JP3302053B2 - Gene involved in production of oxetanosine-A and recombinant DNA containing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention can be used as a marker in the production of oxetanocin-A in a recombinant and the cloning of the recombinant.

[0002]

2. Description of the Related Art Oxetanosine, a nucleoside antibiotic.
A and its producing bacteria (FERM BP-919) are described in JP-A-61-292992 (USP 4,743,689).
Is known by: In addition, the producing bacterium is 51.5 kbp.
It is known that the oxetanocin-A-producing ability is lost in the 51.5 kbp plasmid-free strain. (JP-A-3-147790)

[0003]

It has been observed that the 51.5 kbp plasmid easily drops off during the cultivation, and it is known that the oxetanosin-A-producing ability is lost in this plasmid-free strain. The reason why the oxenosine-A production ability is lost when the oxenosine-A is lost has not been elucidated at all, and this has hindered research on improving oxetanocin-A productivity. Oxetanocin-A
It is not known where and in what form genes involved in the production of oxetanosin-A and / or genes that confer oxetanosin-A tolerance are present. Further, neither a plasmid vector required for breeding oxetanosine-A producing bacteria by a genetic engineering technique nor a method of transforming a recombinant into a host microorganism has been studied.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems and found that oxetanocin-A
Bacillus megaterium NK84-0218 as a producing bacterium
Successfully identified genes involved in the production of oxetanosin-A in a plasmid of about 51.5 Kbp and cloned a DNA fragment containing those genes, and these genes were at least capable of producing oxetanosin-A and oxetanosin-A. A It has been found that it has a function of imparting anti-performance properties. These novel gene DNA fragments are ligated to a vector, the recombinant vector is transferred to a host microorganism, Bacillus megaterium, to obtain a transformant, and the transformant is cultured to produce oxetanocin-A. It succeeded in expressing the controlling functions, that is, the ability to produce oxetanocin-A and the ability to withstand oxetanocin-A. Further, these genes involved in oxetanosin-A production are cloned on a vector plasmid to obtain a recombinant DNA, and other bacteria such as oxetanosin-
Bacillus megaterium ATCC6 not capable of producing A
By transfecting the gene into strain 459 and transforming it, it was found that oxetanocin-A could be produced by the transformant, and the present invention was completed.

That is, the present invention uses a DNA fragment containing a gene involved in the production of oxetanosine-A, a recombinant DNA containing the gene, a transformant transformed with the recombinant DNA, and the transformant. The present invention relates to a method for producing a protein encoded by the gene, a method for producing oxetanocin-A using the protein encoded by the gene, and the transformant. Still further, the present invention relates to a novel plasmid isolated from Bacillus megaterium and a novel vector produced therefrom.

[0006] The gene of the present invention can be further used for the following purposes. For example, an oxetanosin-A resistance gene is converted into a recombinant DNA using an appropriate vector, and the resistance gene is transferred to another strain and transformed to transform another gene fragment using oxetanosin-A resistance as an index. It can also be used for cloning or selection of transformants using oxetanosine-A resistance as an index. Furthermore, by preparing a synthetic probe using the base sequence of the oxetanosin-A producing gene or the oxetanosin-A resistance gene, it becomes possible to screen a oxetanosin-A producing bacterium or an oxetanosin-A resistant bacterium by a hybridization method. .

[0007] Examples of the gene involved in the production of oxetanocin-A in the present invention include a gene directly imparting one or both of oxetanocin-A production ability and oxetanocin-A resistance. Examples of these genes include those having the nucleotide sequences shown in SEQ ID NOs: 2, 3, 4 and 5 obtained from the 51.5 kbp plasmid possessed by the oxetanocin-A producing bacterium Bacillus megaterium NK84-0218. These genes may be partially modified, dropped out or added as long as the above functions are not lost.

[0008] A DNA fragment containing a gene involved in the production of oxetanocin-A in the present invention is produced, for example, using a gene recombination technique as follows. For example, a DNA containing a gene involved in oxetanosin-A production is separated and purified from a microorganism that produces oxetanosin-A. This DNA is digested with a restriction enzyme or the like, and ligated to a plasmid vector or a phage vector which has been cut and made linear by using DNA ligase or the like at the blunt or cohesive end. Then the obtained recombinant DNA
Vector Markers and / or Oxetanocin-A
A microorganism carrying the recombinant DNA vector obtained by screening using the resistance or the production of oxetanocin-A as an indicator is cultured, and the recombinant DN is cultured from the cultured cells.
DNA fragment containing a gene involved in the production and isolation of oxetanosin-A from the recombinant vector, specifically a gene conferring at least one of oxetanosin-A resistance and oxetanosin-A production ability Can be produced by collecting

The microorganism that provides the gene may be any microorganism that produces oxetanocin-A, and is preferably Bacillus megaterium NK84-0218 belonging to the genus Bacillus.
Strain (FERM BP-919). DNA derived from a microorganism as a gene donor is collected as follows. For example, the above strain is cultivated by aeration and stirring in a liquid medium for 1 day, and the resulting culture is centrifuged to collect the cells, which are then lysed to obtain a lysate containing an oxetanosin-A production gene and an oxetanosin-A resistance gene. Prepare the product. As a lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme or a surfactant such as sodium lauryl sulfate is used in combination. From the lysate obtained, D
In order to separate and purify NA, conventional methods are used, for example, by combining methods such as protein denaturation removal treatment by phenol extraction, RNA removal treatment by ribonuclease, DNA precipitation concentration treatment by alcohol, and DNA precipitate recovery by centrifugation. Will be In Bacillus megaterium NK84-0218, the plasmid DNA is preferably separated from the recovered DNA by cesium chloride density gradient centrifugation to obtain a donor DNA for the gene.

The method of cutting the DNA of the separated and purified microorganism can be performed, for example, by treatment with a commercially available restriction enzyme. As the vector, a vector constructed for gene recombination using a phage or a plasmid capable of autonomous replication and propagation in a host microorganism is suitable. When Escherichia coli is used as a host microorganism, lambda phage, M13 phage and the like can be used. When Bacillus megaterium is used as a host microorganism, Bacillus megaterium N
It is preferable to use novel plasmids isolated from the K84-0218 strain and / or those constructed as a vector for gene recombination from these plasmids. When Escherichia coli is used as a host microorganism, pUC18, pUC19, and pBR322 commercially available from Takara Shuzo Co., Ltd. and others are used.
Etc. can be used. Such a vector is prepared by using the oxetanocin-A producing gene and / or oxetanocin-
It is preferable to obtain a vector fragment by digestion with a restriction enzyme that generates the same cohesive end as the restriction enzyme used for digestion of the microorganism DNA that is the A-resistant gene donor.

The method of linking the microbial DNA fragment with the vector fragment may be any method using a known DNA ligase, and a commercially available DNA ligation kit such as D
A NA ligation kit (Ligation Kit (Takara Shuzo Co., Ltd.) can be used. As the host microorganism, a recombinant DNA capable of stably and autonomously replicating and growing, and capable of expressing a foreign DNA trait can be used. For example, when the host microorganism is Bacillus megaterium, Bacillus megaterium NK84-0218, Bacillus megaterium ATCC 6459, etc. can be used.
Oxetanocin-A-producing ability and oxetanocin-induced from Bacillus megaterium NK84-0218
It is a strain that has lost A-resistance. When the host microorganism is Escherichia coli, Escherichia coli JM109 or the like can be used.

As a method for transferring the recombinant DNA to the host microorganism, when the host microorganism is Bacillus megaterium, the recombinant DNA is transferred to protoplastized Bacillus megaterium in the presence of polyethylene glycol. The transfer of the recombinant DNA may be performed in the presence. Selection of the presence or absence of the transfer of the desired recombinant into the host microorganism can be carried out by a) searching for a transformant capable of expressing oxetanosine-A resistance when a gene conferring oxetanosine-A resistance performance is intended. For example, the gene may be grown on a selective medium containing oxetanocin-A. B) When a gene conferring oxetanosin-A-producing ability is intended, a recombinant vector carrying the DNA fragment of interest may be used. A transformant capable of expressing a marker, preferably oxetanosin-A resistance, is selected in accordance with the above, and the transformant is cultured, and those producing oxetanosin-A in the culture solution may be searched. The transformant can be cultured by a known method depending on the host microorganism. For example, when the host is Bacillus megaterium, JP-A-61-293992 (US Pat.
689).

The recombinant having the oxetanosin-A producing gene and / or the oxetanosin-A resistance gene selected in this way can be taken out from a transformed microorganism and transferred to another host microorganism. Further, a recombinant having an oxetanosin-A production gene and / or an oxetanosin-A resistance gene is cleaved with a restriction enzyme or the like to select a part or all of the oxetanosin-A production gene and / or the oxetanosin-A resistance gene. A recombinant having novel characteristics can be prepared by combining with another vector DNA obtained by cleavage by the same method as described above, and can be transferred to another host microorganism.

The transformed microorganism obtained by transferring the recombinant containing the gene of the present invention is specifically described as Bacillus megaterium NK84-0218 (FERM BP-91).
9) Bacillus megaterium ATCC6549 / pOXTB2 transformed with a recombinant plasmid pOXTB2 containing a gene conferring oxetanosin-A-producing ability and oxetanosin-A resistance ability, which was taken out from 9).

The nucleotide sequence of the gene fragment of the present invention is Sci.
ence 214 1205-1210 (1981). The single-stranded DNA used for the dideoxy method is obtained, for example, by incorporating a gene imparting oxetanosin-A production ability and oxetanosin-A resistance ability taken out from Bacillus megaterium NK84-0218 into M13 phage using Escherichia coli as a host microorganism.

[0016]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples. (Example 1) Bacillus megaterium NK84-0218
Preparation of Plasmid Standard from Bacillus megaterium NK84-0218 strain (FERM BP-919) which is an oxetanocin-A producing bacterium
It carries plasmids of different molecular weights. It is necessary to efficiently prepare these plasmids as a donor of the gene of the present invention and for construction of a vector useful for expression of the gene of the present invention. The preparation method is described below.

