JPH099968A - Enhancer dna base sequence of mould and its use - Google Patents

Abstract

PURPOSE: To obtain the subject new base sequence having a specific base sequence, bond to Aspergillus oryzae-derived α-glucosidase gene promoter domain, and capable of efficiently producing useful proteins, peptides using mould as host. CONSTITUTION: This new base sequence is a new enhancer DNA base sequence containing a sequence: -CGGNNATTTA-. This base sequence is incorporated into the promoter domain of an α-glucosidase gene derived from mould such as Aspergillus oryzae and binds a marker gene suitable for selecting the transformants of the mould as host, a terminator and a DNA domain replicable by Escherichia coli, and then incorporated into the host to enable useful proteins and peptides to be produced in high efficiency. This enhancer DNA base sequence is obtained by isolating the α-glucosidase gene promoter and by conducting a retrieval of this base sequence by using a gene-analyzing software.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel enhancer DNA nucleotide sequence effective for efficiently producing useful proteins and peptides using filamentous fungi as a host, a promoter into which the nucleotide sequence is introduced, an expression plasmid containing the promoter, and The present invention relates to a useful protein and peptide production method using the same.

[0002]

2. Description of the Related Art Escherichia coli and yeast have been mainly used as hosts for useful proteins by gene recombination. However, in E. coli, when a heterologous protein is highly expressed, insoluble proteins are formed in the cells,
There is a drawback that post-translational modifications such as addition of sugar chains, which are characteristic of eukaryotic proteins, do not occur, and it is not a little difficult to obtain active proteins. Further, in yeast, although sugar chain addition occurred, there was a problem that the production amount was low.

On the other hand, filamentous fungi secrete and produce a large amount of various enzyme proteins outside the cells. For example, in industrially used strains of Aspergillus niger, 20 g or more of glucoamylase is added per liter of culture solution, , Aspergillus oryzae is also said to produce about 50 g of α-amylase per kg in solid culture, and the cellulase-producing bacterium Trichoderma reesei also has the same level of enzyme productivity, so that it can be used not only for the same species but for heterologous proteins. Is expected to have high secretory production potential. In addition, Aspergillus oryzae and Aspergillus
Many of them are used in the production of brewed foods and the production of enzymes for food processing such as niger, and these filamentous fungi are safe as hosts in various related organizations, for example, the US Food and Drug Administration (FDA). It has been evaluated and it is considered that useful protein production by these filamentous fungi is likely to be approved.

From these points, production of various proteins has recently been reported as a microorganism replacing Escherichia coli or yeast, using a filamentous fungus as a host for genetic engineering. When the target protein is produced by genetic engineering, the production efficiency depends on the transcription amount by the promoter used, the translation efficiency after transcription, post-translational modification such as glycosylation of the expressed protein, the gene copy number, and the host protease. It is regulated by various factors such as protein degradation, but first of all, if the transcription amount of the target protein is not large, expression and secretion in excess of the transcription amount cannot be expected. From this point of view, how to obtain a strong promoter becomes a big problem, and therefore various promoters have been isolated in filamentous fungi, and production of a target protein using these has been reported. As a typical example, the promoter of Aspergillus niger glucoamylase gene [Biotechnology, 5, 368 (1987)], Aspergillus niger
Oryzae α-amylase gene promoter [Biotec
hnology, 6, 1419 (1988)], the promoter of the cellobiohydrolase I gene of Trichoderma reesei [Biotec
hnology, 7, 596 (1989)] and the like.

However, research on the genetic engineering of filamentous fungi has been delayed compared to other microorganisms, and the gene expression mechanism of the promoter has not been clarified yet, and the number of reported cases has finally increased in the last few years. It is the current situation. For example, it is found in the catabolite repressor gene [Mol. Microbiol., 7, 847-857 (1993)], which is involved in negative expression regulation of genes related to the utilization of carbon source, or widely in eukaryotic gene expression regulatory regions. CCA
AT sequence binding factor [Mol. Gen. Genet., 235, 81 (199
2)] is only mentioned. Therefore, the production of useful proteins by filamentous fungi depends on the original expression control of the promoter to be used, and the present situation is to select and use a promoter according to the purpose, and to control the expression regulation of the promoter. Until now, no attempts have been made to improve and obtain more useful promoters. Therefore, if a technique for improving a promoter is provided, it is possible to obtain a more useful promoter by improving an existing promoter without isolating a novel high expression promoter, and to control the expression of the promoter. Also becomes possible, and more efficient production of useful proteins becomes possible.

[0006]

An object of the present invention is to analyze promoter expression control, for example, to identify an enhancer nucleic acid nucleotide sequence and to elucidate the function of this enhancer sequence. Further, it is to develop a protein expression system by improving a promoter using this, constructing an expression plasmid using this promoter, and transforming a filamentous fungus with this plasmid.

[0007]

[Means for Solving the Problems] In order to achieve the above object, the inventors of the present invention have conducted extensive studies, isolated the promoter of the α-glucosidase gene (agdA) of Aspergillus oryzae, and analyzed the promoter in detail. As a result, we found a novel enhancer nucleobase sequence that is deeply involved in positive expression regulation, and by introducing multiple enhancer sequences into the promoter, it was possible to increase the transcriptional activity of the promoter and eliminate the dependence on the carbon source. Successful. Furthermore, a novel expression plasmid for efficiently expressing useful proteins and peptides was constructed using this improved promoter and the nitrate reductase gene (niaD) as a selectable marker. In addition, a filamentous fungus is transformed with this plasmid, and a useful protein, for example,
The present invention has been completed by succeeding in obtaining a high-producing strain of α-glucosidase or a transformant having arbitrary productivity.

That is, the present invention provides (1) Sequence-CGG
An enhancer DNA base sequence containing NNATTTA-, (2) an enhancer DNA base sequence according to (1), wherein NN is GC, (3) sequence-CCAATCAGC
GT-containing enhancer DNA nucleotide sequence, (4)
(1) or the enhancer DNA base sequence containing the sequence described in (3), (5) the enhancer DNA base sequence described in (4) wherein NN is GC, and (6)
An improved promoter comprising one or more enhancer DNA nucleotide sequences according to (1) or (3) introduced into a promoter region that functions in filamentous fungi,
(7) The improved promoter according to (6), wherein the promoter region is a promoter of a hydrolase gene derived from filamentous fungus or a glycolytic enzyme gene, (8) the promoter region is Aspergillus represented by SEQ ID NO: 2. An improved promoter according to (6), which is a promoter region of an α-glucosidase gene derived from oryzae (Aspergillus oryzae), or a promoter region containing a partial sequence thereof, and an improved promoter according to (9) (6), A plasmid for expressing a polypeptide in a filamentous fungus, which has a marker gene suitable for selecting a transformant of a host filamentous fungus, has a terminator, and has a DNA region capable of replicating in Escherichia coli, (10) the marker gene is a filamentous fungus Origin reductase gene, ornithine carbamoyl transferase gene The plasmid of (9), which is a tryptophan synthase gene or an acetamidase gene, (11), the plasmid of (9), wherein the marker gene is a nitrate reductase gene derived from Aspergillus, (12) the terminator is SEQ ID NO: The terminator of the α-glucosidase gene derived from Aspergillus oryzae shown in 3 or a terminator containing a partial sequence thereof (9)
(13) The plasmid according to (9), which has a DNA encoding the polypeptide between a promoter and a terminator, and (14) (1)
3) A method for producing a polypeptide, which comprises introducing the plasmid described in a filamentous fungus and culturing the obtained transformant.
I will provide a. In the sequences described in the claims and the detailed description of the invention, N represents a nucleotide, A represents adenine, T represents thymine, C represents cytosine, and G represents guanine. In addition, in the present specification, in these sequences, the left side is shown as the 5'-terminal side and the right side is shown as the 3'-terminal side, but even if the sequences are in the reverse direction, the enhancer function of the present invention is exerted. Yes, this notation indicates both enhancer sequences unless otherwise noted.
Hereinafter, the present invention will be described in detail.

First, the promoter of the α-glucosidase gene (agdA) of Aspergillus oryzae was isolated. That is, the plasmid pTGF-1 containing an α-glucosidase gene [JP-A-6-62868] was used as a material to determine the entire nucleotide sequence of the α-glucosidase gene (agdA) containing a promoter and terminator. Clones were prepared using a Kilo-sequencing deletion kit (Takara Shuzo), and the nucleotide sequences of these clones were determined by the dideoxy method. From the determined entire nucleotide sequence, the promoter region (SEQ ID NO: 2), α
-After determining the glucosidase coding region (SEQ ID NO: 1) and the terminator region (SEQ ID NO: 3), respectively, search for enhancer nucleic acid nucleotide sequence candidates in the promoter region using gene analysis software, and use the Aspergillus oryzae α-amylase gene. (AmyB) [Biosci. Biotech. Bio
chem., 56, 1849-1853 (1992)] or glucoamylase gene (glaA) [Curr. Genet., 22, 85-91 (199
2)] compared with the promoter.

In order to confirm the function of the enhancer candidate sequence found as a result, first, subcloning of the promoter of the agdA gene and various deletion promoters (FIG. 4) were constructed. Next, in order to identify an enhancer sequence required for expression of the agdA promoter using the various deletion promoters constructed, β-glucuronidase (GUS) gene (uidA) of E. coli as a reporter gene was added to the various deletion promoters. We constructed a fusion gene in which
Various promoter activity measuring plasmids (FIG. 2) were constructed, which consist of a terminator and an Aspergillus oryzae nitrate reductase gene (niaD) as a selection marker. Using this plasmid, a nitrate reductase-deficient strain of Aspergillus oryzae was obtained by a known method [Agric. Biol. Che.
m., 51, 323-328 (1987)] and Southern analysis of the resulting transformant was performed.
Transformants were selected that were able to accurately measure promoter activity without being affected by the position effect of integrating one copy homologously at the gene locus and integrating into the chromosome and the copy number introduced. The β-glucuronidase (GUS) activity of these selected transformants was determined by a known method [P
roc. Natl. Sci. USA, 83, 8447-8451 (1986)], and the enhancer sequence on the agdA promoter was identified.

That is, from the results shown in FIG. 4, there is an enhancer sequence in the XhoI-EcoRV region on the agdA promoter, and an enhancer candidate sequence is contained in this region. Since the activity of agdA deletion promoters 6 and 7 is drastically reduced when the downstream sequence is specifically deleted,
The sequence deleted in this deletion promoter (FIG. 1) was identified as the enhancer nucleobase sequence. That is, the DNA base sequence unit B (5′-CGGGCAT
TTA-3 ') and nucleotide sequence unit C (5'-CCAA
It has come to provide an enhancer nucleobase sequence including TCAGCGGT-3 ′).

