JPH0468909B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing a bacterial strain containing a tryptophan repressor-corresponding gene (TrpR) belonging to the genus Escherichia, and more specifically, to obtain bacterial cells with a high content of tryptophan synthase in the bacterial cells and a high yield. The present invention relates to a method for culturing a Trp-carrying strain belonging to the genus Escherichia that can be used for culturing.Tryptophan synthase is a substance useful as an enzyme catalyst in the production of L-tryptophan from indole and L or DL-serine.L-tryptophan is It can be used as an additive to feeds and drinks, or for various industrial uses, but to date no industrially inexpensive manufacturing method has been developed, and these uses are limited. Therefore, it is desired to establish an industrial method for producing L-tryptophan. By the way, several methods have been proposed so far as a method for producing L-tryptophan. For example, (1) sugar A method for directly fermenting Brevibacterium flavum and purifying and collecting L-tryptophan produced during the culture using a lactobacillus (Japanese Unexamined Patent Publication No. 174096/1983); (2) Contains indole or anthranilic acid and carbohydrates. A method of culturing Bacillus subtilis in a culture solution, producing and accumulating L-tryptophan in the culture solution, and purifying and collecting L-tryptophan from the culture solution (Japanese Patent Publication No. 35152/1983); (3)
A method for producing L-tryptophan by an enzymatic reaction from indole and serine or indole, pyruvate and ammonium ion using an enzyme produced by a microorganism, tryptophan synthase or tryptophanase (Japanese Patent Laid-Open No. 47-39693) , Special Publication No. 49-46917) have been proposed. The present inventors have conducted research focusing on the production of L-tryptophan from indole and serine by an enzymatic method. The key point in the production of L-tryptophan by this enzymatic method is how to produce the tryptophan synthase responsible for the catalytic action at low cost and in large quantities. This tryptophan synthase is produced by microorganisms, so in order to produce tryptophan synthase in large quantities at low cost, it is necessary to increase the content of tryptophan synthase in the microbial cells and at the same time during cultivation. It is essential to develop a culture method that can obtain viable cells in high yield. Gene amplification using gene recombination technology is considered to be one of the effective methods for increasing the content of specific enzymes in microbial cells. However, when using the tryptophan promoter and operator region, even in strains that have the above-mentioned gene amplification effect,
Tryptophan promoter activity is strongly inhibited by the action of the tryptophan repressor gene, which is a tryptophan repression mechanism located on the main chromosome, and as a result, the tryptophan synthase content in the strain is suppressed to a certain level. I ended up
It is difficult to accumulate a significant amount of tryptophan synthase in bacterial cells. It is also possible to use a strain in which the gene TrpR, which encodes the tryptophan repressor, which is the suppressor, is deleted; It has been reported that upon conversion, the plasmid in the strain is extremely unstable, and the entire plasmid may be dropped or partially deleted [Journal of General
Microbiology, 118 , 253-261 (1980)] There is an industrial problem. On the other hand, as a method to suppress the function of tryptophan repressor, indoleacrylic acid, a tryptophan derivative, is added to the culture medium at a concentration of 10 μg/ml or less to inactivate tryptophan repressor. It has been reported that the tryptophan synthase content in the bacterial strain can be increased [Pro.Nat.Acad.Sci.USA,
Vol.71, No.9, pp3455-3459 (1974)]
Since the instructions require that a minimal nutrient medium, that is, a synthetic medium, be used as a medium, the yield of bacterial strains per unit culture is low, and even if the content of tryptophan synthase in one bacterial cell is high, The production amount of tryptophan synthase per unit culture solution is low, and this method is not suitable as a method for obtaining the enzyme at low cost industrially. In addition, a recombinant gene consisting of a gene, vector, and promoter that encodes a peptide derived from the true cell.
