WO2008053019A2

WO2008053019A2 - Method for reducing the expression of a gene in a filamentous fungal cell

Info

Publication number: WO2008053019A2
Application number: PCT/EP2007/061768
Authority: WO
Inventors: VAN Noël Nicolaas Maria Elisabeth PEIJ; Lucie Parenicova; Mojca Bencina; Karolina Ivicak
Original assignee: Dsm Ip Assets B.V.
Priority date: 2006-11-02
Filing date: 2007-10-31
Publication date: 2008-05-08
Also published as: WO2008053019A3

Abstract

The present invention relates to a method for reducing the expression of a gene in a filamentous fungal cell. The invention further relates to a modified filamentous fungal cell suitable for the production of a compound of interest, wherein, compared to the parental cell the modified cell derives from, said modified cell exhibits improved abilities for RNAi mediated reduction of gene expression of a target gene. In addition, the invention relates to a method for the production of a compound of interest in said modified filamentous fungal cell.

Description

METHOD FOR REDUCING THE EXPRESSION OF A GENE IN A FILAMENTOUS

FUNGAL CELL

Field of the invention

The present invention relates to reducing expression of a target gene in a filamentous fungal cell.

Background of the invention

Filamentous fungi are widely used for the production on industrial scale of compounds like enzymes and metabolites. Over years, the filamentous fungal strains used have continuously been optimised for improving the yield of the compound produced. Optimisation of the yield can be performed by classical strain improvement or recombinant DNA techniques. Optimisation is an ongoing process wherein one will try to achieve the highest yield possible of the compound(s) produced. However, in addition to the production of the compound of interest, the host cell may produce other compounds that interfere by e.g. degradation of the compound of interest or co- purification with the compound of interest. Instead of acting downstream of the production process by improving purification and/or recovery, it is advantageous to decrease the production of an undesired compound. Lowering production of an undesired compound may be performed by decreasing expression of a target gene associated with the production of such undesired compound. Several methods are known in the art for modification or inactivation of a target gene. Modification or inactivation may be performed by e.g. specific or random mutagenesis, site-directed mutagenesis, PCR generated mutagenesis, nucleotide insertion and/or deletion and/or substitution, gene interruption or gene replacement techniques, catalytic RNA molecules (ribozymes), catalytic DNA molecules (DNAzymes) and antisense vectors. However, inactivation or modification by the mentioned techniques may be difficult to achieve due to lack of specificity or poor targeting to the target gene. When multiple target genes are to be modified or inactivated, the process may be very laborious. Recently, a gene silencing technique was described in eukaryotes and more recently in filamentous fungi. The technique, called RNA interference (RNAi), is a process wherein double stranded RNA (dsRNA) silences gene expression, either post-transcriptionally by activation of a sequence-specific RNA degradation process or by inhibiting translation.

WO2005/056772 describes the principle of RNAi for reducing the expression of one or more genes in a filamentous fungal cell.

It would be an advantage in the art to have alternative RNAi methods and/or improved RNAi methods for reducing the expression of one or more target genes for strain development or strain improvement, functional genomics and pathway engineering of filamentous fungal cells, wherein the efficiency of the reduction of expression of one or more genes does not entirely rely on the native expression levels of the host cell RNAi components.

It is the object of the present invention to provide alternative RNAi methods and/or improved RNAi methods for reducing the expression of one or more target genes in a filamentous fungal cell, wherein the efficiency of the reduction of expression of one or more genes does not entirely rely on the native expression levels of the host cell RNAi components.

Description of the Figures

Figure 1. Plasmid map of expression vector pMOJ004.

Depicted is the plasmid map of the pMOJ004 expression vector containing the A. nidulans gpdA promoter (gpdAp) and the trpC terminator (trpCt).

Figure 2. Plasmid map of expression vector pMOJ004-lucA.

Depicted is the plasmid map of the pMOJ004-lucA expression vector containing the gpdA promoter (gpdAp), the renilla luciferase gene (rluc) and the trpC terminator (trpCt).

Figure 3. Plasmid map of expression vector pMOJ009.

Depicted is the plasmid map of the pMOJ009 expression vector containing the xlnD promoter (xlnDp) and the trpC terminator (trpCt). Figure 4. Plasmid map of expression vector pMOJ009-siR.

Depicted is the plasmid map of the pMOJ009-siR expression vector containing the xlnD promoter (xlnDp), the siRNA multiple cloning site (siRNA MCS) and the trpC terminator (trpCp).

Figure 5. Schematic representation of shRNAi molecules.

A. shRNAi molecule containing a single gene target.

B. shRNAi molecule containing a double gene target. C. shRNAi molecule containing a triple gene target directly linked to each other.

D. shRNAi molecule containing a triple gene target separated by a small loop. S (1-3) - sense oligonucleotide, AS (1-3) - antisense oligonucleotide, L (1-3) - small loop.

Figure 6. Plasmid map of expression vector pGWE5B.

Depicted is the plasmid map of pGWE5B, which is a plasmid containing a pBlueScript KS vector with insertion of Xhol blunt end orotidine-5-phosphate decarboxylase fragment (pyrA- sequence X96734) inserted into an EcoRV restriction site of pBlueScript.

Figure 7: Ratio of the renilla luciferase activities

Ratio of the renilla luciferase activities grown on xylose versus glucose in strains transformed with pMOJ012-siRNA-X (see Table 4 for strain names). Renilla luciferase activity is per gram of total protein of the strain indicated, grown on 1 % xylose or 1 % glucose media for 24 hours.

Figure 8. Plasmid map of expression vector pGBTOPGLA-1.

Depicted is pGBTOPGLA-1 , which is an integrative expression vector containing a promoter in operative association with a coding sequence of a gene of interest. A gene of interest can be cloned for example into the Pad, Asc\, Hind\\\ cloning sites. Prior to transformation of the A. niger strains, the E. coli vector DNA can be removed from this expression vector by digestion with restriction enzyme Not\. The depicted vector is representative of pGBTOP-based expression vectors. Figure 9. Plasmid map of expression vector pGBFINDCR-1 , a Dicer overexpression construct.

Depicted is an example of a pGBFIN-based expression vector, such as pGBFIN-23, with the dcrA gene cloned. Indicated are the g/aA flanking regions relative to the glaA promoter and Hinύ\\\-Xho\ cloning sites for the dcrA gene encoding Dicer. The E. coli DNA can be removed by digestion with restriction enzyme Not\, prior to transformation of the A. niger strains.

Figure 10: Ratio of the renilla luciferase activities. Ratio of the renilla luciferase activities grown on xylose versus glucose in strains transformed with pGBFINDCR-1 and pGBFINRDR-1 (see Example 7 for constructs). Renilla luciferase activity is per gram of total protein of the strain indicated, grown on 1 % xylose or 1 % glucose media for 24 hours.

Detailed description of the invention

In a first aspect of the present invention, there is provided a modified filamentous fungal cell suitable for the production of a compound of interest, wherein, compared to the parental cell the modified cell is derived from, said modified cell exhibits improved abilities for RNAi mediated reduction of gene expression of a target gene and/or homologues thereof, said target gene encoding a biological compound.

In the RNAi methods known from the prior art as reflected by WO2005056772, the components of the RNAi machinery of the host cell remains unaltered; i.e. the efficiency of reducing the expression of one or more genes relies entirely on the native expression levels of the host cell RNAi components, encoded by RNAi related genes.

In the context of the invention, "RNAi related genes" are encoding polypeptides associated with the process of RNAi mediated reduction of gene expression. These RNAi polypeptides, also depicted as "RNAi components" include, but are not limited to: DICER (dsRNA processing enzyme), RdRP (RNA dependent RNA polymerase), polypeptides of the RISC (RNA induced silencing complex) - nuclease complex containing translation initiation factor, RNA-DNA helicase, specific nuclease, RNA binding proteins. Preferred polypeptides are selected from the group consisting of SEQ ID NO: (3 + 3n), or SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or homologue thereof. More preferred polypeptides are the dcrA protein (SEQ ID NO: 12) and the qdeA protein (SEQ ID NO: 30), or a homologue thereof. The modified filamentous fungal cell according to the present invention provides improved opportunities for strain development and improvement, functional genomics, and pathway engineering in filamentous fungal cells. For example, the present modified filamentous fungal cell can be used for efficient reduction of expression of a highly expressed target gene encoding an undesired compound, which is important for the development of the filamentous fungal cell as a production host for a compound of interest. The present invention is particularly is useful when a gene is resistant to inactivation by standard methods known in the art such as gene knockout and provides a solution to reduce the expression of such a gene. The present invention is also useful in reduction of the expression of multiple genes that are highly homologous to each other. Furthermore, the present invention is particularly useful if one envisages achieving a variable reduction of the expression of a biological compound. This variability is especially important where a complete knockout of a gene encoding a biological compound would be lethal to a particular filamentous fungal strain.

The term "modified" is defined herein as any genetic modification of the filamentous fungal cell to result in improved abilities for RNAi mediated reduction of gene expression.

The term "RNAi mediated reduction of gene expression" is defined herein as a process wherein double stranded RNA (dsRNA) reduces gene expression, either post- transcriptionally by activation of a sequence-specific RNA degradation process or by inhibiting translation. The term "gene" is defined herein as a DNA sequence encoding a biological compound, irrespective whether the DNA sequence is a cDNA or a genomic DNA sequence, which may contain one or more introns.

The term "expression" is defined herein as to include any step involved in the production of the biological compound encoded by the gene including, but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term "improved abilities for RNAi mediated reduction of gene expression" is defined herein as the amplitude of RNAi mediated reduction of gene expression in the modified filamentous fungal cell being higher and/or being able to be dynamically changed in time as compared to the parental, unmodified filamentous fungal cell. Preferably, the amplitude of RNAi mediated reduction of gene expression in the modified filamentous fungal cell is at least 50% higher, compared to the parental, unmodified filamentous fungal cell, more preferably, the reduction is at least 100% higher, even more preferably, the reduction is at least 300% higher, even more preferably, the reduction is at least 500% higher, even more preferably, the reduction is at least 700% higher, and most preferably, the reduction is at least 1000% higher. Preferably, a parental, unmodified filamentous fungal cell is Aspergillus niger CBS513.88, or Penicillium chrysogenum CBS455.95, both deposited at the Centraal Bureau voor Schimmelcultures, the Netherlands (CBS), or Aspergillus niger A645, deposited at the Microbial Culture collection of the National Institute of Chemistry of Slovenia (MKZI). Dynamic changes in the amplitude of RNAi mediated reduction of gene expression are preferably achieved by placing an RNAi related gene under control of an inducible promoter or a medium or strong promoter. This can be reached by introducing an RNAi related gene under control of an inducible promoter or a medium or strong promoter. Alternatively, this can be performed by replacing the endogenous regulatory regions of the RNAi related gene by new regulatory regions, preferably by using a repressible or regulatable promoter, more preferably by using a promoter that can be switched on/off: by glucose repression, or ammonia repression, or pH repression. Examples of glucose-repressed promoters are the Penicillium chrysogenum pcbAB promoter (Martin JF, Casqueiro J, Kosalkova K, Marcos AT, Gutierrez S. Penicillin and cephalosporin biosynthesis: mechanism of carbon catabolite regulation of penicillin production. Antonie Van Leeuwenhoek. 1999 Jan-Feb;75(1-2):21-31. Review.) or the Aspergillus niger endoxylanase (xlnA), beta-xylosidase (xlnD), alcohol dehydrogenase (alcA) or glucoamylase (glaA) promoter. Examples of on/off switchable promoters are described in the following publications:

- An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast: Belli et al, (1998) Nucl. Acid Research, vol 26, n.4:942-947,

- A light-switchable gene promoter system: Shimizu-Sato_et al, (2002) Nat. Biotech. VoI 20, no 10:1041-1044. The ratio between the amount of expression of the target gene subjected to RNAi mediated reduction of gene expression in the modified filamentous fungal cell and the amount of the corresponding gene in the parental filamentous fungal cell is a measure for the difference in amplitude of RNAi mediated reduction of gene expression between the modified and parental cell. Preferably, the amplitude of RNAi mediated reduction of gene expression is determined by activity assay measurement of the amount of gene product of the target gene Examples of other assays include, but are not limited to, Northern blot to quantitatively determine the amount of full-length RNA transcripts of the target gene, quantitative PCR, quantitative real time PCR assays to determine the amount of full-length RNA transcripts of the target gene, Western blot to determine the amount of translated gene product, DNA microarray to determine the specificity of the RNAi mediated reduction of gene expression. The skilled person will know which assay is appropriate for which gene product. Alternatively, the amplitude of RNAi mediated reduction of gene expression is determined using a reporter assay. Preferred examples of these techniques are described in the experimental section.

