EP1078080A1 - Methods for producing polypeptides in filamentous fungal mutant cells - Google Patents

Methods for producing polypeptides in filamentous fungal mutant cells

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Publication number
EP1078080A1
EP1078080A1 EP99925633A EP99925633A EP1078080A1 EP 1078080 A1 EP1078080 A1 EP 1078080A1 EP 99925633 A EP99925633 A EP 99925633A EP 99925633 A EP99925633 A EP 99925633A EP 1078080 A1 EP1078080 A1 EP 1078080A1
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EP
European Patent Office
Prior art keywords
seq
nucleic acid
acid sequence
polypeptide
nucleotides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99925633A
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German (de)
French (fr)
Inventor
Jill Wahleithner
Tove Christensen
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Novozymes AS
Novozymes Inc
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Novozymes AS
Novo Nordisk Biotech Inc
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Publication of EP1078080A1 publication Critical patent/EP1078080A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi

Definitions

  • the present invention relates to methods for producing polypeptides in mutants of filamentous fungal cells, mutants of filamentous fungal cells, and methods for obtaining the mutants.
  • a widely used method for increasing production of a polypeptide is amplification to produce multiple copies of a gene encoding the polypeptide.
  • U.S. Patent No. 5,578,461 discloses the inclusion via homologous recombination of an amplifiable selectable marker gene in tandem with a gene where cells containing amplified copies of the selectable marker can be selected for by culturing the cells in the presence of the appropriate selectable agent.
  • polypeptides has been increased by replacing one promoter with a different promoter or one signal peptide coding region with another. See, e.g., U.S. Patent No. 5,641.670.
  • the secretion of polypeptides has also been modified by overproduction of secretion proteins (Ruohonen et al, 1997, Yeast, 13: 337- 351), and producing a super-secreting cell (U.S. Patent No. 5,312,735).
  • polypeptides also has been increased by disrupting DNA sequences encoding protease capable of hydro lyzing the polypeptide under the conditions for producing the polypeptide.
  • the present invention relates to methods for producing a polypeptide, comprising:
  • mutant cell cultivating a mutant cell of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide in which the mutant cell produces more of the polypeptide than the parent cell when cultivated under the same conditions, wherein the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a modification of one or more second nucleic acid sequences selected from the group consisting of:
  • nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
  • nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4;
  • nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4. (b) a subsequence of (a) of at least 100 nucleotides. or (c) a complementary strand of (a) or (b): riv) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids; (vj an allelic variant of (i), (ii), or (iii); and
  • the present invention also relates to mutants of filamentous fungal cells and methods for obtaining the mutant cells.
  • the present invention also relates to the isolated second nucleic acid sequences
  • DDC2 or DDC3 polypeptides encoding DDC2 or DDC3 polypeptides.
  • Figures 1A and IB show the genomic nucleic acid sequence and the deduced amino acid sequence of DDC2 (SEQ ID NOs. 1 and 2. respectively).
  • Figures 2A and 2B show the genomic nucleic acid sequence and the deduced amino acid sequence of DDC2 (SEQ ID NOs. 4 and 5. respectively).
  • Figure 3 shows a restriction map of pToC391.
  • Figure 4 shows a restriction map of pToC401.
  • the present invention relates to methods for producing a polypeptide in an enhanced amount, comprising cultivating a mutant cell of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide and isolating the polypeptide from the cultivation medium of the mutant cell, wherein the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a modification, e.g.. disruption or deletion, of one or more second nucleic acid sequences endogenous to the parent cell where the modification enhances production of the polypeptide by the mutant cell compared to the parent cell when cultivated under the same conditions.
  • the one or more second nucleic acid sequences have:
  • nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
  • nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4:
  • nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4. (b) a subsequence of (a) of at least 100 nucleotides, or (c) a complementary strand of (a) or (b);
  • nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
  • DDC2 and or DDC3 polypeptide activity is defined herein as an activity (or activities) which when reduced and more preferably eliminated increases or enhances the production of a polypeptide.
  • the second nucleic acid sequences encode polypeptides having an amino acid sequence which has a degree of identity to amino acids 19 to 64 of SEQ ID NO. 2 (i.e.. the mature polypeptide) of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have DCC2 polypeptide activity (hereinafter "DCC2 homologous polypeptides" or "DCC2 polypeptides”), or a degree of identity to amino acids 21 to 83 of SEQ ID NO.
  • DCC2 homologous polypeptides or "DCC2 polypeptides”
  • the mature polypeptide of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%. even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%.
  • DCC3 homologous polypeptides or "DCC3 polypeptides”.
  • the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
  • the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENETM MEGALIGNTM software (DNASTAR, Inc., Madison. WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters were
  • the second nucleic acid sequences encode polypeptides that comprise the amino acid sequence of SEQ ID NO. 2 or an allelic variant thereof; or a fragment thereof that has DCC2 polypeptide activity.
  • the second nucleic acid sequence encodes a DCC2 polypeptide that comprises the amino acid sequence of SEQ ID NO. 2.
  • the second nucleic acid sequence encodes a polypeptide that comprises amino acids 19 to 64 of SEQ ID NO. 2. or an allelic variant thereof; or a fragment thereof that has DCC2 polypeptide activity.
  • the second nucleic acid sequence encodes a polypeptide that comprises amino acids 19 to 64 of SEQ ID NO. 2.
  • the second nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 2 or an allelic variant thereof: or a fragment thereof that has DCC2 poiypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 2. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of amino acids 19 to 64 of SEQ ID NO. 2 or an allelic variant thereof; or a fragment thereof that has DDC2 polypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of amino acids 19 to 64 of SEQ ID NO. 2.
  • the second nucleic acid sequences encode polypeptides that comprise the amino acid sequence of SEQ ID NO. 5 or an allelic variant thereof: or a fragment thereof that has DCC3 polypeptide activity.
  • the second nucleic acid sequence encodes a DCC3 polypeptide that comprises the amino acid sequence of SEQ ID NO. 5.
  • the second nucleic acid sequence encodes a polypeptide that comprises amino acids 21 to 83 of SEQ ID NO. 5. or an allelic variant thereof; or a fragment thereof that has DCC3 polypeptide activity.
  • the second nucleic acid sequence encodes a polypeptide that comprises amino acids 21 to 83 of SEQ ID NO. 5.
  • the second nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 5 or an allelic variant thereof; or a fragment thereof that has DCC3 polypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 2. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of amino acids 21 to 83 of SEQ ID NO. 5 or an allelic variant thereof; or a fragment thereof that has DDC3 polypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide 5 that consists of amino acids 19 to 64 of SEQ ID NO. 5.
  • the second nucleic acid sequences also encompass nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5. which differ from SEQ ID NO. 1 or SEQ ID NO. 4. respectively, by virtue of the degeneracy of the genetic code.
  • the present invention also relates to subsequences of 0 SEQ ID NO. 1 which encode fragments of SEQ ID NO. 2 which have DDC2 polypeptide activity and subsequences of SEQ ID NO. 4 which encode fragments of SEQ ID NO. 5 which have DDC3 polypeptide activity.
  • a subsequence of SEQ ID NO. 1 or SEQ ID NO. 4 is a nucleic acid sequence encompassed by SEQ ID NO. 1 or SEQ ID NO. 4, respectively, except that one or more 5 nucleotides from the 5' and/or 3' end have been deleted.
  • a subsequence of SEQ ID NO. 1 contains at least 114 nucleotides, more preferably at least 138 nucleotides. and most preferably at least 162 nucleotides.
  • a subsequence of SEQ ID NO. 4 contains at least 159 nucleotides, more preferably at least 189 nucleotides. and most preferably at least 219 nucleotides.
  • a fragment of SEQ ID NO. 2 or SEQ ID NO. 5 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence.
  • a fragment of SEQ ID NO. 2 contains at least 38 amino acid residues, more preferably at least 46 amino acid residues, and most preferably at least 54 amino acid residues.
  • a fragment of SEQ ID NO. 5 contains at least 53 amino 5 acid residues, more preferably at least 63 amino acid residues, and most preferably at least 73 amino acid residues.
  • allelic variant denotes any of two or more alternative forms of a gene occupying the same chomosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be o silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • the allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
  • the second nucleic acid sequences have a degree of homology to the mature polypeptide coding sequence of SEQ ID NO. 1 (i.e., nucleotides 1014 to 1151) of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which encode an active DDC2 polypeptide; or allelic variants and subsequences of SEQ ID NO.
  • polypeptide fragments which have DDC2 polypeptide activity or a degree of homology to the mature polypeptide coding sequence of SEQ ID NO. 4 (i.e., nucleotides 1041 to 1229) of at least about 50%>, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%. most preferably at least about 95%, and even most preferably at least about 97%, which encode an active DDC3 polypeptide; or allelic variants and subsequences of SEQ ID NO. 4 which encode polypeptide fragments which have DDC3 polypeptide activity.
  • the second nucleic acid sequences hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (b) a subsequence of (a) of at least 100 nucleotides. or (c) a complementary strand of (a) or (b) (J. Sambrook, E.F. Fritsch, and T. Maniatus.
  • the subsequence of SEQ ID NO. 1 or SEQ ID NO. 4 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has DDC2 or DDC3 polypeptide activity.
  • SUBST ⁇ UTE SHEET (RULE 26)
  • the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 4, or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having DDC2 or DDC3 polypeptide activity from strains of different genera or species according to methods well known in the art.
  • probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25. and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 2 P. ⁇ , 35 S, biotin. or avidin). Such probes are encompassed by the present invention.
  • genomic DNA or cDNA library prepared from such other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having DDC2 or DDC3 polypeptide activity.
  • Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis. or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is used in a Southern blot.
  • hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleic acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 5, its complementary strand, or a subsequence thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.
  • the nucleic acid probe is SEQ ID NO. 1.
  • the nucleic acid probe is a nucleic acid sequence which encodes the polypeptide of SEQ ID NO. 2, or a subsequence thereof.
  • the nucleic acid probe is nucleotides 1014 to 1151 of SEQ ID NO. 1. which encodes a mature polypeptide having DDC2 polypeptide activity.
  • the nucleic acid probe is the nucleic acid sequence contained in cosmid
  • nucleic acid probe is the mature DDC2 polypeptide coding region contained in cosmid 18H7 which is contained in Escherichia coli DSM 12060.
  • the nucleic acid probe is SEQ ID NO. 4.
  • the nucleic acid probe is a nucleic acid sequence which encodes the polypeptide of SEQ ID NO. 5, or a subsequence thereof.
  • the nucleic acid probe is nucleotides 1041 to 1229 of SEQ ID NO. 4, which encodes a mature polypeptide having DDC3 polypeptide activity.
  • the nucleic acid probe is the nucleic acid sequence contained in cosmid 34G12 contained in E. coli DSM 11924, wherein the nucleic acid sequence encodes a polypeptide having DDC3 polypeptide activity.
  • the nucleic acid probe is the mature DDC3 polypeptide coding region contained in cosmid 34G12 contained in E. coli DSM 11924.
  • very low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS. 200 ⁇ g/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies. 35% formamide for medium and medium- high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures.
  • the carrier material is finally washed three times each for 15 minutes using 2X SSC. 0.2%) SDS preferably at least at 45°C (very low stringency), more preferably at least at 50°C (low stringency), more preferably at least at 55°C (medium stringency), more preferably at least at 60°C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70°C (very high stringency).
  • stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5°C to about 10°C below the calculated T m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl. 0.09 M Tris-HCl pH 7.6, 6 mM EDTA. 0.5% NP-40, IX Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.
  • the carrier material is washed once in 6X SCC plus 0.1 % SDS for 15 minutes and twice each for 15 minutes using 6X SSC at about 5°C to about 10°C below the calculated T m .
  • the second nucleic acid sequences may be obtained by (a) hybridizing a DNA under very low. low, medium, medium-high, high, or very high stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1, (ii) a subsequence of (i). or (iii) a complementary strand of (i) or (ii), or (i) nucleotides 1041 to 1229 of SEQ ID NO. 4. (ii) a subsequence of (i). or (iii) a complementary strand of (i) or (ii): and (b) isolating the nucieic acid sequence.
  • the subsequence is preferably a sequence of at least 100 nucleotides such as a sequence which encodes a polypeptide fragment which has DDC2 or DDC3 polypeptide activity.
  • the second nucleic acid sequences encode variants of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids.
  • the amino acid sequences of the variant polypeptides may differ from the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5, or the mature polypeptide thereof, by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues.
  • amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terrninal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within the group of basic amino acids (arginine. lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine). hydrophobic amino acids (leucine. isoleucine and valine). aromatic amino acids (phenylalanine. tryptophan and tyrosine),
  • SUBST ⁇ UTE SHEET (RULE 26) and small amino acids (glycine, alanine, serine. threonine and methionine). Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979. In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu. Thr/Ser, Ala/Gly, Ala/Thr. Ser/Asn. Ala/Val, Ser/Gly, Tyr Phe, Ala/Pro, Lys/Arg, Asp/Asn. Leu/Ile. LeuVal. Ala/Glu. and Asp/Gly as well as these in reverse.
  • the variant sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of SEQ ID NO. 1 or SEQ ID NO. 4, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the polypeptide, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence.
  • nucleotide substitution see. e.g.. Ford et al, 1991, Protein Expression and Purification 2: 95-107.
  • amino acid residues essential to the activity of the polypeptide encoded by the second nucleic acid sequence may be identified according to procedures known in the art. such as site-directed mutagenesis or alanine-scanning mutagenesis (see. e.g.. Cunningham and Wells. 1989. Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for DDC2 or DDC3 polypeptide activity to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see. e.g.. de Vos et al. 1992. Science 255: 306-312; Smith et al, 1992. Journal of Molecular Biology 224: 899-904; Wlodaver et al. 1992, FEES letters 309: 59-64).
  • two second nucleic acid sequences are modified which encode a DDC2 and DDC3 polypeptide.
  • the second nucleic acid sequences may be obtained from microorganisms of any genus.
  • the term "obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted.
  • the polypeptide encoded by the second nucleic acid sequence is secreted extracellularly.
  • the second nucleic acid sequences may be obtained from a fungal source, and more preferably from a yeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain; or more preferably from a filamentous fungal strain such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus. Filibasidium. Fusarium. Gibberella. Humicola. Magnaporthe. Mucor. Myceliophthora. Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces. Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain.
  • a yeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizos
  • the second nucleic acid sequences are obtained from a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii. Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis strain.
  • the second nucleic acid sequences are obtained from an Aspergillus aculeatus, Aspergillus awamori. Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans. Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides. Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi.
  • Fusarium oxysporum Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum.
  • Fusarium sporotrichioides Fusarium sulphureum.
  • Fusarium torulosum Fusarium trichothecioides, Fusarium venenatum, Humicola insolens. Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa.
  • DCC2 polypeptide is obtained from an Aspergillus oryzae strain, and most preferably from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the polypeptide with the amino acid sequence of SEQ ID NO. 2.
  • the nucleic acid sequence is the sequence contained in cosmid 18H7 which is contained in Escherichia coli DSM 12060.
  • the nucleic acid sequence is nucleotides 1014 to 1151 of SEQ ID NO. 1
  • the second nucleic acid sequence encoding a DDC3 polypeptide is obtained from an Aspergillus oryzae strain, and most preferably from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the polypeptide with the amino acid sequence of SEQ ID NO. 2.
  • the nucleic acid sequence is the sequence contained in cosmid 34G12 contained in E. coli DSM 11924.
  • the nucleic acid sequence is nucleotides 1041 to 1229 of SEQ ID NO. 4.
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs. regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • the polypeptides may be obtained from microorganisms which are taxonomic equivalents of Aspergillus as defined by Raper, K.D. and Fennel. D.I.. 1965. The Genus Aspergillus.
  • Aspergilli are mitosporic fungi characterized by an aspergillum comprised of a conidiospore stipe with no known teleomorphic states terminating in a vesicle, which in turn bears one or two layers of synchronously formed specialized cells, variously referred to as sterigmata or phialides, and asexually formed spores referred to as conidia.
  • Known teleomorphs of Aspergillus include Eurotium, Neosartorya, and Emericella.
  • SUBSTTTUTE SHEET (RULE 26) microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art.
  • the nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see. e.g.. Sambrook et al, 1989. supra).
  • the second nucleic acid sequences may be mutant nucleic acid sequences comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO. 1. in which the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 19 to 64 of SEQ ID NO. 2. or at least one mutation in the mature polypeptide coding sequence of SEQ ID NO. 4, in which the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 1 to 83 of SEQ ID NO. 5.
  • the techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA. or a combination thereof.
  • the cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g.. Innis et al. 1990. PCR: A Guide to Methods and Application. Academic Press. New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR). ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligated activated transcription
  • NASBA nucleic acid sequence-based amplification
  • the second nucleic acid sequence may be cloned from a strain of Aspergillus. or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.
  • the mutant filamentous fungal cell may be constructed by reducing or eliminating expression of one or more of the second nucleic acid sequences described herein using methods well known in the art, for example, insertions, disruptions. replacements, or deletions.
  • the second nucleic acid sequence(s) to be modified or inactivated may be. for example, the coding region or a part thereof essential for activity, or a regulatory or control element required for the expression of the coding region.
  • An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i. e.. a part which is sufficient for affecting expression of the nucleic acid sequence.
