JPH09173070A - Plasmid, microorganism containing plasmid transduced thereinto, their production and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a new plasmid, replicable in at least one microorganism, having no selective marker gene expressible in the microorganism and capable of providing a transformed microorganism capable of improving the production efficiency when producing substances with the microorganism.

SOLUTION: This plasmid is a new one, replicable in at least one microorganism and having no selective marker gene expressible in the microorganism and is capable of reducing the energy load of a host cell and improving the production efficiency in producing substances with the microorganism as compared with those of a plasmid containing the selective marker. Since the medicinal resistance is not imparted to the host cell, the product is converted into a more preferred form when the product is a food, a medicine, etc. The plasmid is obtained by inactivating or deleting a selective marker gene on a plasmid used as a vector, preparing a recombinant plasmid, transducing the resultant recombinant plasmid together with the selective marker gene into a host cell and then making only the latter fall off.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plasmid having no selectable marker gene, a microorganism into which the plasmid has been introduced, a method for producing them, and a method for producing a useful substance using the plasmid-introduced microorganism.

[0002]

2. Description of the Related Art Recently, various microorganisms have been used as hosts.
The development of vector systems has made it possible to mass-produce useful substances by incorporating genetic engineering techniques. For example, in the system of Bacillus subtilis and a plasmid vector for Bacillus subtilis, a recombinant plasmid in which the interferon-α2 gene is inserted into a plasmid vector constructed for protein secretion is introduced into Bacillus subtilis, and by culturing the same,
It enables secretory expression of interferon-α (P
alva, I.D. et al. , Gene, 22, 229
(1983)). With the gene manipulation technology established today, it is possible to easily select the microorganism into which the plasmid used as a vector has been introduced by the active expression of the selectable marker gene. As a typical example, the expression of a drug resistance gene naturally encoded in a plasmid or artificially integrated prevents expression of a plasmid-introduced microorganism in a selective medium containing a drug, but Conditions under which only the introduced microorganism can grow can be set. In this way, by applying selective pressure to the medium, it is possible to prevent the growth of microorganisms that do not have a plasmid, and to stabilize the expression product of the gene on the plasmid or the mass production of the substance produced by the involvement of the expression product. You can

However, when the plasmid is stably retained in the host cells without depending on the selective pressure of the medium, it is not always necessary to apply selective pressure to the medium. In this case, the active expression of the selectable marker gene on the plasmid is not an essential condition, and the host cell consumes extra energy for the expression of the gene. Also,
The gene only increases the size of the plasmid without meaning, which also increases the energy burden of the host cell, and is a useful substance to be obtained in large quantities by growing the plasmid-introducing microorganism or culturing the plasmid-introducing microorganism. Can be thought to work against the productivity of. Further, when the selectable marker gene is a drug resistance gene, using a plasmid in which the gene is inactivated or a plasmid in which the gene is not present makes it possible to produce useful substances in fields such as food and pharmaceuticals. When provided, it is a more preferable embodiment.

[0004]

As described above, in the plasmid used as a vector, it is very significant to use a plasmid in which a DNA region not involved in the replication of the plasmid is inactivated or deleted. But,
On the other hand, it becomes very difficult to obtain a microorganism into which a plasmid in which the selectable marker gene is not actively expressed is introduced. This is because it is not possible to separate the microorganisms into which the plasmid has not been introduced and the introduced microorganisms on the medium by using the growth as an index. In particular, this problem is serious in host cells with low plasmid transfer efficiency. further,
When the usual plasmid introduction operation is performed on the reaction product immediately after ligation with the plasmid vector and the inserted DNA fragment, the introduction efficiency of the desired plasmid is further reduced by the amount obtained by multiplying the ligation efficiency. Therefore, when the target plasmid does not actively express the selectable marker gene, it takes a lot of labor to select the microorganism into which the plasmid has been introduced on the medium.

The use of microorganisms in mass production of useful substances is a powerful means, and a system capable of efficiently introducing a plasmid having no selectable marker gene into microorganisms will greatly contribute to the development of the microorganism application industry. . However, to date, there is no known method for efficiently obtaining such a plasmid-introduced microorganism. Therefore, such a plasmid-introduced microorganism and the plasmid itself are not known. Therefore, it is an object of the present invention to provide a plasmid for a microorganism that does not have a selection marker gene and a microorganism into which the plasmid has been introduced. Also,
It is an object of the present invention to provide an efficient production method of a useful substance using the plasmid-introduced microorganism.

[0006]

The present inventors have attempted to prepare a recombinant plasmid in which a selectable marker gene on a plasmid used as a vector has been inactivated or deleted and a DNA fragment has been inserted. After selecting two types of plasmid-introduced microorganisms in which the plasmid was introduced into a host cell together with a plasmid having a selectable marker gene, only the latter plasmid was eliminated from the two types of plasmid-introduced microorganisms, and the microorganism containing only the former recombinant plasmid was efficiently prepared. Came to develop a way to get. As a result of culturing a microorganism containing only the former recombinant plasmid, the productivity of the target substance derived from the inserted DNA fragment is improved as compared with the case of using a recombinant plasmid that does not inactivate or delete the selectable marker gene. It was confirmed. That is, the present invention provides the following.

[1] A plasmid which is replicable in at least one kind of microorganism and has no selectable marker gene expressed in the microorganism. [2] The plasmid according to [1] above, wherein the microorganism is bacteria or yeast. [3] Microorganisms include Bacillus , Escherichia , and Pseud
omonas ) or Saccharomyces ( Saccharomyc
es ) The plasmid according to [1] above, which is a genus yeast.

[4] The plasmid according to any one of the above [1] to [3], which has a gene that can be expressed in at least one kind of microorganism capable of replicating the plasmid. [5] The plasmid according to [4] above, wherein the gene is a protein gene. [6] The plasmid according to [4] above, wherein the gene is an enzyme gene. [7] The plasmid according to [4] above, wherein the gene is a hydrolase gene. [8] The plasmid according to the above [4], wherein the gene is a protease gene or a lipase gene. [9] Gene Bacillus (Bacillus) bacteria, Esusherishia (Escherichia) bacteria or Pseudomonas (P
The plasmid according to any one of [4] to [8] above, which is derived from a bacterium belonging to the genus seudomonas .

