JPH0595787A - Phosphoglycerate kinase gene promoter, its terminator or/and gene expression unit composed of the same, genome gene of phosphoglycerate kinase and cdna gene - Google Patents

Abstract

PURPOSE:To provide the subject promoter derived from genome gene of phosphoglycerate kinase of Aspergillus oryze, capable of mass-producing phosphoglycerate kinase and capable of efficiently producing various kinds of useful substances. CONSTITUTION:Aspergillus oryze R11340 strain is seeded in a culture medium, cultured at 30 deg.C for 3 day, subsequently collected, ground, suspended in a buffer solution, treated with phenol-chloroform and subjected to ethanol precipitation to isolate chromosome DNA. The isolated chromosome DNA is treated with a restriction enzyme and the resultant respective fragments are bonded to a vector to form a gene library of Aspergillus oryze chromosome DNA. The obtained gene library is subsequently screened by using a partial base sequence of phosphoglycerate kinase gene as a probe to select positive clones. From the selected positive clones, the plasmid is recovered and treated with a restriction enzyme, thus obtaining the objective phosphoglycerate kinase gene promoter.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel promoter of Aspergillus oryzae-derived phosphoglycerate kinase, a terminator thereof, and / or a gene expression unit comprising these, a genomic gene of phosphoglycerate kinase, and mRNA. Derived cDNA gene.

The present invention can be used not only for the large-scale production of phosphoglycerate kinase, but also for the above promoter,
It is possible to construct a useful expression vector in fungi and other microorganisms by connecting a terminator, or / and an expression unit consisting of these to an appropriate vector. Therefore, various useful substances using a host-vector system such as fungi can be used. It can also be used for production, and thus the present invention greatly contributes to the production of various useful substances such as physiologically active substances by genetic engineering, in addition to the production of the enzyme.

[0003]

2. Description of the Related Art For the production of substances using genetic engineering, it is extremely important industrially to efficiently express a foreign gene and obtain a large amount of active protein. Escherichia coli, Bacillus subtilis, yeast, animal cells, etc. have been studied as hosts for this purpose, but each has its own advantages and disadvantages. In particular, proteins that undergo post-transcriptional modification and proteins that have sugar chains, and certain three-dimensional proteins Proteins having a structure are often unsuitable for E. coli, Bacillus subtilis, and yeast. In addition, the cost of culturing animal cells is high, and there are many problems in actual production. Therefore, attention has been paid to expression systems using fungi as hosts. For example, reports such as Upshall Bio Tec
hology , Vol. 5,1301-1304 (1
987), TPA (Tissue Plasmin that was not expressed in an active form in E. coli or yeast).
gen Activator is a fungus ( Asper )
gillus nidulans ) is said to be expressed in an active form.

Under these circumstances, the development of promoters, terminators, etc. which function effectively in the host-vector system of fungi has been of great significance, and already, glucoamylase of Aspergillus niger and Aspergillus oryzae have been developed. Utilization of the promoter of the α-amylase gene has been reported. However, these are all inducibly expressed by oligosaccharides such as starch and maltose.

On the other hand, phosphoglycerate kinase (EC: EC), which is one of the glycolytic enzymes, is a gene that is non-inducibly highly expressed in a medium containing normal glucose as a carbon source.
2.7.2.3. ATP: 3-phospho-D-
glycerate 1-phosphotransf
erase) gene. The present enzyme is an enzyme that catalyzes a reaction that produces 3-phosphoglyceric acid and ATP from 1,3-bisphosphoglycerin and ADP and its reverse reaction. This enzyme is also known to be one of the enzymes with a very high expression level, and in yeast Saccharomyces cerevisiae, 5% of mRNA and cytosolic soluble protein (Methods in Enzymol
gy, Vol. 185, 329-341 (1990))
It is reported to occupy.

Conventionally, Aspergillus oryzae which is a kind of Aspergillus oryzae
The structure of the derived phosphoglycerate kinase gene was completely unknown, and it was the actual situation that the gene was not even isolated. Furthermore, it was the actual state of the art that the expression system of the gene, such as its promoter and terminator, had no clue to its elucidation.

[0007]

The object of the present invention is to obtain a genomic gene of phosphoglycerate kinase of Aspergillus oryzae of Aspergillus oryzae and its cDNA, and analyze the structure to determine the promoter and terminator regions of the genomic gene. A promoter that functions effectively in the mold host-vector system from the genomic gene,
It is intended to newly obtain and provide a terminator and a gene expression unit comprising the promoter or / and the terminator.

