USRE37767E1 - Highly stable recombinant yeasts for the production of recombinant proteins - Google Patents

Highly stable recombinant yeasts for the production of recombinant proteins Download PDF

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USRE37767E1
USRE37767E1 US09/640,304 US64030400A USRE37767E US RE37767 E1 USRE37767 E1 US RE37767E1 US 64030400 A US64030400 A US 64030400A US RE37767 E USRE37767 E US RE37767E
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host
gene
group
vector
vector pair
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Reinhard Fleer
Alain Fournier
Patrice Yeh
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Aventis Pharma SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to that of industrial fermentations by recombinant microorganisms.
  • a host/vector pair which is highly stable in a complex medium, its preparation and its use in industrial fermentation.
  • the most common solution used in the laboratory consists of inserting a gene for resistance to an antibiotic into the plasmid used, which endows the bacteria with the capacity to survive and grow in a selective medium containing said antibiotic.
  • the most commonly used method consists of culturing cells with a defective pathway for the biosynthesis of amino acids (Trp, Leu, His) or of purine (adenine) or pyrimidine (uracil) bases, said cells being transformed by a vector containing a gene which is capable of complementing this defect.
  • a solution has been proposed to avoid the use of a synthetic medium or of antibiotic resistance genes, which consists of (i) mutating a gene which is essential for survival in a complex medium in the host cell and (ii) introducing an intact copy of said gene into the expansion plasmid used.
  • This system the principle of which is to force the host cell to retain its plasmid, has enabled the stability of the host/vector pair to be increased. This system has, in particular, been described for E.
  • Application WO 86/01224 describes a similar selection system which is suitable for the yeast S. cerevisiae.
  • This system uses the yeast S. cerevisiae which has a mutation in 2 genes which are involved in the biosynthesis of uracil. It consists of (i) inactivating one of the genes for the synthesis of uracil, (ii) transforming said cell with a vector carrying the active gene, and (iii) blocking the other metabolic pathway by mutagenesis.
  • Another approach for obtaining expression systems which are stable in complex media consists of using vectors which are integrated into the genome of the host cell.
  • this system enables only a small number of copies of the vector to be obtained per recombinant cell, and furthermore, the transformation frequency is low. Under these conditions, the levels of expression of heterologous genes are not always satisfactory.
  • a method enabling amplification of a gene which is integrated into the genome has, moreover, been developed in S. cerevisiae, by directing integration towards the genes encoding ribosomal proteins, said genes being present in multiple copies in the genome.
  • this system proves to be unstable when the integrated genes are expressed at high levels, whether they are homologous or heterologous genes.
  • yeasts which are taxonomically related to the Kluyveromyces genus appear to possess a particularly advantageous capacity for secreting recombinant proteins. This has been observed in particular in the case of the yeast K. lactis, for the production of chymosin (EP 96430), IL-1 ⁇ or human serum albumin (EP 361991).
  • yeast K. lactis for the production of chymosin (EP 96430), IL-1 ⁇ or human serum albumin (EP 361991).
  • no sufficiently stable multicopy expression vectors exist in this organism to permit its use in industrial processes.
  • One embodiment of the invention consists of a host/vector pair which is highly stable in a complex medium, characterised in that the host is a yeast of the Kluyveromyces genus in which a gene which is essential for its growth in said medium is nonfunctional, and in that the vector carries a functional copy of said gene.
  • complex medium is understood to mean any medium for industrial fermentation which is compatible with the economic constraints of a large-scale operation.
  • media containing industrial-type raw materials maize soluble extract, yeast extract, molasses or “distillers”, for example, as opposed to defined synthetic media which are supplemented (for example with antibiotics).
  • disillers for example, as opposed to defined synthetic media which are supplemented (for example with antibiotics).
  • the present invention may also be used on synthetic media, although this embodiment is less advantageous.
  • the functional gene which is present in the vector may be a homologous or heterologous gene.