  • Other control sequences for possible modification include, but are not limited to. a leader, polyadenylation sequence, propeptide sequence, signal 5 sequence, transcription terminator, and transcriptional activator.
  • Modification or inactivation of the second nucleic acid sequence(s) may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the second nucleic acid sequence(s) has been reduced or eliminated.
  • the mutagenesis which may be specific or random, may be performed, for example, by ic use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide. or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods.
  • Examples of a physical or chemical mutagenizing agent suitable for the present is purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), O-methyl hydroxylamine. nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
  • the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice
  • Modification or inactivation of the second nucleic acid sequence(s) may also be accomplished by introduction, substitution, or removal of one or more nucleotides in the sequence or a regulatory element required for the transcription or translation thereof.
  • nucleotides may be inserted or removed so as to result in the introduction of a stop codon. the removal of the start codon. or a change of the open reading frame.
  • a modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art.
  • the modification may be performed in vivo, i.e.. directly on the cell expressing
  • the modification be performed in vitro as exemplified below.
  • nucleic acid sequence(s) by a filamentous fungal cell of choice is based on techniques of gene replacement, gene deletion, or gene disruption.
  • a nucleic acid sequence corresponding to the endogenous gene or gene fragment of interest is mutagenized in vitro to produce a defective nucleic acid sequence which is then transformed into the parent cell to produce a defective gene.
  • the defective nucleic acid sequence replaces the endogenous gene or gene fragment. It may be desirable that the defective gene or gene fragment also encodes a marker which may be used for selection of transformants in which the nucleic acid sequence has been modified or destroyed.
  • modification or inactivation of the second nucleic acid sequence(s) may be performed by established anti-sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the gene. More specifically, expression of the gene by a filamentous fungal cell may be reduced or eliminated by introducing a nucleotide sequence complementary to the second nucleic acid sequence(s) which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA. the amount of protein translated is thus reduced or eliminated.
  • a nucleic acid sequence homologous or complementary to the second nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 4 may be obtained from any microbial source as described infra.
  • the choice of the source of the nucleic acid sequence will depend on the filamentous fungal cell, but preferred sources are fungal sources, e.g.. yeast and filamentous fungi.
  • Preferred filamentous fungal sources include, but are not limited to. species of Acremonium. Aspergillus. Fusarium, Humicola. Myceliophthora. Mucor. Neurospora. Penicillium, Phanerochaete, Thielavia. Tolypocladium, and Trichoderma.
  • Preferred yeast sources include, but are not limited to. species of Candida. Hansenula. Kluyveromyces. Pichia. Saccharomyces. Schizosaccharomyces. and Yarrowia.
  • the nucleic acid sequence may be native to the filamentous fungal cell.
  • the methods of the present invention are not limited to a particular order for obtaining the mutant filamentous fungal cell.
  • the modification of the second nucleic acid sequence(s) as described herein may be introduced into the parent cell at any step in the construction of the cell for the production of a polypeptide.
  • the second nucleic acid sequence(s) of the mutant filamentous fungal cell has already been modified prior to the introduction of a first nucleic acid sequence encoding a heterologous polypeptide.
  • the filamentous fungal cell may be a wild-type cell or a mutant thereof.
  • the filamentous fungal cell is an Acremonium, Aspergillus. Aureobasidium. Cryptococcus. Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe. Mucor. Myceliophthora, Myrothecium, Neocallimastix. Neurospora. Paecilomyces. Penicillium. Piromyces, Schizophyllum, Talaromyces. Thermoascus, Thielavia. Tolypocladium. or Trichoderma cell.
  • the filamentous fungal cell is an Aspergillus aculeatus. Aspergillus awamori, Aspergillus foetidus. Aspergillus japonicus, Aspergillus nidulans. Aspergillus niger, ox Aspergillus oryzae cell.
  • the filamentous fungal cell is a Fusarium bactridioides.
  • Fusarium crookwellense (synonym of Fusarium cerealis), Fusarium culmorum. Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi. Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium solani, Fusarium sporotrichioides. Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides.
  • the filamentous fungal cell is a Gibberella pulicaris. Gibberella zeae. Humicola insolens. Humicola lanuginosa, Mucor miehei. Myceliophthora thermophila, Myrothecium roridin. Neurospora crassa, or Penicillium purpurogenum cell.
  • the filamentous fungal cell is a Trichoderma harzianum. Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
  • the mutant filamentous fungal cell is cultivated in a nutrient medium suitable for production of the polypeptide encoded by the first nucleic acid sequence using methods known in the art.
  • the cell may be cultivated by shake flask cultivation.
  • small-scale or large-scale fermentation including continuous, batch, fed-batch, or solid state fermentations
  • laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the heterologous polypeptide to be expressed
  • SUBST ⁇ UTE SHEET (RULE 26) and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g.. in catalogues of the American Type Culture Collection).
  • the polypeptide can be recovered directly from the medium if secreted.
  • the polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods may include use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate. SDS-PAGE. or any other method known in the art. For example, an enzyme assay may be used to determine the activity of the polypeptide. Procedures for determining enzyme activity are known in the art for many enzymes.
  • the resulting polypeptide may be isolated by methods known in the an.
  • the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation. filtration, extraction, spray- drying, evaporation, or precipitation.
  • the isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g.. ammonium sulfate precipitation), or extraction (see, e.g.. Protein Purification. J.-C. Janson and Lars Ryden.
  • the polypeptide encoded by the first nucleic acid sequence may be any polypeptide native or foreign to the mutant filamentous fungal cell.
  • the term '"polypeptide is not meant herein to refer to a specific length of the encoded product and. therefore, encompasses peptides. oligopeptides. and proteins.
  • the term "heterologous polypeptide” is defined herein as a polypeptide which is not native to the filamentous fungal cell.
  • the mutant filamentous fungal cell may contain one or more copies of the nucleic acid sequence encoding the heterologous polypeptide.
  • the polypeptide is a hormone, hormone variant. enzyme, receptor or portion thereof, antibody or portion thereof, or reporter.
  • the polypeptide is an oxidoreductase. transferase. hydrolase. lyase. isomerase. or ligase.
  • the polypeptide is an aminopeptidase. amylase. carbohydrase. carboxypeptidase. catalase. cellulase. chitinase. cutinase, cyclodextrin glycosyltransferase. deoxyribonuclease. esterase.
  • alpha- galactosidase beta-galactosidase, glucoamylase, alpha-glucosidase. beta-glucosidase, invertase. laccase, lipase. mannosidase, mutanase, oxidase. pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease. transglutaminase or xylanase.
  • the first nucleic acid sequence encoding a polypeptide of interest may be obtained from any prokaryotic. eukaryotic. or other source.
  • the mutant filamentous fungal cell may also be used for the recombinant production of polypeptides which are native to the cell.
  • the native polypeptides may be recombinantly produced by, for example, placing a gene encoding the polypeptide under the control of a different promoter to enhance expression of the polypeptide. to expedite export of a native polypeptide of interest outside the cell by use of a signal sequence, and to increase the copy number of a gene encoding the polypeptide normally produced by the cell.
  • the present invention also encompasses, within the scope of the term "heterologous polypeptide " .
  • nucleic acid sequence encoding a polypeptide may be of genomic. cDNA. RNA. semisynthetic. synthetic origin, or any combinations thereof.
  • polypeptides may also be an engineered variant of a polypeptide.
  • the polypeptides may further include fused or hybrid polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof.
  • a fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide.
  • Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the
  • the hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the mutant filamentous fungal cell.
  • An isolated first nucleic acid sequence encoding a polypeptide of interest may be manipulated in a variety of ways to provide for expression of the polypeptide. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to. transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.
  • nucleic acid construct is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.
  • nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence.
  • coding sequence as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide.
  • the boundaries of the coding sequence are generally determined by the ATG start codon located just upstream of the open reading frame at the 5 " end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA.
  • a coding sequence can include, but is not limited to, genomic, cDNA. RNA, semisynthetic. synthetic. recombinant, or any combinations thereof.
  • control sequences is defined herein to include all components which are necessary or advantageous for the expression of a heterologous polypeptide.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide.
  • control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided
  • SUBST ⁇ UTE SHEET (RULE 26) with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a heterologous polypeptide.
  • linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a heterologous polypeptide.
  • operably linked is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a heterologous polypeptide.
  • the control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a filamentous fungal cell for expression of the nucleic acid sequence.
  • the promoter sequence contains transcriptional control sequences which mediate the expression of the heterologous polypeptide.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the filamentous fungal cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the cell.
  • promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase.
  • Aspergillus nidulans acetamidase Aspergillus oryzae acetamidase (amdS). Fusarium oxysporum trypsin-like protease (U.S. Patent No. 4.288,627), and mutant, truncated, and hybrid promoters thereof.
  • Particularly preferred promoters are the NA2- tpi promoters (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase). glucoamylase. and TAKA amylase promoters.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a filamentous fungal cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the heterologous polypeptide. Any terminator which is functional in the filamentous fungal cell may be used in the present invention.
  • Preferred terminators are obtained from the genes encoding Aspergillus oryzae TAKA amylase. Aspergillus niger glucoamylase. Aspergillus nidulans anthranilate synthase. Aspergillus niger alpha-glucosidase. and Fusarium oxysporum trypsin-like protease.
  • the control sequence may also be a suitable leader sequence, a nontranslated region of a mRNA which is important for translation by the filamentous fungal cell.
  • the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the heterologous polypeptide. Any leader sequence which is functional in the filamentous fungal cell may be used in the present invention.
  • Preferred leaders are obtained from the genes encoding Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
  • the control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3 ' terminus of the nucleic acid sequence and which, when transcribed, is recognized by a filamentous fungal cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the filamentous fungal cell may be used in the present invention.
  • Preferred polyadenylation sequences are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.
  • the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of the heterologous polypeptide and directs the encoded polypeptide into the cell's secretory pathway.
  • the 5 " end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide.
  • the 5 * end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence.
  • the foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region.
  • the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the polypeptide.
  • any signal peptide coding region which directs the expressed heterologous polypeptide into the secretory pathway of a filamentous fungal cell may be used in the present invention.
  • Effective signal peptide coding regions for filamentous fungal host cells aie the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase. Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
  • the control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding region may be obtained from the Rhizomucor miehei aspartic proteinase gene, or the Myceliophthora thermophila laccase gene (WO 95/33836).
  • the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
  • the nucleic acid constructs may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous for directing the expression of the heterologous polypeptide, e.g., a transcriptional activator (e.g., a trans- acting factor), chaperone, and processing protease. Any factor that is functional in a filamentous fungal cell may be used in the present invention.
  • the nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the heterologous polypeptide.
  • regulatory sequences which allow the regulation of the expression of the heterologous polypeptide relative to the growth of the filamentous fungal cell.
  • regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences.
  • Other examples of regulatory sequences are those which allow for gene amplification, e.g., the metallothionein genes which are amplified with heavy metals. In these cases, the nucleic
  • SUBST ⁇ UTE SHEET (RULE 26) acid sequence encoding the heterologous polypeptide would be operably linked with the regulatory sequence.
  • nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the heterologous polypeptide at such sites.
  • the nucleic acid sequence encoding the heterologous polypeptide may be expressed by inserting the sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence encoding the heterologous polypeptide.
  • the choice of the vector will typically depend on the compatibility of the vector with the filamentous fungal cell into which the vector is to be introduced.
  • the vectors may be linear or closed circular plasmids.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the filamentous fungal cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the filamentous fungal cell, or a transposon.
  • the vectors preferably contain one or more selectable markers which permit easy selection of transformed filamentous fungal cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophv to auxotrophs. and the like.
  • a selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to. ⁇ mdS (acetamidase). ⁇ rgB (ornithine carbamoyltransferase), bar (phosphinothricin
  • SUBST ⁇ UTE SHEET (RULE 26) acetyltransferase).
  • hygB hygromycin phosphotransferase.
  • niaD nitrate reductase
  • pyrG orotidine-5 " -phosphate decarboxylase
  • sC sulfate adenyltransferase
  • trpC anthranilate synthase
  • the vectors preferably contain an element(s) that permits stable integration of the vector into a filamentous fungal cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.
  • “Introduction” means introducing a vector comprising the nucleic acid sequence into a filamentous fungal cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the chromosome occurs by homologous recombination, non-homologous recombination, or transposition.
  • the introduction of an expression vector into a filamentous fungal cell may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.
  • the vector may rely on the nucleic acid sequence encoding the heterologous polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination.
  • the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the filamentous fungal cell.
  • the additional nucleic acid sequences enable the vector to be integrated into the genome at a precise location(s) in the chromosome(s).
  • the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs.
  • the integrational elements may be any sequences that are homologous with the target sequence in the genome of the filamentous fungal cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the filamentous fungal cell in question.
  • the mutant filamentous fungal cell may additionally contain modifications of one or more third nucleic acid sequences which encode proteins that may be detrimental to the production, recovery, and/or application of the polypeptide of interest.
  • the modification reduces or eliminates expression of the one or more third nucleic acid sequences resulting in a mutant cell which may produce more of the polypeptide than the mutant cell without the modification of the third nucleic acid sequence when cultured under the same conditions.
  • the third nucleic acid sequence may encode any protein or enzyme.
  • the enzyme may be an aminopeptidase. amylase. carbohydrase. carboxypeptidase. catalase, cellulase. chitinase. cutinase.
  • the third nucleic acid sequence preferably encodes a proteolytic enzyme, e.g.. an aminopeptidase. carboxypeptidase, or protease.
  • the present invention also relates to methods for producing a mutant filamentous fungal cell, comprising:
  • SUBST ⁇ UTE SHEET (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
  • nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4;
  • nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (b) a subsequence of (a) of at least 100 nucleotides. or (c) a complementary strand of (a) or (b); (iv) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
  • step (B) identifying the mutant from step (A) comprising the modified nucleic acid sequence.
  • the present invention also relates to mutant filamentous fungal cells for producing a polypeptide which comprise a first nucleic acid sequence encoding the polypeptide and a modification of one or more second nucleic acid sequences having:
  • nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
  • nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4;
  • nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii); (d) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
  • SUBST ⁇ UTE SHEET (RULE 26) (e) an allelic variant of (a), (b). or (c); and
  • the present invention also relates to the isolated DDC2 and DDC3 polypeptides, as well as fragments, allelic variants, and variants thereof encoded by the nucleic acid sequences described herein.
  • the isolated polypeptides are selected from the group consisting of:
  • polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4. (ii) a subsequence of (i) of at least 100 nucleotides. or (iii) a complementary strand of (i), (ii), or (iii); (c) a variant of the polypeptide having an amino acid sequence of SEQ ID
  • SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and or insertion of one or more amino acids
  • the isolated DDC2 and DDC3 polypeptides of the present invention have at least 20%. preferably at least 40%. more preferably at least 60%. even more preferably at least 80%. even more preferably at least 90%), and most preferably at least 100% of the activity of the polypeptide of SEQ ID NO. 2 or SEQ ID NO. 5.
  • the DDC2 and DDC3 polypeptides may be obtained from a fungal source, and more preferably from a yeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces. Schizosaccharomyces, or Yarrowia strain; or a filamentous fungal strain such as an Acremonium. Aspergillus, Aureobasidium.
  • Cryptococcus Filibasidium, Fusarium. Gibberella. Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix. Neurospora, Paecilomyces, Penicillium, Piromyces,
  • a DDC2 polypeptide of the present invention is obtained from an Aspergillus oryzae strain, and most preferably from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the polypeptide with the amino acid sequence of SEQ ID NO. 2.
  • a DDC3 polypeptide of the present invention is obtained from an Aspergillus oryzae strain, and most preferably from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the polypeptide with the amino acid sequence of SEQ ID NO. 2.
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs. regardless of the species name by which they are known as described supra.
  • the DDC2 and DDC3 polypeptides may be isolated using techniques as described herein.
  • an "isolated" polypeptide is a polypeptide which is essentially free of other polypeptides, e.g., at least about 20%) pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90%) pure, and even most preferably about 95%> pure, as determined by SDS-PAGE.
  • the present invention also relates to isolated nucleic acid sequences which encode DDC2 and DDC3 polypeptides, and fragments thereof, as described herein.
  • the nucleic acid sequence is set forth in SEQ ID NO.
  • nucleic acid sequence is the sequence contained in cosmid 18H7 which is contained in Escherichia coli DSM 12060. In another preferred embodiment, the nucleic acid sequence is the polypeptide coding region of SEQ ID NO. 1. In another preferred embodiment, the nucleic acid sequence is the polypeptide coding region contained in cosmid 18H7 which is contained in Escherichia coli DSM 12060.
  • nucleic acid sequence is set forth in SEQ ID NO. 4. In another preferred embodiment, the nucleic acid sequence is the polypeptide coding region of SEQ ID NO. 4. In another preferred embodiment, the nucleic acid sequence is the sequence contained in cosmid 34G12 which is contained in Escherichia coli DSM 11924. In another preferred embodiment, the nucleic acid sequence is the polypeptide coding region contained in cosmid 34G12 which is contained in Escherichia
  • the present invention also relates to isolated mutant nucleic acid sequences comprising at least one mutation in the polypeptide coding sequence of SEQ ID NO. 1 or SEQ ID NO. 4. in which the mutant nucleic acid sequence encodes a polypeptide which consists of SEQ ID NO. 2 or SEQ ID NO. 5, respectively.
