JPS63251087A - Bialaphos-resistant gene and plasmid - Google Patents

Abstract

PURPOSE:To construct a plasmid having bialaphos resistance, by forming a bialaphos-resistant gene to code an enzyme to acetylize dimethylphosphinothricin and phosphinothricin. CONSTITUTION:Total DNA is extracted from a bacterium such as Streptomyces viridochromogenes or a variant thereof capable of producing bialaphos (BA). The DNA is cleft with restriction enzyme Kpn1 to give the aimed gene- containing DNA fragment. The restriction endonuclease cleavage map is as shown by the figure. Then the fragment as a vector is integrated into a proper virus or cyclic plasmid to give a hybrid plasmid. The plasmid having the BA- resistant marker is usable as a vector for recombinant DNA experiment using a ray fungus, a fungus capable of producing a useful substance, especially antibiotics, as a host.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Industrial Application Field The present invention relates to a bialaphos resistance gene and a hybrid plasmid incorporating the same.

Problems to be Solved by the Prior Art and the Invention Actinomycetes are the most important fermenting microorganisms that are widely used in the microbial industry as producers of useful substances such as antibiotics.

By the way, bacteria belonging to the genus Streptomyces, especially Streptomyces bigroscopicus SF 129
3 (Streptomyces hygroscop
icus 5F1293) strain (Japanese Patent Publication No. 51-639) is rsF1.
It is known as ``Substance 293'' or ``Bialaphos,'' and is used as a fungicide for agricultural and horticultural purposes (Japanese Patent Application Laid-Open No. 48-820
No. 28) and as a herbicide (Japanese Patent Publication No. 59-232)
No. 82) is useful. In particular, this compound is expected to find wide use as a herbicide due to its low residual properties. Bialajos also
Helvetica C
hifflica Acta) Vol, 55°Pa5
c, phosphinothricyl-alenyl-alanine described in 1.224-239.1972.
It is the same substance as alanine). Streptomyces viridochromogenes (7M494 (
Deutsche in West Germany as DSM40736
Sama+lung van Mikro-.

It has been deposited with the organization, and this is JCM
4977 (details described later)) produces bialaphos, and naturally this bacterium has self-resistance to bialaphos.

If a DNA fragment containing this bialaphos-resistant gene is isolated, extremely valuable results can be expected for the breeding of actinomycetes, the creation of bialaphos-resistant plants, and genetic analysis.

[Summary of the invention]

Means for Solving the Problems The present inventors developed a DNA containing the bialaphos resistance gene from Streptomyces viridochromogenes chromosomal DNA using a system using Streptomyces as a host and a plasmid derived from Streptomyces viridochromogenes.
We successfully isolated each of the A fragments and constructed a plasmid with a genetic indicator of bialaphos resistance.

That is, firstly, the bialaphos resistance gene according to the present invention has the following characteristics.

(b) This gene is present in Streptomyces and Pyridochromogenes (Streptomyces virldo).
chloro-Ilogenes) DNA with restriction enzyme K.
It is contained in the DNA corresponding to the 9.5 kilobase fragment obtained by cutting with pnI.

(b) The restriction enzyme cleavage map of this gene is shown in Figure 1.

(c) This gene encodes an enzyme that acetylates demethylphosphinothricin and phosphinothricin.

Second, the 9.5 kilobase DNA fragment having the bialaphos resistance gene according to the present invention is obtained by cleaving the DNA of Streptomyces viridochromogenes or its mutant strain with the restriction enzyme KpnI. , is.

Furthermore, thirdly, the hybrid plasmid according to the present invention is one in which a bialaphos resistance gene having the following characteristics is integrated into a vector plasmid.

(b) This gene is derived from Streptomyces viridochromogenes (Streptomyces vlrldo).
chromogenes) DNA with restriction enzyme Kp.
It is contained in the DNA corresponding to the 9.5 kilobase fragment obtained by cutting with nI.

(b) The restriction enzyme cleavage map of this gene is shown in Figure 1.

(c) This gene encodes an enzyme that acetylates demethylphosphinothricin and phosphinodolysine.

