Engineered pesticidal proteins

Abstract

Engineered pesticidal polypeptides that are highly active against a wide range of pests and methods of making such polypeptides are disclosed. The nucleotide sequences encoding the pesticidal polypeptide can be used to transform various prokaryotic and eukaryotic organisms, which organisms can be used to produce the pesticidal polypeptides. The recombinant organisms and/or the polypeptides produced by the recombinant organisms can be used to control pests in various environments.

What is claimed: 
     
         1 . An engineered Vip3 pesticidal polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1 and further comprising at least one amino acid mutation at a position that corresponds to a position selected from the group consisting of M362, F400, R465, L494, 1503, L514, S532, E546, T600, T614, N625, Y629, K650, N682, S683, L701, T724, T743, S744, M747, T756, S761, T764, L766, R773, G778 and V785, wherein the at least one amino acid mutation is alanine (A), and wherein the mutation reduces or eliminates the pesticidal activity of the engineered polypeptide against at least  Ostrinia nubilalis  (European corn borer, ECB) while maintaining or increasing the activity against at least  Spodoptera frugiperda  (fall armyworm, FAW) compared to the ECB activity and the FAW activity of a wild-type Vip3 polypeptide that does not comprise the at least one mutation. 
     
     
         2 . The engineered Vip3 pesticidal polypeptide of  claim 1 , wherein the at least one amino acid mutation corresponds to position R465, wherein the at least one amino acid mutation at a position that corresponds to R465 is A. 
     
     
         3 . The engineered Vip3 pesticidal polypeptide according to  claim 1 , wherein the at least one amino acid mutation is at a position in SEQ ID NO:1 selected from the group consisting of M362, F400, R465, L494, 1503, L514, S532, E546, T600, T614, N625, Y629, K650, N682, S683, L701, T724, T743, S744, M747, T756, S761, T764, L766, R773, G778 and V785, and wherein the at least one amino acid mutation is alanine (A), and wherein the mutation reduces or eliminates the pesticidal activity of the engineered polypeptide against at least  Ostrinia nubilalis  (European corn borer, ECB) while maintaining or increasing the activity against at least  Spodoptera frugiperda  (fall armyworm, FAW) compared to the ECB activity and the FAW activity of a wild-type Vip3 polypeptide that does not comprise the at least one mutation. 
     
     
         4 . The engineered Vip3 pesticidal polypeptide of  claim 3 , wherein the at least one amino acid mutation is at position R465, wherein the at least one amino acid mutation at a position that corresponds to R465 is A. 
     
     
         5 . The engineered Vip3 pesticidal polypeptide of  claim 1 , wherein the at least one amino acid mutation at a position corresponding to
 a) L701A and I721A; or   b) S72A and S781A; or   c) S761A and S744A; or   d) S781A and S744A; or   e) S781A and T750A; or   d) any combination of a)-e).   
     
     
         6 . The engineered Vip3 pesticidal polypeptide of  claim 3 , wherein the at least one amino acid mutation is selected from the group consisting of a mutation of L701A and I721A, S72A and S781A, S761A and S744A, S781A and S744A, S781A and T750A, and any combination thereof. 
     
     
         7 . The engineered Vip3 pesticidal polypeptide of  claim 3 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO:18, the amino acid sequence of SEQ ID NO:20, the amino acid sequence of SEQ ID NO:62, the amino acid sequence of SEQ ID NO:64, the amino acid sequence of SEQ ID NO:66, the amino acid sequence of SEQ ID NO:68, the amino acid sequence of SEQ ID NO:70, the amino acid sequence of SEQ ID NO:72, the amino acid sequence of SEQ ID NO:74, the amino acid sequence of SEQ ID NO:76, the amino acid sequence of SEQ ID NO:78, the amino acid sequence of SEQ ID NO:80, the amino acid sequence of SEQ ID NO:82, the amino acid sequence of SEQ ID NO:84, the amino acid sequence of SEQ ID NO:86, the amino acid sequence of SEQ ID NO:88, the amino acid sequence of SEQ ID NO:90, the amino acid sequence of SEQ ID NO:92, the amino acid sequence of SEQ ID NO:94, the amino acid sequence of SEQ ID NO:96, the amino acid sequence of SEQ ID NO:98, the amino acid sequence of SEQ ID NO:100, the amino acid sequence of SEQ ID NO:102, the amino acid sequence of SEQ ID NO:104, the amino acid sequence of SEQ ID NO:106, the amino acid sequence of SEQ ID NO:121, the amino acid sequence of SEQ ID NO:123, the amino acid sequence of SEQ ID NO:125, the amino acid sequence of SEQ ID NO:127, the amino acid sequence of SEQ ID NO:129, and any combination thereof. 
     