[10] A microorganism having the plasmid according to any one of [1] to [9], wherein the plasmid does not have a selectable marker gene expressed in the microorganism. Microorganisms. [11] A microorganism having two or more kinds of plasmids, wherein at least one kind of plasmid is the plasmid according to any one of the above [1] to [9], which does not have a selectable marker gene expressed in the microorganism. , The other at least 1
A plasmid-introduced microorganism, wherein the seed plasmid has a selectable marker gene expressed in the microorganism. [12] A microorganism having two or more types of plasmids, wherein at least one type of plasmid is the plasmid according to any one of the above [1] to [9], which does not have a selectable marker gene expressed in the microorganism. , The other at least 1
A plasmid-introduced microorganism, wherein the seed plasmid has a selectable marker gene expressed in the microorganism and a temperature-sensitive origin of replication for replication in the microorganism.

[13] The above-mentioned [10], wherein the microorganism is a bacterium.
~ The plasmid-introduced microorganism according to any one of [12]. [14] The plasmid-introduced microorganism according to any of [10] to [12], wherein the microorganism is yeast. [15] The plasmid-introduced microorganism according to any of [10] to [12], wherein the microorganism is a bacterium belonging to the genus Bacillus . [16] The plasmid-introduced microorganism according to any one of [10] to [12] above, wherein the microorganism is a bacterium of the genus Pseudomonas . [17] The plasmid-introduced microorganism according to any of [10] to [12] above, wherein the microorganism is a bacterium belonging to the genus Escherichia . [18] The plasmid-introduced microorganism according to any one of [10] to [12] above, wherein the microorganism is a yeast of the genus Saccharomyces .

[19] PCR using a plasmid DNA having a selectable marker gene as a template and excluding all or part of the DNA encoding the selectable marker gene
A method for producing a plasmid from which a selection marker gene has been removed, which comprises a step of amplifying DNA by the method. [20] A method for obtaining a microorganism having a plasmid (A) that does not contain a selectable marker gene, the method having the following constitution. 1) A plasmid (A) containing no selection marker gene and a plasmid (B) containing a selection marker gene are simultaneously introduced into a microorganism. 2) A plurality of microorganisms having at least the plasmid (B) are selected using the selectable marker gene on the plasmid (B). 3) Select a microorganism having the plasmid (A) from the group of microorganisms obtained in 2). [21] A method for obtaining a microorganism having only the plasmid (A) containing no selectable marker gene as a plasmid, which has the following constitution. 1) A plasmid (A) containing no selection marker gene and a plasmid (B) containing a selection marker gene are simultaneously introduced into a microorganism. 2) A plurality of microorganisms having at least the plasmid (B) are selected using the selectable marker gene on the plasmid (B). 3) Select a microorganism having the plasmid (A) from the group of microorganisms obtained in 2). 4) The plasmid (B) is eliminated from the microorganism obtained in 3). [22] The origin of replication of the plasmid (B) for replication in a microorganism is temperature sensitive, [2]
1] A method for obtaining the microorganism as described above.

[23] A method for producing a microorganism-produced substance by culturing a plasmid-introduced microorganism and recovering the microorganism-produced substance from the culture, wherein the plasmid possessed by the microorganism comprises a selectable marker gene expressed in the microorganism. A method for producing a microbial product, which does not contain a gene involved in the production of the microbial product. [24] A method for producing a protein by culturing a plasmid-introduced microorganism and recovering the protein from the culture, wherein the plasmid contained in the microorganism does not contain a selectable marker gene expressed in the microorganism, and encodes the protein. A method for producing a protein, comprising a gene that controls the expression of the protein or a gene that regulates the expression of the protein. [25] A method for producing a plasmid, which comprises culturing the plasmid-introduced microorganism according to any of [10] to [18] and recovering a plasmid having no selectable marker gene from the culture.

Hereinafter, the present invention will be described in detail. The method for obtaining a microorganism having a plasmid (hereinafter referred to as plasmid (A)) containing no selectable marker gene of the present invention has the following constitution. 1) A plasmid (A) and a plasmid containing a selectable marker gene (hereinafter referred to as plasmid (B)) are simultaneously introduced into a microorganism. 2) A plurality of microorganisms having at least the plasmid (B) are selected using the selectable marker gene on the plasmid (B). 3) Select a microorganism having the plasmid (A) from the group of microorganisms obtained in 2). 4) Drop off the plasmid (B) if necessary.

In the present invention, the selectable marker gene means a gene that allows only the plasmid-introduced microorganism to grow by expressing the gene in a specific medium in which the non-plasmid-introduced microorganism cannot grow.
For example, the selectable marker gene includes a phenotype that imparts resistance to the host cell to a substance that inhibits the growth of the host cell, or a gene that exhibits a phenotype that complements the auxotrophy of the host cell. Antibiotics such as ampicillin, tetracycline, and kanamycin are applicable as substances that inhibit the growth of host cells. Therefore, when a plasmid (A) encodes a protease gene, a lipase gene, an amylase gene or the like that does not impart drug resistance to the host cell and complements auxotrophy, the secretory enzyme that is the expression product of them. The microorganism having the plasmid (A) can be selected by utilizing the clear zone formed by degrading the substrate around the colony on the agar medium, but it does not correspond to the selection marker gene in the present invention.

[Plasmid (A)] The plasmid (A) of the present invention is characterized by being replicable in at least one kind of microorganism and having no selectable marker gene expressed in the microorganism. Such a plasmid having a gene (excluding a selection marker gene) that can be expressed in the microorganism (hereinafter referred to as plasmid (A1))
Can be used for efficiently obtaining an expression product of the gene or a substance produced by the expression product by culturing the microorganism into which the gene has been introduced. Also,
A plasmid having no such gene (hereinafter referred to as plasmid (A2)) can be used as a material for preparing plasmid (A1). Possible situations in which the selectable marker gene is not expressed include the case where the DNA encoding the selectable marker gene is not completely contained and the case where the selectable marker gene is encoded.
May be completely expressed but may not be expressed in the microorganism.