A method of increasing the productivity of the peptide by culturing a microorganism containing DNA and capable of producing the peptide in a medium supplemented with indole acrylic acid was also recently proposed (Japanese Patent Laid-Open No. 141796/1983). However, this method also uses a synthetic medium with a low organic nitrogen source, resulting in low bacterial cell productivity and cannot be used as an inexpensive enzyme source for industrial use. The present inventors used peptones such as polypeptone, tryptone, and peptone, meat extract, yeast extract, malt extract, corn staple liquor, and casamino acid in place of the synthetic medium in the above-mentioned culture method adding indole acrylic acid. In order to investigate the possibility of using natural media containing natural ingredients such as L medium (10 parts by weight of tryptone, 5 parts by weight of yeast extract, 1 part by weight of glucose, 5 parts by weight of NaCl, 1000 parts by weight of pure water, PH
7.2) and added indole acrylic acid at a concentration of 10 μg/ml according to the instructions in the above literature, and attempted to culture E. coli [strain YK2001, (FERM P-7139)], but as described in the above literature, No effect of increasing the tryptophan synthase content in the bacterial cells was observed. Therefore, the present inventors have conducted intensive studies regarding the effect of adding indole acrylic acid in this natural medium from both the amount and timing of addition of indole acrylic acid. When added at a much higher final concentration of at least 25 μg/ml,
Not only does the content of tryptophan synthase in the bacterial cells increase, but the yield of the bacterial cells also improves. Moreover, this effect is not only achieved when indole acrylic acid is added at the early stage of culture; The present invention has been completed based on the discovery that the present invention can be obtained by continuing the culture by adding the product until sufficient growth occurs, that is, until the end of the logarithmic growth phase. Thus, according to the present invention, when culturing a tryptophan repressor-responsive gene (TrpR)-carrying strain belonging to the genus Escherichia in a nutrient medium containing at least 0.1% by weight of an organic nitrogen source in terms of nitrogen atomic weight, At the latest, by the end of the logarithmic growth phase of the strain, indole acrylic acid or its salts are added to the medium at a final concentration of at least 25 μg/ml, and the tryptophan synthase content in the bacterial cells is reduced. Belonging to the genus Escherichia, which is characterized by obtaining the above-mentioned strains with increased
A method for culturing a TrpR-retaining bacterial strain is provided. In the method of the present invention, a so-called natural medium containing a large amount of organic nitrogen source is used as the nutrient medium, unlike a synthetic medium (minimal nutrient medium).
That is, in the present invention, the organic nitrogen source contains at least 0.1% by weight, preferably 0.2% by weight of nitrogen atoms.
~1.0% by weight, more preferably 0.25-0.6% by weight
Use a natural medium containing: As the organic nitrogen source in such a natural medium, those that are conventionally blended as a nitrogen source in natural media used for culturing microorganisms belonging to the genus Escherichia can be used, such as polypeptone,
Peptones such as tryptone and peptone, meat extracts, yeast extracts, malt extracts, cornstarch liquor, organic nitrogen compounds such as casamino acids (amino acids, peptides, vitamins, unknown growth promoting factors, etc.) are used singly or in combination. It is possible to mix and use more than that. In addition to the organic nitrogen sources mentioned above, the culture medium used in the method of the present invention contains inorganic nitrogen sources such as (NH ₄ ) ₂ SO ₂ ,
It is also possible to include NH ₄ Cl, (NH ₄ ) ₂ PO ₄ , (NH ₄ )NO ₃ and the like. Furthermore, the culture medium used in the method of the present invention can contain, as nutritional sources, a carbon source, minerals, vitamins, etc. in addition to the nitrogen source mentioned above. As a carbon source, various carbohydrates such as glycol, glycerol, fructose, sucrose, and blackstrap molasses can be used. The medium containing the nutrients mentioned above will eventually
After adjusting the pH to generally a range of 5 to 9, preferably a range of 6 to 8, the cells can be cultured. The microorganism that can be cultured according to the method of the present invention is a strain capable of producing tryptophan synthase within the microbial cell, and in particular, a tryptophan lipophilic strain belonging to the genus Escherichia that has the ability to produce tryptophan synthase. This is a strain that retains a gene corresponding to Tsar (herein, this gene is abbreviated as "TrpR"). The tryptophan repressor corresponding gene (TrpR) may be encoded in the main chromosome of the strain or may be present in a plasmid, and the present invention applies to any type of strain. Can be applied. Therefore, in the method of the present invention, any TrpR-carrying strain belonging to the genus Escherichia that has the