JPS6328393A - Novel plasmid - Google Patents

Abstract

PURPOSE:To stabilize a host, by combining DNA fragments containing a gene governing the total synthesis of tryptophanase with DNA fragments governing the autonomous replication ability of ColE1 based plasmid and DNA fragments containing a gene governing multiplication control partition system derived from F plasmid. CONSTITUTION:A novel plasmid obtained by constructing from (A) DNA fragments containing at least a promoter of tryptophanase operon, regulation gene governing the promoter and tryptophanase structural gene, (B) DNA fragments governing autonomous replication of ColE1 based plasmid, e.g. DNA fragments derived from pBR322, and (C) DNA fragments (mini-F fragments) governing multiplication control partition systems derived from F plasmid. The DNA fragments (A) are obtained by cutting chromosomic DNA out of Escherichia coli 12 based strain with restriction enzymes BamHI and HindIII.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel plasmid containing a DNA fragment containing a gene responsible for the biosynthesis of tryptoanase, and more specifically, a novel plasmid that has a large number of copies and is highly stable in the host; The gene tnaA, which performs the biosynthesis of tryptoanase, which is not shed during cell proliferation and is surely inherited from the parent cell to the daughter cell, the promoter that can express this gene, and the regulatory gene that controls this promoter. The present invention relates to a plasmid containing a DNA fragment containing a DNA fragment having

In general, with regard to the stability of constructed plasmids within the host, various genetic instabilities have been reported, such as dropout of the plasmid from the host during culture and loss of inserted genes, and countermeasures are being considered.

For example, there is a method of stabilizing the properties of a microorganism containing the plasmid by incorporating a plasmid into a streptomycin-dependent mutant strain of Escherichia that controls the property of being independent of streptomycin. It has been proposed (Japanese Unexamined Patent Publication No. 156591/1983).

However, this method not only has economical problems, but also requires the incorporation of complex functions into the target plasmid, which makes it difficult for the plasmid to remain stable during host division and multiplication.
It is expected that it will be difficult to distribute to daughter cells, and there will be considerable problems in its industrial application.

Therefore, the present inventors investigated the biosynthesis of tryptoanase i.
In order to stabilize the rJ gene in the host, we first conducted intensive research on plasmids that have the function of allowing stable distribution from parent cells to daughter cells over successive generations.
The DNA fragment containing the rfJ gene, which controls the F-factor plasmid's growth control distribution system, has the ability to significantly contribute to plasmid stabilization, and furthermore, the ColEl-based plasmid of Escherichia coli normally has several dozen copies per cell chromosome. Focusing on the fact that Co1El-based plasmids have a large number of
By combining the DNA fragment containing the gene governing the growth control distribution system of the F factor plasmid with the DNA fragment containing the gene governing the biosynthesis of tryptoanase, the number of copies can be increased. They also succeeded in creating a plasmid that can be stably distributed through generations, leading to the completion of the present invention.

Therefore, according to the present invention, (a) a DNA fragment containing at least a promoter in the tryptophanase operon, a regulatory gene that controls this promoter, and a tryptophanase structural gene, and (b) the ability to autonomously reproduce a ColEl-based plasmid. and (c) a DNA fragment containing a gene that controls the proliferation control distribution system derived from the F plasmid.

At least a promoter in the tryptophanase operon, a regulatory gene that controls this promoter, and tryptophanase NII, which constitute the plasmid of the present invention.
"DNA fragment containing a synthetic gene" (hereinafter sometimes abbreviated as "T fragment") is a DNA fragment containing a gene that controls the biosynthesis of tryptoanase, a #element that plays the role of decomposing tryptophan into indole, pyruvate, and ammonia.
In particular, the T fragment used in the present invention includes a DNA fragment (hereinafter referred to as [tryptophanase promoter]) and, if necessary, a regulatory gene that can control this promoter function.
Includes DNA fragments joined with NA fragments. It takes T
Practically speaking, E. coli white rice fragments are preferably used as the fragments.

