JPH06105691A - Dna fragment containing phenyl hydroxylase gene region, recombinant plasmid, transformant and decomposition of trichloroethylene - Google Patents

Abstract

PURPOSE:To provide a new DNA fragment useful for the preparation of a transformant capable of decomposing trichloroethylene. CONSTITUTION:A DNA fragment containing a phenol hydroxylase gene region expressed by the restriction map shown in the drawing (C230 is a part of catechol 2,3-oxygenase gene; PH is phenol hydroxylase gene). It can be prepared by separating a DNA from a chromosomal DNA of e.g. Pseudomonas putida KWI-9 strain (FERM P-13109) and cleaving at the restriction enzyme StuI site and SmaI site.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a DNA fragment containing a phenol hydroxylase gene region, a novel recombinant plasmid containing this DNA fragment, a transformant carrying this recombinant plasmid, and trichloroethylene using this transformant. Regarding the disassembly method of.

[0002]

2. Description of the Related Art Trichlorethylene is widely used as a solvent, but it is toxic and is not easily decomposed by natural microorganisms, so that it causes soil and groundwater pollution in various places.

As a treatment method of trichlorethylene, there is a method of removing it by adsorbing it on activated carbon or the like, but this method merely recovers trichlorethylene and cannot detoxify it, which is not an essential solution. . Although trichlorethylene can be decomposed by an anaerobic treatment, there is a problem in that highly toxic intermediate products such as dichloroethylene and vinyl chloride are produced.

Utilization of an enzyme or a microorganism that decomposes trichlorethylene can be an effective measure for solving such a problem. Conventionally, methane monooxygenase possessed by methane-utilizing bacteria, toluene monooxygenase possessed by toluene-utilizing bacteria, toluene dioxygenase, and ammonia monooxygenase possessed by nitrifying bacteria have been known as enzymes for degrading trichlorethylene.

However, there are the following problems in the decomposition of trichlorethylene using a natural bacterium capable of degrading trichlorethylene. 1) Since natural bacteria are induced by substances such as methane, toluene and phenol, it is essential to add these inducers. Therefore, the added toluene, phenol, etc. are released into the environment. 2) Once induced, the decomposition ability of trichlorethylene is not maintained for a long period of time, and the activity usually disappears in about half a day. 3) Insufficient resolution when used in a reactor.

On the other hand, in Japanese Patent Publication No. 2-503866, Pseudomonas mendocina KR-1 ( Pseudomo
It has been described that a toluene monooxygenase gene (toluene monooxygenase decomposes trichloroethylene) derived from Nas mendocina KR-1) is introduced into a host bacterium, and this transformant is used to decompose trichlorethylene. However, this method utilizes toluene monooxygenase, which is different from the method utilizing the phenol hydroxylase of the present invention.

[0007]

An object of the present invention is to provide a DNA fragment containing the gene region of phenol hydroxylase having trichloroethylene degrading activity. Another object of the present invention is to provide a novel recombinant plasmid incorporating the above DNA fragment. Another object of the present invention is to provide a transformant in which the above recombinant plasmid is introduced into a host bacterium. Still another object of the present invention is to propose a method for decomposing trichlorethylene using the above transformant.

[0008]

The present invention provides a DNA fragment containing the following phenol hydroxylase gene region, a recombinant plasmid, a transformant, and a method for degrading trichlorethylene using the same. (1) A DNA fragment containing the phenol hydroxylase gene region shown in the following restriction enzyme map [1].

[Chemical 2] (In the map, C23O is a part of the catechol 2,3 oxygenase gene, and PH is the phenol hydroxylase gene.) (2) Pseudomonas putida KWI-9 ( Pseu
domonas putida KWI-9) strain chromosomal DNA-derived phenol hydroxylase gene, a drug resistance gene, and a broad host range replication region containing a gene and a replication initiation site required for autonomous replication in Gram-negative bacteria, A recombinant plasmid capable of replicating in Gram-negative bacteria. (3) A transformant obtained by introducing the recombinant plasmid according to (2) above into a host bacterium. (4) A method for decomposing trichlorethylene, which comprises decomposing trichlorethylene using the transformant described in (3) above.

