MXPA00006122A - Expression cloning in filamentous fungi - Google Patents

Abstract

Methods are provided for isolation of DNA sequences encoding proteins with properties of interest by means of expression cloning in filamentous fungal host cells. The isolated DNA sequences are useful in processes for producing the proteins of interest.

Description

CLONING OF EXPRESSION OF FILAMENTARY FUNGI FIELD OF THE INVENTION The present invention relates to methods for the identification of DNA sequences coding for protein isolates by expression cloning using filamentous fungi as hosts.

BACKGROUND OF THE INVENTION A growing number of protein components with interesting properties is produced by recombinant DNA technology. The production technology of recombinant DNA requires the availability of a DNA sequence that codes for the protein component of antibodies. Conventional methods of cloning DNA sequences coding for protein proteins has the disadvantage that each protein component has to be purified to allow the determination of its (partial) amino acid sequence or, alternatively, to allow the generation of specific antibodies. The (partial) amino acid sequences can then be used to design oligonucleotide probes for separation by hybridization. Alternatively, specific antibodies are used to immunoselect libraries of expression in E. coli, such as for example REF. 120837 lambda-gtll. Both methods require the purification and characterization of the protein of interest, which is a time-consuming process. The cloning of novel protein components can therefore be considerably accelerated by the use of a separation method which involves selecting clones expressing a desired protein activation. Such separation methods based on expression cloning have previously been used successfully for the identification of procapotic gene products, in, for example, Bacillus (see US 4,469,791) and E. coli (for example WO 95/18219 and WO). 95/34662). In some cases, eukaryotic gene products have also been identified using cloning of expression in a bacterium such as E. coli (for example WO 97/13853). However, in general, prokaryotes are less suitable hosts for the cloning of expression of eukaryotic genes because many of these genes are not expressed correctly in bacteria. For example, eukaryotic genes often contain monsters that do not splice into bacteria. Although this splicing problem can be avoided by using cDNA from eucalyptus genes for the cloning of expression in bacteria, many eucalyptus gene products are not produced in active form in bacteria because eukaryotic proteins are not They correctly bend in bacteria or those proteins are easily degraded by bacterial proteases. In addition, bacteria are usually unable to efficiently secrete eucaryotic proteins secreted in active form and in contrast to eukaryotes, they do not have the capacity to glycosylate proteins. More recently, a number of these problems have been overcome by the use of yeasts as hosts for the expression of cloning of eukaryotic genes. Trasse et al. (Eur. J. Biochem. (1989) 184; 699-706) have reported the identification of a mycotic α-amylase by cloning of mycotic genomic DNA expression in yeast Saccharomyces cerevisiae. Similarly, WO 93/11249 reports the identification of a nicotic cell by cloning of mycotic cDNA expression S. cerevisiae. Yeasts are, however, known for their poor secretory capacity, particularly when compared to filamentous fungi. A number of secretory heterologous proteins are only poorly secreted from the yeasts, if they are (see for example Kingman et al., 1987 Trends Biotechnol. 5_: 53-57). It is also known that yeasts hyperglycosylate heterologous proteins (Innis, 1989, En: Yeast Genetic Engmeenng, Barr, Brake S Valenzuela (eds), Butter orth, Boston, pp 233-246). Both the poor secretion and the Hyperglycosylation probably interferes with expression cloning in yeast because it can significantly reduce the chance of detecting a given DNA sequence that codes for a protein with properties of interest. This will apply in particular to sequences of DNA that codes for many useful enzymes that are produced by eukaryotes such as filamentous fungi and that are often secreted and glycosylated. There is thus a need for an expression cloning system that optimizes the opportunity to detect DNA sequences that encode secreted and possibly glycosylated proteins, and that is suitable for the identification of DNA sequences that encode proteins and enzymes produced by eukaryotes, of which in particular filamentous fungi. Alternatively, the expression cloning system should also be applicable to the identification of DNA sequences that encode eukaryotic proteins or filamentous fungi that are not secreted.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Construction of an intermediate expression vector, pGBTOP8. The details of this construction route are presented in the text.

Figure 2: Construction of the expression vectors pGBFin2 and pGBFin5. The details of this construction route are presented in the text. Figure 3: Physical map of pGBFinl2. Figure 4: Physical map of pGBFinll. Figure 5: Physical map of pGBFinl3. Figure 6: Physical map of pGBFinl7. Figure 7: Physical map of pGBFinld. Figure 8: Physical map of pGBFin22. Figure 9: Physical map of pGBFinl9. Figure 10: Physical map of pGBFin23. Figure 11: Physical map of pGBFind. Figure 12: Physical map of pAN8-l. Figure 13: Physical map of pGBFinl4. Figure 1: Physical map of pGBFinl5.

DESCRIPTION OF THE INVENTION The present invention relates to a method for isolating DNA sequences encoding an o. more proteins with properties of interest. The method preferably comprises the steps of: (a) preparing, in a suitable cloning vector, a DNA library of an organism suspected to be capable of producing one or more proteins with properties of interest; (b) transform cells host of filamentous fungi with the DNA library; (c) culturing the host cells obtained in (b) under conditions that lead to the expression of the sequence of DNA that codes for the proteins with properties of antibodies present in the DNA library; and (d) separating the clones from the transformed host cells expressing a protein with the properties of antibodies by analyzing the proteins produced in (c). Any cloning vector capable of transforming a filamentous fungal host cell and capable of accepting DNA fragments from a DNA library is suitable for use in the method of the present invention. The cloning vectors for use in the present invention thus comprise integrable cloning vectors which are integrated randomly or at a predetermined target site in the chromosomes of the filamentous fungal host cell, as well as autonomously maintained cloning vectors such as vectors based on the AMA1 sequence. In a preferred aspect of the invention, the integrable cloning vector comprises a DNA fragment which is homologous to a DNA sequence at a predetermined target site in the genome of the filamentous fungal host cell to direct the integration of the cloning vector to your default site. To promote targeted integration, the cloning vector is preferably lmealized before the transformation of the host cell. The lmealization is preferably carried out so that at least one but preferably any end of the cloning vector is flanked by sequences homologous to the target site. The length of the homologous sequences flanking the target site is preferably at least 0.5 kb, more preferably at least 1 kb, more preferably at least 2 kb. Integration of the cloning vector at a predetermined site will promote uniformity of expression levels in individual clones in the library, thereby increasing the opportunity for each clone in the library to be expressed at a desired level. In a more preferred aspect of the invention, the DNA sequence in the cloning vector that is homologous to the target site is derived from a gene which is capable of a high level of expression in the filamentous fungal host cell. A gene capable of a high level of expression, ie a highly expressed gene, is defined herein as a gene whose mRNA can constitute at least 0.5% (w / w) of the total cellular mRNA, for example under induced conditions, or alternatively , a gene whose genetic product can constitute at least 1% (w / w) of the total cellular protein, or, in the case of a secreted gene product, can be secreted at a level of at least 0.1 g / 1.

In yet another preferred aspect of the invention, the cloning vector comprises a promoter for the expression of DNA sequences encoding prine with properties of interest in the library, whereby this promoter is preferably derived from a filamentous fungus gene. highly expressed The person skilled in the art will appreciate the possibility that the DNA sequence homologous to the direction and the promoter sequence coincide in a DNA fragment. A number of highly expressed, preferred mycotic genes are given by way of example: the genes of amylase, glucoamylase, alcohol dehydrogenase, xylanase, glyceraldehyde-phosphate dehydrogenase or cellobiohydrolase of Aspergilli or Trichoderma. The most preferred highly expressed genes for these purposes are a glucoamylase gene from Aspergill us niger, a TAKA-amylase gene from Aspergillus oryzae, a gpdA gene from Aspergillus nidulans or a cellobiohydrolase gene from Trichoderma reesei. These highly expressed genes are suitable as target sites for the integration of cloning vectors and as a source of highly expressed promoters from which fragments of the library are expressed. In another preferred embodiment the uniformity of the expression levels of the individual library clones is provided by the use of a vector of cloning which is maintained autonomously in a filamentous fungus. An example of such an autonomously maintained cloning vector is described in Example 8 which describes the construction and use of a cloning vector containing the AMAl sequence. AMAl is a 6.0 kb genomic DNA fragment isolated from Aspergillus nidulars which is capable of Staying Autonomous in Aspergillus (see for example Aleksenko and Clutterbuck (1997), Fungal Genet. Biol. 21: 373-397). AMAl-based cloning vectors for use in the method of the present invention provide the advantage of higher transformation frequencies compared to integrable cloning vectors. AMAl-based cloning vectors also provide uniform expressions of individual library clones or reasonable levels, which allow for the easy detection of proteins with library properties. However, AMAl-based cloning vectors do not require maintenance of the selection pressure during the growth of the transformants to avoid loss of AMAl-based cloning vectors due to their poor segregation. The cloning vector can optionally further comprise a signal sequence that is oemably linked to the promoter and upstream of a cloning site, to allow the secretion of the proteins encoded by the DNA fragments in the library which are inserted into the cloning site. The secretion can facilitate the detection of proteins. In another embodiment of the invention, the cloning vector contains a gene that codes for a highly secreted protein, such as p? For example, the glucoamylase gene of A. niger. The gene highly secreted in the cloning vector contains a cloning site for the insertion of fragments from the library which are positioned so that the protein encoded by the library fragments are produced as fusion proteins with the highly secreted protein. This will improve its secretion according to EP-A-0 429 628.