(1) Bacillus megaterium NK84-0
Culture of 218 strain YS slant medium containing 10 μg / ml of oxetanocin-A (yeast extract 0.2%, dextrin 1%, agar 1.
5%:% is% by weight, hereinafter the same unless otherwise noted. ), A spore of Bacillus megaterium NK84-0218 strain is inoculated to obtain vegetative cells of this strain. The vegetative cells were placed in a platinum loop using Davis's minimal medium (0.7% dipotassium phosphate, 0.2% phosphate) containing 10 μg / ml of oxetanocin-A, 0.06% of yeast extract, and 0.03% of casamino acid. Monopotassium, 0.01% magnesium sulfate, 0.5% ammonium sulfate, 0.05% sodium citrate, 0.1%
2% glucose) (about 10 L) at 35 ° C., 18
Incubate overnight at 0 rpm.

(2) Preparation of plasmid sample The cells are centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded, and the obtained cells were placed in a 26 ml GTE solution (50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 m
After suspension in MEDTA), 5.2 ml of a 25 mg / ml lysozyme solution is added. This mixture is incubated at 37 ° C. for 10 minutes. This mixture was added to lysis buffer (2% Triton X-100, 40 mM EDTA, 50 mM).
mM Tris · HCl pH 8.0)
Mix and incubate for 30 minutes at room temperature. If the lysis is not complete, it is incubated at 37 ° C. for a further 10 minutes. The mixture is centrifuged at 25000 rpm for 30 minutes. The supernatant is removed, an equal volume of a phenol: chloroform (1: 1) solution is added, and the mixture is gently shaken for 10 minutes to denature the protein (hereinafter referred to as phenol extraction).
The mixture is centrifuged at 12,000 rpm for 10 minutes, and the upper layer is removed. One tenth volume of 3M sodium acetate (pH 5.5) and 2 volumes of ethanol are mixed with the obtained upper layer, and the mixture is allowed to stand at -20 ° C for 1 hour (hereinafter referred to as ethanol precipitation). This is centrifuged at 12,000 rpm for 10 minutes, the supernatant is discarded, and the DNA is recovered as a precipitate.

After drying, the DNA precipitate was washed with 10 ml of TE buffer (10 mM Tris-HCl pH 7.0, 1 mM
EDTA), add 40 μl of ribonuclease solution (25 mg / ml) and incubate at 37 ° C. for 30 minutes. After extracting phenol again by the method described above, DN
A is ethanol precipitated, and centrifuged at 12000 rpm for 10 minutes to collect DNA. This was dissolved in 14.4 ml of TE buffer, and 13.92 g of cesium chloride, 5 mg
/ Ml of ethidium bromide solution at a concentration of 1.595, and then adjust the density to 1.595.
Centrifuge in a vertical rotor for hours. Chromosomal DNA
The band of the plasmid DNA appearing below is collected and collected using a 19-gauge injection needle. The ethidium bromide is removed by extracting four times with isopropanol saturated with sodium chloride. Six times the volume of sterilized water is added to the obtained DNA solution, and two times the total volume of ethanol is further added to precipitate the DNA. After leaving at -20 ° C for 1 hour, 1
Centrifuge at 2000 rpm for 10 minutes to collect DNA. After drying, it is dissolved in TE buffer to obtain a plasmid preparation. This sample was treated with 40 mM Tris acetate pH 8.0, 1 mM
The gel was subjected to 0.8% agarose gel electrophoresis using an EDTA electrophoresis buffer, stained with ethidium bromide and irradiated with UV, and the DNA band was confirmed.
Four types of plasmids having different sizes of 5 kb, 6.0 kb, 9.5 kb, and 4.8 kb were present in this preparation.

(Example 2) Bacillus megaterium NK0
161. JP-A-3-147790
51.5 Kbp of oxetanocin-A producing bacterium prepared in
It is known that a plasmid-eliminated strain does not produce oxetanocin-A (Japanese Patent Application Laid-Open No. 3-147790). According to the study of the present inventors, this plasmid-depleted strain had lost oxetanosine-A production ability and oxetanosine-A tolerance. Therefore, it can be used as a transformed host microorganism when cloning the oxetanosine-A production gene and the oxetanosine-A resistance gene of the present invention. The strain having lost the ability to produce oxetanocin-A and the ability to withstand oxetanocin-A is named Bacillus megaterium NK0161. This strain can be obtained according to the method disclosed in JP-A-3-147790.

Vegetative cells are obtained by culturing the cryopreserved spores of Bacillus megaterium NK0161 strain at 35 ° C. overnight in YS slant. 50 vegetative cells for one platinum loop
50ml SMMP in 0ml triangular corben
(0.5M sucrose, 0.02M malic acid, 0.02M magnesium chloride, 3.5% Difco Antibiotic Medium N
o, 3, pH 6.5) Suspend in solution. 180 rpm, 3
The cells were cultured at 5 ° C. D. _{When 600} became 0.6-0.8, the culture was stopped, and the culture was transferred to 6000 rpm,
Centrifuge for 10 minutes and collect the vegetative cells. The collected vegetative cells were suspended in 5 ml of SMMP solution, and lysozyme solution (20
(mg / ml), mix well, and incubate at 37 ° C for 45 minutes to transform the vegetative cells into protoplasts.

The protoplast solution was subjected to 3000 rpm
centrifuge for 10 minutes to collect protoplasts. This protoplast is gently suspended again in 25 ml of SMMP solution, and then centrifuged again at 3000 rpm for 10 minutes to collect the protoplast. 5 ml of collected protoplasts
The suspension is suspended in an MMP solution, 0.5 ml of the suspension is separated, and 10 to 50 μl of a DNA solution for transformation is added thereto and mixed well. 1.5 ml of an SMMP solution containing 40% polyethylene glycol 6000 is added to the mixture, mixed gently, and left at room temperature for 2 minutes. Further, 5 ml of SMMP solution is added, and the mixture is centrifuged at 3000 rpm for 10 minutes to collect protoplasts. 1 m of collected protoplasts again
1 in SMMP solution.

After this was incubated at 180 rpm and 35 ° C. for 90 minutes, the protoplast solution was
Drug selection DM3 (0.5% casamino acid, 0.5% yeast extract, 0.8% agar, 13.5% sodium succinate, for example) containing 50 μg / ml of oxetanocin-A
0.35% K ₂ HPO ₄ , 0.15% KH ₂ PO ₄ ,
0.5% glucose, 0.02M magnesium chloride,
(0.01% bovine serum albumin). The plate is left at 35 ° C. to regenerate the transformed protoplasts. Transformants are observed as colonies after 24-48 hours. For example, 1 μg of a plasmid preparation containing the 51.5 kb plasmid prepared in Example 1
By transforming Bacillus megaterium NK0161 by this method, it is possible to obtain 50-100 oxetanosin-A-resistant and oxetanosin-A-producing transformants that incorporate the 51.5 kb plasmid.

Example 3 Cloning of Gene Fragment Involved in Oxetanocin-A Production Gene fragments involved in oxetanocin-A production were prepared using the plasmid prepared in Example 1 as a gene donor and vector. Recombinant plasmid p
Cloned on OXTB2. Furthermore, it was confirmed from the construction of the recombinant plasmid pMGOXT that a partial fragment of the plasmid pOXTB2 contained a gene that imparts oxetanocin-A resistance and oxetanocin-A production ability. The details of the experiment are described below.

1) Construction of pOXTB2 5 μg of a sample of the plasmid obtained in Example 1 (mixture of four kinds of plasmids) was mixed with 150 μl of a reaction solution in 150 μl of a reaction solution to obtain a restriction enzyme BglII (Takara Shuzo). Was completely digested, and 15 μl of 250 m
After adding a MEDTA solution to stop the reaction, a phenol extraction treatment and an ethanol precipitation treatment are performed. Obtained DNA
The precipitate is dissolved in 10 μl of TE buffer, and a DNA fragment is ligated according to a DNA ligation kit (Takara Shuzo) to obtain a recombinant. The obtained recombinant was transformed into the host microorganism Bacillus megaterium NK0 by the method of Example 2.
161 was spread on a DM3 plate containing 50 μg / ml of oxetanosine-A.
A transformant to which oxetanocin-A tolerance was imparted was selected.

Next, this oxetanocin-A resistant strain was cultured at 35 ° C. for 5 days (medium, composition: 2% soybean powder, 0.0015% cobalt chloride, 2% calcium carbonate, 10% lactose 0%). .1% dipotassium phosphate), and the presence or absence of oxetanosin-A in the culture solution is examined using HPLC, and a transformant to which oxetanosin-A-producing ability is imparted is selected. After culturing the thus obtained oxetanosin-A-producing ability and oxetanosin-A resistance-tolerant transformant, a recombinant plasmid was prepared in the same manner as in the preparation of the plasmid preparation in Example 1 below. Prepare. This recombinant plasmid has the restriction map of FIG. 1 and this recombinant plasmid is called plasmid pOXTB2
It is named. This plasmid was confirmed to have a 51.5 kb plasmid-derived origin of replication.

2) Construction of pMGOXT pMGOXT was constructed by combining the BglII 3.4 kb fragment of the 4.8 kb plasmid and the BglII 6.8 kb fragment of pOXTB2 in the plasmid preparation obtained in Example 1. . 2) -1 Preparation of 4.8 kb Plasmid and Bg1II Fragment The plasmid preparation prepared in Example 1 was subjected to 0.8% agarose gel electrophoresis, and the four types of plasmids were separated according to their molecular weights. The gel is cut out with a knife and transferred to a dialysis tube containing a minimal amount (about 2 ml) of an electrophoresis buffer. The sample is subjected to electrophoresis again to elute the plasmid DNA from the gel. The DNA eluate is subjected to a phenol treatment and an ethanol precipitation treatment to recover the plasmid DNA, which is used as a 4.8 kb plasmid preparation. 5 μg of the 4.8 kb plasmid thus obtained was added to 20 μl
after complete digestion with the restriction enzyme Bg1II in
Heat treatment was performed at 0 ° C. for 15 minutes to inactivate the enzyme, which was used in 2) -3. This BglII fragment contains a replication origin therein, and can be used as a novel vector by using it together with a marker gene such as an oxetanosine-A resistance gene.

