JPH01265887A - plasmid - Google Patents

Abstract

PURPOSE:To obtain a plasmid capable of improving the productivity of tryptophanase, by incorporating the original plasmid with the DNA domain containing tryptophanase structural gene (TPD) and a specific domain capable of controlling the discovery of TPD. CONSTITUTION:Bacteria produced by culture of e.g. Escherichia coli ATCC 27325 strain in a tryptone-contg. L culture medium is put to an enzyme treatment using e.g. lysozyme, and the resultant chromosome DNA is subjected to restriction enzyme treatment to obtain the TPD-contg. DNA domain (T- domain) and the regulator gene domain (P-domain) containing promotor and operator capable of controlling the discovery of TPD. Thence, a plasmid pBY-502 of Brevibacterium flavum MJ-233 origin is put to restriction enzyme treatment to obtain the DNA domain (B-domain) capable of autonomous growth in the Corynebacterium cells. A plasmid desired is then incorporated with said T, P, B domains etc., thus producing the objective plasmid pCRY-21 tan AI ca. 8.0 megadalton (ca. 12.3Kb) in molecular weight.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel plasmid, more specifically,
The present invention relates to a plasmid that expresses high tryptophanase production ability when a bacterium belonging to coryneform bacteria is transformed, and a microorganism transformed with the plasmid.

Tryptophanase catalyzes α, β-elimination reactions and β-substitution reactions, including L-tryptophan, 5-methyltryptophan, 5-hydroxytryptophan, 5-aminotryptophan, pyruvate, indole, 5-methylindole, 5-hydroxyindole, 5 amino indole,
etc. can be synthesized from the respective corresponding substrates. [Fermentation Association Journal Vol. 32 No. 7 1974 p. 298-
3l7, and Am1no acid and Nucl
eic acid, vol.

31 p, 113-11.8 (1975), etc.]. It can be applied to the quantification of tryptophan by utilizing this property, and to the quantification of pyridoxal pentaphosphate by utilizing the fact that four molecules of pyridoxal pentaphosphate are bound to this enzyme.

Many of the microorganisms that produce tryptophanase, which has high industrial utility value, are the enterobacteriaceae group, and tryptophanase using coryneform bacteria has many advantages in industrial applications and has a proven track record for amino acid and nucleic acid fermentation. The production of tophanase has hitherto been completely unknown.

The present inventors used recombinant DNA technology to introduce a tryptophanase biosynthesis gene derived from Escherichia coli into bacteria belonging to the coryneform bacteria, which do not originally have tryptophanase activity. We investigated the possibility of producing significant amounts of tryptophanase.

As a result, the present inventors found that (a) a DNA region containing the tryptophanase structural gene, (b) a regulatory gene region containing a promoter and operator capable of controlling the expression of the structural gene, and (c) a coryneform bacterium. We created a new plasmid containing at least a DNA region capable of autonomous replication within cells, introduced the plasmid into coryneform bacteria cells, and succeeded in transforming them into coryneform bacteria with tryptophanase activity. They have now made it possible to produce tryptophanase using coryneform bacteria, and have completed the present invention.

As a result, various enzymatic reactions that utilize tryptophanase activity have many industrial advantages, such as improved bacterial cell yield, reaction operability, suppression of bacterial cell-derived debris, and simplified purification and recovery process. was obtained, and it became possible to use coryneform bacteria that have a proven track record in industrial production.

Hereinafter, the structure of the plasmid newly created in the present invention will be explained in more detail.

"DNA region containing tryptophanase structural gene"
(hereinafter sometimes abbreviated as "T region") is a DNA region that encodes tryptophanase, an enzyme responsible for decomposing L-tryptophan into indole, pyruvate, and ammonia. In addition, the "regulatory gene region containing a promoter and operator that can control the expression of the tryptophanase structural gene" (hereinafter sometimes abbreviated as "P region") is
A DNA region that serves as a signal for the strain to produce tryptophanase, such as a DNA region containing a tryptophan promoter derived from Escherichia coli chromosomal DNA and an operator fraction; a tryptophanase promoter derived from Escherichia coli chromosomal DNA; Includes DNA regions that regulate its expression. The above tryptophan promoter and operator fraction can be, for example, a DNA region derived from plasmid pDR720 (manufactured by Pharmacia).

The microorganisms that are the source of the above T region and P region are not particularly limited, but in general, Escherichia coli ATCC23282, Escherichia coli ATCC2
3437, Escherichia coli ATCC 27325, etc. are advantageously used.

Furthermore, [DNA that can autonomously reproduce within coryneform bacterial cells]
"region" (hereinafter sometimes abbreviated as "region B") is a DNA region that can replicate and proliferate within coryneform bacterial cells, and is mainly sourced from plasmids that can exist within coryneform bacterial cells. Can be used. Therefore, as a source of the B region, coryneform bacteria such as Brevibacterium
7 Labam (Brevibacterium flavu)
m) Plasmid pBY-502 derived from M J-233 (Feikoken Kyoiku No. 3068) is preferably used.