  • Genes which are essential for the growth of the host cell in a complex medium include genes which are involved in the metabolism of a carbon source present in the medium (galactose, lactose, glucose and the like), and genes participating in cellular division, in membrane synthesis, in protein synthesis or DNA replication or transcription.
  • the invention consists of a host/vector pair which is highly stable in a complex medium, characterised in that the host is a yeast of the Kluyveromyces genus in which a gene which is involved in glycolysis is nonfunctional, and in that the vector carries a functional copy of said gene.
  • the present invention relates to host/vector pairs in which the host is chosen from the yeasts Kluyveromyces lactis and Kluyveromyces fragilis.
  • glycolysis involves a succession of complex enzymatic and chemical steps leading to the formation of molecules of ATP and ethanol.
  • the main enzymes involved in this pathway are known and some of the genes encoding these enzymes have been identified and cloned: the genes encoding phosphofructokinase (Kopperschlager et al., Eur. J. Biochem. 81 (1977) 317); pyruvate kinase (Aust et al., J. Biol. Chem. 253 (1978) 7508); phosphoglycerate kinase (Scopes, Meth. Enzymol.
  • RAG1 and RAG2 genes which encode a sugar-transporting protein (Goffrini et al., Nucl. Acid. Res. 18 (1990) 5294) and a phosphoglucose isomerase (Wésolowski-Louvel, Nucl. Acid. Res. 16 (1988) 8714), respectively; and genes which encode alcohol dehydrogenases, ADH (Saliola et al., Yeast 6 (1990) 193-204; Saliola et al., Yeast 7 (1991) 391-400).
  • glycolytic genes may be essential for the growth and/or survival of yeasts of the Kluyveromyces genus in a complex medium and that they may enable the production of particularly stable host/vector pairs.
  • stable and efficient systems may be obtained when the glycolytic gene chosen is a single gene whose product is essential for the metabolism of the carbon sources of the medium by this yeast.
  • some glycolysis steps involve activities which may be encoded by several genes. This is the case, especially in S.
  • ENO enolase
  • PFK phosphofructokinase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • ADH alcohol dehydrogenase
  • the gene involved in glycolysis is a single gene whose product is essential for the metabolism of the carbon sources of the medium by the yeast.
  • PGK phosphoglycerate kinase
  • GPM phosoglycerate mutase
  • PYK pyruvate kinase
  • TPI triose phosphate isomerase
  • the selected gene may be rendered nonfunctional in the host yeast in various ways. It is possible to use nonspecific mutagenesis techniques.
  • the yeasts may be treated with physical agents (X rays; ultra violet rays and the like) or chemical agents (intercalating agents, mono- or bialkylating agents and the like). The yeasts thus treated are then selected on various media depending on the desired mutation.
  • auxotrophy ura3, trp1, leu2, and the like
  • the functional copy of the essential gene present in the vector is placed under the control of a weal promoter.
  • This advantageous embodiment enhances the stability of the pair and the increase in the number of copies of the vector per host cell and, consequently, tends to increase the level of expression of a recombinant gene.
  • weak promoters which may be used for this purpose include the bidirectional promoter of the killer toxin gene (Tanguy-Rougeau et al., Gene 91 (1990) 43) or that of a heterologous gene such as the promoter of the acid phosphatase gene of S. cerevisiae under repression conditions (phosphate-containing culture medium).
  • the functional copy of the essential gene which is present in the vector is either completely free of promoter or is placed under the control of a defective promoter, whether as a result of a mutation of the promoter itself or as a result of the inactivation of a gene involved in the transcriptional activation of said promoter.
  • the essential gene present in the vector may be a gene which is defective under certain conditions such as temperature conditions, for example. In particular, it may be a heat-sensitive gene.
  • the vector comprises, in addition, a DNA sequence containing a structural gene encoding at least a desired protein, and signals permitting its expression.
  • the structural gene encodes a protein which is important in the pharmaceutical or agri-foodstuffs industries.