2) -2 Preparation of BglII Fragment from pOXTB2 Using the transformant obtained in the above 1), 20 μg of the plasmid pOXTB2 prepared according to Example 1 was used for 90 minutes.
complete digestion with the restriction enzyme BglII in
After the reaction was stopped by adding 10 μl of a 250 mM EDTA solution, the mixture was subjected to 0.8% agarose gel electrophoresis, and pO
XTB2 was treated with three linear DNAs (1.4 kb, 4.0 kb).
6.8 kb), each band is cut out with a knife together with the gel, and each DNA is collected with a DNA recovery kit.
Recover from the gel using easy trap (Takara Shuzo).

2) -3 Identification of a fragment containing a gene conferring oxetanosin-A tolerance and oxetanosin-A production ability and construction of pMGOXT a) BglII complete digest of the 4.8 kb plasmid prepared in 2) -1 above 1 μg and 1.4 obtained in 2) -2
After mixing 1 μg of the kb DNA fragment and binding using a DNA ligation kit, the mixture was transferred to Bacillus megaterium NK0161 according to Example 2, and oxetanosine-A
It was spread on a DM3 plate containing 50 μg / ml, but no transformant having acquired oxetanosine-A resistance was obtained at all. b) Next, 1 μg of the 4.0 kb DNA fragment obtained in 2) -2 was mixed with 1 μg of the 4.8 kb plasmid BglII complete digest prepared in 2) -1, ligated with a DNA ligation kit, and transformed similarly to oxetanosin. A transformant having acquired -A resistance was searched, but none was obtained.

C) Finally, 6.8 kb DN obtained in 2) -2)
The A fragment was treated in the same manner, transformed, and oxetanocin-A resistant ability was searched. As a result, several tens of oxetanosin-A resistant ability transformants were obtained. The transformant was cultured, and the presence or absence of oxetanocin-A in the culture solution was examined using HPLC. As a result, oxetanocin-A was confirmed. The transformant having the oxetanosin-A tolerance and oxetanosin-A-producing ability is cultured according to Example 1, and the plasmid is separated in the same manner to obtain a recombinant plasmid. The plasmid was completely digested with BglII, 0.8%
When the structure of the BglII fragment was examined on an agarose gel, it was separated into 6.8 kb and 3.4 kb. 6.8k
The BglII fragment of b was derived from pOXTB2, and 3.4 kb was a BglII fragment derived from the 4.8 kb plasmid.
This recombinant plasmid has the restriction map of FIG.
This recombinant plasmid is named pMGOXT.

From the above results, it was shown that the 6.8 kb BglII fragment obtained as a constituent fragment of pOXTB2 contains a gene that imparts oxetanocin-A resistance and oxetanocin-A production. 6.8
A restriction map of the kb fragment is shown in FIG.

Example 4 Mapping of Oxetanocin-A Producing Gene and Oxetanocin-A Resistance Gene to a 6.8 kb BglII Fragment Using the plasmid pOXTB2 obtained in Example 3-1, the four types shown in FIG. pOXTSP, pOXTB
a, pOXTEc, pOXTXb) recombinant plasmid (a plasmid lacking a part of the 6.8 kb fragment which was confirmed to contain a gene imparting oxetanocin-A tolerance and oxetanocin-A production ability in Example 3) Was constructed and incorporated into Bacillus megaterium NK0161, and the oxetanocin-A producing gene and the oxetanocin-A resistance gene were mapped into the 6.8 kb BglII fragment by examining the oxetanocin-A production ability and oxetanocin-A tolerance of each transformant. did. The details are described below.

1) Construction and function of plasmid pOXTSP pOXTSP is derived from pOXTB2 by StuI and PvuII.
The recombinant plasmid was constructed by closing the loop except for the digested fragment 34 bp (base pairs). pOXTB2 is Stu
Each possesses one I and PvuII site, which are present in the 6.8 kb fragment. Therefore pOXT
Complete digestion of B2 with StuI, PvuII gives 34 bp and the remaining two fragments of approximately 12.2 kb. Therefore, 5 μg of pOXTB2 constructed in Example 3-1) and prepared in the same manner as in Example 1 is completely digested with StuI and PvuII in a 30 μl reaction solution. 3
After stopping the reaction by adding μl of 250 mM EDTA solution, the mixture was subjected to 0.8% agarose gel electrophoresis to separate into 34 bp and 12.2 kb bands. The 12.2 kb band was cut out along with the gel, and then a DNA recovery kit was used. A 12.2 kb DNA fragment is recovered from the gel. Both ends of the recovered fragment were ligated and closed using a DNA ligation kit, and the host microorganism N was ligated by the method of Example 2.
Transfer to K0161.

The cells are spread on a plate containing 50 μg / ml of oxetanosin-A (hereinafter referred to as oxetanosin-A selective medium), and a transformant which has acquired oxetanosin-A resistance is selected. Dozens of oxetanosin-A resistant transformants were obtained, and these transformants were cultured to prepare oxetanosin-A.
When the A-producing ability was examined, production of oxetanosine-A was observed. The plasmid of this transformant was isolated according to the plasmid isolation method of Example 1 and digested with StuI and PvuII.
It was confirmed that the recombinant plasmid lacked a 34 bp fragment between OXTB2 and StuI and PvuII, and was named pOXTSP. From the above results, StuI and Pvu among the 6.8 kb BglII fragments shown in FIG.
It was found that there were no genes involved in oxetanosin-A production or genes involved in oxetanosin-A resistance conferring near II.

2) Construction and function of plasmid pOXTBa pOXTBa is a 2.2 kb BamH from pOXTB2.
This is a recombinant plasmid obtained by closing the circle except for the I fragment. pOXTB2 possesses two BamHI sites, which are present in the 6.8 kb fragment. Therefore p
OXTB2 is digested with BamHI to obtain 10
Give fragments of kb and 2.2 kb. Therefore, first, 5 μg of pOXTB2 was added to 30 in the same procedure as in the above 1) of this embodiment.
Complete digestion with the restriction enzyme BamHI is performed in a μl reaction solution. After stopping the reaction by adding 3 μl of 250 mM EDTA solution, the mixture was subjected to 0.8% agarose gel electrophoresis,
Separate into 0 kb and 2.2 kb bands, cut out the 10 kb band together with the gel, and then recover the 10 kb fragment from the gel. Both ends of the recovered fragment were ligated and closed, and the fragment was transferred to a host microorganism NK0161 according to Example 2, spread on an oxetanocin-A selection medium, and oxetanocin-
A. A transformant strain which has acquired the resistance performance is selected.

Ten or more oxetanosin-A-resistant transformants were obtained. These transformants were cultured, and oxetanosin-A in the culture was measured.
Was not recognized at all. Further, the plasmid of this transformant was isolated according to the plasmid isolation method of Example 1. When this plasmid was digested with the restriction enzyme BamHI, the cleavage point was one and the size was 10 kb. Therefore, this plasmid is 2.2k more than pOXTB2.
It was confirmed that the recombinant plasmid lacked the BamHI fragment of b. This recombinant plasmid is called pOXTBa.
It is named. From the above results, BglII 6.8 kb
It was shown that information necessary for oxetanocin-A production was present in the region containing the missing 2.2 kb BamHI fragment among the fragments. In addition, the Ba of the 6.8 kb fragment
Oxetanocin- is added to a region not containing mHI 2.2 kb.
It was shown that the A resistance gene was present.

3) Construction and function of plasmid pOXTEc pOXTEc is 0.8 kb from pOXTB2 and 4.
This is a plasmid constructed by deleting a 7 kb EcoRI fragment. pOXTB2 has four EcoRI sites, two of which are present in the 6.8 kb fragment. This was completely digested with EcoRI to obtain 5.0k.
b, 4.7 kb, 1.7 kb, 0.8 kb fragments are given. Therefore, in the same procedure as in 1) of this embodiment, 5 μg of pO
XTB2 was completely digested with EcoRI in a 30 μl reaction solution, the reaction was stopped by adding 3 μl of a 250 mM EDTA solution, and then subjected to 0.8% agarose gel electrophoresis.
Separated into two different size bands, 5.0 kb and 1.
A 7 kb band is cut out, and each DNA is recovered. The recovered DNAs are mixed, ligated and closed, followed by transformation using NK0161 as a host microorganism according to the method of Example 2, and the transformed strain is selected on an oxetanosine-A selection medium. Several oxetanosin-A resistant transformants were obtained, and the oxetanosin-A production ability of these transformants was examined, but no oxetanosin-A production was observed.

The plasmid of this transformant was prepared in the same manner as in Example 1 and digested with EcoRI. As a result, 5.0 kb and 1.7 kb EcoRI bands appeared. This recombinant plasmid is named pOXTEc. On the other hand, both ends of each fragment were separately bound without mixing the 5.0 kb and 1.7 kb EcoRI fragments, and NK
0161, but no oxetanosine-A resistant strain could be obtained. From the above results, 6.8 kbB
The upstream EcoRI site in the glII fragment (hereinafter EcoRI site)
2.2k from downstream to BglII
It was shown that the oxetanocin-A resistance gene was present between b. It was also shown that the oxetanocin-A producing gene was present at least upstream of EcoRI-1 in the 6.8 kb BglII fragment.

