# Patent

## Title
Fungal transcriptional activators useful in methods for producing a polypeptide

## Abstract

The present invention relates to functional cDNA and genomic sequences encoding PrtT proteins, which have transcriptional activity on a protease promoter, to PrtT proteins and to their use. The invention further relates to two distinct types of filamentous fungal cells. Filamentous fungal cells are transformed to over-express these PrtT proteins: this type of filamentous fungus will be highly suited as protease producer. Alternatively, the endogenous prtT genes of filamentous fungal cells are inactivated: this type of filamentous fungus is highly suited for the production of any polypeptide native or not which is highly sensitive for protease degradation. The PrtT proteins of the invention provide means for identification of functional homologues in other species.

## Background
This application is a U.S. national stage of International Patent Application No. PCT/EP2005/055145, filed 11 Oct. 2005, which designated the U.S. and claims priority benefit of EP 04105001.4, filed 12 Oct. 2004; the entire contents of each of which are hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to polypeptides having transcriptional activation activity on a protease promoter, to the nucleic acid sequences encoding these polypeptides and to several uses of these nucleic acid sequences.
BACKGROUND OF THE INVENTION
Fungal transcriptional activators named PrtT have been recently described in WO 00/20596 and WO 01/68864. These transcriptional activators were isolated from Aspergillus niger (A. niger) and Aspergillus oryzae (A. oryzae). These transcriptional activators of protease genes can be either used to improve a method for producing proteases in a fungal cell or to improve a method for producing a polypeptide in a fungal cell, wherein the polypeptide is sensitive for protease degradation.
The present invention provides novel PrtT fungal transcriptional activators, which have improved properties compared to the ones described in both earlier applications.



DESCRIPTION OF THE FIGURES
FIG. 1 Endoprotease activity in supernatant of the WT1 strain grown in IM supplemented with 2% defatted soy flour.
Activity on the Y-axis is the endoprotease activity determined in U/50 μl supernatant per h. The X-axis indicates culture time in h after transfer to IM.
FIG. 2 Plasmid map of expression vector pGBFIN-23.
Indicated are the glaA flanking regions relative to the glaA promoter and HindIII-XhoI cloning site. The E. coli DNA can be removed by digestion with restriction enzyme NotI, prior to transformation of the A. niger strains.
FIG. 3 Plasmid map of expression vector pGBFINPRT-1.
Indicated are the glaA flanking regions relative to the glaA promoter and the cDNA insert encoding the PrtT transcriptional regulator of the invention in the HindIII-XhoI cloning site. The E. coli DNA can be removed by digestion with restriction enzyme NotI, prior to transformation of the A. niger strains.
FIG. 4 Alignment of the PrtT sequence of the invention (SEQ ID NO 3 is ‘A. niger 1’) and the PrtT sequence (‘A. niger 2’) from WO 00/20596 and WO 01/68864.
The differences between these amino acid sequences are marked in grey. The underlined sequences depict the position of the zinc binuclear cluster Zn(II)2-Cys6 DNA binding domain (47-89) and Leucine zipper (438-461, as counted based on the A. niger 1 sequence), respectively.
FIG. 5 Alignment of the PrtT sequence of the invention (SEQ ID NO 3 is ‘A. niger’) and the A. oryzae PrtT sequence from WO 01/68864 (‘A. oryzae’).
The differences between these amino acids sequences are marked in grey.
FIG. 6 Schematic presentation of integration through single homologous recombination. The expression vector comprises the selectable amdS marker, a glaA promoter connected to a prtT sequence of the protease transcriptional activator of the invention. These features are flanked by homologous regions of the glaA locus (3′ glaA and 3′′ glaA, respectively) to direct integration at the genomic locus.
FIG. 7 Extra-cellular acidic protease activity of PRTT strains during several days of the fermentation. Protease activity measured using BSA as substrate. One Unit=ΔOD280/hour of 50 ul supernatant.
FIG. 8 Plasmid map of replacement vector pGBDEL. Indicated are the multiple cloning sites for cloning the flanking regions relative to the amdS marker.
FIG. 9 Plasmid map of replacement vector pGBDEL-PRT2. Indicated are the 5′ prtT flanking region, the 3′ prtT flanking regions relative to the amdS marker. The sequence of the prtT 3′ sequences overlap at least a few hundred bp. The E. coli DNA was removed by digestion with restriction enzyme BstBI and XmaI, prior to transformation of the A. niger strains.
FIG. 10 Schematic presentation of the prtT deletion. A linear DNA construct of pGBDEL-PRT2, comprising the amdS selection marker flanked by homologous regions (5′ and 3′) of the prtT gene (1), integrates through double homologous recombination (X) at the genomic prtT locus (2) and replaces the genomic prtT gene copy (3). Subsequently, recombination over the direct repeats (U) removes the amdS marker, resulting in precise excision of the prtT gene (4).
FIG. 11 Endoprotease activity in supernatant of the A. niger WT2 strain and dPRTT strains. Activity on the Y-axis is the endoprotease activity determined in U/50 μl per hour. The X-axis indicates culture time (hours) when the supernatant was collected.
FIG. 12 Plasmid map of the PLA2 expression vector pGBFIN-PLA2. Indicated are the glaA flanking regions relative to the glaA promoter, the truncated glaA gene and the pla2 coding sequence. The E. coli DNA was removed by digestion with restriction enzyme NotI, prior to transformation of the A. niger strains.
FIG. 13 Phospoholipase A2 activity measured in the broth of A. niger pGBFIN-PLA2 transformants of WT 2 and dPRTT strains. Phospholipase A2 activities were measured as indicated in Experimental information.
FIG. 14 Alignment of the PrtT sequence of A. niger WT1 (‘A. niger’) as determined from the sequenced cDNA's (SEQ ID NO 3) and the A. oryzae PrtT sequence identified by TBlastn search against the Patent database (‘A. oryzae’). The differences between these two amino acids sequences are marked in grey.
FIG. 15 Alignment of the PrtT sequence of A. oryzae (‘A. oryzae1’) as identified by the TBlastn search against the Patent database (SEQ ID NO 15) and the A. oryzae PrtT sequence from WO 01/68864 (‘A. oryzae 2’). The differences between these two amino acids sequences are marked in grey.
FIG. 16 Alignment of the A. fumigatus PrtT sequence identified by TBlastn search against nucleotide databases (‘A. fumigatus’) and the PrtT sequence of A. niger WT1 (‘A. niger’) as determined from the sequenced cDNA's (SEQ ID NO 3). The differences between these two amino acids sequences are marked in grey.
FIG. 17 CLUSTAL W multiple alignment of four fungal PrtT polypeptides of this invention. The marked boxes depict the position of the zinc binuclear cluster Zn(II)2-Cys6 DNA binding domain (grey) and Leucine zipper (in bold), respectively. The conserved Leu residues in the Leucine zipper box are in italics.

    
    
        * under the alignment signifies the presence of an identical amino acid,
        : means that the type of amino acids is conserved in all three sequences, and
        . means that at least in two sequences a similar type of amino acid is present

The underlined sequence shows where the peptide SEQ ID NO 22 is localized.

    
    


FIG. 18 The comparison of the peptide SEQ ID NO 22 from A. niger WT1 and the corresponding peptide sequences in the other PrtT fungal polypeptides of the invention and those from the patent applications WO 00/20596 and WO 01/68864.
FIG. 19 Plasmid map of pGBFINPRT-1
Indicated are a 200 bp 3′-fragment of the pg/aA promoter fused to the full-length prtT cDNA sequence according to SEQ ID NO: 2. The PCR generated fusion fragment was cloned in pCR-BluntII-TOPO vector from Invitrogen. The fusion fragment can be isolated by restriction enzyme digestion with EcoRI and XhoI. Subsequently the fragment can be ligated into EcoRI and XhoI sites of expression vector pGBFIN-23.



DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Transcriptional Activation Activity on a Protease Promoter
According to a first aspect, the present invention relates to a polypeptide having transcriptional activity on a protease promoter, wherein said polypeptide is selected from the group consisting of:

    
    
        (a) a polypeptide having an amino acid sequence which has at least 50% match percentage, i.e. identity, with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 21 or a polypeptide comprising a peptide fragment, said peptide fragment having at least 50% match percentage with SEQ ID NO:22 or SEQ ID NO: 4 or SEQ ID NO:25 or SEQ ID NO:26 or SEQ ID NO:27; or
        (b) a polypeptide comprising peptide fragments, said peptide fragments having at least 50% match percentage with both SEQ ID NO:22 and SEQ ID NO: 4, or with both SEQ ID NO:22 and SEQ ID NO:25, or with both SEQ ID NO:22 and SEQ ID NO:26, or with both SEQ ID NO:22 and SEQ ID NO:27; or
        (c) a polypeptide having an amino acid sequence according to (a) and (b) or (a) and (c); or
        (d) a polypeptide having an amino acid sequence which has at least 50% match percentage, i.e. identity, with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 21 and comprising a peptide fragment, said peptide fragment having the amino acid sequence: EWAHL(X)5-20ST, wherein “X” represents any amino acid and the range 5-20 represents the number of “X” found or,
        (e) a variant of (a) or (b) or (c) or (d) or (e).
    
    


A polypeptide having transcriptional activity on a protease promoter is a transcriptional activator of a protease promoter. The term “transcriptional activator” as used herein refers to a polypeptide which has the capability to activate transcription from a specific protease promoter or a set of protease promoters, said activator being necessary for the initiation of transcription of the protease(s) encoding sequence to which the protease promoter(s) is (are) operably linked to.
The biological activity of the transcriptional activator is preferably determined through measurement of protease activities as described in the example section herein for determination of the acidic endo-protease activity using Bovine Serum Albumin (BSA) as substrate. A detailed description of this method is also described by van den Hombergh et al., Current Genetics 28: 299-308 (1995). Alternative methods for protease measurements can be found in WO 02/068623. Alternatively, one or more specific protease reporter genes such as the pepstatin sensitive extracellular aspartic protease encoding pepA gene can be used for measuring the activity of the transcriptional activator.
Additionally, the use of a reporter gene under the control of a protease promoter can be considered such that the enzymatic activity of the reporter protein, which is in operative association with the protease promoter is measured. An example of measuring the activity of a lacZ and GFP reporter gene have already been described (Luo, Gene, (1995), 163:127-131 and in Santerre Henriksen A L et al, Microbiology, (1999), 145:729-34).
Alternatively, the biological activity of the transcriptional activator activity can be determined by measuring the mRNA levels of the protease transcripts. The mRNA levels can, for example, be measured through a Northern blot (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).
According to a preferred embodiment, the polypeptide of the present invention does not have the amino acid sequence SEQ ID NO:2 or SEQ ID NO:49 as disclosed in WO 01/68864.
For purposes of the present invention, the degree of identity, i.e. the match percentage, between two polypeptides, respectively two nucleic acid sequences is preferably determined using the optimal global alignment method CDA (Huang, 1994, A Context Dependent Method for Comparing Sequences, Proceedings of the 5th Symposium on Combinatorial Pattern Matching, Lecture Notes in Computer Science 807, Springer-Verlag, 54-63) with the parameters set as follows: (i) for (poly)peptide alignments: Mismatch: −2 GapOpen: 11 GapExtend: 1 ContextLength: 10 MatchBonus: 1, and (ii) for nucleotide sequence alignments Mismatch: −15 GapOpen: 5 GapExtend: 2 ContextLength: 10 MatchBonus: 1.
The terms “degree of identity”, “identity” and “match percentage” are used interchangeably to indicate the degree of identity between two polypeptides or nucleic acid sequences as calculated by the optimal global alignment method indicated above. Examples of alternative programs used for alignments and determination of homology are Clustal method (Higgins, 1989, CABIOS 5: 151-153), the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENETM MEGALIGNTM software (DNASTAR, Inc., Madison, Wis.), BLAST (NCBI), GAP (Huang) for the optimal global alignments, MAP (Huang), MultiBLAST (NCBI), ClustalW, Cap Assembler and Smith Waterman for multiple alignments.
REFERENCES










Pairwise alignment: (1) BLAST, (2) GAP, (3) MAP,


(4) Smith Waterman, and (5) Cap Assembler
















(1) Tatusova T A and
BLAST 2 sequences, a new tool for


Madden T L (1999)
comparing protein and nucleotide



sequences. FEMS Microbiol Lett 174: 247-



50


(2) (3) Huang X (1994)
On global sequence alignment. Comput



Appl Biosci 10: 227-35


(4) Smith T F and
Identification of common molecular


Waterman M S (1981)
subsequences. J Mol Biol 147: 195-197


(5) Huang X (1992)
A contig assembly program based on



sensitive detection of fragment overlaps.



