JPH10271995A - Candida yeast promoter DNA having activity in the presence of glucose, recombinant DNA containing the same, transformant, and method for producing inositol using the same - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To enable economical inositol production. A promoter DNA having activity in the presence of glucose is used to express an enzyme gene during an inositol biosynthesis reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Inositol is an important substance as a kind of vitamin in higher organisms.
Used for feed additives and pharmaceuticals.

The present invention relates to a novel promoter DNA having an activity in the presence of glucose, a method for expressing a gene using the promoter DNA, and a method for expressing an enzyme gene involved in inositol biosynthesis using the promoter gene. And a method for producing inositol using a microorganism.

[0003]

2. Description of the Related Art Conventionally, inositol has been extracted from rice bran, corn steep liquor and the like (Japanese Patent Laid-Open No. 61-5614).
2), a method for culturing and producing baker's yeast (EP 50)
No. 6289 published public information). In addition, it has been revealed that in baker's yeast, the inositol accumulation concentration can be improved by using the inositol-1-phosphate synthase gene.

[0004]

The method of extracting rice bran, corn steep liquor and the like contains many impurities other than inositol, is difficult to purify, and is economically problematic. In addition, the method of culturing and producing baker's yeast has low productivity, is still economically problematic, and has no industrial record. In addition, there is no known microorganism other than baker's yeast that produces inositol outside the cells. We have:
In addition to baker's yeast, we have searched for microorganisms that have the potential to produce inositol, and have extensively investigated microorganisms that secrete inositol extracellularly. I found something. Furthermore, from a series of inositol biosynthesis reactions, it was estimated that inositol-1-phosphate synthase was the rate-limiting reaction, and it was considered that this enzyme gene was highly expressed by a strong promoter gene to promote inositol biosynthesis. However, there are few existing promoters and their effectiveness is low.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors searched for a promoter DNA having activity in the presence of glucose from yeast of the genus Candida and searched for a glycerol-3-phosphate dehydrogenase gene. The promoter DNA was found to be active in the presence of glucose. Using Candida yeast as a host, the acid phosphatase gene of baker's yeast and the inositol-1-phosphate synthase gene of Candida yeast can be expressed under the control of this promoter DNA, and the inositol-1-phosphate synthase gene is expressed. In this case, it was found that the insitol accumulation concentration and the production yield were improved, and the present invention was reached.

That is, the present invention relates to a novel promoter DNA having an activity in the presence of glucose,
The present invention relates to a self-replicatable DNA containing NA, a method for expressing a gene using the promoter, and a method for producing inositol, which comprises expressing an enzyme during an inositol biosynthesis reaction using the promoter DNA.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION A gene acquisition source according to the present invention will be described. As a gene source for obtaining the promoter DNA, a yeast belonging to the genus Candida, more preferably a yeast belonging to the species Voidiny genus is used. As a gene for obtaining the promoter DNA, a gene of an enzyme having an activity when cultured using glucose as a carbon source, that is, a gene such as glyceraldehyde-3-phosphate dehydrogenase is used.

On the other hand, it has been revealed that the gene expressed by using the promoter DNA of the present invention can be expressed not only by the gene of Candida yeast but also by a gene obtained from baker's yeast. Therefore, the promoter DNA of the present invention
The present invention also encompasses a recombinant DNA containing a gene obtained from a source other than a host as a gene expressed using the above, a transformant, and a method for producing inositol using the same.

When a microorganism is used for the production of inositol, a gene of an enzyme used in an inositol biosynthesis reaction, that is, a gene such as inositol-1-phosphate synthase may be used as a gene expressed using a promoter DNA. Used.

The method for isolating DNA of the present invention will be described. Caudida genus Voidinii (hereinafter Candid)
The method for obtaining the chromosomal DNA of a bodinii) is the same as the method for baker's yeast, and includes, for example, the method of Shunji Natori (Shinsei Kagaku Kenkyusho 17 Microbial Experiment Method p245-246).

