US20200087686A1 - Methods and strain - Google Patents

Methods and strain Download PDF

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US20200087686A1
US20200087686A1 US16/469,784 US201716469784A US2020087686A1 US 20200087686 A1 US20200087686 A1 US 20200087686A1 US 201716469784 A US201716469784 A US 201716469784A US 2020087686 A1 US2020087686 A1 US 2020087686A1
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seq
strain
comx
lactococcus
gene
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Christophe Fremaux
Philippe Horvath
Patrick Boyaval
Pascal Hols
David Blandine
Amandine Radziejwoski
Laetitia Fontaine
Frederic Toussaint
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International N&H Denmark ApS
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DuPont Nutrition Biosciences ApS
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    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • 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.
  • Lactococcus lactis is one of the most important lactic acid bacteria used in the dairy industry, in particular as a main dairy starter species in various cheese preparations (e.g. gouda, cheddar, brie, parmesan, roquefort) and fermented milk products (e.g. buttermilk, sour cream).
  • Other applications of L. lactis bacteria include as a host for heterologous protein production or as a delivery platform for therapeutic molecules. While the growth and fermentation properties of L. lactis have been gradually improved by selection and classical methods, there is great potential for further improvement through natural processes or by genetic engineering. Of particular interest are methods to naturally transform L. lactis without the use of genetic engineering, thereby generating new non-GMO strains with useful industrial properties.
  • Lactococcus raffinolactis is present in a wide range of environments, such as foods (meat, fish, milk, vegetable), animals, and plant materials. In the dairy environment, this species has been found in raw milks (cow, ewe, goat, and camel), natural dairy starter cultures, and a great variety of cheeses. The prevalence of this bacterium in foods even if with a “nondominant” status compared to other lactococci could make it a candidate for future development of starter cultures.
  • DNA acquisition by natural transformation is widespread among prokaryotes and has been identified in over 80 species.
  • Various functions are attributed to competence for natural transformation: genome plasticity, DNA repair, and/or nutrition.
  • competence for natural transformation has been well-characterized in Bacillus subtilis and in various species of the genus Streptococcus (e.g. S. pneumoniae, S. mutans , and S. thermophilus ).
  • competence for DNA transformation is induced in response to secreted signalling peptides referred to as competence pheromones/alarmones.
  • the production of this class of cell-to-cell communication molecules is initiated in response to specific environmental stresses or conditions and allows the coordination of physiological functions (e.g. competence, predation, biofilm formation).
  • competence pheromones activate the master regulator ComX (alternative sigma factor ⁇ X ), which ultimately leads to a transcriptional reprogramming of cells (globally known as late competence phase) including the induction of genes strictly required for DNA transformation.
  • ComX binds to a specific DNA sequence named Com-box or Cin-box, which is located at least in the vicinity of promoters of late competence (corn) genes/operons responsible for DNA uptake (e.g.; comG, comF and comE operons), DNA protection (e.g. ssb) and DNA recombination (e.g. recA, dprA, coiA), and positively controls their expression.
  • comG promoters of late competence
  • comE operons e.g. ssb
  • DNA recombination e.g. recA, dprA, coiA
  • Orthologues of comX and of all late corn genes essential for natural transformation have been identified in the genome of L. lactis , although some are present as putative pseudogenes in different strains (Wydau et al., 2006).
  • the present invention provides a method for transforming a strain of the Lactococcus genus with an exogenous DNA polynucleotide comprising the steps of:
  • the step of modulating the production of a ComX protein is performed by expressing a comX gene in said strain or increasing the expression of a comX gene in said strain.
  • the comX gene is an exogenous comX gene.
  • Said exogenous comX gene may be transferred into said strain by conjugation, transduction, or transformation.
  • Said exogenous comX gene may be operably linked to transcription regulator(s).
  • said comX gene is the endogenous comX gene of said strain.
  • the method comprises:
  • 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, SEQ ID NO:22, or has at least 90% identity or at least 90% similarity 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.
  • said ComX protein has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or has at least 90% identity or at least 90% similarity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.
  • 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 has at least 90% 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 or SEQ ID NO:21.
  • said comX gene has the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or has at least 90% identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.
  • the medium of step (c) is a chemically defined medium.
  • the chemically defined medium (CDM) comprises 0.5 g/L NH 4 Cl, 9.0 g/L KH 2 PO 4 , 7.5 g/L K 2 HPO 4 , 0.2 g/L MgCl 2 , 5 mg/L FeCl 2 , 50 mg/L CaCl 2 ), 5 mg/L ZnSO 4 , 2.5 mg/L CoCl 2 , 0.05 g/L tyrosine, 0.1 g/L asparagine, 0.1 g/L cysteine, 0.1 g/L glutamine, 0.1 g/L isoleucine, 0.1 g/L leucine, 0.1 g/L methionine, 0.1 g/L tryptophan, 0.1 g/L valine, 0.1 g/L histidine, 0.2 g/L arginine, 0.2 g/L glycine, 0.2 g/L glycine
  • said strain prior to step (c) said strain is incubated in a pre-culture medium, preferably wherein the pre-culture medium is a complex medium, more preferably wherein the pre-culture medium is M17G or THBG.
  • said strain is incubated with the exogenous DNA polynucleotide for around 4 to 8 hours at around 30° C. and said medium of step (c) is supplemented with an osmo-stablizer, preferably wherein the osmo-stablizer is glycerol or mannitol, more preferably wherein the osmo-stabilizer is 5% [v/v] glycerol or 5% [w/v] mannitol.
  • an osmo-stablizer preferably wherein the osmo-stablizer is glycerol or mannitol, more preferably wherein the osmo-stabilizer is 5% [v/v] glycerol or 5% [w/v] mannitol.
  • said exogenous DNA polynucleotide is from a strain of the Lactococcus lactis species.
  • said exogenous DNA polynucleotide is from a strain of the Lactococcus raffinolactis species.
  • said strain of step (a) is a Lactoccocus lactis subsp. cremoris strain.
  • the present invention provides a strain of the Lactococcus genus obtained or obtainable by the method of the first aspect of the present invention.
  • said strain of the Lactococcus genus is a strain of the Lactococcus lactis or Lactococcus raffinolactis species.
  • the present invention provides a method for identifying a strain of the Lactococcus genus which is transformable through natural competence comprising the steps of:
  • a rate of at least 1 ⁇ 10 ⁇ 6 transformants per ⁇ g of DNA is indicative of a strain which is transformable through natural competence.
  • said strain of step (a) is identified using the method for identifying a strain of the Lactococcus genus which is transformable through natural competence according to the present invention. In some embodiments of the present invention, said strain of step (a) is identified using Assay A.
  • FIG. 1 Table showing the status of genes involved in natural competence for L. lactis strains MG1363, SK11, KW2, IL1403, SL12651 and SL12653.
  • FIG. 2 Graphs displaying the results of luciferase assays which demonstrate the activation of a reporter construct comprising the late promoter P comGA driven by constitutive comX overexpression
  • A Maximum specific luciferase (Lux) activity (RLU OD 600 ⁇ 1 ) emitted by eight independent clones (cl01 to cl08) of the KW2-derived reporter strain (BLD101, P comGA[MG]-lux AB) carrying plasmid pGhP32comX MG compared to the control strain (Ctl) carrying the empty vector pG + host9.
  • B Kinetics of specific Lux activity (solid line) during growth (RLU/OD 600 ; dotted line) for the control strain (Ctl; black lines) and three selected clones (BLD101 [pGhP32comX MG ], cl02, cl04 and cl05; gray lines).
  • C Kinetics of specific luciferase activity (closed symbols) during growth (RLU/OD 600 ; open symbols) of the MG1363+pGhP32comX MG -P comGA[MG] -luc, grown in M17G at 30° C.