The DNA fragment containing the phenol hydroxylase gene region of the present invention is isolated from the chromosomal DNA of Pseudomonas putida KWI-9 strain or the like. Pseudomonas putida KWI-9 strain was sent to the Institute of Microbial Science and Technology, Ministry of International Trade and Industry, Institute of Microbiology,
No. 109 (FERM P-13109) has been deposited as a microorganism accession number, and its bacterial properties and culture method are described in Japanese Patent Application No. 4-228169.

The DNA fragment of the present invention comprises the restriction enzyme Stu I
To Sma I site, and has the following cleavage sites for the following restriction enzymes: Restriction enzyme number of cleavage sites Sac I 1 Sal I 1 Hin dIII 1 Eco RI 1 The relative cleavage sites of the above restriction enzymes are completely digested with a plurality of restriction enzymes, and the generated DNA fragment is analyzed by agarose gel electrophoresis. Can be obtained by

The DNA fragment of the present invention is a Pseudomonas
8.5 ligated to the following two DNA fragments obtained by cutting the chromosomal DNA of Putida KWI-9 strain with a restriction enzyme
It is present in a kb DNA fragment (see Figure 3). DNA fragments of 10kb obtained by cutting with Hin dIII (see Figure 1): Eco of 2.3kb of DNA fragment
The catechol 2,3 oxygenase (C23O) gene is present between the RI site and the Sal I site of 4.8 kb. The transformant containing this DNA fragment was C23.
Since catechol turns yellow by O, it can be easily detected.

6.0 k obtained by cutting with Eco RI
DNA fragment of b (see FIG. 2): 3.7 of this DNA fragment
It does not Hin dIII site of kb 6.0kb of Eco R
I site part, of the DNA fragment Hin of 0kb dII
It overlaps with the I site or the 2.3 kb Eco RI site part. That is, the 0 kb Eco of the DNA fragment
RI site to Hin dIII site of 3.7kb is,
Is an upstream portion of the DNA fragment of DNA fragment of
It can be separated by Southern hybridization using the overlapping portion of both as a probe.

8.5 kb D containing the DNA fragment of the present invention
NA fragment, Hin of 0kb the above DNA fragment dII
To the I site or 4.8 kb Sal I site fragment, the 0 kb Eco RI of the DNA fragment as the upstream part
Downstream of the site to either associates the Hin dIII site fragment of 3.7 kb, or a DNA fragment, the DN
2.3 kb Eco RI site of the A fragment or 4.8 k
It is obtained by ligating the Sal I site fragment of b. The DNA fragment of the present invention is obtained as a fragment obtained by cleaving the 8.5 kb DNA fragment at the Stu I and Sma I sites.

The recombinant plasmid of the present invention has a phenol hydroxylase gene derived from Pseudomonas putida KWI-9 strain chromosomal DNA. Phenol hydroxylase formed from this gene degrades trichlorethylene, and therefore the recombinant plasmid of the present invention can be introduced into a host bacterium, and the transformant obtained can be used to degrade trichlorethylene.

The recombinant plasmid of the present invention can be constructed by ligating the following DNA fragments with ligase. DNA fragment containing broad host range replication region Drug resistance gene (marker) Pseudomonas putida KWI-9 strain chromosome DN
Phenol hydroxylase gene from A

The broad host range replication region is a gene region required for the recombinant plasmid to grow independently and stably be maintained in Gram-negative bacteria.
However, it is not limited to this. RSF1010 contains the genes and origin of replication required for autonomous replication in Gram-negative bacteria,
It is concentrated in a DNA region of about 5.8 kb. This area is not only for RSF1010, but also for pKT240, pMM
It can be separated from a plasmid such as B22 using a nuclease such as a restriction enzyme.

