Patent Abstract:
The inventors have isolated novel genes with a high homology to the  T. lanuginosus  lipase gene and are 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 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).

Full Description:
FIELD OF THE INVENTION  
         [0001]    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  
         [0002]    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.  
           [0003]    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 9522615, WO 9704079 and WO 0032758 disclose the use of mutagenesis of the lipase gene to produce such variants. WO 0032758 also discloses the construction of variants with the backbone from  T. lanuginosus  lipase and C-terminal from  Fusarium oxysporum  phospholipase by PCR reaction.  
           [0004]    Crameri et al, 1998, Nature, 391: 288-291 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 9841623 discloses shuffling of heterologous polynucleotide sequences.  
           [0005]    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).  
           [0006]    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  
         [0007]    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:  
           [0008]    Talthe1M: SEQ ID NO: 3 and 4 from  Talaromyces thermophilus.    
           [0009]    Theiba1M: SEQ ID NO: 5 and 6 from  Thermomyces ibadanensis.    
           [0010]    Taleme1M: SEQ ID NO: 7 and 8 from  Talaromyces emersonii.    
           [0011]    Talbys1M: SEQ ID NO: 9 and 10 from  Talaromyces byssochiamydoides.    
           [0012]    The following known sequences are included for comparison:  
           [0013]    Thelan1M: Lipase from  Thermomyces lanuginosus , SEQ ID NO: 1 and 2.  
           [0014]    Asptub2M: EMBL A84589 Lipase from  Aspergillus tubingensis.    
           [0015]    Aspory3M: EMBL E16314 Phospholipase A1 from  Aspergillus oryzae.    
           [0016]    Aspnig2M: EMBL A90761 Lysophospholipase from  Aspergillus niger.    
           [0017]    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                  
 
           [0018]    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                  
 
           [0019]    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. Ianuginosus  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.  
           [0020]    The invention also provides a polynucleotide comprising a nucleotide sequence encoding a lipolytic enzyme and a lipolytic enzyme (a polypeptide with lipolytic enzyme activity).  
           [0021]    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.  
           [0022]    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  
       [0023]    [0023]FIG. 1 shows a PCR scheme used in Example 7. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
     Genomic DNA Source  
       [0024]    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.  
         [0025]    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 polypep-   Clone de-   Clone deposit            Source organism   tide sequences   posit No.   date                 Talaromyces thermophilus  ATCC   SEQ ID NO: 3 and   DSM 14051   8 Feb.       10518   4       2001         Thermomyces ibadanensis  CBS   SEQ ID NO: 5 and   DSM 14049   8 Feb.       281.67 = ATCC 22716   6       2001         Talaromyces emersonii  UAMH   SEQ ID NO: 7 and   DSM 14048   8 Feb.       5005 = NRRL 3221 = ATCC 16479 =   8       2001       IMI 116815 = CBS 393.64         Talaromyces byssochlamydoides     SEQ ID NO: 9 and   DSM 14047   8 Feb.       CBS 413.71 = IMI 178524 = NRRL   10       2001       3658                  
 
         [0026]    The above source organisms are freely available on commercial terms from the following strain collections:  
         [0027]    ATCC (American Type Culture Collection), 10801 University Boulevard, Manassas, Va. 20110-2209, USA.  
         [0028]    CBS (Centraalbureau voor Schimmelcultures), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.  
         [0029]    UAMH (University of Alberta Mold Herbarium &amp; Culture Collection), Devonian Botanic Garden, Edmonton, Alberta, Canada T6G 3GI.  
         [0030]    IMI: International Mycological Institute, Bakeham Lane, Englefield Green, EGHAM, Surrey TW20 9TY, United Kingdom.  
       Polynucleotides  
       [0031]    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.  
         [0032]    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. 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.  
         [0033]    Any introns present in the genes may optionally be removed before the shuffling.  
       DNA Recombination (Shuffling)  
       [0034]    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.  
         [0035]    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.  
         [0036]    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, WO98/41623, 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.  
         [0037]    The parental DNA may be fragmented by DNA&#39;se I treatment or by restriction endonuclease digests as descriobed by Kikuchi et al (2000a, Gene 236:159-167). Shuffling of two parents may be done by shuffling single stranded parental DNA of the two parents as described in Kikuchi et al (2000b, Gene 243:133-137).  
         [0038]    A particular method of shuffling is to follow the methods described in Crameri et al, 1998, Nature, 391: 288-291 and Ness et al. Nature Biotechnology 17: 893-896. Another format would be the methods described in U.S. Pat. No. 6,159,687: example 1 and 2.  
       Properties of Lipolytic Enzyme  
       [0039]    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).  
         [0040]    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  
       [0041]    The best alignment of the mature parts of SEQ ID NO: 2, 4, 6, 8 and 10 is achieved by inserting a gap of one amino acid between Q249 and P/G250 of SEQ ID NO: 2, 4 and 6. This alignment defines corresponding amino acids.  
         [0042]    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, S. B. and Wunsch, C. D., (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.  
         [0043]    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.  
         [0044]    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  
       [0045]    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.:  
         [0046]    In the pulp and paper industry, to remove pitch or to remove ink from used paper. WO 9213130, WO 9207138, JP 2160984 A, EP 374700.  
         [0047]    Baking. WO 94/04035, WO 00/32758.  
         [0048]    Detergents. WO 97/04079, WO 97/07202, WO 97/41212, WO 98/08939 and WO 97/43375.  
         [0049]    Leather industry. GB 2233665, EP 505920.  
         [0050]    An enzyme with lipase activity may be used for fat hydrolysis and for modification of triglycerides and for production of mono- and diglycerides.  
         [0051]    An enzyme with lipase activity may be used for interesterification of bulk fats, production of frying fats, shortenings and margarine components.  
         [0052]    An enzyme with phospholipase activity (A1, A2) may be used for degumming of vegetable oils and for lysophospholipid production.  
       Improvement of Filtration  
       [0053]    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 hydrolysate, especially a wheat starch hydrolysate 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  
       [0054]    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.  
         [0055]    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/DK WO 98/08939 and WO 97/43375.  
         [0056]    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.  
         [0057]    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.  
         [0058]    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  
       [0059]    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  
       [0060]    The  Aspergillus oryzae  expression plasmid pCaHj 483 (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:  
         [0061]    The pUC19 origin of replication was PCR amplified from pCaHj483 with the primers 142779 (SEQ ID NO: 35) and 142780 (SEQ ID NO: 36).  
         [0062]    Primer 142780 introduces a Bbul 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.  
         [0063]    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).  
         [0064]    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).  
         [0065]    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, 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 μg/l thiamine and 10 mg/l kanamycin.  
         [0066]    A plasmid from a selected transformant was termed pCaHj 527. The Pna2/tpi promoter present on pCaHj527 was subjected to site directed mutagenises by a simple PCR approach.  
         [0067]    Nucleotide 134-144 was altered from SEQ ID NO: 39 to SEQ ID NO: 40 using the mutagenic primer 141223 (SEQ ID NO: 41).  
         [0068]    Nucleotide 423-436 was altered from SEQ ID NO: 42 to SEQ ID NO: 43 using the mutagenic primer 141222 (SEQ ID 44).  
         [0069]    The resulting plasmid was termed pMT2188.  
       Plasmid pENI11861  
       [0070]    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.  
         [0071]    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 46).  
         [0072]    The fragment was cut BssHII and Bgl II, and cloned into pENI11849 which was also cut with BssHII and Bgl 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  
       [0073]    Plasmid pENI1861 was used as template and the following primers with 5′ phosphorylation were used as selection primers: 177996 (SEQ ID 47), 135640 (SEQ ID 48) and 135638 (SEQ ID 49).  
         [0074]    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  
       [0075]    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  
       [0076]    YPG: 4 g/L Yeast extract, 1 g/L KH2PO4, 0.5 g/L MgSO4-7aq, 5 g/L Glucose, pH 6.0.  
       EXAMPLES  
     Example 1  
     Plasmids Harboring Lipolytic Enzyme Genes  
     Genomic DNA Preparation  
       [0077]    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 μl of proteinase K (25 mg/ml) was added. The suspension was incubated at 65° C. for 30-60min. 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  
       [0078]    PCR reactions on each genomic DNA was done with HL 2 and HL12 (SEQ ID NO: 51 and 52) or HL2 and HL6 (SEQ ID NO: 51 and 53) designed based upon alignment lipases.  
         [0079]    Reaction components (2.6 ng /μl of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.1 U/μl 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                      
 
