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 abroad 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 isolated nucleic acid molecule, encoding: an insecticidal protein having an amino acid sequence (i) with at least about 80% 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, SEQ ID NO: 20, and SEQ ID NO: 22; or (ii) with at least about 91% sequence identity to a protein with an amino acid sequence of SEQ ID NO: 8. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein said nucleic acid molecule encodes an insecticidal protein having an amino acid sequence with at least about 90% 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, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein said nucleic acid molecule encodes an insecticidal protein having an amino acid sequence with at least about 95% 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 isolated nucleic acid molecule of  claim 1 , wherein said nucleic acid molecule encodes an insecticidal protein having 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 isolated nucleic acid molecule of  claim 1 , wherein said nucleic acid molecule encodes an insecticidal 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. 
     
     
         6 . The isolated nucleic acid molecule of any one of  claims 1 - 5 , wherein said nucleic acid molecule is codon optimized for expression in a host cell of interest. 
     
     
         7 . The isolated nucleic acid molecule of  claim 6 , wherein said host cell of interest is a plant cell. 
     
     
         8 . The isolated nucleic acid molecule of  claim 1 , wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21. 
     
     
         9 . A nucleotide construct, comprising: a nucleic acid molecule encoding an insecticidal protein having an amino acid sequence (i) with at least about 80% 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, SEQ ID NO: 20, and SEQ ID NO: 22; or (ii) with at least about 91% sequence identity to a protein with an amino acid sequence of SEQ ID NO: 8, said nucleic acid molecule operably linked to a heterologous regulatory element. 
     
     
         10 . The nucleotide construct of  claim 9 , wherein said nucleic acid molecule encodes an insecticidal protein having an amino acid sequence with at least about 90% 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, SEQ ID NO: 20, and SEQ ID NO: 22. 
     
     
         11 . The nucleotide construct of  claim 9 , wherein said nucleic acid molecule encodes an insecticidal protein having an amino acid sequence with at least about 95% 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. 
     
     
         12 . The nucleotide construct of  claim 9 , wherein said nucleic acid molecule encodes an insecticidal protein having 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. 
     
     
         13 . The nucleotide construct of  claim 9 , wherein said nucleic acid molecule encodes an insecticidal 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. 
     
     
         14 . The nucleotide construct of any one of  claims 9 - 13 , wherein said heterologous regulatory element is a promoter. 
     
     
         15 . The nucleotide construct of any one of  claims 9 - 14 , wherein said nucleotide construct is contained in an expression cassette. 
     
     
         16 . The nucleotide construct of any one of  claims 9 - 15 , wherein said heterologous regulatory element is capable of expressing the encoded protein in a plant. 
     
     
         17 . The nucleotide construct of any one of  claims 9 - 16 , wherein said nucleic acid molecule is codon optimized for expression in a host cell of interest. 
     
     
         18 . The nucleotide construct of  claim 17 , wherein said host cell of interest is a plant cell. 
     
     
         19 . The nucleotide construct of any one of  claims 9 - 18 , wherein said nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21. 
     
     
         20 . An expression vector comprising the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         21 . A plasmid comprising the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         22 . A host cell comprising the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         23 . A method of killing an insect, comprising contacting the insect with the host cell of  claim 22 . 
     
     
         24 . A prokaryotic host cell comprising the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         25 . A eukaryotic host cell comprising the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         26 . A plant cell comprising the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         27 . A monocot plant cell comprising the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         28 . A dicot plant cell comprising the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         29 . A plant stably transformed with the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         30 . A seed produced by a plant that has been stably transformed with the nucleotide construct of any one of  claims 9 - 19 . 
     
     
         31 . An isolated insecticidal protein, comprising: an amino acid sequence (i) with at least about 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22; or (ii) with at least about 91% sequence identity to SEQ ID NO: 8. 
     
     
         32 . The isolated insecticidal protein of  claim 31 , comprising: an amino acid sequence with at least about 90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. 
     
     
         33 . The isolated insecticidal protein of  claim 31 , comprising: an amino acid sequence with at least about 95% sequence identity to 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, or SEQ ID NO: 22. 
     
