JPH066059B2 - Yeast enolase promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a DNA containing a promoter having an expression activity of a gene controlling yeast enolase.

[Conventional technology]

In recent years, techniques for producing useful proteins in microorganisms using genetic engineering techniques have rapidly become popular. Escherichia coli is the most widely used microorganism as a host, but one of the new hosts is yeast, which is a nucleated cell organism. This is because yeast is easy to culture and its safety has been proven historically, and more particularly in Saccharomyces yeast, more genetic biochemical analysis has been performed.

On the other hand, in the genetic engineering method, when a useful protein is produced in a host cell, a promoter region (gene expression regulatory site) suitable for the host is essential, and the expression efficiency depends on the strength of the promoter used. It is believed to be dependent. Particularly for yeast promoters, acid phosphatase promoter, α-factor promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, pyruvate kinase promoter, 3-phosphoglycerokinase promoter and the like have been reported.

Since each enzyme in the glycolytic system of yeast generally occupies a few percent of the total protein in yeast, it is considered that those gene promoters have high expression efficiency. Also,
Of the glycolytic enzymes, several types of isozymes often exist, and their expression is regulated differently depending on the type of carbon source and culture conditions.

Enolase is an enzyme that is located relatively low on the glycolytic metabolism map and catalyzes the mutual exchange between 2-phosphoglycerate and phosphoenolpyruvate. There are two types of genes, both of which have already been isolated and a DNA nucleotide sequence containing a promoter region has been reported. (MJ Holland, et al. J. Biol. Chem., 256 , 1385 (198
1)). The ENO 1 gene is a gene that encodes an enzyme (enolase-1) whose synthesis is induced during the practical production of baker's yeast, and the ENO 2 gene is synthesized when grown in normal batch culture using glucose as a carbon source. A gene encoding an inducible enzyme (enolase-2) (L.Mcalist
er and MJ Holland, J. Biol. Chem., 257 , 7181 (1982). )
Of these, ENO1 is considered to be a gene whose expression is induced by aerobically continuous culturing while limiting the supply of carbon sources such as molasses, and is therefore advantageous for practical production of useful proteins. It is guessed that

[Problems to be solved by the invention]

Thus, while yeast is attracting attention as a host, the development of a yeast-compatible promoter that controls the expression of a gene encoding a protein is not yet sufficient.

Further, in the enolase gene promoter, which is one of the glycolytic enzymes, DNA containing the promoter region is also included.
Although the nucleotide sequence has been clarified, it has not been used to construct a heterologous gene expression system in yeast by gene recombination technology, and no study has been made on the complete promoter region. .

[Means for solving problems]

The present inventors have conducted extensive studies in view of the above points, and in addition to the known nucleotide sequence region of the yeast enolase promoter,
Furthermore, DNA base sequence (I) By ligating the region represented by (5) to the upstream of the 5'-terminal side, a promoter usable for expression of a heterologous gene was constructed in a system using yeast as a host, and the present invention was completed.

That is, according to the present invention, from the 5'-end For the yeast enolase promoter represented by the nucleotide sequence of
Regarding NA.

Although the nucleotide sequence of a novel yeast enolase promoter having a high promoter activity (ENO1 promoter may be abbreviated) using the novel yeast enolase promoter DNA of the present invention, up to −352 base pairs thereof has been reported. , TATAAAT sequence, which is considered to be the recognition site of RNA polymerase, is 146 base pairs upstream of ATG (-1
46 bp) and a similar sequence (TATTAAT) is -121
It exists in bp. Immediately below this (-110 to -60), although its significance has not been clarified yet, there is a CT rich region which is often found in glycolytic genes. Furthermore, the transcription start site is (-40) or (-4
It has been clarified to be adenine (A) of 1). Based on the above findings, the known promoter region is defined as the minimum necessary region for promoter activity, the upstream 172 base pairs of ATG are defined as the DS region, and the upstream region -173 to -352 bp is defined as the UPS region. Was synthesized separately.

a) Chemical Synthesis of DS and UPS Regions The present invention begins by evaluating the significance of the promoter activity of the known nucleotide sequence region of the yeast enolase promoter.