         [0080]    Steps 1 to 3 were repeated 30 times.  
         [0081]    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  
       [0082]    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.  
         [0083]    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.  
         [0084]    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 sequence were compared. The sequence of majority is defined as the right nucleotide sequence.  
       Example 2  
     Cloning of Lipase into Aspergillus Expression Vector  
       [0085]    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:  
         [0086]    A: Plasmid with gene from  Talaromyces thermophilus  and oligo 051200j1/051200j8 (SEQ ID NO: 11 and 18).  
         [0087]    B: Plasmid with gene from  Talaromyces emersonii  and oligo 051200j9/051200j16 (SEQ ID NO: 19 and 26).  
         [0088]    C: Plasmid with gene from  Thermomyces Ibadanensis  and oligo 051200j17/051200j24 (SEQ ID NO: 27 and 34).  
         [0089]    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), pENI2147 ( Thermomyces Ibadanensis  lipase gene) and pENI2148 ( Talaromyces thermophilus  lipase gene).  
         [0090]    These were transformed into Jal250 (described in WO 00/39322) and lipase activity identified as mentioned in pat WO 00/24883.  
       Example 3  
     Construction of Intron-Less Lipase Genes  
     Removal of Introns from  Talaromyces Thermophilus  Lipase Gene  
       [0091]    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 oligoes:  
         [0092]    1: 051200j1 and 051200j3 (SEQ ID NO: 11 and 13)  
         [0093]    2: 051200j2 and 051200j5 (SEQ ID NO: 12 and 15)  
         [0094]    3: 051200j4 and 051200j7 (SEQ ID NO: 14 and 17)  
         [0095]    4: 051200j6 and 051200j8 (SEQ ID NO: 16 and 18)  
         [0096]    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 μl, 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).  
         [0097]    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).  
         [0098]    The PCR-fragment was cloned into pENI1960 cut with Scal (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  
       [0099]    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) and the following oligoes:  
         [0100]    1: 051200j9 and 051200j11 (SEQ ID NO: 19 and 21).  
         [0101]    2: 051200j10 and 051200j13 (SEQ ID NO: 20 and 23).  
         [0102]    3: 051200j12 and 051200j15 (SEQ ID NO: 22 and 25).  
         [0103]    4: 051200j14 and 051200j16 (SEQ ID NO: 24 and 26).  
         [0104]    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 NO: 19 and 26, total of 50 μl, 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. 2 min), 72° C. 5 min).  
         [0105]    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.  
         [0106]    The PCR-fragment was cloned into and cloned into pENI1960 cut Scal 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 lbadanensis  Lipase Gene  
       [0107]    4 PCR reactions were run using PWO polymerase and pENI12147 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 oligoes:  
         [0108]    1: 051200j17 and 051200j19 (SEQ ID NO: 27 and 29).  
         [0109]    2: 051200j18 and 051200j21 (SEQ ID NO: 28 and 31).  
         [0110]    3: 051200j20 and 051200j23 (SEQ ID NO: 30 and 33).  
         [0111]    4: 051200j22 and 051200j24 (SEQ ID NO: 32 and 34).  
         [0112]    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 μl, 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. 2 min), 72° C. 5 min).  
         [0113]    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.  
         [0114]    The PCR-fragment was cloned into and cloned into pENI1960 cut Scal using Gateway cloning as recommended by supplier (life technologies) and transformed into  E. coli  DH10b and sequenced, thus creating intron-less  Thermomyces lbadanensis  lipase gene.  
       Example 4  
     Shuffling of Lipolytic Enzyme Genes  
       [0115]    Plasmids containing DNA sequences encoding lipolytic enzymes are mixed in equimolar amounts. The following components where mixed in a microtube:  
         [0116]    2 μl plasmid mixture (0.15 μg/μl), specific primers flanking the gene (1 pmol/μ), 2 μl 2.5 mM dNTP, 2.5 mM MgCl2, 2 μl 10* taq buffer (Perkin Elmer), 0.5 μl taq enzyme in a total volume of 20 μl.  
         [0117]    The tube is set in a Perkin Elmer 2400 thermocycler. The following PCR-program is run:(94° C., 5 minutes) 1 cycle:  
         [0118]    (94° C., 30 seconds, 70° C., 0 seconds) 99 cycles(72° C., 2 minutes, 4° C. indefinite) 1 cycle.  
         [0119]    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).  
         [0120]    The shuffled sequences can then be subcloned from the  E. coli  TA vector into the yeast vector pJSOO26 (WO 9928448) as a BamHI-Xbal 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  
       [0121]    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:  
         [0122]    1 μl PCR mixture (0.1 μg), decamer random primer (300 pmol), 2 μl 10* Klenow buffer (Promega), 0.25 mM dNTP, 2.5 mM MgCl2 in a total volume of 20 μl.  
         [0123]    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 minutes.  
         [0124]    This procedure generates a high number of small DNA polymers originating from all parts of the gene.  
         [0125]    10 μl is taken out for test on agarose gel.  
         [0126]    10 μl PCR mixture (0.25 mM dNTP, 1 μl 10* Taq buffer (Perkin Elmer), 2.5 mM MgCl2, 0.5 μl Taq enzyme) is added to the 10 μl 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. 7 minutes, 4° C. indefinite.  
         [0127]    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.  
         [0128]    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 μl 10* Taq buffer, 2.5 mM MgCl2. 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° 0  C. 7 minutes, 4° C. indefinite.  
         [0129]    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.  
         [0130]    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.  
         [0131]    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  
       [0132]    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.  
         [0133]    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 oligo&#39;s matching each flank of an exon as well as having a DNA sequence, which is homologous to the next neighbour exon.  
         [0134]    These oligoes 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 oligoes flanking the whole gene, as primers.  
         [0135]    The PCR fragment-containing the full length intron-less gene encoding the lipase was cloned into pENI 1960 as described in pat. appl. PCT/DK02/00050.  
         [0136]    The following primers were used to assemble each intron-less gene:  
         [0137]    [0137] Talaromyces thermophilus:  051200j1, 051200J2, 051200J3, 051200J4, 051200J5, 051200J6, 051200J7 and 051200J8 (SEQ ID NO: 11-18), thus creating pENI2178, when cloned into pENI1960.  
         [0138]    [0138] Talaromyces emersonii:  051200J9, 051200J10, 051200J11, 051200J12, 051200J13, 051200J14, 051200J15 and 051200J16 (SEQ ID NO: 19-26), thus creating pENI2159, when cloned into pENI1960.  
         [0139]    [0139] Thermomyces ibadanensis:  051200J17, 051200J18, 051200J19, 051200J20, 051200J21, 051200J22, 051200J23 and 051200J24 (SEQ ID NO: 27-34), thus creating pENI2160, when cloned into pENI1960.  
         [0140]    [0140] Talaromyces byssochlamydoides:  080201P1, 080201P2, 080201P3, 080201P4, 080201 P5, 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  
       [0141]    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) (pat. PCT/DK02/00050).  
         [0142]    The following oligonucleotides are shown in FIG. 1: 1298-taka, 19670, 19672, 115120 and 050401P6 (SEQ ID NO: 62-65 and 68). 050401P1 (SEQ ID NO: 66) hybridises to 5 ′ T. lanuginose  lipase gene. 030501 P1 (SEQ ID NO: 67) hybridises to 5′ of the other 4 lipase genes.  
         [0143]    The final PCR fragment was cut first with BstEII and then with Sfil, as was the vector pENI2376. pENI2376 is a derivative of pENI1861(pat. PCT/DK02/00050).  
         [0144]    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.  
         [0145]    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.  
         [0146]    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-oligoes:                1298-taka:   gcaagcgcgcgcaatacatggtgttttgatcat                   19670:   ccccatcctttaactatagcg               19672:   ccacacttctcttccttcctc               115120:   gctttgtgcagggtaaatc               050401P1:   cggccgggccgcggaggccagggatccaccatgaggagctcccttgtgctg               030501P1:   cggccgggccgcggaggccacaagtttgtacaaaaaagcagg           (hybridises to 5′ of the other 4 lipase genes)               050401P6:   cggccgggtcaccccccatcctttaactatagcg          
 
