JP2003000240A - Dna encoding transcription factor for controlling solid culture-expressing gene of koji bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a DNA encoding transcription factors for controlling a solid culture-expressing gene of koji bacteria, enabling the efficient production of exogenic proteins in a solid culture and also the comprehensive improvement of an enzyme production in the liquid and solid culture of the koji bacteria. SOLUTION: This gene encoding transcription factors (sgbR1, sgbR2 and sgbR3) for controlling the solid culture-expressing gene of koji bacteria is cloned and their base sequences are determined.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein that binds to a solid culture responsive cis factor of Aspergillus oryzae and controls transcription of a gene downstream of the factor, a gene encoding the protein, a recombinant vector containing the gene, and the recombinant vector. A transformant containing a vector, a recombinant koji mold containing the gene, a method for producing a transcription factor protein using the transformant, a method for expressing a protein in the koji mold and controlling the production of a solid culture-expressed gene group Is.

[0002]

2. Description of the Related Art The transcription of genes of all organisms including Aspergillus oryzae is
It is performed by NA polymerase. RNA polymerase uses double-stranded DNA as a template to polymerize ribonucleoside phosphate toward the 3'direction in a primer-independent manner. There are three types of enzymes in eukaryotic RNA polymerase,
Type I controls the synthesis of rRNA, type II controls the synthesis of mRNA, and type III controls the synthesis of tRNA. Eukaryotic RNA polymerases, by themselves, cannot direct specific transcription from the correct position,
It requires a group of basic transcription factors and binds to a region called a promoter involved in maintaining the basic level of transcription. However, the amount of RNA synthesized by RNA polymerase generally fluctuates according to various culture conditions, that is, cell growth and changes in the external environment. The response to this change in the external environment is carried by a transcriptional regulatory factor that positively or negatively controls the initiation of transcription of RNA polymerase by binding to a region called an enhancer of the gene regulatory region.

There are many reports on such gene expression control in filamentous fungi including Aspergillus oryzae. Examples of such a mechanism include an amylase induction mechanism by starch of Aspergillus oryzae, an amylase inhibition mechanism by glucose, and a protease inhibition mechanism by ammonium. This has been clarified mainly by experimental systems centered on liquid culture.

However, Aspergillus oryzae of the genus Aspergillus such as Aspergillus oryzae, Aspergillus soja, and Aspergillus awamori have been conventionally used in sake brewing such as sake, soy sauce, miso, and mirin brewing, and the culture form for its use. Is a solid culture called koji. The feature of this solid culture is that protein productivity is much higher than that of liquid culture. It is known in the brewing industry that many useful proteins are not produced in liquid culture but are produced in large quantities specifically in solid culture.

In recent years, it has been reported that one of the causes is a gene specifically expressed in solid culture. For example, in the production of glucoamylase from Aspergillus,
Glucoamylase gene (gla) expressed in liquid culture
Strong expression of the glaB gene different from A) in solid culture demonstrated that a large amount of enzyme protein was produced in solid culture. However, the control mechanism of gene expression in solid culture, that is, the transcriptional regulatory factor has hardly been analyzed, and it is clear why solid culture-specific genes such as glaB gene are expressed only in solid culture. is not.

[0006] If a transcriptional regulatory factor that controls gene expression in such solid culture can be obtained, the mechanism of high expression in solid culture will be clarified, and it will be possible to study to further increase the expression level. Further, even in liquid culture in which the gene is not originally expressed at all, it becomes possible to express the gene in a large amount. That is, these transcription control factors are important factors that determine gene expression and enzyme production in solid culture,
The acquisition of transcriptional regulatory factors is desired for efficient substance production in Aspergillus.

[0007]

SUMMARY OF THE INVENTION The present invention is intended to solve the above problems. It is an object of the present invention to provide three types of transcription control factors that bind to solid culture responsive cis factor of Aspergillus and control the transcription of genes downstream of the factor. Another object of the present invention is to provide DNAs encoding such three types of transcription control factors. Another object of the present invention is to provide a recombinant vector containing this gene and a transformant containing this recombinant vector. Another object of the present invention is to produce a transcription factor protein using the above transformant and to comprehensively control the production of a solid culture-expressed gene group by expressing the protein in a koji mold.

[0008]

The present invention has been made in order to achieve the above-mentioned object. First, aspergillus (Aspergillus oryzae: Aspergillus).
s oryzae). Aspergillus A. oryzae
Is a filamentous fungus that has been used in Japan's traditional fermentation industry, such as sake, soy sauce, and miso. A feature of this strain is that it produces a very large amount of useful proteins by solid culture in the fermentation industry. Due to the high protein-producing ability of this strain and its safety as a brewing microorganism, it has attracted attention as a host for heterologous protein production (Biotechnology, 6, 6.
1419 (1988), JP-A-62-272988).

However, there are many reports on such heterologous protein production of Aspergillus oryzae by liquid culture. Despite the fact that the culture form in the brewing industry is solid culture, the reason why heterologous protein production is often carried out in liquid culture is
The gene expression mechanism in solid culture has many unclear points compared to liquid culture. However, solid culture produces a larger amount of protein secretion than liquid culture,
In addition, the enzyme group produced in solid culture has a characteristic that there are many kinds. Therefore, if we can elucidate the gene expression mechanism in solid culture at the molecular level,
Since ancient times, it has become possible to produce heterologous proteins through very useful solid culture.

The inventors have already reported that A. oryzae has succeeded in producing a heterologous protein in solid culture using the glaB promoter that is specifically expressed in solid culture.
It was confirmed that the productivity of genes of the near species such as the genus gillus is further increased. In particular, A. or
The yzae gene was cloned into A. It was found that when expressed under the control of the oryzae glaB promoter, a very large amount of protein was produced by solid culture.
(Japanese Patent Application No. 11-154271, Japanese Patent Application 2000-3675)
4) Therefore, if a transcriptional regulatory factor that controls a promoter sequence specifically expressed in solid culture, which is represented by the glaB promoter, can be clarified, it will lead to improvement in productivity of heterologous protein of Aspergillus oryzae using these solid cultures. For the first time, I focused on the point that it has great industrial significance.

The present inventors have investigated the expression conditions of the gene glaB, which is specifically expressed in solid culture of Aspergillus oryzae.
An attempt was made to analyze a reporter in which the E. coli uidA gene was ligated immediately after the laB promoter sequence. As a result, gla
It was revealed that the B promoter was enhanced in solid culture by low water activity, high temperature and impaired hyphal elongation. In addition, environmental factors such as low water activity, high temperature and impaired hyphal elongation during solid culture are gla.
We investigated which region in the B promoter is responsible. Various deletion mutants of the glaB promoter were prepared, and a reporter assay was performed on each of them to identify the region responsive to the above environmental factors.

A site-specific deletion of 97 bp located 350 to 254 bp upstream of the start codon in the glaB promoter abolishes the environmental factor responsiveness of low water activity, high temperature and impaired hyphal elongation in solid culture. This 97
It was suggested that bp is an essential cis factor for the glaB promoter to be expressed specifically in solid culture. In order to confirm this, an experiment was performed to insert this 97 bp region into a heterologous gene promoter. Gla, which is specifically expressed in Aspergillus oryzae in liquid culture and is rarely expressed in solid culture
As a result of inserting the 97 bp region at the -205 position of the A promoter, this chimeric promoter became strongly expressed in solid culture. This indicates that the above 97 bp region is an essential cis factor in order to express specifically in solid culture. Next, it was confirmed whether introduction of a plurality of 97 bp regions into Aspergillus oryzae causes a deficiency of transcriptional regulatory factors and causes a phenomenon called promoter titration.

As a result, after breeding koji mold in which multiple copies of this region were introduced into koji mold, solid culture of rice koji was carried out and various enzyme productivity was examined. As a result, the Aspergillus oryzae produces not only glucoamylase but also α-amylase, acid protease, acid carboxypeptidase, phytase, etc. with an enzyme production amount of 2 per cell relative to the parent strain.
It fell to 0-40%. This 97 bp region is bound by a transcriptional regulatory factor that positively regulates gene expression in solid culture, and the transcriptional regulatory factor that binds to this region is produced not only by glucoamylase but also by other solid culture. It was speculated that the expression of the enzyme group is also controlled. So, this 9
We obtained a new idea that the acquisition of a transcriptional regulatory factor that binds to the 7-bp region will lead to the elucidation of the regulatory mechanism of the genes expressed in solid culture of Aspergillus oryzae.

