IE922007A1 - Cloning and/or expression vectors, preparation and use - Google Patents

Abstract

The invention relates to a new yeast plasmid, to new cloning and/or expression vectors derived from it, and to their use.

Description

The present invention relates to a new yeast plasmid, cloning or expression vectors derived from the plasmid and to the preparation and use of the vectors, in particular for the production of recombinant proteins. The invention also relates to recombinant host cells containing such vectors.

More especially, the present invention relates to all or part of a new plasmid isolated from Kluyveromvces waltii yeast, or of a derivative of this plasmid, and to the cloning or expression vectors constructed from the latter.

During the last ten years, it has become apparent that yeast is a very promising host microorganism for the production of heterologous proteins.

In particular, the demonstration by Beggs et al (Nature 275 (1978) 104) of the 2μ plasmid and of vectors derived from the latter has been one of the key factors in the current development of the genetic and molecular study of Saccharomvces cerevisiae yeast. Since then, the 2 μ system has enabled heterologous genes to be introduced and expressed in yeast in order to obtain proteins of pharmaceutical or agri-foodstuffs interest. However, the 2μ plasmid and its derivatives can replicate efficiently only in yeasts belonging to the species S. cerevisiae and to a few species closely related to the latter. Hence this system cannot be used for genetic manipulation in most species of yeast. In particular, it cannot be used in - 3 yeasts whose physiological properties, differing from those of S. cerevisiae. would permit novel applications and/or industrial exploitation at a higher level of output.

Various laboratories have hence sought other 5 plasmids in miscellaneous species of yeast. Thus, several circular plasmids have been found in yeast of the genus Zvgosaccharomvces. and in particular plasmids pSRl and pSR2 (Toh-έ et al., J. Bacteriol. 151 (1982) 1380); pSBl, pSB2, pSB3 and pSB4 (Toh-e et al., J. Gen. Microbiol., 130 (1984) 2527); and pSMl (Utatsu et al., J. Bacteriol. 169 (1987) 5537). A circular plasmid has also been found in Kluvveromvces drosophilarum: pKDl (Falcone et al., Plasmid 15 (1986) 248).

All these plasmids possess features in common with the 2μ plasmid, and in particular inverted repeat sequences, and existence in two possible isomeric forms due to a site-specific recombination system.

However, these plasmids still possess the drawback of having a narrow host spectrum. Thus, as a result of their specificity with respect to the yeast host species, these plasmids can be used in only a limited number of strains.

The present invention is based on the isolation a natural plasmid of Kluvveromvces waltii yeast. This plasmid, designated pKWl, is the first known natural plasmid of this species of yeast. This plasmid was purified from a K. waltii strain, designated CBS 6430, and mapped by - 4 means of restriction enzymes: the resulting map is presented in Figure 1.

The present invention therefore relates to the plasmid pKWl isolated from K. waltii strain CBS 6430, or a fragment or derivative of the plasmid.

Derivative is understood, in the sense used in the invention, to mean plasmids which, have been modified but retain properties from the starting plasmid. In particular, the modifications can take the form of mutations or deletions of regions up to relatively large size. They can also be insertions or deletions, for example, of cloning sites.

Fragment of pKWl is understood to mean, in particular, the various genetic elements of this plasmid.

More preferably, as genetic elements of pKWl, there may be mentioned, in particular, the structural genes or portions thereof, functional promoter sequences, the inverted repeat (IR) sequences or alternatively the sequences permitting replication (origin of replication) or conferring stability on the plasmid (stability locus). Such fragments will be of sufficient size to be uniquely identifiable and generally contain 20 or more, preferably 30 or more, more preferably 40 or more and most preferably 50 or more bases. Particular example of fragments are those which may be obtained by digestion of the plasmid pKWl with one or more restriction endonuclease enzymes.

In effect, structural study of plasmid pKWl has - 5 enabled analogies with the 2μ plasmid of S. cerevisiae to be demonstrated. Thus, 4 structural genes have been demonstrated (see Figure 2), as well as an origin of replication. Moreover, the cloning of pKWl in E. coli has enabled 4 types of recombinant plasmids to be isolated, corresponding to 2 isomeric forms of plasmid pKWl (A and B forms) cloned into the vector pKan21 in both possible orientations (the vector pKan21 is described in Example 3.1). These two forms are shown in Figure 1. The existence of two isomeric forms indicates the presence of inverted repeat sequences. The study thus showed that plasmid pKWl contains a pair of inverted repeat sequences of 0.3 kb each, and two single sequences of 2.5 and 2.3 kb whose orientation distinguishes the 2 isomeric forms A and B. The molecular size of plasmid pKWl is hence approximately .5 kb. Various restriction sites have been demonstrated, and as an example the following single sites: EcoRI, SphI, Sail, Clal, Nhel and Bgll.

The complete nucleotide sequence of plasmid pKWl has also been determined (Figure 3). The absence of homology between this sequence and that of known plasmids has been demonstrated, in particular by hybridisation experiments (see Example 2). The absence of hybridisation under moderately stringent conditions is characteristic of this difference in sequence.

In a preferred embodiment, the subject of the invention is a plasmid comprising all or part of the - 6 sequence shown in Figure 3 or of a derivative of this sequence.

Moreover, the Applicant has also demonstrated that it is possible to use plasmid pKWl or fragments of the latter to construct especially stable cloning and/or expression vectors.

The present invention further relates to cloning or expression vectors, which comprise the DNA sequence of the plasmid pKWl of K. waltii CBS 6430 shown in Figure 1, or a fragment thereof, or a derivative thereof.

In particular the invention relates to a cloning or expression vector, which comprises at least one genetic element of the plasmid pKWl.

As a result of the host spectrum of plasmid pKWl, 15 the vectors of the invention may be used in species other than the K. waltii natural host.

They may, in particular, be used for the transformation of a wide variety of species, in particular a variety of yeast species.