The known nucleotide sequence regions (DS and UPS) of the ENO 1 promoter can be isolated from natural yeast, fragmented with appropriate restriction enzymes and cloned, and then used for the measurement of the respective promoter activities. In addition, with the recent progress of genetic engineering technology, DNA chemically synthesized according to a known base sequence can also be used. In the case of the present invention, the significance of the promoter activity of the known nucleotide sequence region of the ENO 1 promoter is also included. In such a case, the method by chemical synthesis is particularly preferable. For example, i) In the conventional cloning method, only natural type DNA can be obtained, whereas in the chemically synthesized DNA method, not only the natural type but also the structure-modified DNA can be easily designed according to the purpose. Ii) If you want to analyze the function of a narrow region where there is no restriction enzyme recognition site, artificially adding a restriction enzyme recognition base sequence to the end of that region and chemically synthesizing Can be easily cloned, or each region can be arbitrarily combined.

In order to chemically synthesize DNA, the desired double-stranded DN
A method is used in which both chains of A are divided into several fragments, chemically synthesized, and each fragment is ligated. Each fragment is usually 10-20
It consists of base pairs, and is designed so that each face is overlapped by 7 to 10 bases. The synthesis method of each fragment includes diester method (Science, 203 , 614 (1979)), triester method (Science, 198 , 1056 (1977)), solid phase method (Nuclei
c Acids Research, 8 , 5491 (1980)), liquid phase method or enzyme method (J. Biol. Chem., 241 , 2014 (1966)). However, the solid phase triester method is preferable in terms of synthesis time, yield, purification and the like.

The oligonucleotides thus synthesized are sequentially bound by DNA ligase, but at this time, it is necessary to phosphorylate the 5'-hydroxyl group of the synthetic fragment. For this purpose, polynucleotide kinase is generally used, but chemical phosphorylation is also possible (Nuclei
c Acids Research, 8 , 5753 (1980)).

b) Ligation into vector and cloning Synthetic DNA of DS and UPS regions chemically synthesized by the above-mentioned method and ligated in arbitrary combination are generally available by inserting into a suitable plasmid by a conventional method. The transformants obtained by transforming various host cells are selected, and cloning is confirmed.

c) Separation of a broader promoter region using chemically synthesized DNA Not only the known region of the yeast enolase promoter but also a wider range of the promoter region is used for the purpose of evaluating the promoter activity.
DNA containing a newly known base sequence from yeast chromosomal DNA using the A fragment as a probe and further including a wide range of regions
Get fragments. As a cloning method, yeast chromosomal DNA is digested with an appropriate restriction enzyme and cloned into a plasmid vector by sailboat cancer. Next, colony hybridization method (Grun
Stein M. et al., Proc. Natl. Acad. Sci. USA, 72 , 3961 (1975))
Thus, a clone containing the target DNA fragment is selected.

d) Determination of unknown nucleotide sequence Regarding the unknown region of the yeast enolase promoter obtained by the method c), the known Maxam-Gilbert method (Maxam, AM & Gilbert, W., Proc. Natl.Aca) is used.
d.Sci.USA, 74, 560 (1977) ) or M-13 dideoxy method (Sanger, F., et al., Proc.Natl.Acad.Sci.USA, 74, 5463 (1
977).

The synthetic or cloning DNA fragment in the above methods a) and b) can also be confirmed for synthesis or cloning by measuring the nucleotide sequence by the same method.

e) Search for promoter activity Various types of ENO 1 promoters produced are known plasmid vectors pMC 1403 (Casadaban, M. et al. J. Bacteriol. 1 ).
43, 971 (1980)) and pMC1587 (Casadaban, M., Et al Met
The promoter activity can be indirectly known by cloning into hods in Enzymology, 100 , 293 (1983)) and measuring the enzymatic activity of β-galactosidase encoded downstream of the insertion site. These plasmid vectors are known as promoter activity search vectors, and pMC 1587 contains pMC 1403 containing 2 μm DNA and Leu2.
It is a shuttle vector incorporating a gene. PMC 1403 is used for E. coli and pMC 1587 is used for yeast.

As a method of cloning the ENO1 promoter into pMC1587, a method of cloning the ENO1 promoter into pMC1403 first and then inserting a region containing 2 μm DNA and Leu2 gene is used.

