WO2004061095A2

WO2004061095A2 - Micro-organism strains having an inactive phor/phon operon for carrying out a method for the overproduction of metabolites, in particular exhibiting an antibiotic and enzymatic activity

Info

Publication number: WO2004061095A2
Application number: PCT/FR2003/003751
Authority: WO
Inventors: Marie-Joëlle Virolle; Safiane Ghorbel; Claude Gerbaud
Original assignee: Centre National De La Recherche Scientifique; Universite Paris Sud Xi
Priority date: 2002-12-16
Filing date: 2003-12-16
Publication date: 2004-07-22
Also published as: FR2848567A1; WO2004061095A3

Abstract

The invention relates to the use of micro-organism strains producing useful compounds provided with an inactive phoR/phoP operon for carrying out a method for stimulating the production of said useful compounds by means of said transformed strains.

Description

STRAINS OF MICROORGANISMS OF WHICH LOPERON PHOR / PHOP IS INACTIVE FOR THE IMPLEMENTATION OF A METHOD OF OVERPRODUCTION OF METABOLITES, ESPECIALLY ANTIBIOTIC OR ENZYMATIC ACTIVITY

The present invention relates to the use of strains of microorganisms modified so that said strains no longer produce functional sensory kinase, and / or the regulator responsible for the activation of the Pho regulon, as well as their use in the as part of the implementation of overproduction processes of metabolites normally produced by said unmodified strains, such as antibiotics or enzymes.

In order to improve the production of their strain, manufacturers use random mutagenesis series. During the first mutagenesis, the most productive mutant (s) are retained and are subjected to a second mutagenesis, etc. During a process that can sometimes take more than ten years, manufacturers thus obtain hyper strains. -productrices. The latter are often "poly-mutated" but the genes affected are not known.

This traditional method of improving strains described above is very long and requires many manipulations and tests. It results in the obtaining of poly- mutated strains which grow poorly, have specific medium requirements, and for which a long phase of medium optimization is necessary.

Thoughtful and targeted manipulation of the expression of certain genes (it may be over-expression or inactivation) which has been shown, in related species, to have an effect on the production of antibiotics can lead much faster than traditional methods to obtain strains of industrial interest that are hyper-productive of antibiotics. Since such strains are not poly-mutated, they are likely to have growth characteristics close to the wild strain and should require a shorter environment optimization phase.

It has long been known that in Streptomyces an inorganic phosphate (Pi) deficiency induces the initiation of antibiotic biosynthesis. During its growth, the bacteria consume the Pi present in the culture medium until the concentration of the latter becomes limiting. This low Pi concentration signals a future state of Pi deficiency and the bacteria then starts the expression of a whole set of genes which allows its adaptation to this limitation in Pi. All of these genes, among which may be cited genes coding for phosphatases of different phosphorylated molecules, high affinity Pi transport systems, Pi storage / destocking systems in Pi polymers (including the ppk gene) etc. constitutes the Pho regulon . These different functions make it possible to recover the Pi present at a low concentration in the medium or present in different biological molecules and thus to differ or reduce the extent of the state of Pi deficiency.

In Escherichia coli or Bacillus subtilis, the expression of the Pho regulon genes is under the control of a two-component system comprising an activator and a sensory kinase. Under conditions of limitation in Pi, the latter self-phosphorylates and transfers its phosphate to the regulator which thus becomes capable of interacting with operators (Pho boxes) located in the promoter regions of the various genes of the Pho regulon in order to stimulate their transcription. .

A situation of limitation in Pi triggers the expression of the regulator Pho, a set of genes whose expression makes it possible to delay the appearance of a state of deficiency in Pi. A situation of deficiency in Pi leads to the triggering of adaptive processes different from those induced by a limitation in Pi. These are still poorly understood processes which lead, in Streptomyces, to the initiation of the biosynthesis of antibiotics. It is likely that this massive production of antibiotics is linked to an accumulation in the cell of precursors of antibiotic biosynthesis. In the case of polyketide type antibiotics, these precursors are often acetate, malonate or butyrate units resulting from the catabolism of fatty acids or certain amino acids. In a state of deficiency in Pi, the bacteria would trigger "autophagy" processes, ie active catabolism of these cellular constituents in order to produce the energy which the cell needs via the Krebs cycle. However, due to multiple deficiencies, the Krebs cycle would slow down and an accumulation of acetate, malonate or butyrate units would follow in the cell. This accumulation could be toxic for the latter if it did not induce condensation / polymerization pathways of these units, condensation which leads to the synthesis of the carbon skeleton of polyketide antibiotics.

The present invention stems from the discovery by the inventors that it is possible to create an early Pi deficiency situation by preventing the expression of all of the genes (Pho regulon) which allows the bacteria to adapt to a limitation in Pi in order to defer the appearance of the state of deficiency in Pi.

To do this, the inventors isolated and interrupted the two genes phoR (kinase) and phoP (regulator) which, in Streptomyces lividans, probably constitute the system with two components responsible for the activation of the expression of all the genes of the Pho regulon of Streptomyces. The phoR and phoP mutants thus constructed are incapable of inducing the expression of the various Pi recovery systems mentioned above; they are therefore precociously and severely deficient in Pi and massively overproduce antibiotics.

The object of the invention is to provide a new process aimed at significantly increasing the production of metabolites or enzymes by strains of microorganisms of industrial interest (Actinomycetales or others), by lifting the repression exerted by phosphate on the production of antibiotics or enzymes by strains of microorganisms of industrial interest.

Advantageously, the process of the invention is easily transposable to any industrial strain whose production of enzymes, metabolites or any other product with high commercial value is known to be repressed by inorganic phosphate (Pi).

The invention also aims to provide new strains of microorganisms of industrial interest for the implementation of a process of overproduction of antibiotics, said strains having the advantage of having growth characteristics close to the wild strain.

The invention also aims to provide new nucleotide sequences for obtaining such strains of microorganisms. The present invention relates to the use of strains of microorganisms producing compounds of interest, including the operon phoR / phoP encoding a two-component system, namely a sensory kinase encoded by phoR, and a regulator encoded by phoP, responsible for activating the Pho regulon of said microorganisms comprising a set of genes involved in the adaptive response to a phosphate limitation in said microorganisms, is inactivated, for the implementation of a method of stimulation of the production of these compounds of interest by said transformed strains.

By process for stimulating the production of compounds of interest, as mentioned above, is meant any process by which the production of these compounds is increased, in particular in proportions of about 20% to 50% or more, by compared to the production of these same compounds in strains identical to the previous ones but whose operon phoR / phoP has not been inactivated.

The invention relates more particularly to the aforementioned use of microorganisms whose production of compounds of interest, in particular of secondary metabolites, depends on the concentration of inorganic phosphate inside said microorganisms, the production of said compounds being stimulated by a limitation of the concentration of inorganic phosphate.

The invention relates more particularly to the aforementioned use of microorganisms producing compounds of interest chosen from metabolites with antibiotic, anticancer, immunosuppressive, anthelmintic, antifungal, herbicidal, insecticidal activity, or enzyme-producing microorganisms.