Further, enhancer base sequences B and C
It is shown that the GUS activity of the agdA promoter containing a total of two DNA nucleotide sequence units E is increased by about 3 times by introducing one DNA nucleotide sequence unit E containing DNA (FIG. 1) into the agdA promoter (FIG. 5). Therefore, it was confirmed that this nucleotide sequence unit E also has an enhancer function.

The sequence E (AA) in which the 4th and 5th bases from the 5'end of the enhancer sequence B are replaced with the sequence -AA- present in the amyB promoter or glaA promoter, or a sequence in which any other base is substituted. It was shown that the promoter activity was increased 2-fold or more when either the E (TC) sequence or the sequence E (CG) sequence was introduced (FIG. 5).

From the above results, the base sequences at the 4th and 5th bases from the 5'end of the enhancer sequence B are -GC-
It was confirmed that the compound exhibited an enhancer action regardless of the substitution with any base, without being specified by. That is, the sequence A (5′-CGGNNATTTA containing the fluctuation of the base sequence
It was confirmed that -3 ') has an enhancer function. As described above, a novel enhancer nucleobase sequence was provided.

Furthermore, the present invention provides improvement of promoter activity by introducing the novel enhancer nucleobase sequence provided as described above into a promoter that functions in filamentous fungi.

The promoter for introducing the enhancer sequence is not particularly limited as long as it functions in filamentous fungi, and specifically, α-amylase, glucoamylase, α-glucosidase, protease, lipase, cellulase, cellobio. Examples thereof include hydrolase genes such as hydrase and acetamidase, and promoters of glycolytic enzyme genes such as 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase. Suitable promoters can be isolated from the Aspergillus α-amylase, glucoamylase, α-glucosidase genes. More preferably, the promoter of the α-glucosidase gene of Aspergillus oryzae is mentioned. The entire nucleotide sequence of the promoter is shown by SEQ ID NO: 2, but even a partial sequence thereof is included in the present invention as long as it has a function as a promoter in filamentous fungi.

The site for introducing the enhancer nucleic acid nucleotide sequence into the promoter is not particularly limited as long as it is a promoter region. The introduction method can be performed as follows, for example. First, the promoter region to be introduced is digested with two appropriate types of restriction enzymes [A] and [B]. However, [A] uses an enzyme that produces a blunt end. If not, the end of [A] is blunt-ended using a DNA branching kit (Takara Shuzo). Then 5 '
A fragment containing an enhancer sequence is amplified by PCR using a primer in which restriction enzyme sites are added from the end in the order of [B] and [A] and a primer in which no restriction enzyme sites are added. By inserting the thus amplified fragment into the promoter digested as described above, the blunt end site and the restriction enzyme B site are linked respectively. After binding, the restriction enzyme recognition site at the blunt end site disappears, but the sites [B] and [A] are present at the other site. When two or more enhancer sequences are introduced, it is achieved by digesting the one introduced promoter with restriction enzymes [A] and [B], and then sequentially introducing the enhancer sequences in the same manner. Further, depending on the number of enhancer sequences to be introduced, it becomes possible to construct a promoter having any transcriptional activity. Furthermore, as a specific example, the promoter of the α-glucosidase gene into which an enhancer sequence is introduced as shown in FIG. 6 not only has increased transcriptional activity, but is not affected by glucose repression, and is not affected by a carbon source and is constantly at a high level. Improved promoter to be expressed. That is, they have succeeded in improving the expression control of promoters.

Furthermore, the novel expression plasmid of the present invention is
A DN capable of replicating in Escherichia coli, which has a promoter capable of functioning in a filamentous fungus improved as described above, a terminator, and a marker gene suitable for selection of a host transformant.
It has an A region.

The terminator is not particularly limited as long as it functions in filamentous fungi, and specifically, the terminator of the Aspergillus oryzae α-glucosidase gene shown in SEQ ID NO: 3 or a terminator containing a partial sequence thereof. Are more preferably used.

Preferred selectable markers include genes for nitrate reductase (niaD), ornithine carbamoyl transferase (argB), tryptophan synthase (trpC) and acetamidase (amdS). However, the host filamentous fungus must use a strain that does not have a functional gene for the selected selection marker. A more preferred selectable marker gene is the nitrate reductase gene (niaD). When the niaD gene is used as a selectable marker, a niaD strain of filamentous fungus having a gene defect for this marker must be used as a host.
This niaD mutant strain was obtained by the method of Uncle et al. [Mol. Gen. Gene
t., 218, 99-104 (1989)]. By using this niaD gene as a selection marker, it becomes easy to obtain a transformant that is homologously integrated into the niaD gene locus of the host chromosome.
That is, since the influence of the expression due to the position to be integrated is excluded, the expression of the useful protein of interest is only defined by the copy number to be integrated. Therefore, by adjusting the number of enhancer sequences introduced into the promoter of the expression plasmid of the present invention and examining the copy number of the transformant selected by the niaD gene by Southern analysis, not only high production of useful proteins but also arbitrary It is possible to obtain a transformant having the following productivity. Furthermore, the niaD gene of Aspergillus, such as Aspergillus oryzae, has a function in many filamentous fungi, such as Aspergillus niger, Aspergillus nidulans, and Penicillium chrysogenum [Mol. G.
En. Genet., 218, 99-104 (1989)], various filamentous fungi can be used as hosts.

Construction of this plasmid can be carried out, for example, as follows. E. coli vector, eg pUC1
Plasmid pSTA14 [Mol. Gen. Genet., 218, 99-104 (198
9)] and insert the niaD gene cut out with an appropriate restriction enzyme. Next, the terminator region to be used is cut out with an appropriate restriction enzyme or amplified by PCR method and inserted into the above plasmid. The promoter obtained by introducing the enhancer sequence constructed by the above method into the obtained plasmid is cut out with an appropriate restriction enzyme and inserted adjacent to the terminator region. An expression plasmid can be constructed in this way. Further, specific examples include plasmids pNAG136 and pNAG142 constructed by the procedure shown in FIG. In these plasmids, eight kinds of restriction enzyme recognition sites are introduced for the purpose of increasing the number of restriction enzyme sites that can be used when inserting a DNA fragment encoding the target protein to be expressed between the promoter and the terminator.

The present invention further discloses a method for producing a useful protein or peptide of interest using a filamentous fungus as a host. This is achieved, for example, by the following steps. First, a site-specific mutagenesis method [Proc. Natl.
Sci. USA, 82, 488 (1985)] or a DNA fragment having these restriction enzyme sites at both ends, which was introduced by the PCR method, and used as an expression plasmid such as pNAG136 or pNAG136 or p
Insert into NAG142. Alternatively, when the gene terminator of the target protein functions in filamentous fungi, a DNA fragment containing the protein coding region and the terminator region can be prepared by the same method and ligated to the promoter. Using the transformation vector thus obtained, a filamentous fungus deficient in the gene for the selectable marker contained in this vector can be isolated by a known method [Agric.
iol. Chem., 51, 323-328 (1987)]. Next, after obtaining a transformant that can grow in a selective medium for the selectable marker, this transformant is cultured under an appropriate medium and culture conditions to produce a target protein. The protein of interest produced is qualitatively and quantitatively determined by an appropriate method, and is isolated and purified as required.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples, but the examples of the present invention are not limited thereto. The restriction enzymes and other genetically engineered enzymes used in this example were products of Takara Shuzo or Toyobo, and the reaction conditions for these enzymes were in accordance with the instructions attached when the products were purchased.

Example 1 Expression of α-glucosidase gene
Promoter region and terminators area plasmid pTG inserted into the PstI site of 7.0kb including α- glucosidase gene identification and nucleotide sequence Aspergillus oryzae RIB40 strain
F-1 [JP-A-6-62868] was used as the material.

First, 5.0 kb S containing α-glucosidase
The caI fragment was obtained from pTGF-1, and after adding an EcoRI linker to this fragment, the plasmid pTGS-1 was obtained by inserting the plasmid pUC118 into the EcoRI site in both forward and reverse directions.
In order to determine the entire nucleotide sequence of the α-glucosidase gene using pTGS-2, a deletion clone was prepared using a kilosequencing deletion kit (Takara Shuzo). Specifically, pTGS-1 or pTGS-2
It was digested with SmaI and PstI, digested with exonuclease III, blunt-ended with mung bean nuclease, end-repaired with Klenow fragment, and ligated to prepare a deletion plasmid of each chain length. Single-stranded DNA was prepared from these deletion plasmids,
The dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463
-5467 (1977)] was used to determine the nucleotide sequence using an automatic sequencer 370A (manufactured by Applied Biosystems). It has been confirmed that the coding region of the α-glucosidase gene is present in the approximately 3.2 kb HindIII-EcoRV region in the determined sequence [JP-A-6-62868], and the consensus sequence of introns in filamentous fungi [[The stru
cture and organization of nuclear genes of filamen
tous fungi ”IRL Press, Oxford, pp93-139 (1987)]. From the above results, the entire base sequence including the coding region of the α-glucosidase gene consisting of 985 amino acids shown in SEQ ID NO: 1 was determined.

Next, in order to determine the nucleotide sequence of the promoter region located upstream of the ScaI site located on the 5'-upstream side, the 1.5 kb PstI-SalI fragment of pTGF-1 [JP-A-6-62868] was used as a plasmid pUC118. Alternatively, it was inserted into the PstI and SalI sites of the multiple cloning site of pUC119. Single-stranded DNA was prepared from the plasmid, and the nucleotide sequence was determined by the dideoxy method using an automatic sequencer as in the case of the deletion plasmid. Based on the above results, the base sequence of the 720 bp promoter region shown in SEQ ID NO: 2 was determined.

The nucleotide sequence of the terminator region 3'downstream from the stop codon (TGA) of the α-glucosidase gene was 5.0 k determined by the deletion plasmid.
b ScaI fragment contains a sufficient length, and SEQ ID NO: 3
Shows the nucleotide sequence of the terminator region of 840 bp from the end.

Example 2 Search for Novel Enhancer Candidates In order to identify an unknown enhancer sequence existing in the promoter region of the α-glucosidase gene (agdA), first, a homology search for the promoter was performed by DNASI.
S gene analysis software (manufactured by Hitachi Software Engineering Co., Ltd.) was used. Aspergillus oryzae α-amylase gene (amyB) promoter, which is generally known to be highly expressed and secreted [Biosci. Biotec
h. Biochem., 56, 1849-1853 (1992)] or the promoter of the glucoamylase gene (glaA) [Curr. Gene
t., 22, 85-91 (1992)], the homology as a whole promoter region is similar to that of amyB promoter.
It showed 51% homology with the glaA promoter and 52% homology, but no region with particularly high homology was found. Therefore, next, the agdA promoter was divided into 20-50 bp units and a more detailed homology search was performed. As a result, the sequence of (5′-CGGGCTAAAT-3 ′) shown in FIG. 1 and the sequence of high homology (5′- CGGAAATTTA-3 ')
Was found in the amyB and glaA promoters. In addition, in this process, there are two known regions that are commonly conserved in the amyB promoter and the glaA promoter.
I and II [Curr. Genet., 22, 85-91 (1992)] are also agdA.
It was confirmed to be present in the promoter region.

