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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
  • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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  • 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
  • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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  • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
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  • 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
  • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• the present invention relates in particular to expression and secretion systems of proteins which can be used, in particular in corynebacteria, as well as a method using these systems, as well as new proteins linked to these expression systems.
• Corynebacteria constitute a group of gram positive bacteria, of irregular morphology, represented by a wide variety of strains.
• the invention relates to an expression and secretion system in a corynebacterium type bacterium comprising the secretory elements of two proteins found mainly in the culture supernatant of certain corynebacteria.
• a system of expression / secretion of an amino acid, polypeptide or protein determined by a corynebacterial strain characterized in that the sequence which codes for said amino acid, polypeptide or said protein is located in a region of chromosomal or plasmid DNA where said sequence is transcribed with towards the 5 'end at least part of the sequence coding for the signal sequence of the protein PS 1 or PS2, said part ensuring the secretion of said amino acid, polypeptide or said protein after translation when the system is incorporated into said corynebacterial strain.
• the present invention relates to an expression and secretion system in a corynebactery comprising:
• a secretion cassette containing a first functional DNA sequence for expression in said corynebacterial strain, a second DNA sequence which codes for an amino acid, a polypeptide and / or a protein, and a third DNA sequence inserted between said first and second DNA sequences which code for elements of a protein chosen from PS 1 or PS2, which ensure the secretion of said amino acids, polypeptides and / or proteins by said corynebacterial strain.
• corynebacterie designates not only the strains of the genus Corynebacterium but also related bacteria, such as Brevibacterium.
• the expression system of the present invention can be found on a self-replicating piasmide in corynebacteria and in this case, the piasmide will have an origin of functional replication in the strain, for example in Corynebacterium an origin of replication PBL 1, but may also be carried by a non-replicating piasmid intended in particular for chromosomal integration, in this case the piasmide will include elements allowing recombination and chromosomal integration; in the case of integration, the expression system will ultimately be found in the chromosome of the bacteria in question.
• heterologous expression elements that is to say that they may be elements which already exist in the host bacterium or which, on the contrary, come from a different bacterium.
• These expression elements will essentially comprise a promoter and a ribosome binding site, but it is also possible to provide other elements, in particular of the expression regulation type type.
• the expression elements which can be used in corynebacteria use will be made more particularly of the Ptac promoter which is a strong, hybrid trp / lac promoter inducible by IPTG and which proves to be functional in corynebacteria as in E. coli; but it is possible to use other promoters, for example, as will be described below, promoters or other elements of expression of corynebacterial structure gene, for example the promoter of gdhA. It is also possible to plan to use, for example, the expression elements, in particular the promoter of one of the proteins PS 1 and / or PS2 as they are identified in the context of the present invention.
• the expression elements may also include DNA sequences ensuring the regulation of the expression of downstream genes.
• the expression system of the present invention may finally comprise other elements, in particular elements such as transcription terminators, for example the terminator of the proteins PS 1 and / or PS2, or of gdhA.
• elements such as transcription terminators, for example the terminator of the proteins PS 1 and / or PS2, or of gdhA.
• it may be advantageous to add to the expression and secretion sequences, all or part of the PS 1 protein in order to obtain fused proteins, the secretion and the expression level of which can under these conditions be improved.
• the expression systems according to the invention may include heterologous elements which allow the construction in bacteria different from corynebacteria, for example as has been said previously, an origin of functional replication in E. coli but also other elements such as a -marker gene which can facilitate transfer into Corynebacterium.
• the marker gene can, of course, be of a very varied type as long as it is functional in corynebacteria, it could be a positive or negative selection gene such as a specific resistance, nevertheless in the current state research into these genes is not readily available.
• the celA gene of Clostridium thermocellum cellulase (celA), which confers the CMC + phenotype, will therefore preferably be used, but it is possible to use other marker genes, in particular lacZ of E. coli.
• the transformed bacteria are selected for the CMC + character after insertion of the coding sequence into an appropriate restriction site such as BstXI.
• the marker gene in the method according to the invention, provision will preferably be made for the marker gene to be easily eliminated after verification of the construction, in particular by placing restriction sites between the marker gene and the coding sequence.
• the coding sequence may be natural, synthetic or mixed.
• the expression and secretion system of the present invention is of course intended more particularly for ensuring the production of products of industrial interest, this is why the coding sequences will more particularly code for a peptide, a polypeptide, or a protein d 'industrial interest but it may also be a sequence coding not directly for a protein of industrial interest, but for a protein which may be involved in the maturation and / or the preparation of an amino acid, a polypeptide, or a protein of industrial interest.
• the methods according to the invention are more particularly intended for the expression of amino acid sequences, in particular repetitive sequences, they are therefore mainly synthetic sequences.
• This second DNA sequence coding for these different products may also include certain elements intended to ensure the maturation of the secreted product.
