JPH02119777A - Xylene oxygenase gene - Google Patents

Abstract

NEW MATERIAL:Xylene oxygenase genes Xy1M (having 41631 molecular weight) and Xy1A (having 38697 molecular weight) having DNA coding an amino acid sequence expressed by formula I and formula II. USE:Oxidation to (methyl)benzyl alcohol of toluene or xylene and conversion of indole to indoxyl, further indigo. PREPARATION:DNA fragment containing xylene oxygenase in Sa1 I and Hind III and having 2, 3kb is further cut out from pGSH 2836 cloning DNA fragment containing xylene oxygenase among TOL plasmid pWWO and subjected to subcloning to pUC system or M13mp system, etc. DNA fragment coding xylene oxygenase subcloned to vector is subjected to ring opening with Pstl and BamHI.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to xylene oxygenase genes (XylM and XylA) that have the ability to assimilate toluene or xylene.

(Prior Art and Problems to be Solved by the Invention) Bacteria capable of assimilating toluene or xylene usually possess a plasmid collectively referred to as a TOL plasmid, and assimilation genes are encoded on the plasmid.

The TOL plasmid has genes for enzymes that generate benzoic acid and toleic acid from toluene or xylene, and enzymes that completely decompose it into pyruvate and acetaldehyde or propionaldehyde.

Xylene oxygenase consists of two subunits, xy IM and XylA proteins, and both subunits function together. Xylene oxygenase has low substrate specificity, and in addition to oxidizing toluene or xylene to (methyl)benzyl alcohol, it also converts indole to indoxyl and then to indigo (indigo).

Indigo is an important dye for clothing and is found in the blue flowers of natural plants such as indigo. However, there are quite a few commercially important flower petals that lack blue color, and if blue flowers were introduced to commonly used flower petals, such as hala and chrysanthemum, their commercial value would be great. It is considered a thing.

On the other hand, in recent years, transformation systems for higher plants have been established in many species, and the production of herbicide- and virus-resistant plants has been recognized [Nature 317.7417
44.1985i Science,
232, 738-743, 1986).

If a gene that produces blue flowers is introduced into plants using such a host-hector system, it is possible to make blue flowers bloom in plants that do not have blue pigments. One candidate gene is a gene that produces indigo.

Until now, the only gene known to produce indigo is the naphthalene dioxygenase gene nahA of the naphthalene plasmid NAH7 (Science (S
science), 222. 1671983).

Naphthalene dioxygenase is an enzyme that converts indole to indigo, but the base sequence of nahA has not yet been determined.

Another known gene that produces indigo is the xylene oxygenase gene. This xylene oxygenase is a different enzyme from naphthalene dioxygenase, but it has the function of producing indigo from indole [Bio/Technology]
, 4. 321 324. 1986]. This xylene oxygenase (XylM.

A long fragment containing XylA) was cloned from pWWO, the wild-type plasmid of the TOL plasmid [Journal of the Hacteriology (J, Bac.
t, ) , , , 167. 455-461゜1986
), but its detailed genetic structure has not yet been investigated.

(Means to Solve the Problem) The present inventors have recently conducted repeated studies to obtain the genes encoded by the genes Xy1M and The present invention was completed by determining the amino acid sequence.

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

The present invention will be explained in detail below.

In the present invention, a DNA fragment containing xylene oxygenase of the TOL plasmid pWWO was cloned into an expression vector, pGS142836 [Journal Op the Bacteriology (J, Bacte.
riol, ), 167, 455 461 198
6], a 2.3 kb DNA fragment containing xylene oxygenase was further excised with 5alr and HindIIl,
pUC strain [Gene, ↓9. 259-
268.1982; Gene, 33103
-119.19853 and M13mP strain [Methods in Enzymology]
mol, ), 101. 20-77. 1983)
[Gene, 2835]
1-359, 1984).

The DNA fragment encoding xylene oxygenase subcloned into the vector was transformed into PstL BamH
The ring is opened with I, and sequential deletions are caused by exonuclease ■ and mung bean nuclease. Since SeaFence can only read up to 200,300 bp from the primer position, clones with deletions of 200 bp each are selected in mini-screening. Seafence each cloning, read it, connect the overlapped parts, and read 2.
．． The nucleotide sequence of the entire 3 kb DNA was determined.

This operation was performed in two directions, the front and back sides of the DNA, and the base sequence was confirmed.

The amino acid sequence was obtained by converting the base sequence obtained from Sea Fence into amino acids based on universal codons. As a result, xylene oxygenase is an enzyme consisting of two proteins, and the protein encoded by XylM has a molecular weight of 4L631.
The first 30 amino acids on the N-terminal side are highly hydrophobic, and x
The protein encoded by ylA was found to have a molecular weight of 38.697 (Figure 1).