Effects of the Invention The plasmid having the bialaphos resistance marker according to the present invention can be particularly used as a vector for recombinant DNA experiments using actinomycetes, which are producing bacteria of useful substances such as antibiotics, as a host. Extremely valuable results can be expected by conducting breeding or genetic analysis of actinomycetes through recombinant DNA experiments using such an Ink streptomycete host-vector system. Furthermore, it can be used not only to improve the productivity of bialaphos-producing bacteria, but also to breed bialaphos-resistant plants.

[Detailed description of the invention]

Definition of Genes, etc. The present invention relates to a bialaphos resistance gene, a DNA fragment containing the gene, and a hybrid plasmid that is a specific form of this DNA fragment.

The bialaphos resistance gene itself or as a DNA fragment containing it has an important meaning in the present invention.

The bialaphos (hereinafter referred to as BA) resistance gene according to the present invention is produced by cutting Streptomyces dochromogenes DNA with the restriction enzyme Kpn I.
DNA corresponding to the 9.5 kilobase fragment obtained by
The restriction enzyme cleavage map is as shown in FIG.

Here, "DNAJ, which corresponds to a 9.5 kilobase fragment obtained by cleaving S. pyridochromogenes DNA with the restriction enzyme Kpn I," means that in addition to this K pn I cut fragment itself, there are It also refers to DNA with the same base sequence, degenerate isomers thereof, and genetically equivalent variants thereof.

Specific examples of the latter DNA are KpnI-cleaved fragments of mutant strains of S. pyridochromogenes and synthetic DNA. Here, "degenerate isomers" refer to isomers that differ in the base sequences that have a degenerate relationship, and "genetically equivalent variants" refer to isomers that differ in base sequences even if bases are substituted, added, or deleted. It refers to DNA with virtually no difference in genetic information. Therefore, the bialaphos resistance gene according to the present invention to be included in such NA also has
have similar tolerances.

A preferred specific example of the BA resistance gene according to the present invention is S
．． 9.5 kilobases (
It is included in the fragment of k b ). As mentioned above, the present invention also relates to this 9.5 kb fragment.

This gene has the ability to encode an enzyme that acetylates demethylphosphinothricin, a biosynthetic intermediate of bialaphos, and phosphinothricin, a degradation product of bialaphos. This ability was demonstrated by transforming host Streptomyces with a Streptomyces hybrid plasmid containing this gene.
This is confirmed by obtaining an A-resistant strain and examining the ability of this recombinant to acetylate demethylphosphinothricin and phosphinothricin (details will be described later).

Gene Production The BA resistance gene according to the present invention may be artificially synthesized, but is usually obtained from S. pyridochromogenes or its mutants.

(]) The BA-producing bacterium used as a live-producing bacterium NA donor is Streptomyces viridochromogenes or a mutant strain thereof.

As S. pyridochromogenes, S. There is Pyridochromogenes JCM4977. This bacterium has been deposited as JCM4977 at the RIKEN Microbial System Storage Facility and is available for sale. This bacterium is a known S. As mentioned above, it is the same as Pyridochromogenes Tu494 (DSM40736).

S. In view of the nature of the present invention, the mutant strain of Pyridochromogenes should have a DNA that provides the restriction enzyme cleavage map shown in FIG.

(2) Gene cloning Total DNA was extracted from the cells of the selected BA-producing bacteria using the 5DS-phenol method (M, G, Sm1th et al.
thods 1n Enzymology 12.54
5 (1967)). By cleaving the extracted DNA with the restriction enzyme KpnI, a DNA fragment containing the desired gene can be obtained together with various DNA fragments.

In order to extract the desired gene-containing fragment from the DNA fragment mixture obtained in this manner and to obtain it in large quantities, it is necessary to integrate it into an appropriate viral or circular plasmid as a vector and to transform a host bacterium with the generated hybrid plasmid. Genetic engineering techniques can be utilized, including transduction, transfection, or transfection.

Regarding general genetic recombination techniques, for example, you can refer to "Gene Manipulation Experimental Methods" (Kodansha), edited by Yasutaka Takagi.