Various types of vectors have been constructed from pKWl, differing in respect of the size of the fragment originating from pKWl, and hence in respect of the functional elements emanating from pKWl.

In a still more particular embodiment the invention relates to a cloning or expression vector, which comprises the origin of replication of plasmid pKWl.

Other constructions may be prepared, containing - 7 larger or smaller fragments, enabling the influence of the various elements of PKW1 on the stability of the vectors, their host specificity and their efficacy for the expression of heterologous genes to be studied. In particular, expression vectors may be produced from the various genetic elements of plasmid pKWl (origin of replication, inverted repeat sequences, structural genes, promoter regions, etc.) that may be introduced into known plasmids to improve their performance or confer new properties on them. Similarly, vectors may be obtained by adding elements to plasmid pKWl, or by replacing certain genetic elements of pKWl by elements originating from other plasmids. Thus, vectors may be obtained by replacing, for example, the origin of replication of pKWl by the origin of replication of the 2μ plasmid of S. cerevisiae or of plasmid pKDl of Kluyveromvces. or by a chromosomal replicon (ARS) of a yeast (for example KARS of K. lactis).

Similarly, vectors may be obtained by replacing the stability locus of pKWl by that of the 2μ plasmid of S. cerevisiae or of plasmid pKDl of Kluyveromvces. It can be especially advantageous to produce hybrid vectors comprising elements of plasmids pKDl and pKWl.

Advantageously, the vectors of the invention comprise substantially the whole, preferably the whole, of DNA sequence of plasmid pKWl as shown in Figure 1.

Preferably, the vectors of the invention comprise plasmid pKWl linearised at a functionally neutral - 8 restriction site.

Functionally neutral restriction site is understood, in the sense used in the present invention, to mean a restriction site at which it is possible to interrupt the structure of the plasmid without impairing it properties of replication and of stability.

In particular, the sites in question may be ones present in plasmid pKWl. As an example, there may be mentioned, in particular, the Clal(l), PstI(4608) or EcoRV (3072) site as shown in Figure 1.

The sites in question may also be ones introduced artificially into plasmid pKWl, or rendered unique. In this case, the sites are preferably introduced into intergenic regions of the plasmid, and in particular into the region located between the genes B and D or into that located between the gene D and IR2.

Advantageously, according to the present invention, the DNA of plasmid pKWl is linearised at a single restriction site.

An especially advantageous site in this connection is the single Clal site localised at position 1 in Figure 1. The Applicant has, in effect, shown that this site enabled plasmid pKWl to be used to construct cloning or expression vectors, by introducing, for example, fragments of heterologous DNA at this point, while maintaining stable replication of the vector obtained. This result is surprising, inasmuch as the Clal site is - 9 localised in the structural gene B.

The use of such neutral cloning sites hence makes it possible to obtain very stable vectors capable of being maintained in transformed cells, even in the absence of any selection pressure.

Advantageously, the vectors of the invention contain, in addition, a heterologous DNA sequence comprising at least one structural gene, under the control of signals permitting its expression.

The signals permitting expression of the structural gene or genes can consist of one or more elements chosen from promoters, terminators or secretion signals. It is understood that these signals are chosen in accordance with the host used, the structural gene and the desired result. In particular, it can be preferable in some cases to use a regulable promoter, enabling the growth phase of the host and the expression phase of the said structural gene or genes to be decoupled. Similarly, the use of a signal peptide (secretion signal) can enable the levels of production of the desired protein to be increased and the purification step to be facilitated.

Preferably, the promoters used are derived from yeast genes. Of very special interest are the promoters derived from glycolytic genes of yeasts of the genus Saccharomvces or Kluvveromvces. In particular, the promoters of the genes coding for phosphoglycerate kinase of S. cerevisiae (PGK), glyceraldehyde-3-phosphate - 10 dehydrogenase (GPP), enolases (ENO) or alcohol dehydrogenases fADH) may be mentioned. Promoters derived from strongly expressed genes such as the gene for lactase (LAC4) or for acid phosphatase (PHO5), may also be mentioned.

Moreover, these regions may be modified by mutagenesis, for example to add further elements for controlling transcription, such as, in particular, UAS (upstream activating sequence) regions.

The structural gene which can be introduced into the vectors of the invention preferably codes for a polypeptide of pharmaceutical or agri-foodstuffs interest. As an example, there may be mentioned enzymes (such as, in particular, superoxide dismutase, catalase, amylases, lipases, amidases, chymosin, etc.), blood derivatives (such as serum albumin, alpha- or beta-globin, factor VIII, factor IX, von Willebrand factor, fibronectin, alphajantitrypsin, etc.), insulin and its variants, lymphokines [such as interleukins, interferons, colony stimulating factors (G-CSF, GM-CSF, M-CSF, etc.), TNF, TRF, etc.), growth factors (such as growth hormone, erythropoietin, FGF, EGF, PDGF, TGF, etc.), apolipoproteins or alternatively antigenic polypeptides for the production of vaccines (hepatitis, cytomegalovirus, Epstein-Barr, herpes, etc.).

In a particular embodiment of the invention, the structural gene can be a gene resulting from the fusion of - 11 several DNA sequences. It can be, in particular, a gene coding for a hybrid polypeptide containing, for example, an active portion combined with a stabilising portion. As an example, fusions between albumin or albumin fragments and a virus receptor or a portion of such a receptor (CD4, etc.) may be mentioned.

In another embodiment, the heterologous DNA sequence can comprise several structural genes, and in particular genes participating genetically or biochemically in the biosynthesis of a metabolite. The metabolite can, in particular, be an antibiotic, an amino acid or a vitamin.

In a particular embodiment, the vectors of the invention contain in addition: - an E. coli replicon, and/or - at least one selectable marker.

These elements enable the vectors of the invention to be manipulated much more easily.

Another subject of the invention relates to recombinant cells containing a vector as defined above.