The plasmid provided by the present invention is the T
In addition to the three DNA regions of the region, P region and B region,
Furthermore, if necessary, it can contain a "DNA region that can autonomously reproduce within cells of the genus Escherichia" (hereinafter sometimes abbreviated as "E region"), and this E region can replicate and proliferate within bacterial cells of the genus Escherichia. A possible DNA region,
Plasmids that can mainly exist in Escherichia bacterial cells can be used as a source. Therefore, as a source of the E region, plasmids pH3G39'8, pH3G39
9, pH3G298, pH3G299, pBR325
etc. are preferably used.

The plasmid provided by the present invention further includes a "DNA region containing a gene responsible for drug resistance," which is a gene capable of imparting the trait of drug resistance to a host microorganism.
(hereinafter sometimes abbreviated as "C region"), examples of which include a DNA region containing a chloramphenicol resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, etc. It will be done. This C region can be present in the plasmid of the present invention as a region separate from the T region, P region, B region and E region, or even if it is present in the B region and/or the E region. good.

The plasmid provided by the present invention is the T
Five DNs: area, P area, B area, E area and C area
Of the A region, it contains at least three regions, the T region, the P region, and the B region, as essential components, and a typical example has a molecular weight of 8.0 megadaltons (approximately 1
A plasmid with a molecular weight of 11.7 megadaltons (approximately 1.8 kb), which the inventors designated as plasmid rpCRY-21tnaA I
One example is one named 2J. Methods for preparing these two types of plasmids will be specifically explained in Examples below.

In addition, in this specification, the molecular weight of a plasmid is a value measured by agarose gel electrophoresis.

The plasmid of the present invention can be introduced into microorganisms, particularly bacteria belonging to coryneform bacteria, to express tryptophanase-producing ability.

The microbial strain that can serve as a host for the plasmid of the present invention is not particularly limited as long as it is a Coryneform bacterium, but generally Brevibacterium 1 flavum (Brevibacterium
vibacterium flavum)M J-2
A strain derived from No. 33 is preferably used.

Transformation of the above-mentioned bacterial strain with the plasmid of the present invention can be carried out by a method known per se, the details of which are shown in the Examples below.

17= Next, a method for culturing a strain derived from Brevibacterium flavum MJ-233 transformed with the plasmid of the present invention will be described.

Cultivation can be carried out in a conventional nutrient medium containing carbon sources, nitrogen sources, inorganic salts, etc. Carbon sources include glucose, ethanol, methanol, molasses, etc., and nitrogen sources include ammonia, ammonium sulfate, chloride, etc. Ammonium, ammonium nitrate, urea, etc. can be used alone or in combination. Further, as the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used. In addition, nutrients such as peptone, meat extract, yeast extract, cornstarch liquor, various vitamins such as casamino acid biotin, etc. can be added to the medium.

Culture is usually carried out under aerobic conditions such as aeration, shaking, etc. for about 2 hours.
It can be carried out at a temperature of 0 to about 40°C, preferably 25 to 35°C. The pH during culturing is maintained at around 5 to IO1, preferably around 7 to 8, and the pH during culturing can be adjusted by adding acid or alkali.

The carbon source concentration at the start of culture is preferably 1 to 5% by volume,
More preferably, it is 2 to 3% by volume. In addition, the culture period can generally be 1 to 78 days, with the optimal period being 1 to 3 days.

By collecting bacterial cells from the culture thus obtained, a tryptophanase active fraction can be obtained.

The present invention has been described above, and will be explained more specifically with reference to the following examples. However, it should be understood that the examples are to be considered as an aid to give a concrete understanding of the present invention, and are not intended to limit the scope of the present invention.

Example 1 Δ! ! L medium (tryptone 10 g, yeast extract 5 g, glucose 1 g, NaCl 3 g, distilled water 1 (2; pH 7,
2) 1 OOml was dispensed into a 500ml Erlenmeyer flask and sterilized at 120°C for 15 minutes.

This medium contains Escherichia coli (Eschcrichi).
a c.

1i) Inoculate K12 ATCC27325 and inoculate 370
After culturing at C for 15 hours, 2 ml of this culture solution was taken, inoculated into 100 ml of the above medium, and cultured again at 37° C. for 4 hours.

After culturing, the entire volume of the culture solution was centrifuged (8000Xg
x 15 minutes, 4°0) to collect the bacteria, and the bacterial cells were diluted to 5QmM.
Tris buffer (pH 8,0)-10mM EDTA・
It was suspended in 50ml of 2Na solution. Next, lysotium was added to a final concentration of 2 mg/ml, and after standing for 5 minutes, 6 ml of 10% sodium dodecyl sulfate solution was added.
It was kept warm at 65°C for 30 minutes. Add 5M to this lysate
Add 15 ml of NaCl solution, cool at 0°C for 1 hour, and centrifuge the entire volume (12000Xg, 60 minutes, 4°C.
)L, separate the upper groove fraction, add twice the amount of ethanol,
After mixing, centrifuge (5000Xg, 10 minutes, 4°O)
did. The resulting precipitate was diluted with 10mM) Lys buffer (pH 7).
,5)-Dissolved in 1mM EDTA・2Na solution,
Funinol treatment (protein removal treatment) and RNA degrading enzyme treatment were performed to finally obtain 1.5 mg of DNA.

