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WO1991000357A1 - New strain with filamentous fungi mutants, process for the production of recombinant proteins using said strain, and strains and proteins produced by said process - Google Patents

New strain with filamentous fungi mutants, process for the production of recombinant proteins using said strain, and strains and proteins produced by said process Download PDF

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WO1991000357A1
WO1991000357A1 PCT/FR1990/000479 FR9000479W WO9100357A1 WO 1991000357 A1 WO1991000357 A1 WO 1991000357A1 FR 9000479 W FR9000479 W FR 9000479W WO 9100357 A1 WO9100357 A1 WO 9100357A1
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strain
protein
sequence
plasmid
tolypocladium
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Thierry Calmels
Henri Durand
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Cayla
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • 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
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a new strain of filamentous fungus, as well as the derivatives of this strain obtained by mutation or by genetic manipulation, and to a process for the production of recombinant protein, using said strains.
  • Recombinant proteins are understood to mean the polypeptide molecules synthesized by a microorganism or a cell population as a result of the introduction into the genetic material of said microorganism or of said cells of a gene coding for the protein considered.
  • the gene in question called the heterologous gene, can come from another living organism or be synthesized artificially.
  • the possibility of introducing and expressing heterologous genes was first demonstrated and applied using bacteria, and more particularly Escherichia coli, as host cells. More recently, productions of re ⁇ combining proteins by other microorganisms including yeasts and fungi as well as by cell cultures of higher organisms have been described.
  • Filamentous fungi are commonly used in the fermentation industry, particularly for the manufacture of certain antibiotics (penicillins, cephalosporins) and a large number of enzymes (gluco-amylases, cellulases, proteases, pectinases -).
  • Filamentous fungi have a number of advantages over microorganisms prokaryotes (bacteria) in particular the ability to excrete large quantities and a wide variety of proteins with post-traditional modifications, in particular glycosylations, specific for eukaryotes. Compared to superior eukaryotic cells, fungi are much easier to cultivate on a large scale, the separation of the mycelium and the culture medium after fermentation is also very easy.
  • the present invention relates to a process for the production of recombinant protein using filamentous gables, characterized in that a strain of the genus Tolypocladium is cultivated on an appropriate medium into which a DNA sequence coding has been introduced. for said protein placed under the control of elements ensuring the expression of said sequence in said strain and in that said protein is recovered.
  • the strain used is a Tolypocladium geodes strain. This strain was deposited under the number 1-880 on June 29, 1989 at the National Collection of Culture of Microorganisms - 28, rue du Dondel Roux - 75015 PARIS.
  • the invention also relates to mutants defiant in protease activity and / or hyperexcretor of exogenous protein of this strain.
  • the present invention is based in particular on the isolation of a new strain of filamentous fungus which is particularly suitable for the production of recombinant proteins.
  • the wild strain NC14 was isolated from a sample of soil rich in decomposing organic matter (humus from an undergrowth of the Graulhet region, Tarn, France). It was originally selected for its strong proteolytic activity, and has been found to analyze to produce significant quantities of very extracellular enzymes, chitinases and glucanases in particular.
  • NC14 strain was entrusted for identification to the laboratory of the National Museum of Natural History in Paris. It was subcultured in Petri dishes on two standard culture media, used for the determination of hyphomycetes: Malt-agar 2% and Potato Dextrose Agar (PDA) and then incubated at 25 ° C.
  • PDA Potato Dextrose Agar
  • the culture on Malt develops slowly, barely 1 cm in 3 weeks of culture and forms a tight colony with a wet appearance. Microscopic observation shows a heterogeneous mycelium, with vacuolated cytoplasm. Conidia are very rare and sporogenic cells are few and far between. The PDA environment is more favorable. The culture is slow growing, reaching 13 to 20 mm in 10 days. The thallus is white, flaky on the surface, fairly dense in depth, with an irregular outline. There is a slight beige pigmentation on the reverse, no pigment in the medium. No characteristic odor.
  • sporogenic cells united or grouped by two or three on a short lateral cell of the mycelium.
  • the phialides are 9 to 14 ⁇ m long, are slightly swollen at the base and then elongated towards the neck which is sometimes curved.
  • the spores (conidia) are grouped in heads; very heterogeneous in shape (spherical, cylindrical, oval) with overall ratios between 3.6-0.9 ⁇ m long by 2.9-0.9 ⁇ m wide with two main classes: 3 x 1.5 (cylindrical) and 1.5 x 1.8 (globular).
  • the mycelium is between 1.5 and 2 ⁇ m wide. No chlamydospores.
  • the NC14 strain is finally very close to the geodes species (Gams) by the mode of grouping of the phialides described above, by their form as well as by the cultural aspect.
  • the heterogeneity of the spores which are very heterogeneous in size and shape, 40% of these correspond to the characteristics of Tolypocladium geodes (Gams), that is to say 1.6 by 2.2 ⁇ m.
  • the NC14 strain of Tolypocladium géodes was the subject of a genetic improvement program, consisting in the recurrent selection of mutants which are increasingly strongly producing proteolytic enzymes, then chitinolytic enzymes.
  • the mutagenesis and selection methodologies used are those previously described for Trichoderma reesei (DURAND et al. Enzyme Microb. Technol., 1988, 10: 341) and for Penicillium occitanis (JAIN et al., Submitted for publication in Enzyme Microbiol. Technology).
  • the first selection step was carried out using a spore suspension of the NC14 strain treated with a mutagenic agent, ultraviolet light. A dilution of said suspension of spores was used to inoculate Petri dishes containing an agar medium of the following composition:
  • NC21 was the subject of a new selection step after mutagenesis with 1 thyl-methane sulfonate (EMS) on the medium based on milk powder, to which ammonium sulfate at the concentration of 5 g / l was added as a suppressant for the production of proteases.
  • EMS thyl-methane sulfonate
  • NC28 was thus isolated. From this latter strain, a mutant hyperproducer of chitinolytic activity was sought, after mutagenesis by nitrous acid, on the basic medium previously described, where the milk powder was replaced by chitin colloid- at the concentration of 10 g / 1.
  • This strain has been shown to be hyper-excretory of proteins: concentrations of the order of 10 g / 1 of proteins were obtained in 6 days of fermentation, in a chemically defined medium (basic saline medium according to MANDELS and WEBER, J Adv. Chem. Ser., 1969, 95: 391) containing glucose (continuously added at a rate of 20 g / l per day) as the only source of carbon.
  • the wild strain NC14 produces approximately 0.5 g / l of extracellular proteins.
  • NC46 strain thus isolated is characterized by the absence of proteolytic activity detectable by the azocasein test (TOMARELLI et al., J. Lab. Clin. Med., 1949, 34: 428) in the culture supernatant, under the conditions of production of extracellular proteins mentioned above and described in Example II below.
  • This NC46 strain was subjected to a new mutagenic acid treatment nitrous, then mutants deficient in amino peptidase activity were sought ⁇ chés according to a modification of the technique described by MILLER and MACKINNON (J. Bact., 1974, 120: 355): after growth 4 days on the medium based on powder of milk described above, the colonies are covered with a solution containing: 0.5 mg / ml of L.
  • NC50 A mutant isolated by this technique, no longer exhibits the Leucine aminopeptidase activity detected in the supernatant of cultures of strains NC46 and of the preceding generations.
  • the genealogy of the NC50 strain is summarized in Table 1.
  • the mutant strain NC50 has the same protein excretion capacities from the quantitative point of view as the hyperexcretory strain NC39 mentioned above.
  • An additional advantage of the NC50 strain resides in the absence of detectable proteolytic activity in the culture supernatants, which makes it possible to avoid or at least minimize the degradation of the proteins produced, and particularly of the proteins. heterologists, in the fermentation medium. This is an important aspect of the present invention.
  • NC14 Another aspect is constituted by the demonstration of the capacity of the NC14 and derived strains to be transformed according to a simplified protocol, original compared to those mentioned.
  • Table 1 Genealogy of mutant strains derived from Tolypocladium geodes NC 4
  • the elements ensuring the expression of said DNA sequence in the strain essentially comprise a promoter sequence of a gene of fungal origin.
  • This DNA sequence coding for said protein will be preceded by a signal sequence ensuring excretion of the protein and followed by a transcription terminator sequence.
  • An additional aspect of the present invention consists of the possibility of selecting transformed strains for which the expression of the gene of interest is amplified, thanks to a selection screen based on the use of a constituent gene. a dominant marker, placed downstream of the gene of interest, without promoter or signal for stopping transcription between the two genes. This technique is illustrated in Example V below.
  • This gene constituting the marker codes for resistance to an antibiotic which is preferably a compound of the phleomycin family.
  • a gene coding for resistance to an antibiotic of the phleomycin family this gene is of genomic origin and comes from the genome of actino-mycetes producing said antibiotic.
  • the DNA sequence coding for said protein as well as the elements ensuring the expression of said sequence will be carried by a plasmid which preferably is an autonomous replication plasmid comprising an autonomous replication sequence effective in Tolypo ⁇ cladium TARS
  • the present invention relates to a new strain of filamentous fungus, Tolypocladium géodes NC14, and the mutants resulting from this strain, selected for their capacity to excrete large quantities of proteins in an extremely simple culture medium, consisting essentially of sugars and mineral salts, which facilitates the extraction and purification of said proteins.
  • This exceptional capacity can be exploited for the production of recombinant proteins, which constitutes a first aspect of the present invention.
  • the present invention provides a means of preserving the stability of the proteins excreted in the culture medium by the use of mutants deficient in proteolytic activities.
  • the present invention provides a means of introducing heterologous genes into the strain of Tolypocladium geodes NC14 and its derivatives according to a simplified protocol which does not involve protoplasts.
  • the expression of heterologous genes by Tolypocla ⁇ dium geodes NC14 and its derivatives can be increased by a selection technique using a dominant marker placed downstream of the gene of interest without promoter nor terminator between the two genes.
  • the present invention also relates to proteins obtained by implementing the method according to the invention. Among these proteins coded according to the invention, mention will be made more particularly of pullulanase, human lysozyme and the Sh protein, the nucleotide sequence of this Sh protein being represented in FIG. 2.
  • the present invention likewise relates to the transformed strains of Tolypocladium, obtained during the process for the production of recombinant proteins.
  • the following examples serve to illustrate the present invention without limitation.
  • FIG. 1 is a diagram of the plasmid pUT720 - Figure 2 shows the nucleotide sequence of the Sh protein gene preceded by the synthetic sequence used as an excretion signal
  • FIG. 3 is a diagram of the plasmid pUT715
  • FIG. 4 is a diagram of the plasmid pUT771 - Figure 5 represents the nucleotide sequence of the promoter part of the plasmid pUT771
  • FIG. 6 is a diagram of the plasmid pUT760
  • Figure 7 shows the synthetic nucleotide sequence encoding the human lysozyme
  • Figure 8 is a diagram of the plasmid pUT772
  • Example I Transformation of Tolypocladium geodes
  • the mutant strain NC50 deficient in proteolytic activities, was transformed by the plasmid pUT703 (DURAND et al, Proc. Biochemistry and Genetics of Cellulose Dégradation, JP Aubert Ed., Académie Press, 1988, p 136).
  • This plasmid is characterized by the presence of a gene for resistance to antibiotics of the phleomycin family (ARMAU et al., French Patent No.
  • a fungal promoter the promoter of the gene 3-phosphide glyce'raldehyde dehydrogenase from Aspergillus nidulans (noted promoter gpd) (Van GORCOM et al., Gene, 1986, 48: 211) and bounded downstream by the yeast terminator CYC1 (SMITH et al ., Cell, 1979, 16: 753)
  • the resistance gene comes from an actinomycete producing antibiotics from the phleomycin family; Streptoalloteichus hindus-tanus.
  • the protein encoded by this gene, called Sh protein inactivates antibiotics by question by formation of an equimolecular complex (GATIGNOL et al., FEBS Letters, 1988, 230: 171).
  • Phleomycin and related antibiotics have a high toxicity for Tolypocladium geodes as for practically all living organisms.
  • the resistance gene therefore provides an easy means of selecting the clones transformed by their capacity to develop in the presence of phleomycin.