     
         34 . The isolated insecticidal protein of  claim 31 , comprising: an amino acid sequence with at least about 99% sequence identity to 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, or SEQ ID NO: 22. 
     
     
         35 . The isolated insecticidal protein of  claim 31 , comprising: an amino acid sequence selected from 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, or SEQ ID NO: 22. 
     
     
         36 . A recombinant insecticidal protein, comprising: an amino acid sequence (i) with at least about 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22; or (ii) with at least about 91% sequence identity to SEQ ID NO: 8. 
     
     
         37 . The recombinant insecticidal protein of  claim 36 , comprising: an amino acid sequence with at least about 90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. 
     
     
         38 . The recombinant insecticidal protein of  claim 36 , comprising: an amino acid sequence with at least about 95% sequence identity to 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, or SEQ ID NO: 22. 
     
     
         39 . The recombinant insecticidal protein of  claim 36 , comprising: an amino acid sequence with at least about 99% sequence identity to 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, or SEQ ID NO: 22. 
     
     
         40 . The recombinant insecticidal protein of  claim 36 , comprising: an amino acid sequence selected from 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, or SEQ ID NO: 22. 
     
     
         41 . A transgenic plant cell, comprising:
 a. a DNA construct, comprising: a polynucleotide encoding a polypeptide having an amino acid sequence (i) with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity to 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, SEQ ID NO: 20, and SEQ ID NO: 22; or (ii) with at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity to an amino acid sequence of SEQ ID NO: 8; and a heterologous regulatory sequence operably linked to the polynucleotide.   
     
     
         42 . The transgenic plant cell of  claim 41 , wherein said polynucleotide encodes a polypeptide having 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. 
     
     
         43 . The transgenic plant cell of  claim 41 , wherein said heterologous regulatory element is a promoter. 
     
     
         44 . The transgenic plant cell of any one of  claims 41 - 43 , wherein said cell is from a monocot species. 
     
     
         45 . The transgenic plant cell of any one of  claims 41 - 43 , wherein said cell is from corn, wheat, oat, or rice. 
     
     
         46 . The transgenic plant cell of any one of  claims 41 - 43 , wherein said cell is from a dicot species. 
     
     
         47 . The transgenic plant cell of any one of  claims 41 - 43 , wherein said cell is from cotton, potato, or soybean. 
     
     
         48 . The transgenic plant cell of any one of  claims 41 - 43 , wherein said cell is from an agricultural row crop species. 
     
     
         49 . A transgenic plant stably transformed with a DNA construct, comprising:
 a. a polynucleotide encoding a polypeptide having an amino acid sequence (i) with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity to 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, SEQ ID NO: 20, and SEQ ID NO: 22; or (ii) with at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity to an amino acid sequence of SEQ ID NO: 8; and   b. a heterologous regulatory sequence operable linked to the polynucleotide.   
     
     
         50 . The transgenic plant of  claim 49 , wherein said polynucleotide encodes a polypeptide having 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. 
     
     
         51 . The transgenic plant of  claim 49 , wherein said heterologous regulatory element is a promoter. 
     
     
         52 . The transgenic plant of any one of  claims 49 - 51 , wherein said plant is a monocot species. 
     
     
         53 . The transgenic plant of any one of  claims 49 - 51 , wherein said plant is corn, wheat, oat, or rice. 
     
     
         54 . The transgenic plant of any one of  claims 49 - 51 , wherein said plant is a dicot species. 
     
     
         55 . The transgenic plant of any one of  claims 49 - 51 , wherein said plant is cotton, potato, or soybean. 
     
     
         56 . The transgenic plant of any one of  claims 49 - 51 , wherein said plant is from an agricultural row crop species. 
     
     
         57 . A seed produced by the plant of any one of  claims 49 - 56 . 
     
     
         58 . A progeny plant produced from the plant of any one of  claims 49 - 56 . 
     
     
         59 . The transgenic plant of  claim 49 , further comprising: a DNA construct comprising a polynucleotide encoding a protein selected from the group consisting of: a Monalysin protein,  Pseudomonas  insecticidal protein, Cry protein, Cyt protein, vegetative insecticidal protein, toxin complex protein, and any combination thereof. 
     
