JPH01215280A - Improvement of microorganism - Google Patents

Abstract

PURPOSE:To obtain a large amount of the objective substance produced by microorganism by using a microbial chromosome containing a gene participating in the production of the objective substance and introducing a manifestation controlling sequence for said gene into the chromosome at a specific position in a specific direction. CONSTITUTION:A microorganism having a chromosome containing a gene participating in the production of the objective substance is used as a starting strain. A manifestation controlling sequence (promoter, terminator, SD sequence, operator, etc.) for the above gene is introduced into a chromosome containing said gene at a position and in a direction to enable the control of the manifestation of said gene. The novel microorganism produced thereby stably maintains the gene participating in the objective substance and effects strong manifestation of said gene.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel method for improving useful microorganisms, microorganisms produced by the improved method, and methods for producing useful substances using the microorganisms. The method for improving microorganisms of the present invention includes:
It is characterized by externally introducing an expression control sequence capable of controlling the expression of an existing gene into a chromosome containing the gene.

[Conventional technology]

With the development and progress of recombinant DNA technology, proteins such as hormones, vaccines, and interferons, enzymes, amino acids,
Furthermore, it has become possible to produce large quantities of secondary metabolites such as vitamins and antibiotics in microorganisms. This involves cloning a specific gene involved in substance production onto an appropriate multi-copy plasmid vector, introducing the plasmid into an appropriate microorganism by a transformation method, and expressing the introduced gene involved in substance production. This is achieved by

At this time, gene expression is performed more efficiently by functionally linking the promoter gene and the target gene using a plasmid vector having a highly active promoter gene. However, due to plasmid shedding, mutations or deletions occurring in the plasmid,
Substance production using plasmid vectors is generally unstable and may be inappropriate for stable substance production.

An alternative method is to integrate the target gene into the chromosome of the host microorganism. This method allows the gene introduced from the outside to be stably maintained over multiple generations, but the degree of amplification of the gene is The drawback is that it is difficult to increase the yield, which limits the productivity of the desired product.

[Problem to be solved by the invention]

Therefore, there is a strong need for a microorganism and a method for creating the same, in which the gene for the target substance is stably maintained in the chromosome, the gene is strongly expressed, and the target substance can be efficiently produced.

[Means to solve the problem]

As a result of various studies to solve the above-mentioned problems, the present inventors added a strong promoter capable of enhancing the expression of a gene involved in the production of a specific target substance to the chromosome of a microorganism that already contains the gene. The inventors discovered that by introducing microorganisms that can efficiently produce the 6th substance, they completed the present invention.

Therefore, the present invention provides for the introduction of an expression control sequence for the gene into the chromosome of a microorganism having a chromosome containing a gene involved in the production of a target substance at a position and direction where the expression of the gene can be controlled. Provided is an improved microorganism that has been developed; a method for producing the microorganism; and a method for producing a useful substance, which comprises culturing the microorganism to produce a product related to the gene, and collecting the product. This is what I am trying to do.

[Specific explanation]

The present invention relates to a microorganism that already has a gene related to the production of a target substance in its chromosome and is capable of producing the target substance, and a microorganism that already has a gene related to the production of the target substance in its chromosome but can produce the target substance. The present invention can be applied to any microorganism that is virtually incapable of producing the target substance, but becomes capable of producing the target substance by newly introducing an expression control gene from the outside. Examples of such microorganisms include Bacillus genus, Escherichia genus, and Serratia spp.
tia), Pseudomonas
), the genus Brevibacterium
us) genus, Corynebacterium (Corynebacterium)
Microorganisms belonging to the genus erium and the like can be mentioned. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus stearothermophilus.

The target substances produced by the method of the present invention include proteins or polypeptides that are direct expression products of genes, such as various enzyme types such as protease, amylase, glucose isomerase, cellulase, tryptophan synthetase; peptide hormones such as insulin, growth hormone, enkephalin, somatostatin; various antigens such as hepatitis vaccine, polio vaccine, herpes vaccine; various lymphokines such as interferon, interleukin, etc.

