JP2792052B2 - Xylene oxygenase gene - Google Patents

Description

The present invention relates to xylene oxygenase genes (XylM and XylA) capable of assimilating toluene or xylene.

(Problems to be Solved by the Prior Art and the Invention) Bacteria capable of assimilating toluene or xylene usually have a plasmid generally referred to as a TOL plasmid, and the assimilation gene is encoded on the plasmid.

The TOL plasmid has genes for enzymes that produce benzoic acid and / or toric acid from toluene or xylene, and enzymes that completely degrade pyruvate and acetaldehyde or propionaldehyde.

Xylene oxygenase is composed of two subunits, the XylM and XylA proteins, and both subunits function together. Xylene oxygenase has low substrate specificity and oxidizes toluene or xylene to (methyl) benzyl alcohol, and also converts indole to indoxyl and indigo (indigo).

Indigo is important as a dye for clothing,
It is contained in blue flowers of natural plants such as indigo. However, some plants that are commercially important lack blue, and their commercial value is great if blue flowers are introduced into commonly popular flowers such as roses and chrysanthemums. Is believed to be something.

On the other hand, in recent years, transformation systems for higher plants have been established in many species, and the production of herbicide-resistant and virus-resistant plants has been recognized [Nature, 317 , 741-744, 1985;
Science, 232 , 738-743, 1986]. When a gene exhibiting a blue flower color is introduced into a plant using such a host-vector system, it is possible to cause a plant having no blue pigment to bloom a blue flower. One candidate for the gene is a gene that produces indigo.

Conventionally, indigo producing genes include the naphthalene dioxygenase gene of the naphthalene plasmid NAH7.
nahA is only known [Scienc
e), 222 , 167, 1983]. Naphthalene dioxygenase is an enzyme that converts indole to indigo, but the nucleotide sequence of nahA has not yet been determined.

A xylene oxygenase gene is known as another indigo-producing gene. This xylene oxygenase is an enzyme different from naphthalene dioxygenase, but has the function of producing indigo from indole [Bio / Technology, 4 , 321-32].
4,1986]. This xylene oxygenase (XylM, XylA)
Was cloned from pWWO, the wild-type plasmid of the TOL plasmid [Journal of
The Bacteriology (J. Bact.), 167 , 455-461,198
6], but its detailed gene structure has not yet been investigated.

(Means for Solving the Problems) The present inventors have repeatedly studied to obtain the genes encoded by the genes XylM and XylA corresponding to xylene oxygenase, and as a result, the DNA sequence and Thus, the present invention has been completed.

That is, the gist of the present invention resides in a xylene oxygenase gene having a DNA encoding a specific amino acid sequence.

 Hereinafter, the present invention will be described in detail.

In the present invention, Sal I, Hin is further derived from pGSH2836 [Journal of the Bacteriology (J. Bacteriol.), 167 , 455-461, 1986], in which a DNA fragment containing xylene oxygenase of the TOL plasmid pWWO was cloned into an expression vector. dIII contains xylene oxygenase
A 2.3 kb DNA fragment was cut out, and the pUC strain [Gene (Gen
e), 19 , 259-268, 1982; Gene, 33 , 103-119,
1985] and the M13mp strain [Methods in Enzymol., 101 , 20-77, 1983], etc., and a kilosequence method based on the dideoxy method [Gene, 28 , 351- 359, 1984].

The DNA fragment encoding xylene oxygenase subcloned in the vector is opened with Pst I and Bam HI, and is continuously deleted by exonuclease III and mung bean nuclease. Since the sequence can be read only from the position of the primer to the position of 200 to 300 bp, the cloning in which a deletion by 200 bp is selected in the mini screening is selected. Each cloning was sequenced and read, and the overlapping portions were joined to determine the nucleotide sequence of the entire 2.3 kb DNA. This operation was performed in two directions, front and back of the DNA, to confirm the chloride sequence.

The amino acid sequence was obtained by converting the base sequence obtained from the sequence into amino acids based on universal codons. As a result, xylene oxygenase is an enzyme consisting of two proteins.The protein encoded by XylM has a molecular weight of 41,631, the first 30 amino acids at the N-terminal are highly hydrophobic, and the protein encoded by XylA has a molecular weight of 38,697. (No. 1
Figure).

In addition, it was predicted that toluene, xylene, oxygen, and the like bind to the Xy1M protein, and that the XylA protein is involved in the electron transport system. The N-terminal side of the XylA protein was highly homologous to plant-type ferredoxin, and it was found that a cystine residue binding to Fe ⁺⁺ was present at the same position, confirming this fact.

(Effects of the Invention) The genes encoded by XylM and XylA of the present invention are considered to be effective for producing blue flower plants.

(Example) Hereinafter, the present invention will be specifically described with reference to examples.