     
         60 . A method of killing a target pest, comprising: providing the transgenic plant of any one of  claims 49 - 56  and  59  to an area, wherein said target pest is exposed to the transgenic plant. 
     
     
         61 . The method of  claim 60 , wherein said target pest feeds on the transgenic plant. 
     
     
         62 . A method of killing a target pest that is resistant to a pesticidal protein, comprising: providing the transgenic plant of any one of  claims 49 - 56  and  59  to an area, wherein said target pest is exposed to the transgenic plant, and wherein the target pest is resistant to a protein selected from the group consisting of: a Monalysin protein,  Pseudomonas  insecticidal protein, Cry protein, Cyt protein, vegetative insecticidal protein, toxin complex protein, and any combination thereof. 
     
     
         63 . A method of killing a target pest that is resistant to a pesticidal protein, comprising: providing the transgenic plant of any one of  claims 49 - 56  and  59  to an area, wherein said target pest feeds on the transgenic plant, and wherein the target pest is resistant to a protein selected from the group consisting of: a Monalysin protein,  Pseudomonas  insecticidal protein, Cry protein, Cyt protein, vegetative insecticidal protein, toxin complex protein, and any combination thereof. 
     
     
         64 . A method of killing a target pest, comprising: providing the transgenic plant of any one of  claims 49 - 56  and  59  to an area, wherein said target pest is exposed to the transgenic plant and said target pest is a member of the Order Coleoptera, Diptera, Hymenoptera, Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or Dermaptera. 
     
     
         65 . An insecticidal composition, comprising:
 a. an isolated insecticidal protein having an amino acid sequence (i) with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity to 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, SEQ ID NO: 20, and SEQ ID NO: 22; or (ii) with at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity to an amino acid sequence of SEQ ID NO: 8; and   b. an agriculturally acceptable carrier.   
     
     
         66 . The insecticidal composition of  claim 65 , wherein the isolated 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. 
     
     
         67 . The insecticidal composition of  claim 65 , wherein said isolated insecticidal protein is present in an insecticidally effective amount. 
     
     
         68 . The insecticidal composition of any one of  claims 65 - 67 , 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. 
     
     
         69 . The insecticidal composition of any one of  claims 65 - 68 , formulated as a dry solid. 
     
     
         70 . The insecticidal composition of any one of  claims 65 - 68 , formulated as a liquid. 
     
     
         71 . The insecticidal composition of any one of  claims 65 - 68 , formulated for foliar application. 
     
     
         72 . The insecticidal composition of any one of  claims 65 - 68 , formulated for in-furrow application. 
     
     
         73 . The insecticidal composition of any one of  claims 65 - 68 , formulated as a seed coating or seed treatment. 
     
     
         74 . The insecticidal composition of any one of  claims 65 - 73 , further comprising: at least one additional pesticidal compound. 
     
     
         75 . The insecticidal composition of  claim 74 , wherein the at least one additional pesticidal compound is selected from the group consisting of: a Monalysin protein,  Pseudomonas  insecticidal protein, Cry protein, Cyt protein, vegetative insecticidal protein, toxin complex protein, and any combination thereof. 
     
     
         76 . The insecticidal composition of any one of  claims 65 - 75 , further comprising: an herbicidal compound. 
     
     
         77 . A method of killing a target pest, comprising: applying to said target pest the insecticidal composition of any one of  claims 65 - 76 . 
     
     
         78 . A method of killing a target pest, comprising: applying to a locus the insecticidal composition of any one of  claims 65 - 76 , wherein said target pest comes into contact with said locus. 
     
     
         79 . A method of killing a target pest, comprising: applying to a crop the insecticidal composition of any one of  claims 65 - 76 , wherein said target pest comes into contact with said crop. 
     
     
         80 . A method of killing a target pest, comprising: applying to a crop the insecticidal composition of any one of  claims 65 - 76 , wherein said target pest comes into contact with said crop, and said target pest is a member of the Order Coleoptera, Diptera, Hymenoptera, Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or Dermaptera. 
     
     
         81 . Cell lysate, comprising: an insecticidal protein comprising an amino acid sequence (i) with at least about 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22; or (ii) with at least about 91% sequence identity to SEQ ID NO: 8. 
     