       Example 8  
     Characterization of Lipolytic Enzymes  
       [0147]    Lipolytic enzymes from  Thermomyces ibadanensis  and  Talaromyces thermophilus  were prepared as described above, purified and used for characterization  
         [0148]    The specific lipase activity was determined by the LU method described in WO 0032758, 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.  
         [0149]    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.  
         [0150]    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  
       [0151]    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.  
         [0152]    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.  
     
       
       
         1 
         
           
             53  
           
           
             1  
             918  
             DNA  
             Thermomyces lanuginosus  
             
               CDS  
               (1)..(873)  
             
             
               sig_peptide  
               (1)..(66)  
             
             
               mat_peptide  
               (67)..()  
             
           
            1 

atg agg agc tcc ctt gtg ctg ttc ttt gtc tct gcg tgg acg gcc ttg       48 
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu 
        -20                 -15                 -10 

gcc agt cct att cgt cga gag gtc tcg cag gat ctg ttt aac cag ttc       96 
Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe 
    -5              -1  1               5                   10 

aat ctc ttt gca cag tat tct gca gcc gca tac tgc gga aaa aac aat      144 
Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn 
                15                  20                  25 

gat gcc cca gct ggt aca aac att acg tgc acg gga aat gcc tgc ccc      192 
Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro 
            30                  35                  40 

gag gta gag aag gcg gat gca acg ttt ctc tac tcg ttt gaa gac tct      240 
Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser 
        45                  50                  55 

gga gtg ggc gat gtc acc ggc ttc ctt gct ctc gac aac acg aac aaa      288 
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys 
    60                  65                  70 

ttg atc gtc ctc tct ttc cgt ggc tct cgt tcc ata gag aac tgg atc      336 
Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile 
75                  80                  85                  90 

ggg aat ctt aac ttc gac ttg aaa gaa ata aat gac att tgc tcc ggc      384 
Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly 
                95                  100                 105 

tgc agg gga cat gac ggc ttc act tcg tcc tgg agg tct gta gcc gat      432 
Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp 
            110                 115                 120 

acg tta agg cag aag gtg gag gat gct gtg agg gag cat ccc gac tat      480 
Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr 
        125                 130                 135 

cgc gtg gtg ttt acc gga cat agc ttg ggt ggt gca ttg gca act gtt      528 
Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val 
    140                 145                 150 

gcc gga gca gac ctg cgt gga aat ggg tat gat atc gac gtg ttt tca      576 
Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser 
155                 160                 165                 170 

tat ggc gcc ccc cga gtc gga aac agg gct ttt gca gaa ttc ctg acc      624 
Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr 
                175                 180                 185 

gta cag acc ggc gga aca ctc tac cgc att acc cac acc aat gat att      672 
Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile 
            190                 195                 200 

gtc cct aga ctc ccg ccg cgc gaa ttc ggt tac agc cat tct agc cca      720 
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro 
        205                 210                 215 

gag tac tgg atc aaa tct gga acc ctt gtc ccc gtc acc cga aac gat      768 
Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp 
    220                 225                 230 

atc gtg aag ata gaa ggc atc gat gcc acc ggc ggc aat aac cag cct      816 
Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro 
235                 240                 245                 250 

aac att ccg gat atc cct gcg cac cta tgg tac ttc ggg tta att ggg      864 
Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly 
                255                 260                 265 

aca tgt ctt tagtggccgg cgcggctggg tccgactcta gcgagctcga gatct        918 
Thr Cys Leu 

 
           
             2  
             291  
             PRT  
             Thermomyces lanuginosus  
           
            2 

Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu 
        -20                 -15                 -10 

Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe 
    -5              -1  1               5                   10 

Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn 
                15                  20                  25 

Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro 
            30                  35                  40 

Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser 
        45                  50                  55 

Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys 
    60                  65                  70 

Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile 
75                  80                  85                  90 

Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly 
                95                  100                 105 

Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp 
            110                 115                 120 

Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr 
        125                 130                 135 

Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val 
    140                 145                 150 

Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser 
155                 160                 165                 170 

Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr 
                175                 180                 185 

Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile 
            190                 195                 200 

Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro 
        205                 210                 215 

Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp 
    220                 225                 230 

Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro 
235                 240                 245                 250 

Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly 
                255                 260                 265 

Thr Cys Leu 

 
           
             3  
             1083  
             DNA  
             Talaromyces thermophilus  
             
               CDS  
               (1)..(67)  
             
             
               mat_peptide  
               (67)..()  
             
             
               CDS  
               (139)..(307)  
             
             
               CDS  
               (370)..(703)  
             
             
               CDS  
               (778)..(1080)  
             