(1) Toluene / xylene degradation plasmid pWWO is ATCC
Pseudomonas puida obtained from
tida). Plasmid pGSH2836 is constructed by connecting a fragment containing xylene oxygenase the P _L promoter λ phage from pWWO [Journal of The Bact (J.Bacteriol.), 167, 455-461,198
6] Since, cut from this further pGSH2836 a fragment containing xylene oxidase 2.1kb in Sal I, Hin d III, 0.7 %
After electrophoresis on an agarose gel, it was excised. The excised DNA fragment was electrically recovered from the agarose gel. The DNA was purified by phenol treatment and ethanol treatment, and pUC18,19 [Gene, 33 , 103-119,198
5] Sal I and Hind III. Escherichia coli HB101
Hanahan method [DNA clonin (DNA clonin
g), 1 , 109-136, 1985].
Competent cells were E. coli HB101 with 0.1 M potassium chloride /
Contains 45mM manganese chloride / 10mM calcium chloride / 10mM potassium acetate-10% glycerol / 3mM hexacobalamin hydrochloride
The mixture was treated at pH 6.4 with a buffer solution of pH 6.4, and further prepared by adding dimethyl sulfoxide (DMSO) to a final concentration of 7%. To the competent cells (100 μm) was added 5-10 μm of the DNA solution obtained above, the mixture was allowed to stand on ice for 30 minutes, and then selected on an LB agar medium containing ampicillin (50 μg.ml) to obtain ampicillin-resistant colonies. A plasmid was extracted from this colony, opened with Pst I and Bam HI, and then deleted with a TAKARA kilosequencing deletion kit. That is,
After cutting 5-10 μg of the plasmid with Pst I, Bam HI,
Extract once with an equal volume of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) saturated phenol, centrifuge, transfer the supernatant to another tube, and add an equal volume of chloroform / isoamyl alcohol (24/1, v / v) and extracted once. After centrifugation, the supernatant was transferred to another tube, 2.5 volumes of ethanol were added, and the mixture was centrifuged to collect the precipitate. After washing with 70% ethanol, vacuum drying is performed. Dry DNA to 100 μl Exonuclease II
Dissolve in I (hereinafter abbreviated as "Exo III") buffer (TAKARA). Place mung bean nuclease buffer (TAKA
RA) is put in 100μ. 1 μ (180 units) of Exo III is added to the Exo III buffer containing DNA, stirred with Vortex (manufactured by Sun Entix Industries), and incubated at 37 ° C. And 10μ every minute
Each sample is taken and put into a 100 μm mung bean nuclease buffer in order to stop the reaction. 5 at 65 ° C
Incubate Exo III by incubating for a minute and return to 37 ° C. To this is added 2μ (50 units) of mung bean nuclease. Incubate at 37 ° C for 30-60 minutes. Phenol treatment and chloroform treatment are carried out, and ethanol precipitation is performed, followed by washing with 70% ethanol, followed by vacuum drying. This DN
Dissolve A in 40μ TE buffer. This DNA solution 5-10μ
To the ligation solution (A) of the TAKARA ligation kit. Add 12μ of the ligation solution (B) and stir with Vortex. Perform the reaction overnight at 16 ° C. After ethanol precipitation, vacuum drying and dissolving in water, Bam H
Cut with I and transform directly into competent cells. The colonies obtained by the transformation are subjected to mini-screening to extract DNA, and those having a desired length are selected. That is,
Dozens of colonies were cultured in 1 ml of LB medium at 37 ° C. overnight with shaking tow, and centrifuged at 10,000 × g in an Eppendorf tube.
Collect for 1 minute. The precipitated bacteria were suspended in a 150 μl TELT solution (50 mM Tris-hydrochloric acid (pH 7.5), 62.5 mM EDTA, 0.4% Triton X-100 and 2.5 M lithium chloride), and suspended in 15 μl.
Lysozyme solution (10 mg / ml aqueous solution). The solution was boiled for 1 minute, then cooled on ice for 5 minutes, and centrifuged for 1 minute.
Perform at 10,000 xg for 10 minutes. To the obtained supernatant, 750 µ of 60% isopropanol is added, and the mixture is centrifuged at 4 ° C for 10 minutes to collect a DNA precipitate. The obtained DNA is washed with 500 µ of 70% ethanol, vacuum-dried, and dissolved in 50 µ of water.

The resulting DNA was cleaved with Eco R I, Hin d III, subjected to electrophoresis on a 1.2% agarose gel, 200 between 200bp~2300bp
A colony of DNA in which a deletion has occurred at the bp step is selected. Select about 30 colonies, both on the back and on the front, and use them as material for sequencing.

