JPH0767683A - Acetic acid-assimilating gene and its use - Google Patents

Abstract

PURPOSE:To enable efficient accumulation of an objective substance with reduced accumulation of acetic acid by culturing a microorganism belonging to Escherichia, by increasing the intracellular expression of the gene of a specific protein effectively suppressing the accumulation of acetic acid and collecting the objective substance accumulated in the medium. CONSTITUTION:Escherichia coli cells are cultured to collect wet cell bodies from the culture mixture and chromosomal DNA is isolated from the cell bodies by a usual manner. The DNA is treated with a restriction enzyme to be linked to a vector to prepare a gene library, which is cloned to isolate an acetic acid- assimilating gene (ace). The acetic acid-assimilating gene is linked to a vector together with the gene coding the objective substance to prepare a multi-copying type expression vector. Then, the vector is inserted into a microorganism of Escherichia coli to effect transformation. The transformant is cultured in a medium to increase the intracellular concentration of Ace protein having a specific amino acid sequence as a product of the ace gene and suppress the acetic acid accumulation. Thus, the objective substance is accumulated in the culture and collected to result in efficient production of the objective substance.

Description

Detailed Description of the Invention

[0001]

The present invention relates to Escherichia coli ( Escherichia co
li ) acetic acid-utilizing gene and its use.

[0002]

When glucose is added to the medium during liquid culture of Escherichia coli, acetic acid generated by assimilating this is released to the outside of the cells, and the pH of the medium is increased with the culture time and the growth of the cells. Is known to decrease. p
The decrease in H 2 is a major problem in suppressing the growth of bacteria and producing substances such as amino acids by fermentation. Therefore, several groups have attempted to solve this problem by improving the bacteria. By using a mutant strain of phosphotransacetylase, Bauer et al. Utilized a strain having a defect in the acetic acid synthetic pathway for the fermentative production of IL-2. This strain did not show acetic acid accumulation in the medium and did not inhibit the growth of the fungus (Keith AB et al., Appl. Env.
iron. Microbiol. 56: 1296, 1990). In addition, Matsuyama et al. Isolated a gene ( ackA ) encoding an acetate kinase involved in the acetic acid synthesis pathway of Escherichia coli (Asahi M.
et sl., J. Bacteriol. 171: 577, 1989).

[0003]

Therefore, the problem to be solved by the present invention is to solve the problem of the inhibition of the growth of bacteria caused by the accumulation of acetic acid in the medium in the production of substances by fermentation.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above-mentioned problems, the present inventors have found the existence of a gene that is thought to be involved in the assimilation of acetic acid in Escherichia coli (hereinafter, this gene is referred to as ace The gene and the protein encoded by the gene are referred to as Ace protein.), And the isolation and structural analysis thereof were carried out to complete the present invention. That is, the present invention provides (i) a method for producing a substance in which a microorganism belonging to the genus Escherichia is cultured in a medium to accumulate and produce a target substance, and the target substance is collected. A method for producing the target substance, which comprises increasing the intracellular concentration of a protein having a specified amino acid sequence; (ii) a cell having a protein having the amino acid sequence represented by SEQ ID NO: (1) in the sequence listing The means for increasing the internal concentration is to increase the expression level of the gene encoding the protein (i) a method for producing the target substance; (iii) shown in SEQ ID NO: 1 of the sequence listing A means for increasing the intracellular concentration of a protein having an amino acid sequence is
A method for producing a target substance according to (ii), which increases the copy number of a gene encoding the protein; (iv) encodes a protein having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing The method for producing a target substance according to (iii), wherein the gene has a nucleotide sequence from 1138 to 1140th ACA to 2350 to 2352th AAT in the sequence listing (1). Hereinafter, the present invention will be described in detail.

When Escherichia coli is cultured with glucose as a carbon source, acetic acid is produced as a decomposition product, which is secreted outside the cells, leading to a decrease in the pH of the culture medium, which inhibits the growth of the bacteria. Is well known. This was confirmed in the experiment conducted by the inventor (FIG. 1). Figure 1 shows E. coli JM103 strain with L-brot supplemented with 0.4% glucose.
The growth curve, pH value change, and acetic acid accumulation in the medium when cultured in h or glucose-free L-broth were shown. Acetic acid accumulated in the latter half of the culture (Fig. 1, right).
It is observed that the value is decreasing (the left figure in FIG. 1).