           
            3 

atg agg agc tcg ctc gtg ctg ttc ttc gtt tct gcg tgg acg gcc ttg       48 
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu 
        -20                 -15                 -10 

gcc agt cct gtc cga cga g gtatgtaaat cacggggtat acttttcatg            97 
Ala Ser Pro Val Arg Arg 
    -5              -1 

cattgcatgt cgaacctgct gtactaagat tgcgcgcaca g ag  gtc tcg cag gat    152 
                                              Glu Val Ser Gln Asp 
                                                              5 

ctg ttt gac cag ttc aac ctc ttt gcg cag tac tcg gcg gcc gca tac      200 
Leu Phe Asp Gln Phe Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr 
                10                  15                  20 

tgc gcg aag aac aac gat gcc ccg gca ggt ggg aac gta acg tgc agg      248 
Cys Ala Lys Asn Asn Asp Ala Pro Ala Gly Gly Asn Val Thr Cys Arg 
            25                  30                  35 

gga agt att tgc ccc gag gta gag aag gcg gat gca acg ttt ctc tac      296 
Gly Ser Ile Cys Pro Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr 
        40                  45                  50 

tcg ttt gag ga  gtaggtgtca acaagagtac aggcacccgt agtagaaata          347 
Ser Phe Glu Asp 
    55 

gcagactaac tgggaaatgt ag t tct gga gtt ggc gat gtc acc ggg ttc       397 
                           Ser Gly Val Gly Asp Val Thr Gly Phe 
                                   60                  65 

ctt gct ctc gac aac acg aac aga ctg atc gtc ctc tct ttc cgc ggc      445 
Leu Ala Leu Asp Asn Thr Asn Arg Leu Ile Val Leu Ser Phe Arg Gly 
            70                  75                  80 

tct cgt tcc ctg gaa aac tgg atc ggg aat atc aac ttg gac ttg aaa      493 
Ser Arg Ser Leu Glu Asn Trp Ile Gly Asn Ile Asn Leu Asp Leu Lys 
        85                  90                  95 

gga att gac gac atc tgc tct ggc tgc aag gga cat gac ggc ttc act      541 
Gly Ile Asp Asp Ile Cys Ser Gly Cys Lys Gly His Asp Gly Phe Thr 
    100                 105                 110 

tcc tcc tgg agg tcc gtt gcc aat acc ttg act cag caa gtg cag aat      589 
Ser Ser Trp Arg Ser Val Ala Asn Thr Leu Thr Gln Gln Val Gln Asn 
115                 120                 125                 130 

gct gtg agg gag cat ccc gac tac cgc gtc gtc ttc act ggg cac agc      637 
Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly His Ser 
                135                 140                 145 

ttg ggt ggt gca ttg gca act gtg gcc ggg gca tct ctg cgt gga aat      685 
Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Ser Leu Arg Gly Asn 
            150                 155                 160 

ggg tac gat ata gat gtg gtatgtagga aaaatgatcc ccgtggagcg             733 
Gly Tyr Asp Ile Asp Val 
        165 

gtcatgtgga aatgtgcagg ggtgtctaat acacagacca acag ttc tca tat ggc     789 
                                                 Phe Ser Tyr Gly 
                                                     170 

gct ccc cgc gtc gga aac agg gct ttt gcg gaa ttc ctg acc gca cag      837 
Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Ala Gln 
        175                 180                 185 

acc ggc ggc acc ttg tac cgc atc acc cac acc aat gat att gtc ccc      885 
Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro 
    190                 195                 200 

aga ctc ccg cca cgc gaa ttg ggt tac agc cat tct agc cca gag tat      933 
Arg Leu Pro Pro Arg Glu Leu Gly Tyr Ser His Ser Ser Pro Glu Tyr 
205                 210                 215                 220 

tgg atc acg tct gga acc ctc gtc cca gtg acc aag aac gat atc gtc      981 
Trp Ile Thr Ser Gly Thr Leu Val Pro Val Thr Lys Asn Asp Ile Val 
                225                 230                 235 

aag gtg gag ggc atc gat tcc acc gat gga aac aac cag cca aat acc     1029 
Lys Val Glu Gly Ile Asp Ser Thr Asp Gly Asn Asn Gln Pro Asn Thr 
            240                 245                 250 

ccg gac att gct gcg cac cta tgg tac ttc ggg tca atg gcg acg tgt     1077 
Pro Asp Ile Ala Ala His Leu Trp Tyr Phe Gly Ser Met Ala Thr Cys 
        255                 260                 265 

ttg taa                                                             1083 
Leu  
           
             4  
             291  
             PRT  
             Talaromyces thermophilus  
           
            4 

Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu 
        -20                 -15                 -10 

Ala Ser Pro Val Arg Arg Glu Val Ser Gln Asp Leu Phe Asp Gln Phe 
    -5              -1  1               5                   10 

Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Ala Lys Asn Asn 
                15                  20                  25 

Asp Ala Pro Ala Gly Gly Asn Val Thr Cys Arg Gly Ser Ile Cys Pro 
            30                  35                  40 

Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser 
        45                  50                  55 

Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Arg 
    60                  65                  70 

Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Leu Glu Asn Trp Ile 
75                  80                  85                  90 

Gly Asn Ile Asn Leu Asp Leu Lys Gly Ile Asp Asp Ile Cys Ser Gly 
                95                  100                 105 

Cys Lys Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asn 
            110                 115                 120 

Thr Leu Thr Gln Gln Val Gln Asn Ala Val Arg Glu His Pro Asp Tyr 
        125                 130                 135 

Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val 
    140                 145                 150 

Ala Gly Ala Ser Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser 
155                 160                 165                 170 

Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr 
                175                 180                 185 

Ala Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile 
            190                 195                 200 

Val Pro Arg Leu Pro Pro Arg Glu Leu Gly Tyr Ser His Ser Ser Pro 
        205                 210                 215 

Glu Tyr Trp Ile Thr Ser Gly Thr Leu Val Pro Val Thr Lys Asn Asp 
    220                 225                 230 

Ile Val Lys Val Glu Gly Ile Asp Ser Thr Asp Gly Asn Asn Gln Pro 
235                 240                 245                 250 

Asn Thr Pro Asp Ile Ala Ala His Leu Trp Tyr Phe Gly Ser Met Ala 
                255                 260                 265 

Thr Cys Leu 

 
           
             5  
             1070  
             DNA  
             Thermomyces ibadanensis  
             
               CDS  
               (1)..(67)  
             
             
               mat_peptide  
               (67)..()  
             
             
               CDS  
               (128)..(296)  
             
             
               CDS  
               (357)..(690)  
             
             
               CDS  
               (765)..(1067)  
             