As the drug resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, kanamycin resistance gene, tetracycline resistance gene and the like can be adopted, and pACYC184, pKK223-3, pTrc.
It can be isolated from plasmids such as 99A and pKT240.
Pseudomonas putida KWI-9 strain chromosomal DNA
As the derived phenol hydroxylase gene,
The DNA fragment of the present invention can be used.

The recombinant plasmid of the present invention can express phenol hydroxylase through various different promoter-terminator systems. Therefore, the improvement of trichlorethylene decomposing ability can be easily performed by improving the promoter. Also, lacIq
By incorporating a repressor gene such as, it becomes possible to control by an inducer such as isopropylthiogalactoside (IPTG). Therefore, addition of phenol and toluene is unnecessary. Furthermore, by deleting the repressor gene, it is not necessary to add an inducer, and it is possible to allow phenol hydroxylase to act as a constituent enzyme. Therefore, trichloroethylene decomposing ability can be maintained for a long time. The lacIq gene / promoter / operator / terminator system is pTr
It can be separated from a plasmid such as c99A using a nucleolytic enzyme such as a restriction enzyme.

Introduction of the recombinant plasmid into the host can be carried out very easily by electroporation, calcium and rubidium treatment, transfer using a helper plasmid, and the like.
Gram-negative bacteria, in which the recombinant plasmid is stably maintained, are used as the host. Pseudomonas bacteria are preferable, and Pseudomonas putida KWI-9 strain and mutants thereof are preferable.

Further, according to the present invention, trichlorethylene can be decomposed using a transformant into which a recombinant plasmid has been introduced. Degradation of trichlorethylene can be performed by a method such as aerobically culturing the transformant in a medium containing trichlorethylene, and complete dehalogenation can be performed.

Cultivation of the transformant into which the recombinant plasmid has been introduced is carried out under conditions suitable for the growth of the host. Peptone, tryptone, yeast extract and the like as carbon and nitrogen sources, sodium chloride and potassium chloride as inorganic salts are used. Etc., pH of the medium is 5 to 8.5, preferably 6 to 7, temperature 1
It is desirable to culture aerobically at 5 to 35 ° C, preferably around 30 ° C.

[0022]

EXAMPLES Next, examples of the present invention will be described. The plasmids and microorganisms used in the examples are as follows. 1) An expression vector used for expressing the pKK223-3 protein. It has a strong tac promoter that can be regulated by the lac repressor and induces transcription when an inducer such as IPTG inactivates the lac repressor. Multiple cloning site (MCS) immediately downstream of tac promoter
And a strong rrn ribosomal terminator is present. This plasmid is commercially available from Pharmacia and has the product code number 27-4935-01.

2) A derivative of pTrc99A pKK233-2, which has a long multicloning site and a strong terminator (rrB) immediately downstream of the strong trc promoter.
Also no lacIq (powerful ones of the lac repressor) in pKK233-2 for containing the gene, E the lac repressor is lost. E. coli can be used as a host. The trc promoter is driven by the addition of IPTG. N of the multi-cloning site
The coI restriction enzyme cleavage site has a translation initiation codon ATG, and it can be expressed by inserting a gene lacking this codon. This plasmid is commercially available from Pharmacia and has a product code number of 27-5007-.
01.

3) pACYC184 E. coli cloning vector having a size of 4244 bp. The first guanine (G) on the Eco RI site
Is the 1st in the nucleotide sequence, it has a tetracycline resistance gene at 1580 to 2770th position and a chloramphenicol resistance gene (Cm) at 3804th to 219th position.

4) It has a broad host range replication region derived from pKT240 plasmid RSF1010 and can be stably maintained in many Gram-negative bacteria.
It is a 2.9 kb plasmid. It has an ampicillin resistance gene and a kanamycin resistance gene. ATCC
Is commercially available from. ATCC No. 37258 (A
TCC Catalog of Recombin
ant DNA Materials 2nd edi
Tion, 1991).

5) E. coli DH5α Easily transformed by a large plasmid. Commercially available from Bethesda Research, Inc. (BRL). Product number is 8
262 SA. 6) Pseudomonas putida KWI-9 strain A trichlorethylene-degrading strain that has a phenol-utilizing ability and not a toluene-utilizing ability, and has been deposited under the above-mentioned microorganism deposit number. Degradability of trichlorethylene is significantly inhibited by the coexistence of phenol.

