Description of the Related Art Many microorganisms are able to grow over a wide pH range. This ability requires an efficient pH homeostatic mechanism that protects intracellular processes against the extremes of pH and a means for insuring that activities undertaken beyond the boundaries of pH homeostasis are only attempted at appropriate ambient pH.

The pacC gene encodes a protein designated PacC which is a sequence-specific DNA binding protein that activates the expression of genes whose products are synthesized preferentially at alkaline pH and represses the synthesis of gene products <BR> <BR> <BR> <BR> appropriate to acidic growth conditions. Tilburn et al., 1995, EMBO Journal 14: 779- 790, have proposed that PacC in Aspergillus nidulans activates transcription of alkaline- <BR> <BR> <BR> <BR> expressed genes in the presence of a signal mediated by the products of the palA, B, C, F, H, and I genes in response to alkaline ambient pH. The palA, B, C, F, H, and I genes are known as the PacC pH signal transduction pathway. Therefore, at alkaline ambient pH the expression of alkaline genes is elevated while the expression of acidic genes is diminished. Under acidic conditions PacC is in a non-functional form and its level is diminished leading to derepression of acid-expressed gene products and lack of activation of alkaline-expressed genes including pacC itself.

Some mutations of the pacC gene or the genes of the PacC pH signal transduction pathway can mimic the effects of growth at a pH other than the actual <BR> <BR> <BR> <BR> ambient pH (Caddick et al., 1986, Molecular General Genetics 203: 346-353; Shah et<BR> <BR> <BR> <BR> <BR> <BR> al., 1991, FEMS Microbiological Letters 77: 209-212; Espeso et al., 1993, EMBO Journal 12: 3947-3956; Arst et al., 1994, Molecular General Genetics 245: 787-790).

Some mutations in any of the six genes palA, B, C, F, H, and I mimic the effects of growth at acidic pH and result, for example, in elevated levels of acid phosphatase and reduced levels of alkaline phosphatase. In contrast, some mutations in the pacC gene

mimic the effects of growth at alkaline pH and result, for example, in elevated levels of alkaline phosphatase and reduced levels of acid phosphatase. These pacC gene mutations that mimic growth under alkaline conditions remove an acidic C-terminal segment which modulates its activity. When the acidic C-terminal domain is removed, PacC is active and can activate expression of genes required for growth under alkaline conditions and repress expression of genes required for growth under acidic conditions.

Such mutations of the pacC gene obviate the need for the signal transduction pathway, leading to constitutivity of alkaline-expressed genes and super-repression of acid- expressed genes. pacC genes have been cloned from Aspergillus nidulans (Tilburn et al., 1995, <BR> <BR> <BR> <BR> supra), Aspergillus niger (Maccabe et al., 1996, Molecular General Genetics 250: 367- 374), Aspergillus parasiticus (Pinero and Keller, 1997, Phytopathology 87: S78), and Penicillium chrysogenum (Suarez et al., 1996, Molecular Microbiology 20: 529-540).

Genes in the PacC pH signal transduction pathway have been disclosed including <BR> <BR> <BR> <BR> the Aspergillus nidulans palA (Negrete-Urtasun et al., 1997, Journal of Bacteriology 179: 1832-1835); Aspergillus gene (Denison et al., 1995, Journal of Biological Chemistry 270: 28519-28522); Aspergillus nidulans gene (Maccheroni <BR> <BR> <BR> <BR> et al., 1997, Gene 194: 163-167); and Aspergillus nidulans pull gene (Arst et al., 1994, supra).

PacC pH signal transduction mutants have been described by Caddick et al., <BR> <BR> <BR> <BR> 1986, Molecular General Genetics 203: 346: 352, and Arst et al., 1994, Molecular General Genetics 245: 787-790.

The ability to reduce or eliminate proteolytic activity that is detrimental to the production of a protein of interest, particularly in a recombinant cell, without the need to disrupt or delete each gene responsible for the overall proteolytic activity would provide a very attractive alternative currently unavailable in the art.

It is an object of the present invention to provide improved methods for producing polypeptides in fungal cells.

Summary of the Invention 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 <BR> <BR> <BR> <BR> acid sequence comprising a modification of at least one of the 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.

Brief Description of Figures Figure 1 shows the genomic nucleic acid sequence and deduced amino acid sequence of an Aspergillus oryzae palB gene (SEQ ID NOS: 1 and 2, respectively).

Figure 2 shows a restriction map of pJaL400.

Figure 3 shows a restriction map of pMT1935.

Figure 4 shows a restriction map of pJaL394.

Figure 5 shows a restriction map of pMT1931.

Figure 6 shows a restriction map of pMT1936.

Figure 7 shows the partial genomic nucleic acid sequence and deduced amino <BR> <BR> <BR> <BR> acid sequence of an Aspergillus oryzae palA gene (SEQ ID NOS: 3 and 4, respectively).

Figure 8 shows a restriction map of pBMla.

Figure 9 shows a restriction map of pBM7.

Figure 10 shows a restriction map of pBM8.

Detailed Description of the Invention 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 fungal cell relates to the parent cell by a modification, e. g., disruption or deletion, of one or more genes of a pacC pH signal transduction pathway or homologues thereof ; and (b) isolating the polypeptide from the cultivation medium of the mutant cell.

The phrase"pacC pH signal transduction pathway"is defined herein as a pathway that senses the ambient pH of the growth environment of a microorganism and <BR> <BR> <BR> <BR> through a group of cellular proteins encoded by the genes including palA, palB, palC, palF, palH, and pall, promotes proteolytically processing of PacC into an active transcription factor. Activated PacC turns on expression of genes required for growth under alkaline conditions and turns off the expression of genes required for growth under

acidic conditions. It will be understood that homologues of the palA, palB, palC, palF, palH, and pali genets are encompassed by the methods of the present invention.

In the methods of the present invention, any gene of a fungal cell involved in the pacC pH signal transduction pathway may be modified including, but not limited to, the palA, palB, palC, palF, palH, and/or palI genes of filamentous fungi or similar homologues of yeast, e. g., Rim9p (Denison et al., 1998, Molecular Microbiology 30: 259-264). In a preferred embodiment, the gene is a palA gene. In another preferred embodiment, the gene is a palB gene. In a more preferred embodiment, the gene is the Aspergillus oryzae palA gene having the nucleic acid sequence of SEQ ID NO: 1. In another more preferred embodiment, the gene is the Aspergillus oryzae palB gene having the nucleic acid sequence of SEQ ID NO: 3.

As shown below, the modification of one or more genes in a pacC pH signal transduction pathway of a fungal cell reduces the amount of proteolytic activity produced by the cell. One or more proteases may be responsible for the proteolytic activity. The deficiency results from a decrease in expression of the specific protease genes under alkaline conditions since PacC does not activate transcription of alkaline-expressed genes due to the absence of a signal mediated by the group of gene products encoded by the palA, B, C, F, H, and I genes.

The pacC pH signal transduction pathway mutant cell may be constructed by reducing or eliminating expression of one or more of the genes described above using methods well known in the art. For example, the gene may be modified or inactivated by altering the coding region or a part thereof essential for activity, or a regulatory function required for the expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i. e., a part which is sufficient for affecting expression of the nucleic acid sequence. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal sequence, transcription terminator, and transcriptional activator.

Modification or inactivation of the gene may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells which can grow at a pH different than the standard growth pH of the parent cell followed by measurement of the proteolytic activity of the mutant cells versus the parent cell. The mutagenesis, which

may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening for mutant cells exhibiting reduced or no expression of a gene of the pacC pH signal transduction pathway.

Modification or inactivation of one or more of the genes in the pacC pH signal transduction pathway may be accomplished by introduction, substitution, or removal of one or more nucleotides in the gene or a regulatory element required for the transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change of the open reading frame. Such modification or inactivation may be accomplished by site- directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i. e., directly on the cell expressing the gene to be modified, it is preferred that the modification be performed in vitro as exemplified below.

An example of a convenient way to inactivate the pacC pH signal transduction pathway is based on techniques of gene replacement, gene deletion, or gene disruption.

In the gene disruption method, a nucleic acid sequence corresponding to the endogenous gene or gene fragment of interest is mutagenized in vitro to produce a defective nucleic acid sequence which is then transformed into the parent cell to produce a defective gene.