  • D Kinetics of specific luciferase activity (closed symbols) during growth (RLU/OD 600 ; open symbols) of IL1403+pGhP32comX IO -P comGA[IO] -luc strains, grown in M17G at 30° C.
  • FIG. 3 Graphs displaying the results of luciferase assays which demonstrate the impact of growth medium on P comGA activation
  • FIG. 4 Results of a transformation assay implemented on a L. lactis subsp. cremoris KW 2 constitutively expressing comX contacted with a DNA consisting of a mutated allele of the rpsL gene as exogenous DNA polynucleotide
  • the dollar sign at position 167 indicates the point mutation (A ⁇ T; strA1 allele) responsible for the streptomycin-resistance phenotype; the pound sign at position 156 highlights a nucleotide that is naturally different between MG1363 and KW2 (T in KW2, A in MG1363); the arobase sign at position 39 indicates a silent nucleotide substitution (T ⁇ G) which is found in the streptomycin-resistant clone derived from MG1363.
  • B DNA transformation with the strA1 allele was assessed for L. lactis strains constitutively expressing ComX.
  • Transformation rate (white bars) and maximum specific luciferase (Lux) activity (black diamonds, RLU OD 600 ⁇ 1 , as reported in FIG. 2 ) of eight clones (cl01 to cl08) of the reporter strain (BLD101, P comGA[MG] -luxAB) carrying plasmid pGhP32comX MG compared to the negative control strain (Ctl ⁇ ) carrying the empty vector (BLD101 [pG + host9]).
  • FIG. 5 Graphs displaying the results of transformation rate of the KW2 derivative BLD101 [pGhP32comX MG ] obtained with overlap PCR products (comEC, mecA, ciaRH, covRS and clpC) and strA1 (rpsL*)-donor DNA.
  • the threshold represents the theoretical transformation rate to obtain only one transformant.
  • FIG. 6 Graphs depicting the results of transformation assays for a L. lactis subsp. cremoris deleted in its comEC gene and constitutively expressing comX.
  • FIG. 7 Graphs displaying natural competence in Lactococcus lactis subsp. lactis SL12651 and 12653 strains.
  • A Transformation rate of L. lactis subsp. lactis SL12651 and 12653 strains in M17G medium, with rpsL* donor DNA (+DNA) or without donor DNA ( ⁇ DNA);
  • B DNA transformation with increasing initial concentration of donor DNA assessed in SL12653 strain;
  • C Comparison of transformation rates between wild-type (WT) SL12653 strain and a SL12653 strain deleted for the comX gene (ComX ⁇ ); transformation rate of three clones of the ComX-deficient strain compared to the WT strain, in presence (+DNA) or in absence ( ⁇ DNA) of donor DNA.
  • the present invention is based on the observation that overexpression of ComX in a strain of the Lactococcus genus allowed to transform this strain by natural competence.
  • a L. lactis strain was generated by natural transformation with an exogenous DNA polynucleotide.
  • these results are the first demonstration of transformation of a L. lactis strain by natural competence.
  • existence of natural competence in the Lactococcus genus has been confirmed in two strains of the Lactococcus raffinolactis species and two Lactococcus lactis species.
  • the present invention provides a method for transforming a strain of the Lactococcus genus with an exogenous DNA polynucleotide comprising the steps of:
  • step (b) and step c) can be carried out sequentially [i.e., step (b) and then step (c)] or in another embodiment step (b) and step (c) can be carried out simultaneously.
  • the present invention relates to a method for transforming a strain of the Lactococcus genus, a Gram-positive bacterium.
  • Lactococcus strains are known as lactic acid bacteria (LAB) for their ability to convert carbohydrate to lactic acid.
  • LAB lactic acid bacteria
  • a strain of the Lactococcus genus and Lactococcus strain are used herein interchangeably.
  • the Lactococcus genus comprises, but is not limited to the following species: Lactococcus chungangensis, Lactococcus fujiensis, Lactococcus garvieae, Lactococcus lactis, Lactococcus piscium, Lactococcus plantarum and Lactococcus raffinolactis . Any strain of one of these species may be used in the current invention, provided that this strain is transformable through natural competence as defined herein.
  • said strain of the Lactococcus genus of step a) is a strain of the Lactococcus lactis species or a strain of the Lactococcus raffinolactis species.
  • said strain of the Lactococcus genus of step a) is a strain of the Lactococcus lactis species.
  • the species Lactococcus lactis comprises several subspecies.
  • said strain is selected in the group consisting of Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. gagtae .
  • a strain of the Lactococcus lactis species is understood to be a genetic variant or subtype of any L. lactis species or subspecies.
  • the different Lactococcus lactis subspecies disclosed here, and in particular the lactis and the cremoris subspecies, are defined herein based on DNA sequences coding for 16S ribosomal RNA [Ward et al., 1998].
  • the present invention provides a method for transforming a strain of the Lactococcus lactis species with an exogenous DNA polynucleotide comprising the steps of:
  • the strain of step (a) is a Lactococcus lactis subsp. cremoris strain or a Lactococcus lactis subsp. lactis strain. Both subspecies have been identified and characterised with full genome sequences see, e.g., Wegmann et al. (2007) J. Bacteriol. 189:3256-3270 and Bolotin et al. (2001) Genome Res. 11:731-753. With regards to the dairy industry, L. lactis subsp. lactis (previously known as Streptococcus lactis ) is preferred for making soft cheese while L. lactis subsp. cremoris (previously known as Streptococcus cremoris ) is preferred for hard cheese production.
  • the strain of step (a) is Lactococcus lactis subsp. cremoris strain.
  • the strain of step (a) is Lactococcus lactis subsp. lactis strain.
  • said strain of the Lactococcus genus of step a) is a strain of the Lactococcus raffinolactis species.
  • the present invention provides a method for transforming a strain of the Lactococcus raffinolactis species with an exogenous DNA polynucleotide comprising the steps of:
  • said strain of the Lactococcus genus of step a) is a strain of the Lactococcus plantarum species.
  • the present invention provides a method for transforming a strain of the Lactococcus plantarum species with an exogenous DNA polynucleotide comprising the steps of:
  • said strain of the Lactococcus genus of step a) is a strain of the Lactococcus piscium species.
  • the present invention provides a method for transforming a strain of the Lactococcus piscium species with an exogenous DNA polynucleotide comprising the steps of:
  • said strain of the Lactococcus genus of step a) is a strain of the Lactococcus garvieae species.
  • the present invention provides a method for transforming a strain of the Lactococcus garvieae species with an exogenous DNA polynucleotide comprising the steps of:
  • said strain of the Lactococcus genus of step a) is a strain of the Lactococcus fujiensis species.
  • the present invention provides a method for transforming a strain of the Lactococcus fujiensis species with an exogenous DNA polynucleotide comprising the steps of:
  • said strain of the Lactococcus genus of step a) is a strain of the Lactococcus chungangensis species.
  • the present invention provides a method for transforming a strain of the Lactococcus chungangensis species with an exogenous DNA polynucleotide comprising the steps of:
  • Bacteria may naturally acquire exogenous DNA via one of three possible mechanisms: transformation, conjugation, or transduction.
  • transformation refers to the uptake of exogenous genetic material (e.g. a DNA polynucleotide) from the external medium. Since transformation requires that genetic material cross the bacterial cell wall and membrane and the uptake of exogenous genetic material is energetically costly, the process is tightly regulated. Accordingly, bacterial cells may only be transformed under certain conditions. Bacterial cells which are in a transformable state are said to be competent.
  • exogenous genetic material e.g. a DNA polynucleotide
  • Competence may be artificially induced in the laboratory, e.g. by electroporation or exposure to divalent cations (e.g. CaCl 2 )) and heat shock.
