JPH0226955B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to anthranilate synthase (hereinafter referred to as A
The present invention relates to a method for producing Escherichia coli with high tryptophan productivity, which has a plasmid containing a tryptophan operon in which the feedback inhibition by tryptophan for (referred to as Sase) has been removed, and has been recombined with this plasmid by genetic manipulation. It is well known that amino acids and the like have been conventionally produced by fermentation using microorganisms, particularly artificial mutant strains with high productivity. However, for tryptophan among amino acids, conventional fermentation methods have low productivity and an industrial production method has not yet been established.
On the other hand, with the recent development of genetic engineering, it has become possible to transfer genes from higher animals and plants into Escherichia coli to produce useful substances, such as insulin and growth hormones, by fermentation techniques. The present inventors succeeded in producing highly tryptophan-producing E. coli that was endowed with tryptophan synthesis activity by transforming E. coli through genetic manipulation. The present invention is as follows. (1) A recombinant plasmid containing a wild-type tryptophan operon is transferred into a host E. coli, and then this E. coli is subjected to mutation treatment, and at this time, the host E. coli is deficient in tryptophan repressor and tryptophanase. If this is the case, the transformation described below is not performed and the host E. coli is not transformed, and if the host E. coli is not such a type, a mutant plasmid is taken from the mutated E. coli and this is transformed into a cell that is deficient in tryptophan repressor and tryptophanase. Transformation is performed by transferring to another host E. coli, and thus the recombinant plasmid has a tryptophan operon in which the tryptophan feedback inhibition of anthranilate synthase has been released, and the tryptophan repressor and A method for producing Escherichia coli with high tryptophan production, which comprises producing Escherichia coli lacking tryptophanase. (2) The production method according to claim 1, wherein the host E. coli is E. coli in which the chromosomal trpE gene has been deleted. (3) The production method according to claim 1, wherein the plasmid is derived from pSC101. In the present invention, a plasmid in which feedback inhibition of A Sase by tryptophan, which is carried by E. coli subjected to mutation treatment, is released will be described. A, the first enzyme in the tryptophan synthesis system
Sase is subject to feedback inhibition by tryptophan, but strains resistant to tryptophan analogues such as 5-methyltryptophan include those in which the feedback inhibition of tryptophan against A Sase is released. Therefore, as described later, E. coli strains
Escherichia coli W 3110 trpAE1 (pSC101−
(trp) That is, a strain containing a plasmid having a tryptophan operon is subjected to mutation treatment by mutation means, and among them, a strain whose A Sase activity is not inhibited by tryptophan is selected for 5-methyltryptophan-resistant bacteria. Plasmids extracted from these (e.g. pSC101-trp・I15)
has a tryptophan operon whose feedback inhibition by tryptophan has been released. The plasmid containing the tryptophan operon used in the present invention, for example, pSC101-trp,
It can be obtained by the method described in Genetics, Vol. 1, pp. 141-152 (1977), or by an improved method thereof. The E. coli produced by the present invention is E. coli that has been transformed by transferring the above-mentioned plasmid, and at the same time, the feedback inhibition by tryptophan has been released, and at the same time, the tryptophan repressor is deleted and the tryptophanase is also missing. When the Escherichia coli produced according to the present invention is cultured in a nutrient medium, particularly an anthranilic acid-supplemented medium, with the addition of a tetracycline antibiotic if necessary, L-tryptophan is collected from the culture solution by a method known per se. can be collected with high productivity. The cultivation and collection methods in this case are the same as in the case of normal fermentation methods. The present invention will be explained in detail below. As the host E. coli in the present invention, for example, the following strains are used. (1) Escherichia coli W 3110 trp AE1 [AE1] (2) Escherichia coli W 3110 trp AE1 trp
Ram 27〔Ram〕 (3) Escherichia coli W 3110 trp AE1 trp R
tna A [Tna] The above Escherichia coli (hereinafter referred to as E.coli)
W3110 is a λ non-lysogenic (λ ^- ) strain without sex factor F (F ^- );
Derived from E. coli K-12 strain with sex factor F
It is ATCC27325 strain. That is, in the present invention,
Any Escherichia coli derived from the E. coli K12 strain can serve as host bacteria, and if the purpose is to produce tryptophan, these bacteria can be infected with tryptophan operon deletion mutations (trp AE1), tryptophan Escherichia coli endowed with a fan repressor-deficient mutant (trp R) and a tryptophanase-deficient mutant (trp A) are preferably used. Next, the characteristics that the three strains mentioned above should have as host E. coli will be described. trp AE1 (deletion mutation of tryptophan operon) The tryptophan operon of E. coli is located on the chromosome.