By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene or gene fragment. It may be desirable that the defective gene or gene fragment also encodes a marker which may be used for selection of transformants in which the nucleic acid sequence has been modified or destroyed.

Alternatively, modification or inactivation of one or more genes in the pacC pH

signal transduction pathway may be performed by established anti-sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the gene.

More specifically, expression of the gene may be reduced or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell.

Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.

A nucleic acid sequence complementary or homologous to the nucleic acid sequence of a gene involved in the pacC pH signal transduction pathway in a fungal cell may be obtained from microbial sources which contain such genes. A preferred source <BR> <BR> <BR> <BR> for a palA gene having a nucleic acid sequence complementary or homologous to the nucleic acid sequence of SEQ ID NO: 1 of Aspergillus oryzae is Aspergillus nidulans. A <BR> <BR> <BR> <BR> <BR> preferred source for a palB gene having a nucleic acid sequence complementary or<BR> <BR> <BR> <BR> <BR> <BR> <BR> homologous to the nucleic acid sequence of SEQ ID NO: 3 of Aspergillus oryzae is Aspergillus nidulans.

Preferred filamentous fungal sources for other genes in the pacC pH signal transduction pathway which may be complementary or homologous to the nucleic acid sequence of the corresponding genes of a fungal cell of choice include, but are not <BR> <BR> <BR> <BR> <BR> limited to, thepalFgene fromAspergillus nidulans (Maccheroni etal., 1997, supra); and<BR> <BR> <BR> <BR> <BR> <BR> <BR> the palI gene from Aspergillus nidulans (Arst et al., 1994, supra). Furthermore, the nucleic acid sequences may be native to the fungal cell.

Preferred yeast sources for homologues of the genes in the pacC pH signal transduction pathway which may be complementary to the nucleic acid sequence of the corresponding genes of a fungal cell of choice include, but are not limited to, Saccharomyces cerevisiae (Rim9p; Denison et al., 1998, supra).

In a preferred embodiment, the mutant fungal cell further comprises two or more copies of a pacC gene. The two or more copies of the pacC gene prevent extragenic suppression of a pal mutant phenotype. The extragenic suppressors have been shown to be truncations of pacC, which lead to a constitutively active PacC.

The pacC gene may be native or heterologous to the fungal cell. Preferred sources for the pacC gene from filamentous fungi include, but are not limited to, <BR> <BR> <BR> Aspergillus nidulans (Tilburn et al., 1995, supra), Aspergillus niger (Maccabe et al.,

1996, supra), Aspergillus parasiticus (Pinero and Keller, 1997, supra), and Penicillium chrysogenum (Suarez et al., 1996, supra). Preferred sources for pacC gene homologues <BR> <BR> <BR> <BR> from yeast include, but are not limited to, Yarrowia lipolytica (Lambert et al., 1997, Molecular and Cellular Biology 17: 3966-3976) and Saccharomyces cerevisiae (Su and Mitchell, 1993, Nucleic Acids Research 21: 3789-3797).

In the methods of the present invention, the modification of the PacC pH signal transduction pathway results in a reduction or elimination of production of one or more proteases.

The phrase"one or more proteases"is defined herein as one or more exopeptidases and/or endopeptidases. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of a substrate, whereas endopeptidases cleave peptide bonds distant from the termini of the substrate (Rao, 1998, Microbiology and Molecular Biology Reviews 62: 597-635). The exopeptidase may be an aminopeptidase or a carboxypeptidase. The exopeptidase or endopeptidase may be a serine protease, metalloprotease, aspartic protease, or cysteine protease (Rao et al., 1998, supra; North, 1982, Microbiological Reviews 46: 308-340; Otto and Schirmeister, 1997, Chemical Reviews 97: 133-171). In a preferred embodiment, the one or more proteases are serine proteases. In another preferred embodiment, the one or more proteases are metalloproteases. In another preferred embodiment, the one or more proteases are aspartic proteases. In another preferred embodiment, the one or more proteases are cysteine proteases. In another preferred embodiment, the one or more proteases are aminopeptidases. In another preferred embodiment, the one or more proteases are carboxypeptidases. In another preferred embodiment, the one or more proteases are a serine protease, metalloprotease, aspartic protease, cysteine protease, aminopeptidase, and/or carboxypeptidase.

The activity of the proteases may be determined using any method well known in the art. Identification of the type of proteolytic activity may also be determined using protease inhibitors well known in the art which are specific for a serine protease, metalloprotease, aspartic protease, or cysteine protease. See, for example, North, 1982, supra; Otto and Schirmeister, 1997, supra; and Rao et al., 1998, supra.

In the methods of the present invention, the mutant fungal cell preferably produces at least about 25% less, more preferably at least about 50% less, even more

preferably at least about 75% less, and most preferably at least about 95% less of one or more proteases compared to a corresponding parent fungal cell when cultured under identical conditions. In a most preferred embodiment, the mutant fungal cell produces no detectable proteases compared to a corresponding parent fungal cell when cultured under identical conditions. The parent and mutant cells may be compared with regard to production of the one or more proteases under conditions conducive for the production of a polypeptide of interest or under conditions conducive for the production of the one or more proteases.

In the methods of the present invention, the polypeptide may be any polypeptide whether native or heterologous to the mutant fungal cell of interest.

The term"polypeptide"is not meant herein to refer to a. specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The term"heterologous polypeptide"is defined herein as a polypeptide which is not native to the fungal cell, a native polypeptide in which modifications have been made to alter the native sequence, or a native polypeptide whose expression is quantitatively altered as a result of a manipulation of the fungal cell by recombinant DNA techniques. For example, a native polypeptide may be recombinantly produced by, e. g., placing a gene encoding the polypeptide under the control of a different promoter to enhance expression of the polypeptide, to expedite export of a native polypeptide of interest outside the cell by use of a signal sequence, and to increase the copy number of a gene encoding the polypeptide normally produced by the cell. The mutant fungal cell may contain one or more copies of the nucleic acid sequence encoding the polypeptide.

Preferably, the polypeptide is a hormone or variant thereof, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter. In a more preferred embodiment, the polypeptide is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In an even more preferred embodiment, the polypeptide is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha- galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The nucleic acid sequence encoding a polypeptide of interest that can be expressed in a fungal cell may be obtained from any prokaryotic, eukaryotic, or other source. For purposes of the present invention, the term"obtained from"as used herein in connection with a given source shall mean that the polypeptide is produced by the source or by a cell in which a gene from the source has been inserted.

The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide of interest are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequence from such genomic DNA can be effected, e. g., by using the well known polymerase chain reaction (PCR). See, for example, Innis et al., 1990, PCR Protocols : A Guide to Methods and Application, Academic Press, New York. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into the mutant fungal cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

In the methods of the present invention, the polypeptide may also include a fused or hybrid polypeptide in which another polypeptide is fused at the N-terminus or the C- terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide. Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter (s) and terminator. The hybrid polypeptide may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the mutant fungal cell.

An isolated nucleic acid sequence encoding a polypeptide of interest may be manipulated in a variety of ways to provide for expression of the polypeptide.

Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification,

translation, post-translational modification, and secretion. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.

"Nucleic acid construct"is defined herein as a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid combined and juxtaposed in a manner that would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term"coding sequence"is defined herein as a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5'end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3'end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term"control sequences"is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide of interest. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term"operably linked"is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a fungal cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences which

mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the fungal cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the fungal cell.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs in a filamentous fungal cell are promoters obtained from the genes encoding <BR> <BR> <BR> Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, <BR> <BR> <BR> Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase,<BR> <BR> <BR> <BR> <BR> Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (WO 96/00787), NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof.

In a yeast cell, useful promoters are obtained from the Saccharomyces cerevisiae <BR> <BR> <BR> enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3- phosphoglycerate kinase gene. Other useful promoters for yeast cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a fungal cell to terminate transcription. The terminator sequence is operably linked to the 3'terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the fungal cell of choice may be used in the present invention.

Preferred terminators for filamentous fungal cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), or

Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the fungal cell. The leader sequence is operably linked to the 5'terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the fungal cell of choice may be used in the present invention.

Preferred leaders for filamentous fungal cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Suitable leaders for yeast cells are obtained from the genes encoding Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'terminus of the nucleic acid sequence and which, when transcribed, is recognized by the fungal cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the fungal cell of choice may be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin- like protease, and Aspergillus niger alpha-glucosidase.

