AU658630B2 - Yeast promoter and use thereof - Google Patents

Abstract

The invention relates to DNA sequences comprising all or part of the promoter of the K. lactis PGK gene or a derivative thereof, and possessing a transcriptional promoter activity. It also relates to the use of these sequences for the expression of recombinant genes. <IMAGE>

Description

OPI DATE 21/12/92 /bOJP DATE 28/01/93 APPLN. ID 17892/92 IIIII 1111111 PCT NUMBER PCT/FR92/00375 I Ii I I ai. *m AU9217892 -VETS (PC-fl (51) Classification internationale des brevets 5 (11) Numeiro de publication internationale: WO 92/19751 C12N 15/81,l1/19 Al C12P 21/02 (43) Date de publication internationale: 12 novembre 1992 (12.11.92) (21) Num~ro de la demnande internationale: PGT/FR92/00375 (22) Date de dep6t international: 28 avril 1992 (28.04.92) Donn~es relatives i la priorit6: 91/05294 30 avril1 1991 (30.04.9 1) FR (71) Deposant (pour fous les Etats d~signi's sauf US): RHONE- POULENC RORER S.A. [FR/FR]; 20, avenue Raymond-Aron, F-92 160 Antony (FR).

(72) Inventeurs; et Inventeurs/Diposants (US seutlement) FLEER, Reinhard [DE/FRI; 1, all~e Port-Royal, Residence de l'Abbaye, F- 91190 Gif-sur-Yvette FOURNIER, Alain [FR/ FR]; 28, avenue Roger-Salengro, F-92000 Chatenay-Malabry MAYAUX, Jean-Franqois [FR/FR]; 21 ter, boulevard de la R~publique, F-92260 Fontenay-aux- Roses YEH, Patrice [FR/FR]; 13, rue Linn&, BAt.

B, F-75005 Paris (FR).

(74) Mandataire: BECKER, Philippe; Rhone-Poulenc Rorer Direction Brevets, 20 avenue Raymond Aron, F- 92165 Antony C~dex (FR).

(81) Etats disignis: AT (brevet europ~en), AU, BE (brevet europ~en), CA, CH (brevet europ~en), DE (brevet europ~en), DK (brevet europ~en), ES (brevet europ~en), FI, FR (brevet europ~en), GB (brevet europ~en), GR (brevet europ~en), HU, IT (brevet europ~en), JP, KR, LU (brevet europ~en), MC (brevet europ~en), NL (brevet europ~en), NO, SE (brevet europ~en), US.

Ai'ec rapport de recherche internationale.

~~863O (54) Title: YEAST PROMOTER AND USE THEREOF (54) Titre: PROMOTEUR DE LEVURE ET SON UTILISATION (57) Abstract DNA sequences including all or part of the promoter of the PGK gene of Ki1actis, or a derivative thereof, and having transcriptional promoter activity. The use of said sequences for expressing recombinant genes is also described.

(57) Abriege L'invention concerne des sequences d'ADN comprenant tout ou partie du promoteur du gene PGK de K~lactis, ou d'un d&riv6 de celui-ci, et poss~dant une activit6 de promoteur transcriptionnel. Elie concerne 6galement l'utilisation de ces sequences pour l'expression dle genes recombines.

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i WO 92/19751 1 PCT/FR92/00375 YEAST PROMOTER AND USE THEREOF The present invention relates to the field of molecular biology. More particularly, it relates to a novel DNA sequence having a transcriptional promoter activity, to expression vectors containing this sequence and to the use thereof for the production of proteins, for example heterologous proteins. The invention also relates to recombinant cells containing this DNA sequence.

The progress accomplished in the field of molecular biology has enabled microorganisms to be modified in order to make them produce heterologous proteins. In particular, many genetic studies have been carried out on the bacterium E. coli. However, the industrial application of these novel modes of production is still limited, in particular by problems of efficiency of gene expression in these recombinant microorganisms. Accordingly, research studies have been carried out with the aim of increasing the efficiency of these production systems in order to isolate strong promoters enabling high levels of expression of heterologous proteins to be obtained. In E. coli, operon tryptophan and operon lactose promoters may be mentioned in particular.