The target substance produced by the method of the invention can also be a substance produced by the catalysis of one or more enzymes that is a direct expression product of a gene. Examples of such substances include L-)liptophan, which is synthesized by multiple tryptophan synthesis-related enzymes, monothreonine, which is synthesized by multiple threonine synthesis-related enzymes, and inosine and guanine, which are synthesized by multiple purine nucleotide synthases. etc. can be mentioned. Genes involved in the production of such target substances may be genes that originally exist in the chromosome of the host microorganism, or genes that have been artificially inserted into the chromosome of the host microorganism in advance. The former gene has an expression control sequence naturally associated with it, and by inserting an additional expression frJ1m sequence in addition to this by the method of the present invention, production of the target substance by the host can be enhanced. Alternatively, the ability to produce the target substance can be imparted to a host that originally did not substantially produce the target substance.
In the case of f&, in many cases, the gene has an expression control region associated with its structural gene, and the above-mentioned effects can be obtained by introducing a strong expression control sequence using the method of the present invention. If the previously inserted gene is not accompanied by its expression control sequence, the host microorganism will not be able to produce the target substance as it is, but by artificially inserting the expression control sequence using the method of the present invention, the host microorganism will not be able to produce the desired substance. Host microorganisms can be given the ability to produce a target substance. As a specific example of a microorganism containing such a gene, a gene related to tryptophan synthesis in Bacillus subtilis and a chloramphenicol resistance gene were ligated in vitro, and both genes were integrated into the chromosome of Bacillus subtilis, a tryptophan-producing bacterium. Examples of microorganisms that stably produce both gene products and tryptophan are created by
-85184, and JP-A-61-88873).

Expression control sequences include, for example, promoters, terminator-1SD sequences, and operators, and depending on the specific expression control sequence, these are usually located upstream or downstream of a structural gene involved in the production of the target substance, which is pre-existing in the chromosome. , is inserted in accordance with the transcription direction of the structural gene. A typical example of said control sequence is a promoter,
This is generally inserted upstream of the structural gene in accordance with the direction of transcription of the structural gene. For example, for a particular host microorganism, promoters that are cloned from naturally occurring promoters in the microorganism, or cloned from promoters naturally occurring in phages of the microorganism, or derived from these promoters. Hybrid promoters and the like can be used. Promoters may also be chemically synthesized.

The expression control region to be inserted is usually introduced by a plasmid that can be amplified in the host microorganism, or as circular or linear DNA that cannot be amplified in the host microorganism. As a method for introducing the desired DNA into a host microorganism, any of the methods commonly used for inserting DNA into cells, such as the calcium cell method (Reference J, Bacterio 1.., 10) 2 (19
) 4)), competent cell method (Reference Gene, 1, 1
53 (19] 7)), protoplast transformation method (Mo
1ec, Gen, Genet, 168, 111 (197
9) ) etc. can be used.

So-called homologous crossover is generally used as a method for integrating an expression control sequence such as a promoter into the chromosome of a host microorganism. For this purpose, the control sequence to be inserted preferably has at one or both ends an NA sequence homologous to the DNA sequence of a gene involved in the production of the target product already present in the chromosome.

In this specification, as a specific example, Bacillus amyloliquefaciens is used as the host microorganism, genes constituting the tryptophan operon are used as genes involved in the production of the target substance, and Bacillus amyloliquefaciens is used as the expression control sequence.
A promoter derived from Amyloliquefaciens or a promoter derived from the 5PO2 phage that infects Bacillus subtilis is used. This specific example will be described below as a real-male side.

In addition, the enzyme reaction conditions in the Examples were approximately as follows.

Old ndlll digestion Reaction medium: 100mM Tris
-HCl (pH 7,5).

50mM NaCl, 5mM MgCZ2 enzyme amount
: 5 units per 1 μg of DNA Reaction conditions = 11indllI partial digestion at 37°C for 60 minutes Reaction medium: 100n+M Tris-HCl (pH 7
, 5>.

50mM NaCl, 5mM HgCl2 Enzyme amount: D
0.1 unit to 1 unit per 1 μg of NA Reaction conditions: Sea I digestion at 37°C for 60 minutes Reaction medium: 1 (LaM Tri
s-IC1 (pH 8,0).

20w+M KCl, 7mM MgClz enzyme amount: D
10 units per μg of NA Reaction conditions: Xba I digestion for 60 minutes at 37°C Reaction conditions: 100mM Tri
s-HCl (pH 7,5).

