JPS63304985A - Novel plasmid - Google Patents

Abstract

PURPOSE:To obtain a novel plasmid containing a gene to code AlaR and D-ATA derived from a thermostable bacterium useful for enzymatic synthesis of D-amino acid. CONSTITUTION:DNA fragment containing a gene to code AlaR (alanine recemase) derived from chromosome DNA of Bacillus stearothermophilus IFO12550 and DNA fragment containing a gene to code D-ATA (D-amino acid transaminase) derived from chromosome DNA of Bacillus sp. YM-1 (FERM P-8057) are prepared. The fragments and vector plasmid pUC18 are digested with ECoRI or SacI and linked with ligase T4 to constitute the aimed plasmid PICRT111. The plasmid is transduced to Escherichia coli HB101, etc., and a transformant is cultivated in a medium to produce AlaR and D-ATA.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention is extremely useful as a modifier for antibiotics and agricultural chemicals, and is particularly effectively used for the enzymatic synthesis of monoamino acids.
The present invention relates to a novel plasmid having genes encoding alanine racemase (hereinafter abbreviated as AIaR) and D-amino acid transaminase (hereinafter abbreviated as D-ATA).

(Prior art) Conventional Bacillus stearothermophilus IF01255
Plasmid pICR4 (Biochemistry (Bioch.
emistry) 25, 3268-3274 (1
Plasmid p containing a gene encoding heat-stable D-ATA derived from Bacillus sp.
lcT113 (Japanese Patent Application No. 62-39173) has been constructed.

(Problems to be solved by the invention) However, using genetic recombination technology, AlaR and D-A
When preparing two TA enzymes and using the two enzymes at the same time, transform hosts separately with two separate plasmids carrying the genes encoding the two enzymes described above, select transformed strains, and then transform them. It is necessary to prepare the two E9g by culturing separately.

This method requires complicated operations, takes time, costs, etc., and is not economically advantageous.

(Means for Solving the Problems) Therefore, the present invention constructs genes encoding the two enzymes on one plasmid, thereby allowing the two enzymes to be prepared at the same time by transformation, culture, etc. The purpose is to make it possible to save energy, labor, and time.

That is, the present invention is a plasmid having genes encoding AlaR and D-ATA.

In order to obtain the plasmid of the present invention, for example, a DNA fragment containing a gene encoding AlaR and a DNA fragment containing a gene encoding D-ATA are obtained, and the two obtained DNA fragments and a DNA fragment having a role as a vector are obtained. D.N.
A, Journal of Molecular Biology (
Journal of Molecular Biolo
OV>96.171-184 (1975), by digesting with restriction enzymes and then ligating with ligase.

The gene encoding AlaR preferably used in the present invention includes, for example, a gene derived from the chromosomal DNA of a thermophilic microorganism in terms of heat resistance, stability, and the like.

Among these, a gene derived from Bacillus stear0 thermophilus with high AlaR activity is desirable. Specific examples of these include IFOI2550 and ATCC7953.

In addition, the chromosomal DNA of thermophilic microorganisms is used as the gene encoding D-ATA due to its heat resistance and stability.
Examples include genes derived from A. Among these, genes derived from moderately hyperthermic bacteria of the genus Bacillus are preferred.

Specific examples of these include Bacillus sp. YM-1,
Bacillus sp. YM-2 (Feikoken Bacillus No. 8058
(No.), etc.

Furthermore, as a plasmid that functions as a vector, plasmid pUc18 is particularly preferred. Further, examples of the υ1 restriction enzyme include l:coRI.

Examples of ligase include T4.
Examples include DNA ligase.

As the plasmid of the present invention, for example, by the method described above,
Plasmid pUc18 contains AlaR derived from the chromosomal DNA of Bacillus stearothermophilus IFO12550.
The gene encoding Bacillus sp. YM-1
An example is plasmid pICRT111 into which a gene encoding D-ATA derived from the chromosomal DNA of

The plasmid of the present invention can be introduced, for example, into bacteria that grow at room temperature, and transformed bacteria can be obtained by introducing this plasmid. Examples of bacteria that grow at room temperature include Escherichia
Bacteria belonging to the genus ichia are mentioned, with Escherichia coli being preferred. Among these, Escherichia Cori H[3101 is particularly preferred.

