Abstract:
The inventors have isolated novel lipolytic enzyme genes with a high homology to the  T. lanuginosus  lipase gene and thus well suited for use in gene shuffling. Accordingly, the invention provides a method of generating genetic diversity into lipolytic enzymes by family shuffling of two or more homologous genes which encode lipolytic enzymes. The DNA shuffling technique is used to create a library of shuffled genes, and this is expressed in a suitable expression system and the expressed proteins are screened for lipolytic enzyme activity. The invention also provides a polynucleotide comprising a nucleotide sequence encoding a lipolytic enzyme and a lipolytic enzyme (a polypeptide with lipolytic enzyme activity).

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 10/250,824 filed Jul. 2, 2003, which is a 35 U.S.C. 371 national application of PCT/DK02/00124 filed 25 Feb. 2002, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2001 00304 filed 23 Feb. 2001 and U.S. provisional application No. 60/271,385 filed 26 Feb. 2001, the contents of which are fully incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a method of generating diversity into lipolytic enzymes by the use of the so-called family shuffling of homologous genes. The invention also relates to polynucleotides for use in the method, and to lipolytic enzymes encoded by the polynucleotides. 
     BACKGROUND OF THE INVENTION 
     The lipase of  Thermomyces lanuginosus  (also known as  Humicola lanuginosa ) is known to be useful for various industrial purposes such as detergents and baking (EP 258068, WO 9404035). Its amino acid and DNA sequences are shown in U.S. Pat. No. 5,869,438. 
     The prior art describes the modification of the amino acid sequence of the  T. lanuginosus  lipase to create variants with the aim of modifying the enzyme properties. Thus, U.S. Pat. No. 5,869,438, WO 95/22615, WO 97/04079 and WO 00/32758 disclose the use of mutagenesis of the lipase gene to produce such variants. WO 00/32758 also discloses the construction of variants with the backbone from  T. lanuginosus  lipase and C-terminal from  Fusarium oxysporum  phospholipase by PCR reaction. 
     Crameri et al., Nature, 391: 288-291 (1998) discloses DNA shuffling of a family of naturally occurring homologous genes from diverse species to create diversity into proteins. U.S. Pat. No. 6,159,687 discloses shuffling of genes encoding variants of the  T. lanuginosus  lipase. WO 98/41623 discloses shuffling of heterologous polynucleotide sequences. 
     The following published sequences of lipolytic enzymes from  Aspergillus  have amino acid identities of 49-51% to the  T. lanuginosus  lipase: Lysophospholipase from  A. foetidus  (EMBL A93428, U.S. Pat. No. 6,140,094), lipase from  A. tubingensis  (EMBL A84589, WO 9845453), phospholipase A1 from  A. oryzae  (EMBL E16314, EP 575133, JP 10155493 A) and Lysophospholipase from  A. niger  (EMBL A90761, WO 98/31790). 
     R. Lattmann et al., Biocatalysis, 3 (1-2): 137-144 (1990) disclose an esterase from  Talaromyces thermophilus.  V. W. Ogundero, Mycologia, 72 (1): 118-126 (1980) describes the lipase activity of  Talaromyces thermophilus . U.S. Pat. No. 4,275,011 and EP 258068 refer to a lipase from  Thermomyces ibadanensis . B. A. Oso, Canadian Journal of Botany, 56: 1840-1843 (1978) describes the lipase activity of  Talaromyces emersonii.    
     SUMMARY OF THE INVENTION 
     The inventors have isolated novel lipolytic enzyme genes with a high homology to the  T. lanuginosus  lipase gene and are thus well suited for use in gene shuffling. The novel genes are shown as SEQ ID NO: 3, 5, 7, 9 and 11. Identity tables for some protein and DNA sequences are shown below. The novel sequences are identified as follows:
         Talthe1M: SEQ ID NO: 3 and 4 from  Talaromyces thermophilus.      Theiba1M: SEQ ID NO: 5 and 6 from  Thermomyces ibadanensis.      Taleme1M: SEQ ID NO: 7 and 8 from  Talaromyces emersonii.      Talbys1M: SEQ ID NO: 9 and 10 from  Talaromyces byssochlamydoides.          

     The following known sequences are included for comparison:
         Thelan1M: Lipase from  Thermomyces lanuginosus , SEQ ID NO: 1 and 2.   Asptub2M: EMBL A84589 Lipase from  Aspergillus tubingensis.      Aspory3M: EMBL E16314 Phospholipase A1 from  Aspergilus oryzae.      Aspnig2M: EMBL A90761 Lysophospholipase from  Aspergillus niger.          

     The following is an identity table of the mature proteins: 
     
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Thelan1 
                 Talthe1 
                 Theiba1 
                 Taleme1 
                 Talbys1 
                 Asptub2 
                 Aspory3 
                 Aspnig2 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Thelan1M 
                 100.0 
                 88.1 
                 78.1 
                 61.9 
                 57.4 
                 50.6 
                 50.4 
                 49.1 
               
               
                 Talthe1M 
                 88.1 
                 100.0 
                 78.8 
                 61.5 
                 59.2 
                 48.7 
                 47.8 
                 48.0 
               
               
                 Theiba1M 
                 78.1 
                 78.8 
                 100.0 
                 61.8 
                 58.0 
                 49.4 
                 50.4 
                 48.0 
               
               
                 Taleme1M 
                 61.9 
                 61.5 
                 61.8 
                 100.0 
                 83.1 
                 54.8 
                 56.1 
                 53.7 
               
               
                 Talbys1M 
                 57.4 
                 59.2 
                 58.0 
                 83.1 
                 100.0 
                 50.9 
                 54.9 
                 49.1 
               