50mM NaCj! , 5n+M t4gc12 enzyme amount: 5 units per 1 μg of DNA Reaction conditions = Digested with EcoRI for 60 minutes at 37°C Reaction medium: 10 (1mM Tris
-IC1 (pH 7,5).

50mM NaCl, 5μlM HgCl2 Enzyme amount: 5 units per 1μg of DNA Reaction conditions = Bam1l I digestion at 37°C for 60 minutes Reaction medium: 100mM Tri
s-tlci' (pH 7,5).

50+++M NaCl, 5mM HgCl2 enzyme amount
: 10 units per 1 μg of DNA Reaction conditions = 60 minutes at 37°C DNA polymerase I Klenow fragment (pol I) treatment 1100ei Tris-IICl
(pH 7,5), 50mM NaCl, 5nM t4g
For 1 μs of DNA (20 μm) digested in c1z buffer, add 3 units (1 μm) of DNA polymerase ■ Klenow fragment.
l), add 2n mole of each dXTP (1 μN of 2mM solution) and incubate at room temperature for 3
Incubate for 0 minutes.

Bacterial alkaline phosphatase (BAP) treatment Add 0.3 units of BAP to 1 μg DNA solution digested with restriction enzymes and incubate at 55°C for 30 minutes.

Ligation with T4 DN^ligase 100mM Tris-HCi' (pH
After mixing each DNA solution digested in 7,5>, 50mM NaC1,5+sM MgC1z, ATP was added to 100μH, 30 units of T4 DN^ligase was added, and the mixture was incubated overnight at 15°C.

First, promoter search vector pGR71 (Natsu
re.

293.309 (1981) Goldfarb, D.
S, et al.) (This vector is widely used in the industry and is easily available.)
was digested with the restriction enzyme old ncln and mixed in a test tube with the chromosomal DNA of Bacillus amyloliquefaciens M 1521, which had also been digested with the restriction enzyme old ndlll, and a ligation reaction was performed using T4 DN^ ligase. After that, the protoplast transformation method (S, Chang & S, N.

Cohen, Molec, gen, Genet, 168
, 111 (1979)), and a large number of transformants that grew on agar plates containing 25 ppm of chloramphenicol were obtained.

Next, the chloramphenicol-resistant dust and chloramphenicol acetyltransferase activities of this transformant were measured, and the transformant with the highest activity, Bacillus subtilis TFKfu56, was cloned with a highly active promoter. I chose it as a stock.

A plasmid was isolated and purified from TFK756, and the old ndIII fragment D of 0.3HD size was cloned.
NA was named sequence P756 having promoter activity,
pGR71 into which P2S5 was cloned is pSDK 7
It was named 56.

Next, in order to integrate the tetracycline resistance gene as a marker upstream of the P756 promoter of pSDK 756, Bacillus subtilis plasmid pTP5 was added to the restriction enzyme port ndI[I
This was mixed with pSDK 756, which had also been partially digested with the restriction enzyme ndI[I, and a ligation reaction was performed using T4 DN^ligase, followed by transformation into Escherichia coli C600, which was resistant to tetracycline. A transformant that was also resistant to chloramphenicol was obtained. This creates a plasmid psDK in which a 1.5HD tetracycline resistance gene is inserted upstream of P756 of pSDK 756.
27364 was produced.

Furthermore, as shown in Figure 1, pSDK 27364 was first partially digested with the restriction enzyme ll1ndlI [Klenow
Next, blunt ends were created using Frag+5ent and 4 types of XTP, mixed with puc ts that had been digested with Sn+a I and dephosphorylated with BAP, and a ligation reaction was performed using T4 DN^ ligase, resulting in E. coli JM. Transformants containing plasmid pSDK27365 and exhibiting resistance to ampicillin and tetracycline were screened. As a result, the DNA containing P)56 was given a marker for tetracycline resistance.
A plasmid was obtained from which fragments could be prepared.

The plasmid pSDK2F365 was digested with EcoRI and Xba1, separated by agarose electrophoresis,
1,8HD EcoRI-Xba containing the tetracycline resistance gene and promoter P756 was purified by phenol extraction and ethanol precipitation.
I. Fragments were prepared.