One of the methods for producing the plasmid (A) of the present invention is improvement of existing plasmids.
The original plasmid to be improved (hereinafter referred to as plasmid (O)) may be a plasmid isolated from the natural world, an artificially modified one thereof, or an artificially prepared one. Any plasmid can be used as long as it is a plasmid that autonomously replicates independently of. Also, a plasmid in which two or more kinds of plasmids are linked can be used. However, the plasmid (O) of interest here includes a DNA region that is not involved in plasmid replication. DN like this
As the A region, a coding region such as a selectable marker gene and a spacer region between genes are applicable. The plasmid (A) of the present invention can be obtained by inactivating or deleting the selectable marker gene on the plasmid (O), but other genes are not particularly required for the growth of host cells and replication of the plasmid. It is also beneficial to inactivate or delete the substance or delete the spacer region as much as possible because the energy burden of the host cell can be reduced. The method for inactivating or deleting these DNA regions is shown below.

In the plasmid (O), a method of inactivating a gene that is not involved in plasmid replication is as follows:
Gene non-expression due to inactivation of promoter and SD sequence, inactivation of gene product due to mutation (base substitution, insertion, deletion) in the structural gene, and the like. These are general gene manipulation techniques. Can be easily achieved by using.

In the plasmid (O), a method for deleting a DNA region that is not involved in plasmid replication is as follows:
Removal of the DNA region by restriction enzyme treatment, or PCR (polymerase chain reaction) method (Saiki, RK et al., Science,
239, 487 (1988)) and amplifying a region other than the DNA region, but not limited to these methods. However, it is desirable to confirm that the DNA region to be deleted does not have a factor involved in stability of the plasmid replication system or in the host cell. An example of the method for removing the DNA region using the PCR method is as follows. As shown in FIG. 1a, two primers (each facing outward) corresponding to a portion immediately outside the DNA region to be removed on the plasmid are prepared, and PCR is performed using the plasmid DNA as a template. As a result, linear DNA in a portion other than the DNA region to be removed can be obtained.
If this is circularized as it is, a plasmid DNA in which only the portion to be removed is removed as compared with the original plasmid can be obtained. However, when another DNA fragment is inserted, the D
It is also possible to circularize the DNA fragment by ligating it with the NA fragment, or once ligating it with another plasmid DNA to form a shuttle vector, and then inserting the target DNA fragment.

Although the plasmid (A2) can be obtained by the above operation, it is necessary to further incorporate a DNA fragment in order to prepare the plasmid (A1). In addition,
As another method for preparing the plasmid (A1), DNA
DN not involved in plasmid replication after insertion of fragment
There is a method of inactivating or deleting the A region. In either method, a plurality of DNA fragments can be incorporated on the plasmid. The DNA fragment to be inserted preferably contains a gene. In addition to the structural gene, this gene preferably has a promoter, an SD sequence, a terminator, other expression control regions, etc., and is preferably constructed so that expression and, in some cases, secretion can proceed without defect. Furthermore, it is preferable that this gene has a certain phenotype and facilitates detection of a plasmid-introduced microorganism, and in particular, it is desirable to have a gene for a useful enzyme secreted and produced outside the host cell. Absent.

For example, in the case of a phenotype of secretory production of protease, the plasmid-introduced microorganism can be easily detected by the size of the clear zone formed around the colony on the medium containing skim milk or the like. Similarly, when making secretory production of lipase phenotype, detection can be easily performed by using a medium containing emulsified olive oil and the like. Even if the secretory production of enzymes such as amylase, cellulase, xylanase, and catalase is phenotype, a suitable substrate component for each enzyme is mixed in the medium, and the change due to the enzyme reaction is confirmed around the colony to detect the plasmid-introduced microorganism. Is possible. Also, the inserted DN
Even when the expression product of the gene encoded by the A fragment is involved in the production of the target substance, it is desirable that the target substance has the property of being easily detected. Included in the category of this expression product are an enzyme that produces a target substance and a substance that controls the activity expression of the target substance. It should be noted that the term “easily detectable” here means that it is simpler than the method of directly confirming the presence or absence of a plasmid by preparing DNA one by one, and it may not be effective depending on the situation. is there. For example, the plasmid (A
In the case of introducing only 1) into a microorganism, there is no method for growing only the plasmid-introduced microorganism, and therefore it has to be detected from a very large number of colonies, which is by no means easy. A method for solving this problem is to obtain a microorganism having a plasmid (A) containing no selectable marker gene of the present invention.

In addition, it is desirable that the plasmid (A) is not integrated with the chromosomal DNA of the host cell and can stably exist in the host cell. Also,
When the plasmid (A) is produced through a ligation operation, it is necessary to obtain a plasmid-introduced microorganism by the method described below in order to separate and obtain these plasmids.

[Host cell] The host cell is a microorganism into which the plasmid (A) can be introduced, and if the plasmid to be introduced is replicable in the host cell, it may be a eukaryote, a prokaryote or the like. Any microorganism can be used regardless of species. In addition, as a combination of the host cell and the plasmid (A), it is desirable that the plasmid (A) is uniformly distributed to the daughter cells in the division process of the host cell, and the plasmid (A) is unlikely to drop out.

Specific examples of the microorganism that can be used as a host cell include Bacillus bacteria, Escherichia bacteria, and Pseudomonas ( Ps).
bacteria such as eudomonas ) and Saccharomyces ( Saccha
romyces ) and other yeasts, preferably Bacillus subtilis , Bacillus subtilis
Bacillus amyloliquefacie
ns ), Bacillus licheni
formis), Bacillus Famasu (Bacillusfirmus),
Bacillus lentus , Bacillus
Bacillus alkalophilus , E. coli, or Saccharomyce
s cerevisiae ).

