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  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/385—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Penicillium
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
  • C12N9/2457—Pullulanase (3.2.1.41)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2477—Hemicellulases not provided in a preceding group
  • C12N9/248—Xylanases
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
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  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01073—Feruloyl esterase (3.1.1.73)

Definitions

• the present invention relates to a recombinant Penicillium funiculosum strain for the production of homologous or heterologous proteins, and a transformation method for obtaining such recombinant P. funiculosum.
• functional cassettes for expression of homologous and heterologous genes are also encompassed by the present invention.
• Micro-organisms are known as producers of a broad spectrum of extracellular enzymes useful in a variety of industrial applications, such as in the baking industry, the wine and juice industry, the textile industry, and also for improving the digestibility of vegetable sources, essentially in animal feed.
• Filamentous fungi produce enzymes used in a variety of these industrial processes, but also produce antibiotics, for example penicillin. Genetic transformation of filamentous fungi was generally performed in order to improve the production of penicillin or other high value compounds (EP 0 235951 and EP 0 260762).
• enzymes used in industrial processes are of lower commercial value and their production has to combine high yields and low cost. In consequence, there is a need for a filamentous fungus able to industrially produce proteins of interest used in industrial processes, and particularly in animal feed and human food.
• the filamentous fungus P. funiculosum is of great economical importance for its ability to produce a mixture of enzymes which can be used to increase the digestibility of animal feed.
• a better yield and control of enzyme production using P. funiculosum could be obtained by transforming P. funiculosum strains with specific transcription activating sequences functional in P. funiculosum to allow the expression of heterologous genes or to alter expression of homologous genes.
• a set of tools are needed. These tools include a method for transforming P. funiculosum, a selection marker for selecting transformants, and expression cassettes comprising regulatory elements functional in P. funiculosum.
• auxotrophic complementation requires the possession of an auxotrophic strain carrying a mutation in a gene involved in a metabolic pathway and the corresponding gene which should complement the auxotrophic strain to prototrophy. This method was described in Yelton et al. (1984 ; Proc. Natl. Acad. Sci. USA ; Vol.
• auxotrophic complementation is the use of dominant selection markers.
• Numerous dominant selection markers are available and have been used to perform the transformation of several fungi, like hygromycin B phosphotransferase (Punt et al., 1987; Gene, 56(1): 117-24), streptothricin acetyltransferase, phleomycin-resistance polypeptide and benomyl resistant beta-tubulin (Gold et al., 1994; Gene, 142(2):225-30), acetamidase (Beri and Turner, 1987; Curr Genet, 11(8):639-41), bialaphos-acetyltransferase (Avalos et al., 1989; Curr Genet, 16(5-6):369-72), but have not yet been used on R. funiculosum.
• the advantage of dominant selection markers is that they can instantly be used without previous selection for auxotrophic mutant strains.
• Efficient regulatory elements are promoter and terminator sequences allowing expression of the desired nucleic acid sequences of interest in a selected organism.
• Other genetic elements of interest are nucleic acid sequences encoding signal peptides that direct the secretion of the said protein of interest into the culture medium. To achieve this goal, such genetic elements need to be functional in the organism to be transformed, and may be isolated from it. Some genes have already been isolated from P. funiculosum, but no regulatory genetic tools have been obtained.
• the technical problem to be solved by the present invention is the preparation of a recombinant R. funiculosum in order to improve and control the industrial production of homologous or heterologous proteins.
• the present invention relates to a recombinant P. funiculosum strain comprising at least one expression cassette functional in R. funiculosum stably integrated into its genome for the production of homologous or heterologous proteins.
• the term "recombinant" applied to an organism means that this organism may contain at least one heterologous gene stably integrated into its genome, one extra copy of an homologous gene stably integrated into its genome, or one homologous gene whose regulation has been artificially modified. In the particular case of this invention, this means that the recombinant R. funiculosum contains at least one expression cassette stably integrated into its genome.
• the expression cassette is necessarily integrated by an artificial genetic mean using molecular and cellular biology techniques known to the one skilled in the art, artificial meaning different than nature-based methods such as hybridisation and selection.
• An expression cassette of the invention contains genetic elements, such as at least a promoter, a coding sequence and a terminator region operably linked in such a way that they are functional in P. funiculosum. Accordingly, the genetic elements constituting this expression cassette may each originate from R. funiculosum or may originate from other organisms provided that they are functional in R. funiculosum. Functional means allowing the expression of the coding sequence when the recombinant R. funiculosum is placed in conditions suitable for the expression of said expression cassette.
• an expression cassette of the invention can comprise regulatory genetic elements, i.e. a promoter, a sequence encoding a signal peptide, and a terminator, originating from a R. funiculosum gene, operably linked to a coding sequence from another species, i.e. encoding a heterologous protein.
