USRE37447E1 - Modified Kluyveromyces yeasts, their preparation and use - Google Patents

Modified Kluyveromyces yeasts, their preparation and use Download PDF

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USRE37447E1
USRE37447E1 US09/640,305 US64030500A USRE37447E US RE37447 E1 USRE37447 E1 US RE37447E1 US 64030500 A US64030500 A US 64030500A US RE37447 E USRE37447 E US RE37447E
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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
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast

Definitions

  • the present invention relates to new genetically modified yeasts belonging to the genus Kluyveromyces and to their use to produce advantageously recombinant proteins.
  • the applicant has now shown that it is possible to improve the levels of production of the said recombinant proteins, that is to say in their integral form, in Kluyveromyces yeasts, by modifying at least one gene encoding a cellular protease, and especially a protease transiting through the secretory pathway.
  • the applicant has furthermore shown that such modifications are particularly advantageous since they make it possible to increase the levels of production of recombinant proteins, and this is all the more advantageous since the said modification is without apparent effect on the growth rate and the viability of the modified cells under industrial fermentation conditions.
  • the applicant has also shown that the said modifications do not affect the stability of the transformant yeasts, which makes it possible to use the said yeasts in a particularly advantageous manner to produce recombinant proteins.
  • the subject of the present invention is therefore yeasts of the genus Kluyveromyces having one or more genetic modifications of at least one gene encoding a protease, modifying the proteolytic activity of the said yeasts.
  • the genetic modification(s) render the said gene partially or totally incapable of encoding the natural protease.
  • the gene(s) thus genetically modified encode a non-functional protease, or a mutant having a modified proteolytic activity spectrum.
  • the gene(s) encoding the said proteases are placed under the control of a regulated promoter.
  • the yeasts of the genus Kluyveromyces according to the invention comprise the yeasts as defined by van der Walt [in: The Yeasts (1987) N. J. W. Kregervan Rij (ed): Elsevier: p.224], and preferably the yeasts K.marxianus var.lactis (K.lactis), K.marxianus var. marxianus (K.fragilis), K.marxianus var. drosophilarum (K.drosophilarum), K.waltii, and the like.
  • Genetic modification should be understood to mean more particularly any suppression, substitution, deletion or addition of one or more bases in the gene(s) considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or alternatively by exposing the said yeasts to a treatment by means of mutagenic agents.
  • mutagenic agents there my be mentioned for example physical agents such as energetic radiation (X, g, ultra violet rays and the like), or chemical agents capable of reacting with various functional groups of the bases of DNA, and for example alkylating agents [ethyl methanesulphonate(EMS), N-methyl-N′-nitro-nitrosoguanidine, N-nitroquinoline 1-oxide (NQO)], bialkylating agents, intercalating agents and the like. Deletion is understood to mean any suppression of the gene considered. It may relate in particular to a part of the region encoding the said proteases and/or of all or part of the transcriptional promoter region.
  • EMS ethyl methanesulphonate
  • NQO N-nitroquinoline 1-oxide
  • the genetic modifications can also be obtained by genes disruption, for example according to the procedure initially described by Rothstein [Meth. Enzymol. 101 (1983) 202].
  • the entire coding sequence will be preferably disrupted so as to allow the replacement, by homologous recombination, of the wild-type genomic sequence by a non-functional or mutant sequence.
  • the said genetic modification(s) may be located in the gene encoding the said proteases, or outside the region encoding the said proteases, for example in the regions responsible for the transcriptional expression and/or regulation of the said genes.
  • the inability of the said genes to encode the natural proteases can manifest itself either by the production of a protein which is inactive because of structural or conformational modifications, or by the absence of production, or by thy production of a protease having a modified enzymatic activity, or alternatively by the production of the natural protease at an attenuated level or according to a desired mode of regulation.
  • modifications such as point mutations are by nature capable of being corrected or attenuated by cellular mechanisms, for example during the replication of DNA preceding cell division. Such genetic modifications are thereby of limited interest at the industrial level since the phenotypic properties resulting therefrom are not perfectly stable.
  • the applicant has now developed a process which makes it possible to prepare Kluyveromyces yeasts having one or more genetic modifications of at least one gene encoding a protease, the said modification(s) being segregationally stable and/or non-reversible.
  • the yeasts having such modifications are particularly advantageous as cellular host for the production of recombinant proteins.
  • the invention also makes it possible to produce yeasts in which the modification(s) made render the gene(s) considered totally or only partially incapable of producing a functional protease.
  • the yeasts according to the invention have one or more segregationally stable genetic modifications. Still according to a preferred embodiment, the genetic modification(s) its non-reversible. Still according to a preferred embodiment of the invention, the genetic modification(s) leave(s) no residual activity for the gens considered.
  • the gene(s) encoding one or more proteases are chosen from the genes encoding proteases transiting through the secretorypathway of Kluyveromyces.
  • proteases may be located in the endoplasmic reticulum, the compartment of the Golgi apparatus, the post-Golgi compartment, and for example the cellular vacuoles, the vesicles of the endosome, the secretion vesicles, or the extracellular medium.
  • Kluyveromyces genes encoding a protease chosen from the families comprising protease A, protease B, or a carboxypeptidase (and for example carboxypeptidase Y or carboxypeptidase S), or alternatively endopeptidase KEX1 of K. lactis or a protease with similar activity, and for example protease YAP3 [Egel-Mitani et al., Yeast 6 (1990) 127], or more generally any other protease involved in the maturation of certain secreted proteins.
  • the considered gene(s) encode proteases which are not involved in the cleavage of the signal peptide of the recombinant proteins expressed in the form of preproteins.