• the sequence expressed codes for a polypeptide of structure (ala-gln) 20 and (ala-gln-lys) 1 0 .
• the ala-gln or ala-gln-lys sequence can be released by subsequent enzymatic treatment. It is possible to provide other polymers of this type such as Ala-Gln-Tyr or Ala-Gln-Met which can be released by enzymatic or chemical treatment.
• codons of the coding sequence can influence expression in corynebacteria, it is preferable to provide a sequence having a richness in GC of the order of 50 to 60%.
• the percentage of GC is of the order of 75%, which may constitute a limitation, this is why the use of polymers comprising 3 amino acids is envisaged, the third of which is rich in A and T for reduce the percentage to 55%.
• Tyr, Lys and Met have two A or T in the first two bases of their codon and therefore allow the percentage of GC to be lowered from 75% to around 60%, which becomes closer to the percentage of GC found in corynebacteria.
• the industrial interest for these two amino acids in the COOH-terminal position of glutamine (Q) has been considered and exists.
• the present invention also relates to the strains of corynebacteria comprising an expression and secretion system as it has been described previously, and more particularly when the strain is a Brevibacterium, in particular a strain of Brevibacterium lactofermentum.
• the present invention relates to a process for obtaining amino acids, polypeptides, or proteins, characterized in that a transformed corynebacterial strain as described above is cultivated in a culture medium, in which the second DNA sequence codes for said amino acids, said polypeptide, and / or said protein and in that said product is optionally separated after culture from the culture medium.
• the product of interest has been secreted and is therefore found in the culture medium from which it can be isolated by known methods, whether separation techniques such as chromatography, precipitation selective for example, this obviously having to be adapted to the nature of the molecule produced.
• PS 1 and PS2 being wall proteins, part of the proteins secreted with this system remain anchored in the wall, which can facilitate their separation because with certain detergents the bacteria is not lysed.
• corynebacteria by plasmids is preferably carried out by electroporation (Bonamy C., Guyonvarch A., Reyes O., David F. and Leblon G. (1990) FEMS Microbiology Letters 66: 263-270) or by any other method appropriate.
• the present invention also relates to expression systems comprising all or part of the expression signals of csp1, csp2 and gdhA or all or part of these three genes, as well as the strains expressing this type of system, in particular the strains of corynebacteria.
• the expression / secretion of the determined amino acid, polypeptide or protein will be regulated by the temperature, the culture medium and / or the nature of the sugars for PS 1 and PS 2 and the concentration of salts (NH 4 + in particular), metabolites (glutamate) and sugars (glucose / fructose) for gdhA systems.
• the present invention also relates to proteins comprising all or part of the sequence of PS 1 or PS2, in particular comprising one or more antigenic sites of these proteins.
• Said proteins can be used as an atypical element, in particular in diagnostic kits, as can the corresponding antibodies.
• the invention also relates to the strains of corynebacteria in which the determined protein is anchored on the wall by the part of PS 1 or PS2 fulfilling this anchoring function or in which the antigenic epitopes of PS 1 or PS2 are exposed on the wall .
• FIG. 1 Diagram of the pCGL612 piasmid.
• PUN121-derived plasmid (Nilsson, B., Uhlén, M., Josephson, S., Gatenbeck, S., and Philipson, L. (1983) An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis. Nucleic Acids Res 11: 8019-8029.) Containing a 2.6 kb fragment of C. melassecola ATCC17965 carrying the entire cspl gene allowing the synthesis of the PS 1 protein.
• FIG. 1 Nucleotide sequence and corresponding amino acid sequence of the cspl gene of Corynebacterium glutamicum called Corynebacterium melassecola ATCC 17965. The numbering of the nucleotides is present on the right side of the figure. The repeated nucleotide sequences are surrounded. The probable Shine-Dalgarno sequence is underlined. A 24 bp palindrome that presumably corresponds to the transcription terminator is indicated by reverse arrows. This sequence appears in the EMBL nucleotide sequence database under the access number X66078.
• FIG. 4 Alignment of the sequences of the PS 1 protein of C. glutamicum and the proteins of the antigenic complex 85 of Mycobacterium.
• 85B M. k. represents M. kansaii 85-B antigen (MIPSG16235).
• 85B M. b. represents M. bovis antigen 85-B (MIPSC83179).
• 85B M. I represents M. leprae's 85-B antigen
• 85A M. t. represents V antigen 85-A of M. tuberculosis (MIPS 160062).
• the sequences were aligned using the FastA program from the "Genetics Computer Group” (University of Wisconsin, USA). The numbering of the residues is given for each protein at the beginning of each line. Similar amino acid residues found between different proteins are surrounded. The residues cosidered as similar are the following; acids or amides (D, E, N, Q); basic (H, K, R); polar (P, A, G, S, T); non-polar (I, L, M, V) and aromatic (F, W, Y). The identical amino acid residues between the seven proteins are indicated by a star above the relevant residues.
