NZ242543A - Dna sequence from kluyveromyces lactis having transcriptional promoter activity - 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

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NEW ZEALAND PATENTS ACT, 1953 No.: : ...

. * * • Date: ' COMPLETE SPECIFICATION YEAST IMPROVER AND ITS USE We, RHONE-POULENC RORER SA, a French body corporate, of 20 Avenue Raymond Aron, F 92160, Antony, France hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to molecular biology. More particularly, it relates to DNA sequences having transcriptional promoter activity, to expression vectors containing such sequences and to their use in the 5 production of proteins, especially heterologous proteins. The invention also relates to recombinant cells containing such DNA sequences.

The progress accomplished in the field of molecular biology has enabled microorganisms to be modified 10 in order to make them produce heterologous proteins. In particular, many genetic studies have been carried out using the bacterium Escherichia coli. However, the industrial application of these novel modes of production is still limited in particular by problems of efficiency of 15 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 20 obtained. In E. coli. the tryptophan operon and the lactose promoter operon may be mentioned in particular.

More recently, in the yeast Saccharomvces cerevisiae. studies have been carried out on promoters derived from genes involved in glycolysis. There may be 25 mentioned in 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 GADPH gene (Holland et al., J. Biol. Chem. 254 . 1979, 9830; Musti el al., Gene 25, 1983, 5 133), on that of the alcohol dehydrogenase 1 ADH1 gene (Bennentzen et al., J. Biol. Chem. 257. 1982, 3018; Denis et al., J. Biol. Chem. 2.5, 1983, 1165), or on that of the enolase 1 ENOl gene (Uemura et al., Gene 45, 1986, 65).

Recently, genetic tools have been developed in 10 order to make use of the yeast Kluvveromvces as host cell for the production of recombinant proteins. The discovery of a 2-micron type plasmid derived from K. drosophilarum (plasmid pKDl - see European Specification EP 241,435) has enabled a very efficient host/vector system for the 15 production of recombinant proteins (see EP 361,991) to be established. However, the promoters used in this system have never been optimised. In particular, the promoters involved are essentially heterologous promoters, that is to say derived from other microorganisms such as in particular 20 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) , it may exhibit a certain toxicity for the host 25 cell due to an absence of regulation, or it may affect the stability of the vector.

Under these conditions, the lack of strong 2 4 ; " 4 homologous promoters in Kluvverorivces constitutes a limiting factor in the industrial exploitation of this expression system.

The Applicant has now identified, cloned and 5 sequenced a region of the Kluvveromvces lactis genome having transcriptional promoter activity (see Figure 1). More specifically, this region corresponds to the promoter of the K. lactis PGK gene. This region, or derivatives of fragments thereof, may be used efficiently for the 10 production of recombinant proteins in yeasts of the Kluvveromvces genus. It is to be understood that this sequence may also be used in other host organisms.

Moreover, an analysis of the region of the Kluvveromvces genome obtained has enabled 2 reading frames 15 to be identified in 2 opposite directions (see Figure 2). This observation shows that the complementary strand of the region presented in Figure 1, also possesses promoter activity acting in the other direction.

One subject of the present invention therefore 20 lies in an isolated DNA sequence comprising all or part of the sequence presented in Figure 1, or an isolated sequence of each complementary strand, or of a derivative of these, and possessing transcriptional promoter activity.

Within the context of the present invention, 25 derivative means any sequence obtained from the sequence given in Figure 1, by structural modifications (mutations, deletions, substitutions, additions, restrictions and the 24 2 5 4 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 10 skilled in the art may obviously be used to this effect.

A more specific subject of the invention is an isolated 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 is a recombinant 15 DNA comprising a DNA sequence as defined above. 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 a "portable" promoter. 20 Preferably, this recombinant DNA contains, in 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). 25 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 (pKDl, 2 ixf and the 5 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 Kluvveromvces lactis by means of a heterologous probe derived from the S. cerevisiae PGK gene. The Applicant has shown that it is possible to clone a promoter region in 15 Kluvveromvces. by hybridisation using heterologous probes corresponding to a S. cerevisiae gene. Details on the cloning of the sequence are given in the Examples below. The intergenic region may then i-e isolated from this sequence, in particular by restriction site insertion using 20 the PCR amplification technique as indicated in the Examples.

