JP2014226090A - Bacterium of genus rhodococcus of which genes encoding proteins participating with cleavage of foreign dna are deleted or inactivated - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a Rhodococcus bacterium whose transformation efficiency is enhanced to efficiently carry out genetic engineering of Rhodococcus bacteria.SOLUTION: Provided is a Rhodococcus bacterium whose genes encoding proteins participating with cleavage of foreign DNA are deleted or inactivated. In some embodiments, the proteins are five nucleases of specified amino acid sequences, proteins having one or a few amino acids deleted, substituted or added in the specified amino acid sequences, or proteins of which amino acid sequences are 65% or more homology to these specified proteins.

Description

  The present invention relates to a Rhodococcus bacterium in which a gene encoding a protein involved in the cleavage of foreign DNA has been deleted or inactivated, and a transformant using the bacterium.

  Rhodococcus bacteria are known as industrially useful microbial catalysts because of their strong physical strength and the ability to accumulate a large amount of enzymes in the cells. It is used for the production of amides or acids by summation or enzymatic hydrolysis (Patent Documents 1 and 2).

  For example, Rhodococcus rhodochrous J1 strain (J1 strain) has been used for industrial production of acrylamide, and attempts have been made to improve the enzyme activity of the microbial catalyst by genetic recombination technology. (Patent Documents 3 to 5).

  In addition, in order to advance genetic manipulation of Rhodococcus bacteria efficiently, development of a host-vector system has been advanced, and search for novel plasmids (Patent Documents 6 to 8 and 16) and vector development (Patent Document 9). To 11 and Non-Patent Document 1) are also performed.

  As a method for transforming Rhodococcus bacteria, the electric pulse method (Patent Documents 12 to 14) and the protoplast method (Non-Patent Documents 2 and 3) are used, and many transformants including Rhodococcus rhodochrous ATCC12274 have been obtained so far. It has been.

  The present inventors have found that by using a plasmid prepared from a Rhodococcus bacterium that is different from the Rhodococcus bacterium to be transformed or a related bacterium, the transformation efficiency of the plasmid to the transformation subject is improved. (Patent Document 15). Further, the present inventors have found a type II restriction enzyme (RrhJ1I) having GCCGGC as a recognition sequence in J1 bacterium, and transformed by modification of a plasmid using a modification enzyme corresponding to the restriction enzyme or deletion of the gene. It has been found that the efficiency is improved (Patent Literature 17, Patent Literature 18).

JP-A-2-470 Japanese Patent Laid-Open No. 3-251192 Japanese Patent Laid-Open No. 4-211379 JP-A-6-25296 JP-A-6-303971 Japanese Patent Laid-Open No. 4-148685 JP-A-4-330287 JP-A-7-255484 JP-A-5-64589 JP-A-8-56669 US Patent No. 4920054 Japanese Patent Laid-Open No. 10-248578 JP-A-9-28380 Japanese Patent Laid-Open No. 5-68566 Japanese Patent Laying-Open No. 2005-095041 JP 2006-050967 A JP 2007-228934 A JP 2013-005792 A

Journal of Bacteriology 170, 638-645 (1988) J. et al. Basic Microbiol. 1998; 38 (2): 101-6. J. et al. Bacteriol. 1988 Feb; 170 (2): 638-45.

  However, the transformation of Rhodococcus bacteria by such a technique is complicated and difficult to carry out simply.

  Therefore, a main object of the present invention is to provide a transformation method that can achieve transformation in Rhodococcus bacteria more easily and with high efficiency.

  As a result of intensive studies to solve the above problems, the present inventors have eliminated or inactivated the activity of a protein involved in the cleavage of foreign DNA in the microbial cells, and thus highly efficient Rhodococcus bacteria. Has been found to be capable of transformation, and the present invention has been completed.

  That is, the present invention relates to Rhodococcus bacteria in which a gene encoding a protein involved in the cleavage of foreign DNA has been deleted or inactivated, and is described in more detail as follows.

The aforementioned Rhodococcus bacterium, wherein the protein having a function involved in the cleavage of foreign DNA is any one of the following proteins (a) to (o):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) a protein comprising an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 (c ) A protein comprising an amino acid sequence having 65% or more homology with the protein comprising the amino acid sequence represented by SEQ ID NO: 2.
(D) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (e) a protein comprising an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence represented by SEQ ID NO: 4 (f ) A protein comprising an amino acid sequence having 65% or more homology with the protein comprising the amino acid sequence represented by SEQ ID NO: 4.
(G) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 (h) a protein comprising the amino acid sequence represented by SEQ ID NO: 6, wherein one or several amino acids are deleted, substituted or added (i ) A protein comprising an amino acid sequence having 65% or more homology with the protein comprising the amino acid sequence represented by SEQ ID NO: 6.
(J) a protein comprising the amino acid sequence represented by SEQ ID NO: 8 (k) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 8 (l ) A protein comprising an amino acid sequence having 65% or more homology with the protein comprising the amino acid sequence represented by SEQ ID NO: 8.
(M) a protein comprising the amino acid sequence represented by SEQ ID NO: 10 (n) a protein comprising an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence represented by SEQ ID NO: 10 (o ) A protein comprising an amino acid sequence having 65% or more homology with the protein comprising the amino acid sequence represented by SEQ ID NO: 10.

  The present invention provides a Rhodococcus bacterium in which the activity of a protein involved in the cleavage of foreign DNA is inactivated, and a transformant thereof. The transformation efficiency of Rhodococcus bacteria provided by the present invention is significantly improved over conventional Rhodococcus bacteria, and thus genetic manipulation of Rhodococcus bacteria can be performed easily and efficiently. According to the present invention, new properties are imparted to Rhodococcus bacteria by the effect of the introduced DNA, and industrially useful microbial catalysts and the like can be developed.

It is a schematic diagram for demonstrating an example (the gene deletion method by conjugation transmission) of deleting the predetermined area | region from the genome of Rhodococcus genus bacteria. It is a schematic diagram which shows the structure of plasmid pK19mobSacB1. It is a figure which shows the amino acid sequence homology of RrhJ1III. It is a figure which shows the amino acid sequence homology of RrhJ1IV. It is a figure which shows the amino acid sequence homology of RrhJ1V. It is a figure which shows the amino acid sequence homology of RrhJ1VI. It is a figure which shows the amino acid sequence homology of RrhJ1VII. It is a schematic diagram which shows the structure of plasmid pK4.

(1) Rhodococcus bacteria in which a gene encoding a protein involved in the cleavage of foreign DNA is deleted or inactivated A gene encoding a protein involved in the cleavage of foreign DNA according to the present invention is deleted or inactivated The Rhodococcus bacterium (hereinafter sometimes referred to as “the Rhodococcus bacterium of the present invention”) is a protein involved in the cleavage of the foreign DNA in the Rhodococcus bacterium having the activity of a protein involved in the cleavage of the foreign DNA. Rhodococcus bacterium in which the gene encoding is deleted or inactivated.

(1-1) Rhodococcus bacterium The Rhodococcus bacterium (target microorganism) used in the present invention is a Rhodococcus bacterium having an activity (enzyme activity) of a protein involved in the cleavage of foreign DNA, and the kind thereof is particularly limited. Not done. For example, Rhodococcus rhodochrous (Rhodococcus rhodochrous), Rhodococcus erythropolis (Rhodococcus erythropolis), Rhodococcus Pirijinoboransu (Rhodococcus pyridinivorans), Rhodococcus Opakasu (Rhodococcus opacus), Rhodococcus Josuti (Rhodococcus jostii), Rhodococcus equi (Rhodococcus equi), Rhodococcus louvers (Rhodococcus ruber ), Rhodococcus imtechensis, Rhodococcus chinshengii, Rhodococcus sp (Rho) dococcus sp.) and the like. Of these, Rhodococcus rhodochrous is more preferable.

  Examples of Rhodococcus rhodochrous include ATCC 999 strain, ATCC 12674 strain, ATCC 17895 strain, ATCC 15998 strain, ATCC 33275 strain, ATCC 184, ATCC 4001 strain, ATCC 4273 strain, ATCC 4276 strain, ATCC 9356 strain, ATCC 12483 strain, ATCC 14345 strain, ATCC 14347 strain, ATCC 14347 strain, ATCC 14347 strain, ATCC 14347 strain ATCC 15998, ATCC 17041, ATCC 19149, ATCC 19150, ATCC 21197, ATCC 21243, ATCC 29670, ATCC 29672, ATCC 29675, ATCC 33258, ATCC 13808, ATCC 17043, ATCC 19067, ATCC 21999, A In addition to CC21291, ATCC21785, ATCC21924, IFO14894, IFO3338, NCIMB11215, NCIMB11216, JCM3202, NCIMB41164 (International Publication No. 05/054456), J1 (FERM BP-1478), M8 (SU173318) M33 (VKM Ac-1515D) and the like are preferable, and Rhodococcus rhodochrous J1 is more preferable.

  As the Rhodococcus erythropolis, Rhodococcus erythropolis PR4, Rhodococcus erythropolis SK121 and the like are preferable. As Rhodococcus pyridinoborans, Rhodococcus pyridinoborans AK37 and the like are preferable. As the Rhodococcus opacus, Rhodococcus opacus B4, Rhodococcus opacus M213 and the like are preferable. Rhodococcus josti is preferably Rhodococcus josti RHA1. As Rhodococcus equi, Rhodococcus equi ATCC 33737, Rhodococcus equi 103S, etc. are preferable. The Rhodococcus louver is preferably Rhodococcus louver BKS 20-38. Rhodococcus imtekensis is preferably Rhodococcus imtekensis RKJ300. Rhodococcus chinsheggy is preferably Rhodococcus chinsheggy BKS20-40. As the Rhodococcus sp, for example, Rhodococcus sp P14, Rhodococcus sp AW25M09, Rhodococcus sp JVH1, and the like are preferable.

(1-2) Gene encoding a protein involved in the cleavage of foreign DNA Examples of the protein involved in the cleavage of foreign DNA to be inactivated in the present invention include nucleases. Although the kind of nuclease is not specifically limited, For example, RrhJ1III, RrhJ1IV, RrhJ1V, RrhJ1VI, RrhJ1VII etc. can be mentioned. Each of these enzymes is a nuclease isolated from Rhodococcus rhodochrous J-1 strain, and has the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, respectively.

  Examples of proteins involved in the cleavage of foreign DNA to be inactivated in the present invention include not only the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, In the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, one or several amino acids are deleted, substituted, or added. Also included are proteins that contain sequences and have nicking enzyme activity.

