Classifications

• • 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)
  • C12N9/2405—Glucanases
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
  • C12N9/2411—Amylases
  • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
• • 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.
• the present invention also relates to isolated promoters and to nucleic acid constructs, vectors, and host cells comprising the promoters operably linked to nucleic acid sequences encoding polypeptides.
• the recombinant production of a heterologous protein in a fungal host cell may provide for a more desirable vehicle for producing the protein in commercially relevant quantities.
• Recombinant production of a heterologous protein is accomplished by constructing an expression cassette in which the DNA coding for the protein is placed under the expression control of a promoter, excised from a regulated gene, suitable for the host cell.
• the expression cassette is introduced into the host cell, usually by plasmid- mediated transformation.
• Production of the heterologous protein is then achieved by culturing the transformed host cell under inducing conditions necessary for the proper functioning of the promoter contained on the expression cassette.
• the present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide operably linked to a second nucleic acid sequence comprising a promoter foreign to the nucleic acid sequence, wherein the promoter comprises a sequence selected from the group consisting of nucleotides 1 to 3949 of SEQ ID NO. 1, nucleotides 1 to 938 of SEQ ID NO. 2, and nucleotides 1 to 3060 of SEQ ID NO. 3, and subsequences thereof; and mutant, hybrid, and tandem promoters thereof; and (b) isolating the polypeptide from the cultivation medium.
• the present invention also relates to isolated promoters sequences and to constructs, vectors, and fungal host cells comprising one or more of the promoters operably linked to a nucleic acid sequence encoding a polypeptide.
• the present invention also relates to methods for obtaining mutant promoters of nucleotides 1 to 3949 of SEQ ID NO. 1, nucleotides 1 to 938 of SEQ ID NO. 2, and nucleotides 1 to 3060 of SEQ ID NO. 3.
• the present invention also relates to isolated nucleic acid sequences, selected from the group consisting of:
• nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 22 to 581 of SEQ ID NO. 4, at least 65% identity with amino acids 19 to 200 of SEQ ID NO. 5, or at least 65% identity with amino acids 1 to 187 of SEQ ID NO. 6;
• nucleic acid sequence having at least 65% homology with nucleotides encoding a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 22 to 581 of SEQ ID NO. 4, at least 65% identity with amino acids 19 to 200 of SEQ ID NO. 5, or at least 65% identity with amino acids 1 to 187 of SEQ ID NO. 6;
• nucleic acid sequence which hybridizes under very low, low, medium, medium-high, high, or very high stringency conditions with (i) nucleotides 4013 to 5743 of SEQ ID NO. 1, nucleotides 993 to 1593 of SEQ ID NO. 2, or nucleotides 3061 to
• nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 4, SEQ ID. NO. 5, or SEQ ID NO. 6 comprising a substitution, deletion, and/or insertion of one or more amino acids;
• the present invention further relates to constructs, vectors, and recombinant host cells of the nucleic acid sequences.
• Figures 1A-F shows the genomic DNA sequence and the deduced amino acid sequence of a Fusarium venenatum glucoamylase gene (SEQ ID NOs. 1 and 4, respectively).
• Figures 2A-C shows the genomic DNA sequence and the deduced amino acid sequence of a Fusarium venenatum gene designated "Daria” (SEQ ID NOs. 2 and 5, respectively).
• Figures 3A-D shows the genomic DNA sequence and the deduced amino acid sequence of a Fusarium venenatum gene designated "Quinn” (SEQ ID NOs. 3 and 6, respectively).
• Figure 4 shows a restriction map of pDM181.
• Figure 5 shows a restriction map of pSheBl .
• Figure 6 shows a restriction map of pDM194.
• Figure 7 shows a restriction map of pJRoy35.
• Figure 8 shows a restriction map of pDM218.
• Figure 9 shows a restriction map of pEJG25A.
• Figure 10 shows a restriction map of pMWR60.
• Figure 11 shows a restriction map of pRaMB62.
• Figure 12 shows a restriction map of pRaMB64.
• Figure 13 shows a restriction map of pRaMB66.
• Figure 14 shows comparative expression of a Humicola lanuginosa lipase reporter gene in Fusarium venenatum under control of Fusarium venenatum amyloglucosidase (pAMG) and Fusarium oxysporum trypsin (pSP387) promoters.
• pAMG Fusarium venenatum amyloglucosidase
• pSP387 Fusarium oxysporum trypsin
• the present invention relates to methods for producing a polypeptide, comprising (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide operably linked to a second nucleic acid sequence comprising a promoter foreign to the first nucleic acid sequence, wherein the promoter comprises a sequence selected from the group consisting of nucleotides 1 to 3949 of SEQ ID NO. 1, nucleotides 1 to 938 of SEQ ID NO. 2, and nucleotides 1 to 3060 of SEQ ID NO. 3, and subsequences thereof; and mutant, hybrid, and tandem promoters thereof; and (b) isolating the polypeptide from the cultivation medium.
• the cells are cultivated in a nutrient medium suitable for production of the polypeptide 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) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
• the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
• the polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate.
• the resulting polypeptide may be recovered by methods known in the art.
• the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray- drying, evaporation, or precipitation.
• polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS- PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
• chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
• electrophoretic procedures e.g., preparative isoelectric focusing
• differential solubility e.g., ammonium sulfate precipitation
• SDS- PAGE or extraction
• promoter is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcriptional start site of a nucleic acid sequence encoding a polypeptide to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region.
• promoter will also be understood to include the 5' non-coding region (between promoter and translation start) for translation after transcription into mRNA, cw-acting transcription control elements such as enhancers, and other nucleotide sequences capable of interacting with transcription factors.
• mutant promoter is defined herein as a promoter having a nucleotide sequence comprising a substitution, deletion, and/or insertion of one or more nucleotides of a parent promoter, wherein the mutant promoter has more or less promoter activity than the corresponding parent promoter.
• mutant promoter will also encompass natural variants and in vitro generated variants obtained using methods well known in the art such as classical mutagenesis, site-directed mutagenesis, and DNA shuffling.
• hybrid promoter is defined herein as parts of two more promoters that are fused together to generate a sequence that is a fusion of the two or more promoters, which is operably linked to a coding sequence and mediates the transcription of the coding sequence into mRNA.
• tandem promoter is defined herein as two or more promoter sequences each of which is operably linked to a coding sequence and mediates the transcription of the coding sequence into mRNA.
• operably linked is defined herein as a configuration in which a control sequence, e.g., a promoter sequence, is appropriately placed at a position relative to a coding sequence such that the control sequence directs the production of a polypeptide encoded by the coding sequence.
• a control sequence e.g., a promoter sequence
• coding sequence is defined herein as a nucleic acid sequence that is transcribed into mRNA which is translated into a polypeptide when placed under the control of the appropriate control sequences.
• the boundaries of the coding sequence are generally determined by the 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 DNA, cDNA, semisynthetic, synthetic, and recombinant nucleic acid sequences.
• the promoter has the nucleic acid sequence of nucleotides 1 to 3949 of SEQ ID NO. 1, or a subsequence thereof.
• the subsequence preferably contains at least about 2100 nucleotides, more preferably at least about 2400 nucleotides, and most preferably at least about 2700 nucleotides.
• the promoter has the nucleic acid sequence of nucleotides 1 to 938 of SEQ ID NO. 2, or a subsequence thereof.
• the subsequence preferably contains at least about 840 nucleotides, more preferably at least about 870 nucleotides, and most preferably at least about 900 nucleotides.
• the promoter has the nucleic acid sequence of nucleotides 1 to 3060 of SEQ ID NO. 3, or a subsequence thereof.
• the subsequence preferably contains at least about 2100 nucleotides, more preferably at least about 2400 nucleotides, and most preferably at least about 2700 nucleotides.
• the promoter is the nucleic acid sequence contained in plasmid pECO3 which is contained in Escherichia coli NRRL B-30067. In another preferred embodiment, the promoter is the nucleic acid sequence contained in plasmid pFAMG which is contained in Escherichia coli NRRL B-30071. In another a preferred embodiment, the promoter is the nucleic acid contained in plasmid pQUINN which is contained in Escherichia coli NRRL B-30075.
• the promoter may also be a mutant of the promoters described above having a substitution, deletion, and/or insertion of one or more nucleotides in the nucleic acid sequence of nucleotides 1 to 3949 of SEQ ID NO. 1, nucleotides 1 to 938 of SEQ ID NO. 2, or nucleotides 1 to 3060 of SEQ ID NO. 3.