Example 3 α-Glucosidase Gene Pro
Motor of subcloning Ningu and various de configuration Promo
Ta of construction homology search in an array that was found (5'-CGGGCT
In order to identify the presence or absence of the enhancer action of AAAT-3 ′) or the enhancer site other than that, ag
Subcloning of the dA promoter was performed using the PCR method.

As an upstream primer with a PstI site added to amplify 702 bp of agdA promoter positions 17 to 718 shown in SEQ ID NO: 2, as a downstream primer with a 5SP: 5'-GGCTGCAGTCATGGCACCACTAGAGATG-3 'SalI site added, 3ASP: 5'-CCGTCGACCGTGGTCCGCCAAGTTGATT-3 'was synthesized using a DNA synthesizer model 391 PCR-MATE (manufactured by Applied Biosystems). Using the above two primers and the plasmid pTGF-1 as a template, a PstI-SalI fragment of the agdA promoter was obtained by thermal cycler (manufactured by Perkin Elmer Japan), and this fragment was inserted into the multiple cloning site of pUC118. The plasmid pAGP1 (FIG. 1) containing the agdA promoter was obtained.

Next, various deletion promoters of agdA as shown in FIG. 4 were constructed using this pAGP1. Specifically, for example, after digesting pAGP1 with PstI and EcoT14I, about 3.8 kb Ps separated by 0.8% agarose electrophoresis.
Use the tI-EcoT14I fragment with GeneClean Kit (Bio 101
(Manufactured by the company). This fragment was blunt-ended with a DNA branching kit (Takara Shuzo) and self-ligated to obtain a plasmid pAGP2 containing agdA deletion promoter 2 (P-agdA2). Below, pAGP1 is referred to as PstI and XhoI, EcoRV, or FbaI.
Plasmid pAGP containing agdA deletion promoter 3, 4 or 5 in the same manner after double digestion with
Acquired 3, 4, or 5, respectively.

The agdA deletion promoter 6 (P-agdA6) obtained by deleting only the base sequence (5'-CGGGCTAAAT-3 ') having high homology to the amyB promoter and glaA promoter found by the homology search was a combination PCR. Method [“PCR Technology”, Stocton
Press, New York, pp. 61-70 (1989)]. Specifically, 5ISP: 5'-GCGTCGTGTCGGGGGATGGACCAATCAGC-3 'was first synthesized as an internal upstream primer lacking a high homology sequence, and 3IASP: 5'-CATCCCCCGACACGACGCTTGAGCCCTGA-3' was also synthesized as an internal downstream primer.

Next, PCR product 1 was amplified using upstream primer 5SP and internal downstream primer 3IASP synthesized during subcloning of agdA promoter, and PC was also amplified using internal upstream primer 5ISP and downstream primer 3ASP.
R product 2 was amplified. The resulting PCR products 1 and 2 were mixed to form a template, and the third PCR reaction was performed using the upstream primer 5SP and the downstream primer 3ASP to generate Gene Amp ^™ k.
It was carried out in the following reaction solution using it (manufactured by Perkin Elmer Japan). H ₂ O 7 0.5 μl 10x reaction solution 10 μl dNTP mix (1,25 mM) 16 μl Upstream primer 5SP (0.1 mM) 1 μl Downstream primer 3 ASP (0.1 mM) 1 μl PCR product 1 1 μl (5-100 ng) PCR product 2 1 μl (5-100 ng) ) Amplitaq ^™ DNA Polymerase 0.5 μl Reaction conditions are (94 ℃, 0.5 minutes) → (55 ℃, 1 minute) → (72 ℃,
2 minutes) for 25 cycles. The reaction solution after PCR amplification was treated with phenol / chloroform, recovered by ethanol precipitation, treated with PstI and SalI, and then subjected to 0.8% agarose electrophoresis to obtain agdA deletion promoter 6 (P-ag
dA6) was isolated and purified. This deletion promoter was inserted into pUC118 treated with PstI and SalI to obtain a plasmid pAGP6 containing P-agdA6.

Using the same combination PCR method, agdA deletion promoter 7 (P-agdA7) in which only the immediately downstream region 3 and the immediately upstream region 1 of the highly homologous nucleotide sequence as shown in FIG. 1 was deleted Alternatively, 8 (P-agdA
8) was obtained and inserted into pUC118 treated with PstI and SalI, respectively, to obtain pAGP7 or pAGP8. In addition, the deletion promoter obtained by the combination PCR method was confirmed to lack the target sequence by examining the nucleotide sequence by the dideoxy method.

Example 4 Plus for measuring promoter activity
Construction of Mido using various constructed agdA deletion promoters
To identify the enhancer sequences required for expression of the agdA promoter, the nitrate reductase gene of aspergillus oryzae (nia) as a selectable marker and the agdA terminator (nia
D) and β of E. coli as a reporter gene
-A plasmid pNAGT4 (Fig. 2) for measuring promoter activity, which consists of a glucuronidase (GUS) gene (uidA), was constructed.

First, the plasmid pSTA14 [Mol. Gen. Genet., 218, 99-104 (19) containing the nitrate reductase gene (niaD)]
89)] was digested with XhoI and HindIII and subjected to 0.8% agarose electrophoresis to isolate and purify a 4.4 kb XhoI-HindIII fragment containing niaD. This fragment was blunt-ended using a DNA blanching kit (Takara Shuzo), and then inserted into pUC118 which had been treated with EcoRI and similarly blunt-ended to obtain pNR10.

Next, β-glucuronidase (GU
S) gene (uidA) to insert pBI221 [Proc. N
atl. Acad. Sci. USA, 83, 8447-8451 (1986)] was digested at the 5'upstream SmaI site of the GUS gene, and a SalI linker was added to the digested site, followed by 3'downstream SacI. After digestion at the site, it was blunt-ended, XbaI linker was added, and self-ligation was performed in the same manner to obtain pBI221-SX. Approximately 1.9 kb SalI- containing the GUS gene from this pBI221-SX
The XbaI fragment was similarly isolated and purified using agarose electrophoresis, and then SalI and X of the pNR10 multicloning site were isolated.
It was inserted into the baI site to obtain the plasmid pNRG1.

In this pNRG1, the α-glucosidase gene (a
gdA) terminator (T-agdA)
First, the terminator was obtained by the PCR method. Of the positions 8 to 837 of the agdA terminator shown in SEQ ID NO: 3
5TSP: 5'-CCTCTAGAAGCGTAACAGGATAGCCT as an upstream primer with an XbaI site added to amplify 830 bp
3TASP: 5'-GGCCCGGGAGTAACCCATTCCCGGTTCT-3 'was synthesized using a DNA synthesizer model 391 PCR-MATE (manufactured by Applied Biosystems) as a downstream primer having an AG-3' SmaI site added. An XbaI-SmaI fragment of an agdA terminator was obtained by a thermal cycler (manufactured by Perkin Elmer Japan) using the above two primers and the plasmid pTGF-1 as a template. This fragment was inserted into pNRG1 digested with XbaI and SmaI to obtain a plasmid pNAGT4 for measuring promoter activity.

[0039] to obtain a transformant of Aspergillus oryzae using a plasmid obtained by inserting various agdA de configuration promoter pNAGT4 obtained as acquisition above Example 5 transformants.

First, among the above-mentioned plasmids containing various agdA deletion promoters, pAGP2, 3, 4, and 5 are SalI
After digestion with and NaeI, the SalI-NaeI fragment containing the deletion promoter was isolated and purified using agarose gel electrophoresis. In addition, SalI-PstI containing a deletion promoter was similarly treated from pAGP1, 6, 7, and 8 treated with SalI and PstI.
The fragment was isolated and purified.

Next, pNAGT4 was digested with SalI or NaeI, or digested with SalI or PstI, and pNAGT4 similarly treated with the respective restriction enzymes was isolated and purified. Eight types of plasmids, pNAGG1-1 to pNAGG1-8, were obtained by introducing the promoter fragments into pNAGT4, respectively. FIG. 2 shows an example thereof.

Next, Aspergillus oryzae was transformed with the plasmid of the pNAGG1 series obtained above. Nitrate reductase-deficient strain of Aspergillus oryzae (ni
aD ^-..), for example, NiaD14 strain [Mol Gen. Genet, 218, 99
-104 (1989)] with dextrin-peptone medium (2% dextrin, 1% polypeptone, 0.5% KH ₂ PO ₄ , 0.05% MgSO ₄ .
₄ · 7H ₂ O) at 30 ° C., it was cultured for 3 days while shaking, and washed the resulting cells with sterilized water. The cell wall lysate [10 mM
It was then suspended in a phosphate buffer, pH 6.0, 0.8 M NaCl, 20 mg / ml Yatalase (manufactured by Takara Shuzo Co., Ltd.) and gently shaken at 30 ° C. for 2 to 3 hours to form protoplasts. The resulting protoplasts were filtered with a glass filter to remove the remaining bacterial cells.

Next, using this protoplast, competent cells were prepared and transformed by the method of Gomi et al. [Agric. Biol. Chem., 51, 323-328 (1987)], and nitric acid was contained as a single nitrogen source. Medium, for example Czapek-Dox medium (0.2% NaNO ₃ , 0.1% K ₂ HPO ₄ , 0.05% KCl, 0.
_{05% MgSO 4 · 7H 2 O} , 2% glucose, viable transformants obtained each 12 share one by a total of 96 strains per each plasmid in pH 5.5].

Example 6 Various deletion promoters
Roh β- Gurukuronida over subjected to Southern analysis of peptidase activity resulting various transformants from them, homologously one copy, select the integrated strains was measured β- glucuronidase (GUS) activity.