A method for isolating DNA encoding glyceraldehyde-3-phosphate dehydrogenase is performed by using a primer using an appropriate site of a glyceraldehyde-3-phosphate dehydrogenase protein-encoding portion, and using genomic DNA as a template and genomic DNA as a template. A method of amplifying a gene in a DNA sequence-specific manner by a race chain reaction method (hereinafter, PCR method) is desirable.

Specifically, primers are synthesized based on a highly conserved sequence of DNA sequences from the protein coding portion of the glyceraldehyde-3-phosphate dehydrogenase gene of baker's yeast and human, and the genomic DNA library is used as a template. The protein coding portion of the gene is amplified by PCR. The amplified DNA fragment is p
After insertion into a vector for Escherichia coli such as UC18, the DNA sequence is confirmed by the dideoxy method to confirm homology with other glyceraldehyde-3-phosphate dehydrogenase genes. Based on the obtained glyceraldehyde-3-phosphate dehydrogenase gene sequence, a primer is synthesized so as to amplify the upstream portion of the protein coding portion. Using the genomic DNA library as a template, the promoter DNA was again amplified by the PCR method, the obtained DNA fragment was inserted into an E. coli vector such as pUC18, and the DNA sequence was confirmed by the dideoxy method.
DN having the same DNA sequence as the protein coding portion of the glyceraldehyde-3-phosphate dehydrogenase gene
An A fragment is obtained, and the upstream portion thereof is obtained as a promoter DNA.

Next, a method for transforming Candida boidiniii will be described. As a host, a strain using the URA3 gene-deficient strain of the strain as a marker is used. As the vector, a vector for the strain which holds the URA3 gene or the like is used, and for example, pYSN01 (FIG. 3) is used. The DNA encoding inositol-1-phosphate synthase obtained as described above is inserted into an appropriate restriction enzyme cleavage site of such a vector to prepare a DNA for transformation. Transformation can be performed by a conventional method such as a protoplast method or a lithium acetic acid method.

The restriction enzyme map, base sequence and the like of the obtained DNA are determined according to a commonly used method.

The culturing method of the present invention will be described. The medium for producing inositol is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic trace components.

Examples of the carbon source include glucose, fructose, hydrolysates of starch and cellulose, sugars such as molasses, organic acids such as fumaric acid, citric acid and succinic acid, and alcohols such as methanol, ethanol and glycerol. 0.1 to 4.0% of an organic ammonium salt such as ammonium acetate, an inorganic ammonium salt such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonia gas, aqueous ammonia, urea, etc. as a nitrogen source. As the organic trace component, the required substance such as biotin is 0.00%.
A medium containing 0001% to 0.1%, and if necessary, 0 to 5% of corn steep liquor, peptone, yeast extract and the like, respectively, is appropriately used. In addition to these,
Potassium phosphate, magnesium sulfate, calcium chloride,
Sodium chloride, zinc sulfate, copper sulfate, ferrous sulfate, etc. are added as trace components. Preferably, an antifoaming agent is also added to stabilize the culture conditions.

The culture is performed under aerobic conditions. During the culture, the pH of the medium was adjusted to 3-8, the temperature was adjusted to 20-35 ° C, and
Preferred results can be obtained by culturing with shaking or aeration and agitation for 20 hours.

The inositol secreted and accumulated in the culture solution can be used as feed without isolation and collection as it is. In addition, inositol can be collected from a culture solution or a reaction solution by a known method. For example, crystals can be obtained by removing cells by centrifugation or the like, removing ionic substances with a cation and anion exchange resin, and concentrating.

[0019]

The present invention will be described below in detail with reference to examples.

Example 1 (Preparation of probe DNA) Candida Boyidiny T
Chromosomal DNA was obtained from R-1 (FERM BP-5076) by the method of Shunji Natori (Shinsei Kagaku Kenkyusho 17 Microbial Experiment Method p245-246). On the other hand, Saccharomyces cerevisiae and human glyceraldehyde-3-phosphate dehydrogenase gene were examined for highly conserved regions of amino acid sequences.
A primer (mix A primer, mix B primer) was synthesized, and a polymerase chain reaction (PCR) was performed using the chromosomal DNA of Candida boidinii TR-1 as a template, and the synthesized DNA was added to a vector pUC18 (Takara Shuzo). Connected.
Analyze the base sequence of the probe DNA and confirm that the Saccharomyces
As a result of comparison with the corresponding sequence of S. cerevisiae, there was 75.9% homology at the DNA level as shown in FIGS.

