CN1268746C - Methods for producing polypeptides in fungal cells - Google Patents

Abstract

The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a mutant of a parent fungal cell under conditions conducive for the production of the polypeptide, wherein the mutant cell comprises a first nucleic acid sequence comprising a modification of one or more genes of a pacC pH signal transduction pathway or homologues thereof, and a second nucleic acid sequence encoding the polypeptide; and (b) isolating the polypeptide from the cultivation medium.

Description

Be used for producing the method for polypeptide the fungal cell

Background of invention

Invention field

The present invention relates to be used for producing the method for polypeptide at the cell of proteolytic enzyme defective.

Description of related art

Many microorganisms can be in wide pH scope growth.This ability cell internal procedure that needs protection is avoided effective pH homeostatic mechanism of extreme pH influence and is guaranteed to be positioned at the method that activity outside the pH stable state boundary only betides suitable environment pH.

The pacC genes encoding is called the protein of PacC, and PacC is that a kind of sequence-specific DNA is conjugated protein, activates preferentially the expression of gene at alkaline pH synthetic product, and suppresses to be suitable for gene product synthetic under acid growth conditions.People such as Tilburn (1995, Europe molecular biology magazine (EMBO Journal) 14:779-790) proposes, when having the signal that is mediated by the palA that replys alkaline environment pH, B, C, F, H and I gene product, the PacC in the Aspergillus nidulans (Aspergillus nidulans) activates transcribing of alkaline expressing gene.Known palA, B, C, F, H and I gene belong to PacC pH signal transduction pathway.Thus, when being in alkaline environment pH, alkaline expression of gene raises, and acid expression of gene reduces.Under acidic conditions, PacC is in the NOT-function form and its level reduces, and causes the derepression of sour expressing gene product and alkaline expressing gene (comprising pacC self) activated to lack.

Some sudden changes of pacC gene or Pac pH signal transduction pathway gene can be imitated growth result (people such as Caddick, 1986, molecule General Genetics (the Molecular General Genetics) 203:346-353 when being in outside the actual environment pH; People such as Shah, 1991, FEMS microbiology communication (FEMS Microbiological Letters) 77:209-212; People such as Espeso, 1993, European molecular biology magazine (EMBO Journal) 12:3947-3956; People such as Arst, 1994, molecule General Genetics (Molecular GeneralGenetics) 245:87-790).6 kinds of gene palA, B, C, F, H and I some sudden change imitations in any are in the growth result of acid pH, and cause that for example acid phosphatase (ester) enzyme level raises and the reduction of alkaline phosphatase (ester) enzyme level.On the contrary, some the sudden change imitations in the pacC gene are in the growth result of alkaline pH, and cause for example alkaline phosphatase (ester) enzyme level rising and acid phosphatase (ester) enzyme level to reduce.These pacC transgenations of imitation alkaline condition growth have been removed and have been regulated its active C end fragment.After having removed acid C end structure territory, PacC is activated, can activate grow required expression of gene and suppress the acidic conditions required expression of gene of growing of alkaline condition.The needs to signal transduction pathway have been avoided in these sudden changes of pacC gene, cause the formation of alkaline expressing gene and the super inhibition of acid expressing gene.

By Aspergillus nidulans (people such as Tilburn, 1995, see above), aspergillus niger (Aspergillus niger) (people such as Maccabe, 1996, molecule General Genetics (MolecularGeneral Genetics) 250:367-374), Aspergillus parasiticus (Aspergillus parasiticus) (Pinero and Keller, 1997, plant pathology (Phytopathology) 87:S78), with produce malicious mould (Penicillium chrysogenum) (people such as Suarez, 1996, molecular microbiology (Molecular Microbiology) 20:529-540) cloned the pacC gene.

Gene in the PacC pH signal transduction pathway is disclosed, comprise Aspergillus nidulans palA (people such as Negrete-Urtasun, 1997, bacteriology magazine (Journal of Bacteriology) 179:1832-1835), Aspergillus nidulans palB gene (people such as Denison, 1995, journal of biological chemistry (Journal of Biological Chemistry) 270:28519-28522); Aspergillus nidulans palF gene (people such as Maccheroni, 1997, gene (Gene) 194:163-167; ) and Aspergillus nidulans palI gene (people such as Arst, 1994, see above).

People such as Caddick (1986, molecule General Genetics (Molecular GeneralGenetics) 203:346-352) and people (1994, molecule General Genetics (Molecular General Genetics) 245:787-790) such as Arst PacC pH signal transduction mutant has been described.

Under the situation that does not need to destroy or delete each gene of being responsible for the total protein hydrolytic activity, reduce or eliminate ability (particularly in reconstitution cell) the deleterious proteolytic activity of production of proteins of interest matter, yet with the surrogate that provides this area can not obtain to have a great attraction at present.

An object of the present invention is to provide and be used in fungal cell's production protein improvement method.

Summary of the invention

The present invention relates to be used to produce the method for polypeptide, comprise: the mutant of (a) under the condition that is suitable for polypeptide production, cultivating the parent fungal cell, wherein mutant cell comprises two kinds of nucleotide sequences, first kind of nucleotide sequence comprises at least a gene of pacC pH signal transduction pathway or the modification of its homologue, second kind of nucleic acid sequence encoding polypeptide; (b) by the nutrient solution isolated polypeptide.

The accompanying drawing summary

Fig. 1 has shown the genomic nucleic acid sequence and the deduced amino acid (being respectively SEQ ID NO:1 and 2) of aspergillus oryzae (Aspergillus orzae) palB gene.

Fig. 2 has shown the restriction map of pJaL400.

Fig. 3 has shown the restriction map of pMT1935.

Fig. 4 has shown the restriction map of pJaL394.

Fig. 5 has shown the restriction map of pMT1931.

Fig. 6 has shown the restriction map of pMT1936.

Fig. 7 has shown the portion gene group nucleotide sequence and the deduced amino acid (being respectively SEQ ID NO:3 and 4) of aspergillus oryzae palA gene.

Fig. 8 has shown the restriction map of pBM1a.

Fig. 9 has shown the restriction map of pBM7.

Figure 10 has shown the restriction map of pBM8.

Detailed Description Of The Invention

The present invention relates to the method for the production of polypeptide, comprise: the mutant of (a) under the condition that is suitable for polypeptide production, cultivating the parent fungal cell, wherein the saltant fungal cell is relevant with parental cell by modifying, such as destruction or the deletion of one or more genes or its homologue of pacC pH signal transduction pathway; (b) by mutant cell nutrient solution isolated polypeptide.

Term " pacC pH signal transduction pathway " is defined as the environment pH of sensation microorganism growth environment herein, and, promote PacC to be transformed into the approach of the proteolysis processing of activated transcription factor by one group of intracellular protein that gene palA, palB, palC, palF, palH and palI encode.Activated PacC opens the alkaline condition required expression of gene of growing, and closes the acidic conditions required expression of gene of growing.Be appreciated that method of the present invention comprises the homologue of palA, palB, palC, palF, palH and palI gene.

In the method for the invention, any fungal cell's gene that participates in pacC pH signal transduction pathway be can modify, filamentous fungus or similar homology yeast included, but is not limited to (as Rim9p; People such as Denison, 1998, molecular microbiology (Molecular Microbiology) 30:259-264) palA, palB, palC, palF, palH and/or palI gene.In preferred embodiments, gene is the palA gene.In another preferred embodiment, gene is the palB gene.In a more preferred embodiment, gene is the aspergillus oryzae palA gene with nucleic acid sequence SEQ IDNO:1.In another preferred embodiment, gene is the aspergillus oryzae palB gene with nucleic acid sequence SEQ ID NO:3.

Shown in hereinafter, the modification of one or more genes in the fungal cell pacC pH signal transduction pathway has reduced the amount of the proteolytic activity that is produced by cell.One or more proteolytic enzyme may be responsible for proteolytic activity.The specific protein enzyme gene expression that is caused by PacC under alkaline condition reduces the defective that produces, owing to lack the signal by the gene product group mediation of palA, B, C, F, H and I genes encoding, does not activate transcribing of alkaline expressing gene.

Use method well-known in the art,, can make up pacC pH signal transduction pathway mutant cell by reducing or eliminate the expression of one or more said gene.For example, by changing coding region or its part or the expression coding region required adjusting function essential, can modify or the deactivation gene activity.The example of this regulation and control or control sequence can be promoter sequence or its functional part, promptly is enough to influence the part that nucleotide sequence is expressed.Other control sequence that may modify includes, but is not limited to leader sequence, polyadenylic acid sequence, propeptide sequence, signal peptide sequence, transcription terminator and transcription activator.

The modification of gene or deactivation can followingly be carried out, and parental cell is carried out mutagenesis, and the mutant cell that selection can be grown outside parental cell standard growth pH is measured the proteolytic activity of mutant cell to parental cell subsequently.For example, can pass through to use suitable physics or chemical mutagen, or by the suitable oligonucleotide of use, or, carry out mutagenesis special or at random by dna sequence dna is carried out PCR mutagenesis.In addition, can carry out mutagenesis by the arbitrary combination of using these mutagenic compound.

Be suitable for the physics of the object of the invention or the example of chemical mutagen and comprise ultraviolet ray (UV) irradiation, azanol, N-methyl-N '-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethylmethane sulfonate (EMS), sodium bisulfite, formic acid and nucleotide analog.

When using these reagent, the common following mutagenesis of carrying out, under conditions suitable, when mutagenic compound were selected in existence, incubation was treated the parental cell of mutagenesis, and the mutant cell of the genetic expression reduction of pacC pH signal transduction pathway is showed in screening.

Can realize the modification or the deactivation of one or more genes in the pacC pH signal transduction pathway by transcribing or translate importing in the required controlling element, substitute or remove one or more Nucleotide at gene or its.For example, can insert or remove Nucleotide, thereby the terminator codon of importing, remove initiator codon or change opening code-reading frame.Can by site-directed mutagenesis or PCR mutagenesis, realize these modifications or deactivation according to the currently known methods of this area.Though in theory, can be in vivo (promptly directly on the cell of expressing gene to be finished) modify, be preferably as follows and civilian illustratively modify external.

The example that makes things convenient for method of deactivation pacC pH signal transduction pathway is based on the technology of gene replacement, gene elmination or gene disruption.In the method for gene disruption, external nucleotide sequence corresponding to interested native gene or gene fragment is carried out mutagenesis to produce the defective type nucleotide sequence, transform parental cell to produce defective gene with this sequence then.By homologous recombination, the defective type nucleotide sequence has replaced native gene or gene fragment.May expect that defective gene or gene fragment are also encoded can be used for selecting nucleotide sequence wherein to be modified or the mark of destructive transformant.

Perhaps, can be by the antisense technology of having set up, the nucleotide sequence of the nucleic acid array complementation of use and gene carries out the modification or the deactivation of one or more genes in the pacC pH signal transduction pathway.In particular, can by import with the nucleic acid array complementation of gene, can record at transit cell, and can with the nucleotide sequence of the mRNA hybridization that produces in the cell, reduce or eliminate expression of gene.Under the condition that can make complementary antisense base sequences and mRNA hybridization, the proteinic amount of translation is lowered thus or eliminates.

With nucleic acid array complementation that relates to the gene of pacC pH signal transduction pathway among the fungal cell or homologous nucleotide sequence can be by the microbe-derived acquisition that comprises these genes.The preferred source that has with the palA gene of aspergillus oryzae nucleic acid sequence SEQ ID NO:1 complementation or homologous nucleotide sequence is an Aspergillus nidulans.The preferred source that has with the palB gene of aspergillus oryzae nucleic acid sequence SEQ ID NO:3 complementation or homologous nucleotide sequence is an Aspergillus nidulans.

The preferred filamentous fungus source of other gene of pacC pH signal transduction pathway (may with the nucleic acid array complementation or the homology of selected fungal cell's corresponding gene) includes, but is not limited to the palF gene (people such as Maccheroni from Aspergillus nidulans, 1997, see above) and from the palI gene (people such as Arst of Aspergillus nidulans, 1994, see above).In addition, nucleotide sequence may be natural for the fungal cell.

The preferred yeast source of pacC pH signal transduction pathway dna homolog thing (may with the nucleic acid array complementation or the homology of selected fungal cell's corresponding gene) includes, but is not limited to yeast saccharomyces cerevisiae (Rim9p; People such as Denison, 1998, see above).

In preferred embodiments, the mutant fungal cell also comprises the pacC gene of two or more copies.The pacC gene of two or more copies has prevented the gene of pal mutant phenotype to suppress outward.Gene is outer to be suppressed to be shown as and causes that the active pacC's of PacC composition is truncate.

PacC can be natural or allogenic for the fungal cell.Preferred source from the pacC gene of filamentous fungus includes, but is not limited to Aspergillus nidulans (people such as Tilburn, 1995, see above), aspergillus niger (people such as Maccabe, 1996, see above), Aspergillus parasiticus (Pinero and Keller, 1997, see above) and produce malicious mould (people such as Suarez, 1996, see above).Preferred source from zymic pacC dna homolog thing includes, but is not limited to Yarrowia lipolytica (people such as Lambert, 1997, molecule and cytobiology (Molecularand Cellular Biology) 17:3966-3976) and yeast saccharomyces cerevisiae (Su and Mitchell, 1993, nucleic acids research (Nucleic Acids Research) 21:3789-3797).

In the method for the invention, the modification of PacC pH signal transduction condition causes the output of one or more proteolytic enzyme to reduce or eliminates.

Term " one or more proteolytic enzyme " is defined as one or more exopeptidases and/or endopeptidase herein.The exopeptidase cutting is near the peptide bond of substrate amino or C-terminal, and the endopeptidase cutting is away from the peptide bond (Rao of substrate end, 1998, microbiology and molecular biology are looked back (Microbiology and Molecular Biology Reviews) 62:597-635).Exopeptidase can be aminopeptidase or carboxypeptidase.Exopeptidase or endopeptidase can be serine protease, metalloprotease, aspartate protease or L-Cysteine HCL Anhydrous (people such as Rao, 1998, see above North, 1982, microbiology is looked back (Microbiological Reviews) 46:308-340; Otto and Schirmeister, 1997, chemical review (Chemical Reviews) 97:133-171).In preferred embodiments, one or more proteolytic enzyme are serine proteases.In another preferred embodiment, one or more proteolytic enzyme are metalloproteases.In another preferred embodiment, one or more proteolytic enzyme are aspartate proteases.In another preferred embodiment, one or more proteolytic enzyme are L-Cysteine HCL Anhydrouss.In another preferred embodiment, one or more proteolytic enzyme are aminopeptidases.In another preferred embodiment, one or more proteolytic enzyme are carboxypeptidases.In another preferred embodiment, one or more proteolytic enzyme are serine protease, metalloprotease, aspartate protease, L-Cysteine HCL Anhydrous, aminopeptidase and/or carboxypeptidase.

