# US 2013/0157309 A1 · Methods of obtaining genetic competence in bacillus cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0157309 A1 - Document ID: US20130157309A1 - Status: Published application - Kind: A1 - Application number: 13/760,763 - Filed: 2013-02-06 - Published: 2013-06-20 - Earliest claimed: 2006-12-21 - Assignees: Novozymes, Inc. - Inventors: Randy Berka; Michelle Maranta; Maria Tang; Barbara Cherry - Web page: https://patentgrep.com/patent/US20130157309A1 ## CPC - C12N 15/75: for Bacillus - C07K 14/32: from Bacillus (G) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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) ## Abstract The present invention relates to methods of obtaining genetic competence in non-competent Bacillus cells for their transformation with exogenous DNA. ## Claims ### Claim 1 A method of obtaining a Bacillus licheniformis transformant, comprising: (a) transforming an exogenous DNA into a Bacillus licheniformis host cell made competent by at least one copy of an introduced first nucleic acid construct comprising a promoter region operably linked to a polynucleotide encoding a ComS polypeptide, wherein the polynucleotide encoding the ComS polypeptide is foreign to the Bacillus licheniformis host cell that was non-competent prior to introduction of the first nucleic acid construct; and (b) isolating a transformant of the Bacillus licheniformis host cell comprising the DNA. ### Claim 2 (depends on 1) The method of claim 1, wherein the ComS polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4. ### Claim 3 (depends on 1) The method of claim 1, wherein the ComS polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4. ### Claim 4 (depends on 1) The method of claim 1, wherein the ComS polypeptide comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 4. ### Claim 5 (depends on 1) The method of claim 1, wherein the ComS polypeptide comprises the amino acid sequence of SEQ ID NO: 4. ### Claim 6 (depends on 1) The method of claim 1, wherein the polynucleotide encoding the ComS polypeptide comprises the nucleotide sequence of SEQ ID NO: 5. ### Claim 7 (depends on 1) The method of claim 1, wherein the competent Bacillus licheniformis host cell further comprises at least one copy of an introduced second nucleic acid construct comprising a promoter region operably linked to a polynucleotide encoding a ComK polypeptide. ### Claim 8 (depends on 7) The method of claim 7, wherein the ComK polypeptide comprises an amino acid sequence of having at least 90% sequence identity with SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, or SEQ ID NO: 50. ### Claim 9 (depends on 7) The method of claim 7, wherein the ComK polypeptide comprises an amino acid sequence of having at least 95% sequence identity with SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, or SEQ ID NO: 50. ### Claim 10 (depends on 7) The method of claim 7, wherein the ComK polypeptide comprises the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, or SEQ ID NO: 50. ### Claim 11 A method of obtaining a competent Bacillus licheniformis host cell, comprising: (a) introducing into a non-competent Bacillus licheniformis host cell at least one copy of a first nucleic acid construct comprising a promoter region operably linked to a polynucleotide encoding a ComS polypeptide, wherein the polynucleotide encoding the ComS polypeptide is foreign to the Bacillus licheniformis host cell; and (b) isolating a competent Bacillus licheniformis host cell comprising the polynucleotide encoding the ComS polypeptide. ### Claim 12 (depends on 10) The method of claim 10, wherein the competent Bacillus licheniformis host cell further comprises at least one copy of an introduced second nucleic acid construct comprising a promoter region operably linked to a polynucleotide encoding a ComK polypeptide. ### Claim 13 A competent Bacillus licheniformis host cell comprising at least one copy of an introduced first nucleic acid construct comprising a promoter region operably linked to a polynucleotide encoding a ComS polypeptide, wherein the polynucleotide encoding the ComS polypeptide is foreign to the Bacillus licheniformis host cell that was non-competent prior to introduction of the first nucleic acid construct. ### Claim 14 (depends on 13) The competent Bacillus licheniformis host cell of claim 13, wherein the competent Bacillus licheniformis host cell further comprises at least one copy of an introduced second nucleic acid construct comprising a promoter region operably linked to a polynucleotide encoding a ComK polypeptide. ### Claim 15 (depends on 13) The competent Bacillus licheniformis host cell of claim 13, which has been transformed with an exogenous DNA. ### Claim 16 (depends on 13) A method of producing a biological substance, comprising: (a) cultivating the Bacillus licheniformis host cell of claim 13 transformed with an exogenous DNA encoding or involved in the expression of a substance having biological activity under conditions conducive for production of the substance; and (b) recovering the substance having biological activity. ### Claim 17 (depends on 13) A method of producing a mutant of a Bacillus licheniformis cell, comprising: (a) transforming into the Bacillus licheniformis cell of claim 13 an exogenous DNA capable of modifying a gene encoding a polypeptide, which results in a mutant cell producing less of the polypeptide; and (b) isolating the mutant cell.