AU6323800A - Novel means of transformation of fungi and their use for heterologous protein production - Google Patents

Description

WO 01/09352 PCT/NLOO/00544 Novel means of transformation of fungi- and their use for heterologous protein production. SUMMARY OF THE INVENTION 5 The invention relates to novel means of transformation of fungi and to their use for production of heterologous proteins. The means involve genetically engineered fungi belonging to the taxonomic group Aspergillus sojae. Suggestions have been provided in the past to use Aspergillus sojae as a host strain for transformation. However to date no data are provided on successful transformation and/or expression of heterologous proteins. 10 In addition it has been found, that so far certain proteins, such as phytase which were difficult to express in large amounts, due to several reasons including proteolytic degradation in expression hosts other than Aspergillus sojae, can surprisingly be expressed in Aspergillus sojae. Production levels for heterologous proteins in Aspergillus sojae have been found to exceed those levels achieved for the same proteins in Aspergillus niger and 15 Aspergillus awamori. In addition to the above, the subject of the invention further covers a process for obtaining improved Aspergillus sojae strains for expression purposes, characterized by, on the one hand, a decreased proteolytic activity, and, on the other hand improved fermentation characteristics related to the morphology of the fungi. 20 BACKGROUND OF THE INVENTION Suggestions have been provided in the past to use Aspergillus sojae as a host for transformation. However, to date no data are provided on successful transformation and/or production of heterologous proteins and, more specifically, nothing is revealed concerning expression of phytase. Previously, expression levels were too low in expression hosts other 25 than Aspergillus sojae, mainly due to proteolytic degradation. We have now found expression levels for the protein in Aspergillus sojae that exceed those levels achieved for the same protein in other strains, e.g. Aspergillus niger, Aspergillus awamori and Trichoderma. It is surprising to find such an improvement in closely related strains. Thus, prior art disclosures concerning phytase production exhibit shortcomings. Prior art 30 disclosures on the use of Aspergillus sojae for expressing heterologous proteins or polypeptides were inadequate. The fact that until now hardly any successful attempts for A. sojae transformation have been reported is remarkable in view of the fact that numerous successful transformations of closely related strains of the taxonomic group Aspergillus oryzae have WO 01/09352 PCT/NLOO/00544 2 been described in the past. On the basis of this close relationship the skilled person would anticipate (and in fact did anticipate) that analogous methods to those used for Aspergillus oryzae are also applicable for Aspergillus sojae strains. W097/04108 for example describes the isolation of a protease encoding nucleic 5 acid sequence, specifically a leucine aminopeptidase encoding sequence, and the transformation of a variety of host organisms, i.a. Aspergillus sojae, with a leucine aminopeptidase encoding sequence. However no illustration of this particular transformation actually having been carried out is provided. It is merely suggested as one possibility among many other strains such as Trichoderma reesei, Aspergillus niger, 10 Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus phoenicis, and Aspergillus oryzae as a potential host strain to be used for transformation. In the cited document, 3 transformation protocols readily used in the art are suggested for the strains. Specifically any of the selection markers acetamidase S (=amdS) (e.g. as maintained on vector p3SR2), argB or hygromycin B (e.g. using the vector pAN7-1) are suggested as 15 being suitable markers to be used according to the transformation protocols described therein. The use of vector p3SR2 with the amdS marker has frequently been described in the literature as being useful for transforming various strains, for example Aspergillus oryzae (in EP 0.238.023), Trichoderma reesei (in EP 0.244.234) and Aspergillus niger 20 (EMBO Journal 4, pages 475-479). Consequently, the analogous use for transforming Aspergillus in general is put forward in W097/04108 on the basis of these previous publications. Quite specifically on page 17 of W097/04108 it is described that Aspergilli and Trichoderma that prior to transformation grew slowly on minimal medium comprising 25 solely the substrate acetamide as source of nitrogen could be selected after transformation with the vector p3SR2 due to a clear growth advantage. Subsequently, the thus obtained transformants would need to be further subjected to selection for leucine aminopeptidase (=LAP) productivity in order to find a desired transformant. As stated above this is merely put forward as speculative means of transformation applicable over the two 30 aforementioned genera in toto based on a few successful transformations of strains other than Aspergillus sojae. The suggested transformation protocol is, however, unsuccessful with Aspergillus sojae. The selection criteria described in the prior art are insufficient to ensure practical selection of desirable transformants when using the vector p3SR2. We have conducted the WO 01/09352 PCT/NLOO/00544 3 experiments and found the described method inoperable due to excessive background growth eliminating practical selectability. Another routinely used selection method for fungal transformants is that of transformation of orotidine-5-monophosphate decarboxylase (=PyrG) mutants. Mattern et 5 al. in Mol. Gen. Genet. 210, pages 460-461 disclose transformation of Aspergillus oryzae using the Aspergillus niger pyrG gene. Standard practice is to isolate pyrG mutants based on direct resistance to fluoro-orotic acid as a positive selection marker. This has resulted in isolation of numerous pyrG mutants for a variety of fungi to date. From experience with a number of different filamentous fungi, the auxotrophic 10 pyrG-based system has many favourable characteristics. Experiments were carried out to obtain A. sojae pyrG mutant strains, using a standard procedure based on direct selection for resistance to fluoro-orotic acid (FOA) on plates containing uridine to support growth of the mutant strain (Van Hartingsveldt et al. in Mol. Gen. Genet. (1987) 206, pages 71-75). However, use of the analogous method on Aspergillus sojae strains did not lead to pyrG 15 mutants. The usual method did lead to fluoro-orotic acid resistant strains but all the strains were able to grow without uridine. Thus, none of these strains were pyrG mutants. Normally, the isolation of the pyrG mutants can be done directly from the fluoro-orotic acid resistant strains on a uridine selection medium. For Aspergillus sojae this method however turned out to be inoperable. 20 Clearly, Aspergillus sojae exhibits different traits than the closely related Aspergillus oryzae when it comes down to transformation. The standard protocols using amdS or pyrG as selectable markers do not suffice. Unfortunately, the method of argB as selectable marker is not an attractive option either, since this requires isolation of a corresponding argB mutant for every host strain one wishes to use. This is an arduous task based on trial 25 and error. The required argB mutant can be obtained through random mutagenesis followed by screening of tens of thousands of colonies. The situation for pyrG is better in that the mutant is itself selectable. In the case of amdS no mutant is required as the presence of amdS works as dominant selectable marker. Additional problems dissuading the skilled person from use of Aspergillus sojae as 30 expression host for recombinant proteins or polypeptides exist. In JP-A-02-234666 for example an ArgB based selection of Aspergillus sojae is described using an analogous protocol to that described for other fungi. Such a process has been described for Aspergillus oryzae in Biotechnology (1988) 6, pages 1419-1422. The cited article also refers to successful analogous transformation of Aspergillus nidulans and Aspergillus WO 01/09352 PCT/NLOO/00544 4 niger. However, when the Aspergillus sojae strain ATCC42251 disclosed in the Japanese patent application was analysed, an undesirable protease profile was found. The protease profile of this strain is incompatible for application as a production host. So even though a transformation protocol has been suggested in the prior art for this particular Aspergillus 5 sojae strain it could not possibly lead to a high level of expression of heterologous protein even if the protocol for transformation was successful. It is in fact due precisely to the explicit characteristics of Aspergillus sojae strains to produce excessive amounts of alkaline proteases and amylases that they currently find application in practice. They are used specifically in processes requiring degradation of 10 complex polymeric substrates. It was thus at best to be expected that any transformants of Aspergillus sojae that are finally successful will not lead to good expression levels unless the product is an Aspergillus sojae protein that is impervious to its own proteases. In summary the problems facing the skilled person in finding a means to use Aspergillus sojae strains for expressing heterologous recombinant proteins on an industrial 15 scale are manifold. Firstly, a number of processes for introducing the desired nucleic acid material to be expressed are not applicable in the manner used for other fungi. This includes pyrG- and amdS-based processes that are useful for the closely related Aspergillus oryzae. Secondly, it remained to be seen whether high level production of heterologous proteins would be feasible despite the known excessive proteolytic activity of the host 20 strain Aspergillus sojae. Unexpectedly, it has been found that the problems addressed above can be solved, thus resulting in novel expression hosts for producing proteins and novel methods of production of heterologous proteins. We describe transformation of A. sojae strains with the amdS and pyrG selection markers. In addition efficient gene expression is described, 25 including expression of a phytase gene. DESCRIPTION OF THE INVENTION As stated the subject invention is directed at Aspergillus sojae strains and the application thereof for production of recombinant proteins and polypeptides. Firstly, a 30 description of Aspergillus sojae strains is provided. Aspergillus sojae determination. The fungal taxonomy is a complex issue. The Aspergillus genus comprises Aspergillus sojae in the Flavi/Tamarii section (see Table 1). A. sojae is clearly shown to be WO 01/09352 PCT/NLOO/00544 5 distinct from A. oryzae which is located in the same section (see Table 2). Currently, strains belonging to Aspergillus sojae can be distinguished from taxonomically closely related Aspergillus oryzae and also closely related Aspergillus parasiticus strains in a number of manners recognised in the art. Reference is made to the random PCR fragments, 5 ver-1, aflR and rDNA sequences as described, respectively, in Ushijama et al. (1981), Chang et al. (1995), Yuan et al. (1995), Kusomoto et al. (1998) and Watson et al. (1999). In addition it has been found that Aspergillus oryzae further differs from Aspergillus sojae upon comparison of the alpA sequence of these strains. Inter alia (there are other sequence differences between A. oryzae and A. sojae alpA which could be used as a determination 10 tool), it has been found that Aspergillus sojae comprises an XmnI restriction site at a specific location in the alpA gene. The corresponding location in the alpA gene of several Aspergillus oryzae strains does not possess such a restriction site. Thus, this provides an additional discrimination point between the two types of fungal strains. Consequently, numerous methods are available to the skilled person to assess whether a strain is an 15 Aspergillus sojae. Currently more than 10 strains are deposited with the ATCC that are defined as Aspergillus sojae. The 10 oldest deposits have been analysed. Two out of 10 did not pass the lastly mentioned determination test. One of them is the ATCC20235 which according to Ushijama et al. (1981) also did not fulfil the requirements for classification as an Aspergillus sojae on the basis of morphological parameters. The other is ATCC46250. 20 The definition of Aspergillus sojae as used throughout the patent application is meant to imply a strain that preferably fulfils all the requirements described in the cited references in combination with the presence of the XmnI restriction site in the alpA gene. Specific homologous primers for both the Aspergillus oryzae and Aspergillus sojae sequences are also provided. They can be used to test for the presence of the XmnI restriction site by way 25 of example of a screening test useful for distinguishing Aspergillus oryzae from Aspergillus sojae (Primer sequences are SEQ ID No.1 MBL1784: 5' CGGAATTCGAGCGCAACTACAAGATCAA-3' and SEQ ID No.2 MBL1785: 5' CGGAATTCAGCCCAGTTGAAGCCGTC-3'). They are derived from the coding region of the alpA gene. It will be obvious to the skilled person on the basis of the known 30 sequence data that alternative probes or primers are conceivable. PCR amplification using these primers on Aspergillus DNA, followed by restriction enzyme digestion of the resulting DNA fragments with XmnI provides a way to discriminate A. sojae strains from A. oryzae strains. Having established the definition of Aspergillus sojae strains we can proceed further with the detailed description of the invention.

WO 01/09352 PCT/NL00/00544 6 The invention in one aspect covers a recombinant Aspergillus sojae comprising an introduced acetamidase S (amdS) gene as a selectable marker. Such an A. sojae is selectable on a medium comprising a substrate for the introduced amdS protein as sole source of nitrogen, said medium further comprising a carbon substrate and said medium 5 being free of endogenous amdS inducing substrate. A suitable medium comprises acrylamide as substrate for the introduced amdS as sole source of nitrogen. A suitable medium at least further comprises minimum substrates required for growth of Aspergillus sojae. A suitable category of A. sojae according to the invention is formed by A. sojae that are not selectable on acetamide comprising medium. An A. sojae according to the 10 invention is suitably an A. sojae selectable on a medium free of glucose, i.e. a medium wherein the carbon source is not glucose. Such a medium can be a medium having sorbitol as carbon source. Best results in the case of sorbitol are achieved when sorbitol is the sole carbon source. An Aspergillus sojae according to the invention may comprise a further introduced 15 nucleic acid sequence, said further introduced sequence preferably encoding a protein or polypeptide. The further introduced sequence may be adapted for optimised codon usage to the host strain codon usage or may have the original codons from the host from which it has been derived. The introduced sequence is in principle any sequence the skilled person wishes to express. The introduced sequence can suitably be heterologous, i.e. foreign to the 20 Aspergillus sojae into which it is introduced. It can also be native but introduced in the form of one or more additional copies. One of the subjects of the invention is aimed at expressing phytase or proteins having phytase activity. Numerous sequences are known to the skilled person concerning sequence data of phytases. We refer to and incorporate by reference the contents of EP 25 684.313, EP 897.010, WO 99/49022, EP 911.416 and EP 897.985. These documents describe various natural and modified phytase sequences. They also describe a consensus sequence. A suitable embodiment is formed by phytase sequences from Peniophora being either the natural sequences or modified versions thereof. The new system is more flexible than prior systems and thus heterologous sequences, including heterologous sequences 30 encoding phytase or proteins having phytase activity that were difficult to express in the prior art fungal systems can be expressed in the novel system according to the invention. An Aspergillus sojae according to the invention as defined in any of the embodiments defined above comprising an introduced amdS gene as selectable marker may suitably have no active endogenous amdS gene. The Aspergillus sojae according to such an embodiment WO 01/09352 PCT/NL00/00544 may by way of example have an endogenous amdS gene comprising an endogenous amdS inactivating mutation. Any type of inactivating mutation known or conceivable to the skilled person may have occurred. A suitable example of such inactivating mutation may be a deletion or disruption. The mutation may inactivate the gene or the gene product. The 5 skilled person will realise that numerous options are available to achieve this and that they can readily be achieved. In an alternative embodiment the invention is also directed at a recombinant Aspergillus sojae free of an active endogenous amdS gene and further comprising an introduced amdS gene as selectable marker. The recombinant Aspergillus sojae according 10 to the invention is selectable on a medium comprising a substrate for the amdS as sole source of nitrogen, said medium further comprising a carbon substrate. A suitable medium at least further comprises minimum substrates required for growth of A. sojae. In a suitable embodiment the endogenous amdS gene can for example have been inactivated. This inactivation can be any type of inactivation known or conceivable to a person skilled in the 15 art that still leaves the A. sojae viable. By way of example the endogenous amdS gene can comprise an inactivating mutation in the form of a substitution, deletion or insertion of the gene or part thereof, or by virtue of a mutation affecting expression of the gene such as to render it inactive. The complete endogenous amdS gene can also be absent. An Aspergillus sojae in any of the described embodiments according to the invention 20 may be an A. sojae into an amdS gene has been introduced. This can be achieved e.g. by transformation or transfection. The resulting Aspergillus sojae according to the invention must then subsequently have been separated from non transformed or transfected A. sojae. Any of the embodiments described above as such or in combination are covered by the invention. 25 The invention not only covers Aspergillus sojae as such, but also covers a method of introducing a nucleic acid sequence into A. sojae. The method comprises subjecting Aspergillus sojae to introduction of a nucleic acid sequence in a manner known per se for introduction of a nucleic acid sequence into a fungus. Such a manner can e.g. be trans formation or transfection of the A. sojae. The method comprises the introduction of the 30 amdS gene as the nucleic acid sequence followed by selection of the resulting transformed or transfected A. sojae on a medium free of endogenous amdS inducing substrate, said medium further comprising a substrate for the introduced amdS as sole source of nitrogen and said medium further comprising a carbon substrate, said medium enabling the desired A. sojae comprising introduced amdS gene to grow whilst eliminating growth of A. sojae WO 01/09352 PCT/NLOO/00544 8 devoid of a functional amdS gene. A suitable embodiment of such a method involves applying a medium comprising a substrate for amdS other than acetamide. Suitably, such a medium comprises acrylamide as substrate for the introduced amdS as sole source of nitrogen. Suitably, a medium for the method according to the invention comprises a carbon 5 source other than glucose. Suitably, a medium for use in a method according to the invention comprises sorbitol as carbon source, preferably as sole carbon source. A suitable medium at least further comprises minimum substrates required for growth of A. sojae. A method according to the invention as defined above in any of the embodiments comprises introduction of an additional nucleic acid sequence besides the amdS gene. The 10 additional nucleic acid sequence for example encodes a protein or polypeptide, such as a phytase or proteins having phytase activity. The sequence does not necessarily have to be a non Aspergillus sojae sequence, but can also include A. sojae derived sequences It is however intended to indicate that the sequence that is introduced is absent in the non transformed strain or else is present in a lower copy number than in the A. sojae according 15 to the invention. Naturally, the subject invention also covers any Aspergillus sojae obtained by the method described above. Basically, the method is directed at introducing a sequence capable of realising the presence of sufficient active amdS to function as selectable marker as opposed to the A. sojae into which the sequence is introduced which cannot for some 20 reason or another produce sufficient active amdS to enable growth on a substrate for amdS as sole source of nitrogen. A method of selecting transformed or transfected A. sojae also falls within the scope of the invention. The method comprises subjecting A. sojae (with no active endogenous amdS gene as defined according to any of the embodiments described) to a method of 25 transformation or transfection of the A. sojae in a manner known per se for transformation or transfection of fungi with a nucleic acid sequence. The method comprises the introduction of an amdS gene as the nucleic acid sequence, followed by selection of the resulting transformed or transfected A. sojae, said selection occurring on a medium comprising a substrate for the introduced amdS as sole source of nitrogen, said medium 30 further comprising a carbon substrate, said medium enabling the desired A. sojae to grow whilst eliminating growth of non transformed or transfected A. sojae due to inability of such to grow without the introduced amdS gene on the selection medium. A suitable medium at least further comprises minimum substrates required for growth of A. sojae. The invention is also directed at a method for producing recombinant Aspergillus WO 01/09352 PCT/NLOO/00544 9 sojae. This method comprises introducing a desired nucleic acid sequence e.g. by transformation or transfection in a manner known per se into an A. sojae, said desired nucleic acid sequence being flanked by sections of the endogenous amdS gene of a length and homology sufficient to ensure recombination. The introduction is followed by 5 selection of the recombinant A. sojae having the desired nucleic acid sequence. The selection occurs for a selectable marker comprised in or transformed in cotransformation with the desired nucleic acid sequence, said selectable marker being absent in the A. sojae prior to introduction of the desired nucleic acid sequence. The flanking sequences may also be sequences corresponding to the endogenous amdS gene sufficient to ensure 10 recombination. The skilled person can readily assess which sequences will suffice on the basis of hybridisation knowledge and the sequence data of the endogenous amdS gene. The recombination event eliminates the endogenous amdS activity in both cases. The selectable marker can quite suitably be pyrG, with, however, uracil instead of uridin in the selection medium. 15 A further embodiment of the invention comprises Aspergillus sojae exhibiting growth on medium comprising uracil and fluoro-orotic acid, said A. sojae further not exhibiting growth on medium comprising uridine and fluoro-orotic acid. This means that the A. sojae exhibits uracil auxotrophy, is unable to utilize uridine, is pyrG negative and exhibits resistance to fluoro-orotic acid. The uracil auxotrophy and the fluoro-orotic acid resistance 20 are relievable upon complementation with an active introduced pyrG gene. Such an A. sojae according to the invention can be free of active endogenous pyrG genes. The pyrG negative A. sojae according to the invention may comprise an endogenous pyrG gene with a mutation inactivating it. The mutation can be any mutation known or conceivable to a person skilled in the art, said mutation inactivating a pyrG gene or the expression product 25 thereof. Such a mutation can by way of example be in the form of an insert of a nucleic acid sequence in the gene, a substitution of a part of the encoding sequence of the gene, a deletion of a part of the encoding sequence of the gene or a deletion of the whole encoding sequence of the gene. The mutation can also occur in the regulating part of the gene. In the case of Aspergillus sojae according to the invention with a mutated pyrG gene, said 30 Aspergillus sojae can have a nucleic acid sequence for the mutated pyrG gene different to that of the wild type A. sojae pyrG gene. A further embodiment comprises pyrG negative A. sojae according to the invention as described in any of the above embodiments which further comprise any of the characteristics described for any of the amdS variant A. sojae according to the invention as such or in combination.