When cultured in L-broth supplemented with glucose, L-broth supplemented with no glucose was added.
It is also observed that the growth is poorer than that in the case of culturing in the above (Fig. 1, left), but it is clear from the following points that this is due to the decrease in pH. That is, by keeping the pH value at 7.0 with the sodium phosphate buffer under the culture conditions of FIG. 1, growth inhibition is not seen and accumulated acetic acid is assimilated (data not shown). .

Therefore, a buffer solution was added to the L-broth medium to adjust the pH.
As shown in Figure 2, E. coli JM1
Strain 03 shows two-stage growth in glucose-supplemented medium.
This is very similar to the two-step growth exhibited by E. coli in medium supplemented with lactose and glucose. The inventor found that this phenomenon was
It was considered that this reflects the fact that acetic acid secreted into the medium was used as the next carbon source to start the growth again. In other words, it was considered that there are genes (groups) involved in acetic acid utilization in Escherichia coli, which are induced to be expressed together with glucose depletion and acetic acid accumulation. As for the gene product induced by acetic acid, only the one reported by the present inventor at the 1991 Japanese Society of Agricultural Chemistry is known.
Therefore, the inventor obtained new knowledge on acetic acid assimilation, as well as new knowledge on acetic acid assimilation mechanism, by analyzing genes (groups) other than the genes reported at the 1991 Japanese Society of Agricultural Chemistry. Therefore, isolation and structure determination of these genes were performed.

As a method for isolating these genes, a recombinant DNA obtained by preparing a gene library of Escherichia coli chromosome using a plasmid vector as a vector
The mixture was introduced into E. coli. This transformant was inoculated on an agar L medium supplemented with glucose. When this was cultured at 37 ° C., transformants capable of forming large colonies, that is, colonies not affected by growth inhibition due to acetic acid accumulation could be obtained.

[0009] Actually, this transformant (hereinafter referred to as strain 3-11) accumulates acetic acid and causes p
It did not result in a decrease in H 2 (Fig. 3, right panel). In addition, the growth of strain 3-11 under the same culture conditions was confirmed by vector plasmid D.
It was better than the strain transformed only with NA (pUC19) (data not shown). Furthermore, the growth of strain 3-11 in acetic acid medium was significantly superior to the strain transformed with vector plasmid DNA (pUC19) alone (data not s).
hown). Therefore, in the recombinant DNA carried by this transformant, genes involved in acetic acid utilization (hereinafter referred to as ace
Called. ) Has been inserted. The method for isolating and determining the structure of the gene will be described in detail below.

First, the preparation of DNA containing the ace gene will be described. First, wild-type E. coli such as E. coli W
The 3110 strain is cultured to obtain a culture. In order to cultivate the above-mentioned microorganism, it may be cultivated by a usual solid culturing method, but it is preferable to adopt a liquid culturing method. Examples of the medium include one or more kinds of nitrogen sources such as yeast extract, peptone, meat extract, corn swipe liquor, soybean or wheat leachate, and the like, potassium monophosphate, dipotassium phosphate phosphate, magnesium sulfate, and chloride. A product obtained by adding at least one inorganic salt such as sodium, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate, and further optionally adding a sugar raw material, vitamins and the like is used. It is appropriate to adjust the initial pH of the medium to 7-8. Culturing is carried out at 30 to 42 ° C., preferably around 37 ° C. for 4 to 24 hours by aeration and agitation deep culture, shaking culture, static culture and the like. The culture thus obtained,
For example, centrifuge at 3,000 rpm for 5 minutes to transform E. coli W3110.
Obtain strain cells.

From this cell, for example, the method of Saito and Miura (Biochem. Biophys. Acta. 72: 619,1963), KS Kir
Chromosomal DNA can be obtained by a method such as by method (Biochem. J. 64: 405, 1956).