The recombinant cells are preferably chosen from yeasts.

The Applicant has shown that the vectors of the invention could, in effect, be used not only in K. waltii (the natural host of pKWl) but also in yeasts of different species or even of different genera. In particular, they may be used in other Kluyveromyces species or in Saccharomvces. Moreover, when K. waltii strain CBS 6430 is - 12 used as a host cell, homologous recombinations between the vectors of the invention and the resident plasmid pKWl can affect the stability of the vectors and can thereby decrease the performance of the host/vector system. In order to improve further the stability of the vectors of the invention in such a host/vector system, the Applicant has prepared a K. waltii strain pKWl (KW18). This strain, which does not contain pKWl, enables the industrial use of the vectors of the invention to be optimised (see Examples 4 and 5).

Various techniques may be used to introduce the vectors of the invention into the host cells. In particular, transformation (Bianchi et al., Curr. Genet. 12 (1978) 185) and electroporation (Delorme, Appl. Environ.

Microbiol. 155 (1989) 2242) give good results. It is nevertheless clear that the invention is not limited to a particular technique.

The invention also relates to a process for preparing a polypeptide, according to which a recombinant cell as defined above is cultured and the polypeptide produced is recovered. More especially, the process of the invention permits the production of proteins of pharmaceutical or agri-foodstuffs interest, such as those mentioned above. More specifically, the process of the invention is suited to the production of human albumin and its variants or precursors.

In the case where the structural genes - 13 participate in the biosynthesis of a metabolite, the recombinant cells may also be used directly in a bioconversion process.

Other advantages of the present invention will 5 become apparent on reading the examples which follow, which are to be considered as illustrative and non-limiting.

LEGENDS TO THE FIGURES Figure 1: Restriction map of plasmid pKWl. The inverted repeat sequences as well as the structural genes A-D are indicated. The positions indicated for the restriction sites correspond to the first nucleotide recognised by the enzyme.

Figure 2: Study of the open reading frames of pKWl. The genetic elements indicated are localised at the following positions with respect to the sequence presented in Figure 3: Gene A: nucleotides 1454 to 2755; Gene B: nucleotides 4948 to 54; Gene C: nucleotides 389 to 1309 on the complementary strand; Gene D: nucleotides 3444 to 4313 on the complementary strand; IR1: nucleotides 53 to 368; IR2: nucleotides 2713 to 3028.

Figure 3: Nucleotide sequence of plasmid pKWl. The sequence shown corresponds to the B form of the plasmid. Position 1 corresponds to the first nucleotide of the sequence recognised by the enzyme Clal. Plasmid pKWl was fragmented with restriction enzymes and the fragments were cloned into pTZ18R (Pharmacia). The sequences of the cloned segments - 14 ~ were determined by Sanger's method on both strands.

Figure 4: Restriction maps of the shuttle plasmids YIP5 and pKan21. Ap: Ampicillin resistance gene; Tc: Tetracycline resistance gene; Km: Kanamycin (G418) resistance gene; LacZ: β-Galactosidase structural gene.

Figure 5: Strategy for construction of the vectors pBNAl, pNEA2, pBNBl/A3, pNEBl, pXXY2 and pXXK3. See also Table 1. Figure 6: Restriction map of the vector pXXK3.

Figure 7: Strategy for construction of the vectors pKWCll, 10 pKWSl and pKWS14.

Figure 8: Study of stability of the vectors pKWCll and pXXK3 in K. waltii strain KW18. In each case, a transformed clone was cultured in non-selective YPG medium for the number of generations indicated, and culture 15 aliquots were then plated out on dishes of YPG agar medium with and without G418 in order to determine the total number of cells and the number of G418-resistant cells. The stability corresponds to the % of resistant cells.

Figure 9: Strategy for construction of the expression vector pXPHO5.

Abbreviations: P = promoter, T = terminator, ss = secretion signal, CIP = calf intestinal phosphatase, Km = kanamycin, E = EcoRI, H = Hindlll, S = Sail, B = BamHI, Sm = Smal. Figure 10: Restriction maps of the vectors pXKN18 and pXPHO5. Legend: see Figure 9.

Figure 11: Immunological detection of IL-1B produced by K. waltii. The molecular weight (KDa) markers are indicated at - 15 the left. Well l: Reference IL-lB (100 ng); Well 2: Culture supernatant of the transformant pXKNIS (without IL-1B cassette); Well 3: Culture supernatant of the transformant pXPHO5 treated with endo-N-acetylglucosaminidase H; Wells 4 and 5: Culture supernatants of the transformants pXPHO5 in LPi and HPi medium, respectively.

Figure 12: Strategy for construction of plasmid pYG65.

Figure 13: Strategy for construction of plasmid pYG70.

Figure 14: Strategy for construction of plasmid pYG141. aph: gene coding for aminoglycoside 3'-phosphotransferase, conferring kanamycin resistance; bla: gene coding for βlactamase, conferring ampicillin resistance.

Figure 15: Strategy for construction of plasmid pYG142. Tables 1 and 2: Composition of vectors derived from pKWl according to the invention. (*) Cloning sites.

Tables 3, 4 and 5: Transformation of S.cerevisiae. K.waltii and various strains of Kluyveromvces, respectively, with vectors of the invention. The stability of the transformants is expressed as the percentage of Ura+ cells after 10 generations of growth in YPD non-selective medium. The isonuclear strains K.waltii pKWl+ and pKWl- are CBS 6430 and KW18, respectively.

GENERAL CLONING TECHNIQUES The conventional methods of molecular biology, such as caesium chloride/ethidium bromide gradient centrifugation of plasmid DNA, digestion with restriction - 16 enzymes, gel electrophoresis, electroelution of DNA fragments from agarose gels, transformation in E.coli. etc., are described in the literature (Maniatis et al., Molecular Cloning : a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986; Ausubel et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987).