The selection marker gene in the cloning vector can be selected from a number of marker genes that are useful for the transformation of filamentous fungi. By way of example those markers include, but are not limited to lacetamidase genes) amdS, auxotrophic marker genes such as the argB, trpC, or pyrG genes and resistance to antibiotics that provide resistance against, for example, phleomycin, hygromycin B or G418 In a preferred aspect of the invention, the cloning vector comprises a selection marker gene which is expressed by the mycotic host cell at sufficient levels during the selection of transformants to avoid a deviation of the transformants in copies il multiple cloning vector integrated in the genome of the host cell. A preferred selection marker gene for this purpose is the amdS coding sequence of A. nidulans fused to the gpdA promoter of A. nidulans. The host cell of the present invention is a fungal fungus, which is capable of being transformed with a cloning vector. For most of the filamentous fungi tested in this way it was found that they could be transformed using the transformation protocols developed for Aspergillus (derived from inter alia Tilburn et al., 1983, Gene 26: 205-221). Those skilled in the art will recognize that the successful transformation of host species of filamentous fungi is not limited to the use of the vectors, selection marker systems, promoters and transformation protocols specifically exemplified herein. A filamentous fungus is defined here as a eucapotic microorganism of the Eumycotma subdivision in the form of filaments, that is to say the vegetative growth that occurs due to the lengthening of the hypha at the beginning. The preferred filamentous fungal host cells are selected from the group consisting of the genera Aspergillus, Trichoderma, Fusarium, Penicilliumum, and Acremomi um In another preferred embodiment, for example when the protein of antibodies is a thermophilic protein, the preferred filamentous fungal host cells are selected from the group of thermophilic fungi consisting of the genera Talaromyces, Thielavia, Myceliophtora, Thermoascus, Sporotrichum, Chaetomi um, Ctenomyces, and Scytalidi um. In a more preferred embodiment of the invention, the filamentous fungal host cell is selected from the group consisting of A. nidulans, A oryzae, A. sojae, Aspergilli Group of A. niger and Trichoderma reeseí. The A niger Group is defined here according to Raper and Fennell (1965, In: The Genus Aspergillus, The Williams &Wilkins Company, Baltimoer, pp. 293-344) and includes all Aspergillus (blacks) included in the by those authors. In a further preferred aspect of the invention the filamentous fungal host cell, at least when used in the method of the invention in combination with an integrable cloning vector comprising a DNA fragment which is homologous to a DNA sequence. in a predetermined target site, it comprises multiple copies of the predetermined target site. More preferably, the host cell comprises multiple copies of a target site comprising a highly expressed gene, such as the highly expressed mycotic genes exemplified above. The advantage of Is host cells with copies Multiple of the target site is that the use of those host cells increases the frequency of integrable directed transformation, thereby increasing the opportunity to obtain efficient expression transformants for each individual clone in the library. The suspect organism produces one or more proteins with ingrown properties usually is an aucapote, preferably a fungus, of which the majority is preferably a filamentous fungus. It is known that these organisms produce a wide variety of proteins that are useful for industrial applications. In the method according to the invention, the library of DNA fragments of an organism that is suspected to produce one or more proteins with the properties of antibodies can be a genomic library or a cDNA library. However, a cDNA library is preferably used to avoid problems with the recognition of promoters or splicing signals in the host organism. The cDNA library is preferably prepared from mRNA isolated from a source organism when it grows under conditions that lead to the expression of the proteins with the properties of the antibodies. The method according to the invention can be applied to the isolation of DNA sequences that code for any protein with properties of interests if it exists an assay available for the detection of the protein when expressed by the fungal host cell. Preferably, the protein with properties of interest is an enzyme. Examples of enzymes that can be identified by the method of the invention are carbohydrases, for example cellulases such as endoglucanases, β-glucanases, cellobiohydrolases or β-glucosidases, hemicellulases -, or pectinolytic enzymes such as xylanases, xylosidases, manases, galactonases, galactosidases, rhamnogalacturonases, arabanasas, galacturonasas, liasas, or amiloliticas enzymes; phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases, transferases, or isomerases. After the transformation of the host cells of filamentous fungi with the DNA library the transformed clones are separated for the expression of the protein with properties of interest. Depending on the assay required for the detection of the protein with the properties of interest, the transformed clones are propagated and stored as colonies on solid media such as agar plates or in liquid media, whereby the clones of the individual libraries are grown , store and / or test in wells of microtiter plates.

A wide variety of protein detection systems with antisense properties are known to those skilled in the art. Because library clones can grow on solid as well as liquid media, the detection systems include any possible assay for protein detection or enzymatic activity. By way of example these test systems include but are not limited to tests based on clear zones around the colonies on solid media, as well as colopometric, photometric, turbidimetic, viscosimetic, mmunological, biological, chromatographic and other available assays. The person skilled in the art will understand that the usual adaptations to the cloning methods known in the art can equally apply to the method of the present invention. The adaptations include, but are not limited to, for example, separation of groups of library clones, separation of the same library for a number of different proteins with properties of interest, as well as resection, reisolation and cloning of positive clones to ensure better results. A variety of methods are available to those skilled in the art for the isolation of the DNA sequence encoding the protein with the properties of the transformed host cell identified in the method of separation, and for the subsequent characterization of the isolated DNA sequence The DNA sequences isolated by the separation method of the invention as described above are used to produce, or to improve the production of, a protein with encoded coding properties. by the DNA sequence. Advantageously, the filamentous fungal host cell transformed as isolated by the separation method described above is used directly in a process for the production of the protein with antiserum properties by culturing the transformed host cell under conditions which lead to the expression of the protein of interest and, optionally, recover the protein. However, often the initial transformed host cell isolated in the separation method of the invention will have an expression level which is satisfactory for separation purposes but which can be significantly improved for purposes of economic production. Up to this point, the DNA sequence is inserted into an expression vector which is subsequently used to transform a suitable host cell. In the expression vector the DNA sequence is operably linked to appropriate expression signals, such as a promoter, optionally a signal sequence and a thermometer, which are capable of directing the expression of the protein in the host organism.

A suitable host cell for the production of the protein is preferably a yeast or a filamentous fungus. The preferred yeast host cells are selected from the group consisting of the genera Sa ccharomyces, Kluyveromyces, Yarrowia, Pichia, and Hansenula. Host cells of preferred filamentous fungi are selected from the same genus listed above as the preferred host cells for the separation method. The most preferred filamentous fungal host cells are selected from the group consisting of Aspergilli from Group A. niger and Tpchoderma reesei. The suitable host cell is transformed with the expression vector by any of the different protocols available to those skilled in the art. The transformed host cell is subsequently used in a process to produce the protein of interest by culturing the transformed host cell under conditions that lead to the expression of the DNA sequence encoding the protein, and recovery of the protein. The present invention is better illustrated with the following examples.

EXAMPLES PhyA gene nomenclature phyA of A. ficuum, which codes for phytase. XylA gene xylA from A. tubigensis, which codes for the xylanase amdS gene amdS from A. niduians, which codes for acetamidase (Corrick et al., 1987 Gene 53: 63-71). GLOBE GLASS OF A. niger coding for the gpdA glucoamylase gene gpdA of A. nidulans, which codes for glyceraldehyde 3-phosphate dehydrogenase (Punt et al., 1988 Gene 69: 49-57) Pgp? promoter of gpdA Pgla? glade promoter for amdS terminator Tgla? Glala terminator GLA protein glucoamylase from A. niger Abbreviations kb kilo base Pb base pairs oligo oligonucleotide PCR Polymerase Chain Reaction PDA Potato Agar and Dextrose Oligonucleotides used The oligonucleotides used in Examples 1-3 are listed in the LIST OF SEQUENCES.

MATERIALS AND METHODS General Procedures Standard molecular cloning techniques, such as DNA isolation, gel electrophoresis, modifications by enzymatic restriction of nucleic acids, Southern analysis, transformation of E. coli, etc., were carried out according to what was described by Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratories, Cold Spring Harbor, New York and Innis et al. (1990) "PCR protocols, a guide for methods and applications" Academic Press, San Diego. Synthetic oligo deoxynucleotides were obtained from ISOGEN Bioscience (Maarssen, The Netherlands). DNA sequence analyzes were performed on an Applied Biosystems 373A DNA sequencer, according to the distributor's instructions.

Marker and DNA Hybridization The labeling and hybridization of the DNA were according to the ECLMR direct nucleic acid labeling and detection systems (Amersham LIFE SCIENCE, Little Chalfont, England or according to standard radioactive labeling techniques as described in Sambrooke et al 1989).

Transformation of Aspergillus niger. The transformation of A. niger was carried out according to the method described by Tilburn, J. et al. (1983) Gene 26, 205-221 and Kelly, J. & Hynes, M. (1985) EMBO J., 4, 475-479 with the following modifications: Spores were grown for 16 hours at 30 ° C on a rotary shaker at 300 rpm in minimal medium for Aspergillus. The minimum medium for Aspergillus contains per liter: 6g of NaN? 3, 0.52 g of KCl, 1. 52 g of KH2PO ", 1.12 ml of 4 M KOH, 0.52 g of MgSOj.7H20, 10 g of glucose, 1 g of casamino acids, 22 mg of ZnS0, | .7H20, 11 mg of H3BO3, 5 mg of FeS04.7H20 , 1.7 mg of CoCl2.6H20, 1.6 mg of CuS04.5H20, 5 mg of MnCl2.2H20, 1.5 mg of Na2Mo04.2H20, 50 mg of EDTA, 2 mg of riboflavin, 2 mg of thiamine-HCl, 2 mg of nicotinamide , 1 mg of pipdoxin-HCl, 0.2 mg of pantothenic acid, 4 g of biotin, 10 ml of Penicillin solution (5000 IU / ml) Streptomycin (5000 GU / ml) (Gibco). 91 Novozim 234MR (Novo Industries) was used in place of the helicase for the preparation of protoplasts; after the formation of the protoplast (60-90 minutes), KC buffer (0.8 M KCl, 9.5 mM citric acid, pH 6.2) was added to a final volume of 45 ml, the protoplast suspension was centrifuged for 10 minutes at 3000 rpm at 4 C on a rotating cube rotor. The protoplasts were resuspended in 20 ml of KC buffer and subsequently 25 ml of STC buffer (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 50 mM CaCl 2) were added. The protoplast suspension was centrifuged for 10 minutes at 3000 rpm at 4 C in a rotating bucket rotor, washed in STC buffer and resuspended in STC buffer at a concentration of 108 protoplasts / ml; - to 200 1 of the protoplast suspension, the DNA fragment was added, dissolved in 10 1 of TE buffer (10 mM Tris-HCl at pH 7.5, 0.1 mM EDTA) and 100 1 of a PEG solution (PEG 4000) 20% (Merck), 0.8 M sorbitol, 10 mM Tris-HCl at pH 7.5, 50 mM CaCl2); - after incubation of the suspension of DNA-protoplasts for 10 minutes at room temperature, 1.5 ml of PEG solution (20% PEG 4000 (Merck), 0.8 M sorbitol, 10 mM Tris-HCl at pH 7.5, 50 mM CaCl2) was added slowly, with repeated mixing of the tubes. After incubating for 20 minutes at room temperature, the suspensions were diluted with 5 ml of 1.2 M sorbitol, mixed by inversion and centrifuged for 10 minutes at 4000 rpm at room temperature. The protoplasts were gently resuspended in 1 ml of 1.2 M sorbitol and cultured on selective regeneration medium consisting of minimal medium for Aspergillus without riboflavin, thiamine, HCL, nicotmamide, pyridoxine, pantothenic acid, biotin, casammoacids and glucose, in the case of the selection of acetamide supplemented with 10 mM acetamide as the sole source of nitrogen and 1 M sucrose as an osmotic source and of C, or, on PDA supplemented with 1-30 μg / ml of fleomycin and sucrose ÍM as osmotic in the case of the selection of fleomicma. The regeneration plates were solidified using 2% Oxoid No. 1 agar. After incubating for 6-10 days at 30 ° C, the copdiospores of transformants were transferred to plates consisting of Aspergi II us selective medium (minimum medium that contained acetamide as the sole nitrogen source in the case of the selection of acetamide or PDA supplemented with 1-30 μg / ml of phleomycin in the case of the selection of phleomycin) with 2% glucose instead of sucrose and 1.5% agarose instead of agar and incubated for 5-10 days at 30 ° C. Unique transformants were isolated and this step of selective purification was repeated once, after which the purified transformants were stored.