Can use any method well-known in the art to measure protease activities.Can also use type well-known in the art, as the special proteinase inhibitor of serine protease, metalloprotease, aspartate protease or L-Cysteine HCL Anhydrous to be identified proteolytic activity.Consult for example North, 1982, see above; Otto and Schirmeister, 1997, see above; With people such as Rao, 1998, see above.

In the method for the invention, when cultivating under the same conditions, compare with corresponding parent fungal cell, one or more proteolytic enzyme that preferred mutant fungal cell produces less about at least 25%, more preferably about at least 50%, even more preferably about at least 75%, most preferably about at least 95%.In the most preferred embodiment, when cultivating under the same conditions, compare with corresponding parent fungal cell, mutant fungal cell does not produce detectable proteolytic enzyme.Under the condition that is suitable for the production polypeptide of interest, perhaps be suitable for producing under the condition of one or more proteolytic enzyme, can aspect one or more proteinic productions, compare parent and mutant cell.

In the method for the invention, polypeptide can be to be natural or allogenic any polypeptide for interested mutant fungal cell.

Term " polypeptide " is not the coded product that refers to length-specific herein, therefore comprises peptide, oligopeptides and protein.Term " heterologous polypeptide " thus be defined as herein for the fungal cell be not natural polypeptide, modified and changed native sequences natural polypeptides or since to the fungal cell carried out recombinant DNA technology operation its be expressed in the natural polypeptides that changes quantitatively taken place.For example, can the recombinant production natural polypeptides, as under the control that places different promoters by gene with coded polypeptide to strengthen polypeptide expression, by using signal sequence promoting the interested natural outer extracellular that is discharged to, and increase the copy number of coding by the gene of the normal polypeptide that produces of cell.Mutant fungal cell can comprise the nucleic acid encoding sequence of one or more copies.

The preferred hormone of polypeptide or its variant, enzyme, acceptor or its part, antibody or its part or reporter gene.In a more preferred embodiment, polypeptide is oxydo-reductase, transferring enzyme, lytic enzyme, lyase, isomerase or ligase enzyme.In addition preferred embodiment in, polypeptide is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, at, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, MUTANASE (mutanase), oxydase, pectin decomposing enzyme, peroxidase, Phospholipid hydrolase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, or zytase.

Can be by any protokaryon, eucaryon or other nucleotide sequence of originating and obtaining the interested polypeptide of coding, can in the fungal cell, express.For the purposes of the present invention, term " by ... obtain " as herein when specifying the source to use, refer to that polypeptide is produced by this source or the cell that inserted from the gene in this source.

Be used to separate or the technology of the nucleotide sequence of clones coding polypeptide of interest is known in this area, and comprise by the separation method of genomic dna, by preparation method or its combination of cDNA.For example pass through to use well-known polymerase chain reaction (PCR), can be by this genomic dna cloning nucleotide sequence.Consult people such as Innis, 1990, " PCR method and application guide " (PCR:A Guide to Method and Application), AcademicPress, New York.Clone's step may relate to cutting and separate the expectation nucleic acid fragment that comprises the nucleic acid encoding sequence, fragment is inserted carrier molecule, and recombinant vectors is mixed and will duplicate the mutant fungal cell of a plurality of copies or clone's nucleotide sequence.Nucleotide sequence can be genome, cDNA, RNA, semi-synthetic, the synthetic source or its arbitrary combination.

In the method for the invention, polypeptide can also comprise fusion or hybrid polypeptide, and wherein another kind of polypeptide merges at polypeptide or its segmental N end or C end.Produce fusion polypeptide by the nucleotide sequence (or its part) of the peptide species of will encoding and nucleotide sequence (or its part) fusion of the another kind of polypeptide of coding.The technology that is used to produce fusion polypeptide is known in this area, and comprises the encoding sequence that connects each peptide species of coding, make them be in same reading frame, and the expression of fusion polypeptide is subjected to the control of identical promoters and terminator.Hybrid polypeptide can comprise at least two kinds of not combinations of the partial or complete peptide sequence of homopolypeptide, and wherein one or more polypeptide may be allogenic for the mutant fungal cell.

Can operate the separated nucleic acid sequence of coding polypeptide of interest in many ways, so that polypeptide expression to be provided.Expression is understood to include and relates to any step that polypeptide is produced, and includes, but is not limited to transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.It may be that wish or essential before inserting carrier nucleotide sequence being operated, and this depends on expression vector.The technology of utilizing cloning process to be used for the modification of nucleic acids sequence is well known in the art.

" nucleic acid construct thing " is defined as by making up and the strand or the double chain acid molecule of nucleic acid fragment arranged side by side the gene isolation of natural generation or modified comprising in the non-existent mode of occurring in nature herein.When the nucleic acid construct thing comprises encoding sequence of the present invention and expresses all required control sequences, term " nucleic acid construct thing " and term " expression cassette " synonym.Term " encoding sequence " is defined as the nucleotide sequence of the aminoacid sequence of direct its protein of indication herein.The border of genome encoding sequence is determined by ATG initiator codon (eukaryote) that just in time is positioned at mRNA opening code-reading frame 5 ' terminal upstream and the Transcription Termination subsequence that just in time is positioned at mRNA opening code-reading frame 3 ' terminal downstream usually.Encoding sequence can include, but is not limited to DNA, cDNA and recombinant nucleic acid sequence.

Term " control sequence " is defined as herein and comprises the essential or favourable all the components to the expression of polypeptide of interest.Each control sequence can be natural or external for the nucleic acid encoding sequence.These control sequences include, but is not limited to leader sequence, polyadenylic acid sequence, propeptide sequence, promotor, signal peptide sequence and transcription terminator.Bottom line, control sequence comprise promotor and transcribe and the translation termination signal.Help to provide control sequence with joint in order to import with control sequence and the special restriction site that nucleic acid encoding sequence encoding district is connected.Term " can be operatively connected " and be defined as herein wherein that control sequence is correctly placed the position with respect to the dna sequence encoding sequence to make control sequence instruct a kind of like this conformation of expression of polypeptides.

Control sequence can be appropriate promoter sequence, i.e. the nucleotide sequence of being discerned by the fungal cell who is used for the express nucleic acid sequence.Promoter sequence comprises the transcriptional control sequence that mediates expression of polypeptides.Promotor can be any nucleotide sequence that shows transcriptional activity in selected fungal cell, the promotor that comprises sudden change, brachymemma and heterozygosis, and can be that the gene of polypeptide in the outer or born of the same parents of homologous or allogenic born of the same parents obtains for the fungal cell by coding.

Being used for instructing the example of the suitable promotor that the nucleic acid construct thing transcribes at filamentous fungal cells is TAKA amylase by the coding aspergillus oryzae, the aspartate protease of Rhizomucor miehei (Rhizomnucor miehei), the neutral α-Dian Fenmei of aspergillus niger, α-Dian Fenmei is stablized in the acid of aspergillus niger, the glucoamylase (glaA) of aspergillus niger or Aspergillus awamori (Aspergillus awamori), the lipase of Rhizomucor miehei, the Sumizyme MP of aspergillus oryzae, the triosephosphate isomerase of aspergillus oryzae, the acetamidase of Aspergillus nidulans, the promotor that the trypsin-like proteolytic enzyme (WO 96/00787) of point sickle spore (Fusarium oxysporum) obtains, and NA2-tpi promotor (heterozygote of the neutral alpha-amylase gene of aspergillus niger and the phosphotriose isomerase gene promotor of aspergillus oryzae), and sudden change, brachymemma, promotor with heterozygosis.

In yeast host, useful promotor is obtained by Hydratase, phosphoenolpyruvate (ENO-1) gene of yeast saccharomyces cerevisiae, galactokinase (GAL1) gene of yeast saccharomyces cerevisiae, alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) gene of yeast saccharomyces cerevisiae and the 3-phoshoglyceric acid kinase gene of yeast saccharomyces cerevisiae.People such as Romanos (1992, yeast (Yeast) 8:423-488) have described other the useful promotor that is used for yeast host cell.

Control sequence can also be suitable Transcription Termination subsequence, promptly by the sequence of fungal cell's identification to stop transcribing.3 ' end of terminator sequence and nucleic acid encoding sequence can be operatively connected.Any terminator of performance function may be used to the present invention in selected fungal cell.

The preferred terminator that is used for filamentous fungal cells is obtained by the gene of the trypsin-like proteolytic enzyme of the alpha-glycosidase of the o-amino benzoyl acid synthase of the glucoamylase of the TAKA amylase of coding aspergillus oryzae, aspergillus niger, Aspergillus nidulans, aspergillus niger and sharp sickle spore.

The preferred terminator that is used for yeast cell is obtained by the gene of the glyceraldehyde-3-phosphate dehydrogenase of the cytochrome C (CYC1) of the Hydratase, phosphoenolpyruvate of coding yeast saccharomyces cerevisiae, yeast saccharomyces cerevisiae or yeast saccharomyces cerevisiae.People such as Romanos (1992, see above) have described other the useful terminator that is used for yeast cell.

Control sequence can also be suitable leader sequence, i.e. the important mRNA non-translational region of translation that carries out for the fungal cell.5 ' end of leader sequence and nucleic acid encoding sequence can be operatively connected.Any leader sequence of performance function may be used to the present invention in selected fungal cell.

The preferred leader sequence that is used for filamentous fungal cells is obtained by the gene of the triosephosphate isomerase of the TAKA amylase of coding aspergillus oryzae and Aspergillus nidulans.

The suitable leader sequence that is used for yeast cell is obtained by the gene of the alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) of the α-factor of the glycerol 3-phosphate acid kinase of the Hydratase, phosphoenolpyruvate (ENO-1) of coding yeast saccharomyces cerevisiae, yeast saccharomyces cerevisiae, yeast saccharomyces cerevisiae and yeast saccharomyces cerevisiae.

Control sequence can also be the polyadenylic acid sequence, and promptly 3 ' end with nucleotide sequence can be operatively connected, be discerned as the sequence of adding the signal of polyadenylic acid residue to the mRNA that transcribes by the fungal cell when transcribing.Any polyadenylic acid sequence of performance function may be used to the present invention in selected fungal cell.

The preferred polyadenylic acid sequence that is used for filamentous fungal cells is obtained by the gene of the alpha-glycosidase of the trypsin-like proteolytic enzyme of the TAKA amylase of coding aspergillus oryzae, the glucoamylase of aspergillus niger, the o-amino benzoyl acid synthase of Aspergillus nidulans, sharp sickle spore and aspergillus niger.

Guo and Sherman (1995, molecular cytobiology (Molecular CellularBiology) 15:5983-5990) have described the useful polyadenylic acid sequence that is used for yeast cell.

Control sequence can also be that coding is connected with the N-terminal of polypeptide and guides encoded polypeptides to enter the signal peptide coding region of the aminoacid sequence of emiocytosis approach.5 ' end of the encoding sequence of nucleotide sequence may inherent be included in the translation reading frame the natural signal peptide coding region that is connected in coding region with the coding secrete polypeptide.Perhaps, may to comprise for encoding sequence be the signal peptide coding region of external source to 5 ' of encoding sequence end.When encoding sequence is natural when not conforming to signal peptide coding region, may need the external source signal peptide coding region.Perhaps, in order to strengthen the secretion of polypeptide, can only replace the natural signals peptide-coding region with the external source signal peptide coding region.But any signal peptide coding region that the guiding polypeptide expressed enters selected fungal cell's Secretory Pathway may be used to the present invention.

The useful signal peptide-coding region that is used for filamentous fungal cells is the signal peptide coding region by the gene acquisition of the lipase of the cellulase of the aspartate protease of the glucoamylase of the neutral starch enzyme of the TAKA amylase of coding aspergillus oryzae, aspergillus niger, aspergillus niger, Rhizomucor miehei, Humicola insolens and Humicolalanuginosa.

The useful signal peptide that is used for yeast cell is obtained by the gene of the saccharase of the α-factor of coding yeast saccharomyces cerevisiae and yeast saccharomyces cerevisiae.People such as Romanos (1992, see above) have described other useful signal peptide coding region.

Control sequence can also be the preceding peptide-coding region that coding is positioned at the aminoterminal aminoacid sequence of polypeptide.The polypeptide that produces is called preferment or preceding polypeptide (perhaps being called proenzyme in some cases).Before polypeptide non-activity normally, and can be transformed into the sophisticated active polypeptide that has by the catalysis or the autocatalysis cutting of preceding polypeptide by propetide.Preceding peptide-coding region can be obtained by the α-factor gene of yeast saccharomyces cerevisiae, the aspartate protease gene and the thermophilic laccase gene (WO 95/33836) of ruining silk mould (Myceliophthora thermophila) of Rhizomucor miehei.

When signal peptide and propetide district all were present in the N-terminal of polypeptide, the propetide district was near the N-terminal of polypeptide, and the signal peptide district is near the N-terminal in propetide district.

May also need to add can be with respect to the regulating and controlling sequence of fungal cell's adjusting and controlling growth expression of polypeptides.The example of regulator control system is to reply the regulator control system that chemistry or physical stimulation (comprise and have regulating compound) cause that genetic expression is opened or closed.Regulator control system in the prokaryotic system comprises lac, tac and trp operon system.In yeast, can use ADH2 system or GAL1 system.In filamentous fungus, the glucoamylase promotor that can use the glucoamylase promotor of TAKA α-Dian Fenmei promotor, aspergillus niger and aspergillus oryzae is as regulating and controlling sequence.Other example of regulating and controlling sequence is the regulating and controlling sequence that can be used in amplification gene.In eukaryotic system, comprise dihydrofolate reductase gene that when having methotrexate, increases and the metallothionein gene that when having heavy metal, increases.In these situations, the nucleic acid encoding sequence will can be operatively connected with regulating and controlling sequence.

In the method for the invention, the nucleic acid construct thing that can use the native gene that is used to change the coding polypeptide of interest to express.This construction can comprise the composition that the change native gene is expressed required minimal number.In one embodiment, this nucleic acid construct thing preferably comprises (a) target sequence, (b) regulating and controlling sequence, (c) exon and (d) donor splicing site.Behind nucleic acid construct thing transfered cell, construction is by the native gene site in the homologous recombination insertion cellular genome.The sequence-directed element of target (a)-(d) is incorporated in the native gene, makes element (b)-(d) and native gene to be operatively connected.In another embodiment, the nucleic acid construct thing comprises (a) target sequence, (b) regulating and controlling sequence, (c) exon, (d) donor splicing site, (e) intron and (f) acceptor splicing site, the wherein integration of the sequence-directed element of target (a)-(f) makes element (b)-(f) and native gene to be operatively connected.But construction can comprise other composition, such as selective marker.

In these two embodiments, the importing of these compositions has produced native gene and has expressed altered new transcription unit.In itself, new transcription unit is by the sequence of target construction importing and the fusion product of native gene.In the altered embodiment of native gene, gene is activated.In this embodiment, use homologous recombination, insertion will cause the regulating and controlling sequence of gene, thereby substitute, destroy or regulating and controlling sequence that deactivation and parental cell native gene normally link to each other with the horizontal expression that is higher than corresponding parental cell.Use method well-known in the art (consulting U.S. Patent number 5,641,670), by comprise the selective marker that can increase in construction, the activated gene can further increase.In altered another embodiment of native gene, genetic expression has reduced.

