MXPA02001061A - Use of aspergillus. - 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

NEW MEANS TO TRANSFORM FUNGI AND ITS USE FOR THE PRODUCTION OF HETEROLOGICAL PROTEINS BRIEF DESCRIPTION OF THE INVENTION The invention relates to novel means for transforming fungi and their use for the production of heterologous proteins. The means involve genetically manipulating fungi belonging to the taxonomic group Aspergillus sojae. In the past, suggestions have been made to use Aspergillus sojae as a host strain for transformation. However, to date no data have been provided on the successful transformation and / or expression of heterologous proteins. Furthermore, it has been found that with respect to certain proteins, such as phytase, which have been difficult to express in large amounts due to various reasons including proteolytic degradation in the expression host other than Aspergillus sojae, they can be expressed in a surprising manner in Aspergillus sojae It has been found that the levels of production with respect to heterologous proteins in Aspergillus sojae far exceed the levels obtained for the same proteins in Aspergillus awa orí. In addition to the above, the subject matter of the invention also covers a method for obtaining improved strains of Aspergillus sojae for expression purposes, characterized in that, on the one hand, they have a reduced proteolytic activity, and on the other hand, they have fermentation characteristics. improvements related to the morphology of the fungus.

BACKGROUND OF THE INVENTION In the past suggestions have been made to use Aspergillus sojae as a host for transformation. However, to date no data have been provided on the successful transformation and / or production of heterologous proteins and, more specifically, nothing has been revealed regarding the expression of phytase. Previously, expression levels have been very low in host species other than Aspergillus sojae, mainly due to proteolytic degradation. At present, expression levels have been discovered for the protein in Aspergillus sojae that far surpass those levels achieved for the same protein in other strains, for example Aspergillus niger, Aspergillus awamori and Trichoderma. It is surprising to discover such an improvement in closely related strains. Therefore, prior art disclosures concerning the production of phytase present difficulties. The prior art disclosures about the use of Aspergillus sojae for the expression of heterologous proteins or polypeptides are inadequate. The fact that to date hardly any of the successful attempts to transform A. sojae has been reported, in view of the fact that numerous successful transformations of closely related strains of the taxonomic group Aspergillus oryzae have been reported in the past . Based on this closeness in the relationship, the person skilled in the art could anticipate, and in fact anticipates, that methods analogous to those used for Aspergillus oryzae for strains of Aspergillus sojae can also be applied. For example, WO97 / 04108 describes the isolation of a nucleic acid sequence encoding a protease, in specific a sequence encoding leucine aminopeptidase and the transformation of a variety of host organisms, among others Aspergillus soj a e, with a sequence encoding leucine aminopeptidase. However, an illustration of this particular transformation that actually takes place is not provided. It is only suggested as a possibility among many other strains such as Trichoderma reesei, Aspergi llus niger, Aspergillus awamori, Aspergi llus foe ti dus, Aspergi ll us j aponi cus, Aspergi ll us phoeniai and Aspergi ll us oryzae as a potential host strain that will be used for the transformation. In the cited document, three transformation protocols widely used in the technique for strains are suggested. Specifically, any of the acetamidase selection markers S (= amdS) (for example, those maintained in the vector p3SR2) are suggested., argB or hygromycin B (for example using the vector pAN7-l) as markers that are suitable for use in accordance with the transformation protocols described in said document. The use of the vector p3SR2 with the marker has often been written in the literature amdS as a useful method to transform various strains, for example Aspergi ll us ori za e (in EP 0.238.023), Tri choderma rees ei (in EP 0.244.234) and Aspergillus niger (EMBO Journal -é., pages 475- 479). Accordingly, analogous use for transforming Aspergillus in general is indicated in WO97 / 04108 on the basis of these prior publications. In completely specific form on page 17 of WO97 / 04108 it is described that Aspergi II us and Tri choderma, which before the transformation grows slowly in minimum medium comprising only the acetamide substrate as nitrogen source, could be selected after the transformation with the p3SR2 vector due to a clear growth advantage. Subsequently, the transformants obtained in this way need to be subjected to an additional selection with respect to the production capacity of leucine aminopeptidase (= LAP) in order to find a desired transformant. As indicated above, this is only indicated as speculative transformation media that can be applied in two of the aforementioned genres "in toto" on the basis of how many successful transformations of different strains of Aspergi llus sojae. However, the suggested transformation protocol does not succeed with Aspergi ll u s soj ae. The selection criteria described in the prior art are insufficient to ensure the practical selection of desirable transformants when the p3SR2 vector is used. Experiments have been conducted and it has been found that the described method does not work due to excessive background growth which eliminates the practical selection ability. Another method of selection used routinely for fungal transformants is the transformation of mutants of orotidine-5-monophosphate decarboxylase (= PyrG). Mattern et al. in Mol. Gen. Genet. 210, pages 460-461, describe the transformation of Aspergi l l u s oryza e using the pyrG gene from Aspergi l l us niger. The normal practice is to isolate pyrG mutants based on direct resistance to fluoro-orotonic acid as a positive selection marker. To date, this has resulted in the isolation of numerous pyrG mutants for a variety of fungi.

Based on experience with a number of different filamentous fungi, the pyrG-based auxotrophic system has many favorable characteristics. Experiments were performed to obtain the mutant pyrG strains of A. soj ae, using a standard procedure based on direct selection with respect to resistance to fluoro-orotic acid (FOA) in plates containing uridine to support the growth of the mutant strain (Van Hartingsveldt et al., in Mol. Gen. Genet (1987) 206, pages 71-75). However, the use of the analogous method in strains of Aspergi l l us soj a e does not lead to pyrG mutants. The usual method leads to strains resistant to fluoro-orotic acid, but all strains are able to grow without uridine. Therefore, none of these strains is a pyrG mutant. Normally, the isolation of the pyrG mutants can be carried out directly from strains resistant to fluoroorotic acid in a selection medium with uridine. For Aspergi l lus soj ae however, this method is not functional. Clearly, Aspergi l l us soj a e has different characteristics than those of Aspergi l l us oryzae closely related when it reaches the point of transformation. Standard protocols using amdS or pyrG as selectable markers is not enough.

Unfortunately, the argB method as a selectable marker is not an attractive option because it requires the isolation of a corresponding argB mutant for each host strain that is to be used. This is an arduous task taking based on trial and error. The required argB mutant can be obtained through random mutagenesis followed by the selection of tens of thousands of colonies. The situation for pyrG is even better in the sense that the mutant can be selected by itself. In the case of amdS the mutant is not required because the presence of amdS functions as a dominant selectable marker. There are additional problems which discourage the person skilled in the art from using Aspergi l lus soj a e as an expression host for recombinant proteins or polypeptides. The document JP-A-02-234666 for example, describes a selection of Aspergillus soj e based on ArgB using a protocol analogous to that described for other fungi. Such a procedure has been described for Aspergillus oryzae in Biotechnology (1988) 6, pages 1419-1422. The cited article also refers to the successful analogous transformation of Aspergillus nidulans and Aspergillus niger. However, when the strain of Aspergillus sojae ATCC42251 described in the Japanese patent application is analyzed, an undesirable protease profile is found. The protease profile of this strain is incompatible for it to be applied as a host for production. Although a transformation protocol has been suggested in the prior art for this strain of Aspergillus sojae it may not be possible to obtain a high level of heterologous protein expression even if the protocol for transformation is successful. This is precisely due to the fact that due to the explicit characteristics of the strains of Aspergillus sojae to produce excessive amounts of alkaline proteases and amylases, these currently find an application in practice, specifically these are used in procedures that require the degradation of complex polymeric substrates, so the most that can be expected is that any of the Aspergillus transformants sojae that eventually Successful results do not produce good levels of expression unless the product is an Aspergillus protein so that it is impermeable to its own proteases. In summary, there are many problems faced by the person skilled in the art to find means to use strains of Aspergi l lus soj a e to express heterologous recombinant proteins on an industrial scale. First, a number of procedures can not be applied to introduce the desired nucleic acid material to be expressed in the form used for other fungi. This includes the procedures based on pyrG and amdS that are useful for Aspergill us oryza e closely related. Secondly, it remains to be seen whether high levels of heterologous protein production are possible despite the known excessive proteolytic activity of the Aspergi strain, the soya and host. Unexpectedly it has been discovered that the problems referred to above can be solved, thus resulting in novel expression hosts to produce novel proteins and methods for production of heterologous proteins. The transformation of A. sojae strains with the amdS and pyrG selection markers is described. Efficient gene expression is also described, including the expression of a gene for phytase.

BRIEF DESCRIPTION OF THE INVENTION As indicated, the present invention is directed to strains of Aspergillus sojae and to the application thereof to produce recombinant proteins and polypeptides. First, a description of the strains of Aspergí 1 lus sojae is given.

Determination of Aspergillus sojae The taxonomy of fungi is a complex issue. The genus Aspergillus comprises Aspergillus sojae in the Flavi / Tamarii section (see table 1). It is clearly shown that A. sojae is distinct from A. oryzae which is located in the same section (see table 2). At present, the strains belonging to Aspergillus sojae of Aspergillus strains can be taxonomically differentiated closely related oryzae and closely related Aspergillus parasiticus in a number of ways recognized in the art. Reference is made to the random PCR fragments, ver-1, aflR and to the 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, Aspergillus oryzae has also been found to differ from Aspergillus sojae after comparing the alpA sequence of these strains. Among others (there are other sequence differences between A. oryzae and A. sojae alpA that could be used as a determination tool), Aspergillus sojae has been found to comprise an Xmnl restriction site at a specific site in the alpA gene. The corresponding site in the -gen alpA of several strains of Aspergillus oryzae does not possess such a restriction site. In this way, an additional point of differentiation between the two types of fungal strains is provided. Therefore, several methods are available to assess whether a strain is an Aspergillus sojae or not. Currently more than 10 strains are deposited in the ATCC that are de-finidas such as Aspergillus sojae. Were analyzed the 10 oldest deposits. Two out of 10 do not pass the aforementioned determination test. One of them is strain ATCC20235 which according to Ushijama et al. (1981) also does not meet the requirements for it to be classified as an Aspergillus sojae based on the morphological parameters. The other one is strain ATCC46250. The definition of Aspergillus sojae as used through the patent application is intended to involve a strain that preferably meets all the requirements described in the cited references in combination with the presence of the X nl restriction site in the alpA gene . Specific homologous primers are also provided for both sequences of Aspergillus oryzae and Aspergillus sojae. These can be used to evaluate for the presence of the Xmnl restriction site as an example of a selection test useful for distinguishing Aspergillus oryzae from Aspergillus sojae (the primer sequences are SEQ ID No. 1 MBL1784: 5 '-CGGAATTCGAGCGCAACTACAAGATCAA- 3 'and SEQ ID No. 2 MBL1785: 5' - CGGAATTCAGCCCAGTTGAAGCCGTC- 3 X). These are derived from the coding region of the -gen alpA. It will be apparent to the person skilled in the art with Based on the known sequence data, alternative probes or initiators can be devised. Amplification of PCR using these primers on Aspergillus DNA, followed by restriction enzyme digestion of the resulting DNA fragments with Xmnl provides a way to discriminate the Aspergillus sojae strains from the A. oryzae strains. Having established the definition of the strains of Aspergillus sojae one can advance with the detailed description of the invention. The invention in one aspect covers a recombinant Aspergillus sojae comprising an acetamidase S (amdS) gene introduced as a selectable marker. Such A. sojae may be selected in a medium comprising a substrate for the amdS protein introduced as the sole source of nitrogen, said medium also comprising a carbon substrate and said substrate-free medium being capable of inducing endogenous amdS. A suitable medium comprises acrylamide as the substrate for the amdS introduced as the sole source of nitrogen. An additional suitable medium comprises at least minimum substrates required for the growth of Aspergillus soj a e. An appropriate category of A. Soj in accordance with the invention is formed by A. soj that can not be selected in medium containing acetamide. A . Soj e in accordance with the invention is suitably an A. so that it can be selected in a glucose-free medium, ie a medium in which the carbon source is not glucose. Said medium can be a medium having sorbitol as the carbon source. Better results are achieved in the case of sorbitol when sorbitol is the only carbon source. An Aspergillus soj e in accordance with the invention may comprise a further sequence of introduced nucleic acid, in which said introduced sequence encodes a protein or polypeptide. The additional sequence introduced can be adapted to optimize codon usage for the use of codons by the host strain or it can have the original codons from the host from which it was derived. The sequence introduced in principle is any sequence that wishes to be expressed by the person skilled in the art. The introduced sequence can be appropriately heterologous, that is, foreign to the Aspergillus soj e in which it is introduced. This can also be native but introduced in the form of one or more additional copies. One of the objects of the invention is directed to the expression of phytase or proteins having phytase activity. A number of sequences relating to the phytase sequence data are known to the person skilled in the art. The contents of documents EP 684.313 are referenced and incorporated for reference., EP 897,010, WO 99/49022, EP 911,416 and EP 897,985. These documents describe various natural and modified phytase sequences. These also describe a consensus sequence. An appropriate modality is formed by phytase sequences from Peni ophora which are natural sequences or modified versions thereof. The novel system is more flexible than previous systems and therefore heterologous sequences, including heterologous sequences encoding phytase or for proteins having phytase activity that were difficult to express in fungal systems of the prior art, can be expressed in the novel system according to the invention.