  • the protocol used for the transformation is as follows: spores obtained by culture of the strain NC50 on PDA medium for 7 days at 27 ° C. are suspended in MnP medium of the following composition: basic saline medium according to Mandels added with sucrose 100 g / 1, MES buffer 5 g / 1, pH 5.5. After filtration on sintered glass No. 2 to remove the mycelium, the suspension is centrifuged (10 minutes, 10,000 rpm) and the residue is taken up in the same MnP medium supplemented with Caylase C3 (lytic enzyme marketed by CAYLA) at 10 mg / ml.
  • Caylase C3 lytic enzyme marketed by CAYLA
  • Q croscopic to the hematimeter that is to say about 10 / ml.
  • a solution of plasmid DNA (5 to 10 ⁇ g of DNA in a volume of 5 to 20 ⁇ l) is added.
  • 50 ⁇ l of MPC solution is added (MOPS 10 mM pH 5.8, PEG 6000 60% pv, CaCl 2 75 mM) and the mixture is incubated for 30 minutes in ice, then added 2.5 ml of MPC.
  • the transformation rates obtained using the plasmid pUT703 were of the order of 2000 to 5000 transformants per microgram of DNA.
  • the stability of the transformants was tested by subculturing the colonies on PDA medium containing 30 ⁇ g / ml of phleomycin (the minimum inhibitory concentration - MIC - for the non-transformed NC50 strain is approximately 10 ⁇ g / ml under these conditions).
  • About 90% of the transplanted colonies confirmed their resistance character.
  • Example II Expression and excretion of a heterologous protein.
  • obtaining transforming clones resistant to phleomycin indicates that the gene coding for the Sh protein is expressed in the strain MC50 of Tolypocladium geodes.
  • the protein could not be demonstrated in the culture supernatants of the transformed strains and is therefore probably not excreted in detectable quantities.
  • the strain NC50 was transformed according to the protocol described in Example I, using the plasmid pUT720, the map of which is shown in FIG. 1.
  • This plasmid is characterized by the presence, under dependence of the gpd promoter, of the Sh protein gene upstream of which a synthetic excretion signal sequence has been added (FIG. 2).
  • this culture made it possible to inoculate a fermenter. of 14 liters of total volume (Chemap) containing 8 liters of the same medium.
  • the fermentation lasted 6 days at 27 ° C with stirring of 500 rpm and aeration of 0.5 vvm, the pH being regulated above 5.0 with ammonia.
  • a sterile glucose solution at 200 g / 1 was introduced continuously at the rate of 0.8 liters per 24 hours, or approximately 20 grams of glucose per liter per day. After 6 days, the culture was harvested and centrifuged.
  • the SDS-PAGE electrophoresis analysis of the fractions corresponding to this peak revealed the presence in the practically pure state of a protein of approximately 14,000 kd of molecular weight, which corresponds to the size of protein Sh (GATIGNOL et al., FEBS Letters, 1988, 230: 171).
  • the presence in the culture supernatant of protein inactivating phleomycin was confirmed by the following test: on paper discs impregnated with phleomycin solution at different concentrations (from 10 to 300 ⁇ g / ml) different volumes of culture supernatant (from 1 to 50 ⁇ l) are deposited. The discs are then placed on the surface of an agar medium (medium No.
  • Example III i Isolation of a promoter sequence
  • the plasmid has been linearized by double BamHI-AsuII cleavage (the two unique restriction sites are located in the multisite sequence located immediately upstream of the coding part of the Sh gene).
  • the hybrid plasmid population thus obtained was used to transform the Escherichia coli DH5 ov strain, with selection on the basis of resistance to ampicillin.
  • the plasmid DNA extracted from the "pool" of E. coli transformants was used to transform the NC14 strain of
  • One of the transformations led to the appearance of clones highly resistant to phleomycin (MIC> 250 ⁇ g / ml) with a frequency of the order of 5000 transformants per microgram of DNA. For two other transformations, the frequency and the level of resistance of the transformants were found to be much lower (some transformants / ⁇ g, MIC ⁇ 100 ⁇ g / ml). Finally four transformations gave no result.
  • the plasmid which has allowed the most efficient transformation of T. geodes called pUT771 has been analyzed, it comprises an insertion of a size of approximately 1.8 kb; the mapping of pUT771 is represented in FIG. 4.
  • the nucleotide sequence of the promoter part is represented in FIG. 5.
  • Example IV Co-transformation of T. geodes with a plasmid carrying the marker for resistance to phleomycin and a plasmid carrying a non-selectable gene.
  • a transformation experiment was carried out according to the protocol described in Example I above, with a mixture containing approximately 1 ⁇ g of plasmid DNA from pUT771 described in Example II and 10 ⁇ g of DNA plas- pUT760 midic, the map of which is represented in FIG. 6.
  • the plasmid pUT760 is characterized by the presence of the Klebsiella pneumoniae ATCC 15050 gene coding for mature pullulanase (MICHAELIS et al., J. Bact., 1985, 164: 633 ), under the dependency of the gpd pro ⁇ motor of Aspergillus nidulans and preceded by the synthetic signal sequence described in Example I (FIG. 2).
  • the 10 transforming clones appearing to have the strongest pullulanase activity were recovered from the PDA-phleomycin medium and cultured in shaken flasks in the medium. according to Mandels supplemented with glucose 30 g / 1, yeast extract 1 g / 1 and buffered with potassium phthalate at 5 g / 1.
  • the cultures were centrifuged and the pullulanase activity was assayed in the supernatants according to the following protocol: to 0.5 ml of a 10 g / l pullulan solution in 0.1 M acetate buffer pH 5.5, 0.5 ml of enzymatic solution (supernatant optionally diluted in the same buffer) is added. After incubation for 60 minutes at 55 ° C, the released glucose is measured by the MILLER method (Analytical
  • This example is intended to illustrate the possibility of preferentially selecting recombinant clones of T. geodes NC14 and of its derivatives which are highly producers of a heterologous protein by means of plasmid constructs calling upon the principle of transcription. polycistronic.
  • the expression of several adjacent genes under the dependence of the same promoter and by the intermediary of the same messenger RNA, known as polycistronic, is a well known phenomenon in bacteria. On the other hand, it is generally accepted that this phenomenon does not exist in eukaryotes. However, it has been reported recently
  • a synthetic gene for human lysozyme was prepared pa * ⁇ General assembly with 10 oligonucleotides syn ⁇ thesis.
  • the sequence represented in FIG. 7 comprises 14 base pairs preceding the initiation codon, followed by 18 codons starting with an ATG corresponding to the amino acids of the signal peptide of human lysozyme with the exception of the 17th codon (replacement of glutamine by leucine) (CHUNG, KESHAU and GORDON, Proc. Natl. Acad. Sci. 1988, 85: 6227) then 130 codons of amino acids of human lysozyme (JOLLES and JOLLES, Mol. Cell. Biochem., 1984, 63 , 165) and finally a stop codon followed by 6 base pairs.
  • the DNA fragment bordered by the EcoRV-BamHI restriction sites was cloned at the EcoRV site of the plasmid pUT771 mentioned in Example II to give, in one of the 2 possible orientations, the plasmid pUT772 (FIG. 8 ).
  • the NC50 strain of T. geodes was transformed with the plasmid pUT772 according to the protocol described in example X with the only difference that the selection of the transformants was carried out with a lower concentration of phleomycin (15 ⁇ g / ml).
  • the colonies obtained with a frequency 50 to 100 times less than that provided by the DNA pUT771 used as a control were subcultured on PDA medium and individually examined for the level of resistance to phleomycin ⁇ and checked for the production of lysozyme.
  • the determination of the minimum phleomycin inhibitory concentrations of the transformants by pUT772 showed that the resistance levels of the clones were low (2 to 10 times the MIC compared to the untransformed strain) while the transformants by ⁇ UT771 were on the contrary very high (30 to 200 times the MIC).
  • lysozyme is verified indirectly by the method of agar cylinders for the production of a lytic activity against Micrococcus lyso-deikticus on PDA.
  • 61 produced an activity detectable by the presence of an inhibition halo around the agar cylinders.
  • no colony resulting from transformation with pUT771 exhibited this activity (55 tested).
  • Clonal analysis showed that the production character was retained for 2/3 of the positive strains after a sporulation stage.
  • the strain NC50 PUT772-28 selected from the most active strains by the box test was more particularly studied.
  • the lysozyme activity was determined by a turbidimetric assay from lyophilized cells of Micro ⁇ coccus lysodeikticus (SHUGAR, Biochim. Biophys., 1952, Acta 8: 302).
  • the proteins of the culture supernatants were fractionated on a MonoS Pharmacia column

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Abstract

The invention relates to a process for the production of recombinant protein using fibrous fungi, characterised by the fact that a strain of the genus Tolypocladium, into which a DNA sequence coding for said protein which has been placed under the control of elements guaranteeing the expression of said sequence in said strain, has been introduced, is cultivated on an appropriate medium and by the fact that said protein is recovered. The invention also relates to the proteins and strains obtained by putting said process into effect.

Description

NOUVELLE SOUCHE ET SES MUTANTS DE CHAMPIGNONS FILAMEN¬ TEUX, PROCEDE DE PRODUCTION DE PROTEINES RECOMBINANTES A L'AIDE DE LADITE SOUCHE ET SOUCHES ET PROTEINES OBTE¬ NUES SELON CE PROCEDE.NOVEL STRAIN AND ITS MUTANTS OF FILAMENTAL MUSHROOMS, PROCESS FOR PRODUCING RECOMBINANT PROTEINS USING SAID STRAIN AND STRAINS AND PROTEINS OBTAINED ACCORDING TO THIS PROCESS.
La présente invention concerne une souche nou¬ velle de champignon filamenteux, ainsi que les dérivés de cette souche obtenus par mutation ou par manipulation génétique, et un procédé pour la production de protéine recombinante, à l'aide desdites souches.The present invention relates to a new strain of filamentous fungus, as well as the derivatives of this strain obtained by mutation or by genetic manipulation, and to a process for the production of recombinant protein, using said strains.
On entend par protéines recombinantes les molé¬ cules polypeptidiques synthétisées par un microorganisme ou une population cellulaire par suite de 1*introduction dans le matériel génétique dudit microorganisme ou des- dites cellules d'un gène codant pour la protéine considé¬ rée. Le gène en question, appelé gène hétérologue, peut provenir d'un autre organisme vivant ou être synthétisé artificiellement. La possibilité d'introduire et de faire exprimer des gènes hétérologues a été tout d'abord mise en évidence et appliquée en utilisant des bactéries, et plus particulièrement Escherichia coli, comme cellules hôtes. Plus récemment, des productions de protéines re¬ combinantes par d'autres microorganismes dont des levures et des champignons ainsi que par des cultures de cellules d'organismes supérieurs ont été décrites.Recombinant proteins are understood to mean the polypeptide molecules synthesized by a microorganism or a cell population as a result of the introduction into the genetic material of said microorganism or of said cells of a gene coding for the protein considered. The gene in question, called the heterologous gene, can come from another living organism or be synthesized artificially. The possibility of introducing and expressing heterologous genes was first demonstrated and applied using bacteria, and more particularly Escherichia coli, as host cells. More recently, productions of re¬ combining proteins by other microorganisms including yeasts and fungi as well as by cell cultures of higher organisms have been described.
Les champignons filamenteux sont utilisés cou.- ramment dans l'industrie des fermentations notamment pour la fabrication de certains antibiotiques (pénicillines, céphalosporines) et d'un grand nombre d'enzymes (gluco- amylases, cellulases, protéases, pectinases —).Filamentous fungi are commonly used in the fermentation industry, particularly for the manufacture of certain antibiotics (penicillins, cephalosporins) and a large number of enzymes (gluco-amylases, cellulases, proteases, pectinases -).