There are no particular restrictions on the source of this T fragment, but Escherichia coli ATCC23282, Escherichia coli ATCC23437, Escherichia coli ATCC2
3461 etc. are advantageously used.

From these source microorganisms, tna A suitable for the purpose of the present invention
A detailed method for preparing a DNA fragment in which the lypto-7anase promoter and the regulatory gene that controls this promoter are combined is shown in (A) of Example 1 below, but the basic procedure is to Extracting the chromosomal gene of E. coli that has a tryptophanase operon in the gene, and cutting out the tryptophanase operon DNA fragment using restriction enzymes Bawl-11, II 1ndIII, controls the tnaA and tryptophanase promoters and this promoter. A DNA fragment containing the regulatory gene is obtained.

As described above, one feature of the present invention is that the above-mentioned T fragment is combined with a DNA fragment containing a gene that controls the growth control distribution system of a plasmid derived from the F factor plasmid. The F factor plasmid is, for example, [Protein 4f acid
Figure 1 on page 98 of 1sLXJJ Vol. 27, No. 1t [1982]
The molecular weight is 94.5 kb (62 x 10
Gluton), which is present in enterobacteria such as Escherichia coli, usually at a number of 1 to 2 copies per cell chromosome, and this plasmid is accurately transmitted into each daughter cell after cell division. (In this way, 5Lrin8 has a mechanism to keep the copy number at a low level and increase the base accurately with the proliferation of the host.
enL growth control), such 5triBenL growth control in the F factor plasmid (5triBenL growth control)
! 1 has a molecular weight of 9.2 kb called wini-F.
It has already been found that the fragments that can autonomously reproduce ["Mo1cular &General"]
genes”196.185-193 (1984
)], it is also known that this m1ni-F can be excised from the F factor plasmid using the restriction enzyme EcoRI.

The present invention utilizes the growth control distribution system carried by this m1ni-F, and thus "DNA fragment containing the gene controlling the growth control distribution system derived from the F factor plasmid"
(hereinafter sometimes abbreviated as "F fragment") means a gene fraction equipped with a mechanism for accurately transmitting the F factor plasmid to daughter cells, as described above. is m1ni-F with a molecular weight of approximately 9°2 kb.
Fragments are mentioned.

Furthermore, in the present invention, a "DNA fragment containing a gene that controls autonomous replication derived from a colEi-based plasmid" (hereinafter referred to as "S fragment") is used in combination with the T fragment and F fragment described above.
(sometimes abbreviated as ) refers to a DNA fragment containing a gene that controls the autonomous replication of Co1El-based plasmids with a copy number of 20 to 30 copies per cell chromosome, and a typical example of an S fragment is approximately Examples include the S fragment of plasmid pBR322 Yume, which has a length of 4.3 kb, and S fragments derived from plasmid pBR322 and the like.

The plasmid provided by the present invention is the T
A DNA fragment that carries the genetic information of Ike, as long as it has three essential DNA fragments: F fragment, F fragment, and S fragment, such as a DNA fragment containing the ambicillin resistance gene, which is an antibiotic resistance marker, and a DNA fragment containing the kanamycin resistance gene. Typical examples include T fragments, F fragments, etc.
It consists essentially of three DNAIEII fragments, the fragment and the S fragment, and has a molecular weight of approximately 10.7 megadaltons (approximately 16.4 k
In the plasmid b), the inventors [plasmid pM
The two plasmids were named plasmid pMTP-IRJ and plasmid pMTP-IRJ.
IR.

Plasmid pMTPi has the same structure as plasmid pMTPi except that the mi old-F fragment part is arranged in the opposite direction6. In addition, in this specification, the molecular weight of plasmid is the value measured by agarose del electrophoresis. It is.

Hereinafter, this plasmid pMTP-1 and pMTP
- IR will be explained in more detail.

The susceptibilities (number of recognition sites) of plasmids pMTP-1 and pMTP-IH to the following restriction sequences (number of recognition sites) and the length (kb) of fragments degraded by the restriction enzymes are shown in the table below.

