FR2818991A1 - STRAINS OF MICRO-ORGANISMS FOR THE IMPLEMENTATION OF A PROCESS FOR THE OVERPRODUCTION OF METABOLITES, IN PARTICULAR WITH ANTIBIOTIC OR ENZYMATIC ACTIVITY - Google Patents

Abstract

The invention concerns the use of micro-organism strains producing compounds of interest, whereof the <i>ppk</i> gene encoding polyphosphate kinase is inactivated, for implementing a method designed to stimulate the production of said compounds of interest by said transformed strains.

Description

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STRAINS OF MICRO-ORGANISMS FOR THE IMPLEMENTATION OF A PROCESS FOR THE OVERPRODUCTION OF METABOLITES, IN PARTICULAR WITH ANTIBIOTIC OR ENZYMATIC ACTIVITY
The present invention relates to the use of strains of microorganisms modified such that said strains no longer produce functional polyphosphate kinase, as well as their use in the context of the implementation of processes for the overproduction of normally produced metabolites. by said unmodified strains, such as antibiotics or enzymes.

Streptomyces are filamentous bacteria that produce more than 75% of antibiotics used in human or veterinary medicine. The production of antibiotics is subject to a number of complex regulations which have not been fully understood, among which we can cite repression by easily assimilable sources of carbon, nitrogen or phosphate.

In Streptomyces, it has long been known that exogenous inorganic phosphate (Pi), namely the inorganic phosphate present in the culture medium, has a very strong inhibitory effect on the biosynthesis of antibiotics.

dégradation des polyP peut être résumé par le schéma ci-dessous :

ppk polyP phosphatases nATP------. polyPn +nADP---------. nPi

Le métabolisme des polyPs a été tout d'abord essentiellement étudié chez E. coli par deux laboratoires américains, celui d'A. Komberg (A. Komberg et al., 1999, Annu. Rev. Many bacterial species, and in particular strains of Streptomyces, are capable of building up reserves of inorganic phosphate in the form of phosphate polymers, polyphosphates (polyP), made up of a few tens to several hundred phosphate residues linked by phospho-anhydride bond. . In E. coli, the main enzyme responsible for the biosynthesis of this polymer (polyP) is the polyphosphate kinase encoded by the ppk gene. This enzyme catalyzes the synthesis of polyP from FATP generated inside the cell. The polyPs can then be degraded to Pi by polyP phosphatases. Polyphosphates therefore constitute an endogenous source of Pi. The mechanism of synthesis and

degradation of polyP can be summarized by the diagram below:

ppk polyP phosphatases nATP ------. polyPn + nADP ---------. nPi

The metabolism of polyPs was first of all essentially studied in E. coli by two American laboratories, that of A. Komberg (A. Komberg et al., 1999, Annu. Rev.

Biochem., 68: 89-125) at Standford, and that of Keasling (Van Dien and Keasling, 1999, Biotechnol. Prog., 15: 587-593) at Berkeley. Then interest was focused on the study of this metabolism in bacteria involved in the processes of depollution of contaminated water.

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en Pi telles qu'Acinetobacter, Pseudomonas aeruginosa, Klebsiella aerogenes, Propionibacterium shermanii.

in Pi such as Acinetobacter, Pseudomonas aeruginosa, Klebsiella aerogenes, Propionibacterium shermanii.

In the various bacterial species mentioned above, the genes responsible for the synthesis (polyP kinase, Ppk) and degradation (polyP phosphatase, Ppx) of the polyPs have been cloned. Interruption or modulation of the level of expression of these genes has enabled the study of the role of polyPs in various biological phenomena.

PolyPs have been found to be involved: - in the metabolism of Pi (in yeast and E. coli). The ability to synthesize polyPs from Pi in some bacteria (mentioned above) is used to reduce Pi pollution in wastewater (Kortstee and van Veen, 1999, Progress in Molecular and
Subcellular Biology, 23,275-97), - in the regeneration of NTP from NDP thanks to the nucleoside diphosphate kinase activity of Ppk (Tzeng and Komberg, 2000, J. of Biol. Chem. 275: 3977-3983), - in various metabolic reactions catalyzed by kinases as a substitute for ATP (Phillips et al., 1999, Progress in Molecular and Subcellular Biology, 23: 101-25), - in the mobility and therefore the virulence of certain pathogenic strains (Rashid et al., 2000,
J. Bacteriol., 182: 225-227), - in the storage and tolerance (chelation / detoxification) to heavy metals (Keasling and Van Dien, 1998, Biotechnol. Bioeng., 58: 231-9). Due to their ability to chelate the divalent cations often necessary for bacterial life, polyPs have a certain antibacterial action (Maier et al., 1999, Appl. Env. Microbiol., 65: 3942-3949), - as a buffer system for alkalis in the green alga Dunaliella salina (Pick and
Weiss, 1991, Plant. Physiol., 97: 1234-1240), in association with polyhydroxybutyrate and Ca in membrane pores which would play a role in the entry of DNA (competence) or ions into the cell (RN

Reusch, 2000, Biochemistry (Moscow), 65: 335-352), in resistance to various stresses (nutritional, osmotic, thermal, oxidative) and in stationary phase survival in E. coli. The polyPs would play the role of alarm signaling a state of stress or deficiency (Ault-Riché et al., 1998, J.

Bacteriol., 180: 1841-1847), - in the regulation of development in different bacteria or fungi.

Although no link has ever been established between the metabolism of polyphosphates and the production of antibiotics whether in Streptomyces bacteria or in other bacteria, it could be assumed that whatever its origin (exogenous or endogenous from of

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hydrolysis of polyP), Pi being found inside the cell, it would exert its repressive effect on the biosynthesis of antibiotics. The inventors, by the present invention have demonstrated that the inorganic phosphate (Pi), produced internally by the strain of Streptomyces lividans from the hydrolysis of polyP, has a strong inhibitory effect on the biosynthesis of antibiotics.