Example 1 It is considered that the phenol hydroxylase produced by Pseudomonas putida KWI-9 strain is degrading trichlorethylene, and the C23O gene coexists with the phenol hydroxylase gene in one operon, and the C23O gene is present. It was considered that the phenol hydroxylase gene was present in the upper stream of the enzyme, and an attempt was made to separate the phenol hydroxylase gene using C23O as a marker. In addition, C23O is catechol as 2-hydroxymuconic semialdehyde (2-Hydroxymuc).
It oxidizes to onic semidehyde) and turns yellow.

LB medium was used for liquid culture of Escherichia coli DH5α, and the culture temperature was set at 37 ° C. Antibiotics are ampicillin and chloramphenicol 100 μg /
ml, used at a concentration of 50 μg / ml. E. coli DH5
The method of Hanahan was used for the introduction of the plasmid into α. The Southern hybridization method, the ligation method, and other basic methods related to molecular biology are
Second of all "Molecular cloning"
According to the edition.

[0029] was cut with Hin dIII, 5 'to the ends were dephosphorylated processing pKK223-3, Pseudomonas putida KWI-9 limits the chromosome of strain enzyme Hin dIII
The DNA fragment cleaved with was incorporated. This plasmid was introduced into Escherichia coli DH5α and transformed. As a result, about 1
2000 colonies were obtained, of which 9 colonies turned yellow by spraying with catechol. All of these transformants incorporated the same 10 kb DNA fragment. A restriction enzyme map of a plasmid containing this DNA fragment is shown in FIG. This DNA fragment contains 2.3 kb of Ec
o The C23O gene was present between the RI site and the Sal I site.

[0030] The pKK223-3 incorporating the Hin dIII DNA fragments of the above 10kb was introduced into E. coli DH5α, were subjected to the decomposition test of phenol by this E. coli, phenol was not degraded at all. Therefore, a further upstream DNA fragment was obtained by the following procedure.

The chromosome of Pseudomonas putida KWI-9 strain was digested with Eco RI and electrophoresed on an agarose gel to excise an agarose gel portion containing a 6.0 kb DNA fragment. The DNA fragment contained in this was taken out by a DNA cell (DNA CELL, trademark) manufactured by Daiichi Pure Chemicals. This 6.0 kb Eco RI D
The NA fragment was incorporated into the Eco RI site of pTrc99A to obtain about 14,000 transformants. 300 of them
10 kb of the Hin dIII DNA
It does not Hin dIII site of 0kb of fragments of 2.3kb
Southern hybridization was performed using up to the Eco RI site as a probe. As a result, 6.0 kb
A transformant having a plasmid incorporating the Eco RI DNA fragment was obtained. The restriction enzyme map of this plasmid is shown in FIG. This plasmid contains 10 kb of H
The 0 kb Hin dIII site of the in dIII DNA fragment was present at the 3.7 kb position.

The above two kinds of DNA fragments (10 kb of Hi
n dIII DNA fragment, Eco RI DN of 6.0kb
A fragment) is ligated as shown in FIG. 3, and the downstream site is C2.
8.5 kb D up to Sal I site containing 3O gene
The NA fragment was incorporated into pTrc99A. The restriction enzyme site in FIG. 3 was determined using a restriction enzyme that does not cleave pTrc99A.

The restriction enzyme site at the upstream site of this plasmid was deleted stepwise to prepare plasmids of various lengths, which were introduced into Escherichia coli DH5α, and colony-derived phenol hydroxylase and C2 were introduced.
An expression test of 3O was performed. C23O was 0.1 M catechol, and in the case of phenol hydroxylase, 0.1 M phenol was used. These were dropped onto the colonies, and the color change of these colonies was observed. When the phenol hydroxylase gene is correctly integrated, the C23O gene is present in the downstream, so that after phenol is oxidized to catechol by phenol hydroxylase, a yellow substance is continuously produced by C23O. Yellowing. Therefore, the detection is easy.