ability to produce tryptophan synthase can be used, but in particular, the tryptophan promoter and operator region and the gene corresponding to tryptophan synthase can be used. An Escherichia coli strain whose ability to produce tryptophan synthase has been amplified by transformation with a plasmid having a tryptophan operene, more preferably a plasmid having a tryptophan operene, is advantageously used. E. coli strains containing such plasmids include, for example, the fraction containing the gene governing the biosynthesis of tryptophan synthase, which was previously created and proposed by the present inventors, and the F factor plasmid growth control fraction system. It is possible to effectively use an E. coli strain transformed with a novel plasmid containing a fraction containing a gene controlling Therefore, this new plasmid will be explained in more detail. "Fraction containing the gene governing the biosynthesis of tryptophan synthase" that constitutes the above plasmid
(hereinafter sometimes abbreviated as "T fraction") is
It refers to the fraction containing the gene responsible for the biosynthesis of L-tryptophan from indole and L- or DL-serine, and the T fraction contains the tryptophan operon or a large gene fraction containing the tryptophan operon. fraction, or a fraction in which promoters and operators are linked to TrpA and TrpB, which are genes that control the biosynthesis of tryptophan synthase in the tryptophan operon, or TrpA and TrpB
This includes a fraction in which a promoter and an operator are bound to at least one of TrpC, TrpD, and TrpE (for example, a fraction in which a promoter and an operator are bound to TrpA, TrpB, TrpC, and TrpE), and the like. As these T fractions, those derived from Escherichia coli are preferably used, and among them, the tryptophan operon itself and the phage φ80pt
A fraction containing the tryptophan operon having a molecular weight of about 22.6 kb obtained by cleaving the gene with the restriction enzyme BamHI is advantageously used. On the other hand, the F factor plasmid has a structure as shown in the genetic map in Figure 1 on page 98 of "Protein Nucleic Acids Enzymes" Vol. 27, No. 1 (1982) and the physical map by Eco RI, and has a molecular weight of 94.5 kb ( 62× ¹⁰⁶
dolton), and is normally present in enterobacteria such as Escherichia coli at a copy number of 1 to 2 copies per cell chromosome. Therefore, if there are two chromosomes per cell, it cannot be an F factor plasmid. Only 2 to 4 are carried. This plasmid is equipped with a mechanism that allows it to double just as the cell divides and to be transmitted accurately to each daughter cell (thus, keeping the copy number at a low level while accurately controlling the host's growth and pace). The mechanism that increases the number of cells together is called stringent growth control). This stringent growth control function in the F factor plasmid is
It has already been determined that mini-F is carried by a fragment with a molecular weight of 9.1 kb that can autonomously reproduce, and from this mini-F factor plasmid, restriction enzymes
It is also known that it can be excised using EcoRI. The above-mentioned new plasmid utilizes the growth control distribution system carried by this mini-F,
However, the "fraction containing genes that control the growth control distribution system of the F factor plasmid" (hereinafter sometimes abbreviated as F fraction) refers to the mechanism that accurately transmits the F factor plasmid to daughter cells as described above. A typical example of such an F fraction is the mini-F fraction, which has a molecular weight of about 9.1 kb. In addition to the T and F fractions described above, the new plasmid contains other gene fractions, such as kanamycin resistance, which controls drug resistance in various bacteria. It may also contain a gene fraction etc. Typical examples of such plasmids include tryptophan operon fraction and F factor plasmids.
It contains the mini-F fraction, has a molecular weight of approximately 31.7 kb, and exhibits the following sensitivity to the following restriction enzymes, namely (a) recognition sites for EcoRI at 5 sites, and (b) recognition for BamHI. (c) Plasmid pMTY-1 is characterized in that it has two sites and three recognition sites for (c) Hind. This plasmid pMTY-1 is approximately
It has a molecular weight of 31.7 kb, which was determined by agarose gel electrophoresis. Furthermore, the sensitivity of plasmid pMTY-1 to various restriction enzymes is as shown in (a) to (c) above, and the molecular weights (kb) of fragments degraded by each restriction enzyme are shown in more detail as follows. (a) EcoRI: 9.1, 7.0, 6.4, 6.1, 3.1 (b) BamHI: 22.6, 9.1 (c) Hind: 18.0, 10.8, 2.9 Plasmids with the above characteristics
pMTY-1 can be produced, for example, as follows. First, the preparation of the tryptophan operon fraction is
For example, Escherichia coli that has tryptophan operon in its chromosomal gene, such as Escherichia coliK-
12 (IFO3301, ATCC10798, ATCC e23562), etc., for example, phage φ80
(ATCC11456a-B ₁ ) etc., and by utilizing lysogenization and induction phenomena, we prepared a large amount of phages incorporating the tryptophan operon into the phage DNA [RMDenney, C. Yanofsky; J.