           
            5 

atg cgg agc tcc ctc gtg ctg ttc ttc ctc tct gcg tgg acg gcc ttg       48 
Met Arg Ser Ser Leu Val Leu Phe Phe Leu Ser Ala Trp Thr Ala Leu 
        -20                 -15                 -10 

gcg cgg cct gtt cga cga g gtatgtagca agggacacta ttacatgttg            97 
Ala Arg Pro Val Arg Arg 
    -5              -1 

accttggtga ttctaagact gcatgcgcag cg  gtt ccg caa gat ctg ctc gac     150 
                                 Ala Val Pro Gln Asp Leu Leu Asp 
                                                 5 

cag ttt gaa ctc ttt tca caa tat tcg gcg gcc gca tac tgt gcg gca      198 
Gln Phe Glu Leu Phe Ser Gln Tyr Ser Ala Ala Ala Tyr Cys Ala Ala 
    10                  15                  20 

aac aat cat gct cca gtg ggc tca gac gta acg tgc tcg gag aat gtc      246 
Asn Asn His Ala Pro Val Gly Ser Asp Val Thr Cys Ser Glu Asn Val 
25                  30                  35                  40 

tgc cct gag gta gat gcg gcg gac gca acg ttt ctc tat tct ttt gaa      294 
Cys Pro Glu Val Asp Ala Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu 
                45                  50                  55 

ga  gtgggtgtcg acaaagcaca gagacagtag tagagacagc agtctaactg           346 
Asp 

agatgtgcag t tct gga tta ggc gat gtt acc ggc ctt ctc gct ctc gac     396 
             Ser Gly Leu Gly Asp Val Thr Gly Leu Leu Ala Leu Asp 
                     60                  65                  70 

aac acg aat aaa ctg atc gtc ctc tct ttc cgc ggc tct cgc tca gta      444 
Asn Thr Asn Lys Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Val 
                75                  80                  85 

gag aac tgg atc gcg aac ctc gcc gcc gac ctg aca gaa ata tct gac      492 
Glu Asn Trp Ile Ala Asn Leu Ala Ala Asp Leu Thr Glu Ile Ser Asp 
            90                  95                  100 

atc tgc tcc ggc tgc gag ggg cat gtc ggc ttc gtt act tct tgg agg      540 
Ile Cys Ser Gly Cys Glu Gly His Val Gly Phe Val Thr Ser Trp Arg 
        105                 110                 115 

tct gta gcc gac act ata agg gag cag gtg cag aat gcc gtg aac gag      588 
Ser Val Ala Asp Thr Ile Arg Glu Gln Val Gln Asn Ala Val Asn Glu 
    120                 125                 130 

cat ccc gat tac cgc gtg gtc ttt acc gga cat agc ttg gga ggc gca      636 
His Pro Asp Tyr Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala 
135                 140                 145                 150 

ctg gca act att gcc gca gca gct ctg cga gga aat gga tac aat atc      684 
Leu Ala Thr Ile Ala Ala Ala Ala Leu Arg Gly Asn Gly Tyr Asn Ile 
                155                 160                 165 

gac gtg gtatgtggga agaagccacc cagacaaaca attatgtgga aacatgcaag       740 
Asp Val 

gatggctaat acacggtcca acag ttc tca tat ggc gcg ccc cgc gtc ggt       791 
                           Phe Ser Tyr Gly Ala Pro Arg Val Gly 
                               170                 175 

aac agg gca ttt gca gaa ttc ctg acc gca cag acg ggc ggc acc ctg      839 
Asn Arg Ala Phe Ala Glu Phe Leu Thr Ala Gln Thr Gly Gly Thr Leu 
        180                 185                 190 

tat cgc atc acc cat acc aat gat atc gtc cct aga ctc cct cct cga      887 
Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro Arg 
    195                 200                 205 

gac tgg ggt tac agc cac tct agc ccg gag tac tgg gtc acg tct ggt      935 
Asp Trp Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Val Thr Ser Gly 
210                 215                 220                 225 

aac gac gtc cca gtg acc gca aac gac atc acc gtc gtg gag ggc atc      983 
Asn Asp Val Pro Val Thr Ala Asn Asp Ile Thr Val Val Glu Gly Ile 
                230                 235                 240 

gat tcc acc gac ggg aac aac cag ggg aat atc cca gac atc cct tcg     1031 
Asp Ser Thr Asp Gly Asn Asn Gln Gly Asn Ile Pro Asp Ile Pro Ser 
            245                 250                 255 

cat cta tgg tat ttc ggt ccc att tca gag tgt gat tag                 1070 
His Leu Trp Tyr Phe Gly Pro Ile Ser Glu Cys Asp 
        260                 265 

 
           
             6  
             291  
             PRT  
             Thermomyces ibadanensis  
           
            6 

Met Arg Ser Ser Leu Val Leu Phe Phe Leu Ser Ala Trp Thr Ala Leu 
        -20                 -15                 -10 

Ala Arg Pro Val Arg Arg Ala Val Pro Gln Asp Leu Leu Asp Gln Phe 
    -5              -1  1               5                   10 

Glu Leu Phe Ser Gln Tyr Ser Ala Ala Ala Tyr Cys Ala Ala Asn Asn 
                15                  20                  25 

His Ala Pro Val Gly Ser Asp Val Thr Cys Ser Glu Asn Val Cys Pro 
            30                  35                  40 

Glu Val Asp Ala Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser 
        45                  50                  55 

Gly Leu Gly Asp Val Thr Gly Leu Leu Ala Leu Asp Asn Thr Asn Lys 
    60                  65                  70 

Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Val Glu Asn Trp Ile 
75                  80                  85                  90 

Ala Asn Leu Ala Ala Asp Leu Thr Glu Ile Ser Asp Ile Cys Ser Gly 
                95                  100                 105 

Cys Glu Gly His Val Gly Phe Val Thr Ser Trp Arg Ser Val Ala Asp 
            110                 115                 120 

Thr Ile Arg Glu Gln Val Gln Asn Ala Val Asn Glu His Pro Asp Tyr 
        125                 130                 135 

Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Ile 
    140                 145                 150 

Ala Ala Ala Ala Leu Arg Gly Asn Gly Tyr Asn Ile Asp Val Phe Ser 
155                 160                 165                 170 

Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr 
                175                 180                 185 

Ala Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile 
            190                 195                 200 

Val Pro Arg Leu Pro Pro Arg Asp Trp Gly Tyr Ser His Ser Ser Pro 
        205                 210                 215 

Glu Tyr Trp Val Thr Ser Gly Asn Asp Val Pro Val Thr Ala Asn Asp 
    220                 225                 230 

Ile Thr Val Val Glu Gly Ile Asp Ser Thr Asp Gly Asn Asn Gln Gly 
235                 240                 245                 250 

Asn Ile Pro Asp Ile Pro Ser His Leu Trp Tyr Phe Gly Pro Ile Ser 
                255                 260                 265 

Glu Cys Asp 

 
           
             7  
             1064  
             DNA  
             Talaromyces emersonii  
             
               CDS  
               (1)..(88)  
             
             
               mat_peptide  
               (88)..()  
             
             
               CDS  
               (142)..(310)  
             
             
               CDS  
               (362)..(695)  
             
             
               CDS  
               (756)..(1061)  
             