  • proteases which are not involved in the cleavage of the signal peptide of the recombinant proteins expressed in the form of preproteins.
  • the said protease(s) possess(es) a signal peptidase activity and the said genetic modification(s) allow their overexpression, which is particularly advantageous in the case or this step is a limiting step of the secretorypathway.
  • the subject of the invention is also any Kluyveromyces yeast as defined above into which an exogenous DNA sequence comprising one or more genes encoding a protein of interest which it is desired to express and/or secrete in the said yeast, has been introduced.
  • exogenous DNA sequence is understood to mean any DNA sequence introduced artificially into the yeast and encoding one or more proteins of interest.
  • this may be complementary DNA (cDNA) sequences, artificial or hybrid sequences, or alternatively synthetic or semi-synthetic sequences, which are included in an expression cassette permitting synthesis in the said yeasts of the said protein(s) of interest.
  • this exogenous DNA sequence may include a region for initiation of transcription, regulated or otherwise in Kluyveromyces, so as to direct, when desirable, the expression of the said proteins of interest.
  • the exogenous DNA sequence is included in a vector, which may be capable of autonomous replication in the yeast considered, or of the integrative type.
  • autonomously replicating vectors can be prepared from autonomously replicating sequences in Kluyveromyces, and for example this may be the plasmid pKD1 [Falcone et al., Plasmids 15 (1986) 248; Chen et al., Nucl. Acids Res. 14 (1986) 4471] characterized by a high segregational stability and especially in the various varieties of K. marxianus, or the plasmid pEW1 isolated in K. waltii [Chen et al., J. General Microbiol. 138 (1992) 337].
  • Autonomously replicating vectors can also be prepared from chromosomal sequences (ARS).
  • ARS chromosomal sequences
  • these can be prepared from chromosomal sequences homologous to the said host yeast, so as to flank the genetic sequence encoding the said proteins of interest, and a genetic selectable marker, so as to orient the integration of the whole by homologous recombination.
  • the said homologous sequences correspond to genetic sequences derived from the coding region of the said protease, which makes it possible to replace by homologous recombination the original sequence of the said protease by the selectable marker and the exogenous DNA sequence, while permitting gene disruption of the said protease.
  • the expression cassette is integrated at the locus encoding the ribosomal RNAs (rDNA) permitting gens amplification of the said expression cassette [Bergkamp et al., Curr. Genet. 21 (1992) 365]. Still in another embodiment, the exogenous DNA sequence is integrated into the chromosome of the said host yeasts by non-homologous recombination.
  • rDNA ribosomal RNAs
  • the exogenous DNA sequence can be introduced into the yeast by the techniques practised by persons skilled in the art, and for example recombinant DNA techniques, genetic crossings, protoplast fusion, and the like.
  • the exogenous DNA sequence is introduced into Kluyveromyces yeasts by transformation, electroporation, conjugation, or any other technique described in the literature.
  • transformation of the Kluyveromyces yeasts the technique described by Ito et al. [J. Bacteriol. 153 (1983) 163] can be used.
  • the transformation technique described by Durrens et al. [Curr. Genet. 18 (1990) 7] using ethylene glycol and dimethyl sulphoxide is also effective. It is also possible to transform the yeasts by electroporation, according to the method described by Karube et al. [FEBS Letters 182 (1985) 90].
  • An alternative procedure is also described in Patent Application EP 361 991.
  • the said Kluyveromyces yeasts modified for their protease content by the techniques described above are advantageously used as host cells to produce recombinant proteins, and for example heterologous proteins of pharmaceutical or dietary interest.
  • the said host yeasts are particularly advantageous since they make it possible to increase the quality and quantity of recombinant proteins which it is desired to produce and/or secrete, and since the said genetic modifications of the said cells do not affect the genetic and mitotic stability of the vectors for expression of the said recombinant proteins.
  • Another subject of the invention therefore lies in a process for producing recombinant proteins according to which a yeast as defined above is cultured under conditions for expressing the protein(s) encoded by the exogenous DNA sequence, and the protein(s) of interest is (are) recovered.
  • the said proteins of interest are secreted into the culture medium.
  • naturally occurring proteins or artificial proteins, and for example hybrid proteins.
  • the use of yeast cells having a modified protease content is particularly advantageous because of the exposure of the hinge region between the various protein domains of the chimera.
  • the said artificial protein contains a peptide fused to one of the ends of the chimera and is particularly sensitive, for example during transit in the secretory pathway, to a proteolytic degradation by an N- or C-terminal exoprotease, and for example a carboxypeptidase.
  • proteolytic degradation of the protein of interest can also result from any cellular protease, and for example cytoplasmic protease, released into the external medium because of an undesirable cell lysis during the fermentation process of the said recombinant yeasts. Genetic modification of the nucleotide sequence encoding such proteases can therefore also result in a particularly advantageous process for producing the said proteins of interest and is also claimed.
  • the process according to the invention allows the production of proteins of pharmaceutical or dietary interest.
  • enzymes such as in particular superoxide dismutase, catalase, amylases, lipases, amidases, chymosin and the like, or any fragment or derivative thereof
  • blood derivatives such as serum albumin, alpha- or beta-globin, coagulation factors, and for example factor VIII, factor IX, von Willebrand's factor, fibronectin, alpha-1 antitrypsin, and the like, or any fragment or derivative thereof
  • insulin and its variants lymphokines [such as interleukins, interferons, colony-stimulating factors (G-CSF, GM-CSF, M-CSF and the like), TNF and the like, or any fragment or derivative thereof]
  • growth factors such as growth hormone, erythropoietin, FGF, EGF, PDGF, TGF, and the like, or any fragment or derivative thereof
  • apolipoproteins such as in particular superoxide
  • Another subject of the present invention lies in a Kluyveromyces DNA fragment encoding a protease.