[0008]

As a result of earnest research, the present inventors newly obtained a genomic gene for phosphoglycerate kinase from chromosomal DNA of Aspergillus oryzae, and further obtained c from mRNA of Aspergillus oryzae.
DNA was obtained, from which cDNA of the phosphoglycerate kinase gene was obtained, its structure was analyzed, and the promoter region upstream of the genomic gene 5 ′ side and the terminator region downstream of the 3 ′ side were successfully analyzed. Completed the invention. Hereinafter, the present invention will be described in detail. The donor of genomic DNA and mRNA used in the present invention is Aspergillus oryzae, and specifically, for example, RIB40 strain.

First, the genomic DNA will be explained. To extract the genomic DNA from the present strain, for example, Agric . Biol . Chem . , Vol. 51,
323-328 (1987), and the method for extracting Aspergillus oryzae chromosomal DNA.

Next, the obtained genomic DNA is treated with an appropriate restriction enzyme, partially digested, and fractionated by sucrose density gradient ultracentrifugation to obtain a fraction of 5.0-15.0 Kbp. ..
A plasmid vector treated with a restriction enzyme (for example, a plasmid vector treated with a restriction enzyme that produces the same sticky end) (for example,
A DNA fragment is inserted into pRBG-1) to prepare a chromosomal gene library. The subclone is
hods in Enzymology, Vol. 15
3, 3-11 (1987), pUC118 and pU
C119 was used.

When the gene is isolated from the chromosomal gene library obtained above, Aspergillus
DNA sequence of the phosphoglycerate kinase gene of Aspergillus nidulans, which belongs to the same genus as Orise ( Gene ,
Vol. 44, 97-105 (1986)), a synthetic oligo DNA probe is prepared, and the probe is used for colony hybridization to clone the DNA of the Aspergillus oryzae phosphoglycerate kinase gene. The obtained DNA fragment is
It had the restriction enzyme fragment map of FIG.

The DNA fragment obtained above was designated as pUC119.
To subclone into pPGF. Then, as a vector of Aspergillus oryzae, for example, pMTAG1 ( A having the argB gene as a marker)
gric . Biol . Chem . , Vol. 55, 19
39-1941 (1991)) and the argB gene fragment is isolated and inserted into the above pPGF. This plasmid DNA is transferred into Aspergillus oryzae of an argB- deficient strain to obtain a transformant. arg B deficient strain as specifically M-2-3 strain (Agric. Biol. Che
m . , Vol. 51, 2549 (1989)). As a transformation method, a known method such as Agri
c . Biol . Chem . , Vol. 51,323-3
28 (1987) or a method similar thereto. A transformant is obtained by this method.

Regarding the transformant and the host phosphoglycerate kinase enzyme of Aspergillus oryzae, known methods such as those described in Appl . Microbi
ol . Biotechnol . , Vol. 34,765
-771 (1991), Methods of enz
ymatic analysis, Vol. 2,2nd
edn. , P502, Academic Pres
S., New York (1974), and crude enzyme extraction from the bacterial cells and measurement of enzyme activity were performed according to the method described in S., New York (1974).

Further, in the present invention, Aspergillus
Extraction of RNA from ORIZE and cDNA based on it
The A library is prepared by the method described below.

That is, from Aspergillus oryzae to DN
A , Vol. Total RNA is extracted by a method according to the method described in 2,329-335 (1983). From the obtained total RNA, protein nucleic acid enzyme, Vol. 35,225
According to the method described in 7-2265 (1990), poly A-containing RNA is purified by using oligo (dT) -latex (manufactured by Nippon Synthetic Rubber Co., Ltd.) to obtain mRNA. This mRN
CDNA from Gene A , Vol. 25,263-2
69 (1983).

From the gene library obtained above,
The cloning of the phosphoglycerate kinase gene cDNA is carried out by using the information on the nucleotide sequence of the above-mentioned Aspergillus oryzae genomic gene and the DNA sequence of the phosphoglycerate kinase gene of Aspergillus nidulans of the same genus as Aspergillus oryzae ( Gene , Vo.
l. 44, 97-105 (1986)), a forward primer containing a transcription initiation point and a reverse primer containing a transcription termination point were synthesized, and PCR (Polymerase) using the above cDNA gene library as a template.
It was carried out by the Chain Reaction method). At this time, in order to make it convenient for DNA recombination,
It is possible to use those prepared by adding known restriction enzyme recognition sequences to both ends of this sequence.