Les champignons filamenteux présentent un cer¬ tain nombre d'avantages par rapport aux microorganismes procaryotes (bactéries) en particulier la capacité d'ex¬ créter de grandes quantités et une grande variété de protéines présentant des modifications post tradition¬ nelles, notamment des glycosylations, spécifiques des eucaryotes. Par rapport aux cellules d'eucaryotes supé¬ rieurs, les champignons sont beaucoup plus faciles à cultiver à grande échelle, la séparation du mycélium et du milieu de culture après la fermentation est aussi très facile. La présente invention se rapporte à un procédé de production de protéine recombinante à l'aide de cham¬ pignons filamenteux, caractérisé en ce qu'on cultive sur un milieu approprié une souche du genre Tolypocladium dans laquelle on a introduit une séquence d'ADN codant pour ladite protéine placée sous le contrôle d'éléments assurant l'expression de ladite séquence dans ladite souche et en ce qu'on récupère ladite protéine.Filamentous fungi have a number of advantages over microorganisms prokaryotes (bacteria) in particular the ability to excrete large quantities and a wide variety of proteins with post-traditional modifications, in particular glycosylations, specific for eukaryotes. Compared to superior eukaryotic cells, fungi are much easier to cultivate on a large scale, the separation of the mycelium and the culture medium after fermentation is also very easy. The present invention relates to a process for the production of recombinant protein using filamentous gables, characterized in that a strain of the genus Tolypocladium is cultivated on an appropriate medium into which a DNA sequence coding has been introduced. for said protein placed under the control of elements ensuring the expression of said sequence in said strain and in that said protein is recovered.
Plus précisément, la souche utilisée est une souche Tolypocladium géodes. Cette souche a été déposée sous le n° 1-880 le 29 juin 1989 à la Collection Natio¬ nale de Culture des Microorganismes - 28, rue du Docteur Roux - 75015 PARIS.More specifically, the strain used is a Tolypocladium geodes strain. This strain was deposited under the number 1-880 on June 29, 1989 at the National Collection of Culture of Microorganisms - 28, rue du Docteur Roux - 75015 PARIS.
L'invention concerne également les mutants défi¬ cients en activité protéasique et/ou hyperexcréteur de protéine exogène de cette souche.The invention also relates to mutants defiant in protease activity and / or hyperexcretor of exogenous protein of this strain.
La présente invention repose notamment sur l'i¬ solement d'une nouvelle souche de champignon filamenteux particulièrement adaptée à la production de protéines recombinantes. La souche sauvage NC14 a été isolée à partir d'un échantillon de terre riche en matières organiques en décomposition (humus d'un sous-bois de la région de Graulhet, Tarn, France) . Elle a été primitivement sélec¬ tionnée pour sa forte activité protéolytique, et s'est avérée à l'analyse produire des quantités notables d'au- tres enzymes extracellulaires, chitinases et -gluca- nases en particulier.The present invention is based in particular on the isolation of a new strain of filamentous fungus which is particularly suitable for the production of recombinant proteins. The wild strain NC14 was isolated from a sample of soil rich in decomposing organic matter (humus from an undergrowth of the Graulhet region, Tarn, France). It was originally selected for its strong proteolytic activity, and has been found to analyze to produce significant quantities of very extracellular enzymes, chitinases and glucanases in particular.
La souche NC14 a été confiée pour identification au laboratoire du Muséum National d'Histoire Naturelle à Paris. Elle a été repiquée en boîtes de Pétri sur deux milieux de culture standard, utilisés pour la détermina¬ tion des hyphomycètes : Malt-agar 2 % et Potato Dextrose Agar (PDA) puis incubée à 25°C.The NC14 strain was entrusted for identification to the laboratory of the National Museum of Natural History in Paris. It was subcultured in Petri dishes on two standard culture media, used for the determination of hyphomycetes: Malt-agar 2% and Potato Dextrose Agar (PDA) and then incubated at 25 ° C.
La culture sur Malt se développe lentement, à peine 1 cm en 3 semaines de culture et forme une colonie serrée d'aspect humide. L'observation microscopique mon¬ tre un mycélium hétérogène, au cytoplasme vacuolisé. Les conidies sont très rares et les cellules sporogènes peu nombreuses et dispersées. Le milieu PDA est plus favorable. La culture a une croissance lente, atteignant 13 à 20 mm en 10 jours . Le thalle est blanc, floconneux en surface, assez dense en profondeur, au contour irrégulier. On observe une lé¬ gère pigmentation beige au revers, aucun pigment ne dif- fuse dans le milieu. Pas d'odeur caractéristique.The culture on Malt develops slowly, barely 1 cm in 3 weeks of culture and forms a tight colony with a wet appearance. Microscopic observation shows a heterogeneous mycelium, with vacuolated cytoplasm. Conidia are very rare and sporogenic cells are few and far between. The PDA environment is more favorable. The culture is slow growing, reaching 13 to 20 mm in 10 days. The thallus is white, flaky on the surface, fairly dense in depth, with an irregular outline. There is a slight beige pigmentation on the reverse, no pigment in the medium. No characteristic odor.
L'observation microscopique montre des cellules sporogènes (phialides) solidaires ou groupées par deux ou trois sur une courte cellule latérale du mycélium. Les phialides mesurent de 9 à 14 μm de long, sont légè- rement renflées à la base puis allongées vers le col qui est quelquefois recourbé. Les spores (conidies) sont groupées en têtes ; de forme très hétérogène (sphériques, cylindriques, ovales) avec des rapports globalement entre 3,6-0,9 μm de long sur 2,9-0,9 μm de large avec deux classes principales : 3 x 1,5 (cylindriques) et 1,5 x 1,8 (globuleuses) . Le mycélium mesure entre 1,5 et 2 μm de large. Pas de chlamydospores.Microscopic observation shows sporogenic cells (phialides) united or grouped by two or three on a short lateral cell of the mycelium. The phialides are 9 to 14 μm long, are slightly swollen at the base and then elongated towards the neck which is sometimes curved. The spores (conidia) are grouped in heads; very heterogeneous in shape (spherical, cylindrical, oval) with overall ratios between 3.6-0.9 μm long by 2.9-0.9 μm wide with two main classes: 3 x 1.5 (cylindrical) and 1.5 x 1.8 (globular). The mycelium is between 1.5 and 2 μm wide. No chlamydospores.
L'aspect cultural, la vitesse de développement et la morphologie de l'appareil sporogène font que cette espèce appartient au genre Tolypocladium (Gams) par la forme, la taille et le groupement des phialides beaucoup plus renflées et groupées en verticilles chez inflatum. Elle diffère aussi de T.c lindrosporum (Gams) par la forme des phialides mais surtout par la forme des spores régulièrement cylindriques chez T.cylindrosporum. La souche NC14 est finalement très proche de l'espèce géodes (Gams) par le mode de groupement des phialides décrit plus haut, par leur forme ainsi que par l'aspect cultural. Malgré l'hétérogénéité des spores, de taille et de forme très hétérogènes, 40 % de celles-ci corres¬ pondent aux caractéristiques de Tolypocladium géodes (Gams) c'est-à-dire 1,6 sur 2,2 μm.The cultural aspect, the speed of development and the morphology of the sporogenic apparatus make this species belongs to the genus Tolypocladium (Gams) by the shape, size and grouping of phialides much more swollen and grouped in whorls in inflatum. It also differs from Tc lindrosporum (Gams) in the form of phialides but especially in the form of regularly cylindrical spores in T. cylindrosporum. The NC14 strain is finally very close to the geodes species (Gams) by the mode of grouping of the phialides described above, by their form as well as by the cultural aspect. Despite the heterogeneity of the spores, which are very heterogeneous in size and shape, 40% of these correspond to the characteristics of Tolypocladium geodes (Gams), that is to say 1.6 by 2.2 μm.
Il s'agit donc d'une souche de Tolypocladium géodes dont la morphologie des spores est quelque peu atypique.It is therefore a strain of Tolypocladium geodes whose spore morphology is somewhat atypical.
La souche NC14 de Tolypocladium géodes a fait l'objet d'un programme d'amélioration génétique, consis¬ tant à sélectionner de façon récurrente des mutants de plus en plus fortement producteurs d'enzymes protéoly- tiques, puis d'enzymes chitinolytiques. Les méthodolo¬ gies de mutagénèse et de sélection utilisées sont celles précédemment décrites pour Trichoderma reesei (DURAND et al.. Enzyme Microb. Technol., 1988, 10 : 341) et pour Pénicillium occitanis (JAIN et al., soumis à publication dans Enzyme Microbiol. Technology) . La première étape de sélection a été effectuée à partir d'une suspension de spores de la souche NC14 traitée par un agent mutagène, la lumière ultraviolette. Une dilution de ladite suspen¬ sion de spores a servi à inoculer des boîtes de Pétri contenant un;milieu gélose de composition suivante :The NC14 strain of Tolypocladium géodes was the subject of a genetic improvement program, consisting in the recurrent selection of mutants which are increasingly strongly producing proteolytic enzymes, then chitinolytic enzymes. The mutagenesis and selection methodologies used are those previously described for Trichoderma reesei (DURAND et al. Enzyme Microb. Technol., 1988, 10: 341) and for Penicillium occitanis (JAIN et al., Submitted for publication in Enzyme Microbiol. Technology). The first selection step was carried out using a spore suspension of the NC14 strain treated with a mutagenic agent, ultraviolet light. A dilution of said suspension of spores was used to inoculate Petri dishes containing an agar medium of the following composition:
- poudre de lait écrémé 10 g- skim milk powder 10 g
- phosphate monopotassique 2 g- monopotassium phosphate 2 g
- sulfate de magnésium 0,3 g- magnesium sulfate 0.3 g
- chlorure de calcium 0,2 g - sulfate de fer 0,005 g - extrait de levure : 1 g- calcium chloride 0.2 g - iron sulfate 0.005 g - yeast extract: 1 g
- agar agar : 15- agar agar: 15
- eau désionisée qsp : 1 litre pH ajusté à 7,0 avec de la soude Après incubation 5 jours à 27°C, les colonies apparues sur ce milieu sont entourées d'un halo clair, dû à l'hydrolyse des protéines du lait par les enzymes protéolytiques excrétées par la souche. Ce crible de sé¬ lection, qui avait déjà été utilisé pour l'isolement de la souche NC14, a permis la recherche de mutants meilleurs producteurs de proteases; ceux-ci sont détectés par l'ap¬ parition d'un halo d'hydrolyse d'un diamètre nettement supérieur à la moyenne de ceux observés autour des colo¬ nies de la souche de départ. Un mutant, appelé NC18, ca- ractérisé par un halo d'hydrolyse très significativement augmenté, a été isolé. A partir de la souche NC18, une deuxième étape de sélection a été effectuée, après muta- génèse par la N-méthyl-N'-nitro-N-nitrosoguanidine (NTG) sur le milieu mentionné ci-dessus auquel a été rajouté de l'hydrolysat acide de caséine (Biokar) à la concentra¬ tion de 5 g/1. Dans ces conditions, la production de pro¬ teases par la souche NC18 est réprimée, ce qui se tra¬ duit par une diminution très nette de la taille des ha¬ los d'hydrolyse. Une colonie entourée d'un large halo a été sélectionnée; le mutant ainsi isolé, NC21 a fait l'objet d'une nouvelle étape de sélection après mutagé- nèse par 1' thyl-méthane sulfonate (EMS) sur le milieu à base de poudre de lait, auquel du sulfate d'ammonium à la concentration de 5 g/1 a été ajouté comme répresseur de la production de proteases. Un mutant dé-réprimé, NC28, a ainsi été isolé. A partir de cette dernière souche, un mutant hyperproducteur d'activité chitinoly- tique a été recherché, après mutagénèse par l'acide ni- treux, sur le milieu de base précédemment décrit, où la poudre de lait a été remplacée par de la chitine colloï- dale à la concentration de 10 g/1. Un mutant hyperchiti- nolytique, NC35, sélectionné sur la base de la taille du halo d'hydrolyse de la chitine, a été soumis à une nou¬ velle étape de sélection sur le même milieu à base de chitine, contenant en plus 10 g/1 de glucose comme ré¬ presseur. Une souche mutante isolée sur ce milieu, NC39 a été retenue. Cette souche s'est révélée hyper- excrétrice de protéines : des concentrations de l'ordre de 10 g/1 de protéines ont été obtenues en 6 jours de fermentation, dans un milieu chimiquement défini (milieu salin de base selon MANDELS et WEBER, J. Adv. Chem. Ser., 1969, 95 : 391) contenant du glucose (ajouté en continu à raison de 20 g/1 par jour) comme seule source de car¬ bone. Dans les mêmes conditions, la souche sauvage NC14 produit environ 0,5 g/1 de protéines extracellulaires.- deionized water qs: 1 liter pH adjusted to 7.0 with sodium hydroxide After incubation for 5 days at 27 ° C., the colonies which appear on this medium are surrounded by a clear halo, due to the hydrolysis of milk proteins by proteolytic enzymes excreted by the strain. This selection screen, which had already been used for the isolation of the NC14 strain, enabled the search for mutants which are better producers of proteases; these are detected by the appearance of a hydrolysis halo with a diameter much greater than the average of those observed around the colonies of the starting strain. A mutant, called NC18, characterized by a very significantly increased hydrolysis halo, was isolated. From the NC18 strain, a second selection step was carried out, after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) on the medium mentioned above to which was added l 'casein acid hydrolyzate (Biokar) at the concentration of 5 g / 1. Under these conditions, the production of proteases by the NC18 strain is suppressed, which results in a very marked reduction in the size of the hydrolysis ha¬ los. A colony surrounded by a large halo was selected; the mutant thus isolated, NC21 was the subject of a new selection step after mutagenesis with 1 thyl-methane sulfonate (EMS) on the medium based on milk powder, to which ammonium sulfate at the concentration of 5 g / l was added as a suppressant for the production of proteases. A depressed suppressed mutant, NC28, was thus isolated. From this latter strain, a mutant hyperproducer of chitinolytic activity was sought, after mutagenesis by nitrous acid, on the basic medium previously described, where the milk powder was replaced by chitin colloid- at the concentration of 10 g / 1. A hyperchitinolytic mutant, NC35, selected on the basis of the size of the hydrolysis halo of chitin, was subjected to a new selection step on the same medium based on chitin, containing in addition 10 g / 1 of glucose as a presser. A mutant strain isolated on this medium, NC39 was retained. This strain has been shown to be hyper-excretory of proteins: concentrations of the order of 10 g / 1 of proteins were obtained in 6 days of fermentation, in a chemically defined medium (basic saline medium according to MANDELS and WEBER, J Adv. Chem. Ser., 1969, 95: 391) containing glucose (continuously added at a rate of 20 g / l per day) as the only source of carbon. Under the same conditions, the wild strain NC14 produces approximately 0.5 g / l of extracellular proteins.
