# US 2020/0399654 A1 · Insecticidal protein discovery platform and insecticidal proteins discovered therefrom > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399654 A1 - Document ID: US20200399654A1 - Status: Published application - Kind: A1 - Application number: 17/009,438 - Filed: 2020-09-01 - Published: 2020-12-24 - Earliest claimed: 2018-03-02 - Assignees: Zymergen Inc. - Applicants: Zymergen Inc. - Inventors: William W. HWANG; Jeffrey Kim; Oliver Liu; Jennifer Shock - Web page: https://patentgrep.com/patent/US20200399654A1 ## CPC - C12N 15/8286: {for insect resistance} - A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46) - C07K 14/195: from bacteria - C07K 14/21: from Pseudomonadaceae (F) - C07K 14/32: from Bacillus (G) - C07K 14/325: Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins - C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups} - C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host - Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## 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. ## Claims ### Claim 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. ### Claim at position 2 -8. (canceled) ### Claim 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. ### Claim 10 (depends on 9) 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. ### Claim 11 (depends on 9) 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. ### Claim 12 (depends on 9) 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. ### Claim 13 (depends on 9) 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. ### Claim 14 (depends on 9) The nucleotide construct of claim 9, wherein said heterologous regulatory element is a promoter. ### Claim 15 (depends on 9) The nucleotide construct of claim 9, wherein said nucleotide construct is contained in an expression cassette. ### Claim 16 (depends on 9) The nucleotide construct of claim 9, wherein said heterologous regulatory element is capable of expressing the encoded protein in a plant. ### Claim 17 (depends on 9) The nucleotide construct of claim 9, wherein said nucleic acid molecule is codon optimized for expression in a host cell of interest. ### Claim 18 (depends on 17) The nucleotide construct of claim 17, wherein said host cell of interest is a plant cell. ### Claim 19 (depends on 9) The nucleotide construct of claim 9, 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. ### Claim 20 (depends on 9) An expression vector comprising the nucleotide construct of claim 9. ### Claim 21 (depends on 9) A plasmid comprising the nucleotide construct of claim 9. ### Claim 22 (depends on 9) A host cell comprising the nucleotide construct of claim 9. ### Claim 23 (depends on 22) A method of killing an insect, comprising contacting the insect with the host cell of claim 22. ### Claim 24 (depends on 9) A prokaryotic host cell comprising the nucleotide construct of claim 9. ### Claim 25 (depends on 9) A eukaryotic host cell comprising the nucleotide construct of claim 9. ### Claim 26 (depends on 9) A plant cell comprising the nucleotide construct of claim 9. ### Claim 27 (depends on 9) A monocot plant cell comprising the nucleotide construct of claim 9. ### Claim 28 (depends on 9) A dicot plant cell comprising the nucleotide construct of claim 9. ### Claim 29 (depends on 9) A plant stably transformed with the nucleotide construct of claim 9. ### Claim 30 (depends on 9) A seed produced by a plant that has been stably transformed with the nucleotide construct of claim 9. ### Claim 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. ### Claim at position 26 -40. (canceled) ### Claim 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. ### Claim at position 28 -103. (canceled)