The nucleotide sequence of DNA was analyzed by a known method (for example, the kilosequencing method, the dideoxy method).

The coating region contained in the gene cDNA of Aspergillus oryzae phosphoglycerate kinase of the present invention obtained and determined as described above has the nucleotide sequence of SEQ ID NO: 4 in the sequence listing. The genomic gene has the restriction enzyme fragment map of FIG. 1 and the base sequence of SEQ ID NO: 3 in the sequence listing, and has two intron sequences in addition to the promoter and terminator region of the present invention. (Indicated as INT1 and INT2 in the figure)

The promoter of the present invention is present in the upstream region of the 5'side of the above-mentioned genomic gene and has a DN of 1632 bp.
Have A. The base sequence is shown in SEQ ID NO: 1 of the sequence listing, which corresponds to the base sequence numbers 1-1632 of the genomic gene of SEQ ID NO: 3.

Furthermore, the terminator of the present invention is characterized in that the above-mentioned genomic gene is present in the downstream region on the 3'side and has a DNA of 617 bp.
have. The base sequence thereof is shown in SEQ ID NO: 2 in the sequence listing, which corresponds to base numbers 3069-3635 in the base sequence of the genomic gene of SEQ ID NO: 3.

By connecting this promoter or terminator to an appropriate plasmid vector, it can be used as a vector for expression in Aspergillus oryzae.

DNAs having the full length of these promoters of the present invention or a part thereof deleted
A region having a sequence, or / and a region having a DNA sequence having the entire length of the terminator or a part thereof deleted, is a promoter or / and a terminator of another source (for example, a promoter of α-amylase). , Terminator) and used as a gene expression unit.

Genes such as human lysozyme, chymosin, interferon α, human interferon γ and its derivatives are inserted into the obtained expression vector, and further, for example, Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus. -The target compound can be efficiently obtained by transforming a host such as awamori, Aspergillus soyer and the like and culturing with the transformant.

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, a phosphoglycerate kinase promoter, a terminator, and / or an expression unit comprising these, and a phosphoglycerate kinase cDNA and a genomic gene can be newly provided. Particularly, the above-mentioned promoter, terminator or / and an expression unit comprising these are useful in fungi such as Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus soyer and the like by connecting to an appropriate plasmid vector. It is possible to construct expression vectors and the present invention makes a major contribution in the production of useful substances using the fungal host-vector system.

As described above, according to the present invention, not only can phosphoglycerate kinase be efficiently produced, but by utilizing the expression unit according to the present invention, various useful substances such as physiologically active substances can be efficiently produced. The present invention also plays an important role in the technical field of genetic engineering.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0027]

Example 1. Preparation of gene library of Aspergillus oryzae chromosomal DNA] Aspergillus oryzae chromosomal DNA was extracted by the method described below. First, a conidia of Aspergillus oryzae RIB40 strain is removed from a slant with one platinum loop, and 100 ml of DP medium (1
% Polypeptone, 2% Dextrin, 0.5% K
H ₂ PO ₄ , 0.1% MgSO ₄ · 7H ₂ O),
The cells were cultured at 30 ° C for 3 days. Collect bacteria with glass filter 3
The collected bacterial cells are washed with distilled water and further washed with TE buffer (10 mM Tris-hydrochloric acid (pH 8.0), 1 mM EDTA). The cells removed of water with filter paper were crushed in liquid nitrogen using a mortar precooled. 10 ml of TE buffer containing 1% SDS was added to the cells and suspended, and the suspension was left at room temperature for 5 minutes. Phenol / chloroform treatment was carried out 3 times to perform ethanol precipitation. Precipitate 5 μg / ml RNas
Suspended in 1 ml of TE buffer containing e.
Reacted for minutes. 2.5 times the amount of ethanol was added to the solution after the phenol / chloroform treatment, and the chromosomal DNA generated at the interface was wound up with a Pasteur pipette. The wound DNA is rinsed with 70% ethanol, dried, and then T
It was dissolved in E buffer to prepare chromosomal DNA of Aspergillus oryzae. This DNA is partially digested with the restriction enzyme Pst I and then fractionated by sucrose density gradient ultracentrifugation to obtain a fraction of 5.0-15.0 Kbp. The Pst I fragment of the obtained Aspergillus oryzae chromosomal DNA was introduced into the Pst I site of the plasmid vector pRBG1 to construct a gene library of Aspergillus oryzae chromosomal DNA.