Directed mutagenesis in vitro with oligodeoxynucleotides is performed according to the method developed by Taylor et al. (Nucleic Acids Res. 13 (1985) 8749-8764), using the kit distributed by Amersham. Nucleotide sequencing is carried out according to the dideoxy technique described by Sanger et al. (Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467). Enzymatic amplification of specific DNA fragments is performed by a PCR reaction (polymerase-catalysed chain reaction) under the conditions described by Mullis and Faloona (Meth. Enzym., 155 (1987) 335-350) and Saiki et al (Science 230 (1985) 1350-1354), using a DNA thermal cycler (Perkin Elmer Cetus) according to the manufacturer's recommendations.

EXAMPLES 1) Isolation and purification of pKWl The strain CBS 6430 is cultured in 2 litres of YPG medium (1 % yeast extract, 1 % Bacto-peptone, 2 % glucose) with agitation at 26°C for approximately 18 hours - 17 The cells in early stationary phase are harvested by centrifugation. 13 to 15 grains of cell mass are usually obtained per litre. The cells are washed with 150 ml of 1M sorbitol containing 30 mg of Zymolyase 20T (Kirin Breweries Co., Tokyo). After incubation at 30°C for 1 hour, 5 ml of 10 % sodium dodecyl sulphate and 5 ml of 0.5M EDTA, pH 7.0, are added to the suspension of photoplasts. The mixture is immediately agitated vigorously and incubated at 50eC for 1 to 2 hours. Potassium acetate is added to the lysate to a final concentration of 1M, and the mixture is kept in ice for 2 hours. The precipitates formed are removed by centrifugation (Sorvall SS34, 15,000 rpm, 30 minutes). 2 volumes of 95 % ethanol are added to the supernatant, which is cooled in ice in order to precipitate the nucleic acids.

The precipitates are collected by centrifugation washed with 70 % ethanol, dried under vacuum and finally dissolved in 40 ml of 5 x TE (lx TE is 10 mM Tris-HCl, ImM EDTA, pH8). After the addition of 40 g of CsCl and 5 ml of ethidium bromide (stock solution, 10 mg/ml), the mixture is centrifuged at 60,000 rpm for 6 hours (Beckman 60 Ti rotor). The fluorescent band of the plasmid DNA lies below the major band of chromosomal DNA. The plasmid DNA is collected and subjected to a second cycle of CsCl/ethidium bromide centrifugation. The collected plasmid DNA is mixed with one volume of isopropanol equilibrated beforehand with 4M CsCl to remove the ethidium bromide. After several extractions with isopropanol, the DNA solution is dialysed - 18 against 1 x TE. The volume of the solution may be reduced by dialysis against polyethylene glycol 6,000 in flake form. 2) Sequencing and studies of homology 5 Plasmid pKWl was sequenced using the method described by Sanger et al (Proc. Natl. Acad. Sci. USA 74. (1977) 5463-5467). The complete sequence is shown in Figure 3.

The absence of homology between this sequence and 10 that of known plasmids was determined by molecular hybridisation experiments: pKWl is labelled with 32P and hybridised with the following plasmids, which are immobilised beforehand on a nitrocellulose filter: - 2μ plasmid of S.cerevisiae. - pSRl, pSB3 and pSB4 of Z.rouxii. - pSBl and pSB2 of Z.bailii. and - pKDl of K.drosophilarum.

Hybridisation was carried out under moderately stringent conditions (0.6 M Na+, 65°C, 18 hours); none of the plasmids gave a positive hybridisation signal.

The other circular plasmids (pSBl, pSB4) whose sequences have not yet been described also differ from pKWl, in molecular size, in the length of the reverse repeats and in their host species. 3) Construction of cloning vectors derived from pKWl Two types of recombinant molecules were - 19 constructed from pKWl. 3.1. In the first type, various fragments of pKWl (corresponding, for example, to the genetic elements of pKWl) were introduced into shuttle vectors, and in particular into the vectors YIp5 (Struhl et al., Proc. Nat. Acad, sci USA 76 (1979) 1035) and pKan21, shown in Figure. 4.

Plasmid pKan21 was constructed by insertion of the aph gene (Genblock, Pharmacia) conferring kanamycin (G418) resistance, in the form of a 1.25-kbp, AccI fragment, into the inner Narl site of plasmid pUC19 (Viera et Messing, Gene 19 (1982) 259). pKan21 hence contains, in addition to aph. the bla gene conferring ampicillin resistance and the origin of replication of ColEl permitting replication in E.coli. Yeasts transformed with vectors derived from pKan21 may be detected by their growth on medium containing 200 Mg/ml of Geneticin (G418).

Plasmid YIp5 is a derivative of plasmid pBR322 in which the URA3 gene of S.cerevisiae has been inserted as a selectable marker. Yeast transformed with YIp5 derivatives is detected by its growth on a uracil-free medium. In the latter case, the host yeast is an auxotroph deficient in orotidinemonophosphate carboxylase of the pathway of uracil synthesis.

The vectors of this first type are pBNAl, pNEA2, PBNB1/A3, pNEBl, pXXY2 and pXXK3 (Table 1 and Figure 4).

- Vector pBNAl - 20 pKWl is digested with BG1II and Nhel (see Figure 5), and the DNA fragments are separated by electrophoresis. The 2.4-kbp fragment is recovered and inserted by ligation between the single BamHI and Nhel sites into the tetracycline resistance gene of plasmid YIp5 (Yanish-Peron et al., Gene 33, 1985, 103-119), the latter being digested beforehand with BamHI and Nhel and repurified. The Bglll and BamHI ends are compatible for specific ligation. E.coli transformed with the ligation mixture is selected on LB agar medium containing ampicillin. The insertion is verified by replicating the transformants on LB agar medium containing tetracycline, these transformants being sensitive to this antibiotic. The structure of the plasmid is verified by extracting the DNA from individual transformants and analysing it with restriction enzymes. As an example, plasmid pBNAl digested with Pstl produces 3 fragments of 3.28, 3.15 and 1.36 kbp; Pstl/Nhel double digestion gives 4 fragments, of 3.15, 2.3, 1.36 and 1.0 kbp.