• accession number is specified associated with the name of the database in question and appear in parentheses.
• Figure 5. Interruption of the cspl gene.
• pCGL613 is non-replicative in C. glutamicum, it contains cspl (blackened area) interrupted by the aphA3 (Km) gene, wt, wild B. lactofermentum and Acspl, integrating it containing interrupted csp1.
• the pCSI616 piasmid corresponds to the pCGL125 piasmide with the C. glutamicum cspl gene.
• FIG. 8 Construction of the pCGL1030 piasmid.
• the region named A in the scheme contains the cspl promoter followed by the DNA region corresponding to the signal sequence of PS 1 and the first 30 amino acids of its mature sequence.
• FIG. 9 Construction of piasmid 1031. The region named A is described in Figure 8. The junction region between PS 1 and EGA has been sequenced and the details of this sequence are presented.
• FIG. 10 Construction of piasmid 1032. The region named A is described in Figure 8. The junction region between PS 1, (AQK) 10 and EGA has been sequenced and the details of this sequence are presented.
• FIG. 1 Construction of piasmid 1033. The region named A is described in Figure 8. The junction region between PS 1, (AQ) 19 and EGA has been sequenced and the details of this sequence are presented.
• ATCC 17965 The numbering of the nucleotides is shown on the right side of the figure. The probable Shine-Dalgarno sequence is underlined. A 22 bp palindrome that presumably corresponds to the transcription terminator is indicated by reverse arrows.
• the non-replicative pCGL830 piasmid in C. glutamicum carries the csp2 gene interrupted by the aphlll gene.
• the meaning of transcription of the genes aphIII and csp2 is represented by an arrow on the piasmid pCGL830.
• Wt represents the strain B. lactofermentum 15 and csp2 :: aphIll, integrating it with the interrupted csp2 gene.
• Figure 17 Complete sequence of the Nhel-Blgl fragment containing the gdhA gene of C. melassecola.
• FIG. 1 Construction of pCGL141 and pCGL142, fusion vectors between the promoter of the gdhA gene and the lacZ gene.
• Figure 21 Detail of construction placing the synthetic gene between ptac and the celA gene - a) construction placing ceLA under the control of ptac, b) becoming of the construction after introduction of the AQ polypeptide.
• RBS ribosome binding site
• DNA sequence equivalent to part of the EGA signal sequence DNA sequence equivalent to the coding sequence of EGA
• FIG. 22 Structure of pCGL125.
• Example 1 Identification of PS1 and PS2 in the culture supernatant and in the wall of Corynebacterium slutamicum.
• the concentrations of PS 1 and PS2 follow the growth curve of the bacteria and reach their maximum in the stationary phase.
• An important secreted fraction of these proteins and especially for PS2 is also located in the wall of the bacteria.
• an SDS treatment of bacteria is used, which does not cause significant lysis of the bacteria.
• PS 1 and PS2 it is therefore possible to combine the two secreted fractions, culture supernatant and parietal fraction, and to obtain a final preparation where PS 1 and PS2 are strongly in the majority.
• Polyclonal antibodies have been prepared against PS 1 and PS2, there is no cross immunological reaction between the two proteins which clearly shows that these proteins are different.
• Proteins with strong immunological cross reactions with PS 1 and PS2 have been found in the culture supernatant of bacterial strains related to Corynebacterium melassecola ATCC 17965 such as the strain Brevibacterium lactofermentum 15 (Bonnassie, S., Oreglja, J., Trautwetter, A ., and Sicard, AM (1990) Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum. FEMS Microbiol Letters 72: 143-146.), Brevibacterium lactofermentum ATCC21086 and Brevibacterium flavum ATCC 14067.
• PS 1 and PS2 have been tested for several enzymatic activities including invertase, pectinase, nuclease, collagenase, amylase, bacteriocin, endoglucanase and protease broad spectrum activity. None of these enzymatic activities could be associated with PS 1 or PS2.
• Example 2. Evidence of the functionality of the signal peptide of PS1 in Escherichia coli ( Figure 1).
• the Western blotting analysis of a crude extract of this recombinant with anti-PS 1 antibodies reveals the presence of a major protein which has the same molecular mass as the PS 1 protein present in the culture supernatant of C. melassecola.
• a minor protein of slightly higher molecular weight is also detected.
• the major protein band corresponds to the mature form of PS 1 (without signal sequence) and the minor protein band to the precursor form of PS 1 (with the signal sequence).
• FIG. 3 represents the restriction map of this sequenced region.
• the putative translation start (GAGAAGGAAAACTTCATG) and transcription start (TACATA (-35) and TAAGAT (-10) signals have been identified.
• the AGAAGGA sequence extracted from the ribosome binding site described above is complementary (underline ) from the 3 'end of the rRNA of Gram-positive bacteria Staphylococcus aureus and Steptomyces' lividans (5'-GAUCACCUCCUUUC UOH-3') (McLaughlin, JR, Murray, CL, and Rabinowitz, JC (1981) Unique features of the ribosome binding site sequence of the Gram positive Staphylococcus aureus ⁇ -lactamase gene. J. Biol Chem.