Plus LLpMTP-1 CcoRI 2 9.2.7.2B
an) II 3
9.9, 4.1, 2.4Hind[I[2
8,4,8,0 Aval 2 3.9.12.
5PstI 6 4,9.4.0.3.
8.1.8.1.5.0.4 SalI 2
12.7.3.7EcoRI 2
9.2.7.2nig) (I
3 10.7, 3.3, 2.4Hind
II 2
0.8, 15.6 AvaI 2
3.9.12.5Pstl
6 4.9.4.9.2.9.1.8.1.5
．． 0.4SalI2 12,6
．． 3.8 Also, plasmids pMTP-1 and pMTP-
The IR restriction enzyme cleavage map is attached in Figure 1! J is shown in Figure 2.

Plasmid pMT of the present invention having the characteristics as described above
P-1 and pMTP-IR can be produced, for example, as follows.

First, DNAI!A fragment in which a trypto-7anase promoter and a regulatory gene that controls this promoter are linked to the lla A fragment! The Ii fragment (T fragment) can be prepared using, for example, Escherichia coli that has a tryptophanase operon in its chromosomal gene, such as Escherichia eoliK-1.
2 (IFO3301, ATCC10798, ATCCe
23562), etc., and then extracted using a conventional method [E, F, Fr1Lseh, Sambr.

OK, Mo1ecular atoning'' (19
82) p, 164-165, Cold S print
gHarbor L aborat.

Restriction enzymes BaIIIHI and 1TR according to Rye
Tryptophanase operon DN using indllr
When the A fragment is excised, a T fragment containing the approximately 3.2 kb trypto-7 anase promoter, the regulatory gene that controls this promoter, and tnaA is excised.

On the other hand, the regulation of the m1ni-F fragment can be carried out, for example, by microorganisms carrying the F factor 9 plasmid, such as Escherichia coli (Ecoli).
) K-12 strain (ATCCI 5153, ATCCe2
3589, ATCC c23590) W in a manner known per se, for example P, Guerry+ DL.

1-e Blanc, S, Falkow; J, Ba
cLo, 11 G,.

1064 (1973), etc., to extract the F factor plasmid, and then use the restriction enzyme Ec.

It can be prepared by cutting out the F-F fragment with a molecular weight of about 9.2 kb using R1.

On the other hand, it is convenient to use plasmid pBR322, which is a typical Co1E1 plasmid, as a source of a DNA fragment containing a gene that controls autonomous replication of the Co1E1 plasmid.

The T fragment prepared as above was treated with the restriction enzyme BamHI.
Plasmid pB R32 treated with [7Hind[[
2 and treated with T4' DNAase to create a plasmid in which the above T fragment is integrated into plasmid pBR322. This plasmid was then transformed into EcoR
Open with I! m1ni prepared as above.
The target plasmid pMTP is combined with the -F fragment and ligated with T4 DNAase.
-1 and pM T P -IR can be obtained.

In addition, plasmids pMTP-1 and pMTP-
The method for preparing IR will be described in more detail in Example 1 below.

In this way, key 5! The plasmid of the present invention has excellent characteristics such as having a large number of fuvies (75C), being stable and less likely to be shed when passed on to daughter cells during cell division of the host.

Therefore, it is highly anticipated that the plasmid of the present invention will be industrially applied in the production of tryptophan or tryptophan-permeable conductors. For the production of tryptophan, a host is transformed with the plasmid of the present invention. Escherichia coli is preferred as a host bacterium that can be used for this transformation.

In addition, the plasmid of the present invention can be introduced into these host bacteria using methods known per se, such as M, M ande.
l, A, HiHa; J, Mol, Biol, 53
．． 159 (1970) and the like.

By culturing the host fungus transformed in this manner by a method known per se, tryptoanase can be sufficiently produced and accumulated within the microbial cell.

