FR2693475A1 - Identification and opt. cloning of gene promoters - using DNA library contg. transposon bearing selectable marker - Google Patents

Abstract

Process for identifying and/or cloning functional transcriptional promoters in a host comprises: (a) preparing a DNA library of a starting cell; (b) inserting a transposon bearing a selectable marker into the DNA; (c) selecting clones expressing the marker; and (d) isolating DNA fragments comprising the promoter regions of the selected clones. Also claimed are a process for producing a recombinant protein by expression of a DNA sequence coding for the protein under the control of a promoter obtained by the above process and a Kluyveromyces DNA library into which a MiniMu transposon contg. a selectable marker has been inserted. USE/ADVANTAGE - The promoters may be used for expression of genes coding for pharmaceutical or food proteins or genes for the biosynthesis of metabolites, e.g. amino acids, vitamins or antibiotics. The process can be applied to all types of starting cells without prior knowledge of the function or structure of the gene naturally expressed by the promoter.

Description

IDENTIFICATION AND / OR CLONING OF PROMOTERS
TRANSCRIPTIONAL. AND USE OF THESE PROMOTERS FOR
THE EXPRESSION OF GENES
The present invention relates to the field of genetic engineering. More particularly, the invention relates to a method for the identification and cloning of transcriptional promoters.

 Advances in molecular biology have made it possible to modify microorganisms to make them produce heterologous proteins. In particular, numerous genetic studies have focused on the bacteria E. coli and on the yeast Saccharomyces. However, the industrial application of these new modes of production is still limited, in particular by the problems of efficiency of expression of genes in these recombinant microorganisms. Also, in order to increase the performance of these production systems, it is necessary to be able to have strong promoters, making it possible to obtain high levels of expression of heterologous proteins. It may also be desirable to have a large number of varied promoters, active under different physiological conditions.

At Ecoli, certain strong promoters have been described (promoters of the tryptophan and lactose operons). Similarly, in the yeast S. cereuisiae, studies have focused on promoters derived from genes involved in glycolysis. Mention may in particular be made of the work on the promoter of the gene for the 3-phosphoglycerate kinase PGK (Dobson et al., Nucleic Acid Res. IQ, 1982, 2625; Hitzeman et al., Nucleic Acid Research 1982, 7791), on that of GAPDH glyceraldehyde-3-phosphate dehydrogenase gene (Holland et al.,
J. Biol. Chem. 254, 1979, 9839; Musti et al., Gene X, 1983, 133), on that of the alcohol dehydrogenase 1 gene ADH1 (Boulezzen et al., J. Biol. Chem.

257 1982, 3018; Denis et al., J. Biol. Chem. X, 1983, 1165), on that of the enolase 1 gene SEND (Uemura et al., Gene 45 1986, 65), on that of the gene
GAL1 / GAL10 Uohnston and Davis, Mol. Cell. Biol. 4 (1984) 1440) or that of the CYC1 gene (Guarente and Ptashne, PNAS 78 (1981) 2199).

 However, it is clear that the genome of these cells could not be studied completely, and that it potentially contains other strong promoters which have not yet been identified. Likewise, more and more, recombinant methods aim today to use new types of host cells (animal, plant cells, yeasts, fungi or bacteria).

Thus, genetic tools have been developed in order to use the Kluyveromyces yeast as a host cell for the production of recombinant proteins (EP 361,991). Studies have also been undertaken with
Bacillus subtilis (Saunders et al., J. Bacteriol. 169 (1987) 2917), with brewer's yeast (EP 201 239), or also with Pichia pastoris yeast (EP 344 459),
Schizosaccharomyces pombe (EP 385,391) or Schwanniomyces (EP 394,538).

 However, the promoters used in these new systems have not yet been optimized. In particular, they are often heterologous promoters, that is to say from other microorganisms, which can cause various drawbacks, and in particular limit the activity of the promoter because of the absence of certain elements of the transcriptional machinery (for example of trans-activators), present a certain toxicity for the host cell due to a lack of regulation, or affect the stability of the vector.