Cette aptitude exceptionnelle de la souche NC39 à excréter des protéines dans des conditions et un milieu de culture extrêmement simples, qui constitue un des as¬ pects de la présente invention, a conduit à envisager de l'utiliser pour la production de protéines hétérologues. Cependant, dans cette optique, la forte activité protéo- lytique des souches constitue un inconvénient. Aussi à partir de la souche NC39, des mutants déficients en pro¬ teases ont été recherchés. Après mutagénèse à la NTG, puis à l'EMS, des colonies ne produisant pas de halo d'hydrolyse sur milieu à base de poudre de lait addition¬ né de glucose (2 g/1) et de sulfate d'ammonium (0,5 g/1) ont été sélectionnées en deux étapes. La souche NC46 ain¬ si isolée se caractérise par l'absente d'activité protéo- lytique détectable par le test à l'azocaséine (TOMARELLI et al., J. Lab. Clin. Med., 1949, 34 : 428) dans le sur¬ nageant de culture, dans les conditions de production de protéines extracellulaires mentionnées plus haut et dé¬ crites dans l'exemple II ci-après. Cette souche NC46 a été soumise à un nouveau traitement mutagène par l'acide nitreux, puis des mutants déficients en activité amino peptidase ont été recher~chés selon une modification de la technique décrite par MILLER et MACKINNON (J.Bact., 1974, 120 : 355) : après croissance 4 jours sur le milieu à base de poudre de lait décrit ci-dessus, les colonies sont recouvertes d'une solution contenant : 0,5 mg/ml de L. Leucine- β -naphtylamide et 2 mg/ml de Fast Garnet GBC (Sigma) . La présence d'activité aminopeptidase est visualisée par l'apparition, après quelques minutes, d'une coloration brune autour des colonies. Les colonies ne produisant pas de coloration sont récupérées et re¬ testées, selon le même protocole.This exceptional ability of the NC39 strain to excrete proteins under extremely simple conditions and culture medium, which constitutes one of the aspects of the present invention, has led to consideration of using it for the production of heterologous proteins. However, from this perspective, the high proteolytic activity of the strains constitutes a drawback. Also from the strain NC39, mutants deficient in proteases have been sought. After mutagenesis at NTG, then at EMS, colonies which do not produce a hydrolysis halo on a medium based on powdered milk with added glucose (2 g / 1) and ammonium sulfate (0, 5 g / 1) were selected in two stages. The NC46 strain thus isolated is characterized by the absence of proteolytic activity detectable by the azocasein test (TOMARELLI et al., J. Lab. Clin. Med., 1949, 34: 428) in the culture supernatant, under the conditions of production of extracellular proteins mentioned above and described in Example II below. This NC46 strain was subjected to a new mutagenic acid treatment nitrous, then mutants deficient in amino peptidase activity were sought ~ chés according to a modification of the technique described by MILLER and MACKINNON (J. Bact., 1974, 120: 355): after growth 4 days on the medium based on powder of milk described above, the colonies are covered with a solution containing: 0.5 mg / ml of L. Leucine- β-naphthylamide and 2 mg / ml of Fast Garnet GBC (Sigma). The presence of aminopeptidase activity is visualized by the appearance, after a few minutes, of a brown coloration around the colonies. Colonies which do not produce coloration are recovered and re¬ tested, according to the same protocol.
Un mutant isolé par cette technique, NC50, ne présente plus l'activité Leucine aminopeptidase détectée dans le surnageant des cultures des souches NC46 et des générations précédentes.A mutant isolated by this technique, NC50, no longer exhibits the Leucine aminopeptidase activity detected in the supernatant of cultures of strains NC46 and of the preceding generations.
La généalogie de la souche NC50 est résumée sur le tableau 1.The genealogy of the NC50 strain is summarized in Table 1.
La souche mutante NC50 possède les mêmes capa- cités d'excrétion de protéines du point de vue quantita¬ tif, que la souche hyperexcrétrice NC39 mentionnée ci- dessus. Un avantage supplémentaire de la souche NC50 ré¬ side dans l'absence d'activité protéolytique détectable dans les surnageants de culture, ce qui permet d'éviter ou tout au moins de minimiser la dégradation des pro¬ téines .produites, et particulièrement des protéines hété- rologues, dans le milieu de fermentation. Ceci constitue un aspect important de la présente invention.The mutant strain NC50 has the same protein excretion capacities from the quantitative point of view as the hyperexcretory strain NC39 mentioned above. An additional advantage of the NC50 strain resides in the absence of detectable proteolytic activity in the culture supernatants, which makes it possible to avoid or at least minimize the degradation of the proteins produced, and particularly of the proteins. heterologists, in the fermentation medium. This is an important aspect of the present invention.
Un autre aspect est constitué par la mise en évidence de la capacité des souches NC14 et dérivées à être transformées selon un protocole simplifié, original par rapport à ceux mentionnés . Tableau 1 : Généalogie des souches mutantes dérivées de Tolypocladium géodes NC 4Another aspect is constituted by the demonstration of the capacity of the NC14 and derived strains to be transformed according to a simplified protocol, original compared to those mentioned. Table 1: Genealogy of mutant strains derived from Tolypocladium geodes NC 4
Figure imgf000010_0001
antérieurement pour d'autres champignons comme Aspergil- lus nidulans (BALLANCE et al., Biochem. Bioph s. Res. Commun, 1983, 112 : 284) ou Trichoderma reesei (KNO LES et al.. Brevet Européen n° EP 0244234 A2, 1987). L'ori¬ ginalité du protocole réside en particulier dans le fait qu'il ne fait pas appel à l'utilisation de protoplastes, dont la formation et la régénération constituent souvent une étape fastidieuse dans la mise au point d'un protocole de transformation. Dans le cas de la souche NC14 et de ses dérivés, il a été trouvé que de l'ADN étranger pou¬ vait être introduit, avec une fréquence élevée, dans des spores traitées de façon ménagée avec une enzyme lytique de telle sorte que leur résistance à la pression osmotique et leur capacité de germination ne soient pas altérées. La mise au point du protocole de transformation a été effectuée en utilisant un ADN plasmidique portant un marqueur dominant sélectionable : un gène conférant la résistance à la phléomycine (ARMAU et al., Brevet
Figure imgf000010_0001
previously for other fungi such as Aspergillus nidulans (BALLANCE et al., Biochem. Bioph s. Res. Commun, 1983, 112: 284) or Trichoderma reesei (KNO LES et al .. European patent n ° EP 0244234 A2, 1987). The originality of the protocol lies in particular in the fact that it does not call for the use of protoplasts, the formation and regeneration of which often constitute a tedious step in the development of a protocol of transformation. In the case of the NC14 strain and its derivatives, it has been found that foreign DNA could be introduced, with a high frequency, into spores treated in a gentle manner with a lytic enzyme so that their resistance at osmotic pressure and their ability to germinate is not impaired. The transformation protocol was developed using a plasmid DNA carrying a selectable dominant marker: a gene conferring resistance to phleomycin (ARMAU et al., Patent
Français, n° 2 569 723. Ce protocole est décrit dans l'e¬ xemple I ci-après.French, no. 2,569,723. This protocol is described in Example I below.
Selon la présente invention, les éléments assu¬ rant l'expression de ladite séquence d'ADN dans la souche comprennent essentiellement une séquence promotrice d'un gène d'origine fongique . Cette séquence d'ADN codant pour ladite protéine sera précédée d'une séquence signal assu¬ rant l'excrétion de la protéine et suivie d'une séquence terminateur de transcription. Un aspect supplémentaire de la présente inven¬ tion est constituée par la possibilité de sélectionner des souches transformées pour lesquelles l'expression du gène d'intérêt est amplifié, grâce à un crible de sélec¬ tion basé sur l'utilisation d'un gène constituant un marqueur dominant, placé en aval du gène d'intérêt, sans promoteur ni signal d'arrêt de transcription entre les deux gènes. Cette technique est illustrée dans l'exemple V ci-après.According to the present invention, the elements ensuring the expression of said DNA sequence in the strain essentially comprise a promoter sequence of a gene of fungal origin. This DNA sequence coding for said protein will be preceded by a signal sequence ensuring excretion of the protein and followed by a transcription terminator sequence. An additional aspect of the present invention consists of the possibility of selecting transformed strains for which the expression of the gene of interest is amplified, thanks to a selection screen based on the use of a constituent gene. a dominant marker, placed downstream of the gene of interest, without promoter or signal for stopping transcription between the two genes. This technique is illustrated in Example V below.
Ce gène constituant le marqueur code pour la ré- sistance à un antibiotique qui est de préférence un com¬ posé de la famille des phléomycines.This gene constituting the marker codes for resistance to an antibiotic which is preferably a compound of the phleomycin family.
Dans le cas d'un gène codant pour la résistance à un antibiotique de la famille des phléomycines, ce gène est d'origine génomique et provient du génome des actino- mycètes producteur dudit antibiotique. La séquence d'ADN codant pour ladite protéine ainsi que les éléments assurant l'expression de ladite séquence seront portés par un plasmide qui de préférence est un plasmide à réplication autonome comportant une séquence de réplication autonome efficace chez Tolypo¬ cladium T.A.R.S.In the case of a gene coding for resistance to an antibiotic of the phleomycin family, this gene is of genomic origin and comes from the genome of actino-mycetes producing said antibiotic. The DNA sequence coding for said protein as well as the elements ensuring the expression of said sequence will be carried by a plasmid which preferably is an autonomous replication plasmid comprising an autonomous replication sequence effective in Tolypo¬ cladium TARS
Selon l'invention, ledit plasmide assure l'in¬ tégration chromosomique des séquences d'ADN en cause. En résumé, la présente invention concerne une nouvelle souche de champignon filamenteux, Tolypocladium géodes NC14, et les mutants issus de cette souche, sélec¬ tionnés pour leur capacité à excréter de grandes quanti¬ tés de protéines dans un milieu de culture extrêmement simple, constitué essentiellement de sucres et de sels minéraux, ce qui facilite l'extraction et la purification desdites protéines. Cette capacité exceptionnelle peut être exploitée pour la production de protéines recombi¬ nantes ce qui constitue un premier aspect de la présente invention. Selon un deuxième aspect, la présente invention fournit un moyen de préserver la stabilité des protéines excrétées dans le milieu de culture par l'utilisation de mutants déficients en activités protéolytiques.According to the invention, said plasmid ensures the chromosomal integration of the DNA sequences in question. In summary, the present invention relates to a new strain of filamentous fungus, Tolypocladium géodes NC14, and the mutants resulting from this strain, selected for their capacity to excrete large quantities of proteins in an extremely simple culture medium, consisting essentially of sugars and mineral salts, which facilitates the extraction and purification of said proteins. This exceptional capacity can be exploited for the production of recombinant proteins, which constitutes a first aspect of the present invention. According to a second aspect, the present invention provides a means of preserving the stability of the proteins excreted in the culture medium by the use of mutants deficient in proteolytic activities.