An Aspergillus in accordance with the invention as defined in any of the embodiments described above comprising an amdS gene introduced as a selectable marker may in appropriate form not have an active endogenous amdS gene. The Aspergillus soj e in accordance with such modality could have, by way of example, an endogenous amdS gene comprising a mutation that inactivates endogenous amdS. Any type of deactivating mutation known or conceivable by the person skilled in the art can be presented. An appropriate example of such a deactivating mutation could be deletion or disruption. The mutation could in-activate the gene or the gene product. It is obvious to the person skilled in the art that numerous options are available to achieve this and that these can be readily available. In an alternative embodiment the invention is also directed to an Aspergillus-free recombinant Aspergillus-an active endogenous amdS gene and also comprises an amdS gene introduced as a selectable marker. Aspergillus and recombinant soybean according to the invention can be selected in a medium that contains a substrate for amdS as the sole source of nitrogen, said medium also comprising a carbon substrate. An appropriate medium comprises at least the minimum substrates required for the growth of A. soj a e. For example, in an appropriate modality the gene for ? k amdS endogenous may be disabled. This deactivation can be any type of deactivation known or that can be conceived by an expert in the art that allows A. Soj ae remains viable. By way of example, the f) gene for endogenous amdS may comprise an inactivating mutation in the form of a substitution, deletion or insertion of the gene or part thereof, or due to a mutation that affects the expression of the gene in such a way that it renders it inactive. The gene for amdS _ endogenous tifc complete may also be absent. An Aspergillus l sous of any of the 20 embodiments described in accordance with the invention could be an A. soj e in which a gene for an amdS has been introduced. This can be achieved, for example by transformation or transfection. The Aspergi l l us soj a e resulting from 25 according to the invention could then subsequently be separated from the A. sojae not transformed or transfected. Any of the embodiments described above as such or in combination are covered by the invention. The invention not only encompasses Aspergillus sojae as such, but also covers a method for introducing a nucleic acid sequence into an A. sojae. The method comprises submitting Aspergillus sojae to the introduction of a nucleic acid sequence in a manner known per se for the introduction of a nucleic acid sequence in a fungus. Such a method of introduction may be, for example, by transformation or transfection A. sojae. The method comprises introducing the gene for amdS as the nucleic acid sequence followed by selection of the transformed or transfected A. sojae in a substrate-free medium that induces endogenous amdS, said media further comprising a substrate for the amdS introduced as the only nitrogen source and said medium also comprising a carbon substrate, said medium allowing the desired A. sojae containing the introduced amdS gene to grow and at the same time eliminate the growth of A. sojae that lack a gene for functional amdS. An appropriate embodiment of said method involves applying a medium containing a substrate for amdS other than acetamide. Suitably, such a medium comprises acrylamide as the substrate for the amdS introduced as the sole source of nitrogen. Suitably, a means for the method according to the invention comprises a carbon source other than glucose. Appropriately, a means for use in a method according to the invention comprises sorbitol as the carbon source, preferably as the sole carbon source. A suitable medium also comprises at least the minimum substrates required for the growth of A. soj a e. A method according to the invention as defined above in any of the embodiments comprises the introduction of an additional nucleic acid sequence in addition to the gene for amdS. The additional nucleic acid sequence for example encodes a protein or polypeptide, such as a phytase x >; for proteins that have phytase activity. The sequence does not necessarily have to be a sequence of type not Aspergi l lu s soj a e, r but q e "can includesequences derived from A. sojae. However, it is intended to indicate that the sequence that is introduced is absent in the untransformed strain or is otherwise present in a smaller number of copies than in that of A. sojae according to the invention. Of course, the present invention also covers any Aspergillus sojae obtained by the method described above. Basically, the method is aimed at introducing a sequence that can effect the presence of active amdS sufficient to function as a selectable marker as opposed to A. sojae in which it was introduced which, for some reason, can not produce active amdS enough to allow growth in a substrate for amdS as the sole source of nitrogen. A method for selecting transformed or transfected A. sojae also falls within the scope of the invention. The method comprises subjecting A. sojae (without the active endogenous amdS gene as defined according to any of the described modalities) to a method of transformation or transfection of A. sojae in a manner known per se for transformation or fungal transfection with a nucleic acid sequence. The method comprises the introduction of a gene for amdS as the nucleic acid sequence, followed by selection of the resulting transformed or transfected A. sojae, said selection being presented in a medium containing a substrate for the amdS introduced as the sole nitrogen source, said medium also comprises a carbon source, said medium allowing the desired A. sojae to grow and at the same time eliminate the growth of untransformed or transfected A. sojae due to the inability of said A. sojae to grow without the amdS gene introduced in the selection medium. A suitable medium also comprises at least the minimum substrates required for the growth of A. sojae. The invention is also directed to a method for producing recombinant Aspergillus sojae. This method comprises introducing a desired nucleic acid sequence, for example 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 homology and enough length to ensure the recombination The introduction is followed by the selection of A. recombinant soya ae having the desired nucleic acid sequence. The selection is presented for a selectable marker comprised in or transformed in cotransformation with the desired nucleic acid sequence, said selectable marker being absent in A. Soj before the introduction of the desired nucleic acid sequence. The flanking sequences could also be sequences corresponding to the gene for endogenous amdS sufficient to ensure recombination. The person skilled in the art can easily assess which sequences "will be sufficient based on the knowledge of the hybridization and sequence data of the gene for endogenous amdS." The recombination event eliminates the activity of endogenous amdS in "both cases. The selectable marker can be quite appropriately pyrG, with uracil instead of uridine in the selection medium. A further embodiment of the invention comprises Aspergillus soya that grows in a medium containing uracil and fluoroorotic acid, without said A. Soj a e present additional growth in a medium that contain uridine and f luoro-orotic acid. This means that the A. soybean has auxotrophy to uracil, can not use uridine, is negative to pyrG and has resistance to fluoro-orotic acid. Urocyte auxotrophy and resistance to fluoro-orotonic acid can be released after supplementing with a gene for pyrG introduced. Said A. Soj a e in accordance with the invention can be free of genes for active endogenous pyrG. The A . soybean and pyrG-negative according to the invention may comprise a gene for endogenous pyrG with a mutation that inactivates it. The mutation can be any mutation known or that can be conceived by a person skilled in the art., said mutation inactivating a gene for pyrG or the expression of the product thereof. Said mutation may be, by way of example, an insert of a nucleic acid sequence in the gene, a substitution of a part of the coding sequence of the gene, a deletion of a part of the coding sequence of the gene or a deletion of the coding sequence of the complete gene. The mutation can also occur in the regulatory part of the gene. In the case of Aspergi l l us soj ae in accordance with the invention he has a gene for mutated pyrG, said Aspergi llu s so a can have a nucleic acid sequence for the gene for pyrG mutated different from that of the pyrG gene of A. soj a e wild type. An additional embodiment comprises A. Soj a pyrG-negativo in accordance with the invention as described in any of the above modalities which also comprises any of the characteristics described for any of the A. The amd S variants according to the invention as such or in combination. A method for selecting Aspergillus soya transformed or transfected also falls within the scope of the invention. The method comprises subjecting A. soj e of the pyrG negative type in accordance with 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 a gene for active pyrG into the A . soj a and pyrG negative in a way known per se to transform or transfect. The introduction step is then followed by selection of the resulting transformed or transfected A. soya in a free medium of uracil and fluoro- orotic, said means also comprising at least the minimum substrates required for the growth of A. soj ae, allowing said means that the A. Soj e desired to grow and at the same time 5 eliminate the growth of A. Soj is not transformed or transfected due to its inability to grow without the uracil caused by the gene for inactivated pyrG. In an appropriate embodiment of said method the gene for active pyrG which is introduces is flanked by identical nucleic acid sequence fragments and A. Soj e pyrG positive resulting from the introduction of the gene for pyrG and the flanking sequences is selected in a medium free of uracil and acid fluoro - orotic. Subsequently, the A is cultivated. soj ae pyrG positive in medium comprising uracil and fluoro-orotic acid, whereby the gene for pyrG that has been introduced is eliminated and in this way an A is obtained. soj a e pyrG negative that can be selected by growth in medium containing uracil and resistance to fluoroorotic acid. In an appropriate embodiment of the aforementioned method, the flanking sequences and the gene for pyrG are also flanked by sequences that direct the integration of the gene for pyrG and the flanking sequences at a specific site, due to the fact that the integration directing sequences are homologous to a specific sequence of A. Soj a e that will be transformed. This allows the deletion (if desired) of the gene associated with the specific sequence. The process of creating mutants with deleted genes as such is well known to the person skilled in the art. Any of the modalities of the selection method described above could comprise the step in which the Aspergillus is transformed or transfected with an additional heterologous nucleic acid sequence. The additional heterologous nucleic acid sequence preferably encodes A prstin or polypeptide and the same comments are valid here as in any of the other parts of the disclosure regarding the nature of such additional nucleic acid sequences for the other embodiments of the invention. Aspergi llus soj aey of filamentous fungi in general in accordance with the invention. The additional sequence can be introduced with the gene for active pyrG in the same vector or by cotransformation with the gene for active pyrG that is introduced. The method of selection for A. Transformed or transfected soya as described could also be carried out in combination with the method for introducing a nucleic acid comprising the introduction of a gene for heterod amdS in any of the embodiments according to the invention mf described above for the same. Of course, the invention encompasses any A. soybean and recombinant obtained using the selection method of A. 10 soj e transformed or transfected in accordance with it. The invention is also directed to a method for producing Aspergi l l us soya a and recombinante, said method comprising Transformation or transfection in a manner known per se from Aspergillus soj e pyrG positive with a nucleic acid sequence comprising the sequence to be introduced, which is flanked by sections of the gene for pyrG or the sequences corresponded to a sufficient length and homology to ensure recombination, eliminating the gene for pyrG and introducing the desired sequence, followed by selection of Aspergi l l us so y a and recombinante with the sequence desired by selecting with respect to A. soj a e with a pyrG phenotype negative. The determination of the corresponding sequences is within the scope of the person skilled in the art due to his knowledge of the hybridization procedures with nucleic acid sequences and the knowledge of the required sequence data of the genes for pyrG. In particular, the invention also covers # such Aspergillus sojae that presents the characteristics of Aspergillus sojae variant of amdS in accordance with the invention as defined above. In this way any strain of • Aspergillus sojae obtained by any of the introduction methods of amdS and / or pyrG according to the invention is a novel strain which falls within the scope of the invention and as is any subsequent use of such novel strain. Such a novel strain may comprise nucleic acid sequences that do not occur in • the corresponding Aspergillus sojae strain or even not present in Aspergillus sojae, in the genus Aspergilli or in filamentous fungi. The sequences may be of mammalian origin or they may be derived from any animal, plant or microbe. You can also express nucleic acid sequences that are present naturally in the Aspergillus sojae strain but which are present in a smaller number of copies in the corresponding untransformed A. sojae. In this way, the production of homologous proteins is also covered by the invention, when Aspergillus sojae strains of pyrG and / or amdS are involved according to the invention. A preferred embodiment is one in which the particular protein or polypeptide to be produced is absent in the corresponding untreated A. sojae and / or is present in a smaller number of copies in the corresponding untreated A. sojae, i.e. A. Sojae before the 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 to produce protein or polypeptide in a fungus thus covers both the precursor sequence for the strain and that which is foreign to the strain. Basically, only the original A. sojae not transformed or transfected from the protection is excluded. A production process comprises cultivating the fungus under appropriate conditions so that the expression of the desired sequence. The production process optionally includes the step of isolating the resulting polypeptide or protein in a manner known per se for the production of protein or polypeptide using filamentous fungi. Preferably the protein or polypeptide w will be secreted into the culture medium. A preferred protein or polypeptide is a protein or polypeptide that can degrade after the expression by Aspergi l l us ni ger or Aspergi ll us awamori. A number of such proteins and • polypeptides have been previously described in the prior art and a large number remains to be determined. However, this determination is a matter of routine for the person skilled in the art. Another preferred embodiment of the protein or polypeptide to be expressed is one in which the protein or polypeptide differs from a protease and amylase from Aspergi l l us so a e. One modality Preferred involves a protein or polypeptide that does not come from Aspergill us soj a e. A particularly interesting embodiment comprises a combination of the two methods for introducing nucleic acid sequences of according to the invention as described previously. The advantage thereof is based on the fact that the transformation frequency obtained with the pyrG marker is clearly much higher than that of the amdS marker. However, the secondary selection of the pyrG + strains that show the best growth in selective acrylamide plates allows the identification of those • Aspergillus and recombinant soybean strains that have the highest number of copies and therefore much more likely the highest level of gene expression.

• As indicated in the examples, homologous and heterologous sequences can be used that regulate the expression by Aspergi l l us soj a e, es say sequences that are presented Originally in the same strain or sequences foreign to the strain. Therefore, the transformants according to the invention can comprise any of said regulatory sequences. The selection of the region The appropriate regulator is a matter of choice that falls within the range of the normal skills of the expert in the art and will depend on the particular application. The regulatory sequences can be constitutive or inducible.