 Under these conditions, the exploitation of these production systems requires having suitable promoters, preferably homologous, controllable and strong. It is therefore important to be able to have an efficient, simple and reliable means for finding and isolating promoters from any cellular host.

The Applicant has now developed, and this constitutes an object of the present invention, a particularly advantageous method making it possible to identify and clone transcriptional promoters. More specifically, the invention relates to a method for identifying and / or cloning functional transcriptional promoters in a given host, according to which the following steps are carried out:
- a DNA bank of a determined starting cell is prepared,
- an insertional mutagenesis is carried out by transposition on this bank making it possible to insert into the DNA of the starting cell a transposon carrying a reporter gene whose expression in the given host can be demonstrated,
the clone (s) expressing, under determined culture conditions, said reporter gene in the given host is selected, and,
- the DNA fragment comprising the promoter region of the
(of) clones thus selected.

 The cloning method according to the invention is particularly advantageous since it can be applied to any starting cell, and it is not necessary to know the function and / or the structure of the gene naturally expressed by this promoter. This process thus makes it possible to clone perfectly unknown promoters. Furthermore, another advantage of the method of the invention is that it makes it possible to clone promoters active under determined culture conditions, and therefore to orient the selection of promoters according for example to a type of regulation. The method of the invention is also advantageous since it can allow rapid identification of the gene placed under the control of the isolated promoter by determination of a short fragment of sequence at the junction between the transposon and the fragment d Of the starting strain by means of a transposon-specific oligonucleotide.

 As regards the first step of the method of the invention, it is applicable to any type of cell, and in particular to eukaryotic and prokaryotic cells.

Among the eukaryotic cells, mention may be made of plant and animal cells, yeasts or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces,
Pichia, Schwanniomyces, or Hansenula. As regards animal cells, mention may be made of COS, CHO, Cl27 cells, etc. Among the fungi capable of being used in the present invention, there may be mentioned more particularly
Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, bacteria such as Escherichia coli, or those belonging to the genera Corynebacterium, Bacillus, or Streptomyces can be used.

 The choice of the starting cell can be dictated by different criteria (ease of manipulation, high level of protein expression, analysis of a little studied cell, etc.).

 The DNA library of the starting cell can be a genomic library or a cDNA library. These can be prepared by any technique known to those skilled in the art. In particular, in the case of a genomic library, it can be prepared by breaking the cells, digesting the DNA by means of restriction enzymes and inserting the DNA thus digested into a vector. Generally, digestion conditions (enzymes, reaction time, etc.) are used which make it possible to obtain fragments of a size of approximately 4 to 8 kb. Being a cDNA library, it can be prepared from RNAs, by reverse transcription.

 For reasons of convenience, the library is generally prepared in E. coli, in pBR322 type vectors. Furthermore, to allow the transfer of the bank from the initial strain (E.coli) to the host in which the expression is sought, it is possible to use either shuttle vectors capable of replication in the 2 types of hosts, or to construct transposons having the elements necessary for replication and selection in the host in which the expression is sought. This second alternative has the advantage of not using too large vectors to carry out the bank.

 As regards the second step of the method of the invention, the reporter gene is a gene whose expression can be demonstrated in the given host. It may be a gene inducing staining of the strains (lacZ gene in particular) or giving them a luminescent character (luxA gene in particular), or else a gene conferring resistance to the strain [gene conferring resistance to G418 (g gene) or copper (COUP1 gene) in particular j. It may also be a gene essential for the growth of the strain under certain culture conditions, etc.

 As an example of a reporter gene which is particularly advantageous for the Kiactis lac4 yeasts, the lacZ gene of E. coli coding for 13-galadosidase allows selection on lactose medium (Chen et al., J. Basic Microbiol. 28 (1988) 211).

 The reporter gene must be present in the transposon in a form which allows its expression only by translational fusion with a gene from the given host. In particular, it must not be preceded by stop codons or other signals which could prevent its expression.

