JPH089977A - Yeast promotor - Google Patents

Abstract

PURPOSE:To obtain a yeast promotor having a specific DNA sequence or a DNA sequence homologous to it and having promotor activity, exhibiting higher promotor activity in a low temperature region than at normal temperature and capable of producing an objective protein in large quantity by gene engineering technology. CONSTITUTION:This is a new yeast promotor having a DNA sequence of the formula or a DNA sequence homologous to it and having promotor activity and capable of exhibiting higher promotor activity in a low temperature region (e.g. <=10 deg.C) than at normal temperature. The promotor can produce an objective protein in large quantity by gene engineering technology as follows: an extraneous gene is combined at the downstream side of the DNA segment or this promotor and a vector is further combined to obtain a recombinant DNA; the recombinant DNA is transduced into a host yeast; and the extraneous gene is expressed in the yeast. The DNA segment for the promotor is obtained by screening a yeast chromosomal DNA library with a probe having a synthesized fraction of DNA coding a protein which is produced by a yeast at a low temperature and subjecting the DNA of an obtained clone to restriction enzyme treatment.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel yeast promoter, and more specifically, to a low temperature region (for example, 10 ° C or lower) from room temperature (about 30 ° C).
Brewer's yeast (Saccharo
myces cerevisiae) -derived promoter.

[0002] The background of the technology in recent years, by applying the techniques of genetic engineering in yeast,
Attempts have been made to construct yeast strains that produce the target protein in large quantities. In this attempt, the target protein itself may be useful, or the target protein may be produced to improve the properties of yeast and enhance the usefulness of the yeast. Specific examples of the latter possibility include alcohol production using yeast, production of metabolites such as amino acid production, fermented foods (for example, bread, miso, soy sauce) or fermented beverages (for example,
Beer, wine, sake, shochu, spirits) production.

[0003] To date, the "promoter" has been vigorously developed for the purpose of expressing a large amount of a target protein in yeast by using the technology of genetic engineering. To date, several strong promoters have been found and shown to be useful for expression of foreign genes in yeast. For example, Saccharomyces cerevisiae gene ADC1
Promoters such as (also known as ADH), GAPDH (also known as GPD) and PGK genes, which were obtained from yeast cultivated at around 30 ° C. as having high promoter activity, were used for expression of foreign genes in yeast. ing.

On the other hand, brewing including beer brewing is
Generally, it is performed at a low temperature of about 10 ° C. or lower. A promoter that is specifically and efficiently expressed at such temperatures has not been obtained. In addition, the development of an expression system using a promoter specific to brewer's yeast for the purpose of expressing the target protein in brewer's yeast has not been reported so far. In addition, experimental yeast (Saccharomyces cere
in visiae), the optimum temperature for culturing the yeast from 30 ° C to 10
As a gene induced by cold shock to ℃,
Up to now, two types of genes, TIP1 and NSR1, have been obtained, but the promoter itself has not been evaluated for these (protein, nucleic acid, enzyme Vol.39 No.5 (19
94)).

[0005]

SUMMARY OF THE INVENTION The present inventors have conducted extensive studies to obtain a promoter having a promoter activity in a low temperature range of 10 ° C. or lower. As a result, we succeeded in obtaining a DNA fragment containing the promoter from the yeast chromosome.

[0006] Therefore, the present invention comprises a D containing a promoter having a promoter activity in a low temperature range of 10 ° C or lower.
The purpose is to provide NA fragments.

The DNA fragment according to the present invention is the DNA sequence shown in SEQ ID NO: 2, or the sequence having homology to the DNA sequence shown in SEQ ID NO: 2 and retaining promoter activity. The expression vector according to the present invention comprises the above DNA fragment and a foreign gene located downstream thereof. Furthermore, the host according to the present invention is one transformed with the above expression vector or the like.

The DNA fragment according to the present invention contains a promoter having a high promoter activity in a low temperature region (for example, 10 ° C. or lower) than at room temperature. Therefore, by introducing into a host a vector or the like in which the DNA fragment according to the present invention is linked with a foreign gene, the foreign gene can be expressed at a high rate in the low temperature range. In addition, by controlling the culture temperature (for example, from room temperature to low temperature),
The expression level of the foreign gene can be controlled.

[0009]

[Detailed Description of the Invention]

<DNA fragment having promoter activity> The DNA fragment having promoter activity according to the present invention is represented by SEQ ID NO: 2. This DNA fragment is located upstream of the structural gene of a protein (p12 protein described later) expressed in yeast at a low temperature range and contains a promoter region. The base sequence containing the coding region and the promoter region of the protein expressed in this low temperature region is shown in SEQ ID NO: 1.

The promoter contained in the DNA fragment of the present invention has a higher promoter activity at 4 to 10 ° C than at room temperature (about 30 ° C). Therefore, according to the present invention, a foreign gene can be expressed at a high temperature at a temperature of 10 ° C. or lower than normal temperature.