As a method for measuring the enzyme activity of β-galactosidase, qualitatively, X-ga which is one of the synthetic substrates of this enzyme is used.
l (5-bromo-4-chloro-3-indolyl-β-D
-Galactoside) is mixed in the medium to produce β produced by the fungus.
-A method of knowing by the blue coloration of 5-bromo-4-chloro-3-indigo generated by decomposition by galactosidase (Miller, J. "Experiments in Molecular Biology" Col
d Spring Harbor Laboratory, CSH New York, 119721)
Is used.

In addition, as a quantitative measurement method, the cells contained in the cell extract are thawed by freezing and thawing the cells and disrupting them with glass beads.
-As with X-gal, the galactosidase activity is measured by the method in which the yellow color developed by the decomposition of ONPG (ortho-nitrophenyl-β-D-galactopyranoside), which is one of the synthetic substrates, is measured at an absorbance of 420 nm. To be The activity unit is represented by the number of moles of ONPG in which a unit protein (mg) is decomposed per unit time (minute). (Rose, M et al.
Proc.Natl.Acad.Sci.USA, 78 , 2460 (1981)).

Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to these.

Example 1 Chemical Synthesis of Yeast Enolase Promoter (1-1) Chemical Synthesis of DS Region As shown in FIG. 2, the DS region of the ENO1 promoter has 22 DNAs each having a length of 11 to 19 bases.
Divided as oligomers, each oligomer was synthesized by a known solid-phase triester method.

The synthesized DNA oligomer was bound during the ligation process shown in FIG. First, 2 μg (about 400 pmole) of the oligomers constituting each block were mixed in an amount equivalent to T ₄ −.
Polynucleotide kinase buffer, [γ- ³² P] ATP in an amount equivalent to 1.5% of the total number of 5'-OH, and total 5'-OH
1 unit of T ₄ -polynucleotidease (Takara Shuzo) was added to 50 times amount of ATP and total 5′-OH 1000 pmole,
The reaction volume was set to 50 μm and the reaction was carried out at 37 ° C. for 60 minutes. After phenol extraction and ether extraction, DNA was precipitated with 2.5 volumes of ethanol. DNA again, _{T 4} -
Dissolve in DNA ligase buffer and add 0.
Twenty-one units of T ₄ -DNA ligase (Nippon Gene) was added to make the reaction volume 100 μm, and the reaction was carried out at 11 ° C. for 15 hours. After phenol extraction and ether extraction, DNA was precipitated with 2.5 volumes of ethanol. Each ligation product is
Separation was performed by 15% polyacrylamide gel electrophoresis containing 8.3 M urea, the reaction results were confirmed by autoradiography with ³² P, and the production of the DNA of the final target chain length was also confirmed in the same manner. What is DS-I
-1).

In the same manner as described above, for the DS region, 1) the restriction enzyme Cla was placed immediately upstream of the translation initiation codon ATG.
A recognition sequence of I is provided to allow direct expression of a structural gene through this site (Fig. 3, hereinafter abbreviated as DS-II).

2) Designed for the same purpose as in 1) and deleting two bases immediately upstream of the recognition sequence site of the restriction enzyme Cla I (4th
Figure, hereinafter abbreviated as DS-III).

3) CT rich region is 34 bases longer than natural (Fig. 7, hereinafter abbreviated as DS-I-2) 4) CT rich region is 32 bases longer than natural, longer CT rich C → T mutations occur in 3 regions,
Furthermore, the 5'end of the CT rich region (oligomer number 9
Deletion of TA at the 5'end (Fig. 8, hereinafter D)
Abbreviated as S-I-3).

A total of 5 types of DS regions were synthesized. However, C in 3) and 4)
DS-I-2 and DS-I-3 with longer T rich regions are synthesized by chemically synthesizing DNA oligomers by the ligation process shown in Fig. 6, and by changing the molar ratio of the oligomers used. Was done.

(1-2) Chemical Synthesis of UPS Region The UPS region of ENO1 promoter has 22 DNs each having a length of 11 to 19 bases as shown in FIG.
The oligomer was divided into A oligomers, and each oligomer was synthesized according to the method shown in FIG.

