WO2001009352A2 - Use of aspergillus sojae as host for recombinant protein production - Google Patents

Abstract

A recombinant Aspergillus sojae comprising an introduced acetamidase S (amdS) gene as selectable marker is disclosed. An Aspergillus sojae exhibiting growth with medium comprising uracil and fluoro-orotic acid, said Aspergillus sojae further not exhibiting growth on medium comprising 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, is described. The Aspergillus sojae further comprises a nucleic acid sequence encoding a phytase or a protein having phytase activity or any other heterologous protein or polypeptide and can be used for the biotechnological production of said phytase or said other heterologous proteins or polypeptides. Additional mutants exhibiting amended morphology are also disclosed. Methods of producing such expression hosts are described.

Description

Novel means of transformation of fungi and their use for heterologous protein production.

SUMMARY OF THE INVENTION 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. 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 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.

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 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 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 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.

WO97/04108 for example describes the isolation of a protease encoding nucleic 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, 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-l) are suggested as 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 (EMBO Journal 4, pages 475-479). Consequently, the analogous use for transforming Aspergillus in general is put forward in WO97/04108 on the basis of these previous publications.

Quite specifically on page 17 of WO97/04108 it is described that Aspergilli and Trichoderma that prior to transformation grew slowly on minimal medium comprising 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 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 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 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 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 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. 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 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 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 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 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 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 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 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, 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 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 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, 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 tool), it has been found that Aspergillus sojae comprises an Xmnl 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 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. 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 Xmnl 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 Xmnl restriction site by way of example of a screening test useful for distinguishing Aspergillus oryzae from Aspergillus sojae (Primer sequences are SEQ ID No.l 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 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 Xmnl 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. 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 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 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 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 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 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 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 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 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 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 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 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. 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 transformation or transfection of the A. sojae. The method comprises the introduction of the 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 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 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 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 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 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 sojae (with no active endogenous amdS gene as defined according to any of the embodiments described) to a method of 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 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 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 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 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. 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 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 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 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. 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 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 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. 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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. 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. 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, ATCC 11906 and ATCC20387. The subject description thus reveals that some known 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 ATCC42251. 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 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. 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 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 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 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 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 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 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 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 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 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, 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 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 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. 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 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 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 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 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 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 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 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 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 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. 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 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. EXPERIMENTAL DETAILS CONCERNING THE INVENTION

CONSTRUCTION OF AN Aspergillus sojae GENE LIBRARY.

Genomic DNA of A. sojae was isolated from protoplasts obtained from 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 Mbol 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, was constructed by ligation of a 3 kb BamRl-Hindlll fragment from pANsCosl (Osiewacs,

1994) and a 3.2 kb Acc65l-Hindlll fragment from pAO4.2 (De Ruiter- Jacobs, 1989) in

Acc65l-Bamϋl digested pHELPl (Gems et al., 1991). This cosmid vector carries the A. oryzae pyrG selection marker and is self-replicating in filamentous fungi.

Mbol digested genomic DNA was ligated to ifømHI-digested pAOpyrGcosarpl, and the ligation mixture was packaged into phage particles using the Stratagene Supercosl 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. 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 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 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 selective acetamide medium. Clearly, the acetamide selection conditions proposed for A. sojae amdS transformants as reported in WO97/04108, 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 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. 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 WO97/04108 and the improved acrylamide selection medium described in Table 3. Further transformation experiments with the three selected A. sojae strains revealed that protoplasting efficiencies for ATCC 11906 and ATCC20387 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 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 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 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 10.000) were isolated from the ATCC11906 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 am dS gene was replaced by a re-usable pyrG selection marker to generate an amdS replacement vector. Transformation of this vector to Aspergillus sojae ATCC11906PyrG 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 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 generate pyrG mutants as described in the introduction resulted in numerous fiuo-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.

(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 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 Clal fragment carrying part of the pyrG coding region was deleted (Figure 4). The Xbal 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.

(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 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. 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 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. 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 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 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 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-l. However, subsequent plating of spores of selected pAB4-l and pAB4-Irep 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 only the small 0.7 kb repeat region at the locus of integration, while in the pAB4-l strains the A. niger gene was still present and had presumably acquired a mutation resulting in the pyrG-negative phenotype.

EXPRESSION HOSTS: STRAIN SELECTION. 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 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 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 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 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 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, ATCC11906 and ATCC20387 culture fluids. In general, the strains with the lowest phytase degradation also show low BSA degradation under the 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 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, ATCC 11906 and ATCC20387 showed low phytase degrading activity. ATCC9362 showed phytase degradation in rich 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, ATCC 11906 and ATCC20387. Thus, A. sojae can cleary be used as expression host for a range of proteins and provides a series of 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 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 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. Development of protease deficient mutants

To obtain protease deficient A. sojae strains two approaches were followed. In a first approach spores from ATCCl 1906 and ATCCl 1906-derived strains were mutagenized with UV. In a second approach gene disruption of the major alkaline protease was carried out.

UV mutants

Freshly harvested spores from A. sojae ATCCl 1906 or one of its pyrG derivatives was UV-mutagenized in a Biorad UV-chamber with a dose resulting in 20-50 % survival. Serial dilutions were plated onto skim-milk plates (Mattern et al., 1992). From 5000 UN- surviving strains four mutant strains with a considerably reduced milk-clearing halo were obtained

AlpA gene disruption In this approach the endogenous alpA (alkaline protease) gene cloned from

ATCCl 1906 was disrupted using a disruption vector carrying the re-usable pyrG selection marker as described in this description.

An ATCCl 1906 cosmid library (in a PyrG cosmid) was constructed. From 10.000 independent cosmid clones initially 4 were found to hybridize under homologous conditions with an A. sojae alp A 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 Hindlll 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 constructed as described in Figure 6. For transformation of an ATCCl 1906pyrG 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 ATCCl 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 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 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".

Analysis of protease activity in UV and disruption mutants

To analyse the levels of protease production in the different low protease derivatives of ATCCl 1906 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 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.

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 strain. Various patent applications teach that low viscosity mutants can be isolated by various ways of screening. WO96/02653 and WO97/26330 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 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.

(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 KEX1 and the Xenopus laevis PC2 genes was carried out. 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 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 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 pclA (from proprotein convertase-/ike). Southern analysis of genomic digests of A. niger revealed that the pclA 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 length genomic copy of the pclA 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 pclA 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 were present (note that the PCR fragment (SΕQ 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 pel A 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 pclA gene is given in Figure 7.