• Another expression cassette of the invention can comprise regulatory genetic elements originating from other species, provided that they are functional in R. funiculosum, operably linked to the coding sequence of a P. funiculosum gene, i.e. encoding a homologous protein, or to the coding sequence of a gene of another species, i.e. encoding a heterologous protein.
• the design and construction of such expression cassettes use standard molecular biology techniques known to the person skilled in the art (Sambrook et al., 1989, Molecular Cloning : A Labratory Manual).
• the expression cassette contained in the recombinant P. funiculosum comprises, at least, operably linked in the direction of transcription, a promoter functional in P. funiculosum, a nucleic acid sequence encoding a homologous or heterologous protein and a terminator region functional in R. funiculosum.
• a homologous protein is a P. funiculosum protein
• a heterologous protein is a protein originating from an organism different than R. funiculosum.
• the term "operably linked" means that the genetic elements are associated in a functional manner allowing the expression of the coding sequence.
• the promoter is a promoter of a P. funiculosum gene.
• the promoter comprises a nucleic acid sequence selected from the sequences disclosed in SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, or a nucleic acid sequence hybridising to any one of these sequences.
• “hybridising” and “hybridisation” refer to nucleic acid sequences that are able to selectively hybridise with any one of the identified sequences (SEQ ID NO) at a level significantly higher than the background noise, and that are sharing the same function as said identified sequences.
• the background noise can be due to hybridisation of other nucleic acid sequences, in particular cDNA or genomic fragments present in cDNA or genomic libraries.
• the level of the signal generated by the interaction between the sequences able to selectively hybridise and the identified sequences (SEQ ID NO) of the invention is generally 10 times, preferably 100 times higher than that resulting from the interaction of the identified sequences with the sequences generating the background noise.
• the interaction level can be measured, for example, by labelling a hybridisation probe with the radio-element 32 P, the hybridisation probe being a sequence identified in the present invention or a fragment thereof.
• Selective hybridisation is generally obtained using stringent conditions (for example NaPO 4 0,5M, 7% SDS at 60°C-70°C and pH 7,0).
• nucleic acid sequences hybridising to any one of the identified sequences are similar to said identified sequences.
• "similar” apply to nucleic acid sequences sharing one or more nucleotide modification compared to the identified sequences (SEQ ID NO). These modifications may be obtained by standard mutation methods, or chosen for the design of artificial oligonucleotide sequences used as probes in a hybridisation process or primers in polymerase chain reaction (PCR). The degree of similarity is expressed by the percentage of identical nucleotides between a similar sequence and an identified sequence.
• nucleic acid sequence similar to a promoter of the invention keeps the same specific promoter function while sharing nucleotide differences with said promoter of the invention.
• the terminator region is a terminator region of a P. funiculosum gene.
• the terminator region comprises a nucleic acid sequence selected from the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a nucleic acid sequence hybridising to any one of these sequences.
• the promoter disclosed in SEQ ID NO: 1 and the terminator region disclosed in SEQ ID NO: 4 derive from the Histone H4b gene. Expression cassettes constructed with this promoter permit a constitutive expression of homologous or heterologous proteins in recombinant R. funiculosum.
• the promoter disclosed in SEQ ID NO: 2 and the terminator region disclosed in SEQ ID NO: 5 derive from the Acidic Aspartyl Protease gene. Expression cassettes constructed with this promoter permit a casein-regulated expression of homologous or heterologous proteins in recombinant R. funiculosum, said casein strongly increasing the expression.
• ID NO: 6 derive from the csl31 gene.
• Expression cassettes constructed with this promoter permit a Corn Steep Liquor (CSL)- regulated expression of homologous or heterologous proteins in recombinant R. funiculosum, said CSL strongly increasing the expression.
• CSL Corn Steep Liquor
• the recombinant P. funiculosum of the invention contains an expression cassette comprising a promoter and a terminator region of the same gene.
• the recombinant R. funiculosum of the present invention contains an expression cassette comprising a nucleic acid sequence encoding any homologous protein.
• the nucleic acid sequence encoding a homologous protein may encode a xylanase, a cellulase, a ⁇ -glucanase, or a ferulic acid esterase, or any cell-wall degrading enzyme isolated from R. funiculosum.
• the recombinant R. funiculosum of the present invention may also contain an expression cassette comprising a nucleic acid sequence encoding any heterologous protein.
• the heterologous protein may be a fungal protein, a bacterial protein, a plant protein, an animal protein, or a protein from unidentified origin.
• the nucleic acid sequence may encode a heterologous protein of interest such as a xylanase, a cellulase, a ⁇ -glucanase, a ferulic acid esterase, a pullulanase, an amidase, a phosphatase, a phytase, a mannanase, a milk-cloating enzyme isolated from any species.
