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WO2025073578A1 - Souches bactériennes à résistance aux phages - Google Patents

Souches bactériennes à résistance aux phages Download PDF

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WO2025073578A1
WO2025073578A1 PCT/EP2024/077105 EP2024077105W WO2025073578A1 WO 2025073578 A1 WO2025073578 A1 WO 2025073578A1 EP 2024077105 W EP2024077105 W EP 2024077105W WO 2025073578 A1 WO2025073578 A1 WO 2025073578A1
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ynba
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lactic acid
acid bacterium
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Anne M. Millen
Dennis A. Romero
Laura SIMDON
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International N&H Denmark ApS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present invention relates to strains of lactic acid bacteria, such as of the genus Lacto- coccus having increased resistance to phage infection, methods for the production of a variant strain of a lactic acid bacterium, such as of the genus Lactococcus; compositions comprising such strain, as well as their use as a starter culture, during fermentation, or as host cells in the pro- duction of biomolecules.
  • Lactococcus lactis and Lactococcus cremoris represent a diverse group of strains that are used extensively in starter cultures for fermented dairy products.
  • phages virulent bacterial viruses known as bacteriophages
  • phages are com- monly found in the industrial environment (Sturino and Klaenhammer, 2006). If left uncon- trolled, phages will reduce the quality of the final fermented product, and if the infection is se- vere, they can cause complete fermentation failure. Lactococcal phages have been classified into ten groups, from which three types are pre- dominantly problematic in the dairy fermentation industry: c2 (Ceduovirus genus), 936 (Skuna- virus genus), and P335 (Deveau et.al., 2006).
  • DGCC13630 maintained the milk coagulation properties equivalent to parent DGCC12640, which are both inconsistent with CWPS mutation. Therefore, DGCC13630 potentially harbors a novel, uncharacterized phage-resistance mechanism.
  • the present inventors have identified the uncharacterized phage resistance encoded by DGCC13630.
  • Whole genome sequencing found a mutation in the ynbA gene of DGCC13630. Complementation and deletion experiments confirmed that this mutation in ynbA was responsible for phage resistance.
  • ynbA targeted deletion strategy example (cremoris species).
  • Gray arrows indicate CDS.
  • White and black arrows indicate regions of homology used to delete ynbA.
  • Figure 3: iCinac milk acidification activity test comparison of parent DGCC12640 to DGCC13630 at 32°C. Feature points: Ta indicates the lag time. t5.2 time to pH 5.2. m6-5 is the slope of the acidification curve.
  • Figure 4 Alignment of DGCC12640 and DGCC13630 ynbA CDS. Dark gray bar indicates identity. Light gray arrows indicate CDS. Zoomed in view of mutation.
  • Figure 5 ynbA deletion gels.
  • A PCR amplicons of the deletion region in DGCC12640 and DGCC12697 ynbA deletion mutants. Amplicon sizes of mutants are consistent with the desired deletion.
  • B Plasmid profiles confirm the DGCC12640 and DGCC12697 deletion mutants are de- rived from the parental genotype.
  • SUMMARY OF THE INVENTION It is an object of embodiments of the invention to provide bacterial strains with increased resistance to phage infections.
  • the present invention relates in a broad aspect to strains of a lactic acid bacterium, such as of the genus Lactococcus having increased resistance to phage infection, such as strains func- tionally inactive with respect to phage infection.
  • the present invention relates to a strain of a lactic acid bac- terium, such as of the genus Lactococcus having the ynbA gene or the homologue thereof encod- ing an endogenous YnbA protein or a homologue thereof disrupted, such as deleted or compris- ing a modification to make the YnbA protein or a homologue thereof functionally inactive with re- spect to phage infection.
  • a strain of a lactic acid bac- terium such as of the genus Lactococcus having the ynbA gene or the homologue thereof encod- ing an endogenous YnbA protein or a homologue thereof disrupted, such as deleted or compris- ing a modification to make the YnbA protein or a homologue thereof functionally inactive with re- spect to phage infection.