  • isolated nucleic acid sequence refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20%) pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90%> pure as determined by agarose electrophoresis.
  • an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced.
  • the cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated.
  • the nucleic acid sequence may be of genomic. cDNA. RNA. semisynthetic. synthetic origin, or any combinations thereof.
  • Modification of a nucleic acid sequence encoding a DDC2 or DDC3 polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the DDC2 or DDC3 polypeptide.
  • the term "substantially similar" to the DDC2 or DDC3 polypeptide refers to non-naturally occurring forms of the polypeptide.
  • These polypeptides may differ in some engineered way from the DDC2 or DDC3 polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like.
  • the variant polypeptides may be constructed as described earlier.
  • the present invention also relates to isolated nucleic acid sequences produced by (a) hybridizing a DNA under very low, low. medium, medium-high, high, or very high stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1, (ii) a subsequence of (i). or (iii) a complementary strand of (i) or (ii); or with (i) nucleotides 1041 to 1229 of SEQ ID NO. 4.
  • the subsequence is preferably a sequence of at least 100 nucleotides such as a sequence which encodes a polypeptide fragment which has DDC2 or DDC3 polypeptide activity.
  • the present invention further relates to methods for producing a mutant nucleic acid sequence, comprising introducing at least one mutation into the polypeptide coding sequence of SEQ ID NO. 1 or SEQ ID NO. 4, or a subsequence thereof, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of SEQ ID NO. 2 or SEQ ID NO. 5. respectively, or a fragment thereof which has DDC2 or DDC3 polypeptide activity.
  • the introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure which utilizes a supercoiled. double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation.
  • the oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with Dpnl which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA.
  • the present invention also relates to nucleic acid constructs, recombinant expression vectors, and host cells containing the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 4, subsequences or homologues thereof as described herein, for expression of the sequences.
  • the constructs and vectors may be constructed as described herein.
  • the host cell may be any cell suitable for the expression of the nucleic acid sequence, and preferably is a fungal cell, and more preferably a filamentous fungal cell selected from the group described herein.
  • the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the present invention also relates to methods for producing a DDC2 or DDC3 polypeptide comprising (a) cultivating a strain, which in its wild-type form is capable of producing the polypeptide. under conditions conducive for production of the DDC2 or DDC3 polypeptide: and (b) recovering the polypeptide from the cultivation medium.
  • the strain is of the genus Aspergillus, and more preferably Aspergillus oryzae.
  • the present invention also relates to methods for producing a DDC2 or DDC3 polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the DDC2 or DDC3 polypeptide; and (b) recovering the DDC2 or DDC3 polypeptide from the cultivation medium.
  • the cells are cultivated in a nutrient medium suitable for production of the DDC2 or DDC3 polypeptide using methods known in the art as described herein.
  • the resulting DDC2 or DDC3 polypeptide may be recovered and purified by methods known in the art as described herein.
  • the present invention also relates to nucleic acid constructs comprising a gene encoding a protein operably linked to a nucleic acid sequence consisting of nucleotides 960 to 1013 of SEQ ID NO:l encoding a signal peptide consisting of amino acids 1 to 18 of SEQ ID NO:2 or nucleotides 981 to 1040 of SEQ ID NO. 4 encoding a signal peptide consisting of amino acids 1 to 20 of SEQ ID NO. 5, wherein the gene is foreign to the nucleic acid sequence.
  • the present invention also relates to recombinant expression vectors and recombinant host cells comprising such nucleic acid constructs.
  • the present invention also relates to methods for producing a protein comprising (a) cultivating such a recombinant host cell under conditions suitable for production of the protein; and (b) recovering the protein.
  • the nucleic acid sequences may be operably linked to foreign genes with other control sequences. Such other control sequences are described above.
  • the protein may be native or heterologous to a host cell.
  • the term “protein” also encompasses two or more polypeptides combined to form the encoded product.
  • the proteins also encompasses two or more polypeptides combined to form the encoded product.
  • SUBST ⁇ SHEET include hybrid polypeptides which comprise a combination of partial or complete polypeptide sequences obtained from at least two different proteins wherein one or more may be heterologous or native to the host cell. Proteins further include naturally occurring allelic and engineered variations of the above mentioned proteins and hybrid proteins.
  • the protein is a hormone, hormone variant, enzyme, receptor or a portion thereof, antibody or a portion thereof, or reporter.
  • the protein is an oxidoreductase. transferase, hydrolase. lyase, isomerase, or ligase.
  • the protein is an aminopeptidase, amylase. carbohvdrase, carboxypeptidase, catalase. cellulase. chitinase. cutinase, cyclodextrin glycosyltransferase. deoxyribonuclease. esterase. alpha-galactosidase, beta- galactosidase.
  • glucoamylase alpha-glucosidase. beta-glucosidase. invertase. laccase. lipase. mannosidase. mutanase. oxidase, pectinolytic enzyme, peroxidase, phytase. polyphenoloxidase. proteolytic enzyme, ribonuclease. transglutaminase. or xylanase.
  • the gene may be obtained from any prokaryotic. eukaryotic, or other source.
  • Chemicals used as buffers and substrates were commercial products of at least reagent grade.
  • Example 1 Production of CAREZYMETM by Aspergillus oryzae strains HC4.01 and 27
  • CAREZYMETM An Aspergillus oryzae strain producing an extracellular beta-l,4-endoglucanase (CAREZYMETM) was constructed as described in WO 91/17243.
  • CAREZYMETM is a Trichoderma harzianum cellulase produced by Novo Nordisk A/S, Bagsvaerd. Denmark.
  • Transformant no. 27 of pSX320 in Aspergillus oryzae A1560-T40 was selected for mutagenesis in order to create a superior strain.
  • Transformant No. 27 was
  • HC4.01 was shown to have an increase in CAREZYMETM yield in shake flask fermentations as well as in tank fermentations.
  • mRNA preparations from the two strains were compared by the differential display method.
  • Aspergillus oryzae strains HC4.01 and 27 were grown side-by-side at 34°C, pH 7, 800-1100 rpm for 5 days in 2 liter fed-batch fermentations composed of Nutriose. yeast extract. MgS0 4 -7H 2 O, citric acid, K 2 SO 4 , KH 2 PO 4 , urea, trimethyl glycine. and trace metals solution.
  • CAREZYMETM activity was measured as a viscosity change (reduction) of a 1%
  • SUBST ⁇ UTE SHEET (RULE 26) strains HC4.01 and 27 were mixed in two separate reactions with 1.0 ⁇ M of primer, 15 ⁇ l of 5x first strand synthesis buffer (Life Technologies. Gaithersburg, MD), 10 ⁇ M dithiothreitol, and 20 ⁇ M dNTP in a final volume of 75 ⁇ l.
  • the solution was incubated at 65°C for 5 minutes, quick chilled on ice. and 500 units of Superscript IITM reverse s transcriptase (Life Technologies, Gaithersburg, MD) was added. After a 1 hour incubation at 37°C. the reaction was stopped by heat treatment for 5 minutes at 95°C and the first strand samples were stored at -20°C.
  • PCR amplification reactions were set up in triplicate for both control and mutant RNAs with each primer pair (240 different primer pairs). The amplifications were composed of 1.0 ⁇ l of the cDNA reaction. 2.0 ⁇ l of 10 x PCR buffer (500 mM KCl; 100 mM Tris-HCl pH 9.0; 15 mM MgCl 2 ; 1% (w/v) gelatin; 1% (v/v) Triton X-100), 1.5 ⁇ l of 25 mM dNTP.
  • 10 x PCR buffer 500 mM KCl; 100 mM Tris-HCl pH 9.0; 15 mM MgCl 2 ; 1% (w/v) gelatin; 1% (v/v) Triton X-100
  • the film and gel were aligned using the trace from the fluorescent ruler. Differentially displayed bands were marked and excised from the gel with a scalpel.
  • the gel slice including the Whatman filter paper, was soaked for 10 minutes at ambient temperature, then incubated for 15 minutes at 95°C in 0.1 ml of water in a 1.5 ml
  • PCR amplified bands were ligated into pCR2.1 (Invitrogen, La Jolla, CA) that had been linearized with EcoRV and TA-tailed following the protocol described in Hadjeb and Berkowitz, 1996, Biotechniques 20: 20-22.
  • Ligations were transformed into competent Escherichia coli DH5 ⁇ cells, and the resulting colonies were screened by blue/white selection on agarose plates containing X-gal (5-bromo-4-chloro-3-indolyl- ⁇ - D-galactopyranoside). For each differentially displayed fragment, six colonies were picked for further analysis.
  • Example 3 Screening of differentially displayed fragments Each of the groups of six colonies described in Example 2 were grown overnight in 3 ml of Luria broth (1% bactotryptone-0.5% yeast extract-0.5% sodium chloride) supplemented with 100 ⁇ g of ampicillin per ml. The cells were collected by centrifugation. The cell pellets were resuspended in 0.3 ml of 10 mM EDTA-50 mM Tris-HCl pH 8.0 buffer containing 100 ⁇ g of RNase A per ml, and lysed by the subsequent addition of 0.3 ml of 1% SDS-200 mM NaOH. After a 5 minute incubation at ambient temperature. 0.3 ml of 2.0 M potassium acetate pH 5.5 was added.
  • the lysate was clarified by a 10 minute centrifugation at 15,0000 x g at 4°C. The supernatant was transferred to an Eppendorf tube and then 0.7 ml of isopropanol was added to precipitate the DNA. The DNA was collected by a 15 minute centrifugation at 15,000 x g at 4°C. The pellet was resuspended in 0.1 ml of 1 mM EDTA-10 mM Tris-HCl pH 8.0 and the sample stored at -20°C.
  • the cloned fragments were initially screened for differential expression by rehybridization back to their originating PCR reaction (the unused portion of the reaction that was stored at -20°C).
  • the prepared DNA from each colony was diluted 1 :20 with H 2 0 and then applied to duplicate nylon membranes using a slot blot apparatus.
  • Each duplicate membrane carried clones from those fragments identified using four separate primer pairs (i.e.. fragments excised from lanes containing the amplicons from oligo E and primers 01, 03. 06 and 08).
  • the membranes were baked for 1-2 hours at 80°C in a vacuum oven, and then hybridized under high stringency conditions (50% formamide, 6xSSC, 42°C).
  • the hybridization probe for one filter comprised the denatured products from the triplicate reactions amplified using cDNA from the mutant strain and the four primer pairs corresponding to the colonies be screened; for the other filter the cDNA was synthesized from the control strain. If the colonies were obtained from fragments produced using oligo D and primers 01, 03, 06, and 08 and Aspergillus oryzae HC4.01 cDNA, then those PCR reactions were added to the hybridization mix. Membranes were washed once for 15 minutes in 0.5 x SSC, 0.1%) SDS at 65°C, and analyzed on a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA).
  • the DNA was radiolabeled by nick translation (Maniatis et al, supra) with ⁇ [ 32 P]dCTP (Amersham. Arlington Heights, IL) and added to the hybridization buffer at an activity of approximately 1 x 10 6 cpm per ml of buffer.
  • Example 4 Isolation and characterization of cDNA clones for DDC2 and DDC3
  • the DDC2 insert from pToC367 (approximately 250 bp) was sequenced on an
  • ABI Automated DNA Sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer ' s instructions to obtain a partial cDNA sequence, which contained a stretch of adenosine residues, the so-called "polyA tail" at one end, indicating the direction of transcription of the gene.
  • Double stranded cDNA was synthesized according to the following protocol.
  • the reaction was centrifuged for 30 seconds in a microfuge, and then the following were added: 2.0 ⁇ l of 5x first strand synthesis buffer transcriptase (Life Technologies, Gaithersburg, MD), 1.0 ⁇ l of 20 mM dithiothreitol, 1.0 ⁇ l of 10 mM dNTP, and 200 units of SuperscriptTM II reverse transcriptase (Life Technologies,
  • Second strand cDNA was prepared by PCR amplification in which 2 ⁇ l of the first strand reaction was mixed with 10 ⁇ l of lOx PCR buffer, 2 ⁇ l of 10 mM dNTP, 2 ⁇ l of 5' PCR primer, 2 ⁇ l of CDS/3' PCR primer, and 2 ⁇ l of 50x Advantage KlenTaq polymerase mix (Clontech, Palo Alto, CA). Amplification was performed in a thermocycler programmed for 1 cycle at 95°C for 1 minute, 20 cycles each at 95°C for 15 seconds and 68°C for 5 minutes.
  • an oligonucleotide specific for DDC2 was synthesized to isolate cDNA clones.
  • the primer was oriented opposite to transcription of the genes.
  • the primer had the following sequence:
  • oligonucleotides specific for DDC3 were synthesized to isolate cDNA clones.
  • the primers were oriented opposite to transcription of the genes.
  • the primers had the following sequences:
  • nested PCR was run.
  • the first PCR reaction was run with primer 26183 and the 5' primer from the Cap Finder Kit (Clontech. Palo Alto. CA) for 30 cycles with an annealing temperature of 60°C.
  • the 5 ' primer is part of the Clontech CAP Finder Kit which will hybridize to the 5 ' end of the cDNA created with this kit.
  • An aliquot from this PCR reaction was used as template for a new reaction with primer 26184 and the 5 'Cap Finder primer. Again the reaction was run for 30 cycles with an annealing temperature of 60°C.
  • the second PCR reaction a fragment of approximately 400 bp was obtained.
  • the fragments were individually cloned into the pCRII vector (Invitrogen. San Diego CA) and sequenced on an ABI Automated DNA Sequencer according to the manufacturer ' s instructions.
  • the DDC2 full-length cDNA sequence and deduced amino acid sequence obtained are shown in SEQ ID NOs. 3 and 2, respectively.
  • the DDC3 full-length cDNA sequence and deduced amino acid sequence obtained are shown in SEQ ID NOs. 6 and 5, respectively.
  • the DDC3 polypeptide contained three repeats of 18 amino acids of the following sequence: DDGAIRIPVKGVPEPEKR (SEQ ID NO. 5).
  • the third repeat showed some deviation at the C-terminal where) the two last amino acids in the repeat are Lys and Arg.
  • This dibasic site is known to be a cleavage site for the kex2 protease involved in maturation of secreted proteins in yeast and fungi.
  • the gene structure suggested that DDC3 encodes a protein that is secreted and processed into possibly 3 small peptides.
  • the gene structure of DDC3 resembles the structure of the alpha-factor gene of Saccharomyces cerevisae (Kurjan et al, 1982, Cell 30: 933-943).
  • the cDNA clone of DDC2 was radiolabeled by nick translation (Sambrook et al. , supra) with ⁇ [ 32 P]dCTP (Amersham, Arlington Heights, IL) and added to the hybridization buffer at an activity of approximately 1 x 10 6 cpm per ml of buffer and used as a probe against a cosmid library of Aspergillus oryzae IFO 4177.
  • the cosmid library was constructed using the SuperCosl Cosmid Vector Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.
  • Genomic DNA of Aspergillus oryzae IFO 4177 was prepared from protoplasts made by standard procedures (Christensen et. al, 1988, Biotechnology 6: 1419-1422). After the protoplasts were isolated, they were pelleted by centrifugation at 2500 rpm for 5 minutes in a Labofuge T (Heto, Denmark). The pellet was then suspended in 10 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 ⁇ g/ml proteinase K and 0.5% SDS as stated in the manual from the SuperCosl Kit. The remaining steps of the DNA preparation were performed according to the manufacturer's instructions accompanying the kit.
  • the size of the genomic DNA was analyzed by electrophoresis using a CHEF- gel apparatus (BioRad Laboratories, Hercules, CA). A 1% agarose gel was run for 20 hours at 200 volts with a 10-50 second pulse. The gel was stained with ethidium bromide and photographed. The DNA was found to be from about 50 to greater than 100 kb in size. The DNA was partially digested with Sau3A. The size of the digested DNA obtained was 20-50 kb as determined by the same type of CHEF-gel analysis as above. A CsCl gradient banded SuperCosl cosmid was prepared according to the manufacturer's instructions. Ligation and packaging was likewise performed according to the manufacturer ' s instructions. After titration of the library, all of the packaging mix from one ligation and packaging was transfected into E. coli XL 1 -Blue MR (Stratagene,
  • Plasmid DNA was prepared from cosmid 18H7 and sequenced on an ABI
  • Plasmid DNA was also prepared and from cosmid 34G12 and sequenced on an
  • New primers were synthesized in order to obtain sequence further upstream and downstream.
  • the resulting genomic nucleic acid sequences and the deduced amino acid sequences of DDC2 and DCC3 are shown in Figure 1 (SEQ ID NOs. 1 and 2, respectively) and Figure 2 (SEQ ID NOs. 4 and 5, respectively), respectively.
  • Example 6 Construction of a deletion plasmid for DDC2 A plasmid designed for deletion of the DDC2 gene in Aspergillus oryzae was constructed by cloning 1 kb of sequence upstream of the start codon and 1 kb of sequence downstream of the stop codon into a vector separated by the Aspergillus oryzae pyrG gene.