• the nucleic acid sequence of interest may also have been obtained after screening of DNA libraries, encompassing cDNA or genomic DNA from various sources, including DNA libraries obtained by combinatorial or conventional mutagenesis of a given sequence or after directed molecular evolution (Arnold et Nolkov, 1999, Current Opinion in Chemical Biology 3 : 54-59 ; Skandalis et al, 1997, Chemistry & Biology 4 : 8889-898; Crameri et al, 1998, Nature 391 : 288-291) or after screening of libraries made with DNA from soil or other environmental samples.
• the nucleic acid sequence may encode a bacterial pullulanase like the one described in Yamashita et al.
• the recombinant P. funiculosum further contains an expression cassette comprising a signal sequence and a pro-sequence inserted between the promoter and the nucleic acid sequence encoding a homologous or heterologous protein.
• the signal sequence encodes a signal peptide which is directing the secretion of the expressed protein into the culture medium
• the pro-sequence encodes a pro-peptide which, when it is linked to an enzyme, maintains the enzyme in an inactive state until it is cut off by a protease.
• the association of such genetic elements to the sequences encoding the desired homologous and heterologous proteins is very useful to direct the secretion of the protein into the culture medium and control the timing of its biological activity.
• these genetic elements are from a P. funiculosum gene. Most preferably, they are isolated from the Acidic Aspartyl Protease gene and correspond to the nucleic acid sequence disclosed in SEQ ID NO: 7, or from the csl31 gene and correspond to the nucleic acid sequences disclosed in SEQ ID NO: 8 or SEQ ID NO: 9, or from nucleic acid sequences hybridising to SEQ ID NO: 7, SEQ ID NO:8,. SEQ ID NO:9.
• the recombinant R. funiculosum of the invention may also comprise an expression cassette containing a nucleic acid sequence encoding a dominant selection marker as homologous or heterologous protein. Selection markers permit the selection of the transformed R. funiculosum, i.e.
• the nucleic acid sequence encoding a dominant selection marker of the invention encodes an enzyme degrading a compound with an antibiotic or a fungicide activity.
• the nucleic acid sequence encoding a dominant selection marker degrading a compound with antibiotic activity encodes an enzyme degrading hygromycin, kanamycin, oligomycin, streptothricm, or phleomycin.
• the nucleic acid sequence encoding a dominant selection marker degrading a compound with fungicide activity encodes an enzyme degrading bialaphos (Avalos et al., 1989; Curr Genet, 16:369-72) or a beta-tubulin resistant against benomyl (Orbach et al., 1986; Mol Cell Biol, 6(7):2452-61).
• the recombinant R. funiculosum of the invention may also comprise an expression cassette containing a nucleic acid sequence encoding a protein capable of complementing an auxotrophic P. funiculosum to prototrophy as homologous or heterologous protein.
• An auxotrophic R. funiculosum is a mutant carrying a mutation in a gene encoding an enzyme involved in a metabolic pathway. In order to restore prototrophy, such auxotrophic R. funiculosum needs to be complemented by genetic transformation with the functional gene corresponding to that carrying the mutation. Said functional gene may also share a mutation, but a mutation different from that shared by the auxotrophic P. funiculosum.
• the nucleic acid sequence encoding a protein capable of complementing an auxotrophic P. funiculosum to prototrophy encodes an enzyme implied in nucleoside biosynthesis or an enzyme implied in amino-acid biosynthesis.
• the nucleic acid sequence is selected from pyrA, pyrB, pyrG, pyr4 (Buxton et Radford ; 1983 ; Mol. Gen. Genet. ; 190 ; 403-405), arg4, argB (Berse et al.
• the recombinant P. funiculosum of the invention may also comprise an expression cassette containing a nucleic acid sequence comprising the amdS gene (Corrick et al., 1987; Gene, 53:63-71 ; Medline: 87248110) allowing said recombinant P. funiculosum to grow on acetamide as a single N-source.
• the present invention also relates to an expression cassette comprising, at least, operably linked in the direction of transcription, a promoter functional in R. funiculosum, a nucleic acid sequence encoding a homologous or heterologous protein and a terminator region functional in P. funiculosum.
• the promoter and the terminator region are as described above.
• the expression cassette of the invention comprises a promoter and a terminator region of the same gene.
• the expression cassette of the invention comprises a nucleic acid sequence encoding any homologous or heterologous protein as described above.
• the expression cassette further comprises a signal sequence and a pro-sequence inserted between the promoter and the nucleic acid sequence encoding a homologous or heterologous protein.
• Said signal sequence and a pro-sequence are as described above.
• the expression cassette of the invention may also contain a nucleic acid sequence encoding a dominant selection marker, or a nucleic acid sequence encoding a protein capable of complementing an auxotrophic P. funiculosum to prototrophy as described above.