The inventors started from the hypothesis according to which, after depletion of Pi from the culture medium, it is the hydrolysis of the polyPs which provides the Pi necessary for the life of the cell.

A mutant strain for the ppk gene would therefore be incapable of synthesizing polyPs and therefore of building up reserves of Pi. The inventors have demonstrated that when the external medium in Pi is exhausted, the aforementioned mutant strain (in this case a strain of Streptomyces lividans in which the ppk gene, encoding a polyphosphate kinase, is interrupted) is very early and severely deficient in Pi, and this extreme deficiency leads to massive induction of antibiotic biosynthesis (up to 20 to 30 times more antibiotic than the wild strain on an appropriate medium, such as solid R2YE medium).

This result is corroborated by the fact that the Inventors have also demonstrated that the overproductive phenotype of the interruption mutant of ppk is attenuated in the presence of a high concentration of Pi in the medium, and is inversely accentuated when the medium is deficient. in Pi.

The present invention relates to the use of strains of microorganisms producing one or more compounds of interest, and whose biosynthesis is capable of being inhibited by inorganic phosphate of endogenous and / or exogenous origin, said microorganisms being modified so that the ppk gene encoding the polyphosphate kinase is inactivated, for the implementation of a process for stimulating the production of these compounds of interest by said strains of microorganisms thus modified.

Advantageously, said strains of microorganisms are modified by inactivation of the ppk gene, so that they no longer synthesize polyP, and therefore indirectly they no longer produce internal inorganic phosphate (Pi), or produce it in quantities less than those of the strains identical to the previous ones but in which the ppk gene is not inactivated, so that the production of compounds of interest by said modified strains is greater than that of the unmodified strains (in particular by a factor of d 'about 20% to 50% or more).

By method of stimulating the production of compounds of interest, as mentioned above, is meant any method according to which the production of these compounds is increased,

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in particular in proportions of approximately 20% to 50% or more, relative to the production of these same compounds in strains identical to the previous ones but in which the ppk gene has not been inactivated.

The invention relates more particularly to the above-mentioned use of microorganisms producing compounds of interest chosen from metabolites with antibiotic, anticancer, antihelmintic, antifungal, herbicide, insecticide, or enzyme-producing microorganisms.

A more particular subject of the invention is the above-mentioned use of strains of microorganisms of the order Actinomycetales, in particular those producing metabolites with antibiotic activity.

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

The invention relates more particularly to the above-mentioned use of strains listed in Table 1 below in the context of stimulating the production of the antibiotics also listed in this Table I.

Again 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, rgalactnectinectinases, methyl esterases, pectinecthamecturonases, methylesterases, pactanectoguronases, pecthamectinectinases, p-lactoguronases, pyl-lactoguronases, pyl-acetoguronases, p-lactoguronases , cellobiohydrolases, transglutaminases produced by different bacterial genera such as ErniKM, Pseudomonas, Klebsiella, Xanthomonas, and by different species of Bacilli, fungi, yeasts or Actinomycetes, are likely to be produced in greater quantity by microorganisms modified according to the invention, insofar as the synthesis of these enzymes is regulated by the inorganic phosphate.

A more particular subject of the invention is the above-mentioned use of strains of microorganisms in which the ppk gene encoding the polyphosphate kinase is modified by

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addition and / or substitution and / or deletion of at least one nucleotide, such that the ppk gene thus modified no longer codes for an active polyphosphate kinase.

The invention also relates to the use of recombinant nucleotide sequences comprising all or part of the ppk gene encoding polyphosphate kinase in microorganisms producing compounds of interest, such as those defined above, said gene being modified by addition and / or substitution and / or deletion of at least one nucleotide, for the transformation of strains of said microorganisms, with a view to obtaining strains of these microorganisms which no longer produce active polyphosphate kinase.

A more particular subject of the invention is the above-mentioned use of recombinant nucleotide sequences as defined above, comprising a fragment of the ppk gene, the size of this fragment advantageously representing at least approximately 50% of the size of the entire ppk gene, said fragment being modified by addition and / or substitution and / or deletion of at least one nucleotide.

A subject of the invention is also the above-mentioned use of nucleotide sequences derived from the recombinant sequences as defined above, advantageously having a percentage of homology with these recombinant sequences of at least about 70% (namely a percentage capable of allow homologous recombination).

NO : 1), ou comprenant tout ou partie de toute séquence ayant un pourcentage d'homologie avec le gène susmentionné d'au moins environ 70%. The invention relates more particularly to the aforementioned use of recombinant nucleotide sequences as defined above comprising all or part of the ppk gene of Streptomyces coelicolor, said gene being delimited by the nucleotides located at positions 1001 and 3325 of the sequence shown in Figure figure 1 (also called SEQ ID

NO: 1), or comprising all or part of any sequence having a percentage homology with the aforementioned gene of at least about 70%.

A more particular subject of the invention is the above-mentioned use of the recombinant nucleotide sequence delimited by the nucleotides located at positions 744 and 2869 of the sequence represented in FIG. 1, and modified as indicated above.

Advantageously, the recombinant nucleotide sequences comprising all or part of the ppk gene of Streptomyces coelicolor, as defined above, are used for the transformation of any strain of microorganisms in which the ppk gene has a

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percentage homology with the Streptomyces coelicolor ppk gene of at least about 70%.

lividans, S. actuosus, S. bikiniensis, S. glaucesens, S. graminofaciens, S. griseofuscus, S. lincolnensis, S. parvulus, S. pristinaespiralis, S. tendae, S. venezuelae, S. violaceoniger, S. wadayamensis, S. ambofaciens ATCC 23877 et S. ambofaciens ATCC 15154. As such, the recombinant nucleotide sequences comprising all or part of the ppk gene of Streptomyces coelicolor, as defined above, are used not only for the transformation of the S. coelicolor strain itself, but also for the transformation of any strain of Streptomyces, and more particularly of Streptomyces

lividans, S. actuosus, S. bikiniensis, S. glaucesens, S. graminofaciens, S. griseofuscus, S. lincolnensis, S. parvulus, S. pristinaespiralis, S. tendae, S. venezuelae, S. violaceoniger, S. wadayamensis, S. ambofaciens ATCC 23877 and S. ambofaciens ATCC 15154.