The term "filamentous fungal cell" is defined herein as a cell from a strain belonging to the group of filamentous fungi, which includes all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelia wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.

The term "target gene" is defined herein as the gene subjected to reduction of gene expression by the RNAi process. A target gene may be any one or more genes encoding a biological compound. Used in the specific context of the target gene, the term "homologues" is defined herein as those genes, which share common ancestral origin and exhibit a high degree of sequence similarity. Preferably, the sequence similarity is at least 50%, preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98% and most preferably at least 99%.

The "biological compound" may be RNA; the biological compound may also be any biopolymer or metabolite. The biological compound may be encoded by a single gene or a series of genes composing a biosynthetic or metabolic pathway or may be the direct result of the product of a single gene or products of a series of genes. The biological compound may be native to the filamentous fungal cell or heterologous.

The term "heterologous biological compound" is defined herein as a biological compound which is not native to the cell; or a native biological compound in which structural modifications have been made to alter the native biological compound.

The term "biopolymer" is defined herein as a chain (or polymer) of identical, similar, or dissimilar subunits (monomers). The biopolymer may be any biopolymer. The biopolymer may for example be, but is not limited to, a nucleic acid, polyamine, polyol, polypeptide (or polyamide), or polysaccharide.

The biopolymer may be a polypeptide. The polypeptide may be any polypeptide having a biological activity 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 "polypeptide" also encompasses two or more polypeptides combined to form the encoded product. Polypeptides also include hybrid polypeptides, which 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 filamentous fungal cell. Polypeptides further include naturally occurring allelic and engineered variations of the above- mentioned polypeptides and hybrid polypeptides. The polypeptide may be a collagen or gelatin, or a variant or hybrid thereof. The polypeptide may be an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide, intracellular protein. The intracellular protein may be an enzyme such as, a protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase. The polypeptide may be an enzyme secreted extracellularly. Such enzymes may belong to the groups of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase. The enzyme may be a carbohydrase, e.g. cellulases such as endoglucanases, β-glucanases, cellobiohydrolases or β-glucosidases, hemicellulases or pectinolytic enzymes such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endo polygalacturonases, exopolygalacturonases rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases, transferases, or isomerases. The enzyme may be a phytase. The enzyme may be an aminopeptidase, amylase, carbohydrase, carboxypeptidase, endo-protease, metallo- protease, serine-protease catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, proteolytic enzyme, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, polyphenoloxidase, ribonuclease, transglutaminase, or glucose oxidase, hexose oxidase, monooxygenase.

The biopolymer may be a polysaccharide. The polysaccharide may be any polysaccharide, including, but not limited to, a mucopolysaccharide^, g., heparin and hyaluronic acid) and nitrogen-containing polysaccharide (eg., chitin). In a more preferred option, the polysaccharide is hyaluronic acid.

The term "metabolite" encompasses both primary and secondary metabolites; the metabolite may be any metabolite. A preferred metabolite is citric acid. The metabolite may be encoded by one or more genes, such as in a biosynthetic or metabolic pathway. Primary metabolites are products of primary or general metabolism of a cell, which are concerned with energy metabolism, growth, and structure. Secondary metabolites are products of secondary metabolism (see, for example, R. B. Herbert, The Biosynthesis of Secondary Metabolites, Chapman and Hall, New York, 1981 ). The primary metabolite may be, but is not limited to, an amino acid, fatty acid, nucleoside, nucleotide, sugar, triglyceride, or vitamin.

The secondary metabolite may be, but is not limited to, an alkaloid, coumarin, flavonoid, polyketide, quinine, steroid, peptide, or terpene. The secondary metabolite may be an antibiotic, antifeedant, attractant, bacteriocide, fungicide, hormone, insecticide, or rodenticide. Preferred antibiotics are cephalosporins and beta-lactams.

The biological compound may also be the product of a selectable marker. 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. Selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleomycin resistance protein), as well as equivalents thereof.

In order to perform the present invention, it may be necessary to isolate the target gene. The techniques used to isolate a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The isolation of the target gene of the present invention from such genomic DNA can be effected, e.g., by using methods based on polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features (See, e.g., lnnis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.). Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the target gene encoding a biological compound, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a modified filamentous 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, semi-synthetic, synthetic origin, or any combinations thereof.

The modified filamentous fungal host cell of the present invention may be a cell obtained by classical genetic techniques or may be a recombinant cell.

According to a preferred embodiment, the modified filamentous fungal cell of the present invention is a recombinant filamentous fungal cell. The term "recombinant" is defined herein as any genetic modification not involving naturally occurring processes and/or genetic modifications induced by subjecting the filamentous fungal cell to random mutagenesis.

According to another preferred embodiment, the expression of at least one RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof is modulated in the modified filamentous fungal cell of the present invention. Preferably, the RNAi related gene in step (a) is the dcrA gene (SEQ ID NO: 10) or the qdeA gene (SEQ ID NO: 28), or a homologue thereof, or a gene encoding the respective dcrA or qdeA polypeptides, or a homologue thereof.

SEQ ID NO: (1 + 3n) represents the gene sequence and corresponds to SEQ ID

NO: (2 + 3n) which represents the coding sequence and which corresponds to SEQ ID NO: (3 + 3n) which represents the amino acid sequence, for a certain n value.

Likewise, SEQ ID NO: (1 19 + 3m) represents the gene sequence and corresponds to SEQ ID NO: (120 + 3m) which represents the coding sequence and which corresponds to SEQ ID NO: (121 + 3m) which represents the amino acid sequence, for a certain m value.

The notation "SEQ ID NOs: (1 + 3n), n being an integer selected from the set of

0<= n <= 19" is used herein to depict the pool of SEQ ID NO's: 1 , 4, 7, 10, etc. to 58, and the notation "SEQ ID NOs: (1 19 + 3m), m being an integer selected from the set of

0<= n <= 18" is used herein to depict the pool of SEQ ID NO's:1 19, 122, 125, 128, etc. to 173.

Likewise, the notation "SEQ ID NOs: (2 + 3n), n being an integer selected from the set of 0<= n <= 19" is used herein to depict the pool of SEQ ID NO's: 2, 5, 8, 1 1 , etc. to 59, and the notation "SEQ ID NOs: (120 + 3m), m being an integer selected from the set of 0<= n <= 18" is used herein to depict the pool of SEQ ID NO's:120, 123, 126,

129, etc. to 174.

Likewise, the notation "SEQ ID NOs: (3 + 3n), n being an integer selected from the set of 0<= n <= 19" is used herein to depict the pool of SEQ ID NO's: 3, 6, 9, 24, etc. to 60, and the notation "SEQ ID NOs: (121 + 3m), m being an integer selected from the set of 0<= n <= 18" is used herein to depict the pool of SEQ ID NO's:121 , 124, 127,

130, etc. to 175.

The group of DNA sequences as specified above consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, is comprised of genomic DNA sequences. The skilled person will know that the corresponding cDNA sequences (consisting of SEQ ID NO: (2 + 3n), SEQ ID NO: (120 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18,), or homologues thereof, can be used alternatively or in combination with genomic DNA sequences. Within the context of the present invention it is herein defined that the genomic and cDNA counterparts can be utilized interchangeably to perform the present invention, and that throughout the description of the present invention genomic DNA and cDNA sequences can be read interchangeably.

The term "modulated" is defined herein as altered expression of at least one of the RNAi related genes compared to the parental cell the filamentous fungal cell derives from. Modulated expression in the context of the present invention can be increased expression of at least one of the RNAi related gene as compared to the parental cell. Alternatively, modulated expression in the context of the present invention can be reduced expression of at least one of the RNAi related gene as compared to the parental cell. Furthermore, modulated expression in the context of the present invention can be a dynamically changing expression level of at least one of the RNAi related gene as compared to the parental cell. A preferred parental filamentous fungal cell of the present invention is Aspergillus niger CBS513.88, or Aspergillus niger A645, or Penicillium chrysogenum CBS455.95; modulated expression (i.e. increased-, decreased- or dynamically changing expression level) of the preferred modified filamentous fungal cell (i.e. modified CBS513.88, CBS455.95 or A645) is preferentially compared to said CBS513.88, CBS455.95 or A645, respectively.

The term "increased expression of at least one of the RNAi related genes" is defined herein as a resulting increased mRNA concentration and/or gene product (i.e. the RNAi component) in the modified filamentous fungal cell as compared to the parental filamentous fungal cell the modified filamentous fungal cell derives from. Preferably, the mRNA concentration and/or gene product is increased with more than 50 %, more than 100 %, 250 %, 500 %, more than 1000 % as measured by Northern analysis or Q-PCR for mRNA; the amount of gene product is preferably measured by Western blotting using a specific antibody against the protein encoded. Preferably, the expression of the RNAi related gene is increased at least by a factor 10, more preferably, by at least a factor of 100, even more preferably, by at least 3 log, even more preferably, by at least 4 log, and most preferably, the expression of the RNAi related gene is increased by at least 5 log. Likewise, the term "reduced expression of at least one of the RNAi related genes" is defined herein as a resulting decreased mRNA concentration in a strain and/or amount of gene product (i.e. the RNAi component) in the modified filamentous fungal cell as compared to the parental filamentous fungal cell the modified filamentous fungal cell derives from. Preferably, the expression of the RNAi related gene or gene product is reduced by at least a factor of 10, more preferably, by at least 2 log, even more preferably, by at least 3 log, even more preferably, by at least 4 log, and most preferably, the expression of the RNAi related gene is reduced by at least 5 log. It follows that the "term dynamically changing expression level of at least one of the RNAi related gene" is defined herein as a resulting higher or lower amount of mRNA or gene product in the modified filamentous fungal cell as compared to the parental filamentous fungal cell the modified filamentous fungal cell derives from, and the amount of gene product dynamically changing over time. Preferably, the expression of the RNAi related gene is dynamically changing between at least 1 log, more preferably, between at least 2 log, even more preferably, between at least 3 log, even more preferably, between at least 4 log, and most preferably, the expression of the RNAi related gene is dynamically changing between at least 5 log. The expression of an RNAi related gene can be determined directly by techniques known to the skilled person. Preferably, the expression of an RNAi related gene is determined using quantitative real time PCR. Examples of other techniques are, but are not limited to, Northern blot, Western blot, quantitative PCR, quantitative NASBA. Alternatively, the expression of a RNAi related gene can be determined by comparing the amplitude of RNAi mediated reduction of gene expression in the modified filamentous fungal cell as compared to the parental filamentous fungal cell the modified filamentous fungal cell derives from, as described above in "improved abilities for RNAi mediated reduction of gene expression".

According to yet another preferred embodiment, the modified filamentous fungal cell of the present invention comprises a RNAi nucleic acid construct comprising a promoter operably linked to an RNAi unit of a target gene, said RNAi unit comprising: a. a first transcriptionally competent nucleotide sequence comprising a first transcriptionally competent homologous region of a target gene, and b. a second transcriptionally competent nucleotide sequence comprising a second transcriptionally competent homologous region of said target gene, wherein the first and second transcriptionally competent homologous region of a target gene are reverse-complementary relative to each other, and optionally c. a third transcriptionally competent nucleotide sequence separating the first and second transcriptionally competent nucleotide sequence. The term "nucleic acid construct" in the context of the invention is defined herein as a nucleic acid molecule, either single-or double-stranded, which is either isolated from a naturally occurring gene or which was modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains all the control sequences required for expression of a (coding) sequence.