(B) Preparation of DNA region containing tryptophanase structural gene 25 μg of the chromosomal DNA prepared in (A) above was treated with restriction enzymes Hindll and BamHI (5 units each).
) was used to cut the chromosomal DNA in a 30°011 hour reaction.
A HindlI and BamHI decomposition product solution was prepared.

Plasmid pBR3251Ig was added to this digest solution using restriction enzymes H1ndlI and BamHI (each l unit
Mix the decomposition product solution obtained by reaction at 30°a for 1 hour using 50mM Tris buffer (pH 7).

6), 10mM dithiothreitol, 1mM ATP, 1
Add 0 mM MgCl2 and 1 uniL of T4 ligase (concentrations of each component are final limits), 16
The reaction was carried out at ℃ for 15 hours to achieve binding.

Using this solution, a conventional method [M, MandeLA, Hig.
a; J, Mo1. Biol,, 53.159 (
1970)] according to Escherichia Colli (Esc.
hcrichia1l-coli) Kl2 strain (tryptophanase-deficient mutant and tryptophan auxotrophic mutant) was transformed, and selective medium (K2HPO47I, KH2PO422, (
NH, )250.17, Mgs O, 7H200゜1
2. Casamino acid 5g, DL-5-methyl-tryptophan 50mg, glycerin 22, agar 202, pure water 1 (l
smeared on. The growing strain on this medium was inoculated into L medium containing ampicillin at a final concentration of 50 μg/ml.
After culturing at 7°C for 7 hours, the bacterial cells were centrifuged (8000
Xg for 10 minutes at 4°C). Tryptophanase activity was examined using this bacterial cell.

50 mg of wet bacterial cells was added to 1 ml of the reaction solution (0.1 M phosphate buffer pH 8, 0.10 mM tryptophan, 0.1% Triton X-100) and reacted at 37°C for 30 minutes. When the produced indole was colored using p-dimethylbenzaldehyde, a red colored strain was obtained. From this strain, the alkaline-3DS method [T, Man
iatis, E., F., Fritsch.

J, Sambrook;
(1982) p. 90-91], and the plasmid was incubated with BamHI and H
When pBR325 was cut with HindllI and Ba was examined for molecular weight using agarose gel.
Approximately 3.2 kb of DNA was inserted into the mHI site (
pB R325-tnaA).

Furthermore, when the above host was retransformed using this plasmid solution, a strain that grew on the selective medium at a frequency of about 105 cells/μg DNA appeared.

(C) Plasmid pB R325 prepared in (B) above
10 μg of tnaA and 23 μg of plasmid pCRY-2 prepared in the reference example described later were treated with the restriction enzyme H1ndllI (2
After inactivating the restriction enzyme by inactivating it by heating at 65°C for 110 minutes, the final concentration of each component in the inactivation solution was 50mM)! JS buffer pH 7.

6.10mM MgCI 2.10mM dithiothreitol, 1mM ATP and T4 ligase units
Each component was strengthened so that

(D) Using the DNA conjugate prepared in (C) above, Escherichia coli (
Escl) crichia coli) K-12 strain (tryptophanase-deficient mutant and tryptophan auxotrophic mutant) was transformed. A plasmid was extracted from the strain grown on the aforementioned selective medium using the alkaline-3DS method, the plasmid was cut using the restriction enzyme HindI[I, and the cut fragment was examined using agarose gel electrophoresis. A plasmid was obtained in which about 4.1 kb of DNA was inserted into the HindI site of tnaA. The molecular weights obtained when this plasmid was digested with various restriction enzymes are shown in Table 1 below.

Table 1 BamHr 2 3.8 (5,8) 4.2 (6,5
) Hind IIT 2 2.7 (4,1) 5.3
(L2) Sma I 1 8.0 (12,3) E c
oRI l 8.0 (12,3) Sal I
3 0.8 (1,3) 3.0 (4,5) 4.2
(6,5) P st I 2 3.1 (4,8) 4
．． 9(7,5)*() indicates kb.

The plasmid characterized by the above restriction enzymes was pCRY-
It was named 21tnaAl. This plasmid pCRY-
The restriction enzyme map of 21tnaA1 is shown in Table 1.