• a mutant promoter may have one or more mutations. Each mutation is an independent substitution, deletion, and/or insertion of a nucleotide.
• the introduction of a substitution, deletion, and/or insertion of a nucleotide into the promoter may be accomplished using any of the methods known in the art such as classical mutagenesis, site-directed mutagenesis, or DNA shuffling. Particularly useful is a 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 promoter is a mutant of nucleotides 1 to 3949 of SEQ ID NO. 1. In another preferred embodiment, the promoter is a mutant of nucleotides 1 to 938 of SEQ ID NO. 2. In another preferred embodiment, the promoter is a mutant of nucleotides 1 to 3060 of SEQ ID NO. 3.
• the promoter may also be a hybrid promoter comprising a portion of one or more promoters of the present invention; a portion of a promoter of the present invention and a portion of another promoter, e.g., a leader sequence of one promoter and the transcription start site from the other promoter; or a portion of one or more promoters of the present invention and a portion of one or more other promoters.
• the other promoter may be any promoter sequence which shows transcriptional activity in the fungal host cell of choice including a mutant, truncated, and hybrid promoter, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
• the other promoter sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide and native or foreign to the cell.
• Examples of other promoters useful in the construction of hybrid promoters with the promoters of the present invention include the promoters obtained from the genes for
• Aspergillus oryzae TAKA amylase Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,
• NA2-tpi promoter a hybrid of the promoters from the genes for
• yeast host cells Saccharomyces cerevisiae enolase
• GAL1 Saccharomyces cerevisiae galactokinase
• ADH2/GAP Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3 -phosphate dehydrogenase
• Other useful promoters for yeast host cells are described by Romanos et al, 1992, Yeast 8: 423-488.
• the promoter may also be a tandem promoter comprising two more promoters of the present invention or alternatively one or more promoters of the present invention and one or more other promoters, such as those exemplified above.
• the two or more promoter sequences of the tandem promoter may simultaneously promote the transcription of the nucleic acid sequence.
• one or more of the promoter sequences of the tandem promoter may promote the transcription of the nucleic acid sequence at different stages of growth of the cell.
• the promoter is foreign to the nucleic acid sequence encoding a polypeptide of interest, but the promoter or nucleic acid sequence may be native to the fungal host cell.
• a mutant, hybrid, or tandem promoter of the present invention will be understood to be foreign to a nucleic acid sequence encoding a polypeptide even if the wild-type promoter is native to the nucleic acid sequence.
• a mutant, hybrid, or tandem promoter of the present invention has at least about 20%, preferably at least about 40%, more preferably at least about 60%, more preferably at least about 80%. more preferably at least about 90%, more preferably at least about 100%, even more preferably at least about 200%, most preferably at least about 300%, and even most preferably at least about 400% of the promoter activity of the promoter of nucleotides 1 to 3949 of SEQ ID NO. 1, nucleotides 1 to 938 of SEQ ID NO. 2, or nucleotides 1 to 3060 of SEQ ID NO. 3.
• polypeptide Encoding Nucleic Acid Sequences The polypeptide encoded by the nucleic acid sequence may be native or heterologous to the fungal host cell of interest.
• polypeptide is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins.
• heterologous polypeptide is defined herein as a polypeptide which is not native to the fungal cell, a native polypeptide in which modifications have been made to alter the native sequence, or a native polypeptide whose expression is quantitatively altered as a result of a manipulation of the fungal cell by recombinant DNA techniques.
• a native polypeptide may be recombinantly produced by, e.g., placing a gene encoding the polypeptide under the control of a promoter of the present invention 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 fungal cell may contain one or more copies of the nucleic acid sequence encoding the polypeptide.
• the polypeptide is a hormone or variant thereof, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter.
• the polypeptide is secreted extracellularly.
• 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 nucleic acid sequence encoding a polypeptide of interest may be obtained from any prokaryotic, eukaryotic, or other source.
• the term "obtained from” as used herein in connection with a given source shall mean that the polypeptide is produced by the source or by a cell in which a gene from the source has been inserted.
• PCR polymerase chain reaction
• the cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into the mutant fungal cell where multiple copies or clones of the nucleic acid sequence will be replicated.
• the nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
• the polypeptide may also include a fused or hybrid polypeptide in which another polypeptide is fused at the N-terminus or the C- terminus of the polypeptide or fragment thereof.
• a fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide.
• Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter(s) and terminator.
• the hybrid polypeptide may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the mutant fungal cell.
• the present invention also relates nucleic acid constructs comprising a nucleic acid sequence encoding a polypeptide operably linked to a promoter of the present invention and one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
• Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
• Nucleic acid construct is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid combined and juxtaposed in a manner that would not otherwise exist in nature.
• the term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains a coding sequence and all the control sequences required for expression of the coding sequence.
• An isolated nucleic acid sequence encoding a polypeptide may be further manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector.
• the nucleic acid sequence may comprise one or more native control sequences or one or more of the native control sequences may be replaced with one or more control sequences foreign to the nucleic acid sequence for improving expression of the coding sequence in a host cell.
• control sequences is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide of the present invention.
• 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, polyadenylation sequence, propeptide sequence, promoter of the present invention, signal peptide sequence, and transcription terminator.
• the control sequences include a promoter of the present invention, and transcriptional and translational stop signals.
• the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
• the control sequence may be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription.
• the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
• Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,
• Aspergillus nidulans anthranilate synthase
• Aspergillus niger alpha-glucosidase Aspergillus niger alpha-glucosidase
• Fusarium oxysporum trypsin-like protease Aspergillus nidulans anthranilate synthase
• Aspergillus niger alpha-glucosidase Aspergillus niger alpha-glucosidase
• Fusarium oxysporum trypsin-like protease Fusarium oxysporum trypsin-like protease
• Preferred terminators for yeast host cells are obtained from the genes for
• the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell.
• the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
• Preferred leaders for filamentous fungal host cells are obtained from the genes for
• Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
• Suitable leaders for yeast host cells are obtained from the genes for
• Saccharomyces cerevisiae enolase ENO-1
• Saccharomyces cerevisiae 3- phosphoglycerate kinase Saccharomyces cerevisiae alpha-factor
• the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
• Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin- like protease, and Aspergillus niger alpha-glucosidase.
• the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell'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 naturally contain a signal peptide coding region.
• the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide.
• any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.
• Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
• Useful signal peptides for yeast host cells are obtained from the genes for
• Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase are described by Romanos et al, 1992, supra.
• the control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
• the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
• a propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
• the propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
• regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host 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.
• Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.
• yeast the ADH2 system or GAL1 system may be used.
• filamentous fungi 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.
• nucleic acid sequence encoding the polypeptide would be operably linked with the regulatory sequence.
• the present invention also relates to nucleic acid constructs for altering the expression of a gene encoding a polypeptide which is endogenous to a host cell.
• the constructs may contain the minimal number of components necessary for altering expression of the endogenous gene.
• the nucleic acid constructs preferably contain (a) a targeting sequence, (b) a promoter of the present invention, (c) an exon, and (d) a splice-donor site.
• the construct Upon introduction of the nucleic acid construct into a cell, the construct inserts by homologous recombination into the cellular genome at the endogenous gene site.
• the targeting sequence directs the integration of elements (a)-(d) into the endogenous gene such that elements (b)-(d) are operably linked to the endogenous gene.
• the nucleic acid constructs contain (a) a targeting sequence, (b) a promoter of the present invention, (c) an exon, (d) a splice- donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of elements (a)-(f) such that elements (b)-(f) are operably linked to the endogenous gene.
• the constructs may contain additional components such as a selectable marker. In both embodiments, the introduction of these components results in production of a new transcription unit in which expression of the endogenous gene is altered. In essence, the new transcription unit is a fusion product of the sequences introduced by the targeting constructs and the endogenous gene.
• the gene is activated.
• homologous recombination is used to replace, disrupt, or disable the regulatory region normally associated with the endogenous gene of a parent cell through the insertion of a regulatory sequence which causes the gene to be expressed at higher levels than evident in the corresponding parent cell.
• the activated gene can be further amplified by the inclusion of an amplifiable selectable marker gene in the construct using methods well known in the art (see, for example, U.S. Patent No. 5,641,670).
• expression of the gene is reduced.
