JP2003518921A - Use of Aspergillus sojae as a host for recombinant protein production - Google Patents

Abstract

  (57) [Summary] Disclosed is a recombinant Aspergillus sojae, which comprises the introduced acetamidase S (amdS) gene as a selectable marker. Aspergillus sojae exhibiting growth on a medium comprising uracil and fluoroorotic acid (the Aspergillus sojae does not further express growth on a medium comprising uridine and fluoroorotic acid, ie, The Aspergillus sojae indicates uracil auxotrophy, the Aspergillus sojae cannot utilize uridine, the Aspergillus sojae is Aspergillus sojae, and the aspergillus sophie is pyger, (Aspergillus sojae) indicates resistance to fluoroorotic acid, and the uracil nutritional requirement Sex and the fluoroorotic acid resistant describe can be relieved upon complementation pyrG genes introduced active). The Aspergillus sojae may further comprise a phytase or a protein having phytase activity, or a nucleic acid sequence encoding any other heterologous protein or polypeptide, and wherein the phytase or the other heterologous protein or It can be used for biotechnological production of polypeptides. Additional mutants that exhibit modified morphology are also disclosed. A method for producing such an expression host will be described.

Description

Detailed Description of the Invention

The present invention relates to novel means of transforming fungi and their use for the production of heterologous proteins. The means is the taxon Aspergillus Soje.
It requires a genetically engineered fungus belonging to the illus sojae). Suggestions have been provided in the past for using Aspergillus sojae as a host strain for transformation. However, to date, no data has been provided on successful transformation and / or expression of heterologous proteins. In addition, to date, Aspergillus soja for several reasons, including proteolytic degradation.
Surprisingly, certain proteins such as phytase, which were difficult to express in large amounts in expression hosts other than e), were surprisingly converted to Aspergillus soje (Asperg).
It has been found that it can be expressed in illus sojae). The production level of the heterologous protein in Aspergillus sojae was determined by the Aspergillus niger.
) And Aspergillus awamori
Was found to exceed the levels achieved for the same protein in.
In addition to the above, the subject of the present invention is, for the purpose of expression, an improved Aspergillus soge (Aspergillus) characterized by reduced proteolytic activity on the one hand and improved fermentation characteristics related on the other hand to fungal morphology. Sojae) strains are further included.

BACKGROUND OF THE INVENTION Aspergillus soje as a host strain for transformation.
Suggestions for using sojae) have been provided in the past. However, to date, no data has been provided on the successful transformation and / or production of heterologous proteins, and more specifically nothing on the expression of phytase. Previously, Aspergillus
In expression hosts other than illus sojae) expression levels were too low, mainly due to proteolytic degradation. We now have other strains, such as Aspergillus niger, Aspergillus awamori and Trichoderma.
above the level achieved for the same protein in choderma),
The expression level of the protein in Aspergillus sojae was found. It is surprising to find such improvements in closely related strains. Thus, the prior art disclosures regarding phytase production represent a drawback. Prior art disclosures regarding the use of Aspergillus sojae to express heterologous proteins or polypeptides have been inadequate.

To date, A. The fact that few successful attempts at transformation of A. sojae have been reported is due to the large number of successful transformations of closely related strains of the taxon Aspergillus oryzae in the past. It should be noted in light of the fact that it is described in. Based on this close relationship, one skilled in the art would appreciate that Aspergillus oryzae
illus oryzae) and methods similar to those used in Aspergillus
It could be expected (and indeed was expected) that it will also be applicable to the Aspergillus sojae strain.

For example, International Patent Application No. WO 97/04108 describes the isolation of nucleic acid sequences encoding proteases, in particular sequences encoding leucine aminopeptidases, and diverse host organisms having sequences encoding leucine aminopeptidases. Body (i.a. Aspergillus sojae)
) Transformation. However, no concrete explanation of this particular transformation carried out in practice is provided. It is used as a potential host strain to be used for transformation, as Trichoderma reesei, Aspergillus niger, Aspergillus.
Aspergillus awamori, Aspergillus feetidus, Aspergillus japonicus, Aspergillus
It has simply been suggested as one possibility among many other strains, such as Aspergillus phoenicis and Aspergillus oryzae. In the cited documents, three transformation protocols readily used in the art are suggested for these strains. In particular, the selection marker acetamidase S (= amdS) (
Either argB or hygromycin B (eg, as maintained on vector p3SR2) is a suitable marker to be used according to the transformation protocol described therein (eg, using vector pAN7-1). Is suggested as.

The use of the vector p3SR2 with the amdS marker has been described in various strains, for example Aspergillus oryzae (European Patent No. E.
P 0.238.023), Trichoderma reesei (Tri.
choderma reesei (in EP 0.244.234) and Aspergillus nige.
r) (EMBO Journal 4, 475-479) is frequently described in the literature as useful for transforming. As a result, similar uses for transforming Aspergillus in general have been proposed in International Patent Application No. WO 97/04108 based on these previous publications.

[0006] Quite specifically, International Patent Application No. WO 97/04108, page 17, shows that Aspergilli grown slowly on minimal medium simply comprising the substrate acetamide as a source of nitrogen before transformation. ) And Trichoderma could be selected by their distinct growth advantage after transformation with the vector p3SR2. afterwards,
The transformants thus obtained may need to be further exposed to selection for leucine aminopeptidase (= LAP) productivity in order to find the desired transformants. As mentioned above, this is based solely on the successful transformation of some strains other than Aspergillus sojae, as a whole as a speculative means of transformation applicable across the two aforementioned genera as a whole. Proposed.

However, the suggested transformation protocol is Aspergillus soje (A
unsuccessful in spergillus sojae). The selection criteria described in the prior art are insufficient to ensure the actual selection of the desired transformants when using the vector p3SR2. We carried out experiments and found that the described method was inoperable due to excessive background growth that eliminated the actual selectability.

Another convention method used for fungal transformants is that of transforming mutants of orotidine-5-monophosphate decarboxylase (= PyrG). Mol.
Gen. Genet. 210, pages 460-461, Matern et al., Discloses transformation of Aspergillus oryzae using the pyrG gene of Aspergillus niger. Standard practice is to isolate pyrG mutants based on their direct resistance to fluoroorotic acid as a positive selectable marker.
This has, to date, led to the isolation of a large number of pyrG mutants of diverse fungi.

From experiments with a large number of diverse filamentous fungi, an auxotrophic pyrG-based system has many advantageous features. Using standard procedures based on direct selection for resistance to fluoroorotic acid (FOA) on plates containing uridine that support the growth of mutant strains, A. Experiments were performed to obtain a pyrG mutant strain of A. sojae (Mol. Gen. Genet. (198).
7) 206, pp. 71-75, Van Hartingsveldt et al.).
However, Aspergillus sojae
The use of similar methods in strains did not lead to pyrG mutants. Conventional methods did not lead to fluoroorotic acid resistant strains, but all strains were able to grow without uridine. Therefore, neither of these strains was a pyrG mutant. Usually, isolation of pyrG mutants can be done directly from fluoroorotic acid resistant strains on uridine selective medium. Aspergillus Soje (
For Aspergillus sojae), however, this method proved inoperable.

Clearly, Aspergillus sojae
) Represents a characteristic that differs from the closely related Aspergillus oryzae when it comes to the problem of transformation. Standard protocols using amdS or pyrG as selectable markers are not sufficient. Unfortunately, the method of argB as a selectable marker is also not an attractive option. Because this is for every host strain that you want to use,
This is because it requires isolation of the corresponding argB mutant. This is a difficult task based on trial and error. The required argB mutant can be obtained by random mutagenesis followed by screening of tens of thousands of colonies. py
The situation for rG is better because the mutant is itself selectable. In the case of amdS, no mutant is required because the presence of amdS serves as the dominant selectable marker.

Aspergillus as an expression host for recombinant proteins or polypeptides
There are additional problems that discourage the person skilled in the art from using the Soger (Aspergillus sojae). For example, Japanese Patent Application No. JP-A-02-23
4666, A of Aspergillus sojae, using a protocol similar to that described for other fungi.
Selection based on rgB is described. Aspergillus oryzae (
Aspergillus oryzae) Biotechnology
(1988) 6, pages 1419-1422. The cited papers are
Aspergillus nidulans
Reference is also made to the successful analogous transformation of Aspergillus niger. However, the Aspergillus sojae strain ATCC disclosed in the Japanese patent application
Unwanted protease characteristics were found when 42251 was analyzed.
The protease characteristics of this strain are incompatible with use as a production host. Therefore, one transformation protocol is that this particular Aspergillus soje
Even if suggested in the prior art for the illus sojae strain, it cannot lead to high level expression of heterologous proteins even if the protocol for transformation was successful.

In fact, the Aspergillus sojae strains currently find use in practice because of their distinctive characteristic of producing, precisely, excess amounts of alkaline proteases and amylases. They are used in particular in methods that require the degradation of complex polymeric substrates. Therefore, the finally successful Aspergillus sauger (Aspergillus s)
It was at best expected that no transformant of Ojae) would reach good expression levels unless the product was a protein of Aspergillus sojae, which is insensitive to its own protease.

In summary, the problems faced by those skilled in the art in finding means for using the Aspergillus sojae strain to express a heterologous recombinant protein on an industrial scale are diverse. First, many methods of introducing the desired nucleic acid material to be expressed are not applicable in the manner used by other fungi. This is a closely related Aspergillus oryzae (Asperg
illus oryzae) and methods based on pyrG and amdS. Second, the high level production of heterologous proteins is due to the host strain Aspergillus
It remained to be investigated whether it could be viable despite the known excess proteolytic activity of Soger (Aspergillus sojae).

It was unexpectedly found that the problems posed above can be overcome, thus leading to new expression hosts for producing proteins and new methods for producing heterologous proteins. We have shown that A. elegans with amdS and pyrG selectable markers. The transformation of the A. sojae strain is described. In addition, efficient gene expression is described, including expression of the phytase gene.

Description of the Invention As stated, the subject invention is based on Aspergis Soge.
L. sojae) strain and its use for the production of recombinant proteins and polypeptides. First, Aspergillus Soge
A description of the r. gills sojae) strain is provided. The decision of Aspergillus sojae.

The taxonomy of fungi is a complex problem. Aspergillus
s) is a Flavi / Tamarii ward (secio)
n) comprises Aspergillus sojae (see Table 1). A. A. sojae are A. sojae arranged in the same section. It is clearly shown to be different from A. oryzae (see Table 2). Currently, Aspergillus
strains belonging to the group Sojae) are closely related taxonomically in a number of art-recognized manners, Aspergillus oryza.
e) and also closely related Aspergillus parasitics (Aspergi
illus parasiticus strain. U respectively
shijima et al. (1981), Chang et al. (1995), Yuan et al. (19).
95), Kusumoto et al. (1998) and Watson et al. (1999), random PCR fragments, ver-1, aflR and rD.
NA sequences are mentioned. In addition, Aspergillus oryzae (Aspergill)
us oryzae) was found to be further different from Aspergillus sojae upon comparison of the alpA sequences of these strains. In particular (there are other sequence differences between A. oryzae and A. sojae alpA that can be used as decision tools), Aspergillus soje (Aspergillus)
Sojae) was found to comprise an XmnI restriction site at specific positions in the alpA gene. Several Aspergillus oryzae
The corresponding position in the alpA gene of the S. oryzae strain does not possess these restriction sites. Therefore, it provides an additional distinction between the two types of fungal strains.
As a result, one strain was identified as Aspergillus soge.
Numerous methods are available to those skilled in the art for assessing whether or not Currently, more than 10 strains, defined as Aspergillus sojae, have been deposited with the ATCC. The 10 oldest deposits have been analyzed. Two out of ten failed the recently listed decision tests.
One of them is ATCC20235, which is described in Ushijima et al. (19
81), Aspergillus soje (Asp) based on morphological parameters.
The requirements for classification as ergillus sojae) were also not met. The other is ATCC 46250. The definition of Aspergillus sojae as used throughout this patent application is al.
It is meant to include strains that preferably meet all the requirements described in the cited references, as well as the presence of an XmnI restriction site in the pA gene. Specific homologous primers for both the Aspergillus oryzae and Aspergillus sojae sequences are also provided. Using them, you can use Aspergillus Soge (A
spergillus sojae) and Aspergillus oryzae (Asperg
As an example of a screening test useful for identifying illus oryzae, one can test for the presence of an XmnI restriction site (the primer sequence is
SEQ ID NO: 1 MBL1784: 5'-CGGAATTCGAGCGCAACTA
CAAGATCAA-3 'and SEQ ID NO: 2 MBL1785: 5'-CGGA
ATTCAGCCCAGTTTGAAGCCGTC-3 '). They are al
It is derived from the coding region of the pA gene. It will be apparent to those skilled in the art that alternative probes or primers are possible based on the known sequence data. PCR amplification with Aspergillus DNA using these primers, followed by restriction enzyme digestion of the resulting DNA fragment with XmnI was performed on A. A. oryzae strain and A. oryzae strain. One method of distinguishing A. sojae strains is provided. Aspergillus
Once the definition of the S. sojae strain is established, further progress can be made using the detailed description of the invention.