DIRECT PCR ON MYCOTIC MICELLY Transformants were incubated on plates containing PDA for two days at 30 ° C. Approximately one third of a colony was incubated for 2 hours at 37 ° C in 50 1 KC buffer (60 g / 1 KCl, 2 g / 1 citric acid, bp 6.2), supplemented with 5 mg / ml Novozyrti * 234. Subsequently, 100 1 (Tris lOmM, 50 mM EDTA, 150 mM NaCl, 1% SDS, pH 8) and 400 1 of QIAquick ™ PB buffer (Qiagen Inc., Chatsworth, USA) were added. The extracts were gently suspended and loaded onto a rotating column of QIAquic A The columns were centrifuged at i minute at 13000 rpm in a microcentrifuge and washed once with 500 1 of QIAquickA buffer cushion. Traces of ethanol were removed with a final rapid centrifugation. . The chromosomal DNA (PCR standard) was eluted from the column by the addition of 50 1 H20 and the subsequent centrifugation for 1 minute at 13,000 rpm. The PCR reactions had 10 1 of buffer B of eL0NGaseMP (Life Technologies, Breda, The Netherlands), 14 1 of dNTP s (1.25 mM each), 1 1 of Enzymatic Mixture of eLONGaseA 1 1 of standard, and 10-30 pmol of each oligo, in a final volume of 50 1. The optimal amount of oligos was determined Experimentally with each batch purchased. On average, 10 to 30 pmol were used. The reactions were carried out with the following conditions of the cycle: Ix (2 minutes' 94 ° C, lOx (15 seconds at 94 A, 30 seconds at 55 ° C, 4 minutes at 68 ° C), 20x (15 seconds at 94 ° C, 30 seconds, 55 ° C for 4 minutes, starting at an incline of 20 seconds per cycle , at 68 ° C), Ix (10 minutes at 68 ° C). The samples were loaded on agarose gels to analyze the PCR products. Fermentations in flasks with Aspergillus niger agitation. A large batch of spores of A. niger strains was synthesized and controlled by culturing spores or mycelia on plates of selective medium or plates of PDA (Potato Agar and Dextrose, Oxoid), prepared according to the distributor's instructions. After growing for 3-7 days at 30 ° C the spores were collected after adding 0.01% Triton X-100 to the plates. After washing with sterile demystified water, approximately 107 spores of selected control transformant strains were inoculated in shake flasks, containing 20 ml of liquid pre-culture medium containing per liter: 30 g of maltose, H20, 5 g of yeast extract, g of hydrolyzed casein, 1 g of KH2P04, 0.5 g of MgS04.7H20, 0.03 g of ZnCl2, 0.02 g of CaCl2, 0.01 g of MnS04.4H20, 0.3 g of FeS04.7 H20, 3 g of Tween 80, 10 ml of penicillin (5000 IU / ml) / Streptomycin (5000 UG / ml), pH 5.5 and 1-30 μg / ml of phleomycin in the case of the selection of phleomycin. These cultures were grown at 34 ° C for 20-24 hours, 10 ml of this culture were inoculated in 100 ml of a fermentation medium of A. niger containing per liter: 70 g of glucose, 25 g of hydrolyzed casein, 12.5 g of yeast extract, 1 g of KH2P04, 2 g of K2S04, 0.5 g of MgS04.7H20, 0.03 g of ZnCl2, 0.02 g of CaCl2, 0.01 g of MnS04.4H, 0.3 g of FeS04.7H, 0 , 10 ml of penicillin (5000 IU / ml) / Streptomycin (5000 UG / ml), adjusted to pH 5.6 with 4N H2SO4, and 1-30 μg / ml of phleomycin in the case of the selection of phleomycin. Those cultures were grown at 34 ° C for 6 days. The samples taken from the fermentation broth were centrifuged (10 ', 5,000 rpm in a rotating cube centrifuge) and the supernatant was collected. Xylanase activity assays were performed 0 phytase (see below) on these supernatants.

Phytase Activity Assay 20 μl of supernatant (diluted when necessary) of fermentations of Aspergill us niger from the shake flask (as reference 20 1 of demystified water) were added to 30 1 of the substrate mixture, which contained acetate buffer. sodium 0.25 M, pH 5.5, phytic acid 1 mM (sodium salt, Sigma P-3168), in a microtiter disk of 96 wells, and incubated for 25 minutes at room temperature. The reaction was stopped by the addition of 150 1 of interruption mixture (14.6 g of FeS04.7h20 in 300 ml of 0. 67% of (NH4) eMo ^ 024.4H20, 2"of H, S04, 3.3% of Trichloroacetic acid). After incubation at room temperature for 5 minutes, the absorbance of the blue color was measured spectrophotopically at 690 nm in an Anthosreader (Proton and Wilton). The measurements are indicative of phytase activity in the range of 0-175 U / ml. The activity of the phytase was measured according to that described in EPO 0 420 358 TO.

Xylanase activity assay The supernatant (prediluted when necessary) was diluted 5 times in 0.25 M sodium acetate buffer, pH 4.5. 20 μl of diluted supernatant was transferred to the microtiter discs and 50 μl of substrate (4% w / v RBB-Xylan, Remazol Brilliant Blue, dissolved at 70 ° C in demineralized water) were added and mixed thoroughly by pipetting up and down. The reaction is incubated for 30 minutes at room temperature.

The reaction was stopped by the addition of 200 1 of 96% ethanol and incubated for 10 minutes at room temperature.

After having finished the reaction the plates Microtitrators were centrifuged for 10 minutes at 2500 rpm in a Beckman GPK centrifuge at a warm temperature. 100 1 of the supernatant was transferred to a new microtiter disk and the absorbance of the blue color was measured spectrophotometrically at 620 nm in an Anthosreader (Proton and Wilton). The specific activity was calculated on a calibration curve using a standard xylanase dissolved in a 0.25 M sodium acetate buffer, pH 4.5. The measurements are indicative of a xylanase activity in the range of 0-150 EXU / ml. The units of xylanase activity were defined as in EP 0 463 706.

Isolation of RNA A strain of A. tubigensis DS116813 (CBS323.90) was grown in minimal medium for Apergillus (per liter 6 g of NaN03, 0.52 g of KCl, 1.52 g of KH2P04, 1.12 ml of 4 M KOH, 0. 52 g of MgS04.7H20, 22 mg of ZnS04.7H20, 11 mg of H3B03, 5 mg of FeS04.7H20, 1.7 mg of CoCl2.6H20, 1.6 mg CuS0 .5H20, 5 mg of MnCl2.2H20, 1.5 mg of Na, Mo04.2H20, 50 mg of EDTA, 10 g of glucose) supplemented with 0.1% yeast extract and 3% xylan taken from oats (Serva). 100 ml of medium were inoculated with 2.10 spores and cultured in a rotary incubator at 30 A and 300 rpm for 48 hours. The mycelium was harvested by filtration using a Miracloth filtration wrap, washed thoroughly with water Dexneralized and squeezed between paper towels to remove excess water. The mycelium was immediately frozen in liquid nitrogen and crushed to a fine powder using a mortar and pestle. The resulting powder was transferred to a sterile 50-μm tupe and weighed, after which for every 1-1.2 g of crushed mycelium 10 were added. my reagent TRIzol (Gioco / BRL) (a maximum of 25 ml per tube). The mycelium powder was immediately solubilized by mixing vigorously (forming a vortex, 1 minute), followed by a 5 minute incubation at room temperature with occasional mixing. A volume of 0 2 (original TRIzol) of chloroform (thus 2 ml per 10 ml of TRIzol originally used) was used, vortexed and left at room temperature for 10 minutes. Subsequently, the mixture was centrifuged at 4 ° C, 6000 g for 30 minutes. The aqueous phase was transferred to a fresh tube and the total RNA was precipitated by adding a volume of 0.5 (original TRIzol) of isopropyl alcohol (thus 5 ml of isopropyl alcohol per 10 ml of TRIzol originally used). After 10 minutes of precipitation at room temperature, the RNA was recovered by centrifugation for 30 minutes at 6000 g. After removal of the supernatant, the RNA pellet was rinsed with a volume of 70% ethanol. After the removal of ethanol, the RNA pellet was air dried. The dried RNA pellet was dissolved in 3 ml of GTS buffer (100 mM Tris-Cl, pH 7.5, 4M guanidinium thiocyanate, 0.5% sodium lauryl sarcosinate.) 10 μl of RNA solution was used to determine the quality and concentration of nucleic acids.

RNA purification via centrifugation in CsCl solution The isolated RNA was further purified by a modification of the method described by Sambrooke et al. (Molecular cloning, second edition Cold Spring Harbor Laboratory, 1989). A solution of CsCl / EDTA was prepared by dissolving 96 g of CsCl in 70 ml of EDTA lOmM, pH 7.5. DEPC was added to a final concentration of 0.1%. The solution was left for 30 minutes at room temperature and then placed in an autoclave for 20 pounds per square inch (psi) (137.89 kPa) over a liquid cycle. After cooling the solution the volume was adjusted to 100 ml with water treated with DEPC. 1.5 ml of this CsCl / EDTA solution was added to each Polyallomer ultracentrifuge tube (2"x 0.5" (5.8 x 1.27 cm), 5 ml capacity). 3 ml of RNA samples (in GTS) were placed on a 1.5 ml mattress of CsCl / EDTA. The ultracentrifuge tubes were filled up to 5 mm from the top with GTS. The filled ultracentrifuge tubes were exactly balanced with GTS and placed in even ultracentrifuge cubes. Ultracentrifuge tubes were centrifuged at 35,000 rpm for 18 h at 20 ° C with slow acceleration and braking for deceleration. After centrifugation the top layer on top of the mattress CsCl, and part of the mattress were removed with clean pasteur pipettes, respectively (0.5 cm of mattress C? C1 was left in the tube). The bottom of the tube was cut with a hot doctor blade, after which the remaining fluid was removed. The bottom of the tube was filled with 70% ethanol at room temperature. The bottom of the tube was inverted and the RNA pellet was air dried. The RNA pellet was dissolved in 1 ml of TE (elution buffer of the PHARMACIA mRNA purification kit, see mRNA isolation). Again, 10 μl was taken to verify the quality and quantity.