Useful polyadenylation sequences for yeast cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5'end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5'end of the coding

sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a fungal cell of choice may be used in the present invention.

An effective signal peptide coding region for filamentous fungal cells is the signal peptide coding region obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, or Humicola lanuginosa lipase.

Useful signal peptides for yeast cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).

A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the Saccharomyces cerevisiae alpha-factor gene, Rhizomucor miehei aspartic proteinase gene, or Myceliophthora thermophila laccase gene (WO 95/33836).

Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of the polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the fungal cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems would include the lac, tac, and

trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase <BR> <BR> <BR> <BR> promoter, and the Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide would be operably linked with the regulatory sequence.

In the methods of the present invention nucleic acid constructs for altering the expression of an endogenous gene encoding a polypeptide of interest may be used. The constructs may contain the minimal number of components necessary for altering expression of the endogenous gene. In one embodiment, the nucleic acid constructs preferably contain (a) a targeting sequence, (b) a regulatory sequence, (c) an exon, and (d) a splice-donor site. Upon introduction of the nucleic acid construct into a cell, the construct inserts by homologous recombination into the cellular genome at the endogenous gene site. The targeting sequence directs the integration of elements (a)- (d) into the endogenous gene such that elements (b)- (d) are operably linked to the endogenous gene. In another embodiment, the nucleic acid constructs contain (a) a targeting sequence, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of elements (a)- (f) such that elements (b)- (f) are operably linked to the endogenous gene. However, the constructs may contain additional components such as a selectable marker.

In both embodiments, the introduction of these components results in production of a new transcription unit in which expression of the endogenous gene is altered. In essence, the new transcription unit is a fusion product of the sequences introduced by the targeting constructs and the endogenous gene. In one embodiment in which the endogenous gene is altered, the gene is activated. In this embodiment, homologous recombination is used to replace, disrupt, or disable the regulatory region normally associated with the endogenous gene of a parent cell through the insertion of a regulatory sequence which causes the gene to be expressed at higher levels than evident in the corresponding parent cell. The activated gene can be further amplified by the inclusion

of an amplifiable selectable marker gene in the construct using methods well known in the art (see, for example, U. S. Patent No. 5,641,670). In another embodiment in which the endogenous gene is altered, expression of the gene is reduced.

The targeting sequence can be within the endogenous gene, immediately adjacent to the gene, within an upstream gene, or upstream of and at a distance from the endogenous gene. One or more targeting sequences can be used. For example, a circular plasmid or DNA fragment preferably employs a single targeting sequence, while a linear plasmid or DNA fragment preferably employs two targeting sequences.

The regulatory sequence of the construct can be comprised of one or more promoters, enhancers, scaffold-attachment regions or matrix attachment sites, negative regulatory elements, transcription binding sites, or combinations of these sequences.

The constructs further contain one or more exons of the endogenous gene. An exon is defined as a DNA sequence which is copied into RNA and is present in a mature mRNA molecule such that the exon sequence is in-frame with the coding region of the endogenous gene. The exons can, optionally, contain DNA which encodes one or more amino acids and/or partially encodes an amino acid. Alternatively, the exon contains DNA which corresponds to a 5'non-encoding region. Where the exogenous exon or exons encode one or more amino acids and/or a portion of an amino acid, the nucleic acid construct is designed such that, upon transcription and splicing, the reading frame is in-frame with the coding region of the endogenous gene so that the appropriate reading frame of the portion of the mRNA derived from the second exon is unchanged.

The splice-donor site of the constructs directs the splicing of one exon to another exon. Typically, the first exon lies 5'of the second exon, and the splice-donor site overlapping and flanking the first exon on its 3'side recognizes a splice-acceptor site flanking the second exon on the 5'side of the second exon. A splice-acceptor site, like a splice-donor site, is a sequence which directs the splicing of one exon to another exon.

Acting in conjunction with a splice-donor site, the splicing apparatus uses a splice- acceptor site to effect the removal of an intron.

In another aspect of the present invention, the mutant fungal cell may additionally contain modifications of one or more third nucleic acid sequences that encode proteins which may be detrimental to the production, recovery, and/or application of the polypeptide of interest. The modification reduces or eliminates expression of the

one or more third nucleic acid sequences resulting in a mutant cell with a modified third nucleic acid sequence which may produce more of the polypeptide than the mutant cell without the modification of the third nucleic acid sequence when cultured under the same conditions. The third nucleic acid sequence may encode any protein or enzyme. For example, the enzyme may be an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence encoding the polypeptide may be expressed by inserting the sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.

In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.

The recombinant expression vector may be any vector (e. g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence encoding the polypeptide. The choice of the vector will typically depend on the compatibility of the vector with the fungal cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i. e., a vector which exists as an extrachromosomal entity, the replication of which is independent of <BR> <BR> <BR> <BR> chromosomal replication, e. g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the fungal cell, is integrated into the genome and replicated together with the chromosome (s) into which it has been integrated. The vector system may be a single

vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the fungal cell, or a transposon.

The vectors preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Suitable markers for yeast cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal cell <BR> <BR> <BR> <BR> include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin <BR> <BR> <BR> <BR> phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate<BR> <BR> <BR> <BR> <BR> <BR> <BR> decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well<BR> <BR> <BR> <BR> <BR> <BR> as equivalents thereof. Preferred for use in a filamentous fungal cell are the amdS and<BR> <BR> <BR> <BR> <BR> <BR> pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

The vectors preferably contain an element (s) that permits stable integration of the vector into a cell's genome or autonomous replication of the vector in the cell independent of the genome.

"Introduction"means introducing a vector comprising the nucleic acid sequence encoding a polypeptide of interest into a cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the chromosome occurs by homologous recombination, non-homologous recombination, or transposition.

The introduction of an expression vector into a fungal cell may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of <BR> <BR> <BR> <BR> Aspergillus cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming <BR> <BR> <BR> <BR> Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press,

Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

For integration into the genome of a cell, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the cell. The additional nucleic acid sequences enable the vector to be integrated into the genome at a precise location (s) in the chromosome (s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination.

The integrational elements may be any sequences that are homologous with the target sequence in the genome of the cell. Furthermore, the integrational elements may be non- encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the cell in question.

Examples of origins of replication for use in a yeast cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS I and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the fungal cell (see, e. g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).

It will be understood that the methods of the present invention are not limited to a particular order for obtaining the mutant fungal cell. The modification of a gene involved in the pacC pH signal transduction pathway may be introduced into the parent cell at any step in the construction of the cell for the production of a polypeptide. It is preferable that a gene in the pacC pH signal transduction pathway of the fungal mutant cell has already been modified using the methods of the present invention prior to the introduction of a gene encoding a polypeptide.

The procedures used to ligate the elements described herein to construct the

recombinant expression vectors are well known to one skilled in the art (see, e. g., J.

Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

In the methods of the present invention, the fungal cell may be a wild-type cell or a mutant thereof.

"Fungi"as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).

In a preferred embodiment, the fungal cell is a yeast cell."Yeast"as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

In a more preferred embodiment, the yeast cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

In a most preferred embodiment, the yeast cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast cell is a Yarrowia lipolytica cell.

In another preferred embodiment, the fungal cell is a filamentous fungal cell.

"Filamentous fungi"include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

In a more preferred embodiment, the filamentous fungal cell is an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma cell.

In a most preferred embodiment, the filamentous fungal cell is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae cell.

In another most preferred embodiment, the filamentous fungal cell is a Fusarium bactridioides, Fusarium crookwellense (synonym of Fusarium cerealis), Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium solani, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell.

The Fusarium venenatum cell is most preferably Fusarium venenatum A3/5, which was originally deposited as Fusarium graminearum ATCC 20334 and recently reclassified as Fusarium venenatum by Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57- 67; as well as taxonomic equivalents of Fusarium venenatum regardless of the species name by which they are currently known. In another preferred embodiment, the Fusarium venenatum cell is a morphological mutant of Fusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO 97/26330.

In another most preferred embodiment, the filamentous fungal cell is a Gibberella pulicaris, Gibberella zeae, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Myrothecium roridin, Neurospora crassa, or Penicillium purpurogenum cell.

