IP Library › Granted Patent US 12,297,448
Granted Patent B2
US 12,297,448 · App. 18/112,968 · Granted May 13, 2025

Methods and strain

Inventors: Christophe Fremaux (Poitiers, FR); Philippe Horvath (Chatellerault, FR); Patrick Boyaval (La Meziere, FR); Pascal Hols (Vedrin, BE); David Blandine (Floreffe, BE); Amandine Radziejwoski (Sterrebeek, BE); Laetitia Fontaine (Louvain-la-Neuve, BE); Frederic Toussaint (Rixensart, BE)
Assignee: International N&H Denmark ApS
C12N15/902C07K7/00C12Q1/02
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Quick Facts
Patent No.
US 12,297,448
App. No.
18/112,968
Granted
May 13, 2025
Kind
B2
Abstract

The present invention relates to a method for transforming a strain of the Lactococcus genus through natural competence. The present invention further relates to strains obtained or obtainable by said method. The present invention also relates to a method for identifying a strain of the Lactococcus genus which is transformable through natural competence.

Claims (16)

1. A method for transforming a strain of the Lactococcus lactis species with an exogenous DNA polynucleotide comprising the steps of:

(a) providing a strain of the Lactococcus lactis species, wherein said strain is transformable through natural competence;

(b) modulating the production of a ComX protein in said strain, by expressing a comX gene in said strain or increasing the expression of a comX gene in said strain;

(c) contacting said strain of step (b) with an exogenous DNA polynucleotide in a medium and incubating the resulting mixture for integration of the exogenous DNA polynucleotide into the genome of said strain; and

(d) selecting a strain which has integrated the exogenous DNA polynucleotide into its genome.

2. A method according to claim 1 , wherein said comX gene is an exogenous comX gene.

3. A method according to claim 2 , wherein said exogenous comX gene is transferred into said strain by conjugation, transduction, or transformation.

4. A method according to claim 1 , wherein said comX gene is the endogenous comX gene of said strain.

5. A method according to claim 4 , wherein the method comprises carrying out step (b) and then carrying out step (c) or comprises carrying out step (b) and step (c) simultaneously.

6. A method according to claim 1 , wherein said ComX protein has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10, 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 an amino acid sequence having at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, 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 an amino acid sequence having at least 90%, at least 95%, at least 97%, or at NO:22.

7. A method according to claim 1 , wherein said comX gene has the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 or a nucleotide sequence having at least 90%, at least 95%, at least 97% or at least 99% identity to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21.

8. A method according to claim 1 , wherein said medium of step (c) is a chemically defined medium.

9. A method according to claim 1 , wherein prior to step (c) said strain is incubated in a pre-culture medium.

10. A method according to claim 1 , wherein said strain is incubated with the exogenous DNA polynucleotide for around 4-8 hours at around 30° C. and said medium of step (c) is supplemented with an osmo-stabilizer.

11. A method according to claim 1 , wherein said exogenous DNA polynucleotide used in step (c) is from a strain of the same species.

12. The method according to claim 10 , wherein the osmo-stabilizer is glycerol or mannitol.

Assignments (1)
CHANGE OF NAME Recorded Feb 6, 2024
From: DUPONT NUTRITION BIOSCIENCES APS
To: INTERNATIONAL N&H DENMARK APS
Reel/Frame 066494/0814 →
Priority Claims (1)
EP 16205055 · Dec 19, 2016 · regional
Continuity (2)
Continuation 16469784
Related Publication 20230332183A1 · Oct 19, 2023
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