(E) Preparation of protoplasts Brevibacterium flavum MJ233 plasmid-depleted strain was cultured in 100 tJ of the above A medium until the early logarithmic growth phase, penicillin G was added at 0.2 units/ml, and further 2 After culturing with shaking for an hour, the bacterial cells were collected by centrifugation, and the bacterial cells were treated with 0.5M thorium succinate.
0mM) Lys buffer, 20mM calcium chloride and 20mM)
TSMC consisting of mM magnesium chloride (pH 7,5)
Wash with 3MC buffer 5 and then 4 +nFi/ttJ
Suspend in 10+J of 73MC buffer containing lysozyme, 100OU/+nβ and achromopeptidase at 30°C.
Protoplast formation was attempted by reaction at C for 16 hours. After the reaction, after centrifugation at 3000 rpm for 10 minutes, the protoplasts were washed with TSMC8MC buffer 2 and 3LI
resuspended in lβ TSMC buffer.

(F) 200 protoplasts obtained in (E) above and plasmid pCRY-21tna obtained in (D) above.
After mixing Al and cooling with water, polyethylene glycol 600
0 to a final concentration of 20%, cooled on ice for about 3 minutes, added TSMC5MC buffer, and centrifuged (30%).
00 rpm, 10 minutes), the cells were resuspended in 3 mN of A medium containing 5 M sucrose at 0°, and cultured at 30° for 0.2 hours.

This culture solution was mixed with regeneration medium (urea 22, (NH4) S
O472, KH2'PO40,51, 2HPO40,
! l, Mg5O, 7H200,52, Fe50.・7
H7H2O6, Mg5Ot・4-6H206mg, yeast extract 2.5i Casamino acid 7,52, biotin 200
μg 1 thiamin hydrochloride 1. Omg, sucrose 171
2. Glucose 52, Gelatin 152, Agar (DIFC)
○) 82, the total volume was made 112 with pure water) was inoculated, and 30
Colonies that appeared after culturing for 0.3 to 15 days were inoculated into medium A containing 7 μg/mλ of chloramphenicol, and chloramphenicol resistance was confirmed. The obtained chloramphenicol-resistant strain was tested for indole production in the reaction solution shown below.

50 mg of wet bacterial cells was added to 1 ml of the reaction solution (0.1 M phosphate buffer pH 8, 0.10 mM tryptophan, 0.1% Triton X-100) and reacted at 37°C for 30 minutes. When the produced indole was colored using p-dimethylbenzaldehyde, a red colored strain was obtained. A plasmid was extracted from this strain by the alkaline-5DS method, cut with HindIII, and examined using agarose gel electrophoresis.
About 4.1 kb of DNA was inserted at the H1ndl[[ site.

When this plasmid was digested with the same various restriction enzymes as shown in Table 1 above, the same results as in Table 1 were obtained. This led to Escherichia coli (E.

Plasmid pcRY − constructed using coli)
It was confirmed that 21tnaA 1 replicates in Brevibacterium flavum, a coryneform bacterium, and expresses tryptophanase derived from E. coli chromosomal DNA.

Novel plasmid pCR obtained as described above
The Brevibacterium flavum MJ233-derived strain harboring Y-21tnaA1 was named Brevibacterium flavum MJ233GEIO03 and was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Higashi 1-1-3, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, in the Showa era. It has been deposited on January 8, 1963, with accession number: FERM P-9804.

Example 2 (A) Preparation of plasmid pMTP-3 Medium (Tryptone 10g1 Yeast extract 5g1 Glucose
7.1g, NaC15gs distilled water 14; pH7.2)
Dispense loOml into a 500ml Erlenmeyer flask,
Sterilization was performed at 120°C for 15 minutes.

Ampicillin was added to this medium to a final concentration of 50 μg/lβ, and Escherichia coli (Es
chcrichia coli) YK 3 0
0 5 [FE RMBP-1736 (FE
RM p-9622)] and cultured at 37°C for 15 hours, 2ml of this culture solution was taken and inoculated into 100ml of the above medium, and cultured again at 37°C for 4 hours. .

After culturing, the entire volume of the culture solution was centrifuged (8000Xg
, 15 minutes, 4°C) to collect bacteria, and remove alkaline from the bacterial cells.
3DS method [T, Maniatis, E, F
.

Fritsch, J., Sambrook
; “Molecular cloning” (
1982) p90-91] to obtain pMTP'-3.

(B) Plasmid pMTP-3I prepared in (A) above
Oμg and plasmid pCRY prepared in the reference example below
-23 μg was added to the restriction enzyme EcoRI (20 units
) for 1 hour at 37°C, and inactivated the restriction enzyme by heating at 65°C for 10 minutes. 50mM Tris buffer pH 7, 6, 10mM MgC
I2.10mM dithiothreitol, 1mM ATP
and T4 ligase, each component was strengthened to 1 unit, and DNA was bound by incubating at 16°C for 15 hours.

-19= (C) Preparation of protoplasts Brevibacterium flavum MJ233 plasmid-depleted strain was cultured in the above A medium at 100 + oj2 until the early logarithmic growth phase, and penicillin G was added at a concentration of 0.2 units)/mβ. , further shake culture for 2 hours, collect the bacterial cells by centrifugation, and add 0.5M sodium succinate,
20mM Tris buffer, 20mM calcium chloride, 20mM
TS consisting of mM magnesium chloride and (pH 7,5)
MC3MC buffer 50 washes, then 4 mg/ml
Lysozyme, 1000 units/ml! Suspend in 1.0 mβ of TSMC buffer containing achromopeptidase,
Protoplast formation was attempted by reaction at °C for 16 hours.