  More specifically, (i) in the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, several, for example, 1 to 20 (preferably 1 to 10, (Ii) an amino acid sequence in which amino acids are deleted (preferably 1-5, more preferably 1-2), (ii) shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 An amino acid sequence in which several amino acids, for example, 1 to 20 (preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 2) of amino acids are substituted with other amino acids, (iii) ) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or several amino acid sequences shown in SEQ ID NO: 10, for example, 1-20 (preferably 1-10, more preferably 1-5) Even more preferred (Iv) several amino acids in the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, for example, 1 to 20 An amino acid sequence in which amino acids (preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 2) are inserted, (v) a combination of any of (i) to (iv) above Amino acid sequences.

  In addition, the protein involved in the cleavage of foreign DNA to be inactivated in the present invention consists of the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10. A homology of 65% or more, preferably 70% or more, more preferably 75% or more, more preferably 80% or more, still more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more with a protein. A protein having nicking enzyme activity is also included.

  These proteins are also annotated as nucleases in a plurality of strains of Rhodococcus bacteria other than the J1 bacterium, and encode proteins having high homology with the nuclease of the J1 bacterium. These are also presumed to be involved in the cleavage of foreign DNA in each strain. The alignment of the amino acid sequence of the nuclease homologue of each strain is shown in FIGS.

  The amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, respectively. It is encoded by the nucleotide sequence. Therefore, in the present invention, the gene to be deleted or inactivated includes, in addition to these genes, an ortholog of a nuclease gene possessed by a Rhodococcus bacterium to be transformed, or a DNA sequence recognized by the nuclease. A nuclease gene that recognizes the same DNA sequence is preferred.

  For Rhodococcus bacteria other than the J1 bacterium, orthologs corresponding to the RrJ1III nuclease gene, RrhJ1IV nuclease gene, RrhJ1V nuclease gene, RrhJ1VI nuclease gene, or RrhJ1VII nuclease gene of the J1 bacterium can be deleted or inactivated.

  Here, the “ortholog” is a related gene encoding a protein having a homologous function that exists in different organisms. For example, in the case of the RrhJ1III nuclease gene, the ortholog is RrJ1III in organisms other than the J1 bacterium. A related gene encoding a protein having nuclease activity.

  The orthologs of the above-mentioned RrhJ1III nuclease gene, RrhJ1IV nuclease gene, RrhJ1V nuclease gene, RrhJ1VI nuclease gene, and RrhJ1VII nuclease gene are respectively RrhJ1III nuclease gene, RrhJ1IV nuclease gene, RrhJ1V nuclease gene, RrhJ1VI nuclease gene, and RrhJ1V Have homology. Therefore, in addition to the genes having the base sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, these sequences and about 60% or more, preferably about 70% or more, and more DNA having a base sequence having a homology (identity) of preferably about 80% or more, particularly preferably about 90% or more, and even more preferably about 98% or more is also included in the nuclease gene of the present invention.

  In addition, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequences described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, respectively. As long as it encodes a protein having nuclease activity such as RrhJ1III nuclease, RrhJ1IV nuclease, RrhJ1V nuclease, RrhJ1VI nuclease or RrhJ1VII nuclease, it is included in the nuclease gene of the present invention.

  In addition, as stringent conditions, for example, a nylon membrane on which DNA is immobilized is 6 × SSC (1 × SSC is 8.76 g of sodium chloride, 4.41 g of sodium citrate dissolved in 1 liter of water), 1 Hybridization is carried out by incubation with a probe at 65 ° C. for 20 hours in a solution containing% SDS, 100 μg / mL salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone and 0.1% ficoll. Although conditions can be mentioned, it is not necessarily limited to this. A person skilled in the art sets hybridization conditions in consideration of other conditions such as probe concentration, probe length, reaction time, etc. in addition to such conditions as salt concentration and temperature of the buffer solution. be able to. Here, “%” in this specification indicates “w / v”.

(1-3) Rhodococcus bacteria in which a gene encoding a protein involved in cleavage of foreign DNA has been deleted or inactivated The Rhodococcus bacterium according to the present invention has one of the nuclease genes described above deleted. Alternatively, it may be inactivated, or a plurality of nuclease genes deleted or inactivated.

  The “deletion” means that part or all of the nuclease gene is deleted, and as a result, part or all of the nuclease activity of the protein encoded by the gene is lost. “Inactivation” means that part or all of the nuclease activity of the protein encoded by the gene is lost for reasons other than deletion of the nuclease gene (for example, modification of the regulatory region described later). means.

  Here, “nuclease activity” means an activity of cleaving DNA. The DNA to be cleaved may be a double-stranded DNA or a single-stranded DNA, and the cleaving sites include those that are cleaved sequentially from the inside or from the end. Furthermore, the nuclease activity of the present invention includes a nicking activity for cleaving one strand of double-stranded DNA.

  The method for measuring the nuclease activity of the Rhodococcus bacterium according to the present invention can be evaluated, for example, by contacting the nuclease with DNA and measuring the molecular weight or the number of DNA fragments after the contact. A person skilled in the art can set conditions such as substrate DNA, enzyme amount at the time of contact, temperature, solution composition or contact time. The molecular weight of DNA can be measured, for example, by agarose gel electrophoresis. The nuclease activity can be evaluated by comparing the molecular weight of DNA before contact with the molecular weight of DNA after contact, or the number of DNA fragments before contact and the number of DNA fragments after contact.

(2) Production of Rhodococcus bacteria in which a gene encoding a protein involved in cleavage of foreign DNA has been deleted or inactivated (2-1) Deletion of a gene encoding a protein involved in cleavage of foreign DNA or Inactivation The Rhodococcus bacterium of the present invention is one in which the originally possessed nuclease gene has been deleted or inactivated. Here, the deletion or inactivation of a gene (target gene) means that the nuclease gene encoded in the genomic DNA of the target microorganism is such that part or all of the nuclease activity of the protein encoded by the gene is lost. Deletion, substitution, addition of part or all; Deletion, substitution, addition of part or all of promoter sequence; Deletion, substitution, addition, etc. of part or all of a gene related to expression regulation. These deleted, substituted, and added regions may be used alone or in combination.

  For example, when the target Rhodococcus genus bacterium is Rhodococcus rhodochrous J1 strain, it has nuclease genes RrhJ1III, RrhJ1IV, RrhJ1V, RrhJ1VI, and RrhJ1VII, and any one of these may be used as a target gene. But you can. In the case of a plurality, the combination or number is not limited.

  A technique for specifically deleting or inactivating the nuclease gene is not particularly limited, and a known gene recombination technique can be used.

  As described above, deletion or inactivation of a nuclease gene can be achieved by deleting the target nuclease gene from the genome, inserting another DNA fragment into the target nuclease gene, or the transcription / translation initiation region of the gene. This is achieved by giving mutations to.

  The Rhodococcus bacterium according to the present invention may be one in which the target nuclease gene is intentionally deleted or inactivated, and the nuclease gene is deleted as a result of randomly mutating the gene. Alternatively, it may be inactivated.

  Examples of the method for deleting or inactivating the nuclease gene include a method using homologous recombination.

  Specifically, a drug resistance marker gene or the like is inserted into the base sequence of the target gene by homologous recombination so that the target gene does not function or (the target gene does not include part or all of the gene) It is a method of inhibiting the expression itself by deleting the target gene itself (by replacing it with a sequence).

(2-2) Gene Deletion Method by Conjugate Transfer As an example of a method for deleting or inactivating a nuclease gene, a gene deletion method by conjugation transfer will be described with reference to FIG. The present method is a method for deleting or inactivating a nuclease gene comprising using a transformation method utilizing conjugative transfer from a donor microorganism to a recipient microorganism having a nuclease activity (Rhodococcus genus), The following steps (a) to (d) are included.

(A) a step of producing a Rhodococcus bacterium having enhanced resistance to a drug to which the donor microorganism used for conjugation transmission is sensitive;
(B) As a donor microorganism, the following (i) to (v):
(I) a sequence obtained by deleting or inactivating the resistance gene in a base sequence including a nuclease gene (target gene) in a recipient microorganism and a base sequence in the vicinity thereof,
(Ii) a junction transmission initiation region that functions in the donor microorganism;
(Iii) a replication initiation region that functions in the donor microorganism;
(Iv) a resistance gene for a drug to which the recipient microorganism is sensitive; and
(V) producing a microorganism transformed with a plasmid for genetic modification containing a lethal gene for the recipient microorganism;
(C) producing a transformant of the recipient microorganism by performing conjugative transfer from the donor microorganism produced in step (b) to the recipient microorganism produced in step (a); and (d ) A step of culturing the transformant prepared in the step (c) under a culture condition that allows the lethal gene to function.

  The recipient microorganism used in the deletion or inactivation method is not limited as long as it is a Rhodococcus bacterium having a nuclease gene. Specific examples of Rhodococcus bacteria and the like include those described above.

  On the other hand, the donor microorganism is not limited as long as it is a microorganism that can be conjugated and transferred to the recipient microorganism. For example, E. coli is preferred.

・ Process (a)
In the step (a), Rhodococcus bacteria having enhanced resistance to a drug to which a donor microorganism used for conjugation transmission is sensitive are produced as recipient microorganisms used for conjugation transmission. “Strengthening resistance to drugs” refers to imparting drug resistance if the recipient microorganism does not have drug resistance, and if the recipient microorganism has poor drug resistance, the resistance Is to make it stronger.

  When the conjugation transfer method is used, a drug resistance marker is required for Rhodococcus bacteria that are recipient microorganisms. Therefore, when a Rhodococcus bacterium having no drug resistance or a Rhodococcus bacterium having a low drug resistance is used as a recipient microorganism, it is necessary to produce a strain having a drug resistance sufficient to select a drug. Here, considering the use of the junction transfer method, the agent is preferably an agent that is sensitive to the donor microorganism used for the junction transfer. As the drug, chloramphenicol, ampicillin, kanamycin, trimethoprim, gentamicin, naldicric acid, carbenicine, thiostrepton, tetracycline, streptomycin, etc. are preferable, and chloramphenicol and ampicillin are more preferable.

  The method for producing a recipient microorganism with enhanced drug resistance as described above is not particularly limited. For example, by using a natural mutagenesis method, a mutagenesis method using ultraviolet irradiation or a mutagen, a random mutagenesis tool such as EZ-TN5 (manufactured by Epicenter), a drug resistance gene that originally has no recipient Preferably, a method for artificially introducing the gene into the genome of the microorganism, a method for introducing a plasmid for acquiring antibiotic resistance separately from the plasmid for genetic modification in advance, and the like are preferable. Among these, the natural mutagenesis method is more preferable.