  • the Applicant has indeed detected, isolated and characterized certain Kluyveromyces proteases and especially proteases transiting through the secretcry pathway. More preferably, one of the subjects of the invention relates to a Kluyveromyces protease chosen especially from proteases A, B, carboxypeptidase Y, as well as the family of seine proteases of the subtilisin type and of which one representative is the K. lactis KEX1 protease [Wésolowski-Louvel et al., Yeast A (1988) 71]. By way of example, the nucleotide sequences of the K.
  • lactis genes encoding proteases A, B and carboxypeptidase Y were determined by the Applicant and are presented SEQ ID No. 5, 1 and 2 respectively.
  • a restriction map of a chromosomal fragment encoding K. lactis protease A is also presented in FIG. 10 .
  • the said variations may be of natural origin, but may also be obtained in situ or in vitro by genetic engineering techniques, or after treating the cells with a mutagenic agent, and include in particular point or multiple mutations, deletions, additions, insertions, hybrid proteases and the like.
  • the genetic variations may also relate to the regions for controlling the expression of the said proteases, for example so as to modify their levels of expression or their mode of regulation.
  • the subject of the invention is also any protein resulting from the expression of an exogenous DNA fragment as defined above.
  • the subject of the invention is also a process for preparing a genetically modified Kluyveromyces yeast and its advantageous use for producing proteins of interest.
  • the process of the invention consists in replacing the chromosomal gene(s) considered by a version modified in vitro.
  • FIGS. 2 A and B Hybridization of the S. cerevisiae PRB1 probe with restriction fragments of the K. lactis genomic DNA.
  • Top panelPanel A photo of the agarose gel (1%) stained with ethidiumbromide and before transfer onto nylon filter
  • bottom panelPanel B schematic representation of the signals obtained after hybridization of the filter with the radioactive probe.
  • FIGS. 3 A and B Hybridization of the S. cerevisiae PRB 1 probe with the 34 minipreparations of DNA (mixture of 10 clones each) after double restriction with the enzymes EcoRI and BglIII.
  • Panel A photo of the agarose gel (1%) stained with ethidiumbromide and before transfer onto nylon filter;
  • Panel B autoradiography of the nylon filter after hybridization with the S. cerevisiae PRB1 probe; subfractions “d”, “e” and “f” corresponding to an aliquot of the K. lactis genomic DNA fragments after total digestion with EcoRI+BglII and size fractionation by electroelution are indicated.
  • FIGS. 4 A and B Control hybridization of the positive clones 18-3 and 31-L
  • Panel A photo after running on 1% agarose gel of EcoRI+BglII digests of the DNA of clones 31-I (well no. 1), 18-3 (well no. 2) and 31-K (negative control, well no. 3).
  • Panel B autoradiography of the nylon filter corresponding to the preceding gel after hybridization with the S. cerevisiae PRB1 probe.
  • P corresponds to the location of the undigested plasmid (pYG1224); the position of the BglII-EcoRI restriction fragment of about 0.7 kb hybridizing with the radioactive probe is indicated.
  • FIG. 5 Comparison of the protein sequence of the BglII-EcoRI fragment of K. lactis genomic DNA (residues Arg 308 to Phe 531 of the peptide sequence SEQ ID No. 1) with the corresponding part of the S. cerevisiae PRB1 gene (SEQ ID NO. 8) (residues Arg 105 to Leu328). The asterisks indicate the amino acids conserved between the two sequences.
  • FIGS. 6 A-D Restriction maps of the genomic inserts of plasmids pYG1224, pYG1226 and pYG1227 (panel A); pYG1231 (panel B); pYG1237, pYG1238, pYG1239, pYG1240, pYG1241 and pYG1242 (panel C).
  • Panel D location of the coding phase of the K. lactis PRB1 gene; the vertical arrow indicates the rough position of the N-terminal end of the mature protein. The position of the codon presumed to be for initiation of translation and the position of the translational stop codon are indicated by an asterisk.
  • FIG. 7 Restriction map of the insert of the plasmid pC34.
  • the box corresponds to the K. lactis genomic insert and the line corresponds to the sequences of the vector KEp6.
  • the arrow indicates the position of the K. lactis PRC1 gene.
  • FIG. 8 Restriction map of the insert of the plasmid pA25/1.
  • the box corresponds to the K. lactis genomic insert and the line corresponds to the sequences of the vector KEp6.
  • the arrow indicates the rough position of the PRA1 gene as indicated by a Southern blot hybridization by means of specific 3′ and 5′ probes.
  • Panel B Disruption of the K. lactis PRB1 gene by the selectable marker URA3 from S. cerevisiae. This Southern blot corresponds to the genomic DNA of K.
  • lactis CBS 294.91 (uraA) after transformation by the BglII-EcoRI fragment of panel A and selection in the absence of uracil.
  • Wells 1 to 3 genomic DNA of three transformants after BglII+EcoRI double restriction; the strain of well 3 is K. lactis Y750;
  • well 4 genomic DNA of the strain CBS 294.91 after BglII+EcoRI double restriction.
  • the radioactive probe used corresponds to the BglII-EcoRI fragment of the plasmid pYG1224 (FIG. 6 A).
  • FIGS. 10 A-D Restriction map of the inserts of the plasmids pC34 [vector KEp6; panel a)], pYG154 [vector pIC-20R; panel b)] and pYG155 [vector pIC-20R; panel c)].