• the targeting sequence can be within the endogenous gene, immediately adjacent to the gene, within an upstream gene, or upstream of and at a distance from the endogenous gene.
• One or more targeting sequences can be used.
• a circular plasmid or DNA fragment preferably employs a single targeting sequence
• a linear plasmid or DNA fragment preferably employs two targeting sequences.
• the constructs further contain one or more exons of the endogenous gene.
• An exon is defined as a DNA sequence which is copied into RNA and is present in a mature mRNA molecule such that the exon sequence is in-frame with the coding region of the endogenous gene.
• the exons can, optionally, contain DNA which encodes one or more amino acids and/or partially encodes an amino acid.
• the exon contains DNA which corresponds to a 5' non-encoding region.
• the exogenous exon or exons encode one or more amino acids and/or a portion of an amino acid
• the nucleic acid construct is designed such that, upon transcription and splicing, the reading frame is in- frame with the coding region of the endogenous gene so that the appropriate reading frame of the portion of the mRNA derived from the second exon is unchanged.
• the splice-donor site of the constructs directs the splicing of one exon to another exon.
• the first exon lies 5' of the second exon
• the splice-donor site overlapping and flanking the first exon on its 3' side recognizes a splice-acceptor site flanking the second exon on the 5' side of the second exon.
• a splice-acceptor site like a splice-donor site, is a sequence which directs the splicing of one exon to another exon. Acting in conjunction with a splice-donor site, the splicing apparatus uses a splice- acceptor site to effect the removal of an intron.
• the present invention further relates to methods for producing a polypeptide comprising (a) cultivating a homologously recombinant cell, having incorporated therein a new transcription unit comprising a promoter of the present invention, an exon, and/or a splice donor site operably linked to a second exon of an endogenous nucleic acid sequence encoding the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
• the methods are based on the use of gene activation technology, for example, as described in U.S. Patent No. 5,641,670.
• the present invention also relates to recombinant expression vectors comprising a promoter of the present invention, a nucleic acid sequence encoding a polypeptide, and transcriptional and translational stop signals.
• the various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the promoter and/or nucleic acid sequence encoding the polypeptide at such sites.
• the nucleic acid sequence may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the promoter and/or sequence into an appropriate vector for expression.
• the coding sequence is located in the vector so that the coding sequence is operably linked with a promoter of the present invention and one or more appropriate control sequences for expression.
• the recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence.
• the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
• the vectors may be linear or closed circular plasmids.
• the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
• the vector may contain any means for assuring self-replication.
• the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
• 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 host cell, or a transposon may be used.
• the vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells.
• a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
• Suitable markers for yeast host cells are ADE2,
• Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB
• the vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
• the vector may rely on the nucleic acid sequence encoding the 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 host cell.
• the additional nucleic acid sequences enable the vector to be integrated into the host cell 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, 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 sequence that is homologous with the target sequence in the genome of the host cell.
• the integrational elements may be non-encoding or encoding nucleic acid sequences.
• the vector may be integrated into the genome of the host cell by non- homologous recombination.
• the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
• origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARSl, ARS4, the combination of ARSl and CEN3, and the combination of
• the origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978,
• More than one copy of a nucleic acid sequence encoding a polypeptide may be inserted into the host cell to increase production of the gene product.
• An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
• the present invention also relates to recombinant host cells, comprising a promoter of the present invention operably linked to a nucleic acid sequence encoding a polypeptide, which are advantageously used in the recombinant production of the polypeptides.
• a vector comprising a promoter of the present invention operably linked to a nucleic acid sequence encoding a polypeptide is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described earlier.
• 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 host cell may be any fungal cell useful in the methods of the present invention.
• "Fungi” as used herein includes the phyla Ascomycota, Basidiomycota,
• Chytridiomycota, and Zygomycota as defined by Hawksworth et al, In, Ainsworth and
• the fungal host cell is a yeast cell.
• yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and
• the yeast host cell is a Candida, Hansenula,
• Kluyveromyces Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
• the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Sac
• the yeast host cell is a
• the fungal host cell is a filamentous fungal cell.
• Filamentous fungi include all filamentous forms of the subdivision Eumycota and
• Oomycota (as defined by Hawksworth et al, 1995, supra).
• the filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as
• Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
• the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.
• the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell.
• the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell.
• the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
• the Fusarium venenatum cell is Fusarium venenatum A3/5, which was originally deposited as Fusarium graminearum ATCC 20334 and recently reclassified as Fusarium venenatum by Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnell et al, 1998, Fungal Genetics and Biology 23: 57-67; as well as taxonomic equivalents of Fusarium venenatum regardless of the species name by which they are currently known.
• the Fusarium venenatum cell is a mo ⁇ hological mutant of Fusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO 97/26330.
• Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al, 1984, Proceedings of the National Academy of Sciences USA 81 : 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al, 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
• the present invention further relates to methods for obtaining a mutant promoter, comprising (a) introducing at least one mutation into the sequence of nucleotides 1 to
• the present invention also relates to methods for obtaining a mutant promoter by
• the mutant promoter may be isolated using methods known in the art such as restriction enzyme digestion or PCR amplification.
• the present invention also relates to isolated nucleic acid sequences, selected from the group consisting of: (a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 22 to 581 of SEQ ID NO. 4, at least 65% identity with amino acids 19 to 200 of SEQ ID NO. 5, or at least 65% identity with amino acids 1 to 187 of SEQ ID NO. 6;
• nucleic acid sequence having at least 65% homology with nucleotides 4013 to 5743 of SEQ ID NO. 1, at least 65% homology with nucleotides 993 to 1593 of
• SEQ ID NO. 2 or at least 65% homology with nucleotides 3061 to 3678 of SEQ ID NO. 3;
• nucleic acid sequence which hybridizes under very low, low, medium, medium-high, high, or very high stringency conditions with (i) nucleotides 4013 to 5743 of SEQ ID NO. 1, nucleotides 993 to 1593 of SEQ ID NO. 2, or nucleotides 3061 to 3678 of SEQ ID NO. 3; (ii) the cDNA sequence contained in nucleotides 4013 to 5743 of SEQ ID NO. 1, nucleotides 993 to 1593 of SEQ ID NO. 2, or nucleotides 3061 to 3678 of SEQ ID NO. 3; (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
• nucleic acid sequence encoding a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 4, SEQ ID. NO. 5, or SEQ ID NO. 6 comprising a substitution, deletion, and/or insertion of one or more amino acids;
• 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 inco ⁇ oration 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.
• the present invention relates to isolated nucleic acid sequences encoding polypeptides having an amino acid sequence which has a degree of identity to amino acids 22 to 581 of SEQ ID NO. 4, amino acids 19 to 200 of SEQ ID NO. 5, or amino acids 1 to 187 of SEQ ID NO. 6 (i.e., the mature polypeptide) of at least about 65%, 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% (hereinafter "homologous 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 22 to 581 of SEQ ID NO. 4, amino acids 19 to 200 of SEQ ID NO. 5, or amino acids 1 to 187 of SEQ ID NO. 6.
• nucleic acid sequences of the present invention encode polypeptides that comprise the amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5, or
• nucleic acid sequence of the present invention encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6.
• nucleic acid sequence of the present invention encodes a polypeptide that comprises amino acids 22 to 581 of SEQ ID NO. 4, amino acids 19 to 200 of SEQ ID NO. 5, or amino acids 1 to 187 of SEQ ID NO. 6; or an allelic variant thereof; or a fragment thereof.
• nucleic acid sequence of the present invention encodes a polypeptide that comprises amino acids 22 to 581 of SEQ ID NO.
• nucleic acid sequence of the present invention encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6; or an allelic variant thereof; or a fragment thereof.
• nucleic acid sequence of the present invention encodes a polypeptide that consists of the amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6.
• nucleic acid sequence of the present invention encodes a polypeptide that consists of amino acids 22 to 581 of SEQ ID NO. 4, amino acids 19 to 200 of SEQ ID NO.
• nucleic acid sequence of the present invention encodes a polypeptide that consists of amino acids 22 to 581 of SEQ ID NO. 4, amino acids 19 to 200 of SEQ ID NO. 5, or amino acids 1 to 187 of SEQ ID NO. 6.
• the present invention also encompasses nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6, which differ from SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3, respectively, by virtue of the degeneracy of the genetic code.
• the present invention also relates to subsequences of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 which encode fragments of SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.