A more particular subject of the invention is the aforementioned use of strains of microorganisms of the order of Actinomycetals, in particular those producing metabolites with antibiotic activity. As such, the invention relates to the aforementioned use of strains of Bacillus, of

Cephalosporium, and Penicillium producing metabolites with antibiotic activity, and strains of Actinomycetes producing metabolites with antibiotic activity chosen from strains of Streptomyces, Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Corynebacterium, Actinoplanes, Actinomadura, Dactylosporangium, and Mycobacterium.

The invention relates more particularly to the aforementioned use of strains listed in Table I below in the context of stimulation of the production of the antibiotics also listed in this Table I.

Advantageously also, the strains of microorganisms used in the context of the invention are strains of microorganisms producing enzymes.

The production of enzymes such as that of proteases, cellulases, endoglucanases, .beta.-glucanases, hemicellulases, lipases, peroxidases, laccases, .alpha.-amylases, glucoamylases, chitinase, cutinases, agarase, ligninase, nuclase, pectinases, reductases , oxidases, phenoloxidases, ligninases, pullulanases, arabinosidases, mannanases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, pectin methylesterases, cellobiohydrolases, different genera bacteria, transglutaminases Ps Xanthomonas, and by different species of Bacilli, fungi, yeasts or Actinomycetales, are capable of being produced in greater quantity by the microorganisms modified according to the invention, insofar as the synthesis of these enzymes is regulated by inorganic phosphate.

The strains of microorganisms used in the context of the present invention are also chosen from those transformed with the aid of vectors containing sequences coding for the above-mentioned compounds of interest. A more particular subject of the invention is the aforementioned use of strains of microorganisms, including the phoR / phoP operon. or the promoter region of this operon, is modified by addition and / or substitution and / or deletion of at least one nucleotide, so that said operon thus modified no longer codes for the active sensory kinase, and / or for the regulator (or activator) transcription of the genes of the Pho regulon.

The invention relates more particularly to the aforementioned use of strains of microorganisms whose promoter region of the phoR / phoP operon, and / or the phoR gene, and / or the phoP gene, are modified by addition and / or substitution and / or deletion of at least one nucleotide. A more particular subject of the invention is the aforementioned use of strains of microorganisms whose promoter region of the phoR / phoP operon, or at least the phoP gene, are modified by addition and / or substitution and / or deletion of at least one nucleotide.

The invention more particularly still relates to the aforementioned use of strains of microorganisms whose promoter region of the phoR / phoP operon, and / or the phoR gene, and / or the phoP gene, are modified by insertion of a cassette inside this promoter region, or of these genes, with or without deletion of all or part of this promoter region, or of these genes.

The invention relates more particularly to the aforementioned use of strains of microorganisms as defined above comprising, instead of their promoter region of the phoR / phoP operon, and / or their phoR gene, and / or their wild-type phoP gene, a corresponding nucleotide sequence:

- the promoter region of the phoR / phoP operon, and / or the phoR gene, and / or the phoP gene, wild modified by addition and / or substitution and / or deletion of at least one nucleotide, - or the promoter region of the phoR / phoP operon, and / or the phoR gene, and / or the phoP gene, exogenous modified by addition and / or substitution and / or deletion of at least one nucleotide, and originating from a strain of microorganism other than that initially containing the promoter region of the phoR / phoP operon, and / or the phoR gene, and / or the phoP gene, wild. Advantageously, in the case where the abovementioned strains of microorganisms contain a promoter region of the phoR / phoP operon, and / or a phoR gene, and / or a modified exogenous phoP gene, these code for proteins having a percentage of identity with the corresponding wild proteins of at least about 50%. The invention also relates to the use of recombinant nucleotide sequences comprising a phoR / phoP operon, or the promoter region of this operon, originating from microorganisms producing compounds of interest, such as those defined above, said phoR operon / phoP, or its promoter region, being modified by addition and / or substitution and / or deletion of at least one nucleotide, for the -transformation of strains of microorganisms, with a view to obtaining strains of these microorganisms producing more active sensory kinase, and / or transcriptional regulator of the genes of the Pho regulon, for implementing a process for stimulating the production of these compounds of interest by said strains of microorganisms thus modified. The invention relates more particularly to the above-mentioned use of recombinant nucleotide sequences comprising:

- a nucleotide sequence. corresponding to the promoter region of the phoR / phoP operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the nucleotide sequence SEQ ID NO: 1 shown in FIG. 1, or corresponding to a nucleotide sequence derived from the region promoter mentioned above and corresponding to a promoter region of the phoR / phoP operon in microorganisms other than Streptomyces, as mentioned above, said promoter region, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- And / or a nucleotide sequence corresponding to the phoR gene of Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoR gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoR gene of at least about 50%, said derived sequence corresponding to a phoR gene in microorganisms other than Streptomyces, as mentioned above, said phoR gene, or its sequence derivative, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- And / or a nucleotide sequence corresponding to the phoP gene of Streptomyces delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoP gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoP gene of at least about 50%, said derived sequence corresponding to a phoP gene in microorganisms other than Streptomyces, as mentioned above, said phoP gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide. A more particular subject of the invention is the aforementioned use of recombinant nucleotide sequences comprising:

a nucleotide sequence corresponding to the promoter region of the phoR / phoP operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the promoter region mentioned above and corresponding to a promoter region of the phoR / phoP operon in microorganisms other than Streptomyces, as mentioned above, said promoter region, or its derived sequence, being modified by addition and / or substitution and / or deletion of '' at least one nucleotide, - or a nucleotide sequence corresponding to the Streptomyces phoP gene delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoP gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoP gene of at least about 50%, said derived sequence corresponding to a phoP gene in microorganisms other than Streptomyces, as mentioned above, said phoP gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide ,

- and, where appropriate, a nucleotide sequence corresponding to the phoR gene from Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ LD NO: 1, or corresponding to a nucleotide sequence derived from the phoR gene and coding for a protein having a percentage identity with the protein encoded by the aforementioned phoR gene of at least about 50%, said derived sequence corresponding to a phoR gene in microorganisms other than Streptomycess, as mentioned above, said phoR gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide.

A subject of the invention is also the aforementioned use of recombinant nucleotide sequences comprising:

- the promoter region of the phoR / phoP operon of Streptomyces lividans, or a derived sequence, - and / or the phoR gene of Streptomyces lividans, or a derived sequence,

- And / or the phoP gene of Streptomyces lividans, or a derived sequence, the promoter region or the aforementioned genes being modified by insertion of a heterogeneous sequence at a given endogenous or exogenous restriction site in the gene sequence. A more particular subject of the invention is the aforementioned use, of recombinant nucleotide sequences in which the promoter region or the phoR and phoP genes, are modified by insertion at a determined restriction site of said sequence, of a cassette containing a marker, such as a marker for resistance to an antibiotic, in particular apramycin.