A subject of the invention is also any recombinant nucleotide sequence comprising all or part of the ppk gene encoding polyphosphate kinase in microorganisms producing metabolites or enzymes, such as those defined in Table I, said gene 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, characterized in that it comprises a fragment of the ppk gene, the size of this fragment advantageously representing at least approximately 50% of the size of the ppk gene. entire, said fragment being modified by addition and / or substitution and / or deletion of at least one nucleotide.

The invention relates more particularly to any recombinant nucleotide sequence as defined above, characterized in that the ppk gene is modified by inserting a heterogeneous sequence at a restriction site determined in the sequence of the gene.

A subject of the invention is also any recombinant nucleotide sequence as defined above, characterized in that the nucleotide sequence encoding the polyphosphate kinase is 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 hygromycin.

The invention also relates to any nucleotide sequence as defined above, comprising all or part of the ppk gene of Streptomyces coelicolor, said gene being delimited by the nucleotides located at positions 1001 and 3325 of the sequence shown in FIG. 1, or comprising all or part of any sequence having a percentage homology with the aforementioned gene of at least about 70%.

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A more particular subject of the invention is any nucleotide sequence as defined above, delimited by the nucleotides located at positions 744 and 2869 of the sequence shown in FIG. 1.

The invention also relates to any nucleotide sequence as defined above, characterized in that the gene or the fragment of the ppk gene is modified by insertion at the level of the StuI site delimited by positions 1352 and 1357 of the sequence represented in FIG. 1. , of a heterogeneous sequence, such as a cassette containing a marker, in particular a marker for resistance to an antibiotic, such as hygromycin (in particular the 2300 nucleotide cassette comprising a marker for resistance to hygromycin as described in Blondelet-Rouault etal., 1997).

A subject of the invention is also any vector, in particular any plasmid, where appropriate with thermosensitive 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 ppk gene encoding the polyphosphate kinase 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 the order Actinomycetales, such as those defined in Table I.

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

A more particular subject of the invention is the above-mentioned strains of microorganisms, as obtained by transformation of said microorganisms with a vector defined above.

A 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 all or part of the ppk gene of the strain of microorganism that it is desired to modify, and the amplification of a nucleotide sequence such as

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as defined above containing all or part of the ppk gene of said strain, using two oligonucleotide primers chosen from known sequences located in the ppk gene, or located on either side of this gene, - modification of the nucleotide sequence containing all or part of the ppk gene of strains of microorganisms, such as that obtained in 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 aforementioned recombinant nucleotide sequence instead of the nucleotide sequence of the original ppk gene.

According to a particularly advantageous embodiment of the method of the invention, the nucleotide sequence as defined above containing all or part of the ppk gene, is modified by inserting a cassette comprising a marker, in particular a marker for resistance to a. antibiotic, in order to select the modified strains obtained. The recombinant sequence thus obtained is then cloned in an appropriate vector, in particular in 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 aforementioned vector, the selection of the modified strains is carried out directly by observing the presence or absence 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 heat-sensitive replication, or is not replicative.

l'invention, la séquence nucléotidique telle que définie ci-dessus contenant tout ou partie du gène ppk, est modifiée par addition ou délétion d'au moins un nucléotide. Dans ce cas, il est préférable d'utiliser un vecteur à réplication thermosensible, et, après passage à haute température, on repère les colonies ayant perdu le plasmide (colonies sensibles à l'antibiotique auquel le plasmide confère d'ordinaire la résistance) et qui surproduisent l'antibiotique sur un milieu approprié. Ces colonies pourront être repérées sur boîte de milieu de culture approprié en les faisant pousser en présence d'une souche sensible à l'antibiotique produit. According to another particularly advantageous embodiment of the method of

the invention, the nucleotide sequence as defined above containing all or part of the ppk gene, 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 passage at high temperature, the colonies which have lost the plasmid are identified (colonies sensitive to the antibiotic to which the plasmid usually confers resistance) and which overproduce the antibiotic on a suitable medium. These colonies can be identified on a dish 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. Firstly, the nucleotide sequence as defined above containing all or part of the ppk gene, is modified by insertion of an aforementioned cassette conferring resistance to an antibiotic, then this sequence modified by insertion is replaced by a version

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délétée de cette séquence. Dans ce cas, on repère les colonies redevenues sensibles à l'antibiotique auquel la cassette confère d'ordinaire la résistance.

deleted from this sequence. In this case, the colonies which have become again sensitive to the antibiotic to which the cassette usually confers resistance are identified.

A subject of the invention is also a method for stimulating the production of metabolites, in particular of metabolites with antibiotic or other activity, or of enzymes, by microorganisms used for the production of these compounds, said method comprising the implementation culture of strains of modified microorganisms as defined above, in an appropriate 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 as a source of carbon and nitrogen) with an osmotic pressure equivalent to that conferred by 103 g / 1 of sucrose and without the addition of exogenous inorganic phosphate.