More recently, in the yeast S. cerevisiae, studies have been carried out on promoters derived from genes involved in glycolysis. There may be mentioned in 4 I I O AI j

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2 particular the work on the promoter of the 3-phosphoglycerate kinase PGK gene (Dobson et al., Nucleic Acid Res. 10, 1982, 2625; Hitzeman et al., Nucleic Acid Research 1982, 7791), on that of the glyceraldehyde-3-phosphate dehydrogenase GAPDH gene (Holland et al., J. Biol. Chem. 254, 1979, 9839; Musti el al., Gene 25, 1983, 133), on that of the alcohol dehydrogenase 1 ADH1 gene (Bennentzen et al., J. Biol.

Chem. 257, 1982, 3018; Denis et al., J. Biol. Chem. 1983, 1165), or on that of the enolase 1 ENO1 gene (Uemura et al., Gene 45, 1986, Recently, genetic tools have been developed in order to make use of the yeast Kluyveromyces as host cell for the production of recombinant proteins. The discovery of a 2-micron type plasmid derived from K. drosophilarum (plasmid pKD1 EP 241,435) has enabled a very efficient host/vector system for the production of recombinant proteins (EP 361,991) to be established. However, the promoters used in this system have never been optimized. In particular, the promoters involved are essentially heterologous promoters, that is to say derived from other microorganisms, such as in particular S. cerevisiae. This situation may lead to various disadvantages and, in particular, it may limit the activity of the promoter because of the absence of certain elements of the transcriptional machinery (for example, of trans-activators), exhibit a certain toxicity for the host cell due to an absence of LiU 0f 3 regulation, or affect the stability of the vector.

Under these conditions, the lack of strong homologous promoters in Kluyveromyces constitutes a limiting factor in the industrial exploitation of this expression system.

The Applicant has now identified, cloned and sequenced a region of the Kluyveromyces lactis genome having a transcriptional promoter activity (see Figure More specifically, this region corresponds to the promoter of the K. lactis PGK gene. This region, or derivatives or fragments thereof, may be used efficiently for the production of recombinant proteins in yeasts of the Kluyveromyces genus. It is understood that this sequence may also be used in other host organisms.

Moreover, an analysis of the region of the Kluyveromyces genome obtained has enabled 2 reading frames to be identified in the 2 opposite directions (see Figure This observation shows that the complementary strand of the region presented in Figure 1, also possesses a promoter activity acting in the other direction.

One subject of the present invention therefore lies in a DNA sequence comprising all or part of the sequence presented in Figure 1, or a sequence of its complementary strand, or of a derivative of these, and possessing a promoter activity.

Within the context of the present invention,

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4yu i 4 derivative is understood to mean any sequence obtained from the sequence given in Figure 1, by structural modifications (mutations, deletions, substitutions, additions, restrictions and the like) which conserve promoter activity. In particular, the mutations may involve one or more nucleotides, and the additions and/or substitutions may involve regulatory elements or activator regions such as UASs.

When a derivative is produced, its transcriptional promoter activity may be demonstrated in several ways and in particular by placing a resistance gene or a complementation marker under the control of the sequence studied. Any other technique known to a person skilled in the art may obviously be used to this effect.

A more specific subject of the invention relates to a DNA sequence corresponding to the region between the 2 open reading frames ORF PGK and ORF X, as presented in Figure 6.

Another subject of the invention relates to a recombinant DNA comprising a DNA sequence as defined above.

J This recombinant DNA may contain for example the promoter sequence presented in Figure 1 or a derivative thereof, in which a restriction site is inserted, facilitating the use of this sequence as "portable" promoter.

Preferably, this recombinant DNA contains, in

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9 7 Ofi addition, one or more structural genes.

Still more preferably, the recombinant DNA also contains signals permitting the secretion of the expression product of the said structural gene(s).

In a specific embodiment of the invention, the recombinant DNA is part of an expression plasmid which may be of autonomous or integrative replication.

In particular, autonomously replicating vectors may be obtained by using autonomously replicating sequences (ARS) in the host selected. In yeast in particular, replication origins derived from known plasmids (pKD1, 2 p, and the like) may be involved.

Integrative vectors may be obtained in particular by using homologous sequences at certain regions of the host genome which permit integration of the vector by homologous recombination.

The sequence presented in Figure 1 was obtained by screening a total genomic DNA library from Kluyveromyces lactis by means of a heterologous probe derived from the S. cerevisiae PGK gene. The Applicant has indeed shown that it is possible to clone a promoter region in Kluyveromyces, by hybridisation using heterologous probes corresponding to a S. cerevisiae gene. Details on the cloning of the sequence are given in the examples. The intergenic region may then be isolated from this sequence, in particular by restriction site insertion using the PCR W_0 6 amplification technique as indicated in the examples.