 As examples of transposons which can be used in the context of the invention, mention may be made more particularly of the MiniMu transposon and the Tn transposons (Tn3, Tn5, Tn7, Tn10, Tnbla, etc.).

The third step of the process of the invention consists in selecting the clone (s) expressing the reporter gene in the given host, under determined culture conditions. In the case where the library is produced in a cell different from the given host in which the expression is sought, the library must first be transferred to the given host, before proceeding to the selection of the clones expressing the reporter gene. In this case, a first selection is made to isolate the transformants having received the DNA from the bank. This selection can be made using any auxotrophy or resistance marker. The transfer of DNA from the bank can be carried out by any technique known to a person skilled in the art, and in particular by transformation, electroporation, or conjugation. With regard to transformation in yeast, different protocols have been described in prior art. In particular, it can be carried out by treating the whole cells in the presence of lithium acetate and of polyethylene glycol according to the technique described by Ito et al. g. Bacteriol. 153 (1983) 163-168), or in the presence of ethylene glycol and dimethyl sulfoxide according to the technique of
Durrens et al. (Curr. Genet. 18 (1990) 7). An alternative protocol has also been described in patent application EP 361 991. It is also possible to transform yeasts by electroporation, according to the method described by
Karube et al. (FEBS Letters 182 (1985) 901. The selection of the clones expressing the reporter gene in the given host is carried out after transformation, under different culture conditions according to the characteristics of the promoter sought (regulatable promoter, strong promoter, etc.). , according to the reporter gene used, the clones having a promoter region active under the conditions of selection are those generating a certain coloration or luminescence, those exhibiting resistance to a given compound (resistance gene) or those capable of growing in certain environments (genes complementing an auxotrophy such as the i, URA3 or LEU2 genes, in particular), etc.

 In an advantageous embodiment of the invention, it is also possible to simultaneously carry out the selection of transformants and the selection of clones expressing the reporter gene.

During the fourth step of the process of the invention, the isolation of the DNA fragment comprising the promoter region is generally carried out by excision using restriction enzymes, optionally followed by digestion with nucleases or a chemical synthesis to reconstruct a missing part. Preferably, the excision is preceded by a step for delimiting the promoter region carried by the DNA fragment. This is generally carried out in several stages:
- determination of a restriction card for the insert carried by the clone. This step is nonetheless optional,
- localization of the 3 'end of the promoter region: This step consists in identifying the ATG codon. It is generally carried out by sequencing the junction between the transposon and the DNA fragment of the starting strain. This end of sequence can then make it possible, by searching for homologies with known sequences, to locate the ATG. If the search for homology does not give results, ATG can be identified by complete sequencing of the DNA fragment of the starting strain carrying the promoter region.

 - localization of the 5 'end of the promoter region: This can be demonstrated by sequential deletions on the DNA fragment of the starting strain carrying the promoter region and subcloning in expression vectors.

 The promoters obtained by the process of the invention can be used for the expression of any gene either in the starting cell, or in the given host, or even in any other cell in which they are functional. They can in particular be used for the expression of genes coding for recombinant proteins such as for example proteins of pharmaceutical or agrifood interest, or also for the expression of genes for biosynthesis of metabolites, such as for example biosynthesis genes amino acids, vitamins, antibiotics, etc.

 Furthermore, these DNA fragments can also be modified before their use, for example to prepare portable promoters, to adapt them to expression in a particular type of vector or host, to reduce their size, to increase their transciption promoter activity, to generate inducible promoters, improve their level of regulation, or even change the nature of their regulation. Such modifications can be made for example by in vitro mutagenesis, by introduction of additional control elements or synthetic sequences, or by deletions or substitutions of the original control elements.

 The subject of the invention is also a method of producing a recombinant protein by expression of a DNA sequence coding for said protein in a cellular host, according to which the expression of the DNA sequence is under the control of 'a promoter obtained by the process described above.

 Other advantages of the present invention will appear on reading the following examples, which should be considered as illustrative and not limiting.