Based on the restriction map given in Figure 7 the complete DNA sequence of the pclA gene was determined from the EcoRI and EcoRV subclones (SΕQ 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 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. 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 pclA gene is a functional gene encoding a functional protein, the construction of strains devoid of the pclA gene was attempted. Therefore, pPCLlA, a pclA deletion vector, in which a large part of the pel A 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- 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 pclA gene. All strains displaying wild-type growth were shown to carry a copy of the replacement fragment integrated adjacent to the wild-type pclA gene or at a non-homologous position. 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. To further improve the yields of fusion proteins controlled batch and fed-batch fermentations were also carried out. Surprisingly the fermentation characteristics of pclA mutant strains were clearly superior to those of the parent strain resulting in a much reduced viscosity/biomass ratio, without loss of productivity.

(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 ATCCl 1906 cosmid library were isolated, which comprised the A. sojae proprotein processing protease pclA gene. Partial sequence analysis of the isolated sequences SΕQ ID No. 4 confirmed the 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 pclA gene, a replacement vector was generated using the EcoRV-site in the A. sojae pclA gene to clone the re-usable pyrG marker as a Smal fragment inside (Figure 11). The resulting vector was partially digested with Clal to obtain the delta-pel 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 pel A mutants in ATCCl 1906 and ATCCl 1906 derivatives. The resulting strains were used for the expression of homologous and 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 of fungal genes homologous to Aspergillus pclA Based on the comparison of amino acid sequences inferred from the A. niger and A. sojae pclA 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 (SΕQ ID Nos. 10 to 16).

These oligonucleotide mixtures were used in PCR with chromosomal DNA from 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 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 pclA homologues from these different fungi. Figure 12 gives the inferred aminoacid sequences corresponding with the various DNA fragments (SΕQ ID Nos. 5 to 9).

(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. 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 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 η (in centipoise = cP) is plotted against shear rate γ (1/s) and gives the viscosity profile of the fermentation broth.

Viscosity ranges have been determined for fermentations using the specified fungal organism using the above procedure (Table 7).

Biomass:

Biomass is determined by the following procedure: Pre weigh 5.5 cm filter paper (Whatman 54) in an aluminium weighing dish. Filter 25.0 ml 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/1) is equal to the difference between the initial and final weights multiplied by 40.

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 fungal organisms and reflect actual commercial data.

A fungal strain such as A. sojae IfvA 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 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. The high protein production with low biomass concentration produced by A. sojae IjvA 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 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 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 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.

(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 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 alp A (alkaline protease; inducible), amyA (amylase; inducible) and gpdA (glyceraldehyde-3-phosphate dehydrogenase; constitutive) was attempted using primers based on sequences available 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 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.

The complete sequence of the cloned genes was determined. As shown in Figure 14 the A. sojae ATCCl 1906 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.

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 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.

Phytase production

DNA fragments carrying various fungal phytases (Wyss et al. (1999) Appl. Environ. Microbiol. 65, 359-366) were ligated as 5' Ncol or BspHl sites introduced at the ATG codon - 3' blunt-ended fragments downstream of the A.nidulans gpdA promoter in pAΝ52-lΝotI. 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.

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) 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. 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 pAN56-l (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. 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- 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 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 control of the A. nidulans gpdA promoter was introduced into A. sojae ATCCl 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 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. 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 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 these transformants. SDS-PAGE and Western analysis of culture supematants 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 (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 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 supematants from selected, shakeflask- 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 supematants also the secreted GFP showed significant fluorescence.

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), 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. 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 Figure 2a provides the background growth of the A. sojae strain described in patent WO97/04108 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 WO97/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention. Figure 2b provides the background growth of the A. sojae strain ATCCl 1906 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 WO97/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention. 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 WO97/04108 and the bottom right picture shows the results using improved medium (acrylamide) according to the invention.

Figure 3 (a and b): This figure provides a comparison of A. sojae ATCCl 1906 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.

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-l via isolation ofXhol fragment and Hindlll 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-StwI fragment from pASl-1 with the ATCCl 1906 genomic fragment, the 2.6 kb Smal-Ncol fragment from pAB4-lrep and the 4.4 kb NcoI-EcoRI fragment from pASl-2A are ligated in a 3 way ligation thus providing pASl-deltaalp.

Figure 7: This figure provides the restriction map of the DΝA fragment carrying the A. niger pclA gene.

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 at the right end is a D/Ε rich region.

Figure 9: This figure illustrates growth phenotype of an A. niger pclA mutant strain.

Figure 10: This figure provides a DΝA sequence comparison between the A. sojae and A. niger pclA genes. A vertical bar indicates identity; : indicates 5; • indicates 1. 72.139%) similarity and 12.013% identity were found.

Figure 11 : The construction of the pclA gene replacement vector is disclosed in this figure. A 7.6 kb Clal fragment, which is a ATCCl 1906 genomic fragment, was cloned into pMTL23p. In this constmct the 2.6 kb Smal fragment from pAB4-lrep was cloned into the EcoRV-site, thus providing pAS2-delta pel.

Figure 12: This figure shows the amino acid sequence comparison of the various PclA homolous from S. cerevisiae (Sckex2), K. lactis (Klkexl), A. sojae (Aspcla), A. niger (A. niger), P. chrysogenum (Penpcll), A. bisporus (Agarmbll29), 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 ~ . Figure 13: Sequence data are provided in figure 13a for the A. oryzae alpA promoter sequences (Q11755). 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 promoter sequences (EP0.436.858 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 ATCCl 1906, A. oryzae, A. niger and A. nidulans. Asterisks indicate the putative intron present in the 5' untranslated region of the promoters. 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.

SEQUENCE LISTING

SEQ ID No. 1

MBL1784: 5'-CGGAATTCGAGCGCAACTACAAGATCAA-3'

SEQ ID No. 2

MBL 1785 : 5'-CGGAATTCAGCCCAGTTGAAGCCGTC-3'

SEQ ID No. 3

The sequence of the Aspergillus niger gene encoding proprotein convertase 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