Isolation of mRNA A modified protocol (using gravity flow instead of centrifugation) of the PHARMACIA purification kit (Cat # 27-9258-02) was used for mRNA isolation.

The PHARMACIA column was completely resuspended by repeated inversion, after which the column was packed via gravity flow. The column was placed at a temperature of 50 ° C and washed with 1 ml of High Buffer in Sales. The mRNA solution (in TE) was heated at 65 ° C for 5 minutes, after which 200 μl of sample buffer and the solution were added.

RNA was loaded onto the column. The flow was collected and loaded again on the column. The column was washed 3 times with 0.5 ml of High Buffer in Sales and then several times with 0.5 of Low Buffer in Sales until no UV absorbing material was eluted from the column. The pol? (A) + RNA was eluted with preheated Elusion damper (65 ° C) from which 4-5 fractions 0.25 ml were collected. The concentrations of the different fractions were determined spectrophotometrically and fractions with an O.D ratio 260/280 of at least 1.5 were pooled. We added 0.1 volume of Sample Buffer and 2 volumes of absolute ethanol and the solution was incubated overnight at -20 C.

Northen Analysis The RNA was separated by electrophoresis on a 1% agarose gel containing 6% formaldehyde and using 1 x MOPS (20 mM MOPS / pH7.0, 1 mM EDTA) as an electrophoresis buffer. Samples (approximately 10 g of total RNA or 1 g of mRNA) were dissolved in a total volume of 20 1 of charge buffer (final concentrations: MOPS 20 Mm / pH 7.0, EDTA 1 Mm, 6% formaldehyde, 50% formamide, 0. 05 g of ethidium bromide) and denatured by heating at 68 ° C for 10 minutes. After electrophoresis for 3-4 hours at 100 Volts, the RNA was visualized using a UV illuminator. For the Northen analysis the gel was washed for 20 minutes in demineralized water and transferred to a Hybond-N + nylon membrane (Amersham) by capillary staining. The RNA was fixed to the membrane by baking at 80 ° C for 2 hours. Specific transcripts were detected using the ECLMR system or standard radioactive labeling technique as described in Sambrooke et al. 1989 CDNA analysis by electrophoresis on an alkaline agarose gel This step of control analysis revealed the size of cDNA synthesized and was used to verify the potential presence of the hairpin structure. Since the specific activity of the second synthetic strand was much smaller than that of the synthesis of the first strand, the volume of the synthesis of the second strand used was 10 times that of the synthesis of the first strand. A thin 1% agarose gel was prepared, melting 0.6 of agarose in 54 ml of water, cooling to 55 ° C, adding 6 ml of 10X of alkaline buffer (0.3 M NaOH, EDTA 20 mM), mixing and emptying. Samples were mixed (1: 1) with 2X alkaline gel loading buffer (30 mM NaOH, 20% glycerol, 1/10 volumes of saturated bromophenol blue). The samples were run (along with molecular weight markers marked with 32P) in IX of alkaline buffer. The gel was fixed for 30 minutes in 7% acetic acid and stained on Whatman 3MM paper and dried. The dried gel was exposed to an X-ray film which was revealed in an automatic film processor.

CDNA Synthesis For the cDNA synthesis both the Superscript ™ choice system (Gibco-BRL) and the STRATAGENE cDNA synthesis kit have been used. When the cDNA was synthesized with the Superscript ™ choice system, 5 μg of mRNA was used according to the manufacturer's instructions, except that oligonucleotide 6967 was used for the synthesis of the first strand and oligonucleotides 7676 (phosphorylated at the position 5') and 7677 (non-phosphorylated) were used as adapters. Annealing of oligonucleotides 7676 and 7677 was achieved by mixing equimolar amounts of both oligonucleotides in 10 mM Tris-HCl / pH 7.5, 1 mM EDTA, 1 mM MgCl 2. The mixture was covered in a water bath at 80 ° C for 10 minutes after which the water was allowed to cool slowly to room temperature. For the synthesis of the cDNA with the Strategene cDNA Synthesis Kit, the protocol had to be optimized to clone the described pGBFIN vectors. The main changes were: 1) the amount of cDNA synthesized was quantified by TCA precipitation. 2) the phosphorylation of the ends of the cDNAs was omitted and the cDNAs were ligated to the vector DNA with the phosphorylated ends. 3) the cDNA was extracted with phenol / chloroform after digestion with XhoI instead of fractionation by size. 4) the use of both synthesis of the first strand MMVL-RT (STRATAGENE) and TERMOSCRIPT (Gibco / BRL) resulted in consistent cDNA with greater lengths than the use of any single enzyme. 5) control reactions treated with [alpha32P] dATP (800 Ci / mmol to prevent interference with the synthesis) were carried out together for quality control; The components of the first strand and the poly (A) + RNA components were combined and mixed according to the protocol and left for 10 minutes at room temperature to allow annealing of the primer pattern. 1.5 μl of MMLV-RT (50 U / μl) and 1 μl of THERMOSCRIPT (15 U / μl, GibcoBRL) were added to the reaction of the first strand to obtain a final reaction volume of 50 μl. After mixing, 5 μl of the reaction mixture was taken and added to 0.5 μl of [alpha32P] dATP (800 Ci / mmol) to obtain a radioactive control reaction of the first strand. Synthesis reactions of the first strand were incubated at 35 ° C for 0.5 hours followed by 55 ° C for 0.5 hours. The non-radioactive synthesis reaction of the first strand was placed on ice and 20 μl of 10X of the second strand remover, 6 μl of the second strand dNTP mixture, 114 μl of sterile distilled water, were added to it. μl of [alpha32P] dATP (800 Ci / mmol), 2 μl of RNase H (1.5 U / μl) and 11 μl of DNA polymerase I (9.0 U / μl). After mixing the reaction mixture was incubated at 16 ° C for 2.5 hours. After incubation, 10 μl was removed and frozen.

Estimation of the amount of cDNA synthesized by TCA precipitation 1 μl of the radioactive reaction (control) of the first strand was mixed with 20 μl of water. In a similar manner, 2 μl of the synthesis reaction of the second strand was mixed with 200 μl of water. 5 μl of the solution thus obtained (4X for each control solution) was stained on a Whatmann glass fiber filter (GF / C or GF / A, 23 mm in diameter) and air dried. The filters were transferred to 200 ml of ice cold 5% trichloroacetic acid (TCA) and 20 mM sodium pyrophosphate. The ice-cooled TCA / sodium pyrophosphate solution was changed 3-4 times every 2 minutes. The filters were rinsed with 70% ethanol at room temperature for 2 minutes. Each filter was inserted into a flare flask, 10 ml of flasher was added and the radioactive material was counted, after which the specific activity of the ADCc was calculated.

Blunting of the terminal end of the cDNA and ligation of adapters The synthesis reaction of the second strand was added 23 ul of blunt dNTP mixture and 2 ul of Pfu DNA polymerase (2.5 U / ul), after which the mixture of reaction was incubated at 72 ° C for 30 minutes. The reaction mixture was extracted with phenol / chloroform [200 ul of solution 1: 1 (volume / volume), pH 7-8], extracted with chloroform [200 ul] and the cDNA was precipitated by the addition of 20 ul of 3 M sodium acetate and 400 ul absolute ethanol followed by incubation overnight at -20 ° C. He CDNA was collected via centrifugation, washed with ethanol 70% and the cDNA pellet obtained was dried with air and resuspended in 8 ul of adapter solution. 1 ul of 10X ligase buffer, 1 ul of rATP and 1 ul of T4 DNA ligase were added and the ligation mixture was incubated either at 8 ° C overnight or at 4 ° C for 2 days. Then, the ligase was inactivated by incubation at 70 ° C for 30 minutes.

Restriction enzyme digestion of the cDNAs and size fractionation 10 μl of sterile water was added (to compensate the volume in the omitted phosphorylation step), 28 ul of restriction enzyme buffer and 3 ul of restriction enzyme (40 U / ul) to the cDNA. The reaction was incubated at 37 ° C for 1.5 hours. After adding 30 ul of sterile water and 10 ul of 10X STE the reaction mixture was extracted with 100 ul of phenol / chloroform followed by an extraction with 100 ul of chloroform. The cDNAs were collected via centrifugation after adding 200 ul of absolute ethanol (and overnight precipitation at -20 ° C), they were dried and resuspended in 14 ul of IX STE to which 3.5 ul of column loading dye had been added. The matrix of SEPHAROSE CL-2B and STE buffer were equilibrated at room temperature, resuspended and used to empty a column in a glass pipette. 1 mi. After sedimentation of the SEPHAROSE matrix, the column was washed with 10-15 ml of STE. The sample was loaded, after which 3 ml of STE were added and fractions of 0.3 ml were collected (verification of the entire process with Geiger counter). The radioactivity in each fraction was estimated by measuring a flashing counter.

Analysis by electrophoresis on non-denaturing gel 3 ml of 10X TBE, 5 ml of 30% acrylamide [(weight / volume), 29: 1 of acplamide: b? S-acr? Lam? Da) and 22 ml of water were mixed. , they were degassed, after which 150 ul of fresh 10% ammonium persulfate and 20 ul of TEMED were added. The solution was applied to the assembled gel modules and allowed to settle. 8 ul of each fraction (collected from the column) containing radioactivity was taken and mixed with 2 u of 5X of buffer of cargo. The samples were loaded together with a radioactive molecular weight marker and subjected to electrophoresis. After electrophoresis, the gels were fixed in 100 ml of acetic acid in 7% for 20-30 minutes, dried on Whatmann 3MM paper and exposed to a X-ray film Processing of the cDNA fractions Based on the results of the non-denaturing gel electrophoresis, the fractions containing the desired size distribution were pooled. (Normally, fractions with cDNAs above 0.5 kb are collected, if desired, the sublibraries can be reconstructed by ligation of the different size fractions selected with the vector). 2 ul of the collected fractions were removed and stained on a GF / C Whatman filter. The filter was washed 3 times with 10 ml of TCA solution cooled in ice / pyrophosphate, rinsed with 10 ml of 70% ethanol, dried and counted with a liquid flasher to estimate the amount of cDNA present. The collected fractions were precipitated overnight at -20 ° C adding 2 volumes of absolute ethanol and collected via centrifugation. The precipitation was aided by the addition of purified tRNA at 10 ug / ml as carriers. After washing, the sediment was air-dried and resuspended in TE or sterile water at 10-20 ng / ul. The cDNAs were ligated to the vector DNA with an excess at a molar ratio of 5: 1.