Tryptoanase is an agricultural
ndB iological Cbemistry
V of 36, N013, P2523-2528 (
1972) and Japanese Patent Publication No. 49-46917, indoles such as indole, 5-hydroxyindole, 5-7 mifindole, 5-methylindole, pyruvic acid, oxaloacetic acid, from at least one organic acid such as malic acid, hepatomaric acid, glyoxylic acid, lactic acid, and ammonium ion; or from the above indoles and at least one amino acid such as cysteine, cystine, S-methylcysteine, serine, etc. , L-) Librifan or L
-) It can be used in enzymatic reactions during the production of Sub7-an derivatives.

When using the cultured bacterial cells for the enzymatic reaction, the bacterial cells can be used as they are, but they can be crushed by ultrasonication or the like, or Furthermore, it can be used in the form of an extract extracted with water or the like, or a crude N product obtained by further treating the extract with ammonium sulfate or the like to precipitate oxygen components, and furthermore, the bacterial cells or the treated product can be used in the form of a crude N product. It can also be used after being solidified if necessary.

The reaction between indole, pyruvic acid or its salt, and ammonium ion in the presence of the microbial cells or their processed material is carried out in the same way as a normal enzyme reaction, for example, using 1 ffl of 0°1 M' heptacid buffer (pH 7,5- 10,0) or water (pi
(7,5-10.0), about 20 to about 50°
C1 preferably at a temperature of about 30 to about 40°C and usually about 10
It takes about 72 hours.

There is no particular restriction on the amount of indole, pyruvate or its salt, and ammonium ion used in the reaction, as long as the concentration does not inhibit the enzymatic reaction, but generally each is used at 0.1-20% (wt/vol). ) is suitable for use within the concentration range. Although the amount of the bacterial cells or the processed product thereof is not limited, it is generally 1 to 1.
It can be used at a concentration of 0% (wt/vol).

The culture of the above-mentioned transformed bacterium varies depending on the type of host bacterium, but in general, it can be carried out using commonly used synthetic or natural media. As a carbon source, various carbonized substances such as gluten □, glycerol, 7-lactose, sucrose, and molasses can be used. As a nitrogen source, tryptone, yeast extract,
Natural nitrogen sources such as corn steep liquor and casein hydrolyzate can be used. Many natural organic nitrogen sources can serve as carbon sources as well as nitrogen sources. In addition, the medium contains L-
) lipdoan and/or 5-methyltryptophan,
It is preferable that a tryptophan derivative such as 5-oxytryptophan be present at a concentration of usually 0.01% (4 t/vol) or more, preferably about 0.1% to 1.0% resistance/vo1.

Cultivation can be carried out under aerobic conditions such as shaking culture or submerged culture with aeration. Culture temperature is generally 20~s
p)-1 of the medium in the culture medium is preferably maintained near neutral or slightly alkaline, and the culture period is usually 1 to 5 days.

1) Separation of L-tryptophan produced in the reaction solution obtained by reacting indole, pyruvic acid or its salt, and ammonium ion using the bacterial cells obtained by the above-mentioned culture method or a treated product thereof. - Preparation can be carried out using known methods such as adsorption and desorption treatment using ion exchange membranes (N, activated carbon, etc.).

Furthermore, the host bacteria transformed with the plasmid of the present invention is L-
It can also be used to produce tryptophan by fermentation. That is, when a host transformed with the plasmid "C" of the present invention is cultured in a medium "C" containing indole, L-)lip)7an is produced and accumulated in the medium, and by collecting it, L-)-lipdooran can be obtained. can be manufactured.

The preparation of the plasmid of the present invention will be explained in more detail using Examples for dogs.

Example 1 Niblasmid MTP-1 [7MTP-11 x] - A)
a co↓YuL medium (10 g of tryptone, 5 g of yeast extract, 1 g of glucose, 15 g of NaC, J' distilled water IN,
pH7,2) 100 (to) 1 to 1500 so 1 triangle 7
The mixture was dispensed into Raphs and sterilized at 120°C for 15 minutes.