The present invention, in one aspect, comprises a recombinant Aspergillus soge (s) comprising the introduced acetamidase S (amdS) gene as a selectable marker.
Aspergillus sojae). Such A. Soje (A.
sojae) is selectable on a medium comprising a substrate of the introduced amdS protein as the sole source of nitrogen, said medium further comprising a carbon substrate, and said medium comprising endogenous amdS. Contains no inducing substrate. A suitable medium comprises acrylamide as a substrate for the introduced amdS as the sole source of nitrogen. A suitable medium is Aspergillus
U.S. sojae) at least further comprising the minimal substrate required for growth. A. of the present invention A suitable category of A. sojae is A. sojae, which is not selectable on medium comprising acetamide. It is formed by A. sojae. A. of the present invention A. sojae is suitably A. sojae, which is selectable on glucose-free medium, i.e. medium whose carbon source is not glucose. Soje (A
． Sojae). Such a medium can be a medium having sorbitol as a carbon source. Best results with sorbitol are achieved when sorbitol is the only carbon source.

The Aspergillus sojae of the present invention may comprise an additional introduced nucleic acid sequence, said further introduced sequence preferably encoding a protein or polypeptide. The additional introduced sequences may be adapted to codon usage optimized for the codon usage of the host strain, or may have the original codons from the host from which it was derived. The introduced sequence is, in principle, any sequence that one skilled in the art would like to express. The introduced sequence may suitably be heterologous, ie foreign to the Aspergillus sojae into which it is introduced. It can also be natural, but can be introduced in the form of one or more additional copies.

One of the subjects of the present invention is aimed at the expression of phytase or proteins having phytase activity. Numerous sequences are known to those of skill in the art for phytase sequence data. We have European patents EP684.313 and EP897.
010, International Patent Application No. WO 99/49022, European Patent No. EP911.
No. 416 and EP 897.985, which are incorporated by reference. These documents describe a variety of natural and modified phytase sequences. They also describe consensus sequences. One suitable embodiment is formed by the phytase sequence from Peniophora, either the native sequence or a modified version thereof. The new system is more versatile than conventional systems, and thus contains heterologous sequences, including those encoding phytase or proteins with phytase activity that were difficult to express in prior art fungal systems, It can be expressed in the novel system of the present invention.

The Aspergillus sojae (A) of the invention as defined in any of the embodiments defined above comprising an introduced amdS gene as a selectable marker.
The spergillus sojae) may suitably not have the active endogenous amdS gene. One such aspect of Aspergillus soje (Asp
ergillus sojae) can have, by way of example, an endogenous amdS gene comprising a mutation that inactivates the endogenous amdS. There may be any type of inactivating mutation known or considered to one of skill in the art. One suitable example of such an inactivating mutation is a deletion or disruption.
)). The mutation can inactivate a gene or gene product. Those of ordinary skill in the art will appreciate that numerous options are available to accomplish this, and that they can be easily accomplished.

In an alternative embodiment, the present invention provides a recombinant Aspergillus sojae that is free of the active endogenous amdS gene and further comprises the introduced amdS gene as a selectable marker. Is also directed. The recombinant Aspergillus soje of the present invention
lus sojae) is selectable on a medium comprising a substrate of amdS as the sole source of nitrogen, said medium further comprising a carbon substrate. A suitable medium is A. It further comprises at least a minimal substrate required for the growth of A. sojae. In a suitable embodiment, the endogenous amdS gene can be inactivated, for example. This inactivation is due to A. Soje (A.so
It can be any type of inactivation known or considered by one of skill in the art that still leaves jae) viable. By way of example, an endogenous amdS gene may be expressed in the form of a substitution, deletion or insertion of the gene or part thereof, or by a mutation affecting the expression of the gene, such as to render it inactive, It can comprise an inactivating mutation. The complete endogenous amdS gene may also be absent.

In any of the described embodiments of the invention Aspergillus soje (Asp
ergillus sojae) is an A. cerevisiae in which the amdS gene has been introduced. It can be a sojae. This can be achieved, for example, by transformation or transfection. The resulting Aspergillus sojae of the present invention is then transformed into A. non-transformed or transfected. Must be separated from A. sojae. Any of the above-described aspects, either on their own or in combination, are encompassed by the present invention.

The present invention relates to Aspergillus sojae.
Not only does it include itself, but A. Also included are methods of introducing a nucleic acid sequence into A. sojae. The method is based on Aspergillus
rgillus sojae) comprising exposing the nucleic acid sequence to a fungus in a manner known per se for the introduction of the nucleic acid sequence. Such modalities are described, for example, in A. It can be transformation or transfection of A. sojae. The method comprises introducing the amdS gene as a nucleic acid sequence,
The resulting transformed or transfected A. pneumoniae is then grown on medium lacking the substrate that induces endogenous amdS. Comprising the selection of A. sojae, said medium further comprising a substrate of introduced amdS as the sole source of nitrogen, and said medium further comprising a carbon substrate, The medium is
The desired A. lactis comprising the introduced amdS gene. While allowing A. sojae to grow, A. sojae lacks a functional amdS gene. Soje (A.
sojae) growth is eliminated. One suitable aspect of such a method involves applying a medium comprising a substrate for amdS other than acetamide. Suitably, such a medium comprises acrylamide as the sole source of nitrogen, as a substrate for the introduced amdS. Suitably, the medium of the method of the present invention comprises a carbon source other than glucose. Suitably, the medium for use in the method of the invention comprises a carbon source, preferably sorbitol as the sole carbon source. A suitable medium is A. It further comprises at least a minimal substrate required for the growth of A. sojae.

The method of the invention as defined above in any of the embodiments comprises the introduction of an additional nucleic acid sequence in addition to the amdS gene. The additional nucleic acid sequence encodes a protein or polypeptide, such as a phytase or a protein having phytase activity. The sequence is not necessarily non-Aspergillus soja (As
pergillus sojae) sequence, but A. It is also possible to include sequences from A. sojae. However, the introduced sequence is either not present in the untransformed strain or is otherwise A.
It is intended to indicate that it exists in a lower copy number than in A. sojae.

Of course, the subject invention also encompasses any Aspergillus sojae obtained by the method described above. fundamentally,
The method is introduced where the sequence is introduced where the a.d.s. In contrast to A. sojae, it is directed to the introduction of sequences capable of achieving the presence of sufficiently active amdS to function as a selectable marker.

The transformed or transfected A. A. sojae
The selection method of is also within the scope of the present invention. The method is carried out according to A. (without the active endogenous amdS gene as defined by any of the described embodiments). A. sojae is transformed into A. sojae in a manner known per se for transformation or transfection of fungi with nucleic acid sequences. A. sojae
Exposure to the method of transformation or transfection. The method involves the introduction of the amdS gene as a nucleic acid sequence, followed by the resulting transformed or transfected A. Comprising selection of A. sojae, said selection occurring on a medium comprising a substrate of introduced amdS as the sole source of nitrogen, said medium further comprising a carbon substrate. , The medium is the desired A. While allowing A. sojae to grow, A. sojae that was not transformed or transfected due to the inability of these to grow without the introduced amdS gene on selective medium. A. soja
Eliminate the growth of e). A suitable medium is A. It further comprises at least a minimal substrate required for the growth of A. sojae.

The present invention provides a recombinant Aspergillus sorghum.
It is also directed to a method of manufacturing ojae). The method is for example performed by transformation or transfection in a manner known per se. Soje (A.
sojae) comprising the desired nucleic acid sequence, which is flanked by sections of the endogenous amdS gene of sufficient length and homology to ensure recombination. After the introduction, the recombinant A.
The selection of A. sojae continues. The selection takes place with respect to a selectable marker contained in the desired nucleic acid sequence or transformed by co-transformation therewith, said selectable marker being introduced into said A. Soje (A
． Sojae). The flanking sequences can also be sequences that correspond to an endogenous amdS gene sufficient to ensure recombination. One of skill in the art can readily assess which sequences can suffice based on knowledge of hybridization and sequence data for the endogenous amdS gene. The recombination event eliminates the activity of endogenous amdS in both cases. The selectable marker may very suitably be pyrG, however, uracil is used instead of uridine in the selection medium.

[0028] A further aspect of the invention is Aspergillus soj exhibiting growth on a medium comprising uracil and fluoroorotic acid.
ae), wherein said A. A. sojae does not show further growth on media comprising uridine and fluoroorotic acid. This is It is meant that A. sojae exhibits uracil auxotrophy, is incapable of utilizing uridine, is pyrG negative and exhibits resistance to fluoroorotic acid. Uracil auxotrophy and fluoroorotic acid resistance can be rescued upon complementation with the active transduced pyrG gene. Such A. A. sojae may not contain an active endogenous pyrG gene. The pyrG-negative A. A. sojae can contain an endogenous pyrG gene with a mutation that inactivates it. The mutation can be any mutation known or considered to one of skill in the art,
Said mutation inactivates the pyrG gene or its expression product. Such mutations are, for example, the insertion of a nucleic acid sequence in a gene, the replacement of a part of the coding sequence of the gene, the deletion of a part of the coding sequence of the gene, or the deletion of the entire coding sequence of the gene. It can be in the form. Mutations can also be in the regulatory part of the gene. In the case of the Aspergillus sojae of the present invention having a mutated pyrG gene, the Aspergillus sojae is a wild type A. It may have a mutated nucleic acid sequence of the pyrG gene different from that of the A. sojae pyrG gene. A further aspect is
As such or in combination, the A. Soje (A.
sojae), wherein the pyrG-negative A. pneumoniae of the invention as described in some of the above embodiments further comprises any of the features described for any of the Soje (
A. sojae).

Transformed or transfected Aspergillus soje (As
The method of selecting pergillus sojae) is also within the scope of the invention. The method may comprise the pyrG-negative A. Comprising exposing A. sojae to a method of transformation or transfection with a nucleic acid sequence, the method comprising pyrG-negative A in a manner known per se for transformation or transfection. ． Soje (A
． Sojae), which comprises introducing an active pyrG gene. After the introducing step, the resulting transformed or transfected A. lactis on medium lacking uracil and fluoroorotic acid. The selection of A. sojae followed, and the medium was A. sojae. Further comprising at least the minimal substrate required for the growth of A. sojae, said medium comprising the desired A. sojae. Soje (A.s.
ojae) is allowed to grow, while it is not transformed or transfected by the inability of these to grow without uracil due to the inactivated pyrG gene. Eliminate the growth of A. sojae. In a suitable embodiment of such a method, the introduced active pyrG gene is flanked by identical nucleic acid sequence fragments, and the pyrG positive A. A. sojae is selected on medium without uracil and fluoroorotic acid. Then pyr
G-positive A. A. sojae was cultivated on a medium comprising uracil and fluoroorotic acid, thereby eliminating the pyrG gene introduced thereby and thus growing on a medium comprising uracil and fluoroorotic acid resistance PyrG negative A. Brings A. sojae.
In a suitable embodiment of the aforementioned method, the flanking sequences and the pyrG gene are the A. Due to the fact that it is homologous to a particular sequence in A. sojae, it is further flanked by sequences which direct the integration of the pyrG gene and flanking sequences into a particular position. This allows knockout of genes with specific sequences (if desired). Methods of creating knockout mutants per se are known to those skilled in the art. Any of the aspects of the selection method just described are Aspergillus sojae.
Can be further transformed or transfected with an additional heterologous nucleic acid sequence. The additional heterologous nucleic acid sequence preferably encodes a protein or polypeptide, and the Aspergillus soja (A) of the invention.
The same remarks are made here as to other aspects of the spergillus sojae) and the nature of these additional nucleic acid sequences in fungi in general, as made elsewhere in this description. The additional sequences can be introduced with the active pyrG gene either on the same vector or by co-transformation with the active pyrG gene to be introduced. Transformed or transfected A. The method of selecting A. sojae can also be carried out in combination with a method of introducing a nucleic acid comprising the introduction of a heterologous amdS gene in any of the aspects of the invention disclosed above. Naturally, the present invention is directed to the transformed or transfected A. Soje (A.s.
Ajae) of any recombinant obtained by the selection method of A. Soje (A.so
jae) is also included.