  • Structural genes include, but are not limited to, enzymes (such as, in particular, superoxide dismutase, catalase, amylases, lipases, amidases, chymosin and the like), blood derivatives (such as serum albumin or variants or precursors thereof, alpha- or beta-globin, factor VIII, factor IX, the von Willebrand factor or portions thereof, 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, MIP 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, Epstein-Barr, herpes and the like
  • the DNA sequence comprises, in addition, signals enabling the secretion of the recombinant protein.
  • signals may correspond to the natural signals for the secretion of the protein in question, but they may also be of a heterologous origin.
  • secretion signals derived from yeast genes such as those from the killer toxin or alpha pheromone gene may be used.
  • the structural gene encodes human serum albumin, its precursors or its molecular variants.
  • “Molecular variants” of albumin is understood to mean the natural variants resulting from the polymorphism of albumin, the structural derivatives possessing an albumin-type activity, the truncated forms of albumin, or any albumin-based hybrid protein.
  • the structural gene(s) encodes(s) polypeptides which are involved at the genetic or biochemical level in the biosynthesis of a metabolite.
  • they may be genes which are involved in the biosynthesis of amino acids, antibiotics or vitamins.
  • the signals enabling the expression of the structural gene are chosen from transcription promoters and terminators. It is understood that these signals are chosen as a function of the structural gene and of the desired result. In particular, it may be preferable to use in certain cases a promoter which can be regulated so as to be able to uncouple the host growth phase(s) from the gene expression phase. Likewise, for reasons related to strength and compatibility, it may be preferable to use, in certain cases, the natural promoters for the structural genes and, in other cases, promoters of a different origin.
  • the promoters used are derived from yeast genes and, still more preferably, from yeast glycolytic genes.
  • the promoters derived from the glycolytic genes of yeasts of the Saccharomyces or Kluyveromyces genus are of very particular importance.
  • examples include the promoters of genes which encode phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), enolases (ENO) or alcohol dehydrogenases (ADH).
  • Promoters derived from genes which are strongly expressed such as the lactase gene (LAC4), acid phosphatase gene (PHO5) or gene for elongation factors (TEF) may also be mentioned.
  • these promoter regions may be modified by mutagenesis, for example, so as to add additional elements for the control of transcription, such as in particular UAS regions (“Upstream Activating Sequence”).
  • UAS regions Upstream Activating Sequence
  • a hybrid promoter between the promoters of the PGK and GAL1/GAL10 genes of S. cerevisiae gives good results.
  • the host/vector pairs of the invention may be used in methods for producing recombinant proteins. They thus enable particularly efficient production systems to be achieved in yeasts of the Kluyveromyces genus.
  • another embodiment of the invention relates to a method for producing a recombinant protein in which a host/vector pair as defined above, comprising the structural gene which encodes said protein under the control of signals permitting its expression, is cultured and the protein produced is recovered.
  • Such a method enables the production of proteins which are important in the pharmaceutical or agri-foodstuffs industries, such as those stated above. It is particularly suitable for the production of human serum albumin, its precursors and molecular variants, although not limited thereto.
  • the host/vector pairs of the invention may also be used directly as catalysts in bioconversion reactions.
  • Another subject of the invention relates to the expression vectors for yeasts of the Kluyveromyces genus carrying a functional copy of a gene which is essential for the growth of the Kluyveromyces yeast on a complex medium.
  • the essential gene is a gene which is involved in one of the above-mentioned functions.
  • This gene may be obtained by any method known to a person skilled in the art (hybridisation cloning using heterologous probes, mutant complementation cloning, and the like).
  • the vectors of the invention are free of any bacterial sequences. It has indeed been shown that it is possible to transform Kluyveromyces yeasts with such vectors in vitro. This system has the advantage of enabling the use of vectors which are smaller and therefore easier to manipulate and capable of accepting larger recombinant DNA sequences.
  • vectors include, in particular, the vectors pYG1023, pYG1033 and pYG1033 ⁇ SfiI, which are described in the examples.
  • FIG. 1 Restriction map of the plasmid pYG600.
  • bla ⁇ -lactamase gene which confers resistance to ampicillin.