In another most preferred embodiment, the filamentous fungal cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

In the methods of the present invention, the mutant fungal cell is cultivated in a nutrient medium suitable for production of a polypeptide of interest using methods

known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.

The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e. g., in catalogues of the American Type Culture Collection). The secreted polypeptide can be recovered directly from the medium.

The polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods may include use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or SDS-PAGE.

For example, an enzyme assay may be used to determine the activity of the polypeptide.

Procedures for determining enzyme activity are known in the art for many enzymes.

The resulting polypeptide may be isolated by methods known in the art. For example, the polypeptide may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray- drying, evaporation, or precipitation. The isolated polypeptide may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e. g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e. g., preparative isoelectric focusing), differential solubility (e. g., ammonium sulfate precipitation), or extraction (see, e. g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Examples Materials Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Media and Solutions PDA plates contained 39 g/1 Potato Dextrose Agar (Difco) and were supplemented with 10 mM uridine for pyrG auxotrophs.

MY25 medium at pH 6.5 was composed per liter of 25 g of maltose, 2.0 g of MgS04-7H20,10 g of KH2PO4,2.0 g of citric acid, 10 g of yeast extract, 2.0 g of K2SO4, 2.0 g of urea, and 0.5 ml of trace metals solution. MY25 shake flask medium was diluted 1: 100 or 1: 1000 with glass distilled water for use in microtiter growth experiments (MY25/100 or MY25/1000). Cultures were grown at 34°C.

2X MY Salts pH 6.5 solution was composed per liter of 4 g of MgSO4 7H2O, 4 g of K2SO4,20 g of KH2PO4,4 g of citric acid, 1 ml of trace metals, and 2 ml of CaCl2 2H2O (100 g/1 stock solution.

Minimal medium transformation plates were composed per liter of 6 g of NaN03, 0.52 g of KCI, 1.52 g of KH2PO4, 1 ml of trace metals solution, 1 g of glucose, 500 mg of MgSO4-7H2O, 342.3 g of sucrose and 20 g of Noble agar per liter (pH 6.5). Minimal medium transfer plates (pH 6.5) were composed per liter of 6 g of NaN03,0.52 g of KCI, 1.52 g of KH2PO4, 1 ml of trace elements, 10 g of glucose, 500 mg of MgSO4-7H2O, and 20 g Noble agar.

The trace metals solution (1000X) was composed per liter of 22 g of ZnS04-7H20,11 g of H3BO3,5 g of MnCl24H2O, 5 g of FeS04-7H20,1.6 g of COC'2-5H2011.6 g of (NH4) 6Mo7024, and 50 g of Na4EDTA.

COVE plates were composed per liter of 343.3 g of sucrose, 20 ml of COVE salts solution, 10 ml of 1 M acetamide, 10 ml of 3 M CsCl, and 25 g of Nobel agar. The COVE salts (50X) solution was comprised of 26 g of KCI, 26 g of MgSO4 7H2O, 76 g of KH2PO4, and 50 ml of COVE trace metals solution. COVE trace metals solution was composed of (per liter): 0.04 g of NaB407lOH2O, 0.040 g of CUS04-5H20,0.70 g of FeS04-H20,0.80 g of Na2Mo02-2H20, and 10 g of ZnSO4.

NZY plates were composed per liter of 5 g of NaCl, 2 g of MgSO4-7H2O, 5 g of yeast extract, 10 g of NZ amine, and 15 g of Bacto agar.

YEG medium was composed per liter of 5 g yeast extract and 20 g dextrose.

STC was composed of 1.2 M sorbitol-10 mM CaCl2-10 mM Tris pH 7.5.

SM buffer was composed per liter of 5.8 g of NaCl, 2 g of MgSO4 7H2O, 50 ml of 1 M Tris-HCl pH 7.5, and 5 ml of 2% gelatin.

Example 1: Aspergillus oryzae HowB430 transformation with pDSY82 Aspergillus oryzae HowB430 was constructed as described in WO 98/11203.

Protoplasts of Aspergillus oryzae HowB430 were prepared according to the following protocol. Aspergillus oryzae HowB430 was grown in 100 ml of 1% yeast extract-2% peptone-1% glucose at 32°C for 16-18 hours with agitation at 150 rpm. The mycelia were recovered by filtration through a 0.45 mm filter until approximately 10 ml remained on the filter, washed with 25 ml of 1.0-1.2 M MgSO4-10 mM sodium phosphate pH 6.5, filtered as before, washed again as before until 10 ml remained, and then resuspended in 10 ml of 5 mg/ml NOVOZYM 234TM (Novo Nordisk A/S, Bagsværd, Denmark) in 1.2 M MgSO4-10 mM sodium phosphate pH 6.5 (0.45 mm filtered) in a 125 ml Ehrlenmeyer flask. The suspension was incubated with gentle agitation at 50 rpm for approximately one hour at 37°C to generate protoplasts. A volume of 10 ml of the protoplast/mycelia preparation was added to a 30 ml Corex centrifuge tube, overlaid with 5 ml of 0.6 M sorbitol-10 mM Tris-HCl pH 7.5, and centrifuged at 3600 x g for 15 minutes in a swinging bucket rotor to recover the protoplasts. The protoplasts were recovered from the buffer interface with a Pasteur pipet. The protoplasts were then washed with five volumes of STC, centrifuged, and then rewashed and centrifuged as before. The protoplasts were resuspended in STC to a final concentration of 2 x 107 protoplasts per ml.

A 5-15 ul aliquot of pDSY82 (WO 98/11203) linearized with 15 U of BamHI was added to 0.1 ml of the protoplasts at a concentration of 2 x 107 protoplasts per ml in a 14 ml Falcon polypropylene tube followed by 250 il of 60% PEG 4000-10 mM CaCl2-10 mM Tris-HCl pH 7, gently mixed, and incubated at 37°C for 30 minutes. The suspension was mixed with 12 ml of molten overlay agar (1X COVE salts, 1% NZ amine, 0.8 M sucrose, 0.6% Noble agar) or 3 ml of STC and the suspension was poured onto a Minimal medium plate. The plates were incubated at 37°C for 3-5 days.

The transformation frequencies of the BamHI REMI pDSY82 transformations ranged from about 80 to 110 transformants per ig of DNA. A BamHI REMI library of -27,000 DNA-tagged transformants of Aspergillus oryzae HowB430 was obtained.

The Aspergillus oryzae HowB430 tagged mutant library pool was designated"b" for pDSY82 digested with BamHI with subsequent transformation in the presence of BamHI. The library was pooled into groups of-1000 transformants and stored in 10%

glycerol at-80°C.

Example 2: Lipase expression screening The Aspergillus oryzae HowB430 tagged mutant library"b"pools described in Example 1 were assayed for lipase expression.

For 96-well plate screens, MY25 medium was diluted 1000-fold using a diluent made of equal volumes of sterile water and 2X MY Salts pH 6.5 solution. For 24-well plate methods, MY25 medium was diluted 100-fold using a diluent made of equal volumes of sterile water and 2X MY Salts pH 6.5 solution.

Primary 96-well plate screens involved the dilution of spores from distinct pools into MY25/1000 so that one spore on average was inoculated per well when 50 ml of medium was dispensed into the wells. After inoculation, the 96-well plates were grown for 7 days at 34°C under static conditions. Cultures were then assayed for lipase activity as described below. Mutants of interest were inoculated directly into 24-well plates containing MY25/100 and were grown for 7 days at 34°C. Cultures were then assayed for lipase activity as described below. Mutants of interest were then plated on COVE plates to produce spores, spread on PDA plates to produce single colonies, and then 4 single colonies from each isolate were tested in the 24-well plate method described above.

The lipase assay substrate was prepared by diluting 1: 50 a p-nitrophenylbutyrate stock substrate (21 ml of p-nitrophenylbutyrate/ml DMSO) into MC buffer (4 mM CaCl2-100 mM MOPS pH 7.5) immediately before use. Standard lipase (LIPOLASETM, Novo Nordisk A/S, Bagsvaerd, Denmark) was prepared to contain 40 LU/ml of MC buffer containing 0.02% alpha olefin sulfate (AOS) detergent. The standard was stored at 4°C until use. Standard lipase was diluted 1/40 in MC buffer just before use.