Furthermore, in addition to the sequence represented by SEQ ID NO: 2, the DNA fragment having promoter activity according to the present invention has homology to the DNA sequence described in SEQ ID NO: 2 and retains the promoter activity. Arrays are also included. Such sequences include deletions, substitutions, and additions of any of the bases in the sequence of SEQ ID NO: 2 and its partial sequences, and combinations thereof, but also include sequences that retain their promoter activity. Should be interpreted as meaning. Here, “to retain the promoter activity” is used to mean a promoter activity still higher than the promoter activity at 30 ° C. of the sequence represented by SEQ ID NO: 2 in a low temperature range of 10 ° C. or lower. .

In the present invention, the term "promoter" means, when linked to a structural gene in a host cell, to increase the transcription, translation or mRNA stability of the structural gene in the absence of the promoter. Means any DNA sequence. Also, "foreign gene", regardless of whether the gene is derived from a specific organism,
It means any gene that does not functionally coexist with a particular promoter in nature.

DN containing the above yeast promoter of the present invention
The A fragment may be wholly synthesized by a commonly used nucleic acid synthesis method, or may be obtained from the following deposited microorganism.

<Deposition of microorganism> A DNA fragment containing a promoter gene according to the present invention is a plasmid pC described below.
The E. coli EKB502 strain contained in SP-P8 (see Example 4) and containing this plasmid pCSP-P8 was
F on June 15, 2014 at the Institute of Biotechnology, Institute of Biotechnology, AIST.
It has been deposited under the accession number of ERM BP-4698.

<Transformation and Expression of Foreign Gene> By introducing a foreign gene (hereinafter sometimes referred to as “promoter-linked foreign gene”) into which a DNA fragment containing a promoter according to the present invention is linked upstream,
Yeast in which the expression of a foreign gene is controlled by the promoter according to the present invention can be obtained.

As a method for introducing this promoter-linked foreign gene into yeast, ANALYTICAL BIOCHEMISTRY 163,39
1 (1987), a method commonly used in the art can be used. Specific examples thereof include a method of incorporating a promoter-linked foreign gene into a vector and introducing the vector into yeast, and a method of directly introducing into yeast.

According to the present invention, there is provided an expression vector comprising a DNA fragment containing a promoter gene and a foreign gene located downstream thereof. An example of such an expression vector is an expression plasmid in which a DNA fragment containing a DNA fragment containing a promoter gene and a foreign gene located downstream thereof is linked to the vector.

The pramid used for constructing the expression vector includes, for example, YRp system (a yeast multicopy vector having an ARS sequence of the yeast chromosome as an origin of replication), Y
Ep system (multicopy vector having origin of replication of 2 μm DNA of yeast), YCp system (single copy vector for yeast having origin of replication of yeast chromosome ARS sequence and having DNA sequence of centromere of yeast chromosome), Y
Examples thereof include those used as yeast vectors such as the Ip system (vector for integrating yeast chromosome that does not have a replication origin of yeast). The production of these vectors and recombinants thereof are described in "New Biotechnology of Yeast", p284, published by Medical Publishing Center.

Although the foreign gene to be linked is not particularly limited, for example, α-acetolactate decarboxylase gene, alcohol acetyltransferase gene, glucoamylase gene and the like can be used. The foreign gene to be linked is not limited to the purpose of expressing the gene product encoded by the foreign gene, and the antisense R
Control of expression by NA is also included in that purpose.

The expression plasmid contains a marker gene for selection of transformants (eg G418 resistance gene, cerulenin resistance gene, copper resistance gene), a terminator (eg PGK gene, etc.) according to the type of host to be transformed.
ADC gene, GPD gene terminator) and the like can be linked.

As a transformation method using the expressed pramid,
For example, the lithium acetate method may be mentioned, but the method is not limited thereto. Further, as a method for directly introducing DNA into yeast, a cotransformation method in which yeast is simultaneously transformed with a plasmid having a marker gene such as a drug resistance gene and the gene DNA to be introduced (Japanese Patent Publication No. 5-60918).
No.) can be mentioned.

The host transformed with the expression vector is preferably yeast. Specific examples include experimental yeast, brewer's yeast, wine yeast, sake yeast, whiskey yeast, brewing yeast such as shochu yeast, and bread. Food-grade yeast such as yeast and industrial yeast such as alcohol-producing yeast can be used.

The protein can be produced by culturing the transformed host according to the present invention. As the protein that can be expressed, for example, in brewer's yeast, the α-acetolactate decarboxylase gene, alcohol acetyltransferase gene, glucoamylase gene and the like can be preferably expressed. The culture conditions may be appropriately determined depending on the host, but since the promoter according to the present invention has a high activity in the low temperature range, the culture is preferably performed at 10 ° C or lower, more preferably 4 ° C.