[0028]

Example 2. Aspergillus oryzae chromosomal DNA
Cloning of Phosphoglycerate Kinase Gene from Gene Library] Generally, if the nucleotide sequence of a gene is already known in another organism, if the gene is cloned in another species, its nucleotide information is well preserved. Cloning is carried out by hybridization using a sequence that seems to exist as a probe. In the practice of the present invention, based on the information of the phosphoglycerate kinase gene that has already been clarified, for example, the phosphoglycerate kinase gene ( Gene) of Aspergillus nidulans of the same genus as Aspergillus oryzae shown in Chemical Formula 1 below ( Gene , Vol. 44, 97-105 (1
986)) is prepared using a DNA synthesizer of Applied Biosystems Co., Ltd. using a base of 29 bases corresponding to the 43rd to 52nd amino acids from the N-terminus as a probe, and a known method (for example, Molecular Clonin).
g, 2nd, edn. , P12.21-12.23, C
old Spring Harbor Laborat
Ory (1989)), colony hybridization was performed, and 5 positive clones were obtained from about 15,000 colonies.

[0029]

[Chemical 1]

The resulting plasmids of the 5 positive clones all contained the common 3.6 Kbp Pst I fragment. FIG. 1 shows a map of these restriction enzyme fragments by observing the fragment patterns by agarose gel electrophoresis after digestion with several restriction enzymes. When Southern hybridization was carried out on the positive clone using the above-mentioned probe, it hybridized with the 0.8 Kbp fragment cut out by Sal I and Xho I in the figure. Therefore, the 3.6 Kbp Pst I fragment contained in this common fragment was hybridized. Was determined to sufficiently encode the genomic gene for phosphoglycerate kinase.

[0031]

Example 3. Investigation of Copy Number of Phosphoglycerate Kinase Gene in Aspergillus oryzae The following experiments were conducted to examine the copy number of the phosphoglycerate kinase gene of the present invention in Aspergillus oryzae.

The 3.6 Kbp Pst I fragment obtained in the previous section was inserted into the Pst I site of pUC119 to newly prepare pPGF. The chromosomal DNA of Aspergillus oryzae RIB40 strain was digested with some restriction enzymes (for example, Eco RI, Pst I, etc.) and then separated by agarose gel electrophoresis. After the electrophoresis, the DNA was blotted on a nylon membrane filter (manufactured by Amersham Co.) by Southern transfer method.
Southern hybridization was performed on this membrane filter using a fragment obtained by digesting pPGF with the same enzyme as a restriction enzyme that digested chromosomal DNA,
Bands were detected by the ECL detection system of Amersham Co., Ltd., which applies the principle of detecting chemiluminescence by peroxidase-catalyzed luminol oxidation reaction. It was revealed that only one band hybridized with the digested product with the major 6-base recognition restriction enzyme, and it was revealed that only one phosphoglycerate kinase gene of this clone exists in Aspergillus oryzae. I chose

[0033]

Example 4. DN of Aspergillus oryzae phosphoglycerate kinase gene isolated from chromosomal DNA
A sequence: The pPGF obtained in the previous section was digested with several restriction enzymes and then the fragment pattern was observed by agarose gel electrophoresis to prepare a restriction enzyme fragment map. The result is shown in FIG. Therefore, as shown in the figure, the DNA sequence was determined using an appropriate restriction enzyme site and the deletion clone of pPGF described below. The deletion clone is described in Gene , Vol. Two
8,351-359 (1984) and Gene , Vo.
l. 33, 103-119 (1985), the 3.6 Kbp insert on pPGF is treated with exonuclease III and mung bean nuclease to shorten the chain, and part of the insert fragment is shed to give a different chain length. Various clones having the same were prepared. In this process, a Kilo-sequencing deletion kit (Takara Shuzo Co., Ltd.) was used. The inserts of the various clones obtained were subjected to the dideoxy method ( Science , Vol.
-1210 (1981)) using an automated DNA sequencer of Applied Biosystems Co., Ltd.
The NA sequence was determined. As a result, the DNA sequences of the entire coding region of the Aspergillus oryzae phosphoglycerate kinase gene and its intron region, as well as the promoter region at the 5'side upstream and the terminator region at the 3'downstream region, are shown in Table 1 below. Table 5
The sequence shown in SEQ ID NO: 3 in the sequence listing shown in was found.

