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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
• • C—CHEMISTRY; METALLURGY
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  • C12N1/00—Microorganisms; 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/102—Mutagenizing nucleic acids
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• • 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
• • 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 new strains of lactic acid bacteria having resistance to stress, the process for obtaining said strains as well as their use in particular in fermentation processes.
• the subject of the present invention is new bacterial strains which exhibit increased resistance to stress.
• bacterial growth in laboratories has been studied for decades and it is known that the growth of bacteria in the laboratory. follows a sigoid curve, thus, when inoculating a fresh medium, there is a latency period during which the bacteria prepares for growth, followed by cell multiplication in which the cells grow exponentially up to 'that the environment becomes a limiting condition for growth. It is at this point that the cells enter what is called the stationary phase during which multiplication is stopped and the metabolism of the cells decreases. Survival during these three growth phases, i.e. the latency, exponential and stationary phases. can vary very significantly depending on the specific growth conditions and the type of strain used.
• laboratory conditions are optimal conditions for growth, in industrial fermentation processes one can find conditions that lead to the death of bacteria. Below are given examples in which the survival of lactic acid bacteria depends on the growth conditions.
• L. lactis like other lactic acid bacteria, acidify the medium during their growth and are able to withstand acidic conditions up to pH 4, which is reached during the stationary phase. However, if the culture medium is brutally acidified during the exponential phase, the cells die quickly.
• the cells can be adapted to low pH by first exposing them to intermediate pH, between pH 5 and 6 for example, before moving to lower pH. In this case, better survival is obtained.
• L. lactis cells die if they are exposed to high temperatures (eg 55 ° C). However, if they are exposed to intermediate temperatures (37 ° C) before a thermal shock, there is a better survival of the cells.
• the heat shock genes include in particular dnaJ, grpE-dnaK, groELS and h ⁇ B
• the genes to involve in repairing damage caused by oxygen include in particular recA and sodA
• each bacterial species has a specific niche and must therefore be adapted to its particular environment Under these conditions, the regulators of the stress response genes may be different for different organizations
• control There is a great deal of information concerning the control of stress responses in different bacteria Essentially two types of control seem to exist * one type of control which induces genes in response only to specific stress, the other type of control resulting in the expression of many genes that will protect the cell against different types of stress In bacteria, the first type of control can be done via activators, repressors or metabolites, while the second type of control can be carried out by transcription enzymes, RNA polymerase (by ⁇ factors)
• the present invention relates to the discovery of genes involved in stress resistance and whose mutation leads to an increase in stress resistance of the corresponding bacteria
• the present invention relates to bacteria having an improved resistance to stress compared to the parental strain, characterized in that they comprise at least one mutation in a gene which alters the normal activity of said gene, this gene being involved.
• GTP GTP, (p) ppGpp which we will call GP in the rest of the text, the genes of the clone RI 1 (pstS), RI 4 (arll), RI 5 (glnP), RI 7 (carB), RI 8 ( glnP), RI 14 (arl2), RI 16 (glnP), RI 17 (glnQ), RI 20 (arl3), R2 6 (pstB), R2 9 (reciV), R2 1 1 (arl4), R2 15 (arl5 ), R2 17 (arlT), R2 20 (arl8,
• the mutation in question may also be a mutation by insertion of a DNA sequence, deletion of a DNA sequence and / or point mutation by means for example of a mutagenic agent or by spontaneous mutation
• insertion of a DNA sequence use will be made of the introduction of mobile genetic elements such as insertion sequences as will be described in the example with ISS /
• the DNA when the DNA is inserted, it will be DNA coming from the same species, this so as to keep the acceptability on the agri-food level of the strain thus obtained
• lactic acid bacteria in particular Lactobacilhts, Lactococcus and Streptococcus
• the bacteria thus obtained exhibit an increase in resistance to certain stresses or to a set of stress conditions. It is possible to envisage multiple mutations on different genes For each specific problem posed, it is preferable to compose a set of bacteria mutated in different genes and to select the most appropriate mutants for the stress conditions which are likely to arise during the fermentation process.