• a subsequence is a nucleic acid sequence encompassed by SEQ ID NO. 1 , SEQ
• a subsequence of SEQ ID NO. 1 contains at least 1410 nucleotides, more preferably at least 1500 nucleotides, and most preferably at least 1590 nucleotides.
• a subsequence of SEQ ID NO. 2 contains at least 450 nucleotides, more preferably at least 480 nucleotides, and most preferably at least 510 nucleotides.
• a fragment of SEQ ID NO. 4, SEQ ID NO. 6, or SEQ ID NO. 6 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. 4 contains at least 470 amino acid residues, more preferably at least 500 amino acid residues, and most preferably at least 530 amino acid residues.
• a fragment of SEQ ID NO. 5 contains at least 150 amino acid residues, more preferably at least 160 amino acid residues, and most preferably at least 170 amino acid residues.
• a fragment of SEQ ID NO. 6 contains at least 155 amino acid residues, more preferably at least 165 amino acid residues, and most preferably at least 175 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 polymo ⁇ hism within populations. Gene mutations can be 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 present invention relates to isolated nucleic acid sequences which have a degree of homology to nucleotides 4013 to 5743 of SEQ ID NO. 1, nucleotides 4013 to 1593 of SEQ ID NO. 2, or nucleotides 3061 to 3678 of SEQ ID NO. 3 (i.e., mature polypeptide coding region) of at least about 65%, preferably about 70%, preferably about 80%, more preferably about 90%, even more preferably about 95%. and most preferably about 97% homology, which encode an active polypeptide; or allelic variants and subsequences thereof.
• the degree of homology between two nucleic acid sequences is determined by the Wilbur-
• the present invention relates to isolated nucleic acid sequences encoding polypeptides which 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 (i) nucleotides 4013 to 5743 of SEQ ID NO. 1, nucleotides 993 to 1593 of SEQ ID NO. 2, or nucleotides 3061 to 3678 of SEQ ID NO. 3; (ii) the cDNA sequence contained in nucleotides 4013 to 5743 of SEQ ID NO.
• SEQ ID NO. 1 nucleotides 993 to 1593 of SEQ ID NO. 2, or nucleotides 3061 to 3678 of SEQ ID NO. 3; (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (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, SEQ ID NO. 2, or SEQ ID NO. 3 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment.
• nucleic acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3, or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5. or SEQ ID NO. 6, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides 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.
• 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 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
• 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. 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, SEQ ID NO. 2, or SEQ ID NO. 3, their complementary strands, or subsequences 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 the nucleic acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3. In another preferred embodiment, the nucleic acid probe is a nucleic acid sequence which encodes the polypeptide of SEQ ID NO.
• nucleic acid probe is SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3.
• the nucleic acid probe is nucleotides 4013 to 5743 of
• nucleic acid probe is nucleotides 993 to 5743 of SEQ ID NO. 2, which encodes a mature polypeptide.
• nucleic acid probe is nucleotides 3061 to 3678 of SEQ ID NO. 3, which encodes a mature polypeptide.
• nucleic acid probe is the nucleic acid sequence contained in plasmid pECO3 which is contained in Escherichia coli NRRL B-30067.
• nucleic acid probe is the nucleic acid sequence contained in plasmid pFAMG which is contained in Escherichia coli NRRL B-30071.
• nucleic acid probe is the nucleic acid sequence contained in plasmid pQUINN which is contained in Escherichia coli NRRL B-30076.
• the nucleic acid probe is the mature polypeptide coding region contained in plasmid pECO3 which is contained in Escherichia coli NRRL B-
• the nucleic acid probe is the mature polypeptide coding region contained in plasmid pFAMG which is contained in
• the nucleic acid probe is the mature polypeptide coding region contained in plasmid pQUINN which is contained in Escherichia coli NRRL B-30076.
• 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 2 x 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).
• 2 x 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 5°C to 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 5°C to 10°C below the calculated T m .
• 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 4013 to 5743 of SEQ ID NO. 1, nucleotides 993 to 1593 of SEQ ID NO. 2, or nucleotides 3061 to 3678 of SEQ ID NO. 3, (ii) the cDNA sequence contained in nucleotides 4013 to 5743 of SEQ ID NO. 1, nucleotides 993 to 1593 of SEQ ID NO. 2, nucleotides 3061 to 3678 of SEQ ID NO.
• the present invention relates to isolated nucleic acid sequences which encode variants of the polypeptide having an amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6 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. 4, SEQ ID NO. 5, or SEQ ID NO. 6, or the mature polypeptides 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-terminal 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
• 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), 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,
• nucleic acid sequences of the present invention may be obtained from microorganisms of any genus.
• the term "obtained from” is used as defined herein.
• the polypeptide encoded by a nucleic acid sequence of the present invention is secreted extracellularly.
• the 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, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain.
• a yeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowi
• the nucleic acid sequences are obtained from a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis strain.
• the nucleic acid sequences are obtained from an
• Aspergillus aculeatus Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain.
• the nucleic acid sequences are obtained from a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum strain.
• the nucleic acid sequences are obtained from a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium gramin
• Fusarium venenatum and most preferably from Fusarium venenatum ATCC 20334, e.g., the nucleic acid sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3.
• nucleic acid sequence of SEQ ID NO. 1 is the sequence contained in plasmid pECO3 which is contained in Escherichia coli NRRL B-
• NRRL B-30071 In another more preferred embodiment, the nucleic acid sequence of
• SEQ ID NO. 3 is the sequence contained in plasmid pQUINN which is contained in Escherichia coli NRRL B-30076.
• the nucleic acid sequence is nucleotides 4013 to 5743 of SEQ ID NO. 1, which encodes a mature polypeptide.
• the nucleic acid sequence is nucleotides 993 to 1593 of SEQ ID NO. 2, which encodes a mature polypeptide.
• the nucleic acid sequence is nucleotides 3061 to 3678 of SEQ ID NO. 3, which encodes a mature polypeptide.
• the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamo ⁇ hs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these species 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 microorganisms isolated from nature (e.g.
• 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 present invention also relates to mutant nucleic acid sequences comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO. 1, SEQ ID NO.
• mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 22 to 581 of SEQ ID NO. 4, amino acids 19 to 200 of SEQ ID NO. 5, or amino acids 1 to 187 of SEQ ID NO. 6, respectively.
• the present invention also relates to nucleic acid constructs, recombinant vectors, and host cells containing the nucleic acid sequences described above. The same methods may be used as described earlier for their construction.
• the present invention further relates to the polypeptides encoded by the nucleic acid sequences described above as well as methods of production of the polypeptides using the methods described herein.
• Chemicals used as buffers and substrates were commercial products of at least reagent grade.
• COVE trace metals solution was composed per liter of 0.04 g of NaB 4 O 7 -10H 2 O, 0.4 g of CuSO 4 -5H 2 O, 1.2 g of FeSO 4 -7H 2 O, 0.7 g of MnSO 4 -H 2 O, 0.8 g of Na 2 MoO 2 -2H 2 0, and 10 g of ZnSO 4 -7H 2 O.
• COVE medium was composed per liter of 342.3 g of sucrose, 20 ml of 50X COVE salt solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl 2 , and 25 g of Noble agar.
• 50X Vogels medium was composed per liter of 150 g of sodium citrate, 250 g of KH 2 PO 4 , 10 g of MgSO 4 -7H 2 O, 10 g of CaCl 2 -2H 2 O, 2.5 ml of biotin stock solution, and
• COVE top agarose was composed per liter of 20 ml of 50X COVE salts, 0.8 M sucrose, 1.5 M cesium chloride, 1.0 M acetamide, and 10 g of low melt agarose, pH adjusted to 6.0.
• B ASTA top agarose was composed of COVE top agarose supplemented with 10 mg/ml of the herbicide BastaTM (Hoechst Schering, Rodovre, Denmark).
• RA sporulation medium was composed per liter of 50 g of succinic acid, 12.1 g of
• NaNO 3 1 g of glucose, 20 ml of 50X Vogels, and 0.5 ml of a 10 mg/ml NaMoO 4 stock solution, pH to 6.0.
• YEPG medium was composed per liter of 10 g of yeast extract, 20 g of peptone, and 20 g of glucose.
• STC was composed of 0.8 M sorbitol, 25 mM Tris pH 8, 25 mM CaCl 2 .