Bacteriol., 118 , 505 (1974)], then conventional methods [EFFritsch, Sambrook, “Molecular
cloning” (1982) p.164-165, Cold Spin
Harbor Laboratory)
Extract the DNA and use a restriction enzyme, e.g. BamHI,
This can be done by cutting out a gene fraction containing the tryptophan operon using EcoRI or the like. On the other hand, the preparation of the miniF fraction can be carried out using microorganisms carrying the F factor plasmid, such as Escherichia coli (E.
coli) K-12 strain (ATCC15153, ATCC e23589,
ATCC e23590) etc. in a manner known per se,
For example, P. Guerry, DLLeBlanc, S. Falkow; J.
Bact., 116 , 1064 (1973), etc., by removing the F factor plasmid, and then excising the miniF fragment with a molecular weight of about 9.1 kb using the restriction enzyme EcoRI. The miniF fragment thus prepared was treated with the same restriction enzyme used to excise the gene fraction containing the tryptophan operon, and then combined with the gene fraction containing the tryptophan operon prepared above. The target plasmid pMTY-1 can be obtained by ligation using a ligase such as _T4 phage-derived ligase. A specific method for preparing plasmid pMTY-1 is described in detail in Reference Example 1 below. Although the plasmid prepared in this manner has a small number of copies, it has the excellent property that it is accurately inherited by daughter cells during cell division of the host and is rarely shed. Therefore, when this plasmid is applied industrially in the production of tryptophan, a host is transformed with the plasmid. As a host strain that can be used for this transformation, for example, Escherichia coli, particularly a tryptophan auxotrophic mutant strain, is particularly preferred. The above-mentioned plasmids can be introduced into these host bacteria using methods known per se, such as M. Mandel, A.
This can be carried out by the method described in literature such as Higa; J. Mol. Biol, 53 , 159 (1970). When culturing the above-mentioned bacterial strain in a natural medium as described above, the method of the present invention includes adding indole acrylic acid or a salt thereof (for example, indole acrylic acid Na salt, K salt, NH ₄ salt, etc.) to the medium. ) is added in such an amount that a final concentration of at least 25 μg/ml is achieved. The addition of this specific high concentration of indole acrylic acid or its salt inactivates the tryptophan repressor and derepresses the tryptophan promoter and operator, making the strain highly productive of tryptophan synthase. However, it is thought to be generated. Therefore, the amount of indole acrylic acid or its salt added to the medium is the final amount.
It is added at a rate of 25 μg/ml or more.
There is no particular upper limit to the amount added, but it is meaningless to add more than the solubility in the medium, and generally
It can be 500μg/ml or less. The suitable range of addition amount depends on the strain used, but it is usually 80~
200 μg/ml, particularly preferably in the range of 90 to 150 μg/ml. Indole acrylic acid or its salt may be added in the above amount at once, or may be added in several portions as the case may be. In addition, indole acrylic acid or its salts should be added until the bacterial cells have fully grown, that is, until the end of the logarithmic growth phase. It was found that the effect of increasing sales could be achieved. Thus, indoleacrylic acid or its salt may be added at the beginning of the culture (at the beginning), or may be added just before the end of the logarithmic growth phase of the bacterial cells, or alternatively, it may be added several times during the above period. It may be added separately. In any case, the total amount of indoleacrylic acid or its salt added to the medium at the end of the logarithmic growth phase should ultimately be 25 μg/ml or more. The strain can be cultured by a method known per se, except for adding indoleacrylic acid or a salt thereof as described above.
For example, the culture can be carried out under aerobic conditions such as shaking culture or submerged culture with aeration and agitation. The culture temperature can generally range from 20 to 50°C,
The PH of the medium is around neutral or slightly alkaline, usually PH
It is desirable to maintain it in the range of 6-8. Cultivation can be continued until enough tryptophan synthase accumulates within the bacterial cells, and the cultivation period is usually 4 to 4 hours.