  • a 5' fragment was obtained by PCR using cosmid 18H7 as template with the following primers :
  • 102012 GAAGATCTTGGGGGCAGTCAGTGACGGG (SEQ ID NO. 46)
  • 102013 TCCCCCGGGTATGATTTGATTAGGATG (SEQ ID NO. 47)
  • the 3 ' fragment was obtained by PCR using cosmid 18H7 as template under the same conditions described above with the following primers: 102014: TCCCCCGGGAGTGTTATTAATAAGGAGG (SEQ ID NO. 48) 102015: TGCACTGCAGGTATCTGTATCCCAGTCAGC (SEQ ID NO. 49)
  • the 5' PCR fragment was cut with restriction enzymes Smal and Bgl ⁇ l and the cut fragment was purified from an agarose gel.
  • the 3 ' PCR fragment was cut with Smal and Pstl and the restricted fragment was purified from an agarose gel.
  • the 5' fragment and the 3 ' fragment were cloned into pIC19H (Marsh et al.
  • pToC391 can be linearized with Pvul for transformation of Aspergillus oryzae pyrG' strains to produce transformants in which the DDC2 gene is replaced by the pyrG gene.
  • DDC2 mutant strains can be found, e.g., by Southern analysis or by PCR methods.
  • the DDC2 mutant strains can be used as expression hosts for any heterologous protein.
  • the DDC2 mutant strain so isolated can easily be made pyrG' by selection of resistance to fluororonic acid due to the 400 bp repeat flanking the yrG gene on pJaL335.
  • a plasmid designed for deletion of DDC3 in Aspergillus oryzae was constructed by cloning approximately 1 kb of sequence upstream of the start codon and approximately 1 kb of sequence downstream of the stop codon into a vector in which the two insertions were separated by the Aspergillus oryzae pyrG gene.
  • the cosmid 34G12 did not contain enough upstream sequence to allow for contruction of a deletion plasmid.
  • Southern analysis of genomic DNA from Aspergillus oryzae IFO 4177 (the parent strain of Aspergillus oryzae A1560-T40) with a 32 P-labelled DDC3 probe revealed an approximately 2.5 kb Nsil fragment.
  • the genomic DNA was digested with Nsil, ligated, and used as template for the inverse PCR reaction using the following primers: 30985: CTATTTCTCAGCGGGTGGTTCG (SEQ ID NO. 45) 101449: CTCTATGTAACCAACTCCTGC (SEQ ID NO. 50)
  • a fragment of the expected size (2.3 kb) was obtained and cloned into the vector pCRII (Invitrogen, San Diego CA). The fragment was sequenced on an ABI Automated DNA Sequencer.
  • the sequence information was used to design the primers for construction of the deletion plasmid.
  • the primers used to obtain the 5 ' fragment were as follows: 116497: CGAAAGCTTACTCTCTGGAACAGG (SEQ ID NO. 51) 118141 : CATGGAGCTCGATGGCCAAATGACTGATTCC (SEQ ID NO. 52)
  • the primers used to obtain the 3 ' fragment were as follows:
  • Two PCR reactions were run using IFO4177 genomic DNA as template and either the primer pair 116497/118141 or the pair 116494/116495.
  • the 5' PCR fragment was cut with restriction enzymes S ⁇ cl and Hindlll, the 3 ' fragment was restricted with Hindlll and Xhol, and both cut fragments were purified from an agarose gel.
  • the fragments were cloned into the vector pBluescript (Stratagene, La Jolla, CA) restricted with Sacl and Xhol.
  • plasmid pToC401 can be linearized with Sacl for transformation of Aspergillus oryzae (pyrG ' ) strains to produce transformants in which the DDC3 gene is replaced by the pyrG gene).
  • pyrG ' Aspergillus oryzae
  • the DDC3 mutant strains can be used as expression hosts for any heterologous protein.
  • the DDC3 mutant strains isolated by this procedure can easily be made pyrG' by selection of resistance to fluororonic acid due to the 400 bp repeat flanking the pyrG gene on pJaL335.
  • the strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. ⁇ 1.14 and 35 U.S.C. ⁇ 122.
  • the deposit represents a substantially pure culture of the deposited strain.
  • the deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

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Abstract

The present invention relates to methods for producing a polypeptide in enhanced amounts, comprising (a) cultivating a mutant of a parent filamentous fungal cell in a nutrient medium suitable for production of the polypeptide, wherein (i) the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a modification of one or more second nucleic acid sequences encoding DDC2 and/or DDC3 polypeptides, or homologues thereof, and (ii) the mutant cell produces more of the polypeptide than the parent cell when cultured under the same conditions; and (b) recovering the polypeptide from the nutrient medium of the mutant cell. The present invention also relates to the second nucleic acid sequences, polypeptides encoded by the second nucleic acid sequences, and nucleic acid constructs, recombinant expression vectors, and host cells comprising the sequences. The present invention further relates to mutants of filamentous fungal cells and methods for obtaining the mutant cells.

Description

METHODS FOR PRODUCING POLYPEPTIDES
IN FILAMENTOUS FUNGAL MUTANT CELLS
Background of the Invention
Field of the Invention
The present invention relates to methods for producing polypeptides in mutants of filamentous fungal cells, mutants of filamentous fungal cells, and methods for obtaining the mutants.
Description of the Related Art
Several methods have been used to modify the production of polypeptides by mutagenizing cells. For example, the production of proteins has been altered by producing mutant cells by classical mutagenesis which involves treating cells with chemical, physical, and biological agents as mutagenic (mutation inducing) agents to increase the frequency of mutational events.
A widely used method for increasing production of a polypeptide is amplification to produce multiple copies of a gene encoding the polypeptide. For example, U.S. Patent No. 5,578,461 discloses the inclusion via homologous recombination of an amplifiable selectable marker gene in tandem with a gene where cells containing amplified copies of the selectable marker can be selected for by culturing the cells in the presence of the appropriate selectable agent.
In addition, the production of polypeptides has been increased by replacing one promoter with a different promoter or one signal peptide coding region with another. See, e.g., U.S. Patent No. 5,641.670. The secretion of polypeptides has also been modified by overproduction of secretion proteins (Ruohonen et al, 1997, Yeast, 13: 337- 351), and producing a super-secreting cell (U.S. Patent No. 5,312,735).
The production of polypeptides also has been increased by disrupting DNA sequences encoding protease capable of hydro lyzing the polypeptide under the conditions for producing the polypeptide.
It is an object of the present invention to provide improved methods for increasing production of polypeptides in mutant filamentous fungal strains to yield commercially significant quantities of the polypeptides.
Summary of the Invention
The present invention relates to methods for producing a polypeptide, comprising:
(A) cultivating a mutant cell of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide in which the mutant cell produces more of the polypeptide than the parent cell when cultivated under the same conditions, wherein the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a modification of one or more second nucleic acid sequences selected from the group consisting of:
(i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
(ii) a nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4;
(iii) a nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4. (b) a subsequence of (a) of at least 100 nucleotides. or (c) a complementary strand of (a) or (b): riv) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids; (vj an allelic variant of (i), (ii), or (iii); and
(vi) a subsequence of (i), (ii), (iii), or (v), wherein the subsequence encodes a polypeptide fragment having DDC2 or DDC3 polypeptide activity: and
(B) recovering the polypeptide from the cultivation medium of the mutant cell. The present invention also relates to mutants of filamentous fungal cells and methods for obtaining the mutant cells.
The present invention also relates to the isolated second nucleic acid sequences
- 2 - SUBSTTTUTE SHEET (RULE 26) encoding DDC2 or DDC3 polypeptides. isolated DDC2 or DDC3 polypeptides encoded by the second nucleic acid sequences, and nucleic acid constructs, recombinant expression vectors, and host cells comprising the second nucleic acid sequences.
Brief Description of Figures
Figures 1A and IB show the genomic nucleic acid sequence and the deduced amino acid sequence of DDC2 (SEQ ID NOs. 1 and 2. respectively).
Figures 2A and 2B show the genomic nucleic acid sequence and the deduced amino acid sequence of DDC2 (SEQ ID NOs. 4 and 5. respectively). Figure 3 shows a restriction map of pToC391. Figure 4 shows a restriction map of pToC401.
Detailed Description of the Invention
The present invention relates to methods for producing a polypeptide in an enhanced amount, comprising cultivating a mutant cell of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide and isolating the polypeptide from the cultivation medium of the mutant cell, wherein the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a modification, e.g.. disruption or deletion, of one or more second nucleic acid sequences endogenous to the parent cell where the modification enhances production of the polypeptide by the mutant cell compared to the parent cell when cultivated under the same conditions.
In the methods of the present invention, the one or more second nucleic acid sequences have:
(i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
(ii) a nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4:
(iii) a nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4. (b) a subsequence of (a) of at least 100 nucleotides, or (c) a complementary strand of (a) or (b);
(iv) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids; (v) an allelic variant of (i), (ii), or (iii); and
(vi) a subsequence of (i), (ii), (iii), or (v), wherein the subsequence encodes a polypeptide fragment having DDC2 or DDC3 polypeptide activity.
DDC2 and or DDC3 polypeptide activity is defined herein as an activity (or activities) which when reduced and more preferably eliminated increases or enhances the production of a polypeptide.
In a first embodiment, the second nucleic acid sequences encode polypeptides having an amino acid sequence which has a degree of identity to amino acids 19 to 64 of SEQ ID NO. 2 (i.e.. the mature polypeptide) of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have DCC2 polypeptide activity (hereinafter "DCC2 homologous polypeptides" or "DCC2 polypeptides"), or a degree of identity to amino acids 21 to 83 of SEQ ID NO. 5 (i.e., the mature polypeptide) of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%. even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%. which have DCC3 polypeptide activity (hereinafter "DCC3 homologous polypeptides" or "DCC3 polypeptides". In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5. For purposes of the present invention, the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison. WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters were
- 4 -
SUBSTΓΓUTE SHEET (RULE 26) Ktuple=l. gap penalty=3, windows=5, and diagonals=5.
Preferably, the second nucleic acid sequences encode polypeptides that comprise the amino acid sequence of SEQ ID NO. 2 or an allelic variant thereof; or a fragment thereof that has DCC2 polypeptide activity. In a more preferred embodiment, the second nucleic acid sequence encodes a DCC2 polypeptide that comprises the amino acid sequence of SEQ ID NO. 2. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that comprises amino acids 19 to 64 of SEQ ID NO. 2. or an allelic variant thereof; or a fragment thereof that has DCC2 polypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that comprises amino acids 19 to 64 of SEQ ID NO. 2. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 2 or an allelic variant thereof: or a fragment thereof that has DCC2 poiypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 2. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of amino acids 19 to 64 of SEQ ID NO. 2 or an allelic variant thereof; or a fragment thereof that has DDC2 polypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of amino acids 19 to 64 of SEQ ID NO. 2. Preferably, the second nucleic acid sequences encode polypeptides that comprise the amino acid sequence of SEQ ID NO. 5 or an allelic variant thereof: or a fragment thereof that has DCC3 polypeptide activity. In a more preferred embodiment, the second nucleic acid sequence encodes a DCC3 polypeptide that comprises the amino acid sequence of SEQ ID NO. 5. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that comprises amino acids 21 to 83 of SEQ ID NO. 5. or an allelic variant thereof; or a fragment thereof that has DCC3 polypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that comprises amino acids 21 to 83 of SEQ ID NO. 5. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 5 or an allelic variant thereof; or a fragment thereof that has DCC3 polypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 2. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide that consists of amino acids 21 to 83 of SEQ ID NO. 5 or an allelic variant thereof; or a fragment thereof that has DDC3 polypeptide activity. In another preferred embodiment, the second nucleic acid sequence encodes a polypeptide 5 that consists of amino acids 19 to 64 of SEQ ID NO. 5.
The second nucleic acid sequences also encompass nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5. which differ from SEQ ID NO. 1 or SEQ ID NO. 4. respectively, by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of 0 SEQ ID NO. 1 which encode fragments of SEQ ID NO. 2 which have DDC2 polypeptide activity and subsequences of SEQ ID NO. 4 which encode fragments of SEQ ID NO. 5 which have DDC3 polypeptide activity.
A subsequence of SEQ ID NO. 1 or SEQ ID NO. 4 is a nucleic acid sequence encompassed by SEQ ID NO. 1 or SEQ ID NO. 4, respectively, except that one or more 5 nucleotides from the 5' and/or 3' end have been deleted. Preferably, a subsequence of SEQ ID NO. 1 contains at least 114 nucleotides, more preferably at least 138 nucleotides. and most preferably at least 162 nucleotides. Preferably, a subsequence of SEQ ID NO. 4 contains at least 159 nucleotides, more preferably at least 189 nucleotides. and most preferably at least 219 nucleotides. 0 A fragment of SEQ ID NO. 2 or SEQ ID NO. 5 is a polypeptide having one or more amino acids deleted from the amino and/or carboxy terminus of this amino acid sequence. Preferably, a fragment of SEQ ID NO. 2 contains at least 38 amino acid residues, more preferably at least 46 amino acid residues, and most preferably at least 54 amino acid residues. Preferably, a fragment of SEQ ID NO. 5 contains at least 53 amino 5 acid residues, more preferably at least 63 amino acid residues, and most preferably at least 73 amino acid residues.
An allelic variant denotes any of two or more alternative forms of a gene occupying the same chomosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be o silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. The allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
- 6 -
SUBSTΓΓUTE SHEET (RULE 26) In a second embodiment, the second nucleic acid sequences have a degree of homology to the mature polypeptide coding sequence of SEQ ID NO. 1 (i.e., nucleotides 1014 to 1151) of at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which encode an active DDC2 polypeptide; or allelic variants and subsequences of SEQ ID NO. 1 which encode polypeptide fragments which have DDC2 polypeptide activity, or a degree of homology to the mature polypeptide coding sequence of SEQ ID NO. 4 (i.e., nucleotides 1041 to 1229) of at least about 50%>, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%. most preferably at least about 95%, and even most preferably at least about 97%, which encode an active DDC3 polypeptide; or allelic variants and subsequences of SEQ ID NO. 4 which encode polypeptide fragments which have DDC3 polypeptide activity. For purposes of the present invention, the degree of homology between two nucleic acid sequences is determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters were Ktuple=3, gap penalty=3, and windows=20.
In a third embodiment, the second nucleic acid sequences hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (b) a subsequence of (a) of at least 100 nucleotides. or (c) a complementary strand of (a) or (b) (J. Sambrook, E.F. Fritsch, and T. Maniatus. 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). The subsequence of SEQ ID NO. 1 or SEQ ID NO. 4 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has DDC2 or DDC3 polypeptide activity.
- 7 -
SUBSTΓΓUTE SHEET (RULE 26) The nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 4, or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having DDC2 or DDC3 polypeptide activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25. and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 2P. Η, 35S, biotin. or avidin). Such probes are encompassed by the present invention.
Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having DDC2 or DDC3 polypeptide activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis. or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA which is homologous with SEQ ID NO. 1 or SEQ ID NO. 4. or a subsequence thereof, the carrier material is used in a Southern blot. For purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleic acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 5, its complementary strand, or a subsequence thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.
In a preferred embodiment, the nucleic acid probe is SEQ ID NO. 1. In another preferred embodiment, the nucleic acid probe is a nucleic acid sequence which encodes the polypeptide of SEQ ID NO. 2, or a subsequence thereof. In another preferred embodiment, the nucleic acid probe is nucleotides 1014 to 1151 of SEQ ID NO. 1. which encodes a mature polypeptide having DDC2 polypeptide activity. In another preferred
- 8 -
SUBSTΓΓUTE SHEET (RULE 26) embodiment, the nucleic acid probe is the nucleic acid sequence contained in cosmid
18H7 which is contained in Escherichia coli DSM 12060, wherein the nucleic acid sequence encodes a polypeptide having DDC2 polypeptide activity. In another preferred embodiment, the nucleic acid probe is the mature DDC2 polypeptide coding region contained in cosmid 18H7 which is contained in Escherichia coli DSM 12060.
In a preferred embodiment, the nucleic acid probe is SEQ ID NO. 4. In another preferred embodiment, the nucleic acid probe is a nucleic acid sequence which encodes the polypeptide of SEQ ID NO. 5, or a subsequence thereof. In another preferred embodiment, the nucleic acid probe is nucleotides 1041 to 1229 of SEQ ID NO. 4, which encodes a mature polypeptide having DDC3 polypeptide activity. In another preferred embodiment, the nucleic acid probe is the nucleic acid sequence contained in cosmid 34G12 contained in E. coli DSM 11924, wherein the nucleic acid sequence encodes a polypeptide having DDC3 polypeptide activity. In another preferred embodiment, the nucleic acid probe is the mature DDC3 polypeptide coding region contained in cosmid 34G12 contained in E. coli DSM 11924.
For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS. 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies. 35% formamide for medium and medium- high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures.
For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2X SSC. 0.2%) SDS preferably at least at 45°C (very low stringency), more preferably at least at 50°C (low stringency), more preferably at least at 55°C (medium stringency), more preferably at least at 60°C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70°C (very high stringency).
For shoπ probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5°C to about 10°C below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl. 0.09 M Tris-HCl pH 7.6, 6 mM EDTA. 0.5% NP-40, IX Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.
For short probes which are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6X SCC plus 0.1 % SDS for 15 minutes and twice each for 15 minutes using 6X SSC at about 5°C to about 10°C below the calculated Tm.
