FR2848567A1 - Using microorganisms with modified phoK/phoR operons for production of metabolites, e.g. antibiotics, provides increased yields by inducing deficiency of inorganic phosphate - Google Patents

Abstract

Use of a recombinant nucleotide sequence (I) containing at least one modified promoter region of the phoK/phoR operon of Streptomyces and/or modified phoK or phoR genes for transformation of microorganisms that produce compounds of interest (II), is new. Use of a recombinant nucleotide sequence (I) containing at least one modified promoter region of the phoK/phoR operon of Streptomyces and/or modified phoK or phoR genes for transformation of microorganisms that produce compounds of interest (II). The promoter region is between positions 420 and 630 of a 3540 bp sequence (1; reproduced); the phoK gene is between positions 632 and 1909 and the phoR gene between positions 1917 and 2587, and all are modified by addition, substitution and/or deletion of at least one nucleotide. The transformed strains no longer produce the kinase and/or regulator encoded by the phoK/phoR operon and are used to stimulate production of (II). Independent claims are also included for: (1) (I) as new sequences; (2) vector that contains (I); and (3) strains of (II)-producing microorganisms transformed with the vector of (2).

Description

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STRAINS OF MICROORGANISMS INCLUDING OPERON PHOK / PHOR FOR THE IMPLEMENTATION OF A PROCESS FOR THE OVERPRODUCTION OF METABOLITES, IN PARTICULAR ANTIBIOTIC OR ENZYMATIC ACTIVITY
The present invention relates to the use of modified microorganism strains such 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 in the context 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, the industrialists thus obtain strains hyper-productive. The latter are often "poly-mutated" but the affected genes are not known.

 This traditional method of improving strains described above is very long and requires many manipulations and tests. It results in the production of polymerized strains that grow poorly, have particular environmental requirements, and for which a long phase of optimization of the medium 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 antibiotic production can drive much faster than traditional methods to obtain strains of industrial interest hyperproductive antibiotic. Since such strains are not poly-mutated, they are likely to have growth characteristics close to the wild-type strain and should require a shorter phase of optimization of the medium.

 It has long been known that Streptomyces deficiency of inorganic phosphate (Pi) induces the initiation of antibiotic biosynthesis. During its growth, the bacterium consumes the Pi present in the culture medium until the concentration of the latter becomes limiting. This low concentration of Pi signals a future state of deficiency in Pi and the bacterium then starts the expression of a whole set of genes that allows its adaptation to this limitation in Pi. The set of these genes, among

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 which include genes encoding phosphatases of different phosphorylated molecules, high affinity Pi transport systems, Pi storage / retrieval systems in Pi polymers (including the ppk gene) and the like. Pho. 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 defer or reduce the extent of the deficiency state Pi.

 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 different genes of the Pho regulon in order to stimulate their transcription. .

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

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

 In order to do this, the inventors have isolated and interrupted the two phoK (kinase) and phoR (regulator) genes which, in Streptomyces lividans, are probably the system

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 two-component system responsible for the activation of the expression of all Streptomyces Pho regulon genes. The mutants phoK and phoR 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 overproduce antibiotics in a massive way.

 The object of the invention is to provide a novel process for significantly increasing the production of metabolites or enzymes by strains of microorganisms of industrial interest (Actinomycétales or others), by removing the repression that is exerted phosphate on the production of antibiotics or enzymes by strains of microorganisms of industrial interest.

 Advantageously, the process of the invention is easily transferable 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 method 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 subject of the present invention is the use of strains of microorganisms producing compounds of interest, including the phoK / phoR operon encoding a two-component system, namely a phoK encoded sensory kinase, and a regulator encoded by phoR, which is responsible for the activation of the Pho regulon of said microorganisms comprising a set of genes involved in the adaptive response to phosphate limitation in said microorganisms, is inactivated, for the implementation of a stimulation method the production of these compounds of interest by said transformed strains.

 By the process of stimulating the production of compounds of interest, as mentioned above, is meant any process according to which the production of these compounds is increased, especially in proportions of about 20% to 50% or more, with respect to the production of these same compounds in strains identical to the previous ones but whose operon phoK / phoR was not inactivated.

 The invention relates more particularly to the aforementioned use of microorganisms producing compounds of interest selected from the metabolites with activity

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 antibiotic, anticancer, immunosuppressive, antihehnetic, antifungal, herbicide, insecticide, or enzyme-producing microorganisms.

 The invention more particularly relates to the above-mentioned use of strains of microorganisms of the order Actinomycetales, especially those producing metabolites with antibiotic activity.

 As such, the invention relates to the aforementioned use of Actinomycetes strains producing metabolites with antibiotic activity chosen from the strains of Streptomyces, Bacillus, Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Cephalosporium, Penicillium , Corynebacterium, Actinoplanes, Actinomadura, Dactylosporangium, and Mycobacterium.

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

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

 The production of enzymes such as proteases, cellulases, endoglucanases,. beta-glucanases, hemicellulases, lipases, peroxidases, laccases, .alpha.-amylases, glucoamylases, chitinase, cutinases, agarase, ligninase, nuclease, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, arabinosidases, mannanases, xyloglucanases, xylanases , pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, pectin methylesterases, cellobiohydrolases, transglutaminases produced by different bacterial genera such as Erwinia, Pseudomonas, Klebsiella, Xanthomonas, and by different species of Bacillus, fungi, yeasts or Actinomycetes, are likely to be produced in greater quantities by the microorganisms modified according to the invention, to the extent that the synthesis of these enzymes is regulated by inorganic phosphate.

 The microorganism strains used in the context of the present invention are also chosen from those transformed using vectors containing sequences coding for the compounds of interest mentioned above.

 The invention more particularly relates to the aforementioned use of strains of microorganisms whose operon phoK / phoR, or the promoter region of this operon, is modified by addition and / or substitution and / or deletion of at least a nucleotide, such that said operon thus modified no longer codes for the active sensory kinase, and / or for the transcriptional regulator (or activator) of the Pho regulon genes.