DNA extraction for sequencing was performed as follows. That is, 1.5 ml of LB medium (1% w / v bacto-tryptone,
The bacteria cultured overnight with 0.5% w / v yeast extract, 1% w / v sodium chloride) were collected by centrifugation at 10,000 xg for 2 minutes, and then
Was suspended in a 50 mM glucose, 25 mM Tris (pH 8.0), 10 mM EDTA solution, and a 40 μl lysozyme solution (10 mg / ml above solution) was added thereto, followed by standing at room temperature for 5 minutes. Next, 0.2N sodium hydroxide and a 1% SDS solution are added, and the mixture is allowed to stand on ice for 5 minutes. A 150 μM 5M potassium acetate solution (pH 4.8) is added, and the mixture is placed on ice for 5 minutes. Centrifuge at 12,000 xg, 4 ° C for 10 minutes to obtain supernatant. The supernatant is gently mixed with an equal volume of a TE-saturated phenol solution for 5 minutes, and centrifuged at 12,000 × g for 5 minutes to collect an aqueous layer. To further remove phenol, mix the aqueous layer with an equal volume of chloroform / isoamyl alcohol (24: 1) and add 12,000
Collect the aqueous layer again by centrifugation at xg for 5 minutes. Add twice the volume of ethanol to the aqueous layer and centrifuge at 12,000 xg for 10 minutes at 4 ° C. The obtained DNA precipitate is washed with 70% ethanol, dried under vacuum, and dissolved in a 50 µ TE solution (pH 8.0). To this solution, 1 μm of 0.5 mg / ml RNaseA is added and reacted at 37 ° C. for 30 minutes. After RNase treatment, add 30μ of 20% polyethylene glycol 6000,2.5M sodium chloride solution, and place on ice for 1 hour.
The mixture is left standing for 1 hour, centrifuged at 12,000 × g for 5 minutes, washed with 70% ethanol, and dried in vacuum. Dissolve the DNA in 20 µ of distilled water to use as a sample for sequencing.

The sequence was performed using TAKARA 7-DEAZA sequence kit. A plasmid of a colony to be sequenced was prepared by the above method, and its DNA solution (10 μm) was prepared.
μg DNA) with 2μ of 2N sodium hydroxide and 2μ2mM E
DTA and 6 µ weight water are added and left at 37 ° C for 5 minutes to carry out alkali denaturation. 8μ of 5M ammonium acetate (pH4.5)
And then neutralized by adding 100 μl of ethanol.
Centrifuge at 10 ° C for 10 minutes to precipitate DNA. The precipitate is washed with 70% ethanol and dried under vacuum. Next, this DNA was dissolved in 9.5 µ of distilled water, and 1.5 ml of 10 × buffer (TAKARA) and 1 µ
The primer (〜0.5 pmol) is added, heated at 60 ° C. for 20 minutes, and allowed to stand at room temperature to anneal the primer and DNA. [Α- ³² P] -dCTP (400
(Ci / mmol) and 1 μl (2 units) of Klenow enzyme are added, mixed, and centrifuged for 1 minute. 4 kinds of different prepared
The above solution is added to the dNTP-ddNTP mixed solution (2 µ) in 3.5 µ each and mixed. After leaving at 37 ° C. for 20 minutes, add 1 μm of the chase mixture and let stand for another 20 minutes. Add 6μ formamide stop solution, mix, heat at 95 ° C for 3 minutes, then quench.

Next, electrophoresis is performed on a sequence gel (8% acrylamide gel). The sample is overlaid on the gel by 2 to 4 μm, and electrophoresis is performed at a constant voltage of 2000 V. After the electrophoresis, the gel is transferred to Whatman 3MM, dried in a gel dryer, and an autoradiogram is taken in a freezer at 20 ° C. After 24 hours exposure, develop and read the DNA sequence.

Conversion of the DNA sequence to amino acids was performed using the lift of GENETYX (Tokyo).

[Brief description of the drawings]

FIG. 1 is a drawing showing the base sequence (2,352 bp) of the xylene oxygenase gene obtained in the example. In the figure,
The 27th to 1136th represent the coding region of XylM, and the 1286 to 23rd
The 38th shows the XylA coding region. The 27th to 1136th and the 1268th to 1278th represent ribosome binding positions. FIGS. 2 (a) and (b) are drawings showing the amino acid sequences of XylM and XylA, respectively.

Continuation of front page (56) References BACTERIOL (1987) Vol. 167, no. 2, P. 455-461 J.C. BACTERIOL (1987) Vol. 169, no. 2, P. 764-770 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15/52-15/61 C12N 9/02-9/12 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ (G (ENETYX)

Claims (2)

(57) [Claims] 1. A xylene oxygenase gene encoding a protein having an amino acid sequence represented by the following formula (I) or (II): 2. A xylene oxygenase gene having a base sequence represented by the following formula (III) or (IV).