  • Panel d) fragment used for the disruption. The restriction sites in brackets were destroyed with the Klenow enzyme or with T4 DNA polymerase as indicated in the text. The box corresponds to the K. lactis genomic insert and the line corresponds to the sequences of the vectors.
  • FIG. 11 Restriction map of the plasmid pYG105 and strategy for constructing the plasmid pYG1212.
  • P K. lactis LAC4 promoter T, transcriptional terminator
  • IR inverted repeat sequences of the plasmid pKD1
  • LP prepro region of HSA: Ap r and Km r designate respectively the genes for resistance to ampicillin (E. coli) and to G418 (Kluyveromyces).
  • FIG. 12 Comparison of the capacities of secretion of a truncated variant of human albumin in the K. lactis CBS 293.91 strains (wells 2, 4, 6, 8 and 10) or its disrupted mutant for the gene for protease B (strain Y750; wells 1, 3, 5, 7 and 9), after transformation with the plasmid pYG1212.
  • the transformant cells are cultured in Erlenmeyer flasks in the presence of G418 (200 mg/l) for 2 days (wells 1, 2, 7 and 8), 4 days (wells 3, 4, 9 and 10), or 7 days (wells 5 and 6); wells 1 to 6 correspond to growth in YPD medium, and wells 7 to 10 correspond to growth in YPL medium.
  • the spots are equivalent to 50 ml of culture supernatents.
  • the restriction enzymes were provided by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used according to the recommendations of the suppliers.
  • the pBR322 and pUC type plasmids and the phages of the M13 series are of commercial origin (Bethesda Research Laboratories).
  • the pIC type plasmids have been described by Marsh et al. [Gene 32. (1984) 481].
  • the DNA fragments are separated according to their size by electrophoresis on agarose or acrylamide gels, extracted with phenol or with a phenol/chloroformmixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the supplier.
  • the filling of the protruding 5′ ends is carried out by the Klenow fragment of DNA polymerase I of E. coli (Biolabs) according to the specifications of the supplier.
  • the destruction of the protruding 3′ ends is carried out in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer.
  • the destruction of the protruding 5′ ends is carried out by a controlled treatment with S1 nuclease.
  • the exonuclease Ba131 is used according to the recommendations of the supplier (Biolabs).
  • the oligodeoxynucleotides are synthesized chemically according to the phosphoramidite method using ⁇ -cyanoethyl protective groups [Sinha et al., Nucleic Acids Res. 12 (1984) 4539]. After synthesis, the protective groups are removed by treatment with ammonium hydroxide and two precipitations with butanol make it possible to purify and concentrate the oligodeoxynucleotides [Sawadogo and Van Dyke, Nucleic Acids Res. 19 (1991) 674]. The DNA concentration is determined by measuring the optical density at 260 nm.
  • the DNA fragment used to serve as molecular probe on the K. lactis genomic DNA is amplified in vitro by the PCR technique [Polymerase-catalysed Chain Reaction, Saiki R. K. et al., Science 230 (1985) 1350; Mullis K. B. and Faloona F. A., Meth. Enzym. 155 (1987) 335] on the S. cerevisiae DNA.
  • the amplification is automated (40 amplification cycles) and is carried out in a Perkin Elmer Cetus apparatus (DNA thermal cycler) Using the Taq polymerase (isolated from the archaebacterium Thermophilus aquaticus) provided by the company Perkin Elmer.
  • Each amplification cycle comprises three stages:
  • T 1/2 melting temperature of the oligodeoxynucleotides
  • radioactive nucleotide probes The preparation of the radioactive nucleotide probes is carried out by incorporation of radioactive adCTP (phosphorus 32) along the length of the molecule neosynthesized from 20 ng of DNA using the “Random Primed DNA Labeling” kit marketed by the firm Boehringer.
  • radioactive adCTP phosphorus 32
  • the transfers of DNA onto nylon membrane (Biodyne, Pall. St Germain en Laye) or nitrocellulose (Schleicher & Schuell, Dassel) are carried out according to the method initially developed by Southern [J. Mol. Biol. 98 (1979) 503].
  • the hybridization and washing conditions used depend on the nature of the probe used: under heterologous conditions (K. lactis genomic DNA hybridized with a probe from S. cerevisiae for example), the hybridization and the washes are carried out under conditions which are not very stringent (hybridization for 15 hours at 40° C. without formamide in 5X SSC/5X Denhart, the filter is then washed 3 times in 5X SSC/1% SDS at 40° C.
  • bacteria strains used are E. coli MC 1060 (lacIPOZYA, X74, galU, galK, strA r ), E. coli TG1 (lac, proA, B, supE, thi,hsdDS/FtraD36, proA + B + , lacI 1 ,lacZ, M15), or E. coli JM101 [Messing et al., Nucl. Acids Res. 9 (1981) 309].
  • the yeast strains used belong to the budding yeasts and more particularly to yeasts of the genus Kluyveromyces.
  • the K. lactis MW98-8C (a, uraA, arq, lys, K + , pKD1°), K. lactis CBS 293.91, K. lactis CBS 294.91 (uraA), and K. lactis CBS 2359/152 [a, metA, (k1, k2); Wésolowski et al., Yeast 4 (1988) 71] strains were particularly used; a sample of the MW98-8C strain was deposited on 16 Sep. 1988 at Centraalbureau voor Schimmelkulturen (CBS) at Baarn (The Netherlands) where it was registered under the number CBS579.88.
  • CBS Centraalbureau voor Schimmelkulturen
  • yeast genomic DNA is essentially derived from the technique by Hoffman and Winston [Gene 57 (1987) 267] and is described in detail in the text.
