JPS63112978A - Escherichia coli - Google Patents

Abstract

PURPOSE:To make it possible to readily prepare two enzymes in a single operation, by preparing Escherichia coli having the ability to produce L-alanine dehydrogenase and D-amino acid transaminase. CONSTITUTION:A DNA fragment containing a gene capable of coding L-alanine dehydrogenase and a DNA fragment containing a gene capable of coding L- amino acid transaminase are obtained. The obtained DNA having both the genes and role as a vector is digested with a restriction enzyme and linked with a ligase to prepare a recombinant plasmid, which is then brought into contact with Escherichia coli treated with calcium chloride at about 0 deg.C to afford a transformed Escherichia coli. The resultant Escherichia coli is extremely useful for producing D-amino acids, etc.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to L-alanine dehydrogenation resistant yarn (hereinafter A1a D
The present invention relates to a novel Escherichia coli that has the ability to produce both enzymes (abbreviated as H) and amino acid transaminase (hereinafter abbreviated as D-ATA).

(Prior art) ′ Conventionally, Bacillus sudealothermophilus (Bac
-illus 5tcarotharIllophil
Escherichia coli (Japanese Unexamined Patent Publication No. 180580/1983) transformed with plasmid plcR301 containing the gene encoding the thermostable AI aDH derived from US),
Bacillus sp. YM-1
1) A plasmid carrying the gene encoding the thermostable D-AT8 derived from E. p.
Cori (Japan Agricultural Chemistry Society 60th Annual Conference Abstracts P, 343)
It has been known.

(Problems to be Solved by the Invention) However, when two enzymes, Al aDH and D-ATA, are prepared using genetic engineering technology and used at the same time, the two enzymes encoding the aforementioned two enzymes are separately transforming E. coli with two separate plasmids carrying the genes;
After selecting the transformed strains, it is necessary to culture them separately to prepare the two enzymes. This method requires complicated operations, takes time, costs, etc., and is not advantageous in terms of performance.

The present invention provides a microorganism that simultaneously produces the two enzymes.
The purpose of this method is to easily prepare the two enzymes at once.

(Means for Solving the Problems) The present inventors have discovered that a microorganism that can simultaneously produce the two enzymes can be obtained by transforming Escherichia coli, leading to the present invention.

That is, the present invention is Escherichia coli that has the ability to simultaneously produce A I aDH and D-ATA.

The Escherichia coli of the present invention may be endowed with the ability to produce two yeast cords by any method, and an example thereof is Escherichia coli obtained by genetic recombination technology. Among them, Escherichia coli transformed with a plasmid capable of producing the two enzymes is particularly mentioned. This Escherichia coli may be transformed with two plasmids, a plasmid having a gene encoding AlaDH-1 and a plasmid having a gene encoding D-ATA, or a gene encoding AlaDH and a plasmid having a gene encoding D-ATA. It may be transformed with a single plasmid containing the gene encoding D-ATA.

The above plasmid may be a natural plasmid extracted from a microorganism, as long as it has a gene encoding the enzyme, or a plasmid in which the gene encoding the enzyme is ligated to an appropriate vector (hereinafter referred to as a recombinant plasmid). ) is also acceptable.

Among these, Escherichia coli transformed with a recombinant plasmid is most preferred from the viewpoint of productivity of the two enzymes, economic efficiency, etc.

To obtain the recombinant plasmid used in the present invention, for example, a DNA fragment containing the gene encoding AlaDH,
A DNA fragment containing the gene encoding D-ATA was obtained, and both of the obtained genes and the DNA serving as a vector were published in the Journal of Molecular Biology.
oloQV) 96.171-184 (1975), by digesting with restriction enzymes and then ligating with ligavi.

The gene encoding AlaDH that is 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 stearothermophilus with high Al aDH activity is preferred. Specific examples of these include IFO12550 and ArCC7953. Also, the gene encoding D-ATA is the chromosome D of thermophilic microorganisms due to its heat resistance and stability.
Examples include genes derived from NA. Among these, genes derived from moderate thermophiles of the genus Bacillus are preferred. Specific examples of these include Bacillus sp. YM-1, Bacillus sp.
1/ M-2 (Bacillus sp, YH-2
, Microtechnical Research Institute No. 8058).

Moreover, as a plasmid having a role as a vector, plasmid I) IJC18 is particularly preferable. Further, as restriction enzymes, for example, l-1ind m, Hin
Examples of the ligase include T4ONΔ ligase.

As a dark blue plasmid for transforming Escherichia coli of the present invention, for example, the plasmid p
Bacillus stare in UC18 [ ] Thermophilus IFO
The gene encoding HaIaDH derived from the chromosomal DNA of B. 12550 and the chromosomal DNA of Bacillus sp. YM-1
An example of this is plasmid plcDT111 into which a gene encoding D-ATA derived from the plant was introduced.

