JP2858460B2 - DNA fragment containing gene responsible for stabilizing plasmid in host microorganism - Google Patents

Description

The present invention relates to a DNA fragment containing a gene responsible for stabilizing a plasmid in a host microorganism, and more particularly to Brevibacterium stathionis.
The present invention relates to a DNA fragment containing a gene having a stabilizing function and derived from plasmid pBY503 carried by IFO12144, and its use.

Coryneform bacteria, including bacteria of the genus Brevibacterium, are industrially useful microorganisms that produce amino acids, organic acids, purine nucleotides, etc. In particular, the development of vectors that are industrially useful and have excellent stability using coryneform bacteria as hosts is strongly desired.

Generally, regarding the stability of the constructed plasmid in the host,
Hitherto, various genetic instabilities such as dropout of a plasmid from a host and deletion of an inserted gene during culture have been reported, and countermeasures have been studied.

For example, a plasmid into which a chromosomal gene DNA fragment responsible for the property of being independent of streptomycin derived from Escherichia coli is incorporated into a streptomycin-dependent mutant strain of the genus Escherichia to stabilize the properties of the microorganism containing the plasmid. A method has been proposed (JP-A-55-156591).

However, such a method is not only economically problematic, but also requires the incorporation of a complex function into the target plasmid, so that it is expected that the plasmid will not be stably distributed to daughter cells during division and propagation of the host. There are considerable problems with practical application.

Naturally-occurring plasmids obtained from coryneform bacteria, including industrially useful bacteria of the genus Brevibacterium, have the potential to genetically improve this genus, but most can be used as plasmid markers. It is a plasmid that does not have a drug-resistant gene and does not have a cloning site to which a useful gene can be ligated, and is unsuitable for improving the breeding of coryneform bacteria if it is a natural plasmid.

The present inventors have previously described the native plasmid pBY502 or pB
Drug resistance gene that can be a plasmid marker in Y503,
By introducing cloning sites and gene regions necessary for replication in Escherichia coli, industrially useful plasmid vectors such as pCRY2 and pCRY3 were created and proposed (Japanese Patent Application No. 63-13659). reference).

However, the plasmid vector has considerable instability in the cells, and has a drawback that the efficiency is not sufficient when a gene product is produced in a large amount in the cells.

Therefore, it is desired to develop a method for stabilizing a plasmid which is stably and reliably distributed from a parent cell to a daughter cell in a transformed microbial cell.

Thus, the present inventors have conducted intensive studies and have found that Brevibacterium stationis can replicate and grow in coryneform bacteria.
A gene involved in a function of stably retaining a plasmid capable of replicating and growing in at least a Coryneform bacterium of the genus Brevibacterium on pBY503 derived from IFO12144 (hereinafter referred to as a "stabilizing function") ( Hereinafter, this is sometimes referred to as “stabilizing gene” for the sake of convenience.) The present inventors succeeded in isolating a DNA fragment containing this stabilizing gene from the above-mentioned pBY503, and completed the present invention. Was.

Thus, according to the present invention, at least Brevibacterium stationis containing a gene involved in a function of stably retaining a plasmid capable of replicating and growing in a coryneform bacterium belonging to the genus Brevibacterium. 2.) A DNA fragment derived from plasmid pBY503 carried by IFO12144 is provided.

The use of the DNA fragment of the present invention makes it possible to construct a vector DNA capable of stably multiplying in Brevibacterium or other coryneform bacteria. Transformation of pasteurization makes it possible to improve the breeding of industrially useful coryneform bacteria.

Hereinafter, the DNA fragment of the present invention will be described in more detail.

A DNA fragment containing the stabilized gene of the present invention (hereinafter referred to as “stabilized DNA fragment”) is a Brevibacterium
Stationis (Brevibacterium stationis) IFO12144
(BYK No. 10136), plasmid pBY503
(Approximately 15 kb-see Japanese Patent Application Laid-Open No. 1-95785), which can be separated and obtained from the cleaved fragments generated by cleaving this pBY503 with an appropriate restriction enzyme by the method described below. .

That is, plasmid pBY503 containing a stabilized DNA fragment
Is digested with an appropriate restriction enzyme, and the resulting DNA fragment is inserted into a vector plasmid which is unstable in a coryneform bacterium and retains a drug resistance marker, and this vector plasmid is transformed by an electric pulse method to obtain a coryneform bacterium. To be introduced.

Plasmid DNA was extracted from the resulting transformant, and the DN from pBY503 was inserted by analysis with restriction enzymes.
Examine the A fragment. Each recombinant plasmid-holding strain is cultured for several tens of generations under non-selective pressure, and those whose plasmid retention rate after culture is higher than that of the original vector plasmid are stabilized DAN.
Isolate as a recombinant plasmid containing the fragment.

One of the stabilized DAN fragments thus obtained is a DNA fragment having a size of about 7.4 kb, which is obtained by cutting out the plasmid pBY503 with a restriction enzyme KpnI.

Table 1 below shows the number of recognition sites and the size of the cleaved fragment when the stabilized DNA fragment of about 7.4 kb was cleaved with various restriction enzymes.
Shown in

In this specification, the “number of recognition sites” by a restriction enzyme refers to a DNA fragment or a plasmid which is completely degraded in the presence of an excessive restriction enzyme, and the degraded product is subjected to a 1% agarose gel according to a known method. It is subjected to electrophoresis and polyacrylamide gel electrophoresis, and can be determined from the number of separable fragments.

In addition, when agarose gel electrophoresis is used, the “size of the cut fragment” and the size of the plasmid have a known molecular weight obtained by cutting the DNA of lambdaphage (λphage) of Escherichia coli with the restriction enzyme HindIII. Based on the standard line drawn by the migration distance of the DNA fragment on the same agarose gel, and when using polyacrylamide gel electrophoresis, the E. coli
Based on the standard line drawn by the migration distance on the same polyacrylamide gel of a DNA fragment of known molecular weight obtained by cleaving DNA of 174 phage (φx174phage) with restriction enzyme Hae III, the cut DNA fragment or each DNA fragment of plasmid Is calculated. The size of the plasmid is determined by adding the sizes of the cut fragments. In the determination of the size of each DNA fragment, the size of the fragment
% Agarose gel electrophoresis, 4% for fragments of about 0.1 kb to less than 1 kb
The results obtained by polyacrylamide gel electrophoresis were employed.

In Table 1 above, it was confirmed that the 6.8 kb SacI digestion fragment, the 6.4 kb SmaI digestion fragment, the 2.1 kb EcoRI digestion fragment and the 1.5 kb XbaI digestion fragment also have a stabilizing function. Therefore, these truncated fragments are also included in the stabilized DNA fragment of the present invention.

Thus, the stabilized gene is a DNA fragment of about 2.1 kb obtained by cutting the plasmid pBY503 with the restriction enzymes KpnI and EcoRI, and the size obtained by cutting the plasmid with KpnI and XbaI. About 1.5kb
Is considered to be contained in the DNA fragment.