Another subject of the invention is the recombinant cells which contain a DNA sequence as defined above. Advantageously, such cells are chosen from yeasts, 25 and still more preferably from yeasts of the Kluvveromvces genus. It is to be understood, however, that the invention covers all recombinant ce.l Is 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, 5 electroporation or any other technique known to a person skilled in the art.

Another subject of the invention is the use of a sequence as defined above, for the expression of recombinant genes. As illustrated in the Examples below, 10 the DNA sequences ofthe invention permit high levels of production of recombinant proteins.

Moreover, the bidirectional promoter activity of the sequences of the invention permit a particularly advantageous use. In particular, it is possible to use 15 these sequences in the 2 directions possible, for the simultaneous expression of a plurality of structural genes. Advantageously, the invention relates to the use of a sequence as defined above for the simultaneous expression of recombinant genes in the 2 opposite directions. 20 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, 25 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-l-antitrypsin and the like), insulin and its variants, lymphokines (such as interlukins, interferons, colony stimulating factors (G-CSF, GM-CSF, M-CSF and tha like), TNF, TRF and the like), group factors (such as 5 growth hormone, erythropoietin, FGF, EGF, PDGF, TGF and the like), apolipoproteins, antigenic polypeptides for the product of vaccines (hepatitis, cytomegalovirus, Epstein-Barr, herpes and the like) or alternatively polypeptide fusions such as in particular fusions 10 comprising an active part fused with a stabilising 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 is more completely described in the 15 following Examples which should be considered as illustrative and nonlimiting.

In the accompanying drawings, the Figures are as follows: 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.

Figure 2: Analyses of the open reading frames. The vertical half-lines represent codons of initiation of 25 translation. The full vertical lines represent stop codons. The clair 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 Xbal fragment.

Figure 5: Sequence and location of the oligodeoxynucleotides used in the PCR reaction for inserting a Hindlll site in -6 of the ATG of the sequence in Figure 1. The oligodeoxynucleotides are represented in 10 italics. ATG corresponds to the codon of initiation of translation of the PGK gene.

Figure 6: Nucleotide sequence of the intergenic region of the 2.2-kb fragment. 6(a): oligodeoxynucleotides used in the PCR reaction. 6(b): Sal(I)-Hindlll fragment 15 corresponding to the nucleotides 1343 to 2246 on the sequence in Figure 1.

Figure 7: Strategy for the construction of the plasmid pYG45.

Figure 8: Strategy for the construction of cassettes for the expression of human serum albumin.

Figure 9: Strategy for the construction of the plasmid pYG65.

Figure 10: Strategy for the construction of the plasmid pYG70.

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

Figure 12: Strategy for the construction of the vector y -■v pYG621.

Figure 13: Visualisation, by Northern blotting, of the expression of the human albumin chain under the control of the K. lactis PGK promoter. The samples correspond to 10 nq 5 of total RNA. 18S and 28S are the positions of the 18S and 28S ribosomal RNAs. ALB = fragments recognised by the probe corresponding to the albumin gene; URA = fragments recognised by the probe corresponding to the K. lactis URA A gene serving as loading reference.

Figure 14: Visualisation of albumin production in strains transformed by the expression vector pYG621 containing the K. lactis PGK promoter. The samples correspond to 30 jul of culture supernatant; the bands are the level of the 66 kd marker correspond to albumin. 15 M = molecular weight markers: bovine carbonic anhydrase (31 kd), ovalbumin (45 kd), BSA (66 kd), rabbit phosphorylase b (92 kd).

EXAMPLES 1. Isolation of the promoter region of the K. lactis 20 PGK gene.

The sequence presented in Figure 1 was obtained by screening a total genomic DNA library from Kluvveromvces lactis CBS2359 using a heterologous probe derived from the S. cerevisiae PGK gene (Dobson et al., Nucleic Acid Res. 25 1982, 10, 2625) . More specifically, the probe used corresponds to the 1.3-kb N-terminal PvuI-EcoRI fragment of c i- -lithe S. cerevisiae PGK gene.

In Southern blotting (Southern et al., J. Biol. Chem., 1975, 98, 503), the probe used hybridises two different fragments when the genomic DNA is digested with 5 Xbal. One of them, of about 2.5 kb, was isolated by screening a small genomic library from K,. lactis CBS2359, consisting of Xbal-cut DNA fragments of between 2 and 3 kb in size, which were introduced inside the plasmid pUC18 at the Xbal. A library with 500 clones was thus produced and 10 then screened with the heterologous probe described above.