I) Organismes producteurs d'aminocyclitols

ORGANISME ANTIBIOTIQUE Bacillus (espèces variées) Ammocyclitols Mjtromonospora (espèces variées) Gentamycines Saccharopolyspora (espèces variées) Aminocyclitols Streptomyces albogriseolus Neomycines albus var. metamycinus Metamycine aquacanus N-methyl hygromycine B atrofaciens Hygromycines bikiniensis Streptomycine bluensis var. bluensis bluensis var. bluensis Bluensomycine canus Ribosyl paromamine catenulae Catenuline chrestomyceticus aminosidine crystallinus hygromycine A erythrochromogenes streptomycine var. narutoensis eurocidicus A16316-C fradiae hybrimycines et neomycine fradiae var. italicus aminosidine galbus streptomycine griseus streptomycine griseoflavus MA 1267 hofuensis seldomycine complexe hygroscopicus hygromycinse hyg-roscopicusforma leucanicidine et hygrolidine glebosus glebomycine Table 1 List of antibiotic producing organizations

I) Organisms producing aminocyclitols

ANTIBIOTIC ORGANISM Bacillus (various species) Ammocyclitols Mjtromonospora (various species) Gentamycins Saccharopolyspora (various species) Aminocyclitols Streptomyces albogriseolus Neomycins albus var. metamycinus Metamycin aquacanus N-methyl hygromycin B atrofaciens Hygromycins bikiniensis Streptomycin bluensis var. bluensis bluensis var. bluensis Bluensomycin canus Ribosyl paromamine catenulae Catenulin chrestomyceticus aminosidin crystallinus hygromycin A erythrochromogenes streptomycin var. narutoensis eurocidicus A16316-C fradiae hybrimycins and neomycin fradiae var. italicus aminosidin galbus streptomycin griseus streptomycin griseoflavus MA 1267 hofuensis seldomycin complex hygroscopicus hygromycinse hyg-roscopicusforma leucanicidin and hygrolidine glebosus glebomycin

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hygroscopicus var. limoneus validamycines hygroscopicus var. sagamiensis spectinomycine kanamyceticus kanamycine A et B kasuqaensis kasugamycines kasugaspinus kasugamycins lavendulae neomycine lavendulae neomvome lividus lividus lividomycines mashuensis rnashuensis streptomycine microsporeus SF-767 netropsis LL-AM31 noboritoensis hygromycines olzvaceus olivaceus streptomycine olivoreticuli var. cellulophilus destomycine A poolensis poolensis streptomycine rameus streptomycine ribosidificus SF733 rimofaciens rimofaciens destomycine A rimosus forma paromomycinus paromomycines, catenuline spectabilis spectinomycine tenebrarius tenebrarius tobramycine et apramycine Streptoverticilliumflavopersieus spectinomycine

II) Organismes producteurs d'Ansamycine

ORGANISME ANTIBIOTIQUE Micromonospora (espèces variées) ansamycmes Nocardia mediterranei rifamycine Streptomyces collinus ansatrienes, napthomycines diastochromogenes ansatrienes, napthomycines galbus subsp. griseosporeus napthomycine B hygroscopicus herbimycine hygroscopicus var. geldanus geldamycine var. nova nigellus 21-hydroxy-25-demethyl 25-methylthioprotostreptovaricine rishiriensis mycotrienes sp. E/784 actamycin and mycotriene sp. E88 mycotrienes spectabilis streptovaricines tolypophorous tolypomycine

III) Organismes producteurs d'Anthracycline et de Quinone

ORGANISME ANTIBIOTIQUE Streptomyces caespitosus mitomycines A, B, et C coelicolor actmorllodme coelicolor actinorhodine coeruleorubidicus daunomycine cyaneus cyan eus ditrisarubicine flavogriseus cyanocycline A

hygroscopicus var. limoneus validamycins hygroscopicus var. sagamiensis spectinomycin kanamyceticus kanamycin A and B kasuqaensis kasugamycins kasugaspinus kasugamycins lavendulae neomycin lavendulae neomvome lividus lividus lividomycins mashuensis rnashuensis streptomycins olaceusi oleuszieus7 streptomycin-colobycin-colobycins7 nashuensis lividomycins1 streptomycin 767 losporziuensis streptomycin 76731-losporiveus7 nashuensis lividomycins mashuensis streptomycin-7 31ospreus7-lospreus7 lospreusi-vizicis-712 lospreusvidomycinsis lividomycins lividomycinsi-vareusi-vareusi-ostreus7-serosi cellulophilus destomycin A poolensis poolensis streptomycin rameus streptomycin ribosidificus SF733 rimofaciens rimofaciens destomycin A rimosus forma paromomycinus paromomycins, catenulin spectabilis spectinomycin tenebrarius tenebrarius tobramycin and apramycinsomycin Streptycin and apramycinus

II) Ansamycin producing organisms

ANTIBIOTIC ORGANISM Micromonospora (various species) ansamycms 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-methylthioprotostreptovaricine rishiriensis mycotrienes sp. E / 784 actamycin and mycotriene sp. E88 mycotrienes spectabilis streptovaricins tolypophorous tolypomycin

III) Organisms producing Anthracycline and Quinone

ANTIBIOTIC ORGANISM Streptomyces caespitosus mitomycins A, B, and C coelicolor actmorllodme coelicolor actinorhodine coeruleorubidicus daunomycin cyaneus cyan eus ditrisarubicine flavogriseus cyanocycline A

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galilaeus aclacinomycine A, auramycines, sulfurmycines lusitanus napthyridinomycine peuceticus daunomycine, adriamycine violochromoqenes arugomycine

IV) Organismes producteurs de Beta Lactame

ORGANISME ANTIBIOTIQUE Cephalosporium (espèces variées) beta-lactames Nocardia lactamadurans cephamycine C Penicillium (espèces variées) beta-lactames Streptomyces antibioticus acide clavulanique argenteolus asparenomycine A, MM 4550, MM 13902 cattleya thienamycine chartreusis SF 1623, cephamycine A and B cinnamonensis cephamycine A and B clavuligerus PA-32413-1, cephamycine C, A16886A, acide clavulanique, et autres clavames fimbriatus cephamycine A and B flavovirens MM 4550 et MM 13902 flavus MM 4550 et MM 13902 fulvoviridis MM 4550 et MM 13902 griseus cephamycine A et B halstedi cephamycine A et B heteromorphus C2081X, cephamycine A, B hygroscopicus deacetoxycephalosporine C lipmanii penicilline N, 7-methoxycephalosporine C, Al 6884, MM 4550, et MM 13902 olivaceus (MM 17880) epithienamycine F, MM 4550, et MM 13902 panayensis C2081X, cephamycine A, B pluracidomyceticus pluracidomycine A rochei cephamycine A et B sioyaensis MM 4550 et MM 13902 sp. OA-6129 OA-6129A sp. KC-6643 carpetimycine A tokunomensis asparenomycine A viridochromogenes cephamycine A et B wadayamensis WS-3442-D

galilaeus aclacinomycin A, auramycins, sulfurmycins lusitanus napthyridinomycin peuceticus daunomycin, adriamycin violochromoqenes arugomycin