Table 2 below shows the number of recognition sites and the size of the cleaved fragment when the DNA fragment of about 2.1 kb was further cleaved with various restriction enzymes.
Shown in

On the other hand, for a DNA fragment having a size of about 1.5 kb obtained by cutting out plasmid pBY503 with KpnI and XbaI, its base sequence is converted to plasmid pVC18 or pVC19 [Mess
ing, J. and Vieira, J., Gene 19 , 269 (1982)].
rmination) method [Sanger, F.et.al., Proc. Natl. Acad. Sci.
USA 74 , 5463 (1977)]. The nucleotide sequence of the above-mentioned DNA fragment of about 1.5 kb thus determined is as follows and is composed of 1763 bases: Although it has not been determined exactly which part of the base sequence is responsible for the stabilizing gene, it is presumed that it is contained in the latter half of the base sequence.

The stabilized DNA fragment of the present invention having the above-mentioned base sequence is not limited to one isolated from a natural plasmid, but may be a commonly used DNA synthesizer such as System-1 manufactured by Beckman.
It may be synthesized using Plus.

Further, as described above, the stabilized DNA fragment of the present invention obtained from the plasmid pBY503, even if some bases of the base sequence are replaced with other bases, as long as the stabilizing function is not substantially impaired. It may be well or deleted, or a new base may be inserted, furthermore, a part of the base sequence may be transposed, and any of these derivatives may be used in the present invention. It is included in the stabilized DNA fragment.

FIGS. 1 and 2 show the cleavage point maps of the plasmid pBY503 and the stabilized DNA fragment described above in detail by restriction enzymes, respectively.
Shown in the figure.

The stabilized DNA fragment of the present invention is a vector DNA having excellent retention stability in a coryneform bacterium by introducing it into a DNA fragment or a plasmid containing a gene responsible for the replication (autonomous) growth function in coryneform bacterium. Can be obtained.

Examples of plasmid vectors containing a gene capable of integrating and replicating in a coryneform bacterium into which the stabilized DNA fragment of the present invention can be incorporated include plasmids pCRY2 and pCRY3 disclosed in Japanese Patent Application No. 63-13659; JP-A-58-676
No. 79, pAM330; JP-A-58-77895, pHM1519; JP-A-58-192900, pAJ655, pA
J611 and pAJ1844; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; pCG4 and pCG11 described in JP-A-57-183799.

Among them, plasmids used in a host vector system of a Coryneform bacterium of the genus Brevibacterium, such as pAM330, pH
M1519, pCG1, pCG4, pBY502, pBY503, especially pBY502 and p
It is preferable to include a gene derived from BY503 that controls the replication growth function. For example, plasmids pCRY2 and pCRY3 are suitably used.

As a method for constructing the plasmid vectors pCRY2 and pCRY3, Brevibacterium flavum (Brevibac
terium flavum) MJ-233 (FERM BP-1497) and B. stationis IFO12144.
(FERM P-10136; plasmids pBY502 and pBY503 DNA were extracted from an international deposit based on the Budapest Treaty on July 12, 1999 (transferred to FERM, BP-2515), and the restriction enzyme Hin was extracted.
4.1 kb and 6 kb DNA fragments are obtained by dIII or KpnI treatment. Next, the DNA fragment and plasmid pHSG398 (Takara Shuzo)
Are combined with those treated with the same restriction enzymes to produce pCRY-2 and pCRY-3 (Japanese Patent Application No. 63-1365).
No. 9).

Further, from the plasmid vector as described above, a DNA fragment containing a gene responsible for the replication growth function in coryneform bacteria is cut out as described above, and this is ligated with the stabilized DNA fragment of the present invention, so that the coryneform bacterium can be used in coryneform bacteria. Can be used as a vector DNA capable of replicating and proliferating with excellent retention stability.

A plasmid vector as described above, such as pCRY2 or pCR
As a method for introducing the stabilized DNA fragment of the present invention into Y3, for example, plasmid vector pCRY2 or pCRY3 is completely or partially digested with restriction enzymes KpnI or KpnI and EocRI or KpnI and XbaI. Cleavage, and the above-mentioned stabilized DNA fragment of the present invention (about 7.4 kb, about 2.1 kb or 1763 bp)
Fragment) by a ligation enzyme treatment.

The vector DNA that can be replicated and propagated in a coryneform bacterium containing the stabilized gene thus created can incorporate a DNA fragment containing a structural gene or the like encoding various industrially useful substances. As described above, by introducing a vector DNA recombined with a useful gene or the like into a host microorganism and culturing it, a useful substance encoded by the useful gene can be stably and efficiently produced.

Examples of the DNA containing a useful structural gene or the like that can be incorporated into the vector DNA according to the present invention include, for example, at least a tryptophan synthase gene (a DNA fragment containing trp A and trp B, a promoter and an operator that controls the expression of the gene; Examples include a threonine biosynthesis gene, a DNA fragment containing an amino acid biosynthesis gene of an isoleucine production / synthesis gene, and the like.

On the other hand, host microorganisms that can be transformed with a vector DNA recombined with a useful gene or the like include coryneform bacteria such as Brevibacterium flavum MJ-233 (FERM BP-1).
497), Brevibacterium flavum MJ233-AB-41
(FERM BP-1498), Brevibacterium flavum MJ
233-ABT-11 (FERM BP-15PP), Brevibacterium flavum MJ233-ABD-21 (FERM BP-1499) and the like.

The above strain of FERM BP-1498 is FERM BP-14
This strain is an ethanol-assimilating microorganism that has been positively imparted with DL-α-aminobutyric acid resistance using the strain No. 97 as a parent strain.
59-28398, columns 3 and 4) and FERM BP-1
The strain No. 500 was prepared by using the strain of FERM BP-1497 as a parent strain.
-Α-aminobutyric acid transaminase high activity mutant (see Japanese Patent Application Laid-Open No. 62-51998). In addition, FERM BP
The strain of -1499 was a D- strain having the strain of FERM BP-1497 as a parent strain.
α-Aminobutyric acid deaminase is a highly active mutant (see JP-A-61-177993).

In addition to these microorganisms, Brevibacterium ammoniagenes ATCC687
1, ATCC13745, ATCC13746, Brevibacterium
Debarikatam (Brevibacterium divaricatum) ATCC1402
0 or the like can be used as the host microorganism.

When Brevibacterium flavum MJ-223 is used as a host, the plasmid pBY502 carried by this strain is used.
(Refer to JP-A-63-36787), which may make transformation difficult. In such a case, it is desirable to remove plasmid pBY502 from the present strain in advance.
Such a plasmid can be naturally dropped out by repeating subculture, for example, or artificially removed by the method described in, for example, Bact. Rev., 36 , 361-405 (1972). It is also possible.

An example of a method for artificially removing a plasmid is specifically described below.

Acridine orange at a concentration that incompletely inhibits the growth of the host Brevibacterium flavum MJ-233 (concentration: 0.2
~ 50μg / ml) or ethidium bromide (concentration: 0.2 ~
50 μg / ml), and inoculated at about 10 cells per ml.
Incubate at ° C. After diluting the culture solution, apply it to the agar medium, and
For about 2 days. A plasmid extraction operation is performed independently from each of the appearing colonies, and a strain from which the plasmid has been removed is selected. By this operation, a strain derived from Brevibacterium flavum MJ-233 from which pBY502 has been removed is obtained.