It has been suggested that such a transcription control factor recognizes environmental factors such as low water activity, high temperature and impaired hyphal elongation in solid culture. This factor is stressed by temperature and drought and hyphal elongation. The possibility of responding to distress stress was suggested. It has already been found that in plant Arabidopsis thaliana, many structurally C ₂ H ₂ type zinc finger proteins are responsive to stress. Further, it is said that about 1% of all genes in mice are C ₂ H ₂ type zinc finger proteins. Therefore, we extracted the C ₂ H ₂ type zinc finger protein from the Aspergillus oryzae EST library.

As a result, three types of C ₂ H ₂ type zinc fin which exist only in solid culture using wheat bran
The ger protein was successfully extracted from the EST clone. One is C, which controls the stress response of Saccharomyces cerevisiae.
_It showed homology to MSN2, which is a ₂ H ₂ type zinc finger protein, and was named sgbR1. The other is a C ₂ H ₂ -type zinc finger that binds to the upstream region of the Fusarium cutinase gene.
It showed homology with the er protein and was named sgbR2. The last one is the fission yeast Schizosacc
indicates unknown function C ₂ H ₂ type of zinc finger protein homologous aromyces pombe, and was named SgbR3. These three C ₂
In order to examine whether the H ₂ -type zinc finger protein binds to the 97 bp region,
The protein was expressed in E. coli.

As a result of gel shift assay using the expressed protein, sgbR1 and sgbR1
It was confirmed that two factors of gbR2 bind. In addition, the co-suppression method was used to determine the functions of these three transcription control factors, and koji mold in which the expression of each gene was suppressed was constructed. As a result of performing solid culture,
Not only glucoamylase, but also α-amylase, acid protease, acid carboxypeptidase,
With respect to phytase and the like, the enzyme production per bacterial cell was reduced to 30-70% with respect to the parent strain. Therefore, it was revealed that both transcription control factors positively control the expression of the gene group specific to solid culture. The present inventors have found a new viewpoint that gene expression in solid culture can be freely controlled by regulating the activity of these transcription factors.

The details of the present invention will be described below.

The present inventors have already identified the glucoamylase gene gla as a gene that is specifically expressed in solid culture of Aspergillus oryzae.
It was reported that B was discovered (Japanese Patent Laid-Open No. 10-8496).
8). Furthermore, in order to determine the environmental factor for expressing this gene in solid culture, expression conditions of the glaB promoter were examined using Escherichia coli-derived β-glucuronidase (GUS) as a reporter (H. Ishida et al., Curr. Genet., 37,
p.373-379, 2000).

That is, as a promoter analysis system for developing a high expression system, specifically, FIG.
Promoter analysis plasmid pNGUS as shown in
Was used. This plasmid is A. oryzae niaD gene (ES Unk
le et al., Mol. Gen. Genet. 218, p.
99-104, 1989) and a reporter gene E. coli β-glucuronidase (GUS) encoding the uidA gene (RA Jefferson et al., Pr.
oc. Natl. Acad. Sci. , P. 8447-
8451, 1986). Uid of this plasmid
Upstream region of A gene (for example, Sa1I and PstI sites)
Various gene promoters to be studied or a part thereof were inserted into A. oryzae (Aspergillus
oryzae O-1013: This strain was transferred to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (now, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) under FERM P-
16528 already deposited), niaD mutant strain (nitrate-utilizing strain, Nitrate Reduct)
ase deficiency: For example, Aspergillus oryz
ae 1013-niaD: This strain is FERM P-17 at the Institute of Biotechnology, Institute of Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center).
(Deposited as 707), and a transformant in which only one copy of the introduced plasmid was introduced into niaD locus of the host chromosome was selected. Then, the GUS activity of these transformants was measured and used as an index of the promoter activity. The transformant obtained by introducing the promoter analysis plasmid pNGUS into E. coli is
It has been named Escherichia coli IN 113, and has been deposited as FERM P-18254 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) of the Ministry of Economy, Trade and Industry.

As a result of examining the expression conditions of the glaB promoter using the above analysis system, the glaB gene was found to be
It was clarified that its expression is enhanced by factors of low water activity, high temperature, and hyphal elongation disorder in solid culture (JP-A-11-225746). In order to identify the region responding to environmental factors of low water activity, high temperature, and hyphal elongation disorder in solid culture, various deletion mutants of glaB promoter were prepared. As a result, due to the deletion of 97 bp existing upstream from the start codon 350 to 254 bp, the glaB promoter activity in solid culture was completely lost, and this 97 bp was an environmental factor for low water activity, high temperature and impaired hyphal elongation in solid culture. It was identified to be a response area (Japanese Patent Laid-Open No. 11-243965).

Next, it was examined whether the 97 bp region (the nucleotide sequence of which is shown in SEQ ID NO: 7 (FIG. 21) of the sequence listing) actually positively regulates gene expression in solid culture. This region has a weak expression in solid culture. oryza
It was inserted into the -205 bp position of the e-derived glaA promoter, and the resulting chimeric promoter was subjected to GUS reporter assay (Fig. 1). As a result, the GUS expression level of the chimeric promoter in solid culture was increased about 50 times that of the native one. Therefore, it was revealed that this 97 bp region actually positively controls gene expression in solid culture.

Next, a promoter titration experiment was carried out by introducing multiple copies of this 97 bp region into Aspergillus oryzae. The fragment in which the 97 bp region was repeated in 8 copy tandem was A. nigerans derived argB marker together with A. oryzae M-2
-3 (arg-). As a result of Southern analysis, this book 9
A transformant having 80-300 copies of the 7-bp region was obtained. As a result of solid culture of 5 strains among these transformants, as shown in FIG. 2, not only glucoamylase but also α-amylase, acid protease,
Regarding acid carboxypeptidase, phytase, etc., the enzyme production amount per bacterial cell with respect to the parent strain was 20-40.
Fell to%. It was speculated that this is because the transcription control factor was insufficient because a large number of transcription control factors bound to the artificially introduced 97 bp region, and as a result, the enzyme production amount decreased.

From these results, a transcription control factor that positively controls gene expression in solid culture binds to this 97 bp region, and the transcription control factor that binds to this region is not only glucoamylase but also other It was presumed that the expression of the enzyme group produced in the solid culture of the above was also controlled. Therefore, the present inventors considered that the acquisition of a transcriptional regulatory factor that binds to this 97 bp region would lead to the elucidation of the regulatory mechanism of the genes expressed in solid culture of Aspergillus oryzae.

It has been suggested by the present inventors that such a transcription control factor recognizes environmental factors such as low water activity, high temperature and impaired hyphal elongation in solid culture. It was suggested that it may respond to such stress and hyphal elongation disorder stress. Already in plants, Arabidopsis is structurally a C ₂ H ₂ type of zinc finger.
It has been found that many er proteins are responsive to stress. Therefore, we selected a C ₂ H ₂ type zinc finger from the Aspergillus oryzae EST library.
The er protein was extracted. As a result, they succeeded for the first time in extracting EST clones of three types of C ₂ H ₂ -type zinc finger proteins that exist only in solid culture using wheat bran.

The first is the C ₂ H ₂ type zinc of budding yeast.
It shows homology with MSN2 which is a finger protein. It is known that MSN2 binds to a sequence called STRE in Saccharomyces cerevisiae and positively regulates the expression of multiple stress-responsive genes. The other is Fusar
C ₂ H ₂ binding to upstream region of cutinase gene of ium
It showed homology with the type of zinc finger protein. This is a transcriptional regulatory factor that positively regulates the expression of cutinase secreted when Fusarium genus infects plant leaves, and is considered to be a factor in which Fusarium genus responds to an adhesion signal to plant leaves. The last one is the fission yeast Schizosaccharomyces
C ₂ H ₂ type zinc with unknown function of s pombe
It showed homology with the finger protein. The partial cDNAs of the three C ₂ H ₂ type zincfinger proteins were used as probes. A genomic gene was cloned from the oryzae λEMBL3 library.