51 TTCACCAAAA CTCTCCCCCA AACGATGCGT CTTACAGGTG GTGTCGCTGC 101 GGCTCTGGGC CTCTGCGCTG CTGCCTCCGC TTCTCTCCAT CCCCATCGTT

151 CCTACGAGAC CCATGATTAC TTCGCTCTAC ACCTTGATGA ATCCACCTCG 201 CCGGCCGACG TCGCCCAACG ACTAGGTGCT CGCCACGAAG GCCCCGTCGG

251 AGAATTACCC TCACATCATA CCTTCTCGAT ACCCCGTGAA AACAGTGACG

301 ATGTCCATGC GCTGCTGGAT CAATTGCGCG ATCGTCGGAG GTTACGCCGC

351 CGCTCCGGAG ATGACGCCGC TGTCCTTCCC TCCTTGGTCG GGCGAGACGA

401 AGGTCTAGGT GGCATTCTTT GGTCCGAGAA GCTGGCTCCC CAGAGAAAGC 451 TCCATAAAAG AGTGCCGCCG ACAGGATATG CTGCCAGATC GCCCGTCAAC

501 ACTCAGAATG ACCCCCAAGC GCTTGCGGCG CAGAAACGCA TTGCCTCGGA

551 ATTGGGCATC GCGGACCCCA TCTTCGGCGA ACAATGGCAT TTGTATAATA

601 CTGTTCAGTT GGGCCATGAT CTTAACGTGA CGGGTATCTG GCTGGAGGGC

651 GTTACAGGGC AGGGTGTCAC GACGGCCATT GTCGATGACG GTTTGGACAT 701 GTACAGCAAC GATCTTAGGC CGAACTATTT TGCGGCGGGT TCTTATGACT

751 ATAACGACAA AGTACCAGAG CCGAGGCCGC GCTTGAGCGA TGACCGCCAC

801 GGTACTAGAT GCGCGGGTGA AATCGGTGCG GCGAAGAACG ACGTGTGCGG

851 GGTTGGTGTT GCGTATGATA GTCGCATCGC TGGTATTCGG ATTCTCTCCG

901 CACCCATCGA TGACACTGAT GAGGCTGCGG CTATTAACTA CGCCTATCAG 951 GAGAACGATA TCTACTCGTG TTCCTGGGGT CCCTATGATG ATGGCGCCAC

1001 AATGGAAGCC CCGGGCACTC TGATCAAGCG GGCCATGGTC AATGGTATCC

1051 AAAATGGTCG AGGTGGAAAA GGCTCGGTTT TTGTATTTGC GGCTGGTAAC

1101 GGTGCCATTC ATGACGATAA CTGTAACTTT GACGGTTACA CCAACAGTAT 1151 CTACAGCATC ACGGTGGGTG CCATTGATCG GGAGGGTAAC CATCCTCCGT 1201 ATTCGGAATC CTGCTCGGCG CAACTGGTGG TTGCCTACAG CAGCGGCGCC 1251 AGTGATGCAA TTCATACCAC GGACGTCGGC ACAGACAAGT GCTCGACTAC 1301 CCATGGTGGA ACTTCGGCGG CCGGCCCGCT CGCTGCGGGA ACCGTGGCGC

1351 TGGCCCTCAG TGTGCGGCCG GAACTCACCT GGCGTGACGT TCAGTATTTG 1401 ATGATTGAGG CGGCAGTGCC TGTTCATGAA GATGATGGAA GCTGGCAGGA

1451 CACTAAGAAC GGGAAGAAGT TCAGCCATGA CTGGGGATAT GGTAAGGTCG

1501 ACACATATAC GCTGGTGAAA CGGGCAGAGA CCTGGGATCT GGTGAAGCCT

1551 CAAGCCTGGC TCCATTCCCC CTGGCAGCGG GTTGAGCATG AGATCCCACA

1601 GGGCGAGCAG GGCTTGGCTA GTTCGTACGA GGTGACGGAG GATATGTTGA 1651 AGGGAGCCAA CCTGGAACGG CTGGAGCATG TCACGGTCAC CATGAATGTT

1701 AACCACACCC GCCGAGGCGA TCTCAGCGTG GAGTTACGGA GCCCTGATGG

1751 TCGGGTCAGT CACCTCAGTA CGCCCCGGCG GCCAGATAAT CAAGAGGTGG

1801 GCTATGTTGA TTGGACCTTC ATGAGCGTTG CTCACTGqta agtaaaaact

1851 ttttctcgqt tqtcgqttct tctqctaata catatctagG GGCGAGTCCG 1901 GGATTGGCAA ATGGACTGTG ATTGTCAAGG ACACCAATGT CAACGAGCAT

1951 ACTGGGCAAT TCATCGATTG GCGACTCAAC TTGTGGGGCG AGGCGATTGA

2001 CGGAGCCGAG CAGCCTCTCC ACCCCATGCC TACTGAACAC GATGACGACC

2051 ACAGCTATGA GGAAGGAAAC GTGGCTACCA CGAGCATCAG CGCCGTTCCC

2101 ACGAAAACCG AGCTGCCTGA CAAGCCCACT GGTGCGTTG ATCGCCCGGT 2151 GAACGTTAAG CCTACAACAT CCGCGATGCC GACCGGTAGT CTTACAGAGC

2201 CCATCGATGA TGAAGAACTC CAGAAGACCC CTAGTACAGA GGCAAGCTCA 2251 ACACCAAGTC CTTCTCCGAC CACCGCGTCA GATAGTATCC TGCCTTCCTT

2301 CTTCCCCACG TTCGGTGCGT CGAAGCGGAC CGAAGTTTGG ATCTACGCTG 2351 CGATCGGCTC CATCATTGTG TTCTGCATTG GCCTGGGCGT CTACTTCCAT

2401 GTGCAGCGCC GCAAACGTAT TCGCGACGAC AGCCGGGATG ACTACGATTT

2451 CGAGATGATC GAGGACGAGG ATGAGCTACA GGCAATGAAC GGACGGTCGA

2501 ACCGTTCACG TCGCCGGGGT GGCGAGCTGT ACAATGCTTT TGCGGGCGAG

2551 AGCGATGAGG AACCATTATT CAGTGATGAG GATGATGAAC CGTATCGGGA 2601 TCGGGGGATC AGCGGCGAAC AAGAACGGGA GGGCGCAGAT GGAGAGCATT

2651 CTCGGAGATG AAAGTGCAGT AGATGAGGGT TGACTTTATT TCGGACAGTG

2701 TTTCTAACTT GTTGGATGAC CTGCGTTGAA CAATATTTCT GCTGTGTATG

2751 CTGCATAGAG AAGCGTGTAT ATACCATGTA TGTGTGCATC ATCGTGATCG

2801 GGTTTATCAT TCTTCATCTG CCATGGTTTG TGATCTCCGG AATAGTACCA 2851 AAGGAACACT AAATTAAGGG TCTTGGCGAT GACGCTTCCC GTCGCTGCTT

2901 TTGACTTCCT CCGCATCTCG TCTCTCCTGC TGTTGACCGC GCGCCAACCA

2951 ACCTCCATCT CCTCACTCCT CCCACCTTAA TCTTGCTGTG CTGCTTCTAG

3001 AACCCCCCAG TTTAATTTAA AAACCGGCTT TTCCTAGCTC CACGTATTGT 3051 ACCTCGCACT GATCCCCATC TCCGCCCACT CCAACGCTAC CGACCCAGGC 3101 TTCTCTGGCG GCTCCAGGCG GCAGGCAATC AAACCAACCC CTCGATGGAT