Mix A primer: 5 'AA (CT) GGITT (CT) GGI (A
C) GIAT (ACT) GG 3 'mix B primer: 5' ACIGC (TC) TTIGCIGCICCIGT 3 '

Example 2 (Preparation of chromosomal DNA library) 20 μg of the chromosomal DNA obtained from Candida boidinii TR-1 (FERM BP-5076) in Example 1 was subjected to the restriction enzyme Sau3AI.
Was partially decomposed at 37 ° C. for 5 minutes using 1.4 units.
A DNA fragment near 5 kbp was purified by electrophoresis, and PCR was performed using this DNA fragment as a template in the same manner as in Example 1. As a result, a 626 bp DNA fragment having the same size as that in Example 1 was obtained. Glyceraldehyde-3-
It was confirmed that the phosphate dehydrogenase gene was present. The purified DNA fragment of about 5 kbp is λ DNA (STRA
TAGENE λ ZAP Express Vector)
A library was created.

Example 3 (Acquisition of glyceraldehyde-3-phosphate dehydrogenase promoter DNA by two-step PCR) Example 2
Using the chromosomal DNA library created in
P proA primer and M4 prim present in λ DNA sequence
The first amplification by PCR was performed using er. Subsequently, a second PCR was performed using the first PCR reaction solution as a template and G3P proB primer and T7 primer present in the λ DNA sequence. The amplified DNA fragment was purified by electrophoresis, introduced into pUC18, and subjected to DNA sequence analysis to obtain a 766 bp DNA sequence represented by SEQ ID NO: 1. In addition, a restriction enzyme map as shown in FIG. 6 was created.

G3P proA primer: 5 'CTTGTATTGTTTGTGGGT
GGAA 3 'G3P proB primer: 5' AAGCAGCGTAATCAGCAGCAA 3 'M4 primer: 5' GTAAAACGACGGCCAGT 3 'T7 primer: 5' GTAATACGACTCACTATAGGGC 3 '

Example 4 (Construction of Acid Phosphatase Expression Vector) Example 3
Using the promoter DNA obtained in step 1 as a template, phoA prime
PCR was performed by synthesizing r and phoB primer, and a restriction enzyme recognition sequence of SacI was introduced at the 5 ′ end of the promoter DNA and a restriction enzyme recognition sequence of BglII was introduced at the 3 ′ end. As shown in FIG. 7, an acid phosphatase gene PHO5 derived from baker's yeast was located downstream of the promoter.
[EC 3.1.3.2] was connected to the terminator DNA of the alcohol oxidase gene, and a recombinant DNA (pH30) into which the URA3 gene was further inserted was prepared.

PhoA primer: 5 'CCCGAGCTCGATCTACTGCCGT
TCTTGCT 3 'phoB primer: 5' GGGAAGATCTGTTTGTTTGTAAGTTTATAT 3 '

Example 5 (Construction of Inositol-1-Phosphate Synthase Expression Vector) Using the promoter DNA obtained in Example 3 as a template, inoA primer and inoB primer were synthesized, PCR was performed, and 5 ′ of the promoter DNA was obtained. A restriction enzyme recognition sequence for EcoRI was introduced at the terminal side and a restriction enzyme recognition sequence for NotI at the 3 ′ terminal side. As shown in FIG. 8, the promoter DNA of the shuttle vector pYSN01 of Escherichia coli and Candida boidini was replaced, and Candida boidinii DGR1-14 was placed downstream of the promoter DNA.
A recombinant DNA (pYSN602) into which the inositol-1-phosphate synthase gene of (FERM BP-5070) was introduced was prepared.