An example of such a recombinant technique is as follows. That is, when the above donor DNA is cleaved with Kpn I, the vector plasmid is allowed to coexist and this vector plasmid is also cleaved at the Kpn1 site at the same time, or this vector plasmid is separately cleaved with Kpn I and then the above The vector plasmid is mixed with the KpnI cut of the donor DNA and treated with a suitable gauze, e.g. T4 ligase.
A plasmid mixture containing a chimeric plasmid incorporating the M4977 DNA fragment is obtained. As the vector plasmid used in this case, one that has a KpnI cleavage site and provides a propagable fragment when cut with KpnI is generally suitable. Its origin is actinomycetes,
It can be E. coli, Bacillus subtilis, or other purpose-oriented microorganisms, but a specific example is pIJ70, which is derived from actinomycetes.
2 (Available from John Innes Institute. Literature: J, General lllIfcrob4o1o
gy, 129.2703-2714 (1983))
and p I J 680 (available from John Innes Laboratory. Reference: GeneLlc Mani
Strepto-myces
, a 1 laboratory LII annual.

(1985)), and others. To obtain a target plasmid from a host bacterium, the techniques described in the above-mentioned documents and other techniques commonly used for genetic recombination may be followed.

Next, host bacteria are transformed with the obtained plasmid mixture. Depending on the type of vector used, suitable host bacteria may be selected from among actinomycetes, Escherichia coli, Bacillus subtilis, and other microorganisms, particularly those with high transformation efficiency and high sensitivity to BA. When the vector is an actinomycete plasmid, an actinobacterium is used as the host bacterium, and specific examples in this case include S. Lividans 66 (Feikoken Bacteria No. 6982).

In order to introduce a vector incorporating a DNA fragment of a BA-producing bacterium into a host bacterium, the most efficient method is selected depending on the host bacterium and the type of vector. The most common method is to transform bacterial protoplasts.

The selection of recombinants obtained by transformation involves genetic indicators possessed by the vector used, such as antibiotic resistance, pock formation (M, J, Bibb et al.
: Mofec, Gen, Ger+et, 154.
155 (1977), Nature274, 39g
(197g)) etc. can be used.

In order to select those containing the BA resistance gene from the recombinants thus obtained, it is most efficient to select BA-resistant strains produced by expression of BA resistance. To do this, first, the minimum inhibitory concentration of BA against the host bacteria is determined, and clones that grow on a selective medium containing BA at or above this concentration are selected.

The obtained BA-resistant clones were cultured and cultured using known methods (Hansen et al.: J, Bacte.
riol, 135°227, (1978)'] and inserted D by restriction enzyme treatment.
The NA fragment can be removed and a DNA fragment containing the BA resistance gene can be isolated by agarose gel electrophoresis. In addition, confirmation that it is a clone of the BA resistance gene may be performed by reintroducing the isolated plasmid into the host bacterium and examining the expression of BA resistance.

By further cutting the obtained DNA fragments with selected restriction enzymes and analyzing the size of each fragment by performing agarose gel pneumophoresis, it is possible to create a restriction enzyme cleavage map. Incorporate the fragment into an appropriate vector,
By recloning, the structure of the BAA gene can be analyzed and its characteristics can be clarified. As mentioned above, the restriction enzyme cleavage map of the DNA containing the BAA gene of the present invention has the fold shown in FIG.

Hybrid Plasmid The hybrid plasmid according to the present invention contains the BAA gene described in 1-1 above.

Specific examples of the hybrid plasmid of the present invention in which the vector Blasmii into which the BA exogenous gene is to be integrated are actinomycete plasmids are as already described above in connection with cloning.

One specific example of a hybrid plasmid according to the invention is pMS853-1. pMS 853-co is a B.
9.5kb DNA 1lf containing AA gene
Actinomycete plasmid (14,
7kb) (Figure 2). This plasmid has been deposited as a bacterium transformed with it at the Microtech Institute (details will be described later).