The invention relates more particularly to the abovementioned use of recombinant nucleotide sequences chosen from:

the sequence SEQ ID NO: 3 shown in FIG. 2, comprising the interruption of the phoR gene by insertion at the Ncol restriction site of the gene, located at positions 1389 to 1394 of the sequence SEQ ID ΝO: 1, of a cassette containing a apramycin resistance marker, and delimited by the amino acids located at positions 1425 and 3626 - of the sequence SEQ ÏÏ3 ΝO: 3, -

the sequence SEQ ID NO: 4 shown in FIG. 3, comprising the interruption of the phoP gene by insertion at the Notl restriction site of the phoP gene, located at positions 2111 to 2118 of the sequence SEQ ID ΝO: 1, d '' a cassette containing a marker for resistance to apramycin, and delimited by the amino acids located at positions 2148 and 3920 of the sequence SEQ ID 4O: 4,

the sequence SEQ ID ΝO: 5 shown in FIG. 4, comprising the interruption of the phoR and phoP genes by insertion at the above-mentioned Ncol and NotI restriction sites, with deletion of the Ncol / NotI fragment, of a cassette containing a apramycin resistance marker, and delimited by the amino acids located at positions 1425 and 3626 of the sequence SEQ ID ΝO: 5.

The invention also relates to any recombinant nucleotide sequence comprising: - a nucleotide sequence corresponding to the promoter region of the phoR / phoP operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the sequence SEQ ID ΝO: 1, or corresponding to a nucleotide sequence derived from the aforementioned promoter region and corresponding to a promoter region of the phoR / phoP operon in microorganisms other than Streptomyces, chosen from strains of Cephalosporium and Penicillium producing metabolites with antibiotic activity, and strains of Actinomycetes producing metabolites with antibiotic activity selected from the strains of Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Corynebacterium, Actinoplanes, dΑctinomadura, and Dactylosporangium, said region promoter, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- And / or a nucleotide sequence corresponding to the phoR gene of Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoR gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoR gene of at least about 50%, said derived sequence corresponding to a phoR gene in microorganisms other than Streptomyces, as mentioned above, said phoR gene, or its derived sequence , being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- And / or a nucleotide sequence corresponding to the phoP gene of Streptomyces delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoP gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoP gene of at least about 50%, said derived sequence corresponding to a phόP gene in microorganisms other than Streptomyces, as mentioned above, said phoP gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

The invention more particularly relates to any recombinant nucleotide sequence as defined above, comprising:

- A nucleotide sequence corresponding to the promoter region of the operon. phoR / phoP of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the sequence SEQ LD NO: 1, or corresponding to a nucleotide sequence derived from the above-mentioned promoter region and corresponding to a promoter region of the phoR / phoP operon in microorganisms other than Streptomyces, as mentioned above, said promoter region, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- or a nucleotide sequence corresponding to the phoP gene of Streptomyces delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoP gene and coding for a protein having a percentage of identity with the protein encoded by the above-mentioned phoP gene of at least about 50%, said derived sequence corresponding to a phoP gene in microorganisms other than Streptomyces, as mentioned above, said phoP gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- and, where appropriate, a nucleotide sequence corresponding to the phoR gene from Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoR gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoR gene of at least about 50%, said derived sequence corresponding to a phoR gene in microorganisms other than Streptomyces, as mentioned above, said gene phoR, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide.

The subject of the invention is more particularly any recombinant nucleotide sequence as defined above, comprising: ^• - • ^{• •} - ^• ••• ^• -

- And / or the phoP gene of Streptomyces lividans, or a derived sequence, the promoter region or the aforementioned genes being modified by insertion of a heterogeneous sequence at a given endogenous or exogenous restriction site in the gene sequence. The invention relates more particularly to any recombinant nucleotide sequence as defined above, in which the promoter or the phoR and phoP genes, are modified by insertion at a determined restriction site of said sequence, of a cassette containing a marker, such as a marker for resistance to an antibiotic, in particular apramycin. The invention also relates to any recombinant nucleotide sequence as defined above, chosen from:

the sequence SEQ ID NO: 3, comprising the interruption of the phoR gene by insertion at the Ncol restriction site of the gene, located at positions 1389 to 1394 of the sequence SEQ ID ΝO: 1, of a cassette containing a marker resistance to apramycin, and delimited by the amino acids located at positions 1425 and 3626 of the sequence SEQ ID ΝO: 3,

the sequence SEQ ID ΝO: 4, comprising the interruption of the phoP gene by insertion at the Notl restriction site of the phoP gene, located at positions 2111 to 2118 of the sequence SEQ ID ΝO: 1, of a cassette containing a apramycin resistance marker, and bounded by the amino acids located at positions 2148 and 3920 of the sequence SEQ ID

NO: 4,

- the sequence SEQ ID NO: 5, comprising the interruption of the phoR and phoP genes by insertion at the Ncol and NotI restriction sites mentioned above, with deletion of the Ncol / Notl fragment, of a cassette containing a resistance marker to 'apramycin, and delimited by the amino acids located at positions 1425 and 3626 of the sequence SEQ ID ΝO:

5.

The subject of the invention is also any vector, in particular any plasmid, if applicable with heat-sensitive replication, containing a recombinant nucleotide sequence as defined above.

The invention also relates to the strains of microorganisms producing metabolites or enzymes, said microorganisms being modified so that the phoR / phoP operon coding for a two-component system, namely a sensory kinase coded by phoR, and a pavphoP coded regulator, responsible for activating the Pho regulon comprising a set of genes involved in the biosynthesis of phosphates in said microorganisms, is inactivated.

The invention relates more particularly to the strains of modified microorganisms as defined above, said strains being producers of compounds of interest as defined above. A more particular subject of the invention is the strains of modified microorganisms as defined above, characterized in that they are chosen from strains of Cephalosporium and Penicillium producing metabolites with antibiotic activity, and strains of Actinomycetes producing metabolites with antibiotic activity chosen from the strains of Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Corynebacterium, Actinoplanes, Actinomadura, and

Dactylosporangium, such as those defined in Table I.

The invention also relates to the strains of modified microorganisms as defined above, said strains producing enzymes, and being advantageously chosen from the strains mentioned above. A more particular subject of the invention is the strains of microorganisms mentioned above, as obtained by transformation of said microorganisms with a vector defined above.

