Patent Publication Number: US-2011059862-A1

Title: Expression Cloning Methods In Filamentous Fungi

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 10/504,646 filed Aug. 13, 2004, which is a 35 U.S.C. 371 national application of PCT/DK03/00106 filed Feb. 18, 2003, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2002 00256 filed Feb. 19, 2002 and U.S. provisional application No. 60/359,256 filed Feb. 21, 2002, the contents of which are fully incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Several methods for the construction of libraries of polynucleotide sequences of interest in yeast have been disclosed in which the libraries are screened in yeast prior to transformation of an industrially relevant filamentous fungal host cell with a selected polynucleotide. 
     Often however, a polynucleotide sequence identified by screening in yeast or bacteria cannot be expressed or is expressed at low levels when transformed into production relevant filamentous fungal cells. This may be due any number of reasons, including differences in codon usage, regulation of mRNA levels, translocation apparatus, post-translational modification machinery (e.g., cysteine bridges, glycosylation and acylation patterns), etc. 
     A. Aleksenko and A. J. Clutterbuck (1997. Fungal Genetics and Biology 21:373-387) disclose the use of autonomous replicative vectors, or autonomously replicating sequences (ARS), for gene cloning and expression studies. AMA1 (autonomous maintenance in  Aspergillus ) is one of the plasmid replicator elements discussed. It consists of two inverted copies of a genomic repeat designated MATE1 (mobile  Aspergillus  transformation enhancer) separated by a 0.3 kb central spacer. AMA1 promotes plasmid replication without rearrangement, multimerization or chromosomal integration. AMA1-based plasmids provide two advantages in gene cloning in filamentous fungi. The first is a high frequency of transformation which both increases the potential library size and can eliminate the need for library amplification in an intermediate host, e.g.,  E. coli , so that a recipient  Aspergillus  strain can be transformed directly with a ligation mixture. Secondly, by providing a stable and standard environment for gene expression, the properties of the transformants will be uniform (WO 00/24883; Novozymes A/S). 
     Kozak, 1981 , Nucleic Acids Research  9: 5233-5252, proposed the following “consensus” sequence for initiation of translation in higher eukaryotes: 
                            Aa Acc  aug  G            
In this sequence, often referred to as a “consensus Kozak”, the most highly conserved nucleotides are the purines, adenine (A) and guanine (G), shown in capital letters above; the start-codon of the gene to be translated is underlined in the above. Mutational analysis confirmed that these two positions have the strongest influence on initiation (Kozak, 1987 , Molecular Cell Biology  7: 3438-3445). Kozak also determined that alterations in the sequence upstream of the consensus Kozak can effect translation (Kozak, 1986 , Proceedings of the National Academy of Sciences USA  83: 2850-2854).
 
     WO 94/11523 and WO 01/51646 disclose expression vectors comprising a fully impaired consensus Kozak or “crippled” consensus Kozak sequence. 
     SUMMARY OF THE INVENTION 
     Expression cloning as such in filamentous fungi is presently part of the standard methodology in the art, however the use of such methods is of such industrial relevance that even minor increments in efficiency, performance or economy is of great interest. Until now expression cloning in filamentous fungi may have provided an interesting polypeptide candidate, whereupon the encoding gene would typically have been sub-cloned into a more suitable expression vector to achieve polypeptide yields of sufficient quantity to further characterize the polypeptide of interest, before setting up expensive larger scale trial productions. A problem to be solved is how to screen a polynucleotide library for a polypeptide with a property of interest in a filamentous fungal host cell in a manner which allows quick and easy characterization of the subsequent polypeptide. 
     An aspect of the present invention relates to methods for isolating a recombinant polypeptide of interest, the methods comprising the steps of:
         a) providing a polynucleotide library derived from an organism capable of producing one or more polypeptides of interest, wherein the library was prepared in an expression cloning vector comprising at least the following elements:
           i) a polynucleotide encoding a selectable marker in which the translation initiation start site of the marker-encoding sequence comprises the following sequence:   
               

     
       
         
           
               
               
            
               
                   
                 −4 
               
            
           
           
               
               
               
            
               
                   
                 N YNN  ATG  YNN 
                 (SEQ ID NO: 1) 
               
            
           
         
       
         
         
           
             
               
                  wherein “Y” in position −3 is a pyrimidin (Cytidine or Thymidine/Uridine), “N” is any nucleotide, and the numerical designations are relative to the first nucleotide in the start-codon “ATG” (in bold) of the marker; 
                 ii) a fungal replication initiation sequence, preferably an automously replicating sequence (ARS), more preferably an AMA1-sequence or a functional derivative thereof; and 
                 iii) a polynucleotide comprising in sequential order: a promoter derived from a filementous fungal cell, a cloning-site into which the library is cloned, and a transcription terminator; 
               
             
             b) transforming a filamentous fungal host cell with the library; 
             c) culturing the transformed host cell obtained in (b) under conditions suitable for expression of the polynucleotide library; and 
             d) selecting a transformed host cell which produces the polypeptide of interest. 
           
