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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/90—Isomerases (5.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • 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 invention relates to the regulation of the production of exocellular heteropolysaccharides by lactic acid bacteria.
• polysaccharides are widely used as additives in food, but also in cosmetics and pharmaceuticals, for example as thickening agents and / or gelling agents, texture stabilizers, fat substitutes, etc.
• cosmetics and pharmaceuticals for example as thickening agents and / or gelling agents, texture stabilizers, fat substitutes, etc.
• polysaccharides used in this way particular mention will be made of those produced by microorganisms in particular bacteria, such as dextrans, xanthans, gellans, pullulans, etc.
• lactococci such as Lactococcus lactis, Leuconostoc such as Leuconostoc mesen teroide, streptococci such as Streptococcus thermophilus, and lactobacilli such as Lactobacillus casei, Lactobacillus sake, Lactobacillus rhamnosus achamusus Lhamobusus achamusus Lhamobusus achamusus subsp. bulgaricus, Lactobacillus helveticus, etc. produce polysaccharides.
• homopolysaccharides such as dextrans, which result from the polymerization of a single sugar
• heteropolysaccharides of complex structure, associating basic units made up of 2 or more different sugars ( frequently D-galactose, D-glucose and L-rhamnose).
• the heteropolysaccharides of lactic acid bacteria are usually designated by the general term EPS (for exopolysaccharides), which will also be used below. They play a major role in the development of the texture, the perception in the mouth and the rheology of fermented dairy products. In addition, it was observed that some of them possessed biological activities by which they could exert various effects health benefits, [for review, cf. DE VUYST, and DEGEEST, FEMS Microbiology Reviews, 23, 153-177, (1999)].
• heteropolysaccharides produced by lactic acid bacteria is generally low (of the order of 10 to 200 mg per liter of fermented product).
• manufacturers of fermented products add other texturing agents such as stabilizers (modified starches, carragheen, guar, pectin, gelatin ).
• stabilizers modified starches, carragheen, guar, pectin, gelatin .
• these additions are not always allowed (for example in plain yogurt), and generally affect the taste and aroma of the product.
• Optimized production of exocellular heteropolysaccharides (EPS) in the product is therefore preferable.
• the inventors have set themselves the goal of obtaining mutants of lactic acid bacteria which can be controlled and in particular increase the capacity to use sugars available in the medium, and in particular galactose, to produce EPS.
• the inventors have now managed to clone and characterize a pgm gene, coding for an ⁇ -PGM of Streptococcus thermophilus.
• the subject of the present invention is a nucleic acid sequence coding for an ⁇ -PGM whose amino acid sequence has at least 70% identity or at least 85% similarity, preferably 80% identity or at least 90% similarity, advantageously at least 90% identity or at least 95% similarity, and most preferably at least 95% identity or at least 99% similarity, with the ⁇ -PGM represented by the sequence SEQ ID NO: 2, as well as any fragment of more than 20 bp of said sequence.
• the percentage identity of a sequence with a reference sequence is defined here as the percentage of residues of this sequence which are identical with those of the reference sequence when the 2 sequences are aligned for a maximum correspondence between the positions of the residues .
• a polypeptide whose amino acid sequence has at least X% identity with a reference sequence can thus comprise up to 100-X modifications per 100 amino acids of the reference sequence. These modifications include the deletion, substitution, or insertion of amino acid residues, consecutive or not.
• the percentage similarity of a sequence with a reference sequence is defined here as the percentage of residues of this sequence which are identical to those of the reference sequence or which differ from it only by one conservative substitution, when the 2 sequences are aligned for a maximum correspondence between the positions of the residues.
• a polypeptide whose amino acid sequence has at least X% similarity to a reference sequence can thus include up to 100-X non-conservative modifications per 100 amino acids of the reference sequence. These modifications include deletion, non-conservative substitution, or insertion of amino acid residues, consecutive or not.
• the polypeptides thus exhibiting the highest percentages of identity or similarity with the sequence SEQ ID NO: 2, identified by search on the "GENBANK nr" database using the BLASTp software [ALTSCHUL et al. , Nucleic Acids Res. , 25, 3389-3402, (1997)], with the default settings, are as follows:
• Strepto / nyces coelicolor 42% identity, and 55% similarity
• the inventors have carried out site-directed mutagenesis of the pgm gene of S. thermophil us, and found that, from surprisingly, the total or partial inactivation of this gene led to an increase in the production of EPS.