When these BAA plasmids are transformed into corresponding host bacteria, they can cause the host bacteria to express BA resistance. For example, when Streptomyces lividans 66 is transformed with pMS85B-1 (Fig. 2), in which a DNA fragment containing the BAA gene of the present invention is integrated into Streptomyces plasmid p I J 680, 1000 μg of Streptomyces lividans 66 is transformed.
A recombinant showing BA resistance of 1/ml was obtained.

Example 1 Streptomyces viridochromogenes JCM497
7 in S-1 medium (2% soluble starch, 1.0% peptone, 0.3% meat extract, 0.05% K
2 HP O4, pH 7,0)] inoculated into Om1, 28
After culturing at ℃ for 24-48 hours, this was used as a miscellaneous bacteria, and Myc
The seeds were planted at a 5% inoculum level. MYG medium is 1% malt extract, 0.4% yeast extract, 2.0% glucose, pH 7.0 (80 ml) with 2% glycine added. This was incubated with shaking at 28°C for 24 hours.
．． 1. Bacteria were collected by centrifugation at 10,000×g/10 minutes. From this bacterial cell, the known method of Smith et al.
s in Enzymology 12.545. (
15, TESL buffer (10mM Tris-HCI pH8,
It was dialyzed against 0.1mM EDTA, 2.5mM NaCl) to obtain donor DNA.

0 μg of the donor DNA obtained in this way was added to a total of ff12
00μg20mM Tris-HCI pH7,4
, 10mM MgCl2. Restriction enzyme K p n in a reaction solution with a composition of 10 mM β-mercaptoethanol.
10 units of I were added and reacted at 37°C for 2 hours.

Plasmid pIJ680 DNA 2μg, total amount 40
μg of 20mM Tris-HCI pH 7.4,
Restriction enzyme KpnI in a reaction solution with a composition of 10mM M g Cl 2.10mM β-mercaptoethanol
Two units were added and reacted at 37°C for 2 hours.

This was heated at 70°C for 10 minutes to inactivate the enzyme, and then 1/20 amount of IM Tris-HCI pH9
, 0, calf, intestin, alkaline,
10 units of phosphatase (c, i, a, p,) were added and the reaction was carried out at 37°C for 60 minutes.

TESH buffer (0.2M Tris-HCI) was added to each of the donor DNA and plasmid DNA reaction solutions.
pH8, 0, 20mM EDTA, 50mM NaCl
) was added in equal volumes, stirred for 1 minute, and both solutions were combined. This is 10010000
After centrifugation for X minutes, the second (aqueous) layer was removed with a Pasteur pipette.

This aqueous layer containing DNA was subjected to ethyl ether extraction to remove phenol, then an equal amount of ethanol was added, and after being left at -80°C for 2 hours, the DNA was precipitated by centrifugation at 10010000X minutes.

The DNA thus obtained was washed with 500 μg of ethanol, dried under reduced pressure, and then dissolved in 50 Mg of heat-sterilized pure water. This DNA was heat-treated at 70°C for 10 minutes, then slowly cooled to room temperature, and buffer (0.66M
Tris-HCI pH7,6,66mM
M g CI 2.0.1M dithiothreitol, 10
5 μg of mM ATP) and 5 μN of T4 ligase (1
unit) and reacted at 22°C for 4 hours to obtain p I
Recombinant DNA of J680 and Streptomyces viridochromogenes DNA was prepared.

The host bacterium Streptomyces lividans 66 (LolIlovskaya et at:
Genetlcs.

88, 341. (1971)) was transformed using the known method of Chater et al. (Curr, Topics Mic
robfol, rmmar+o1. .