WO 01/09352 PCT/NLOO/00544 10 A method of selecting transformed or transfected Aspergillus sojae also falls within the scope of the invention. The method comprises subjecting A. sojae of the pyrG negative type according to any of the embodiments of the invention as described above to a method of transformation or transfection with a nucleic acid sequence, said method comprising 5 introducing an active pyrG gene into the pyrG negative A. sojae in a manner known per se for transformation or transfection. The introduction step is then followed by selection of the resulting transformed or transfected A. sojae on a medium free of uracil and fluoro orotic acid, said medium at least further comprising minimum substrates required for growth of A. sojae, said medium enabling the desired A. sojae to grow whilst eliminating 10 growth of non-transformed or -transfected A. sojae due to inability of such to grow without uracil due to the inactivated pyrG gene. In a suitable embodiment of such a method the active pyrG gene that is introduced is flanked by identical nucleic acid sequence fragments, and the pyrG positive A. sojae resulting from the introduction of the pyrG gene and the flanking sequences is selected on a medium free of uracil and fluoro-orotic acid. 15 Subsequently the pyrG positive A. sojae is cultivated on medium comprising uracil and fluoro-orotic acid, thereby eliminating the pyrG gene that had been introduced and thus resulting in a pyrG negative A. sojae that is selectable by growth on uracil comprising medium and fluoro-orotic acid resistance. In a suitable embodiment of the aforementioned method the flanking sequences and the pyrG gene are further flanked by sequences that 20 direct integration of the pyrG gene and the flanking sequences into a specific location, due to the fact that the integration directing sequences are homologous to a specific sequence of the A. sojae to be transformed. This enables knock out (if desired) of the gene associated with the specific sequence. The process of knock-out mutant creation as such is well known to the person skilled in the art. Any of the embodiments of the selection method just 25 described may further comprise the step wherein the Aspergillus sojae is transformed or transfected with a further heterologous nucleic acid sequence. The further heterologous nucleic acid sequence preferably encodes a protein or polypeptide and the same remarks are valid here as made elsewhere in this description for the nature of such further nucleic acid sequences for the other embodiments of Aspergillus sojae and fungi in general 30 according to the invention. The further sequence can be introduced with the active pyrG gene either on the same vector or by cotransformation with the active pyrG gene that is introduced. The method of selecting transformed or transfected A. sojae as described may also be carried out in combination with the method for introducing a nucleic acid comprising introduction of a heterologous amdS gene in any of the embodiments according WO 01/09352 PCT/NLOO/00544 11 to the invention disclosed therefore above. Naturally, the invention covers any recombinant A. sojae obtained by the method of selecting transformed or transfected A. sojae according to the invention. The invention is also directed at a method for producing recombinant Aspergillus 5 sojae, said method comprising transformation or transfection in a manner known per se of a pyrG positive Aspergillus sojae with a nucleic acid sequence comprising the sequence to be introduced flanked by sections of the pyrG gene or corresponding sequences of a length and homology sufficient to ensure recombination eliminating the pyrG gene and introducing the desired sequence, followed by selection of the recombinant Aspergillus 10 sojae with the desired sequence by selecting for the A sojae with a pyrG negative phenotype. Determination of the corresponding sequences lies within the reach of the skilled person by virtue of their knowledge of hybridisation processes with nucleic acid sequences and their knowledge of required sequence data of the pyrG genes. In particular the invention also covers such Aspergillus sojae exhibiting the 15 characteristics of the amdS variant A sojae according to the invention as defined above. Thus any Aspergillus sojae strain obtained by either the amdS and/or pyrG introduction method according to the invention is a novel strain falling within the scope of the invention as is any subsequent use of such a novel strain. Such a novel strain can comprise nucleic acid sequences that do not occur in the original corresponding Aspergillus sojae strain or 20 even do not occur in Aspergillus sojae, Aspergilli or fungi. The sequences can be of mammalian origin or derived from any animal, plant or microbe. Nucleic acid sequences can also be expressed that are naturally present in the Aspergillus sojae strain but that are present in a lower copy number in the corresponding non-transformed A sojae. Thus the production of homologous proteins is also covered by the invention when pyrG and/or 25 amdS Aspergillus sojae strains according to the invention are involved. A preferred embodiment is that wherein the particular protein or polypeptide to be produced is absent in the corresponding non-treated A. sojae and/or is present in a lower copy number in the corresponding non-treated A. sojae, i.e. the A. sojae prior to introduction of the nucleic acid sequence. Expression of heterologous proteins by any of the novel strains of 30 Aspergillus sojae in a manner known per se for producing protein or polypeptide in a fungus thus covers both sequence native to the strain and foreign to the strain. Basically, only the native non-transformed or -transfected A sojae is excluded from protection. A process of production comprises cultivating the fungus under suitable conditions for expression of the desired sequence to occur. The process of production optionally includes WO 01/09352 PCT/NLOO/00544 12 the step of isolation of the resulting polypeptide or protein in a manner known per se for protein or polypeptide production by fungi. Preferably the protein or polypeptide will be secreted into the culture medium. A preferred protein or polypeptide is a protein or polypeptide susceptible to 5 degradation upon expression by Aspergillus niger or Aspergillus awamori. A number of such proteins and polypeptides have already been disclosed in the prior art and a large number remain yet to be determined. Such determination is however a matter of routine for the skilled person. Another preferred embodiment of the protein or polypeptide to be expressed is one whereby the protein or polypeptide differs from an Aspergillus sojae 10 protease and amylase. A preferred embodiment involves a non Aspergillus sojae protein or polypeptide. A particularly interesting embodiment comprises a combination of the two processes for introducing nucleic acid sequences according to the invention as described above. The advantage thereof lies in the fact that the frequency of transformation obtained 15 with the pyrG marker is clearly much higher than that of the amdS marker. However, secondary screening of the pyrG+ strains for the best growth on acrylamide selective plates allows the identification of those recombinant Aspergillus sojae showing the highest copy number and thus most likely the highest level of gene expression. As indicated in the examples homologous and heterologous expression regulating 20 sequences can be used by Aspergillus sojae i.e. natively occurring sequences of the strain itself or sequences foreign to the strain can be used. Thus the transformants according to the invention can comprise any such regulatory sequences. The selection of the suitable regulatory region is a matter of choice that lies well within the range of the standard capabilities of the skilled person and will depend on the particular application. The 25 regulating sequences can be constitutive or inducible. The regulating sequences can be fungal or non-fungal. A broad range are exemplified in the examples. A large number of expression regulating sequences are regularly used in the art for other systems, in particular fungal systems such as Aspergilli, and can routinely be applied without undue burden in the Aspergilli according to the invention. 30 For introducing the desired nucleic acid sequences into Aspergillus sojae any vector may be used that is suitable for introducing nucleic acid sequences into fungal host cells. Numerous examples are available in the art. In particular vectors that have been found suitable for transformation, transfection or expression in Aspergilli such as Aspergillus niger, Aspergillus awamori and Aspergillus oryzae can suitably be applied.

WO 01/09352 PCT/NLOO/00544 13 In addition to the above the subject invention describes efficient protein production for recombinant Aspergillus sojae. Such efficient production is disclosed in those strains having a protease profile superior to ATCC42251 or at least as good as any of ATCC9362, ATCC11906 and ATCC20387. The subject description thus reveals that some known 5 strains of A. sojae are well suited already as such for production of proteins, polypeptides and metabolites. These Aspergillus sojae strains exhibit a lower proteolytic activity than the reference strain A. sojae ATCC4225 1. In particular the two known strains ATCC 11906 and ATCC20387 are well suited. So preferred A. sojae strains for production of proteins, polypeptides and metabolites will be those expressing equal to or less proteolytic activity 10 than the two preferred strains. Strain ATCC 11906 is the best embodiment of the deposited ATCC A. sojae strains according to the prior art. Suitable proteins or polypeptides will be produced. Now that the subject invention has enabled introduction of nucleic acid sequences, such can serve to provide any protein or polypeptide of choice using an A. sojae as expression host. 15 The subject invention offers an improvement over existing expression systems. A number of existing protein production systems exhibit expression problems due to proteolysis. In particular the new system is better than the currently frequently applied expression systems Aspergillus niger and Aspergillus awamori. The subject invention now renders it possible to provide a recombinant Aspergillus sojae comprising a introduced 20 nucleic acid sequence encoding a protein or a polypeptide for expression, said protein or polypeptide being susceptible to degradation upon expression by A. niger or A. awamori. The invention also provides a recombinant A. sojae comprising an introduced nucleic acid sequence encoding a protein or polypeptide for expression, said protein or polypeptide being other than A. sojae protease and amylase. A preferred embodiment is that wherein 25 the introduced nucleic acid sequence encodes a non-A. sojae protein or polypeptide. Such recombinant A. sojae strains also fall within the scope of the invention. In addition, illustration of Aspergillus sojae strains that have been modified in order to enhance their suitability as expression hosts is currently provided. These modifications can be reduced proteolytic activity as induced by any means. Specifically, the use of UV 30 random mutagenesis is illustrated. Also specific mutation of one or more protease genes is illustrated. The means by which mutations can be introduced are common knowledge to the skilled person, and numerous alternative embodiments are thus readily available to arrive at the desired mutants. A suitable embodiment is formed by mutants in which alkaline proteolytic activity has been reduced. In particular elimination of activity of WO 01/09352 PCT/NLOO/00544 14 specifically the major 35 kDa alkaline protease is illustrated as ensuring increased expression of proteins and polypeptides. Specifically the invention thus also covers novel strains exhibiting reduced proteolytic activity, specifically reduced alkaline proteolytic activity. Such strains are obtainable using any specific mutation route known or 5 conceivable to the skilled person. A preferred embodiment of such expression hosts exhibiting reduced proteolytic activity as described above further comprises a selectable marker. Quite suitably the selectable marker will be amdS, pyrG or a combination thereof. The invention in particular covers a method of producing protease deficient mutants of A. sojae by knocking out the 35 kDa alkaline protease gene. There are 10 numerous ways in which this can potentially be achieved on the basis of the sequence data provided for this gene. In particular a method using recombination with a pyrG selection marker linked to two flanking regions eliciting cross over of the 35 kDa alkaline protease gene, whereby the resulting strain has the pyrG selection marker and misses the 35 kDa alkaline protease gene is an elegant one. Subsequently the pyrG selection marker can be 15 eliminated, thus providing a 35 kDa alkaline protease negative Aspergillus sojae mutant that can be used for expression purposes of any desired sequence to be introduced therein. Naturally, the sequence to be introduced can have been incorporated in the previous steps already either on the same vector as the pyrG marker or in a cotransformation event. Also the method can be carried out analogously where a different protease gene than the 35 kDa 20 alkaline protease gene is to be knocked out. The analogous measures to be taken are obvious to the skilled person on the basis of the illustration provided herein in combination with knowledge of other protease sequences. Also analogously the amdS selectable marker can be used in accordance with the invention as described elsewhere in this description. Mutant fungi exhibiting improved fermentation characteristics are also provided as 25 an additional aspect of the subject invention. Specifically, the invention is directed at a fungus comprising a mutation inhibiting the activity of proprotein convertase or an equivalent protein. Numerous proprotein convertases are known in the art. In particular we refer to Figure 1 providing sequence data of a number of such proteins. A fungus according to the invention is suitably selected from Agaricus, Aspergillus, Trichoderma, Rhizopus, 30 Mucor, Phanerochaete, Trametes, Penicillium, Cephalosporium, Neurospora, Tolypocladium and Thielavia. Particularly suitable fungi are Aspergillus niger, Aspergillus foetidus, Aspergillus sojae, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysosporum, Cephalosporium acremonium, Neurospora crassa, Tolypocladium geodes and Thielavia terrestris. A preferred embodiment covers the mutant WO 01/09352 PCT/NLOO/00544 15 when it is an Aspergillus sojae, most particular preference is extended to Aspergillus sojae as defined above according to the invention, i.e. comprising heterologous nucleic acid sequences, e.g. in combination with the selectable markers amdS and/or pyrG. A suitable equivalent of a proprotein convertase is a protein or polypeptide 5 exhibiting an amino acid sequence with more than 40%, preferably more than 45% similarity or identity with the inferred amino acid sequences of the DNA sequences given in SEQ ID No. 3 (= gene fragment encoding A. niger proprotein convertase amino acid sequence), SEQ ID No. 4 (= partial gene fragment encoding Aspergillus sojae proprotein convertase amino acid sequence) or with any of the sequences given in Seq ID Nos. 5 to 9. 10 The functionally equivalent protein may suitably have a nucleic acid sequence capable of hybridising under stringent conditions to a nucleic acid sequence according to SEQ ID Nos. 3 to 9. Stringent hybridisation conditions can readily be determined by the skilled person. A suitable example of stringent hybridisation conditions are hybridisation at 50*C and preferably at 56*C and final washes at 3xSCC. PE4, PCL1 and PCL2 are specifically 15 mentioned as examples of suitable oligonucleotide mixtures corresponding to the coding strand (i.e. SEQ ID Nos. 10, 11 and 12). For the noncoding strand PE6, PCL2-rev, PCL3 and PCL4 are mentioned (i.e. SEQ ID Nos. 13, 14, 15 and 16, respectively). Use of these primers in amplification procedures common in the art will provide equivalent sequences and such use and the resulting newly found sequences and application thereof in the 20 manner analogous to that described in the subject description fall within the scope of the invention. The sequences for which the oligonucleotides were made are well conserved as could be determined from comparison of the various amino acid sequences for the proteins provided (see Figure 1). Any other nucleic acid sequences exhibiting the same or higher degree of identity, similarity or homology with the sequences provided in the subject 25 patent application for the proteins or relevant active parts thereof are covered by the invention as is the use thereof as primers or probes to find other proprotein convertase or equivalent protein encoding sequences and/or for subsequently introducing mutations in such protein encoding sequences. By way of example Maniatis et al. (1982) Molecular Cloning, A Laboratory manual, Cold Spring Harbor Laboratory, New York or any other 30 handbook on cloning and/or screening nucleic acid sequences has been referred to. The equivalent protein or polypeptide will exhibit the activity of a proprotein convertase as the one having an amino acid sequence according to SEQ ID Nos. 3 to 9. The mutant fungus can comprise a substitution, insertion or deletion in the encoding sequence of the proprotein convertase or equivalent protein. The mutant fungus can suitably comprise a WO 01/09352 PCT/NLOO/00544 16 mutation in the regulation of the expression of the gene encoding proprotein convertase or equivalent protein. A mutant fungus according to the invention in a suitable embodiment exhibits reduced viscosity vis a vis the corresponding non mutated fungus under equivalent cultivation conditions. A mutant fungus according to any of the above embodiments 5 exhibiting increased expression of a desired introduced nucleic acid sequence encoding a protein or polypeptide is included within the scope of the invention, said fungus exhibiting increased production of a protein or polypeptide under equivalent conditions vis a vis the corresponding wild type fungus. The activity site for the A. sojae proprotein convertase has been ascertained to be comprised within the amino acid sequences inferred by SEQ ID 10 Nos. 3 and 4. A process for producing a phytase or protein having phytase activity or any other protein or polypeptide, preferably a recombinant phytase or any other heterologous protein or any other polypeptide, said process comprising cultivating a mutant fungus according to any of the embodiments described above falls within the scope of the invention. A process 15 for obtaining the resulting protein or polypeptide either from the cell as such or after secretion thereof from the cell is also included. The use of any of the described novel strains for transformation of any nucleic acid sequence encoding a phytase or protein having phytase activity or any other protein or polypeptide thereto and any subsequent expression of any nucleic acid sequence 20 introduced therein and also optionally any following processing and/or secretion and/or isolation is covered by the invention. Any phytase or phytase-like or any other heterologous protein or polypeptide encoding sequence can suitably be used. This can be of fungal or non-fungal origin. A preferred embodiment is formed by acid labile protein or polypeptide encoding sequences. 25 Suitably the protein encoding sequence encodes non protease-like proteins. The examples show a phytase sequence and a number of heterologous sequences suitable for use in transformation and also for expression in Aspergillus sojae hosts. Further examples of suitable proteins to be expressed are obvious to a person skilled in the art. The invention is further illustrated by the examples below. The examples are not to 30 be considered restrictive to the interpretation of the scope of the invention. Alternative embodiments are readily envisageable to the skilled person on the basis of the description and knowledge of the relevant field of technology. The content of references cited in the description are incorporated by reference. The claims serve to illustrate the intended scope of the invention.