The second nucleic acid sequences may be obtained by (a) hybridizing a DNA under very low. low, medium, medium-high, high, or very high stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1, (ii) a subsequence of (i). or (iii) a complementary strand of (i) or (ii), or (i) nucleotides 1041 to 1229 of SEQ ID NO. 4. (ii) a subsequence of (i). or (iii) a complementary strand of (i) or (ii): and (b) isolating the nucieic acid sequence. The subsequence is preferably a sequence of at least 100 nucleotides such as a sequence which encodes a polypeptide fragment which has DDC2 or DDC3 polypeptide activity.
In a fourth embodiment, the second nucleic acid sequences encode variants of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids.
The amino acid sequences of the variant polypeptides may differ from the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5, or the mature polypeptide thereof, by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terrninal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the group of basic amino acids (arginine. lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine). hydrophobic amino acids (leucine. isoleucine and valine). aromatic amino acids (phenylalanine. tryptophan and tyrosine),
- 10 -
SUBSTΓΓUTE SHEET (RULE 26) and small amino acids (glycine, alanine, serine. threonine and methionine). Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979. In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu. Thr/Ser, Ala/Gly, Ala/Thr. Ser/Asn. Ala/Val, Ser/Gly, Tyr Phe, Ala/Pro, Lys/Arg, Asp/Asn. Leu/Ile. LeuVal. Ala/Glu. and Asp/Gly as well as these in reverse.
The variant sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of SEQ ID NO. 1 or SEQ ID NO. 4, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the polypeptide, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see. e.g.. Ford et al, 1991, Protein Expression and Purification 2: 95-107.
It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by the second nucleic acid sequence, and therefore preferably not subject to substitution, may be identified according to procedures known in the art. such as site-directed mutagenesis or alanine-scanning mutagenesis (see. e.g.. Cunningham and Wells. 1989. Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for DDC2 or DDC3 polypeptide activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see. e.g.. de Vos et al. 1992. Science 255: 306-312; Smith et al, 1992. Journal of Molecular Biology 224: 899-904; Wlodaver et al. 1992, FEES letters 309: 59-64).
In a preferred embodiment, two second nucleic acid sequences are modified which encode a DDC2 and DDC3 polypeptide. In a more preferred embodiment, the DCC2 and DDC3 nucleic acid sequences contained in SEQ ID NO. 1 and SEQ ID NO.
4, respectively, are modified.
The second nucleic acid sequences may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted. In a preferred embodiment, the polypeptide encoded by the second nucleic acid sequence is secreted extracellularly.
The second nucleic acid sequences may be obtained from a fungal source, and more preferably from a yeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain; or more preferably from a filamentous fungal strain such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus. Filibasidium. Fusarium. Gibberella. Humicola. Magnaporthe. Mucor. Myceliophthora. Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces. Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain.
In a preferred embodiment, the second nucleic acid sequences are obtained from a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii. Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis strain.
In another preferred embodiment, the second nucleic acid sequences are obtained from an Aspergillus aculeatus, Aspergillus awamori. Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans. Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides. Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi. Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum. Fusarium sporotrichioides, Fusarium sulphureum. Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens. Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa. Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain. In a more preferred embodiment, the second nucleic acid sequence encoding a
DCC2 polypeptide is obtained from an Aspergillus oryzae strain, and most preferably from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the polypeptide with the amino acid sequence of SEQ ID NO. 2. In another more preferred embodiment, the nucleic acid sequence is the sequence contained in cosmid 18H7 which is contained in Escherichia coli DSM 12060. In another preferred embodiment, the nucleic acid sequence is nucleotides 1014 to 1151 of SEQ ID NO. 1
In another more preferred embodiment, the second nucleic acid sequence encoding a DDC3 polypeptide is obtained from an Aspergillus oryzae strain, and most preferably from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the polypeptide with the amino acid sequence of SEQ ID NO. 2. In another more preferred embodiment, the nucleic acid sequence is the sequence contained in cosmid 34G12 contained in E. coli DSM 11924. In another preferred embodiment, the nucleic acid sequence is nucleotides 1041 to 1229 of SEQ ID NO. 4. It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs. regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. For example, the polypeptides may be obtained from microorganisms which are taxonomic equivalents of Aspergillus as defined by Raper, K.D. and Fennel. D.I.. 1965. The Genus Aspergillus.
The Wilkins Company. Baltimore, regardless of the species name by which they are known. Aspergilli are mitosporic fungi characterized by an aspergillum comprised of a conidiospore stipe with no known teleomorphic states terminating in a vesicle, which in turn bears one or two layers of synchronously formed specialized cells, variously referred to as sterigmata or phialides, and asexually formed spores referred to as conidia. Known teleomorphs of Aspergillus include Eurotium, Neosartorya, and Emericella. Strains of Aspergillus and teleomorphs thereof are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC). Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection. Northern Regional Research Center (NRRL). Furthermore, such nucleic acid sequences may be identified and obtained from other sources including
- 13 - SUBSTTTUTE SHEET (RULE 26) microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see. e.g.. Sambrook et al, 1989. supra).
The second nucleic acid sequences may be mutant nucleic acid sequences comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO. 1. in which the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 19 to 64 of SEQ ID NO. 2. or at least one mutation in the mature polypeptide coding sequence of SEQ ID NO. 4, in which the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 1 to 83 of SEQ ID NO. 5. The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA. or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g.. Innis et al. 1990. PCR: A Guide to Methods and Application. Academic Press. New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR). ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The second nucleic acid sequence may be cloned from a strain of Aspergillus. or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.
The mutant filamentous fungal cell may be constructed by reducing or eliminating expression of one or more of the second nucleic acid sequences described herein using methods well known in the art, for example, insertions, disruptions. replacements, or deletions. The second nucleic acid sequence(s) to be modified or inactivated may be. for example, the coding region or a part thereof essential for activity, or a regulatory or control element required for the expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i. e.. a part which is sufficient for affecting expression of the nucleic acid sequence. Other control sequences for possible modification include, but are not limited to. a leader, polyadenylation sequence, propeptide sequence, signal 5 sequence, transcription terminator, and transcriptional activator.
Modification or inactivation of the second nucleic acid sequence(s) may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the second nucleic acid sequence(s) has been reduced or eliminated. The mutagenesis. which may be specific or random, may be performed, for example, by ic use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide. or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods.
Examples of a physical or chemical mutagenizing agent suitable for the present is purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), O-methyl hydroxylamine. nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice
20 under suitable conditions, and selecting for mutant cells exhibiting reduced or no expression of the second nucleic acid sequence(s).
Modification or inactivation of the second nucleic acid sequence(s) may also be accomplished by introduction, substitution, or removal of one or more nucleotides in the sequence or a regulatory element required for the transcription or translation thereof. For
25 example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon. the removal of the start codon. or a change of the open reading frame. Such a modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i.e.. directly on the cell expressing
3 o the second nucleic acid sequence(s) to be modified, it is preferred that the modification be performed in vitro as exemplified below.
An example of a convenient way to reduce or eliminate expression of the second
- 15 - SUBSTTTUTE SHEET (RULE 26) nucleic acid sequence(s) by a filamentous fungal cell of choice is based on techniques of gene replacement, gene deletion, or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous gene or gene fragment of interest is mutagenized in vitro to produce a defective nucleic acid sequence which is then transformed into the parent cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene or gene fragment. It may be desirable that the defective gene or gene fragment also encodes a marker which may be used for selection of transformants in which the nucleic acid sequence has been modified or destroyed. Alternatively, modification or inactivation of the second nucleic acid sequence(s) may be performed by established anti-sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the gene. More specifically, expression of the gene by a filamentous fungal cell may be reduced or eliminated by introducing a nucleotide sequence complementary to the second nucleic acid sequence(s) which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA. the amount of protein translated is thus reduced or eliminated.
A nucleic acid sequence homologous or complementary to the second nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 4 may be obtained from any microbial source as described infra. The choice of the source of the nucleic acid sequence will depend on the filamentous fungal cell, but preferred sources are fungal sources, e.g.. yeast and filamentous fungi. Preferred filamentous fungal sources include, but are not limited to. species of Acremonium. Aspergillus. Fusarium, Humicola. Myceliophthora. Mucor. Neurospora. Penicillium, Phanerochaete, Thielavia. Tolypocladium, and Trichoderma. Preferred yeast sources include, but are not limited to. species of Candida. Hansenula. Kluyveromyces. Pichia. Saccharomyces. Schizosaccharomyces. and Yarrowia. Furthermore, the nucleic acid sequence may be native to the filamentous fungal cell.
It will be understood that the methods of the present invention are not limited to a particular order for obtaining the mutant filamentous fungal cell. The modification of the second nucleic acid sequence(s) as described herein may be introduced into the parent cell at any step in the construction of the cell for the production of a polypeptide.
- 16 - It is preferable that the second nucleic acid sequence(s) of the mutant filamentous fungal cell has already been modified prior to the introduction of a first nucleic acid sequence encoding a heterologous polypeptide.
In the methods of the present invention, the filamentous fungal cell may be a wild-type cell or a mutant thereof. Preferably, the filamentous fungal cell is an Acremonium, Aspergillus. Aureobasidium. Cryptococcus. Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe. Mucor. Myceliophthora, Myrothecium, Neocallimastix. Neurospora. Paecilomyces. Penicillium. Piromyces, Schizophyllum, Talaromyces. Thermoascus, Thielavia. Tolypocladium. or Trichoderma cell. In a preferred embodiment, the filamentous fungal cell is an Aspergillus aculeatus. Aspergillus awamori, Aspergillus foetidus. Aspergillus japonicus, Aspergillus nidulans. Aspergillus niger, ox Aspergillus oryzae cell.
In another preferred embodiment, the filamentous fungal cell is a Fusarium bactridioides. Fusarium crookwellense (synonym of Fusarium cerealis), Fusarium culmorum. Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi. Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium solani, Fusarium sporotrichioides. Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides. or Fusarium venenatum cell. In another preferred embodiment, the filamentous fungal cell is a Gibberella pulicaris. Gibberella zeae. Humicola insolens. Humicola lanuginosa, Mucor miehei. Myceliophthora thermophila, Myrothecium roridin. Neurospora crassa, or Penicillium purpurogenum cell.
In another preferred embodiment, the filamentous fungal cell is a Trichoderma harzianum. Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
The mutant filamentous fungal cell is cultivated in a nutrient medium suitable for production of the polypeptide encoded by the first nucleic acid sequence using methods known in the art. For example, the cell may be cultivated by shake flask cultivation. small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the heterologous polypeptide to be expressed
- 17 -
SUBSTΓΓUTE SHEET (RULE 26) and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g.. in catalogues of the American Type Culture Collection). The polypeptide can be recovered directly from the medium if secreted.
The polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods may include use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate. SDS-PAGE. or any other method known in the art. For example, an enzyme assay may be used to determine the activity of the polypeptide. Procedures for determining enzyme activity are known in the art for many enzymes.
The resulting polypeptide may be isolated by methods known in the an. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation. filtration, extraction, spray- drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g.. ammonium sulfate precipitation), or extraction (see, e.g.. Protein Purification. J.-C. Janson and Lars Ryden. editors, VCH Publishers. New York. 1989). The polypeptide encoded by the first nucleic acid sequence may be any polypeptide native or foreign to the mutant filamentous fungal cell. The term '"polypeptide" is not meant herein to refer to a specific length of the encoded product and. therefore, encompasses peptides. oligopeptides. and proteins. The term "heterologous polypeptide" is defined herein as a polypeptide which is not native to the filamentous fungal cell. The mutant filamentous fungal cell may contain one or more copies of the nucleic acid sequence encoding the heterologous polypeptide.
In a preferred embodiment, the polypeptide is a hormone, hormone variant. enzyme, receptor or portion thereof, antibody or portion thereof, or reporter. In a more preferred embodiment, the polypeptide is an oxidoreductase. transferase. hydrolase. lyase. isomerase. or ligase. In an even more preferred embodiment, the polypeptide is an aminopeptidase. amylase. carbohydrase. carboxypeptidase. catalase. cellulase. chitinase. cutinase, cyclodextrin glycosyltransferase. deoxyribonuclease. esterase. alpha- galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase. beta-glucosidase, invertase. laccase, lipase. mannosidase, mutanase, oxidase. pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease. transglutaminase or xylanase.
The first nucleic acid sequence encoding a polypeptide of interest may be obtained from any prokaryotic. eukaryotic. or other source.
In the methods of the present invention, the mutant filamentous fungal cell may also be used for the recombinant production of polypeptides which are native to the cell. The native polypeptides may be recombinantly produced by, for example, placing a gene encoding the polypeptide under the control of a different promoter to enhance expression of the polypeptide. to expedite export of a native polypeptide of interest outside the cell by use of a signal sequence, and to increase the copy number of a gene encoding the polypeptide normally produced by the cell. The present invention also encompasses, within the scope of the term "heterologous polypeptide". such recombinant production of endogenous polypeptides native to the filamentous fungal cell, to the extent that such expression involves the use of genetic elements not native to the cell, or use of native elements which have been manipulated to function in a manner that do not normally occur in the host cell. The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are described herein. The nucleic acid sequence encoding the polypeptide may be of genomic. cDNA. RNA. semisynthetic. synthetic origin, or any combinations thereof.
In the methods of the present invention, the polypeptides may also be an engineered variant of a polypeptide.
In the methods of the present invention, the polypeptides may further include fused or hybrid polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide. Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the
- 19 - SUBSTTTUTE SHEET (RULE 26) fused polypeptide is under control of the same promoter(s) and terminator. The hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the mutant filamentous fungal cell. An isolated first nucleic acid sequence encoding a polypeptide of interest may be manipulated in a variety of ways to provide for expression of the polypeptide. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to. transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.
"Nucleic acid construct" is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence.
The term "coding sequence" as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide. The boundaries of the coding sequence are generally determined by the ATG start codon located just upstream of the open reading frame at the 5" end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, genomic, cDNA. RNA, semisynthetic. synthetic. recombinant, or any combinations thereof.
The term "control sequences" is defined herein to include all components which are necessary or advantageous for the expression of a heterologous polypeptide. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided
- 20 -
SUBSTΓΓUTE SHEET (RULE 26) with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a heterologous polypeptide. The term "operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a heterologous polypeptide.
The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a filamentous fungal cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences which mediate the expression of the heterologous polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the filamentous fungal cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the cell. Examples of suitable promoters for directing the transcription of the nucleic acid constructs in the methods of the present invention are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase. Aspergillus nidulans acetamidase. Aspergillus oryzae acetamidase (amdS). Fusarium oxysporum trypsin-like protease (U.S. Patent No. 4.288,627), and mutant, truncated, and hybrid promoters thereof. Particularly preferred promoters are the NA2- tpi promoters (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase). glucoamylase. and TAKA amylase promoters.
The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a filamentous fungal cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the heterologous polypeptide. Any terminator which is functional in the filamentous fungal cell may be used in the present invention.
Preferred terminators are obtained from the genes encoding Aspergillus oryzae TAKA amylase. Aspergillus niger glucoamylase. Aspergillus nidulans anthranilate synthase. Aspergillus niger alpha-glucosidase. and Fusarium oxysporum trypsin-like protease.
The control sequence may also be a suitable leader sequence, a nontranslated region of a mRNA which is important for translation by the filamentous fungal cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the heterologous polypeptide. Any leader sequence which is functional in the filamentous fungal cell may be used in the present invention.
Preferred leaders are obtained from the genes encoding Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by a filamentous fungal cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the filamentous fungal cell may be used in the present invention.
Preferred polyadenylation sequences are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.
The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of the heterologous polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5" end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5* end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the polypeptide. However, any signal peptide coding region which directs the expressed heterologous polypeptide into the secretory pathway of a filamentous fungal cell may be used in the present invention.
- 22 -
SUBSTΓΓUTE SHEET (RULE 26) Effective signal peptide coding regions for filamentous fungal host cells aie the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase. Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. The control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the Rhizomucor miehei aspartic proteinase gene, or the Myceliophthora thermophila laccase gene (WO 95/33836).
Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
The nucleic acid constructs may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous for directing the expression of the heterologous polypeptide, e.g., a transcriptional activator (e.g., a trans- acting factor), chaperone, and processing protease. Any factor that is functional in a filamentous fungal cell may be used in the present invention. The nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the heterologous polypeptide.
It may also be desirable to add regulatory sequences which allow the regulation of the expression of the heterologous polypeptide relative to the growth of the filamentous fungal cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. The TAKA alpha-amylase promoter. Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow for gene amplification, e.g., the metallothionein genes which are amplified with heavy metals. In these cases, the nucleic
- 23 -
SUBSTΓΓUTE SHEET (RULE 26) acid sequence encoding the heterologous polypeptide would be operably linked with the regulatory sequence.
The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the heterologous polypeptide at such sites. Alternatively, the nucleic acid sequence encoding the heterologous polypeptide may be expressed by inserting the sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence encoding the heterologous polypeptide. The choice of the vector will typically depend on the compatibility of the vector with the filamentous fungal cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the filamentous fungal cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the filamentous fungal cell, or a transposon.
The vectors preferably contain one or more selectable markers which permit easy selection of transformed filamentous fungal cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophv to auxotrophs. and the like. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to. αmdS (acetamidase). αrgB (ornithine carbamoyltransferase), bar (phosphinothricin
- 24 -
SUBSTΓΓUTE SHEET (RULE 26) acetyltransferase). hygB (hygromycin phosphotransferase). niaD (nitrate reductase), pyrG (orotidine-5" -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents from other species. Preferred for use in a filamentous fungal cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
The vectors preferably contain an element(s) that permits stable integration of the vector into a filamentous fungal cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.