This medium contains Escherichia coli (Espberichi).
After inoculating K 12ATCC27325 and culturing at 37°C for 15 hours, this culture 1! Solution 2 and Volume 1 were found, newly inoculated into 100 ml of the above medium, and cultured again at 37°C with opening at 4 o'clock.

After culturing, this culture 1! Centrifuge the entire liquid (8000x
8.15 minutes at 4°C) to collect bacteria, and collect 50t*M of bacterial cells.
) Squirrel supplement a (pH 8,0)-10mM E
DTA・2Na solution 50+ell: Suspended. Next, rhizonaum was added to the final concentration of 2o+g/ml, and after the mixture was allowed to stand for 5 minutes, 10% sodium dodecyl sulfate solution A was added at 6-1, and the mixture was kept at 65°C for 30 minutes. Add 15 ml of 5M NaCl solution to this lysate and heat at 0°C.
Cool for 1 hour and centrifuge the entire volume (12,000 x g, 6
0 minutes, 4'C) L, separate the supernatant fraction, add twice the amount of ethanol, mix, and centrifuge (5000Xg,
The resulting precipitate was diluted to 10mM) for 10 min at 4°C.
Squirrel buffer (pH 7,5)-1+sMEDT A・2N
The DNA was dissolved in A78 solution and subjected to 7 el/-el treatment (protein removal treatment) and treatment with RNA degrading enzyme to finally obtain 1.5 mg of DNA.

B) ) Ribto-7 anase was purified (A
25 μg of chromosomal DNA prepared in ) was treated with restriction enzyme X) [1
The H
indl and Bagu HT decomposition product solution? Made by A.

If) 1 volume of plasmid pBR3221
gg with restriction enzyme 2f-(ind II! and 13am
The decomposition product solution obtained by cleavage using I4I (1 unit each) at 30°C for 11 hours was mixed with 50 to 1 Tris-enriched solution (pH 7.6>, 10 mM), 1,000 osthreitol, and 1 ml. ATP, 10mM! 'ii g
Cl 2 and 1 ml of T4 sugar were added (the concentrations of each component were the final concentrations), and the mixture was allowed to react at 15:00 at 16° C. for binding.

Using this solution, use the conventional method [M-Mandcl, A) (ig
a; J, Mof, Biol, , Dan, 15
9 (1970) ref. 1, Esierichia coli (
E5cbcrichia coli) K12 strain (tryptoanase-deficient mutant and tryptoanase auxotrophic mutant) was transformed, and selective medium (KzHPO478,
K.H., Po.

2g, (NH+)2sOn1g, MgSO4・7H20
0.1g, casamino acid, sFl, DL-5-methyl-tryptophan 50B, glycerin 2g, agar 20g,
It was smeared on pure water 11). Transfer the live 'strain on this medium to L medium with ampicillin at a final concentration of 50 μg/+n l'7
After culturing and weighting at 37°C for 7 hours, the m bodies were incubated by centrifugation (8000×g, 10 minutes, 4°C). Using this bacterial cell, Y ribdoor 7nase activity was examined.

Reaction solution (0,IM phosphate buffer p[(8,0,10cm
) 17 7' ) 7 7 :/ ,
0, 1%) 'J) N X-100
) 50 mg of 7gm was added to 1 ml and reacted at 37°C for 30 minutes. The indole produced is p-nomethylben:! : When colored using aldehyde, a red colored strain was obtained. From this strain, Alkali-3DS
method [T, Maniatis+E, F, Fr1tsch+
J, Sambrook;
eloieng" (1982) p90-91 reference 1, the plasmid was cut with BawHI and Hind[[, and the molecular weight was examined using a 7 galose del.
wHI? Approximately 3.2 kb of DNA was inserted at position fK.

Furthermore, when the above host was retransformed using this plasmid solution, approximately 10' cells/μg DNA
Strains that grew on the selective medium appeared at a frequency of .