Example 2 Ligation into vector and cloning (2-1) Ligation reaction product obtained in Example 1 (DS-I-
1) was inserted and ligated into the EcoRI and BamHI sites of the plasmid vector pBR322 to transform E. coli C-600 strain.

The ligation reaction was performed with pBR322 treated with a restriction enzyme equivalent to 0.2 μg.
And the ligation product shown in Example 1 with 4 units of T
_4-DNA ligase was added, so that the reaction volume is _50.mu., T 4-DNA ligase buffer was added and allowed to react for 15 hours at 11 ° C.. Transformation was carried out according to a conventional method, that is, 0.2 ml of the bacterial solution obtained by treatment with calcium chloride was mixed with 0.1 ml of the ligation reaction solution. For a transformant resistant to ampicillin and sensitive to tetracycline, plasmid DNA was prepared according to a conventional method, and digested with a restriction enzyme.
The size of the inserted DNA fragment was confirmed by 8% polyacrylamide gel electrophoresis. Furthermore, the nucleotide sequence of this inserted DNA fragment was determined by the Maxam-Gilbert method or the M-13 dideoxy method.

By inserting each region of the ENO1 promoter obtained in Example 1 by the above method into EcoRI and BamHI sites of pBR322, pBR322-ENO / DS-I-1, p containing each promoter region was inserted.
BR322-ENO / DS-I-2, pBR322-ENO / DS-I-3, pBR322-ENO / DS
-II, pBR322-ENO / DS-III and pBR322-ENO / UPS, a total of 6 types of plasmids were obtained.

(2-2) Next, cloned DS region into pBR322-E
Digestion of NO / DS-I-1 with EcoRI and BamHI, separation by polyacrylamide gel electrophoresis, extraction from gel,
Recovered. And the vector pM for searching promoter activity
The insertion of the DS-I-1 region into C1403 was inserted and ligated into the EoRI and BamHI sites according to FIG. That is, 0.2 μg of pMC1
403 restriction enzyme treatment solution, 0.5 μg of DS, DNA fragment, and T ₄ -DNA ligase buffer were added so that 2 units of T ₄ -DNA ligase were contained in 20 μ,
Reacted for hours.

For transformation, M known as 1ac operon-deficient strain
C1061 strain (Δlac (IPO2YA) × 74, gal U, gal K, strA ^r , hsd
^{R -, Δ (ava, ieu} ) ] (M.Casadaban et al, J.Mol.Biol, 138, 1
79 (1980)) as a host, and according to a conventional method, 0.1 ml of the ligation reaction solution was added to 0.2 ml of MC1061 treated with calcium chloride, and X-gal (2% dimethylformamide solution) was added on top.
Mixed with agar and sown. For ampicillin-resistant colonies that decompose X-gal to give a blue color, according to a conventional method,
Plasmid DNA was prepared and cloning was confirmed by restriction enzyme treatment.

The promoter activity search vector pMC1403-ENO / DS-I-1, which has the DS region of the ENO1 promoter, was prepared by the above method.
pMC1403-ENO / DS-I-2, pMC1403-ENO / DS-I-3, pMC1403-EN
O / DS-II and pMC1403-ENO / DS-III were obtained.

Example 3 Ligation and cloning of UPS and DS Ligation of UPS and DS was performed according to the procedure shown in FIG. First, 10 μg of pB obtained in Example 2 (2-1)
R322-ENO / UPS was double digested with 25 units of EcoRI and 25 units of Sau3A at 37 ° C for 2 hours. On the other hand, 10μ
37 g of pBR322-ENO / DS-I-1 with 25 units of San3A
Digested at ℃ for 2 hours. The inserted DNA fragments were separated by 5% polyacrylamide gel electrophoresis and extracted and recovered. The obtained DNA fragment was dissolved in 20 μl of T ₄ -DNA lyase buffer, 2 units of T ₄ -DNA ligase was added, and the mixture was reacted at 11 ° C. for 15 hours. 0.2μ of ligation reaction
ligated with pMC1403 EcoRI and BamHI digested DNA of
The E. coli.MC1061 strain was transformed. UPS and DS- from transformants showing blue color after decomposing X-gal by ampicillin resistance
The presence of pMC1403 cloned from the I-1 ligated DNA was confirmed, and this plasmid was named pMC1403-ENO / UPS + DS-I-1.