           
            7 

atg ttc aaa tcg gcc gct gtg cgg gcc att gct gcc ctc gga ctg act       48 
Met Phe Lys Ser Ala Ala Val Arg Ala Ile Ala Ala Leu Gly Leu Thr 
                -25                 -20                 -15 

gcg tca gtc ttg gct gct cct gtt gaa ctg ggc cgt cga g gtaaggaagc      98 
Ala Ser Val Leu Ala Ala Pro Val Glu Leu Gly Arg Arg 
            -10                 -5              -1 

atgacggaga gaacaccctg tgcgacctgc tgacatcctt cag at  gtt tct cag      152 
                                                Asp Val Ser Gln 

gac ctc ttc gac cag ctc aat ctt ttc gag cag tac tcg gcg gct gcg      200 
Asp Leu Phe Asp Gln Leu Asn Leu Phe Glu Gln Tyr Ser Ala Ala Ala 
5                   10                  15                  20 

tac tgt tca gct aac aat gag gcc tct gcc ggc acg gca atc tct tgc      248 
Tyr Cys Ser Ala Asn Asn Glu Ala Ser Ala Gly Thr Ala Ile Ser Cys 
                25                  30                  35 

tcc gca ggc aat tgc ccg ttg gtc cag cag gct gga gca acc atc ctg      296 
Ser Ala Gly Asn Cys Pro Leu Val Gln Gln Ala Gly Ala Thr Ile Leu 
            40                  45                  50 

tat tca ttc aac aa  gtgggtgtca cggaaaagat tgttgatacc aacatgttga      350 
Tyr Ser Phe Asn Asn 
        55 

cgtgttgtca g c att ggc tct ggc gat gtg acg ggt ttt ctc gct ctc       398 
               Ile Gly Ser Gly Asp Val Thr Gly Phe Leu Ala Leu 
                       60                  65 

gac tcg acg aat caa ttg atc gtc ttg tca ttc cgg gga tca gag act      446 
Asp Ser Thr Asn Gln Leu Ile Val Leu Ser Phe Arg Gly Ser Glu Thr 
70                  75                  80                  85 

ctc gaa aac tgg atc gct gac ctg gaa gct gac ctg gtc gat gcc tct      494 
Leu Glu Asn Trp Ile Ala Asp Leu Glu Ala Asp Leu Val Asp Ala Ser 
                90                  95                  100 

gcc atc tgt tcc ggc tgt gaa gca cac gat ggg ttc ctt tca tcc tgg      542 
Ala Ile Cys Ser Gly Cys Glu Ala His Asp Gly Phe Leu Ser Ser Trp 
            105                 110                 115 

aat tca gtc gcc agc act ctg aca tcc aaa atc tcg tcg gcc gtc aac      590 
Asn Ser Val Ala Ser Thr Leu Thr Ser Lys Ile Ser Ser Ala Val Asn 
        120                 125                 130 

gaa cat ccc agc tac aag ctg gtc ttc acc ggc cac agt ctc gga gcc      638 
Glu His Pro Ser Tyr Lys Leu Val Phe Thr Gly His Ser Leu Gly Ala 
    135                 140                 145 

gcc ttg gct aca ctt gga gcc gtt tct ctt aga gag agc gga tat aat      686 
Ala Leu Ala Thr Leu Gly Ala Val Ser Leu Arg Glu Ser Gly Tyr Asn 
150                 155                 160                 165 

att gac ctc gtaagtttcc ggcacgggcg tcgtcatcat cgagcggaaa              735 
Ile Asp Leu 

gactgaccgg ttaactgcag tac aat tat ggc tgc ccc cgg gtc ggt aac acc    788 
                      Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asn Thr 
                          170                 175 

gcg ctc gca gac ttc atc acc acg caa tcc gga ggc aca aat tac cgc      836 
Ala Leu Ala Asp Phe Ile Thr Thr Gln Ser Gly Gly Thr Asn Tyr Arg 
180                 185                 190                 195 

gtc acg cat tcc gat gac cct gtc ccc aag ctg cct ccc agg agt ttt      884 
Val Thr His Ser Asp Asp Pro Val Pro Lys Leu Pro Pro Arg Ser Phe 
                200                 205                 210 

gga tac agc caa ccg agc cca gag tac tgg atc acc tca ggg aac aat      932 
Gly Tyr Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Ser Gly Asn Asn 
            215                 220                 225 

gta act gtt caa ccg tcc gac atc gag gtc atc gaa ggc gtc gac tcc      980 
Val Thr Val Gln Pro Ser Asp Ile Glu Val Ile Glu Gly Val Asp Ser 
        230                 235                 240 

act gca ggc aac gac ggc acc cct gct ggc ctt gac att gat gct cat     1028 
Thr Ala Gly Asn Asp Gly Thr Pro Ala Gly Leu Asp Ile Asp Ala His 
    245                 250                 255 

cgg tgg tac ttt gga ccc att agc gca tgt tcg tga                     1064 
Arg Trp Tyr Phe Gly Pro Ile Ser Ala Cys Ser 
260                 265                 270 

 
           
             8  
             299  
             PRT  
             Talaromyces emersonii  
           
            8 

Met Phe Lys Ser Ala Ala Val Arg Ala Ile Ala Ala Leu Gly Leu Thr 
                -25                 -20                 -15 

Ala Ser Val Leu Ala Ala Pro Val Glu Leu Gly Arg Arg Asp Val Ser 
            -10                 -5              -1  1 

Gln Asp Leu Phe Asp Gln Leu Asn Leu Phe Glu Gln Tyr Ser Ala Ala 
    5                   10                  15 

Ala Tyr Cys Ser Ala Asn Asn Glu Ala Ser Ala Gly Thr Ala Ile Ser 
20                  25                  30                  35 

Cys Ser Ala Gly Asn Cys Pro Leu Val Gln Gln Ala Gly Ala Thr Ile 
                40                  45                  50 

Leu Tyr Ser Phe Asn Asn Ile Gly Ser Gly Asp Val Thr Gly Phe Leu 
            55                  60                  65 

Ala Leu Asp Ser Thr Asn Gln Leu Ile Val Leu Ser Phe Arg Gly Ser 
        70                  75                  80 

Glu Thr Leu Glu Asn Trp Ile Ala Asp Leu Glu Ala Asp Leu Val Asp 
    85                  90                  95 

Ala Ser Ala Ile Cys Ser Gly Cys Glu Ala His Asp Gly Phe Leu Ser 
100                 105                 110                 115 

Ser Trp Asn Ser Val Ala Ser Thr Leu Thr Ser Lys Ile Ser Ser Ala 
                120                 125                 130 

Val Asn Glu His Pro Ser Tyr Lys Leu Val Phe Thr Gly His Ser Leu 
            135                 140                 145 

Gly Ala Ala Leu Ala Thr Leu Gly Ala Val Ser Leu Arg Glu Ser Gly 
        150                 155                 160 

Tyr Asn Ile Asp Leu Tyr Asn Tyr Gly Cys Pro Arg Val Gly Asn Thr 
    165                 170                 175 

Ala Leu Ala Asp Phe Ile Thr Thr Gln Ser Gly Gly Thr Asn Tyr Arg 
180                 185                 190                 195 

Val Thr His Ser Asp Asp Pro Val Pro Lys Leu Pro Pro Arg Ser Phe 
                200                 205                 210 

Gly Tyr Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Ser Gly Asn Asn 
            215                 220                 225 

Val Thr Val Gln Pro Ser Asp Ile Glu Val Ile Glu Gly Val Asp Ser 
        230                 235                 240 

Thr Ala Gly Asn Asp Gly Thr Pro Ala Gly Leu Asp Ile Asp Ala His 
    245                 250                 255 

Arg Trp Tyr Phe Gly Pro Ile Ser Ala Cys Ser 
260                 265                 270 

 
           
             9  
             1074  
             DNA  
             Talaromyces byssochlamydoides  
             