  • divalent cations e.g. CaCl 2
  • some species of bacteria express a proteinaceous machinery that provides natural competence; this system of natural competence has been widely studied in streptococci.
  • conjugation refers to the transfer of genetic material between bacterial cells.
  • transduction refers to the transfer of genetic material from a virus (e.g. a bacteriophage) or a viral vector into bacterial cell.
  • the method of the present invention comprises the step of modulating the production of a ComX protein in said strain.
  • ComX protein is an alternative sigma factor, also known as ⁇ x , which acts as master regulator for the late corn genes and is responsible for transcriptional reprogramming of cells including the induction of genes strictly required for DNA transformation (Lee et al., 1989; Petersen et al. 2004).
  • ComX may bind to a specific target sequence (or box) termed the Com-box (or Cin-box).
  • Com-boxes are located in the vicinity of the promoters of late competence (corn) genes/operons responsible for DNA uptake (e.g., comG, comF, and comE operons), DNA protection (e.g. ssb) and DNA recombination (e.g. recA, dprA, coiA), and positively controls their expression (Campbell et al., 1998; Luo and Morrison, 2003).
  • the production of the ComX protein in a strain of interest may be increased relatively to an appropriate control strain, i.e., the Lactococcus strain in which the production of the ComX protein has not been modulated.
  • ComX protein may be produced (expressed) following modulation as compared to an appropriate control strain, i.e., the Lactococcus strain in which the ComX protein is not produced.
  • the production of the ComX protein is constitutive or inducible.
  • ComX protein may be monitored using any method known in the art. For example, by western blotting using an antibody specific for the ComX protein. Alternatively, comX gene mRNA transcript levels may be measured by qPCR.
  • the ComX protein may be monitored using a reporter construct polynucleotide, e.g. as described in the Example 1 and Materials and Methods.
  • the reporter construct polynucleotide may comprise genes encoding one or more reporter proteins, preferably the genes encoding the reporter proteins are operably linked to a promoter comprising a Com-box sequence.
  • the reporter proteins may be LuxAB or Luc. Accordingly, ComX expression (and activity) may be detected and measured using a luciferase assay (Fontaine et al., 2010).
  • the step of modulating the production of a ComX protein is performed by expressing a comX gene in said strain or increasing the expression of a comX gene in said strain.
  • the step of modulating the production of a ComX protein is performed by expressing a comX gene in said strain in some growth conditions, whereas said strain does not express the ComX protein outside of these growth conditions.
  • the step of modulating the production of a ComX protein is performed by increasing the expression of a comX gene in said strain in some growth conditions.
  • the comX gene may be an exogenous comX gene.
  • an “exogenous comX gene” is understood to be a comX gene which is brought into the cytoplasm of the Lactococcus strain of step a), in order to be expressed.
  • the exogenous comX gene may have the same sequence as the comX gene found in the genome of the Lactococcus strain of step a) or may have a different sequence from the comX gene found in the genome of the Lactococcus strain of step a).
  • the comX gene may be derived from a strain of a different species, a different subspecies or a different strain of Lactococcus.
  • the exogenous comX gene may be integrated within the genome of said Lactococcus strain.
  • the exogenous comX gene may be located within a vector.
  • the vector may be selected from a plasmid, a viral vector (e.g. a phage), a cosmid, or a bacterial artificial chromosome.
  • Said plasmid may be transferred into said Lactococcus strain by conjugation, transformation or transduction. Said plasmid may be auto-replicative in the transformed Lactococcus strain or not.
  • the exogenous comX gene may be operably linked to transcription regulator(s).
  • the exogenous comX gene may be located in a linear or circular polynucleotide.
  • the comX gene is the endogenous comX gene of said Lactococcus strain.
  • the endogenous comX gene of said strain is understood to be a comX gene that is naturally present in the genome of said strain.
  • said comX gene is a Lactococcus comX gene. In an embodiment, said comX gene is a Lactococcus lactis comX gene. In a particular embodiment, said comX gene is a Lactococcus lactis subsp. lactis comX gene. In a particular embodiment, said comX gene is a Lactococcus lactis subsp. cremoris comX gene.
  • the comX gene may comprise or consist of a nucleotide sequence selected from the group consisting of:
  • said comX gene has the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or is a variant of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 encoding respectively the ComX protein of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 or is a variant of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 encoding respectively a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.
  • the comX gene comprises the nucleotide sequence of SEQ ID NO:1, any sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1, a variant of SEQ ID NO:1 encoding the ComX protein of SEQ ID NO:2 or a variant of SEQ ID NO:1 encoding a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:2.
  • the comX gene comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:5, any sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:3 or SEQ ID NO:5 or a variant of SEQ ID NO:3 or SEQ ID NO:5 encoding respectively the ComX protein of SEQ ID NO:4 or SEQ ID NO:6 or a variant of SEQ ID NO:3 or SEQ ID NO:5 encoding respectively a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:4 or SEQ ID NO:6.
  • said comX gene has the nucleotide sequence of SEQ ID NO:7 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO:7 or is a variant of SEQ ID NO:7 encoding the ComX protein of SEQ ID NO:8, or is a variant of SEQ ID NO:7 encoding a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:8.
  • said comX gene is used when the Lactococcus strain in step a) is a Lactococcus raffinolactis strain.
  • said comX gene has the nucleotide sequence of SEQ ID NO:9 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO:9 or is a variant of SEQ ID NO:9 encoding the ComX protein of SEQ ID NO:10, or is a variant of SEQ ID NO:9 encoding a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:10.
  • said comX gene is used when the Lactococcus strain in step a) is a Lactococcus plantarum strain.
  • said comX gene has the nucleotide sequence of SEQ ID NO:11 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO:11 or is a variant of SEQ ID NO:11 encoding the ComX protein of SEQ ID NO:12, or is a variant of SEQ ID NO:11 encoding a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:12.
  • said comX gene is used when the Lactococcus strain in step a) is a Lactococcus piscium strain.
  • said comX gene has the nucleotide sequence of SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17, any sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or a variant of SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 encoding respectively the ComX protein of SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or a variant of SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 encoding respectively a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18.
  • said comX gene is
  • said comX gene has the nucleotide sequence of SEQ ID NO:19 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO:19 or is a variant of SEQ ID NO:19 encoding the ComX protein of SEQ ID NO:20, or is a variant of SEQ ID NO:19 encoding a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:20.
  • said comX gene is used when the Lactococcus strain in step a) is a Lactococcus fujiensis strain.
  • said comX gene has the nucleotide sequence of SEQ ID NO:21 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO:21 or is a variant of SEQ ID NO:21 encoding the ComX protein of SEQ ID NO:22, or is a variant of SEQ ID NO:21 encoding a functional ComX protein having at least 90% identity or at least 90% similarity to a ComX protein of SEQ ID NO:22.
  • said comX gene is used when the Lactococcus strain in step a) is a Lactococcus chungangensis strain.
  • a comX gene is specified as having a particular nucleotide sequence
  • the comX gene comprises said nucleotide sequence.
  • the comX gene consists of said nucleotide sequence.
  • variants as defined herein of comX genes are selected from the list of DNA sequences disclosed in Table 1 below:
  • lactis Il1403 AE005176.1 From 2223528 to 2224019 (reverse) Lactococcus lactis subsp. lactis IO-1 DNA AP012281.1 From 2287126 to 2287617 (reverse) Lactococcus lactis subsp. lactis JCM 7638 BBAP01000017.1 From 34164 to 34656 (forward) Lactococcus lactis subsp. lactis K231 LKLE01000041.1 From 32159 to 32650 (forward) Lactococcus lactis subsp. lactis K337 LKLF01000041.1 From 34909 to 35400 (forward) Lactococcus lactis subsp.
  • a comX gene is understood to be a gene that encodes a functional ComX protein in the strain where it is expressed.
