JPH03119992A - New dna - Google Patents

Abstract

PURPOSE:To obtain DNA regions (a) containing a gene governing replicating and proliferating function of a plasmid in a strain cell belonging to a coryneform bacterium (A) and a plasmid vector, replicative and proliferative in the coryneform bacterium (A) by introducing the aforementioned DNA regions (a) thereinto. CONSTITUTION:The following new DNA regions and plasmid vector are obtained, a DNA region (a) of 1.1kb whole length, derived from a plasmid pBY502 and containing a gene governing replicating and proliferating function of a plasmid in a strain of Brevibacterium.flavum MJ-233 which is a coryneform bacterium (A), a DNA region (a) of 2.9kb whole length derived from a plasmid pBY503 and a plasmid vector, replicative and proliferative in the coryneform bacterium (A) in which the DNA regions (a) are introduced. A DNA fragment, etc., containing a structural gene capable of coding various industrially useful substances are integrated into the plasmid vector, introduced into a host microorganism and cultured to enable stable and efficient production of the useful substances coded with the useful gene.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel NA region that controls the replication and propagation function of plasmids in coryneform bacteria.

(Prior art) Coryneform bacteria including Brevibacterium bacteria are industrially useful microorganisms that produce amino acids, organic acids, purine nucleotides, etc., but breeding and improvement of bacterial strains by introducing recombinant DNA technology has not been possible. , Escherichia coli, etc., and the development of industrially useful plasmid vectors that host coryneform bacteria is particularly desired.

In general, with regard to the stability of constructed plasmids within the host, a number of genetic instability cases have been reported, such as plasmid shedding from the host during culture and loss of inserted genes. The reason for this is the size of the recombinant plasmid, and depending on the useful gene to be inserted, it may be 8 kb long, such as Tributov7 Oberon, making it difficult to clone into a vector using a restriction enzyme that does not exist in the inserted gene. becomes. Therefore, the development of miniaturized vector plasmids is desired.

In addition, transborane-like sequences present on plasmids (
G, Grandi, M, Motles and V,
Sgaramella: plasmid, 6. p99(
1981) and some DNA regions are known to cause unstable replication and proliferation, and it is desired to purify only the DNA region on the plasmid that controls the replication and proliferation function.

[Problem to be solved by the invention]

Therefore, as a result of intensive research, the present inventors found that the DNA region that controls the replication and propagation function of plasmids in coryneform bacteria is a 1°1 kb DNA region derived from plasmid pBY502.
The present invention has been completed by discovering that the present invention exists within the A fragment or within a 2.9 kb full-length DNA fragment derived from plasmid pBY503, and that plasmids constructed by incorporating these DNA fragments can replicate and proliferate within coryneform bacteria. reached.

(Means for Solving the Problems) Thus, according to the present invention, plasmid pBY502, which contains a gene governing the replication and proliferation function of the plasmid, is produced in a strain derived from Brevibacterium flavum MJ-233, which is a coryneform bacterium (A). The total length of the origin is 1. i kb DN
A region (a) and a full-length 2.9 kb DNA region (a) derived from plasmid pBY503, and these DNAs
A plasmid hector capable of replicating and propagating in a coryneform bacterium (A) into which region (a) has been introduced is provided.

Plasmid p serving as the source of the DNA region (a) of the present invention
B'1502 was developed by the inventors of the present invention from Brevibacterium flavum.
vam) MJ-233 (a plasmid newly isolated from FERN BP-14971, the details of which are disclosed in JP-A No. 63-36787.
Plasmid pBY503 was created by the inventors of the present invention using Brevibacterium stathionis.
ium 5tationis) 1FOI2144 (
FERM BP-2515), and its details are disclosed in JP-A-1-95785.