Although it is adjacent to the ton B gene, ton B is necessary for phage T ₁ to adsorb to E. coli, so if a mutation occurs in ton B, phage T ₁ cannot adsorb. Therefore, E. coli becomes resistant to T ₁ phage (T ₁ ^r ) due to mutation in ton B. When the tryptophan auxotrophy was assayed for the T ₁ phage-resistant mutant strain (T ₁ ^r ) obtained by ultraviolet (UV) irradiation, most of the strains that became T ₁ ^r and Trp ^- acquired tryptophan from ton B on the chromosome. The DNA fragment spanning the Juan operon was partially or completely deleted. These mutation treatments and confirmation methods are described in detail in, for example, Genetics, Vol. 49, pp. 267-278 (1964). trp Ram27, trp R (tryptophan repressor mutation) The tryptophan repressor mutant has a resistance to tryptophan analogs (e.g., 5-methyltryptophan, 6-methyltryptophan, etc.). Make your next selection. Analog-resistant strains include (1) strains in which repression has been lifted, (2) strains in which feedback inhibition has been lifted, and (3) strains in which the analog cannot be taken into cells due to changes in membrane structure. is possible. Therefore, even if an analog-resistant strain is cultured in a medium containing tryptophan (a condition in which the tryptophan synthesis enzyme is hardly produced due to repression in the wild strain), if the tryptophan synthesis enzyme is constitutively produced, repression will be released. This means that it has been done. As to whether this is due to a repressor defect or an operator mutation, the analog resistance trait is linked to the thr (threonine auxotrophic) gene or the trp (tryptophan auxotrophic) gene by transduction with Phage PIKc (a universal transducing phage of Escherichia coli). If it is linked to the thr gene, it can be confirmed that a repressor mutation has occurred, and it can be used in the present invention. Note that trp Ram27 is a nonsense mutation lacking tryptophan repressor, and trp R is a missense mutation. For details, see J. Mol. Biol. No. 44.
Volume 185-193 (1969), Genetics Vol. 52, 1303-
See p. 1316 (1965), etc. tna A (tryptophanase-deficient mutation) Wild E. coli has tryptophanase activity, so it can grow using tryptophan as a single carbon source (tryptophan → indole + pyruvate + NH ₃ ) However, the tryptophanase-deficient strain
Cannot grow using tryptophan as the sole carbon source. Using this physiology, tryptophanase-deficient strains can be isolated. For example, UV irradiation,
Mutation treatment is performed with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl-methane-sulfonate (EMS), and colonies are formed on a minimal agar medium using glucose as a carbon source. This is replicated on a minimal agar medium with tryptophan as the sole carbon source to select strains that grow when the carbon source is glucose but cannot grow when tryptophan is used. The absence of tryptophanase activity can be confirmed by adding an indole reagent [same as the one used for tryptophan synthase (T Sase) activity measurement] to the culture medium cultured in L-broth;
[color reaction to indole] and confirm that the red color specific to indole does not occur. See, for example, J. Bacteriology, Vol. 89, pp. 680-686 (1965). To obtain E.coli W3110 trpAE1 trpR tnaA strain,
This can be done by combining the above methods, but specifically, it can be carried out by the following procedure. (1) Add trpR mutation to E.coli W3110 cysB strain. (2) cysB trpR strain was transduced with PIKc lysate from E. coli W3110 trpAE1 strain to produce W3110trpAE1.
Obtain trpR strain. (3) Add tnaA mutation to (2). In the following, plasmids such as pSC101-
Tryptophan synthase (T
Sase) and anthranilate synthase (A Sase)
The activity of A by tryptophan is shown in Table 1.
Table 2 shows the inhibition rate of Sase activity, and Table 3 shows the tryptophan productivity.

[Table] In Table 1, the activity unit is unit/mg protein. The results were obtained by measuring the activity of a disrupted bacterial cell solution (crude enzyme solution) cultured in medium-A, which will be described later.For A Sase, tryptophan was not added (-Trp) and tryptophan was added to 0.2.
Measurements were made for the case where mM was added (+Trp). T Sase activity and A Sase activity are greater in the tryptophan repressor-deficient strains Ram and Tna strains than in the AE1 strain. None of A Sase is inhibited by tryptophan.

[Table] According to Table 2, pSC101−trp・I5 or pSC101
The tryptophan concentration that inhibits A Sase activity by 50% was determined for the AE1 strain transformed with pSC101-trp・I5.