As a result of performing these DNA sequences,
A transcription factor homologous to MSN2 has its amino acid sequence shown in SEQ ID NO: 1 (FIGS. 6 to 8) and its nucleotide sequence shown in SEQ ID NO: 2 (FIGS. 9 and 10). As shown, it was a gene encoding 717 amino acid residues and having no intron. In addition, this gene is S. c
ST of various stress response genes of Erevisiae
It shows homology with MSN2 binding to the RE sequence. S this
It was named gbR1.

On the other hand, the transcription factor clone having homology with the Fusarium cutinase regulatory factor has its amino acid sequence shown in SEQ ID NO: 3 (FIGS. 11 to 13) and its nucleotide sequence shown in SEQ ID NO: 4 (FIG. 14, FIG. As shown in 15), it was a gene encoding 597 amino acid residues and having one intron as shown in SEQ ID NOs: 3 and 4. This was named sgbR2.

The last fission yeast Schizosaccharomyces pomb
be) Function of unknown C ₂ H ₂ type zinc finger
The transcription factor clone showing homology with the er protein has its amino acid sequence shown in SEQ ID NO: 5 (FIGS. 16 to 18),
Its base sequence is shown in SEQ ID NO: 6 (FIGS. 19 and 20), and as shown in SEQ ID NOs: 5 and 6, it was a gene encoding 468 amino acid residues and having two introns. This was named sgbR3.

In order to determine the function of these factors, the koji mold in which the expression of each gene was suppressed was constructed by using the cossablation method. SgbR is downstream of the histoneH2A promoter that is constitutively highly expressed in Aspergillus oryzae.
1, 500 from the start codon of sgbR2 and sgbR3
The partial fragment of bp was ligated, and this was nidulan
Using the sC marker of s, it was introduced into Koji mold in multiple copies. As a result of carrying out solid culture using the obtained koji mold, not only glucoamylase but also α-amylase, acid protease, acid carboxylpeptidase, phytase, etc. as shown in FIG. In all cases fell to 30-70%. Therefore, it was revealed that these transcription control factors are factors that positively control the expression of gene groups specific to solid culture.

Next, it was confirmed by gel shift assay whether these genes have the binding ability to 97 bp which is a cis factor of the glaB promoter. sgb
MR prepared from solid-cultured cells using steamed rice to obtain cDNAs for R1, sgbR2 and sgbR3
RT-PCR was performed based on NA. The restriction enzyme site BamHI (sg
bR1), EcoRI (sgbR2), SmaI (sg
bR3) was added. The obtained clone was designated as pUC118.
Of the expression vector (pSGBR1, pSGBR1,
(named pSGBR2 and pSGBR3) was used for Escherichia coli JM10
Transformed into 9. The transformants obtained by culturing in an ampicillin-containing medium and selecting an ampicillin-resistant strain were respectively transformed into Escherichia coli (Escherichia coli).
coli) SGBR1, SGBR2, SGBR3
And named it, National Institute of Advanced Industrial Science and Technology
FERM P-1 at the Patent Biological Deposit Center
8357, FERM P-18358, FERMP-1
Deposited as 8359. This transformant is sgbR
1, which contains genes encoding sgbR2 and sgbR3 proteins, and by IPTG induction, lacZ
SgbR1, sgbR2, sgb under the control of promoter
It was confirmed that the R3 protein was expressed.

In the gel shift assay, a cell-free preparation of Escherichia coli was used as the protein, and gla was used as the probe.
97 bp double-stranded D which is a cis factor of B promoter
The dimer of NA was labeled with fluorescein. After incubating at room temperature for 30 minutes, polyacrylamide gel electrophoresis was performed. The electrophoresis gel was alkali-transferred to a positively charged membrane, and fluorescein was detected by an antigen-antibody reaction. As shown in Figure 5, sgbR
A delayed band was confirmed by binding of 1 and sgbR2 with 97 bp which is a cis factor of the glaB promoter. Also, this band disappeared due to the addition of excess unlabeled probe. Therefore, it was confirmed that both sgbR1 and sgbR2 have the ability to bind to 97 bp, which is the cis factor of the glaB promoter.

From the above results, it was revealed that sgbR1, sgbR2 and sgbR3 are transcriptional control factors that positively control protein production in solid culture of Aspergillus oryzae.

By obtaining sgbR1, sgbR2 and sgbR3, which are the present transcriptional regulatory factors (sometimes referred to as transcription factors), it is possible to comprehensively improve enzyme production in liquid culture and solid culture of Aspergillus oryzae. Therefore, it was suggested that the gene is very promising industrially.

[0034]

Example 1 Identification of a region essential for high expression in solid culture in the glaB promoter It was clarified that a region essential for high expression in solid culture of the glaB promoter was 97 bp from the start codon 350 to 254 bp upstream by deletion mutation. . Next, an attempt was made to identify a region essential for imparting the function of high expression in solid culture.
97, 61, and 27 bp were inserted downstream from the start codon 350 of the glaB promoter into the glaA promoter, which expression is weak in solid culture, and the high expression ability of the chimeric promoter in solid culture was compared. The insertion position is glaA
It was determined at a position 205 bp upstream of the initiation codon in which the upstream end of Region IIIa, which is a cis factor essential for starch induction of the promoter, is present.

The above three kinds of fragments were inserted at the −205 position of the glaA promoter, and the resulting chimeric promoter was used as a plasmid pNGUS for promoter analysis.
Subcloning into the SalI site of (Fig. 22)
After transformation into Aspergillus oryzae using iaD as a marker, GUS assay in solid culture was performed. As a result, the chimeric promoter having 97 bp inserted showed a GUS expression level about 50 times that of the native one. Therefore, 97 from 350 to 254 upstream of the start codon of the glaB promoter.
It was proved that bp (SEQ ID NO: 7, FIG. 21) is a cis factor essential for high expression in solid culture (FIG. 1). In the figure,
GUS activity shows GUS activity (U / mg-protein), Glc is glucose and St is starch as a single carbon source.

[0036]

Example 2 GlaB promoter cis factor 97 bp
A promoter titration assay was carried out in order to confirm whether or not the transcription control factor involved in high expression in solid culture that binds to the cis factor 97 bp of the glaB promoter controls genes other than the glaB gene. A restriction enzyme site of SalI was added to the upstream side of 97 bp, and a restriction enzyme site of XhoI was added to the downstream side, and subcloning was performed into pBluescript II SK ⁺ . New 97bp on this XhoI site
By repeating the operation for binding the SalI site of 3 times, a 97 bp octamer was obtained. A. n
A. duluans-derived argB as a marker. or
It was introduced into yzae M-2-3 (argB-). As a result of Southern analysis, a transformant into which 80 to 300 copies of the 97 bp region were introduced was obtained.

As a result of solid culture of 5 of these transformants, as shown in FIG. 2, not only glucoamylase but also α-amylase, acid protease, acid carboxypeptidase, phytase, etc. 20-40% of enzyme production per body
Fell to. This is because a transcriptional regulatory factor that positively regulates gene expression in solid culture binds to the artificially introduced 97 bp region, and due to the lack of this transcriptional regulatory factor, efficient transcription of the enzyme gene group is not performed. It is thought to be a tame. Therefore, it was speculated that the transcription control factor binding to this region controls not only glucoamylase but also gene expression of other enzyme groups produced in solid culture.

[0038]

Example 3 Cloning of C ₂ H ₂ Type Zinc Finger Protein from EST Library A transcription factor that binds to 97 bp recognizes environmental factors such as low water activity, high temperature and impaired hyphal elongation in solid culture. It was suggested that this factor may be a factor that responds to stress such as temperature, drought and impaired hyphal elongation. It has already been found that in Arabidopsis plants, many structurally C ₂ H ₂ type zinc finger proteins are responsive to stress. Therefore, we selected C from the koji mold EST library.
Extraction of ₂ H ₂ type zinc finger protein was performed. As a result, three types of C ₂ H ₂ type zinc fingering that exist only in solid culture using wheat bran
An EST clone of the er protein was extracted.

These three C ₂ H ₂ type zinc fi
Using a partial cDNA of the nger protein as a probe,
Using the Gene Image kit (manufactured by Amersham Pharmacia), Aspergillus oryzae O-101
About 3000 lambda EMBL3 genomic DNA libraries of 3 strains (FERM P-16528) were screened by plaque hybridization to obtain positive clones.