         
       
    
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to a method of the first aspect of the invention for isolating a recombinant polypeptide of interest, the method comprising the steps of:
         a) providing a polynucleotide library derived from an organism capable of producing one or more polypeptides of interest, wherein the library was prepared in an expression cloning vector comprising at least the following elements:
           i) a polynucleotide encoding a selectable marker in which the translation initiation start site of the marker-encoding sequence comprises the following sequence:   
               

     
       
         
           
               
               
            
               
                   
                 −4 
               
            
           
           
               
               
               
            
               
                   
                 N YNN  ATG  YNN 
                 (SEQ ID NO: 1) 
               
            
           
         
       
         
         
           
             
               
                  wherein “Y” in position −3 is a pyrimidin (Cytidine or Thymidine/Uridine), “N” is any nucleotide, and the numerical designations are relative to the first nucleotide in the start-codon “ATG” (in bold) of the marker; 
                 ii) a fungal replication initiation sequence, preferably an automously replicating sequence (ARS), more preferably an AMA1-sequence or a functional derivative thereof; and 
                 iii) a polynucleotide comprising in sequential order: a promoter derived from a filementous fungal cell, a cloning-site into which the library is cloned, and a transcription terminator; 
               
             
             b) transforming a filamentous fungal host cell with the library; 
             c) culturing the transformed host cell obtained in (b) under conditions suitable for expression of the polynucleotide library; and 
             d) selecting a transformed host cell which produces the polypeptide of interest. 
           
         
       