• a sequence at the NH2 end of the protein has the characteristics of a signal sequence of Gram-positive bacteria (Watson, MEE (1984) Compilation of published signal sequences. Nucleic Acids Res .12: 5145-5164.).
• This signal sequence comprises an excess of positive charge in NH2-terminal position (7 amino acids with positive charge in the first 18 amino acids), followed by a sequence with an excess of non-polar amino acids (18 non-polar amino acids in the next 23 amino acids) then two putative amino acid sequences of a signal sequence cleavage site (pro thr ala ile ala, in position 28 to 32) (pro met ala ser ala, in position 39 to 43) .
• the second pro met ala ser ala at position 39 to 43 seems the most likely; in fact, the PS 1 protein has been purified from the culture supernatant of
• the calculated molecular mass corresponding to the 657 amino acids contained in the open reading phase is 70,874.
• the molecular mass of the most probable signal sequence site of cleavage between amino acid 42 and 43 is 4411, which gives a molecular mass calculated for the mature protein of 66463 and which is very close to the value of 67000 estimated on denaturing polyacrylamide gel
• Example 4 Sequence homologies between PS1 of Corynebacterium glutamicum and the proteins of the antigenic complex 85 of Mycobacterium. ( Figure 4). The NH2 half of the PS 1 protein is very similar to the three secreted mycobacterial antigens named 85-A, 85-B and 85-C (Closs, O, Harboe, M., Axelsen-Christensen, NH, and Magnussen, M. ( 1980) The antigens of Mycobacterium bovis, strain BCG, studied by crossed immuno-electrophoresis: a reference System Scand J.
• Mycobacterium bovis BCG1173P2 Mycobacterium tuberculosis antigen 85-A (Borremans, M., De Wit, L., Volckaert, G., Ooms, J., De Bruyn , J., Huygen, K., Van Vooren, J.-P., Stelandre, M., Verhofstadt, R., and Content, J. (1989) Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis.
• Bacteriol 170: 3847-3854. (Matsuo, K., Yamaguchi, R., Yamazak i, A., Tasaka, H., Terasaka, K., and Yamada, T. (1990) Cloning and expression of the gene for the cross-reactive a antigen of Mycobacterium kansaii. Infect Immun 58: 550-556.) (De Mendonça Lima, L., Content, J., Van Heuverswyn, H., and Degrave, W. (1991) Nucleotide sequence of the gene coding for the 85-B antigen of Mycobacterium leprae.
• Protocol 1 PS1 and PS2 purification protocols used for the determination of the N-terminal sequence. Protocol 1:
• the proteins PS 1 and PS2 were purified from the culture supernatant of C. glutamicum ATCC 17965 by preparative electrophoresis on polyacrylamide gel and electroelution.
• the bacteria cultivated in 200 ml of LB rich medium at 34 ° C., are harvested in the stationary growth phase by centrifugation at 8000 g for 15 minutes and at 4 ° C.
• the proteins of the culture supernatant are then precipitated with 60% ammonium sulphate and harvested by centrifugation at 13000 g for 15 minutes at 4 ° C.
• the pellet is dissolved in 4 ml of 10 mM Tris HCl buffer pH 6.8 and the solution is then dialyzed for 24 hours at 4 ° C. in this same buffer.
• the dialysis protein extract obtained after precipitation with ammonium sulphate is deposited on an electrophoresis gel of format 16 ⁇ 20 ⁇ 0.75 cm.
• the electrophoresis is carried out according to the protocol described by Laemmli (1970) using a 4% concentration gel and a 7.5% separation gel. The migration is done in fifteen hours at 40mA. The gels are then stained with copper chloride according to the protocol described by Lee et al (1987). The protein bands corresponding to the proteins PS 1 and PS2 are cut out and then completely discolored. The proteins are then electroeluted from the gel for 5 hours at 48mA and at 4 ° C., then dialyzed several times in a 10 mM Tris HCl buffer pH 6.8 before being distributed into several aliquots and frozen at ⁇ 20 ° C. The purification yield is of the order of 25% with a purity greater than 90%.
• the proteins PS 1 and PS2 are purified from the culture supernatant of C. glutamicum ATCC 17965 by ultrafiltration, electrophoresis and transfer to a PVDF membrane.
• the bacteria cultivated in the LB rich medium at 34 ° C., are harvested in the stationary growth phase by centrifugation at 8000 g for 15 minutes and at 4 ° C. 4 ml of supernatant are diluted 50 times in a 50 mM phosphate buffer pH 7.0 before being centrifuged on an ultrafiltration membrane whose cutoff threshold is 30 kD. This step makes it possible to obtain a protein extract of 80 ⁇ l which is then deposited on an electrophoresis gel composed of a concentration gel at 4% and a separation gel at 7.5%.