First, in order to perform Southern analysis, chromosome D
NA was prepared. Conidiospores of transformants 1 Platinum loops were added to dextrin / peptone medium (2% dextrin, 1% polypeptone, 0.5% KH ₂ PO ₄ , 0.1% NaNO ₃ , 0.05% MgSO ₄
7H ₂ O) inoculated into 100 ml and shake-cultured at 30 ° C. for 3 days, the obtained bacterial cells were collected with a 3G1 glass filter and washed with sterile water. After removing the water content of the bacterial cells with a filter paper, the cells were ground in liquid nitrogen using a mortar precooled to -80 ° C. This crushed cell product is a TE solution (10 mM Tris-HCL, 1 mM
After suspending in EDTA, pH 8.0), lysing solution (0.5% SDS, 5
After adding an equal amount of 0 mM EDTA) and gently stirring, the mixture was left at 37 ° C. for 30 minutes. The obtained lysate was centrifuged at 3000 rpm for 20 minutes, and the supernatant was obtained. The supernatant was treated twice with an equal amount of a phenol / chloroform mixed solution to remove contaminating proteins, and then 2.5 volumes of cold ethanol was added to precipitate DNA. This precipitate was added to TE containing 0.1 mg / ml RNase.
It was slowly dissolved in the solution and reacted at 30 ° C for 30 minutes. After this solution was treated with phenol / chloroform, 2.5 volumes of cold ethanol was added, and the resulting chromosomal DNA was wound up with a Pasteur pipette. After drying the wound DNA,
It was dissolved in TE solution to prepare a chromosomal DNA solution. The resulting chromosomal DNA is digested with SalI and then separated by agarose gel electrophoresis to obtain a nylon membrane such as Hybond N.
(Between Amersham) and then plasmid pUC1
Southern analysis was performed using 18 as a probe. At this time, EC
Labeling of probes and detection of signals were performed using an L random prime DNA labeling / detection system (manufactured by Amersham). As a result, detection of a single band of 9.3 kb to 8.9 kb depending on the size of each deletion promoter contained in the plasmid used for transformation, or, in addition to this single band, 11.0 kb to 10.6 kb corresponding to the size of the plasmid There were two patterns in the case where a total of two bands were detected by hybridizing also to kb. This result is 1 as shown in FIG.
1 copy for a 2 band band, 2 for a 2 band band
More than one copy means homologous integration into the host niaD locus.

The plasmid used for transformation contains the niaD gene as a selection marker gene, and when transformed with this selection marker alone, it is homologously integrated at a high frequency [Mol. Gen. Genet., 218 , 99-104 (19
89)] has been clarified, but when transformed with a plasmid containing another gene to be newly introduced, niaD is not affected by the introduced gene.
It was revealed that the loci were integrated homologously. Also, there was a pattern in which recombination occurred at the niaD locus due to double crossover and only the niaD gene was integrated.

From the results of Southern analysis, a transformant suitable for the measurement of promoter activity, that is, a homologously integrated one copy, was not affected by the position effect at the time of integration into the chromosome and the copy number introduced. In addition, 16 transformants capable of accurately measuring the promoter activity were arbitrarily selected, 2 strains per plasmid used, for a total of 16 strains.

Next, the selected 16 strains of conidia ⁵ × 10 ⁵
Pieces of Czapek'dox-polypeptone medium (1% _{polypeptone, 0.1% K 2 HPO 4,} 0.05% MgSO 4 · 7H 2 O, 0.05%
15 ml of KCL, 0.01% FeSO ₄ , 2% glucose, pH 5.5) was inoculated, and after shaking culture at 30 ° C. for 3 days, the obtained bacterial cells were collected with a 3G1 glass filter. After removing the water content of the cells with a filter paper, about 0.2 g of the cells was ground in liquid nitrogen using a mortar cooled to -80 ° C. 0.8 ml of extraction solution (50 mM phosphate buffer, pH 7.0, 10 mM EDTA,
After suspending in 1% Triton X-100, 0.1% sarkosyl, 10 mM β-mercaptoethanol), the enzyme was extracted by vigorously stirring for about 1 minute, and the bacterial cell residue was removed by centrifugation at 15000 rpm for 15 minutes. The supernatant was used as the enzyme solution. The β-glucuronidase (GUS) activity in the obtained enzyme solution was determined by a known method [Proc. Natl. Acad. Sci. USA, 83, 8447-8451 (198).
6)], 1 ml of reaction solution (50 mM phosphate buffer, pH
7.0, 0.1% Triton X-100, 10 mM β-mercaptoethanol, 1 mM p-nitrophenyl glucuronide) at 37 ° C
The reaction was carried out by reacting 5 μl to 50 μl of the enzyme solution with and measuring the absorbance at 415 nm. The activity at this time was shown as a relative activity with the GUS activity produced by the original non-degraded α-glucosidase promoter (P-agdA1) as 100%. FIG. 4 shows the results.

When the agdA promoter was deleted from the 5'upstream region, the region between P-agdA1 and P-agdA2 (PstI-EcoT1
4I) and 2 of the region (XhoI-EcoRV) between P-agdA3 and P-agdA4
A large decrease in activity was observed at several places. Among them, PstI-EcoT1
In the 4I region, a known conserved sequence RegionI [Curr. Genet.,
22, 85-91 (1992)] existed. Also, XhoI-EcoRV
The region contained the base sequence B (5′-CGGGCATTTA-3 ′) found as a result of the homology search. Furthermore, since the activity of the deletion promoter (P-agdA6) lacking only the above sequence B is also drastically reduced, the nucleotide sequence B present in the agdA promoter is
It was confirmed to be a new enhancer. Moreover, among promoters P-agdA7 and P-agdA8 in which only the nucleotide sequences before and after enhancer sequence B were deleted, respectively, the relative activity of P-agdA7 showed only 19%.
It was confirmed that (5'-CCAATCAGCGT-3 ') is also an enhancer sequence.

Example 7 Diversity of novel enhancer sequences
The nucleotide sequence B (5′-CGGGCATTTA-3 ′) confirmed to be a novel enhancer sequence and a highly conserved sequence are the results of the homology search performed in Example 2,
It has been confirmed that it also exists in the α-amylase promoter and the glucoamylase promoter, and in order to confirm whether or not these sequences also have an enhancer function, a base part different from sequence B was replaced with an arbitrary base. A promoter was prepared by inserting the sequence into the agdA promoter, and the promoter activity was measured.

First, the plasmid pAGP1 containing the agdA promoter (0.7 kb PstI-SalI fragment) obtained in Example 3 was used to treat the EcoRV and ClaI sites of the agdA promoter with a restriction enzyme, and then a DNA branching kit (Takara Shuzo) was used. )
Was made blunt-ended with and the EcoRV-ClaI fragment containing no enhancer sequence was removed by self-ligation to remove the agdA deletion promoter 131 (P-agdA13
A plasmid pAGP131 containing 1) was obtained (FIG. 5). Next, DN containing the enhancer sequences B and C shown in FIG.
Ec from which A base sequence E (specific sequence is shown in FIG. 4) is removed
This sequence E was obtained by PCR for insertion in place of the oRV-ClaI fragment. Specifically, as an upstream primer, 5SPE: 5'-TCAAGCGTCGTGTCGGGCATT-3 'As a downstream primer with a ClaI site added, 3ASPE: 5'-CCATCGATGATATCCCTACGCTGATTGG-3' is a DNA synthesizer model 391 PCR-MATE (Applied Biosystems). (Manufactured by the company). Amplification was performed by a thermal cycler (manufactured by Perkin Elmer Japan) using the above two primers and the plasmid pTGF-1 as a template. In order to insert this amplified sequence E into the agdA promoter, agd was inserted in the same manner as in P-agdA131.
The A promoter was digested with EcoRV and ClaI and the EcoRV-ClaI fragment was removed using agarose gel electrophoresis. This and array E
Plasmid p containing the promoter 131GC (P-agdA132GC) having the sequence E inserted by ligating
AGP132-GC was obtained.

Next, a sequence (5 ') in which the fourth and fifth bases from the 5'end of enhancer sequence B are replaced with -AA-
-CGGAAATTTA-3 '), that is, the sequence E (AA) containing a sequence common to the α-amylase (amyB) promoter and the glucoamylase (glaA) promoter is added to P-ag.
Similar to dA131GC, PCR amplification of sequence E (AA) was performed for insertion into the agdA promoter. As the upstream primer, 5SPE-AA: 5'-TCAAGCGTCGTGTCGGAAATT-3 'was synthesized, and the downstream primer was amplified by the thermal cycler as before using the primer 3ASPE used in the sequence E amplification. This amplified sequence E (AA)
Was inserted into the agdA promoter in the same manner as for P-agdA132GC to obtain a plasmid pAGP132-AA containing the promoter 132AA (P-agdA132AA). In addition to the above, the 4th base from the 5'end of enhancer sequence B and the 5th base
Sequence E (TC) or sequence E (CG) containing a sequence whose base is replaced by -TC- or -CG- is amplified by PCR in the same manner as above, and then inserted into the agdA promoter. The promoter 132TC (P-agdA13
2TC) or promoter 132CG (P-agdA132C
G) containing plasmid pAGP132-TC or pAGP132-CG
Got

The 5 kinds of plasmids obtained above were designated as PstI,
PstI containing various modified agdA promoters after treatment with SalI
-The SalI fragment was isolated and purified by agarose gel electrophoresis. This fragment was inserted into the PstI and SalI sites of the promoter activity measurement plasmid pNAGT4 (FIG. 2) constructed in Example 4 to obtain pNAGG1-131, -132GC, -132AA, -132TC and -1.
I got 32 CG. Next, using these plasmids, a total of 30 Aspergillus oryzae transformants, 6 strains for each plasmid, were obtained by the method shown in Example 5. Southern analysis of these transformants was performed by the method of Example 6 to determine the effect of one copy, which was homologously integrated at the nitrate reductase gene (niaD) locus, integrated into the chromosome, and the effect of the introduced copy number. Transformants not receiving 2 strains were arbitrarily selected. According to the method of Example 6, the selected transformant was cultured in a Czapek-Dox polypeptone medium, and then the cells were disrupted with liquid nitrogen, the enzyme was extracted from the disrupted cells, and the β-glucuronidase (GUS) of the extracted enzyme solution was added. ) The activity of the modified promoter was measured by measuring the activity.

As a result, the GUS activity of the modified promoter 132GC (P-agdA132GC) obtained by introducing the DNA base sequence E containing the enhancer sequences B and C into the agdA promoter was 2.7 times higher than that of the control P-agdA131. It was confirmed that the sequence E including the enhancer sequences B and C has the ability to enhance the promoter activity. Further, the sequence E (AA) in which the 4th and 5th bases from the 5'end of the enhancer sequence B are replaced with the sequence -AA- present in the amyB promoter and glaA promoter, or
Sequence E (TC) and sequence E (C) substituted with any other base
It was shown that the promoter activity was increased 2-fold or more when any of the sequences of G) was introduced. based on the above results,
It was confirmed that the base sequences at the 4th and 5th bases from the 5'end of the enhancer sequence B exhibit an enhancer action regardless of which base is substituted without being specified by -GC-. That is, the sequence A (5'- containing the fluctuation of the base sequence
CGGNNATTTA-3 ′) was shown to have an enhancer function.

Example 8 By introducing enhancer sequence
Enhancer sequence A (5'-CG containing improved 2 base fluctuation of promoter
The promoter activity was improved by introducing a plurality of DNA base sequences E containing GNNATTTA-3 ′) and enhancer sequence C into the agdA promoter.