  • functional ComX protein it is meant a protein which induces or is able to induce the expression of genes regulated by the Com-box, and at least one of the late competence genes selected from comFA, comFA, comGA, dprA, coiA, ssbA, radA, radC, recA, and recX.
  • the ComX protein may have 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, 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 91%, at least 92%, at least 93%, at least 94%, at least 95%,
  • the ComX protein may have the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the amino acid sequence of SEQ ID NO:2.
  • said ComX protein is used when the Lactococcus strain in step a) is a Lactococcus lactis subsp. lactis strain.
  • the ComX protein may have the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6 or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.
  • said ComX protein is used when the Lactococcus strain in step a) is a Lactococcus lactis subsp. cremoris strain.
  • the ComX protein may have the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the amino acid sequence of SEQ ID NO:8.
  • said ComX protein is used when the Lactococcus strain in step a) is a Lactococcus raffinolactis strain.
  • the ComX protein may have the amino acid sequence of SEQ ID NO:10, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the amino acid sequence of SEQ ID NO:10.
  • said ComX protein is used when the Lactococcus strain in step a) is a Lactococcus plantarum strain.
  • the ComX protein may have the amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the amino acid sequence of SEQ ID NO:12.
  • said ComX protein is used when the Lactococcus strain in step a) is a Lactococcus piscium strain.
  • the ComX protein may have the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18 or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the amino acid sequence of SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18.
  • said ComX protein is used when the Lactococcus strain in step a) is a Lactococcus garvieae strain.
  • the ComX protein may have the amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the amino acid sequence of SEQ ID NO:20.
  • said ComX protein is used when the Lactococcus strain in step a) is a Lactococcus fujiensis strain.
  • the ComX protein may have the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO:22 or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the amino acid sequence of SEQ ID NO:22.
  • said ComX protein is used when the Lactococcus strain in step a) is a Lactococcus chungangensis strain.
  • a ComX protein when a ComX protein is defined by its amino acid sequence having a percentage of identity or percentage of similarity to a specific SEQ ID, said ComX protein is a functional ComX protein as defined herein.
  • a ComX protein comprises said amino acid sequence.
  • the ComX protein consists of said amino acid sequence.
  • ComX proteins having percentage of identity or percentage of similarity as defined herein are selected from the list of protein sequences derived, after translation, from the list of DNA sequences disclosed in Table 1 above.
  • reference to a sequence which has a percentage identity or similarity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity or similarity with the SEQ ID NO referred to, over the entire length of the two sequences.
  • Percentage (%) sequence identity is defined as the percentage of amino acids or nucleotides in a candidate sequence that are identical to the amino acids or nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Percentage (%) sequence similarity is defined as the percentage of amino acids in a candidate sequence that are similar to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence similarity.
  • Similarity between amino acids is based on established amino acid substitution matrices such as the PAM series (Point Accepted Mutation; e.g. PAM30, PAM70, and PAM250) or the BLOSUM series (BLOck SUbstitution Matrix; e.g. BLOSUM45, BLOSUM50, BLOSUM62, BLOSUM80, and BLOSUM90). Alignment for purposes of determining percent sequence identity or similarity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as CLUSTALW, CLUSTALX, CLUSTAL Omega, BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • PAM series Point Accepted Mutation
  • BLOSUM series BLOck SUbstitution Matrix
  • Alignment for purposes of determining percent sequence identity or similarity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as CLUSTALW, CLUS
  • similarity between amino acids is determined using the BLASTp software with the BLOSUM62 matrix.
  • Appropriate parameters for measuring alignment including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, or gap penalties to be introduced, can be determined by known methods.
  • step b) when the modulation in step b) results from an exogenous comX gene, said exogenous comX gene is (obtained) from a strain of the same species, in particular of the same subspecies, as the strain provided in step a).
  • the exogenous comX gene needs to be functional, in particular needs to encode a functional ComX protein, as defined herein in the strain provided in step a).
  • the method of the present invention comprises the step of contacting the strain of step b) with an exogenous DNA polynucleotide in a medium and incubating the resulting mixture for integration of the exogenous DNA into the genome of said strain [step c].
  • exogenous DNA polynucleotide refers to a DNA polynucleotide that is brought into the cytoplasm of said strain, in order to be integrated into the genome of said strain (target sequence).
  • the method comprises carrying out step (b) [ComX modulation] and then carrying out step (c) [contact with the exogenous DNA polynucleotide] [i.e., that step (c) is carried out on a strain obtained following step (b)].
  • step (b) [ComX modulation]
  • step (c) [contact with the exogenous DNA polynucleotide]
  • the method comprises carrying out simultaneously step (b) [ComX modulation] and step (c) [contact with the exogenous DNA polynucleotide].
  • step (b) [ComX modulation]
  • step (c) [contact with the exogenous DNA polynucleotide].
  • the sequence of the exogenous DNA polynucleotide used in step c) share some similarities or identities with the genome of the Lactococcus strain to be transformed (of step a).
  • the exogenous DNA polynucleotide used in step c) is designed such that its 5′ part and its 3′ part are identical or highly similar to parts of the genome of the Lactococcus strain to be transformed (of step a), while its central part can be different from the genome of the Lactococcus strain to be transformed (of step a).
  • the high similarity of the arms with the regions surrounding the target sequence can be determined by the person skilled in the art using common general knowledge, in particular by reference to homologous recombination.
  • the exogenous DNA polynucleotide used in step c) is designed such that:
  • each of the 5′ part and 3′ part is long enough to ensure efficient recombination.
  • each of the 5′ part and 3′ part is from 0.5 to 5 kb in length.
  • the size of the arms can be determined by the person skilled in the art using common general knowledge, in particular by reference to homologous recombination.
  • the exogenous DNA polynucleotide used in step c) is (obtained) from a strain of the Lactococcus genus.
  • said exogenous DNA polynucleotide used in step (c) is (obtained) from a strain of the same species, in particular of the same subspecies, as the strain provided in step (a).
  • the exogenous DNA polynucleotide used in step c) is from a strain of the Lactococcus lactis species. In a particular embodiment, the exogenous DNA polynucleotide used in step c) is from a strain of the same Lactococcus lactis subspecies as the strain provided in step a). In a particular embodiment, the exogenous DNA polynucleotide used in step c) is from a strain of a Lactococcus lactis subspecies which is different from the strain provided in step a).
  • the exogenous DNA polynucleotide used in step c) is from a strain of the Lactococcus raffinolactis species
  • the exogenous DNA polynucleotide may encode part of a gene sequence, a gene sequence, or a plurality of gene sequences.
  • the gene sequence may be operably linked to transcription regulator(s).
  • the exogenous DNA polynucleotide is linear.
  • the exogenous DNA polynucleotide may be designed to facilitate its incorporation within the genome of the L. lactis strain by homologous recombination (e.g. the exogenous DNA polynucleotide may comprise one or more recombination arms).
  • the exogenous DNA polynucleotide may be a single stranded linear DNA.
  • the exogenous DNA polynucleotide when incorporated into the genome of said Lactococcus strain leads to genetic modification of the strain such as gene replacement (to add or to remove a mutation), gene addition (to add a new gene or to duplicate an existing gene), gene deletion (to remove part or the totality of a gene), modification of non-coding region (to modulate expression of a gene).
  • the exogenous DNA polynucleotide when incorporated into the genome of said Lactococcus strain confers an interesting or useful phenotype, e.g. modified kinetic of acidification, improved resistance to bacteriophage, modified capability to grow in milk, modified texturing properties, improved safety of the strain.
  • improved bacteriophage resistance could be achieved by incorporating genes coding for a restriction/modification system into the strain genome or by introducing a mutation or a deletion into the pip gene.
  • a L. lactis strain in milk could be improved by inserting into the chromosome the prtP and prtM genes that allow casein hydrolysis and better nitrogen nutrition; alternatively, these genes could be inactivated to reduced milk proteolysis in cheese.