    
     In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide, and under conditions that select for multiple copies of the selectable marker, using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, 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. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). 
     If the polypeptide of interest is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates. 
     The polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. The polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. 
     The polypeptides may be 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), SDS-PAGE, or extraction (see, e.g.,  Protein Purification , J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). 
     Crippled Translational Initiator Sequences 
     The term “translational initiator sequence” is defined herein as the ten nucleotides immediately upstream of the initiator or start codon of the open reading frame of a polypeptide-encoding nucleic acid sequence. The initiator codon encodes for the amino acid methionine, the so-called “start” codon. The initiator codon is typically an ATG, but may also be any functional start codon such as GTG. It is well known in the art that uracil (uridine), U, replaces the deoxynucleotide thymine (thymidine), T, in RNA. 
     The term “crippled translational initiator sequence” is defined herein as the ten nucleotides immediately upstream of the initiator codon of the open reading frame of a polypeptide-encoding nucleic acid sequence, wherein the initiator sequence comprises a T at the −3 position and a T at one or more of the −1, −2, and −4 positions. 
     Accordingly, a preferred embodiment of the invention relates to a method of the first aspect, wherein the sequence SEQ ID NO:1 comprises a Thymidin (Uridin) in the −3 position; even more preferably the sequence SEQ ID NO:1 further comprises a Thymidin (Uridin) in one more of the positions −1, −2, and −4. 
     The term “operably linked” is defined herein as a configuration in which a control sequence, e.g., a crippled translational initiator sequence, is appropriately placed at a position relative to a coding sequence such that the control sequence directs the production of a polypeptide encoded by the coding sequence. 
     The term “coding sequence” is defined herein as a nucleic acid sequence that is transcribed into mRNA which is translated into a polypeptide when placed under the control of the appropriate control sequences. The boundaries of the coding sequence are generally determined by the start codon located at the beginning of the open reading frame of the 5′ end of the mRNA and a stop codon located at the 3′ end of the open reading frame of the mRNA. A coding sequence can include, but is not limited to, genomic DNA, cDNA, semisynthetic, synthetic, and recombinant nucleic acid sequences. 
     In the methods of the present invention, the crippled translational initiator sequence is foreign to the gene encoding a selectable marker. 
     The crippled translational sequence results in inefficient translation of the gene encoding the selectable marker. When a fungal host cell harbouring an expression vector comprising a polynucleotide encoding a polypeptide of interest physically linked with a second polynucleotide comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker, is cultured under conditions that select for multiple copies of the selectable marker, the copy number of the polypeptide-encoding polynucleotide cloned into the vector is also increased. 
     The term “selectable marker” is defined herein as a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like, which permits easy selection of transformed cells. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an  Aspergillus  cell are the amdS and pyrG genes of  Aspergillus nidulans  or  Aspergillus oryzae  and the bar gene of  Streptomyces hygroscopicus . Functional derivatives of these selectable markers are also of interest in the present invention, in particular those functional derivatives which have decreased activity or decreased stability, thereby enabling a selection for a higher copy-number of the expression vector without increasing the concentration of the selective substance(s). 
     Accordingly, a preferred embodiment is a method of the first aspect, wherein the selectable marker of step (i) is selected from the group of markers consisting of amdS, argB, bar, hygB, niaD, pyrG, sC, and trpC; preferably the selectable marker of step (i) is pyrG or a functional derivative thereof, more preferably the selectable marker of step (i) is a functional derivative of pyrG which comprises a substitution of one or more amino acids, and most preferably the derivative comprises the amino acid substitution T102N. 
     The term “copy number” is defined herein as the number of molecules, per genome, of a gene which is contained in a cell. Methods for determining the copy number of a gene are will known in the art and include Southern analysis, quantitative PCR, or real time PCR. 
     The fungal host cell preferably contains at least two copies, more preferably at least ten copies, even more preferably at least one hundred copies, most preferably at least five hundred copies, and even most preferably at least one thousand copies of the expression cloning vector. 
     Polypeptide Encoding Polynucleotides 
     The polypeptide of interest may be native or heterologous to the filamentous fungal host cell of interest. The term “heterologous polypeptide” is defined herein as a polypeptide which is not native to the fungal cell, a native polypeptide in which modifications have been made to alter the native sequence, or a native polypeptide whose expression is quantitatively altered as a result of a manipulation of the fungal cell by recombinant DNA techniques. The polynucleotide encoding the polypeptide of interest may originate from any organism capable of producing the polypeptide of interest, including multicellular organisms and microorganisms e.g. bacteria and fungi. 
     A preferred embodiment of the invention relates to methods of the first aspect, wherein the organism of step (a) capable of producing one or more polypeptides of interest is a eukaryote, preferably the eukaryote is a fungus, and most preferably a filamentous fungus. 
     The term “polypeptide” is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. 
     Preferably, the polypeptide of interest is an enzyme, an enzyme variant, or a functional derivative thereof, more preferably the enzyme or enzyme variant is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase; and most preferably the enzyme or enzyme variant is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, a pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. 
     Preferably, the polypeptide is a hormone or hormone variant or a functional derivative thereof, a receptor or receptor variant or a functional derivative thereof, an antibody or antibody variant or a functional derivative thereof, or a reporter. 
     In a preferred embodiment, the polypeptide is secreted extracellularly. In a more preferred embodiment, the polypeptide is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In an even more preferred embodiment, the polypeptide is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. 
     The nucleic acid sequence encoding a polypeptide of interest may be obtained from any prokaryotic, eukaryotic, or other source. 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. 
     The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide of interest are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequence from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR). See, for example, Innis et al., 1990 , PCR Protocols: A Guide to Methods and Application , Academic Press, New York. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into the mutant fungal cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof. 
     In the methods of the present invention, the polypeptide 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 polypeptide 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. 
     Once a transformed host cell has been selected which produces the polypeptide of interest according to the methods of the invention, the encoding polynucleotide can be isolated from the selected transformed host cell, and a further optimized expression system can be designed. 