Subsequently, the ligation mixture was transformed to XLIO-Gold bacterial cells (STRATAGENE) according to the protocol (corresponding).

EXAMPLE 1 1.1 Description and construction of the expression vector pGBFin 2 1.1.a Rationale Separation by expression in A. niger can be improved by a number of factors, which when used in combination probably produce the most optimal result. The effective transformation system is preferred to obtain a sufficient number of mycotic transformants. Care should be taken to keep the cDNAs in the library intact during the cloning procedure. In addition, the separation will be more successful when the expression levels of the cDNA gene product are sufficiently high. Therefore, in the expression cloning constructs the functionalities used to direct the expression of the cDNAs were derived from a gene that is highly expressed. In the integrable system the cassette expression is preferably directed to a place which is highly expressed and which, even more preferably, has been amplified in the genome. In this example, glaA was chosen, which is present in 3 copies in the genome of A. niger strain DS2978 (deposited on April 8, 1997 in Centraalbureau voor Schimmelcultures, Baarn, The Netherlands under accession number CBS 646.97). Several expression vectors, designed for efficient management at this site and allowing different DNA cloning strategies were constructed and tested (see examples 1-7). 1. 1.b Basic design of integrable expression vectors Linear DNA molecules are preferred for targeted integration in the genome of filamentous fungi. In addition, both 5 'and 3' ends (flanking) preferably consist of DNA homologs at the desired integration site. Fragments of the transformation, therefore, comprise the expression cassette (the gene of interest regulated by a suitable promoter and terminator), as well as a selection marker flanked by the 5 'and 3' address domains. These fragments are cloned into an E. coli vector for propagation of the plasmid. The resulting expression vectors are designed so that the sequences of E. ^ já? ^ coli are removed during the linearization and isolation of the transformation fragment. For the selection of transformants, the amdS selection marker was used, the expression of which is controlled by the gpdA promoter of A. nidulane The use of the strong gpdA promoter will predominantly result in transformants in a copy. To achieve high levels of expression the cDNA was fused to the glaA promoter. A number of unique restriction site combinations were introduced for the enzymes (rare cut) (for example pací and AscI [Example 1]), EcoRI and Xhol [Examples 4 and 6] or HmdlI I and Xhol [Example 7]) and -1 a set of integrable expression vectors at the proposed initial transcription point of the glaA promoter. Since the targeted insertion (direction) of the rDNA molecules in the genome occurs through homologous recombination, the rDNA cassettes are preferably flanked by DNA fragments homologous to the target site of the genome. Therefore the integration cassette is flanked at both 5 'and 3' ends by approximately 2 kb of the DNA sequence homologous to the glaA site. To facilitate the removal of E DNA. coli of the construct, unique Notl sites were introduced (Notl restriction sites are rare, reducing thereby minimizing the risk of undesirable digestion of the introduced cDNA). 1. 1. c Construction of an intermediate expression vector, pGBTOP8 Oligonucleotides AB5358 and AB5359 were annealed in equimolar amounts and ligated into the restriction sites of the coRI and pinciIII of pTZ18R, thereby introducing a NotI-XhoI-coRI-SnaBI polylinker. -fímdIII (the £ coRI site was not restored). The resulting plasmid was named pGBTOPl. An XhoI-coRI fragment of 1.8 kb was isolated., comprising the promoter region of the glaA gel, of plasmid pAB6-l (containing the entire A niger glaA site on a 15.5 kb JilindlII fragment, cloned into pUC19 as described in one of our previous patents, EP-A -0 635 574) and cloned into the Xhol and EcoRI sites of the plasmid pGBTOPl, producing the plasmid pBGTOP2. To mediate the direction of constructs to the 3 'non-coding region of the glaU, two different portions of this region were cloned on either side of the expression cassette. These parts were designated as 3"glaA and 3" glaA, the last being the most downstream part of the region. The fragment 3"glaA was generated by PCR using the oligonucleotides AB5291 and AB5292 (the oligo AB5291 was designed to disrupt an undesirable EcoRI site). The generated PCR fragment was used as a standard in a second PCR reaction using the oligonucleotides AB5361 and AB5292, thereby generating a? Phot site in the fragment. The PCR fragment was digested with? Fotl and Xhol and cloned into the corresponding restriction sites of plasmid pGBT0P2, yielding pGBT0P5. The undesirable EcoRI sites in the 3 'non-coding region of the glaU were disturbed using a PCR method. A fusion PCR reaction was carried out using the oligo AB5288 (5 '), AB5293 (3' inverted), AB5290 (internal, inverted) and AB5289 (internal, encoder). Oligos AB5290 and 5289 were complementary oligos designed to disrupt the £ coRI site in that position while oligo AB5293 was designed to disturb a second coRI site. The resulting fusion PCR product was digested with SnaBI and HindIII and cloned into the corresponding sites of pGBTOP2, resulting in pGBTOP6. PGTOP6 was used as a standard in a second PCR reaction using oligonucleotides AB5363 and AB5567. The resulting PCR product was digested with SnaBI and fiindIII and cloned into the sites corresponding to pGBTOP5, resulting in plasmid pGBT0P8 (see Figure 1). 1. 1.a Construction of pGBFin2. Using oligonucleotides 6963 and 7266, and 10 ng of the vector pAB6-l (EP-AA 635 574) as a standard, a specific PCR fragment PgiaA- was generated. This fragment was digested with EcoRi and Smal and introduced into the pGBTOP-8 vector. digested with EcoRi and SnaBI, resulting in the pGBFinl vector. The introduced PCR fragment sequence was confirmed by sequence analysis. Xhol sites were introduced to the Pgpds-amd? by PCR. Oligonucleotides 7423 and 7424 were used and the plasmid pGBAA [alpha] (EP-A-0 635 574) as a template was used to generate a fragment of 3.1 kb. This fragment was digested with EcoRI and introduced into the EcoRI site of pTZ19R, resulting in plasmids pTZamdSX-1. The 2.6 kb Xhol-Clai of pTZamdSX-1 was replaced by the corresponding fragment of plasmid pGBAAS-1 to avoid mutations caused by the PCR process. The 0.5 kb Kpnl-Clal of pTZamdSX-1 was replaced by the corresponding fragment of plasmid pTZamdSX-1 to avoid mutations caused by the PCR process. Sequence analysis of the remaining 0.5 kb fragment of the resulting pTZamdSX-2 plasmid revealed a single mutation in the PgPdA- fragment The 3.1 kb Xhol fragment, comprising the selection cassette PgpdA-amdS, was isolated from the vector PTZamdSX-2 and introduced into the unique Xhol site of pGBFinl, resulting in the pGBFin2 vector (see Figure 2). 1. 2 Expression of the phytase used for the expression vector pGBFin2 1.2.a Rationale Both the direction and the efficient expression of the construct to the glacia loci site of A. niger DS2978 and a sufficiently high expression level of the cDNA of interest are preferred for optimal application of separation by A. niger expression. Therefore, the properties of the expression construct were tested using a model gene, phyA, for which the expected production of protein by the gene copy integrated in a glaA site had previously been established. 1. 2.b Construction of the phytase expression vector, pGBFin5 A phyA fragment was generated by PCR using the oligonucleotides 6964 and 6965 and the plasmid pAF-2S (described in EP-A-0 420 358) as a standard. The PCR fragment was cloned into the Smal site of vector pTZ18R, resulting in pTZFytl. Sequence analysis of the insert of pTZFytl did not reveal deviations from the sequence present in pAF2-2S. An Ascl-Pacl fragment was isolated from 1. 7 kb comprising the complete phyA sequence of pTZFytl and cloned into the pBSFin2 digested with Ascl-Pacl, resulting in the pGBFin 5 vector (see Figure 2). 1.2.C Transformation of Aspergillus piger DS2978 with pGBFin5 Plasmid pGBFin 5 (100 g) was digested with Notl (150 Units, 4 hours at 37 ° C). The protein was removed by extraction with an equal volume of Phenol-Chloroform-10-Alisoisyl (24: 23: 1). The DNA was concentrated by precipitation with alcohol and used for transformation of A. niger DA2978 as described. The transformants were purified on plates of minimal selective medium and stored later. 15 1.2.d Analysis of the transformants of pGBFin5 The direction of the integration cassette of the glaA site was analyzed for 24 independent transformants, using oligonucleotides 5454 and 5456, and for the presence of the phyA gene using the oligonucleotides specific to phyA 6964 and 665. A PCR product indicative of the correct direction of the integration cassette pGBFin5 was found to a glaA site in a high number of transformants (12 of 24 = 50%), although all the transformants showed a product of the PCR indicative of the presence of a copy of phyA in its genome. Six positive transformants were analyzed for the production of phytase in a bottle-shake fermentation experiment. The phytase activity for all transformants was 140-180 U / ml. Such a production level is indicative of the integration of a copy of pGBFin5 in each transformant. It was concluded that both of the direction frequencies and expression levels were sufficient to use the expression system designed in the expression cloning experiments.

EXAMPLE 2 2.1 Construction and analysis of a cDNA library 2.1.a Background Expression libraries were constructed from a pool of mRNA that was expected to comprise the expression transcripts of interest. For this reason it is preferable, although usually not necessary, to isolate the mRNA from the isolated mycelium of a culture grown under inducing conditions. The isolated mRNA was analyzed for the presence of the transcript of interest and for the quality of the mRNA. If the mRNA is intact and comprises the transcript of Intents can be used for the synthesis of cDNA. The cloning of the cDNA into the pGBFm2 expression vector requires the presence of a PacI site on the 5-terminus and of an AscI site at the 3 'end of the cDNA. Therefore, the oligonucleotide primer of the first strand and the adapter sequences used were designed to satisfy those prerequisites. The adapter was designed in such a way that it is compatible with the Pací site in the pGBFm2 while the Pací site is not restored after the ligation of the cDNA into the vector. This makes possible the discrimination between vector molecules comprising a cDNA insert and vector molecules without insert. 2. 2 Preparation of a cDNA library of A. tubigensis. MRNA induced for xylanase activity. A. tubigensis DS116813 (CBS 323.90) was grown under inducing conditions. Samples of medium were taken at different times and analyzed for xylanase activity. Maximum activity was reached after 66 hours of culture, while the levels of xylanase activity remained constant until 7 days after the start of the experiment. Mycelium samples were taken at different times and the total RNA was isolated from these samples. The presence of specific transcripts was analyzed of xylA in a Northern spotting experiment using a specific xylanase probe. The maximum levels of xylA mRNA were determined after 48 hours of induction although the xylA mRNA was still detectable 66 hours later. After prolonged incubation of the mycelium in inducer medium there was no detectable xylA mRNA. In all cases, the specific transcript of xylA was apparently intact. The mRNA was isolated from the total RNA isolated after 48 hours of induction. After the Northern analysis, which shows that xylA mRNA was intact, this mRNA was used for cDNA synthesis (according to the Superscript ™ choice system [Gibco-BRL]) using oligonucleotide 6967 as a primer for the synthesis of the first strand. After annealing of a specific PacI binder, the cDNA was digested with AscI and size-separated using Sephacryl columns supplied with the cDNA synthesis kit [Superscript ™ choice system [GibcoBRL]). Both mRNA and cDNA were analyzed for the presence of intact xylA in the samples using Northern and Southern blot analysis respectively and by PCR analysis. The resulting cDNA was ligated into pGBFin 2 digested with Ascl-Pacl and introduced by electroporation into E. coli resulting in a primary library of approximately 17,000 transformants.