LEGEND OF FIGURES
Figure 1: Preparation of the Mini Mu MudIIZK1 transposon.

Figure 2: Restriction map of the Mini Mu MudIIZK1 transposon.

Figure 3: Sequence of the junction between the promoter of the PGK gene of Kadis and the lacZ gene of E. coli (mini mu MudIIZK1) on the clone 4B3.

Figure 4: Sequence of the junction between the promoter of the ADHII gene of K. tactis and the lacZ gene of Ecoli (mini mu MudIIZK1) on clone 10B1.

Figure 5: Restriction map of the insert of clone 1D12.

Figure 6: Sequence of the junction between the promoter of the PDC1 gene of K. lactis and the lacZ gene of E. coli (mini mu MudIIZK1) on the clone lu12.

GENERAL CLONING TECHNIQUES.

Classically used methods in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoeesis on agarose or acrylamide gels, purification of DNA fragments by electroelution, the extraction of proteins with phenol or phenolchloroform, the precipitation of DNA in a saline medium with ethanol or isopropanol, the transformation in Eschwichia coli, etc., are well known to those skilled in the art and are abundantly described in the literature [Maniatis
T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, 1982; Ausubel FM et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].

 Restriction enzymes have been supplied by New England Biolabs (Biolabs) or Pharmacia and are used as recommended by the suppliers.

 The pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories).

 For ligatures, the DNA fragments are separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of DNA. phage T4 ligase (Boehringer) according to supplier's recommendations.

 The filling of the prominent 5 ′ ends is carried out by the Klenow fragment of DNA Polymerase I of E. coli (Boehringer) according to the supplier's specifications. The destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations. The destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease S1.

Mutagenesis directed in vitro by synthetic oligodeoxynucleotides is carried out according to the method developed by Taylor et al. [Nucleic Acids
Res. 13 (1985) 8749-8764].

The enzymatic amplification of DNA fragments by the technique called PCR Eolymerase-catalyzed Chain Reaction, Saiki RK et al., Science 230 (1985) 1350-1354; Mullis KB and Faloona FA, Meth. Enzym. 155 (1987) 335-350] is performed using a "DNA thermal cycler" (Perkin Elmer
Cetus) according to the manufacturer's specifications.

 Verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467].

 The transformations of K tacts are carried out by any technique known to those skilled in the art, an example of which is given in the text.

Unless otherwise specified, the bacterial strains used are Ecoli
DH1 (Hanahan D., J. Mol. Biol. / 166 (1983) 557) or Ecoli JM109: :( Mucts) (Daignan-fornier and Bolotin-Fukuhara, Gene 62 (1988) 45).

 The yeast strains used belong to budding yeasts and more particularly to yeasts of the genus Kluyveromyces.

The strains K lactis 2359/152 and K lactis SD6 were particularly used.

 The yeast strains transformed by the plasmids are cultivated in Erlenmeyer flasks or in pilot fermenters from 21 (SETRIC, France) at 28 "C in rich medium (YPD: 1% yeast extract, 2% Bactopeptone, 2% glucose; or YPL: 1 % yeast extract, 2% Bactopeptone, 2% lactose) with constant stirring.

EXAMPLES
EXAMPLE OF CLONING OF KLuYvERoMYCE PROMOTERS USING
AS REPORTER GENE THE LACZ GENE OF ECOLI AND AS A SYSTEM OF
MUTAGENESIS TRANSPOSrrION.

1. Preparation of the Mini Mu MudIIZK1 transposon (Figures 1 and 2)
The Mini Mu MudIIZK1 was built from the Mini Mu MudIIZZ1 described by Daignan-Fornier and Bolotin-Fukuhara (Gene 62 (1988) 45). It was obtained by replacing the origin of replication of the mini transposon MudIIZZ1 with an origin of functional replication in Kluyveromyces: the origin of replication of the plasmid pKD1 (EP231,435).