Then, the chromosomal DNA was digested with a restriction enzyme,
For example, Sau 3AI is used at a temperature of 30 ° C or higher, preferably 37 ° C.
℃, enzyme concentration 1 ~ 10 units / ml, various time 1 min ~
Digested by acting for 2 hours, partially decomposed and various chromosome D
A NA fragment mixture is obtained. On the other hand, as the vector DNA that can be used in the present invention, a plasmid vector DNA is preferable, and specifically pUC19
Etc. Chromosomal DNA in addition to the above vector DNA
The restriction enzyme Bam HI to generate a restriction enzyme Sau 3AI same terminal base sequence used for cutting, the temperature 30 ° C. or higher, the enzyme concentration 10 to 1,000 units / ml for 1 hour or more, and preferably allowed to act for 1-3 hours Completely digested, cleaved and cleaved D
Get NA. Then E. coli W31 obtained as described above
Mixtures of 10 strains containing a DNA fragment containing the acetate P gene and vector DNA cleaved and cleaved were mixed with DNA ligase, preferably T4 DNA.
A ligase is allowed to act at a temperature of 4 to 16 ° C. and an enzyme concentration of 1 to 100 units for 1 hour or more, preferably 6 to 24 hours to obtain a recombinant DNA.

Using this recombinant DNA, for example, Escherichia coli K-12 strain, preferably JM103 strain, etc. is transformed to obtain a strain. This transformation is performed by DM Morrison (Meth
ods in Enzymology 68: 326, 1979). From the above strain, D containing the ace gene
E. coli carrying the recombinant DNA with NA inserted in the vector DNA can be isolated by the method described in paragraph No. 0008. The method for recovering the recombinant DNA containing the ace gene from Escherichia coli can be carried out by a conventional method. For the method of recovering recombinant DNA, see P. Guer
ry et al. (J. Bacteriol., 116: 1064, 1973), DB
The method of Clewell (J. Bacteriol., 110: 667, 1972) and the like are used.

The passenger DNA inserted into the recovered recombinant DNA is deleted to various extents from both the 3'side and the 5'side to prepare shortened DNA. To make the shortened DNA at this time, Mung Bean Nuclease, E
There are methods using xonuclease III etc. This shortened DNA was ligated to a vector to transform Escherichia coli again, and the ability of the transformant to form a large colony on L-Broth agar medium containing glucose was used as an index to determine the region of the gene. Can be limited.

DNA cloned as described above,
Alternatively, a recombinant DNA containing a shortened DNA in which the gene region is limited can be used as a sample to determine the entire base sequence of a DNA containing the ace gene by the dideoxy method, which is a conventional base sequence determination method. . The structure of DNA was analyzed based on the obtained nucleotide sequence information, and then the open reading frame was deduced from this entire nucleotide sequence, and the amino acid sequence of the polypeptide defined by this was deduced (SEQ ID NO: 1)). The gene encoding the amino acid sequence thus determined is the ac of the present invention.
e gene. Specifically, the sequence listing sequence number (1)
1138 to 1140th ACA to 2350 to 2352nd AAT base sequences.

In recent years, a method for producing a substance has been reported in which a microorganism belonging to the genus Escherichia is cultured in a medium, various substances are accumulated and produced in the medium, and the target substance is collected. At this time, the target substances are bioactive proteins produced by human cells (interferon, interleukin, tissue-specific plasminogen activator, etc.), bioactive proteins produced by animal cells (bovine growth hormone, etc.), enzymes, amino acids. , Organic acids, antibiotics, etc.
The problem at this time is acetic acid accumulation. This is because acetic acid accumulation impairs the growth of microorganisms and reduces the productivity of the target substance. ace gene or Ace protei
n can be used to solve this problem.

In order to suppress acetic acid accumulation, it is effective to increase the copy number of the ace gene.
It is clear from the process of obtaining the gene. However,
It is clear that the principle of acetic acid accumulation suppression is due to the increase in intracellular concentration of Ace protein, and thus it is not limited to this. As long as the intracellular concentration of Ace protein can be increased, any method can prevent the productivity of the target substance from decreasing due to acetic acid accumulation.

For example, the means for increasing the intracellular concentration of Ace protein may be one which increases the expression level of the gene encoding the protein. This is achieved by strengthening the promoter of the gene. Also, by removing the operator, constitutive expression of the gene is achieved, resulting in an increase in the intracellular concentration of Ace protein.