  • FIG. 2 Strategy for constructing the plasmid pYG70.
  • FIG. 3 Strategy for constructing the plasmid pYG70-2. See FIG. 2 for the legend. The sites marked with an (*) possess ends which are co mpatible with the corresponding sites without restoring, after ligation, cleavage sites recognised by said enzymes.
  • FIG. 4 Sequence of synthetic oligonucleotides A-H (SEQ ID NOS: 1-8) which were used in constructing the adaptors 1 to 3 and in the PCR reaction for checking the genotype of the pgk mutants (Example 3.2.(ii)).
  • FIG. 6 Strategy for constructing the plasmid pYG1003. See FIG. 2 for the legend.
  • FIG. 7 Strategy for constructing the plasmid pYG1023. See FIG. 2 for the legend.
  • FIG. 8 Strategy for constructing the plasmid pYG1033.
  • FIG. 9 Representation of the vectors pYG1033 and pYG1033 ⁇ SfiI. See FIG. 2 for the legend.
  • FIG. 10 Strategy for cloning and modifying the URA3 gene of K. lactis CBS2359.
  • FIG. 11 Strategy for constructing the plasmid pYG1012.
  • FIG. 12 Strategy for constructing the plasmid pYG1013. and representation of the EcoRI-SpeI fragment carrying the modified PGK K. 1 gene.
  • FIG. 13 This figure represents the production of human albumin by the plasmid pYG1023-transformed yeast FB05D, over 200 culture generations in an industrial-type complex medium, together with the stability of the plasmid pYG1023 in this yeast during the same period.
  • the stability and the production of albumin are defined in the corresponding examples.
  • Site-directed mutagenesis in vitro with oligodeoxynucleotides is carried out according to the method developed by Taylor et al. (Nucleic Acids Res. 13 (1985) 8749-8764) using the kit distributed by Amersham.
  • the sequencing of nucleotides is performed according to the dideoxy technique described by Sanger et al, (Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467).
  • the enzymatic amplification of specific DNA fragments is carried out by the PCR reaction (“Polymerase 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 (Perking Elmer Cetus) in accordance with the recommendations of the manufacturer.
  • the pair thus obtained is stable in an industrial-type complex medium such as defined above.
  • the PGK gene was isolated from K. lactis CBS2359 by screening a partial genomic library with a heterologous probe corresponding to the N-terminal portion of the S. cerevisiae PGK gene (Dobson et al., Nucl. Acid. Res. 10 (1982) 2625-2637). More specifically, the probe used corresponds to the 1.3-kb PvuI-EcoRI fragment.
  • the plasmid pYG70 is derived from the plasmid pKan707 (see EP 361 991) by removal of EcoRI fragments containing the URA3 gene and the sequence of the pKD1 plasmid, and of the unique HindIII site present in the aph gene, so as to facilitate subsequent cloning steps.
  • the aph gene encodes aminoglycoside 3′-phosphotransferase (I) (Oka et al., J. Mol. Biol. 147 (1981) 217), and is used as marker for geneticin (G418) resistance in yeast.
  • the PstI fragment of the plasmid pKan707 containing the aph gene was subcloned into the bacteriophage M13mp7.
  • the HindIII site present in this gene was then destroyed by site-directed mutagenesis according to the method described by Taylor et al. (cf. general cloning techniques), in order to generate the plasmid pYG65 (see FIG. 2 ).
  • the following oligodeoxynucleotide was used to perform this mutagenesis:
  • This oligodeoxynucleotide enabled the triplet CTT which encodes leucine 185 to be replaced by CTC. This change does not modify the resulting protein sequence.
  • the portion containing the bacterial replicon from pKan707 was isolated by digestion with the EcoRI enzyme and recircularised with T4 DNA ligase to form the plasmid pYG69 (FIG. 2 ). The PstI fragment present in the latter, containing the aph gene, was then replaced with the equivalent mutated fragment derived from the pYG65.
  • the plasmid pYG70 thus obtained therefore contains:
  • a selection marker for yeast (mutated aph gene) under the control of the killer toxin promoter k1.