• the present invention also relates to a mutant of an EPS-producing lactic acid bacteria in which the pgm gene for alpha-phosphoglucomutase is totally or partially inactivated.
• Said lactic acid bacteria will preferably be a mesophilic or thermophilic bacterium, chosen from streptococci and lactobacilli.
• it may be La ctococcus la cti s, Streptococcus thermophil us, Leuconos toc mesen teroide, La ctoba cill us casei, La ctoba ci l l us delbrueckii, La ctoba ci ll us sake, etc.
• Inactivation of the pgm gene can be obtained by carrying out one or more mutations at the level of the sequence coding for ⁇ -PGM, and / or at the level of sequences controlling its expression.
• an exogenous sequence for example a transposon.
• one or more modification, by insertion, deletion, or substitution, of one or more nucleotides, consecutive or not, is carried out within a sequence identical to that of the region of the gene which one wishes to mutate. .
• the mutated sequence is inserted into a vector allowing integration by recombination (simple crossing-over) between the bacterial DNA fragment cloned into the vector and the homologous region of the bacterial genome. Excision of vector sequences can be performed following a second recombination event (double crossing-over), which results in the substitution of the wild chromosomal form by the modified form.
• Vectors allowing the integration of an exogenous sequence into the chromosome of a lactic acid bacteria are known in themselves, and available for most species of lactic acid bacteria; they may, for example, be non-replicative vectors, unstable or conditional replicative vectors, vectors carrying insertion sequences, etc. It is also possible, in certain cases, to carry out the direct transformation of the bacteria with the DNA carrying the mutated sequence which it is desired to insert.
• Mutants of the pgm gene in accordance with the invention can also be obtained by random mutagenesis (for example by chemical mutagenesis or by radiation); they may also be natural mutants selected from cultures of lactic acid bacteria by screening on the basis of their phenotypic properties.
• mutants in which the ⁇ -PGM is partially or completely inactive have normal growth on lactose, and very slow growth on glucose or galactose alone. Such mutants can therefore, whether they are natural mutants, or whether they result from mutagenesis, be selected directly on the basis of this property.
• EPS production it is also possible, if desired, to increase or decrease EPS production at will as a function, for example, of culture conditions, by placing the pgm gene under transcriptional control of an inducible promoter.
• the inventors have also cloned and characterized the gal U gene which codes for glucose-1-phosphate uridyl transferase from S. thermophilus. This gene is represented in the sequence list in the appendices under the number SEQ ID NO: 3.
• the GalU protein encoded by this gene is represented under the number SEQ ID NO: 4.
• the present invention also relates to a nucleic acid sequence coding for a glucose-1-phosphate uridyl transferase whose amino acid sequence has at least 90% identity or at least 95% similarity, and advantageously at least 95% identity or at least 99% similarity with glucose-1-phosphate uridyl transferase represented by the sequence SEQ ID NO: 4.
• the polypeptides thus having the highest percentages of identity or similarity with the sequence SEQ ID NO: 4, identified by research on the "GENBANK nr" database using the BLASTp software [ALTSCHUL et al. , Nucleic Acids Res., 25, 3389-3402, (1997)] are as follows: Streptococcus mutans glucose-1-phosphate uridyltransferase: 87% identity, and 93% similarity;
• Streptococcus pyogenes 84% identity, and 90% similarity
• Cap3C protein of Streptococcus pneumoniae 76% identity, and 88% similarity
• UDP-glucose pyrophosphorylase from Bacillus subtil is: 55% identity, and 74% similarity.
• the overexpression of the gal U gene makes it possible to increase the synthesis of EPS, by increasing the quantity of their precursors.
• the present invention therefore also relates to a mutant of an EPS-producing lactic acid bacteria in which the gal U gene is overexpressed.
• Such a mutant can in particular be obtained by introducing one or more copies of this gene into a lactic acid bacterium, and / or by replacing its promoter with a strong promoter.
• said mutant is obtained from a lactic acid bacterium also having a partially or completely inactive ⁇ -PGM.
• Mutant strains in accordance with the invention can in particular be obtained from S. thermophil us, and in particular from strains of S. thermophilus selected for their growth capacity on galactose.
• another limiting factor for the synthesis of EPS may come from the fact that a large number of strains do not use galactose efficiently, either for their growth or for the synthesis of EPS.
• gal + strains of S. thermophil us selected for their growth capacity on galactose could synthesize a larger amount of EPS. From these strains capable of using galactose for their growth, it is possible to carry out a second selection of the strains producing the colonies of larger appearance, which translates a more important synthesis of EPS.