  • the present invention relates to a strain of a lactic acid bacterium, which is selected from the list consisting of a strain deposited under accession number DSM34734 on 9 August 2023 at the DSMZ or a mutant thereof; and a strain deposited under accession number DSM34735 on 9 August 2023 at the DSMZ or a mu- tant thereof.
  • the present invention relates to a method for the production of a variant strain of a lactic acid bacterium, such as of the genus Lactococcus, resistant to phages, such as phages of the genus Skunavirus, which method comprises the step of either deleting the ynbA gene or homologue thereof or making a suitable modification of the ynbA gene or homologue thereof disrupting or abolishing the expression of a functional YnbA protein.
  • a composition such as a food or feed composition comprising a strain of a lactic acid bacterium according to the invention, or a bacte- riophage resistant variant strain produced using the method of the invention.
  • the present invention relates to methods for preparing such food or feed, starter cultures, fer- mentation methods, as well as the use of a lactic acid bacterium according to the invention for use as a host bacterium in the production of an antimicrobial, a biomolecule, or a recombinant proteins including enzymes.
  • DETAILED DESCRIPTION OF THE INVENTION Lactococcus cremoris DGCC13630 (DSM34734) is a bacteriophage insensitive mutant (BIM) of DGCC12640, isolated from challenge with a Skunavirus.
  • DGCC13630 harbors a previ- ously uncharacterized resistance mechanism against skunaviruses.
  • some bacterial cells exhibit "low susceptibility to bacte- riophage multiplication”. This term refers to the level of bacteriophage multiplication in a bacte- rium being below a level that would cause a deleterious effect to a culture in a given period of time.
  • Such deleterious effects on a culture include, but are not limited to, no coagulation of milk during production of fermented milk products (e.g., yogurt or cheese), inadequate or slow lower- ing of the pH during production of fermented milk products (e.g., yogurt or cheese), slow ripen- ing of cheese, and/or deterioration of a food's texture to the point where it is unappetizing or un- suitable for consumption.
  • bacteria has its conventional meaning as understood in the art, i.e. a virus that infects bacteria. Many bacteriophages are specific to a particular genus or spe- cies or strain of bacteria. The term “bacteriophage” is synonymous with the term “phage”.
  • bacteriophages selected from a P335-type phage, phages of the Ceduovirus genus (formerly known as c2-type phages), such as 7140hy , phages of the Skuna- virus genus (formerly known as 936-type phages), such as any one of GL7, FB3, FB6, RH10, FB10, RH6, FB14, GP14, GP13, GP15, M7671 and D7457.
  • bacteriophage or phage referred to herein is a phage of the Skunavirus genus selected from M7671 and D7457.
  • the term "disrupted,” as applied to the gene ynbA, refers to any genetic modification that de- creases or eliminates the expression of the gene and/or the functional activity of the correspond- ing gene product (mRNA and/or protein). Genetic modifications include complete or partial inacti- vation, suppression, deletion, interruption, blockage, or down-regulation of a gene. This can be accomplished, for example, by gene “knockout,” inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or by use of inhibitory RNAs (e.g., sense, antisense, or RNAi technology). A disruption may encompass all or part of a gene's coding sequence.
  • YnbA gene refers to the gene encoding YnbA protein or homologs, such as a gene identified by any one of SEQ ID Nos 1, 3, 5, 7, 9, 11 or a corresponding homolog or polymorphic sequence thereof.
  • YnbA protein refers to the protein encoded by the ynbA gene. The protein may also be referred to as site-specific tyrosine recombinase, Tyrosine recombinase XerD-like or just XerD.
  • the gene name ynbA is not intended to be limiting, but is intended to encompass homologs (i.e., which may be endogenous to a related microbial organism) and polymorphic var- iants. Homologs and variants can be identified based on sequence identity and/or similar biologi- cal (e.g., enzymatic) activity.