• the present invention also relates to vectors containing an expression cassette according to the invention.
• Such vectors may be plasmids, phages or viruses, and they are used in the genetic transformation of host cells.
• Host cells containing these vectors are also encompassed by the present invention. These host cells may be fungal cells, bacterial cells, plant cells, or animal cells. A preferred fungal cell is a R. funiculosum cell.
• the present invention also concerns a method for producing recombinant R. funiculosum.
• This method is a co-transformation method, that is a method leading to the uptake of at least one non-selected DNA, i.e. a plasmid or a DNA containing at least one expression cassette, and at least one other DNA fragment used for selection, i.e. plasmid or a DNA containing a gene encoding a dominant selection marker or a protein capable of complementing an auxotrophic R. funiculosum to prototrophy.
• the method also encompasses transformation which is essentially similar to co-transformation except that the DNA used for selection and the expression cassette are carried on the same piece of DNA.
• this method consists in generating protoplasts of P. funiculosum.
• Generating protoplasts consists in the removing of the fungal cell wall by incubating the mycelia with specific enzymes using standard techniques well known to those skilled in the art.
• protoplasts are transformed in the presence of polyethyleneglycol (PEG) with at least two vectors containing an expression cassette according to the invention, one of these vectors containing a gene encoding a dominant selection marker.
• PEG polyethyleneglycol
• only one piece of DNA can be used provided it contains both an expression cassette according to the invention and a gene encoding a dominant selection marker.
• recombinant P. funiculosum are selected with the selection agent corresponding to the dominant selection marker, and recovered recombinant P. funiculosum contain at least one expression cassette encoding the dominant selection marker and one expression cassette encoding a homologous or heterologous protein stably integrated into their genome.
• protoplasts are transformed in the presence of polyethyleneglycol (PEG) with at least two vectors containing an expression cassette according to the invention, one of these vectors containing a gene encoding a protein capable of complementing an auxotrophic P. funiculosum to prototrophy.
• PEG polyethyleneglycol
• only one piece of DNA can be used provided it contains both an expression cassette according to the invention and a gene encoding a protein capable of complementing an auxotrophic R. funiculosum to prototrophy.
• recombinant P. funiculosum are selected with culture conditions corresponding to said prototrophy, and recovered recombinant R. funiculosum contain at least one expression cassette encoding the protein capable of complementing an auxotrophic P. funiculosum to prototrophy and one expression cassette encoding a homologous or heterologous protein stably integrated into their genome.
• protoplasts are transformed in the presence of polyethyleneglycol (PEG) with at least two vectors containing an expression cassette according to the invention, one of these vectors containing the amdS gene.
• PEG polyethyleneglycol
• only one piece of DNA can be used provided it contains both an expression cassette according to the invention and a the amdS gene.
• recombinant P. funiculosum are selected on culture medium containing acetamide as single N-source, and recovered recombinant R. funiculosum contain at least one expression cassette containing the amdS gene and one expression cassette encoding a homologous or heterologous protein stably integrated into their genome.
• co-transformation frequencies up to 95% are obtained.
• co-transformations with a 1 :1 ratio or a 1 :3 ratio (selectable : non-selectable DNA) are performed.
• the first method is described in Sanchez et al. (1998 ; Mol. Gen. Genet. ; 258 ; 89-94) for the transformation of Aspergillus. It is called the restriction enzyme-mediated integration (REMI).
• REMI restriction enzyme-mediated integration
• This method consists in approximately the same method as the one described above which is the subject of the present invention, except that a mixture of restriction enzymes is added to the protoplasts, the DNA and the PEG during the transformation process. This method has led to an increased transformation frequency (20-60 fold) when applied to Aspergillus (Sanchez et al., 1998 ; Mol. Gen. Genet. ; 258 ; 89-94). Applying the REMI transformation method to P.
• the second known transformation method is that disclosed in the European Patent n° EP 0260762.
• This method was applied to P. funiculosum but, after regeneration and selection, no candidate colonies were able to grow, i.e. were transformed, instead of nearly all with the method developed in the present invention. Accordingly, the transformation method disclosed in the European Patent n° EP 0260762 is not efficient in transforming P. funiculosum, while the transformation method disclosed in the present invention is particularly adapted to the transformation of P. funiculosum. Examples
• the fungus is cultivated on LYMM agar plates for 5 days at 25°C. During this time, sufficient amounts of conidia are produced. They were harvested in 0,01% Triton X-100 by pipetting up and down. 11 Erlenmeyer flask with vexations containing 200ml of MN-Uri broth (Punt, P.J. and van den Hondel, C.A.M.J.J. (1992) Methods in Enzymology 216, 447-457) was inoculated with 0,5-1,0 ml of the harvested conidia, to a final concentration of 10 6 conidia/ml. Temperature of incubation was 28°C for 24-28hrs with an agitation of 220 ⁇ m.