WO 01/09352 PCT/NLOO/00544 17 EXPERIMENTAL DETAILS CONCERNING THE INVENTION CONSTRUCTION OF AN Aspergillus sojae GENE LIBRARY. Genomic DNA of A. sojae was isolated from protoplasts obtained from 5 ATCC 11906 using a previously described protocol (Punt, van den Hondel, 1992). After isolation, DNA was extracted from the protoplasts using the protocol described by Kolar et al., 1988. Subsequently the DNA was partially digested with MboI to result in DNA fragments of an average size of 30-50 kb. Vector pAOpyrGcosarpl, which was used for the construction of the gene library, 10 was constructed by ligation of a 3 kb BamHI-HindIII fragment from pANsCos1 (Osiewacs, 1994) and a 3.2 kb Acc65I-HindIII fragment from pAO4.2 (De Ruiter-Jacobs, 1989) in Acc65I-BamHI digested pHELP1 (Gems et al., 1991). This cosmid vector carries the A. oryzae pyrG selection marker and is self-replicating in filamentous fungi. A4boI digested genomic DNA was ligated to BamHI-digested pAOpyrGcosarpl, and the 15 ligation mixture was packaged into phage particles using the Stratagene Supercos1 vector kit. In total 30,000 individual clones were obtained representing an approximate 30-fold representation of the A. sojae genome. Stocks (in 15% glycerol) of pools of the resulting clones were stored at -80'C for later use. 20 AMDS TRANSFORMATION METHOD AND TRANSFORMANTS. Two currently used protoplasting protocols and transformation protocols [the modified OM-method (Yelton et al., P.N.A.S. 81 (1984) 1470-1474) and the NaCl-method (Punt and Van den Hondel, Meth. Enzym. 216 (1993) 447-457)] were tested on the Aspergillus sojae strain ATCC9362. Both methods resulted in protoplasts, but the yield of 25 viable protoplasts with the OM-method was clearly better. The overall yields were lower than normally obtained for A. niger. A pilot protoplasting/transformation experiment was carried out with all A. sojae strains using the OM method. For transformation, vector p3SR2 (carrying the amdS marker) was used in combination with pAOpyrGcosARPl. This latter vector is a derivative of the 30 autonomously replicating Aspergillus vector Arpl, which in all Aspergillus species tested so far, resulted in highly increased numbers of (instable) transformants when used as a cotransforming vector. For nearly all strains sufficient protoplasts (about 10E6-10E7 per transformation) were obtained. Analysis of appropriate AmdS selection conditions for the various A. sojae strains revealed vigorous growth of most strains on the commonly used WO 01/09352 PCT/NLOO/00544 18 selective acetamide medium. Clearly, the acetamide selection conditions proposed for A. sojae amdS transformants as reported in W097/04108, were not appropriate for the selection of A. sojae transformants. Our experiments revealed, surprisingly, that AmdS+ transformants could only be selected with acrylamide selection. Even on selective 5 acrylamide plates, a considerable background from non-transformed protoplasts was observed. Selection of primary transformants requires around three weeks and many of the initially selected putative transformants turned out to be false positives, only showing background growth after transfer to fresh selective acrylamide plates. To optimize selection of transformants attempts had to be made to reduce this background growth. 10 Improved results were obtained by omitting glucose from the selective plates. In Table 3 the composition of the improved selection medium and the usual media is given. Figures 2a, b and c show the background growth observed for selected strains on the selection medium described in W097/04108 and the improved acrylamide selection medium described in Table 3. 15 Further transformation experiments with the three selected A. sojae strains revealed that protoplasting efficiencies for ATCC 11906 and ATCC203 87 were better using the NaCl-method. Successful protoplasting was obtained using various commercially available protoplasting enzyme preparations such as NOVOZYM, Caylase, Glucanex, etc. Based on the NaCl transformation protocol the three selected A. sojae strains were transformed with 20 amdS selection vector p3SR2 or derivatives thereof. Using the modified acrylamide selection plates a number of vigorously growing transformants were obtained, while no growth was observed in a control transformation without DNA. Another approach to circumvent background growth of non-transformed mycelium is the elimination of the activity of the wild type A. sojae amdS gene. This can be achieved for example by 25 disruption of the A. sojae amdS gene. As a first step specific DNA fragments carrying ATCC 11906 amdS sequences were PCR-amplified using primers derived from published A. oryzae amdS sequences (Gomi et al.; 1991, Gene 108, 91-98). Previous experiments had shown that cloning by stringent hybridisation would be unsuccessful due to a low level of sequence conservation between A. nidulans and A. sojae amdS sequences. The expected 30 fragment of about 1.6 kb, which should carry most of the coding region of the amdS gene, was obtained. Sequence analysis from both ends of the cloned PCR fragment (Figures 3a and 3b) confirmed the cloning of a part of the A. sojae amdS gene. The stringent hybridisation occurred at 56*C with final washes at 3xSSC. The cloned sequence was very similar to the published A. oryzae amdS sequence. Several hybridising clones (7 out of WO 01/09352 PCT/NL00/00544 19 10.000) were isolated from the ATCC1 1906 cosmid library in pAOpyrGcosarpl using the cloned ATCC 11906 amdS fragment as a probe. After subcloning a fragment carrying the complete amdS gene, a part of the amdS gene was replaced by a re-usable pyrG selection marker to generate an amdS replacement vector. Transformation of this vector to 5 Aspergillus sojae ATCC1 1906PyrG resulted in pyrG+ transformants. After subsequent analysis of these transformants on acetamide and acrylamide selection plates several of these transformants showed reduced background growth. Southern analysis of a few of these strains revealed that the expected gene replacement had occurred. One of these strains was used for subsequent transformation with the A. nidulans amdS gene using 10 acrylamide selection plates and resulted in a number of amdS+ transformants. PYRG TRANSFORMATION METHOD AND TRANSFORMANTS. (1) Initial experiments For A. sojae, the standard experiments used in the prior art for other fungi to 15 generate pyrG mutants as described in the introduction resulted in numerous fluo-orotic acid (FOA)-resistant strains. However, all of these strains were able to grow on medium without uridine and were therefore not considered pyrG mutants. With our final aim to isolate the appropriate mutant strains a number of alternative approaches were followed. 20 (2) Near-homologous gene disruption Based on the expectation that the pyrG genes from A. sojae and A. oryzae are very similar in sequence (which was confirmed by Southern hybridisation carried out under stringent conditions), experiments were carried out to disrupt the A. sojae pyrG gene with a mutant version of the A. oryzae pyrG gene using an approach previously described by 25 Gouka et al. (1996). The stringent hybridisation occurred at 65*C with final washes at 0.3 x SSC. An A. oryzae pyrG disruption vector was constructed in which an 0.5 kb ClaI fragment carrying part of the pyrG coding region was deleted (Figure 4). The XbaI pyrG fragment from this new vector was used for transformation and direct selection for FOA resistant transformants. None of the FOA resistant colonies obtained was uridine requiring. 30 (3) UV mutagenesis and filtration enrichment Another approach to improve the yield of specific mutant strains is the use of a filtration-enrichment step (Bos et al. 1986, Thesis, Agricultural University Wageningen). The UV mutagenized spores are used for inoculation of a minimal medium (MM) liquid WO 01/09352 PCT/NL00/00544 20 culture. From the resulting repeated overnight culture those spores unable to germinate in minimal medium (a.o. pyrG mutant spores) are separated from the grown mycelium by filtration through myracloth. The spores obtained after several enrichment steps were tested for their PyrG phenotype, by inoculating the spores on plates containing FOA. 5 Again none of the resulting FOA resistant colonies was uridine requiring. Also none of the colonies obtained after this enrichment on MM plates containing uridine was shown to be uridine requiring. (4) Modified selection conditions 10 Our previous attempts to isolate pyrG mutants from A. sojae had failed suggesting the inability of the required pyrG mutants to utilize exogenous uridine, which is used in the FOA selection medium for the analysis of uridine auxotrophy. A modified selective FOA medium, now containing uracil next to uridine, was used in a new isolation attempt. From this approach several FOA resistant mutants were obtained which were uracil requiring. 15 Retesting of these strains showed that these were unable to grow on uridine supplemented minimal medium. Subsequent transformation experiments with some of the uracil requiring strains showed that these mutants could indeed be complemented with a fungal pyrG gene (e.g. vector pAB4. 1; A. niger pyrG). The inability of pyrG mutants to grow in minimal medium supplemented with only uridine was an unprecedented observation for 20 related Aspergillus species (A. nidulans, A. niger, A. oryzae) and various other fungal species. (5) Re-usable selection marker Versatile genetic modification of A. sojae requires the possibility to modify, disrupt 25 and express a number of different genes in a single fungal strain, which would require the availability of a (series) of different selection markers. However, the availability of a marker such as pyrG, which allows selection of both the mutant (FOA selection) and the transformant (Uracil-less medium), provides the possibility of repeated use of the same marker in subsequent experiments. For this approach a pyrG marker gene was designed, in 30 which the complementing sequence was flanked by a direct repeat sequence originating from the 3' flanking end of the pyrG gene. The resulting plasmid is pAB4-lrep. The construction of this vector is detailed in Figure 5. The full sequence of the vector is given in SEQ ID No. 17. Transformation of A. sojae pyrG mutants with this vector results in a similar number of PyrG+ transformants as with the vector pAB4-1. However, subsequent WO 01/09352 PCT/NLOO/00544 21 plating of spores of selected pAB4-1 and pAB4-Trrep transtormants to FOA selection plates resulted in many more FOA resistant/uracil requiring colonies for the pAB4-lrep transformant. Southern analysis of these FOA resistant/uracil requiring clones showed that in most of the pAB4-lrep strains the A. niger pyrG marker gene had been deleted leaving 5 only the small 0.7 kb repeat region at the locus of integration, while in the pAB4-1 strains the A. niger gene was still present and had presumably acquired a mutation resulting in the pyrG-negative phenotype. EXPRESSION HOSTS: STRAIN SELECTION. 10 Protease production Very important characteristics of a fungal expression system are the level and type of fungal proteases produced under various culture conditions. Sometimes strains which can be readily transformed are not suitable as expression hosts due to production of proteases or acidification of the culture media which is detrimental to the expressed 15 product. Analysis of the growth behaviour of the various A. sojae strains revealed that, in contrast to what was observed for A. niger, acidification of the culture medium did not occur either on agar based plates (MacConkey) or in shake flask cultures. In fact in shake flask cultures, irrespective of the three medium types analyzed (Table 4), in most cases even an alkaline pH was obtained in the cultures. Based on these results and literature data 20 it is thus expected that primarily alkaline proteases will be present in the A. sojae culture fluid. To analyse protease activity of the culture fluids of the various strains, a milk clearing assay was performed. In addition medium samples were incubated with different proteins (e.g. bovine serum albumin (BSA)), and degradation of these proteins was followed in time in order to assess the suitability of the tested strains as expression hosts 25 for a range of products. BSA was chosen as in our previous experiments with A. niger. This protein was shown to be very susceptible to proteases. A. terreus phytase was chosen as example of another proteolytically instable protein. Degradation of milk proteins as shown by the formation of a milk clearing zone at the periphery of growing colonies is a generally accepted criterion for protease activity. Detection of BSA was carried out by 30 Coomassie staining of SDS-PAGE gels. For phytase, Western analysis using specific antibodies, was carried out. As shown in Table 4, clear differences of degradation in A. niger culture fluid are evident when this is compared with that in A. sojae culture fluid. In A. niger culture fluid (pH 3-4) rapid degradation of BSA occurs. In A. sojae culture fluids WO 01/09352 PCT/NLOO/00544 22 from richer media, degradation of BSA occurs, albeit less than in A. niger culture fluid. In most A. sojae culture fluids (pH 7-8) rapid degradation of A. terreus phytase occurs, with the exception of ATCC9362, ATCC1 1906 and ATCC20387 culture fluids. In general, the strains with the lowest phytase degradation also show low BSA degradation under the 5 conditions tested. In particular the two A. oryzae strains ATCC20235 and ATCC46250 show much higher proteolytic activity than most A. sojae strains. To exclude that differences in the pH of the culture fluid cause the observed effects, similar degradation experiments were also carried out with culture fluids of which the pH was adjusted to pH 4.5 (50 mM Na/HAc), pH 5.8 (50 mM Na/HAc) and pH 8.3 (50 mM 10 Tris/HCl). Table 5 gives the degradation data obtained with these samples. As can be seen in the table A. oryzae ATCC20235, which had the highest proteolytic activity at pH 7-8 also shows high proteolysis at other pH values. Degradation of A. terreus phytase occurs primarily at pH 8. Similarly to what was found before, ATCC11906 and ATCC20387 showed low phytase degrading activity. ATCC9362 showed phytase degradation in rich 15 media. BSA degradation by A. sojae showed no significant differences with the data presented in Table 4. In conclusion, these protease assays resulted in the identification of three low protease A. sojae strains, namely ATCC9362, ATCC11906 and ATCC20387. Thus, A. sojae can cleary be used as expression host for a range of proteins and provides a series of 20 advantages over prior art transformation and expression systems. STRAIN IMPROVEMENT Once the potential of transformability and expression had been ascertained for Aspergillus sojae, means by which additional strains could be created with enhanced 25 characteristics for expression were considered. Two different approaches which can be used as such or in combination were developed to provide novel improved strains for expression of proteins. On the one hand the possibility of developing protease deficient mutants was investigated and the impact of such on levels of expression was assessed. On the other 30 hand strains with amended morphology were developed with a view to improve fermentation characteristics. To achieve this a hitherto non-disclosed or suggested route was followed which is applicable not only to Aspergillus sojae but to Aspergilli and in fact to fungi in general.