  • the yeast strains transformed with the expression plasmids encoding the proteins of the present invention are cultured in Erlenmeyer flasks or in 2 l pilot fermenters (SETRIC, France) at 28° C. in rich medium (YPD: 1% yeast extract, 2% Bactopeptone, 2% glucose; or YPL: 1% yeast extract, 2% Bactopeptone, 2% lactose) with constant stirring.
  • YPD 1% yeast extract, 2% Bactopeptone, 2% glucose
  • YPL 1% yeast extract, 2% Bactopeptone, 2% lactose
  • 10 ml dNTP dATP+dCTP+dGTP+dTTP, each at a concentration of 10 mM
  • the addition of a drop of paraffin oil makes it possible to avoid
  • a DNA fragment of 1.039 base pairs is obtained, whose identity is verified by analysis of the positions of certain restriction sites and corresponding to virtually the entire amino acid sequence of the mature form of protease B (Asp 3 to Ile 348 ). This fragment is then purified by electroelution after migration on a 0.8% agarose gel and is used to prepare the radioactive probe according to the “Random Priming” method.
  • the yeasts are then ground in the presence of phenolchloroform by the mechanical action of glass beads added to the mixture which is vortexed for 2 minutes.
  • the aqueous phase is then recovered after centrifugation and precipitated by addition of 2.5 volumes of ethanol.
  • the DNA is taken up in TE so as to be subjected to purification on a caesium gradient. Starting with 1 litre of culture, 1 mg of high molecular weight genomic DNA (>>20kb) is obtained.
  • the genomic DNA preparation is subjected to total digestion with restriction enzymes whose sites are present in the multiple cloning site of the vector pIC-20H.
  • restriction enzymes whose sites are present in the multiple cloning site of the vector pIC-20H.
  • a preliminary result shows that only conditions which are not very stringent (hybridization for 15 hours at 40° C. without formamide in 5X SSC/SX Denhart, then 3 washes in 5X SSC/1% SDS at 40° C. for 15 minutes, then 1 wash in 0.25X SSC/1% SDS for 10 minutes) make it possible to visualize an EcoRI fragment of 1.8 kb hybridizing with the S. cerevisiae PRB1 probe.
  • each well contains 12 mg of genomic DNA cleaved for 15 hours with 20 units of EcoRI and 20 units of a second restriction enzyme (FIG. 2 ).
  • a genomic fragment of about 700 bp and derived from the EcoRI+BglII double digestion hybridizes with the S. cerevisiae PRB1 probe (FIG. 2, well no. 3).
  • a BglII restriction fragment of greater size (about 1.3 kb) hybridizes with this probe (FIG. 2, well no. 2).
  • the fragment of about 700 bp detected after digestion of the genomic DNA with EcoRI+BglII is therefore a BglII-EcoRI restriction fragment.
  • the fraction of the gel comprising the fragments whose size is between 500 and 1,000 bp is then cut into three subfractions (subfraction ⁇ fraction (500/700) ⁇ bp; subfraction “e”: ⁇ fraction (700/800) ⁇ bp; subfraction “d”: ⁇ fraction (800/1000) ⁇ bp; FIG. 3 ).
  • a Southern blotting carried out after running an aliquot of these subfractions and hybridization with the S. cerevisiae PRB1 probe shows a hybridization signal of the expected size (700 bp) and particularly intense with the subfraction “e” ( ⁇ fraction (700/800) ⁇ bp).
  • a genomic library restricted to the BglII-EcoRI fragments of this subfraction is therefore constructed by cloning the BglII-EcoRI restriction fragments into the corresponding sites of the vector pIC-20H.
  • the transformation of the ligation in E. coli gives 90% of white clones in LB dishes supplemented with ampicillin and X-gal.
  • 340 clones (including about 30 blue clones) are then subcultured on the same medium so as to isolate them.
  • the 340 clones of the restricted library are then divided into 34 mixtures each corresponding to 10 different clones and their DNA is digested with EcoRI+BglII, run on agarose gel, transferred onto membrane so as to then be hybridized with the S.
  • FIG. 3 shows this Southern blot where two mixtures (no. 18 and no. 31) show a hybridization signal of the expected size (about 700 bp). The same operation is carried out in order to analyse separately the 10 clones of mixtures no. 18 and no. 31: clone no. 3 is the only clone of the mixture no. 18 to have a hybridization signal at the expected size (clone 18-3);. in a similar manner, only clone I of the mixture 31 (clone 31-I) gives a hybridization signal at the expected size.
  • the nucleotide sequence of the BglII-EcoRI fragment of clone 18-3 is produced in order to demonstrate that this fragment indeed corresponds to a fraction of the K. lactis PRB1 gene.
  • a rough restriction map of the plasmid pYG1224 from clone 18-3 is first produced and reveals the presence of an apparently unique SalI site at the centre of the BglII-EcoRI fragment.
  • the BglII-SalI (about 350 bp) and SalI-EcoRI (about 300 bp) fragments of the plasmid pYG1224 are then cloned into the vector pUC19, which generates the plasmids pYG1226 and pYG1227 respectively (FIG. 6, panel A).
  • the inserts of these plasmids are then sequenced in full using “universal primers”. As indicated in FIG.
  • the BglII-EcoRI fragment of the plasmid pYG1224 contains an open reading frame (225 residues) which exhibits sequence homologies with a fragment of the S. cerevisiae PRB1 gene (Arg 105 to Leu 328 ).
  • the presence of such a homology, as well as the strict conservation of the amino acids invariably found in the serine proteases of the subtilisin family demonstrate that the genomic DNA fragment carried by the plasmid pYG1224 indeed corresponds to a fragment of the K. lactis PRB1 gene.