3151 CAGCACGACG ACTTCGACAG SGTCTCGTGG AGGCATGACC CGGACAGCGA

3201 TCTCTCGCGA CCCACGAACT CCGGAACAGA CACAGAGGAA CAGGCGCCAT

3251 ACACTCACGA TGTCAATGGC AAACGGAGGA TGAGCAACCG CTCAAGAAAG

3301 CCCTCAGGCT GGACCACTGG CGGATGCCGT CGACCTGGCG GGCATCGCGA 3351 CGGCGTACTA GAGTGTCGGG TAGATTCACC GTTGAAGGAG AATATGGACG

3401 AAAGACGCTT ATATCTCCTA TTTGGTACAC TACTAGGTGG GTATCTTACC

3451 TCAGTGATCT CAGATGGA

SEQ ID No. 4

The partial sequence in the coding region of the Aspergillus sojae gene encoding proprotein convertase

CGCGGATCCA TGGAACACGA TGTGCGGGTG AAATTGGAGC AGCTAGGAAT

51 GATGTCTGTG GAGTAGGTGT TGCATACGAC AGCCAAGTTG CCGGAATTCG

101 GATTTTGTCC GCACCCATTG ACGACGCAGA TGAGGCTGCT GCCATCAACT 151 ATGGCTTCCA GCGCAATGAT ATATATTCAT GCTCCTGGGG CCCTCCGGAT

201 GATGGCGCCA CGATGGAGGC GCCAGGGATT CTTATCAAAC GAGCTATGGT

251 CAACGGTATC CAAAATGGCC GAGGAGGTAA AGGTTCTATC TTCGTCTTTG

301 CAGCTGGAAA TGGTGCAGGG TACGATGACA ACTGCAATTT CGACGGTTAT

351 ACAAACAGCA TTTACAGCAT CACCGTCGGC GCTATTGATC GAGAGGGCAA 401 ACATCCCAGC TACTCGGAAT CATGCTCTGC CCAGTTGGTT GTCGCTTATA

451 GCAGTGGCTC GAGTGACGCG ATTCATACCA CCGACGTTGG AACTGATAAA

501 TGTTATTCAC TNTCACGGGC GGAACTTCTG CAACTGGACC GCTAGCTGCG

551 GGTACTATTG CCCTCGCTCT TAGTGCCCGA CCGGAACTAA CTTGGCGAGA

601 TGCCCAGTAC CTGATGATAG AGACCGCAGT TCCCGTCCAC GAAGACGACG 651 GGAGCTGGCA GACTACCAAA ATGGGGAAGA AGTTTAGCCA TGACTGGGGT

701 TTTGGGAAAG TAGATGCATA TTCACTGGTC CAGCTGGCCA AGACGTGGGA 751 GCTGGTGAAA CCACAGGCGT GGTTCCACTC ACCGTGGCTG CGGGTGAAGC

801 ATGAAATCCC ACAAGGTGAC CAGGGCCTTG CCAGCTCATA CGAAATTACC

851 AAGGATATGA TGTACCAGGC CAATGTCGAG AAATTGGAAC ATGTCACTGT

901 GACCATGAAT GTAAATCACA CTCGCCGAGG CGATATCAGC GTGGAGTTGC

951 GCAGCCCCGA AGGTATCGTC AGTCATCTGA GTACAGCGCG GCGGTCAGAT

1001 AATGCAAAGG CTGGCTATGA AGATTGGACG TTTATGACTG TGGCTCATTG

1051 GTATGTATTT GCTCCCGTAA TTTAGTTTTC GTGCTCAGTC CTGACATTTA

1101 CATTTAGGGG TGAGTCCGGT GTTGGAAAGT GGACGGTCAT TGTGAAGGAT

1151 ACCAATGTCA ATGATCATGT TGGAGAATTC ATCGACTGGC GGCTCAACCT

1201 CTGGGGACTT TCGATCGACG GCTCCAGCCA GCCCCTTCAT CCTATGCCCG

1251 ATGAGCATGA CGATGACCAC TCGATTGAAG ATGCCATTGT TGTTACCACT

1301 AGTGTTGACC CTATCCCAAC TAAGACTGAA GCCCCACCTG TCCCAACTGA

1351 TCCCGTGGAT CGTCCTGTGA ACGCAAAGCC ATCTGCGCAG CCAACGATGC

1401 CTTCAGAGGC TCCTGCTCAA GAGACATCTG AAGTTCCCAC CCCGACGAAA

1451 CCTAGTTCTA CTGAATCACC TTCTTACCAC CTCCTCTGCG GATAGCTTTT

1501 TGCCATCCTT CTTCCCCACG TTCGGTGCGT CGTGAGGATC CAAGCTTGGG

1551 TACGT

SEQ ID No.5

The partial sequence in the coding region ofthe Trichoderma reesei QM9414 gene encoding proprotein convertase

1 GCTGTCCGCA CTGATGCGTG CGGCCTTGGC GTTGCCTACG ACTCCAAGAT 51 TGCTGGCATC CGCATCCTTA GTAGTGCCAT CAGCGATGCG GACGAGGCCG

101 AGGCCATGAT TTACAAGTTC CAGGACAACC AGATCTACTC GTGCTCCTGG

151 GGGCCTCCCG ACGATGGGAG GTCCATGGAA GCCCCCGACG TCCTGATTCG

201 ACGAGCCATG CTCAAGGGCG TCCAGGAGGG ACGAGGAGGC CTCNGCTCCA 251 TCTACGNCTT TGCTAGTGGT AACGGTGCCG CCAGTGGCGA TAACTGCAAC

301 TNCGACGGAT ACNCAAACA

SEQ ID No. 6

The partial sequence in the coding region of the Fusarium venenatum ATCC20334 gene encoding proprotein convertase

1 GGTTTNNCCG TTGGTGTTGC TACGACTCCA AGTCGCCGGA ATCCGTATTC

51 TCAGCAAACT GATCAGCGAC GCCGACGAAG CAGAAGCGCT TATGTACAAG

101 TACCATGACA ACCATATTTA CTCTTGCTCA TGGGGTCCTT CCGATGATGG

151 CCAGACTATG GAGGCACCCG ATGTTGTCAT TCGACGAGCA ATGCTTAAGG

201 CGATTCAGGA GGGACGTAAT GGTCTTGGCT CTGTCTACGT CTTTGCCAGT 251 GGAAACGGTG CAGGCCAAGG AGATAACTGC AACTNCGACG GATCCACCAA

301 ACA

SEQ ID No. 7

The partial sequence in the coding region of the Penicillium chrysogenum P2 gene encoding for proprotein convertase 1 GTGGGTGTTG CCTATGACAG CAAGGTGTCA GGTATCCGGA TTCTGTCCAA