"Introduction" means introducing a vector comprising the nucleic acid sequence into a filamentous fungal cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the chromosome occurs by homologous recombination, non-homologous recombination, or transposition. The introduction of an expression vector into a filamentous fungal cell may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al, 1984. Proceedings of the National Academy of Sciences USA 81 : 1470-1474. A suitable method of transforming Fusarium species is described by Malardier et al. 1989, Gene
78: 147-156 and WO 96/00787.
For integration into genome of a filamentous fungal cell, the vector may rely on the nucleic acid sequence encoding the heterologous polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the filamentous fungal cell. The additional nucleic acid sequences enable the vector to be integrated into the genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs. preferably 400 to 1.500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequences that are homologous with the target sequence in the genome of the filamentous fungal cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the filamentous fungal cell in question.
The procedures used to ligate the elements described herein to construct the recombinant expression vectors are well known to one skilled in the art (see. e.g., J. Sambrook. E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual. 2d edition. Cold Spring Harbor, New York).
In another aspect of the present invention, the mutant filamentous fungal cell may additionally contain modifications of one or more third nucleic acid sequences which encode proteins that may be detrimental to the production, recovery, and/or application of the polypeptide of interest. The modification reduces or eliminates expression of the one or more third nucleic acid sequences resulting in a mutant cell which may produce more of the polypeptide than the mutant cell without the modification of the third nucleic acid sequence when cultured under the same conditions. The third nucleic acid sequence may encode any protein or enzyme. For example, the enzyme may be an aminopeptidase. amylase. carbohydrase. carboxypeptidase. catalase, cellulase. chitinase. cutinase. cyclodextrin glycosyltransferase. deoxyribonuclease, esterase. alpha-galactosidase. beta-galactosidase. glucoamylase. alpha-glucosidase. beta-glucosidase. invertase. laccase. lipase. mannosidase. mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase. phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease. transglutaminase, or xylanase. The third nucleic acid sequence preferably encodes a proteolytic enzyme, e.g.. an aminopeptidase. carboxypeptidase, or protease.
The present invention also relates to methods for producing a mutant filamentous fungal cell, comprising:
(A) modifying one or more nucleic acid sequences of a parent filamentous fungal cell having:
- 26 -
SUBSTΓΓUTE SHEET (RULE 26) (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
(ii) a nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4;
(iii) a nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (b) a subsequence of (a) of at least 100 nucleotides. or (c) a complementary strand of (a) or (b); (iv) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(v) an allelic variant of (i). (ii). or (iii); and
(vi) a subsequence of (i), (ii), (iii). or (v), wherein the subsequence encodes a polypeptide fragment having DDC2 or DDC3 polypeptide activity; and
(B) identifying the mutant from step (A) comprising the modified nucleic acid sequence.
The present invention also relates to mutant filamentous fungal cells for producing a polypeptide which comprise a first nucleic acid sequence encoding the polypeptide and a modification of one or more second nucleic acid sequences having:
(a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
(b) a nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4;
(c) a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii); (d) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
- 27 -
SUBSTΓΓUTE SHEET (RULE 26) (e) an allelic variant of (a), (b). or (c); and
(f) a subsequence of (a), (b), (c), or (e), wherein the subsequence encodes a polypeptide fragment having DDC2 or DDC3 polypeptide activity.
The present invention also relates to the isolated DDC2 and DDC3 polypeptides, as well as fragments, allelic variants, and variants thereof encoded by the nucleic acid sequences described herein. The isolated polypeptides are selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 50%) identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4. (ii) a subsequence of (i) of at least 100 nucleotides. or (iii) a complementary strand of (i), (ii), or (iii); (c) a variant of the polypeptide having an amino acid sequence of SEQ ID
NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and or insertion of one or more amino acids;
(d) an allelic variant of (a) or (b); and
(e) a fragment of (a), (b). or (d). wherein the fragment has DDC2 or DDC3 polypeptide activity.
The isolated DDC2 and DDC3 polypeptides of the present invention have at least 20%. preferably at least 40%. more preferably at least 60%. even more preferably at least 80%. even more preferably at least 90%), and most preferably at least 100% of the activity of the polypeptide of SEQ ID NO. 2 or SEQ ID NO. 5. The DDC2 and DDC3 polypeptides may be obtained from a fungal source, and more preferably from a yeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces. Schizosaccharomyces, or Yarrowia strain; or a filamentous fungal strain such as an Acremonium. Aspergillus, Aureobasidium. Cryptococcus, Filibasidium, Fusarium. Gibberella. Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix. Neurospora, Paecilomyces, Penicillium, Piromyces,
Schizophyllum. Talaromyces. Thermoascus. Thielavia. Tolypocladium, or Trichoderma strain.
- 28 - SUBSTTTUTE SHEET (RULE 26) In a more preferred embodiment, a DDC2 polypeptide of the present invention is obtained from an Aspergillus oryzae strain, and most preferably from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the polypeptide with the amino acid sequence of SEQ ID NO. 2. In another more preferred embodiment, a DDC3 polypeptide of the present invention is obtained from an Aspergillus oryzae strain, and most preferably from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the polypeptide with the amino acid sequence of SEQ ID NO. 2.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs. regardless of the species name by which they are known as described supra.
The DDC2 and DDC3 polypeptides may be isolated using techniques as described herein. As defined herein, an "isolated" polypeptide is a polypeptide which is essentially free of other polypeptides, e.g., at least about 20%) pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90%) pure, and even most preferably about 95%> pure, as determined by SDS-PAGE.
The present invention also relates to isolated nucleic acid sequences which encode DDC2 and DDC3 polypeptides, and fragments thereof, as described herein. In a preferred embodiment, the nucleic acid sequence is set forth in SEQ ID NO.
1. In another preferred embodiment, the nucleic acid sequence is the sequence contained in cosmid 18H7 which is contained in Escherichia coli DSM 12060. In another preferred embodiment, the nucleic acid sequence is the polypeptide coding region of SEQ ID NO. 1. In another preferred embodiment, the nucleic acid sequence is the polypeptide coding region contained in cosmid 18H7 which is contained in Escherichia coli DSM 12060.
In another preferred embodiment, the nucleic acid sequence is set forth in SEQ ID NO. 4. In another preferred embodiment, the nucleic acid sequence is the polypeptide coding region of SEQ ID NO. 4. In another preferred embodiment, the nucleic acid sequence is the sequence contained in cosmid 34G12 which is contained in Escherichia coli DSM 11924. In another preferred embodiment, the nucleic acid sequence is the polypeptide coding region contained in cosmid 34G12 which is contained in Escherichia
- 29 -
SUBSTΓΓUTE SHEET (RULE 26) coli DSM 11924.
The present invention also relates to isolated mutant nucleic acid sequences comprising at least one mutation in the polypeptide coding sequence of SEQ ID NO. 1 or SEQ ID NO. 4. in which the mutant nucleic acid sequence encodes a polypeptide which consists of SEQ ID NO. 2 or SEQ ID NO. 5, respectively.
The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and described herein.
The term "isolated nucleic acid sequence" as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20%) pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90%> pure as determined by agarose electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic. cDNA. RNA. semisynthetic. synthetic origin, or any combinations thereof.
Modification of a nucleic acid sequence encoding a DDC2 or DDC3 polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the DDC2 or DDC3 polypeptide. The term "substantially similar" to the DDC2 or DDC3 polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the DDC2 or DDC3 polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variant polypeptides may be constructed as described earlier.
The present invention also relates to isolated nucleic acid sequences produced by (a) hybridizing a DNA under very low, low. medium, medium-high, high, or very high stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1, (ii) a subsequence of (i). or (iii) a complementary strand of (i) or (ii); or with (i) nucleotides 1041 to 1229 of SEQ ID NO. 4. (ii) a subsequence of (i), or (iii) a complementary strand of (i) or (ii); and (b) isolating the nucleic acid sequence from the DNA The subsequence is preferably a sequence of at least 100 nucleotides such as a sequence which encodes a polypeptide fragment which has DDC2 or DDC3 polypeptide activity. The present invention further relates to methods for producing a mutant nucleic acid sequence, comprising introducing at least one mutation into the polypeptide coding sequence of SEQ ID NO. 1 or SEQ ID NO. 4, or a subsequence thereof, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of SEQ ID NO. 2 or SEQ ID NO. 5. respectively, or a fragment thereof which has DDC2 or DDC3 polypeptide activity.
The introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure which utilizes a supercoiled. double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with Dpnl which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA.
Other procedures known in the art may also be used.
The present invention also relates to nucleic acid constructs, recombinant expression vectors, and host cells containing the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 4, subsequences or homologues thereof as described herein, for expression of the sequences. The constructs and vectors may be constructed as described herein. The host cell may be any cell suitable for the expression of the nucleic acid sequence, and preferably is a fungal cell, and more preferably a filamentous fungal cell selected from the group described herein. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
The present invention also relates to methods for producing a DDC2 or DDC3 polypeptide comprising (a) cultivating a strain, which in its wild-type form is capable of producing the polypeptide. under conditions conducive for production of the DDC2 or DDC3 polypeptide: and (b) recovering the polypeptide from the cultivation medium. Preferably, the strain is of the genus Aspergillus, and more preferably Aspergillus oryzae. The present invention also relates to methods for producing a DDC2 or DDC3 polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the DDC2 or DDC3 polypeptide; and (b) recovering the DDC2 or DDC3 polypeptide from the cultivation medium.
In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the DDC2 or DDC3 polypeptide using methods known in the art as described herein. The resulting DDC2 or DDC3 polypeptide may be recovered and purified by methods known in the art as described herein.
Signal Peptides
The present invention also relates to nucleic acid constructs comprising a gene encoding a protein operably linked to a nucleic acid sequence consisting of nucleotides 960 to 1013 of SEQ ID NO:l encoding a signal peptide consisting of amino acids 1 to 18 of SEQ ID NO:2 or nucleotides 981 to 1040 of SEQ ID NO. 4 encoding a signal peptide consisting of amino acids 1 to 20 of SEQ ID NO. 5, wherein the gene is foreign to the nucleic acid sequence.
The present invention also relates to recombinant expression vectors and recombinant host cells comprising such nucleic acid constructs.
The present invention also relates to methods for producing a protein comprising (a) cultivating such a recombinant host cell under conditions suitable for production of the protein; and (b) recovering the protein.
The nucleic acid sequences may be operably linked to foreign genes with other control sequences. Such other control sequences are described above.
The protein may be native or heterologous to a host cell. The term "protein" is not meant herein to refer to a specific length of the encoded product and. therefore, encompasses peptides. oligopeptides. and proteins. The term "protein" also encompasses two or more polypeptides combined to form the encoded product. The proteins also
- 32 -
SUBSTΓΠΠΈ SHEET (RULE 26) include hybrid polypeptides which comprise a combination of partial or complete polypeptide sequences obtained from at least two different proteins wherein one or more may be heterologous or native to the host cell. Proteins further include naturally occurring allelic and engineered variations of the above mentioned proteins and hybrid proteins.
Preferably, the protein is a hormone, hormone variant, enzyme, receptor or a portion thereof, antibody or a portion thereof, or reporter. In a more preferred embodiment, the protein is an oxidoreductase. transferase, hydrolase. lyase, isomerase, or ligase. In an even more preferred embodiment, the protein is an aminopeptidase, amylase. carbohvdrase, carboxypeptidase, catalase. cellulase. chitinase. cutinase, cyclodextrin glycosyltransferase. deoxyribonuclease. esterase. alpha-galactosidase, beta- galactosidase. glucoamylase, alpha-glucosidase. beta-glucosidase. invertase. laccase. lipase. mannosidase. mutanase. oxidase, pectinolytic enzyme, peroxidase, phytase. polyphenoloxidase. proteolytic enzyme, ribonuclease. transglutaminase. or xylanase. The gene may be obtained from any prokaryotic. eukaryotic, or other source.
The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.
Examples
Materials
Chemicals used as buffers and substrates were commercial products of at least reagent grade.
Example 1: Production of CAREZYME™ by Aspergillus oryzae strains HC4.01 and 27
An Aspergillus oryzae strain producing an extracellular beta-l,4-endoglucanase (CAREZYME™) was constructed as described in WO 91/17243. CAREZYME™ is a Trichoderma harzianum cellulase produced by Novo Nordisk A/S, Bagsvaerd. Denmark.
Transformant no. 27 of pSX320 in Aspergillus oryzae A1560-T40 (WO 91/17243) was selected for mutagenesis in order to create a superior strain. Transformant No. 27 was
- J5 J> - mutagenized by NTG and strains with an altered plate morphology were isolated. One of these named HC4.01 was shown to have an increase in CAREZYME™ yield in shake flask fermentations as well as in tank fermentations. In an attempt to further characterize the difference between Aspergillus oryzae strains HC4.01 and no. 27, mRNA preparations from the two strains were compared by the differential display method.
Aspergillus oryzae strains HC4.01 and 27 were grown side-by-side at 34°C, pH 7, 800-1100 rpm for 5 days in 2 liter fed-batch fermentations composed of Nutriose. yeast extract. MgS04-7H2O, citric acid, K2SO4, KH2PO4, urea, trimethyl glycine. and trace metals solution. CAREZYME™ activity was measured as a viscosity change (reduction) of a 1%
CMC solution at pH 7 compared to a purified CAREZYME™ standard obtained from Novo Nordisk A/S. Bagsvasrd. Denmark.
At 48 hours, the CAREZYME™ levels produced by the two strains began to differ.
Example 2: Differential Display
The genetic basis for differences in the phenotype of Aspergillus oryzae strains HC4.01 and 27 was analyzed by differential mRNA display. The primers used in these experiments are listed in Table 1. Primers contained 4 nucleotides at the 5" end. followed by a Hindlll restriction enzyme site. Using the set of oligo(dT12N2) primers, the mRNA populations of the two cell lines at 48 hours were divided into 12 subpopulations and cDNA was produced. This cDNA was then further subdivided by PCR amplification using the same set of oligo(dT12N2) primers and a set of random primers. The amplicons from these reactions were then resolved by denaturing polyacrvlamide gel electrophoresis. Genes that were differentially expressed between the two cell lines were identified by the absence of bands in one of the lanes when the footprints were compared side-by-side.
RNA was prepared from frozen mycelia of each cell line at 48 hours according to the procedure described by Wahleithner et al, 1996, Current Genetics 29: 395-403. Genomic DNA fragments were removed by DNase treatment using a MessageClean Kit
(GenHunter Corp.. Nashville, TN) according to manufacturer's instructions.
For each anchored dT12N2 primer, 3.75 μg of total RNA from Aspergillus oryzae
- 34 -
SUBSTΓΓUTE SHEET (RULE 26) strains HC4.01 and 27 were mixed in two separate reactions with 1.0 μM of primer, 15 μl of 5x first strand synthesis buffer (Life Technologies. Gaithersburg, MD), 10 μM dithiothreitol, and 20 μM dNTP in a final volume of 75 μl. The solution was incubated at 65°C for 5 minutes, quick chilled on ice. and 500 units of Superscript II™ reverse s transcriptase (Life Technologies, Gaithersburg, MD) was added. After a 1 hour incubation at 37°C. the reaction was stopped by heat treatment for 5 minutes at 95°C and the first strand samples were stored at -20°C.