As a result of cloning the above-mentioned genomic gene,
Transcription factors homologous to MSN2 include SEQ ID NO: 1 (amino acid sequence: FIGS. 6 to 8), SEQ ID NO: 2 (base sequence: FIG. 9,
As shown in 10), it was a gene encoding 717 amino acid residues and having no intron. In addition, this gene is S. It shows homology with MSN2 binding to the STRE sequences of various stress response genes of S. cerevisiae. This was named sgbR1. On the other hand, Fusari
A transcription factor clone homologous to the um genus cutinase regulatory factor is SEQ ID NO: 3 (amino acid sequence: FIGS. 11 to 13),
As shown in SEQ ID NO: 4 (base sequence: FIGS. 14 and 15),
It was a gene encoding 597 amino acid residues and having one intron. This was named sgbR2.
The last fission yeast Schizosaccharomyces
C ₂ H ₂ type zinc with unknown function of s pombe
Transcription factor clones showing homology with the finger protein are 4 as shown in SEQ ID NO: 5 (amino acid sequence: FIGS. 16 to 18) and SEQ ID NO: 6 (base sequence: FIGS. 19 and 20).
It was a gene encoding 68 amino acid residues and having two introns. This was named sgbR3.

[0041]

Example 4 Northern Analysis of sgbR1, sgbR2 and sgbR3 Northern analysis was performed to analyze the expression conditions of sgbR1, sgbR2 and sgbR3. Liquid culture (2% dextrin, 1% peptone, 0.5% yeast extract,
0.05% MgSO ₄ , 0.1% KCl, 0.1%
K ₂ HPO ₄ , 0.001% FeSO ₄ ), solid culture using wheat bran, and RNA extraction from solid culture cells using steamed rice. ISOGEN for RNA extraction
(Manufactured by Nippon Gene Co., Ltd.) was used.

20 μg of each of the obtained RNAs was subjected to 1% formaldehyde-denaturing agarose gel electrophoresis. After electrophoresis, Hybond N ⁺ membrane (manufactured by Amersham Pharmacia) was alkali-transferred, and then Gene I
Northern analysis was performed using a image kit (manufactured by Amersham Pharmacia). As shown in FIG. 3 (photograph of electrophoretic pattern: photograph in lieu of drawing), expression of both genes was observed in liquid culture, solid culture using wheat bran, and solid culture using steamed rice.

[0043]

Example 5 Analysis of Gene Silencing Strains by Cosuppression Method of sgbR1, sgbR2 and sgbR3 Using the cosuppression method to determine the functions of sgbR1, sgbR2 and sgbR3, the expression of both genes was suppressed in Aspergillus oryzae. Considered construction. A. which is constitutively highly expressed in Aspergillus oryzae. oryzae-derived histone H2A
SgbR1 and sgb downstream of the promoter (685 bp)
Initiation codons of R2 and sgbR3 (in SEQ ID NOS: 2, 4, and 6, 186th, 1288th, 50th, respectively)
A partial fragment of 500 bp from the 6th ATG) was ligated, and this was ligated to Aspergillus nidulans (A. nidu).
was introduced into koji mold in multiple copies using the sC marker of lans).

As a result of carrying out solid culture using the obtained koji mold, as shown in FIG. 4, not only glucoamylase but α
-For amylase, acid protease, acid carboxypeptidase, phytase, etc., the enzyme production per cell was reduced to 30-70% with respect to the parent strain. Therefore, it was revealed that these transcription control factors are factors that positively control the expression of gene groups specific to solid culture.

[0045]

Example 6 Gel shift assay of sgbR1, sgbR2 and sgbR3 with 97 bp cis factor These transcription factors are ci of the glaB promoter.
It was confirmed by gel shift assay whether or not it has the binding ability to 97 bp which is an s factor. sgbR1, sgb
In order to obtain the R2 and sgbR3 cDNAs, RT-PCR was performed based on the mRNA prepared from solid-cultured bacterial cells using steamed rice. The restriction enzyme sites BamHI (sgbR1), E
coRI (sgbR2) and SmaI (sgbR3) were added. The obtained clone was lacZ of pUC118.
Expression vectors (pSGBR1, pSGBR2, pSG) obtained by subcloning in the positive direction of the promoter
BR3) was transformed into E. coli JM109. Culture in ampicillin-containing medium, select ampicillin resistant strain,
The resulting transformant was Escherichia co
li SGBR1, Escherichia coli
SGBR2, Escherichia coli S
Named GBR3 and named it FERM P-183 at National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center.
57, FERM P-18358, FERM P-18
359, respectively.

The transformants are the transcription factors sgbR1 and sg.
All genes encoding bR2 and sgbR3 proteins
Contained. In this respect, the transformants were OD₆₆₀But,
After culturing at 37 ℃ until 0.6, the final 1 mM IP
TG and final 1 mM ZnSO _FourIs added, and further 30 ° C
Culturing was carried out for 6 hours, and by this IPTG induction, lac
SbgR1, sgbR2 and s under the control of the Z promoter
This was confirmed by expressing the gbR3 protein.

The gel shift assay was performed using binding buffer (final 1 mM MgCl ₂ , 0.5 mM) as protein.
EDTA, 0.5 mM DTT, 50 mM KCl,
5 mM Tris-HCl pH 7.5, 0.05 μg
/ Μl polydIdC, 4% glycerol)
10 μg of a cell-free preparation of Escherichia coli dialyzed against was used as a probe, and a dimer of 97 bp double-stranded DNA which is a cis factor of the glaB promoter was used as a gene.
0.8 pmol of fluorescein-labeled with Image kit (manufactured by Amersham Pharmacia) was used. Both were buffered (final 1 mM MgCl ₂ , 0.
5 mM EDTA, 0.5 mM DTT, 50 mM K
Cl, 5 mM Tris-HCl pH 7.5, 0.0
5 μg / μl polydIdC, 4% glycer
2) after incubation at room temperature for 30 minutes in
15% gradient polyacrylamide gel electrophoresis was performed at 120C at 4 ° C. The electrophoretic gel was alkali-transferred to a positively charged membrane, and fluorescein was detected by an antigen-antibody reaction using a Gene Image kit (manufactured by Amersham Pharmacia).

As shown in FIG. 5 (photograph of pattern showing results of gel shift assay: photograph substituting for drawing), sgbR1
A delayed band was confirmed by binding of sgbR2 and 97 bp, which is a cis factor of the glaB promoter. Also, this band disappeared due to the addition of excess unlabeled probe. Therefore, it was confirmed that both sgbR1 and sgbR2 have the ability to bind to 97 bp, which is the cis factor of the glaB promoter. sgbR3 is 97
Although the binding to bp could not be confirmed, the expression of the enzyme gene group in solid culture was reduced by cosuppression. Therefore, the enzyme gene expression in solid culture is controlled by interacting with sgbR1 and sgbR2. It has been suggested.

[0049]

EFFECTS OF THE INVENTION Solid culture is a culture form of the brewing industry, and has a characteristic that the amount of secretory protein produced is larger than that of liquid culture, and that the enzyme groups produced in a culture-specific manner have a great variety. According to the present invention, a transcription control factor involved in gene expression in this solid culture was clarified for the first time. Solid culture is a culture method that has been put to practical use in various industries.
If the expression mechanism in the present culturing method can be elucidated at the molecular level, it will not only significantly contribute to the development of the industry but also enable heterologous protein production in solid culture. Further, in the present invention, since the enzyme of koji mold is produced by koji mold,
The enzyme protein produced is extremely safe and is an epoch-making technology that can be applied to the food, pharmaceutical, cosmetics industries, etc. Therefore, it was possible to comprehensively improve enzyme production in liquid culture and solid culture of Aspergillus oryzae by obtaining this transcriptional regulatory factor, and it was suggested that this gene is a very promising gene in industry. .