     Accordingly, a preferred embodiment relates to methods of the first aspect, wherein subsequently to step (d) the polynucleotide coding for the polypeptide of interest is isolated from the selected transformed host cell of step (d). 
     Fungal Replication Initiating Sequences 
     As used herein, the term “fungal replication initiating sequence” is defined as a nucleic acid sequence which is capable of supporting autonomous replication of an extrachromosomal molecule, e.g., a DNA vector such as a plasmid, in a filamentous fungal host cell, normally without structural rearrangement of the DNA-vector or integration into the host cell genome. The replication initiating sequence may be of any origin as long as it is capable of mediating replication initiating activity in a fungal cell. For instance the replication initiating sequence may be a telomer of human origin which confer to the plasmid the ability to replicate in  Aspergillus  (Aleksenko and Ivanova, Mol. Gen. Genet. 260 (1998) 159-164). Preferably, the replication initiating sequence is obtained from a filamentous fungal cell, more preferably a strain of  Aspergillus, Fusarium  or  Alternaria , and even more preferably, a strain of  A. nidulans, A. oryzae, A. niger, F. oxysporum  or  Alternaria altenata.    
     A fungal replication initiating sequence may be identified by methods well-known in the art. For instance, the sequence may be identified among genomic fragments derived from the organism in question as a sequence capable of sustaining autonomous replication in yeast, (Ballance and Turner, Gene, 36 (1985), 321-331), an indication of a capability of autonomous replication in filamentous fungal cells. The replication initiating activity in fungi of a given sequence may also be determined by transforming fungi with contemplated plasmid replicators and selecting for colonies having an irregular morphology, indicating loss of a sectorial plasmid which in turn would lead to lack of growth on selective medium when selecting for a gene found on the plasmid (Gems et al, Gene, 98 (1991) 61-67). AMA1 was isolated in this way. An alternative way to isolate a replication initiating sequence is to isolate natural occurring plasmids (eg as disclosed by Tsuge et al., Genetics 146 (1997) 111-120 for  Alternaria atemata ). 
     Examples of fungal replication initiating sequences include, but are not limited to, the ANSI and AMA1 sequences of  Aspergillus nidulans , e.g., as described, respectively, by Cullen, D., et al. (1987, Nucleic Acids Res. 15:9163-9175) and Gems, D., et al. (1991, Gene 98:61-67). 
     Preferred embodiments relate to methods of the first aspect of the invention, wherein the fungal replication initiation sequence of step (ii) comprises the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 of WO 00/24883, or is a functional derivative thereof, preferably the functional derivative is at least 80% identical to SEQ ID NO:1 or SEQ ID NO: 2 of WO 00/24883. 
     The term “replication initiating activity” is used herein in its conventional meaning, i.e. to indicate that the sequence is capable of supporting autonomous replication of an extrachromosomal molecule, such as a plasmid or a DNA vector in a fungal cell. 
     The term “without structural rearrangement of the plasmid” is used herein to mean that no part of the plasmid is deleted or inserted into another part of the plasmid, nor is any host genomic DNA inserted into the plasmid. The replication initiating sequence to be used in the methods of the present invention is a nucleotide sequence having at least 50% identity with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2 of WO 00/24883, and is capable of initiating replication in a fungal cell; or a subsequence of (a) or (b), wherein the subsequence is capable of initiating replication in a fungal cell. 
     In a preferred embodiment, the nucleotide sequence has a degree of identity to the nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 of WO 00/24883 of at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 97% identity (hereinafter “homologous polynucleotide”). The homologous polynucleotide also encompasses a subsequence of SEQ ID NO:1 or SEQ ID NO:2 of WO 00/24883 which has replication initiating activity in fungal cells. For purposes of the present invention, the degree of identity may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following settings for polynucleotide sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3. 
     The techniques used to isolate or clone a nucleic acid sequence having replication initiating activity are known in the art and include isolation from genomic DNA or cDNA. The cloning from such DNA can be effected, e.g., by using methods based on polymerase chain reaction (PCR) 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) may be used. 
     In preferred embodiment, the replication initiating sequence has the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 of WO 00/24883, or a respective functional subsequence thereof. For instance, a functional subsequence of SEQ ID NO:1 of WO 00/24883 is a nucleic acid sequence encompassed by SEQ ID NO:1 or SEQ ID NO 2 of WO 00/24883 except that one or more nucleotides from the 5′ and/or 3′ end have been deleted. Preferably, a subsequence contains at least 100 nucleotides, more preferably at least 1000 nucleotides, and most preferably at least 2000 nucleotides. In a more preferred embodiment, a subsequence of SEQ ID NO:1 of WO 00/24883 contains at least the nucleic acid sequence shown in SEQ ID NO:2 of WO 00/24883. 
     Nucleic Acid Constructs 
     The present invention also relates to nucleic acid constructs comprising a polynucleotide comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker in which the 3′ end of the crippled translational initiator sequence is immediately upstream of the initiator codon of the gene encoding the selectable marker. The polynucleotides are operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. 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. 
     “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 combined and juxtaposed in a manner that would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression vector when the nucleic acid construct comprises a second polynucleotide encoding a polypeptide of interest and all the control sequences required for its expression. 
     An isolated polynucleotide encoding a polypeptide may be further manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence 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 recombinant DNA methods are well known in the art. 
     In the methods of the present invention, the nucleic acid sequences may comprise one or more native control sequences or one or more of the native control sequences may be replaced with one or more control sequences foreign to the nucleic acid sequence for improving expression of the coding sequence in a host cell. 
     The term “control sequences” is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide of interest. 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, polyadenylation sequence, propeptide sequence, crippled translational initiator sequence of the present invention, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include translational initiator sequences, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites or cloning sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding 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 host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. 
     Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for  Aspergillus oryzae  TAKA amylase,  Rhizomucor miehei  aspartic proteinase,  Aspergillus niger  neutral alpha-amylase,  Aspergillus niger  acid stable alpha-amylase,  Aspergillus niger  or  Aspergillus awamori  glucoamylase (gIaA),  Rhizomucor miehei  lipase,  Aspergillus oryzae  alkaline protease,  Aspergillus oryzae  triose phosphate isomerase,  Aspergillus nidulans acetamidase, Fusarium venenatum  amyloglucosidase,  Fusarium oxysporum  trypsin-like protease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for  Aspergillus niger  neutral alpha-amylase and  Aspergillus oryzae  triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof. 