WO 01/09352 PCT/NLOO/00544 23 Development ofprotease deficient mutants To obtain protease deficient A. sojae strains two approaches were followed. In a first approach spores from ATCC11906 and ATCC11906-derived strains were mutagenized with UV. In a second approach gene disruption of the major alkaline protease 5 was carried out. UV mutants Freshly harvested spores from A. sojae ATCC 1906 or one of its pyrG derivatives was UV-mutagenized in a Biorad UV-chamber with a dose resulting in 20-50 % survival. 10 Serial dilutions were plated onto skim-milk plates (Mattern et al., 1992). From 5000 UV surviving strains four mutant strains with a considerably reduced milk-clearing halo were obtained AlpA gene disruption 15 In this approach the endogenous alpA (alkaline protease) gene cloned from ATCC1 1906 was disrupted using a disruption vector carrying the re-usable pyrG selection marker as described in this description. An ATCC1 1906 cosmid library (in a PyrG cosmid) was constructed. From 10.000 independent cosmid clones initially 4 were found to hybridize under homologous 20 conditions with an A. sojae alpA fragment obtained by PCR with primers MBL1784 and MBL1785. Rescreening of the 4 clones revealed only strong hybridisation with one clone. A 4 kb EcoRI and a 2.5 kb HindIII fragment from this clone, together expected to carry the complete gene, were subcloned and characterised by restriction enzyme digestion and sequence analysis. Based on these subclones a new gene-replacement vector was 25 constructed as described in Figure 6. For transformation of an ATCC l1906pyrG derivative the vector was digested with EcoRI, and the 8.7 kb alpA deletion fragment was used for transformation (see Figure 6). Transformation of the replacement cassette to ATCC1 1906pyrG5 resulted in a number of transformants with a reduced milk-halo. Southern analysis of these strains revealed the successful deletion of the alpA gene. To 30 allow subsequent use of the pyrG marker for transformation of one of these strains, spores from this strain were plated on FOA containing medium selective for pyrG mutants. From strains with the correctly integrated disruption cassette with the re-usable pyrG marker a large number of FOA resistant colonies were obtained. In contrast to the results obtained for spontaneous FOA resistant mutants of wild type strains, the FOA strains obtained from WO 01/09352 PCT/NLOO/00544 24 these disruption strains were virtually all uracil requiring and turned out to be PyrG negative again. Southern analysis was used to confirm the desired removal of the pyrG marker gene at the alpA locus, leaving only the 700 bp "footprint". 5 Analysis ofprotease activity in UV and disruption mutants To analyse the levels of protease production in the different low protease derivatives of ATCC 11906 controlled batch fermentation experiments were carried out. From the resulting culture supernatants protease activities were determined at various pH values. Deletion of the alpA gene resulted in a strong reduction of proteolytic activity at 10 alkaline pH. Analysis of the protease activity in one of the UV mutants showed almost complete absence of proteolytic activity at both pH 6 and pH 8. Consequently the level of proteolysis towards secreted proteins produced in these strains was considerably less than observed for the parent strain. 15 Development of low viscosity mutants Initial controlled batch or fed batch fermentation trials with A. sojae resulted in considerable biomass yield, however both the culture viscosity and sporulation phenomena in the fermenter vessel represented less favourable characteristics. Therefore attempts were made to improve these characteristics in the desired host 20 strain. Various patent applications teach that low viscosity mutants can be isolated by various ways of screening. W096/02653 and W097/26330 describe non defined mutants exhibiting low viscosity. However here we describe a new unexpected case of a completely characterised and fully defined low viscosity mutant from A. sojae. It was found that a proprotein processing mutant from this organism had an unexpected aberrant growth 25 phenotype (hyper-branching) while no detrimental effect on the productivity of proteins was observed. Controlled fermentation experiments with this strain revealed that increased biomass concentrations were obtained at considerably lower viscosity values. The observed characteristics were not only present in A. sojae but other fungi as well, e.g. in A. niger. 30 (1) Construction of an A. niger proprotein processing mutant To clone the proprotein convertase encoding gene from A. niger, heterologous hybridisation using specific probes from the Saccharomyces cerevisiae KEX2, Schizosaccharomyces pombe KEX and the Xenopus laevis PC2 genes was carried out.

WO 01/09352 PCT/NL00/00544 25 However, no specific hybridisation signals were obtained even at very low stringency hybridisation conditions (47*C, washes at 6xSSC), precluding the use of this approach to clone the corresponding A. niger gene. As an alternative approach to clone the proprotein convertase encoding gene from 5 A. niger, PCR was used. Based on the comparison of various proprotein convertase genes from various yeast species and higher eukaryotes (Figure 1) different PCR primers were designed (SEQ ID Nos. 10, 13 and 18-23) which are degenerated, respectively, 2048, 49152, 4, 2, 2, 512, 2048, and 4608 times. From the amplification using primers PE4 and PE6, two individual clones were obtained of which the encoded protein sequence did show 10 significant homology to the S. cerevisiae KEX2 sequence (SEQ ID No. 24). These clones were used for further experiments. Based on the observed homology to other proprotein convertase genes of the cloned PCR fragment, the corresponding A. niger gene was designated pcA (from proprotein convertase-like). Southern analysis of genomic digests of A. niger revealed that the pcA 15 gene was a single copy gene with no closely related genes in the A. niger genome, as even at stringent hybridisation conditions (50*C; washes at 6xSSC), no additional hybridisation signals were evident. A first screening of an EMBL3 genomic library of A. niger N401 (van Hartingsveldt et al., 1987) did not result in any positively hybridising plaques although about 10-20 genome equivalents were screened. In a second screening a full 20 length genomic copy of the pc/A gene was isolated from an A. niger N400 genomic library in EMBL4 (Goosen et al., 1987). Of the 8 hybridising plaques which were obtained after screening 5-10 genome equivalents, 6 were left after a first rescreening. All these 6 clones most likely carried a full copy of the pcA gene, as in all clones (as was observed for the genomic DNA) with the PCR fragment two hybridising EcoRV fragments of 3 and 4 kb 25 were present (note that the PCR fragment (SEQ ID No. 24) contains an EcoRV restriction site). Based on a comparison of the size of other proprotein convertases, together these fragments will contain the complete pcA gene with 5'- and 3'-flanking sequences. The two EcoRV fragments and an overlapping 5 kb EcoRI fragment were subcloned for further characterisation. A detailed restriction map of the DNA fragment carrying the pcA gene is 30 given in Figure 7. Based on the restriction map given in Figure 7 the complete DNA sequence of the pcA gene was determined from the EcoRI and EcoRV subclones (SEQ ID No. 3). Analysis of the obtained sequence revealed an open reading frame with considerable similarity to that of the S. cerevisiae KEX2 gene and other proprotein convertases. Based WO 01/09352 PCT/NLOO/00544 26 on further comparison two putative intron sequences (SEQ ID No. 3, from position 1838 to 1889 and from 2132 to 2181) were identified in the coding region. Subsequent PCR analysis with primers flanking the putative introns, on a pEMBLyex based A. niger cDNA library revealed that only the most 5' of these two sequences represented an actual intron. 5 The general structure of the encoded PclA protein was clearly similar to that of other proprotein convertases (SEQ ID No. 25 and Figure 8). The overall similarity of the PclA protein with the other proprotein convertases was about 50% (Figure 1). To demonstrate that the cloned pcA gene is a functional gene encoding a functional protein, the construction of strains devoid of the pcA gene was attempted. Therefore, 10 pPCL1A, a pcA deletion vector, in which a large part of the pcA coding region was replaced with the A. oryzae pyrG selection marker gene, was generated. Subsequently, from this vector the 5 kb EcoRI insert fragment was used for transformation of various A. niger strains. From these transformations (based on pyrG selection) numerous transformants were obtained. Interestingly, a fraction of the transformants (varying from 1 15 50%) displayed a very distinct aberrant phenotype (Figure 9). Southern analysis of several wild-type and aberrant transformants revealed that these aberrant transformants, which displayed a severely restricted growth phenotype, had lost the pcA gene. All strains displaying wild-type growth were shown to carry a copy of the replacement fragment integrated adjacent to the wild-type pcA gene or at a non-homologous position. 20 Analysis of protein production in selected pclA mutant strains carrying various glucoamylase fusion genes revealed the production of unprocessed fusion protein. The production of high levels of unprocessed glucoamylase-interleukin-6 fusion protein in a pclA mutant was achieved. Protein analysis revealed that in pclA mutant strains also no fully processed endogenous glucoamylase is formed but only pro-glucoamylase. 25 To further improve the yields of fusion proteins controlled batch and fed-batch fermentations were also carried out. Surprisingly the fermentation characteristics of pelA mutant strains were clearly superior to those of the parent strain resulting in a much reduced viscosity/biomass ratio, without loss of productivity. 30 (2) Construction of an A. sojae proprotein processing mutant To construct the corresponding mutant in A. sojae, functional complementation of the low-viscosity mutant of A. niger genomic cosmid clones from the ATCC 11906 cosmid library were isolated, which comprised the A. sojae proprotein processing protease pcA gene. Partial sequence analysis of the isolated sequences SEQ ID No. 4 confirmed the WO 01/09352 PCT/NLOO/00544 27 cloning of the A. sojae pclA gene. Figure 10 shows the comparison of the DNA sequences of part of the A. niger and A. sojae pclA genes. Based on the A. sojae sequence and a partial restriction map with the coding region of the A. sojae pcA gene, a replacement vector was generated using the EcoRV-site in the A. sojae pcA gene to clone the re-usable 5 pyrG marker as a SmaI fragment inside (Figure 11). The resulting vector was partially digested with Clal to obtain the delta-pcl fragment of 10.5 kb (see Figure 11). This fragment was isolated to be used for transformation of A.sojae pyrG strains. The gene replacement vector was used to generate pclA mutants in ATCC 11906 and ATCC 11906 derivatives. The resulting strains were used for the expression of homologous and 10 heterologous proteins. Controlled fermentation experiments with some of the resulting transformants revealed improved fermentation characteristics, in particular a lower viscosity/biomass ratio of the culture. (3) Cloning offungal genes homologous to Aspergillus pcA 15 Based on the comparison of amino acid sequences inferred from the A. niger and A. sojae pcA genes with those of other proprotein processing enzymes (Figure 1) several oligonucleotide mixtures corresponding to the coding or non-coding strand of well conserved sequences were designed (SEQ ID Nos. 10 to 16). These oligonucleotide mixtures were used in PCR with chromosomal DNA from 20 Trichoderma reesei QM9414, Fusarium venenatum ATCC20334, Penicillium chrysogenum P2, Trametes versicolor, Rhizopus oryzae ATCC200076, and Agaricus bisporus HORST. Depending on the template DNA used, PCR amplifications (30 cycles; 1 min 94*C; 1 min 40*C; 2 min 68*C) with one or more of the combinations of coding and non-coding strand oligonucleotides resulted in specific PCR products. Table 6 gives the 25 results of the various amplification reactions. Sequence analysis was carried out with a number of the obtained PCR fragments using either of the two oligonucleotide mixtures used for amplification. These analyses resulted in the identification of the various pcA homologues from these different fungi. Figure 12 gives the inferred aminoacid sequences corresponding with the various DNA fragments (SEQ ID Nos. 5 to 9). 30 (4) Examples of biomass and viscosity determinations The following operating parameter data ranges have been determined for fungal fermentations using a number of different fungal strains.

WO 01/09352 PCT/NLOO/00544 28 Viscosity: Viscosity is determined on a Haake Viscotester VT500 using sensor system MV DIN (vessel number 7), operated at 20*C. Obtain a fresh sample of fermentation broth and place 40 ml of the broth in the measuring cell. After a small period of equilibration (4 min) at 5 a set spindle speed the viscosity is measured. This measurement is repeated for ten different spindle speeds. Multiplication of the spindle speed with the measuring cell factor results in the shear rate. Viscosity rl (in centipoise = cP) is plotted against shear rate y (1/s) and gives the viscosity profile of the fermentation broth. Viscosity ranges have been determined for fermentations using the specified fungal 10 organism using the above procedure (Table 7). Biomass: Biomass is determined by the following procedure: Preweigh 5.5 cm filter paper (Whatman 54) in an aluminium weighing dish. Filter 25.0 ml 15 whole broth through the 5.5 cm paper on a Buchner funnel, wash the filter cake with 25.0 ml deionised water, place the washed cake and filter in a weighing pan and dry overnight at 60'C. Finish drying at 100*C for 1 hour, then place in desiccator to cool. Measure the weight of dried material. Total biomass (g/l) is equal to the difference between the initial and final weights multiplied by 40. 20 Protein: Protein levels were determined using the BioRad Assay Procedure. The data presented above represent values determined 48 hours into the fermentation process until fermentation end; all values of Aspergilli and Trichoderma are for commercially relevant 25 fungal organisms and reflect actual commercial data. A fungal strain such as A. sojae lfvA and A. sojae pclA has the advantage that the low viscosity permits the use of lower power input and/or shear the in the fermentation to meet oxygen demands for those cases where shear stress on the product may be detrimental to productivity due to physical damage of the product molecule. The lower biomass production 30 at high protein production indicates a more efficient organism in the conversion of fermentation media to product. Thus A.sojae mutants provides better biomass and viscosity data whilst also delivering at least as much protein, and in fact a lot more protein than the two commercially used systems which obviously are better than for typically deposited Aspergillus or Trichoderma reesei strains in general public collections.

WO 01/09352 PCT/NLOO/00544 29 The high protein production with low biomass concentration produced by A. sojae lfvA would allow development of fermentation conditions with higher multiples of increase in biomass, if increasing biomass results in increased productivity, for the desired product before reaching limiting fermentation conditions. The present high levels of biomass and viscosity 5 produced by the T. longibrachiatum and A. niger organisms restrict the increase of biomass as the present levels of biomass and viscosity are near limiting practical fermentation conditions. EFFICIENT GENE EXPRESSION (1) Heterologous regulatory sequences 10 The three selected A. sojae strains were cotransformed with three GUS reporter vectors carrying different fungal expression signals (A. nidulans PgpdA; pGUS54, A. niger PglaA; pGUS64, A. niger PbipA; pBIPGUS) and the amdS selection vector p3SR2 or derivatives thereof. Expression of the GUS gene was analysed in representative transformants (Table 8). From the results it is clear that under the conditions tested the 15 gpdA promoter was by far the best promoter resulting in about 5000 U GUS/mg protein. This corresponds to about 5% of the total amount of cellular protein. The bipA promoter results in about 30% of the gpdA activity, which corresponds to expression data obtained in A. niger. Surprisingly, the glaA promoter which is very active in A. niger (at least as active as gpdA) results in less than 1% of the gpdA activity in A. sojae. 20 (2) A. sojae regulatory sequences We also isolated an Aspergillus sojae homologous promoter and assessed the applicability of such in an expression system. In some instances of expression it will be preferable to use a homologous promoter rather than a heterologous promoter. It was also 25 interesting to assess whether the homologous promoter would be more efficient than a heterologous one. PCR cloning of three efficiently expressed A. sojae genes, namely alpA (alkaline protease; inducible), amyA (amylase; inducible) and gpdA (glyceraldehyde-3-phosphate dehydrogenase; constitutive) was attempted using primers based on sequences available 30 from A. oryzae (SEQ ID Nos. 26 to 31). Figures 13 a, b and c give the sequences and the position in published A. oryzae sequences of the various PCR primers used for this approach. Genomic template DNA from A. sojae ATCC 11906 was used for PCR amplification. Initial PCR amplifications (30 cycles; 1 min 94*C; 1 min 40*C; 2 min 68*C) resulted in a specific PCR product of the expected size (400 bp) for the gpdA. For the other WO 01/09352 PCT/NLOO/00544 30 two PCR reactions no product was obtained. Therefore, for alpA PCR conditions were made less stringent (10 cycles; 1 min 94*C; 1 min 25*C; 2 min 68*C + 20 cycles; 1 min 94*C; 1 min 40*C; 2 min 68*C), which resulted in a specific alpA PCR product of about 1000 bp. 5 The complete sequence of the cloned genes was determined. As shown in Figure 14 the A. sojae ATCC 11906 gpdA promoter region has a very high homology with other gpdA promoter sequences and the alpA promoter was virtually identical to the A. oryzae alpA promoter (SEQ ID Nos. 32 and 33). Expression vectors carrying expression cassettes comprising these A. sojae promoters show significant levels of gene expression. 10 HETEROLOGOUS PROTEIN PRODUCTION A number of heterologous proteins were tested which were known to be susceptible to acidic proteolysis and thus could not be expressed efficiently in other well known 15 expression systems. Also proteins that are already efficiently expressed in alternative systems were tested in order to assess by way of comparison the levels of expression achieved with Aspergillus sojae vis a vis other known expression systems such as Aspergillus niger. 20 Phytase production DNA fragments carrying various fungal phytases (Wyss et al. (1999) Appl. Environ. Microbiol. 65, 359-366) were ligated as 5' NcoI or BspHI sites introduced at the ATG codon - 3' blunt-ended fragments downstream of the A.nidulans gpdA promoter in pAN52-1NotI. The resulting vectors were used in cotransformation experiments of A. 25 sojae using the amdS and or pyrG selection marker. Phytase producing transformants were screened using the described phytase plate-assay. Further improved phytase expression vectors were generated using a multicopy cosmid approach. In this approach several copies of a phytase expression cassette, recloned in a multiple cloning site vector (pMTL series, Chambers et al., (1988) Gene 68, 139-149) 30 to allow its isolation as a EcoRI fragment. Several copies of these EcoRI fragments were cloned into cosmid vector pAN4cosl through packaging (Verdoes et al. (1993) Transgenic Research 2, 84-92), resulting in cosmid clones carrying a number of expression cassettes. The resulting clones were introduced into A. sojae using the amdS selection marker. AmdS+ clones were screened for phytase production using the phytase plate-assay.

WO 01/09352 PCT/NLOO/00544 31 Further phytase expression vectors were generated using the GLA fusion approach (e.g. Broekhuijsen et al. (1993) J. Biotech. 31, 135-145). To this end phytase gene fragments, encoding the mature A. fumigatus phytase protein were fused, using convenient restriction sites and fusion PCR, to the 3'-end of the glucoamylase carrier gene in vector 5 pAN56-1 (Genbank accession number Z32700). Between the glucoamylase and phytase part of the gene-fusion a sequence encoding a proprotein processing site (Asn-Val-Ile-Ser Lys-Arg) was introduced. The resulting vectors were used in cotransformation experiments of A. sojae using the amdS and/or pyrG selection marker. Phytase producing transformants were screened using the described phytase plate-assay. 10 Shake flask fermentation was carried out resulting in significant levels of active phytase. Yield were significantly higher than those reported in literature for A. niger (van Hartingsveldt et al. (1993) Gene 127, 87-94; Van Gorcom et al. (1991) EP420358). On average, the levels obtained with the multicopy cosmid vectors were higher than those obtained with the single copy vectors. Phytase levels obtained with the glucoamylase 15 phytase fusion vectors resulted in high levels of both glucoamylase and phytase. Controlled batch and fedbatch fermentations from a selected number of phytase producing A. sojae transformants revealed a further increased level of phytase. Glucoamylase production 20 An example of an efficiently produced fungal protein is provided by the expression of the A. niger glaA gene. Vector pGLA6S (Figure 15) is derived from pGLA6 (Punt et al. (1991) J. Biotech. 17, 19-334) by introducing a 5 kb EcoRI fragment carrying the A. nidulans amdS gene as selection marker into the unique EcoRI site of pGLA6. Vector pGLA6S (Figure 15) carrying the amdS selection marker and the glucoamlyase gene under 25 control of the A. nidulans gpdA promoter was introduced into A. sojae ATCC1 1906pyrG using cotransformation with vector pAB4. 1. Starch plate-assays demonstrated the production of increased levels of amylolytic activity in these transformants. From the resulting transformants those showing proficient growth on acrylamide medium were analysed for glucoamylase production. On a Coomassie Briljant Blue-stained SDS PAGE 30 gel from the culture supernatant of some of these transformants a single dominant protein band corresponding to glucoamylase was observed . Western analysis using a monoclonal antibody against glucoamylase (Verdoes et al. (1993) Transgenic Research 2, 84-92) was used to confirm the identity of this protein band.