The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of mRNA and / or a polypeptide. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, optimal translation initiation sequences (as described in Kozak, 1991 , J. Biol. Chem. 266:19867- 19870), a polyadenylation sequence, a pro-peptide sequence, a pre-pro-peptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be optimized to their specific purpose. Preferred optimized control sequences used in the present invention are those described in WO2006/077258, which is herein incorporated by reference.

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 production of a polypeptide. The control sequence may be an appropriate promoter sequence, a nucleic acid sequence, which is recognized by a host 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 cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the cell.

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

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

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

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

Other control sequences may be isolated from the Penicillium IPNS gene, or pcbC gene, the beta tubulin gene. All the control sequences cited in WO 01/21779 are herewith incorporated by reference.

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

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

The term "promoter" is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcriptional start site of a nucleic acid sequence encoding a biological compound to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of a coding region. The term "promoter" will also be understood to include the 5'-non-coding region (between promoter and translation start) for translation after transcription into mRNA, cis-acting transcription control elements such as enhancers, and other nucleotide sequences capable of interacting with transcription factors. The promoter may be any appropriate promoter sequence, which shows transcriptional activity in the cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extra-cellular or intracellular polypeptides either homologous (native) or heterologous (foreign) to the cell. The promoter may be a constitutive or inducible promoter. Examples of inducible promoters that can be used are a starch-, copper-, oleic acid- inducible promoters. The promoter may be selected from the group, which includes but is not limited to promoters obtained from the genes encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA), R. miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase, the NA2-tpi promoter (a hybrid of the promoters from the genes encoding A. niger neutral alpha- amylase and A. oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Particularly preferred promoters for use in filamentous fungal cells are a promoter, or a functional part thereof, from a protease gene ;e. g., from the F. oxysporum trypsin-like protease gene (U. S. 4, 288, 627), A. oryzae alkaline protease gene(alp), A. niger pacA gene, A. oryzae alkaline protease gene, A. oryzae neutral metalloprotease gene, A. niger aspergillopepsin protease pepA gene, or F. venenatum trypsin gene, A. niger aspartic protease pepB gene. Other preferred promoters are the promoters described in WO2006/092396 and WO2005/100573, which are herein incorporated by reference.

The term "operably linked" is defined herein as a configuration in which a promoter sequence is appropriately placed at a position relative to a nucleic acid sequence such that the promoter sequence directs the transcription of a nucleic acid sequence.

The term "RNAi unit" is defined herein as a double stranded, transcriptionally competent nucleotide sequence, comprising a first, second and optionally third transcriptionally competent nucleotide sequence. The optional third transcriptionally competent sequence separates first and second nucleotide sequences for stability purposes during construction and cloning and has little or no homology to the first and second nucleotide sequences of the RNAi unit. Transcription of an RNAi unit results in a small hairpin RNA (shRNA). The respective first and second transcriptionally competent nucleotide sequences of the RNAi unit comprise respective first and second transcriptionally competent homologous regions of a target gene, wherein the first and second regions are reverse complementary relative to each other. The RNAi unit of the present invention may comprise one, two, three or more sets of first and second transcriptionally competent homologous regions of a target gene, separated by optional third transcriptionally competent nucleotide sequences. Each set of first and second transcriptionally competent nucleotide sequences of a target gene are homologous to distinct regions of the target gene. Transcription of said RNAi unit comprising one, two or more sets of first and second transcriptionally competent homologous regions of a target gene results in a small hairpin RNA (shRNA) directed to one, two or more target regions within the target gene.

The optional third transcriptionally competent sequence can be any nucleotide sequence having little or no homology to the first and second nucleotide sequences and having preferably little or no homology to sequences in the genome of the cell to avoid nonspecific effects, e.g. non-specific targeting and/or non-specific recombination. Preferably, the optional third transcriptionally competent sequence has at least less than 50% percent homology with the first and second transcriptionally competent sequences of the RNAi unit and sequences in the genome of the cell, more preferably at least less than 40%, more preferably at least less than 30%, more preferably at least less than 20%, more preferably at least less than 10%, and most preferably, the optional third transcriptionally competent sequence has 0% percent homology with the first and second transcriptionally competent sequences and sequences in the genome of the cell. Homology is preferably determined using ClustalW, this technique and alternative techniques are described below in definition of "degree of identity". Preferably, the third transcriptionally competent sequence itself does not comprise an intra-molecular secondary structure.

Preferably, intra-molecular structure is determined using the Zuker algoritm (Mfold web server for nucleic acid folding and hybridization prediction, M. Zuker, Nucleic Acids Res. 2003 JuI 1 ;31 (13):3406-15).

The term "transcriptionally competent nucleotide sequence" is defined herein as a nucleotide sequence, which is capable of being transcribed into an RNA, which may or may not be translated into a biological compound. Examples of RNA are, but are not limited to ncRNA (non-coding RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), miRNA (micro RNA). The nucleotide sequence can include, but is not limited to, genomic DNA, cDNA, semi-synthetic, synthetic and recombinant nucleic acid molecules. The term "transcriptionally competent homologous region of a target gene" is defined herein as a nucleotide sequence, which is capable of being transcribed into an RNA, and is identical or homologous to a corresponding region of the open reading frame, or a part thereof, of the target gene. The degree of identity, i.e. match percentage, between a homologous region and the corresponding region of a target gene will be of influence on the amplitude of RNAi mediated reduction of gene expression. A higher match percentage will likely result in a higher amplitude of RNAi mediated reduction of gene expression. This amplitudes of RNAi mediated reduction of gene expression of RNAi units comprising homologous regions of distinct match percentages can be determined using the assay described above in "improved abilities for RNAi mediated reduction of gene expression". Preferably, the degree of identity of a transcriptionally competent homologous region of a target gene to the corresponding region is at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%, and most preferably 100%. For purposes of the present invention, the "degree of identity", i.e. the "match percentage", between two polypeptides, respectively two nucleic acid sequences is preferably determined using Clustal W as described below.

The terms "degree of identity", "identity" and "match percentage" are used interchangeably to indicate the degree of identity between two polypeptides or nucleic acid sequences as calculated by the optimal global alignment method indicated above.

Examples of alternative programs used for alignments and determination of homology are Clustal method (Higgins, 1989, CABIOS 5 : 151-153) , the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LAS E RG E N E. TM. MEGALIGN.TM. software (DNASTAR, Inc., Madison, Wis.), BLAST (NCBI), GAP (Huang) for the optimal global alignments, MAP (Huang), MultiBLAST (NCBI), ClustalW, Cap Assembler and Smith Waterman for multiple alignments. References:

! Pairwise alignment: (1) BLAST, (2) GAP, (3) MAP, (4) Smith Waterman, and (5) Cap

Alternatively, the ability of a transcriptionally competent homologous region of a target gene and the corresponding region to hybridize to each other under various stringency conditions can provide an indication of the match percentage required for reduction of expression of a target gene. However, it should be recognized that the lower the stringency conditions required to achieve hybridization between a transcriptionally competent homologous region of a target gene and the corresponding region, the less efficient the reduction of the expression of the target gene will likely be. Preferably, a transcriptionally competent homologous region of a target gene and the corresponding region hybridize under low stringency conditions. More preferably, a transcriptionally competent homologous region of a target gene and the corresponding region hybridize under medium stringency conditions. Even more preferably, a transcriptionally competent homologous region of a target gene and the corresponding region hybridize under medium-high stringency conditions. Even more preferably, a transcriptionally competent homologous region of a target gene and the corresponding region hybridize under high stringency conditions. Most preferably, a transcriptionally competent homologous region of a target gene and the corresponding region hybridize under very high stringency conditions. For nucleotide sequences of at least 100 nucleotides in length, very low to very high stringency conditions are defined as pre-hybridization and hybridization at 42 ⁰C in 5X SSPE, 0.3% SDS, 200ug/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

For nucleotide sequences of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at least at 45 ⁰C for very low stringency, at 50 ⁰C for low stringency, at 55 ⁰C for medium stringency, at 60 ⁰C for medium-high stringency, at 65 ⁰C for high stringency, and at 70 ⁰C for very high stringency.

For nucleotide sequences of approximately 15 nucleotides to approximately 70 nucleotides in length, stringency conditions are defined as pre-hybridization, hybridization, and washing post-hybridization at approximately 5 ⁰C to 10 ⁰C below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48: 1390) in 0.9 M NaCI, 0.09M Tris-HCI pH 7.6, 6 mM EDTA, 0.5% NP- 40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally. For nucleotide sequences of approximately 15 nucleotides to approximately 70 nucleotides in length, the carrier material is washed once in 6X SCC, 0.2% SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5 ⁰C to10 ⁰C below the calculated Tm.

The respective first and second transcriptionally competent homologous regions of a target gene, comprised in the respective first and second transcriptionally competent sequences of the RNAi unit, wherein first and second regions are reverse complementary relative to each other, may be identically matching or homologous to each other. Identically matching is defined herein as 100% match identity of the complementary nucleic acid strands. Preferably, a second homologous region is 100% identically matching a first homologous region, and reverse complementary relative to a first homologous region. In another preferred option, a second homologous region is a part of a first homologous region, wherein the part is 100% identically matching the corresponding part of a first homologous region. In another preferred option, a second homologous region is a homologue of the corresponding part of a first homologous region. In another preferred option, a second homologous region is a homologue part of the corresponding part of a first homologous region. The homologous region or homologue part is at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97% identically matching the corresponding part of the first homologous region.

The respective first and second transcriptionally competent homologous regions of a target gene, comprised in the respective first and second transcriptionally competent sequences of the RNAi unit, may be derived from any homologous transcriptionally competent part of a target gene, such as the 5'-untranslated region, the coding sequence, or the 3'-untranslated region of the target gene or combinations thereof. Preferably, a second homologous region corresponds to the coding sequence of a target gene, or a part thereof. In another preferred option, a second homologous region corresponds to the 5'- untranslated region of a target gene, or a part thereof. In another preferred option, a second homologous region corresponds to the 3'-untranslated region of a target gene, or a part thereof.

According to a more preferred embodiment, the modified filamentous fungal cell of the invention comprises an RNAi nucleic acid construct comprising a promoter operably linked to an RNAi unit of a target gene, said RNAi unit comprising: a. a first transcriptionally competent homologous region of a target gene comprising at least 19 nucleotides of a target gene, and/or b. a second transcriptionally competent homologous region comprising at least 19 nucleotides of the first homologous region, wherein said nucleotides are in reverse-complementary orientation relative to the first homologous region, and/or c. a optional third transcriptionally competent nucleotide sequence comprising at least 5 nucleotides.

A first transcriptionally competent homologous region of a target gene preferably comprises at least 19 nucleotides, more preferably at least 40 nucleotides of a target gene, more preferably at least 60 nucleotides of a target gene, more preferably at least 80 nucleotides of a target gene, even more preferably at least 100 nucleotides of a target gene, and most preferably 200 nucleotides of a target gene. In another preferred option, the first transcriptionally competent homologous region comprises the entire open reading frame of a target gene or homologue thereof.

A second transcriptionally competent homologous region of a target gene preferably comprises at least 19 nucleotides, more preferably at least 40 nucleotides of a target gene, more preferably at least 60 nucleotides of a target gene, more preferably at least 80 nucleotides of a target gene, even more preferably at least 100 nucleotides of a target gene, and most preferably 200 nucleotides of a target gene. In another preferred option, a second transcriptionally competent homologous region comprises the entire open reading frame of a target gene or homologue thereof. An optional third transcriptionally competent region preferably comprises at least 5 nucleotides, more preferably at least 10 nucleotides, more preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, even more preferably at least 50 nucleotides and most preferably at least 100 nucleotides. According to another more preferred embodiment, the modified filamentous fungal cell of the invention comprises an RNAi nucleic acid construct comprising a promoter operably linked to an RNAi unit of a target gene, said RNAi unit comprising: a. a first transcriptionally competent homologous region comprises at most 19, preferably at most 18, more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most 14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 1 1 , even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, and most preferably at most 7 nucleotides of a target gene, and/or b. a second transcriptionally competent homologous region comprises at most 19, preferably at most 18, more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most 14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 1 1 , even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, and most preferably at most 7 nucleotides of the first homologous region, wherein said nucleotides are in reverse- complementary orientation relative to the first homologous region and/or c. a optional third transcriptionally competent nucleotide sequence comprises between 5 and 9 nucleotides. An optional third transcriptionally competent region preferably comprises at most 9 nucleotides, more preferably at most 8 nucleotides, more preferably at most 7 nucleotides, even more preferably at most 6 nucleotides, and most preferably at most 5 nucleotides.