Transformation of the host microorganism with the recombinant vector DNA can be performed by a method known per se, for example, Calvin, NMand
Hanawalt, PC, Journal of Bacteriology, 170, 2796 (1
988); Ioo, K., Nishida, T. and Izaki, K., Agricultural a
nd Biological Chemistry, 52, 293 (1988), etc., for example, by applying a pulse wave to a host microorganism.

Next, the present invention will be described more specifically with reference to examples.
However, the following examples are given only as a help to obtain a specific understanding of the present invention, and the scope of the present invention is not limited by these examples.

Example 1 Construction Method and Properties of Composite Plasmid pCRY2 Consisting of Plasmid pBY502 and Plasmid pHSG398 A) Preparation of Plasmid pBY502 Plasmid pBY502 was prepared from Brevibacterium flavum MJ-233 (FERM BP-1497).
Molecular weight approximately 30 megadaltons (approximately 45k
b) The plasmid described in JP-A-63-36787. Plasmid pBY502 was prepared as follows.

Semi-synthetic medium A medium [Composition: 2 g of urea, 7 g of (NH ₄ ) ₂ SO ₄ , K
_{2 HPO 4 0.5g, KH 2 PO} 4 0.5g, MgSO 4 0.5g, FeSO 4 · 7H 2 O 6m
_{g, MnSO 4 4~6H 2 O 6mg} , yeast extract 2.5g, casamino acids 5
g, 200 μg of bition, 200 μg of thiamine hydrochloride, 20 g of glucose, and pure water 1] were cultured with Brevibacterium flavum MJ-233 (FERM BP-1497) until the late logarithmic growth phase, and the cells were collected. The obtained cells were buffered with lysozyme at a concentration of 10 mg / ml [25 mM Tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose] 20 m
l and reacted at 37 ° C for 1 hour. 40 ml of an alkali-SDS solution [0.2 N NaOH, 1% (w / v) SDS] was added to the reaction solution, mixed gently, and allowed to stand at room temperature for 15 minutes.

Next, 30 ml of a potassium acetate solution [a mixture of 60 ml of a 5M potassium acetate solution, 11.5 ml of acetic acid, and 28.5 ml of pure water] was added to the reaction solution, mixed well, and allowed to stand in ice water for 15 minutes.

Transfer the whole lysate to a centrifuge tube, 15,000 x 10 minutes at 4 ° C.
g was centrifuged to obtain a supernatant.

An equal volume of a phenol-chloroform solution (phenol / chloroform 1/1 mixed solution) was added thereto, suspended, transferred to a centrifuge tube, and centrifuged at 15,000 × g at room temperature for 5 minutes.
The aqueous layer was collected. Add two volumes of ethanol to the aqueous layer,
After leaving at 20 ° C. for 1 hour, the mixture was centrifuged at 15,000 × g at 4 ° C. for 10 minutes to collect a precipitate.

After drying the precipitate under reduced pressure, a TE buffer [Tris 10 mM, EDTA, 1 mM
PH was adjusted to 8.0 with M and HCl]. 15 ml of a cesium chloride solution [a solution in which 170 g of cesium chloride was dissolved in 100 ml of a 5-fold concentration of TE buffer] and 1 ml of a 10 mg / ml ethidium bromide solution were added to the solution, and the density was adjusted to 1.392 g / ml. This solution was centrifuged at 116,000 × g at 12 ° C. for 42 hours.

Plasmid pBY502 is found in the centrifuge tube as a lower band by UV irradiation. The band was removed from the side of the centrifuge tube with a syringe to obtain plasmid pBY502.
Was obtained.

This fraction was then added to an equivalent amount of isoamyl alcohol for 4 hours.
To remove ethidium bromide by extraction
Dialysis was performed against TE buffer. To the dialysate containing the thus obtained plasmid pBY502, a 3M sodium acetate solution was added to a final concentration of 30 mM, and then a 2-fold amount of ethanol was added, followed by standing at -20 ° C for 1 hour. 15,000 × g of this solution
DNA is precipitated by centrifugation of plasmid pBY502
Was obtained in an amount of about 20 μg.

B) Preparation of plasmid pHSG398 Plasmid pHSG398 was prepared from Escherichia coli.
This is a plasmid having a molecular weight of about 1.4 megadalton that replicates in chia coli and expresses chloramphenicol resistance, and can be purchased from Takara Shuzo as a commercial product.

C) Construction of composite plasmid pCRY2 Plasmid pHSG398 (manufactured by Takara Shuzo) was added to 0.5 μg of the restriction enzyme Hi.
nd III (5 units) was reacted at 37 ° C for 1 hour,
A was completely decomposed.

2 μg of the plasmid pBY502 prepared in the above A) was reacted with a restriction enzyme Hind III (1 unit) at 37 ° C. for 30 minutes to partially degrade the plasmid DNA.

After mixing both plasmid DNA digests and heating at 65 ° C. for 10 minutes in order to inactivate the restriction enzyme, the components in the inactivated solution were each brought to a final concentration of 50 mM Tris buffer pH7.
6, 10 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP and T4 ligase Enrich each component to 1 unit, 16 ℃
For 15 hours. This solution was used to transform Escherichia coli JM109 competent cells (Takara Shuzo).

The transformants were 30 μg / ml (final concentration) chloramphenicol, 100 μg / ml (final concentration) 1PTG (isopropyl-
L medium (β-D-galactopyranoside), 100 μg / ml (final concentration) X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (tryptone 10 g, yeast 5 g extract, 5 g NaCl, 1 pure water, pH 7.2)
The cells were cultured at 37 ° C. for 24 hours and obtained as growth strains. Among these growing strains, those growing in white colonies were selected, and each plasmid was subjected to the alkali-SDS method [T. Maniatis, E.
F. Fritsch, J. Sambrook, “Molecular cloning” (198
2) 90-91].

D) Transformation of complex plasmid into coryneform bacterium The electric pulse method was used for transformation. Brevibacterium flavum MJ-233 (FE
RM BP-1497) The plasmid-free strain was cultured in 100 ml of the above-mentioned A medium until the early logarithmic growth phase, penicillin G was added at 1 unit / ml, and the cells were further cultured with shaking for 2 hours.
The cells were collected by centrifugation, the cells pulsed solution _{[272mM Sucrose, 7mMKH 2 PO 4} 1mM MgCl 2: pH7.4] of 20ml and washed with. Further, the cells were collected by centrifugation, suspended in 5 ml of a pulse solution, and 0.75 ml of the cells and the DNA obtained in the above C).
Mix 50 ml of the solution and leave on ice for 20 minutes. 2500 volts, 25μ using Gene Pulser (manufactured by Bio-Rad)
After qualifying as FD, apply pulse and let stand for 20 minutes in ice. The whole amount was transferred to 3 ml of the above-mentioned A medium, and cultured at 30 ° C. for 1 hour.
Inoculate the agar medium and culture at 30 ° C for 2-3 days. A plasmid was obtained from the emerged chloramphenicol-resistant strain by the method described in A) above. This plasmid was cleaved with various restriction enzymes and the molecular weight was measured. The results are shown in Table 3 below.