Selon un troisième aspect, la présente invention fournit un moyen d'introduire des gènes hétérologues dans la souche de Tolypocladium géodes NC14 et ses dérivés se¬ lon un protocole simplifié ne faisant pas intervenir les protoplastes.According to a third aspect, the present invention provides a means of introducing heterologous genes into the strain of Tolypocladium geodes NC14 and its derivatives according to a simplified protocol which does not involve protoplasts.
Selon un quatrième aspect de la présente inven- tion, l'expression des gènes hétérologues par Tolypocla¬ dium géodes NC14 et ses dérivés peut être augmentée par une technique de sélection faisant appel à un marqueur dominant placé en aval du gène d'intérêt sans promoteur ni terminateur entre les deux gènes. La présente invention se rapporte également aux protéines obtenues par la mise en oeuvre du procédé se¬ lon l'invention. Parmi ces protéines codées selon l'in¬ vention, on citera plus particulièrement la pullulanase, le lysozyme humain et la protéine Sh, la séquence nucléo- tidique de cette protéine Sh étant représentée en figure 2.According to a fourth aspect of the present invention, the expression of heterologous genes by Tolypocla¬ dium geodes NC14 and its derivatives can be increased by a selection technique using a dominant marker placed downstream of the gene of interest without promoter nor terminator between the two genes. The present invention also relates to proteins obtained by implementing the method according to the invention. Among these proteins coded according to the invention, mention will be made more particularly of pullulanase, human lysozyme and the Sh protein, the nucleotide sequence of this Sh protein being represented in FIG. 2.
La présente invention concerne de même les sou¬ ches de Tolypocladium transformées, obtenues lors du pro¬ cédé de production de protéines recombinantes. Les exemples suivants servent à illustrer la- présente invention de manière non limitative.The present invention likewise relates to the transformed strains of Tolypocladium, obtained during the process for the production of recombinant proteins. The following examples serve to illustrate the present invention without limitation.
Ces exemples seront décrits en se référant aux figures sur lesquelles :These examples will be described with reference to the figures in which:
- la figure 1 est un schéma du plasmide pUT720 - la figure 2 représente la séquence nucléotidique du gène de la protéine Sh précédé de la séquence synthé¬ tique utilisée comme signal d'excrétion- Figure 1 is a diagram of the plasmid pUT720 - Figure 2 shows the nucleotide sequence of the Sh protein gene preceded by the synthetic sequence used as an excretion signal
- la figure 3 est un schéma du plasmide pUT715- Figure 3 is a diagram of the plasmid pUT715
- la figure 4 est un schéma du plasmide pUT771 - la figure 5 représente la séquence nucléotidique de la partie promotrice du plasmide pUT771- Figure 4 is a diagram of the plasmid pUT771 - Figure 5 represents the nucleotide sequence of the promoter part of the plasmid pUT771
- la figure 6 est un schéma du plasmide pUT760- Figure 6 is a diagram of the plasmid pUT760
- la figure 7 représente la séquence nucléotidique syn¬ thétique codant pour le lysozyme humain - la figure 8 est un schéma du plasmide pUT772- Figure 7 shows the synthetic nucleotide sequence encoding the human lysozyme - Figure 8 is a diagram of the plasmid pUT772
Les abréviations utilisées sur ces figures sont considérées comme connues ou seront explicitées dans la description.The abbreviations used in these figures are considered to be known or will be explained in the description.
Sauf indication contraire, les différents pror cédés et produits mentionnés sont mis en oeuvre selon les techniques connues et/ou préconisées par le fabricant, Exemple I : : Transformation de Tolypocladium géodesUnless otherwise indicated, the various pror ceded and products mentioned are used according to techniques known and / or recommended by the manufacturer, Example I:: Transformation of Tolypocladium geodes
La souche mutante NC50, déficiente en activités protéolytiques, a été transformée par le plasmide pUT703 (DURAND et al, Proc. Biochemistry and Genetics of Cellulose Dégradation, JP Aubert Ed., Académie Press, 1988, p 136). Ce plasmide se caractérise par la présence d'un gène de résistance aux antibiotiques de la famille des phléomycines (ARMAU et al., Brevet Français n° 2 569 723 placé sous la dépendance d'un promoteur fongi¬ que : le promoteur du gène de la glyce'raldéhyde 3-phos- phate deshydrogénase d'Aspergillus nidulans (noté promo¬ teur gpd) (Van GORCOM et al., Gène, 1986, 48 : 211) et borné en aval par le terminateur CYC1 de levure (SMITH et al., Cell, 1979, 16 : 753). Le gène de résistance provient d'un actinomycète producteur d'antibiotiques de la famille des phléomycines; Streptoalloteichus hindus- tanus. La protéine codée par ce gène, appelée protéine Sh, inactive les antibiotiques en question par formation d'un complexe équimoléculaire (GATIGNOL et al., FEBS Letters, 1988, 230 : 171).The mutant strain NC50, deficient in proteolytic activities, was transformed by the plasmid pUT703 (DURAND et al, Proc. Biochemistry and Genetics of Cellulose Dégradation, JP Aubert Ed., Académie Press, 1988, p 136). This plasmid is characterized by the presence of a gene for resistance to antibiotics of the phleomycin family (ARMAU et al., French Patent No. 2,569,723 placed under the dependence of a fungal promoter: the promoter of the gene 3-phosphide glyce'raldehyde dehydrogenase from Aspergillus nidulans (noted promoter gpd) (Van GORCOM et al., Gene, 1986, 48: 211) and bounded downstream by the yeast terminator CYC1 (SMITH et al ., Cell, 1979, 16: 753) The resistance gene comes from an actinomycete producing antibiotics from the phleomycin family; Streptoalloteichus hindus-tanus. The protein encoded by this gene, called Sh protein, inactivates antibiotics by question by formation of an equimolecular complex (GATIGNOL et al., FEBS Letters, 1988, 230: 171).
La phléomycine et les antibiotiques apparentés présentent une toxicité élevée pour Tolypocladium géodes comme pour pratiquement tous les organismes vivants. Le gène de résistance fournit donc un moyen facile de sé¬ lectionner les clones transformés par leur capacité à se développer en présence de phléomycine.Phleomycin and related antibiotics have a high toxicity for Tolypocladium geodes as for practically all living organisms. The resistance gene therefore provides an easy means of selecting the clones transformed by their capacity to develop in the presence of phleomycin.
Le protocole utilisé pour la transformation est le suivant : des spores obtenues par culture de la souche NC50 sur milieu PDA pendant 7 jours à 27°C sont suspendues dans du milieu MnP de composition suivante : milieu salin de base selon Mandels additionné de saccharose 100 g/1, tampon MES 5 g/1, pH 5,5. Après filtration sur verre fritte n° 2 pour éliminer le mycélium, la suspension est centrifugée (10 minutes, 10 000 rpm) et le culot est re¬ pris par le même milieu MnP additionné de Caylase C3 (enzyme lytique commercialisée par CAYLA) à 10 mg/ml. Après 4 heures d'incubation sur table agitée à 32°C, 100 rpm la suspension est à nouveau centrifugée, et le culot est repris par du milieu MnP additionné de chlorure de calcium, 50 mM, dans un volume tel que le titre de la suspension de spores obtenue, déterminé par comptage mi-The protocol used for the transformation is as follows: spores obtained by culture of the strain NC50 on PDA medium for 7 days at 27 ° C. are suspended in MnP medium of the following composition: basic saline medium according to Mandels added with sucrose 100 g / 1, MES buffer 5 g / 1, pH 5.5. After filtration on sintered glass No. 2 to remove the mycelium, the suspension is centrifuged (10 minutes, 10,000 rpm) and the residue is taken up in the same MnP medium supplemented with Caylase C3 (lytic enzyme marketed by CAYLA) at 10 mg / ml. After 4 hours of incubation on a table stirred at 32 ° C., 100 rpm, the suspension is again centrifuged, and the pellet is taken up in MnP medium supplemented with chloride. calcium, 50 mM, in a volume such as the titer of the suspension of spores obtained, determined by mid-count
Q croscopique à 1'hématimètre, soit d'environ 10 /ml. A 200 μl de cette suspension, une solution d'ADN plasmidique (5 à 10 μg d'ADN dans un volume de 5 à 20 μl) est ajoutée. Après 10 minutes à température am¬ biante, on ajoute 50 μl de solution MPC (MOPS 10 mM pH 5,8, PEG 6000 60 % p.v., CaCl2 75 mM) et on incube le mélange 30 minutes dans la glace, puis on rajoute 2,5 ml de MPC. Après 15 minutes à température ambiante, des aliquots de 0,5 ml de ce mélange sont introduits dans des tubes contenant chacun 3 ml de gélose molle MnR (milieu salin selon Mandels additionné de saccharose 150 g/1, glucose 2,5 g/1, extrait de levure 2,5 g/1, agar 6 g/1) maintenu en surfusion à 45°C. Le contenu de cha¬ que tube est ensuite versé à la surface d'une boîte de Pétri contenant le même milieu MnR, gélose à 15 g/1 et additionné de 30 μg/ml de phléomycine (commercialisée par CAYLA) comme agent de sélection des clones transfor- mants. Ceux-ci apparaissent sous la forme de petites colonies après 4 jours d'incubation à 27°C.Q croscopic to the hematimeter, that is to say about 10 / ml. At 200 μl of this suspension, a solution of plasmid DNA (5 to 10 μg of DNA in a volume of 5 to 20 μl) is added. After 10 minutes at room temperature, 50 μl of MPC solution is added (MOPS 10 mM pH 5.8, PEG 6000 60% pv, CaCl 2 75 mM) and the mixture is incubated for 30 minutes in ice, then added 2.5 ml of MPC. After 15 minutes at room temperature, aliquots of 0.5 ml of this mixture are introduced into tubes each containing 3 ml of MnR soft agar (saline medium according to Mandels added with sucrose 150 g / 1, glucose 2.5 g / 1 , yeast extract 2.5 g / 1, agar 6 g / 1) maintained in supercooling at 45 ° C. The content of each tube is then poured onto the surface of a Petri dish containing the same MnR medium, 15 g / l agar and supplemented with 30 μg / ml of phleomycin (marketed by CAYLA) as agent for the selection of transforming clones. These appear as small colonies after 4 days of incubation at 27 ° C.
Les taux de transformation obtenus en utilisant le plasmide pUT703 ont été de l'ordre de 2000 à 5000 transformants par microgramme d'ADN. La stabilité des transformants a été testée par repiquage des colonies sur milieu PDA contenant 30 μg/ml de phléomycine (la concentration minimale inhibitrice - CMI - pour la souche NC50 non transformée est d'environ 10 μg/ml dans ces conditions). Environ 90 % des colonies repiquées ont confirmé leur caractère de résistance.The transformation rates obtained using the plasmid pUT703 were of the order of 2000 to 5000 transformants per microgram of DNA. The stability of the transformants was tested by subculturing the colonies on PDA medium containing 30 μg / ml of phleomycin (the minimum inhibitory concentration - MIC - for the non-transformed NC50 strain is approximately 10 μg / ml under these conditions). About 90% of the transplanted colonies confirmed their resistance character.
