Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
  • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins

Definitions

• the present invention relates to a method of producing insecticidal endotoxins and recombinant cells that express such endotoxins.
• 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 (I), Lepidoptera- and Diptera-specific (II), Coleoptera- specific (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 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. Ill : 521-530 (1989); Visick & Whiteley, J. Bacteriol. 173: 1748-1756 (1991)). Studies have shown that in E. coli and B.
• 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)).
• 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 &
• 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)).
• Crickmore & Ellar Mol. Microbiol. 6: 1533-1537 (1992); Crickmore et al , Aspects Appl. Biol. 24: 17-24 (1990)
• expression of the 20 kDa protein gene in E. coli enhances the production of some endotoxin genes, but not others (Yoshisue et al., Biophys. Bioiechnol. Biochem. 56: 1429-1433 (1992)).
• the ability of the 20 kDa protein to enhance production of insecticidal endotoxins is therefore unpredictable.
• the 20 kDa protein gene surprisingly can be used to enhance the production of Lepidoptera-specific proteins (Cry IB and CrylC), Lepidoptera- and Diptera-specific proteins (Cry II A), and Coleoptera-specific proteins (CrylllA).
• the present invention provides a method of enhancing production of the insecticidal endotoxins CryllA, CrylHA, CrylB, and CrylC by introducing 20 kDa protein gene into cells that are competent to express these insecticidal endotoxins.
• 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 cryllA, cry IIA, cry IB, 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.
• 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. lenebrionis, or B. thuringiensis subsp. israelensis strain 4Q7.
• the insecticidal endotoxin is expressed endogenously in B. thuringiensis. In a further embodiment, the endogenously expressed insecticidal endotoxin is CryllA or CrylHA.
• the host cell is transformed with a gene encoding an insecticidal endotoxin, and in a further embodiment the endotoxin is CryllA or CrylUA.
• the host cell transformed with an exogenous endotoxin gene is B. thuringiensis or E. coli.
• the 20 kDa protein gene is operably linked to a cry ⁇ A(c) promoter.
• the method of enhancing production of an insecticidal endotoxin in B. thuringiensis is composed of two steps: First, transfo ⁇ ning B. thuringiensis with a 20 kDa protein gene operably linked to a cry ⁇ A(c) promoter.
• the transformed B. thuringiensis is competent to express CryllA, CrylllA, CryJJB, or CrylC.
• 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 20 kDa protein gene.
• 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.
• Lhe 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.
• 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 non-naturally occurring analogs thereof.
• 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 (i.e. , A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U” replaces "T.”
• 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.
• recombinant polynucleotides polynucleotides synthesized or otherwise manipulated in vitro
• an cloned polynucleotide may be inserted into a suitable expression vector, such as a bacterial plasmid, and the plasmid c
• 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, e.g. , a "recombinant protein. "
• a recombinant polynucleotide may serve a non-coding function (e.g. , promoter, origin of replication, ribosome-binding site, etc.) as well.
• 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, e.g., 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, e.g., 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.
• the expression cassette includes a promoter and a heterologous or native polynucleotide sequence that is transcribed.
• Expression cassettes may also include, e.g., transcription termination signals, polyadenylation signals, and enhancer elements.
• a “promoter” is an array of nucleic acid control sequences, e.g, the cry ⁇ A(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.
• the term “operably linked” refers to a functional relationship between two parts in which the activity of one part (e.g. , the ability to regulate transcription) results in an action on the other part (e.g.
• 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 (e.g. , a native or a heterologous polynucleotide), where the expression control sequence directs transcription of the polynucleotide.
• a polynucleotide expression control sequence such as a promoter or other transcription regulation sequences
• a second polynucleotide sequence e.g. , a native or a heterologous polynucleotide
• Bactus 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, e.g. , 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, e.g. , CryllA, CrylHA, CrylB, CrylC (see Hofte & Whiteley, Microbiol. Rev. 53: 242-255 (1989)).