The subject of the invention is also a method for preparing strains of microorganisms as defined above, characterized in that it comprises the following steps: - if necessary, the cloning of the phoR / phoP operon, or of the promoter region of this operon, of the strain of microorganism which it is desired to modify, and the amplification of a nucleotide sequence as defined above containing the phoR / phoP operon, or its promoter region, using two oligonucleotide primers chosen from known sequences located in this operon or its promoter region, or located on either side of this operon or its promoter region,

- modification of the nucleotide sequence containing this operon or its promoter region, such as that obtained during the previous step, by addition and / or substitution and / or deletion of at least one nucleotide, - transformation of strains of microorganisms using vectors containing the recombinant nucleotide sequence obtained in the previous step,

selection of the strains of transformed microorganisms having integrated into their genome the above-mentioned recombinant nucleotide sequence in place of the nucleotide sequence corresponding to the wild-type operon or promoter region. According to a particularly advantageous embodiment of the method of the invention, the nucleotide sequence as defined above containing the phoR / phoP operon, or its promoter region, is modified by insertion of a cassette comprising a marker, in particular a antibiotic resistance marker, in order to select the modified strains obtained. The recombinant sequence thus obtained is then cloned into an appropriate vector, in particular into a plasmid capable of interspecific transfer and capable or not of replicating in a heat-sensitive manner in the strain of the microorganism to be modified. After transformation of the strains with the abovementioned vector, the selection of the modified strains is done directly by observation of the presence or not of the marker carried by the cassette in the genome of the modified strains, after passage or not at high temperature depending on whether the plasmid is thermosensitive replication, or is not replicative.

According to another particularly advantageous embodiment of the method of the invention, the nucleotide sequence as defined above containing the operon phoR / phoP, or its promoter region, is modified by addition or deletion of at least one nucleotide. In this case, it is preferable to use a thermosensitive replication vector, and, after passing to high temperature, the colonies having lost the plasmid are identified (colonies sensitive to the antibiotic to which the plasmid usually confers resistance) and who overproduce the antibiotic on an appropriate medium. These colonies can be identified on a box of appropriate culture medium by growing them in the presence of a strain sensitive to the antibiotic produced. As a variant, this latter process can also be carried out in two stages. First, the nucleotide sequence as defined above containing the operon phoR / phoP, or its promoter region, is modified by insertion of a cassette mentioned above conferring resistance to an antibiotic, then this sequence modified by insertion is replaced by a deleted version of this sequence. In this case, the colonies once again become sensitive to the antibiotic to which the cassette usually confers resistance.

A subject of the invention is also a process for stimulating the production of metabolites, in particular metabolites with antibiotic or other activity, or enzymes, by microorganisms used for the production of these compounds, said process comprising culture of modified microorganism strains as defined above, in an appropriate culture medium.

Advantageously, the above-mentioned process of the invention is characterized in that the culture is carried out in a rich medium (namely a medium containing amino acids as source of carbon and nitrogen) with an osmotic pressure equivalent to that imparted by 103 g / 1 of sucrose and without the addition of exogenous inorganic phosphate.

Table I List of organisms producing antibiotics

I) Aminocyclitol producing organisms

ANTIBIOTIC ORGANISM

Bacillus (various species) Aminocyclitols Micromonospora (various species) Gentamycins Saccharopolyspora (various species) Aminocyclitols Streptomyces albogriseolus Neomycines albus var. metamycinus Metamycin aquacanus N-methyl hygromycin B atrofaciens Hygromycines bikiniensis Streptomycin bluensis var. bluensis Bluensomycin canus Ribosyl paromamine catenulae Catenulin chrestomyceticus aminosidine crystallinus hygromycin A erythrochromogenes streptomycin var. narutoensis eurocidicus A16316-C fradiae hybrimycins and neomycine fradiae var. italians aminosidine galbus streptomycin griseus streptomycin griseoflavus MA 1267 hofuensis seldomycin complex hygroscopicus hygromycinse hygroscopicus forma leucanicidine and hygrolidine glebosus glebomycin hygroscopicus var. limoneus validamycins hygroscopicus var. sa ^' gamiensis spectinomycin kanamyceticus kanamycin A and B kasuqaensis kasugamycins kasugaspinus kasugamycins lavendulae neomycine lividus lividomycines mashuensis streptomycin microsporeus SF-767 netropsis LL-AM31 nomororinoborinines cellulophilus destomycin A po 'oleήsis ^' '^' streptomycin rameus streptomycin ribosidificus SF733 rimofaciens destomycin A rimosus forma paromomycinus paromomycins, catenulin spectabilis spectinomycin tenebrarius tobramycin and apramycin streptycin Streptycin

II) Ansamycin producing organisms

ANTIBIOTIC ORGANISM

Micromonospora (various species) ansamycins Nocardia mediterranei rifamycin Streptomyces collinus ansatrienes, napthomycins diastochromogenes ansatrienes, napthomycins galbus subsp. griseosporeus napthomycin B hygroscopicus herbimycin hygroscopicus var. geldanus geldamycin var. nova nigellus 21 -hydroxy-25-demethyl

25-methylthioproto-streptovaricin rishiriensis mycotrienes sp. E / 784 actamycin and mycotriene sp. E88 mycotrienes spectabilis streptovaricins tolypophorous tolypomycin

III) Organisms producing Anthracycine and Quinone

ANTIBIOTIC ORGANISM

Streptomyces caespitosus mitomycins A, B, and C coèlicolor actinorhodine coeruleorubidicus daunomycin cyaneus ditrisarubicin flavogriseus cyanocycline A galilaeus aclacinomycin A, auramycins, sulfurmycins lusitanus napthyridinomycin peuceticus daunomycin, adriamycin violochromoqenes arugomycin

IV) Beta Lactame producing organisms

ANTIBIOTIC ORGANISM

Cephalosporium (various species) beta-lactams Nocardia lactamadurans cephamycin C Penicillium (various species) beta-lactams Streptomyces antibioticus clavulanic acid argenteolus asparenomycinë A, MM 4550, MM 13902 cattleya thienamycine chartreusis SF 1623, cephamycin -32413-I, cephamycin C, A16886A, clavulanic acid, and other clavames fimbriatus cephamycin A and B flavovirens MM 4550 and MM 13902 flavus MM 4550 and MM 13902 fulvoviridis MM 4550 and MM 13902 griseus cephamycin A and B halstedi cephamycin A and B halstedi cephamycin A and B C2081X, cephamycin A, B hygroscopicus deacetoxycephalosporin C lipmanii penicillin N, 7-methoxycephalosporin C, A16884, MM 4550, and MM 13902 olivaceus (MM 17880) epithienamycin F, MM 4550, and MM 13902 panayensis C2081ycin Aycidin B20 rochei cephamycin A and B sioyaensis MM 4550 and MM 13902 sp. OA-6129 OA-6129A sp. KC-6643 carpetimycin A tokunomensis asparenomycinë A viridochromogenes cephamycin A and B wadayamensis WS-3442-D