[0024]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1 Brewer's yeast is produced at low temperature
Identification of protein to be (1) Preparation of protein produced by brewer's yeast Brewer's yeast with 50 ml of minimal medium A (0.67% yeast nitrogen base, 2% glucose, 10 μm)
g / ml adenyl sulfate, 10 μg / ml uracil,
Furthermore, L-tryptophan, L-histidine hydrochloride, L-
Arginine hydrochloride, L-tyrosine, L-leucine, L-isoleucine, L-lysine hydrochloride, L-phenylalanine,
L-glutamic acid, L-aspartic acid, L-valine,
L-threonine, L-serine, L-cysteine, L-alanine and L-glycine are each contained at 12.5 μg / ml)
One platinum loop was inoculated and cultured with shaking at 30 ° C. overnight. From this, 10 ml each was inoculated into three 1000 ml minimal mediums A and statically cultured at 30 ° C. until the OD at 600 nm was about 4 (culture liquid 1).

45 ml of this culture broth was collected and collected to collect 45 ml of a minimal medium A (final concentration: 1.75 mg / ml).
To which L-methionine was added). After static culture at 30 ° C. for 1 hour, the bacterial cells were collected and used as 30 ° C. cultured bacterial cells. Also, the culture solution 1 to 900
After collecting the cells, the cells were collected and resuspended in 900 ml of the minimal medium A, followed by static culture at 10 ° C. overnight. The medium was replaced with an equal volume of fresh minimal medium A, and static culture was carried out at 4 ° C. overnight. This medium was added to an equal volume of new minimal medium A (final concentration 1.75 mg
L-methionine was added to adjust the amount to 1 ml / ml), the mixture was allowed to stand for 1 hour, and the cells were collected.
The cells were cultured at ℃. 50mM of all bacterial cells after harvesting
After washing with Tris-HCl buffer (pH 6.8), -8
Stored at 0 ° C.

The glass beads method was used to obtain proteins from yeast cells. After suspending the bacterial cells in buffer A (20 mM Tris-HCl buffer (pH 8.8), 2 mM CaCl ₂ ) containing 1/40 volume of the original medium, glass beads having a diameter of 0.4 mm were suspended. It was added to 1 mm below the liquid surface and cooled in ice. After vigorous stirring for 60 seconds using a vortex, the operation of leaving it in ice for 60 seconds was repeated 8 times. The solution was recovered and used as a cell disruption solution. Micrococcal nuclease (manufactured by Sigma) was added to this so that the final concentration was 50 μg / ml, and the temperature was 4 ° C.
Left for 30 minutes. Next, SDS-2ME solution (2%
SDS, 10% 2-mercaptoethanol) and DN
ase-RNase solution (1 mg / ml deoxyribonuclease I, 2 mg / ml ribonuclease A,
500 mM Tris-HCl buffer (pH 7.5), 50 m
M MgCl ₂ ) was added to each 1/10 volume of the disrupted cell suspension, and the mixture was allowed to stand at 4 ° C. for 30 minutes and stored as a yeast protein sample at −20 ° C. until use. The protein concentration of the yeast protein sample was measured with a protein assay kit (manufactured by Bio-Rad).

(2) Identification of protein produced by brewer's yeast at low temperature The protein produced by brewer's yeast at low temperature is described in Zuisei Chemistry Laboratory Course 2 Protein Chemistry (above) (Tokyo Kagaku Dojin), pages 15-27. Two-dimensional electrophoresis method (O'Far
(Rell method). Ampholine pH5-8 (manufactured by Pharmacia) was used as the first-dimensional amphoteric carrier for isoelectric focusing. Two-dimensional electrophoresis was performed on 500 μg of cell proteins prepared from 30 ° C.-cultured cells or 4 ° C.-cultured cells, and Coomassie Brilliant Blue staining was performed, and protein migration patterns were compared. As a result, a protein having a molecular weight of 12 kDa (hereinafter referred to as “p12”, which is not detected in the migration pattern of the 30 ° C.-cultured bacterial protein, appears in the migration pattern of the 4 ° C.-cultured bacterial protein.
Spotted "protein").

Example 2 Partial amino acid of p12 protein
Acid sequence determination Partial amino acid sequence determination was performed by Iwamatsu (Biochemistry 63, p13
9 (1991)) polyvinylidene difluoride (PVD
F) It was carried out according to the method using a membrane. Example 1
In the same manner as (2), two-dimensional electrophoresis of bacterial cell proteins cultured at 4 ° C was performed, and after electrophoresis, p1 was obtained by electroblotting.
2 proteins from the gel into PVDF membrane
t "(manufactured by Applied Biosystems). As an electroblotting apparatus, a Sartoblot type IIs (manufactured by Sartorius) was used, and "electroblotting was performed according to Example 1 for sample pretreatment method of protein sequencer" edited by Shimadzu Corporation.
It was carried out at 60 mA for 1 hour. After transfer, JP-A-6-6284
The peptidase-digested peptide fragment of p12 protein was obtained by the method described in Japanese Patent Publication No. 9, and the amino acid sequence determination test was performed by an automated Edman degradation method using a gas phase protein sequencer type 470 manufactured by Applied Biosystems according to the manual. It was The amino acid sequence obtained is as follows.