• 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 cryWD operon that encodes a protein of approximately 20 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 cryTV 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. II. 20 kDa protein gene and insecticidal endotoxin genes
• host cells are transformed with the 20 kDa protein gene, which encodes a known protein (Frutos et al., supra; Visick & Whitely, supra), to enhance the production of specific insecticidal endotoxins.
• 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 20 kDa protein gene (Adams et al. , supra; Visick et al., supra).
• 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.
• recombinant vectors that express specific endotoxins are provided.
• the genes for cryllA, crylHA, 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, crylHA, crylB, or crylC.
• CryllA and crylHA are preferred insecticidal endotoxin genes.
• 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 e.g. , 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.
• 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.
• a method e.g. , 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 inco ⁇ orated 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.
• the 20 kDa protein gene is initially isolated as a restriction enzyme fragment from the plasmid pMl (Galjart et al , Curr. Microbiol 16: 171-177 (1987); see Example
• 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 e.g. , 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, e.g., 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 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.
• 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.
• the 20 kDa protein gene is operably linked to the Btl and Btll promoters ("the cry ⁇ A(c) promoter") of the cry ⁇ A(c) gene, creating a heterologous nucleic acid operably linked to a promoter.
• the cry ⁇ A(c) promoter is highly active in growth conditions that induce sporulation.
• the cryHIA 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.
• the 20 kDa protein gene expression cassette includes a native hairpin transcription terminator (see Example I, plasmid pWP41), and in another embodiment, the hairpm transcription te ⁇ ninator has been deleted from me 20 kDa protein gene expression cassette (see Example I, plasmid pWP61). IV. Expression of the 20 kDa protein gene and enhanced crystal protein production
• the recombinant expression vector 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.
• 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.g. , 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 oi Bacillus thuringiensis (see, e.g., Microbial Control of Pests and Plant Diseases 1970- 1989 (Burges ed. , 1981)).
• Preferred subspecies of B. thuringiensis include, e.g., 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.
• 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, e.g. , glucose.
• a carbohydrate source e.g. , glucose.
• Suitable conditions for incubation include a temperature in the range of 15-45°C, preferably 30°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, e.g., L-agar.
• an inoculum from a stock host cell culture is grown on PWYE medium (5% peptone, 0.1 % yeast extract, 0.5 % NaCl, pH 7.5; Herrnstadt et al. , Biotechnology 4: 305-308 (1986)).
• PWYE medium 5% peptone, 0.1 % yeast extract, 0.5 % NaCl, pH 7.5; Herrnstadt et al. , Biotechnology 4: 305-308 (1986)
• 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 CryllA, CrylHA, CrylB, and CrylC is observed after host cells competent to express these genes are transformed wim 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 I), 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, e.g., 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.
• control hosts are otherwise genetically identical 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.
• parasporal bodies are isolated by centrifugation from lysed cultures and are examined by SDS-PAGE gels stained with Coomassie blue.
• 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.
• B. thuringiensis subsp. kurstaki which expresses CryllA, is useful to control caterpillar pests (Frankenhuyzen, supra) including, e.g. , the corn earworm (Heliothis zia), the cabbage looper (Trichoplusia ni), and the fall army worm (Spodoptera frugiperda) .
• caterpillar pests including, e.g. , the corn earworm (Heliothis zia), the cabbage looper (Trichoplusia ni), and the fall army worm (Spodoptera frugiperda) .
• B. thuringiensis subsp. morrisoni is active, e.g., against coleopteran pests such as the Colorado potato beetle (Leptinotarsa decemlineat ⁇ ), and the cottonwood leaf beetle (Chrysomela script ⁇ ).
• a preferred recombinant host cell for control of insects is B. thuringiensis subsp. kurstaki.
• Insecticidal compositions can be produced, e.g.
