DE10123857A1 - Process for the production of heterologous proteins in a homothallic fungus of the Sordariaceae family - Google Patents

Abstract

The invention relates to a method for producing a heterologous protein in a filamentous fungus and to suitable promoters therefor. The invention also relates to vectors and host cells and to a kit and the use thereof. The aim of the invention is to provide a method for producing a heterologous protein in a filamentous fungus, which allows the efficient production of heterologous proteins and in which a contamination of the created protein by vegetative spores is prevented. To achieve this, the invention discloses a method for producing a heterologous protein in a filamentous fungus, which comprises (a) the cultivation of a homothallic fungus of the Sordariaceae family, which contains an expression cassette with the following elements in a functional combination: a promoter that is active in the fungus of the Sordariaceae family; a heterologous gene and a terminator that is active in the fungus of the Sordariaceae family and (b) the harvesting of the created protein in a manner known per se.

Description

The present invention relates to methods for producing heterologous protein in a filamentous fungus and suitable promoters. Furthermore, the present Invention vectors and host cells and a kit and their use.

Genetic engineering methods allow the establishment of expression systems for the Production of recombinant proteins. In the approximately 20-year tradition of industrial Genetic engineering became different microorganisms or cell lines as hosts systems developed and used. The determination of a host was based on criteria availability, the economy of a production process based on it, the area of application and the properties of the protein to be produced. As an ex pression systems served different gram-positive and gram- negative bacteria, mammalian cells and various types of fungi, including yeast and fi lamentous mushrooms.

The production of mammalian proteins is of particular economic importance and especially human proteins that are incorporated into pharmaceutical products be set. For the production of such proteins, which are often a complex glycosyly bacterial expression systems such as E. coli are often not suitable because bacteria neither secrete such heterologous proteins nor those with the Secretion pathway of higher (eukaryotic) organisms related modifications of Can form polypeptides such as glycosylation. The deposition of recombinant Products in inclusion bodies very often lead to the formation of insoluble and biologically inactive protein aggregates. A heterologous product must therefore often fig in a complex de-and renaturation process in a biologically active form be transferred.

For the production of proteins that require complex mammalian glycosylation, therefore mammalian cells are used. However, mammalian cell systems are very expensive; the cells are also a potential target for pathogenic viruses. Yeast and filamentous Fungi, on the other hand, have a glycosyly which differs from the complex type of mammal patterns, but they can be used for a variety of products. she are capable of secretion as eukaryotic microorganisms and can act like bacteria  fermented on inexpensive media in large cell densities. Because the culture media the serum produced can contain neither serum nor other potential contaminants heterologous protein can be cleaned easily and inexpensively. Examples of established ex Pression systems are yeasts such as S. cerevisiae and the methylotrophic yeast Hansenula polymorpha.

Within the group of filamentous mushrooms considered to be better "secretors" various Aspergillus species and Neurospora crassa for heterologous gene expression used. However, these mushrooms have so far been used almost exclusively for industrial manufacture the use of technical enzymes because of the presence of vegetative spores that are found in large amounts occur in the culture and are difficult to remove their use for the production of pharmaceutical products, because spore-free preparations prevented can only be produced with great effort. This is especially true of those who use it frequently added species Aspergillus nidulans, which is sexual (through ascospores) and especially ase xuell can multiply (through conidiospores).

Another problem in the production of heterologous proteins in filamentous fungi represent the inactivation systems for heterologous DNA, which are particularly effective become sam when heterologous DNA is inserted into the genome of the fungus. The gen inactivation z. B. very good for Neurospora crassa or Ascobolus immersus was written at different levels, i.e. the transcriptional or the post-transcriptional level. Similar phenomena were also found at transge NEN plants described. An overview of this can be found at Cogoni and Macino (1999).

Neurospora crassa became the so-called RIP phenomenon after DNA transformation observed (Singer et al., 1995; Selker, 1997), in which multiple copies of heterologous DNA be inactivated by point mutations or DNA modifications (e.g. methylation). As a result, foreign genes in Neurospora crassa cannot cross after meiotic crossing expressed efficiently.

The object of the present invention is therefore to provide a method for producing he to provide terological protein in a filamentous fungus, which is an efficient produc tion of heterologous proteins allowed and contamination of the protein produced is avoided by vegetative spores.  

According to the invention, this object is achieved by a method for producing heterologous protein in a filamentous fungus, which comprises cultivating a homothallic fungus of the Sordariaceae family which contains an expression cassette which contains the following elements in functional combination:

• A promoter active in the fungus of the Sordariaceae family,
• - a heterologous gene and
• A terminator active in the fungus of the Sordariaceae family,

and harvesting the protein produced in a manner known per se.

For the purposes of the invention, a “heterologous gene” denotes a coding nucleic acid sequence that comes from a gene other than that contained in the expression cassette tene promoter.

Homothallic mushrooms of the Sordariaceae family form, unlike heterothallic species this family or other filamentous mushrooms like Aspergillus nidulans or Podospora anserina, neither macroconidia nor microconidia, since they are exclusive multiply xuell. Athrospores, which are formed by the decay of the hyphae, are also not to be found. Thus, contamination of the protein produced with vegetati ven spores involved in the production of pharmaceutically relevant proteins with fila avoided a big problem.

Furthermore, it has surprisingly been found that this is the case with Neurospora crassa and other filamentous fungi observed RIP phenomenon after DNA transformation in homothallic mushrooms of the Sordariaceae family and especially in Sordaria macrospora does not occur. Sordaria macrospora has no genetic mechanisms, for repetitive sequences e.g. B. inactivate by methylation or point mutation. Consequently, heterologous DNA, which after transformation into the genome of the fungus in a lot copies are available, unlike Neurospora crassa and other filamentö not inactivated. Post-transcriptional gene inactivation is at homothalli Fungi of the Sordariaceae family are not known.