• the electrophoresis is carried out according to the protocol described by Laemmli with the following modifications.
• the solutions used to prepare the gels as well as the migration buffer are degassed and contain 0.1 M of thioglycolate.
• the separation gel is subjected to a "pre-run" before its use. All these precautions are taken in order to avoid as much as possible the formation of free radicals which could lead to modifications of the N-terminal end of the proteins and consequently to a possible blocking of this end.
• the proteins are transferred onto a PVDF membrane. This step is carried out in a 50 mM Tris, 50 mM borate pH 8.0 buffer for 60 minutes and at 50V. The membrane is then stained with amidoblack, which makes it possible to locate and cut the bands corresponding to the proteins PS 1 and PS2.
• Example 6 Obtaining a strain of Corynebacterium glutamicum no longer synthesizing PS1, called PS1-. ( Figure 5).
• the C. glutamicum strain called Brevibacterium lactofermentum 15 is permissive to the modified DNA of E. coli K12 (Bonnassie, S., Oreglia, J., Trautwetter, A., and Sicard, AM (1990) Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum. FEMS Microbiol Letters 72: 143-146), while the C. glutamicum strain called C. melassecola 'ATCC17965 is a very restrictive strain with regard to the DNA of E. coli ( Reyes O., Guyonvarch, A., Bonamy, C, Salti, V., David, F., and Leblon, G.
• the cspl gene is contained in a fragment of approximately 7.5 kb in the wild strain, while the integrating pCGL613 'contains a fragment of approximately 9 kb corresponding to the 1.5 kb aphA3 gene inserted into the cspl gene.
• the BamHI-EcoRI digestion confirms the structure of the integrant presented in FIG. 5.
• the integrating Km r -Tets was also analyzed by Western blotting for the production of PS 1 using anti-PS 1 polyclonal antibodies. There is no PS 1 protein, neither in the culture supernatant, nor in the crude extract of this strain. This confirms that the cspl gene cloned in ⁇ gt11 corresponds to a single gene which codes well for PS 1 in C. glutamicum.
• This C. glutamicum PS 1- strain is completely viable and does not seem to be affected in its growth rate. This result shows that it is possible to use the region of the cspl gene as a target for integration of homologous or heterologous DNA into a strain of C. glutamicum, without a priori affecting its viability.
• Example 7 Expression in C. elutamicum of the csul gene in multicopy. Analysis of the important regions of PS1, necessary for its synthesis and its secretion.
• the pCAM616 piasmid was constructed. It contains the entire cspl gene and was constructed from the piasmid pCGL125, corresponding to the piasmid pBL1 (Santamaria, R., Gil, JA, Mesas, JM and JF Martin (1984) Characterization of an endogenous plasmid and development of cloning vectors and a transformation System in Brevibacterium lactofermentum, J. Gen. Microbiol. 130: 2237-2246.), replicative in C.
• glutamicum provided with a cloning cassette comprising the aphA3 gene of Streptococcus faecalis and the piasmid pCSP1G, replicative in E. coli, containing the cspl gene.
• the piasmid, pCGL616, resulting from this construction ( Figure 6) is replicative in C. glutamicum. Restoration of the synthesis of PS1 in a strain of C. glutamicum PS1-.
• the largest deletion allowing this result corresponds to the deletion of the Ncol-BspEl (BspM11) fragment of approximately 1.3 kb from the cspl gene (pCGL1041) giving a size of protein precursor for PS 1 equal to approximately 29 kD and to approximately 24kD for the mature form secreted.
• B. lactofermentum 15 PS 1 strain harboring this piasmid pCGL1041 it is well detected by Western blotting with anti-PS 1 antibodies a protein of approximately 23 kD.
• Example 8 Construction of an expression and secretion vector in C. glutamicum named pCGL1030 based on the cspl system. ( Figure 8. 9, 10, 11)
• This replicative piasmid in C. glutamicum (contains the piasmid pBL1 of C. glutamicum) carries the promoter of the cspl gene of C. glutamicum and the DNA region of this gene corresponding to the signal sequence plus the first 30 amino acids of the sequence mature of PS 1.
• a mutiple cloning site (polylinker 2 in FIG. 8) was placed immediately behind the 30 th amino acid of the mature sequence of PS 1, in order to allow easy phase cloning of any heterologous gene to be expressed in C. glutamicum.
• this piasmid is endowed with the PS 1 elements necessary for secretion and therefore corresponds to a tool for expression and secretion.
• the celA gene from C. thermocellum (Cornet, P., Millet, J., Béguin, P. and J.-P. Aubert (1983) Characterization of two cel (cellulose degradation) genes of Clostridium thermocellum coding for endoglucanases. Bio / Technology 1: 589-594.) Encoding an endoglucanase called endoglucanase A or EGA, was cloned into the vector pCGL1030 at the Sma I site, giving rise to the piasmid pCGL 1031 (FIG. 9).