The target sequence can be positioned at native gene, directly adjacent with gene, be positioned at upstream gene or be positioned at the native gene upstream and certain distance apart.Can use one or more target sequences.For example, cyclic plasmid or dna fragmentation preferably adopt single target sequence, and linear plasmid or dna fragmentation preferably adopt two target sequences.

The regulating and controlling sequence of construction can comprise one or more promotors, enhanser, scaffold attached region or matrix binding site, negative regulatory element, transcribe the combination of binding site or these sequences.

Construction also comprises one or more exons of native gene.Exon is defined as and copies into RNA and be present in dna sequence dna in the ripe mRNA molecule, so the coding region of exon sequence and native gene is in same reading frame.The DNA that comprises one or more amino acid of coding and/or part coded amino acid that exon can be chosen wantonly.Perhaps, exon comprises the DNA corresponding to 5 ' non-translational region.When external source exon or one or more amino acid of exons coding and/or partial amino-acid, the nucleic acid construct thing be designed to transcribe with montage after, reading frame and native gene coding region are in same reading frame, and therefore the correct reading frame by second exon deutero-mRNA part does not change.

The donor splicing site of construction instructs the montage of an exon and another exon.Usually, first exon is positioned at 5 ' of second exon, and is positioned at first exon 3 ' flank and have the donor splicing site identification of overlapping to be positioned at the acceptor splicing site of second exon 5 ' flank.Acceptor splicing site is the same with donor splicing site, is the sequence that instructs an exon and another exon generation montage.Splicing system utilizes acceptor splicing site and donor splicing site combined action to remove intron.

In another aspect of the present invention, mutant fungal cell can also comprise the modification of production, recovery that one or more codings may be harmful in polypeptide of interest and/or proteinic the third nucleotide sequence of using.Modify the expression that reduces or eliminated one or more the third nucleotide sequences, when causing cultivating under the same conditions, the mutant cell that comprises the third modified nucleotide sequence can produce more polypeptide than the mutant cell of the modification that does not contain the third nucleotide sequence.The third nucleotide sequence can encode any protein or enzyme.For example, enzyme can be aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, at, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, MUTANASE, oxydase, pectin decomposing enzyme, peroxidase, Phospholipid hydrolase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases or zytase.

Above-mentioned various nucleic acid and control sequence can be linked together, to produce recombinant expression vector, this carrier can comprise one or more restriction sites easily, makes it possible to insert or replacement nucleic acid encoding sequence in these sites.Sequence that perhaps can be by will comprising this nucleotide sequence or nucleic acid construct thing insert the suitable carrier that is used to express and express the nucleic acid encoding sequence.In the process of construction of expression vector, encoding sequence is placed carrier, make encoding sequence and the suitable control sequence that is used for expression (with possible secretion) to be operatively connected.

Recombinant expression vector can be any carrier (as plasmid or virus) that can carry out the recombinant DNA step easily and can express the nucleic acid encoding sequence.Carrier and the consistency that will import the fungal cell of this carrier are depended in the selection of carrier usually.Carrier can be linearity or closed loop plasmid.Carrier can be the carrier of self-replicating, and promptly as the carrier of the outer entity existence of karyomit(e), it duplicates and does not rely on THE REPLICATION OF CHROMOSOME, as plasmid, extra-chromosomal element, minute chromosome or artificial chromosome.Carrier can comprise any means of guaranteeing self-replacation.Perhaps carrier can be to be incorporated in the genome after importing the fungal cell and carrier that the karyomit(e) of integrating with it duplicates.Carrier system can be single carrier or plasmid or two or more carrier or plasmid (they comprise total DNA that will import in fungal cell's genome jointly) or transposon.

Carrier preferably comprises one or more can select selective marker through transformant easily.Selective marker be its product biocide or virus resistance, heavy metal resistance are provided, make auxotroph become prototroph, or the like gene.The appropriate flags that is used for yeast cell is ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.The selective marker that is used for filamentous fungal cells includes, but is not limited to amdS (acetamidase), argB (ornithine carbamyl transferase), bar (phosphinothricin acetyltransferase), hygB (hygromix phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5 '-phosphate decarboxylase), sC (sulfate adenylyl transferase) and trpC (o-amino benzoyl acid synthase) and equivalent thereof.What be preferred for filamentous fungal cells is the amdS of Aspergillus nidulans or aspergillus oryzae and the bar gene of pyrG gene and suction Streptomycin sulphate (Streptomyces hygroscopicus).

Carrier preferably comprises can make the carrier stable integration maybe can make carrier not rely on genome and the element of self-replicating in cell in cellular genome.

" importing " refers to and will comprise the carrier transfered cell of the nucleotide sequence of the polypeptide of interest of encoding, make carrier obtain to keep as the outer carrier of the karyomit(e) of chromosomal integration vector or self-replacation.It has been generally acknowledged that it is favourable integrating, because nucleotide sequence more likely exists at cell inner stablity like this.Vector integration takes place by homologous recombination, non-homogeneous reorganization or swivel base to the process in the karyomit(e).

Expression vector is incorporated into may relate to the process of forming by with the regeneration of the conversion of the forming of the protoplastis of form known per se, protoplastis and cell walls among the fungal cell.The appropriate steps that is used to integrate the Aspergillus cell is described in people such as EP 238023 and Yelton, and 1984, progress (the Proceedings of National Academy of Sciences USA) 81:1470-1474 of NAS.The appropriate method that is used to transform the fusarium species is described in people such as Malardier, and 1989, gene (Gene) 78:147-156 and WO 96/00787.(J.N.Abelson and M.I.Simon compile can to use Becker and Guarente, yeast genetics and molecular biology guide (Guide to Yeast Genetics and Molecular Biology), Enzymology method (Methods in Enzymology), the 194th volume, the 182-187 page or leaf, Academic Press company, New York; People such as Ito, 1983, bacteriology magazine (Journal ofBacteriology) 153:163; With people such as Hinnen, 1978, progress (Proceedings of the National Academy of Sciences USA) 75:1920 of NAS) the step transformed yeast of describing.

In order to be incorporated in the cellular genome, carrier can rely on the nucleic acid encoding sequence or be used for by homology or non-homogeneous reorganization carrier stable integration any other element to genome.Perhaps carrier can comprise other nucleotide sequence that is used for being incorporated into by the homologous recombination guidance cellular genome.Above-mentioned other nucleotide sequence makes carrier to be incorporated in the genome in the accurate site in karyomit(e).In order to improve the possibility of integrating in accurate site, integrated element should preferably comprise the nucleic acid of enough numbers, such as 100-1,500 base pairs, preferred 400-1,500 base pairs, most preferably 800-1,500 base pairs, these nucleotide sequences and the probability of corresponding target sequence height homology with the enhancing homologous recombination.Integrated element can be with cellular genome in any sequence of target sequence homologous.In addition, integrated element can be the nucleotide sequence of non-coding or coding.On the other hand, carrier can be by non-homogeneous recombination and integration in cellular genome.

For self-replicating, carrier can also comprise make carrier can be in described cell the replication orgin of self-replicating.The example that is used for the replication orgin that uses at yeast cell is the combination of 2 μ replication orgin, ARS1, ARS4, ARS1 and CEN3 and the combination of ARS4 and CEN6.Replication orgin can be to have the replication orgin that to make its function in the fungal cell be the sudden change of responsive to temperature type (to consult Ehrlich, 1978, progress (the Proceedings of the National Academy of Sciences USA) 75:1433 of NAS).

Be appreciated that method of the present invention is not limited to be used to obtain mutant fungal cell's specific order.Can be used for producing any step of process of the cell of polypeptide at structure, will be referred to the modification transfered cell of the gene of pacC pH signal transduction pathway.Preferably, before the gene that imports coded polypeptide, the gene in the pacC pH signal transduction pathway of fungi mutant cell is modified by method of the present invention.

Being used to connect said elements is well-knownly (to consult J.Sambrook with the step that makes up recombinant expression vector for those skilled in the art, E.F.Fritsch, and T.Maniatus, 1989, molecular cloning: laboratory manual (Molecular Cloning, A LaboratoryManual), the 2nd edition, the cold spring port, New York).

In the method for the invention, the fungal cell can be wild-type cell or mutant cell.

" fungi ", as used herein, comprise Ascomycota (Ascomycota), Basidiomycota (Basidiomycota), chytrid door (Chytridiomycota), and Zygomycota (Zygomycota) (is defined in " Ainsworth and BisbyShi fungi dictionary " (Ainsworth and Bisby ' s Dictionary of The Fungi) by people such as Kawksworth, the 8th edition, 1995, CAB International, Universlty Press, Cambridge, Britain), and oomycetes door (Oomycota) (writes down as people such as Hawksworth, 1995, see above the 171st page) and all mitospore fungies (people such as Hawksworth, 1995, see above).

In preferred embodiments, the fungal cell is a yeast cell." yeast " as used herein, comprises ascosporogenous yeast (Endomycetale (Endomycetales)), produces the load yeast and belongs to the yeast (gemma guiding principle (Blastomycete)) of deuteromycetes.Because the zymic classification may change in the future, for the purposes of the present invention, should be as " zymic biology and activity " (Biology and Activities of Yeast) (F.A.Skinner, S.M.Passmore and R.R.Davenport volume, the 9th symposium of SAB (Soc.App.Bacteriol.Symposium Series NO.9), 1980) described definition yeast.

In a more preferred embodiment, yeast cell is the cell that mycocandida (Candida), Hansenula (Hansenula), genus kluyveromyces (Kluyveromyces), Pichia (Pichia), yeast belong (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces) or Yarrowia belong to.

In the most preferred embodiment, yeast cell is saccharomyces carlsbergensis (Saccharomycescarlsbergensis), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces diastaticus (Saccharomyces diastaticus), Douglas yeast (Saccharomycesdouglasii), Crewe not yeast (Saccharomyces kluyveri), promise ground yeast (Saccharomyces norbensis) or ellipsoideus yeast (Saccharomyces oviformis) cell.In another the most preferred embodiment, yeast cell is Kluyveromyces lactis (Kluyveromyces lactis) cell.In another the most preferred embodiment, yeast cell is a Yarrowia lipolytica cell.

In another preferred embodiment, the fungal cell is a filamentous fungal cells." filamentous fungus " comprises all thread forms of the subphylum of Mycophyta (Eumycota) and oomycetes door (Oomycota) (as people such as Hawksworth definition, 1995, see above).The characteristics of filamentous fungus are the mycelia wall be made up of chitin, Mierocrystalline cellulose, dextran, chitosan, mannosans and other complicated polysaccharide usually.Prolongation by mycelia is nourished and grown, and the carbon metabolism is obligate aerobic.On the contrary, the nourishing and growing of yeast (such as yeast saccharomyces cerevisiae) is to be undertaken by sprouting of unicellular thalline, and the carbon metabolism can be fermentable.

In a more preferred embodiment, filamentous fungal cells is the mould genus of top spore (Acremonium), Aspergillus (Aspergillus), aureobasidium genus (Aureobasidium), Cryptococcus (Cryptococcus), Filobasidiella (Filibasidium), fusarium (Fusarium), Gibberella (Gibberella), Humicola (Humicola), Magnaporthe belongs to, Mucor (Mucor), myceliophthora (Myceliophthora), Myrothecium (Myrothecium), Neocallimastix belongs to, the mould genus of arteries and veins spore (Neurospora), paecilomyces (Paecilomyces), Penicillium (Penicillium), Piromyces belongs to, Schizophyllum (Schizophyllum), Talaromyces (Talaromyces), heater capsule Pseudomonas (Thermoascus), Thielavia (Thielavia), Tolypocladium belongs to, or the cell of Trichoderma (Trichoderma).

In the most preferred embodiment, filamentous fungal cells is microorganism Aspergillus aculeatus (Aspergillusaculeatus), Aspergillus awamori, smelly aspergillus (Aspergillus foetidus), aspergillus japonicus (Aspergillus japonicus), Aspergillus nidulans, aspergillus niger or aspergillus oryzae cell.

In another the most preferred embodiment, filamentous fungal cells is bar spore shape sickle spore, Fusarium crookwellense (synonym of Fusarium cerealis), yellow sickle spore, F.graminearum schw, the red sickle spore of standing grain, different spore sickle spore, albizzia sickle spore, sharp sickle spore, racemosus sickle spore, pink sickle spore, Williams Elder Twig sickle spore, colour of skin sickle spore, fusariun solani, side spore sample sickle spore, sulphur look sickle spore, Fusarium torulosum, Fusarium trichothecioides or Fusariumvenenatum cell.

Fusarium venenatum optimum cell is selected Fusarium venenatum A3/5, at first as F.graminearum schw ATCC 20334 preservations, recently by Yoder and Christianson (1998, fungi genetics and biology (Fungal Genetics and Biology) 23:62-80) and people (1998, fungi genetics and biology (Fungal Genetics andBiology) 23:57-67) such as O ' Donnell be re-classified as Fusarium venenatum; And the taxonomy coordinator of Fusariumvenenatum, no matter the present title of these species is how.In another preferred embodiment, Fusarium venenatum cell is the morphology mutant of Fusariumvenenatum A3/5 or Fusarium venenatum ATCC 20334, is disclosed in WO 97/26330.

In another the most preferred embodiment, filamentous fungal cells is the conspicuous Mucor (Mucor miehei) of lice shape red mould (Gibberella pulicaris), Gibberella zeae (Gibberella zeae), Humicolainsolens, Humicols lanuginosa, rice, thermophilicly ruin that silk is mould, the cell of myrothecium roidium (Myrothecium roridin), Neuraspora crassa (Neurosporacrassa) or penicillium purpurogenum (Penicillium purpurogenum).

In another the most preferred embodiment, filamentous fungal cells is the cell of Trichodermaharzianum, healthy and free from worry wood mould (Trichoderma koningii), Trichodermalongibrachiatum, Trichoderma reesei or viride (Trichoderma viride).

In the method for the invention, use methods known in the art, in the substratum that is suitable for the production polypeptide of interest, cultivate the mutant fungal cell.For example, can be by shake-flask culture and a small amount of of in laboratory or industrial fermentation jar, carrying out or bulk fermentation (comprise continuously, in batch, fed-batch or solid state fermentation), in suitable medium and can express and/or the condition of isolated polypeptide under, culturing cell.In the suitable culture medium that comprises carbon source, nitrogenous source and inorganic salt, use step known in the art to cultivate.Suitable medium can be bought by suppliers, perhaps can be according to the moiety preparation of delivering (as the catalogue of U.S. typical case's culture collecting center).Can directly reclaim secreted polypeptides by substratum.