Regulatory sequences can be of origin fungal or non-fungal. In the examples, a wide range is illustrated. In the art, a large number of expression regulatory sequences are used on a regular basis for other systems, in particular filamentous fungal systems such as Aspergilli, and can be applied routinely without undue burden on the Aspergilli according to the invention. . To enter the desired nucleic acid sequences in Aspergillus sojae, any vector that is suitable for introducing nucleic acid sequences into fungal host cells can be used. Several examples are available in the art. In particular, vectors that have been shown to be suitable for transformation, transfection or expression in Aspergilli, such as Aspergillus niger, Aspergillus awamori and Aspergillus oryzae, can be applied appropriately. In addition to the above, the present invention describes the efficient production of protein for recombinant Aspergillus' sojae. Such efficient production is described in those strains that have a superior protease profile for ATCC42251 or at least as good as any of ATCC9362, ATCC11906 and ATCC2023B7. Thus the present disclosure reveals that some known strains of A. Soybeans are well suited as such for the production of proteins-, polypeptides and metabolites. These strains of Aspergi l l us so so ae have a lower proteolytic activity than the reference strain A. soj ae ATCC42251. In particular the two known strains ATCC11906 and ATCC20387 are well suited. The strains of A. Most preferred for the production of proteins, polypeptides and metabolites will be those that express a proteolytic activity equal to or less than that of the two preferred strains. Strain ATCC11906 - is the best modality of strains of A. They are deposited in the ATCC in accordance with the -an technique. Suitable proteins or polypeptides will be produced. Now that the present invention has allowed the introduction of nucleic acid sequences, it can serve to provide any protein or polypeptide of choice using an A. soj e as the host of expression. The present invention offers an improvement with respect to existing expression systems. Several of the existing protein production systems present problems of expression due to proteolysis. In particular, the novel system is better than the expression systems of Aspergillus niger and Aspergillus awamori frequently applied today. The present invention now makes it possible to provide a recombinant Aspergillus sojae comprising an introduced nucleic acid sequence encoding a protein or polypeptide to be expressed, said protein or polypeptide being susceptible to degradation after being expressed by A. niger or A. awamori. The invention also provides recombinant A. sojae comprising an introduced nucleic acid sequence encoding a protein or polypeptide to be expressed, said protein or polypeptide being different from the protease and amylase of A. sojae. A preferred embodiment is one in which the introduced nucleic acid sequence codes for a protein or polypeptide that does not belong to A. sojae. Said recombinant A. sojae strains also fall within the scope of the invention. In addition, the illustration of Aspergillus sojae strains that have been modified in order to intensify their appropriate character as expression hosts is also provided. These modifications they can reduce proteolytic activity as induced by any means. Specifically, the use of random mutagenesis by UV is illustrated. The specific mutation of one or more genes for protease is also illustrated. The means by which mutations can be introduced are commonly known to those skilled in the art, and thus numerous alternative modalities are readily available to reach the desired mutants. An appropriate embodiment is formed by mutants in which the alkaline proteolytic activity has been reduced. In particular, the elimination of the activity of the main 35 kDa alkaline protease to ensure the increased expression of proteins and polypeptides in specific form is illustrated. Specifically, the invention thus also covers novel strains which exhibit reduced proteolytic activity, specifically reduced alkaline proteolytic activity. Such strains can be obtained using any specific mutation route known or which can be conceived by the person skilled in the art. A preferred embodiment of such expression hosts exhibiting reduced proteolytic activity as described previously also comprises a selectable marker. In completely appropriate form the selectable marker will be amdS, pyrG or a combination thereof. In particular, the invention covers a method for producing deficient A protease mutants. Soj a and suppressing the gene for 35 kDa alkaline protease. There are numerous ways in which this can potentially be achieved based on the sequence data provided for this gene. In particular, a method using recombination with a pyrG selection marker linked to two flanking regions that present the crosslinking of the 35 kDa alkaline protease gene, whereby the resulting strain has the pyrG selection marker and suppresses the gene for protease 35 kDa alkaline, whereby the resulting strain has the pyrG selection marker and suppresses the 35 kDa alkaline protease gene is an elegant method. Subsequently, the "pyrG selection marker" can be eliminated, thus providing the Aspergi llus soj mutant alkaline protease -negative 35 kDa that can be used for expression purposes of any desired sequence that will be introduced therein. then the sequence which will be introduced can be incorporated in the previous steps either in the same vector as the pyrG marker or in a cotransformation event. In addition, a method in which a gene for protease other than the gene for 35 kDa alkaline protease will be deleted can be performed analogously. The analogous measures that will be taken are obvious to the person skilled in the art based on the illustration provided in the present invention in combination with the knowledge of other sequences for protease. The amdS selectable marker according to the invention can also be used analogously as described elsewhere in this description. As a further aspect of the invention, mutant filamentous fungi having improved fermentation characteristics are also provided. Specifically, the invention is directed to a filamentous fungus comprising a mutation that inhibits the activity of the pro-protein convertase or an equivalent protein. Numerous pro-protein convertases are known in the art. In particular reference is made to Figure 1 which provides the sequence data of a number of such proteins. A filamentous fungus in accordance with the invention is appropriately selected from Agaricus, Aspergillus, Trichoderma, Rhizopus, Mucor, Phanerochaete, Trametes, Penicillium, Cephalosporiu, Neurospora, Tolypocladium and Thielavia. Particularly suitable filamentous 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, more particularly it extends to Aspergillus sojae as defined above according to the invention, i.e. comprising heterologous nucleic acid sequences, for example in combination with the selectable markers amdS and / or pyrG. An appropriate equivalent of a pro-protein convertase is a protein or polypeptide having 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 cited in SEQ. ID No. 3 (= gene fragment that codes for the amino acid sequence of "pro-protein convertase from A. niger), SEQ ID No. 4 (= a partial gene fragment encoding the amino acid sequence of the pro-protein convertase from Aspergi ll us soj ae) or with either of the sequences indicated in SEQ ID Nos. 5 to 9. The functionally equivalent protein could appropriately have a nucleic acid sequence that can hybridize under stringent conditions to a nucleic acid sequence in accordance with SEQ ID Nos. 3 to 9. The conditions of astringent hybridization can be easily determined by the person skilled in the art. »An appropriate example of astringent hybridization conditions are hybridization at 50 ° C and preferably at 56 ° C and final washes at 3xSCC . PE4, PCL1 and PCL2 are specifically mentioned as examples of appropriate oligonucleotide mixtures corresponding to the coding strand (ie SEQ ID Nos. 10, 11 and 12). They are mentioned for the non-coding string PE6, PCL2-rev, PCL3 and PCL4 ie SEQ ID Nos. 13, 14, 15 and 16, respectively). The use of these primers in amplification procedures common in the art allows to obtain equivalent sequences and such use and the resulting sequences newly discovered and their application in the analogous manner described in the present description fall within the scope of the invention The sequences for which the oligonucleotides are prepared are well preserved as can be determined from the comparison of various sequences of amino acid for the proteins provided (see Figure 1) Any of the nucleic acid sequences having an equal or greater degree of identity, similarity or homology with the sequences provided in the present patent application for the relevant proteins or active parts of they are covered by the invention as well as the use thereof as primers or probes to discover other proprotein convertases or equivalent proteins that code for sequences and / or to subsequently introduce mutations in such protein coding sequences. makes reference to Maniatis et al. (1982) Molecular Cloning, AL aboratory manual, Cold Spring Harbor Laboratory, New York or any other manual on cloning and / or selection of nucleic acid sequences. The equivalent protein or polypeptide will exhibit the activity of a pro-protein convertase as that of the one having an amino acid sequence according to SEQ ID Nos. 3 to 9. The mutant fungus may comprise a substitution, insertion or deletion in the coding sequence of the pro-protein convertase or equivalent protein. The fungus, mutant may appropriately comprise a mutation in the regulation of the expression of the gene encoding the pro-protein convertase or equivalent protein. A mutant fungus according to the invention in an appropriate embodiment has reduced viscosity compared to the corresponding non-mutated fungus under equivalent culture conditions. A mutant fungus according to any of the foregoing modalities 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 increasing the production of a protein or protein. polypeptide under equivalent conditions compared to the corresponding wild-type fungus. It has been determined that the activity site for the pro-protein convertase of A. Soj a e is included within the sequences of amino acids 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 and any other heterologous protein or protein, falls within the scope of the invention. any other polypeptide, said method comprising culturing a mutant fungus in accordance with any of the embodiments above described. A method for obtaining the resulting protein or polypeptide either from the cell as such or after it is also included is also included. secret of it. It is covered by the invention the use of Any of the novel strains for transforming any nucleic acid sequence encoding a phytase or protein having phytase activity or any other protein or polypeptide thereof and any subsequent expression of Any nucleic acid sequence introduced therein and also optionally any subsequent process and / or secretion and / or isolation. Any sequence that codes for a protein can be used appropriately phytase or phytase protein or any protein or heterologous polypeptide. This can be of fungal or non-fungal origin. A preferred embodiment is formed by sequences encoding the acid-labile protein or polypeptide. In appropriate form the protein coding sequence codes for non-protease type proteins. The examples show a sequence of phytase and a number of appropriate heterologous sequences to be used in the transformation and also for the expression in Aspergi hosts l l u s soj e e. Additional examples of suitable proteins to be expressed are apparent to the person skilled in the art. The invention is also illustrated by the following examples. The examples should not be considered as restrictions for the interpretation of the field of the invention. Alternative modalities based on the description and knowledge of the relevant technology field can be easily contemplated. The content of the references cited in the description is incorporated for reference. The claims serve to illustrate the intended field of the invention.

EXPERIMENTAL DETAILS CONCERNING THE INVENTION Construction of a library of Aspergi l lus soj ae Genomic DNA from A. soj to e was isolated from protoplasts obtained from ATCC11906 using a previously described protocol (Punt, van den Hondel, 1992). After isolation, the DNA was extracted from the protoplasts using the protocol described by Kol et al. , 1988. Subsequently, the DNA was partially digested with MobI to obtain DNA fragments with an average size of 30-50 kb. The pAOpyrGcosarpl vector was constructed, which was used for the construction of the library, by ligation of a 3 kb BaüiHI-Hindl II fragment from pANsCosl (Osiewacs, 1994) and a 3.2 kb Acc65 I-HindI II fragment from pA04.2 (De Rui ter-Jacobs, 1989) in? cc65I-.Ba-mHI digested with pHELPl (Gems et al., 1991). This cosmid vector carries the selection marker pyrG of A. oryzae and is self-replicating in filamentous fungi. Genomic DNA digested with MobI was ligated to pAOpyrGcosarpl digested with BamHl, and the ligation mixture was packed into phage particles using the Stratagene Supercosl vector kit. In total, 30,000 individual clones were obtained representing approximately 30 times the genome of A. soj a e. The stocks (in 15% glycerol) of the resulting clone mixtures were stored at -80 ° C for later use.

Transformation method with amdS and transformants Two protocols were evaluated to form protoplasts currently in use and transformation protocols [the modified OM method (Yelton et al., PNAS 81 (1984) 1470-1474) and the sodium chloride method (Pun and Van del Hondel, Meth. Enzym. 216 (1993) 447-457)] in strain ATCC9362 of Aspergi ll us soj ae. Both methods resulted in protoplasts, the yield of reliable protoplasts with the OM method was clearly better. The total yields were lower than those normally obtained for A. ni ger. A pilot experiment was carried out to form protoplasts / transformation with all strains of TO . Soj a e using the OM method. For the transformation, the vector p3SR2 (carrying the amdS marker) was used in combination with pAOpyrGcosARPl. This last vector is a derivative of the Arpl vector of Aspergillus that replicates autonomously, which in all the Aspergillus species evaluated so far gave as results quite increased numbers of (unsta transformants when used as a vector of contransformation. Almost for all strains sufficient protoplasts were obtained (approximately 10E6-10E7 per transformation). The analysis of the selection conditions of AmdS appropriate for the different strains of A. soj ae revealed the vigorous growth of most strains in the selective acetamide medium used in common form. Clearly, the selection conditions with acetamide proposed for the amdS transformants of A. Soj as reported in WO97 / 04108, were not appropriate for the selection of A transformants. soj a e. The experiments reveal, surprisingly, that the AmdS + transformants could not only be selected with the acrylamide selection. Even in acrylamide plates selective, a considerable background is observed coming from the non-transformed protoplasts. The selection of primary transformants requires about three weeks and many of the putative transformants initially selected turn out to be false positives, demonstrating only background growth after transfer to new selective acrylamide plates. To optimize the selection of transformants attempts have been made to reduce this background growth. Improved results were obtained by omitting the glucose from the selective plates. In Table 3, the composition of the improved selection means and the usual means are indicated. Figures 2a, b and c demonstrate the background growth observed for the selected strains in the selection medium described in WO97 / 04108 and the selection medium with improved acrylamide described in Table 3. Further transformation experiments with the three strains of TO . Selected soybeans reveal that the efficiencies to form protoplasts from ATCC119-06 and ATCC20387 were better using the sodium chloride method. Successful protoplast formation is obtained using various enzyme preparations to form commercially available protoplasts such as NOVOZYM, Caylase, Glucanex, etc. Based on the transformation protocol with sodium chloride, the three strains of A were transformed. Soj e are selected with the selection vector p3SR2 for amdS or derivatives thereof. Using the selection plates - with modified acrylamide a number of transformants with vigorous growth was obtained, while no growth was observed in the control transformation in DNA. Another method to overcome the background growth of untransformed mycelium is the elimination of the gene activity for amdS from A. soj a e wild type. This can be achieved for example by disrupting the gene for amdS of A. soj ae As a first step, specific DNA fragments carrying the sequences for amdS of strain ATCC11906 are amplified by PCR using primers derived from the sequences for amdS of A. published oryzae (Gomi et al., -1991, Gene 108, 91-98). Previous experiments have shown that cloning using astringent hybridization may not be successful due to a low level of sequence conservation between the sequences for mdS of A. nor sweet ans and A. soj a e. The expected fragment of approximately 1.6 kb was obtained, which could carry the majority of the coding region for the amdS gene. Sequence analysis from both ends of the cloned PCR fragment (Figures 3a and 3b) confirmed the cloning of a part of the gene for amdS from A. soj a e. The astringent hybridization was presented at 56 ° C with final washes at 3xSSC. The cloned sequence was very similar to the sequence published for amdS of A. oryzae Several hybridization clones (7 of 10,000) were isolated from the cosmid library for ATCC11906 in pAOpyrGcosarpl using the amdS fragment of ATCC11906 co or a probe. After subcloning a fragment carrying the complete amdS gene, one part of the gene for amdS was replaced with a reusable pyrG selection marker to generate an amdS replacement vector. Transformation of this vector to Aspergillus ATCC119CL-6PyrG ll soya e resulted in pyrG + transformants. After further analysis of these transformants on acetamide and acrylamide selection plates, several of these transformants demonstrated reduced background growth. He Southern analysis of a few of these strains revealed that the expected gene replacement occurs. One of these strains was used for the subsequent transformation with the amdS gene of A. nor dul ans using selection plates with acrylamide and resulted in a number of amdS + transformants.