The expression of the gene from the Stu I site was carried out as follows. Expression of the gene from the Stu I site caused growth failure in E. coli and did not form colonies. This was due to the Nc of pTrc99A.
Since it was found that the protein synthesized from ATG at the o I site was the cause of the inhibition, a new plasmid pTrc100 obtained by removing the ATG portion from pTrc99A was used instead of pTrc99A. This plasmid was cut pTrc99A at Nco I, creating the exposed CATG Remove with Mung bean Nuclease.

The expression results are shown in FIG. From FIG. 4, Stu I
It can be seen that the phenol hydroxylase gene exists between the site and the second Sma I site, and the C23O gene exists between the second Eco RI site and the second Sal I site. It should be noted that those showing phenol hydroxylase activity also showed trichloroethylene degrading activity, and it was revealed that phenol hydroxylase degrades trichlorethylene. Locations of the phenol hydroxylase gene and the C23O gene are shown in FIG.

Example 2 DN from Stu I to the last Sal I site containing phenol hydroxylase gene and C23O gene
A recombinant plasmid incorporating the A fragment (see FIG. 5) was introduced into Pseudomonas putida KWI-9 strain, and a trichlorethylene degradation test was conducted as follows.

The following SOB medium was used for liquid culture of Pseudomonas putida KWI-9 strain. As the agar medium, SOB agar medium was used. Antibiotics were ampicillin and chloramphenicol at 100 μg / ml and 50, respectively.
Used at a concentration of μg / ml. The culture temperature was set to 30 ° C. The introduction of the plasmid into Pseudomonas putida KWI-9 strain was carried out by the electroporation method using Gene Pulser manufactured by Bio-Rad (BIO-RAD).

SOB medium: Bacto tryptone 20 g Bacto yeast extract 5 g NaCl 0.5 g 250 mM KCl 10 ml Distilled water to a total volume of 990 ml pH 7.0 Sterilized by autoclaving the above solution and cooling to room temperature , 2M Mg ²⁺ solution sterilized by autoclave in another bottle (1M MgSO ₄ · 7H ₂ O + 1M M
10 ml of gCl ₂ .6H ₂ O) is added.

First, the RSF1010 replicon, which is widely used in Pseudomonas, the chloramphenicol resistance gene of pACYC184, and the l of pTrc100.
Using the parts from acIq to the terminator, the plasmid pRCL100 for Pseudomonas that can be controlled by IPTG shown in FIG. 6 was prepared.

[0040] In other words, Pvu II and P from pKT240
about 5.8kb replicon site to be cut out by st I (this replicon of RSF1010 Pvu II (bp194
8) -corresponds to Pst I (bp 7768))
And Bst BI (bp3716) of pACYC184 ~
The Bsa AI (bp310) portion was treated with T ₄ DNA polymerase and then ligated with T ₄ DNA ligase to prepare a plasmid. Next, the Xmn I of RSF1010
This plasmid was cleaved using the (bp2030) site, and p was treated with T ₄ DNA polymerase at this site.
Trc100 of Bsa AI (bp2769) ~ Bsp H
I (bp793) (where lacIq is present in this part) was incorporated to create a new plasmid pRCL100.

Next, since there is no Bam HI site in the DNA fragment from the Stu I to Sal I sites containing the phenol hydroxylase gene and the C23O gene (see FIG. 5), Bam HI linkers are added to both ends of this DNA fragment. It was ligated and modified so that all the DNA fragments could be excised with a single enzyme. Then, pRCL100 is Bam H
It was cleaved with I, and a DNA fragment containing the phenol hydroxylase gene and the C23O gene was inserted into that portion to prepare a new plasmid pNEM101 shown in FIG. 7.
This plasmid is Pseudomonas putida KWI-
When introduced into 9 strains, it remained stable.