  The natural mutagenesis method is a method in which a target microorganism is originally subcultured in a medium containing a desired drug to induce spontaneous mutation in a microorganism that is originally impossible or difficult to grow in the medium. This is a method for obtaining a strain that can grow even in the medium containing a drug of a concentration. The degree to which drug resistance is enhanced depends on the recipient microorganism, donor microorganism, and drug to be selected, but the drug concentration is selected so that the growth of the recipient microorganism is not suppressed and the growth of the donor microorganism is inhibited. It is preferable. For example, when Rhodococcus bacterium is used as the recipient microorganism, Escherichia coli is used as the donor microorganism, and chloramphenicol is used as the selection agent, chloramphenicol is 1 to 200 mg / L, preferably 10 to 100 mg / L by spontaneous mutation. It is desirable to obtain a recipient microorganism (chloramphenicol resistant strain) capable of growing in a medium containing

・ Process (b)
In step (b), a microorganism transformed with a predetermined gene modifying plasmid is prepared as a donor microorganism to be used for conjugation transmission. As a plasmid for gene modification, that is, plasmid DNA for modifying a nuclease gene in a recipient microorganism, a plasmid containing the structures (gene / base sequence) of (i) to (v) described above is used.

  Here, the sequence of (i) is a sequence obtained by deleting or inactivating the nuclease gene in a base sequence including a nuclease gene in a recipient microorganism to be modified and a base sequence around the gene. It is. The sequence can be prepared by using a known technique such as isolation (cloning) of a base sequence including a nuclease gene and a base sequence around the gene from the genome of a recipient microorganism, gene library preparation, PCR, or the like. it can.

  The base sequence around the nuclease gene is not limited. For example, in addition to the gene, homologous regions upstream and downstream of a gene related to expression regulation are preferably sequences each containing a base sequence of 100 to 3000 bp from both ends. More preferably, it is a sequence comprising a base sequence of 500 to 2000 bp. The method for preparing the above (i) using the isolated base sequence is not particularly limited, and can be performed using a known technique such as PCR method or excision or replacement of a target gene portion using a restriction enzyme.

  The junction transmission start region (ii) described above is not limited as long as it includes a base sequence serving as a junction transmission start point in the donor microorganism used. For example, oriT derived from plasmid PR4 is preferable.

  The replication start region of (iii) is not limited as long as it contains a base sequence that can function as a self-replication origin of the gene-modifying plasmid in the donor microorganism used. For example, oriV is preferable.

  The drug resistance gene of the above (iv) is not limited as long as it is a resistance gene for a drug produced in the above-mentioned step (a) to which the recipient microorganism used for conjugation transmission is sensitive. For example, ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, trimethoprim resistance gene, gentamicin resistance gene, naldic acid resistance gene, carbenicin resistance gene, thiostrepton resistance gene and the like are preferable.

  The lethal gene of (v) is not limited as long as it is a gene that can have an action of causing the microorganism to die when introduced into the genome of the recipient microorganism. For example, sacB gene, rpsL gene, ccdB gene and the like can be mentioned, and sacB gene is preferable. The sacB gene is an enzyme (a levansucrase) that produces a harmful substance having a lethal action on a microorganism using sucrose as a substrate when a microorganism (for example, Rhodococcus bacterium) possessing and expressing the gene is cultured in a sucrose-containing medium. ).

  The plasmid for gene modification used for conjugation transfer may be constructed based on any vector, and is not limited. For example, when the recipient microorganism is a genus Rhodococcus, it is preferable to use a pK19mob vector (Schaefer et al, Genel, vol. 45, p. 69-73 (1994)).

  The gene modification plasmids (i) to (v) are composed of, for example, in order from the upstream, the sequence (i), the resistance gene (iv), the lethal gene (v), and (ii) Are preferably arranged in the order of the junction transmission start region and the replication start region described in (iii) above. Construction of a plasmid for gene modification including the above-described configurations (i) to (v) can be performed using a known gene recombination technique.

  The plasmid for gene modification having the above-described constitutions is introduced into a donor microorganism and transformed to produce a donor microorganism to be used for conjugation transfer. In that case, as a transformation method, a known method can be used as a transformation method of microorganisms. For example, when Escherichia coli is used as a donor microorganism, an electroporation method, a calcium method, or the like can be used.

・ Process (c)
In the step (c), the junction transfer from the donor microorganism produced in the step (b) to the recipient microorganism produced in the step (a) is performed. Usually, the cell suspensions of the donor microorganism and the recipient microorganism are mixed and spread evenly on a suitable plate medium (LB medium or the like) to allow the two microorganisms to join.

  In this conjugation, the gene modification plasmid in the donor microorganism moves into the recipient microorganism, a double crossover occurs in the homologous sequence of the recipient microorganism's genome and the above plasmid, and the nuclease gene in the genome is deleted. The By this conjugation, a transformant of the recipient microorganism is produced. That is, in the transformant, a part of the gene modification plasmid in the donor microorganism is introduced into the genome of the recipient microorganism by homologous recombination.

  Whether or not the desired transformant is desired can be confirmed using the drug resistance of the recipient microorganism itself and the drug resistance derived from the aforementioned gene modification plasmid. Specifically, a desired transformant can be selected by culturing the microorganism after conjugation in a medium containing both drugs (for example, a medium containing kanamycin and chloramphenicol).

・ Process (d)
In the step (d), the recipient microorganism transformant (transformed microorganism) produced in the step (c) is cultured under subculture conditions in which the lethal gene derived from the gene-modifying plasmid can function (subculture). To do. The culture conditions under which the lethal gene can function are not limited. For example, when the lethal gene is the sacB gene, culture using a sucrose-containing medium is preferable.

  In this culture, since the transformed microorganism having the lethal gene is difficult to grow, the base sequence region containing the lethal gene is removed spontaneously by homologous recombination from the genome of the microorganism by subculture. A transformed (dropped) transformed microorganism can be obtained.

  However, among the obtained microorganisms, the nuclease gene in the recipient microorganism is deleted or inactivated as originally intended, and the nuclease gene is not (as a result of homologous recombination at the time of dropping). The nuclease gene function has been restored).

  Therefore, it is usually possible to extract the genomic DNA of the obtained microorganism, confirm that the nuclease gene has been deleted as originally intended, for example by PCR, and select a desired transformed microorganism. More preferred.

(3) Method for transforming Rhodococcus bacteria in which the nuclease gene has been deleted or inactivated The plasmid for introduction into the Rhodococcus bacteria of the present invention has a DNA region capable of replicating and growing in Rhodococcus bacteria. Any plasmid may be used.

  For example, examples of the DNA region that can replicate and proliferate in Rhodococcus bacteria include DNA regions that can replicate and proliferate included in plasmids pAN12, pNC903, pFAJ2600, pRC10, pRC20, pRC001, pRC002, pRC003, or pRC004. Preferably, a DNA region capable of replicating proliferation contained in plasmids pRC001, pRC002, pRC003 or pRC004 can be mentioned. The plasmids pRC001, pRC002, pRC003 or pRC004 are derived from Rhodococcus rhodochrous ATCC4276, ATCC14349, ATCC14348, and IFO3338 strains, respectively, and disclosed in JP-A-2-84198, JP-A-4-148585, and JP-A-5-64589. JP-A-5-68566.

  Moreover, pSJ023 (Japanese Patent Laid-Open No. 10-337185) and pSJ034, which are vectors containing the above pRC004, can also be used.

As the plasmid, a plasmid prepared by the alkali-SDS method can be used, and high-purity DNA with few impurities is preferably used. As a method for purifying the vector with high purity, a density gradient centrifugation method or a commercially available purification kit can be used. In the density gradient centrifugation, for example, CsCl or CF ₃ COOCs is used, and after centrifugation at 100000 G for 2 to 48 hours, the target DNA band is extracted to purify the vector.

  The host used for preparing the plasmid is not particularly limited. For example, bacteria such as Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), actinomycetes, yeast, mold, animal cells, plant cells, insects Examples include cells. The preferred host for plasmid preparation is E. coli.

  The host bacterium used for the transformation is a nuclease-deficient or inactivated Rhodococcus bacterium prepared by the above-mentioned method 2. The host bacteria can increase the efficiency of introducing plasmid DNA by washing the culture solution and using it as it is, or by changing the structure of the cell wall. As a method of changing the structure of the cell wall, a method of treating with glycine, penicillin G or isonicotinic acid hydrazide during culture is preferable. Further, a method of culturing by adding 10% to 20% sucrose of the culture solution can also be used. The host bacteria prepared in this way are called competent cells.

  Transformation is performed using the plasmid DNA and host bacteria prepared as described above. The transformation method is not particularly limited as long as it is a transformation method known to those skilled in the art, and an electroporation method, a method using calcium ions, a spheroplast method, a lithium acetate method, a calcium phosphate method, a lipofection method, etc. are used. be able to.

  For example, using an electric pulse method, an appropriate amount of plasmid DNA and competent cells are mixed and placed in a cuvette, and an electric pulse is applied. The application conditions of the electric pulse are 10 to 25 KV / cm, preferably 20 KV / cm as the voltage, and 50 to 400Ω, preferably 100 to 200Ω as the resistance value. After applying the electric pulse, heat shock is performed at 37 ° C. for several minutes, and then an appropriate medium is added in an appropriate amount, followed by incubation at about 30 ° C. for several hours. By performing this culture, the cell wall changed by the addition of the drug as described above is restored to a normal structure, and the expression of the plasmid-derived drug resistance gene occurs. The culture time is preferably 1 hour or longer, more preferably 12 hours or longer.

  The marker drug for confirming the transformation or the selective medium is not particularly limited as long as the transformant can be obtained and the host bacterium can be distinguished from the transformant. Examples include kanamycin, ampicillin, chloramphenicol, trimethoprim, tetracycline, streptomycin and the like.

  In order to obtain a transformant efficiently, the drug concentration is preferably set to the minimum necessary amount with respect to the selective medium. This concentration can be set by plating host bacteria on an agar medium containing various concentrations of drugs and examining the presence or absence of colony growth. For example, when pK1, pK2, pK3, pK4, pSJ023, pSJ002 or the like is used as the plasmid DNA, the kanamycin concentration is 1 to 100 μg / mL, preferably 10 to 50 μg / mL.

  The transformation efficiency is calculated by, for example, the number of transformant colonies per amount of vector used for transformation (cfu) (= cfu / μg) or the amount of transformant per amount of host used for transformation. can do.