The plasmid characterized by the restriction fragment was named "pCRY2".

In addition, Brevibacterium flavum (Brevibacterium flavu) transformed by the composite plasmid pCRY2 was used.
m) MJ-233GE101 was sent to the Research Institute of Microorganisms and Industrial Technology, 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture, on January 8, 1988.
Accession number by date: No.9801
01), and transferred to the International Deposit under the Budapest Treaty on July 12, 1999 (FERM, BP-2512).

Example 2 Construction Method and Properties of Composite Plasmid pCRY3 Consisting of Plasmid pBY503 and Plasmid pHSG398 A) Preparation of Plasmid pBY503 Plasmid pBY503 was prepared from Brevibacterium stationis IFO12144 [July 18, 1988 It has been deposited with the National Institute of Advanced Industrial Science and Technology as Microbial Laboratory Bacteria No. 10136 (FERM P-10136),
And transferred to an international deposit (FERM, BP-2515) based on the Budapest Treaty on July 12, 1999]. The plasmid having a molecular weight of about 10 megadaltons (about 15 kb) was newly obtained. -95785. Plasmid pBY503 was prepared as follows.

Semi-synthetic medium A medium [urea 2 g, (NH ₄ ) ₂ SO ₄ 7 g, K ₂ HPO
_{4 0.5g, KH 2 PO 4 0.5g} , MgSO 4 0.5g, FeSO 4 · 7H 2 O 6mg, MnS
O ₄ 4-6H ₂ O 6 mg, yeast extract 2.5 g, casamino acid 5 g, bithion 200 μg, thiamine hydrochloride 200 μg, glucose 20 g,
Brevibacterium stationis IFO12144 was cultured in pure water 1] 1 until the late logarithmic growth phase, and the cells were collected. 10 mg / ml of the obtained cells
Was suspended in 20 ml of a buffer solution containing lysozyme at a concentration of [25 mM Tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose], and reacted at 37 ° C. for 1 hour. Add 40 ml of alkali-SDS solution [0.2N NaOH, 1% (w / v) SDS] to the reaction solution.
Was added, mixed gently, and allowed to stand at room temperature for 15 minutes.

Next, 30 ml of a potassium acetate solution [a mixture of 60 ml of a 5M potassium acetate solution, 11.5 ml of acetic acid, and 28.5 ml of pure water] was added to the reaction solution, mixed well, and allowed to stand in ice water for 15 minutes.

Transfer the whole lysate to a centrifuge tube, 15,000 × for 10 minutes at 4 ° C.
g was centrifuged to obtain a supernatant.

An equal volume of a phenol-chloroform solution (phenol / chloroform 1/1 mixed solution) was added thereto, suspended, transferred to a centrifuge tube, and centrifuged at 15,000 × g at room temperature for 5 minutes.
The aqueous layer was collected. Add two volumes of ethanol to the aqueous layer,
After leaving at 20 ° C. for 1 hour, the mixture was centrifuged at 15,000 × g at 4 ° C. for 10 minutes to collect a precipitate.

After drying the precipitate under reduced pressure, a TE buffer [Tris 10 mM, EDTA, 1 mM
PH was adjusted to 8.0 with M and HCl]. 15 ml of a cesium chloride solution [a solution in which 170 g of cesium chloride was dissolved in 100 ml of a 5-fold concentration of TE buffer] and 1 ml of a 10 mg / ml ethidium bromide solution were added to the solution, and the density was adjusted to 1.392 g / ml. This solution was centrifuged at 116,000 × g at 12 ° C. for 42 hours.

Plasmid pBY503 is found in the centrifuge tube as a lower band by UV irradiation. The band was removed from the side of the centrifuge tube with a syringe to obtain plasmid pBY503.
Was obtained.

This fraction was then added to an equivalent amount of isoamyl alcohol for 4 hours.
To remove ethidium bromide by extraction
Dialysis was performed against TE buffer. To the dialysate containing the thus obtained plasmid pBY503, a 3M sodium acetate solution was added to a final concentration of 30 mM, and then a 2-fold amount of ethanol was added, followed by standing at -20 ° C for 1 hour. 15,000 × g of this solution
DNA is precipitated by centrifugation of plasmid pBY503.
Was obtained in an amount of about 50 μg.

B) Preparation of plasmid pHSG398 Plasmid pHSG398 was prepared from Escherichia coli.
This is a plasmid having a molecular weight of about 1.4 megadalton that replicates in chia coli and expresses chloramphenicol resistance, and can be purchased from Takara Shuzo as a commercial product.

C) Construction of composite plasmid pCRY3 Plasmid pHSG398 (manufactured by Takara Shuzo) was added to 0.5 μg of restriction enzyme Kp.
n I (5 units) was reacted at 37 ° C. for 1 hour to completely degrade the plasmid DNA.

2 μg of the plasmid pBY503 prepared in the above A) was reacted with the restriction enzyme Kpn I (1 unit) at 37 ° C. for 30 minutes to partially decompose the plasmid DNA.

After mixing both plasmid DNA digests and heating at 65 ° C. for 10 minutes in order to inactivate the restriction enzyme, the components in the inactivated solution were each brought to a final concentration of 50 mM Tris buffer pH7.
6, 10 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP and T4 ligase Enrich each component to 1 unit, 16 ℃
For 15 hours. This solution was used to transform Escherichia coli JM109 competent cells (Takara Shuzo).

The transformants were 30 μg / ml (final concentration) of chloramphenicol, 100 μg / ml (final concentration) of IPTG (isopropyl-
L medium (β-D-galactopyranoside), 100 μg / ml (final concentration) X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (tryptone 10 g, yeast 5 g extract, 5 g NaCl, 1 pure water, pH 7.2)
The cells were cultured at 37 ° C. for 24 hours and obtained as growth strains. Among these growing strains, those growing in white colonies were selected, and each plasmid was subjected to the alkali-SDS method [T. Maniatis, E.
F. Fritsch, J. Sambrook, “Molecular cloning” (198
2) 90-91].

D) Transformation of complex plasmid into coryneform bacterium The electric pulse method described in Example 1, D) was used for transformation. A plasmid was obtained from the emerged chloramphenicol resistant strain by the method described in A) above. This plasmid was cleaved with various restriction enzymes and the molecular weight was measured. The results are shown in Table 4 below.

The plasmid characterized by the restriction fragment was named "pCRY3".

In addition, Brevibacterium flavum (Brevibacterium flavum) transformed with the composite plasmid pCRY3 was used.
m) MJ-233GE102 was transferred to the Research Institute of Microorganisms and Industrial Technology, 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture, on January 8, 1988.
Accession number by date: No.9802
02), and transferred to the International Deposit under the Budapest Treaty on July 12, 1999 (FERM, BP-2512).

Example 3 Responsible for stabilizing plasmids pCRY2 and pCRY3 (7.4 k
b) Cloning of DNA Fragment A) Preparation of 7.4 kb DNA Fragment Responsible for Stabilizing Function Plasmid pBY503 was prepared from Brevibacterium stationis IFO12144 (FERM BP
-2515) by the method described in Example 2, A).