  • the BglII-EcoRI fragment of the plasmid pYG1224 contains a unique KpnI restriction site located downstream of the BglII site (FIG. 6, panel A).
  • the KpnI-EcoRI restriction subfragment of about 665 nucleotides is therefore generated from this fragment, isolated by electroelution after running on a 1% agarose gel and radioactively labelled by the “random priming” method. This radioactive probe is then used to determine the size of the restriction fragments of the K. lactis genomic DNA which include it.
  • a KpnI-BglII fragment of about 1.2 kb is thus detected after hybridization and washing under stringent conditions (hybridization for 15 hours at 40° C.
  • a restricted library of K. lactis genomic DNA (KpnI-BglII restriction fragments of between 1 and 1.5 kb in size) is then constructed according to Example E1.3. and the restriction fragment hybridizing with the probe is cloned between the KpnI and BamHI sites of the vector pIC-20H, which generates the plasmid pYG1231 (FIG. 6, panel B).
  • the genomic insert of this plasmid is then sequenced using the oligodeoxynucleotide Sq2101 (5′-GACCTATGGGGTAAGGATTAC-3′) (SEQ ID NO. 11) as primer.
  • This oligodeoxynucleotide corresponds to a nucleotide sequence present in the BglII-EcoRI fragment of the plasmid pYG1224 and located at about 30 nucleotides from the EcoRI site. It therefore makes it possible to determine the nucleotide sequence situated in 3′ of this restriction site, and especially the sequence located between the EcoRI site and the translational stop codon of the messenger RNA corresponding to the K. lactis PRB1 gene.
  • the nucleotide sequence produced in E.1.5. demonstrates the existence of a HindIII restriction site located between the EcoRI site and the translational stop codon.
  • the use of the KpnI-EcoRI restriction fragment corresponding to the C-terminal part of the K. lactis PRB1 gene as radioactive probe on the K. lactis genomic DNA digested with HindIII and a second enzyme makes it possible to identify, by Southern blotting, a HindIII-EcoRV fragment of about 1.7 kb which hybridizes with this probe. This restriction fragment is first cloned between the EcoRV and HindIII sites of the vector pIC-20R, thereby generating the plasmid pYG1237.
  • a restriction map of the genomic DNA insert contained in the plasmid pYG1237 is produced (FIG. 6, panel C), and the following plasmids are generated: pYG1238 (plasmid pYG1237 deleted in relation to its PstI fragment), pYG1239 (PstI fragment of pYG1237 in the vector pUC19), pYG1240 (plasmid pYG1237 deleted in relation to its KpnI fragment), pYG1241 (plasmid pYG1237 deleted in relation to its ClaI fragment) and pYG1242 (plasmid pYG1237 deleted in relation to its SalI fragment; FIG. 6, panel C).
  • genomic inserts of these various plasmids are then sequenced with the aid of universal primers and the oligodeoxynucleotide Sq2148 (5′-GCTCGGCAACATATTCG-3′) (SEQ ID NO. 12) which makes it possible to sequence the region situated immediately in 5′ of the BglII site.
  • Sq2148 5′-GCTCGGCAACATATTCG-3′
  • This strategy makes it possible to obtain overlapping sequences demonstrating the uniqueness of the BglII, ClaI and PstI restriction sites and making it possible to identify the probable ATG for initiation of translation of the K. lactis PRB1 gene.
  • Example 1 The general strategy described in Example 1 is repeated for the cloning of the carboxypeptidase Y gene of K. lactis CBS 2359/152.
  • a preparation of genomic DNA of the strain S. cerevisiae S288C [Mortimer and Johnston, Genetics 113 (1986) 35] is first carried out according to Example E1.2.
  • a PCR amplification of this genomic DNA preparation is then carried out with the oligonucleotides 5′-CTTCTTGGAGTT GTTCTTCG-3′ and 5′-TGGCAAGACATCC GTCCACGCCTTATT-ACC-3′, Specific for the PRC1 gene.
  • An amplified fragment of the expected size (699 bp) is thus obtained which corresponds to positions 696-1395 (the ATG initiation codon being numbered +1) of the open reading frame of the S. Cerevisiae PRC1 gens [Valls et al., Cell 48 (1987) 887].
  • This fragment is then purified by electroelution and radiolabelled according to the “Random Priming” technique.
  • the K. lactis PRC1 gene is obtained by screening the K. lactis genomic library constructed Wésolowski-Louvel [Yeast A (1988) 71] from the strain 2359/152 in the cloning vector KEp6 [Chen et al., J. Basic. Microbiol. 28 (1988) 211].
  • the stain E. coli JM101 is transformed with the DNA from the library and the transformants are plated on LB medium supplemented with ampicillin (50 mg/l). 15,000 clones are then transferred onto nitrocellulose filters and the filters are hybridized with the probe described in Example E.2.1.
  • the hybridization and washing conditions are those of Example E.1.3.
  • pC34 12 positive clones are thus isolated and one of them, designated pC34, is selected for the rest of the study.
  • the hybridization of the plasmid pC34 with the probe corresponding to the S. cerevisiae PRC1 gene is confirmed by Southern blotting.
  • a restriction map of the genomic insert (about 6.9 kb)of the plasmid pC34 is given in FIG. 7 .
  • the sequence of the 2.5 kb EcoRI-SalI fragment comprising the K. lactis PRC1 gene is then determined on the 2 strands. This sequence is presented SEQ ID No.3.