• SPTC was composed of 40% PEG 4000, 0.8 M sorbitol, 25 mM Tris pH 8, 25 mM CaCl 2 .
• M400Da medium was composed per liter of 50 g of maltodextrin, 2 g of
• Example 1 Production of Fusarium venenatum mycelial tissue
• Fusarium venenatum CC1-3 a mo ⁇ hological mutant of Fusarium strain ATCC 20334 (Wiebe et al, 1991, Mycological Research 95: 1284-1288), was grown in a two- liter lab-scale fermentor using a fed-batch fermentation scheme with NUTRIOSETM (Roquette Freres, S.A., Beinheim, France) as the carbon source and yeast extract. Ammonium phosphate was provided in the feed. The pH was maintained at 6 to 6.5, and the temperature was kept at 30°C with positive dissolved oxygen.
• Example 1 according to the method of Timberlake and Barnard (1981, Cell 26: 29-37), and the RNA samples were analyzed by Northern hybridization after blotting from 1% formaldehyde-agarose gels (Davis et al, 1986, Basic Methods in Molecular Biology,
• Double-stranded cDNA was synthesized using approximately 5 ⁇ g of poly(A)+ mRNA according to the method of Gubler and Hoffman (1983, Gene 25: 263-269) except a N ⁇ tI-(dT)18 primer (Pharmacia Biotech, Inc., Piscataway, ⁇ J) was used to initiate first strand synthesis.
• the cD ⁇ A was treated with mung bean nuclease (Boehringer Mannheim Co ⁇ oration, Indianapolis, IN) and the ends were made blunt with T4 DNA polymerase (New England Biolabs, Beverly, MA).
• the cDNA was digested with Notl, size selected by agarose gel electrophoresis
• Two independent directional cDNA libraries were constructed using the plasmid cloning vector pZ ⁇ rO-2.1.
• Library A was made using mRNA from mycelia harvested at four days, and Library B was constructed with mRNA from the six day time point.
• Neither cDNA library was amplified in order to examine a representative "snapshot" of the gene expression profile in the cells. Instead the libraries were plated, titered, and independent clones from each were analyzed by DNA sequencing.
• Library A (4 day cells) consisted of about 7.5 x 10 4 independent clones and
• the primary microtiter plates were stored frozen at -80°C.
• the secondary deep-dish plates were incubated at 37°C overnight with vigorous agitation (300 ⁇ ra) on rotary shaker.
• each secondary culture plate was covered with a polypropylene pad (Advanced Genetic Technologies Co ⁇ oration, Gaithersburg, MD) and a plastic microtiter dish cover. DNA was isolated from each well using the 96-well Miniprep Kit protocol of
• Nucleotide sequence data were scrutinized for quality, and samples giving improper spacing less than or equal to 9.2 or ambiguity levels exceeding 3% were discarded or re-run.
• Vector sequences were trimmed with assistance of FACTURATM software (Perkin-Elmer Applied Biosystems, Inc., Foster City, CA).
• FACTURATM software Perkin-Elmer Applied Biosystems, Inc., Foster City, CA.
• sequences were truncated at the end of each sample when the number of ambiguous base calls increased. All sequences were compared to each other to construct overlapping contigs using TIGR Assembler software (Sutton, G.G. et al, 1995, Genome Science and
• Putative glucoamylase clones were identified by partial sequencing of random cDNA clones using an Applied Biosystems Model 377 XL Automated DNA Sequencer according to the manufacturer's instructions and comparison of the deduced amino acid sequence to the sequences in the NRDB as described in Example 4. From more than 2000 cDNA sequences analyzed, two clones from Library A and nine clones from Library B showed amino acid sequence homology to glucoamylase proteins from other fungi and yeasts.
• glucoamylase cDNA clones discovered in this manner, one was estimated to be full-length (encoding the complete protein) on the basis of its alignment to the Neurospora crassa (Geneseq protein accession number R71034) and Humicola grisea (Trembl accession number Q 12623) glucoamylase amino acid sequences and the presence of a possible signal peptide, detected using the Signal-P computer program (Nielsen, et al, 1997, Protein Engineering 10: 1-6).
• This clone designated E. coli FA0401 containing plasmid pFA0401 was selected for use as a probe to clone the corresponding glucoamylase genomic DNA sequence from Fusarium venenatum (see Example 7).
• Example 6 Construction a library of Fusarium venenatum genomic DNA
• Genomic libraries were constructed in ⁇ ZipLox as specified by the procedure of Berka et al. (1998, Appl. Environ. Microbiol. 64, 4423-4427). Briefly, Fusarium venenatum total cellular DNA was partially digested with Tsp5Q91 and size-fractionated on 1% agarose gels. DNA fragments migrating in the size range 3-7 kb were excised and eluted from the agarose gel slices using Prep-a-Gene reagents (BioRad, Hercules,
• the eluted DNA fragments were ligated with EcoRI-cleaved and dephosphorylated ⁇ ZipLox vector arms (Life Technologies, Gaithersburg, MD), and the ligation mixtures were packaged using commercial packaging extracts (Stratagene, La Jolla, CA).
• the packaged DNA libraries were plated and amplified on Escherichia coli Y1090ZL cells and stored at 4°C using standard methods (Davis, R.W., Botstein, D., and Roth, J.R.,
• Example 7 Isolation, nucleotide sequencing and characterization of a genomic DNA segment encoding Fusarium venenatum glucoamylase
• plaques from the Fusarium venenatum genomic DNA library were screened by hybridization (Davis et al, 1980, supra) with a radiolabeled probe fragment comprising the cloned cDNA insert from pFA0401 (Example 5) using high stringency conditions (i.e., hybridization at 45°C in 50%) formamide, 5 x SSPE, 0.3%> SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA; filters washed once in 0.2 X SSPE with 0.1% SDS at 45°C followed by two washes in 0.2 X SSPE with no SDS at the same temperature).
• high stringency conditions i.e., hybridization at 45°C in 50%
• Plaques which gave hybridization signals were purified twice on E. coli Y1090ZL cells, and the individual clones were subsequently excised from the ⁇ ZipLox vector as pZLl -derivatives (D'Alessio et al, 1992, Focus® 14: 76).
• DNA sequence analysis revealed that one of these clones, containing a plasmid designated pFAMG, comprised the entire glucoamylase coding region as well as 3.9 kb of 5'-flanking and 0.3 kb of 3'-flanking DNA encompassing the promoter and terminator regions, respectively (see Figure 1).
• DNA sequencing of the cloned insert in pFAMG was done with an Applied
• genomic sequence data By comparing the genomic sequence data to the contig of glucoamylase cDNA sequences, it was determined that the genomic DNA segment encoding Fusarium venenatum glucoamylase contained an open reading frame of 1743 bp interrupted by an intron of 51 bp and encoding a polypeptide of 581 amino acids.
• the nucleotide sequence The nucleotide sequence
• Example 8 Amino terminal sequence analysis of Fusarium venenatum glucoamylase
• the PVDF membrane was stained with 0.1 % Commassie Blue R-250 in 40% methanol/1% acetic acid for 20 seconds and destained in 50% ethanol to observe the protein bands.
• the major polypeptide species migrating at approximately 65 kDa was excised and subjected to N-terminal sequencing on an Applied Biosystems 476A Protein Sequencer (Perkin Elmer/Applied Biosystems Division, Foster City, CA) with on-line HPLC and liquid phase trifluoroacetic acid (TFA) delivery. Sequencing reagents were from Perkin Elmer/Applied Biosystems Division. (Foster City, CA).
• Detection of phenylthiohydantoin-amino acids was accomplished by on-line HPLC using Buffer A containing 3.5% tetrahydrofuran in water with 18 ml of the Premix concentrate (Perkin Elmer/ Applied Biosystems Division, Foster City, CA) containing acetic acid, sodium acetate, and sodium hexanesulfonate and Buffer B containing Acetonitrile. Data was collected and analyzed on a Macintosh Ilsi using Applied Biosystems 610 Data Analysis
• Example 9 Identification of abundant cDNA clones encoding an putative vacuolar associated protein and an unknown secreted gene product Two additional cDNA species were selected based on their relative abundance among the clones present in Libraries A and B (Example 2).