     
         8 . A recombinant nucleic acid molecule comprising a nucleotide sequence encoding the engineered Vip3 polypeptide of  claim 1 . 
     
     
         9 . The recombinant nucleic acid molecule of  claim 8 , wherein the nucleotide sequence is selected from the group consisting of the nucleotide sequence of SEQ ID NO:19, the nucleotide sequence of SEQ ID NO:21, the nucleotide sequence of SEQ ID NO:63, the nucleotide sequence of SEQ ID NO:65, the nucleotide sequence of SEQ ID NO:67, the nucleotide sequence of SEQ ID NO:69, the nucleotide sequence of SEQ ID NO:71, the nucleotide sequence of SEQ ID NO:73, the nucleotide sequence of SEQ ID NO:75, the nucleotide sequence of SEQ ID NO:77, the nucleotide sequence of SEQ ID NO:79, the nucleotide sequence of SEQ ID NO:81, the nucleotide sequence of SEQ ID NO:83, the nucleotide sequence of SEQ ID NO:85, the nucleotide sequence of SEQ ID NO:87, the nucleotide sequence of SEQ ID NO:89, the nucleotide sequence of SEQ ID NO:91, the nucleotide sequence of SEQ ID NO:93, the nucleotide sequence of SEQ ID NO:95, the nucleotide sequence of SEQ ID NO:97, the nucleotide sequence of SEQ ID NO:99, the nucleotide sequence of SEQ ID NO:101, the nucleotide sequence of SEQ ID NO:103, the nucleotide sequence of SEQ ID NO:105, the nucleotide sequence of SEQ ID NO:107, the nucleotide sequence of SEQ ID NO:122, the nucleotide sequence of SEQ ID NO:124, the nucleotide sequence of SEQ ID NO:126, the nucleotide sequence of SEQ ID NO:128, the nucleotide sequence of SEQ ID NO:130, and any combination thereof. 
     
     
         10 . An expression cassette comprising the recombinant nucleic acid molecule of  claim 8 , wherein the nucleotide sequence is operatively associated with a heterologous promoter. 
     
     
         11 . A recombinant vector comprising the expression cassette of  claim 8 . 
     
     
         12 . A transgenic non-human host cell comprising the nucleic acid molecule of  claim 8 . 
     
     
         13 . The transgenic non-human host cell according to  claim 12 , which is a bacterial cell or a plant cell, wherein the transgenic host cell comprises the nucleic acid molecule or the engineered Vip3 pesticidal polypeptide. 
     
     
         14 . A transgenic plant comprising the plant cell of  claim 13 . 
     
     
         15 . The transgenic plant of  claim 14 , wherein the plant is selected from the group consisting of sorghum, wheat, sunflower, tomato, cole crops, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley, oilseed rape and maize. 
     
     
         16 . A product harvested from the transgenic plant of  claim 15 , wherein the product comprises the nucleic acid molecule or the engineered Vip3 pesticidal polypeptide. 
     
     
         17 . A processed product produced from the harvested product of  claim 16 , wherein the processed product comprises the nucleic acid molecule or the engineered Vip3 pesticidal polypeptide. 
     
     
         18 . A crop comprising a plurality of the plants of  claim 14  planted together in an agricultural field. 
     
     
         19 . A transgenic seed comprising the recombinant nucleic acid molecule of  claim 8 . 
     
     
         20 . A composition comprising the engineered Vip3 pesticidal polypeptide of  claim 1  in an agriculturally acceptable carrier.