Genomics 14: 18-25


(5) Huang X (1996)
An improved sequence assembly program.



Genomics 33: 21-31


(6) Thompson J D, Higgins
CLUSTAL W: improving the sensitivity of


D G, and Gibson T J (1994)
progressive multiple sequence alignment



through sequence weighting, positions-



specific gap penalties and weight matrix



choice. Nucleic Acids Research 22: 4673-



4680









In a most preferred embodiment, the polypeptide has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 21 or a polypeptide obtainable by expression of the prtT cDNA contained in pGBFINPRT-1 shown in FIG. 3, deposited under accession number CBS118680 or a polypeptide obtainable by expression of the prtT cDNA contained in pGBPRT-1 shown in FIG. 19, deposited under accession number CBS118681.
According to a more preferred embodiment, the polypeptide has an amino acid sequence which has at least 50% match percentage, i.e. identity, with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 21 and comprises a peptide fragment, said peptide fragment having at least 50% match percentage, i.e. identity, with SEQ ID NO: 22 or SEQ ID NO: 4 or SEQ ID NO: 25 or SEQ ID NO:26 or SEQ ID NO:27.
According to another more preferred embodiment, the polypeptide has an amino acid sequence which has at least 50% match percentage, i.e. identity, with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 21 and comprises peptide fragments, said peptide fragments having at least 50% match percentage with both SEQ ID NO:22 and SEQ ID NO: 4, or with both SEQ ID NO:22 and SEQ ID NO:25, or with both SEQ ID NO:22 and SEQ ID NO:26, or with both SEQ ID NO:22 and SEQ ID NO:27.
According to another preferred embodiment, the polypeptide has an amino acid sequence which has at least 50% match percentage, i.e. identity, with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 21 and comprises a peptide fragment, said peptide fragment having the amino acid sequence: EWAHL(X)5-20ST, wherein “X” represents any amino acid and the number represents the number of “X” found. An example of such a peptide fragment is represented by the SEQ ID NO: 22.
Preferably, the match percentage, i.e. identity, is at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, even more preferably at least about 93%, even more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97%, even more preferably at least about 98%, most preferably at least about 99%.
According to another preferred embodiment, the polypeptide is a variant of any one of the polypeptide sequences defined before. Modification of the polypeptide of the present invention may be necessary for the synthesis of variant polypeptides. The term variant preferably refers to non-naturally occurring forms of the polypeptide. These polypeptide variants may differ in some engineered way from the polypeptide isolated from its native source. For example, it may be of interest to synthesize variants of the polypeptide where the variants differ in specific activity, binding specificity and/or affinity, or the like by, e.g., site-directed mutagenesis. The variant sequence may be constructed on the basis of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21 or on the basis of the nucleic acid sequence presented as the polypeptide encoding part of SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 17 or SEQ ID NO: 20.
A variant of SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21 is preferably a polypeptide having:

    
    
        one or more amino acids deleted preferably from the amino and/or carboxy terminus of this amino acid sequence and/or
        one or more amino acid residues inserted and/or
        one or more amino acid residues replaced by one or more different amino acid residues
        combinations of the variations mentioned above.
    
    


According to another preferred embodiment, a variant of SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21 contains at least the polypeptide sequence shown in SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21.
In a preferred embodiment, the polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21 or SEQ ID NO: 22.
According to another preferred embodiment, a polypeptide variant is a transcriptional activator isolated from other organisms and/or another family member of the transcriptional activator initially isolated and present in the same organism.
According to a preferred embodiment, the polypeptide variant contains mutations that do not alter the biological function of the encoded polypeptide. Such polypeptides differ in amino acid sequence from SEQ ID NO 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21, yet retain at least one of their biological activities and preferably, these proteins are not the ones having SEQ ID NO: 2 or 49 as disclosed in WO 01/68864. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., Science 247:1306-1310 (1990).
According to a preferred embodiment, the polypeptide variant exhibits a particular function of the transcriptional activator of a protease promoter. This transcriptional activator variant exhibits at least the function of transcriptional activator on at least one protease promoter. This variant may contain only conservative substitutions of one or more amino acids of sequences having SEQ ID NO 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21, or substitutions, insertions or deletions of non-essential amino acids. Accordingly, a non-essential amino acid is a residue that can be altered in one of these sequences without substantially altering the biological function. For example, amino acid residues that are conserved among the transcriptional activator of the present invention are predicted to be particularly unamenable to alteration. The term “conservative substitution” is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and hystidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).
Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Example of mutagenesis procedures are the QuickChangeTM site-directed mutagenesis kit (Stratagene Cloning Systems, La Jolla, Calif.), the ‘The Altered Sites® II in vitro Mutagenesis Systems’ (Promega Corporation) or by overlap extension using PCR as described in Gene. Apr. 15, 1989; 77(1):51-9. (Ho S N, Hunt H D, Horton R M, Pullen J K, Pease L R “Site-directed mutagenesis by overlap extension using the polymerase chain reaction”) or using PCR as described in Molecular Biology: Current Innovations and Future Trends. (Eds. A. M. Griffin and H. G. Griffin. ISBN 1-898486-01-8; 1995 Horizon Scientific Press, PO Box 1, Wymondham, Norfolk, U.K.).
The transcriptional activator of a protease promoter of the invention may be obtained from any filamentous fungus.
“Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelia wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma. 
In a more preferred embodiment, the transcriptional activator of a protease promoter of the present invention is obtained from a strain of Aspergillus, such as A. awamori, A. nidulans, A. niger, A. oryzae, A. sojae or A. fumigatus. Preferably, the transcriptional activator is obtained from a strain of A. niger or A. oryzae or A. fumigatus. Even more preferably, the transcriptional activator is obtained from an isolate of a strain of A. fumigatus; e.g., the polypeptide sequence set forth in SEQ ID NO: 18. Even more preferably, the transcriptional activator is obtained from an isolate of a strain of A. oryzae; e.g., the polypeptide sequence set forth in SEQ ID NO: 15. Even more preferably, the transcriptional activator is obtained from an isolate of a strain of A. niger; e.g., the polypeptide sequence set forth in SEQ ID NO: 3.
In another preferred embodiment, the transcriptional activator is obtained from an isolate of a strain of Penicillium, such as Penicillium chrysogenum; e.g., the polypeptide sequence set forth in SEQ ID NO: 21.
According to another preferred embodiment, the transcriptional activator of the invention is an orthologue of the A. niger, A. oryzae, A. fumigatus, or P. chrysogenum transcriptional activator. Orthologues of these polypeptides are polypeptides that can be isolated from other strains or species and possess a similar or identical biological activity. Such orthologues can readily be identified as comprising an amino acid sequence that is substantially homologous to SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:18 or SEQ ID NO:21.
The term “substantially homologous” refers to a first polypeptide or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with similar side chain) amino acids or nucleotides to a second polypeptide or nucleotide sequence such that the first and the second polypeptide or nucleotide sequences have a common peptide or nucleic acid fragment. For example, polypeptide or nucleotide sequences which contain a common peptide or nucleic acid fragment having about 90%, preferably about 92%, preferably about 93%, preferably about 95%, more preferably about 97%, even more preferably about 99%, identity or more are defined herein as sufficiently identical. An example of such a common domain is the one depicted in SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27.
In a preferred embodiment, the transcriptional activator of protease promoters has an amino acid sequence according to SEQ ID NO 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21. In another embodiment, the polypeptide or a peptide derived thereof is substantially homologous to the amino acid sequence according to SEQ ID NO 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21, SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and retains at least one biological activity of a polypeptide according to SEQ ID NO 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21, yet differs in amino acid sequence due to natural variation or mutagenesis as described above. Preferably, the transcriptional activator of protease promoters of the invention are not the ones depicted in WO 01/68864 and having SEQ ID NO: 2 or 49.
In another preferred embodiment, the transcriptional activator of the present invention is obtained from a strain of Fusarium, such as F. oxysporum. Preferably, the strain is a strain of F. venenatum. 
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. For example, the polypeptides may be obtained from micro-organisms, which are taxonomic equivalents of Aspergillus as defined by Raper, K. D. and Fennel, D. I. (1965. The Genus Aspergillus, The Wilkins Company, Baltimore Md.) regardless of the species name by which they are known.
Aspergilli are mitosporic fungi characterized by an aspergillum comprised of a conidiospore stipe with no known teleomorphic states terminating in a vesicle, which in turn bears one or two layers of synchronously formed specialized cells, variously referred to as sterigmata or phialides, and asexually formed spores referred to as conidia. Known teleomorphs of Aspergillus include Eurotium, Neosartorya, and Emericella. 
Strains of Aspergillus and teleomorphs thereof are readily accessible to the public in a number of culture collections. According to a preferred embodiment, the strains of filamentous fungus used to isolate the transcriptional activator is Aspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, Aspergillus fumigatus AF293 (CBS101355), P. chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense ATCC44006 and derivatives thereof.
Furthermore, such transcriptional activators of protease promoters may be identified and obtained from other sources including micro-organisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating micro-organisms from natural habitats are well known in the art. The nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another micro-organism. Once a nucleic acid sequence encoding a transcriptional activator of protease promoter has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., J. Sambrook, et al. 1989,).
Deteriorated Transcriptional Activator of a Protease Promoter
In another preferred embodiment of the invention, variants of transcriptional activator of a protease promoter are deteriorated transcriptional activators of a protease promoter. Deteriorated transcriptional activators of a protease promoters are polypeptides, wherein at least one of their biological activities is decreased compared to the biological activity of their wild type counterpart measured in a given filamentous fungal host using a given assay or an in vitro assay as defined earlier in the description. For example, such polypeptide may have less transcriptional activity than its wild type counterpart on at least one protease promoter as measured in a given filamentous fungal host using one of the given assays. Alternatively, the deteriorated polypeptide may have lost its transcriptional activity as compared to the activity of its wild type counterpart on at least one specific protease promoter and retained the activity of its wild type counterpart on at least another protease promoter as measured in a given filamentous fungal host using one of the given assays. According to a preferred embodiment, the deteriorated transcriptional activator does not have any detectable transcriptional activating activity on any protease promoter tested by comparison with the activity of its wild type counterpart as measured in a given filamentous fungal host using one of the given assays. A preferred assay for measuring the transcriptional activity of a deteriorated transcriptional activator is by measuring the acidic endo-protease activity using Bovine Serum Albumin (BSA) as substrate as described herein. Preferably, the filamentous fungal host used to perform the protease assay is one of the deposited strains as described earlier on in the description. All these deteriorated transcriptional activators may be used to replace their wild type counterpart in a given filamentous fungal host cell. Such a cell is highly suited for producing any polypeptide subject to protease degradation (see section host cell). Such a cell, especially Aspergillus fumigatus may also have a reduced pathogenicity.
Such polypeptide may be obtained by randomly introducing mutations along all or part of their coding sequence (using SEQ ID NO:2, SEQ ID NO: 14, SEQ ID NO: 17 or SEQ ID NO: 20,), such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity using one of the assays mentioned in the previous paragraphs. For instance, the art provides standard assays for measuring the transcriptional activity and thus transcriptional activator with deteriorated transcriptional activity may easily be selected.
Transcriptional Activator of a Protease Promoter with Enhanced Activity
According to another preferred embodiment of the invention, variants of transcriptional activators of a protease promoter are transcriptional activators of a protease promoter with enhanced activity. Transcriptional activators of a protease promoter with enhanced activity are polypeptides, wherein at least one of their biological activities is increased compared to the biological activity of their wild type counterpart measured in a given filamentous fungal host using a given assay or an in vitro assay. For example, such polypeptide may have at least more transcriptional activity than its wild type counterpart on at least one protease promoter as measured in a given filamentous fungal host using one of the assays mentioned in the previous paragraphs. Alternatively, the enhanced polypeptide may have more transcriptional activity on protease promoter than its wild type counterpart on at least one specific protease promoter and retained the activity of its wild type counterpart on at least one other protease promoter as measured in a given filamentous fungus using one of the given assays. According to a preferred embodiment, the enhanced transcriptional activator has an enhanced transcriptional activating activity on any protease promoter tested by comparison to the activity of its wild type counterpart as measured in a given filamentous fungus host using one of the assays described earlier. A preferred assay for measuring the transcriptional activity of a deteriorated transcriptional activator is by measuring the acidic endo-protease activity using Bovine Serum Albumin (BSA) as substrate as described herein. Preferably, the filamentous fungal host used to perform the protease assay is one of the deposited strains as described earlier on in the description. All these enhanced transcriptional activator may be used to replace their wild type counterpart in a given filamentous fungal host cell. Such a cell is highly suited for producing any polypeptide subject to transcription regulation by such a transcriptional activator (see section host cell). Such enhanced transcriptional activator may be obtained using the same strategy as the one described for obtaining deteriorated transcriptional activator.
Nucleic Acid Sequence Encoding a Transcriptional Activator of a Protease Promoter
According to a further aspect, the invention provides a nucleic acid sequence coding for the polypeptide as defined in the former sections.
In a preferred embodiment, the nucleic acid sequence is selected from the group consisting of:

    
    
        (a) a nucleic acid sequence having at least 50% match percentage, i.e. identity with the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14 or SEQ ID NO: 17, or SEQ ID NO: 20; or
        (b) a nucleic acid sequence comprising a fragment, said fragment having at least 45% match percentage, i.e. identity with the fragment consisting of base pair number 1267 till base pair number 1302 of nucleic acid sequence of SEQ ID NO:2; or
        (c) a nucleic acid sequence having at least 50% match percentage, i.e. identity with the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14 or SEQ ID NO: 17, or SEQ ID NO: 20; and comprising a fragment, said fragment having at least 45% match percentage, i.e. identity with the fragment consisting of base pair number 1267 till base pair number 1302 of nucleic acid sequence of SEQ ID NO:2; or
        (d) a variant of (a), (b), or (c); or
        (e) a subsequence of (a), (b), (c) or (d).
    