Transformation method with pyrG and transformants 1) Initial experiments For A. soj a e, standard experiments used in the prior art for other fungi to generate pyrG mutants as described in the introduction resulted in numerous strains resistant to fluoro-orotic acid (FQA). However, all these strains were able to grow in medium without uridine and therefore are not considered as pyrG mutants. With the ultimate goal of isolating the appropriate mutant strains a number of alternative methods were followed. 2) Disruption of gene near homology Based on the expectation that the genes for pyrG from _ A. soj a e y A. oryzae have very similar sequences (which was confirmed by Southern hybridization performed under stringent conditions), experiments were performed to upset the gene for pyrG of A. Soj e with a mutant version of the gene for pyrG from A. oryzae using a method previously described by Gouka et al. (nineteen ninety six) . The astringent hybridization is presented at 65 ° C with final washes at 0.3 x SSC. A pyrG disruption vector of A. oryzae was constructed in which a 0.5 kb Clal fragment carrying part of the coding region for pyrG was deleted (figure 4). The Xbal pyrG fragment from this new vector was used for transformation and direct selection with respect to FOA-resistant transformants. None of the FOA-resistant colonies obtained is uridine-dependent. 3) UV mutagenesis and enrichment by filtration Another method to improve the performance of specific mutant strains is the use of a step of enrichment by filtration (Bos et al., 1986, Thesis, Agricultural University Wageningen). Spores subjected to mutation with UV are used to inoculate a liquid culture in minimal medium (MM). Spores that can not germinate in minimal medium (spores of mutants pyrG a. Or.) Are separated from the culture repeated throughout the night resulting from the mycelium developed by filtration through myracloth. The spores obtained after several enrichment steps are evaluated with respect to their PyrG phenotype, inoculating the spores in plates containing FOA. Again, none of the resulting FOA resistant colonies was uridine dependent. In addition none of the colonies obtained after this enrichment in MM plates containing uridine proved to be uridine dependent. 4) Modified selection conditions Previous attempts to isolate pyrG mutants from A. Soj e could not suggest the lack of capacity of the pyrG mutants required to use exogenous uridine, which is used in the FOA selection medium for the analysis of auxotrophy to uridine. A modified selective FOA medium is used, which on this occasion contains uracil followed by uridine, in a new attempt at isolation. From this one In this method, several FOA-resistant mutants are obtained, which are dependent on uracil. The revaluation of these strains showed that they can not grow in minimal medium supplemented with uridine. Subsequent transformation experiments with some of the uracil dependent strains demonstrate that these mutants can indeed be complemented with a gene for fungal pyrG (eg the vector pAB4.1; pyrG from A. ni ger). The lack of ability of the mutants to grow in minimal medium supplemented only with uridine was an unprecedented observation for the related Aspergi species (A. ni dulans, A. ni ger, A. oryzae) and various other fungal species.

) Reusable selection marker The versatile genetic modification of A. Soj e requires the possibility - to modify, disturb and express a number of different genes in a single fungal strain, which may require the availability of one (series) of different selection markers. However, the availability of a marker such as pyrG, which allows the selection of both the mutant (selection with FOA) As the transformant (medium without uracil), it offers the possibility of repeated use of the same marker in subsequent experiments. For this method a gene for the pyrG marker was designed, in which the complementary sequence is flanked by a direct repeat sequence that originates from the 3 'flanking end of the gene for pyrG. The resulting plasmid is pAB4-lrep. The construction of this vector is described in detail in Figure 5. The complete sequence of the vector is given in SEQ ID No. 17. The transformation of the pyrG mutants of A. soj ae results in a similar number of transformants PyrG-t- that with the vector pAB4-l. Nevertheless, the subsequent plating of spores of the transformants pAB-4-1 and pAB4-lrep selected in FOA selection plates results in many more FOA / uracil dependent colonies for the transformant pAB4-lrep. Southern analysis of these FOA / uracil dependent clones demonstrates that in most strains pAB4-lrep the gene for the pyrG marker of A. niger has been deleted leaving only the 0.7 kb repeat region in the integration locus, whereas in the strains pAB4-l the gene for A. niger is still present and presumably acquires a mutation that results in the pyrG-negative phenotype.

Expression hosts: selection of the strain Protease production The very important characteristics of a fungal expression system are the level and type of fungal proteases produced under various culture conditions. Sometimes strains that can be easily transformed are not suitable as expression hosts due to the production of proteases or acidification of the culture medium which is detrimental to the expressed product. The analysis of the growth behavior of the various strains of A. Soj e reveals that, in contrast to what is observed for A. niger, the acidification of the culture medium does not occur either on agar-based plates (MacConkey) or in shake flask culture. In fact, in cultures with shake flask, without taking into account the three types of medium analyzed (table 4), in most cases in crops an even alkaline pH. Based on these results and data from the literature, it is therefore expected that alkaline proteases will mainly be present in the culture fluid of A. soj ae To analyze the protease activity of the culture fluids of the various strains, a milk elimination test is carried out. Samples are incubated in addition medium with different proteins (for example bovine serum albumin (BSA)), and the degradation of these proteins is followed over time in order to evaluate the appropriateness of the strains evaluated as expression hosts for a range of products. BSA was chosen in the previous experiments with A. niger This protein proves to be very susceptible to proteases. The phytase of A was chosen. t erreus as an example of another proteolytically unstable protein. The degradation of milk proteins, as demonstrated by the formation of a milk removal zone at the periphery of the growing colonies, is a generally accepted criterion regarding protease activity. The detection of BSA is performed by Coomassie staining of SDS-PAGE gels. For the phytase, Western analysis was carried out using specific antibodies. As shown in Table 4, the clear differences of degradation in the A. niger culture fluid are evident when this is compared to that obtained in the culture fluid of A. sojae. A rapid degradation of BSA occurs in the culture fluid of A. niger (pH 3-4). In the culture fluids of A. sojae from more enriched media degradation of BSA occurs, although to a lesser extent than in the culture fluid of A. niger. Rapid degradation of A. terreus phytase occurs in most culture fluids of A. sojae (pH 7-8), with the exception of the culture fluids of ATCC9362, ATCC11906 and ATCC20387. In general, strains with the lowest degradation of phytase also demonstrate a minor degradation of BSA under the conditions evaluated. In particular, the two strains ATCC20235 and ATCC46250 of A. oryzae demonstrate a much higher proteolytic activity than most strains of A. sojae. To exclude that the differences in the pH of the culture fluid cause the observed effects, experiments of similar degradation with culture fluids in which the pH is 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 table A. oryza e ATCC20235, which exhibits the highest proteolytic activity "at pH 7-8 also demonstrates high proteolysis at other pH values." The degradation of A. terreu s phytase occurs mainly at pH 8. In a similar way to what previously found, ATCC11906 and ATCC20387 demonstrate a low phytase degradation activity, ATCC9362 presents degradation of phytase in enriched media.Degradation of BSA by A. soj ae does not present significant differences with the data presented in table 4J In conclusion, these protease tests result in the identification of three strains of A. soj ae with low protease content, in specific ATCC9362, ATCC11906 and ATCC20387. Therefore, A can be clearly used. Soj e as host of expression for a range of proteins and provides a series of advantages with respect to the transformation and expression systems of the prior art.

Improvement in the strain Once the potential for transformation and expression capacity for Aspergillus soya e is established, means are considered through which additional strains can be created with improved characteristics with respect to expression. Two different methods were developed which can be used alone or in combination to provide novel improved strains regarding protein expression. On the one hand, the possibility of developing protease-deficient mutants and the impact of this on expression levels was investigated. On the other hand strains with amended morphology were developed with the intention of improving - the fermentation characteristics. In order to obtain this, a route was followed that until now has not been described nor suggested, which can be applied not only to Aspergillus soils but also to Aspergillus and, in fact, to filamentous fungi in general.

Development of protease-deficient mutants To obtain strains of A. soj e e deficient Two methods are followed in protease. In a first method spores from strains derived from ATCC11906 and ATCC11906 with UV are subjected to mutation. In a second method, the gene disruption of the main alkaline protease is effected.

JSF Mutants by UV Freshly harvested spores from A are mutated. soj a e ATCC11906 or one of its pyrG derivatives with UV in a chamber for UV Biorad with a dose that results in 20-50% survival. Serial dilutions are plated on plates with skim milk (Mattern et al., 1992). From 5,000 surviving strains of UV four mutant strains with a substantially reduced milk elimination halo are obtained.

Disruption of the AlpA gene In this method the endogenous gene is disrupted alpA (alkaline protease) from ATCC11906 using a disruption vector carrying the selection marker pyrG usable as described in this description. A cosmid library of ATCC11906 (in a pyrG cosmid) is constructed. Starting from 10,000 independent cosmid clones it was initially found that 4 of these hybridize under homologous conditions with an alpA fragment of A. soj ae obtained by PCR with primers MBL1784 and MBL1785. Reselection of the four clones revealed only strong hybridization with one clone. A 4 kb EcoRI fragment and a 2.5 kb HindlII fragment from this clone, which are expected to carry the complete gene together, were subcloned and characterized by restriction enzyme digestion and sequence analysis. Based on these subclones, a new gene replacement vector is constructed as described in figure 6. For the transformation of an ATCC11906pyrG derivative, the vector is digested with BcoRI, and the 8.7 kb alpA deletion fragment is used for the transformation. (see figure 6). The transformation of the replacement cassette to ATCC11906pyrG5 results in a number of transformants with a reduced milk halo. Southern analysis of these strains reveals the successful deletion of the gene for alpA. To allow the subsequent use of the ^ p rG marker for the transformation of one of these strains, spore plates of this strain are seeded in selective medium that contains FOA for the pyrG mutants. From the strains with the disruption cassette integrated in a corrected manner, the reusable pyrG marker yields a large number of FOA-resistant colonies. In contrast to the results obtained for the spontaneous FOA-resistant mutants of wild type strains, the FOA strains obtained from these disruption strains were virtually all dependent on uracil and again turned out to be pyrG negative. Southern analysis was used to confirm the desired removal of the pyrG marker gene at the alpA locus, leaving only the "footprint" of 700 bp.

Analysis of protease activity in UV mutants and by disruption _ To analyze the levels of low protease production in the different protease derivatives of ATCC11906, controlled intermittent fermentation experiments were carried out. The protease activities at various pH values were determined from the resulting culture supernatants. The deletion of the alpA gene results in a strong reduction of proteolytic activity at alkaline pH. The analysis of Protease activity in one of the UV mutants showed an almost complete absence of tannoly proteolytic activity "at * pH '6 as at pH 8. Accordingly, the level of proteolysis towards the secreted proteins produced in these strains was considerably less than the observed for the precursor strain Development of mutants with low viscosity Controlled or intermittent intermittent fermentation with initial feeding with A. Soya e resulted in a considerable biomass yield, however both the viscosity of the culture and the sporulation phenomenon in the fermenting vessel represented less favorable characteristics. Therefore attempts are made to improve these characteristics in the desired host strain. Several patent applications teach that loamy viscosity mutants can be isolated using various forms of selection. WO96 / 02653 and WO97 / 26330 describe undefined mutants having low viscosity. However, in the present invention an unexpected novel case of a low mutant is described.