The present invention also provides a recombinant Aspergillus soge.
Sojae), which comprises the division of the pyrG gene,
Or a nucleic acid sequence comprising a sequence to be introduced flanked by corresponding sequences of sufficient length and homology to exclude the pyrG gene and ensure recombination to introduce the desired sequence, pyrG positive Aspergillus soje (Asp
transformation or transfection in a manner known per se of S. ergillus sojae), followed by A soge with a pyrG negative phenotype (
Comprising selection of recombinant Aspergillus sojae with the desired sequence by selecting for As. The determination of said corresponding sequences lies within the reach of the person skilled in the art, with their knowledge of the method of hybridization with nucleic acid sequences and their knowledge of the required sequence data of the pyrG gene.

In particular, the invention also relates to the A of the amdS variant of the invention as defined above.
Such Aspergillus soje (A sojae) characteristic
spergillus sojae). Thus, any Aspergillus sojae strain obtained by the method of introduction of either amdS and / or pyrG of the invention is within the scope of the invention, as is any subsequent use of such novel strains. It is a new strain. These new strains are based on the original corresponding Aspergillus Soje.
L. sojae strain or Aspergillus soje (As
pergillus sojae), Aspergilli
Alternatively, it can include nucleic acid sequences that are not even present in the fungus. The sequence can be of mammalian origin or can be from any animal, plant or microbial origin. The Aspergillus
S. sojae) A naturally occurring in S. but not corresponding transformed A
Nucleic acid sequences that are present in lower copy numbers in A sojae can also be expressed. Thus, where the pyrG and / or amdS Aspergillus sojae strains of the invention are required, the production of homologous proteins is also encompassed by the invention. In a preferred embodiment, the particular protein or polypeptide to be produced is the corresponding unprocessed A. A. sojae and / or corresponding unprocessed A. sojae. A. sojae, that is, A. sojae before introduction of the nucleic acid sequence. It exists in a smaller copy number in A. sojae. Aspergillus soja in a manner known per se for expressing proteins or polypeptides in fungi
Expression of the heterologous protein by any of the novel strains of e) includes sequences both native to the strain and foreign to the strain. Basically, only A sojae that are not naturally transformed or transfected are excluded from protection. The method of production comprises culturing the fungus under conditions suitable for the expression of the desired sequence to occur. The method of manufacture optionally includes a step of isolation of the resulting polypeptide or protein in a manner known per se for the production of the protein or polypeptide by a fungus. Preferably, the protein or polypeptide can be secreted into the medium.

A preferred protein or polypeptide is Black Aspergillus (Aspe).
rgillus niger) or Aspergillus awamori (Asper
It is a protein or polypeptide that is susceptible to degradation upon expression by gillus awamori. Many such proteins and polypeptides have already been disclosed in the prior art, and many remain to be determined. Such a determination, however, is a matter of routine to those skilled in the art. Another preferred embodiment of the protein or polypeptide to be expressed is that the protein or polypeptide is Aspergillus soge.
Ojae) protease and amylase. One preferred embodiment requires a non-Aspergillus sojae protein or polypeptide.

One embodiment of particular interest comprises the combination of two methods of introduction of the nucleic acid sequences of the invention as described above. Its advantage lies in the fact that the frequency of transformation obtained with the pyrG marker is clearly much higher than that of the amdS marker. However, secondary screening of the pyrG + strain for best growth on acrylamide selection plates revealed that recombinant Aspergillus sojae showed the highest copy number, and thus most likely the highest level of gene expression. Enables identification of

As shown in the examples, homologous and heterologous expression control sequences are used in Aspergillus
It can be used by Soger (Aspergillus sojae). That is, a naturally existing sequence of the strain itself or a sequence foreign to the strain can be used. Thus, the transformants of the present invention can include any such regulatory sequences. The choice of a suitable regulatory region is a matter of choice that lies well within the standard capabilities of the person skilled in the art, and can depend on the particular application. The regulatory sequences can be constitutive or inducible. The regulatory sequence can be fungal or non-fungal. A wide range is illustrated in the examples. A large number of expression control sequences are used uniformly in the art in other systems, especially fungal systems such as Aspergilli, and are customary in the present invention without undue burden. Can be applied to.

In order to introduce the desired nucleic acid sequence into Aspergillus sojae, any vector suitable for introducing the nucleic acid sequence into the fungal host cell can be used. Many examples are available in the art. In particular, black Aspergillus niger,
Vectors found to be suitable for transformation, transfection or expression in the genus Aspergilli such as Aspergillus awamori and Aspergillus oryzae can be suitably applied.

In addition to the above, the subject invention is a recombinant Aspergillus soje.
The efficient protein production of L. sojae) is described. Such efficient production is superior to ATCC42251, or ATCC9362, AT
Disclosed in strains that have at least as good a protease profile as either CC11906 and ATCC20387. The subject description is therefore based on A.I. of several known strains. We show that A. sojae is already well suited by itself for the production of proteins, polypeptides and metabolites. These Aspergillus sojae strains are based on the reference strain A.
It exhibits a lower proteolytic activity than A. sojae ATCC 42251. In particular, two known strains, ATCC 11906 and ATCC 2038.
7 is well suited. Therefore, the preferred A. cerevisiae for the production of proteins, polypeptides and metabolites. The A. sojae strain can be one that exhibits proteolytic activity that is comparable to or less than the two preferred strains. Stock A
TCC11906 is a prior art deposited ATCCA. A. soja
e) The best mode of strain. Suitable proteins or polypeptides can be produced. Since the subject invention allowed the introduction of nucleic acid sequences, these were used as expression hosts in A. A. sojae can be used to provide any protein or polypeptide to be selected.

The subject invention provides improvements over existing expression systems. Many existing protein production systems represent proteolytic expression problems. Among other things, the new system is the currently frequently applied expression system Aspergill.
us niger and Aspergillus
better than aawamori). The subject invention now makes it possible to provide a recombinant Aspergillus sojae comprising an introduced nucleic acid sequence encoding a protein or polypeptide for expression, said protein or polypeptide Is black Aspergillus (A. niger) or A. niger. It is susceptible to degradation during expression by A. awamori. The present invention also relates to recombinant A. cerevisiae comprising an introduced nucleic acid sequence encoding a protein or polypeptide for expression. Soje (
A. sojae) and said protein or polypeptide is A. Other than A. sojae protease and amylase. In a preferred embodiment, the introduced nucleic acid sequence is non-A. It encodes an A. sojae protein or polypeptide. The A. Soje (A.
sojae) strains are also within the scope of the invention.

In addition, a specific description of Aspergillus sojae strains modified to enhance their suitability as expression hosts is now provided. These modifications can reduce proteolytic activity when induced by any means. In particular, the use of UV random mutagenesis is illustrated. Specific mutations of one or more protease genes are also specifically described. The means by which mutations can be introduced are universal knowledge to those skilled in the art, and numerous alternative embodiments are therefore readily available to arrive at the desired mutation. One suitable aspect is formed by mutants with reduced alkaline proteolytic activity. Above all,
Elimination of the activity of the major 35 kDa alkaline protease is specifically illustrated as ensuring increased expression of proteins and polypeptides. In particular, the invention therefore also comprises new strains which exhibit a reduced proteolytic activity, in particular a reduced alkaline proteolytic activity. Such strains can be obtained using any particular mutation pathway known or considered to those of skill in the art. A preferred embodiment of such an expression host exhibiting reduced proteolytic activity as described above further comprises a selectable marker. Quite suitably, the selectable marker can be amdS, pyrG or a combination thereof.

The present invention provides, inter alia, A. pneumoniae by knocking out the 35 kDa alkaline protease gene. A method for producing mutants defective in the protease of A. sojae is included. There are numerous ways in which this could potentially be achieved based on the sequence data provided for this gene. Above all,
A method using recombination with a pyrG selectable marker linked to two flanking regions leading to a crossover of the 35 kDa alkaline protease gene, wherein the resulting strain has the pyrG selectable marker and loses the 35 kDa alkaline protease gene belongs to. Thereafter, the 35 kDa alkaline protease negative Aspergillus soje (Asperg) Soger (Asperg), which can be used for the purpose of excluding the pyrG selectable marker and thus thus expressing any desired sequence to be introduced therein.
illus sojae) mutants can be provided. Of course, the sequence to be introduced can already have been integrated in the previous step either on the same vector as the pyrG marker or in a cotransformation event. Also, the method is 3
An analogous procedure can be carried out if a protease gene different from the 5 kDa alkaline protease gene is to be knocked out. Similar measures to be taken will be apparent to those skilled in the art based on the specific description provided herein in combination with knowledge of other protease sequences. Also, similarly, the selectable marker for amdS can be used in accordance with the present invention as described elsewhere in this description.

Mutant fungi exhibiting improved fermentation characteristics are also provided as an additional aspect of the subject invention. In particular, the present invention relates to proprotein convertase (co
nvertase) or an equivalent protein is directed to the fungus comprising a mutation that inhibits the activity of the protein. Many proprotein convertases are known in the art. Among other things, we refer to Figure 1, which provides sequence data for many such proteins. The fungus of the invention is suitably of the genus Agaricus.
us), Aspergillus, Trichoderma (Tr)
ichoderma), Rhizopus genus, Mucor genus (Muc)
or), Phanetochaete, Trametes (T)
Rametes), Penicillium, Cephalosporium, Neurospora
ora), the genus Tolypocladium and the genus Thielavia. Particularly suitable fungi include Black Aspergillus niger, Aspergillus foetidus, Aspergillus sojae, Aspergillus sojae.
ergillus awamori), Aspergillus oryzae (Asperg
illus oryzae), Trichoderma reesei (Trichoderu)
ma reesei), Penicillium chrysosporum (Penicilliu)
m chrysosporum), cephalosporium acremonium (Ce
phallosporium acremonium), Neurospora crassa, Tolypocladium geodes (To)
Lypocladium geodes and Chielavia terrestris (T
Hieravia terrestris). A preferred embodiment comprises the mutant when it is Aspergillus sojae, the most particular preference being as defined above in the present invention, ie for example the selectable markers amdS and / or Alternatively, Aspergillus soge comprising an heterologous nucleic acid sequence in combination with pyrG.
s sojae).