The Sordariaceae family belongs to the class of Ascomycetes (tubular mushrooms), according to Strasburger (textbook of botany) in the order of the Sphaeriales (Sordaria les) is classified. As an example for the life cycle of homothallic mushrooms of the The Sordariaceae family can be the life cycle of Sordaria macrospora as follows  write: This mushroom is a haplo-dikaryote that develops from eventually sexually increased. Vegetative spores, e.g. B. Conidiospores are from this Mushroom not formed. The fungus is homothallic, i. H. with him there is a monocia that does not is overlaid by incompatibility and therefore he can become sexual by selfing increase (Esser 2000). The process of fertilization, that of sexual reproduction preceded is referred to as autogamous. The ones in the Ascogon share haploid nuclei conjugate and thus initiate the dikaryotic phase. The sexual Ascospores are formed in Asci (tubes). Several Asci (approx. 100) mature at the same time early in fruiting bodies, the so-called perithecia. This type of fruiting body is typical of the representatives of the Sordariales. After ripening, the ascospores become active hurled out of the perithecies. In nature, the ascospores are created by the Food is ingested by herbivores and reaches a gastrointestinal Passage outside again. The gastrointestinal passage is a prerequisite for the subsequent Eating spore germination on the herbivore manure.

The homothallic fungus of the Sordariaceae family is preferably which is used in the method according to the invention to a fungus of the genus Sordaria. Homothallic mushrooms particularly preferred for carrying out the invention the Sordariaceae family are Sordaria macrospora or Sordaria fimicola; other ho mothallic mushrooms of the same family, which are responsible for carrying out the fiction suitable methods are Neurospora linoleata, Neurospora africana, Neurospora dodgei, Neurospora galapagosensis, Neurospora pannonica and Neurospora terricola.

Sordaria macrospora, like Sordaria fimicola, is a coprophilic saprophyte that occurs on herbivore dung grows. The hyphae is taxonomic in the department of Classify Eumycota and is therefore one of the higher fungi that have chitin walls Zen. The entire life cycle of Sordaria macrospora is under laboratory conditions completed within seven days (Esser, 2000). So that's the life cycle considerably shorter than the four-week cycle of Neurospora crassa. molecular biology Stem development work from S. macrospora can therefore be considerably shorter Time to be done.

For S. macrospora is both the formal genetic and the molecular genetic Analysis well developed. Formal genetic analysis takes advantage of the fact that sterile mutants of S. macrospora cannot form self-aspirations that however, two mutants that have different defects sexually in crosses  are reproducible. I.e. Meiosis takes place in so-called ascogenic hyphae leads to the formation of tetrads. The result is Asci with eight linearly arranged Spores, two of which are genetically identical. The size of the spores (18 × 28 µm) and the linearity of the arrangement allow formal genetics due to the orderly tetrads technically easy. In addition, a variety of Mutants are described that have both physiological and morphological properties affect the fungus (Esser and Straub, 1958; Nowrousian et al., 1999; Masloff et al., 1999; Le Chevanton and Leblon, 1989). An indexed cosmid library that contains the genes isolation from S. macrospora was made possible by Pöggeler et al. (1997).

For optimal expression of heterologous genes, codon usage is in the host organism crucial. From the list of the most common amino acid codons inside semi protein coding genes in Table 1 it can be seen that between the most frequently most used amino acid codons in Sordaria macrospora, Drosophila melanoga and primates an amazing match exists. In contrast, read There are clear differences between E. coli and Saccharomyces cerevisiae. Due to the similarity of codon usage, S. macrospora highlights one host system for the production of heterologous proteins of human origin.

Homothallic mushrooms of the family Sordariaceae can be easily and inexpensively in La boreal cultures are attracted. As eukaryotic microorganisms, they are also in the Location, post-translational modifications of recombinant eukaryotic proteins make. For the biotechnical use of homothallic mushrooms from the family Sordariaceae also speaks that this is not a human, animal or plant pathogenic organisms. Furthermore, recombinant strains of Sordaria macrospora through genetic engineering, also in combination with classic (con conventional) crossings are produced, a considerable advantage over imper perfect mushrooms of the genus Aspergillus.

The only spores from homothallic fungi of the Sordariaceae family and ins Special forms of S. macrospora or S. fimicola are so-called Asco spores that occur in fruiting bodies and perithecia in the course of sexual reproduction to step. Such ascospores are much larger than the conidiospores, heavier and are formed in significantly fewer numbers. You therefore have a very low spreadability. In submerged cultures or in shake cultures in the Usually no ascospores are formed. Should the heterologous protein produced be absolutely spo  to be free of ren, in a particularly preferred embodiment of the invention According to the method for producing heterologous protein, a sterile mutant of homothallic mushroom of the family Sordariaceae used. Sterile mutant strains, for example, by mutagenesis of protoplasts with EMS or by radiation can be obtained with UV light, have no propagation structures and per therefore do not induce ascospores. For example, in the invention Process for the production of heterologous protein sterile mutants of S. macrospora (Esser and Straub, 1958; Masloff et al., 1999; Nowrosian et al., 1999) can be used.

The cultivation of the for the production of heterologous protein is preferably carried out set homothallic mushrooms at a temperature of 27 ± 2 ° C. This wax Optimum is significantly lower than that of Neurospora crassa, which is above 30 ° C lies. As an essential safety-relevant aspect, it should be noted that S. macrospora dies at temperatures above 32 ° C.

According to a preferred embodiment of the invention, the in the mushroom derives from Family Sordariaceae active promoter, under whose control the heterologous gene ex is primed by a filamentous fungus. This promoter can be, for example, the gpd Aspergillus nidulans promoter, but preferably the promoter is a pro engine from Sordaria macrospora. Particularly preferred promoters are the acl1-Pro motor, the ppg1 promoter, the cpc2 promoter or the ndk1 promoter from Sordaria macrospora. The S. macrospora acl1 gene was developed by Nowrousian et al. (1999) be wrote. The ppg1 gene from S. macrospora was described by Pöggeler (2000). The S. macrospora cpc2 promoter and ndk1 promoter are shown below described.

The terminator active in the fungus of the Sordariaceae family preferably comes from a filamentous mushroom. The terminator can, for example, be the trpC terminator Aspergillus nidulans (Mullaney et al., 1985) or a terminator from Sordaria macrospora his. Particularly preferred terminators from S. macrospora are the acl1 terminator (Nowrosian et al., 1999), the ppg1 terminator (Pöggeler, 2000), the cpc2 terminator or the ndk1 terminator.

