Compositions and Methods for Pesticide Degradation

Abstract

Compositions and methods related to anucelate cells (e.g., bacterial minicells) for pesticide degradation applications including related cells, polypeptides, and vectors.

What is claimed is: 
     
         1 . A composition comprising a plurality of intact, bacterially-derived minicells, wherein each minicell of said plurality comprises a biologically active polypeptide displayed on the surface of the bacterial minicell, wherein said biologically active polypeptide has pesticide degrading activity. 
     
     
         2 . The composition of  claim 1 , wherein the bacterially-derived minicells are produced from a strain of  Escherichia coli, Bacillus  spp.,  Salmonella  spp.,  Listeria  spp.,  Mycobacterium  spp.,  Shigella  spp., or  Yersinia  spp. 
     
     
         3 . The composition of  claim 1 , wherein the pesticide is selected from the group consisting of organophosphates, glyphosates, neonicotinoids, carbamates, organochlorines, pyrethroids, Stribilurins, anilenes, thiophthlamadies (Captan), aromatic hydrocarbons, nitriles, cyanoimidazoles, demethylation inhibitors, phenylpyrroles, carboximides, phosphonates, dicarboximides, phenylamides, polyoxins, benzimidazoles, aryloxyphenoxy propionates, cyclohexanediones, phenylpyrazolin, imidazolinones, sulfonylamino-carbonyltriazolinones, amides, sulfonylureas, pyraxoles, triazolpyramidines, triazolones, dinitroanilines, benzoic acids, carboxylic acids, piconolinic acids, phenoxys, phenyl-carbamates, triazines, triazinones, uracils, benzthiadiazoles, ureas, thiocarbamates, triazoles, aryl triazones, chloroacetamides, pyrazoles, benzofuranyl alkylsulfonates, semicarbazones, bipyridyliums, and benzopyrazoles. 
     
     
         4 . The composition of  claim 1 , wherein the pesticide is an organophosphate selected from the group consisting of paraoxon, malathion, parathion, diazinon, naled, fenthion, dichlorvos, chlorpyrifos, phosmet, acephate, ethion, soman, sarin, tabun, VX, echothiophate, isoflurophate, trichlorfon, tetrachlorvinphos, azamethiphos, and azinphos-methyl. 
     
     
         5 . The composition of  claim 4 , wherein the pesticide comprises malathion. 
     
     
         6 . The composition of any one of  claims 1 - 5 , wherein the biologically active polypeptide comprises a fusion protein. 
     
     
         7 . The composition of  claim 6 , wherein the fusion protein comprises at least one surface-expressing moiety, and at least one enzyme moiety. 
     
     
         8 . The composition of  claim 7 , wherein the at least one surface-expressing moiety comprises an ice nucleation protein (INPNC) or an exported bacterial protein, including wild type or mutant versions thereof. 
     
     
         9 . The composition of  claim 8 , wherein the exported bacterial protein is selected from the group consisting of LamB (λ receptor), OprF, OmpA (3a, II), Lpp (lipoprotein), MalE (maltose-binding protein), PhoA (alkaline phosphatase), Bla (TEM-1 β-lactamse, f1 or M13 major coat, f1 or M13 minor coat, and any wild type or mutant versions thereof. 
     
     
         10 . The composition of  claim 9 , wherein said at least one enzyme moiety is selected from the group consisting of hydrolases, lipases, phospholipases, transacylases, transaminases, pectinase, proteases, amylases, cellulases, cutinases, esterases, acylases, invertases, isomerases, lyases, glucosidases, oxidoreductases, transferases, ligases, amidases, manganese peroxidase, laccase, lignin peroxidase, horseradish peroxidase or phosphonate dehydrogenase, cytosolic aldehyde oxidase, carbaryl hydrolases, and amidases, HCH dehydrochlorinase, haloalkane dehalogenase, pyrethroid hydrolases (PytH, EspA) and permethrinase. 
     
     
         11 . The composition of  claim 9 , wherein said at least one enzyme moiety comprises an organophosphate hydrolase. 
     
     
         12 . The composition of  claim 11 , wherein the organophosphate hydrolase is selected from the group consisting of phosphotriesterases, methyl parathion hydrolases, and organophosphorus acid anhydrolases. 
     
     
         13 . The composition of anyone of  claims 1 - 12 , further comprising a second polypeptide displayed on the surface of the bacterial minicell, to increase adhesion to plants. 
     
     
         14 . The composition of  claim 13 , wherein the second polypeptide comprises a fusion protein. 
     
     
         15 . The composition of  claim 14 , wherein the fusion protein comprises at least one surface-expressing moiety and at least one plant cell adhesion moiety. 
     
     
         16 . The composition of  claim 15 , wherein said at least one surface-expressing moiety comprises an exported bacterial protein selected from the group consisting of LamB (λ receptor), OprF, OmpA (3a, II), Lpp (lipoprotein), MalE (maltose-binding protein), PhoA (alkaline phosphatase), Bla (TEM-1 β-lactamse, f1 or M13 major coat, f1 or M13 minor coat and any wild type or mutant versions thereof. 
     
     
         17 . The composition of  claim 16 , wherein said at least one plant cell adhesion moiety comprises a cellulose binding domain. 
     
     
         18 . The composition of any one of  claims 1 - 17 , wherein the minicells have at least 90-95% purity. 
     
     
         19 . The composition of any one of  claims 1 - 18 , wherein the minicells are about 0.4 μm-0.5 μm in diameter. 
     
     
         20 . A method for pesticide degradation, the method comprising contacting a crop or an agricultural product with the composition of any one of  claims 1 - 19 , wherein the composition mediates degradation of the pesticide to yield inert by-products. 
     
     
         21 . A method for bioremediation, the method comprising contacting a crop or an agricultural product with the composition of any one of  claims 1 - 19 , wherein the composition mediates degradation of the pesticide to yield inert by-products. 
     
     
         22 . A method for reducing the half-life of a pesticide, the method comprising contacting a crop or agricultural product with the composition of any one of  claims 1 - 19 . 
     
     
         23 . The method of  claim 22 , wherein the pesticide comprises malathion. 
     
     
         24 . The method of  claim 22  or  claim 23 , wherein the half-life is about 4 hours. 
     
     
         25 . The method of any one of  claims 20 - 24 , wherein the crop or agricultural product is selected from the group consisting of grapes, peaches, apples, sweet bell peppers, celery, nectarines, strawberries, cherries, pears, spinach, lettuce, and potatoes 
     
     
         26 . The method of any one of  claims 20 - 25 , wherein the crop or agricultural product comprises grapes.