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 The invention relates more particularly to the above-mentioned use of microorganism strains in which the promoter region of the phoK / phoR operon, and / or the phoK gene, and / or the phoR gene, are modified by addition and / or substitution. and / or deletion of at least one nucleotide.

 The invention more particularly relates to the above-mentioned use of strains of microorganisms whose promoter region of the phoK / phoR operon, or at least the phoR gene, are modified by addition and / or substitution and / or deletion. at least one nucleotide.

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

 The invention relates more particularly to the aforementioned use of microorganism strains as defined above comprising instead of their promoter region of the phoK / phoR operon, and / or their phoK gene, and / or of their wild-type phoR gene, a nucleotide sequence corresponding to: - the promoter region of the phoK / phoR operon, and / or the phoK gene, and / or the phoR gene, wild-type modified by addition and / or substitution and / or deletion of at least one nucleotide, or the promoter region of the phoK / phoR operon, and / or phoK gene, and / or phoR 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 phoK / phoR operon, and / or the phoK gene, and / or the phoR gene, wild type.

 Advantageously, in the case where the aforementioned microorganism strains contain a promoter region of the phoK / phoR operon, and / or a phoK gene, and / or a modified exogenous phoR gene, these code for proteins having a percentage identity with the corresponding wild proteins of at least about 50%.

 The invention also relates to the use of recombinant nucleotide sequences comprising a phoK / phoR operon, or the promoter region of this operon, originating from microorganisms producing compounds of interest, such as those defined above, said phoK operon / phoR, 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 which produce no more than

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 active sensory kinase, and / or transcriptional regulator of the Pho regulon genes, for the implementation of 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 aforementioned use of recombinant nucleotide sequences comprising: a nucleotide sequence corresponding to the promoter region of the phoK / phoR operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the nucleotide sequence SEQ ID NO: 1 represented in FIG. 1, or corresponding to a nucleotide sequence derived from the abovementioned promoter region and corresponding to a promoter region of the phoK / phoR 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 phoK 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 a derivative of the phoK gene and encoding a protein having a percentage identity with the protein encoded by the abovementioned phoK gene of at least about 50%, said derived sequence corresponding to a phoK gene in microorganisms other than Streptomyces, as mentioned above, said phoK 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 Streptomyces phoR 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 phoR gene and coding for a protein having a percentage identity with the protein encoded by the abovementioned phoR gene of 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.

 The invention more particularly relates to the aforementioned use of recombinant nucleotide sequences comprising: a nucleotide sequence corresponding to the promoter region of the phoK / phoR 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 aforementioned promoter region and corresponding to a promoter region of the phoK / phoR operon

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 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 corresponding nucleotide sequence to the phoR 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 phoR gene and coding for a protein having a percentage of 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 above, said phoR gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide, and, if appropriate, a nucleotide sequence corresponding to the phoK gene of Streptomyces deli mit 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 phoK gene and encoding a protein having a percentage identity with the protein encoded by the abovementioned phoK gene d at least about 50%, said derived sequence corresponding to a phoK gene in microorganisms other than Streptomycess, as mentioned above, said genephoK, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide.

 The invention also relates to the aforementioned use of recombinant nucleotide sequences comprising: the promoter region of the phoK / phoR operon of Streptomyces lividans, or a derived sequence, and / or the Streptomyces lividans gene phoK, or a sequence derivative, and / or the Streptomyces lividansphosphoric gene, or a derived sequence, the promoter region or the aforementioned genes being modified by insertion of a heterogeneous sequence at an endogenous or exogenous restriction site determined in the gene sequence .

 The invention more particularly relates to the aforementioned use of recombinant nucleotide sequences in which the promoter region or the phoK and phoR genes are modified by insertion at a specific restriction site of said sequence, of a cassette containing a marker, such as an antibiotic resistance marker, including apramycin.

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 The invention relates more particularly to the aforementioned use of recombinant nucleotide sequences chosen from: the sequence SEQ ID NO: 3 represented in FIG. 2, comprising the interruption of the phoK gene by insertion at the NcoI restriction site of the gene , located at positions 1389 to 1394 of the sequence SEQ ID NO: 1, of a cassette containing an apramycin resistance marker, and delimited by the amino acids at positions 1425 and 3626 of the sequence SEQ ID NO: 3 the sequence SEQ ID NO: 4 represented in FIG. 3, comprising the interruption of the phoR gene by insertion at the NotI restriction site of the phoR gene, located at positions 2111 to 2118 of the sequence SEQ ID NO: 1, of a cassette containing an apramycin resistance marker, and delimited by the amino acids at the positions 2148 and 3920 of the sequence SEQ ID NO: 4, the sequence SEQ ID NO: 5 represented in FIG. 4, comprising disruption of the phoK and phoR genes by insertion at the aforementioned NcoI and NotI restriction sites, with deletion of the NcoI / NotI fragment, of a cassette containing an apramycin resistance marker, and delimited by the amino acids located at positions 1425 and 3626 of the sequence SEQ ID NO: 5.

 The invention also relates to any recombinant nucleotide sequence comprising: a nucleotide sequence corresponding to the promoter region of the phoK / phoR 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 aforementioned promoter region and corresponding to a promoter region of the phoK / phoR 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 phoK 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 phoK gene and coding for a protein having a percentage of identity with the protein encoded by the aforementioned phoK gene of at least about 50%, said derived sequence corresponding to a phoK gene in microorganisms other than Streptomyces, as mentioned above, said phoK gene, or its derived sequence , being modified by addition and / or substitution and / or deletion of at least one nucleotide,

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and / or a nucleotide sequence corresponding to the phoR 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 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 above, said phoR 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 phoK / phoR 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 aforementioned promoter region and corresponding to a promoter region of the phoK / phoR 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 phoR 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 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 above, said phoR gene or its derived sequence being modified by addition and / or substitution and / or deletion of at least one nucleotide, and, if appropriate, a nucleotide sequence corresponding to the phoK 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 phoK gene and encoding a protein having a percentage identity with the protein encoded by the abovementioned phoK gene of at least about 50%, said sequence derivative corresponding to a phoK gene in microorganisms other than Streptomyces, as mentioned above, said phoK gene, or its derived sequence, being modified by addition and / or substitution and / or deletion of at least one nucleotide.