In addition, in order to transform the above-mentioned bacteria that grow at room temperature with the plasmid of the present invention, the above-mentioned bacterium treated with calcium chloride at around 0°C and the present invention may be used, for example, according to the method of Journal Molecular Biology 53, 159-162 (1970). This can be done by contacting the plasmid with the plasmid.

An example of a bacterium transformed as described above is the Escherichia coli 1-IB101/pICRT111 strain into which plasmid plCRT111 has been introduced. This strain has heat-resistant AlaR production ability, heat-resistant D-A
It has the same mycological properties as the known Escherichia coli HBlol except that it has TA production ability and ampicillin resistance. This bacterial cell maintains safety without changing from non-transmissible to transmissible or from non-pathogenic to pathogenic.

The plasmid of the present invention can be used for the synthesis of D-amino acids shown in the following formula 1, that is, AIaDH, D-ATA, A
A catalytic amount of OL-alanine (hereinafter referred to as 0L-
It can be particularly effectively used in the method of synthesizing D-amino acids from formic acid, ammonia and α-keto acids (Japanese Patent Application No. 61-48233) in the presence of AlaR and NAD. - It facilitates the preparation of ATA.

Formula 1 (Effect of the invention) As mentioned above, the plasmid of the present invention can be used as a useful microorganism,
For example, by transforming bacteria that grow at room temperature,
Bacteria can be endowed with the ability to produce AlaR,D,-ATA at the same time, and AIaR and D-ATA can be obtained from the transformed bacteria in large quantities easily and at the same time. Very useful for the production of amino acids, etc.

(Examples) Next, the present invention will be described in detail based on Examples, but the present invention is not limited thereto.

(1) Preparation of plasmid pICR113 (subcloned from plcR4) carrying the gene encoding thermostable AIaR and plasmid plcT113 carrying the gene encoding thermostable D-ATA Plasmid plcR113 was added to vector pBR322 using Bacillus stearothermophila. IFO12550
This is a plasmid into which a gene encoding AlaR derived from the chromosomal DNA of plasmid p[cT113
is vector pUC18, Bacillus sp. YM
This is a plasmid into which a gene encoding D-ATA derived from the chromosomal DNA of -1 was introduced.

Escherichia coli 8810, which was infected with plasmid pICR113 by the same method as described in (5) below.
One strain was incubated with 11 super broth containing 50 μg/M1 ampicillin (15 g polypeptone, 20 g yeast extract, 5 afl glycerol, 100 m 1M potassium phosphate mII solution (p117.6) in Ij) at 37 ml.
After culturing at ℃ for 2 to 3 hours, chloramphenicol solution (1
- add 6af of 34II (1g of 34II to ethanol),
The cells were further cultured for 16 hours.

After culturing, the bacterial cells were collected by centrifugation, and solution I (50 m
M glucose, 25mM Tris-HCl buffer (1) H8
, O), 10mM EDTA, 5q/d lysozyme) 20d and left for 30 minutes. Then solution I
Add 40ml of I (0,2N NaOH, 1% 5DS (sodium dodecyl sulfate)), stir gently and leave on ice for 10 minutes, then add 5M potassium acetate buffer (pH 4,
8) was added for 30 minutes, stirred vigorously, left on ice for 10 minutes, and centrifuged to remove proteins, RNA, and chromosomal DNA.
The precipitate containing the supernatant was separated from the supernatant. Next, 110 d of cold ethanol was added to the supernatant containing the crude plasmid, and after being left at -80°C for 30 minutes, the plasmid DNA was recovered as an ethanol precipitate by centrifugation. After drying the precipitate, 16dTE (1
0mM Tris-HCl buffer (pH 8, O), 1mM
EDTA>, ribonuclease A (RNase A, Seikagaku Corporation, 10 gw/d) 200.11 was added and the mixture was heated at 37°C.
The mixture was reacted for 1 hour with phenol-chloroform (1:1
) Remove the denatured protein by extraction, add 2xM ethanol and leave at -80°C for 30 minutes to extract the plasmid D.
NA was recovered as an ethanol precipitate. Next, the pellet-shaped plasmid was dissolved in a small amount of TE, and cesium chloride (
Add 1 g of plasmid solution to 1 d of plasmid solution) and ethidium bromide (add 0.8 d of ethidium bromide solution (10N 1 g/d) to 10 d of cesium chloride solution), ultracentrifuge at 45,000 rpm for 12 hours, and c c
c (covalently closed cir)
After collecting only the DNA band (Cular) and removing ethidium bromide by extraction with butanol, T
Purified pICR113 was obtained by thorough dialysis against E.