Another subject of the invention relates to the recombinant cells which contain a DNA sequence as defined above.

Advantageously, cells are chosen from yeasts, and still more preferably from yeasts of the Kluvveromvces genus. It is understood, however, that the invention covers all the recombinant cells in which the promoter regions of the invention are active.

These cells may be obtained by any method enabling a foreign DNA to be introduced into a cell.

The methods involved may be in particular transformation, electroporation or any other technique known to a person skilled in the art.

Another subject of the invention relates to the use of a sequence as defined above, for the expression of recombinant genes.

As illustrated by the examples, the DNA sequences according to the invention indeed permit high levels of production of recombinant proteins.

Moreover, the bidirectional promoter activity of the sequences of the invention permits a particularly advantageous use. In particular, it is possible to use these sequences in the 2 directions possible, for the simultaneous expression of several structural genes.

Advantageously, the invention relates to the use of a sequence as defined above for the simultaneous r 1 7 expression of recombinant genes in the 2 opposite directions.

Advantageously, the sequences of the invention may be used for the expression of genes encoding proteins of interest in the pharmaceutical or foodstuffs sector. By way of example, there may be mentioned enzymes (such as in particular superoxide dismutase, catalase, amylases, lipases, amidases, chymosin and the like), blood derivatives (such as serum albumin, alpha- or beta-globin, factor VIII, factor IX, van Willebrand factor, fibronectin, alpha- 1-antitrypsin and the like), insulin and its variants, lymphokines (such as interleukins, interferons, colony stimulating factors (G-CSF, GM-CSF, M-CSF and the like), TNF, TRF and the like), growth factors (such as growth hormone, erythropoietin, FGF, EGF, PDGF, TGF and the like), apolipoproteins, antigenic polypeptides for the production of vaccines (hepatitis, cytomegalovirus, Eppstein-Barr, herpes and the like) or alternatively polypeptide fusions such as in particular fusions comprising an active part fused with a stabilizing part (for example fusions between albumin or fragments of albumin and the virus receptor or part of a virus receptor (CD4, and the like)).

The invention will be more completely described by means of the following examples which should be considered as illustrative and nonlimiting.

K o0 fc^J U I 8 LEGEND TO THE FIGURES Figure 1: Nucleotide sequence of the 2.2-kb region of the chromosomal fragment situated upstream of the initiation codon for translation of the K. lactis PGK gene having the promoter activity.

Fiqure 2: Analysis of the open reading frames. The vertical half-lines represent initiation codons for translation. The full vertical lines represent stop codons. The clear regions show the open reading frames (ORF X and ORF PGK).

Figure 3: Restriction map of the plasmid pYG610. The black region corresponds to the region isolated from the K. lactis genome.

Figure 4: Strategy for sequencing the 2.5-kb XbaI fragment.

Figure 5: Sequence and location of the oligodeoxynucleotides used in the PCR reaction for inserting a HindIII site in -6 of the ATG of the sequence in Figure 1. The oligodeoxynucleotides are represented in italics. ATG corresponds to the initiation codon for translation of the PGK gene.

Figure 6: Nucleotide sequence of the intergenic region of the 2.2-kb fragment. oligodeoxynucleotides used in the PCR reaction. Sal(I)-HindIII fragment corresponding to the nucleotides 1343 to 2246 on the sequence in Figure 1.

Fiure 7: Strategy for the construction of the plasmid i 9 Figure 8: Strategy for the construction of human serum albumin expression cassettes.

Figure 9: Strategy for the construction of the plasmid Figure 10: Strategy for the construction of the plasmid Figure 11: Strategy for the construction of the plasmid pYG72.

Ficure 12: Strategy for the construction of the vector pYG621.

Figure 13: Visualization, by Northern blotting, of the expression of the human albumin gene under the control of the K. lactis PGK promoter. The samples correspond to 10 pg of total RNA. 18S and 28S are the positions of the 18S and 28S ribosomal RNAs. ALB fragments recognized by the probe corresponding to the albumin gene; URA fragments recognized by the probe corresponding to the K. lactis URA A gene serving as loading reference.

Fiqure 14: Visualization of albumin production in strains transformed by the expression vector pYG621 Scontaining the K. lactis PGK promoter. The samples correspond to 30 il of culture supernatant; the bands at the level of the 66 kd marker correspond to albumin.