• This celA gene comes from the piasmid pCGL1008 where a restriction site BstX1 has been introduced artificially, very close to the translation start-up site of the protein.
• EGA for chimeric construction purposes (see Figure 10, 11).
• CMC carboxymethylcellulose
• This CMC test will be used to confirm the synthesis of the EGA protein of C. thermocellum in C. glutamicum.
• a CMC test of activity on a dish carried out on whole cells or on the culture supernatant, in rich medium highlights in both cases, the endoglucanase activity of 'a strain of C. glutamicum called Brevibacterium lactofermentum 15 harboring the piasmide pCGL1031.
• Brevibacterium lactofermentum 15 harboring the piasmide pCGL1031.
• We will note a stronger activity on LB medium + fructose, or + glucose, indicating a stimulating effect of these two sugars on the expression of celA under control of the promoter cspl.
• a synthetic gene corresponding to the alanine-glutamine-lysine polypeptide repeated 10 times was chemically synthesized and cloned at the BstX1 site of the piasmide pCGL1008, giving rise to the piasmide pCGL1017.
• the EcoRl fragment of the piasmid pCGL1017 was cloned at the Smal site of the piasmid pCGL1030, located downstream of the cspl promoter of the signal sequence and of the first 30 amino acids of PS 1 (and upstream of the reporter gene celA), giving rise to the piasmid pCGL1032 ( Figure 10).
• the detection of the chimeric protein PS1- (AQK) 10-EGA is carried out as described above, by CMC test on dish by zymogram or by Western blotting. Use of the csp1 system for the expression and secretion of the synthetic polypeptide (AQ) 19 ( Figure 11).
• a synthetic gene corresponding to the alanine-glutamine polypeptide repeated 20 times has been chemically synthesized and cloned at the BstXI site of the piasmide pCGL1008, giving rise to the piasmide pCGL1002.
• the ERI co fragment of the pCGL1002 piasmid was cloned at the Smal site of the PCGL1030 piasmid, located downstream of the cspl promoter of the signal sequence and of the first 30 amino acids of PS 1 (and upstream of the reporter gene celA), giving rise to the piasmid pCGL1033 (FIG. 11).
• the detection of the chimeric protein PS1- (AQ) 19-EGA is carried out as described above, by CMC test on dish, by zymogram or by Western blotting.
• the sequence in B. lactofermentum in the piasmide pCGL1033 revealed the loss of an AQ coding sequence (passage from AQ 20 to AQ 19 during cloning in B. lactofermentum).
• glutamicum the production of EGA or chimeric proteins (AQK) 10-EGA or (AQ) 19-EGA, is better on LB medium than on BHI medium, it is strongly stimulated by glucose or fructose on LB medium.
• the cspl promoter of C. glutamicum seems stronger than the natural promoter of celA from C. thermocellum; indeed, the so-called B. lactofermentum 15 strain hosting the piasmide pCGL602, which contains the natural promoter of celA exhibits significantly less endoglucanase activity than this same strain hosting the piasmid pCGL1031, where this celA is under the control of the cspl promoter of C. glutamicum .
• a Shine Dalgarno type sequence has been identified, AAGGAG, just upstream from the translation start codon (-12 to -17).
• the proposed signal sequence of 30 amino acids reveals a glutamine (position 31) as the first amino acid of the mature sequence, easily converted into pyroglutamic acid making it impossible to amino-terminal sequencing of the protein by the technique of Edman.
• a putative independent rho-type terminator site is found in the 3 'region of the gene 76 nucleotides from the stop codon.
• Example 10 Obtaining a Corynebacterium glutamicum strain no longer synthesizing PS2. called PS2-. ( Figure 14).
• the interruption of the csp2 gene was carried out in C. glutamicum called B. lactofermentum 15 using the vector pCGL830 ( Figure 14), a non-replicating vector in corynebacteria, and carrying a copy of the csp2 gene inactivated by the insertion of the aphlll gene (cloning of the aphlll gene at the unique Nru I site of cspl carried by the plasmid pCGL811).
• No PS2 signal was demonstrated by immunological detection with anti-PS2 polyclonal antibodies, on cell extracts from the E.
• Example 12 Construction of a Corynebacterium melassecola ATCC 17965 chromosomal DNA library and cloning of the gdhA gene
• the chromosomal DNA of the C. melassecola ATCC 17965 strain was prepared according to the method described by Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (Eds) ((1987) Current protocols in Molecular Biology. John Wiley and Sons, New York).
• the cloning piasmide pUN121 (Nilsson B, Uhlen M, Josephson S, Gatenberg S, Philipson L (1983) An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis. Nucleic Acids Res 11: 8019-8030) was prepared by the method de Birnboim HC, Doly J ((1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513-1523), from the strain of E. coli GM 2929 freely available from Dr. B. Bachmann. The piasmid was linearized by the restriction endonuclease Bcl I (Boehringer).