InoA primer: 5 'CCGGAATTCGATCTACTGCCGT
TCTTGCT 3 'inoB primer: 5' AAGGAAAAAAGCGGCCGCTGAAGAAGTTTTTGTT
TGTT 3 '

Example 6 (Measurement of Promoter Activity) Using the expression vector of Example 4, Candida voidinii δ-65 (FERMP-157
From 39), Y. J. SAKAI et al. Bacterio
l. 173: 7458-7463 Host Candida voidinii FOAR6 transformed into a URA3 gene-deficient strain
5-4 was transformed by the protoplast method (Yeast Molecular Genetics Experiment Method (Society Press Center) p120-121, edited by Taiji Oshima).

The recombinant was inoculated into an 18 mm diameter test tube containing 5 ml of a medium having the composition shown in Table 1 and cultured for 2 days. The cells were washed with physiological saline, and 5 ml of a medium having the composition shown in Table 2 was added. The absorbance at 660 nm is 0.05
It was inoculated. After culturing until the absorbance at 660 nm becomes 1.0, the cells are washed with physiological saline, and
It was suspended in a 5 M aqueous acetic acid solution (pH 4.0).

Paranitrophenyl phosphate 32 mg / 0.
800 μl of 50 ml of a 05M acetic acid aqueous solution (pH 4.0)
Was added to 200 μl of the bacterial cell suspension, reacted at 37 ° C. for 10 minutes, added with 1 ml of a 10% aqueous solution of trichloroacetic acid, stirred, and added with 1 ml of a saturated aqueous solution of disodium carbonate. It was measured. The amount of paranitrophenol phosphate reacted using paranitrophenol / 0.05M acetic acid aqueous solution (pH 4.0) as a standard solution was calculated, and the acid phosphatase activity was measured. As shown in Table 3, the acid phosphatase activity was improved. The obtained recombinant TF2 strain was obtained.

[0032]

[Table 1]

[0033]

[Table 2]

[0034]

[Table 3]

Example 7 (Expression of inositol-1-phosphate synthase) A protoplast method (yeast molecular genetics) was performed using the expression vector prepared in Example 5 for the host Candida boidinii FOAR65-4 (a URA3-deficient strain). Experimental Method (Academic Publishing Center) p120-1
21, edited by Taiji Oshima))).

Recombinants were prepared using the medium containing 5 ml of the medium shown in Table 4.
After inoculating into a 2 mm diameter test tube and culturing for 2 days, 5% was inoculated into a 500 ml baffled flask containing 50 ml of the medium shown in Table 4. Culturing 40 hours of cells by ultrasonication,
The activity of inositol-1-phosphate synthase was measured in Japanese Patent Application No. 8-28.
As a result, the recombinant TF163 strain having improved enzyme activity was obtained as shown in Table 5.

[0037]

[Table 4]

[0038]

[Table 5]

Example 8 (Inositol fermentation using recombinant) Using the recombinant TF163 strain obtained in Example 7, inositol fermentation was performed.

The recombinant was inoculated into a 22 mm diameter test tube containing 5 ml of the medium shown in Table 4, and cultured for 1.5 days.
5% was inoculated into a 500 ml baffled flask containing 0 ml. 1.5 days after the start of culture, 50 g of glucose /
As a result of adding L and culturing for a total of 5 days, the accumulated concentration was improved as shown in Table 6.

[0041]

[Table 6]

[0042]

According to the present invention, the enzyme gene can be expressed in the presence of glucose by using the promoter DNA of the present invention, and the enzyme activity can be improved. Furthermore, by expressing the enzyme gene during the inositol biosynthesis reaction using the promoter DNA of the present invention, it is possible to improve the inositol fermentation ability.