  • the plasmid pYG70 was digested with SphI and the cohesive ends were then removed by digestion in the presence of phage T4 DNA polymerase. After ligation in the presence of ligase, the plasmid pYG70 ⁇ SphI was obtained (see FIG. 3 ).
  • the adaptor 1 was obtained by hybridisation of the synthetic oligodeoxynucleotides A and B (SEQ ID NOS: 1 and 2) presented in FIG. 4 .
  • 2 ⁇ g of each oligodeoxynucleotide were incubated in a 20 ⁇ l of hybridisation buffer (30 mM Tris-HCl buffer, pH 7.5; 30 mM NaCl; 7.5 mM MgCl 2 ; 0.25 mM ATP; 2 mM DDT; 0.2 mM EDTA), and then the temperature was raised to 80° C. for 10 minutes, and slowly reduced to room temperature.
  • the adaptor thus obtained contains cleavage sites for the following enzymes: SacI, SalI, MluI, BssHII and SfiI, and enables the SalI site present in the plasmid pYG70 ⁇ SphI to be removed during its introduction.
  • This adaptor was introduced by ligation into the plasmid pYG70 ⁇ SphI, previously digested with the enzymes SalI and SacI.
  • the plasmid obtained is called pYG70-1.
  • the adaptor 2 was produced by following the procedure described for the adaptor 1 , using the oligodeoxynucleotides C and D (SEQ ID NOS: 3 and 4) described in FIG. 4 .
  • This adaptor contains cleavage sites for the following enzymes: SfiI; AatII, SphI; NarI and SacI and enables the EcoRI site present in the plasmid pYG70-1 to be removed during its introduction. It was introduced by ligation into the plasmid pYG70-1, previously digested with the enzymes EcoRI and SacI, to form the plasmid pYG70-2 (FIG. 3 ).
  • the human serum albumin expression cassette used comprises:
  • This cassette was isolated from the plasmid pYG404 (EP 361 991 incorporated herein by reference) in the form of a SalI-SacI fragment and then introduced by ligation into the plasmid pYG70-2 previously digested with the corresponding enzymes.
  • the plasmid obtained is called pYG70-3 (FIG. 5 ).
  • the K. lactis PGK gene was isolated from the plasmid pYG600 (FIG. 1 ), subcloned into the plasmid pYG1002 in order to generate the plasmid pYG1003, and then isolated from the latter in the form of a MluI-BssHII fragment.
  • the subcloning into pYG1002 enabled the K. lactis PGK gene to be obtained free of its promoter and in the form of an MluI-BssHII fragment.
  • the plasmid pYG1003 was obtained in the following manner (FIG. 6 ):
  • the plasmid pIC20H (Marsh et al., Gene 32 (1984) 481) was digested with BglII and EcoRI so as to introduce the adaptor 3 .
  • This adaptor which provides the EcoRI, BssHII, ClaI, NheI, MluI and BglII sites, was obtained as described above (2.2.(ii)), by hybridisation of the oligodeoxynucleotides E and F (SEQ ID NOS: 5 and 6; FIG. 4 ).
  • the resulting plasmid is called pYG1002.
  • lactis PGK gene was introduced into this new plasmid in the form of a ClaI-NheI fragment derived from the plasmid pYG600.
  • the plasmid obtained is called pYG1003 (FIG. 6 ).
  • the MluI-BssHII fragment derived from the plasmid pYG1003 carrying the K. lactis PGK gene was then introduced into the corresponding sites on the plasmid pYG70-3 in order to generate the plasmid pYG70-4 (FIG. 7 ).
  • the K. lactis PGK gene is thereafter placed under the control of the killer toxin bidirectional promoter k1.
  • the plasmids pYG70-4 (FIG. 7) and pKD1 (EP 361 991) were digested with SphI and ligated together in the presence of ligase. After this ligation, 4 vectors may be obtained depending on the conformation of the plasmid pKD1 (A form or B form) and the orientation of the portion corresponding to the plasmid pYG70-4 relative to pKD1.