Can use the special means known in the art of polypeptide are detected polypeptide.These detection methods can comprise the use of specific antibody, the formation of enzyme product, the disappearance or the SDS-PAGE of enzyme substrates.For example, the enzyme experiment can be used to measure polypeptide active.The step that is used to measure the enzymic activity of many enzymes is known in this area.

Can separate the polypeptide that produces by the currently known methods of this area.For example, can be by conventional procedures by the nutrient solution isolated polypeptide, include, but is not limited to centrifugal, filtration, extraction, spraying drying, evaporation or precipitation.Can pass through the polypeptide of the multiple known steps purifies and separates of this area then, include, but is not limited to chromatography (as ion-exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis step (as the isoelectrofocusing of preparation property), difference dissolving (as ammonium sulfate precipitation) or extracting and (consult " protein purification " (Protein Purification), J.-C.Janson and Lars Ryden compile, VCH Publishers, New York, 1989).

The present invention is further described by the following example, and these embodiment not should be understood to the restriction of the scope of the invention.

Embodiment

Material

The pharmaceutical chemicals that uses as damping fluid and substrate is the commodity of SILVER REAGENT at least.

Material and solution

The PDA flat board contains 39g/L potato dextrose agar (Difco), and interpolation 10mM uridine is used for pyrG auxotroph.

MY25 substratum (pH6.5) contains 25g maltose, 2.0g MgSO for every liter ₄7H ₂O, 10g KH ₂PO ₄, 2.0g citric acid, 10g yeast extract, 2.0g K ₂SO ₄, 2.0g urea and 0.5ml trace metal solution.MY25 shake-flask culture base was diluted to be used for microtitration culture experiment (MY25/100 or MY25/1000) with glass distilled water in 1: 100 or 1: 1000.With culture in 34 ℃ of cultivations.

2X MY salts solution (pH6.5) contains 4g MgSO for every liter ₄7H ₂O, 4g K ₂SO ₄, 20g KH ₂PO ₄, 4g citric acid, 1ml trace metal solution and 2ml CaCl ₂2H ₂O (100g/L stoste).

Minimum medium transforms dull and stereotyped (pH6.5) every liter and contains 6g NaNO ₃, 0.52g KCl, 1.52g KH ₂PO ₄, 1ml trace metal solution, 1g glucose, 500mg MgSO ₄7H ₂O, 342.3g sucrose and 20g Noble agar.Minimum medium shifts dull and stereotyped (pH6.5) every liter and contains 6g NaNO ₃, 0.52g KCl, 1.52g KH ₂PO ₄, 1ml trace element, 10g glucose, 500mg MgSO ₄7H ₂O and 20g Noble agar.

Trace metal solution (1000X) contains 22g ZnSO for every liter ₄7H ₂O, 11g H ₃BO ₃, 5g MnCl ₂4H ₂O, 5g FeSO ₄7H ₂O, 1.6g CoCl ₂5H ₂O, 1.6g (NH ₄) ₆Mo ₇O ₂₄, and 50g Na ₄EDTA.

COVE contains 343.3g sucrose, 20ml COVE salts solution, 10ml 1M ethanamide, 10ml 3M CsCl and 25g Nobel agar for dull and stereotyped every liter.COVE salts solution (50X) contains 26g KCl, 26g MgSO for every liter ₄7H ₂O, 76g KH ₂PO ₄, and 50ml COVE trace metal solution.COVE trace metal solution contains 0.04g NaB for every liter ₄O ₇10H ₂O, 0.040g CuSO ₄5H ₂O, 0.70g FeSO ₄H ₂O, 0.80g Na ₂MoO ₂2H ₂O and 10g ZnSO ₄

NZY contains 5g NaCl, 2g MgSO for dull and stereotyped every liter ₄7H ₂O, 5g yeast extract, 10g NZ amine and 15g Bacto agar.

The YEG substratum contains 5g yeast extract and 20g dextrose for every liter.

STC contains 1.2M sorbyl alcohol/10mM CaCl ₂/ 10mM Tris (pH7.5).

The SM damping fluid contains 5.8g NaCl, 2g MgSO for every liter ₄7H ₂O, 50ml 1MTris-HCl (pH7.5) and 5ml 2% gelatin.

Embodiment 1: transform aspergillus oryzae HowB430 with pDSY82

As structure aspergillus oryzae HowB430 as described in the WO 98/11203.

The protoplastis for preparing aspergillus oryzae HowB430 according to following scheme.Aspergillus oryzae HowB430 was cultivated 16-18 hour with the 150rpm jolting in 32 ℃ in 100ml substratum (1% yeast extract/2% peptone/1% glucose).By 0.45mm filter filtered and recycled mattress filament remaining about 10ml on filter, with 25m1 1.0-1.2M MgSO ₄/ 10mM sodium phosphate (pH6.5) cleans, as preceding filtration, once more as preceding cleaning until remaining 10ml, be resuspended in the 10ml 5mg/ml NOVOZYM234 that is contained in the 125ml Ehrlenmeyer flask then ^TM(Novo Nordisk A/S,

, Denmark) and (be dissolved in 1.2M MgSO ₄/ 10mM sodium phosphate (pH6.5), 0.45mm filters).With suspension with the gentle jolting of 50rpm in 37 ℃ of incubations 1 hour to produce protoplastis.10ml protoplastis/mycelium prepared product is added in the 30ml Corex centrifuge tube, with 5ml 0.6M sorbyl alcohol/10mM Tris-HCl (pH7.5) capping, and in the rotary head of rotation tubbiness with centrifugal 15 minutes of 3600xg to reclaim protoplastis.Use the pasteur transfer pipet to reclaim protoplastis by buffer interface.Then the STC of protoplastis with 5 times of volumes cleaned, centrifugal, as preceding cleaning and centrifugal.Protoplastis is resuspended in STC to final concentration 2 * 10 ⁷Protoplastis/ml.

Adding 0.1ml concentration in 14ml Falcon polypropylene tube is 2 * 10 ⁷The protoplastis of protoplastis/ml, 5-15 μ l linearizing pDSY82 of 15U BamHI (WO98/11203) and 250 μ l 60%PEG4000/10mM CaCl ₂/ 10mM Tris-HCl (pH7), gentle mixing, and in 37 ℃ of incubations 30 minutes.Top-layer agar (1X COVE salt/1%NZ amine/0.8% sucrose/0.6%Nobel agar) or 3ml STC with the 12ml fusing mixes with suspension, and suspension is poured on the minimum medium flat board.With flat board in 37 ℃ of incubation 3-5 days.

The scope of the transformation efficiency that BamHI REMI pDSY82 transforms is about 80-100 transformant/μ g DNA.Obtain the BamHI REMI library of aspergillus oryzae HowB430, contained about 27,000 DNA label transformant.

The set of aspergillus oryzae HowB430 label sudden change storehouse is called " b ", because pDSY82 digests through BamHI, and has BamHI during subsequent transformation, and the library is merged into the group of about 1000 transformant and is stored in-80 ℃ in 10% glycerine.

Embodiment 2: the lipase expression screening

The sudden change storehouse of HowB430 label described in the embodiment 1 " b " set test lipase is expressed.

For the screening of 96 orifice plates, the diluent that uses equal-volume sterilized water and 2X MY salts solution (pH6.5) to make dilutes 1000 times with the MY25 nutrient solution.For the screening of 24 orifice plates, the diluent that uses equal-volume sterilized water and 2X MY salts solution (pH6.5) to make dilutes 100 times with the MY25 nutrient solution.

Initial 96 orifice plates screening comprises with MY25//1000 dilutes the spore that converges body from difference, makes that 1 spore is on average inoculated in each hole when installing to 50ml nutrient solution branch in the hole.After the inoculation, with 96 orifice plates in 34 ℃ of static cultivations 7 days.The lipase activity of test culture as mentioned below then.Directly in 24 orifice plates of MY25/100 are housed, inoculate interested mutant, and cultivated 7 days in 34 ℃.The lipase activity of test culture as mentioned below then.Then interested mutant is coated on the COVE flat board to produce spore, spreads on the PDA flat board, use 4 single bacterium colonies of every kind of isolate of above-mentioned 24 orifice plate methods test then to produce single bacterium colony.

Face use before with right-oil of mirbane butyric acid substrate stoste (21ml is right-usefulness MC damping fluid (the 4mM CaCl of oil of mirbane butyric acid/mlDMSO) ₂/ 100mM MOPS (pH7.5)) lipase test substrate is prepared in 1:50 dilution thus.With standard lipase (LIPOLASE ^TM, NovoNordisk A/S,

, Denmark) and be mixed with 40LU/ml with the MC damping fluid that contains 0.02% alpha-olefin sulphonate (AOS) stain remover.Reference liquid is stored in 4 ℃ until use.Before facing use standard lipase is diluted with MC damping fluid 1/40.The nutrient solution sample is diluted with the damping fluid that contains the 0.02%AOS stain remover, and 20 μ l branch is filled in the hole of 96 orifice plates, add 200 μ l subsequently through the dilution substrate.Use 2 readings of plate reader record 405nm light absorption value, about 1 minute at interval.With respect to lipase criterion calculation lipase unit/ml (LU/ml).

96 orifice plates screenings and subsequently 24 orifice plate results of screening identified called after DEBY10.3 the DNA tagged mutant to be used for further evaluation, the lipase level comparison that this bacterial strain produces is according to bacterial strain aspergillus oryzae HowB430 height.

Embodiment 3: the bottle that shakes of aspergillus oryzae DEBY10.3 is estimated

Aspergillus oryzae DEBY10.3 described in the embodiment 2 is coated on the spore that is used to shake the bottle evaluation on the COVE flat board with generation.

Shake bottle and estimate following carrying out, inoculate with 300-500ml spore suspension (0.02% tween 80 and from the spore of COVE flat board) and be contained in 125ml and shake 25ml MY25 substratum (pH6.5) in the bottle.To shake bottle in 34 ℃ with 200rpm incubation 3 days.The 2nd day and sampling in the 3rd day, and measure lipase activity as described in example 2 above.

Gained the results are shown in hereinafter table 1, and the lipase output standard of wherein inciting somebody to action aspergillus oryzae HowB430 in contrast turns to 1.0.

The lipase of table 1.DNA tagged mutant is expressed

Bacterial strain is described	Make up	Set	The screening of #96 orifice plate	24 orifice plate results (LU/ml)	Shake a bottle result (LU/ml)
						HowB430 DEBY10.3	HowB 425+pBANe8 pDSY81+BamHI	NA b1	NA 808	1.0 1.7	1.0 2.2

As shown in table 1, the lipase comparison that aspergillus oryzae DEBY10.3 produces when cultivating in shaking bottle is according to much about 2.2 times of bacterial strain aspergillus oryzae HowB430.

Embodiment 4: by aspergillus oryzae DEBY10.3 rescue plasmid DNA and flanking DNA

Separate pDSY82DNA and genomic flanking gene seat by aspergillus oryzae DEBY10.3.

According to the following steps by aspergillus oryzae DEBY10.3 isolation of genomic DNA.With the spore original seed of every kind of mutant inoculation 150ml YEG substratum, and in 37 ℃ with the 250rpm overnight incubation.(Calbiochem, La Jolla is CA) by filtering by every kind of culture results mycelium, and with (TE) rinsing 2 times of 10mM Tris/0.1mM EDTA (pH8) to use Miracloth.Wear into segmentation then with the quick freezing in liquid nitrogen of mycelium prepared product, and with mortar and pestle.Every kind of powdery mycelium prepared product is transferred in the 50ml pipe, and added the 20ml lysis buffer.In every kind of prepared product, add the RNA enzyme to final concentration 20 μ g/ml, and with prepared product in 37 ℃ of incubations 30 minutes.In every kind of prepared product, add Proteinase K then to final concentration 0.1mg/ml, and with prepared product in 50 ℃ of incubations 1 hour.Then with prepared product with 15, centrifugal 20 minutes of 000xg is with the precipitation insoluble substance.With every kind of supernatant liquor be added to the QBT equilibrated Qiagen MAXI post that provides with manufacturers (Qiagen, Chatsworth, CA) on.The QC that provides with 30ml manufacturers cleans pillar then.The QF that provides with 15ml manufacturers is by the pillar eluted dna, the isopropanol precipitating by adding 0.7 times of volume and with 15 then, and 000xg reclaimed in centrifugal 20 minutes.To precipitate at last with 5ml 70% ethanol and clean, dry air, and be dissolved in 200 μ l TE.

Get 2 μ g aspergillus oryzae DEBY10.3 genomic dna prepared products, respectively with BalI, HpaI, NarI, NdeI, SphI and StuI digestion.Restriction enzyme does not cut pDSY82, makes it possible to separate the plasmid and the flanking genomic dna of integration.To in 20 μ l reaction systems, use T through the genomic dna of digestion then ₄Dna ligase connects.

With every kind of DNA prepared product Transformed E .coli HB101 through connecting.Following then screening transformant, extract plasmid DNA by transformant, restrictive diges-tion is inserted fragment to confirm that they are derived from pDSY82, and the use Applied Biosystems 373A of company type automatic sequencer (Applied Biosystems Model 373A Automated DNA Sequencer) (Applied Biosystems company, Foster City, CA), use primer walking technology and dyestuff to stop thing chemical method (people such as Giesecke, 1992, virological method magazine (Journalof Virol.Methods) 38:47-60), use M13 reverse primer (48) and M13 forward primer (20) (New England Biolabs, Beverly, MA), use the primer special, on two chains, check order inserting fragment to pDSY82.

Transformant intestinal bacteria HB101-pDSY109 comprises the locus that is reclaimed by aspergillus oryzae DEBY10.3 SphI.

Embodiment 5: the qualitative evaluation of aspergillus oryzae DEBY10.3 rescue locus pDSY109

Use the APPlied Biosystems 373A of company type automatic sequencer, use primer walking technology and dyestuff to stop the thing chemical method, use M13 reverse primer (48) and M13 forward primer (20), the special primer of sequenced dna is treated in use, on two chains is checked order in 3.4 and 2.2kb zone of aspergillus oryzae DEBY10.3 recovery locus pDSY109 integration incident both sides.Nucleotide sequence explanation integration incident betides in the opening code-reading frame of palB gene.The palB genes encoding relates to the L-Cysteine HCL Anhydrous of pacC pH signal transduction pathway, pacC pH signal transduction pathway delivery context pH signal.

Use scheme described in the embodiment 4, separate the genomic dna of aspergillus oryzae HowB430.Make up aspergillus oryzae HowB430 genomic library by at first partly digesting aspergillus oryzae HowB430 genomic dna with Tsp509I.Use the condition of manufacturer recommendation, use the Tsp509 of 4 units to digest 3.5 μ g aspergillus oryzae HowB430 genomic dnas.React on 65 ℃ and carry out, by sampling in 5 minutes at interval in 0-50 minute.Reaction solution is placed on ice, and stop by adding EDTA to 10mM.Then these digests are contained electrophoresis on 1% sepharose of ethidium bromide, and downcutting the gel area that contains 3-9kb DNA.Use β-gelase I then, (scheme that MA) provides is by the gel piece purify DNA for New England Biolabs, Beverly to use manufacturers.(indication MO) is connected in the λ ZipLox EcoRI arm in 16 ℃ of DNA that will select according to size that spend the night for Life Technologies company, Gaithersburg according to manufacturers then.(CA), according to the indication of manufacturers, packing and titration connect product for Stratagene, La Jolla to use Gigapack GoldIII package kit (Gigapack GoldIII Packaging Kit).Amount to, obtained 8 * 10 ⁶Individual reorganization plaque, and the scheme amplification library of using manufacturers to provide.