               
                 Asptub2M 
                 50.6 
                 48.7 
                 49.4 
                 54.8 
                 50.9 
                 100.0 
                 55.9 
                 93.7 
               
               
                 Aspory3M 
                 50.4 
                 47.8 
                 50.4 
                 56.1 
                 54.9 
                 55.9 
                 100.0 
                 53.7 
               
               
                 Aspnig2M 
                 49.1 
                 48.0 
                 48.0 
                 53.7 
                 49.1 
                 93.7 
                 53.7 
                 100.0 
               
               
                   
               
             
          
         
       
     
     The following is an identity table of DNA sequences coding for the mature proteins (stop codons omitted): 
     
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Thelan1 
                 Talthe1 
                 Theiba1 
                 Taleme1 
                 Talbys1 
                 Asptub2 
                 Aspory3 
                 Aspnig2 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Thelan1M 
                 100.0 
                 86.0 
                 79.3 
                 62.0 
                 58.4 
                 57.0 
                 55.6 
                 56.2 
               
               
                 Talthe1M 
                 86.0 
                 100.0 
                 79.1 
                 62.6 
                 60.0 
                 57.8 
                 55.7 
                 57.1 
               
               
                 Theiba1M 
                 79.3 
                 79.1 
                 100.0 
                 63.5 
                 60.4 
                 56.6 
                 57.8 
                 55.6 
               
               
                 Taleme1M 
                 62.0 
                 62.6 
                 63.5 
                 100.0 
                 84.1 
                 58.2 
                 58.4 
                 58.7 
               
               
                 Talbys1M 
                 58.4 
                 60.0 
                 60.4 
                 84.1 
                 100.0 
                 57.5 
                 56.5 
                 56.8 
               
               
                 Asptub2M 
                 57.0 
                 57.8 
                 56.6 
                 58.2 
                 57.5 
                 100.0 
                 58.7 
                 91.7 
               
               
                 Aspory3M 
                 55.6 
                 55.7 
                 57.8 
                 58.4 
                 56.5 
                 58.7 
                 100.0 
                 56.5 
               
               
                 Aspnig2M 
                 56.2 
                 57.1 
                 55.6 
                 58.7 
                 56.8 
                 91.7 
                 56.5 
                 100.0 
               
               
                   
               
             
          
         
       
     
     Accordingly, the invention provides a method of generating genetic diversity into lipolytic enzymes by family shuffling of two or more homologous genes which encode lipolytic enzymes. One gene encodes a lipolytic enzyme with at least 90% identity to the  T. lanuginosus  lipase, and another gene encodes a lipolytic enzyme with 55-90% identity to the  T. lanuginosus  lipase. The DNA shuffling technique is used to create a library of chimeric shuffled genes, and this is expressed in a suitable expression system and the expressed proteins are screened for lipolytic enzyme activity. The expressed proteins may further be screened to identify lipolytic enzymes with improved properties. 
     The invention also provides a polynucleotide comprising a nucleotide sequence encoding a lipolytic enzyme and a lipolytic enzyme (a polypeptide with lipolytic enzyme activity). 
     The polynucleotide may be a DNA sequence cloned into a plasmid present in  E. coli  deposit number DSM 14047, 14048, 14049, or 14051, the DNA sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9 or one that can be derived therefrom by substitution, deletion, and/or insertion of one or more nucleotides. The polynucleotide may have at least 90% identity with the DNA sequence encoding a mature peptide shown in SEQ ID NO: 3, at least 80% identity with the DNA sequence encoding a mature peptide shown in SEQ ID NO: 5, at least 65% identity with the DNA sequence encoding a mature peptide shown in SEQ ID NO: 7, or at least 60% identity with the DNA sequence encoding a mature peptide shown in SEQ ID NO: 9. It may also be an allelic variant of the DNA sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9; or it may hybridize under high stringency conditions with a complementary strand of the nucleic acid sequence encoding a mature peptide shown in SEQ ID NO: 3, 5, 7 or 9, or a subsequence thereof having at least 100 nucleotides. 
     The lipolytic enzyme may be encoded by a DNA sequence cloned into a plasmid present in  E. coli  deposit number DSM 14047 or 14049, or may have an amino acid sequence which is the mature peptide of SEQ ID NO: 6 or 10, or one that can be derived therefrom by substitution, deletion, and/or insertion of one or more amino acids. The lipolytic enzyme may have an amino acid sequence which has at least 80% identity with the mature peptide of SEQ ID NO: 6 or at least 60% identity with the mature peptide of SEQ ID NO: 10. The lipolytic enzyme may further be immunologically reactive with an antibody raised against the mature peptide of SEQ ID NO: 6 or 10 in purified form, be an allelic variant of the mature peptide of SEQ ID NO: 6 or 10; or be encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the nucleic acid sequence encoding a mature peptide shown in SEQ ID NO: 5 or 9, or a subsequence thereof having at least 100 nucleotides. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  shows a PCR scheme used in Example 7. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Genomic DNA Source 
     Lipolytic enzyme genes of the invention may be derived from strains of  Talaromyces  or  Thermomyces , particularly  Talaromyces thermophilus, Thermomyces ibadanensis, Talaromyces emersonii  or  Talaromyces byssochlamydoides , using probes designed on the basis of the DNA sequences in this specification. 
     Thus, genes and polypeptides shown in the sequence listing were isolated from the organisms indicated below. Strains of  Escherichia coli  containing the genes were deposited by the inventors under the terms of the Budapest Treaty with the DSMZ—Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE as follows: 
     
       
         
               
               
               
               
             
           
               
                   
               
               