A suitable equivalent of proprotein convertase is SEQ ID NO: 3 (= gene fragment encoding the amino acid sequence of the proprotein convertase of black Aspergillus (A. niger)), SEQ ID NO: 4 (= Aspergillus soge
40% or more of the deduced amino acid sequence of the DNA sequence shown in (partial gene fragment encoding the amino acid sequence of proprotein convertase of illus sojae) or any of the sequences shown in SEQ ID NOs: 5 to 9, preferably Is a protein or polypeptide representing an amino acid sequence having 45% or more similarity or identity. A functionally equivalent protein can suitably have a nucleic acid sequence capable of hybridizing under stringent conditions to the nucleic acid sequences of SEQ ID NOs: 3-9. Stringent hybridization conditions can be easily determined by those skilled in the art. One suitable example of stringent hybridization conditions is hybridization at 50 ° C. and preferably 56 ° C., and a final wash with 3 × SCC. PE4, PCL1
And PCL2 is the coding strand (ie SEQ ID NOS: 10, 11 and 12)
Among the examples of suitable oligonucleotide mixtures corresponding to PE6, PCL2-rev, PCL3 and PCL4 for the non-coding strand
(Ie SEQ ID NOS: 13, 14, 15 and 16, respectively). Use of these primers in amplification procedures universal in the art can provide equivalent sequences, and also to such uses and the resulting newly found sequences and those described in the subject description. Their use in a similar fashion is within the scope of the invention. In contrast, the sequences from which the oligonucleotides were made are well conserved so that they can be determined from a comparison of the various amino acid sequences for the provided proteins (see Figure 1). Any other nucleic acid sequence exhibiting the same or a higher degree of identity, similarity or homology with the sequences provided in the subject patent application for the protein or their associated active portion is the other proprotein. As with their use as primers or probes for finding sequences encoding invertases or equivalent proteins and / or subsequently introducing mutations in the sequences encoding such proteins, the present invention Included by As an example, Maniatis et al. (1982
) Molecular Cloning, A Laboratory manu
al, Cold Spring Harbor Laboratory (Cold Spring)
g Harbor Laboratory), New York, or any other guide to cloning and / or screening of nucleic acid sequences. Equivalent proteins or polypeptides can exhibit proprotein convertase activity, such as those having the amino acid sequences of SEQ ID NOs: 3-9. Mutant fungi can contain substitutions, insertions or deletions in the coding sequence of the proprotein convertase or equivalent protein.
The mutant fungus may suitably contain a mutation in the regulation of expression of the gene encoding the proprotein convertase or equivalent protein. The mutant fungi of the present invention in a suitable aspect exhibit reduced viscosity with respect to the corresponding non-mutated fungus under comparable culture conditions. Mutant fungi of any of the above embodiments which exhibit increased expression of the desired introduced nucleic acid sequence encoding a protein or polypeptide are included within the scope of the invention, said fungi being the corresponding wild-type. With respect to type fungi, it represents increased production of the protein or polypeptide under comparable conditions. A. It has been determined that the active site of the A. sojae proprotein convertase is contained within the amino acid sequence deduced by SEQ ID NOs: 3 and 4.

A method for producing a phytase or a protein having phytase activity, or any other protein or polypeptide, preferably a recombinant phytase or any other heterologous protein or any other polypeptide. Comprising culturing a mutant fungus of any of the embodiments described above) is within the scope of the invention. Also included are methods of obtaining the resulting protein or polypeptide, either from the cells themselves or after their secretion from the cells.

Transformation of any nucleic acid sequence encoding phytase or a protein having phytase activity, or any other protein or polypeptide thereto, and any nucleic acid sequence introduced therein. Subsequent expression of, and optionally also any subsequent use of any of the described novel strains for processing and / or secretion / and / or isolation is encompassed by the present invention.

Sequences encoding any phytase or phytase-like, or any other heterologous protein or polypeptide may be suitably used. It can be of fungal or non-fungal origin. A preferred embodiment is formed by sequences encoding acid labile proteins or polypeptides. Suitably, the protein-encoding sequence encodes a non-protease-like protein. Examples include phytase sequences, as well as Aspergillus soje (
Aspergillus sojae) shows a number of heterologous sequences suitable for use in transformation and also expression in a host. Further examples of suitable proteins to be expressed will be apparent to those of skill in the art.

The present invention is more specifically illustrated by the examples below. The examples should not be considered limiting to the interpretation of the scope of the invention. Alternative embodiments will be readily foreseen by those skilled in the art based on the description and knowledge of the relevant arts of the technology. The contents of the references cited in the description are incorporated by reference. The claims serve to illustrate the intended scope of the invention. Experimental Details for the Invention Construction of the Aspergillus sojae gene library.

A. Genomic DNA of A. sojae was labeled with ATCC1 using the protocol previously described (Punt, van den Hondal, 1992).
Isolated from protoplasts obtained from 1906. After isolation, Kolar et al., 1
DNA was extracted from protoplasts using the protocol described by 988. The DNA was then partially digested with MboI resulting in an average size DNA fragment of 30-50 kb.

The vector pAOpyrGcosarp used for the construction of the gene library
1 is Acc65I-BamHI digested pHELP1 (Gems et al., 199).
3 kb Ba from pANsCos1 (Osiewacs, 1994) in 1)
mHI-HindIII fragment and pAO4.2 (De Ruiter)
3.2 kb Acc65I-HindIII from Jacobs, 1989).
Constructed by ligation of fragments. This cosmid vector is an A. Orize (A
． oryzae) carrying the pyrG selection marker and self-replicating in filamentous fungi. The genomic DNA digested with MboI was digested with pAOpyrGco digested with BamHI.
It was ligated to sarpl and the ligation mixture was packaged into phage particles using the Stratagene Supercos 1 vector kit. A. Approximately 3 of the A. sojae genome
A total of 30,000 individual clones were obtained, corresponding to a 0-fold representation. The stock of pools of resulting clones (in 15% glycerol) was used -80 for later use.
Stored at ° C. amdS transformation method and transformants.

Two currently used protoplasting and transformation protocols [modified OM method (Yelton et al., P.N.A.
． S. 81 (1984) 1470-1474) and the NaCl method (Punt and V
an den Handel, Meth. Enzym. 216 (1993) 44
7-457)] to Aspergillus soj
ae) Strain ATCC 9362 tested. Both methods resulted in protoplasts, but the yield of viable protoplasts with the OM method was clearly better. The overall yield was lower than that normally obtained for black A. niger. Pilot protoplast formation / transformation experiments are based on OM
All A. It was carried out in A. sojae strain.

For transformation, the vector p3SR2 (carrying the amdS marker) was transformed into p
Used with AOpyrGcosARP1. This latter vector resulted in a highly increased number of (unstable) transformants when used as a cotransformation vector in all Aspergillus species tested to date, Aspergillus that replicates autonomously
Is a derivative of the vector Arp1. Sufficient protoplasts (approximately 10E6-10E7 per transformation) were obtained for almost all strains. Diverse A. Soje (A.s.
Analysis of the appropriate AmdS selection conditions for O. jae) strains showed vigorous growth of most strains on the universally used selective acetamide medium. Apparently, as described in International Patent Application No. WO 97/04108, A. Soje (
A. The proposed acetamide selection conditions for the amdS transformants of S. It was not suitable for selection of A. sojae transformants. Surprisingly, our experiments showed that only AmdS + transformants could be selected using acrylamide selection. Significant background from untransformed protoplasts was observed even on selective acrylamide plates. Selection of primary transformants required about 3 weeks, and many of the putative transformants initially selected were found to be false positives, showing background after transfer to fresh selective acrylamide plates. Only showed growth.
In order to optimize the selection of transformants, attempts had to be made to reduce the growth of this background. Improved results were obtained by omitting glucose from the selection plate. Table 3 shows the composition of the improved selective medium and the normal medium.
Figures 2a, b and c show background growth observed for selected strains on selection medium as described in WO 97/04108 and modified acrylamide selection medium as described in Table 3. Indicates.

Three selected A. Further transformation experiments with the A. sojae strain showed that the efficiency of protoplast formation for ATCC 11906 and ATCC 20387 was better using the NaCl method. Successful protoplast formation is achieved by NOVOZYM, Caylas.
e), obtained using various commercially available protoplast-forming enzyme preparations such as Glucanex and the like. Based on the NaCl transformation protocol, three selected A. The A. sojae strain was transformed with the amdS selection vector p3SR2 or its derivatives. A large number of actively growing transformants were obtained using modified acrylamide selection plates, while DNA
No growth was observed in the control transformation without. Another approach to avoid background growth of untransformed mycelium is wild type A. Elimination of the activity of the amdS gene of A. sojae. This is, for example,
A. It can be achieved by disruption of the amdS gene of A. sojae. As a first step, a specific D carrying the amdS sequence of ATCC 11906
The NA fragment was labeled with the published A. PCR amplified using primers derived from the amdS sequence of A. oryzae (Gomi et al .; 1991, Gen.
e 108, 91-98). Previous experiments have shown that cloning by stringent hybridization has been described in A. A. nidulans and A. nidulans. It was shown to be unsuccessful due to the low level of sequence conservation between the A. sojae amdS sequence. We obtained the expected fragment of approximately 1.6 kb, which should carry the majority of the coding region of the amdS gene.
Sequence analysis from both ends of the cloned PCR fragment (Figs. 3a and 3b
) Is A. The cloning of part of the amdS gene of A. sojae was confirmed. Stringent hybridization occurred at 56 ° C with a final wash in 3xSSC. The cloned sequence was published in A. It was very similar to the amdS sequence of A. oryzae. Using the cloned amdS fragment of ATCC 11906 as a probe, pAO
Several hybridizing clones (7 out of 10.000) were isolated from the cosmid library of ATCC 11906 in pyrGcosarp1. After subcloning the fragment carrying the complete amdS gene, the amdS
A portion of the gene was replaced with a reusable pyrG selectable marker to generate an amdS replacement vector. Aspergillus
sojae) Transformation of this vector into ATCC 11906PyrG
This resulted in G + transformants. After subsequent analysis of these transformants on acetamide and acrylamide selection plates, some of these transformants showed reduced background growth. Southern analysis of several of these strains
It was shown that the expected gene replacement had occurred. One of these strains was transformed into A. cerevisiae using acrylamide selection plates. A of A. nidulans
Used for subsequent transformation with the mdS gene and resulted in a large number of amdS + transformants. pyrG transformation method and transformants. (1) Initial experiment A. For A. sojae, pyrG as described in the introduction.
Standard experiments used in the prior art on other fungi to generate mutants have resulted in a large number of fluoroorotic acid (FOA) resistant strains. However, all of these strains are capable of growing on uridine-free medium,
And therefore was not considered a pyrG mutant. With our ultimate goal of isolating the appropriate mutant strains, we followed a number of alternative approaches. (2) Near homologous gene disruption A. sojae and A. sojae. The pyrG gene from A. oryzae is based on the expectation that it will be very similar in sequence (confirmed by Southern hybridization performed under stringent conditions).
ouka et al. (1996), using the approach previously described by A. A mutant version of the pyrG gene of A. oryzae was used. Experiments were performed to disrupt the pyrG gene of A. sojae. Stringent hybridization occurred at 65 ° C with a final wash in 0.3xSSC. The 0.5 kb ClaI fragment-bearing portion of the pyrG coding region was deleted, A. An A. oryzae pyrG disruption vector was constructed (FIG. 4). The XbaI pyrG fragment from this new vector was used for transformation and direct selection of FOA resistant transformants. None of the resulting FOA resistant colonies were uridine auxotrophic. (3) UV Mutagenesis and Filtration Concentration Another approach to improve the yield of certain mutant strains is the use of a filtration concentration step (Bos et al. 1986, dissertation dissertation, Wageningen Agricultural University (Ag.
critical University Wageningen)). U
V-mutagenized spores are used to inoculate a minimal medium (MM) liquid culture.
From the resulting repeated overnight cultures, spores that were unable to germinate in minimal medium (
a. o. (pyrG mutant spores) are separated from the mycelium grown by filtration through myraclos. The spores obtained after several enrichment steps were treated with their Pyr by inoculating the spores onto plates containing FOA.
Tested for G phenotype. Again, none of the resulting FOA resistant colonies were uridine auxotrophic. Also, none of the colonies obtained after this concentration on MM plates containing uridine were shown to be uridine auxotrophic.
(4) Modification selection conditions A. Our previous attempts to isolate the pyrG mutant from A. sojae failed, suggesting the inability to utilize the required exogenous uridine of the pyrG mutant, It is used for analysis of uridine auxotrophy in FOA selective medium. Modified selected FO now containing uracil next to uridine
Medium A was used in a new isolation attempt. From this approach, we obtained several FOA resistant mutants that were uracil auxotrophic. Retesting of these strains showed that they were unable to grow on minimal medium supplemented with uridine. Subsequent transformation experiments with some of the uracil auxotrophs showed that these mutants were in fact supplemented with the fungal pyrG gene (eg vector pAB4.1; black Aspergillus pyrG). It was shown that it was possible. The inability of the pyrG mutants to grow in minimal medium supplemented with uridine alone is due to the related species of Aspergillus (A
． A. nidulans, Black Aspergillus (A. nige)
r), A. It was an unprecedented observation for A. oryzae) and a variety of other fungal species. (5) Reusable selection marker A. The versatile genetic modification of A. sojae requires the possibility of modifying, disrupting and expressing a number of different genes in a single fungal strain, which makes use of a diverse array of selectable markers. It seems to need sex. However, the availability of a marker such as pyrG that allows for selection of both mutants (FOA selection) and transformants (medium lacking uracil) has been shown to repeat the same marker in subsequent experiments. Provide the possibility of use. For this approach, a pyrG marker gene was designed in which the complementary sequences were flanked by direct repeat sequences emanating from the 3'flanking ends of the pyrG gene. The resulting plasmid is pAB4-1rep. The construction of this vector is detailed in FIG. The complete sequence of the vector is shown in SEQ ID NO: 17. A. in this vector A. soj
Transformation of the pyrG mutant of ae) results in a similar number of PyrG + mutants with vector pAB4-1. However, subsequent plate culture of spores of selected pAB4-1 and pAB4-1rep transformants on FOA selection plates resulted in many more FOA resistant / pAB4-1rep transformants.
This resulted in uracil-requiring colonies. Southern analysis of these FOA resistant / uracil auxotrophic clones showed that in most of the pAB4-1rep strains, the black A. niger pyrG marker gene was deleted and a small 0.7 kb repeat at the integrated locus was deleted. In the pAB4-1 strain, the black Aspergillus (A. niger) gene is still present and pyrG
It was shown that they had probably acquired a mutation that resulted in a negative phenotype. Expression host: strain selection. Protease Production A very important feature of fungal expression systems is the level and type of fungal proteases produced under a variety of culture conditions. Occasionally, easily transformable strains are not suitable as expression hosts due to the production of proteases or acidification of the medium that are deleterious to the expressed product. Diverse A. Analysis of growth behavior of A. sojae strains, in contrast to what was observed for black A. niger, showed that acidification of the medium was based on agar-based plates (Macconkey (MacConkey).
Conkey)) did not occur either on or in shake flask cultures. In fact, in shake flask cultures, in most cases even an alkaline pH was obtained in culture, irrespective of the three media types analyzed (Table 4). Based on these results and the literature data, therefore, mainly alkaline proteases from A. It is expected that it can be present in A. sojae broth. A milk clearing assay was performed to analyze the protease activity of cultures of various strains. in addition,
To assess the suitability of the tested strain as an expression host for a range of products, media samples were tested for various proteins (eg bovine serum albumin (BSA)).
Were incubated with and the degradation of these proteins was followed at just the right time. BSA was selected as in our previous experiment with Black A. niger. This protein has been shown to be very sensitive to proteases. A. A. terreus phytase was chosen as an example of another proteolytically unstable protein. Degradation of milk proteins, as evidenced by the formation of milky areas around growing colonies, is a generally accepted measure of protease activity. Detection of BSA is SDS-P
It was performed by Coomassie staining of AGE gel. For phytase, Western analysis was performed using specific antibodies. As shown in Table 4, a clear difference in degradation in black Aspergillus (A. niger) broth is due to this. Soje (A
． sojae) apparent when compared to that in culture. Rapid decomposition of BSA occurs in black A. niger culture (pH 3-4). A. from a richer media. Soje (A.s.
BSA degradation occurs in Ojae broth but nevertheless less than in black A. niger broth. Most A. ATCC 9362, ATCC in A. sojae culture solution (pH 7 to 8)
11906 and ATCC20387 except for A. Teleus (A.ter
Reus) phytase undergoes rapid degradation. In general, strains with minimal phytase degradation also show low BSA degradation under the conditions tested. In particular, two A. A. oryzae strains ATCC20235 and ATCC462
50 is most of the A. It shows much greater proteolytic activity than the A. sojae strain.