- Vector pNEA2 The 2.0-kbp Nhel-EcoRI fragment of pKWl (see Figure 5) is isolated and inserted between the two single NHel and EcoRI sites into the tetracycline resistance gene of YIp5, the latter being digested beforehand with these two enzymes. The ligation product is introduced into E.coli. and the ampicillin-resistant and tetracyclinesensitive transformants are isolated. The plasmid is - 21 isolated as in the case of pBNAl from one of these transformants. The structure of the recombinant plasmid obtained is verified by restriction. As an example, Pstl digestion produces 2 fragments, of 4.35 and 3.15 kbp; Pstl/Xhol double digestion produces 4 fragments of 3.15, 1.8, 1.75 and 0.7 kbp.

- Vector pXXY2 The 545-bp Xhol-Xbal fragment of pKWl (see Figure 5) is isolated and inserted between the single Sall-Nhel sites into the tetracycline resistance gene of plasmid YIp5. The Xhol and Sail ends on the one hand, and Xbal and Nhel ends on the other hand, are compatible for specific ligation. The ligation product is introduced into E.coli. and the recombinant plasmid is isolated as in the case of pBNAl. The structure of the plasmid obtained is verified by restriction. As an example, EcoRI + Nrul digestion produces two fragments, of 4.5 and 0.96 kbp. - 22 - Vector pXXK3 The 545-bp Xhol-Xbal fragment of pKWl (see figure 5) is isolated and inserted between the single Sall-Xbal sites (N-terminal polylinker of LacZ) of plasmid pKan21.

The ligation mixture is introduced into E.coli. and the transformants, plated out on LB medium containingX-gal and IPTG, are isolated as white colonies among the blue ones. When replicated on LB medium con-taining kanamycin, they grow well. These colonies are individually analysed for their plasmid content. The plasmid obtained from one of the transformants possesses the structure shown in Figure 6, verified by restriction. As an example, BamHI + Pstl digestion produces four fragments, of 2.5, 1.3, 0.55 and 0.19 kbp.

- Vector pBNBl/A3 The 1.9-kbp BGIII-Nhel fragment of pKWl (see Figure 5) is isolated and inserted between the BamHI-Nhel sites of YIp5. The recombinant plasmid is isolated as in the case of pBNAl. The structure of the plasmid is verified by restriction. As an example, Pstl digestion produces 3 fragments, of 3.15, 2.8 and 1.36 kbp; Pstl-Nhel double digestion produces 4 fragments, of 3.15, 1.8, 1.36 and 1.0 kbp.

- Vector pNEBl The 2.5-kbp Nhel-EcoRI fragment of pKWl (see Figure 5) is isolated and inserted between the NHel-EcoRI sites of YIp5. The recombinant plasmid is isolated as in - 23 the case of pBNAl. The structure of the plasmid is verified by restriction. As an example, Pstl digestion produces 2 fragments, of 4.65 and 3.15 kbp; Pstl/Xhol double digestion produces 3 fragments, of 3.15, 2.9 and 1.75 kbp. 3.2. The second type of recombinant molecule contains the whole of the pKWl sequence. To obtain these vectors, pKWl is linearised by single cleavage at one restriction site, enabling segments of heterologous DNA to be introduced. Such segments can contain structural genes included, for example, in expression cassettes, and/or complete shuttle vectors such as, in particular, pKan21 or YIp5 (Figure 4).

The examples of this type of vector are pKWCll, pKWSl and pKWS14 (Table 2 and Figure 7).

- Vector pKWS14 The DNA of pKWl is digested with the restriction enzyme Sail. Plasmid pKan21, described above, is also digested with Sail (the single Sail site is localised in the cloning multisite in the LacZ gene). The two plasmids are ligated with DNA ligase. The ligation mixture is used for the transformation of E.coli JM83 as in the previous case. The suspension of transformed cells is plated out on LB agar containing X-gal and IPTG. The white colonies among the blue ones are recovered individually. They are resistant to ampicillin and to kanamycin. Their plasmid content is analysed on DNA minipreparations as before. The plasmid, pKWS14, isolated from one of the transformants, - 24 contains the A form of plasmid pKWl and possesses the structure shown in Figure 7. This structure is verified by restriction. As an example, BamHI digestion produces 3 fragments, of 5.6, 2.6 and 1.2 kbp.

- Vector pKWCll Plasmid pKWl is digested with Clal. Plasmid pKan21 is digested with Accl. They are repurified by phenol treatment and ethanol precipitation. The DNAs of the two plasmids are mixed in approximately equal quantities and subjected to the ligation reaction with DNA ligase overnight. The ligation product is amplified in E.coli. The colonies of the transformants are white on LB medium containing X-gal and IPTG. They are resistant to kanamycin and ampicillin on medium containing one or other of these antibiotics. The plasmid, pKWCll, isolated from one of the transformants, contains the A form of plasmid pKWl and possesses the structure shown in Figure 7. This structure is verified by restriction. As an example, BamHI digestion of the plasmid produces 3 fragments, of 5.6, 2.9 and 0.9 kbp.

- Vector pKWSl Plasmid pKWl and plasmid YIp5 are digested with Sail. The mixture is repurified and subjected to the ligation reaction. The ligation product is introduced into E.coli. Ampicillin-resistant and tetracycline-sensitive transformants are obtained. The plasmid, pKWSl, isolated from one of them, contains the A form of plasmid pKWl and - 25 possesses the structure shown in Figure 7. As an example, EcoRI digestion of the plasmid gives two fragments, of 8.3 and 2.7 kbp. 4) Construction of a strain CBS 6430 pKWl- K.waltii CBS 6430 was first transformed with the recombinant plasmid pKWS14 (Table 2 and Figure 7). The method of transformation used is essentially that described by Chen and Fukuhara (Gene 69, 181 (1988)) using protoplasts. The transformants obtained are maintained for 75 generations on YPD-agar medium, 1 mg/ml G418 (YPD medium : yeast extract 10 g/1; peptone 20 g/1; glucose 20 g/1). They are then transferred to YPD liquid medium without antibiotic and maintained for 10 generations.