The heterologous gene preferably codes for a glyco after expression in eukaryotes sylated protein. For example, the heterologous gene may be one Growth factor, a cytokine, a coagulation factor, an industrial protein or a  act technical enzyme. The heterologous gene particularly preferably codes for one of the following proteins: G-CSF, GM-CSF, IL-1, IL-2, IL-4, IL-6, IL-1ra, IFN-α, IFN-β, IFN-γ, erythropoietin, glucoamylase, coagulation factor VIII, coagulation factor XII, gerin Factor XIII, human serum albumin.

In a further embodiment of the method according to the invention, between the Promoter and the heterologous gene in reading frame with the heterologous gene a Se quenz arranged that a signal in the fungus of the family Sordariaceae functioning sequence encoded. The signal sequence is preferably one of a filamentous fungus derived signal sequence, for example the signal sequence of glucoamylase from Aspergillus niger (Gordon et al. 2000; Gouka et al. 1997). Signal sequences from Sordaria macrospora, e.g. B. the signal sequence of the ppg1 gene from S. macrospora are special prefers.

Another object of the invention is to provide promoters which in the Process according to the invention used for the production of heterologous protein can be.

This task is accomplished by a nucleic acid molecule that:

• 1. a promoter active in a homothallic fungus of the Sordariaceae family, which is selected from the following nucleic acids:
  • a) a nucleic acid with the sequence given in SEQ ID NO: 1;
  • b) a nucleic acid with the sequence given in SEQ ID NO: 2;
  • c) a nucleic acid with a sequence which has at least 50% identity with one of the sequences given in (a) or (b);
  • d) a nucleic acid which hybridizes with the counter strand of one of the nucleic acids given in (a) or (b);
  • e) a derivative of one of the nucleic acids specified in (a) or (b) obtained by substitution, addition and / or deletion of one or more nucleotides;
  • f) a fragment of one of the nucleic acids given in (a) to (e), which retains the function of the promoter active in the fungus of the Sordariaceae family;
  • g) a combination of several of the nucleic acids specified in (a) to (f), it being possible for the sequences of the nucleic acids to be the same or different; or
• 2. a nucleic acid with a sequence that corresponds to the sequence of one of (a) to (g) specified nucleic acids is complementary,

includes.

For the purposes of the invention, the term “% identity” refers to identity on DNA Level that according to known methods, e.g. B. the computer-aided sequence comparisons (Altschul et al., 1990).

The term "identity" known to those skilled in the art denotes the degree of relatedness shaft between two or more DNA molecules, which is determined by the match between the sequences is determined. The percentage of "identity" results from the Percentage of identical areas in two or more sequences taking into account of gaps or other sequence peculiarities.

The identity of related DNA molecules can be determined using known methods be determined. As a rule, special computer programs are used algorithms that meet their requirements. Preferred procedures to determine identity first generate the greatest match between the sequences examined. Computer programs for determining identity between two sequences, but are not limited to that GCG program package, including GAP (Devereux et al., 1984; Genetics Computer Group University of Wisconsin, Madison, (WI)); BLASTP, BLASTN and FASTA (Altschul at al., 1990). The BLAST X program can be obtained from the National Center for Biotechnology In formation (NCBI) and from other sources (BLAST Handbook, Altschul S. et al., NCB NLM NIH Bethesda MD 20894; Altschul et al., 1990). Even the well-known Smith Waterman's algorithm can be used to determine identity.  

Preferred parameters for sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol 48: 443-453 (1970)
Comparison matrix: Match (matches) = + 10, mismatch = 0
Gap penalty: 15
Gap Length Penalty: 1

The GAP program is also suitable for use with the above parameters net. The above parameters are the default parameters for Nucleic acid sequence comparisons.

Further exemplary algorithms, gap opening penalties, Gap extension penalties, comparison matrices included those mentioned in the Program Guide, Wisconsin Package, Version 9, September 1997 can be used. The selection will depend on the comparison to be made depend and continue whether the comparison between sequence pairs, where GAP or Best Fit are preferred, or between a sequence and an extensive Se quenz database, with FASTA or BLAST being preferred.

The feature "sequence that matches the opposite strand of a sequence according to (a) or (b) hy bridisiert "indicates a sequence which, under stringent conditions, corresponds to the Ge gene strand of a sequence with the features specified under (a) or (b) hybridized. For example, the hybridizations can be carried out at 68 ° C. in 2 × SSC.

The sequences given in SEQ ID NO: 1 and SEQ ID NO: 2 correspond to the Pro motor sequences of the ndk1 and cpc2 genes isolated from S. macrospora. The in (c) Given nucleic acid can also have at least 60%, 70%, 80% or 90% identity have one of the sequences given in (a) or (b). Particularly preferably points they have 95% identity with one of these sequences.

The invention further provides a vector for transforming a homothallic fungus of the Sordariaceae family, which contains the following elements in functional connection with one another:

• A promoter active in the fungus of the Sordariaceae family,  
• - a heterologous gene,
• - a terminator active in the fungus of the Sordariaceae family and
• - a selection marker.

The promoter of the Vek according to the invention active in the fungus of the Sordariaceae family tors can be the gpd promoter from Aspergillus nidulans. The acl1- from Sordaria macrospora or the ppg1 promoter from Sordaria macrospora are however a promoter particularly preferred. The nucleic acids described above under (1) are as Promoters particularly preferred.

The terminator contained in the vector according to the invention can be the trpC terminator from Aspergillus nidulans, the acl1 terminator, the ppg1 terminator, the cpc2 terminator or the ndk1 terminator from Sordaria macrospora are particularly preferred, however. The ura5 gene from Sordaria macrospora, for example, is suitable as a selection marker (Le Chevanton and Leblon, 1989) or Podospora anserina (Turcq and Begueret, 1987). A hygromycin B resistance gene (see, for example, Kaster et al., 1983).

The invention also provides a host organism. With this host organism is a homothallic mushroom of the Sordariaceae family that invented one contains inventive vector. The host organism preferably belongs to the genus Sordaria, the host organism Sordaria macrospora or is particularly preferred Sordaria fimicola. In a particularly preferred embodiment, it is the host organism around a sterile strain that is neither asexual nor sexual nor sexual Meiospores forms.