                   
                 Gene and 
                   
                   
               
               
                   
                 polypeptide 
                 Clone deposit 
                 Clone deposit 
               
               
                 Source organism 
                 sequences 
                 No. 
                 date 
               
               
                   
               
             
             
               
                   Talaromyces thermophilus  ATCC 
                 SEQ ID NOS: 3 
                 DSM 14051 
                 8 Feb. 2001 
               
               
                 10518 
                 and 4 
               
               
                   Thermomyces ibadanensis  CBS 
                 SEQ ID NOS: 5 
                 DSM 14049 
                 8 Feb. 2001 
               
               
                 281.67 = ATCC 22716 
                 and 6 
               
               
                   Talaromyces emersonii  UAMH 5005 = NRRL 
                 SEQ ID NOS: 7 
                 DSM 14048 
                 8 Feb. 2001 
               
               
                 3221 = ATCC 16479 = IMI 
                 and 8 
               
               
                 116815 = CBS 393.64 
               
               
                 
                   Talaromyces byssochlamydoides 
                 
                 SEQ ID NOS: 9 
                 DSM 14047 
                 8 Feb. 2001 
               
               
                 CBS 413.71 = IMI 178524 = NRRL 
                 and 10 
               
               
                 3658 
               
               
                   
               
             
          
         
       
     
     The above source organisms are freely available on commercial terms from the following strain collections: 
     ATCC (American Type Culture Collection), 10801 University Boulevard, Manassas, Va. 20110-2209, USA. 
     CBS (Centraalbureau voor Schimmelcultures), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. 
     UAMH (University of Alberta Mold Herbarium &amp; Culture Collection), Devonian Botanic Garden, Edmonton, Alberta, Canada T6G 3GI. 
     IMI: International Mycological Institute, Bakeham Lane, Englefield Green, EGHAM, Surrey TW20 9TY, United Kingdom. 
     Polynucleotides 
     The polynucleotides to be used for recombination (shuffling) are two or more genes encoding lipolytic enzymes, including one with at least 90% identity and one with 55-90% identity to the  T. lanuginosus  lipase (SEQ ID NO: 2). The poloynucleotides differ in at least one nucleotide. 
     The starting material may include the mature part of two or more (e.g., three, four or five) of SEQ ID NO: 1, 3, 5, 7 and/or 9. It may also include genes encoding two or more (e.g., three, four or five) of variants of SEQ ID NO: 2, 4, 6, 8 or 10 obtained by deleting, substituting and/or inserting one or more amino acids and/or by attaching a peptide extension at the N- and/or C- terminal C-terminal. Examples of variants of the  T. lanuginosus  lipase are described, e.g., in U.S. Pat. No. 5,869,438, WO 9522615, WO 9704079 and WO 0032758, and similar variants can be made by altering corresponding amino acids in the other sequences. 
     Any introns present in the genes may optionally be removed before the shuffling. 
     DNA Recombination (Shuffling) 
     Shuffling between two or more homologous input polynucleotides (starting-point polynucleotides) may involve fragmenting the polynucleotides and recombining the fragments, to obtain output polynucleotides (i.e., polynucleotides that have been subjected to a shuffling cycle) wherein a number of nucleotide fragments are exchanged in comparison to the input polynucleotides. 
     DNA recombination or shuffling may be a (partially) random process in which a library of chimeric genes is generated from two or more starting genes. A number of known formats can be used to carry out this shuffling or recombination process. 
     The process may involve random fragmentation of parental DNA followed by reassembly by PCR to new full length genes, e.g., as presented in U.S. Pat. No. 5,605,793, U.S. Pat. No. 5,811,238, U.S. Pat. No. 5,830,721, U.S. Pat. No. 6,117,679. In-vitro recombination of genes may be carried out, e.g., as described in U.S. Pat. No. 6,159,687, WO 98/41623, U.S. Pat. No. 6,159,688, U.S. Pat. No. 5,965,408, U.S. Pat. No. 6,153,510. The recombination process may take place in vivo in a living cell, e.g., as described in WO 97/07205 and WO 98/28416. 
     The parental DNA may be fragmented by DNA&#39;se I treatment or by restriction endonuclease digests as described by Kikuchi et al. (Gene 236:159-167 (2000)). Shuffling of two parents may be done by shuffling single stranded parental DNA of the two parents as described in Kikuchi et al. (Gene 243:133-137 (2000)). 
     A particular method of shuffling is to follow the methods described in Crameri et al., Nature, 391: 288-291 (1998) and Ness et al., Nature Biotechnology 17: 893-896. Another format would be the methods described in U.S. Pat. No. 6,159,687: Examples 1 and 2. 
     Properties of Lipolytic Enzyme 
     The lipolytic enzyme obtained by the invention is able to hydrolyze carboxylic ester bonds and is classified as EC 3.1.1 according to Enzyme Nomenclature 1992, Academic Press, Inc. It may particularly have activity as a lipase (triacylglycerol lipase) (EC 3.1.1.3), phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), cholesterol esterase (EC 3.1.1.13) and/or galactolipase (EC 3.1.1.26). 
     The thermostability was evaluated by means of Differential Scanning Calorimetry (DSC). The denaturation peak (T d ) when heated at 90 deg/hr at pH 5 is slightly above 75° C. for the lipolytic enzyme from  T. ibadanensis , compared to slightly above 70° C. for the prior-art  T. lanuginosus  lipase. The lipolytic enzyme from  T. ibadanensis  has optimum activity at alkaline pH (similar to the  T. lanuginosus  lipase) and has an isoelectric point of about 4.3 (slightly lower than the  T. lanuginosus  lipase). 
     Homology and Alignment 
     The best alignment of the mature parts of SEQ ID NOS: 2, 4, 6, 8 and 10 is achieved by inserting a gap of one amino acid between Q249 and P/G250 of SEQ ID NOS: 2, 4 and 6. This alignment defines corresponding amino acids. 
     The degree of homology may be 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 and Wunsch, 1970, Journal of Molecular Biology, 48: 443-45), using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. 
     The determination of homology may also be made using Align from the fasta package version v20u6. Align is a Needleman-Wunsch alignment (i.e., global alignment), useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is −12 for proteins and −16 for DNA. While the penalty for additional residues in a gap is −2 for proteins and −4 for DNA. 
     The homologies discussed in this specification may correspond to at least 60% identity, in particular to at least 70% or at least 80% identity, e.g., at least 90% or at least 95% identity. 
     Use of Lipolytic Enzyme 
     Depending on the substrate specificity, the enzyme of the invention can be used, e.g., in filtration improvement, vegetable oil treatment, baking, detergents, or preparation of lysophospholipid. Thus, it may be used in known applications of lipolytic enzymes by analogy with the prior art, e.g.:
         In the pulp and paper industry, to remove pitch or to remove ink from used paper. WO 92/13130, WO 92/07138, JP 2160984 A, EP 374700.   Baking. WO 94/04035, WO 00/32758.   Detergents. WO 97/04079, WO 97/07202, WO 97/41212, WO 98/08939 and WO 97/43375.   Leather industry. GB 2233665, EP 505920.   An enzyme with lipase activity may be used for fat hydrolysis and for modification of triglycerides and for production of mono- and diglycerides.   An enzyme with lipase activity may be used for interesterification of bulk fats, production of frying fats, shortenings and margarine components.   An enzyme with phospholipase activity (A1, A2) may be used for degumming of vegetable oils and for lysophospholipid production.
 