Purified pICT113 was also prepared in the same manner as pICR113.

(2) EC0RI-sac of plasmid pICR113
pICR11340μ9 obtained in r fragment preparation (1) was digested with restriction enzyme 3
Sac I buffer (10mM Tris-HCl W buffer (pH 8, O) containing 300U of acl (manufactured by Takara Shuzo)
, 7mM MCJCI, 7mM 2-ME,
After reaction and digestion for 5 hours in 400 µm of 0.01% BSA, 5 M NaC 18 µm, cold ethanol 850 µm.
μm was added, and the DNA was recovered as an ethanol precipitate by centrifugation. The recovered DNA pellet was dissolved in 20 μm TE, and ECORI buffer (50 mM Tris-salt 11111i1(1)117.5) containing 150 U of restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.), 7 mM
MgCI, 100mM NaCI, 7mM
2-ME, 0.01% BSA) at 37°C in 400 μβ;
After reacting for 5 hours, 800 μm of cold ethanol was added to collect the DNA as an ethanol precipitate. This DNA was dissolved in 20 μm TH and 0.7% LMP (
low melting point) agarose (
Bethesda Research Laborato
ries@) at 100 μm at 4°C.
Electrophoresis was performed for 6 hours, a 1.9 Kb ECORl-8ac (fragment) containing the gene encoding Δ1aR was separated, the agarose containing this fragment was cut out, 5 times the volume of TE was added, and the mixture was heated at 65°C for 5 minutes. This solution was subjected to phenol extraction, phenol-chloroform (1:1) extraction, and chloroform extraction once each to recover DNA fragments as ethanol precipitation.
The A fragment was dissolved in low ω TE, purified by reverse phase liquid chromatography (NENSOR31M20, DuPont), eluted with 50% methanol, and dried to prepare a purified EcoRl-8ac I fragment of plasmid plcR113. .

(3) EC0R (-8aC) of plasmid plcT113
Plasmid plCT11330μ9 obtained in Preparation of I fragment (1) was mixed with 400μ of 3acI buffer containing 1300U of Sac.
After reaction and digestion for 5 hours at 37°C in p.p., 5M N
18 μm of aC and 850 μm of cold ethanol were added, and DNA was recovered as an ethanol precipitate by centrifugation. The recovered DNA pellet was dissolved in 20 μm TE and subjected to EC.
After reaction and digestion for 5 hours in 400 μm of EC0RI buffer containing 1150 U of 0R, 800 μm of cold ethanol was added and the DNA was recovered as an ethanol precipitate by centrifugation.

This plasmid pICT113 [coRI-8ac
The T fragment is a portion derived from vector pUC18.

(4) Plasmid I) EC0RI-8a of ICT113
cI fragment and plasmid picR113 once 0RI-
The EcoRl-8ac I fragment of plcT113, which is the part derived from vector pUc18 prepared in ligation (3) of the 3aCI fragment, was added to 20μ
plcR113 ECORl-8ac I fragment prepared in (2) was dissolved in 20 μm TE. pICT113 coRl-8ac I fragment solution 11. 8 μm of EcoRl-8ac I fragment solution of pICR113 was treated with T4 DNA ligase (Takara Shuzo Co., Ltd.) 700
T4 DNA ligase buffer containing LJ (66mM Tris-salt 1lll buffer (1) I7.6), 6.6mM
MgCI2.10mM dithiothreitol (DT
T), 1iHATP) DNA was reacted overnight in 20 μρ.
The fragments were ligated.

<5) Transformation of Escherichia coli HBlol with the DNA ligated in (4) Escherichia coli HBlol was transformed into 100 d of YT medium (1 g of polypeptone, 0.5 g of yeast extract, NaCl0
．． 2-3 in 100Id medium containing 5g, pH 7,2)
Incubate for an hour, collect the bacterial cells by centrifugation, and cool at 100 m
After washing with M M gCl2, cold 100 mM Ca
The mixture was suspended in Cl2 and left on ice for 1 hour. Next, after removing the supernatant by centrifugation, the cells were resuspended in 25 mL of cold 100 mM CaCl to obtain competent cells.