According to an embodiment, the RNAi nucleic acid construct of the modified filamentous fungal cell of the present invention comprises at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 and most preferably at least 6 repeats of the RNAi unit, each RNAi unit being operably linked to a promoter.

According to an embodiment, the RNAi nucleic acid construct of the modified filamentous fungal cell of the present invention comprises one promoter operably linked to at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 and most preferably at least 6 repeats of the RNAi units.

RNAi units may be separated by transcriptionally competent separating nucleotide sequences. A separating nucleotide sequence may or may not be in frame with the RNAi units. Preferably, a separating nucleotide sequence comprises at most 96 nucleotides, more preferably at most 60 nucleotides, more preferably at most 48 nucleotides, more preferably at most 36 nucleotides, even more preferably at most 24 nucleotides and most preferably at most 12, 10, 8, 6, 5, 4, 3, 2, 1 nucleotides. In a most preferred option, the RNAi units are separated by 0 nucleotides.

A separating nucleotide sequence can be any nucleotide sequence having little or no homology to an RNAi unit and having preferably little or no homology to sequences in the genome of the cell to avoid non-specific effects, e.g. non-specific targeting and/or nonspecific recombination. Preferably, a separating nucleotide sequence has at least less than 50% percent homology with an RNAi unit and sequences in the genome of the cell, more preferably at least less than 40%, more preferably at least less than 30%, more preferably at least less than 20%, more preferably at least less than 10%, and most preferably, a separating nucleotide sequence has 0% percent homology with an RNAi unit and sequences in the genome of the cell. Preferably, a separating nucleotide sequence itself does not comprise an intra-molecular secondary structure. According to an embodiment, the RNAi nucleic acid construct of the modified filamentous fungal cell of the present invention comprises at least two RNAi units of distinct target genes. Multiple RNAi units of distinct target genes within a single RNAi nucleic acid construct allow simultaneous RNAi mediated reduction of gene expression of multiple distinct target genes.

According to an embodiment, the modified filamentous fungal cell of the present invention further comprises a gene encoding a compound of interest to be produced. The compound of interest to be produced can be any endogenous or heterologous biological compound (e.g. a polypeptide or a metabolite), such as described earlier in the definition of a biological compound. In addition to the description of the biological compound, the compound of interest can be human insulin or an analogue thereof, human growth hormone, erythropoietin, tissue plasminogen activator (tPA) or insulinotropin.

A gene encoding a heterologous compound of interest 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 compound of interest is produced by the source or by a cell in which a gene from the source has been inserted.

The modified filamentous fungal host cell of the present invention may also be used for the recombinant production of polypeptides, which are native to the cell. The native polypeptides 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 present invention also encompasses, within the scope of the term "heterologous polypeptide", such recombinant production of polypeptides native to the cell, to the extent that such expression involves the use of genetic elements not native to the cell, or use of native elements which have been manipulated to function in a manner that do not normally occur in the filamentous fungal cell. The techniques used to isolate or clone a nucleic acid sequence encoding a heterologous polypeptide are known to the skilled person and include isolation from genomic DNA, preparation from cDNA, or a combination thereof.

Alternatively, or in combination with isolation and cloning, the gene or polynucleotide encoding the polypeptide of interest may be a synthetic nucleic acid sequence. The synthetic nucleic acid may be optimized in its codon use, preferably according to the methods described in WO2006/077258 and/or PCT/EP2007/055943, which are herein incorporated by reference. PCT/EP2007/055943 addresses codon-pair optimization. Codon-pair optimisation is a method wherein the nucleotide sequences encoding a polypeptide have been modified with respect to their codon-usage, in particular the codon- pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the encoded polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.

In the context of the present invention, heterologous polypeptides 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 polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the mutant fungal cell. An isolated nucleic acid sequence encoding a heterologous polypeptide of interest may be manipulated in a variety of ways to provide for expression of the polypeptide.

In order to perform the invention, it may be necessary to isolate the gene encoding the compound of interest. Methods for performing such isolation are well known in the art and are described in the previous paragraph for isolation of a target gene encoding a biological compound.

According to a preferred embodiment, the modified filamentous fungal cell of the present invention is a cell belonging to a species of the Aspergillus, Penicillium, Fusarium, Mortierella or Trichoderma genus. More preferably, the modified filamentous fungal host cell is a cell selected from the group consisting of: Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus sojae, Penicillium chrysogenum, Trichoderma reesei, Fusarium oxysporum, Mortierella alpina. Even more preferably, the modified filamentous fungal cell is a cell selected from the group consisting of: A.niger, P. chrysogenum. Most preferably, the modified filamentous fungal cell is a cell selected from the group consisting of: A. niger CBS513.88, A. niger A645, or P. chrysogenum CBS455.95.

Optionally, the modified filamentous fungal cell of the present invention comprises an elevated unfolded protein response (UPR) compared to the wild type cell to enhance production abilities of a compound of interest. UPR may be increased by techniques described in US2004/0186070A1 and/or US2001/0034045A1 and/or WO01/72783A2. More specifically, the protein level of HAC1 and/or IRE1 and/or PTC2 has been modulated in order to obtain a host cell having an elevated UPR.

Alternatively, or in combination with an elevated UPR, the modified filamentous fungal cell of the present invention cell is genetically modified to obtain a phenotype displaying lower protease expression and/or protease secretion compared to the wild type cell in order to enhance production abilities of a compound of interest. Such phenotype may be obtained by deletion and/or modification and/or inactivation of a transcriptional regulator of expression of proteases. Such a transcriptional regulator is e.g. prtT. Lowering expression of proteases by modulation of prtT is preferable performed by techniques described in US2004/0191864A1 , WO2006/04312 and WO2007/062936.

Alternatively, or in combination with an elevated UPR and/or a phenotype displaying lower protease expression and/or protease secretion, the modified filamentous fungal cell of the present invention cell displays an oxalate deficient phenotype in order to enhance the yield of production of a compound of interest. An oxalate deficient phenotype is preferable obtained by techniques described in WO2004/070022, which is herein enclosed by reference.

Alternatively, or in combination with an elevated UPR and/or a phenotype displaying lower protease expression and/or protease secretion and/or oxalate deficiency, the modified filamentous fungal cell of the present invention cell displays a combination of phenotypic differences compared to the wild type cell to enhance the yield of production of the compound of interest. These differences may include, but are not limited to, lowered expression of glucoamylase and/or neutral alpha-amylase A and/or neutral alpha-amylase B, protease, and oxalic acid hydrolase. Said phenotypic differences displayed by the modified filamentous fungal cell of the present invention cell may be obtained by genetic modification according to the techniques described in US2004/0191864A1. In a second aspect, the present invention provides a method for the construction of the modified filamentous fungal cell of the first aspect.

According to an embodiment, the modified filamentous fungal cell is prepared by a method comprising: a. mutation of a filamentous fungal cell by random mutagenesis, and b. screening from the pool resulting from (a) for a modified filamentous fungal cell wherein abilities for RNAi mediated reduction of gene expression of a target gene encoding a biological compound are improved compared to the parental filamentous fungal cell, and c. optionally isolating the modified filamentous fungal cell of (b)

Mutation of a filamentous fungal cell is preferably performed by random mutagenesis. Random mutagenesis may be performed by methods known in the art. An example of such method is, but is not limited to, use of a suitable physical or chemical mutagenizing agent. Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet(W) irradiation, hydroxylamine,N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), 0-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 selecting for mutant cells exhibiting the required feature. In the context of the present invention, the required feature of the filamentous fungal cell is: improved abilities for RNAi mediated reduction of gene expression of a target gene and/or homologues thereof, said target gene encoding a biological compound. The definition of "improved abilities for RNAi mediated reduction of gene expression" is described earlier; likewise the preferred assay to determine the amplitude of RNAi mediated reduction of gene expression is described earlier.

According to another embodiment, the recombinant modified filamentous fungal cell of the first aspect is prepared by: a. inserting into the filamentous fungal cell at least one nucleic acid construct comprising, operably linked to a promoter, at least one RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n),

SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof, and/or b. rendering inactive at least one native RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof by specific mutagenesis, site-directed mutagenesis, PCR generated mutagenesis, nucleotide insertion and/or deletion and/or substitution, gene interruption or gene replacement techniques, or a combination thereof. Preferably, the RNAi related gene in step (a) is the dcrA gene (SEQ ID NO: 10) or the qdeA gene (SEQ ID NO: 28), or a homologue thereof, or a gene encoding the respective dcrA or qdeA polypeptides, or a homologue thereof.

In order to insert the nucleic acid construct comprising the RNAi related gene into the cell it may be necessary to isolate the RNAi related gene. Methods to perform such isolation are known in the art and are described in a previous paragraph concerning isolation of a target gene.

In order to accomplish expression of the RNAi related gene, it may be expressed by inserting the nucleic acid construct comprising the RNAi related gene into an appropriate vector. 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 filamentous fungal cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i. e., a vector, which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. An autonomously maintained cloning vector may comprise the AM A1 -sequence (see e.g. Aleksenko and Clutterbuck (1997), Fungal Genet. Biol. 21 : 373- 397). Alternatively, the vector may be one which, when introduced into the filamentous fungal cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The integrative cloning vector may integrate at random or at a predetermined target locus in the chromosomes of the filamentous fungal cell. In a preferred embodiment of the invention, the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of the filamentous fungal cell for targeting the integration of the cloning vector to this predetermined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 30 bp, preferably at least 50 bp, preferably at least 0.1 kb, even preferably at least 0.2 kb, more preferably at least 0.5 kb, even more preferably at least 1 kb, most preferably at least 2 kb. Preferably, the efficiency of targeted integration into the genome of the host cell, i.e. integration in a predetermined target locus, is increased by augmented homologous recombination abilities of the host cell. Such phenotype of the cell preferably involves a deficient ku70 gene as described in WO2005/095624. WO2005/095624 discloses a preferred method to obtain a filamentous fungal cell comprising increased efficiency of targeted integration. Preferably, the homologous flanking DNA sequences in the cloning vector, which are homologous to the target locus, are derived from a highly expressed locus meaning that they are derived from a gene, which is capable of high expression level in the filamentous fungal cell. A gene capable of high expression level, i.e. a highly expressed gene, is herein defined as a gene whose mRNA can make up at least 0.5% (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 1% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.1 g/l (as described in EP 357 127 B1 ). A number of preferred highly expressed fungal genes are given by way of example: the amylase, glucoamylase, alcohol dehydrogenase, xylanase, glyceraldehyde-phosphate dehydrogenase or cellobiohydrolase (cbh) genes from Aspergilli or Trichoderma. Most preferred highly expressed genes for these purposes are a glucoamylase gene, preferably an A. niger glucoamylase gene, an A. oryzae TAKA-amylase gene, an A. nidulans gpdA gene, a Trichoderma reesei cbh gene, preferably cbh 1. More than one copy of a nucleic acid sequence may be inserted into the cell to increase production of the gene product. This can be done, preferably by integrating into its genome copies of the DNA sequence, more preferably by targeting the integration of the DNA sequence at one of the highly expressed locus defined in the former paragraph. Alternatively, this can be done by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. To increase even more the number of copies of the DNA sequence to be over expressed the technique of gene conversion as described in WO98/46772 may be used.