Fully defined and fully characterized viscosity from A. soj a e. It was found that a mutant that processes pro-protein from this organism has an unexpected aberrant growth phenotype (hyperramification) while no deleterious effect on protein productivity is observed. Fermentation experiments controlled with this strain reveal that increased biomass concentrations are obtained with considerably lower viscosity values. The observed characteristics were not only present in A. soj a e but also in other mushrooms, for example in A. nor ge. 1) Construction of a mutant that processes pro-protein from A. ni ger _ To clone the gene coding for the pro-protein convertase from A. In addition, several heterologous hybridizations were carried out using specific probes for the genes from Sa ccharomyces cerevi si ae KEX2, Schi zosaccharomyces pombe KEXl and Xenopu s l a evi s PC2. However, no signals were obtained specific hybridization even at conditions of very low astringency hybridization (47 ° C, washed at 6xSSC), which avoids the use of this method to clone the A gene. corresponding niger. PCR was used as an alternative method to clone the gene coding for pro-protein convertase from A. Niger. Based on the comparison of several genes for proprotein convertase from several yeast species and higher euricariontes (figure 1), different PCR primers were designed (SEQ ID Nos. 10, 13 6 18-23), which are degenerate, respectively , 2048, 49152, 4, 2, 2, 512, 2048 and 4608 times. From the amplification using the primers PE4 and PE6, two individual clones were obtained in which the encoded protein sequence shows significant homology with the S sequence. cerevi si ae KEX2 (SEQ ID No. 24). These clones were used for subsequent experiments. Based on the observed homology with respect to other pro-protein convertase genes of the cloned PCR fragment, the A gene was designed. nor ger corresponding pclA (of pro-protein convertase). The Southern analysis of genomic digests of A. niger reveals that the pclA gene was a single copy gene with no closely related genes in the genome of A. nor ger, even at astringent hybridization conditions (50 ° C, washed at 6xSSC), no further hybridization signals were evident. A first selection of an EMBL3 genomic library of A. niger N401 (van Hartingsveldt et al., 1987) does not result in any positive hybridization plates although approximately 10-20 genomic equivalents were selected. In a second selection, a full-length genomic copy of the pclA gene was isolated from the genomic library of A. niger N400 in EMBL4 (Goosen et al., 1987). Of the 8 hybridization plates that were obtained after selecting 5-10 genomic equivalents, 6 were left after a first reselection. All these 6 clones probably carry a complete copy of the pclA gene, because they are present in all clones (as observed for genomic DNA) with the EcoRV fragments that hybridize to the 3 4 kb PCR fragments (caution that the PCR fragment (SEQ ID No. 24) contains an EcoRV restriction site). Based on a comparison of the size of other pro-protein convertases, these fragments together contain the complete pclA gene with 5 'and 3' flanking sequences. The two EcoRX fragments in an overlapping 5 kb EcoRI fragment were subcloned for further characterization. A detailed restriction map of the DNA fragment carrying the pclA gene is shown in Figure 7. Based on the restriction map given in Figure 7, the complete DNA sequence of the pclA gene is determined from the EcoRI and EcoRV subclones (SEQ ID No. 3). The analysis of the obtained sequence reveals an open reading frame with a considerable similarity with that of the KEX2 gene of S. cerevi si ae and other pro-protein convertases. Based on subsequent comparisons, two putative intron sequences were identified (SEQ ID No. 3, from position 1838 to 1889 and from 2132 to 2181) in the coding region. The subsequent PCR analysis with the primers flanking the putative introns, in a cDNA library of A. niger based on pEMBLyex revealed that only the sequence with the 5 'end of these two sequences represents a current intron. The general structure of the encoded PclA protein was clearly similar with that of other pro-protein convertases (SEQ ID No. 25 and Figure 8). The general similarity of the PclA protein with the other pro-protein convertases was approximately 50% (Figure 1). To demonstrate that the cloned pclA gene is a functional gene coding for a functional protein, the construction of strains lacking the pclA gene was attempted. Therefore, a pclA deletion vector, pPCLlA, was generated in which a large part of the coding region of pclA is replaced with the pyrG selection marker gene of A. oryza e. Subsequently, from this vector, the fragment of the EcoRI insert of 5 kb was used for the transformation of various strains of A. niger Starting from these transformations (based on the pyrG selection) numerous transformants are obtained. Interestingly, a fraction of the transformants (ranging from 1 to 50%) showed a different aberrant phenotype (Figure 9). Southern analysis of several wild-type and aberrant transformants reveals that these aberrant transformants, which display a severely restricted growth phenotype, have lost the pclA gene. All strains showing wild-type growth demonstrated carrying a copy of the integrated replacement fragment adjacent to the wild-type pclA gene or in 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-1 fusion protein in a pclA mutant was achieved. Protein analysis revealed that completely mutated endogenous glucoamylase is not formed in the pclA mutant strains but only pro-glucoamylase. In order to further improve the yields of the fusion protein, intermittent and intermittent fermentation with feeding is also carried out. Surprisingly, the fermentation characteristics of the pclA mutant strains were clearly superior to those of the precursor strain, which results in a reduced viscosity / biomass ratio, without loss of productivity. 2) Construction of a mutant to process pro-protein of A. sojae To construct the corresponding mutant in A. sojae, the functional complement of the low viscosity mutant of A. niger, genomic cosmids are isolated from the cosmid library ATCC11906 , which comprises the pclA gene for pro-protein processing of A. sojae. The partial sequence analysis of the isolated sequence SEQ ID No. 4 confirms the cloning of the pclA gene of A. sojae. Figure 10 demonstrates the comparison of the DNA sequences of a part of the pclA genes of A. niger and A. sojae. Based on the sequence of A. sojae and a partial restriction map with the coding region of the pclA gene of A. sojae, a replacement vector is generated using the EcoRV site in the pclA gene of A. sojae to clone the reusable pyrG marker as an inner Smal fragment (figure 11). The resulting vector is partially digested with Clal to obtain the 10.5 kb delta-pei fragment (see Figure 11). This fragment is isolated to be used in the transformation of the pyrG strains of A. sojae. The gene replacement vector was used to generate pclA mutants in derivatives of ATCC11906 and ATCC11906. The resulting strains were used for the expression of homologous and heterologous proteins. The 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 homologous fungal genes with the pclA gene of Aspergi l l us ^ __ Based on the comparison of amino acid sequences inferred from the pclA genes of A. niger and A. Soj e with those of other pro-protein processing enzymes (Figure 1), several mixtures of oligonucleotide corresponding to the coding or non-coding strand of well conserved sequences were designed (SEQ ID Nos. 10 to 16). These oligonucleotide mixtures were used in PCR with chromosomal DNA from Tri choderma reesei and QM9414, Fusarium um venom tum ATCC20334, Peni ci l i um chrysogenum P2, Trame tes versi color, Rhi zopus oryzae ATCC200076 and Agari cus bi sporus HORST. Depending on the DNA template used, the amplifications with PCR (30 cycles, 1 minute 94 °, 1 minute 40 ° C, 2 minutes 68 ° C) with one or more combinations of coding and non-coding oligonucleotide chains resulted in specific PCR products . Table 6 gives the results of the various amplification reactions. Sequence analysis was performed with a number of the PCR fragments obtained from either of the oligonucleotide mixtures used for amplification. These analyzes resulted in the identification of various pclA homologs from these different fungi. Figure 12 indicates the inferred amino acid sequences corresponding to the various DNA fragments (SEQ ID Nos. 5 to 9). 4) Example of determinations of biomass and viscosity __ ... _. The following ranges of operation parameter data for fungal fermentations were determined using a number of different fungal strains.

Viscosity Viscosity is determined in a Viscotester VT500 from Haake using the MV DIN detector system (container number 7), operated at 20 ° C. A new sample of fermentation broth is obtained and 40 ml of the broth are placed in the measuring cell. The viscosity is measured after a small period of equilibrium (4 minutes) at a fixed spindle speed. This measurement is repeated for 10 different speeds of use. The multiplication of the speed of use with the cell measurement factor results in the tangential stress velocity. The viscosity? (in centipoise = cp) is plotted against the speed of tangential stress? (l / s) and allows obtaining the viscosity profile of the fermentation broth. The viscosity ranges for the fermentations were determined using the fungal organisms specified using the previous procedure (Table 7).

Biomass Biomass is determined by the following procedure: 5.5 cm filter paper (Whatman 54) is tared on an aluminum tray for weight. 25.0 ml of full broth are filtered through the 5.5 cm paper in a Buchner funnel, washed the filter cake with 25 ml of deionized water, place the washed cake and the filter in a tray for weight and dry overnight at 60 ° C. The drying is concluded at 100 ° C for 1 hour, then it is placed in a desiccator to cool it, the weight of the dry material is determined, the total biomass (g / l) is equal to the difference between the initial and final multiplied by 40.

Protein Protein levels are determined using the BioRad test procedure. The data presented above represent values determined 48 hours in the fermentation process until it ends; All values of Aspergillus and Tri-choderma are for fungal organisms of commercial relevance and reflect the actual commercial data. A fungal strain such as A. soj a e l fvA y A. soj ae pclA has the advantage that the low viscosity allows the use of a lower power supply and / or a tangential stress in the fermentation to meet the oxygen demands in those cases in which the shear stress on the product could be harmful to productivity due to physical damage to the product molecule. The production of biomass less than a higher protein production indicates a more efficient organism in the conversion of the fermentation medium to product. Therefore the mutants of A. soda ae provide better biomass and viscosity data and at the same time also supply at least the same amount of protein, and in fact a much higher amount of protein than the two commercially used systems which obviously are better than for the strains typically deposited from Aspergi ll use Tri choderma reesei in general public deposits. High protein production with low biomass concentration produced by A. soj ae lFvA could allow the development of fermentation conditions with multiples of increment higher in biomass, if the increasing biomass results in an increased productivity, for the desired product before reaching the limiting conditions of the fermentation. The high levels of biomass and viscosity produced by the present invention by T organisms. l ongibra chi a t um y A. ni ger they restrict the increase of biomass because the current levels of viscosity and biomass are close to the conditions practically limiting the fermentation.

Efficient expression of gene 1) Heterologous regulatory sequences The three strains of A were cotransformed. Soj e were selected with three GUS reporter vectors that carry different fungal expression signals (A. no PgpdA ans, pGUS54, A. niger PglaA, pGUS64, A. or ger PbipA, pBIPGUS) and p3SR2 vector for selection of amdS or derivatives thereof. The expression of the GUS gene in representative transformants was analyzed (Table 8). From these results it is clear that under the evaluated conditions the gpdA promoter was by far the best promoter resulting in approximately 5000 U of GUS / mg of protein. This corresponds to approximately 5% of the total amount of cellular protein. The bipA promoter results in approximately 30% of the activity of gpdA, which corresponds to expression data obtained in A. niger Surprisingly, the gl aA 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. soj a e. 2) Regulatory sequences of A. The homologous promoter of Aspergi ll us soj ae was also isolated and the ability of its application in an expression system was evaluated. In some cases of expression it will be preferable to use a homologous promoter instead of a heterologous promoter. It is also interesting to evaluate if the homologous promoter will be more efficient or not than a heterologous one. The cloning by PCR of three A genes was attempted. Soj a and expressed in an efficient way, in specific alpA (alkaline protease, inducible), amyA (amylase, inducible) and gpdA (iceralde dodo-3-phosphate dehydrogenase, constitutive) using primers based on the available sequences from A. oryzae (SEQ ID Nos. 26 to 31). Figures 13a, b and c give the sequences and position in the published sequences of A. oryza e of the various PCR primers used for this method. Genomic template DNA from A is used. soj ae ATCC11906 for PCR amplification. The initial PCR amplifications (30 cycles, 1 minute 94 ° C, 1 minute 40 ° C, 2 minutes 68 ° C) result in a specific PCR product of the expected size (400 bp) for the gpdA. No product is obtained for the other two PCR reactions. Therefore, for alpA, the PCR conditions were less astringent (10 cycles, 1 minute 94 ° C, 1 minute 25 ° C, 2 minutes 68 ° C + 20 cycles, 1 minute 94 ° C, 1 minute 40 ° C 2 minutes 68 ° C), which resulted in a specific lpA PCR product of approximately 1000 bp. The complete sequence of the cloned genes was determined. As shown in Figure 14, the gpdA promoter region of A. Soj e ATCC11906 has a very high homology with other gpdA promoter sequences and the promoter at lpA was virtually identical with the alpA promoter of A. oryza e (SEQ ID Nos. 32 and 33). Expression vectors carrying expression cassettes comprising these A promoters. Soybeans demonstrate significant levels of gene expression.

Production of heterologous protein A number of proteins were evaluated heterologous which are known to be susceptible to proteolysis in acid medium and therefore could not be expressed efficiently in other well-known expression systems. Proteins that are efficiently expressed in alternative systems were also evaluated in order to evaluate, as a comparison, the expression levels obtained with Aspergillus so as a compared to other known expression systems such as Aspergi l ls u 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 BspHI sites introduced into the blunt end fragments of the ATG codon. -3 'to the 3' end of the gpdA promoter of A. ni du l ans in pA? 52-l? otI. The resulting vectors were used in A cotransformation experiments. Soj e using the selection marker amdS and / or pyrG. The phytase-producing transformants were selected using the plate test for phytase described. Expression vectors were generated additional improved phytase using a multiple copy cosmid method. In this method, several copies of a phytase expression cassette are cloned back into a multiple cloning site vector (pMTL series, Chambers et al., (1988) Gen 68, 139-149) to allow its isolation as a EcoRI fragment. Several copies of these EcoRI fragments were cloned into the cosmid vector pAN4cosl through packaging (Verdoes et al (1993) Transgenic Research 2, 84-92), which results in cosmid clones carrying a number of expression cassettes. . The resulting clones are introduced in A. soj e using the amdS selection marker. AmdS + clones are selected for phytase production using the phytase plate test. Additional expression vectors for phytase were generated using the GLA fusion method (eg Broekhuij sen et al (1993) J. Biotech, 31, 135-145). For this purpose fragments of the gene for phytase are fused, which code for the mature phytase protein of A. fumigate your, using convenient restriction sites and fusion PCR, to the 3 'end of the carrier gene glucoamylase in vector pAN56-l (access number Z32700 of Genbank). A sequence encoding a pro-protein processing site (Asn-Val-lie-Ser-Lys -Arg) is introduced between the glucoamylase part and the phytase part of the gene fusion. The resulting vectors were used in A cotransformation experiments. Soj e using the selection marker amdS and / or pyrG. The phytase-producing transformants were selected using the plate test for phytase described. The fermentation was performed in stirred flask resulting in significant levels of active phytase. The yield was significantly higher than those reported in the 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 multiple copy cosmid vectors were higher than those obtained with the single copy vectors. The levels of phytase obtained with the glucoamylase-phytase fusion vectors resulted in high levels of both glucoamylase and phytase. Intermittent and intermittent fermentations fed controlled from a selected number of transformants of A. Soya phytase producers revealed an even higher level of phytase.