[0019]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 Chromosome D of Escherichia coli W3110 strain
Preparation of NA) E. coli W3110 strain was mixed with TY medium [1% Bacto-
trypton (Difco), 0.5% Bacto-yeastextract (Difco), 0.
5% NaCl: (pH7.2)] 100 ml and incubate at 37 ° C.
After culturing for a time, a culture was obtained. 3,000 rp of this culture
Centrifuge for 15 minutes at m.
After obtaining g, the method of Saito and Miura (Biochem.
Actio., 72: 619, 1963) to obtain chromosomal DNA. Then, 60 μg of this chromosomal DNA and the restriction enzyme
Sau 3AI, 3 units of 10 mM Tris-HCl buffer (50 m
Each of them was mixed with M NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol (pH 7.4) and reacted at a temperature of 37 ° C. for 30 minutes. By a conventional method The reaction-terminated liquid, and phenol extraction treatment to obtain chromosomal DNA fragments 50μg of ethanol precipitation treated E. coli strain W3110 was digested with Sau 3AI and.

Example 2 Preparation of E. coli W3110 Strain Gene Library Using Plasmid Vector DNA 20 μg of plasmid vector DNA (pUC19) and restriction enzyme Bam HI200 unit were added to 50 mM Tris-HCl buffer (100 mM NaCl and 10 mM magnesium sulfate). Contains: pH 7.
The mixture was mixed with 4) and reacted at a temperature of 37 ° C. for 2 hours to obtain a digestive juice, which was subjected to phenol extraction and ethanol precipitation treatment by a conventional method. After this, DNA from the plasmid vector
Molecu to prevent the fragments from rejoining
Bacterial Alkaline Phosph in the way of lar Cloningp133
The DNA fragment was dephosphorylated by atase treatment, phenol-extracted by an ordinary method, and ethanol-precipitated.

1 μl of pUC 19 digested with this Bam HI
g, 1 μg of the chromosomal DNA fragment of Escherichia coli W3110 strain digested with Sau 3AI obtained in the above item (1), and 2 units of T4 DNA ligase (Takara Shuzo) were added to 66 mM magnesium chloride, 10 mM dithiothreitol and 10 mM. A
It was added to 66 mM Tris-HCl buffer (pH 7.5) containing TP and reacted at a temperature of 16 ° C. for 16 hours to ligate the DNA. Then, E. coli JM1 was mixed with the DNA mixture by a conventional method.
The strain 03 was transformed and plated on L agar medium containing 100 μg / ml of ampicillin to obtain about 5,000 colonies, which was used as a gene library.

Example 3 Recovery of Recombinant DNA from Gene Library Recombinant DNA was recovered from about 5,000 colonies described above. The 5,000 colonies were divided into 100 colonies each to make 50 batches, and then DNA was collected. The method of recovery is shown above
Followed the method of P. Guerry et al.

Example 4 Transformation of Escherichia coli JM103 Strain The recombinant DNA mixture divided into 50 batches was introduced into the JM103 strain according to the conventional transformation method described above. The transformant was plated on glucose-containing agar L medium and statically cultured at 37 ° C. Of these, one strain that formed colonies larger than the others was selected and named 3-11 strain. This strain did not undergo growth inhibition due to pH decrease in glucose-containing L liquid medium (FIG. 3).

Example 5 Assay of Acetogenic Utilization of Strains Carrying Multiple Copies of ace Gene To confirm that the ace gene obtained in the present invention encodes a protein involved in acetic acid utilization, LB + 3-11 strain (strain that retains multiple copies of ace gene) and JM103 / pUC19 on glucose medium
The strain (the strain carrying pUC19) was investigated for glucose consumption and acetic acid accumulation. Similar to the JM109 strain shown in FIG. 1, the JM103 / pUC19 strain accumulated acetic acid with the consumption of glucose (data
Although not shown), strain 3-11 consumed acetic acid as a carbon source after glucose depletion (the right diagram in FIG. 3). From this result, it was confirmed that holding multiple copies of ace enhances the ability of Escherichia coli to utilize acetate.