96, 69 (1981)). That is, 80 ml of YEME medium (34% sucrose, 0.3%
Yeast extract, 0. 5% bactopeptone,
0.3% malt extract, 1% glucose, 5m
Streptomyces φlividans 66 was inoculated into MgC (1 MgC, 0.5% glycine, pH 7,0) at 32°
After culturing with shaking for 36 hours at C, mycelia were collected by centrifugation at 110,000X and washed once with 10% sucrose solution, followed by 10ml of P medium (0.3M sucrose, 1.4mM KSO, 10mM gCI2). .0.4mM2
4ゝKHPO25mM CaCl2.25mM2 4ゝTBS buffer, pH 7.2, 11500 (trace element solution). Next, egg white and zozyme were added to the final concentration of 1 mg/ml, and the mixture was kept at 32° C. for 60 minutes to form protoplasts. Mycelia that did not form into protoplasts were removed by filtration with cotton. 800X, /7
Protoplasts were collected by centrifugation for 1 min, washed once with P medium and then suspended in 2 ml of P medium (composition of trace element solution (in 1 liter): 40 mg Zn
1 C, 200mg FeC1・6H0, 10mg
CuCl2・2H20, 10mg Mn C12・
4 H20, 10mg Na2B407e 10H20
-10 mg (NH4)6Mo7024.4H2o). Protoplast solution 100 μN, P- prepared at 3/24 degrees
Maleate buffer n (2.5% sucrose, 1.4mM K2S
o4.11500 amount trace element solution, 100mMcac1
2.50mM Tris-maleic acid, pH 8.0)
100μg, recombinant DNA solution 50μg and 375
Mix µg of polyethylene glycol solution (polyethylene glycol #1000 33%/P-maleic acid buffer), let stand at room temperature for 1 minute, dilute with 5 ml of P medium,
Protoplasts were collected by centrifugation at 00 × g/10 min and suspended in 2 ml of P medium. This protoplast solution was poured onto 20 R2YE agar plates (0, 0,
3M sucrose, 1.4mM KSO50rnM Mg C12, 1% gluco2 4 ml, 0.01% Casamino acids, 1150 trace element solution, 0.4mM KH2PO4, 20mMCa C12
, 0.3% proline, 25mM TES buffer, pH 7,
2.5mM NaOH.

0.5% yeast extract, 2.2% agar) and cultured at 31° C. for 1 day, then overlaid with sterile water containing 200 μg/ml of thiostrepton and cultured for an additional 4 days. Bacteria regenerated from protoplasts on an agar medium were grown in a minimal medium containing 100 μg/ml of bialaphos (0
, 75% soluble starch, 0.075% KH2PO
4,0,0025%Mg5O-7H,0,0,13%
N a N O3, 0,000025% CoC1・6
H20゜1/400 amount trace element solution, 1.5% agar, p
H7,0) using velvet cloth and cultured for 2 days at 31°C. As a result, a total of 16 bialaphos-resistant clones were obtained, and each of these colonies on the selective medium was scraped off with Aze and mixed with 0.1-0.2 ml of sterile water using a glass Botter homogenizer.
This was spread on R2YE medium and cultured at 31°C for 5 days.

16 strains of Bialaphos M Streptomyces lividans 66 were each inoculated into 80 ml of YEME medium, and the cultured cells were shaken for 21 minutes at 31°C using the known method by Hansen et al. (J, Baeteriol.

135, 227 (1978)). When Streptomyces lividans 60 was transformed again with these plasmids, resistance to bialaphos was expressed in all cases. 1 plasmid obtained from 16 strains
When cleaved with the restriction enzyme K pn I and examined by 0.8% agarose electrophoresis, in addition to the 5.2 kb plJ680 (however, the K pn I small fragment is missing), the 9.5 kb A common NA fragment was observed. Furthermore, these plasmids were transformed into BaIHI, Ps
When I cut it with a restriction enzyme such as tI and examined it in detail, I found that 9
．． The 5kb DNA fragment is plasmid pIJ6
The directionality built in for 80 was not constant. However, the resistance to bialaphos of Streptomyces lividans 66 transformed with two types of plasmids with different insertion directions of 1.9.5 kb DNA was 1000 μz / ml, and the resistance to bialaphos was 1000 μz / ml. There was no change.

The bialaphos resistance gene encoded by the 9.5kb DNA is shown in Figure 1 as the bialaphos resistance gene.
The restriction enzyme map of the fragment and pIJ680 into which this DNA fragment was incorporated was named pMS B 53-1, and its restriction enzyme map is shown in FIG.

