanced production is determined through comparison with "net" endotoxin amounts from control hosts that are not transformed with the 20 kDa protein gene. Net endotoxin amounts refers to the amount of endotoxin in parasporal bodies or crystals. The control hosts are otherwise genetically identical WO 97/39623 PCT/S97/14052 12 with the transformed hosts and grown on comparative media. Enhancement is any statistically significant increase in endotoxin production, and is preferably 5-10% greater and most preferably 10-300% greater. In a preferred embodiment, parasporal bodies are isolated by centrifugation from lysed cultures and are examined by SDS-PAGE gels stained with Coomassie blue.

V. Use as an insecticide Host cells that have been transformed with the 20 kDa protein gene and produced enhanced amounts of insecticidal endotoxins are used as insecticides to control insects. Insects that can be controlled with insecticidal endotoxins include, e.g., lepidopterans, dipterans, and coleopterans. For example, B. thuringiensis subsp.

kurstaki, which expresses CryIIA, is useful to control caterpillar pests (Frankenhuyzen, supra) including, the corn earworm (Heliothis zia), the cabbage looper (Trichoplusia ni), and the fall army worm (Spodoptera frugiperda). B. thuringiensis subsp. israelensis is useful to control dipteran pests such as mosquito and blackfly larvae.

B. thuringiensis subsp. morrisoni is active, against coleopteran pests such as the Colorado potato beetle (Leptinotarsa decemlineata), and the cottonwood leaf beetle (Chrysomela scripta). A preferred recombinant host cell for control of insects is B.

thuringiensis subsp. kurstaki.

Insecticidal compositions can be produced, from live recombinant bacterium that express the 20 kDa protein and an insecticidal endotoxin, lysed host cells, spores, isolated parasporal bodies, and isolated insecticidal endotoxins. The compositions may contain agriculturally-acceptable adjuvants for topical application to the desired plant, soil or other locus. The compositions can take any form known in the art for the formulation of insecticides, in a suspension, a dispersion, an aqueous emulsion, a dusting powder, a dispersible powder, an emulsifiable concentrate, or granules. Moreover, the composition can be in a suitable form for direct application to a locus or as a concentrate of primary composition which requires dilution with a suitable quantity of water or other diluent before application.

The compositions are applied in an insecticidally effective amount, which will vary depending on such factors as, for example, the specific lepidopteran, dipteran or coleopteran insects to be controlled, the specific plant to be treated, and the method of applying the insecticidally active compositions. Some compositions may be composed of WT OT97/t963 PrT/TSQ'7/14 13 almost pure insecticidal endotoxin. The concentration of insecticidally active endotoxin is preferably within the range from about 0.5 to about 75 by weight, especially about to 40 by weight. The compositions can be applied, for example, by spraying or dusting a locus such as a plant or soil that is infested or liable to be infested.

EXAMPLES

The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

Example I: Construction and expression of a 20 kDa protein gene to enhance production of endogenously expressed CryIIA and CryIIIA A. Materials and Methods 1. Bacterial subspecies and strains The subspecies and strains of B. thuringiensis used in this example and their sources are listed below in Table 1.

TABLE 1 Subspecies Strain Source B.t. subsp. kurstaki HD-1 USDA, Brownsville, Texas B.t. subsp. kurstaki NB75 Novo Nordisk, Davis, CA B.t. subsp. tenebrionis DSM 2803 Novo Nordisk, Davis, CA B.t. subsp. tenebrionis NB176 Novo Nordisk, Davis, CA The HD-1 and NB75 strains of B.t. subsp. kurstaki are those used, respectively, in the commercial products Dipel and Biobit. Strain DSM 2803 is the original isolate of B.t. subsp. tenebrionis, while strain NB176 is a mutant strain derived WO 97/39623 PCT/US97/14052 14 from the original by mutagenesis. Strain NB176 produces 2-3 fold the amount of CryIIIA produced by the original isolate, DSM 2803.