Next, 10 μm of the ligated DNA solution obtained in (4) and 200 μΩ of the competent cell suspension were mixed at 0°C, left on ice for 60 minutes with occasional stirring, then left at 42°C for 2 minutes, and then placed on ice. It was rapidly cooled.

Next, 1 d of YT medium was added to this suspension, and after culturing with shaking at 37°C for 1 hour, ampicillin-containing (50 μg/d)
The colonies were plated on YT-agar medium (20 g of agar/1) and cultured overnight at 37°C, and the formed colonies were re-plated on YT-agar medium containing ampicillin and cultured overnight at 37°C. The resulting colonies were used as transformed strains.

(6) Selection of transformed strains possessing the desired plasmid Ten of the 15 transformed strains obtained in (5) were cultured overnight in 5 d of YT medium containing ampicillin (100 μg/d). Each plasmid was isolated on a small scale according to the same method as in (1).

When these plasmids were digested with ECOR1 and Sac I according to method (2), all 7 lasmids were cut at one site each, resulting in a single band of approximately 4.5 Kb. Furthermore, double digestion with EcoRl and 5aCI produced two bands of approximately 2.7 Kb and approximately 1.9 Kb, indicating that all plasmids were isolated from the vector p
EcoR of pICT113, a portion derived from LJC18
It was found that one I-8ac I fragment and one EcoRl-8ac I fragment of plcR113 containing the gene encoding AIaR were combined. These plasmids were named plcR114.

(7) EcoRI-EC of plasmid pICT113
1) ICT113 40μ obtained in preparation of ORI fragment (1) was
ECORI buffer 400μρ containing RI 150U
After reaction and digestion at 37° C. for 5 hours, 800 μm of cold ethanol was added and centrifuged to recover as an ethanol precipitate. This DNA was dissolved in 20 μm TH and (3
) After separation by LMP agarose electrophoresis, approximately 1.7 Kb containing the gene encoding D-ATA was obtained.
The DNA fragment was excised and purified in the same manner as in (4),
It was dissolved in 20 μm TE to obtain an EcoRI-EcoRI fragment solution of plasmid plcT113.

(8) A portion of pICR114 (approximately 4 μg) produced in W4 by digestion of plasmid pICR114 with ECORI (6)
For EC0RI including 10tJ EC0RI! After reacting and digesting in 50μp of 1 buffer at 37°C for 5 hours,
0 μm cold ethanol was added, centrifugation was performed to collect the ethanol precipitate, and the mixture was dissolved in 10 μm TE to obtain an EcoRI digest solution of plasmid pICR114.

(9) EcoRI-Eco of plasmid plcT113
Ec of plasmid pICT113 prepared by ligation (7) of RI fragment and pICR114 l:coRIfi
8 μm oRI-EcoRI fragment solution and 0.5 μm ECORI digest solution of pICR114 prepared in (10)
The DNA was reacted overnight at 16°C in 20 μp of T4 DNA ligase buffer containing 700 U of T4 DNA ligase.
The fragments were ligated.

(10) Escherichia using DNA linked in (9)
200μ of competent cells prepared in the same manner as for transformation of E. coli HBloI (5)
m and 10 μm of the reaction solution containing the DNA linked in (9) were mixed, and a transformed strain was obtained in the same manner as in (5).

(11) Selection of transformants with D-ATA activity (10) 1 out of about 2000 transformants obtained in (10)
68 colonies were added to 500μ' of Buffer I (10mM
Potassium phosphate SaW solution (pl+7.2), 0.01
%2-ME, 50 μM pyridoxal = 5°-phosphoric acid (
D-ATA activity was measured using an enzyme solution that was suspended in PLP) and disrupted by ultrasonication for 30 seconds.

<Method for measuring D-ATA activity> Tris-HCl buffer (pH 8,1) 50 μmol, P
LP50nmo I, a-'yt”J Lutal WJ10
A reaction solution of 11d containing μmol, D-Arani>25, cZmol, lactic acid 11R water JR enzyme (LDH, manufactured by Boehringer Mannheim Yamanouchi) 5U1NADH0.2μmol and enzyme was reacted in a cuvette at 50°C.
The decrease in absorbance at 340 nm due to the decrease in NADH was measured. Note that 1U is 1 μmol of N per minute under these conditions.
It was defined as the amount of enzyme that catalyzes the reduction of ADH. As a result of measuring the D-ATA activity of 168 colonies according to the above method, D-ATA activity was observed in two colonies.