Des expériences d'hybridation de l'ADN des sou¬ ches transformées par une sonde marquée correspondant à une partie du gène de résistance à la phléomycine ont montré que ce gène était intégré dans le chromosome des transformants; le ou les sites d'intégration et le nombre de copies varient d'une souche à l'autre. Exemple II : Expression et excrétion d'une protéine hétérologue. Dans l'exemple I ci-dessus, l'obtention de clones transformants résistants à la phléomycine indique que le gène codant pour la protéine Sh s'exprime dans la souche MC50 de Tolypocladium géodes. Cependant, la pro¬ téine n'a pas pu être mise en évidence dans les surna¬ geants de culture des souches transformées et n'est donc vraisemblablement pas excrétée en quantité détectable. Dans l'exemple suivant, la souche NC50 a été transformée selon le protocole décrit dans l'exemple I, en utilisant le plasmide pUT720 dont la carte est re¬ présentée sur la figure 1. Ce plasmide se caractérise par la présence, sous la dépendance du promoteur gpd, du gène de la protéine Sh en amont duquel a été ajoutée une séquence signal synthétique d'excrétion (figure 2) .Hybridization experiments on the DNA of strains transformed by a labeled probe corresponding to a part of the phleomycin resistance gene have shown that this gene is integrated into the chromosome of the transformants; the integration site (s) and the number copies vary from strain to strain. Example II: Expression and excretion of a heterologous protein. In Example I above, obtaining transforming clones resistant to phleomycin indicates that the gene coding for the Sh protein is expressed in the strain MC50 of Tolypocladium geodes. However, the protein could not be demonstrated in the culture supernatants of the transformed strains and is therefore probably not excreted in detectable quantities. In the following example, the strain NC50 was transformed according to the protocol described in Example I, using the plasmid pUT720, the map of which is shown in FIG. 1. This plasmid is characterized by the presence, under dependence of the gpd promoter, of the Sh protein gene upstream of which a synthetic excretion signal sequence has been added (FIG. 2).
Parmi les colonies résistantes, obtenues avec une fréquence de 3000 transformants par microgramme d' ADN, une centaine ont été repiquées sur des boîtes de PDA contenant des concentrations croissantes de phléo¬ mycine, de 25 à 1000 μg/ml. 3 souches résistantes à plus de 1000 μg/ml de phléomycine ont ainsi été sélec¬ tionnées; l'une d'elles a été cultivée en fermenteur de laboratoire dans les conditions suivantes : les spores provenant d'une boîte de PDA incubée 7 jours à 27°C ont servi à inoculer 500 ml de milieu selon Mandels conte¬ nant 20 g/1 de glucose et 1 g/1 d'extrait de levure, ré¬ parti dans deux erlenmeyers de 1 1. Après incubation 48 heures sur table agitée à 27°C, 200 rpm, cette cul¬ ture a permis d'inoculer un fermenteur de 14 litres de volume total (Chemap) contenant 8 litres du même milieu. La fermentation a duré 6 jours à 27°C avec une agitation de 500 rpm et une aération de 0,5 v.v.m., le pH étant régulé supérieur à 5,0 avec de l'ammoniaque. A partir du 2ème jour, une solution stérile de glucose à 200 g/1 a été introduite en continu à raison de 0,8 litre par 24 heures, soit environ 20 grammes de glucose par litre et par jour. Après 6 jours, la culture a été récoltée et centrifugée.Among the resistant colonies, obtained with a frequency of 3000 transformants per microgram of DNA, a hundred were subcultured on PDA dishes containing increasing concentrations of phleomycin, from 25 to 1000 μg / ml. 3 strains resistant to more than 1000 μg / ml of phleomycin were thus selected; one of them was cultivated in a laboratory fermenter under the following conditions: the spores from a PDA box incubated for 7 days at 27 ° C. were used to inoculate 500 ml of medium according to Mandels containing 20 g / 1 of glucose and 1 g / 1 of yeast extract, distributed in two 1 1 Erlenmeyer flasks. After incubation for 48 hours on a table stirred at 27 ° C., 200 rpm, this culture made it possible to inoculate a fermenter. of 14 liters of total volume (Chemap) containing 8 liters of the same medium. The fermentation lasted 6 days at 27 ° C with stirring of 500 rpm and aeration of 0.5 vvm, the pH being regulated above 5.0 with ammonia. From the 2nd day, a sterile glucose solution at 200 g / 1 was introduced continuously at the rate of 0.8 liters per 24 hours, or approximately 20 grams of glucose per liter per day. After 6 days, the culture was harvested and centrifuged.
La concentration totale en protéines, détermi¬ née selon la méthode de LO RY (LO RY et al., J. Biol. Chem., 1951, 193 : 265) a été trouvée égale à 9,6 g/1. La présence de protéine Sh dans le milieu de culture a été mise en évidence par chromatographieThe total protein concentration, determined according to the LO RY method (LO RY et al., J. Biol. Chem., 1951, 193: 265) was found to be 9.6 g / l. The presence of Sh protein in the culture medium was demonstrated by chromatography
(FPLC) sur colonne échangeuse d'anions MonoQ, Pharmacia (tampon Tris-HCl 20 mM pH 7,0 - élution gradient liné¬ aire 0 - 0,6 M NaCl) . En comparaison avec le surnageant d'une culture effectuée dans les mêmes conditions avec la souche non transformée, on constate l'apparition d'un pic supplémentaire de protéines fortement retenues sur la colonne MonoQ, élue à 0,45 M. L'aire de ce pic repré¬ sente environ 20 % de l'aire totale du chromatogramme. L'analyse en électrophorèse SDS-PAGE, des fractions cor- respondant à ce pic a révélé la présence à l'état prati¬ quement pur d'une protéine d'environ 14 000 kd de poids moléculaire, ce qui correspond à la taille de la protéine Sh (GATIGNOL et al., FEBS Letters, 1988, 230 : 171). La présence dans le surnageant de culture de protéine inactivant la phléomycine a été confirmée par le test suivant : sur des disques de papier imprégnés de solution de phléomycine à différentes concentrations (de 10 à 300 μg/ml) différents volumes de surnageant de cul¬ ture (de 1 à 50 μl) sont déposés. Les disques sont en- suite posés à la surface d'un milieu gélose (milieu n° 2 pour l'essai des antibiotiques, BioMérieux) ensemencé par une souche d'Escherichia coli sensible à la phléomy¬ cine : HB101. Après une nuit d'incubation à 37°C, des halos d'inhibition du voile bactérien sont visibles au- tour des disques contenant la phléomycine seule. Lorsque des quantités connues de protéine Sh purifiée sont ajoutées, on constate une diminution du diamètre des halos, et une disparition totale de ceux-ci pour un rapport en moles protéines/antibiotiques de 1/1 soit environ 10/1 en poids. Ce test permet donc un dosage semi-quantitatif de .la protéine Sh dans une solution de concentration inconnue. Dans le présent exemple, la con¬ centration de protéine Sh dans le surnageant de culture a été trouvée voisine de 2 g/1. II a donc été possible par fermentation d'un transformant de la souche de Tolypocladium géodes NC50 par le plasmide pUT720 de produire environ 2 g/1 de pro¬ téine recombinante extracellulaire en 6 jours. Exemple III i Isolement d'une séquence promotrice Le plasmide pUT715, dont la carte est présentée sur la figure 3 a été utilisé comme vecteur-sonde pour la recherche de séquences promotrices fongiques. Le plas¬ mide a été linéarisé par double coupure BamHI-AsuII (les deux sites uniques de restriction sont situés dans la séquence multisites située immédiatement en amont de la partie codante du gène Sh) . Après purification par élec- troélution, le grand fragment a été mis en présence d'ADN chromosomique de Trichoderma reesei partiellement digérée par les enzymes Mbol et Taql pour générer des fragments compris entre 1 et 5 kilobases et d'ADN ligase. La population de plasmides hybrides ainsi obtenue a ser¬ vi à transformer la souche d'Escherichia coli DH5 ov, avec sélection sur la base de la résistance à l'ampicil- line. L'ADN plasmidique extrait du "pool" des transfor- mants d'E.coli a servi à transformer la souche NC14 de(FPLC) on an anion exchange column MonoQ, Pharmacia (Tris-HCl buffer 20 mM pH 7.0 - linear gradient elution 0 - 0.6 M NaCl). In comparison with the supernatant of a culture carried out under the same conditions with the untransformed strain, there is the appearance of an additional peak of proteins strongly retained on the MonoQ column, eluted at 0.45 M. The area of this peak represents approximately 20% of the total area of the chromatogram. The SDS-PAGE electrophoresis analysis of the fractions corresponding to this peak revealed the presence in the practically pure state of a protein of approximately 14,000 kd of molecular weight, which corresponds to the size of protein Sh (GATIGNOL et al., FEBS Letters, 1988, 230: 171). The presence in the culture supernatant of protein inactivating phleomycin was confirmed by the following test: on paper discs impregnated with phleomycin solution at different concentrations (from 10 to 300 μg / ml) different volumes of culture supernatant (from 1 to 50 μl) are deposited. The discs are then placed on the surface of an agar medium (medium No. 2 for testing antibiotics, BioMérieux) seeded with a strain of Escherichia coli sensitive to phleomy¬ cine: HB101. After an overnight incubation at 37 ° C., halos of inhibition of the bacterial veil are visible around the discs containing phleomycin alone. When known quantities of purified Sh protein are added, there is a decrease in the diameter of the halos, and a total disappearance of these for a mole protein / antibiotic ratio of 1/1 or about 10/1 by weight. This test therefore allows a semi-quantitative assay of the Sh protein in a solution of unknown concentration. In the present example, the concentration of Sh protein in the culture supernatant was found to be close to 2 g / l. It was therefore possible by fermentation of a transformant of the Tolypocladium geodes NC50 strain with the plasmid pUT720 to produce approximately 2 g / 1 of extracellular recombinant protein in 6 days. Example III i Isolation of a promoter sequence The plasmid pUT715, the map of which is presented in FIG. 3, was used as vector-probe for the search for fungal promoter sequences. The plasmid has been linearized by double BamHI-AsuII cleavage (the two unique restriction sites are located in the multisite sequence located immediately upstream of the coding part of the Sh gene). After purification by electroelution, the large fragment was placed in the presence of chromosomal DNA from Trichoderma reesei partially digested with the enzymes Mbol and Taql to generate fragments of between 1 and 5 kilobases and DNA ligase. The hybrid plasmid population thus obtained was used to transform the Escherichia coli DH5 ov strain, with selection on the basis of resistance to ampicillin. The plasmid DNA extracted from the "pool" of E. coli transformants was used to transform the NC14 strain of
T.géodes selon le protocole décrit dans l'exemple I ci- dessus. 50 transformants fortement résistants à la phléo¬ mycine (CMI > 250 μg/ml) ont été sélectionnés et répartis en 10 groupes de 5 clones. L'ADN de chaque groupe a été extrait, digéré par l'enzyme BstEII et soumis à ligation. Avec chaque préparation une transformation de E.coli DH5 crt a été effectuée.T. geodes according to the protocol described in Example I above. 50 transformants highly resistant to phleomycin (MIC> 250 μg / ml) were selected and divided into 10 groups of 5 clones. The DNA of each group was extracted, digested with the enzyme BstEII and subjected to ligation. With each preparation, a transformation of E.coli DH5 crt was carried out.
L'ADN plasmidique de 6 colonies d'E.coli résul¬ tant de chaque transformation soit 60 transformants au total a été analysé individuellement par électrophorèse en gel d'agarose après micro-extraction. 7 plasmides de tailles différentes et supérieures à celle du vecteur pUT715 ont été choisis. Chacun de ces plasmides, extrait de la souche d'E.coli correspondante, a servi à transfor- mer T.géodes NC14. L'une des transformations a conduit à l'apparition de clones fortement résistants à la phléo¬ mycine (CMI > 250 μg/ml) avec une fréquence de l'ordre de 5000 transformants par microgramme d'ADN. Pour deux autres transformations, la fréquence et le niveau de ré- sistance des transformants ont été trouvées beaucoup plus faibles (quelques transformants/μg, CMI < 100 μg/ml). Enfin quatre transformations n'ont donné aucun résultat.The plasmid DNA of 6 E. coli colonies resulting from each transformation, ie 60 transformants in total, was analyzed individually by agarose gel electrophoresis after micro-extraction. 7 plasmids of different sizes and larger than that of the vector pUT715 were chosen. Each of these plasmids, extracted from the corresponding E. coli strain, was used to transform T. geodes NC14. One of the transformations led to the appearance of clones highly resistant to phleomycin (MIC> 250 μg / ml) with a frequency of the order of 5000 transformants per microgram of DNA. For two other transformations, the frequency and the level of resistance of the transformants were found to be much lower (some transformants / μg, MIC <100 μg / ml). Finally four transformations gave no result.