[0034]

[Table 1]

[0035]

[Table 2]

[0036]

[Table 3]

[0037]

[Table 4]

[0038]

[Table 5]

[0039]

Example 5. Preparation of Aspergillus oryzae cDNA Library] Known methods (for example, DNA , Vol.
2,329-335 (1983)), mRNA was obtained from Aspergillus oryzae RIB40 strain. Specifically, Aspergillus oryzae RIB40
10 strains in a 500 ml baffled Erlenmeyer flask
Medium 0 ml (2% maltose, 1% _{polypeptone, 0.1% KH 2 PO 4,} MgSO 4 · 0.05%
7H ₂ O, 0.05% KCl, 0.001% FeS
After culturing with shaking in O ₄ .7H ₂ O) at 30 ° C. for 2 days, the cells were collected with a glass filter 3G1, washed with distilled water, and then water was removed with a filter paper. The cells were freeze-crushed in liquid nitrogen in a mortar, and 5M Guanidine isothi
The fraction suspended in ocyanate and precipitated with 3M LiCl was obtained as total RNA. 3 more times phenol
After a series of operations including chloroform treatment once with chloroform, ethanol precipitation, ethanol rinsing, air drying and suspension in TE buffer, about 7.0 mg of total RNA was finally obtained from 2.6 g of wet bacterial cells. 4 of this obtained RNA
40 μg is 2 mg (100 μm) manufactured by Nippon Synthetic Rubber Co., Ltd.
1) Oligo (dT) -latex is mixed with a known method (for example, protein nucleic acid enzyme, Vol. 35, 2257).
-2265 (1990)), about 2 μg of poly
(A) + RNA was obtained.

Next, cDNA was prepared from this mRNA by the following method. The principle is Mol . Cell . Bio
l . , Vol. 2, 161-170 (1982) and G
ene , Vol. 25,263-269 (1983), and the cDNA synthesis system plus manufactured by Amersham Co., Ltd. was used for the actual operation.
Andom Hexaprimer as a primer,
Reverse Transcriptase, RNa
cDNA was synthesized using each enzyme of se H and DNA Polymerase I.

[0041]

Example 6. Aspergillus oryzae phosphoglycerate kinase gene cDNA from a cDNA library
Cloning of NA] From the gene library obtained above, the phosphoglycerate kinase gene cDNA
The cloning of A was carried out by using the information on the nucleotide sequence of the above-mentioned Aspergillus oryzae genomic gene and the DNA sequence of the phosphoglycerate kinase gene of Aspergillus nidulans of the same genus as Aspergillus oryzae ( Gen.
e , Vol. 44,97-105 (1986)), a forward primer (5'-CG ATCGAT GTCTCTC) containing a restriction enzyme Cla I site and a transcription initiation point.
TCCACCAAGCTTGCC-3 ', but underlined is C
la I site), the restriction enzyme Bgl II site, and the reverse primer containing the transcription termination point (5'GC AGATCT TA
CTTACTTGACAGAGCATCA-3 ', where underlined synthesized using Bgl II site) of Applied Biosystems Co. DNA synthesizer, the cDN
A known method using the A gene library as a template (eg, Cell Engineering, Vol. 8, 545-553 (198
9)) according to the PCR method (Polymerase C).
The chain reaction method) was used. This fragment is from the probe nucleotide sequences using, for having Bgl II site in the 'restriction enzyme Cla I site at the end side and the 3' 5 end side, which of pUC118 and pUC119 Ac
Plasmid p inserted into c I site and Bam HI site
CP8 and pCP9 were created.