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 The invention more particularly relates to any recombinant nucleotide sequence as defined above, comprising: the promoter region of the phoK / phoR operon of Streptomyces lividans, or a derived sequence, and / or the phoK gene of Streptomyces lividans, or a derived sequence, and / or the Streptomyces lividansphoR gene, or a derived sequence, the aforementioned promoter region or genes being modified by insertion of a heterogeneous sequence at an endogenous or exogenous restriction site determined in the gene sequence.

 The invention relates more particularly to any recombinant nucleotide sequence as defined above, in which the promoter or the phoK and phoR genes are modified by insertion at a particular restriction site of said sequence, of a cassette containing a marker, such as an antibiotic resistance marker, especially 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 phoK gene by insertion at the NcoI restriction site of the gene, located at the positions 1389 to 1394 of the sequence SEQ ID NO: 1, a cassette containing an apramycin resistance marker, and delimited by the amino acids at positions 1425 and 3626 of the sequence SEQ ID NO: 3, the sequence SEQ ID NO: 4, comprising the interruption of the phoR gene by insertion at the NotI restriction site of the phoR gene, located at positions 2111 to 2118 of the sequence SEQ ID NO: 1, of a cassette containing a resistance marker at apramycin, and delimited by the amino acids at positions 2148 and 3920 of the sequence SEQ ID NO: 4, the sequence SEQ ID NO: 5, comprising the interruption of the phoK and phoR genes by insertion at the sites of restriction Ncol e The above-mentioned examples, with deletion of the NcoI / NotI fragment, of a cassette containing an apramycin resistance marker and delimited by the amino acids at positions 1425 and 3626 of the sequence SEQ ID NO: 5.

 The subject of the invention is also any vector, in particular any plasmid, optionally thermally sensitive replication, containing a recombinant nucleotide sequence as defined above.

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

 The invention more particularly relates to strains of modified microorganisms as defined above, said strains producing compounds of interest as defined above.

 The subject of the invention is more particularly the strains of modified microorganisms as defined above, characterized in that they are chosen from the strains of the order Actinomycetales, such as those defined in Table I.

 The invention also relates to strains of modified microorganisms as defined above, said strains being producing enzymes, and being advantageously chosen from the strains mentioned above.

 The invention more particularly relates to the aforementioned microorganism strains, as obtained by transformation of said microorganisms with a vector defined above.

 The subject of the invention is also a process for the preparation of strains of microorganisms as defined above, characterized in that it comprises the following steps: where appropriate, the cloning of the phoK / phoR operon, or the promoter region of this operon, the strain of microorganism that is to be modified, and the amplification of a nucleotide sequence as defined above containing the phoK / phoR operon, or its promoter region, at 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 in the preceding step, by addition and / or substitution and / or deletion of at least one nucleotide, - transformation of microorganism strains using vector tails the recombinant nucleotide sequence obtained in the previous step, - selection of the transformed microorganism strains having integrated in their genome the aforementioned recombinant nucleotide sequence in place of the nucleotide sequence corresponding to the wild-type operon or promoter region .

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 According to a particularly advantageous embodiment of the method of the invention, the nucleotide sequence as defined above containing the phoK / phoR operon, or its promoter region, is modified by insertion of a cassette comprising a marker, in particular a resistance marker to an antibiotic, in order to select the modified strains obtained.

The recombinant sequence thus obtained is then cloned in a suitable vector, in particular in a plasmid capable of interspecific transfer and capable or not of replicating in a thermosensitive 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 observing the presence or absence of the marker carried by the cassette in the genome of the modified strains, after a passage or not at high temperature depending on whether the plasmid is with heat-sensitive 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 phoK / phoR operon, or its promoter region, is modified by the addition or deletion of at least one nucleotide. In this case, it is preferable to use a thermosensitive replication vector, and, after passage at high temperature, the colonies which have lost the plasmid (colonies sensitive to the antibiotic to which the plasmid ordinarily confers the resistance) are identified. which overproduce the antibiotic on a suitable 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 product.

 Alternatively, the latter method can also be carried out in two steps. In a first step, the nucleotide sequence as defined above containing the phoK / phoR operon, or its promoter region, is modified by insertion of a aforementioned cassette conferring resistance to an antibiotic, then this modified sequence by insertion is replaced by a deleted version of this sequence. In this case, colonies are identified that have become sensitive to the antibiotic to which the cassette usually confers resistance.

 The invention also relates to 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 the implementation of culture of strains of modified microorganisms as defined above, in a suitable culture medium.

 Advantageously, the aforementioned method of the invention is characterized in that the culture is carried out in a rich medium (namely a medium containing amino acids

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 as a source of carbon and nitrogen) with an osmotic pressure equivalent to that conferred by 103 g / l of sucrose and without addition of exogenous inorganic phosphate.

Table 1
List of Antibiotic Producing Organizations I) Aminocyclitol Producing Organizations