Restriction enzyme Kpn I (20 units) is added to 20 μg of plasmid pBY503 DNA.
s) was reacted at 37 ° C. for 2 hours to completely degrade the DNA of the plasmid. The digest was separated by 0.8% agarose gel electrophoresis, and a DNA fragment fraction of about 7.4 kb was cut out from the gel. DNA was extracted and purified from the fraction to obtain a DNA of about 5 μm.

B) 7.4 kb DNA for stabilizing plasmid pCRY2
Cloning of fragment The restriction enzyme K was added to 1 μg of the plasmid pCRY2DNA obtained in Example 1.
Incubate pn I (5 units) at 37 ° C for 2 hours,
The DNA was completely degraded and 2 μg of the DNA fragment prepared in the above A) was used.
After heating at 65 ° C. for 10 minutes to inactivate the restriction enzyme, the components in the inactivated solution were each adjusted to a final concentration of 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , and 10 mM dithiothreitol. , 1 mM ATP and 1 unit of T4 ligase, each component was enriched and incubated at 16 ° C. for 15 hours.
Using this solution, Escherichia co
li) HB101 competent cells (Takara Shuzo) were transformed.

The transformed strain was an L medium containing 30 μg / ml (final concentration) of chloramphenicol [10 g of tryptone, 5 g of yeast extract, N
ag, pure water 1, pH 7.2] at 37 ° C for 24 hours to obtain a growing strain. Plasmids were isolated from the obtained growth strains by the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sam.
brook, "Molecular cloning" (1982) 90-91].

C) Transformation of Plasmid pCRY21 into Coryneform Bacteria For the transformation, the strain described in Example 1, D) and the electric pulse method were used. A plasmid was obtained from the emerged chloramphenicol resistant strain by the method described in Example 1. A) above. The plasmid was cleaved with various restriction enzymes and the molecular weight was measured. The results are shown in Table 5 below.

The plasmid characterized by the restriction fragments was named "pCRY21".

D) 7.4 kb DNA that stabilizes plasmid pCRY3
Cloning of fragment The restriction enzyme K was added to 1 μg of the plasmid pCRY3DNA obtained in Example 2.
Incubate pn I (5 units) at 37 ° C for 15 minutes.
A is partially decomposed, mixed with 2 μg of the DNA fragment prepared in the above A), and heat-treated at 65 ° C. for 10 minutes to inactivate the restriction enzyme.
Each component was enriched to 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP and T4 ligase (1 unit), and incubated at 16 ° C. for 15 minutes. Using this solution, Escherichia coli
coli) HB101 competent cells (Takara Shuzo) were transformed.

The transformed strain was an L medium containing 30 μm / ml (final concentration) chloramphenicol [10 g of tryptone, 5 g of yeast extract, N
ag, pure water 1, pH 7.2] at 37 ° C for 24 hours to obtain a growing strain. Plasmids were isolated from the obtained growth strains by the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sam.
brook, "Molecular cloning" (1982) 90-91].

E) Transformation of plasmid pCRY31 into coryneform bacteria For the transformation, the strain described in Example 1, D) and the electric pulse method were used. A plasmid was obtained from the emerged chloramphenicol-resistant strain by the method described in Example 1.A). This plasmid was cleaved with various restriction enzymes and the molecular weight was measured. The results are shown in Table 6 below.

A plasmid characterized by the above restriction fragments was named "pCRY31".

Example 4 2.1 kb of D having a stabilizing function to plasmids pCRY2 and pCRY3
Cloning of NA Fragment A) Preparation of 2.1 kb DNA Fragment Responsible for Stabilizing Function Plasmid pBY503 was prepared from Brevibacterium stationis IFO12144 (FERM BP
-2515) by the method described in Example 1, section A).

Restriction enzymes Kpn I and EcoR were added to 20 μg of plasmid pBY503 DNA.
I (20 units each) was reacted at 37 ° C. for 2 hours to completely degrade the plasmid DNA. The digest was separated by 0.8% agarose gel electrophoresis, and a DNA fragment fraction of about 2.1 kb was cut out from the gel. DNA is extracted and purified from the fractions for about 5
μg of DNA was obtained.

B) 2.1 kb DNA that has a stabilizing function for plasmid pCRY2
Cloning of fragment The restriction enzyme K was added to 1 μg of the plasmid pCRY2DNA obtained in Example 1.
Reaction of pn I and EcoR I (5 units) at 37 ° C for 2 hours,
Plasmid DNA is completely degraded and the DNA prepared in A) above
Mix with 2 μg of the fragment and add 65 ° C to inactivate the restriction enzyme.
After the heat treatment for 10 minutes at 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 mM
mM dithiothreitol, 1 mM ATP and T4 ligase 1 unit
Each component was fortified and incubated at 16 ° C. for 15 hours. Using this solution, Escherichia coli (Esch
E. coli) HB101 competent cells (Takara Shuzo) were transformed.

The transformed strain was an L medium containing 30 μg / ml (final concentration) of chloramphenicol [10 g of tryptone, 5 g of yeast extract, N
ag, pure water 1, pH 7.2] at 37 ° C for 24 hours to obtain a growing strain. Plasmids were isolated from the obtained growth strains by the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sam.
brook, "Molecular cloning" (1982) 90-91].

C) Transformation of plasmid pCRY2KE into coryneform bacteria The transformation described in Examples 1, D) and the electric pulse method were used. A plasmid was obtained from the emerged chloramphenicol resistant strain by the method described in Example 1. A) above. The plasmid was cleaved with various restriction enzymes and the molecular weight was measured. The results are shown in Table 7 below.

The plasmid characterized by the restriction fragment was named "pCRY2KE".

D) 2.1 kb DNA responsible for stabilizing plasmid pCRY3
Cloning of fragment The restriction enzyme K was added to 1 μg of the plasmid pCRY3DNA obtained in Example 1.
pn I and EcoRI (5 units) were reacted at 37 ° C. for 15 minutes to partially degrade the plasmid DNA, mixed with 2 μg of the DNA fragment prepared in the above section A), and mixed at 65 ° C. to inactivate the restriction enzyme.
After the heat treatment for 10 minutes, the components in the inactivation solution were each adjusted to a final concentration of 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 mM
Dithiothreitol, 1 mM ATP and T4 ligase (1uni
Each component was fortified as in t) and incubated at 16 ° C. for 15 minutes. Using the solution, Escherichia coli (Es
cherichia coli) HB101 competent cell (Takara Shuzo)
Was transformed.

The transformed strain was an L medium containing 30 μm / ml (final concentration) chloramphenicol [10 g of tryptone, 5 g of yeast extract, N
ag, pure water 1, pH 7.2] at 37 ° C for 24 hours to obtain a growing strain. Plasmids were isolated from the obtained growth strains by the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sam.
brook, "Molecular cloning" (1982) 90-91].

E) Transformation of plasmid pCRY3KE into coryneform bacteria For the transformation, the strain described in Example 1, D) and the electric pulse method were used. A plasmid was obtained from the emerged chloramphenicol-resistant strain by the method described in Example 1.A). This plasmid was cleaved with various restriction enzymes and the molecular weight was measured. The results are shown in Table 8 below.