About 48 hours is sufficient. By the culture method of the present invention described above, it is possible to obtain a high yield of bacterial cells with a high content of tryptophan synthase in the bacterial cells, and the production amount of tryptophan synthase per unit medium is It is possible to increase the amount by about 2 to 10 times as much as when culturing is carried out without adding acrylic acid or its salt. Therefore, according to the method of the present invention, it is possible to provide tryptophan synthase industrially at low cost and in large quantities, and it is possible to provide tryptophan synthase industrially at low cost and in large quantities.
This will greatly contribute to the industrial production of L-tryptophan from - or DL-serine. When using the bacterial cells obtained by the method of the present invention for the production of L-tryptophan, the bacterial cells can be separated from the culture and used as they are; It may be used after crushing,
Alternatively, it can be used in the form of an extract obtained by further extracting the crushed product with water or the like, or in the form of a crude product obtained by further treating the extract with ammonium sulfate or the like to precipitate the enzyme component. Bacterial cells or treated products thereof can be used after being immobilized if necessary. The bacterial cells prepared in this manner or their treated products are used as a catalyst for the reaction between indole and L- or DL-serine. The reaction between indole and L- or DL-serine in the presence of the bacterial cells or their processed material can be carried out in the same manner as a normal enzymatic reaction, for example.
0.1M phosphate buffer (PH7.0-9.0) or water (PH
7.0 to 9.0) at a temperature of about 20 to about 50°C, preferably about 30 to about 40°C, for about 10 to about 72 hours. Although there is no particular restriction on the amount of indole and L- or LD-serine used in the reaction, it is generally appropriate to use each in a concentration range of 0.1 to 20% (wt/vol). In addition, the amount of the bacterial cells or their processed material to be used is not particularly limited, but generally 1
Can be used at concentrations of ~10% (wt/vol). Indole and L-, or
The separation and purification of L-tryptophan produced in the reaction solution obtained by reacting DL-serine is as follows:
This can be carried out by known methods such as adsorption and desorption treatment using ion exchange resins, activated carbon, etc. Next, the present invention will be further explained with reference to Examples and Reference Examples. Reference example 1: Preparation of plasmid pMTY-1 (A) Preparation of miniF fraction E. coli K-12 strain (ATCC15153)
1 L medium (Bacto tryptone 10g, yeast extract 5g, NaCl 5g, glucose 1g, water 1
; pH 7.2) and cultured with shaking at about 37°C for about 4 hours, the bacterial cells were collected, treated with lysotium, and lysed by adding sodium dodecyl sulfate (SDS). This lysate was heated at 32,000×g.
After centrifugation for 40 minutes, the supernatant was fractionated, followed by cesium chloride-ethidium bromide equilibrium density gradient centrifugation, followed by dialysis to separate a solution containing the F factor plasmid.
This solution was subjected to ethanol precipitation, and approximately 20 μg of factor F plasmid was finally collected. Next, when preparing the miniF fraction, the above F
Take 5 μg of factor plasmid and use restriction enzyme.
The F factor plasmid DNA strand was cleaved by applying EcoRI at 37° C. for 1 hour to prepare a DNA solution containing a miniF fragment of approximately 9.1 kb. (B) Preparation of Phage φ80pt 100% of E. coli K-12 strain (IFO3301)
Inoculate 37 ml of L medium (composition is the same as above).
0.2 ml of _the culture shaken for about ⁴ hours at
ml) in L-medium soft agar (L-medium + agar droplet), and then layered on an L-medium agar plate. When the plate is cultured at 37°C for about 5 hours, plaques (lytic plaques) are formed, and when the culture is continued at 37°C for an additional 2 to 3 days, growing colonies of Phage φ80 lysogenic bacteria are formed in the plaques. After culturing the lysogenic bacteria in L medium at 37°C for 4 hours, they were spread on the same L medium agar plate as above, and then irradiated with ultraviolet light (400 to 800 ergs/mm ² , 10 to
Prepare phage φ80pt (phage DNA containing the tryptophan operon) by inducing lysogenic phages (20 seconds). (C) Preparation of tryptophan operon fraction Escherichia coli (E. coli) K-12 strain (IFO3301) was
L medium (composition is the same as above) was inoculated, and approx.