V) Producing organisms of Macrolide, Lincosamide, and Streptogramin

ANTIBIOTIC ORGANISM

Micromonospora rosaria rosaramicin Streptomyces albireticuli carbomycin albogriseolus mikonomycin albus albomycetin albus var. coilmyceticus coleimycine ambofaciens spiramycin, foromaçidine D antibioticus oleandomycin avermitilis avermectins bikiniensis chalcomycin bruneogriseus albocycline caelestis Ml 88 celesticetine cinerochromogenes cineromycine B cirratus cirramycine DeltaE deltamycines djakartensis niddamycin erythreus erythromycins eurocidicus methymycin eurythermus angolamycin fasciculus amaromycin felleus argomycine and picromycin fimbriatus amaromycin flavochromogenes amaromycin, and shincomycines fradiae tylosin fungicidicus NA-181 fungicidicus var. espinomyceticus espinomycetines furdicidicus mydecamycin goshikiensis bandamycin griseofaciens PA133A and B griseoβavus acumycin griseofuscus buridline griseolus griseomycin griseospiralis relomycin griseus borrelidine griseus ssp. sulphurus bafilomycins halstedi carbomycin, leucanicidine hygroscopicus tylosin hygroscopicus subsp aureolacrimosus milbemycines kitastoensis leucomycin A.sub.3, and josamycin lavendulae aldgamycin lincolnensis lincomycin loidensom vernycin maizeus mgramycme mycarofaciens acetyl-leukomycin, and espinomycin narbonensis josamycin and narbomycin narbonensis var. josamyceticus leucomycin A, josamycin olivochromogenes oleandomycin platensis platenomycin rimosus tylosin and neutramycin rochei lankacidine and borrelidine rochei var. volubilis T2636 roseochromogenes albocyline roseocitreus albocycline spinichromoenes var.suragaoensis kujimycins tendae carbomycin thermotolerans carbomycin venezuelae methymycins violaceoniger lankacidines, lankamycin

VI) Streptomyces species producing various antibiotics a) Amino acid analogues

Streptomyces sp. Cycloserine b) Containing cyclopentane rings

Streptomyces coelicolor methylenomycin A erythrochromogenes sarkomycin violaceoruber methylenomycin A c) Containing nitrogen

Streptomyces venezuelae chlorampheήicol d) Polyenes

Streptomyces griseus candicidin nodosus amphotericin B noursei nystatin e) Tetracyclines

Streptomyces aureofaciens tetracycline, chlortetra-cycline, demethyltetra-cycline, and demethylchlortetra-cycline rimosus oxytetracycline

VII) Nucleoside producing organisms

ANTIBIOTIC ORGANISM

Corynebacterium michiganese pv. rathayi tunicamycin analogs Nocardia candidus pyrazofurine Streptomyces antibioticus ara-A chartreusis tunicamycin griseoflavus var. streptoviridans thuringiensis griseolus sinefungin

VIII) Peptide producing organisms

ANTIBIOTIC ORGANISM

Actinoplanes missouriensis actaplanine teichomyceticus teicoplanin Bacillus (various species) bacitracin, polymixin, and colistin

Nocardia candidus A-35512 and avoparcin lurida ristocetine orientalis vancomycin Streptomyces antibioticus actinomycin aureus thiostreptone canus amphomycin eburosporeus LL-AM374 haranomachiensis vancomycin pristinaespiralis pristinamycin roseistorus A109 virginensia10Aginia34 virginensine10

IX) Antibiotic producing organisms

ANTIBIOTIC ORGANISM

Actinomadura (various species) various polyethers Dactylosporangium (various species) various polyethers Nocardia (various species) various polyethers Streptomyces albus A204, A28695A and B, and salinomycin aureofaciens narasine cacaoi var. asoensis lysocellme chartreusis A23187 cinnamonensis monensine conglobatus ionpmycine eurocidicus var. asterocidicus laidlomycin flaveolus CP38936 gallinarius RP 30504 griseus grisorixine hygroscopicus A218, emericide, DE3936,

A120A.A28695A and B, etheromycin, dianemycin lasaliensis lasalocide longwoodensis lysocelline mutabilis S-1173a ribosidificus lonomycin violaceoniger nigericin Streptoverticillium (various species) various polyethers The invention will be further detailed with the aid of the examples below.

Example 1

Inactivation of the Pho regulon of Streptomyces lividans

As was specified above, a situation of limitation in Pi triggers the expression of the regulator Pho, a set of genes whose expression makes it possible to delay the onset of a state of deficiency in Pi. If this adaptive response does not may take place, a situation of Pi deficiency follows. Deficiency in Pi leads to the triggering of adaptive processes different from those induced by a limitation in Pi and it is these still poorly understood processes which lead, in Streptomyces, to initiation of biosynthesis of antibiotics. -

By interrupting the two genes ^' phoR (kinase) and phoP (regulator) which, in Streptomyces lividans, probably constitute the two-component system responsible for activating the expression of all the genes of the Pho regulon of Streptomyces, The inventors early created a situation of severe Pi deficiency. These mutants massively overproduce antibiotics.

A) Principle of the method:

The inventors have isolated and interrupted the two genes phoR (kinase) and phoP (regulator) which, in Streptomyces lividans, probably constitute the two-component system responsible for activating the expression of all the genes of the Pho regulon. of Streptomyces. The phoR and phoP mutants constructed are incapable of inducing the expression of the various Pi recovery systems mentioned above, they are therefore precociously and severely deficient in Pi and massively overproduce antibiotics on all the media tested.

a) Characterization of the phoR / phoP genes: 1 In silico: Two-component systems related to phoR (kinase) couples

/ phoP (regulator) of Bacillus subtilis and phoR (kinase) / phoB (regulator) of Escherichia coli were sought in the fully sequenced genome of Streptomyces coelicolor (a species very close to S. lividans but which is much more easily manipulated by classical molecular biology techniques than the latter) using BLAST software. Several putative proteins of S. coelicolor have been shown to have significant homology with these proteins. Similarity searches for these S. coelicolor proteins were then carried out in the genomes of B. subtilis and E. coli (BLAST system back) and revealed that only five kinase / regulator pairs of S. coelicolor exhibited significant similarity. with the initial couples.

2_In vivo: The genes encoding these proteins were amplified by PCR from the chromosomal DNA of S. coelicolor and served as a probe to detect by "Northern blot" techniques the corresponding transcripts. RNAs had been prepared under conditions of abundance or limitation in Pi in a wild and mutant context for the ppk gene. Indeed, we knew that the interruption of the ppk gene led to an early limitation in Pi and therefore probably to the triggering of the expression of the Pho regulon. Out of the five candidates, only one exhibited the type of regulation expected, ie an increase in its level of expression under conditions of limitation in Pi. This increase was much greater in a mutated context for ppk. In Streptomyces, this pair has been called phoR (kinase) / pboP (regulator).

b) Cloning of the phoRIphoP operon:

The protocol for cloning and interrupting the phoR andphoP genes described below is applicable to all strains of Streptomyces and to other bacterial species of industrial interest. lA fragment of S. lividans of about 3.2 kb containing the operon phoR-phoP and 500bp upstream and downstream of the latter was amplified using two oligonucleotides suitably chosen from the sequence of the equivalent region of S. coelicolor .

2 A cassette conferring resistance to apramycin was cloned into a NotI site located in the center of the phoP gene and brought to a clean edge using the Klenow fragment so as to interrupt it. An excisable cassette conferring resistance to apramycin was cloned into the NcoI site located at the center of the phoR gene and brought to a free edge using the Klenow fragment, so as to interrupt the latter. It should be noted that, for scientific reasons, we used to interrupt phoR an excisable cassette because phoR and phoP being co-transcribed, the interruption dephoR has a polar effect on the expression dephoP.