  • An inner fragment of 449 bp of the PRA1 gene (or PEP4 gene) from S. cerevisiae is first amplified by the PCR technique starting with the plasmid CBZIB1 [Woolford et al., Mol. Cell. Biol. 6 (1986) 2500] provided by Dr E. Jones (Carnegie-Mellon University, Pittsburgh, Pa., USA) and the oligodeoxynucleotides 5′-CTGTTGATAAGGTGGTCC-3′ (SEQ ID NO. 15) and 5′CAAGCGTGTAATCGTATGGC-3′ (SEQ ID NO. 16).
  • the amplified fragment obtained corresponds to positions 617-1066 of the open reading frame of the S. cerevisiae PRA1 gene, the ATG initiation codon being numbered +1. This fragment is then purified by electroelution and radiolabelled according to the “Random Priming” technique.
  • the PRA1 gene is obtained by screening the K. lactis genomic library constructed by Wéselowski-Louvel [Yeast 4 (1988) 71] starting with the strain 2359/152 in the cloning vector KEp6 [Chen et al., J. Basic Microbiol. 28 (1988) 211]. After transforming the library in E. coli JM101 and selecting in the presence of ampicillin (50 mg/l). 15,000 clones are then transferred onto nitrocellulose filters and the filters are hybridized with the probe described in Example E3.1. The hybridization and washing conditions are those of Example E.1.3. Only 1 positive clone was thus isolated and designated pA25/1.
  • the hybridization of the plasmid pA25/1 with the probe corresponding to the S. cerevisiae PRA1 gene is confirmed by Southern blotting.
  • a restriction map of the genomic insert (about 7.5 kb) of this plasmid is represented in FIG. 8 .
  • the sequence of the 1.6 kb ClaI-EcoRI fragment comprising the K. lactis PRA1 gene is then determined on the 2 strands. This sequence is presented SEQ ID No. 5.
  • the transformation of the yeasts belonging to the genus Kluyveromyces, and in particular the strains K. lactis MW98-8C, CBS 293.91 and CBS 294.91 (uraA) is carried out for example by the technique for treating whole cells with lithium acetate [Ito H. et al., J. Bacteriol. 153 (1983) 163-168], modified as follows. The growth of the cells occurs at 28° C.
  • YPD medium 50 ml of YPD medium, with stirring and up to an optical density of 600 nm (OD600) of between 0.6 and 0.8; the cells are then harvested by low-speed centrifugation, washed in a sterile solution of TE (10 mMTris HCl pH 7.4; 1 mM EDTA), resuspended in 3-4 ml of lithium acetate (0.1 M in TE) in order to obtain a cell density of about 2 ⁇ 10 8 cells/ml, and then incubated at 30° C. for 1 hour with gentle stirring. 0.1 ml aliquots of the resulting suspension of competent cells are incubated at 30° C.
  • the cells are washed twice, then resuspended in 0.2 ml sterile water.
  • the selectable marker is the S. cerevisiae URA3 gene
  • the cells are directly plated on YNB (Yeast Nitrogen Base; Difco)/glucose (20 g/l)/agar.
  • the selectable marker is the aph gene of the transposon Tn903, the cells are first incubated for 16 hours at 28° C.
  • the plasmid pYG1229 is constructed by cloning the HindIII-EcoRI fragment (including the BglII-EcoRI fragment of about 700 bp and corresponding to the C-terminal part of the K. Lactis PRB1 gene) of the plasmid pYG1224 between the corresponding sites of the plasmid pUC9.
  • the plasmid pYG1228 is constructed by cloning the HindIII fragment of 1.1 kb and corresponding to the S. cerevisiae URA3 genes derived from the plasmid pCG3 [Gerbaud et al., Curt.
  • the plasmid pYG1228 therefore makes it possible to have a SalI-XhoI restriction fragment of about 1.1 kb and containing the entire HindIII fragment containing the S. cerevisiae URA3 gens. This restriction fragment is then cloned into the SalI site of the plasmid pYG1229 which generates the plasmid pYG1232 (2 possible orientations). The digestion of this plasmid with the BglII and EcoRI enzymes makes it possible to generate a restriction fragment of about 1.8 kb corresponding to the S. Cerevisiae URA3 gene bordered by K.
  • lactis genomic sequences derived from the PRB1 gene (FIG. 9, panel A).
  • the transformation of the K. lactis uraA mutants with the BglII-EcoRI fragment of the plasmid pYG1232 generates transformed clones (complemented by the S. cerevisiae URA3 gens) corresponding to the integration of this fragment in to the chromosome.
  • Panel B of FIG. 9 shows the integration of this fragment into the genomic DNA of the K. lactis CBS 294.91 strain (uraA) after non-homologous recombination (well 1) or after homologous recombination in the PRB1 gene (well 3, this disruptant is noted Y750).
  • the disruption of the wild-type allel of the PRB1 gene does not modify the growth characteristics of the strain.
  • the 4.4 kb SalI-SphI fragment derived from the plasmid pC34 is first subcloned into the corresponding sites of the vector pIC-20R, which generates the plasmid pYG154 (FIG. 12, panel b).
  • This plasmid is then digested with the SphI enzyme, then treated with phage T4 DNA polymerase I in the presence of calf intestinal phosphatase (CIP).
  • CIP calf intestinal phosphatase
  • the plasmid obtained is then ligated to the EcoRI fragment of 1.6 kb carrying the S. cerevisiae URA3 gene derived from the plasmid pKan707 (EP 361 991), previously treated with the Klenow fragment of DNA polymerase I of E. coli.
  • the plasmid obtained is designated pYG155 (FIG. 10, panel c).