• Strains of lactic acid bacteria according to the invention can advantageously be used for the manufacture of fermented products, in particular food products, the EPS content of which it is desired to control, and in particular to increase, as well as for the production of EPS.
• the growths are carried out as follows: overnight precultures in 10 ml of BELLIKER medium
• Figure 1 represents the growth on different sugars, of the industrial strain JIM7459 (1A) and of a gal + mutant selected from this strain (1B).
• FIG. 2 represents the growth on different sugars, of a gal + (2A) mutant selected as described in A above, from a culture of a strain industrial (JIM7446, DANONE collection), and a gal ++ mutant (2B) selected from a culture of this gal + mutant, based on the size of the colonies.
• the bacterial pellet of the gal + mutants is more filamentous than that of the wild-type strains, which may reflect a difference in the production of EPS between the mutants obtained and their strain. parental.
• the production of EPS of these strains was determined from culture in 80 ml of chemically defined medium [SISSLER et al. , Proc. Natl. Acad. Sci. USA, 96, no. 16, 8985-8990, (1999)].
• the cultures are centrifuged at 16,000 g for 10 min at 4 ° C.
• the cell pellet is eliminated, and the supernatant is precipitated (2 volumes of 100% ethanol for one volume of supernatant) for 24 hours at 4 ° C.
• centrifugation is carried out at 16,000 g for 15 minutes at 4 ° C.
• the supernatant is removed and the residue is resuspended in 60 ml of water.
• Dialysis is performed against water for 4 days by changing the water 3 to 4 times a day.
• the EPS are determined by the phenol / sulfuric acid method [DUBOIS et al. , Analytical Chemistry, 28, 350-356, (1956)].
• EXAMPLE 2 CLONING OF THE PGM GENE OF STREPTOCOCCUS THERMOPHILUS AND CONSTRUCTION OF MUTANTS IN WHICH THIS GENE IS INACTIVE.
• the pgm gene of Streptococcus thermophilus can be obtained by reverse PCR [OCHMAN et al. , Biotechnology
• thermophil us is digested with restriction enzymes (BamHI, EcoRI, HindI I I, Ncol, Psi, Xhl) then the cleavage products are circularized then amplified by PCR using the primers complementary to the opposite strand OST15, OST16,
• the bands obtained are extracted from the gel and sequenced.
• the sequence of the cloned fragment, comprising the pgm gene and its flanking regions is represented in the sequence list in the appendix under the number SEQ ID NO: 1.
• the size of the open reading frame for the pgm gene from Streptococcus thermophil us is 1350 bp.
• thermally sensitive replication plasmids containing fragments internal to the pgm gene were constructed from the thermally sensitive replication vector pG + host [BISWAS et al. , J. Bacteriol., 175, 11, 3628-3635, (1993); PCT application WO / 181164].
• the 650 bp fragment carried by pST28 is central to the gene, it is therefore expected that its insertion will produce a total inactivation of the gene.
• the 835 bp fragment carried by pST29 contains the 5 ′ part of the gene with the translation start codon, but in the absence of its ribosome binding site. It is expected that the insertion of pST29 into the chromosome causes a very significant decrease in the translation of pgm by allowing only a very weak basic expression.
• the plasmids pG + host, pST28 and pST29 were introduced by transformation into the strains of S. thermophil us JIM7446 and JIM7459 at 30 ° C, then integrated by simple crossing-over as shown schematically in Figure 3.
• Streptococcus thermophil us strains containing the plasmid pG + host or its derivatives are grown overnight at 30 ° C in the presence of erythromycin, then diluted 50 times in the same medium. They are then transferred to 42 ° C for 6 hours. The samples are then diluted and spread on the one hand at 42 ° C on M17 dishes containing erythromycin to detect integration events, and on the other hand at 30 ° C on M17 dishes without antibiotics to detect the total number of viable cells. The integration frequency per cell is calculated by reporting these counts.
• the integration frequencies of these plasmids are respectively 3.5 x 10 "3 , 3 x 10 " 3 and 10 "2 for pG + host, PST28 and pST29. Clones from each of these integrations were isolated to give the strains STJ3, STJ1 and STJ2 respectively. STJ1 (pST28), STJ2 (pST29) and STJ3 strains
• the STJ1 and STJ2 clones therefore do not grow on glucose or galactose alone, but normally on lactose or on a glucose and galactose mixture. This shows that the metabolism of glucose and galactose has indeed been decoupled in this strain and that the gene whose activity has been affected is indeed pgm.