Le plasmide ayant permis la transformation de T.géodes la plus efficace appelée pUT771 , a été analysé, il comporte une insertion d'une taille d'environ 1,8 kb ; la cartographie de pUT771 est représentée sur la figure 4. La séquence nucléotidique de la partie promotrice est représentée sur la figure 5.The plasmid which has allowed the most efficient transformation of T. geodes called pUT771, has been analyzed, it comprises an insertion of a size of approximately 1.8 kb; the mapping of pUT771 is represented in FIG. 4. The nucleotide sequence of the promoter part is represented in FIG. 5.
Au cours d'expériences comparatives, le plasmide pUT771 a toujours conduit à des taux de transformation supérieurs d'un facteur deux environ à ceux obtenus dans les mêmes conditions avec pUT703.In comparative experiments, the plasmid pUT771 always led to transformation rates greater by a factor of about two than those obtained under the same conditions with pUT703.
Exemple IV : Co-transformation de T.géodes avec un plas¬ mide portant le marqueur de résistance à la phléomycine et un plasmide portant un gène non sélectionable. Une expérience de transformation a été réalisée selon le protocole décrit dans l'exemple I ci-dessus, avec un mélange contenant environ 1 μg d'ADN plasmidique de pUT771 décrit dans l'exemple II et 10 μg d'ADN plas- midique de pUT760, dont la carte est représentée sur la figure 6. Le plasmide pUT760 se caractérise par la présence du gène de Klebsiella pneumoniae ATCC 15050 codant pour la pullulanase mature (MICHAELIS et al., J. Bact., 1985, 164 : 633), sous la dépendance du pro¬ moteur gpd d'Aspergillus nidulans et précédé par la sé¬ quence signal synthétique décrite dans l'exemple I (fi¬ gure 2) .Example IV: Co-transformation of T. geodes with a plasmid carrying the marker for resistance to phleomycin and a plasmid carrying a non-selectable gene. A transformation experiment was carried out according to the protocol described in Example I above, with a mixture containing approximately 1 μg of plasmid DNA from pUT771 described in Example II and 10 μg of DNA plas- pUT760 midic, the map of which is represented in FIG. 6. The plasmid pUT760 is characterized by the presence of the Klebsiella pneumoniae ATCC 15050 gene coding for mature pullulanase (MICHAELIS et al., J. Bact., 1985, 164: 633 ), under the dependency of the gpd pro¬ motor of Aspergillus nidulans and preceded by the synthetic signal sequence described in Example I (FIG. 2).
Environ 500 transformants sélectionnés sur la base de la résistance à la phléomycine ont été repiqués individuellement d'une part sur un milieu PDA contenant 30 μg/ml de phléomycine et d'autre part sur un milieu gélose contenant : glucose 5 g/1, sulfate d'ammonium 1 g/1, phosphate monopotassique 2 g/1, sulfate de magné- sium 0,3 g/1, chlorure de calcium 0,2 g/1, extrait de levure 1 g/1, pullulane (Sigma) 10 g/1, agar 15 g/1. Après 7 jours d'incubation à 27°C, la surface de ce der¬ nier milieu a été recouverte d'alcool éthylique, et les boîtes de Pétri ont été placées à -20°C pendant une heure. A cette température et en présence d'alcool, le pullulane précipite, rendant le milieu opaque. Environ 2 % des co¬ lonies testées étaient entourées d'un halo clair plus ou moins large, dû vraisemblablement à l'hydrolyse du pullu¬ lane. Aucun halo n'a été détecté autour des colonies de la souche NC50 non transformée testées dans les mêmes conditions. Les 10 clones transformants semblant présen¬ ter la plus forte activité pullulanase, c'est-à-dire ceux correspondant aux colonies entourées des halos les plus larges ont été récupérés sur le milieu PDA-phléomy- cine et cultivés en fioles agitées dans le milieu selon Mandels additionné de glucose 30 g/1, extrait de levure 1 g/1 et tamponné par le phtalate de potassium à 5 g/1. Après 5 jours d'incubation à 27°C, 200 rpm, les cultures ont été centrifugées et l'activité pullulanase a été dosée dans les surnageants selon le protocole suivant : à 0,5 ml d'une solution de pullulane à 10 g/1 dans du tampon acétate 0,1 M pH 5,5, on ajoute 0,5 ml de solution enzymatique (surnageant éventuellement dilué dans le même tampon) . Après incubation 60 minutes à 55°C, le glucose libéré est dosé par la méthode de MILLER (AnalyticalAbout 500 transformants selected on the basis of resistance to phleomycin were individually subcultured on the one hand on a PDA medium containing 30 μg / ml of phleomycin and on the other hand on an agar medium containing: glucose 5 g / 1, sulfate ammonium 1 g / 1, monopotassium phosphate 2 g / 1, magnesium sulfate 0.3 g / 1, calcium chloride 0.2 g / 1, yeast extract 1 g / 1, pullulan (Sigma) 10 g / 1, agar 15 g / 1. After 7 days of incubation at 27 ° C, the surface of the latter medium was covered with ethyl alcohol, and the petri dishes were placed at -20 ° C for one hour. At this temperature and in the presence of alcohol, the pullulan precipitates, making the medium opaque. About 2% of the colonies tested were surrounded by a more or less wide clear halo, probably due to the hydrolysis of the pullu¬ lane. No halo was detected around the colonies of the non-transformed NC50 strain tested under the same conditions. The 10 transforming clones appearing to have the strongest pullulanase activity, that is to say those corresponding to the colonies surrounded by the largest halos, were recovered from the PDA-phleomycin medium and cultured in shaken flasks in the medium. according to Mandels supplemented with glucose 30 g / 1, yeast extract 1 g / 1 and buffered with potassium phthalate at 5 g / 1. After 5 days of incubation at 27 ° C, 200 rpm, the cultures were centrifuged and the pullulanase activity was assayed in the supernatants according to the following protocol: to 0.5 ml of a 10 g / l pullulan solution in 0.1 M acetate buffer pH 5.5, 0.5 ml of enzymatic solution (supernatant optionally diluted in the same buffer) is added. After incubation for 60 minutes at 55 ° C, the released glucose is measured by the MILLER method (Analytical
Che . , 1959, 31 : 426). L'activité est exprimée en unités internationales (u.i) correspondant au nombre de micro- • moles de glucose libérées par minute. Dans les surnageants des 10 cultures testées, des activités pullulanse de 12 u.i/ml à 350 u.i/ml ont été trouvées. Aucune activité n'a été détectée dans les surnageants de la souche non transformée cultivée dans les mêmes conditions. Exemple V :Che. , 1959, 31: 426). Activity is expressed in International Units (es) corresponding to the number of micro • moles of glucose released per minute. In the supernatants of the 10 cultures tested, pullulans activities from 12 IU / ml to 350 IU / ml were found. No activity was detected in the supernatants of the untransformed strain cultivated under the same conditions. Example V:
Cet exemple est destiné à illustrer la possibi- lité de sélectionner préférentiellement des clones recom¬ binants de T.géodes NC14 et de ses dérivés qui soient fortement producteurs d'une protéine hétérologue grâce à des constructions plasmidiques faisant appel au prin¬ cipe de la transcription polycistronique. L'expression de plusieurs gènes adjacents sous la dépendance du même promoteur et par 1'intermédiaire du même ARN messager, dit polycistronique, est un phéno¬ mène bien connu chez les bactéries. En revanche, il est généralement admis que ce phénomène n'existe pas chez les eucaryotes. Cependant, il a été fait état récemmentThis example is intended to illustrate the possibility of preferentially selecting recombinant clones of T. geodes NC14 and of its derivatives which are highly producers of a heterologous protein by means of plasmid constructs calling upon the principle of transcription. polycistronic. The expression of several adjacent genes under the dependence of the same promoter and by the intermediary of the same messenger RNA, known as polycistronic, is a well known phenomenon in bacteria. On the other hand, it is generally accepted that this phenomenon does not exist in eukaryotes. However, it has been reported recently
(KAUFMAN et al., EMBO J. , 1987, 6 : 187) d'une expression par des cellules animales en culture du gène de la dihy- drofolate réductase permettant la résistance au métho- trexate,dans des constructions où ledit gène était placé immédiatement en aval d'un autre gène non sélectionnable, ce dernier étant précédé d'un promoteur. Ceci permet une • sélection indirecte des clones exprimant fortement le gène non sélectionnable sur la base du niveau de résis¬ tance conféré par le gène placé en aval. II a été montré par les auteurs de la présente invention que ce principe pouvait s'appliquer à T.géodes, en utilisant par exemple comme parqueur sélectionnable le gène Sh de résistance à la phléomycine.(KAUFMAN et al., EMBO J., 1987, 6: 187) of an expression by animal cells in culture of the dihydrofolate reductase gene allowing resistance to methotrexate, in constructions where said gene was placed immediately downstream of another non-selectable gene, the latter being preceded by a promoter. This allows • indirect selection of clones strongly expressing the selectable gene not based on the level of résis¬ tance conferred by the gene placed downstream. It has been shown by the authors of this invention that this principle could be applied to T. geodes, using for example as a selectable marker the Sh gene for resistance to phleomycin.