After the reaction, centrifuge at 3000 rpm for 10 minutes,
Wash the protoplasts with TSMC buffer 20-3m
Resuspend in N 13MC buffer.

200 μl of this protoplast and the DN obtained in (C) above
A solution 10CI! After cooling with water, add polyethylene glycol 6000 to a final concentration of 20%, cool on ice for about 3 minutes, add TSMC=20-Buffer 5mj2, and centrifuge (3000r
p.m.

10 minutes), then A medium 3 containing 0.5M sucrose.
The cells were resuspended in A-β and cultured at 30°C for 2 hours.

This culture solution was mixed with regeneration medium (urea 211 (NH4)S) containing chloramphenicol 7 μg/rnβ (final concentration).
O472, KH2PO40,s9, K2HPO40,5
9, MgSO4' 7H200,59, Fe50. '
7H206mliis MnS O4・4~6 H20
6mgz Yeast extract 2.5i Casamino acids 7.5i Biotin 200μg 1 Thiamine hydrochloride 10mg, Sucrose 1712, Glucose 5. ? , gelatin 152, agar (DIFCO) 87, and pure water to make the total volume 1a) and cultured at 30° for 0.3 to 15 days. The chloramphenicol resistance was confirmed.

These chloramphenicol-resistant strains were tested for indole production in the reaction solution shown below.

Reaction solution (0.1M phosphate buffer pH 8, 0.10mM tryptophan, 0.1% Triton X-100)], ml
50 mg of wet bacterial cells were added to the mixture and reacted at 37°C for 30 minutes. When the produced indole was colored using p-dimethylbenzaldehyde, a red colored strain was obtained.

(D) A plasmid was obtained from the strain obtained in (C) above that was resistant to chloramphenicol and exhibited tryptophanase activity according to the method described in (A) above. This plasmid was cut with the restriction enzymes shown below, and the molecular weight was measured using agarose gel electrophoresis.

Table 2 Restriction enzyme Recognition site number Molecular weight Megadalton H1ndI
II 2 2.7.9.0EcoRI
3 13.3.8.6.6K pn I
3 2.0.4.2.5.5P st I
6 0.45.1.0.1.2.1.4
．． 2.4.5,25 Saffl 5 0.7.0.9.2.3
．． 3.6.4.2 SmaI 4 0.35, ■, 25.4
, 1.6.0 The plasmid characterized by the above restriction enzyme was named pCRY-22tnaA2.

(E) Introduction of plasmid pCRY-22tnaA2 into E. coli Using 1 μg of plasmid pCRY-22LnaA2, a conventional method [M, Mandel, A, H4ga; J
, Mol.

Biol, 53, 159 (1970)].
E.coli) K12 strain (tryptophanase-deficient mutant and tryptophan auxotrophic mutant) was transformed into a selective medium containing chloramphenicol at a final concentration of 30 μg/m4 (7 g of K2HOP4, 2 g of KH2PO4).

(NH4)2 S○+1g, MgS○, 7H200
゜1g, casamino acid 5g, DL-5-methyl-tryptophan 50 mgs glycerin 2gs agar 20g1
When smeared on pure water (112) and cultured at 37°C for 24 hours, approximately 10'ce1 per 1 μg of plasmid DNA was found.
One virulently added strain was obtained.

Ten of these transformed strains were treated with alkaline-3DS.
When the plasmid was extracted by the method, cut with various restriction enzymes, and its molecular weight was measured by agarose gel electrophoresis, it was found that Brevibacterium flavum MJ233GE
Plasmid pCRY-22tnaA obtained from 1001
2 (Table 2). This means pC
It was shown that RY-22tnaA 2 is a complex plasmid that replicates in Escherichia coli and a strain derived from Brevibacterium flavum MJ233, a type of coryneform bacterium, and that the tryptophanase gene is present on the plasmid. This suggests that there is.

The strain derived from Brevibacterium flavum MJ233 carrying this novel plasmid pCRY-22tnaA2 is Brevibacterium flavum MJ233GE1.
It was named 001 and was installed at the Institute of Microbial Technology, Agency of Industrial Science and Technology, Higashi 1-1-3, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, in 1988.
As of October 20, 2015, the accession number is: Microtechnology Research Institute No. 9665 (
FERM P-9665).

Example 3 =24- Medium (0.4% urea, 1.4 g ammonium sulfate.