[0043]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 766 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism name: Candida boidiny Strain name: TR-1 Sequence characteristics symbols represent the characteristics: promoter a promoter existing position: 1-766 method to determine the characteristics: E SEQ GATCTACTGC CGTTCTTGCT GGTAGTGCGG GTCTCCTGGA AGGCAGCTTA CAAATTCGCA 60 ATTCTGTCAA CAACGGCGGC TTATTCTCTG CCAGACCCAG TGGTTTTCGC CACCAGGCGC 120 AATATAGTCA ATACACCGGG CCAGGAGGGC CCTGAGCGAG TGCTCTCGGA GGCACTGCAG 180 GCAAGCGAAA CCAATAACAG ATACTACAGC GGTCGCCACG TTAAAGAGCG GCGCCTGCAT 240 GATGAAGCAC ATAGATGCAG AACATACAGA CACACCACGC CAATACCGTA CAACACATTG 300 AACACATGCA TTCATATAAC ACAACCTAAC TGCAAAAGAT AATATTTTAA CCAACAACTC 360 CACTTTCTCT CTCCTCTGTC AATTACTGTT TCAAGTGGAA TTTTAATTTT AATTAATTCATT TATTATTGATTAATTAATTTAATT ATATT ATATT ATTAT G 540 AGAAGGAGGT CTTAACAGTT TTCAATTTTG GTTTTTTTGC TGATTGTCCT TACCCAGAAA 600 TTTTAATATA AATATTTGAC AATTGCCCCT ATTTCTAAAT CAATTTAATT TGTATCAATT 660 TATTATTATA TTTTTTTCTC CTTTCATCATTATCATATACATACATACATACAA 720

SEQ ID NO: 2 Sequence length: 526 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide (protein) sequence Met Thr Tyr Gln Phe Val Pro Thr Val Lys Val Asp Asn Asp Asn 1 5 10 15 Cys Thr Tyr Thr Asp Ser Glu Leu Val Thr Lys Tyr Thr Tyr Lys 20 25 30 Asn Ser Val Val Thr Lys Glu Ser Asn Gly Ser Phe Ser Ile Lys 35 40 45 Pro Lys Thr Glu Asp Tyr Glu Phe Lys Val Asp Leu Lys Val Pro 50 55 60 Lys Leu Gly Val Met Leu Val Gly Leu Gly Gly Asn Asn Gly Thr 65 70 75 Thr Phe Cys Ala Ala His Phe Ala Asn Lys His Asn Ile Glu Phe 80 85 90 Asn Thr Lys Glu Gly Val Val Lys Pro Asn Tyr Tyr Gly Ser Val 95 100 105 Thr Gln Ser Ala Thr Ile Lys Leu Gly Ile Asp Glu Glu Gly Leu 110 115 120 Asp Val Tyr Ala Pro Phe Asn Ser Leu Leu Pro Phe Thr Asn Pro 125 130 135 Asn Asp Phe Val Ile Ser Gly Trp Asp Ile Ser Gly Ile Asn Gln 140 145 150 Tyr Asp Ala Met Val Arg Ala Glu Val Leu Glu Tyr Asp Leu Gln 155 160 165 Gln Lys Leu Lys Pro Tyr Met Glu Thr Ile Lys Pro Leu Pro Ser 170 175 180 Ile Tyr Tyr Pro Asp Phe Ile Ala Ala Asn Gln Asp Gly Arg Ala 185 190 195 Asp Asn Cys Tyr Asn Arg Lys Gly Asn Glu Pro Ala Ser Thr Lys 200 205 210 Asp Lys Trp Ala His Val Glu Gln Ile Arg Lys Asp Ile Arg Glu 215 220 225 Phe Lys Lys Ser Asn Asn Leu Asp Lys Val Ile Val Leu Trp Thr 230 235 240 Ala Asn Thr Glu Arg Tyr Cys Asp Leu Ile Glu Gly Val Asn Asp 245 250 255 Thr Ala Asp Asn Leu Val Asn Ala Ile Lys Asn Asp His Ala Glu 260 265 270 Val Ser Pro Ser Thr Val Phe Ala Ile Ala Ser Ile Leu Glu Asn 275 280 285 Thr Pro Tyr Ile Asn Gly Ser Pro Gln Asn Thr Phe Val Pro Gly 290 295 300 Leu Leu Glu Leu Ala Glu Lys Glu Asn Thr Phe Ile Gly Gly Asp 305 310 315 Asp Phe Lys Ser Gly Gln Thr Lys Phe Lys Ser Val Ile Ala Gln 320 325 330 Phe Leu Val Asp Ala Gly Ile Arg Pro Ile Ser Ile Ala Ser Tyr 335 340 345 Asn His Leu Gly Asn Asn Asp Gly Tyr Asn Leu Ser Ser Pro Lys 350 355 360 Gln Phe Arg Ser Lys Glu Ile Ser Lys Ala Ser Val Val Asp Asp 365 370 375 Ile Ile Lys SerAsn Glu Ile Leu Tyr Asn Asp Lys Thr Gly Arg 380 385 390 Lys Val Asp His Cys Ile Val Ile Lys His Met Ser Ala Val Gly 395 400 405 Asp Ser Lys Val Ala Met Asp Glu Tyr Tyr Ser Glu Leu Met Leu 410 415 420 Gly Gly His Asn Arg Ile Ser Cys His Asn Val Cys Glu Asp Ser 425 430 435 Leu Leu Ala Thr Pro Leu Ile Ile Asp Leu Ile Ile Met Thr Glu 440 445 450 Phe Phe Ser Arg Val Ser Tyr Lys Lys Ala Glu Asp Asn Ala Ser 455 460 465 Ala Tyr Asp Lys Met Tyr Ser Val Leu Ser Phe Leu Ser Tyr Trp 470 475 480 Leu Lys Ala Pro Leu Thr Arg Pro Gly Tyr Glu Ala Ile Asn Gly 485 490 495 Leu Asn Lys Gln Arg Ala Gly Val Asp Asn Phe Leu Arg Leu Leu 500 505 510 Ile Gly Leu Pro Ala Leu Asp Glu Leu Arg Phe Glu Glu Arg Leu 515 520 525 Lys 526