No significant difference was observed in the inhibition of A Sase activity by tryptophan between pSC101-trp.I15, demonstrating the universality of the plasmid in the present invention. E. coli transformed in the present invention, e.g.
The Tna (pSC101-trp/I15) strain is free from inhibition by tryptophan and is deficient in tryptophan repressor and tryptophanase, and a strain that satisfies these conditions is the first to achieve high tryptophan levels. Concentration accumulation is achieved. This is clear from Table 3 below.

[Table] Suppression and release of inhibition by tryptophan are essential for high concentration accumulation of tryptophan.
Deficiency of tryptophanase is also required.
The productivity of Tna (pSC101−trp・I15) was determined by the chromosomal double mutant strain (repression ⁻ ,
inhibition ^- ) [180 μg/ml: Journal of Technology, Vol. 43, pp. 302-306 (1965)],
This seems to be the result of the gene amplification effect of the plasmid being reflected in the amount of final product produced. In addition, the above Escherichia coli W3110 trpAE1
trpR tnaA (pSC101−trp・I15) is a member of the AMERICAN TYPE CULTURE collection.
COLLECTION) on October 28, 1980, and has the deposit number (ATCC31743). As a medium for culturing E. coli, a nutrient medium containing a carbon source, a nitrogen source, inorganic substances, etc., and
Examples include a medium additionally containing a resistant drug and/or anthranilic acid imparted during the production of the microorganism used. Either synthetic or natural media can be used. As the carbon source and nitrogen source used in the culture medium, any type of carbon source and nitrogen source that can be used by the bacteria used may be used. Carbon sources include glucose, glycerol,
Carbohydrates such as fructose, sucrose, maltose, mannose, starch, starch hydrolyzate, and molasses can be used. Nitrogen sources include inorganic or organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium phosphate, and ammonium acetate, or meat extract, yeast extract, corn steep liquor, casein hydrolyzate, and defatted soybean meal. Natural organic nitrogen sources can be used, such as its digestate. Many natural organic nitrogen sources can be both nitrogen and carbon sources. Furthermore, as inorganic substances, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate,
Sodium chloride, ferrous sulfate, calcium chloride,
Zinc chloride, copper sulfate, manganese chloride, cobalt chloride, ammonium molybdate, boric acid, etc. may be used as necessary. If Escherichia coli is endowed with antibacterial drug resistance, contaminating bacteria can be prevented by adding the appropriate antibacterial agent to the culture medium. Furthermore, addition of anthranilic acid, which is a precursor of tryptophan, is also advantageous for increasing the yield of tryptophan. Cultivation is performed under aerobic conditions such as shaking culture or submerged culture with aeration and stirring. Culture temperature is usually in the range of 20-50°C. It is desirable to maintain the pH of the medium during culture near neutrality, and the culture period is usually 1 to 3 days. The produced and accumulated tryptophan can be collected by a method known per se, such as using an ion exchange resin. Next, the present invention will be explained with reference to Examples, but the present invention is not limited thereto. Production example (recombinant plasmid with wild-type tryptophan operon) A type of drug-resistant plasmid for E. coli.
pSC101 (tetracycline resistance: ^Tcr , copy number per chromosome 5-10) is used as a vector, and special transducing phage trp EA60-3 is used as a tryptophan operon donor of Escherichia coli. (1) Restriction enzyme EcoRI treatment λtrp EA60-3 DNA (approximately 0.1 μg) and pSC101 plasmid DNA (approximately 1.9 μg) are reacted at 37° C. for 1 hour in the following reaction solution. Thereafter, it is heated at 65°C for 5 minutes to inactivate EcoRI and stop the reaction. EcoRI reaction solution (total volume 50μ) Tris-HCL PH7.4 90mM MgCl ₂ 10mM DNA 2μg EcoRI 2 units The above EcoRI is a commercially available product (Miles Laboratories
Inc., Bethesda Research Laboratories Inc.
etc.). (2) T4-DNA ligase treatment For T4-DNA ligase treatment, add the following reagents to the above EcoRI reaction solution and make 4.