51 GGCGATTGAC GACGTCGACG AAGCAGCTGC CATCAACTTT GCCTTCCAAG 101 ATAACGATAT ATACTCCTGC TCGTGGGGTC CTCCTGATGA TGGTGCGACC

151 ATGGATGCGC CGGGCTTGTT GATCAAGCGG GCGATGGTCA ATGGTGTGCA

201 NGAGGGACGA GGTGGAAAGG GTTCGATCTT CGTGTTNGCC GCAGGCAACG 251 GTGCTCTTTT TGGCGACAAC TGCAACTTCG ACGGATACAA CAAACA

SEQ ID No.8

The partial sequence in the coding region of the Rhizopus oryzae ATCC200076 gene encoding proprotein convertase

1 ACTNGGGGCA TTGGTGAAAT NTTGCTTGTG GNTTGGTGTT GCTTACGACG 51 CAAAAATATC TGGTATACGT ATATTATCAG GTGAAATCAC AGAGGCAGAC

101 GAGGCTGCTG CTTTGAATTA CAAATATCAA GAAAATCAAA TCTACTCCTG 151 CTCNTGGGGC CCA

SEQ ID No. 9

The partial sequence in the coding region of the Agaricus bisporus HORST gene encoding proprotein convertase

1 ATGTGGTCTT GGTCTCGCCT ACGAATCCAA GGTCGCTGGT GTTCGCATAT 51 TGTCTGGTCC CATAACGGAC GTCGATGAAG CGACTGCGCT CAACTATGGT

101 TTCCAAAATG TATCTATCTT CAGCTGTAGT TGGGGCCCAC CTGACAATGG

151 TATGTCCATG GAAGGCCCAG GATACCTCAT CAAAAAAGCT GTCGTCAACG 201 GCATTAACCA GGGACGTGGC GGGAAGGGCT CCATTTTCGT CTTCGCCAGT

251 GGCAACGGCG CTGCTTCGGA TGACCAATGC AACTACGACG GATACACAAA 301 CA

SEQ ID No. 10 coding strand itømHI-site is underlined PE4 5*- CG CGGATC CACT/C) GGX ACX (C/A)GX TG(T/C) GCX GG -3' degenerated 2048 times

SEQ ID No. 11 coding strand 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 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 non coding strand BamRl-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

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 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

SEQ ID No. 16 non coding strand

PCL4 5'-GC XGC XGA XGT XCC XCC (A/G)TG-3' degenerated 2048 times

SEQ ID No. 17

The sequence of pAB4-lrep 59-499 bp 0.4 kb Hindϊll fragment of pAB4-l 1-58 bp 500-513 bp 2873-2930 bp polylinker sequence of pMTL24

(indicated with underlined small letters)