Culture with shaking at 37℃ for about 3 hours, and 25% in logarithmic growth phase.
Add 10 ml of (w/v) glucose solution and the Phage φ80pt solution prepared above at a concentration of 10 ^{to 11} cells/ml (moi 2.0), continue shaking for 5 hours, and then add Phage φ80pt as usual by adding chloroform. Prepared in large quantities [T. Maniatis, EFFritsch,
Sambrook; “Molecular cloning” (1982)
See p.76-80 Cold Spring Harbor Laboratory]. Next, the obtained Phage φ80pt solution was dialyzed against Tris buffer (PH7.8), and then DNA extraction was performed using the phenol method [above “Molecular Clon-
ing” p. 85], and added the restriction enzyme BamHI to it at 30°C.
The reaction was allowed to proceed for 30 minutes, and a gene fraction containing the tryptophan operon was obtained. (D) Combining the tryptophan operon fraction and the miniF fraction. Add restriction enzyme BamHI to the miniF fraction obtained in (A) above.
was reacted at 30℃ for 30 minutes, then at 60℃ for 5 minutes.
After inactivating BamHI by heat treatment for a minute,
Add and mix the tryptophan operon fraction obtained in (C), add ATP (adenosine triphosphate) and dithiothreitol, and add DNA ligase derived from T4 phage (having DNA binding activity; manufactured by Takara Shuzo Co., Ltd.). "T4DNA ligase")
After adding , the reaction was carried out at 12°C for 17 hours.
Next, the DNA ligase was inactivated by heat treatment at 60° C. for 5 minutes, and then plasmid pMTY-1 was collected by ethanol precipitation. The molecular weight of the obtained plasmid pMTY-1 is
It was measured by agarose gel electrophoresis.
The agarose gel electrophoresis device used was a flat agarose gel electrophoresis device KS-8422FAE manufactured by Marisol Sangyo Co., Ltd. The electrophoresis conditions were as follows: 0.6% agar gel was prepared, buffer solution (0.04M Tris, 0.04M Sodium Acetate, 0.008M
A mixture of DNA fragments obtained by degrading plasmid pMTY-1 using a limited enzyme (EcoRI, BamHI or Hind) was placed in a solution (adjusted to pH 8.3 with EDTA and acetic acid) and electrophoresed at 80V for 4 to 5 hours. . After electrophoresis, the agar gel was taken out, stained with ethidium bromide, and irradiated with 254 nm UV light.
Detects the position of DNA movement and DNA with known molecular weight
Fragment (obtained by digesting λDNA with Hind)
DNA: “λDNA-Hind” manufactured by Wako Pure Chemical Industries, Ltd.
Molecular weights were determined by comparison with ``digests''). Reference Example 2: Transformation of E. coli K-12 mutant strain with plasmid pMTY-1 (A) Preparation of host bacteria NTG (N-methyl- Tryptophan auxotrophic mutants were prepared by treatment with N'-nitro-N-nitroguanidine [EA
Adelberg et al., Biochem, Biophys, Res.
Comm., 18 , 788 (1965)]. That is, the above E. coli K-12 strain was cultured in L medium until the mid-logarithmic growth phase, collected by centrifugation, and the strain was added to Tris-maleate buffer (PH6.0).
After suspending in water, add 100 μg/ml to 200 μg/ml NTG and treat for 15 to 30 minutes. After washing the treated product with the same buffer, it was added to L medium and cultured with shaking at 37°C. A tryptophan auxotrophic mutant strain was isolated from the culture using a conventional method in combination with a penicillin enrichment method and a replica method. (B) Transformation The E. coli K-12 strain (tryptophan auxotrophic mutant) obtained in (A) above was inoculated into 10 ml of the same L medium used in Reference Example 1, and cultured with shaking at 37°C. After growing to the mid- or late-logarithmic phase, the bacteria were collected by centrifugation and suspended in an aqueous solution of 0.1M MgCl ₂ and 0.1MCaCl ₂ under ice-cooling at 0°C. Tent fungus) was prepared. Plasmid pMTY-1 obtained in Reference Example 1 was added to this, and the mixture was allowed to react under ice-cooling at 0°C for 40 minutes, then heated at 40°C for 3 minutes, and then again under ice-cooling at 0°C. The reaction was allowed to proceed for 2 hours. Next, L medium was inoculated with the above-mentioned treated bacterial cells, cultured with shaking at 37°C for 1 hour and 30 minutes, collected by centrifugation, and washed with minimal medium (Davis medium: K ₂
HPO ₄ 7g, KH ₂ PO ₄ 2g, MgSO ₄・7H ₂ O0.1
g, (NH ₄ ) ₂ SO ₄ 1 g, glucose 2 g, pure water 1) was plated on a plate medium prepared, cultured at 37°C, grown colonies were purified again on the same medium, and then transformed. strain (loss of Trp auxotrophy,
That is, a strain was obtained that was capable of biosynthesis using the tryptophan operon on the plasmid and was able to grow on a minimal medium. This transformed strain was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology, 1-1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, in July 1982.