               CDS  
               (1)..(85)  
             
             
               mat_peptide  
               (85)..()  
             
             
               CDS  
               (150)..(318)  
             
             
               CDS  
               (376)..(709)  
             
             
               CDS  
               (760)..(1071)  
             
           
            9 

atg ttc aaa tca act gtc cgg gcc atc gcc gcc ctc gga ctg acc tcg       48 
Met Phe Lys Ser Thr Val Arg Ala Ile Ala Ala Leu Gly Leu Thr Ser 
            -25                 -20                 -15 

tca gtc ttt gct gct cct atc gaa ctg ggc cgt cga g gtaaggggca          95 
Ser Val Phe Ala Ala Pro Ile Glu Leu Gly Arg Arg 
        -10                 -5              -1 

tgaaaactcc ctgtatggca tctcatctgg cagcatatct actgacatcc tcag at       151 
                                                            Asp 

gtt tcg gag cag ctc ttc aac cag ttc aat ctc ttc gag cag tat tcc      199 
Val Ser Glu Gln Leu Phe Asn Gln Phe Asn Leu Phe Glu Gln Tyr Ser 
            5                   10                  15 

gcg gct gcg tac tgt cca gcc aac ttt gag tcc gct tcc ggc gcg gca      247 
Ala Ala Ala Tyr Cys Pro Ala Asn Phe Glu Ser Ala Ser Gly Ala Ala 
        20                  25                  30 

att tct tgt tcc aca ggc aat tgc ccg ctc gtc caa cag gct ggc gca      295 
Ile Ser Cys Ser Thr Gly Asn Cys Pro Leu Val Gln Gln Ala Gly Ala 
    35                  40                  45 

acc acc ctg tat gca ttc aac aa  gtgagtgtca tggaaaggct tgttggtaca     348 
Thr Thr Leu Tyr Ala Phe Asn Asn 
50                  55 

ccgtacgggt atgttgactg tcatcag c atc ggc tct ggc gat gtg acg ggt      400 
                                Ile Gly Ser Gly Asp Val Thr Gly 
                                        60                  65 

ttt ctt gct gtc gat ccg acc aac cga ctc atc gtc ttg tcg ttc cgg      448 
Phe Leu Ala Val Asp Pro Thr Asn Arg Leu Ile Val Leu Ser Phe Arg 
                70                  75                  80 

ggg tca gag agt ctc gag aac tgg atc act aat ctc agc gcc gac ctg      496 
Gly Ser Glu Ser Leu Glu Asn Trp Ile Thr Asn Leu Ser Ala Asp Leu 
            85                  90                  95 

gtc gat gcc tct gca atc tgt tcc ggg tgt gaa gcc cat gac gga ttc      544 
Val Asp Ala Ser Ala Ile Cys Ser Gly Cys Glu Ala His Asp Gly Phe 
        100                 105                 110 

tat tcg tct tgg caa tca gtt gcc agc act ctg acc tcc caa atc tcg      592 
Tyr Ser Ser Trp Gln Ser Val Ala Ser Thr Leu Thr Ser Gln Ile Ser 
    115                 120                 125 

tcg gcc ctc tcg gca tat cca aac tac aag ctg gtc ttc acc ggc cac      640 
Ser Ala Leu Ser Ala Tyr Pro Asn Tyr Lys Leu Val Phe Thr Gly His 
130                 135                 140                 145 

agt ctc gga gcc gcc tta gct aca ctt gga gct gtc tct ctc agg gag      688 
Ser Leu Gly Ala Ala Leu Ala Thr Leu Gly Ala Val Ser Leu Arg Glu 
                150                 155                 160 

agt gga tac aat atc gac ctc gtaagttcct ggcattgcca tcatggaaag         739 
Ser Gly Tyr Asn Ile Asp Leu 
            165 

agactcacag ttaactgtag tac aac ttt ggc tgt ccc cgg gtc ggc aac act    792 
                      Tyr Asn Phe Gly Cys Pro Arg Val Gly Asn Thr 
                          170                 175 

gcg ctc gca gac ttt att acc aac caa acc ggt ggc aca aat tac cgg      840 
Ala Leu Ala Asp Phe Ile Thr Asn Gln Thr Gly Gly Thr Asn Tyr Arg 
180                 185                 190                 195 

gta acg cat tac gag gac cct gtc ccc aag ctg cct ccc agg agt ttt      888 
Val Thr His Tyr Glu Asp Pro Val Pro Lys Leu Pro Pro Arg Ser Phe 
                200                 205                 210 

gga tac agc caa cct agc ccg gaa tac tgg atc acg tcg gga aac aat      936 
Gly Tyr Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Ser Gly Asn Asn 
            215                 220                 225 

gtg act gtg act tcg tcc gac atc gat gtc gtc gtg ggt gtc gac tcg      984 
Val Thr Val Thr Ser Ser Asp Ile Asp Val Val Val Gly Val Asp Ser 
        230                 235                 240 

act gca ggc aac gac ggg acg cct gat ggc ctt gac act gct gcc cat     1032 
Thr Ala Gly Asn Asp Gly Thr Pro Asp Gly Leu Asp Thr Ala Ala His 
    245                 250                 255 

agg tgg tat ttt gga cct act acc gaa tgt tcg tcg tca tga             1074 
Arg Trp Tyr Phe Gly Pro Thr Thr Glu Cys Ser Ser Ser 
260                 265                 270 

 
           
             10  
             300  
             PRT  
             Talaromyces byssochlamydoides  
           
            10 

Met Phe Lys Ser Thr Val Arg Ala Ile Ala Ala Leu Gly Leu Thr Ser 
            -25                 -20                 -15 

Ser Val Phe Ala Ala Pro Ile Glu Leu Gly Arg Arg Asp Val Ser Glu 
        -10                 -5              -1  1 

Gln Leu Phe Asn Gln Phe Asn Leu Phe Glu Gln Tyr Ser Ala Ala Ala 
5                   10                  15                  20 

Tyr Cys Pro Ala Asn Phe Glu Ser Ala Ser Gly Ala Ala Ile Ser Cys 
                25                  30                  35 

Ser Thr Gly Asn Cys Pro Leu Val Gln Gln Ala Gly Ala Thr Thr Leu 
            40                  45                  50 

Tyr Ala Phe Asn Asn Ile Gly Ser Gly Asp Val Thr Gly Phe Leu Ala 
        55                  60                  65 

Val Asp Pro Thr Asn Arg Leu Ile Val Leu Ser Phe Arg Gly Ser Glu 
    70                  75                  80 

Ser Leu Glu Asn Trp Ile Thr Asn Leu Ser Ala Asp Leu Val Asp Ala 
85                  90                  95                  100 

Ser Ala Ile Cys Ser Gly Cys Glu Ala His Asp Gly Phe Tyr Ser Ser 
                105                 110                 115 

Trp Gln Ser Val Ala Ser Thr Leu Thr Ser Gln Ile Ser Ser Ala Leu 
            120                 125                 130 

Ser Ala Tyr Pro Asn Tyr Lys Leu Val Phe Thr Gly His Ser Leu Gly 
        135                 140                 145 