The invention further provides a kit that:

• a) a vector according to the invention and
• b) a homothallic fungus suitable for producing heterologous protein of the Sordariaceae family

includes.

The nucleic acid molecule, the vector, the host organism and the kit according to the Erfin can express a heterologous gene under the control of the promoter  or used to make one or more proteins.

The following figures and examples illustrate the invention.

Fig. 1 shows the nucleotide sequence of the Ndk1 gene (partial fragment) of S. macrospora, incl. The promoter region of about 1.4 Kb.

Fig. 2 shows the nucleotide sequence of the gene of Sordaria macrospora CPC2 and the amino acid sequences derived therefrom. The intron sequences (4) are marked by underlining, the promoter is located in the section of nucleotide 1-2611, the termination sequence can be found in the region between nucleotide 4258 and nucleotide 4539.

Figure 3 shows the cloning scheme for the expression vector pMN110.

Fig. 4 shows the nucleotide sequence (promoter and terminator) of the plasmid pMN110. The sequences are from the acl1 gene from S. macrospora.

Figure 5 shows the cloning scheme for the expression vector pMN112.

Fig. 6 shows the physical and genetic map of the plasmid pMN112.

Figure 7 shows the cloning scheme for the expression vector pSMY3.

Fig. 8 shows the physical and genetic map of the plasmid pPROM1.

Fig. 9 shows the physical and genetic map of the plasmid pTERM1.

Fig. 10 shows the physical and genetic map of the plasmid pSMY2.

Fig. 11 shows the physical and genetic map of the plasmid pSMY3.

Fig. 12 shows the nucleotide sequence of the insert fragment of the plasmid pSMY3.

Fig. 13 shows the autoradiograph of a Northern hybridization. In the individual tracks, 5 µg of S. macrospora mRNA was applied, in the odd tracks from the wild-type strain, in the even tracks from the sterile mutant inf. The acl1 gene (lane 1, 2), the cpc2 gene (lane 3, 4), the ndk1 gene (lane 5, 6) and the ppg1 gene (lane 7, 8) were used as the probe. In lanes 1, 2 there is a 2.7 kb acl transcript, in lanes 3, 4 a cpc2 transcript of 1.7 kb, in lanes 5, 6 an ndk1 transcript of 1.5 kb and a ppg1 transcript of 0.65 kb can be seen in lanes 7, 8.

Fig. 14 shows the plasmid pEGFP / gpd / tel, which served as starting plasmid for the cloning of plasmids and pSM1 PSM2.

Figure 15 shows the cloning scheme of plasmid pSM1.

Figure 16 shows the cloning scheme of plasmid pSM2.

Fig. 17 shows the fluorescence microscopic analysis of asci with ascospores of S. macrospora transformants carrying the plasmid pEGFP / gpd / tel. The strain shown comes from a cross between the S. macrospora transformant T-EG2 and the color-saving mutant of S. macrospora FUS1. The latter does not carry a gfp gene. To interpret the fluorescence microscope image (bottom), the corresponding light microscope image is shown above.

Fig. 18 shows the physical and genetic map of the plasmid pSMY1-1.

Fig. 19 shows the physical and genetic map of the plasmid pSMY1-2.

Fig. 20 shows the nucleotide sequence near the ATG start codon of the lacZ gene of the plasmid pSMY1.

Fig. 21 shows the physical and genetic map of the plasmid pSMY5-1.

Fig. 22 shows the physical and genetic map of the plasmid pSMY5-2.

Fig. 23 shows the physical and genetic map of the plasmid pSMY4-1.

Fig. 24 shows the physical and genetic map of the plasmid pSMY4-2.

Examples material and methods A. Cultivation of S. macrospora 1) Culture media

• - BMM (fructification medium): 0.8% organic salt in maize flour extract, pH 6.5
• - BMM + NaAc (spore germ medium): BMM + 0.5% NaAc, pH 6.0
• CCM: 0.3% sucrose, 0.05% NaCl, 0.5% K ₂ HPO ₄ , 0.05% MgSO ₄ , 0.001% FeSO ₄ , 0.5% Tryptic Soy Broth, 0.1% yeast extract, 0.1% meat extract, 1.5% dextrin pH 7.0
• - GM (minimal medium): 2% glucose, 0.7% Bacto Yeast Nitrogen Base, 0.4 µM biotin, 25 µl / L mineral concentrate (5% ascorbic acid, 5% ZnSO ₄ × 7H ₂ O, 1% Fe (NH ₄ ) ₂ (SO ₄ ) ₂ × 6H ₂ O, 0.25% CuSO ₄ × 5H ₂ O, 0.05% MnSO ₄ × 1 H ₂ O, 0.05% H ₃ BO ₄ , 0.05% Na ₂ MoO ₄ , 1% chloroform, pH 6.0 (Zickler et al. 1984)
• GMU: GM with 10 mM uridine
• - GMS: GM with 10% sucrose
• - LB: 1% Bacto-Trypton, 0.5% yeast extract, 0.5% NaCl, pH 7.2
• - CM: 0.15% KH ₂ PO ₄ , 0.05% KCl, 0.05% MgSO ₄ , 0.37% NH ₄ Cl, 1% glucose, 0.2% tryptone, 0.2% yeast extract, trace elements , pH 6.4-6.6
• - CMS: CM medium with 10.8% sucrose
• - MM (minimal medium): 55.5 mM glucose, 1.8 mM KH ₂ PO ₄ , 1.7 mM K ₂ HPO ₄ , 8.3 mM urea, 1 mM MgSO ₄ , 5 µM biotin, 0.1 ml / l mineral concentrate (see GM medium)
• MMU: MM with 10 mM uridine
• - Solid media: 1.5% agar each, GM top agar: 0.4% agar

2) Cultivation conditions

As a rule, cultivation takes place at 27 ° C. S. macrospora is used for fructification Fixed media attracted; The fructification can be observed after only 7 days of culture become. S. macrospora is drawn onto liquid media for nucleic acid preparations genes, usually in stand cultures in Fernbach flasks.  