  • hisDC and tyrDC are genes known to be responsible for biogenic amine production (histamine and tyramine, respectively) in a diversity of lactic acid bacteria; disruption or mutation of these genes could help to prevent safety issues related to cheese consumption.
  • the exogenous DNA polynucleotide has a minimal size selected from the group consisting of 100 bp, 200 bp, 500 bp, 1 kb, 2 kb and 5 kb, and a maximal size selected from the group consisting of 500 bp, 1 kb, 2 kb, 5 kb, 10 kb, 20 kb and 50 kb.
  • the size of the exogenous DNA polynucleotide may be between 100 bp and 50 kb, more preferably between 500 bp to 20 kb, even more preferably between 1 kb to 10 kb.
  • the concentration of exogenous DNA polynucleotide in the medium of step (c) may be between 0.5 mg/L and 1 g/L, preferably between 1 mg/L and 500 mg/L, more preferably between 5 mg/L and 100 mg/L, even more preferably between 10 mg/L and 50 mg/L of medium.
  • the method of the present invention comprises the step of selecting a strain which has integrated the exogenous DNA polynucleotide into its genome [step d)].
  • step c) when the exogenous DNA polynucleotide used in step c) provides a particular phenotype that the Lactococcus strain of step a) does not display (either a new phenotype or restoring a lost phenotype), it is possible to select strains which have integrated the exogenous DNA polynucleotide into their genome by selecting strains expressing the phenotype. This is the case for a strain having integrated in its genome an exogenous DNA polynucleotide mutated for the pip gene (that provides resistance to some bacteriophages).
  • step c) when the exogenous DNA polynucleotide used in step c) leads once integrated to a loss of a phenotype initially displayed by the Lactococcus strain of step a), it is possible to select strains which have integrated the exogenous DNA polynucleotide into their genome by selecting strains which do not display the phenotype any more. This is the case for an exogenous DNA polynucleotide bearing a mutated hisDC or tyrDC gene, which suppresses or decreases the production of histamine or tyramine, respectively.
  • the exogenous DNA polynucleotide may bear an antibiotic resistance gene.
  • a Lactococcus strain which has integrated the exogenous DNA polynucleotide into its genome may be selected by plating onto a medium comprising said antibiotic. Only strains that express the appropriate antibiotic resistance gene, as a result of a successful transformation with the exogenous DNA polynucleotide, will multiply.
  • Example 3 a positive effect on natural competence induction in L. lactis strains was observed when cells were pre-cultured in a complex medium before transferring the cells to a chemically defined medium ( FIG. 3 ).
  • the medium of step (c) is a chemically defined medium.
  • the term “chemically defined medium” (CDM) refers to a medium for which the exact chemical composition is known.
  • the CDM may have the composition of the CDM set out in Sissler et al. (1999, Proc Natl Acad Sci USA 96:8985-8990).
  • the chemically defined medium comprises 0.5 g/L NH 4 Cl, 9.0 g/L KH 2 PO 4 , 7.5 g/L K 2 HPO 4 , 0.2 g/L MgCl 2 , 5 mg/L FeCl 2 , 50 mg/L CaCl 2 ), 5 mg/L ZnSO 4 , 2.5 mg/L CoCl 2 , 0.05 g/L tyrosine, 0.1 g/L asparagine, 0.1 g/L cysteine, 0.1 g/L glutamine, 0.1 g/L isoleucine, 0.1 g/L leucine, 0.1 g/L methionine, 0.1 g/L tryptophan, 0.1 g/L valine, 0.1 g/L histidine, 0.2 g/L arginine, 0.2 g/L glycine, 0.2 g/L lysine, 0.2 g/L phenylalan
  • said strain prior to step (c) said strain is incubated in a pre-culture medium, preferably wherein the pre-culture medium is a complex medium, more preferably wherein the pre-culture medium is M17G (i.e., the M17 medium supplemented with glucose) or THBG (i.e., the THB medium supplemented with glucose).
  • M17G i.e., the M17 medium supplemented with glucose
  • THBG i.e., the THB medium supplemented with glucose
  • the complex medium may be Todd Hewitt broth (THB) (Todd and Hewitt, 1932; Updyke and Nickle, 1954) or M17 broth (Terzaghi and Sandine, 1975).
  • THB may comprise 500 g/L beef heart infusion, 20 g/L peptic digest of animal tissue, 2 g/L dextrose, 2 g/L sodium chloride, 0.4 g/L sodium phosphate, 2.5 g/L sodium carbonate.
  • M17 broth may comprise: 0.5 g/L ascorbic acid, 5 g/L lactose, 0.25 g/L magnesium sulfate, 5 g/L meat extract, 2.5 g/L meat peptone (peptic), 19 g/L sodium glycerophosphate, 5 g/L soya peptone (papainic), 2.5 g/L tryptone, 2.5 g/L yeast extract.
  • the present invention relates to a method for identifying a strain of the Lactococcus genus which is transformable through natural competence. Said method comprises the following steps:
  • a rate of at least 1 ⁇ 10 ⁇ 6 transformants per ⁇ g of DNA is indicative of a strain which is transformable through natural competence.
  • rate of recombination events may be used interchangeably with the term “transformation rate”.
  • the rate of recombination events is calculated by determining the ratio of the number of cells having integrated the exogenous marker DNA polynucleotide over the total number of viable cells. A rate of at least 10 ⁇ 6 was selected as a threshold, based on the observation that the level of spontaneous mutation in lactococci is less than 10 ⁇ 6 , typically around 10 ⁇ 7 mutants per ⁇ g of DNA [spontaneous means with no comX expression or overexpression].
  • At least 90% identity to the endogenous comX gene of said strain it is meant—as particular embodiments of the method—at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • said comX gene has 100% identity to the endogenous comX gene of said strain.
  • said method is implemented with a strain of the Lactococcus genus selected from the group consisting of Lactococcus lactis, Lactococcus raffinolactis, Lactococcus plantarum, Lactococcus piscium, Lactococcus garivieae, Lactococcus fujiensis and Lactococcus chungangensis.
  • a strain of the Lactococcus genus selected from the group consisting of Lactococcus lactis, Lactococcus raffinolactis, Lactococcus plantarum, Lactococcus piscium, Lactococcus garivieae, Lactococcus fujiensis and Lactococcus chungangensis.
  • said method is implemented with a strain of the Lactococcus lactis species. Said method comprises the following steps:
  • a rate of at least 1 ⁇ 10 ⁇ 6 transformants per ⁇ g of DNA is indicative of a strain of the Lactococcus lactis species which is transformable through natural competence.
  • said method is implemented with a strain of the Lactococcus raffinolactis species. Said method comprises the following steps:
  • the comX gene is from a strain of the same species, in particular of the same subspecies, as the strain provided in step a). In some embodiments, the comX gene is identical (100% identity) to the polynucleotide sequence of the endogenous comX gene of the strain of step a).
  • the strain of step a) is a Lactococcus lactis subsp. lactis strain
  • the comX gene has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:1.
  • the strain of step a) is a Lactococcus lactis subsp. cremoris strain
  • the comX gene has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:3 or SEQ ID NO:5.
  • the strain of step a) is a Lactococcus raffinolactis strain
  • the comX gene has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:7.
  • the comX gene has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:9.
  • the strain of step a) is a Lactococcus piscium strain
  • the comX gene has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:11.
  • the comX gene when the strain of step a) is a Lactococcus garvieae strain the comX gene has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17.
  • the strain of step a) is a Lactococcus fujiensis strain
  • the comX gene has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:19.
  • the comX gene when the strain of step a) is a Lactococcus chungangensis strain the comX gene has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:21.