Glucoamylase production An example of a fungal protein produced efficiently by the expression of the glaA gene of A. nígrer is provided. The pGLA6S vector (Figure 15) is derived from pGLA6 (Punt et al (1991) J. Biotech 17, 19-334) by introducing a 5 kb EcoRI fragment carrying the amdS gene of A. ni dulans as the selection marker in the unique EcoRI site of pGLA6. The vector pGLA6S (FIG. 15) carrying the amdS selection marker and the glucoamylase gene under the control of the gpdA promoter from A. nidul ans is introduced into A. soj and ATCC11906pyrG using cotransformation with the vector pAB4.1. Plaque starch tests demonstrate the production of increased levels of amylolytic activity in these transformants. From the resulting transformants, those that show a proficient growth in acrylamide medium with respect to glucoamylase production are analyzed. On a SDS PAGE gel dyed with blue Coomassie brightness from the culture supernatant of some of these transformants shows a single band of dominant protein corresponding to glucoamylase. Western analysis was used using a monoclonal antibody against glucoamylase (Verdoes et al (1993) Transgenic Research 2, 84-92) to confirm the identity of this protein band.

Production of interleukin 6 The production of interleukin 6, which is an example of a protein quite sensitive to 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 IL-6 yields were only a few mg per liter of culture fluid. The introduction of IL-6 vector pAN56-4 (Broekhui j sen et al. (1993) J. Biotech 31, 134-145) in A. soj ae by cotransformation with the pyrG or amdS marker resulted in transformants expressing the IL-6 fusion gene present in this vector. Starting from the resulting transformants, some were selected how many for later analysis? Fermentation experiments were carried out in stirred flask with these transformants. The SDS-PAGE and Western analyzes of the culture supernatants of several of these strains showed surprisingly well-processed levels of IL-6 that were approximately 5-10 times higher than the levels obtained in the best cases reported in A. ni ger. The use of the various types of mutants protease deficient and with fermentation optimization from A. Soj e also increased the level of IL-6 production that will be obtained from controlled fermentations (Broekhui j sen et al (1993) J. Biotech, 31, 134-145).

Green fluorescent protein (GFP) Another type of acid labile protein that was tried to produce in -A. soj ae is GFP from the jellyfish Aequoria vi c toria. This protein is not only proteolytically sensitive but also loses its activity at acid pH values. Vectors were introduced that carry the GFP fusion genes or GLA-GFP (controlled by the giddA promoter of anidula) in A. soj ae by cotransformation using any of the selection markers pyrG or amdS. The expression resulted in bright fluorescent A. soj transformants for both types of vector. Based on the observed fluorescence and subsequent analysis of culture supernatants from transformants grown in shaken flask selected using SDS-PAGE and Western analysis, it was established that the yields of cytoplasmic GFP and intact secreted GLA-GFP are much higher than those obtained in the protease deficient hosts of A. niger (Siedenberg et al .. Biotechn Prog (1999) 15, 43-50; Gordon et al., Microbiology (2000) 146, 415-426). In contrast to the situation in the culture supernatants of A. Niger also secreted GFP showed significant fluorescence.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: this figure provides a comparison of amino acid sequences of KEX2-type processing proteases from X. laevi s (XENPC2 and XNFURIN), S. cerevi siae (SCKEX2), K. l a c ti s (KLKEX1), C. albi cans (CAKEX2), 17 S. pombe (SPKRP) and Y. l ipolyti ca (YLKEX2). Initiators are indicated, which code for the amino acid sequences with the highest overall identity (indicated by boxes in light blue): MBL793, MBL1208, MBL7"94, MBL1158, PE6, PCL1, PCL2 (rev), PE6, PCL3 , MBL789, PCL4 and MBL1219. Global identity regions (4 of 7 entries) are indicated by purple boxes, spaces are indicated by., Non-sequence data are indicated by ~, asterisks indicate stop codon. of the protein Figure 2: this consists of figures 2a, b and c Figure 2a provides the background growth of the strain of A. soj ae described in patent WO97 / 04108 after 5 days of incubation at 33 ° C. the upper image reveals the growth in a non-selective medium. The lower left image shows the selection means according to WO97 / 04108 and the lower right image presents the results using improved medium (acrylamide) according to the invention. Figure 2B provides the background growth of the strain of A. Soj e ATCC11906 after 5 days of incubation at 33 ° C. The upper image reveals the growth in a non-selective medium. The lower left image presents the selection means according to WO97 / 04108 and the lower right figure presents the results using the improved medium (acrylamide) according to the invention. Figure 2C provides the background growth of the A strain. Soj e ATCC20387 after 5 days of incubation at 33 ° C. The upper image reveals the growth in a non-selective medium. The lower left image presents the selection means according to WO97 / 04108 and the lower right image presents the results using the improved medium (acrylamide) according to the invention. Figure 3 (a and b) -. this figure provides a comparison of A. soj to ATCC11906 and the amdS sequences of A. oryzae from both ends. A and B indicate the two extremes. The cloned sequence of A was used. soj ae of 1.6 kb. The bases in bold and underlined 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 a through pA04-13 and pA0 -13 of taCla. Figure 5: this figure illustrates the construction of pAB4-lrep starting from pAB4-l by isolating the XhoI fragment and the HyndIII fragment followed by cloning into pMTL24. Figure 6: This figure describes the construction of the alpA gene replacement vector. An EcoRI-Stul fragment of 4.4 kb from pASl-1 is ligated with the genomic fragment of ATCC11906, the Smal-Ncol fragment of 2.6 kb from pAB4-lrep and the NcoI-EcoRI fragment of 4.4 kb from pASl-2A in a 3-way ligation thus providing pASl - del taalp. Figure 7: this figure provides the restriction map of the AD fragment? that carries the pclA gene of A. niger Figure 8: this figure provides the structure (functional organization) of the pclA-encoded protein of A. niger. This sample shows preactivity, proactivity and P domains from left to right.

The triangles in light color indicate the sites KR The triangles in dark color indicate the glycosylation sites. The clear picture with vertical stripes is a region rich in S / P / T. The box with dark wavy pattern on the far right is a region rich in D / E. Figure 9: this figure illustrates the growth phenotype of a mutant strain A. n iger pclA. Figure 10: this figure provides a DNA sequence comparison between the pclA genes of A. soj a e y A. niger A vertical bar indicates identity; : indicates 5; 'indicates 1. Found 72,139% similarity 72,073% identity Figure 11: This figure describes the construction of the replacement vector of the pclA gene. A Clal fragment of 7.6 kb, which is a genomic fragment of ATCC11906, is cloned into pMTL23p. In this construction, the 2.6 kb Smal fragment from pAB4-lrep is cloned into the EcoRV site, in order to obtain pAS2-delta pcl. Figure 12: this figure shows the amino acid sequence comparison of the various homologues of PclA from S. cerevisiae (Sckex2), K. lac ti s (Klkex-1), A. soj a e (Aspela), A. ni ger (A. niger), P. Chrysogenum (Penpcll), A. bi sporus (Agarmbll29), T. reesei (Trichpcll), R. oryzae (Rhizpcll), F. poison tum (Fuspcll), S. pombe (Spkrp), C. albi cans (Cakex2) and Y. lipolyti ca (Ylkex2). The identity regions global (8 of 12 entries) are indicated by yellow boxes. The spaces are indicated with ..; non-sequence data are indicated by ~. Figure 13: Figure 13a provides Sequence Data for the alpA promoter sequences of A. oryza e (Q11755). The position of the primer for cloning with PCR is indicated. In FIG. 13b, the sequence data for the amyA promoter sequences of A. oryzae including the initiator positions (A02532) are provided. Figure 13c provides the gpdA promoter sequences derived from A. oryzae ATCC42149 (EPO.436.858 Al) also including the positions of the primer. Figure 14: This figure provides a comparison between various Aspergillus gpdA promoter sequences. - from top to bottom, "A. sojae ATCC11906, A. orizae, A. niger and A. nidulans The asterisks indicate the putative intron present in the 5 'untranslated region of the promoters.The arrowheads indicate the regions rich in CT. underlined and in bold indicate the differences between the sequences of A. orizae and A. sojae Figure 15: this figure shows a map of the vector pGLA6S of 12,700 bp.