[Plasmid (B)] The plasmid that can be used as the plasmid (B) is replicable in the host cell, has a selectable marker gene that is expressed in the host cell, and is in the host cell. It is sufficient that the plasmid (A) and the plasmid (B) are compatible. Further, from the necessity of the operation of dropping only the plasmid (B) in the present invention, it is preferable that the plasmid (B) has a temperature-sensitive replication origin.

[Acquisition of Plasmid-Introduced Microorganisms] 1) In order to obtain a microorganism having a plasmid (A) containing no selectable marker gene of the present invention, first, the plasmid (A) and the plasmid (B) are simultaneously introduced into the microorganism. However, when the ligation reaction product is directly used as the plasmid (A), the ligation reaction product should be used in a large amount as much as possible.
This results in the increased frequency of introduction into the host cell. As a method for introducing a plasmid into a host cell, any method can be used as long as plasmid introduction can be achieved, and a conventional method can also be used. For example, to introduce a plasmid into E. coli, the competent cell method (Hana
han, D.C. , J. et al. Mol. Biol. , 166,55
7 (1983)), electroporation method (Dow
er, W.C. J. et al. , Nucleic Aci
ds Res. , 16, 6127 (1988)) and the like. To introduce a plasmid into a Bacillus bacterium, the protoplast method (Chang, S. and
Cohen, S .; N. , Molec. Gen. Gene
t. , 168, 111 (1979)) and the electroporation method (Brigidi, P. et al., FE).
MS Microbiol. Lett. , 67,135
(1990)) and the like can be used. To introduce a plasmid into yeast such as Saccharomyces, the spheroplast method (Burgers, M. J. and Per) is used.
cival, K .; J. , Anal. Biochem. ,
163, 391 (1987)) and the electroporation method (Becker, DM and Guarent).
e, L. , Meth. Enzymol. , 194, 18
2 (1990)) and the like can be used.

2) Next, a plasmid-introduced microorganism is selected. First, a plurality of microorganisms into which at least the plasmid (B) has been introduced are selected using the selection marker gene on the plasmid (B). That is, when the selectable marker gene is a drug resistance gene, one that grows on an agar medium containing the drug may be selected. When the selectable marker gene complements the auxotrophy, one that grows on the nutrient-deficient agar medium may be selected. By going through this step,
Microorganisms into which no plasmid has been introduced can be eliminated. When a microorganism having a low transforming ability is used as a host, most of the microorganisms have no transforming ability, and naturally, such a microorganism does not contain the plasmid (A). Unnecessary microorganisms can be removed and the detection efficiency can be greatly improved. The plurality of candidates are selected here in order to select a microorganism that also has the plasmid (A) in the later step. Therefore, the number of candidates to be selected here should be a number that includes microorganisms that also have the plasmid (A), but since the number varies depending on the types of plasmid (A) and plasmid (B), experimental conditions, etc. Can't decide Considering human labor, etc., preferably within 10,000 shares, more preferably 10 shares
It is advisable to select plasmids and set experimental conditions so that 100 or less strains can be obtained, and more preferably 100 or less strains can be obtained. In addition, it is safer to actually select a larger number.

3) There are several methods for selecting a plasmid-containing microorganism (A) from the plasmid-introduced microorganisms obtained in 2). For example, there is a method of preparing a plasmid from each strain and checking the presence or absence thereof, or a method of carrying out hybridization using an appropriate probe. Although generally used methods can be applied, in the case of hybridization, it is a necessary condition that a sequence homologous to the probe does not exist on the chromosomal DNA of the host cell. When selecting a microorganism having a plasmid (A) into which a desired DNA fragment has been inserted, a probe corresponding to the DNA fragment can be used.
When it is derived from A, it is necessary to ligate a sequence not included in the chromosomal DNA to the DNA fragment and detect it. In addition, it is desirable that the DNA fragment preferably has a certain phenotype as described above and enables detection of the plasmid (A) -introduced microorganism more easily on the medium. In particular, as a host cell, when the DNA fragment inserted into the plasmid (A) encodes a secretory enzyme, if it has the ability to secrete the secretory enzyme to the outside of the cell, the plasmid (A) -introduced microorganism on the medium is used. Is easy to detect, which is desirable.

4) Next, if necessary, only the plasmid (B) is removed from the microorganism into which the two types of plasmids have been introduced. For example, if the temperature sensitive plasmid is such that the plasmid (B) cannot be replicated when the plasmid (B) -introduced microorganism is cultured at a high temperature of a certain temperature or higher, 2
By selectively culturing the microorganism into which the seed plasmid has been introduced at a high temperature, selective loss of the plasmid (B) can be efficiently achieved. Even when the loss of the plasmid cannot be induced by changing the culture conditions, it is possible to remove only the plasmid (B) by subculturing the microorganism into which the two plasmids have been introduced. However, the method of removing the plasmid (B) from the host cell is not necessarily limited to these methods. By the above operation, the target plasmid-introduced microorganism can be obtained.

[Plasmid-introduced microorganism] The plasmid-introduced microorganism of the present invention is characterized by having the plasmid (A), but may have other plasmids. For example, as described above, a plurality of plasmids are introduced once in order to obtain a microorganism having the plasmid (A). However, two or more types of plasmids are introduced,
It is preferably within 3 types, more preferably 2 types.
In this case, it is necessary that one kind is a plasmid (A) and at least one kind remaining has a selectable marker gene. If it is not necessary to remove a plasmid other than the plasmid (A), or if it is difficult to remove it, it may be left. Moreover, when considering the production of two or more kinds of substances,
It may have more than one type of plasmid (A).

[Use of plasmid-introduced microorganism] A plasmid (A) -introduced microorganism is cultivated, and D on the plasmid (A) is cultivated.
A target substance derived from NA or a target substance generated by the involvement of a substance derived from DNA can be obtained. The medium and culture conditions used to obtain the target substance are not particularly limited, and any medium and culture conditions can be used as long as they can grow the plasmid (A) -introduced microorganism. As a microbial production substance that is the target substance, even if it had the ability to produce microorganisms originally,
It may be one that can be produced as a result of artificially imparting productivity. Examples of microorganism-derived substances include proteins such as enzymes such as protease, amylase, lipase, cellulase, xylanase, catalase and oxidase, sugars, amino acids and lipids. In addition, proteins that are not derived from microorganisms include proteins represented by hormones such as human growth hormone. In addition, by culturing a plasmid (A) -introduced microorganism and preparing DNA therefrom, the plasmid (A)
Can be obtained in large quantities. The obtained plasmid (A)
Can be used for introduction into other microorganisms, or can be effectively used as a material for producing a new plasmid.