• LYMM contained (mg/ml): malt extract (10), yeast extract (1), Na 2 HPO 4 (6), KH 2 PO 4 (4), (NH 4 ) 2 SO 4 (2), MgSO 4 .7H 2 O (0.2), CaCl 2 .7H 2 O (0.001), H 3 BO 3 (0.000001), MnSO 4 .4H 2 O (0.00001), ZnSO 4 .7H 2 O (0.00007), CuSO 4 .5H 2 O (0.00005), (NH 4 ) 6 MO 7 O 24 .4H 2 O (0.0000123), FeSO 4 .7H 2 O (0.0001). Germinated conidia were filtered through an autoclaved Nybold membrane (0 20 ⁇ m;
• DNA of colonies appearing after 5 days on hygromycin B containing agar plates was extracted using standard molecular biology techniques (Sambrook et al., 1989, Molecular Cloning : A Labratory Manual). The DNA was digested with EcoRI, transferred onto a nylon membrane and hybridised with an EcoRI/BamHI DNA fragment of pAN7-l containing the gene hph. After exposition, 14 out of 14 hygromycin B resistant colonies showed at least one integration of the plasmid pAN7-l on the autoradiograph ( Figure 1).
• Example 3 Construction of a P. funiculosum genomic library in ⁇ ZAPH
• LYMM containing glucose (10 g/1) instead of malt extract (CM) was inoculated with 10 8 conidia and incubated with rotation (150 ⁇ m) at 30°C for 3 days. The pH was adjusted to 5,0.
• Genomic DNA was isolated from the mycelia according to Raeder, V. and Broda, P. (1985 ; Lett Appl Microbiol, 1 :17-20), re-suspended in 50 mM MOPS pH 7.0, 0.75M NaCl and applied to a plasmid purification column (QIAgen). The column was washed and the DNA eluted according to the manufacturer's instructions.
• the purified DNA was partially digested with EcoRI (2 U/ ⁇ g DNA) at 37°C for 25 minutes, extracted with phenol :chloro form (1 :1, v/v) and electrophoresed using a 1% TA ⁇ -agarose gel (Sambrook et al., 1989, Molecular Cloning : A Labratory Manual). DNA fragments of length 3.5 to 12 kb were excised from the gel and purified using a QIAex gel extraction kit (Qiagen). The purified fragments were ligated to Ec ⁇ RI/ ⁇ ZAPII vector arms (Stratagene), packaged into the phage and amplified according to the manufacturer's instructions. The final titre was determined at 1.2 X 10 pfu/rnl.
• the P. funiculosum genomic library was screened for the histone H4 gene by PCR using the degenerate primers NB063 (SEQ ID NO: 10) and NB064 (SEQ ID NO:l 1). Positive clones bearing the 7.6 kbp Ec ⁇ RI insert were sub-cloned and the 2.3 kbp insert was sequenced.
• PCR amplification of the H4b promoter region was performed in a 50 ⁇ l volume containing 50 ng of template DNA, 250 nM of primers NB156 (S ⁇ Q ID NO: 12) NB 157(S ⁇ Q ID NO: 13), 200 ⁇ M dNTPs, 5 mM MgCl 2 and reaction buffer supplied by the manufacturer.
• the positive clones were grown in 100 ml LB broth (100 ⁇ g/ml ampicillin) and the plasmid DNA purified using Qiagen 100 columns according to the manufacturer's instructions (QIAgen). Sequencing was performed on an automated DNA sequencer using the Big Dye terminator cycle sequencing kit (Applied Biosystems). PCR amplification and cloning of the H4b terminator region (SEQ ID NO:4) was performed as described above using primers MJA004 (SEQ ID NO: 14) /MJA005(SEQ ID NO: 15) and the restriction enzymes Xhol and Kpnl.
• Example 5 Identification of the Acidic Aspartyl Protease gene and cloning of a genomic DNA fragment carrying the promoter, gene and 3 'terminal region
• PCR amplification of an internal fragment of apf was performed using the primer combination: PEP5' (SEQ ID NO: 16) and PEP3' (SEQ ID NO: 17). These degenerate primers were designed on the basis of those disclosed in Gente et al. (1997; Mol Gen Genet, 256:557- 65) taking into account codon usage to lower the degree of degeneracy.
• the purified DNA fragment was then ligated into the T- vector pGEM-Teasy (Promega).
• the resulting plasmid was named pPEP. Sequencing of the cloned fragment has been performed by Genome Express. Translation of the identified DNA sequence revealed strong homology to the Aspartyl Protease of P. roqueforti.
• the 620bp DNA fragment of pPEP was isolated by an EcoRI digest, purified over gel and labelled with 32 P ⁇ -dCTP using the Megaprime labelling system (Amersham).