Perform at 18°C for 18 hours. The above ligase is a commercially available product. Reagent 50mM MgCl 10μ 100mM dithioerythritol 10μ 0.5mM adenosine triphosphate (ATP)
10 μ water 20 μ T4-DNA ligase 0.2 units (3) Transformation of E. coli Transform E. coli C600rk ^- , mk ^- , Trp ^- strains into 20 ml of L
- in broth (polypeptone 10 g/, yeast extract 5 g/, NaCl 5 g/, pH 7)
Culture at 37℃ until OD ₆₆₀ is 0.4, and after harvesting
Wash with 20ml of 100mM _MgCl2 ,
Suspend in 10 ml of CaCl ₂ and let stand in an ice bath for 20 minutes.
Collect cells again by centrifugation, resuspend in 1ml of 100mM CaCl ₂ , mix 200μ of this cell suspension and 100μ of T4-DNA ligase reaction solution in an ice bath for 30 minutes.
Let stand for a minute. Next, this mixture is treated at 42° C. for 2 minutes, and then left to stand again in an ice bath for 20 minutes. L-
After adding 2.7 ml of broth and incubating at 37°C for 2 hours,
Tc r ^and Trp ⁺ transformants are selected by spreading on medium A (containing 1.5% agar) with the following composition containing 10 μg/ml of tetracycline (Tc). Regarding E.coliC600 mentioned above, the symbol rk indicates that the bacterium uses its own DNA molecules (bases on the DNA are specifically methylated) and external DNA molecules (the methylation sites are different from its own). Function to recognize and restrict external DNA (restriction)
The symbol mk is once introduced into the cell.
It refers to the function of modifying DNA into a self-specific DNA molecule. What is ^rk- and ^mk- ?
Indicates that these functions are missing. Medium-A (Vogel and Bonner minimal agar medium,
PH7) K ₂ HPO ₄ 10g / NaNH ₄ HPO ₄・4H ₂ O 3.5g / MgSO ₄・7H ₂ O 0.2g / Citric acid・H ₂ O 2g / Glucose 2.5g / Casamino acid 0.5g / (4) pSC101− Extraction of trp plasmid DNA The Tc ^r , Trp ⁺ transformant was cultured at 37°C in 100 ml of L-broth until the OD ₆₆₀ reached 0.8. After harvesting, 1 ml of Tris-sucrose [Tris-
HCl PH8 (50mM), sucrose (25%,
wt/Vol)]. Add 0.2 ml of lysozyme (5 mg/ml) in an ice bath, let stand for 5 minutes, and then add 0.2 ml of lysozyme (5 mg/ml).
Add ml of Na ₂ EDTA (250mM, PH8) and let stand for 5 minutes. Then 0.5ml of 5M NaCl and 10%
0.2 ml (wt/Vol) of sodium dodecyl sulfate (SDS) was added continuously, and after stirring, the mixture was allowed to stand overnight in an ice bath. The supernatant obtained by centrifugation (30,000xg) for 30 minutes at 4°C was diluted with 50mM
Increase to 5mg with Na ₂ EDTA (PH8), 4.7
g of CsCl and 1.8 mg of ethidium bromide
(ethidium bromide) to increase the density to 1.57g/
Adjust to ml. This sample was subjected to equilibrium density gradient ultracentrifugation (Hitachi RP65 rotor, 3800 rpm).
40 hours). After centrifugation, the fraction containing plasmid DNA was taken out, ethidium bromide was extracted with butanol, and TE buffer (Tris.
Dialyze against HCl 10mM, Na ₂ EDTA 0.1mM, pH 8). The principle of the above equilibrium density gradient ultracentrifugation is as follows. Plasmid DNA (circular molecule) and chromosomal DNA
(Linear molecules: They are circular molecules inside cells, but due to their large molecular weight, they become linear molecules during the extraction process.) The molecular forms are different. The dye ethidium bromide enters between the bases of DNA, but it enters more into linear molecules, resulting in plasmids
A density difference occurs between DNA and chromosomal DNA.
Plasmid DNA is separated using this density difference. pSC101-trp extracted by the above method has a molecular weight of approximately 16.5×10 ⁶ Daltons. After treatment with the restriction enzyme EcoRI, two DNA bands are observed when subjected to agarose gel electrophoresis. Large molecular weight (molecular mobility,
small) contains the tryptophan operon
A DNA fragment with a small molecular weight (large electrophoretic mobility) of 10.8 × 10 ⁶ daltons was used as a vector.
pSC101 is 5.7 x ¹⁰⁶ daltons. Example 1 Escherichia coli containing a plasmid containing a tryptophan operon whose feedback inhibition by Trp was released was produced as follows. (1) Isolation of 5-methyltryptophan (5MT)-resistant strains 5MT, an analog of tryptophan,
Similar to tryptophan, it inhibits transcription from the tryptophan operon to mesendia RAM (mRAM), and inhibits anthranilate synthase (A
Feedback inhibition of Sase) activity. Normal wild-type strains cannot proliferate in the presence of 5MT, but
Tryptophan repressor is no longer suppressed due to mutations, or A.