[0031]

BEST MODE FOR CARRYING OUT THE INVENTION In one embodiment of the present invention, a bacterium belonging to the genus Bacillus is used as a host, a plasmid gene is a plasmid pUB110 in which a kanamycin resistance gene is deleted and a protease gene is inserted, and a plasmid (B) is pE194. There is one using. That is, the DNA encoding the kanamycin resistance gene (= neomycin resistance gene) of the plasmid pUB110.
DNA with deleted part and coding for protease gene
Is inserted to produce plasmid (A). Next, pE as the obtained plasmid (A) and plasmid (B)
194 is simultaneously introduced into Bacillus bacteria and then grown on agar plates containing erythromycin and skim milk. The strains growing here are at least pE194
Having. Furthermore, among these strains, those forming a large clear zone also have the plasmid (A). By culturing the strain having the plasmid (A) and the plasmid pE194 obtained by the above operation at high temperature, pE194 having a temperature-sensitive replication origin can be eliminated, and a microorganism having only the plasmid (A) can be obtained. The plasmid-introduced microorganism thus obtained can be cultured to perform protease production, or the plasmid (A) can be produced in large quantities.

[0032]

EXAMPLES Specific examples of the present invention are shown below. However, these are merely examples, and the present invention is not limited to only these. [Example 1] The selection marker gene was deleted and the protease gene was cloned.
Preparation of a plasmid into which a DNA fragment to be inserted is inserted. First, a pair of primers (SEQ ID NOs: 1 and 2) having a restriction enzyme recognition sequence added to the 5'end side and plasmid vector pUB110 (McKenzie, T. et al.
et al. , Plasmid, 15, 93 (198
6)), the region excluding the kanamycin resistance gene (Km ^r ) on the pUB110 vector was amplified by the PCR method (FIG. 1a). Here, in the PCR method, Perki
n Elmer Cetus reagent kit and DNA
Using a Thermal Cycler machine, 94 ℃,
1.5 minutes (heat denaturation of double-stranded DNA) → 55 ° C., 3 minutes (annealing of primer and template DNA) → 72 ° C., 3 minutes (primer extension reaction by Taq DNA polymerase) was repeated 30 cycles (hereinafter, The PCR method used was performed under the same conditions).

The amplified DNA fragment was digested with the restriction enzyme Sal.
After digestion with I, the plasmid vector pPD119 (plasmid vector in which the restriction enzyme PstI recognition site in the multiple cloning site of pUC119 (Takara Shuzo Co., Ltd.) was also digested with the restriction enzyme SalI and further treated with alkaline phosphatase) Mixed with T4D
NA ligase treatment was performed. Using this reaction solution, Escherichia coli JM101 strain was transformed by the competent cell method, and 20 ppm of ampicillin (Amp),
A transformant that became a white colony on L agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, 2% agar) containing 0.1 mM IPTG and 40 ppm X-gal. Was selected. Extraction of plasmid DNA from these transformants by a conventional method,
The result of 0.8% agarose gel electrophoresis after purification and digestion with the restriction enzyme SalI confirmed that the target shuttle vector pSV1 was constructed (FIG. 1b).
(A similar procedure was used for the subsequent genetic manipulation). In addition,
In FIGS. 1b and c, Amp ^r represents an ampicillin resistance gene.

In addition, the shuttle vector contains Bacillus NKS-21 strain (accession number FERM BP-93-
The protease gene derived from 1) was inserted into the restriction enzyme PstI recognition site. The protease gene is Cl obtained by the method described in JP-A-1-141596.
The aI digested fragment was treated with exonuclease to have an appropriate length, and a DNA was used in which a linker was ligated so that it could be ligated to a PstI digested end. In addition, an equivalent DNA was prepared using the chromosomal DNA of Bacillus NKS-21 strain as a template.
PC using the DNAs shown in SEQ ID NOs: 3 and 4 as primers
It can also be obtained by performing R. From the results of 0.8% agarose gel electrophoresis after digestion with the restriction enzymes PstI or SacI, the protease gene was identified as pUB110.
Plasmid pSV2 integrated in the same direction as ORFβ of
Was confirmed to have been constructed (Fig. 1c). Next, 20 μg of the plasmid into which this protease gene had been inserted was treated with a restriction enzyme SalI, the region other than pPD119 was recovered by 0.8% agarose gel electrophoresis, and then circularized by treatment with T4 DNA ligase (pKDPI1 was obtained (FIG. 1c). ).

Example 2 Bacillus NKS-21 with pKDPI1 and pE194
PE194 with final ligation reaction products and erythromycin resistance gene was obtained by cotransformation Example 1 (Hor
inouchi, S .; and Weisblum,
B. , J. et al. Bacteriol. , 150, 804 (1
982)) Bacillus NKS-21 strain was transformed by the following method using 500 ng of the mixture.