SEQ ID NO: 3 Sequence length: 1578 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism: Candida Boydinii Strain: DGR1-14 Sequence Characteristic symbol: mat peptide Location: 1. . 1578 method to determine the characteristics: E SEQ ATGACTTACC AATTTGTTCC AACTGTTAAA GTCGATAACG ACAACTGTAC TTATACTGAC 60 TCTGAGTTAG TCACTAAGTA TACTTATAAA AACAGTGTTG TTACCAAGGA AAGTAATGGT 120 TCATTCTCGA TTAAACCAAA GACAGAAGAT TATGAGTTTA AAGTTGATTT AAAAGTTCCA 180 AAATTAGGTG TTATGCTTGT CGGTTTAGGT GGTAACAATG GTACTACTTT CTGTGCTGCT 240 CATTTTGCTA ATAAACATAA TATTGAATTC AATACGAAAG AAGGTGTTGT TAAACCAAAT 300 TATTATGGTT CTGTTACTCA GTCTGCTACT ATTAAATTAG GTATTGATGA AGAAGGTTTG 360 GATGTTTATG CCCCATTTAA CTCTCTTTTA CCATTCACTA ACCCAAATGA TTTTGTTATT 420 TCTGGTTGGG ATATTAGCGG TATTAACCAA TACGATGCTA TGGTTAGAGC TGAAGTCTTA 480 GAATATGACT TACAACAAAA ATTAAAGCCA TACATGGAAA CTATTAAACC ATTACCATCA 540 ATCTACTATC CAGATTTCAT TGCTGCCAAC CAAGATGGAA GAGCTGATAA TTGTTACAAC 600 AGAAAGGGTA ATGAACCAGC TTCTACCAAG GATAAATGGG CCCATGTTGA ACAAATTAGA 660 AAAGATATCA GAGAATTCAA GAAATCTAAT AATTTAGATA AAGTCATTGT CTTATGGACT 720 GCCAATACTG AAAGATATTG TGATCTTATT GAAGGTGTTA ATGATACCGC TGATAATTTA 780 GTCAATGCCA TAAAGAATGA TCATGCTGAA GTTT CACCAT CTACTGTCTT TGCCATTGCT 840 TCAATTTTAG AAAACACTCC ATATATCAAT GGTTCTCCAC AAAACACTTT TGTTCCAGGT 900 CTCTTAGAAT TAGCCGAAAA GGAAAATACT TTTATTGGTG GTGATGATTT TAAGAGTGGT 960 CAAACCAAAT TTAAATCTGT TATTGCTCAA TTTTTAGTTG ATGCTGGTAT TAGACCAATT 1020 TCCATTGCTT CATACAACCA TTTAGGTAAC AATGATGGTT ATAACTTAAG TTCTCCAAAA 1080 CAATTCAGAT CTAAGGAAAT TTCCAAGGCT TCTGTTGTTG ATGATATCAT TAAATCAAAC 1140 GAAATTTTAT ACAACGATAA GACAGGAAGA AAAGTTGATC ATTGTATTGT CATTAAGCAC 1200 ATGAGTGCTG TTGGTGATTC TAAAGTTGCC ATGGATGAAT ATTATTCTGA ATTAATGCTT 1260 GGCGGTCATA ACAGAATTTC ATGTCATAAC GTTTGTGAAG ATTCTTTATT AGCAACACCA 1320 TTAATTATTG ATTTAATTAT TATGACTGAA TTTTTTTCTA GAGTTTCTTA TAAGAAAGCT 1380 GAAGATAATG CATCTGCATA TGATAAGATG TATTCTGTAT TATCATTCCT TTCTTATTGG 1440 TTGAAGGCAC CATTAACTAG ACCAGGTTAT GAAGCCATCA ATGGTTTAAA CAAACAACGT 1500 GCTGGTGTTG ACAATTTCTT AAGATTATTA ATTGGTTTAC CAGCACTTGA TGAGTTAAGA 1560 TTTGAAGAAA GATTAAAA 1578