• the bank was built by ligation with T4 DNA ligase
• the plasmids of all the tetracycline resistant clones were prepared by the method of Birnboim and Doly (1979). All of these plasmids correspond to the DNA library.
• the E. coli strain CLR207 recA (Mattaj IW, McPherson MJ, Wooton JC (1982) Localization of a strongly conserved section of coding sequence in glutamate dehydrogenase genes. FEBS Letters 147: 21-25), deficient in glutamate dehydrogenase activity was transformed with the DNA library of C. melassecola ATCC 17965. A transforming clone of E.
• coli CLR 207 recA capable of growing on minimum selection medium containing 100 ⁇ g / ml of ampicillin was selected.
• This clone carries a recombinant plasmid, pCGL310.
• Glutamate dehydrogenase activity measured according to the method of Meers JL, Tempest DW, Brown CM ((1970) Glutamine (amide): 2-oxoglutarate amino transferase oxido-reductase (NADP), an enzyme involved in the synthesis of glutamate by some bacteria J Gen Microbiol 64: 187-194), is restored in the strain of E. coli CLR207 recA carrying the piasmid pCGL310.
• promoter (nucleotides 1 to 572)
• TTCACA signal having a similarity with the sequence TTGAC (Pu) characteristic of the -35 zone of the promoters of Streptomyces sp. (Strohl)
• the reading phase extending from position 573 to 1913 corresponds to that of glutamate dehydrogenase due to the following data: _
• the protein deduced from this reading phase contains 447 amino acids, with a predicted molecular weight of 48,957 Daltons. This molecular weight is very close to that of the polypeptide (48300 D) observed after denaturing gel electrophoresis of a preparation of glutamate dehydrogenase of the strain of C. melassecola ATCC 17965.
• the primary structure of glutamate dehydrogenase deduced from the nucleotide sequence of the gdh A gene of C. melassecola has strong similarities with the primary structures of glutamate dehydrogenases of other organisms (Teller JK, Smith RJ, McPherson MJ, Engel PC, Guest JR ((1992) The glutamate dehydrogenase gene of Clostridium symbiosum: cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli. Eur J Biochem 206: 151-159).
• the regulation of the expression of the gdh A gene of C. melassecola ATCC 17965 was studied by measuring variations in the specific activity of glutamate dehydrogenase as a function of the nature of the medium in which this strain was cultivated. Glutamate dehydrogenase activity was measured by the method of Meers et al. (1970) from acellular extracts of C. melassecola obtained by ultrasonication.
• the culture media used for this study are synthetic media, the base of which is that described by Liebl W, Klamer R, Schleifer KH (1989) (Requirement of chelating eompounds for the growth of
• the carbon source is either glucose at 11 g / l final (media 1, 2 and 4) or fructose at 10 g / l (media 3).
• the concentration of NH4 + ions is 125 mM in media 1, 3 and 4. It is 1.25 mM in media 2 (limitation in NH4 +).
• Medium 4 contains 50 g / l final L-glutamate.
• the functionality of the gdh A - lac fusion has been shown by demonstrating a ⁇ -galactosidase activity in the strains of C. melassecola ATCC 17965 transformed by pCGL 141 and pCGL142, activity absent from the same strain transformed by pCGL 125.
• the ⁇ -galactosidase activity is demonstrated by culture of the bacteria on a solidified complete medium (BHI, Difco) containing the chromogenic substrate X-gal (5-bromo-4-chloro-3- indolyl ⁇ -D-galactopyranoside). Colonies from bacteria with ⁇ -galactosidase activity turn blue on such a medium.
• NTG mutagenesis of a strain derived from C. melassecola ATCC 17965 was carried out. From this mutagenesis, a first selection was applied on the criterion of resistance to a glutamate analog, 4-fluoroglutamate. Mutants resistant to this analog can belong to different classes including the catabolic non-repression class. Indeed, in such mutants, it can be expected that the increase in specific activity of glutamate dehydrogenase will lead to an overproduction of intracellular glutamate, and thus to a dilution of the toxic analog, hence the resistance phenomenon. Mutants of resistance to 4-fluoroglutamate were pooled and all of the cells were subjected to transformation by pCGL 141.
• the transformed bacteria were spread on solidified medium 1 containing X-Gal and kanamycin. The bacterial colonies showing the most intense blue color were isolated, and cultured in medium 1 liquid containing kanamycin. Glutamate dehydrogenase activity was measured from a cell-free extract, ⁇ -galactosidase activity from whole toluene cells (Miller, 1972). The results obtained for one of the selected mutants are presented below. glutamate dehydrogenase ⁇ -galactosidase activity
• mutant 90 12.1 23.66 The results obtained therefore clearly show that it is possible to select, by phenotypic screening, mutants for regulating the gdh A gene with the constructed tool. It should be noted that it is very easy to remove pCGL 141 and pCGL 142 from the cells after selection, simply by culture in the absence of kanamycin selection pressure.