KH2P0. 0.5%, K2HP0. 0.05%,
MgSO4.7 H200-05%, CaCl222
H202ppms FeSO4・7 H2O2ppm,
Mn5Ot・4-6H202ppm, Zn5O,・
71-120 2ppmsNaCQ 2ppmz Piotine 200 pg/(1, Thiamine-HCQ 11
00p/(1, tryptone 0.1%, yeast extract 0
．． After dispensing 100 mff of 1%) into a 500 m Erlenmeyer flask and sterilizing (pH 7.0 after sterilization), Brevibacterium flavum MJ233GE1001 and Brevibacterium flavum MJ233GE1003 strains were each inoculated, and glucose was aseptically finalized. Concentration 2% (W/V)
, chloramphenicol was added at a concentration of 10 μg/mff, and cultured with shaking at 30° C. for 15 hours.

Next, the main culture medium (ammonium sulfate 2.3%, KH2
PO40.05%, K2HP0. 0.05%, Mg5
O, 7H, OO, 05%, FeSO4 7H2020
ppmXMnSO4-nH202'Oppmx Piotine 200μg/12, Thiamine/HCQ 100μg1
0. , tryptone 0.3%, yeast extract 0.3%)
Pour 2g of ooomp into a stirring tank and sterilize it (120°
C for 120 minutes), then aseptically add glucose to a final concentration of 2%.
(W/V), chloramphenicol was added at 10 μg/mQ, and the preculture was added at 20 mQ each.
Added, rotation speed 1100 () rp, ventilation amount 1 vvm
The cells were cultured for 10 hours at a temperature of 33°C and a pH of 7.6.

Note that glucose was continuously added about every 1 to 2 hours so that the concentration of the medium during culture did not exceed 2% by weight.

After culturing, the cells were collected from 400 mQ of the culture by centrifugation, and the cells were washed twice with demineralized distilled water and added to the reaction solution [indole 5 g, DL-serine 20 g, 1 pyridoxal-5
'-Phosphate lQmg, KCl2 2g.

After suspending in 1000 m4 of 1000 mff of distilled water (adjusted to pH 8.0 with 5N-KOH), the suspension was placed in a 2 g aeration stirring tank, the number of revolutions was 30 Orpm, and the temperature was 37.
The reaction was carried out at ℃ and pH 8.0 for 10 hours. After the reaction was completed, the reaction solution was centrifuged (6000 rpm for 5 minutes,
A supernatant was prepared at 4°C) and L-tryptophan in the supernatant was measured. In addition, 500 mff of the reaction completed liquid was passed through a column of ammonia-type strongly acidic ion exchange resin (Diaio 73 IIBJ, manufactured by Mitsubishi Chemical Corporation), extracted with an alkaline solution, and concentrated to obtain crude L-1-lyptophan. Crystals were precipitated. The results are shown in Table 3.

Pouch 3 As a comparative example, a similar reaction was carried out using a host cell that did not carry a plasmid, and as a result, the production concentration of L-tryptophan was 0.05 g/ff or less. In this case, the medium used for preparing host cells was the main culture medium with chloramphenicol removed.

Example 4 The same reaction solution used in Example 3 was added to the reaction solution [indole
4 mmoQ, sodium pyruvate 200 mmo
ff, ammonium acetate 100 mmoQ, pyridogyza-5'-phosphate Q, 4 mmoQ, total amount 1000 mff with distilled water (5N-
The same operation as in Example 3 was performed except that the solution was changed to (prepared with NaOH)].

Indole was added sequentially so that the concentration in the reaction solution did not exceed 4mM, and the final total amount added was 30mM.
The reaction was carried out until 2 equivalents were obtained.

The results are shown in Table 4.

Table 4 As a comparative example, when host cells (not containing plasmids) were used as in Example 3, no production of L-tryptophan was observed.

Reference Example Construction and Properties (A) Preparation of Plasmid pBY502 Plasmid p
BY502 is Brevibacterium, flavum MJ23
3 [FERM BP-1497 (FERM p
-3068)] is a plasmid with a molecular weight of about 30 megadaltons newly isolated from JP-A-61-17975.
This is a plasmid described in Publication No. 1.

On the other hand, plasmid pBY502 was prepared as follows.

Semi-synthetic medium, A medium (urea 2 gl (NH 4) 2
S O 47g, 2HPO4 0.5g, 'KH
2PO4 0.5g.

Mgso, 0.5g, FeS○+・7H20 6
mg1MnS O 4 ・4-6 H 20 6
mgz Yeast Extract 2.5g1 Casamino Acid 5g, Piotin 200μg1 Thiamine Hydrochloride 200μg1 Glucose 20g1 IQ with Pure Water) Brevibacterium flavum MJ233 [FERM BP-149
7 (FERM P-3068)] was cultured until the late logarithmic growth phase, and the bacterial cells were collected. The obtained bacterial cells were
A buffer containing lysozyme [25 m
M) Lis(hydroxymethyl)aminomethane, 10mM
EDTA.

5QmM glucose]20m0. 1 at 37°C.
Allowed time to react. Alkali = SDS liquid [Q, 2
N NaOH, 1% (W/V) SDS Co 40
mQ was added, mixed gently and allowed to stand at room temperature for 15 minutes.