  The Rhodococcus bacterium of the present invention has a remarkably high transformation efficiency compared to the wild type, and can easily and efficiently introduce a target gene. Thereby, it is possible to impart new properties to Rhodococcus bacteria and develop industrially useful microbial catalysts and the like.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1]
Preparation of plasmids for deletion of RrhJ1III, RrhJ1IV, RrhJ1V, RrhJ1VI, and RrhJ1VII (1) Preparation of conjugation transfer plasmid vector (pK19mobsacB1) Primer SAC to which sacB gene in pDNR-1r (Clontech) was added Amplification was performed by PCR using -01 (SEQ ID NO: 11) and SAC-02 (SEQ ID NO: 12) to obtain a sacB gene fragment of about 1.9 kb. Amplification conditions are as follows.

Reaction solution composition:
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
10 μM SAC-01 (SEQ ID NO: 11) 1 μL
10 μM SAC-02 (SEQ ID NO: 12) 1 μL
pDNR-1r (Clontech) (100-fold dilution) 1 μL
Total volume 50μL
Temperature cycle:
Reactions of 98 ° C .: 10 seconds, 55 ° C .: 5 seconds, and 72 ° C .: 10 seconds with 30 cycle primers:
SAC-01: 5′-GGTTCGAATACCTGCCGGTCACTATTATTTAGTG-3 ′ (SEQ ID NO: 11)
SAC-02: 5'-GGTTCGAATCGGCATTTTCTTTTGCGTTTTTTTTTG-3 '(SEQ ID NO: 12).

  After 2 μL of the PCR product was subjected to 0.7% agarose gel electrophoresis to confirm amplification, the reaction solution was purified by GFX PCR DNA band and GelBand Purification kit (Amersham Bioscience).

The purified sacB gene fragment and pK19mob were digested with restriction enzyme NspV (manufactured by Takara Bio Inc.), and then each cleaved fragment was purified with GFX PCR DNA band and Gel Band Purification kit.
The NspV cleavage fragment of sacB and the NspV cleavage fragment of pK19mob were ligated using DNA ligation kit <Mighty mix> (manufactured by Takara Bio Inc.). The reaction conditions are as follows.

Reaction solution composition:
Ligation light mix (manufactured by Takara Bio Inc.) 5 μL
sacB / NspV cleavage fragment 4 μL
pK19mob / NspV cleavage fragment 1 μL
Total volume 10μL
reaction:
16 ° C., 1 hour.

The total amount of the ligation product was added to 200 μL of Escherichia coli JM109 strain competent cell prepared by the method described below and allowed to stand at 0 ° C. for 30 minutes. Subsequently, the competent cell was subjected to a heat shock at 42 ° C. for 45 seconds and cooled at 0 ° C. for 2 minutes. Then, 1 mL of SOC medium (20 mM glucose, 2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO ₄ , 1 mM MgCl ₂ ) was added and shaken at 37 ° C. for 1 hour. Cultured. 200 μL of the culture solution after culturing is applied to LB Km agar medium (LB medium (1% bactotryptone, 1% NaCl, 0.5% bacto yeast extract) containing kanamycin 50 mg / L, 2% agar). And overnight at 37 ° C. A plurality of transformant colonies grown on an agar medium were cultured overnight at 37 ° C. in 1.5 mL of LB Km medium (LB medium containing kanamycin 50 mg / L). After collecting the obtained culture broth, the recombinant plasmid was recovered using QIAprep miniprep kit (manufactured by QIAGEN). Using a capillary DNA sequencer CEQ2000 (manufactured by Beckman Coulter), the base sequence and direction of the PCR amplification product cloned in the plasmid were analyzed according to the attached manual. The plasmid vector into which the SacB gene was introduced in the forward direction was designated as pK19mobsacB1. FIG. 2 shows the structure of pK19mobsacB1.

  A competent cell of Escherichia coli JM109 strain was prepared by the following method.

E. coli strain JM109 was inoculated into 1 mL of LB medium and pre-cultured aerobically at 37 ° C. for 5 hours. Next, 0.4 mL of the preculture solution is added to 40 mL of SOB medium (2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO ₄ , 1 mM MgCl ₂ ) at 18 ° C. Cultured for 20 hours. The obtained culture was collected by centrifugation (3,700 × g, 10 minutes, 4 ° C.), and then cooled TF solution (20 mM PIPES-KOH (pH 6.0), 200 mM KCl, 10 mM CaCl ₂ , 40 mM MnCl). ₂ ) 13 mL was added, left at 0 ° C. for 10 minutes, and centrifuged again (3,700 × g, 10 minutes, 4 ° C.) to remove the supernatant. The obtained Escherichia coli cells were suspended in 3.2 mL of a cold TF solution, 0.22 mL of dimethyl sulfoxide was added, the mixture was allowed to stand at 0 ° C. for 10 minutes, and then frozen with liquid nitrogen to obtain a competent cell.

(2) Preparation of J1 strain chromosomal DNA Rhodococcus rhodochrous J1 strain was added to 100 mL of MYK medium (0.5% polypeptone, 0.3% bactoeast extract, 0.3% bactomalt extract, 0.2% K). ₂ HPO ₄ , 0.2% KH ₂ PO ₄ , pH 7.0), and cultured with shaking at 30 ° C. for 72 hours.

  After culturing, the cells were collected, and the collected cells were suspended in 4 mL of a Saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl (pH 8.0)). 40 mg of lysozyme was added to the suspension, shaken at 37 ° C. for 1 to 2 hours, and then frozen at −20 ° C.

  Next, 10 mL of Tris-SDS solution (1% SDS, 0.1 M NaCl, 0.1 M Tris-HCl (pH 9.0)) was added with gentle shaking, and 10 μL of proteinase K (Merck) was added (final). (Concentration 10 mg / mL) was added and the mixture was shaken at 37 ° C. for 1 hour.

  Next, an equal amount of TE (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) saturated phenol was added, stirred and centrifuged. After centrifugation, the upper layer was taken, and twice the amount of ethanol was added. Then, the DNA was wound with a glass rod, and phenol was sequentially removed with 90%, 80%, and 70% ethanol.

  Next, DNA was dissolved in 3 mL of TE buffer, ribonuclease A solution (100 ° C., heat-treated for 15 minutes) was added to 10 μg / mL, and the mixture was shaken at 37 ° C. for 30 minutes. Furthermore, after adding proteinase K and shaking at 37 ° C. for 30 minutes, an equal amount of TE saturated phenol was added and centrifuged to separate the upper layer and the lower layer.

  After repeating this operation twice for the upper layer, the same amount of chloroform (containing 4% isoamyl alcohol) was added, and the same extraction operation was repeated. Thereafter, twice the amount of ethanol was added to the upper layer, and the DNA was wound up and collected with a glass rod to obtain a J1 strain chromosomal DNA solution. This chromosomal DNA has the RrhJ1III gene shown in SEQ ID NO: 1, the RrhJ1IV gene shown in SEQ ID NO: 3, the RrhJ1V gene shown in SEQ ID NO: 5, the RrhJ1VI gene shown in SEQ ID NO: 7, and the RrhJ1VII gene shown in SEQ ID NO: 9. .

(3) Construction of plasmid for deleting RrhJ1III gene Cloning of fragment containing RrhJ1III gene A primer for amplifying RrhJ1III gene of J-1 strain by PCR was designed by the following method.

FIG. 3 shows the results of amino acid homology analysis of endonuclease produced by Rhodococcus bacteria. In FIG. 3, two particularly conserved regions were selected and degenerate primers of SEQ ID NO: 13 and SEQ ID NO: 14 were designed, and the J-1 strain chromosomal DNA prepared in Example 1 (2) was used as a template for degeneration. PCR was performed under the following conditions. As a result, amplification of a band of about 0.3 kb was confirmed.

Reaction solution composition
Template DNA (J-1 chromosomal DNA) 1 μL
10 × Ex Buffer (Takara Bio Inc.) 10 μL
150 μM primer DG-01 (SEQ ID NO: 13) 1 μL
150 μM primer DG-02 (SEQ ID NO: 14) 1 μL
2.5 mM dNTP 8 μL
DMSO 10μL
Sterile water 18μL
ExTaq DNA polymerase (Takara Bio) 1 μL
Total volume 50μL
Temperature cycle: 94 ° C. for 30 seconds, 65 ° C. for 30 seconds and 72 ° C. for 1 minute for 30 cycles Primer DG-01: 5′-GGIAG (A / G) TGCTT (A / G) TGGGTG (C / T) GGIAGG- 3 ′ (SEQ ID NO: 13)
DG-02: 5'-CCT (T / C) AACTGICCGCT (T / C) AAGTA-3 '(SEQ ID NO: 14).

  Next, direct sequencing of the amplified sequence was performed using the primers used in PCR. As a result, the sequence shown in SEQ ID NO: 15 was obtained. As a result of homology search, homology with the above-mentioned endonuclease was confirmed.

Genomic Southern hybridization ApaLI, BamHI, BseYI, ClaI, EcoNI, EcoT14I, KpnI, MluI, NcoI, NotI, PvuII, SalI, XbaI RhJ1III probes prepared by the method described below When Southern hybridization was performed, a single signal of about 5.9 kb was obtained from the fragment double-digested with BseYI-EcoNI.

  The probe was prepared as follows.

  The PCR product prepared in Example 1 (3) was purified using the GFX PCR DNA band and Gel Band Purification kit (GE Healthcare Bioscience). The purified PCR product was labeled using the AlkPhos Direct Labeling kit (GE Healthcare Bioscience) according to the attached manual to obtain a probe.

Colony hybridization J-1 chromosomal DNA was digested with restriction enzyme BseYI-EcoNI, separated by 0.7% agarose gel electrophoresis, and GFX PCR DNA band and Gel Band Purification kit (GE Healthcare Bioscience) was removed from the gel. Used to recover about 5.9 kb fragment. The resulting fragment was blunted at both ends according to the attached manual using a Mighty Cloning Kit <Blunt End> (Takara Bio). The blunted DNA fragment was ligated to a pBluescript II SK (+) vector (Stratagene) using a DNA Ligation kit <Mighty mix> (Takara Bio). The reaction conditions are as follows.

Reaction solution composition:
Ligation Mighty mix (manufactured by Takara Bio Inc.) 5 μL
J-1 chromosomal DNA / smoothed fragment 4 μL
pBluescriptII SK (+) / EcoRV cleavage fragment 1 μL
Total volume 10μL
Reaction: 16 ° C., 1 hour.

Escherichia coli JM109 strain competent cells were transformed with the total amount of the ligation product, and color selection with LB AIX plates (LB agar medium containing 100 μg / L ampicillin, 100 μM IPTG, 50 μg / L X-gal) was performed. 94 white recombinant colonies grown on the plate were isolated on a new LB AIX plate for 10 plates per plate. Each plate was inoculated with 2 colonies / plate of JM109 strain transformed with pBluescript II SK (+) without insert. After the colony-isolated plate is left overnight at 37 ° C., the colony is copied onto a Hybond-N ⁺ (GE Healthcare Bioscience) membrane, and colony hybridization is performed using a probe prepared by genomic Southern hybridization. It was.