B. Production of Sterile S. macrospora Strains 1) UV mutagenesis

A protoplast suspension (1.5 × 10 ⁸ protoplasts per ml protoplast buffer) from the Sordaria macrospora wild-type strain ATCC MYA-334 is prepared for UV mutagenesis. The suspension is exposed to UV light (0.05 µW / cm ² ) when shaken gently. The times vary between 10 and 20 minutes. The protoplasts are then placed on CMS solid medium (0.15% KH ₂ PO ₄ , 0.05% KCl, 0.05% MgSO ₄ , 0.37% NH ₄ Cl, 1% glucose, 0.2% trypton, 0 , 2% yeast extract, trace elements, pH 6.4-6.6, 10.8% sucrose, 1.5% agar) and plated and incubated at 27 ° C. for about 48 hours. The regenerated protoplasts are then inoculated onto MB solid medium (0.8% organic salt in maize flour extract, pH 6.5, 1.5% agar). After 1-4 weeks, the clones are phenotypically characterized to identify sterile mutants. These are usually characterized by changes in the formation of fruiting bodies. In order to distinguish mutants from variants, the mitotic stability of the strains is tested by inoculation on MB medium. After a growth distance of approx. 7 cm, the mycelium is transferred again to fresh nutrient medium. This process is repeated three times. After this test of mitotic stability, the sterile strains are genetically checked in cross-over experiments to isolate ascospores. This creates homokaryotic strains.

2) EMS mutagenesis

In EMS mutagenesis, ethyl methyl sulfonate is used as a mutagenic agent. For mutagenesis, 5 × 10 ⁶ protoplasts of the wild-type strain of Sordaria macrospora (see above) in a total volume of 500 μl are treated with EMS. The final concentration of EMS (σM-0880) is 34.1 mg / ml. The mutagenesis is carried out at 27 ° C. for 45 minutes. The protoplasts are then plated out on CMS solid medium as described under 1) above and treated further as described there.

The strains generated are identified below by "inf" (infertile).

C. Transformation of sterile S. macrospora strains

S. macrospora mutants are transformed according to Walz and Kück (1995). Fungal mycelium of the strain to be transformed is 2 days at 27 ° C in CM liquid medium (0.15% KH ₂ PO ₄ , 0.05% KCl, 0.05% MgSO ₄ , 0.37% NH ₄ Cl, 1% Glucose, 0.2% tryptone, 0.2% yeast extract, trace elements, pH 6.4-6.6, 10.8% sucrose, 1.5% agar) was grown as a stand culture. After a washing step of the mycelium in protoplast buffer (13 mM Na ₂ HPO ₄ , 45 mM KH ₂ PO ₄ , 600 mM KCL, pH 6.0, Le Chevanton and Leblon 1989) and subsequent filtration, the mycelium is dissolved in Novozym solution (10 mg Novozym 234 (Novo Nordisk) / ml protoplast buffer) added. After 45 minutes of incubation at 100 rpm and 27 ° C., the protoplasts are separated from the residual mycelium using a glass frit (pore size 1, Schott). After pelleting the protoplasts by centrifugation, the cells are washed twice in protoplast buffer and sedimented again. The protoplasts are then taken up in transformation buffer (1 M sorbitol, 80 mM CaCl ₂ , pH 7.5), so that the protoplast titer is 2 × 10 ⁸ / ml. For the transformation, 2 × 10 ⁷ / ml protoplasts are mixed with 20 μg plasmid DNA or 20 μl cosmid DNA and incubated on ice for 10 min. After the addition of 200 µl 25% PEG (in transformation buffer), incubation for 20 min at RT follows. From the transformation batch, 100-200 µl in different batches are plated directly onto the CMS medium. After a maximum of 12 hours of incubation at 27 ° C., the regenerating protoplasts are overlaid with hygromycin B-containing top agar (0.8 M NaCl, 0.8% agar). The hy gromycin B concentration in the top agar is selected so that a final concentration of 110 U / ml is present in the entire medium. Transformants appear after 2 to 4 days and are inoculated on BMM medium (0.8% biomalt in maize flour extract, pH 6.5), which contains 100 U / ml hygromycin B. The phenotypic investigations of the mutants follow on BMM medium without selection pressure.

example 1 Cloning of the regulatory sequences of the cpc2 and ndk1 genes from S. macrospora

To clone the genes cpc2 and ndk1 from S. macrospora, a set of oligonucleotides was synthesized (Table 2), which made it possible to carry out PCR amplifications. Using S. macrospora genomic DNA, the oligonucleotide primers 1095 and 1096 were used for the PCR amplifications of the cpc2 gene and the oligonucleotide primers 1265 and 1266 for the amplification of the ndk1 gene. The resulting amplificates of 655 bp (cpc2) and 594 bp (ndk1) were then used for DNA sequencing. The DNA sequence of the ndk1 gene, which also contains a 1.4 kb promoter region in front of the putative ATG start codon, is shown in FIG. 1. The ATG start codon is located at position 1384-1386 in this sequence. The nucleic acid sequence and the amino acid sequences of the cpc2 gene derived therefrom are given in FIG. 2.

The gene fragments were then used for so-called Northem hybridizations comparative hybridizations with other genes from S. macrospora were used carried out. From the comparative hybridizations with 10 different ones S. macrospora gene probes show that the cpc2 gene and the ndk1 gene of S. macrospora has a very high transcriptional level. Compared to Hybri this signal level is significantly higher. Around We then used these genes for the construction of expression vectors the complete genomic copies of both genes from an indicated genomi Cosmid gene bank of S. macrospora (Pöggeler et al. 1997) isolated. The screening resulted in the isolation of the cosmid clones VIG10 (cpc2) and VIIG10 (ndk1). to Clear identification of the regulatory sequences were subfragments of both Sequenced cosmid clones. The promoter and terminator sequences of the cpc2 or ndk1 genes were then used for the construction of expression vectors det.

Example 2 Regulatory elements of the acl1 gene from S. macrospora

In animals and fungi, ATP citrate lyase (ACL) is localized in the cytosol during the homologous protein is localized in plants in chloroplast. With all three organisms systems produces ACL Acetyl-CoA, which is primarily used for fatty acids and sterols biosynthesis is used. In mushrooms, the enzyme consists of two subunits, the one of two separate genes (acl1, acl2), which are adjacent to the chromosomal DNA locally are encoded (Nowrousian et al., 2000). In contrast to that, the conti Nuclear polypeptide encoded by a gene in animals.