Next, this reaction solution was added with a potassium acetate solution [5M potassium acetate solution 5Qm12, acetic acid 11.5m12, pure water 28.
5m (mixture of 2), 30mD, was added, mixed well, and left to stand in ice water for 15 minutes. Transfer the entire amount of the lysate to a centrifuge tube, and centrifuge at 15,000 x g for 10 minutes at 4°C. It was subjected to separation to obtain a supernatant.

This was suspended in an equal amount of phenol/chloroform solution (phenol/chloroform mixture in ml), transferred to a centrifuge tube, and centrifuged at 15°000×g for 5 minutes at room temperature to collect the aqueous layer.

15. Add twice the amount of ethanol to the aqueous layer and leave it at -20°C for 1 hour, then at 4°C for 10 minutes. The precipitate was collected by centrifugation in OOOXg.

After drying the precipitate under reduced pressure, TEE buffer [Tris 10mM, ED
TA 1mM, adjusted to pH 8.0 with HCl2 12mQ
, dissolved in. Add cesium chloride solution to the dissolution solution [liquid containing 10 g of 5 times concentrated TE mild buffer solution, 15+nQ and lomg/m+2
Add 1 mQ of ethidium bromide solution to bring the density to 1.
It was adjusted to 392g/mff. This solution was heated at 12°C for 4
Centrifugation was performed at 116,000×g for 2 hours.

Plasmid pBY-502 is found as a lower band in the centrifuge tube upon UV irradiation. By extracting this band from the side of the centrifuge tube with a syringe, a fraction containing plasmid pBY-502 was obtained.

Next, this fractionated solution was treated with an equal volume of isoamyl alcohol four times to extract and remove ethidium bromide, and then dialysis was performed against a TEE solution. After adding 3M sodium acetate solution to the dialysate containing the plasmid pBY-502 obtained in this way to a final concentration of 30mM, 2
Double the volume of ethanol was added, and the mixture was allowed to stand at -20°C for 1 hour. This solution was centrifuged at 15,000×g to
Precipitate A, and approximately 20 μg of plasmid pBY-502
I got it.

(B) Preparation of Plasmid pH3G398 Plasmid pH3G398 is a 1.4 megadalton plasmid that is purified in Escherichia coli and expresses chloramphenicol resistance, and can be purchased from Zenshuzo.

(C) Construction of complex plasmid pCRY-2 Add restriction enzyme H to 0.5 μg of plasmid pH3G398 (manufactured by Zenshuzo Co., Ltd.)
1ndlI [(5 units) at 37°C,
The plasmid DNA was completely degraded by reacting for 1 hour.

Plasmid I) BY5022pg prepared in (A) above
Add restriction enzyme H 1ndl [(l units) to 3
The reaction was carried out at 7°C for 30 minutes to partially degrade the plasmid DNA. Both plasmid DNA digests were mixed and heated at 65°0 for 10 minutes to inactivate the restriction enzymes, and the final concentration of each component in the inactivation solution was 5Qm.
M) Lys buffer pH 7.6, 10mM Mg (12, 10
Each component was enriched with mM dithiothreitol, 1 mM ATP, and 1 unit of T4 ligase, and incubated at 16°C for 15 hours. Escherichia Co. using this solution! JJM109:1-impitent cells (manufactured by Zenshuzo Co., Ltd.) were transformed.

The transformed strain was treated with chloramphenicol (30 μg/mQ final concentration) and 100 μg/mQ (final concentration) I
PTG isopropyl-β-D-galactopyranoside and 100 pg/mQ (final concentration) X-ga12
Medium containing 5-bromo-4-chloro-3-indolyl-β-D galactopyranoside (tryptone 10g, yeast extract 5g, NaCQ 5g 1 pure water 1121) H7
．． 2) was cultured at 37°C for 124 hours to obtain a viable strain. Among these growing strains, those growing in white colonies were selected, and each plasmid was subjected to the alkaline-5DS method (T, Maniatis, E-F, Fr1ts).
ch, J., Sambrook, “Mo1ecu
lar Cloning” (1982) p90~
92).

(D) Preparation of protoplasts Brevibacterium flavum MJ233 plasmid-depleted strain was cultured in Lomff's A medium to the early logarithmic growth phase, penicillin G was added at a concentration of 0.2 units/mQ, and the mixture was further shaken for 2 hours. Culture, collect bacterial cells by centrifugation, and add 0.5 M sodium succinate to 20 m
M Tris buffer, 20mM calcium chloride and 20mM
TSMC3 consisting of 0mM magnesilla chloride) H7,5)
Washed with MC buffer 50 and then suspended in TSMC3MC buffer 1 containing 4 mg/mff lysozyme and 1000 units/mQ macromopeptidase and incubated at 30°C for 16
Glotoplast formation was attempted through a time reaction. After the reaction was completed, the protoplasts were centrifuged at 3000 rpm for IO minutes, and the protoplasts were washed with TSMC3MC buffer 2f. TS
Resuspended in MC buffer.