  • the invention includes a polynucleotide or polypeptide sequence with at least 50%, 60%, 70%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the named gene ynbA or its gene product.
  • Identity in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or sub-sequences that are the same or have a specified percentage of nucleotides or amino acid residues, respectively, that are the same. Percent identity may be de- termined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which may also con- tain gaps to optimize the alignment) for alignment of the two sequences.
  • the se- quence can have a percent identity of at least 50%, 60%, 70%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a specified region to a reference sequence when com- pared and aligned for maximum correspondence over a comparison window, or designated re- gion as measured using a sequence comparison algorithm or by manual alignment and visual in- spection.
  • Alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.
  • the lactic acid bacterium used in the present invention may be a bacterium belonging to the ge- nus Lactococcus, for example, Lactococcus allomyrinae, Lactococcus chungangensis, Lactococcus cremoris, Lactococcus formosensis,.
  • Lactococcus fujiensis Lactococcus garvieae, Lactococcus hir- cilactis, Lactococcus kimchii, Lactococcus lactis, Lactococcus laudensis, Lactococcus nasutiter- mitis, Lactococcus petauri, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolac- tis, Lactococcus reticulitermitis, Lactococcus taiwanensis, Lactococcus termiticola, Lactococcus cremoris subsp. occidentaltae, or Lactococcus lactis subsp.
  • the lactic acid bacterium is a bacterium selected from the species of Lactococcus lactis or Lactococcus cremoris, and Lactococcus lactis subsp. lactis bv. diacetylac- tis. Lactococcus lactis subsp. lactis bv. diacetylactis may also be referred to as Lactococcus lactis bv. Diacetylactis, and Lactococcus lactis ssp. Diacetylactis.
  • an aspect of the present invention relates to a strain of a lactic acid bacte- rium, such as of the genus Lactococcus having the ynbA gene or the homologue thereof encoding an endogenous YnbA protein or a homologue thereof disrupted, such as deleted or comprising a modification to make the YnbA protein or a homologue thereof functionally inactive with respect to phage infection.
  • the lactic acid bacterium is of the genus Lactococcus, such as a strain is selected from the species Lactococcus lactis, Lactococcus cremoris, and Lactococcus lactis subsp. lactis bv. diacetylactis.
  • the YnbA protein or a homologue thereof is selected from a YnbA protein and the putative site-specific tyrosine recombinase (XerD).
  • the YnbA protein or a homologue thereof is functionally inactive with re- spect to phage infection due to deletion of the complete ynbA gene.
  • the YnbA protein or a homologue thereof is functionally inactive with re- spect to phage infection due to a modification introduced into the ynbA gene, such as a mutation introducing a stop codon.
  • the YnbA protein or homologue thereof is expressed by a gene comprising or consisting of a nucleotide sequence selected from i) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:1; ii) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:3 ; iii) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:5 ; iv) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:7; v) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:9; vi) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:11; vii) a nucleotide sequence encoding a YnbA protein at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%
  • the YnbA protein or homologue thereof is expressed by a gene comprising or consisting of a nu- cleotide sequence at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a sequence selected from SEQ ID NO 1, 3, 5, 7, 9, and 11.
  • the modification to make the YnbA protein or a homologue thereof func- tionally inactive with respect to phage infection is selected from the group consisting of a stop codon, a deletion, a mutation, or an insertion, such as an insertion that causes a frame shift, a deletion or a mutation causing a premature stop codon, or any other insertion such as of a gene or transposable element disrupting the ynbA gene.
  • the strain according to the present invention has increased resistance to phages, such as phages of the genus Skunavirus, such as M7671.
  • the strain according to the present invention has increased resistance to phages of the genus Ceduovirus, such as increased resistance to both phages of the genus Skunavirus, such as M7671 and to phages of the genus Ceduovirus.
  • the strain according to the present invention shows a reduction in phage sensitivity as measured by a spot titer assays described herein by a factor of at least 50, such as at least 100, such as at least 500, such as at least 1000, such as at least 10000 for each phage tested.