[0050]

[Sequence list]            SEQUENCE LISTING <110> National Research Institute of Brewing; Gekkeikan Inc., Ltd. <120> DNA-encoding Transfer factor Regulating Expression of Koji-mo ld            Gene in Solid Culture <130> 6431 <141> 2001-6-27 <160> 7 <210> 1 <211> 717 <212> PRT <213> Aspergillus oryzae <400> 1 Met Ser Met Tyr Pro Ser Ile Arg Asn Lys Gly Phe Ile Ser Glu Pro   1 5 10 15 Thr Ser Thr Pro Phe Ile Asn Phe Gln Asp Pro Val Phe Ile Gln Asp              20 25 30 Glu Phe Leu Pro Asp Leu Ser Gln Glu Ala Ala Leu Gln Phe Tyr Asn          35 40 45 Gln Gln Leu Pro Arg Leu Gln Ser Pro Phe Gln Tyr His Ser Gly Leu      50 55 60 Glu Phe Tyr Ser Pro Pro Ser Ser Ala Pro Ser Ser Pro Thr Ser Ser  65 70 75 80 Pro Ala Ser Ser Thr Thr His Leu Pro Ala Thr Thr Ala Val Ala Ala                  85 90 95 Glu Tyr Pro Asn Ser Phe Glu Ala Pro Ser Met Thr Pro Val Ser Ser             100 105 110 Tyr Asn Ser Ala Phe Asn Ile Gln His Thr Ala Thr Pro Pro Ser Ser         115 120 125 Gln Pro Tyr Leu Ser Gln Tyr Pro Gln Tyr Leu Phe Glu Asn Ser Pro     130 135 140 Met Leu Ala Gln Gln Pro Val Ala Asn Thr His Gly Trp Glu Asn Asn 145 150 155 160 Leu Gln Leu Leu Ser Ser Ala Arg Met Gln His Lys Ala Ser Pro Ser                 165 170 175 Thr Ser Ser Thr Arg Ser Ala Pro Ala Gly Ser Ser Tyr Gln Arg Ser             180 185 190 Asn Ala Ser Thr Leu Ser Lys Pro Leu Pro Thr Pro Val Gln Thr Pro         195 200 205 Ile Gln Asn Ser Phe Leu Ala Ala Pro Tyr Gln Gln Asn Tyr Asp Thr     210 215 220 Ser Val His Asp Gly Ser Gln Ala Glu Ala Glu Met Val Arg Arg Ala 225 230 235 240 Val Met Glu Gln Gln Gln Lys Gln Gln Gln Gln Gln Ser His His Gln                 245 250 255 His Gln Pro Ser Asp Tyr Ser Leu Ala Pro Ser Val Ser Ser Val Ser             260 265 270 His Asn Ser Pro Val Thr Pro Gln Ile Lys Pro Glu Glu Leu Asp Glu         275 280 285 Ala Ser Lys Ala Met Val Asn Gly Glu Lys Arg Tyr Pro Asp Ile Asp     290 295 300 Arg Trp Met Asp Asp Tyr Leu His Leu Asp Ala Phe Ala Asp Tyr Asn 305 310 315 320 Asn His Asn Gly Asn Asn Leu Pro Ile Gly Ile Pro Lys Ile Asn Arg                 325 330 335 Thr Met Ser Asp Ile Tyr Gln Asp Glu Leu Tyr Asn Pro Ala Leu Met             340 345 350 Pro Thr Pro Gln Val Ser Lys Gln Thr Thr Asn Gln Gln Asn Leu Leu         355 360 365 Asn Pro Phe Arg Asn Val Phe Ala Asp Arg Leu Gln Ala Ala Asn Gln     370 375 380 Gly His Met Thr Ala Arg Ser His Ser Pro Val Val Asn Met His Arg 385 390 395 400 Asp Arg Ser Pro Phe Arg Gln Asn Ser Pro Leu Ala Ala Glu Tyr Asn                 405 410 415 Asn Gly Phe Gln Gln Pro Gln Met Ala Thr Ser Val Pro Met Thr Gln             420 425 430 Asn Val Gly Gln Ser Gln Gly Glu Gly Glu Pro Lys Thr Met Ser Pro         435 440 445 Lys Asp Ala Leu Leu Asp Phe Asn Glu Gly Asp Asp Ala Gly Ile Pro     450 455 460 Leu Phe Pro Thr Ser Gln Pro Asp Phe Asn Leu Gly Glu Ala Leu Gly 465 470 475 480 Leu Arg Arg Glu Ser Ser Ser Ser Phe Pro Gln Ser Gln Asn Phe Thr                 485 490 495 Ser Met Glu Ser Phe Pro Thr Gln Tyr Thr Thr Pro Asn Gly Leu Pro             500 505 510 Gln Gln Tyr Pro Phe Ala Gln Gln Gln Gln Asp His Gln Gln Gln Gln         515 520 525 Gln Asn Asn Leu Leu His Gln Thr Pro Glu Phe Pro Ala Ser Leu Pro     530 535 540 His Phe Glu Ser Thr Asn Ser Asp Ala Gly Val Asn Asn Gly Val Ala 545 550 555 560 Ser Pro Pro Ala Gln Pro Thr Met Ala Met Arg Pro Val Lys Glu Glu                 565 570 575 Ile Thr Arg Pro Glu Arg Thr Ser Ala Asp Ser Gly Thr Tyr Thr Cys             580 585 590 Thr Tyr His Gly Cys Thr Leu Arg Phe Glu Thr Pro Thr Lys Leu Gln         595 600 605 Lys His Lys Arg Glu Ala His Arg Gln Thr Thr Pro Gly Gly His Leu     610 615 620 Val Gly Arg Asp Thr Ser Ala Arg Asn Ser Gln Ala Gly Pro His Lys 625 630 635 640 Cys Glu Arg Ile Asn Pro Ser Thr Gly Lys Pro Cys Asn Ser Val Phe                 645 650 655 Ser Arg Pro Tyr Asp Leu Thr Arg His Glu Asp Thr Ile His Asn Ala             660 665 670 Arg Lys Gln Lys Val Arg Cys His Leu Cys Thr Glu Glu Lys Thr Phe         675 680 685 Ser Arg Asn Asp Ala Leu Thr Arg His Met Arg Val Val His Pro Glu     690 695 700 Val Asp Trp Pro Gly Lys Gln Arg Arg Arg Gly Arg Glu 705 710 715 717 <210> 2 <211> 2362 <212> DNA <213> Aspergillus oryzae <400> 2 ccacttcata ttcgcagtgt tcggggataa agataatcgg atcttgggcc gttggaactt 60 ttatttggag gcttgggtgc catacctagt acttaacttc tggaacctcc cccagtggcg 120 aatcctctga acttccccat ccactccggc gtcataccct tgacgtccat ttgttgaaag 180 agaacatgtc catgtaccct tcgatacgca ataagggttt tatcagtgaa ccaacatcta 240 cccccttcat caatttccaa gaccccgttt tcatccagga cgagttcctt cccgacttgt 300 cgcaggaggc cgcattacag ttctacaacc agcaactacc caggctccaa tcacctttcc 360 agtaccactc gggtcttgaa ttttactctc cgccatcttc cgcgccgtcg tcaccgactt 420 cctctccagc ctcttccacc acccacctgc cagcgaccac agcggtggct gcggaatacc 480 ccaactcctt tgaggctcct tcgatgacac ccgtgtcctc ctataactct gcattcaaca 540 ttcagcatac cgcgacacca ccgtcctcac agccatactt gtcacaatac ccccaatacc 600 tttttgaaaa ctccccaatg ttggcgcaac agcctgtcgc aaacacccac ggctgggaaa 660 acaacctgca gctgctgagc tcggctcgca tgcagcacaa ggcatcaccg agtacctcgt 720 cgactcgatc tgcacctgca gggagctcgt accaaagatc taacgcttct acgctgtcga 780 agcctctacc cactcccgtc cagacaccta tccagaactc gtttctcgcc gcgccctatc 840 agcagaacta tgacacttcg gtgcatgatg gtagccaagc tgaagcggag atggtgagac 900 gggctgtgat ggaacagcag cagaagcagc aacagcaaca aagtcaccat cagcatcagc 960 caagtgatta ctcactggca ccttcggtat catcagtgag ccacaactca cctgttactc 1020 ctcagataaa accggaggaa cttgacgagg cctcgaaggc gatggtcaat ggtgagaaac 1080 gatatcctga tatagaccga tggatggatg attacttaca tctcgatgcc tttgcagact 1140 acaataacca caacggaaac aaccttccga tcggcattcc caaaatcaac cgaactatgt 1200 cggacatcta ccaagatgag ctctataacc cagctctcat gccgactcct caagtctcca 1260 aacagactac gaaccagcag aatcttctga acccgttcag aaacgtcttt gctgatcgac 1320 tgcaagccgc caatcagggc cacatgactg cacggtctca ttctcctgtt gttaacatgc 1380 accgggatcg gtctcctttc cgacagaact cgcccctggc tgctgagtac aacaatgggt 1440 tccagcagcc ccagatggcg accagtgtgc ctatgactca aaatgtaggc caaagtcaag 1500 gagaaggaga accgaagact atgtctccta aggatgccct gttggacttc aacgagggag 1560 atgacgccgg gattcctttg ttcccaacaa gtcaacctga cttcaacctt ggtgaagcac 1620 tcggcctccg acgtgaaagc tcatcatcct tcccgcagtc gcagaacttt acttccatgg 1680 agtcattccc cacgcaatac actacaccga atggacttcc ccagcagtat cccttcgcgc 1740 agcagcaaca agaccatcag cagcaacaac agaacaacct cctgcaccag acccccgaat 1800 tccctgcatc ccttcctcac tttgagtcta cgaacagtga tgccggggtc aacaacggtg 1860 tggcttctcc accggcacag cccacaatgg cgatgcgccc agtgaaggaa gagatcaccc 1920 gccctgagcg cacttctgcg gacagtggca cttacacttg cacttaccac