Amino acid sequence 1 Asn Ala Asp Ser Gln Gly Glu Ser Phe Ala Asp Gln Ala Arg Asp Tyr Met Gly Ala Ala Lys Amino acid sequence 2 Asp Ala Val Glu Tyr Val Ser Gly Arg Ala His Glu Glu Asp Pro Thr Lys Amino acid sequence 3 Asp Ala Val Glu Tyr Val Ser Gly Arg Val His Gly Glu amino acid sequence 4 Leu Asn Asp Ala Val Glu Tyr Val amino acid sequence 5 Gly Val Phe Gln Gly Val His amino acid sequence 6 Glu Phe Ile Thr

Example 3 D producing p12 protein
Cloning of NA chain from brewer's yeast The DNA chain producing the p12 protein, that is, the Lg-CSP1 gene of brewer's yeast was cloned by the following method. (1) Synthesis and labeling of probe Based on the information of the amino acid sequences 1, 3 and 4 among the partial amino acid sequences obtained in Example 2, attached using a DNA synthesizer "Model 380B" of Applied Biosystems The following synthetic probe was prepared according to the operator's manual. The purified synthetic probes were independently [γ- ³² P] ATP (˜600
0 Ci / mmol) and labeled with T4 polynucleotide kinase.

Probe 3 (derived from amino acid sequence 4) Asn Asp Ala Val Glu Tyr 5 ′ AAT GAT GCT GTT GAA TA 3 ′ C C C C G A A A G G

Probe 3C (from amino acid sequence 3) His Glu Glu Asp Pro Thr 5 ′ CAT GAA GAA GAT CCT AC 3 ′ C G G C C C A G

Probe 1 (derived from amino acid sequence 1) Asp Tyr Met Gly Ala Ala 5 ′ GAT TAT ATG GGT GCT GC 3 ′ C C C C A A G G

(2) Cloning by plaque hybridization About 10,000 clones of a brewer's yeast chromosomal DNA phage library prepared by using the phage vector EMBL3 described in JP-A-6-62849 were cloned into a nylon membrane (Colony / PlaqueScreen).
n (manufactured by NEN) according to the protocol attached thereto, and hybridization is performed using the probe 3 labeled with [γ- ³² P] ATP prepared in (1),
176 positive clones were obtained. Then, among these clones, 6 positive clones that hybridized with probe 1 and probe 3C were obtained. One clone, φCSP3, was used in the following examples.

Example 4 Lg-CSP1 promoter region
Acquisition of DNA fragment containing region (Fig. 3) DNA of phage φCSP3 obtained in Example 3 (2)
Was prepared according to a standard method and hybridized with all of probes 1, 3, and 3C, and a 4.3 kb SalI-Hi.
The ndIII fragment was digested with SalI-HindI of pUC119.
Subcloning between the II sites, plasmid pφ3HS
8 was produced. Figure 1 shows a restriction map of the insert fragment of this plasmid.

Using this plasmid, a 1405 bp nucleotide sequence containing the coding region of p12 protein was determined. The sequence is shown in SEQ ID NO: 1. From this sequence, the coding region and promoter region of p12 protein were deduced. Then 2.3 kb of pφ3HS8
The PstI fragment was subcloned into the PstI site of pUC119 to create plasmid pφ3P19. Next, the 1.7 kb PstI-EcoR of this plasmid
The I fragment was subcloned between the PstI and EcoRI sites of BluescriptII d SK + (manufactured by Stratagene) to prepare plasmid pφ3PE1.

The shortening of the 3'end of the promoter region
It was carried out using the PCR method. The positions of the primers used in the PCR reaction are shown in FIG. The primer on the 5'side of the promoter region (PCR primer 5 ') is a plasmid p
Pst on the 5'side of the promoter region on φ3P19
It was prepared based on the DNA base sequence determined from the I site.
In addition, a primer on the 3'side of the promoter region (PCR
Primer 3 ') is a sequence complementary to the base sequence of 612 to 644 in SEQ ID NO: 1 corresponding to the position immediately before the translation initiation point and H
Contains the recognition sequence for indIII. Specifically, it is the following sequence.