LIST OF SEQUENCES SEQ ID No. 1 MBL1784: 5 '- CGGAATTCGAGCGCAACTACAAGA-TCAA- 3 SEQ ID No MBL1785: 5' - CGGAATTCAGCCCAGTTGAAGCCGTC - 3 ' SEQ ID No. 3 The sequence of the Aspergillus niger gene that codes for the pro-protein convertase. The start codon and the stop codon are indicated with underlined and bold letters. The intron is indicated with lowercase underlined 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 CCT CTCGAT ACCCCGTGAA AACAGTGACG 301 ATGTCCATGC GCTGCTGGAT CAATTGCGCG ATCGTCGGAG GTTACGCCGC 351 CGCTCCGGAG ATGACGCCGC TGTCCTTCCC TCCTTGGTCG GGCGAGACGA 401 AGGTCTAGGT GGCATTCTTT GGTCCGAGAA GCTGGCTCCC CAGAGAAAGC 451 TCCATAAAFTS 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 GC TGAGCGA TGACCGCCAC 801 GGTACTAGAT GCGCGGGTGA AATCGGTGCG GCGAAGAACG ACGTGTGCGG 851 GGTTGGTGTT GCGTATGATA GTCGCATCGC TGGTATTCGG ATTCTCTCCG 901 CACCCATCGñ 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 13S1 TGGCCCTCAG TGTGCGGCCG GAACTCACCT GGCGTGACGT TCAGTATTTG 1401 ATGATTGAGG CGGCAGTGCC TGTTCATGAA QATGATGGAA GCTGGCAGGA 1451 CACTAAGAAC GGGAAG GT TCAGCCATGA CTGGGGA? AT GGTAAGGTCG 1501 ACACATATAC GCTGGTGAAA CGGGCAGAGA CCTGGGATCT GGTGAAGCCT 1551 CAAGCCTGGC TCCATTCCCC CTGGCAGCGG GnGAGCATG 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 aqtaaaaact 1851 ttttctcqqt tgtc qttct tctgctaata catat tagG GGCGAGTCCG 1901 GGATTGGCAA ATGGACTGTG ATTGTCAftGG 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 TTCTGCAT? G GCCTGGGCGT CTACTTCCAT 2401 GTGCAGCGCC GCAAACGTAT TCGCGAOGAC AGCCGGGATG ACTACGATTT 2451 CGAGATGATC GAGGACGAGG ATGñGCTACA GGCAATGAAC GGACGGTCGA 2501 ACCGTTCACG TCGCCGGGGT GGCGAGCTGT AC? ATGCTTT TGCGGGCGAG 2551 AGCGATGAGG AACCATTATT CAGTGATGAG GATGATGAAC CGTATCGGGA 2601 TCGGGGGATC AGCGGCGAAC AAGAACGGGA GGGCGCAGAT GGAGAGCATT 2651 CTCGGAGATG AAAGTGCAGT AGATGAGGGT TGACTTTATT TCGGACAGTG 2701 TTTCTAftCTT GTTGGATGAC CTGCGTTGAA CAATATTTCT GCTGTGTATG 2751 CTGCATAGAG AAGCGTGTAT ATACCATGTA TGTGTGCATC ATCGTGATCG 2601 GGTTTATCAT TCTTCATCTG CCATGGTTTG TGATCTCCGG AATAG ACCA 2651 AAGGAACACT AAATTAAGGG TCTTGGCGAT GACGCTTCCC GTCGCTGCTT 2901 TTGACTTCCT CCGCATCTCG TCTCTCCTGC TGTTGACCGC GCGCCAACCAATCT CCTCACTCCT CCCACCTTAA TCTTGCTGTG CTGCTTCTAG_3001_AACCCCCCAG TTTAATTTAA AAACCGGCTT TTCCTAGCTC CACGTATTGT 3051 ACCTCGCACT GATCCCCATC TCCGCCCACT CCAACGCTAC CGACCCAGGC 3101 TTCTCTGGCG GCTCCAGGCG GCAGGCAATC AAACCAACCC CTCGATGGAT 151 CAGCACGACG ACTTCGACAG SGTCTCGTGG AGGCATGACC CGGACAGCGA 3201 TCTCTCGCGA CCCACGRACT 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 coding for the pro-protein convertase 1 CGCGGATCCA TGGAACACGA TGTGCGGGTG AAATTGGAGC AGCTAGGAAT 51 GATGTCTGTG GAGTAGGTGT TGCATACGAC AGCCAAGTTG CCGGAATTCG 101 GATTTTGTCC GC ACCC ATTG ACGACGCAGA TGAGGCTGCT GCCATCAACT 151 ATGGCTTCCA GCGCAATGAT ATATATTCAT GCTCCTGGGG CCCTCCGGAT 201 GATGGCGCCA CGATGGAGGC GCCAGGGATT CTTATCAAAC GAGCTATGGT 251 CAACGG ATC 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 601 ATGAAATCCC ACAAGGTGAC CAGGGCCTTG CCAGCTCATA CGAAATTACC 951 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 CC ATGCCCG 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. S The partial sequence in the coding region of the Trichoderma reesei QM9414 gene coding for the pro-protein convertase 1 GCTGTCCGCA CTGATGCGTG CGGCCTTGGC GTTGCCTACG ACTCCAAGAT 51 TGCTGGCATC CGCATCCTTA GTAGTGCCAT CAGCGATGCG GACGAGGCCG 01 AGGCCATGAT TTACAAGTTC CAGGACAACC AGATCTACTC GTGCTCCTGG 51 GGGCCTCCCG ACGATGGGAG GTCCATGGAA GCCCCCGACG TCCTGATTCG 201 ACGAGCCATG CTCAAGGGCG TCCAGGAGGG ACGAGGAGGC CTCWGCTCCA 251 TCTACGWCTT TGCTAGTGGT AACGGTGCCG CCAGTGGCGA TAACTGCAAC 301 TNCGACGGAT ACNCAAACA SEQ ID No.6 The partial sequence in the coding region of the Fusarium venenatum gene ATCC20334 coding for the pro-protein 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 P2 gene of Penicilium cht? Sogenum coding for the pro-protein 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 otyzae gene ATCC20 076 coding for the pro-protein convertase 1 ACTNGGGGCA TTGGTGAAAT NTTGCTTGTG GNTTGGTGTT GCTTACGACG 51 CAAAAATATC TGGTATACGT ATATTATCAG GTGAAATCAC AGAGGCAGAC 101 GAGGCTGCTG CTTTGAATTA CAAATATCAA GAAAATCAAA TCTACTCCTG 151 CTCÜ3TGGGGC CCA SEQ ID No. 9 The partial sequence in the coding region of the Agaricus bispoms HORST gene coding for the pro-protein 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 01 GCATTAACCA GGGACGTGGC GGGAAGGGCT CCATTTTCGT CTTCGCCAGT 251 GGCAACGGCG CTGCTTCGGA TGACCAATGC AACTACGACG GATACACAAA 301 CA SEQ ID No. 10 coding chain site B mHÍ is underlined PE4 5'- CG CGGATC CAfT / O GGX ACX (C / A) GX TG (T / C) GCX GG -3 'degenerate 2048 times SEQ ID No. 11 encoder string PCL1 5'-CA (T / C) GGX ACX (C / A) GX TG (T / C) GCX GGX GA-3 'degenerate 8192 times SEQ ID No. 12 coding chain PCL2 5'-AT (C / T / A) TA (T / C) TCX TG (T / C) TCX TGG GGX CC-31 degenerate 768 times SEQ ID No, 13 non-coding string the B m site? 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 'degenerate 49152 times SEQ ID No. 14 non-coding chain PCL2rev 5'-GG XCC CCA XGA (A / G) CA XGA (A / G) TA (A / T / G) AT-3 'degenerate 768 times SEQ ID No. 15 non-coding chain PCL3 5 '- (A / G) TT XGT (A G) TA XCC (A / G) TC (A G) (AJ) A (A / G) TT ~ 3' degenerate 1024 times SEQ ID No. 16 non-coding chain PCL4 5'-GC XGC XGA XGT XCC XCC (A / G) TG-3 'degenerate 2048 times SEQ ID No. 17 The sequence of pAB4-lrep 59-499 bp Hindm fragment. of 0.4 kb 1-58 bp 500-513 bp 2873-2930 bp the polylinker sequence pAB4-l of pMTL24 (indicated with underlined lowercase) 514-2872 bp: fragment XIwl of 2.3 kb of pAB4-l 1 ggccaqtqaa ttcgaqctcq qtacccgqgq atcstctaqa gtcgacctgc 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 catgcctgca ggcCTOGAGC 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 TTGGftGTTGC GAGGGAGTTG ATTTCCTACT TCTAAGTTTG GACTTGAATC 851 CGTGGTGTGA TTGAGGTGAT TGGCGATGTT TGGCTATACC AGCTATATGT 901 AAAATCTCT ACTGTATACT ACTATTCAAC GCATTTTACT ATGCGTGCTG 951 CTAGGGTCGG CAATGACñAT GGCAATCTGA CTGACGTGGT CTATTTCTCC 1 ATGTGCAGCA GGGAATACGA GCTCCAATGG ACCTCGGGAG TGGCACAGTC 1051 AATGGCAAGG AAACTCCGCC TTTGCAGGTG TGGCTGñACC 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 1 S51 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 GCAACGOATA CGGAAGCGGA AACGGACCAA 2351 TGTCGAGCAC GGGTAGTCAG ACTGCGGCAT CGGATGTCCA AACGGTATTG 2401 ATCCTGCAGG CTACTATGGT GTGGCACAAG GATCAATGCG GTACGACGAT 2451 TTGATGCAGA TAAGCAGGCT GCGAAGTAGT AACTCTTGCG TAGAGAAAAT 2501 GGCGACGGGT GGGCTGATAA GGGCGGTGAT AAGCT AATT 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 ja cgtcgactct 2901 gaggatcccc gggtaceqag ctcgaa tcg SEQ ID No. 18 MBL 789 JECORI is underlined 5'- GGAA TTC (A / G) GA ATA (T / A) GG AGG ATG TAG -3 'degenerated 4 times SEQ ID No. 19 MBL 793 B mKl is underlined 5'- CGGATCCG CAG TGG CAC TTG (G / A) TC AAT CCA A -3 'degenerated 2 times SEQ ID No. 20 MBL 794 EcoRl is underlined 5X GGA ATT CTT AAA A (T / G) C CCA AGA ACC TTC A -3 'degenerated 2 times SEQ ID No.21 MBL 1158 £ coRl is underlined 5'- G GAA TTC (T / QTC (T / G) CC (T / G) GC (A / G) CA (C / G) C (T / G) (C / G) GT (T / G) CC (A / G) TG -3 'degenerate 512 times SEQ ID No. 22 MBL 1208 Clal is underlined '- CGG? IC GAÍT / C) GGX ACX (C A) GX TG (T / C) GCX GG -3 'degenerate 2048 times SEQ ID No.23 MBL 1219 Bai? L is underlined 5'- CGG ATC (CmTG XA (G / T / C) (A / G) TC XC (T / G) CCA XGT (C / AG) AG -3 ' degenerate 4608 times SEQ? D No. 24 The restriction sites are in bold The initiators are underlined B mHl initiator PE4 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 initiator PE6 CCCCCGGAAA TGGTGGATCC 320 BamHl SEQ ID No. 25 Sequence of the Aspergillus niger PclA protein 1 Met Arg Leu Thr Gly Gly Val Wing Wing Wing Leu Gly Leu Cys Wing 16 Wing Wing Being Wing Being Leu Hia Pro His Axg Being Tyr Glu Thr His 31 Asp Tyr Phe Ala Leu His Leu Asp Glu Ser Thr Ser Pro Wing Asp 46 Val Ala Gln Arg Leu Gly Ala Arg Hís Glu Gly Pro Val Gly Glu 61 Leu Pro Ser His His Thr Phe Ser He Pro Arg Glu Asn Ser Asp 76 Asp Val His Wing Leu Leu Asp Gln Leu Arg Asp Arg Arg Arg Leu 91 Arg Arg Arg Ser Gly Asp Asp Ala Wing Val Leu Pro Ser Leu Val 06 Gly Arg Asp Glu Gly Leu Gly Gly He Leu Trp Ser Glu Lys Leu 21 Wing Pro Gln Arg Lys Leu His Lys Arg Val Pro Pro Thr Gly Tyr 36 Ala Ala Arg Ser Pro Val Asn Thr G n Asn Asp Pro Gln Ala Leu 51 Wing Wing Gln Lys Arg Wing Wing Ser Glu Leu Gly Wing Wing Asp Pro 66 Xle Phe Gly Glu Gln Trp His Leu Tyr Asn Thr Val Gln Leu Gly 181 His Asp Leu Asn Val Thr Gly He Trp Leu Glu Gly Val Thr Gly 196 Gln Gly Val Thr Thr Ala He Val Asp Asp Gly Leu Asp Met Tyr 211 Ser Asn Aßp Leu Arg Pro Asn Tyr Phe Wing Wing 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 Wing Gly Glu He Gly Wing Wing Lys Asn 256 Asp Val Cys Gly Val Gly Val Wing Tyr Asp Ser Arg He Wing Gly 271 H Arg He Leu Ser Wing Pro He Asp Asp Thr Asp Glu Ala Wing 2S6 Ala He Asn Tyr Ala Tyr Gln. Glu Asn Asp He Tyr Ser Cys Ser 301 Trp Gly Pro Tyr Asp Asp Gly Wing Thr Met Glu Wing Pro Gly Thr 316 Leu He Lys Arg Wing Met Val Asn Gly He Gln Asn Gly Arg Gly 331 Gly Lys Gly Ser Val Phe Val Phe Ala Ala Gly Asp 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 Wing He Asp Arg Glu Gly Asn Hia Pro Pro 376 Tyr Ser Glu Ser Cys Ser Ala Gln Leu Val Val Ala Tyr Ser Ser 391 Gly Ala Ser Asp Ala He His Thr Thr Asp Val Gly Thr Aap Lys 406 Cys ser Thr Thr His Gly Gly Thr Ser Ala Ala Gly Pro Leu Ala 421 Wing Gly Thr Val Wing Leu Wing Leu Ser Val Arg Pro Glu Leu Thr 436 Trp Arg Asp Val Gln Tyr Leu Met He Glu Ala Wing Val Pro Val 451 His Glu Asp Asp Gly Ser Trp Gln Asp Thr Lys Asn Gly Lys Lys 466 Phe Ser His Asp Trp Gly Tyr Gly Lys Val Asp Thr Tyr Thr Leu 481 Val Lys Arg Wing Glu Thr Trp Asp Leu Val Lys Pro Gln Wing Trp 496 Leu His Ser Pro Trp Gln Arg Val Glu His Glu He Pro Glp Gly 511 Glu Gln Gly Leu Wing Being 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 Hie Thr Arg Arg Gly Asp Leu Ser Val Glu Leu Arg 556 Ser Ro Asp Gly Arg Val Ser His Leu Ser Thr Pro Arg Arg Pro 571 Asp Asn Gln Glu Val Gly Tyr Val Asp Trp Thr Phe Mßt Ser Val 586 Wing 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 Gln Phe He Asp Trp 616 Arg Leu Aen Leu Trp Gly Glu Wing He Asp Gly Wing Glu Gln Pro 631 Leu His Pro Met Pro Thr Glu His Asp Asp Asp His Ser Tyr Glu 646 Glu Gly Asn Val Wing Thr Thr Ser He Be Wing 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 Wing Met Pro Thr Gly Ser Leu Thr 691 Glu Pro He Asp Asp Glu Glu Leu Gln Lys Thr Pro Ser Thr Glu 706 Wing Being Being Thr Pro Being Pro Pro Being Thr Thr Wing Being Asp Being 721 He Leu Pro Be Phe Phe Pro Thr Phe Gly Wing 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 Gln Arg Arg Lys Arg I have 766 Arg Asp Asp Ser Arg Asp Asp Tyr Asp Phe Glu Met He Glu Asp 781 Glu Asp Glu Leu Gln Ala Met Asn Gly Arg Ser Asn Arg Ser Arg 796 Arg Arg Gly Glu Glu Leu Tyr Asn Wing Phe Wing Gly Glu Being Asp 811 Glu Glu Pro Leu Phe Ser Asp Glu Asp Asp Glu Pro Tyr Arg Asp 826 Arg Gly He Ser Gly Glu Gln Glu Arg Glu Gly Wing Asp Gly Glu 841 His Ser Arg Arg SEQ ID Nos. 26 to 31 PCR primers for the cloning of the A. sojae promoter The restriction sites are underlined SEQ ID No. 32 The sequence of the gpdA promoter region of Aspergillus sojae 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 alpA promoter region of Aspergiüus sojae 1 GCGGCCGCGG TTATTCTGCG GAAGCGGACCENTOTTCC 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 GA ATCGTTT 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 S 01 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 Aspergi llus (Samson, 1992) SUBGENER GENRE SECTION Selected species "SUBESPECIES" Aspergillus Clrcumdati entii A. wentii (glucosidase) Flavi / Tamarii A.oryzae (amylase, protease) A. sojae (fermented food, protease) A. parasiticustox ___ Nigri A. niger - - A pulverulent (fermented food, A. phoenicis various proteins, A. awamori organic acids) A. foetidus A. awachii A. usamii A. ficuum A. japonicus A. aculeatus (endoglucanase) (glucosidase, galactanase ) A. ellipticus A. tubingensis > "A. niger" Circumdati A. ochraceusJ0 * (xulanasal A. alliaceusto _ Candidi A. candidus (lipase, glucosidase) C emei A.itaconicus (organic acid) Sparsi A. sparsus Aspergillus Aspergillus Aspergillus A. glaucus (fermented food) Restricti A restrictustoJ Fumigati Fumigati A. fumigatusto SUBGENARIO GENDER SECC? ÜN Selected species "SUBESPECIES" Cervini Ornati Clava i Clavati A. giantus Nidulants Nidulans A. nidulans Versicolores A. sydown (lipase) Usti Terrei A. terreustox (glucanase) Flavipedes a For the species selected for this list, either the production of proteins / organic acids / fermented foods (indicated in brackets) and / or a DNA-mediated transformation procedure is described. (indicated with underline), except for A. tamarii, A. sparsus and A. ellipticus The species registered as producing toxins are indicated with "tox". b Based on several methods, names listed as synonyms to the name listed in the SPECIE section may be considered.

TABLE 2 Classification of the different ATCC strains Legenda ND = not determined 11 REF: Ushij ima S, Hayashi K and Murakami H (1982) The current taxonomic status of Aspergillus sojae used in Shoyu fermentation. Agrie.

Biol. Chem, 46.2365-2367, 1981. 2) R? F: 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.

REF: Chang PK, Bhatnagar D, Cleveland TE and Bennett J (1995) Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes specie ^ in Aspergillus section Flavi. Appl.

Environm. Microbiol., 61: 40-43 Conclusion about the classification elaborated by TNO based on the data presented in this table. This strain was deposited in the ATCC as A. oryzae, but was then re-classified as A. sojae based on Yuan et al, 19952 'and Chang et al, 19953'. This strain was deposited in the ATCC as A. parasi ticus, but was later classified as A. sojae based on Ushij ima et al, 19811 'and Yuan et al, 19952') REF: ATCC catalog REF: Liu BH, Chu FS (1998) Appl. Env. Microbiol, 64: 3718-3723.