The analysis of 24 hills randomly revealed 5 plasmids without insert, while the remaining plasmids had insert sizes between 0.5 and 2 kb. The E. coli library was collected by detaching the plates in a total volume of 25 ml of 2xTY medium. 10 ml of medium was used to prepare glycerol standards while 2xTY was added to the rest of the E. coli suspension to a final volume of 100 mi. The plasmid DNA was isolated from this culture after 2 hours of growth at 37 ° C.

EXAMPLE 3 3.1 Construction and analysis of an expression library in A. niger 3.1.a Foundation A was transformed. niger DS2978 using the DNA isolated from the cDNA library in E. coli, as described in Example 2.2 above. The transformants were selected by the presence of the amdS selection marker growing on acetamide as the sole source of N. Since both the amdS selection marker and the cDNA expression cassette are present on the integration fragment growing on acetamide this is indicative of the presence of a cDNA expression cassette. The conidiospores of the amdS positive transformants are - * - * "- * transferred to plates of selective medium to avoid isolation of false positives and subsequently transferred to microtiter plates comprising slices of solidified PDA. This master library was used for the selection of the production of enzymes of interest, for example xylanase. Since it would be useful if transformants that produce enzymes could be used directly for the production of enzymes on a large scale it is of interest to determine the levels of enzyme production in fermentations in jars with agitation. 3. 2 Transformation of A. niger DS2978. The DNA was isolated from the amplified E. coli cDNA library as described. The total plasmid DNA (100 g) was digested for 4 hours at 37 ° C with α fotl (150 U) to remove the plasmid sequences derived from E. coli and with Pací (30 U). After purification of the DNA by extraction with an equal volume of phenol: chloroform: isoamyl alcohol (24: 23: 1) the DNA was recovered by precipitation with alcohol and dissolved in 100 1 of sterile demineralized water. Multiple transformations of A. niger DS2978 were performed using 2107 protoplasts and 10 g of plasmid DNA. After approximately 10 days of incubation at 30 ° C, 1900 transformants were removed and the conidiospores were transferred to plates containing a selective medium.

After 3 days of incubation at 30 ° C, the conidiospores of each transformant were transferred to individual wells in a 96-well microtiter disk, each well containing approximately 100 1 of solidified PDA. 3. 3 Analysis of the expression library of A. niger. The conidiospores of individual transformants were transferred to xylanase detection plates made of minimal medium for Aspergillus (6 g of NaN03, 0.52 g of KCl, 1.52 g of KH2P04, 1.12 ml of 4 M KOH, 0.52 g of MgSO4.7H20, 22 mg of ZnS04.7H20, 11 mg of H3B03, 5 mg of FeS04 7H20, 1.7 mg of CoCl2.6H20, 1.6 mg of CuS04.5H20, 5 mg of MnCl2.2H20, 1.5 mg of Na2Mo04.2H20, 50 mg of EDTA, 10 g glucose) supplemented with 2% of 2% oat dextrose xylan and 2% bacteriological agar (Oxoid, England), which had a cloudy appearance due to the presence of dissolved xylan After 2 days of incubation 30 ° C the formation of a halo of 10 colonies could be observed, indicating the degradation of xylan by xylanases. Conidiospores of positive transformants were isolated and used to inoculate PDA plates. The DNA was isolated from the single colonies and analyzed by PCR for the integration of the expression plasmid in the glaA site ("direction") using the oligonucleotides 5454 and 5456. 8 of the 17 colonies showed to be directed to one of the glaA sites (47%). 3. 4 Analysis of xylanase production levels in the transformants The xylanase-producing transformants, identified in the xylanase plate assay, were grown in bottle fermentation with shaking. Media samples were taken after 5 days of fermentation and analyzed for the activity of the xylanase. The results are presented in Table I. 3. 5 Genetic analysis of strains that produce xylanase 3.5.a Rationale Multiple genes encoding xylanase in fungi have been found. Therefore, it was of interest to determine whether each xylanase-producing strain identified in the expression cloning experiment contains an identical cDNA. In addition, clear differences were found between the individual strains that produce xylanase. These differences could be caused both by the presence of different genes encoding the xylanase and by differences in the 5 'non-coding region of the cDNA. The latter could be due to a partial degradation of the mRNA during the ^ dfita ^ Ui ^ RNA or mRNA isolation procedure or due to incomplete synthesis of cDNA. To investigate this, the 5 'sequences of the introduced cDNAs were determined. 3. 5.b Analysis of the clones that produce xylanase. PCR standards were prepared for each transformant that produces xylanase as described. Transformants were analyzed for the presence of an expression construct comprising xylA cDNA in a PCR experiment using oligonucleotides 6856 (internal xylA) and 6963. { PgiaA) - Transformants # 5C2 and # 7A8 were shown to comprise an expression cassette with the xylA gene fused to Pg? AA. Using oligonucleotides 6963 (specific PgiaA) and 6967 (specific for the 3 'end cDNA), PCR fragments were generated, which were expected to comprise all the cDNA as well as 200 bp of PgiaA- A partial DNA sequence of the fragments was determined. PCR using a nucleotide 6963 for six transformants. Sequences indicative of the presence of both of the xylA gene (2 clones) and the xylB (4 clones) were detected (the DNA sequences of xylA and xylB are described in our earlier patent applications EP-A-0 463 706 and WO 94 / 14965, respectively). Different lengths of the 'untranslated region. However, no relationship could be observed between the length of the untranslated region 5 'of the CDNA and xylanase production levels of different xylB transformants In contrast, the short 5 'untranslated region found in transformant # SC2 positive to xylA resulted in a significant reduction in XYIA activity. However, it is clear that production levels were still sufficient to identify this transformant in a plaque assay.

Table I. Analysis of the transformants that produce xylanase. The positive transformants were analyzed to determine the levels of xylanase production in a fermentation experiment. The identity of the genes encoding the xylanase was determined by partially sequencing the cDNA insert. The details are described in the text.

Table 1 Table 1 (continued) nd = not determined EXAMPLE 4 4. 1 Construction and analysis of an integrable expression vector applicable for the cloning of cDNA (pGBFINll) mediated by EcoRI-Xhol 4.1.a Rationale Expression libraries of cDNA assembly were constructed. The cDNA encoding the desired activity was separated (detected) via a separation format described above. Since the exact characteristics of the cDNA (for example the restriction enzyme sites present within the cDNA) in most cases are not known before actual identification due to the absence of restriction sites in the cDNA. Therefore, there is a possibility that in the construction as described in Example 2 the cDNA desired does contain an internal AscI site and thus be cloned as an inactive clone of non-complete length that can not be separated.

As a consequence, the plasmid pGBFINII has been constructed, which allows the cloning of cDNA with EcoRI-Xhol cohesive ends without avoiding the danger of internal restriction sites. The 3 'primer used for the synthesis of the first strand of cDNA contains an Xhol site (unmethylated) while methylated dCTP is used during the synthesis of cDNA. As a consequence, the cDNAs can be digested with XhoI avoiding fragmentation of the cDNAs due to the internal Xhol sites (these Xhol sites are methylated and thus undigested). PGBFINll is a vector derived from pGBFIN2 in which the Xhol and Existing EcoRIs have been removed after which the cDNA cloning site has been changed from Pacl-Ascl to EcoRI-Xhol. Thus, all the features and functionalities in the expression vector are identical except for the restriction sites used for the cloning of the cDNAs 4. 1.b Construction of vector pGBFINll In a first step Xhol, HindIII, Seal and EcoRI present at the 5 'end of the gpdA promoter were removed via PCR and a SnaBI (rare) site was introduced, resulting in the intermediate construct PGBFIN12 In a second step of the PCR, the existing glaA promoter and the cDNA cloning site were adjusted in such a way that I) the Pacl-Ascl cDNA cloning site was changed to the EcoRI-? HoI, II cloning site) At the same time the EcoRI in the promoter was activated, III) at the same time the promoter was shortened (starting from the SalI site at position 6084 in pGBFIN2 instead of starting from the Xhol site at position 5289 in pGBFIN2) and IV ) at the same time the Xhol site present at position 5289 was inactivated and introduced a (second) rare cutting restriction enzyme.

The resulting plasmid (pGBFINll) is described in Figure 34. 2 Expression of phytase using the pGBFINll vector 4.2.a Rationale In the pGBFINll a test gene (for example phytase) has been inserted in a similar manner as described in example 1.2 for the vector pGBFIN2. The resulting vector, pGBFIN13, has been tested together with the pGBFIN5 vector to demonstrate the functionality of this pGBFINll type vector. 4. 2.b Construction of a phytase expression vector, pGBFIN13 Similar to the situation described for the pGBFIN2 vector (example 1; 1.2.b), the functionality of the pGBFINll vector was also tested via the use of a model gene, phyA . 4. 2.C Transformation of Aspergillus niger with pGBFIN13 Similar to the situation described for vector pGBFIN2 (example 1; 1.2.c) the vector pGBFIN13 was digested with? Jotl to generate the linear fragment that could be used for direction during transformation .