  A culture solution obtained by culturing the detected colonies was collected, and then the recombinant plasmid was recovered using QIAprep miniprep kit (manufactured by QIAGEN). Using a capillary DNA sequencer CEQ2000 (manufactured by Beckman Coulter), the base sequence of the chromosomal DNA fragment cloned in the plasmid was analyzed according to the attached manual. As a result, the base sequence represented by SEQ ID NO: 16 was obtained. In the nucleotide sequence shown in SEQ ID NO: 16, a 393 bp open reading frame (ORF1) shown in SEQ ID NO: 1 and a 384 bp open reading frame (ORF2) shown in SEQ ID NO: 17 were found. Since the amino acid sequences encoded by these ORF1 and ORF2 have 68 to 94% homology with the endonucleases derived from Rhodococcus bacteria A to D shown in FIG. 3, ORF1 and ORF2 are endonucleases. Therefore, the endonucleases encoded by ORF1 and ORF2 were designated as RrhJ1III nuclease (1st) and RrhJ1III nuclease (2nd), respectively. In addition, the plasmid containing ORF1 and ORF2 obtained in this example was named pBRrhJ1III.

Cloning of the sequence around the RrhJ1III gene In order to obtain a sequence including the upstream region and the downstream region of the RrhJ1III gene, PCR was performed using the reaction solution composition and primers shown below.

Reaction solution composition:
Template DNA (pBRrhJ1III) 1 μL
10 μM primer JM64 (SEQ ID NO: 18) 1 μL
10 μM primer JM65 (SEQ ID NO: 19) 1 μL
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50μL
Temperature cycle:
30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds and 72 ° C. for 10 seconds:
JM64: 5′-GGtctagaACAAGATCTCACCGGGACGCC-3 ′ (SEQ ID NO: 18)
JM65: 5′-GCacttagGGACCGTGCTTCGAGGACGAG-3 ′ (SEQ ID NO: 19)
Similarly, PCR was performed under the same conditions as described above using primers JM66 (SEQ ID NO: 20) and JM67 (SEQ ID NO: 21).

JM66: 5′-GCactagtCAGCCGGTGATCACGAAGACCACT-3 ′ (SEQ ID NO: 20)
JM67: 5′-gccctgcaggCTTCCCCGTCGCCATCGTCCGGA-3 ′ (SEQ ID NO: 21)
After completion of PCR, 2 μL of each reaction solution was subjected to 0.7% agarose gel electrophoresis, and a PCR product of about 2 kbp was detected. After confirming the PCR product, the reaction solution was purified with GFX PCR DNA band and GelBand Purification kit (Amersham Bioscience).

Next, in order to link the two PCR fragments amplified as described above, assembly PCR was performed under the following conditions.

Reaction solution composition:
Template DNA1 (Amplification product of JM64 and JM65) 0.5 μL
Template DNA2 (amplification product of JM66 and JM67) 0.5 μL
Primer JM64 (SEQ ID NO: 18) 1 μL
Primer JM67 (SEQ ID NO: 21) 1 μL
Sterile water 22 μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50 μL
Temperature cycle:
30 cycles of reaction at 98 ° C for 10 seconds, 55 ° C for 5 seconds and 72 ° C for 20 seconds.

  After completion of the PCR, an amplification product of about 4 kbp was confirmed by 0.7% agarose electrophoresis, and ligated to pUC118 using a Mighty Cloning Kit (Blunt End) (manufactured by Takara Bio Inc.). The plasmid thus obtained was named pUC118 / ΔRrhJ1III.

Subsequently, the plasmid pUC118 / ΔRrhJ1III and the plasmid vector pK19mobsacB1 were digested with restriction enzymes XbaI and Sse8387I.

Reaction solution composition 1:
Plasmid pUC118 / ΔRrhJ1III 20 μL
XbaI (Takara Bio Inc.) 1μL
Sse8387I (Takara Bio Inc.) 1μL
10 × M Buffer (attached to the enzyme) 5 μL
10 × BSA (attached to the enzyme) 5 μL
Sterile water 18μL
Total volume 50μL

Reaction solution composition 2:
Plasmid pK19mobsacB1 5 μL
XbaI (Takara Bio Inc.) 0.5μL
Sse8387I (Takara Bio Inc.) 0.5μL
10 × M Buffer (attached to the enzyme) 2 μL
10 x BSA (attached to the enzyme) 2 μL
Sterile water 10 μL
Total volume 20 μL
Reaction: 37 degreeC, 2.5 hours.

  After completion of the reaction, the plasmid pUC118 / ΔRrhJ1III was subjected to 0.7% agarose gel electrophoresis to cut out an approximately 4 kb fragment, and recovered with GFX PCR DNA band and GelBand Purification kit (Amersham Biosciences). ΔRrhJ1III / XbaI-Sse8387I.

  For the plasmid vector pK19mobsacB1, 2 μL of 3M sodium acetate and 50 μL of 99.5% ethanol were added to the reaction mixture, mixed well, and cooled at −20 ° C. for 1 hour. The solution after cooling was centrifuged at 15,000 rpm, 4 ° C. for 10 minutes to remove the supernatant, and the solvent was removed by drying under reduced pressure. Thereafter, 50 μL of sterilized water was added to the dried pellet and dissolved again to obtain a vector solution of pK19mobsacB1 / XbaI-Sse8387I.

Next, the recovered ΔRrhJ1III / XbaI-Sse8387I was ligated with the vector pK19movsacB1 / XbaI-Sse8387I.

Reaction solution composition:
Ligation light mix (manufactured by Takara Bio Inc.) 5 μL
ΔRrhJ1III / XbaI-Sse8387I 4 μL
pK19mobsacB1 / XbaI-Sse8387I 1 μL
Total volume 10μL
Reaction: 16 ° C., 1 hour After completion of the reaction, E. coli JM109 strain was transformed in the same manner as in (1) to obtain a RrhJ1III gene-deleted plasmid. This plasmid was named pK19ΔRrhJ1III.

(4) Construction of RrhJ1IV Gene Deletion Plasmid Cloning of Fragment Containing RrhJ1IV Gene Primers for amplifying RrJ1IV gene of J-1 strain by PCR were designed by the following method.

FIG. 4 shows the results of amino acid homology analysis of endonuclease produced by Rhodococcus bacteria. In FIG. 4, two particularly conserved regions were selected and degenerate primers of SEQ ID NO: 22 and SEQ ID NO: 23 were designed, and the J-1 strain chromosomal DNA prepared in Example 1 (2) was used as a template. Degenerate PCR was performed as in 1 (3). As a result, amplification of a band of about 0.6 kb was confirmed.
Primer DG-03: 5′-tggggc (c / g) cg (g / c) ac (c / g) tacct (c / g) aaggcatg-3 ′ (SEQ ID NO: 22)
DG-04: 5'-gcc (g / c) aggccgtgctcctt (g / c) ag (g / c) ag-3 '(SEQ ID NO: 23).

  Next, direct sequencing of the amplified sequence was performed using the primers used in PCR. As a result, the sequence shown in SEQ ID NO: 24 was obtained. As a result of homology search, homology with the above-mentioned endonuclease was confirmed.

Genomic Southern hybridization ApaI, ApaLI, BamHI, BclI, ClaI, Eco52I, KpnI, NcoI, NotI, PvuI, SacI, XbaI PCR products obtained by the above degenerate PCR As a result of Southern hybridization using a probe prepared in the same manner as in Example 1 (3), a single signal of about 6.3 kb was obtained from the fragment digested with BclI.

Colony hybridization J-1 chromosomal DNA was digested with the restriction enzyme BclI, and a fragment of about 6.4 kb was recovered and ligated to the BamH site of the pBluescript II SK (+) vector in the same manner as in Example 1 (3).

  Colony hybridization was performed on 940 transformants obtained here in the same manner as in Example 1 (3) using the probe obtained in Example 1 (4).

  A recombinant plasmid was recovered from the detected colony in the same manner as in Example 1 (3), and the base sequence of the chromosomal DNA fragment cloned in the plasmid was analyzed. As a result, the base sequence represented by SEQ ID NO: 25 was obtained. The 939 bp open reading frame (ORF3) shown in SEQ ID NO: 3 was found in the base sequence shown in SEQ ID NO: 25. Since the amino acid sequence encoded by ORF3 has 55 to 99% homology with the endonucleases derived from Rhodococcus bacteria of A to E shown in FIG. 4, ORF3 encodes the endonuclease. Therefore, the endonuclease encoded by ORF3 was named RrhJ1IV nuclease. In addition, the plasmid containing ORF3 obtained in this example was named pBRrhJ1IV.

Cloning of RhrJ1IV Gene Peripheral Sequence In order to obtain a sequence including the upstream region and the downstream region of the RrhJ1IV gene, PCR was performed using the following reaction solution composition and primers.

Reaction solution composition:
Template DNA (pBRrhJ1IV) 1 μL
10 μM primer JM72 (SEQ ID NO: 26) 1 μL
10 μM primer JM73 (SEQ ID NO: 27) 1 μL
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50μL
Temperature cycle:
30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds and 72 ° C. for 20 seconds:
JM72: 5'-CCtctagaAAGATCAGGGAACCAGCTCGAACATTCTC-3 '(SEQ ID NO: 26)
JM73: 5'-GGcctgcaggATCGCGCTTCTCGATGTACTCGAACAT-3 '(SEQ ID NO: 27).

  After completion of PCR, a PCR fragment of about 3.9 kb was detected in the same manner as in Example 1 (3), and ligated to plasmid vector pUC118 in the same manner as in Example 1 (3). The plasmid obtained here was named pUC118 / RrhJ1IV.

Next, in order to obtain a sequence containing only the upstream region and the downstream region of the RrhJ1IV gene, PCR was performed using the following reaction solution composition and primers.

Reaction solution composition:
Template DNA (plasmid pUC118 / RrhJ1IV) 1 μL
Primer JM84 (SEQ ID NO: 28) 1 μL
Primer JM85 (SEQ ID NO: 29) 1 μL
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50μL
Temperature cycle:
Reactions of 98 ° C .: 10 seconds, 55 ° C .: 5 seconds, and 72 ° C .: 30 seconds, 30 cycle primers:
JM84: 5′-GGAAGGTCCCGCGTAGCGCCGTCGCG-3 ′ (SEQ ID NO: 28)
JM85: 5'-CTACCGCGACCTCCCCCTAACGTCGC-3 '(SEQ ID NO: 29).