[Brief description of the drawings]

FIG. 1 is a diagram showing a restriction enzyme cleavage map of a promoter DNA of the present invention.

FIG. 2 is a diagram showing a restriction enzyme cleavage map of DNA encoding the inositol-1-phosphate synthase of the present invention.

FIG. 3 is a diagram showing an example of a vector used in the present invention.

FIG. 4 shows the relationship between the probe DNA obtained in Example 1 of the present invention and
Glyceraldehyde-3- of Saccharomyces cerevisiae
It is a figure which compared the sequence of DNA of the protein part of a phosphate dehydrogenase gene protein.

FIG. 5 shows the probe DNA obtained in Example 1 of the present invention and
Glyceraldehyde-3- of Saccharomyces cerevisiae
It is a figure which compared the sequence of DNA of the protein part of a phosphate dehydrogenase gene protein.

FIG. 6 is a diagram showing a restriction map of a promoter DNA obtained in Example 3 of the present invention.

FIG. 7 is a diagram showing the construction of an acid phosphatase expression vector obtained in Example 4 of the present invention.

FIG. 8 is a diagram showing the construction of an inositol-1-phosphate synthase expression vector obtained in Example 5 of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 7/22 C12P 7/22 // (C12N 15/09 ZNA C12R 1:72) (C12N 1/19 C12R 1:72) ( C12P 7/22 C12R 1:72) (72) Inventor Makoto Shirai 1 Nagoya Office, Minato-ku, Nagoya City, Aichi Prefecture 1 Toray Industries, Inc. Nagoya Office 9 Toray Co., Ltd. Nagoya Office

Claims (26)