     A preferred embodiment relates to methods of the first aspect, wherein the promoter of step (iii) is the promoter from the neutral amylase encoding gene (NA2) from  Aspergillus niger  disclosed in WO 89/01969. 
     The control sequence may be a suitable transcription terminator sequence, a sequence recognized by a host 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 host cell of choice may be used in the present invention. 
     Preferred terminators for filamentous fungal host cells are obtained from the genes for  Aspergillus oryzae  TAKA amylase,  Aspergillus niger glucoamylase, Aspergillus nidulans  anthranilate synthase,  Aspergillus niger  alpha-glucosidase, and  Fusarium oxysporum  trypsin-like protease. 
     A preferred embodiment relates to methods of the first aspect, wherein the transcription terminator of step (iii) is the terminator from the glucoamylase encoding gene (AMG) from  Aspergillus niger  (Boel, E.; Hjort, I.; Svensson, B.; Norris, F.; Norris, K. E.; FiiI, N. P., Glucoamylases G1 and G2 from  Aspergillus niger  are synthesized from two different but closely related mRNAs. EMBO J. 3:1097 (1984)). 
     The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention. 
     Preferred leaders for filamentous fungal host cells are obtained from the genes for  Aspergillus oryzae  TAKA amylase and  Aspergillus nidulans  triose phosphate isomerase. 
     A preferred embodiment relates to methods of the first aspect, wherein the promoter is operably linked, upstream of the cloning-site of step (iii), to the polynucleotide encoding the leader peptide of triose phosphate isomerase (tpiA) from  Aspergillus nidulans . (Mcknight G. L., O&#39;Hara P. J., Parker M. L., “Nucleotide sequence of the triosephosphate isomerase gene from  Aspergillus nidulans : Implications for a differential loss of introns”, Cell 46:143-147 (1986)). 
     The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention. 
     Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for  Aspergillus oryzae  TAKA amylase,  Aspergillus niger glucoamylase, Aspergillus nidulans  anthranilate synthase,  Fusarium oxysporum  trypsin-like protease, and  Aspergillus niger  alpha-glucosidase. 
     The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell&#39;s secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention. 
     Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for  Aspergillus oryzae  TAKA amylase,  Aspergillus niger  neutral amylase,  Aspergillus niger glucoamylase, Rhizomucor miehei  aspartic proteinase,  Humicola insolens  cellulase, and  Humicola lanuginosa  lipase. 
     The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for  Bacillus subtilis  alkaline protease (aprE),  Bacillus subtilis  neutral protease (nprT),  Saccharomyces cerevisiae  alpha-factor,  Rhizomucor miehei  aspartic proteinase, and  Myceliophthora thermophila  laccase (WO 95/33836). 
     Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region. 
     Expression Vectors 
     The present invention also relates to recombinant expression vectors comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker in which the 3′ end of the crippled translational initiator sequence is immediately upstream of the initiator codon of the gene encoding the selectable marker and a nucleic acid sequence encoding a polypeptide of interest as well as any control sequences involved in the expression of the sequences. 
     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 promoter and/or nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the crippled translational initiator sequence and/or 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 a crippled translational initiator sequence of the present invention and one or more appropriate control sequences for expression. 
     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 a nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host 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. The vector may contain any means for assuring self-replication. 
     The vectors of the present invention also contain one or more selectable markers which permit easy selection of transformed cells as described earlier. 
     For autonomous replication, the vector further comprises an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978 , Proceedings of the National Academy of Sciences USA  75: 1433). 
     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 
     The host cell may be any fungal cell useful in the methods of the present invention. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,  Ainsworth and Bisby&#39;s Dictionary of The Fungi,  8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). 
     In a preferred embodiment, the fungal host cell is a filamentous fungal cell. “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 mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as  Saccharomyces cerevisiae  is by budding of a unicellular thallus and carbon catabolism may be fermentative. 
     In a preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to,  Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium , or  Trichoderma.    
     In a more preferred embodiment, the filamentous fungal host cell is an  Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger  or  Aspergillus oryzae  cell. In another most preferred embodiment, the filamentous fungal host cell is a  Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides , or  Fusarium venenatum  cell. In another most preferred embodiment, the filamentous fungal host cell is a  Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei , or  Trichoderma viride  cell. In an even most preferred embodiment, the  Fusarium venenatum  cell is  Fusarium venenatum  A3/5, which was originally deposited as  Fusarium graminearum  ATCC 20334 and recently reclassified as  Fusarium venenatum  by Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80 and O&#39;Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; as well as taxonomic equivalents of  Fusarium venenatum  regardless of the species name by which they are currently known. In another preferred embodiment, the  Fusarium venenatum  cell is a morphological mutant of  Fusarium venenatum  A3/5 or  Fusarium venenatum  ATCC 20334, as disclosed in WO 97/26330. 
     Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of  Aspergillus  host cells are described in EP 238 023 and Yelton et al., 1984 , Proceedings of the National Academy of Sciences USA  81: 1470-1474. Suitable methods for transforming  Fusarium  species are described by Malardier et al., 1989 , Gene  78: 147-156 and WO 96/00787. 
     The present invention is further described by the following examples which should not be construed as limiting the scope of the invention. 
     EXAMPLES 
     Example 1 
     In order to improve expression of a gene of interest on an expression plasmid, it may be desirable to reduce the expression of the selection gene, exemplified here by the pyrG gene. By cultivating a host cell harbouring an expression plasmid comprising a selection gene, that has reduced expression, under normal selective pressure results in a selection for a host cell which has an increased plasmid copy number, thus achieving the total expression level of the selection gene necessary for survival. The higher plasmid copy-number however also results in an increased expression of the gene of interest. 
     One way of decreasing the expression level of the selection gene is to lower the mRNA level by either using a poorly transcribed promoter or decreasing the functional halflife of the mRNA. Another way is to reduce translation efficiency of the mRNA. One way to do this is to mutate the Kozak-region. This is a region just upstream of the initiation codon (ATG), which is important for the initiation of translation. 
     Plasmid pENI2155 comprises a bad kozak region upstream of the pyrG gene, and is constructed as follows: 
     Using plasmid pENI1861 (the construction of which is described below) as template, and PWO polymerase (conditions as recommended by manufacturer); two PCR-reactions were made using primer 141200j1 and 270999J9 in the one PCR-reaction and primers 141200J2 and 290999J8 in another PCR-reaction: 
     