.514-2872 bp. 2.3 kbXhoI fragment of pAB4-l

1 qqccaqtqaa ttcqaqctcq qtacccqqqq atcctctaqa qtcqacctgc

51 aqqcatqcAA GCTTGGTCAG CAGTACCAGA CGCCCGGATC GGCTATCGGC

101 CGGGGTGCTG ACTTCATTAT CGCGGGTCGC GGTATCTACG CCGCGCCGGA

151 TCCGGTGCAG GCTGCGCAAC AGTATCAGAA GGAGGGGTGG GAAGCCTACC

201 TGGCCCGTGT CGGCGGAAAC TAATACTATA AAAGGAGGAT CGAAGTTCTG 251 ATGGTTATGA ATGATATAGA AATGCAACTT GCCGCAACGG ATACGGAAGC

301 GGAAACGGAC CAATGTCGAG CACGGGTAGT CAGACTGCGG CATCGGATGT

351 CCAAACGGTA TTGATCCTGC AGGCTACTAT GGTGTGGCAC AAGGATCAAT

401 GCGGTACGAC GATTTGATGC AGATAAGCAG GCTGCGAAGT AGTAACTCTT

451 GCGTAGAGAA AATGGCGACG GGTGGGCTGA TAAGGGCGGT GATAAGCTT_ 501 catgcctqca ggcCTCGAGC TAACATACAT TCCGAACCGT GCAGCCCAAG

551 GCCGAGCAGT TCAACTGCGC TCAGCGCGCT CATGCCAACT TCCTTGAGAA

601 CTCCAGCCAA ACTATGCTCT TCCTCCTGGT AGCTGGACTG AAGTACCCCC

651 AGTTGGCGAC TGGCCTCGGA AGCATCTGGG TCCTCGGTCG CTCACTGTTC

701 CTTTACGGAT ATGTGTACTC CGGCAAGCCG CGGGGTCGCG GTCGTTTGTA

751 CGGCAGCTTC TACTTGCTTG CACAGGGAGC TCTCTGGGGC NTGACGTCTT

801 TTGGAGTTGC GAGGGAGTTG ATTTCCTACT TCTAAGTTTG GACTTGAATC 851 CGTGGTGTGA TTGAGGTGAT TGGCGATGTT TGGCTATACC AGCTATATGT

901 AATAATCTCT ACTGTATACT ACTATTCAAC GCATTTTACT ATGCGTGCTG

951 CTAGGGTCGG CAATGACAAT GGCAATCTGA CTGACGTGGT CTATTTCTCC

1001 ATGTGCAGCA GGGAATACGA GCTCCAATGG ACCTCGGGAG TGGCACAGTC

1051 AATGGCAAGG AAACTCCGCC TTTGCAGGTG TGGCTGAACC CCACGGGTCG 1101 GAGGCGGAGC AATCCACCCC CGATGTGGCT GGTGCGTGGA GGGGCTCGCG

1151 ATGATTTTAC TGAGCTTGCT TTTCTTGTCG ACATTGAACA TTGTCCTTGG

1201 TCTTCCTTCA GATTTAAGGG TCAGTCACTG CTACATTTCT CAGTAGTATC

1251 CGCGCACGTC TCTGGATTTA CGAATCAGGG TCCACCAGTC GAAACTTCGA

1301 ACTACTCTCA TTATACAATC CTCTTTCCAT TCCCGCATTA ACCCCTCCAT 1351 CAACACCATG TCCTCCAAGT CGCAATTGAC CTACACTGCC CGTGCCAGCA

1401 AGCATCCCAA TGCTCTGGCG AAGAGGCTGT TCGAGATTGC CGAGGCCAAG

1451 AAGACCAATG TGACTGTCTC GGCTGACGTT ACCACCACTA AGGAGCTACT

1501 AGATCTTGCT GACCGTAGGC CGACCCGCTA CTCTGCCTGA TTATGCTGCA

1551 TGCAAACTTA TTAACGGTGA TACCGGACTG CAGGTCTCGG TCCCTACATT 1601 GCCGTGATCA AAACCCACAT CGATATCCTC TCTGATTTCA GCAACGAGAC

1651 CATTGAGGGA CTTAAGGCTC TCGCGCAGAA GCACAACTTT CTCATCTTCG

1701 AGGACCGCAA GtTCATTGAC ATCGGCAACA CGGTCCAGAA GCAATACCAC

1751 GGCGGTACCC TCCGTATCTC GGAATGGGCC CACATCATCA ACTGCAGCAT 1801 TCTCCCTGGT GAGGGTATCG TCGAGGCTCT CGCTCAGACG GCGTCTGCAC

1851 CGGACTTCGC CTACGGCCCC GAACGCGGTC TGTTGATCTT GGCAGAGATG

1901 ACCTCTAAGG GCTCCTTGGC TACCGGCCAG TACACTACTT CCTCGGTCGA

1951 TTATGCCCGG AAATACAAGA ACTTCGTTAT GGGATTCGTG TCGACGCGCG

2001 CGTTGGGTGA GGTGCAGTCG GAAGTCAGCT CTCCTTCGGA TGAGGAGGAC 2051 TTTGTGGTCT TCACGACTGG TGTGAACATT TCTTCCAAGG GAGATAAGCT

2101 TGGTCAGCAG TACCAGACGC CCGGATCGGC TATCGGCCGG GGTGCTGACT

2151 TCATTATCGC GGGTCGCGGT ATCTACGCCG CGCCGGATCC GGTGCAGGCT

2201 GCGCAACAGT ATCAGAAGGA GGGGTGGGAA GCCTACCTGG CCCGTGTCGG

2251 CGGAAACTAA TACTATAAAA GGAGGATCGA AGTTCTGATG GTTATGAATG 2301 ATATAGAAAT GCAACTTGCC GCAACGGATA CGGAAGCGGA AACGGACCAA

2351 TGTCGAGCAC GGGTAGTCAG ACTGCGGCAT CGGATGTCCA AACGGTATTG

2401 ATCCTGCAGG CTACTATGGT GTGGCACAAG GATCAATGCG GTACGACGAT

2451 TTGATGCAGA TAAGCAGGCT GCGAAGTAGT AACTCTTGCG TAGAGAAAAT

2501 GGCGACGGGT GGGCTGATAA GGGCGGTGAT AAGCTTAATT GTCATCGCAG 2551 ATAAGCACTG CTGTCTTGCA TCCAAGTCAG CGTCAGCAGA AATACGGGAC

2601 TTCCGAAAGT ATATGGCAAA ATTAAAGAAC TTGACTCTCC AGCAATGTTT 2651 TGCCCTGACC GTCGCTAAAA CGTTACTACC CCTATACCCG TCTGTTTGTC

2701 CCAGCCCGAG GCATTAGGTC TGACTGACAG CACGGCGCCA TGCGGGCTTG 2751 GGACGCCATG TCCGTCGCGT GATAAGGGTT GATCCATGCA GCTACTATCC

2801 TTCCATCGTT CCATTCCCAT CCTTGTCCTA TCTCCATCCT TGAAACTTTA

2851 CTAGTTTAGT TGGATGCTCG AGatctccat qqacqcqtqa cqtcqactct 2901 gaqqatcccc q qtaccqaq ctcgaattcg

SEQ ID No.18 MBL 789 EcoRI is underlined

5'- GGAA TTC (A/G)GA ATA (T/A)GG AGG ATG TAG -3' degenerated 4 times

SΕQ ID No. 19 MBL 793 BamHl is underlined

5'- CGGATCCG CAG TGG CAC TTG (G/A)TC AAT CCA A -3' degenerated 2 times

SΕQ ID No. 20 MBL 794 EcoRI is underlined

5'- GGA ATT CTT AAA A(T/G)C CCA AGA ACC TTC A -3" degenerated 2 times

SΕQ ID No. 21 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

SΕQ ID No. 22

MBL 1208 Clal is underlined 5'- CGG ATC GAXT/C) GGX ACX (C/A)GX TGTT/C) GCX GG -3' degenerated 2048 times

SEQ ID No. 23 MBL 1219 BamHl is underlined

5'- CGG ATC fC/DTG XA(G/T/C) (A/G)TC XC(T/G) CCA XGT (C/A/G)AG -3' degenerated 4608 times

SEQ ID No. 24

Restriction sites are bold Primers are underlined

BamHI PE4 primer GGATCCATGG CACGAGATGT GCAGGTGAAA TCGGTGCGGC GAAAGAAAAC AACGTGTGCG 60

GGGTTGGTGT TGCGTATGAT AGTCGCATCG CTGGTATTCG GATTCTCTCC ACACCCATCG 120 EcoRV

ATGACACTGA TGAGGCTGCG GCTATTAACT ACGCCTATCA GGAGAACGAT ATCTACTCGT 180

GTTCCTGGGG TCCCTATGAT GATGGCGCCA CAATGGAAGC CCCGGGCACT CTGATCAAGC 240

GGGCCATGGT CAATGGTATC CAAAATGGTC GAGGTGGAAA AGGCTCGGTT TTTGTCTGCG 300

PE6 primer CCCCCGGAAA TGGTGGATCC 320

BamHI SEQ ID No.25

Aspergillus niger PclA protein sequence Met Arg Leu Thr Gly Gly Val Ala Ala Ala Leu Gly Leu Cys Ala