• Clone FA0035 containing pFA0035, encoded a primary translation product of 200 amino acids with a possible signal peptide of 18 amino acids that was detected using the Signal-P computer program (Nielsen, et al, 1997, supra). Clone FA0035 was represented by approximately 1.9% of the cDNA clones in Library A and 0.8% of Library B. Comparison of the deduced amino acid sequence of clone FA0035 to the sequences in the NRDB as described in Example 4 revealed no significant homology to any known protein sequences, and it is therefore categorized as an unknown open reading frame (ORF). The name "Daria" was arbitrarily assigned to this clone.
• Clone FA0759 containing plasmid pFA0759, encoded a polypeptide of 187 amino acids with 72% amino acid sequence identity to a putative subunit of the vacuolar associated protein from Neurospora crassa (Trembl accession number P87252). This gene product did not appear to be a secreted protein as no signal peptide was detected using Signal-P software. Clone FA0759 was represented by approximately 2.0% of the cDNA clones in Library A and 1.5% of Library B.
• Example 10 Cloning and nucleotide sequence analysis of genomic DNA segments encoding unknown genes "Daria" and the putative vacuolar associated protein
• Fusarium venenatum genomic DNA segments encoding the unknown secreted protein "Daria" and the putative vacuolar associated protein were isolated by screening the ⁇ ZipLox library described in Examples 6 and 7. Radiolabeled cDNA inserts from plasmids pFA0035 and pFA0759 were used as probes to screen the library. Plaques which hybridized strongly to either probe using the same conditions in Example 7 were purified twice on E. coli Y1090ZL cells, and the individual clones were subsequently excised from the ⁇ ZipLox vector as pZLl -derivatives (D'Alessio et al, 1992, supra).
• DNA sequence analysis revealed that one of these clones, containing a plasmid designated as pECO3, comprised the entire "Daria" coding region as well as 0.9 kb of 5'-flanking and 0.9 kb of 3'-flanking DNA encompassing the promoter and terminator regions, respectively.
• the nucleotide sequence (SEQ ID NO. 2) and deduced amino acid sequence (SEQ ID NO. 5) are shown in Figure 2.
• the coding region was punctuated by a single 55 bp intron.
• a genomic DNA clone was isolated through screening of the ⁇ ZipLox library with a radiolabeled probe derived from the cDNA insert of pFA0579.
• the genomic clone designated as pQUINN, encoded the entire vacuolar associated protein subunit plus 3.0 kb of 5'-flanking and 0.3 kb of 3'-flanking DNA comprising the promoter and terminator regions, respectively.
• the nucleotide sequence SEQ ID NO. 3
• deduced amino acid sequence SEQ ID NO. 6
• Nucleotide sequence comparisons between the genomic and cDNA clones revealed the presence of a 594 bp intron in the 5 '-untranslated region of the putative vacuolar associated protein subunit.
• the coding region contained a 77 bp intron.
• Plasmid pDM181 was constructed using the technique of splice overlap extension to fuse the 1.2 kb Fusarium oxysporum trypsin promoter to the 1.1 kb Fusarium oxysporum trypsin terminator.
• a polylinker containing Swal, Kp ⁇ and Pad restriction sites was inserted between the promoter and terminator as part of the overlapping PCR strategy.
• a Xho site was added and the native EcoRl site was preserved.
• Hindlll and Nsil sites were inco ⁇ orated by the PCR reaction.
• a PCR fragment containing -1208 to -1 of the Fusarium oxysporum trypsin promoter plus a 25 base pair polylinker was generated from plasmid pJRoy20 (Royer et al, 1995, Biotechnology 13: 1479-1483) using the following primers:
• Primer 1 (sense): 5'-GAGCTCGAGGAATTCTTACAAACCTTCAAC-3' (SEQ ID NO. 7)
• the 100 ⁇ l PCR reaction contained 10 ng of pJRoy20, 50 pmol of each primer,
• PCR conditions were 95°C for 3 minutes followed by 25 cycles at 95°C for 30 seconds, 50°C for 1 minute, and 72°C for 1 minute. The final extension cycle was at 72°C for 5 minutes.
• the final 2.3 kb overlapping PCR fragment which contained -1208 to -1 of the Fusarium oxysporum trypsin promoter, the 25 base pair polylinker and 1060 base pairs of the Fusarium oxysporum trypsin terminator was obtained using 0.2 ⁇ l of the first PCR
• the Fusarium venenatum expression vector pSheBl ( Figure 5) was generated by modification of pDM181.
• the modifications included (a) removal of two Ncol sites within the pDM181 sequence, and (b) restoration of the natural translation start of the
• Fusarium oxysporum trypsin promoter (reconstruction of an Ncol site at the ATG start codon).
• Example 13 Construction of pDM194 and pDM218
• the 7.8 kb plasmid pDM194 (7.8 kb) used to obtain expression of Thermomyces lanuginosus (formerly designated Humicola lanuginosa) lipase in Fusarium venenatum has the following gene elements:
• E. coli vector pUC19 and a 1.8 kB fragment comprising the Aspergillus nidulans amdS gene promoter (Hynes et al, 1988, Mol. Cell. Biol. 8: 2589-2596), Streptomyces hygroscopicus phosphinothricin acetyltransferase (bar) gene (Thompson et al., 1987, EMBO Journal 6: 2519-2514), and Aspergillus niger AMG terminator.
• the Sw ⁇ llP ⁇ cl lipase fragment was generated by PCR.
• Plasmid pMHan37 which contains the cDNA of the Thermomyces lanuginosus lipase gene (EP 305 216) was used as the template.
• PCR primers 5 and 6 shown below were used to introduce a Swal site at the 5' end and a Pad site at the 3' end of the lipase coding region.
• IX PCR buffer Perkin-Elmer Corp., Branchburg, NJ
• 250 ⁇ M each of dATP, dCTP, dGTP, and dTTP, and 5 units of Taq DNA polymerase Perkin-Elmer Co ⁇ ., Branchburg, NJ
• the PCR conditions used were one cycle at 95°C for five minutes followed by 30 cycles at 95°C for 1 minute, 55°C for one minute, and 72°C for two minutes.
• the 0.9 kb PCR product was subcloned into pCRII of the TA Cloning Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
• Plasmid DNA was isolated using a Qiagen Maxiprep Kit (Qiagen, Santa Clarita, CA), digested with Swal and Pad, and electrophoresed on a 1% agarose gel for one hour at 100 volts.
• the 0.9 kb fragment was excised, purified using a SpinBind Kit (FMC, Rockland, ME), and cloned into pBANe ⁇
• pBANe ⁇ was digested with Swal and Pad and the 0.9 kb lipase fragment was ligated to Swal/ Pad digested pDM181 yielding lipase expression plasmid pDM194
• pDM218 was constructed from pJRoy35 ( Figure 7). Notl and Emel restriction sites were introduced at the 5' end of the Fusarium oxysporum trypsin promoter by PCR.
• a 362 bp amplicon containing the 5' end of the promoter was produced using pDM194 as a template with the following primers: multil : 5'-ATAAGAATGCGGCCGCTAGTTTAAACTTACAAACCTTCAACAGTG-
• the 100 ⁇ l PCR reaction contained 150 ng of 3 kb EcoRI fragment, 50 pmol of each primer, IX Pwo buffer, 200 ⁇ M each of dATP, dCTP, dGTP, and dTTP, and 5 units of Pwo DNA polymerase.
• the PCR conditions used were one cycle at 95°C for 3 minutes, 30 cycles at 95°C for 1 minute, 50°C for 1 minute, and 72°C for 1.5 minutes; followed by a 5 minute extension at 72°C.
• the amplicon was subcloned into vector pCR-Blunt (Invitrogen, Carlsbad, CA). A 0.25 kb NotllBcul fragment was electrophoresed on a 1% agarose gel for one hour at 100 volts, excised, and purified using a QIAquick Gel Extraction Kit (Qiagen, Santa Clarita, CA).
• Hpal, Snd&l, and PpuM sites were introduced at the 3' end of the Fusarium oxysporum trypsin terminator by PCR.
• a 714 bp amplicon containing the 3' end of the trypsin terminator was produced using the 3 kb EcoRI fragment as template with the following primers: multi3: 5'-GTGTGCAGTGACCCAGAAT-3' (S ⁇ Q ID NO.21) multi4: 5'-GATTGGGTCCCTACGTAGTTAACACTATAGGCCATCGTTTAC-3'
• the 100 ⁇ l PCR reaction contained 50 pmol of each primer, 150 ng of the 3 kb
• a 2.3 kb BcullNhel fragment containing the 3' end of the Fusarium oxysporum trypsin promoter, the Humicola lanuginosa lipase gene (Geneseq nucleotide accession number N91076), and the 5' end of the Fusarium oxysporum trypsin terminator was isolated from pDM194.