IV) Beta Lactam producing organizations

ANTIBIOTIC ORGANISM Cephalosporium (various species) beta-lactams Nocardia lactamadurans cephamycin C Penicillium (various species) beta-lactams Streptomyces antibioticus clavulanic acid argenteolus asparenomycin A, MM 4550, MM 13902 cattleya thienamycin 1623 and cephamycin cephamycin and cephamycin Sf clavuligerus PA-32413-1, cephamycin C, A16886A, clavulanic acid, and other clavams fimbriatus cephamycin A and B flavovirens MM 4550 and MM 13902 flavus MM 4550 and MM 13902 fulvoviridis MM cephamycin A and MM 13902 griseus cephamycin A and B halstedi B heteromorphus C2081X, cephamycin A, B hygroscopicus deacetoxycephalosporin C lipmanii penicillin N, 7-methoxycephalosporin C, Al 6884, MM 4550, and MM 13902 olivaceus (MM 17880) epithien cepamycin F, MM 4550, and MM 13902 panXayensis, Bychamensis pluracidomyceticus pluracidomycin A rochei cephamycin A and B sioyaensis MM 4550 and MM 13902 sp. OA-6129 OA-6129A sp. KC-6643 carpetimycin A tokunomensis asparenomycin A viridochromogenes cephamycin A and B wadayamensis WS-3442-D

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V) Organismes producteurs de Macrolide, Lincosamide, et Streptogramine

ORGANISME ANTIBIOTIQUE Micromonospora rosaria rosaramicine Streptomyces albireticuli carbomycine aoMeo mikonomycine albus albomycetine 17 mlkonomyome albus var. coilmyceticus coleimycine ambofaczens ambofaciens spiramycine, foromacidine D antibioticus antibiotieus oleandomycine avermitilis avermitilis avermectines 6'nM chalcomycine bruneogriseus albocycline cae/eM M188, celesticetme cinerochromogenes cmeromycine B cirratus cirramycine deltae deltamycines dyakartensis djakartensis niddamycine erythreus erythromycines eurocidicus methymycine eurythermus angolamycine fasciculus amaromycine felleus argomycine et picromycine amaromycine flavochromogenes amaromycine, et shincomycines fradiae tylosine fungicidicus NA-181 fungicidicus var. espinomyceticus espinomycetines furdicidicus furdicidicus mydecamycine goshikiensis bandamycine griseofaciens PA133A et B griseoflavus acumycme griseofuscus bundline griseolus griseomycine griseospiralis relomycine griseus borrelidine griseus ssp. sulphurus bafilomycines halstedi carbomycine, leucanicidine hygroscopicus tylosine hygroscopicus subsp aureolacrimosus milbemycines oeM leucomycine A. sub. 3, etjosamycine lavendulae aldgamycine Izncolnensis lincolnensis lincomycine loidensis vemamycine A et B macrosporeus carbomycine maizeus ingrainycine

V) Organisms producing Macrolide, Lincosamide, and Streptogramin

ANTIBIOTIC ORGANISM Micromonospora rosaria rosaramicin Streptomyces albireticuli carbomycin aoMeo mikonomycin albus albomycetine 17 mlkonomyome albus var. coilmyceticus coleimycine ambofaczens ambofaciens spiramycin, oleandomycin foromacidine D antibioticus antibiotieus avermitilis avermitilis avermectins 6'nM chalcomycin bruneogriseus albocycline cae / eM M188, celesticetme cinerochromogenes cmeromycine B cirratus cirramycine DeltaE deltamycines dyakartensis djakartensis niddamycin erythreus erythromycins eurocidicus methymycin eurythermus angolamycin fasciculus amaromycin felleus argomycine and picromycin amaromycin flavochromogenes amaromycin, and shincomycins fradiae tylosin fungicidicus NA-181 fungicidicus var. espinomyceticus espinomycetines furdicidicus furdicidicus mydecamycin goshikiensis bandamycin griseofaciens PA133A and B griseoflavus acumycme griseofuscus bundline griseolus griseomycin griseospiralis relomycin griseus borrelidin griseus ssp. sulphurus bafilomycins halstedi carbomycin, leucanicidin hygroscopicus tylosin hygroscopicus subsp aureolacrimosus milbemycines oeM leucomycin A. sub. 3, etjosamycin lavendulae aldgamycin Izncolnensis lincolnensis lincomycin loidensis vemamycin A and B macrosporeus carbomycin maizeus ingrainycin

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mycarofaciens acetyl-leukomycine, et espinomycine narbonensis josamycine et narbomycine narbonensis var. josamyceticus leucornycine A, josamycine olivochromogenes oleandomycine platensis platenomycine rimosus tylosine et neutramycine rochei lankacidine et borrelidine rocheivar. volubilis T2636 roseochromogenes albocyline roseocitreus albocycline spinichromoenes var. suragaoensis kujirnycines tendae carbomycine thermotolerans carbomycine venezuelae methymycines violaceoniger lankacidines, lankamycine