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

The vectors preferably contain one or more selectable markers, which permit easy selection of transformed 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. A selectable marker for use in a filamentous fungal cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), bleA (phleomycin binding), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5¹- phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents from other species. Preferred for use in an Aspergillus and Penicillium cell are the amdS (EP 635574 B1 , WO 97/06261 ) and pyrG genes of A. nidulans or A. oryzae and the bar gene of Streptomyces hygroscopicus. More preferably an amdS gene is used, even more preferably an amdS gene from A. nidulans or A. niger. A most preferred selection marker gene is the A.nidulans amdS coding sequence fused to the A.nidulans gpdA promoter (see EP 635574 B1). Other preferred AmdS markers are those described in WO2006/040358. AmdS genes from other filamentous fungi may also be used (WO 97/06261 ). The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., supra). The introduction of an expression vector or a nucleic acid construct into a cell may be performed using commonly known techniques. It may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81 : 1470-1474. Other methods can be applied such as a method using biolistic transformation as described in: Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei. Christiansen SK, Knudsen S, Giese H. Curr Genet. 1995 Dec; 29(1 ): 100-2. In addition to recombinant expression of an RNAi related gene, the endogenous

RNAi related gene is preferably inactivated by specific or, site-directed mutagenesis, PCR generated mutagenesis, nucleotide insertion and/or deletion and/or substitution, gene interruption or gene replacement techniques, anti-sense techniques, or combinations thereof. Methods include, but are not limited to: subjecting the parent cell to mutagenesis and selecting for mutant cells in which the capability to produce an RNAi related gene product with reduced activity by comparison to the parental cell. The mutagenesis may be performed, for example, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis.

Alternatively, the sequences of a recombinant RNAi related gene are used to inactivate the endogenous copy (or copies) of an RNAi related gene in the genome of the filamentous fungal cell. To this extend, an inactivation vector can be constructed using the sequences of the RNAi related gene to target the vector to an endogenous copy of the gene by homologous recombination. The inactivation can then be accomplished either by replacement of, or by insertion into the endogenous RNAi related gene. Gene replacement techniques are known in the art. An example of gene replacement technique is described in EP357127B1.

According to an embodiment, the modified filamentous fungal cell of the present invention is prepared by inserting into the filamentous fungal cell a nucleic acid construct comprising a promoter operably linked to an RNAi unit of a target gene. An RNAi nucleic acid construct comprising an RNAi unit of a target gene may be prepared by molecular biological techniques known in the art, or combinations thereof. Examples of the techniques include, but are not limited to, PCR, excision with appropriate enzyme, ligation. An RNAi nucleic acid construct comprising an RNAi unit may be synthetic or partly synthetic.

Insertion of the RNAi nucleic acid construct comprising an RNAi unit into the filamentous fungal cell may require inserting the RNAi nucleic acid construct into an appropriate vector. Methods to perform this are well known in the art and are described in a previous paragraph concerning expression of an RNAi related gene. Likewise, transformation of a filamentous fungal cell is described herein.

In an embodiment, the modified filamentous fungal cell of the present invention further comprises a gene encoding a compound of interest. It may be necessary to insert a gene encoding a compound of interest into the cell. In order to insert a nucleic acid construct comprising the gene encoding the compound of interest into the cell it may be necessary to isolate the gene. Methods to perform such isolation are known in the art and are described in a previous paragraph concerning isolation of a target gene.

Insertion of the nucleic acid construct comprising a gene encoding a compound of interest into the filamentous fungal cell may require inserting the nucleic acid construct into an appropriate vector. Methods to perform this are well known in the art and are described in a previous paragraph concerning expression of a gene encoding a compound of interest.

Likewise, transformation of a filamentous fungal cell is described herein.

In a preferred embodiment, the modified filamentous fungal cell of the present invention is prepared by: a. mutation of a filamentous fungal cell by random mutagenesis, and b. screening from the pool resulting from (a) for a modified filamentous fungal cell wherein abilities for RNAi mediated reduction of gene expression of a target gene encoding a biological compound are improved compared to the parental filamentous fungal cell, and c. optionally isolating the modified filamentous fungal cell of (b), and/or d. inserting into the filamentous fungal cell at least one nucleic acid construct comprising, operably linked to a promoter, at least one RNAi related gene selected from the the group consisting of SEQ ID NO: (1 +

3n), SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof, and/or e. rendering inactive at least one native RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (1 19 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof by specific mutagenesis, site-directed mutagenesis, PCR generated mutagenesis, nucleotide insertion and/or deletion and/or substitution, gene interruption or gene replacement techniques, or a combination thereof, and/or inserting into the filamentous fungal cell a nucleic acid construct comprising a promoter operably linked to an RNAi unit of a target gene, and/or inserting into the filamentous fungal cell a gene encoding a compound of interest. Preferably, the RNAi related gene in step (d) is the dcrA gene (SEQ ID NO: 10) or the qdeA gene (SEQ ID NO: 28), or a homologue thereof, or a gene encoding the respective dcrA or qdeA polypeptides, or a homologue thereof.

In a more preferred embodiment, the modified filamentous fungal cell to be prepared in this second aspect of the present invention belongs to a species of the Aspergillus,

Penicillium, Fusarium, Mortierella, or Trichoderma genus. Even more preferably, the modified filamentous fungal host cell is a cell of the group consisting of: Aspergillus niger,

Aspergillus nidulans, Aspergillus oryzae, Aspergillus sqjae, Penicillium chrysogenum,

Fusarium oxysporum, Mortierella alpina, Trichoderma reesei. Even more preferably, the modified filamentous fungal cell is an A. niger cell. Most preferably, the modified filamentous fungal cell is a cell selected from the group consisting of: A. niger CBS513.88, A. niger

A645, or P. chrysogenum CBS455.95.

In a third aspect, the present invention provides a method for reducing the expression of a target gene and/or homologue thereof in the modified filamentous fungal cell of the first aspect.

In a preferred embodiment, the reduced expression of a target gene and/or homologue thereof in the modified filamentous fungal cell of the first aspect is mediated by RNAi, i.e. a process wherein double stranded RNA (dsRNA) reduces gene expression, either post-transcriptionally by activation of a sequence-specific RNA degradation process or by inhibiting translation. Preferably, the method for reducing the expression of a target gene and/or homologue thereof in the modified filamentous fungal cell of the first aspect comprises: a. inducing recombinant expression of at least one RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof, by culturing the modified filamentous fungal cell under conditions conducive to expression of the RNAi related gene, and/or b. inducing expression of both an RNAi unit into an interfering RNA and expression of a target gene into RNA transcripts, by culturing the modified filamentous fungal cell under conditions conducive to production of interfering RNA, wherein the interfering RNA interacts with the RNA transcripts of the target gene encoding the biological compound to reduce the expression of the target gene encoding the biological compound. Preferably, the RNAi related gene in step (a) is the dcrA gene (SEQ ID NO: 10) or the qdeA gene (SEQ ID NO: 28), or a homologue thereof, or a gene encoding the respective dcrA or qdeA polypeptides, or a homologue thereof.

Culturing the modified filamentous fungal cell of the first aspect is preferably performed in a nutrient medium suitable for recombinant expression of a RNAi related gene and/or expression of both an RNAi unit into interfering RNA and expression of the target gene into RNA transcripts using methods known in the art. For example, the cells 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 appropriate genes to be expressed. The culture takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e. g., Bennett, J. W. and LaSure, L.,eds., More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared using published compositions (e. g., in catalogues of the American Type Culture Collection).

In a preferred embodiment, the expression of a target gene or homologue thereof is reduced by at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60 %, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, more preferably by at least 95%, more preferably by at least 98%, more preferably by at least 99%, and most preferably by 100%. The reduction of gene expression is preferably determined by mRNA measurement through Quantitative real time PCR and/or activity assay measurement of the amount of gene product, as described earlier.

In a fourth aspect, the present invention provides a nucleotide sequence derivable from a filamentous fungus encoding SEQ ID NO: (3 + 3n), SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof, wherein the nucleotide sequence is selected from the group consisting of: a. SEQ ID NO: (1 + 3n), SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof b. fragments or mutants of any one of the nucleotide sequences of (a). Preferably, the RNAi related gene of (a) is the dcrA gene (SEQ ID NO: 10) or the qdeA gene (SEQ ID NO: 28), or a homologue thereof, or a gene encoding the respective dcrA or qdeA polypeptides, or a homologue thereof.

The term "fragment" is herein defined as a nucleotide sequence consisting of at least one part of a parent sequence. A fragment may be obtained by methods well known in the art such as isolation and purification of nucleic acids, electrophoresis of nucleic acids, enzymatic modification, cleavage and/or amplification of nucleic acids. The term "mutant" is defined herein as a nucleotide sequence comprising a substitution, deletion, and/or insertion of one or more nucleotides of a parent sequence. The term "mutant" encompasses in vitro generated mutants obtained using methods well known in the art such as classical mutagenesis, site-directed mutagenesis, and DNA shuffling. In a preferred embodiment, an RNAi component encoded by the sequence according to (a) or (b) comprises an amino acid sequence, wherein the amino acid positional identity of the respective sequences SEQ ID NO: (3 + 3n), SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18 is more than 40%. Preferably, the match percentage, i.e. positional identity is at least about 50%, more preferably at least about 60%, even more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 97%, even more preferably at least about 98%, even more preferably at least about 99% identity, and most preferably, the match percentage i.e. identity is equal to 100%. Match percentage is preferably determined using ClustalW, as described earlier in the definition of "degree of identity".

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein can be readily used to isolate the complete gene from filamentous fungi, in particular A. niger which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a nucleic acid sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.

In a fifth aspect, the present invention provides a nucleic acid construct comprising a nucleotide sequence according to the fourth aspect of the invention. Preferably, the nucleic acid construct comprises a promoter, which is native to the nucleotide sequence. Alternatively, the nucleic acid construct comprises a promoter, which is foreign to the nucleotide sequence.

In another preferred embodiment, the nucleic acid construct comprises direct repeats suitable for gene replacement by double cross-over mediated integration of the nucleic acid construct. Preferably, gene replacement is performed to replace or inactivate an endogenous RNAi related gene. Gene replacement techniques are well known in the art. An example of a gene replacement technique, which should not be construed as limiting to the present invention, is described in EP357127B1.

In a sixth aspect, the present invention provides an RNAi nucleic acid construct comprising a promoter operably linked to an RNAi unit of a target gene, said RNAi unit comprising: a. a first transcriptionally competent nucleotide sequence comprising a first transcriptionally competent homologous region of a target gene, and b. a second transcriptionally competent nucleotide sequence comprising a second transcriptionally competent homologous region of said target gene, wherein the first and second homologous transcriptionally competent region are reverse-complementary relative to each other, and optionally c. a third transcriptionally competent nucleotide sequence separating the first and second transcriptionally competent nucleotide sequence.

In a preferred embodiment, a. a first transcriptionally competent homologous region comprises at least 19 nucleotides of a target gene, and/or b. a second transcriptionally competent homologous region comprises at least 19 nucleotides of the first homologous region, wherein said nucleotides are in reverse-complementary orientation relative to the first homologous region, and/or c. a optional third transcriptionally competent nucleotide sequence comprises at least 5 nucleotides. In a another preferred embodiment, a. a first transcriptionally competent homologous region comprises at most 19, preferably at most 18, more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most 14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 11 , even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, and most preferably at most 7 nucleotides of a target gene, and/or b. a second transcriptionally competent homologous region comprises at most 19, preferably at most 18, more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most

14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 11 , even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, and most preferably at most 7 nucleotides of the first homologous region, wherein said nucleotides are in reverse-complementary orientation relative to the first homologous region and/or c. a optional third transcriptionally competent nucleotide sequence comprises between 5 and 9 nucleotides.

In a more preferred embodiment, the RNAi nucleic acid construct comprises at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 and most preferably at least 6 repeats of the RNAi unit, each RNAi unit being operably linked to a promoter. More preferably, the RNAi nucleic acid construct comprises at least two RNAi units of distinct target genes. The promoter may be native to the RNAi unit. Alternatively, the promoter may be foreign to the RNAi unit. In yet another preferred embodiment, the RNAi nucleic acid construct according to the invention comprises one promoter operably linked to at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 and most preferably at least 6 repeats of the RNAi units. More preferably, the RNAi nucleic acid construct comprises at least two RNAi units of distinct target genes. The promoter may be native to the RNAi unit. Alternatively, the promoter may be foreign to the RNAi unit.