Screening-gene group library is to obtain the palB genomic clone.Described in the scheme that provides with λ ZipLox arm, genomic library is suitably diluted to obtain 7000 plaques/150mm flat board.Use standard scheme (people such as Sambrook, 1989, see above) with the phagocytosis spot to Hybond N ⁺Filter membrane (Amersham, Cleveland, OH) on.Use the crosslinked fixedly filter membrane of UV, and in DIG Easy Hyb in 42 ℃ of prehybridizations.0.25kb palB probe hybridization with filter membrane and DIG mark.Use following primer through the pcr amplification probe, these primers use Applied Biosystems company's 394 type DNA/RNA synthesizers (AppliedBiosystems Model 394DNA/RNA Synthesizer) synthetic according to the indication of manufacturers, and use Genius test kit (Boehringer Mannheim, Indianapolis, IN) with diosgenin (dioxygenin) mark:

5’-CTGCCGTCGAAGGTGTCCAAG-3’(SEQ ID NO：5)

5’-ATTGTGGCCCCTATGTGGATT-3’(SEQ ID NO：6)

Use about 0.2 μ g pDSY109 as template for preparing 100 μ l amplification reaction systems.Each reaction system comprises following composition: 0.2 μ g plasmid DNA, 48.4pmol forward primer and 48.4pmol reverse primer, 1mM dATP, dCTP, dGTP and dTTP, 1X Taq polymerase buffer, with 2.5U Taq polysaccharase (Perkin-Elmer company, Branchburg, NJ).With reaction system incubation in Ericomp Twin Block System Easy Cycler, it is as follows to programme: 95 ℃ of 5min, 1 circulation; 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2min, 30 circulations.

Cleaning filter membranes, and use the scheme that provides with the Genius test kit to carry out back hybridization.Identify several positive plaques, and used standard scheme (people such as Sambrook, 1989, see above) to be purified to homogeneous.

Measured a palB clone's nucleotide sequence according to method described in the embodiment 1.Fig. 1 has shown nucleotide sequence (SEQ ID NO:1) and deduced amino acid (SEQ IDNO:2).The opening code-reading frame of palB genes encoding 4700bp, 854 amino acid whose polypeptide of this reading frame coding.Opening code-reading frame is interrupted by 3 introns.

Use Clustal method (Higgins, 1989, CABIOS 5:151-153), use LASERGENE ^TMMEGALIGN ^TM(DNASTAR company, Madison WI), to parameter, have carried out the comparative comparison of palB aminoacid sequence: breach point penalty 10, notch length point penalty 10 with identity table and following multiple ratio to software.The comparison parameter is Ktuple=1 in pairs, breach point penalty=3, frame=5, diagonal lines=5.

Comparative comparison shows that aspergillus oryzae PalB albumen (SEQ ID NO:2) and Aspergillus nidulans PalB albumen (people such as Denison, 1995, see above) have 66.4% identity.

Prepared as mentioned above through the aspergillus oryzae DEBY10.3 of BalII digestion and the Southern trace of aspergillus oryzae HowB101 genomic dna.Detect trace to confirm that the flanking DNA that reclaims is a splitted gene among the aspergillus oryzae DEBY10.3 with the 0.25kb palB probe of DIG mark.

From about 7.5kb BalII band and the probe hybridization of aspergillus oryzae HowB101, simultaneously from the 12kb band of aspergillus oryzae DEBY10.3 also with probe hybridization.Difference in size meets the expection size of integrating a plasmid copy, thereby has confirmed that the recovery locus divides in aspergillus oryzae DEBY10.3.

Because the integration incident among the prediction aspergillus oryzae DEBY10.3 will cause PalB albumen to lose function, therefore test is cultivated aspergillus oryzae DEBY10.3 at pH8.0 and pH6.5.Aspergillus nidulans palB ^-Bacterial strain can not be grown at pH8.0, but can grow at pH6.5.In the minimum medium that adds the 10mM uridine, cultivate aspergillus oryzae HowB430 and aspergillus oryzae DEBY10.3 at pH8.0 and pH6.5.As what predict, aspergillus oryzae DEBY10.3 can not grow at pH8.0.

The structure of embodiment 6:pMT1936

Use according to the following primer of indication synthetic of manufacturers, has made up the pTM1936 that comprises palB division box on Applied Biosystems company 394 type DNA/RNA synthesizers:

100752：

5’-GGTTGCATGCTCTAGACTTCGTCACCTTATTAGCCC-3’

(SEQ ID NO：7)

100753：

5’-TTCGCGCGCATCAGTCTCGAGATCGTGTGTCGCGAGTACG

-3’(SEQ ID NO：8)

100754：

5’-GATCTCGAGACTAGTGCGCGCGAACAGACATCACAGGAAC

C-3’(SEQ ID NO：9)

100755：

5’-CAACATATGCGGCCGCGAATTCACTTCATTCCCACTGCGT

GG-3’(SEQ ID NO：10)

By according to the aspergillus oryzae A1560 of scheme described in the embodiment 4 preparation (people such as Christensen, 1988, biotechnology (Bio/Technology) 6:1419-1422) genomic dna is through the sequence of pcr amplification aspergillus oryzae palB 5 ' flanking sequence and coding palB product N end parts.Used about 0.5 μ g genomic dna and two kinds of primers 100754 and 100755 each 5pmol. to use polysaccharase Pwo, as manufacturers (Boehringer Mannheim, Indianapolis, IN) the described amplification.40 circulations have been carried out in amplification.The partial reaction product is carried out phenol take out, ethanol sedimentation, EcoRI and XhoI digestion, and the fragment of having separated about 1.05kb by agarose gel electrophoresis.

Should obtain the sequence of aspergillus oryzae palB 3 ' flanking sequence and coding palB gene product C end parts with primer 100752 and 100753 as mentioned above, and, carry out the fragment that about 1.5kb has been reclaimed in gel electrophoresis then with XhoI and XbaI digestion PCR product.

With above-mentioned two kinds through digestion be connected with carrying out three fragments with the PCR fragment of purifying from the purified 2.7kb EcoRI-XbaI fragment of carrier pJaL400 (Fig. 2), the generation pMT1935 (Fig. 3).The palB 5 ' of pMT1935 and 3 ' flank are separated by BssHII, SpeI and the XhoI site through PCR primer 100754 and 100753 importings.

To contain the 3.5kb HindIII fragment cloning of pJaL394 (Fig. 4) of repeated fragment in the pBIuescriptII SK (-) at the pyrG flank with HindIII cutting, dephosphorylation and purifying.Obtained to comprise the insertion fragment plasmid of two kinds of orientations.Selected a kind of plasmid pMT1931 (Fig. 5), wherein the SpeI site of pBluescript polylinker is positioned at the downstream of pyrG gene, and the XhoI site is positioned at the upstream of pyrG gene.Separate the pyrG gene with 3.5kb SpeI-XhoI fragment, and insert pMT1935, produce division plasmid pMT1936 (Fig. 6) through SpeI and XhoI digestion and purifying.

Can separate pyrG with 6.2kb NotI fragment (NotI in the cutting polylinker) or 5.5kb AseI-PvuI fragment (palB 5 ' and the interior AseI and PvuI of 3 ' flanking sequence that cutting is actual) by pMT1936 and can select palB division box.

Embodiment 7: use the AseI/PvuI palB division box from pMT1936 to transform aspergillus oryzae

Use same conversion scheme described in the embodiment 1, use the 5.5kbAseI-PvuI fragment that obtains by pMT1936 to transform aspergillus oryzae HowB430.(Qiagen, Chatsworth CA) according to indication isolated fragment on 1% sepharose of manufacturers, separate the linear fragment that is used to transform thus by digesting pMT1936 with AseI and PvuI and using the Qia PhastGel to extract test kit.The growth of tested transformant on the minimum medium flat board of pH6.5 or pH8.0 then.

The result shows that 13 have palB in 128 transformant of test ^-Phenotype shows as and can not grow at pH8.0.With 13 kinds of palB ^-Bacterial strain and 13 kinds can carry out the spore purifying in the transformant of pH8.0 growth.

To from aspergillus oryzae palB ^-Mutant, aspergillus oryzae palB ⁺The genomic dna of bacterial strain and aspergillus oryzae HowB430 carries out the Southern trace, is incorporated in the palB locus as removing replacement to confirm AsnI-PvuI transfering DNA fragment.Prepare genomic dna according to step described in the embodiment 4, with PvuI digestion, and on 0.8% sepharose electrophoresis.Use 0.4NNaOH and capillary action that DNA is transferred to Hybond N ⁺On the filter membrane.UV is crosslinked with trace, then in 65 ℃ of prehybridizations in Rapid Hyb.Detect trace with containing then from the segmental probe of 0.9kb AsnI-SpeI of pMT1936.Behind electrophoresis on 1% sepharose, use Qia fast rotational post (QiaQuick spin column) to separate the 0.9kb fragment.Use and cause labelling kit Vistra ECF Random Prime Labeling Kit labeled fragment at random.With the trace prehybridization, and in 65 ℃ Rapid Hyb (Amersham, Cleverland, OH) in hybridization, in 2X SSC/0.1%SDS, clean 2 times then in 65 ℃ of 5min, in 0.2X SSC/0.1%SDS, clean 2 times in 65 ℃ of 10min.After the cleaning, (OH) (MolecularDynamics, Sunnyvale CA) detect trace with imaging system STORM860Imaging System for Amersham, Cleveland to use signal to amplify test kit Vistra ECF Signal Amplification Kit.

S0uthern trace result has proved probe and has hybridized from the 6kb band of aspergillus oryzae HowB430.Estimating clearly to divide (fragment) will hybridize with about 8kb PvuI band.Southern trace result has also shown some palB ^-Bacterial strain has clear division (fragment), and other bacterial strain does not then have.

Embodiment 8: the extracellular protease of aspergillus oryzae Δ palB bacterial strain generates

With 3 kinds of aspergillus oryzae palB described in the embodiment 7 ^-With 3 kinds of aspergillus oryzae palB ⁺Bacterial strain is being equipped with substratum (Nutriose, yeast extract, (NH ₄) ₂HPO ₄, MgSO ₄7H ₂O, citric acid, K ₂SO ₄, CaCl ₂H ₂O and trace metal solution) 2 liters of fermentor tanks in cultivated 8 days in 34 ℃, pH7,1000-2000rpm.The outer nutrient solution sampling of pair cell every day, and used hereinafter described FTC-casein test method that sample is test total extracellular protease activity at the 6th day.

FTC-casein test method is following to carry out.In the enzyme solution that 10 μ l suitably dilute with 0.1M MOPS damping fluid (pH7.0), add 1: 1 mixed FTC-casein (Twining of 40 μ l and 0.1M MOPS damping fluid (pH7.0), 1984, analysis of biochemical (AnalyticalBiochemistry) 143:30-34) open and begin to react.Reaction system in 37 ℃ of incubations 2 hours, is reacted with 150 μ l, 5% trichoroacetic acid(TCA) quencher subsequently.It is centrifugal then 10 minutes that quencher is reflected at 5 ℃ of underlyings 2 hours.Transfer in the test tube that 2ml 0.5M borate buffer solution (pH9.0) is housed 20 μ l supernatant liquors also mixed.Then with 200 μ l sample transfer of this solution at the bottom of the black U-shaped 96 orifice plates (Dynatech company, Chantiily, VA) in, with borate buffer solution as blank with instrument mark zero.(Dynatech company, Chantiily VA) use with reference to the channel 3 and the 4.1V bulb voltage that are provided with 1176, measure fluorescence to use Fluorolite 1000 instruments.Instrument dynamic range is between the 0-4000 flat fluorescent, and best " Kong Yukong " repeatability is between the 400-3500 unit.

Result displayed is pointed out aspergillus oryzae palB in the table 2 ^-The extracellular protease that bacterial strain produces is than aspergillus oryzae palB ⁺On average low 10 times of bacterial strains.Also have serpin PMSF or inhibitors of metalloproteinase 1,10-tests during phenanthrolene.Shown in the table 2 by shown in the active per-cent of total protease that suppresses of inhibitor.Aspergillus oryzae palB ⁺And palB ^-The suppression mode of sample is similar, illustrates that the palB deletion influences the generation of serine protease and metalloprotease.

Table 2.palB ^-With the extracellular protein enzyme level in the wild type strain

Bacterial strain	The proteolytic enzyme flat fluorescent+	%PMSF suppresses **	%1, the 10-phenanthrolene suppresses **
				DEBY10.3palB - DEBY10.3palB - DEBY10.3palB - DEBY10.3palB + DEBY10.3palB + DEBY10.3palB +	1120 1370 810 11100 9785 13050	73 77 69 87 83 84	4 15 3 37 34 37

+ in 37 ℃ and pH7 incubation 2 hours. ^*Inhibitor concentration is 1mM.

As mentioned above also with other 3 kinds of aspergillus oryzae palB ^-With other 3 kinds of aspergillus oryzae palB ⁺Bacterial strain was cultivated 8 days in 2 liters of fermentor tanks, and carried out the extracellular sampling every day.When existing or not having inhibitor, test sample the 6th angel with above-mentioned FTC-casein test method.

Result displayed is pointed out aspergillus oryzae palB in the table 3 ^-The total protease specific activity aspergillus oryzae palB that bacterial strain produces ⁺On average low 10 times of bacterial strains.

Table 3. aspergillus oryzae palB ^-With the extracellular protein enzyme level in the wild type strain

Bacterial strain	The proteolytic enzyme flat fluorescent+	%PMSF suppresses **	%1, the 10-phenanthrolene suppresses **
				DEBY10.3palB - DEBY10.3palB - DEBY10.3palB - DEBY10.3palB + HowB430palB + HowB430palB +	485 345 540 4580 5025 3765	77 48 52 92 86 77	35 0 0 23 39 37

+ in 37 ℃ and pH7 incubation 1 hour. ^*Inhibitor concentration is 1mM.

As mentioned above also with 2 kinds of aspergillus oryzae palB ^-Bacterial strain and a kind of aspergillus oryzae palB ⁺Bacterial strain was cultivated 8 days in 2 liters of fermentor tanks.Aspergillus oryzae palB ^-Bacterial strain is the clear division of palB gene.Carry out the extracellular sampling 8 day every day.When existing or not having inhibitor, test sample the 6th angel with above-mentioned FTC-casein test method.The results are shown in hereinafter table 4.Aspergillus oryzae palB ^-The extracellular protease activity that bacterial strain produces is than aspergillus oryzae palB ⁺On average low 20 times of bacterial strains.