Table I. Primers used for differential display reactions oligo (dT12N2) primers: 0 A GCGCAAGCTTTTTTTTTTTTCT (SEQ ID NO. 7)
B GCGCAAGCTTTTTTTTTTTTCC (SEQ ID NO. 8)
C GCGCAAGCTTTTTTTTTTTTCG (SEQ ID NO. 9)
D GCGCAAGCTTTTTTTTTTTTGT (SEQ ID NO. 10)
E GCGCAAGCTTTTTTTTTTTTGG (SEQ ID NO. 11 ) s F GCGCAAGCTTTTTTTTTTTTGA (SEQ ID NO. 12)
G GCGCAAGCTTTTTTTTTTTTAT (SEQ ID NO. 13)
H GCGCAAGCTTTTTTTTTTTTAC (SEQ ID NO. 14)
I GCGCAAGCTTTTTTTTTTTTAG (SEQ ID NO. 15)
K GCGCAAGCTTTTTTTTTTTTAA (SEQ ID NO. 16) o L GCGCAAGCTTTTTTTTTTTTCA (SEQ ID NO. 17)
M GCGCAAGCTTTTTTTTTTTTGC (SEQ ID NO. 18) Random primers:
01 CGGGAAGCTTATCGACTCCAAG (SEQ ID NO. 19)
02 CGGGAAGCTTTAGCTAGCATGG (SEQ ID NO. 20) 5 03 CGGGAAGCTTGCTAAGACTAGC (SEQ ID NO. 21)
04 CGGGAAGCTTTGCAGTGTGTGA (SEQ ID NO. 22)
05 CGGGAAGCTTGTGACCATTGCA (SEQ ID NO. 23)
06 CGGGAAGCTTGTCTGCTAGGTA (SEQ ID NO. 24)
07 CGGGAAGCTTGCATGGTAGTCT (SEQ ID NO. 25) o 08 CGGGAAGCTTGTGTTGCACCAT (SEQ ID NO. 26)
09 CGGGAAGCTTAGACGCTAGTGT (SEQ ID NO. 27)
10 CGGGAAGCTTTAGCTAGCAGAC (SEQ ID NO. 28) 11 CGGGAAGCTTCATGATGCTACC (SEQ IDNO.29)
12 CGGGAAGCTTACTCCATGACTC (SEQ IDNO.30)
13 CGGGAAGCTTATTACAACGAGG (SEQ IDNO.31)
14 CGGGAAGCTTATTGGATTGGTC (SEQ IDNO.32) 15 CGGGAAGCTTATCTTTCTACCC (SEQ IDNO.33)
16 CGGGAAGCTTATTTTTGGCTCC (SEQ IDNO.34)
17 CGGGAAGCTTATCGATACAGG (SEQ ID NO.35)
18 CGGGAAGCTTTATGGTAAAGGG (SEQ ID NO.36)
19 CGGGAAGCTTTATCGGTCATAG (SEQ ID NO.37) 20 CGGGAAGCTTTAGGTACTAAGG (SEQ ID NO.38)
PCR amplification reactions were set up in triplicate for both control and mutant RNAs with each primer pair (240 different primer pairs). The amplifications were composed of 1.0 μl of the cDNA reaction. 2.0 μl of 10 x PCR buffer (500 mM KCl; 100 mM Tris-HCl pH 9.0; 15 mM MgCl2; 1% (w/v) gelatin; 1% (v/v) Triton X-100), 1.5 μl of 25 mM dNTP. 2.5 μl of 10 mM 5' arbitrary primer, 1.25 μl of 10 mM T-rich primer, 0.15 μl of 32P-dATP (2000 Ci/mM, New England Nuclear - DuPont, Boston, MA), and 2.5 units of Taq DNA polymerase (Perkin-Elmer Corp., Branchburg, NJ) with the volume adjusted to 20 μl with H2O. Reactions were conducted using a hermocycler programmed for one cycle of 98°C for 10 seconds; four cycles each at 94°C for 30 seconds. 42°C for 60 seconds, and 72°C for 30 seconds; and 25 cycles each at 94°C for
30 seconds. 60°C for 60 seconds, and 72°C for 30 seconds.
A 3.5 μl aliquot of each PCR reaction was denatured in formamide sequencing dye solution and resolved on a 6% polyacrylamide, 7 M urea sequencing gel. The triplicate control and mutant PCR reactions from one primer pair were electrophoresed in adjacent rows for comparison of band patterns. Gels were dried onto Whatman 3M paper, marked for orientation with fluorescent ruler tape, and exposed to a sheet of medical X-ray film overnight. The unused portions of the differential display reactions were frozen at -20°C for use in fragment screening.
The film and gel were aligned using the trace from the fluorescent ruler. Differentially displayed bands were marked and excised from the gel with a scalpel. The gel slice, including the Whatman filter paper, was soaked for 10 minutes at ambient temperature, then incubated for 15 minutes at 95°C in 0.1 ml of water in a 1.5 ml
- 36 - SUBSTTTUTE SHEET (RULE 26) Eppendorf microfuge tube. The eluted DNA was precipitated using a solution of 10 μl of 3 M sodium acetate, 5 μl of 10 mg/ml glycogen, and 0.45 μl of 100% ethanol. The DNA pellet was resuspended in 10 μl of water and reamplified with 40 PCR rounds using the same primer pair initially used for amplification and under the same conditions described above but using a 60°C annealing temperature.
The PCR amplified bands were ligated into pCR2.1 (Invitrogen, La Jolla, CA) that had been linearized with EcoRV and TA-tailed following the protocol described in Hadjeb and Berkowitz, 1996, Biotechniques 20: 20-22. Ligations were transformed into competent Escherichia coli DH5α cells, and the resulting colonies were screened by blue/white selection on agarose plates containing X-gal (5-bromo-4-chloro-3-indolyl-β- D-galactopyranoside). For each differentially displayed fragment, six colonies were picked for further analysis.
Example 3: Screening of differentially displayed fragments Each of the groups of six colonies described in Example 2 were grown overnight in 3 ml of Luria broth (1% bactotryptone-0.5% yeast extract-0.5% sodium chloride) supplemented with 100 μg of ampicillin per ml. The cells were collected by centrifugation. The cell pellets were resuspended in 0.3 ml of 10 mM EDTA-50 mM Tris-HCl pH 8.0 buffer containing 100 μg of RNase A per ml, and lysed by the subsequent addition of 0.3 ml of 1% SDS-200 mM NaOH. After a 5 minute incubation at ambient temperature. 0.3 ml of 2.0 M potassium acetate pH 5.5 was added. The lysate was clarified by a 10 minute centrifugation at 15,0000 x g at 4°C. The supernatant was transferred to an Eppendorf tube and then 0.7 ml of isopropanol was added to precipitate the DNA. The DNA was collected by a 15 minute centrifugation at 15,000 x g at 4°C. The pellet was resuspended in 0.1 ml of 1 mM EDTA-10 mM Tris-HCl pH 8.0 and the sample stored at -20°C.
The cloned fragments were initially screened for differential expression by rehybridization back to their originating PCR reaction (the unused portion of the reaction that was stored at -20°C). The prepared DNA from each colony was diluted 1 :20 with H20 and then applied to duplicate nylon membranes using a slot blot apparatus. Each duplicate membrane carried clones from those fragments identified using four separate primer pairs (i.e.. fragments excised from lanes containing the amplicons from oligo E and primers 01, 03. 06 and 08). The membranes were baked for 1-2 hours at 80°C in a vacuum oven, and then hybridized under high stringency conditions (50% formamide, 6xSSC, 42°C). The hybridization probe for one filter comprised the denatured products from the triplicate reactions amplified using cDNA from the mutant strain and the four primer pairs corresponding to the colonies be screened; for the other filter the cDNA was synthesized from the control strain. If the colonies were obtained from fragments produced using oligo D and primers 01, 03, 06, and 08 and Aspergillus oryzae HC4.01 cDNA, then those PCR reactions were added to the hybridization mix. Membranes were washed once for 15 minutes in 0.5 x SSC, 0.1%) SDS at 65°C, and analyzed on a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA). If a fragment was amplified from Aspergillus oryzae HC4.01 cDNA but not Aspergillus oryzae 27 cDNA, the clone should only hybridize back to the Aspergillus oryzae HC4.01 PCR reaction. Clones hybridizing with only one of the two probes in agreement with the original differential display were stored and their inserts used as probes in Northern analysis. Northern blot analyses were performed according to Maniatis et al, 1982,
Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York, for formaldehyde gel electrophoresis. Filters containing total RNA from Aspergillus oryzae strains 27 and HC4.01, respectively, were hybridized under high stringency conditions (50% formamide, 6xSSC at 42°C) to radiolabeled agarose gel purified DNA from those clones that were positive after the initial slot blot screening.
The DNA was radiolabeled by nick translation (Maniatis et al, supra) with α[32P]dCTP (Amersham. Arlington Heights, IL) and added to the hybridization buffer at an activity of approximately 1 x 106 cpm per ml of buffer.
Two different inserts showed differential hybridization in which they hybridized to Aspergillus oryzae 27 RNA and not to Aspergillus oryzae HC4.01 RNA. These plasmids were designated pToC367 with the insert designated DDC2 and pToC370 with the insert designated DDC3.
Example 4: Isolation and characterization of cDNA clones for DDC2 and DDC3 The DDC2 insert from pToC367 (approximately 250 bp) was sequenced on an
ABI Automated DNA Sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions to obtain a partial cDNA sequence, which contained a stretch of adenosine residues, the so-called "polyA tail" at one end, indicating the direction of transcription of the gene.
Double stranded cDNA was synthesized according to the following protocol. To create the first strand cDNA, 1.0 μg of total RNA from Aspergillus oryzae 27 (day 2) was added with 1 mM CapSwitch™ oligonucleotide (Clontech, Palo Alto, CA) and 1 mM CDS/3' PCR primer (Oligo(dT)30N,N and N,=A, C or G), (Clontech, Palo Alto, CA) in a final volume of 5 μl, incubated at 72°C for 2 minutes, and then chilled on ice for 2 minutes. The reaction was centrifuged for 30 seconds in a microfuge, and then the following were added: 2.0 μl of 5x first strand synthesis buffer transcriptase (Life Technologies, Gaithersburg, MD), 1.0 μl of 20 mM dithiothreitol, 1.0 μl of 10 mM dNTP, and 200 units of Superscript™ II reverse transcriptase (Life Technologies,
Gaithersburg, MD). After incubation at 42°C for 1 hour, the cDNA was stored at -20°C.
Second strand cDNA was prepared by PCR amplification in which 2 μl of the first strand reaction was mixed with 10 μl of lOx PCR buffer, 2 μl of 10 mM dNTP, 2 μl of 5' PCR primer, 2 μl of CDS/3' PCR primer, and 2 μl of 50x Advantage KlenTaq polymerase mix (Clontech, Palo Alto, CA). Amplification was performed in a thermocycler programmed for 1 cycle at 95°C for 1 minute, 20 cycles each at 95°C for 15 seconds and 68°C for 5 minutes.
Based on the sequence data of the insert obtained from pToC367, an oligonucleotide specific for DDC2 was synthesized to isolate cDNA clones. The primer was oriented opposite to transcription of the genes. The primer had the following sequence:
26186: CATCTCATTATCAGCCATTCC (SEQ ID NO. 39)
Based on the sequence data of the insert obtained from pToC370, oligonucleotides specific for DDC3 were synthesized to isolate cDNA clones. The primers were oriented opposite to transcription of the genes. The primers had the following sequences:
26183: CCCAACATACCCGGAAATCG (SEQ IDNO.40) 26184: GCGGGTGGTTCGGGAACACC (SEQ IDNO.41) PCR reactions were run on the Aspergillus oryzae no. 27 cDNA with primer
26186 (DDC2) and the 5' primer from the CAP Finder Kit (Clontech, Palo Alto, CA). Thirty PCR cycles were run with an anealing temperature of 60°C. The 5' primer is part of the CAP Finder Kit which will hybridize to the 5' end of the cDNA created with this kit. A fragment of approximately 400 bp was obtained.
In order to obtain a DDC3 PCR product, nested PCR was run. The first PCR reaction was run with primer 26183 and the 5' primer from the Cap Finder Kit (Clontech. Palo Alto. CA) for 30 cycles with an annealing temperature of 60°C. The 5' primer is part of the Clontech CAP Finder Kit which will hybridize to the 5 'end of the cDNA created with this kit. An aliquot from this PCR reaction was used as template for a new reaction with primer 26184 and the 5 'Cap Finder primer. Again the reaction was run for 30 cycles with an annealing temperature of 60°C. In the second PCR reaction a fragment of approximately 400 bp was obtained.
The fragments were individually cloned into the pCRII vector (Invitrogen. San Diego CA) and sequenced on an ABI Automated DNA Sequencer according to the manufacturer's instructions. The DDC2 full-length cDNA sequence and deduced amino acid sequence obtained are shown in SEQ ID NOs. 3 and 2, respectively. The DDC3 full-length cDNA sequence and deduced amino acid sequence obtained are shown in SEQ ID NOs. 6 and 5, respectively.
Computer analysis for coding regions by Testcode and Codonpreference (Genetics Computer Group, Inc., Madison, WI) and translation of the cDNA for DDC2 suggested an open reading frame encoding a polypeptide of 64 amino acids (SEQ ID NO. 2) and for DDC3 suggested an open reading frame encoding a polypeptide of 83 amino acids (SEQ ID NO. 5). The program Sigcleave (Genetics Computer Group, Inc.. Madison. WI) predicted the presence of a secretion signal of 18 amino acids for the DCC2 polypeptide and a secretion signal of 20 amino acids for the DCC3 polypeptide. suggesting that the polypeptides are secreted. The DDC3 polypeptide contained three repeats of 18 amino acids of the following sequence: DDGAIRIPVKGVPEPEKR (SEQ ID NO. 5). The third repeat showed some deviation at the C-terminal where) the two last amino acids in the repeat are Lys and Arg. This dibasic site is known to be a cleavage site for the kex2 protease involved in maturation of secreted proteins in yeast and fungi. The gene structure suggested that DDC3 encodes a protein that is secreted and processed into possibly 3 small peptides. The gene structure of DDC3 resembles the structure of the alpha-factor gene of Saccharomyces cerevisae (Kurjan et al, 1982, Cell 30: 933-943).
- 40 -
SUBSTΓΓUTE SHEET RULE 26) The deduced amino acid sequences of the DDC2 and DDC3 polypeptides (SEQ
ID NOs. 2 and 5, respectively) were compared to the sequences in GeneBank by various search algorithms such as Fasta (Lipman and Pearson, 1988, Proceedings of the National Academy of Sciences USA 85: 2444) and Blast (Altschul et al, 1990, Journal of Molecular Biology 215: 403). No homologies were found.
Example 5: Isolation of DDC2 and DDC3 genomic clones
The cDNA clone of DDC2 was radiolabeled by nick translation (Sambrook et al. , supra) with α[32P]dCTP (Amersham, Arlington Heights, IL) and added to the hybridization buffer at an activity of approximately 1 x 106 cpm per ml of buffer and used as a probe against a cosmid library of Aspergillus oryzae IFO 4177. The cosmid library was constructed using the SuperCosl Cosmid Vector Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.
Genomic DNA of Aspergillus oryzae IFO 4177 was prepared from protoplasts made by standard procedures (Christensen et. al, 1988, Biotechnology 6: 1419-1422). After the protoplasts were isolated, they were pelleted by centrifugation at 2500 rpm for 5 minutes in a Labofuge T (Heto, Denmark). The pellet was then suspended in 10 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 μg/ml proteinase K and 0.5% SDS as stated in the manual from the SuperCosl Kit. The remaining steps of the DNA preparation were performed according to the manufacturer's instructions accompanying the kit. The size of the genomic DNA was analyzed by electrophoresis using a CHEF- gel apparatus (BioRad Laboratories, Hercules, CA). A 1% agarose gel was run for 20 hours at 200 volts with a 10-50 second pulse. The gel was stained with ethidium bromide and photographed. The DNA was found to be from about 50 to greater than 100 kb in size. The DNA was partially digested with Sau3A. The size of the digested DNA obtained was 20-50 kb as determined by the same type of CHEF-gel analysis as above. A CsCl gradient banded SuperCosl cosmid was prepared according to the manufacturer's instructions. Ligation and packaging was likewise performed according to the manufacturer's instructions. After titration of the library, all of the packaging mix from one ligation and packaging was transfected into E. coli XL 1 -Blue MR (Stratagene,
La Jolla, CA) and plated on LB plates supplemented with 50 μg of ampicillin per ml. Approximately 3800 colonies were obtained. Cosmid preparation from ten colonies indicated that all had inserts of the expected size. The colonies were picked individually and inoculated into microtiter plate wells containing 100 μl of Luria Broth medium supplemented with 100 μg of ampicillin per ml and incubated at 37°C overnight. One hundred μl of 50%> glycerol was added to each well and the entire library was frozen at -80°C. A total of 3822 colonies were stored. This represented an approximate 4.4-fold amplification of the Aspergillus oryzae genome.
The entire library was spotted at high density onto nylon membranes (Genome
Systems. Inc., St. Louis, MO). 32P-labelled DDC2 cDNA was hybridized under high stringency conditions (2xSSC, 65°C) to cosmid 18H7. 2P-labelled DDC3 cDNA was hybridized under high stringency conditions (2X SSC, 65°C) and cosmid 34G12 showed a strong hybridization signal.
Plasmid DNA was prepared from cosmid 18H7 and sequenced on an ABI
Automatic DNA Sequencer according to the manufacturer's instructions using the following primers both of which are contained within the cDNA sequence:
30982: TGCAGATCTCCTGGTTTGCC (SEQ ID NO. 42)
30983: CTCCCTAAGGAATGCAAATGG (SEQ ID NO. 43)
Plasmid DNA was also prepared and from cosmid 34G12 and sequenced on an
ABI Automatic DNA Sequencer according to the manufacturer's instructions using the following primers made from the cDNA sequence:
30984: CCATGAAGCTCTTCTCTACC (SEQ ID NO. 44)
30985: CTATTTCTCAGCGGGTGGTTCG (SEQ ID NO. 45)
New primers were synthesized in order to obtain sequence further upstream and downstream. The resulting genomic nucleic acid sequences and the deduced amino acid sequences of DDC2 and DCC3 are shown in Figure 1 (SEQ ID NOs. 1 and 2, respectively) and Figure 2 (SEQ ID NOs. 4 and 5, respectively), respectively.
Example 6: Construction of a deletion plasmid for DDC2 A plasmid designed for deletion of the DDC2 gene in Aspergillus oryzae was constructed by cloning 1 kb of sequence upstream of the start codon and 1 kb of sequence downstream of the stop codon into a vector separated by the Aspergillus oryzae pyrG gene.