After transformation, randomly selected transformants were purified to allow subsequent analysis. 4. 2.d Analysis of the transformants pGBFIN13 Again, similarly to the situation described for the vector pGBFIN2 (example 1; 1.2.0), the purified pGBFIN13 transformants were tested for the direction of the constructs at the correct site for the expression of the phytase . Both of the direction and expression frequencies of the phytase were in the range of what has been described above for the pGBFIN2 transformants. Thus, it was concluded that for the pGBFINll vector both the direction frequency and the expression levels were sufficient for the use of the expression system designed in cloning by cDNA expression with EcoRI-Xhol cohesive ends. . 1.a Background After the demonstration of the functionality of the pGBFINll vector, the complete expression cloning system based on this type of vector was tested (EcoRI-Xhol cohesive ends, since the introduction of an EcoRI cDNA cloning site) Xhol allowed the use of the equipment cloning of STRATAGENE cDNA, the applicability of this system (which has the benefit of avoiding the digestion of intact cDNAs during restriction digestion to generate the 3 'cohesive cloning site) was tested in combination with the new pGBFINll vector. In a manner similar to what has been described in Examples 2 and 3, a set of RNA derived from A was used. niger with the STRATAGENE protocol optimized for cloning into pGBFIN vectors as detailed in the materials and methods, a set of cDNAs (with EcoRI-Xhol cohesive ends). This cDNA assembly was cloned into the pGBFINll vector to generate an E. coli library. Subsequently, the efficiencies of the cloning were compared with the previous construction of the library in the vector pGBFIN2. . 2.b. Preparation of a cDNA library of a culture of Aspergillus induced with xylanase The mycelium, from which (as has been described above) it was known that xylanases expressed at the time of harvest was used to subtract the total RNA as detailed in the Materials and Methods. Subsequently, the total RNA collected was further purified by centrifugation through a CsCl mattress. After verifying RNA quality, the mRNA was isolated via a modified protocol with the Pharmacia Purification Team. For the synthesis of cDNA, the Stratagene DNA Synthesis Kit was used. The corresponding cDNA synthesis protocol was adapted to optimize cloning in pGBFm vectors. Major adaptations included; 1) Quantities of cDNA were quantified via TCA precipitation; 2) Phosphorylation of the ends of the cDNAs was omitted and the cDNAs were ligated to the vector DNA, which was not dephosphopel. This prevents the ligation of multiple inserts in the vector (which could prevent the expression of several if not all of the inserts present in that vector). 3) The cDNA was extracted with phenol / chloroform after digestion with XhoI instead of after fractionation by size. 4) Both of the MMLV-RT and the Thermoscppt were used in the synthesis of the first strand, which resulted in cDNA with longer lengths than with the use of any single enzyme. 5) The control reactions were plotted with [alpha32P] dATP (800 Ci / mmol, to prevent interference with the synthesis) for quality control. A cDNA assembly was constructed according to the protocol thus modified. For pGBFmll, a set of poorly digested pGBFinll (EcoRI-Xhol) vectors was prepared (background ligation <1%). The generated cDNA set was ligated into the vector pGBFmll and transformed to E. coil bacterial cells XLIO-Gold to generate a library. . 1 C. Analysis of the E. coli cDNA library (in the pGBFmll vector) The procedure described already in this example resulted in a significant increase in the efficiency of ligation and transformation. With the cDNA set isolated according to the optimized procedure it was possible to obtain in combination with the double digested pGBFmll vector set to obtain an E. coli library of size 106-107 starting with 1 ug of pGBFmll. Next, via hybridization experiments, the cDNA frequency of gpdA and xylB was established in the E cDNA library. with . Since the gpdA gene represents a relatively long gene and the xylB gene is relatively short, the comparison of the total length percentages of the clones could clarify the quality of the cDNAs generated and the identity if there were differences in the efficiency of generation of cDNA of full length between short and long mRNAs. After the identification of clones xylB and gpdA positive, a selected number was sequenced to determine the percentages of clones with full length within the cDNA population originating from those particular genes. Both cDNAs of gpdA and xylB showed that the percentage of full-length clones was higher than 85% In addition, sequencing showed that none of the clones contained multiple inserts. Thus, it was concluded that the protocol for RNA purification, cDNA synthesis and optimized cloning resulted in a considerable improvement in the efficiency and quality of the construction of the cDNA library (in terms of the size and frequencies of the libraries, in terms of the full-length percentages and in terms of the cloning only one cDNA insert in the expression vector). . 1.d. Transformation of pGBFinll constructs containing xylB to A. niger and separation by xylanase activity A number of xylB clones identified (and analyzed in 5.1.c) were transformed to A. niger (similarly to what has been described for the vectors pGBFm5 and pGBFml3). After purification of a selected number of transformants those transformants were separated on plate to determine the xylanase activity. All transformants tested were positive in the plaque assay for xylanase, demonstrating the applicability of the pGBFinll vector for purposes of cloning by expression in A. Niger.

EXAMPLE 6 6. 1 Construction of a second integrable expression vector applicable for the cloning of cDNA (pGBFin22) mediated by EcóRT-Xhol 6.1.a Rationale During the construction of the pGBFinll vector, the second PCR fragment (used to inactivate the EcoRI site in the glucoamylase promoter, among the other modifications listed in example 4) to prove the correct modification. This demonstrated correct modification of the indicated restriction sites but also showed a number of small PCR errors in the parts upstream of the glucoamylase promoter. Therefore, based on the promoter region of glycoamylase without change in pGBFinl2, a new vector was constructed in which the errors introduced by the PCR were absent and which was adequate to clone cDNA with EcoRI-Xhol cohesive ends. 6. 1.b Construction of the expression vector pGBF? N22 In the pGBF? Nl2 (Figure 3) the remaining Xnol site was activated after digestion with Xhol via filling the end with T4 DNA polymerase and the retroligation resulted in pGBFml7 ( see Figure 6). In pGBF? Nl7 the remaining £ coRI site was similarly removed (digestion with £ coRI followed by end filling with T4 DNA polymerase and retroligation), which resulted in plasmid pGBFmld (see Figure 7). Primers containing £ coRI and XhoI restriction sites and (after annealing together) containing cohesive ends for PacI and AscI were annealed. The primers were constructed in such a way that after the cloning of the primers annealed in the pGBFml8 digested with Pací and AscI, no ATG (extra) was generated in the cloning site of the cDNAs. Thus, by cloning the annealed primers described in pBiFinlβ digested with Paci and AscI, a cloning site was generated for the cDNAs with the EcoRI-Xhol cohesive ends. The plasmid thus obtained was named pGBFm22 (see Figure 8). 6. 2 Expression of the phytase using the vector pGBFin22 6.2.a Background In the vector pGBFin22 a test gene (for example of the phytase) has been inserted in a manner similar to that which has been described in example 4 for the vector pGBFinll.

The resulting vector, pGBFin25, has been tested for the production of phytase as well as its functionality. 6. 2.b Construction of a phytase expression vector, pGBFin25 pGBFinl3 was digested with £ coRI to release the phytase gene. This gene fragment encoding the phytase was cloned into pGBFin22. After the identification of a clone with the correct orientation of the phytase gene, this clone was designated pGBFin25. 6. 2.c Transformation of A. niger with pGBFin25 and analysis of pGBFin25 transformants pGBFin25 was used for transformation to A. niger and the subsequent analysis of the transformants as detailed in Examples 1 and 4 for the transformants of pGBFin5 and pGBFinl3 respectively. The results were similar as has been indicated for the pGBFinl3 transformants which demonstrate the Applicability of the pGBFin22 vector for expression cloning purposes.

EXAMPLE 7 7. 1 Construction of an integrable expression vector applicable for the cloning of HipdlII-X ol mediated cDNA, pGBFin23 7.1.a Rationale After obtaining the set of integrable expression cloning vectors described above it was recognized that the availability of a cloning vector by expression that could be useful for cloning cDNA with 5 'cohesive ends of HindIII could be useful. Both in terms of being able to use cDNA pools that were already constructed for other purposes of cohesive ends of HindlII-X or I and due to the fact that in this method no changes need to be made to the glucoamylase promoter. 7. 1.b Construction of a phytase expression vector applicable for the cloning of HindIII-XhoI mediated cDNA, pGBFin23 In pGBFinl7 the remaining HindlII site was removed (digestion of Fíindlll followed by filling of the end with T4 DNA polymerase and retroligation), which resulted in the plasmid pGBFinl9 (see Figure 9). Two primers containing restriction sites were annealed HindI II and Xhol and (after annealing them together) that contained cohesive ends for Pací and Ascl. The primers were constructed in such a way that after the cloning of the primers annealed in the pBiFinl9 digested with Paci and AscI no ATG (extra) was generated in the cloning site of the CDNA In this way, by cloning the annealed primers described in pBiFinl9 digested with Paci and AscI, a cloning site was generated for the cDNAs with the cohesive ends of Hindi I I-Xhol. The plasmid thus obtained was named pGBFin23 (see Figure 10). 7. 2 Expression of phytase using the vector pGBFin23 7.2.a Background In the pGBFin23 vector a test gene (for example of the phytase) has been inserted in a manner similar to what has been described in example 4 for the vector pGBFinll. The resulting vector, pGBFin26 has been tested for the production of phytase to demonstrate its functionality. 7. 2.b Construction of a phytase expression vector, pGBF? N26 In this example, the phytase gene was PCRed with a 5 'oligo which contained a fi llelll site and an oligo 3' containing an X? OI site. After digestion with Hmdl I I and Xhol this fragment was cloned directly into pGBFm23, thus generating pGBFm26. After the isolation of a number of clones containing the phytase gene, the phytase inserts were sequenced to verify the introduction of putative PCR errors. Finally, a correct pGBFm26 plasmid (without changes in the coding protein sequence) was selected and used for the transformation and subsequent analysis. 7. 2.C Transformation of A niger with pGBFm26 and analysis of pGBFm26 transformants pGBFm26 was tested for transformation to A. niger and the subsequent analysis of the transformants as detailed in Examples 1, 4 and 6 for the transformants of pGBF? n5, pGBFml3 and pGBF? n25, respectively. The results were similar to what has been indicated for the pGBFml3 transformants, which demonstrated the applicability of the pGBFm23 vector for expression cloning purposes.