VI) Espèces de Streptomyces producteurs d'antibiotiques variés

a) Analogues d'aminoacides Streptomyces sp. Cycloserine b) Contenant des noyaux cyclopentanes Streptomyces coelicolor methylenomycine A erythrochromogenes sarkomycine violaceoruber methylenomycine A c) Containing de l'azote Streptomyces venezuelae chloramphenicol d) Polyènes Streptomyces griseus candicidine nodosus amphotericine B noursei nystatine e) Tétracyclines Streptomyces aureofaciens tetracycline, chlortetracycline, demethyltetracycline, et demethylchlortetra-cycline rimosus oxytetracycline

VII) Organismes producteurs de Nucléosides

ORGANISME ANTIBIOTIQUE Corynebacterium michiganese pv. rathayi analogues de tunicamycine Nocardia candidus pyrazofurine Streptomyces antibioticus ara-A chartreusis tunicamycine

mycarofaciens acetyl-leukomycin, and espinomycin narbonensis josamycin and narbomycin narbonensis var. josamyceticus leucornycin A, josamycin olivochromogenes oleandomycin platensis platenomycin rimosus tylosin and neutramycin rochei lankacidin and borrelidin rocheivar. volubilis T2636 roseochromogenes albocyline roseocitreus albocycline spinichromoenes var. suragaoensis kujirnycines tendae carbomycin thermotolerans carbomycin venezuelae methymycins violaceoniger lankacidins, lankamycin

VI) Streptomyces species producing various antibiotics

a) Amino acid analogues Streptomyces sp. Cycloserine b) Containing nuclei cyclopentanes Streptomyces coelicolor methylenomycin A erythrochromogenes sarkomycine violaceoruber methylenomycin A c) Containing the Streptomyces venezuelae chloramphenicol nitrogen d) polyenes Streptomyces griseus candicidin nodosus amphotericin B noursei nystatin e) Tetracyclines Streptomyces aureofaciens tetracycline, chlortetracycline, demethyltetracycline, and demethylchlortetra -cycline rimosus oxytetracycline

VII) Nucleoside producing organisms

ANTIBIOTIC ORGANISM Corynebacterium michiganese pv. rathayi tunicamycin analogues Nocardia candidus pyrazofurin Streptomyces antibioticus ara-A chartreusis tunicamycin

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VIII) Organismes producteurs de Peptides

ORGANISME ANTIBIOTIQUE Actinoplanes missouriensis actaplanine teichomyceticus teicoplanine Bacillus (espèces variées) bacitracine, polymixine, et colistine Nocardia candidus A-35512 et avoparcine lurida ristocetine orientalis vancomycme Streptomyces antibioticus actinomycine aureus thiostreptone canus amphomycine eburosporeus LL-AM374 haranomachiensis vancomycine pristinaespiralis pristinamycine roseosporus lipopeptides, tel A21978C toyocaensis A47934 virginiae A1030

IX) Organismes producteurs d'Antibiotiques

ORGANISME ANTIBIOTIQUE Actinomadura (espèces variées) polyéthers variés Dactylosporangium (espèces variées) polyéthers variés Nocardia (espèces variées) polyéthers variés Stre Streptomyces albus A204, A28695A et B, et salinomycine aureofaciens narasine cacaoi var. asoensis lysocelline chartreusis A23187 cmnamonensis monensine conglobatus ioilomycine eurocidicus var. asterocidicus laidlomycine flaveolus CP38936 gallinarius RP 30504 griseus grisorixine hygroscopicus A218, emericide, DE3936, A120A, A28695A et B, etheromycine, dianemycine lasaliensis lasalocide longwoodensis lysocelline mutabilis S-1173 a ribosidificus lonomycine violaceoniger nigericine Streptoverticillium (espèces variées) polyéthers variés griseoflavus var. streptoviridans thuringiensis griseolus sinefungin

VIII) Peptide-producing organisms

Organization ANTIBIOTIC Actinoplanes missouriensis actaplanin teichomyceticus teicoplanin Bacillus (various species) bacitracin, polymyxin, and colistin Nocardia candidus A-35512 and avoparcin lurida ristocetin orientalis vancomycme Streptomyces antibioticus actinomycin aureus thiostrepton canus amphomycin eburosporeus LL-AM374 haranomachiensis vancomycin pristinaespiralis pristinamycin roseosporus lipopeptides, such as 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 lysocelline chartreusis A23187 cmnamonensis monensine conglobatus ioilomycin eurocidicus var. asterocidicus laidlomycin flaveolus CP38936 gallinarius RP 30504 griseus grisorixin hygroscopicus A218, emericide, DE3936, A120A, A28695A and B, etheromycin, dianemycin lasaliensis lasalocid longwoodensis lysocellin mutabigosin S-1173 varicidicetium lysocellin mutabigabilisin S-1173 varicillicosidium nycellabilisin S-1173 nycellabilisin mutigerosidisinifericillicidium nycigabilisin S-1173 varietigerosigerosigerosigerosigerosigerosigerosigerosigero-1173 mutigabilisin mutigabilisin (S-1173)

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The invention will be further detailed with the aid of the following description of the cloning and disruption of the ppk gene of Streptomyces lividans: A gene encoding a protein having 57% identity with the polyphosphate kinase of E. coli (Akiyama et al., 1992) was found in the genome undergoing sequencing of Streptomyces coelicolor, a species closely related to S. lividans. This gene codes for a putative protein of 774 amino acids.

- A unique StuI (blunt-edged enzyme) site located in the first third of the gene was identified (it is underlined on the sequence shown in Figure 1) and considered to be well located to serve as an insertion site for a cassette interruption.

- Two oligos A and B (respectively represented by the nucleotides located at positions 744 and 763, and those located at positions 2850 and 2869 of the sequence shown in Figure 1) were then designed so that the StuI site is located in the center of a 2106 bp fragment after PCR amplification.