To exclude that the difference in pH of the cultures causes the observed effect, p
H to pH 4.5 (50 mM Na / HAc), pH 5.8 (50 mM Na / H)
Ac) and a culture solution adjusted to pH 8.3 (50 mM Tris / hydrochloric acid),
Similar decomposition experiments were also performed. Table 5 shows the degradation data obtained with these samples. As can be seen in the table, the A. elegans with the highest proteolytic activity at pH 7-8. A. oryzae ATCC 20235 also shows high proteolysis at other pH values. A. Degradation of A. terreus phytase occurs predominantly at pH 8. AT, similar to what was previously found
CC11906 and ATCC20387 showed low phytase degrading activity.
ATCC 9362 showed phytase degradation in rich media. A. BSA degradation by A. sojae showed no significant differences from the data presented in Table 4.

In conclusion, these protease assays consist of three low protease A. This resulted in the identification of the A. sojae strains ATCC9632, ACTT11906 and ATCC20387. Therefore, A. A. soja
e) can obviously be used as an expression host for a range of proteins and offers a series of advantages over prior art transformation and expression systems. Strain Improvement Since the transformability and expression potential of Aspergillus sojae was confirmed, it was considered a means by which additional strains with enhanced characteristics for expression could be created. .
In order to provide new and improved strains for the expression of proteins, two different approaches, which can be used on their own or in combination, have been developed.

On the one hand, the possibility of developing mutants defective in proteases was investigated and the effect of these on the level of expression was evaluated. On the other hand, strains with modified morphology were developed to improve the characteristics of fermentation. To accomplish this, a previously undisclosed or unsuccessful pathway that is applicable not only to Aspergillus sojae but also to Aspergilli and in general to fungi was followed. Development of Protease Deficient Mutants A. 2 to obtain A. sojae strain
The species approach was followed. In the first approach, ATCC 11906
And spores from strains derived from ATCC 11906 were UV mutagenized. In the second approach, a major alkaline protease gene disruption was performed. UV mutant A. Freshly harvested spores from A. sojae ATCC 11906 or one of its pyrG derivatives were UV mutagenized in a Biorad UV chamber using a dose that resulted in 20-50% survival.
Serial dilutions were plated on skim milk plates (Mattern et al., 1992). From the 5000 UV-surviving strains, four mutant strains were obtained with a significantly reduced emulsion loss halo. alpA gene disruption In this approach, the endogenous alpA (alkaline protease) gene cloned from ATCC 11906 was disrupted using a disruption vector carrying a reusable pyrG selectable marker as described in this description. did.

The ATCC 11906 cosmid library (in the pyrG cosmid) was constructed. From 10.000 independent clones, first 4 were primer MB
A. coli obtained by PCR with L1784 and MBL1785. Soje (A
． It was found to hybridize with the alpA fragment of Sojae) under homologous conditions. Rescreening of the 4 clones showed only strong hybridization with 1 clone. 4 kb EcoRI and 2.5 kb Hind from this clone, expected to carry the complete gene together
The III fragment was subcloned and characterized by restriction enzyme digestion and sequence analysis. Based on these subclones, new gene replacement vectors were constructed as described in FIG. For transformation of the ATCC 11906pyrG derivative, the vector was digested with EcoRI and the 8.7 kb alp
The A deletion fragment was used for transformation (see Figure 6). ATCC1
Transformation of the replacement cassette into 1906pyrG5 resulted in a large number of transformants with reduced milky areola. Southern analysis of these strains showed successful deletion of the alpA gene. To allow subsequent use of the pyrG marker to transform one of these strains, spores from this strain were plated on FOA-containing medium selective for the pyrG mutant. A large number of FOA resistant colonies were obtained from strains with a correctly integrated disruption cassette with a reusable pyrG marker. In contrast to the results obtained with spontaneous FOA resistant mutants of wild type strains, the FOA strains obtained from these disrupted strains are virtually all uracil auxotrophic and may again become pyrG negative. all right. Using Southern analysis, a
Confirmed the desired removal of the pyrG marker gene at the lpA locus, 700 bp
I left only the "footprint". Analysis of Protease Activity in UV and Disruption Mutants Controlled batch fermentation experiments were performed to analyze the level of protease production in various low protease derivatives of ATCC 11906. Protease activity was measured at various pH values from the resulting culture supernatant. The deletion of the alpA gene
It resulted in a strong decrease in proteolytic activity at alkaline pH. Analysis of protease activity with one of the UV mutants showed almost complete absence of proteolytic activity at both pH 6 and pH 8. As a result, the level of proteolysis on secreted proteins produced in these strains was significantly lower than that observed for the parent strains. Development of low viscosity mutants A. Initial controlled or fed-batch fermentation trials with A. sojae resulted in significant biomass yields, however, both the viscosity of the medium and the sporulation phenomenon in the fermentation vessel were less favorable properties. Was represented.

Therefore, attempts were made to improve these characteristics in the desired host strain.
Various patent applications teach that low viscosity mutants can be isolated by various screening methods. International patent applications WO 96/02653 and WO 97/26330 describe undefined mutants that exhibit low viscosity. However, here we are A. sojae
) Describes a new and unexpected example of a fully characterized and well-defined low viscosity mutant. A proprotein processing mutant from this organism has an unexpected aberrant growth phenotype (hyper-branch
ing)) while no detrimental effect on protein productivity was observed. Controlled fermentation experiments with this strain showed that increased biomass concentrations were obtained at much lower viscosity values. The observed features are A
． It was present not only in A. sojae, but also in other fungi, such as A. niger. (1) Construction of Proprotein Processing Mutant of Black Aspergillus (A. niger) In order to clone a gene encoding a proprotein converting enzyme from Black Aspergillus (A. niger), brewer's yeast (Saccharomyc) was cloned.
es cerevisiae) KEX2, Schizosaccharomyces pombe (Sc
hizosaccharomyces pombe) KEX1 and xenopus
Heterologous hybridization was performed using a specific probe from the Eenopus laevis PC2 gene. However, very low stringency hybridization conditions (47 ° C., 6 × SSC wash)
Even at, no specific hybridization signal was obtained, precluding the use of this approach to clone the corresponding black A. niger gene.

PCR was used as an alternative approach to clone the gene encoding the proprotein convertase from Black Aspergillus (A. niger). Based on a comparison of various proprotein convertase genes from various yeast species and higher eukaryotes (Figure 1), various PCR primers were designed (SEQ ID NO: 10, 1
3 and 18-23), which are respectively 2048, 49152, 4, 2, 2,
It is degenerated 512, 2048 and 4608 times. Primers PE4 and PE6
Two separate clones were obtained from amplification using S. cerevisiae KEX2 sequence (SEQ ID NO: 24).
). These clones were used for further experiments.

Based on the observed homologies of the cloned PCR fragments to other proprotein convertase genes, the corresponding A. niger
r) gene to pclA (proprotein convertase-like (proprotein c
(from the overtase-like)). Black Aspergillus (A.
Southern analysis of the genomic digest of A. niger showed that the pclA gene was a single copy gene with no closely related genes in the A. niger genome. This is because no additional hybridization signal was apparent, even under stringent hybridization conditions (50 ° C .; 6 × SSC wash). Black Aspergillus (A.
niger) N401 EMBL3 genomic library (van Hartin
The first screen of gsveldt et al., 1987) did not result in any positively hybridizing plaques, but screened approximately 10-20 genomic equivalents. In the second screen, a full-length genomic copy of the pclA gene was cloned into black Aspergillus (A. niger) in EMBL4.
It was isolated from the N400 genomic library (Goosen et al., 1987). 5
Of the 8 hybridizing plaques obtained after screening -10 genomic equivalents, 6 remained after the initial screen. All these 6 clones most likely carried one complete copy of the pclA gene. This is because there were two hybridizing EcoRV fragments of 3 and 4 kb (as observed for genomic DNA) in all clones carrying the PCR fragment (PCR fragment (SEQ ID NO: 24) was EcoRV restricted). Note that it contains sites). Based on size comparisons of other proprotein convertases, these fragments together yield 5
It can contain the complete pclA gene with the'and 3'flanking sequences.
The two EcoRV fragments and the 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 FIG.