[0042]

Example 7. Determination of the DNA sequence of the coding region contained in the Aspergillus oryzae phosphoglycerate kinase cDNA The information on the DNA sequence of the genomic gene of Aspergillus oryzae phosphoglycerate kinase obtained in Example 4 was used as shown in FIG. Using the restriction enzyme sites, pCP8 and pCP9 were self-cloned, and the nucleotide sequence of the phosphoglycerate kinase gene cDNA was determined in the same manner as in Example 4.
(SEQ ID NO: 4 in the sequence listing shown in Tables 6 to 8 below)

[0043]

[Table 6]

[0044]

[Table 7]

[0045]

[Table 8]

[0046]

Example 8. Production of Aspergillus oryzae Producing Phosphoglycerate Kinase of Active Aspergillus oryzae First, according to the production of the deletion clone of Example 4, the 5'insertion site of the phosphoglycerate kinase kinase gene of Aspergillus oryzae of pPGF was prepared. A plasmid pPGFd1 in which the Pst I site was deleted by deletion was prepared. This pPGFd
In 1, there is only one Pst I site on the 3'side of the inserted gene.

As a vector of Aspergillus oryzae, for example, pMTAG1 having the argB gene as a marker
( Agric . Biol . Chem ., Vol. 55,
1939-1941 (1991)), the marker ar
The gB gene is isolated and Pst I linkers are ligated at both ends. This Pst I fragment was labeled with pPGFd1 and pUC11.
Inserted into Pst I site of pPAd1 and pPad1 respectively.
Make MAR1. These plasmids are called argB
A transformant is obtained by introducing into the defective strain Aspergillus oryzae. Specific examples of the argB- deficient strain include M-2-3 strain ( Agric . Biol . Chem ., Vol. 51,
2549-2555 (1987)). As a transformation method, a known method, for example, Agric . Bio
l . Chem . , Vol. 51, 323-328 (19
87) or a method analogous thereto. A transformant is obtained by this method.

[0048]

Example 9. Measurement of Aspergillus oryzae phosphoglycerate kinase activity] The transformant of the phosphoglycerate kinase gene obtained in the preceding section and the crude extraction method and activity measurement method of the host Aspergillus oryzae phosphoglycerate kinase enzyme are known. Method (eg, Appl . Microbiol . Biotechn) .
ol . , Vol. 34, 765-771 (1991),
Methods of enzymatic analysis
sis, Vol. 2,2nd edn. It was performed as follows according to p502).

Host or transformed Aspergillus oryzae was treated with 10 ml of medium (3% glucose, 1%
Polypeptone, 0.1% KH ₂ PO ₄ , 0.05% M
_{gSO 4 · 7H 2 O, 0.05} % KCl, 0.001%
FeSO ₄ · 7H ₂ O) with 1 platinum loop inoculum and 30 days at 30 ℃
The culture was carried out with shaking. Bacteria collection is glass filter 3G1
The collected bacterial cells were washed with distilled water, and further dissolved in a lysis buffer (50 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA,
20 μM Phenylmethylsulfonyl
Wash with Fluoride). The cells removed of water with filter paper were crushed in liquid nitrogen using a mortar precooled. The cells are suspended in 2 ml of lysis buffer, and the centrifugation supernatant is used as a crude extract.

The protein concentration is quantified by the Lowry method (for example, Methods in Enzymology,
Vol. 3, 451-1454).

The phosphoglycerate kinase activity was measured as follows. Phosphoglycerate kinase is an enzyme that catalyzes the reaction shown in Chemical formula 2 below, and catalyzes this reaction in both directions. Therefore, the activity of phosphoglycerate kinase is determined by coupling the substrate 3-phosphoglycerate through 1,3-diphosphoglycerin by coupling with glyceraldehyde 3-phosphate dehydrogenase as shown in Chemical formula 2 below. The decrease in NADH that occurs in the reaction leading to cellaldehyde 3-phosphate is measured by the decrease in absorbance at 340 nm. In addition, 1 unit of phosphoglycerate kinase (1
unit) is the amount for converting the substrate 3-phosphoglyceric acid into 1 μmol per minute at 30 ° C.

[0052]

[Chemical 2]

As a result, the first table shown in Table 13 below is obtained.
A table was obtained, and by introducing this gene, Aspergillus
Orise phosphoglycerate kinase was found to be elevated.

[0054]

[Table 13]

[0055]

Example 10. Construction of expression vector] Tables 9 to 10 below
A region having the full length of the promoter region represented by SEQ ID NO: 1 in the sequence listing shown by or a region having a DNA sequence in which a part thereof is deleted and SEQ ID NO: 2 in the sequence listing shown in Tables 11 and 12 below. By inserting a tandem sequence having a DNA sequence that has the entire length of the terminator region shown or a part thereof is deleted, for example, by inserting this as a vector of Aspergillus oryzae into pMAR1 having the argB gene as a marker, The vector was constructed.