(12) Confirmation of plasmids in strains retaining D-ATA activity Plasmids were isolated on a small scale from the two strains selected in (11) in the same manner as in (1). These plasmids,
Digestion with 5aCl yielded a single band of approximately 6°2 Kb, and digestion with ECORI yielded a band of approximately 4.5 Kb, the same as the EcoRI digest of pICR114, and pICR114.
Approximately 1 same as EcoRI-EcoRI fragment of lcT113
．． 7, a band b was generated. Also ECORI and 3ac
FCORI-E of pICT113 by double digestion of i
Approximately 1.7 Kb band, pICR1, same as CORI fragment
Approximately 1.9 Kb, same as the ECORl-3ac i fragment of 14
band and plcT113 ECORl-8ac
Since a band of about 2.7 Kb, which is the same as that of 1 fragment, was generated,
These plasmids were found to be the EcoRI-EcoRI fragment of plcT113 fused to the EcoRI digest of plcR114.

These plasmids were named pICRTllll.

Therefore, the obtained plasmid plcRT111 is Al
This is a plasmid that simultaneously contains a gene encoding aR and a gene encoding D-ATA.

The restriction enzyme cleavage map of plasmid pICRT111 is
As shown in the figure.

(13) Measurement of Al aRSD-ATA activity Niji I transformed with plasmids pLIC18, pICR114, pICT113, and pICRT1ll, respectively.
Lichia coli H8101 was cultured at 37°C overnight in 1.5j of ampicillin-containing YT medium (50Ug/ml),
．． Bacterial cells were collected by centrifugation at OOOrpm for 5 minutes. The wet weight of the collected national polity was 4.8g and 5.7g, respectively.
g, 7.2 g, and 5.1 g.

5ml of each bacterial body! buffer (10mM phosphate buffer (p
H7,2), 0.01% 2-ME, 50UM PL
P) and sonicated for 10 minutes at 18,000 rpm.
The supernatant obtained was dialyzed against the above buffer to obtain a cell-free extract.

Using this cell-free extract, each enzyme activity was measured according to the following method.

In addition, proteins were prepared using the method of Lowry et al. (Journal
of biological chemistry deafournal
of biolo 1cal chemist)
193, 265 (1951)).

The results are shown in Table-1.

<Method for measuring D-ATA activity> This was done in the same manner as described above.

<Method for measuring AI aR activity> Glycine-KC1-KOH! 1 buffer solution (pH 9,0) 10
0 μmol, PLP50 nmol I, D-alanine 50 μmol I, NAD+ 2.5 μmol,
Al aDH (Bacillus stearothermophilus IF
A 1 ml reaction solution containing 10 U (derived from OI 2550) and the enzyme was reacted in a cuvette at 50° C., and the increase in absorbance at 340 nm due to the increase in NADH was measured. Sho 1
U is the amount of enzyme that catalyzes an increase in NADH by 1 μmol per minute under these conditions.

Table-1

[Brief explanation of drawings]

FIG. 1 represents a restriction enzyme cleavage map of plasmid plcRT111. In the figure, the single line in the circle represents the part derived from vector pLJ018, and 22 represents the part encoding the target enzyme.Patent applicant
Daicel Chemical Industries, Ltd. Figure 1

Claims (4)

[Claims] (1) Plasmid containing genes encoding alanine racemase and D-amino acid transaminase (2) The plasmid according to claim 1, wherein the genes encoding alanine racemase and D-amino acid transaminase are both derived from heat-resistant bacteria. (3) Heat-resistant bacteria are Bacillus (￥Bacillus￥)
The plasmid according to claim 2, which is a bacterium of the genus (4) The gene encoding alanine racemase is derived from Bacillus stearothermophilus (￥Bacillus￥
¥stearothermophilus¥)IFO1
2550, and the gene encoding D-amino acid transaminase is derived from Bacillus sp.
Bac-illus\sp. ) The plasmid according to claim 1, which is derived from YM-1 (Feikoken Bibori No. 8057)