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AssigneeMonsanto Company
InventorsWilliam P. Donovan, Yuping Tan
Estimating the term…
The present invention is based on the discovery, isolation, sequencing and characterization of two protease genes present in B.t. One is an alkaline protease gene generally designated "apr." The other is a neutral protease gene generally designated "npr." The invention also includes genetically disabling the apr and the npr genes to reduce proteolytic activity of the proteases encoded by the genes, and using the disabled genes in insecticidal B.t. constructs along with one or more B.t. protein toxin genes. These resulting B.t. constructs are useful for the enhanced production of B.t. toxin protein. The B.t. toxin protein recovered from the fermentation production of these B.t. constructs also exhibits improved stability with respect to its insecticidal activity.
A method of reducing the proteolytic degradation of a Bacillus thuringiensis insecticidal toxic protein in a composition comprising the toxic protein, the method comprising including in the composition a Bacillus thuringiensis protease gene disabled from coding for protease capable of degrading the toxic protein, the protease gene before being disabled being selected from the group consisting of an alkaline protease gene and a neutral protease gene, the alkaline protease gene before being disabled having a nucleotide base sequence coding for the amino acid sequence identified in SEQ ID NO:2, and the neutral protease gene before being disabled has a nucleotide base sequence coding for the amino acid sequence identified in SEO ID NO:4, whereby protease expressed by an organism with in the composition containing the disabled protease gene has reduced proteolytic activity against the toxic protein compared to protease encoded by a counterpart protease gene that has not been disabled, thereby resulting in reduced proteolytic activity against the toxic protein.
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From Bacillus: bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
Bacillus: b. thuringiensis
Derived from bacteria or Archaea: bacteria being Bacillus
Peptides
Term ended 31 Mar 2015 (estimated)
USPTO file history isn’t available for this patent, so only indexed dates are shown.
The present invention relates to an alkaline protease gene (apr) and a neutral protease gene (npr) derived from a Bacillus thuringiensis (B.t.) strain, genetically disabled apr and npr genes, insecticidal B.t. strain constructs containing a genetically disabled apr gene and/or npr gene, an insecticide composition containing the disabled genes or the constructs including the disabled genes, and a method for using the disabled genes and their constructs.
Bacillus thuringiensis ( "B.t.") is a gram-positive soil bacterium that produces proteinaceous crystalline inclusions during sporulation. These B.t. crystal proteins are often highly toxic to specific insects. Insecticidal activities have been identified for crystal proteins from various B.t. strains against insect larvae from the insect orders Lepidoptera (caterpillars), Diptera (mosquitos, flies) and Coleoptera (beetles).
Recently, certain B.t. strains and B.t. crystal proteins have been reported as having activity against non-insect species such as nematodes. The term "insecticidal," as used herein with reference to B.t. strains and their crystal proteins, is intended to include such pathogenic activities against non-insect species.
FIG. 1 consists of FIGS. 1A and 1B and depicts the nucleotide sequence for the apr gene, the B.t. alkaline protease gene (SEQ ID NO:1) of this invention. The deduced amino acid sequence of the Apr alkaline protease protein (SEQ ID NO:2), encoded by the open reading frame extending from nucleotide base positions 225 to 1415 (excluding the. terminal stop codon), is also shown. The restriction endonuclease cleavage site for SstI at nucleotide position 954 within the apr gene is also shown.
FIG. 2 consists of FIGS. 2A through 2C and depicts the nucleotide sequence for the npr gene, the B.t. neutral protease gene (SEQ ID NO:3) of this invention. The deduced amino acid sequence of the Npr neutral protease protein (SEQ ID NO:4), encoded by the open reading frame extending from nucleotide base positions 182 to 1879 (excluding the terminal stop codon), is also shown. The restriction endonuclease cleavage sites for NdeI within the npr gene are also shown.
FIG. 3 is a linear structural map of plasmid pEG1140, a 14.7 kilobase (kb) plasmid containing the cloned apr gene on a 12 kb EcoRI DNA fragment inserted into plasmid vector pUC18, shown by the labeled open segment. The location and orientation of the apr gene are shown by the arrow. Locations of various restriction endonuclease cleavage sites are shown in this Figure and in subsequent Figures.
To assure the availability of materials to those interested members of the public upon issuance of a patent on the present application, deposits of the microorganism listed in the following Table 1 were made prior to filing the present application with the ARS Patent Collection, Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604.
These microorganism deposits were made under the provisions of the "Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure." All restrictions on the availability to the public of these deposited microorganisms will be irrevocably removed upon issuance of a United States patent based on this application.
TABLE 1 ______________________________________ Cell Recombinant NRRL Accession Date of Type Strain Plasmid Number Deposit ______________________________________ B. t. EG2371 none NRRL B-18209 23 Apr '87 B. t. EG7283 pEG1111 NRRL B-21111 9 Jun '93 (cryET5.sup.+ apr.sup.+) B. t. EG10368 none NRRL B-21125 30 Jun '93 E. coli EG7940 pEG1194 (apr.sup.+) NRRL B-21342 11 Oct '94 B. t. EG7950 pEG1212 (apr1) NRRL B-21343 11 Oct '94 B. t. EG10654 none NRRL B-21344 11 Oct '94 E. coli EG7978 pEG1233 (npr2) NRRL B-21345 11 Oct '94 E. coli EG11447 pEG1245 (npr3) NRRL B-21346 11 Oct '94 B. t. EG10624 none NRRL B-21347 11 Oct '94 E. coli EG11466 pEG1283 (npr.sup.+) NRRL B-21358 29 Nov '94 ______________________________________