  • the exogenous marker DNA polynucleotide bears a gene encoding a luciferase gene. Accordingly, a Lactococcus strain which has integrated the exogenous DNA polynucleotide into its genome may be selected for expression of the luciferase. Only strains that express the luciferase gene (i.e., integrated) will be detectable by bioluminescence.
  • the exogenous marker DNA polynucleotide bears an antibiotic resistance gene. Accordingly, a Lactococcus strain which has integrated the exogenous DNA polynucleotide into its genome may be selected by plating the cells onto a medium comprising said antibiotic.
  • Test A An example of a method for identifying a strain of the Lactococcus genus which is transformable through natural competence according to the present invention (Assay A) may be performed using the following steps:
  • the transformation rate may be calculated as the number of antibiotic-resistance CFU mL ⁇ 1 divided by the total number of viable CFU mL ⁇ 1 .
  • Example 1 Induction of the comGA Promoter by Constitutive comX Expression in Various Strains of the Lactococcus Species
  • a constitutive comX expression plasmid (pGhP32comX MG ) was created by cloning the comX gene from strain MG1363, under the control of the lactococcal P 32 promoter on the thermosensitive plasmid pG + host9. The latter was introduced in strain KW2 that contains a chromosomally-encoded P comGA[MG] -luxAB transcriptional fusion (BLD101).
  • the promoter of the late competence gene comGA (P comGA ) contains a putative ComX-binding motif and is used here as proxy for competence activation in the ComX strain.
  • a portable luminescent reporter system was also constructed.
  • This replicative plasmid carries the luminescent reporter P comGA[MG] -luc with the P 32 -comX MG cassette.
  • the pGhP32comX MG -P comGA[MG] -luc plasmid was transformed in strain MG1363.
  • Specific P comGA[MG] -luc/luxAB activities were monitored for the different strains constructed.
  • the luminescent assays were performed in rich (M17G) and/or CDM media comparing the luciferase activity between the overexpressing comX strain and its related negative control (no additional comX copy).
  • a constitutive comX expression plasmid (pGhP32comX IO ) was created by cloning the comX gene from strain IO-1, under the control of the lactococcal P 32 promoter on the thermosensitive plasmid pG + host9.
  • a portable luminescent reporter system was also constructed; this replicative plasmid carries the luminescent reporter P comGA[IO] -luc with the P 32 -comX IO cassette.
  • the promoter of the late competence gene comGA (P comGA ) contains a putative ComX-binding motif and is used here as proxy for competence activation in the ComX + strain.
  • This replicative plasmid pGhP32comX IO -P comGA[IO] -luc was transformed in strain IL1403 and specific P comGA[IO] -luc activities were monitored.
  • One of the IL1403 transformants produced specific P comGA[IO] -luc activities confirming that ComX induces the comG operon ( FIG. 2D ).
  • lactis IL1403 strain is concerned, its dprA gene contains nonsense mutations probably impairing its ability to transform DNA by competence.
  • strain KW2 of plant origin corn fermentation
  • strain KW2 of plant origin contains the whole set of known essential late genes required to fulfil natural DNA transformation, making it the best candidate to further study the functionality of competence in the cremoris subspecies.
  • the transforming PCR fragments used encompass the mutated rpsL allele of a spontaneous streptomycin-resistant (Str r ) clone of L. lactis subsp. cremoris MG1363 (strA1 allele, also called rpsL*).
  • This mutated allele bears an A ⁇ T substitution at position 167 [resulting in the altered ribosomal protein S12 with mutation K56I] as compared to the sequence of the wild-type, streptomycin-sensitive MG1363.
  • the two rpsL alleles differ by a silent nucleotide substitution at position 39 (T ⁇ G).
  • the sequence of the rpsL (wild-type) and rpsL* (conferring streptomycin resistance) alleles are disclosed respectively as SEQ ID NO:23 and NO:24 ( FIG. 4A ).
  • the rpsL alleles of KW2 and MG1363 differ by a nucleotide substitution at position 156 (A in MG1363, T in KW2).
  • the rpsL allele of KW2 is disclosed as SEQ ID NO:25 ( FIG. 4A ).
  • the transforming PCR product also contains upstream and downstream recombination arms of ⁇ 1.85 kb surrounding the strA1 mutation. Transformation assays were performed with the eight previously selected clones of the ComX + reporter strain (BLD101 [pGhP32comX MG ]) and the control strain (BLD101 [pG + host9], empty vector) using the standard protocol reported in Material and Methods. Validation of natural transformation is made by sequencing the rpsL region covering the point mutations from the donor DNA conferring streptomycin resistance using primers RpsL Univ UP and RpsL Univ DN.
  • the ComX + clones 02 and 04 that displayed the highest P comGA activation yielded mutation frequencies ⁇ 15-fold higher than the background level of spontaneous mutation that was calculated in the absence of DNA ( FIG. 4B ).
  • a transformation rate of up to 4 ⁇ 10 ⁇ 5 transformants per ⁇ g of DNA was obtained for clone 02 which displays the highest P comGA activation.
  • the negative control strain had a spontaneous mutation rate of ⁇ 1 ⁇ 10 ⁇ 7 transformants per ⁇ g of DNA.
  • the rpsL ORF of 10 Str r -derivatives of cl02 was amplified by PCR and sequenced. In all cases, we observed the co-transfer of strA1 (mutation A ⁇ T at position 167 of the rpsL gene) and the closely-located T ⁇ A mutation at position 156. In some cases, the T ⁇ G mutation at position 39 was also co-transferred with strA1.
  • the chimeric nature of rpsL in some Str r ComX + derivatives of KW2 i.e. presence of both mutations at positions 156 and 167 without the mutation at position 39 ultimately demonstrates that a recombination process occurred between the exogenous and chromosomal DNA ( FIG. 4A ).
  • exogenous DNA polynucleotides containing P 32 -cat surrounded by KW2-specific recombination arms were assembled in vitro by overlapping PCR to target the comEC, mecA, ciaRH, covRS or clpC gene (see Materials and Methods for details) and transferred by natural transformation in the ComX + strain (cl02).
  • Validation of natural transformation is made by sequencing the targeted region (comEC, mecA, ciaRH, covRS or clpC, which should contain the chloramphenicol resistance cassette P 32 -cat) using primers listed in Table 3.
  • the transformation rate observed for overlap PCR products was ⁇ 1.2 ⁇ 10 ⁇ 6 to 1.1 ⁇ 10 ⁇ 4 transformants per ⁇ g of DNA for the different overlap DNA fragments that were tested (see FIG. 5 ).
  • these rates are relatively high for DNA double recombination deletion/replacement.
  • clone 02 of the ComX + reporter strain was grown in CDM conditions in presence of PCR products encompassing the comEC gene disrupted by the insertion of the chloramphenicol resistance cassette P 32 -cat (see Materials and Methods).
  • Four mutants with disrupted comEC (BLD102 [pGhP32comX MG ] cl01 to cl04) were validated by PCR for P 32 -cat insertion in comEC.
  • Transformation assays with the mutated rpsL allele showed that the frequencies of appearance for Str r clones in all tested ⁇ comEC derivatives were similar to the background level of spontaneous rpsL mutation frequencies ( ⁇ 10 ⁇ 7 ) ( FIG. 6 ). Although heterogeneity in P comGA activation was observed between clones as previously reported for the WT ComX + reporter strain, half of the ⁇ comEC derivative clones (i.e. cl01 and cl03) displayed maximum specific Lux activity similar to the transformable WT strains (>1.0 ⁇ 10 6 RLU OD 600 ⁇ 1 ) ( FIG. 4B ). This shows that the transformation defect in these ⁇ comEC clones does not result from a too low production of ComX.
  • Lactococcus lactis subsp. lactis strains SL12651 and SL12653, carrying all the essential late corn genes ( FIG. 1 ) were tested.