A clone was identified by colony hybridisation 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 15 the same insert in the opposite direction (see Figure 8).

In a second stage, the 2.5-kb fragment thus isolated was sequenced using the Sanger method (Sanger et al., Proc. Nat. Acad. Sci 24., 1977, 5463). For that, the fragment derived from pYG611 was first subcloned in the 20 bacteriophages M13tgl30 and M13_tgl31. The strategy for the sequencing of the fragment is schematically represented in Figure 4.

Analyses of the sequence obtained shows that the fragment isolated contains a part encoding the N-terminal 25 region of the protein Pgk from Kluvveromvces lactis (0.3 kb), and 2.2 kb corresponding to the promoter region situated upstream of the site of initiation of translation. / t' /*.

It shows furthermore that a second reading frame, situated about 0.9 kb upstream of 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 10 from those previously described, from the point of view of its structure and consequently from the point of view of its regulation. 2. Construction of expression vectors for the production of heterologous 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 enables subsequently 20 any gene, which it is desired to express, to be introduced downstream of the promoter obtained. For consideration of compatibility with existing expression vectors (EP 361,991), "portable" promoters were constructed in the form of Sall-Hindlll fragments.

A Hindlll 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, 5 were used for this purpose.

The oligodeoxynucleotide A corresponds to the sequence situated at 467 bp upstream of the ATG codon, at the level of a Hindlll site which will thus be replaced by a Sail site during the amplification. The 10 oligodeoxynucleotide B corresponds to the sequence upstream of the initiation site, and enables a Hindlll site to be introduced in position -6.

The fragment obtained PCR was inserted between the Sail and Hindlll sites of the bacteriophage M13tgl30 in 15 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 bp recombinant DNA obtained above was 20 introduced at the level of the Sail and Hindlll sites, inside the plasmid pYG45 (Figure 7), in order to obtain the vector pYG614 (Figure 8). The plasmid pYG45 contains an expression cassette consisting of the promoter and the terminator of the S. cerevisiae PGK gene between which the 25 prepro-human serum albumin-encoding gene (prepro-HSA sequence) is inserted at the level of an Hindlll site. £• ' * " pYG45 is derived from pYG18 (see European Patent 361,991) by subcloning the Sall-BamHI fragment, containing the HSA expression cassette into the sites corresponding to the vector plC-20RDH (Figure 7). pIC-20RDH is obtained by 5 digesting the plasmid pIC-20R (March et al., Gene 32., 1984, 481) with the enzyme Hindlll. filling the ends using the Klenow fragment of E. coli polymerase I and recircularisation with T4 DNA ligase.