• Genomic DNA of IMI 134756 has been isolated and digested with the enzymes Bglll, Pstl, Bglll/Pstl, Mlul, Ncol, SacII, NcoI/SacII and applied onto a 1% agarose gel.
• the gel was blotted onto a Hybond N Nylon membrane (Amersham) following the described protocols in Sambrook et al. (1989, Molecular Cloning : A Labratory Manual).
• Hybridisation using the labelled 620bp EcoRI fragment as probe has been carried out over night at 65°C in 0,5M NaPO 4 , 7% SDS; pH7,0 Hybridisation buffer. Washing was performed twice at 65°C for lOmin with 0,1M NaPO 4 , 1% SDS; pH 7,0 Washing buffer. The film (Hyperfilm MP; Amersham) was exposed for 3hrs. After development only one single signal was clearly visible in each lane, indicating that apf is indeed a single copy gene.
• the screen of our genomic lambda ZAPII library using a 450bp EcoRI/Kpnl fragment of the 620bp PCR apf product as probe comprehended approximately 90 000 phages which led to the identification of 15-20 phages carrying a DNA hybridising with the apf probe.
• the isolated plasmid has a size of l lkb, carrying a genomic EcoRI insert of 8kb.
• Several restriction enzymes have been used to draw a restriction map of this EcoRI fragment ( Figure 2).
• Proteins of the supernatant separated on a SDS-PAGE deriving from a two days old culture of minimal medium, containing only CSL and Casein revealed the presence of a prominent protein of 31 kDa.
• proteins of a set of different minimal media were analyzed two days after inoculation.
• As a control minimal medium containing Casein and CSL incubated without P. funiculosum was used ( Figure 3, lane 1).
• Figure 3, lane 4 shows that CSL as substrate induces the production of this fungal protein.
• a slight reduction of the protein could be noted in a minimal medium containing CSL and Ammonium ( Figure 3, lane 3).
• Protein sequencing of the N-terminus had been initiated. An aliquot of filtrated supernatant form cultures incubated for 48hrs at 28°C, 190 ⁇ m was applied onto a 10% SDS-PAGE. The proteins were transferred from the gel onto a PNDF membrane using the semi-dry ProBlott system (Applied Biosystems). The membrane was Amido black stained (45% Methanol; 1% acetic acid; 0,1% amido black) and washed in water. After drying, the stained protein band was cut out of the membrane and sent for micro-sequencing. Protein micro-sequencing was performed by the Institute Pasteur (Paris; see analyze 98C1148 as reference).
• the resulting sequence was: X X Y Q T R I F E A G T T F G (SEQ ID NO: 18).
• a 3 ' RACE rapid amplification of cDNA ends
• the principle of the 3 'RACE is the use of a polydT anchor primer (QT: SEQ ID NO: 19), following a reverse transcription of mRNA.
• QT polydT anchor primer
• the outer part of this QT primer represent an annealing site for the primer QO (SEQ ID NO:20).
• the corresponding primer in the N-terminal of Csl31 covers the sequence QTRIF (GSP12: SEQ ID NO:21).
• the estimated size of the csl31 mRNA is about 900bp ( Figure 5).
• Genomic DNA of the strain P. funiculosum IMI 134756 has been partially digested with 0,8U Sail for 20 and 40 minutes and with 2,8U for 40 minutes at 37°C. Fragments had been separated on a CHEF gel for 20 hours (5N/cm; switch time of 8sec and a migration angle of 120°). D ⁇ A of the size between 25 and 60kb has been cut out, ⁇ -Agarase digested and ligated into the Xhol site of the vector pMOCosX (M.Orbach; Gene (1994), 150, 159-162).
• a 600bp EcoRI/Bglll fragment (deriving from the RACE screen) containing the 3 ' end of the csl31 gene was used as probe to screen the cosmid library.
• the pool filters and the individual filters from each microtiter plate were hybridized first, hybridizing the first 20 filters at the same time with the csl31 RACE probe.
• One clear signal could be obtained for filter no 18.
• DNA of the respective E.coli clone (B5) was isolated, digested and analyzed on Southern blot ( Figure 7). The obtained signal pattern was identical to that previously observed on the genomic Southern blot. Following the established restriction map, a 3,6kb Sphl fragment carrying the csl31 gene in the middle was cloned.
• the 3,6kb Sphl genomic subclone of csl31 was cloned and entirely sequenced. This fragment consists of 1350bp promoter region (SEQ ID NO: 3), 912bp csl31 open reading frame and 1350bp terminal region (SEQ ID NO:6).
• H4b promoter plasmid pMJAOOl was constructed by assembling the amplified PCR fragment containing the H4b promoter region into Bluescript SK plasmid (Stratagene Ltd, Cambridge, U.K.).