Example 6 Preparation of DNA Escherichia coli JM103 strain (3-11 containing the recombinant DNA obtained above)
Strain), tryptone 1%, yeast extract 0.5% and NaC
l Pre-incubate 20 ml of 0.5% medium at 37 ℃ for 24 hours, inoculate 20 liters of the obtained culture solution into 1 liter of the same composition as above, and incubate at 37 ℃ for 3 hours.
Chloramphenicol (0.2 g) was added, and the mixture was further cultured at the same temperature for 20 hours to obtain a culture solution. Then, this culture solution was centrifuged at 3,000 rpm for 10 minutes by a conventional method to obtain 2 g of each wet microbial cell, which was added to 20 ml of a 350 mM Tris-hydrochloric acid buffer solution (pH 8. After suspending in 0), lysozyme (manufactured by Sigma) 10
mg, 0.25 M EDTA solution (pH 8.0) 8 ml and 20% sodium dodecyl sulfate solution 8 ml were added respectively, and the temperature was 60 ° C.
The solution was kept warm for 30 minutes to lyse and a lysate was obtained. To this lysate, 13 ml of 5M NaCl solution was added, and the mixture was treated at a temperature of 4 ° C for 16 hours and then centrifuged at 15,000 rpm for 30 minutes by a conventional method. The supernatant was subjected to a phenol extraction treatment and an ethanol precipitation treatment by a conventional method to precipitate DNA.

This precipitate was dried under reduced pressure to obtain 1 m
10 mM Tris-HCl buffer containing M EDTA (pH 7.5)
The product was dissolved in 6 ml, and 6 g of cesium chloride and 0.2 ml of ethidium bromide (19 mg / ml) were added to the solution, which was then subjected to equilibrium density gradient centrifugation using an ultracentrifuge at 39,000 rpm for 42 hours by a conventional method. Treatment, isolation of DNA and further removal of ethidium bromide using n-butanol followed by 10 mM containing 1 mM EDTA.
It was dialyzed against Tris-HCl buffer (pH 7.5) to obtain about 500 μg of purified recombinant DNA p3-11.

Example 7 DNA containing the ace gene
Analysis of base sequence of recombinant DN obtained in Example 6
A was denatured with alkali to prepare single-stranded DNA.

Sequencing with the obtained single-stranded DNA was carried out using M-13 Sequencing Kit (Takara Shuzo).
It was done according to the method of Sanger. The nucleotide sequence of the obtained DNA containing the ace gene is as shown in the sequence listing. The open reading frame was deduced, and the amino acid sequence of the product deduced from the nucleotide sequence is also shown in the sequence listing. This protein was designated as Ace protein. It is well known that the methionine residue at the N-terminal of the protein is removed by the action of post-translational peptidase. This is because the N-terminal methionine is derived from the translation initiation codon, ATG, and is often irrelevant to the original function of the protein. In the case of the Ace protein of the present invention, it is possible that the methionine residue has been removed.

The nucleotide sequences and amino acid sequences were compared for homology with known sequences. The database used is Swiss-prot. As a result, the protein shown in SEQ ID NO: 1 of the Sequence Listing was a previously reported protein. The protein is Mol. Microbiol. Vol. 3 (1989).
505-515; Biochemistry & Cell Biology vol. 68 (199
0) 123-137; Mol. Gen. Genet. Vol. 219 (1989) 97-105
, It is described as NagC protein or NagR protein. However, there is no description in any of these documents suggesting that the protein is involved in acetic acid-utilizing ability. Further, as shown in FIG. 8, cloned DNA fragments p3-11 is, nag R (nag C, ace ) in addition to n
It was found that it also contains part of the ag A gene and part of the nag D gene.

Example 7 Preparation of Truncated DNA 3-11
Recombinant DNA was recovered from the strain and the gene region was limited. That is, the open reading frame estimated in Example 7 and the protein (Aceprotein, Nag R) encoded by the open reading frame.
A truncated DNA was prepared to confirm whether the assumption that protein) is involved in acetic acid utilization is really correct. DN of SEQ ID NO: 1 inserted in p3-11
Of the A fragments, the EcoRV-EcoRV 700bp fragment (Figs. 5 and 6)
(Shaded portion of) was removed to prepare a plasmid. Escherichia coli JM103 strain was transformed again with this shortened DNA, and it was examined whether or not the transformant could exert the acetic acid assimilating ability in L medium containing glucose. As a result, it was proved that the transformant carrying this plasmid did not exhibit the ability to assimilate acetic acid, and the estimation of the open reading frame and the protein encoded by it was performed by the present inventor.