The Bacillus NKS-21 strain was mixed with L liquid medium (1
% Polypeptone, 0.5% yeast extract,
PH 9.5 with 0.5% sodium chloride and sodium carbonate
The culture was performed overnight at 37 ° C. with shaking. This culture broth was treated with SPI medium (0.2% ammonium sulfate, 1.4% dipotassium hydrogen phosphate, 0.6% potassium dihydrogen phosphate,
0.1% sodium citrate dihydrate, 0.02% magnesium sulfate heptahydrate, 0.5% glucose, 0.02
% Casamino acid, 0.1% yeast extract, 50
ppm L-leucine, 50 ppm L-methionine)
Inoculate 1% by volume, and 37
Shaking culture was performed at ℃. Glycerin was added to this culture solution to a final concentration of 12.5%, and SPII medium (0.2% ammonium sulfate, 1.4% dipotassium hydrogen phosphate, 0.6% potassium dihydrogen phosphate, 0.1% sodium citrate dihydrate, 0.02% magnesium sulfate heptahydrate, 0.5% glucose, 0.02% casamino acid, 0.1% yeast extract, 5 ppm L-
After being diluted 7.5 times with leucine and 5 ppm L-methionine), the mixture was shake-cultured at 37 ° C. for 90 minutes to prepare a competent cell suspension. To 1 ml of this competent cell suspension, add the above-mentioned plasmid mixture and
After shaking for 2 minutes, 2 ml of L liquid medium (pH 9.5)
Was added, and the mixture was further shaken at 30 ° C. for 60 minutes. This culture solution was applied to 50 L-agar medium (adjusted to pH 9.5 with sodium carbonate) containing 10 ppm erythromycin and 2% skim milk, and after culturing at 30 ° C. for 30 hours, it showed erythromycin resistance and a large clear zone. A colony that formed p., That is, a cotransformant with pKDPI1 and pE194 was selected (Table 1).

[0037]

[Table 1]

[Example 3] pE194 from the cotransformant obtained in Example 2
Selective shedding The cotransformant prepared in Example 2 was cultured in L liquid medium at 50 ° C. for 20 hours. The culture solution was appropriately diluted with physiological saline, spread on L agar medium (pH 9.5) containing 2% skim milk, and cultured at 37 ° C. for 20 hours. 100 colonies forming a larger clear zone were selected from the colonies formed on the L agar medium, and only L agar medium (pH 9.5) containing 2% skim milk and 10 ppm erythromycin and 2% skim milk were selected. Were transferred to L agar medium (pH 9.5) containing Furthermore, after culturing these L agar media at 37 ° C. for 20 hours, they cannot grow on the former L agar media,
In the latter L agar medium, 98 strains forming a large clear zone were obtained. Next, work was performed to confirm that only pE194 was selectively shed from these strains. 10 strains randomly selected from the 98 strains were prepared from the inside of the cells by a conventional method, treated with the restriction enzyme PstI, and then subjected to 0.8% agarose gel electrophoresis. As a result, pKD for all 10 strains
Only PI1 could be confirmed. Further, Southern blotting was performed on a nitrocellulose filter using this agarose gel after electrophoresis, and hybridization was also performed using pE194 as a probe, but no DNA band hybridizing with the probe was detected. From this, it was concluded that pE194 was completely shed from the cells.

[Example 4] Inactivation of a selectable marker gene to give a protease gene
Construction of a plasmid into which a DNA fragment coding for is inserted. First, a pair of primers (SEQ ID NOs: 1 and 5) having a restriction enzyme recognition sequence added to the 5'end and pU as a template DNA.
Using B110, the entire region of pUB110 was amplified by the PCR method. The amplified DNA fragment is treated with the restriction enzyme SalI.
It was inserted into pPD119 at the recognition site. Furthermore, the Bacillus NKS-21 used in Example 1 was added to this shuttle vector.
The strain-derived protease gene was inserted at the restriction enzyme PstI recognition site so that it was integrated in the same direction as the Pre gene of pUB110. Inactivation of the kanamycin resistance gene was performed on this plasmid. Here, the site-specific mutation was introduced after the start codon of the gene to make the stop codon appear to inactivate the gene. The site-specific mutation was performed according to the Kunkel method as follows.

E. coli BW313 strain (H
frKL16PO / 45 [lysA (61-62)],
dut1, nug1, thy-1, relA1) according to a conventional method, and Escherichia coli BW31 having the above plasmid.
3 strains were obtained. Furthermore, from this E. coli,
Et al. (Vieira, J. and Messin).
g, J. , Method in Enzymolog
y, 153, 3 (1987)), a single-stranded DNA was prepared, and a single-stranded DNA in which a part of deoxythiamine (dT) in the DNA was replaced with deoxyuracil (dU) was obtained. In addition, the oligonucleotide shown in SEQ ID NO: 6 was chemically synthesized, and its 5 ′ end was phosphorylated with T4 polynucleotide kinase. Using this oligonucleotide, the above single-stranded DNA is annealed in a buffer (20 mM).
The mixture was mixed in Tris hydrochloride (pH 8.0), 10 mM magnesium chloride, 50 mM sodium chloride, 1 mM DTT), allowed to stand at 65 ° C. for 15 minutes, and further allowed to stand at 37 ° C. for 15 minutes to change the oligonucleotide. The site was annealed. Next, 2.5 times as much extension buffer (50 mM Tris hydrochloride (pH 8.0), 60 mM ammonium acetate, 5 mM magnesium chloride, 5 mM dithiothreitol, 1 mM nicotinamide adenine dinucleotide, 0.5 mM d was added to this DNA mixture.
ATP, 0.5 mM dCTP, 0.5 mM dGTP,
0.5 mM dTTP) was added, E. coli DNA ligase and T4 DNA polymerase were further added, and the mixture was allowed to stand at 25 ° C. for 2 hours to synthesize a complementary strand. Then, this DNA was introduced into Escherichia coli BMH71-18mutS strain by a conventional method, and an ampicillin-resistant transformant was selected. Single-stranded DNA is obtained from the obtained transformant according to a conventional method.
Was prepared and the nucleotide sequence was determined by the dideoxy method to obtain a plasmid into which the desired mutation was introduced. Next, 20 μg of this plasmid was treated with the restriction enzyme SalI to give 0.8%
After recovering the region other than pPD119 by agarose gel electrophoresis, it was circularized by treatment with T4 DNA ligase (pKIPI1, FIG. 2). In FIG. 2, Km ′ represents an inactivated kanamycin resistance gene.

Example 5 Bacillus NKS-21 with pKIPI1 and pE194
By the same procedure as in Example 2.
The co-transformed strain of 4 was obtained. In addition, this co-transformant is a kanamycin-containing L agar medium (pH 9.5).
However, it was confirmed that the kanamycin resistance gene was inactivated.