• the EPS assay is carried out as described in Example 1 above.
• EPS of the strain affected in the expression of the pgm gene is 20 mg / 1. This production, compared to the 8 mg / 1 obtained for the parental strain 7446
• Gal ++ therefore corresponds to an increase of two and a half times.
• EXAMPLE 3 PRODUCTION OF EPS BY MUTANTS OF THE pgm GENE DERIVATIVES OF INDUSTRIAL STRAINS
• the plasmid pST29 described in Example 2 above was used to transform the industrial strains (DANONE collection) Ext 1.1 (JIM7455) and Ext 1.10. (JIM7464). These strains produce EPS of different composition (1- glu: 2-gal: 1-galNac for Extl.l and 3-gal: l-rha for Extl.10), and which are also different from that produced by the strain JIM7446 (3-glu: 4-gal).
• the cells are spread at 30 ° C for 12 hours and then the dishes are set at 42 ° C in order to force the integration of the plasmid pST29 into the pgm gene by homologous recombination between the pgm sequences present on the plasmid and those present on the chromosome.
• Colonies of mutated bacteria capable of growing at 42 ° C in the presence of erythromycin were obtained. These bacteria are also capable of growing on MCD (chemically defined medium), in the presence of glucose and galactose, but not in the presence of glucose alone or galactose alone.
• the strains transformed or not were cultured in 10 ml of MCD lactose 2%, casitone 1.5%, urea 10 M, in the presence of erythromycin (5 ⁇ g / ml) for the strains transformed by the plasmid pST29.
• the EPS produced by the different strains were assayed after 67 h 30 min of culture at
• strains whose pgm gene is inactivated in accordance with the invention produce, at equal biomass, 2 to 2.5 times more EPS than the original strains.
• a vector called pSTJ ⁇ containing a pgm gene inactivated by internal mutation, was constructed by reverse PCR amplification using the plasmid pST29 as template, and the following primers, derived from the sequence of the pgm gene by point mutations, inducing in particular the creation of 'an Nsil site:
• the PCR product is cleaved by Nsil and religated on itself to give the vector pSTJ ⁇ .
• This vector is introduced by transformation into strain JIM7446 of S. thermophilus.
• the colonies are spread at 30 ° C for 12 H, then grown at 42 ° C for 24 H, in order to force the integration of the vector into the chromosome by a first crossing-over by recombination between the pgm sequences present on the plasmid and those present on the chromosome.
• the colonies thus obtained, resistant to erythromycin and capable of growing at 42 "C, are then grown at 30 ° C in order to promote the excision of the sequences of the plasmid pG + host by a second crossing-over by recombination between the pgm sequences present on either side of the pG + host sequences pG + host is then eliminated from the cell by a growth step at 42 ° C. in the absence of selection by erythromycin.
• the strain thus obtained does not contain more sequences of the pG + host vector, and in particular, it does not contain an antibiotic resistance gene.
• the mutated strain has the phenotype characteristic of the pgm mutants, described in Examples 2 and 3 above. It is able to grow on MCD in the presence of glucose and galactose but not in the presence of glucose alone or galactose alone.
• the increase in EPS production by this strain compared to the parental strain is comparable to that observed in the case of strains transformed with pST29, described in Examples 2 and 3.
• the entire gal U gene can be obtained by reverse PCR [OCHMAN et al. , (1990), publication cited above] from the chromosomal DNA of S. thermophil us, using primers complementary to the opposite strand (OST13,
• the bands obtained are extracted from the gel and sequenced.
• sequence of the cloned fragment, comprising the gal U gene is represented in the sequence list in the appendix under the number SEQ ID NO: 3.
• the size of the gal U gene is 914 bp.
• the gal U gene (with its terminator and its ribosome binding site) was amplified with the oligonucleotides OST44 and OST45 (Table VI) and cloned downstream of the promoter p45 [SIBAKOV et al. , Appl. About. Microbiol.,
• the plasmid pGKV259 [VAN DER VOSSEN et al. , Appl. About. Microbiol., 53, 2452-2457, (1987)] expressing the gal U gene under the control of the p45 promoter is called pSTJ4 ( Figure 4).
• This plasmid is introduced by transformation into the strain S. thermophil us JIM7446, JIM7459 or in the strain containing a mutation in the pgm gene (STJ2).

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