4) Construction and function of plasmid pOXTXb pOXTXb is derived from pOXTB2 by 2.7 kbp and 3.
This plasmid was constructed by deleting the XbaI fragment of 05 kbp. pOXTB2 has three Xbal sites and is 6.45 kb by complete digestion with Xbal.
This gives a fragment of 3.05 kbp, 2.7 kbp.
The 6.45 kbp fragment contains about half of the 6.8 kbp BglII fragment. Therefore, 5 μg of pOXTB2 was added to Xba in 30 μl of the reaction solution in the same procedure as in 1) of this example.
After complete digestion with I, the reaction was stopped by adding 3 μl of 250 mM EDTA solution, the mixture was subjected to 0.8% agarose gel electrophoresis, and separated into three bands of different sizes.
The 45 kbp band is cut out and the DNA is recovered. After binding both ends of the recovered DNA and closing the ring,
Transformation is performed using K0161 as a host microorganism, and the transformed strain is selected on an oxetanosine-A selection medium. Ten or more oxetanosin-A resistant transformants were obtained, and the oxetanosin-A production ability of these transformants was examined, but no oxetanosin-A production was observed. Also,
A plasmid of this transformant was prepared in the same manner as in Example 1, and digested with XbaI. As a result, it was confirmed that the plasmid had one XbaI breakpoint and had a size of 6.45 kbp. This recombinant plasmid is named pOXTXb. From the above results, BglII shown in FIG.
It was shown that the oxetanocin-A production gene was present in the vicinity of the 6.8 kbp fragment including the XbaI site located at the approximate center of the restriction enzyme map. It was also shown that an oxetanocin-A resistance gene was present downstream from the XbaI site.

Example 5 Base Sequence of Fragment Containing Gene Involved in Oxetanosin-A Production and Gene Involved in Oxetanosin-A Resistance Provision pOXTB2 obtained in Example 4-1) was replaced with restriction enzyme Bg
After complete digestion with II, the mixture is subjected to 0.8% agarose gel electrophoresis, and a DNA fragment of about 6.8 Kbp is cut out along with the gel and collected. The recovered fragment was digested with restriction enzymes PvuII, X
baI, BamHI, SacI, KpnI, ScaI,
HpaI, SpeI, EcoRI, HaeIII, Ba
Separately or by combining a plurality of fragments with lI or the like, and ligating them into M13 phage vectors mp18 and mp18.
19 (both manufactured by Takara Shuzo Co., Ltd.)
Of the bBglII fragment, single-stranded DNA of both positive and negative strands was prepared over the region where the gene of the present invention shown in Example 4 is present, that is, a region of about 6.1 kb from PvuII to BglII downstream, and dideoxy. Law (Scien
ce, 214, 1205-1210 (1981)). The result is shown in SEQ ID NO: 1. The 6.1 kb fragment containing the gene of the present invention has four open reading frames (ORFs) as shown in FIG.
a, b, c, d, respectively, 582 bp, 2232
bp, 975 bp, and 648 bp.

The nucleotide sequences of these four ORFs and the amino acid sequences encoded thereby are shown in SEQ ID NOs: 2, 3, 4 and 5, respectively, in the order of a, b, c and d.
Those having all of a, b, c and d of these ORFs have resistance to oxetanocin-A and produce oxetanocin-A, so that those a, b, c
It can be said that the four genes d and d are involved in the production of oxetanosine-A.

The plasmid pOXT of Example 4-2)
Ba can be said to have lost oxetanosine-A producing ability due to lack of open reading frames a and b.
In addition, it can be said that the plasmid pOXTEc of Example 4-3) and the plasmid pOXTXb of Example 4-4) also lacked the oxetanocin-A producing ability due to lack of the open reading frames a and b. pOXTBa, pO
XTEc and pOXTXb have both open reading frames c and d, indicating oxetanosine-A resistance. From the above results, the open reading frames a, b, c, and d may be oxetanocin-A production genes, and at least one or both of a and b are oxetanocin-A production genes. In addition, it can be said that at least both or one of the open reading frames c and d is an oxetanosine-A resistance gene.

Example 6 Bacillus megaterium ATC
Oxetanocin-A production in strain C6459 The oxetanosin-A-producing transformant is transformed by transferring the oxetanosin A-producing gene and oxetanosin-A resistance gene obtained in the present invention into a strain having no oxetanosin-A production ability. Created. The Bacillus megaterium ATCC 6459 strain purchased from the ATCC (American Type Culture Collection) was used in accordance with Example 3 to obtain the plasmid p obtained in Example 3-1).
Transformation is performed using OXTB2, the obtained transformant is cultured for 5 days, and the culture is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is collected. A 10-fold volume of methanol was added to this supernatant, and the supernatant obtained by centrifugation at 10,000 rpm for 10 minutes was analyzed by HPLC. As a result, production of oxetanocin-A was confirmed. When the antibacterial activity of the same culture solution was examined, oxetanocin-A antibacterial activity was detected. As a result, the gene fragment that imparts oxetanocin-A-producing ability and oxetanocin-A-resistant ability according to the present invention functions in a host microorganism different from oxetanocin-A-producing bacterium Bacillus megaterium NK84-0218 to produce oxetanocin-A. It was shown that.

(Example 7) Expression of the gene of the present invention in an oxetanosin-A producing strain 1) Detection of a protein specifically expressed in a strain having oxetanosin-A producing ability As a strain having oxetanosin-A producing ability A transformant of NK0161 by pOXTB2 obtained in Bacillus megaterium NK84-0218, Example 3-1) (NK0
161 / pOXTB2), a transformant of NK0161 by pOXTSP obtained in Example 4-1) (N
K0161 / pOXTSP) and NK0161 as a strain having no oxetanosine-A producing ability.
And pOXTBa obtained in Examples 4-2) and 4-3),
Transformants of NK0161 with pOXTEc (NK0161 / pOXTBa, NK0161 / pOX, respectively)
TEc) was individually added to 30 ml of a liquid medium (1%
Yeast extract, 0.5% ammonium chloride, 0.0015
% Cobalt chloride, 10% lactose, 0.3% dipotassium phosphate) in a 500 ml triangular corben at 35 ° C, 1
Incubate at 80 rpm for 2 days.

The culture was centrifuged at 10,000 rpm for 8 minutes to collect the cells, and the supernatant was discarded.
M Tris · HCl pH 7.5, 5 mM EDTA, 0.2
M sucrose) to suspend the cells. The suspension is sonicated for 30 seconds while cooling on ice to disrupt the bacteria. 1 crushed liquid
After centrifugation at 2000 rpm for 10 minutes, the supernatant is collected as a lysate. 5 μl of the lysate was prepared according to the method of Laemmli et al. (Nature, 227, 685-688 (197)
0)), the mixture was subjected to 10% polyacrylamide gel electrophoresis containing SDS, and the protein in the lysate was examined by staining with Coomassie brilliant blue (CBB). At a position between the molecular weight markers 96K and 67K, a band of a protein specifically expressed in a strain capable of producing oxetanosine-A was detected. Strains without the ability to produce oxetanocin-A have in common that they lack the open reading frames a and b shown in Example 5,
It is suggested that the protein is the gene product of open reading frame a or b.

2) Determination of the N-terminal amino acid sequence of the detected protein The NK0161 / pOXTB2 strain lysate obtained in 1) was subjected to 10% polyacrylamide gel electrophoresis in the same manner as in Example 1). With 10% methanol in CAPS (3-cyclohexylamino-1--
The protein in the gel was immersed in a propane-sulfonic acid buffer (CAPS 1.1 g / L, pH 11) for 5 minutes, and the protein in the gel was immersed in a ProBlott membrane (manufactured by Applied Biosystems) previously immersed in 100% methanol for 10 seconds. CA including
Electrotransfer in PS buffer (200 mA, 3
0 minutes). After the transfer, the membrane was washed with deionized water for 5 minutes.
B. B. After staining, wash and dry again with deionized water. 1)
The protein band detected in the above step was cut out with scissors, and this membrane piece was placed on an amino acid sequencer, and the amino acid sequence was determined from the N-terminal side. Xaa-Gln-Thr-T
yr-Leu. This amino acid sequence coincided with the N-terminal amino acid sequence encoded by the nucleotide sequence of open reading frame b in Example 5. Therefore, it was confirmed that at least the protein encoded by the open reading frame b was expressed in a strain having the ability to produce oxetanosine-A.

[0047]

According to the present invention, production of oxetanosine-A in a genetically modified microorganism has become possible for the first time. Furthermore, it can be used for cloning of a new gene fragment using the host-vector system of the present invention, a gene, and the like, selection of a new transformant, and preparation of a probe using the nucleotide sequence of the present gene. Oxetanocin-A
Producing bacteria and / or oxetanosine-A resistant bacterial DNA
Can be identified by the hybridization method.