First, the promoter P- constructed in Example 7
agdA132GC is a promoter having one sequence E introduced, and the 5'end of the sequence E introduced at this time binds with a blunt end, and the EcoRV recognition site disappears after the binding. on the other hand,
The 3'end has a P in addition to the EcoRV site originally contained in sequence E.
Since the ClaI site is added at the time of CR, there are two restriction enzyme sites after binding as shown in FIG. 5 or 6. Using the EcoRV and ClaI sites, the sequence E was successively introduced in tandem. Specifically, the plasmid pAGP132-GC containing P-agdA132GC was EcoRV,
It was digested with ClaI, and the sequence E having EcoRV and ClaI sites at the 3'end, which was PCR-amplified in Example 7, was introduced into pAGP1.
Built 33. This pAGP133 is digested with EcoRV and ClaI, and then the sequence E is sequentially introduced into the promoter in the same manner,
A maximum of 11 plasmids were introduced, and a total of 12 plasmids containing the promoter P-agdA142 in which the sequence E was present in tandem were constructed. 4 of these plasmids are sequence E,
Plasmid pA containing 6 or 12 existing promoters
After digesting GP134, 136, 142 with PstI and SalI, these improved promoters were isolated and purified by agarose gel electrophoresis, and
Plasmid pNAGT4 for measuring promoter activity, which was constructed in Example 4 with an improved promoter having tI and SalI sites at both ends.
PNAGG1-134, -1 inserted into the PstI and SalI sites of (Fig. 2)
Acquired 36 and 142.

Next, as a control of the improved agdA promoter, the promoter of the glucoamylase gene (agdA) of Aspergillus oryzae was used, and a plasmid in which the GUS gene as a reporter gene was ligated thereto was agdA.
Constructed as for the promoter. Specifically, first, a known glaA promoter nucleotide sequence [Curr. Genet.,
22, 85-91 (1992)] and the following primers were synthesized to subclone the glaA promoter (P-glaA). As the upstream primer with PstI site added, 5SGP: 5'-GGCTGCAGAGCCTACGCTAAAGCAAAGT-3 'As the downstream primer with SalI site added, 5ASGP: 5'-CCGTCGACTGCTTCGACTTCGTTTGCTG-3' The glaA promoter (P-gla
A) -containing plasmids such as pRGA-1 [Gene, 108, 14
5-150 (1991)] as a template to perform PCR amplification to obtain a 0.7 kb PstI-SalI fragment of the galA promoter (P-glaA), and the PstI and SalI sites of the promoter activity measurement plasmid pNAGT4 constructed in Example 4. Insert into pNGA
I built G1. Next, using these plasmids, transformants were obtained by the method shown in Example 5 in a total of 24 strains, 6 strains for each plasmid in total, and Southern analysis of these transformants was performed by the method of Example 6. Then, two transformants, which were homologously integrated at the niaD gene locus and integrated, were randomly selected in two strains. The modified transformant was cultured according to the method of Example 6 and the enzyme was extracted, and the β-glucuronidase (GUS) activity of the extracted enzyme solution was measured to measure the activity of the improved promoter.

As a result, as shown in FIG. 6, the GUS activity increased as the sequence E was introduced in tandem into the promoter, and P-agdA142 had 3.5 times the GUS activity as compared with P-agdA131 having one sequence E. It was confirmed that the promoter activity was enhanced.
Furthermore, it was confirmed that the method of introducing an enhancer sequence into a promoter is very effective, since it exhibits a promoter activity three times or more that of the glaA promoter which is originally useful as a high expression promoter.

Example 9 Comparison of improved promoter with carbon source
In order to test the effectiveness of the promoter P-agdA142 highly expressed by introducing a plurality of efficiency enhancer sequences E in tandem against carbon sources, GUS of cells grown in media containing various carbon sources was tested. The activity was measured.

The transformant in which one copy of the fusion gene in which the GUS gene was ligated to the original unmodified agdA promoter (P-agdA1) used in Example 6 was integrated, and the improved promoter used in Example 8 (P -agdA142) or a transformant in which one copy of the fusion gene of glaA promoter (P-gla) and GUS gene was integrated,
1 x 10 ⁶ conidia of 2 strains each, total 6 strains
Dox Polypeptone Medium (1% Polypeptone, 0.1%
_{K 2 HPO 4, 0.05% MgSO} 4 · 7H 2 O, 0.05% KCl, 0.01% FeS
O ₄ , 2% glucose, pH 5.5) or 15 ml of medium in which glucose in this medium is replaced with various carbon sources shown in Table 1.
And shake culture at 30 ° C. for 3 days, followed by disruption of cells and enzyme extraction according to the method of Example 6 to extract GUS of the enzyme solution.
The activity was measured. Table 1 shows the results.

[0061]

[Table 1]

The improved promoter (P-agdA142) showed 10 times or more higher activity in glucose and 5 to 6 times higher activity in other carbon sources than the original promoter (P-agdA1). Furthermore, P-agdA142 showed high activity at any of the carbon sources tested, compared to the glaA promoter (P-glaA). In addition, the glaA promoter or the original agdA promoter has a characteristic that the activity when it is a carbon source glucose is extremely lowered because it is affected by glucose repression, but the improved promoter has about the same level as other carbon sources in glucose. It retained 80% relative activity. This result means that the promoter P-agdA142 improved by introducing a plurality of enhancer sequences is not affected by the carbon source and is constantly highly expressed, and its usefulness is very high. The expression control function of the promoter is also
It shows that it can be improved.

Example 10 Northern of Improved Promoter
Analysis The improved promoter P-agdA142 constructed in Example 8 confirmed a significant increase in GUS activity. This increased activity resulted in an increase in the transcriptional activity of the promoter, that is, an increase in the mRNA of the GUS gene (uidA). I checked whether it was because of it.

The enhancer sequence E constructed in Example 8 was
Promoter with only one gene (P-agdA131) Promoter with four genes (P-agdA134) or improved promoter with 12 genes (P-agdA142) and one copy of GUS gene fusion gene, integrated transformant 1 × 10 ⁶ conidia of the above were inoculated into 100 ml of Czapek-Dox-polypeptone medium, shake-cultured at 30 ° C. for 3 days, harvested with a glass filter, and washed with sterile water. Washed cells about 2.
After transferring 0 g to 100 ml of Czapek Dox Polypeptone medium containing glucose or maltose as a carbon source,
Further, the cells that had been shake-cultured at 30 ° C. for 12 hours were collected with a glass filter and washed with sterile water. After removing the water content of the washed cells with filter paper, grind about 2.0 g of the cells in liquid nitrogen using a mortar cooled to -80 ° C.
l guanidine isothiocyanate solution (5M guanidine isothiocyanate, 10 mM EDTA, 50 mM Tris-HCl, pH
7.5) and 0.6 ml of β-mercaptoethanol were added to prepare a suspension solution, and then RNA was prepared according to a known method [DNA, 2, 329-335 (1983)]. After separating 20 μg of this RNA by formaldehyde-agarose gel electrophoresis, nylon membrane such as Hibond N (Amersham)
To the membrane by trans-blotting and then irradiating with ultraviolet light using a trans-illuminator.
A was fixed. Next, the GUS gene (uidA) of the 1.9 kb XbaI-SalI fragment prepared from the plasmid pBI221-SX (Fig. 2) was hybridized with the membrane (5xSSPE, 5x).
Denhardt's solution, 50% formamide, 0.5% SDS, 100 μg
/ ml heat-denatured salmon sperm DNA) at 42 ° C. for 17 hours, and then the membrane was washed with an appropriate washing solution such as 6 × SSC or 0.2 × SSC, 0.1% SDS. The signal hybridized with this membrane was detected on X-ray film.

As a result, as shown in FIG. 7, in both cases of the carbon source glucose and maltose, the promoter (P-agdA142) having 12 enhancer sequences was found to be better than the promoter (P-agdA131) having one enhancer sequence. , A large amount of GUS gene (uidA) mRNA was detected. This means that the transcription activity of the promoter was increased by introducing multiple enhancer sequences into the agdA promoter, and the increase in GUS activity may be the result of the improved promoter function of P-agdA142. confirmed.

Example 11 High Expression Vectors pN AG136 and pNA
Construction of G142 Having a nitrate reductase gene (niaD) as a selectable marker, high expression promoter P-agdA136 or P-agdA142 and agdA
Multi-cloning site (MC
A versatile high expression vector pNAG136 or pNAG142 into which S) was introduced was constructed by the procedure shown in FIG.

First, in order to amplify 578 bp from the agdA terminator position 8 to 585 shown in SEQ ID NO: 3, the upstream primer 5T added with the XbaI site synthesized in Example 4 was used.
A downstream primer, 3TASP-2: 5'-GGGAGGTGTACGCTTGGTAAAGT-3 ', to which SP and SmaI sites were added, was synthesized using a DNA synthesizer model 391 PCR-MATE (manufactured by Applied Biosystems). PCR amplification was performed using these primers with the plasmid pTGF-1 [JP-A-6-62868] as a template to obtain a 0.6 kb XbaI-SmaI fragment of the agdA terminator. This fragment was inserted into the XbaI and SmaI sites of the multicloning site (MCS) of the plasmid pNR10 containing the niaD gene constructed in Example 4 to construct the plasmid pNRT10. At the PstI and SalI sites of this plasmid, a high expression promoter Pa having 6 or 12 enhancer sequences constructed in Example 8 was used.
The PstI-SalI fragment of gdA136 or P-agdA142 was inserted to obtain pNAG136xs or pNAG142xs.

Next, the agdA terminator (T-agdA) and the highly expressed agdA promoter (P-agdA136 or P-agdA14
In order to introduce a multi-cloning site between 2), we first searched for restriction enzymes that can be used as a multi-cloning site. That is, the niaD gene and agdA terminator (T-ag
dA), high expression promoter (P-agdA136 or P-agd
A142) and pUC118 were searched for restriction enzymes, and as a result, in addition to XbaI and SalI which can be used in pNAG136xs or pNAG142xs, NdeI, NotI, PmaCI and Spe were newly added.
It was confirmed that I can be used. Also, if HindIII and SphI existing at the 5'end of the highly expressed agdA promoter are removed, these restriction enzymes can also be used, so pNAG136x
s or pNAG142xs was digested with HindIII and SphI to give DN
After blunting with A branching kit (Takara Shuzo), self-ligation was performed to obtain Hind.
III and SphI sites were removed. Next, as a result of the above-mentioned search, 8 types of usable restriction enzymes (XbaI and SalI at both ends)
The multi-cloning site consisting of (Fig. 8) was used to synthesize a sense strand and an antisense strand using a DNA synthesizer, and after mixing these, (94 ° C, 1 minute) → (55 ° C, 10 minutes) and incubation, It was annealed by cooling with ice to obtain a double-stranded multi-cloning site. The synthesized single-stranded DNA is shown below.