  • donor DNA PCR fragments which encompass the mutated rpsL allele (rpsL*) of a spontaneous streptomycin-resistant (Str r ) clone of L. lactis subsp. lactis IL1403 was used.
  • Cells were pre-cultured overnight in a complex medium supplemented with glucose (e.g. M17G) at 30° C.
  • the negative control in absence of donor DNA had a spontaneous mutation rate of 6 ⁇ 10 ⁇ 9 ( FIG. 7A ; ⁇ DNA).
  • the transformants were validated by sequencing the rpsL region covering the point mutation from the donor DNA conferring streptomycin resistance.
  • the SL12653 strain was assayed in the same conditions with variable quantity of donor DNA (0.5, 2.5, 5 and 25 ⁇ g mL ⁇ 1 ). It has been shown that the transformation rate obtained is directly correlated to the initial quantity of donor DNA, yielding up to a transformation rate of 5 ⁇ 10 ⁇ 6 ( FIG. 7B ).
  • the comX gene of SL12653 was knocked-out (as described in example 5 above).
  • Three mutants of SL12653 with disrupted comX gene were designed by inserting PCR products encompassing the comX gene disrupted by the insertion of the chloramphenicol resistance cassette P 32 -cat and validated by PCR for P 32 -cat insertion. Transformation assays with rpsL* as donor DNA in all ⁇ comX clones (ComX ⁇ ) showed that the frequencies of appearance of Str r clones were similar to the background level of spontaneous mutation frequencies ( FIG. 7C ). These results confirm that in SL12653, the transformation is dependent on the expression of the endogenous comX gene.
  • an inducible comX expression plasmid [pGhPxylTcomX IO ] was constructed by cloning the comX gene from strain L. lactis subsp. lactis IO-1 under the control of the P xyIT promoter from strain IO-1 on the thermosensitive plasmid pG + host9.
  • This plasmid is a variant of pGhPxylTcomX MG (pGIFPT001) described in David et al., 2017. The transformation procedure described in David et al (2017) was followed.
  • SL12653 [pGhPxylTcomX IO ] yielded a transformation rate at least 20-fold higher than in absence of xylose, confirming that the overexpression of comX in SL12653 increased its transformability by natural competence.
  • the bacterial strains and plasmids used in this application are listed in Table 2.
  • Plasmid 11 260-263 IO-1 Wild-type isolate from water in the Ishizaki A, Osajima K, Nakamura K, Katsunori drain pit of a kitchen sink K, Hara T, and Ezaki T. 1990. J. Gen. Appl.
  • Escherichia coli was grown with shaking at 37° C. in Lysogeny-Broth (LB) broth. Plasmids derived from pMG36e and pG + host9 were constructed in E. coli strains TG1 and EC1000, respectively. L. lactis and L. raffinolactis were cultivated in M17 (Becton, Dickinson, and Company), Todd Hewitt broth (THB) (Becton, Dickinson, and Company) or CDM at 30° C. without agitation. M17 and THB were supplemented with 0.5% (w/v) of glucose (M17G and THBG, respectively). Solid agar plates were prepared by adding 2% (w/v) agar to the medium.
  • erythromycin When required, 5 ⁇ g ml ⁇ 1 of erythromycin, 1 mg ml ⁇ 1 of streptomycin, and/or 10 ⁇ g ml ⁇ 1 of chloramphenicol were added to the medium for L. lactis and L. raffinolactis ; and 250 ⁇ g ml ⁇ 1 of erythromycin, 250 ⁇ g ml ⁇ 1 of ampicillin, 10 ⁇ g ml ⁇ 1 of chloramphenicol for E. coli.
  • luciferase activity is expressed in relative light units (RLU) and the specific luciferase activity in RLU OD 600 ⁇ 1 .
  • the comX gene from the laboratory strain MG1363 was initially chosen. ComX proteins of this subspecies are highly conserved with at least 98% of identity.
  • the comX gene was amplified by PCR using primers BID_ComXSDLLCup/BID_ComXSDLLCdown and inserted into plasmid pMG36eT under the control of the constitutive P 32 promoter by SacI/PstI cloning, yielding plasmid pMGP32comX MG .
  • the P 32 -comX MG fusion from pMGP32comX MG was amplified by PCR with primers BID_pMGP32UpMfeI/BID_pMGTerDown, digested by MfeI/KpnI, and cloned in the EcoRI/KpnI-digested thermosensitive pG + host9 vector.
  • the resulting plasmid was named pGhP32comX MG .
  • the comX gene from the IO-1 strain was chosen.
  • the comX gene was amplified by PCR using primers BID_ComXSDLLLup/BID_ComXSDLLLdown and inserted into plasmid pMG36eT under the control of the constitutive P 32 promoter by SacI/PstI cloning, yielding plasmid pMGP32comX IO .
  • the P 32 -comX IO fusion from pMGP32comX IO was amplified by PCR with primers BID_pMGP32UpMfeI/BID_pMGTerDown, digested by MfeI/KpnI, and cloned in the EcoRI/KpnI-digested thermosensitive pG + host9 vector.
  • the resulting plasmid was named pGhP32comX IO .
  • the Core part of the resolution site (IRS) recognized by the TnpI recombinase from Tn4430 was assembled by using the complementary primers DD-pGhost-CoreUp/DD-pGhost-CoreDW.
  • the resulting DNA fragment was cloned between HindIII and EcoRI sites in plasmid pG + host9.
  • the resulting plasmid, named pGhost-Core was transformed in E. coli harbouring plasmid pGIV004 (TnpI + ) for obtaining multimeric forms (Vanhooff V, Galloy C, Agaisse H, Lereclus D, Revet B, Hallet B. Mol Microbiol. 2006 May; 60(3):617-29).
  • the P comGA[MG] promoter was amplified by PCR from chromosomal DNA of L. lactis MG1363 (identical nucleotide sequence between MG1363 and KW2) with primers BID_LuxLLCf1/BID_LuxLLCr1 (PCR1 product).
  • the luxAB genes were amplified by PCR from plasmid pJIM4900 with primers BID_LuxLLCf2/BID_LuxLLCr2 (PCR2 product).
  • the P comGA[MG] -luxAB fusion was created by overlapping PCR using PCR1 and PCR2 products and primers BID_LuxLLCf1/BID_LuxLLCr2.
  • plasmid pSEUDOPusp45GFP using restriction enzymes XhoI and BamHI, yielding plasmid pSEUDOPusp45PcomGAluxAB.
  • the entire vector except the P usp45 promoter was amplified by inverse PCR with primers BID_P3pseudoLLC/BID_LuxLLCf1 and self-ligated after XhoI digestion, leading to plasmid pSEUDOPcomGAluxAB.
  • the insertion cassette llmg_pseudo_10::P comGA[MG] -luxAB was excised from plasmid pSEUDOPcomGAluxAB and cloned into the pG + host9 thermosensitive vector using restriction enzymes KpnI/EagI.
  • the resulting plasmid pGhPcomGAluxAB was then electro-transformed in strain KW2 and used to integrate the P comGA[MG] -luxAB cassette at locus kw2_0563 (llmg_pseudo_10 in MG1363) by double homologous recombination, resulting in the reporter strain KW2 kw2_0563::P comGA[MG] -luxAB (strain BLD101).
  • the P comGA[MG] promoter was amplified by PCR from chromosomal DNA of L. lactis MG1363 with primers BID_LuxLLCf1/BID_LucLLCr1 (PCR1 product).
  • the luc gene was amplified by PCR from plasmid pXL with primers BID_LucLLCf2/BID_LucLLCr2 (PCR2 product).
  • the P comGA[MG] -luc fusion was created by overlapping PCR using PCR1 and PCR2 products and primers BID_LuxLLCf1/BID_LucLLCr2.