       
         
           
               
               
            
               
                   
                 141200J1 (SEQ ID NO: 2): 
               
               
                   
                 5′ atcggttttatgtcttccaagtcgcaattg 
               
               
                   
                   
               
               
                   
                 141200J2 (SEQ ID NO: 3): 
               
               
                   
                 5′ cttggaagacataaaaccgatggaggggtagcg 
               
               
                   
                   
               
               
                   
                 270999J8 (SEQ ID NO: 4): 
               
               
                   
                 5′ tctgtgaggcctatggatctcagaac 
               
               
                   
                   
               
               
                   
                 270999J9 (SEQ ID NO: 5): 
               
               
                   
                 5′ gatgctgcatgcacaactgcacctcag 
               
            
           
         
       
     
     The PCR fragments were purified from a 1% agarose gel using QIAGEN™ spin columns. A second PCR-reaction was run using the two fragments as template along with the primers 270999J8 and 270999J9. The PCR-fragment from this reaction was purified from a 1% agarose gel as described; the fragment and the vector pENI1849 (containing a lipase gene as expression reporter) were cut with the restriction enzymes StuI and SphI, the resulting fragments were purified from a 1% agarose gel as described previously. 
     The purified fragments were ligated and transformed into the  E. coli  strain DH10B. Plasmid DNA from one of the transformants was isolated and sequenced to confirm the introduction of a mutated Kozak region: ggttttatg (rather than the wildtype: gccaacatg). This Plasmid was denoted: pENI2155. 
       Aspergillus  cells were transformed with plasmid pENi1849 (control wildtype plasmid), and pENi2155 (mutated Kozak region upstream of the pyrG gene). Approximately 1 microgram of pENI1849 and pENi2155 were transformed into  A. oryzae  Ja1355 (JaL355 is a derivative of  A. oryzae  A1560 wherein the pyrG gene has been inactivated, as described in WO 98/01470; transformation protocol as described in WO 00/24883). The transformants were incubated for 4 days at 37° C. 
     24 transformants from the pENi2155 transformation and 12 transformants from pENI1849 were inoculated in a 96 well microtiter plate containing 1*Vogel medium and 2% maltose (Methods in Enzymology, vol. 17, p. 84). After 4 days growth at 34° C., the culture broth was assayed for lipase activity using pnp-valerate as a lipase substrate. 
     A 10 microliter aliquot of media from each well was added to a microtiter well containing 200 microliter of a lipase substrate of 0.018% p-nitrophenylvalerate, 0.1% Triton X™-100, 10 mM CaCl 2 , 50 mM Tris pH 7.5. Lipase activity was assayed spectrophotometrically at 15-second intervals over a five minute period, using a kinetic microplate reader (Molecular Device Corp., Sunnyvale Calif.), using a standard enzymology protocol (e.g., Enzyme Kinetics, Paul C. Engel, ed., 1981, Chapman and Hall Ltd.). Briefly, product formation is measured during the initial rate of substrate turnover and is defined as the slope of the curve calculated from the absorbance at 405 nm every 15 seconds for 5 minutes. The arbitrary lipase activity units were normalized against the transformant showing the highest lipase activity. For each group of thirty transformants an average value and the standard deviations were calculated. Given in arbitrary units the average lipase activity and relative standard deviation was: 
     1849 Transformant: 65±14 
     2155 Transformant: 120±22 
     Clearly there is nearly a doubling of lipase expression in the 2155 transformant, wherein the mutated Kozak region was introduced in front of the selection gene pyrG. 
     Plasmid pENI1861 was made in order to have the state of the art  Aspergillus  promoter in the expression plasmid, as well as a number of unique restriction sites for cloning. A PCR fragment (Approx. 620 bp) was made using plasmid pMT2188 (the construction of pMT2188 is described below) as template and the following primers: 
     
       
         
           
               
            
               
                 051199J1 (SEQ ID NO: 6): 
               
               
                 5′ cctctagatctcgagctcggtcaccggtggcctccgcggccgctg 
               
               
                   
               
               
                 gatccccagttgtg 
               
               
                   
               
               
                 1298TAKA (SEQ ID NO: 7): 
               
               
                 5′ gcaagcgcgcgcaatacatggtgttttgatcat 
               
            
           
         
       
     
     The fragment was cut with BssHII and BgIII, and cloned into pENI1849 which was also cut with BssHII and Bgl II. The cloning was verified by sequencing. 
     Plasmid pENI1849 was made in order to truncate the pyrG gene to the essential sequences for pyrG expression, in order to decrease the size of the plasmid, thus improving transformation frequency. A PCR fragment (Approx. 1800 bp) was made using pENI1299 (described in WO 00/24883 FIG. 2 and Example 1) as template and the following primers: 270999J8 (SEQ ID NO:3), and 270999J9 (SEQ ID NO:4) 
     The PCR-fragment was cut with the restriction enzymes Stul and SphI, and cloned into pENI1298 (described in WO 00/24883 FIG. 1 and Example 1), also cut with Stul and SphI; the cloning was verified by sequencing. 
     Plasmid pMT2188 was based on the  Aspergillus  expression plasmid pCaHj 483 (described in WO 98/00529) which consists of an expression cassette based on the  Aspergillus niger  neutral amylase II promoter fused to the  Aspergillus nidulans  triose phosphate isomerase non translated leader sequence (Pna2/tpi) and the  A. niger  amyloglycosidase terminater (Tamg). Also present on the pCaHj483 is the  Aspergillus  selective marker amdS from  A. nidulans  enabling growth on acetamide as sole nitrogen source. These elements are cloned into the  E. coli  vector pUC19 (New England Biolabs). The ampicillin resistance marker enabling selection in  E. coli  of pUC19 was replaced with the URA3 marker of  Saccharomyces cerevisiae  that can complement a pyrF mutation in  E. coli , the replacement was done in the following way: 
     The pUC19 origin of replication was PCR amplified from pCaHj483 with the primers: 
     
       
         
           
               
               
            
               
                   
                 142779 (SEQ ID NO: 8): 
               
               
                   
                 5′ ttgaattgaaaatagattgatttaaaacttc 
               
               
                   
                   
               
               
                   
                 142780 (SEQ ID NO: 9): 
               
               
                   