M molecular weight markers: bovine carbonic anhydrase (31 kd), ovalbumin (45 kd), BSA (66 kd), rabbit phosphorylase b (92 kd).

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EXAMPLES

1/ Isolation of the promoter region of the K. lactis PGK gene.

The sequence presented in Figure 1 was obtained by screening a total genomic DNA library from Kluyveromyces lactis CBS2359 using a heterologous probe derived from the S. cerevisiae PGK gene (Dobson et al., Nucleic Acid Res. 1982, 10, 2625). More specifically, the probe used corresponds to the 1.3-kb N-terminal PvuI-EcoRI fragment of the S. cerevisiae PGK gene.

In Southern blotting (Southern et al., J.

Biol. Chem., 1975, 98, 503), the probe used hybridizes to two different fragments when the genomic DNA is digested with XbaI. One of them, of about 2.5 kb, was isolated by screening a small genomic library from K. lactis CBS2359 consisting of XbaI-cut DNA fragments of between 2 and 3 kb in size, which were introduced inside the plasmid pUC18 at the XbaI site. A library with 500 clones was thus produced and then screened with the heterologous probe described above.

A clone was identified by colony hybridization and its plasmid DNA was prepared. This plasmid (pYG610) contains a 2.5-kb genomic DNA fragment whose restriction map is presented in Figure 3. The plasmid pYG611 contains the same insert in the opposite direction (see Figure 8).

L U 11 In a second stage, the 2.5-kb fragment thus isolated was sequenced using the Sanger method (Sanger et al., Proc. Nat. Acad. Sci 74, 1977, 5463). For that, the fragment derived from pYG611 was first subcloned in the bacteriophages M13tgl30 and M13_tg31. The strategy for the sequencing of the fragment is schematically represented in Figure 4.

Analysis of the sequence obtained shows that the fragment isolated contains a part encoding the N-terminal region of the protein Pgk from Kluyveromyces lactis (0.3 kb), and 2.2 kb corresponding to the promoter region situated upstream of the site of initiation of translation. It shows furthermore 1'at a second reading frame, situated about 0.9 kb upstream of the ATG of the PGK gene, is situated in the opposite direction relative to the PGK gene (Figure 2).

Comparison of this sequence with that of the promoter of the S. cerevisiae PGK gene reveals the absence of specific homology, especially with its regulatory element. This sequence therefore corresponds to a completely novel promoter region, which is very distinct from those previously described, from the point of view of its structure and consequently from the point of view of its regulation.

1O)IC 12 2/ Construction of expression vectors for the production of heteroloqous proteins: This example illustrates the use of the promoter capabilities of the 2.2-kb sequence of the sequence in Figure 1 and of derived sequences.

a) Insertion of a restriction site in -6 from

ATG.

The insertion of this site subsequently enables any gene, which it is desired to express, to be introduced downstream of the promoter obtained. For reasons of compatibility with existing expression vectors (EP 361,991), "portable" promoters were constructed in the form of SalI-HindIII fragments.

A HindIII site was introduced in position -6 relative to the site of initiation of translation (ATG) of the PGK gene using the PCR amplification technique (Mullis et al., Meth. Enzymol. 155, 1987, 335).

2 oligodeoxynucleotides, which are presented in Figure 5, were used for this purpose.

The oligodeoxynucleotide A corresponds to the sequence situated at 467 pb upstream of the ATG codon, at the level of a HindIII site which will thus be replaced by a SalI site during the amplification. The oligodeoxynucleotide B corresponds to the sequence upstream of the initiation site, and enables a HindIII 13 site to be introduced in position -6.

The fragment obtained by PCR was inserted between the SalI and HindIII sites of the bacteriophage M13tg130 in order to verify, by sequencing, that mutations did not occur during the amplification.

b) Construction of human serum albumin expression cassettes: Figure 8.

The 474-pb recombinant DN\ obtained above was introduced at the level of the Sail and HindIII sites, inside the plasmid pYG45 (Figure in order to obtain the vector pYG614 (Figure The plasmid contains an expression cassette consisting of the promoter and the terminator of the S. cerevisiae PGK gene between which the prepro-human serum albuminencoding gene (prepro-HSA sequence) is inserted at the level of a HindIII site. pYG45 is derived from pYG18 (see Patent EP 361,991) by subcloning the SalI-BamHI fragment containing the HSA expression cassette into the sites corresponding to the vector (Figure pIC-20RDH is obtained by digesting the Splasmid pIC-20R (March et al., Gene 32, 1984, 481) with the enzyme HindIII, filling the ends using the Klenow fragment of E. coli polymerase I and recircularization with T4 DNA ligase.