<Tb>
<tb> ORGANISM <SEP> ANTIBIOTICS
<tb> Bacillus <SEP> (various <SEP> species) <SEP> Aminocyclitols
<tb> Micromonospora <SEP> (various <SEP> species) <SEP> Gentamycins
<tb> Saccharopolyspora <SEP> (various <SEP> species) <SEP> Aminocyclitols
<tb> Streptomyces <SEP> albogriseolus <SEP> Neomycin
<tb> albus <SEP> var. <SEP> metamycinus <SEP> Metamycin
<tb> aquacanus <SEP> N-methyl <SEP> hygromycin <SEP> B
<tb> atrofaciens <SEP> Hygromycines
<tb> bikiniensis <SEP> Streptomycin
<tb> bluensis <SEP> var. <SEP> bluensis <SEP> Bluensomycin
<tb> canus <SEP> Ribosyl <SEP> paromamine
<tb> catenulae <SEP> Catenuline
<tb> chrestomyceticus <SEP> aminosidine
<tb> crystallinus <SEP> hygromycin <SEP> A
<tb> erythrochromogenes <SEP> streptomycin
<tb> var. <SEP> narutoensis <SEP> eurocidicus <SEP> A16316-C
<tb> fradiae <SEP> hybrimycins <SEP> and <SEP> neomycin
<tb> fradiae <SEP> var. <SEP> italicus <SEP> aminosidine
<tb> galbus <SEP> streptomycin
<tb> griseus <SEP> streptomycin
<tb> griseoflavus <SEP> MA <SEP> 1267
<tb> hofuensis <SEP> seldomycin <SEP> complex
<tb> hygroscopicus <SEP> hygromycinse
<tb> hygroscopicus <SEP> forma <SEP> leucanicidine <SEP> and <SEP> hygrolidine
<tb> glebosus <SEP> glebomycin
<tb> hygroscopicus <SEP> var. <SEP> limoneus <SEP> validamycine
<tb> hygroscopicus <SEP> var. <SEP> sagamiensis <SEP> spectinomycin
<tb> kanamyceticus <SEP> kanamycin <SEP> A <SEP> and <SEP> B
<tb> kasuqaensis <SEP> kasugamycins
<tb> kasugaspinus <SEP> kasugamycins
<tb> lavendulae <SEP> neomycin
<tb> lividus <SEP> lividomycins
<tb> mashuensis <SEP> streptomycin
<tb> m <SEP> icrosporeus <SEP> SF-767
<tb> netropsis <SEP> LL-AM31
<tb> noboritoensis <SEP> hygromycine
<tb> olivaceus <SEP> streptomycin
<tb> olivoreticuli <SEP> var. <SEQ> cellulophilus <SEP> destomycin <SEP> A
<tb> poolensis <SEP> streptomycin
<Tb>

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<Tb>
<tb> rameus <SEP> Streptomycin
<tb> ribosidificus <SEP> SF733
<tb> rimofaciens <SEP> destomycin <SEP> A
<tb> rimosus <SEP> forma <SEP> paromomycinus <SEP> paromomycin, <SEP> catenulin
<tb> spectabilis <SEP> spectinomycin
<tb> tenebrarius <SEP> tobramycin <SEP> and <SEP> apramycin
<tb> Streptoverticillium <SEP> flavopersicus <SEP> spectinomycin
<tb> II) <SEP> Ansamycin organisms <SEP> producers <SEP>
<tb> ORGANISM <SEP> ANTIBIOTICS
<tb> Micromonospora <SEP> (various <SEP> species) <SEP> ansamycins
<tb> Nocardia <SEP> mediterranei <SEP> rifamycin
<tb> Streptomyces <SEP> collinus <SEP> ansatrienes, <SEP> napthomycin
<tb> diastochromogenes <SEP> ansatrienes, <SEP> napthomycin
<tb> galbus <SEP> subsp. <SEP> griseosporeus <SEP> napthomycin <SEP> B
<tb> hygroscopicus <SEP> herbimycin
<tb> hygroscopicus <SEP> var. <SEP> geldanus <SEP> geldamycin
<tb> var. <SEP> nova <SEP> nigellus <SEP> 21-hydroxy-25-demethyl
<tb> 25-methylthioprotostreptovaricin
<tb> rishiriensis <SEP> mycotrienes
<tb> sp. <SEP> E / 784 <SEP> actamycin <SEP> and <SEP> mycotriene
<tb> sp. <SEP> E88 <SEP> mycotrienes
<tb> spectabilis <SEP> Streptovaricins
<tb> tolypophorous <SEP> tolypomycin
<tb> III) <SEP> Organisms <SEP> producers <SEP> of Anthracycline <SEP> and <SEP> of <SEP> Quinone
<tb> ORGANISM <SEP> ANTIBIOTICS
<tb> Streptomyces <SEP> caespitosus <SEP> mitomycin <SEP> A, <SEP> B, <SEP> and <SEP> C
<tb> coelicolor <SEP> actinorhodin
<tb> coeruleorubidicus <SEP> daunomycin
<tb> cyaneus <SEP> ditrisarubicin
<tb> flavogriseus <SEP> cyanocycline <SEP> A
<tb> galilaeus <SEP> aclacinomycin <SEP> A,
<tb> auramycins, <SEP> sulfurmycines
<tb> Lusitanus <SEP> napthyridinomycin
<tb> peuceticus <SEP> daunomycin, <SEP> adriamycin
<tb> violochromoqenes <SEP> arugomycin
<tb> IV) <SEP> Bodies <SEP> Producers <SEP> of <SEP> Beta <SEP> Lactam
<tb> ORGANISM <SEP> ANTIBIOTICS
<tb> Cephalosporium <SEP> (various <SEP> species) <SEP> beta-lactams
<tb> Nocardia <SEP> lactamadurans <SEP> cephamycin <SEP> C
<tb> Penicillium <SEP> (various <SEP> species) <SEP> beta-lactams
<tb> Streptomyces <SEP> antibioticus <SEP> acid <SEP> clavulanic
<tb> argenteolus <SEP> asparenomycin <SEP> A,
<Tb>