Sense strand: 5'-CTAGAGCATGCCATATGAC
TAGTCACGTGGCGGCCGCAAGCTTG-3 'Antisense strand: 5'-TCGACAAGCTTGCGGCCGCCACGTGACTAGT
CATATGGCATGCT-3 '

The 5'end of the multi-cloning site obtained here is phosphorylated using T4 polynucleotide kinase (Takara Shuzo), and then inserted into the XbaI and SalI sites of pNAG136xs and pNAG142xs from which HindIII and SphI have been removed. As a result, pNAG136 and pNAG142 were constructed.

Example 12 α of Aspergillus oryzae
-Acquisition of glucosidase high-producing strain P-agdA136 improved to a high expression promoter by introducing a plurality of enhancer sequences E constructed in Example 8 into the agdA promoter, or α-glucosidase (AGL) high production by P-agdA142 In order to breed the strain, first, the transformation plasmid pNAGL136 or pNAGL142
Was built. The construction procedure is shown in FIG.

Improved promoter P-ag constructed in Example 8
The plasmid pAGP136 or pAGP142 containing dA136 or P-agdA142 was digested with CpoI and BamHI, and then isolated and purified by agarose electrophoresis. Next, a plasmid pTGF-1 containing an α-glucosidase gene (agdA) [JP-A-6-6286]
8] was treated with CpoI and BamHI, and a 3.8 kb CpoI-BamHI fragment containing the coding region and terminator region of agdA was similarly isolated and purified. This fragment was inserted into the above-mentioned plasmid treated with CpoI and BamHI to construct pAGL136 or pAGL142. After digesting these plasmids with PstI and SmaI,
The PstI-SmaI fragment of the agdA gene linked to the improved agdA promoter of 4.4 kb or 4.7 kb was isolated and purified, and this fragment contained the nitrate reductase gene (niaD) as a selection marker constructed in Example 4. PstI, S of plasmid pNR10
By inserting it into the maI site, the transformation plasmid p
NAGL136 or pNAGL142 was constructed.

Next, using these plasmids, the nitrate reductase deficient strain of Aspergillus oryzae (nia14) was transformed by the method shown in Example 5, and after obtaining transformants of about 300 strains, The production strain was screened by the method described below. The conidial 1 platinum loop of the transformant was treated with 0.05% 4-nitrophenyl-α-D-glucoside (4N
PG) containing Czapek-Dox agar (0.2% NaN
_{O 3, 0.1% K 2 HPO} 4, 0.05% KCl, 0.05% MgSO 4 · 7H 2 O, 2%
Glucose, 1.5% agar, pH 5.5) was inoculated, and the color around the colonies of the grown bacterial cells was caused by the action of secreted α-glucosidase to decompose the substrate 4NPG 4-
Screening was performed by using the time and the color intensity at which the color started to change to yellow due to nitrophenol as an index. By this screening, a dozen or more strains with strong activity were selected, and then Southern analysis of these transformants was performed to screen the strain with the highest copy number. At the same time, Southern analysis was also performed on arbitrary strains with weak activity. The chromosomal DNA was prepared according to the method of Example 6. The chromosomal DNA obtained here is digested with XhoI and SalI, separated by agarose electrophoresis, and blotted on a nylon membrane, for example, Hybond N (manufactured by Amersham), and then the agdA gene of 3.8 kb CpoI-BamHI fragment is probed. Southern analysis was carried out. At this time, probe labeling and signal detection were performed using an ECL random prime DNA labeling / detection kit (manufactured by Amersham).

As a result, as shown in FIG. 10, 7.1 kb corresponding to agdA originally possessed by Aspergillus oryzae.
Band was commonly detected, and other than that, 8.0 kb or 8.9 depending on the length of the high expression promoter used.
kb band is detected and there are two signals in total ni
A strain homologous to the aD locus and integrated, or these two signals, in addition to the transforming plasmid (pNAGL136 or pNAGL1) used.
A band of 12.0 kb or 12.3 kb corresponding to the size of 42) was also detected, and two patterns of homologous 2 copies or more and integrated strains in which a total of 3 signals were present were detected. In addition, the transformants that had strong activity in the plate screening had integrated 2 copies or more, and among them, the strain AGL136-60 having the strongest signal of 12.0 kb had 4 to 5 copies, 12.
Strain AGL142-72, which had the strongest signal of 3 kb, was estimated to have integrated 3 to 4 copies, respectively.

Next, the above AGL136-60 strain and AGL142-72 strain,
In addition, 2 copies each of 1 copy and 2 copy strains obtained from the plasmid used for transformation were selected, and α-glucosidase (AGL) activity of the transformants of these 10 strains was measured.

Two conidia 2 platinum loops of the selected 10 strains and the parent strain niaD14 were treated with dextrin-peptone (DP) medium (2%
Dextrin, 1% polypeptone, 0.5% KH ₂ PO ₄ , 0.05%
MgSO ₄ · 7H ₂ O) or carbon source 2% glucose or 2%
Maltose Czapek Docks Polypeptone (C
DP medium (1% polypeptone, 0.1% K ₂ HPO ₄ , 0.05%
_{MgSO 4 · 7H 2 O, 0.05} % KCl, 0.01% FeSO 4, 2% carbon source, pH
Inoculated into 15 ml of the three kinds of medium of 5.5), shake culture was carried out at 30 ° C. for 3 days, and the culture filtrate was prepared by removing the cells using a glass filter. The α-glucosidase (AGL) activity in this culture filtrate was measured by the reaction solution (20 mM acetate buffer, pH 5.
0, 0.2% 4-nitrophenyl-α-D-glucoside (4NP
(G)), an appropriate amount, for example, 0.1 ml of the culture filtrate is added to make 1 ml, and after the reaction at 37 ° C. for an arbitrary time (1 to 60 minutes), 2.0 ml of 0.
The reaction was stopped by adding a 5M Na ₂ CO ₃ solution, and 4-nitrophenol (4NP) liberated in the reaction solution was measured by an increase in absorbance at 405 nm. One unit (U) was defined as the activity of releasing 1 μmol of 4NP in 1 minute. The protein concentration in the culture filtrate was quantified with a protein assay staining solution (manufactured by Bio-Rad). Table 2 shows the measurement results
Shown in

[0077]

[Table 2]

The AGL activity of the transformant was much higher than that of the parent strain when cultured in any medium. For example, 1 copy of AGL142-9 was only integrated in DP medium.
It increased more than 40 times in 5 strains. Further, AGL136 strain, in any of the transformants of the AGL142 strain, the higher the integrated copy number, the higher the activity, as a result of Southern analysis, the most copy number AGL136-60 strain, AGL142-72 strain,
80 times more in DP medium, when glucose in CD-P medium
The activity was increased more than 130 times and that of maltose was increased more than 25 times. In addition, the activity of the improved promoter used was P-ag
P-agdA142 was higher than dA136, and the AGL142 strain was more active among the transformants having the same copy number, reflecting this.

The above results show that when the niaD gene is used as a selection marker, it is homologously integrated and therefore is not affected by the position effect when it is integrated into the chromosome. Therefore, the strength of the promoter to be used and the integration are not affected. It means that it is possible to obtain a transformant having any enzyme activity depending on the copy number. Furthermore, the relative activity of glucose on the carbon source maltose of AGL of the transformants was 70-80% in most of the strains, compared to only 15% in the parent strain, which is the AGL produced by the improved promoter. Means that there is little effect of glucose suppression.

The culture filtrates of these transformants were subjected to SDS-polyacrylamide gel electrophoresis,
It was confirmed that a protein having the same molecular weight as α-glucosidase was clearly increased.

[0081]

By introducing the enhancer DNA nucleotide sequence of the present invention into a promoter that functions in filamentous fungi,
Not only the transcription activity of the promoter is increased, but also a promoter having an arbitrary strength can be constructed, and further, its expression control function can be improved, and the existing promoter can be improved to a more useful promoter. In addition, by using an expression plasmid containing the promoter thus improved, industrially useful enzymes, proteins, polypeptides, etc. can be more efficiently produced by using a filamentous fungus having high safety and high secretion ability as a host. It has become possible to manufacture in large quantities.
Further, analysis of the promoter relating to the enhancer sequence of the present invention contributes to the elucidation of the expression control mechanism of filamentous fungi to some extent.

[0082]

[Sequence list]