  After completion of PCR, 2 μL of the reaction solution was subjected to 0.7% agarose gel electrophoresis, and a PCR product of about 6 kbp was detected. After confirming the PCR product, JM109 strain was transformed with 2 μL of the reaction solution to obtain a plasmid containing only the upstream region and the downstream region of the RrhJ1IV gene. This plasmid was named pUC118 / ΔRrhJ1IV.

  Subsequently, using the plasmid pUC118 / ΔRrhJ1IV and the plasmid vector pK19mobsacB1, the RrhJ1IV deletion fragment was obtained by ligating the RrhJ1IV deletion fragment to the XbaI-Sse8387I site of the plasmid vector pK19mobsacB1 in the same manner as in Example 1 (3). The plasmid for use was named pK19ΔRrhJ1IV.

(5) Construction of RrhJ1V gene deletion plasmid Cloning of fragment containing RrhJ1V gene Primers for amplifying RrJ1V gene of J-1 strain by PCR were designed by the following method.

  FIG. 5 shows the results of amino acid homology analysis of endonuclease produced by Rhodococcus bacteria. In FIG. 5, two conserved regions were selected and degenerate primers of SEQ ID NO: 30 and SEQ ID NO: 31 were designed, and the J-1 strain chromosomal DNA prepared in Example 1 (2) was used as a template. Degenerate PCR was performed as in 1 (3). As a result, amplification of a band of about 0.5 kb was confirmed.

Primer DG-05: 5′-aagcg (g / c) cg (g / c) gt (c / g) ct (c / g) ct (c / g) ct (c / g) aac-3 ′ (sequence) Number 30)
DG-06: 5'-aa (g / c) gc (g / c) gcgccctcgcc (c / g) aggta (c / g) gg-3 '(SEQ ID NO: 31).

  Next, direct sequencing of the amplified sequence was performed using the primers used in PCR. As a result, the sequence shown in SEQ ID NO: 32 was obtained. As a result of homology search, homology with the above-mentioned endonuclease was confirmed.

Genomic Southern hybridization Obtained by the above degenerate PCR for J-1 chromosomal DNA digested with ApaI, ApaLI, BamHI, BclI, BbvCI, ClaI, Eco52I, KpnI, NcoI, NotI, NspV, PvuI, SacI, XbaI. Using the obtained PCR product, Southern hybridization was performed using a probe prepared by the same method as in Example 1 (3). As a result, it was found that the fragment obtained by double digestion with BbvCI and NspV two enzymes was about 6. A single signal of 1 kb was obtained.

Colony hybridization After decomposing J-1 chromosomal DNA with the restriction enzyme BbvCI-NspV and collecting a fragment of about 6.1 kb in the same manner as in Example 1 (3), a terminal fragment was obtained in the same manner as in Example 1 (3). Smoothing was performed and ligated to the EcoRV site of the pBluescript II SK (+) vector (Stratagene) to obtain a transformant of E. coli JM109 in the same manner as in Example 1 (3).

  Colony hybridization was performed on 940 transformants obtained here in the same manner as in Example 1 (3) using the probe obtained in Example 1 (5).

  A recombinant plasmid was recovered from the detected colony in the same manner as in Example 1 (3), and the base sequence of the chromosomal DNA fragment cloned in the plasmid was analyzed. As a result, the base sequence represented by SEQ ID NO: 33 was obtained. In the nucleotide sequence shown in SEQ ID NO: 33, an 1155 bp open reading frame (ORF4) shown in SEQ ID NO: 5 was found. Since the amino acid sequence encoded by ORF4 has 71 to 99% homology with the endonucleases derived from Rhodococcus bacteria A to E shown in FIG. 5, ORF4 encodes an endonuclease. Therefore, the endonuclease encoded by ORF4 was named RrhJ1V nuclease. In addition, the plasmid containing ORF4 obtained in this example was named pBRrhJ1V.

-Cloning of the sequence around the RrhJ1V gene In order to obtain a sequence including the upstream region and the downstream region of the RrhJ1V gene, PCR was performed using the following reaction solution composition and primers.

Reaction solution composition:
Template DNA (pBRrhJ1V) 1 μL
10 μM primer JM76 (SEQ ID NO: 34) 1 μL
10 μM primer JM77 (SEQ ID NO: 35) 1 μL
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50μL
Temperature cycle: reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds and 72 ° C. for 40 seconds for 30 cycles Primer:
JM76: 5′-CCtctagaGTGGAAGTCGAAGGCGAGAT-3 ′ (SEQ ID NO: 34)
JM77: 5'-GGaagcttTACCTGTGCCCCTGCACAC-3 '(SEQ ID NO: 35).

  After completion of PCR, a PCR fragment of about 4.1 kb was detected in the same manner as in Example 1 (3) and ligated to plasmid vector pUC118 in the same manner as in Example 1 (3). The plasmid obtained here was named pUC118 / RrhJ1V.

Next, in order to obtain a sequence containing only the upstream region and the downstream region of the RrhJ1IV gene, PCR was performed using the following reaction solution composition and primers.

Reaction solution composition:
Template DNA (plasmid pUC118 / RrhJ1V) 1 μL
Primer JM88 (SEQ ID NO: 36) 1 μL
Primer JM89 (SEQ ID NO: 37) 1 μL
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50μL
Temperature cycle: 98 ° C .: 10 seconds, 55 ° C .: 5 seconds and 72 ° C .: 30 seconds reaction for 30 cycles Primer:
JM88: 5'-CGACGAGGTCGGGCCGTTCTGCGGGG-3 '(SEQ ID NO: 36)
JM89: 5'-CGGCCCGAACTCGTCCGACTACATGAAC-3 '(SEQ ID NO: 37).

  After completion of PCR, the JM109 strain was transformed in the same manner as in Example 1 (4) to obtain a plasmid containing only the upstream and downstream regions of the RrhJ1V gene. This plasmid was named pUC118 / ΔRrhJ1V.

  Subsequently, using the plasmid pUC118 / ΔRrhJ1V and the plasmid vector pK19mobsacB1, the RrhJ1V deletion fragment was ligated to the XbaI-HindIII site of the plasmid vector pK19mobsacB1 in the same manner as in Example 1 (3). The plasmid for use was named pK19ΔRrhJ1V.

(6) Construction of RrhJ1VI gene deletion plasmid Cloning of fragment containing RrhJ1VI gene Primers for amplifying RrJ1VI gene of J-1 strain by PCR were designed by the following method.

FIG. 6 shows the results of amino acid homology analysis of endonuclease produced by Rhodococcus bacteria. In FIG. 6, two particularly conserved regions were selected and degenerate primers of SEQ ID NO: 38 and SEQ ID NO: 39 were designed, and the J-1 strain chromosomal DNA prepared in Example 1 (2) was used as a template for degeneration. PCR was performed under the following conditions. As a result, amplification of a band of about 1.0 kb was confirmed.

Reaction solution composition
Template DNA (J-1 chromosomal DNA) 1 μL
10 × Ex Buffer (Takara Bio Inc.) 10 μL
150 μM primer DG-07 (SEQ ID NO: 38) 1 μL
150 μM primer DG-08 (SEQ ID NO: 39) 1 μL
2.5 mM dNTP 8 μL
DMSO 10μL
Sterile water 18μL
ExTaq DNA polymerase (Takara Bio) 1 μL
Total volume 50μL
Temperature cycle: reaction at 94 ° C. for 30 seconds, 65 ° C. for 30 seconds and 72 ° C. for 1 minute for 30 cycles Primer DG-07: 5′-aagataggc (c / g) aacct (c / g) gacggc-3 ′ (SEQ ID NO: 38 )
DG-08: 5'-aa (g / c) gt (c / g) cgggtgccagcc (g / c) aggta-3 '(SEQ ID NO: 39).

  Next, direct sequencing of the amplified sequence was performed using the primers used in PCR. As a result, the sequence shown in SEQ ID NO: 40 was obtained. As a result of homology search, homology with the above-mentioned endonuclease was confirmed.

Genomic Southern Hybridization ApaI, ApaLI, BamHI, ClaI, Eco52I, KpnI, MluI, NcoI, NotI, NsiI, PvuI, SacI, XbaI were used for the above degenerate PCR products. Southern hybridization was performed using a probe prepared in the same manner as in Example 1 (3), and a single signal of about 0.7 kb was obtained from the fragment double-digested with both BamHI and NsiI enzymes. It was.

Colony hybridization J-1 chromosomal DNA was double digested with the restriction enzyme BamHI-NsiI, and a fragment of about 6.4 kb was recovered in the same manner as in Example 1 (3). The recovered DNA fragment was ligated to the PstI-BamHI site of pBluescript II SK (+) vector (Stratagene), and then transformed into E. coli JM109 strain competent cells. Colony hybridization was performed on 940 transformants obtained in the same manner as in Example 1 (3), using a probe prepared by genomic Southern hybridization.

  A recombinant plasmid was recovered from the detected colony in the same manner as in Example 1 (3), and the base sequence of the chromosomal DNA fragment cloned in the plasmid was analyzed. As a result, the base sequence represented by SEQ ID NO: 41 was obtained. A 1008 bp open reading frame (ORF5) shown in SEQ ID NO: 7 was found in the base sequence shown in SEQ ID NO: 41. Since the amino acid sequence encoded by ORF5 has 62 to 83% homology with endonucleases derived from Rhodococcus bacteria A to E shown in FIG. 6, ORF5 encodes an endonuclease. Therefore, the endonuclease encoded by ORF5 was named RrhJ1VI nuclease. Moreover, the plasmid containing ORF5 obtained in this Example was named pBRrhJ1VI.

-Cloning of the sequence around the RrhJ1VI gene In order to obtain the sequence including the upstream region and the downstream region of the RrhJ1VI gene, PCR was performed using the following reaction solution composition and primers.

Reaction solution composition:
Template DNA (pBRrhJ1VI) 1 μL
10 μM primer JM68 (SEQ ID NO: 42) 1 μL
10 μM primer JM69 (SEQ ID NO: 43) 1 μL
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50μL
Temperature cycle:
30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds and 72 ° C. for 10 seconds:
JM68: 5′-GCggatccGTTACCAGAACATCCGTTG-3 ′ (SEQ ID NO: 42)
JM69: 5'-gcacttagGGCTCCAGAAAGTCACTGAG-3 '(SEQ ID NO: 43).

  Similarly, primers JM70 (SEQ ID NO: 44) and JM71 (SEQ ID NO: 45) were used, and PCR was performed under the same conditions as described above.

JM70: 5'-GCactagtAGGGGACGATAGAGAATCATGG-3 '(SEQ ID NO: 44)
JM71: 5'-GCcctgcaggTCGAACTACATCGGACAGACC-3 '(SEQ ID NO: 45).