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

- pHCl was digested with StuI (unique site in the plasmid) and the Q Hygro cassette which confers resistance to hygromycin (Blondelet-Rouault et al., 1997) was cloned into this site, in the form of a fragment BamHI klenowté to give pHC2.

- The insert of pHC2 was transferred in the form of a B. g711 fragment into the BamHI site of pGM160 (Muth et al., 1989). pGM160 is an E. coli / S shuttle vector. thermosensitive replication lividans in S. lividans. It carries the bla and tsr genes which confer resistance to ampicillin in E. coli and to nosiheptide in S. lividans respectively, as well as the aacC7 gene which confers resistance to apramycin in E. coli and S. lividgns. This new plasmid pHC3 was 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-type ppk gene with its interrupted allele, a colony of S. lividans TK24 containing pHC3 and therefore resistant to nosiheptide was crushed and grown for 24 h at 30 C in 2 ml of TSB medium (Hopwood et al., 1985 containing nosiheptide at 20 ng / 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 hygromycin at 50 ag / ml.

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- La culture a ensuite été mise à pousser à 41 C pendant 48 heures afin de permettre l'élimination du plasmide réplicatif.

- The culture was then grown at 41 ° C. for 48 hours in order to allow the elimination of the replicative plasmid.

mycéliens correspondant à une seule cellule. Ces derniers ont été étalés en dilution sur des boîtes de milieu HT agar (Hopwood et al., 1985) contenant de l'hygromycine à 50 Jlglml afin d'avoir des colonies bien séparées. - The mycelium was then harvested, crushed, and sonicated so as to have fragments

mycelia corresponding to a single cell. The latter were spread in dilution on plates of HT agar medium (Hopwood et al., 1985) containing hygromycin at 50 μlglml in order to have well separated colonies.

- The sporulated colonies were then replicated on HT medium and HT medium containing nosiheptide at 20 μlglml in order to identify the colonies sensitive to nosiheptide. About 33% of the hygromycin resistant colonies were sensitive to nosiheptide. These had lost the replicative plasmid.

The chromosomal DNA structure of four independent colonies resistant to hygromycin and sensitive to nosiheptide was then determined by the Southem blot technique (Hopwood et al., 1985). Chromosomal DNA was prepared from these four colonies, was digested with XhoI (which does not cut in the Qhygro cassette), migrated on 1% agarose gel, transferred to Hybond membrane and hybridized with the fragmentppk amplified by PCR.

The results show (FIG. 2) that in colony n 4, the size of the initial XhoI chromosomal fragment of 1.8 kb carrying the ppk gene is increased by 2.3 kb (size of the Q2ro cassette), it is therefore 4.1 kb. In this case, we did have the replacement of the wild-type ppk gene by the interrupted allele.

QUANTIFICATION We quantified the production of actinorhodin (blue compound) as follows: About 106 spores of the wild-type reference strain and of the strain interrupted for the ppk gene were plated on a dish of rich standard R2YE gel medium at the surface of which had been deposited a cellophane membrane allowing nutrients to pass through.

These boxes were grown for about two weeks at 30 C.

By lifting the cellophane membrane, cores of gelose medium were taken from each of the two types of dishes using a suitable punch.

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Each of two types of carrots was dissolved in 1ml of H2O by heating for 1 Omn at 90 C.

The absorbance of the solutions thus obtained was determined at 600 nm (wavelength at which the antibiotic actinorhodin, blue compound, absorbs) using a spectrophotometer against a blank consisting of H2O.

Solutions where the carrots were from the medium where the strain inactivated for the ppk gene had grown exhibited 20 to 30 times greater uptake than those of carrots from the medium where the wild strain had grown.

This detection method is valid in this case because we are dealing with a colored antibiotic, but this situation is rare. In most cases, the detection of overproducing strains will be carried out in the presence of a strain of microorganism (called the indicator strain) sensitive to the antibiotic produced.

sucrose : 103g K2SO4 : 0. 25g

MgCl2. 6H20 : 10. 12g

Glucose : 10g Casaminoacids Difco : O. lag Solution d'éléments minéraux* : 2ml Extrait de levure Difco : 5g

Tampon TES : 5. 73g

Agar : 22g Autoclaver et ajouter après autoclavage : KH2P04à0. 5% : 10ml R2YE MEDIUM Composition of the R2YE medium (Hopwood et al., 1985) used for the test of the actinorhoddine hyper-production phenotype of the interruption mutant of the ppk gene of Streptomyces lividans (P9) For 1 liter:

sucrose: 103g K2SO4: 0.25g

MgCl2. 6:20 am 10. 12g

Glucose: 10g Casaminoacids Difco: O. lag Solution of mineral elements *: 2ml Difco yeast extract: 5g

TES buffer: 5.73g

Agar: 22g Autoclave and add after autoclaving: KH2P04à0. 5%: 10ml

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CaCl2. 2H20 à 5M : 4ml L-prolineà 20% : 15ml NaOh à 1M : 7ml Solution d'éléments minéraux pour 1 litre : # -ZnCl2 : 40mg

.-FeCl3. 6H20 : 200mg .-CuCl2. 2H20 : 10mg

'-MnCl2. 4H20 : 10mg

*-Na2B407. 10H20 : 10mg *- (NH4) 6Mo7024. 4H20 : 10mg 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. JBiol Chem. 267 (31) : 22556-61.

CaCl2. 2H20 at 5M: 4ml L-proline at 20%: 15ml NaOh at 1M: 7ml Solution of mineral elements for 1 liter: # -ZnCl2: 40mg

.-FeCl3. 6H20: 200mg.-CuCl2. 2H20: 10mg

'-MnCl2. 4H20: 10mg

* -Na2B407. 10H20: 10mg * - (NH4) 6Mo7024. 4:20: 10mg 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. JBiol 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. 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 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. Uncomfortable. 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.