C) F plasmid white rice m1ni-F-refusal γ! Escherichia coli (E 5 chcri
chiaColi) Kl 2 YK2004 (FER
M-P-7838) was inoculated, and plasmid pMTY-2 was extracted by the alkali-6DS method. This plasmid pMTY-22 μg and the plasmid pBR322-
tnaA1/jH with restriction enzyme EeoRT 5 un
Eco
After inactivating the RI, the components in the inactivation solution were adjusted to a final concentration of 50 μM Tris buffer pH 7.6, lon+, respectively.
MMgC+□, 10uM nothiothreitol, 1mMA
TP, T41J, and each component were strengthened to form f-gel and kept warm at 16°C for 15 hours. Using this solution, Escherichia and coli K12 strains (tryptoanase-deficient mutants and tryptophan auxotrophic mutants) were transformed according to a conventional method, plated on the above selective medium, and cultured at 37°C for 2 days. did. Plasmids were extracted from the grown strains by the alkaline-3DS method, and restriction enzyme EeoR1 (5 uni
The plasmid was cut using plasmid pMTY-2 and EcoH1ndlII (5 units), and the molecular weight was measured using agarose del electrophoresis.
By integrating into the RIe position, 5 (%) rL plasmids were obtained. Furthermore, Hindll of these five plasmids
When the cleavage pattern was examined, it was confirmed that there was one plasmid in which the 1ini-F fragment was linked in a different coordination. These plasmids were transformed into pMTP-1 and pMTP
-IR, and their restriction enzyme cleavage maps are as shown in FIGS. 1 and 2.

L medium (tryptone 10g, yeast extract 58, NaCl
5g, Glutoose 1g, distilled water 11p87.2) 100
Dispense ml into an Erlenmeyer flask with a capacity of 500 II + l, and add 1
Sterilization was performed at 20°C for 15 minutes. In this medium, Esehcrichia coli (Esehcrichia eoli) K.
12ATCC27325 strain was treated with a known method [Jikken Agricultural Chemistry (Part 2) fjS3 edition P226-230 (31j Kyoto University Faculty of Agriculture S Art Chemistry Department, edited by Asakura Shoten, May 25, 1976) Reference 1 Mutant strain () Reply door unrequested,
37
After 15 hours of incubation at °C, 2 ml of this culture
100ml of the above medium was newly inoculated, and 37ml was added again.
Culture was performed at ℃ for 2 hours. After culturing, 30ml of this culture was aseptically centrifuged (8000Xg, 5 minutes).
4°C) to collect bacteria.

Sterilized 100mM MgCl2 solution 3
After suspending at 0ml, centrifugation (8000xg for 5 minutes)
Do C). It was resuspended in sterilized 100mM CaCl210a+I that had been previously cooled to 0''C, and this suspension was cooled in water for 1 hour.

After cooling, this suspension i ([plasmid p
MTP-10.5 μg or pMTP-IRo.

5 μg was added and cooled in water for 30 minutes. Next 42
Incubate at °C for 2 minutes and use selective medium (K2HPOiag,
KH2PO42g, (NH4)2SOi 1g, Mg6
0.7HzOO, Ig, Casamino Acid 58, DL-
Methyltryptophan 50mg, adenine hydrochloride 50B,
Glycerin 2g1 Anbinlin 201I+g, Agar 20
g, pure water 12) was plated and cultured at 37°C for 24 hours to obtain a raw bacterial strain. The strain transformed with plasmid pMTP-1 was named YK3002, and the strain transformed with plasmid pMTP-1R was named YK3003.

Out” y-coffin m Tsui 1 Oi Z15 t 戊J-pMTP-
1105 pM105p F< 1 0' Control group O 12 YK3002 was transferred to the transformed strain Escherichia coli carrying this plasmid pMTP-1. on July 10, 1985, and the transformed strain Escherichia F17 K12 YK3 carrying the plasmid pMTP-IR was designated
003 is also the accession number dated July 10, 1986:
Microtechnology Research Institute PTFJ8845 (FERM P-884
It has been deposited as 5).