Based on the restriction map shown in FIG. 7, the complete DNA sequence of the pclA gene was Ec
Determined from oRI and EcoRV subclones (SEQ ID NO: 3). Analysis of the sequences obtained revealed an open reading frame with considerable similarity to that of the S. cerevisiae KEX2 gene and other proprotein convertases. Based on further comparison, two putative intron sequences (SEQ ID NO: 3
, Positions 1838 to 1889 and 2132 to 2181) were identified in the coding region. Black Aspergillus based on pEMBLyex (A.
Subsequent PCR analysis on the Niger) cDNA library with primers flanking the putative intron showed that only the majority 5'of these two sequences represented the actual intron. The general structure of the encoded PclA protein was clearly similar to that of other proprotein convertases (SEQ ID NO: 25 and Figure 8). The overall similarity of the PclA protein with other proprotein convertases was approximately 50% (Figure 1).

To prove that the cloned pclA gene is a functional gene encoding a functional protein, an attempt was made to construct a strain lacking the pclA gene. Therefore, most of the pclA coding region is A. Py of A. oryzae
pPCL1A (pclA deletion vector) replaced with the rG selectable marker gene was generated. The 5 kb EcoRI insert fragment was then used from this vector to transform various black A. niger strains.
A number of transformants (based on pyrG selection) were obtained from these transformations. Interestingly, only a few transformants (variable from 1 to 50%) displayed a very different aberrant phenotype (Figure 9). Southern analysis of some wild type and aberrant transformants showed that these aberrant transformants, which displayed a severely restricted growth phenotype, were missing the pclA gene. All strains displaying wild-type growth were shown to carry one copy of the replacement fragment integrated adjacent to the wild-type pclA gene or in a non-homologous position.

Analysis of protein production in selected pclA mutant strains carrying diverse glucoamylase fusion genes showed production of unprocessed fusion proteins. High levels of production of unprocessed glucoamylase-interleukin-6 fusion protein in the pclA mutant were achieved. Protein analysis showed that the fully processed endogenous glucoamylase was also not formed in the pclA mutant strain, but only proglucoamylase was formed.

Controlled and fed-batch fermentations were also performed to further improve the yield of fusion protein. Surprisingly, the fermentation characteristics of the pclA mutant strain were clearly superior to those of the parent strain, resulting in a much reduced viscosity / biomass ratio with no loss of productivity. (2) A. Construction of Proprotein Processing Mutants of A. sojae To construct the corresponding mutants in A. sojae, AT
CC11906 Black Aspergillus (A. nigés) from cosmid library
Functional complementation of the low viscosity mutant of the genomic cosmid clone of r) (compl)
element), which is isolated from A. The proprotein processing protease pclA gene of A. sojae was included. Partial sequence analysis of the isolated sequence, SEQ ID NO: 4 was carried out according to A. The cloning of the pclA gene of A. sojae was confirmed. Figure 10 shows the black Aspergillus (A. niger)
) And A. 1 shows a comparison of DNA sequences of a portion of the pclA gene of A. sojae. A. The sequence of A. sojae, and A. sojae. Soje (A.s.
Ojae) based on a partial restriction map containing the coding region of the pclA gene, a reusable pyrG marker was cloned as an inner SmaI fragment for cloning. The EcoRV site in the pclA gene of A. sojae was used to generate a replacement vector (Figure 11). The resulting vector is ClaI
It was partially digested with to give a 10.5 kb Δpcl fragment (see Figure 11). This fragment is A. It was isolated for use in transformation of the A. sojae pyrG strain. AT using the gene replacement vector
The pclA mutation was generated in CC11906 and ATCC11906 derivatives. The resulting strain was used for expression of homologous and heterologous proteins. Controlled fermentation experiments with some of the resulting transformants showed improved fermentation characteristics, in particular a lower viscosity / biomass ratio of the culture. (3) Cloning of fungal gene homologous to pclA of Aspergillus (A. niger) and A. niger. A. sojae
Based on a comparison of the amino acid sequence deduced from the pclA gene with those of other proprotein processing enzymes (FIG. 1), several oligonucleotides corresponding to the coding or non-coding strand of the well-conserved sequence A mixture was designed (SEQ ID NO: 10-16).

These mixtures of oligonucleotides were added to Trichoderma reesei (Trich
odema reesei) QM9414, Fusarium venenatum (Fus)
Aridium venenatum) ATCC20334, Penicillium chrysogenum P2, Trametes versicolor, and Rhizopus oryzae of Rhizopus oryzae sorghum (Rhizopus oryzae) ATCC200076. PCR amplification with one or more combinations of oligonucleotides of the coding and non-coding strands (30 cycles; 1 min 94 ° C; 1 min 40 ° C; 2 min 68 ° C, depending on the template DNA used)
) Resulted in a specific PCR product. Table 6 shows the results of various amplification reactions. Sequence analysis was performed with a number of the resulting PCR fragments using either of the two oligonucleotide mixtures used for amplification. These analyzes have led to the identification of diverse pclA homologues from these diverse fungi. FIG. 12 shows the deduced amino acid sequences corresponding to the various DNA fragments (SEQ ID NOs: 5-9). (4) Example of Determination of Biomass and Viscosity The following operating parameter data range was determined for fungal fermentation using a number of different fungal strains. Viscosity: Viscosity is measured using a sensor system MV DIN (container number 7) operated at 20 ° C., Haake Viscotester VT50.
Measure at 0. A fresh sample of fermentation medium is obtained and 40 ml of medium is placed in the measuring cell. Viscosity is measured after a short period (4 minutes) of equilibration at the set spindle speed. This measurement is repeated for 10 different spindle velocities. The spindle speed is multiplied by the measured cell coefficient to yield the shear rate. The viscosity η (in centipoise = cP) is plotted against the shear rate γ (1 / s) and yields the viscosity profile of the fermentation medium.

Viscosity ranges were measured for fermentations using the specified fungal organisms using the above procedure (Table 7). Biomass: Biomass is measured by the following procedure: 5.5 cm filter paper (Whatman 54) in an aluminum weighing dish.
Is weighed in advance. 25.0 ml of total medium was filtered on a Buchner funnel through 5.5 cm filter paper, the filter cake was washed with 25.0 ml of deionized water, the washed cake and filter were placed in a weighing dish and at 60 ° C. overnight. dry. Drying is completed at 100 ° C. for 1 hour, and then placed in a desiccator and cooled. Weigh the dried material. Total biomass (g / l) is equal to the difference between the initial and final weight multiplied by 40. Protein: Protein levels were measured using the BioRad assay procedure. The data presented above represent values measured during the fermentation process up to the end of fermentation for 48 hours; Aspergilli and Trichoderma (
All values for Trichoderma) are for commercially relevant fungal organisms and reflect actual commercial data.

A. A. sojae lfvA and A. sojae. A. sojae
Fungal strains, such as pclA, have low viscosity but low power input (low power i).
nput) and / or shear during fermentation has the advantage of meeting the oxygen demand where shear stress to the product may be detrimental to productivity due to physical damage to the product molecules. Lower biomass production with high protein production indicates a more efficient organism in the conversion of fermentation medium to product. Therefore, A.
The A. sojae mutants are also clearly better for two typically deposited Aspergillus or Trichoderma reesei strains in common public collections. It delivers better biomass and viscosity data while delivering at least as much protein, and indeed much more, than the systems used commercially.

A. The high protein production with low biomass concentration produced by A. sojae lfvA results in the production of biomass prior to reaching the fermentation conditions where the desired product limits where the increased biomass results in increased productivity. It appears to allow the development of fermentation conditions with increasing numbers of multiples. T. This high level of biomass and viscosity produced by T. longibrachiatum and A. niger organisms increases the biomass because this level of biomass and viscosity almost limit actual fermentation conditions. To limit. Efficient gene expression (1) Heterologous regulatory sequences Three selected A. Various strains of fungal expression signals (A. nidulans PgpdA; pGUS54, black Aspergillus (A. niger) PglaA; pGUS64, black Aspergillus (A. niger) PbipA; pBIPGUS) were used for S. sojae strain. 3 types of GU to carry
S-reporter vector, and amdS selection vector p3SR2 or its derivative (s) were cotransformed. Expression of the GUS gene was analyzed in representative transformants (Table 8). From the results it is clear that under the conditions tested, the gpdA promoter was by far the best promoter yielding about 5000 U GUS / mg protein. This corresponds to about 5% of the total cellular protein. The bipA promoter confers about 30% of gpdA activity, which corresponds to expression data obtained with A. niger. Surprisingly, the glaA promoter, which is highly active in Aspergillus niger (A. niger) (at least as active as gpdA), is expressed in A. Soje (A
． Sojae) resulted in less than 1% of gpdA activity. (2) A. Regulatory sequences of A. sojae We also know that Aspergillus soj
The cognate promoter of ae) was also isolated and the applicability of these in expression systems was also evaluated. In some instances of expression, it may be preferable to use a cognate promoter rather than a heterologous promoter. It was also interesting to evaluate whether a homologous promoter could be more efficient than a heterologous one.

Three efficiently expressed A. A. sojae gene, ie a
P of lpA (alkaline protease; inducible), amyA (amylase; inducible) and gpdA (glyceraldehyde-3-phosphate dehydrogenase; constitutive)
CR cloning was performed using A. Attempts were made using primers based on the sequences available from A. oryzae (SEQ ID NOs: 26-31). Figures 13a, b and c show the sequences of various PCR primers used in this approach, and published A. The position in the A. oryzae sequence is indicated. A. Soje (
A. Sojae) Genomic template DNA from ATCC 11906 was used for PCR amplification. First PCR amplification (30 cycles; 1 minute 94 ° C; 1 minute 40 ° C; 2 minutes 68 ° C)
) Resulted in a specific PCR product of the expected size of gpdA (400 bp). No product was obtained for the other two PCR reactions. Therefore, alpA
For, the PCR conditions were made less stringent (10 cycles; 1 min 94
1 minute 25 ° C; 2 minutes 68 ° C and 20 cycles; 1 minute 94 ° C; 1 minute 40 ° C; 2 minutes 6
8 ° C.), which resulted in a specific alpA PCR product of approximately 1000 bp.

The complete sequence of the cloned gene was determined. As shown in FIG.
A. The gpdA promoter region of A. sojae ATCC 11906 has very high homology with other gpdA promoter sequences and has alpA homology.
The promoter is A. It was virtually identical to the alpA promoter of A. oryzae (SEQ ID NOs: 32 and 33). These A. A. soj
ae) An expression vector carrying an expression cassette comprising a promoter shows a significant level of gene expression. Heterologous Protein Production A number of heterologous proteins known to be sensitive to acidic proteolysis and therefore unable to be efficiently expressed in other known expression systems were tested. Also, the proteins that were already efficiently expressed in the alternative system may be compared with other known expression systems, such as Aspergillus niger, by comparison with Aspergillus soge (Aspergillus soge).
jae) was tested to assess the level of expression achieved. Phytase Production A DNA fragment carrying various fungal phytase (Wyss et al. (199
9) Appl. Environ. Microbiol. 65, 359-366)
At the 5'NcoI or BspHI site-A. The 3 ′ downstream of the A. nidulans gpdA promoter was ligated into pAN52-1NotI as a blunt-ended fragment. The resulting vector was cloned into A. coli using the amdS and / or pyrG selectable markers. Used in A. sojae cotransformation experiments. Transformants producing phytase were screened using the phytase plate assay described.

A further improved phytase expression vector was generated using the multicopy cosmid approach. In this approach, several copies of the phytase expression cassette are added to the multi-cloning site vector (pMTL series, Chamber).
rs et al. (1988) Gene 68, 139-149) and recloned into E.
Allowed its isolation as a coRI fragment. Several copies of these Ecs
packaging the oRI fragment (Verdoes et al. (1993) Tra
nsgenic Research 2, 84-92) and cloned into the cosmid vector pAN4cos1 resulting in a cosmid clone carrying multiple expression cassettes. The resulting clones were cloned into A.
It was introduced into A. sojae. AmdS + clones were screened for phytase production using the phytase plate assay.