A plasmid characterized by the above restriction fragments was named "pCRY3KE".

Example 5 1763 bp responsible for stabilizing plasmids pCRY2 and pCRY3
A) Preparation of 1763 bp DNA Fragment Responsible for Stabilizing Function Plasmid pBY503 was prepared from Brevibacterium stationis IFO12144 (FERM BP
-2515) by the method described in Example 1, A).

Restriction enzymes Kpn I and Xba were added to 20 μg of plasmid pBY503 DNA.
I (20 units each) was reacted at 37 ° C. for 2 hours to completely degrade the plasmid DNA. The digest was separated by 0.8% agarose gel electrophoresis, and a 1763 bp DNA fragment fraction was cut out from the gel. DNA is extracted and purified from the fraction to about 5μ.
g of DNA was obtained.

For the DNA obtained above, pUC18 or pUC19 [Messing,
Dideo using J. and Vieira, J, Gene 19 , p269 (1982)
xy chain termination method [Sanger.F, etal., Proc.Natl.A
cad Sci. USA 74 , p5463 (1977)].

B) Cloning of 1763 bp DNA Fragment Responsible for Drink Stabilization into Plasmid pCRY2 1 μg of plasmid pCRY2 DNA obtained in Example 1 was digested with restriction enzyme K
pn I and Xba I (5 units) were reacted at 37 ° C for 2 hours,
DNA prepared in the above A) by completely decomposing the plasmid DNA
Mix with 2 μg of the fragment and add 65 ° C to inactivate the restriction enzyme.
After the heat treatment for 10 minutes at 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 mM
mM dithiothreitol, 1 mM ATP and T4 ligase 1 unit
Each component was fortified and incubated at 16 ° C. for 15 hours. Using this solution, Escherichia coli (Esch
E. coli) HB101 competent cells (Takara Shuzo) were transformed.

The transformed strain was an L medium containing 30 μg / ml (final concentration) of chloramphenicol [10 g of tryptone, 5 g of yeast extract, N
ag, pure water 1, pH 7.2] at 37 ° C for 24 hours to obtain a growing strain. Plasmids were isolated from the obtained growth strains by the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sam.
brook, "Molecular cloning" (1982) 90-91].

C) Transformation of plasmid pCRY2KX into coryneform bacterium The strain described in Example 1, D) and the electric pulse method were used for transformation. A plasmid was obtained from the emerged chloramphenicol resistant strain by the method described in Example 1. A) above. The plasmid was cleaved with various restriction enzymes and the molecular weight was measured. The results are shown in Table 9 below.

The plasmid characterized by the restriction enzyme cleavage fragment was named "pCRY2KX".

D) 1763 kb DNA which has a stabilizing function to plasmid pCRY3
Cloning of fragment The restriction enzyme K was added to 1 μg of the plasmid pCRY3DNA obtained in Example 2.
pn I and Xba I (5 units) were reacted at 37 ° C. for 2 hours to completely decompose the plasmid DNA.
After incubating at 65 ° C. for 10 minutes to inactivate the restriction enzyme, the components in the inactivated solution were adjusted to a final concentration of 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , and 10 mM dithiothreate, respectively. Each component was enriched to Toll, 1 mM ATP and T4 ligase (1 unit) and incubated at 16 ° C. for 15 minutes. Using the solution, Escheric
hia coli) HB101 competent cells (Takara Shuzo) were transformed.

The transformed strain was an L medium containing 30 μm / ml (final concentration) chloramphenicol [10 g of tryptone, 5 g of yeast extract, N
ag, pure water 1, pH 7.2] at 37 ° C for 24 hours to obtain a growing strain. Plasmids were isolated from the obtained growth strains by the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sam.
brook, "Molecular cloning" (1982) 90-91].

E) Transformation of plasmid pCRY3KX into coryneform bacteria For the transformation, the strain described in Example 1, D) and the electric pulse method were used. A plasmid was obtained from the emerged chloramphenicol-resistant strain by the method described in Example 1.A). This plasmid was cleaved with various restriction enzymes and the molecular weight was measured. The results are shown in Table 10 below.

The plasmid characterized by the restriction fragments was named "pCRY3KX".

Example 4 Stability of constructed plasmids Plasmids pCRY2, pCRY2 obtained in Examples 1 to 5
1, pCRY2KE, pCRY2KX, pCRY3, pCRY3K7, pCRY3KE and pC
Brevibacterium flavum each holding RY3KX (B
revibacterium flavum) MJ233-derived strain is inoculated into the above-mentioned A medium containing 5 μg / ml (final concentration) of chloramphenicol, and precultured at 30 ° C. for 15 hours. Next, add 1 ml / ml
Subculture was carried out at a ratio of 0 ⁴ cells, and the cells were cultured for about 100 generations. Table 11 below shows the results of percentages of the number of chloramphenicol-resistant strains and the number of strains retaining the sunawachi plasmid in the culture solution to the total number of bacteria.

Next, specific examples of the improvement of breeding of coryneform bacteria using the plasmid vector of the present invention will be described. Of course, the following examples are given only as an aid to gain a specific understanding of the application of the present invention, and the scope of the present invention is not limited by these examples.

Example A Cloning of a DNA region containing a gene (Trp gene) responsible for tryptophan synthase biosynthesis into plasmids pCRY31, pCRY3KE and pCRY3KX, and production of L-tryptophan by coryneform bacteria transformed with those plasmids ( 1) Preparation of DNA region containing gene (Trp gene) responsible for tryptophan synthase biosynthesis A) Preparation of phage φ80pt Escherichia coli K-12 strain (IFO3301) in 100 ml of L medium (composition) was inoculated into the same) and the, and the culture 0.2ml was shaken for about 4 hours at 37 ° C., 0.1 ml of phage φ80 (ATCC11456a-B1) solution ⁽¹⁰⁵ Ke / ml)
Are mixed in L medium soft agar (L medium + agar) and then layered on an L medium agar plate. Plate 37
Cultivation for about 5 hours at ℃ produces plaque
When cultivation is continued at 37 ° C. for a further 2 to 3 days, a growing colony of phage φ80 lysogen is formed in the plaque. The lysogen was cultured in L medium at 37 ° C. for 4 hours, spread on the same L medium agar plate as described above, and then irradiated with ultraviolet light (400 to 8).
A phage φ80pt (a phage DNA containing a tryptophan operon) is prepared by inducing a lysogen phage with 00ergs / mm ² , 10 to 20 seconds.

B) Preparation of tryptophan operon fraction Escherichia coli K-12 strain (IFO3301) was inoculated into 1 L medium (the composition is the same as above), and cultured with shaking at about 37 ° C. for about 3 hours. , 25% in log phase
(W / v) 10 ml of glucose solution and the phage φ80pt solution prepared above were added at a concentration of 10 ¹¹ / ml (moi 20).
After continued shaking for a long time, a large amount of phage φ80pt was prepared by adding chloroform as usual [T. Maniatis, EFFri
tsch, Sambrook; "Molecular cloning" (1982) 76-80C.
old Spring Harboy Laborat ory].