This pNEM101 was introduced into Pseudomonas putida KWI-9 strain, and a trichlorethylene decomposition test was conducted as follows. 1 to 2 ml of the preculture liquid of the recombinant Pseudomonas putida KWI-9 strain was added to 10
After inoculating 0 ml of SOB medium, culturing at 30 ° C.,
Absorbance at 00 nm (hereinafter referred to as A ₆₀₀ ) is 0.5 to
When it reached 0.7, after adding 5 mM of IPTG,
Induction was carried out by culturing for 2 hours. Then collect the cells by centrifugation,
The cells were suspended in the following inorganic medium so that the A ₆₀₀ was 2.0.

Inorganic medium: Na ₂ HPO ₄ + KH ₂ PO ₄ (1M, pH6.8) 40ml Huntner's vitamin-free mineral base * 1 20ml (NH ₄ ) ₂ SO ₄ 1g distilled water 840ml * 1 Huntner's vitamin-free mineral base; Nitrilotriacetic acid 10.0 g MgSO ₄ 14.45 g CaCl ₂・ 2H ₂ O 3.335 g (NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O 9.25 mg FeSO ₄・ 7H ₂ O 99 mg Metals “44” * 2 50 ml distilled water total volume to 1000 ml * 2 Metals "44" (Metals "44"); ethylenediaminetetraacetic acid _{250.0mg ZnSO 4 · 7H 2 O 1095.0mg} (250mg Zn) FeSO 4 · 7H 2 O 500.0mg (100mg Fe) MnSO _{4 · H 2 O 154.0mg (50mg} Mn) CuSO 4 · 5H 2 O 39.2mg (10mg Cu) Co (NO 3) 2 · 6H 2 O 24.8mg (5mg Co) Na 2 B 4 O 7 · 10H 2 O 17.7 mg (2mg B) Add a few drops of sulfuric acid to prevent precipitation 100ml distilled water

10 ml of this bacterial solution was placed in a 125 ml vial bottle manufactured by GL Sciences, 10 mg / l of trichlorethylene (concentration when all were dissolved in the liquid) was added, and a Teflon-coated butyl rubber stopper was added.
Sealed with aluminum cap. This vial was subjected to shaking culture at 30 ° C. and 200 rpm, and 100 μl of the gas phase was periodically sampled with a gas tight cylinder to carry out a decomposition test of trichlorethylene. As a control,
A bacterial solution that was not induced with IPTG was used.

The results are shown in FIG. From FIG. 8, pRCL1
When IPTG that activates the 00 promoter is added, trichlorethylene is significantly decomposed as compared with the case where it is not added, and the phenol hydroxylase gene inserted downstream of the trc promoter effectively acts,
It was revealed that active hydrolysis of trichlorethylene was carried out by phenol hydroxylase.

Example 3 In Example 2, lacI was used instead of pNEM101.
Using plasmid pNEM201 deficient in q, IP
A trichlorethylene decomposition test was conducted in the same manner as in Example 2 without adding TG.

PNEM201 was prepared as follows. First replicon site from about 5.8kb to be cut out from the pKT240 with Pvu II and Pst I (Pvu II (bp1948 of the replicon RSF1010) ~ Pst I
(Corresponding to part (bp7768)) and pACYC1
84 Bst BI (bp 3716) to BsaAI (bp
A portion (310) was treated with T ₄ DNA polymerase and then ligated with T ₄ DNA ligase to prepare a plasmid. Next, RSF1010's Xmn I (bp 2030)
This plasmid was cleaved using the site and the P of pTrc100 treated with T ₄ DNA polymerase was digested at this site.
vu II (bp4096) to Bsp HI (bp793)
(No lacIq is present in this part) was incorporated to create a new plasmid pRCT200.

Next, D from Stu I to Sal I containing the phenol hydroxylase gene and the C23O gene
Since there is no Bam HI site in the NA fragment (see FIG. 5), Bam HI linkers were ligated to both ends of this DNA fragment so that all the DNA fragments could be excised with a single enzyme. Then, pRCL200 was cleaved with Bam HI, and a DNA fragment containing the phenol hydroxylase gene and the C23O gene was incorporated into that portion to create a new plasmid pNEM201 shown in FIG. 9. This plasmid is derived from pNEM101 used in Example 2.
It was deficient in acIq and was stably maintained when introduced into Pseudomonas putida KWI-9 strain.