It has been deposited on May 6th under accession number: FERM P-7139. Reference Example 3: Stability of Transformed Strain 100 ml of the aforementioned L medium was dispensed into a 500 ml Erlenmeyer flask, sterilized at 120°C for 15 minutes, and the transformed strain obtained in Reference Example 2 was inoculated, After culturing with shaking at 37°C for 24 hours, 2 ml of culture was subcultured into L medium prepared in the same manner, and cultured with shaking at 37°C for 24 hours. This operation was repeated for a total of 5 times. Culture was carried out. At the end of each culture (24-hour culture), collect the bacteria, wash the cells, and add 20 L-tryptophan to the minimal medium.
A fixed amount of the mixture was plated on a plate medium prepared by adding mg/mg/ml, and cultured at 37°C for 1 day. 200 growing colonies (random selection) are inoculated into a minimal medium containing no tryptophan, and after culturing at 37°C for 2 days, they are inoculated again into the same minimal medium and the growing colonies are counted. As a result, it was confirmed that all the colonies that grew on the tryptophan-supplemented medium also grew on the minimal medium that did not contain tryptophan, that is, the high stability of the plasmid was confirmed. In addition. As a test to confirm the presence of the plasmid in the transformed strain, a Trp auxotrophic strain appeared by treating the transformed strain with acridine orange, and the above-mentioned
Plasmid confirmation was simultaneously performed using the SDS-lysozyme method and the cesium chloride-ethidium bromide method, and it was confirmed that there was no change in the molecular weight of the plasmid. Example 1 The following composition Bacto tryptone 10 parts by weight Yeast extract 5 Glucose 1 NaCl 5 Pure water 1000 PH 7.2 L medium was dispensed into test tubes with a volume of 5 ml and a diameter of 18 mm.
One platinum loop of the transformed strain (FERM P-7139) created in Reference Example 2 was inoculated into a medium that had been sterilized at 120°C for 15 minutes, and cultured with shaking at 37°C for 18 hours. Next, add 100 ml of L medium with the same composition as above to a volume of 500
2 ml of the above preculture was inoculated into a culture solution that had been sterilized at 120°C for 15 minutes in a shaking flask, and at the same time, indole acrylic acid was added in the amount shown in Table 1 below, followed by shaking culture at 37°C for 7 hours. did. After completion of the culture, the bacteria were collected by centrifugation, and the bacteria were collected using the method of Yanofsky et al. [Method in Enzy-
mology, vol.5, 794 (1962)], 0.2
g (wet bacterial cells) was weighed, suspended in 100 mM Tris (hydroxymethylaminomethane) buffer solution pH 7.8, and the bacterial cells were destroyed by ultrasonication. This solution was centrifuged at 12,000 rpm for 40 minutes, and the supernatant was used as a crude enzyme solution. The reaction solution composition was 0.4 μmole of indole, 80 μmole of DL-serine, 180 μmole of sodium chloride, and pyridoxal pentaphosphate.