Table 4. aspergillus oryzae palB ^-With the extracellular protein enzyme level in the wild type strain

Bacterial strain	The proteolytic enzyme flat fluorescent+	%PMSF suppresses **	%1, the 10-phenanthrolene suppresses **
				DEBY10.3palB - HowB430ΔpalB 76-11-1 HowB430ΔpalB 76-11-1 pLRF2-10(palB +)	1095 715 16360	35 44 91	0 3.5 36

+ in 37 ℃ and pH7 incubation 2 hours. ^*Inhibitor concentration is 1mM.

Embodiment 9: the pcr amplification of Aspergillus nidulans palA gene

By the genomic dna for preparing as described in example 4 above, use PCR and following primer, amplification is from the palA gene of Aspergillus nidulans.

palA2540R：5’-TCGCGCAGTCGTGATTCAAAG-3’(SEQ ID NO：11)

pal172：5’-CCGCACTGGAGTAAATAACAT-3’(SEQ ID NO：11)

Reaction system comprises 50ng Aspergillus nidulans genomic dna, palA2540R and each 50pmol of palA172, Perkin Elmer PCR damping fluid, 1mM dNTP and 0.5U Taq archaeal dna polymerase.Reaction system is circulated in Ericomp Twin Block System Easy Cycler, and it is as follows to programme: 95 ℃ of 3min, 1 circulation; 95 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min, 1 circulation.Get a part of reaction solution electrophoresis on sepharose, and obtained the expection product of about 2.5kb.With half reaction solution electrophoresis on preparation property gel, downcut the gel piece that contains the product of wanting, and use the Qia PhastGel to extract test kit by gel separation DNA.

(Stratagene, La Jolla CA) according to the indication of manufacturers, use to use initiation test kit Prime-It Kit ³²The gel separation PCR product of P-dCTP mark 1/10.TEMidi G-50 post (5 ' to 3 ', Boulder, CO) after the reaction of enterprising row labels, purifying palA probe.

Embodiment 10: the Southern that carries out the aspergillus oryzae genomic dna with Aspergillus nidulans palA probe

Analyze

With the genomic dna of the aspergillus oryzae HowB430 of preparation as described in example 4 above with BamHI, EcoRI or HindII digestion, and on sepharose electrophoresis.As described in example 7 above, under alkaline condition, DNA is transferred to Hybond N ⁺On the filter membrane.With identical 3 traces in low, moderate and highly rigorous degree hybridization buffer in 42 ℃ of hybridization 1 hour (prehybridization and hybridization 5X SSPE/0.3% SDS/200 μ g/ml cut off and the sex change salmon sperm dna in carry out, low, moderate and highly reach very high rigorous degree and contain 25,35 or 50% methane amide respectively).Add described in the embodiment 10 ³²The palA probe of P-dCTP mark, and trace spent the night in 4 ℃ of hybridization.With trace in 42 ℃ in 2X SSC/0.1%SDS 5min clean 2 times, 10min cleans 2 times in 0.2X SSC/0.1%SDS.Observe specific band on the trace of in moderate and low rigorous degree damping fluid, hybridizing.

The separation of embodiment 11:palA aspergillus oryzae genomic clone

In λ ZipLox, make up the genomic library of aspergillus oryzae HowB425 according to the indication of manufacturers.630,000 plaque-forming units are contained in the library, and wherein 73% contains the insertion fragment.Average insertion clip size is 3.5kb.(Life Technologies, Gaithersburg MD) are coated on the NZY massive plate together with about 7000 recombinant phages and intestinal bacteria Y1090.Use standard scheme with the phagocytosis spot to Hybond N ⁺On the filter membrane.UV is crosslinked with filter membrane, and as described in example 10 above in 42 ℃ of prehybridizations 1 hour in medium rigorous degree hybridization buffer.Add ³²The Aspergillus nidulans palA probe of P mark, and filter membrane spent the night in 42 ℃ of hybridization.Cleaning filter membranes and make the x-ray film exposure as mentioned above.

2 positive colonies have been obtained.The picking positive plaque and in 1ml SM damping fluid wash-out.With elutriant dilution 10 ⁴, and get 50 or 100 μ l and be coated on the NZY massive plate with intestinal bacteria Y1090 cell.Get plaque and handle filter membrane as preceding point.By purifying positive plaque of each plate isolation.By (Life Technologies company, Gaithersburg MD) are coated with intestinal bacteria DH10B cell, downcut plasmid DNA by positive colony according to the scheme that provides.

The analysis of embodiment 12:palA aspergillus oryzae genomic clone

(Qiagen, Chatsworth is CA) by the isolated plasmid dna of 2 positive colonies (palA5A and palA6A) described in the embodiment 11 to use Qia to prepare test kit Qiaquick Prep8 Kit fast.Comprise the insertion fragment with PstI digested plasmid DNA to confirm them.Use the applying biological 377XL of system type automated DNA sequenator (AppliedBiosystems Model 377XL Automated DNA Sequencer), use M13 forward and reverse primer, carry out the part nucleotide sequencing of genomic clone.The part order-checking has confirmed that the clone comprises aspergillus oryzae palA homologue.Measured the nucleotide sequence of maximum clone palA5A.Use the applying biological 377XL of system type automated DNA sequenator, use dyestuff to stop the thing chemical method, carry out dna sequencing.(Foster City CA) has produced the contig sequence for primer island swivel base test kit Primer Island Trahsposition Kit, Perkin-Elmer/Applied Biosystems company to use transposon to insert strategy.Through order-checking, it is 5.3 that the 2.9kb of palA5A inserts segmental average degree.

PalA5A clone's nucleotide sequence (SEQ ID NO:3) proves the part clone who has separated palA, according to Aspergillus nidulans palB gene (people such as Negrete-Urtasun, 1997, see above) homology, last about 245 the amino acid whose DNA of disappearance coding palA.Partial nucleotide sequence (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) are shown in Fig. 7.The opening code-reading frame of palA genes encoding 2855bp, 549 amino acid whose polypeptide of this reading frame coding.Opening code-reading frame is interrupted by 4 introns, and being positioned between Aspergillus nidulans and the aspergillus oryzae clone of they is strict conservative.

Use Clustal method (Higgins, 1989, CABIOS 5:151-153), use LASERGENE ^TMMEGALIGN ^TM(DNASTAR company, Madison WI), to parameter, have carried out the comparative comparison of part aspergillus oryzae PalA derivation aminoacid sequence and other PalA aminoacid sequence: breach point penalty 10, notch length point penalty 10 with identity table and following multiple ratio to software.The comparison parameter is Ktuple=1 in pairs, breach point penalty=3, frame=5, diagonal lines=5.

Comparative comparison display part aspergillus oryzae PalA albumen (SEQ ID NO:2) and Aspergillus nidulans PalA albumen (people such as Negrete-Urtasun, 1997, see above) have 88% identity.

Embodiment 13: the deletion of palA in the aspergillus oryzae strain

Make up the plasmid pBM8 that replaces 600bp palA opening code-reading frame with the pyrG gene.

Use applying biological system's 394 type DNA/RNA synthesizers (Applied BiosystermsModel 394DNA/RNA Synthesizer), according to the indication of manufacturers, synthesized and be designed for the segmental following synthetic oligonucleotide primer thing of two kinds of palA that separate of pcr amplification.First group comprises 5 ' primer that is designed for adding XhoI site and the 3 ' primer that is used to add the HindIII site.

First group:

Primer 980163:5 '-AGTAGCCGTCTATCTCTCCAGCAGTAGTGT-3 '

(SEQ ID NO：13)

Primer 980164:5 '-AAGCTTACTCGCAATTAGCCTTCTCGGTGAA

TCG-3’(SEQ ID NO：14)

Second group comprises 5 ' primer that is designed for adding HindIII site and the 3 ' primer that is used to add the NotI site.

Second group:

Primer 980165:5 '-AAGCTTTACGATGTCATGCTGGGCAACGGTC

AAG-3’(SEQ ID NO：15)

Primer 980166:5 '-GCGGCCGCCTCTGCGTGATACTCTAGGGTCTGG

-3’(SEQ ID NO：16)

Bold-type letter presentation code sequence.

Set up the PCR reaction system of 100 μ l, comprise that 50ng palA5A dna profiling, each 50pmol of various primer, 1X PCR damping fluid contain 2mM MgSO ₄(BoehringerMannheim), 1mM dNTP and 2.5 Taq of unit archaeal dna polymerases (BoehringerMannheim).Reaction system is circulated in Ericomp Twin Block System Easy Cycler, and it is as follows to programme: 95 ℃ of 5min, 1 circulation; 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, 30 circulations; 95 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 3min, 1 circulation.

Get 10 μ l PCR reaction systems on 1.0% sepharose in 100 volts of electrophoresis 1 hour.Downcut the primary product of first group of about 1100bp of primer and the primary product of second group of about 730bp of primer by gel, and use the Qia PhastGel to extract test kit (Qiaquick GelExtraction Kit) purifying.Subsequently according to the indication of manufacturers, with purified PCR product cloning to plasmid pCR2.1-TOPO (Invitrogen, San Diego, CA) in, and transform the TOP10 cell (Invitrogen, San Diego, CA).

Obtained 2 clones, 1100bp inserts segmental called after intestinal bacteria pPalA1, and 730bp inserts segmental called after intestinal bacteria pPalA2.Use the Perkin-Elmer applying biological 377XL of system type sequenator (Perkin-Elmer Applied Biosystems Model 377Sequencer XL), use dyestuff to stop the thing chemical method, use lac forward sequencing primer and lac reverse sequencing primer, analyze this two clones by order-checking.

With HindIII digestion pBluescript KS-, and it is flat terminal to handle generation with the Klenow fragment.With DNA electrophoresis on preparation property gel, and downcut the band that contains through the cutting plasmid DNA.Use the Qia PhastGel to extract test kit by gel separation DNA.Then plasmid is connected, transformed into escherichia coli DH5 α, and by several bacterium colony isolated plasmid dnas.By the disappearance of HindIII restrictive diges-tion to colony screening HindII site.A clone called after pBM1a (Fig. 8) with disappearance HindII site.(Qiagen, Chatsworth CA) prepare plasmid DNA by pBM1a to use Qiaquick Prep8 scheme.With 0.5 μ l XhoI and NotI (every kind of 10U/ μ l) digestion pBM1a.With 0.5 μ l XhoI and HindIII (every kind of 10U/ μ l) digestion pPalA1.With 0.5 μ l NotI and HindIII (every kind of 10U/ μ l) digestion pPalA2.Will through dna digestion on 1% sepharose in 100 volts of electrophoresis 1 hour.Downcut 1100bp fragment, the 730bp fragment of pPalA2 and the 2.9kb fragment of pBM1a of pPalA1 by gel, and be resuspended in 200 μ l 10mM Tris (pH7.5).Subsequently three kinds of dna fragmentations are linked together, and transformed into escherichia coli DH5 α competent cell.The plasmid called after pBM7 (Fig. 9) that produces.

With the DNA of 1 μ l HindIII (10U/ μ l) digestion from pJal394.Will through dna digestion on 0.7% sepharose in 100 volts of electrophoresis 1 hour, produce the 3539bp band.Downcut the 3539bp fragment by gel, and be resuspended in 200 μ l 10mM Tris (pH7.5).

With the DNA of 1 μ l HindIII (10U/ μ l) digestion from pBM7.In 65 ℃ of hot deactivations 10 minutes, (Boehringer Mannheim, Indianopolis was IN) according to the indication dephosphorylation of manufacturers to use shrimp alkaline phosphotase subsequently with HindIII.Will through dna digestion on 0.7% sepharose in 100 volts of electrophoresis 1 hour, produce the 3630bp band.Downcut the 3630bp fragment by gel, and be resuspended in 300 μ l 10mM Tris (pH7.5).Two kinds of dna fragmentations are linked together, and transformed into escherichia coli DH5 α competent cell.The clone's called after pBM8 (Figure 10) that produces.

In order to separate the deletion fragment of pBM8, with NotI and XhoI digested plasmid, and with digest electrophoresis on preparation property gel.Use the Qia PhastGel to extract test kit by gel separation 5.3kb deletion fragment.Prepare aspergillus oryzae HowB430 protoplastis as described in example 1 above with the fragment conversion, and on the minimum medium flat board, select.Total has obtained 93 transformant.The pal of tested transformant on the minimum medium flat board of pH8.0 ^-Phenotype.Can not grow at pH8.0 for 3 in 93.The Southern analysis confirmation 3 bacterial strains be clear division.

Embodiment 14: the extracellular protease in the aspergillus oryzae Δ palA bacterial strain generates

With 2 Aspergillus oryzae palA described in the embodiment 13 ⁺With 2 Aspergillus oryzae palA ^-Bacterial strain was cultivated 8 days in 34 ℃ in 2 liters of fermentor tanks as described in example 8 above.Use FTC-casein test method described in the embodiment 8 to measure total extracellular protease activity of the 6th day sample.

Hereinafter result shown in the table 5 proves aspergillus oryzae palA ^-The extracellular protein enzyme level that bacterial strain produces is aspergillus oryzae palA ⁺About 1/10 of bacterial strain.

Table 5.HowB430 Δ palA bacterial strain and extracellular protease

Bacterial strain	The proteolytic enzyme flat fluorescent
		DEBY599.3(wt) ΔpalA 1-3 palA 6-1(wt) ΔpalA 7-1	7450 710 5150 565

The preservation of biomaterial

Following biologic material is preserved in agricultural research institute preservation center (Agricultural Research Service Patent Culture Collection) according to budapest treaty, northern area research centre (Northern Regional Research Center), Peoria, Illinois (Illinois), preserving number is as follows:

The preservation thing	Numbering	Preservation date
			Intestinal bacteria HB101pDSY109 intestinal bacteria XL-1Blue pDSY174	NRRL B-21623 NRRL B-30089	On January 28,1999 on the 5th September in 1996

These bacterial strains have carried out preservation, at the pending trial of this patent application in the phase, guarantee can obtain this culture according to patent and the determined people of the trade mark council that 37C.F.R. § 1.14 and 35U.S.C. § 122 authorize.This preservation thing has been represented the pure basically culture of preservation strain.In the country of the copy of having submitted this application to or its subsequent application, can provide this preservation thing according to the requirement of this state's patent law.But, be understood that the availability of preservation thing does not constitute implementing permission of the present invention to invade the patent right of being authorized by action by government.

Because particular embodiment disclosed herein is intended to the illustration as the many aspects of the invention of describing and requiring herein, so the present invention is in no way limited within the scope of these embodiments.Any suitable embodiment all belongs within the scope of the present invention.Even those skilled in the art according to foregoing description obviously can understand the present invention except shown here and described various variations.These variations also belong within the scope of claims.When clashing, comprise that with this specification sheets definition is as the criterion.