• the present invention also relates to a fermentation process using a bacterium according to the present invention and in particular the fermentation processes using milk or milk by-products as the fermentation medium.
• a fermentation process using a bacterium according to the present invention and in particular the fermentation processes using milk or milk by-products as the fermentation medium.
• fermented milk products such as yogurts and equivalent products as well as the preparation of cheeses.
• the bacteria according to the present invention can also be used for the preservation of food products or for probiotic purposes.
• Figure 1 illustrates the mutagenesis system via the plasmid pGh: ISS /.
• Figure 2 illustrates the obtaining of mutants lacking foreign DNA.
• Figure 3 illustrates the identification process of the mute gene
• Figure 4 represents the biosynthetic pathway of GP SEQ ID N ° 1 to 34 represent the sequences of the mutated genes, at the origin of multiresistance, at the junctions of the insertion plasmid, the microorganisms concerned being from what has been called Selection 1 in the example
• SEQ ID N ° 35 to 44 represent the sequences of the mutated genes, at the origin of multiresistance, at the junctions of plasmid insertion, the microorganisms concerned being from what has been called Selection 2 in the example
• SEQ ID N ° 45 to 52 represent the sequences of mutated genes by site-directed mutagenesis
• SEQ ID N ° 53 to 71 represent a list of the sequences of the mutated genes, in the presence of an acid resistance superior to the parental strain (data not shown) at the junctions of the plasmid insertion, the mutants being derived from the
• FIG. 1A represents a microorganism cell with its chromosome, into which the plasmid pGh ISS / which contains the replicon pG + host (Ts on), an antibiotic resistance gene (Ab * ⁇ ) and a mobile element ISS / At 30 ° C, the plasmid replicates in the microorganism cell
• FIG. 1B represents the population of mutant bacterial cells resulting from the integration of the plasmid into the bacterial chromosome after duplication of the ISS / Indeed, at 37 ° C., the plasmid repation is inactive and the plasmid is lost unless the bacteria undergoes transposition event
• FIG. 2 A represents the chromosomal structure of a mutant obtained by transposition of the ISS / The duplicated ISS elements /, frame the plasmid pG + host At 30 ° C., the rephcation of the pG + host is activated and stimulates the recombination homologous between ISS / duplicate sequences
• Figure 2B shows the homologous recombination taking place between the two ISS sequences / leading to the excision of the plasmid pGh ISS / which is then lost when the strain is spread at a non-permissive temperature of 37 ° C.
• the mutated strain contains only one copy of ITSS / which is a sequence originating from the mutated microorganism, which therefore contains no trace of foreign DNA
• FIG. 3 represents the cloning of the junctions between the ISS / and the chromosome
• the Hindlll and EcoR ⁇ restriction sites are unique in the transposed structure and are located on either side of the plasmid pG + host
• Digestion by Hindlll of the Chromosomal DNA of the mutant produces a fragment consisting of the pG + host and the right junction ISS / - chromosome After circularization, this fragment is established as a plasmid in E. coli or other bacteria.
• the junction is then sequenced by means of primers corresponding to the sequence of the ISS / or that of the pG + host.
• the left junction ISS / - chromosome is obtained by applying the same procedure after digestion with
• SEQ ID Nos. 1 to 34 represent the sequences of the mutated genes as they could be determined in accordance with the protocol illustrated in FIG. 3 An "R" code was given to identify each gene concerned Each sequenced junction was compared, in terms of homology, to the sequences of already known microorganisms, and when one of them was identified, it was mentioned NS means that no significant homology with an already known gene could be established The data numeric appearing (for each junction identified), corresponds to the probability that the sequence represented will not be identical to the already known genes mentioned
• SEQ ID N ° 35 to 44 represent the sequences of the mutated genes as determined in accordance with the protocol illustrated in Figure 3 (unless otherwise indicated)
• SEQ ID N ° 45 to 52 represent the sequences of internal fragment of certain genes whose junction sequence had already been determined in accordance with SEQ ID N ° 1 to 34.