3 The fragments carrying the interrupted phoR and phoP genes were cloned into pGM160 vector with heat-sensitive replication. These vectors were introduced into S. lividans and a replacement of wild phoR and phoP genes by their interrupted copy was carried out by a standard protocol. Given the strong sequence homology detected by Southern between the phoR / phoP genes of S. lividans and that of various strains of Streptomyces producing antibiotics, it is not necessary to re-clone the corresponding genes to construct mutants of interruption in these species. In fact, the phoR / phoP genes of the different strains of Streptomyces are sufficiently similar to that of S. lividans so that the double recombination between homologous sequences, necessary for the creation of the mutant by gene replacement, is possible.

We have shown that a strain of Streptomyces lividans in which the phoR and / or phoP genes were interrupted produced much more antibiotics than the wild-type strain on all the media tested. The phoP gene codes for the transcriptional activator of the Pho regulon of Streptomyces and the latter requires phosphorylation by phoR to fulfill its activating function. In a mutant of interruption of the phoR and / or phoP genes, the induction of the genes necessary for the adaptation of the bacteria to a limiting concentration in Pi does not take place. The latter is therefore precociously and severely deficient in Pi and there follows a massive induction of the production of antibiotics.

B) Detailed experimental method

Cloning and interruption of the phoP, phoR genes and the phoR-phoP operon from Streptomyces lividans:

- An operon encoding a two-component system consisting of a PhoR sensory kinase (427 AA) and a PhoP regulator (224 AA) having 67% identity with their respective counterparts PhoR and PhoB of E. coli was found in the fully sequenced genome of Streptomyces coelicolor, a closely related species of S. lividans.

- two unique sites Ncol and Notl (enzymes giving 5 'outgoing ends) located respectively towards the 3' end of the phoR gene and towards the 5 'end of the phoP gene have been identified (they are underlined in italics and in bold on the sequence SΕQ ID ΝO: 1 of FIG. 1) and considered to be well located to serve as insertion sites for an interruption cassette.

- Two oligos A and B (in bold on the sequence of the figurel) were then designed so as to n aτmnnp1liiffiife.rr n paarr P PfCTRR t toonuttee l laa r réégriioonn .. - The fragment amplified using these oligos was then cloned into the SmaI site of the plasmid pIJ2925 (Janssen and Bibb, 1993) to give pSG1.

- pSGl was digested with Ncol (unique site in the plasmid). The Ncol site was brought to the clear edge by the Klenow fragment and the Qaac exisable cassette which confers resistance to apramycin (Blondelet-Rouault et al., 1997) was cloned into this site, in the form of an EcoRY fragment to interrupt phoR and give pSG2.

- pSGl was digested with NotI (unique site in the plasmid). The Notl site was brought to the clear edge by the Klenow fragment and the Ωααc cassette which confers resistance to apramycin (Blondelet-Rouault et al., 1997) was cloned into this site, in the form of a Smal fragment to interrupt phoP and give pSG3.

- pSGl was digested with Notl and Ncol. The Notl-Ncol fragment from the insert was eliminated and the Ncol and Notl sites were brought to the clear edge by the deleted Klenow fragment and the Ωaac cassette which confers resistance to apramycin (Blondelet-Rouault et a, 1997) was cloned as a SmaI fragment in place of the deleted Ncol-NotI fragment to jointly interrupt phoR andphoP. and to give pSG4.

- The insert of pSG2 was cloned in the form of an EcoRI-Xbal fragment (brought to the free edge by the Klenow fragment) in the BamΗI site (brought to the free edge by the fragment

Klenow) of pGM160, to give pSG7.

_. pGM160 is a E. coli I S. lividans shuttle vector with heat-sensitive replication in S. j lividans. It carries the bla and tsr genes which confer resistance to ampicillin in E. coli and to the nosiheptide in S. lividans respectively, as well as the aacCl gene which confers resistance to apramycin in E. coli and S. lividans

- The inserts of pSG3 and pSG4 were transferred in the form of BglI fragments into the BamHI site of pGM160 (Muth et al, 1989) to give the plasmids pSG5 and pSG6 respectively. - These plasmids pSG5, pSG6 and pSG7 were introduced into S. lividans by the usual transformation techniques (Hopwood et al, 1985). The transformed colomas were selected in the presence of nosiheptide.

- In order to replace wild phoR and phoP genes with their interrupted alleles, colonies independent of S. lividans TK24 containing pSG5, pSG6 or pSG7 and therefore resistant to nosiheptide were ground and grown independently for 24 h to 30 ° C in 2 ml of TSB medium (Hopwood et al, 1985 containing apramycin at 25 μg / ml.

- The mycelium thus produced was then potterized and the ground material was used to inoculate at low density 2 ml of TSB medium containing apramycin at 25 μg / ml.

- The cultures were grown at 41 ° C for 48 hours to allow the elimination of the replicative plasmid.

- The mycelium was then harvested, ground, and sonicated so as to have mycelial fragments corresponding to a single cell. The latter were spread in dilution on dishes of HT agar medium (Hopwood et al, 1985) containing apramycin at 50 μg / ml in order to have well separated colonies.

- The spore-forming colonies were then replicated on HT medium and HT medium containing nosiheptide at 20 μg / ml in order to identify the colonies sensitive to nosiheptide.

About 15% of apramycin-resistant colonies were susceptible to nosiheptide. The latter having lost the replicative plasmid were likely to be the expected double recombinants.

- The overproduction of antibiotic, actinorhodin, by the mutated strain for phoP is observed visually (Figure 5); indeed in liquid medium, the cultures of S. lividans mutated for phoP appear much darker (Figure 5 on the left) than those of the wild strain (Figure 5 on the right) due to the accentuated production of a blue compound, l 'actinorhodin.

- Alternatively, it is possible to take carrots of agar nutrient medium used for the growth of S. lividans, mutated and not mutated, to dissolve them in water by heating to 90 ° C., then measuring the absorbance at 600 nm of the solutions thus obtained in order to quantify the actinorhodin having diffused in the agar medium.

Example 2 Inactivation of the Pho Regon of Streptomyces Ambofaciens

Streptomyces ambofaciens produces at least two antibiotics, spiramycin and congocidine. In order to analyze in this strain the effects of the inactivation of the operon Pho, the phoR and phoP genes of S. ambofaciens were inactivated, according to the protocol described in Example 1, using the plasmids pSG5 and pSG6.

The mutated strain of S. ambofaciens obtained is tested for its capacity for overproduction of spiramycin and congocidine relative to the corresponding wild strain.

The overproduction of spiramycin is demonstrated by a biological assay, as described in Gourmelen et al. (1998) Antimicrob. chemotherapies. 42: 2612-2619.