WO 01/09352 PCT/NLOO/00544 32 Interleukin-6 production Production of interleukin-6, which is an example of a highly sensitive protein for proteolytic degradation, was shown to be virtually impossible in A. niger without the use of the gla-fusion strategy and protease deficient strains. Even with all these improvements the 5 yields of IL-6 were only a few mg per litre culture fluid. Introduction of the IL-6 vector pAN56-4 (Broekhuijsen et al. (1993) J. Biotech. 31, 134-145) into A. sojae by cotransformation with the pyrG or amdS marker resulted in transformants expressing the IL-6-fusion gene present in this vector. From the resulting transformants a few were selected for further analysis. Shake flask fermentation experiments were carried out with 10 these transformants. SDS-PAGE and Western analysis of culture supernatants of several of these strains surprisingly showed levels of correctly processed IL-6 which were about 5-10 fold higher than the levels obtained in the best reported cases in A. niger. The use of the various types of protease deficient and fermentation-optimized mutants from A. sojae further increased the level of IL-6 production to be obtained from controlled fermentations 15 (Broekhuijsen et al. (1993) J. Biotech. 31, 134-145). Green fluorescent protein (GFP) Another type of acid labile protein we have attempted to produce in A.sojae is GFP from the jelly fish Aequoria victoria. This protein is not only proteolytically sensitive but 20 furthermore it loses its activity at acid pH. Vectors carrying GFP or GLA-GFP fusion genes (driven by the A. nidulans gpdA promoter) were introduced into A. sojae by cotransformation using either the pyrG or amdS selection marker. Expression resulted in brightly fluorescent A. sojae transformants for both vector types. Based on the observed fluorescence and the subsequent analysis of culture supernatants from selected, shakeflask 25 cultured transformants using SDS-PAGE and Western analysis it was ascertained that the yields of intact cytoplasmic GFP and secreted GLA-GFP are much higher than those obtained in A. niger protease deficient hosts (Siedenberg et al. Biotechn. Prog. (1999) 15, 43-50; Gordon et al., Microbiology (2000) 146, 415-426). In contrast to the situation in A. niger culture supernatants also the secreted GFP showed significant fluorescence. 30 DESCRIPTION OF THE FIGURES Figure 1: This figure provides a comparison of amino acid sequences of KEX2-like processing proteases from X laevis (XENPC2 and XNFURIN), S. cerevisiae (SCKEX2), WO 01/09352 PCT/NLOO/00544 33 K lactis (KLKEX1), C. albicans (CAKEX2), S. pombe (SPKRP) and Y. lipolytica (YLKEX2). The primers, which encode for the amino acid sequences with the highest overall identity (indicated with lightblue boxes), are indicated: MBL793, MBL1208, MBL 794, MBL1158, PE6, PCL1, PCL2(rev), PE6, PCL3, MBL789, PCL4 and MBL1219. 5 Regions of overall identity (4 out of 7 entries) are indicated with purple boxes. Gaps are indicated with. ; no sequence data are indicated with ~ ; asteriks indicate the stop codon of the protein. Figure 2: This consists of 2a, b and c 10 Figure 2a provides the background growth of the A. sojae strain described in patent W097/04108 after 5 days of incubation at 33*C. The top picture reveals growth on non selection medium. The bottom left picture shows selection medium according to W097/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention. 15 Figure 2b provides the background growth of the A. sojae strain ATCC 11906 after 5 days of incubation at 33*C. The top picture reveals growth on non selection medium. The bottom left picture shows selection medium according to W097/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention. 20 Figure 2c provides the background growth of the A. sojae strain ATCC20387 after 5 days of incubation at 33*C. The top picture reveals growth on non selection medium. The bottom left picture shows selection medium according to W097/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention. 25 Figure 3 (a and b): This figure provides a comparison of A. sojae ATCC 11906 and A. oryzae amdS sequences from both ends. A and B indicate the two ends. The cloned 1.6 kb A. sojae sequence was used. Underlined bold bases differ between species/strains. Intron I sequences are indicated in small letters. 30 Figure 4 (a and b): This figure illustrates the construction of a pyrG disruption vector via pAO4-13 and pAO4-13deltaCla. Figure 5: This figure illustrates the construction of pAB4-lrep going from pAB4-1 via WO 01/09352 PCT/NLOO/00544 34 isolation of XhoI fragment and HindIII fragment followed by cloning into pMTL24. Figure 6: The construction of the alpA gene replacement vector is disclosed in this figure. A 4.4 kb EcoRI-StuI fragment from pAS1-1 with the ATCC 11906 genomic fragment, the 5 2.6 kb SmaI-NcoI fragment from pAB4-lrep and the 4.4 kb NcoI-EcoRI fragment from pAS 1 -2A are ligated in a 3 way ligation thus providing pAS 1 -deltaalp. Figure 7: This figure provides the restriction map of the DNA fragment carrying the A. niger pc/A gene. 10 Figure 8: This figure provides the structure (functional organisation) of the A. niger pclA encoded protein. It shows pre, pro, activity and P domains from left to right. The light coloured triangles indicate KR sites. The dark coloured triangles indicate glycosylation sites. The vertically striped light box is an S/P/T rich region. The dark weavepatterned box 15 at the right end is a D/E rich region. Figure 9: This figure illustrates growth phenotype of an A. niger pcA mutant strain. Figure 10: This figure provides a DNA sequence comparison between the A. sojae and A. 20 niger pcA genes. A vertical bar indicates identity; : indicates 5; - indicates 1. 72.139% similarity and 72.073% identity were found. Figure 11: The construction of the pcA gene replacement vector is disclosed in this figure. A 7.6 kb ClaI fragment, which is a ATCC 11906 genomic fragment, was cloned into 25 pMTL23p. In this construct the 2.6 kb SmaI fragment from pAB4-lrep was cloned into the EcoRV-site, thus providing pAS2-delta pcl. Figure 12: This figure shows the amino acid sequence comparison of the various PclA homolous from S. cerevisiae (Sckex2), K lactis (Klkex1), A. sojae (Aspcla), A. niger (A. 30 niger), P. chrysogenum (Penpcll), A. bisporus (Agarmbl129), T. reesei (Trichpcll), R. oryzae (Rhizpcll), F. venenatum (Fuspcll), S. pombe (Spkrp), C. albicans (Cakex2) and Y lipolytica (Ylkex2). Regions of overall identity (8 out of 12 entries) are indicated with yellow boxes. Gaps are indicated with.. ; no sequence data are indicated with ~ .