The DNA region (a) of the present invention is the plasmid pBY described above.
502 or plasmid pB'1503 with an appropriate restriction enzyme, and is not particularly limited as long as it has the function of replicating and propagating the plasmid in coryneform bacteria. As a specific example, for example, restriction enzyme 5ac1 and Bam from plasmid pB'1502
A DNA fragment with a length of L 1 kb was excised by restriction enzymes Sma and Sp from plasmid pBY503.
The DNA fragment may be a 2.9 kb long DNA fragment excised by the DNA fragment, or the entire DNA fragment, or a partial fragment thereof. 1, +kb of DNA above
Table 1 shows the number of sites recognized by various restriction enzymes on the fragment and the molecular weight (length) of the cleaved fragment.
Table 2 shows the length. In addition, the molecular weight (length) of the plasmid or DNA fragment described in this specification is the value measured by the 7-galose gel electrophoresis method.

Table 1 Restriction enzyme recognition site number Molecular weight [megadalton] 5t)
hl+0.6(0,9)”
,01(0,2)BaJ21 1 0.
4(0,6), 0J(0,5)Cffal
1 0.6(1,0), O,I(0,
I)=() indicates the length (kb).

Table 2 Restriction enzyme recognition site number Molecular weight [megadalton] Pst
l 2 1.4(2,2), 0.5(0,
7) Xbal 3 1J (2,0), 0.
5 (0,8).

0゜+(0,1) 0: The value in parentheses indicates the length (kb).

As a method for constructing plasmid hector using the above DNA fragment, Brevibacterium flavum MJ-
233 (FERN BP-1497) to plasmid p
BY502DNA was extracted and treated with restriction enzymes BamHl and SacI to obtain a DNA fragment with a length of 1 and 1 kb, or Brevibacterium stationis 1FOI.
Plasmid pBY503DNA was extracted from 2+44 (FERM BP-2515), and restriction enzyme Smal
A DNA fragment with a length of 2.9 kb is obtained by the and Sp old treatment. Next, add any of the DNA fragments obtained above and plasmid pH5G398C (manufactured by Takara Shuzo) or plasmid pH
It can be produced by combining 5G298 (manufactured by Takara Shuzo) with a substance treated with a similar restriction enzyme through enzyme treatment.

The plasmid vector constructed in this way, which contains a gene governing the replication and proliferation function and is capable of autonomous replication in coryneform bacteria, further contains a DNA fragment containing a drug resistance gene,
Industrially useful plasmid vectors that incorporate ()NA fragments containing stabilizing genes, DNA fragments containing structural genes encoding various industrially useful substances, DNA fragments containing genes that control the expression of these structural genes, etc. It can be done. By introducing a plasmid vector recombined with this useful gene etc. into a host microorganism and culturing it,
It becomes possible to stably and efficiently produce useful substances encoded by useful genes.

The host microorganisms that can be transformed with the plasmid vector of the present invention described above include coryneform bacteria, such as Brevibacterium flavum MJ-233 (FERN
BP-1497), Brevibacterium flavum MJ
-233-8B-41 (FERN BP-1498),
Brevibacterium flavum MJ-233-ABT-
11 (FERN HP-1500), Brevibacterium flavum MJ-233-ABD-21 (FERMB
P-1499), etc.

The above FERN BP-1498 strain is FERN BP-1498.
OL-α-7 using the RN BP-1497 strain as the parent strain
It is an ethanol-assimilating microorganism that has been actively given resistance to minobutyric acid (see columns 3 and 4 of Japanese Patent Publication No. 59-28398).

In addition, the strain of FERN BP-1500 is FERN
It is a mutant strain with high L-α-7minobutyric acid trans7minase activity using the BP-1497 strain as the parent strain (Japanese Patent Application Laid-open No. 62
(Refer to Publication No.-51998). Furthermore, FERN BP-
The strain No. 1499 is a D-α-7minobutyric acid deaminase high activity mutant strain using the FERN 8P-1497 strain as the parent strain (see Japanese Patent Laid-Open Publication No. 177993/1983).

Brevibacterium flavum MJ- as a host microorganism
When using 233, transformation may be difficult due to the plasmid p8Y502 (see Japanese Patent Application Laid-Open No. 63-36787) that this strain possesses. It is desirable to remove. This plasmid is, for example,
It is possible to drop it naturally by repeating subculturing, or it is possible to artificially remove it by repeating subculturing, for example, Bact, Rev., 3
By the method described in 6.361-405 (1972),
It is also possible to remove it.