Briefly, the presence of spiramaycine in the culture medium of S. ambofaciens is measured by taking a sample of the culture medium with which a disk of absorbent paper of the Whatman type is soaked. This disk is then placed on a culture dish. on which an indicator bacterial strain sensitive to spiramycin and resistant to congocidine was previously seeded. The diameter of bacterial growth inhibition observed around the paper disc, indicative of the amount of antibiotic present in the culture medium, is then measured.

The presence of congocidine is similarly measured, using an indicator bacterial strain sensitive to congocidine and resistant to spiramycin.

Legend of figures

Figure 1: Nucleotide sequence SEQ -D NO: 1 of a fragment of 3541bp from S. coelicolor containing the operon phoR phoP encoding the sensory kinase and the regulator of the Phosphate regulon. The start and end translation codons are in italics and the aa sequences are provided. The oligonucleotides A and B used for the PCR amplification of the DNA fragment which was used to interrupt the phoR phoP operon of S. lividans are in bold. The Notl and Ncol sites are underlined in bold and in italics. The Pstl and BspEl sites are underlined and in bold.

Figure 2: nucleotide sequence SEQ ID ΝO: 3: Mutant phoRr.Ωaac

Figure 3: nucleotide sequence SEQ ID ΝO: 4: Mutant phoPr.Ωaac

Figure 4: nucleotide sequence SEQ ID ΝO: 5: Double mutant phoR / phoPr.Ωaac

Figure 5: Photograph of a culture of S. lividans mutated for phoP (left) and of a culture of wild S. lividans (right). The culture of S. lividans mutated according to the invention (on the left) appears much darker due to the overproduction of a blue antibiotic, actinorhodine.

REFERENCES:

1. Akiyama, M., E. Crooke, and A. Kornberg. 1992. The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein. J Biol Chem. 267 (31): 22556-61.

2. Blondelet-Rouault, M. H., J. eiser, A. Lebrihi, P. Branny, and J. L. Pernodet. 1997. Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces. Uncomfortable. 190 (2): 315-7.

3. Hopwood DA, B. M., Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM and schrempf H. 1985. Genetic Manipulation of Streptomyces: a laboratory Manual. , Norwich: John Innés Foundation.

4. Janssen, G. R ,, and M. J. Bibb. 1993. Derivatives of pUC18 that have BglII sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of Escherichia coli colonies. Uncomfortable. 124 (1): 133-4. 5. Muth, G., B. Nussbaumer, W. Wohlleben, and A. Pϋlher. 1989. A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes. Mol Gen Broom. 219: 341-348.

Claims

1. Use of strains of microorganisms producing compounds of interest, including the operon phoR / phoP encoding a two-component system, namely a sensory kinase encoded by phoR, and a regulator encoded by phoP, responsible for activation of the Pho regulon of said microorganisms comprising a set of genes involved in the adaptive response to a phosphate limitation in said microorganisms, is inactivated, for the implementation of a process for stimulating the production of these compounds d interest by said transformed strains.

2. Use according to claim 1, of strains of microorganisms producing compounds. of interest. chosen from among. metabolites with antibiotic, anticancer, immunosuppressive, anthelmintic, antifungal, herbicidal, insecticidal, or enzymatic activity.

3. Use according to claim 1 or 2, strains of Bacillus, Cephalosporium, and Penicillium producing metabolites with antibiotic activity, and strains of Actinomycetes producing metabolites with antibiotic activity chosen from strains of Streptomyces, of Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Corynebacterium, to? Actinoplanes, Actinomadura, Dactylosporangium, and Mycobacterium.

4. Use according to one of claims 1 to 3, strains of microorganisms whose operon phoR / phoP, or the promoter region of this operon, is modified by addition and / or substitution and / or deletion of at minus one nucleotide, so that said operon thus modified no longer codes for the active sensory kinase, and / or for the transcriptional regulator (or activator) of the genes of the Pho regulon.

5. Use according to one of claims 1 to 4, strains of microorganisms whose promoter region of the phoR / phoP operon, and / or the phoR gene, and / or the phoP gene, are modified by addition and / or substitution and / or deletion of at least one.nucleotide.

6. Use according to one of claims 1 to 5, strains of microorganisms whose promoter region of the phoR / phoP operon, or at least the phoP gene, are modified by addition and / or substitution and / or deletion at least one nucleotide.

7. Use according to one of claims 1 to 6, strains of microorganisms whose promoter region of the phoR / phoP operon, and / or the phoR gene, and / or the phoP gene, are modified by insertion of a cassette inside this promoter region, or of these genes, with or without deletion of all or part of this promoter region, or of these genes.

8. Use of recombinant nucleotide sequences comprising:

a nucleotide sequence corresponding to the promoter region of the phoR / phoP operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the nucleotide sequence SEQ ID NO: 1 shown in FIG. 1, or corresponding to a derived nucleotide sequence of the aforementioned promoter region and corresponding to a region. promoter ..of the phoR / phoP operon. at. of. microorganisms other than •

Streptomyces, as mentioned in claim 3, said promoter region, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- And / or a nucleotide sequence corresponding to the phoR gene of Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoR gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoR gene of at least about 50%, said derived sequence corresponding to a phoR gene in microorganisms other than Streptomyces, as mentioned in claim 3, said phoR gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

_. - And / or a nucleotide sequence corresponding to the phoP gene of Streptomyces delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoP gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoP gene of at least about 50%, said derived sequence corresponding to a phoP gene in microorganisms other than Streptomyces, as mentioned in claim 3, said phoP gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide, for the transformation of strains of microorganisms producing compounds of interest, with a view to obtaining strains of these micro -organisms no longer producing the sensory kinase and / or the regulator coded by the phoR / phoP operon, for the implementation a method of stimulating the production of these compounds of interest by said strains of microorganisms thus modified.

9. Use according to claim 8, of recombinant nucleotide sequences comprising:

a nucleotide sequence corresponding to the promoter region of the phoR / phoP operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the sequence SEQ LD NO: 1, or corresponding to a nucleotide sequence derived from the promoter region mentioned above and corresponding to a promoter region of the phoR / phoP operon in microorganisms other than Streptomyces, as mentioned in claim 3, said promoter region, or its derived sequence, being modified by addition and / or substitution and or. deletion of at least one nucleotide,.

- or a nucleotide sequence corresponding to the phoP gene of Streptomyces delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoP gene and coding for a protein having a percentage of identity with the protein coded by the above-mentioned phoP gene of at least about 50%, said derived sequence corresponding to a phoP gene in microorganisms other than Streptomyces, as mentioned in claim 3, said phoP gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- And, if necessary, a nucleotide sequence corresponding to the phoR gene from Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoR gene and coding for, a protein having a percentage identity with the protein encoded by the aforementioned phoR gene of at least about 50%, said derived sequence corresponding to a phoR gene in microorganisms other than Streptomyces, as mentioned in claim 3, said phoR gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide.

10. Use according to claim 9 or 8, of recombinant nucleotide sequences comprising:

- the promoter region of the phoR / phoP operon of Streptomyces lividans, or a derived sequence,

- and / or the phoR gene from Streptomyces lividans, or a derived sequence, - And / or the phoP gene of Streptomyces lividans, or a derived sequence, the promoter region or the aforementioned genes being modified by insertion of a heterogeneous sequence at a given endogenous or exogenous restriction site in the gene sequence.