SEQ ID NO: 1 Sequence length: 3207 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Sequence: TCCGTTTCTT AAAAGAACAA CTACAACACG GTCCGGAATC AACTTGGCGG ACCACGAC 58 ATG GCC GGT CTA AAA AGC TTC CTT GCC AGT TCT TGG CTG CTA CCA GTG 106 Met Ala Gly Leu Lys Ser Phe Leu Ala Ser Ser Trp Leu Leu Pro Val 1 5 10 15 GCT TGC GGG GCG AGT CAA TCT ATC GTT CCT AGC ACT TCG GCA ACA GCG 154 Ala Cys Gly Ala Ser Gln Ser Ile Val Pro Ser Thr Ser Ala Thr Ala 20 25 30 GCA TAC TCG CAG TTC ACC ATT CCC GCC TCT GCC GAT GTG GGC GCG AAT 202 Ala Tyr Ser Gln Phe Thr Ile Pro Ala Ser Ala Asp Val Gly Ala Asn 35 40 45 TTG GTC GCC AAC ATT GAT GAC CCC CAA GCG GTC AAC GCG CAA TCT GTC 250 Leu Val Ala Asn Ile Asp Asp Pro Gln Ala Val Asn Ala Gln Ser Val 50 55 60 TGT CCG GGC TAC AAG GCC TCC GAT GTG AAA CAT TCC TCC CAG GGT TTC 298 Cys Pro Gly Tyr Lys Ala Ser Asp Val Lys His Ser Ser Gln Gly Phe 65 70 75 80 ACC GCT AGC CTG GAG TTG GCT GGA GAC CCT TGT AA T GTT TAC GGA ACG 346 Thr Ala Ser Leu Glu Leu Ala Gly Asp Pro Cys Asn Val Tyr Gly Thr 85 90 95 GAC GTC GAT TCG TTG ACC CTG ACC GTG GAA TAC CAG GCA AAG GAT CGT 394 Asp Val Asp Ser Leu Thr Leu Thr Val Glu Tyr Gln Ala Lys Asp Arg 100 105 110 TTG AAC ATC CAG ATT GTT CCG ACG TAT TTT GAC GCC TCC AAT GCA TCT 442 Leu Asn Ile Gln Ile Val Pro Thr Tyr Phe Asp Ala Ser Asn Ala Ser 115 120 125 TGG TAC ATT CTT TCG GAA GAG CTA GTG CCC AGA CCA AAG GCT TCC CAA 490 Trp Tyr Ile Leu Ser Glu Glu Leu Val Pro Arg Pro Lys Ala Ser Gln 130 135 140 AAT GCA TCG GTT CCT CAG AGT GAT TTT GTT GTC TCT TGG TCC AAC GAA 538 Asn Ala Ser Val Pro Gln Ser Asp Phe Val Val Ser Trp Ser Asn Glu 145 150 155 160 CCT TCT TTC AAC TTT AAG GTG ATC CGA AAA GCT ACT GGT GAC GTG CTA 586 Pro Ser Phe Asn Phe Lys Val Ile Arg Lys Ala Thr Gly Asp Val Leu 165 170 175 TTC AAC ACA AAG GGC TCT ACC TTA GTC TAC GAG AAT CAG TTC ATA GAA 634 Phe Asn Thr Lys Gly Ser Thr Leu Val Tyr Glu Asn Gln Phe Ile Glu 180 185 190 TTT GTC ACG TTG TTG CCT GAA GAA TAT A AC CTA TAT GGC TTG GGA GAG 682 Phe Val Thr Leu Leu Pro Glu Glu Tyr Asn Leu Tyr Gly Leu Gly Glu 195 200 205 CGG ATG AAC CAG CTG CGG CTA CTG GAG AAC GCT AAT TTG ACG CTA TAT 730 Arg Met Asn Gln Leu Arg Leu Leu Glu Asn Ala Asn Leu Thr Leu Tyr 210 215 220 GCC GCA GAT ATC GCA GAT CCC ATT GAC GA gtacgtatct ggcatttggt gc 781 Ala Ala Asp Ile Ala Asp Pro Ile Asp As 225 230 ggcccatt aggttttagg cTAatccat GGAc 837 p Asn Ile Tyr Gly His His 235 240 GCA TTT TAC TTG GAT ACA AGG TAC TAC AAG GTG GGT GGT CAG AAT AAG 885 Ala Phe Tyr Leu Asp Thr Arg Tyr Tyr Lys Val Gly Gly Gln Asn Lys 245 250 255 AGC CAT ACC ATA GTC AAG AGC AGC GAA GCG GAA CCA TCT CAA GAA TAC 933 Ser His Thr Ile Val Lys Ser Ser Glu Ala Glu Pro Ser Gln Glu Tyr 260 265 270 GTC TCA TAT TCT CAC GGA GTG TTC CTC AGA AAT GCC CAT GGA CAG GAG 981 Val Ser Tyr Ser His Gly Val Phe Leu Arg Asn Ala His Gly Gln Glu 275 280 285 ATC CTC CTG CGG GAT CAA AAG TTG ATC TGG CGC ACT CTG GGA GGA AGC 1029 Ile Leu Leu Arg Asp Gln Lys L eu Ile Trp Arg Thr Leu Gly Gly Ser 290 295 300 GTT GAT CTG ACA TTC TAC TCT GGC CCA ACG CAA GCC GAG GTC ACC AAG 1077 Val Asp Leu Thr Phe Tyr Ser Gly Pro Thr Gln Ala Glu Val Thr Lys 305 310 315 320 CAA TAT CAG CTC AGC ACC GTG GGA CTG CCT GCC ATG CAG CAA TAT AAC 1125 Gln Tyr Gln Leu Ser Thr Val Gly Leu Pro Ala Met Gln Gln Tyr Asn 325 330 335 ACG CTC GGA TTT CAC CAG TGC CGC TGG GGC TAT AAC AAC TGG TCC GAA 1173 Thr Leu Gly Phe His Gln Cys Arg Trp Gly Tyr Asn Asn Trp Ser Glu 340 345 350 TTT GAA GAC GTA CTT GCC AAT TTC GAG AGA TTC GAG ATT CCT TTG GAG 1221 Phe Glu Asp Val Leu Ala Asn Phe Glu Arg Phe Glu Ile Pro Leu Glu 355 360 365 TAC CTC TG gtaagaaaca tgttcgtcgc tcattcggct tccttctaac gcctatat 1277 Tyr Leu Tr 370 gc ag G GCC GAT ATC GAT TAC ATG CAT GGA TAT CGC AAT TTT GAC 1321 p Aly Tyr Arp Asp Asp 375 380 AAT GAC CAA CAT CGC TTT TCG TAT GAA GAA GGT GAA AAG TTC CTC AAC 1369 Asn Asp Gln His Arg Phe Ser Tyr Glu Glu Gly Glu Lys Phe Leu Asn 385 390 395 400 AAG CTT C AC GCC GGT GGA CGT CGC TGG GTC CCA ATC GTT GAC GGA GCT 1417 Lys Leu His Ala Gly Gly Arg Arg Trp Val Pro Ile Val Asp Gly Ala 405 410 415 CTT TAT ATT CCC AAT CCG GAG AAC GCT TCT GAT GC gtaagtggcc Legtctt 1 Tyr Ile Pro Asn Pro Glu Asn Ala Ser Asp Al 420 425 ccaca tactcttgcc cgtgaacgaa gactcaccgt gattatag T TAC GAA ACT TAT 1523 a Tyr Glu Thr Tyr 430 GAC AGA GGC GCC AAG GAC GAT GTT TTC ATC AAG AAT CCC GAC GGC Arg AGT Ala Lys Asp Asp Val Phe Ile Lys Asn Pro Asp Gly Ser 435 440 445 CTA TAC ATT GGC GCT GTC TGG CCT GGC TAT ACT GTC TAC CCC GAC TGG 1619 Leu Tyr Ile Gly Ala Val Trp Pro Gly Tyr Thr Val Tyr Pro Asp Trp 450 455 460 CAT CAT CCT AAG GCC TCC GAT TTC TGG GCT AAT GAG CTG GTC ACC TGG 1667 His His Pro Lys Ala Ser Asp Phe Trp Ala Asn Glu Leu Val Thr Trp 465 470 475 480 TGG AAC AAG CTG CAT TAT GAT GGG GTC TGG TAC GAC ATG GCT GAA GTT 1715 Trp Asn Lys Leu His Tyr Asp Gly Val Trp Tyr Asp Met Ala Glu Val 485 490 495 TCT TCC TTC TGC GTA GGG AGC TGC GGA ACT GGC AAT CTG TCA ATG AAC 1763 Ser Ser Phe Cys Val Gly Ser Cys Gly Thr Gly Asn Leu Ser Met Asn 500 505 510 CCG GCT CAT CCA CCG TTC GCT CTC CCC GGC GAA CCA GGG AAC GTC GTC 1811 Pro Ala His Pro Pro Phe Ala Leu Pro Gly Glu Pro Gly Asn Val Val 515 520 525 TAT GAT TAT CCA GAG GGC TTT AAC ATC ACC AAT GCT ACG GAA GCA GCC 1859 Tyr Asp Tyr Pro Glu Gly Phe Asn Ile Thr Asn Ala Thr Glu Ala Ala 530 535 540 TCA GCA TCC GCT GGG GCG GCA AGC CAA TCC GCA GCG GCA TCA TCC ACA 1907 Ser Ala Ser Ala Gly Ala Ala Ser Gln Ser Ala Ala Ala Ser Ser Thr 545 550 555 560 ACT ACA TCA GCC CCC TAC CTG CGT ACA ACA CCT ACC CCC GGA GTT CGT 1955 Thr Thr Ser Ala Pro Tyr Leu Arg Thr Thr Pro Thr Pro Gly Val Arg 565 570 575 AAT GTT GAC CAC CCT CCT TAT GTT ATC AAC CAT GTC CAA CCT GGC CAC 2003 Asn Val Asp His Pro Pro Tyr Val Ile Asn His Val Gln Pro Gly His 580 585 590 GAC CTG AGC GTT CAC GCC ATC TCA CCA AAT TCT ACT CAC TCG GAT GGG 2051 Asp Leu Ser Val His Ala Ile Ser Pro Asn Ser Thr His Ser Asp Gly 595 600 605 GTC CAG GAG TAT GAT GTA CAC AGT CTT TAC GGC CAC CAA GGC ATA AAT 2099 Val Gln Glu Tyr Asp Val His Ser Leu Tyr Gly His Gln Gly Ile Asn 610 615 620 GCA ACC TAT CAC GGA TTG CTC AAG GTG TGG GAG AAC AAA CGC CCC TTT 2147 Ala Thr Tyr His Gly Leu Leu Lys Val Trp Glu Asn Lys Arg Pro Phe 625 630 635 640 ATC ATC GCA CGC TCT ACA TTT TCC GGC TCT GGG AAA TGG GCC GGC CAC 2195 Ile Ile Ala Arg Ser Thr Phe Ser Gly Ser Gly Lys Trp Ala Gly His 645 650 655 TGG GGT GGT GAT AAC TTC TCC AAA TGG GGA TCG ATG TTC TTT TCG ATC 2243 Trp Gly Gly Asp Asn Phe Ser Lys Trp Gly Ser Met Phe Phe Ser Ile 660 665 670 TCG CAG GCC CTC CAG TTC TCG CTC TTT GGC ATC CCT ATG TTT GGT GTT 2291 Ser Gln Ala Leu Gln Phe Ser Leu Phe Gly Ile Pro Met Phe Gly Val 675 680 685 GAC ACC TGT GGT TTC AAT GGA AAC ACG GAT GAG GAG CTA TGC AAC CGA 2339 Asp Thr Cys Gly Phe Asn Gly Asn Thr Asp Glu Glu Leu Cys Asn Arg 690 695 700 TGG ATG CAG CTC TCG GCC TTT TTC CCT TTC TAC CGC AAC CAT AAT GTT 2387 Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr Arg Asn His Asn Val 705 710 715 720 CTC TCT GCA ATC CCA C AA GAG CCC TAT CGG TGG GCG TCC GTG ATC GAT 2435 Leu Ser Ala Ile Pro Gln Glu Pro Tyr Arg Trp Ala Ser Val Ile Asp 725 730 735 GCC ACG AAG GCG GCA ATG AAC ATT CGA TAC GCT ATT TTG CCG TAC TTT 2483 Ala Thr Lys Ala Ala Met Asn Ile Arg Tyr Ala Ile Leu Pro Tyr Phe 740 745 750 TAC ACC CTG TTC CAT TTG GCC CAC ACC ACT GGA TCT ACG GTC ATG CGC 2531 Tyr Thr Leu Phe His Leu Ala His Thr Thr Gly Ser Thr Val Met Arg 755 760 765 GCA CTT GCG TGG GAG TTC CCG AAT GAC CCC TCC CTA GCT GCT GTC GGC 2579 Ala Leu Ala Trp Glu Phe Pro Asn Asp Pro Ser Leu Ala Ala Val Gly 770 775 780 ACC CAA TTT CTT GTC GGT CCC TCG GTC ATG GTG ATT CCT GTT CTT GAG 2627 Thr Gln Phe Leu Val Gly Pro Ser Val Met Val Ile Pro Val Leu Glu 785 790 795 800 CCA CAG GTA GAT ACT GTC CAG GGT GTC TTC CCA GGT GTT GGA CAT GGG 2675 Pro Gln Val Asp Thr Val Gln Gly Val Phe Pro Gly Val Gly His Gly 805 810 815 GAA GTC TGG TAC GAC TGG TAC TCT CAA ACA GCT GTT GAT GCA AAG CCC 2723 Glu Val Trp Tyr Asp Trp Tyr Ser Gln Thr Ala Val Asp Ala Lys Pro 820 825 830 GG T GTC AAC ACA ACA ATC TCA GCG CCA CTG GGC CAC ATT CCG GTT TTC 2771 Gly Val Asn Thr Thr Ile Ser Ala Pro Leu Gly His Ile Pro Val Phe 835 840 845 GTT CGT GGT GGT AGC ATT CTG CCC ATG CAG GAG GTT GCG CTG ACC ACT 2819 Val Arg Gly Gly Ser Ile Leu Pro Met Gln Glu Val Ala Leu Thr Thr 850 855 860 CGC GAC GCT CGC AAG ACC CCC TGG TCT TTG CTC GCG TCG CTG AGC AGT 2867 Arg Asp Ala Arg Lys Thr Pro Trp Ser Leu Leu Ala Ser Leu Ser Ser 865 870 875 880 AAT GGA ACT GCC TCT GGC CAG CTC TAC CTC GAT GAT GGA GAA AGT GTC 2915 Asn Gly Thr Ala Ser Gly Gln Leu Tyr Leu Asp Asp Gly Glu Ser Val 885 890 895 TAC CCC GAG GAT ACG CTT TCT GTG GAC TTC CTG GCG TCT CGC TCC ACT 2963 Tyr Pro Glu Asp Thr Leu Ser Val Asp Phe Leu Ala Ser Arg Ser Thr 900 905 910 CTC CGA GCC TCT GCG CGG GGT ACT TGG AAG GAG GCG AAT CCA CTA GCG 3011 Leu Arg Ala Ser Ala Arg Gly Thr Trp Lys Glu Ala Asn Pro Leu Ala 915 920 925 AAT GTG ACG GTA CTT GGT GTG ACT GAG AAG CCA TCC TCA GTG ACA CTC 3059 Asn Val Thr Val Leu Gly Val Thr Glu Lys Pro Ser Ser Val Thr Leu 930 935 940 AAT GGC GAG ACG CTC TCC TCC GAC TCT GTG AAG TAT AAC GCC ACC TCA 3107 Asn Gly Glu Thr Leu Ser Ser Asp Ser Val Lys Tyr Asn Ala Thr Ser 945 950 955 960 CAC GTT CTC CAC GTT GGT GGC TTG CAG AAG CAC ACA GCG GAT GGA GCA 3155 His Val Leu His Val Gly Gly Leu Gln Lys His Thr Ala Asp Gly Ala 965 970 975 TGG GCG AAG GAC TGG GTA CTG AAA TGG TGAAGGAAGC GTAACAGGAT AGCCT 3207 Trp Ala Lys Asp Trp Val Leu Lys Lys 985