[0056]

[Table 9]

[0057]

[Table 10]

[0058]

[Table 11]

[0059]

[Table 12]

Specifically, it was manufactured according to the strategy shown in FIGS. First, after synthesizing the primer shown in the following chemical formula 3 with a DNA synthesizer manufactured by Applied Biosystems Co., Ltd., Example 6. Similarly, using pPGF as a template, primers # 1 and # 2 were used to define the promoter region, and primers # 3 and # 4 were used.
Was used to synthesize the terminator region by PCR. The synthesized promoter region is 5'from the primer.
Since the restriction enzyme Eco RI site is at the end and the restriction enzyme Kpn I site is the only site at the 3'end, this was digested with Eco RI and Kpn I and inserted into the same site of pUC118 to prepare pPGP. .. In addition, the terminator region synthesized in the same manner had a Bam at its 5'end.
The HI site has a Pst I site at the 3'end as the only site, so this is the Bam HI and Pst I site.
The fragment cut out in (4) was inserted into the same site of pUC118 to prepare pPGT, and this fragment was inserted into the same site of pPGP to prepare pPGPT. pPGP, pPGT and pPGPT is, a cut from pMAR1 to this site by having only the Pst I site in the Pst I
Inserting the rgB region, pPAP, pPAT,
pPAPT was made. These plasmid vectors have unique restriction enzyme sites for Kpn I, Sma I, and Bam HI from the 5 ′ side downstream of the promoter and / or upstream of the terminator. pPAPT is a vector that can be expressed by inserting a heterologous gene into this site. In addition, in pPAP, by inserting a heterologous gene-terminator-3 'into this site, pPAP
AT is a vector capable of expressing a heterologous gene by inserting a 5'-promoter-heterologous gene into this site.

[0061]

[Chemical 3]

[Brief description of drawings]

FIG. 1 is a restriction enzyme fragment map of a phosphoglycerate kinase genomic gene.

FIG. 2 shows the phosphoglycerate kinase cDNA gene
A restriction enzyme fragment map of Cla I- Bgl II fragment.

3 to 5 are construction diagrams of expression vectors.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location (C12N 9/12 C12R 1:69) (72) Inventor Katsuya Gomi 2 Takinogawa, Kita-ku, Tokyo 6-30, National Tax Agency, Brewing Laboratory (72) Inventor, Katsuhiko Kitamoto, 2-6, Takinogawa, Kita-ku, Tokyo, 6-23, National Tax Agency, Brewing Laboratory (72) Inventor, Eiko Kumagaya, 2 Takinogawa, Kita-ku, Tokyo No. 6-30, National Bureau of Taxation, Brewing Laboratory (54) [Title of Invention] Phosphoglycerate kinase gene promoter, the same terminator or / and a gene expression unit comprising these, and phospho Glyceric acid kinase genomic and cDNA genes

Claims (10)

[Claims] 1. A phosphoglycerate kinase gene promoter derived from the Aspergillus oryzae phosphoglycerate kinase genomic gene. 2. The phosphoglycerate kinase gene promoter according to claim 1, which has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. 3. A phosphoglycerate kinase gene terminator derived from the Aspergillus oryzae genomic gene. 4. The phosphoglycerate kinase gene terminator according to claim 3, which has the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. 5. The phosphoglycerate kinase gene promoter according to claim 1 or 2 having the full length,
Alternatively, a region having a DNA sequence in which a part thereof is deleted, and / or a region having the full length of the phosphoglycerate kinase gene terminator according to claim 3 or 4, or a region having a DNA sequence in which a part thereof is deleted. A gene expression unit consisting of. 6. A genomic gene for phosphoglycerate kinase of Aspergillus oryzae, which comprises the phosphoglycerate kinase gene promoter region according to claim 1 and the phosphoglycerate kinase gene terminator region according to claim 3 and contains an intron sequence. 7. The phosphoglycerate kinase genomic gene according to claim 6, which has two intron sequences. 8. The phosphoglycerate kinase genomic gene according to claim 6 or 7, which has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing. 9. A cDNA gene for Aspergillus oryzae phosphoglycerate kinase. 10. The phosphoglycerate kinase c according to claim 9, which has the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing.
DNA gene.