On the other hand, plasmid psDT1111, in which the tryptophan synthesis system gene of Bacillus amyloliquefaciens was cloned into Escherichia coli plasmid pBR322, was digested with restriction enzymes EcoRI and Xba I, separated by agarose gel electrophoresis, and subjected to phenol extraction and ethanol precipitation. By purification, 2,5H containing the upstream part of the tryptophan synthesis system gene
An EcoRI-Xba 1 fragment of size D was obtained. The E. coli containing the plasmid psDT1111 has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology as FERMP-7861.

Next, approximately 1 μg of both fragments were mixed, and T4D
A ligation reaction was carried out using N^ligase, and the ligation reaction was used to transform cells of Bacillus 5D30, a tryptophan-producing strain of Bacillus amyloliquefaciens, in the following manner.

First, TBA [l agar medium CD1fco, Bacto
Tryptoselog, Bacto Beef
Extract 3g% NaC15g1Bact.

Bacillus 5D30 streak-cultured on ^gar 15g; 112011') was cultured in CI medium (211P0414g).
, KH2PO46g, (NH4)2S0゜2g, sodium citrate・2H201g, MgSO4・7H2
05-, glucose 5g, casamino acid 0.2g, L-)
Liptophan 50ppa+, H2O11) to 0066
Inoculated so that 0 becomes 0.05, cultured with shaking at 37°C,
When 00660 becomes approximately 0.5, centrifuge (400
0 rpm for 10 minutes), and the precipitate was transferred to cn medium (K2HP
O414g, KH2PO46g, (NH4)2S04
2g, sodium citrate/2H201g, MgSO4
・7H205gM, glucose 5g, casamino acid 0.1
g, L-tryptophan (5 ppm) so as to be diluted 2 times. Further, the shaking culture was continued at 37°C.
After 30 minutes, add the ligated DNA solution and
Shake at 7°C for 1 hour and add 50% tetracycline.
It was spread on a TBAB agar medium containing ppm.

After overnight culture at 37°C, tetracycline-resistant transformants were obtained.

In this way, promoter P756
was integrated immediately upstream of the tryptophan synthesis gene on the chromosome, and the desired tryptophan-producing strain was obtained. The results of this homologous crossover are schematically shown in FIG.

(Figure 3) The starting plasmid psD[l 136 shown in Figure 3 is a promoter (P2O3) derived from Bacillus subtilis phage 5PO2.
Contains an EcoRI fragment of 0,17HD containing the restriction enzyme cleavage point Bam1l l downstream of it,
Contains Sal I and Pst I, and further downstream thereof, Bacillus pumi!
It is a 5HD-sized plasmid containing the structural gene for the chloramphenicol resistance gene derived from U.S.

The plasmid is pPL708 ((:ene).

16.199 (1981) > (Bacillus Ge
netic 5touch Center.

Commercially available from Ohio State University. ) was digested with EcoRl and Bgl I[, pBR3 digested with EcoRI and Bam1ll]
After mixing with 22 and ligating with T4 DN^ ligase, E. coli c600 is transformed, and the resulting chloramphenicol-resistant transformant is prepared.

Plasmid pSDB 136 was digested with the restriction enzyme BamHI and then treated with the Klenow fragment of E. coli DNA polymerase. On the other hand, plasmid pSDT1
11 was digested with the restriction enzyme EcoR (then E. coli DNA
Treated with Klenow fragment of polymerase II. After mixing both DNA fragments, they were ligated using T4 DN^ ligase, and this product was used to transform tryptophan-requiring Escherichia coli J^221. Plasmids were extracted from the resulting tryptophan-independent, ampicillin-resistant, and chloramphenicol-resistant transformants and analyzed to determine whether the 5PO2 phage-derived promoter and tryptophan synthesis gene-containing fragment were functionally linked. Recombinant plasmid of S10HD, 5EY1213
was gotten.

This plasmid psEY1213 was used to transform competent cells of the tryptophan-producing strain Bacillus 5D-30 of Bacillus amyloliquefaciens in the same manner as described in Example 1. In this way, the promoter P2O1 was integrated into the chromosome immediately upstream of the tryptophan synthesis gene through homologous crossover, and the desired tryptophan-producing strain was obtained. The results of this homologous crossover are schematically shown in FIG.

1 case (tryptophan type)
The activities of enzymes tryptophan synthetase and anthranyl synthetase produced by Bacillus 5D1034 and Bacillus 5D1035, in which a promoter sequence was inserted near the tryptophan gene on the bacterial chromosome, were measured by measuring the activity of both enzymes.