In a seventh aspect, the present invention provides a polypeptide encoded by a nucleotide sequence of the fourth aspect, wherein the amino acid sequence is selected from the group consisting of: a. SEQ ID NO: (3 + 3n), SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, and b. fragments or mutants of any one of the amino acid sequences of (a).

A preferred polypeptides of (a) is the dcrA protein (SEQ ID NO: 12) and/or the qdeA protein (SEQ ID NO: 30), or a fragment or mutant thereof.

The term "fragment" is herein defined as an amino acid sequence consisting of at least one part of a parent sequence. The term "mutant" is defined herein as a amino acid sequence comprising a substitution, deletion, and/or insertion of one or more amino acids of a parent sequence. The term "mutant" also encompasses natural mutants and in vitro generated mutants obtained using methods well known in the art such as classical mutagenesis, and site- directed mutagenesis Preferably, an RNAi component of the amino acid sequence according to (a) or (b) comprises an amino acid sequence, wherein the amino acid positional identity of the respective sequences SEQ ID NO: (3 + 3n), SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18: is more than 40%. Preferably, the match percentage, i.e. positional identity is at least about 50%, more preferably at least about 60%, even more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 97%, even more preferably at least about 98%, even more preferably at least about 99% identity, and most preferably, the match percentage i.e. identity is equal to 100%. In an eight aspect, the present invention provides a method for the production of a compound of interest in a modified filamentous fungal cell of the first aspect of the invention, said method comprising: a. reducing the expression of a target gene and/or homologues thereof according to the method of the third aspect of the present invention, and b. culturing the modified filamentous fungal cell under conditions conducive to the production of the compound of interest, and c. optionally recovering the compound of interest from the culture medium, and d. optionally purifying the compound of interest.

Culturing the modified filamentous fungal cell of the first aspect of the invention is described in a previous paragraph concerning a method for reducing the expression of a target gene and/or homologues thereof and can be applied likewise in the method for production of a compound of interest. If the compound of interest is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the compound of interest is not secreted, it is recovered from cell lysates.

The resulting compound of interest may be isolated by methods known in the art. For example, the compound of interest 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 compound of interest 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).

According to a preferred embodiment, the modified filamentous fungal cell of the method for the production of a compound of interest in a modified filamentous fungal cell belongs to a species of the Aspergillus, Penicillium, Fusarium, Morteirella, or Trichoderma genus. Even more preferably, the modified filamentous fungal host cell is a cell of the group consisting of: Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus sojae, Penicillium chrysogenum, Trichoderma reesei, Mortierella alpina or Fusarium oxysporum. Even more preferably, the modified filamentous fungal cell is a cell selected from the group consisting of: A. niger, P. chrysogenum. Most preferably, the modified filamentous fungal cell is a cell selected from the group consisting of: A. niger CBS513.88, A. niger A645, or P.chrysogenum CBS455.95.

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

The present invention is further illustrated by the following examples.

Examples

Experimental information

In the examples described herein, standard molecular cloning techniques such as isolation and purification of nucleic acids, electrophoresis of nucleic acids, enzymatic modification, cleavage and/or amplification of nucleic acids, transformation of E.coli, etc., were performed as described in the literature (see, e.g., Sambrook & Russell,

"Molecular Cloning: A Laboratory Manual", 3rd Ed., CSHL Press, Cold Spring Harbor,

NY, 2001 ; and Ausubel et al., "Current Protocols in Molecular Biology", Wiley InterScience, NY, 1995; and lnnis et al. (eds.) (1990) "PCR protocols, a guide to methods and applications" Academic Press, San Diego).

Strains

WT 1 : This A. niger strain is a wild-type strain. This strain is deposited at the CBS Institute under the deposit number CBS 513.88.

WT 2: This A. niger strain is a descendent of wild-type strain CBS 513.88 and has a pyrA genotype. This means that medium should be complemented with uridine / uracil. WT 3: This Penicillium chrysogenum strain is used as a wild-type strain. This strain is deposited at the CBS Institute under the deposit number CBS 455.95.

Transformation Linear or circular DNA was isolated and used to transform the Aspergillus niger strains WT1 , 2 or 3 or derivatives using a method earlier described (Biotechnology of Filamentous fungi: Technology and Products. (1992) Reed Publishing (USA); Chapter 6: Transformation pages 1 13 to 156). Transformation and subsequent selection of transformants for these strains essentially was done according the protocol as described in WO199932617 and WO199846772. Transformants were selected on selective media (containing acetamide, hygromycin or without uridin/uracil, dependent on marker(s) used) and colony purified according to standard procedures. Spores optionally were plated on fluoro-acetamide media to select strains, which lost the amdS marker (EP 635574B). Growing colonies were diagnosed by PCR and Southern analysis for integration of genes of interest.

A. niger shake flask fermentations

Fermentation with A. niger strains is performed in 500 ml flasks with baffle with 100 ml fermentation broth as indicated (MM-X or MM-G) at 30⁰C and 150 rpm for 16-20 h.

Induction medium (MM-X) contains per liter: 20 g D-xylose, 6 g NaNO3, 0.25 g KCI, 1.5 g KH₂PO₄, 1.13 ml 4M KOH, 0.5 g MgSO₄JH₂O, 1 ml of stock trace elements (stock trace elements per liter: 22 g ZnSO₄JH₂O, 1 1 g H₃BO₃, 5 g FeSO₄JH₂O, 1.7 g CoCI₂.6H₂O, 1.6 g CuSO₄.5H₂O, 5 g MnCI₂.4H₂O, 1.5 g Na₂MoO₄.2H₂O, 50 g EDTA, adjust the pH to 6.5 with 4M KOH, filter sterilize and store in the dark at 4°C) and adjusted to pH 5.6.

MM-G repressing medium contains the same ingredients as MM-X except for 20 g of D- glucose instead of the D-xylose.

PCR reactions

Amplification reactions generally were carried out in 50μl and were composed of 1x Platinum Pfx buffer, 1x Mg²⁺ (1 mM) (Invitrogen), 0,2 mM dNTPs, 50 ng plasmid DNA, 20 pmol sense primer, 20 pmol antisense primer, 2 units Platinum Pfx polymerase (Invitrogen).

Protein extraction

A. niger strains were grown in 100 ml liquid media MM-X or MM-G at 3O⁰C and 150 rpm, collected by vacuum filtration through a Buchner funnel, washed with physiological solution and ground to a fine powder under liquid nitrogen. Powder was transferred to a micro centrifuge tube and 1 ml protein extraction buffer was added per 0.2 g mycelia powder. The mixture was well mixed and centrifuged for 5 minutes at 13,000 rpm.

Protein extraction buffer composition was 25 mM Tris, 0.15 M sodium chloride, 50% glycerol (v/v) buffer at pH = 7.2. Furthermore 10μl_ of protease inhibitor coctail (Sigma P-8215) was added to 10 ml. of extraction buffer.

Protein concentration

The protein determination was performed using BCA assay (SIGMA) according to the manufacturer's instructions. BSA standards were prepared by dissolving BSA in protein extraction buffer. Blank was protein extraction buffer. The absorption at 465 nm was measured using Mithras (Berthold Technologies) or Beckman spectrophotometer. Standard curve was constructed and used to determine the concentration of the unknown samples.

Luminometry Luminometry was done using Mithras (Berthold Technologies) luminometer with two injectors. Flat bottom 96 well microtiter plates were used with each well capacity of 350 μl.

Renilla luciferase assay In vitro assay

Protein samples containing 20 μg total proteins in protein extraction buffer MM-X or MM-G were prepared. Aliquots were loaded into a 96 well plate. Luminescence was measured at 3O⁰C using a flash assay. A 100 μl injection of 2.5 μM coelenterazine was given and 1 s after injection two consecutive measurements were taken to determine renilla luciferase present in the protein sample. Blank wells were used to estimate background light levels. The measurement with highest relative light units reading was used and presented as RLU per g of protein.

Example 1. Construction of expression vector with renilla luciferase

The Renilla luciferase reporter plasmid was introduced in strains to be able to measure RNAi mediated reduction of gene expression using a specific enzymatic assay.

1.1 Construction of pMOJ004

Cloning techniques and plasmid DNA isolation were done according to known principles and routine plasmid isolation techniques (Sambrook, et al., supra). Expression vector pMOJ004 was constructed for transcription of reporter proteins. The Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter, gpdA, was amplified by PCR using A. nidulans genomic DNA as template and the gpdA- specific oligonucleotides SEQ ID NO: 61 and SEQ ID NO: 62, which additionally lead to the introduction of Sstl and Xbal - Ndel sites at the 5' and 3' ends of the gpdA promoter fragment, respectively. In a similar way the A. nidulans glutamine amidotransferase Il terminator, trpC, was amplified by PCR using oligonucleotides SEQ ID NO: 63 and SEQ ID NO: 64, respectively, leading to an introduction of Xbal - BamHI and EcoRI at the 5' and 3'ends of the trpC terminator, respectively.

The resulting PCR fragments were digested with Sstl and Xbal for the gpdA promoter fragment and Xbal and EcoRI for the trpC terminator fragment, gel purified, followed by a purification of a 0.88 kbp and 0.51 kbp products using a QIAquick GEL Extraction Kit (QIAGEN) according to manufacturer's instructions.

Consequently, the two PCR products were ligated into pBlueScript (Stratagene) digested with the same restriction enzymes using T4 DNA ligase (New England Biolabs, Promega) to generate pMOJ004 (Figure 1 ). The sequence of the PCR fragments in pMOJ004 comprising the gpdA and trpC regions were confirmed by sequence analysis.

1.2 Construction of pMOJ004-lucA Expression vector pMOJ004-lucA was constructed for the expression of Renilla luciferase. A fragment containing the luciferase gene sequence (AF362545 pGL4 Luciferase Reporter Vectors; Promega) was constructed by PCR amplification using 50 ng pGL4 as template and primers SEQ ID NO: 65 and SEQ ID NO: 66, which additionally lead to the introduction of Nde\ and SamHI sites at the respective ends of the fragment. The resulting PCR fragment was digested with Nde\ and SamHI, gel purified, followed by a purification of a the 0.94 kb DNA fragment using a QIAquick GEL Extraction Kit (QIAGEN), according to manufacturer's instructions. Consequently, the Nde\ and SamHI cleaved luciferase fragment was ligated into pMOJ004 digested with the same restriction enzymes using T4 DNA ligase to generate pMOJ004-lucA (Figure 2). The sequence of the renilla luciferase fragment in pMOJ004- lucA was confirmed by sequence analysis.

Example 2: Construction of vector for expression of RNAi molecules

2.1 Construction of PMOJ009

Expression vector pMOJ009 was constructed for transcription of small hairpin (sh) RNAi molecules.

The Aspergillus niger xylanase D promoter xlnD was amplified by PCR using 100 ng genomic DNA of WT1 as template and the xlnD-specific oligonucleotides SEQ ID NO:67 and SEQ ID NO: 68 and which additionally lead to the introduction of Sst\ at the 5' end and Xba\ - Λ/col sites and 3' end, respectively. In a similar way the A. nidulans glutamine amidotransferase Il terminator, trpC, was amplified by PCR using A. nidulans genomic DNA as template and oligonucleotides SEQ ID NO: 69 and SEQ ID NO: 70, leading to an introduction of Xba\ - EcoRI at the 5' end and Xho\ at the 3' ends of the terminator, respectively.

The resulting PCR fragments were digested with Sst\ and Xba\ for the xlnD promoter fragment and Xba\ and Xho\ for the trpC terminator fragment, gel purified, followed by a purification of a 0.86 kbp and 0.51 kbp products using a QIAquick GEL Extraction Kit (QIAGEN) according to manufacturer's instructions.

Consequently, the two PCR products were ligated into pBlueScript (Stratagene) digested with the same restriction enzymes using T4 DNA ligase (New England Biolabs, Promega) to generate pMOJ009 (Figure 3). The sequence of the PCR fragments in pMOJ009 comprising the xlnD promoter and trpC terminator were confirmed by sequence analysis.