A specific example of a method for artificially removing a plasmid is as follows.

7crisine orange (concentration:
0.2 to 50 μ9/m12) or ethidium bromide (concentration = 0.2 to 50 μ9/mj:l), etc., inoculated at approximately 10 cells/m42 to completely inhibit growth. Incubate at 35°C for about 24 hours. After diluting the culture solution, spread it on an agar medium and culture at 35°C for about 2 days. Plasmid extraction is performed independently from each colony that appears, and strains from which the plasmid has been removed are selected.

Through this operation, a Brevibacterium flavum MJ-233-derived strain from which pB'1502 has been removed is obtained.

The transformation of the host microorganism with the plasmid vector of the present invention described above can be carried out by methods known per se, for example, Ca1
vin, N., M. and Hanawalt, P.C.
Journal of Bacteriology, 1
70.2796 (1988); Ito, K., N.
15 hida.

T and Izaki, K. -Agricultu
ral and Biological Chemist
This can be carried out by, for example, applying a pulse wave to the host microorganism according to the method described in the literature such as R.R.Y., 52.293 (+988).

Next, the present invention will be explained in more detail with reference to Examples. However, the following examples are given only to help gain a concrete understanding of the present invention, and the scope of the present invention is not limited thereby.

Construction of pcRYZA A) Preparation of plasmid payso2 Plasmid pBY502 is a plasmid newly isolated from Brevibacterium flavum HJ-233 (FERN BP-1497) and has a molecular weight of about 30 megadaltons. This is the plasmid described in the specification. Plasmid pBY502 was prepared as follows.

Semi-synthetic medium A medium [composition: urea 29, (NH4) 2S
O479, 2HPO40,59, KH2PO40,5
9, M9SO, 0.59, Fe5O4H7H206mg
x Mn50.4-6 N206 mgx yeast extract 2
．． 59, Casamino acid 59, Bithion 200μ9, Thiamine hydrochloride 200μ9, Glucose 209, Pure water IJ2]1
β is Brevibacterium flavum Mu-233 (F
ERM BP1497) was cultured until the late logarithmic growth phase,
Bacterial cells were collected. The obtained bacterial cells were mixed with a buffer containing lysozyme at a concentration of 10 mg/mJ2 [25 mHt-lis(hydroxymethyl)aminemethane, 1 OmM EDTA,
50mM glucose] suspended in 20ml and heated at 37°C.
The mixture was reacted for 1 hour. Flukali-5OS solution [0
, 2NNa, and 1% (w/v) SDSI40mf2 were 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 60 mJ2, acetic acid 11.5 mJ2, pure water 28.5
130 mj2 of a mixed solution of mt2 was added, thoroughly mixed, and then allowed to stand in ice water for 15 minutes.

Transfer the entire lysate to a centrifuge tube and incubate at +5.0°C for 10 minutes at 4°C.
The supernatant was obtained by centrifugation in OOX 9.

Add an equal amount of phenol-chloroform solution (phenol/chloroform = 1/1 mixture) to this, suspend it, transfer it to a centrifuge tube, centrifuge at 15,000 x 9 for 5 minutes at room temperature, and remove the aqueous layer. Recovered. Add twice the amount of ethanol to the aqueous layer, let stand at -20'C for 1 hour, then stand at 4°C for 10 minutes.
The precipitate was collected by centrifugation at 5.0 OOX 9.

After drying the precipitate under reduced pressure, TE buffer [tris(hydroxymethyl)aminemethane 10 mM, EDTA 1 mM
;Dissolved in HC (adjusted to pH 8.0 with l)2-.

Add cesium chloride solution [5x TE buffer 10
Solution of cesium chloride 17o9 dissolved in 0mf2] +
51 and 10 mg/m, g edidium bromide solution 1 m
1 was added to adjust the density to 1.3929/m4. This solution was heated to 116.000 x 9 for 42 hours at 12°C.
(1) a Heart separation was performed.