WO 01/09352 PCT/NLOO/00544 35 Figure 13: Sequence data are provided in figure 13a for the A. oryzae alpA promoter sequences (Q1 1755). The primer position for PCR cloning is indicated. In figure 13b the sequence data are provided for the A. oryzae amyA promoter sequences also including primer positions (A02532). Figure 13c provides the ATCC42149 A. oryzae derived gpdA 5 promoter sequences (EPO.436.858 al) also including primer positions. Figure 14: This figure provides a comparison between various gpdA promoter sequences of Aspergillus: From top to bottom, A sojae ATCC1 1906, A. oryzae, A. niger and A. nidulans. Asterisks indicate the putative intron present in the 5' untranslated region of the promoters. 10 Arrowhats indicate the CT rich regions. Bold underlined letters indicate the differences between the A. oryzae and A. sojae sequences. Figure 15: This figure shows a map of the vector pGLA6S of 12700bp. 15 SEQUENCE LISTING SEQ ID No. 1 MBL1784: 5'-CGGAATTCGAGCGCAACTACAAGATCAA-3' 20 SEQ ID No. 2 MBL 1785: 5'-CGGAATTCAGCCCAGTTGAAGCCGTC-3' 25 SEQ ID No. 3 The sequence of the Aspergillus niger gene encoding proprotein convertase 30 The start codon and the stop codon are indicated with bold underlined letters The intron is indicated with underlined small letters 1 CCATGGCAAG CCTCCTACTT GGCCTGATTA CATCGTCCTG AGAGAGAGAG 35 51 TTCACCAAAA CTCTCCCCCA AACGATGCGT CTTACAGGTG GTGTCGCTGC WO 01/09352 PCT/NLOO/00544 36 101 GGCTCTGGGC CTCTGCGCTG CTGCCTCCGC TTCTCTCCAT CCCCATCGTT 151 CCTACGAGAC CCATGATTAC TTCGCTCTAC ACCTTGATGA ATCCACCTCG 5 201 CCGGCCGACG TCGCCCAACG ACTAGGTGCT CGCCACGAAG GCCCCGTCGG 251 AGAATTACCC TCACATCATA CCTTCTCGAT ACCCCGTGAA AACAGTGACG 301 ATGTCCATGC GCTGCTGGAT CAATTGCGCG ATCGTCGGAG GTTACGCCGC 10 351 CGCTCCGGAG ATGACGCCGC TGTCCTTCCC TCCTTGGTCG GGCGAGACGA 401 AGGTCTAGGT GGCATTCTTT GGTCCGAGAA GCTGGCTCCC CAGAGAAAGC 15 451 TCCATAAAAG AGTGCCGCCG ACAGGATATG CTGCCAGATC GCCCGTCAAC 501 ACTCAGAATG ACCCCCAAGC GCTTGCGGCG CAGAAACGCA TTGCCTCGGA 551 ATTGGGCATC GCGGACCCCA TCTTCGGCGA ACAATGGCAT TTGTATAATA 20 601 CTGTTCAGTT GGGCCATGAT CTTAACGTGA CGGGTATCTG GCTGGAGGGC 651 GTTACAGGGC AGGGTGTCAC GACGGCCATT GTCGATGACG GTTTGGACAT 25 701 GTACAGCAAC GATCTTAGGC CGAACTATTT TGCGGCGGGT TCTTATGACT 751 ATAACGACAA AGTACCAGAG CCGAGGCCGC GCTTGAGCGA TGACCGCCAC 801 GGTACTAGAT GCGCGGGTGA AATCGGTGCG GCGAAGAACG ACGTGTGCGG 30 851 GGTTGGTGTT GCGTATGATA GTCGCATCGC TGGTATTCGG ATTCTCTCCG 901 CACCCATCGA TGACACTGAT GAGGCTGCGG CTATTAACTA CGCCTATCAG 35 951 GAGAACGATA TCTACTCGTG TTCCTGGGGT CCCTATGATG ATGGCGCCAC 1001 AATGGAAGCC CCGGGCACTC TGATCAAGCG GGCCATGGTC AATGGTATCC 1051 AAAATGGTCG AGGTGGAAAA GGCTCGGTTT TTGTATTTGC GGCTGGTAAC 40 1101 GGTGCCATTC ATGACGATAA CTGTAACTTT GACGGTTACA CCAACAGTAT 1151 CTACAGCATC ACGGTGGGTG CCATTGATCG GGAGGGTAAC CATCCTCCGT WO 01/09352 PCT/NLOO/00544 37 1201 ATTCGGAATC CTGCTCGGCG CAACTGGTGG TTGCCTACAG CAGCGGCGCC 1251 AGTGATGCAA TTCATACCAC GGACGTCGGC ACAGACAAGT GCTCGACTAC 5 1301 CCATGGTGGA ACTTCGGCGG CCGGCCCGCT CGCTGCGGGA ACCGTGGCGC 1351 TGGCCCTCAG TGTGCGGCCG GAACTCACCT GGCGTGACGT TCAGTATTTG 10 1401 ATGATTGAGG CGGCAGTGCC TGTTCATGAA GATGATGGAA GCTGGCAGGA 1451 CACTAAGAAC GGGAAGAAGT TCAGCCATGA CTGGGGATAT GGTAAGGTCG 1501 ACACATATAC GCTGGTGAAA CGGGCAGAGA CCTGGGATCT GGTGAAGCCT 15 1551 CAAGCCTGGC TCCATTCCCC CTGGCAGCGG GTTGAGCATG AGATCCCACA 1601 GGGCGAGCAG GGCTTGGCTA GTTCGTACGA GGTGACGGAG GATATGTTGA 20 1651 AGGGAGCCAA CCTGGAACGG CTGGAGCATG TCACGGTCAC CATGAATGTT 1701 AACCACACCC GCCGAGGCGA TCTCAGCGTG GAGTTACGGA GCCCTGATGG 1751 TCGGGTCAGT CACCTCAGTA CGCCCCGGCG GCCAGATAAT CAAGAGGTGG 25 1801 GCTATGTTGA TTGGACCTTC ATGAGCGTTG CTCACTGga aqtaaaaact 1851 ttttctcgqt tqtcqqttct tctqctaata catatctaqG GGCGAGTCCG 30 1901 GGATTGGCAA ATGGACTGTG ATTGTCAAGG ACACCAATGT CAACGAGCAT 1951 ACTGGGCAAT TCATCGATTG GCGACTCAAC TTGTGGGGCG AGGCGATTGA 2001 CGGAGCCGAG CAGCCTCTCC ACCCCATGCC TACTGAACAC GATGACGACC 35 2051 ACAGCTATGA GGAAGGAAAC GTGGCTACCA CGAGCATCAG CGCCGTTCCC 2101 ACGAAAACCG AGCTGCCTGA CAAGCCCACT GGTGCGTTG ATCGCCCGGT 40 2151 GAACGTTAAG CCTACAACAT CCGCGATGCC GACCGGTAGT CTTACAGAGC 2201 CCATCGATGA TGAAGAACTC CAGAAGACCC CTAGTACAGA GGCAAGCTCA WO 01/09352 PCT/NLOO/00544 38 2251 ACACCAAGTC CTTCTCCGAC CACCGCGTCA GATAGTATCC TGCCTTCCTT 2301 CTTCCCCACG TTCGGTGCGT CGAAGCGGAC CGAAGTTTGG ATCTACGCTG 5 2351 CGATCGGCTC CATCATTGTG TTCTGCATTG GCCTGGGCGT CTACTTCCAT 2401 GTGCAGCGCC GCAAACGTAT TCGCGACGAC AGCCGGGATG ACTACGATTT 2451 CGAGATGATC GAGGACGAGG ATGAGCTACA GGCAATGAAC GGACGGTCGA 10 2501 ACCGTTCACG TCGCCGGGGT GGCGAGCTGT ACAATGCTTT TGCGGGCGAG 2551 AGCGATGAGG AACCATTATT CAGTGATGAG GATGATGAAC CGTATCGGGA 15 2601 TCGGGGGATC AGCGGCGAAC AAGAACGGGA GGGCGCAGAT GGAGAGCATT 2651 CTCGGAGATG AAAGTGCAGT AGATGAGGGT TGACTTTATT TCGGACAGTG 2701 TTTCTAACTT GTTGGATGAC CTGCGTTGAA CAATATTTCT GCTGTGTATG 20 2751 CTGCATAGAG AAGCGTGTAT ATACCATGTA TGTGTGCATC ATCGTGATCG 2801 GGTTTATCAT TCTTCATCTG CCATGGTTTG TGATCTCCGG AATAGTACCA 25 2851 AAGGAACACT AAATTAAGGG TCTTGGCGAT GACGCTTCCC GTCGCTGCTT 2901 TTGACTTCCT CCGCATCTCG TCTCTCCTGC TGTTGACCGC GCGCCAACCA 2951 ACCTCCATCT CCTCACTCCT CCCACCTTAA TCTTGCTGTG CTGCTTCTAG 30 3001 AACCCCCCAG TTTAATTTAA AAACCGGCTT TTCCTAGCTC CACGTATTGT 3051 ACCTCGCACT GATCCCCATC TCCGCCCACT CCAACGCTAC CGACCCAGGC 35 3101 TTCTCTGGCG GCTCCAGGCG GCAGGCAATC AAACCAACCC CTCGATGGAT 3151 CAGCACGACG ACTTCGACAG SGTCTCGTGG AGGCATGACC CGGACAGCGA 3201 TCTCTCGCGA CCCACGAACT CCGGAACAGA CACAGAGGAA CAGGCGCCAT 40 3251 ACACTCACGA TGTCAATGGC AAACGGAGGA TGAGCAACCG CTCAAGAAAG 3301 CCCTCAGGCT GGACCACTGG CGGATGCCGT CGACCTGGCG GGCATCGCGA WO 01/09352 PCT/NLOO/00544 39 3351 CGGCGTACTA GAGTGTCGGG TAGATTCACC GTTGAAGGAG AATATGGACG 3401 AAAGACGCTT ATATCTCCTA TTTGGTACAC TACTAGGTGG GTATCTTACC 5 3451 TCAGTGATCT CAGATGGA SEQ ID No. 4 10 The partial sequence in the coding region of the Aspergillus sojae gene encoding proprotein convertase 1 CGCGGATCCA TGGAACACGA TGTGCGGGTG AAATTGGAGC AGCTAGGAAT 15 51 GATGTCTGTG GAGTAGGTGT TGCATACGAC AGCCAAGTTG CCGGAATTCG 101 GATTTTGTCC GCACCCATTG ACGACGCAGA TGAGGCTGCT GCCATCAACT 20 151 ATGGCTTCCA GCGCAATGAT ATATATTCAT GCTCCTGGGG CCCTCCGGAT 201 GATGGCGCCA CGATGGAGGC GCCAGGGATT CTTATCAAAC GAGCTATGGT 251 CAACGGTATC CAAAATGGCC GAGGAGGTAA AGGTTCTATC TTCGTCTTTG 25 301 CAGCTGGAAA TGGTGCAGGG TACGATGACA ACTGCAATTT CGACGGTTAT 351 ACAAACAGCA TTTACAGCAT CACCGTCGGC GCTATTGATC GAGAGGGCAA 30 401 ACATCCCAGC TACTCGGAAT CATGCTCTGC CCAGTTGGTT GTCGCTTATA 451 GCAGTGGCTC GAGTGACGCG ATTCATACCA CCGACGTTGG AACTGATAAA 501 TGTTATTCAC TNTCACGGGC GGAACTTCTG CAACTGGACC GCTAGCTGCG 35 551 GGTACTATTG CCCTCGCTCT TAGTGCCCGA CCGGAACTAA CTTGGCGAGA 601 TGCCCAGTAC CTGATGATAG AGACCGCAGT TCCCGTCCAC GAAGACGACG 40 651 GGAGCTGGCA GACTACCAAA ATGGGGAAGA AGTTTAGCCA TGACTGGGGT 701 TTTGGGAAAG TAGATGCATA TTCACTGGTC CAGCTGGCCA AGACGTGGGA WO 01/09352 PCT/NLOO/00544 40 751 GCTGGTGAAA CCACAGGCGT GGTTCCACTC ACCGTGGCTG CGGGTGAAGC 801 ATGAAATCCC ACAAGGTGAC CAGGGCCTTG CCAGCTCATA CGAAATTACC 5 851 AAGGATATGA TGTACCAGGC CAATGTCGAG AAATTGGAAC ATGTCACTGT 901 GACCATGAAT GTAAATCACA CTCGCCGAGG CGATATCAGC GTGGAGTTGC 10 951 GCAGCCCCGA AGGTATCGTC AGTCATCTGA GTACAGCGCG GCGGTCAGAT 1001 AATGCAAAGG CTGGCTATGA AGATTGGACG TTTATGACTG TGGCTCATTG 1051 GTATGTATTT GCTCCCGTAA TTTAGTTTTC GTGCTCAGTC CTGACATTTA 15 1101 CATTTAGGGG TGAGTCCGGT GTTGGAAAGT GGACGGTCAT TGTGAAGGAT 1151 ACCAATGTCA ATGATCATGT TGGAGAATTC ATCGACTGGC GGCTCAACCT 20 1201 CTGGGGACTT TCGATCGACG GCTCCAGCCA GCCCCTTCAT CCTATGCCCG 1251 ATGAGCATGA CGATGACCAC TCGATTGAAG ATGCCATTGT TGTTACCACT 1301 AGTGTTGACC CTATCCCAAC TAAGACTGAA GCCCCACCTG TCCCAACTGA 25 1351 TCCCGTGGAT CGTCCTGTGA ACGCAAAGCC ATCTGCGCAG CCAACGATGC 1401 CTTCAGAGGC TCCTGCTCAA GAGACATCTG AAGTTCCCAC CCCGACGAAA 30 1451 CCTAGTTCTA CTGAATCACC TTCTTACCAC CTCCTCTGCG GATAGCTTTT 1501 TGCCATCCTT CTTCCCCACG TTCGGTGCGT CGTGAGGATC CAAGCTTGGG 1551 TACGT 35 SEQ ID No. 5 The partial sequence in the coding region of the Trichoderma reesei QM9414 gene 40 encoding proprotein convertase 1 GCTGTCCGCA CTGATGCGTG CGGCCTTGGC GTTGCCTACG ACTCCAAGAT WO 01/09352 PCT/NLOO/00544 41 51 TGCTGGCATC CGCATCCTTA GTAGTGCCAT CAGCGATGCG GACGAGGCCG 101 AGGCCATGAT TTACAAGTTC CAGGACAACC AGATCTACTC GTGCTCCTGG 5 151 GGGCCTCCCG ACGATGGGAG GTCCATGGAA GCCCCCGACG TCCTGATTCG 201 ACGAGCCATG CTCAAGGGCG TCCAGGAGGG ACGAGGAGGC CTCNGCTCCA 10 251 TCTACGNCTT TGCTAGTGGT AACGGTGCCG CCAGTGGCGA TAACTGCAAC 301 TNCGACGGAT ACNCAAACA 15 SEQ ID No. 6 The partial sequence in the coding region of the Fusarium venenatum ATCC20334 gene encoding proprotein convertase 20 1 GGTTTNNCCG TTGGTGTTGC TACGACTCCA AGTCGCCGGA ATCCGTATTC 51 TCAGCAAACT GATCAGCGAC GCCGACGAAG CAGAAGCGCT TATGTACAAG 101 TACCATGACA ACCATATTTA CTCTTGCTCA TGGGGTCCTT CCGATGATGG 25 151 CCAGACTATG GAGGCACCCG ATGTTGTCAT TCGACGAGCA ATGCTTAAGG 201 CGATTCAGGA GGGACGTAAT GGTCTTGGCT CTGTCTACGT CTTTGCCAGT 30 251 GGAAACGGTG CAGGCCAAGG AGATAACTGC AACTNCGACG GATCCACCAA 301 ACA 35 SEQ ID No. 7 The partial sequence in the coding region of the Penicillium chrysogenum P2 gene encoding for proprotein convertase WO 01/09352 PCT/NL00/00544 42 1 GTGGGTGTTG CCTATGACAG CAAGGTGTCA GGTATCCGGA TTCTGTCCAA 51 GGCGATTGAC GACGTCGACG AAGCAGCTGC CATCAACTTT GCCTTCCAAG 5 101 ATAACGATAT ATACTCCTGC TCGTGGGGTC CTCCTGATGA TGGTGCGACC 151 ATGGATGCGC CGGGCTTGTT GATCAAGCGG GCGATGGTCA ATGGTGTGCA 201 NGAGGGACGA GGTGGAAAGG GTTCGATCTT CGTGTTNGCC GCAGGCAACG 10 251 GTGCTCTTTT TGGCGACAAC TGCAACTTCG ACGGATACAA CAAACA SEQ ID No. 8 15 The partial sequence in the coding region of the Rhizopus oryzae ATCC200076 gene encoding proprotein convertase 1 ACTNGGGGCA TTGGTGAAAT NTTGCTTGTG GNTTGGTGTT GCTTACGACG 20 51 CAAAAATATC TGGTATACGT ATATTATCAG GTGAAATCAC AGAGGCAGAC 101 GAGGCTGCTG CTTTGAATTA CAAATATCAA GAAAATCAAA TCTACTCCTG 25 151 CTCNTGGGGC CCA SEQ ID No. 9 30 The partial sequence in the coding region of the Agaricus bisporus HORST gene encoding proprotein convertase 1 ATGTGGTCTT GGTCTCGCCT ACGAATCCAA GGTCGCTGGT GTTCGCATAT 35 51 TGTCTGGTCC CATAACGGAC GTCGATGAAG CGACTGCGCT CAACTATGGT 101 TTCCAAAATG TATCTATCTT CAGCTGTAGT TGGGGCCCAC CTGACAATGG 151 TATGTCCATG GAAGGCCCAG GATACCTCAT CAAAAAAGCT GTCGTCAACG 40 WO 01/09352 PCT/NLOO/00544 43' 201 GCATTAACCA GGGACGTGGC GGGAAGGGCT CCATTTTCGT CTTCGCCAGT 251 GGCAACGGCG CTGCTTCGGA TGACCAATGC AACTACGACG GATACACAAA 5 301 CA SEQ ID No. 10 coding strand BamHI-site is underlined 10 PE4 5'- CG CGGATC CA(T/C) GGX ACX (C/A)GX TG(T/C) GCX GG -3' degenerated 2048 times SEQ ID No. 11 coding strand 15 PCL1 5'-CA(T/C) GGX ACX (C/A)GX TG(T/C) GCX GGX GA-3' degenerated 8192 times SEQ ID No. 12 coding strand 20 PCL2 5'-AT(C/T/A) TA(T/C) TCX TG(T/C) TCX TGG GGX CC-3' degenerated 768 times SEQ ID No. 13 25 non coding strand BamHI-site is underlined PE6 5'- CGC GGA TCC XCC (A/G)TT XCC X(C/G)(A/G) XGC (G/A/C)(C/A)A XAC -3' degenerated 49152 times 30 SEQ ID No. 14 non coding strand PCL2rev 5'-GG XCC CCA XGA (A/G)CA XGA (A/G)TA (A/T/G)AT-3' degenerated 768 times WO 01/09352 PCT/NLOO/00544 44 SEQ ID No. 15 non coding strand PCL3 5'-(A/G)TT XGT (A/G)TA XCC (A/G)TC (A/G)(A/T)A (A/G)TT-3' degenerated 1024 times 5 SEQ ID No. 16 non coding strand PCL4 5'-GC XGC XGA XGT XCC XCC (A/G)TG-3' degenerated 2048 times 10 SEQ ID No. 17 The sequence of pAB4-lrep ........ 59-499 bp................................................. 0.4 kb HindIII fragment of pAB4-1 15 1-58 bp........500-513 bp.............2873-2930 bp: polylinker sequence of pMTL24 (indicated with underlined small letters) ................................... 514-2872 bp.................... 2.3 kb Xhol fragment of pAB4-1 20 1 qqccaqtgaa ttcgagctcg 9tacccqqqq atcctctaga qtcgacctgc 51 aqqcatqcAA GCTTGGTCAG CAGTACCAGA CGCCCGGATC GGCTATCGGC 101 CGGGGTGCTG ACTTCATTAT CGCGGGTCGC GGTATCTACG CCGCGCCGGA 25 151 TCCGGTGCAG GCTGCGCAAC AGTATCAGAA GGAGGGGTGG GAAGCCTACC 201 TGGCCCGTGT CGGCGGAAAC TAATACTATA AAAGGAGGAT CGAAGTTCTG 30 251 ATGGTTATGA ATGATATAGA AATGCAACTT GCCGCAACGG ATACGGAAGC 301 GGAAACGGAC CAATGTCGAG CACGGGTAGT CAGACTGCGG CATCGGATGT 351 CCAAACGGTA TTGATCCTGC AGGCTACTAT GGTGTGGCAC AAGGATCAAT 35 401 GCGGTACGAC GATTTGATGC AGATAAGCAG GCTGCGAAGT AGTAACTCTT 451 GCGTAGAGAA AATGGCGACG GGTGGGCTGA TAAGGGCGGT GATAAGCTTg WO 01/09352 PCT/NLOO/00544 45 501 catqcctqca gccCTCGAGC TAACATACAT TCCGAACCGT GCAGCCCAAG 551 GCCGAGCAGT TCAACTGCGC TCAGCGCGCT CATGCCAACT TCCTTGAGAA 5 601 CTCCAGCCAA ACTATGCTCT TCCTCCTGGT AGCTGGACTG AAGTACCCCC 651 AGTTGGCGAC TGGCCTCGGA AGCATCTGGG TCCTCGGTCG CTCACTGTTC 701 CTTTACGGAT ATGTGTACTC CGGCAAGCCG CGGGGTCGCG GTCGTTTGTA 10 751 CGGCAGCTTC TACTTGCTTG CACAGGGAGC TCTCTGGGGC NTGACGTCTT 801 TTGGAGTTGC GAGGGAGTTG ATTTCCTACT TCTAAGTTTG GACTTGAATC 15 851 CGTGGTGTGA TTGAGGTGAT TGGCGATGTT TGGCTATACC AGCTATATGT 901 AATAATCTCT ACTGTATACT ACTATTCAAC GCATTTTACT ATGCGTGCTG 951 CTAGGGTCGG CAATGACAAT GGCAATCTGA CTGACGTGGT CTATTTCTCC 20 1001 ATGTGCAGCA GGGAATACGA GCTCCAATGG ACCTCGGGAG TGGCACAGTC 1051 AATGGCAAGG AAACTCCGCC TTTGCAGGTG TGGCTGAACC CCACGGGTCG 25 1101 GAGGCGGAGC AATCCACCCC CGATGTGGCT GGTGCGTGGA GGGGCTCGCG 1151 ATGATTTTAC TGAGCTTGCT TTTCTTGTCG ACATTGAACA TTGTCCTTGG 1201 TCTTCCTTCA GATTTAAGGG TCAGTCACTG CTACATTTCT CAGTAGTATC 30 1251 CGCGCACGTC TCTGGATTTA CGAATCAGGG TCCACCAGTC GAAACTTCGA 1301 ACTACTCTCA TTATACAATC CTCTTTCCAT TCCCGCATTA ACCCCTCCAT 35 1351 CAACACCATG TCCTCCAAGT CGCAATTGAC CTACACTGCC CGTGCCAGCA 1401 AGCATCCCAA TGCTCTGGCG AAGAGGCTGT TCGAGATTGC CGAGGCCAAG 1451 AAGACCAATG TGACTGTCTC GGCTGACGTT ACCACCACTA AGGAGCTACT 40 1501 AGATCTTGCT GACCGTAGGC CGACCCGCTA CTCTGCCTGA TTATGCTGCA 1551 TGCAAACTTA TTAACGGTGA TACCGGACTG CAGGTCTCGG TCCCTACATT WO 01/09352 PCT/NLOO/00544 46 1601 GCCGTGATCA AAACCCACAT CGATATCCTC TCTGATTTCA GCAACGAGAC 1651 CATTGAGGGA CTTAAGGCTC TCGCGCAGAA GCACAACTTT CTCATCTTCG 5 1701 AGGACCGCAA GtTCATTGAC ATCGGCAACA CGGTCCAGAA GCAATACCAC 1751 GGCGGTACCC TCCGTATCTC GGAATGGGCC CACATCATCA ACTGCAGCAT 10 1801 TCTCCCTGGT GAGGGTATCG TCGAGGCTCT CGCTCAGACG GCGTCTGCAC 1851 CGGACTTCGC CTACGGCCCC GAACGCGGTC TGTTGATCTT GGCAGAGATG 1901 ACCTCTAAGG GCTCCTTGGC TACCGGCCAG TACACTACTT CCTCGGTCGA 15 1951 TTATGCCCGG AAATACAAGA ACTTCGTTAT GGGATTCGTG TCGACGCGCG 2001 CGTTGGGTGA GGTGCAGTCG GAAGTCAGCT CTCCTTCGGA TGAGGAGGAC 20 2051 TTTGTGGTCT TCACGACTGG TGTGAACATT TCTTCCAAGG GAGATAAGCT 2101 TGGTCAGCAG TACCAGACGC CCGGATCGGC TATCGGCCGG GGTGCTGACT 2151 TCATTATCGC GGGTCGCGGT ATCTACGCCG CGCCGGATCC GGTGCAGGCT 25 2201 GCGCAACAGT ATCAGAAGGA GGGGTGGGAA GCCTACCTGG CCCGTGTCGG 2251 CGGAAACTAA TACTATAAAA GGAGGATCGA AGTTCTGATG GTTATGAATG 30 2301 ATATAGAAAT GCAACTTGCC GCAACGGATA CGGAAGCGGA AACGGACCAA 2351 TGTCGAGCAC GGGTAGTCAG ACTGCGGCAT CGGATGTCCA AACGGTATTG 2401 ATCCTGCAGG CTACTATGGT GTGGCACAAG GATCAATGCG GTACGACGAT 35 2451 TTGATGCAGA TAAGCAGGCT GCGAAGTAGT AACTCTTGCG TAGAGAAAAT 2501 GGCGACGGGT GGGCTGATAA GGGCGGTGAT AAGCTTAATT GTCATCGCAG 40 2551 ATAAGCACTG CTGTCTTGCA TCCAAGTCAG CGTCAGCAGA AATACGGGAC 2601 TTCCGAAAGT ATATGGCAAA ATTAAAGAAC TTGACTCTCC AGCAATGTTT WO 01/09352 PCT/NLOO/00544 47 2651 TGCCCTGACC GTCGCTAAAA CGTTACTACC CCTATACCCG TCTGTTTGTC 2701 CCAGCCCGAG GCATTAGGTC TGACTGACAG CACGGCGCCA TGCGGGCTTG 5 2751 GGACGCCATG TCCGTCGCGT GATAAGGGTT GATCCATGCA GCTACTATCC 2801 TTCCATCGTT CCATTCCCAT CCTTGTCCTA TCTCCATCCT TGAAACTTTA 2851 CTAGTTTAGT TGGATGCTCG AGatctccat qqacqcqtga cqtcqactct 10 2901 gaggatcccc qqqtaccqaq ctcgaattcq SEQ ID No. 18 15 MBL 789 EcoRI is underlined 5'- GGAA TTC (A/G)GA ATA (T/A)GG AGG ATG TAG -3' degenerated 4 times SEQ ID No. 19 20 MBL 793 BamHI is underlined 5'- CGGATCCG CAG TGG CAC TTG (G/A)TC AAT CCA A -3' degenerated 2 times SEQ ID No. 20 25 MBL 794 EcoRI is underlined 5'- GGA ATT CTT AAA A(T/G)C CCA AGA ACC TTC A -3' degenerated 2 times SEQ ID No. 21 30 MBL 1158 EcoRI is underlined 5'- G GAA TTC (T/C)TC (T/G)CC (T/G)GC (A/G)CA (C/G)C(T/G) (C/G)GT (T/G)CC (A/G)TG -3' degenerated 512 times 35 SEQ ID No. 22 MBL 1208 ClaI is underlined WO 01/09352 PCT/NLOO/00544 48 5'- CGG ATC GA(T/C) GGX ACX (C/A)GX TG(T/C) GCX GG -3' degenerated 2048 times SEQ ID No. 23 5 MBL 1219 BamHI is underlined 5'- CGG ATC (C/T)TG XA(G/T/C) (A/G)TC XC(T/G) CCA XGT (C/A/G)AG -3' degenerated 4608 times 10 SEQ ID No. 24 Restriction sites are bold Primers are underlined BamHI PE4 primer 15 GGATCCATGG CACGAGATGT GCAGGTGAAA TCGGTGCGGC GAAAGAAAAC AACGTGTGCG 60 GGGTTGGTGT TGCGTATGAT AGTCGCATCG CTGGTATTCG GATTCTCTCC ACACCCATCG 120 20 EcoRV ATGACACTGA TGAGGCTGCG GCTATTAACT ACGCCTATCA GGAGAACGAT ATCTACTCGT 180 GTTCCTGGGG TCCCTATGAT GATGGCGCCA CAATGGAAGC CCCGGGCACT 25 CTGATCAAGC 240 GGGCCATGGT CAATGGTATC CAAAATGGTC GAGGTGGAAA AGGCTCGGTT TTTGTCTGCG 300 PE6 primer 30 CCCCCGGAAA TGGTGGATCC 320 BamHI 35 SEQ ID No. 25 Aspergillus niger PclA protein sequence WO 01/09352 PCT/NLOO/00544 49 1 Met Arg Leu Thr Gly Gly Val Ala Ala Ala Leu Gly Leu Cys Ala 16 Ala Ala Ser Ala Ser Leu His Pro His Arg Ser Tyr Glu Thr 5 His 31 Asp Tyr Phe Ala Leu His Leu Asp Glu Ser Thr Ser Pro Ala Asp 10 46 Val Ala Gln Arg Leu Gly Ala Arg His Glu Gly Pro Val Gly Glu 61 Leu Pro Ser His His Thr Phe Ser Ile Pro Arg Glu Asn Ser Asp 15 76 Asp Val His Ala Leu Leu Asp Gln Leu Arg Asp Arg Arg Arg Leu 91 Arg Arg Arg Ser Gly Asp Asp Ala Ala Val Leu Pro Ser Leu 20 Val 106 Gly Arg Asp Glu Gly Leu Gly Gly Ile Leu Trp Ser Glu Lys Leu 25 121 Ala Pro Gln Arg Lys Leu His Lys Arg Val Pro Pro Thr Gly Tyr 136 Ala Ala Arg Ser Pro Val Asn Thr Gln Asn Asp Pro Gln Ala Leu 30 151 Ala Ala Gln Lys Arg Ile Ala Ser Glu Leu Gly Ile Ala Asp Pro 166 Ile Phe Gly Glu Gln Trp His Leu Tyr Asn Thr Val Gln Leu 35 Gly 181 His Asp Leu Asn Val Thr Gly Ile Trp Leu Glu Gly Val Thr Gly 40 196 Gln Gly Val Thr Thr Ala Ile Val Asp Asp Gly Leu Asp Met WO 01/09352 PCT/NLOO/00544 50 Tyr 211 Ser Asn Asp Leu Arg Pro Asn Tyr Phe Ala Ala Gly Ser Tyr Asp 5 226 Tyr Asn Asp Lys Val Pro Glu Pro Arg Pro Arg Leu Ser Asp Asp 241 Arg His Gly Thr Arg Cys Ala Gly Glu Ile Gly Ala Ala Lys 10 Asn 256 Asp Val Cys Gly Val Gly Val Ala Tyr Asp Ser Arg Ile Ala Gly 15 271 Ile Arg Ile Leu Ser Ala Pro Ile Asp Asp Thr Asp Glu Ala Ala 286 Ala Ile Asn Tyr Ala Tyr Gln Glu Asn Asp Ile Tyr Ser Cys Ser 20 301 Trp Gly Pro Tyr Asp Asp Gly Ala Thr Met Glu Ala Pro Gly Thr 316 Leu Ile Lys Arg Ala Met Val Asn Gly Ile Gln Asn Gly Arg 25 Gly 331 Gly Lys Gly Ser Val Phe Val Phe Ala Ala Gly Asn Gly Ala Ile 30 346 His Asp Asp Asn Cys Asn Phe Asp Gly Tyr Thr Asn Ser Ile Tyr 361 Ser Ile Thr Val Gly Ala Ile Asp Arg Glu Gly Asn His Pro Pro 35 376 Tyr Ser Glu Ser Cys Ser Ala Gln Leu Val Val Ala Tyr Ser Ser 391 Gly Ala Ser Asp Ala Ile His Thr Thr Asp Val Gly Thr Asp 40 Lys WO 01/09352 PCT/NLOO/00544 51 406 Cys Ser Thr Thr His Gly Gly Thr Ser Ala Ala Gly Pro Leu Ala 5 421 Ala Gly Thr Val Ala Leu Ala Leu Ser Val Arg Pro Glu Leu Thr 436 Trp Arg Asp Val Gln Tyr Leu Met Ile Glu Ala Ala Val Pro Val 10 451 His Glu Asp Asp Gly Ser Trp Gln Asp Thr Lys Asn Gly Lys Lys 466 Phe Ser His Asp Trp Gly Tyr Gly Lys Val Asp Thr Tyr Thr 15 Leu 481 Val Lys Arg Ala Glu Thr Trp Asp Leu Val Lys Pro Gln Ala Trp 20 496 Leu His Ser Pro Trp Gln Arg Val Glu His Glu Ile Pro Gln Gly 511 Glu Gln Gly Leu Ala Ser Ser Tyr Glu Val Thr Glu Asp Met Leu 25 526 Lys Gly Ala Asn Leu Glu Arg Leu Glu His Val Thr Val Thr Met 541 Asn Val Asn His Thr Arg Arg Gly Asp Leu Ser Val Glu Leu 30 Arg 556 Ser Pro Asp Gly Arg Val Ser His Leu Ser Thr Pro Arg Arg Pro 35 571 Asp Asn Gln Glu Val Gly Tyr Val Asp Trp Thr Phe Met Ser Val 586 Ala His Trp Gly Glu Ser Gly Ile Gly Lys Trp Thr Val Ile Val 40 WO 01/09352 PCT/NLOO/00544 52 601 Lys Asp Thr Asn Val Asn Glu His Thr Gly Gln Phe Ile Asp Trp 616 Arg Leu Asn Leu Trp Gly Glu Ala Ile Asp Gly Ala Glu Gln 5 Pro 631 Leu His Pro Met Pro Thr Glu His Asp Asp Asp His Ser Tyr Glu 10 646 Glu Gly Asn Val Ala Thr Thr Ser Ile Ser Ala Val Pro Thr Lys 661 Thr Glu Leu Pro Asp Lys Pro Thr Gly Gly Val Asp Arg Pro Val 15 676 Asn Val Lys Pro Thr Thr Ser Ala Met Pro Thr Gly Ser Leu Thr 691 Glu Pro Ile Asp Asp Glu Glu Leu Gln Lys Thr Pro Ser Thr 20 Glu 706 Ala Ser Ser Thr Pro Ser Pro Ser Pro Thr Thr Ala Ser Asp Ser 25 721 Ile Leu Pro Ser Phe Phe Pro Thr Phe Gly Ala Ser Lys Arg Thr 736 Glu Val Trp Ile Tyr Ala Ala Ile Gly Ser Ile Ile Val Phe Cys 30 751 Ile Gly Leu Gly Val Tyr Phe His Val Gln Arg Arg Lys Arg Ile 766 Arg Asp Asp Ser Arg Asp Asp Tyr Asp Phe Glu Met Ile Glu 35 Asp 781 Glu Asp Glu Leu Gln Ala Met Asn Gly Arg Ser Asn Arg Ser Arg 40 796 Arg Arg Gly Gly Glu Leu Tyr Asn Ala Phe Ala Gly Glu Ser WO 01/09352 PCT/NLOO/00544 53 Asp 811 Glu Glu Pro Leu Phe Ser Asp Glu Asp Asp Glu Pro Tyr Arg Asp 5 826 Arg Gly Ile Ser Gly Glu Gln Glu Arg Glu Gly Ala Asp Gly Glu 841 His Ser Arg Arg 10 SEQ ID Nos. 26 to 31 15 PCR-primers for A.sojae promoter cloning Restriction sites are underlined Primer Sequence (5' - 3') SEQ ID Alp-1 GGAATTCGCGGCCGCGGTTATTCTGCGGAAGC No. 26 G EcoRI Noti SEQ ID Alp-2 GGAATTCCCATGGTGAGAAGATTGTAAAG No. 27 EcoRI Ncol SEQ ID Amy-1 GGAATTCGCGGCCGCAGATCTGCCCTTATAAA No. 28 TCTCC EcoR] Noti SEQ ID Amy-2 GGAATTCCCATGGATGCCTTCTGTGGGG No. 29 EcoRI NcoI SEQ ID AOGPDA GGAATTCGCGGCCGCCTATGAAACCGGAAAG No. 30 -1 EcoRI Noti SEQ ID AOGPDA GGAATTCTAGCCATGGTTTAGATGTG No. 31 -2 EcoRI Nco] 20 SEQ ID No. 32 The sequence of the Aspergillus sojae gpdA promoter region WO 01/09352 PCT/NLOO/00544 54 1 AATTGCGGCC GCTATGAAAC CGGAAAGGGC TGCTGAGAGC TGGGGAACGG 51 CGCAAGCCGG GAAAACAGCT GACAAGGACC CATTTCACTC TGGATCTTGA 5 101 GGAGAGCTGT AGCTTTTGCC CCGTCTGTCC ACCCGGTGAC TGGATTAGTG 151 ACCTGGTCGT TGCGTCAGTC AACATTGCTC TTTTTTTATC TCCCCCTCCC 201 CCGCCGTCCG ACTTTTCTCC CCTTTTCTAC TCTCTTCGTA TACTCACCAC 10 251 TGCAATCATC TTATCCCTTT GTCTTCTTAC TTAAAGTGAG TCGTCTCCCG 301 CCCATCGTTC CCTTTGAACC TTGTAAATCA GAGCCACTTT CAAGTGTCTA 15 351 CCGTTTCCTT TCCACATAGA TTGACTGACA GCTACCCCGC CACACCAGCA 401 GACACATCTA AACCATGG 20 SEQ ID No. 33 The sequence of the Aspergillus sojae alpA promoter region 1 GCGGCCGCGG TTATTCTGCG GAAGCGGACC CCCCCCTTCC GCCCAAACAG 25 51 GGCGAATGTG CCCAAGTTCT GATACTATCA GAAGACCTCC AGGAGCACAT 101 GCCTGTTCGC ATAACCCTGG TGTAGCACCA GGAATTGCTT AGCTTAGCTT 30 151 CTTCGACTGA GGGGCCAGAA AGTGCTTATC GCAAAGATCC CACTTCTTTG 201 TGTGATAGCC CCTCCCGCGG CCCTTGATCA AGCCGTTCTC GCTATCCAAT 251 ATTGAAAGCG TGATATTATA GGTGCACATG GTTATTATCC TTTTTCTTTT 35 301 TCTCTTTCTT TGCTTTTCAT GCAACCCCAT ACGTTGCCGA ATTTGGCTAC 351 ACCTTGGGGC TCATTCTTCG AAGTTTAGAT TCCGACAAGA CCTCACCACC 40 401 CAATCAAAAC CCTTGATTCC TGATAAAAGA CGTGGAAAGA AGCGGATATC 451 GCGTGAGGAT GCCAAGCAAA GGGAATGGGT CACATTGATC TCTGTCGCGT WO 01/09352 PCT/NLOO/00544 55 501 TGTTAGGATG ATCTTCACTC CTAAAGGCAT CGCCCGCGGC ACTAGGTCCT 551 TCCTGTCCAG GATATCGTTT ACTCCTCTCA TTATGGCGAG CTACTTTGTG 5 601 AATTAATTGA CTGAGGGATA TACCACCTTC CCTTTGAAGG TACCAAGCCA 651 CTACCTTGAG CGTTAGTTAC TTTTTCGAGG AAAGCGTCCT ATGCTGGTCT 701 CCGCCAAACC CTCGACAACT TGCCATAGCC TTGTGTTCTT CATGGTCTAT 10 751 CGGAGTACCC GTTCATGACT GAAGCGGGTC AGCGTCCGTG GTGGTCATCA 801 TCATTCTCAT CTTTCATCAT GCCCGCTGAT TGATAGAGTA ATTTCCGGTG 15 851 GAGCACAACG CCGTCCTCTG AGATGCAATG TCACCCTGTA AGTTTCAACT 901 ACACTCTGTA GTACAGAGCA TCCTTGCCAT TGCATGCTGT GCAAGTGATC 951 TAAATCCGTA GAATCTGCTC GAGAACGGGG AAATATAGAA CTCCTGAAGG 20 1001 TTATAAATAC CACATGCATC CCTCGTCCAT CCTCATTTCC ATCATCAAGC 1051 CAGCGGTTTC TATCCTCCGA CTTGAGTCGT TCTCGCGCAT CTTTACAATC 25 1101 TTCTCACCAT GG WO 01/09352 PCT/NLOO/00544 56 Table 1. Taxonomic scheme of the genus Aspergillus (Samson, 1992) GENUS SUBGENUS SECTION SELECTED SPECIESa "SUBSPECIESES"b 5 Aspergillus Circumdati Wentii A. wentii (glucosidase) Flavi/Tamarii A.oryzae (amylase, protease) A. tamariitox A. sojae (fermented food, protease) 10 A. parasiticus t ox A. flavustox Nigri A. niger ---------------------- >A. pulverulentes (fermented food, A. phoenicis various proteins, A. awamori 15 organic acids) A. foetidus A. kawachii A. usamii A. ficuum A. japonicus---------- A. aculeatus 20 (endoglucanase) (glucosidase, galactanase) A. ellipticus A. tubingensis ------------ "A. niger" Circumdati A. ochraceusto' (xulanase) 25 A. alliaceus t ox Candidi A. candidus (lipase, glucosidase) Cremei A.itaconicus (organic acid) Sparsi A. sparsus Aspergillus Aspergillus Aspergillus A. glaucus (fermented food) 30 Restricti A. restrictusto' Fumigati Fumigati A. fumigatuso Cervini Oaati Clavati Clavati A. giganteus t o'~ WO 01/09352 PCT/NLOO/00544 57 Nidulantes Nidulantes A. nidulanstox Versicolores A. sydowii (lipase) Usti Terrei A. terreustox (glucansae) 5 Flavipedes a For the species selected for this list either the production of proteins/organic 10 acids/fermented foods (indicated between brackets) and/or a DNA-mediated transformation procedure (indicated by underlining) is described, except for A. tamarii, A. sparsus and A. ellipticus. Species recorded to produce toxins are indicated by t*x b Based on various methods the listed names may be considered synonymous to the given SPECIES name. 15 20 25 30 WO 01/09352 PCT/NLOO/00544 58 Table 2. The classification of the different ATCC strains Strains Morphology') Aflatoxin RAPD) PCRaflR ) PCRalA Classification 4 ) Production ATCC ND no A. sojae A. sojae A. sojae A. sojae 9362') type I ATCC A. sojae no '7 A. sojae ND A. sojae A. sojae 119066) type I ATCC A. oryzae no) A. sojae ND A. A. oryzae 20235 type II oryzae ATCC A. sojae no A. sojae A. sojae A. sojae A. sojae 20245 type I ATCC A. sojae no ND ND A. sojae A. sojae 20387 ATCC A. sojae no ND ND A. sojae A. sojae 20388 ATCC A. sojae no A. sojae ND A. sojae A. sojae 42249 type II ATCC A. sojae no ND ND A. sojae A. sojae 42250 ATCC A. sojae no) ND A. sojae A. sojae A. sojae 42251 ATCC ND ND ND ND A. A. oryzae 46250 oryzae IFO 4177 A. oryzae No' ND ND A. A. oryzae (CBS oryzae 205.89) WO 01/09352 PCT/NLOO/00544 59 Legend: ND = not determined ' REF: Ushijima S, Hayashi K and Murakami H (1982) The current taxonomic status of Aspergillus sojae 5 used in Shoyu fermentation. Agric. Biol. Chem., 46:2365-2367, 1981. ) REF: Yuan GF, Liu CS and Chen CC (1995) Differentiation of Aspergillusparasiticus from Aspergillus sojae by Random Amplification of Polymorphic DNA. Apple. Environm. Microbiol., 61:2384-2387. 3 REF: Chang PK, Bhatnagar D, Cleveland TE and Bennett JW (1995) Sequence variability 10 in homologs of the aflatoxin pathway gene afR distinguishes species in Aspergillus section Flavi. Appl. Environm. Microbiol., 61:40-43. 4) Conclusion on classification drawn by TNO based on data presented in this table 5 This strain was deposited at ATCC as A. oryzae, but later reclassified as A. sojae based on Yuan et al, 19952) and Chang et al, 1995 3 15 6) This strain was deposited at ATCC as A. parasiticus, but later reclassified as A. sojae based on Ushijima et al, 1981) and Yuan et al, 19952) 7 REF: ATCC catalogue ) REF: Liu BH, Chu FS (1998) Appl. Env. Microbiol., 64:3718-3723.