PCR Primer 5 ′ 5 ′ CAGGTCCGACGGATCTCGATGGGGGTCGCCCCATAT SalI ACTGT 3 ′

PCR Primer 3 ′ 5 ′ CCAAGCTTGTTTTGTTTTTTTGTTTGTTTTAGTTG HindIII TTGTTTGA 3 ′

1. Prepared from plasmid pφ3P19
PC using 7 kb PstI-EcoRI fragment as a template
The PCR product obtained using R primer 5 ′ and PCR primer 3 ′ was cleaved with EcoT14I and HindIII to obtain a 0.5 kb EcoT14I-HindIII fragment. This fragment was added to plasmid pφ3PE1 of 4.
It was ligated with the 1 kb EcoT14I-HindIII fragment to prepare the plasmid pCSP1-P8 containing only the Lg-CSP1 promoter region. The nucleotide sequence of the PCR product was confirmed to be the same as the nucleotide sequence determined using the plasmid pφ3HS8 as a template by nucleotide sequence determination using T3 primer (manufactured by Stratagene) and the following three types of primers.

Primer 3 for PCR product nucleotide sequence confirmation (complementary sequence of 218-238 of SEQ ID NO: 1 ) 5'CCGCGCCCCTTAAAGAATAGC 3 '

Primer 6 for PCR product nucleotide sequence confirmation (complementary sequence of 502 to 522 of SEQ ID NO: 1 ) 5 ′ CTTAGGGGTGGTTATTTATACGT 3 ′

Primer 7 for PCR product nucleotide sequence confirmation (complementary sequence of 362-382 of SEQ ID NO: 1 ) 5 ′ ACCGCCCACCACCTAGCAATA 3 ′

Example 5 Integration of Lg-CSP promoter
Preparation of ligated expression vector (1) Ligation of Lg-CSP1 promoter and reporter gene (FIG. 4) After cleaving pCPS-P8 with SmaI, XhoI linker (CCTCGAGG, Takara Shuzo) was ligated, and plasmid p
CSP1-P8SX was produced. The 1.6 kb XhoI-HindIII fragment obtained from this plasmid was pA
8.4 kb of X50G2 (Japanese Patent Publication No. 5-60918)
XhoI-HindIII fragment of Lg-CSP
1 promoter and reporter gene (ALDC gene)
Plasmid YEpCPA having a DNA fragment ligated with
L9 was produced. 2.9 kb Xh from this plasmid
The oI-SalI fragment was obtained and SalI of pRS415 was obtained.
YCpCPAL3 was prepared by inserting into the site.

(2) Shortening of Lg-CSP1 promoter (FIG. 5) 1.6 kb PstI-H obtained from pCPS-P8
The indIII fragment was added to pUC119 PstI-Hind.
It was inserted between the III sites to create the plasmid pUCP1. This plasmid was cleaved with BamHI and treated with Klenow fragment to self-ligate to prepare plasmid pUCP1dB1. This plasmid was digested with EcoRI and treated with Klenow fragment,
BamHI linker (CGGATCCG) is ligated,
The plasmid pUCP1EB was created. The shortened DNA fragment from the 5'side of the Lg-CSP1 promoter region of this plasmid is De for Kilo-Sequence.
It was prepared using a region Kit (Takara Shuzo).
Specifically, pUCP1EB was digested with KpnI and SalI and then digested with exonuclease III.
Those treated with mung bean nuclease and Klenow fragment were self-ligated to prepare plasmids pdCP47 and pdCP50.
pdCP47 is a plasmid having a sequence of 56 to 644 of SEQ ID NO: 1 and a nucleotide sequence having a HindIII recognition site on the 3'side. pdCP50 is 4 of SEQ ID NO: 3
It is a plasmid having a sequence of 91 to 644 and a nucleotide sequence having a HindIII recognition site on the 3'side.

(3) Preparation of plasmid for promoter activity assay (FIG. 6) pdCP47 and pdCP50 were replaced with BamHI and Hin.
After cutting with dIII, a BamHI-HindIII fragment containing the Lg-CSP1 promoter sequence was obtained, and the 7.4 kb Ba obtained from YCpCPAL3 prepared in (1) was obtained.
Ligated with mHI-HindIII fragment, YCpdCPAL
47 and YCpdCPAL50 were made. G418
The resistance genes are pAX50G2 in Hind III and Sac.
It was prepared by ligating the 8.5 kb fragment obtained by digesting with I with Klenow fragment and the 1.1 kb fragment obtained by digesting pUC4K (manufactured by Pharmacia) with PstI and XhoI and treating with Klenow fragment. The obtained plasmid pGPDNEO was obtained. The 2.5 kb SalI fragment (G418 resistance gene cassette) obtained from pGPDNEO was inserted into the XhoI site of YCpdCPAL47 to prepare YCpdCPALG47.