• the plasmid pMWR60 was derived from the expression vector pEJG25A.
• pEJG25A was constructed by insertion of a phytase coding sequence from Peniophora lycii (WO 98/28408) into pDM181 (Example 11) as follows: First, two synthetic oligonucleotide primers shown below were designed to amplify the Peniophora lyci phytase coding sequence from plasmid pAlphy2 (WO
• Reverse primer 5'-TTAATTAACTATTCCGACGGAACAAAGC-3' (SEQ ID NO.
• Each 100 ⁇ l Pwo polymerase reaction contained 50 pmol of each primer, 1 ng of template DNA, 2 ⁇ l of 10 mM dNTPs, IX Pwo polymerase buffer, and 2.5 units of Pwo polymerase. The reactions were incubated in a Perkin Elmer Model 9600 Thermal
• Cycler programmed as follows: One cycle at 94°C for 2 minutes, 55°C for 30 seconds, and 72°C for 1 minute; 9 cycles at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 1 minute; 15 cycles at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 1 minute, with an extension of 20 seconds per cycle; a final cycle at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 7 minutes; and a soak cycle at 4°C.
• the reaction product was electrophoresed on a 1% agarose gel for one hour at 100 volts. The 1.3 kb band was excised and purified using Qiaex II.
• the purified PCR product was subsequently cloned into the plasmid pCR2.1 (Invitrogen, Carlsbad, CA) and used to transform E. coli TOP 10 cells (Invitrogen, Carlsbad, CA). Plasmid DNA was isolated from several of the transformant colonies and analyzed by DNA sequencing to ensure that no mutations were introduced during the PCR. Subsequently one phytase clone was digested with restriction endonucleases Swal and Pad. The fragments were electrophoresed on a 1% agarose gel for one hour at 100 volts, excised, and purified using Qiaex II. The 1.3 kb phytase gene fragment was ligated with pDM181 (Example
• pEJG25A was mutagenized using the Quick-ChangeTM site-directed mutagenesis kit in combination with the following oligonucleotide primers to obtain the vector pMWR60-Int2: primer A: 5'-CTCTTGGATATCTATCTCTTCACCATGGTTTCTTCGGCATTCGC-3' (SEQ ID NO. 25) primer B:
• the purified fragment was ligated with pSheBl (described in Example 12) which had been previously digested with Notl.
• the resulting intermediate was designated as pMWR60-Int4a.
• pMWR60- Int4a was then cut with Bspl ⁇ Jl ll plus Nbel, and a 5.25 kb segment was purified as above.
• This isolated segment was ligated with pSheBl, which had also been digested with -9-spLUl lI plus Nbel, and purified as above.
• the resulting vector product was named pMWR60 ( Figure 10).
• Example 15 Generation of a lipase reporter gene
• the gene encoding the LIPOLASETM enzyme was obtained from pAFIL (WO 92/05249).
• the Seal site of the ampicillin gene of pAHL was changed to a Mlul site by use of the primer 7258, thus changing the Seal site found in the ampicillin resistance gene and used for cutting to a
• Primer 7258 5'-GAATGACTTGGTTGACGCGTCACCAGTCAC-3' (SEQ ID NO. 27)
• the pAHL vector comprising the LIPOLASETM gene was then used as a template for DNA polymerase with oligos 7258 and 7770, thus changing the Seal site found in the
• Primer 7770 5'-TCTAGCCCAGAATACTGGATCAAATC-3' (SEQ ID NO. 28)
• the desired mutation (e.g., the introduction of a cysteine residue) was introduced into the LIPOLASETM gene by addition of an appropriate oligos comprising the desired mutation.
• Primer 101782 shown below was used to introduce SPPCGRRP (-E).
• Primer 101782 5'-GGACGGCCTTGGCTAGCCCTCCGTGCGGCCGCCGGCCGGTCTCAGGATC
• Primer 9639 shown below was used to introduce E239C.
• Primer 9639 5'-ATATCGTGAAGATATGCGGCATTGATGCCACC-3' (SEQ ID NO.
• Primer 8829 5'-GGCGGCAATAACCGGCCGAACATTCCGGATATCCC-3' (SEQ ID NO: 1
• Example 16 Construction of pRaMB60 Plasmid pRaMB60 was constructed by ligating the 6.5 kb Sful-Nhel vector fragment from pMWR60 with a 0.5 kb Sful-Nhel fragment from pSheBl . All fragments were isolated by agarose gel electrophoresis and purified from gel slices using BioRad Prep-a-Gene reagents (BioRad Laboratories, Hercules, CA).
• Example 17 Construction of plasmid pRaMB62, an expression vector containing the glucoamylase promoter
• a unique BspUJl ll site was generated by site-directed mutagenesis of plasmid pFAMG using the following primers in combination with the Quick-ChangeTM mutagenesis kit : primer 1 : 5'-dCACTGCTATCACCAACATGTTTACTCAAGTCC-3' (SEQ ID NO. 33) primer 2: 5'-dGGACTTGAGTAAACATGTTGGTGATAGCAGTG-3' (SEQ ID NO.
• the lipase reporter gene described in Example 15 was amplified by PCR using plasmid pEVil l63 as template with the following primers which introduced a
• the PCR reaction was composed of 1 ⁇ l of template DNA (10 ng), 1 ⁇ l of forward primer (77 pmol), 1 ⁇ l of reverse primer (81 pmol), 10 ⁇ l of 10X Pwo polymerase buffer, 16 ⁇ l of 1.25 mM dNTP mix, and 1 ⁇ l (2.5 units) of Pwo polymerase.
• the reaction was incubated in a Perkin-Elmer Model 480 thermocycler using the following temperature settings: One cycle at 95°C for 5 minutes; 30 cycles at 95°C for 1 minute. 47°C for 1 minute, and 68°C for 2 minutes; and a 4°C soak cycle.
• the amplified product was then digested with BspHl plus E ⁇ cl, purified by agarose gel electrophoresis (Example 16), and used in a three-part ligation with the 5.8 kb Pmel-Ncol fragment of pRaMB60 and the 2.1 kb Stz.I--5-.pLUl II fragment from pMWR62-intl .
• the resulting vector, designated pRaMB62 ( Figure 11) contained the lipase reporter gene under the transcriptional control of the Fusarium venenatum glucoamylase promoter.
• Example 18 Construction of plasmid pRaMB64, an expression vector containing the "Daria” promoter
• the promoter region from pECO3 (Example 10), termed the "Daria” promoter, was amplified using PCR in combination with the following primer pair that were designed to introduce Sw ⁇ l and Bspl ⁇ Jl ll sites at the 5' and 3' ends of the promoter segment, respectively: primer 1 : 5'-GCATTTAAATTACTACTGTGATGTG-3 * (SEQ ID NO. 37) primer 2: 5'-GATTGATGTGAAACACATGTTGATG-3' (SEQ ID NO. 38)
• the PCR reaction was composed of 1 ⁇ l of pECO3 (10 ng), 1 ⁇ l of forward primer (50 pmol), 1 ⁇ l of reverse primer (50 pmol), 10 ⁇ l of 10X Pwo polymerase buffer, 16 ⁇ l of 1.25 mM dNTP mix, and 1 ⁇ l (2.5 units) of Pwo polymerase.
• the reaction was incubated in a Perkin-Elmer Model 480 thermocycler using the following temperature settings: One cycle at 95°C for 5 minutes; 30 cycles at 95°C for 1 minute, 47°C for 1 minute, and 68°C for 2 minutes; and a 4°C soak cycle.
• the 0.9 kb PCR product was electrophoresed on a 1% agarose gel for one hour at 100 volts, excised, and purified using Qiaex II.
• the amplified DNA segment was subcloned into pCR2.1 (Invitrogen, Carlsbad, CA) and analyzed by restriction enzyme cleavage with Sw ⁇ l, .RspLUl lI, EcoRI, and Xhol to verify that it was correct.
• the plasmid generated in this manner termed p ⁇ CO4, was digested with Swal plus Bspl ⁇ Jl ll, and the 0.9 kb promoter fragment was purified by gel electrophoresis as described in Example 16.