Parent strain Bacillus 5D30 without promoter introduction
, as well as the strain Bacillus 5D into which the promoter has been introduced.
1034 and Bacillus 5D1035 were pre-cultured on TBAB agar medium (Difco), and inoculated onto 5pizizen minimal medium 100 so that OD <660ns) was approximately 0.03, and ○D (660nm) was approximately 0.03 at 37°C. 0
．． The cells were cultured with shaking until the number of cells reached 5. 5000rpm,
Perform refrigerated centrifugation for 15 minutes, and transfer the precipitate to buffer 1 (0
,0258KH2PO4,0,075MK2HPO,,
After washing and centrifugation with pH 7,3,0,018L-glutamine, 10% glycerin), two companies' buffer ■-(0
,025M KH3PO,,0,075M K2)IP
O,, pH 7,3-0,01ML-glutamine, 4mM
MgCl2 (22.40% glycerol). Added 0.5 mg of lysozyme and 5 μg of DNase, 37
Incubated for 30 minutes at °C.

30. The mixture was centrifuged at OOOrpm for 30 minutes, and the supernatant was used as a crude enzyme solution.

Method in Engymology, 5, 79
4 (1962) and the results of tryptophan synthetase activity measurements performed according to Genetics, 52.
1303 (1965).

Strain Tryptophan Anthranyl Synthetase Activity Acid Synthetase 5D30 100 100SD
1034 250 300SD10
35 300 320 Bacillus 5D
In No. 1034, the upstream part of the tryptophan synthesis gene containing the P756 promoter and anthranilate synthetase gene was introduced into the vicinity of the avian 1) fan synthesis gene on the chromosome of the host bacterium, and as a result, the chromosomal The expression of the tryptophan synthesis system gene originally present in the genus has been enhanced, and an additional anthranilate synthetase gene has been introduced.

Therefore, the tryptophan synthetase activity is approximately 2.5
The anthranilate synthetase activity was enhanced approximately 3.0 times. On the other hand, in Bacillus 5D1035, a diploid of the tryptophan synthesis system gene is formed, and P2O1 promoter sequence DNA is introduced near the 1-ributophane synthesis system gene on one side, resulting in anthranilate synthetase. Activity and Tryptophan Synthetase Example 5. L-trypto-2-anjuan glucose 5%, ammonium sulfate 0.2%, K211PO. 1.4%, K1
2P040. f3%, sodium citrate/2H201
g,agsQ.・7H20 0.02%, F
eS04・7H20 1 ppm, MlIS
800 ppm of anthranilic acid was added to 2 L of a medium (pH 7.0) containing 041 ppm, and the parent strain Bacillus SD30 in which no promoter had been introduced, Bacillus SD1034 and Bacillus SD1035 in which the promoter had been introduced were inoculated. Culture in a 5L jar fermenter at 35°C with aeration. During the culture, when the anthranilic acid concentration decreased to 50 pp+s or less, anthranilic acid was added as appropriate so that the concentration of anthranilic acid became approximately toopp-. During the culturing, 100 g of glucose was added, and the pH of the medium was adjusted by adding ammonia water. 7.
The cells were cultured for 15 hours while maintaining the temperature at 0±0.4. The amount of L-tryptophan accumulated in the culture solution was measured using high performance liquid chroma I.
・The results measured by graphing are shown below.

S030 4.7 SD1034 8.9SD1035
15.1 As is clear from the table above, the expression of the tryptophan synthesis system gene on the chromosome was enhanced by the introduction of the promoter of the present invention in Bacillus SD1.
034 accumulated about twice as much tryptophan as the parent strain SD30. On the other hand, Bacillus SD103 into which an additional tryptophan synthesis gene was introduced in addition to the promoter
5 accumulated about three times as much tryptophan as the parent strain SD30.

Example 61 It was confirmed by the following Southern hybridization method that the promoter sequence DNA was introduced in the vicinity of the tryptophan gene in Bacillus SD1034 and Bacillus SD1035.

Bacillus SD1034 and Bacillus SD1035 L
Culture with shaking in 300 ml of medium at 35°C overnight, and then perform the usual DNA extraction method (Biochem.Biophys.^
eta 72,619.