• H4b promoter + H4b terminator plasmid pMJA007 was constructed by assembling the amplified PCR fragment containing the H4b terminator region into pMJAOOl plasmid.
• a MCS Multiple cloning site containing the following restriction sequences: EcoRN, Hind III, Cla I, Bsp ⁇ 06 I, Sal I, Ace I, Hinc II was inserted between the promoter and terminator sequences of H4b.
• the plasmid pMJA003 containing the Xylanase ORF and the histone H4b promoter and terminator was constructed by assembling the PCR amplified fragment containing the Xylanase ORF into the pMJA007.
• the P. funiculosum phage genomic library was screened with the redundant primers
• Furniss 3 (SEQ ID NO:24) and 5 (SEQ ID NO:25).
• the 2.9 kbp Xylanase C sub-fragment was contained in a 8 kbp EcoRI insert. Essentially, PCR amplification and cloning of the
• Xylanase C ORF region was performed using primers MJA001 (SEQ ID NO:26) /MJA003 (SEQ ID NO: 28) and the restriction enzymes EcoRI and Xhol.
• the plasmid pMJA008 containing the Histone H4b promoter, Xylanase ORF and Xylanase terminator was constructed by assembling the PCR amplified fragment containing the Xylanase ORF and Xylanase terminator into pMJAOOl .
• PCR amplification and cloning of the Xylanase C gene was using primers MJA001(SEQ ID NO:26)/MJA002(SEQ ID NO:27) and the restriction enzymes EcoRI and Xhol.
• the construction of the histone driven uidA reporter gene plasmids started with the construction of the H4b- vector.
• PCRs were performed with pBS-H4B (the original library clone containing the H4B gene) as template and NB116(S ⁇ Q ID NO:29)/ NB117(SEQ ID NO:30) for the H4B promoter or NB118(SEQ ID NO:31)/T7 sequencing primer for the H4B terminator.
• the PCR products were gel electrophoresed and DNA fragments corresponding to predicted sizes purified and ligated to pGEM-T-EASY.
• the resultant plasmids (pNJB61 containing H4b promoter and pNJB62 containing H4b terminator) were checked by restriction digests.
• pNJB62 was digested with BamHl and the resultant 2.9 kbp fragment containing H4b terminator was gel purified and ligated to BamHl cut/phosphatased pNJB61 to give pNJB63.
• the unwanted BamHl site in pNJB63 was removed by digesting with Xbal/Pstl, blunt-ending with T4 DNA polymerase and re-ligating to give pNJB64.
• a H4b promoter + uidA + H4b terminator vector was constructed. Plasmid pAN52-7 carrying the E. coli uidA gene on a Ncol fragment was gel purified and the uidA fragment blunted by Mung bean digestion. The product was then ligated to BamHl cut/Mung bean blunt ended pNJB64 to give pNJB68. The success of all ligations was checked by sequencing.
• pNJB68 was digested with Spel and Ncol and after gel purification, re-ligated originating the p ⁇ JB69.
• the construction of the expression cassette pBAG was initiated by PCR, using the plasmid p ⁇ p (8kb EcoRI) as template.
• This plasmid contains the whole 8kb apf genomic sequence including 2,2kb promoter region.
• the promoter region was amplified using the primer M13(-20) as standard universal primer and ApfA (SEQ ID NO:32) cutting off the putative pre-pro sequence of Apf at position aa 68.
• the altered aa sequence is therefore SAASM instead of SAAAS.
• PCR conditions were lx Taq Pol. Buffer; lpmol of each primer; 200 ⁇ M dNTPs; 5U Taq Polymerase (Appligen) in 50 ⁇ l Volume.
• the resulting 2,4kb PCR fragment was ligated into pGEM T-easy.
• the plasmid containing this insert was named pPl .
• the terminator region of apf was amplified by PCR using the vector pBIISK- containing a 4,2kb Sail genomic subclone of apf.
• primer again the primer M13(-20) could be used and the primer ApfB (SEQ ID NO:33) carrying the Stop codon (TAA) of apf.
• TAA Stop codon
• the uidA gene deriving from the vector pNOM102 (Roberts et al., 1989; Curr.Genet.l_5, 177-180) was used as Ncol fragment.
• pUC19 was digested by EcoRI/Sall.
• the plasmid pPl carrying the promoter region was restricted by NcoI/EcoRI and the fragment was purified.
• the plasmid pT7 containing the terminator region of apf was digested with Sall/Ncol and this 2,2kb DNA fragment was also purified.
• the identified 3,6kb Sphl fragment ligated into pUC19 encompassing the csl31 gene served as template for the construction of the csl31 expression cassette.
• Two primer combinations were used to amplify the promoter and terminator region of csl31.