  • This vector comprises:
  • a human serum albumin expression cassette containing the structural gene which encodes the prepro form under the control of the inducible promoter of the K. lactis LAC4 gene, and of the terminator of the S. cerevisiae PGK gene,
  • a construct derived from the plasmid pYG1023 was produced with the aim of obtaining, on introduction into yeast, expression vectors free of bacterial replicon and markers for resistance to ampicillin and to geneticin, and in which the k1 promoter situated upstream of the PGK gene is removed.
  • This construct was produced in the following manner:
  • This step is optional. However, it was carried out so as to avoid any risk of recombination within the expression vector itself, which would lead to a reduction in the host/vector pair-producing capacities.
  • the fragment used as terminator inside the plasmid pYG1023 contains the S. cerevisiae PGK terminator and also the C-terminal portion of the S. cerevisiae PGK structural gene and, as a result, exhibits a homology with the corresponding region of the K. lactis PGK gene used as selection marker. This terminator was therefore modified in the following manner:
  • the 3.6-kb PvuII fragment isolated from pYG1023 was, in the first instance, subcloned into the corresponding sites of the vector pUC18 to form the plasmid pYG1027.
  • the 427-bp SmaI-HindIII fragment of this plasmid, carrying the PGK terminator region was then replaced with a 325-bp fragment corresponding to the noncoding 3′ region of the S. cerevisiae PGK gene (that is to say containing no element of the structural gene).
  • This 325-pb fragment was obtained by PCR amplification reaction (cf. general cloning techniques) on the PGK terminator present in the plasmid pYG1023, by using the following oligodeoxynucleotides:
  • the vector obtained was designated pYG1028 (FIG. 8 ). Digestion of the latter with the enzymes AvrII and AflII enables a 1.1-kb fragment to be isolated, which was used to replace the corresponding fragment in pYG1023.
  • the plasmid obtained was designated pYG1033 (FIG. 9 ).
  • the plasmid pYG1033 was then subjected to digestion in the presence of the enzyme SfiI in order to excise the 3.5-kb fragment containing the bacterial replicon and the resistance markers, and then to ligation in the presence of ligase so as to generate the plasmid pYG1033 ⁇ SfiI (FIG. 9 ).
  • This example described the preparation of a pgk mutant from a wild K. lactis strain by avoiding the use of genes for resistance to antibiotics. Two steps were carried out successively:
  • This mutagenesis technique makes it possible to avoid the use of nonspecific mutagenic agents which may affect other regions of the cell genome. It also makes it possible to avoid any genetic reversion event which carries the risk of correcting the modifications carried out on the PGK gene.
  • the K. lactis URA3 gene which encodes orotidine-5-phosphate decarboxylase was cloned in the form of a 1.2-kb BamHI-PstI fragment using the PCR technique (cf. general cloning techniques), starting with a K. lactis CBS2359 genomic DNA extract (Rose et al., “Methods in Yeast Genetics” Cold Spring Harbor Laboratory Press, N.Y., 1990), by means of the following oligodeoxynucleotides:
  • the fragment obtained was then subcloned into the BamHI and PstI sites of the plasmid pIC20H to give the plasmid pYG1007 (FIG. 10 ).
  • the URA3 gene was then modified by deletion of a StyI fragment inside ORF, comprising 286 bp. This was carried out on pYG1007 by digestion with the enzyme StyI followed by ligation in the presence of ligase. This new plasmid is called pYG1010 (FIG. 10 ).
  • the CBS 293.91 strain was transformed according to the procedure described by Durrens et al. (Curr. Genet. 18 (1990) 7) with 10 ⁇ g of the PstI-BamHI fragment isolated by electroelution from the plasmid pYG1010, which contains the deleted URA3 gene. After a sudden rise in temperature to 42° C. (heat shock) and 2 successive washes with water, 600 ⁇ l of YPD medium (10 g/I yeast extract; 20 g/l peptone; 20 g/l glucose) were added and the cells were incubated overnight.