ggctgcacgc 1980 ttcggtttga aacgcctacg aagctgcaga agcacaagcg cgaggcccac cgtcagacta 2040 cgcctggcgg ccacttggtt gggcgcgata cttccgcacg caactctcag gccggtcccc 2100 acaaatgtga gcggatcaat ccgtctacgg gaaagccatg caattctgtt ttctcccggc 2160 cttacgatct tacccggcac gaggacacga tccacaacgc acgcaagcag aaggtccggt 2220 gtcatctgtg cacggaagaa aagacctttt cgcgtaatga tgcgctgacc cggcacatgc 2280 gagtggtgca ccctgaggtc gattggcccg gcaagcaaag aaggagaggc agggagtaag 2340 cctgaatggg gttgtggtgt ca 2362 <210> 3 <211> 597 <212> PRT <213> Aspergillus oryzae <400> 3 Met Ala Pro Thr Val Gln Gly Gln Pro Ser Phe Ala Tyr Tyr Pro Thr   1 5 10 15 Ala Asp Ser Gln Arg Gln Gln Tyr Thr Ser His Pro Ala Glu Pro Gln              20 25 30 Pro Tyr Tyr Gly Gln Ile Gln Ala Phe Pro Gln Gln His Cys Leu Pro          35 40 45 Glu Gln Gln Pro Val Tyr Asn Ala Gln Pro Met Met Asn Met His Gln      50 55 60 Met Ala Thr Thr Asn Ala Phe Arg Gly Ala Met Asn Met Thr Pro Ile  65 70 75 80 Ala Ser Pro Gln Pro Ser His Leu Lys Pro Thr Ile Val Val Gln Gln                  85 90 95 Gly Ser Pro Ala Leu Met Pro Leu Asp Thr Arg Phe Val Gly Asp Tyr             100 105 110 Tyr Ser Phe Pro Ser Thr Pro Pro Leu Ser Thr Ala Gly Ser Ser Ile         115 120 125 Ser Ser Pro Pro Ser Ser Ser Gly Thr Leu His Thr Pro Ile Asn Asp     130 135 140 Cys Phe Phe Ser Phe Glu Lys Val Glu Gly Val Lys Glu Gly Cys Glu 145 150 155 160 Ser Asp Val His Ala Glu Leu Leu Ala Ser Ala Asp Trp Thr Arg Ser                 165 170 175 Ala Thr Ser Pro Pro Met Thr Pro Val Phe Ile Asn Ala Asn Ser Leu             180 185 190 Thr Ala Ser Gln Ser Ser Asp Phe Leu Ser Ala His Gly Ser Cys Pro         195 200 205 Ser Leu Ser Pro Ser Pro Leu Leu Val Ser Ser Val Phe Thr Pro Ser     210 215 220 Gln Ser Ala Phe Pro Val Glu Gln Ala Thr Ser Asp Phe Cys Asp Pro 225 230 235 240 Arg Gln Leu Thr Val Glu Ser Ser Val Asn Thr Ser Ser Pro Ala Glu                 245 250 255 Leu Pro Pro Leu Pro Thr Leu Ser Cys Asp Glu Glu Glu Pro Lys Val             260 265 270 Val Leu Gly Ser Glu Ala Val Thr Leu Pro Val His Glu Asp Pro Ser         275 280 285 Pro Ala Tyr Thr Ser Ser Thr Glu Asp Pro Leu Ser Ser Leu Pro Thr     290 295 300 Phe Asp Ser Phe Ser Asp Leu Asp Ser Glu Asp Glu Phe Val Asn Arg 305 310 315 320 Leu Val Asp Phe His Pro Ser Gly Asn Thr Tyr Tyr Val Gly Glu Lys                 325 330 335 Arg Gln Arg Leu Ser Ala Tyr Ser Phe Asp Asp Glu Glu Phe Leu Ser             340 345 350 Glu His Ser Leu Glu Asp Ser Ser Asp Asp Leu Glu Leu Ala His Ser         355 360 365 Gly Leu Ser Phe Leu Gly Cys Ala Asp Phe Ala Pro Ala Gln Ser Asp     370 375 380 Ala Ser Glu Thr Ala Asp Glu Met Lys Thr Lys Lys Arg Ser Asn Ser 385 390 395 400 Arg Lys Ser Leu Lys Arg Ala Asn Ser Glu Asp Gln Asp Ala Leu Lys                 405 410 415 Lys Ala Gln Ala Pro Ile Asn Ser Arg Ala Asn Ser Thr Glu Ala Asn             420 425 430 Val Ala Gln Gln Ala Ala Ala Pro Ser Cys Ser Ala Ser Glu Ala Asn         435 445 440 Val Ser Ser Ser Cys Glu Ala Pro Ser Val Pro Val Ser Val Asn Arg     450 455 460 Arg Gly Arg Lys Gln Ser Leu Thr Asp Asp Pro Ser Lys Thr Phe Val 465 470 475 480 Cys Ser Leu Cys Ser Arg Arg Phe Arg Arg Gln Glu His Leu Lys Arg                 485 490 495 His Tyr Arg Ser Leu His Thr Gln Asp Lys Pro Phe Glu Cys Asn Glu             500 505 510 Cys Gly Lys Lys Phe Ser Arg Ser Asp Asn Leu Ala Gln His Ala Arg         515 520 525 Thr His Ala Gly Gly Ser Ile Val Met Gly Val Leu Asp Thr Asn Asn     530 535 540 Ala Ser Glu Arg Tyr Asp Asn Arg Asp Ala Ser Thr Met Gly Ala Val 545 550 555 560 Leu Tyr Glu Ala Ala Thr Leu Arg Arg Pro Ser Arg Arg Leu Ala Asn                 565 570 575 His Pro Lys Met Gly Phe Leu Thr His Pro His Arg Ser Ser Ala Arg             580 585 590 Gln Glu Ala Gln Ala         595 597 <210> 4 <211> 3167 <212> DNA <213> Aspergillus oryzae <400> 4 ggggccggtt gggggccctc ccggggttgg gttacgggaa aagtttcaag ggccaaagga 60 aaaaaaaggt aaaaaagatt tagggaaaaa aaggccaaaa aaaagggcct tggaaaaagg 120 aattcccaag aacctttccc ccaaaaggtt aattggggta ggggaaccat tggggtttct 180 taccaaggtt tttcccttta tcctttgccc ccttcaggga aaaatttact tattatccta 240 gggaatttgg ccccccccag gggggggatc cctgggattt aagggggggt gccccccggc 300 ccccccctaa gggggttccc ccctttttag ggattttttt ttttttggaa attaaatttc 360 ctttcccttt ttttttaaaa aaaataaaat cttttcggtt ttattttatt ttttataata 420 aaaaaattcc taattataat tataaatccg ggtattttta gggataaatt ctaggtttta 480 actaccctcc ctcccttttc cccgggcccc cccccctcca aatagacaag acggactaaa 540 actaaaaagt acggctccag tcctacgact aactctcact acgcacggcg ctactaagtc 600 gtagcacaag cagatcagtc tctcttttat ccactgtcca tccttctggc cgtctgtccc 660 tcttcccttt cccatatcgg ccctgtctct cgtttccccg cgactgtcca ttagtcctct 720 ttatttctta gtcttttcct tttttccctt tttagacttg tctcgattct ctgtccttcc 780 cttcgttatt tcttattttt tggctttgat ccccatctct ctcttctctc tcttctttta 840 ctcatcatct gtctccggta cctttgatcg tgatcgtccg aatccaaaag gtggctcgta 900 tcgaccagag gttcctgtcc ttccttctat cagatataac tataccgtac attcacactc 960 agaagcccag aagacgttgc atattttata aaccctaatt cggactgcac tcttctgcct 1020 tcagtcattc attcatccag aacaagagac atctacattc attcaaccta cagtgctcga 1080 catctatcat tagcaccaag ctgcctcgac atcaatctgg aacatcctga cttagactaa 1140 acatacttaa taatacccaa gaggttaagc agtgttcgct tgatcctcat cacattcatc 1200 attggttcgt ggaaggatga ctatatatca tacaggatgc ccatactgat ccctcgtctt 1260 ctagtcacaa tggacgctac atacaccatg gcaccaacgg tgcaaggaca accatcattt 1320 gcatactacc ccactgctga ttcacaaaga caacaataca caagccaccc ggctgagccc 1380 cagccatact atggacagat tcaggccttc cctcagcaac actgcctacc agagcaacaa 1440 ccggtctaca atgctcaacc catgatgaac atgcatcaga tggctaccac caatgccttc 1500 cgcggagcaa tgaacatgac tcccatcgcc tctccccaac catctcacct gaagcccaca 1560 atcgtcgtcc agcagggttc cccagccctc atgcctttgg acacaaggtt cgtcggtgac 1620 tactatagct tcccttctac ccctccactc tccaccgcgg gaagttctat cagcagcccg 1680 ccatccagca gtggcacctt gcacacccca atcaacgact gcttcttctc attcgaaaag 1740 gtggagggtg taaaggaggg ttgtgaaagt gacgtccatg ccgagctttt ggccagtgcc 1800 gactggactc ggtcagcaac ttcgcctccc atgactcctg gtatgttttc ttctcatcag 1860 ttgagtaggg ggttggaacc agtctaactt cttctagtct tcatcaacgc caattctctc 1920 accgccagtc agagctcaga cttcctctct gcccacgggt cttgcccctc tctttctcct 1980 tcgcctctcc tggtatcttc cgtgtttact ccgtctcagt cggctttccc tgttgagcag 2040 tcgcctctcc tggtatcttc cgtgtttact ccgtctcagt cggctttccc tgttgagcag 2040 gccacctccg acttctgtga ccctcggcag ctgactgtcg agtcttccgt caacacatcc 2100 tctcccgctg agcttcctcc tttgcccact ctctcctgcg atgaggagga accgaaggtg 2160 gtcctgggaa gcgaggctgt gactttgcca gttcatgagg acccatcacc cgcttacact 2220 agctctaccg aggaccccct