[Example 6] pE194 from the cotransformant obtained in Example 5
Selective shedding pE from the co-transformed strain by the same procedure as in Example 3.
194 was eliminated and a transformant consisting of pKIPI1 alone was obtained.

[Example 7] A DNA fragment encoding a protease gene was inserted.
Preparation of transformant strain using plasmid First, a plasmid in which a protease gene was inserted into a shuttle vector in which pUB110 and pPD119 were ligated was constructed in the same manner as in Example 4. Next, this plasmid 2μ
The pUC119 portion was removed from g by treatment with the restriction enzyme SalI, the remaining region was recovered by 0.8% agarose gel electrophoresis, and circularized by treatment with T4 DNA ligase (pPI1, FIG. 3). Note that Km ^r in FIG.
Same as. The thus constructed plasmid was used to transform the Bacillus NKS-21 strain. For detection of the transformant, 10 L agar medium (pH 9.5) containing 20 ppm of kanamycin was applied and cultured at 37 ° C. for 20 hours, and then grown colonies were obtained. A plasmid was prepared from the inside of the cells of the 5 colony strains by a conventional method, treated with the restriction enzyme PstI, and then subjected to 0.8% agarose gel electrophoresis. As a result, the target plasmid was confirmed in all 5 strains.

[Example 8] Cultivation and isolation of the transformants prepared in Examples 3, 6 and 7.
Measurement of accumulation activity of secretory protease Bacillus NKS-21 and the transformants obtained in Examples 3, 6 and 7 were respectively used in C liquid medium (1% casein, 1% meat extract, 1% polypeptone, sodium carbonate, pH 9.5). It was cultivated by shaking in 300 ml at 37 ° C. for 50 hours to produce protease in the medium. This culture solution was centrifuged at 4 ° C., and the supernatant was used as a crude enzyme solution. Next, the activity of protease was measured as follows. 50m
50 μl of the appropriately diluted crude enzyme solution was added to 500 μl of M borate buffer (pH 10) and preincubated at 30 ° C. for 5 minutes. To this solution, 500 μl of 2% Hammer Stencasein solution was added, and after incubation at 30 ° C. for 10 minutes, 2 ml of trichloroacetic acid solution was further added to stop the reaction. This is 10 at 30 ℃
After standing for more than one minute, filtration was performed using filter paper. 0.4 ml of sodium carbonate was added to 1 ml of this filtrate.
l, add 1 ml of 6-fold diluted phenol reagent, 30 ℃
After allowing it to stand for 20 minutes for color development, the absorbance at 660 nm was measured. Enzyme unit is defined as 30 ℃, pH1
When casein was used as a substrate at 0 and the reaction was performed in the trichloroacetic acid-soluble fraction for 1 second, 660 equivalent to 1 mol of tyrosine was obtained.
The protease activity that produces a degradation product exhibiting a color of nm was defined as 1 katal. Bacillus NKS-2
The protease activity of the supernatant of the culture broth when one strain was cultivated in a liquid C medium was defined as 100, and pKDPI1 and pKIP
Table 2 shows the results showing the relative protease activity of the supernatant of the culture broth in the case of culturing the transformant strain with I1 and pPI1.
Summarized in

[0045]

[Table 2]

From this result, it is found that the protease productivity of the transformant strain with the plasmid in which the kanamycin resistance gene is deleted is improved by about 1.3 times as compared with the case of using the plasmid in which the kanamycin resistance gene is actively expressed. all right. Further, it was found that the protease productivity of the transformant strain using the plasmid in which the kanamycin resistance gene was inactivated was improved by about 1.1 times as compared with the case of using the plasmid in which the kanamycin resistance gene was actively expressed. Further, the plasmid retention rate of the host cells was examined as follows. The transformant strain with pKDPI1 or pKIPI1 was shake-cultured in a C liquid medium at 37 ° C. for 50 hours, and this culture solution was appropriately diluted with physiological saline.
It was spread on L agar medium (pH 9.5). When 1000 colonies generated after culturing at 37 ° C. were transferred to a nitrocellulose filter and subjected to colony hybridization using pUB110 as a probe, 99% or more colonies of both transformants showed a positive signal, It has been found to hold stable.

[0047]

As described above in detail, the productivity of the target substance can be improved by culturing the microorganism into which the plasmid of the present invention which does not contain the selectable marker gene is introduced. The use of the above plasmid reduces the energy burden on the host cell as compared with the case of using a plasmid containing a selectable marker gene. Therefore,
It is particularly advantageous for production when a large amount of a target substance is obtained by culturing the above plasmid-introduced microorganism. Furthermore, when the selectable marker gene phenotypes drug resistance, a plasmid in which the gene is inactivated or a plasmid in which the gene is not present does not impart drug resistance to the plasmid-introduced cells, and thus is produced by that. It is a more preferable embodiment when the product is used for foods, pharmaceuticals and the like. In addition, the method for obtaining a microorganism having a plasmid that does not contain the selectable marker gene of the present invention can efficiently obtain the microorganism. In addition, the P of the present invention
By the method for producing a plasmid using the CR method, the plasmid not containing the selectable marker gene of the present invention can be efficiently produced.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 26 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGGAGCTCGG CCCGTTTGTT GAACTA 26

SEQ ID NO: 2 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGGAGCTCCT GCAGCTCTTG TATCTTTTTT AT 32

SEQ ID NO: 3 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGCTGCAGAT AACGGTCCAG GTATTC 26

SEQ ID NO: 4 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGCTGCAGGT ATGCGAAAGT TGAGTT 26

SEQ ID NO: 5 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGGAGCTCCT GCAGAGGTCT CTCGTATCTT TT 32

SEQ ID NO: 6 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AATAGTGAAT TGACCAATAA T

[Brief description of the drawings]

FIG. 1a: Process of construction of plasmid pKDPI1.

FIG. 1b: Process of construction of plasmid pKDPI1.

FIG. 1c: Process of construction of plasmid pKDPI1.

FIG. 2. Structure of plasmid pKIPI1.