    


Preferably the match percentage, i.e. identity is of at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, most preferably at least about 90%, even most preferably at least about 93%, even most preferably at least about 95%, even most preferably at least about 96%, even most preferably at least about 97%, even most preferably at least about 98%, even most preferably at least about 99% identity.
In an even more preferred embodiment, the nucleic acid sequence encoding a transcriptional activator of a protease promoter has a nucleic acid sequence as set forth in SEQ ID NO: 2, or SEQ ID NO: 14, or SEQ ID NO: 17 or SEQ ID NO: 20, or the XhoI/HindIII fragment of plasmid pGBFINPRT-1 depicted in FIG. 3, deposited under accession number CBS118680 or the prtT cDNA contained in plasmid pGBPRT-1 depicted in FIG. 19, deposited under accession number CBS118681.
According to another preferred embodiment, the nucleic acid sequence of the invention is not the nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 48 as disclosed in WO 01/68864.
According to another preferred embodiment, the nucleic acid sequence of the invention is a variant of any of the nucleic acid sequences as defined before. Modification of the nucleic acid sequence of the present invention may be necessary for the synthesis of variant polypeptides (as defined earlier in the description). The nucleic acid sequence variant may be constructed on the basis of the nucleic acid sequence SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 17 or SEQ ID NO: 20 or the XhoI/HindIII fragment of plasmid pGBFINPRT-1 depicted in FIG. 3, deposited under accession number CBS118680 or the prtT cDNA contained in plasmid pGBPRT-1 depicted in FIG. 19, deposited under accession number CBS118681.
A nucleic acid sequence variant may be a fragment of the native nucleic acid sequence cited in the former paragraph. A preferred nucleic acid sequence variant is a nucleic acid sequence, which contains silent mutations. Alternatively or in combination, a nucleic acid sequence variant may also be obtained by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
Preferably, the nucleic acid variant is such that starting from any one of the nucleic acid sequences cited in the former paragraph, one or more nucleotides from the 5′ and/or 3′ end have been deleted. More preferably, the variant encodes a polypeptide fragment, which has transcriptional activation activity on a protease promoter.
Alternatively or in combination, a nucleic acid sequence variant is preferably a nucleic acid sequence encoding a transcriptional activator isolated from other organisms and/or another family member of the transcriptional activator initially isolated and present in the same organism. All these variants can be obtained in a typical approach, using cDNA or genomic DNA libraries constructed from organisms, e.g. filamentous fungi, in particular from the species Aspergillus by screening them by hybridisation (standard Southern blotting procedures) under low to medium to high stringency conditions with one of the following nucleic acid sequences which can be used to design probes:

    
    
        SEQ ID NO: 2,
        SEQ ID NO: 14,
        SEQ ID NO: 17,
        SEQ ID NO: 20,
        the XhoI/HindIII fragment of plasmid pGBFINPRT-1 depicted in FIG. 3, deposited under accession number CBS118680,
        the prtT cDNA contained in plasmid pGBPRT-1 depicted in FIG. 19, deposited under accession number CBS118681.
        or a fragment of any one of these sequences, preferably the fragment consisting of base pair number 1267 till base pair number 1302 of nucleic acid sequence of SEQ ID NO:2,
        their complementary strand.
    
    


Low to medium to high stringency conditions means prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 pg/ml sheared and denatured salmon sperm DNA, and either 25%, 35% or 50% formamide for low to medium to high stringencies, respectively. Subsequently, the hybridization reaction is washed three times for 30 minutes each using 2×SSC, 0.2% SDS and either 55° C., 65° C. or 75° C. for low to medium to high stringencies.
The designed probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 40 nucleotides in length. Additionally, such probes can be used to amplify DNA probes through PCR. Both DNA, RNA and Peptide Nucleid Acid (PNA) probes can be used for hybridisation. Such probes are encompassed by the present invention. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 33P, 3H, 35S, biotin, avidin or a fluorescent marker). For example, molecules to which a 32P, 33P, 3H- or 35S-labelled oligonucleotide probe hybridizes may be detected by use of X-ray film or Phospho-Image analysis.
A variant of the nucleic acid sequence may also be a paralogous of the transcriptional activator of a protease promoter. In the context of the invention, paralogous means nucleic acid sequence homologous of SEQ ID NO: 2, the nucleic acid sequence consisting of base pair number 1267 till base pair number 1302 of nucleic acid sequence of SEQ ID NO:2, SEQ ID NO: 14, SEQ ID NO: 17 or SEQ ID NO: 20 and derived from A. niger or A. oryzae or A. fumigatus or P. chrysogenum. 
For example, Aspergillus strains can be screened for homologous nucleic acid sequences coding for the transcriptional activator of a protease promoter by Northern blot analysis. Upon detection of transcripts homologous to nucleic acid sequences according to the invention, cDNA libraries can be constructed from RNA isolated from the appropriate strain, utilizing standard techniques well known to those of skill in the art. Alternatively, a total genomic DNA library can be screened using a probe that hybridises to a nucleic acid sequence according to the invention.
The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a nucleic acid sequence according to the invention. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the nucleic acid sequences of a new transcriptional activator of a protease promoter, or a functional equivalent thereof. The PCR fragment can then be used to isolate a full-length cDNA clone by a variety of known methods. PCR technology also can be used to isolate full-length cDNA sequences from other organisms. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source. The resulting RNA/DNA hybrid can then be “tailed” (e.g., with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C primer). For a review of useful cloning strategies, see e.g. Sambrook et al., 1989; and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, 1995.
According to another preferred embodiment, a nucleic acid variant is an allelic variant. An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (i.e., no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. In a preferred embodiment, the nucleic acid sequence encoding a transcriptional activator of protease promoter of the present invention is an allelic variant of one of the nucleic acid sequences defined earlier in the description.
Nucleic acid variant may also be nucleic acid sequences, which differ from:

    
    
        SEQ ID NO:2,
        SEQ ID NO: 14,
        SEQ ID NO: 17 or
        SEQ ID NO: 20,
        the XhoI/HindIII fragment of plasmid pGBFINPRT-1 (depicted in FIG. 3), deposited under accession number CBS118680,
        the prtT cDNA contained in plasmid pGBPRT-1 depicted in FIG. 19, deposited under accession number CBS118681

by virtue of the degeneracy of the genetic code.

    
    


Nucleic acid variant may also be nucleic acid sequences, which comprises a variant of the fragment consisting of base pair number 1267 till base pair number 1302 of nucleic acid sequence of SEQ ID NO:2. Variant is given the same meaning as earlier on in this section.
The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using methods based on polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features (See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.). Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used.
In another preferred embodiment, the nucleic acid sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, or SEQ ID NO: 15, or SEQ ID NO: 18 or SEQ ID NO: 21 or a fragment thereof, which has transcriptional activation activity on a protease promoter. In another preferred embodiment, the nucleic acid sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, or SEQ ID NO: 25, or SEQ ID NO:26, or SEQ ID NO: 27 and/or SEQ ID NO: 22.
According to another preferred embodiment, the invention relates to nucleic acid sequences having SEQ ID NO: 16 and SEQ ID NO: 19. These nucleic acid sequences are genomic nucleic acid sequences from A. fumigatus and P. chrysogenum respectively.
Sequence Errors
The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein can be readily used to isolate the complete gene from filamentous fungi, in particular A. niger which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.
Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a nucleic acid sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.
It is to be understood that the present invention does not comprise the prtT nucleic acid sequences and the PrtT polypeptide sequences as disclosed in WO 01/68864 and having respectively SEQ ID NO: 1, or 48 and 2, or 49.
Nucleic Acid Constructs
Another aspect of the present invention relates to nucleic acid constructs comprising a nucleic acid sequence encoding a transcriptional activator of a protease promoter of the invention as isolated or with either deteriorated or enhanced transcriptional activity on a protease promoter, said nucleic acid sequence being operably linked to one or more control sequences, which direct the production of the transcriptional activator of a protease promoter in a suitable expression host. In a preferred embodiment, the nucleic acid sequence is SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 17 or SEQ ID NO: 20, encoding the polypeptide shown in SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 21, respectively.
Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Manipulation of the nucleic acid sequence encoding a polypeptide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.
“Nucleic acid construct” is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence. The term “coding sequence” as defined herein is a sequence, which is transcribed into mRNA and translated into a transcriptional activator of a protease promoter of the invention. The boundaries of the coding sequence are generally determined by the ATG start codon at the 5′ end of the mRNA and a translation stop codon sequence terminating the open reading frame at the 3′ end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
The term “control sequences” is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, optimal translation initiation sequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), a polyadenylation sequence, a pro-peptide sequence, a pre-pro-peptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals.
The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term “operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a polypeptide.
The control sequence may be an appropriate promoter sequence, a nucleic acid sequence, which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence, which shows transcriptional activity in the cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the cell.
The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a filamentous fungal cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide. Any terminator, which is functional in the cell, may be used in the present invention.
Preferred terminators for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, A. niger glucoamylase (glaA), A. nidulans anthranilate synthase, A. niger alpha-glucosidase, trpC gene and Fusarium oxysporum trypsin-like protease.
The control sequence may also be a suitable leader sequence, a non-translated region of a mRNA which is important for translation by the filamentous fungal cell. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence, which is functional in the cell, may be used in the present invention.
Preferred leaders for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase and A. nidulans triose phosphate isomerase and A. niger glaA.
Other control sequences may be isolated from the Penicillium IPNS gene, or pcbC gene, the beta tubulin gene. All the control sequences cited in WO 01/21779 are herewith incorporated by reference.
The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the filamentous fungal cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence, which is functional in the cell, may be used in the present invention.
Preferred polyadenylation sequences for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, A. niger glucoamylase, A. nidulans anthranilate synthase, Fusarium oxyporum trypsin-like protease and A. niger alpha-glucosidase.
Expression Vectors
The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites.
Alternatively, the nucleic acid sequence encoding the polypeptide may be expressed by inserting the sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.
The recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence encoding the polypeptide. The choice of the vector will typically depend on the compatibility of the vector with the filamentous fungal cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. An autonomously maintained cloning vector may comprise the AMA1-sequence (see e.g. Aleksenko and Clutterbuck (1997), Fungal Genet. Biol. 21: 373-397).
Alternatively, the vector may be one which, when introduced into the filamentous fungal cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The integrative cloning vector may integrate at random or at a predetermined target locus in the chromosomes of the filamentous fungal host cell. In a preferred embodiment of the invention, the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of the filamentous fungal host cell for targeting the integration of the cloning vector to this predetermined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the host cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 30 bp, preferably at least 50 bp, preferably at least 0.1 kb, even preferably at least 0.2 kb, more preferably at least 0.5 kb, even more preferably at least 1 kb, most preferably at least 2 kb. Preferably, the DNA sequence in the cloning vector, which is homologous to the target locus is derived from a highly expressed locus meaning that it is derived from a gene, which is capable of high expression level in the filamentous fungal host cell. A gene capable of high expression level, i.e. a highly expressed gene, is herein defined as a gene whose mRNA can make up at least 0.5% (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 1% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.1 g/l (as described in EP 357 127 B1). A number of preferred highly expressed fungal genes are given by way of example: the amylase, glucoamylase, alcohol dehydrogenase, xylanase, glyceraldehyde-phosphate dehydrogenase or cellobiohydrolase (cbh) genes from Aspergilli or Trichoderma. Most preferred highly expressed genes for these purposes are a glucoamylase gene, preferably an A. niger glucoamylase gene, an A. oryzae TAKA-amylase gene, an A. nidulans gpdA gene, a Trichoderma reesei cbh gene, preferably cbh1. According to another preferred embodiment, the highly expressed genes are the loci of SEQ ID NO 1, SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO: 19. More than one copy of a nucleic acid sequence encoding a polypeptide may be inserted into the host cell to increase production of the gene product. This can be done, preferably by integrating into its genome copies of the DNA sequence, more preferably by targeting the integration of the DNA sequence at one of the highly expressed locus defined in the former paragraph. Alternatively, this can be done by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. To increase even more the number of copies of the DNA sequence to be over expressed the technique of gene conversion as described in WO98/46772 may be used.
The vector system may be a single vector or plasmid or two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the filamentous fungal cell, or a transposon.
The vectors preferably contain one or more selectable markers, which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. A selectable marker for use in a filamentous fungal cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), bleA (phleomycin binding), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents from other species. Preferred for use in an Aspergillus and Penicillium cell are the amdS (EP 635574 B1, WO 97/06261) and pyrG genes of A. nidulans or A. oryzae and the bar gene of Streptomyces hygroscopicus. More preferably an amdS gene is used, even more preferably an amdS gene from A. nidulans or A. niger. A most preferred selection marker gene is the A. nidulans amdS coding sequence fused to the A. nidulans gpdA promoter (see EP 635574 B1). AmdS genes from other filamentous fungi may also be used (WO 97/06261).
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
Host Cells
All the transcriptional activators of the invention are preferably used for designing two types of host cells:

    
    
        first type of host cell would be highly suited for producing a desired polypeptide, said desired polypeptide being sensitive to protease degradation, and
        second type of host cell would be highly suited for producing a polypeptide, said polypeptide being under the control of the transcriptional activator of the invention.
    
    


Optionally, both types of host cells additionally comprise an expression construct or a nucleic acid construct comprising a nucleic acid sequence coding for a polypeptide to be produced: polypeptide sensitive to protease degradation or polypeptide being under the control of the transcriptional activator or the transcriptional activator itself.
Optionally, the host cell comprises an elevated unfolded protein response (UPR) to enhance production abilities of a polypeptide of interest. UPR may be increased by techniques described in US2004/0186070A1 and/or US2001/0034045A1 and/or WO01/72783A2. More specifically, the protein level of HAC1 and/or IRE1 and/or PTC2 has been modulated in order to obtain a host cell having an elevated UPR.
The choice of a host cell in the invention will to a large extent depend upon the source of the nucleic acid sequence encoding the desired polypeptide to be produced. Preferably, the host cell is a filamentous fungus as defined earlier in section nucleic acid sequences encoding transcriptional activators of a protease promoter or in WO 01/68864 or WO 00/20596.
The introduction of an expression vector or a nucleic acid construct into a filamentous fungal cell may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. A suitable method of transforming Fusarium species is described by Malardier et. al., 1989, Gene 78: 147156 or in WO 96/00787. The expression vector or nucleic acid construct that can be used were already described under the corresponding sections.
In a more preferred embodiment, a transcriptional activator of a protease promoter of the invention is obtained from an A. niger strain, more preferably from Aspergillus niger AB4. 1 (van Hartingsveldt, W., et al., 1987. Mol. Gen. Genet. 206: 71-75), and most preferably from A. niger CBS 513.88 or a mutant strain thereof, harbouring, e.g., the polypeptide with the amino acid sequence of SEQ ID NO: 3.
According to another preferred embodiment, a transcriptional activator of a protease promoter of the invention is obtained from one of the following deposited strains: A. oryzae ATCC 20423, IFO 4177, ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, A. fumigatus Af 293 (CBS 101355), or P. chrysogenum CBS 455.95 harbouring, e.g., the polypeptide with the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 21, respectively.
Host Cell Suited for Production of a Polypeptide Sensitive to Protease Degradation
According to a preferred embodiment, the invention relates to a filamentous fungal host cell which is a mutant of a parent filamentous fungal cell useful for the production of a polypeptide sensitive to protease degradation, in which the parent cell comprises one or more nucleic acid (DNA) sequences encoding a protease, the transcription of which is activated by a transcriptional activator of the invention, and the mutant cell produces less of the transcriptional activator and/or the protease(s) than the parent cell when cultured under the same conditions as preferably measured by a protease activity assay as described in the description earlier on or as follows.
A preferred method for measurement of protease activity in a host cell is described in the example section herein for determination of the acidic endo-protease activity using Bovine Serum Albumin (BSA) as substrate. A detailed description of this method is also described by van den Hombergh et al., Current Genetics 28: 299-308 (1995). Measurement of protease(s) also may be assayed using other known methods. In one such method, an aliquot of a 48 hour culture media is incubated with 3H-labelled sperm whale myoglobin at pH 4.0 and the radioactivity in the TCA-soluble fraction is measured (van Noort, J. M., et al., 1991. Anal. Biochem 198: 385-390). Other methods have been described for identifying, e.g., aspartic proteinase A. of A. niger (Takahashi, K., 1991. Meth. in Enzymol. 248: 146-155), endopeptidases (Morihara, K., 1995. Meth. in Enzymol. 248: 242-253), carboxypeptidases (Reminton, J., and Breddam, K., 1994. Meth. in Enzymol. 244: 231-248), dipeptidyl peptidase (Ikehara, Y., et al., 244: 215-227), and aminopeptidases (Little, G., et al., 1976. Meth. in Enzymol. 45: 495-503). Alternatively other protease assays may be used such as the one described in WO 02/068623. Alternatively, the assay used may be a northern blotting (in Molecular Cloning: A Laboratory Manual, Sambrook et al 1989), the use of a reporter gene under the control of a protease promoter, or a western blotting or a DNA array analysis (Eisen, M. B. and Brown, P. O. DNA arrays for analysis of gene expression. Methods Enzymol. 1999:303:179-205) as also described herein.
According to a preferred embodiment, the mutant cell has a modified or an inactivated endogenous transcriptional activator of a protease promoter or an endogenous transcriptional activator, which has been replaced with a deteriorated transcriptional activator. According to another preferred embodiment, the mutant expresses a transcriptional activator of a protease promoter, which transcriptional activity can be modulated.
According to another preferred embodiment, the mutant cell A. niger produces less of the transcriptional activator and/or less protease(s) than the deposited cell CBS 513.88 as measured by any one of the given assays. According to another preferred embodiment, the mutant cell Aspergillus oryzae produces less of the transcriptional activator and/or less protease(s) than the deposited A. oryzae cited earlier. According to another preferred embodiment, the mutant cell Penicillium chrysogenum produces less of the transcriptional activator and/or less protease(s) than CBS 455.95. According to another preferred embodiment, the mutant cell Aspergillus fumigatus produces less of the transcriptional activator and/or less protease(s) than Aspergillus fumigatus AF293 (CBS101355).
Such a mutant cell may be obtained by genetic manipulation by one of the following techniques or by a combination thereof:

    
    
        a. using recombinant genetic manipulation techniques,
        b. submitting the filamentous fungus to mutagenesis.
    
    