According to the above method, each DS region and UPS are linked to each other to obtain pMC1403-ENO / UPS + DS-I-2, pMC1403-ENO / UPS + DS-I-
3, pMC1403-ENO / UPS + DS-III, a total of 4 types of plasmids were obtained.

Example 4 Cloning into yeast plasmid Cloning of the yeast enolase promoter into the yeast plasmid was performed according to FIG. That is, in a reaction volume of 50 μ, 3 μg of pMC1587 was added to 7.5 units of EcoRI.
Partially decomposed by reacting at 37 ℃ for 15 minutes,
DN of the region containing 2 μm DNA and Leu2 gene (about 6 Kb)
I was able to obtain A. 0.8% of these partially decomposed products
After separation by agarose gel electrophoresis, a DNA corresponding to about 6 Kb was cut out, and a known method (Dcdonell, MW et al. JM) was used.
Ol.Biol, 110 , 119 (1977)) was electrophoretically eluted and collected in a dialysis tube.

PMC1403 0.05 μg incorporating various ENO1 promoters
To 0.5 μg of 6 Kb fragment was added to 2 units of T ₄
-A reaction was carried out with a DNA ligase in a 20 µ reaction solution at 11 ° C for 15 hours.

The E. coli C-600 strain was transformed by a conventional method, and ampicillin-resistant bacteria were threonine (50 μg / ml) and thiamine (1 μm).
g / ml) and glucose (0.1%) supplemented with M-9 medium (M
iller, J., “Experiments in Molecular Genetics”, 431
-433 Cold Spring Harbor Laboratory, New York (197
2)) was replanted with a sterilized "toothpick" to obtain a leucine-producing strain. For the ampicillin-resistant leucine-producing strain, plasmid DNA was prepared according to a conventional method, and cloning was confirmed by restriction enzyme treatment.

As a result, pMC1587-ENO / DS-I-1, pMC1587-ENO / DS-I-2,
pMC1587-ENO / DS-I-3, pMC1587-ENO / DS-II, pMC1587-ENO
/ DS-III, pMC1587-ENO / UPS + DS-I-1 and pMC1587-ENO /
I got UPS + DS-1II.

(The above promoters are collectively referred to as pMC1587-ENO.) Example 5 Introduction of pMC1587 / ENO into Yeast Each pMC158 cloned by introducing into E. coli C600 strain.
7 / ENO was transformed into yeast S. cerevisiae AH22 (aleu2-3, leu2-112, his
4, can1] (for example, Hinnen, A. et al., Proc.
Natl. Acad. Sci. USA 75 , 1929 (1978)). As the medium, amino acids other than leucine (20 ml /: L-Trp, L-Hi
s, L-Arg, L-Met, 30mg /: L-Tyr, L-Ile, L-Lys, 50mg /:
L-Phe, 100mg /: L-Glu, L-Aps, 150mg /: L-Va1,200mg
/: L-Thr, 375mg /: L-Ser) and 20mg / g Adenine
SD medium containing Sulfate and Uracil was used.

Example 6 Qualitative evaluation by X-gal In order to qualitatively evaluate the activity of β-galactosidase produced by the ENO1 promoter reconstructed based on the known nucleotide sequence, an SD plate of pH 7.0 containing X-gal was used. , P
The MC1587 / ENO-introduced AH22 strain was transferred, and the degree of coloration to blue was evaluated, but the strains carrying any promoter showed almost no coloration. Therefore, when the cells were transferred onto a nitrocellulose filter, freeze-thawed to destroy the cells, and then measured with the same system, a slightly blue color was exhibited. And, the degree of coloration was slightly higher in the promoter linked with UPS. That is, several ENO1 promoters reconstructed based on known nucleotide sequences functioned as promoters in yeast, but their activity was weak.

Example 7 Cloning of ENO1 promoter containing unknown nucleotide sequence region Report by Holland et al. (Holland, MJ et al., J. Biol. Chem., 256 ,
1385 (1981)), a 1.6 kb DNA fragment obtained by treating the yeast chromosome with the restriction enzyme EcoRI, contains a part of the structural gene of the ENO1 gene and about 720 bp of the 5'untranslated region upstream thereof. Known to cover. Therefore, a known colony hybridization method (Grunstein, M. et al., Proc. Natl. Acad was used as a probe using ENO / UPS + DS-I-1 synthesized based on the previously cloned known nucleotide sequence. .Sci.USA, 72 , 3961 (1
975)) and an EcoRI fragment of about 1.6 kb was cloned.