Under these conditions (without selection pressure), plasmid pKWS14 is gradually lost. The G418sensitive colonies which appear are removed and tested individually for the presence of plasmids. The test consists in extracting the cell DNA, followed by electrophoresis of these DNAs on a agarous gel. The presence of plasmids is visualised by ethidium bromide staining. Among colonies which became G418-sensitive, 25 % proved to be bereft of any plasmid. One of these colonies was kept as a strain of K.waltii devoid of plasmid pKWl and designated KW18.

) Transformation of various yeasts .1. Transformation of S.cerevisiae Among the various vectors described in Tables 1 - 26 and 2 and in Figures 4 and 5, some containing the marker URA3 were used to transform a ura3 auxotrophic strain of S.cerevisiae (the strain S150—2B: Mat a, ura3. leu2. trol. his3, 2μ).

The method of transformation is essentially that described by Sherman et al (Yeast Genetics, Cold Spring Harbor, NY, 1986).

The results obtained are shown in Table 3. They show that the vectors of the invention are capable of transforming yeasts of the genus Saccharomvces. .2. Transformation of K.waltii Transformation of K.waltii was carried out with vectors carrying the kanamycin resistance marker.

The method of transformation used is essentially 15 that described by Chen and Fukuhara (Gene 69 (1988) 181) using protoplasts. It is clear that any other technique enabling DNA fragments to be introduced into a microorganism may be used.

The results obtained are presented in Table 4.

They show that the vectors of the invention are capable of transforming K.waltii yeast with a high frequency.

Moreover, the stability study described in Figure 8 shows that vectors may be obtained from pKWl, possessing 100 % stability after 50 generations of growth in a nonselective medium. This is fully illustrated by the vector pKWCll. This study also shows that it is preferable, in - 27 order to obtain a relatively high stability, to use vectors containing only the origin of replication of plasmid pKWl in host cells possessing a resident pKWl plasmid. .3 Transformation of other yeasts The vector pKWCll, which is highly stable and autonomous in K.waltii. was used to test transformability of various species of yeasts, and in particular those belonging to genus Kluyveromvces.

The results are presented in Table 5.

The presence of the vector pKWCll in the transformants was verified by electrophoresis.

These results collectively show that the range of species which are hosts for the vectors of the invention can be very wide, extending beyond the genus Kluyveromvces. 6) Use of the vectors of the invention for the production of heterologous proteins 6.1. Interleukin-l/?: 6.1.1. Construction of a vector derived from pKWl for the expression and secretion of IL-1/3 (Figures 9 and ).

- The vector pXXK3 (Table 1, Figure 6) is linearised with EcoRI, and the ends are filled in with the Klenow fragment of E.coli DNA polymerase I. A synthetic linker (5'-GCGGCCGC-3') forming a restriction site recognised by the enzyme Notl is added by means of T4 ligase, and the vector thereby obtained (pXKN18) is purified after its amplification in E.coli (Figure 10). - 28 - An IL-10 expression cassette is produced, composed of (a) the regulated promoter PHO5 originating from S.cerevisiae (Bajwa et al., Nucl.Acid.Res.12. (1984) 7721-7739), (b) the human IL-10 gene (Jung et al., Ann.Inst.Pasteur/Microbiol. 139 (1988) 129-146) preceded by (c) a synthetic sequence corresponding to the signal sequence of the killer toxin of pGKLl of K.lactis (preregion of the gene for the alpha subunit) (Stark and Boyd, EMBO J. 5, (1986) 1995-2002), and (d) the PHO5 terminator. The expression cassette was isolated from the vector pSPHO5-IL14, the construction of which is described in Patent EP 361,991. The cassettee was produced in the following manner: The following synthetic sequence, in the form of an EcoRI fragment: MetAsnllePheTyrllePheLeuPheLeuLeuSerPheValGlnGlyLysArg '-AATTATGAATATATTTTACATATTTTTGTTTTTGCTGTCATTCGTTCAAGGTAAAAG-3' 3'-TACTTATATAAAATGTATAAAAACAAAAACGACAGTAAGCAAGTTCCATTTTCTTAA-5' is inserted at the 5' end of the gene coding for the mature portion of IL-10.

The final codons added (Lys and Arg) form a potential cleavage site recognised by the endopeptidase Kexl of K.lactis (Tanguy-Rougeau et al; FEBS Lett.234 (1988) 464). This sequence was fused to the IL-10 gene via the EcoRI site, forming the following junction: Gly Lys Arg lie His Met Ala ·...GGT AAA AGA ATT CAT ATG GCA ....3· The alanine (GCA) corresponds to the first amino acid of - 29 mature IL-1/3. Arg-Ile-His-Met corresponds to an EcoRI-Ndel linker introduced in order to facilitate cloning (see EP 361,991).

The whole of the cassette is made into the form 5 of a Notl fragment by adding a corresponding linker (5'GCGGCCGC-3').