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LEGEND TO THE FIGURES Figure 1: Nucleotide sequence of a 3780bp fragment of S. coelicolor containing the ppk gene encoding polyphosphate kinase. Translation start and stop codons are in bold. The oligonucleotides A and B used for PCR amplification of the DNA fragment which served to interrupt the ppk gene of S. lividans are in bold and italic characters. The StuI site and the two XhoI sites internal to ppk are respectively underlined and double underlined.

Figure 2: The chromosomal DNAs of the wild strain TK24 (well TK24) and of 4 colonies derived from this strain (wells 1, 2, 3 and 4) but having undergone a standard gene replacement procedure described above were digested by XhoI, migrated on 1% agarose gel, transferred to Hybond membrane and hybridized with the fragment pk amplified by PCR. The kb well contains a scale of size markers.

Claims (22)

1. Use of strains of microorganisms producing compounds of interest, in which the ppk gene encoding the polyphosphate kinase is inactivated, for the implementation of a process for stimulating the production of these compounds of interest by said strains transformed. 2. Use according to claim 1, of strains of microorganisms producing compounds of interest chosen from metabolites with antibiotic, anticancer, antihelmintic, antifungal, herbicide, insecticide or enzymatic activity. 3. Use according to claim 1 or 2, of strains of Actinomycetes producing metabolites with antibiotic activity chosen from strains of Streptomyces, Bacillus, Micromonospora, Saccharopolyspora, Streptoverticillium, Nocardia, Cephalosporium, Penicillium, Corynebacterium, Actinoplanes, Actinomadura, Dactylosporangium, and Mycobacterium. 4. Use according to one of claims 1 to 3, of strains of microorganisms in which the ppk gene encoding the polyphosphate kinase is modified by addition and / or substitution and / or deletion of at least one nucleotide, in such a way that the ppk gene thus modified no longer codes for an active polyphosphate kinase.

5. Use of recombinant nucleotide sequences comprising all or part of the ppk gene encoding polyphosphate kinase in microorganisms producing compounds of interest, such as those defined in claim 2 or 3, said gene being modified by addition and / or substitution and / or deletion of at least one nucleotide, for the transformation of strains of said microorganisms, with a view to obtaining strains of these microorganisms which no longer produce active polyphosphate kinase. 6. Use according to claim 5, of recombinant nucleotide sequences comprising a fragment of the ppk gene, the size of this fragment representing at least about 50% of the size of the entire ppk gene, said fragment being modified by addition and / or substitution and / or deletion of at least one nucleotide. <Desc / Clms Page number 21> 7. Use according to claim 5 or 6, of nucleotide sequences derived from the recombinant sequences as defined in claims 5 and 6, advantageously having a percentage of homology with these recombinant sequences of at least about 70%. 8. Use according to one of claims 5 to 7, of recombinant nucleotide sequences comprising all or part of the ppk gene of Streptomyces coelicolor, said gene being delimited by the nucleotides located at positions 1001 and 3325 of the sequence shown in FIG. 1, or comprising all or part of any sequence having a percentage homology with the aforementioned gene of at least about 70%. 9. Use according to one of claims 5 to 8, of the nucleotide sequence delimited by the nucleotides located at positions 744 and 2869 of the sequence shown in Figure 1. 10. Recombinant nucleotide sequence comprising all or part of the ppk gene encoding polyphosphate kinase in microorganisms producing compounds of interest, such as those defined in claim 2 or 3, said gene being modified by addition and / or substitution. and / or deletion of at least one nucleotide. 11. Recombinant nucleotide sequence according to claim 10, characterized in that it comprises a fragment of the ppk gene, the size of this fragment representing at least about 50% of the size of the entire ppk gene, said fragment being modified by addition and / or substitution and / or deletion of at least one nucleotide. 12. Recombinant nucleotide sequence according to claim 10 or 11, characterized in that the ppk gene is modified by insertion of a heterogeneous sequence at a restriction site determined in the sequence of the gene. 13. Recombinant nucleotide sequence according to one of claims 10 to 12, characterized in that the nucleotide sequence encoding the polyphosphate kinase is <Desc / Clms Page number 22> modified by inserting, at a determined restriction site of said sequence, a cassette containing a marker, such as a marker for resistance to an antibiotic, in particular hygromycin. 14. Recombinant nucleotide sequences according to one of claims 10 to 13, comprising all or part of the ppk gene of Streptomyces coelicolor, said gene being delimited by the nucleotides located at positions 1001 and 3325 of the sequence shown in FIG. 1, or comprising all or part of any sequence having a percentage homology with the aforementioned gene of at least about 70%. 15. Recombinant nucleotide sequence according to one of claims 10 to 14, delimited by the nucleotides located at positions 744 and 2869 of the sequence shown in Figure 1. 16. Recombinant nucleotide sequence according to claim 14 or 15, characterized in that the gene or the fragment of the ppk gene is modified by insertion at the level of the Stuhl site delimited by positions 1352 and 1357 of sequence shown in FIG. 1, d a heterogeneous sequence, such as a cassette containing a marker, in particular a marker for resistance to an antibiotic, such as hygromycin. 17. Vector containing a recombinant nucleotide sequence according to one of claims 10 to 16. 18. Strains of microorganisms producing compounds of interest, said microorganisms being modified so that the ppk gene encoding the polyphosphate kinase is inactivated. 19. Strains of microorganisms according to claim 18, said strains being producers of compounds of interest defined in claim 2. 20. Strains of microorganisms according to claim 18 or 19, characterized in that they are chosen from strains of the order Actinomycetales, such as those defined in claim 3. <Desc / Clms Page number 23> 21. Strains of microorganisms according to one of claims 18 to 20, as obtained by transforming said microorganisms with a vector according to claim 17. 22. A method of stimulating the production of compounds of interest, in particular of compounds defined in claim 2, by microorganisms usually used for the production of these compounds, said method comprising the cultivation of strains of these micro- organisms. transformed organisms as defined in one of claims 18 to 21 in a suitable culture medium.