Un gène synthétique pour le lysozyme humain a été préparé pa*ς ssemblage de 10 oligonucléotides de syn¬ thèse. La séquence représentée dans la figure 7 comporte 14 paires de base précédant le codon d'initiation, suivi de 18 codons débutant par un ATG correspondant aux acides aminés du peptide signal du lysozyme humain à l'exception du 17ème codon (remplacement de la glutamine par une leu¬ cine) (CHUNG, KESHAU et GORDON, Proc. Natl. Acad. Sci. 1988, 85 : 6227) puis 130 codons des acides aminés du lysozyme humain (JOLLES et JOLLES, Mol. Cell. Biochem., 1984, 63, 165) et enfin un codon stop suivi de 6 paires de bases. Le fragment d'ADN bordé par les sites de res¬ triction EcoRV-BamHI a été clone au site EcoRV du plas¬ mide pUT771 mentionné dans l'exemple II pour donner dans l'une des 2 orientations possibles, le plasmide pUT772 (figure 8) . La souche NC50 de T.géodes a été transformée par le plasmide pUT772 selon le protocole décrit dans l'e¬ xemple I avec la seule différence que la sélection des transformants a été réalisée avec une concentration moindre de phléomycine (15 μg/ml). Les colonies obtenues avec une fréquence 50 à 100 fois moindre à celle fournie par l'ADN pUT771 utilisé comme contrôle ont été repiquées sur milieu PDA et individuellement examinées pour le ni¬ veau de résistance à la phléomycine^et vérifiée pour la production de lysozyme. La détermination des concentra- tions minimales inhibitrices de phléomycine des transfor¬ mants par pUT772 a montré que les niveaux de résistances des clones étaient faibles (2 à 10 fois la CMI par rap¬ port à la souche non transformée) alors que les transfor¬ mants par ρUT771 étaient au contraire très élevés (30 à 200 fois la CMI) . La production de lysozyme est vérifiée indirec¬ tement par la méthode des cylindres d'agar pour la pro¬ duction d'une activité lytique contre Micrococcus lyso- deikticus sur PDA. Parmi 83 souches testées provenant de la transformation par pUT772, 61 produisaient une ac¬ tivité détectable par la présence d'une auréole d'inhibi¬ tion autour des cylindres d'agar. En contraste aucune colonie issue de la transformation par pUT771 ne présen¬ tait cette activité (55 testées) . Une analyse clonale a montré que le caractère de production était conservé pour 2/3 des souches posi¬ tives après une étape de sporulation. La souche NC50 PUT772-28 sélectionnée parmi les souches les plus actives par le test sur boîte a été plus particulièrement étudiée. Par cultures successives sur milieu solide PDA contenant des concentrations croissantes de phléomycine, un variant de la souche NC50/pUT772-28 a été sélectionné. Ce variant noté NC50/28R4 résiste à 100 μg/ml de phléo¬ mycine alors que la concentration maximale de croissance pour le parent est de 20 μg/ml. Les auréoles d'inhibi¬ tion sur un voile de Micrococcus lysodeikticus sont éga¬ lement supérieures pour la souche NC50/28R4 par comparai¬ son au parent NC50/pUT772-28. Les deux souches ont été cultivées en fiole agitée dans les conditions décrites dans 1'exemple ,IV. Après cinq jours d'incubation à 27°C l'activité lysozyme a été déterminée par un dosage turbi- dimétrique à partir de cellules lyophilisées de Micro¬ coccus lysodeikticus (SHUGAR, Biochim. Biophys., 1952, Acta 8 : 302) . Les protéines des surnageants des cultures ont été fractionnées sur une colonne MonoS PharmaciaA synthetic gene for human lysozyme was prepared pa * ς General assembly with 10 oligonucleotides syn¬ thesis. The sequence represented in FIG. 7 comprises 14 base pairs preceding the initiation codon, followed by 18 codons starting with an ATG corresponding to the amino acids of the signal peptide of human lysozyme with the exception of the 17th codon (replacement of glutamine by leucine) (CHUNG, KESHAU and GORDON, Proc. Natl. Acad. Sci. 1988, 85: 6227) then 130 codons of amino acids of human lysozyme (JOLLES and JOLLES, Mol. Cell. Biochem., 1984, 63 , 165) and finally a stop codon followed by 6 base pairs. The DNA fragment bordered by the EcoRV-BamHI restriction sites was cloned at the EcoRV site of the plasmid pUT771 mentioned in Example II to give, in one of the 2 possible orientations, the plasmid pUT772 (FIG. 8 ). The NC50 strain of T. geodes was transformed with the plasmid pUT772 according to the protocol described in example X with the only difference that the selection of the transformants was carried out with a lower concentration of phleomycin (15 μg / ml). The colonies obtained with a frequency 50 to 100 times less than that provided by the DNA pUT771 used as a control were subcultured on PDA medium and individually examined for the level of resistance to phleomycin ^ and checked for the production of lysozyme. The determination of the minimum phleomycin inhibitory concentrations of the transformants by pUT772 showed that the resistance levels of the clones were low (2 to 10 times the MIC compared to the untransformed strain) while the transformants by ρUT771 were on the contrary very high (30 to 200 times the MIC). The production of lysozyme is verified indirectly by the method of agar cylinders for the production of a lytic activity against Micrococcus lyso-deikticus on PDA. Among 83 strains tested originating from the transformation with pUT772, 61 produced an activity detectable by the presence of an inhibition halo around the agar cylinders. In contrast, no colony resulting from transformation with pUT771 exhibited this activity (55 tested). Clonal analysis showed that the production character was retained for 2/3 of the positive strains after a sporulation stage. The strain NC50 PUT772-28 selected from the most active strains by the box test was more particularly studied. By successive cultures on solid PDA medium containing increasing concentrations of phleomycin, a variant of the strain NC50 / pUT772-28 was selected. This variant noted NC50 / 28R4 resists 100 μg / ml of phleomycin while the maximum growth concentration for the parent is 20 μg / ml. The halos of inhibition on a veil of Micrococcus lysodeikticus are also superior for the strain NC50 / 28R4 by comparison with its parent NC50 / pUT772-28. The two strains were cultivated in a shaken flask under the conditions described in example, IV. After five days of incubation at 27 ° C. the lysozyme activity was determined by a turbidimetric assay from lyophilized cells of Micro¬ coccus lysodeikticus (SHUGAR, Biochim. Biophys., 1952, Acta 8: 302). The proteins of the culture supernatants were fractionated on a MonoS Pharmacia column
(tampon phosphate 50 mM pH 7,2 - élution gradient liné¬ aire de 0 à 1 M NaCl) . Un pic protéique élevé à 0,25 M est présent dans les deux souches transformées et absent chez le parent NC50. Par le test d'activité biologique, identification immunologique avec un anticorps antilyso— zyme humain et electrophorèse en gel d'acrylamide, il a été vérifié par comparaison avec un échantillon authen¬ tique de lysozyme humain que les protéines éluées à 0,25 M renferment du lysozyme. Les concentrations finales dans les surnageants de culture ont été estimées à 25 μg/ml et 200 μg/ml de lysozyme recombinant respecti¬ vement pour les souches NC50 pUT772-28 et NC50-28R4. (50 mM phosphate buffer pH 7.2 - linear gradient elution from 0 to 1 M NaCl). A protein peak raised to 0.25 M is present in the two transformed strains and absent in the parent NC50. By biological activity test, immunological identification with an antilyso- human zyme and acrylamide gel electrophoresis, it was verified by comparison with an authentic sample of human lysozyme that the proteins eluted at 0.25 M contain lysozyme. The final concentrations in the culture supernatants were estimated at 25 μg / ml and 200 μg / ml of recombinant lysozyme respectively for the NC50 strains pUT772-28 and NC50-28R4.

Claims

- REVENDICATIONS -- CLAIMS -
1 - Procédé de production de protéine recombi¬ nante à l'aide de champignons filamenteux, caractérisé en ce qu'on cultive sur un milieu approprié une souche du genre Tolypocladium dans laquelle on a introduit une séquence d'ADN codant pour ladite protéine placée sous le contrôle d'éléments assurant l'expression de ladite séquence dans ladite souche et en ce qu'on récupère la¬ dite protéine. 2 - Procédé selon la revendication 1, caracté¬ risé en ce que la souche est une souche de Tolypocladium géodes.1 - Process for the production of recombinant protein using filamentous fungi, characterized in that a strain of the genus Tolypocladium is cultivated on an appropriate medium into which a DNA sequence coding for said protein placed under the control of elements ensuring the expression of said sequence in said strain and in that la¬ said protein is recovered. 2 - Method according to claim 1, caracté¬ ized in that the strain is a strain of Tolypocladium geodes.
3 - Procédé selon la revendication 1 , caracté¬ risé en ce que la souche est une souche de Tolypocladium géodes déposée sous le n° 1-880, le 29 juin 1989 à la Collection Nationale de Culture des Microorganismes ou l'un de ses mutants déficients en activité protéasique et/ou hyperexcréteur de protéine exogène.3 - Method according to claim 1, caracté¬ ized in that the strain is a strain of Tolypocladium geodes deposited under the number 1-880, June 29, 1989 at the National Collection of Culture of Microorganisms or one of its mutants deficient in protease activity and / or hyperexcretor of exogenous protein.
4 - Procédé selon l'une des revendications 1 à 3, caractérisé en ce que les éléments assurant l'expres¬ sion comprennent essentiellement une séquence promotrice d'un gène d'origine fongique.4 - Method according to one of claims 1 to 3, characterized in that the elements ensuring the expression essentially comprise a promoter sequence of a gene of fungal origin.
5 - Procédé selon l'une des revendications 1 à5 - Method according to one of claims 1 to
4, caractérisé en ce que la séquence d'ADN codant pour ladite protéine est précédée d'une séquence signal assu¬ rant l'excrétion de la protéine.4, characterized in that the DNA sequence coding for said protein is preceded by a signal sequence ensuring the excretion of the protein.
6 - Procédé selon l'une des revendications 1 à6 - Method according to one of claims 1 to
5, caractérisé en ce que la séquence d'ADN codant pour ladite protéine est suivie d'un gène marqueur sous le contrôle des mêmes éléments d'expression.5, characterized in that the DNA sequence coding for said protein is followed by a marker gene under the control of the same expression elements.
7 - Procédé selon la revendication 6, caracté¬ risé en ce que ce gène marqueur code pour la résistance à un antibiotique.7 - Process according to claim 6, caracté¬ ized in that this marker gene codes for resistance to an antibiotic.
8 - Procédé selon la revendication 7, caracté- risé en ce que l'antibiotique est un composé de la fa- mille des phléomycines.8 - Process according to claim 7, characterized in that the antibiotic is a compound of the fa- thousand of phleomycins.
9 - Procédé selon la revendication 7, caracté¬ risé en ce que le gène codant pour la résistance à un antibiotique de la famille des phléomycines est d'origi- ne génomique et provient du génome des actinomycètes producteur dudit antibiotique.9 - Process according to claim 7, characterized in that the gene coding for resistance to an antibiotic of the phleomycin family is of genomic origin and comes from the genome of actinomycetes producing said antibiotic.
10 - Procédé selon l'une des revendications 1 à10 - Method according to one of claims 1 to
9, caractérisé en ce que la protéine codée est choisie parmi la pullulanase et le lysozyme humain. 11 - Procédé selon l'une des revendications 5 à9, characterized in that the encoded protein is chosen from pullulanase and human lysozyme. 11 - Method according to one of claims 5 to
10, caractérisé en ce que la séquence signal est la sé¬ quence signal synthétique figurant à la figure 2.10, characterized in that the signal sequence is the synthetic signal sequence appearing in FIG. 2.
12 - Procédé selon l'une des revendications 1 à12 - Method according to one of claims 1 to
11, caractérisé en ce que la séquence promotrice est la séquence promotrice figurant à la figure 5.11, characterized in that the promoter sequence is the promoter sequence shown in FIG. 5.
13 - Procédé selon l'une des revendications 1 à13 - Method according to one of claims 1 to
12, caractérisé en ce que la séquence d'ADN codant pour ladite protéine est suivie d'une séquence terminateur de transcription. 14 - Procédé selon l'une des revendications 1 à12, characterized in that the DNA sequence coding for said protein is followed by a transcription terminator sequence. 14 - Method according to one of claims 1 to
13, caractérisé en ce que la séquence d'ADN codant pour ladite protéine ainsi que les éléments assurant l'ex¬ pression de ladite séquence sont portés par un plasmide.13, characterized in that the DNA sequence coding for said protein as well as the elements ensuring the expression of said sequence are carried by a plasmid.
15 - Procédé selon la revendication 14, caracté- risé en ce que ledit plasmide est un plasmide à réplica¬ tion autonome comportant une séquence de réplication au¬ tonome efficace chez Tolypocladium : T-ARS.15 - Process according to claim 14, characterized in that said plasmid is a plasmid with autonomous replica¬ tion comprising an au¬ tonome replication sequence effective in Tolypocladium: T-ARS.
16 - Procédé selon la revendication 14 ou 15, caractérisé en ce que ledit plasmide assure 1'intégra- tion chromosomique des séquences d'ADN en cause.16 - Process according to claim 14 or 15, characterized in that said plasmid ensures the chromosomal integration of the DNA sequences in question.
17 - Protéine obtenue par la mise en oeuvre du procédé selon l'une des revendications 1 à 16.17 - Protein obtained by the implementation of the method according to one of claims 1 to 16.
18 - Protéine Sh caractérisée en ce qu'elle est obtenue par la mise en oeuvre du procédé selon l'une des revendications 1 à 16. 19 - Protéine Sh selon la revendication 18, caractérisée en ce qu'elle comporte la séquence d'acides aminés représentée en figure 2.18 - Sh protein characterized in that it is obtained by the implementation of the method according to one of claims 1 to 16. 19 - Sh protein according to claim 18, characterized in that it comprises the amino acid sequence shown in FIG. 2.
20 - Souche de Tolypocladium obtenue au cours de la mise en oeuvre du procédé selon l'une des reven¬ dications 1 à 16.20 - Tolypocladium strain obtained during the implementation of the method according to one of claims 1 to 16.
21 - Souche de Tolypocladium déposée sous le n° 1-880 et ses mutants déficients en activité protéasique et/ou hyperexcréteur de protéine exogène. 21 - Tolypocladium strain deposited under n ° 1-880 and its mutants deficient in protease activity and / or exogenous protein hyperexcretor.
PCT/FR1990/000479 1989-06-30 1990-06-28 New strain with filamentous fungi mutants, process for the production of recombinant proteins using said strain, and strains and proteins produced by said process WO1991000357A1 (en)

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FR89/08838 1989-06-30

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FR2649120B1 (en) 1994-01-28
EP0433431A1 (en) 1991-06-26
FR2649120A1 (en) 1991-01-04

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