In order to investigate the resistance mechanism of bialaphos resistance gene, Streptomyces lividans 66 (pMS
853-1) (Feikoken Bibori No. 9278) to 80nnl
Cultured with shaking in YEME medium at 31'C for 2 days.
, 3 g of wet bacterial cells were obtained by centrifugation. The cells were soaked in a buffer solution (50mM Tris-HCI, pH 7).
, 0.5 μPv1 β-mercaptoethanol), suspended in 7.5 ml of the same buffer, treated with an ultrasonic crusher for 3 minutes, and centrifuged at 30,000 x g for 30 minutes. The supernatant was used as the crude enzyme solution. did. The acetylase activity of this crude enzyme solution was examined using demethylphosphinothricin and phosphinothricin as substrates. That is, O,
IM! -Lis hydrochloric acid, pH 7, 5, 0, 4 mg, z'm1
5. 5'-dithiobis-2-nitrobenzoic acid, 0
．． 1mM acetyl coenzyme A1Q, lm
When the reaction was carried out at 37°C in a reaction system containing M-demethylphosphinothricin or phosphinothricin and a crude enzyme solution, acetyltransferase activity could be detected.

Deposit of related microorganisms The microorganism transformed by the plasmid incorporating the bialaphos resistance gene according to the present invention, namely Streptomyces lividans 66 (pMS853-1), was submitted to the Agency of Industrial Science and Technology Microbiology Technology as No. 9278. It has been deposited at the Research Institute (Feikoken). The mycological properties of this microorganism are the same as those of the host microorganism Streptomyces lividans 66, except for those caused by the introduced plasmid pMS853-1. The mycological properties of Streptomyces lividans 66 were published in Japanese Unexamined Patent Publication No. 5
9-175889 and 'J, orVlrolo
gy, 9, 258 (1972), and this microorganism has been deposited with the National Institute of Fine Arts and Technology as No. 6982.

Streptomyces viridochromogenes JCM4977, which is the donor of the bialaphos resistance gene according to the present invention, has been deposited at the RIKEN Microbial System Collection Facility, and
It is available for sale.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing a restriction enzyme cleavage map of the bialaphos resistance gene according to the present invention. FIG. 2 shows the plasmid according to the invention I) M S 85
FIG. 3 is an explanatory diagram showing a restriction enzyme cleavage map of No. 3-1.

Claims (1)

[Claims] 1. A bialaphos resistance gene having the following characteristics. (b) This gene is derived from Streptomyces viridochromogenes (Streptomyces viridochromogenes).
hro-mogenes) DNA with the restriction enzyme Kpn.
It is contained in the DNA corresponding to the 9.5 kilobase fragment obtained by cutting with I. (b) The restriction enzyme cleavage map of this gene is shown in Figure 1. (c) This gene encodes an enzyme that acetylates demethylphosphinothricin and phosphinothricin. 2. The bialaphos resistance gene according to claim 1, wherein the DNA containing the gene is derived from Streptomyces viridochromogenes or a mutant strain thereof. 3. A 9.5 kilobase DNA fragment containing a bialaphos resistance gene obtained by cleaving the DNA of Streptomyces viridochromogenes or its mutant strain with the restriction enzyme Kpn I. 4. A hybrid plasmid obtained by incorporating a bialaphos resistance gene having the following characteristics into a vector plasmid. (b) This gene is derived from Streptomyces viridochromogenes (Streptomyces viridochromogenes).
romo-genes) using the restriction enzyme Kpn I
It is included in the DNA corresponding to the 9.5 kilobase fragment obtained by cutting with. (b) The restriction enzyme cleavage map of this gene is shown in Figure 1. (c) This gene encodes an enzyme that acetylates demethylphosphinothricin and phosphinothricin. 5. The hybrid plasmid according to claim 4, wherein the vector plasmid is an actinomycete plasmid. 6. A patent claim in which the bialaphos resistance gene is incorporated in the form of a 9.5 kilobase DNA fragment obtained by cleaving the DNA of Streptomyces viridochromogenes or its mutant strain with the restriction enzyme Kpn I. The hybrid plasmid according to any one of items 4 to 5.