Use 0.03 μmole, 100 μmole of tris (hydroxymethylaminomethane), 0.1 ml of assembled enzyme solution, and make a total volume of 1 ml (PH7.8) with pure water and 1N hydrochloric acid. The reaction is carried out at 37°C for 20 minutes to stop the 2N sodium hydroxide reaction. Then, 4ml of toluene
was added to extract the remaining indole, and 1 ml of it was added to a coloring solution (p-dimethylbenzaldehyde 3.6% (w/
v), 2N hydrochloric acid 1.8% (v/v), ethanol solution)
Add 1.5 ml and 3 ml of ethanol, stir, let stand for 40 minutes, and perform colorimetric determination using As540 nm. A calibration curve for indole was determined by the same method, and the tryptophan synthase content was determined therefrom. The obtained results are shown in Table 1 below as relative contents, with the tryptophan synthase content when no indole acrylic acid was added being 1.

[Table] *Concentration per ml of medium. Example 2 Same as above Example 2 Six shake flasks each inoculated with 2 ml of preculture were prepared in the same manner as in Example 1, and each flask was inoculated with 37
Incubate with shaking at ℃ for 7 hours. On the other hand, 100 μg/ml of indole acrylic acid was added to each flask after the time shown in Table 2 below from the start of culture, and the relationship between the timing of addition of indole acrylic acid and the tryptophan synthase content was investigated. The results are shown in Table 2 below. In addition, when determining the timing of addition, the growth curve of the strain used was determined. That is, 5 ml of L medium was dispensed into test tubes with a diameter of 18 mm, and after sterilization at 120°C for 15 minutes,
One loopful of this bacterial strain was inoculated and cultured with shaking at 37°C for 18 hours. 100 ml of L medium was placed in a 500 ml shaking flask and sterilized at 120°C for 15 minutes. 2 ml of the above culture was inoculated into the medium, samples were taken over time, and the amount of bacterial cells was measured by turbidimetry (OD610). The results obtained are shown in FIG.

[Table] * Same meaning as Example 1.
Example 3 Culture was carried out in the same manner as in Example 1, except that the culture medium having various compositions shown in Table 3 below was used, and the results shown in Table 4 below were obtained. However, indole acrylic acid has a concentration of 20, 25, 30, 50 and 50, respectively, at the start of main culture.
It was added at a concentration of 100 μg/ml.

[Table] Percentage of

[Table] Reference Example 4: Production of L-tryptophan Weighed 200 mg of each of the bacterial cells obtained in Example 2,
The following components were suspended in an aqueous reaction solution containing the following concentrations to make a total volume of 10 ml, and the mixture was reacted at 37°C for 2 hours. Reaction liquid composition: Indole 0.1% L-serine 1% Pyridoxal 5p 0.01% PH 7.9 After the reaction was completed, L-tryptophan in the reaction liquid was determined by high performance liquid chromatography. The results are shown in Table 5 below.

【table】

【table】 [Brief explanation of drawings]

FIG. 1 is a growth curve of the strain used in the above example.

Claims (1)

[Scope of Claims] 1. A bacterial strain possessing a tryptophan repressor corresponding gene (TrpR) belonging to the genus Escherichia has an organic nitrogen source of at least 0.1 in terms of nitrogen atomic weight.
When culturing in a nutrient medium containing % by weight, indole acrylic acid or a salt thereof is added to the medium at the latest by the end of the logarithmic growth phase of the strain.
A method for culturing a TrpR-retaining strain belonging to the genus Escherichia, which comprises adding the above-mentioned strain at a final concentration of 25 μg/ml and culturing to obtain the above-mentioned strain in which the content of tryptophan synthase in the bacterial body is increased. 2. The method according to claim 1, wherein the TrpR-retaining bacterial strain belongs to the genus Escherichia and contains a plasmid having at least a tryptophan promoter and an operator region, and a gene corresponding to tryptophan synthase. 3. Claim 1, wherein the TrpR-retaining bacterial strain is a TrpR-retaining bacterial strain belonging to the genus Escherichia that contains a plasmid having a tryptophan operon.
The method described in section. 4. The TrpR-carrying strain belongs to the genus Escherichia, which has a gene corresponding to tryptophan repressor (TrpR) on the chromosome and/or in the plasmid.
The method according to claim 1, which is a TrpR-retaining bacterial strain. 5. The method of claim 1, wherein the organic nitrogen source is selected from peptone sources, meat extracts, yeast extracts, malt extracts, cornstap liquor and casamino acids. 6 Indole acrylic acid or its salt from 80 to
2. A method according to claim 1, in which the agent is added at a final concentration of 200 μg/ml.