cagttcgttg cccacttttg acagcttctc cgaccttgat 2280 tcggaggacg agtttgtcaa ccgcttggtt gacttccatc ccagcggtaa tacttactac 2340 gtgggcgaaa agagacagcg cctcagcgca tactccttcg atgacgagga gttcttgagc 2400 gagcacagct tggaagattc ttcagacgac ctggagttgg cccactctgg cctttctttc 2460 ctgggatgcg ccgatttcgc tccggctcaa agtgatgcca gcgagaccgc tgacgagatg 2520 aagacaaaga agcgaagcaa ctcccgcaag tctctcaaga gagcaaactc tgaggaccag 2580 gatgctctca agaaggcaca ggcaccgatc aacagccggg ccaacagcac agaggccaac 2640 gtggctcagc aggcagctgc tccgtcctgc tcagcctcgg aagccaatgt atctagctcc 2700 tgcgaggccc catctgttcc tgtgtcggtc aaccgccggg gtcgtaagca atccctgacg 2760 gatgatcctt ccaagacctt tgtctgctct ctctgctctc gtcgtttccg tcgtcaggag 2820 caccttaagc gtcactaccg ctctctgcac acccaggaca agcccttcga gtgcaacgag 2880 tgtggtaaga agttttctcg gagtgacaac cttgcgcagc atgccaggac ccatgccggt 2940 ggctctatcg tgatgggtgt cctggacacg aacaacgcat ctgaacggta tgataatcgc 3000 gacgcgagta ccatgggtgc tgtgctctat gaagctgcca cgctgcggcg accaagtcga 3060 cgactagcga atcatccgaa gatggggttt ctgacgcacc ctcatcgatc gtcggcccgc 3120 caagaagcgc aagcgtgacg agcactttag ttgcccccat tgccggt 3167 <210> 5 <211> 468 <212> PRT <213> Aspergillus oryzae <400> 5 Met Ala Ser Ile Asp Pro Asn Val Pro Thr Thr Thr Leu Pro Tyr Thr   1 5 10 15 Cys Asn Thr Cys Leu Val Ala Phe Arg Gly Ser Asp Ala Gln Arg Asp              20 25 30 His Met Arg Lys Asp Trp His Leu Tyr Asn Met Lys Arg Arg Ile Ala          35 40 45 Ser Leu Pro Pro Val Ser Gln Glu Val Phe Asn Asp Lys Val Leu Ala      50 55 60 Ala Lys Ala Thr Thr Ser Ala Ala Ala Ala Lys Ala Ser Phe Glu Lys  65 70 75 80 Thr Cys Val Ala Cys Gln Lys Thr Phe Phe Ser Glu Asn Ser Tyr Gln                  85 90 95 Asn His Val Lys Ser Ser Lys His Lys Ala Arg Glu Ala Gln Met Leu             100 105 110 Arg Asp Ser Ala Asp Asp Ala Ser Ser Val Met Ser Ser Thr Phe Ser         115 120 125 Leu Gly Glu Pro Val Asn Lys Pro Arg Glu Arg Ser Glu Val Ser Lys     130 135 140 Val Thr Glu Ser Leu Lys Asn Ala Thr Ile Glu Glu Asp Asp Glu Asp 145 150 155 160 Glu Glu Met Glu Glu Gln Gly Phe Ser Ala Ser Arg Cys Leu Phe Cys                 165 170 175 Asn Glu Lys Ser Ser Asp Leu Gln Gln Asn Thr Glu His Met Phe Lys             180 185 190 Thr His Gly Met Phe Ile Pro Glu Lys Asp Tyr Leu Val Asp Leu Glu         195 200 205 Gly Leu Val His Tyr Leu Tyr Arg Lys Ile Asn Glu Asn Ser Glu Cys     210 215 220 Leu Tyr Cys His Ala Val Arg Asn Asn Pro Glu Gly Ala Arg Thr His 225 230 235 240 Met Arg Asp Lys Gly His Cys Met Ile Ala Phe Glu Lys Gln Asp Glu                 245 250 255 Gln Val Glu Ile Gly Gln Phe Tyr Asp Phe Arg Ser Thr Tyr Ser Asp             260 265 270 Gly Glu Gly Glu Asp Glu Glu Asp Ser Ile Met Glu Asp Gly Gly Val         275 280 285 Lys Val Asp Gly Glu Asp Asp Glu Gly Trp Glu Thr Glu Thr Ser Ala     290 295 300 Ser Ser Met Asp Asp Asp Glu Asp Glu Leu Asp Asp Thr Lys Gly Gln 305 310 315 320 Val Tyr Ala Thr Glu Phe Glu Leu His Leu Pro Ser Gly Arg Thr Ala                 325 330 335 Gly His Arg Ser Leu Ala Lys Tyr Tyr Arg Gln Asn Leu Arg Asn Tyr             340 345 350 Pro Thr Ala Glu Glu Arg Ala Ala Arg Gln Leu Ala Ile Glu Asn Gly         355 360 365 Glu Ile Glu Glu Glu Glu Pro Lys Pro Arg Gly Arg Asp Leu Asn Arg     370 375 380 Ala Val Pro Glu Ala Phe Ala Ala Glu Leu Glu Arg Lys Asp Arg Thr 385 390 395 400 Arg Ala Gln Arg Gln Glu Lys Arg Tyr Thr Ala Arg Val Asn Arg Ala                 405 410 415 Ala Asn Asn Gln Lys His Phe Arg Val Cys Phe Ile Pro Leu Leu Ile             420 425 430 Ile Arg Gln Ala Asn Thr Pro Ser Arg Ile Pro Ser Cys Ser Arg Phe         435 440 445 Leu Tyr Leu Ile Cys Leu Arg Ala Cys Trp His Cys Leu Ala Lys Ser     450 455 460 Trp Ser Tyr Gly 465 468 <210> 6 <211> 2337 <212> DNA <213> Aspergillus oryzae <400> 6 tggtcgacgg cccgggctgg tatcatacgg actgacacac cagcctcagg cgccaagcat 60 gaggccggct gactaacatt aaatggaaga agtatgatcc gatcgttgat gcactaggct 120 tgatatagat agggccttcc cagacaggat tagtggaggg gcacgtgcaa tcaacgtcat 180 gttctctaaa agcggcatgg gcaatggttt tcaatgtgtg gtccgggtta tcggcgacat 240 cgattactag cccgatcttt tctgtcgagg ggcaatattg cgcccgcttc actttctcgc 300 tagatacttc ctaccggctt ttatttctaa tcaggcccaa aactttctgt gacgactacg 360 cctggttttg ctttttcatt gctcttgcaa aaacgaatac ccccgcgcaa tatatattcc 420 cacaaccgat cgccttttca tcaaatttat ttctgtgata cccacctttt ttttttttgt 480 tccatatcag gaactgtcag aaatcatggc ctccatcgat ccaaatgtac caacaactac 540 gttgccatat acctgcaaca cctgtctcgt tgcttttcgc ggtagcgatg ctcagcggga 600 tcatatgcgc aaggactggc agtaagtttg ttggaaaata tacgactcag aatgcattac 660 taatgttcgg tcgactcaag tctctataac atgaagcgcc gcatcgcgtc tctgccccca 720 gtgtcccagg aggtttttaa cgacaaggtc cttgctgcca aagccacaac tagtgctgcc 780 gccgccaaag cttcctttga gaagacatgt gtcgcctgcc agaagacatt cttcagcgag 840 aactcgtatc agaaccacgt gaagagttcc aagcacaagg cccgtgaggc acagatgctt 900 agagacagcg ccgatgatgc atcgtctgtc atgagttcta ctttctctct gggcgagcca 960 gtcaacaagc ctcgtgagcg gtcggaggtg tcgaaagtta cggagagtct caagaacgct 1020 accatcgaag aggatgacga agatgaggaa atggaagagc agggcttctc ggcctcccgt 1080 tgtcttttct gtaatgagaa atcttcagat cttcaacaaa acaccgagca catgttcaag 1140 acccacggca tgttcatacc agagaaggac tatcttgttg atttggaggg acttgtccac 1200 tatctttatc ggaagatcaa tgagaacagt gaatgtttat actgccatgc cgtccgaaac 1260 ggtgagggtg aggatgagga ggactcgatc atggaggatg gtggtgtcaa ggtggatggc 1440 gaggatgatg agggatggga aaccgaaact tctgcatctt ctatggatga cgatgaggat 1500 gagctcgacg acacgaaggg acaggtctat gcgacagagt ttgagctgca tttgccctca 1560 ggccgcactg caggtcaccg atcgctcgcc aaatactacc gccagaactt gcgtaactac 1620 cctacagcgg aagaacgggc agctcgtcag ctggctattg aaaatggcga gattgaggag 1680 gaggaaccga aacccagggg tcgcgacctc aatcgggccg ttgttagccg tggcaatggc 1740 ggtattgggt atgatcggtg ctactgacag ccagaagcat ttgctgctga gcttgaacgc 1800 aaagaccgga ctcgtgccca gcgacaagag aagcggtaca cagctcgggt caaccgggct 1860 gccaataacc agaagcactt cagggtatgt ttcatccctt tactgataat aagacaagct 1920 aacaccccct ctaggatccc ctcctgcagt agattcctgt atctgatttg tttgcgtgca 1980 tgttggcatt gtttggcaaa atcatggagt tacggctgaa tgacctgtag atactcatgg 2040 caagataaaa agtgattatt aagaagatga aggatattac ttcgcattcc tgctatcttt 2100 actattttag acagtgctgc atcttatgac ggatataccg aaggtctacc agactgaagg 2160 tgaatcaagt cctacatcta accccccgac caaaccctaa gtcaccacat ctcacagaac 2220 cgatagcaaa cttacaatca agcaccataa caacaaacca ggaccacaaa taataatata 2280 tgaacatcat atcactttac cagcccgggc cgtcgaccac gcgtgcccta tagtgag 2337 <210> 7 <211> 97 <212> DNA <213> Artificial Sequence <400> 7 gagaactaag agaatggcgg cacgggcaga tgtcgggatg atcactttta gtcectccga 60 cgcaatatcg atttcaattg gatccgccac gctgcat 97