<Desc / Clms Page number 15>

<Tb>
<tb> MM <SEP> 4550, <SEP> MM <SEP> 13902
<tb> cattleya <SEP> thienamycin
<tb> chartreusis <SEP> SF <SEP> 1623, <SEP>
<tb> cephamycin <SEP> A <SEP> and <SEP> B
<tb> cinnamonensis <SEP> cephamycin <SEP> A <SEP> and <SEP> B
<tb> clavuligerus <SEP> PA-32413-I,
<tb> cephamycin <SEP> C, <SEP> A16886A,
<tb> clavulanic <SEP> acid, <SEP> and
<tb> other <SEP> clavames
<tb> fimbriatus <SEP> cephamycin <SEP> A <SEP> and <SEP> B
<tb> flavovirens <SEP> MM <SEP> 4550 <SEP> and <SEP> MM <SEP> 13902
<tb> flavus <SEP> MM <SEP> 4550 <SEP> and <SEP> MM <SEP> 13902
<tb> fulvoviridis <SEP> MM <SEP> 4550 <SEP> and <SEP> MM <SEP> 13902
<tb> griseus <SEP> cephamycin <SEP> A <SEP> and <SEP> B
<tb> halstedi <SEP> cephamycin <SEP> A <SEP> and <SEP> B
<tb> heteromorphus <SEP> C2081X, <SEP> cephamycin <SEP> A, B
<tb> hygroscopicus <SEP> deacetoxycephalosporin <SEP> C
<tb> lipmanii <SEP> penicillin <SEP> N,
<tb> 7-methoxycephalosporin
<tb> C, <SEP> A16884, <SEP> MM <SEP> 4550, <SEP> and
<tb> MM <SEP> 13902
<tb> olivaceus <SEP> (MM <SEP> 17880)
<tb> epithienamycin <SEP> F,
<tb> MM <SEP> 4550, <SEP> and <SEP> MM <SEP> 13902
<tb> panayensis <SEP> C2081X, <SEP> cephamycin <SEP> A, B
<tb> pluracidomyceticus <SEP> pluracidomycin <SEP> A
<tb> rochei <SEP> cephamycin <SEP> A <SEP> and <SEP> B
<tb> sioyaensis <SEP> MM <SEP> 4550 <SEP> and <SEP> MM <SEP> 13902
<tb> sp. <SEP> OA-6129 <SEP> OA-6129A
<tb> sp. <SEP> KC-6643 <SEP> carpetimycin <SEP> A
<tb> tokunomensis <SEP> asparenomycin <SEP> A
<tb> viridochromogenes <SEP> cephamycin <SEP> A <SEP> and <SEP> B
<tb> wadayamensis <SEP> WS-3442-D
<Tb>
V) Macrolide, Lincosamide, and Streptogramin producing organisms

<Tb>
<tb> ORGANISM <SEP> ANTIBIOTICS
<tb> Micromonospora <SEP> rosaria <SEP> rosaramicin
<tb> Streptomyces <SEP> albireticuli <SEP> carbomycin
<tb> albogriseolus <SEP> mikonomycine
<tb> albus <SEP> albomycetin
<tb> albus <SEP> var. <SEP> coilmyceticus <SEP> coleimycin
<tb> ambofaciens <SEP> spiramycin,
<tb> foromacidine <SEP> D
<tb> antibioticus <SEP> oleandomycin
<tb> avermitilis <SEP> avermectins
<tb> bikiniensis <SEP> chalcomycin
<Tb>

<Desc / Clms Page number 16>

<Tb>
<tb> brownogriseus <SEP> albocycline
<tb> caelestis <SEP> M188, <SEP> celesticetin
<tb> cinerochromogenes <SEP> cineromycin <SEP> B
<tb> cirratus <SEP> cirramycin
<tb> deltae <SEP> deltamycins
<tb> djakartensis <SEP> niddamycin
<tb> erythreus <SEP> erythromycin
<tb> eurocidicus <SEP> methymycin
<tb> eurythermus <SEP> angolamycin
<tb> fasciculus <SEP> amaromycin
<tb> felleus <SEP> argomycin <SEP> and <SEP> picromycin
<tb> fimbriatus <SEP> amaromycin
<tb> flavochromogenes <SEP> amaromycin,
<tb> and <SEP> shincomycins
<tb> fradiae <SEP> tylosin
<tb> fungicidicus <SEP> NA-181
<tb> fungicidicus <SEP> var. <SEP> espinomyceticus <SEP> espinomycetins
<tb> furdicidicus <SEP> mydecamycin
<tb> goshikiensis <SEP> bandamycin
<tb> griseofaciens <SEP> PA133A <SEP> and <SEP> B
<tb> griseoflavus <SEP> acumycin
<tb> griseofuscus <SEP> bundline
<tb> griseolus <SEP> griseomycin
<tb> griseospiralis <SEP> relomycin
<tb> griseus <SEP> borrelidin
<tb> griseus <SEP> ssp. <SEP> sulphurus <SEP> bafilomycin
<tb> halstedi <SEP> carbomycin, <SEP> leucanicidin
<tb> hygroscopicus <SEP> tylosin
<tb> hygroscopicus <SEP> subsp <SEP> aureolacrimosus <SEP> milbemycine
<tb> kitastoensis <SEP> leucomycin <SEP> A.sub.3,
<tb> etjosamycin
<tb> lavendulae <SEP> aldgamycin
<tb> lincolnensis <SEP> lincomycin
<tb> loidensis <SEP> vernamycin <SEP> A <SEP> and <SEP> B
<tb> macrosporeus <SEP> carbomycin
<tb> maizeus <SEP> ingramycin
<tb> mycarofaciens <SEP> acetyl-leukomycin,
<tb> and <SEP> espinomycin
<tb> narbonensis <SEP> josamycin <SEP> and <SEP> narbomycin
<tb> narbonensis <SEP> var. <SEP> josamyceticus <SEP> leucomycin <SEP> A, <SEP> josamycin
<tb> olivochromogenes <SEP> oleandomycin
<tb> platensis <SEP> platenomycin
<tb> rimosus <SEP> tylosin <SEP> and <SEP> neutramycin
<tb> rochei <SEP> lankacidine <SEP> and <SEP> Borrelidin
<tb> rochei <SEP> var. <SEP> volubilis <SEP> T2636
<tb> roseochromogenes <SEP> albocyline
<tb> roseocitreus <SEP> albocycline
<tb> spinichromoenes <SEP> var.suragaoensis <SEP> kujimycin
<tb> tendae <SEP> carbomycin
<tb> thermotolerans <SEP> carbomycin
<Tb>