• the purified fragment was mixed in a three-part ligation with the 5.8 kb Pmel-Ncol fragment of pRaMB60 and the 0.9 kb BspUl-Pacl segment encoding the lipase reporter gene.
• Example 19 Construction of plasmid pRaMB66, an expression vector containing the promoter derived from the vacuolar associated protein gene
• the promoter segment from pQUINN (Example 10), encoding a putative vacuolar associated protein, was amplified by PCR with the following primers which introduced a Sm ⁇ l site and an Ncol site at the 5'and 3' ends of the promoter, respectively: primer 1 : 5'-dCGACCCGGGAATTAGAGAGGTTAGG-3' (SEQ ID NO. 39) primer 2: 5'-dCGTATAACCCATGGTGGACTTGTCGGAC-3' (SEQ ID NO. 40)
• the PCR reaction was composed of 1 ⁇ l of pQUINN (10 ng), 1 ⁇ l of forward primer (50 pmol), 1 ⁇ l of reverse primer (50 pmol), 10 ⁇ l of 10X Pwo polymerase buffer, 16 ⁇ l of 1.25 mM dNTP mix, and 1 ⁇ l (2.5 units) of Pw polymerase.
• the reaction was incubated in a Perkin-Elmer Model 480 thermocycler using the following temperature settings: One cycle at 95°C for 5 minutes; 30 cycles at 95°C for 1 minute, 47°C for 1 minute, and 68°C for 2 minutes; and a 4°C soak cycle.
• the 3.1 kb amplified DNA fragment was electrophoresed on a 1%> agarose gel for one hour at 100 volts, excised, and purified using Qiaex II.
• the 3.1 kb amplified DNA fragment was subcloned into pCR-Script (Stratagene, La Jolla, CA) to generate the intermediate plasmid pQUINN-promoterA.
• Two restriction fragments were isolated from this plasmid; a 2.4 kb Smal-Ndel fragment, and a 0.7 kb Ndel-Ncol fragment (together these segments span the entire promoter region).
• the isolated fragments were combined in a four-part ligation with the 8 kb Pmel-Ncol fragment of pRaMB60 and the 0.9 kb BspHl-Pad segment encoding the lipase reporter gene described in the previous examples.
• the product of this ligation, pRaMB66 ( Figure 13), contained the lipase reporter gene under the transcriptional control of the putative vacuolar associated protein gene promoter.
• Example 20 Expression of the lipase reporter gene in Fusarium venenatum under control of the AMG, "Daria" and vacuolar associated protein promoters
• Spores of Fusarium venenatum CC1-3 were generated by inoculating a flask containing 500 ml of RA sporulation medium with 10 plugs from a IX Vogels medium plate (2.5% Noble agar) supplemented with 2.5% glucose and 2.5 mM sodium nitrate and incubating at 28°C, 150 ⁇ m for 2 to 3 days. Spores were harvested through Miracloth (Calbiochem, San Diego, CA) and centrifuged 20 minutes at 7000 ⁇ m in a
• Sorvall RC-5B centrifuge E. I. DuPont De Nemours and Co., Wilmington, DE.
• Pelleted spores were washed twice with sterile distilled water, resuspended in a small volume of water, and then counted using a hemocytometer.
• Protoplasts were prepared by inoculating 100 ml of YEPG medium with 4 X 10 7 spores of Fusarium venenatum CC1-3 and incubating for 16 hours at 24°C and 150 ⁇ m.
• the culture was centrifuged for 7 minutes at 3500 ⁇ m in a Sorvall RT 6000D (E. I.
• the pellet was resuspended, washed twice with STC, and centrifuged at 2000 ⁇ m for 10 minutes to pellet the protoplasts.
• Protoplasts were counted with a hemocytometer and resuspended in an 8:2:0.1 solution of STC:SPTC:DMSO to a final concentration of 1.25 x 10 7 protoplasts/ml.
• the protoplasts were stored at -80°C, after controlled-rate freezing in a Nalgene Cryo 1°C Freezing Container (VWR Scientific, Inc., San Francisco, CA).
• Frozen protoplasts of Fusarium venenatum CC1-3 were thawed on ice.
• Five to ten ⁇ g of pRaMB62, pRaMB64, and pRaMB66 (described in Examples 17-19) and 5 ⁇ l of heparin (5 mg per ml of STC) were added to separate 50 ml sterile polypropylene tubes.
• One hundred ⁇ l of protoplasts was added to each tube, mixed gently, and incubated on ice for 30 minutes.
• One ml of SPTC was added and incubated 20 minutes at room temperature.
• BASTATM is phosphinothricin.
• BASTATM was obtained from AgrEvo (Hoechst Schering, Rodovre, Denmark) and was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1), and once with chloroform:isoamyl alcohol (24: 1) before use.
• the transformants were picked directly from the selection plates (COVE underlay with COVE-BASTATM overlay) into 125 ml shake flasks containing 25 ml of M400Da medium supplemented with 1 mM CaCl 2 and 100 ⁇ g/ml ampicillin (to prevent bacterial contamination) and incubated at 28°C, 200 ⁇ m on a platform shaker for 7 days.
• the untransformed recipient strain was also included as a negative control.
• Flasks were sampled at 7 days. Cells were removed by centrifugation, and 10 ⁇ l of each supernatant sample was heated to 95 °C for 5 minutes with an equal volume of Tris-glycine sample buffer (Novex Experimental Technology, San Diego, CA). The denatured supernatant proteins were separated on a 10-20% Tris-glycine gradient gel (Novex Experimental Technology, San Diego, CA) and stained with Coomassie blue. SDS-PAGE analysis showed that the lipase-producing transformants secrete a prominent polypeptide with an apparent molecular weight of approximately 43 kDa.
• Example 21 Comparative expression of the Humicola lanuginosa lipase reporter gene under control of Fusarium venenatum amyloglucosidase and Fusarium oxysporum trypsin promoters Fusarium venenatum CC1-3 transformants containing either pRamB64 (Example
• Figure 14 shows comparative expression of the Humicola lanuginosa lipase reporter gene in Fusarium venenatum under control of either the Fusarium venenatum amyloglucosidase (pAMG) promoter or Fusarium oxysporum trypsin (pSP387) promoter.
• the results demonstrated higher levels of lipase activity with the Fusarium venenatum amyloglucosidase promoter than the Fusarium oxysporum trypsin promoter whether urea or ammonium phosphate was used as the source of nitrogen in the feed.
• higher levels of lipase activity were observed using ammonium phosphate in the feed
• E. coli DH10B (pQUINN) NRRL B-30076 October 27, 1998
• E. coli TOP 10 (pFB0346) NRRL B-30073 October 27, 1998
• the strains have been deposited under conditions that assure that access to the culture swill 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 deposits represent substantially pure cultures of the deposited strains. The deposits are available as required by foreign patent laws in countries wherein counte ⁇ arts of the subject application, or its progeny are filed.

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• 2000
  • 2000-03-22 AU AU40257/00A patent/AU4025700A/en not_active Abandoned
  • 2000-03-22 DK DK10180302.1T patent/DK2278016T3/da active
  • 2000-03-22 JP JP2000606759A patent/JP4620253B2/ja not_active Expired - Fee Related
  • 2000-03-22 CN CN 200410031492 patent/CN1526820B/zh not_active Expired - Fee Related
  • 2000-03-22 CN CNB2004100314931A patent/CN100532561C/zh not_active Expired - Fee Related
  • 2000-03-22 CN CNB008053324A patent/CN100482801C/zh not_active Expired - Fee Related
  • 2000-03-22 EP EP00919596A patent/EP1194572A2/en not_active Withdrawn
  • 2000-03-22 CN CNB2004100314912A patent/CN100510096C/zh not_active Expired - Fee Related
  • 2000-03-22 CN CNA2006100999266A patent/CN1940067A/zh active Pending
  • 2000-03-22 WO PCT/US2000/007815 patent/WO2000056900A2/en active Application Filing
  • 2000-03-22 EP EP10180302A patent/EP2278016B1/en not_active Expired - Lifetime
• 2010
  • 2010-05-12 JP JP2010109995A patent/JP5264825B2/ja not_active Expired - Fee Related
• 2013
  • 2013-02-06 JP JP2013021124A patent/JP2013121360A/ja active Pending
  • 2013-02-06 JP JP2013021169A patent/JP2013121361A/ja not_active Ceased

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