Example 3: U-transformation moxibustion growth 1 100 ml of the above selective medium was dispensed into a 500 ml Erlenmeyer flask, sterilized at 120°C for 15 minutes, and the transformed strain obtained in Example 2 was added to each of the flasks. After inoculating and culturing with shaking at 37°C for 24 hours, L medium 1001 prepared in the same manner was dispensed into a 500ml triangular plastic tube.
050 per 101 sterilized at 120'C for 15 minutes
Connect the base so that it is 1st to the left of cells, and also 37°
After culturing with shaking for 24 hours at
A certain amount was spread on a plate culture medium prepared as L medium and L medium without additives at a ratio of 1/1 and 1 at 37°C.
Count the viable colonies after a day of culture.

As a result, it was found that the number of colonies that grew on the ampyridine-added and non-ampyridine medium was the same for both transformed strains, and that all L-medium-grown colonies grew on the selective medium used in Example 1. The high degree of stability of the plasmid was confirmed.

Example 3 Nitribut 7 Anai Ton 1-O'Mimi Medium 10
Dispense 0 so 1 into a 500+ al Erlenmeyer flask, 120
After sterilization at 15°C for 15 minutes, the transformed strain obtained in Example 1 was inoculated, and after cultured with shaking at 37°C for 1 day, tryptophan prepared in the same manner was added to 200 pFi/ml.
0.2 ml was inoculated into 100 ml of a culture medium containing a concentration of 0.2 ml, and cultured with shaking at 37°C for 8 hours. Said culture 1
! By centrifuging the liquid, the bacterial cells were isolated to 100+
M) After washing with 50 IllI of Squirrel slow solution (pH 8, 0), centrifuging again and collecting, 20,011 g of the wet body was collected, and 1 infusion of 100-M Tris buffer [(pH 8, O)
was suspended in water and subjected to ultrasonication. After treatment, the crushed bacterial cells were diluted appropriately with 100 mN/1 Tris buffer (p! 48.0), and 100 μmole in 1 solution was added.
Squirrel ff1itia (pH 8,0), 100 μ
It was added to a reaction solution containing mole K Cl, 10 μmole L-tryptophan, and 0.03 μmole pyridoxal-5-phosphate and reacted at 37°C for 15 minutes, followed by a conventional method [0,H,S+5iLh and C,Y
anofasky:Methods in E nz
ymology”, A academic+N
ew York (1962), vol. 5, p.
794-8061, the indole produced was quantified.

As a result, plasmid pMTP-1 or pMT of the present invention
E. coli harboring P-IR (FERM P-8844
and FERM P-8845), the activity was about 150 units (1 unit = 0,
As a control, m1ni-
When the activity was examined by the above method using a rectal colon carrying the F-free plasmid pB R322-LnaA, it showed an activity value of about 70 units.

Example 4: L-) Ribdoor Anne's 1 force - Dispense 50 ml of a medium having the following composition into a 5001111 volume Erlenmeyer flask,
The above Example 2 was sterilized at 120°C for 15 minutes.
-12 (FPR
MP-8844 and VF゛PRM P-8845)
Inoculate and culture with shaking at 37°C for 1 day! ! Thereafter, the cells were brought into 2ω1 contact with 100a+l of L medium containing L-) ribdoan prepared in the same manner at a concentration of 200 pg/ml, and cultured with shaking at 37°C for 8 hours.

Shi Yoshikuni's group (Tryptone 10g Yeast extract 5g NaCl 5g Glucose 1g Distilled water 12 pH 7.2 Collect the bacterial cells using a centrifuge and indole 1
．． 5 g of sodium pyruvate, 1.5 g of ammonium acetate, 0.5 g of pyridoxal phosphate, and 5 g of Triton
The reaction was carried out for 5 hours. After the reaction is complete, add 1 portion of the reactant with water.
After diluting to 0 times, the supernatant obtained by centrifugation was analyzed for L-) lipoan produced by high performance liquid chromatography, and it was found that 2.1B/ 111 L-) Lipdore 7
Formation of ions was observed.