(1963)) to extract and purify chromosomal DNA.
mg was obtained. Add μg of each DNA using restriction enzyme Bawl.
It was completely digested with E, EcoR I and Xba I, respectively, and subjected to agarose electrophoresis. After alkaline denaturation and neutralization according to conventional methods, the DNA was transferred to a nitrocellulose filter.
was transferred. After cleaning the filter. 80℃
It was heat-treated for 2 hours.

5HD EcoRI fragment containing tryptophan gene purified separately and P756 as probe DNA
0.3HD old ndl containing promoter [0.18MD Eco containing fragment and P201 promoter]
The RT fragment was labeled with [γ-32P] dCTP using the nick translation method and the specific activity was 40μ.
Probe DNA of Ci/100 nH was prepared.

Filter the aforementioned prehybridization solution (
6X SSC, 5x with 40 μCi of probe DNA after 2 h incubation at 42°C in 5x Denhardt's solution.
Hybridization solution containing 0% formamide (
Incubation was performed overnight at 42°C in 6X SSC and 2X Denhardt's solution. Then filter twice and once at 37°C.
Incubate in buffer (2XSSC) for 5 min, transfer to low salt buffer (0.IXSSC), 2x, 3x
Washed at 7°C for 5 minutes. The filter was wiped dry and autoradiography was performed at -80°C for 3 hours using Ricodak XAR5 film.

As a result, in the case of Bacillus 5D1034, Bam1l
A signal was generated with both the fragment probe containing the tryptophan gene around the size of 7HD digested with I and the fragment probe containing P2S5. Also, Eco
A signal was generated in both the fragment probe containing the tryptophan gene and the fragment probe containing P2S5 in the vicinity of the size of 4,3HD digested with RI, and
Signals were also generated near the size of 4,3HD digested with baT for both the tryptophan gene-containing fragment probe and the P2S5-containing fragment probe. Therefore, the structure near the tryptophan gene of Bacillus 5D1034 was as shown in FIG. 2, and it was confirmed that the promoter sequence was introduced near the tryptophan gene.

In the case of Bacillus 5D1035, both the fragment probe containing the tryptophan gene and the fragment probe containing P2O1 produced signals around the size of 10 MD digested with Xba I, and as a result of homologous crossover, the promoter It was confirmed that the sequence was introduced near the tryptophan gene.

[Effects of the present invention]

According to the present invention, unlike gene amplification using plasmids, it becomes possible to stably industrially produce large quantities of specific substances produced by microorganisms.

Furthermore, in gene amplification, the purpose cannot be achieved unless the target gene is completely intact, but in the present invention, the upstream portion of the target gene and any promoter DNA
If you do, you can easily achieve your goal.

[Brief explanation of the drawing]

FIG. 1 shows a method for cloning promoter sequence DNA. FIG. 2 shows a method for introducing promoter sequence DNA and the upstream portion of the tryptophan gene into bacteria. FIG. 3 shows a method for preparing DNA into which a hetributophane gene downstream of the promoter sequence is integrated, and a method for introducing it into bacteria.

Claims (1)

[Scope of Claims] 1. In the chromosome of a microorganism that has a chromosome containing a gene involved in the production of the target substance, an expression control sequence for the gene is located at a position and in a direction where the expression of the gene can be controlled. Improved microorganisms introduced in 2. The microorganism according to claim 1, wherein the microorganism is a microorganism belonging to the genus Bacillus. 3. The microorganism is Bacillus subtilis.
ussubtilis) or Bacillus amyloliquefaciens (Bacillus amylolique)
3. The microorganism according to claim 2, which is a microorganism (E. faciens). 4. Claim 1, wherein the expression control sequence is a promoter, and the promoter is inserted upstream of the gene.
The microorganism according to any one of 3 to 3. 5. The microorganism according to claim 4, wherein the promoter is a promoter of a microorganism belonging to the genus Bacillus or a promoter of a phage of a microorganism belonging to the genus Bacillus, and the promoter is inserted upstream of the gene. 6. The microorganism according to any one of claims 1 to 5, wherein the gene is a gene related to tryptophan synthesis. 7. Introducing an expression control region for the gene into the chromosome of a microorganism having a chromosome containing a gene involved in the production of the target substance at a position and direction that can enhance the expression of the gene. An improved method for producing microorganisms. 8. A method for producing a useful substance, which comprises culturing the microorganism according to claim 1 to produce a product related to the gene, and collecting the product.