Additional phytase expression vectors were generated using the approach of GLA fusion (eg Broekhuijsen et al. (1993) J. Biotech.
31, 135-145). To this end, mature A. Fumigatus (A. fumi)
phytase gene fragment encoding the phytase protein of
It was fused to the 3'end of the glucoamylase carrier gene in Genbank accession number Z32700). Between the glucoamylase and phytase parts of the gene fusion, a proprotein processing site (Asn-Val-Ile-
A sequence encoding Ser-Lys-Arg) was introduced. The resulting vector is a
A. using mdS and / or pyrG selectable markers. Soje (A.so
jae) used in co-transformation experiments. Transformants producing phytase were screened using the phytase plate assay described.

Shake flask fermentations were performed resulting in significant levels of active phytase. Yields are based on the literature on A. niger (van H).
artingsveldt et al. (1993) Gene 127, 87-94; Va.
n Gorcom et al. (1991) EP 420 358) was significantly higher. On average, the levels obtained with the multicopy cosmid vector were higher than those obtained with the single copy vector. The levels of phytase obtained with the glucoamylase-phytase fusion vector resulted in high levels of both glucoamylase and phytase. A. which produces a selected number of phytases. Controlled and fed-batch fermentations from A. sojae transformants showed even increased levels of phytase. Glucoamylase Production One example of an efficiently produced fungal protein is Black Aspergillus (A. ni.
ger) provided by expression of the glaA gene. Vector pGLA6S (Fig. 15
) Is A. as a selection marker. Amd of A. nidulans
The 5 kb EcoRI fragment carrying the S gene was cloned into pGLA6 (Punt
(1991) J. Biotech. 17, 19-334) only EcoRI
It is generated from pGLA6 by introducing it into the site. A. Nidulans (A
． nidulans) gpdA promoter under the control of the amdS selectable marker and the glucoamylase gene carrying the vector pGLA6S (FIG. 15) using A. A. sojae
) Introduced into ATCC 11906pyrG. The starch plate assay demonstrated the production of increased levels of amylose degrading activity in these transformants. The resulting transformants, which showed good growth on acrylamide medium, were analyzed for glucoamylase production. A single predominant protein band corresponding to glucoamylase was observed on Coomassie Brilliant Blue stained SDS PAGE gels from the culture supernatants of some of these transformants. Monoclonal antibody against glucoamylase (Verdoes et al. (1993) Tra
Western analysis using nsgenic Research 2, 84-92) was used to confirm the identity of this protein band.
Production of Interleukin-6 Interleukin-6, an example of a protein that is highly sensitive to proteolytic degradation, produces interleukin-6 without the use of gla fusion strategies and strains defective in proteases. ) Has shown that it is virtually impossible. Even with all these improvements, the yield of IL-6 was only a few mg / l of culture. A. by cotransformation with the pyrG or amdS markers. IL-6 vector pAN56-4 (for A. sojae)
Broekhuijsen et al. (1993) J. Am. Biotech. 31,134-
Introduction of 145) resulted in transformants expressing the IL-6 fusion gene present in this vector. From the resulting transformants, several were selected for further analysis. Shake flask fermentation experiments were performed with these transformations. SDS-PAGE and Western analysis of the culture supernatants of some of these strains surprisingly revealed that they were approximately 5-10 fold above the levels obtained in the best reported case with A. niger. Higher, correctly processed IL-
6 levels were shown. A. The use of mutants defective in various types of proteases from A. sojae and optimized for fermentation further increased the level of IL-6 production that should be obtained from controlled fermentation ( Broekhuijse
(1993) J. et al. Biotech. 31, 134-145). Green Fluorescent Protein (GFP) Another type of acid-labile protein that was attempted to be produced in A. sojae was a jellyfish, Aequoria Victoria (Aequor).
GFP from ia victoria). This protein is not only proteolytically sensitive, but additionally it loses its activity at acidic pH. GFP or GLA-GFP fusion protein (A. nidulans (A
． nidulans), which is driven by the gpdA promoter), was transformed with A. It was introduced into A. sojae. Expression is bright and fluorescent for both vector types. This resulted in A. sojae transformants. Based on the observed fluorescence and subsequent analysis of culture supernatants from selected shake flask cultured transformants using SDS-PAGE and Western analysis, intact cytoplasmic GFP and secretory GLA-. The yield of GFP was found to be due to a host (Sieden) defective in a black A. niger protease.
berg et al. Biotech. Prog. (1999) 15, 43-50; Go.
rdon et al., Microbiology (2000) 146, 415-426).
It was confirmed to be much larger than that obtained in. In contrast to the situation in black A. niger culture supernatant, secreted GFP also showed significant fluorescence.

[0071]

[Table 1]

[0072]

[Table 2]

[0073]

[Table 3]

[0074]

[Table 4]

[0075]

[Table 5]

[0076]

[Table 6]

[0077]

[Table 7]

[0078]

[Table 8]

[0079]

[Table 9]

[0080]

[Table 10]

[0081]

[Table 11]

[0082]

[Table 12]

[0083]

[Table 13]

[0084]

[Table 14]

[0085]

[Table 15]

[0086]

[Table 16]

[0087]

[Table 17]

[0088]

[Table 18]

[0089]

[Table 19]

[0090]

[Table 20]

[0091]

[Table 21]

[0092]

[Table 22]

[0093]

[Table 23]

[0094]

[Table 24]

[0095]

[Table 25]

[0096]

[Table 26]

[0097]

[Table 27]

[0098]

[Table 28]

[0099]

[Table 29]

[0100]

[Table 30]

[0101]

[Table 31]

[Sequence list]

[Brief description of drawings]

FIG. 1 shows an X. X. laevis (XENPC2 and XN
FURIN), brewer's yeast (S. cerevisiae) (SCKEX2),
K. K. lactis (KLKEX1), C.I. Albicans (C.
albicans) (CAKEX2), S. S. pombe (SPK
RP) and Y. Y. lipolytica (YLKEX2)
10 provides a comparison of the amino acid sequences of KEX2-like processing proteases from.
The primers encoding the amino acid sequences with the highest overall identity (indicated by the light blue box) are shown: MBL793, MBL1208, MBL794, MBL1.
158, PE6, PCL1, PCL2 (rev), PE6, PCL3, MBL7
89, PCL4 and MBL1219. Area of overall identity (7 kinds of recording (en
4 types of try) are shown in a purple box. The gap is. Is shown; the sequence data is absent; * is the stop codon of the protein.

[Fig. 2]   It consists of 2a, b and c according to the invention.

FIG. 2a: Patent WO97 / 04 according to the invention after 5 days incubation at 33 ° C.
A.108. Provides background growth of A. sojae strains. The top picture shows growth on non-selective medium. The picture on the bottom left is W
The O97 / 04108 selection medium is shown, and the lower right picture shows the results using the modified medium of the present invention (acrylamide).

FIG. 2b: In accordance with the invention, after A. Soje (A.so
jae) provides background growth of strain ATCC 11906. The top picture shows growth on non-selective medium. The lower left picture shows the selection medium of WO 97/04108, and the lower right picture shows the results using the modified medium of the present invention (acrylamide).

FIG. 2c: In accordance with the invention, after A. Soje (A.so
jae) provides background growth of strain ATCC20387. The top picture shows growth on non-selective medium. The lower left picture shows the selection medium of WO 97/04108, and the lower right picture shows the results using the modified medium of the present invention (acrylamide).

Figure 3 (a and b) A. from both ends according to the invention. A. sojae ATCC119
06 and A. A comparison of the amdS sequences of A. oryzae is provided.
A and B indicate two ends. The 1.6 kb cloned A. Soje (A.
sojae) sequence was used. Bold bases underlined differ between species / strains. The sequence of intron I is shown in lower case.

FIG. 4 (a and b): Inventive pyr mediated by pAO4-13 and pAO4-13ΔCla.
The construction of the G disruption vector will be specifically described.

FIG. 5: pA proceeding from pAB4-1 via isolation of XhoI and HindIII fragments according to the invention, followed by cloning into pMTL24.
The construction of B4-1rep will be specifically described.

FIG. 6 discloses in this figure the construction of the alpA gene replacement vector according to the invention. AT
4.4 kb Ec from pAS1-1 containing CC11906 genomic fragment
oRI-StuI fragment, 2.6 kb Sma from pAB4-1rep
I-NcoI fragment and the 4.4 kb NcoI from pAS1-2A
-The EcoRI fragment is ligated in a 3-way ligation, thus providing pAS1-Δalp.

Figure 7 provides a restriction map of a DNA fragment carrying the black A. niger pclA gene according to the invention.

Figure 8 provides the structure (functional composition) of a protein encoded by A. niger pclA in black according to the invention. From left to right, pre, professional,
The activity and P domain are indicated. Lightly colored triangles indicate KR sites. Darkly colored triangles indicate glycosylation sites. The thin box with vertical stripes is S /
This is an area rich in P / T. The darkly corrugated box on the far right is the D / E rich region.

Figure 9 illustrates the growth phenotype of a black A. niger pclA mutant strain according to the invention.

FIG. 10 is a schematic diagram of an A. A. sojae and Black Aspergillus (A.n)
iger) of the pclA gene. Vertical bars indicate identity ;: indicates 5; -indicates 1. 72.139% similarity and 72.
A 073% identity was found.

FIG. 11 Discloses in this figure the construction of the pclA gene replacement vector according to the invention. AT
The 7.6 kb ClaI fragment, a CC11906 genomic fragment, was cloned into pMTL23p. In this construct, the 2.6 kb SmaI fragment from pAB4-1rep was cloned into the EcoRV site, thus providing pAS2-Δpcl.

FIG. 12: S. cerevisiae (Sckex2) according to the invention
, K. K. lactis (Klkex1), A. Soje (A.so
jae) (Aspcla), black Aspergillus (A. niger) (A. ni
ger), P.G. P. chrysogenum (Penpcl1
), A. A. bisporus (Agarmbl129), T.
T. reesei (Trichpcl1), R. Orize (R.or
yzae) (Rhizpcll), F.I. F. venenatum
(Fuspcl1), S. S. pombe (Spkrp), C.I. C. albicans (Cakex2) and Y. Lipolytica (
Y. lipolytica) (Ylkex2) from various PclA homolases (
The comparison of the amino acid sequences of The areas of overall identity (8 out of 12 recordings) are shown in yellow boxes. The gap is. ． Indicated by; if there is no sequence data, indicated by.

FIG. 13 is a schematic diagram of an A. Sequence data for the A. oryzae alpA promoter sequence (Q11755) is provided in Figure 13a. Primer positions for PCR cloning are indicated. In Figure 13b, the A.
Sequence data for the amyA promoter sequence of A. oryzae (A02532) is provided. Figure 13c shows ATCC4, which also includes primer positions.
2149 A. GpdA promoter sequence derived from A. oryzae (
European Patent Application EP 0.436.858 a1) is provided.

FIG. 14: Diverse gpd of Aspergillus according to the invention.
A comparison between A promoter sequences is provided: from top to bottom, A soge (A so
jae) ATCC 11906, A. A. oryzae, black Aspergillus (A. niger) and A. oryzae. A. nidulans.
* Indicates a putative intron present in the 5'untranslated region of the promoter. Yajiri (
Arrowhats) indicate CT-rich regions. Bold, underlined letters indicate A. A. oryzae and A. oryzae. The differences between the sequences of A. sojae are shown.