(E) Transformation of the complex plasmid into coryneform bacteria. Mix 200 μα of the protoplasts obtained in (D) above with the plasmid DNA obtained in (C) above, and after cooling with water, add polyethylene glycol 6000 to a final concentration of 20 %, cool on ice for about 3 minutes, and add TSMC3M.
Add C buffer 2 and centrifuge (3000 rpm, l
0mln), then A medium 3 containing 0.5M sucrose.
The cells were resuspended in m4 and cultured at 300C for 12 hours.

This culture solution was mixed with regeneration medium containing chloramphenicol 7 μg/mQ (final concentration) (urea 2g1 (NH4)so,
7gXKH2PO40.5g, to 2HPO40.5
g, Mg5O<-7H200,5gXFeSO4・7
H206mg, Mn5O, ・4-6HzO6mg5 Yeast extract 2.5g1 Casamino acid 7.5g, Biotin 20
0 μg 1 Thiamine hydrochloride 10 mg, Shoe 70-su 171
g, glucose 5g, gelatin 15g, agar (DIFC)
8g of O), the total volume was made up to 1Q with pure water), and incubated at 30°
C. Colonies that appeared after 3 to 15 days of culture were inoculated into medium A containing chloramphenicol 7 μg/m+2, and chloramphenicol resistance was confirmed.

(F) A plasmid was obtained from the chloramphenicol-resistant strain obtained in (E) above using the method described in (A) above. The molecular weight of this plasmid was measured using various restriction enzymes (Table 5).

Table 5 Restriction enzyme recognition site number Molecular weight water megadalton H1nd
I[[22,7(4-1) 1.4(2-2)K pn
I l 4.1 (6,3) EcoR
I l 4.1 (6,3) BamHI
2 2.2(3,4) 1.9(2,9)S
ma I2 4.0 (6,1)
A plasmid characterized by the above restriction enzyme was pCR
It was named Y-2.

(G) Escherichia coli HB 101 competent cells (manufactured by Takara Shuzo) were transformed using the plasmid pcRY-2 obtained in (F) above.

When the transformed strain was selected on a medium containing floramphenicol at a final concentration of 30 μg/mQ, it was found that pCRY-2
Transformants exhibiting chloramphenicol resistance were obtained at a frequency of 105 cells/μg. Plasmids were extracted from 10 of these transformed strains by the alkaline-3DS method, cut with various restriction enzymes, and their molecular weights were measured by agarose gel electrophoresis. It was equivalent to the obtained plasmid pCRY-2 (Table 5).

This indicates that pcRY-2 is a complex plasmid that replicates in Brevibacterium flavum MJ233, a type of coryneform bacterium, and Escherichia coli. This shows that the vector is expressed between both bacterial genera, and is considered to be an industrially useful vector.

In addition, Brevibacterium flavum MJ233GE101 transformed with complex plasmid pCRY-2
The strain was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Higashi 1-1-3, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, on January 8, 1986, with accession number: FERM P-
9801).

(H) Preparation of plasmid pc RY-2 Semi-synthetic medium A medium (2 g of urea, 47 g of (NH4)2SO)
1, 2HPO40.5g, KH2P0. 0.5g5M
g5O,' 7H200,5g, Fe50.・7H27
H2O6MnSO4' 4-f3) I2o 6mg5
Yeast extract 2.5g, Casamino acid 5gs Bithion 2
00μg1 thiamine hydrochloride 200μg, glucose 20g
1 Pure water 112) 100 m (2) was dispensed into a 500 mQ Erlenmeyer flask and sterilized at 120°C for 15 minutes.

Add chloramphenicol to this medium at a final concentration of 15 μg/
mQ, and further add Brevibacterium.
Flavum MJ233GE101 (FERM P-9
801) and cultured with shaking at 30°C for 20 hours. After culturing, the entire volume of the culture solution was centrifuged (8
000Xg for 15 minutes at 40C) to collect the bacteria, extract the plasmid from the bacteria by the alkaline-3DS method, and transform it into pCR.
Y-2 was obtained.

[Brief explanation of the drawing]

FIG. 1 is a restriction enzyme map of plasmid pCRY-21tnaA1. −40=

Claims (1)

[Claims] 1. (a) DNA containing a tryptophanase structural gene
(b) a regulatory gene region containing a promoter and operator capable of controlling the discovery of the tryptophanase structural gene; and (c) a DNA region capable of autonomous replication in coryneform bacterial cells. Plasmid. 2. (d) DN capable of autonomous proliferation within Escherichia cells
The plasmid according to claim 1, further comprising an A region. 3. The plasmid according to claim 1 or 2, which has a DNA region containing a gene responsible for drug resistance. 4. The plasmid according to any one of claims 1 to 3, which is pCRY-21tnaA1. 5. The plasmid according to any one of claims 1 to 3, which is pCRY-22tnaA2. 6. Plasmid pCRY-21tnaA1 or pCRY
Brevibacterium flavum MJ233 transformed with -22tnaA2.