• the internal fragment of the gene in question was amplified by PCR then sequenced on the two strands. These fragments were then used to inactivate, by homologous recombination, the corresponding genes in the wild strain so as to verify that the multidrug resistance phenotypes were indeed due to the inactivation of these genes.
• SEQ ID Nos. 53 to 71 represent only the sequences of the genes whose inactivation has made it possible to demonstrate at least one resistance to the acid of the strains concerned.
• Lactococcus lactis is a bacterium which, in the wild state, has the following characteristics: on a medium at 30 ° C., whether the pH is 7 or 5.5, it has the same spreading efficiency; on a medium at 37 ° C, the spreading efficiency on pH 7 is 10 ⁇ times higher than that obtained on pH 5.5 or pH 5.
• a group of mutants comes from the wild strain MG1363 of .Lactococcus lactis and consists of mutants having at 37 ° C a spreading efficiency on a medium at 37 ° C at pH 5.5 between 1 and 0.01 relative to pH 7. Thirty mutants were thus isolated.
• a second group of mutants comes from the wild strain MG1363 of Lactococcus lactis previously mutated in the recA gene making it sensitive to ultraviolet radiation.
• This strain called VEL1 122, is sensitive to temperature. It was found that at the temperature of 39.3 ° C. (with a pH of the medium of approximately 7 but not determining) the strain recA had a spreading efficiency of less than 10 " - ⁇ when the wild strain MG1363 had a spreading efficiency of 1. Mutants were selected from the VEL1 122 strain capable of growing at a temperature of 39.3 ° C. About twenty mutants were thus isolated.
• thermosensitive plasmid pGh ISS / (cf. reference 16).
• This plasmid consists of the replicon pG + host (Ts ori), a gene for resistance to an antibiotic (in the present case erythromycin) and the mobile element ISS / (cf. FIG. 1).
• This plasmid has the particularity of replicating in cells at 30 ° C, but not at 37 ° C or more.
• This plasmid can integrate into the chromosome of bacteria via an ISS / transposition event. During transposition, the entire plasmid, flanked on either side by 1TSS / is integrated into the chromosome.
• the transposon integration sites are random.
• the bacteria that have integrated the plasmid pGh: ISS / into their chromosome therefore retain resistance to erythromycin at 37 ° C or more. Consequently, among the bacteria having undergone the above mutagenesis, those which are still capable of growing in the presence of antibiotics on a medium at 37 ° C. or higher are selected. These bacteria have undergone a transposition event which generates random mutations.
• mutants more resistant to stress were selected according to the methods previously indicated (Selection 1 or 2).
• the mutants resulting from Selections 1 and 2 are subjected to a temperature of 30 ° C. which activates the rephcation of the pG + host and stimulates the homologous recombination between the ISS / duplicated sequences which results in the excision of the plasmid pGh: ISS /.
• the bacteria in question therefore contain, inserted in their chromosome, only one copy of the ISS / (cf. Figure 2)
• the final strain mutated by insertion of the ISS / is stable and does not contain DNA foreign to Lactococcus, it therefore constitutes a food-grade mutant.
• Lactococcus lactis bacteria thus obtained (Selection 1 or Selection 2 mutants) are then subjected to different stress conditions in respect of which their resistance has been tested compared to that of the parental strain subjected to the same conditions.
• the genes identified in Table I correspond to SEQ ID Nos. 1 to 34.
• microorganisms grown at 30 ° C, are directly subjected to a thermal stress of 55 ° C.
• microorganisms, cultured at 30 ° C are subjected for 15 minutes to a temperature of 37 ° C and then to a thermal stress of 55 ° C. actual survival of the parental strain; this value is then reduced to 1.
• NAME / KEY RI. 1-EcoRI junction: clone SS80 L. lactis (3e-14)
• NAME / KEY RI. 1-Hindlll junction: clone SS80 L. lactis (3e-47)
• NAME / KEY RI. 5-EcoRI junction: GlnQ. E. coli (3. e-1!