  • the 4.5 kb SalI-BamHI fragment of the plasmid pYG155 is then purified by electroelution and used to transform the K. lactis CBS 294.91 strain (uraA).
  • the transformants are selected for the Ura + phenotype and a few clones are then analysed by Southern blotting in order to check the site of integration of the URA3 marker.
  • the clone Y797 is thus identified in which the chromosomal PRC1 gene has been replaced, by homologous recombination, by the disrupted allel constructed in vitro. The disruption of the wild-type allele of the PRC1 gene does not modify the growth characteristics of the strain.
  • genes for the proteins of interest which it is desired to secrete and/or express are first inserted, “in the productive orientation” (defined as the orientation which places the N-terminal region of the protein proximally relative to the transcription promoter), under the control of regulatable or constitutive functional promoters such as for example those present in the plasmids pYG105 (K- lactis LAC4 promoter), pYG106 (S. cerevisiae PGK promoter), pYG536 (S. cerevisiae PHO5 promoter), or hybrid promoters such as those described in patent application EP 361 991.
  • regulatable or constitutive functional promoters such as for example those present in the plasmids pYG105 (K- lactis LAC4 promoter), pYG106 (S. cerevisiae PGK promoter), pYG536 (S. cerevisiae PHO5 promoter), or hybrid promoters such as those described in patent application EP 361 991.
  • the plasmids pYG105 and pYG106 are particularly useful because they allow the expression of genes included in HindIII restriction fragments from regulatable (pYG105) or constitutive (pYG106) promoters which are functional in K. lactis.
  • the plasmid pYG105 corresponds to the plasmid pKan707 described in patent application EP 361 991 in which the unique HindIII restriction site located in the gene for resistance to geneticin (G418) has been destroyed by site-directed mutagenesis, conserving a protein unchanged (oligodeoxynucleotide 5′GAAATGCATAAGCT CTTGCCATTCTCACCG-3′) (SEQ ID NO. 17).
  • the SalI-SacI fragment encoding the URA3 gene of the plasmid thus mutated was then replaced by a Sail-Sac 1 restriction fragment containing an expression cassette consisting of the K.
  • lactis LAC4 promoter [in the form of a SalI-HindIII fragment derived from the plasmid pYG1075 Fleer et al., Bio/Technology 9 (1991) 968]) and the S. cerevisiae PGK gene terminator [in the form of a HindIII-SacI fragment; Fleer et al., Bio/Technology 9 (1991) 968].
  • the plasmid pYG105 is mitotically very stable in Kluyveromyces yeasts and a restriction map is given in FIG. 11 .
  • the plasmids pYG105 and pYG106 differ from each other only in the nature of the transcription promoter encoded by the SalI-HindIII fragment.
  • the protein encoded by the plasmid pYG1212 corresponds approximately to the first two domains of human serum albumin (HSA).
  • HSA human serum albumin
  • the HindIII-MstII restriction fragment of the plasmid YP40 corresponding to residues 1 to 403 of RSA (the ATG for initiation of translation is noted +1) is ligated to the MstII-HindIII fragment of the plasmid pYG221 [Yeh et al., Proc. Natl. Acad. Sci. USA 89 (1992) 1904], which generates a HindIII fragment including the 403 N-terminal residues of HSA followed by the last three residues of HSA (residues Leu-Gly-Leu) and a translational stop codon [truncated variant noted HSA (1-403) ].
  • the HindIII fragment is then cloned in the productive orientation into the plasmid pYG105, which generates the plasmid pYG1212 (FIG. 11 ).
  • the yeasts K. Lactis CS 293.91 and Y750 are transformed with the plasmid pYG1212. After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the protein HSA (1-403) . A few clones are incubated in YPD or YPL medium at 28° C. The culture supernatents are recovered by centrifugation when the cells reach the stationary growth phase, concentrated 10 fold by precipitation for 30 minutes at ⁇ 20° C. in a final concentration of 60% ethanol, then tested after electrophoresis on an 8.5% SDS-PAGE gel and staining of the gel with coomassie blue. The results presented in FIG.
  • lactis protease C gene “K1.PRC1” 3 GAATTCTGTC AACTGGATAC GGAAGACAAT AGAATGGACA CATAATGGTC TCAATACGAC 60 AATTCAACGG CTCTTAGAAG GTGAGTTATT CTTGACATTT TCATGGCTCT TCGAGCATGC 120 TTTCTAAGAT GACGCGGAAG GTGAAAAAGA TTAGAAAACG GCCATTCACG TGAATATCAC 180 GTGAACTACA AATTCATGAT ATATTACCGC CAATAGTATT GGTGGTTACC CGATCGTATC 240 GAATGTACTG ACTTCGAAAA TATGAATAGT CCTCTTTAAA ACAAAGGGTT TTCAGTGACC 300 CTTACTCCAT CATCCTTA GTATTTGGTC TACAGACTCG CCATTGCCGT ATATTCAGGG 360 TAGTAGTCAG TACATCGGTG TCTGCC ATG GTT TCG ATA AAG TTT CTT TTA TCT 413 Met Val Ser Ile Lys P

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JP2000078978A (ja) * 1998-09-04 2000-03-21 Kirin Brewery Co Ltd プロテアーゼ活性の低下したカンジダ・ボイジニ株及び異種タンパク質製造用宿主としてのその使用
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AR107872A1 (es) * 2016-03-16 2018-06-13 Godo Sushei Co Ltd Proteinasa b y solución de lactasa usando sus propiedades y método para producirlo
WO2018141872A1 (fr) * 2017-02-02 2018-08-09 Lallemand Hungary Liquidity Management Llc Expression de protéase hétérologue pour améliorer la fermentation alcoolique
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