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  • 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
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  • C12N1/00—Microorganisms; 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
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  • 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
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  • 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
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  • 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
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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)
  • C12N9/2405—Glucanases
  • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
  • C12N9/2457—Pullulanase (3.2.1.41)
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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)
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  • 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
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  • 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
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  • 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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