Ala Ala Leu Ala Thr Leu Gly Ala Val Ser Leu Arg Glu Ser Gly Tyr 
    150                 155                 160 

Asn Ile Asp Leu Tyr Asn Phe Gly Cys Pro Arg Val Gly Asn Thr Ala 
165                 170                 175                 180 

Leu Ala Asp Phe Ile Thr Asn Gln Thr Gly Gly Thr Asn Tyr Arg Val 
                185                 190                 195 

Thr His Tyr Glu Asp Pro Val Pro Lys Leu Pro Pro Arg Ser Phe Gly 
            200                 205                 210 

Tyr Ser Gln Pro Ser Pro Glu Tyr Trp Ile Thr Ser Gly Asn Asn Val 
        215                 220                 225 

Thr Val Thr Ser Ser Asp Ile Asp Val Val Val Gly Val Asp Ser Thr 
    230                 235                 240 

Ala Gly Asn Asp Gly Thr Pro Asp Gly Leu Asp Thr Ala Ala His Arg 
245                 250                 255                 260 

Trp Tyr Phe Gly Pro Thr Thr Glu Cys Ser Ser Ser 
                265                 270 

 
           
             11  
             51  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200j1  
             
           
            11 

ggggacaagt ttgtacaaaa aagcaggacc atgaggagct cgctcgtgct g              51 

 
           
             12  
             39  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J2  
             
           
            12 

ccagtcctgt ccgacgagag gtctcgcagg atctgtttg                            39 

 
           
             13  
             39  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J3  
             
           
            13 

caaacagatc ctgcgagacc tctcgtcgga caggactgg                            39 

 
           
             14  
             39  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J4  
             
           
            14 

tctctactcg tttgaggatt ctggagttgg cgatgtcac                            39 

 
           
             15  
             36  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J5  
             
           
            15 

acatcgccaa ctccagaatc ctcaaacgag tagaga                               36 

 
           
             16  
             36  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J6  
             
           
            16 

gggtacgata tagatgtgtt ctcatatggc gctccc                               36 

 
           
             17  
             36  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J7  
             
           
            17 

gggagcgcca tatgagaaca catctatatc gtaccc                               36 

 
           
             18  
             48  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J8  
             
           
            18 

ggggaccact ttgtacaaga aagctggtta caaacacgtc gccattga                  48 

 
           
             19  
             51  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J9  
             
           
            19 

ggggacaagt ttgtacaaaa aagcaggacc atgttcaaat cggccgctgt g              51 

 
           
             20  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J10  
             
           
            20 

ctgttgaact gggccgtcga gatgtttctc aggacctctt cg                        42 

 
           
             21  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J11  
             
           
            21 

cgaagaggtc ctgagaaaca tctcgacggc ccagttcaac ag                        42 

 
           
             22  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J12  
             
           
            22 

catcctgtat tcattcaaca acattggctc tggcgatgtg ac                        42 

 
           
             23  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J13  
             
           
            23 

gtcacatcgc cagagccaat gttgttgaat gaatacagga tg                        42 

 
           
             24  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J14  
             
           
            24 

agcggatata atattgacct ctacaattat ggctgccccc gg                        42 

 
           
             25  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J15  
             
           
            25 

ccgggggcag ccataattgt agaggtcaat attatatccg ct                        42 

 
           
             26  
             48  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J16  
             
           
            26 

ggggaccact ttgtacaaga aagctggtca cgaacatgcg ctaatggg                  48 

 
           
             27  
             51  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J17  
             
           
            27 

ggggacaagt ttgtacaaaa aagcaggacc atgcggagct ccctcgtgct g              51 

 
           
             28  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J18  
             
           
            28 

tggcgcggcc tgttcgacga gcggttccgc aagatctgct cg                        42 

 
           
             29  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J19  
             
           
            29 

cgagcagatc ttgcggaacc gctcgtcgaa caggccgcgc ca                        42 

 
           
             30  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J20  
             
           
            30 

gtttctctat tcttttgaag attctggatt aggcgatgtt ac                        42 

 
           
             31  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J21  
             
           
            31 

gtaacatcgc ctaatccaga atcttcaaaa gaatagagaa ac                        42 

 
           
             32  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J22  
             
           
            32 

aatggataca atatcgacgt gttctcatat ggcgcgcccc gc                        42 

 
           
             33  
             42  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J23  
             
           
            33 

gcggggcgcg ccatatgaga acacgtcgat attgtatcca tt                        42 

 
           
             34  
             48  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051200J24  
             
           
            34 

ggggaccact ttgtacaaga aagctggcta atcacactct gaaatggg                  48 

 
           
             35  
             31  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               142779  
             
           
            35 

ttgaattgaa aatagattga tttaaaactt c                                    31 

 
           
             36  
             25  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               142780  
             
           
            36 

ttgcatgcgt aatcatggtc atagc                                           25 

 
           
             37  
             26  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               140288  
             
           
            37 

ttgaattcat gggtaataac tgatat                                          26 

 
           
             38  
             32  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               142778  
             
           
            38 

aaatcaatct attttcaatt caattcatca tt                                   32 

 
           
             39  
             11  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               gtactaaaacc  
             
           
            39 

gtactaaaac c                                                          11 

 
           
             40  
             11  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               ccgttaaattt  
             
           
            40 

ccgttaaatt t                                                          11 

 
           
             41  
             45  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               141223  
             
           
            41 

ggatgctgtt gactccggaa atttaacggt ttggtcttgc atccc                     45 

 
           
             42  
             14  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               atgcaatttaaact  
             
           
            42 

atgcaattta aact                                                       14 

 
           
             43  
             14  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               cggcaatttaacgg  
             
           
            43 

cggcaattta acgg                                                       14 

 
           
             44  
             44  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               141222  
             
           
            44 

ggtattgtcc tgcagacggc aatttaacgg cttctgcgaa tcgc                      44 

 
           
             45  
             59  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               051199J1  
             
           
            45 

cctctagatc tcgagctcgg tcaccggtgg cctccgcggc cgctggatcc ccagttgtg      59 

 
           
             46  
             33  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               1298TAKA  
             
           
            46 

gcaagcgcgc gcaatacatg gtgttttgat cat                                  33 

 
           
             47  
             30  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               177996  
             
           
            47 

gaatgacttg gttgacgcgt caccagtcac                                      30 

 
           
             48  
             25  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               135640  
             
           
            48 

cttattagta ggttggtact tcgag                                           25 

 
           
             49  
             37  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               135638  
             
           
            49 

gtccccagag tagtgtcact atgtcgaggc agttaag                              37 

 
           
             50  
             64  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               080399J19  
             
           
            50 

gtatgtccct tgacaatgcg atgtatcaca tgatataatt actagcaagg gaagccgtgc     60 

ttgg                                                                  64 

 
           
             51  
             18  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               HL 2  
             
           
            51 

wsngcngcng cntaytgy                                                   18 

 
           
             52  
             28  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               HL 12  
             
           
            52 

ggnacnrkrt crttnnnrtg ngtnaync                                        28 

 
           
             53  
             26  
             DNA  
             Artificial Sequence  
             
               misc_feature  
               HL 6  
             
           
            53 

avngcnccnc cnarnswrtg nccngt                                          26

Technology Classification (CPC): 2