WO 01/09352 PCT/NLOO/00544 60 Table 3. Composition of selection media Non-selection Selection Acrylamide Improved medium medium selection medium acrylamide Composition (W097/041 selection medium 08)

KH

2

PO

4 1.5 g/l 1.5 g/l 1.5 g/l 1.5 g/l KCI 0.5 g/l 0.5 g/l 0.5 g/l 0.5 g/l MgSO 4 .7H 2 0 0.5 g/l 0.5 g/l 0.5 g/l 0.5 g/l NaNO 3 6 g/l glucose 10 g/l 10 g/l 10 g/l sorbitol 1.2 M ---- 1.2 M 1.2 M saccharose ---- 1 M ---- mineral solution') 0.1% v/v 0.1% v/v 0.1% v/v 0.1% v/v acetamide ---- 10 mM acrylamide ---- ---- 10 mM 10 mM CsCI ---- 15 mM 15 mM 15 mM agar 15 g/l 15 g/l 15 g/ 15 g/l 5 ' mineral solution: CusO 4 .5H 2 0 0.16 g/l FeSO 4 .7H 2 0 0.5 g/l ZnSO 4 .7H 2 0 2.2 g/l MnC1 2 .4H 2 0 0.5 g/l CoCl 2 .6H 2 0 0.17 g/l 10 Na 2 MoO 4 .2H 2 0 0.15 g/l

H

3 B0 3 1.1 g/l EDTA 5 g/l WO 01/09352 PCT/NLOO/00544 61 S00 r N N Nl r (~e C-) "o 0- 00 w l 0 0 - c C.) CD 0 0 N Nq N 00

C-

WO 01/09352 PCT/NLOO/00544 62 Legend: + (partial) degradation of proteins after 4 hours incubation, large milk clearing zone - no degradation of proteins after 4 hours incubation, small/no milk 5 clearing zone Incubation at 30*C: 27 l medium sample 2.5 pl BSA (25 mg/ml) 0.5 pl Phytase (A.terreus, 3-4 g/l) 0 BSA and phytase were added after the mediumsample was taken from the culture. This sample was incubated at 30*C and after certain timepoints the sample was analysed for the degradation of BSA and phytase.