     
         82 . The cell lysate of  claim 81 , comprising: an insecticidal protein comprising an amino acid sequence with at least about 90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. 
     
     
         83 . The cell lysate of  claim 81 , comprising: an insecticidal protein comprising an amino acid sequence with at least about 95% sequence identity to 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, or SEQ ID NO: 22. 
     
     
         84 . The cell lysate of  claim 81 , comprising: an insecticidal protein comprising an amino acid sequence with at least about 99% sequence identity to 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, or SEQ ID NO: 22. 
     
     
         85 . The cell lysate of  claim 81 , comprising: an insecticidal protein comprising an amino acid sequence selected from 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. 
     
     
         86 . A method of killing a target pest, comprising: applying to said target pest the cell lysate of any one of  claims 81 - 85 . 
     
     
         87 . 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 about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity to 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, SEQ ID NO: 20, and SEQ ID NO: 22; or (ii) with at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity to an amino acid sequence of SEQ ID NO: 8. 
     
     
         88 . The method of  claim 87 , wherein the host cell expresses a polynucleotide that encodes a polypeptide having 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. 
     
     
         89 . The method of  claim 87 , wherein the host cell is a Prokaryotic host cell. 
     
     
         90 . The method of  claim 87 , wherein the host cell naturally expresses the polynucleotide. 
     
     
         91 . The method of  claim 87 , wherein the host cell is from the genus  Pseudomonas.    
     
     
         92 . The method of any one of  claims 87 - 91 , wherein said target pest is a member of the Order Coleoptera, Diptera, Hymenoptera, Lepidoptera, Hemiptera, Orthroptera, Thysanoptera, or Dermaptera. 
     
     
         93 . A method for constructing a genomic library, enriched for DNA from  Pseudomonas  encoding insecticidal proteins, comprising:
 a. providing an initial sample comprising one or more microorganisms;   b. exposing the initial sample to a solid nutrient limiting media that enriches for growth of species from the genus  Pseudomonas , which results in a subsequent sample enriched for  Pseudomonas  sp.;   c. isolating DNA from the subsequent enriched sample;   d. extracting DNA from the isolated DNA and performing degenerate PCR with primers selected to amplify target insecticidal protein genes;   e. cloning the PCR-amplified DNA into a plasmid; and   f. sequencing the cloned DNA from said plasmid.   
     
     
         94 . The method of  claim 93 , further comprising: assembling the sequenced DNA into a genomic library. 
     
     
         95 . The method of  claim 93 , further comprising: identifying insecticidal protein genes within the sequenced DNA. 
     
     
         96 . The method of  claim 93 , further comprising: identifying insecticidal protein genes within the sequenced DNA, wherein said identified insecticidal protein genes are unknown. 
     
     
         97 . The method of any one of  claims 93 ,  95 , and  96  further comprising: utilizing a Hidden Markov model to identify insecticidal protein genes within the sequenced DNA. 
     
     
         98 . The method of  claim 93 , further comprising: identifying insecticidal protein genes within the sequenced DNA, wherein said identified insecticidal protein genes comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 7, 11, 13, 15, 17, 19, and 21. 
     
     
         99 . The method of  claim 93 , further comprising: identifying insecticidal protein genes within the sequenced DNA, wherein said identified insecticidal protein genes encode a protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 8, 12, 14, 16, 18, 20, and 22. 
     
     
         100 . The method of  claim 93 , wherein the primers are selected to amplify target insecticidal protein genes that encode a protein with an amino acid sequence with at least 50% sequence identity to SEQ ID NO: 87. 
     
     
         101 . The method of  claim 93 , wherein the initial sample is from soil. 
     
     
         102 . An insecticidal genomic library enriched for DNA from  Pseudomonas  encoding insecticidal proteins, as constructed by the method of  claim 93 . 
     
     
         103 . An insecticidal protein, comprising: a) an amino acid sequence that scores at or above a bit score of 521.5; and/or b) an amino acid sequence that matches at an E-value of less than or equal to 7.9e−161, when scored or matched using the HMNM in Table 6.