Insecticidal protein discovery platform and insecticidal proteins discovered therefrom

Abstract

The disclosure presents a platform for discovering novel insecticidal proteins from highly heterogeneous environmental sources. The methodology utilizes metagenomic enrichment procedures and unique genetic amplification techniques, which enables access to a broad class of unknown microbial diversity and their resultant proteome. The disclosed insecticidal protein discovery platform (IPDP) can be computationally driven and is able to integrate molecular biology, automation, and advanced machine learning protocols. The platform will enable researchers to rapidly and accurately access the vast repertoire of untapped insecticidal proteins produced by uncharacterized and complex microbial environmental samples. Also presented herein are a group of newly discovered pore-forming toxins (PFT) from a rare class of insecticidal proteins, which were discovered utilizing the insecticidal protein discovery platform.

What is claimed is: 
     
         1 . An insecticidal composition, comprising:
 a) an insecticidal protein having an amino acid sequence (i) with at least 97% sequence identity to a protein with the amino acid sequence of SEQ ID NO: 20; or (ii) with at least 96% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 22; or (iii) with at least 94% sequence identity to a protein with the amino acid sequence of SEQ ID NO: 8; and   b) an agriculturally acceptable carrier.   
     
     
         2 . The insecticidal composition of  claim 1 , wherein said insecticidal protein has an amino acid sequence with at least about 97% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 22. 
     
     
         3 . The insecticidal composition of  claim 1 , wherein said insecticidal protein has an amino acid sequence with at least about 98% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         4 . The insecticidal composition of  claim 1 , wherein said insecticidal protein has an amino acid sequence with at least about 99% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         5 . The insecticidal composition of  claim 1 , wherein said insecticidal protein has an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         6 . The insecticidal composition of  claim 1 , wherein said isolated insecticidal protein is present in an insecticidally effective amount. 
     
     
         7 . The insecticidal composition of  claim 1 , wherein said agriculturally acceptable carrier is selected from the group consisting of: adjuvants, inert components, dispersants, surfactants, sticking agents, tackifiers, binders, natural or regenerated mineral substances, solvents, wetting agents, fertilizers, and combinations thereof. 
     
     
         8 . The insecticidal composition of  claim 1 , formulated as a dry solid. 
     
     
         9 . The insecticidal composition of  claim 1 , formulated as a liquid. 
     
     
         10 . The insecticidal composition of  claim 1 , formulated for foliar application. 
     
     
         11 . The insecticidal composition of  claim 1 , formulated for in-furrow application. 
     
     
         12 . The insecticidal composition of  claim 1 , formulated as a seed coating or seed treatment. 
     
     
         13 . The insecticidal composition of  claim 1 , further comprising an additional pesticidal compound. 
     
     
         14 . The insecticidal composition of  claim 13 , wherein the additional pesticidal compound is selected from the group consisting of: a Monalysin protein, a  Pseudomonas  insecticidal protein, Cry protein, Cyt protein, vegetative insecticidal protein, toxin complex protein, and any combination thereof. 
     
     
         15 . The insecticidal composition of  claim 1 , further comprising an herbicidal compound. 
     
     
         16 . A method of killing a target pest, comprising: applying to said target pest the insecticidal composition of  claim 1 . 
     
     
         17 . A method of killing a target pest, comprising: applying to a locus the insecticidal composition of  claim 1 , wherein said target pest comes into contact with said locus. 
     
     
         18 . The method of  claim 17 , wherein the locus is a crop. 
     
     
         19 . The method of  claim 17 , wherein said target pest is a member of the order Coleoptera, Diptera, Hymenoptera, Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or Dermaptera. 
     
     
         20 . A cell lysate, comprising: an insecticidal protein having an amino acid sequence (i) with at least 97% sequence identity to a protein with the amino acid sequence of SEQ ID NO: 20; or (ii) with at least 96% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 22; or (iii) with at least 94% sequence identity to a protein with the amino acid sequence of SEQ ID NO: 8. 
     
     
         21 . The cell lysate of  claim 20 , wherein said insecticidal protein has an amino acid sequence with at least about 97% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 22. 
     
     
         22 . The cell lysate of  claim 20 , wherein said insecticidal protein has an amino acid sequence with at least about 98% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         23 . The cell lysate of  claim 20 , wherein said insecticidal protein has an amino acid sequence with at least about 99% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         24 . The cell lysate of  claim 20 , wherein said insecticidal protein has an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         25 . A method of killing a target pest, comprising: applying to said target pest the cell lysate of  claim 20 ; or applying to a locus the cell lysate of  claim 20 , wherein said target pest comes into contact with said locus. 
     
     
         26 . The method of  claim 25 , wherein the locus is a crop. 
     
     
         27 . The method of  claim 25 , wherein said target pest is a member of the order Coleoptera, Diptera, Hymenoptera, Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or Dermaptera. 
     
     
         28 . A method of killing a target pest, comprising: applying to said target pest a host cell that expresses a polynucleotide encoding a polypeptide having an amino acid sequence (i) with at least 97% sequence identity to a protein with the amino acid sequence of SEQ ID NO: 20; or (ii) with at least 96% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 22; or (iii) with at least 94% sequence identity to a protein with the amino acid sequence of SEQ ID NO: 8. 
     
     
         29 . The method of  claim 28 , wherein said polypeptide has an amino acid sequence with at least about 97% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 22. 
     
     
         30 . The method of  claim 28 , wherein said polypeptide has an amino acid sequence with at least about 98% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         31 . The method of  claim 28 , wherein said polypeptide has an amino acid sequence with at least about 99% sequence identity to a protein with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         32 . The method of  claim 28 , wherein said polypeptide has an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         33 . The method of  claim 28 , wherein the host cell is a Prokaryotic host cell. 
     
     
         34 . The method of  claim 28 , wherein the host cell naturally expresses the polynucleotide. 
     
     
         35 . The method of  claim 28 , wherein the host cell is from the genus  Pseudomonas.    
     
     
         36 . The method of  claim 28 , wherein said target pest is a member of the order Coleoptera, Diptera, Hymenoptera, Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or Dermaptera.