Using the transformant thus obtained, a trichlorethylene decomposition test was conducted in the same manner as in Example 2.
The results are shown in Fig. 10. From FIG. 10, the inducer (IPT
It can be seen that decomposition of trichlorethylene occurs even without addition of G), and the ability is maintained for a long time of 50 hours or more. That is, when lacIq is present, an inducer is required as shown in Example 2, but when this lacIq is deleted, the repressor is not produced, and therefore, decomposition of trichlorethylene is not required even if the inducer is not added. Is possible.

[0050]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a DNA fragment containing the gene for phenol hydroxylase degrading trichlorethylene can be obtained. In addition, the above DNA
A novel recombinant plasmid containing the fragment, which can be used as a vector when preparing a transformant degrading trichlorethylene and has a high trichlorethylene degrading activity, can be obtained.

Furthermore, a transformant can be obtained which retains the above recombinant plasmid and can be utilized for the decomposition of trichlorethylene. Furthermore, by utilizing the above transformant, trichlorethylene can be decomposed easily and efficiently.

[Brief description of drawings]

FIG. 1 is a restriction map of the plasmid prepared in Example 1.

FIG. 2 is a restriction enzyme map of the plasmid prepared in Example 1.

FIG. 3 is a restriction enzyme map of a plasmid containing the DNA fragment of the present invention.

FIG. 4 is a diagram showing test results of Example 1.

FIG. 5 is a restriction map of a DNA fragment containing the DNA fragment of the present invention.

FIG. 6 is a restriction enzyme map of the plasmid prepared in Example 2.

FIG. 7 is a restriction enzyme map of the plasmid prepared in Example 2.

FIG. 8 is a graph showing the test results of Example 2.

FIG. 9 is a restriction enzyme map of the plasmid prepared in Example 3.

FIG. 10 is a graph showing the test results of Example 3.

[Explanation of symbols]

 P promoter PH phenol hydroxylase gene C23O catechol 2,3 oxygenase gene Cm chloramphenicol resistance gene 5ST1T2, T terminator IPTG isopropyl thiogalactoside

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[Procedure amendment]

[Submission date] August 11, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0048

[Correction method] Change

[Correction content]

Next, D from Stu I to Sal I containing the phenol hydroxylase gene and the C23O gene
Since there is no Bam HI site in the NA fragment (see FIG. 5), Bam HI linkers were ligated to both ends of this DNA fragment so that all the DNA fragments could be excised with a single enzyme. Then, pRCT 200 was cleaved with Bam HI, and a DNA fragment containing the phenol hydroxylase gene and the C23O gene was incorporated into that portion to construct a new plasmid pNEM201 shown in FIG. This plasmid is derived from pNEM101 used in Example 2.
It was deficient in acIq and was stably maintained when introduced into Pseudomonas putida KWI-9 strain.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 1/21 7236-4B // (C12N 15/53 C12R 1:40) (C12N 1/21 C12R 1:19)

Claims (4)

[Claims] 1. A DNA fragment containing a phenol hydroxylase gene region shown in the following restriction enzyme map [1]. [Chemical 1] (In the map, C23O is a part of the catechol 2,3 oxygenase gene, and PH is the phenol hydroxylase gene.) 2. Pseudomonas putida KWI-9
( Pseudomonas putida KWI-
9) It has a phenol hydroxylase gene derived from strain chromosomal DNA, a drug resistance gene, a gene necessary for autonomous replication in Gram-negative bacteria, and a broad host range replication region containing a replication initiation site. A recombinant plasmid capable of replicating in. 3. A transformant obtained by introducing the recombinant plasmid according to claim 2 into a host bacterium. 4. A method for decomposing trichlorethylene, which comprises decomposing trichlorethylene using the transformant according to claim 3.