                 5′ ttgcatgcgtaatcatggtcatagc 
               
            
           
         
       
     
     Primer 142780 introduces a BbuI site in the PCR fragment. The Expand™ PCR system (Roche Molecular Biochemicals, Basel, Switserland) was used for the amplification following the manufacturers instructions for this and the subsequent PCR amplifications. 
     The URA3 gene was amplified from the general  S. cerevisiae  cloning vector pYES2 (Invitrogen corporation, Carlsbad, Calif., USA) using the primers: 
     
       
         
           
               
               
            
               
                   
                 140288 (SEQ ID NO: 10): 
               
               
                   
                 5′ ttgaattcatgggtaataactgatat 
               
               
                   
                   
               
               
                   
                 142778 (SEQ ID NO: 11): 
               
               
                   
                 5′ aaatcaatctattttcaattcaattcatcatt 
               
            
           
         
       
     
     Primer 140288 introduces an EcoRI site in the PCR fragment. The two PCR fragments were fused by mixing them and amplifying using the primers 142780 and 140288 in the splicing by overlap method (Horton et al (1989) Gene, 77, 61-68). 
     The resulting fragment was digested with EcoRI and BbuI and ligated to the largest fragment of pCaHj 483 digested with the same enzymes. The ligation mixture was used to transform the pyrE  E. coli  strain DB6507 (ATCC 35673) made competent by the method of Mandel and Higa (Mandel, M. and A. Higa (1970) J. Mol. Biol. 45, 154). Transformants were selected on solid M9 medium (Sambrook et. al (1989) Molecular cloning, a laboratory manual, 2. edition, Cold Spring Harbor Laboratory Press) supplemented with 1 g/l casaminoacids, 500 microgram/I thiamine and 10 mg/l kanamycin. A plasmid from a selected transformant was termed pCaHj527. ThePna2/tpi promoter present on pCaHj527 was subjected to site directed mutagenises by a simple PCR approach. Nucleotide 134-144 was altered from GTACTAAAACC to CCGTTAAATTT using the mutagenic primer 141223. Nucleotide 423-436 was altered from ATGCAATTTAAACT to CGGCAATTTAACGG using the mutagenic primer 141222. The resulting plasmid was termed pMT2188. 
     
       
         
           
               
            
               
                 Primer 141223 (SEQ ID NO: 12): 
               
               
                 5′ ggatgctgttgactccggaaatttaacggtttggtcttgcatccc 
               
               
                   
               
               
                 Primer 141222 (SEQ ID NO: 13): 
               
               
                 5′ ggtattgtcctgcagacggcaatttaacggcttctgcgaatcgc 
               
            
           
         
       
     
     Example 2 
     In order to improve expression of a gene of interest from a plasmid, it may be desirable to reduce the stability and/or the activity of the protein encoded by the selection gene (for instance the pyrG gene) as already mentioned in Example 1. 
     One way of decreasing the stability of the protein encoded by the selection gene is to add a “degron” motif to the protein (Dohmen R. J., Wu P., Varshaysky A., (1994) Science vol 263 p. 1273-1276). Another way is to identify structurally important conserved amino acid residues, based on alignment to homologous proteins or based on a model-structure of the protein (if available). These amino acids may then be mutated to decrease the stability and/or the activity of the enzyme. 
     A protein alignment was made with the protein sequence: swissprot_dcop_aspng (the OMP decarboxylase encoded by the pyrG gene on plasmid pENI2155) to the following database entries: Swissprot_dcop-aspor, geneseqp_r05224, geneseqp_y99702, tremblnew_aag34761, swissprot_dcop_phybl, remtermbl_aab01165, remtembl_aab16845, and sptrembl_q9uvz5. 
     The alignment was done using the program ClustalW (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). 
     Based on these alignments and the structure of the related  Bacillus subtilis  OMP decarboxylase (Appleby t., Kinsland C., Begley T. P., Ealick S. E. (2000), Proc. Natl. Acad. Sci. USA, vol 97 p. 2005-2010) the following conserved residues were identified as potentially structurally important, and as such suitable targets for mutation: P50, F91, F96, N101, T102, G128, G222, D223, G239. A number of mutagenic primers were constructed, and were phosphorylated using T4 polynucleotide kinase (New England Biolabs). 
     
       
         
           
               
               
            
               
                   
                 P50-260301j1 (SEQ ID NO: 14): 
               
               
                   
                 5′ acaggactcggt ncg tacattgccgtg 
               
               
                   
                   
               
               
                   
                 F91-260301j2 (SEQ ID NO: 15): 
               
               
                   
                 5′ aatttcctcatc tnc gaagatcgcaag 
               
               
                   
                   
               
               
                   
                 F96-260301j3 (SEQ ID NO: 16): 
               
               
                   
                 5′ gaagatcgcaag tnc atcgatatcgga 
               
               
                   
                   
               
               
                   
                 N101, T102-260301j4 (SEQ ID NO: 17): 
               
               
                   
                 5′ atcgatatcgga nacanc gtccaaaagcag 
               
               
                   
                   
               
               
                   
                 G128-260301j5 (SEQ ID NO: 18): 
               
               
                   
                 5′ agtattctgccc gnt gagggtatcgtc 
               
               
                   
                   
               
               
                   