SEQ ID NO: 2 Sequence length: 720 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Sequence: GCCATCGGAT GCTCCCGTCA TGGCACCACT AGAGATGGCG TGGAAAACCC TCACCGGCAC 60 ACCGGAGGGG TTTAGGCACC TTGGAATATG AGGTGGGGAA GCCAGTATTG 120 ACTCTGGTGA ATGGATCTCT CGAGAAATAC TACCTTTTCA GGGCTCAAGC GTCGTGTCGG 180 GCATTTATCG GGGGATGGAC CAATCAGCGT AGGGATATCA GATGATCGCC AGCATTGGTC 240 AGGAACGTTT CCAATTTCCG GACACGGAAG TACTGTAACT GCTCCCAAGA ATCAACACAC 300 TCTTTTCCGG TCTCGTCCTT TGCTCGGCAG AGATTCATCT CCCATCGTCG GCTTAACCGG 360 TACTCTTTCG TCACGTTCCA AAAGGCTTGA TCATGCTGTC CCCACTCCGT GCGGGTGAAG 420 CCACCTCATT GCTGCGTAGG ACCTATACCC TTCAACTAGC GTGACTTCTT CCCCTCTCAT 480 GGTCGAGAGA TTGCAGGCAA TGCCCCTCGG ACGTTTGACG GGGAATGTTT TGCCTTCACG 540 GCAGGTAGCA CAAATCGATG GGAACGGGAC GGGCCATCAA TTGTGAGGGA TTTCCCGTGG 600 ACACCTGGTT CGTCAAGACA TATACATCTA GCTACAATTC CGGTTCGGAG ACGGCAGAGG 660 GGTCCGTTTC TTAAAAGAAC AACTACAACA CGGTCCGGAGG ACCATG GAC 720

SEQ ID NO: 3 Sequence length: 840 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Sequence: TGAAGGAAGC GTAACAGGAT AGCCTAGACC CACATACTAT CTGTATACAA CTCCGCAATA 60 TGAAGTGATG AATGCAAACT AGCAGCGAAT CGGATATCAG TAATCGGTAA 120 GCGAGTTGCC CGCGCAAGCG AGTTGCCCAC CACACGCGTT TTCAACGCGC CTCAATTTCT 180 TAGATGATTA AAACATCAGC CATACCACCA AAAATACCTA AATCAAAAAA ATCACGCGTT 240 GAGCAGGAAG GCAGAATCCT ATTAGCTATA TCAGCTATAA AAAAACAAGA AATCAGCAGT 300 TTTAGAAAGG CAGCTGAAAT TTTTAATATA CCTATCGCTA CACTACGTTA TCGTCTAAAT 360 GGAGGTTCCT TTCGAAATGA TACTCGTGCC AATAGTTATA AAATAACTTC TAGTGAAGAG 420 AAATCGCTTA AAAAATAGAT TCTATCACTA GATAAACGTG GAGCACCTCC TCGGCCTGTA 480 CACGTACGAG AAATAGCCAA TATCCTGCTT TTAAAGCGTA ATACTACCTC CCCCCCTACT 540 ACTGTAGAGG AGAAATGGGT ATACAACTTT ACCAAGCGTA CACCTAAGCT TAAATTCTGC 600 TTTGCACGTC GCTACAACTA TCAGCATGCC AAGGTAGAGG ATCCTAAGGT TCTAGGTACT 660 TAGTTTAAGC AGGTAAATAA GGTTATTCAG AAGTACGGTA TAGCTTCAAG CGATAC TAC 720 AATTTTAATA AAACGGGGTT TATAATGGGC CTAATAGCTA CAGCCAAAGT TGTTACTAGA 780 TCTAATATGC CAGGGAAACT ATTTTTATTA CAGCTAGAGA ACCGGGAATG GGTTACTGCC 840

[Brief description of drawings]

FIG. 1 shows a restriction enzyme map of the promoter region of the α-glucosidase gene, an enhancer sequence, and a base sequence around it.

FIG. 2 shows the construction procedure of plasmids pNAGT4 and pNAGG1-1.

FIG. 3 shows the homologous integration pattern of the GUS gene at the niaD locus.

FIG. 4 shows relative activities of various deletion promoters of α-glucosidase gene.

FIG. 5 shows promoter activity when 2 base pairs in the fluctuation portion of enhancer base sequence B are replaced with arbitrary bases.

FIG. 6 shows the promoter activity when the enhancer nucleotide sequence E is introduced into the promoter.

FIG. 7 is a photograph as a substitute for a drawing showing electrophoresis, showing Northern blot analysis of a promoter into which an enhancer nucleotide sequence E has been introduced.

FIG. 8 shows the procedure for constructing plasmids pNAG136 and pNAG142.

FIG. 9 shows the procedure for constructing plasmids pNAGL136 and pNAGL142.

FIG. 10 shows the homologous integration pattern of the α-glucosidase gene at the niaD locus.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location C12R 1:69) (72) Inventor Akinaka Kanda 4-9 Imazudeike-cho, Nishinomiya-shi, Hyogo Ozeki Co., Ltd. General Research Institute (72) Inventor Yataro Nunokawa 4-9 Imazu Izaikecho, Nishinomiya-shi, Hyogo Prefecture Ozeki Co., Ltd. General Research Institute (72) Inventor Katsuya Gomi 2-6 RC101 Takinogawa, Kita-ku, Tokyo Inventor Katsuhiko Kitamoto 3-14-18 Kamishashida Ushiku-shi, Ibaraki (72) Inventor Kojiro Takahashi 2-6-30 Takinogawa, Kita-ku, Tokyo Inside the National Tax Agency Brewing Laboratory (72) Inventor Manzo Tamura Tokyo 2-17-12 Sanno, Ota-ku

Claims (14)

[Claims] 1. An enhancer DNA base sequence containing the sequence -CGGNNATTTA-. 2. The enhancer DNA nucleotide sequence according to claim 1, wherein NN is GC. 3. An enhancer DNA base sequence containing the sequence -CCAATCAGCGT-. 4. An enhancer DNA base sequence containing the sequence according to claim 1 or 3. 5. The enhancer DNA nucleotide sequence according to claim 4, wherein NN is GC. 6. The enhancer D according to claim 1 or 3.
An improved promoter comprising one or more NA base sequences introduced into a promoter region that functions in filamentous fungi. 7. The improved promoter according to claim 6, wherein the promoter region is a promoter region of a hydrolase gene derived from a filamentous fungus or a glycolytic enzyme gene. 8. A promoter region has an Aspergillus oryzae represented by SEQ ID NO: 2.
The promoter region of the derived α-glucosidase gene,
Alternatively, the improved promoter according to claim 6, which is a promoter region containing a partial sequence thereof. 9. A filamentous product having the improved promoter according to claim 6, a marker gene suitable for selecting a transformant of a host filamentous fungus, a terminator, and a DNA region replicable in Escherichia coli. A plasmid for expressing a polypeptide in a bacterium. 10. The plasmid according to claim 9, wherein the marker gene is a filamentous fungus-derived nitrate reductase gene, ornithine carbamoyl transferase gene, tryptophan synthase gene, or acetamidase gene. 11. The plasmid according to claim 9, wherein the marker gene is a nitrate reductase gene derived from the genus Aspergillus. 12. The plasmid according to claim 9, wherein the terminator is a terminator of the α-glucosidase gene derived from Aspergillus oryzae represented by SEQ ID NO: 3, or a terminator containing a partial sequence thereof. 13. A method for carrying a DNA encoding a polypeptide between a promoter and a terminator.
The described plasmid. 14. A method for producing a polypeptide, which comprises introducing the plasmid according to claim 13 into a filamentous fungus and culturing the obtained transformant.