2.2 Construction of pMOJ009-siR Expression vector pMOJ009-siR was prepared by insertion of a multiple cloning site in the vector pMOJ009 to create a basis construct for expression of RNAi molecules. A double stranded DNA fragment MCS1 was obtained by annealing the following oligonucleotides with sequence SEQ ID NO: 71 and SEQ ID NO: 72. These oligonucleotides were mixed at a same molar ratio, heated to 95⁰C for 5 minutes and cooled down slowly. An example of the layout of the MCS is given below.

Dsal Muni BamHI PIeI Apol

Ncol Mfel NIaIV MboI I Hinfl MaeII EcoRI

Styl SspBI Real

BsaJI BsrGI Acil BgIII BspHI BsiWI Acsl CCATGGAATG TACATCCAAT TGCGGATCCT TCAAGAGAAG ATCTTACTCA TGAGTCGTAC GTTGAATTC GGTACCTTAC ATGTAGGTTA ACGCCTAGGA AGTTCTCTTC TAGAATGAGT ACTCAGCATG CAACTTAAG

Vector pMOJ009-siR was constructed by ligation of the MCS1 dsDNA into pMOJ009 digested with Λ/col and EcoRI using T4 DNA ligase (Figure 4). The sequence of the MCS fragment in pMOJ009-siR was confirmed by sequence analysis.

2.3 Construction of renilla luciferase silencing vectors

Different types of silencing vectors can be constructed. In this example various shRNA's are constructed for triggering RNAi in filamentous fungi. All these constructs contain short gene specific oligonucleotides. Oligonucleotides were designed using either siRNA selector (Wistar Institute) or siRNA design (GeneScript) or BLOCK-iT™ RNAi Designer (Invitrogen).

2.3.1 Construction of silencing vector expressing single oligonucleotide gene target To construct a specific shRNA molecule containing a single 26 bp target renilla luciferase oligonucleotide, the sequences in Table 1 were used. The sense (S) and antisense (AS) sequences separated by the loop (L) are reverse complementary to each other. Table 1. Specific renilla luciferase oligonucleotide sequences for single target shRNA molecule. The underlined sequences represent the loops.

Name of Sequence of oligonucleotides carrying one gene target sequence of SEQ ID oligo renilla luciferase NO: s iRNA- CATGGTGGTGGGCTCGCTGCAAGCAAATGAATTCAAGAGATTCATTTGCTTG 73 H S CAGCGAGCCCACCAG siRNA- AATTCTGGTGGGCTCGCTGCAAGCAAATGAATCTCTTGAATTCATTTGCTTG 74

1 IAS CAGCGAGCCCACCAC siRNA- CATGGGGCCTCGCGAGATCCCTCTCGTTAAGTTCAAGAGACTTAACGAGAGG 75

12S GATCTCGCGAGGCCG siRNA- AATTCGGCCTCGCGAGATCCCTCTCGTTAAGTCTCTTGAACTTAACGAGAGG 76 12AS GATCTCGCGAGGCCC

The pMOJ012-siRNA-1 1 and pMOJ012-siRNA-12 vectors expressing the hairpin double strand renilla luciferase specific RNA molecule were obtained by annealing the corresponding sense and antisense oligonucleotides (Table 1 ) as describe above. Subsequently the vector pMOJ009 was digested with Ncol and EcoRI and the annealed double strand oligonucleotide sequence was ligated together with the vector using T4 DNA ligase (see Fig. 5A). The sequence of the pMOJ012-siRNA-1 1 and pMOJ012- siRNA-12 inserts was confirmed by sequence analysis.

2.3.2 Construction of silencing vector expressing double oligonucleotide gene target

For the construction of the pMOJ012-siRNA-14 shRNA double target expression vector the pMOJ009-siR basic vector was used. The two renilla luciferase specific oligonucleotide target sequences (each of 27 bp separated by a short 4 bp long spacer) were separated by a loop sequence present in the pMOJ009-siR vector, as indicated in the graph below (see Fig. 5B). The oligonucleotide sequences used to create the shRNA molecule with double gene target are shown in Table 2.

Table 2. Specific renilla luciferase oligonucleotide sequences for double target shRNAi molecule. The underlined sequence represents the 4 bp spacer. The loop is shown in bold. Name Sequence of oligonucleotides carrying double gene target SEQ ID sequence of renilla luciferase NO: s iRNA- GTACATGGTGGGCTCGCTGCAAGCAAATGAACCGAAGGCCTCGCGAG 77

14 S 1 +S2 ATCCCTCTCGTTAAGGG forward siRNA- GATCCCCTTAACGAGAGGGATCTCGCGAGGCCTTCGGTTCATTTGCT 78

14S1+S2 TGCAGCGAGCCCACCAT reverse siRNA- GATCTCCTTAACGAGAGGGATCTCGCGAGGCCCGAAGTTCATTTGCT 79

14AS2+AS1 TGCAGCGAGCCCACCAC forward siRNA- GTACGTGGTGGGCTCGCTGCAAGCAAATGAACTTCGGGCCTCGCGAG 80

14AS2+AS1 ATCCCTCTCGTTAAGGA reverse

The 5'> 3' sequence of the siRNA-14 molecule, SEQ ID NO: 81

CCATGGAATGTACATGGTGGGCTCGCTGCAAGCAAATGAACCGAAGGCCTCGCGAGATCCCTCTCGTTAAGGG

GATCC TTCSAGaGSAGATCT

CACCACGTACGTTGAATTC

The dsDNA oligonucleotides to create the siRNA-14 shRNA molecule (14S1 +S2 forward and reverse and 14AS2+AS1 forward and reverse) were obtained by mixing at the same molar range the corresponding forward and reverse oligonucleotides, heating to 95⁰C for 5 minutes and slowly cooling down as also described in example 2.2 above. The annealed dsDNA oligonucleotides, as indicated in the Table 2 above, were ligated into pMOJ009-siR, which was digested with BsrGI and BamHI, generating an intermediate construct. This intermediate construct was digested with BgIII and BsiWI and subsequently the anti-sense dsDNA oligonucleotide was ligated in the intermediate construct, generating the silencing construct pMOJ012-siRNA-14 (no picture included). The sequence of the pMOJ012-siRNA-14 insert was confirmed by sequence analysis.

2.3.3 Construction of silencing vector expressing triple oligonucleotide gene target To test the effectiveness of three oligonucleotide gene targets in one shRNA molecule the following double stranded DNA sequences were synthesized (see Table 3). The structure of the molecules is depicted in Fig. 5C and 5D. Table 3. Specific renilla luciferase oligonucleotide sequences for triple target shRNAi molecule. The underlined sequence represents the 4 bp spacer. The loop is shown in bold.

To create the expression vectors pMOJ012-siRNA-3533 and pMOJ012-siRNA-3534 the vector pMOJ009 and the synthesized dsDNA molecule (Table 3) were digested with Ncol and EcoRI and they were ligated together using T4 DNA ligase. The sequence was confirmed by sequencing the inserts of the final constructs.

Example 3. Overexpression of luciferase reporter gene in A. niger

In order to introduce the reporter constructs in A. niger WT2, a co-transformation and subsequent selection of transformants was carried out as described in WO98/46772 and WO99/32617. To obtain the luciferase reporter strains, DNA of vector pAN7-1 (containing the hygromycin B phosphotransferase gene - pAN7-1 : accession no. Z32698) was mixed with pMOJ004-lucA (Example 1 - see also Figure 2), and used to transform A. niger strain WT2. Transformants were selected for hygromycin resistance and colonies were purified according standard procedures. For all strains, a PCR analysis and Southern analysis were carried out to determine positive co-transformants and copy numbers.

The primary renilla luciferase transformants of A. niger strains transformed with pMOJ004-lucA and pAN7-1 were submitted to a renilla luciferase activity assay as described above. A strain having a high luciferase activity, a similar phenotype for growth as the parental WT2 strain and a single pMOJ004-lucA copy number was chosen as reporter strain for RNA silencing experiments. This strain was named LUC-A.

Example 4. Introduction of silencing vectors in A. niger luciferase reporter strain

In order to introduce the respective silencing constructs in A. niger strain LUC-A, a co- transformation and subsequent selection of transformants was carried out as described in WO98/46772 and WO99/32617. To obtain luciferase reporter strains with a copy of an appropriate RNA interference construct, DNA of vector pGWE5B (Figure 6) was mixed with a co-transforming plasmid as indicated below in Table 4 and used to transform the A. niger luciferase strain LUC-A.

Table 4. Transformation scheme for strains and constructs indicated.

Transformants for all plasmid combinations were selected for uridin / uracil prototrophy (plates without uridin / uracil) and colonies were purified according standard procedures. For all strains, a PCR analysis and Southern analysis were carried out to determine positive co-transformants and low copy numbers. Representative strains were named and numbered as indicated in Table 4.

Example 5. Silencing of luciferase reporter in LUC-A derived silencing strains

A number of selected transformants as indicated in Table 4, were cultured in shake flask for 24 to 26 h as indicated before in MM-G media (siRNA repressing condition) and MM-X media (siRNA inducing condition). Mycelia were collected and proteins were extracted from powdered mycelia with the extraction buffer as described before. Renilla luciferase activities per gram of total proteins of A. niger were measured for all transformants for both growth conditions. The ratio of the luciferase activity of the respective strains when grown on xylose medium versus glucose is indicated in Figure 7. The activities measured clearly indicate that under shRNA inducing condition, a substantially reduced renilla luciferase activity is measured. This is a clear indication for silencing. All silencing vectors tested show a reduction although the vector with a triple target sequence for the target gene seems to work best.

Example 6. Identification of RNAi related genes in filamentous fungi.

The following examples describe the modulation of RNAi related genes in an A. niger host. After construction, the ability for RNAi mediated reduction of gene expression is measured for a host strain containing the renilla luciferase protein and a silencing vector.

Genomic DNA's of Aspergillus niger strain WT1 and Penicillium chrysogenum strain WT3 were sequenced and analyzed. A number of RNAi related genes were identified. The all have translated proteins associated with the process of RNAi mediated reduction of gene expression. These polypeptides include, but are not limited to: DICER (dsRNA processing enzyme), RdRP (RNA dependent RNA polymerase), polypeptides of the RISC (RNA induced silencing complex) - nuclease complex containing translation initiation factor, RNA-DNA helicase, specific nuclease, RNA binding proteins. In the following Table 5, the A. niger sequences for RNAi related genes, comprising the genes (Gene - with introns) with 5'- and 3'-ends of the genes, coding sequences (CDS) and encoded proteins are indicated with their reference to the sequence listing.

Table 5. RNAi related genes from A. niger.

In the following table 6, the P. chrysogenum sequences for RNAi related genes, comprising the genes (Gene - with introns) with 5'- and 3'-ends of the genes, coding sequences (CDS) and encoded proteins are indicated with their reference to the sequence listing. Table 6. RNAi related genes from P. chrysogenum.

Example 7. Construction of overexpression vectors for RNAi related genes.

Overexpression vectors for the genes mentioned above were designed according to known principles and constructed according to routine cloning procedures. Examples of the general design of expression vectors and the use of expression vectors for gene over expression can be found in WO199932617, WO200121779 and WO2005100573. In essence, expression vectors comprise at least a promoter and terminator for proper expression of a gene. The genomic DNA or cDNA can be used for cloning and expression of the RNAi related genes. A selection marker for transformation, such as the A. nidulans bi-directional amdS selection marker can be on the vector or can be used as separated vector in co-transformation. Examples of pGBTOP-based or pGBFIN-based expression vectors can be found in figure 8 or 9. A number of A. niger genes listed in Table 5 were cloned in a pGBFIN-based overexpression vector. An example of such an overexpression vector for Dicer (dcrA) is found in figure 8, which is named pGBFINDCR-1 . Another example of such an overexpression vector for RNA- dependent RNA polymerase (qdeA) is named pGBFINRDR-1 (plasmid map not shown).

Example 8. Construction of A niger strains with improved abilities for RNAi mediated reduction of gene expression.