Plasmid p8'1502 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 pBY502 was obtained.

This fraction was then treated four times with an equivalent amount of in7milfurcol to extract and remove ethidium bromide, and then treated with T
Dialysis was performed against Ei buffer.

After adding 3M sodium acetate solution to the dialysate containing the plasmid pBY502 obtained in this way to a final concentration of 30%, double the amount of ethanol was added, and the mixture was heated at -20℃ for 1 hour.
Let it stand for a while. This solution was centrifuged at 15,000×9 to precipitate the DNA, and the plasmid pB'150
Approximately 20μ9 of 2 was obtained.

B) Preparation of Plasmid pH5G39B Plasmid pH3G398 is a plasmid with a molecular weight of about 1.4 megadaltons that replicates in Escherichia coli and expresses chloramphenicol resistance, and can be purchased as a commercial product from Takara Shuzo.

C) Construction of plasmid pcRV2A Plasmid pH56398 (manufactured by Takara Shuzo) 0.5μ9 was reacted with restriction enzymes Sacl and Bam)+1<7 units each) at 37°C for 1 hour to completely degrade the plasmid DNA.

The plasmid pBY5022μ9 prepared in the above section A) was reacted with restriction enzymes 5acl and BamHl (1 unit each) at 37°C for 30 minutes to partially degrade the plasmid DNA.

Both plasmid DNA digests were mixed and heated at 65°C for 10 minutes to inactivate the restriction enzymes, and then the components in the inactivation solution were added to a final concentration of 50mM Tris buffer pH 7, 6, and 10mM M9Cβ2, respectively. .10mM dithiothreitol, lmM ATP and T4 ligase 1
Each component was strengthened to form a unit and kept at 16° C. for 15 hours. Using this solution, Enelichhu-Koli JM
+09 competent cells (manufactured by Takara Shuzo) were transformed.

The transformed strain was treated with chloramphenicol at 30μ9/mJ2 (final concentration) and 100μc+/mff1 (final concentration).
IPTG (inpropyl-β-D-galactopyranoside), 100μ9/mf (final concentration) X-98β
(5-bromo-4-chloro-3indolyl-β-D-galactopyranoside) in Kuramushi medium (tryptone 109,
Yeast extract 59, NaCn 59, pure water IJ2; p) 1
7.2) was cultured at 37°C for 24 hours to obtain a viable strain. Among these growing strains, those that grew as white colonies were selected, and each plasmid DNA was alkali-inoculated.
5DS method [Ding, Man at is.

E., F., Fr1tsch, J., Sambrook, “M.
o1ecular cloning” (+982) 9
0 to 91].

Transformation was then performed using the electric pulse method.

First, Brevibacterium flavum MJ-233 (FE
RNBP-1497) The plasmid-depleted strain was cultured in 100 m2 of the above A medium until the early logarithmic growth phase, and then treated with penicillin G.
was added at a concentration of 1 unit/mβ, cultured with shaking for another 2 hours, and the bacterial cells were collected by centrifugation.
j2 pulse solution [272mH5ucrose.

7mM KH2PO41mM M9CJ22: p
Wash with H7.4]. Furthermore, the bacterial cells were collected by centrifugation, suspended in 5 m12 of pulse solution, and 0.75 mj2
The cells were mixed with 50 μβ of the plasmid DNA solution obtained above, and the mixture was left standing in water for 20 minutes. Using Sheen Pulsar (manufactured by Bio-Rad), 2.500 volts, 25 μF
After applying the pulse, leave it in water for 20 minutes.

The whole amount is transferred to the above A medium of 3mj2 and cultured at 30°C for 1 hour, then inoculated onto the above A agar medium containing chloramphenicol 3 μ9/nu (final concentration) and cultured at 30°C for 2 to 3 days. A plasmid was obtained from the chloramphenicol-resistant strain that appeared using the method described in section A) above. This plasmid was cut with various restriction enzymes and its molecular weight was measured.

The results are shown in Table 3 below.