Example 6 Promoter in Experimental Yeast
Activity test YCpd, which is a YCp type plasmid prepared in Example 5 (3) and having the shortened Lg-CSP1 promoter linked to the ALDC gene coding region as a reporter gene.
Experimental yeast TD4 (a, his, ura, leu, trp) was treated with CPAL47 and YCpdCPAL50, respectively, by the lithium acetate method (J. Bacteriol. 153, 163 (1983)).
Transformed by. Experimental yeast TD4 is Saccharomyces cerevisiae S288C (α, suc2, ma
1, gal2, CUP1: ATCC26108). The resulting two transformants (YCpdCPA
The activity of the Lg-CSP1 promoter in L47 / TD4, YCpdCPAL50 / TD4) was evaluated by measuring the ALDC activity encoded by the reporter gene.

Two transformants were added to 11 ml of minimal medium B.
(12.5 μg / in the minimum medium A shown in Example 1 (1)
One platinum loop was inoculated into a medium to which methionine was added so that the amount became ml, and the mixture was cultured with shaking at 30 ° C. overnight. From this, inoculate 6 ml each into three 250 ml minimal mediums B, 600 nm
The cells were cultivated at 30 ° C. overnight until the OD was about 2.5. From each of the culture broths, 5 ml was collected and the cells were collected to obtain cells at 30 ° C. for 0 hours. In addition, cells were collected from the rest of the culture solution and resuspended in 200 ml of the minimal medium B to obtain 10
The cells were divided into 0 ml each, and statically cultured at 10 ° C. and 30 ° C. overnight, and the collected cells were designated as 10 ° C.-24 hours and 30 ° C.-24 hours. The ALDC activity was measured by the method described in Japanese Patent Publication No. 5-60918 using an enzyme solution obtained by crushing these cells using glass beads. The result was as shown in FIG. 7. YCpdC
In TD4 with PAL50, no difference in ALDC activity was observed between 30 ° C and 10 ° C cultures.
In the experimental yeast TD4 having CpdCPAL47, increased ALDC activity at low temperature was observed.

Example 7 Promoter in brewer's yeast
-Activity test It was examined whether the activity of the Lg-CSP1 promoter possessed by YCpdCPAL47 was observed not only in experimental yeast but also in brewer's yeast.

The conventional brewer's yeast was transformed with YCpdCPALG47 using the method for transforming brewer's yeast described in JP-A-6-62849. The obtained transformant was mixed with 10 ml of YPD medium containing 10 μg / ml of G418 (2% bactotryptone,
1 platinum loop was inoculated into 1% yeast extract and 2% glucose), and the mixture was cultured with shaking at 30 ° C. overnight. 1.5 ml from this is 250 containing 10 μg / ml G418.
The cells were inoculated in ml of YPD medium and statically cultured at 30 ° C. overnight until the OD at 600 nm was about 4. From each of the culture broths, 5 ml was collected and the cells were collected to obtain cells at 30 ° C. for 0 hours. In addition, cells were collected from the rest of the culture solution,
The cells were resuspended in 200 ml of YPD medium containing μg / ml of G418, divided into 100 ml, and cultured at 10 ° C. and 30 ° C. overnight, respectively, and then the bacterial cells were collected, respectively, 10 ° C.-24 hours. The cells were used at 30 ° C. for 24 hours.
The ALDC activity was measured by the method described in Japanese Patent Publication No. 5-60918 using an enzyme solution obtained by crushing these cells using glass beads. The result was as shown in FIG. Lg-CSP1 possessed by YCpdCPALG47
The promoter had an activity of expressing a gene linked to the downstream thereof in brewer's yeast and also at a high rate at a temperature lower than room temperature.