• 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 me art for the formulation of insecticides, e.g., in a suspension, a dispersion, an aqueous emulsion, a dusting powder, a dispersible powder, an emulsifiable concentrate, or granules.
• 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 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 20 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.
• Example I Construction and expression of a 20 kDa protein gene to enhance production of endogenously expressed CryllA and CrylHA
• 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 from the original by mutagenesis. Strain NB176 produces 2-3 fold the amount of CrylHA produced by the original isolate, DSM 2803.
• cry ⁇ A(c) gene was removed from the eighth codon onward and replaced with the portion of the cryTVD operon extending from the 51st terminal codon through to the Clal site, 154 bases after the end of the gene, creating construct WF40.
• this construct was removed from M13 DNA and cloned into the Escherichia coli-B. thuringiensis shuttle vector pHT3101 using the restriction sites Sphl and Sail to produce plasmid pWF41.
• the putative weak hairpin terminator that exists between the 3' end of cryP/D and the 5' end of the 20 kDa ORF was removed to create plasmid pWF61.
• construct WF40 in which the hairpin structure is located between two Ndel sites 300 bp apart, was partially digested with Ndel and then religated.
• WF40 there are five Ndel sites, two in the Bt DNA and three in the M 13 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 tne Bt DNA Ndel fragment containing the hairpin structure was absent.
• the latter recombinant construct, designated WF60 was differentiated from WF40 on the basis of size.
• construct WF60 was cloned from M13 into pHT3101 using the Sphl and Sail sites to yield ⁇ WF61.
• 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. 4. Growth conditions
• a starter culture was prepared by inoculating 1 ml of PWYE medium (5% peptone, 0.1 % yeast extract, 0.5% NaCl, pH7.5; Herrnstadt et al , Biotechnology 4: 305-308 (1986)) with cells from stock cultures, and growing this inoculum overnight at 30 °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.
• PWYE medium 5% peptone, 0.1 % yeast extract, 0.5% NaCl, pH7.5; Herrnstadt et al , Biotechnology 4: 305-308 (1986)
• 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°C in a shaker (250 rpm) until sporulation and cell lysis were at least 95% complete, which usually required 3-4 days.
• sporulation and cell lysis were at least 95% complete, which usually required 3-4 days.
• 25 mg/ml of erythromycin was added to the medium.
• 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 -70°C.
• SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
• parasporal crystals were purified from spores and cell debris by centrifugation on sodium bromide gradients (Ibarra & Federici, /. Bacteriol. 165: 527- 533 (1986)), and also quantified by SDS-PAGE.
• Molecular mass marker proteins used in the same gels were myosin (200 kDa), /3-galactosidase, (116 kDa), phosphorylase B (97 kDa), serum albumin (66 kDa), and ovalbumin (45 kDa).
• the first experiment examined the effect of the 20 kDa protein on production of CryllA in the HD-1 strain provided by the USDA.
• HD-1 transformed with either pWF41 or pWF61 produced increased yields of endogenously expressed CryllA protein.
• the increase obtained with pWF41 was approximately 100%, whereas the increase obtained with pWF61 was about 50% .
• Very similar results were obtained with the NB75 strain derived from HD-1.
• the yield of CryllA was about 20% greater in the USDA HD-1 strain transformed with pWF61.
• Example II Enhanced production of CrylHA protein expressed from a recombinant plasmid
• a recombinant crylHA expression vector and the 20 kDa protein gene expression vector are used to co-transform Bacillus thuringiensis according to the methods described above, with the following changes.
• the recombinant crylHA 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 crylllA 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 me crylllA expression vector, and enhanced production of CrylHA protein from an exogenous gene is observed as described in Example I.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Crystallography & Structural Chemistry (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Agricultural Chemicals And Associated Chemicals (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=24354061

Family Applications (1)

Country Status (5)

Families Citing this family (3)

• 1997
  • 1997-01-17 EP EP97938210A patent/EP0896619A2/de not_active Withdrawn
  • 1997-01-17 CA CA 2242544 patent/CA2242544A1/en not_active Abandoned
  • 1997-01-17 WO PCT/US1997/014052 patent/WO1997039623A2/en not_active Application Discontinuation
  • 1997-01-17 AU AU40590/97A patent/AU711493B2/en not_active Ceased
  • 1997-01-17 JP JP9533863A patent/JP2000506739A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events