2. Construction of 20 kDa protein gene expression vectors The 20 kDa protein gene was placed under control of the BtI and BtII promoters of the crylA(c) gene and cloned into an expression vector (see Wu Federici (1993), supra). First, most of the coding sequence in a M13 clone of the crylA(c) gene was replaced with a fragment of the crylVD operon that contained a portion of the 3' end of the cryIVD gene and the entire 20 kDa ORF. Specifically, the crylA(c) gene was removed from the eighth codon onward and replaced with the portion of the crylVD operon extending from the 51st terminal codon through to the Clal site, 154 bases after the end of the gene, creating construct WF40. Second, this construct was removed from M13 DNA and cloned into the Escherichia coli-B. thuringiensis shuttle vector pHT3101 using the restriction sites SphI and Sall to produce plasmid pWF41.

In an attempt to increase production of the 20 kDa protein, the putative weak hairpin terminator that exists between the 3' end of crylVD and the 5' end of the kDa ORF was removed to create plasmid pWF61. First, construct WF40, in which the hairpin structure is located between two NdeI sites 300 bp apart, was partially digested with NdeI and then religated. In WF40 there are five Ndel sites, two in the Bt DNA and three in the M13 vector DNA. Deletions in the M13 portions destroy the ability of the phage DNA to replicate, and therefore the only possible recombinants that could be obtained were religations that restored the original structure or those in which the Bt DNA NdeI fragment containing the hairpin structure was absent. The latter recombinant construct, designated WF60, was differentiated from WF40 on the basis of size. Second, construct WF60 was cloned from M13 into pHT3101 using the SphI and Sall sites to yield pWF61.

3. Transformation of Bt cells The modified pHT3101 plasmids designated pWF41 and pWF61 were transformed into the various strains of B. thuringiensis by electroporation as described by Wu et al. (1994), supra.

WO 97/39623 PCT/US97/14052 4. Growth conditions For each strain, a starter culture was prepared by inoculating 1 ml of PWYE medium peptone, 0.1% yeast extract, 0.5% NaC1, pH7.5; Herrstadt et al., Biotechnology 4: 305-308 (1986)) with cells from stock cultures, and growing this inoculum overnight at 30 0 C in a shaker (250 rpm). The overnight culture was then diluted 1:10 in the same medium for 30 minutes to ensure there was no cell clumping.

While the cells were in log-phase growth, the culture was diluted 1:100 with either nutrient broth or G-Tris medium and grown in 100 ml of medium in 200 ml flasks at 30 0 C in a shaker (250 rpm) until sporulation and cell lysis were at least 95% complete, which usually required 3-4 days. To the cultures of cells transformed with the two different constructs (pWF41 and pWF61) used to produce the 20-kDa protein, 25 mg/ml of erythromycin was added to the medium.

5. Quantification of endotoxin yield To quantify the differences in parasporal body crystal production between non-transformed cells and cells transformed with constructs used to express the protein gene, crystals were harvested from lysed cultures and quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see Wu Federici (1995), supra). For most strains, 1 ml of the lysed culture was collected and pipetted into a 1.5 ml Eppendorf tube. The crystals and spores were sedimented by centrifugation, and the pellets were immediately subjected to SDS-PAGE or stored at In addition, parasporal crystals were purified from spores and cell debris by centrifugation on sodium bromide gradients (Ibarra Federici, J. Bacteriol. 165: 527- 533 (1986)), and also quantified by SDS-PAGE.

6. Sodium dodecvl sulfate-polvacrylamide gel electrophoresis (SDS-PAGE) Leammli sample buffer (200 1l of 0.125 M Tris-HCI pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol and 20 mM EDTA) was added to the pellets prepared as described above. The samples were then boiled for 5 min or until the pellets were completely dissolved, and the proteins separated on acrylamide gels (Leammli, Nature 227: 680-685 (1970). Protein compositions were analyzed by polyacrylamide gel electrophoresis as described by Leammli, supra. Molecular mass wn Q7/36on PCT/ TS9'7/14052 16 marker proteins used in the same gels were myosin (200 kDa), 0-galactosidase, (116 kDa), phosphorylase B (97 kDa), serum albumin (66 kDa), and ovalbumin (45 kDa).

B. Results 1. Enhanced production of CrvIIA in B. thuringiensis subsp. kurstaki.

The first experiment examined the effect of the 20 kDa protein on production of CryIIA in the HD-1 strain provided by the USDA. In comparison to the untransformed control strain, HD-1 transformed with either pWF41 or pWF61 produced increased yields of endogenously expressed CryIIA protein. Based on the width and density of the protein CryIIA band separated by SDS-PAGE, the increase obtained with pWF41 was approximately 100%, whereas the increase obtained with pWF61 was about Very similar results were obtained with the NB75 strain derived from HD-1.