In addition, in order to transform Escherichia coli using this dark blue plasmid, for example, the Journal of Fist Molecular Biology 53゜159-162 <19
This can be carried out by bringing the recombinant plasmid into contact with E. coli treated with calcium chloride at around 0° C. according to the method of 70).

As an example of Escherichia coli of the present invention transformed as described above, Nizielidia coli HB101-plcDT11 into which plasmid pICD111 has been introduced is
You can give one share. This strain has the same mycological properties as the known E. coli HB101, except that it has a heat-resistant AIaDH production ability, a heat-resistant D-ATA production ability, and ampicillin resistance.

This bacterial cell maintains safety without changing from non-transmissible to transmissible or from non-pathogenic to pathogenic.

Escherichia coli of the present invention is capable of synthesizing the D-amino acid represented by the following formula 1, namely, AIaDH, D-AT.
A, DL-alanine (OL-△la) of catalyst m and NA
Method for synthesizing D-amino acids from formic acid, ammonia and α-keto acids in the presence of D” (Patent application No. 61-48233
), etc., and can be used for A I aDH and D-
Facilitates the preparation of ATA.

(Formula 1) (Effect of the invention) As mentioned above, Esieritia coli of the present invention is A I
Since it has the ability to produce aDHID-ATA and can produce AlaD[1 and D-ATA simultaneously, easily and simultaneously, it is very useful for the above-mentioned IFJ production of D-amino acids.

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

(Example) (1) Plasmid pICD112 having a gene encoding a heat-stable Al aDH (plasmid having a gene encoding a heat-stable Al aDH derived from Bacillus stearothermophilus, JP-A-60-18059)
plasmid plcT113 (same as plasmid plcR301 described in 0) and plasmid plcT113 (same as plasmid plcR301 described in 0) and plasmid plcT113 (same as plasmid plcT113, which has a gene encoding heat-stable D-ATA derived from Bacillus sp. YM-1).
Plasmid linked to U018, summary of the 60th annual meeting of the Japanese Society of Agricultural Chemistry! Plasmid plcR described in AP, 343
E. coli HB 1 into which plasmid plcD112 was introduced in the same manner as described in (5) above after the preparation of E. coli HB 1 (subcloned version of 11).
01 strain, 50μsI/! 1N Super Broth containing lIi ampicillin (15 g polypeptone, 20 g yeast extract, 5 d glycerol.

1M potassium phosphate buffer (pH 7,6) 100 r
After culturing at 37° C. for 2 to 3 hours in 11 medium containing d), a chloramphenicol solution (34 mg added to 1-ethanol) was added for 6 hours, and the cells were further cultured for 16 hours.

After culturing, the bacterial cells were collected by centrifugation and diluted with a solution (50 m
M glucose, 25111M Tris-HCl buffer (pH
8,0), 10+aHEDTA1 lysozyme blQ7
11! The mixture was mixed vigorously with 20 ae of water and allowed to stand for 30 minutes. Next, solution U (0,2N Na01-1.1% SO8
('/dium dodecyl sulfate)) was added, stirred gently and left on ice for 10 minutes, then added 30ml of 5M potassium acetate buffer (pH 4,8), stirred vigorously, left on ice for 10 minutes, and then centrifuged. protein, RNA,
The precipitate containing chromosomal DNA was separated from the supernatant.

110 all of cold ethanol was added to the supernatant containing the crude plasmid, and after incubation at -80°C for 30 minutes, J:ri plasmid DNA was recovered as an ethanol precipitate by centrifugation. After drying the precipitate, 16m! TE (10mM Tris-salt mmw
solution (D118.O), 1mHEDTA), ribonuclease (RNaseA, manufactured by Sigma, 10#lSF/
me) Add 200 μl of ethanol and react at 37°C for 1 hour. Remove denatured proteins by extraction with Uphenoroborum (1:1), add 2x h1 of cold ethanol,
℃T: 30 minutes and collected as ethanol precipitate by centrifugation. Dissolve the pellet-shaped plasmid in a small amount of TE and add cesium chloride (1!7 to 1 m of plasmid solution).
) and ethidium bromide (0.8 ai! added to 10 d of cesium chloride solution). OO
Ultracentrifuged for 12 hours at Orpm, c c c (co
valcntly closed cir-cular
) Only the DNA band was collected, ethidium bromide was removed by n-butanol extraction, and then thoroughly dialyzed against TE to obtain purified pICD112.

Purified plcT113 was also prepared using the same procedure as ρlCD112.