[Brief description of drawings]

FIG. 1 shows the identification of a region essential for high expression in solid culture in the glaB promoter.

FIG. 2 shows a promoter titration assay using the glaB promoter cis factor.

FIG. 3 is a drawing-substituting photograph showing a pattern for Northern analysis of sgbR1, sgbR2, and sgbR3.

FIG. 4 shows cosuppression assays for sgbR1, sgbR2 and sgbR3.

FIG. 5 is a drawing-substituting photograph showing a pattern of a gel shift assay of sgbR1, sgbR2, and sgbR3.

FIG. 6 shows the amino acid sequence of the present transcription factor sgbR1 protein.

FIG. 7 shows the continuation of the above.

FIG. 8 shows the continuation of the above.

FIG. 9 shows the nucleotide sequence of the present transcription factor sgbR1 gene.

FIG. 10 shows the continuation of the above.

FIG. 11 shows the amino acid sequence of the present transcription factor sgbR2 protein.

FIG. 12 shows the continuation of the above.

FIG. 13 shows the continuation of the above.

FIG. 14 shows the nucleotide sequence of the transcription factor sgbR2 gene.

FIG. 15 shows the continuation of the above.

FIG. 16 shows the amino acid sequence of the present transcription factor sgbR3 protein.

FIG. 17 shows the continuation of the above.

FIG. 18 shows the continuation of the above.

FIG. 19 shows the nucleotide sequence of the transcription factor sgbR3 gene.

FIG. 20 shows the continuation of the above.

FIG. 21: Solid culture expression gene cis factor (97 bp)
The base sequence of is shown.

FIG. 22 shows a plasmid pNGUS for promoter analysis.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12N 15/00 A C12P 21/02 5/00 A (72) Inventor Yoji Hata Fushimi, Kyoto 300 Katahara-cho, Gekkei-cho Co., Ltd. in the General Research Institute (72) Inventor Shoji Kawado 24 Shimotoba Koyanagi-cho, Fushimi-ku, Kyoto City In the General Research Institute, Keikei-sha Co., Ltd. (72) Ken Akao 3-7-1 Kagamiyama, Higashihiroshima City, Hiroshima Prefecture No. F-term in the National Institute of Alcoholic Beverages (Reference) 4B024 AA20 BA80 DA11 FA02 4B064 AG01 CA19 CC24 4B065 AA26X AA63X AA63Y AB01 CA24 4H045 AA10 AA20 BA10 CA15 FA74

Claims (8)

[Claims] 1. A protein which controls the transcription of a gene downstream of a cis factor which is responsive to solid-state culture, and which has at least one of the amino acid sequences shown in SEQ ID NOs: 1, 3 and 5, respectively. 2. A solid-culture responsive c having at least one of the nucleotide sequences shown in SEQ ID NOs: 2, 4, and 6, respectively.
DNA of a gene encoding a protein that controls the transcription of a gene downstream of the is factor. 3. A recombinant vector comprising at least a coding region of the DNA according to claim 2. 4. A recombinant vector pSGBR1 or pS
GBR2, or pSGBR3. 5. A transformant obtained by introducing the recombinant vector according to claim 3 or 4. 6. A transformant, Escherichia
coli SGBR1 (FERM P-18357),
Or Escherichia coli SGBR2
(FERM P-18358), or Escheric
hia coli SGBR3 (FERM P-183
59). 7. A method for producing a protein which controls transcription of a gene downstream of a cis factor responsive to solid culture in the koji mold into which the DNA according to claim 2 is introduced. 8. A method of controlling the production of a solid culture-expressed gene group by expressing the protein according to claim 1 in Aspergillus oryzae.