If Sase undergoes a mutation and is no longer inhibited,
It becomes possible to grow by synthesizing the minimum amount of tryptophan (5MT ^r ). Now, with the pSC101-trpDNA, E.coli
The W3110trpAE1 (Trp ^- , [AE1], in which the tryptophan operon on the chromosome has been completely deleted) was transformed, and the Tc ^r , Trp ⁺ transformant strain AE1 (pSC101) was transformed.
−trp) was obtained. This AE1 (pSC101−trp) strain
Treated with NTG (200 μg/ml) and 5MT (200 μg/ml).
ml) containing agar medium-A (15 g/mL in medium-A)
Spread about ¹⁰ cells (containing agar) on
Cultured overnight at 37°C. Approximately 70 strains were obtained using this method.
The 5MT ^r strain was isolated independently. (2) Isolation of strains in which feedback inhibition has been released Seventy 5MT ^r strains were each placed in 100 ml of medium-A.
After culturing at 37℃ for 16 hours and collecting bacteria, the bacterial cells were reduced to 0.9%
Wash with NaCl and add 5 ml of 100 mM Tris HCl.
(PH7.8). Ultrasonic treatment for 20 seconds 3 times (ultrasonic generator, model 4280, Kaijo
Denki CO., 10KHz) to crush the bacterial cells,
The mixture was centrifuged at 18,000 rpm for 30 minutes (Sorval SS-34 rotor), and the supernatant was used as a crude enzyme solution. The A Sase activity of this crude enzyme solution was measured to examine whether the A Sase activity would be inhibited even if 0.2 mM tryptophan was present in the reaction solution. As a result, the A Sase activities of the two strains I5 and I15 were not inhibited at all by 0.2 mM tryptophan and were resistant to feedback inhibition. Next, a higher concentration of 5-5-
By isolating bacteria resistant to MT, we isolated a more effective Escherichia coli strain. (3) Preparation of plasmid with tryptophan operon with feedback inhibition released From each strain I5 and I15, plasmid
pSC101-trp·I5 and pSC101-trp·I15 were obtained. pSC101−trp・I5, pSC101−trp・
When each I15 DNA was treated with EcoRI and subjected to agarose gel electrophoresis, two bands of 10.8 x 10 ⁶ daltons and 5.7 x 10 ⁶ daltons were observed, similar to pSC101-trp. Transformation of E. coli with the mutant plasmid The plasmid obtained above was transformed into E. coli W3110 by the same method as described in the above production example.
trpAE1 trpR TnaA (tryptophan operon-deficient, tryptophan repressor-deficient, tryptophanase-deficient [Tna]) was transferred and transformed. The E. coli strain contained a recombinant plasmid with a tryptophan operon in which the tryptophan operon had been deleted, and was deficient in the tryptophan repressor and tryptophanase.
AE1 strain, E.coli by trp・I15 plasmid
W3110 trpAE1 trpRam27 (tryptophan operon deletion, tryptophan repressor deletion [Ram]), E.coli W3110 trpAE1 trpR tnaA
(Tryptophan operon deletion, tryptophan repressor deletion, tryptophanase deletion [Tna]) were each transformed. Selected Tc ^r , Trp ⁺ pSC101−trp・I5 or
Regarding the pSC101-trp·I15 holding strain, a crude enzyme solution was prepared by the method described above, and the A Sase activity was measured. In all cases, the A Sase activity was not inhibited by tryptophan.
Therefore, the trait of resistance to feedback inhibition is
This is due to mutations in the tryptophan operon on the pSC101-trp·I5 or pSC101-trp·I15 plasmid DNA. Production example (tryptophan) Strain Tna obtained by transforming Tna strain with plasmid pSC101-trp/I15 (pSC101-trp/I15)
First, as a pre-culture, 20 μg/satracycline was added.
One platinum loop was inoculated from the agar preservation medium into L-broth with the following composition containing 1.5 ml of L-broth and cultured at 37° C. for 9 to 15 hours.
Next, inoculate 0.5 ml of the above preculture solution (0.5% inoculation) into 100 ml of medium-B (500 ml flask) with the following composition.