 1.1. Construction of a cassette carrying the origin of replication of the plasmid pKD1 (fragment S11).

In order to facilitate subsequent manipulations, the S11 fragment (carrying the origin of replication of the plasmid pKD1) was put into the form of a NotI cassette. For this, a derivative of the plasmid pUC18 was constructed, in which the external sites of the cloning multisite (HindIII and EcoRI sites) were changed into NotI sites. This was done by digestion with the corresponding enzyme, action of the Klenow enzyme and ligation with a synthetic oligonucleotide corresponding to a NotI site [oligo d (AGCGGCCGCT); Biolabsl. The plasmid obtained is designated pGM67. The 960 bp S11 fragment obtained by digestion with the enzyme Sau3A of the plasmid KEp6 (Chen et al, Nucl. Acids Res. 114 (1986) 4471) was then inserted at the BamHI compatible site of the plasmid pGM67. The plasmid thus obtained, designated pGM68, contains in the form of a NotI cassette the fragment
S11.

1.2. Suppression of the origin of 2p replication of the transposon
MudIIZZ1.

 The plasmid pGM15 carrying the mini Mu MudIIZZ1 (Daignan-Fornier and Bolotin-Fukuhara above) was deleted from the 2p regions by digestion using the enzyme SalI. The unique SalI site thus obtained was then transformed into a NotI site by ligation of a synthetic oligonucleotide corresponding to a NotI site after the action of the Klenow enzyme. The resulting plasmid is called pGM59.

1.3. Insertion of the S11 fragment
The NotI cassette bearing the origin of replication of the plasmid pKD1 (fragment S11), originating from the modified plasmid pUC18 was then introduced into the unique NotI site of the plasmid pGM59.

The plasmid obtained, designated pGM83, carries a mini Mu, called
MudIIZK1, which is adapted to the yeast Kluyveromyces lactis, as well as a functional copy of the LEU2 gene from S.cerevisiae capable of complementing a leu2 mutation in Kiactis (vamper et al, Curr. Genet. 19 (1991) 109) (Figure 2 ).

2. Introduction of the Mini Mu MudIIZK1 into the E. coli strain carrying the Mu helper JM109: :( Mucts): Obtaining the strain JM109: :( Mucts): :( MudIIZK1).

 The strain JM109: :( Mucts) was transformed by the plasmid pGM83 containing the mini mu MudIIZK1 in the presence of calcium chloride. After transformation, the transposition was induced by thermal shock according to the technique described by Castilho et al. (J. Bacteriol. 158 (1984) 488). The phage lysate obtained after induction is then used to superinfect the JM109 strain: :( Mucts). The strain JM109: :( Mucts) being recA, the linear DNA packaged by the phage cannot be closed to give a plasmid replicatff. The integrants [strain JM109: :( Mucts): :( MudIIZK1) j are therefore selected as resistant chloramphenicol clones (CmR), sensitive ampicillin (AmpS).

3. Preparation of the Klactis genomic library in E.coU DH1
The high molecular weight DNA was prepared from the Kiactis 2359/152 strain, and partially digested with the enzyme Sau3A. The fragments of a size of 4 to 8 kb were recovered on LMP agarose gel ("Low
Melting Point ", SEAKEM) and cloned into the plasmid pBR322 linearized by
BamHI and dephosphorylated by the action of calf intestinal phosphate (Biolabs). 35 pools of 1000 resistant ampicillin (AmpR) and tetracycline sensitive (TetS) colonies in E. coli DH1 were thus produced.

4. Preparation of the fusion bank
4.1. Introduction of the ltlactis genomic bank into the JM109 strain: :( Mucts): :( MudIIZK1).

 The plasmid DNA of each pool produced in DH1 is extracted (Maniatis). This DNA is then used to transform the strain JM109: :( Mucts): :( MudIIZK1) in the presence of calcium chloride. To be representative of the 1000 colonies contained in each pool of the genomic library, more than 3000 clones per pool were recovered in the strain JM109: :( Mucts): :( MudIIZK1) allowing the transduction.