[Sequence list]

SEQ ID NO: 1 Sequence length: 6163 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: other nucleic acid Plasmid DNA Origin Organism: Bacillus megaterium
m) strain name: NK84-0218 sequence CAGCTGCTAA ACCAACGGGA TTTAAAAAAT CCAATCCCCA GTACGAGGAT TTAAGTTTAG 60 GGTCGTTAGT TGTCATCTTT TTATTATACT GGTCTCTTAC AAAAGAATTT GATTGCATTA 120 TTTTCAGAGC CCTAATACTT AAATTATGTG CTCTTTCTGG ATCTACATTA AATAGCATTG 180 GACGGACAGT AGAATAAACC ATTAGATAAT CTCACTTTCT ATGTAATCTT TATTTTCTGG 240 ATAAGATTTC AGATTGAATT ACATTAAAAT AAAATGTCTT AATCAATCTA TAAGTAATTA 300 GAAAATCACT TTCTAATTAT AAGATTATAA CAAAAGGGTA TTCTATAAAA TCCCTAAATT 360 GTAAAATTAT AAAAATGAGA CTAAGGAAAT AATACTAAAT TGTATCCAAA TCTTTTTTAT 420 GTAATTTATC TAGATATATA AGACTAATTT AAGAGAGTTT ATTTTTCACT TAGAAAATCA 480 GCGCTAATTT TTATAAGAAT GAGTTTTTAA AAAAATAGGA TTTTTTTATA TTTGTTTAAA 540 AGAACATTTA GGAAGGTGCA ATTTATTAGG TGCTAGAAGC CCTTGATAAG AGAGTTTTTT 600 AGTAAGCTAT TGTCAATAAC TTGCCTTATG GAAACTAAAC ATAAATATCT TCTATATTTT 660 AGTGAGTTAA GAGCCATCAC AGTCAGCTAA CTGATTATTT GTTGCTCTAT AGAAGTGATA 720 GTAAACCTTC TTGGTCATAT GCTTTTCTTT CATATTTAAT GCATATTGAA AAATTATTGG 780 ATCCATTCAG TAGGTTTTTC TATGTATGGA GTTTTAGTAA CTCTAAAAAA TTTAACAAGT 840 AGCTGTTTAC TTTTTTAAGT CTGTTAATTT TATGTGCTAT TTATTTAAAT GTTAATCTCT 900 ATAAAAAAGT TACATACCTG AAAACATAGA CTAATTTCTG CAAGTATTTG TAACAGATCC 960 ATTTATTATA TTAAAGAGCT CCAATTATCA TTTTATGGAT ATAAAAGGGT TATTTTACCT 1020 TGTAATAATA AACCATATAT GTTAATTATA TAATTGGAAT ATTTTTTCAG GAGAGGTGTA 1080 GCAATGAGTA GTCTATTAGA CATTATTTAT CAGCTAAGAC AGGTTCCGCG GTGGGATGGC 1140 TCTTTTCAAT TTGAAAAAGA AGATGTATCT CAGCATAGTT TCTCAGTAAT AGCAATATCT 1200 CATATACTTT GTGAGTTAAA AGAAACCCTT GAAGGTAAAA AAATCAACAA AGAAAAACTC 1261 TTGTTATATG CACTTTATCA TGATGTTACA GAAGTCGTTT CTACTCATAT TATTAGTCCT 1320 GTGAAGAAGA ATAGTATATT AAAAGATCCT TTTAATGCTT TTAGGGAGCA GATTAAAAAT 1380 AGTTTGTTCG ATAATTTGCC AATAACATTG TCAGACACAT TATCAACCAT CTTAAATAAT 1440 AACGATTTAG AGATACAAGA AATTGTGGAG CATGCAGACC ATGTTGATGC GTACTGCAAA 1500 TCATGTATAG AAGTACATAG AGGAAATAAA GATTTTATCT CTATTCAGAG AAGCCTTGGA 1560 GATAAACTAG ATAACTTGAC TAAAGAATAT CCATACCTAA AAGAATTTCA AAATCTATTT 1620 CTTAAGGATT TTCCCTTAGA AAATAAAAAT TACAGGTACC TTAACTGAG G AGGATAAAAA 1680 TGCAGACTTA TTTATCAACT AAAAGTATTG AGTACTATTT AAAGGAGCTT AAGGAGATAT 1740 TTTCACAAAT ATGGTTGAAA CCTTCTGAGA TAGAAAAAAG GTGTGAAGAG CTTTTTAAAC 1800 GTAGTAAAGA ATTTGATTAT AAAAGGATAC TTGTATCAGG GGAAACTGAT AACACAACCC 1860 TCTACGTAAT AGAAGATAGT TCCAAGATAC ATGTTTTTTC ACCTAATAGA GATTTAAGAG 1920 AAAACCCTCT ATTAATGCGT TGGCACCCTT CCTGGTATGA GATTGAGAGC AAAGAAATTT 1980 ACTATAAATG CTTCCTGAGT TGTGAAGAAT TATATGAACA TTTAGAACTT CCTACTGTAA 2040 CATTAGTAAA TTTATGCGTA ATAGAAAATT TCCCCATTCC AAGATTAAAC TTAAGTACGG 2100 GAACTTTATC TTCTTACTTA AGAAAAGAAC AATTAGCTAA AGTTGAGTTA ATTGATATGC 2160 AAGTAGGGAC TACAATTAAC CAAATTATAA AAAACCTCTT GGATAGTCAG CCTGATATTA 2220 TTGGCTTATC TGTGAATTTT GGGCAAAAGA AGTTAGCCTT CGAGATTTTA GATTTGATTT 2280 ATTCACATAT AGAGAATGGA GACTTATCTT CAATCATTAC TGTAGGAAAC GTAATTCCTT 2340 CATTCTCTCC AGAACAATTC TTTGAAAGGT ACCCTTCATT GCTGATTTGT GATAAGGAGG 2400 GTGAATATAC ACTAAGAGAC CTGATAAAAA TGCTAAAAAA AGAACTTAAA CTTGATGAAG 2460 TAAATGGAAT ATCATATGTA GATGAGTCTG GAGAAGTAAA GCATAACGTA GCTG AAACTG 2520 TTAACTTTAA GGAAGAAGTC CCCACACCTT CACTGGATAT TTTAGGCGAA ATATCGAAGT 2580 TTAGGGGAGC ATTGACACTA GAAACTAGTA GGGGTTGTGA TTATTCTCGA TGTACCTTCT 2640 GCCCAAGAGA TCATAAATTA AGATCATGGC GACCATTATC TGTCGAACAA ACTCTTAAAC 2700 AATTAGATGA TATTTTAAGA GCCGGTAAAC ATTTCAATAT AAAGCCCCAT ATTTATATGG 2760 CTGACGAAGA ATTTATTGGT GAGCTACCAA ATGGAACAGA GGCTCAGAGA ATTATTGATA 2820 TATGTGAGGG GTTATTAAAA AGAGAGGAAA AAATAAAATT TGATTTTGCT GCTAGAGCAG 2880 ACTCTGTGTA TGAACCAAAA AGAACAAAAG AGTGGAACGT TGAAAGGCTA AAAATGTGGC 2940 ATTACTGCGC TCTGGCAGGT GCTGATAGAA TCTTTATTGG AGTAGAATCA GGATCCAATC 3000 AACAATTGAA GAGATATGGG AAGGGCACCA CATCAGAACA AAATATAATT GCTTTGAGAT 3060 TAGTAAGTGC ATTGGGTATC AATTTGAGAA TTGGTTTTAT CATGTTTGAT CAATTAATGA 3120 AGGGATTAGA TAACCTCAAA GAAAACCTAG ATTTTCTAGA AAGAACAGAT GCTCTCATGA 3180 AACCTATTGA TATTGGTGAT ATGACATATG AAGAATTATA TGACAAACTT TTAAACGACA 3240 AAGAATTTAT TGAAAAACAT AAAACAGGTA AACCTGTCTA TACTATTGTT TCATATATGT 3300 TAGCTTCTAT GGAAATTCTG ATGAATACCC CTTATTCAAG AATGGTTCAA TTAACAGAAC 3360 GAAAAGAAGA AGTGAACCTT ATAATGAATG ATGGAAAGCC GGATATGAAC ATGGGAAGAT 3420 ATGCAACTTC CTTTGTAGAT AAAACAAACG GTAATTTAAG TGAGGCTTGT CAAATGTGGA 3480 TAGATAGTAA CTTTGGGGTA ATGTACACAA TTAAGAGTTT GCATAAAGTT GCTAATCCTA 3540 GAGAAAAGAA AAAGTTGTAT AGTTATATGG AAACACATAG AGAGATTAGT CATTTTCTTT 3600 TAAAGTATCT TGTATATAAT CTTAGTCCCG ATAAGGAAAG TCAAATTATC TTATCAGATT 3660 TTCTAAGAAT GCATTCAATG GAACACATTT TAGATAATTC TAAAATAAAT GTGGGGGATG 3720 GTTCTAAAGA AAATATTTTG AATGTAATGA CAAATTGGCA ACTAATTATG GAAAAACTTT 3780 TAAGAGATGT AGAGGCTGAT TTAAATAAAG GGATTATTAC TGATAGCGAA GATCATAGAC 3840 TCCATAATAC GTTAAAACGA TGGTTTAGTG ATATGGGTAA CTGGTCTTTA ATAAATGCTT 3900 ACGAGCTAAA CTAAAGGCAG TGGGAATTCT ATATTCCCAC TGCTTTTACA TAGGTACCCT 3960 TTTCACATTT ATGATAAGCC TCTATAGTAA ATTGTTTAAT CTGTGGTGAA ATGGAAGAGG 4020 CGCACTCAAC TTTCTTCTGA TTATTAACTA ATGGATCTAT TTCCCTCATT TTTATTTTAG 4080 AGCTATATTC ATAGTCTTCT TTTGATTCTA TAATATGATT ATGATTAAGA TCAAACTTAC 4140 TAATTAAGTT TATTATTTGT TTATCTCTAC TAGAAGTTAA TTTTTCCATT ACAGTAAAAT 4200 CGTCTGTAAG GAAGTCATCT AAATGAATAA TTTCTTTTTC AAGAGCCAAG GTTAGAATTT 4260 TAGTTAAATG TCTATTCGAG AAGACATTTA ATGGATGTAA AAAGAAATCT ATTACTTCTT 4320 TATAATATAA TTTAGTAAAC CATTCTGCCG ACTCAATATT CTTTATCACT ATCTTATTAT 4380 TGATAACATG AAGGTTATTT AAAAATTTTT GAATATCCTC TAAATTTTCG CCTTGATAAT 4440 AATACATATC CCTCAACGTA TAATCAATTC TGTCTGCACA AGGTAAAGGC AAATCTTGTT 4500 CTAAAATTCC CCAACAACTG TGGTCGAGAA TTAAATTAAT ATCAATATCA TATTTTTTTA 4560 ATATACTTGG AATTTCTGAT GAAAAAACAA TTTTATCTAC AATCTTTTCA TGATAATCTT 4620 CCTCATCATT ATCTAATACA TAGTCTACTA CGTGGGAAAA GGCCGTATGT GAGATGTCAT 4680 GAAGTAAACC AGCAATCTGT TCTAATATAC TTCCATTTAA ATATCTTATT AACAACATTA 4740 CTCCTACGGA ATGATCGTAA CGAGTAACAT CCCACTTGCC ATTAATTAAA TAACATCCCC 4800 CTGCCTGGTG TATTTTTTTT AAACGTTGAA CAGGCTTTGT ATGTATTAGT TCTGTTAAAA 4860 CTGGTTCCAA ATCATATTCT CCATAAATTG GATCAAATAT TTTCATTATG TTCCCCCTTT 4920 AAAATAAAAT TTTAAAATAA TCTTCTTTCT ATTTAATTAT GGAATAATTA GGGATTTTGT 4980 ACAACTATTT TAAAATATAT TTTAAATATT AGCTACTCCA TTACCGATAT TTACCTAAAA 5040 ATAAGT TTTT TTACATTATA TATAGATATT TCCTTCTGAC TATATATAGC TACTATACAT 5100 ACATTATTAA CGCTGGGGAG TAATATACAT TGTTATTTTT TATACTTAAC GTAATCAAAA 5160 TTATAGGAAC GAAAAGAAGC TCAAGAACTA GTTAAGTAGT CCTTGAGCTC TTTTTCTCCA 5220 GTGTCCTGGA GTCGCTAAAG CAGAGGGCAA CTTTGTATGT AAATCGCCTT TTGCTGCATT 5280 CACCTGAGCT TGGGTAAGGA AAATACTTCC TGTAAGATCA GCACCACTTA AGTCAGCATC 5340 TCTGAAGTCT GCTCCAATAA GATCCGTTAA TCTGAGGTCA GCTTCTCTAA GATCAGCAGC 5400 TATAAATAAT GCTCCCTTTA AATTAGCTCC TCTAAGATTA GCTCTTCTTA ACTTAGCCCC 5460 AATATAGTCA CTACTACGCT TAAATTTCTT TCCTAACTTT TTGCTAGATG TTTTGGGACG 5520 AACCAATTCA CTTGTACGTA AAAGTAATTC TCTGACTTTC ATTCGATGCC CATGCACATC 5580 AATTTTTAAA ATGTTTTCTG GTGTTTGTTT AATTAAACGT TCCGTTTCTT CAAAGGAAAT 5640 CCTTAAATCA TCATGAATGG AATTCGTAGC TTGAAATTCA AGTGCCTCGC TTAAATACCA 5700 AAGTATTTCA TGCAATTGCT GAACAATTGG AAACACATTA AACATTTCTT TAGCTCTTGC 5760 TTTATTACCT CTCCAATCAT TTCCTTCATA AATCGTTTGA GAAACCCTTT GGCCAGCACC 5820 GAAACATTCA TATCCCACAC AGCCTTTAAA TCCCGTAGTG ATAAGATTCT TGTGAATGGA 5880 ACATGAATAG T TTGACTGTA AATGACGGCA AGGAGTTCCA CCATCTTTGT CAAAGGCAAA 5940 ATCAGCAGAT TTTGCGTAAG GTAAAGCTAC GCAGCACAAA CCAAAACAAT TTGAGCAGTC 6000 CGAACGTAAA TTTTCTCGAA CTTTATTATC TATCTCTAGT TTGTTAGGCA AGAAAACACA 6060 TTCCTTTCTT ATAATTTCCT GTAATTTTAT AATAATTGGA ATTATAAGAA AAATAAAGGT 6120 TGTTACTGAT TTAGATTTAT TACTGTATAA GATATACAGA TCT 6163