Linear DNA of overexpression vectors, such as pGBFINDCR-1 and pGBFINRDR-1 constructed in Example 7, was isolated and used to transform Aspergillus niger LUC-A and LUC-siR-3533-435 using the method earlier described. This linear DNA can integrate into the genome and transformants were selected on acetamide media and colony purified according to standard procedures as described in EP635574A2. Growing colonies were diagnosed by PCR for integration of one of the respective genes of interest and candidate strains were tested by Southern analyses for single copy introduction of the respective genes. Strains LUC-DCR-1 and LUC-siR-3533-435- DCR-1 were selected as representative strains for overexpression of the dcrA gene (SEQ ID NO: 10), encoding a Dicer protein (SEQ ID NO: 12) in their respective backgrounds. In a similar way, strains LUC-RDR-1 and LUC-siR-3533-435-RDR-1 were selected as representative strains for overexpression of the qdeA gene (SEQ ID NO: 28), encoding a RNA-dependent RNA polymerase protein (SEQ ID NO: 30).

Example 9. Improved silencing of luciferase reporter using overexpression of A niger RNAi related genes

All selected transformants as constructed earlier and indicated below, were cultured in shake flask as indicated before for 24 h in MM-G media (siRNA repressing condition) and MM-X media (siRNA inducing condition). Mycelia were collected and proteins were extracted from powdered mycelia with the extraction buffer as described before. Renilla luciferase activities per gram of total proteins of A. niger were measured for all transformants for both growth conditions. The ratio of the luciferase activity of the respective strains when grown on glucose medium versus xylose is indicated in Figure 10.

Surprisingly, renilla luciferase activities measured clearly indicate that overexpression of RNAi related genes, results in a substantially reduced renilla luciferase activity compared to a normal silencing condition. This is a clear indication that silencing can be improved drastically by overexpression of RNAi related genes, such as a Dicer protein or a RNA-dependent RNA polymerase protein.

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Claims

1. A modified filamentous fungal cell suitable for the production of a compound of interest, wherein, compared to the parental cell the modified cell derives from, said modified cell exhibits improved abilities for RNAi mediated reduction of gene expression of a target gene and/or homologues thereof, said target gene encoding a biological compound.

2. The modified filamentous fungal cell according to claim 1 , wherein said cell is a recombinant filamentous fungal cell.

3. The modified filamentous fungal cell according to any one of claims 1 or 2, wherein the expression of at least one RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof is modulated.

4. The modified filamentous fungal cell according to any one of claims 1 to 3, comprising a RNAi nucleic acid construct comprising a promoter operably linked to an RNAi unit of a target gene, said RNAi unit comprising: a. a first transcriptionally competent nucleotide sequence comprising a first transcriptionally competent homologous region of a target gene, and b. a second transcriptionally competent nucleotide sequence comprising a second transcriptionally competent homologous region of said target gene, wherein the first and second transcriptionally competent homologous region of a target gene are reverse-complementary relative to each other, and optionally c. a third transcriptionally competent nucleotide sequence separating the first and second transcriptionally competent nucleotide sequence.

5. The modified filamentous fungal cell according to claim 4, wherein: a. a first transcriptionally competent homologous region of a target gene comprises at least 19 nucleotides, and/or b. a second homologous transcriptionally competent region comprises at least 19 nucleotides of the first homologous region, wherein said nucleotides are in reverse-complementary orientation relative to the first homologous region, and/or c. an optional third transcriptionally competent nucleotide sequence comprises at least 5 nucleotides.

6. The modified filamentous fungal cell according to claim 4, wherein: a. a first transcriptionally competent homologous region comprises at most 19, preferably at most 18, more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most 14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 1 1 , even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, and most preferably at most 7 nucleotides of a target gene, and/or b. a second transcriptionally competent homologous region comprises at most 19, preferably at most 18, more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most 14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 1 1 , even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, and most preferably at most 7 nucleotides of the first homologous region, wherein said nucleotides are in reverse- complementary orientation relative to the first homologous region and/or c. an optional third transcriptionally competent nucleotide sequence comprises between 5 and 9 nucleotides.

7. The modified filamentous fungal cell according to any one of claims 4 to 6, wherein the RNAi nucleic acid construct comprises at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 and most preferably at least 6 repeats of the RNAi unit, each RNAi unit being operably linked to a promoter.

8. The modified filamentous fungal cell according to any one of claims claim 4 to 6, wherein the RNAi nucleic acid construct comprises one promoter operably linked to at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 and most preferably at least 6 repeats of the RNAi units.

9. The modified filamentous fungal cell according to any one of claims 7 or 8, wherein the nucleic acid construct comprises at least two RNAi units of distinct target genes.

10. The modified filamentous fungal cell according to any one of claims 1 to 9, further comprising a gene encoding a compound of interest to be produced.

1 1. The filamentous fungal cell according to any one of claims 1 to 10, wherein the modified filamentous fungal cell is a cell selected from the group consisting of: Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus sojae, Penicillium chrysogenum, Trichoderma reesei, Fusarium oxysporum, Mortierella alpina.

12. A method for preparing the modified filamentous fungal cell according to any one of claims 1 or 3, said method comprising: a. mutation of a filamentous fungal cell by random mutagenesis, and b. screening from the pool resulting from (a) for a modified filamentous fungal cell wherein abilities for RNAi mediated reduction of gene expression of a target gene encoding a biological compound are improved compared to the parental filamentous fungal cell, and c. optionally isolating the modified filamentous fungal cell of (b)

13. A method for preparing the modified filamentous fungal cell according to claim 3, said method comprising: a. inserting into the filamentous fungal cell at least one nucleic acid construct comprising, operably linked to a promoter, at least one RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (1 19 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof, and/or b. rendering inactive at least one native RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (1 19 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof by specific mutagenesis, site-directed mutagenesis, PCR generated mutagenesis, nucleotide insertion and/or deletion and/or substitution, gene interruption or gene replacement techniques, or a combination thereof.

14. A method for preparing the modified filamentous fungal cell according to any of claims 4 to 1 1.

15. The method according to any one of claims 12 to 14, wherein the modified filamentous fungal cell is a cell selected from the group consisting of:

Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus sojae, Penicillium chrysogenum, Trichoderma reesei, Fusarium oxysporum, Mortierella alpina.

16. A method for reducing the expression of a target gene and/or homologue thereof in the modified filamentous fungal cell according to any of claims 1 to 1 1.

17. A method for reducing the expression of a target gene and/or homologue thereof in the modified filamentous fungal cell according to claim 16, said method comprising: a. inducing recombinant expression of at least one RNAi related gene selected from the group consisting of SEQ ID NO: (1 + 3n), SEQ ID NO: (119 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof, by culturing the modified filamentous fungal cell under conditions conducive to expression of the RNAi related gene, and/or b. inducing expression of both an RNAi unit into an interfering RNA and expression of a target gene into RNA transcripts, by culturing the modified filamentous fungal cell under conditions conducive to production of interfering RNA, wherein the interfering RNA interacts with the RNA transcripts of the target gene encoding the biological compound to reduce the expression of the target gene encoding the biological compound.

18. The method for reducing the expression of a target gene or homologue thereof according to any one of claims 16 to 17, wherein the expression of the target gene and/or homologues thereof is reduced by at least 50 %, more preferably at least 60 %, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, more preferably by at least 95%, more preferably by at least 98%, more preferably by at least 99%, and most preferably by 100%.

19. A nucleotide sequence derivable from a filamentous fungus encoding SEQ ID NO: (3 + 3n), SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof, wherein the nucleotide sequence is selected from the group consisting of: a. SEQ ID NO: (1 + 3n), SEQ ID NO: (1 19 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, or a homologue thereof; and, b. fragments or mutants of any one of the nucleotide sequences of (a)

20. The nucleotide sequence encoding SEQ ID NO: (3 + 3n), SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18 according to claim 19, said nucleotide sequence encoding a polypeptide, wherein the amino acid positional identity with any one of the sequences of the group consisting of SEQ ID NO: (3 + 3n), SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18 is more than

40% and preferably is at least about 50%, more preferably at least about 60%, even more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 97%, even more preferably at least about 98%, even more preferably at least about 99% identity, and most preferably, is equal to 100%..

21. A nucleic acid construct comprising a nucleotide sequence according to claim 20

22. The nucleic acid construct according to claim 21 comprising a promoter, which is native to the nucleotide sequence.

23. The nucleic acid construct according to claim 21 comprising a promoter, which is foreign to the nucleotide sequence.

24. The nucleic acid construct according to any one of claims 21 to 23 comprising direct repeats suitable for gene replacement by double cross-over mediated integration of the nucleic acid construct.

25. An RNAi nucleic acid construct comprising a promoter operably linked to an RNAi unit of a target gene, said RNAi unit comprising: a. a first transcriptionally competent nucleotide sequence comprising a first transcriptionally competent homologous region of a target gene, and b. a second transcriptionally competent nucleotide sequence comprising a second transcriptionally competent homologous region of said target gene, wherein the first and second transcriptionally competent homologous region are reverse-complementary relative to each other, and optionally c. a third transcriptionally competent nucleotide sequence separating the first and second transcriptionally competent nucleotide sequence.

26. The RNAi nucleic acid construct according to claim 25, wherein a. a first transcriptionally competent homologous region of a target gene comprises at least 19 nucleotides of a target gene, and/or b. a second transcriptionally competent homologous region comprises at least 19 nucleotides of the first homologous region, wherein said nucleotides are in reverse-complementary orientation relative to the first homologous region, and/or c. an optional third transcriptionally competent nucleotide sequence comprises at least 5 nucleotides.

27. The RNAi nucleic acid construct according to claim 25, wherein: a. a first transcriptionally competent homologous region comprises at most 19, preferably at most 18, more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most 14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 1 1 , even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, and most preferably at most 7 nucleotides of a target gene, and/or b. a second transcriptionally competent homologous region comprises at most 19, preferably at most 18, more preferably at most 17, even more preferably at most 16, even more preferably at most 15, even more preferably at most 14, even more preferably at most 13, even more preferably at most 12, even more preferably at most 1 1 , even more preferably at most 10, even more preferably at most 9, even more preferably at most 8, and most preferably at most 7 nucleotides of the first homologous region, wherein said nucleotides are in reverse- complementary orientation relative to the first homologous region and/or c. a optional third transcriptionally competent nucleotide sequence comprises between 5 and 9 nucleotides.

28. The RNAi nucleic acid construct according to any one of claims 25 to 27, comprising at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 and most preferably at least 6 repeats of the RNAi unit, each RNAi unit being operably linked to a promoter.

29. The RNAi nucleic acid construct according to any one of claims 25 to 27, comprising one promoter operably linked to at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 and most preferably at least 6 repeats of the RNAi units.

30. The RNAi nucleic acid construct according to any one of claims 28 or 29, comprising at least two RNAi units of distinct target genes.

31. The RNAi nucleic acid construct according to any one of claims 25 to 30 comprising a promoter, which is native to the RNAi unit.

32. The RNAi nucleic acid construct according to any one of claims 25 to 30 comprising a promoter, which is foreign to the RNAi unit.

33. A polypeptide encoded by the nucleic acid sequence according to any one of claims 19 or 20, wherein the amino acid sequence is selected from the group consisting of: a. SEQ ID NO: (3 + 3n), SEQ ID NO: (121 + 3m), n being an integer selected from the set of 0<= n <= 19 and m being an integer selected from the set of 0<= m <= 18, and b. fragments or mutants of any one of the amino acid sequences of (a)

34. A method for the production of a compound of interest in the modified filamentous fungal cell of claim 10, said method comprising: a. reducing the expression of a target gene and/or homologues thereof according to any one of claims 16 or 18, and b. culturing the modified filamentous fungal cell under conditions conducive to the production of the compound of interest, and c. optionally recovering the compound of interest from the culture medium, and d. optionally purifying the compound of interest.

35. The method of claim 34, wherein the modified filamentous fungal cell is a cell selected from the group consisting of: Aspergillus niger, Aspergillus nidulans,

Aspergillus oryzae, Aspergillus sojae, Penicillium chrysogenum, Trichoderma reesei, Fusarium oxysporum, Mortierella alpina.