[Claims] 1. A Candida yeast promoter DNA which is active in the presence of glucose. 2. The promoter DNA according to claim 1, wherein the restriction enzyme cleavage map is shown in FIG. 3. The promoter DNA according to claim 2, wherein the nucleotide sequence is SEQ ID NO: 1 or a nucleotide sequence complementary thereto. 4. The promoter DNA according to claim 3, wherein one or more nucleotides in the nucleotide sequence are substituted with other nucleotides. 5. The promoter DNA according to claim 4, wherein one or two or more base sequences are substituted with other bases, and one or more restriction enzyme recognition sequences are introduced. 6. The promoter DNA according to any one of claims 1 to 5, wherein the yeast is a yeast belonging to the species Voidiny of the genus Candida. 7. A DNA encoding a Candida yeast promoter DNA and an enzyme having activity in the presence of glucose.
A, a replicable recombinant DNA comprising a terminator DNA, an autonomously replicable vector. 8. A Candida yeast promoter DNA and an enzyme-encoding DN which are active in the presence of glucose.
A, a recombinant DNA that can be integrated into chromosomal DNA, comprising a terminator DNA and a vector that can be integrated into the chromosomal DNA of Candida yeast. 9. The promoter DNA according to any one of claims 1 to 6, wherein the promoter DNA is the promoter DNA according to any one of claims 1 to 6.
Or the recombinant DNA according to 8. 10. An inositol-1-promoter DNA of a Candida yeast having activity in the presence of glucose.
DNA encoding phosphate synthase, terminator D
NA, a replicable recombinant DNA comprising an autonomously replicable vector. 11. Inositol-1-promoter Candida yeast promoter DNA which is active in the presence of glucose.
DNA encoding phosphate synthase, terminator D
NA, a recombinant DNA that can be integrated into chromosomal DNA, comprising a vector that can be integrated into chromosomal DNA of Candida yeast. 12. The recombinant DNA according to claim 10, wherein the restriction enzyme cleavage map of the DNA encoding inositol-1-phosphate synthase is shown in FIG. 13. The set according to claim 10, wherein the amino acid sequence of inositol-1-phosphate synthase translated from the DNA encoding inositol-1-phosphate synthase is the amino acid sequence shown in SEQ ID NO: 2. Replacement DNA. 14. The recombinant DNA according to any one of claims 10 to 13, wherein the DNA encoding inositol-1-phosphate synthase has the nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence complementary thereto. . 15. The DNA encoding inositol-1-phosphate synthase has one or more base sequences of SEQ ID NO: 3 substituted with other bases without changing the amino acid sequence of SEQ ID NO: 2. The recombinant DNA according to any one of claims 10 to 14, which is a DNA. 16. The recombinant DNA according to any one of claims 10 to 15, wherein the DNA encoding inositol-1-phosphate synthase is DNA derived from yeast belonging to the genus Candida sp. 17. The recombinant DNA according to any one of claims 10 to 16, wherein the promoter DNA is the promoter DNA according to any one of claims 1 to 6. 18. A Candida yeast promoter DNA which is active in the presence of glucose, and a DNA encoding an enzyme.
A transformant obtained by appropriately introducing a replicable recombinant DNA containing NA, terminator DNA, and an autonomously replicable vector into a host. 19. A Candida yeast promoter DNA which is active in the presence of glucose, and D which codes for an enzyme.
A transformant obtained by appropriately introducing a recombinant DNA that can be incorporated into chromosomal DNA into a host, comprising a vector that can be incorporated into chromosomal DNA of NA, terminator DNA, and Candida yeast. 20. The transformant according to claim 18 or 19, wherein the recombinant DNA is the recombinant DNA according to any one of claims 10 to 17. 21. The host according to claim 18, wherein the host is Escherichia coli.
The transformant according to any one of the above. 22. The transformant according to any one of claims 18 to 20, wherein the host is a yeast belonging to the genus Candida. 23. The transformant according to claim 22, wherein the host is a yeast belonging to the genus Codyda Boyidny. 24. A Candida yeast promoter DNA which is active in the presence of glucose, and D which codes for an enzyme.
A method for producing inositol using a transformant obtained by appropriately introducing a replicable recombinant DNA containing NA, terminator DNA, and an autonomously replicable vector into a host. 25. A Candida yeast promoter DNA which is active in the presence of glucose, and D which codes for an enzyme.
Production of inositol using a transformant obtained by appropriately introducing into a host a recombinant DNA that can be incorporated into chromosomal DNA, comprising a vector that can be incorporated into chromosomal DNA of NA, terminator DNA, and Candida yeast Method. 26. The transformant according to any one of claims 18 to 23, wherein the transformant is the transformant according to any one of claims 18 to 23.
6. The method for producing inositol according to 5.