Interestingly, the yield of CryIIA was about 20% greater in the USDA HD-1 strain transformed with pWF61.

To determine whether the increases in endotoxin yields in the transformed cells were due to net increases per cell rather than in increase in the number of cells per ml, the growth rates of the standard cells and transformed cells were compared by measuring optical density at A6 from the initiation of a log-phase growth until sporulation was complete. No significant differences were observed between the control untransformed HD-1 cells and cells transformed with either pWF41 or pWF61.

2. Enhanced production of CryIIIA in B. thuringiensis subsp. tenebronis Both strains of B. thuringiensis subsp. tenebrionis transformed with either pWF41 or pWF61 showed a marked increase in endogenously expressed CryIIIA production in comparison to the untransformed control strain. The CryIIIA yield from DSM 2803 cells (the original Btt isolate) transformed with pWF61 was about 100% greater than that of non-transformed cells of the same strain. A similar increase in CryIIIA crystal yield was obtained with the mutant NB176 strain transformed with pWF41, but the yield of this strain transformed with pWF61 did not appear, based on SDS gels, to be any greater than that obtained with control untransformed NB176 cells.

The control and transformed Btt cells all grew and sporulated normally.

When examined by phase microscopy, NB176 transformed with pWF61 produced the largest crystals among all of the transformed and non-transformed strains of B.

WO 97/19623 PCT/nS97/14t52 17 thuringiensis subsp. tenebrionis tested. However, autolysis of this strain was markedly slower than that of the other strains, and even as late as five days after culture initiation, only 50-60% of the cells had, lysed. Thus, the yield indicated by SDS-PAGE may be under estimated in this construct by as much as The above experiments show that expression of the 20 kDa protein gene can substantially increase the yield of endotoxin proteins in strains of B. thuringiensis, including commercial strains. These results indicate that typical increases for CryIIlA subsp. tenebrionis) endotoxin is in the range of 50-100%. Moreover, in the case of the Cryll endotoxin subsp. kurstaki), increases in endotoxin yields of up to 50%, in some cases higher, could be obtained in the presence of the 20 kDa protein. In these experiments, the quantification of endotoxin yield was based on the width and intensity of protein bands in SDS-polyacrylamide gels stained with Coomassie Brilliant Blue.

Example II: Enhanced production of CryIIIA protein expressed from a recombinant plasmid The use of the 20 kDa protein gene to enhance production of an exogenous CrylIIA endotoxin is examined. A recombinant crylllA expression vector and the kDa protein gene expression vector are used to co-transform Bacillus thuringiensis according to the methods described above, with the following changes. The recombinant cryllA expression vector was constructed as described above, substituting the crylllA gene for the 20 kDa protein gene. Bacillus thuringiensis subsp. israelensis strain 4Q7, an acrystalliferous strain, is used for co-transformation with the cryIIA and 20 kDa protein gene expression vectors. This strain has been cured of all native crystal protein plasmids and is useful for expression of exogenous crystal protein genes.

20 kDa protein gene expression vectors pWF41 and pWF61 are separately co-transformed with the crylIIA expression vector, and enhanced production of CryIIIA protein from an exogenous gene is observed as described in Example I.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily WO 97/39623 PCT/US97/14052 18 apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims (23)

1. A method of enhancing production of an insecticidal endotoxin in a host cell, said method comprising the steps of: a. transforming the host cell with a gene encoding a crylVD operon 20 kDa protein, wherein the host cell is competent to express an insecticidal endotoxin selected from the group consisting of CryIIA, CryIIIA, CryIB, and CryIC; and b. expressing the gene encoding the crylVD operon 20 kDa protein in the host cell; whereby expression of the gene encoding the cryIVD operon 20 kDa protein in the host cell enhances production of the insecticidal endotoxin as compared to production of insecticidal endotoxin in a host cell that is not transformed with the gene encoding the crylVD operon 20 kDa protein.

2. The method of claim 1, wherein the host cell is selected from the group consisting of Bacillus thuringiensis and E. coli.