(2) HindI of plasmid pICD112 [[
-E coRV fragment preparation (1) 40 μ9 of plcD112 was mixed with a buffer containing 40 U of restriction enzyme Hindll (manufactured by Takara Shuzo Co., Ltd.) (
10fflH Tris-HCl! 1 buffer (1) 117.5)
, 71118MOC12, 60mHNaCI) 300
Plasmid DNA digested by reacting with uR at 37°C for 3 hours
A to 5HNaCI 3uR1 cold ethanol 6504M
was added and centrifuged to perform ethanol precipitation and recovery. The recovered DNA pellet was dissolved in 50μρ TE,
ml containing 40 U of restriction enzyme EcoRV (manufactured by Takara Shuzo Co., Ltd.)
solution (10 mH Tris salt M!l buffer (pH 7,5), 7 m
HMOCI, 150 mH NaCl, 7 mN 2-
Mercaptoethanol, 0.01% bovine serum albumin)
React overnight at 37°C in 300 μi, add cold ethanol for 6 hrs.
50μ! DNA was recovered as an ethanol precipitate. This DNA pellet is coated with a T of 40 μρ.
Dissolve in E and add 1% LMP (low melting
point) Agarose (BethesdaRes)
(manufactured by arch Laboratories)
Electrophoresis was performed at 100°C for 2 to 3 hours, and A
A 1.4 kilobase pair Hind m-EcoRV fragment containing the gene encoding laDH was isolated, the agarose containing this fragment was cut out, and 5 times the volume of TE was added.
The agarose was dissolved by heating at 5° C. for 5 minutes.

This solution was subjected to phenol extraction twice, phenol-chloroform (1:1) extraction twice, and chloroform extraction twice to recover DNA fragments as ethanol precipitates. This D
The NA fragment was dissolved in a small amount of IE and subjected to reverse phase liquid chromatography (NENSORBTH20, manufactured by DUr'ONT).
The purified p[cD112 t-1ind m-E coRV
Fragments were prepared.

(3) HindI [[ of plasmid plcT113
- 1 inc [20μ of plasmid pICT113 prepared in fragment preparation (1)]
HindIII (manufactured by Takara Shuzo Co., Ltd.>20U, Hinc■
(Takara Shuzosha'!!J) Buffer solution (10ml) containing 24U
Tris-J! 2 pull 'Ii ui liquid (pH 7,5), 1
After reaction and digestion overnight at 37°C in 300μj of 8MqCI2.60mHNaC+, 5HNaC13μ
! , 650 μl of cold ethanol was added to collect the ethanol precipitate, and after dissolving in TE, LM
A purified fragment was obtained by purification by P-agarose electrophoresis in the same manner as in 4.2 (2) containing the gene encoding vector puc18 and D-ATΔ.

(4) Ratio of plasmid pICT113 1ndll-
Specific rib CI [fragment and specific rib d of plasmid pICD112
m-EcoRV fragment (7) ligation 1) ICD112 HindI[-EcollV fragment prepared in (2) was dissolved in 20 μl of TE, plcT1 prepared in (3)
40 μl of Hind m-Hinc II fragment of 13
Dissolved in TE. 1” NA ligase of pICD112 (
700U of Takara Shuzo Co., Ltd. buffer solution (66mM Tris-
Hydrochloric acid buffer (pH 7,6), 6.6mM MgCl,
10mHDTT, 1mHATP) 40μ! The DNA fragments were ligated by reacting overnight at 17°C.

(5) E. coli H by linked DNA
Transformation of B101 (4) 100 μβ of cold ethanol was added to the reaction solution of ligation reaction (4) to precipitate DNA, which was collected by centrifugation and dissolved in 90 μJTE.

Next, add 10% of the host bacterium Nigielidea coli 1'B 101.
Culture for 2 to 3 hours in 0 d YT medium (100 d medium pH 7,2 containing 1 g of polypeptone, 0.5 g of Ajffl extract, and 0.5 g of NaCl), collect by centrifugation, wash with cold 100 mH 1vloc12, and incubate with cold 100 mH.
It was suspended in HCaCI2 and left on ice for 1 hour. Next, after removing the supernatant by centrifugation, add cold 100n + Hc a Cl
The cells were resuspended for 25 d to become competent cells.

Next, 90 μl of the ligated plasmid DNA solution and 200 μl of the competent cell suspension were mixed at 0° C., kept on ice for 60 minutes with occasional stirring, then left at 42° C. for 2 minutes, and rapidly cooled with ice water. Next, 1 d of YT medium was added to this suspension, and after culturing at 37°C for 1 hour, a plate of YT medium containing 50 μg/Idl of ampicillin (agar 2 (1 d)
/l) and plated. This plate was cultured at 37°C for 16 hours, and the formed colonies were again grown on ampicillin-containing (50 μ9/d) YT medium (agar 2
0 g/l) and cultured in the same manner as the -th time to obtain a transformed strain.