Next, the obtained phage φpt solution was washed with Tris buffer (pH 7.
After dialysis in 8), the phage DNA was extracted by the phenol method (see "Molecular cloning" p.85).
Was extracted and purified, and a restriction enzyme BamHI was added thereto and reacted at 30 ° C. for 30 minutes to obtain a tryptophan operon gene fraction.

C) Preparation of plasmid pBR322trp The tryptophan operon fraction obtained in B) was allowed to react with restriction enzymes Sal I and Xho I at 37 ° C. for 1 hour and then at 65 ° C.
For 5 minutes to inactivate the restriction enzyme, and then added and mixed the plasmid pBR322 similarly treated with the restriction enzyme SalI. The components in the quenching solution were then brought to a final concentration of 50
mM Tris buffer pH 7.6, 10 MgCl _2, 10 mM dithiothreitol, DNA by strengthening the components to be 1 mM ATP and T ₄ ligase 1unit, incubated 15 hours at 16 ° C.
Was combined.

Escherichia coli (Escheri
chia coli) K-12 strain (tryptophan auxotroph, A
TCC23718) according to a conventional method, and the transformed strain [Tr
p auxotrophic loss, i.e. TrpA on the plasmid allows biosynthesized by TrpB genes, minimal medium (K ₂ HPO ₄ 7g, KH
₂ g of ₄ PO ₄ , 0.1 g of MgSO ₄ .7H ₂ O, 1 g of (NH ₄ ) ₂ SO ₃ , 2 g of glucose, and a strain capable of growing on pure water 1)]. This strain was subjected to liquid culture according to a conventional method, and plasmid pBR322trp was separated and purified from the culture solution.

D) Cloning of trpAB Fraction into Plasmid pDR720 Plasmid pBR322trp obtained in C) above was replaced with the restriction enzyme SacI
Cleavage with I and Sal I for 1 hour at 37 ° C. yielded a fraction containing the trpAB gene. Then, it was partially digested with a restriction enzyme Hinc II at 37 ° C. to obtain a minimum fraction containing trpAB. S to this fragment
mixing al I linker, it is bonded through T ₄ DNA ligase,
A trpAB fraction having Sal I sites at both ends was obtained.

Next, the plasmid pDR720 (Pharmacia) was reacted with the restriction enzyme Sal I at 37 ° C. for 1 hour, and heated at 65 ° C. for 5 minutes to inactivate the restriction enzyme. The trpAB fraction was added and mixed.

The components in the quenching solution were each 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM as final concentration.
Enhancing the various components as will become mM ATP and T ₄ ligase 1unit, by incubation for 15 hours at 16 ° C., it was bound to DNA.

Escherichia coli (Escheri
chia coli) K-12 strain (tryptophan auxotroph, A
TCC23718) according to a conventional method, and the transformed strain [Tr
p auxotrophic loss, i.e. trpA on the plasmid allows biosynthesized by trpB genes, minimal medium (K ₂ HPO ₄ 7g, KH
₂ g of ₄ PO ₄ , 0.1 g of MgSO ₄ .7H ₂ O, 1 g of (NH ₄ ) ₂ SO ₄ , 2 g of glucose, and a strain capable of growing on 1) pure water 1). This strain was subjected to liquid culture according to a conventional method, and plasmid pDR720trpAB was separated and purified from the culture solution.

E) Cloning of trpAB gene into plasmid pUC9 The plasmid pDR720trpAB fraction obtained in the above D) was reacted with the restriction enzyme Ecol I at 37 ° C for 1 hour, and inactivated by heat treatment at 65 ° C for 5 minutes. Similarly, plasmid pUC9 (manufactured by Pharmacia) was treated with restriction enzyme Ecol I, and ligated in the same manner as described above. The religated DNA was transformed as described above. From the obtained strains, those in which the trpAB fraction was inserted into the plasmid pUC9 were selected, and the plasmid pU was used in a conventional manner.
C9trpAB was extracted.

(2) Cloning of DNA region containing gene (Trp gene) responsible for biosynthesis of tryptophan synthase into plasmids pCRY3I, pCRY3KE and pCRY3KX, and transformation of coryneform bacteria with these plasmids A) Plasmids pCRY3Itrp1, pCRY3KEtrp1 and pCRY3KXtr
Construction of p1 Plasmid pUC9trpABμ prepared in (1) and D) above
g and plasmids pCRY31 and pCRY prepared in Examples 3-5.
3 μg of each of Y3KE and pCRY3KX was ligated with the restriction enzyme BamHI.
C. Cleavage by reacting for 1 hour, and inactivating the restriction enzyme by heating at 65.degree. C. for 10 minutes.The components in the inactivated solution are each 50 mM Tris buffer pH 7.
6,10mM MgCl _2, 10mM dithiothreitol, to strengthen the component to be 1 mM ATP and T ₄ ligase 1unit, 16 ° C.
The DNA was bound by incubation for 15 hours in Escherichia coli, and Escherichia coli HB
101 competent cells (Takara Shuzo) were transformed. As a control, the same operation as described above was performed for the plasmid pCRY3 prepared in Example 2.

The transformed strain was an L medium containing 30 μg / ml (final concentration) of chloramphenicol (10 g of tryptone, 5 g of yeast extract, N
The cells were cultured for 24 hours at 37 ° C. in 5 g of aCl, pure water 1, pH 7.2) and obtained as growth strains. Plasmids were isolated from the obtained growth strains by the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sam.
brook; "Molecular cloning" (1982) 90-91].

B) Transformation of coryneform bacterium with constructed plasmid Coryneform bacterium was transformed with the plasmid constructed in the above section A) using the strain described in Example 1.D) and the electric pulse method. A plasmid was obtained from the emerged chloramphenicol resistant strain by the method described in A) above. The plasmid obtained by cloning the Trp gene into the plasmid pCRY31 was digested with various restriction enzymes, and the molecular weight was measured. The results are shown in Table 12 below.

Plasmid characterized by the above restriction enzymes is called "pCRY31
trp1 ". Brevibacterium flavum (Brevib) transformed with the plasmid pCRY31trp1 was used.
acterium flavum) MJ233GE1004 was submitted to the Research Institute of Microbial Industry and Technology at 1-3-1 Higashi, Tsukuba, Tsukuba, Ibaraki Prefecture on May 26, 1988, under the accession number:
No. 5 (FERM P-10035; transferred to the International Deposit under the Budapest Treaty on July 12, 1999 (FERM BP-2514)).

In addition, the plasmid obtained when the trp gene was cloned into the plasmid pCRY3KE was cut with various restriction enzymes, and the molecular weight was measured. The results are shown in Table 13 below.

A plasmid characterized by the above-mentioned restriction fragment was named "pCRY3KEtrp1".

Furthermore, the plasmid obtained when the trp gene was cloned into the plasmid pCRY3KX was digested with various restriction enzymes, and the molecular weight was measured. The results are shown in Table 14 below.

The plasmid characterized by the restriction enzyme cleavage fragment was named "pCRY3KXtrp1".