[0032]

INDUSTRIAL APPLICABILITY As described above, the present invention relates to the acetic acid-utilizing gene ( ace gene) of Escherichia coli. By introducing multiple copies of this gene into Escherichia coli, an increase in acetic acid assimilating ability of E. coli is observed. Therefore, the present invention becomes a problem when producing a substance using E. coli,
It is intended to solve the inhibition of the growth of bacteria due to the decrease in pH.

[0033]

[Sequence list]

 SEQ ID NO: (1) Sequence length: 2556 bp Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: GenomicDNA Sequence features Characteristic symbols: mat peptide Location: 1135-2352 GATCTTTACC GGCCACGAAT TTCTTGATGA CCACGCGGTT GTTATCGCTG ATGGCCTGAT 60 TAAAAGCGTC TGTCCGGTAG CGGAACTGCC GCCAGAGATC GAACAACGTT CACTGAACGG 120 GGCCATTCTC TCCCCCGGTT TTATCGATGT GCAGTTAAAC GGCTGCGGCG GCGTACAGTT 180 TAACGACACC GCTGAAGCGG TCAGCGTGGA AACGCTGGAA ATCATGCAGA AAGCCAATGA 240 GAAATCAGGC TGTACTAACT ATCTGCCGAC GCTTATCACC ACCAGCGATG AGCTGATGAA 300 ACAGGGCGTG CGCGTTATGC GCGAGTACCT GGCAAAACAT CCGAATCAGG CGTTAGGTCT 360 GCATCTGGAA GGTCCGTGGC TGAATCTGGT AAAAAAAGGC ACCCATAATC CGAATTTTGT 420 GCGTAAGCCT GATGCCGCGC TGGTCGATTT CCTGTGTGAA AACGCCGACG TCATTACCAA 480 AGTGACCCTG GCACCGGAAA TGGTTCCTGC GGAAGTCATC AGCAAACTGG CAAATGCCGG 540 GATTGTGGTT TCTGCCGGTC ACTCCAACGC GACGTTGAAA GAAGCAAAAG CCGGTTTCCG 600 CGCGGCCCATCATCTAGC CAACGCGATG CCGTATATTA CCGGTCGTGA 660 ACCTGGCCTG GCGGGCGCGA TCCTCGACGA AGCTGACATT TATTGCGGTA TTATTGCTGA 720 TGGCCTGCAT GTTGATTACG CCAACATTCG CAACGCTAAA CGTCTGAAAG GCGACAAACT 780 GTGTCTGGTT ACTGACGCCA CCGCGCCAGC AGGTGCCAAC ATTGAACAGT TCATTTTTGC 840 GGGTAAAACA ATATACTACC GTAACGGACT TTGTGTGGAT GAGAACGGTA CGTTAAGCGG 900 TTCATCCTTA ACCATGATTG AAGGCGTGCG TAATCTGGTC GAACATTGCG GTATCGCACT 960 GGATGAAGTG CTACGTATGG CGACGCTCTA TCCGGCGCGT GCGATTGGCG TTGAGAAACG 1020 TCTCGGCACA CTCGCCGCAG GTAAAGTAGC CAACCTGACT GCATTCACAC CTGATTTTAA 1080 AATCACCAAG ACCATCGTTA ACGGTAACGA GGTCGTAACT CAATAAGAGA AAGT ATG 1137 Met 1 ACA CCA GGC GGA CAA GCT CAG ATA GGT AAT GTT GAT CTC GTA AAA CAG 1185 Thrn Glu Asl Gn Asl AAC AGC GCG GCG GTT TAT CGC CTG ATT GAC CAG TAC GGG CCA ATC 1233 Leu Asn Ser Ala Ala Val Tyr Arg Leu Ile Asp Gln Tyr Gly Pro Ile 20 25 30 TCG CGG ATT CAG ATT GCC GAG CAA AGC CAG CTT GCC CCC GCC AGC GTA 1281 Ser Arg Ile Gln Ile Ala Glu Gln Ser Gln Leu Ala Pro Ala Ser Val 35 40 45 ACC AAA ATT ACG CGT CAG CTT ATC GAA CGC GGG CTG ATC AAA GAA GTT 1329 Thr Lys Ile Thr Arg Gln Leu Ile Glu Arg Gly Leu Ile Lys Glu Val 50 55 60 65 GAT CAG CAG GCC TCC ACC GGG GGC CGC CGC GCT ATC TCC ATC GTC ACC 1377 Asp Gln Gln Ala Ser Thr Gly Gly Arg Arg Ala Ile Ser Ile Val Thr 70 75 80 GAA ACC CGC AAT TTC CAC GCA ATC GGC GTA CGG CTT GGT CGT CAT GAC 1425 Glu Thr Arg Asn Phe His Ala Ile Gly Val Arg Leu Gly Arg His Asp 85 90 95 GCC ACC ATC ACT CTG TTT GAT CTC AGC