SEQ ID NO: 2 Sequence length: 582 (nucleic acid), 194 (amino acid) Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Plasmid DNA Origin Organism name : Bacillus megateriu
m) Strain name: NK84-0218 sequence ATG AGT AGT CTA TTA GAC ATT ATT TAT CAG CTA AGA CAG GTT CCG CGG 48 Met Ser Ser Leu Leu Asp Ile Ile Tyr Gln Leu Arg Gln Val Pro Arg 16 TGG GAT GGC TCT TTT CAA TTT GAA AAA GAA GAT GTA TCT CAG CAT AGT 96 Trp Asp Gly Ser Phe Gln Phe Glu Lys Glu Asp Val Ser Gln His Ser 32 TTC TCA GTA ATA GCA ATA TCT CAT ATA CTT TGT GAG TTA AAA GAA ACC 144 Phe Ser Val Ile Ala Ile Ser His Ile Leu Cys Glu Leu Lys Glu Thr 48 CTT GAA GGT AAA AAA ATC AAC AAA GAA AAA CTC TTG TTA TAT GCA CTT 192 Leu Glu Gly Lys Lys Ile Asn Lys Glu Lys Leu Leu Leu Tyr Ala Leu 64 TAT CAT GAT GTT ACA GAA GTC GTT TCT ACT CAT ATT ATT AGT CCT GTG 240 Tyr His Asp Val Thr Glu Val Val Ser Thr His Ile Ile Ser Pro Val 80 AAG AAG AAT AGT ATA TTA AAA GAT CCT TTT AAT GCT TTT AGG GAG CAG 288 Lys Lys Asn Ser Ile Leu Lys Asp Pro Phe Asn Ala Phe Arg Glu Gln 96 ATT AAA AAT AGT TTG TTC GAT AAT TTG CCA ATA ACA TTG TCA GAC ACA 336 Ile Lys Asn Ser Leu Phe Asp Asn Leu Pro Ile Thr Leu Ser Asp Thr 112 TTA TCA A CC ATC TTA AAT AAT AAC GAT TTA GAG ATA CAA GAA ATT GTG 384 Leu Ser Thr Ile Leu Asn Asn Asn Asp Leu Glu Ile Gln Glu Ile Val 128 GAG CAT GCA GAC CAT GTT GAT GCG TAC TGC AAA TCA TGT ATA GAA GTA 432 Glu His Ala Asp His Val Asp Ala Tyr Cys Lys Ser Cys Ile Glu Val 144 CAT AGA GGA AAT AAA GAT TTT ATC TCT ATT CAG AGA AGC CTT GGA GAT 480 His Arg Gly Asn Lys Asp Phe Ile Ser Ile Gln Arg Ser Leu Gly Asp 160 AAA CTA GAT AAC TTG ACT AAA GAA TAT CCA TAC CTA AAA GAA TTT CAA 528 Lys Leu Asp Asn Leu Thr Lys Glu Tyr Pro Tyr Leu Lys Glu Phe Gln 176 AAT CTA TTT CTT AAG GAT TTT CCC TTA GAA AAT AAA AAT TAC AGG TAC 576 Asn Leu Phe Leu Lys Asp Phe Pro Leu Glu Asn Lys Asn Tyr Arg Tyr 192 CTT AAC 582 Leu Asn 194