Ala Ala Ser Ala Ser Leu His Pro His Arg Ser Tyr Glu Thr His

Asp Tyr Phe Ala Leu His Leu Asp Glu Ser Thr Ser Pro Ala Asp

Val Ala Gin Arg Leu Gly Ala Arg His Glu Gly Pro Val Gly Glu

Leu Pro Ser His His Thr Phe Ser lie Pro Arg Glu Asn Ser Asp

Asp Val His Ala Leu Leu Asp Gin Leu Arg Asp Arg Arg Arg

Leu

Arg Arg Arg Ser Gly Asp Asp Ala Ala Val Leu Pro Ser Leu Val

Gly Arg Asp Glu Gly Leu Gly Gly lie Leu Trp Ser Glu Lys Leu

Ala Pro Gin Arg Lys Leu His Lys Arg Val Pro Pro Thr Gly Tyr

Ala Ala Arg Ser Pro Val Asn Thr Gin Asn Asp Pro Gin Ala Leu

Ala Ala Gin Lys Arg lie Ala Ser Glu Leu Gly lie Ala Asp Pro

lie Phe Gly Glu Gin Trp His Leu Tyr Asn Thr Val Gin Leu Gly

His Asp Leu Asn Val Thr Gly lie Trp Leu Glu Gly Val Thr Gly

Gin Gly Val Thr Thr Ala lie Val Asp Asp Gly Leu Asp Met Tyr

211 Ser Asn Asp Leu Arg Pro Asn Tyr Phe Ala Ala Gly Ser Tyr Asp

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 lie Gly Ala Ala Lys Asn

256 Asp Val Cys Gly Val Gly Val Ala Tyr Asp Ser Arg lie Ala Gly

271 lie Arg lie Leu Ser Ala Pro lie Asp Asp Thr Asp Glu Ala Ala

286 Ala lie Asn Tyr Ala Tyr Gin Glu Asn Asp lie Tyr Ser Cys

Ser

301 Trp Gly Pro Tyr Asp Asp Gly Ala Thr Met Glu Ala Pro Gly

Thr

316 Leu lie Lys Arg Ala Met Val Asn Gly lie Gin Asn Gly Arg Gly

331 Gly Lys Gly Ser Val Phe Val Phe Ala Ala Gly Asn Gly Ala He

346 His Asp Asp Asn Cys Asn Phe Asp Gly Tyr Thr Asn Ser He Tyr

361 Ser He Thr Val Gly Ala He Asp Arg Glu Gly Asn His Pro

Pro

376 Tyr Ser Glu Ser Cys Ser Ala Gin Leu Val Val Ala Tyr Ser

Ser

391 Gly Ala Ser Asp Ala He His Thr Thr Asp Val Gly Thr Asp Lys 406 Cys Ser Thr Thr His Gly Gly Thr Ser Ala Ala Gly Pro Leu Ala

421 Ala Gly Thr Val Ala Leu Ala Leu Ser Val Arg Pro Glu Leu Thr

436 Trp Arg Asp Val Gin Tyr Leu Met He Glu Ala Ala Val Pro

Val

451 His Glu Asp Asp Gly Ser Trp Gin Asp Thr Lys Asn Gly Lys

Lys

466 Phe Ser His Asp Trp Gly Tyr Gly Lys Val Asp Thr Tyr Thr Leu

481 Val Lys Arg Ala Glu Thr Trp Asp Leu Val Lys Pro Gin Ala Trp

496 Leu His Ser Pro Trp Gin Arg Val Glu His Glu He Pro Gin Gly

511 Glu Gin Gly Leu Ala Ser Ser Tyr Glu Val Thr Glu Asp Met

Leu

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 Arg

556 Ser Pro Asp Gly Arg Val Ser His Leu Ser Thr Pro Arg Arg Pro

571 Asp Asn Gin Glu Val Gly Tyr Val Asp Trp Thr Phe Met Ser Val

586 Ala His Trp Gly Glu Ser Gly He Gly Lys Trp Thr Val He Val 601 Lys Asp Thr Asn Val Asn Glu His Thr Gly Gin Phe He Asp Trp

616 Arg Leu Asn Leu Trp Gly Glu Ala He Asp Gly Ala Glu Gin Pro

631 Leu His Pro Met Pro Thr Glu His Asp Asp Asp His Ser Tyr Glu

646 Glu Gly Asn Val Ala Thr Thr Ser He Ser Ala Val Pro Thr Lys

661 Thr Glu Leu Pro Asp Lys Pro Thr Gly Gly Val Asp Arg Pro

Val

676 Asn Val Lys Pro Thr Thr Ser Ala Met Pro Thr Gly Ser Leu

Thr

691 Glu Pro He Asp Asp Glu Glu Leu Gin Lys Thr Pro Ser Thr Glu

706 Ala Ser Ser Thr Pro Ser Pro Ser Pro Thr Thr Ala Ser Asp Ser

721 He Leu Pro Ser Phe Phe Pro Thr Phe Gly Ala Ser Lys Arg Thr

736 Glu Val Trp He Tyr Ala Ala He Gly Ser He He Val Phe

Cys

751 He Gly Leu Gly Val Tyr Phe His Val Gin Arg Arg Lys Arg

He

766 Arg Asp Asp Ser Arg Asp Asp Tyr Asp Phe Glu Met He Glu Asp

781 Glu Asp Glu Leu Gin Ala Met Asn Gly Arg Ser Asn Arg Ser Arg

796 Arg Arg Gly Gly Glu Leu Tyr Asn Ala Phe Ala Gly Glu Ser Asp

811 Glu Glu Pro Leu Phe Ser Asp Glu Asp Asp Glu Pro Tyr Arg Asp

826 Arg Gly He Ser Gly Glu Gin Glu Arg Glu Gly Ala Asp Gly Glu

841 His Ser Arg Arg

SEQ ID Nos.26 to 31 PCR-primers for A. sojae promoter cloning Restriction sites are underlined

SEQ ID No. 32

The sequence of the Aspergillus sojae gpdA promoter region 1 AATTGCGGCC GCTATGAAAC CGGAAAGGGC TGCTGAGAGC TGGGGAACGG