(6) Selection of a transformant that produces both IaDH and D-ATA for heat resistance.) A replica of the transformant obtained in (5) was created on L'r- (Na5G manufactured by ToyoMi Paper Co., Ltd.). Proceedings of the Academy of Natural Sciences
Science of Nis Nis Nis (Proc, N
atl, Acad, Sci, USA) 72, 22
74-2278 (1975), 5Q
IIM glycine-KCI-KOH buffer pHIO, 5,
50mHL-alanine, 1.3e)l NAD+, 0
．． 128mMPMS (phenazine methosulfate)
, 0.48 mHNBT and troblue tetrazolium) were reacted with reaction solution 2d containing A I aDH by color development.
Transformants with activity were identified and isolated. Transfer the colony of this isolated transformant to 50 μl of 1QmH potassium phosphate! It was suspended in ll1i solution (pH 7,2) and disrupted by ultrasonication for 3 minutes. This suspension was centrifuged, and the A1aDH activity and D-A-rA activity of the resulting supernatant were measured.

■A I aDl → Activity measurement method Glycine-KCI-KOH buffer (pHlo, 7) 1
00μm101, L-alanine 50μmol, NA
D"25 μmol and the reaction solution of 1- containing enzyme
The reaction was carried out in a cuvette at 0° C., and the increase in absorbance at 340 dm resulting from the formation of N A D H was measured.

Note that 1U is 1 μnof of NAD per minute under these conditions.
1] was designated as enzyme m that catalyzes the production of.

■D-ATA activity measurement method Tris-salt m buffer (11118,1) 50 μmo
l, PLP (pyridoxal 5'-phosphorus fi) 50n
m.

1. 1# containing 10 μmol of α-ketoglutaric acid, 25 μmol of [)-alanine, 5LJ of lactic acid dehydration MM element (manufactured by Boehringer Mannheim Yamanouchi), 12 μsol of NADHO, and enzyme! i! The reaction solution was reacted in a cuvette at 50°C, and the decrease in absorbance at 340 dm due to the decrease in NADH was measured. Note that 1 U was the enzyme m that catalyzed a decrease in N A D +1 of 1 μnof in 1 minute under these conditions.

When the AlaDH and D-ATA activities of the selected transformed strain were measured according to the above method, the transformed strain
E. coli strain 88101 (E, coli H8101-1) ICD containing plasmid plcD112
112) Al aDH activity and plasmid plcT
Esieritia coli H [3101 strain (
E, D- of coli 88101-plcT1t3)
ATA activity was maintained at the same level and at the same time. The plasmid contained in this transformed strain was named pICDTlll.

(7) Properties of plasmid DNA plcDTlll Transformant strain obtained in (6) (E, coli II
BIOI-1) ICDTll) was cultured on a small scale according to the method of (1) and Ji1 to obtain pure pICDTll.
I got l. As a result of cutting this plasmid with various restriction M chords, the cutting sites were found to be two for 3maI, one for 5acI, and one for HindDl. In addition, the size of this plasmid is approximately 5.6 kilobase pairs, and in conjunction with the result of (6), plCDTll is the I'I of plCT113.
[It was confirmed that the 1-EcoRV fragments were combined one by one. A restriction enzyme cleavage map of this plasmid plcDT111 is shown in FIG.

[Brief explanation of the drawing]

FIG. 1 is a restriction enzyme cleavage map of the plasmid I) ICDTll of the present invention. In the figure, F3eCj represents a gene encoding Δl aDH, and d-ata represents a gene encoding D-ATA. ←−→ are a, 2d and d-ata, respectively
The single line in the circle indicates the location of plasmid ptJc1.
The part 8 above, 222, indicates the inserted DNA fragment. Patent applicant: Daicel Chemical Industries, Ltd. Agent: Patent attorney: Yang Yue

Claims (4)

[Claims] (1) Escherichia coli (￥E
scherichia coli￥) (2) Escherichia coli according to claim 1 transformed with a plasmid having both a gene encoding L-alanine dehydrogenase and a gene encoding D-amino acid transaminase. (3) The gene encoding L-alanine dehydrogenase is derived from the chromosomal DNA of a thermophilic microorganism, and the gene encoding D-amino acid transaminase is derived from the chromosomal DNA of a thermophilic microorganism. Escherichia coli according to claim 2, which is a gene encoding D-amino acid transaminase derived from chromosomal DNA. (4) Both thermophilic microorganisms are Bacillus (￥Bacillus).
Escherichia coli according to claim 3, which is a bacterium belonging to the genus