The Sall-SacI fragment may be isolated from the 10 plasmid pYG614 by digestion. It contains: a 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. 15 Another expression cassette may be obtained from the plasmid pYG614 by cloning the Af111-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 20 generates the plasmid pYG615. The Sall-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 2 5 expression cassette using the promoter sequence of the invention. y . ' L "» - ■* 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 5 (Figure 10). In particular, an expression vector was obtained (vector pYG621) by inserting the Sall-SacI fragment from pYG615, containing the albumin expression cassette, inside the vector pYG72 (see Figure 10). This vector corresponds to the plasmid pKan 707 (see EP 361,991) 10 from which the SacI fragment, containing the URA3 gene, has been removed, as well as the unique Hindlll site present in the aph gene, so as to facilitate subsequent constructions. The aph gene encodes aminoglycoside 3'-phosphotransferase (I) (Oka et al., J. Mol. Biol. 147. 15 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 9). The Hindlll site present inside this gene was destroyed by 20 site directed mutagenesis according to the method described by Taylor et al., (Nucleic Acid Res. 13., 1985, 8749). The resultant plasmid was called pTY65 (Figure 9). The oligodeoxynucleotide used for the mutagenesis had the sequence 5'-GAA ATG CAT AAG CTC TTG CCA TTC TCA CCG -3' and 25 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 iw. ' containing the bacterial replicon of the vector pKan 707 was isolated by digestion with the enzyme EcoRI and recircularisation with T4 DNA ligase so as to obtain pYG69. The PstI fragment present in this latter vector, containing 5 the aph gene, was replaced by the equivalent mutant fragment derived from pYG65. This construct was called pYG70. The 4.7 kb pKDl sequence between the EcoRI and SacI sites was introduced inside this latter vector so as to obtain pYG72. The vector pYG621 (Figure 11) was obtained by 10 insertion of the Sall-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 Sail 15 site and a Hindlll 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 20 using, on the one hand, the oligodeoxynucleotide A which introduces a Sail 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 Hindlll site in the -1 position relative to the site of 25 initiation of translation of the X gene (see Figure 6(a)). 3 PCR reactions were then carried out using, at each stage, -lithe plasmid pYG610 as template, so as to remove a Hindlll site present in the promoter region: - the first 2, to amplify the regions on either side of the Hindlll site using the oligodeoxynucleotides A and B coupled to the oligodeoxynucleotides C and D respectively (Figure 6). These last 2 are complementary and they enable a point mutation to be introduced at the level of the inner Hindlll 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 than 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, 7). This 20 strain is derived from the wild strain CBS2359 and is of the genotype: Mat_, uraA, lvsA. araA. K+, cir. The transformant yeasts are selected for the G418-resistance phenotype which the plasmid pYG621 confers on YPD medium (10 g/1 yeast extract, 20 g/1 peptone, 20 g/1 glucose) 25 containing 0.2 g/1 of geneticin. The plasmid pYG72- transformed strains not containing the expression cassette ' " J 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 to that 5 from S. cerevisiae. The vector pYG19 is similar to the vector pYG621 expect that the albumin gene is under the control of the S. cerevisiae (EP 361,991). a) Analysis of the mRNAs: The cells are cultured at 28°C in selective YPD 10 medium (10 g/1 yeast extract, 20 g/1 peptone, 20 g/1 glucose) containing 0.2 g/1 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 hybridised to 15 a probe corresponding to the albumin structural gene (1.9-kb Hindlll-Hindlll fragment) derived from the vector pYG18 (Figure 7) following the Northern blot method (Maniatis et al., 1982 Molecular cloning, Cold Spring Harbor, Laboratory Press). Autoradiography clearly shows a 20 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 25 S. cerevisiae PGK promoter (pYG19).

■■■• J b) Analysis of the proteins: The cells are cultured in Erlenmeyer flasks in a selective YPD medium (10 g/1 yeast extract, 20 g/1 peptone, 20 g/1 glucose) containing 0.2 g/1 geneticin, at 28°C with 5 shaking. After 96 hours of culture, 30 /il of supernatant are collected and mixed with an equivalent volume of 2x Laemmli butter (Laemmli, 1970, Nature 227, 680). After heating at 96°C for 10 minutes, the sample proteins are then separated on 8.5 % SDS-polyacrylamide gel. The 10 production of albumin is then visualised 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 15 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 20 promoter. This region, or reduced forms or derivatives thereof, constitute an important industrial tool for microbiological, and more particularly eucaryotic, production systems. 2.4 l b 4 3

Claims (16)

WHAT WE CLAIM IS:

1. An isolated DNA sequence comprising all or part of the sequence presented in Figure 1 or an isolated sequence of each conplenentary strand, or of a derivative thereof, and possessing 5 transcriptional promoter activity.

2. An isolated 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 10 according to claim 1 or 2.

4. Recombinant DNA according to claim 3, which contains, in addition, one or more structural genes.

5. Recombinant DNA according to claim 4, which also contains signals permitting the secretion of the 15 expression product of the said structural gene(s).

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

7. A recombinant cell containing a DNA sequence 20 or recombinant DNA according to any one of the preceding claims.

8. A recombinant cell according to claim 7, which is a yeast.

9. A recombinant cell according to claim 8, 25 which is a yeast of the Kluvveromvces genus. ^

10. Use of a DNA sequence according to claim 1 cv>> , "" \\\ ' 24 rr '' ''O - 21 - or 2 or of recombinant DNA according to any one of claims 3 to 6 for the expression of recombinant genes.

11. Use according to claim 10, for the simultaneous expression of a plurality of recombinant genes 5 in 2 opposite directions.

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

13. Process for the preparation of a recombinant 10 protein or peptide which comprises expressing its gene in a cellular host under the control of a DNA sequence according to claim 1 or 2.

14. Process according to claim 13 in which the said protein is human serum albumin.

15 15. An expression plasmid comprising recombinant DNA according to any one of claims 3 to 5 and substantially as hereinbefore described.

16. Process according to claim 13 substantially as hereinbefore described. , > / /