[0052]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1405 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin: Organism name: Saccharomyces cerevisiae
s cerevisiae) Sequence features Characteristic symbol: CDS Location: 655. ． How to determine the 978 features: E SEQ GGGGAGGATC AACTCCGACA GCAAAAGTCC GGACGTTGAC GGAGCCGGTG CACGGCACGG 60 ACTAAAGCCC AAAAAAAAGG GGCGGTGACG TGTGGGTGGC GCCCGGCGGT AGCGATGACG 120 ACTTGTTGAC GGCCCTTGGC TCTTTGCGTA AGGGACAAAA CCAGAACTTG AGGGTGGTAA 180 AAATAGCAAT TTGGATTTGG TAACGGATGC CAGCTATGCT ATTCTTTAAG GGGCGCGGTG 240 TAGGTTGTAG GGGGCTACTA GTAGAAAGTA AAAAACAACC CGGAGAGGGT GAAAGTGGTG 300 ATGTGGCAAC TAGAAGAAGA AGAAGAAAGA AAAAAAAAAA GACACAAACA CAAACAAGAC 360 GTATTGCTAG GTGGTGGGCG GTAAAGTGAT CTACAGCCCC TGGGAATTGC TTTCGGGCGA 420 AGAACAGTTC CCTTCGTCCC TCTCTCTTCT CCTTCTTCCC CCCTCTTAAT CTGGAATCTA 480 GTCCTTGAAT CCTGGATTCA AACGTATAAA TACCACCCTA AGTTGCTTTC TTCTCTCTCA 540 CTTGTATTCT TTCCTCTTGT TTATGTTCTC TCTCATATAA TAAAAAAAAA CCATCTGATT 600 ATTTCATAAT CTCAAACAAC AACTAAAACA AACAAAAAAC AAACTAATAT AACA ATG 657 Met 1 TCT GAC ACA GGT AGA AAA GGA TTC GGC GAC AAG GCT TCT GAG GCT TTG 705 Ser Asp Thr Gly Ser Lys Gly Phe Gly Asp Lys Ala Ser Glu Ala Leu 5 10 15 AAA CCA GAC TCT CAA AAA TCA TAC GCC GAA CAA GGT AAG GAA TTC ATC 753 Lys Pro Asp Ser Gln Lys Ser Tyr Ala Glu Gln Gly Lys Glu Phe Ile 20 25 30 ACC GAC AAG GCC GAC AAG GTC GCC GGT AAG GTC GAA AGC AAC GAC AAC 801 Thr Asp Lys Ala Asp Lys Val Ala Gly Lys Val Glu Ser Asn Asp Asn 35 40 45 AAG GGT GTC TTC CAA GGT GTT CAC GAC TCC GCT CAA CAA GGT AAG GAC 849 Lys Gly Val Phe Gln Gly Val His Asp Ser Ala Gln Gln Gly Lys Asp 50 55 60 65 AAC GCT GAC AGC CAA GGT GAA TCT TTT GCC GAC CAA GCC AGA GAC TAC 897 Asn Ala Asp Ser Gln Gly Glu Ser Phe Ala Asp Gln Ala Ser Asp Tyr 70 75 80 ATG GGT GCC GCC AAG TCC AAG TTG AAC GAC GCT GTT GAA TAC GTT TCC 945 Met Gly Ala Ala Lys Ser Lys Leu Asn Asp Ala Val Glu Tyr Val Ser 85 90 95 GGA CGT GCT CAC GAA GAA GAC CCA ACC AAG AAG TA AACTCGCTAG 990 Gly Arg Ala His Glu Glu Asp Pro Thr Lys Lys 100 105 CGAGTCTTCTCT TTGTGGTTAC AAGGTCTTTT TTTTTCCTTG TGATGTGTGA TGTTCCTTAA 1050 TATTATTCGA CGAATAGCAG AATAATGAAA AACTAAAGAA AAAGAGAAAA AAAACAGTTG 1110 AAACAAATGC GGAATTTTAT ATGTATATGT ATTTATGTGT ATATTCTCCT AGCTTAGTTT 1170 A AAAATTAAT GGTTTTTTTA TTTCTAGTTT TTTTTATTTT TATTTTTATT GTACGTACGT 1230 GCTTCGTTCT AGTCTAGTTC TAGCTTAAAC ATGCTACTCG ACTCCTCGTCCTCGATCCTGATCCTCCGCAGGAAGAGCTCCTGTAG

SEQ ID NO: 2 Sequence length: 589 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin: Organism: Saccharomyces cerevisiae
s cerevisiae) promoter region sequence of feature Lg-CSP1 gene sequence CACGGACTAA AGCCCAAAAA AAAGGGGCGG TGACGTGTGG GTGGCGCCCG GCGGTAGCGA 60 TGACGACTTG TTGACGGCCC TTGGCTCTTT GCGTAAGGGA CAAAACCAGA ACTTGAGGGT 120 GGTAAAAATA GCAATTTGGA TTTGGTAACG GATGCCAGCT ATGCTATTCT TTAAGGGGCG 180 CGGTGTAGGT TGTAGGGGGC TACTAGTAGA AAGTAAAAAA CAACCCGGAG AGGGTGAAAG 240 TGGTGATGTG GCAACTAGAA GAAGAAGAAG AAAGAAAAAA AAAAAGACAC AAACACAAAC 300 AAGACGTATT GCTAGGTGGT GGGCGGTAAA GTGATCTACA GCCCCTGGGA ATTGCTTTCG 360 GGCGAAGAAC AGTTCCCTTC GTCCCTCTCT CTTCTCCTTC TTCCCCCCTC TTAATCTGGA 420 ATCTAGTCCT TGAATCCTGG ATTCAAACGT ATAAATACCA CCCTAAGTTG CTTTCTTCTC 480 TCTCACTTGT ATTCTTTCCT CTTGTTTATG TTCTCTCTCA TATAATAAAA AAAAACCATC 540 TGATTATTTC ATAATCTCAA ACAACAACTA AAACAAACAA AAAACAAAC 589