Table 3 Restriction enzyme recognition site number Molecular weight [megadalton] Sac
l I 2.2(3,3) 'B
am old + 2.2 (3,3) Sph l
2 1.6 (2,4L O,6(0
,9) Baj21 I 2.2(3
,3) CJ2al l 2.2(3
, 3) 1: () indicates the length (kb).

The plasmid characterized by the cleavage fragment of the above restriction enzyme was named pCR'/2A.

From the results, 161 derived from plasmid pBY502
It was confirmed that the gene responsible for the replication and propagation function of the plasmid was present within the kb DNA fragment.

Example 2 Evaluation of replication and proliferation ability of plasmids pcRY2 and pCR'/2A The replication and proliferation ability of plasmid pcRV2 and plasmid pcRY2A constructed in Example 1 was evaluated using chloramphenicol acetyltransferase (CAT) encoded on these plasmids. ) was compared using the activity as an index.

The above-mentioned plasmid pcRY2 is a plasmid vector newly created by the inventors of the present application and proposed earlier, and contains a DNA region controlling the replication and proliferation function with a total length of 4.1 kb derived from plasmid pB'1502 and plasmid pH5G3.
It has a DNA region derived from No. 98 (see the specification of JP-A-1-19168ε). This plasmid corresponds to the parent plasmid of plasmid ρCR'/2A constructed in Example 1.

Brevibacterium flavum MJ233 strain harboring plasmid pcRY2 or pCRY2A was inoculated into semi-synthetic A medium (described in Example 1) supplemented with chloramphenicol 3 μ9/nu (final concentration), and after culturing in the evening. , 1% ν/V was inoculated into a 50012 Erlenmeyer flask containing 100 nl of the same medium, and cultured for 6 hours.
It was used to measure AT activity. CAT activity is determined by IJ, V,
The method of 5ha et al. (J.

Bacteriology Jan, (1968)
28-36).

The results are shown in Table 4 as relative activities, with the activity of the strain carrying plasmid pCR'/2 being taken as 1.

Table 4 Bacterial strain CA activity (relative activity) pcRY2A
Owned 1.5ρCRV2 Owned
1 From Table 4, 1. By downsizing to + kb, it was confirmed that it retained 1.5 times the replication and proliferation ability of the parent plasmid.

A) Preparation of plasmid ρBV503 Plasmid pBY503 was prepared from Brevibacterium stationis IFOI2144 (FERN Bρ-2515
) and has a molecular weight of approximately 10 megadaltons, and is a plasmid described in JP-A-1-95785. Plasmid ρBY503 was prepared as follows.

Semi-synthetic medium A medium [composition: urea 29, (NH4) 2S
0479,K2HPO,0,59,KN,,PO40,
59,14clsO,0,59,,FeSO4・7H2
06 mg, MnSO44-6) 120 6 mgs yeast extract 2.59% casamino acids 59, bithion 200μ
Brevibacterium stathionis tFG12144 (FERN BP-2515) was cultured in 9, thiamine hydrochloride 200 μ9, glucose 209, pure water 142] until the late logarithmic growth phase, and the bacterial cells were collected. The obtained bacterial cells were added to a slow IIR solution containing lysozyme at a concentration of 10 mg/mJ2 [25 mM tris(hydroxymethyl)7minomethane,
10mM EDTA, 50mM glucose co2
The suspension was suspended in 0ml and reacted at 37°C for 1 hour. reaction j
? i+, near/l, force IJ-SDS liquid [0,2NNa, 1% (w/v) SOS] 401! 1J2 was added, mixed well and allowed to stand at room temperature for 15 minutes.

Next, a potassium acetate solution [5M potassium acetate solution 60ml, acetic acid 11.5+nj2, pure water 28.5ml] was added to this reaction solution.
130 mj2 of the m+2 mixed solution was added, thoroughly mixed, and then left standing in ice water for 15 minutes.

Transfer the entire lysate to a centrifuge tube and incubate at 4°C for 10 min.
The supernatant was obtained by centrifugation at 000X 9.