FIG. 3: Structure of plasmid pPI1.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C12N 9/50 C12N 9/50 C12P 21/02 C12P 21/02 C C12Q 1/68 7823-4B C12Q 1/68 A // (C12N 1/19 C12R 1:85) (C12N 1/21 C12R 1:07) (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1:38) (C12N 9 / 20 C12R 1:07) (C12N 9/20 C12R 1:19) (C12N 9/20 C12R 1:38) (C12N 9/20 C12R 1:85) (C12N 9/50 C12R 1:07) (C12N 9 / 50 C12R 1:19) (C12N 9/50 C12R 1:38) (C12N 9/50 C12R 1:85) (C12P 21/02 C12R 1:07) (C12P 21/02 C12R 1:19) (C12P 21 / 02 C12R 1:38) ( 12P 21/02 C12R 1:85) (72) Inventor Shiro Fukuyama 1-1-1, Onodai, Midori-ku, Chiba-shi, Chiba Showa Denko KK Research Institute (72) Inventor Yoshiaki Miyoda Onodai, Midori-ku, Chiba-shi 1-1-1 Showa Denko KK Research Institute (72) Inventor Tadashi Yoneda 1-1-1 Onodai, Midori-ku, Chiba-shi, Chiba Showa Denko KK Research Institute

Claims (25)

[Claims] 1. A plasmid capable of replicating in at least one kind of microorganism and having no selectable marker gene expressed in the microorganism. 2. The microorganism is a bacterium or a yeast.
The described plasmid. 3. A bacterium of the genus Bacillus ,
Escherichia spp, Pseudomonas spp , or Saccharomyces ( Sa
The plasmid according to claim 1, which is a yeast of the genus ccharomyces ). 4. The plasmid according to any one of claims 1 to 3, which has a gene that can be expressed in at least one microorganism of the replicable microorganisms of the plasmid. 5. The plasmid according to claim 4, wherein the gene is a protein gene. 6. The plasmid according to claim 4, wherein the gene is an enzyme gene. 7. The plasmid according to claim 4, wherein the gene is a hydrolase gene. 8. The plasmid according to claim 4, wherein the gene is a protease gene or a lipase gene. 9. A bacterium of the genus Bacillus having a gene,
Esusherishia (Escherichia) bacteria or Pseudomonas (Pseudomonas) bacteria from a which claims 4-8
The plasmid according to any one of 1. 10. A microorganism having the plasmid according to any one of claims 1 to 9, wherein the plasmid does not have a selectable marker gene expressed in the microorganism. 11. A microorganism having two or more kinds of plasmids, wherein at least one kind of plasmid does not have a selectable marker gene expressed in the microorganism.
5. The plasmid-introduced microorganism, which is the plasmid according to any one of claims 1 to 3, wherein at least one other plasmid has a selectable marker gene that is expressed in the microorganism. 12. A microorganism having two or more kinds of plasmids, wherein at least one kind of plasmid does not have a selectable marker gene expressed in the microorganism.
5. The plasmid-introduced microorganism, characterized in that the at least one other plasmid has a selectable marker gene expressed in the microorganism and a temperature-sensitive replication origin for replication in the microorganism. . 13. The microorganism according to claim 10, which is a bacterium.
The plasmid-introduced microorganism according to any one of 1. 14. The microorganism according to claim 10, which is yeast.
The plasmid-introduced microorganism according to any one of 1. 15. The plasmid-introduced microorganism according to claim 10, wherein the microorganism is a bacterium of the genus Bacillus . 16. The microorganism is Pseudomona.
The plasmid-introduced microorganism according to any one of claims 10 to 12, which is a s ) bacterium. 17. The microorganism is Escherichi.
The plasmid-introduced microorganism according to any one of claims 10 to 12, which is a genus bacterium. 18. The microorganism is Saccharomyces.
ces ) yeast, The plasmid-introduced microorganism according to any one of claims 10 to 12. 19. A method comprising the step of amplifying a DNA by a PCR method using a plasmid DNA having a selectable marker gene as a template and removing all or part of the DNA of the portion encoding the selectable marker gene. A method for producing a plasmid from which a marker gene has been removed. 20. A method for obtaining a microorganism having a plasmid (A) containing no selectable marker gene, which comprises:
A method consisting of the following configurations. 1) A plasmid (A) containing no selection marker gene and a plasmid (B) containing a selection marker gene are simultaneously introduced into a microorganism. 2) A plurality of microorganisms having at least the plasmid (B) are selected using the selectable marker gene on the plasmid (B). 3) Select a microorganism having the plasmid (A) from the group of microorganisms obtained in 2). 21. A method for obtaining a microorganism having only a plasmid (A) containing no selectable marker gene as a plasmid, which comprises the following constitution: 1) A plasmid (A) containing no selection marker gene and a plasmid (B) containing a selection marker gene are simultaneously introduced into a microorganism. 2) A plurality of microorganisms having at least the plasmid (B) are selected using the selectable marker gene on the plasmid (B). 3) Select a microorganism having the plasmid (A) from the group of microorganisms obtained in 2). 4) The plasmid (B) is eliminated from the microorganism obtained in 3). 22. The method for obtaining a microorganism according to claim 21, wherein the origin of replication of the plasmid (B) for replication in the microorganism is temperature sensitive. 23. A method for producing a microorganism-producing substance by culturing a plasmid-introduced microorganism and recovering the microorganism-producing substance from the culture, wherein the plasmid contained in the microorganism contains a selectable marker gene expressed in the microorganism. No
A method for producing a microbial product, comprising a gene involved in the production of the microbial product. 24. A method for producing a protein by culturing a plasmid-introduced microorganism and recovering the protein from the culture, wherein the plasmid contained in the microorganism does not contain a selectable marker gene expressed in the microorganism. A method for producing a protein, which comprises a gene encoding the protein or a gene that regulates the expression of the protein. 25. A method for producing a plasmid, which comprises culturing the plasmid-introduced microorganism according to any one of claims 10 to 18 and recovering a plasmid having no selectable marker gene from the culture.