TABLE 3 Composition of the selection medium i) Mineral solution: CUS04-5H20 0.16 g / l FeS04-7H20 s .5 g / l ZnS04"7H20 2.2 g / l MnCl2-4H20 0.5 g / l C? Cl2'6H20 0.17 g / l Na2Mo04'2H20 0.15 g / l l H3BO3 rl g / l EDTA 5 g / l TABLE 4 Protease activity in different media fifteen Caption: + (partial) degradation of proteins after 4 hours of incubation, large milk elimination zone. no protein degradation after 4 hours of incubation, small milk elimination zone / no elimination Incubation at 30 ° C: 27 μl sample medium. 2.5 μl of BSA (25 mg / ml) 0.5 μl of Fitasa (A. terreus, 3-4 g / l) BSA and phytase are added after taking the culture medium sample. This sample is incubated at 30 ° C and after certain times the sample is analyzed for the degradation of BSA and phytase.

TABLE 5 Protease activity at different pH values Legend: + degradation (partial) of proteins after 4 hours of incubation. there is no degradation of proteins after 4 hours of incubation. Incubation at 30 ° C: 25 μl sample medium. 2 μl of buffer solution (50mM) [50 mM NaAc, pH = 4.2; 50 mM NaAc, pH = 5.8; 50 mM Tris / HCl, pH = 8.3]. 2.5 μl of BSA (25 mg / ml) 0.5 μl of Fitasa (A. terreus, 3-4 g / l) BSA, phytase and buffer are added after taking the culture medium sample.

This sample is incubated at 30 ° C and after certain times the sample is analyzed for the degradation of BSA and phytase.

TABLE 6 PCR results for the cloning of fungal pclA genes Legend: + Specific PCR product Non-specific product or does not come from PCR this PCR product was used to determine the sequence TABLE 7 Viscosity intervals of the various strains of A, sojae TABLE 8 Strength of the promoter in A. sojae transformants

Claims (35)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered a novelty and therefore the content of the following is claimed as property:
2. CLAIMS 1.- A recombinant Aspergillus sojae comprising a gene for acetamidase S (amdS) introduced as a selectable marker. 2. An Aspergillus sojae according to claim 1, characterized in that said Aspergillus sojae can be selected in a medium comprising a substrate for the amdS introduced as the sole source of nitrogen, the medium also comprises a carbon substrate and said medium it is free of substrate that induces endogenous amdS.
3. 3. An Aspergillus sojae according to claim 1 or 2, characterized in that the nitrogen source is acrylamide.
4. 4. An Aspergillus sojae according to any of the preceding claims characterized in that Aspergillus sojae does not have an active gene for endogenous amdS, for example because the gene for endogenous amdS comprises a mutation that inactivates the endogenous amdS, by example, a deletion or disruption.
5. 5. A method for introducing a nucleic acid sequence in Aspergillus sojae, characterized in that said method comprises subjecting Aspergillus sojae to a method for introducing a nucleic acid sequence for example, transformation or transfection of Aspergillus sojae in a manner known per se for introducing a nucleic acid sequence into fungi, said method comprising introducing the gene for amdS as the nucleic acid sequence (hereinafter the gene for amdS introduced) followed by selection of the transformed or transfected Aspergillus sojae resulting in a free medium substrate that induces endogenous amdS, "the medium also comprises a substrate for the amdS introduced as the sole source of nitrogen and the medium also comprises a substrate for carbon, said medium allows the desired Aspergillus sojae comprising the nucleic acid sequence to grow and at the same time eliminates the growth of Aspergillus sojae free of the so-called introduced nucleic acid sequence due to the inability of such Aspergillus sojae to grow without the amdS gene introduced into the selection medium, said medium suitably comprises a substrate for amdS other than acetamide, for example acrylamide as a substrate for the amdS introduced as the sole source of nitrogen.
6. 6. An Aspergillus sojae obtained by the method according to claim 5.
7. 7. A method for selecting transformed or transfected Aspergillus sojae, characterized in that said method comprises subjecting Aspergillus sojae according to any of claims 1-4 and 6. to a method of transformation or transfection of Aspergillus sojae in a manner known per se for transformation or transfection of fungi with a nucleic acid sequence, said method comprises introducing the gene for amdS as the nucleic acid sequence followed by selection of Aspergillus sojae Transformed or transfected resulting in a medium comprising a substrate for amdS introduced as the sole source of nitrogen and the medium also comprises a substrate for carbon, said medium allows the desired Aspergillus sojae to grow and at the same time eliminates the growth of Aspergillus sojae not transformed or not transfected due to the i ncapacity of the same to grow without the gene for amdS introduced in the selection medium.
8. 8. A method for producing recombinant Aspergillus soju ae, characterized in that said method comprises introducing a nucleic acid sequence in an Aspergillus soju for example, by transformation or transfection in a manner known per se in accordance with any of claims 1-4 and 6, said nucleic acid sequence comprises a desired sequence to be introduced flanked by sections of a gene for endogenous amdS or corresponding sequences having sufficient length and homology to ensure recombination thereby eliminating, simultaneously, the gene for endogenous amdS and introducing the desired sequence, followed by selection of the recombinant Aspergi llus soj ae with the desired sequence by selecting with respect to a selectable marker comprised in or transformed into cotransformation with the desired sequence, said selectable marker it is absent in the Aspergi llus soj e before introducing the nucleic acid sequence, appropriately the selectable marker is pyrG.
9. 9. - An Aspergillus sojae that presents growth with a medium comprising uracil and fluoro-orotic acid, in addition said Aspergillus does not present growth in medium comprising uridine and fluoro-orotic acid, that is, said Aspergillus sojae presents auxotrophy to uracil, said Aspergillus sojae can not use uridine, said Aspergillus sojae is pyrG negative, said
10. Aspergillus sojae exhibits resistance to fluoro-orotic acid, said auxotrophy to uracil and said resistance to fluoroorotic acid can be released after supplementing with a gene introduced for active PyrG, appropriately said Aspergillus sojae is free of genes for active endogenous pyrG; for example, the gene for endogenous pyrG of Aspergillus sojae comprises a mutation in the form of an insertion, substitution or deletion in the gene or in a regulatory sequence of the gene, for example, a deletion of the complete 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. 11. - A method for selecting transformed or transfected Aspergillus sojae, characterized in that said method comprises subjecting Aspergillus sojae according to claim 9 or 10 to a transformation or transfection method with a nucleic acid sequence, said method comprises introducing an active pyrG gene into Aspergillus sojae in a manner known per se for the transformation or transfection of fungi followed by selection of the transformed or transfected Aspergillus sojae in a free medium of uracil and fluoro-orotic acid, said medium also comprising at least the minimum substrates required for the growth of Aspergillus sojae, said medium allows the desired Aspergillus sojae to grow and at the same time eliminates the growth of untransformed or untransfected Aspergillus sojae due to the inability thereof to grow without uracil caused by the inactive pyrG gene.
12. 12. - A method according to claim 11, further characterized in that the active pyrG gene that is introduced is flanked by identical fragments of the nucleic acid sequence, and the Aspergillus sojae pyrG positive that resulting from the introduction of the pyrG gene and the flanking sequences is selected in a free medium of uracil and fluoro-orotic acid and subsequently the Aspergillus soj e pyrG positive is grown in a medium comprising uracil and fluoro-orotic acid with which eliminates the pyrG gene that has been introduced and in this way an Aspergi llus soj e pyrG negative is obtained which can be selected by growth in a medium comprising uracil and by means of resistance to fluoro-orotic acid, in appropriate form the sequences The flanking and the pyrG gene are also flanked by sequences that direct the integration of the p rG gene and the flanking sequences to a specific site due to the fact that the sequences that direct integration are homologous with respect to a specific sequence of Aspergi llus soj aea to be transformed, which allows the removal, if desired, of the gene associated with the specific sequence.
13. 13. - A method according to claim 11 or 12, further characterized in that the Aspergi l lus soj e in accordance with claim 9 or 10 has an additional nucleic acid sequence introduced therein, In preference to said additional nucleic acid sequence encoding a protein or polypeptide, said additional nucleic acid sequence is introduced with the active pyrG gene either in the same vector or by cotransformation with the active pyrG gene that is introduced.
14. 14. A method for selecting Aspergi llus eoj ae transformed or transfected by carrying out the method according to any of claims 11-13 in combination with the method according to claim 5.
15. 15.- A method for producing Aspergi ll us sojae recombinant, characterized in that said method comprises introducing a nucleic acid sequence into a positive Aspergillus soj and pyrG, for example, by transformation or transfection in a manner known per se, said nucleic acid sequence comprises the desired sequence flanked by sections of the gene pyrG or corresponding sequences having sufficient length and homology to ensure recombination, eliminating the pyrG gene and introducing the desired sequence, followed by selection of the recombinant Aspergi llus sojae with the desired sequence by selecting with respect to Aspergi llus soj ae with a pyrG negative phenotype.
16. 16. A recombinant Aspergillus sojae obtained by a method according to any of claims 11-15, further characterized in that it optionally also comprises the characteristics of an Aspergillus sojae according to any of claims 1-4, 6, 9 and 10.
17. 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 after being expressed by Aspergillus niger or Aspergillus awamori.
18. 18. A recombinant Aspergillus sojae comprising an introduced nucleic acid sequence encoding a protein or polypeptide to be expressed, said protein or polypeptide being different from the amylase and the Aspergillus protease sojae, said protein or polypeptide preferably they are a protein or polypeptide that does not belong to Aspergillus sojae.
19. 19. A mutant Aspergillus sojae or recombinant comprising a mutation that inactivates a gene for protease, properly a gene for alkaline protease.
20. 20. A mutant or recombinant Aspergillus mutant that comprises a mutation that inactivates the main gene for protease, properly a mutation that inactivates the main gene for alkaline protease, for example, the gene that codes for the main protease gene alkaline of 35 kDa.
21. 21. A method for producing Aspergi l l u s so y a and recombinante, characterized in that said A. Recombinant soybean exhibits reduced proteolytic activity, said method comprises introducing into an A. soj e, e.g., by transformation or transfection in a manner known per se, a nucleic acid sequence comprising a selectable marker encoding the sequence to be introduced flanked by sections of the protease gene to be deleted and further flanking sequence and the selectable marker encoding the sequence are comprised within sequences having sufficient length and homology to ensure recombination in the gene for protease thereby eliminating, simultaneously, the gene for protease and introducing the desired selectable marker encoding the sequence, the introduction is followed by selection of A. and recombinant soya by selecting with respect to the selectable marker, whereby A. Soj before the introduction of the nucleic acid sequence, for example, by transformation or transfection, is free of the selectable marker that will be introduced, for example, A. Soj is mutated before introducing the nucleic acid sequence in such a way that A. Soj e can not produce the active selectable marker, appropriately the selectable marker is the gene for pyrG, the method is properly carried out together with the method according to any of claims 11-15.
22. 22. A recombinant Aspergillus soj and obtained according to the method according to claim 21.
23. 23. An Aspergillus or mutant or recombinant Aspergillus according to any of claims 17-20 or 22 comprising a selectable marker, preferably amdS as defined in any one of claims 1-4 or 6 and / or pyrG as defined in claims 9, 10 or 16.
24. 24.- A recombinant Aspergillus soju ae according to any 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. 25. A method for expressing a nucleic acid sequence -introduced that encodes a protein or polypeptide comprised in a recombinant Aspergi llus soj ae or mutant as defined in any of claims 1-4, 6, 9, 10, 16 -20, 22-24 or which is obtained through a method according to any of claims 5, 7, 8, 11-15 and 21, further characterized in that said method comprises cultivating the A. Recombinant or mutant soya, suitably the introduced nucleic acid sequence encoding a protein or polypeptide is absent in A. Soja to the untransformed or wild type and / or is present in a smaller number of copies.
26. 26.- A recombinant fungus that comprises a mutation in a gene encoding a proprotein convertase or a functionally equivalent protein.
27. 27. A fungus according to claim 26, further characterized by having increased production of a protein, polypeptide or metabolite under equivalent conditions when compared to the corresponding wild-type h-ongo. -
28. 28.- A fungus according to claims 26 or 27, further characterized in that said mutation is obtained by specific modification of the gene using transformation or transfection in a manner known per se.
29. 29. A fungus according to claims 26-28, further characterized in that said proprotein convertase or functionally equivalent protein is encoded by a nucleotide sequence from which a fragment can be amplified by amplification of DNA in vi tro using any of two mixtures of nucleotides indicated in SEQ ID Nos. 10 to 16.
30. 30.- A fungus as described in claim 27, further characterized in that said proprotein convertase or functionally equivalent protein is encoded by a nucleotide sequence that permits functional complementation of the growth phenotype of a mutant Aspergi l lus niger comprising a mutation that inhibits the activity of a proprotein convertase or a functionally equivalent protein.
31. 31. A fungus according to any of claims 26-30, further characterized in that said fungus is an Aspergi l l u s soj e e.
32. 32. A fungus according to any of claims 26-30, further characterized in that said fungus also contains a gene for amdS or a gene for pyrG introduced.
33. 33.- A method for expressing a protein or polypeptide, preferably a recombinant protein or polypeptide, encoded by a nucleotide sequence, characterized in that said method comprises culturing a fungus according to any of claims 26-32.
34. 34. - A method for producing a protein or polypeptide, preferably a recombinant protein or polypeptide, characterized in that said method comprises an expression method according to claim 33, optionally including the processing and / or secretion and / or isolation of the expressed protein or polypeptide.
35. 35.- A process for producing a phytase or a protein having phytase activity, preferably a recombinant phytase or a recombinase protein having phytase activity, further characterized in that said method comprises an expression method according to claim 33, optionally including the processing and / or secretion and / or isolation of the phytase or expressed protein having phytase activity.