AGC AAA GTG CTG GCA GAA GAA 1473 Ala Thr Ile Thr Leu Phe Asp Leu Ser Ser Lys Val Leu Ala Glu Glu 100 105 110 CAT TAC CCG CTG CCG GAA CGT ACC CAG CAA ACG CTG GAA CAT GCC CTG 1521 His Tyr Pro Leu Pro Glu Arg Thr Gln Gln Thr Leu Glu His Ala Leu 115 120 125 TTG AAT GCC ATT GCT CAG TTT ATT GAT AGC TAC CAG CGC AAA CTG CGC 1569 Leu Asn Ala Ile Ala Gln Phe Ile Asp Ser Tyr Gln Arg Lys Leu Arg 130 135 140 145 GAG CTG ATC GCG ATT TCG GTG ATC CTG CCA GGG CTT GTT GAC CCG GAC 1617 Glu Leu Ile Ala Ile Ser Val Ile Le u Pro Gly Leu Val Asp Pro Asp 150 155 160 AGC GGC AAA ATT CAT TAC ATG CCG CAT ATT CAG GTA GAA AAC TGG GGG 1665 Ser Gly Lys Ile His Tyr Met Pro His Ile Gln Val Glu Asn Trp Gly 165 170 175 CTG GTA GAA GCT CTG GAA GAA CGT TTT AAA GTG ACC TGT TTC GTT GGT 1713 Leu Val Glu Ala Leu Glu Glu Arg Phe Lys Val Thr Cys Phe Val Gly 180 185 190 CAC GAT ATC CGT AGT CTG GCG CTG GCG GAC GAC TAC TTC GGT GCA AGT 1761 His Asp Ile Arg Ser Leu Ala Leu Ala Asp Asp Tyr Phe Gly Ala Ser 195 200 205 CAG GAT TGC GAA GAC TCC ATT CTG GTG CGT GTC CAT CGC GGA ACC GGG 1809 Gln Asp Cys Glu Asp Ser Ile Leu Val Arg Val His Arg Gly Thr Gly 210 215 220 225 GCC GGG ATT ATC TCT AAC GGG CGC ATT TTT ATT GGC CGC AAC GGC AAC 1857 Ala Gly Ile Ile Ser Asn Gly Arg Ile Phe Ile Gly Arg Asn Gly Asn 230 235 240 GTC GGT GAA ATT GGC CAT ATT CAG GTC GAA CCG CTG GGT GAA CGC TGC 1905 Val Gly Glu Ile Gly His Ile Gln Val Glu Pro Leu Gly Glu Arg Cys 245 250 255 CAC TGC GGC AAC TTT GGC TGC CTG GAA ACT ATC GCT GCC AAC GCT GCC 1953 His Cys Gly Asn Phe Gly Cys Leu Glu Thr Ile Ala Ala Asn Ala Ala 260 265 270 ATT GAA CAA CGG GTG TTG AAT CTG TTA AAG CAG GGC TAC CAG AGC CGC 2001 Ile Glu Gln Arg Val Leu Asn Leu Leu Lys Gln Gly Tyr Gln Ser Arg 275 280 285 GTG CCG CTG GAC GAC TGC ACC ATC AAA ACT ATC TGC AAA GCC GCG AAC 2049 Val Pro Leu Asp Asp Cys Thr Ile Lys Thr Ile Cys Lys Ala Ala Asn 290 295 300 305 AAA GGC GAT AGT CTG GCG TCG GAA GTA ATT GAG TAT GTC GGT CGT CAT 2097 Lys Gly Asp Ser Leu Ala Ser Glu Val Ile Glu Tyr Val Gly Arg His 310 315 320 CTG GGT AAA ACC ATC GCC ATT GCT ATC AAC TTA TTT AAT CCG CAA AAA 2145 Leu Gly Lys Thr Ile Ala Ile Ala Ile Asn Leu Phe Asn Pro Gln Lys 325 330 335 ATT GTT ATT GCC GGT GAA ATC ACC GAA GCC GAT AAA GTG CTG CTC CCT 2193 Ile Val Ile Ala Gly Glu Ile Thr Glu Ala Asp Lys Val Leu Leu Pro 340 345 350 GCT ATT GAA AGC TGC ATT AAT ACC CAG GCG CTG AAG GCG TTT CGC ACT 2241 Ala Ile Glu Ser Cys Ile Asn Thr Gln Ala Leu Lys Ala Phe Arg Thr 355 360 365 AAT CTG CCG GTG GTA CGT TCT GAG CTG GAT CAC CGC TCG GCA ATC GGC 2 289 Asn Leu Pro Val Val Arg Ser Glu Leu Asp His Arg Ser Ala Ile Gly 370 375 380 385 GCT TTT GCG CTG GTA AAA CGC GCC ATG CTC AAC GGT ATT TTG CTC CAG 2337 Ala Phe Ala Leu Val Lys Arg Ala Met Leu Asn Gly Ile Leu Leu Gln 390 395 400 CAT TTG CTG GAA AAT TAATGTGCTT TTATAGTGGC GCTTATTGTT GTCAATATTC 2392 His Leu Leu Glu Asn 405 TG