<Desc / Clms Page number 17>

 venezuelae methymycines violaceoniger lankacidines, lankamycin VI) Streptomyces species of various antibiotic producers a) Amino acid analogues Streptomyces sp. Cycloserine b) Containing cyclopentane nuclei Streptomyces coelicolor methylenomycin A erythrochromogenes sarkomycin violaceoruber methylenomycin A c) Containing nitrogen Streptomyces venezuelae chloramphenicol d) Polyenes Streptomyces griseus candicidin nodosus amphotericin B noursei nystatin e) Tetracyclines Streptomyces aureofaciens tetracycline, chlortetra-cyclin, demethyltetra- cycline, and demethylchlortetra-cyclin rimosus oxytetracycline VII) Nucleoside-Producing Organisms ANTIBIOTIC ORGANISM Corynebacterium michiganese pv. rathayi tunicamycin analogues Nocardia candidus pyrazofurin Streptomyces antibioticus ara-A chartreusis tunicamycin griseoflavus var. streptoviridans thuringiensis griseolus sinefungine VIII) Peptides producing organisms ANTIBIOTIC ORGANISM Actinoplanes missouriensis actaplanin teichomyceticus teicoplanin Bacillus (various species) bacitracin, polymixin, and colistin Nocardia candidus A-35512 and avoparcine lurida ristocetine

<Desc / Clms Page number 18>

vancomycin orientalis Streptomyces antibioticus actinomycin aureus thiostreptone canus amphomycin eburosporeus LL-AM374 haranomachiensis vancomycin pristinaespiralis pristinamycin roseosporus lipopeptides, such A21978C toyocaensis A47934 virginiae A1030 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 narasin cacaoi var. asoensis lysocellin chartreusis A23187 cinnamonensis monensin conglobatus ionomycin eurocidicus var. asterocidicus uglylcine flaveolus CP38936 gallinarius RP 30504 griseus grayorixin hygroscopicus A218, emericide, DE3936,
A120A, A28695A and B, etheromycin, dianemycin lasaliensis lasalocidis longwoodensis lysocellin mutabilis S-1173a ribosidificus lonomycin violaceoniger nigericin Streptoverticillium (various species) various polyethers
The invention will be further detailed with the aid of the following description of the cloning and disruption of Streptomyces lividans pppk gene.

 As has been specified above, a limiting situation in Pi triggers the expression of the Pho regulon, a set of genes the expression of which makes it possible to delay the onset of a deficiency state Pi. can occur, it follows a situation of deficiency in Pi. The deficiency in Pi causes the triggering of adaptive processes different from those induced by a limitation in Pi and it is these processes still

<Desc / Clms Page number 19>

 poorly known diseases that lead to Streptomyces triggering the biosynthesis of antibiotics.

 By interrupting the two genes phoK (kinase) and phoR (regulator) which, in Streptomyces lividans, are probably the two-component system responsible for the activation of the expression of all the genes of the Streptomyces Pho regulon, the inventors have early created a situation of severe deficiency in Pi.

 These mutants overwhelmingly overproduce antibiotics.

A) Principle of the method:
The inventors have isolated and interrupted the two genes phoK (kinase) and phoR (regulator) which, in Streptomyces lividans, are probably the two-component system responsible for the activation of the expression of all the genes of the Phon regulon. Streptomyces. The mutants phoK and phoR constructed are incapable of inducing the expression of the various Pi recovery systems mentioned above, they are therefore early and severely deficient Pi and overproduce massive antibiotics on all media tested. a) Characterization of the phoK / phoR Senes:
In silico: The two-component systems related to the phoR (kinase) / phoP (regulator) pairs of Escherichia coli Bacillus subtilis and phoR (kinase) / phoB (regulator) were searched in the fully sequenced genome of Streptomyces coelicolor ( species very close to S. lividans but which is much more easily manipulated by conventional molecular biology techniques than the latter) using the BLAST software.

Several putative S. coelicolor proteins have been found to have significant homology with these proteins. Similarity searches for these S. coelicolor proteins were then made in the genomes of B. subtilis and E. coli (back BLAST system) and revealed that only five kinase / regulatory pairs of S. coelicolor showed significant similarity with the initial couples.

 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 "Northem blot" techniques the corresponding transcripts. RNAs were prepared under conditions of abundance or P1 restriction 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

<Desc / Clms Page number 20>

no doubt at the outbreak of the expression of the regulon Pho. Of the five candidates, only one had the type of regulation expected, ie an increase in its expression level under the limiting condition in Pi. This increase was much greater in a context mutated for ppk. In Streptomyces, this pair was called phoK (kinase) / phoR (regulator), a more logical nomenclature than that used in B. subtilis and E. coli. b) Cloning of the phoKIphor operon:
The phoK and phoR gene cloning and disruption protocol described below is applicable to all strains of Streptomyces and other bacterial species of industrial interest.

 1 A fragment of S. lividans of about 3.2 kb containing the phoK-phoR operon and 500bp upstream and downstream of the latter was amplified by two oligonucleotides suitably selected from the sequence of the equivalent region of S. coelicolor.

 A cassette conferring apramycin resistance was cloned into a NotI site located at the center of the phoR gene and brought to freeboard with the Klenow fragment to interrupt it. An excisable cassette conferring resistance to apramycin was cloned into the NcoI site located at the center of the phoK gene and brought to the brink with the Klenow fragment, so as to interrupt the latter. It should be noted that, for scientific reasons, we used to interrupt phoK an excisable cassette because phoK and phoR being cotranscribed, the interruption of phoK has a polar effect on the expression of phoR.

 Fragments harboring the phoK and phoR genes interrupted were cloned into pGM160 thermosensitive replication vector. These vectors were introduced into S. lividans and a replacement of wild phoK and phoR genes by their interrupted copy was performed by a standard protocol.

 Given the strong sequence homology detected by Southern between the S. lividans phoK / phoR genes and that of various strains of antibiotic-producing Streptomyces, there is no need to re-clone the corresponding genes to construct mutants. interruption in these species. Indeed, the phoK / phoR 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 phoK and / or phoR genes were interrupted produced much more antibiotic than the wild-type strain on all media tested. The phoR gene encodes the transcriptional activator of the regulon

<Desc / Clms Page number 21>

 Pho Streptomyces and the latter requires phoK phosphorylation to fulfill its activating function. In an interrupt mutant of the phoK and / orphor genes, the induction of the genes necessary for the adaptation of the bacterium to a limiting concentration in Pi does not take place. The latter is therefore early and severely deficient Pi and it follows a massive induction of antibiotic production.