Alternatively or in combination with above-mentioned techniques and according to another preferred embodiment, the mutant may be obtained by submitting the filamentous fungus to an inhibiting compound/composition.
The filamentous fungus obtained may be subsequently selected by monitoring the expression level of the nucleic acid sequence of the invention and/or the nucleic acid sequence of any protease known to be under control of the transcriptional activator of the invention. Optionally, the filamentous fungus is subsequently selected by measuring the expression level of a given gene of interest to be expressed in the host cell.
More preferably, the mutant is made with recombinant genetic manipulation techniques such as defined in step a. to obtain a recombinant filamentous fungus. Most preferably step a. comprises deleting the DNA sequence encoding the transcriptional activator, even most preferably the deleted DNA sequence is replaced by a non-functional variant thereof, and even most preferably the deletion and replacement are made by gene replacement preferably as described in EP 357127 B.
In a preferred embodiment the mutant cell is obtained by modification or inactivation of a nucleic acid sequence present in the cell and necessary for expression of the transcriptional activator.
In another preferred embodiment the reduced expression of the transcriptional activator and/or protease in the mutant cell is obtained by modification or inactivation of a control sequence required for the expression of the transcriptional activator. The term “control sequence” is defined, supra, in the section entitled “Nucleic Acid Constructs”. In a more preferred embodiment the control sequence in the mutant cell is a promoter sequence or a functional part thereof, i.e., a part, which is sufficient for affecting expression of the nucleic acid sequence. Other control sequences for possible modification include, but are not limited to, a leader, a polyadenylation sequence, a regulatory sequence and a transcription terminator.
In yet another preferred embodiment the reduced expression of the transcriptional activator and/or protease in the mutant cell is obtained by modification of the initiation codon (ATG) into a sub-optimal initiation codon.
Modification or inactivation of the nucleic acid sequence encoding the transcriptional activator of the invention may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which the capability to produce a transcriptional activator has been reduced by comparison to the parental cell. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.
Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet(W) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 0-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutant cells exhibiting reduced expression of the gene.
Alternatively, modification or inactivation of the gene may be performed by established anti-sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the gene. More specifically, expression of the gene by a filamentous fungal cell may be reduced or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence, which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated. An example of expressing an antisense-RNA is shown in Appl Environ Microbiol. February 2000; 66(2):775-82. (Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. Ngiam C, Jeenes D J, Punt P J, Van Den Hondel C A, Archer D B) or (Zrenner R, Willmitzer L, Sonnewald U. Analysis of the expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA. Planta. (1993);190(2):247-52.).
Furthermore, modification, downregulation or inactivation of the gene may be obtained via the RNA interference (RNAi) technique (FEMS Microb. Lett. 237 (2004): 317-324). In this method identical sense and antisense parts of the nucleotide sequence, which expression is to be affected, are cloned behind each other with a nucleotide spacer in between, and inserted into an expression vector. After such a molecule is transcribed, formation of small (21-23) nucleotide fragments will lead to a targeted degradation of the mRNA, which is to be affected. The elimination of the specific mRNA can be to various extends. The RNA interference techniques described in WO2005/05672A1 and/or WO2005/026356A1 may be used for downregulation, modification or inactivation of the gene.
In another preferred embodiment, the filamentous fungus mutant cell harbours a nucleic acid sequence, which has been modified or inactivated by any of the methods described above and produces less of a protease or a combination of proteases than the filamentous fungus parent cell when cultured under identical conditions as measured using the same assays as defined before. The mutant cell produces preferably at least about 25% less, more preferably at least about 50% less, even more preferably at least about 75% less, and even more preferably at least about 95% less of a protease or a combination of proteases than the parent cell when cultured under identical conditions using the same assays as defined before. According to a preferred embodiment, the filamentous fungus Aspergillus niger or Aspergillus oryzae or Aspergillus fumigatus or Penicillium chrysogenum mutant cell produces less of a protease or a combination of protease than the corresponding deposited filamentous fungus cell cited earlier when cultured under identical conditions using the same assays as defined before.
In an even more preferred embodiment, the filamentous fungus mutant cell produces essentially undetectable amounts of a protease or combination of proteases than the parent cell when cultured under identical conditions using the same assays as defined before.
In a most preferred embodiment, the filamentous fungus mutant cell produces less or essentially undetectable amounts of a protease or combination of proteases than the parent cell when cultured under identical conditions as described above using the assay for determination of the acidic endo-protease activity using Bovine Serum Albumin (BSA) as substrate as defined and referenced before herein.
In another preferred embodiment, the filamentous fungus mutant cell harbours at least one copy of a nucleic acid sequence encoding a polypeptide of interest (see section producing a polypeptide).
Host Cell Suited for Protease Production
According to another preferred embodiment, the invention relates to a host cell highly suited for the production of a polypeptide wherein the host cell is a mutant of a parent cell in which the mutant (a) produces more of the transcriptional activator of the present invention as compared to the parent cell when cultured under the same conditions and using the same assay as defined in the former section; and (b) comprises a DNA sequence encoding the polypeptide, the transcription of which is activated by the transcriptional activator.
According to another preferred embodiment, the mutant cell A. niger produces more of the transcriptional activator and/or more protease(s) than the deposited cell CBS 513.88 when cultured under identical conditions and as measured by one of the given assays defined in the former section. A preferred method for measurement of protease activity in a host cell is described in the example section herein for determination of the acidic endo-protease activity using Bovine Serum Albumin (BSA) as substrate. According to another preferred embodiment, the mutant cell Aspergillus oryzae produces more of the transcriptional activator and/or more protease(s) than the deposited A. oryzae cited earlier when cultured under identical conditions and as measured by one of the given assays defined in the former section. According to another preferred embodiment, the mutant cell Penicillium chrysogenum produces more of the transcriptional activator and/or more protease(s) than CBS 455.95 when cultured under identical conditions and as measured by one of the given assays defined in the former section. According to another preferred embodiment, the mutant cell Aspergillus fumigatus produces more of the transcriptional activator and/or more protease(s) than the deposited A. fumigatus cited earlier when cultured under identical conditions and as measured by one of the given assays defined in the former section.
In a preferred embodiment, the filamentous fungal host cell produces more of the transcriptional activator than the parent cell and/or more than any cited deposited parent cell when cultured under the same conditions by introducing into the parent cell one or more copies of (i) a nucleic acid sequence encoding a transcriptional activator of a protease promoter as isolated or having enhanced transcriptional activity on a protease promoter, (ii) a nucleic acid construct comprising a nucleic acid sequence encoding a transcriptional activator of a protease promoter, (iii) an expression vector as defined above in the section “Expression Vectors”.
In a more preferred embodiment, the nucleic acid sequence encoding the transcriptional activator is operably linked to a promoter, or a functional part thereof, which is stronger than the corresponding promoter of the filamentous fungal parent cell. In an even more preferred embodiment, the promoter, or a functional part thereof, mediates the expression of a gene encoding an extracellular protease, such as the A. oryzae alkaline protease, A. oryzae neutral metalloprotease, A. niger aspergillopepsin protease, Fusarium oxysporum trypsin-like protease or F. venenatum trypsin.
The present invention also relates to a filamentous fungal host cell useful for the production of a polypeptide wherein the filamentous fungal host cell is a mutant of a parent filamentous fungal cell in which the mutant comprises
a) a modification or inactivation of a transcriptional activator of the present invention, or a regulatory sequence thereof, and
b) (i) an inducible promoter operably linked to a nucleic acid sequence encoding a transcriptional activator of the present invention, and (ii) a promoter sequence to which the transcriptional activator can bind, operably linked to a nucleic acid sequence encoding the polypeptide, wherein (i) and (ii) can be introduced simultaneously or sequentially.
The inactive form of the transcriptional activator in (a) above can be obtained as described in the former section.
The inducible promoter sequence in (b) above may be any promoter sequence, or a functional part thereof, wherein the transcription initiation activity of the promoter can be induced according to the fermentation conditions. Preferably, the induction is mediated by a carbon or nitrogen catabolite. In a preferred embodiment, the promoter is the amdS promoter of A. nidulans or A. oryzae, the niaD promoter of A. nidulans or A. niger, A. oryzae or A. niger, the niiA promoter of Aspergillus species, the alkaline phosphatase promoter of Aspergillus sp., the acid phosphatase promoter of Aspergillus sp., or the alcA promoter of A. niger, A. tubingensis xylanase (xInA) promoter.
In another preferred embodiment, the filamentous fungal host cell further comprises a promoter sequence, wherein the promoter sequence can be activated by the transcriptional activator and is operably linked to the nucleic acid sequence encoding the polypeptide.
The promoter sequence activated by the transcriptional activator of the present invention may be any promoter sequence, or a functional part thereof. Preferably, the promoter is from a protease gene. More preferably, the promoter is selected from the group, which includes but is not limited to promoters obtained from the genes encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase,
A. niger or A. awamori glucoamylase (glaA), R. miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase, the NA2-tpi promoter (a hybrid of the promoters from the genes encoding A. niger neutral alpha-amylase and A. oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Particularly preferred promoters for use in filamentous fungal cells are a promoter, or a functional part thereof, from a protease gene; e.g., from the F. oxysporum trypsin-like protease gene (U.S. Pat. No. 4,288,627), A. oryzae alkaline protease gene(alp), A. niger pacA gene, A. oryzae alkaline protease gene, A. oryzae neutral metalloprotease gene, A. niger aspergillopepsin protease pepA gene, or F. venenatum trypsin gene. A. niger aspartic protease pepB gene.
In another preferred embodiment, the filamentous fungal host cell harbours at least one copy of a nucleic acid sequence encoding a polypeptide.
The nucleic acid constructs described herein may be introduced into a parent fungal cell according to any of the methods as described supra in the section, “Host Cells” to obtain a host cell useful for the production of a polypeptide.
It will be understood that the methods of the present invention are not limited to a particular order for obtaining the mutant filamentous fungal cell. The modification of the second nucleic acid sequence may be introduced into the parent cell at any step in the construction of the cell for the production of a polypeptide.
Producing a Polypeptide
Another aspect of the present invention relates to methods of producing a polypeptide in a filamentous fungal host cell of the present invention, comprising:

    
    
        (a) cultivating the filamentous fungal host cell which harbours a gene encoding the polypeptide in a nutrient medium suitable for production of the polypeptide and optionally;
        (b) recovering the polypeptide from the nutrient medium of the filamentous fungal host cell.
    
    