[Brief description of drawings]

FIG. 1 left diagram shows the growth of JM103 strain. A glucose-added L medium and a glucose-free L medium were used as the medium. In addition to the growth curve, the graph also shows the change in pH of the medium over time. The right diagram of FIG. 1 shows the glucose concentration and the acetic acid concentration in the medium when the JM103 strain was cultured in a glucose-added L medium.

FIG. 2 shows a growth curve when Escherichia coli JM103 strain was cultured in an L medium supplemented with glucose.
The medium is maintained at pH 7.2 with sodium phosphate buffer. Two-step growth is observed only in the medium supplemented with glucose.

FIG. 3 shows that 3 obtained by shotgun cloning.
-JM103, which retains the acetic acid assimilation ability of strain 11 only in vector
/ Compared with pUC19 strain. Glucose-added L medium was used for each medium. When culturing the JM103 / pUC19 strain, a sodium phosphate buffer was added to the medium to keep the pH value at 7.0. The glucose concentration and the acetic acid concentration in the medium of each of strains 3-11 and JM103 / pUC19 were measured over time, and the results are shown in the graph.

FIG. 4 shows the structure and the restriction enzyme cleavage position of the DNA fragment shown in SEQ ID NO: 1 of the Sequence Listing.

FIG. 5 shows the structure and restriction enzyme cleavage position of the DNA fragment shown in SEQ ID NO: 1 of the Sequence Listing.

FIG. 6 shows the structure and restriction enzyme cleavage position of the DNA fragment shown in SEQ ID NO: (1) of the Sequence Listing.

FIG. 7 shows the structure and restriction enzyme cleavage position of the DNA fragment shown in SEQ ID NO: (1) of the Sequence Listing.

FIG. 8 shows a group of genes contained on the DNA fragment inserted into the plasmid p3-11.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 13, 1993

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 3

[Correction method] Change

[Correction content]

[Figure 3]

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Correction target item name] Figure 8

[Correction method] Change

[Correction content]

[Figure 8]

Claims (4)

[Claims] 1. A method for producing a substance, comprising culturing a microorganism belonging to the genus Escherichia in a medium, accumulating and producing the target substance in the medium, and collecting the target substance, which is represented by SEQ ID NO: 1 in the sequence listing. A method for producing the target substance, which comprises increasing the intracellular concentration of a protein having an amino acid sequence. 2. The means for increasing the intracellular concentration of a protein having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is to increase the expression level of the gene encoding the protein. 1. A method for producing a target substance according to 1. 3. The means for increasing the intracellular concentration of a protein having the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing is increasing the copy number of the gene encoding the protein. 2. A method for producing a target substance according to 2. 4. A gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 1 of Sequence Listing is 2350 from ACA 1138 to 1140 of SEQ ID NO: 1 in Sequence Listing.
4. The method for producing a target substance according to claim 3, which has the base sequence of the 2352nd AAT.