Broth samples were diluted in MC buffer containing 0.02% AOS detergent and 20 ul aliquots were dispensed to wells in 96-well plates followed by 200 Vtl of diluted substrate. Using a plate reader, the absorbance at 405 nm was recorded as the difference of two readings taken at approximately 1 minute intervals. Lipase units/ml (LU/ml) were calculated relative to the lipase standard.

The results of the 96-well screen followed by the 24-well screen identified for further evaluation a DNA tagged mutant designated Aspergillus oryzae DEBY10.3

which produced higher levels of lipase than the control strain Aspergillus oryzae HowB430.

Example 3: Shake flask evaluation of Aspergillus oryzae DEBY10.3 Aspergillus oryzae DEBY10.3 described in Example 2 was then plated onto COVE plates to produce spores for shake flask evaluation.

Shake flask evaluations were performed by inoculating 300-500 ml of a spore suspension (0.02% Tween-80 plus spores from the COVE plates) into 25 ml of MY25 medium at pH 6.5 in a 125 ml shake flask. The shake flasks were incubated at 34°C for 3 days at 200 rpm. Samples were taken at day 2 and day 3 and lipase activity was measured as described in Example 2.

The results obtained are shown in Table 1 below where the lipase yield of Aspergillus oryzae HowB430 as a control was normalized to 1.0.

Table 1. Lipase Expression by DNA Tagged Mutants Strain Construction Pool # Screened 24-well Shake Flask Description in 96-well Plate Results <BR> <BR> Plates Results (LU/ml)<BR> <BR> <BR> <BR> <BR> (LU/ml) HowB430 HowB425 + pBANe8 NA NA 1.0 1.0 DEBY10.3 pDSY81 + BamHI bl 808 1.7 2.2 As shown in Table 1, Aspergillus oryzae DEBY10.3 produced approximately 2.2-fold more lipase than the control strain Aspergillus oryzae HowB430 when grown in shake flasks.

Example 4: Rescue of plasmid DNA and flanking DNA from Aspergillus oryzae DEBY10.3 The pDSY82 DNA and genomic flanking loci were isolated from Aspergillus oryzae DEBY10.3.

Genomic DNA was isolated from Aspergillus oryzae DEBY10.3 according to the following procedure. Spore stocks of each mutant were inoculated into 150 ml of YEG medium and were grown overnight at 37°C and 250 rpm. The mycelia were harvested from each culture by filtration through Miracloth (Calbiochem, La Jolla, CA) and rinsed twice with 10 mM Tris-0.1 mM EDTA pH 8 (TE). The mycelia preparations were then frozen quickly in liquid nitrogen and ground to a fine powder with a mortar and pestle.

The powdered mycelia preparations were each transferred to a 50 ml tube and 20 ml of lysis buffer was added. RNAse was added to each preparation to a final concentration of 20 tg/ml, and the preparations was incubated at 37°C for 30 minutes. Protease K was then added to each preparation to a final concentration of 0.1 mg/ml, and the preparations were incubated at 50°C for 1 hour. The preparations were then centrifuged at 15,000 x g for 20 minutes to pellet the insoluble material. Each supernatant was applied to a Qiagen MAXI column (Qiagen, Chatsworth, CA) which was equilibrated with QBT provided by the manufacturer. The columns were then washed with 30 ml of QC provided by the manufacturer. DNA was eluted from each column with 15 ml of QF provided by the manufacturer and then recovered by precipitation with a 0.7 volume of isopropanol and centrifugation at 15,000 x g for 20 minutes. The pellets were finally washed with 5 ml of 70% ethanol, air-dried, and dissolved in 200 ll of TE.

Two ug aliquots from the Aspergillus oryzae DEBY10.3 genomic DNA preparation were digested separately with BglSI HpaI, NarI, NdeI, SphI, and StuI. The restriction endonucleases did not cut pDSY82 which allowed the isolation of the integrated plasmid and the flanking genomic DNA. The digested genomic DNAs were then ligated in a 20 ul reaction with T4 DNA ligase.

The ligated DNA preparations were each transformed into E. coli HB101. The transformants were then screened by extracting plasmid DNA from the transformants, restriction digesting the inserts to confirm they were derived from pDSY82, and sequencing the inserts with an Applied Biosystems Model 373A Automated DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) on both strands using the primer walking technique with dye-terminator chemistry (Giesecke et al., 1992, Journal of Virol. Methods 38: 47-60) using the M13 reverse (-48) and M13 forward (-20) primers (New England Biolabs, Beverly, MA) and using primers specific to pDSY82.

Transformant E. coli HB101-pDSY109 contained the SphI rescued locus from Aspergillus oryzae DEBY10.3.

Example 5: Characterization of Aspergillus oryzae DEBY10.3 rescued locus pDSY109 The 3.4 and 2.2 kb regions on either side of the integration event of the Aspergillus oryzae DEBY10.3 rescued locus pDSY109 were sequenced with an Applied

Biosystems Model 373A Automated DNA Sequencer on both strands using the primer walking technique with dye-terminator chemistry using the M13 reverse (-48) and M13 forward (-20) primers and primers unique to the DNA being sequenced. The nucleic acid sequence suggested that the integration event occurred within the open reading frame of a palB gene. palB genes encode a cysteine protease involved in the pacC pH signal transduction pathway that signals ambient pH.

Genomic DNA of Aspergillus oryzae HowB430 was isolated using the protocol described in Example 4. A genomic library of Aspergillus oryzae HowB430 was constructed by first partially digesting Aspergillus oryzae HowB430 genomic DNA with Tsp509I. Four units of Tsp509 were used to digest 3.5 ug of Aspergillus oryzae HowB430 genomic DNA using conditions recommended by the manufacturer. The reaction was carried out at 65°C, and samples were taken at 5 minute intervals (from 0 to 50 minutes). The reactions were placed on ice and stopped by the addition of EDTA to 10 mM. These digests were then run on a 1% agarose gel with ethidium bromide, and the region of the gel containing DNA from 3 kb to 9 kb was excised. The DNA was then purified from the gel slice using Beta-Agarase I using the protocol provided by the manufacturer (New England Biolabs, Beverly, MA). The size-selected DNA was then ligated into Lambda ZipLox EcoRI arms according to the manufacturer's instructions (Life Technologies, Inc., Gaithersburg, MD) at 16°C overnight. The ligation reaction was packaged and titered using a Gigapack GoldIII Packaging Kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Totally, 8 X 106 recombinant plaques were obtained, and the library was amplified using a protocol provided by the manufacturer.

The genomic library was screened to obtain a genomic clone of palB.

Appropriate dilutions of the genomic library were made to obtain 7000 plaques per 150 mm petri plate as described in the protocols provided with the Lambda ZipLox arms.

The plaques were lifted to Hybond N+ filters (Amersham, Cleveland, OH) using standard protocols (Sambrook et al., 1989, supra). The filters were fixed using UV crosslinking, and prehybridized at 42°C in DIG Easy Hyb. The filters were hybridized with a DIG- labeled 0.25 kb palB probe. The probe was PCR amplified with the following primers synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions and labeled with dioxygenin using a Genius Kit

(Boehringer Mannheim, Indianapolis, IN): 5'-CTGCCGTCGAAGGTGTCCAAG-3' (SEQ ID NO: 5) 5'-ATTGTGGCCCCTATGTGGATT-3' (SEQ ID NO: 6) The amplification reactions (100 p1) were prepared using approximately 0.2 zig of pDSY109 as the template. Each reaction contained the following components: 0.2 u. g of plasmid DNA, 48.4 pmol of the forward primer, 48.4 pmol of the reverse primer, 1 mM each of dATP, dCTP, dGTP, and dTTP, 1 x Taq polymerase buffer, and 2.5 U of Taq polymerase (Perkin-Elmer Corp., Branchburg, NJ). The reactions were incubated in an Ericomp Twin Block System Easy Cycler programmed for 1 cycle at 95°C for 5 minutes followed by 30 cycles each at 95°C for 1 minute, 55°C for 1 minute and 72°C for 2 minutes.

The filters were washed and processed post-hybridization using protocols provided with the Genius Kit. Several positive plaques were identified and purified to homogeneity using standard protocols (Sambrook et al., 1989, supra).

The nucleotide sequence was determined for one of the palB clones according to the method described in Example 1. The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) are shown in Figure 1. The palB gene encoded an open reading frame of 4700 bp encoding a polypeptide of 854 amino acids.