3. The method of claim 2, wherein the host cell is Bacillus thuringiensis.

4. The method of claim 2, wherein the insecticidal endotoxin is expressed endogenously in Bacillus thuringiensis.

5. The method of claim 4, wherein the insecticidal endotoxin is selected from the group consisting of CryIIA and CryIIIA.

6. The method of claim 1, further comprising the step of transforming the host cell with a gene encoding an insecticidal. endotoxin.

7. The method of claim 6, wherein the host cell is selected from the group consisting of Bacillus thuringiensis and E. coli. WO 97/39623 PCT/US97/14052

8. The method of claim 7, wherein the insecticidal endotoxin is selected from the group consisting of CryllA and CrylIIA.

9. The method of claim 1, wherein the host cell is a commercial strain of Bacillus thuringiensis.

The method of claim 9, wherein Bacillus thuringiensis is selected from the group consisting of Bacillus thuringiensis subsp. kurstaki, Bacillus thuringiensis subsp. tenebrionis, and Bacillus thuringiensis subsp. israelensis (strain 4Q7).

11. The method of claim 1, wherein the gene encoding the cryIVD operon 20 kDa protein is operably linked to a crylA(c) promoter.

12. A method of enhancing production of an insecticidal endotoxin in Bacillus thuringiensis, said method comprising the steps of: a. transforming Bacillus thuringiensis with a gene encoding a crylVD operon 20 kDa protein operably linked to a crylA(c) promoter, wherein the Bacillus thuringiensis is competent to express an insecticidal endotoxin selected from the group consisting of CryllA, CrylIIA, CryIB, and CryIC; and b. expressing the gene encoding the crylVD operon 20 kDa protein operably linked to a crylA(c) promoter in Bacillus thuringiensis; whereby expression of the gene encoding the cryIVD operon 20 kDa protein operably linked to the crylA(c) promoter enhances production of the insecticidal endotoxin as compared to production of insecticidal endotoxin in Bacillus thuringiensis that is not transformed with the gene encoding the cryIVD operon 20 kDa protein.

13. A method of controlling insects, said method comprising applying to a locus an insecticidally effective amount of an insecticidal endotoxin prepared according to the method of claim 1.

14. A method of creating a recombinant bacterium, said method comprising the steps of: WO 97/39623 PCT[US97/14052 21 a. transforming the recombinant bacterium with a gene encoding a crylVD operon 20 kDa protein, wherein the recombinant bacterium is competent to express an insecticidal endotoxin selected from the group consisting of CryIIA, CrylIIA, CryIB, and CryIC; and b. expressing the gene encoding the cryIVD operon 20 kDa protein in the recombinant bacterium; whereby expression of the cryIVD operon 20 kDa protein in the recombinant bacterium enhances production of the insecticidal endotoxin as compared to production of insecticidal endotoxin in a bacterium that is not transformed with the gene encoding the crylVD operon 20 kDa protein.

The method of claim 14, wherein the recombinant bacterium is selected from the group consisting of Bacillus thuringiensis and E. coli.

16. The method of claim 15, wherein the insecticidal endotoxin is expressed endogenously in Bacillus thuringiensis.

17. The method of claim 16, wherein the insecticidal endotoxin is selected from the group consisting of CryllA and CryIIlA.

18. The method of claim 14, further comprising the step of transforming the recombinant bacterium with a gene encoding an insecticidal endotoxin.

19. The method of claim 18, wherein the recombinant bacterium is selected from the group consisting of Bacillus thuringiensis and E. coli.

The method of claim 19, wherein the insecticidal endotoxin is selected from the group consisting of CryIIA and CryIlA.

21. The method of claim 14, wherein the recombinant bacterium is a commercial strain of Bacillus thuringiensis. WO 97/39623 PCT1US97/14052 22

22. The method of claimn 21, wherein Bacillus thuringiensis is selected from the group consisting of Bacillus thuringiensis subsp. kurstaki, Bacillus thuringiensis subsp. tenebrionis, and Bacillus thuringiensis subsp. israelensis (strain 4Q7).

23. The method of claim 14, wherein the gene encoding the cryIVL) operon 20 kDa protein is operably linked to a crylA(c) promoter.