  • the strain according to the present invention has the endogenous gene ynbA deleted.
  • the truncated YnbA is 90-aa vs 248-aa of DGCC12640: MPSLTTLQNIMKLPNEIEEYLVSRNFSENTRSNYYYDLVYLQAFFEDKSVTEEALE- LYKHQLSKLSLLRHNGEKFRVPTNIFYFYMKIKK 717-nt (SEQ ID NO:7) Wild-type ynbA gene in DGCC12640: ATGAAATTACCAAACGAGATTGAGGAGTATTTAGTAAGTAGAAACTTTTCTGAAAA- TACTCGCTCAAATTATTATTATGACTTAGTCTATTTGCAGGCTTTTTTTGAAGATAAGTCTGTCACAGAA GAAGCACTAGAACTTTACAAACACCAGCTTAGTAAACTCTCTCCTGCGGCACAAC- GGCGAAAAATTTCGAGTGCCAACCAATATTTTCTATTTTTATATGAAAATAAAAAAAATAAACCAGTTTTT CAAAATTAAGCAAGTTGTTCAGAAAAAAACT
  • the truncated YnbA is 90-aa vs 238-aa of DGCC12640: MKLPNEIEEYLVSRNFSENTRSNYYYDLVYLQAFFEDKSVTEEALE- LYKHQLSKLSLLRHNGEKFRVPTNIFYFYMKIKK EXAMPLES EXAMPLE 1 Bacterial Strains and Phages Exemplary bacteria and phages used to illustrate the present invention are listed in Table 1.
  • Table 1 Bacteria and phages used Biological Material Relevant Characteristics Reference Bacteria Lactococcus cremoris DGCC12640 phage-sensitive starter strain This example DGCC13630 Skunavirus-resistant mutant of This example DGCC12640 12640 ⁇ ynbA 1-3 DGCC12640 with deletion in ynbA This example 13630-989 DGCC13630 + pTRK989 This example 13630-YnbA 2 and 3 DGCC13630 + pTRK989-ynbA This example DGCC14127 Ceduovirus-resistant mutant of This example (DSM34735) DGCC13630 Lactococcus lactis DGCC12697 Skunavirus-sensitive starter strain This example 12697 ⁇ ynbA DGCC12697 with deletion in ynbA This example Escherichia coli TG1 RepA+ supE hsd ⁇ 5 thi ⁇ (kac-proAB) F’ Duwat et al., (
  • Escherichia coli was propagated aerobically in LB broth (BD Difco, USA) at 37°C.
  • antibiotics were added to the media as follows: chloramphenicol (Cm; 5 ⁇ g/mL), erythromycin (Em; 5 ⁇ g/mL for lactococci; 150 ⁇ g/mL for E. coli).
  • Phage lysates were prepared as described by Terzaghi and Sandine (1975). High titer phage ly- sates were passed through a 0.45 ⁇ m sterilized filter and stored at 4°C. Spot titer assays and plaque assays were performed as previously described (Terzaghi and Sandine, 1975) on MRS medium (BD Difco, USA).
  • Phage adsorption tests were performed as previously described (Sand- ers and Klaenhammer, 1980) on MRS medium.
  • Phage Mutagenesis A single-step phage challenge was performed by exposing DGCC12640 to M7671 and DGCC13630 to phage 7140hy at varying multiplicities of infection followed by plating in MRS overlay. Colonies that grew in the presence of phage were randomly selected and streaked onto FSDA II medium (Sandine 1985), with the trimagnesium phosphate substituted with ⁇ -glycer- ophosphate to evaluate their ability to acidify and then subsequently inoculated into milk over- night to evaluate their ability to clot milk.