B) Detailed experimental method
Cloning and disruption of the phoR, phoK and phoK-phoR operon genes of Streptomyces lividans: - An operon coding for a two-component system consisting of a PhoK (427 AA) sensory kinase and a PhoR (224 AA) regulator with 67 % identity with their respective PhoR and PhoB counterparts of E. coli was found in the genome entirely sequence of Streptomyces coelicolor, very closely related species of S. lividans.

 - two unique sites NcoI and NotI (enzymes giving 5 'outward ends) located respectively towards the 3' end of the phoK gene and towards the 5 'end of the phoR gene have been identified (they are underlined in italics and in bold on the sequence SEQ ID NO: 1 of FIG. 1) and considered to be well located to serve as insertion sites for an interrupt cassette.

 - Two oligos A and B (in bold on the sequence of the figurel) were then designed so as to PCR amplify the whole region.

 The fragment amplified with these oligos was then cloned into the SmaI site of plasmid pIJ2925 (Janssen and Bibb, 1993) to give pSG1.

 pSG1 was digested with NcoI (single site in the plasmid). The NcoI site was brought aboard by the Klenow fragment and the exable #aac cassette which confers resistance to apramycin (Blondelet-Rouault et al., 1997) was cloned into this site, in the form of a fragment. EcoRV to interrupt phoK and give pSG2.

<Desc / Clms Page number 22>

 pSG1 was digested with NotI (single site in the plasmid). The NotI site was brought aboard by the Klenow fragment and the Qaac cassette which confers resistance to apramycin (Blondelet-Rouault et al., 1997) was cloned into this site, in the form of a SmaI fragment for interrupt phoR and give pSG3.

 pSG1 was digested with NotI and NcoI. The NotI-NcoI fragment of the insert was removed and the NcoI and NotI sites were brought aboard by the deleted Klenow fragment and the Qaac cassette which confers resistance to apramycin (Blondelet-Rouault et al., 1997). ) was cloned as a SmaI fragment in place of the NcoI-NotI fragment deleted to jointly terminate phoK and phoR.et to give pSG4.

 The pSG2 insert was cloned as an EcoRI-XbaI fragment (brought aboard by the Klenow fragment) into the BamHI site (brought aboard by the Klenow fragment) of pGM160, to give pSG7. pGM160 is a thermosensitive replication E. coli / S. lividans shuttle vector in S. lividans. It carries the bla and tsr genes that confer ampicillin resistance in E. coli and nosiheptide in S. lividans respectively, as well as the aacCl gene that confers resistance to apramycin to E. coli and S. lividans - The inserts of pSG3 and pSG4 were transferred as BglII fragments into the BamHI site of pGM160 (Muth et al., 1989) to give 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 colonies were selected in the presence of nosiheptide.

 - In order to replace the wild phoK and phoR genes by their interrupted alleles, colonies independent of S. lividans TK24 containing pSG5, pSG6 or pSG7 and therefore resistant to nosiheptide were crushed and grown independantly for 24 h to 30 hrs. In 2 ml of TSB medium (Hopwood et al., 1985 containing apramycin at 25 μg / ml.

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

<Desc / Clms Page number 23>

 Cultures were grown at 41 C for 48 hours to allow removal of the replicative plasmid.

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

 The sporulated 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 the apramycin resistant colonies were nosiheptide sensitive.

 The latter having lost the replicative plasmid were likely to be the expected double recombinants.

 BRIEF DESCRIPTION OF THE FIGURES FIG. 1: nucleotide SEQ ID NO: 1 of a 3541bp fragment of S. coelicolor containing the PhoK PhoR operon encoding the sensory kinase and regulator of the phosphate regulon. The start and end codons of translation are in italics and the sequences in aa are provided. Oligonucleotides A and B used for PCR amplification of the DNA fragment which served to interrupt the PhoK PhoR operon of S. lividans are in bold. The NotI and Ncol sites are underlined in bold and italic. The PstI and BspEI sites are underlined and in bold.

Figure 2: nucleotide sequence SEQ ID NO: 3: mutant phoK :: # aac
Figure 3: nucleotide sequence SEQ ID NO: 4: mutant phoR :: # aac
FIG. 4: nucleotide sequence SEQ ID NO: 5: PhoK / phoR :: mutant double mutant

<Desc / Clms Page number 24>

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

2. Blondelet-Rouault, M. H., J. Weiser, 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. Gene. 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: laboratory Manual. , Norwich: John Innes 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. Gene. 124 (1): 133-4.

5. Muth, G., B. Nussbaumer, W. Wohlleben, and A. Pülher. 1989. A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes. Mol Gen Genet. 219: 341-348.

Claims (15)