  • plasmid pSEUDOPusp45GFP using restriction enzymes XhoI and BamHI, yielding plasmid pSEUDOPusp45PcomGAluc.
  • the entire vector except the P usp45 promoter was amplified by inverse PCR with primers BID_P3pseudoLLC/BID_LuxLLCf1 and self-ligated after XhoI digestion, leading to plasmid pSEUDOPcomGAluc.
  • the reporter cassette P comGA[MG] -luc was amplified by PCR from pSEUDOPcomGAluc (primers BID_PcomGALLCF1*/BID_IucR1*) and cloned between XmaI and EagI into the pGhP32comX MG plasmid.
  • the resulting reporter plasmid was named pGhP32comX MG -P comGA[MG] -luc.
  • the P comGA[IO] promoter was amplified from the IO-1 chromosome (primers BID_IuxLLLf1/BID_IucLLLr1) and the luciferase gene (luc) was amplified from plasmid pXL (primers BID_IucLLLf2/BID_IucLLLr2).
  • the cassette P comGA[IO] -luc was created by overlapping PCR with primers BID_IuxLLLf1/BID_IucLLLr2.
  • the cassette P comGA[IO] -luc was then amplified from the overlapping PCR product with primers BID_PcomGALLLF1*/BID_IucR1* for XmaI/EagI cloning into pGhP32comX IO .
  • the resulting reporter plasmid was named pGhP32comX IO -P comGA[IO] -luc.
  • a 3.7-kb fragment containing the rpsL mutated gene (strA1 allele) was amplified by PCR with primers BID_LLcdacARpsL/BID_LLIcfusARpsL and cloned into the pGEM®-T easy vector (Promega), yielding plasmid pGEMrpsL*.
  • This plasmid was used as template to generate the 3.7-kb PCR product with primers BID_LLcdacARpsL/BID_LLIcfusARpsL that was used as donor DNA in natural transformation assays of strain KW2.
  • the BLD101 reporter strain carrying the pGhP32comX MG plasmid (BLD101 [pGhP32comX MG ]) was grown overnight in M17G at 30° C. Then, 1.5 ml of the pre-culture was diluted in 8.5 ml of fresh M17G medium to restart the culture. After 2 hours of growth, cells were washed twice in distilled water and OD 600 was adjusted to 0.05 in CDM containing erythromycin (5 ⁇ g ml ⁇ 1 ) and supplemented with either 5% (v/v) glycerol or 5% (w/v) mannitol used as potential osmo-stabilizers.
  • a comEC-containing DNA fragment of ⁇ 3.2 kb was amplified by PCR with primers BID_ComECLLCUp/BID_ComECLLCDown. Then, the PCR product was digested by SacI/NheI and cloned into the SacI/XbaI-digested suicide plasmid pUC18Ery (van Kranenburg et al., 1997), yielding plasmid pUCcomEC. To generate a comEC disruption cassette that allows the selection of double crossing-over recombinants, the P 32 -cat fusion conferring resistance to chloramphenicol was cloned in the middle of the comEC gene.
  • the P 32 -cat cassette was amplified by PCR from plasmid pNZ5319 (Lambert et al., 2007, Appl. Environ. Microbiol. 73:1126-1135) with primers BID_CatUpSpeI/BID_CatDownSpeI.
  • the amplification product was digested by SpeI and cloned into the XbaI-digested pUCcomEC, yielding plasmid pUCcomECcat.
  • This suicide plasmid was used to generate high quantity of donor DNA by PCR amplification for comEC disruption by natural transformation.
  • the insertion of the P 32 -cat cassette in the comEC gene of KW2 transformants was validated by PCR (primers in Table 3).
  • the mecA, ciaRH, covRS, and clpC genes were similarly inactivated by the exchange of their ORFs by the P 32 -cat cassette using double crossing-over events.
  • overlapping PCR products containing the P 32 -cat cassette flanked by two recombination arms of ⁇ 1.5 kb (upstream and downstream homologous regions) were generated as previously reported.
  • upstream, downstream, and P 32 -cat fragments were separately amplified by PCR, purified, mixed in equimolar concentration, and assembled by overlapping PCR by using the most external primers (see list of primers in Table 3).
  • thermosensitive vector pGhP32comX MG was cured by growing the strains overnight at 37° C. without erythromycin. The cultures were subsequently diluted and plated on M17G agar without erythromycin at 30° C. The resulting colonies were streaked in parallel on M17G plates with and without erythromycin. Absence of plasmid pGhP32comX MG in Ery S clones was validated by PCR.
  • Wild-type Lactococcus raffinolactis i.e., L. raffinolactis strains which have not been previously engineered for the overproduction of the comX gene
  • M17G i.e., L. raffinolactis strains which have not been previously engineered for the overproduction of the comX gene
  • 1.5 ml of the pre-culture was diluted in 8.5 ml of fresh M17G medium to restart the culture. After 2 hours of growth, cells were washed twice in distilled water and OD 600 was adjusted to 0.05 in CDM supplemented with either 5% (v/v) glycerol or 5% (w/v) mannitol used as potential osmo-stabilizers.
  • the L. lactis subsp. lactis SL12653 and 12651 strains were grown overnight at 30° C. Cells were washed twice in distilled water and OD 600 was adjusted to 0.05 in M17G. Typically, 5 ⁇ g of donor DNA was added in 200 ⁇ l of inoculated medium (25 ⁇ g/ml) and the culture was further incubated during 24 hours at 30° C. Cells were then spread on M17G agar plates supplemented with appropriate antibiotics and CFUs were counted after 48 hours of incubation at 30° C. The transformation frequency calculated exactly as described above (see Standard natural transformation assay).
  • the comX gene and the promoter of the xylT gene from the IO-1 strain were chosen.
  • the comX gene was amplified by PCR using primers FT_comXIOrecfw and FT_comXIOrecry (PCR1), both containing overlapping sequences.
  • the xylT promoter region was amplified by PCR using primers FT_PxylTIOsacllfw and FT_PxylTIOrv (PCR2).
  • the carrying vector was amplified from plasmid pGhP32comX MG and amplified by PCR using primers FT_pGhPxylcomXIOsacllrv and FT_pGhPxylcomX (PCR3).
  • the three PCR products were purified, mixed in an equimolar concentration and assembled by overlapping PCR using the most external primers, containing a SacII restriction site.
  • the amplification product was digested by SacI I and self-ligated.
  • the resulting plasmid was named pGhPxylTcomX IO .
  • the comX gene of SL12653 was inactivated by exchange of their ORF by the P 32 -cat cassette using double crossing-over events.
  • overlapping PCR products containing the P 32 -cat cassette flanked by two recombination arms of ⁇ 1.5 kb (upstream and downstream homologous regions) were generated as previously reported.
  • upstream, downstream, and the P 32 -cat fragments were separately amplified by PCR, purified and mixed in equimolar concentration, and assembled by overlapping PCR by using the most external primers (see primers in Table 3). 5 ⁇ g of the obtained PCR product was used as donor DNA for natural transformation of strain SL12653 [pGhPxylTcomX IO ] (ComX + ).
  • thermosensitive vector pGhPxylTcomX IO was cured by growing the strains overnight at 37° C. without erythromycin.
  • the L. lactis subsp. lactis SL12653 [pGhPxylTcomX IO ] was grown overnight at 30° C. Cells were washed twice in distilled water and OD600 was adjusted to 0.05 in M17 supplemented with 1% (w/v) xylose. Typically, 5 ⁇ g of DNA was added in 200 ⁇ l of inoculated medium and the culture was further incubated during 24 hours at 30° C. Cells were then spread on M17G agar plates supplemented with appropriate antibiotics and CFUs were counted after 48 hours of incubation at 30° C. The transformation frequency was calculated exactly as described above (see Standard natural transformation assay).

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