The open reading frame was interrupted by 3 introns.

A comparative alignment of PalB amino acid sequences was undertaken using the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENETM MEGALIGNTM software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10, and gap length penalty of 10. Pairwise alignment parameters were Ktuple=l, gap penalty=3, windows=5, and diagonals=5.

The comparative alignment showed that the Aspergillus oryzae PalB protein (SEQ ID NO: 2) shared 66.4% identity with the Aspergillus nidulans PalB protein (Denison et al., 1995, supra).

A Southern blot of Aspergillus oryzae DEBY10.3 and Aspergillus oryzae HowB101 genomic DNA digested with Bgll9I was prepared as described above. The blot was probed with the DIG-labeled 0.25 kb palB probe to confirm that the rescued flanking DNA was the gene disrupted inAspergillus oryzae DEBY10.3.

A BglII band of-7.5 kb from Aspergillus oryzae HowB101 hybridized to the probe while a band of 12 kb from Aspergillus oryzae DEBY10.3 hybridized to the probe.

The size difference was the expected size for one plasmid copy being integrated confirming the locus rescued was disrupted inAspergillus oryzae DEBY10.3.

Because the integration event in Aspergillus oryzae DEBY10.3 would be <BR> <BR> <BR> <BR> predicted to lead to a nonfunctional PalB protein, Aspergillus oryzae DEBY10.3 was<BR> <BR> <BR> <BR> <BR> <BR> tested for growth at pH 8.0 and pH 6.5. Aspergillus nidulans palB minus strains are<BR> <BR> <BR> <BR> <BR> <BR> unable to grow at pH 8.0 but are able to grow at pH 6.5. Aspergillus oryzae HowB430 and Aspergillus oryzae DEBY10. 3 were grown in Minimal medium supplemented with 10 mM uridine at either pH 8.0 or pH 6.5. As predicted, Aspergillus oryzae DEBY10.3 was unable to grow at pH 8.0.

Example 6: Construction of pMT1936 pMT1936 was constructed to contain a disruption cassette of palB using the following primers synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions.

100752: 5'-GGTTGCATGCTCTAGACTTCGTCACCTTATTAGCCC-3' (SEQ ID NO: 7) 100753: 5'-TTCGCGCGCATCAGTCTCGAGATCGTGTGTCGCGAGTACG-3' (SEQ ID NO: 8) 100754: 5'-GATCTCGAGACTAGTGCGCGCGAACAGACATCACAGGAACC-3' (SEQ ID NO: 9) 100755: 5'-CAACATATGCGGCCGCGAATTCACTTCATTCCCACTGCGTGG-3' (SEQ ID NO: 10) <BR> <BR> <BR> <BR> The Aspergillus oryzae palB 5'flanking sequence and the sequence encoding the<BR> <BR> <BR> <BR> <BR> <BR> N-terminal part of the palB product were PCR amplified from genomic DNA of<BR> <BR> <BR> <BR> <BR> <BR> Aspergillus oryzae A1560 (Christensen et al., 1988, BiolTechnology 6: 1419-1422) prepared according to the protocol described in Example 4. Approximately 0.05 llg of genomic DNA and 5 pmole of each of the two primers 100754 and 100755 were used.

Amplification was performed with the polymerase Pwo as described by the manufacturer (Boehringer Mannheim, Indianapolis, IN). Amplification proceeded through 40 cycles.

Part of the reaction product was phenol extracted, ethanol precipitated, digested with

EcoRI and XhoI and a fragment of approximately 1.05 kb was isolated by agarose gel electrophoresis.

The Aspergillus oryzae palB 3'flanking sequence and the sequence encoding the C-terminal part of the palB gene product were obtained as described above except that primers 100752 and 100753 were used for amplification and the PCR product was digested with XhoI and XbaI before gel electrophoresis to recover a fragment of approximately 1.5 kb.

The two digested and purified PCR fragments described above were ligated in a three part ligation with the purified 2.7 kb EcoRI-XbaI fragment from the vector pJaL400 (Figure 2) to produce pMT1935 (Figure 3). The palB 5'and 3'flanks of pMT1935 were separated by BssHII, SpeI, and Xhol sites introduced via PCR primers 100754 and 100753.

The 3.5 kb Hindlll fragment of pJaL394 (Figure 4) containing the repeat flanked pyrG gene was cloned into HindIII cut, dephosphorylated, and purified pBluescript II SK (-). Plasmids with inserts in either orientation were obtained. One plasmid, pMT1931 (Figure 5), was selected in which the SpeI site of the pBluescript polylinker was downstream of the pyrG gene and the Xhol site was upstream of the pyrG gene. The pyrG gene was isolated as a 3.5 kb SpeI-XhoI fragment and inserted in SpeI and XhoI digested and purified pMT1935 to produce the disruption plasmid pMT1936 (Figure 6).

ThepyrG selectablepalB disruption cassette can be isolated from pMT1936 as a 6.2 kb NotI fragment (NotI cutting in polylinkers) or as a 5.5 kb AseI-PvuI fragment (AseI and PvuI cutting within the actual palB 5'and 3'flanking sequences).

Example 7: Aspergillus oryzae transformation with AseI/PvuI palB disruption cassette from pMT1936 Aspergillus oryzae HowB430 was transformed using the same transformation procedure described in Example 1 with a 5.5 kb AseIlPvuI fragment obtained from pMT1936. The linear fragment for transformation was isolated by digestion of pMT1936 with AseI and PvuI and separation of the fragment on a 1 % agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. The transformants were then tested for growth on Minimal medium plates at pH 6.5 or pH 8.0.

The results showed that 13 of the 128 transformants tested possessed the palB minus phenotype as indicated by the inability to grow at pH 8.0. The 13 palB minus strains and 13 of the transformants that were able to grow at pH 8.0 were spore purified.

Southern blots of the genomic DNA from an Aspergillus oryzae palB minus mutant, an Aspergillus oryzae palB plus strain, and Aspergillus oryzae HowB430 were performed to determine if the AsnIlPvuI transforming DNA fragment had integrated as a clean replacement into the palB locus. The genomic DNAs were prepared according to the procedure described in Example 4, digested with PvuI, and electrophoresed on a 0.8% agarose gel. The DNAs were transferred to a Hybond N+ filter using 0.4 N NaOH and capillary action. The blot was UV crosslinked prior to prehybridization at 65°C in Rapid Hyb. The blot was then probed with a 0.9 kb AsnIlSpeI fragment from pMT1936.

The 0.9 kb fragment was isolated from an agarose gel slice using QiaQuick spin column after electrophoreses on a 1% agarose gel. The fragment was labeled using Vistra ECF Random Prime Labeling Kit. The blots were prehybridized and hybridized at 65°C in Rapid Hyb (Amersham, Cleveland, OH), and then washed twice for 5 minutes in 2X SSC, 0.1% SDS at 65°C and twice for 10 minutes in 0.2X SSC, 0.1% SDS at 65°C.

Following the washes, the blot was processed for detection using the Vistra ECF Signal Amplification Kit (Amersham, Cleveland, OH) and the STORM860 Imaging System (Molecular Dynamics, Sunnyvale, CA).

The Southern blot results demonstrated that the probe hybridized to a band of 6 kb from Aspergillus oryzae HowB430. A clean disruption would be expected to hybridize to about an 8 kb PvuI band. The Southern blot results further showed that some of the palB minus strains had clean disruptions while others did not. <BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>Example 8: Extracellular protease production of Aspergillus oryzae apalB strains Three Aspergillus oryzae palB minus and three Aspergillus oryzae palB plus strains described in Example 7 were grown at 34°C, pH 7,1000-2000 rpm for 8 days in 2 liter fermentors containing medium composed of Nutriose, yeast extract, (NH4) 2HPO4, MgSO47H2O, citric acid, K2SO4, CaCl2H2O, and trace metals solution. Samples of the extracellular medium were taken every day, and the day 6 samples were assayed for total extracellular protease activity using the FTC-casein assay described below.