The SalI-SacI fragment may be isolated from the plasmid pYG614 by digestion. It contains: a i ,v 1 1 I 14 promoter region derived from the sequence in Figure 1, the albumin gene and the terminator of the S. cerevisiae PGK gene. It constitutes an expression cassette which may be inserted inside a plasmid so as to constitute an expression vector.

Another expression cassette may be obtained from the plasmid pYG614 by cloning the AflII-SacI fragment containing part of the PGK promoter of the invention, the albumin gene (prepro-HSA) and the S. cerevisiae PGK terminator inside the plasmid pYG611 described above. This generates the plasmid pYG615. The SalI-SacI fragment containing: the complete promoter region in Figure 1, the prepro-serum albumin-encoding gene, and the terminator of the S. cerevisiae PGK gene, may then be isolated by digestion. This fragment constitutes a second albumin expression cassette using the promoter sequence of the invention.

c) Construction of albumin expression vectors.

Albumin expression vectors may be constructed by inserting the expression cassettes obtained above inside K. lactis/E. coli shuttle plasmids such as pYG72 (Figure 10). In particular, an expression vector was obtained (vector pYG621) by inserting the SalI-SacI fragment from pYG615, containing the albumin expression cassette, inside the vector pYG72 (see Figure 10). This /7) U q l vector corresponds to the plasmid pKan 707 (see EP 361,991) from which the Sad fragment, containing the URA3 gene, has been removed, as well as the unique HindIII site present in the aDh gene, so as to facilitate subsequent constructions. The aph gene encodes aminoglycoside 3'-phosphotransferase (Oka et al., J. Mol. Biol. 147, 1981, 217) and is used, in yeast, as marker for resistance to G418. The PstI fragment of the plasmid pKan 707, containing the aph gene, was subcloned into the bacteriophage M13mp7 to give the vector pYG64 (Figure The HindIII site present inside this gene was destroyed by site-directed mutagenesis according to the method described by Taylor et al., (Nucleic Acid Res. 13, 1985, 8749). The resultant plasmid was called pYG65 (Figure The oligodeoxynucleotide used for the mutagenesis had the sequence 5'-GAA ATG CAT AAG CTC TTG CCA TTC TCA CCG -3' and transformed the triplet CTT encoding the amino acid 185 (Leu) to CTC. This change does not modify the resultant protein sequence. To construct the plasmid pYG72, the part containing the bacterial replican of the vector pKan 707 was isolated by digestion with the -enzyme EcoRI and recircularization with T4 DNA ligase so as to obtain pYG69. The PstI fragment present in this latter vector, containing the aph gene, was replaced by the equivalent mutant fragment derived from This construct was called pYG70. The 4.7-kb pKDl sequence between the EcoRI and SacI sites was 16 introduced inside this latter vector so as to obtain pYG72. The vector pYG621 (Figure 11) was obtained by insertion of the SalI-SacI fragment containing the albumin expression cassette derived from pYG615.

3/ Construction of a cassette enabling the promoter region to be used in the 2 directions.

This construct was obtained by introducing a SalI site and a HindIII site on either side of the region between the 2 open reading frames identified in Figure 2: ORF PGK and ORK X, that is, at the level of the nucleotides 1343 and 2246 in Figure 1.

This construct was produced by the PCR technique using, on the one hand, the oligodeoxynucleotide A which introduces a SalI site in the -1 position relative to the site of initiation of translation of the PGK gene and, on the other hand, the oligodeoxynucleotide B which introduces a HindIII site in the -1 position relative to the site of initiation of translation of the X gene (see Figure 3 PCR reactions were then carried out using, at each stage, /the plasmid pYG610 as template, so as to remove a HindIII site present in the promoter region: the first 2, to amplify the regions on either side of the HindIII site using the oligodeoxynucleotides A and B coupled to the oligodeoxynucleotides C and D respectively (Figure 6).

L r I 17 These last 2 are complementary and they enable a point mutation to be introduced at the level of the inner HindIII site; the last one, to generate the final fragment containing the modified promoter region, using the previous 2 amplification products as primer.

This region may then be introduced inside the vectors described in Example 2, and used as bidirectional promoter.