1. Use of recombinant nucleotide sequences comprising: a nucleotide sequence corresponding to the promoter region of the phoK / phoR operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the nucleotide sequence SEQ ID NO: 1 represented in FIG. 1, said promoter region being modified by addition and / or substitution and / or deletion of at least one nucleotide, and / or a nucleotide sequence corresponding to the phoK gene of Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, said phoK gene 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 1917 to 2587 of the sequence SEQ ID NO: 1, said phoR gene being modified by addition and / or substitution and / or deletion of at least a nucleotide, for the transformation of strains of microorganisms producing compounds of interest, with a view to obtaining strains of these microorganisms no longer producing the sensory kinase and / or the regulator encoded by the phoK operon phoR, for the implementation of a process for stimulating the production of these compounds of interest by said strains of microorganisms thus modified.  2. Use according to claim 1, of recombinant nucleotide sequences comprising: a nucleotide sequence corresponding to the promoter region of the phoK / phoR operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the sequence SEQ ID NO: 1 , said promoter region being modified by addition and / or substitution and / or deletion of at least one nucleotide, or a nucleotide sequence corresponding to the phoR gene of Streptomyces delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO : 1, said phoR gene being modified by addition and / or substitution and / or deletion of at least one nucleotide, and, if appropriate, a nucleotide sequence corresponding to the phoK gene of Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, said phoK gene being modified by addition and / or substitution and / or deletion of at least one nude cléotide. <Desc / Clms Page number 26>  3. Use according to claim 1 or 2, of recombinant nucleotide sequences comprising: the promoter region of the phoK / phoR operon of Streptomyces lividans, and / or the phoK gene of Streptomyces lividans, and / or the phoR gene of Streptomyces lividans, the promoter region or the aforementioned genes being modified by insertion of a heterogeneous sequence at an endogenous or exogenous restriction site determined in the sequence of the gene.  4. Use according to one of claims 1 to 3, of recombinant nucleotide sequences in which the promoter region or phoK and phoR genes, are modified by insertion at a specific restriction site of said sequence, a cassette containing a marker, such as an antibiotic resistance marker, including apramycin.  5. Use according to one of claims 1 to 4, of recombinant nucleotide sequences chosen from: - the nucleotide sequence SEQ ID NO: 3 shown in Figure 2, comprising the interruption of the phoK gene by insertion at the restriction site NcoI of the gene, located at positions 1389 to 1394 of the sequence SEQ ID NO: 1, of a cassette containing an apramycin resistance marker and delimited by the nucleotides at positions 1425 and 3626 of the sequence SEQ ID NO : 3, - the nucleotide sequence SEQ ID NO: 4 represented in FIG. 3, comprising the interruption of the phoR gene by insertion at the NotI restriction site of the phoR gene, located at positions 2111 to 2118 of the sequence SEQ ID NO : 1, a cassette containing an apramycin resistance marker, and delimited by the nucleotides at positions 2148 and 3920 of the sequence SEQ ID NO: 4, - the nucleotide sequence SEQ ID NO: 5 represents 4, comprising the interruption of the phoK and phoR genes by insertion at the abovementioned NcoI and NotI restriction sites, with deletion of the Ncol / NotI fragment, of a cassette containing an apramycin resistance marker, and delimited by the nucleotides at positions 1425 and 3626 of the sequence SEQ ID NO: 5. <Desc / Clms Page number 27>  6. A recombinant nucleotide sequence comprising: a nucleotide sequence corresponding to the promoter region of the phoK / phoR operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the nucleotide sequence SEQ ID NO: 1 represented in FIG. said promoter region being modified by addition and / or substitution and / or deletion of at least one nucleotide, and / or a nucleotide sequence corresponding to the phoK gene of Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, said phoK gene 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 1917 to 2587 of the sequence SEQ ID NO: 1, said phoR 'gene being modified by addition and / or substitution and / or deletion of at least one nucleotide.  7. A recombinant nucleotide sequence according to claim 6 comprising: a nucleotide sequence corresponding to the promoter region of the phoK / phoR operon of Streptomyces delimited by the nucleotides located at positions 420 to 630 of the sequence SEQ ID NO: 1, said region promoter being modified by addition and / or substitution and / or deletion of at least one nucleotide, or a nucleotide sequence corresponding to the phoR gene of Streptomyces delimited by the nucleotides located at positions 1917 to 2587 of the sequence SEQ ID NO: 1, said phoR gene being modified by addition and / or substitution and / or deletion of at least one nucleotide, and, where appropriate, a nucleotide sequence corresponding to the phoK gene of Streptomyces delimited by the nucleotides located at positions 632 to 1909 of the sequence SEQ ID NO: 1, said phoK ene being modified by addition and / or substitution and / or deletion of at least one nucleotide e.  8. A recombinant nucleotide sequence according to claim 6 or 7, comprising: the promoter region of the phoK / phoR operon of Streptomyces lividans, and / or the Streptomyces lividans gene phoK, and / or the phoR gene of Streptomyces lividans, the promoter region or the aforementioned genes being modified by insertion of a heterogeneous sequence at an endogenous or exogenous restriction site determined in the gene sequence. <Desc / Clms Page number 28>  9. The recombinant nucleotide sequence according to claim 6, in which the promoter or the phoK and phoR genes are modified by insertion at a specific restriction site of said sequence of a cassette containing a marker. such as an antibiotic resistance marker, especially apramycin.  10. The recombinant nucleotide sequence of one of claims 6 to 9, chosen from: the sequence SEQ ID NO: 3, comprising the interruption of the phoK gene by insertion at the NcoI restriction site of the gene, located at positions 1389 to 1394 of the sequence SEQ ID NO: 1, a cassette containing an apramycin resistance marker, and delimited by the nucleotides at positions 1425 and 3626 of the sequence SEQ ID NO: 3, the sequence SEQ ID NO : 4, comprising interrupting the phoR gene by insertion at the NotI restriction site of the phoR gene, located at positions 2111 to 2118 of the sequence SEQ ID NO: 1, of a cassette containing a resistance marker at apramycin, and delimited by the nucleotides at positions 2148 and 3920 of the sequence SEQ ID NO: 4, the sequence SEQ ID NO: 5, comprising the interruption of the phoK and phoR genes by insertion at the NcoI restriction sites and NotI mentioned above, with deletion Ncol / NotI fragment, a cassette containing an apramycin resistance marker, and delimited by the nucleotides at positions 1425 and 3626 of the sequence SEQ ID NO: 5.  Vector containing a recombinant nucleotide sequence according to one of claims 6 to 10.  12. Microorganism strains producing compounds of interest, said microorganisms being modified by transformation with a vector according to claim 11.  13. Microorganism strains according to claim 12, said strains being producing compounds of interest selected from metabolites with antibiotic, anticancer, immunosuppressive, anthelmintic, antifungal, herbicidal, insecticidal or enzymatic activity.  14. Strains of microorganisms according to claim 12 or 13, characterized in that they are selected from the strains of the order Actinomycetales, such as strains <Desc / Clms Page number 29>  Streptomyces, Bacillus, Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Cephalosporium, Penicillium, Corynebacterium, Actinoplanes, Actinomadura, Dactylosporangium, and Mycobacterium.  15. Process for stimulating the production of compounds of interest, in particular compounds defined in claim 13, by microorganisms normally used for the production of these compounds, said process comprising culturing strains of these microorganisms. Transformed organisms as defined in any one of claims 12 to 14 in a suitable culture medium.