  After completion of PCR, each PCR product was detected and purified in the same manner as in Example 1 (3).

Next, in order to link the two PCR fragments amplified as described above, assembly PCR was performed under the following conditions.

Reaction solution composition:
Template DNA1 (amplification product of JM68 and JM69) 0.5 μL
Template DNA2 (amplification product of JM70 and JM71) 0.5 μL
Primer JM68 (SEQ ID NO: 42) 1 μL
Primer JM71 (SEQ ID NO: 43) 1 μL
Sterile water 22 μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50 μL
Temperature cycle:
30 cycles of reaction at 98 ° C for 10 seconds, 55 ° C for 5 seconds and 72 ° C for 40 seconds.

  After completion of PCR, an amplification product of about 4 kbp was detected and ligated to pUC118 in the same manner as in Example 1 (3). The plasmid thus obtained was named pUC118 / ΔRrhJ1VI.

  Subsequently, the RrhJ1VI gene deletion fragment was ligated to the BamHI-Sse8387I site of the plasmid vector pK19movsacB1 using the plasmid pUC118 / ΔRrhJ1VI and the plasmid vector pK19movsacB1 in the same manner as in Example 1 (3). This plasmid was named pK19ΔRrhJ1VI.

(7) Preparation of RrhJ1VII deletion plasmid Cloning of fragment containing RrhJ1VII gene A primer for amplifying RrJ1VII gene of J-1 strain by PCR was designed by the following method.

FIG. 7 shows the results of amino acid homology analysis of a putative endonuclease produced by Rhodococcus bacteria. In FIG. 7, two regions that are particularly conserved are selected and degenerate primers of SEQ ID NO: 46 and SEQ ID NO: 47 are designed, and the J-1 strain chromosomal DNA prepared in Example 1 (2) is used as a template. Degenerate PCR was performed as in 1 (3). As a result, amplification of a band of about 0.3 kb was confirmed.
Primer DG-09: 5′-atggt (c / g) gagcagcc (g / c) gc (c / g) aaggt (c / g) atg-3 ′ (SEQ ID NO: 46)
DG-10: 5'-ctg (c / g) cgcatctgctc (g / c) agctg-3 '(SEQ ID NO: 47).

  Next, direct sequencing of the amplified sequence was performed using the primers used in PCR. As a result, the sequence shown in SEQ ID NO: 48 was obtained. As a result of homology search, homology with the above-mentioned putative endonuclease was confirmed.

Genomic Southern hybridization ApaI, ApaLI, BamHI, ClaI, Eco52I, KpnI, NcoI, NotI, NspV, PvuI, SacI, XbaI PCR products obtained by the above degenerate PCR Southern hybridization was performed using the probe prepared in the same manner as in Example 1 (3), and a single fragment of about 7.8 kb was obtained from the double digested fragment of ClaI and NotI. A signal was obtained.

Colony hybridization J-1 chromosomal DNA was digested with restriction enzyme ClaI-NotI, and a fragment of about 7.8 kb was recovered in the same manner as in Example 1 (3), and pBluescript II SK (as in Example 1 (3)) +) A transformant of E. coli JM109 was obtained in the same manner as in Example 1 (3) by ligation to the ClaI-NotI site of a vector (Stratagene).

  Colony hybridization was performed on 940 transformants obtained here in the same manner as in Example 1 (3) using the probe obtained in Example 1 (6).

  A recombinant plasmid was recovered from the detected colony in the same manner as in Example 1 (3), and the base sequence of the chromosomal DNA fragment cloned in the plasmid was analyzed. As a result, the base sequence represented by SEQ ID NO: 49 was obtained. The 585 bp open reading frame (ORF6) shown in SEQ ID NO: 9 was found in the base sequence shown in SEQ ID NO: 49. Since the amino acid sequence encoded by ORF5 has 69-95% homology with the putative endonucleases derived from Rhodococcus bacteria of A to E shown in FIG. 7, ORF6 encodes the endonuclease. Therefore, the endonuclease encoded by ORF6 was named RrhJ1VII nuclease. In addition, the plasmid containing ORF6 obtained in this example was named pBRrhJ1VII.

-Cloning of the sequence around the RrhJ1VII gene In order to obtain the sequence including the upstream region and the downstream region of the RrhJ1VII gene, PCR was performed using the reaction solution composition and primers shown below.

Reaction solution composition:
Template DNA (pBRrhJ1VII) 1 μL
10 μM primer JM82 (SEQ ID NO: 50) 1 μL
10 μM primer JM83 (SEQ ID NO: 51) 1 μL
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50μL
Temperature cycle:
30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds and 72 ° C. for 40 seconds:
JM82: 5'-aatctagaACGCCCGGTGCACCTGCGCT-3 '(SEQ ID NO: 50)
JM83: 5'-ttcctgcaggAATCTGTGCCGCCTCATCGA-3 '(SEQ ID NO: 51).

  After completion of PCR, a PCR fragment of about 3.5 kb was detected in the same manner as in Example 1 (3), and ligated to plasmid vector pUC118 in the same manner as in Example 1 (3). The plasmid obtained here was named pUC118 / RrhJ1VII.

Next, in order to obtain a sequence containing only the upstream region and the downstream region of the RrhJ1IVII gene, PCR was performed using the following reaction solution composition and primers.

Reaction solution composition:
Template DNA (plasmid pUC118 / RrhJ1VII) 1 μL
Primer JM94 (SEQ ID NO: 52) 1 μL
Primer JM119 (SEQ ID NO: 53) 1 μL
Sterile water 22μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 25 μL
Total volume 50μL
Temperature cycle:
Reactions of 98 ° C .: 10 seconds, 55 ° C .: 5 seconds, and 72 ° C .: 30 seconds, 30 cycle primers:
JM94: 5′-AGGCGGGAGCTCTCATTCTCCCTTTC-3 ′ (SEQ ID NO: 52)
JM119: 5'-ATGGAGCCTCCCGCCGGGTACCGCCCC-3 '(SEQ ID NO: 53).

  After completion of PCR, the JM109 strain was transformed in the same manner as in Example 1 (4) to obtain a plasmid containing only the upstream and downstream regions of the RrhJ1VII gene. This plasmid was named pUC118 / ΔRrhJ1VII.

  Subsequently, using the above plasmid pUC118 / ΔRrhJ1VII and the plasmid vector pK19mobsacB1, the RrhJ1VII deletion fragment obtained by ligating the RrhJ1VII deletion fragment to the XbaI-Sse8387I site of the plasmid vector pK19mobsacB1 in the same manner as in Example 1 (3). The plasmid for use was named pK19ΔRrhJ1VII.

[Example 2]
Preparation of J1 strain having drug resistance The donor used for conjugation transmission needs drug resistance for selection of gene deletion strains. Therefore, attempts were made to obtain various drug-resistant strains, and mutant strains of the J1 strain having chloramphenicol resistance were obtained by the following method.

MYK plate containing 2 mg / L chloramphenicol (0.5% polypeptone, 0.3% bacto yeast extract, 0.3% malt extract, 0.2% KH ₂ PO ₄ , 0.2% K ₂ HPO ₍₄ , 2% agar) was streaked with J1 strain and kept at 30 ° C. until colonies grew. About 2 weeks later, the chloramphenicol resistant strain that had grown was streaked again on a 2 mg / L chloramphenicol plate and kept warm at 30 ° C.

  Next, the colonies grown from the 2 mg / L chloramphenicol plate were streaked on the 5 mg / L chloramphenicol plate and incubated at 30 ° C. until a resistant strain appeared. Thereafter, the same operation was repeated to increase the chloramphenicol concentration to 10 mg / L, and a chloramphenicol resistant strain (J1-Cm strain) that grew at a chloramphenicol concentration of 10 mg / L was obtained.

[Example 3]
Deletion of nuclease gene by conjugation transfer (1) Preparation of donors In a test tube sterilized by dry heat, 20 μL each of competent cells of Escherichia coli S17-1λpir were added with plasmids pK19ΔRrhJ1III, pK19ΔRrhJ1IV, pK19ΔRrhJ1V, pK19ΔRrhJ1VI, or pK19ΔRrhJ1VI, or ice on pK19ΔRrhJ1VI Let stand for 30 minutes. After heat shock at 42 ° C. for 30 seconds, 180 μL of SOC medium was added, and shaking culture was performed at 37 ° C. for 1 hour. Then, it apply | coated to LB Km plate and left still at 37 degreeC overnight.

  On the next day, colonies that grew on each plate were collected with 1 mL of each LB medium, and the cells were collected by centrifugation, and the centrifuged supernatant was removed. The same operation was repeated once again, and finally 0.5 mL of LB medium was added to prepare each cell suspension. Each cell suspension was used as a donor solution.

(2) Preparation of recipient The J1-Cm strain was streaked on a MYK plate and grown at 30 ° C. for 2 days. The grown colonies were collected and washed in the same manner as in Example 3 (1) to prepare a bacterial cell suspension serving as a recipient.

(3) Conjugate transfer 100 μL each of the donor solution prepared in Example 3 (1) and the recipient solution prepared in (2) were applied to a MYK plate containing no antibiotics, and allowed to stand at 30 ° C. overnight. I put it.

  On the next day, the grown colonies were recovered with 1 mL of LB medium, and 100 μL was applied to a MYK plate containing 10 mg / L of kanamycin and 10 mg / L of chloramphenicol. The coated plate was kept at 30 ° C. for 1 week until a recombinant bacterial colony appeared.

One week later, each one of the colonies that appeared on each plate was designated as # RrhJ1III, # RrhJ1IV, # RrhJ1V, # RrhJ1VI, and # RrhJ1VII, and used in subsequent experiments.
(4) Deletion of nuclease gene Recombinant bacteria # RrhJ1III, # RrhJ1IV, # RrhJ1V, # RrhJ1VI, and # RrhJ1VII obtained by conjugation transfer are RrhJ1III gene, RrhJ1IV gene, RrhJ1V gene, RrhJ1V gene on the genome, respectively. Alternatively, each plasmid is inserted into the region of the RrhJ1VII gene by homologous recombination, but two steps of homologous recombination are required to delete each gene. Therefore, next, selection using the sacB gene was performed.

Prepare a MYK plate containing 10% sucrose, apply a solution prepared by suspending colonies of recombinant bacteria # RrhJ1III, # RrhJ1IV, # RrhJ1V, # RrhJ1VI, and # RrhJ1VII in sterile water. And left at 30 ° C. Colony PCR was performed on 14 colonies grown on each plate, and the size of the PCR fragment was examined by electrophoresis.