That is, S. cerevisiae AH 22 strain was treated with 500 ml of YPD medium (1%
30 with yeast extract, 2% polypeptone, 2% glucose)
After shaking culture at 0 ° C. overnight, the obtained strain was isolated by the method of Crey et al. (Method in Cell Biology, 12 , 39 (1975)). 150 μg of this DNA is added to 450 units
It was cut by treating with EcoRI at 37 ° C. for 2 hours. After the reaction, separate by 1.5% agarose gel electrophoresis and
Cut out around the band corresponding to Kb. Known method (Dc
donell, MW et al., J. Mol. Biol. 110 , 119 (1977))
The DNA was eluted and collected in the dialysis tube. Recovered 1.
The EcoRI fragment of around 6 Kb was used as a known plasmid vector pA.
It was inserted and ligated into the EcoRI site of CYC184, and E. coli. C-600 strain was transformed. The tetracycline-resistant and kanamycin-sensitive transformant strain 3600 was subjected to colony hybridization with 366 bp UPS + DS-I-1 radiolabeled with ³² P to obtain two positive clones. For these two strains, plasmid DNA was prepared by a conventional method, a restriction enzyme map was prepared, and compared with the previously reported patterns to obtain the desired DNA.
Was cloned into pACYC184-ENO62.

Example 8 Preparation of long chain synthetic promoter A long chain synthetic promoter was prepared by the method shown in FIG. That is, pACYC 184-EN obtained in Example 7
O62 is a part of ENO1 structural gene and about 720bp upstream of it.
Was incorporated with a DNA covering the untranslated region of E. coli.

The 5'untranslated region of about 720 bp has an unknown nucleotide sequence of about 370 bp upstream of the 5'end of the known nucleotide sequence region of 352 bp. Therefore, this unknown base sequence region
In order to ligate upstream of the known nucleotide sequence region, the cloned EcoRI DNA fragment of about 1.6 kb was digested with restriction enzyme Hinf I and ligated via the restriction enzyme Hinf I site located in the UPS region to obtain long-chain synthesis. Created a promoter. Hereinafter, a DNA fragment 5 ′ upstream of Hinf I of the naturally-occurring ENO1 promoter is abbreviated as UAS.

pACYC 184-ENO62 10 μg with 25 units EcoRI
Digested at 37 ℃ for 2 hours and electrophoresed on 1% agarose gel.
A DNA fragment of about 1.6 Kb was separated and purified. 2 μg of the obtained 1.6 Kb DNA fragment was treated with 4 units of Hinf I at 37 ° C. for 2
After digestion for a while, the mixture was separated by 5% polyacrylamide gel electrophoresis, the UAS fragment having a size of about 0.5 Kb was excised from the three resulting DNA fragments, and the DNA was recovered.

On the other hand, pMC1403-ENO / UPS + DS-I-1 20 μg was treated with 25 units of EcoRI and 30 units of BamHI at 37 ° C. for 2 hours, 2 hours.
After double digestion and separation by 5% poeacrylamide gel electrophoresis, the larger of the two resulting DNA fragments was recovered.

The two kinds of DNA fragments thus obtained were mixed in a reaction volume 2
The ligation reaction was carried out with T ₄ -DNA ligase at 0 μm to obtain a long chain synthetic promoter containing about 720 bp. This long-chain synthetic promoter was inserted into the EcoRI and BamHI sites of plasmid pMC1403 in the same manner as in Fig. 11, and the Ec
The oli MC1061 strain was transformed.

Among the transformants showing amphicillin resistance and degrading X-gal to give a blue color, pMC1403-ENO / UA into which the target DNA was incorporated was used in accordance with the conventional method according to the restriction enzyme cleavage pattern.
S + UPS + DS-I-1 was obtained.

By combining each UPS + DS region with the UAS region by the above method, pMC1403-ENO / UAS + UPS + DS-I-2, pMC1403-ENO /
Four types of plasmids, UAS + UPS + DS-I-3 and pMC1403-ENO / UAS + UPS + DS-III, were obtained in total.