SEQ ID NO: 3 Sequence length: 2232 (nucleic acid), 744 (amino acid) Sequence type: nucleic acid Number of chains: double-stranded Topology: linear Sequence type: other nucleic acid Plasmid DNA Origin Organism name : Bacillus megateriu
m) Strain name: NK84-0218 sequence ATG CAG ACT TAT TTA TCA ACT AAA AGT ATT GAG TAC TAT TTA AAG GAG 48 Met Gln Thr Tyr Leu Ser Thr Lys Ser Ile Glu Tyr Tyr Leu Lys Glu 16 CTT AAG GAG ATA TTT TCA CAA ATA TGG TTG AAA CCT TCT GAG ATA GAA 96 Leu Lys Glu Ile Phe Ser Gln Ile Trp Leu Lys Pro Ser Glu Ile Glu 32 AAA AGG TGT GAA GAG CTT TTT AAA CGT AGT AAA GAA TTT GAT TAT AAA 144 Lys Arg Cys Glu Glu Leu Phe Lys Arg Ser Lys Glu Phe Asp Tyr Lys 48 AGG ATA CTT GTA TCA GGG GAA ACT GAT AAC ACA ACC CTC TAC GTA ATA 192 Arg Ile Leu Val Ser Gly Glu Thr Asp Asn Thr Thr Leu Tyr Val Ile 64 GAA GAT AGT TCC AAG ATA CAT GTT TTT TCA CCT AAT AGA GAT TTA AGA 240 Glu Asp Ser Ser Lys Ile His Val Phe Ser Pro Asn Arg Asp Leu Arg 80 GAA AAC CCT CTA TTA ATG CGT TGG CAC CCT TCC TGG TAT GAG ATT GAG 288 Glu Asn Pro Leu Leu Met Arg Trp His Pro Ser Trp Tyr Glu Ile Glu 96 AGC AAA GAA ATT TAC TAT AAA TGC TTC CTG AGT TGT GAA GAA TTA TAT 336 Ser Lys Glu Ile Tyr Tyr Lys Cys Phe Leu Ser Cys Glu Glu Leu Tyr 112 GAA CAT T TA GAA CTT CCT ACT GTA ACA TTA GTA AAT TTA TGC GTA ATA 384 Glu His Leu Glu Leu Pro Thr Val Thr Leu Val Asn Leu Cys Val Ile 128 GAA AAT TTC CCC ATT CCA AGA TTA AAC TTA AGT ACG GGA ACT TTA TCT 432 Glu Asn Phe Pro Ile Pro Arg Leu Asn Leu Ser Thr Gly Thr Leu Ser 144 TCT TAC TTA AGA AAA GAA CAA TTA GCT AAA GTT GAG TTA ATT GAT ATG 480 Ser Tyr Leu Arg Lys Glu Gln Leu Ala Lys Val Glu Leu Ile Asp Met 160 CAA GTA GGG ACT ACA ATT AAC CAA ATT ATA AAA AAC CTC TTG GAT AGT 528 Gln Val Gly Thr Thr Ile Asn Gln Ile Ile Lys Asn Leu Leu Asp Ser 176 CAG CCT GAT ATT ATT GGC TTA TCT GTG AAT TTT GGG CAA AAG AAG TTA 576 Gln Pro Asp Ile Ile Gly Leu Ser Val Asn Phe Gly Gln Lys Lys Leu 192 GCC TTC GAG ATT TTA GAT TTG ATT TAT TCA CAT ATA GAG AAT GGA GAC 624 Ala Phe Glu Ile Leu Asp Leu Ile Tyr Ser His Ile Glu Asn Gly Asp 208 TTA TCT TCA ATC ATT ACT GTA GGA AAC GTA ATT CCT TCA TTC TCT CCA 672 Leu Ser Ser Ile Ile Thr Val Gly Asn Val Ile Pro Ser Phe Ser Pro 224 GAA CAA TTC TTT GAA AGG TAC CCT TCA TTG CTG ATT TGT GAT A AG GAG 720 Glu Gln Phe Phe Glu Arg Tyr Pro Ser Leu Leu Ile Cys Asp Lys Glu 240 GGT GAA TAT ACA CTA AGA GAC CTG ATA AAA ATG CTA AAA AAA GAA CTT 768 Gly Glu Tyr Thr Leu Arg Asp Leu Ile Lys Met Leu Lys Lys Glu Leu 256 AAA CTT GAT GAA GTA AAT GGA ATA TCA TAT GTA GAT GAG TCT GGA GAA 816 Lys Leu Asp Glu Val Asn Gly Ile Ser Tyr Val Asp Glu Ser Gly Glu 272 GTA AAG CAT AAC GTA GCT GAA ACT GTT AAC TTT AAG GAA GAA GTC CCC 864 Val Lys His Asn Val Ala Glu Thr Val Asn Phe Lys Glu Glu Val Pro 288 ACA CCT TCA CTG GAT ATT TTA GGC GAA ATA TCG AAG TTT AGG GGA GCA 912 Thr Pro Ser Leu Asp Ile Leu Gly Glu Ile Ser Lys Phe Arg Gly Ala 304 TTG ACA CTA GAA ACT AGT AGG GGT TGT GAT TAT TCT CGA TGT ACC TTC 960 Leu Thr Leu Glu Thr Ser Arg Gly Cys Asp Tyr Ser Arg Cys Thr Phe 320 TGC CCA AGA GAT CAT AAA TTA AGA TCA TGG CGA CCA TTA TCT GTC GAA 1008 Cys Pro Arg Asp His Lys Leu Arg Ser Trp Arg Pro Leu Ser Val Glu 336 CAA ACT CTT AAA CAA TTA GAT GAT ATT TTA AGA GCC GGT AAA CAT TTC 1056 Gln Thr Leu Lys Gln Leu Asp Asp Ile Leu Arg Ala Gly Lys His Phe 352 AAT ATA AAG CCC CAT ATT TAT ATG GCT GAC GAA GAA TTT ATT GGT GAG 1104 Asn Ile Lys Pro His Ile Tyr Met Ala Asp Glu Glu Phe Ile Gly Glu 368 CTA CCA AAT GGA ACA GAG GCT CAG AGA ATT ATT GAT ATA TGT GAG GGG 1152 Leu Pro Asn Gly Thr Glu Ala Gln Arg Ile Ile Asp Ile Cys Glu Gly 384 TTA TTA AAA AGA GAG GAA AAA ATA AAA TTT GAT TTT GCT GCT AGA GCA 1200 Leu Leu Lys Arg Glu Glu Lys Ile Lys Phe Asp Phe Ala Ala Arg Ala 400 GAC TCT GTG TAT GAA CCA AAA AGA ACA AAA GAG TGG AAC GTT GAA AGG 1248 Asp Ser Val Tyr Glu Pro Lys Arg Thr Lys Glu Trp Asn Val Glu Arg 416 CTA AAA ATG TGG CAT TAC TAC TGC GCT CTG GCA GGT GCT GAT AGA ATC TTT 1296 Leu Lys Met Trp His Tyr Cys Ala Leu Ala Gly Ala Asp Arg Ile Phe 432 ATT GGA GTA GAA TCA GGA TCC AAT CAA CAA TTG AAG AGA TAT GGG AAG 1344 Ile Gly Val Glu Ser Gly Ser Asn Gln Gln Leu Lys Arg Tyr Gly Lys 448 GGC ACC ACA TCA GAA CAA AAT ATA ATT GCT TTG AGA TTA GTA AGT GCA 1392 Gly Thr Thr Ser Glu Gln Asn Ile Ile Ala Leu Arg Leu Val Ser Ala 464 TTG GGT ATC AAT TTG AGA ATT GGT TTT ATC ATG TTT GAT CAA TTA ATG 1440 Leu Gly Ile Asn Leu Arg Ile Gly Phe Ile Met Phe Asp Gln Leu Met 480 AAG GGA TTA GAT AAC CTC AAA GAA AAC CTA GAT TTT CTA GAA AGA ACA 1488 Gly Leu Asp Asn Leu Lys Glu Asn Leu Asp Phe Leu Glu Arg Thr 496 GAT GCT CTC ATG AAA CCT ATT GAT ATT GGT GAT ATG ACA TAT GAA GAA 1536 Asp Ala Leu Met Lys Pro Ile Asp Ile Gly Asp Met Thr Tyr Glu Glu 512 TTA TAT GAC AAA CTT TTA AAC GAC AAA GAA TTT ATT GAA AAA CAT AAA 1584 Leu Tyr Asp Lys Leu Leu Asu Asn Asp Lys Glu Phe Ile Glu Lys His Lys 528 ACA GGT AAA CCT GTC TAT ACT ATT GTT TCA TAT ATG TTA GCT TCT ATG 1632 Thr Gly Lys Pro Val Tyr Thr Ile Val Ser Tyr Met Leu Ala Ser Met 544 GAA ATT CTG ATG AAT ACC CCT TAT TCA AGA ATG GTT CAA TTA ACA GAA 1680 Glu Ile Leu Met Asn Thr Pro Tyr Ser Arg Met Val Gln Leu Thr Glu 560 CGA AAA GAA GAA GTG AAC CTT ATA ATG AAT GAT GGA AAG CCG GAT ATG 1728 Arg Lys Glu Glu Val Asn Leu Ile Met Asn Asp Gly Lys Pro Asp Met 576 AAC ATG GGA AGA TAT GCA ACT TCC TTT GTA GAT AAA A CA AAC GGT AAT 1776 Asn Met Gly Arg Tyr Ala Thr Ser Phe Val Asp Lys Thr Asn Gly Asn 592 TTA AGT GAG GCT TGT CAA ATG TGG ATA GAT AGT AAC TTT GGG GTA ATG 1824 Leu Ser Glu Ala Cys Gln Met Trp Ile Asp Ser Asn Phe Gly Val Met 608 TAC ACA ATT AAG AGT TTG CAT AAA GTT GCT AAT CCT AGA GAA AAG AAA 1872 Tyr Thr Ile Lys Ser Leu His Lys Val Ala Asn Pro Arg Glu Lys Lys 624 AAG TTG TAT AGT TAT ATG GAA ACA CAT AGA GAG ATT AGT CAT TTT CTT 1920 Lys Leu Tyr Ser Tyr Met Glu Thr His Arg Glu Ile Ser His Phe Leu 640 TTA AAG TAT CTT GTA TAT AAT CTT AGT CCC GAT AAG GAA AGT CAA ATT 1968 Leu Lys Tyr Leu Val Tyr Asn Leu Ser Pro Asp Lys Glu Ser Gln Ile 656 ATC TTA TCA GAT TTT CTA AGA ATG CAT TCA ATG GAA CAC ATT TTA GAT 2016 Ile Leu Ser Asp Phe Leu Arg Met His Ser Met Glu His Ile Leu Asp 672 AAT TCT AAA ATA AAT GTG GGG GAT GGT TCT AAA GAA AAT ATT TTG AAT 2064 Asn Ser Lys Ile Asn Val Gly Asp Gly Ser Lys Glu Asn Ile Leu Asn 688 GTA ATG ACA AAT TGG CAA CTA ATT ATG GAA AAA CTT TTA AGA GAT GTA 2112 Val Met Thr Asn Trp G ln Leu Ile Met Glu Lys Leu Leu Arg Asp Val 704 GAG GCT GAT TTA AAT AAA GGG ATT ATT ACT GAT AGC GAA GAT CAT AGA 2160 Glu Ala Asp Leu Asn Lys Gly Ile Ile Thr Asp Ser Glu Asp His Arg 720 CTC CAT AAT ACG TTA AAA CGA TGG TTT AGT GAT ATG GGT AAC TGG TCT 2208 Leu His Asn Thr Leu Lys Arg Trp Phe Ser Asp Met Gly Asn Trp Ser 736 TTA ATA AAT GCT TAC GAG CTA AAC 2232 Leu Ile Asn Ala Tyr Glu Leu Asn 744