Furthermore, it was predicted that the X71M protein binds toluene, xylene, oxygen, etc., and that the xy IA protein is a protein involved in the electron transport system. This was supported by the fact that the N-terminal side of the XYIA protein was highly homologous to plant-type ferredoxin, and a cystine residue that binds to Fe'' was found to exist at the same position.

(Effects of the Invention) The genes of the present invention (7) Xy IM and Xy IA co-1:' are considered to be effective in producing plants with blue flowers.

(Example) The present invention will be specifically described below with reference to Examples.

(1) Toluene/xylene degrading plasmid pWWO is A
Pseudomonas putida (Pseudomonas) obtained from TCC
domonas utida). A fragment containing xylene oxygenase from pWWO was added to P of λ phage.
A plasmid pGSH2836 linked to the promoter has been constructed [Journal of the Hacteriology (J, BacLeriol), 167455
-'461.1986), so this time we also added pGS H28.
5all fragments containing xylene oxidase from 36 to 2.1 kb were excised with HindIII and 0.7%
After performing electrophoresis on agarose gel, it was cut out. The excised DNA fragments were electrically recovered from the agarose gel. DNA by phenol treatment and ethanol treatment
was purified and pUc11319 [Gene
, 33, 103-119.

5all, HindIII of 1985). Ilanahan's method using competent cells of Escherichia coli HBIO
Cloning (DNA cloning), 1.
109-136.1.985). Competent cells are E. coli HB 101 0.1
pl+6.M potassium chloride/45mM manganese chloride/10mM calcium chloride/10mM potassium acetate 10% glycerol/3mM hexacobalamin hydrochloride.
4 was treated with O'C, and further dimethyl sulfoxide (DMSO) was added to a final concentration of 7%. The DNA volume obtained from Combitea 1-cell LOOul-J2 was 510μ! 30 in water.
After standing for a minute, ampicillin (50 μg, mj2)
Ampicillin-resistant colonies were obtained by selection on an LB agar medium containing .

A plasmid was extracted from this colony and PstlBam
After opening the ring with HI, it was deleted using TAKARA's Kiro Sea Fence Plesion Kit. That is, Ps t I, B
After cutting 5 to 10 μg of plasmid with amHI, an equal amount of TE (lomMl Lis-HCl (pH 8,0), 1 m
Extract once with saturated phenol (M EDTA), centrifuge and transfer the supernatant to another tube, and add an equal volume of chloroborum/isoamyl alcohol (24/l, v/v
) and extracted once.

After centrifugation, the supernatant was transferred to another tube, 2.5 times the volume of ethanol was added, and the mixture was centrifuged to collect the precipitate. After washing with 70% ethanol, vacuum drying is performed. 100 pieces of dry DNA
It is dissolved in μi exonuclease ■ (hereinafter abbreviated as "EXo■") buffer (TAKARA). In a separate tube, add mung bean nuclease buffer (TAKARA) at 100%
μ! I'll put it in. Add 1 μl (180 units) of Exoll to the ExoIII buffer solution containing DNA, and
Stir with rtex (manufactured by Santific Industries) and incubate at 37°C. Then, 10 μβ were sampled every minute, and the sample was gradually added to 100 μl of mung bean nuclease buffer to stop the reaction. Incubate for 5 minutes at 65 °C.
Inactivate xoll and return to 37°C. 2μ for this
Add 1 (50 units) of mung bean nuclease. 37
Incubate for 30-60 minutes at °C. 7. Perform phenol treatment, chloroform treatment, and ethanol precipitation.
After washing with 0% ethanol, vacuum dry. Dissolve this DNA in 40 μl of TE buffer. This DNA solution 5-1
0μ! Add to the ligation solution (A) of the TAKARA ligation kit. Add 12μ2 of ligation solution (B) and stir by Vortex. 16
Perform the reaction overnight at C. Perform ethanol precipitation, vacuum dry, dissolve in water, cut with BamHI, and directly transform into competent cells. The colonies obtained by transformation are subjected to mini screening to extract DNA. extract and select one of desired length, i.e. several dozen colonies at one time.
Shake and culture in ml of I and B medium overnight at 37°C.
10.01)
Collect bacteria by using Ox g for 1 minute. 15 precipitated bacteria
'1゛rg L T solution (50mM l-Lis-HCl (pH 7.5 > 62.5mM EDTA, 0.4%
Tosu1henX, -100 and 2.5M lithium chloride)
Suspend at 15μ! Lysozyme solution (10mg/
aqueous solution). The solution was boiled for 1 minute, then cooled on ice for 5 minutes, and then centrifuged for 10 minutes. Run at OOOxg for 10 minutes.

Add 750IP 60% isopropanol to the resulting supernatant, centrifuge at 4°C for 10 minutes, and collect the DNA precipitate. 500μ of the obtained DNA! After washing with 70% ethanol, vacuum dry and dissolve in 50 μl of water.