Reaction completed f! Add 50 ml of 10 times diluted solution 500 mackerel to an ammonia-type strongly acidic ion exchange resin (Diaion 5K-I).
B.Mitsubishi! ! After absorbing N through a column manufactured by L-) Ribbed Anne, L-) Ribbed Anne was eluted with an alkaline solution and concentrated to precipitate crude crystals of L-) Ribbed Anne. This was washed with amethane and dried to obtain 0.0% L-tributophane crystals.
7g was obtained.

As a control, a similar bi(L-) ribdoane was produced using a transformed strain containing the plasmid pB R322-tnaA, which does not contain the mi old-F fragment. Only 0.3g of was obtained.

[Brief explanation of drawings]

fpJ1 diagram and Figure 2 are respectively plasmid pMTP-
1 and pMTP-IR restriction enzyme cleavage maps.

Claims (1)

[Scope of Claims] 1. (a) A DNA fragment containing at least a promoter in the tryptoanase operon, a regulatory gene that controls this promoter, and a tryptoanase structural gene, and (b) a DNA that controls the autonomous replication ability of ColEl-based plasmids.
A novel plasmid characterized in that it consists of an A fragment and (c) a DNA fragment containing a gene controlling a growth control distribution system derived from the F plasmid. 2. DNA governing autonomous replication ability of ColEl-based plasmids
The plasmid according to claim 1, wherein fragment (b) is derived from pBR322. 3. Gene controlling the F plasmid growth control distribution system (c)
Claim 1 or 2, wherein is a miniF fragment.
Plasmids described in section. 4. A DNA fragment (a) containing at least a promoter of the tryptoanase operon, a regulatory gene that controls this promoter, and a tryptoanase structural gene,
The chromosomal DNA of B. enterica K12 strain was digested with the restriction enzyme BamH.
The plasmid according to any one of claims 1 to 3, which is a DNA fragment having a molecular weight of about 3.2 Kb obtained by excision with HindIII and HindIII. 5. The plasmid according to any one of claims 1 to 4, which is plasmid pMTP-1 or pMTP-1R. 6. The molecular weight is about 10.7 megadaltons, the cleavage sites for the restriction enzymes BamHI, HindIII, and EcoRI are 3, 2, and 2, respectively, and the molecular weight of the fragment cleaved by EcoRI is about 6. 0 megadaltons and about 4.7 megadaltons, and BamH
The cleavage fragments by I are about 6.4 megadaltons, about 2.7 megadaltons and about 1.6 megadaltons, and H
The plasmid according to claim 5, which is a plasmid pMTP-1, characterized in that the indIII cleavage fragments are about 5.5 megadaltons and about 5.2 megadaltons. 7. The molecular weight is approximately 10.7 megadaltons, the cleavage sites for the restriction enzymes BamHI, HindIII, and EcoRI are 3, 2, and 2, respectively, and the molecular weight of the fragment cleaved by EcoRI is approximately 6.0. megadalton and approximately 4.7 megadalton, BamH I
The cleavage fragments by Hi
6. The plasmid according to claim 5, which is a plasmid pMTP-1R, characterized in that the ndIII cleavage fragments are approximately 10.2 megadaltons and approximately 0.5 megadaltons. 8. Escherichia coli K12 microorganism transformed with plasmid pMTP-1 or pMTP-1R. 9. A DNA fragment containing at least a promoter in the tryptophanase operon, a regulatory gene that controls this promoter, and a tryptophanase structural gene;
In the presence of a culture of Escherichia coli transformed with a plasmid consisting of a DNA fragment controlling the autonomous propagation ability of the olE1-based plasmid and a DNA fragment containing a gene controlling the growth control distribution system derived from the F plasmid, or a processed product thereof. A method for producing L-tryptophan, which comprises reacting indole, pyruvic acid or a salt thereof, and ammonia or ammonium ion.