The promoter element of the acl1 gene from S. macrospora (Nowrousian et al. 1999) was used for the construction of the expression plasmid pMN110 (see FIG. 3). To amplify the promoter sequence, the oligonucleotides 1197 and 1199 (Table 2) were used together with the genomic DNA from S. macrospora as template DNA. The cloning of the expected fragment in a size of 2.3 Kb he followed in the plasmid pMON 38201 (Borovkov and Rivkin, 1997). The resulting plasmid was named pMN95. The terminator sequence of the acl1 gene was then amplified and cloned. In this case too, the S. macrospora genomic DNA served as template DNA in order to generate a fragment of 0.6 Kb in size by PCR with the oligonucleotides 1194 and 1200. The resulting plasmid was named pMN102. The terminator sequence was then cloned from the plasmid pMN102 into the vector pKS +. For this purpose, the plasmid pMN102 was hydrolyzed with the enzymes NotI and SacI. The resulting 0.6 Kb restriction fragment was ligated into the NotI and SacI restricted vector pKS +. The resulting plasmid was named pMN109. This plasmid was then restricted with the enzymes HindIII and NotI and ligated with the 2.3 Kb fragment of the plasmid pMN95. The resulting plasmid was named pMN110 and was used for further cloning. The cloning strategy is shown in FIG. 3 and the corresponding sequence of the insert DNA of the plasmid pMN110 is shown in FIG. 4.

In the next cloning step, the plasmid pMN112 was constructed, which is suitable for the transformation of S. macrospora and for the transformation of E. coli (see FIG. 5). For this purpose, the plasmid pBCHygro (Silar, 1995) was hydrolyzed with NotI and the corresponding restriction ends were filled in using Klenow polymerase. The resulting linear plasmid with NotI ends filled in was restricted with the enzyme ClaI. This creates a linear vector molecule that is terminated by a "blunt" end or by a ClaI cut. This vector molecule treated in this way was used in a ligation in which a 2.9 Kb fragment from the plasmid pMN110 was used. This fragment was generated by linearizing the plasmid pMN110 with the HindIII enzyme. The protruding restriction ends were then filled in with Klenow polymerase in order to generate “blunt” ends. This restriction fragment treated in this way was then restricted with the enzyme ClaI and eluted after gel electrophoresis in order to be used for the ligation discussed above. The cloning strategy is shown in FIG. 5 and the resulting plasmid pMN112 is shown in FIG. 6. It has a total size of 9.605 Kb.

The expression plasmid pMN112 can be used for the transformation of S. macrospora and E. coli can be used. In this plasmid is the acl1 promoter with the acl1 termi nator connected by a NotI restriction cut. This unique NotI restriction cut is suitable for the insertion of foreign DNA which is under the control of the acl1- Promoter should be expressed.  

Example 3 Regulatory elements of the ppg1 gene

The ppg1 gene for the sex pheromone from S. macrospora codes for a preproprotein of 277 amino acids. Included here is a leader peptide of 16 amino acids (Pöggeler, 2000), which can be used as a signal sequence for protein secretion.

For the construction of the expression plasmid pSMY3, the promoter sequence, the sequence encoding the leader peptide and the termination sequence of the ppg1 gene were used (see FIG. 7).

For the cloning of the promoter sequence together with the leader peptide coding Sequence, the oligonucleotides ppg1-1 and ppg1-2 were used. Both oligonucleos tides were expanded by sequences for restriction endonucleases (see Table 2). In the case of the oligonucleotide ppg1-1, the recognition sequence for the enzyme SacI used, in the case of the oligonucleotide ppg1-2, for the enzyme NotI. For the amplifica tion with these two oligonucleotides was the S. macrospora genomic DNA used. The amplification gave a 1.8 Kb fragment.

The termination sequence of the ppg1 gene was also amplified by the ent speaking sequence won. For this purpose the oligonucleotides ppg1-3 as well ppg1-4 used. Both oligonucleotides also contain sequence extensions for the enzymes NotI (ppg1-3), or for the enzyme BamHI (ppg1-4). The amplification with these two oligonucleotides were in turn made using genomic DNA by S. macrospora and delivered an 880 bp DNA fragment.

The two amplificates were inserted into the vector pMON38201 linearized with XcmI as described above. The plasmids resulting from the cloning were called pPROM1 (contains the promoter region) or pTERM1 (contains the terminator sequence). The physico-genetic map of the plasmids and pPROM1 pTERM1 is shown in Fig. 8 and Fig. 9.

The promoter sequence was then cloned into the transformation vector pCB1004 (Carroll et al., 1994). For this purpose, the plasmid pPROM1 was restricted with SacI and NotI, and inserted into the SacI / NotI hydrolyzed vector pCB1004. The corresponding recombinant plasmid was named pSMY2. This plasmid was then hydrolyzed with the enzymes NotI and BamHI and the NotI and BamHI restriction fragment from the plasmid pTERM1 was inserted into the vector pSMY2 restricted with NotI and BamHI ( FIG. 10). The resulting plasmid was named pSMY3 ( FIG. 11) and contains both the promoter sequence, including the sequence encoding the leader peptide, and the terminator sequence of the ppg1 gene. The promoter sequence is linked to the terminator sequence by a NotI restriction cut. This restriction cut is unique in the plasmid pSMY3 and can therefore be used for the insertion of heterologous DNA. The DNA sequence of the insert in the plasmid pSMY3 is shown in FIG. 12.

Example 4 Comparison of the transcriptional expression of the genes ppg1, ndk1, cpc2 and acl1 in Sordaria macrospora

In order to compare the transcriptional expression of the above-mentioned genes, the different transcript frequencies for different strains were determined in a Northern hybridization. For this purpose, 5 μg mRNA from the wild-type strain or from the sterile mutant inf were applied and hybridized with the respective probes which were specific for the genes mentioned above. In the autoradiogram shown in FIG. 13 it can be seen that the ppg1 gene gives the weakest signals compared to so-called "house-keeping" genes (such as α- or β-tubules) however, the ppg1 gene is heavily transcribed. The signal for the acl1 gene is significantly stronger, the strongest signals were found for the cpc2 and ndk1 genes. The autoradiogram makes it clear that the four genes are suitable for the construction of expression vectors due to their high transcriptional expression.