FIG. 15   1 shows a map of the 12700 bp vector pGLA6S according to the invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) (C12N 9/16 C12R 1:66) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, Z, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Herikhuizen, Margrate Netherlands Enuel-3705 H.E.H. Zeist Johann Van Olden Barnebel Trahn 52-1 (72) Inventor Van Den Hondell, Cornelis Antonius Maria Jacobs Johannes Netherlands Enuel-2804 Pieszt Gouda Waterrelly 124 (72) Tomorrow Punt, Peter Jan The Netherlands Enuel-3994 Etsxstay Houten Vogttorzkersgilde 1 (72) Inventor Van Bizen, Nikolaas Adrián Netherlands Enuel-3939 Diar Houten Futenbaide 16 (72) Inventor Albers , Alwines René Netherlands Enuel-3706 Giebe Zeist Ran Bambolenhobe 1535 (72) Inventor Vogel, Kurt Swiss Seeh-4102 Binengen Drenbatsuhyu Trase 91 F term (reference) 4B024 AA11 BA11 CA04 CA07 DA11 EA04 FA10 GA11 GA25 GA27 4B050 CC03 DD03 LL10 4B065 AA60X AA60Y AB01 AC20 BA02 BA18 BA23 BA25 CA31

Claims (35)

[Claims] 1. Recombinant Aspergillus soge comprising the introduced acetamidase S (amdS) gene as a selectable marker.
rgillus sojae). 2. Selectable on a medium comprising a substrate of introduced amdS as the sole source of nitrogen, said medium further comprising a carbon substrate, and said medium being endogenous amdS. The Aspergillus sojae according to claim 1, which does not contain a substrate that induces. 3. The method according to claim 1, wherein the nitrogen source is acrylamide.
Aspergillus sojae described in 1. 4. Aspergillus soj
ae) is, for example, active endogenous amdS because the endogenous amdS gene comprises a mutation (eg deletion or disruption) that inactivates the endogenous amdS.
An Aspergillus sojae according to any of the preceding claims, which does not have a gene. 5. Aspergillus soj
ae) is a method of introduction of nucleic acid sequences (eg Aspergillus in a manner known per se for introduction of nucleic acid sequences into fungi).
S. sojae) transformation or transfection), wherein the method comprises the introduction of the amdS gene as the nucleic acid sequence (futurely introduced amdS gene), followed by a substrate for inducing endogenous amdS. Resulting Transformed or Transfected Aspergillus on Free Medium
Comprising the selection of Soger (Aspergillus sojae), the medium further comprising a substrate of introduced amdS as the sole source of nitrogen, and the medium further comprising a carbon substrate, the medium comprising , While allowing the desired Aspergillus sojae comprising said nucleic acid sequence to grow, such Aspergillus sojae (Aspergillus sojae)
The growth of Aspergillus sojae, which does not contain so-called introduced nucleic acid sequences, is eliminated due to the inability to grow on selective medium without the introduced amdS gene of G. A method of introducing a nucleic acid sequence into Aspergillus sojae, suitably comprising a substrate for amdS other than acetamide (eg acrylamide as a substrate for the introduced amdS) as the sole source of A. 6. An Aspergillus sojae obtained by the method according to claim 5. 7. Aspergillus according to any one of claims 1-4 and 6.
Comprising exposing Soger (Aspergillus sojae) to a method of transforming or transfecting Aspergillus sojae with a nucleic acid sequence in a manner known per se for fungal transformation or transfection. The method comprises introducing an amdS gene as the nucleic acid sequence, then resulting transformed or transfected Aspergillus soj on a medium comprising the introduced amdS substrate as the sole source of nitrogen.
ae) and said medium further comprises a carbon substrate, said medium comprising the desired Aspergillus sojae.
), While Aspergillus soge was not transformed or transfected due to the inability of these to grow on selective media without the introduced amdS gene.
A method for selecting transformed or transfected Aspergillus sojae that eliminates the growth of jae). 8. Introduction of a nucleic acid sequence into an Aspergillus sojae, for example by transformation or transfection in a manner known per se according to any of claims 1-4 and 6.
The nucleic acid sequence is sufficiently long and homologous to segment the endogenous amdS gene, or to eliminate recombination at the same time to ensure recombination and to introduce the desired sequence. A desired sequence to be introduced flanked by sequences that are desired to be introduced), and then by selecting for a selectable marker contained in or transformed by co-transformation with the desired sequence. Recombinant Aspergillus soge with the sequence
jae), wherein said selectable marker is not present in Aspergillus sojae prior to the introduction of said nucleic acid sequence, suitably the selectable marker is pyrG. Aspergillus sojae manufacturing method. 9. No further growth on the medium comprising uridine and fluoroorotic acid (ie uracil auxotrophy), no availability of uridine, pyrG negative and fluoro. Represents resistance to orotate (the uracil auxotrophy and fluoroorotate resistance can be rescued upon complementation with the active PyrG gene introduced), suitably without the active endogenous PyrG gene; For example, the Aspergillus Soger
The endogenous PyrG gene of (illus sojae) comprises uracil and fluoro, comprising mutations in the form of insertions, substitutions or deletions in the gene or gene regulatory sequences (eg deletions of the entire coding sequence of the gene). Aspergillus soje (Aspergillus) expressing growth in a medium containing orotic acid
s sojae). 10. An Aspergillus sojae according to claim 9 in combination with the features of Aspergillus sojae according to any of claims 1-4 and 6.
. 11. The Aspergillus sojae according to claim 9 or 10.
Exposing Aspergillus sojae) to a method of transformation or transfection with a nucleic acid sequence, said method being in a manner known per se for transformation or transfection of fungi, Aspergillus sojae. ) To the active pyrG gene, followed by selection of the resulting transformed or transfected Aspergillus sojae on medium lacking uracil and fluoroorotic acid, said medium comprising Aspergillus sojae. (Aspergillus sojae), further comprising at least a minimal substrate required for the growth of Aspergillus sojae, said medium comprising illus sojae is allowed to grow, while the inactivated pyrG gene prevents the growth of Aspergillus sojae which is not transformed or transfected by the inability of these to grow without uracil. Method for selecting transformed or transfected Aspergillus sojae to eliminate. 12. The active pyrG gene to be introduced is flanked by identical nucleic acid sequence fragments, and the pyrG-positive Aspergillus sojae resulting from the introduction of the pyrG gene and adjacent sequences.
) Was selected on medium free of uracil and fluoroorotic acid, and was then pyrG positive Aspergillus soge.
jae) is cultivated on a medium comprising uracil and fluoroorotic acid to eliminate the pyrG gene introduced thereby, thus growing and fluoroorotic acid resistance on a medium comprising uracil. Can be selected by
results in a rG-negative Aspergillus sojae, suitably flanking sequences and the pyrG gene are the Aspergillus sojae into which the integration instruction sequence is to be transformed.
) By a sequence which directs the integration of the pyrG gene and flanking sequences at a particular position by virtue of the fact that it is homologous to the particular sequence and thereby allows the knockout of a gene related to that particular sequence if desired. Claim 1 further adjacent
The method according to 1. 13. The Aspergillus sojae according to claim 9 or 10.
Aspergillus sojae) has one additional nucleic acid sequence introduced into it, preferably said additional nucleic acid sequence encoding one protein or polypeptide, said additional nucleic acid sequence being identical with the active pyrG gene. 13. A method according to claim 11 or 12, which is introduced either on the vector of claim 1 or by co-transformation with the active pyrG gene introduced. 14. A method for selecting transformed or transfected Aspergillus sojae by carrying out the method according to any one of claims 11 to 13 in combination with the method according to claim 5. 15. A pyrG-positive Aspergillus soge (Aspe), for example by transformation or transfection in a manner known per se.
Introducing a nucleic acid sequence into r. gills sojae (wherein the nucleic acid sequence is p
a segment of the yrG gene, or comprising a desired sequence flanked by corresponding sequences of sufficient length and homology to ensure recombination that excludes the pyrG gene and introduces the desired sequence), A recombinant Aspergillus soje having the desired sequence is then selected by selecting for Aspergillus sojae with a pyrG negative phenotype.
us sojae) comprising recombinant Aspergillus sojae
Aspergillus sojae) manufacturing method. 16. A method according to any of claims 11-15, optionally further comprising the features of Aspergillus sojae according to any of claims 1-4, 6, 9 and 10. Recombinant Aspergillus sojae obtained by the method
). 17. A degraded nucleic acid sequence comprising an introduced nucleic acid sequence encoding a protein or polypeptide for expression, wherein said protein or polypeptide is degraded upon expression by Aspergillus niger or Aspergillus awamori. Responsive to Recombinant Aspergillus Soje
lus sojae). 18. An introduced nucleic acid sequence encoding a protein or polypeptide for expression, said protein or polypeptide other than Aspergillus sojae protease and amylase, wherein said protein or polypeptide The recombinant Aspergillus sojae (Asp), wherein the peptide is preferably a non-Aspergillus sojae protein or polypeptide
ergillus sojae). 19. A mutant or recombinant Aspergillus sojae comprising a mutation that inactivates a protease gene, suitably an alkaline protease gene. 20. A mutation that inactivates the major protease gene, suitably a mutation that inactivates the major alkaline protease gene (eg, the gene encoding the major alkaline protease gene of 35 kDa). ,
Mutant or recombinant Aspergillus
s sojae). 21. Aspergillus soge, for example by transformation or transfection in a manner known per se.
sojae) the introduction of a nucleic acid sequence comprising a sequence encoding a selectable marker to be introduced flanked by sections of the protease gene to be eliminated, and further comprising said flanking sequences and The sequence encoding the selectable marker is of sufficient length to ensure recombination with the protease gene and at the same time eliminate the protease gene and introduce the sequence encoding the desired selectable marker. And A. of the recombinant by inclusion within the homologous sequence and selecting for the selectable marker after the introduction. A. soja
The selection of e) is followed, whereby A. A. is introduced before introduction of said nucleic acid sequence, for example by transformation or transfection. A. sojae does not contain a selectable marker to be introduced (eg, A. soge before introduction of the nucleic acid sequence).
ojae) is an A. A. sojae is mutated such that it cannot produce an active selectable marker), suitably the selectable marker is p
the recombinant A. yrG gene, which is suitable for use with recombinant A. A. sojae (A of the recombinant)
． A method for producing proteolytic activity with reduced A. sojae. 22. A recombinant Aspergillus sojae obtained according to the method of claim 21. 23. A selectable marker, preferably 1-4 or 6
21. 20 or 22 comprising amdS as defined in any of the above and / or pyrG as defined in claim 9, 10 or 16.
The mutant or recombinant Aspergillus sojé (A
spergillus sojae). 24. An incorporated nucleic acid sequence encoding a phytase or a protein having phytase activity, claims 1-4,6,9,10,1.
6-20, 22 and 23, wherein the recombinant Aspergillus
Soje (Aspergillus sojae). 25. A recombinant or mutant Aspergillus sojé (
Aspergillus sojae), wherein the introduced nucleic acid sequence, suitably encoding a protein or polypeptide, comprises a corresponding untransformed or wild type form of A. 1-4, 6, 9, 10, not present in A. sojae and / or present in a lower copy number.
16-20, 22-24 or any of claims 5, 7, 8, 1
1-15 and 21. The recombinant or mutant A. A method for expressing an introduced nucleic acid sequence encoding a protein or polypeptide contained in A. sojae. 26. A recombinant fungus comprising a mutation in the gene encoding a proprotein convertase or a functionally equivalent protein. 27. Demonstrating increased production of a protein, polypeptide or metabolite under comparable conditions when compared to the corresponding wild type fungus.
Fungus according to. 28. The fungus according to claim 26 or 27, wherein the mutation is obtained by modification of a particular gene using transformation or transfection in a manner known per se. 29. The proprotein convertase or functionally equivalent protein can be amplified by in vitro DNA amplification using any of the two mixtures of nucleotides set forth in SEQ ID NOs: 10-16. 29. The fungus of claims 26-28, encoded by a nucleotide sequence. 30. Aspergillus ni, wherein the proprotein convertase or functionally equivalent protein comprises a mutation that inhibits the activity of the proprotein convertase or functionally equivalent protein.
ger) a fungus as described in claim 27, encoded by a nucleotide sequence that permits functional complementation of the growth phenotype of the mutant. 31. The fungus is Aspergill.
The fungus according to any of claims 26-30, which is Us sojae). 32. The amdS gene or pyrG introduced with the fungus.
31. The fungus of any of claims 26-30, which further comprises a gene. 33. A method of expressing a protein or polypeptide encoded by a nucleotide sequence, preferably a recombinant protein or polypeptide, comprising culturing a fungus according to any of claims 26-32. 34. A protein or polypeptide, preferably comprising a method of expression according to claim 33, optionally comprising processing and / or secretion and / or isolation of the expressed protein or polypeptide. A method for producing a recombinant protein or polypeptide. 35. A phytase or phytase activity comprising an expression method according to claim 33, optionally comprising processing and / or secretion and / or isolation of the expressed phytase or protein having phytase activity. A method for producing a protein having the above, preferably a recombinant phytase or a recombinant protein having a phytase activity.