Improvement of Filtration
       

     An enzyme with lysophospholipase activity can be used to improve the filterability of an aqueous solution or slurry of carbohydrate origin by treating it with the variant. This is particularly applicable to a solution or slurry containing a starch hydrolyzate, especially a wheat starch hydrolyzate since this tends to be difficult to filter and to give cloudy filtrates. The treatment can be done in analogy with EP 219,269 (CPC International). 
     Detergents 
     The lipolytic enzyme produced by the invention may be used as a detergent additive, e.g., at a concentration (expressed as pure enzyme protein) of 0.001-10 (e.g., 0.01-1) mg per gram of detergent or 0.001-100 (e.g., 0.01-10) mg per liter of wash liquor. 
     The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations. In a laundry detergent, the variant may be effective for the removal of fatty stains, for whiteness maintenance and for dingy cleanup. A laundry detergent composition may be formulated as described in WO 97/04079, WO 97/07202, WO 97/41212, PCT/DK98/08939 and WO 97/43375. 
     The detergent composition of the invention may particularly be formulated for hand or machine dishwashing operations. e.g., as described in GB 2,247,025 (Unilever) or WO 99/01531 (Procter &amp; Gamble). In a dishwashing composition, the variant may be effective for removal of greasy/oily stains, for prevention of the staining/discoloration of the dishware and plastic components of the dishwasher by highly colored components and the avoidance of lime soap deposits on the dishware. 
     The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous. 
     The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight, e.g., 0.5-40%, such as 1-30%, typically 1.5-20%. 
     Dough and Baked Products 
     The lipolytic enzyme can be used in the preparation of dough and baked products made from dough, such as bread and cakes, e.g., to increase dough stability and dough handling properties, or to improve the elasticity of the bread or cake. Thus, it can be used in a process for making bread, comprising adding it to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with U.S. Pat. No. 4,567,046 (Kyowa Hakko), JP-A 60-78529 (QP Corp.), JP-A 62-111629 (QP Corp.), JP-A 63-258528 (QP Corp.) or EP 426211 (Unilever). The lipolytic enzyme may be used together with an anti-staling amylase, particularly an endo-amylase such as a maltogenic amylase in analogy with WO 99/53769 (Novo Nordisk). Thus, the lipolytic enzyme may be incorporated in a flour composition such as a dough or a premix for dough. 
     MATERIALS AND METHODS 
     Strains and Plasmids: 
     Plasmid pMT2188 
     The  Aspergillus oryzae  expression plasmid pCaHj483 (WO 98/00529) 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 terminator (Tamg). Also present on the plasmid 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 this plasmid 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: 35) and 142780 (SEQ ID NO: 36). 
     Primer 142780 introduces a BbuI site in the PCR fragment. The Expand PCR system (Roche Molecular Biochemicals, Basel, Switzerland) 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: 37) and 142778 (SEQ ID NO: 38). 
     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., Gene, 77:61-68 (1989)). 
     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 pyrF  E. coli  strain DB6507 (ATCC 35673) made competent by the method of Mandel and Higa (Mandel and Higa, J. Mol. Biol. 45: 154 (1970)). Transformants were selected on solid M9 medium (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press (1989)) supplemented with 1 g/l casaminoacids, 500 micrograms/l thiamine and 10 mg/l kanamycin. 
     A plasmid from a selected transformant was termed pCaHj527. The Pna2/tpi promoter present on pCaHj527 was subjected to site directed mutagenesis by a simple PCR approach. 
     Nucleotides 134-144 were altered from SEQ ID NO: 39 to SEQ ID NO: 40 using the mutagenic primer 141223 (SEQ ID NO: 41). 
     Nucleotides 423-436 were altered from SEQ ID NO: 42 to SEQ ID NO: 43 using the mutagenic primer 141222 (SEQ ID NO: 44). 
     The resulting plasmid was termed pMT2188. 
     Plasmid pENI1861 
     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 (app. 620 bp) was made using pMT2188 (see above) as template and the primers 051199J1 (SEQ ID 45) and 1298TAKA (SEQ ID NO: 46). 
     The fragment was cut BssHII and BgI II, and cloned into pENI1849 which was also cut with BssHII and BgI II. The cloning was verified by sequencing. Plasmid pENI1902 was made in order to have a promoter that works in both  E. coli  and  Aspergillus . This was done by unique site elimination using the “Chameleon double stranded site-directed mutagenesis kit” as recommended by Stratagene®. 
     Plasmid pENI1861 
     Plasmid pENI1861 was used as template and the following primers with 5′ phosphorylation were used as selection primers: 177996 (SEQ ID NO: 47), 135640 (SEQ ID NO: 48) and 135638 (SEQ ID NO: 49). 
     The 080399J19 primer (SEQ ID NO: 50) with 5′ phosphorylation was used as mutagenic primer to introduce a −35 and −10 promoter consensus sequence (from  E. coli ) in the  Aspergillus  expression promoter. Introduction of the mutations was verified by sequencing. 
     Plasmid pENI1960 
     Plasmid pENI1960 was made using the Gateway Vector™ conversion system (Lifetechnology® cat no. 11828-019) by cutting pENI1902 with BamHI, filling the DNA ends using Klenow fragment polymerase and nucleotides (thus making blunt ends) followed by ligation to reading frame A Gateway™ PCR fragment. The cloning in the correct orientation was confirmed by sequencing. 
     Media and Substrates 
     YPG: 4 g/L Yeast extract, 1 g/L KH 2 PO 4 , 0.5 g/L MgSO 4 -7aq, 5 g/L Glucose, pH 6.0. 
     EXAMPLES 
     Example 1  
     Plasmids Harboring Lipolytic Enzyme Genes 
     Genomic DNA Preparation 
     Strains of  Thermomyces ibadanensis, Talaromyces emersonii, Talaromyces byssochlamydoides , and  Talaromyces thermophilus  were used as a genomic DNA supplier. Each strain was cultivated in 100 ml of YPG at appropriate temperature for several days. Mycelia was harvested and ground in liquid N 2 . It was suspended with 2 ml of 50 mM Tris-HCl (pH8.0) buffer including 100 mM NaCl, 25 mM EDTA, and 1% SDS and then 12 microliters of proteinase K (25 mg/ml) was added. The suspension was incubated at 65° C. for 30-60 min. Phenol extraction was done to remove proteins and DNA was precipitated by 0.7 volume of isopropanol. The precipitate was dissolved with sterilized water and RNase was added. After Phenol/isoamylalcohol extraction, DNA was precipitated by EtOH. 
     PCR Screening of Lipolytic Enzyme Genes 
     PCR reactions on each genomic DNA were done with HL2 and HL12 (SEQ ID NOS: 51 and 52) or HL2 and HL6 (SEQ ID NOS: 51 and 53) designed based upon alignment lipases. 
     Reaction components (2.6 ng/microliter of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.1 U/microliter of Taq polymerase in 1× buffer (Roche Diagnostics, Japan)) were mixed and submitted for PCR under the following conditions. 
     