According to a first preferred embodiment, the polypeptide produced is the transcriptional activator of the invention.
According to a second preferred embodiment, the polypeptide produced is a polypeptide sensitive to protease degradation. In this case, the first type of host cell (as described in “Host cell suited for production of a polypeptide sensitive to protease degradation”) will be used.
According to a third preferred embodiment, the polypeptide to be produced is a polypeptide, whose expression is activated by the transcriptional activator of the invention. In this case, the second type (as described in “Host cell suited for protease production”) of host cell will be used.
The filamentous fungal host cells of the present invention are cultivated in a nutrient medium suitable for production of the polypeptide of interest using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fedbatch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., Bennett, J. W. and LaSure, L., eds., More Gene Manipulations in Fungi, Academic Press, Calif., 1991). Suitable media are available from commercial suppliers or may be prepared using published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it is recovered from cell lysates.
The resulting polypeptide may be isolated by methods known in the art. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing, differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
The polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods may include use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or SDS PAGE. For example, an enzyme assay may be used to determine the activity of the polypeptide. Procedures for determining enzyme activity are known in the art for many enzymes.
In the methods of the present invention, the filamentous fungal host cell produces at least about 20% more, preferably at least about 50% more, more preferably at least about 100% more, even more preferably at least about 200% more, and most preferably at least about 300% more of the polypeptide than a corresponding parent cell when cultivated under the same conditions using one of the given assays. More preferably, the parent cell is one of the deposited strains cited earlier.
The polypeptide may be any polypeptide whether native or heterologous to the filamentous fungal cell. The term “heterologous polypeptide” is defined herein as a polypeptide, which is not produced by a wild-type filamentous fungal cell. The term “polypeptide” is not meant herein to refer to a specific length of the encoded produce and therefore encompasses peptides, oligopeptides and proteins. The polypeptide may also be a recombinant polypeptide, which is a polypeptide native to a cell, which is encoded by a nucleic acid sequence, which comprises one or more control sequences, foreign to the nucleic acid sequence, which is involved in the production of the polypeptide. The polypeptide may be a wild-type polypeptide or a variant thereof. The polypeptide may also be a hybrid polypeptide, which contains a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides where one or more of the polypeptides may be heterologous to the cell. Polypeptides further include naturally occurring allelic and engineered variations of the above-mentioned polypeptides.
In a preferred embodiment, the polypeptide is an antibody or portions thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or portions thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, intracellular protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor. In a preferred embodiment, the polypeptide is secreted extracellularly.
In a more preferred embodiment, the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase.
In an even more preferred embodiment, the polypeptide is a carbohydrase, e.g. cellulases such as endoglucanases, β-glucanases, cellobiohydrolases or β-glucosidases, hemicellulases or pectinolytic enzymes such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endo polygalacturonases, exopolygalacturonases rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases, transferases, or isomerases. More preferably, the desired gene encodes a phytase. In an even more preferred embodiment, the polypeptide is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine-protease catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, proteolytic enzyme, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, polyphenoloxidase, ribonuclease, transglutaminase, or glucose oxidase, hexose oxidase, monooxygenase.
In another even more preferred embodiment, the polypeptide is human insulin or an analog thereof, human growth hormone, erythropoietin, tissue plasminogen activator (tPA) or insulinotropin.
The nucleic acid sequence encoding a heterologous polypeptide may be obtained from any prokaryotic, eukaryotic, or other source.
Alternatively the polypeptide may be an intracellular protein or enzyme such as for example a chaperone, protease or transcription factor. An example of this is described in Appl Microbiol Biotechnol. October 1998; 50(4):447-54 (“Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli. Punt P J, van Gemeren I A, Drint-Kuijvenhoven J, Hessing J G, van Muijlwijk-Harteveld G M, Beijersbergen A, Verrips C T, van den Hondel C A). This can be used for example to improve the efficiency of a host cell as protein producer if this polypeptide, such as a chaperone, protease or transcription factor, was known to be a limiting factor in protein production.
For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide is produced by the source or by a cell in which a gene from the source has been inserted.
In the methods of the present invention, the filamentous fungal cells may also be used for the recombinant production of polypeptides, which are native to the cell. The native polypeptides may be recombinantly produced by, e.g., placing a gene encoding the polypeptide under the control of a different promoter to enhance expression of the polypeptide, to expedite export of a native polypeptide of interest outside the cell by use of a signal sequence, and to increase the copy number of a gene encoding the polypeptide normally produced by the cell. The present invention also encompasses, within the scope of the term “heterologous polypeptide”, such recombinant production of polypeptides native to the cell, to the extent that such expression involves the use of genetic elements not native to the cell, or use of native elements which have been manipulated to function in a manner that do not normally occur in the filamentous fungal cell. The techniques used to isolate or clone a nucleic acid sequence encoding a heterologous polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof.
In the methods of the present invention, heterologous polypeptides may also include a fused or hybrid polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide.
Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter(s) and terminator. The hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the mutant fungal cell. An isolated nucleic acid sequence encoding a heterologous polypeptide of interest may be manipulated in a variety of ways to provide for expression of the polypeptide. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, posttranscriptional modification, translation, post-translational modification, and secretion. Manipulation of the nucleic acid sequence encoding a polypeptide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.
The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.
EXAMPLES
Experimental Information
Strains
WT 1: This A. niger strain is used as a wild-type strain. This strain is deposited at the CBS Institute under the deposit number CBS 513.88.
WT 2: This A. niger strain is a WT 1 strain comprising a deletion of the gene encoding glucoamylase (glaA). WT 2 was constructed by using the “MARKER-GENE FREE” approach as described in EP 0 635 574 B1. In this patent it is extensively described how to delete glaA specific DNA sequences in the genome of CBS 513.88. The procedure resulted in a MARKER-GENE FREE ? glaA recombinant A. niger CBS 513.88 strain, possessing finally no foreign DNA sequences at all.
WT 3: This Penicillium chrysogenum strain is used as a wild-type strain. This strain is deposited at the CBS Institute under the deposit number CBS 455.95.
Plasmids
pGBFINPRT-1: This prtT expression construct (depicted in FIG. 3) was deposited at the CBS Institute under the accession number CBS118680.
pGBPRT-1: This prtT cDNA vector (depicted in FIG. 19) was deposited at the CBS Institute under accession number CBS118681.
A. niger Shake Flask Fermentations
A. niger strains were precultured in 20 ml preculture medium as described in the Examples: “Aspergillus niger shake flask fermentations” section of WO99/32617. After overnight growth, 10 ml of this culture was transferred to fermentation medium 1 (FM1) with 7% glucose as described in WO99/32617. This FM1 contains per liter: 25 g Caseinhydrolysate, 12.5 g Yeast extract, 1 g KH2PO4, 2 g K2SO4, 0.5 g MgSO4.7H2O, 0.03 g ZnCl2, 0.02 g CaCl2, 0.01 g MnSO4.4H2O, 0.3 g FeSO4.7H2O, 10 ml Pen-Strep (5000 IU/ml Pen-5 mg/ml Strep), adjusted to pH 5.6 with 4 N H2SO4. Fermentation is performed in 500 ml flasks with baffle with 100 ml fermentation broth at 34° C. and 170 rpm for the number of days indicated.
For protease induction, mycelia were harvested after culturing for 16-24 h in FM1, washed at room temperature with Induction Medium (IM) and transferred to IM with C-source as indicated.
Induction Medium (IM) Contains Per Liter:
6 g NaNO3, 0.5 g KCl, 1.5 g KH2PO4, 1.13 ml 4M KOH, 0.5 g MgSO4.7H2O, 0.01% (w/v) casamino acids, 0.1% (w/v) yeast extract, 1 ml of stock trace elements (stock trace elements per liter: 22 g ZnSO4.7H2O, 11 g H3BO3, 5 g FeSO4.7H2O, 1.7 g CoCl2.6H2O, 1.6 g CuSO4.5H2O, 5 g MnCl2.4H2O, 1.5 g Na2MoO4.2H2O, 50 g EDTA, adjust the pH to 6.5 with 4M KOH, filter sterilize and store in the dark at 4° C.), 10 ml of stock vitamins (stock vitamins per liter: 200 mg riboflavin, 200 mg thiamine.HCl, 200 mg nicotinamide, 100 mg pyridoxine.HCl, 20 mg panthotenic acid, 0.4 mg biotin, adjust to pH 6 with 4M NaOH, filter sterilize and store in the dark at 4° C.), and adjusted to pH 5.6. Fermentation medium 2 (FM2) is used for PLA2 fermentations and contains per liter: 82.5 g Glucose.1H2O, 25 g Maldex 15 (Boom Meppel, Netherlands), 2 g Citric acid, 4.5 g NaH2PO4.1H2O, 9 g KH2PO4, 15 g (NH4)2SO4, 0.02 g ZnCl2, 0.1 g MnSO4.1H2O, 0.015 g CuSO4.5H2O, 0.015 g CoCl2.6H2O, 1 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 0.3 g FeSO4.7H2O, 30 g MES (2-[N-Morpholino]ethanesulfonic acid), pH=6.
Oligonucleotide Sequences
All primers used in the experiments are described in the sequence listing under the SEQ ID numbers 5-12 and 23-24.
Protease Activity Assays
Total acidic endo-protease activities in culture supernatants were determined as the amount of degraded BSA. 450 μl 1% (w/v) BSA in 0.1 M NaAc pH 4.0 was incubated with 50 μl culture supernatant at 37 degrees Celsius for different time intervals. At the end of the incubation period, the remainder of the BSA was precipitated with 500 μl 10% (w/v) trichloracetic acid (TCA) and followed by incubation on ice for 10 min. The precipitate was centrifuged for 10 min at 13000 rpm in an Eppendorf centrifuge. The absorbance of the supernatant was measured at 280 nm. One unit of protease activity was defined as the change in absorbance units at 280 nm per hour (Anson assay). A more detailed description and references for this method is also described by van den Hombergh et al., Current Genetics 28: 299-308 (1995).
Exo-protease activities in culture supernatants were determined using specific peptides attached to a colour group (p-nitroanilide (pNA) or 3-(2-furyl)acryloyl (FA)). Exo-protease activity releases the pNA or FA from the peptide, which causes a change in absorbance. 450 μl of the 0.2 mM substrate solution in water was incubated with 50 μl culture supernatant at room temperature. pNA-substrates were measured at 405 nm (pH 6-7); FA-substrates were measured at 332 nm ( ̃pH 5). The absorbance of pNA-substrates increases upon release of the pNA-group; the absorbance of FA-substrates decreases upon release of the FA-group. The protease activity in units is calculated as the change in absorbance per hour.
Assaying proteolytic activity and the different protease activities in general is described in WO 02/068623.
Protease Plate Assay
To screen for enhanced or decreased proteases expression, minimal medium plates containing dialyzed skim milk were used as described by Mattern et al. (Mol. Gen. Genet. 1992, 234:332-336). The A. niger WT 1 and WT 2 are producing a clear halo on this medium after 4 days incubation at 30° C.
PLA2 Phospholipase Activity
To determine phospholipase PLA2 activity (PLA2) in Aspergillus niger culture broth spectrophotometrically, an artificial substrate is used: 1,2-dithiodioctanoyl phophatidylcholine (diC8, substrate). PLA2 hydrolyses the sulphide bond at the A2 position, dissociating thio-octanoic acid. Thio-octanoic acid reacts with 4,4 dithiopyridine (color reagent, 4-DTDP), forming 4-thiopyridone. 4-Thiopyridone is in tautomeric equilibrium with 4-mercaptopyridine, which absorbs radiation having a wavelength of 334 nm. The extinction change at that wavelength is measured. One unit is the amount of enzyme that liberates of 1 nmol thio-octanoic acid from 1,2-dithiodioctanoyl phosphatidylcholine per minute at 37° C. and pH 4.0.
The substrate solution is prepared by dissolving 1 g diC8 crystals per 66 ml ethanol and add 264 ml acetate buffer. The acetate buffer comprises 0.1 M Acetate buffer pH 3.85 containing 0.2% Triton-X100. The colour reagent is a 11 mM 4,4-dithiodipyridine solution. It was prepared by weighting 5.0 mg 4,4-dithiodipyridine in a 2 ml eppendorf sample cup and dissolving in 1.00 ml ethanol. 1.00 ml of milli-Q water was added.
Example 1
Construction of an A. niger cDNA Expression Library and Isolation of a prtT cDNA Clone
This example describes the construction of an expression library in an expression vector. The pool of mRNA's is isolated from mycelium grown under inducing conditions for protease activity. After construction, the expression library is used for isolation of a prtT cDNA clone.
1.1 Construction of a cDNA Library Induced for Protease Activity
In the following example, the induction of the proteolytic system in A. niger is determined by measuring a number of proteolytic activities in the culture broth.
A. niger strain WT 1 was used to perform shake flask experiments in 100 ml of the medium as described herein at 34° C. and 170 rpm in an incubator shaker using a 500 ml baffled shake flask. A. niger WT 1 was pre-cultured overnight and subsequently the mycelium was transferred to Fermentation Medium 1 (FM1). After 20 h of growth the mycelium was shifted to Induction Medium (IM), containing 1% (w/v) collagen or 2% (w/v) defatted soy flour (see Experimental Information). The growth was continued for 4 days. Samples were collected to determine protease activities, as described above. In FIG. 1, endoprotease activities for the soy flour culture are given. A clear induction of endoproteases is shown upon growth on soy flour. A similar profile was found for the collagen culture (data not shown).
Additionally, exo-protease activities were measured for different time-points for the two C-sources. For both collagen and soy flour a clear induction of exo-proteases was found after 2 days of growth (data not shown).
Mycelia harvested 18, 28 and 48 h after the shift to IM containing 1% (w/v) collagen or 2% (w/v) defatted soy flour were used for RNA extractions. The RNA extractions and mRNA isolations were performed as described in detail in WO99/32617. The construction of a cDNA expression library comprising a.o. the cDNA synthesis, the ligation of linkers and E. coli transformation is described as well in WO99/32617. Linkers used for the cDNA reactions consisted of a HindIII and XhoI restriction sites. The resulting cDNA pools were ligated in the HindIII-XhoI digested pGBFIN-23 vector, which construction and use is described in WO99/32617. A physical map of pGBFIN-23 can be found in FIG. 2. The ligation mixtures were used to transform DH10B electrocompetent cells (Invitrogen) resulting in the generation of over 105 colonies per cDNA library obtained from both the soy flour and the collagen induced mycelium. Random sequencing of 96 clones of each of the two libraries indicated a low percentage of vectors without insert. The insert sizes for the clones sequenced were between 0.5-4.7 kb with an average of 1.7 kb. To enable an efficient screening format, the library was constructed in pools of 103 clones. For each of these pools, glycerol stocks were made and stored for later use.
1.2 Transformation of A. niger WT 2 with an Expression Library
For 20 pools of the cDNA library (10 originating from the soy flour and 10 from the collagen induced library), plasmid DNA was isolated according to known principles and routine plasmid isolation techniques (Sambrook, J. et al., 1989). For each of the pools, 5 μg of total plasmid DNA was digested for 4 hours at 37° C. with NotI (20 U), to remove E. coli derived plasmid sequences.
For each of the 20 pools, an A. niger WT 2 transformation was performed using the E. coli-free linear fragments containing A. niger cDNA clones. Per pool, 1000 colonies were purified on selective medium containing acetamide and transferred to individual wells in a 96 well microtiter dish, all as described in WO99/32617
1.3 Analysis of the A. niger Expression Library
All individual transformants were tested using the protease plate assay as described above. Conidiospores of individual transformants were transferred to the minimal medium plates containing dialyzed skim milk. After 2-3 days of incubation at 30° C., halo formation could be observed for 29 colonies, indicating increased protein degradation. For all other transformants a halo started to appear after 4-6 days. At day 5, the halo for the 29 colonies was also larger in size when compared to WT 2.
The 29 positively identified transformants originated from 14 different pools of the library. To be able to analyze independent transformants, conidiospores for 14 positive transformants originating from different pools of the expression library were isolated and used to inoculate PDA plates.
For strain WT 2 and the 14 selected transformants, shake flask experiments were performed as described in more detail in EP 635 574 B1. Essentially, mycelium was grown in 100 ml medium at 34° C. and 170 rpm in an incubator shaker using a 500 ml baffled shake flask. After 2 and 4 days of fermentation, samples were taken to determine protease activities as described in Experimental Information. For all selected transformants, the total acid extracellular protease activity was increased compared to WT 2 in both time points examined (data not shown).
For the 14 selected transformants, genomic DNA was isolated from single colonies.
1.4 Isolation of a cDNA Expression Clone Containing a Protease Transcriptional Activator PrtT
In essence, the pGBFIN-23 based expression vector used in the construction of the expression library (FIG. 2) comprises the glucoamylase promoter, a variable cDNA coding sequence operably linked to the promoter and the glucoamylase terminator region, flanked by the 3′ and 3′′ glaA targeting sites, and the amdS selection marker in an E. coli vector. Therefore, specific, but unknown, cDNA sequences in a WT 2 transformant, which carries a deletion of the endogenous glucoamylase region, can be identified using PCR and two glucoamylase-specific primers. One based on the standard glucoamylase promoter and another on the glucoamylase terminator region. Using 100 ng of the genomic DNA, PCR was performed with the 14 selected A. niger transformants using the glucoamylase-specific oligonucleotides identified as SEQ ID NO 23 and SEQ ID NO 24. For 11 of the transformants, a band could be amplified using PCR. These bands were cloned in pCR2.1-TOPO (Invitrogen) and sequenced. Six of the cDNA clones contained an identical cDNA sequence. The ORF of this cDNA sequence is shown in the sequence listings under the SEQ ID NO 2. To the polypeptide sequence encoded by SEQ ID NO 2, the SEQ ID NO 3 has been assigned. Using 10 ng of plasmid DNA of six positive pools and cDNA-specific oligonucleotides identified as SEQ ID NO 5 and SEQ ID NO 6, all pools tested were found positive showing the presence of a band of about 500 bp using PCR.
Subsequently, the glycerol stocks for a number of positive pools were used to plate colonies. These colonies were screened by colony hybridization and the 500 bp probe and the clones containing a hybridizing insert, named prtT, were isolated according standard techniques as described in Sambrook et al., 1989 (see above). In FIG. 3, a physical map for a representative isolated clone, which is named pGBFINPRT-1, is presented. Sequences of several prtT clones obtained by sequencing of pGBFINPRT plasmids were aligned (data not shown). The alignment showed that all the sequenced plasmids contained the cDNA insert (identified as SEQ ID NO 2) encoding the identical protein PrtT (identified as SEQ ID NO 3).
Example 2
Identification of prtT Genes
Genomic DNAs of the A. niger WT 1 and the P. chrysogenum WT 3 strains were sequenced and analyzed. Using the SEQ ID NO 2 in a search against these genomes, the genomic sequences of the A. niger prtT and P. chrysogenum prtT genes were determined. The SEQ ID NO 1 was assigned to the A. niger prtT genomic sequence and the SEQ ID NO 19 was assigned to the P. chrysogenum prtT genomic sequence. The genomic sequence of the A. niger prtT locus comprises the ORF and approximately 3000 bp of the 5′ untranslated region (UTR) and 1700 bp of the 3′ UTR and the P. chrysogenum genomic sequence contains the ORF and 574 bp of the 5′ UTR and 238 bp of the 3′ UTR. The nucleotide sequence encoding the PrtT protein of P. chrysogenum is shown in the sequence listings under the SEQ ID NO 20. The translated sequence of the SEQ ID NO 20 is assigned as the SEQ ID NO 21 and it represents the amino acid sequence of the transcriptional activator PrtT of P. chrysogenum WT 3.
Example 3
Alignment of Novel cDNA Sequences and the Encoded Proteins
Example 3.1
Alignment of Polypeptide Sequences with the PrtT Polypeptide of SEQ ID NO 3
A search was performed against a nucleotide sequence patent database with the SEQ ID NO 2. Two publications (WO 00/20596 and WO 01/68864) were identified, which deal with PrtT. In order to examine the extent of the sequence identity among the PrtT proteins, the PrtT polypeptide sequences of A. niger and A. oryzae described in above mentioned publications were aligned with the PrtT sequence SEQ ID NO 3 of this invention (see FIG. 4 and FIG. 5). Surprisingly, it became clear from these alignments that the SEQ ID NO 3 of PrtT of A. niger WT 1 is different from both polypeptide sequences published earlier. The alignment presented in FIG. 4 identified 45 amino acid differences between the A. niger WT 1 PrtT sequence and the A. niger polypeptide sequence published in WO 00/20596 and WO 01/68864. These differences were caused by one amino acid substitution in the N-terminal part and by differences in the identified ORFs, i.e. the A. niger WT 1 prtT sequence comprises an additional intron in the 3′ of the coding sequence, and the A. niger WT1 prtT sequence is missing the last exon identified in the A. niger prtT sequence of WO 00/20596 and WO 01/68864.
More substantial differences were found between PrtT of A. niger WT 1 and A. oryzae PrtT sequence published in WO 01/68864 (see FIG. 5). The polypeptide sequences in FIG. 4 have 93% match percentage, i.e. identity, as identified using the CDA method (Huang, 1994) with settings as described in the text and the sequences in the FIG. 5 have 49% match percentage, i.e. identity. See FIG. 5 for more detail.
Example 3.2
In silico Analysis of the A. niger PrtT Polypeptide
In order to obtain a prediction of functional domains of the PrtT transcriptional activator of A. niger WT 1, the SEQ ID NO 3 was analyzed using several web domain databases: Two regions were predicted:

    
    
        (i) using Pfam (Sonnhamer E L et al, (1997), Pfam: a comprehensive database of protein families based on seed alignments. Proteins. 28:405-420), a motif having similarity to a Zn(II)2-Cys6 binuclear cluster DNA binding motif was found, and
        (ii) using Prosite (Bairoch A, et al, (1996), The PROSITE database, its status in 1995. Nucleic Acids Res., 24:189-196) a Leucine zipper motif was found (see FIG. 4).
    
    


The later motif is known to be a functional domain responsible for dimerization that is found in several transcription factor families (Bauer-Bornberg, E., Rivals, E., and Vingron, M. (1998) Nucl. Acid Res. 26 (11): 2740-2746). The SEQ ID NO 4 is assigned to the predicted Zn(II)2-Cys6 binuclear cluster DNA binding domain of the PrtT transcriptional activator of A. niger WT 1.
Example 4
Overexpression of the prtT Gene in A. niger By Transformation with the pGBFINPRT-1 Construct
In the following example, an expression construct is introduced in a fungal host cell by transformation.
In order to introduce the pGBFINPRT-1 vector (FIG. 3) in A. niger WT 2, a transformation and subsequent selection of transformants was carried out as described in WO98/46772 and WO99/32617. In principle, linear DNA of vector pGBFINPRT-1 was isolated and used to transform A. niger. Transformants were selected on acetamide media and colony purified according standard procedures. Growing colonies were diagnosed for integration at the glaA locus and for the copy number. An example of this is shown in FIG. 6. Transformants of pGBFINPRT-1 with similar estimated copy number were selected and named PRTT.
Additionally, the selectable marker gene and the gene of the invention could have been on two constructs. The vector with the gene of the invention would have been co-transformed with an amdS selectable marker-gene containing vector, which is designated pGBAAS-1 (constructed as described in EP 635574B1). Both vectors comprise two DNA domains homologous to the glaA locus of A. niger host strain to direct targeting to the truncated glaA locus in WT 2. In the case of co-transformation, spores of transformants are plated on fluoro-acetamide media to select strains, which lost the amdS marker.
Example 5
Enhanced Protease Expression in prtT Transformants Compared to WT Strains
In the following example, the effect of the overexpression of a representative cDNA prtT clone (pGBFINPRT-1) comprising the sequence as identified in the SEQ ID NO 2 on the activity of the protease spectrum is determined.
Protease activities for a number of selected PRTT transformants (generated in example 4) are determined in the culture broth and compared to WT strains.
In a first step, the prtT overexpression was confirmed by Northern blot analysis of a number of PRTT transformants of A. niger WT 2 and the prtT mRNA levels were compared to A. niger WT 1 and WT 2. The RNA samples were obtained from the mycelium grown in 100 ml of the medium at 34° C. and 170 rpm in an incubator shaker using a 500 ml baffled shake flask as described above and in more detail in EP 635 574 B1. After 2 and 4 days of fermentation mycelium was collected and used for isolation of RNA (protocol see Example 1) and Northern blot analysis following the standard procedures of Northern blot analysis (Sambrook et al., 1989). For a number of PRTT transformants, increased prtT expression was seen after visualization of the Northern blot (data not shown).
The PRTT transformants of WT 2 with increased prtT expression and both strains WT 1 and WT 2 were used to perform shake flask experiments as described above. After 2, 3, 4 and 5 days of fermentation, samples were taken to determine protease activities as described in Experimental Information. Results are shown in FIG. 7. The total acid extracellular protease activity in the selected PRTT transformants of WT 2 was increased compared to WT 1 and WT 2 in almost all time points examined. Additionally the samples obtained by the incubation of BSA with the culture supernatant were analyzed on SDS-PAGE gels (data not shown). These data clearly demonstrated an enhanced degradation of BSA in the samples of PRTT transformants leading amongst others to formation of low molecular weight peptides.
This data prove that the cDNA prtT clone of pGBFINPRT-1 encodes a functional protease transcriptional activator. Therefore, the nucleotide sequence provided in SEQ ID NO 2 is encoding a functional transcriptional activator protein PrtT.
Example 6
Inactivation of the prtT gene in Aspergillus niger 
A gene replacement vector for the prtT gene encoding the protease regulator of the invention was designed according to known principles and constructed according to routine cloning procedures (Sambrook et al. (1989)). In essence, these vectors comprise approximately 1000-3000 bp flanking regions of an prtT ORF for homologous recombination at the predestined genomic locus. In addition, it contains a bi-directional amdS selection marker, in-between direct repeats. The general design of these deletion vectors was previously described in EP635574 B and WO 98/46772.
Genomic DNA of A. niger WT 1 was sequenced and analyzed as described above. Using the oligonucleotides identified as SEQ ID NO 11 and identified as SEQ ID NO 12 and genomic DNA of A. niger WT 2 as a template, PCR was used to amplify a 1.5 kb prtT downstream flanking region and introduce KpnI and XmaI restriction sites at the ends, to allow cloning in pGBDEL (FIG. 8). This 1.5 kb prtT downstream flanking fragment was digested with KpnI and XmaI and introduced in a KpnI and XmaI digested vector pGBDEL, generating pGBDEL-PRT1.
Using the oligonucleotides identified as SEQ ID NO 7 and identified as SEQ ID NO 8 and genomic DNA of A. niger WT 2 as a template, a 3 kb prtT upstream flanking region, identified as a fragment A, was amplified by PCR. Additionally, a BstBI restriction site was attached to the 5′-end and an overlapping sequence of the prtT downstream region at the 3′-end of the fragment A. Using oligonucleotides identified as SEQ ID NO 9 and as SEQ ID NO 10 and genomic DNA of A. niger WT 2 as a template, a 500 bp prtT downstream flanking region, identified as a fragment B, was amplified by PCR. Both resulting fragments, A and B, were fused by sequence overlap extension (SOE-PCR, as described in Gene. Apr. 15, 1989; 77(1):51-9. Ho S N, Hunt H D, Horton R M, Pullen J K, Pease L R “Site-directed mutagenesis by overlap extension using the polymerase chain reaction”) using PCR, oligonucleotides identified as SEQ ID NO 7 and SEQ ID NO 10 and fragments A and B; generating a 3.5 kb fragment C. This fragment C was digested with BstBI and AscI and introduced in a BstBI and AscI digested vector pGBDEL-PRT1, generating pGBDEL-PRT2 (FIG. 9). The sequence of the introduced PCR fragments comprising the upstream and downstream regions of the prtT gene were confirmed by sequence analysis.
Linear DNA of BstBI/XmaI-digested deletion vector pGBDEL-PRT2 was isolated and used to transform A. niger WT 2. This linear DNA can integrate into the genome at the prtT locus, thus substituting the prtT coding sequence with the construct containing amdS (see FIG. 10). Transformants were selected on acetamide media and colony purified according to standard procedures. Growing colonies were diagnosed by PCR for integration at the prtT locus. Deletion of the prtT gene was detectable by amplification of a band, with a size specific for the pGBDEL-PRT2 insert and loss of a band specific for the wild-type prtT locus. Spores were plated on fluoro-acetamide media to select strains, which lost the amdS marker. Candidate strains were tested using Southern analysis for proper deletion of the prtT gene. Strains dPRTT were selected as representative strains with the prtT gene inactivated (see FIG. 10).
Example 7
Comparison of the Protease Production in WT 2 and dPRTT A. niger Strains
The selected dPRTT strains (proper pGBDEL-PRT2 transformants of WT 2, isolated in example 6) and the strain A. niger WT 2 were used to perform shake flask experiments in 100 ml of the medium as described in EP 635 574 B1 at 34° C. and 170 rpm in an incubator shaker using a 500 ml baffled shake flask. After 1, 3, 6 and 8 days of fermentation, samples were taken to determine the endoprotease activity. In FIG. 11, endoprotease activity of WT 2 and dPRTT strains is shown. The endoprotease activity in the selected dPRTT transformants of WT 2 was clearly decreased compared to the one of A. niger WT 2 at all time points measured. We concluded that inactivation of the protease regulator PrtT was successful and resulted in the decrease of expression of extracellular proteases.
Example 8
Increased Production of a Protease Sensitive Protein in the dPRTT A. niger Strain
Proteolytic degradation is a well-known problem when over-expressing a (heterologous) protein in A. niger. This example demonstrates how increased yields of a protein of interest can be obtained by manipulating the protease spectrum of A. niger through the disruption of prtT.
Porcine phospholipase A2 (PLA2) protein was selected as a model protein. It has been shown earlier that this protein is susceptible to protease degradation (Roberts I. N., Jeenes D. J., MacKenzie D. A., Wilkinson A. P., Sumner I. G. and Archer D. B. (1992). Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic phospholipase A2 fusion protein is secreted and processed to yield mature enzyme. Gene 122: 155-161.). The fragment for overexpression of PLA2 was made as a fusion of proPLA2 with a native glucoamylase A gene of A. niger and was prepared in principle as described by Roberts et al. (1992). This glaA-pla2 fusion gene was cloned into pGBFIN23 using the same technique as described in example 2.1, resulting in pGBFIN-PLA2 (FIG. 12). In order to introduce the pGBFIN-PLA2 vector in A. niger WT 2 and A. niger dPRTT strains (as constructed in Example 6), a transformation and subsequent selection of transformants was carried out as described in WO98/46772 and WO99/32617. In principle, linear DNA of vector pGBFIN-PLA2 was isolated and used to transform A. niger. Transformants were selected on acetamide media and colony purified according standard procedures. Growing colonies were diagnosed for integration at the glaA locus and for the copy number. An example of this is shown in FIG. 6. Several A. niger WT 2 and dPRTT transformants having one copy of the pGBFIN-PLA2 plasmid integrated in the glaA locus were selected to perform shake flask experiments in 100 ml of the Fermentation Medium 2 (FM2) as described in experimental procedures at 34° C. and 170 rpm in an incubator shaker using a 500 ml baffled shake flask. The culture broth was collected during 9 days of the cultivation and the PLA2 activity was measured as described above. FIG. 13, shows the PLA2 activity as measured in an A. niger dPRTT pGBFIN-PLA2 transformant and in an A. niger WT 2 pGBFIN-PLA2 transformant. It is clear that in the strain with the deleted prtT gene, a clear increase of the PLA2 activity is seen, whereas in the strain having the intact copy of prtT almost no PLA2 activity could be measured.
Example 9
Isolation of Nucleotide Sequences Encoding Proteins with Similarity to A. niger PrtT By Performing TBlastn Search in Nucleotide Sequence Databases
In this example, we show how the A. niger PrtT protein sequence (SEQ ID NO 3) can be used to identify functional homologues in other organisms. We present data of two searches—(i) a search in a nucleotide sequence patent database, such as GENESEQTM (Aspergillus oryzae case), and (ii) in nucleotide sequence databases accessible for instance via National Centrum for Biotechnology Information (NCBI) (Aspergillus fumigatus case).
ad (i) The PrtT sequence of SEQ ID NO 3 was used to perform a TBlastn (protein query vs. translated database) search against the nucleotide sequence patent databases. A protein sequence of 624 amino acids was identified, which has 71% match percentage, i.e. identity, with the A. niger PrtT sequence SEQ ID NO 3 (see FIG. 14). To the corresponding cDNA sequence, the SEQ ID NO 14 was assigned, which describes the nucleotide sequence encoding the A. oryzae PrtT protease transcriptional activator. The deduced protein sequence is described in SEQ ID NO 15 and the genomic sequence under the SEQ ID NO 13. Surprisingly, the protein under the SEQ ID NO 15, which was identified using the functional A. niger WT 1 PrtT protein differs from the A. oryzae protein described by the authors of WO 01/68864 in the C-terminal part and by one amino acid substitution in the N-terminal part of the protein (see FIG. 15). We have previously demonstrated the functionality of SEQ ID NO 3 (Example 5), therefore the A. oryzae PrtT protein sequence as described under the SEQ ID NO 15 should represent the functional homolog of A. niger PrtT in A. oryzae. 
ad (ii) We have performed a similar search as described above against eukaryotic nucleotide sequence databases. A polypeptide sequence (621 amino acids) with a high match percentage, i.e. identity, to PrtT of A. niger has been identified in A. fumigatus (see FIG. 16). The PrtT sequence of A. fumigatus has 66% of match percentage, i.e. identity, with the A. niger PrtT SEQ ID NO 3. The A. fumigatus sequence of the A. niger PrtT homolog has SEQ ID NO 18, the cDNA encoding this polypeptide SEQ ID NO 17, and the prtT genomic sequence of A. fumigatus is under the SEQ ID NO 16.
Four fungal PrtT polypeptide sequences of this invention were aligned using CLUSTAL W (reference see above in the text). Through out the alignment all the sequences show a high degree of amino acid identity (see the conserved boxes of amino acid sequences in FIG. 17). This example strengthens further the fact that the isolated PrtT polypeptides as described in SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO: 21 indeed encode functional homologues of the A. niger PrtT, the protease transcriptional regulator. To the zinc binuclear cluster Zn(II)2-Cys6 DNA binding domain of the PrtT polypeptides of A. oryzae, A. fumigatus and P. chrysogenum the SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27 were assigned, respectively.
FIG. 18 depicts the differences found between several PrtT polypeptide sequences in their C-terminal part: an alignment was performed along 12 amino acids derived from the functional A. niger PrtT polypeptide of the invention and having the SEQ ID NO: 22.
Alignment of the following PrtT was made: the A. niger and A. oryzae PrtT sequences of applications WO 00/20596 and WO 01/68864 and the PrtT polypeptides of the invention. It was found that the A. oryzae PrtT sequence of the application WO 01/68864 terminates preliminary. As underlined in FIG. 17, this preliminary termination of this PrtT polypeptide is close to the Leu-zipper domain and therefore it might affect its functionality. The A. oryzae PrtT sequence of the application WO 01/68864 completely misses the Leu zipper domain. Furthermore, the A. niger PrtT sequence of WO 00/20596 and WO 01/68864 applications comprise 17 additional amino acids in this region, caused by an unrecognized intron sequence as mentioned above in Example 3.1. This insertion can affect the topology of the protein domains (e.g. the subsequent Leu zipper domain as depicted in FIG. 4) and therefore the functionality of the PrtT protein.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein enclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure including definitions will control.