51 CGCAAGCCGG GAAAACAGCT GACAAGGACC CATTTCACTC TGGATCTTGA 101 GGAGAGCTGT AGCTTTTGCC CCGTCTGTCC ACCCGGTGAC TGGATTAGTG

151 ACCTGGTCGT TGCGTCAGTC AACATTGCTC TTTTTTTATC TCCCCCTCCC

201 CCGCCGTCCG ACTTTTCTCC CCTTTTCTAC TCTCTTCGTA TACTCACCAC

251 TGCAATCATC TTATCCCTTT GTCTTCTTAC TTAAAGTGAG TCGTCTCCCG

301 CCCATCGTTC CCTTTGAACC TTGTAAATCA GAGCCACTTT CAAGTGTCTA 351 CCGTTTCCTT TCCACATAGA TTGACTGACA GCTACCCCGC CACACCAGCA

401 GACACATCTA AACCATGG

SEQ ID No. 33

The sequence of the Aspergillus sojae alp A promoter region

GCGGCCGCGG TTATTCTGCG GAAGCGGACC CCCCCCTTCC GCCCAAACAG

51 GGCGAATGTG CCCAAGTTCT GATACTATCA GAAGACCTCC AGGAGCACAT

101 GCCTGTTCGC ATAACCCTGG TGTAGCACCA GGAATTGCTT AGCTTAGCTT 151 CTTCGACTGA GGGGCCAGAA AGTGCTTATC GCAAAGATCC CACTTCTTTG

201 TGTGATAGCC CCTCCCGCGG CCCTTGATCA AGCCGTTCTC GCTATCCAAT

251 ATTGAAAGCG TGATATTATA GGTGCACATG GTTATTATCC TTTTTCTTTT

301 TCTCTTTCTT TGCTTTTCAT GCAACCCCAT ACGTTGCCGA ATTTGGCTAC

351 ACCTTGGGGC TCATTCTTCG AAGTTTAGAT TCCGACAAGA CCTCACCACC 401 CAATCAAAAC CCTTGATTCC TGATAAAAGA CGTGGAAAGA AGCGGATATC

451 GCGTGAGGAT GCCAAGCAAA GGGAATGGGT CACATTGATC TCTGTCGCGT 501 TGTTAGGATG ATCTTCACTC CTAAAGGCAT CGCCCGCGGC ACTAGGTCCT

551 TCCTGTCCAG GATATCGTTT ACTCCTCTCA TTATGGCGAG CTACTTTGTG

601 AATTAATTGA CTGAGGGATA TACCACCTTC CCTTTGAAGG TACCAAGCCA

651 CTACCTTGAG CGTTAGTTAC TTTTTCGAGG AAAGCGTCCT ATGCTGGTCT

701 CCGCCAAACC CTCGACAACT TGCCATAGCC TTGTGTTCTT CATGGTCTAT

751 CGGAGTACCC GTTCATGACT GAAGCGGGTC AGCGTCCGTG GTGGTCATCA

801 TCATTCTCAT CTTTCATCAT GCCCGCTGAT TGATAGAGTA ATTTCCGGTG 851 GAGCACAACG CCGTCCTCTG AGATGCAATG TCACCCTGTA AGTTTCAACT

901 ACACTCTGTA GTACAGAGCA TCCTTGCCAT TGCATGCTGT GCAAGTGATC

951 TAAATCCGTA GAATCTGCTC GAGAACGGGG AAATATAGAA CTCCTGAAGG

1001 TTATAAATAC CACATGCATC CCTCGTCCAT CCTCATTTCC ATCATCAAGC 1051 CAGCGGTTTC TATCCTCCGA CTTGAGTCGT TCTCGCGCAT CTTTACAATC 1101 TTCTCACCAT GG

Table 1. Taxonomic scheme of the genus Aspergillus (Samson, 1992)

GENUS SUBGENUS SECTION SELECTED SPECIES³ "SUBSPECIESES^"b

Aspergillus Circumdati Wentii A. wentii (glucosidase)

Flavi/Tamarii A.orvzae (amylase, protease)

A. tamarii^tox

A. sojae (fermented food, protease)

A. parasiticus^tox

A. flavus^tox

Nigri A. niger - A. pulverulentes

(fermented food, A. phoenicis various proteins, A. awamori organic acids) A. foetidus A. kawachii

A. usamii A. ficuum

A. japomcus -> A. aculeatus

(endoglucanase) (glucosidase. galactanase)

A. ellipticus

A. tubingensis -> "A. niger"

Circumdati A. ochraceus^tox (xulanase A. alliaceus^tox

Candidi A. candidus (lipase, glucosidase)

Cremei A.itaconicus (organic acid)

Sparsi A. sparsus

Aspergillus Aspergillus A. glaucus (fermented food)

Restricti A. restrictus^tox

Fumigati Fumigati A. fumigatus^tox Cervini

Omati

Clavati Clavati A. giganteus^to Nidulantes Nidulantes A. nidulans^tox

Versicolores A. sydowii (lipase)

Usti

Terrei A. terreus^tox (glucansae)

Flavipedes

^a For the species selected for this list either the production of proteins/organic 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 ^{tox b} Based on various methods the listed names may be considered synonymous to the given

SPECIES name.

Table 2. The classification of the different ATCC strains

Legend ND = not determined

" REF Ushijima S, Hayashi K and Murakami H (1982) The current taxonomic status of Aspergillus sojae used in Shoyu fermentation Agric Biol Chem , 46 236S-2367, 1981

²⁾ REF Yuan GF, Liu CS and Chen CC (1995) Differentiation of Aspergillus parasiticus from

Aspergillus sojae by Random Amplification of Polymorphic DNA Appl Environm Microbiol , 61 2384-2387

³⁾ REF Chang PK, Bhatnagar D, Cleveland TE and Bennett JW (1995) Sequence variability in homologs of the aflatoxin pathway gene aflR 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" > 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⁾ 7¹ REF ATCC catalogue 8^,REF Liu BH, Chu FS (1998) Appl Env Microbiol , 64 3718-3723

Table 3. Composition of selection media

'• mineral solution CuS0₄5H₂0 016 g/1

FeS0₄7H₂0 05 g1

ZnS0₄7H₂0 22 g/1

MπCl₂4H₂0 05 g1

CoCl₂6H₂0 017 g1

Na₂Mo0₄2H₂0 015 g/1

H,B0₃ U g1

EDTA 5 g1

Table 4. Protease activity in different media

Legend : + (partial) degradation of proteins after 4 hours incubation, large milk clearing zone no degradation of proteins after 4 hours incubation, small/no milk clearing zone

Incubation at 30°C: 27 μl medium sample 2.5 μl BSA (25 mg/ml) 0.5 μl Phytase (A.terreus, 3-4 g/1) 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.

Table 5. Protease activity at different pH values

Legend + (partial) degradation of proteins after 4 hours incubation no degradation of proteins after 4 hours incubation

Incubation at 30°C: 25 μl mediumsample 50mM NaAc pH=42

2 μl buffer (50mM) -» 50mM NaAc pH=5 8

2.5 μl BSA (25 mg/ml) 50mM Tπs/HCl pH=83 0.5 μl Phytase (A.terreus, 3-4 g/1)

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.

Table 6. PCR results for cloning fungal pclA genes

Legend: + specific PCR product aspecific or no PCR product fU this PCR product was used for sequencing Table 7. The viscosity ranges of the various A. sojae strains

Table 8. Promoter strength in A. sojae transformants

Claims

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

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 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. 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 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 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.

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

7. A method of selecting transformed or transfected Aspergillus sojae said method 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 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 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 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 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 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 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 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 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- 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 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 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 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 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.

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 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 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 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 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 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 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 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 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 t e pyrG 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 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 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, 1, 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 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 corresponding wild-type fungus.

28. A fungus according to claims 26 or 27 said mutation being obtained by specific gene 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 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 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 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 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 phytase activity.