The obtained DNA was cut with EcoRI Hindll, electrophoresed on a 1.2% agarose gel, and 200b
Colonies with DNA in which deletions have occurred at 200 bp intervals between p and 2300 bp are selected. - Select about 30 colonies (back and front) and use them as +A materials for the sea fence.

DNA extraction for sea fence was performed as follows. That is, 1.5 mn of LB medium (1% W/V Bacto I
・Lipton, 0.5% w/V yeast extract, 1% w
10, O
Harvest bacteria by centrifugation for 2 minutes at OOxg, and add 50mM at 60μ℃.
Glucose, 25mM Tris (pH 8°0), 10mM
Suspend in EDTA solution and add 40μ! Add lysozyme solution (L Omg/me of the above solution) and let stand at room temperature for 5 minutes. Next, 0.2N sodium hydroxide, 1
% SDS solution and let it stand for 5 minutes in water, add 150 μl of 5M potassium acetate solution (pH 4,8) and let it stand in water for 5 minutes.
Leave it for a minute. Centrifuge at 12,000xg and 4°C for 10 minutes to obtain a supernatant. The supernatant is gently mixed with an equal volume of TE-saturated phenol solution for 5 minutes, centrifuged at 12,000 xg for 5 minutes, and the aqueous layer is collected. To further remove phenol, the aqueous layer was diluted with an equal volume of chloroborum/isoamyl alcohol (24=
, 1) and centrifuged at 12,000xg for 5 minutes to collect the aqueous layer again. Add twice the amount of ethanol to the aqueous layer,
Centrifuge at 12,000 χ for 10 minutes at °C. The obtained DNA precipitate is washed with 70% ethanol, dried in vacuo, and dissolved in a TE warm solution of 50 IP (pl+ 8.0). 1μ in this solution! 0,5 mg/ml of RNas
Add eA and carry out the reaction at 37°C for 30 minutes. RNas
After the e-treatment, 30 tt 1 of 20% polyethylene glycol 6000, 2.5M thorium chloride solution was added, left to stand on ice for 1 hour, centrifuged at 12,000xg for 5 minutes, and washed with 70% ethanol. Vacuum dry. D.N.
20 A’s! Dissolve it in distilled water and use it as a sea fence sample.

Sea fencing was performed using the TAKARA 7-DEAZA sequencing kit. Prepare a plasmid for a colony to be sea fenced using the above method, and add 10μ of the DNA solution! (~2μg DNA) with 2μP of 2N thorium hydroxide and 2μ! 2mM EDTA and 6μ! Add weight water and let stand at 37°C for 5 minutes to perform alkaline denaturation. 5M ammonium acetate (pH 4,5) 8μ! After denaturation, neutralize by adding 100μ of ethanol! add 4°
Centrifuge at C for 10 minutes to precipitate the DNA. 70 for precipitation
% ethanol and vacuum dry. Next, add this DNA to 9.5μ! of distilled water and 10x buffer (TAKA
RA) 1.5 ml and tμp primer (~0.5
pmol) was added, heated at 60°C for 20 minutes, and left at room temperature to perform annealing between the primer and the DNA. [α-32P]d CTP (40
0Ci/mmol) 2p E and old enow enzyme 17
Add 1ffi (2 units), mix and centrifuge for 1 minute. Add 3.5μβ of the above solution to each 2μ of the four types of dNTl)-ddNTP mixture prepared separately and mix. After leaving at 37°C for 20 minutes, add 1
Add μβ at a time and leave for another 20 minutes. 6μ! Add formamide stop solution, mix, heat at 95°C for 3 minutes, and then rapidly cool.

Next, electrophoresis is performed on Seefence gel (8% acrylamide gel). Layer 2 to 4 μβ of each sample on the gel,
Electrophoresis is performed at a constant voltage of 2000V.

After the electrophoresis, the gel was transferred to Wara 1-Man 3MM, dried in a gel dryer, and an autoradiogram was taken in a 20°C freezer. After 24 hours of exposure, it is developed and the DNA sequence is read.

Conversion of DNA sequences into amino acids is performed using GENETYX (T
This was done using a lift from OKYO.

[Brief explanation of 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 indicate the xyIM code region, and the 1286th to 2338th indicate the xylA code region. Also, the 27th to 1136th and 12th
Positions 68 to 1278 indicate bosome binding positions. Figures 2(a) and (b) show X)l IM and Xy, respectively.
1 is a drawing showing the amino acid sequence of IA.

Claims (2)

[Claims] (1) A xylene oxygenase gene having DNA encoding the amino acid sequences represented by the following formulas (I) and (II). Formula (I) [There is a gene sequence] Formula (II) [There is a gene sequence] (2) A xylene oxygenase gene having a base sequence represented by the following formula (III) and formula (IV). Formula (III) [There is a gene sequence] Formula (IV) [There is a gene sequence]