11. Use according to claim 10, of recombinant nucleotide sequences in which the promoter region or the phoR and phoP genes, are modified by insertion at a determined restriction site of said sequence, of a cassette containing a marker, such as '' an antibiotic resistance marker, especially apramycin.

12. Use according to claim 10 or 11, of recombinant nucleotide sequences chosen from:

the nucleotide sequence SEQ ID NO: 3 shown in FIG. 2, comprising the interruption of the phoR gene by insertion at the Ncol restriction site of the gene, located at positions 1389 to 1394 of the sequence SEQ ID ΝO: 1, d 'a cassette containing a marker for resistance to apramycin, and delimited by the amino acids located at positions 1425 and 3626 of the sequence SEQ ID ΝO: 3,

the nucleotide sequence SEQ LD ΝO: 4 shown in FIG. 3, comprising the interruption of the phoP gene by insertion at the Notl restriction site of the phoP gene, located at positions 2111 to 2118 of the sequence SEQ ID ΝO: 1, a cassette containing a apramycin resistance marker, and delimited by the amino acids located at positions 2148 and 3920 of the sequence SEQ ID ΝO: 4,

the nucleotide sequence SEQ ID ΝO: 5 shown in FIG. 4, comprising the interruption of the phoR and phoP genes by insertion at the above-mentioned Ncol and NotI restriction sites, with deletion of the Ncol / NotI fragment, of a cassette containing a marker for resistance to apramycin, and delimited by the amino acids located at positions 1425 and 3626 of the sequence SEQ ID ΝO: 5.

13. Recombinant nucleotide sequence comprising: - a nucleotide sequence corresponding to the promoter region of the phoR / phoP operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the sequence SEQ ID ΝO: 1, or corresponding to a nucleotide sequence derived from the aforementioned promoter region and corresponding to a promoter region of the phoR / phoP operon in microorganisms other than Streptomyces, chosen from strains of Cephalosporium and Penicillium producing metabolites with antibiotic activity, and strains of Actinomycetes producing metabolites with antibiotic activity chosen from strains of Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Corynebacterium, Actinoplanes, and Actinomadura, and Dactylosporangium, said promoter region, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- And / or a nucleotide sequence corresponding to the phoR gene of Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoR gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoR gene of at least about 50%, said derived sequence corresponding to a phoR gene in microorganisms, other than. Streptomyces,. As mentioned above, said phoR _y gene. or its derivative sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide, - and / or a nucleotide sequence corresponding to the phoP gene of Streptomyces delimited by the nucleotides located in positions 1917 to 2587 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoP gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoP gene of at least about 50%, said derived sequence corresponding to a phoP gene in microorganisms other than Streptomyces, as mentioned above, said phoP gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

14. Recombinant nucleotide sequence according to claim 13 comprising: - a nucleotide sequence corresponding to the promoter region of the phoR / phoP operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the above-mentioned promoter region and corresponding to a promoter region of the phoR / phoP operon in microorganisms other than Streptomyces, as mentioned in claim 3, said promoter region, or its derivative sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- or a nucleotide sequence corresponding to the Streptomyces phoP gene delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO: 1, or corresponding to a nucleotide sequence derived from the phoP gene and coding for a protein having a percentage identity with the protein encoded by the above-mentioned phoP gene of at least about 50%, said derived sequence corresponding to a phoP gene in microorganisms other than Streptomyces, as mentioned in claim 13, said phoP gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide,

- and, where appropriate, a nucleotide sequence corresponding to the phoR gene from Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ DD NO: 1, or corresponding to a nucleotide sequence derived from the phoR gene and coding for a protein having a percentage identity with the protein encoded by the aforementioned phoR gene of at least about 50%, said derived sequence corresponding to a phoR gene in microorganisms other than Streptomyces, as mentioned in the reyendicatiqn, 13., . _said,. geneE> boJ. ,, or, its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide.

15. Recombinant nucleotide sequence according to claim 13 or 14, comprising:

- and / or the phoR gene from Streptomyces lividans, or a derived sequence,

- And / or the phoP gene of Streptomyces lividans, or a derived sequence, the promoter region or the aforementioned genes being modified by insertion of a heterogeneous sequence at a given endogenous or exogenous restriction site in the gene sequence.

16. Recombinant nucleotide sequence according to claim 15, in which the promoter or the phoR and phoP genes are modified by insertion, at a determined restriction site of said sequence, of a cassette containing a marker, such as a marker for resistance to an antibiotic, in particular apramycin.

17. Recombinant nucleotide sequence according to claim 15 or 16, chosen from:

the sequence SEQ ID NO: 3, comprising the interruption of the phoR gene by insertion at the Ncol restriction site of the gene, located at positions 1389 to 1394 of the sequence SEQ ID ΝO: 1, of a cassette containing a marker resistance to apramycin, and delimited by the amino acids located at positions 1425 and 3626 of the sequence SEQ ID NO:

3

the sequence SEQ ID NO: 4, comprising the interruption of the phoP gene by insertion at the NotI restriction site of the phoP gene, located at positions 2111 to 2118 of the sequence SEQ ID ΝO: 1, of a cassette containing a apramycin resistance marker, and delimited by the amino acids located at positions 2148 and 3920 of the sequence SEQ ID ΝQ: 4,

- the sequence SEQ ID NO: 5, comprising the interruption of the phoR and phoP genes by insertion at the Ncol and NotI restriction sites mentioned above, with deletion of the Ncol / Notl fragment, of a cassette containing a resistance marker to 'apramycin, and delimited by the amino acids located at positions 1425 and 3626 of the sequence SEQ ID ΝO: 5.

18. Vector containing a recombinant nucleotide sequence according to one of claims 13 to 17.

19. Strains of microorganisms producing compounds of interest, said microorganisms being modified so that the operon phoR / phoP encoding a two-component system, namely a sensory kinase encoded by phoR, and a regulator encoded by phoP, responsible for activating the Pho regulon comprising a set of genes involved in the biosynthesis of phosphates in said microorganisms, is inactivated.

20. Strains of microorganisms according to claim 19, said strains producing compounds of interest defined in claim 2. j

21. Strains of microorganisms according to claim 19 or 20, characterized in that they are chosen from strains of Cephalosporium and Penicillium producing metabolites with antibiotic activity, and strains of Actinomycetes producing metabolites with antibiotic activity chosen from strains of Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Corynebacterium, Actinoplanes, Actinomadura, and Dactylosporangium.

22. Strains of microorganisms according to one of claims 19 to 21, as obtained by transformation of said microorganisms with a vector according to claim 18.

23. A method of stimulating the production of compounds of interest, in particular of the compounds defined in claim 2, by microorganisms usually used for the production of these compounds, said method comprising culturing strains of these micro- transformed organisms as defined in one of claims 19 to 22 in an appropriate culture medium.