SEQ ID NO: 3 Sequence length: 154 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin: Organism name: Saccharomyces cerevisiae
s cerevisiae) Sequence characteristics Part of the promoter region of Lg-CSP1 gene CCTGGATTCA AACGTATAAA TACCACCCTA AGTTGCTTTC TTCTCTCTCA CTTGTATTCT 60 TTCCTCTTGT TTATGTTCTC TCTCATATAA TAAAAAAAAA CCATCTGATT ATTTCATAAC 120AAAAAAAAAC ACAAAAAAACAAC

SEQ ID NO: 4 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Other information: Partial amino acid sequence of p12 protein Sequence Asn Ala Asp Ser Gln Gly Glu Ser Phe Ala Asp Gln Ala Arg Asp Tyr Met Gly 1 5 10 15 Ala Ala Lys 20

SEQ ID NO: 5 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Other information: Partial amino acid sequence of p12 protein Sequence Asp Ala Val Glu Tyr Val Ser Gly Arg Ala His Glu Glu Asp Pro Thr Lys 1 5 10 15

SEQ ID NO: 6 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Other information: Partial amino acid sequence of p12 protein Sequence Asp Ala Val Glu Tyr Val Ser Gly Arg Val His Gly Glu 1 5 10

SEQ ID NO: 7 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Other information: Partial amino acid sequence of p12 protein

SEQ ID NO: 8 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Other information: Partial amino acid sequence of p12 protein

SEQ ID NO: 9 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Other information: Partial amino acid sequence of p12 protein

SEQ ID NO: 10 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other information: Sequence used as probe AATGATGCTG TTGAATA 17

SEQ ID NO: 11 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other information: Sequence used as probe CATGAAGAAG ATCCTAC 17

SEQ ID NO: 12 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other information: Sequence used as probe GATTATATGG GTGCTGC 17

SEQ ID NO: 13 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other information: PCR primer 5 ' Sequence CAGGTCGACG GATCTCGATG GGGTCGCCCC TAT 33

SEQ ID NO: 14 Sequence length: 41 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other information: PCR primer 3 ' Sequence CCAAGCTTGT TTGTTTTTTG TTTGTTTTAG TTGTTGTTTG A 41

SEQ ID NO: 15 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other information: For confirmation of nucleotide sequence Primer sequence CCGCGCCCCT TAAAGAATAG C 21

SEQ ID NO: 16 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other information: For confirmation of base sequence Primer sequence CTTAGGGTGG TATTTATACG T 21

SEQ ID NO: 17 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Other information: For confirmation of nucleotide sequence Primer sequence ACCGCCCACC ACCTAGCAAT A 21

[Brief description of drawings]

FIG. 1 Lg-C subcloned into pφ3HS
It is the figure which showed the restriction map of SP1 gene DNA fragment.

FIG. 2 P for preparing the Lg-CSP1 promoter region in the PstI-PstI fragment of pφ3P19
It is the figure which showed the position of CR primer.

FIG. 3 is a diagram showing a step of preparing an Lg-CSP1 promoter region.

FIG. 4 Lg-CSP1 promoter and reporter (A
It is the figure which showed the ligation process with LDC) gene.

FIG. 5 is a diagram showing a step of preparing an Lg-CSP1 shortened promoter.

FIG. 6 is a diagram showing a step of preparing a plasmid to be introduced into brewer's yeast. The transformed brewer's yeast is used for a promoter activity test.

FIG. 7. Truncated Lg-CS in experimental yeast TD4
It is the figure which showed the evaluation by ALDC activity of P1 promoter.

FIG. 8. Truncated Lg-CSP1 in brewer's yeast.
It is the figure which showed the evaluation by the ALDC activity of a promoter.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1: 865) (C12P 21/02 C12R 1: 865) (72) Inventor Akira Iwamatsu Hiko Kanagawa 1-13-5, Fukuura, Kanazawa-ku, Yokohama-shi, Kirin Brewery Co., Ltd.

Claims (8)

[Claims] 1. A DNA fragment having the DNA sequence set forth in SEQ ID NO: 2 or a sequence having homology to the DNA sequence set forth in SEQ ID NO: 2 and retaining promoter activity. 2. An expression vector comprising the DNA fragment according to claim 1 and a foreign gene located downstream thereof. 3. A DNA fragment comprising the DNA fragment according to claim 1 and a foreign gene located downstream thereof. 4. A host transformed with the expression vector according to claim 2 or the DNA fragment according to claim 3. 5. The host according to claim 4, wherein the host is yeast. 6. A method for producing a protein, which comprises culturing the host according to claim 4 or 5. 7. The method for producing a protein according to claim 6, wherein the culture temperature is 10 ° C. or lower. 8. A DNA fragment having the DNA sequence set forth in SEQ ID NO: 1.