Reaction solution composition:
10 μM Forward primer 0.1 μL
10 μM Reverse primer 0.1 μL
Sterile water 4.8μL
2 × PrimeStar Max (manufactured by Takara Bio Inc.) 5 μL
Total volume 10 μL
Temperature cycle:
Primer sets using 30 cycles of 98 ° C .: 10 seconds, 55 ° C .: 5 seconds, and 72 ° C .: 20 seconds are as follows.
For confirmation of RrhJ1III gene deletion:
Forward primer: JM64 (SEQ ID NO: 18)
Reverse primer: JM67 (SEQ ID NO: 19)
For confirmation of RrhJ1IV gene deletion:
Forward primer: JM72 (SEQ ID NO: 26)
Reverse primer: JM73 (SEQ ID NO: 27)
For confirmation of RrhJ1V gene deletion:
Forward primer: JM76 (SEQ ID NO: 34)
Reverse primer: JM77 (SEQ ID NO: 35)
Forward primer for confirmation of RrhJ1VI gene deletion: JM68 (SEQ ID NO: 42)
Reverse primer: JM71 (SEQ ID NO: 43)
Forward primer for confirmation of RrhJ1VII gene deletion: JM82 (SEQ ID NO: 50)
Reverse primer: JM83 (SEQ ID NO: 51).

As a result, it was confirmed that the RrJ1III gene, the RrhJ1IV gene, the RrhJ1V gene, the RrhJ1VI gene, or the RrhJ1VII gene was deleted in each of the plurality of colonies on each plate. The obtained deletion strains of each nuclease gene were designated as ΔRrhJ1III, ΔRrhJ1IV, ΔRrhJ1V, ΔRrhJ1VI, and ΔRrhJ1VII, respectively.
[Example 4]
Evaluation of transformation efficiency (1) ΔRrhJ1III, ΔRrhJ1IV, ΔRrhJ1V, ΔRrhJ1VI, ΔRrhJ1VII, and production of competent cells of J1 MYKG medium (0.5% polypeptone, 0.3% bacteast extract, 0.3% (Maltz extract, 0.2% KH ₂ PO ₄ , 0.2% K ₂ HPO ₄ , 1% glucose) was dispensed into Φ24 mm × 165 mm test tubes at 10 mL each, and ΔRrhJ1III, ΔRrhJ1IV, ΔRrhJ1V, ΔRrhJ1VI, ΔRrhJ1VII to each test tube And the colony of J1 stock used as a control was inoculated, respectively. After culturing at 30 ° C. and 350 rpm for 1 day, 3 mL of each of the obtained culture solutions was inoculated into MYKG-20% sucrose medium (100 mL in a 500 mL Erlenmeyer flask), and cultured at 30 ° C. and 230 rpm for 20 hours. The obtained culture solution was transferred to a tube with a total volume of 50 mL, and each cell was collected by centrifugation (5000 rpm, 10 minutes, 4 ° C.). After each collected bacterial cell was washed twice with 10 mL Electroporation Buffer (2 mM K ₂ HPO ₄ , 10% sucrose, pH 8.3; hereinafter sometimes abbreviated as EB), the bacterial concentration was the same. Each was suspended in EB and frozen at −80 ° C. to obtain competent cells.

(2) Preparation of plasmid pK4 Plasmid pK4 (FIG. 8) used for evaluating transformation efficiency was prepared by the following method. E. coli SCS110 strain (manufactured by Stratagene) and JM109 strain competent cells were prepared by the method described in Example 1 (1).

  2 μL each of plasmid pK4 was added to 100 μL each of competent cells of E. coli SCS110 strain and JM109 strain, and transformed by the method described in Example 1 (1). Each culture solution after transformation was applied to an LB Km agar medium (LB medium containing kanamycin 50 mg / L, 2% agar) and cultured at 37 ° C. overnight. A colony of each transformant that appeared on the agar medium was inoculated into a 2 mL LB Km liquid medium and cultured with shaking at 37 ° C. and 180 rpm overnight. Plasmids were collected from the total amount of each culture solution after culture using QIAprep Spin Miniprep Kit (manufactured by QIAGEN), and each was used as a plasmid for evaluating transformation efficiency.

(3) Transformation into ΔRrhJ1III, ΔRrhJ1IV, ΔRrhJ1V, ΔRrhJ1VI, ΔRrhJ1VII 2 μL of plasmid pK4 was added to 10 μL of each competent cell prepared in Example 4 (1), and the total amount was adjusted to 20 μL with sterile water. And left on ice for 20 minutes. Subsequently, the entire amount of the competent cell was transferred to an electroporation cuvette with a gap of 1 mm and electroporated under application conditions of 1.5 kV, 400Ω, and 25 μF. Then, it was left on ice for 10 minutes, and then left at 37 ° C. for 10 minutes. To each cuvette, 0.5 mL of each MYK liquid medium was added and mixed well. The suspension was transferred to a Φ10 × 105 mm Wasselmann test tube and shaken overnight at 30 ° C. and 180 rpm for reincubation. The whole culture solution after reversion culture was applied to a MYK agar medium (containing 10 mg / L kanamycin) and cultured at 30 ° C. for 3 days. Table 1 shows the transformation efficiency calculated by counting the number of colonies that appeared on the agar medium.

  The transformation efficiency was calculated by the following formula.

      Transformation efficiency = number of appearance colonies ÷ {added DNA (plasmid) amount [μg]}

  As shown in Table 1, .DELTA.RrhJ1III, .DELTA.RrhJ1IV, .DELTA.RrhJ1V, .DELTA.RrhJ1VI, and .DELTA.RrhJ1VII lacking the nuclease showed significantly higher transformation efficiency than the wild type (J1).

  The present invention provides a method for transforming Rhodococcus bacteria. Since the transformant of Rhodococcus bacteria obtained by the transformation method according to the present invention can impart new properties due to the effect of the introduced DNA, it is used for the development of industrially useful microbial catalysts and the like. it can.

Sequence number 11: Primer SAC-01
Sequence number 12: Primer SAC-02
Sequence number 13: Primer DG-01
SEQ ID NO: 14: Primer DG-02
Sequence number 18: Primer JM64
Sequence number 19: Primer JM65
Sequence number 20: Primer JM66
Sequence number 21: Primer JM67
Sequence number 22: Primer DG-03
SEQ ID NO: 23: Primer DG-04
SEQ ID NO: 26: Primer JM72
SEQ ID NO: 27: Primer JM73
SEQ ID NO: 28: Primer JM84
Sequence number 29: Primer JM85
Sequence number 30: Primer DG-05
Sequence number 31: Primer DG-06
SEQ ID NO: 34: Primer JM76
Sequence number 35: Primer JM77
SEQ ID NO: 36: Primer JM88
SEQ ID NO: 37: Primer JM89
SEQ ID NO: 38: Primer DG-07
SEQ ID NO: 39: Primer DG-08
SEQ ID NO: 42: Primer JM68
SEQ ID NO: 43: Primer JM69
SEQ ID NO: 44: Primer JM70
Sequence number 45: Primer JM71
SEQ ID NO: 46: Primer DG-09
SEQ ID NO: 47: Primer DG-10
Sequence number 50: Primer JM82
Sequence number 51: Primer JM83
SEQ ID NO: 52: Primer JM94
SEQ ID NO: 53: Primer JM119

Claims (7)

  A Rhodococcus bacterium in which a gene encoding a protein involved in the cleavage of foreign DNA is deleted or inactivated. The Rhodococcus bacterium according to claim 1, wherein the protein involved in the cleavage of foreign DNA is any one of the following proteins (a) to (o).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) a protein comprising an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 (c ) A protein comprising an amino acid sequence having 65% or more homology with the protein comprising the amino acid sequence represented by SEQ ID NO: 2 (d) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 (e) the amino acid represented by SEQ ID NO: 4 A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence (f) From an amino acid sequence having 65% or more homology with a protein comprising the amino acid sequence represented by SEQ ID NO: 4 A protein (g) consisting of an amino acid sequence represented by SEQ ID NO: 6 A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in No. 6 (i) 65% or more homology with a protein consisting of the amino acid sequence shown in SEQ ID No. 6 (J) a protein comprising the amino acid sequence having sex (j) a protein comprising the amino acid sequence represented by SEQ ID NO: 8 (k) in the amino acid sequence represented by SEQ ID NO: 8, wherein one or several amino acids are deleted, substituted or added (1) a protein comprising the amino acid sequence represented by SEQ ID NO: 8 and a protein comprising the amino acid sequence having 65% homology or more (m) a protein comprising the amino acid sequence represented by SEQ ID NO: 10 (n ) 1 or several amino acids in the amino acid sequence shown in SEQ ID NO: 10 (O) a protein comprising the amino acid sequence represented by SEQ ID NO: 10 and a protein comprising an amino acid sequence having 65% or more homology The Rhodococcus bacterium according to claim 1 or 2, wherein the gene encoding the protein involved in the cleavage of foreign DNA is the following (a) to (o) or an ortholog thereof.
(A) DNA comprising the base sequence represented by SEQ ID NO: 1
(B) DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1.
(C) DNA comprising a base sequence having a homology of 60% or more with the base sequence described in SEQ ID NO: 1
(D) DNA containing the base sequence represented by SEQ ID NO: 3
(E) DNA that hybridizes under stringent conditions with DNA containing a base sequence complementary to the base sequence represented by SEQ ID NO: 3
(F) DNA comprising a base sequence having a homology of 60% or more with the base sequence described in SEQ ID NO: 3
(G) DNA containing the base sequence represented by SEQ ID NO: 5
(H) a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 5
(I) DNA comprising a base sequence having a homology of 60% or more with the base sequence described in SEQ ID NO: 5
(J) DNA containing the base sequence represented by SEQ ID NO: 7
(K) DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 7
(L) DNA comprising a base sequence having a homology of 60% or more with the base sequence described in SEQ ID NO: 7
(M) DNA comprising the base sequence represented by SEQ ID NO: 9
(N) DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 9
(O) DNA consisting of a base sequence having a homology of 60% or more with the base sequence of SEQ ID NO: 9   The Rhodococcus bacterium according to any one of claims 1 to 3, wherein the Rhodococcus bacterium is Rhodococcus rhodochrous J1 strain or a mutant thereof.   A transformant obtained by transforming the Rhodococcus bacterium according to any one of claims 1 to 4.   Rhodococcus bacteria trait comprising a step of producing Rhodococcus bacteria in which a gene encoding a protein involved in cleavage of foreign DNA is deleted or inactivated, and a step of transforming the obtained Rhodococcus bacteria A method for producing a converter.   A method for improving the transformation efficiency of Rhodococcus bacteria, comprising deleting or inactivating a gene encoding a protein involved in the cleavage of foreign DNA.

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