Add an equal amount of phenol-chloroform solution (phenol/chloroform = 1/1 mixture) to this, suspend it, transfer it to a centrifuge tube, centrifuge at 15,000 x 9 for 5 minutes at room temperature, and remove the aqueous layer. Recovered. Add twice the amount of ethanol to the aqueous layer, let it stand at -20℃ for 1 hour, and then keep it at 4℃ for 10 minutes for 15 minutes.
．． The precipitate was collected by centrifugation at 000X 9.

After drying the precipitate under reduced pressure, TEI! Solution [Tris(hydroxymethyl)7minomethane 10 mH, EDTA 1 mM
;) Adjusted to pH 8.0 with 1cJ2] Dissolved in 2m+2. Add 15 ml of cesium chloride solution [a solution of cesium chloride 1709 dissolved in 100 ml of 5-fold concentrated TE buffer] and 10 mg/mj2 ethidium bromide solution 111IJ2 to the solution to adjust the density to 1.392 q/m.
Adjusted to A. This solution was heated at 12°C for 42 hours at 116,
Centrifugation was performed at 000×(l).

Plasmid p8Y5 (13) was found as a lower band in the centrifuge tube by ultraviolet irradiation. By extracting this band from the side of the centrifuge tube with a syringe, a fraction containing plasmid pB'1503 was obtained.

This fraction was then treated with an equivalent amount of isoamyl alcohol four times to extract and remove ethidium bromide, and then treated with T.
E[Dialysis was performed against a buffer solution.

A 3M sodium acetate solution was added to the dialysate containing the plasmid pBY503 thus obtained to a final concentration of 3QmM, then 2 times the amount of ethanol was added, and the mixture was allowed to stand at -20°C for 1 hour. This solution was centrifuged at 15,000×9 to precipitate the DNA, and the plasmid pBY50
Approximately 20μ9 of 3 was obtained.

B) Preparation of Plasmid pH5G398 Plasmid pH5G398 is a plasmid with approximately 1.4 megadalton molecule I that replicates in Escherichia coli and expresses chloramphenicol resistance, and can be purchased as a commercial product from Takara Shuzo.

C) Construction of Fu7 Sumito pCR'/2.9 Plasmid pH5G39B (manufactured by Takara Shuzo) 0.5μ9 was reacted with restriction enzymes a1 and 5phl (1 unit each) at 37°C for 1 hour to completely degrade the plasmid DNA.

Restriction enzymes Smal and 5phl (1 unit each) were added to the plasmid p8Y5032μ9 prepared in section A) above.
The reaction was carried out at 7°C for 30 minutes to partially degrade the plasmid DNA.

After mixing both plasmid DNA digests and heating at 65°C for 10 minutes to inactivate the restriction enzymes, the components in the inactivation solution were mixed in a final concentration of 50111M Tris buffer pH7, 6, and 10mM M9CJ22, respectively. .10m
Each component was strengthened to 1 unit of M dithiothreitol, 1 m+4 ATP, and T4 ligase, and kept at 16° C. for 15 hours. Escherichia coli JMI09 competent cells (manufactured by Takara Shuzo) were transformed using this solution.

The transformed strain was treated with chloramphenicol at 30 μ9/mJ2 (final concentration) and 100 μq/mJ2 (i% F concentration) I
PτG (isopropyl-β-D-galactopyranoside)
, 100u9/ml! (Final concentration)×-gaf
(5-bromo-4-chloro-3indolyl-β-D-galactopyranoside) (tryptone 109,
Yeast extract 59, NaCj259, pure water 1u; pH 7,
2) was cultured at 37°C for 24 hours to obtain a viable strain. Among these growing strains, those that grew as white colonies were selected, and each plasmid DNA was injected into alkaline-5D.
S method [T, Maniatis.

E., F., Fr1tsch, J., Sambrook, “
Mo1ecular cloning” (1982)
90-91].

Transformation was then performed using the electric pulse method.