- The IL-l/S secretion cassette is inserted at the Notl site into pXKN18. The resulting vector is referred to as pXPHO5 (Figure 10). 6.1.2. K.waltii strain CBS 6430 is transformed with the vector pXPHO5 under the conditions described in Example 5.2. 6.1.3. Expression of IL-1/3: The transformed cells are cultured at 28°C in the absence of G418, in LPi liquid medium (having a low inorganic phosphate content) and HPi medium (having a high phosphate content), prepared according to Chen and Fukuhara (Gene 69 (1988) 181-192), for 4 days. 50 ml of culture are centrifuged, and the supernatants filtered through a Millipore membrane (0.22 gm). The proteins are precipitated by adding ethanol to a final concentration of 60 %. The precipitates are dissolved in 2 ml of Laemmli buffer (Nature 227 (1970) 680-685) and 20 μΐ samples are used for SDS-PAGE analysis according to Laemmli (document cited above). After electrophoresis, the proteins are transferred onto a nitrocellulose sheet and treated with a rabbit anti-human IL-1/3 polyclonal antiserum. The blot is then - 30 treated with a 2nd, biotinylated anti-rabbit polyclonal antibody (Vectastain ABC ImmunoPeroxidase Kit, Vector Laboratories). The antigen-antibody complex is visualised according to the protocol of the supplier.

Figure 11 shows that a protein of apparent molecular weight 21 kDa is secreted by the yeast transformed with pXPHO5. The protein is specifically recognised by anti-IL-10 antiserum. This protein is not synthesised by yeast transformed with the control vector pXKN18 (without an IL-10 cassette). The secreted protein corresponds to the glycosylated form of IL-10, which is demonstrated by reduction of the apparent MW after treatment with the enzyme endo-N-glucosaminidase H (Figure 11, lane 3). This K.waltii/pXPHO5 host/vector system, not yet optimised, secretes approximately 5 mg of IL-10 per litre of culture. The level of secretion of IL-10 by K.waltii is higher in LPi medium than in HPi medium, suggesting that the activity of the pHO5 promoter is regulated by phosphate in K.waltii as well as in S.cerevisiae. 6.2. Human serum albumin: 6.2.1. Construction of plasmid pYG140 (Figures 12-14).

A plasmid was constructed comprising: - an E.coli repiicon, - the aph gene under the control of the kl - 31 promoter of killer toxin of K.lactis (EP 361,911), in which the Hindlll site has been removed by directed mutagenesis, and - the bla gene, conferring ampicillin resistance. 5 The aph gene under the control of the kl promoter is isolated from plasmid pKan707 (EP 361,991) in the form of a Pstl fragment, which is cloned into the equivalent site of the phage M13mp7. The resulting plasmid is referred to as pYG64 (Figure 12). The Hindlll site present in this gene was destroyed by directed mutagenesis according to the method described by Taylor et al. (Nucl.Acid.Res. 13 (1985) 8749). The resulting plasmid is referred to as pYG65. The oligodeoxynucleotide used for the mutagenesis has the following sequence: '-GAAATGCATAAGCTCTTGCCATTCTCACCG-3', and enabled the CTT triplet coding for leucine 185 to be converted to CTC. To construct plasmid pYG70, the portion containing the bacterial replicon of the vector pKan707 was isolated by digestion with the enzyme EcoRI and recircularisation with T4 DNA ligase to obtain pYG69. The Pstl fragment present in the latter vector containing the aph gene was then replaced by the mutated equivalent fragment originating from pYG65. The resulting plasmid is referred to as pYG70 (Figure 13).

This plasmid is then digested with EcoRI and religated in the presence of an adaptor EcoRI-Narl-EcoRI containing the following sequence: ·-AATTCGGCGCCG-3·. - 32 The plasmid obtained is referred to as pYG140 (Figure 14). 6.2.2. Introduction of an albumin expression cassette (Figure 14).

The gene coding for prepro-HSA under the control of the promoter and the terminator of the PGK gene of S.cerevisiae was isolated in the form of a Sall-Sacl fragment from the expression vector pYG19 (EP 361,991).

This fragment was introduced into the corresponding sites of plasmid pYG140 to generate plasmid pYG141. 6.2.3. Construction of the expression vector pYG142 (Figure 15).

Plasmids pYG141 and pKWl are digested with the enzymes Narl and Clal, respectively. After ligation, 4 recombinant plasmids are obtained, as a result of the existence of the 2 forms A and B of pKWl and of the orientation of the pKWl portion relative to the pYG141 portion.

Figure 15 describes the restriction map of one of these 4 plasmids: pYG142, containing the form B of pKWl.

The other plasmids are referred to as pYG143, pYG144 and pYG145.

A sample of K.lactis strain CBS 6430 was deposited with the CBS at Baarn (Netherlands) according to the conditions of the Budapest Treaty on 4th June 1991 under number CBS 290.91.

TABLE 1 VECTOR FRAGMENT OF pKWl SHUTTLE VECTOR MARKER pBNAl Bglll-Nhel 2.4 kb YIp5 *BamHI-NheI URA3 pNEA2 Nhel-EcoRI 2.0 kb YIp5 Nhel-EcoRI URA3 pXXY2 Xhol-Xbal 0.55 kb YIp5 Sall-Nhel URA3 pXXK3 Xhol-Xbal 0.55 kb pKan21 Sall-Xbal KanR PBNB1/A3 Bgllll-Nhel 1.9 kb YIp5 BamHI-Nhel URA3 pNEBl Nhel-EcoRI 2.5 kb YIp5 Nhel-EcoRI URA3 TABLE 2 VECTOR SITE OF LINEARISATION OF pKWl SHUTTLE VECTOR MARKER pKWSl4 Sail pKan21* (Sail) KanR pKWCll Clal pKan21 (AccI) KanR pKWSl Sail YIp5 (Sail) URA3 TABLE 3 Transformation of Saccharomyces cerevisiae with vectors derived from pKWl.

VECTOR Ura+ transformants per yg of DNA pKWSl 4,400 pBNAl 1,200 10 PBNB1/A3 7,600 pXXY2 4,000 pSKl 4,500 TABLE 4 Transformation of Kluyveromvces waltii with vectors derived from pKWl.