4.2. Transposition of the Mini Mu MudIIZK1
The fusion bank is created by extensive transposition of the Mini
Mu MudIIZK1 on the plasmids forming the genomic DNA library of Kiactis. The mini-muductions were made according to the protocol described by
Castilho et al. g. Bacteriol. 158 (1984) 488) and the transductants were selected on selective LBAC medium (LB medium (Gibco, BRL) supplemented with 50 mg / l of ampicillin and 30 mg / l of chloramphenicol), the marker
AmpR being provided by the plasmid, and the CmR marker by the mini-mu.

For each pool, transpositions are made in series, and between 10,000 and 20,000 transductants are recovered per pool. The DNA of the transductants is then extracted from a 100 ml preparation, purified by precipitation with polyethylene glycol (Maniatis et al, 1989) and resuspended in 100 μl of water. This
DNA was then used to transform Kiactis and to clone promoters.

5. Search for promoters at Klactis
The fusion DNA prepared above was used to transform, by electroporation, a receptor strain of K. lacfis. This receptor strain, designated SD6, carries the mutations leu2 (corresponding to the selection marker of the mini-mu MudIIZK1) and lacS8. This last mutation prevents the strain from growing on a medium containing lactose as the only carbon source, but it can be complemented by the overexpression of the lacZ gene of E. coli coding for ss-galactosidase (Chen et al., J. Basic Microbiol. 28 (1988) 211). Therefore, the expression of a protein fused to ss-galactosidase must allow the growth of the strain SD6 on lactose after transformation. This positive screen was used to rapidly select clones carrying strong promoters.

 5.1. Construction of the receptor strain K.lacfis SD6.

This strain must carry the following markers:
- auxotrophy marker complemented by the mini mu marker (LEU2)
- Ade + marker to avoid reading problems in the middle
Xgal due to the red coloration of the Ade- allele,
- lac4 marker (mutated for ss-galactosidase). The use of this marker must make it possible, in the case of a strong promoter and therefore of a strongly expressed fusion protein, to complement the lacole phenotype of the strain.

The strain SD6 (Chen et al., Mol. Gen. Genet. 233 (1992) 97) was obtained by crossing the strain CXJ1-7A (a, lac4-8, uraA, adel-1, K1, K2, pKD1 ) (Chen and Fukuhara, Gene 69 (1988) 18187) with the strain AWJ-137 (leu2, trpl, homothallic) (vamper et al, Curr. Genet. 19 (1991) 109), and selection of spores having the ADE + genotype , uraA, leu2, lacS8. As the spores obtained were not capable of regenerating after transformation by protoplasts, a back crossing was made with the strain CXJ1-7A. After mass sporulation, the spores of the genotype chosen were tested by transformation with lithium chloride with the KEp6 plasmid according to a technique derived from that described by Ito et al. U. Bacteriol. 153 (1983) 163) (the LiCl concentration is 20 mM, 10 times less than that used by
Ito for S.cerevisiae). The strain CXJ1-7A served as a transformation control.

 The strain SD6, selected on these criteria, is transformed correctly: 1 to 3. 104 transformants per pg of DNA; and the transformants have satisfactory stability: 30 to 40% of the colonies retain the phenotype [Ura + i after 6 generations in non-selective medium.