       
         
               
               
               
             
           
               
                   
               
               
                 Step 
                 Temperature 
                 Time 
               
               
                   
               
             
             
               
                 1 
                 94° C. 
                  1 min 
               
               
                 3 
                 50° C. 
                  1 min 
               
               
                 4 
                 72° C. 
                  2 min 
               
               
                 5 
                 72° C. 
                 10 min 
               
               
                 6 
                  4° C. 
                 forever 
               
               
                   
               
             
          
         
       
     
     Steps 1 to 3 were repeated 30 times. 
     540 bp of fragment and 380 bp of fragment were amplified from primer sets of HL2/HL12 and HL2/HL6, respectively. They were gel-purified with GFX™ PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech). Each DNA was sequenced and compared to the lipase, showing that a clone encodes the internal part of the lipase. 
     Cloning of Lipase Genes 
     All lipase genes were cloned using LA PCR™ in vitro Cloning Kit (TaKaRa) according to the manufacturer&#39;s instructions. Thus, genomic DNA was cut with various restriction enzymes and each DNA was ligated with the appropriate cassette of the kit. Each ligation solution was applied to PCR with the primers of the one designed from internal sequence and a cassette primer of the kit. Amplified DAN fragment was sequenced. This step was repeated till ORF was determined. 
     The fidelity of LA- taq polymerase of the kit is not good so in order to get the right sequence whole gene was amplified by Expand high fidelity polymerase according to the manufacturer&#39;s instructions. 
     Amplified DNA fragment was gel-purified with GFX™ PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech) and ligated into a pT7Blue vector or pST BLue-1 AccepTor vector (Novagen) with ligation high (TOYOBO, Japan). The ligation mixtures were transformed into  E. coli  JM109 or DH5α. The sequence of four plasmids of each gene was determined and their sequences were compared. The sequence of majority is defined as the right nucleotide sequence. 
     Example 2  
     Cloning of Lipase into  Aspergillus  Expression Vector 
     3 different PCR reaction were run using PWO polymerase in the following reaction 94° C. 5 min, 30* (94° C. 30 sec., 50° C. 30 sec, 72° C. 2 min, 72° C. 5 min). In each case, the template was a plasmid harboring a lipolytic enzyme gene prepared as in Example 1, and the following primers were used: 
     A: Plasmid with gene from  Talaromyces thermophilus  and oligo 051200j1/051200j8 (SEQ ID NOS: 11 and 18). 
     B: Plasmid with gene from  Talaromyces emersonii  and oligo 051200j9/051200j16 (SEQ ID NOS: 19 and 26). 
     C: Plasmid with gene from  Thermomyces Ibadanensis  and oligo 051200j17/051200j24 (SEQ ID NOS: 27 and 34). 
     The PCR fragments were run and purified from a 1% agarose gel and cloned into pENI1960 (see above) using Gateway cloning as recommended by the supplier (Life Technologies) and transformed into  E. coli  DH10b (Life Technologies, Gaithersburg, Md.) and sequenced, thus creating pENI 2146 ( Talaromyces emersonii  lipase gene), pEN12147 ( Thernomyces Ibadanensis  lipase gene) and pENI2148 ( Talaromyces thermophilus  lipase gene). 
     These were transformed into JaI250 (described in WO 00/39322) and lipase activity identified as mentioned in WO 00/24883. 
     Example 3 
     Construction of Intron-Less Lipase Genes 
     Removal of Introns from  Talaromyces thermophilus  Lipase Gene 
     4 PCR reactions were run using PWO polymerase and pENI2148 as template (94° C. 5 min, 30* (94° C. 30 sec., 50° C. 30 sec, 72° C. 1 min), 72° C. 5 min) and the following oligos: 
     1: 051200j1 and 051200j3 (SEQ ID NOS: 11 and 13) 
     2: 051200j2 and 051200j5 (SEQ ID NOS: 12 and 15) 
     3: 051200j4 and 051200j7 (SEQ ID NOS: 14 and 17) 
     4: 051200j6 and 051200j8 (SEQ ID NOS: 16 and 18) 
     The specific bands were run and purified from a 1.5% agarose gel. Equal amounts of PCR fragments were mixed along with PWO polymerase, buffer, dNTP, oligo 051200j1 and 051200j8 (SEQ ID NO: 11 and 18, total of 50 microliters, as recommended by the supplier Boehringer Mannheim) and a second PCR was run (94° C. 5 min, 30* (94° C. 30 sec., 50° C. 30 sec, 72° C. 2 min), 72° C. 5 min). 
     The correct band size was checked on a 1.5% agarose gel (app. 900 bp) and the rest of the PCR-fragment was purified using Biorad spin columns (cat no.732-6225) 