The FTC-Casein assay was conducted as follows. The reaction was initiated by

the addition of 40 ul of FTC-casein (Twining, 1984, Analytical Biochemistry 143: 30-34) mixed 1: 1 with 0.1 M MOPS pH 7.0 buffer to 10 p1 of enzyme solution diluted in 0.1 M MOPS pH 7.0 buffer as appropriate. The reaction was incubated at 37°C for 2 hours followed by quenching of the reaction with 150 ul of 5% trichloroacetic acid. The quenched reaction was placed at 5°C for 2 hours and then centrifuged for 10 minutes. A 20 pu ouf the supernatant was transferred into a test tube containing 2 ml of 0.5 M borate pH 9.0 buffer and mixed. A 200 RI sample of this solution was then transferred into a black"U"bottom 96 well plate (Dynatech, Inc., Chantiily, VA). The borate buffer was used as a blank to zero the instrument. The fluorescence was measured using a Fluorolite 1000 instrument (Dynatech, Inc., Chantiily, VA) using channel 3 at a reference setting of 1176 and a lamp voltage at 4.1 V. The instrument's dynamic range was between 0 and 4000 fluorescence units with best well-to-well reproducibility between 400 and 3500 units.

The results shown in Table 2 indicated that the Aspergillus oryzae palB minus strains produced on average 10-fold less extracellular protease as compared to the Aspergillus oryzae palB plus strains. The assays were also performed in the presence of either the serine protease inhibitor PMSF or the metalloprotease inhibitor 1,10- phenanthroline. The percent of the total protease activity that was inhibited by the indicated inhibitor is shown in Table 2. The inhibition patterns for the Aspergillus oryzae palB plus and palB minus samples were similar suggesting that the palB deletion affects the production of both serine and metalloproteases.

Table 2. Extracellular Protease Levels in palB Minus and Wildtype Strains Strain Protease % Inhibition % Inhibition by 1,10- Fluorescent by PMSF* * phenanthroline* * Units+ DEBY10.3 palB minus 1120 73 4 DEBY10.3 palB minus 1370 77 15 DEBY10.3 palB minus 810 69 3 DEBY10.3 palB plus 11100 87 37 DEBY10.3 palB plus 9785 83 34 DEBY10.3 palB plus 13050 84 37 + 2 hour incubation at 37°C at pH 7. ** Inhibitor concentrations were 1 mM.

Three additional Aspergillus oryzae palB minus and three additional palB plus strains were also run in 2 liter fermentors for 8 days as described above, and extracellular samples were taken daily. The day 6 samples were assayed using the FTC-Casein assay described above in both the presence and absence of the inhibitors.

The results as shown below in Table 3 indicated that the Aspergillus oryzae palB minus strains produced on average 10-fold less total protease activity as compared to the Aspergillus oryzae palB plus strains.

Table 3. Extracellular Protease Levels in Aspergillus oryzae palB Minus and Wildtype Strains Strain Protease % Inhibition % Inhibition by Fluorescent by PMSF** 1, 10-phenanthroline** Units+ DEBY10.3 palB minus 485 77 35 DEBY10.3 palB minus 345 48 0 DEBY10.3 palB minus 540 52 0 DEBY10.3 palB plus 4580 23 HowB430 palB plus 5025 86 39 HowB430 palB plus 3765 77 37 + 1 hour incubation at 37°C at pH 7.

** Inhibitor concentrations were 1 mM.

Two Aspergillus oryzae palB minus and one Aspergillus oryzae palB plus strain were also grown in 2 liter fermentors for 8 days as described above. The Aspergillus oryzae palB minus strains were clean disruptions of the palB gene. Extracellular samples were taken daily for 8 days. The day 6 samples were assayed using the FTC- Casein assay described above in both the presence and absence of the inhibitors. The results are shown below in Table 4. The Aspergillus oryzae palB minus strains produced on average 20-fold less extracellular protease than the Aspergillus oryzae palB plus strain. Table 4. Extracellular Protease Levels in Aspergillus oryzae palB Minus and Wildtype Strains Strain Protease % Inhibition by % Inhibition by Fluorescent Units+ PMSF** 1,10- phenanthroline** DEBY10.3 palB minus 1095 35 0 HowB430 ApalB 76-1-1 715 44 3. 5 HowB430 ApalB 76-1-1 16360 91 36 pLRF2-10 (palB plus) _

+ 2 hour incubation at 37e (: at pH/.

* * Inhibitor concentrations were 1 mM Example 9: PCR amplification of Aspergillus gene The palA gene from Aspergillus nidulans was amplified from genomic DNA prepared as described in Example 4 using PCR and the following primers: paIA2540R: 5'-TCGCGCAGTCGTGATTCAAAG-3' (SEQ ID NO: 11) palA172: 5'-CCGCACTGGAGTAAATAACAT-3' (SEQ ID N0: 12) The reaction contained 50 ng of Aspergillus nidulans genomic DNA, 50 pmole each of paIA254OR and palA172, Perkin Elmer PCR Buffer, 1 mM dNTPs, and 0.5 U Taq DNA polymerase. The reactions were cycled in an Ericomp Twin Block System Easy Cycler programmed for 1 cycle at 95°C for 3 minutes; 30 cycles each at 95°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute; and 1 cycle at 72°C for 5 minutes. An aliquot of the reaction was electrophoresed on an agarose gel, and the expected product of-2.4 kb was obtained. Half of the reaction was run on a preparative gel, a gel slice containing the desired product was excised, and DNA was isolated from the gel using a Qiaquick Gel Extraction Kit.

One-tenth of the gel-isolated PCR product was labeled with 32P-dCTP using a Prime-It Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The palA probe was purified following the labeling reaction on a TE Midi G-50 column (5' to 3', Boulder, CO).

Example 10: Southern analysis of Aspergillus oryzae genomic DNA with Aspergillus

nidulans palA probe Aspergillus oryzae HowB430 genomic DNA prepared as described in Example 4 <BR> <BR> <BR> <BR> was digested with BamHI, EcoRI or HindlII and electrophoresed on an agarose gel. The DNA was transferred to Hybond N'filters under alkaline conditions as described in Example 7. Identical thirds of the blot were prehybridized for 1 hour in low, medium and high stringency hybridization buffer at 42°C (prehybridization and hybridization in 5X SSPE, 0.3% SDS, 200 Zg/ml sheared and denatured salmon sperm DNA, and either 25,35 or 50% formamide for low, medium, and high and very high stringencies, respectively). The P32-CTP labeled palA probe described in Example 10 was added and the blots were hybridized overnight at 42°C. The blots were washed at 42°C for 5 minutes in 2X SSC, 0.1% SDS twice and for 10 minutes in 0.2X SSC, 0.1% SDS twice.

Specific bands were observed on the blots that hybridized in medium and low stringency buffers. <BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>Example 11: Isolation of a palA Aspergilllus oryzae genomic clone<BR> <BR> <BR> <BR> <BR> <BR> An Aspergillus oryzae HowB425 genomic library in XZipLox was constructed according to the manufacturer's instructions. The library contained 630,000 plaque forming units with 73% containing inserts. The average insert size was 3.5 kb.

Approximately 7000 recombinant phage were plated with E. coli Y1090 (Life Technologies, Gaithersburg, MD) on large NZY plates. The plaques were lifted to Hybond N+ filters using standard protocols. The filters were UV crosslinked and prehybridized at 42°C for 1 hour in medium stringency hybridization buffer as described <BR> <BR> <BR> <BR> in Example 10. The 32P-labeled Aspergillus nidulans palA probe was added and the filters were hybridized overnight at 42°C. The filters were washed as described above and exposed to X-ray film.

Two positive clones were obtained. The positive plaques were picked and eluted in 1 ml of SM buffer. The eluate was diluted 104, and 50 or 100 ul aliquots were plated with E. coli Y1090 cells on large NZY plates. The plaques were lifted and the filters were processed as before. One purified positive plaque was isolated from each plate. <BR> <BR> <BR> <BR> <P> Plasmid DNA was excised from the positive clones by plating with E. coli DH1OB cells following the protocol provided (Life Technologies, Inc., Gaithersburg, MD).