5.2. Isolation of strong promoters
The strain SD6 was transformed by electroporation according to Becker and
Guarante (in Methods in Enzymology vol194 (1991) 182) (Jouan apparatus; 2500 V / cm; 80-100 ng of DNA / transformation) with the DNA of i1 pools of transductants obtained in 4. (corresponding to a library of 11,000 clones in E. coli). After 5 hours of regeneration in YPD medium (yeast extract: 10g / l; peptone: 10g / l; glucose 20g / l), the cells were spread on minimum lactose medium. The transformants capable of growing on lactose were restricted and, for each clone, the plasmid was extracted, amplified in E. coli, and, after rapid verification of the restriction map of the vector and of the minimu, used to transform the yeast SD6 .After retransformation, different Kiactis clones capable of growing on lactose have been identified. Each of these clones carries a promoter region capable of expressing a heterologous gene in the Kiactis yeast. Among the clones thus identified, several were analyzed by restriction and by sequencing the junctions between the Ktactis protein and R-galactosidase. Indeed, the sequence of junctions from the lacZ end of the mini-mu (double-stranded sequence) can be determined by sequencing using an oligonucleotide complementary to the ends of the mini-mu.It is in particular possible to use the oligonucleotides having the following sequence:
(i) upstream oligonucleotide: 5'CTGTTTCATTTGAAGCGCG-3 '
(il) downstream oligonucleotide: 5'-CCCACCAAATCTAATCCC-3 '
In this example, the sequence was carried out using the oligonucleotide (i) located at -59 nucleotides from the junction. This sequencing step makes it possible to verify the insertion of the mini-mu in phase in an open reading phase (an ORF is awaited in frame -1) and, when this is the case, to analyze the protein sequence deduced from the nucleotide sequence by comparison with the protein bank sequences of other yeasts or eukaryotes (Genbank, MIPS, EMBL, etc.).

 Analysis of the junction sequence made it possible to show that the clone 4B3 carried the promoter of the Kiactîs phospho-glycerate kinase gene (FIG. 3), that the clone lOBI carried the promoter of the alcohol dehydrogenase II gene of Kiactis (Figure 4), and that the clone 1D12 carried the promoter of the pyruvate decarboxylase gene from K. lactis (Figures 5 and 6).

Claims (13)

 1. Method for identifying and / or cloning functional transcriptional promoters in a given host, characterized in that the following steps are carried out:  - a DNA bank of a determined starting cell is prepared,  - an insertional mutagenesis is carried out by transposition on this bank making it possible to insert into the DNA of the starting cell a transposon carrying a reporter gene whose expression in the given host can be demonstrated,  the clone (s) expressing, under determined culture conditions, said reporter gene in the given host is selected, and,  - The DNA fragment comprising the promoter region of the clone (s) thus selected is isolated.  2. Method according to claim 1 characterized in that the starting cell is a eukaryotic or prokaryotic cell.  3. Method according to claim 1 characterized in that the library of the starting cell is a genomic library prepared by breaking of the cells, digestion of the DNA by means of restriction enzymes and insertion of the DNA thus digested in a vector.  4. Method according to claim 3 characterized in that digestion conditions are used making it possible to obtain fragments of a size of approximately 4 to 8 kb.  5. Method according to claim 1 characterized in that the library of the starting cell is a cDNA library prepared from the RNAs by reverse transcription.  6. Method according to claim 1 characterized in that the reporter gene is a gene whose expression induces a coloration of the given host, or gives it a luminescent character, or gives it resistance, or is a gene essential for the growth of the given host under certain culture conditions.  7. Method according to claim 6 characterized in that the reporter gene is chosen from the LacZ, LuxA, CUP1 and aph gene.  8. Method according to claim 6 or 7 characterized in that the reporter gene is present in the transposon in a form allowing its expression only by translational fusion with a gene of the given host.  9. Method according to claim 1 characterized in that the transposon is chosen from the mini mu transposon and the Tn transposons.  10. Use of a promoter obtained by the method according to any one of claims 1 to 9 for the expression of genes.  11. Use according to claim 10 for the expression of genes coding for recombinant proteins such as for example proteins of pharmaceutical or agrifood interest, or also for the expression of genes for biosynthesis of metabolites, such as for example genes biosynthesis of amino acids, vitamins, or antibiotics.  12. Method for producing a recombinant protein by expression of a DNA sequence coding for said protein in a cellular host characterized in that the expression of the DNA sequence is under the control of a promoter obtained by the method according to any one of claims 1 to 9.  13. DNA bank known as a fusion bank, consisting of a Kluyveromyces DNA bank on which insertional mutagenesis has been carried out by means of a MiniMu transposon comprising a reporter gene.