EXAMPLE 8 8. 1 Construction of AMAI-based plasmid expression vectors suitable for expression cloning of cDNA (pGBFin6 and pGBFinl5) 8.1.a Rationale In another example, the functionalities were used to direct high expression of the cloned cDNAs and a selectable marker in plasmids which also contain a sequence called AMAl. As a result, an expression cloning plasmid was generated which was autonomously maintained in Aspergillus. In this type of expression cloning vectors were combined the highly efficient transformation frequencies obtainable with vectors based on the AMA1 type and the functionalities that direct the high expression of the cloned cDNAs. Two expression vectors were constructed which differed in the selection marker gene used for the selection of the transformants in Aspergillus and both designed for expression cloning systems based on AMAl. 8. 1.b Construction of pGBFin6 vector pTZamdSX-2 (see Figure 2) was linearized with HindIII after which the AMA 1 HindIII fragment of . 2 of A. nidulans (as described by Aleksenko and Clutterbuck, 1998) was cloned into it, resulting in the intermediate plasmid pAMAamdS. Next, the pAMAamdS was digested with Knpl and BglII and a fragment containing AMA 1 of approximately 9 kb was isolated. After digestion of the pGBFin2 vector (see Figure 2) with Knpl and BglII, the fragment containing the 5.2 kb glucoamylase promoter was isolated. The 5.2 kb fragmetne derived from pGBFin2 was cloned into the 9 kb fragment of pAMAamdS resulting in the expression cloning plasmid based on the selection of AMA 1 and acetamide pGBFind (see Figure 11). 8. 1.b Construction of pGBFin 15 vector pGBFin6 was digested with Xhol and the glucoamylase promoter containing the fragment was isolated. Next, the plasmid pAN8-l (see Figure 12), containing the functional ble gene (encoding for resistance to phleomycin) derived by a gpdA A. nidulans promoter and terminated by the trpC terminator using as a template in a PCR reaction. The PCR primers were designed in such a way that a fragment was generated that it contained a truncated (but still fully functional) gpdA promoter, the ble gene, and a truncated (but still fully functional) trpC terminator which also contained at both ends of the fragment a functional Xhol site. In addition, primer 5"contained a HindIII site which was necessary for the additional cloning steps (as described in detail in the construction of pGBFinl5.) After Xhol digestion of the PCR product of approximately 1.9 kb, it was cloned. in the Xhol fragment previously isolated from pGBFin2 The resulting plasmid (pGBFinl4, see Figure 13) was verified by the correct orientation via restriction analysis and by PCR errors via sequencing.PGBfínl4 was linearized with HindIII after which inserted the HindIII fragment of AMAl of 5.2 kb, resulting in plasmid pGBFml5 (see Figure 14). 8. 2 Expression of phytase in vectors based on AMAl 8.2.a Background The expression constructs based on AMAl were tested by the expression of a phytase in a manner similar to what has been described for the integral expression vectors. Again a test gene (for example phytase) was inserted in a similar manner as described in Example 1 for the pGBFind vector. The vectors ^^^^^ The results were tested for the production of phytase to demonstrate the functionality and applicability of expression vectors based on AMAl. 8. 2.b Construction of pGBFm7 and pGBF? Nl6 Both pGBFinβ and pGBFinld vectors were linearized via a double digestion with Pací and Ascl. Next, plasmid pGBFm5 was digested with Pací and AscI to release the fragment encoding the phytase gene (with cohesive ends of Pací and AscI). This fragment of phytase was cloned directly into the vectors pGBFmd and pGBFml5 digested to generate, pGBFm7 and pGBFinlß, respectively. 8. 2. c Transformation of Aspergillis niger with pGBFm7 and pGBFml6 pGBFm7 and pGBFmlβ were transformed to A. niger according to the procedures described in the previous examples and as best described in Materials and Methods. Transformants of pGBFm7 were selected on media containing acetamide as the sole nitrogen source, while transformants of pGBFml6 were selected on media containing phleomycin. Both plasmids demonstrated a significantly increased transformation sequence compared to the type integrable of the expression vectors; the transformation frequencies of AMAl-based plasmids were up to 105 transformants per ug of plasmid. The positive transformants were purified by replanting single colonies on selective medium and finally storing. 8. 2.d Analysis of phytase expression in pGBFinl6 transformants After purification, 20 randomly selected pGBFinld transformants were fermented in shaking flasks using the same medium (in this case supplemented with phleomycin) as described for the integrable vectors . The fermentation samples were tested for the production of phytase which was demonstrated in a range in all cases (except for one) of approximately 40 U / ml at 60 U / ml. In a particular case, the expression was 117 U / ml, which was probably a result of the integration of the plasmid pGBFinl6 into the genome (see also the comments in example 1; 1.2.d). These results demonstrate that AMAl-based plasmids as described in this example can be used to direct cloning by expression in Aspergillus. Due to the use of functionality it is in glaA -aaa "-''- 'J-' which are capable of directing high expression levels of cloned cDNA production, although reduced in comparison to expression after integration into a high expression site, the expression is still certainly high enough for efficient separation in an expression library containing AMAl, especially when the significantly increased frequency of transformation is taken into account. A further advantage of the vectors based on AMAl is provided by the fact that the recovery (isolation) of these filament fungal expression guest hosts is simplified in comparison with the integrable plasmids. The direct transformation of E. coli with the total DNA isolated from the host in question will be sufficient in this regard. 7c LIST OF SEQUENCES < 110 > Gist-brocades B.V. < 120 > separation by expression in filamentous fungi < 130 > 2865-p < 140 > < 141 > < 160 > 22 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 52 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5288 < 400 > 1 tagtacgtag cgcccacaat caaccattt cgctatagtt aaaggatgcg ga 52 < 210 > 2 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5289 < 400 > 2 gatcaggatc tccggatcaa tactcccggcg tat 33 < 210 > 3 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5290 < 400 > 3 atacgccgga ctattcatcc ggagatcctg ate 33 < 210 > 4 < 211 > 84 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5291 < 400 > 4 cggaaagctt cactgacgta accaggaccc ggcggcttat ccatcatggg aaacaacacc 60 tacaaatccg ccacaatact ctcg 84 < 210 > 5 < 211 > 45 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5292 < 400 > 5 gcaatcctcg aggtcccacc ggcaaacatc tgcccataga agaac 45 < 210 > 6 < 211 > 88 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5293 < 400 > 6 agtgaagctt tccgtggtac taagagagag gttactcacc gatggagccg tattcgccct 60 caagcaccgc gtgaccccac tattcgac 88 < 210 > 7 < 211 > 46 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5258 < 400 > 7 aatttgctcc cgcccgctcg agcggggaat tcccggtacg tacgca 46 < 210 > 8 < 211 > 46 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5359 < 400 > 8 agcttgcgta cgtaccggga attccccgct cgagcgggcg gccgca 46 < 210 > 9 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5361 < 400 > 9 ccaggacgcg gccgcttatc catcatggga 30 < 210 > 10 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5361 < 400 > 10 tagtacgtac aatcaatcca tttcgctat 29 < 210 > 11 < 211 > 44 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5367 < 400 > 11 cccaagcttg cggccgcgtc ctggttacgt cagtgatgtt tccg 44 < 210 > 12 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5454 < 400 > 12 tccgcatgcc agaaagagtc accgg 25 < 210 > 13 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 5456 < 400 > 13 gcatccatcg gccaccgtca ttgga 25 < 210 > 14 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 6856 < 400 > 14 cggcagagta ggtgatagcg ttagaagaac cagtggtcc 39 < 210 > 15 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 6963 < 400 > 15 acggaattca agctagatgc taagcgatat tgc 33 < 210 > 16 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 6964 < 400 > 16 ttaattaact cataggcatc atgggcgtc 29 < 210 > 17 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 6965 < 400 > 17 ggcgcgccga gtgtgattgt ttaaagggtg at 32 < 210 > 18 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 6967 < 400 > 18 atcatcggcg cgcctttttt tttttttttt tt 32 < 210 > 19 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 7423 < 400 > 19 ggaattctcg aggccgcaag ctcagcgtcc aattc 35 < 210 > twenty < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 7424 < 400 > 20 ggaattctcg agcacgcatg gttgagtgg tatgg 35 < 210 > 21 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 7676 < 400 > 21 taggcccatat gggccat • 17 < 210 > 22 < 211 > 15 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > oligonucleotide 7677 < 400 > 22 ggcccatatg gccta 15 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (14)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. 1. A method for isolating a DNA sequence encoding a protein with properties of interest, the method is characterized in that it comprises the steps of: (a) preparing, in a suitable cloning vector, a DNA library of an organism that it is suspected that it is capable of producing one or more proteins with properties of interest; (b) transforming filamentous fungal host cells with the DNA library; (c) culturing the host cells obtained in (b) under conditions that lead to the expression of the DNA sequence encoding the proteins with properties of interest present in the DNA library; and (d) separating the clones from the transformed host cells expressing a protein with properties of interest by analyzing the proteins produced in (c).
2. 2. The method according to claim 1, characterized in that the cloning block comprises a DNA fragment which is homologous to a DNA sequence. at a predetermined target site in the genome of the filamentous fungal host cell.
3. 3. The method according to claim 2, characterized in that the predetermined target site comprises a highly expressed gene.
4. 4. The method according to claim 2 or 3, characterized in that the filamentous fungal host cell comprises more than one copy of the predetermined target site.
5. 5. The method of compliance with the claim 1, characterized in that cloning vector is a vector which is able to remain autonomous in a host cell of filamentous fungus.
6. 6. The method according to claim 5, characterized in that the vector is able to remain autonomous in a vector comprising a sequence of AMA1.
7. The method according to any of claims 1 to 6, characterized in that the DNA sequence encoding the protein with properties of interest is expressed from a promoter which is derived from a highly expressed filamentous fungus gene.
8. The method according to any of claims 1 to 7, characterized in that the filamentous fungal host cell is of a species of the genus Aspergillus or Trichoderma.
9. 9. The method according to any of claims 1 to 8, characterized in that the filamentous fungal host cell is from a species of the genus Aspergillus nidulans, Aspergillus oryzae, Aspergillus sojae, the species of the group Aspergillus and Tp choderma reesei.
10. 10. The method according to any of claims 1 to 9, characterized in that the suspect organism is capable of producing one or more proteins with the properties of antibodies is a eukaryote.
11. The method according to any of claims 1 to 10, characterized in that the eukaryote is a fungus, preferably a filamentous fungus.
12. 12. The method according to any of claims 1 to 11, characterized in that the protein with the properties of antibodies is an enzyme.
13. 13. A process for producing a protein with the properties of antibodies in a suitable host cell, the process is characterized in that it comprises the steps of transforming the host cell with a DNA sequence that encodes the protein with the properties of the DNA, the DNA sequence. has been isolated by the method according to any of claims 1-12, culturing the transformed host cell ba or conditions that lead to the expression of the DNA sequence encoding the protein, and recovering the protein.
14. 14. A process for producing a protein with properties of interest, the process is characterized in that it comprises the steps of culturing a transformed host cell under conditions that lead to the expression of the DNA sequence encoding the protein, and recovering the protein, so that the transformed host cell is a clone expressing the protein with the properties of interest according to that obtained by the method according to any of claims 1-12.