  • Acidification Testing For a more thorough assessment of acidification properties, overnight 11% NFDM cul- tures were transferred into M17-Lac and grown. After the cultures reached an OD600 ⁇ 0.50, they were inoculated at 0.75% into activity milk (commercial 2% fat Kemp’s). Samples were kept in a 32 ⁇ C water bath overnight with pH probes and iCinac software monitoring acidification of each strain every 2 minutes. Each strain was run in triplicate. DNA preparation, PCR, and sequencing Plasmid DNA was isolated from lactococci as previously described (Anderson and McKay, 1983).
  • PCRs were performed with GoTaq® Colorless Master Mix (Promega Corp., USA) or Phusion HF Mastermix (Thermo Scientific, USA) according to manufacturer’s instructions. Primer sequences are listed in Table 2. Primers were synthesized by Integrated DNA Technologies (USA). PCR products were purified using Wizard SV Gel and PCR Clean-Up System (Promega Corp., USA). Whole genome sequencing of DGCC12640 and DGCC13630 was performed using Illumina technology. Sanger sequencing was performed by Eurofins Genomics (USA), and results were analyzed using Geneious Prime 2021.0.3 (https://www.geneious.com).
  • Fig- ure 1 shows the vector map for pTRK989-YnbA (A).
  • Figure 2 shows an example of the strategy used for deletion in ynbA using homologous recombination. While Figure 2 shows only the vector map for pCremYnbADel (A) and ynbA deletion region in L. cremoris DGCC12640 (B), a similar vector and strategy were used for deletion of ynbA in L. lactis DGCC12697. Fragments used for each assembly are listed in Table 3. Fragments were assembled using NEBuilder HiFi DNA As- sembly MasterMix (New England Biolabs, USA), and assemblies were electroporated into TG1 RepA + according to manufacturer’s instructions.
  • DGCC12640 containing pCremYnbADel and DGCC12697 containing pLacYnbADel were propagated first at 37°C in M17 + Em broth. Because the origin of replication of the vectors is temperature-sensitive, growth at 37°C while keeping antibiotic pressure should force integration of each element into the host chromosome. Cultures were plated after subculturing up to five times at 37°C. Single colonies were checked for integra- tion by PCR using primers check ynbA del-F and check ynbA del-R of their respective species. Colonies giving amplicons consistent with integration of the full vector or the deletion event were propagated in M17 without Em at 30°C with subculturing over 3 days.
  • DGCC13630 was slightly faster to pH 5.2 than parent strain at 32°C ( Figure 3).
  • DGCC14127 (DSM34735) was isolated from mutagenesis of DGCC13630 with Ceduovirus 7140hy .
  • DGCC14127 was confirmed to be fully resistant to 7140hy (Table 4).
  • DGCC13630 encodes a mutation in ynbA Comparison of Illumina draft genomes of DGCC12640 and DGCC13630 identified a muta- tion in ynbA in DGCC13630 (Fig. 4).
  • Representative pTRK989-ynbA + transformants 13630-YnbA 2 and 13630-YnbA 3 were found to be sensitive to M7671 in spot titer assays, while representative pTRK989 transformant 13630- 989 remained phage resistant (Table 4).
  • PCR and sequencing using primers ynbA 2-F and ynbA 2-R confirmed that the chromosomal copy of ynbA in 13630-YnbA 2 and 13630-YnbA 3 remained mutated (data not shown).
  • Targeted deletion of ynbA results in phage resistance
  • Targeted deletion of ynbA in DGCC12640 was performed using homologous recombina- tion.
  • lactis ynbA is also involved in phage sensitivity
  • targeted deletion was performed in L. lactis DGCC12697.
  • pLacYnbADel was successfully assembled in TG1 RepA+ and electroporated into DGCC12697.
  • Colonies containing the desired deletion were iso- lated from propagations at 37°C in the presence of Em followed by propagations at 30°C without Em.
  • a representative colony that contained the desired ynbA deletion was analyzed (Fig. 5). Spot titer assays found that the ynbA deletion provided complete resistance against D7457 in the DGCC12697 mutant (Table 4). Table 4: Phage assays.
  • cremoris ynbA was analysed using HHPred to identify any conserved domains within the protein that may give insight into its func- tion.