     The PCR-fragment was cloned into pENI1960 cut with ScaI (in order to cleave in the ccdB gene) using Gateway cloning as recommended by the supplier (Life Technologies) and transformed into  E. coli  DH10b and sequenced, thus creating intron-less  Talaromyces thermophilus  lipase gene. 
     Removal of Introns from  Talaromyces emersonii  LiPase Gene 
     4 PCR reactions were run using PWO polymerase and pENI2146 as template (94° C. 5 min, 30* (94° C. 30 sec., 50° C. 30 sec, 72° C. 1 min), 72° C. 5 min) 
     1: 051200j9 and 051200j11 (SEQ ID NOS: 19 and 21). 
     2: 051200j10 and 051200j13 (SEQ ID NOS: 20 and 23). 
     3: 051200j12 and 051200j15 (SEQ ID NOS: 22 and 25). 
     4: 051200j14 and 051200j16 (SEQ ID NOS: 24 and 26). 
     The specific bands were run and purified from a 1.5% agarose gel. Equal amounts of PCR fragments were mixed along with PWO polymerase, buffer, dNTP, oligo 051200j9 and 051200j16 (SEQ ID NOS: 19 and 26, total of 50 microliters, as recommended by the supplier) and a second PCR was run (94° C. 5 min, 30* (94° C. 30 sec., 50° C. 30 sec, 72° C. 5 min). 
     The correct band size was checked on a 1.5% agarose gel (app. 900 bp) and the rest of the PCR-fragment was purified using Biorad spin columns. 
     The PCR-fragment was cloned into and cloned into pENI1960 cut ScaI using Gateway cloning as recommended by the supplier (Life Technologies) and transformed into  E. coli  DH10b and sequenced, thus creating an intron-less  Talaromyces emersonii  lipase gene. 
     Removal of Introns from  Thermomyces Ibadanensis  Lipase Gene 
     4 PCR reactions were run using PWO polymerase and pENI2147 as template (94° C. 5 min, 30* (94° C. 30 sec., 50° C. 30 sec, 72° C. 1 min), 72° C. 5 min) 
     1: 051200j17 and 051200j19 (SEQ ID NOS: 27 and 29). 
     2: 051200j18 and 051200j21 (SEQ ID NOS: 28 and 31). 
     3: 051200j20 and 051200j23 (SEQ ID NOS: 30 and 33). 
     4: 051200j22 and 051200j24 (SEQ ID NOS: 32 and 34). 
     The specific bands were run and purified from a 1.5% agarose gel. Equal amounts of PCR fragments were mixed along with PWO polymerase, buffer, dNTP, oligo 051200j17 and 051200j24 (SEQ ID NO: 27 and 34, total of 50 microliters, as recommended by the supplier) and a second PCR was run (94° C. 5 min, 30* (94° C. 30 sec., 50° C. 30 sec, 72°min). 
     The correct band size was checked on a 1.5% agarose gel (app. 900 bp) and the rest of the PCR-fragment was purified using Biorad spin columns 
     The PCR-fragment was cloned into and cloned into pENI1960 cut ScaI using Gateway cloning as recommended by supplier (life technologies) and transformed into  E. coli  DH10b and sequenced, thus creating intron-less  Thermomyces Ibadanensis  lipase gene. 
     Example 4 
     Shuffling of Lipolytic Enzyme Genes 
     Plasmids containing DNA sequences encoding lipolytic enzymes are mixed in equimolar amounts. The following components where mixed in a microtube: 
     2 microliters plasmid mixture (0.15 microgram/microliter), specific primers flanking the gene (1 pmol/μ), 2 microliters 2.5 mM dNTP, 2.5 mM MgCl2, 2 μl 10* taq buffer (Perkin Elmer), 0.5 microliter taq enzyme in a total volume of 20 microliters. 
     The tube is set in a Perkin Elmer 2400 thermocycler. The following PCR-program is run:(94° C., 5 minutes) 1 cycle: 
     (94° C., 30 seconds, 70° C., 0 seconds) 99 cycles(72° C., 2 minutes, 4° 
     The PCR-reaction is run on a 1.5% agarose gel. A DNA-band of the specific expected size is cut out of the agarose gel and purified using JETsorb (from GENOMED Inc.). The purified PCR-product is cloned into a TA-vector (from Invitrogen (the original TA cloning kit). The ligated product is transformed into a standard  Escherichia coli  strain (DH5a). 
     The shuffled sequences can then be subcloned from the  E. coli  TA vector into the yeast vector pJSOO26 (WO 99/28448) as a BamHI-XbaI fragment (see WO 97/07205), and e.g., screened for new shuffled sequences with improved properties, e.g., improved performance in detergents (see WO 97/07205). 
     Example 5  
     Shuffling of Lipolytic Enzyme Genes 
     PCR products of lipolytic enzyme genes are generated as in the previous example and pooled in equimolar amounts. The following mixture is generated in a suitable tube: 
     1 microliter PCR mixture (0.1 microgram), decamer random primer (300 pmol), 2 microliters 10* Klenow buffer (Promega), 0.25 mM dNTP, 2.5 mM MgCl 2  in a total volume of 20 microliters. 
     The mixture is set in a PE2400 thermocycler where the following program is run: 96° C. 5 minutes, 25° C. 5 minutes, 0.5 ml Klenow enzyme is added, 25° C. 60 minutes, 35° C. 90 
     This procedure generates a high number of small DNA polymers originating from all parts of the gene. 
     10 microliters is taken out for test on agarose gel. 
     10 microliters PCR mixture (0.25 mM dNTP, 1 microliter 10* Taq buffer (Perkin Elmer), 2.5 mM MgCl2, 0.5 microliter Taq enzyme) is added to the 10 microliters in the tube in the thermocycler. Then the following standard PCR-program is run: (94° C., 5 minutes) 1 cycle, (94° C. 30 seconds, 45° C., 30 seconds, 72° C. 30 seconds) 25 cycles, 72° C indefinite. 