Main culture was performed at 37°C. The PH was adjusted to 7 with 2N NaOH every 3-5 hours. After 48 hours of culture, the amount of tryptophan accumulated was determined by the xanthydrol method and was 360 μg/ml. L-Brosbactotryptone 10g Yeast extract 5g NaCl 5g/PH7 Medium-B KH ₂ PO ₄ 3g K ₂ HPO ₄ 7g NH ₄ Cl 3g MgSO ₄・7H ₂ O 0.2g/PH7 Glucose 3% Casamino acids 0.1% Production Example (Tryptophan) The above medium-B was inoculated with Tna (pSC101-trp·I15), and anthranilic acid and casamino acid were added as shown in the table below and cultured. The pH was adjusted to 7 with 2N NaOH for up to 48 hours. The tryptophan accumulation amounts shown in the table below were obtained. Anthranilic acid is 500mg/approximately 24 hours, 800mg/
mg/ is completely consumed in about 48 hours. As the concentration of anthranilic acid increases, bacterial growth worsens; 63 hours are required for 1000 mg of anthranilic acid, and 63 hours or more for 1500 mg of anthranilic acid. The consumed anthranilic acid is almost completely converted to tryptophan.

[Table] Production example (tryptophan) In L-broth containing 20 μg/ml of tetracycline, strain Tna (pSC101-trp・I15) at 37℃ for 9 to 15 minutes.
preincubated for hours. 100 ml of this pre-culture solution was inoculated into a jar fermenter (Marubishi Scientific Instruments Institute MD-250 model, 2 volumes) containing 1.5 of medium-C and heated to 37°C.
It was cultured in Stirring was performed at 500 rpm, aeration was performed at 1 v.vm, and the pH was controlled at 7 with 14% NH ₄ OH. The substrate anthranilic acid was added at a rate of 50 mg/hr from the 8th hour of culture. After culturing with xanthidrol for 28 hours, the amount of accumulated tryptophan was determined by the xanthidrol method and was 5.6 g/. Medium - C KH ₂ PO ₄ 3g / K ₂ HPO ₄ 7g / NH ₄ Cl 3g / MgSO ₄・7H ₂ O 0.2g / FeSO ₄・7H ₂ O 0.01g / Anthranilic acid 0.5g / Glucose 50g / Casamino acid 10g / Tetracycline 0.01 g/PH7 The culture solution 1 thus obtained is centrifuged to remove bacterial cells, and the centrifuged supernatant is poured into a column filled with 900 ml of activated carbon for chromatography to adsorb tryptophan. 50 containing tryptophan and 0.7% ammonia after washing with water
% ethanol solution, and the eluate was concentrated under reduced pressure to remove ammonia and ethanol. The resulting tryptophan aqueous solution was adjusted to pH 4.5,
This was passed through a column (10 x 40 cm) made of Dowex 50 x 8 and Na type resin (manufactured by Dow Chemical Co.) buffered with a 0.1 MPH 3.4 citric acid buffer. Then, after flowing 0.1 MPH 5.0 citrate buffer 4.5, tryptophan is eluted with 50% methanol solution containing 0.2% ammonia. This eluate is concentrated to dryness under reduced pressure to obtain a crystalline coarse powder of tryptophan. This was dissolved in a small amount of 50% hot ethanol water, decolorized with a small amount of activated carbon, and after cooling, 3.5 g of white scaly crystals of tryptophan were obtained. As can be seen from the above, in the present invention, by using Escherichia coli transformed by genetic manipulation, tryptophan can be obtained in a shorter time and more efficiently than when using conventional mutant strains. Further efficiency in harvesting can be expected by changing the species or by further improving the resistance.

Claims (1)

[Scope of Claims] 1. A recombinant plasmid having a wild-type tryptophan operon is transferred into a host E. coli, and then this E. coli is mutated, and at this time, the host E. coli has a tryptophan repressor and a tryptophanase. If the host E. coli is defective, the transformation described below is not performed and the host E. coli is not transformed. If the host E. coli is not such a type, a mutant plasmid is taken from the mutated E. coli and transformed with tryptophan repressor and tryptophan. Transformation is carried out by transferring the enzyme-deficient E. coli to another host, and the result is a recombinant plasmid containing a tryptophan operon in which the feedback inhibition of anthranilate synthase by tryptophan has been removed, and A method for producing Escherichia coli with high tryptophan productivity, which comprises producing Escherichia coli that is deficient in repressor and tryptophanase. 2. The production method according to claim 1, wherein the host E. coli is E. coli in which the chromosomal trpE gene has been deleted. 3. The production method according to claim 1, wherein the plasmid is derived from pSC101.