  • Table 5 shows the top ten hits from the HHPred analysis.
  • Top 10 domain hits No Hit name Prob E- P- Score SS aligned Query Template value value cols HMM HMM 1 5C6K_B Integrase; Inte- 98.98 8.90E- 1.40E- 186.1 22.9 238 5- 8-261 (292) grase, tyrosine 30 34 248 recombinase, in- tegration, site- specific recombi- nation, hydrolase; 1.9A ⁇ Enterobacteria phage P2 ⁇ SCOP: d.163.1.1 5VFZ_A Gp33; Bacterio- 98.97 2.20E- 3.40E- 186.2 23.3 240 6- 12-271 phage, Brujita, 29 34 248 (318) DNA-binding, In- tegrase, DNA BINDING PRO- TEIN; HET: ACT, GOL; 1.847A ⁇ Mycobacterium phage Brujita ⁇ 2A3V_C site-specific re- 98.
  • L. cremoris DGCC12640 mutant DGCC13630 was isolated from a phage challenge with Skunavirus M7671. Skunaviruses have been shown to require the host CWPS as their receptor, and mutants against these phages often have mutations in the cwps gene cluster and display decreased phage adsorption. However, these cwps mutations often have a deleterious effect on the strain physiology, including slower growth rates and cell clumping phenotypes (Romero et al., 2020).
  • DGCC13630 did not show a de- crease in phage adsorption, nor did it display impaired acidification, which is inconsistent with what would be expected from a CWPS mutation.
  • DGCC14127 DSM34735 was isolated from mutagenesis of DGCC13630 with a Ceduovirus to further increase the phage ro- bustness of this genotype. Due to the phage resistance phenotype and satisfactory acidification profile of DGCC13630, characterization of its mutation that results in Skunavirus resistance was prioritized. Genome comparisons identified a mutation in the ynbA gene of DGCC13630, a putative tyrosine recombinase.
  • Lactococcus cremoris DGCC13630 and DGCC14127 are Skunavirus-resistant mutants of the DGCC12640 genotype. This resistance was found to result from truncation of a single gene, ynbA. Targeted deletion of the ynbA homolog in L. lactis DGCC12697 also resulted in phage resistance, indicating that ynbA is involved in Skunavirus infection of both lactococcal species, thereby identifying a new target for the construction of phage-resistant lactococcal starter strains.
  • STRAINS AND DEPOSITS DGCC numbers are internal references to DuPont Danisco collection; DSM numbers are the numbers assigned by the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, following deposit under the Budapest Treaty.
  • Dairy lactococcal and streptococcal phage–host interactions an industrial perspective in an evolving phage landscape.
  • the Streptococci Milk Products, p. 5 - 23. In S.E. Gilliland (ed), Bacterial Starter Cultures for Food, CRC Press, Inc. Sanders, M. E., & Klaenhammer, T. R. (1980). Restriction and modification in Group N strepto- cocci: effect of heat on development of modified lytic bacteriophage. Applied Environmental Mi- crobiology, 40, 500-506.

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Abstract

La présente invention concerne des souches de bactérie d'acide lactique, telles que du genre Lactococcus ayant une résistance accrue à l'infection par phage, des procédés de production d'une souche variante d'une bactérie d'acide lactique, telle que du genre Lactococcus ; des compositions comprenant une telle souche, ainsi que leur utilisation en tant que culture de départ, pendant la fermentation, ou en tant que cellules hôtes dans la production de biomolécules.
PCT/EP2024/077105 2023-10-04 2024-09-26 Souches bactériennes à résistance aux phages Pending WO2025073578A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2807446A1 (fr) * 2000-04-11 2001-10-12 Agronomique Inst Nat Rech Genomes de lactococcus lactis, polypeptides et utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2807446A1 (fr) * 2000-04-11 2001-10-12 Agronomique Inst Nat Rech Genomes de lactococcus lactis, polypeptides et utilisations

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