Complete being incorporated herein by reference of many places cited herein reference.

SEQUENCE LISTING

<110>Debbie S.Yaver

<120>Methods For Producing Polypeptides In

Fungal Cells

<130>5693.204-WO

<140>PCT/US00/02864

<141>2000-02-02

<150>09/241,955

<151>1999-02-02

<160>16

<170>FastSEQ for Windows Version 4.0

<210>1

<211>4700

<212>DNA

<213>Aspergillus

<400>1

acagcgactg ggatggtgaa tatctgagcg atagcccccg acacagcacc aagggtaagc 60

tccatagcgg ttccaggtgg cttagatacg ctcttcgatg ccatataaag gcttctaacc 120

acgctgtacc agtagaagta cgcaaagttg gtcgaggcca cgccaagcaa agaaccaacc 180

atcccggaat ataaaccttc aattccctct ttctccacaa tcttgttgat ggcatctagg 240

gtcgactcgt aatgtactac atctccgctt ttcgattcag gtgcgttctt cacttggact 300

tgaagtttgg ttttgacact aaagattagg aacgagtcag tctcagtcta caccactaag 360

ccaactggca aggctatgta cgcacaggtc cagtggatag acgatagcat ttgcgagaac 420

agcaccagtt gcacctgcga cagcactacc ccaaggggag agcgcgggtt tcgattggcc 480

ggccattatg caggatgagc taaagtgcct ctgccaattc cgtcagaaag aatagtataa 540

gagcacaaat actggtaaaa ccaagaccgg cgaatgaggc aggactctgt gcgattcggg 600

gggtttagcg ttcggcttga aggcttaccg gatcgactga tagaaaagtt gaatgccgag 660

taaggtgaaa agaccccagc tcccaagcag caacagtaag tggaagagct taaggataga 720

aaaaataaat taggattaag aaaaaaagaa gaacctcaag actggtcaca cagtcccggc 780

atcctgaacg taaaatgcgg gaaggataga gtcggcaggg ccagggcagt tgcacctcgg 840

cgctctggtt tgcgcatgac gaaatgagcc gaggttcgtt ttttggaggc caatttctga 900

acaccgacct tcgaattccc gttcctcccc accgacacgc tagtgaatga tccagcaagc 960

atacttggtg ttgttttgac ctcattccac tgcgtgtgaa ttagcattaa tttaagttta 1020

tgattaacag tcaattgcta tacgcgaaaa tcatcatcgt cttgattggc ccttcataaa 1080

acttgacaag gaagtttgat cgacctcgga tgtcgcgctt tcggaaattt cacggagccc 1140

ttcggacggg tcacaagcaa ggtgtctgac tgtctcgttt agtcggatag acgctagttg 1200

aactgttatg cctatcgcgg ggaagatctc ggagtgtcac ggtgtttgaa gatcccaggc 1260

gctcgtcaaa atactgcccg gcctgccagt atgtctagac cgaacgcctc agcccagaag 1320

tcctttataa ctcaggcact ggtacttgac cctttttttt tatggttttt tgtttctttc 1380

ttgttacacc ttatttttct tcttctcgtt ttttgtagat aatactgacc actggctaga 1440

aagccgagcg ggatgtatcg tccgccactt ctcaaaggca agctttagaa gctgccattg 1500

atgctgctga acactatatg aaagccttaa atctggcatc tgttcagaaa gacaaacatg 1560

cattggatgc aaagtgtaaa gaatggctca caagagcgga aaagatcaaa gaatctaagg 1620

actggcaagc tgctgcccgt ttccatgaca aaactgttcc agagccacgg ttgcctgtat 1680

ctactcgtaa gctcaccaca cgggaggaga tcattctgct agagggagcc aagttgaatg 1740

gcttcatatt ccctccatgg tccacctccc caggctctga cgagttcaaa cgagaggatg 1800

gtgaatcccc gtttacgtaa gttctggtgg tctgcatcgt caatgttgca tgtataccca 1860

gatgactgct ggatattcta accgataaca gcgacaaacc cgatcttcat ctatcttatc 1920

ctcaaaggaa agtttttgat ggctggaaac gaccttccga gcttctcgcg aaagacacgg 1980

aagatgtgta cacaaaggtg gttcctgtga tgtctgttcc aggaaagaca gatctagtcc 2040

aggatatgct gacggactgt tctgtcgttg ctagcctttg tgctactacg tcaatgctag 2100

aacgcggcca gtgtactgta agaagattga tcccttccgg ctgacctgca tggttcgctg 2160

tgactaatag gtgtagcatt ttcttccaat gatataccct agccggggga gctctcagcc 2220

ttcaccgtca ggcaagtata tatttcgctt ttatttcaat gggtgcttcc ggaaagtcat 2280

cattgacgac cgtttgccat cgtctaagac atcaagatca ctccacgtga tcgaccggaa 2340

aaatcccaat ttcctttggc cggcgctcgt agagaaggcg tatttgaaat tgcgcggagg 2400

ctatgatttt cccggaagca attccgggac agatctctgg gtgctgacag gttggattcc 2460

cgagcaagtc tttctccata atgacgatgt gactggcgac cagctctgga agcgacttta 2520

cagatccttt caccaaggag atgttctctt gactataggt accggtgaac tcactgagag 2580

ggaacaaaga gaactaggcc tcgtgagtga gcatgattat gctattctgg atatgaagga 2640

atctaaaggt cgccgacaat tactcgtgaa aaacccttgg gctggagcag atactgcccc 2700

cggcgacaat ggaagcctct ctgcatcgca ggatttaccc cataacccgc cctcatttga 2760

gccgggtacc ttttggatgg attgcgaaaa gctgcttcaa cattttgaaa acctctattt 2820

gaattggaac cctgagattt tcaaataccg cgaagacgtc cactttacgt gggacctcaa 2880

caacggaga ggtgtagccg gctgttttgt gaataacccg cagttcgcag tgtcaaccga 2940

gaacggtggg attgtctggt tacttctagg caagcatttc agaacaacag ggcagccgga 3000

acgacctctt gacgaatacc aagcgaatga ggagtcggct tttataagca tatatgtctt 3060

taacgcagat ggcaaacggg tctctttgag tgatggggct ctacatcgtg gcccctatgt 3120

ggattcccct aatacgctca tgaggttaga gatgcccccc agaacaacat acacagtcgt 3180

ggtctccgag caatcactgc catctttgaa tcaaaacttt actttgtctg ccttctctac 3240

ctgccctgta cggatggcaa aagcccaaga taaatacatg tgtgtcagga agattcaagg 3300

gtcttggaca ccttcgacgg caggtgggaa tgccgaatct tctcgatatc cactcaaccc 3360

ccaatttagg ttggagatag agaatgacac agatgtttca ctcctgctgg aatgcccaaa 3420

cacggaactc gcgacccatg ttaagttatt ctggtccaat ggaaatcgtg tgtcgcgagt 3480

acgcagtcgc gacataatcg ctgatagtgg tgactatcgc cgtggtggct cccttgtgga 3540

aaagaaggct ctggaaccgg gctcatatac aatcgtctgt tccacattcg cgccggatca 3600

acttggccga ttcacgctct gggtatcctc cttagttcct tgcaagacga gcccgctccc 3660

accagaggca gcaggtcgac gaacggtcat ttcagatatt ggcgtactgc ctcccgggag 3720

agaccgaatg ttagcttctc tgcaagtgcc gcggcttacg aggatcaagc tcatcacccg 3780

aagtaggcaa tccatcatcg ggagccatcc tgttggaccc tcgcccgttt taatgacagt 3840

ggagctcggg caagggccat acaaacagat cctggcgact tcggaagatg gaactcacag 3900

tgatgctgta tcgggggtac gtgttgagga ctttgacttg cagcctgggc tagaggagag 3960

tggtggtatt tggattgtta ttgagaggat tgggggtcct ggagggcagg tagaggacca 4020

ctttgaggtg gaagctttgg ctgaagagag ggttgagatt ggggagtgga tacttgaaga 4080

tgcttgatct cttatcctgc agaagctctg aagctctgga cggtcttagt tgagcttttt 4140

tgatcgtcgt tgtgattagc acgttagagt agaagagcgg aaacaatgat agacatgaat 4200

ttctcttatt gtctctattg gccagaagag aaagaaggca ttaaatcaat acattaaaag 4260

caagagttta tctatagata ccgagcagcc tcaggatttg agctgaggtt tgtcgcgatc 4320

gcgaccgcca aaatggagtt agcttgcttt actccgcata aattaaatcc tcggctcggg 4380

gcccgaattc ccctcccgag tgctttcaac gtcatcccgt ttgctgtgtg cattgtgcct 4440

ccccaccttt aacaattgga gctctgtcca aggacaaatc cttattctcg cggcctccat 4500

ggcgactaat attcctgctg ctctgaagtc tgcggacatt gggcgctttg ccgtcagagc 4560

agctcagctt gaacgtgtaa agcccgtggt cgcctactgg tgtgagtatt gtgtgattat 4620

acccagtacg aactacgagc tgatgagcgg cctctgctga tcgcaggcaa cttctggatc 4680

gtcaaccaga ttattgagaa 4700

<210>2

<211>854

<212>PRT

<213>Aspergillus

<400>2

Met Ser Arg Pro Asn Ala Ser Ala Gln Lys Ser Phe Ile Thr Gln Ala

1 5 10 15

Leu Lys Ala Glu Arg Asp Val Ser Ser Ala Thr Ser Gln Arg Gln Ala

20 25 30

Leu Glu Ala Ala Ile Asp Ala Ala Glu His Tyr Met Lys Ala Leu Asn

35 40 45

Leu Ala Ser Val Gln Lys Asp Lys His Ala Leu Asp Ala Lys Cys Lys

50 55 60

Glu Trp Leu Thr Arg Ala Glu Lys Ile Lys Glu Ser Lys Asp Trp Gln

65 70 75 80

Ala Ala Ala Arg Phe His Asp Lys Thr Val Pro Glu Pro Arg Leu Pro

85 90 95

Val Ser Thr Arg Lys Leu Thr Thr Arg Glu Glu Ile Ile Leu Leu Glu

100 105 110

Gly Ala Lys Leu Asn Gly Phe Ile Phe Pro Pro Trp Ser Thr Ser Pro

115 120 125

Gly Ser Asp Glu Phe Lys Arg Glu Asp Gly Glu Ser Pro Phe Thr Asp

130 135 140

Lys Pro Asp Leu His Leu Ser Tyr Pro Gln Arg Lys Val Phe Asp Gly

145 150 155 160

Trp Lys Arg Pro Ser Glu Leu Leu Ala Lys Asp Thr Glu Asp Val Tyr

165 170 175

Thr Lys Val Val Pro Val Met Ser Val Pro Gly Lys Thr Asp Leu Val

180 185 190

Gln Asp Met Leu Thr Asp Cys Ser Val Val Ala Ser Leu Cys Ala Thr

195 200 205

Thr Ser Met Leu Glu Arg Gly Gln Cys Thr His Phe Leu Pro Met Ile

210 215 220

Tyr Pro Ser Arg Gly Ser Ser Gln Pro Ser Pro Ser Gly Lys Tyr Ile

225 230 235 240

Phe Arg Phe Tyr Phe Asn Gly Cys Phe Arg Lys Val Ile Ile Asp Asp

245 250 255

Arg Leu Pro Ser Ser Lys Thr Ser Arg Ser Leu His Val Ile Asp Arg

260 265 270

Lys Asn Pro Asn Phe Leu Trp Pro Ala Leu Val Glu Lys Ala Tyr Leu

275 280 285

Lys Leu Arg Gly Gly Tyr Asp Phe Pro Gly Ser Asn Ser Gly Thr Asp

290 295 300

Leu Trp Val Leu Thr Gly Trp Ile Pro Glu Gln Val Phe Leu His Asn

305 310 315 320

Asp Asp Val Thr Gly Asp Gln Leu Trp Lys Arg Leu Tyr Arg Ser Phe

325 330 335

His Gln Gly Asp Val Leu Leu Thr Ile Gly Thr Gly Glu Leu Thr Glu

340 345 350

Arg Glu Gln Arg Glu Leu Gly Leu Val Ser Glu His Asp Tyr Ala Ile

355 360 365

Leu Asp Met Lys Glu Ser Lys Gly Arg Arg Gln Leu Leu Val Lys Asn

370 375 380

Pro Trp Ala Gly Ala Asp Thr Ala Pro Gly Asp Asn Gly Ser Leu Ser

385 390 395 400

Ala Ser Gln Asp Leu Pro His Asn Pro Pro Ser Phe Glu Pro Gly Thr

405 410 415

Phe Trp Met Asp Cys Glu Lys Leu Leu Gln His Phe Glu Asn Leu Tyr

420 425 430

Leu Asn Trp Asn Pro Glu Ile Phe Lys Tyr Arg Glu Asp Val His Phe

435 440 445

Thr Trp Asp Leu Asn Asn Gly Arg Gly Val Ala Gly Cys Phe Val Asn

450 455 460

Asn Pro Gln Phe Ala Val Ser Thr Glu Asn Gly Gly Ile Val Trp Leu

465 470 475 480

Leu Leu Gly Lys His Phe Arg Thr Thr Gly Gln Pro Glu Arg Pro Leu

485 490 495

Asp Glu Tyr Gln Ala Asn Glu Glu Ser Ala Phe Ile Ser Ile Tyr Val

500 505 510

Phe Asn Ala Asp Gly Lys Arg Val Ser Leu Ser Asp Gly Ala Leu His

515 520 525

Arg Gly Pro Tyr Val Asp Ser Pro Asn Thr Leu Met Arg Leu Glu Met

530 535 540

Pro Pro Arg Thr Thr Tyr Thr Val Val Val Ser Glu Gln Ser Leu Pro

545 550 555 560

Ser Leu Asn Gln Asn Phe Thr Leu Ser Ala Phe Ser Thr Cys Pro Val

565 570 575

Arg Met Ala Lys Ala Gln Asp Lys Tyr Met Cys Val Arg Lys Ile Gln

580 585 590

Gly Ser Trp Thr Pro Ser Thr Ala Gly Gly Asn Ala Glu Ser Ser Arg

595 600 605

Tyr Pro Leu Asn Pro Gln Phe Arg Leu Glu Ile Glu Asn Asp Thr Asp

610 615 620

Val Ser Leu Leu Leu Glu Cys Pro Asn Thr Glu Leu Ala Thr His Val

625 630 635 640

Lys Leu Phe Trp Ser Asn Gly Asn Arg Val Ser Arg Val Arg Ser Arg

645 650 655

Asp Ile Ile Ala Asp Ser Gly Asp Tyr Arg Arg Gly Gly Ser Leu Val

660 665 670

Glu Lys Lys Ala Leu Glu Pro Gly Ser Tyr Thr Ile Val Cys Ser Thr

675 680 685

Phe Ala Pro Asp Gln Leu Gly Arg Phe Thr Leu Trp Val Ser Ser Leu

690 695 700

Val Pro Cys Lys Thr Ser Pro Leu Pro Pro Glu Ala Ala Gly Arg Arg

705 710 715 720

Thr Val Ile Ser Asp Ile Gly Val Leu Pro Pro Gly Arg Asp Arg Met

725 730 735

Leu Ala Ser Leu Gln Val Pro Arg Leu Thr Arg Ile Lys Leu Ile Thr

740 745 750

Arg Ser Arg Gln Ser Ile Ile Gly Ser His Pro Val Gly Pro Ser Pro

755 760 765

Val Leu Met Thr Val Glu Leu Gly Gln Gly Pro Tyr Lys Gln Ile Leu

770 775 780

Ala Thr Ser Glu Asp Gly Thr His Ser Asp Ala Val Ser Gly Val Arg