## Claims

1. An isolated polypeptide having transcriptional activity on a protease promoter, wherein said polypeptide is selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3;
(b) a polypeptide comprising a peptide fragment, said peptide fragment having at least 80% sequence identity with SEQ ID NO: 22;
(c) a polypeptide comprising peptide fragments, said peptide fragments having at least 80% sequence identity with both SEQ ID NO: 22 and SEQ ID NO: 4; and
(d) a polypeptide comprising (i) an amino acid sequence which has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 and comprising (ii) a peptide fragment, said peptide fragment comprising the amino acid sequence EWAHL(X)5-20ST, wherein “X” represents any amino acid and the range 5-20 represents the number of “X” found.

2. The polypeptide according to claim 1, wherein the polypeptide comprises both (i) an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3 and (ii) a peptide fragment comprising the amino acid sequence EWAHL(X)5-20ST.

3. The polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3, or is a polypeptide obtainable by expression of the prtT cDNA contained in pGBFINPRT-1, deposited under accession number CBS118680, or is a polypeptide obtainable by expression of the prtT cDNA contained in pGBPRT-1, deposited under accession number CBS118681.

4. The polypeptide of claim 1, wherein the polypeptide is obtained from a filamentous fungal strain.

5. The polypeptide of claim 4, wherein the filamentous fungal cell is an Aspergillus strain.

6. The polypeptide of claim 5, wherein the Aspergillus strain is a strain of Aspergillus niger or Aspergillus oryzae or Aspergillus sojae or Aspergillus fumigatus, or a respective synonym or teleomorph thereof.

7. An isolated nucleic acid, wherein the nucleic acid codes for the polypeptide as defined in claim 1.

8. The nucleic acid according to claim 7, wherein the nucleic acid is selected from the group consisting of:
(a) a nucleic acid comprising a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 2;
(b) a nucleic acid comprising a fragment, said fragment having at least 70% sequence identity with base pair number 1267 till base pair number 1302 of the nucleotide sequence of SEQ ID NO: 2; and
(c) a nucleic acid comprising a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 2, said nucleic acid comprising a fragment as defined under (b).

9. The nucleic acid of claim 8, wherein the nucleic acid comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 2.

10. The nucleic acid of claim 8, wherein the nucleic acid comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 2.

11. The nucleic acid according to claim 7, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 2, or the nucleotide sequence of the XhoI/HindIII fragment of plasmid pGBFINPRT-1, deposited under accession number CBS118680, or the nucleotide sequence of the prtT cDNA contained in plasmid pGBPRT-1, deposited under accession number CBS118681.

12. A nucleic acid construct comprising the nucleic acid of claim 7 coding for a polypeptide, said nucleic acid being operably linked to one or more control sequences, which direct the production of the polypeptide in a suitable expression host.

13. An expression vector comprising the nucleic acid construct of claim 12, a promoter, and transcriptional and translational stop signals.

14. A host cell comprising the nucleic acid construct of claim 12 or an expression vector comprising said nucleic acid construct, a promoter, and transcriptional and translational stop signals.

15. A fungal host cell useful for the production of a polypeptide, wherein the cell:
a) is a mutant of a parent fungal cell in which the parent cell comprises one or more DNA sequences encoding a protease, the transcription of which is activated by a transcriptional activator encoded by a nucleic acid of claim 7; and
b) produces less of the transcriptional activator and/or the protease (s) than the parent cell when cultured under the same conditions.

16. The host cell of claim 15, wherein reduced production of the transcriptional activator and/or protease is obtained by modification or inactivation of a nucleotide sequence present in the cell and necessary for expression of the transcriptional activator.

17. The host cell of claim 15, wherein reduced production of the transcriptional activator and/or protease is obtained by modification or inactivation of a control sequence required for the expression of the transcriptional activator.

18. The host cell of claim 17, wherein the control sequence is a promoter sequence, or a functional part thereof.

19. The host cell of claim 15, wherein reduced production of the transcriptional activator and/or protease is obtained by modification or inactivation of the nucleotide sequence coding for the transcriptional activator.

20. The host cell of claim 15, wherein modification or inactivation of a nucleotide or control sequence in the cell is performed by specific or random mutagenesis, site-directed mutagenesis, PCR generated mutagenesis, nucleotide insertion and/or deletion and/or substitution, gene interruption or gene replacement techniques, anti-sense techniques, RNAi techniques, or a combination thereof.

21. The host cell of claim 15, wherein the host cell comprises at least one copy of a nucleotide sequence encoding the polypeptide.

22. The host cell of claim 15, wherein the host cell produces less of a native protease or a combination of native proteases than the parent cell when cultured under identical conditions.

23. A method of producing a polypeptide, comprising:
(a) cultivating the host cell of claim 15, wherein the host cell harbours a gene encoding the polypeptide, in a nutrient medium suitable for production of the polypeptide and optionally; and
(b) recovering the polypeptide from the nutrient medium of the mutant cell.

24. The method of claim 23, wherein the polypeptide is native to the parent cell.

25. The method of claim 23, wherein the polypeptide is heterologous to the parent cell.

26. A fungal host cell useful for the production of a polypeptide, wherein the host cell is a mutant of a parent cell, in which the mutant:
a) produces more of a transcriptional activator encoded by a nucleotide sequence of claim 7 than the parent cell when cultured under the same conditions, and
b) comprises a DNA sequence encoding the polypeptide, the transcription of which is activated by the transcriptional activator.

27. The host cell of claim 26, wherein the host cell produces more of the transcriptional activator than the parent cell when cultured under the same conditions by introducing into the parent cell one or more copies of:
(i) said nucleotide sequence,
(ii) a nucleic acid construct comprising said nucleotide sequence operably linked to one or more control sequences, or
(iii) an expression vector comprising said nucleic acid construct, a promoter, and transcriptional and translational stop signals.

28. The host cell of claim 26, wherein the nucleotide sequence encoding the transcriptional activator is operably linked to a promoter, which is stronger than the corresponding promoter of the parent cell.

29. The host cell according to claim 26, useful for the production of polypeptides, wherein the polypeptides are extracellular proteases, preferably Aspergillus oryzae alkaline protease, A. oryzae neutral metalloprotease or A. niger aspergillopepsin proteases.

30. The host cell of claim 26, which further comprises a promoter sequence, wherein the promoter sequence can be activated by the transcriptional activator and is operably linked to the nucleotide sequence encoding the polypeptide.

31. The host cell of claim 30, wherein the promoter sequence, or a functional part thereof, is from a protease gene.

32. The host cell of claim 31, wherein the protease gene is Fusarium oxysporum trypsin-like protease gene, Aspergillus oryzae alkaline protease gene, Aspergillus niger pacA gene, Aspergillus oryzae alkaline protease gene, A. oryzae neutral metalloprotease gene, A. niger aspergillopepsin protease gene, or F. venenatum trypsin gene.

33. A fungal host cell useful for the production of a polypeptide, wherein the cell is a mutant of a parent cell in which the mutant comprises:
a) a modification or inactivation of a transcriptional activator which is encoded by a native nucleotide sequence of claim 7, or a regulatory sequence thereof, and
b) (i) an inducible promoter operably linked to said nucleotide sequence of, and (ii) a promoter sequence to which a transcriptional activator encoded by said nucleotide sequence of can bind, operably linked to a nucleotide sequence encoding the polypeptide, wherein (i) and (ii) can be introduced simultaneously or sequentially.

34. The host cell of claim 33, wherein the native nucleotide sequence, or a regulatory sequence thereof, is modified or inactivated by specific or random mutagenesis, site-directed mutagensis, PCR generated mutagenesis, nucleotide insertion and/or deletion and/or substitution, gene interruption or gene replacement techniques, anti-sense techniques, RNAi techniques, or a combination thereof.

35. The host cell of claim 33, wherein induction of the inducible promoter is mediated by a carbon or nitrogen catabolite.