• the celA gene available in the form of a HindIII fragment of 3.5 kb containing the promoter region, the gene and the start of another unidentified gene, was subcloned in the form of a HindIII-EcoRI fragment of 2, 6kb deleted from the unknown gene piece, in a replicative vector of E. coli, pMTL23 (Chambers, SP, Prior, SE, Barstow, DA and Minton NP (1988) The pMTL nie - cloning vectors.
• I Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene. 68: 139-149.
• the Eco RI site was introduced by site-directed mutagenesis immediately behind the gene transcription terminator. This intermediate subcloning, taking into account the restriction sites introduced, is necessary for the next stage of cloning; in particular, cloning into the polylinker of pMTL23 allows the introduction of an Ncol restriction site just behind the Eco RI site, which makes it possible to extract the fragment containing the coding region of celA in the form of a Nael-Ncol fragment. This step also makes it possible to have a celA gene lacking unidentified 3 'sequences.
• the cloning of cel A in the form of a HindIII-EcoRI fragment was carried out in the piasmid pMTL23 using the receptor strain of E. coli TGl.
• the strain of E. coli possessing this piasmid is well endowed with the CMC + phenotype associated with the expression of EGA; the analysis of the restriction fragments obtained is in accordance with what is expected.
• This replicative plasmid in E. coli containing the tac promoter (Brossius et al. Gene 27: 161, 1984) is hydrolyzed by EcoRI-NcoI.
• the piasmid thus obtained is called pPROK-celA. It contains the celA gene under the control of the tac promoter separated by a BstXl site introduced using the DGF1 / DGF2 adapters.
• Example 14 Construction of a piasmid allowing the expression of multiple AQ sequences
• DGF5 / DGF6 (FIG. 1.9) which correspond to the synthetic gene is used:
• [AQ] representing the sequence coding for Ala-Gln.
• the ends of the DGF5 and DGF6 sequences are compatible with the BstX1 site and the sequence can therefore be cloned into this site.
• DGF5 and DGF6 are such that, on the one hand, they direct the direction of cloning, and, on the other hand, they destroy the BstXI site following cloning.
• pPROK (AQ) 20 celA having the structure represented in FIG. 20.
• FIG. 21 details in particular the structure of the AQ / EGA fusion site and shows the advantage of the BstX1 site used. This site is structured:
• the insertion of the DGF5 / DGF6 adapter can only be done in one direction and does not introduce any foreign base to the targeted object.
• This piasmid is treated with BamHI and ligated with the restriction product of the pCGL125 piasmid treated with the same enzyme.
• the pCGL125 piasmid (FIG. 22) is a functional piasmid of
• Brevibacterium lactofermentum 15 having an origin of replication pBL1.
• the piasmid pCGL 125- (AQ) 20 -celA
• selection of the transformed strains a strain is obtained according to the present invention.
• the translation begins with a methionine immediately followed by (AQ) 20 ; we also took the precaution of border (AQ) 20 with a methionine in COOH-terminal; the detection of the AQ polypeptide fused or not fused to the celA protein can be done using specific antibodies or by analytical detection after summary purification of the fusion protein and hydrolysis with cyanogen bromide or vice versa.
• AQ border
• Corynebacterium glutamicum Corynebacterium melassecola
• the DH5alpha strain is available in the catalog of
• ATCC strains are available at American Type Culture Collection c / o Sales and Marketing Department, 12301 Parkiawn Drive, Rockville, MD 20852 USA.
• ORGANISM Corynebacterium melassecola
• GCT GGT GAA GTC ACC TAC CCA GAG ATC GCC GGG CTG CCT 689 Ala Gly Glu Val Thr Tyr Pro Glu Ile Ala Gly Leu Pro
• GAG CGC CCA ATC AAG GTG CAG CTG CTG CTT CCG CGT GAC 806 Glu Arg Pro Ile Lys Val Gln Leu Leu Leu Leu Arg Arg
• GGT GCA GAT GAC TTC GGT AAG GAA GAC TCT GTA GCT ATT 1391 Gly Ala Asp Asp Phe Gly Lys Glu Asp Ser Val Ala Ile
• ORGANISM Corynebacterium melassecola
• BIOLOGICAL ACTIVITY precursor of the protein constituting the outer surface layer of the wall and of the antigen
• ORGANISM Corynebacterium melassecola
• FIGURE 8
• FIGURE 9 is a diagrammatic representation of FIGURE 9
• FIGURE 1 0
• FIGURE 22
• Polylinker1 0.001 / Sacll.BstXI.Notl.Xbal.
• Polylinker2 1.531 / Clal.Sall.Aatl.Mlul.Ncol.Bglll.Xhol.Stul.Pstl.
• Polylinker3 1.561 / Xbal.Notl.Sacll.BstXI.

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