     The PCR products are run on a 1.5% agarose gel. A clear unbiased smear is seen. DNA between 400 and 800 bp is isolated from the gel. 
     Half of the purified PCR product is mixed in a tube with two specific primers (40 pmol) flanking the gene of interest, 0.25 mM dNTP, 2 microliters 10* Taq buffer, 2.5 mM MgCl 2 . Then the following standard PCR-program is run: (94° C., 5 minutes) 1 cycle, (94° C., 30 seconds, 50° C., 30 seconds, 72° C. 30 seconds) 25 cycles, 72° C. 7 minutes, 4° C. indefinite. 
     The PCR product is run on a 1.5% agarose gel. A band of the expected size is isolated. Additional PCR is run using specific primers (as mentioned above) in order to amplify the PCR-product before cloning. 
     The PCR-product and the desired vector are cut with the appropriate restriction enzymes (BamHI/XhoI). The vector and the PCR product are run on a 1.5% agarose gel, and purified from the gel. 
     The cut PCR-product and the cut vector are mixed in a ligase buffer with T4 DNA ligase (Promega). After overnight ligation at 16° C. the mixture is transformed into  E. coli  strain DH5a. 
     Example 6  
     Creation of Intron-Less Lipase Genes 
     A number of lipase genes with homology to the  Thermomyces lanuginosus  lipase gene were cloned. These genes were cloned as genomic DNA and were thus known to contain introns. 
     The intention was to shuffle these genes in order to obtain chimeric genes. In order to obtain the highest possible quality of library, the introns had to be removed. This was done by creating DNA oligos matching each flank of an exon as well as having a DNA sequence, which is homologous to the next neighbor exon. 
     These oligos were used in standard PCR (as known to a person skilled in the art), thus creating PCR fragments covering each and every exon (coding sequence) in the gene. These PCR fragments were purified from a 1% agarose gel. The PCR fragments were assembled into a full length gene, in a second PCR using the DNA oligos flanking the whole gene, as primers. 
     The PCR fragment containing the full length intron-less gene encoding the lipase was cloned into pENI 1960 as described in patent application PCT/DK02/00050. 
     The following primers were used to assemble each intron-less gene: 
       Talaromyces thermophilus:  051200j1, 051200J2, 051200J3, 051200J4, 051200J5, 051200J6, 051200J7 and 051200J8 (SEQ ID NO: 11-18), thus creating pENI2178, when cloned into pENI1960. 
       Talaromyces emersonii:  051200J9, 051200J10, 051200J11, 051200J12, 051200J13, 051200J14, 051200J15 and 051200J16 (SEQ ID NO: 19-26), thus creating pENI2159, when cloned into pENI1960. 
       Thermomyces ibadanensis:  051200J17, 051200J18, 051200J19, 051200J20, 051200J21, 051200J22, 051200J23 and 051200J24 (SEQ ID NO: 27-34), thus creating pENI2160, when cloned into pENI1960. 
       Talaromyces byssochlamydoides:  080201P1, 080201P2, 080201P3, 080201P4, 080201P5, 080201P6, 080201P7 and 080201P8 (SEQ ID NO: 54-61), thus creating pENI2230 when cloned into pENI1960. 
     Example 7  
     Shuffling of the Intron-Less Lipase Genes 
     A method using dUTP and uracil-DNA glycosylase was employed in order to make DNA fragments in sufficient quantities for DNA shuffling. The 3 genes  T. lanuginosus, T. thermophilus  and  T. ibadanensis  are quite homologous to each other (thus named Group A) as are  T. emersonii  and  T. byssochlamydoides  (named Group B). Thus in order to improve recombination between the two groups the following PCR scheme (see  FIG. 1 ) was employed, using the following templates: pENI2178, pENI2159, pENI2160, pENI2230, and the  T. lanuginosus  gene cloned into pENI1902 (cut BamHI and SacII) (patent application PCT/DK02/00050). 
     The following oligonucleotides are shown in  FIG. 1 : 1298-taka, 19670, 19672, 115120 and 050401P6 (SEQ ID NOS: 62-65 and 68). 050401P1 (SEQ ID NO: 66) hybridizes to 5′  T lanuginose  lipase gene. 030501P1 (SEQ ID NO: 67) hybridizes to 5″ of the other 4 lipase genes. 
     The final PCR fragment was cut first with BstEII and then with SfiI, as was the vector pENI2376. pENI2376 is a derivative of pENI1861(patent application PCT/DK02/00050) 
     The vector and PCR-fragment was purified from a 1% gel and ligated O/N. The ligated DNA pool was transformed into electro-competent  E. coli  DH10B, thus creating a library of app. 700.000 independent clones. 
     This library can be screened for activity towards various substrates such as Lecithin, DGDG, triglycerides such as tributyrine, olive oil, PNP-valerate or PNP-palmitate at different conditions such as high pH, low pH, high temperature, in presences of detergent, in the presence of ions or in the absence of ions. 
     This can be done in order to find, e.g., a thermo-stable lipase, a detergent phospholipase, a detergent lipase with first-wash performance, and no activity at neutral pH and so forth. 
     DNA-oligos: 
     