Furthermore, the plasmid obtained when the trp gene was cloned into the control plasmid pCRY3 was cleaved with various restriction enzymes, and the molecular weight was measured. The results are shown in Table 15 below.

The plasmid characterized by the restriction fragment was named "pCRY3trp1".

(3) Plasmids pCRY3Itrp1, pCRY3KEtrp1 and pCRY3KX
Stability of trp1 100 ml of the A medium was dispensed into a 500 ml Erlenmeyer flask,
(2), B) after sterilization at 120 ° C for 15 minutes
After inoculating each of the transformed strains obtained in the above and shaking culture at 30 ° C. for 24 hours, 100 ml of A medium prepared in the same manner was added to 500 ml
An aliquot was dispensed into an ml Erlenmeyer flask, sterilized in the same manner as above for 15 minutes, and subcultured at a rate of 50 cells / ml, followed by shaking culture at 30 ° C. for 24 hours. Next, the cells were collected by centrifugation, the cells were washed, and a certain amount was spread on an A medium to which chloramphenicol was added at a rate of 5 μg / ml and a plate medium prepared as an A medium without addition, 30 ℃
After culturing for 1 day, the number of growing colonies is counted.

As a result, the same number of colonies grew on the medium with and without chloramphenicol, and all the colonies growing on the A medium grew on the medium with chloramphenicol, ie, plasmids pCRY3Itrp1, pCRY3KEt.
A high degree of stability of rp1 and pCRY3KXtrp1 was confirmed. Also,
The control plasmid pCRY3trp1 had about 10% of the number of colonies grown in the chloramphenicol-supplemented medium as compared to the non-supplemented medium.

(4) Plasmid pCRY3Itrp1, pCRY3KEtrp1 or pCRY3KX
Production of L-tryptophan by coryneform bacterium transformed with trp1 Medium (0.4% urea, 1.4% ammonium sulfate, KH ₂ PO ₄₀ .
_{5%, K 2 HPO 4 0.05} %, MgSO 4 · 7H 2 O 0.05%, CaCl 2 · 2H 2
_{O 2ppm, FeSO 4 · 7H 2} O 2ppm, MnSO 4 · 4~6H 2 O 2pp
_{m, ZnSO 4 · 7H 2 O} 2ppm, NaCl 2ppm, biotin 200 [mu] g /
, Thiamine / HCl 100μg /, tryptone 0.1%, yeast extract 0.1%) 100ml into a 500ml Erlenmeyer flask,
After sterilization (pH 7.0 after sterilization), plasmid pCRY3Itrp1, p
CRY3KEtrp1, pCRY3KXtrp1 or control plasmid pCRY3trp1
Each of the transformed strains described in (2) B) above is inoculated, and glucose is aseptically added to a final concentration of 2%.
(W / v) and shaking culture was performed at 30 ° C. for 15 hours.

Next, the main culture medium (ammonium sulfate 2.3%, KH ₂ PO
_{4 0.05%, K 2 HPO 4} 0.05%, MgSO 4 · 7H 2 O 0.05%, FeSO 4
・ 7H ₂ O 20ppm, MnSO ₄・ nH ₂ O 20ppm, biotin 200μg /
, Thiamine / HCl 100 μg /, tryptone 0.3%, yeast extract 0.3%) was charged into a 2-volume agitated tank, sterilized (120 ° C, 20 minutes), and then sterilized to a final concentration of 2% (w / v) glucose. So that the culture is
Add 0ml, rotation speed 100rpm, ventilation volume 1vvm, temperature 33 ° C pH
Culture was performed at 7.6 for 18 hours.

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

After cultivation, after collecting cells by centrifugation from 400 ml of culture,
The cells were washed twice with demineralized distilled water and the cells were reacted [Indole
5 g, DL-serine 20 g, pyridoxal-5'-phosphate 10 m
g, KCl 2 g, distilled water 1000 ml (adjusted to pH 8.0 with 5N-KOH) ", and the suspension was charged into a two-volume agitated tank at a rotation speed of 300 rpm, a temperature of 37 ° C. and a pH of 8 And reacted for 10 hours. After the reaction is completed, centrifuge (6000 rp)
m, 15 minutes, 4 ° C.), and L-tryptophan in the supernatant was measured by liquid chromatography.

After the reaction is completed, 500 ml of the solution is treated with an ammonia-type strongly acidic ion-sympathetic resin ("Diaion SK-1B", manufactured by Mitsubishi Chemical Corporation).
After eluted with an alkaline solution, the mixture was concentrated to precipitate crude L-tryptophan crystals.

The results are shown in Table 16 below.

[Brief description of the drawings]

FIG. 1 is a map of cleavage points of plasmid pBY503 by restriction enzymes Kpn I, EcoR I and Sma I, and FIG. 2 is a map of cleavage points of stabilized DNA fragments by various restriction enzymes.

 ──────────────────────────────────────────────────の Continued on the front page Accession number of microorganisms FERM BP-2512 Accession number of microorganisms FERM BP-2513 Accession number of microorganisms FERM BP-2514 Accession number of microorganisms FERM BP-2515 (72) Inventor Hideaki Yukawa Inashiki-gun, Ibaraki 8-3-1, Amicho Chuo, Tsukuba Research Laboratory, Mitsubishi Yuka Corporation

Claims (8)

(57) [Claims] 1. A gene containing at least a gene involved in a function of stably retaining a plasmid capable of replicating and growing in a Coryneform bacterium of the genus Brevibacterium in said bacterium.
Brevibacterium s stationis
tationis) derived from plasmid pBY503 carried by IFO12144
A DNA fragment having a size of about 7.4 kb and showing the number of recognition sites and the size of the cut fragment shown in Table 1 below when cut with restriction enzymes shown in Table 1 below. DNA fragment 2. A gene containing at least a gene involved in a function of stably retaining a plasmid capable of replicating and growing in a Coryneform bacterium belonging to the genus Brevibacterium in said bacterium.
Brevibacterium s stationis
tationis) derived from plasmid pBY503 carried by IFO12144
A DNA fragment having a size of about 2.1 kb and showing the number of recognition sites and the size of the cut fragment shown in Table 2 when cut with a restriction enzyme shown in Table 2 below. DNA fragment 3. The following nucleotide sequence involved in the function of stably maintaining a plasmid capable of replicating and growing in at least a Coryneform bacterium of the genus Brevibacterium: DNA fragment containing 4. A vector capable of replicating and growing in a coryneform bacterium into which the DNA fragment according to claim 1 has been introduced.
DNA. 5. The vector DNA according to claim 4, wherein the vector DNA contains a plasmid-derived gene responsible for a replication-proliferating function used in a host vector system of a Coryneform bacterium belonging to the genus Brevibacterium. 6. The vector DNA according to claim 5, wherein the plasmid is pBY502 or pBY503. 7. The plasmid according to claim 4, which is a plasmid pCRY2 or pCRY3.
7. The vector DNA according to any one of to 6 above. 8. A recombinant plasmid comprising the vector DNA according to claim 4, wherein a DNA fragment containing at least a tryptophan synthase gene and a DNA fragment containing a promoter and an operator controlling the expression of the gene are introduced.