• the oligonucleotids CslDell (starting at the identified N-terminal region of the gene transcribing in upward direction) and CslDel2 (starting at the Stop codon of Csl31 transcribing downstream) are both carrying a Nael site which is thought to be used to cut and religate promoter and terminator region of csl31, thereby deleting the csl31 gene.
• the promoter region was amplified by PCR [94°, lmin; 55°, lmin; 72°, 2min; 30cycles] with the primer combination CslDell(SEQ ID NO:34)/M13reverse.
• the 1,5 kb DNA fragment was purified over gel and cloned into pGEM T-easy (Promega). One clone has been chosen, sequenced and named pP3.
• the terminator region was also amplified by PCR [94°, lmin; 55°, lmin; 72°, 2min; 30cycles] with the primer combination CslDel2 (SEQ ID NO:35)/M13(-20).
• the resulting 1,3 kb DNA fragment was purified over gel and cloned into pGEM T-easy (Promega).
• the plasmid has been named pT2 and sequenced.
• the csl31 cassette was constructed by ligating the Sphl-Spel 1450bp promoter fragment of pP3 together with the Spel-PvuII 1250bp terminator fragment of pT2 into Sphl-Smal pUC19.
• the resulting plasmid was named pUCE2. Sequencing revealed that only the Nael site of CslDell remained in this construct.
• the Hindlll/Smal fragment of pUCEl carrying the promoter and leader sequence of csl31 was ligated into Hindlll/Smal pBIISK- (Stratagene) resulting in the plasmid pBC3.
• the uidA reporter gene was cut out from pNOM102 by Ncol, blunt ended by Mung Bean nuclease and inserted into the Smal site of pBC3 resulting in the plasmid pBCG8.
• the csl31 terminator region from pUCEl was then isolated as Spel/SacII fragment and ligated into Spel/SacII restricted pBCG8.
• the resulting plasmid containing the csl31 expression cassette was named pBCGT (see Figure 9).
• the plasmid pBAG was co-transformed with the plasmid pAN7-l into P. funiculosum IMI 134756.
• Hygromycin resistant colonies have been transferred onto LYMM agar plates containing 200 ⁇ g/ml Hygromycin B and analysed on Southern blot using the uidA gene as probe.
• 11 out of 12 hygromycin-resistant colonies carried the integrated plasmid pBAG.
• Four transformants (no2, no4, no5 and no8 see Figure 10) have been further investigated towards their ⁇ -glucuronidase activity.
• the obtained mycelium was resuspended in 1ml Extraction buffer (50mM sodium phosphate buffer pH7,0; ImM EDTA; 5mM ⁇ - Mercaptoethanol; 0,005% Triton X-100), and sonicated (5min, 50% active cycle, step2). After centrifugation, the clear supernatant was divided in two aliquots (one for ⁇ -glucuronidase determination, the second for a Bradford assay). A Bradford assay has been performed to determine the amount of protein according to the protocol of the manufacturer (Bio-Rad). Due to the presence of oligo-peptides in the filtrate (deriving from the hydrolyses of the Casein) a proper quantification of secreted fungal proteins was not possible.
• Extraction buffer 50mM sodium phosphate buffer pH7,0; ImM EDTA; 5mM ⁇ - Mercaptoethanol; 0,005% Triton X-100
• the predicted leader and pre-pro sequence of the acidic Aspartyl Protease are functional and direct the secretion of the ⁇ -glucuronidase into the medium. Furthermore, the ⁇ -glucuronidase activity measured in the mycelium is almost proportional with the integration number of the plasmid pBAG. Therefore, the values reflect rather the strength of the Aspartyl protease promoter than the appearance of position effects.
• uidA transformants obtained by co-transformation of pNJB68 and pAN7-l, were grown in minimal medium (1% glucose, 0.1M NH 4 C1) at 25°C and the time course for the intracellular expression of uidA driven by the histone 4b promoter measured essentially as described by Roberts et al.,1989.Curr .Genet. 15: 177-180 ( Figure 11).
• pNJB68 was digested with Spel and Ncol and after gel purification, re-ligated originating the p ⁇ JB68.
• the constructs pMJA003 and pMJA008 was co-transformed with a pAN7-l hygromycin vector into P. funiculosum.
• transformants were screened for Xylanase activity by a modified version of the
• Congo Red polysaccharide interaction method routinely used for lambda library screening at The Babraham Institute and originally described by R.M.Teather & P. J. Wood (1982) Applied and Environmental Microbiology, 43(4):777-780.
• soluble xylan is prepared by solubilising oat spelt xylan (lOg - Sigma
• the xylan overlay is then prepared by heating a solution of soluble xylan (0.25%) and agar (1%) in phosphate/citrate buffer (50 mM - pH 6.5). After cooling to c.a. 50°C, the xylan overlay is poured (4 ml) on top of MN-hygromycin B plates previously inoculated with

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