                 G222, D223-260301j6 (SEQ ID NO: 19): 
               
               
                   
                 5′ ctctcctcgaag gntnac aagctgggacag 
               
               
                   
                   
               
               
                   
                 G239-230301j7 (SEQ ID NO: 20): 
               
               
                   
                 5′ gctgttggacgc gnt gccgactttatt 
               
            
           
         
       
     
     Seven individual PCR/ligation reactions were performed (as described by Sawano A., Miyawaki A. (2000) Nucleic Acid Research vol 28 e78) using pENI2155 as template, and 1 microliter DNA from each of the seven libraries was transformed into the  E. coli  strain DH10B. Approximately 1000  E. coli  clones were obtained from each library. DNA preparation was made from each library and the DNA was pooled together (named pBIB16). 
     The  Aspergillus  strain MT2425 (a pyrG minus strain, which gives small transformant-clones, when grown on the selection plates) was transformed with 1 microgram of the pBIB16 DNA and 10 microgram herring sperm DNA (carrier DNA) pr. 100 microliter protoplast using standard procedures. 
     The transformed protoplast were spread on selection plates (2% maltose (inducing small morphology and lipase expression), 10 mM NaNO 3 , 1.2 M sorbitol, 2% bacto agar, and standard salt solution. 
     After 5 days of growth, an overlay (containing 0.004% brilliant green, 2.5% olive oil, 1% agar, 50 mM TRIS pH 7.5 treated with a mixer for 1 min. (Ultrathorax™ Type T25B, IKA Labortechnic, Germany)) was poured onto the  Aspergillus  transformant clones. The plates where incubated over night at room temperature. 
     Twenty of the clones having highest activity towards olive oil were inoculated in to 200 microliter YPM in a 96 well microtiter plate. After 4 days of growth at 34° C., the culture broths were assayed for lipase activity using pnp-valerate as described above. 
     The 6 transformants giving the highest activity in the lipase assay were inoculated in 5 ml YPM. DNA was isolated and transformed into the  E. coli  strain DH10B, thus rescuing the plasmid (as also described in WO 00/24883). Two pyrG variants were identified: 
     1) F96S; the plasmid was denoted pENI2343, and 
     2) T102N; the plasmid was denoted pENI2344. 
     Approx. 2 microgram of each of the plasmids pENI2155, pENI2343 and pENI2344 were transformed into an  Aspergillus oryzae  pyrG-minus mutant denoted Ja1355, and an  Aspergillus niger  pyrG-minus mutant denoted Mbin115, using standard procedures. 
     The transformed protoplasts were spread on selection plates (2% maltose 10 mM NaNO 3 , 1.2 M sorbitol, 2% bacto agar, salt solution. After 4 days of growth, very poor sporulation was seen for the pENI2343 Ja1355 transformants, and no transformants were seen for MBIN115 transformed with pENI2343. 
     6 independent transformants of each plasmid transformation were inoculated into 200 microliter 1*vogel, 2% maltose in a 96-well microtiter plate. After 4 days growth at 34° C., the culture broths were assayed for lipase activity. The results are given in the table below as relative lipase units with relative standard deviation, and are averages of the activity of the independent clones. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Jal355 
                 Mbin115 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 pENI2155 (wt) 
                 48 ± 8% 
                  7 ± 14% 
               
               
                   
                 pENI2343 (F96S) 
                 49 ± 15% 
                 No growth 
               
               
                   
                 pENI2344 (T102N) 
                 71 ± 13% 
                 80 ± 11% 
               
               
                   
                   
               
            
           
         
       
     
     The expression of lipase from the pENI2343 transformants was very high compared to the fungal biomass in the wells, which was very poor (less than 1/10 of the other transformants). An approx. 1.5-fold increase in lipase expression level is seen for the Ja1355 transformants, and an approx. 11-fold increase is seen in the Mbin115 transformants, when comparing the pENI2155 transformants with the pENI2344 transformants. 
     Thus the pyrG T102N mutation leads to an increase in lipase expression, likely due to an increased plasmid copy number, which is selected for because of the unstable, less active OMP decarboxylase encoded by the selection gene pyrG. 
     Example 3 
     In order to evaluate plasmid stability, a screen was set up to evaluate the percentage of spores containing a stably episomaly replicated plasmid (comprising a pyrG selection gene). 
     Two DNA libraries were constructed, the first library was cloned into a plasmid comprising the wildtype pyrG gene as selection gene, whereas the second library was cloned into a plasmid comprising a mutated pyrG gene which comprised a mutated Kozak region as described in Examplel and a T102N mutation as described in Example 2. 
     A spore suspension was made from each library and plated on to growth plates (2% maltose 10 mM NaNO 3 , 1.2 M sorbitol, 2% bacto agar, salts, with or without 20 mM uridine). The plates were grown for 3 days at 37° C. Results are shown in the table below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Selection gene 
                 −uridine 
                 +uridine 
                 % viable spores 
               
               
                   
               
             
            
               
                 Wildtype pyrG 
                 11 
                 83 
                 13 
               
               
                 Mutant (Kozak/T102N) pyrG 
                 36 
                 63 
                 57 
               
               
                   
               
            
           
         
       
     
     Evidently a much larger fraction of the spores contain a plasmid, when using the mutated (Kozak/T102N) pyrG gene.