       
         
               
               
               
             
           
               
                 1298-taka: 
                   
                   
               
               
                 gcaagcgcgcgcaatacatggtgttttgatcat 
                 (SEQ ID NO:62) 
               
               
                   
               
               
                 19670: 
               
               
                 ccccatcctttaactatagcg 
                 (SEQ ID NO:63) 
               
               
                   
               
               
                 19672: 
               
               
                 ccacacttctcttccttcctc 
                 (SEQ ID NO:64) 
               
               
                   
               
               
                 115120: 
               
               
                 gctttgtgcagggtaaatc 
                 (SEQ ID NO:65) 
               
               
                   
               
               
                 050401P1: 
               
               
                 cggccgggccgcggaggccagggatccaccatgagg 
                 (SEQ ID NO:66) 
               
               
                 agctcccttgtgctg 
               
               
                   
               
               
                 030501P1: 
               
               
                 cggccgggccgcggaggccacaagtttgtacaaaaa 
                 (SEQ ID NO:67) 
               
               
                 agcagg 
               
               
                 (hybridizes to 5′ of the other 
               
               
                 4 lipase genes) 
               
               
                   
               
               
                 050401P6: 
               
               
                 cggccgggtcaccccccatcctttaactatagcg 
                 (SEQ ID NO:68) 
               
             
          
         
       
     
     Example 8  
     Characterization of Lipolytic Enzymes 
     Lipolytic enzymes from  Thermomyces ibadanensis  and  Talaromyces thermophilus  were prepared as described above, purified and used for characterization. 
     The specific lipase activity was determined by the LU method described in WO 00/32758, and the amount of enzyme protein was determined from the optical density at 280 nm. The specific activity was found to be 3181 LU/mg for the  Th. ibadanensis  lipase and 1000 LU/mg for the  Tal. thermophilus  lipase. 
     The pH-activity relation was found by determining the lipase by the LU method at pH 5, 6, 7, 8, 9 and 10. Both enzymes were found to have the highest lipase activity at pH 10. The  Th. ibadanensis  lipase showed a broad optimum with more than 50% of maximum activity in the pH range 6-10 whereas the  Tal. thermophilus  lipase showed a stronger activity drop at lower pH with less than 30% of maximum activity at pH 5-8. 
     The thermostability was determined by differential scanning calorimetry (DSC) at pH 5 (50 mM acetate buffer), pH 7 (50 mM HEPES buffer) and pH 10 (50 mM glycine buffer) with a scan rate of 90° C./hr. The temperature at the top of the denaturation peak (T d ) was found to be as follows: 
     
       
         
               
               
               
             
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 T d  (° C.) 
                   
               
             
          
           
               
                 pH 
                 
                   T. ibadanesis 
                 
                 
                   T. thermophilus 
                 
               
               
                   
               
             
          
           
               
                 5 
                 74* 
                 72* 
               
               
                 7 
                 72 
                 75 
               
               
                 10 
                 64 
                 69 
               
               
                   
               
             
          
         
       
     
     Example 9  
     Lysophospholipase Activity 
     Purified lipolytic enzymes from  T. ibadanensis  and  T. thermos  were tested by incubating with lysolecithin as substrate at pH 5 and 7, and the extent of reaction was followed by use of NEFA kit. 
     The results were that the enzyme from  T. ibadanensis  showed high lysophospholipase activity at pH 5 and some activity at pH 7. The enzyme from  T. thermos  showed a slight activity.