• NAME / KEY RI. 5-Hindlll junction: GlnQ. E. coli (6.6e-3)
• NAME / KEY RI. 7-Hindlll junction: carbamoyl-phosphate synthase.B.caldolyticus (3.9e-ll)
• NAME / KEY RI. 8-EcoRI junction: GlnH. E. coli (5.3e-29)
• CGTNCAATTC CGCTTTTAGT GTTAANAATC TTTATCTTCT ANGGGATTCC TAANCTTCTA 420
• CAAATCATT 429 (2) INFORMATION FOR SEQ ID NO: 10:
• CTCGTTTAAC TTATCGTGGC ATAATCTTAT ATCAAGCTAT CTAATCATGT TTTATCACTG 120
• AACACGTTGT TTGTGTCCTC CAGAAAGCAT TCTGGCATCG CGTCTTTTTT GTCTGCCAAG 300
• TTTACTTCAC CAGAAGATTT TGTTCCTCCG TGATAGCTTG ATACGGTCAT AAAATCAGTT 300
• NAME / KEY R2.2-EcoRI junction: Rel. S. equisimilis (1.4e-53)
• NNATCTCAAC TCGGTCCCCT GTTTTTAATT GAACAGATAA AGGTTTCATA CGTCCATTIA 120
• GAGCGGCCGC CACCGCGGTG GGATCCTCTA GAGTCTAGGG ACCTCTTTAG CTCCTTGGAA 540
• AAAATAGTAA AAAGAAAGTC CATGAATTGA TAGCGTGTTT TC ⁇ CCTCCCA AATGATGGTA 540
• GGTTTTTNNN NNNNNNTGAC TTTATTTAGC TTAAGAGCCG AATGNTATAA ACCTTGTATT 60
• NAME / KEY R2.3-Hindi junction 11: GuaA. B. subtilis (2.4e-33)
• TATTACGANC AAACCACCAG CAACTGTTGA GTGGCANTAC CAATAAAAAA ACTGATAGCA 360
• NAME / KEY R2.6-EcoRI junction: Hypothetical ABC transporter.
• GACTATTCAA GCCTGAATAA TATTCGGACG ACAATGACCG AATTGGATAG AATGGCAATT 180
• AACATCTCCT AAAGAATATC ATAATATTAG GGAAAAGAGT AAAAATGAAA GCGCTATAGT 240
• AAAGCAACTT CCTAAAGACT TGTCAAAAAG AATCCTAAAC CTAGTGTACG CCAAAGTCCT 480
• TCAACTCNTA TCATTCCTTT AAGGNGCCCC CATTGACAAT ATACCTTGTA TTTGATTTCN 60
• TAAACTTTGC AACAGAACCC TTGNAAAGTC ATTTTTGGCA GATAAGCGGT CATGGTGTGC 120
• AAGATTGAGC AAAATGAAAG GATATTTATC CCATTCAACG CCAAAGAGGC CAGTCACATT 240
• GACAAACATC CAGACTACTA ⁇ ATACCGAGA TAGGTGATAA TAAAAGCCCA CGAACCGACG 300
• CAACTTTTTC ACGAATATCT TTGATTTGCA TATCAATGAA GTTTTCCATT GACCAGTTCC 300
• CTCGAGGTCG ACGGTATCGA TAAGCTTGAT ATCGAATTCC TGCAGCCCTG AATTAGTCGA 60
• NAME / CLE relA gene (inactive in mutant R2.2) strand 2 sequence (complementary)
• NAME / CLE guaA gene (inactive in mutants R2.3 and R2.14) strand 1 sequence
• NAME / CLE guaA gene (inactive in mutants R2.3 and R2.14) strand 2 sequence (complementary)
• AAATTAAAAA ACGATTATTA ATCGTTTTTTTT TATCTTATAA TGAATTAAAT AATTCTTGAA 240
• CTGTTCAAAA AATTTTTGAA AAAAGCCCTA ANATAAATTC ATCAATCTCC AAGTTCAAAA 660
• GGTTTTTNNN NNNNNNTGAC TTTATTTAGC TTAAGAGCCG AATGNTATAA ACCTTGTATT 60

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