4/ Expression of albumin The vector pYG621 was introduced, by transformation, inside the K. lactis strain MW98-8C (CBS 579.88), using the ethylene glycol/dimethyl sulphoxide technique (Durrens et al., 1990, Curr.

Genet. 18, This strain is derived from the wild strain CBS2359 and is of the genotype: Mat_, uraA, ysA, argA, cir. The transformant yeasts are selected for the G418-resistant phenotype which the plasmid pYG621 confers on the YPD media (10 g/l yeast extract, 20 g/1 peptone, 20 g/l glucose) containing 0.2 g/l of geneticin. Plasmid pYG72-transformed strains not containing an expression cassette were selected to serve as control in the production tests. Moreover, vector pYG19-transformed strains were also selected in order to compare the efficiency of the K. lactis PGK promoter according to the invention with that from i i u

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18 S. cerevisiae. The vector pYG19 is similar to the vector pYG621 except that the albumin gene is under the control of the S. cerevisiae PGK promoter (EP 361,991).

a) Analysis of the mRNAs: The cells are cultured at 28 0 C in selective YPD media (10 g/1 yeast extract, 20 g/l peptone, 20 g/l glucose) containing 0.2 g/l of geneticin. The total RNAs are extracted (Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1986, 143) and separated by electrophoresis on agarose gel. The RNAs are hybridized to a probe corresponding to the albumin structural gene (1.9-kb HindIII-HindIII fragment) derived from the vector pYG18 (Figure 7) following the Northern blot method (Maniatis et al., 1982 Molecular cloning, Cold Spring Harbor, Laboratoty Press).

Autoradiography clearly shows a 2.3-kb band which is specific to albumin (Figure 13). Moreover, it is clearly evident that the level of transcription of the albumin gene is substantially higher in the strains containing a promoter region of the invention (pYG621) than in those containing the intact S. cerevisiae PGK promoter (pYG19).

b) Analysis of the proteins: The cells are cultured in Erlenmeyer flasks ii i 19 in a selective YPD media (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose) containing 0.2 g/l geneticin, at 28"C with shaking. After 96 hours of culture, 30 il of supernatant are collected and mixed with an equivalent volume of 2 x Laemmli buffer (Laemmli, 1970, Nature 227, 680). After heating at 96°C for 10 minutes, the sample proteins are then separated on 8.5 SDSpolyacrylamide gel. The production of albumin is then visualized by staining the gel with coomassie blue, and it is then evaluated for the different vectors used.

Figure 14 shows that the 4 clones which were obtained separately by transforming the strain MW98-8C using the vector pYG621, secrete substantially more albumin than those obtained by transformation using the vector pYG19.

It is evident that the promoter region of the invention permits excellent albumin production by yeast, greater than that obtained with the S. cerevisiae PGK promoter. This region, or reduced forms or derivatives thereof, constitute(s) an important industrial tool for microbiological, and more particularly eucaryotic, production systems.

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Claims (14)

1. DNA sequence comprising all or part of the sequence presented in Figure 1 or its complementary strand, or of a derivative thereof, and possessing a transcriptional promoter activity.

2. DNA sequence according to Claim 1, comprising all or part of the sequence presented in Figure 6(b).

3. Recombinant DNA comprising a DNA sequence according to Claims 1 or 2.

4. Recombinant DNA according to Claim 3, characterized in that it contains, in addition, one or more structural genes.

Recombinant DNA according to Claim 4, characterized in that it also contains signals permitting the secretion of the expression product of the said structural gene(s).

6. Recombinant DNA according to any one of Claims 3 to 5, characterized in that it is part of an expression plasmid, which may be of autonomous or integrative replication.

7. Recombinant cell containing a DNA sequence or a recombinant DNA according to any one of the preceding claims.

8. Recombinant cell according to Claim 7, characterized in that it is a yeast.

9. Recombinant cell according to Claim 8, I 21 characterized in that it is a yeast of the Kluvveromyces genus.

Use of a DNA sequence according to any one of Claims 1 to 6 for the expression of recombinant genes.

11. Use according to Claim 10, for the simultaneous expression of recombinant genes in 2 opposite directions.

12. Use according to one of Claims 10 or 11 for the expression of genes encoding proteins of interest in the pharmaceutical or foodstuffs sector.

13. Process for the preparation of a recombinant protein by expression of its gene in a cellular host characterized in that the expression of the said gene is under the control of a sequence according to Claim 1.

14. Process according to Claim 13, characterized in that the protein is human serum albumin. r1 I 1 B 1 i d*