Example 12: Analysis of palA Aspergillus oryzae genomic clones Plasmid DNA was isolated from the two positive clones (palA5A and palA6A) described in Example 11 using Qiaquick Prep8 kit (Qiagen, Chatsworth, CA). The plasmid DNAs were digested with PstI to confirm they contained inserts. Partial nucleotide sequencing of the genomic clones was performed using an Applied Biosystems Model 377 XL Automated DNA Sequencer M13 with forward and reverse primers. The partial sequencing confirmed that the clones contained an Aspergillus oryzae palA homolog. The nucleotide sequence of the largest clone palA5A was determined. DNA sequencing was done with an Applied Biosystems Model 377 XL Automated DNA Sequencer using dye-terminator chemistry. Contig sequences were generated using a transposon insertion strategy (Primer Island Transposition Kit, Perkin- Elmer/Applied Biosystems, Inc., Foster City, CA). The 2.9 kb insert of palA5A was sequenced to an average redundancy of 5.3.

The nucleotide sequence of the palA5A clone (SEQ ID NO: 3) demonstrated that a partial clone of palA was isolated, missing the DNA coding for approximately the last 245 amino acids of palA based on homology to the Aspergillus nidulans palB gene (Negrete-Urtasun et al., 1997, supra). The partial nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO: 4) are shown in Figure 7. The palA gene encoded an open reading frame of 2855 bp encoding a polypeptide of 549 amino acids.

The open reading frame was interrupted by 4 introns, whose positions are strictly conserved between the Aspergillus nidulans and Aspergillus oryzae clones.

A comparative alignment of the partial Aspergillus oryzae PalA deduced amino acid sequence to other PalA amino acid sequences was undertaken using the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENETM MEGALIGNTM software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10, and gap length penalty of 10.

Pairwise alignment parameters were Ktuple=l, gap penalty=3, windows=5, and diagonals=5.

The comparative alignment showed that the partial Aspergillus oryzae PalA protein (SEQ ID NO: 2) shared 88% identity with the Aspergillus nidulans PalA protein (Negrete-Urtasun et al., 1997, supra).

Example 13: Deletion of palA in an Aspergillus oryzae strain Plasmid pBM8 was constructed in which 600 bp of the palA open reading frame were replaced with the pyrG gene.

The following synthetic oligonucleotide primers designed to PCR amplify two separate palA fragments were synthesized with an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions. The first set has a 5'primer designed to add a XhoI site and a 3'primer to add a HindIII site.

Set 1: Primer: 980163: 5'-AGTAGCCGTCTATCTCTCCAGCAGTAGTGT-3' (SEQ ID NO: 13) Primer 980164: 5'-AAGCTTACTCGCAATTAGCCTTCTCGGTGAATCG-3' (SEQ ID NO: 14) The second set has a 5'primer designed to add a HindIII site and a 3'primer to add a NotI site.

Set 2: Primer: 980165: 5'-AAGCTTTACGATGTCATGCTGGGCAACGGTCAAG-3' (SEQ ID NO: 15) Primer: 980166: 5'-GCGGCCGCCTCTGCGTGATACTCTAGGGTCTGG-3' (SEQ ID NO: 16) Bold letters represent coding sequence.

PCR reactions were set up in 100 pl volumes containing 50 ng of palA5A DNA template, 50 pmoles of each primer, 1X PCR buffer with 2mM MgS04 (Boehringer Mannheim), 1 mM dNTPs, and 2.5 units Taq DNA polymerase (Boehringer Mannheim).

The reactions were cycled in an Ericomp Twin Block System Easy Cycler programmed for 1 cycle at 95°C for 5 minutes; 30 cycles each at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1.5 minutes; and 1 cycle at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 3 minutes.

A 10 J. l volume of the PCR reaction was electrophoresed for 1 hour at 100 volts on a 1.0% agarose gel. The major products at-1100 bp from primer set 1 and at-730 bp from primer set 2 were excised from the gel and purified using a Qiaquick Gel Extraction Kit. The purified PCR product was subsequently cloned into plasmid pCR2.1-TOPO (Invitrogen, San Diego, CA) and transformed into TOP10 cells

(Invitrogen, San Diego, CA) according to the manufacturer's instructions.

Two clones were obtained designated E. coli pPalAl for the 1100 bp insert and E. coli pPalA2 for the 730 bp insert. The two clones were analyzed by DNA sequencing using a Perkin-Elmer Applied Biosystems Model 377 Sequencer XL with dye-terminator chemistry and the lac-forward and lac-reverse sequencing primers. pBluescript KS-was digested with HindIII and treated with Klenow fragment to create blunt ends. The DNA was electrophoresed on a preparative gel, and the band containing the cut plasmid DNA was excised. The DNA was isolated from the gel using a Qiaquick Gel Extraction Kit. The plasmid was then ligated and transformed into E. coli DH5a, and plasmid DNA was isolated from several of the colonies. The clones were screened for the absence of the HindIII site by restriction digestion with HindIII. A clone missing the HindlII site was designated pBMla (Figure 8). Plasmid DNA from pBMla was prepared using the Qiaquick Prep8 protocol (Qiagen, Chatsworth, CA). pBMla was digested with 0.5 jLil ofol and NotI (10 U each per 1). pPalA1 was digested with 0.5 pl of XhoI and HindlII (10 U each per ul). pPalA2 was digested with 0.5 u. l of NotI and HindlII (10 U per u. l). The digested DNAs were electrophoresed for 1 hour at 100 volts on a 1% agarose gel. The 1100 bp fragment for pPalAl, the 730 bp fragment for pPalA2, and the 2.9 kb fragment for pBM I a were excised from the gel and re-suspended in 200 pl of 10 mM Tris pH 7.5. The 3 DNA fragments were subsequently ligated together and transformed into E. coli DHSa competent cells. The resulting plasmid was designated pBM7 (Figure 9).

DNA from plasmid pJal394 was digested with 1 pLl of HindlII (10 U per ul). The digested DNA was electrophoresed for 1 hour at 100 volts on a 0.7% agarose gel yielding a band of 3539 bp. The 3539 bp fragment was excised from the gel and resuspended in 200 ul of 10 mM Tris pH 7.5.

DNA from plasmid pBM7 was digested with 1 ul of HindIII (10 U per til).

HindIII was heat-inactivated at 65°C for 10 minutes followed by dephosphorylation using shrimp alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN) according to manufacturer's suggestions. The digested DNA was electrophoresed for 1 hour at 100 volts on a 0.7% agarose gel yielding a 3630 bp band. The 3630 bp fragment was excised from the gel and re-suspended in 300 u. l of 10 mM Tris pH7.5. The two DNA fragments were ligated and transformed into E. coli DH5a competent cells. The resulting clone

was designated pBM8 (Figure 10).

To isolate the deletion fragment of pBM8 the plasmid was digested with NotI and Xhol and the digest was electrophoresed on a preparative gel. The 5.3 kb deletion fragment was isolated from the gel using a Qiaquick Gel Extraction Kit. Aspergillus oryzae HowB430 protoplasts prepared as described in Example 1 were transformed with the fragment selecting on minimal medium plates. A total of 93 transformants were obtained. The transformants were tested for the pal minus phenotype on pH 8.0 minimal medium plates. Three of the 93 were unable to grow at pH 8.0. Southern analysis confirme the three strains were clean disruptions.

Example 14: Extracellular protease production of the Aspergillus oryzae 4paL4 strains The two Aspergillus oryzae palA plus and two Aspergillus oryzae palA minus strains described in Example 13 were grown for 8 days at 34°C in 2 liter fermentors as described in Example 8. The total extracellular protease activity of the day 6 samples was determined using the FTC-Casein assay described in Example 8.

The results shown below in Table 5 demonstrated that the extracellular protease levels of the Aspergillus oryzae palA minus strains were about one-tenth of the levels produced by the Aspergillus oryzae palA plus strains.

Table 5. HowB430 4paL4 Strains & Extracellular Proteases Strains Protease Fluorescent Units DEBY599.3 (wt) 7450 palA1-3 710 palA 6-1 (wt) 5150 Avala 7-1 565 Deposit of Biological Materials The following biological materials have been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL), Peoria, Illinois, and given the following accession numbers:

Deposit Accession Number Date of Deposit <BR> <BR> <BR> <BR> E. coli HB101 pDSY109 NRRL B-21623 September 5,1996<BR> <BR> <BR> <BR> <BR> <BR> E. coli XL-lBlue pDSY174 NRRL B-30089 January 28,1999 The strains have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C. F. R. §1.14 and 35 U. S. C. §122. The deposits represent substantially pure cultures of the deposited strains. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed.

However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.