785 790 795 800

Val Glu Asp Phe Asp Leu Gln Pro Gly Leu Glu Glu Ser Gly Gly Ile

805 810 815

Trp Ile Val Ile Glu Arg Ile Gly Gly Pro Gly Gly Gln Val Glu Asp

820 825 830

His Phe Glu Val Glu Ala Leu Ala Glu Glu Arg Val Glu Ile Gly Glu

835 840 845

Trp Ile Leu Glu Asp Ala

850

<210>3

<211>2855

<212>DNA

<213>Aspergillus

<400>3

aattgcatct gtttacctga gcttcagcca ctgacttgag taaaggcccc tttgagcttg 60

ctaagaatgt cgttcagacc tcggtcttgg tgtcaaaccg tgcccaggcc tcgccagacg 120

cggtcaatga cccctcattg cgtaacaagc cccgtctcgg caccatcgag gccattcggc 180

agatcgtcca acgttacgga atccgtggcc tgtacaccgg ctttcatctg cacgctctgc 240

gtgatactct agggtctggc ttatacttta gtgtctacga aacggtaaag caagtcgcat 300

cgaaagagct tggcccagac aaatccccgt tcggcggccc catgattgcc ggggcgattt 360

gcagcactgt cccgtggttt tgtgtaagtg ccgcaacaat gatgcctttt tgcgggttac 420

taaccgtcat agacctatcc acttgatact cgaaagaccc gtgcccagag cgtgttgctc 480

ggaaaatcca gcgaagtcgg ggaggcgtcg gctgcggttg ccaagtccag tatgtataaa 540

ggactttcga ttattctcat ccgtactggg gtaaacaata tgatcttgct cagtatcttc 600

gaatacataa agatgaagat caatcaattg gattgatacg atttctggga cattagccga 660

tgcgcagatg gccattggcc ttggggactg ccggagtatg cttttatgat ttatatttta 720

tcgagtacat atgtgcatga gcctgaggct ccacgcaaat acagaatgaa taacttgact 780

gttatttaat tagactaagt cgtgtagcca caaattattg tgcggagtga atctatagaa 840

atacaatatc aagttatgtg gtgcctcagg ccaacacaca acgtcatccg ttgtccggtg 900

gggttgggtt ttcaaacaac atcaacaaaa gttgcccggc gacgatccct ccttcattgc 960

gctcttgacc gttgcccagc atgacatcgt atgtgctgtc tctaaggata tgctgtttca 1020

tcttgattgt ctaggctaat ttattgttca gaaatatcct tcagatcccc tttcgacgct 1080

ctcacaccgt ttccttgtca gatgcgatca ctcagtatat ttcgaccaag tatgaccagc 1140

gtccagatat gttcgcagat gatctattga tcatagatcg cctgcgtaac gaagcgattc 1200

atgtccagga gccacatgtt agtgggatca gtcggttggt aacatatgcc gcgcagttga 1260

aatggcttgg tggaaagttt ccggtcgatg tgagtaaccg tagcatagcg gtgggggaac 1320

tgaccacaaa gccgttatta acacgggagt ctacagattg gagtcgagtt tccatggtac 1380

cctgcgtttg gtttcaatac ctcaagaccg agtatgttaa gagtaattcg gtcactagca 1440

ttgttgctaa cgaatacaag tctcgcagaa caatattcga tttgaacttg ccaatatcct 1500

ctttaacctg gtagcactat actcgcaatt agccttctcg gtgaatcgta caacaccaga 1560

tggactcaaa caggcatgca actacctctg ccaggcagct ggagtcttag cacatctccg 1620

agcggatatc ctccctgatc ttcgtgcctc tcccccagag gatatggatg atatgacact 1680

acagagtctg gaacagcttc ttctcgcgca gggacaagag tgcttctggc agaaggccgt 1740

caaggatggg ctgaaagatg cttccattgc tcggctggcg gccaaggtct ccgattttta 1800

cgcagaagga ggtgactatg cggttcaatc gaatgccatt agtcctgaat ggattcacca 1860

tatgacggcc aagcatcatc attttgctgc cgctgcgcag tatcgccagt ccttggactg 1920

tttggagaag agaaagtatg gtgaagaggt ggctcgtctt cgagacagcg aggtttgtgt 1980

caacgaggcc ttgaaggaat cacgttggat caatcgcact gtgctagggg acttgcaagg 2040

attaaaaaac cgtgtgacgg aggacttaaa gcgcgccgag aaagacaacg atgtaatcta 2100

tctcaatcca gtccctccaa agtcagaatt gaagatcatc gatcgagcat gtatggtcgc 2160

agcgaaggcg ccatctcagg tgactgatgc tatctccatg ttgggagaca acggaccttt 2220

agggcagccg ttattttcaa agctggtgcc atatgccgtt catatcgctg ccagtattta 2280

ctccgaccgc cgtgatcggc ttgtcaacga gacaattatc ggggagctgg agaccatgac 2340

ggacaaatta cgggagtaag tattaaaatc taggctatga tgagcaagat actaactggt 2400

atagtttatt atcatctctg aatcttccgg gctccttgca ggctctggaa aagcctcttg 2460

gattaccgcc tacgttggtc tcccatgcgg aagaaatgcg ccagcaagat gggttgaacc 2520

ggttgcggag atcactcgag gataccgcca gggtgaaggc caatgacaaa gctgcataca 2580

atgagggtgt tgaactactc gccgcagaaa aggcagagga tgactcttcg cgccgcaaat 2640

acggaactga caggtgggcc agagagccct ccgaagcagc agcgtcgaag ctgtacacta 2700

ctgctcgaga gatagacggc tacttttcct ccgctcagag tagcgataat ctggtagagc 2760

agaagctgag agactcggag gcagtcttcc gagttttgac cggtactaac cgggatttgg 2820

agatgtacgt ccctagcagc cggagagcgg caatt 2855

<210>4

<211>549

<212>PRT

<213>Aspergillus

<400>4

Met Thr Ser Asn Ile Leu Gln Ile Pro Phe Arg Arg Ser His Thr Val

1 5 10 15

Ser Leu Ser Asp Ala Ile Thr Gln Tyr Ile Ser Thr Lys Tyr Asp Gln

20 25 30

Arg Pro Asp Met Phe Ala Asp Asp Leu Leu Ile Ile Asp Arg Leu Arg

35 40 45

Asn Glu Ala Ile His Val Gln Glu Pro His Val Ser Gly Ile Ser Arg

50 55 60

Leu Val Thr Tyr Ala Ala Gln Leu Lys Trp Leu Gly Gly Lys Phe Pro

65 70 75 80

Val Asp Ile Gly Val Glu Phe Pro Trp Tyr Pro Ala Phe Gly Phe Asn

85 90 95

Thr Ser Arg Pro Ile Ser Gln Asn Asn Ile Arg Phe Glu Leu Ala Asn

100 105 110

Ile Leu Phe Asn Leu Val Ala Leu Tyr Ser Gln Leu Ala Phe Ser Val

115 120 125

Asn Arg Thr Thr Pro Asp Gly Leu Lys Gln Ala Cys Asn Tyr Leu Cys

130 135 140

Gln Ala Ala Gly Val Leu Ala His Leu Arg Ala Asp Ile Leu Pro Asp

145 150 155 160

Leu Arg Ala Ser Pro Pro Glu Asp Met Asp Asp Met Thr Leu Gln Ser

165 170 175

Leu Glu Gln Leu Leu Leu Ala Gln Gly Gln Glu Cys Phe Trp Gln Lys

180 185 190

Ala Val Lys Asp Gly Leu Lys Asp Ala Ser Ile Ala Arg Leu Ala Ala

195 200 205

Lys Val Ser Asp Phe Tyr Ala Glu Gly Gly Asp Tyr Ala Val Gln Ser

210 215 220

Asn Ala Ile Ser Pro Glu Trp Ile His His Met Thr Ala Lys His His

225 230 235 240

His Phe Ala Ala Ala Ala Gln Tyr Arg Gln Ser Leu Asp Cys Leu Glu

245 250 255

Lys Arg Lys Tyr Gly Glu Glu Val Ala Arg Leu Arg Asp Ser Glu Val

260 265 270

Cys Val Asn Glu Ala Leu Lys Glu Ser Arg Trp Ile Asn Arg Thr Val

275 280 285

Leu Gly Asp Leu Gln Gly Leu Lys Asn Arg Val Thr Glu Asp Leu Lys

290 295 300

Arg Ala Glu Lys Asp Asn Asp Val Ile Tyr Leu Asn Pro Val Pro Pro

305 310 315 320

Lys Ser Glu Leu Lys Ile Ile Asp Arg Ala Cys Met Val Ala Ala Lys

325 330 335

Ala Pro Ser Gln Val Thr Asp Ala Ile Ser Met Leu Gly Asp Asn Gly

340 345 350

Pro Leu Gly Gln Pro Leu Phe Ser Lys Leu Val Pro Tyr Ala Val His

355 360 365

Ile Ala Ala Ser Ile Tyr Ser Asp Arg Arg Asp Arg Leu Val Asn Glu

370 375 380

Thr Ile Ile Gly Glu Leu Glu Thr Met Thr Asp Lys Leu Arg Asp Leu

385 390 395 400

Leu Ser Ser Leu Asn Leu Pro Gly Ser Leu Gln Ala Leu Glu Lys Pro

405 410 415

Leu Gly Leu Pro Pro Thr Leu Val Ser His Ala Glu Glu Met Arg Gln

420 425 430

Gln Asp Gly Leu Asn Arg Leu Arg Arg Ser Leu Glu Asp Thr Ala Arg

435 440 445

Val Lys Ala Asn Asp Lys Ala Ala Tyr Asn Glu Gly Val Glu Leu Leu

450 455 460

Ala Ala Glu Lys Ala Glu Asp Asp Ser Ser Arg Arg Lys Tyr Gly Thr

465 470 475 480

Asp Arg Trp Ala Arg Glu Pro Ser Glu Ala Ala Ala Ser Lys Leu Tyr

485 490 495

Thr Thr Ala Arg Glu Ile Asp Gly Tyr Phe Ser Ser Ala Gln Ser Ser

500 505 510

Asp Asn Leu Val Glu Gln Lys Leu Arg Asp Ser Glu Ala Val Phe Arg

515 520 525

Val Leu Thr Gly Thr Asn Arg Asp Leu Glu Met Tyr Val Pro Ser Ser

530 535 540

Arg Arg Ala Ala Ile

545

<210>5

<211>21

<212>DNA

<213>Aspergillus

<400>5

ctgccgtcga aggtgtccaa g 21

<210>6

<211>21

<212>DNA

<213>Aspergillus

<400>6

attgtggccc ctatgtggat t 21

<210>7

<211>36

<212>DNA

<213>Aspergillus

<400>7

ggttgcatgc tctagacttc gtcaccttat tagccc 36

<210>8

<211>40

<212>DNA

<213>Aspergillus

<400>8

ttcgcgcgca tcagtctcga gatcgtgtgt cgcgagtacg 40

<210>9

<211>41

<212>DNA

<213>Aspergillus

<400>9

gatctcgaga ctagtgcgcg cgaacagaca tcacaggaac c 41

<210>10

<211>42

<212>DNA

<213>Aspergillus

<400>10

caacatatgc ggccgcgaat tcacttcatt cccactgcgt gg 42

<210>11

<211>21

<212>DNA

<213>Aspergillus

<400>11

tcgcgcagtc gtgattcaaa g 21

<210>12

<211>21

<212>DNA

<213>Aspergillus

<400>12

ccgcactgga gtaaataaca t 21

<210>13

<211>30

<212>DNA

<213>Aspergillus

<400>13

agtagccgtc tatctctcca gcagtagtgt 30

<210>14

<211>34

<212>DNA

<213>Aspergillus

<400>14

aagcttactc gcaattagcc ttctcggtga atcg 34

<210>15

<211>34

<212>DNA

<213>Aspergillus

<400>15

aagctttacg atgtcatgct gggcaacggt caag 34

<210>16

<211>33

<212>DNA

<213>Aspergillus

<400>16

gcggccgcct ctgcgtgata ctctagggtc tgg 33

Claims (13)

1. method that is used to produce polypeptide comprises:

A) cultivate the mutant of parent's Aspergillus cell, wherein mutant cell comprises one or both genes that is selected from palA and palB, and it is an inactivation; With the nucleic acid encoding sequence, mutant cell lacks than one or more proteolytic enzyme quantity that parent's Aspergillus cell produces when wherein cultivating under the same conditions;

B) by the nutrient solution isolated polypeptide.

2. the process of claim 1 wherein that polypeptide is natural or allogenic for the Aspergillus cell.

3. the process of claim 1 wherein that one or more proteolytic enzyme are extracellular proteases.

4. the method for claim 3, wherein extracellular protease is serine protease or metalloprotease.

5. the process of claim 1 wherein that one or more proteolytic enzyme that mutant cell produces when cultivating under the same conditions lack 25% at least than parent's Aspergillus cell.

6. the process of claim 1 wherein that polypeptide is hormone, hormone variant, enzyme, acceptor or its part, antibody or its part or reporter molecule.

7. the method for claim 6, wherein enzyme is oxydo-reductase, transferring enzyme, lytic enzyme, lyase, isomerase or ligase enzyme.

8. the method for claim 6, wherein enzyme is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, at, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, MUTANASE, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, or zytase.

9. the polypeptide of producing by each method of claim 1-8.

10. the protease-deficient mutant of parent's Aspergillus cell comprises the gene that one or more are selected from palA and palB, and it is an inactivation; With the nucleic acid encoding sequence, when wherein cultivating under the same conditions, the amount of one or more proteolytic enzyme that mutant cell produces is lacked than parent's Aspergillus cell.

11. the mutant cell of claim 10, wherein polypeptide is natural or allogenic with respect to mutant cell.

12. isolated nucleic acid sequences that comprises the nucleotide sequence of SEQ ID NO.3.

13. the nucleotide sequence of claim 12, this nucleotide sequence are contained among the contained plasmid pDSY174 of intestinal bacteria NRRL B-30089.

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