These plasmids were cloned into a plasmid for yeast according to Example 4 (method of FIG. 12) to obtain pMC1.
587-ENO / UAS + UPS + DS-I-1, pMC1587-ENO / UAS + UPS + DS
-I-2, pMC1587-ENO / UAS + UPS + DS-I-3 were obtained. Furthermore, according to Example 5, the yeast S. cerevisiae AH 22 strain was transformed.

Example 9 Measurement of long-chain synthetic promoter activity The expression activity of the long-chain synthetic promoter of ENO 1 having UAS in yeast was qualitatively evaluated by X-gal according to Example 6. As a result, a vivid blue color appeared around the cells, suggesting the strong promoter activity.

Therefore, the expression activity of the promoter was further analyzed by the ONPG assay method (Mark Rose et al., Proc. Natl. Acad. Sci. USA, 78 , 2460).
(1981)).

The results are shown in Table 1.

The ENO1 promoter not linked to UAS is X-gal
The qualitative activity evaluation method by all showed little activity. The activity value was about 2.2 to 5.3 units based on the quantitative value by the ONGP assay method. As a result of ligating UAS to these, the activity was X-gal.
Both the qualitative evaluation by and the result by the ONPG assay showed a surprising increase. This activity value is obtained by using the GAL1 promoter (Piotr P. Stepien et al. Gene, 24 , 28) which has already been used as a yeast promoter for the expression of heterologous genes.
9 (1983)) measured in the same system (R. West et al., Mol. C.
ell.Biol., 4 , 2467 (1984)).

Example 10 Determination of nucleotide sequence of USA It was suggested that the ligation of USA markedly increased the activity of the ENO1 synthetic promoter and that the region was largely involved in the promoter activity.
M-13 dideoxy method (Sanger, F. et al., Proc. Natl. Aca
d. USA, 74 , 5463 (1977)).

The results are shown in FIG. It covers from 5'end EcoRI recognition site to 3'end Hinf I recognition site,
The region of the known base sequence is located downstream of T at the 373th position from the 5'end, but the base sequence of the region was different from the report at two positions (T at the 444th and 458th positions from the 5'end were mutated to C). . This is considered to be the difference between the strains from which DNA was obtained.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence up to the Hinf I cleavage site containing the nucleotide sequence of the novel portion of the yeast enolase promoter region. FIG. 2 shows a DNA oligomer segmentation fragment in the chemical synthesis of the DS region of the ENO1 promoter. FIG. 3 shows the recognition base sequence of the restriction enzyme Cla I immediately upstream of the translation initiation codon ATG of the DS region, and FIG.
2 shows a fragmented DNA oligomer fragment of an abnormal portion in which two bases immediately upstream of the recognition base sequence of la I are deleted. FIG. 5 shows the ligation reaction procedure of the DS region, and FIG. 6 shows
The ligation reaction procedure of DS-I-2 or DS-I-3 is shown. FIG. 7 shows the mutated portion of DS-I-2, and FIG. 8 shows the mutated portion of DS-I-3. FIG. 9 shows a DNA oligomer segmentation fragment in the chemical synthesis of the UPS region of the ENO1 promoter. FIG. 10 shows recombinant DNA pMC1403-ENO / DS-I-1
Fig. 1 shows recombinant DNApMC1403-ENO / UPS + DS-I-1 and Fig. 12 shows recombinant DNApMC1587-ENO / UPS + DS-I-1.
Furthermore, FIG. 13 shows a process for producing a long-chain synthetic promoter having expression activity.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Hideaki Tanaka, 1-1 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefectural Chemical Research Institute (72) Inventor Satoshi Nakazato 1-1-chome, Yatabe-cho, Tsukuba-gun, Ibaraki Chemical Research In-house (72) Inventor Nobumasa Toshimitsu 1336-28 Hirotsu, Yoshitomi-cho, Tsukigami-gun, Fukuoka (56) References J. Biol. Chem. [256] (1981) P. 1385-1395

Claims (1)

[Claims] 1. From the 5'-terminal side For the yeast enolase promoter represented by the nucleotide sequence of
NA.