Example 5 Expression vector construction and production of the "green fluorescent protein" (GFP)

In order to express the heterologous gfp gene (Clonetech, USA) in Sordaria macrospora, two expression plasmids were constructed in which the gfp gene was placed under the control of various promoters or is free of promoters. The plasmid pEGFP / gpd / tel (Inglis et al., 1999) was used as the starting plasmid (see FIG. 14).

In the plasmid pSM1, the gfp gene is controlled by the Aspergillus nidulans gpd promoter and terminated by the Aspergillus nidulans trpC termination sequence. The plasmid also contains the hygromycin B resistance gene for the selection of fungal transformants. The construction of the plasmid pSM1 is shown in FIG. 15.

Unlike the plasmid pSM1, the plasmid pSM2 does not contain a gpd promoter. In front of the gfp gene there is a multiple cloning site for several enzymes which are suitable for inserting heterologous or homologous promoter sequences and thus for controlling gfp gene expression. The constructions of the plasmid pSM3 is shown in FIG. 16.

The three plasmids, pEGFP / gpd / tel, pSM1 and pSM3, were separately described as above ben transformed into a sterile Sordaria macrospora strain. Then were the transformants selected for hygromycin resistance as described and fluo Analyzed by rescence microscopy. The analysis was carried out with the Zeiss Axiophot microscope when excited with light of wavelength 420 nm. GFP-producing clones were identified concluding for a formal genetic analysis against the wild type strain or others Strains of testers used. The intersection can be used to check the extent to which this heterologous GFP protein in meiosis is passed on stably, and in particular how as far as the GFP expression remains stable in the offspring.

As can be seen from FIG. 17, the GFP gene expression is recognizable both in the vegetative mycelium of the transformants and in the ascospores. The expected 1: 1 splitting can be clearly seen in the eight-pore Asci (4 spores show fluorescence, 4 spores show fluorescence). This GFP gene expression can also make it clear that the heterologous gene expression in S. macrospora after the meiotic cross is not destroyed by inactivation processes (e.g. RIP, MIP, quelling).

Example 6 Galactosidase production

In order to express the bacterial β-galactosidase gene (lacZ) in S. macrospora, the plasmid pSMY1-1 was constructed. The plasmid pSMY1-1 was generated by inserting the lacZ gene into the singular NotI restriction site of the plasmid pMN112. The lacZ gene was generated from the plasmid pSI8.8 (Menne et al., 1994) by PCR amplification. For this purpose, oligonucleotides 1206 and 1215 (Table 2) were used, which have the recognition sequence for the restriction enzyme NotI at the terminal end. The amplificate has a size of 3.0 Kb and was inserted into the unique site of the plasmid pMON 38201 (Borovkov and Rivkin 1997). The resulting plasmid was given the designation pMN104, which was subsequently hydrolyzed with NotI hy. The resulting 3.0 Kb NotI fragment was inserted into the NotI linearized plasmid pMN112 ( Fig. 6). The resulting plasmids pSMY1-1 ( Fig. 18) and pSMY1-2 ( Fig. 19) differ in the orientation of the lacZ gene. In the plasmid pSMY1-1, the lacZ gene is under the control of the acl1 promoter. In pSMY1-2 there is an inverse arrangement of the lacZ gene compared to plasmid pSMY1-1, which means that acl1-promoter-controlled expression is not possible. The plasmid pSMY1-2 can thus be used as a control in expression experiments. The two resulting plasmids are shown in FIGS. 18 and 19. The sequence on the ATG start codon is shown in FIG. 20.

In order to express the β-galactosidase gene under the control of the regulatory sequences of the ppg1 gene, the vector pSMY5 was constructed on the basis of the expression plasmid pSMY3. For this purpose, the lacZ gene was obtained from the plasmid pMN1004 as described above. A 3.0 Kb NotI fragment was then inserted into the vector pSMY3 / NotI. The resulting plasmid was named pSMY5-1 ( Fig. 21). This expression plasmid should not only allow the production of the β-galactosidase in S. macrospora, but should also lead to the secretion of the heterologous β-galactosidase into the culture medium. This secretion should be possible through the appropriate leader peptide, which is encoded by the homologous pheromone gene.

The plasmid pSMY5-2 ( FIG. 22) is used as the control plasmid, which carries the lacZ gene in the opposite direction to the plasmid pSMY5-1.

Example 7 Production of the pre-protein of human serum albumin

To produce the pre-protein of human serum albumin (HSA) in Sordaria, the hsa gene (from plasmid pPreHSA, Rhein Biotech GmbH) was in the express ion vector pMN112 cloned.  

For this purpose, the gene for the pre-protein of human serum albumin (PreHsa) was obtained by amplification. The gene was amplified using the oligonucleotides hsa1 and hsa2 using the plasmid pPreHsa as template DNA. The 1.8 kb amplificate has terminal NotI restriction sites. The PCR fragment was inserted into the XcmI-restricted cloning vector pMON 38201 (Borovkov and Rivkin 1997) by ligation. The resulting plasmid was named pMON-HSA. The insert of the plasmid pMON-HSA was checked by sequencing. This plasmid was then restricted with the enzyme NotI and the resulting 1.8 Kb fragment was inserted into the vector pMN112 restricted with NotI. The plasmid thus generated was given the name pSMY4-1 ( FIG. 23). The vector pSMY4-2 ( FIG. 24), also created by cloning, contains the PreHsa gene in inverse orientation and was used as a negative control for the expression experiments.

All constructs created were checked for their richness by control DNA sequencing activity checked.  

Table 1

Comparison of the most commonly used amino acid codons in E. coli, Saccharomyces cerevisiae (S. c.), Sordaria macrospora (S. m.), Drosophila melanogaster (D. m.) And primates (Prim). Those codons were shaded in the individual columns that are also used most frequently in Sordaria macrospora.