  • YPD medium 10 g/I yeast extract; 20 g/l peptone; 20 g/l glucose
  • the cells were then plated on an SD minimal synthetic medium (6.7 g bacto-yeast nitrogen base without amino acids (Difco); 20 g glucose; 20 g Bacto-agar; 1000 ml distilled water) in the presence of uracil (100 ⁇ g/ml), of uridine (100 ⁇ g/ml) and 5-fluoroorotate (5FO) 15 mM. Clones appeared after 4 to 5 days. They were subcultured on YPD medium so as to obtain isolated colonies.
  • SD minimal synthetic medium 6.7 bacto-yeast nitrogen base without amino acids (Difco); 20 g glucose; 20 g Bacto-agar; 1000 ml distilled water
  • uracil 100 ⁇ g/ml
  • uridine 100 ⁇ g/ml
  • 5FO 5-fluoroorotate
  • the clones derived from the secondary subculture were then tested for the Ura3 ⁇ phenotype using a drop test on SD and SD+uracil medium (Jund and Lacroute, J. of Bact. 102 (1970) 607-615; Bach and Lacroute, Mol. Gen. Genet. 115 (1972) 126-130).
  • the ura3 phenotype of the clones obtained was checked by:
  • the ura3. mutant selected is called K. lactis Y616.
  • a DNA fragment was prepared containing a PGK gene modified by substitution of an inner portion of a gene with a DNA fragment carrying the S. cerevisiae URA3 gene. This fragment was then used to replace the intact genomic copy of the PGK gene by double homologous recombination (Rothstein, mentioned above).
  • the PGK gene was restored, in the first instance, delimited by larger flanking regions.
  • the regions situated upstream of the PGK gene were cloned and then ligated into the fragment carrying the plasmid pYG600 (FIG. 1 ).
  • Example 1 Screening of the library described in Example 1 also enabled on XbaI genomic fragment of about 2.5 kb to be revealed by Southern blot analysis.
  • This fragment was isolated by screening a limited K. lactis CBS2359 genomic library consisting of XbaI-cut DNA fragments of between 2 and 3 kb is size, which were introduced into the XbaI site of the plasmid pUC18.
  • a library with 500 clones was thus prepared and then screened with the heterologous probe used in Example 1.
  • a clone was identified by colony hybridisation and its plasmid DNA isolated.
  • This plasmid, pYG610 (FIG. 11 ) contains a 2.5-kb genomic DNA fragment. Analysis of the sequence of this fragment shows that it contains a portion which encodes the N-terminal region of the K. lactis Pgk protein (0.3 kb), and 2.2 kb corresponding to the region situated upstream of the PGK gene.
  • the AflII-HindIII fragment of plasmid pYG600 and EcoRI-AflII fragment of the plasmid pYG610 were isolated by electroelution and then introduced together, by ligation, into the plasmid pUC9 previously digested with EcoRI and Hind III.
  • the plasmid obtained is called pYG1012.
  • the SalI-SacI fragment inside the PGK gene which is carried by the plasmid pYG1012, was replaced by digestion followed by ligation with the SalI-SacI fragment derived from the plasmid pYG1011, carrying the S. cerevisiae URA3 gene, in order to generate the plasmid pYG1013 (FIG. 12 ).
  • the plasmid pYG1011 was constructed by insertion of a HindIII fragment isolated from the plasmid YEp24 (ATCC No. 37051), containing the S. cerevisial URA3 gene, into the corresponding sites of the bacteriophage M13tg130 (Kieny et al., Gene 26 (1983) 101).
  • the plasmid pYG1013 therefore contains, in the form of an EcoRI-SpeI fragments, the S. cerevisiae URA3 gene, delimited on one side of the noncoding 5′ region of the first 500 pairs of the K. lactis PGK structural gene, and on the other by the last 100 base pairs of the K. lactis PGK structural gene and its terminator (FIG. 12 ).
  • the Y616 strain (ura3) was transformed according to the method described by Durrens et al. (mentioned above), with 10 ⁇ g of the EcoRI-SpeI fragment isolated from the plasmid pYG1013.