SEQ ID NO: 4 Sequence length: 975 (nucleic acid), 325 (amino acid) Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Plasmid DNA Origin Organism : Bacillus megateriu
m) Strain name: NK84-0218 sequence ATG AAA ATA TTT GAT CCA ATT TAT GGA GAA TAT GAT TTG GAA CCA GTT 48 Met Lys Ile Phe Asp Pro Ile Tyr Gly Glu Tyr Asp Leu Glu Pro Val 16 TTA ACA GAA CTA ATA CAT ACA AAG CCT GTT CAA CGT TTA AAA AAA ATA 96 Leu Thr Glu Leu Ile His Thr Lys Pro Val Gln Arg Leu Lys Lys Ile 32 CAC CAG GCA GGG GGA TGT TAT TTA ATT AAT GGC AAG TGG GAT GTT ACT 144 His Gln Ala Gly Gly Cys Tyr Leu Ile Asn Gly Lys Trp Asp Val Thr 48 CGT TAC GAT CAT TCC GTA GGA GTA ATG TTG TTA ATA AGA TAT TTA AAT 192 Arg Tyr Asp His Ser Val Gly Val Met Leu Leu Ile Arg Tyr Leu Asn 64 GGA AGT ATA TTA GAA CAG ATT GCT GGT TTA CTT CAT GAC ATC TCA CAT 240 Gly Ser Ile Leu Glu Gln Ile Ala Gly Leu Leu His Asp Ile Ser His 80 ACG GCC TTT TCC CAC GTA GTA GAC TAT GTA TTA GAT AAT GAT GAG GAA 288 Thr Ala Phe Ser His Val Val Asp Tyr Val Leu Asp Asn Asp Glu Glu 96 GAT TAT CAT GAA AAG ATT GTA GAT AAA ATT GTT TTT TCA TCA GAA ATT 336 Asp Tyr His Glu Lys Ile Val Asp Lys Ile Val Phe Ser Ser Glu Ile 112 CCA AGT A TA TTA AAA AAA TAT GAT ATT GAT ATT AAT TTA ATT CTC GAC 384 Pro Ser Ile Leu Lys Lys Tyr Asp Ile Asp Ile Asn Leu Ile Leu Asp 128 CAC AGT TGT TGG GGA ATT TTA GAA CAA GAT TTG CCT TTA CCT TGT GCA 432 His Ser Cys Trp Gly Ile Leu Glu Gln Asp Leu Pro Leu Pro Cys Ala 144 GAC AGA ATT GAT TAT ACG TTG AGG GAT ATG TAT TAT TAT CAA GGC GAA 480 Asp Arg Ile Asp Tyr Thr Leu Arg Asp Met Tyr Tyr Tyr Gln Gly Glu 160 AAT TTA GAG GAT ATT CAA AAA TTT TTA AAT AAC CTT CAT GTT ATC AAT 528 Asn Leu Glu Asp Ile Gln Lys Phe Leu Asn Asn Leu His Val Ile Asn 176 AAT AAG ATA GTG ATA AAG AAT ATT GAG TCG GCA GAA TGG TTT ACT AAA 576 Asn Lys Ile Val Ile Lys Asn Ile Glu Ser Ala Glu Trp Phe Thr Lys 192 TTA TAT TAT AAA GAA GTA ATA GAT TTC TTT TTA CAT CCA TTA AAT GTC 624 Leu Tyr Tyr Lys Glu Val Ile Asp Phe Phe Leu His Pro Leu Asn Val 208 TTC TCG AAT AGA CAT TTA ACT AAA ATT CTA ACC TTG GCT CTT GAA AAA 672 Phe Ser Asn Arg His Leu Thr Lys Ile Leu Thr Leu Ala Leu Glu Lys 224 GAA ATT ATT CAT CAT TTA GAT GAC TTC CTT ACA GAC GAT TTT ACT G TA ATG 720 Glu Ile Ile His Leu Asp Asp Phe Leu Thr Asp Asp Phe Thr Val Met 240 GAA AAA TTA ACT TCT AGT AGA GAT AAA CAA ATA ATA AAC TTA ATT AGT 768 Glu Lys Leu Thr Ser Ser Arg Asp Lys Gln Ile Ile Asn Leu Ile Ser 256 AAG TTT GAT CTT AAT CAT AAT CAT ATT ATA GAA TCA AAA GAA GAC TAT 816 Lys Phe Asp Leu Asn His Asn His Ile Ile Glu Ser Lys Glu Asp Tyr 272 GAA TAT AGC TCT AAA ATA AAA ATG AGG GAA ATA GAT CCA TTA GTT AAT 864 Glu Tyr Ser Ser Lys Ile Lys Met Arg Glu Ile Asp Pro Leu Val Asn 288 AAT CAG AAG AAA GTT GAG TGC GCC TCT TCC ATT TCA CCA CAG ATT AAA 912 Asn Gln Lys Lys Val Glu Cys Ala Ser Ser Ile Ser Pro Gln Ile Lys 304 CAA TTT ACT ATA GAG GCT TAT CAT AAA TGT GAA AAG GGT ACC TAT GTA 960 Gln Phe Thr Ile Glu Ala Tyr His Lys Cys Glu Lys Gly Thr Tyr Val 320 AAA GCA GTG GGA ATA 975 Lys Ala Val Gly Ile 325

SEQ ID NO: 5 Sequence length: 648 (nucleic acid), 216 (amino acid) Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid Plasmid DNA Origin Organism : Bacillus megateriu
m) Strain name: NK84-0218 sequence GTG GGA TAT GAA TGT TTC GGT GCT GGC CAA AGG GTT TCT CAA ACG ATT 48 Val Gly Tyr Glu Cys Phe Gly Ala Gly Gln Arg Val Ser Gln Thr Ile 16 TAT GAA GGA AAT GAT TGG AGA GGT AAT AAA GCA AGA GCT AAA GAA ATG 96 Tyr Glu Gly Asn Asp Trp Arg Gly Asn Lys Ala Arg Ala Lys Glu Met 32 TTT AAT GTG TTT CCA ATT GTT CAG CAA TTG CAT GAA ATA CTT TGG TAT 144 Phe Asn Val Phe Pro Ile Val Gln Gln Leu His Glu Ile Leu Trp Tyr 48 TTA AGC GAG GCA CTT GAA TTT CAA GCT ACG AAT TCC ATT CAT GAT GAT 192 Leu Ser Glu Ala Leu Glu Phe Gln Ala Thr Asn Ser Ile His Asp Asp 64 TTA AGG ATT TCC TTT GAA GAA ACG GAA CGT TTA ATT AAA CAA ACA CCA 240 Leu Arg Ile Ser Phe Glu Glu Thr Glu Arg Leu Ile Lys Gln Thr Pro 80 GAA AAC ATT TTA AAA ATT GAT GTG CAT GGG CAT CGA ATG AAA GTC AGA 288 Glu Asn Ile Leu Lys Ile Asp Val His Gly His Arg Met Lys Val Arg 96 GAA TTA CTT TTA CGT ACA AGT GAA TTG GTT CGT CCC AAA ACA TCT AGC 336 Glu Leu Leu Leu Leu Arg Thr Ser Glu Leu Val Arg Pro Lys Thr Ser Ser 112 AAA AAG T TA GGA AAG AAA TTT AAG CGT AGT AGT GAC TAT ATT GGG GCT 384 Lys Lys Leu Gly Lys Lys Phe Lys Arg Ser Ser Asp Tyr Ile Gly Ala 128 AAG TTA AGA AGA GCT AAT CTT AGA GGA GCT AAT TTA AAG GGA GCA TTA 432 Lys Leu Arg Arg Ala Asn Leu Arg Gly Ala Asn Leu Lys Gly Ala Leu 144 TTT ATA GCT GCT GAT CTT AGA GAA GCT GAC CTC AGA TTA ACG GAT CTT 480 Phe Ile Ala Ala Asp Leu Arg Glu Ala Asp Leu Arg Leu Thr Asp Leu 160 ATT GGA GCA GAC TTC AGA GAT GCT GAC TTA AGT GGT GCT GAT CTT ACA 528 Ile Gly Ala Asp Phe Arg Asp Ala Asp Leu Ser Gly Ala Asp Leu Thr 176 GGA AGT ATT TTC CTT ACC CAA GCT CAG GTG AAT GCA GCA AAA GGC 576 Gly Ser Ile Phe Leu Thr Gln Ala Gln Val Asn Ala Ala Lys Gly Asp 192 TTA CAT ACA AAG TTG CCC TCT GCT TTA GCG ACT CCA GGA CAC TGG AGA 624 Leu His Thr Lys Leu Pro Ser Ala Leu Ala Thr Pro Gly His Trp Arg 208 AAA AGA GCT CAA GGA CTA CTT AAC 648 Lys Arg Ala Gln Gly Leu Leu Asn 216

[Brief description of the drawings]

FIG. 1 is a restriction map of plasmid pOXTB2,

FIG. 2. Plasmids pOXTB2 and oxetanocin-A
Plasmid pMGO from a 4.8 kb plasmid from the producer strain Bacillus megaterium NK84-0218
XT creation process diagram

FIG. 3 is a restriction map of a 6.8 kb DNA fragment obtained by digesting pOXTB2 with Bg1 II.

FIG. 4: Plasmid pO from plasmid pOXTB2
XTSP, pOXTBa and pOXTEc production process diagram

FIG. 5. Pvμ in a 6.8 kb fragment from pOXTB2
FIG. 4 is a layout diagram of an open reading frame in a II-Bg1II 6.1 kb fragment.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 15/09 (C12P 21/02 C12R 1:11) C12R 1:11) (C12P 21/02 C12N 15/00 ZNAA C12R 1 : 11) C12R 1:11) (56) References JP-A-3-147790 (JP, A) Tani K, et al. , Identification of Germination Gene of Bacillus megaterium. , Biochem. Biophy s. Res. Commun. (1990), Vol. 167, no. 2,. p. 402- 406 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) CA (STN) JICST file (JOIS) WPI / L (QUESTEL) SwissProt / PIR / GeneSeq GenBank / EMBL / DDBJ / GeneSeq PubMed

Claims (3)

(57) [Claims] 1. A DNA encoding a protein represented by the following (1) or (2): (1) a protein producing oxetanocin-A having an amino acid sequence represented by SEQ ID NO: 3 in the sequence listing; and (2) a sequence in the sequence listing. Having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in No. 3, and oxetanocin-A
Protein that has the ability to produce 2. A recombinant plasmid comprising the DNA according to claim 1. 3. A transformant or transductant containing the recombinant plasmid according to claim 2.