A 5' fragment was obtained by PCR using cosmid 18H7 as template with the following primers :
102012: GAAGATCTTGGGGGCAGTCAGTGACGGG (SEQ ID NO. 46) 102013 : TCCCCCGGGTATGATTTGATTAGGATG (SEQ ID NO. 47)
The 3 ' fragment was obtained by PCR using cosmid 18H7 as template under the same conditions described above with the following primers: 102014: TCCCCCGGGAGTGTTATTAATAAGGAGG (SEQ ID NO. 48) 102015: TGCACTGCAGGTATCTGTATCCCAGTCAGC (SEQ ID NO. 49) The 5' PCR fragment was cut with restriction enzymes Smal and Bglϊl and the cut fragment was purified from an agarose gel. The 3 ' PCR fragment was cut with Smal and Pstl and the restricted fragment was purified from an agarose gel. The 5' fragment and the 3 ' fragment were cloned into pIC19H (Marsh et al. 1984, Gene 32: 481-485) restricted with BgRl and Pstl. The resulting plasmid was cut with Smal and dephosphorylated with alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN). A 3.5 kb Hindlll fragment containing the Aspergillus oryzae pyrG gene was isolated from pJaL335 (PCT/DK 96/00528) and blunt ended by incubation with the Klenow fragment of DNA polymerase (Promega, Madison, WI) and dNTP. The two fragments were ligated resulting in plasmid pToC391 as shown in Figure 3. pToC391 can be linearized with Pvul for transformation of Aspergillus oryzae pyrG' strains to produce transformants in which the DDC2 gene is replaced by the pyrG gene. Such DDC2 mutant strains can be found, e.g., by Southern analysis or by PCR methods. The DDC2 mutant strains can be used as expression hosts for any heterologous protein. The DDC2 mutant strain so isolated can easily be made pyrG' by selection of resistance to fluororonic acid due to the 400 bp repeat flanking the yrG gene on pJaL335.
Example 7: Construction of a deletion plasmid for DDC3
A plasmid designed for deletion of DDC3 in Aspergillus oryzae was constructed by cloning approximately 1 kb of sequence upstream of the start codon and approximately 1 kb of sequence downstream of the stop codon into a vector in which the two insertions were separated by the Aspergillus oryzae pyrG gene.
The cosmid 34G12 did not contain enough upstream sequence to allow for contruction of a deletion plasmid. To clone more upstream sequence inverse PCR was used. Southern analysis of genomic DNA from Aspergillus oryzae IFO 4177 (the parent strain of Aspergillus oryzae A1560-T40) with a 32P-labelled DDC3 probe revealed an approximately 2.5 kb Nsil fragment. The genomic DNA was digested with Nsil, ligated, and used as template for the inverse PCR reaction using the following primers: 30985: CTATTTCTCAGCGGGTGGTTCG (SEQ ID NO. 45) 101449: CTCTATGTAACCAACTCCTGC (SEQ ID NO. 50)
A fragment of the expected size (2.3 kb) was obtained and cloned into the vector pCRII (Invitrogen, San Diego CA). The fragment was sequenced on an ABI Automated DNA Sequencer.
The sequence information was used to design the primers for construction of the deletion plasmid. The primers used to obtain the 5 ' fragment were as follows: 116497: CGAAAGCTTACTCTCTGGAACAGG (SEQ ID NO. 51) 118141 : CATGGAGCTCGATGGCCAAATGACTGATTCC (SEQ ID NO. 52) The primers used to obtain the 3 ' fragment were as follows:
116494: GACACTCGAGTAAGATATGCTGCAGAC (SEQ ID NO. 53) 116495: CATAAGCTTTCGAGTGATAATGTCTTGG (SEQ ID NO. 54)
Two PCR reactions were run using IFO4177 genomic DNA as template and either the primer pair 116497/118141 or the pair 116494/116495. The 5' PCR fragment was cut with restriction enzymes Sαcl and Hindlll, the 3' fragment was restricted with Hindlll and Xhol, and both cut fragments were purified from an agarose gel. The fragments were cloned into the vector pBluescript (Stratagene, La Jolla, CA) restricted with Sacl and Xhol. The resulting plasmid was cut with Hindlll, dephosphorylated with alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN), and then ligated with the 3.5 kb Hindlll fragment from pJaL335 (PCT/DK 96/00528) containing the pyrG gene. The two fragments were ligated resulting in plasmid pToC401 as shown in Figure 4. pToC401 can be linearized with Sacl for transformation of Aspergillus oryzae (pyrG') strains to produce transformants in which the DDC3 gene is replaced by the pyrG gene). Such mutant strains can be found, e.g., by Southern analysis or by PCR methods. The DDC3 mutant strains can be used as expression hosts for any heterologous protein. The DDC3 mutant strains isolated by this procedure can easily be made pyrG' by selection of resistance to fluororonic acid due to the 400 bp repeat flanking the pyrG gene on pJaL335.
Deposit of Biological Material
The following biological material has been deposited under the terms of the Budapest Treaty with the Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany and given the following accession numbers:
Deposit Accession Number Date of Deposit
E. coli DH5 + cosmid 18H7 DSM 12060 March 3, 1998 E. coli DH5α + cosmid 34G12 DSM 11924 January 19, 1998
The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.
Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.
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Claims

ClaimsWhat is claimed is:
1. A method for producing a polypeptide, comprising:
(A) cultivating a mutant cell of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide in which the mutant cell produces more of the polypeptide than the parent cell when cultivated under the same conditions, wherein the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a modification of one or more second nucleic acid sequences selected from the group consisting of:
(i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5; (ii) a nucleic acid sequence having at least 50% homology to nucleotides 1014 to
1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4;
(iii) a nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (b) a subsequence of (a) of at least 100 nucleotides, or (c) a complementary strand of (i) or (ii);
(iv) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(v) an allelic variant of (i), (ii), or (iii); and (vi) a subsequence of (i), (ii), (iii), or (v), wherein the subsequence encodes a polypeptide fragment having DDC2 or DDC3 polypeptide activity; and
(B) recovering the polypeptide from the cultivation medium of the mutant cell.
2. The method of claim 1, wherein the first nucleic acid sequence encodes a polypeptide native to the fungal cell.
3. The method of claim 1, wherein the first nucleic acid sequence encodes a polypeptide heterologous to the fungal cell.
4. The method of any of claims 1-3, wherein the polypeptide is a hormone, hormone variant, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter.
5. The method of claim 4, wherein the enzyme is an oxidoreductase. transferase, hydrolase, lyase, isomerase. or ligase.
6. The method of claim 5, wherein the enzyme is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase. oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
7. The method of any of claims 1-6, wherein the filamentous fungal cell is an Acremonium, Aspergillus. Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum. Talaromyces, Thermoascus. Thielavia. Tolypocladium, or Trichoderma cell.
8. The method of claim 7. wherein the filamentous fungal cell is an Aspergillus cell.
9. The method of claim 8, wherein the Aspergillus cell is an Aspergillus oryzae cell.
10. The method of claim 8, wherein the Aspergillus cell is an Aspergillus niger cell.
11. The method of any of claims 1-10, wherein the second nucleic acid sequence is a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
12. The method of claim 11, wherein the second nucleic acid sequence is a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 60% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
13. The method of claim 12, wherein the second nucleic acid sequence is a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 70% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
14. The method of claim 13, wherein the second nucleic acid sequence is a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 80% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
15. The method of claim 14, wherein the second nucleic acid sequence is a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 90% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
16. The method of claim 15, wherein the second nucleic acid sequence is a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 95% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
17. The method of any of claims 1-10, wherein the second nucleic acid sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5.
18. The method of any of claims 1-10, wherein the second nucleic acid sequence encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5; or a fragment thereof having DDC2 or DDC3 polypeptide activity.
19. The method of claim 18, wherein the second nucleic acid sequence encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5.
20. The method of any of claims 1-10, wherein the second nucleic acid sequence encodes a polypeptide consisting of amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
21. The method of any of claims 1-10. wherein the second nucleic acid sequence is a nucleic acid sequence which has at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4.
22. The method of claim 21, wherein the second nucleic acid sequence is a nucleic acid sequence which has at least 60% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4.
23. The method of claim 22, wherein the second nucleic acid sequence is a nucleic acid sequence which has at least 70% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4.
24. The method of claim 23, wherein the second nucleic acid sequence is a nucleic acid sequence which has at least 80% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4.
25. The method of claim 24, wherein the second nucleic acid sequence is a nucleic acid sequence which has at least 90% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4.
26. The method of claim 25, wherein the second nucleic acid sequence is a nucleic acid sequence which has at least 95% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4.
27. The method of any of claims 1-10, wherein the second nucleic acid sequence is the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 4.
28. The method of any of claims 1-10, wherein the second nucleic acid sequence comprises the nucleic acid sequence of nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4.
29. The method of any of claims 1-10, wherein the second nucleic acid sequence is a nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (b) a subsequence of (a) of at least 100 nucleotides. or (c) a complementary strand of (a) or (b). 0
30. The method of claim 29, wherein the second nucleic acid sequence is a nucleic acid sequence which hybridizes under low stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, or (b) a complementary strand of (a). 5
31. The method of any of claims 1-10, wherein the second nucleic acid sequence is a nucleic acid sequence which hybridizes under medium stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (b) a subsequence of (a) of at least 100 nucleotides, or (c) a complementary strand of (a) or 0 (b).
32. The method of claim 31, wherein the second nucleic acid sequence is a nucleic acid sequence which hybridizes under medium stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, or (b) a 5 complementary strand of (a) .
33. The method any of of claims 1-10, wherein the second nucleic acid sequence is a nucleic acid sequence which hybridizes under high stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (b) a o subsequence of (i) of at least 100 nucleotides, or (c) a complementary strand of (a) or (b).
34. The method of claim 33, wherein the second nucleic acid sequence is a nucleic acid sequence which hybridizes under high stringency conditions with (a) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, or (b) a complementary strand of (a).
35. The method of any of claims 1-10, which encodes a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids.
36. The method of any of claims 1-10, wherein the second nucleic acid sequence is the nucleic acid sequence contained in the cosmid 34G12 contained in E. coli DSM 11924.
37. The method of any of claims 1-10, wherein the second nucleic acid sequence is the nucleic acid sequence contained in the cosmid 18H7 which is contained in E coli DSM 12060.
38. The method of any of claims 1-37, wherein the mutant cell further comprises one or more modifications of one or more third nucleic acid sequences, wherein the modification reduces or eliminates expression of the one or more third nucleic acid sequences. 0
39. The method of claim 38, wherein the third nucleic acid sequence encodes an enzyme selected from the group consisting of an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase. cellulase, chitinase, cutinase, esterase, alpha-galactosidase, beta- galactosidase. glucoamylase, alpha-glucosidase, beta-glucosidase, laccase, lipase, 5 mannosidase. mutanase, oxidase, pectinolytic enzyme, peroxidase, proteolytic enzyme, ribonuclease. transglutaminase, and xylanase.
40. A mutant filamentous fungal cell for producing a polypeptide comprising a first nucleic acid sequence encoding the polypeptide and a modification of a second nucleic acid o sequence selected from the group consisting of:
(a) a nucleic acid sequence encoding a polypeptide having an amino acid
-53- SUBSTITUTΕ SHEET (RULE 26) sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
(b) a nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4; (c) a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii);
(d) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(e) an allelic variant of (a), (b), or (c); and
(f) a subsequence of (a), (b), (c), or (e), wherein the subsequence encodes a polypeptide fragment having DDC2 or DDC3 polypeptide activity.
41. The mutant cell of claim 40, further comprising one or more modifications of one or more third nucleic acid sequences, wherein the modification reduces or eliminates expression of the one or more third nucleic acid sequences.
42. The mutant cell of claim 41, wherein the third nucleic acid sequence encodes an enzyme selected from the group consisting of an aminopeptidase, amylase, carbohydrase, carboxypeptidase. catalase. cellulase, chitinase, cutinase, esterase, alpha-galactosidase. beta- galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase. laccase, lipase, mannosidase, mutanase. oxidase, a pectinolytic enzyme, peroxidase, proteolytic enzyme, ribonuclease, transglutaminase. and xylanase.
43. A method for producing the mutant cell of any of claims 40-42, comprising modifying one or more second nucleic acid sequences of a parent filamentous fungal cell selected from the group consisting of: (a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
(b) a nucleic acid sequence having at least 50% homology to nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4;
(c) a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID
NO. 4, (ii) a subsequence of (i) of at least 100 nucleotides, or (iii) a complementary strand of (i) or (ii);
(d) a nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(e) an allelic variant of (a), (b), or (c); and
(f) a subsequence of (a), (b), (c), or (e), wherein the subsequence encodes a polypeptide fragment having DDC2 or DDC3 polypeptide activity.
44. An isolated polypeptide selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5;
(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (ii) a subsequence of (i) of at least 100 nucleotides. or (iii) a complementary strand of (i) or (ii);
(c) a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids; (d) an allelic variant of (a) or (b); and
(e) a fragment of (a), (b), or (d), wherein the fragment has DDC2 or DDC3 polypeptide activity.
45. The polypeptide of claim 44, comprising an amino acid sequence which has at least 50% identity to amino acids 19 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
46. The polypeptide of claim 44, consisting of the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5; or a fragment thereof which has DDC2 or DDC3 polypeptide activity.
47. The polypeptide of claim 46, consisting of the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5.
48. The polypeptide of claim 46, consisting of amino acids 1 to 64 of SEQ ID NO. 2 or amino acids 21 to 83 of SEQ ID NO. 5.
49. The polypeptide of claim 44, which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, (ii) a subsequence of (i) of at least 100 nucleotides. or (iii) a complementary strand of (i) or (ii).
50. The polypeptide of claim 44, which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 1014 to 1151 of SEQ ID NO. 1 or nucleotides 1041 to 1229 of SEQ ID NO. 4, or (ii) a complementary strand of (i).
51. The polypeptide of claims 44, which is a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids.
52. The polypeptide of claim 44, which is encoded by the nucleic acid sequence contained in cosmid 34G12 contained in E. coli DSM 11924.
53. The polypeptide of claim 44, which is encoded by the nucleic acid sequence contained in cosmid 18H7 which is contained in E. coli DSM 12060.
54. An isolated nucleic acid sequence which encodes the polypeptide of claim 44.
55. A nucleic acid construct comprising the nucleic acid sequence of claim 54 operably linked to one or more control sequences which direct the expression of the polypeptide in a suitable expression host.
56. A recombinant expression vector comprising the nucleic acid construct of claim 55.
57. A recombinant host cell comprising the nucleic acid construct of claim 55.
58. A method for producing the polypeptide of claim 44 comprising (a) cultivating a strain under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide from the cultivation medium.
59. A method for producing a polypeptide comprising (a) cultivating the host cell of claim 57 under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide from the cultivation medium.
60. A nucleic acid construct comprising a gene encoding a protein operably linked to a nucleic acid sequence encoding a signal peptide consisting of nucleotides 960 to 1013 of SEQ ID NO. 1 or nucleotides 981 to 1040 of SEQ ID NO. 4, wherein the gene is foreign to the nucleic acid sequence.
61. A recombinant expression vector comprising the nucleic acid construct of claim 60.
62. A recombinant host cell comprising the nucleic acid construct of claim 60.
63. A method for producing a protein comprising (a) cultivating the recombinant host cell of claim 62 under conditions suitable for production of the protein; and (b) recovering the protein.
-57- SUBSTΓTUTE SHEET (RULE 26)
EP99925633A 1998-05-15 1999-05-14 Methods for producing polypeptides in filamentous fungal mutant cells Withdrawn EP1078080A1 (en)

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WO2002002825A1 (en) * 2000-07-03 2002-01-10 Wageningen University Method for the production of proteins and polypeptides using fungal cells, in particular aspergillus vadensis
SI1685244T1 (en) 2003-11-21 2012-06-29 Danisco Us Inc Expression of granular starch hydrolyzing enzymes in trichoderma and process for producing glucose from granular starch substrates
WO2005118795A2 (en) 2004-05-27 2005-12-15 Genencor International, Inc. Aspergillus kawachi acid-stable alpha amylase and applications in granular starch hydrolysis
JP5007879B2 (en) * 2004-08-30 2012-08-22 アサヒグループホールディングス株式会社 Yeast strain releasing mannan protein and method for producing mannan protein
EP2247609A1 (en) * 2008-02-25 2010-11-10 Novozymes A/S Method for increasing expression yield of a protein of interest
CN102762714B (en) 2009-12-18 2015-11-25 诺维信股份有限公司 For producing the method for polypeptide in the Deficient In Extracellular Proteases mutant of Trichoderma
WO2012000886A1 (en) * 2010-06-29 2012-01-05 Dsm Ip Assets B.V. Polypeptide having beta-glucosidase activity and uses thereof
CN102168023B (en) * 2011-01-24 2013-05-01 华东师范大学 Method for screening Aspergillus niger strains for highly producing alpha-glucuronide
WO2013028912A2 (en) * 2011-08-24 2013-02-28 Novozymes, Inc. Methods for producing multiple recombinant polypeptides in a filamentous fungal host cell
WO2013028915A2 (en) * 2011-08-24 2013-02-28 Novozymes, Inc. Methods for obtaining positive transformants of a filamentous fungal host cell
CN105754993B (en) * 2016-04-14 2018-06-29 中国林业科学研究院木材工业研究所 A kind of DNA extraction method for seasoned wood
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CN112522231B (en) * 2020-12-14 2023-03-14 广东省微生物研究所（广东省微生物分析检测中心） Acyltransferase, and coding gene and application thereof

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Publication number Priority date Publication date Assignee Title
CA1333777C (en) * 1988-07-01 1995-01-03 Randy M. Berka Aspartic proteinase deficient filamentous fungi
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