  • the cells containing the functional URA3 gene were selected on an SD minimal synthetic medium in which glucose was replaced by glycerol (3%) and ethanol (2%). This medium permits the growth of the pgk mutants.
  • the transformed colonies thus obtained were then subcultured in a complex YPD medium in which the pgk mutants are unable to grow. 50% of the strains obtained exhibited a Pgk ⁇ phenotype in this test.
  • the amplification was carried out on whole cells exhibiting the Pgk ⁇ phenotype. After 30 amplification cycles, the supernatants (10 ⁇ l) were analyzed on agarose gel (0.8%) electrophoresis in order to determine the size of the bands. The controls were produced by amplification using the same oligodeoxynucleotides on the plasmids pYG600 (intact gene) and pYG1013 (modified gene).
  • the vector pYG1023 was introduced, by transformation, into the K. lactis strain FB05D, using an ethylene glycol/dimethyl sulphoxide technique (Durrens et al., mentioned above). Transformed yeasts were selected for the Pgk + phenotype conferred by the plasmid pYG1023 on a complex YPD medium.
  • the vector pYG1033 ⁇ SfiI was introduced into a K. lactis strain FB05D by electroporation according to the technique described by Meilhoc et al. (Biotechnologie 8 (1990) 223). After transformation, the cells were plated on YPD medium and cultured at 30° C. for 3 days. The cells which are capable of growing on this type of medium (therefore containing a functional copy of the PGK gene) were then tested for their resistance to geneticin. 60% of them are unable to grow on YPD medium in the presence of 200 ⁇ g/ml of G418. These results show that the introduced vector has lost the G418 marker and that it is capable of complementing the pgk mutation of the FB05D strain.
  • the transformed cells were precultured in Erlenmeyer flasks in an M9CS20 industrial-type complex medium [M9 medium (Maniatis et al., mentioned above) supplemented with 20 g/l of maize soluble extract (Solulys. L, Roquette) in the presence of 2 g/l of ammonium acetate and 20 g/l of lactose] at 28° C. with stirring.
  • M9 medium Maniatis et al., mentioned above
  • 20 g/l of maize soluble extract Solulys. L, Roquette
  • the culture in Erlenmeyer flask 2 was then used in its turn to inoculate two other Erlenmeyer flasks at a dilution of 10 ⁇ 3 (Erlenmeyer flask 3 ) and 10 ⁇ 6 (Erlenmeyer flask 4 ).
  • the cells were then cultured as above, for 3 days.
  • the cells in Erlenmeyer flask 3 had undergone 30 cellular divisions (30 generation times), and the cells in Erlenmeyer flask 4 had undergone 40 cellular divisions (40 generation times).
  • YPGE dish (10 g/l yeast extract, 20 g/l peptone, 30 g/l glycerol, 20 g/l ethanol). This medium permits the growth of all the cells.
  • YPD dish in the presence of G418 (200 ⁇ g/ml). Only the cells containing the plasmid pYG1023 which carries the gene for resistance to G418 and the PGK gene are capable of growing on this type of dish.
  • the stability of the plasmid pYG1023 was thus determined for each culture.
  • the results are presented in FIG. 13, in which the stability is defined by the ratio of the number of colonies present on the YPD/G418 dishes to the number of colonies present on the YPGE dishes.
  • FIG. 13 shows the variation of albumin production as the function of the number of culture generations under conditions for induction (medium containing lactose). The values are given in percentage of production relating to the mean production measured over 200 generations.
  • K. lactis Y616 and K. lactis FB05D strains were deposited on Jun. 11, 1991 in Centraalbureau voor Schimmelkulturen (CBS) at Baarn in the Netherlands, in accordance with the conditions of the Budapest treaty under the numbers CBS 294.91 and CBS 295.91, respectively.
  • the K. lactis strain CBS 293.91 corresponds to the strain CBS1065 redeposited on Jun. 11, 1991 in accordance with the conditions of the Budapest treaty.

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