WO 01/09352 PCT/NLOO/00544 63 Table 5. Protease activity at different pH values STRAINS Degradation of proteins in Minimal Medium + Trusoy after incubation pH=4.5 pH=6 pH=8 BSA Phytase BSA Phytase BSA Phytase (A. terreus) (A. terreus) (A. terreus) ATCC 9362 - - - - - + ATCC - - - - 11906 ATCC + - + + + + 20235 (=A. oryzae) ATCC - - - - 20387 Legend: + (partial) degradation of proteins after 4 hours incubation - no degradation of proteins after 4 hours incubation 5 Incubation at 30*C: 25 pl mediumsample 50mM NaAc pH=4.2 2 pl buffer (50mM) - 50mM NaAc pH=5.8 2.5 pl BSA (25 mg/ml) 50mM Tris/HCI pH=8.3 0.5 pl Phytase (A.terreus, 3-4 g/l) 10 BSA, phytase and buffer were added after the mediumsample was taken from the culture. This sample was incubated at 30*C and after certain timepoints the sample was analysed for the degradation of BSA and phytase. 15 20 WO 01/09352 PCT/NL00/00544 64 Table 6. PCR results for cloning fungal pclA genes Primercombination Expected size PCR product 1 pcll + pcl2rev 180 bp 2 pcll + pcl3 350 bp 3 pcll + pcl4 500 bp 4 pcll + MBL1372 300 bp 5 pcl2 + pcl3 200 bp 6 pc12 + pcl4 350 bp 7 pc12 + MBL1372 150 bp 8 MBL1298 + pcl2rev 180 bp 9 MBL1298 + pcl3 350 bp 10 MBL1298 + pcl4 500 bp 5 Strain Primercombination 1 2 3 4 5 6 7 8 9 10 Trichoderma reesei QM9414 + + - - - - - + - Penicillium chrysogenum P2 + + + - + - - + - Fusarium venenatum ATCC20334 + + + - - + - + + Trametes versicolor TV1 - - - - - - - - - Rhizopus oryzae ATCC200076 + - - - - - - + - Agaricus bisporus HORST - + - - - - - - Aspergillussojae ATCCl1906 + + + - - + - + - positive control + + + + + + + + + + Legend: + specific PCR product - aspecific or no PCR product + this PCR product was used for sequencing t0 WO 01/09352 PCT/NLOO/00544 65 Table 7. The viscosity ranges of the various A. sojae strains 5 StrainsViscosity (cP) Biomass (g/l) Shear rate 6.5 1/s Shear rate 83.2 1/s Shear rate 644.4 1/s A. sojae wild type >>2000 1505 155 8.8 and 16.6 A. sojae pcA 2000 751 76 7.6 and 17.2 A. sojae lfvA 1565 94 18 6.9 and 18.8 0 Table 8. Promoter strength in A. sojae transformants Transformants Promoter GUS activity (U/mg) in Minimal Medium 5% 5% 5% 2% 5% xylose glucose maltodextrin starch trusoy ATCC11906 wild ---- 0 0 0 0 0 type ATCC11906[pGUS5 gpdA 9141 6291 6667 6937 3391 4] ATCC11906[pGUS6 glaA 33 50 25 51 176 4] ATCC11906[pBIPG bipA 2914 2849 1642 493 2083

US]

Claims (35)

1. A recombinant Aspergillus sojae comprising an introduced acetamidase S (amdS) gene as selectable marker. 5

2. An Aspergillus sojae according to claim 1, said Aspergillus sojae being selectable on a medium comprising a substrate for the introduced amdS as sole source of nitrogen, said medium further comprising a carbon substrate and said medium being free of endogenous amdS inducing substrate.

3. An Aspergillus sojae according to claim 1 or 2, wherein the source of nitrogen is 0 acrylamide.

4. An Aspergillus sojae according to any of the preceding claims wherein the Aspergillus sojae has no active endogenous amdS gene, for example because the endogenous amdS gene comprises an endogenous amdS inactivating mutation, e.g. a deletion or a disruption.

5 5. A method of introducing a nucleic acid sequence into Aspergillus sojae, said method comprising subjecting Aspergillus sojae to a method of introduction of a nucleic acid sequence e.g. transformation or transfection of the Aspergillus sojae in a manner known per se for introduction of a nucleic acid sequence into fungi, said method comprising the introduction of the amdS gene as the nucleic acid sequence (henceforth 20 the introduced amdS gene) followed by selection of the resulting transformed or transfected Aspergillus sojae on a medium free of endogenous amdS inducing substrate, said medium further comprising a substrate for the introduced amdS as sole source of nitrogen and said medium further comprising a carbon substrate, said medium enabling the desired Aspergillus sojae comprising the nucleic acid sequence Z5 to grow whilst eliminating growth of Aspergillus sojae free of the socalled introduced nucleic acid sequence due to inability of such Aspergillus sojae to grow without the introduced amdS gene on the selection medium, said medium suitably comprising a substrate for amdS other than acetamide, for example acrylamide as substrate for the introduced amdS as sole source of nitrogen. 30

6. An Aspergillus sojae obtained by the method of claim 5.

7. A method of selecting transformed or transfected Aspergillus sojae said method WO 01/09352 PCT/NLOO/00544 67 comprising subjecting Aspergillus sojae according to any of claims 1-4 and 6 to a method of transformation or transfection of the Aspergillus sojae in a manner known per se for transformation or transfection of fungi with a nucleic acid sequence, said method comprising the introduction of the amdS gene as the nucleic acid sequence 5 followed by selection of the resulting transformed or transfected Aspergillus sojae on a medium comprising a substrate for the introduced amdS as sole source of nitrogen and said medium further comprising a carbon substrate, said medium enabling the desired Aspergillus sojae to grow whilst eliminating growth of non-transformed or transfected Aspergillus sojae due to inability of such to grow without the introduced 0 amdS gene on the selection medium.

8. A method for producing recombinant Aspergillus sojae, said method comprising introduction of a nucleic acid sequence into an Aspergillus sojae e.g. by transformation or transfection in a manner known per se according to any of claims 1 4 and 6, said nucleic acid sequence comprising a desired sequence to be introduced .5 flanked by sections of an endogenous amdS gene or corresponding sequences of a length and homology sufficient to ensure recombination thus simultaneously eliminating the endogenous amdS gene and introducing the desired sequence, followed by selection of the recombinant Aspergillus sojae with the desired sequence by selecting for a selectable marker comprised in or transformed in cotransformation 20 with the desired sequence, said selectable marker being absent in the Aspergillus sojae prior to introduction of the nucleic acid sequence, suitably the selectable marker being pyrG.

9. An Aspergillus sojae exhibiting growth with medium comprising uracil and fluoro orotic acid, said Aspergillus further not exhibiting growth on medium comprising 25 uridine and fluoro-orotic acid, i.e. said Aspergillus sojae exhibiting uracil auxotrophy, said Aspergillus sojae being unable to utilize uridine, said Aspergillus sojae being pyrG negative, said Aspergillus sojae exhibiting resistance to fluoro-orotic acid, said uracil auxotrophy and said fluoro-orotic acid resistance being relievable upon complementation with an active introduced pyrG gene, suitably said Aspergillus sojae 30 being free of active endogenous pyrG genes; e.g. the Aspergillus sojae endogenous pyrG gene comprises a mutation in the form of an insertion, substitution or deletion in the gene or in a gene regulating sequence, e.g. a deletion of the whole coding WO 01/09352 PCT/NLOO/00544 68 sequence of the gene.

10. An Aspergillus sojae according to claim 9 in combination with the characteristics of an Aspergillus sojae according to any of claims 1-4 and 6.

11. A method of selecting transformed or transfected Aspergillus sojae, said method 5 comprising subjecting Aspergillus sojae according to claim 9 or 10 to a method of transformation or transfection with a nucleic acid sequence, said method comprising introducing an active pyrG gene into the Aspergillus sojae in a manner known per se for transformation or transfection of fungi followed by selection of the resulting transformed or transfected Aspergillus sojae on a medium free of uracil and fluoro 0 orotic acid, said medium at least further comprising minimum substrates required for growth of Aspergillus sojae, said medium enabling the desired Aspergillus sojae to grow whilst eliminating growth of non-transformed or -transfected Aspergillus sojae due to inability of such to grow without uracil due to the inactivated pyrG gene.

12. A method according to claim 11, wherein the active pyrG gene that is introduced is 15 flanked by identical nucleic acid sequence fragments, and the pyrG positive Aspergillus sojae resulting from the introduction of the pyrG gene and the flanking sequences is selected on a medium free of uracil and fluoro-orotic acid and subsequently the pyrG positive Aspergillus sojae is cultivated on medium comprising uracil and fluoro-orotic acid thereby eliminating the pyrG gene that had been 20 introduced thus resulting in a pyrG negative Aspergillus sojae that is selectable by growth on uracil comprising medium and by fluoro-orotic acid resistance, suitably the flanking sequences and the pyrG gene being further flanked by sequences that direct integration of the pyrG gene and the flanking sequences into a specific location due to the fact that the integration directing sequences are homologous to a specific sequence 25 of the Aspergillus sojae to be transformed, thereby enabling knock out, if desired, of the gene associated with the specific sequence.

13. A method according to claim 11 or 12, wherein the Aspergillus sojae according to claim 9 or 10 has a further nucleic acid sequence introduced therein, preferably said further nucleic acid sequence encoding a protein or polypeptide, said further nucleic 30 acid sequence being introduced with the active pyrG gene either on the same vector or by cotransformation with the active pyrG gene that is introduced. WO 01/09352 PCT/NLOO/00544 69

14. A method of selecting transformed or transfected Aspergillus sojae by carrying out the method according to any of claims 11-13 in combination with the method of claim 5.

15. A method for producing recombinant Aspergillus sojae, said method comprising introducing a nucleic acid sequence into a pyrG positive Aspergillus sojae, e.g. by 5 transformation or transfection in a manner known per se, said nucleic acid sequence comprising the desired sequence flanked by sections of the pyrG gene or corresponding sequences of a length and homology sufficient to ensure recombination eliminating the pyrG gene and introducing the desired sequence, followed by selection of the recombinant Aspergillus sojae with the desired sequence by selecting for 0 Aspergillus sojae with a pyrG negative phenotype.

16. A recombinant Aspergillus sojae obtained by a method according to any of claims 11 15, optionally further comprising the characteristics of an Aspergillus sojae according to any of claims 1-4, 6, 9 and 10.

17. A recombinant Aspergillus sojae comprising an introduced nucleic acid sequence 5 encoding a protein or polypeptide for expression, said protein or polypeptide being susceptible to degradation upon expression by Aspergillus niger or Aspergillus awamori.

18. A recombinant Aspergillus sojae comprising an introduced nucleic acid sequence encoding a protein or polypeptide for expression, said protein or polypeptide being 0 other than Aspergillus sojae protease and amylase, said protein or polypeptide preferably being a non-Aspergillus sojae protein or polypeptide.

19. A mutant or recombinant Aspergillus sojae comprising a mutation inactivating a protease gene, suitably an alkaline protease gene.

20. A mutant or recombinant Aspergillus sojae comprising a mutation inactivating the 5 major protease gene, suitably a mutation inactivating the major alkaline protease gene, e.g. the gene encoding major alkaline protease gene of 35 kDa.

21. A method for producing recombinant Aspergillus sojae, said recombinant A. sojae exhibiting reduced proteolytic activity, said method comprising introduction into an A. sojae, e.g. by transformation or transfection in a manner known per se, of a nucleic 0 acid sequence comprising a selectable marker encoding sequence to be introduced flanked by sections of the protease gene to be eliminated and further said flanking WO 01/09352 PCT/NLOO/00544 70 sequence and the selectable marker encoding sequence being comprised within sequences of a length and homology sufficient to ensure recombination at the protease gene thus simultaneously eliminating the protease gene and introducing the desired selectable marker encoding sequence, the introduction being followed by selection of 5 the recombinant A. sojae by selecting for the selectable marker, whereby the A. sojae prior to the introduction of the nucleic acid sequence, e.g. by transformation or transfection, is free of the selectable marker to be introduced, e.g. the A. sojae prior to the introduction of the nucleic acid sequence being mutated such that the A. sojae cannot produce active selectable marker, suitably the selectable marker being the pyrG 0 gene, suitably the method being carried out together with the method according to any of claims 11-15.

22. A recombinant Aspergillus sojae obtained according to the method of claim 21.

23. A mutant or recombinant Aspergillus sojae according to any of claims 17-20 or 22 comprising a selectable marker, preferably amdS as defined in any of claims 1-4 or 6 5 and/or pyrG as defined in claims 9, 10 or 16.

24. A recombinant Aspergillus sojae according to any one of claims 1-4, 6, 9, 10, 16-20, 22 and 23, comprising an introduced nucleic acid sequence encoding phytase or a protein having phytase activity.

25. A process of expression of an introduced nucleic acid sequence encoding a protein or !0 polypeptide comprised in a recombinant or mutant Aspergillus sojae as defined in any of the claims 1-4, 6, 9, 10, 16-20, 22-24 or obtained via a method according to any of claims 5, 7, 8, 11-15 and 21, said process comprising cultivating the recombinant or mutant A. sojae, suitably the introduced nucleic acid sequence encoding a protein or polypeptide being absent in the corresponding non-transformed or wild-type A. sojae Z5 and/or being present in a lower copy number.

26. A recombinant fungus comprising a mutation in a gene encoding a proprotein convertase or a functionally equivalent protein.

27. A fungus according to claim 26 exhibiting increased production of a protein, polypeptide or metabolite under equivalent conditions when compared to the 30 corresponding wild-type fungus.

28. A fungus according to claims 26 or 27 said mutation being obtained by specific gene WO 01/09352 PCT/NLOO/00544 71 modification using transformation or transfection in a manner known per se.

29. A fungus according to claims 26-28, said proprotein convertase or functionally equivalent protein being encoded by a nucleotide sequence of which a fragment can be amplified by in vitro DNA amplification using any of two mixtures of nucleotides 5 given in SEQ ID Nos. 10 to 16.

30. A fungus as described in claim 27, said proprotein convertase or functionally equivalent protein being encoded by a nucleotide sequence allowing functional complementation of the growth phenotype of an Aspergillus niger mutant comprising a mutation which inhibits the activity of a proprotein convertase or a functionally 0 equivalent protein.

31. A fungus according to any of claims 26-30, said fungus being an Aspergillus sojae.

32. A fungus according to any of claims 26-30, said fungus further containing an introduced amdS gene or pyrG gene.

33. A process for expressing a protein or polypeptide, preferably a recombinant protein or 15 polypeptide, encoded by a nucleotide sequence, said process comprising cultivating a fungus according to any of the claims 26-32.

34. A process for producing a protein or polypeptide, preferably a recombinant protein or polypeptide, said process comprising a process of expression according to claim 33, optionally including processing and/or secretion and/or isolation of the expressed 20 protein or polypeptide.

35. A process for producing a phytase or a protein having phytase activity, preferably a recombinant phytase or recombinant protein having phytase activity, said process comprising a process of expression according to claim 33, optionally including processing and/or secretion and/or isolation of the expressed phytase or protein having 25 phytase activity.