VECTOR Replication carrier G418-resistant transformants per pg of DNA pKWl+ pKWl pKWCll whole pKWl 36,000 (98%) 8,000 (100%) 25 pXXK3 Xbal-Xhol 540bp of pKWl 35,000 (49%) 10,000 (2.8%) pKWSl4 whole pKWl 10,000 (92%) 8,000 (29%) TABLE 5 Transformation of yeasts of the genus Kluyveromvces with the vector pKWCll.

Species Strain GC% Frequency of transformation per pg of DNA Stability of the transformants (*) K.waltii K.thermo- CBS6430 45.6 2,400 100 tolerans CBS6340 46.2 4,000 25

Claims (35)

1. Plasmid pKWl isolated from K.waltii strain CBS 6430, or a fragment or derivative of the plasmid.

2. Fragment according to claim 1, characterised 5 in that it is a genetic element of pKWl or a derivative thereof.

3. Plasmid, characterised in that it comprises the sequence shown in Figure 3 or a fragment or derivative thereof. 10

4. Cloning or expression vector, characterised in that it comprises the DNA sequence of plasmid pKWl of K.waltii CBS 6430 or a fragment thereof or a derivative thereof.

5. Vector according to claim 4, characterised 15 in that it comprises at least one genetic element of plasmid pKWl.

6. Vector according to claim 5, characterised in that it comprises the origin of replication of plasmid pKWl. 20 7. Vector according to claim 6, characterised in that it comprises the whole of plasmid pKWl. 8. Vector according to any one of claims 4 to

7. , characterised in that it comprises all or part of the sequence shown in Figure 3 or a derivative thereof. 25 9. Vector according to any one of claims 4 to

8. , characterised in that plasmid pKWl is linearised at a - 37 functionally neutral restriction site.

9. 10. Vector according to claim 9, characterised in that the restriction site is present in plasmid pKWl, or introduced artificially therein. 5

10. 11. Vector according to claim 10, characterised in that the restriction site is introduced artificially into an intergenic region.

11. 12. Vector according to claim 11, characterised in that the restriction site is introduced into the region 10 located between the genes B and D or into the region located between the gene D and IR2.

12. 13. Vector according to claim 11 or 12, characterised in that plasmid pKWl is linearised at the Clal(l), PstI(4608) or EcoRV(3072) site, as shown in Figure 15 3.

13. 14. Vector according to any one of claims 4 to 13, characterised in that it contains, in addition, a heterologous DNA sequence comprising at least one structural gene, under the control of signals 20 permitting its expression.

14. 15. Vector according to claim 14, characterised in that the expression signals comprise one or more promoters, terminators or secretion signals.

15. 16. Vector according to claim 15, characterised 25 in that the heterologous DNA sequence comprises a regulable promoter.

16. 17. Vector according to claim 15 or 16, - 38 characterised in that the heterologous DNA sequence comprises a promoter derived from a yeast gene.

17. 18. Vector according to claim 17, characterised in that the promoter is derived from a glycolytic yeast 5 gene.

18. 19. Vector according to any one of claims 14 to 18, characterised in that the structural gene codes for a polypeptide of pharmaceutical or agri-foodstuffs interest.

19. 20. Vector according to any one of claims 14 to 10 19, characterised in that the structural gene codes for a hybrid protein.

20. 21. Vector according to any one of claims 14 to 20, characterised in that the structural gene or genes are genes participating genetically or biochemically in the 15 biosynthesis of a metabolite.

21. 22. Vector according to any one of claims 4 to 21, characterised in that it contains, in addition, an E.coli rep1icon.

22. 23. Vector according to any one of claims 4 to 20 22, characterised in that it contains, in addition, at least one selectable marker.

23. 24. Recombinant cell containing a vector according to any one of claims 4 to 23.

24. 25. Cell according to claim 24, characterised in 25 that it is a yeast.

25. 26. Cell according to claim 25, characterised in that it is a yeast of the genus Kluvveromvces or - 39 Saccharomvces.

26. 27. Process for preparing a polypeptide, characterised in that a recombinant cell according to one of claims 23 to 26 is cultured and the polypeptide produced 5 is recovered.

27. 28. Process according to claim 27 characterised in that the polypeptide is an enzyme, a blood derivative, insulin or a variant thereof, a lymphokine, a growth factor, an apolipoprotein or an antigenic polypeptide 10 useful for the production of a vaccine.

28. 29. Process according to claim 28, characterised in that the polypeptide is: an enzyme which is superoxide dismutase, catalase, an amylase, a lipase, an amidase, or chymosin; a blood derivative which is serum 15 albumin, alpha- or beta-globin, factor VIII, factor IX, von Willebrand factor, fibronectin, or alpha,-antitrypsin; insulin or a variant thereof; a lymphokine which is an interleukin, an interferon, a colony stimulating factor (which is G-CSF, GM-CSF or M-CSF) TNF or TRF; a growth 20 factor which is growth hormone, erythropoietin, FGF, EGF, PDGF or TGF; an apolipoprotein; or an antigenic polypeptides useful for the production of a hepatitis, cytomegalovirus, Epstein-Barr or herpes vaccine.

29. 30. Process according to claim 29, characterised 25 in that the polypeptide is human serum albumin or one of its variants or precursors.

30. 31. Use of a recombinant cell according to any - 40 one of claims 24 to 26 as a catalyst in a bioconversion reaction.

31. 32. A plasmid, fragment or derivative according to claim 1 which is substantially as hereinbefore described 5 with reference to Figure 1 or 2.

32. 33. A vector according to claim 4, which is substantially as hereinbefore described with reference to any one of Examples 3 to 6 and/or any one of Figures 1, 2, 5 to 7, 9, 10 or 12 to 15. 10

33. 34. A cell according to claim 24, which is substantially as hereinbefore described with reference to any one of Examples 4, 5 or 6 and/or any one of Figures 1, 2 or 5 to 15.

34.

35. A process according to claim 27, which is 15 substantially as hereinbefore described with reference to Example 6 and/or any one of Figures 1, 2 or 5 to 15.