First, Brevibacterium flavum MJ-233 (FE
RNBP-1497) plasmid removed strain 100m, +
The cells were cultured until the early logarithmic growth phase in the above A medium of 2, and penicillin G was added at 1 unit/m 12, and the cells were further cultured with shaking for 2 hours. The cells were collected by centrifugation, and the cells were pulsed with 2On+J2. solution [272mM 5uc
rose.

7mM K)I2P041mM MgCjL:
Wash at pH 7.4]. Further, the bacterial cells are collected by centrifugation, suspended in 5 mu pulse solution, 0.75 mf cells and 50 μl of the plasmid DNA solution obtained above are mixed, and the mixture is left standing in water for 20 minutes. Using Sheen Pulsar (manufactured by Bio-Rad), 2.500 volts, 25 μFD
After applying the pulse, leave it in water for 20 minutes. Transfer the entire amount to the above A medium of 3 m, +2 and culture at 30°C for 1 hour, then inoculate the above A agar medium containing chloramphenicol 3μ9/m12 (final concentration) and culture at 30°C for 2 to 3 days. . From the emerging chloramphenicol-resistant strains,
A plasmid was obtained using the method described in section A) above. This plasmid was cut with various restriction enzymes and its molecular weight was measured. The results are shown in Table 5 below.

Table 5 Number of restriction enzyme recognition sites Molecule M [megadaltonko Sma
l I 3.4(5,1)sph
l + 3.4(5,1)Pst
l 2 2.9(4,4L D, 5(0
,7)Xbal 3 2.8(4,2),0
．． 5 (0, 8), 0i (0, 1) 8: () is the length (kb
) is shown.

The plasmid characterized by the cleavage fragment of the above restriction enzyme was named pcRV2.9.

From the above results, it was confirmed that a gene controlling the replication and propagation function of the plasmid was present in the 2.9 kb DNA fragment derived from plasmid l)B'l'503.

Example 4 Evaluation of replication and proliferation ability of plasmids pcRY3 and pCRV2゜9 The replication and proliferation ability of plasmid pCR'/3 and plasmid RY2.9 constructed in Example 3 was evaluated using chloramphenicol encoded on these plasmids. A comparative study was conducted using the activity of 7 cetyltransferase (CAT) as an index.

The above plasmid pcRV3 is a plasmid vector newly constructed and previously proposed by the inventors of the present invention, and has a total length of 5.5 mm derived from plasmid pBY503. DNA region governing the replication and proliferation function of Qkb and plasmid pH5G39B
It possesses a DNA region derived from
-191686 specification). This plasmid corresponds to the parent plasmid of plasmid pcRV2.9 constructed in Example 3.

Brevibacterium flavum MJ233 strain carrying plasmid pCR'/3 or pcRV2.9 was inoculated into the above-mentioned semi-synthetic A medium supplemented with chloramphenicol 3 μg/nu (final concentration), and After that, add 1 liter to a soomI2 Erlenmeyer flask containing the same medium IQOnl.
%V/V inoculation, cultured for 6 hours, collected, and CAT
was used to measure the activity of CAT activity is LV, 5ha
- method of et al. (J, 3acteriologyJan,
(1968) 28-36]. The results are shown in Table 6. The activity of the strain carrying plasmid pcRY3 is 1
It is expressed as relative activity.

Table 6 Bacterial strain CAT activity (relative activity) pcRY2.
9 held 1.8pcRY3 held
1 From Table 6, the DNA region governing the replication and proliferation function of the parent plasmid 6. It was confirmed that by reducing the size of Okb to 2.9 kb, it retained 1.8 times the replication and proliferation ability of the parent plasmid.

Claims (4)

[Claims] (1) Strain belonging to coryneform bacteria (A) A DNA region (a
). (2) The DNA region (a) according to claim 1, wherein the DNA region (a) is a DNA fragment with a total length of 1.1 kb derived from plasmid pBY502. (3) The DNA region (a) according to claim 1, wherein the DNA region (a) is a DNA fragment having a total length of 2.9 kb derived from plasmid pBY503. (4) The DNA region (a
) is a plasmid vector capable of replicating and propagating in a coryneform bacterium (A) into which it has been introduced.