Table 2

Oligonucleotides used

Table 3

Recombinant plasmids

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SEQUENCE LISTING

Claims (33)

1. A method for producing heterologous protein in a filamentous fungus, comprising cultivating a homothallic fungus of the Sordariaceae family, which contains an expression cassette which contains the following elements in functional combination:

• a promoter active in the fungus of the Sordariaceae family,
• - a heterologous gene and
• a terminator active in the fungus of the Sordariaceae family,

and harvesting the protein produced in a manner known per se. 2. The method according to claim 1, characterized in that the homothallic Mushroom belonging to the genus Sordaria. 3. The method according to claim 2, characterized in that the homothallic Sordaria macrospora or Sordaria fimicola is fungus. 4. The method according to claim 1, 2 or 3, characterized in that it is the fungus is a sterile mutant. 5. The method according to any one of claims 1 to 4, characterized in that the Cultivation of the homothallic fungus takes place at 27 ± 2 ° C. 6. The method according to any one of claims 1 to 5, characterized in that the in the homothallic fungus of the Sordariaceae family active promoter of one filamentous mushroom. 7. The method according to claim 6, characterized in that the promoter Is a promoter from Sordaria macrospora. 8. The method according to claim 7, characterized in that the promoter of acl1 promoter, the ppg1 promoter, the cpc2 promoter or the ndk1 promoter Sordaria macrospora is.   9. The method according to any one of claims 1 to 8, characterized in that the in the fungus of the Sordariaceae family active terminator from a filamentous fungus comes. 10. The method according to claim 9, characterized in that the terminator Terminator from Sordaria macrospora is. 11. The method according to claim 10, characterized in that the terminator of acl1 terminator, the ppg1 terminator, the cpc2 terminator or the ndk1 termi nator from Sordaria macrospora. 12. The method according to any one of claims 1 to 11, characterized in that the heterologous gene for a protein glycosylated after expression in eukaryotes coded. 13. The method according to any one of claims 1 to 12, characterized in that it the heterologous gene is a growth factor, a cytokine, a gene clotting factor, an industrial protein or a technical enzyme. 14. The method according to claim 13, characterized in that the heterologous gene encodes one of the following proteins: G-CSF, GM-CSF, IL-1, IL-2, IL-4, IL-6, IL-1ra, IFN-α, IFN-β, IFN-γ, erythropoietin, glucoamylase, coagulation factor VIII, Coagulation factor XII, coagulation factor XIII, human serum albumin. 15. The method according to any one of claims 1 to 14, characterized in that between the promoter and the heterologous gene in the reading frame with the hetero logen gene a sequence is arranged, one in the fungus of the Sordaria family ceae functioning signal sequence encoded. 16. The method according to claim 15, characterized in that the signal sequence is a signal sequence from a filamentous fungus. 17. The method according to claim 16, characterized in that the signal sequence is a signal sequence from Sordaria macrospora. 18. A nucleic acid molecule comprising:

• 1. a promoter active in a homothallic fungus of the Sordariaceae family, which is selected from the following nucleic acids:
  • a) a nucleic acid with the sequence given in SEQ ID NO: 1;
  • b) a nucleic acid with the sequence given in SEQ ID NO: 2;
  • c) a nucleic acid with a sequence which has at least 50% identity with one of the sequences given in (a) or (b);
  • d) a nucleic acid which hybridizes with the counter strand of one of the nucleic acids given in (a) or (b);
  • e) a derivative of one of the nucleic acids specified in (a) or (b) obtained by substitution, addition and / or deletion of one or more nucleotides;
  • f) a fragment of one of the nucleic acids given in (a) to (e), which retains the function of the promoter active in the fungus of the Sordariaceae family;
  • g) a combination of several of the nucleic acids specified in (a) to (f), it being possible for the sequences of the nucleic acids to be the same or different; or
• 2. a nucleic acid with a sequence that is complementary to the sequence of one of the nucleic acids specified in (a) to (g).

19. Nucleic acid molecule according to claim 18, characterized in that the under (c) nucleic acid specified at least 70% identity with one of the in (a) or (b) specified sequences. 20. Nucleic acid molecule according to claim 18, characterized in that the under (c) specified nucleic acid at least 90% identity with one of the in (a) or (b) specified sequences. 21. Nucleic acid molecule according to claim 18, characterized in that the under (c) nucleic acid specified at least 95% identity with one of the in (a) or (b)  specified sequences. 22. Vector for the transformation of a homothallic fungus of the Sordariaceae family, characterized in that it contains the following elements in functional connection with one another:

• a promoter active in the fungus of the Sordariaceae family,
• - a heterologous gene,
• - a terminator active in the fungus of the Sordariaceae family and
• - a selection marker.

23. Vector according to claim 22, characterized in that in the mushroom from Fa milie Sordariaceae active promoter the acl1 promoter from Sordaria macrospora or the ppg1 promoter from Sordaria macrospora. 24. Vector according to claim 22, characterized in that the in the homothalli the fungus of the family Sordariaceae active promoter according to a nucleic acid one of claims 18 to 21, alternative (1). 25. Vector according to one of claims 22 to 24, characterized in that the in the fungus of the Sordariaceae family active terminator the acl1 terminator, the ppg1- Terminator, the cpc2 terminator or the ndk1 terminator from Sordaria macrospora is. 26. Vector according to one of claims 22 to 25, characterized in that the Selection marker is a hygromycin B resistance gene. 27. Host organism, characterized in that it is a homothallic fungus The Sordariaceae family is a vector according to any one of claims 22 to 26 contains. 28. Host organism according to claim 27, characterized in that it is the gat tung belongs to Sordaria. 29. Host organism according to claim 28, characterized in that it is Sordaria macrospora or Sordaria fimicola is.   30. Host organism according to claim 27, 28 or 29, characterized in that it is a sterile strain. 31. Kit comprising:

• a) a vector according to any one of claims 22 to 26 and
• b) a homothallic fungus of the Sordariaceae family suitable for producing heterologous protein.

32. Use of a nucleic acid molecule according to one of claims 18 to 21 or an expression vector according to any one of claims 22 to 26 or one Kits according to claim 31 for the expression of a heterologous gene under the con trolls of the promoter. 33. Use of a nucleic acid molecule according to one of claims 18 to 21 or an expression vector according to any one of claims 22 to 26 or one Kits according to claim 31 for the production of one or more proteins.